0 . 6 as the cutoff ( Supplementary file 6 ) .","Among the seven FXR1 target genes identified by ChIP-seq , five showed significant downregulation upon FXR1 knockdown .","The remaining two genes showed moderate downregulation ( Supplementary file 7 ) .","Among the genes whose promoters are occupied by FXR1 , 45 out of 210 genes showed regulation upon FXR1 knockdown in RNA-seq assay ( Figure 5—figure supplement 1D and Supplementary file 7 ) .","RNA-seq revealed significantly more genes regulated by FXR1 than ChIP-seq .","The discrepancy may be attribute primarily to the fact that FXR1 is a well-known mRNA-binding protein , regulating RNA metabolism and stability in addition to the newly discovered function in transcription regulation revealed in this study .","Therefore , FXR1 knockdown could impact on both populations of genes , those regulated by FXR1’s transcriptional activity and those regulated by FXR1’s RNA-binding function .","In addition , the scope and limitation of RNA-seq and ChIP-seq could also contribute to the discrepancy .","In order to assess whether target genes mediate FXR1’s function in proliferation regulation , we utilized siRNAs to knockdown target genes and monitored the change in cell proliferation .","The knockdown efficiency of the target genes by siRNAs was assessed using q-RT-PCR ( Figure 6—figure supplement 1A ) .","Among the seven target genes , knockdown of GLI1 showed the most consistent anti-proliferative effect whereas ARID1A , CITED1 , or SSBP2 knockdown showed less inhibitory effect in a 7-day proliferation assay ( Figure 6—figure supplement 1B ) .","We further evaluated the combinatorial effect on cell proliferation of downregulation of both FXR1 and its target genes .","We performed combinatorial partial knockdowns of both FXR1 and its target genes by applying lower concentration of siRNA ( 5 nM ) and shortening Dox treatment duration ( to 4 days ) .","As shown in the cell image and MTS detection ( Figure 6A ) , the combined knockdown of both the target genes and FXR1 showed an additive effect on cell growth inhibition .","Amongst the target genes , GLI1 downregulation by siRNA showed the most dramatic combinatorial effect with FXR1 knockdown in suppressing cell proliferation .","Upon FXR1 inhibition , cells underwent blebbing , suggesting a role of FXR1 in cell apoptosis .","Following this lead , we found that FXR1 knockdown induced the cleavage of PARP in H358 cells ( Figure 6B ) , a signal event of cell apoptosis .","Furthermore , FXR1 inhibition increased caspase 3/7 activity ( Figure 6B ) .","Consistently , knockdown of FXR1 target gene GLI1 also induced apoptotic signals including the cleavage of PARP and activation of caspase 3/7 in the same cells ( Figure 6C ) .","We observed the similar effect of FXR1 shRNA in inducing PARP cleavage and caspase 3/7 activity in another cell line , HL-60 ( Figure 6—figure supplement 2 ) .","More interestingly , the effect on PARP cleavage could be rescued by ectopic expression of shRNA-resistant full-length FXR1m_a , indicating a direct role of FXR1 in cell apoptosis signaling ( Figure 6—figure supplement 2 ) .","These data suggest that downregulation of FXR1 inhibits cell growth through the activation of cell apoptotic signaling , at least partially through regulating target gene transcription .","Taken together , the findings of our study indicate that FXR1 inhibition suppresses cell proliferation in cancer cells containing TP53 and FXR2 homozygous deletions in a collateral lethality manner .","FXR1 regulates gene transcription , at least in part , to enable control of cell proliferation ."],["The importance of p53 in cancer has led to tremendous efforts to target its activity by therapeutic approaches .","For cancers containing normal TP53 copy number , there has been good progress in interrupting MDM2–p53 interactions to stabilize and promote p53 tumor suppressor activity .","For TP53 mutated cancers , the current strategy is to restore p53 anti-tumor activity ( Khoo et al . , 2014; Soragni et al . , 2016 ) .","No therapeutic strategies have been proposed to target TP53 deletion cancers until a recent report demonstrated that TP53 heterozygous deletion predisposes cancer cells to further suppression of POLR2A , a neighboring gene undergoing concomitant deletion ( Bradner , 2015; Errico , 2015; Liu et al . , 2015 ) .","However , this strategy will not be applicable to cancers with homozygous TP53 deletions in which POLR2A is co-deleted in most cases ( Figure 1—figure supplement 1C ) .","Here , for the first time , we report an approach to target cancers with concomitant homozygous TP53 and FXR2 deletion by inhibiting FXR1 .","The passenger deletion of FXR2 in the TP53 deletion locus renders cancer vulnerable to FXR1 inhibition , which echoes with the recently raised concept of collateral lethality in tumor suppressor gene deletion ( Muller et al . , 2015; Muller et al . , 2012; Nijhawan et al . , 2012 ) .","In this study , several lines of evidence confirmed the redundant function between FXR1 and FXR2:","1 ) both FXR1 and FXR2 can rescue FXR1 shRNA-inhibited cell proliferation ( Figure 1D ) ,","2 ) FXR1 and FXR2 share a significant portion of overlapping binding sites in the genome ( Figure 5I , Figure 5—figure supplement 4B–C ) , and","3 ) knockout of either protein sensitizes TP53 homozygous deletion-containing cancer cells for the inhibition of the remaining member ( Figure 2D , Figure 2—figure supplement 2 ) .","Although another family member , FMR1 , shares structural and functional similarity with FXR1 and FXR2 , our data suggest that FMR1 does not participate in the regulation of proliferation or in rescuing the FXR1 inhibition in the tested cell models .","TP53 homozygous deletion is observed in 1–15% of human tumors , the majority of which have FXR2 homozygous deletions according to the TCGA database .","This study therefore opens the avenue to the development of FXR1-based targeted therapies to treat TP53 homozygous deletion cancers , in which there is a significant unmet medical need .","Interestingly , we have found that TP53 deletion is required for cancer cells to respond to FXR1 inhibition .","This was confirmed in our engineered FXR2 single knockout cell clones which were only sensitive to FXR1 knockdown when p53 was downregulated .","However , the mechanism of action remains elusive .","There have been reports suggesting that p53 deficiency promotes the activation of JAK/STAT signaling , which may lead to cellular dependency on FXR1 because it regulates the localization of STAT1/3 at gene promoters ( Guo et al . , 2014; Spehlmann et al . , 2013 ) .","Considering that FXR1 can both suppress p53 to escape senescence ( Majumder et al . , 2016 ) and activate gene transcription ( evidence from this study ) , p53 deletion may augment FXR1’s role in transcription regulation in controlling cancer cell proliferation .","Given the various critical functions of p53 in governing cellular behavior , its deficiency may create a cell dependency on FXR1 and FXR2 activities .","The underlying molecular mechanism requires further investigation .","Some studies have explored the oncogenic property of FXR1 in lung cancer with FXR1 overexpression or amplification ( Comtesse et al . , 2007; Qian et al . , 2015 ) .","FXR1 copy number gain occurs in some types of cancers , particularly in lung squamous carcinoma ( Comtesse et al . , 2007; Qian et al . , 2015 ) and its occurrence is mutually exclusive with TP53 and FXR2 homozygous deletion according to TCGA database ( Figure 1A ) .","Together with our study , this evidence suggests that FXR1 controls tumor growth in cancers with either TP53/FXR2 homozygous deletion or FXR1 amplification .","Furthermore , we reveal a novel function of FXR1 in transcriptional regulation and elucidate its molecular mechanism in regulating cell proliferation , in addition to its well-established role in mRNA binding .","Our ChIP-MS study revealed that FXR1 interacts with chromatin regulators and transcription factors .","In addition , about a half of FXR1’s peaks in the ChIP-seq assay co-localize with H3K4me3 peaks at gene promoters , suggesting a new role for FXR1 in facilitating active transcription .","FXR1 interacts with and recruits or stabilizes STAT1 or STAT3 at the target gene promoters , suggesting that cooperation with STATs is required for its transcriptional activity .","Indeed , the inhibitor of the STAT upstream kinase JAK suppressed some of FXR1’s target genes expression .","Reciprocally , FXR1 is required for the localization of STATs on its target gene promoters .","Generally , it is believed that JAK-catalyzed STAT1/3 phosphorylation drives their shuttling to the nucleus and localization at gene promoters in order to regulate transcription ( Levy and Darnell , 2002 ) .","Our findings advance the current views about the regulation of transcription by STAT1/3 by revealing the necessity for FXR1 in regulating STAT occupancy at promoters .","STAT is well established in regulating cytokine gene transcription .","We have also observed that FXR1 knockdown inhibited the expression of multiple cytokines ( data not shown ) , consistent with a recent report of FXR1 function in positively regulating cytokine expression in monocytes ( Le Tonqueze et al . , 2016 ) .","Notably , only about 0 . 7–1 . 57% of the STAT1/3 peaks overlap with FXR1 peaks in our ChIP-seq study , suggesting that FXR1 only participates in the transcription of a very small fraction of JAK-STAT target genes .","Although JAK inhibition suppressed the expression of some FXR1 target genes , it had no effect on inhibiting cancer cell proliferation in FXR1 shRNA-sensitive cells ( Figure 5—figure supplement 6 ) .","This is in line with the fact that JAK-STAT signaling pathway governs a much broader spectrum of gene expression and cellular behaviors .","It w worth investigating whether other epigenetic regulators are involved in STAT transcriptional activity .","Among the FXR1 peaks , approximately 25% overlapped with STAT peaks according to our study in H358 cells .","FXR1 may also cooperate with other transcription factors in regulating transcription; examples include BTF ( Ma et al . , 2014 ) which was also identified in an FXR1 protein complex in our ChIP-MS study .","In addition , we identified several other FXR1-interacting proteins that have essential function in transcription regulation , including TOP2A/2B , GTF2A , and FACT80 .","Further investigations are needed to dissect the underlying mechanism of FXR1 in transcription regulation .","FXR1 is an RNA-binding protein that regulates mRNA stability , transportation of RNA from the nucleus to the cytoplasm , and translation through its functional KH domains and RGG box .","Our ChIP-MS data confirmed the established role of FXR1 , which binds to and regulates mRNA processing and translation ( Figure 4B ) .","Furthermore , we confirmed ChIP-MS findings and discovered that FXR1 binds to the mRNA of target genes such as CASC4 , TRAPPC9 , GLI1 , and SSBP2 in the mRNA binding assays ( Figure 6—figure supplement 3 ) .","Our study confirmed that , in addition to its direct function in transcription , FXR1 binds to some but not all of its target gene mRNAs , which may contribute to its role in proliferation regulation .","This raises the possibility that FXR1 has dynamic functions in transcription and subsequent mRNA processing .","The newly discovered N-terminal tandem Tudor domain has a high degree of similarity among the three Tudor family members ( Adams-Cioaba et al . , 2010; Hu et al . , 2015; Myrick et al . , 2015 ) .","The Tudor domain in FMR1 was reported to bind to multiple methylated lysine at histone H3 and to participate in DNA damage repair ( Alpatov et al . , 2014 ) .","Our study shows that the tandem Tudor in FXR1 binds to methylated lysine in histone H3 in a similar manner as that in FMR1 and it is required for FXR1’s function in regulating cancer cell proliferation .","Further investigation is needed to study the amino acid residues and the structure of the aromatic cage within the FXR1 tandem Tudor domain that are responsible for histone binding , which might provide the foundation for development of Tudor domain inhibitors .","The observation that FXR1 knockout mice die shortly after birth and knockdown of FXR1 causes abnormalities in striated muscle and heart in zebrafish ( Mientjes et al . , 2004; Zarnescu and Gregorio , 2013 ) raise potential safety concerns with regard to inhibiting FXR1 in cancer patients .","However , the inducible FXR1 knockout in adult mice didn’t display the lethal phenotype ( Mientjes et al . , 2004 ) , suggesting the potential of FXR1 as a drugging target .","Overall , this study provides an important approach to targeting human cancers with TP53 homozygous deletions .","The novel function of FXR1 in transcription advances our knowledge regarding FXR1’s function in gene expression on top of its known roles in mRNA processing and translation ."],["Copy-number analysis in human tumor tissues was performed using data from The Cancer Genome Atlas ( TCGA ) ( http://www . cbioportal . org/cross_cancer . do ) as previously described ( Gao et al . , 2013 ) .","Gene copy numbers in human cancer cell lines were downloaded from the Cancer Cell Line Encyclopedia database ( CCLE ) ( http://www . broadinstitute . org/ccle ) and analyzed using an R package ComplexHeatmap ( https://bioconductor . org/packages/release/bioc/html/ComplexHeatmap . html ) .","KATOIII , HL-60 , H358 , MKN45 , AGS , HepG2 , A549 , H1299 , MG-63 , SKOV3 , Hep3B , and HEK 293 T cell lines were purchased from American Type Culture Collection ( ATCC , Manassas , Virginia , USA , http://www . atcc . org ) .","The KMS-11 cell line was obtained from Horizon Discovery Ltd . ( Cambridge , UK ) .","The L540 cell line was purchased from Biovector Science Lab , Inc ( Beijing , China ) .","Although L540 is listed as a misidentified cell line by ICLAC ( International Cell Line Authentication Committee ) , we used it as a TP53 single deletion cell model after comfirming the abscence of p53 mRNA and protein expression .","The cell lines were cultured in RMPI 1640 medium ( GIBCO ) or DMEM medium ( GIBCO ) supplemented with 10% fetal bovine serum ( FBS , GIBCO ) and 100 U/mL penicillin-streptomycin ( GIBCO ) at 37°C with 5% CO2 .","All cell lines were authenticated by Short Tandem Repeat ( STR ) profiling and routinely monitored for mycoplasma contamination .","The source and mycoplasma status as well as the Research Resource Identifiers ( RRIDs ) of each cell line are listed in Supplementary file 8 .","Cell proliferation rate was measured by MTS assay .","Cell apoptosis was determined by analyzing Caspase3/7 activity .","The luminescence signal in the Caspase3/7 assay was recorded by an Envision 2104 Multilabel reader ( Perkin Elmer , Waltham , MA ) .","The detailed procedure and information are in the Supplementary methods .","ChIP sample preparation follows the manual of SimpleChIP Enzymatic Chromatin IP Kit ( Magnetic Beads , CST , #9003 ) .","The experimental procedure and reagent information for ChIP-WB , ChIP-MS , ChIP-seq , or ChIP-PCR are in the Supplementary methods .","In cell assays , three independent replicates were included in each experiment .","Student T Test was performed to compare the differences between two groups with normal distribution .","All data were analyzed using GraphPad Prism 6 .","Gene-specific shRNA sequences were obtained from the Sigma website ( http://www . sigmaaldrich . com/ ) , and the hairpin sequences were cloned into the doxycycline ( Dox ) -inducible lentiviral vector pLKO-Tet-On with Puromycinas as a selection marker .","Five shRNAs targeting each gene ( FXR1 , FXR2 and STAT3 ) were screened .","Lentiviral particles were packaged in HEK 293 T cells by transient transfection .","Briefly , 6 µg of shRNA encoding lentiviral plasmid , 0 . 6 µg of helper PCG10 plasmid and 5 . 4 µg of helper PCG41 plasmid were mixed with 30 µl X-tremeGENE HP DNA Transfection Reagent ( Roche , 06366236001 ) and co-transfected into HEK 293 T cells at 70–80% confluency in a 10 cm Petri dish .","At 72 hr post transfection , the supernatant containing virus particles was collected and filtered through a 0 . 45 µm PVDF Millex syringe filter .","Each cell line was subjected to a puromycin tolerance test to determine the optimal concentration for establishing stable cell lines .","To generate a shRNA-expressing stable cell line , cells were infected with lentiviral particles with 8 µg/ml polybrene .","After 48 hr , the cells were selected by 0 . 5–2 µg/ml puromycin ( GIBCO , A11138-03 ) for 7 days .","shRNA expression was induced by 0 . 1–1 µg/ml Dox ( Sigma , D9891 ) , and the knockdown efficiency was tested by WB assay or q-RT-PCR .","Dox concentration was optimized for each stable cell line to achieve the most optimal knockdown efficiency at the minimum dose .","For ectopic expression of proteins of interest , cells were infected with the lentiviral pLenti6 . 3-MCS construct encoding the FXR1 , FXR2 or FMR1 gene open reading frame and selected using 2–6 µg/ml blasticidin ( GIBCO , A11139-03 ) for stable cell line generation .","The inserted open reading frames are: FXR1_a , NM_005087 . 3; FXR1_b , NM_001013438 . 2; FXR1_c , NM_001013439 . 2; FXR2 , – NM_004860 . 3; and FMR1 , NM_002024 . 5 .","The sequence of the FXR1 shRNA resistant mutation was CGCCAGGTTCCATTTAATGAA mutated to CGTCAAGTACCGTTCAACGAG and TTGCGAAGTATTCGTACGAAGTTG mutated to TTACGGAGCATCCGAACAAAATTA .","FXR1 shRNAs were inserted in adenoviral vector pAd/CMV/IRES/GFP .","The adenoviruses were produced in HEK 293 T cells by transient transfection of the plasmids .","Virus titer was determined by counting GFP-positive cells after adenoviral infection .","In the proliferation assay , adenoviruses were added to the cell culture medium and replenished 4 days afterwards to achieve efficient FXR1 knockdown .","The shRNA target sequence is listed in Supplementary file 8 .","Cells were lysed using RIPA lysis buffer ( CST , 9806S ) with complete protease inhibitor cocktail ( Sigma , P8340 ) and phosphatase inhibitor cocktail ( Sigma , P5726 ) .","The protein concentration was detected by BCA kit ( Thermo Scientific , Cat#23225 ) .","The protein lysate was denatured by adding NuPAGE sample reducing agent ( Novex , Invitrogen , NP0009 ) and NuPAGE LDS Sample buffer ( Novex , Invitrogen , NP0007 ) followed by heating at 95°C for 5 min .","Equal amounts of protein lysate was loaded to the NuPAGE Electrophoresis System ( Life Technologies ) according to the manufacturer's protocol .","Proteins in the gel were transferred to the nitrocellulose membrane in iBlot 2 Transfer Stacks ( IB23001 and IB23002 ) using the iBlot 2 Dry Blotting System ( Life Technology , IB21001 ) .","The membrane was blocked in SuperBlock Blocking Buffer in TBS ( Pierce , 37535 ) , incubated with the specific primary antibodies for the protein of followed by reaction with the secondary antibodies .","The protein signals were detected using SuperSignal West Femto Maximum Sensitivity Substrate ( Thermo , 34095 ) , and were scanned and quantified using ChemiDoc MP System ( BIO-RAD ) .","The antibodies and their Research Resource Identifiers ( RRIDs ) used in this study are listed in Supplementary file 8 .","Cell proliferation rate was detected by MTS assay .","Briefly , the cells were seeded in 96-well plates at optimal density in 100 µl medium per well followed by treatment .","Cells were added with CellTiter 96 AQueous One Solution Reagent ( Promega , G3581 ) at the indicated time by following the manual instructions and subjected to absorbance recording at 490 nm using a PowerWave HT Microplate Spectrophotometer ( BioTek ) .","The cell proliferation rate in MTS was determined by measuring absorbance at 490 nm .","In the duplicated experiment , cell images were scanned after fixing the cells with cold methanol and staining with 0 . 1% crystal violet .","In some cases , the cells were seeded on the solidified Matrigel ( BD Bioscience ) layer in 96-well plate ( 50 µl per well ) to measure the organoid growth .","Cell apoptosis was determined by analyzing Caspase3/7 activity using the Caspase-Glo3/7 assay kit ( Promega , G8092 ) according to the kit instructions or by detecting the protein level of cleaved PARP ( antibody from Cell Signaling Technology , 9541 ) in Western blot .","The luminescence signal in the Caspase3/7 assay was recorded by an Envision 2104 Multilabel reader ( Perkin Elmer ) .","The HL-60-derived xenograft model in female BALB/c nude mice was used to determine FXR1’s function in tumor growth .","Briefly , the female BALB/c nude mice ( age 6–8 weeks ) housed under pathogen-free conditions were inoculated subcutaneously in the right flank with HL-60 cells stably expressing control shRNA ( shCtrl ) or Doxycycline-inducible FXR1-sh3 .","Each mouse received 5 × 106 cells in 0 . 2 ml PBS:Matrigel ( 1:1 mixture ) for tumor development .","When tumor size reached approximately 180 mm3 on day 14 , mice were randomly grouped ( n = 6 ) and Doxycycline ( 2 mg/ml ) was added to drinking water .","Doxycycline was replenished twice per week .","Tumor volume and body weight were measured twice a week .","Tumor size was measured using a calliper , and tumor volume was calculated according to the following equation: tumor volume ( mm3 ) = length ( mm ) × width2 ( mm2 ) × 0 . 5 .","The experiment was terminated when the tumor volume reached 2 , 000 mm3 according to animal welfare guidelines .","All animal studies were carried out under IACUC number R20150728-Mouse and Rat approved by the Institutional Animal Care and Use Committee ( IACUC ) of WuXi AppTec .","Summary statistics , including mean and the standard error of the mean ( s . e . m ) , were provided for the tumor volume of each group at each time point .","The A549-derived xenograft model was studied using a similar method .","Guide RNA ( gRNA ) was designed according to the formula of GN20GG or CCN20C in the forward or reverse strand of genomic DNA , respectively ( Cong et al . , 2013; Ran et al . , 2013; Shalem et al . , 2014; Wang et al . , 2013 ) .","Two sets of gRNAs targeting the 5’ and 3’ sequences of the genomic region of interest ( TP53-to-FXR2 , TP53 alone , FXR1 alone , or FXR2 alone ) were designed .","The synthesized double-strand DNA of the gRNA was inserted into the pU6-gRNA_SPycas9-2Acherry vector expressing Cas9 protein ( Mavrakis et al . , 2016 ) .","To test gRNA efficiency , gRNA-expressing plasmids were transfected into cells using XtremeGene reagent ( Roche ) .","Genomic DNA was extracted using the Purelink Genome DNA mini kit ( Invitrogen , K1820-02 ) .","PCR was performed using genomic DNA as the template and the Accuprime Pfx SuperMix PCR system ( Invitrogen , 12344040 ) to amplify the sequence containing the gRNA PAM position , and PCR product was sequenced to determine the successful cut by gRNA-guided Cas9 by monitoring the miscellaneous peaks .","The efficient gRNA target sequences were: TP53-5’-gRNA , GGGCAGCTACGGTTTCCGTCTGG; TP53-3’-gRNA , GGTGTGCGTCAGAAGCACCCAGG; FXR2-5’-gRNA , GGAGGCGGTGGCGGCGCCATGGG; FXR2-3’-gRNA , GGGGGGTGGCACAGCAGCTTGGG; FXR1-5’-gRNA , GTGGAGGTTCGCGGCTCTAA; FXR1-3’-gRNA , GAAAAAAAAGTTGCTGGCTAT .","The donor plasmid contains the recombination sequences compatible with the 5’ and 3’ arms of the genomic region of interest and the GFP-coding sequence in vector pcDNA3 . 1 .","The mixture of gRNA-Cas9 plasmids targeting the 5’ or 3’ sequence of the genomic region and donor plasmid at ratio 1:1:1 was transfected into the candidate cells in a 10 cm Petri dish using XtremeGene .","One week post-transfection , the GFP-expressing cells were sorted and seeded into a 96-well plate at a density of one cell per well using FACSArisII ( BD ) .","The single cell clones were cultured for two weeks to identify and filter out the cells with transient GFP signal .","The clones with strong GFP signal , which would have potential insertion and replacement of the genomic region of interest by the donor sequence , were subjected to knockout validation by genomic PCR-sequencing , q-RT-PCR , and Western blot .","The sequence of genomic PCR primers is listed in Supplementary file 8 .","ChIP sample preparation: ChIP samples were prepared using SimpleChIP Enzymatic Chromatin IP Kit ( Magnetic Beads , CST , #9003 ) according to the manufacturer’s protocol .","Briefly , cells cultured in 15 cm Petri dishes were crosslinked with 1% formaldehyde when reaching 70–80% confluence .","Crosslinking was terminated by adding 10 × glycine followed by rinsing the cells twice with cold PBS , and 4 × 107 cells were collected into one 15 ml conical tube .","The cell pellet was resuspended and incubated in Buffer A on ice for 10 mins for cell lysis .","The nuclei were collected by centrifugation at 3 , 000 rpm for 5 mins at 4°C and resuspended in Buffer B followed by centrifugation and supernatant removal .","The nuclei pellets were resuspended in 1 . 0 ml Buffer B , and transferred to a 1 . 5 ml microcentrifuge tube .","7 . 5 µl of Micrococcal Nuclease was added and incubated for 20 mins at 37°C to digest the DNA to lengths of approximately 150–300 bp .","After the termination of digestion by adding 100 µl of 0 . 5 M EDTA , the nuclei pellets were collected by centrifuging at 13 , 000 rpm for 1 min at 4°C .","The nuclear pellets were resuspended in 1 ml ChIP buffer and separated into two tubes , and incubated on ice for 10 mins followed by subsequent sonication of lysates at 9W , 10 s ON/30 s OFF , nine cycles .","Lysates were collected by centrifugation at 10 , 000 rpm for 10 mins at 4°C .","The supernatant ( single nucleosome lysate ) was transferred to a new tube , and stored at −80°C for the following experiments .","50 µl of lysate was used for the analysis of chromatin digestion .","Pulldown: The single nucleosome lysate was diluted with ChIP buffer with protease inhibitor cocktail ( for each pulldown , 200 μl lysate was added into 300 μl 1 × ChIP Buffer ) .","10 µl of sample was saved as input .","To reduce nonspecific binding , the lysate was pre-cleared with 10% BSA and 30 μl ChIP Grade Protein G Magnetic Beads ( CST , #9006 ) at 4°C with rotation for 1 hr .","The supernatant was collected by placing the tube on a magnetic separation rack .","Antibodies of interest or IgG control were added and incubated on rotation at 4°C overnight followed by incubation with 30 µl of magnetic beads for 2 hr at 4°C .","Beads were washed with low salt wash buffer ( 100 µl 10 × ChIP Buffer in 900 ml water ) three times and with high salt wash buffer ( 100 µl 10 × ChIP Buffer and 70 µl 5 M NaCl in 830 µl water ) once on rotation .","The antibodies used in ChIP pulldown were: anti-FXR1 ( Sigma , HPA018246 , RRID: AB_1849204 ) ; anti-FXR2 ( Invitrogen , PA5-28979 , RRID: AB_2546455 ) ; anti-STAT1 ( Santa Cruz , sc-345 , RRID: AB_675903; CST , 14994S ) ; anti-STAT3 ( Santa Cruz , sc-482 , RRID: AB_632440; CST , 12640S , RRID: AB_2629499 ) ; rabbit IgG ( CST , 2729 , RRID: AB_1031062 ) ; anti-H3K4me3 ( CST , 9727 , RRID: AB_561095 ) ; anti-H3K9me3 ( Active Motif , 39161 , RRID: AB_2532132 ) ; and anti-H3K27me3 ( Millipore , 07–449 , RRID: AB_310624 ) .","For ChIP-WB , protein G beads were added with 30 μl of DTT-containing 2 × SDS sample buffer and denatured at 95°C in thermomixer with gentle vortexing ( 1 , 200 rpm ) for 5 min .","Eluted proteins were separated in SDS-PAGE gel and analyzed by WB .","For ChIP-MS , eluted proteins were separated in SDS-PAGE gel followed by gel fixing in buffer containing methanol , acetic acid , and water at 5:1:4 ratio .","The gel was stained in EZBlue Gel Staining Reagent ( Sigma , G1041 ) and subjected to mass spectrometry ( MS ) analysis .","For ChIP-seq or ChIP-PCR , beads were eluted with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing ( 1 , 200 rpm ) in thermomixer .","Eluted supernatant was incubated with 6 µl 5 M NaCl and 2 µl Proteinase K at 65°C for 2 hr in thermomixer to digest the protein .","DNA was purified using spin columns , and sent for ChIP-seq or ChIP-PCR analysis .","DNA from ChIP pull down complexes was subjected to library preparation according to Illumina's instructions ( Part # 11257047 ) .","Sequencing was performed on Illumina HiSeq 2000 .","ChIP-seq data quality was analyzed using FastQC ( Andrews , 2010 ) .","Reads in the fastq files were trimmed by 5 bp in the 5´-end , and then mapped to the reference human genome ( HG19 ) using BOWTIE2 .","Multiple mapped reads and duplicate reads were removed .","FXR1 binding genomic regions ( called ChIP-seq peaks ) were identified by the Model-based Analysis of ChIP-Seq ( MACS ) algorithm using the default p-value cutoff of 1 × e−5 .","The distribution of FXR1 ChIP-seq peaks were investigated by the annotations on the human genome .","The peaks were binned into eight groups according to the positions relative to annotated genes: 5’ Distal ( up to 100 kb upstream TSS ) , 5’ Proximal ( up to 10 kb upstream TSS ) , 5’ TSS ( 5 kb upstream TSS to 1 kb downstream TSS ) , Intragenic ( 1 kb downstream TSS to 1 kb upstream TTS ) , 3’ TTS ( 1 kb upstream TTS to 5 kb downstream TTS ) , 3’ Proximal ( up to 10 kb downstream TTS ) , 3’ Distal ( up to 100 kb downstream TTS ) , and the remaining sequence defined as Intergenic region .","The aggregation signals of FXR1 , H3K4me3 , STAT1 , and STAT3 at genebody ( TSS to TTS ) plus upstream and downstream 5 kb region were extracted and plotted using in-house perl and MATLAB code .","A hub for the UCSC genome browser was constructed in order to investigate the detailed information of the peaks .","The summit positions of the FXR1 ChIP-seq peaks were extended to 100 bp both upstream and downstream as input region for HOMER ( Heinz et al . , 2010 ) to call known and de novo motifs .","The overlap between ChIP-seq peaks was determined by 1 bp overlap of two peaks that were extended by 500 bp both upstream and downstream .","The TSS associated genes in each ChIP-seq were determined by overlapping peaks to refseq genes coordinates .","Venn diagrams were generated using online web tool VENNY 2 . 1 ( http://bioinfogp . cnb . csic . es/tools/venny/index . html ) .","Protein function enrichment was analyzed using DAVID ( Huang et al . , 2008 , 2009 ) .","The ChIP-seq peaks were visualized using IGV ( http://www . broadinstitute . org/igv/ ) .","The sequence of ChIP-PCR primers is listed in Supplementary file 8 .","FXR1 or IgG control ChIP pulldown complexes were separated in SDS-PAGE gel .","The gel was stained with EZblue ( Sigma , G1041 ) .","The stained gel was cut into multiple parts based on protein molecular weight .","Gel pieces were destained in solution containing 50 mM NH4HCO3 ( Sigma Aldrich ) and Acetonitrile ( ACN ) ( Fisher Scientific ) at 1:1 ratio , dried with SpeedVac , rehydrated in 300 μl of 10 mM DTT ( Sigma Aldrich ) solution at 56°C for 1 hr , and reacted in 25 mM IAA ( Sigma Aldrich ) at 37°C for 30 mins .","After washing with 25 mM NH4HCO3 ( Sigma Aldrich ) for 10 mins , the gel pieces were digested with trypsin solution ( 0 . 01 g/L trypsin in 25 mM NH4HCO3 ) ( Promega ) at 37°C for 12 hr .","The digested peptides were extracted using 300 μl of 50% ACN-0 . 1% FA .","The extracted solution was collected and concentrated with SpeedVac .","The samples were resolved with 0 . 1% FA and analyzed by nanoLC-MS/MS ( Thermofisher ) .","MS/MS was performed in a data-dependent mode in which the top 10 most abundant ions ( S/N > 30 ) for each MS scan were selected for MS/MS analysis by HCD .","Parameters used during searching included enzyme , trypsin; missed cleavages , One; dynamic modifications , oxidation ( M ) ; static modifications , Carbamidomethyl ( C ) ; precursor mass tolerance , 10 ppm; fragment mass tolerance , 0 . 1 Da .","Parameters in data filtration with Prohits included kinds of exclusion proteins: Heat Shock , Ribosomal , Cytoskeleton , Keratin , Artifact Protein , Rib , Nucleoprotein and Albumin; Max SAINT < 0 . 7 ( Liu et al . , 2012; Liu et al . , 2010 ) .","All MS and MS/MS data were searched in the combined mode against Uniprot Human database ( August , 2013 ) using the Proteome Discoverer 1 . 3 software ( Thermofisher ) .","A score greater than 31 ( confidence interval values > 95% ) obtained with the Mascot search engine ( Matrix Science , U . K . ) was considered as significant .","All the searching results were filtered with Prohits software to remove non-specific binding proteins ( Max SAINT < 0 . 7 ) ( Liu et al . , 2010 , 2012 ) .","FXR1 interacting proteins were classified according to the GO ( gene ontology ) database .","Protein function clustering was analyzed with DAVID .","Visualization and network map of FXR1-interacting proteins were delineated using BioGRID in Cytoscape .","Protein–protein interaction was analyzed using the KEGG ( Kyoto Encyclopedia of Genes and Genomes ) pathway database .","Total RNA was extracted using RNeasy Mini Kit ( Qiagen , 74106 ) and Qiagen QIAcube 230 V w . starter Pack-2 machine , and reverse-transcribed using Oligo ( dT ) primer and the SuperScript III First-Strand synthesis system ( Invitrogen , 18080–051 ) .","q-RT-PCR using cDNA product as the template was performed using Power SYBR Green PCR Master Mix ( Applied Biosystems ( AB ) , 4367659 ) and an ABI 7900HT Fast Real-Time PCR-3 Machine with specific primers .","Gene expression was quantified using the comparative Ct ( cycle threshold ) method and the results were normalized to GAPDH .","Sequences of q-RT-PCR primers are listed in Supplementary file 8 .","Each RNA-seq sample had three biological replicates .","RNA isolation was performed using Trizol and RNeasy MinElute Cleanup Kit from cells .","Following extraction , RNA quantity was assessed using Nanodrop and the integrity of total RNA using the RNA 6000 Nano Kit on an Aligent 2100 Bioanalyzer .","An RNA library was constructed using the TruSeq RNA Sample Preparation Kit .","The first step in the workflow involved purifying the poly-A containing mRNA molecules using oligo-dT-attached magnetic beads .","Following purification , mRNA was fragmented into small pieces using divalent cations under elevated temperature .","The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers .","Second-strand cDNA synthesis followed , using DNA PolymeraseI and RNase H . The cDNA fragments then went through an end repair process , involving the addition of a single ‘A’ base , and then ligation of the adapters .","The products were then purified and enriched with PCR to create the final cDNA library .","The clusters of the cDNA library were generated on HiSeq X HD PE flow cell .","Then the clusters were sequenced on a HiSeq X system .","RNA-seq data quality was analyzed using FastQC ( Andrews , 2010 ) .","Reads in the fastq files were trimmed by five base pairs at the 5´-end , and then mapped to the reference human genome ( HG19 ) with the algorithm STAR using default parameters .","Multiple mapped reads and duplicate reads were removed to extract raw reads of refseq genes using HOMER .","Differential expressed genes were identified using R package DESeq2 and determined using log2FC cutoff 0 . 6 and adjusted p value cutoff 0 . 01 .","KEGG pathway enrichment was investigated using GSEA ( Subramanian et al . , 2005; Mootha et al . , 2003 ) .","Histone H3 MLA proteins with methylation at various residues were purchased from Active Motif ( 31208–31222 ) .","His-tagged-FXR1-Tudor ( 2-132 ) protein were expressed and purified from an Escherichia coli system , and pulldown assay was performed as previously described ( Alpatov et al . , 2014 ) .","In brief , in each reaction , 2 µg of MLA protein and 2 µg of FXR1-Tudor protein were added in 500 µl of Histone MLA Binding Buffer ( 50 mM Tris pH 7 . 5 , 1 M NaCl , 2 mM MgCl2 , 0 . 5% Triton X-100 , and 10 mM Imidazole ) ( save 10 µl as input ) , followed by rotation at 4°C for 30 mins .","The reaction solution was spun down ( 10 , 000 g , 2 min ) to collect the supernatant in a new tube .","15 μl of Ni-NTA beads ( Qiagen ) was added to the collected supernatant followed by rotation at 4°C for 1 hr .","Beads were collected by spinning at 1 , 000 g for 2 min , washed with 1 ml of Histone MLA Binding Buffer five times , suspended in 30 μl of 2 × SDS sample buffer and boiled to denature the proteins .","The FXR1-bound histone MLA proteins were detected in a WB assay .","JAK inhibitor S-Ruxolitinib ( dual JAK1/2 inhibitor tool compound ) was purchased from selleck ( http://www . selleck . cn/ ) .","Owing to the instability of the compound , the medium with inhibitor was replaced every 5 days .","Gene-specific siRNA sequence was designed using the software in Hannon Lab ( http://cancan . cshl . edu/RNAi_central/RNAi . cgi ? type=siRNA ) .","The siRNA oligonucleotides were produced by Invitrogen , and the MISSION siRNA Universal Negative Control #2 ( Sigma ) was used as a negative control .","Five siRNAs for each gene were tested .","siRNAs were introduced into cells at a final concentration of 5–20 nM using Lipofectamine RNAiMAX Transfection Reagent ( Invitrogen , 13778–150 ) and Opti-MEM ( Gibco , 31985–062 ) according to the manufacturer’s protocol .","Knockdown efficiency was assessed 48 hr post-transfection by q-RT-PCR or WB .","Sequences of siRNAs are listed in Supplementary file 8 .","Flag-tagged FXR1 or HA-tagged-STAT1 or -STAT3 proteins were expressed in HEK 293 T cells .","Cell lysates were prepared using lysis buffer ( 50 mM Tris-Cl , pH 7 . 5 , 500 mM NaCl , 1% Triton X-100 ) with complete protease inhibitor cocktail ( Sigma , P8340 ) , phosphatase inhibitor cocktail ( Sigma , P5726 ) , and PMSF ( Sigma , 93482–50 ml-F ) .","Lysate was incubated , at 4°C overnight , with 100 μl Flag-M2 magnetic beads ( Sigma , M8823 ) to purify Flag-tagged-FXR1 protein or with 100 μl Influenza Hemagglutinin ( HA ) magnetic beads ( Thermo Fisher , 88837 ) to purify HA-tagged-STAT protein .","Beads were washed with 1 ml lysis buffer three times and with 600 μl elution buffer ( 50 mM Tris-Cl , pH7 . 5 , 500 mM NaCl , 10% Glycerol ) once .","Target protein was eluted with 30 μl of Flag peptide solution ( 5 mg/ml , in elution buffer , Sigma , F4799-4MG ) to obtain Flag-tagged-FXR1 protein or with HA peptide ( 2 mg/ml , in elution buffer , Sigma , I2149-1MG ) to harvest HA-tagged-STAT protein .","For in vitro pulldown , 25 µl purified Flag-tagged-FXR1 protein was incubated with 75 µl purified HA-tagged-STAT1 or -STAT3 protein in 500 µl binding buffer ( 50 mM Tris pH 7 . 5 , 150 mM NaCl , 0 . 1% NP-40 , 1 mM DTT , phosphatase inhibitor and protease inhibitor cocktail ) at 4°C overnight .","30 µl Flag or HA magnetic beads were added and incubated at 4°C for another 3 hr .","Beads were washed with wash buffer ( 50 mM Tris-pH 7 . 5 , 300 mM NaCl , 0 . 1% NP-40 ) three times .","The eluted products in 30 µl elution buffer were subjected to WB analysis .","H358 cells were lysed in lysis buffer ( 100 mM NaCl , 10 mM MgCl2 , 30 mM Tris-HCl , pH 7 . 5 , 1 mM dithiothreitol [DTT] , protease inhibitor cocktail , 100 U/ml RNasin , 0 . 1% Nonidet P40 [NP40] ) and divided into two parts .","2% of lysate was saved as input .","The lysate was pre-cleared by 10% BSA and 30 µl ChIP Grade Protein G Magnetic Beads ( CST , #9006 ) on rotation for 1 hr at 4°C .","The supernatant was collected by a magnetic separation rack , incubated with FXR1 antibody ( Sigma , HPA018246 ) or IgG control ( CST , 2729 ) for 1 hr at 4°C , and incubated with 30 µl of magnetic beads for 1 hr at 4°C on rotation .","Beads were washed with NT2 buffer ( 50 mM Tris-Cl , 150 mM NaCl , 1 mM MgCl2 , and 0 . 05% NP-40 ) five times .","Endogenous RNA protein complexes ( RNP ) were eluted from antibody/Protein G beads by incubation with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing ( 1200 rpm ) in thermomixer .","Proteins in the elution were digested by Proteinase K . RNA was purified using spin columns from RNeasy Mini Kit ( Qiagen , 74106 ) and reverse-transcribed using Oligo ( dT ) primer and the SuperScript III First-Strand synthesis system ( Invitrogen , 18080–051 ) .","The cDNA was subjected to RT-PCR assay using AccuPrime Pfx SuperMix ( Invitrogen , 12344040 ) ( Qian et al . , 2015 ) .","The PCR products are detected by electrophoresis in agarose gels and stained with ethidium bromide , visualized by ChemiDoc .","The non-target genes GAPDH and Actin serve as the negative controls .","ChIP-seq data reported in this paper have been deposited in the Gene Expression Omnibus ( GEO ) database under accession number GSE79707 .","RNA-seq data reported in this paper have been deposited in the Gene Expression Omnibus ( GEO ) database under accession number GSE101754 ."]],"string":"[\n [\n \"p53 , a critical tumor suppressor , primarily serves as a transcription factor to control various cellular stress-response signaling pathways , including cell cycle arrest , DNA repair , senescence and apoptosis ( Vousden and Lane , 2007 ) .\",\n \"Loss of p53 function through mutation or deletion of its encoding TP53 is a common feature in a majority of human cancers , resulting in the escape from tumor-suppressor activities .\",\n \"Numerous strategies have been explored to reverse dysregulated p53 suppressor function , including stabilizing p53 expression by antagonizing the p53–MDM2 interaction in cancers harboring normal TP53 copy number , and restoring p53's tumor suppressor activity in TP53-mutated cancer ( Khoo et al . , 2014; Soragni et al . , 2016 ) .\",\n \"In cancer , tumor suppressor genes are often inactivated by genomic deletions that encompass the deletion of neighboring genes .\",\n \"Such bystander genes often belong to multigene families; therefore , their deletion is tolerated due to genetic redundancy .\",\n \"The newly proposed ‘collateral lethality’ concept suggests that such passenger deletion predisposes cancer cells to vulnerabilities that are induced by further inhibition of the remaining genes in the family , whose functions are essential and redundant ( Muller et al . , 2012 , 2015; Nijhawan et al . , 2012 ) .\",\n \"This concept therefore opens the avenue to anti-cancer drug development targeting cancers containing co-deletion of tumor suppressor genes and neighboring genes without affecting wild-type cells .\",\n \"Drugging TP53-deleted cancers had been a challenging task until a recent report demonstrated inhibition of a neighboring gene POLR2A , which is located about 200 kb downstream of TP53 on chromosome 17 and undergoes heterozygous deletion in colorectal cancers containing TP53 heterozygous deletion ( Liu et al . , 2015 ) .\",\n \"Homozygous deletion , resulting in inactivation of both alleles , occurs less frequently and is more focal than heterozygous deletion .\",\n \"There is no documented therapeutic strategy targeting homozygous TP53-deleted cancers .\",\n \"POLR2A is co-deleted in a majority of tumors with TP53 homozygous deletion , and thus its inhibition would not be relevant .\",\n \"FXR2 ( Fragile X-related Protein 2 , also known as FXR2P ) , located 100 kb downstream of TP53 , is also a neighboring gene of TP53 at chromosome 17p13 . 1 .\",\n \"It belongs to the fragile X gene family that has essential functions in binding and regulating mRNA stability , transportation and translation ( Ascano et al . , 2012; Chen et al . , 2014; Darnell et al . , 2001; Siomi et al . , 1996 ) .\",\n \"In this study , we investigated whether FXR2 passenger deletion at the TP53 homozygous deletion locus would result in subsequent cancer-specific vulnerability to inhibition of its family member , FXR1 ( Fragile X-related Protein 1 , also known as FXR1P ) .\",\n \"The fragile X gene family contains three mammalian members , including fragile X mental retardation protein FMR1 ( also called as FMRP ) and its structural homologs FXR1 and FXR2 ( Siomi et al . , 1993 , 1995 ) .\",\n \"These proteins are highly conserved in many species and share a high degree of sequence similarity in major functional domains , including tandem Tudor , KH and RGG box domains ( Kirkpatrick et al . , 2001 ) .\",\n \"They all participate in RNA-binding , regulation of mRNA metabolism , ribosome-binding , and translation ( Ascano et al . , 2012; Chen et al . , 2014; Darnell et al . , 2001; Siomi et al . , 1996 ) .\",\n \"These proteins contain nuclear localization and nuclear export signals which allow them to be shuttled between cytoplasm and nucleus ( Eberhart et al . , 1996 ) .\",\n \"FMR1 , which is highly expressed in neurons , plays a critical role in synaptic plasticity and its silencing results in Fragile X Syndrome , an inherited intellectual disability and the major cause of autism ( Consortium , 1994; Darnell et al . , 2011; Santoro et al . , 2012; Verkerk et al . , 1991 ) .\",\n \"FXR1 is ubiquitously expressed and has potential roles in cardiac and skeletal muscle development ( Huot et al . , 2005; Mientjes et al . , 2004; Van't Padje et al . , 2009; Whitman et al . , 2011 ) .\",\n \"Increasing evidence suggests that FXR1 and FXR2 possess both common and distinct functions in post-transcriptional regulation ( Ascano et al . , 2012; Cavallaro et al . , 2008; Darnell et al . , 2009; Say et al . , 2010; Xu et al . , 2011 ) .\",\n \"FXR1 copy number was amplified and demonstrated oncogenic activity in lung squamous cell carcinoma ( Comtesse et al . , 2007; Qian et al . , 2015 ) .\",\n \"A recent study suggested that FXR1 downregulates p21 by binding and reducing its mRNA stability and/or by modulating p53 expression to avoid senescence in cancer ( Majumder et al . , 2016 ) .\",\n \"At the N-terminus , all three members of the FMR1 family contain a tandem Tudor domain belonging to the Royal family of chromatin-binding proteins ( Adams-Cioaba et al . , 2010; Hu et al . , 2015; Myrick et al . , 2015 ) .\",\n \"The tandem Tudor domain in FMR1 was reported to recognize methylated lysine on histones at the chromatin interface , thus resulting in the protein's subsequent localization to the DNA damage loci , thereby facilitating the repair process ( Adams-Cioaba et al . , 2010; Alpatov et al . , 2014 ) .\",\n \"Nevertheless , the function of FXR1 in cancer and the underlying molecular mechanisms remain elusive .\",\n \"From an analysis of The Cancer Genome Atlas ( TCGA ) and Cancer Cell Line Encyclopedia ( CCLE ) databases , we observed that FXR2 undergoes concomitant homozygous deletion in combination with TP53 homozygous deletion in a significant proportion of human cancers .\",\n \"Our data show that FXR1 inhibition blocks cell growth in TP53 and FXR2 co-deletion cell lines , but not in copy number normal or single deletion cell lines .\",\n \"The results were further confirmed in CRISPR-Cas9-generated TP53 and FXR2 knockout cell clones .\",\n \"Through a comprehensive analysis of chromatin immunoprecipitation followed by mass spectrometry ( ChIP-MS ) and ChIP coupling with high-throughput sequencing ( ChIP-seq ) , we uncovered the molecular mechanism through which FXR1 regulates cell proliferation .\",\n \"FXR1 is located in the gene promoter region together with histone H3 lysine 4 trimethylation ( H3K4me3 ) , and recruits transcription factor STAT1 or STAT3 at gene promoters to facilitate transcription regulation .\",\n \"In an agreement with a role in the FXR1-assocaited mechanism , inhibition of STAT1 or STAT3 target genes can also reduce cell proliferation .\",\n \"Taken together , our findings not only demonstrate the possibility of inhibiting FXR1 in TP53 and FXR2 homozygous co-deletion cancers as a potential treatment option , but also reveal the novel role of FXR1 in gene transcription .\"\n ],\n [\n \"Copy number data in various human tumors published in TCGA database ( access through cBioPortal ( http://www . cbioportal . org; accessed 18 Aug 2016 ) ( Gao et al . , 2013 ) show that homozygous TP53 genomic deletion occurs in various human cancers , at frequencies ranging from 1% to 15% ( Figure 1A , Figure 1—figure supplement 1A ) .\",\n \"One of the neighboring genes , FXR2 , located around 100 kb downstream of TP53 at chromosome 17p13 . 1 , undergoes concomitant deletion in most tumors carrying TP53 homozygous deletion with only a few exceptions ( Figure 1A and B ) .\",\n \"Consistently , in the CCLE database ( Barretina et al . , 2012 ) , the majority of cancer cell lines carrying TP53 homozygous deletion also contain FXR2 deletion ( Figure 1—figure supplement 1B ) .\",\n \"However , FXR2 single deletion is rarely observed in human tumors .\",\n \"In TP53/FXR2 co-deletion tumors , the copy number of FXR1 and FMR1 ( Figure 1A , Figure 1—figure supplement 1B ) is largely unaltered .\",\n \"Of note , FXR1 or FMR1 copy number gain was also observed in tumors , although this wasmutually exclusive with TP53/FXR2 deletion ( Figure 1A , Figure 1—figure supplement 1B ) .\",\n \"The recent discovery of the collateral lethality concept prompted us to hypothesize that concomitant deletion of passenger FXR2 in TP53-deleted cancer cells might make cell growth dependent on FXR1 .\",\n \"We therefore tested whether TP53/FXR2 co-deletion renders cancer cells sensitive to FXR1 inhibition .\",\n \"We selected four cancer cell lines harboring co-deletion of TP53 and FXR2: KATOIII , HL-60 , H358 , and KMS-11 , as well as four cell lines harboring the normal copy number of TP53 and FXR2: MKN45 , AGS , HepG2 , and A549 .\",\n \"The mRNA and protein level of p53 , FXR2 , FXR1 and FMR1 were assessed using q-RT-PCR and Western Blot ( WB ) , respectively .\",\n \"Our data showed that cancer cells with homozygous co-deletion of TP53 and FXR2 exhibit either absent or significantly lower mRNA and protein levels of p53 and FXR2 .\",\n \"By contrast , the copy-number-normal and co-deleted cell lines have comparable FXR1 and FMR1 levels ( Figure 1—figure supplement 2 ) .\",\n \"It is worth noting that p53 protein levels were assessed under a stress condition induced by doxorubicin .\",\n \"Next , we monitored cell proliferation rate upon FXR1 inhibition using inducible short hairpin RNAs ( shRNAs ) .\",\n \"Five doxycycline ( Dox ) -inducible FXR1 shRNAs were tested and most of them resulted in robust downregulation of FXR1 protein level and exhibited anti-proliferative activity in the TP53/FXR2 co-deleted cancer cell line KATOIII , but not in the copy-number-normal cell line MKN45 ( Figure 1—figure supplement 3A ) .\",\n \"We selected shRNA 2 and 3 ( FXR1-sh2 , FXR1-sh3 ) , which showed profound knockdown efficiency for the following studies .\",\n \"Consistently , FXR1 inhibition only suppressed cell proliferation in the cell lines with TP53/FXR2 co-deletion ( H358 , HL-60 , KATOIII and KMS-11 ) but not in the copy-number-normal cells ( AGS , A549 , MKN45 and HepG2 ) , as indicated in Figure 1C and also in Table 1 and Figure 1—figure supplement 3 .\",\n \"Furthermore , we used cell imaging and Matrigel assays to confirm FXR1-knockdown-induced anti-proliferative effects ( Figure 1—figure supplement 4 ) .\",\n \"To determine whether the observed cell growth reduction is attributed to FXR1 inhibition , we rescued FXR1 expression by ectopic expression of shRNA-resistant FXR1 ( harboring mutations on the shRNA-targeting sequence , FXR1m ) ( Figure 1—figure supplement 5 ) .\",\n \"In multiple cancer cell lines , the growth rate was recovered to a level comparable to that of the control , indicating that FXR1 inhibition was responsible for the growth phenotype ( Figure 1—figure supplement 5 , Figure 1D , Figure 3B ) .\",\n \"Consistent with the FXR1m data , expression of FXR2 also rescued the anti-proliferative phenotype induced by FXR1 inhibition , suggesting that FXR2 and FXR1 have redundant functions ( Figure 1D ) .\",\n \"However , ectopic expression of FXR2 only showed partial rescue compared with FXR1m , suggesting that the proteins may also have some distinct functions .\",\n \"Interestingly , FMR1 did not rescue FXR1 knockdown-induced anti-proliferation ( Figure 1E ) .\",\n \"Furthermore , FMR1 knockdown had no impact on proliferation in both TP53/FXR2 copy-number-normal and co-deleted cancer cells ( Figure 1—figure supplement 6 ) .\",\n \"The observation that cancer cells harboring deletion of both TP53 and FXR2 exhibited sensitivity to FXR1 inhibition suggested a collateral lethality correlation between FXR1 and TP53 deletion .\",\n \"To investigate whether TP53/FXR2 co-deletion is essential in determining cells’ sensitivity to FXR1 inhibition , we monitored cell growth rate upon FXR1 downregulation in cell lines carrying homozygous deletion of either TP53 ( H1299 , L540 , MG-63 , SKOV3 ) or FXR2 ( Hep3B ) alone .\",\n \"The levels of p53 , FXR2 and FXR1 were confirmed by q-RT-PCR and WB ( Figure 1—figure supplement 2 ) .\",\n \"In the tested cell lines , the proliferation rate was unaltered ( Figure 1F ) , suggesting that concomitant deletion of FXR2 and TP53 is necessary for triggering FXR1 inhibition-induced cell lethality .\",\n \"The role of FXR1 in controlling cell proliferation in TP53/FXR2 co-deleted cancer cells was further confirmed in an in vivo xenograft .\",\n \"Dox-induced FXR1 downregulation significantly reduced tumor growth in the TP53/FXR2 co-deleted cell line HL-60-derived xenograft model ( Figure 1G ) , but had no impact on growth in the copy-number-normal cell A549-derived xenograft ( Figure 1—figure supplement 3C ) .\",\n \"To confirm the above observations , we engineered the copy-number-normal cell line AGS that is suitable for use with CRISPR ( clustered regularly interspaced short palindromic repeat ) /Cas9 to generate the isogenic homozygous TP53 and FXR2 double knockout ( DKO ) cell clone , a TP53 single KO ( TP53 KO ) cell clone , and a FXR2 single KO ( FXR2 KO ) cell clone .\",\n \"The knockout strategy is illustrated in Figure 2—figure supplement\",\n \"1 . Both the mRNA and the protein levels of p53 and FXR2 in the individual knockout clones were determined by WB ( Figure 2A ) and q-RT-PCR ( Figure 2—figure supplement 2A ) .\",\n \"Consistent with the phenotypes observed in cancer cell lines , shRNA-induced FXR1 downregulation significantly inhibited cell proliferation only in the TP53 and FXR2 double knockout clones ( Figure 2B ) and had no effect on the TP53 single KO ( Figure 2C ) or FXR2 single KO ( Figure 2D ) clones .\",\n \"The FXR1-inhibition-induced anti-proliferative activity observed in TP53/FXR2 double knockout cells was confirmed in the Matrigel assay ( Figure 2—figure supplement 2B ) .\",\n \"Resistance to FXR1 inhibition in both FXR2 single knockout clones ( Figure 2D ) and the FXR2 homozygous deletion cell line Hep3B ( Figure 1E ) suggested that p53 deficiency is required for FXR1 to function in regulating cell proliferation .\",\n \"We downregulated p53 using RNAi in FXR2 KO cells that stably expressed FXR1 shRNA to further investigate the necessity for p53 deficiency in FXR1’s function in proliferation control .\",\n \"In line with its tumor suppressor function , TP53 knockdown by siRNA enhanced cell growth in the tested cells ( Figure 2—figure supplement 2C ) .\",\n \"Interestingly , TP53 knockdown granted FXR1 the capability to regulate cell proliferation: diminished FXR1 suppressed the growth of TP53 siRNA-treated FXR2 KO cells but not of control siRNA-treated FXR2 KO cells ( Figure 2—figure supplement 2C ) .\",\n \"Moreover , knockout of TP53 and FXR2 didn’t change the expression of FXR1 or FMR1 ( Figure 2—figure supplement 2D ) .\",\n \"To confirm the redundant function shared by FXR1 and FXR2 , we also used the same system to engineer a TP53/FXR1 DKO clone and stably expressed FXR2 shRNA-2 or FXR2 shRNA-3 .\",\n \"We observed that knockdown of FXR2 upon Dox induction suppressed proliferation ( Figure 2—figure supplement 3 ) , further confirming the redundancy between FXR1 and FXR2 .\",\n \"Table 1 summarizes the studies in the cancer cell lines and CRISPR-Cas9 knockout clones .\",\n \"Using an arbitrary 30% proliferation inhibition as a cut off , there was a clear pattern of FXR1 regulation of cell proliferation in TP53 and FXR2 co-deleted cancer cells .\",\n \"Therefore , our data validate the role of FXR1 inhibition in blocking cell proliferation in TP53-deleted cancers in a collateral lethality manner upon passenger deletion of FXR2 .\",\n \"FXR1 contains multiple functional domains including the newly discovered N-terminal tandem Tudor domain , KH domains and the RGG box , as illustrated in Figure 3A .\",\n \"In order to pinpoint the domain responsible for regulating cell proliferation , we examined the ability of FXR1 isoforms to rescue FXR1-inhibition-induced cell growth reduction .\",\n \"There are three major isoforms , including full-length FXR1_a , C-tail truncation FXR1_b , and N-terminal tandem Tudor truncation FXR1_c ( isoform gene codes are listed in Materials and methods ) .\",\n \"Ectopic expression of the shRNA-resistant plasmids encoding the three isoforms restored the expression of individual proteins in cells depleted of endogenousFXR1 ( Figure 3B ) .\",\n \"Interestingly , only the full-length ( FXR1_a ) could rescue FXR1 shRNA-induced anti-proliferation .\",\n \"The Tudor-domain-truncated version FXR1_c completely lost its ability to rescue cell proliferation ( Figure 3B ) .\",\n \"The C-tail truncation FXR1-b only partially rescued cell proliferation ( data not shown ) .\",\n \"These data indicate that the tandem Tudor domain is necessary for FXR1 to regulate cell proliferation .\",\n \"It has recently been reported that the tandem Tudor domain of FMR1 can recognize methylated lysine at histone H3 and recruit DNA repair protein , thus playing a role in DNA damage repair ( Alpatov et al . , 2014 ) .\",\n \"We used the histone methyl lysine analog ( MLA ) protein pulldown assay to examine whether histones are bound to the FXR1’s tandem Tudor domain .\",\n \"The Tudor domain showed binding to histone H3 containing methylated lysine at various positions , especially H3K4me1 , H3K4me3 , H3K9me3 , H3K27me2 , H3K36me1 , and H3K79me2 ( Figure 3C ) .\",\n \"The histone binding pattern of the tandem Tudor domain of FXR1 is similar to that of FMR1 .\",\n \"These data indicate that the tandem Tudor domain can potentially bring FXR1 to the chromatin interface , thus playing a role in proliferation control .\",\n \"We then investigated how FXR1 regulates proliferation in TP53/FXR2 co-deleted cancer cells .\",\n \"In order to obtain a comprehensive assessment of FXR1’s function , we conducted chromatin immunoprecipitation using a validated FXR1-specific antibody , followed by mass spectrometry ( ChIP-MS ) , aiming to capture both the chromatin-associated and chromatin-free protein complexes in the cells .\",\n \"Proteins identified in the FXR1 pull-down complex from both H358 and KATOIII cells were analyzed .\",\n \"They are listed in Supplementary file 1 and exemplified in Figure 4A .\",\n \"The enrichment analysis using the Database for Annotation , Visualization and Integrated Discovery ( DAVID ) clustered the potential FXR1-interacting proteins according to their function ( Supplementary file 2 ) .\",\n \"Proteins involved in mRNA binding , stability , splicing/metabolism , transportation , and translation were most enriched ( Figure 4b , Supplementary file 2 ) , consistent with the known function of FXR1 .\",\n \"Interestingly , proteins participating in chromatin/chromosome organization and transcription signaling were also enriched ( Figure 4B and Supplementary file 2 ) .\",\n \"Next , we confirmed the protein interactions using ChIP followed by WB ( ChIP-WB ) .\",\n \"In agreement with our DAVID enrichment analysis , FXR1 interacted with multiple proteins that are involved in chromatin and transcriptional signaling , including the transcription factors STAT1 and STAT3 , the chromatin regulator CHD4 , SNF2H , histone H2B and H3K4me3 , and DNA topoisomerase TOP2A in H358 cells ( Figure 4C ) .\",\n \"We discovered that phosphorylated STAT1 or STAT3 can also interact with FXR1 from the ChIP-WB analysis in the same cells ( Figure 4C , lower panel ) .\",\n \"Importantly , the in vitro pull-down assay using the purified tagged proteins showed that FXR1 may directly interact with both STAT1 and STAT3 ( Figure 4D ) .\",\n \"Interestingly , FXR1 had no impact on the phosphorylation of STAT1 or STAT3 or on their shuttling from the cytoplasm to the nucleus ( Figure 4—figure supplement 1 ) .\",\n \"When combined together evidence of FXR1's capacity to bind methylated histone H3 , these results suggest that FXR1 may be involved in chromatin signaling and gene transcription .\",\n \"To confirm the role of FXR1 , we conducted chromatin immunoprecipitation coupled with high-throughput sequencing ( ChIP-seq ) in the TP53/FXR2 co-deleted H358 cells using FXR1- , STAT1- , STAT3- , H3K4me3- , H3K9me3- , or H3K27me3-specific antibodies .\",\n \"We identified unfiltered 882 peaks of FXR1 ( Supplementary file 3 ) , primarily located at gene promoters close to transcriptional start sites ( TSS ) ( Figure 5A–B and Supplementary file 3 ) .\",\n \"In agreement with our hypothesis , STAT1 , STAT3 and H3K4me3 were also primarily located at TSS ( Figure 5B and Figure 5—figure supplement 1A and Supplementary file 3 ) .\",\n \"Interestingly , the location of FXR1 significantly overlapped with those of STAT1 , STAT3 , and H3K4me3 at their target genes ( Figure 5C and Supplementary file 3 ) .\",\n \"The percentage of overlapping increased when we focused on TSS ( Figure 5C and Supplementary file 3 ) .\",\n \"The FXR1-H3K4me3- or FXR1-STAT1/3-overlapped peaks primarily located in TSS regions , whereas the non-overlapped peaks primarily located in intergenic regions ( Figure 5—figure supplement 1B and Supplementary file 3 ) .\",\n \"At TSS , a total of 210 peak-associated genes were identified for FXR1 ( Figure 5C and Supplementary file 3 ) .\",\n \"Among them , 145 , 147 and 200 peak-associated genes overlapped with STAT1 , STAT3 and H3K4me3 , respectively ( Figure 5C and Supplementary file 3 ) .\",\n \"FXR1 peaks rarely overlap with H3K9me3 or H3K27me3 ( for examples , see Figure 5D , Figure 5—figure supplement 2 , and Supplementary file 3 ) , indicating FXR1's potential role in facilitating active transcription .\",\n \"Peaks aligning FXR1 , STAT1 , STAT3 and selective histone markers of representative genes — CASC4 , ARID1A , GLI1 , SSBP2 , CITED1 , NPAS3 and TRAPPC9 — are depicted in Figure 5D and Figure 5—figure supplement\",\n \"2 . We selected FXR1-peak-associated putative target genes ( 239 genes listed in Supplementary file 5 ) and examined FXR1’s role in their expression using q-RT-PCR .\",\n \"Upon inducible FXR1 knockdown , genes ARID1A , CASC4 , TRAPPC9 , GLI1 , CITED1 , NPAS3 , and SSBP2 were consistently downregulated in both H358 and KATOIII cells .\",\n \"Data from H358 cells are shown in Figure 5E .\",\n \"Using ChIP-PCR , we further investigated whether FXR1 directly occupies the promoter of these target genes .\",\n \"On the basis of the ChIP-seq peak position , we designed and selected two PCR primer pairs against each target gene’s promoter region ( Figure 5—figure supplement 2B ) to capture FXR1’s localization in the ChIP-PCR assay .\",\n \"The promoter regions of the target gene were detected in the FXR1 ChIP pull-down complex , indicating that FXR1 is located at the target gene promoters ( Figure 5F , shCtrl/Dox- and FXR1-sh3/Dox- ) .\",\n \"Dox treatment did not reduce FXR1’s occupancy in the shRNA control cells ( shCtrl/Dox+ , Figure 5F ) , but it evicted FXR1 from the gene promoter in FXR1-shRNA-expressing H358 cells ( FXR1-sh3/Dox+ , Figure 5F ) , confirming the specificity of the FXR1 ChIP assay .\",\n \"Using the same approach , STAT1 or STAT3 were located in the vicinity of FXR1 at the target gene promoter ( Figure 5F ) .\",\n \"More interestingly , FXR1 knockdown resulted in STAT1 and STAT3 eviction from the gene promoters ( Figure 5F ) , suggesting that FXR1 is required to recruit or stabilize STAT1 and STAT3 at gene promoters .\",\n \"On the other hand , STAT3 did not affect FXR1’s occupancy of sites at gene promoters because its knockdown had no effect on the localization of FXR1 at promoters ( Figure 5G , Figure 5—figure supplement 3 ) .\",\n \"To further confirm the redundant function shared between FXR1 and FXR2 as well as to investigate the interaction between FXR1/2 and STAT1/3 , we conducted FXR1 , FXR2 , STAT1 , STAT3 , and H3K4me3 ChIP-seq in AGS cells .\",\n \"The data indicated that the four proteins were enriched at TSS regions in the genome ( Figure 5H , Figure 5—figure supplement 4A and Supplementary file 3 ) .\",\n \"FXR1 peak-associated genes had 89 . 95% ( 2935/3263 FXR1-peak-associated genes ) overlap with FXR2 peak-associated genes in AGS cells ( Figure 5I ) , and this percentage increased when we focused on TSS ( Figure 5—figure supplement 4B and Supplementary file 3 ) , suggesting a shared function of FXR1 and FXR2 at gene promoters .\",\n \"Only a small percentage of FXR2 binding-site-associated or peak-associated genes overlapped with those similarly associated with FXR1 in AGS cells .\",\n \"The difference is probably due to the weaker capability of the FXR1 ChIP antibody in pull down assay when compared to the FXR2 ChIP antibody , as reflected by the difference in the peak-associated gene numbers for FXR1 and FXR2 ( 3263 and 15 , 346 ) , respectively ( Figure 5I ) .\",\n \"There was also significant overlapbetween FXR1-associated genes and STAT1/3-associated genes , suggesting at least partially , that FXR1 mediates STAT1/3 transcriptional activity in AGS cells ( Figure 5I , Figure 5—figure supplement 4B ) .\",\n \"The ChIP-seq peaks at the promoter region of the representative gene ARID1A in AGS cells are shown in Figure 5—figure supplement 4C .\",\n \"Our ChIP-PCR results revealed a similar localization pattern for FXR2 as for FXR1 , with both proteins occupying the promoters of target genes ( Figure 5J upper panel and Figure 5—figure supplement 4D ) .\",\n \"Furthermore , the expression levels of target genes remained constant after FXR2 knockdown except for a slight inhibition of CASC4 and TRAPPC9 expression , further confirming the redundant function shared by FXR1 and FXR2 ( Figure 5J , lower panel ) .\",\n \"Taken together , our data confirm that FXR1 is required to recruit or stabilize STATs on gene promoters , thus facilitating FXR1’s transcriptional activity , and also show that FXR1 and FXR2 share similar function .\",\n \"Our study showed that only a small portion of STAT1/3 peaks in the genome ( 0 . 7–1 . 57% ) overlap with FXR1 peaks , indicating that FXR1 cooperates with STAT1/3 in a small number of STAT target genes’ transcription in H358 cells ( Supplementary file 3 ) .\",\n \"By contrast , 24 . 38–25 . 74% of FXR1 peaks overlap with STAT1 and STAT3 peaks , suggesting a significant role for STATs in FXR1’s transcriptional activity ( Supplementary file 3 ) .\",\n \"As STAT1 and STAT3 participate in FXR1 transcriptional activity , we hypothesized that a JAK inhibitor could repress FXR1 target gene expression .\",\n \"Indeed , we found that the JAK inhibitor S-Ruxolitinib suppressed STAT1 phosphorylation in a dose-dependent manner and inhibited the expression of some but not all FXR1 target genes in H358 cells ( Figure 5K , Figure 5—figure supplement 5 ) .\",\n \"Note that JAK inhibitor didn’t suppress FXR1 expression ( Figure 5K ) .\",\n \"These data confirm the involvement of STAT in FXR1’s transcriptional activity .\",\n \"We performed RNA-seq to analyze gene expression in H358 cells with biological triplicates .\",\n \"We selected FXR1-knockdown-induced gene expression changes using fold change log2 >0 . 6 as the cutoff ( Supplementary file 6 ) .\",\n \"Among the seven FXR1 target genes identified by ChIP-seq , five showed significant downregulation upon FXR1 knockdown .\",\n \"The remaining two genes showed moderate downregulation ( Supplementary file 7 ) .\",\n \"Among the genes whose promoters are occupied by FXR1 , 45 out of 210 genes showed regulation upon FXR1 knockdown in RNA-seq assay ( Figure 5—figure supplement 1D and Supplementary file 7 ) .\",\n \"RNA-seq revealed significantly more genes regulated by FXR1 than ChIP-seq .\",\n \"The discrepancy may be attribute primarily to the fact that FXR1 is a well-known mRNA-binding protein , regulating RNA metabolism and stability in addition to the newly discovered function in transcription regulation revealed in this study .\",\n \"Therefore , FXR1 knockdown could impact on both populations of genes , those regulated by FXR1’s transcriptional activity and those regulated by FXR1’s RNA-binding function .\",\n \"In addition , the scope and limitation of RNA-seq and ChIP-seq could also contribute to the discrepancy .\",\n \"In order to assess whether target genes mediate FXR1’s function in proliferation regulation , we utilized siRNAs to knockdown target genes and monitored the change in cell proliferation .\",\n \"The knockdown efficiency of the target genes by siRNAs was assessed using q-RT-PCR ( Figure 6—figure supplement 1A ) .\",\n \"Among the seven target genes , knockdown of GLI1 showed the most consistent anti-proliferative effect whereas ARID1A , CITED1 , or SSBP2 knockdown showed less inhibitory effect in a 7-day proliferation assay ( Figure 6—figure supplement 1B ) .\",\n \"We further evaluated the combinatorial effect on cell proliferation of downregulation of both FXR1 and its target genes .\",\n \"We performed combinatorial partial knockdowns of both FXR1 and its target genes by applying lower concentration of siRNA ( 5 nM ) and shortening Dox treatment duration ( to 4 days ) .\",\n \"As shown in the cell image and MTS detection ( Figure 6A ) , the combined knockdown of both the target genes and FXR1 showed an additive effect on cell growth inhibition .\",\n \"Amongst the target genes , GLI1 downregulation by siRNA showed the most dramatic combinatorial effect with FXR1 knockdown in suppressing cell proliferation .\",\n \"Upon FXR1 inhibition , cells underwent blebbing , suggesting a role of FXR1 in cell apoptosis .\",\n \"Following this lead , we found that FXR1 knockdown induced the cleavage of PARP in H358 cells ( Figure 6B ) , a signal event of cell apoptosis .\",\n \"Furthermore , FXR1 inhibition increased caspase 3/7 activity ( Figure 6B ) .\",\n \"Consistently , knockdown of FXR1 target gene GLI1 also induced apoptotic signals including the cleavage of PARP and activation of caspase 3/7 in the same cells ( Figure 6C ) .\",\n \"We observed the similar effect of FXR1 shRNA in inducing PARP cleavage and caspase 3/7 activity in another cell line , HL-60 ( Figure 6—figure supplement 2 ) .\",\n \"More interestingly , the effect on PARP cleavage could be rescued by ectopic expression of shRNA-resistant full-length FXR1m_a , indicating a direct role of FXR1 in cell apoptosis signaling ( Figure 6—figure supplement 2 ) .\",\n \"These data suggest that downregulation of FXR1 inhibits cell growth through the activation of cell apoptotic signaling , at least partially through regulating target gene transcription .\",\n \"Taken together , the findings of our study indicate that FXR1 inhibition suppresses cell proliferation in cancer cells containing TP53 and FXR2 homozygous deletions in a collateral lethality manner .\",\n \"FXR1 regulates gene transcription , at least in part , to enable control of cell proliferation .\"\n ],\n [\n \"The importance of p53 in cancer has led to tremendous efforts to target its activity by therapeutic approaches .\",\n \"For cancers containing normal TP53 copy number , there has been good progress in interrupting MDM2–p53 interactions to stabilize and promote p53 tumor suppressor activity .\",\n \"For TP53 mutated cancers , the current strategy is to restore p53 anti-tumor activity ( Khoo et al . , 2014; Soragni et al . , 2016 ) .\",\n \"No therapeutic strategies have been proposed to target TP53 deletion cancers until a recent report demonstrated that TP53 heterozygous deletion predisposes cancer cells to further suppression of POLR2A , a neighboring gene undergoing concomitant deletion ( Bradner , 2015; Errico , 2015; Liu et al . , 2015 ) .\",\n \"However , this strategy will not be applicable to cancers with homozygous TP53 deletions in which POLR2A is co-deleted in most cases ( Figure 1—figure supplement 1C ) .\",\n \"Here , for the first time , we report an approach to target cancers with concomitant homozygous TP53 and FXR2 deletion by inhibiting FXR1 .\",\n \"The passenger deletion of FXR2 in the TP53 deletion locus renders cancer vulnerable to FXR1 inhibition , which echoes with the recently raised concept of collateral lethality in tumor suppressor gene deletion ( Muller et al . , 2015; Muller et al . , 2012; Nijhawan et al . , 2012 ) .\",\n \"In this study , several lines of evidence confirmed the redundant function between FXR1 and FXR2:\",\n \"1 ) both FXR1 and FXR2 can rescue FXR1 shRNA-inhibited cell proliferation ( Figure 1D ) ,\",\n \"2 ) FXR1 and FXR2 share a significant portion of overlapping binding sites in the genome ( Figure 5I , Figure 5—figure supplement 4B–C ) , and\",\n \"3 ) knockout of either protein sensitizes TP53 homozygous deletion-containing cancer cells for the inhibition of the remaining member ( Figure 2D , Figure 2—figure supplement 2 ) .\",\n \"Although another family member , FMR1 , shares structural and functional similarity with FXR1 and FXR2 , our data suggest that FMR1 does not participate in the regulation of proliferation or in rescuing the FXR1 inhibition in the tested cell models .\",\n \"TP53 homozygous deletion is observed in 1–15% of human tumors , the majority of which have FXR2 homozygous deletions according to the TCGA database .\",\n \"This study therefore opens the avenue to the development of FXR1-based targeted therapies to treat TP53 homozygous deletion cancers , in which there is a significant unmet medical need .\",\n \"Interestingly , we have found that TP53 deletion is required for cancer cells to respond to FXR1 inhibition .\",\n \"This was confirmed in our engineered FXR2 single knockout cell clones which were only sensitive to FXR1 knockdown when p53 was downregulated .\",\n \"However , the mechanism of action remains elusive .\",\n \"There have been reports suggesting that p53 deficiency promotes the activation of JAK/STAT signaling , which may lead to cellular dependency on FXR1 because it regulates the localization of STAT1/3 at gene promoters ( Guo et al . , 2014; Spehlmann et al . , 2013 ) .\",\n \"Considering that FXR1 can both suppress p53 to escape senescence ( Majumder et al . , 2016 ) and activate gene transcription ( evidence from this study ) , p53 deletion may augment FXR1’s role in transcription regulation in controlling cancer cell proliferation .\",\n \"Given the various critical functions of p53 in governing cellular behavior , its deficiency may create a cell dependency on FXR1 and FXR2 activities .\",\n \"The underlying molecular mechanism requires further investigation .\",\n \"Some studies have explored the oncogenic property of FXR1 in lung cancer with FXR1 overexpression or amplification ( Comtesse et al . , 2007; Qian et al . , 2015 ) .\",\n \"FXR1 copy number gain occurs in some types of cancers , particularly in lung squamous carcinoma ( Comtesse et al . , 2007; Qian et al . , 2015 ) and its occurrence is mutually exclusive with TP53 and FXR2 homozygous deletion according to TCGA database ( Figure 1A ) .\",\n \"Together with our study , this evidence suggests that FXR1 controls tumor growth in cancers with either TP53/FXR2 homozygous deletion or FXR1 amplification .\",\n \"Furthermore , we reveal a novel function of FXR1 in transcriptional regulation and elucidate its molecular mechanism in regulating cell proliferation , in addition to its well-established role in mRNA binding .\",\n \"Our ChIP-MS study revealed that FXR1 interacts with chromatin regulators and transcription factors .\",\n \"In addition , about a half of FXR1’s peaks in the ChIP-seq assay co-localize with H3K4me3 peaks at gene promoters , suggesting a new role for FXR1 in facilitating active transcription .\",\n \"FXR1 interacts with and recruits or stabilizes STAT1 or STAT3 at the target gene promoters , suggesting that cooperation with STATs is required for its transcriptional activity .\",\n \"Indeed , the inhibitor of the STAT upstream kinase JAK suppressed some of FXR1’s target genes expression .\",\n \"Reciprocally , FXR1 is required for the localization of STATs on its target gene promoters .\",\n \"Generally , it is believed that JAK-catalyzed STAT1/3 phosphorylation drives their shuttling to the nucleus and localization at gene promoters in order to regulate transcription ( Levy and Darnell , 2002 ) .\",\n \"Our findings advance the current views about the regulation of transcription by STAT1/3 by revealing the necessity for FXR1 in regulating STAT occupancy at promoters .\",\n \"STAT is well established in regulating cytokine gene transcription .\",\n \"We have also observed that FXR1 knockdown inhibited the expression of multiple cytokines ( data not shown ) , consistent with a recent report of FXR1 function in positively regulating cytokine expression in monocytes ( Le Tonqueze et al . , 2016 ) .\",\n \"Notably , only about 0 . 7–1 . 57% of the STAT1/3 peaks overlap with FXR1 peaks in our ChIP-seq study , suggesting that FXR1 only participates in the transcription of a very small fraction of JAK-STAT target genes .\",\n \"Although JAK inhibition suppressed the expression of some FXR1 target genes , it had no effect on inhibiting cancer cell proliferation in FXR1 shRNA-sensitive cells ( Figure 5—figure supplement 6 ) .\",\n \"This is in line with the fact that JAK-STAT signaling pathway governs a much broader spectrum of gene expression and cellular behaviors .\",\n \"It w worth investigating whether other epigenetic regulators are involved in STAT transcriptional activity .\",\n \"Among the FXR1 peaks , approximately 25% overlapped with STAT peaks according to our study in H358 cells .\",\n \"FXR1 may also cooperate with other transcription factors in regulating transcription; examples include BTF ( Ma et al . , 2014 ) which was also identified in an FXR1 protein complex in our ChIP-MS study .\",\n \"In addition , we identified several other FXR1-interacting proteins that have essential function in transcription regulation , including TOP2A/2B , GTF2A , and FACT80 .\",\n \"Further investigations are needed to dissect the underlying mechanism of FXR1 in transcription regulation .\",\n \"FXR1 is an RNA-binding protein that regulates mRNA stability , transportation of RNA from the nucleus to the cytoplasm , and translation through its functional KH domains and RGG box .\",\n \"Our ChIP-MS data confirmed the established role of FXR1 , which binds to and regulates mRNA processing and translation ( Figure 4B ) .\",\n \"Furthermore , we confirmed ChIP-MS findings and discovered that FXR1 binds to the mRNA of target genes such as CASC4 , TRAPPC9 , GLI1 , and SSBP2 in the mRNA binding assays ( Figure 6—figure supplement 3 ) .\",\n \"Our study confirmed that , in addition to its direct function in transcription , FXR1 binds to some but not all of its target gene mRNAs , which may contribute to its role in proliferation regulation .\",\n \"This raises the possibility that FXR1 has dynamic functions in transcription and subsequent mRNA processing .\",\n \"The newly discovered N-terminal tandem Tudor domain has a high degree of similarity among the three Tudor family members ( Adams-Cioaba et al . , 2010; Hu et al . , 2015; Myrick et al . , 2015 ) .\",\n \"The Tudor domain in FMR1 was reported to bind to multiple methylated lysine at histone H3 and to participate in DNA damage repair ( Alpatov et al . , 2014 ) .\",\n \"Our study shows that the tandem Tudor in FXR1 binds to methylated lysine in histone H3 in a similar manner as that in FMR1 and it is required for FXR1’s function in regulating cancer cell proliferation .\",\n \"Further investigation is needed to study the amino acid residues and the structure of the aromatic cage within the FXR1 tandem Tudor domain that are responsible for histone binding , which might provide the foundation for development of Tudor domain inhibitors .\",\n \"The observation that FXR1 knockout mice die shortly after birth and knockdown of FXR1 causes abnormalities in striated muscle and heart in zebrafish ( Mientjes et al . , 2004; Zarnescu and Gregorio , 2013 ) raise potential safety concerns with regard to inhibiting FXR1 in cancer patients .\",\n \"However , the inducible FXR1 knockout in adult mice didn’t display the lethal phenotype ( Mientjes et al . , 2004 ) , suggesting the potential of FXR1 as a drugging target .\",\n \"Overall , this study provides an important approach to targeting human cancers with TP53 homozygous deletions .\",\n \"The novel function of FXR1 in transcription advances our knowledge regarding FXR1’s function in gene expression on top of its known roles in mRNA processing and translation .\"\n ],\n [\n \"Copy-number analysis in human tumor tissues was performed using data from The Cancer Genome Atlas ( TCGA ) ( http://www . cbioportal . org/cross_cancer . do ) as previously described ( Gao et al . , 2013 ) .\",\n \"Gene copy numbers in human cancer cell lines were downloaded from the Cancer Cell Line Encyclopedia database ( CCLE ) ( http://www . broadinstitute . org/ccle ) and analyzed using an R package ComplexHeatmap ( https://bioconductor . org/packages/release/bioc/html/ComplexHeatmap . html ) .\",\n \"KATOIII , HL-60 , H358 , MKN45 , AGS , HepG2 , A549 , H1299 , MG-63 , SKOV3 , Hep3B , and HEK 293 T cell lines were purchased from American Type Culture Collection ( ATCC , Manassas , Virginia , USA , http://www . atcc . org ) .\",\n \"The KMS-11 cell line was obtained from Horizon Discovery Ltd . ( Cambridge , UK ) .\",\n \"The L540 cell line was purchased from Biovector Science Lab , Inc ( Beijing , China ) .\",\n \"Although L540 is listed as a misidentified cell line by ICLAC ( International Cell Line Authentication Committee ) , we used it as a TP53 single deletion cell model after comfirming the abscence of p53 mRNA and protein expression .\",\n \"The cell lines were cultured in RMPI 1640 medium ( GIBCO ) or DMEM medium ( GIBCO ) supplemented with 10% fetal bovine serum ( FBS , GIBCO ) and 100 U/mL penicillin-streptomycin ( GIBCO ) at 37°C with 5% CO2 .\",\n \"All cell lines were authenticated by Short Tandem Repeat ( STR ) profiling and routinely monitored for mycoplasma contamination .\",\n \"The source and mycoplasma status as well as the Research Resource Identifiers ( RRIDs ) of each cell line are listed in Supplementary file 8 .\",\n \"Cell proliferation rate was measured by MTS assay .\",\n \"Cell apoptosis was determined by analyzing Caspase3/7 activity .\",\n \"The luminescence signal in the Caspase3/7 assay was recorded by an Envision 2104 Multilabel reader ( Perkin Elmer , Waltham , MA ) .\",\n \"The detailed procedure and information are in the Supplementary methods .\",\n \"ChIP sample preparation follows the manual of SimpleChIP Enzymatic Chromatin IP Kit ( Magnetic Beads , CST , #9003 ) .\",\n \"The experimental procedure and reagent information for ChIP-WB , ChIP-MS , ChIP-seq , or ChIP-PCR are in the Supplementary methods .\",\n \"In cell assays , three independent replicates were included in each experiment .\",\n \"Student T Test was performed to compare the differences between two groups with normal distribution .\",\n \"All data were analyzed using GraphPad Prism 6 .\",\n \"Gene-specific shRNA sequences were obtained from the Sigma website ( http://www . sigmaaldrich . com/ ) , and the hairpin sequences were cloned into the doxycycline ( Dox ) -inducible lentiviral vector pLKO-Tet-On with Puromycinas as a selection marker .\",\n \"Five shRNAs targeting each gene ( FXR1 , FXR2 and STAT3 ) were screened .\",\n \"Lentiviral particles were packaged in HEK 293 T cells by transient transfection .\",\n \"Briefly , 6 µg of shRNA encoding lentiviral plasmid , 0 . 6 µg of helper PCG10 plasmid and 5 . 4 µg of helper PCG41 plasmid were mixed with 30 µl X-tremeGENE HP DNA Transfection Reagent ( Roche , 06366236001 ) and co-transfected into HEK 293 T cells at 70–80% confluency in a 10 cm Petri dish .\",\n \"At 72 hr post transfection , the supernatant containing virus particles was collected and filtered through a 0 . 45 µm PVDF Millex syringe filter .\",\n \"Each cell line was subjected to a puromycin tolerance test to determine the optimal concentration for establishing stable cell lines .\",\n \"To generate a shRNA-expressing stable cell line , cells were infected with lentiviral particles with 8 µg/ml polybrene .\",\n \"After 48 hr , the cells were selected by 0 . 5–2 µg/ml puromycin ( GIBCO , A11138-03 ) for 7 days .\",\n \"shRNA expression was induced by 0 . 1–1 µg/ml Dox ( Sigma , D9891 ) , and the knockdown efficiency was tested by WB assay or q-RT-PCR .\",\n \"Dox concentration was optimized for each stable cell line to achieve the most optimal knockdown efficiency at the minimum dose .\",\n \"For ectopic expression of proteins of interest , cells were infected with the lentiviral pLenti6 . 3-MCS construct encoding the FXR1 , FXR2 or FMR1 gene open reading frame and selected using 2–6 µg/ml blasticidin ( GIBCO , A11139-03 ) for stable cell line generation .\",\n \"The inserted open reading frames are: FXR1_a , NM_005087 . 3; FXR1_b , NM_001013438 . 2; FXR1_c , NM_001013439 . 2; FXR2 , – NM_004860 . 3; and FMR1 , NM_002024 . 5 .\",\n \"The sequence of the FXR1 shRNA resistant mutation was CGCCAGGTTCCATTTAATGAA mutated to CGTCAAGTACCGTTCAACGAG and TTGCGAAGTATTCGTACGAAGTTG mutated to TTACGGAGCATCCGAACAAAATTA .\",\n \"FXR1 shRNAs were inserted in adenoviral vector pAd/CMV/IRES/GFP .\",\n \"The adenoviruses were produced in HEK 293 T cells by transient transfection of the plasmids .\",\n \"Virus titer was determined by counting GFP-positive cells after adenoviral infection .\",\n \"In the proliferation assay , adenoviruses were added to the cell culture medium and replenished 4 days afterwards to achieve efficient FXR1 knockdown .\",\n \"The shRNA target sequence is listed in Supplementary file 8 .\",\n \"Cells were lysed using RIPA lysis buffer ( CST , 9806S ) with complete protease inhibitor cocktail ( Sigma , P8340 ) and phosphatase inhibitor cocktail ( Sigma , P5726 ) .\",\n \"The protein concentration was detected by BCA kit ( Thermo Scientific , Cat#23225 ) .\",\n \"The protein lysate was denatured by adding NuPAGE sample reducing agent ( Novex , Invitrogen , NP0009 ) and NuPAGE LDS Sample buffer ( Novex , Invitrogen , NP0007 ) followed by heating at 95°C for 5 min .\",\n \"Equal amounts of protein lysate was loaded to the NuPAGE Electrophoresis System ( Life Technologies ) according to the manufacturer's protocol .\",\n \"Proteins in the gel were transferred to the nitrocellulose membrane in iBlot 2 Transfer Stacks ( IB23001 and IB23002 ) using the iBlot 2 Dry Blotting System ( Life Technology , IB21001 ) .\",\n \"The membrane was blocked in SuperBlock Blocking Buffer in TBS ( Pierce , 37535 ) , incubated with the specific primary antibodies for the protein of followed by reaction with the secondary antibodies .\",\n \"The protein signals were detected using SuperSignal West Femto Maximum Sensitivity Substrate ( Thermo , 34095 ) , and were scanned and quantified using ChemiDoc MP System ( BIO-RAD ) .\",\n \"The antibodies and their Research Resource Identifiers ( RRIDs ) used in this study are listed in Supplementary file 8 .\",\n \"Cell proliferation rate was detected by MTS assay .\",\n \"Briefly , the cells were seeded in 96-well plates at optimal density in 100 µl medium per well followed by treatment .\",\n \"Cells were added with CellTiter 96 AQueous One Solution Reagent ( Promega , G3581 ) at the indicated time by following the manual instructions and subjected to absorbance recording at 490 nm using a PowerWave HT Microplate Spectrophotometer ( BioTek ) .\",\n \"The cell proliferation rate in MTS was determined by measuring absorbance at 490 nm .\",\n \"In the duplicated experiment , cell images were scanned after fixing the cells with cold methanol and staining with 0 . 1% crystal violet .\",\n \"In some cases , the cells were seeded on the solidified Matrigel ( BD Bioscience ) layer in 96-well plate ( 50 µl per well ) to measure the organoid growth .\",\n \"Cell apoptosis was determined by analyzing Caspase3/7 activity using the Caspase-Glo3/7 assay kit ( Promega , G8092 ) according to the kit instructions or by detecting the protein level of cleaved PARP ( antibody from Cell Signaling Technology , 9541 ) in Western blot .\",\n \"The luminescence signal in the Caspase3/7 assay was recorded by an Envision 2104 Multilabel reader ( Perkin Elmer ) .\",\n \"The HL-60-derived xenograft model in female BALB/c nude mice was used to determine FXR1’s function in tumor growth .\",\n \"Briefly , the female BALB/c nude mice ( age 6–8 weeks ) housed under pathogen-free conditions were inoculated subcutaneously in the right flank with HL-60 cells stably expressing control shRNA ( shCtrl ) or Doxycycline-inducible FXR1-sh3 .\",\n \"Each mouse received 5 × 106 cells in 0 . 2 ml PBS:Matrigel ( 1:1 mixture ) for tumor development .\",\n \"When tumor size reached approximately 180 mm3 on day 14 , mice were randomly grouped ( n = 6 ) and Doxycycline ( 2 mg/ml ) was added to drinking water .\",\n \"Doxycycline was replenished twice per week .\",\n \"Tumor volume and body weight were measured twice a week .\",\n \"Tumor size was measured using a calliper , and tumor volume was calculated according to the following equation: tumor volume ( mm3 ) = length ( mm ) × width2 ( mm2 ) × 0 . 5 .\",\n \"The experiment was terminated when the tumor volume reached 2 , 000 mm3 according to animal welfare guidelines .\",\n \"All animal studies were carried out under IACUC number R20150728-Mouse and Rat approved by the Institutional Animal Care and Use Committee ( IACUC ) of WuXi AppTec .\",\n \"Summary statistics , including mean and the standard error of the mean ( s . e . m ) , were provided for the tumor volume of each group at each time point .\",\n \"The A549-derived xenograft model was studied using a similar method .\",\n \"Guide RNA ( gRNA ) was designed according to the formula of GN20GG or CCN20C in the forward or reverse strand of genomic DNA , respectively ( Cong et al . , 2013; Ran et al . , 2013; Shalem et al . , 2014; Wang et al . , 2013 ) .\",\n \"Two sets of gRNAs targeting the 5’ and 3’ sequences of the genomic region of interest ( TP53-to-FXR2 , TP53 alone , FXR1 alone , or FXR2 alone ) were designed .\",\n \"The synthesized double-strand DNA of the gRNA was inserted into the pU6-gRNA_SPycas9-2Acherry vector expressing Cas9 protein ( Mavrakis et al . , 2016 ) .\",\n \"To test gRNA efficiency , gRNA-expressing plasmids were transfected into cells using XtremeGene reagent ( Roche ) .\",\n \"Genomic DNA was extracted using the Purelink Genome DNA mini kit ( Invitrogen , K1820-02 ) .\",\n \"PCR was performed using genomic DNA as the template and the Accuprime Pfx SuperMix PCR system ( Invitrogen , 12344040 ) to amplify the sequence containing the gRNA PAM position , and PCR product was sequenced to determine the successful cut by gRNA-guided Cas9 by monitoring the miscellaneous peaks .\",\n \"The efficient gRNA target sequences were: TP53-5’-gRNA , GGGCAGCTACGGTTTCCGTCTGG; TP53-3’-gRNA , GGTGTGCGTCAGAAGCACCCAGG; FXR2-5’-gRNA , GGAGGCGGTGGCGGCGCCATGGG; FXR2-3’-gRNA , GGGGGGTGGCACAGCAGCTTGGG; FXR1-5’-gRNA , GTGGAGGTTCGCGGCTCTAA; FXR1-3’-gRNA , GAAAAAAAAGTTGCTGGCTAT .\",\n \"The donor plasmid contains the recombination sequences compatible with the 5’ and 3’ arms of the genomic region of interest and the GFP-coding sequence in vector pcDNA3 . 1 .\",\n \"The mixture of gRNA-Cas9 plasmids targeting the 5’ or 3’ sequence of the genomic region and donor plasmid at ratio 1:1:1 was transfected into the candidate cells in a 10 cm Petri dish using XtremeGene .\",\n \"One week post-transfection , the GFP-expressing cells were sorted and seeded into a 96-well plate at a density of one cell per well using FACSArisII ( BD ) .\",\n \"The single cell clones were cultured for two weeks to identify and filter out the cells with transient GFP signal .\",\n \"The clones with strong GFP signal , which would have potential insertion and replacement of the genomic region of interest by the donor sequence , were subjected to knockout validation by genomic PCR-sequencing , q-RT-PCR , and Western blot .\",\n \"The sequence of genomic PCR primers is listed in Supplementary file 8 .\",\n \"ChIP sample preparation: ChIP samples were prepared using SimpleChIP Enzymatic Chromatin IP Kit ( Magnetic Beads , CST , #9003 ) according to the manufacturer’s protocol .\",\n \"Briefly , cells cultured in 15 cm Petri dishes were crosslinked with 1% formaldehyde when reaching 70–80% confluence .\",\n \"Crosslinking was terminated by adding 10 × glycine followed by rinsing the cells twice with cold PBS , and 4 × 107 cells were collected into one 15 ml conical tube .\",\n \"The cell pellet was resuspended and incubated in Buffer A on ice for 10 mins for cell lysis .\",\n \"The nuclei were collected by centrifugation at 3 , 000 rpm for 5 mins at 4°C and resuspended in Buffer B followed by centrifugation and supernatant removal .\",\n \"The nuclei pellets were resuspended in 1 . 0 ml Buffer B , and transferred to a 1 . 5 ml microcentrifuge tube .\",\n \"7 . 5 µl of Micrococcal Nuclease was added and incubated for 20 mins at 37°C to digest the DNA to lengths of approximately 150–300 bp .\",\n \"After the termination of digestion by adding 100 µl of 0 . 5 M EDTA , the nuclei pellets were collected by centrifuging at 13 , 000 rpm for 1 min at 4°C .\",\n \"The nuclear pellets were resuspended in 1 ml ChIP buffer and separated into two tubes , and incubated on ice for 10 mins followed by subsequent sonication of lysates at 9W , 10 s ON/30 s OFF , nine cycles .\",\n \"Lysates were collected by centrifugation at 10 , 000 rpm for 10 mins at 4°C .\",\n \"The supernatant ( single nucleosome lysate ) was transferred to a new tube , and stored at −80°C for the following experiments .\",\n \"50 µl of lysate was used for the analysis of chromatin digestion .\",\n \"Pulldown: The single nucleosome lysate was diluted with ChIP buffer with protease inhibitor cocktail ( for each pulldown , 200 μl lysate was added into 300 μl 1 × ChIP Buffer ) .\",\n \"10 µl of sample was saved as input .\",\n \"To reduce nonspecific binding , the lysate was pre-cleared with 10% BSA and 30 μl ChIP Grade Protein G Magnetic Beads ( CST , #9006 ) at 4°C with rotation for 1 hr .\",\n \"The supernatant was collected by placing the tube on a magnetic separation rack .\",\n \"Antibodies of interest or IgG control were added and incubated on rotation at 4°C overnight followed by incubation with 30 µl of magnetic beads for 2 hr at 4°C .\",\n \"Beads were washed with low salt wash buffer ( 100 µl 10 × ChIP Buffer in 900 ml water ) three times and with high salt wash buffer ( 100 µl 10 × ChIP Buffer and 70 µl 5 M NaCl in 830 µl water ) once on rotation .\",\n \"The antibodies used in ChIP pulldown were: anti-FXR1 ( Sigma , HPA018246 , RRID: AB_1849204 ) ; anti-FXR2 ( Invitrogen , PA5-28979 , RRID: AB_2546455 ) ; anti-STAT1 ( Santa Cruz , sc-345 , RRID: AB_675903; CST , 14994S ) ; anti-STAT3 ( Santa Cruz , sc-482 , RRID: AB_632440; CST , 12640S , RRID: AB_2629499 ) ; rabbit IgG ( CST , 2729 , RRID: AB_1031062 ) ; anti-H3K4me3 ( CST , 9727 , RRID: AB_561095 ) ; anti-H3K9me3 ( Active Motif , 39161 , RRID: AB_2532132 ) ; and anti-H3K27me3 ( Millipore , 07–449 , RRID: AB_310624 ) .\",\n \"For ChIP-WB , protein G beads were added with 30 μl of DTT-containing 2 × SDS sample buffer and denatured at 95°C in thermomixer with gentle vortexing ( 1 , 200 rpm ) for 5 min .\",\n \"Eluted proteins were separated in SDS-PAGE gel and analyzed by WB .\",\n \"For ChIP-MS , eluted proteins were separated in SDS-PAGE gel followed by gel fixing in buffer containing methanol , acetic acid , and water at 5:1:4 ratio .\",\n \"The gel was stained in EZBlue Gel Staining Reagent ( Sigma , G1041 ) and subjected to mass spectrometry ( MS ) analysis .\",\n \"For ChIP-seq or ChIP-PCR , beads were eluted with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing ( 1 , 200 rpm ) in thermomixer .\",\n \"Eluted supernatant was incubated with 6 µl 5 M NaCl and 2 µl Proteinase K at 65°C for 2 hr in thermomixer to digest the protein .\",\n \"DNA was purified using spin columns , and sent for ChIP-seq or ChIP-PCR analysis .\",\n \"DNA from ChIP pull down complexes was subjected to library preparation according to Illumina's instructions ( Part # 11257047 ) .\",\n \"Sequencing was performed on Illumina HiSeq 2000 .\",\n \"ChIP-seq data quality was analyzed using FastQC ( Andrews , 2010 ) .\",\n \"Reads in the fastq files were trimmed by 5 bp in the 5´-end , and then mapped to the reference human genome ( HG19 ) using BOWTIE2 .\",\n \"Multiple mapped reads and duplicate reads were removed .\",\n \"FXR1 binding genomic regions ( called ChIP-seq peaks ) were identified by the Model-based Analysis of ChIP-Seq ( MACS ) algorithm using the default p-value cutoff of 1 × e−5 .\",\n \"The distribution of FXR1 ChIP-seq peaks were investigated by the annotations on the human genome .\",\n \"The peaks were binned into eight groups according to the positions relative to annotated genes: 5’ Distal ( up to 100 kb upstream TSS ) , 5’ Proximal ( up to 10 kb upstream TSS ) , 5’ TSS ( 5 kb upstream TSS to 1 kb downstream TSS ) , Intragenic ( 1 kb downstream TSS to 1 kb upstream TTS ) , 3’ TTS ( 1 kb upstream TTS to 5 kb downstream TTS ) , 3’ Proximal ( up to 10 kb downstream TTS ) , 3’ Distal ( up to 100 kb downstream TTS ) , and the remaining sequence defined as Intergenic region .\",\n \"The aggregation signals of FXR1 , H3K4me3 , STAT1 , and STAT3 at genebody ( TSS to TTS ) plus upstream and downstream 5 kb region were extracted and plotted using in-house perl and MATLAB code .\",\n \"A hub for the UCSC genome browser was constructed in order to investigate the detailed information of the peaks .\",\n \"The summit positions of the FXR1 ChIP-seq peaks were extended to 100 bp both upstream and downstream as input region for HOMER ( Heinz et al . , 2010 ) to call known and de novo motifs .\",\n \"The overlap between ChIP-seq peaks was determined by 1 bp overlap of two peaks that were extended by 500 bp both upstream and downstream .\",\n \"The TSS associated genes in each ChIP-seq were determined by overlapping peaks to refseq genes coordinates .\",\n \"Venn diagrams were generated using online web tool VENNY 2 . 1 ( http://bioinfogp . cnb . csic . es/tools/venny/index . html ) .\",\n \"Protein function enrichment was analyzed using DAVID ( Huang et al . , 2008 , 2009 ) .\",\n \"The ChIP-seq peaks were visualized using IGV ( http://www . broadinstitute . org/igv/ ) .\",\n \"The sequence of ChIP-PCR primers is listed in Supplementary file 8 .\",\n \"FXR1 or IgG control ChIP pulldown complexes were separated in SDS-PAGE gel .\",\n \"The gel was stained with EZblue ( Sigma , G1041 ) .\",\n \"The stained gel was cut into multiple parts based on protein molecular weight .\",\n \"Gel pieces were destained in solution containing 50 mM NH4HCO3 ( Sigma Aldrich ) and Acetonitrile ( ACN ) ( Fisher Scientific ) at 1:1 ratio , dried with SpeedVac , rehydrated in 300 μl of 10 mM DTT ( Sigma Aldrich ) solution at 56°C for 1 hr , and reacted in 25 mM IAA ( Sigma Aldrich ) at 37°C for 30 mins .\",\n \"After washing with 25 mM NH4HCO3 ( Sigma Aldrich ) for 10 mins , the gel pieces were digested with trypsin solution ( 0 . 01 g/L trypsin in 25 mM NH4HCO3 ) ( Promega ) at 37°C for 12 hr .\",\n \"The digested peptides were extracted using 300 μl of 50% ACN-0 . 1% FA .\",\n \"The extracted solution was collected and concentrated with SpeedVac .\",\n \"The samples were resolved with 0 . 1% FA and analyzed by nanoLC-MS/MS ( Thermofisher ) .\",\n \"MS/MS was performed in a data-dependent mode in which the top 10 most abundant ions ( S/N > 30 ) for each MS scan were selected for MS/MS analysis by HCD .\",\n \"Parameters used during searching included enzyme , trypsin; missed cleavages , One; dynamic modifications , oxidation ( M ) ; static modifications , Carbamidomethyl ( C ) ; precursor mass tolerance , 10 ppm; fragment mass tolerance , 0 . 1 Da .\",\n \"Parameters in data filtration with Prohits included kinds of exclusion proteins: Heat Shock , Ribosomal , Cytoskeleton , Keratin , Artifact Protein , Rib , Nucleoprotein and Albumin; Max SAINT < 0 . 7 ( Liu et al . , 2012; Liu et al . , 2010 ) .\",\n \"All MS and MS/MS data were searched in the combined mode against Uniprot Human database ( August , 2013 ) using the Proteome Discoverer 1 . 3 software ( Thermofisher ) .\",\n \"A score greater than 31 ( confidence interval values > 95% ) obtained with the Mascot search engine ( Matrix Science , U . K . ) was considered as significant .\",\n \"All the searching results were filtered with Prohits software to remove non-specific binding proteins ( Max SAINT < 0 . 7 ) ( Liu et al . , 2010 , 2012 ) .\",\n \"FXR1 interacting proteins were classified according to the GO ( gene ontology ) database .\",\n \"Protein function clustering was analyzed with DAVID .\",\n \"Visualization and network map of FXR1-interacting proteins were delineated using BioGRID in Cytoscape .\",\n \"Protein–protein interaction was analyzed using the KEGG ( Kyoto Encyclopedia of Genes and Genomes ) pathway database .\",\n \"Total RNA was extracted using RNeasy Mini Kit ( Qiagen , 74106 ) and Qiagen QIAcube 230 V w . starter Pack-2 machine , and reverse-transcribed using Oligo ( dT ) primer and the SuperScript III First-Strand synthesis system ( Invitrogen , 18080–051 ) .\",\n \"q-RT-PCR using cDNA product as the template was performed using Power SYBR Green PCR Master Mix ( Applied Biosystems ( AB ) , 4367659 ) and an ABI 7900HT Fast Real-Time PCR-3 Machine with specific primers .\",\n \"Gene expression was quantified using the comparative Ct ( cycle threshold ) method and the results were normalized to GAPDH .\",\n \"Sequences of q-RT-PCR primers are listed in Supplementary file 8 .\",\n \"Each RNA-seq sample had three biological replicates .\",\n \"RNA isolation was performed using Trizol and RNeasy MinElute Cleanup Kit from cells .\",\n \"Following extraction , RNA quantity was assessed using Nanodrop and the integrity of total RNA using the RNA 6000 Nano Kit on an Aligent 2100 Bioanalyzer .\",\n \"An RNA library was constructed using the TruSeq RNA Sample Preparation Kit .\",\n \"The first step in the workflow involved purifying the poly-A containing mRNA molecules using oligo-dT-attached magnetic beads .\",\n \"Following purification , mRNA was fragmented into small pieces using divalent cations under elevated temperature .\",\n \"The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers .\",\n \"Second-strand cDNA synthesis followed , using DNA PolymeraseI and RNase H . The cDNA fragments then went through an end repair process , involving the addition of a single ‘A’ base , and then ligation of the adapters .\",\n \"The products were then purified and enriched with PCR to create the final cDNA library .\",\n \"The clusters of the cDNA library were generated on HiSeq X HD PE flow cell .\",\n \"Then the clusters were sequenced on a HiSeq X system .\",\n \"RNA-seq data quality was analyzed using FastQC ( Andrews , 2010 ) .\",\n \"Reads in the fastq files were trimmed by five base pairs at the 5´-end , and then mapped to the reference human genome ( HG19 ) with the algorithm STAR using default parameters .\",\n \"Multiple mapped reads and duplicate reads were removed to extract raw reads of refseq genes using HOMER .\",\n \"Differential expressed genes were identified using R package DESeq2 and determined using log2FC cutoff 0 . 6 and adjusted p value cutoff 0 . 01 .\",\n \"KEGG pathway enrichment was investigated using GSEA ( Subramanian et al . , 2005; Mootha et al . , 2003 ) .\",\n \"Histone H3 MLA proteins with methylation at various residues were purchased from Active Motif ( 31208–31222 ) .\",\n \"His-tagged-FXR1-Tudor ( 2-132 ) protein were expressed and purified from an Escherichia coli system , and pulldown assay was performed as previously described ( Alpatov et al . , 2014 ) .\",\n \"In brief , in each reaction , 2 µg of MLA protein and 2 µg of FXR1-Tudor protein were added in 500 µl of Histone MLA Binding Buffer ( 50 mM Tris pH 7 . 5 , 1 M NaCl , 2 mM MgCl2 , 0 . 5% Triton X-100 , and 10 mM Imidazole ) ( save 10 µl as input ) , followed by rotation at 4°C for 30 mins .\",\n \"The reaction solution was spun down ( 10 , 000 g , 2 min ) to collect the supernatant in a new tube .\",\n \"15 μl of Ni-NTA beads ( Qiagen ) was added to the collected supernatant followed by rotation at 4°C for 1 hr .\",\n \"Beads were collected by spinning at 1 , 000 g for 2 min , washed with 1 ml of Histone MLA Binding Buffer five times , suspended in 30 μl of 2 × SDS sample buffer and boiled to denature the proteins .\",\n \"The FXR1-bound histone MLA proteins were detected in a WB assay .\",\n \"JAK inhibitor S-Ruxolitinib ( dual JAK1/2 inhibitor tool compound ) was purchased from selleck ( http://www . selleck . cn/ ) .\",\n \"Owing to the instability of the compound , the medium with inhibitor was replaced every 5 days .\",\n \"Gene-specific siRNA sequence was designed using the software in Hannon Lab ( http://cancan . cshl . edu/RNAi_central/RNAi . cgi ? type=siRNA ) .\",\n \"The siRNA oligonucleotides were produced by Invitrogen , and the MISSION siRNA Universal Negative Control #2 ( Sigma ) was used as a negative control .\",\n \"Five siRNAs for each gene were tested .\",\n \"siRNAs were introduced into cells at a final concentration of 5–20 nM using Lipofectamine RNAiMAX Transfection Reagent ( Invitrogen , 13778–150 ) and Opti-MEM ( Gibco , 31985–062 ) according to the manufacturer’s protocol .\",\n \"Knockdown efficiency was assessed 48 hr post-transfection by q-RT-PCR or WB .\",\n \"Sequences of siRNAs are listed in Supplementary file 8 .\",\n \"Flag-tagged FXR1 or HA-tagged-STAT1 or -STAT3 proteins were expressed in HEK 293 T cells .\",\n \"Cell lysates were prepared using lysis buffer ( 50 mM Tris-Cl , pH 7 . 5 , 500 mM NaCl , 1% Triton X-100 ) with complete protease inhibitor cocktail ( Sigma , P8340 ) , phosphatase inhibitor cocktail ( Sigma , P5726 ) , and PMSF ( Sigma , 93482–50 ml-F ) .\",\n \"Lysate was incubated , at 4°C overnight , with 100 μl Flag-M2 magnetic beads ( Sigma , M8823 ) to purify Flag-tagged-FXR1 protein or with 100 μl Influenza Hemagglutinin ( HA ) magnetic beads ( Thermo Fisher , 88837 ) to purify HA-tagged-STAT protein .\",\n \"Beads were washed with 1 ml lysis buffer three times and with 600 μl elution buffer ( 50 mM Tris-Cl , pH7 . 5 , 500 mM NaCl , 10% Glycerol ) once .\",\n \"Target protein was eluted with 30 μl of Flag peptide solution ( 5 mg/ml , in elution buffer , Sigma , F4799-4MG ) to obtain Flag-tagged-FXR1 protein or with HA peptide ( 2 mg/ml , in elution buffer , Sigma , I2149-1MG ) to harvest HA-tagged-STAT protein .\",\n \"For in vitro pulldown , 25 µl purified Flag-tagged-FXR1 protein was incubated with 75 µl purified HA-tagged-STAT1 or -STAT3 protein in 500 µl binding buffer ( 50 mM Tris pH 7 . 5 , 150 mM NaCl , 0 . 1% NP-40 , 1 mM DTT , phosphatase inhibitor and protease inhibitor cocktail ) at 4°C overnight .\",\n \"30 µl Flag or HA magnetic beads were added and incubated at 4°C for another 3 hr .\",\n \"Beads were washed with wash buffer ( 50 mM Tris-pH 7 . 5 , 300 mM NaCl , 0 . 1% NP-40 ) three times .\",\n \"The eluted products in 30 µl elution buffer were subjected to WB analysis .\",\n \"H358 cells were lysed in lysis buffer ( 100 mM NaCl , 10 mM MgCl2 , 30 mM Tris-HCl , pH 7 . 5 , 1 mM dithiothreitol [DTT] , protease inhibitor cocktail , 100 U/ml RNasin , 0 . 1% Nonidet P40 [NP40] ) and divided into two parts .\",\n \"2% of lysate was saved as input .\",\n \"The lysate was pre-cleared by 10% BSA and 30 µl ChIP Grade Protein G Magnetic Beads ( CST , #9006 ) on rotation for 1 hr at 4°C .\",\n \"The supernatant was collected by a magnetic separation rack , incubated with FXR1 antibody ( Sigma , HPA018246 ) or IgG control ( CST , 2729 ) for 1 hr at 4°C , and incubated with 30 µl of magnetic beads for 1 hr at 4°C on rotation .\",\n \"Beads were washed with NT2 buffer ( 50 mM Tris-Cl , 150 mM NaCl , 1 mM MgCl2 , and 0 . 05% NP-40 ) five times .\",\n \"Endogenous RNA protein complexes ( RNP ) were eluted from antibody/Protein G beads by incubation with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing ( 1200 rpm ) in thermomixer .\",\n \"Proteins in the elution were digested by Proteinase K . RNA was purified using spin columns from RNeasy Mini Kit ( Qiagen , 74106 ) and reverse-transcribed using Oligo ( dT ) primer and the SuperScript III First-Strand synthesis system ( Invitrogen , 18080–051 ) .\",\n \"The cDNA was subjected to RT-PCR assay using AccuPrime Pfx SuperMix ( Invitrogen , 12344040 ) ( Qian et al . , 2015 ) .\",\n \"The PCR products are detected by electrophoresis in agarose gels and stained with ethidium bromide , visualized by ChemiDoc .\",\n \"The non-target genes GAPDH and Actin serve as the negative controls .\",\n \"ChIP-seq data reported in this paper have been deposited in the Gene Expression Omnibus ( GEO ) database under accession number GSE79707 .\",\n \"RNA-seq data reported in this paper have been deposited in the Gene Expression Omnibus ( GEO ) database under accession number GSE101754 .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Tumor suppressor p53 prevents cell transformation by inducing apoptosis and other responses .","Homozygous TP53 deletion occurs in various types of human cancers for which no therapeutic strategies have yet been reported .","TCGA database analysis shows that the TP53 homozygous deletion locus mostly exhibits co-deletion of the neighboring gene FXR2 , which belongs to the Fragile X gene family .","Here , we demonstrate that inhibition of the remaining family member FXR1 selectively blocks cell proliferation in human cancer cells containing homozygous deletion of both TP53 and FXR2 in a collateral lethality manner .","Mechanistically , in addition to its RNA-binding function , FXR1 recruits transcription factor STAT1 or STAT3 to gene promoters at the chromatin interface and regulates transcription thus , at least partially , mediating cell proliferation .","Our study anticipates that inhibition of FXR1 is a potential therapeutic approach to targeting human cancers harboring TP53 homozygous deletion ."],"string":"[\n \"Tumor suppressor p53 prevents cell transformation by inducing apoptosis and other responses .\",\n \"Homozygous TP53 deletion occurs in various types of human cancers for which no therapeutic strategies have yet been reported .\",\n \"TCGA database analysis shows that the TP53 homozygous deletion locus mostly exhibits co-deletion of the neighboring gene FXR2 , which belongs to the Fragile X gene family .\",\n \"Here , we demonstrate that inhibition of the remaining family member FXR1 selectively blocks cell proliferation in human cancer cells containing homozygous deletion of both TP53 and FXR2 in a collateral lethality manner .\",\n \"Mechanistically , in addition to its RNA-binding function , FXR1 recruits transcription factor STAT1 or STAT3 to gene promoters at the chromatin interface and regulates transcription thus , at least partially , mediating cell proliferation .\",\n \"Our study anticipates that inhibition of FXR1 is a potential therapeutic approach to targeting human cancers harboring TP53 homozygous deletion .\"\n]"},"summary":{"kind":"list like","value":["Healthy human cells employ many tricks to avoid becoming cancerous .","For example , they produce proteins known as tumor suppressors , which sense if a cell shows early signs of cancer and instruct the cell to die .","A gene known as TP53 produces one of the most important tumor suppressor proteins , and this gene is inactive or missing in many types of human cancer .","Treating cancers that have completely lost the TP53 gene is particularly difficult .","One way to develop new treatments for these conditions would be to target other proteins that these cancers need to survive; but these proteins first need to be identified .","Fan et al . have now identified one such protein in human cancer cells lacking TP53 .","Searching databases of DNA sequences from human cancer cells revealed that those without the TP53 gene often also lose a neighboring gene called FXR2 .","Cancer cells survive without FXR2 because a similar gene , called FXR1 , can compensate .","Fan et al . therefore decided to experimentally lower the activity of the FXR1 gene and , as expected , cancer cells without TP53 and FXR2 stopped growing .","Normal cells , on the other hand , were unaffected by the deletion of the FXR1 gene since FXR2 is still there .","This phenomenon , in which cancer cells become vulnerable after the loss of certain genes but only because they have already lost important tumor suppressors , is called “collateral lethality” .","Further experiments showed that the protein encoded by FXR1 coordinates with other proteins to activate genes that contribute to cell growth .","These findings suggest new ways to treat human cancers that have lost TP53 .","For example , if scientists can find small molecules that inhibit the protein encoded by FXR1 and show that these molecules can block the growth of tumors lacking TP53 and FXR2 , this could eventually lead to a new anticancer drug .","However , like any new drug , these small molecule inhibitors would also need to be extensively tested before they could be taken into human clinical trials ."],"string":"[\n \"Healthy human cells employ many tricks to avoid becoming cancerous .\",\n \"For example , they produce proteins known as tumor suppressors , which sense if a cell shows early signs of cancer and instruct the cell to die .\",\n \"A gene known as TP53 produces one of the most important tumor suppressor proteins , and this gene is inactive or missing in many types of human cancer .\",\n \"Treating cancers that have completely lost the TP53 gene is particularly difficult .\",\n \"One way to develop new treatments for these conditions would be to target other proteins that these cancers need to survive; but these proteins first need to be identified .\",\n \"Fan et al . have now identified one such protein in human cancer cells lacking TP53 .\",\n \"Searching databases of DNA sequences from human cancer cells revealed that those without the TP53 gene often also lose a neighboring gene called FXR2 .\",\n \"Cancer cells survive without FXR2 because a similar gene , called FXR1 , can compensate .\",\n \"Fan et al . therefore decided to experimentally lower the activity of the FXR1 gene and , as expected , cancer cells without TP53 and FXR2 stopped growing .\",\n \"Normal cells , on the other hand , were unaffected by the deletion of the FXR1 gene since FXR2 is still there .\",\n \"This phenomenon , in which cancer cells become vulnerable after the loss of certain genes but only because they have already lost important tumor suppressors , is called “collateral lethality” .\",\n \"Further experiments showed that the protein encoded by FXR1 coordinates with other proteins to activate genes that contribute to cell growth .\",\n \"These findings suggest new ways to treat human cancers that have lost TP53 .\",\n \"For example , if scientists can find small molecules that inhibit the protein encoded by FXR1 and show that these molecules can block the growth of tumors lacking TP53 and FXR2 , this could eventually lead to a new anticancer drug .\",\n \"However , like any new drug , these small molecule inhibitors would also need to be extensively tested before they could be taken into human clinical trials .\"\n]"},"year":{"kind":"string","value":"2017"}}},{"rowIdx":4182,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["ecology"],"string":"[\n \"ecology\"\n]"},"title":{"kind":"string","value":"Group size and composition influence collective movement in a highly social terrestrial bird"},"id":{"kind":"string","value":"elife-59902-v1"},"sections":{"kind":"list like","value":[["Movement behaviour fundamentally influences how individuals encounter resources that are critical for their fitness .","In highly social animals , movements are determined by how efficiently a group can coordinate their behaviour ( Chapman and Chapman , 2000; Couzin et al . , 2002; Strandburg-Peshkin et al . , 2017; Strandburg-Peshkin et al . , 2015 ) .","Coordination requires effectively deciding where and when to move ( Conradt and Roper , 2005 ) as well as maintaining cohesion during movement ( Gall and Manser , 2017 ) .","Both these factors could be shaped by intragroup characteristics .","For example , it has become increasingly evident that group composition ( Farine et al . , 2015 ) , such as the presence of uniformed individuals ( Couzin et al . , 2011 ) or the mixture of individual personality types ( Aplin et al . , 2014; Jolles et al . , 2017 ) , can shape the behavioural properties of groups .","In societies where individuals form stable groups , group size is another potentially important group characteristic that may also significantly impact fine-scale collective movement patterns .","However , while the balance of benefits ( such as decreased predation risk according to the ‘many eyes hypothesis’; Pulliam , 1973 ) and costs ( increased intra-group competition over resources ) associated with group size have been extensively explored ( Krause and Ruxton , 2002 ) , we still have very little information on whether group size translates to differences in movement behaviour and ranging patterns of naturally occurring groups .","Theory predicts that larger group size can benefit animal groups that move collectively through complex and heterogeneous landscapes .","If group members pool their information , then larger groups should be able to reach more accurate estimates of their environment ( Cantor et al . , 2020; Couzin et al . , 2011 ) , as highlighted by the ‘wisdom of the crowd’ theory ( Galton , 1907 ) .","This process can translate to navigational accuracy , via the ‘many wrongs hypothesis’ , as pooling the imperfect individual estimations of directions can generate accurate group-level estimates that allow groups to move more directly towards resources ( Simons , 2004 ) .","Larger groups are also more likely to contain individuals with knowledge relevant to the current position or situation of a group ( the ‘pool of competence hypothesis’; Giraldeau , 1984 ) .","For example , studies on killer whales ( Brent et al . , 2015 ) and elephants ( McComb et al . , 2001 ) have shown that older individuals become leaders under conditions of extreme resource shortages .","Such mechanisms should therefore allow larger groups to more effectively navigate their environment , and therefore exhibit a larger home-range size .","Effective navigation , and subsequently larger home ranges , should be beneficial for group members .","Larger areas encompass a greater diversity and abundance of resources needed to meet group members’ nutritional needs ( Corriale et al . , 2013; Hubbs and Boonstra , 1998; Maher and Burger , 2011 ) , and moving to more new areas increases the probability of discovering new food resources .","Using larger areas can also allow groups to be more variable in their movements , which could make them more unpredictable to predators ( Roth and Vetter , 2008 ) and thus decreases predation risk ( Richardson et al . , 2018 ) .","Thus , all else being equal , such as environmental characteristics , the members of a group with a relatively larger home range should have higher survival in periods of resource limitations and/or greater reproductive output than an identical group with a smaller home range .","In reality , home-range size is often limited by many factors , including competition for space with other groups ( Christensen and Radford , 2018 ) .","However , many group-living species live in non-territorial societies , where groups can benefit from exploiting a home range without paying the costs of intergroup fights and competition .","In such species , groups can interact affiliatively and preferentially with specific other groups , resulting in a multilevel society .","In such societies , having a larger home-range size might generate more chances to associate with conspecifics from other groups ( Grueter et al . , 2020 ) , which , in turn , can increase mating opportunities ( by increasing contact with members of the opposite sex; Grueter et al . , 2015 ) , dispersal opportunities ( as sub-adults often disperse to neighbouring groups; Städele et al . , 2015 ) and information sharing about food resources or predation risk ( Whitehead et al . , 2012 ) .","Thus , there are many ways that groups can benefit from maximizing their space use , up to the point where the costs of travel and navigation do not override the benefits gained .","However , living in large groups can also introduce challenges , particularly in species that maintain stable group membership .","Collective movement requires maintaining cohesion , a challenge that increases with group size and in more complex environments ( Codling et al . , 2007 ) .","From a movement perspective , coordination among many individuals could result in slower decision-making , due to both coordination challenges as well as larger groups being likely to harbour more conflicts of interest .","For example , one study of collective movement in baboons found that the probability that an individual baboon followed one initiator versus the other was much lower when both disagreement among initiators on where to go and the number of initiators increased ( Strandburg-Peshkin et al . , 2015 ) .","Larger groups are also likely to have slower movement speeds while ‘on-the-go’ .","We all have personal experiences of how much slower it can be to move or make decisions ( such as where to have lunch ) when we are in a large group , as the group generally may have to conform to the slowest members ( Herbert-Read et al . , 2013 ) or satisfy a diversity of preferences ( Strandburg-peshkin et al . , 2017 ) .","Finally , it may be more challenging for individuals to influence larger groups to move to new areas , and thus to extend the group’s home-range .","These factors suggest that the consensus costs borne by larger groups will ultimately limit the size of the home-range that such groups can exploit .","Given that both positive and negative relationships between group size and home-range size may exist , we a priori predict a non-linear relationship between the size of stable groups and collective movement characteristics .","This non-linear relationship is likely to represent an optimal group size for movement that reflects the point where the difference between navigational accuracy and coordination problems is maximized .","To our knowledge , this relationship remains largely unexplored in wild , free-living groups .","A first challenge in quantifying the relationship between home-range size and group size requires long-term tracking across multiple replicated groups and , ideally , replication in time .","A second challenge is that many species that live in stable groups ( at least most of the well-studied species ) either experience increased intra-group competition as group sizes increase ( e . g . baboons; Markham et al . , 2015 ) or they are territorial ( e . g . meerkats; Bateman et al . , 2015 ) .","Territoriality introduces several confounding effects .","For example , the greater resource-holding potential of larger groups ( Bateman et al . , 2015; Markham et al . , 2015 ) can translate to larger home-ranges .","Further , different territorial groups can vary in the density and distribution of resources within their territory , which could make it appear that smaller groups have larger home-ranges if they live in marginal areas with low resource densities .","As a result , the relationship between group movement characteristics and group size has remained relatively unclear .","In this study , we examine the effect of group size on the movement and space use patterns of wild groups of vulturine guineafowl ( Acryllium vulturinum ) , and whether resulting patterns correspond to differences in reproductive output among groups .","Our study species lives in a multilevel society consisting of large , stable groups with multiple breeding units and groups form stable associations with specific other groups ( Papageorgiou et al . , 2019 ) .","The stable social structure and the lack of territoriality—the entire population inhabits a small area of approximately 500 ha and all groups can easily access all parts of our study area—makes vulturine guineafowl an ideal species to investigate how group size influences movement characteristics because groups can range without major constraints on ranging imposed by other groups .","Further , their large body size ( ~1 . 5 kg ) allow guineafowl to be fitted with high-resolution solar-powered GPS tags to collect both fine-scale data on their movement as well as long-term data on their ranging behaviour .","We use data from GPS tags fitted to one individual from a total of 21 distinct groups , collected over five 2-month-long non-breeding seasons ( Figure 1 ) .","We fit a continuous-time movement model ( ctmm ) to each individual GPS dataset ( one bird per group per season ) using GPS locations collected every 5 min .","From each model , we calculated ( 1 ) home-range size , ( 2 ) the tendency for groups to use the same areas on consecutive days , and ( 3 ) daily travel distance .","We then used high-resolution ( 1 Hz ) GPS location data from each individual to quantify ( 4 ) the movement speed of its group while ‘on the go’ .","Because groups move through heterogeneous environments , larger groups should be able to navigate more effectively .","Their large size should also result in larger daily travel distances as they search for the resources needed to survive .","By contrast , moving as a large group also introduces consensus costs , and these could limit the home-range size of the largest groups by affecting their speed or their tendency to move to new areas .","As a result of the interaction between these two effects , we expect that there will be a non-linear relationship between group size and movement , which , if it results in more effective use of available resources , could correspond to an optimal group size for group movement characteristics .","We predict that intermediate groups will have the largest home-range sizes .","This will arise because while larger groups should travel further , intermediate groups will have lower day-to-day home-range fidelity .","We further predict that intermediate size groups will present the fastest speed while ‘on the go’ .","However , these factors may be tempered by group composition .","Groups in our population reproduced several times over our study period , and the presence of young group members could restrict the movements of groups .","Thus , we run our analysis while controlling for the number of chicks present in the groups , and predict that groups with more chicks should travel slower and shorter distances , thereby restricting their home-range size .","Finally , we predict that a non-linear relationship between group size and movement should translate to corresponding peak in fitness , measured as the number of chicks in a group .","Such a pattern would suggest that maximising the difference between navigation and coordination represents an optimal group size at which groups are most efficient in exploiting their environment ."],["Our movement analysis , based on 1 , 274 , 434 five-min GPS positions , spanned 45 seasonal observations of individuals ( groups ) over five 2-month-long seasons ( Figure 1 , see Supplementary file 1A for a summary of the data ) .","Group sizes , recorded during daily census observations , were widely distributed , with intermediate groups being observed less often ( Figure 2 ) .","Fitting generalised estimating equations ( GEEs ) with the number of adults ( group size ) and the number of chicks as predictors ( while accounting for repeated measures of the same groups across seasons ) revealed that home-range size was significantly predicted by group size and its quadratic term , but that this relationship was modulated by the presence of chicks .","Intermediate groups had the largest home-range , but home-range size decreased with increasing number of chicks ( Table 1A , see Supplementary file 1B for all model fits ) .","Average temporal overlap in space use was also significantly predicted by group size and its quadratic term , but was not significantly modulated by the presence of chicks ( Table 1B , see Supplementary file 1C for all model fits ) .","Average daily distance travelled increased linearly with group size ( Table 1C , see Supplementary file 1D for all model fits ) .","Speed ‘on-the-go’ was not explained by group size or by number of chicks in the group ( Table 1D , see Supplementary file 1E for all model fits ) .","Overall , smaller and larger groups expressed smaller home-range sizes and increased average temporal overlap in space use , while larger groups expressed longer daily travel distances than groups of smaller size ( see Figure 3 and Figure 4 ) .","The number of chicks in the group was also significantly predicted by group size and its quadratic term ( Table 1E , Figure 3D , see also Supplementary file 1F for all model fits ) .","We found that intermediate-sized groups had more chicks , with the peak in fitness ( group size = 33 ) closely matching the peak in home-range size ( group size = 36 ) and average daily overlap ( group size = 37 ) .","Number of adults ( group size ) and its quadratic term significantly predicted ( A ) home-range size and ( B ) average temporal overlap in space use .","The presence of chicks modulated the relationship between group size and home-range size , reducing the overall contribution of the number of adults to home-range size ( the negative interaction with number of adults ) and reducing the quadratic effect ( the positive interaction with number of adults squared , which offsets the negative quadratic coefficient ) .","( C ) The average daily distance travelled increased with number of adults ( see Figure 3C ) .","( D ) Speed while travelling was not significantly impacted by group size or its quadratic term .","( E ) The number of chicks per group was significantly predicted by group size and its quadratic term ( see Figure 3D ) .","Parameter estimates , significance , and R2 were generated using the most parsimonious fitted GEEs based on QIC ( see Materials and methods ) .","All models tested are summarized in the Supplementary file 1B to 1F ."],["Our study shows that group size and composition can impact collective movement characteristics .","Groups of vulturine guineafowl that have an intermediate size exhibit larger home-ranges and explore more new areas day-by-day .","However , this relationship exists despite intermediate groups travelling shorter distances each day compared to larger groups .","Even though the presence of chicks does not impact how far or how fast groups travel , having many chicks restricts the home range of their group .","Further , our study also shows that groups of intermediate size have higher fitness as they produce more offspring than larger or smaller groups .","Our results are therefore consistent with our a priori prediction that a non-linear relationship should exist between group size and collective movement , reflecting the increasing navigation ability with increasing group size and the increasing challenges of coordinating movement in larger groups .","The relationship between group size and reproductive output further suggests that intermediate-sized groups are more effective at exploiting space , providing evidence for an optimal group size for collective movement .","The relationships between group size and group movement outcomes provide new insights into how differently sized animal groups interact with their environment .","Larger groups cover larger distances during the day , which is likely to be important for encountering sufficient resources to meet their energetic demands , as proposed by the ecological constraints model ( Chapman and Chapman , 2000; Ganas and Robbins , 2005; Gillespie and Chapman , 2001; Teichroeb and Sicotte , 2009 ) .","However , by exploiting larger areas over a whole season , groups of intermediate size are likely to benefit from having access to a greater abundance and/or diversity of food resources ( Maher and Burger , 2011 ) , they potentially also benefit both from being less predictable to predators ( Richardson et al . , 2018; Roth and Vetter , 2008 ) , and from having access to a more diverse social landscape .","In addition , intermediate-sized groups achieve larger home ranges while travelling shorter distances than larger groups , implying that they have lower energy expenditure .","It is worth noting that we focused our analysis on 2-month periods each capturing similar ecological conditions .","Future studies could investigate whether these non-linear relationships between group-size and movement characteristics change across different environmental conditions and especially in seasons where energy might be under tighter constraints , such as during droughts .","Evidence for this could bring important insights into the drivers of group size variation .","While we predicted that group size would translate to differences in movement while ‘on-the-go’ , our measure of moment-by-moment movement ( speed while travelling ) did not reveal any relationship between group size and movement speed .","One reason for this could be that larger groups could have better knowledge of the environment , meaning that they can offset consensus costs ( i . e . periods of slow movement due to conflict within the group regarding movement directions ) with more effective movement once departed ( Couzin et al . , 2005 ) .","For example , the speed and polarization , and subsequently the directionality of group motion , increases with the size of fish schools ( Tunstrøm et al . , 2013 ) .","As group size increases in locusts , they transition from disordered to ordered motion ( Buhl et al . , 2006 ) .","Thus , it is possible that while the realised movement speed does not differ with group size , the characteristics of how the movement is achieved could vary between smaller and larger groups .","Alternatively , the challenge of reaching a consensus could mean that larger groups end up repeating the same routes more often , which they can achieve by moving faster as individuals all know where they are going .","This explanation is consistent with our findings that larger groups were less variable in where they travelled each day .","Such mechanisms linking collective movement with ranging behaviour warrant further investigation .","Several studies in the past have investigated the relationship between group size and ranging behaviour .","Most notably , Markham et al . , 2015 found that intermediate-sized groups of wild baboons ( Papio cynocephalus ) utilized smaller areas , covered shorter distances per day , and used space less evenly than smaller and larger groups—patterns which are largely opposite to our findings .","Stevenson and Castellanos , 2000 found that woolly monkeys ( Lagothrix lagothricha ) in groups of intermediate size travelled less during the day than larger and smaller groups .","Finally , research on meerkats ( Suricata suricatta ) Bateman et al . , 2015 found no relationship between group size and group movement characteristics .","However , in most previously studied systems , group movement characteristics are likely to be impacted not only by the ability for groups to move , but also by intra-group contest competition and inter-group competition ( Markham et al . , 2015 ) .","For example , smaller groups have the weakest resource-holding potential , meaning that they should have the smallest territories .","At the same time , they might be excluded from the best , most resource-rich , habitats by larger groups ( who need more resources due to intra-group competition ) , meaning that they could also end up having to use equally sized or even larger areas ( Markham et al . , 2015; Stevenson and Castellanos , 2000 ) .","In contrast to previous studies , our evidence for a non-linear relationship between group size and movement characteristics comes from a species that not only presents minimum intergroup competition but also groups use largely overlapping areas .","These features reduce the confounds on the link between group size and group movement behaviours .","Our study is therefore more likely to reveal effects arising from movement-based mechanisms without confounding effects that are exogenous to the group .","Vulturine guineafowl live in a multilevel society , where groups preferentially associate with other groups ( Papageorgiou et al . , 2019 ) .","In such societies , intergroup associations can bring many important benefits , such as information sharing on predators and resources , as well as mating and dispersal opportunities ( Grueter et al . , 2020 ) .","A large home range size in a multilevel society could provide groups with access to more communal roosts ( we have observed up to five groups in a single roost , Papageorgiou et al . , 2019 ) and more glades , and thus more opportunities to associate with more groups .","However , the benefits of associating with other groups might not increase linearly with home range size , as associating with a large number of conspecifics can also imply costs , such as disease transmission or increased competitions for mates ( Krause and Ruxton , 2002 ) .","More work is needed on the interactions between ranging behaviour of groups and the broader social environment in multilevel societies .","We built on existing insights from studies of collective behaviour to develop an a priori prediction that the relationship between group size and movement characteristic should be non-linear .","Our prediction proposed that maximising the use of space ( i . e . a larger home range ) through effective movement ( faster movement and less repetitive ranging ) should result in more efficient exploitation of the resources available to a group .","A key question is whether groups actually benefit from this maximisation ?","By analysing data on the reproductive success across the groups , we found striking evidence for a peak in fitness , with intermediate-sized groups having more chicks .","This peak , which closely matches the group size peaks in movement characteristics ( home range size and average daily overlap ) , suggests that intermediate group sizes might be linked to higher reproductive success .","Our findings therefore provide evidence for an optimal group size in the context of collective movement .","An interesting additional finding from our study is that optimally sized groups ( 33–37 individuals ) were relatively rare .","Instead , the distribution of group size appears to be bimodal with most groups being either smaller or larger than the intermediate group size .","This observation aligns with the hypothesis that while an optimal group size results in maximum individual fitness ( Pride , 2005 ) groups at the optimal size should be unstable and therefore rarely observed ( Sibly , 1983 ) .","Groups in our study grow either through reproduction or through immigration .","We found that intermediate-sized groups have high reproductive success , which naturally pushes them beyond the optimal .","Further , because dispersing individuals should prefer optimal size groups , they should also turn intermediate-sized groups into groups that are larger than the optimal size .","Future studies should examine whether groups near the optimal size for collective movement also highly attract more dispersers .","The observed variation in results between our study and previous studies investigating the effect of group size on ranging behaviour in mammals could also potentially arise due to methodological differences .","For example , we used high-resolution tracks and continuous-time movement modelling to get much more precise estimates of movement characteristics .","By contrast , other studies have used much lower sampling resolutions , often relying on hand-held GPS devices ( Bateman et al . , 2015; Markham et al . , 2015 ) or used simplistic minimum convex polygons to model home-ranges ( Markham et al . , 2015 ) .","Minimum convex polygons are unlikely to be very good estimates of home-ranges as they are sensitive to outliers ( e . g . from transcription errors or poor GPS fixes ) and capture only one element of space use ( the extreme boundaries , which could be influenced by a range of factors such as habitat geometry; He et al . , 2019 ) .","However , despite these potential methodological differences , we believe that conflicting findings are more likely to suggest that the effect of group size on ranging behaviour and collective movement varies according to the properties of the different animal social systems .","For example , we predict that the outcome of the many factors that contribute to the ranging characteristics of social groups will be modulated by the distribution of food resources each species exploits .","Vulturine guineafowl have very little need to defend areas from other groups or compete for resources within groups , as their food source ( insects and small seeds ) are widespread and they roost on the top of dense stands of trees , a habitat feature which is common in the study area .","When competition is decreased , we predict that group size could become a more important driver of collective movement outcomes .","We hope that by collecting more data on this question across species there will soon be sufficient studies to fully test this prediction .","In addition to the effects of the number of adults in the group , we also revealed the impact of having young present in the group .","Vulturine guineafowl are precocial , meaning that they are very small when they join the group , and mature over long periods of time—at 1 year of age they are only approximately 65% of the full adult size .","For a number of months after hatching , chicks are unlikely to contribute to making decisions , but are highly vulnerable to predators—causing them to remain more in cover .","At the same time , chicks are likely to move more slowly , or cover less ground , due to their smaller body size .","We found that having chicks in the group has a profound effect on home-range size .","Our results indicate that groups with more chicks use smaller areas , thereby limiting groups of intermediate sizes from exhibiting larger home-ranges .","The limitations that chicks have on group movements raise interesting questions about the costs of group living .","The fact that groups of vulturine guineafowl remain cohesive across seasons and years suggests that any costs borne from collective movement will be shared by all group members , including by individuals which have failed to reproduce ( vulturine guineafowl are plural breeders; Papageorgiou et al . , 2019 ) or by subadults who have yet to disperse .","Thus , our data reveals a potentially important source of conflict among group members .","Such conflicts may be common in plural-breeding groups , which are commonly found in multilevel societies .","We have shown that using synchronous and high-resolution GPS tracking of multiple social groups that reside in the same area can reveal new insights into the movement and social ecology of species .","Specifically , we demonstrated that group size and composition can predict collective movement and space use characteristics .","Our results are consistent with the prediction that increased navigation accuracy but decreased coordination ability could produce a non-linear relationship between group size and collective movement .","However , the mechanisms that underlie the interaction between coordination maintenance and navigation accuracy in free-living groups of various sizes remain to be discovered .","Further , we have identified dimensions of movement—specifically that intermediate-sized groups have larger home-ranges and use a larger diversity of areas—that translate into intermediate-sized groups having greater efficacy at exploiting their environment , and , therefore , higher fitness .","These results suggest the presence of an optimal group size for collective movement .","A further interesting observation is that the benefits of having an optimal group size are not present when groups have offspring , and that few groups are of an optimal size , highlighting a potentially important feedback between current and future reproductive success .","The ability to develop and test our novel hypotheses relating to collective movement of animals in the wild was only possible by conducting a large-scale multi-group tracking study in a species that does exhibit little intra-group and inter-group competition .","We hope that cheaper , more reliable , and smaller devices , will continue to reveal similar insights into the lives of wild animals across the diversity of social systems ."],["We studied a population of vulturine guineafowl that resides in an area of approximately 500 ha in the southern part of the Mpala Research Conservancy ( MRC ) in Laikipia , Kenya .","Vulturine guineafowl are large , predominantly terrestrial , and highly gregarious birds .","They primarily forage on the ground , feeding on small grass root buds , seeds , fresh grass and small invertebrates , in other words widely dispersed food sources that have been predicted to limit within- and between-group contest competition ( Dammhahn and Kappeler , 2009 ) .","Based on 3 years of observations of colour-marked individuals , we found that they live in social groups , which range in size from 13 to 65 adults , have stable membership across years and remain completely cohesive ( with individuals rarely out of sight of other group members ) during non-breeding seasons ( typically January to March and July to October ) ( Papageorgiou et al . , 2019 ) .","Within a local area , groups form a multilevel society , which includes groups roosting communally ( with roosts containing several hundred birds ) and groups regularly encountering other groups during the day ( at which times between-groups agonistic interactions are rarely observed ) ( Papageorgiou et al . , 2019 ) .","Intergroup encounters and foraging are most common on glades ( Papageorgiou et al . , 2019 ) , which are small deforested areas that were previously used as overnight shelters for cattle and are therefore are rich in nutrients ( Young et al . , 1995 ) .","All vulturine guineafowl groups have access to glades as they are numerous in our study area .","On glades , vulturine guineafowl are prone to predation by raptors , such as martial eagles ( Naude et al . , 2019 ) , while moving through dense vegetation makes them prone to ambush by carnivorous mammals , such as jackals or leopards .","We trapped nearly all the adult members from each of the groups in our study area using large walk-in traps .","All trapped individuals were kept inside covered cages ( 1 m*50 cm*50 cm cage with 2 cm plastic mesh covered by a canvas ) to minimize stress and allow birds to remain standing while awaiting processing .","Birds were removed one by one , measured , and ringed with an individually numbered National Museums of Kenya stainless steel ring ( fitted on the tarsus of the right leg ) and a unique combination of four plastic colour bands fitted on each leg , on the tibiotarsus , for field identification .","For more details on trapping and marking processes , see Papageorgiou et al . , 2019 .","In each group , we fitted high-resolution solar-powered GPS tags ( 15 g Bird Solar , e-obs Digital Telemetry , Grünwald , Germany ) to between one and five individuals .","The devices were elevated using neoprene pads to prevent feathers from covering the tag’s solar panel , and were attached to the body using a backpack harness design ( Teflon ribbon ) , which included a cotton thread safety release mechanism .","GPS tags stayed on the birds from 6 months to 2 years , before the cotton breaks .","Groups were trapped once per year to mark new members and check the condition of the birds which carry GPS tags .","Since the start of the study in 2016 , no bird has ever been found with problems due to the GPS tags .","Each tag ( tag , pads , ribbons , crimps ) weighed approximately 20 . 5 g , far less than the recommended 3% of the birds’ body weight ( Kenward , 2001 ) .","Each device was programmed to record one data point ( date , time , coordinates ) every second when the battery had a high charge ( approximately every 2nd to 3rd day , for up to 8 hr continuously ) .","When the battery was at the next lower threshold , we set tags to record one point per second for the first 10 s of every 5th minute .","If battery charge was very low ( this setting was rarely used during our study period ) , tags were set to record one point every 15 min .","Data were remotely downloaded every 2 or 3 days using a BaseStation II ( e-obs Digital Telemetry , Grünwald , Germany ) .","Field-testing suggested that that the relative spacing of tags was accurate to within 1 m , 95% of the time tracked .","We uploaded all of our data to the Movebank repository ( Wikelski and Kays , 2018 ) , see https://www . movebank . org/cms/webapp ?","gwt_fragment=page=studies , path=study475851705 for further details .","We selected seasons with intermediate rainfall and greenness , as described in Papageorgiou et al . , 2019 .","Vulturine guineafowl groups temporarily split during the breeding ( wet ) season as breeding pairs form , subadults disperse from their natal groups and females lay and incubate their eggs ( Papageorgiou et al . , 2019 ) .","It is therefore challenging to quantify membership accurately during the breeding season .","Breeding seasons can occur twice a year ( during April or May and during October or November ) .","We also excluded periods of drought , as these cause groups to regularly travel to large bodies of water , such as the river that crosses our study area .","This unusual situation ( guineafowl are adapted to rarely require access to standing water under normal conditions ) would make the estimation of home-range size confounded by how far from the river each group roosts .","We thus made our selection of seasons based on the seasonal conditions where groups remain cohesive but do not make extraordinary movements , while remaining blind to the specific movement patterns of the GPS-tagged birds .","We defined seasons as being 2 months , which corresponds to period changes in conditions at our study site .","Given that our entire study population inhabits a small area , approximately 500 ha , and that all groups can easily access all parts of our study area , we assume that groups’ relative ranging behaviour was not restricted by the environment during these intermediate seasons .","Data for this study were collected from May 2017 to February 2020 , and the specific time periods that fulfilled the criteria listed above were categorised as Season A ( 30 . 5 . 2017 to 1 . 8 . 2017 ) , Season B ( 15 . 12 . 2017 to 15 . 2 . 2018 ) , Season C ( 1 . 8 . 2018 to 29 . 9 . 2018 ) , Season D ( 10 . 6 . 2019 to 9 . 8 . 2019 ) , and Season E ( 14 . 1 . 2020 to 16 . 3 . 2020 ) .","Census data was collected daily from a vehicle driving along the roads in our study area .","Every time a group was encountered , the identities of the marked birds were recorded , and a total number of birds present was counted ( including unmarked birds ) .","Each observation of a ‘group’ is defined as being a single or a multiple group , depending on whether the group moves away cohesively or whether individuals move away in different directions .","Counts are split into the number of adults and the number of chicks , with chicks being easily distinguishable until ~ 9 months of age .","Within each season , we selected groups containing one or more GPS tagged individuals for which we had precise group-size counts where observers could clearly see and count all the group members being present ( i . e . groups were counted more than once by more than one observer , the group size was found consistent across counts ) .","We included data on 21 different observational datasets of groups , with some groups replicated in more than one of the seasons ( Supplementary file 1A , Figure 1 ) .","For seasons in which a group had chicks , we calculated the median number of chicks per group , across all observations , in each study season .","We used the GPS data from each group in each season to quantify movement behaviour and resulting home-range characteristics .","In groups containing more than one GPS-tagged individual , we randomly selected one to represent that group’s movement , given that groups move very cohesively ( typically group members are within 29 m of each other 95% of the time tracked; Papageorgiou et al . , 2019 ) .","We calculated ( 1 ) the core home-range size ( 50% auto-correlated kernel density estimate ) , ( 2 ) average temporal overlap in space use , ( 3 ) average daily distance travelled .","We also explored ( 4 ) speed while moving .","We used the built-in features from the Movebank data repository to remove the very rare outliers in our dataset that were falling outside Kenya .","For ( 1 , 2 ) and ( 3 ) we used data points collected on the tenth second of every fifth minute , sub-setting this from high-resolution ( 1 Hz ) data and from data when the tag was set to collect 10 points every 5th minute .","This approach allowed us to ensure that the data points from all GPS-tagged birds were precisely synchronised in time .","For core home-range size ( 1 ) , we calculated home-range sizes by fitting a continuous-time movement model ( ctmm ) to each individual GPS dataset ( one bird per group per season ) .","These models follow a continuous-time stochastic process from which the maximum likelihood Gaussian home-range area can be extracted after the best fitted model is selected based on AIC .","Because ctmms model the actual movement , they better predict home-ranges than classical approaches , such as minimum convex polygons , that are typically prone to being highly influenced by just a few data points ( Fleming et al . , 2015 ) .","We used the maximum likelihood home-ranges to determine the area in which each bird was located 50% of the time .","This procedure was done using the ‘ctmm’ R package ( Calabrese et al . , 2016; Fleming and Calabrese , 2018 ) .","For average temporal overlap in space use ( 2 ) , we measured the inverse of the exploration of new areas , for each bird in each season .","We first fitted a ctmm to the 5-min data from each bird for the first 21 days it was tracked in each season ( longer time spans were not computationally feasible for this particular analysis ) .","We then calculated the overlap of the daily auto-correlated kernel density estimation for home-range of each bird , using the ‘overlap’ function of the ‘ctmm’ R package ( Winner et al . , 2018 ) .","Specifically , we calculated the overlap between daily ranges that were up to 3 days apart .","Finally we calculated the mean of the spatial overlap of one group with itself across each consecutive sets of 3 days , for the entire 21-day period , for each group .","For average daily distance travelled ( 3 ) , we used the 5-min data from above ( one bird per group per season ) and the function ‘speed’ from the ctmm R package , which provides the mean travel distance per day but not the speed while ‘on the go’ .","To estimate the mean speed ‘on the go’ ( 4 ) for each individual in each season we used only a subset of the data representing the times when the GPS tags were collecting one point per second ( 1 Hz ) .","From these data , we created a histogram of the log of the movement speeds ( distance travelled between consecutive points ) , revealing that speeds were multimodally distributed for each individual .","We found that the minima between the second and the third mode corresponded to −3 . 5 ( or 0 . 03 ms−1 ) and classified speeds exceeding this value as ‘moving’ and speeds slower than this value as ‘stationary’ ( Bender et al . , 2011; Wilson et al . , 2015 ) .","We then used the data classified as ‘moving’ to calculate the mean of the distance travelled in consecutive seconds of the 1 Hz data .","We fitted GEEs using the R package ‘geepack’ to test whether group size and number of chicks predicted home-range size , average temporal overlap in space use , average daily distance travelled and speed ‘on the go’ .","Our primary predictor variable was the number of adults in the group .","Because chicks could modulate the relationship between the number of adults ( our measure of group size ) and movement response variables , we also allowed models to include a term for the number of chicks .","Because we expected a non-linear relationship between group size and movement parameters , we also allowed models to include the number of adults squared ( the quadratic term ) .","Further , because we did not know how chicks could modulate group movement , we allowed the number of chicks and the number of chicks squared to be added in interactions with the number of adult and the number of adults squared .","We created all combinations of response variables and interactions ( only between terms containing adults and chicks ) , but ensuring that models contained at least the number of adults as a predictor , and always included the number of chicks when the number of chicks squared was included .","We used QIC to select the most parsimonious models ( lowest QIC ) ( Hocking , 2014 ) and we also calculated the R2 of each GEE to estimate their goodness of fit ( Zheng , 2000 ) .","For all models , we fitted group ID as a repeated measure to account for multiple measures of the same group across seasons .","All calculations and statistical tests were conducted in R version 3 . 5 . 1 ( R Development Core Team , 2018 ) with the significance threshold set at p≤0 . 05 .","We investigated the relationship between group size and reproductive success by fitting a GEE containing group size and it’s quadratic term as predictors of the median number of chicks in the group in a given season .","We limited this analysis to seasons during which there were chicks in our study population .","In Seasons A and D , the study population had no chicks since they had not bred for more than 10 months .","During the rest of the three study seasons ( Seasons B , C and E ) , the chicks where between 3 and 5 months old , and each season represented a different cohort of chicks .","The code to run the ctmms and conduct the statistics is available on https://github . com/DanPapageorgiou/Group_size; Papageorgiou , 2020; copy archived at swh:1:dir:fcd6639f112b1d96bfe8d122f96a2c0ed2a1673b . All data used in this study are stored on Movebank under the study name Avulturinum_Farine: https://www . movebank . org/cms/webapp ?","gwt_fragment=page=studies , path=study475851705 ."]],"string":"[\n [\n \"Movement behaviour fundamentally influences how individuals encounter resources that are critical for their fitness .\",\n \"In highly social animals , movements are determined by how efficiently a group can coordinate their behaviour ( Chapman and Chapman , 2000; Couzin et al . , 2002; Strandburg-Peshkin et al . , 2017; Strandburg-Peshkin et al . , 2015 ) .\",\n \"Coordination requires effectively deciding where and when to move ( Conradt and Roper , 2005 ) as well as maintaining cohesion during movement ( Gall and Manser , 2017 ) .\",\n \"Both these factors could be shaped by intragroup characteristics .\",\n \"For example , it has become increasingly evident that group composition ( Farine et al . , 2015 ) , such as the presence of uniformed individuals ( Couzin et al . , 2011 ) or the mixture of individual personality types ( Aplin et al . , 2014; Jolles et al . , 2017 ) , can shape the behavioural properties of groups .\",\n \"In societies where individuals form stable groups , group size is another potentially important group characteristic that may also significantly impact fine-scale collective movement patterns .\",\n \"However , while the balance of benefits ( such as decreased predation risk according to the ‘many eyes hypothesis’; Pulliam , 1973 ) and costs ( increased intra-group competition over resources ) associated with group size have been extensively explored ( Krause and Ruxton , 2002 ) , we still have very little information on whether group size translates to differences in movement behaviour and ranging patterns of naturally occurring groups .\",\n \"Theory predicts that larger group size can benefit animal groups that move collectively through complex and heterogeneous landscapes .\",\n \"If group members pool their information , then larger groups should be able to reach more accurate estimates of their environment ( Cantor et al . , 2020; Couzin et al . , 2011 ) , as highlighted by the ‘wisdom of the crowd’ theory ( Galton , 1907 ) .\",\n \"This process can translate to navigational accuracy , via the ‘many wrongs hypothesis’ , as pooling the imperfect individual estimations of directions can generate accurate group-level estimates that allow groups to move more directly towards resources ( Simons , 2004 ) .\",\n \"Larger groups are also more likely to contain individuals with knowledge relevant to the current position or situation of a group ( the ‘pool of competence hypothesis’; Giraldeau , 1984 ) .\",\n \"For example , studies on killer whales ( Brent et al . , 2015 ) and elephants ( McComb et al . , 2001 ) have shown that older individuals become leaders under conditions of extreme resource shortages .\",\n \"Such mechanisms should therefore allow larger groups to more effectively navigate their environment , and therefore exhibit a larger home-range size .\",\n \"Effective navigation , and subsequently larger home ranges , should be beneficial for group members .\",\n \"Larger areas encompass a greater diversity and abundance of resources needed to meet group members’ nutritional needs ( Corriale et al . , 2013; Hubbs and Boonstra , 1998; Maher and Burger , 2011 ) , and moving to more new areas increases the probability of discovering new food resources .\",\n \"Using larger areas can also allow groups to be more variable in their movements , which could make them more unpredictable to predators ( Roth and Vetter , 2008 ) and thus decreases predation risk ( Richardson et al . , 2018 ) .\",\n \"Thus , all else being equal , such as environmental characteristics , the members of a group with a relatively larger home range should have higher survival in periods of resource limitations and/or greater reproductive output than an identical group with a smaller home range .\",\n \"In reality , home-range size is often limited by many factors , including competition for space with other groups ( Christensen and Radford , 2018 ) .\",\n \"However , many group-living species live in non-territorial societies , where groups can benefit from exploiting a home range without paying the costs of intergroup fights and competition .\",\n \"In such species , groups can interact affiliatively and preferentially with specific other groups , resulting in a multilevel society .\",\n \"In such societies , having a larger home-range size might generate more chances to associate with conspecifics from other groups ( Grueter et al . , 2020 ) , which , in turn , can increase mating opportunities ( by increasing contact with members of the opposite sex; Grueter et al . , 2015 ) , dispersal opportunities ( as sub-adults often disperse to neighbouring groups; Städele et al . , 2015 ) and information sharing about food resources or predation risk ( Whitehead et al . , 2012 ) .\",\n \"Thus , there are many ways that groups can benefit from maximizing their space use , up to the point where the costs of travel and navigation do not override the benefits gained .\",\n \"However , living in large groups can also introduce challenges , particularly in species that maintain stable group membership .\",\n \"Collective movement requires maintaining cohesion , a challenge that increases with group size and in more complex environments ( Codling et al . , 2007 ) .\",\n \"From a movement perspective , coordination among many individuals could result in slower decision-making , due to both coordination challenges as well as larger groups being likely to harbour more conflicts of interest .\",\n \"For example , one study of collective movement in baboons found that the probability that an individual baboon followed one initiator versus the other was much lower when both disagreement among initiators on where to go and the number of initiators increased ( Strandburg-Peshkin et al . , 2015 ) .\",\n \"Larger groups are also likely to have slower movement speeds while ‘on-the-go’ .\",\n \"We all have personal experiences of how much slower it can be to move or make decisions ( such as where to have lunch ) when we are in a large group , as the group generally may have to conform to the slowest members ( Herbert-Read et al . , 2013 ) or satisfy a diversity of preferences ( Strandburg-peshkin et al . , 2017 ) .\",\n \"Finally , it may be more challenging for individuals to influence larger groups to move to new areas , and thus to extend the group’s home-range .\",\n \"These factors suggest that the consensus costs borne by larger groups will ultimately limit the size of the home-range that such groups can exploit .\",\n \"Given that both positive and negative relationships between group size and home-range size may exist , we a priori predict a non-linear relationship between the size of stable groups and collective movement characteristics .\",\n \"This non-linear relationship is likely to represent an optimal group size for movement that reflects the point where the difference between navigational accuracy and coordination problems is maximized .\",\n \"To our knowledge , this relationship remains largely unexplored in wild , free-living groups .\",\n \"A first challenge in quantifying the relationship between home-range size and group size requires long-term tracking across multiple replicated groups and , ideally , replication in time .\",\n \"A second challenge is that many species that live in stable groups ( at least most of the well-studied species ) either experience increased intra-group competition as group sizes increase ( e . g . baboons; Markham et al . , 2015 ) or they are territorial ( e . g . meerkats; Bateman et al . , 2015 ) .\",\n \"Territoriality introduces several confounding effects .\",\n \"For example , the greater resource-holding potential of larger groups ( Bateman et al . , 2015; Markham et al . , 2015 ) can translate to larger home-ranges .\",\n \"Further , different territorial groups can vary in the density and distribution of resources within their territory , which could make it appear that smaller groups have larger home-ranges if they live in marginal areas with low resource densities .\",\n \"As a result , the relationship between group movement characteristics and group size has remained relatively unclear .\",\n \"In this study , we examine the effect of group size on the movement and space use patterns of wild groups of vulturine guineafowl ( Acryllium vulturinum ) , and whether resulting patterns correspond to differences in reproductive output among groups .\",\n \"Our study species lives in a multilevel society consisting of large , stable groups with multiple breeding units and groups form stable associations with specific other groups ( Papageorgiou et al . , 2019 ) .\",\n \"The stable social structure and the lack of territoriality—the entire population inhabits a small area of approximately 500 ha and all groups can easily access all parts of our study area—makes vulturine guineafowl an ideal species to investigate how group size influences movement characteristics because groups can range without major constraints on ranging imposed by other groups .\",\n \"Further , their large body size ( ~1 . 5 kg ) allow guineafowl to be fitted with high-resolution solar-powered GPS tags to collect both fine-scale data on their movement as well as long-term data on their ranging behaviour .\",\n \"We use data from GPS tags fitted to one individual from a total of 21 distinct groups , collected over five 2-month-long non-breeding seasons ( Figure 1 ) .\",\n \"We fit a continuous-time movement model ( ctmm ) to each individual GPS dataset ( one bird per group per season ) using GPS locations collected every 5 min .\",\n \"From each model , we calculated ( 1 ) home-range size , ( 2 ) the tendency for groups to use the same areas on consecutive days , and ( 3 ) daily travel distance .\",\n \"We then used high-resolution ( 1 Hz ) GPS location data from each individual to quantify ( 4 ) the movement speed of its group while ‘on the go’ .\",\n \"Because groups move through heterogeneous environments , larger groups should be able to navigate more effectively .\",\n \"Their large size should also result in larger daily travel distances as they search for the resources needed to survive .\",\n \"By contrast , moving as a large group also introduces consensus costs , and these could limit the home-range size of the largest groups by affecting their speed or their tendency to move to new areas .\",\n \"As a result of the interaction between these two effects , we expect that there will be a non-linear relationship between group size and movement , which , if it results in more effective use of available resources , could correspond to an optimal group size for group movement characteristics .\",\n \"We predict that intermediate groups will have the largest home-range sizes .\",\n \"This will arise because while larger groups should travel further , intermediate groups will have lower day-to-day home-range fidelity .\",\n \"We further predict that intermediate size groups will present the fastest speed while ‘on the go’ .\",\n \"However , these factors may be tempered by group composition .\",\n \"Groups in our population reproduced several times over our study period , and the presence of young group members could restrict the movements of groups .\",\n \"Thus , we run our analysis while controlling for the number of chicks present in the groups , and predict that groups with more chicks should travel slower and shorter distances , thereby restricting their home-range size .\",\n \"Finally , we predict that a non-linear relationship between group size and movement should translate to corresponding peak in fitness , measured as the number of chicks in a group .\",\n \"Such a pattern would suggest that maximising the difference between navigation and coordination represents an optimal group size at which groups are most efficient in exploiting their environment .\"\n ],\n [\n \"Our movement analysis , based on 1 , 274 , 434 five-min GPS positions , spanned 45 seasonal observations of individuals ( groups ) over five 2-month-long seasons ( Figure 1 , see Supplementary file 1A for a summary of the data ) .\",\n \"Group sizes , recorded during daily census observations , were widely distributed , with intermediate groups being observed less often ( Figure 2 ) .\",\n \"Fitting generalised estimating equations ( GEEs ) with the number of adults ( group size ) and the number of chicks as predictors ( while accounting for repeated measures of the same groups across seasons ) revealed that home-range size was significantly predicted by group size and its quadratic term , but that this relationship was modulated by the presence of chicks .\",\n \"Intermediate groups had the largest home-range , but home-range size decreased with increasing number of chicks ( Table 1A , see Supplementary file 1B for all model fits ) .\",\n \"Average temporal overlap in space use was also significantly predicted by group size and its quadratic term , but was not significantly modulated by the presence of chicks ( Table 1B , see Supplementary file 1C for all model fits ) .\",\n \"Average daily distance travelled increased linearly with group size ( Table 1C , see Supplementary file 1D for all model fits ) .\",\n \"Speed ‘on-the-go’ was not explained by group size or by number of chicks in the group ( Table 1D , see Supplementary file 1E for all model fits ) .\",\n \"Overall , smaller and larger groups expressed smaller home-range sizes and increased average temporal overlap in space use , while larger groups expressed longer daily travel distances than groups of smaller size ( see Figure 3 and Figure 4 ) .\",\n \"The number of chicks in the group was also significantly predicted by group size and its quadratic term ( Table 1E , Figure 3D , see also Supplementary file 1F for all model fits ) .\",\n \"We found that intermediate-sized groups had more chicks , with the peak in fitness ( group size = 33 ) closely matching the peak in home-range size ( group size = 36 ) and average daily overlap ( group size = 37 ) .\",\n \"Number of adults ( group size ) and its quadratic term significantly predicted ( A ) home-range size and ( B ) average temporal overlap in space use .\",\n \"The presence of chicks modulated the relationship between group size and home-range size , reducing the overall contribution of the number of adults to home-range size ( the negative interaction with number of adults ) and reducing the quadratic effect ( the positive interaction with number of adults squared , which offsets the negative quadratic coefficient ) .\",\n \"( C ) The average daily distance travelled increased with number of adults ( see Figure 3C ) .\",\n \"( D ) Speed while travelling was not significantly impacted by group size or its quadratic term .\",\n \"( E ) The number of chicks per group was significantly predicted by group size and its quadratic term ( see Figure 3D ) .\",\n \"Parameter estimates , significance , and R2 were generated using the most parsimonious fitted GEEs based on QIC ( see Materials and methods ) .\",\n \"All models tested are summarized in the Supplementary file 1B to 1F .\"\n ],\n [\n \"Our study shows that group size and composition can impact collective movement characteristics .\",\n \"Groups of vulturine guineafowl that have an intermediate size exhibit larger home-ranges and explore more new areas day-by-day .\",\n \"However , this relationship exists despite intermediate groups travelling shorter distances each day compared to larger groups .\",\n \"Even though the presence of chicks does not impact how far or how fast groups travel , having many chicks restricts the home range of their group .\",\n \"Further , our study also shows that groups of intermediate size have higher fitness as they produce more offspring than larger or smaller groups .\",\n \"Our results are therefore consistent with our a priori prediction that a non-linear relationship should exist between group size and collective movement , reflecting the increasing navigation ability with increasing group size and the increasing challenges of coordinating movement in larger groups .\",\n \"The relationship between group size and reproductive output further suggests that intermediate-sized groups are more effective at exploiting space , providing evidence for an optimal group size for collective movement .\",\n \"The relationships between group size and group movement outcomes provide new insights into how differently sized animal groups interact with their environment .\",\n \"Larger groups cover larger distances during the day , which is likely to be important for encountering sufficient resources to meet their energetic demands , as proposed by the ecological constraints model ( Chapman and Chapman , 2000; Ganas and Robbins , 2005; Gillespie and Chapman , 2001; Teichroeb and Sicotte , 2009 ) .\",\n \"However , by exploiting larger areas over a whole season , groups of intermediate size are likely to benefit from having access to a greater abundance and/or diversity of food resources ( Maher and Burger , 2011 ) , they potentially also benefit both from being less predictable to predators ( Richardson et al . , 2018; Roth and Vetter , 2008 ) , and from having access to a more diverse social landscape .\",\n \"In addition , intermediate-sized groups achieve larger home ranges while travelling shorter distances than larger groups , implying that they have lower energy expenditure .\",\n \"It is worth noting that we focused our analysis on 2-month periods each capturing similar ecological conditions .\",\n \"Future studies could investigate whether these non-linear relationships between group-size and movement characteristics change across different environmental conditions and especially in seasons where energy might be under tighter constraints , such as during droughts .\",\n \"Evidence for this could bring important insights into the drivers of group size variation .\",\n \"While we predicted that group size would translate to differences in movement while ‘on-the-go’ , our measure of moment-by-moment movement ( speed while travelling ) did not reveal any relationship between group size and movement speed .\",\n \"One reason for this could be that larger groups could have better knowledge of the environment , meaning that they can offset consensus costs ( i . e . periods of slow movement due to conflict within the group regarding movement directions ) with more effective movement once departed ( Couzin et al . , 2005 ) .\",\n \"For example , the speed and polarization , and subsequently the directionality of group motion , increases with the size of fish schools ( Tunstrøm et al . , 2013 ) .\",\n \"As group size increases in locusts , they transition from disordered to ordered motion ( Buhl et al . , 2006 ) .\",\n \"Thus , it is possible that while the realised movement speed does not differ with group size , the characteristics of how the movement is achieved could vary between smaller and larger groups .\",\n \"Alternatively , the challenge of reaching a consensus could mean that larger groups end up repeating the same routes more often , which they can achieve by moving faster as individuals all know where they are going .\",\n \"This explanation is consistent with our findings that larger groups were less variable in where they travelled each day .\",\n \"Such mechanisms linking collective movement with ranging behaviour warrant further investigation .\",\n \"Several studies in the past have investigated the relationship between group size and ranging behaviour .\",\n \"Most notably , Markham et al . , 2015 found that intermediate-sized groups of wild baboons ( Papio cynocephalus ) utilized smaller areas , covered shorter distances per day , and used space less evenly than smaller and larger groups—patterns which are largely opposite to our findings .\",\n \"Stevenson and Castellanos , 2000 found that woolly monkeys ( Lagothrix lagothricha ) in groups of intermediate size travelled less during the day than larger and smaller groups .\",\n \"Finally , research on meerkats ( Suricata suricatta ) Bateman et al . , 2015 found no relationship between group size and group movement characteristics .\",\n \"However , in most previously studied systems , group movement characteristics are likely to be impacted not only by the ability for groups to move , but also by intra-group contest competition and inter-group competition ( Markham et al . , 2015 ) .\",\n \"For example , smaller groups have the weakest resource-holding potential , meaning that they should have the smallest territories .\",\n \"At the same time , they might be excluded from the best , most resource-rich , habitats by larger groups ( who need more resources due to intra-group competition ) , meaning that they could also end up having to use equally sized or even larger areas ( Markham et al . , 2015; Stevenson and Castellanos , 2000 ) .\",\n \"In contrast to previous studies , our evidence for a non-linear relationship between group size and movement characteristics comes from a species that not only presents minimum intergroup competition but also groups use largely overlapping areas .\",\n \"These features reduce the confounds on the link between group size and group movement behaviours .\",\n \"Our study is therefore more likely to reveal effects arising from movement-based mechanisms without confounding effects that are exogenous to the group .\",\n \"Vulturine guineafowl live in a multilevel society , where groups preferentially associate with other groups ( Papageorgiou et al . , 2019 ) .\",\n \"In such societies , intergroup associations can bring many important benefits , such as information sharing on predators and resources , as well as mating and dispersal opportunities ( Grueter et al . , 2020 ) .\",\n \"A large home range size in a multilevel society could provide groups with access to more communal roosts ( we have observed up to five groups in a single roost , Papageorgiou et al . , 2019 ) and more glades , and thus more opportunities to associate with more groups .\",\n \"However , the benefits of associating with other groups might not increase linearly with home range size , as associating with a large number of conspecifics can also imply costs , such as disease transmission or increased competitions for mates ( Krause and Ruxton , 2002 ) .\",\n \"More work is needed on the interactions between ranging behaviour of groups and the broader social environment in multilevel societies .\",\n \"We built on existing insights from studies of collective behaviour to develop an a priori prediction that the relationship between group size and movement characteristic should be non-linear .\",\n \"Our prediction proposed that maximising the use of space ( i . e . a larger home range ) through effective movement ( faster movement and less repetitive ranging ) should result in more efficient exploitation of the resources available to a group .\",\n \"A key question is whether groups actually benefit from this maximisation ?\",\n \"By analysing data on the reproductive success across the groups , we found striking evidence for a peak in fitness , with intermediate-sized groups having more chicks .\",\n \"This peak , which closely matches the group size peaks in movement characteristics ( home range size and average daily overlap ) , suggests that intermediate group sizes might be linked to higher reproductive success .\",\n \"Our findings therefore provide evidence for an optimal group size in the context of collective movement .\",\n \"An interesting additional finding from our study is that optimally sized groups ( 33–37 individuals ) were relatively rare .\",\n \"Instead , the distribution of group size appears to be bimodal with most groups being either smaller or larger than the intermediate group size .\",\n \"This observation aligns with the hypothesis that while an optimal group size results in maximum individual fitness ( Pride , 2005 ) groups at the optimal size should be unstable and therefore rarely observed ( Sibly , 1983 ) .\",\n \"Groups in our study grow either through reproduction or through immigration .\",\n \"We found that intermediate-sized groups have high reproductive success , which naturally pushes them beyond the optimal .\",\n \"Further , because dispersing individuals should prefer optimal size groups , they should also turn intermediate-sized groups into groups that are larger than the optimal size .\",\n \"Future studies should examine whether groups near the optimal size for collective movement also highly attract more dispersers .\",\n \"The observed variation in results between our study and previous studies investigating the effect of group size on ranging behaviour in mammals could also potentially arise due to methodological differences .\",\n \"For example , we used high-resolution tracks and continuous-time movement modelling to get much more precise estimates of movement characteristics .\",\n \"By contrast , other studies have used much lower sampling resolutions , often relying on hand-held GPS devices ( Bateman et al . , 2015; Markham et al . , 2015 ) or used simplistic minimum convex polygons to model home-ranges ( Markham et al . , 2015 ) .\",\n \"Minimum convex polygons are unlikely to be very good estimates of home-ranges as they are sensitive to outliers ( e . g . from transcription errors or poor GPS fixes ) and capture only one element of space use ( the extreme boundaries , which could be influenced by a range of factors such as habitat geometry; He et al . , 2019 ) .\",\n \"However , despite these potential methodological differences , we believe that conflicting findings are more likely to suggest that the effect of group size on ranging behaviour and collective movement varies according to the properties of the different animal social systems .\",\n \"For example , we predict that the outcome of the many factors that contribute to the ranging characteristics of social groups will be modulated by the distribution of food resources each species exploits .\",\n \"Vulturine guineafowl have very little need to defend areas from other groups or compete for resources within groups , as their food source ( insects and small seeds ) are widespread and they roost on the top of dense stands of trees , a habitat feature which is common in the study area .\",\n \"When competition is decreased , we predict that group size could become a more important driver of collective movement outcomes .\",\n \"We hope that by collecting more data on this question across species there will soon be sufficient studies to fully test this prediction .\",\n \"In addition to the effects of the number of adults in the group , we also revealed the impact of having young present in the group .\",\n \"Vulturine guineafowl are precocial , meaning that they are very small when they join the group , and mature over long periods of time—at 1 year of age they are only approximately 65% of the full adult size .\",\n \"For a number of months after hatching , chicks are unlikely to contribute to making decisions , but are highly vulnerable to predators—causing them to remain more in cover .\",\n \"At the same time , chicks are likely to move more slowly , or cover less ground , due to their smaller body size .\",\n \"We found that having chicks in the group has a profound effect on home-range size .\",\n \"Our results indicate that groups with more chicks use smaller areas , thereby limiting groups of intermediate sizes from exhibiting larger home-ranges .\",\n \"The limitations that chicks have on group movements raise interesting questions about the costs of group living .\",\n \"The fact that groups of vulturine guineafowl remain cohesive across seasons and years suggests that any costs borne from collective movement will be shared by all group members , including by individuals which have failed to reproduce ( vulturine guineafowl are plural breeders; Papageorgiou et al . , 2019 ) or by subadults who have yet to disperse .\",\n \"Thus , our data reveals a potentially important source of conflict among group members .\",\n \"Such conflicts may be common in plural-breeding groups , which are commonly found in multilevel societies .\",\n \"We have shown that using synchronous and high-resolution GPS tracking of multiple social groups that reside in the same area can reveal new insights into the movement and social ecology of species .\",\n \"Specifically , we demonstrated that group size and composition can predict collective movement and space use characteristics .\",\n \"Our results are consistent with the prediction that increased navigation accuracy but decreased coordination ability could produce a non-linear relationship between group size and collective movement .\",\n \"However , the mechanisms that underlie the interaction between coordination maintenance and navigation accuracy in free-living groups of various sizes remain to be discovered .\",\n \"Further , we have identified dimensions of movement—specifically that intermediate-sized groups have larger home-ranges and use a larger diversity of areas—that translate into intermediate-sized groups having greater efficacy at exploiting their environment , and , therefore , higher fitness .\",\n \"These results suggest the presence of an optimal group size for collective movement .\",\n \"A further interesting observation is that the benefits of having an optimal group size are not present when groups have offspring , and that few groups are of an optimal size , highlighting a potentially important feedback between current and future reproductive success .\",\n \"The ability to develop and test our novel hypotheses relating to collective movement of animals in the wild was only possible by conducting a large-scale multi-group tracking study in a species that does exhibit little intra-group and inter-group competition .\",\n \"We hope that cheaper , more reliable , and smaller devices , will continue to reveal similar insights into the lives of wild animals across the diversity of social systems .\"\n ],\n [\n \"We studied a population of vulturine guineafowl that resides in an area of approximately 500 ha in the southern part of the Mpala Research Conservancy ( MRC ) in Laikipia , Kenya .\",\n \"Vulturine guineafowl are large , predominantly terrestrial , and highly gregarious birds .\",\n \"They primarily forage on the ground , feeding on small grass root buds , seeds , fresh grass and small invertebrates , in other words widely dispersed food sources that have been predicted to limit within- and between-group contest competition ( Dammhahn and Kappeler , 2009 ) .\",\n \"Based on 3 years of observations of colour-marked individuals , we found that they live in social groups , which range in size from 13 to 65 adults , have stable membership across years and remain completely cohesive ( with individuals rarely out of sight of other group members ) during non-breeding seasons ( typically January to March and July to October ) ( Papageorgiou et al . , 2019 ) .\",\n \"Within a local area , groups form a multilevel society , which includes groups roosting communally ( with roosts containing several hundred birds ) and groups regularly encountering other groups during the day ( at which times between-groups agonistic interactions are rarely observed ) ( Papageorgiou et al . , 2019 ) .\",\n \"Intergroup encounters and foraging are most common on glades ( Papageorgiou et al . , 2019 ) , which are small deforested areas that were previously used as overnight shelters for cattle and are therefore are rich in nutrients ( Young et al . , 1995 ) .\",\n \"All vulturine guineafowl groups have access to glades as they are numerous in our study area .\",\n \"On glades , vulturine guineafowl are prone to predation by raptors , such as martial eagles ( Naude et al . , 2019 ) , while moving through dense vegetation makes them prone to ambush by carnivorous mammals , such as jackals or leopards .\",\n \"We trapped nearly all the adult members from each of the groups in our study area using large walk-in traps .\",\n \"All trapped individuals were kept inside covered cages ( 1 m*50 cm*50 cm cage with 2 cm plastic mesh covered by a canvas ) to minimize stress and allow birds to remain standing while awaiting processing .\",\n \"Birds were removed one by one , measured , and ringed with an individually numbered National Museums of Kenya stainless steel ring ( fitted on the tarsus of the right leg ) and a unique combination of four plastic colour bands fitted on each leg , on the tibiotarsus , for field identification .\",\n \"For more details on trapping and marking processes , see Papageorgiou et al . , 2019 .\",\n \"In each group , we fitted high-resolution solar-powered GPS tags ( 15 g Bird Solar , e-obs Digital Telemetry , Grünwald , Germany ) to between one and five individuals .\",\n \"The devices were elevated using neoprene pads to prevent feathers from covering the tag’s solar panel , and were attached to the body using a backpack harness design ( Teflon ribbon ) , which included a cotton thread safety release mechanism .\",\n \"GPS tags stayed on the birds from 6 months to 2 years , before the cotton breaks .\",\n \"Groups were trapped once per year to mark new members and check the condition of the birds which carry GPS tags .\",\n \"Since the start of the study in 2016 , no bird has ever been found with problems due to the GPS tags .\",\n \"Each tag ( tag , pads , ribbons , crimps ) weighed approximately 20 . 5 g , far less than the recommended 3% of the birds’ body weight ( Kenward , 2001 ) .\",\n \"Each device was programmed to record one data point ( date , time , coordinates ) every second when the battery had a high charge ( approximately every 2nd to 3rd day , for up to 8 hr continuously ) .\",\n \"When the battery was at the next lower threshold , we set tags to record one point per second for the first 10 s of every 5th minute .\",\n \"If battery charge was very low ( this setting was rarely used during our study period ) , tags were set to record one point every 15 min .\",\n \"Data were remotely downloaded every 2 or 3 days using a BaseStation II ( e-obs Digital Telemetry , Grünwald , Germany ) .\",\n \"Field-testing suggested that that the relative spacing of tags was accurate to within 1 m , 95% of the time tracked .\",\n \"We uploaded all of our data to the Movebank repository ( Wikelski and Kays , 2018 ) , see https://www . movebank . org/cms/webapp ?\",\n \"gwt_fragment=page=studies , path=study475851705 for further details .\",\n \"We selected seasons with intermediate rainfall and greenness , as described in Papageorgiou et al . , 2019 .\",\n \"Vulturine guineafowl groups temporarily split during the breeding ( wet ) season as breeding pairs form , subadults disperse from their natal groups and females lay and incubate their eggs ( Papageorgiou et al . , 2019 ) .\",\n \"It is therefore challenging to quantify membership accurately during the breeding season .\",\n \"Breeding seasons can occur twice a year ( during April or May and during October or November ) .\",\n \"We also excluded periods of drought , as these cause groups to regularly travel to large bodies of water , such as the river that crosses our study area .\",\n \"This unusual situation ( guineafowl are adapted to rarely require access to standing water under normal conditions ) would make the estimation of home-range size confounded by how far from the river each group roosts .\",\n \"We thus made our selection of seasons based on the seasonal conditions where groups remain cohesive but do not make extraordinary movements , while remaining blind to the specific movement patterns of the GPS-tagged birds .\",\n \"We defined seasons as being 2 months , which corresponds to period changes in conditions at our study site .\",\n \"Given that our entire study population inhabits a small area , approximately 500 ha , and that all groups can easily access all parts of our study area , we assume that groups’ relative ranging behaviour was not restricted by the environment during these intermediate seasons .\",\n \"Data for this study were collected from May 2017 to February 2020 , and the specific time periods that fulfilled the criteria listed above were categorised as Season A ( 30 . 5 . 2017 to 1 . 8 . 2017 ) , Season B ( 15 . 12 . 2017 to 15 . 2 . 2018 ) , Season C ( 1 . 8 . 2018 to 29 . 9 . 2018 ) , Season D ( 10 . 6 . 2019 to 9 . 8 . 2019 ) , and Season E ( 14 . 1 . 2020 to 16 . 3 . 2020 ) .\",\n \"Census data was collected daily from a vehicle driving along the roads in our study area .\",\n \"Every time a group was encountered , the identities of the marked birds were recorded , and a total number of birds present was counted ( including unmarked birds ) .\",\n \"Each observation of a ‘group’ is defined as being a single or a multiple group , depending on whether the group moves away cohesively or whether individuals move away in different directions .\",\n \"Counts are split into the number of adults and the number of chicks , with chicks being easily distinguishable until ~ 9 months of age .\",\n \"Within each season , we selected groups containing one or more GPS tagged individuals for which we had precise group-size counts where observers could clearly see and count all the group members being present ( i . e . groups were counted more than once by more than one observer , the group size was found consistent across counts ) .\",\n \"We included data on 21 different observational datasets of groups , with some groups replicated in more than one of the seasons ( Supplementary file 1A , Figure 1 ) .\",\n \"For seasons in which a group had chicks , we calculated the median number of chicks per group , across all observations , in each study season .\",\n \"We used the GPS data from each group in each season to quantify movement behaviour and resulting home-range characteristics .\",\n \"In groups containing more than one GPS-tagged individual , we randomly selected one to represent that group’s movement , given that groups move very cohesively ( typically group members are within 29 m of each other 95% of the time tracked; Papageorgiou et al . , 2019 ) .\",\n \"We calculated ( 1 ) the core home-range size ( 50% auto-correlated kernel density estimate ) , ( 2 ) average temporal overlap in space use , ( 3 ) average daily distance travelled .\",\n \"We also explored ( 4 ) speed while moving .\",\n \"We used the built-in features from the Movebank data repository to remove the very rare outliers in our dataset that were falling outside Kenya .\",\n \"For ( 1 , 2 ) and ( 3 ) we used data points collected on the tenth second of every fifth minute , sub-setting this from high-resolution ( 1 Hz ) data and from data when the tag was set to collect 10 points every 5th minute .\",\n \"This approach allowed us to ensure that the data points from all GPS-tagged birds were precisely synchronised in time .\",\n \"For core home-range size ( 1 ) , we calculated home-range sizes by fitting a continuous-time movement model ( ctmm ) to each individual GPS dataset ( one bird per group per season ) .\",\n \"These models follow a continuous-time stochastic process from which the maximum likelihood Gaussian home-range area can be extracted after the best fitted model is selected based on AIC .\",\n \"Because ctmms model the actual movement , they better predict home-ranges than classical approaches , such as minimum convex polygons , that are typically prone to being highly influenced by just a few data points ( Fleming et al . , 2015 ) .\",\n \"We used the maximum likelihood home-ranges to determine the area in which each bird was located 50% of the time .\",\n \"This procedure was done using the ‘ctmm’ R package ( Calabrese et al . , 2016; Fleming and Calabrese , 2018 ) .\",\n \"For average temporal overlap in space use ( 2 ) , we measured the inverse of the exploration of new areas , for each bird in each season .\",\n \"We first fitted a ctmm to the 5-min data from each bird for the first 21 days it was tracked in each season ( longer time spans were not computationally feasible for this particular analysis ) .\",\n \"We then calculated the overlap of the daily auto-correlated kernel density estimation for home-range of each bird , using the ‘overlap’ function of the ‘ctmm’ R package ( Winner et al . , 2018 ) .\",\n \"Specifically , we calculated the overlap between daily ranges that were up to 3 days apart .\",\n \"Finally we calculated the mean of the spatial overlap of one group with itself across each consecutive sets of 3 days , for the entire 21-day period , for each group .\",\n \"For average daily distance travelled ( 3 ) , we used the 5-min data from above ( one bird per group per season ) and the function ‘speed’ from the ctmm R package , which provides the mean travel distance per day but not the speed while ‘on the go’ .\",\n \"To estimate the mean speed ‘on the go’ ( 4 ) for each individual in each season we used only a subset of the data representing the times when the GPS tags were collecting one point per second ( 1 Hz ) .\",\n \"From these data , we created a histogram of the log of the movement speeds ( distance travelled between consecutive points ) , revealing that speeds were multimodally distributed for each individual .\",\n \"We found that the minima between the second and the third mode corresponded to −3 . 5 ( or 0 . 03 ms−1 ) and classified speeds exceeding this value as ‘moving’ and speeds slower than this value as ‘stationary’ ( Bender et al . , 2011; Wilson et al . , 2015 ) .\",\n \"We then used the data classified as ‘moving’ to calculate the mean of the distance travelled in consecutive seconds of the 1 Hz data .\",\n \"We fitted GEEs using the R package ‘geepack’ to test whether group size and number of chicks predicted home-range size , average temporal overlap in space use , average daily distance travelled and speed ‘on the go’ .\",\n \"Our primary predictor variable was the number of adults in the group .\",\n \"Because chicks could modulate the relationship between the number of adults ( our measure of group size ) and movement response variables , we also allowed models to include a term for the number of chicks .\",\n \"Because we expected a non-linear relationship between group size and movement parameters , we also allowed models to include the number of adults squared ( the quadratic term ) .\",\n \"Further , because we did not know how chicks could modulate group movement , we allowed the number of chicks and the number of chicks squared to be added in interactions with the number of adult and the number of adults squared .\",\n \"We created all combinations of response variables and interactions ( only between terms containing adults and chicks ) , but ensuring that models contained at least the number of adults as a predictor , and always included the number of chicks when the number of chicks squared was included .\",\n \"We used QIC to select the most parsimonious models ( lowest QIC ) ( Hocking , 2014 ) and we also calculated the R2 of each GEE to estimate their goodness of fit ( Zheng , 2000 ) .\",\n \"For all models , we fitted group ID as a repeated measure to account for multiple measures of the same group across seasons .\",\n \"All calculations and statistical tests were conducted in R version 3 . 5 . 1 ( R Development Core Team , 2018 ) with the significance threshold set at p≤0 . 05 .\",\n \"We investigated the relationship between group size and reproductive success by fitting a GEE containing group size and it’s quadratic term as predictors of the median number of chicks in the group in a given season .\",\n \"We limited this analysis to seasons during which there were chicks in our study population .\",\n \"In Seasons A and D , the study population had no chicks since they had not bred for more than 10 months .\",\n \"During the rest of the three study seasons ( Seasons B , C and E ) , the chicks where between 3 and 5 months old , and each season represented a different cohort of chicks .\",\n \"The code to run the ctmms and conduct the statistics is available on https://github . com/DanPapageorgiou/Group_size; Papageorgiou , 2020; copy archived at swh:1:dir:fcd6639f112b1d96bfe8d122f96a2c0ed2a1673b . All data used in this study are stored on Movebank under the study name Avulturinum_Farine: https://www . movebank . org/cms/webapp ?\",\n \"gwt_fragment=page=studies , path=study475851705 .\"\n ]\n]"},"abstract":{"kind":"list like","value":["A challenge of group-living is to maintain cohesion while navigating through heterogeneous landscapes .","Larger groups benefit from information pooling , translating to greater ‘collective intelligence’ , but face increased coordination challenges .","If these facets interact , we should observe a non-linear relationship between group size and collective movement .","We deployed high-resolution GPS tags to vulturine guineafowl from 21 distinct social groups and used continuous-time movement models to characterize group movements across five seasons .","Our data revealed a quadratic relationship between group size and movement characteristics , with intermediate-sized groups exhibiting the largest home-range size and greater variation in space use .","Intermediate-sized groups also had higher reproductive success , but having more young in the group reduced home-range size .","Our study suggests the presence of an optimal group size , and composition , for collective movement ."],"string":"[\n \"A challenge of group-living is to maintain cohesion while navigating through heterogeneous landscapes .\",\n \"Larger groups benefit from information pooling , translating to greater ‘collective intelligence’ , but face increased coordination challenges .\",\n \"If these facets interact , we should observe a non-linear relationship between group size and collective movement .\",\n \"We deployed high-resolution GPS tags to vulturine guineafowl from 21 distinct social groups and used continuous-time movement models to characterize group movements across five seasons .\",\n \"Our data revealed a quadratic relationship between group size and movement characteristics , with intermediate-sized groups exhibiting the largest home-range size and greater variation in space use .\",\n \"Intermediate-sized groups also had higher reproductive success , but having more young in the group reduced home-range size .\",\n \"Our study suggests the presence of an optimal group size , and composition , for collective movement .\"\n]"},"summary":{"kind":"list like","value":["Many social animals live in stable groups that stay together for years , or even lifetimes .","Being in a group offers a range of benefits , such as safety from predators , information on where to find food or water , and more accurate navigation .","But these benefits come at a cost .","The larger the group , the harder it is to make decisions that balance the needs of each individual .","So , while members of a large group should be better at locating resources and finding their way , they may take longer to decide where to go next .","In nature , groups of the same species can vary greatly in size and can have large or small numbers of offspring .","This raises the question of whether there is an optimal group size where the benefits of living together are maximized relative to the costs ?","To help answer this question , Papageorgiou and Farine studied the group behaviour of vulturine guineafowl , a social , ground-dwelling bird found in the savannahs of East Africa .","A lightweight GPS tracker was fitted to the members of 21 different groups of vulturine guineafowl to see how group size affects the movement of these birds .","The tags collected data every five minutes from dawn until dusk each day , and remained active over five two-month spans of similar weather conditions .","This revealed that groups of intermediate size , which contain 33 to 37 birds , ranged over larger areas allowing them to access more diverse resources , and used less energy by travelling shorter distances .","Birds in these groups also explored more new areas , decreasing their chances of encountering a predator , and produced more chicks , suggesting that their collective behaviour gave them a reproductive advantage .","These findings suggest that intermediate sized groups display an optimal level of movement compared to larger or smaller groups .","Understanding how social groups of different sizes interact with their environment can aid conservation planning .","Future work should focus on how this relationship changes with the seasons .","This could reveal more about the effects of group size during challenging conditions , like drought ."],"string":"[\n \"Many social animals live in stable groups that stay together for years , or even lifetimes .\",\n \"Being in a group offers a range of benefits , such as safety from predators , information on where to find food or water , and more accurate navigation .\",\n \"But these benefits come at a cost .\",\n \"The larger the group , the harder it is to make decisions that balance the needs of each individual .\",\n \"So , while members of a large group should be better at locating resources and finding their way , they may take longer to decide where to go next .\",\n \"In nature , groups of the same species can vary greatly in size and can have large or small numbers of offspring .\",\n \"This raises the question of whether there is an optimal group size where the benefits of living together are maximized relative to the costs ?\",\n \"To help answer this question , Papageorgiou and Farine studied the group behaviour of vulturine guineafowl , a social , ground-dwelling bird found in the savannahs of East Africa .\",\n \"A lightweight GPS tracker was fitted to the members of 21 different groups of vulturine guineafowl to see how group size affects the movement of these birds .\",\n \"The tags collected data every five minutes from dawn until dusk each day , and remained active over five two-month spans of similar weather conditions .\",\n \"This revealed that groups of intermediate size , which contain 33 to 37 birds , ranged over larger areas allowing them to access more diverse resources , and used less energy by travelling shorter distances .\",\n \"Birds in these groups also explored more new areas , decreasing their chances of encountering a predator , and produced more chicks , suggesting that their collective behaviour gave them a reproductive advantage .\",\n \"These findings suggest that intermediate sized groups display an optimal level of movement compared to larger or smaller groups .\",\n \"Understanding how social groups of different sizes interact with their environment can aid conservation planning .\",\n \"Future work should focus on how this relationship changes with the seasons .\",\n \"This could reveal more about the effects of group size during challenging conditions , like drought .\"\n]"},"year":{"kind":"string","value":"2020"}}},{"rowIdx":4183,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience","genetics and genomics"],"string":"[\n \"neuroscience\",\n \"genetics and genomics\"\n]"},"title":{"kind":"string","value":"Taar1 gene variants have a causal role in methamphetamine intake and response and interact with Oprm1"},"id":{"kind":"string","value":"elife-46472-v3"},"sections":{"kind":"list like","value":[["Amphetamine-like stimulants , including methamphetamine ( MA ) , remain the second most common class of illicit drugs used worldwide , behind cannabis-containing drugs ( United Nations Office on Drugs and Crime , 2016 ) .","MA is the most widely abused psychostimulant ( Chomchai and Chomchai , 2015 ) , and the consequences of MA addiction pose major societal and health concerns ( National Institute on Drug Abuse , 2019 ) .","MA-associated deaths in the United States were relatively stable between 2005 and 2013 , but have since been on an increasing trajectory , rising from about 3600 individuals in 2013 to almost 11 , 000 in 2017 ( National Institute on Drug Abuse , 2018 ) .","Discovering and understanding genetic factors that contribute to MA addiction risk , and uncovering the linked molecular and cellular processes , may help to curb this trend and improve interventions and long-term treatment success .","Whereas use of large human cohorts is effective at mapping loci and nominating candidate genes , precise experimental control over genetic composition , development , and drug exposure history is practical only in animal models .","Evidence that mouse models continue to contribute critical information to our understanding of pathological conditions was recently reviewed ( Nadeau and Auwerx , 2019 ) .","Selected lines of rodents have been derived to model genetic risk factors for drug use .","These include , particularly , those bred for various drug intake phenotypes , such as our mice bred for different amounts of voluntary MA intake ( Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .","In previous studies , we demonstrated a strong genetic contribution to level of voluntary MA consumption using multiple replicate sets of mice that we bidirectionally , selectively bred for MA high drinking ( MAHDR ) and MA low drinking ( MALDR ) , which for simplicity are also referred to here as the ‘high’ and ‘low’ lines .","Multiple lines of evidence support the involvement of the trace amine-associated receptor 1 gene , Taar1 , in differences in MA intake , including: quantitative trait locus ( QTL ) mapping in the high and low lines ( Belknap et al . , 2013 ) ; the direct agonist activity of MA at the trace amine-associated receptor 1 ( TAAR1 ) ( Bunzow et al . , 2001; Wolinsky et al . , 2007 ) ; the presence of a single nucleotide polymorphism ( SNP ) in Taar1 ( Taar1m1J ) that renders the receptor it expresses ( TAAR1 ) non-functional in response to MA and to other direct agonists ( Harkness et al . , 2015; Shi et al . , 2016 ) ; fixation of the Taar1m1J allele in high line mice of all five replicates , and retention of the wildtype , Taar1+ allele in all low line replicates ( Harkness et al . , 2015; Reed et al . , 2018 ) ; greater level of MA intake in Taar1 knockout mice compared to wildtype mice ( Harkness et al . , 2015 ) ; strong association of Taar1 genotype with MA intake in multiple genetic models ( Reed et al . , 2018 ) ; and strong association of Taar1 genotype with additional MA-related traits that correspond with level of MA intake , including level of sensitivity to MA-induced hypothermia and conditioned taste aversion ( Harkness et al . , 2015; Reed et al . , 2018 ) .","In the current studies , to demonstrate a causal role for Taar1 , we used CRISPR-Cas9 technology to replace the Taar1m1J allele found in high line mice with the Taar1+ allele , creating a knock-in with functional TAAR1 .","These mice were then tested for MA consumption in comparison to an unaltered high line control , with the prediction that knock-in of Taar1+ would reduce MA intake .","In addition , we tested knock-in and control mice for MA-induced change in body temperature to determine if this gene has a pleiotropic effect on this MA trait .","We predicted that insertion of the Taar1+ allele would increase sensitivity to MA-induced hypothermia in the knock-in mice .","Our existing data indicate that Taar1 genotype has a major impact on MA consumption , and gene mapping results demonstrate that a chromosome 10 QTL , at the location of Taar1 , accounts for as much as 60% of the genetic variance in risk for MA intake ( Belknap et al . , 2013 ) .","Other undefined variants must account for the remaining genetic variance .","We previously examined the µ-opioid receptor 1 gene , Oprm1 , as a quantitative trait gene ( QTG ) residing at the proximal end of mouse chromosome 10 , but it was eliminated as a risk factor for MA intake ( Eastwood et al . , 2018 ) .","Although Oprm1 variants do not predict risk , Oprm1 is involved in MA consumption via regulation by a top-ranked transcription factor network of genes that is differentially expressed between the MA drinking lines .","We found that Oprm1 and its product , the µ-opioid receptor ( MOP-r ) , are more highly expressed in the prefrontal cortex of low line mice , relative to high line mice ( Belknap et al . , 2013; Eastwood et al . , 2018 ) .","In addition , when MOP-r agonists were given to high line mice to test the hypothesis that increased MOP-r-regulated activity reduces MA intake , the acquisition of MA intake was attenuated , simulating the voluntary intake phenotype of the low line ( Eastwood et al . , 2018; Eastwood and Phillips , 2014a ) .","High line mice possess the same mutant Taar1m1J/m1J genotype as their progenitor DBA/2J ( D2 ) strain and are more likely to also possess the Oprm1 variant from the D2 strain , due to the relatively close proximity of Taar1 and Oprm1 on chromosome 10 ( 17 Mb apart ) .","Due to this linkage , we have been unable to explore independent and interactive effects of the Taar1 and Oprm1 genotypes on MA intake or other MA-related traits in the MA drinking lines .","Instead , we explored this in the present studies , using a family of related recombinant inbred mouse strains derived from a C57BL/6J ( B6 ) x D2 inbred strain intercross ( collectively the BXD strains ) in which the linkage is disrupted .","We examined the independent and interactive effects of these genes on MA intake and sensitivity to MA-induced change in body temperature , and performed QTL mapping for both traits using the BXD strain data ."],["We predicted that MA intake would be reduced in high line mice in which the Taar1m1J allele was removed and replaced with the Taar1+ allele .","In the initial repeated measures analyses of variance ( ANOVAs ) , there were no interactions involving sex for either mg/kg MA consumption or total volume consumed , so we performed additional analyses with data for the sexes combined .","For MA consumption ( Figure 1a; Figure 1—source data 1 ) , there was a significant MA concentration x genotype interaction ( F[1 , 36]=66 . 8 , p<0 . 0001 ) .","Replacement of the Taar1m1J allele with the Taar1+ allele converted high MA intake to low MA intake for both MA concentrations .","Control mice , which are homozygous Taar1m1J , consumed significantly more MA at the 40 mg/l concentration , compared to the 20 mg/l concentration , whereas low and comparable levels of MA consumption were found for both MA concentrations in knock-in mice .","For total volume consumed from the water and MA tubes , during the time that MA was available ( Figure 1b; Figure 1—source data 1 ) , there was only a significant main effect of MA concentration ( F[1 , 36]=21 . 4 , p<0 . 0001 ) .","Total volume consumed was greater when mice were offered 40 mg/l MA , compared to 20 mg/l MA .","Thermal response to MA is a genetically-correlated response to selection for level of MA intake; thus , low MA drinking line mice display MA-induced hypothermia , whereas high line mice do not ( Harkness et al . , 2015 ) .","We tested the hypothesis that Taar1 genotype has a causal role .","Knock-in of Taar1+ in high line mice restored a hypothermic response to MA that did not occur in control mice ( Figure 2; Figure 2—source data 1 ) .","In the initial repeated measures ANOVA there were significant independent interactions of sex with genotype ( F[1 , 90]=7 . 1 , p=0 . 009 ) , MA dose ( F[1 , 90]=4 . 9 , p=0 . 03 ) and time ( F[4 , 360]=2 . 9 , p=0 . 02 ) .","However , sex did not determine the pattern of response to saline vs . MA in each genotype across time .","There was a significant 3-way genotype x MA dose x time interaction ( F[4 , 360]=10 . 2 , p<0 . 0001 ) that did not interact with sex .","We next examined the effects of genotype and time within each dose group .","In the saline-treated mice ( Figure 2a ) , there were significant main effects of time ( F[4 , 188]=35 . 9 , p<0 . 0001 ) and genotype ( F[1 , 47]=4 . 7 , p=0 . 04 ) .","Temperature declined by 0 . 3°C over time , regardless of genotype , and knock-in mice had 0 . 25°C higher body temperature than control mice , regardless of time .","In the MA-treated mice ( Figure 2b ) , the genotype x time interaction was significant ( F[4 , 188]=13 . 0 , p<0 . 0001 ) .","Knock-in mice had a significant hypothermic response to MA at T30 compared to T0 , whereas control mice were resistant to the hypothermic effects of MA , instead exhibiting significant MA-induced increases in body temperature at all post-MA treatment time points , compared to T0 .","Body temperatures were significantly higher in knock-in than control mice at baseline , by about 0 . 6°C , but significantly lower by about 1 . 2°C , at T30 .","Initial analyses characterized 18 BXD strains for MA consumption ( Figure 3; Figure 3—source data 1 ) .","There was a significant MA concentration x strain interaction ( F[17 , 215]=17 . 2 , p<0 . 0001 ) .","MA intake differed among the strains for both the 20 ( Figure 3a; p<0 . 0001 ) and 40 ( Figure 3b; p<0 . 0001 ) mg/l MA concentrations , and all Taar1m1J/m1J strains , but not Taar1+/+ strains , exhibited higher levels of MA intake from the 40 mg/l MA concentration than from the 20 mg/l concentration ( p=0 . 0006 to<0 . 0001 ) .","For total volume consumed when mice were offered water vs . MA , there was a main effect of strain ( F[17 , 215]=27 . 7 , p<0 . 0001 ) and a main effect of MA concentration ( F[1 , 215]=58 . 6 , p<0 . 0001 ) .","Total volume consumed differed among the strains by as much as 4 ml ( Figure 3c ) , but the strain distribution pattern for total volume did not correspond with the distributions for MA intake at either MA concentration .","Total volume consumed was significantly less when water and 20 mg/l MA were available , compared to when water and 40 mg/l MA were available ( mean ± SEM = 5 . 3±0 . 1 and 5 . 7 ± 0 . 1 for 20 and 40 mg/l , respectively ) .","We next examined the data from the BXD mice with regard to Taar1 and Oprm1 genotype to detect potential independent and epistatic effects on MA consumption .","First , to identify potential independent effects , we calculated Pearson’s correlations of each genotype with individual 20 and 40 mg/l MA intake amounts .","MA intake was significantly associated with Taar1 genotype ( r = 0 . 74 and 0 . 81 , ps <0 . 0001 , for 20 and 40 mg/l MA , respectively ) , but not Oprm1 genotype ( r = 0 . 06 and 0 . 10 , p=0 . 36 and 0 . 13 , for 20 and 40 mg/l MA , respectively ) .","Next , we considered main and interaction effects by repeated measures ANOVA with MA concentration , Oprm1 genotype , Taar1 genotype , and sex as factors .","There were no significant interactions involving sex , so further analyses were performed with data for the sexes combined .","Data are summarized in Figure 4 ( Figure 4—source data 1 ) , with mice grouped according to their four possible Taar1/Oprm1 genotypes ( Table 1 ) .","For MA consumption ( Figure 4a ) , there was a significant Oprm1 genotype x Taar1 genotype x MA concentration interaction ( F[1 , 225]=17 . 6 , p<0 . 0001 ) .","For consumption of MA from the 20 mg/l MA concentration , main effects of the Oprm1 ( F[1 , 229]=8 . 6 , p=0 . 004 ) and Taar1 ( F[1 , 229]=301 . 3 , p<0 . 0001 ) genotypes indicated that both genes influenced MA intake , with greater MA intake in Taar1m1J/m1J and Oprm1D2/D2 mice , compared to Taar1+/+ and Oprm1B6/B6 mice , respectively .","However , there was no significant Oprm1 genotype x Taar1 genotype interaction .","In contrast , for MA consumption from the 40 mg/l MA concentration , there was a significant Oprm1 genotype x Taar1 genotype interaction ( F[1 , 229]=13 . 9 , p=0 . 0002 ) .","MA intake was significantly higher in Taar1m1J/m1J mice , compared to Taar1+/+ mice , regardless of Oprm1 genotype ( ps <0 . 0001 ) .","However , Taar1m1J/m1J/Oprm1D2/D2 mice consumed significantly more MA than Taar1m1J/m1J/Oprm1B6/B6 mice .","Oprm1 genotype did not significantly impact MA intake in Taar1+/+ mice .","For total volume consumed ( Figure 4b ) , there were significant main effects of Oprm1 genotype ( F[1 , 225]=24 . 8 , p<0 . 0001 ) , Taar1 genotype ( F[1 , 225]=4 . 7 , p=0 . 03 ) , and MA concentration ( F[1 , 225]=75 . 7 , p<0 . 0001 ) , but no significant interactions .","Oprm1D2/D2 mice consumed more total volume than Oprm1B6/B6 mice , and Taar1m1J/m1J mice consumed more than Taar1+/+ mice .","In addition , total volume consumed was greater when mice were offered 40 mg/l MA , compared to 20 mg/l MA .","We used a similar approach to analyze body temperature data that were collected after saline or MA treatment in 15 BXD strains ( Figure 5; Figure 5—source data 1 ) .","For ease of viewing , the data in Figure 5 are separated by saline/MA treatment and Taar1 genotype for strain responses across time .","A repeated measures ANOVA detected a significant strain x MA dose x time interaction ( F[56 , 1172]=10 . 0 , p<0 . 0001 ) .","In saline-treated mice ( Figure 5a , b ) , the BXD strains differed at each time point ( ps = 0 . 04 to<0 . 0001 ) , except T0 , and body temperature differed across time in strains 161 ( Taar1+/+/Oprm1D2/D2 ) , 171 , 194 ( both Taar1m1J/m1J/Oprm1B6/B6 ) , 186 , 210 ( both Taar1m1J/m1J/Oprm1D2/D2 ) , and 218 ( Taar1+/+/Oprm1B6/B6 ) ( ps = 0 . 004 to<0 . 0001 ) .","In MA-treated mice ( Figure 5c , d ) , the BXD strains differed at each time point ( ps <0 . 0001 ) , except T0 , and body temperature differed across time in all strains ( ps = 0 . 007 to<0 . 0001 ) , except 160 , 171 , 194 ( all Taar1m1J/m1J/Oprm1B6/B6 ) , 178 , 186 , and 210 ( all Taar1m1J/m1J/Oprm1D2/D2 ) .","The maximum mean drop in body temperature in saline-treated mice was 0 . 9°C ( Figure 5a , b ) , whereas there was a maximum mean drop of 4°C in MA-treated Taar1+/+ mice ( Figure 5c ) , and a maximum increase of 1°C in MA-treated Taar1m1J/m1J mice ( Figure 5d ) .","We next examined the BXD data with regard to Taar1 and Oprm1 genotype to detect potential independent and epistatic effects on the hypothermic effect of MA .","Again , to identify potential independent effects , we calculated Pearson’s correlations of each genotype with MA-induced change in body temperature from T0 to T30 .","There was a significant correlation of body temperature change with Taar1 genotype ( r = 0 . 71 , p<0 . 0001 ) , but not with Oprm1 genotype ( r = 0 . 11 , p=0 . 16 ) .","Next , we considered main and interaction effects by repeated measures ANOVA with time , Oprm1 genotype , Taar1 genotype , sex and MA dose as factors .","There were significant independent interactions of sex with MA dose ( F[1 , 307]=8 . 0 , p=0 . 006 ) and time ( F[4 , 1228]=5 . 0 , p=0 . 0007 ) .","However , sex did not play a significant role in the pattern of response of each genotype to each dose across time .","There was a significant Oprm1 genotype x Taar1 genotype x MA dose x time interaction ( F[4 , 1228]=8 . 0 , p<0 . 0001 ) that did not interact with sex .","We next examined the effects of genotype and time within each dose group ( Figure 6; Figure 6—source data 1 ) .","In the saline-treated mice ( Figure 6a ) , the Oprm1 genotype x time interaction was significant ( F[4 , 596]=2 . 6 , p=0 . 04 ) .","Both Oprm1B6/B6 and Oprm1D2/D2 mice exhibited reductions in body temperatures at T60-120 ( ps <0 . 001 ) , compared to T0 .","However , these differences in body temperature were of no more than 0 . 3°C on average .","In the MA-treated mice ( Figure 6b ) , there was a significant 3-way interaction of Oprm1 genotype x Taar1 genotype x time ( F[4 , 664]=11 . 4 , p<0 . 0001 ) .","Taar1+/+/Oprm1D2/D2 mice had significantly lower body temperatures at all post-MA treatment time points , compared to Taar1+/+/Oprm1B6/B6 mice .","Taar1+/+/Oprm1B6/B6 mice exhibited a significant hypothermic response to MA at T30-90 , compared to T0 , and recovered to baseline ( T0 ) temperatures by T120 , whereas Taar1+/+/Oprm1D2/D2 mice exhibited a significant hypothermic response to MA at T30-120 , and remained below baseline for the duration of testing .","However , the rate of increase after the initial decrease at T30 was similar for the two genotypes .","Regardless of Oprm1 genotype , Taar1m1J/m1J mice were resistant to the hypothermic effects of MA , exhibiting increases in body temperature at all post-MA treatment time points , compared to T0 .","The QTL results for both traits are displayed in Figure 7 ( Figure 7—source data 1 ) .","A significant QTL in the same region of chromosome 10 emerged for both MA consumption ( Figure 7a ) and body temperature response to MA ( Figure 7b ) .","This QTL is at the location of the Taar1 SNP ( 23 . 9 Mb ) .","The correlations between the strain means and the Taar1 SNP were r = 0 . 94 , p<0 . 0001 for MA consumption and r = 0 . 82 , p=0 . 02 for temperature response to MA .","In addition , suggestive QTLs were detected on chromosomes 17 and 18 for MA effect on body temperature .","There was a strong statistical trend for an increase in the chromosome 10 logarithm of the odds ( LOD ) score for MA intake ( from 8 . 2 to 8 . 8; p=0 . 07 ) , and a significant increase for the chromosome 10 LOD score for temperature response ( from 3 . 7 to 4 . 2; p=0 . 01 ) , when results were compared for interval mapping and composite interval mapping .","Composite mapping identified a suggestive QTL on chromosome four for MA intake that had not reached the suggestive significance threshold in initial mapping; however , the LOD change from 2 . 5 to 2 . 7 was not statistically significant ( Figure 7c ) .","The LOD scores for the suggestive QTLs on chromosomes 17 and 18 for temperature response were comparable for initial and composite mapping ( Figure 7d ) ."],["We leveraged CRISPR-Cas9 technology to replace the mutant Taar1m1J allele with the common Taar1+ allele in selectively bred high line mice .","The patterns of MA consumption in mice with the different Taar1 genotypes mimicked the patterns previously observed in the MA drinking lines ( Harkness et al . , 2015; Shabani et al . , 2011; Wheeler et al . , 2009 ) .","Thus , control mice escalated their MA consumption with increasing MA concentration , similar to previous findings in high line mice , whereas MA consumption was low and not concentration-dependent in the knock-in mice , similar to previous findings in the selectively bred low line mice .","Although oral is not the most common route of administration , some MA users do take MA orally , and when taken via this route , MA has a half-life similar to that for other routes of administration ( Cruickshank and Dyer , 2009 ) .","Furthermore , oral consumption , like other routes of administration , can lead to dependence ( Galloway et al . , 2010 ) .","On average , mice with absent TAAR1 function consume about 6 mg/kg/18h from a 40 mg/l MA solution ( Harkness et al . , 2015; Hitzemann et al . , 2019; Reed et al . , 2018; Shabani et al . , 2011; Wheeler et al . , 2009 ) .","High line mice will consume an average of as much as 14 mg/kg/18h , when MA is offered as a 140 mg/l concentration ( Shabani et al . , 2016 ) .","Daily MA use in humans ranges from 300 to 800 mg/day on average ( Cho et al . , 2001; Simon et al . , 2002 ) , which translates to 3 . 9 to 10 . 4 mg/kg/day in a 77 kg individual .","Whereas the half-life of MA is shorter in rodents compared to humans ( Cho et al . , 2001 ) , relevant levels of MA are attained in our mouse model , with locomotor stimulant effects observed in the high line mice at consumed doses as low as 0 . 4 mg/kg in 1h ( Shabani et al . , 2012a ) .","In rats with functional TAAR1 , TAAR1 agonists delivered systemically or to specific subregions of the mesocorticolimbic system , decreased MA seeking and cocaine seeking ( Liu et al . , 2017; Pei et al . , 2017 ) , suggesting that treatments that increase TAAR1 activity could be beneficial therapeutics .","However , when TAAR1 is non-functional , receptor agonists are not an option , leaving us to consider downstream mechanisms of TAAR1 activation .","Details of the mechanisms enlisted by TAAR1 activation remain elusive .","Expressing TAAR1 using a heterologous expression system to identify its messenger system ( s ) has proved difficult , potentially due to the predominant intracellular localization of the receptor .","Studying the function of TAAR1 is also complicated by the lack of good quality antibodies and selective antagonists ( Liu and Li , 2018; Rutigliano et al . , 2017 ) .","There is one available TAAR1 antagonist , EPPTB ( Bradaia et al . , 2009 ) , but it has a high rate of clearance ( Rutigliano et al . , 2017; Stalder et al . , 2011 ) and poor solubility , which limit in vivo use .","Our attempts to develop better antagonists with chemist collaborators have not yet met with success , and other investigators in the field have had a similar experience ( Lam et al . , 2018 ) .","One strategy that could be considered for reducing MA consumption is to target mechanisms that are impacted by the absence of TAAR1 function .","For example , TAAR1 modulates monoaminergic neurotransmission ( Espinoza et al . , 2015a; Leo et al . , 2014; Lindemann et al . , 2008; Revel et al . , 2011; Xie and Miller , 2008 ) , and recently has been implicated in glutamatergic neurotransmission ( Espinoza et al . , 2018; Espinoza et al . , 2015b ) .","High line mice are hyperglutamatergic at baseline , compared to low line mice , in both the nucleus accumbens ( NAc ) and medial prefrontal cortex ( mPFC ) .","They also exhibit a larger glutamate response to MA in the NAc , but not mPFC ( Lominac et al . , 2016; Szumlinski et al . , 2017 ) .","In addition , compared to low line mice , high line mice have blunted dopamine levels in both the NAc and mPFC , and exhibit a heightened dopaminergic response to MA in the mPFC , but not NAc ( Lominac et al . , 2014 ) .","Whether these differences are related to Taar1 genotype is unknown , but they suggest certain manipulations to be examined for their role in MA intake .","Our previous findings in the MA drinking lines demonstrated that the thermal response to MA is genetically-correlated with level of MA consumption .","Thus , high line mice are resistant to the hypothermic effect of MA , whereas low line mice are highly sensitive to this effect of MA ( Harkness et al . , 2015 ) .","Here , we performed QTL analysis using data from the BXD strains and identified a significant QTL on chromosome 10 for the effect of MA on body temperature .","This QTL is in the SNP-poor region of chromosome 10 ( Shi et al . , 2016 ) , where we mapped the QTL for MA consumption in the MA drinking lines and BXD strains .","QTL mapping was previously performed in BXD strains for the effects of 4 , 8 and 16 mg/kg MA on body temperature recorded 48 min after administration .","Although a QTL was mapped on chromosome 10 for the 4 mg/kg dose , it was considerably distal to the current QTL ( Grisel et al . , 1997 ) , and not likely to be the same one for several reasons .","First , the MA doses and time after administration were considerably different from those in our studies .","But , more importantly , the Grisel et al . ( 1997 ) paper was published well before the Taar1m1J mutation arose in D2 mice ( Reed et al . , 2018 ) ; therefore , all of the BXD strains in that study shared the common Taar1+/+ genotype .","We have confirmed the Taar1+/+ genotype of many of the BXD strains that were derived prior to when the mutation appeared ( Reed et al . , 2018; Shi et al . , 2016 ) .","Sensitivity to MA-induced hypothermia corresponds with low levels of MA consumption in MAHDR-Taar1+/+ knock-in mice , MALDR line mice , the wildtype littermates of Taar1 knockout mice , and BXD strains with the Taar1+/+ genotype .","These data could indicate that Taar1 has independent pleiotropic effects on the two traits .","Another possibility is that the subjective experience of MA-induced hypothermia in mice with functional TAAR1 limits MA consumption , thereby playing a protective role .","Not known is whether MA-induced hypothermia is subjectively unpleasant .","However , hypothermia prolonged the associative period during which aversion could be conditioned in a conditioned taste aversion ( CTA ) procedure ( Misanin et al . , 1998; Misanin et al . , 2002 ) , and mice with functional TAAR1 form MA-induced CTA , whereas those without functional TAAR1 do not ( Harkness et al . , 2015; Shabani et al . , 2012b; Wheeler et al . , 2009 ) .","Low line and high line mice voluntarily consume similar amounts of MA on the first day that MA is offered , but low line mice then decrease consumption on the subsequent day , and remain at low intake levels from then on ( Eastwood et al . , 2014; Shabani et al . , 2012a ) .","It is possible that initial MA consumption induces hypothermia in low line mice , so that initial MA consumption is associated with this potentially unpleasant physiological effect , and reduces the desire to consume more MA .","Additional research is needed to determine if MA drinking produces changes in body temperature , as occurs in response to an IP injection of MA .","Regardless of the chicken-egg question , the Taar1+ allele replacement in high line mice produced MA intake levels like those found in low line mice , indicating that this gene has a major role in determining level of MA intake .","Similarly , both the pattern and magnitude of hypothermic response to MA in the knock-in mice resembled those of low line mice .","Low line mice had a maximal response of 1–2°C ( depending upon replicate line ) after treatment with 2 mg/kg MA ( Harkness et al . , 2015 ) , and the response in the knock-in mice was also maximal at 30 min and of about 1 . 2°C .","Therefore , Taar1 appears also to have a primary role in the hypothermic response to MA .","However , Taar1 genotype does not impact basal body temperature , nor the hypothermic response to ethanol ( Harkness et al . , 2015 ) .","Further , the Taar1 mutation does not appear to affect locomotor activity at baseline or in response to the 2 mg/kg dose of MA used here ( Shabani et al . , 2011; Wheeler et al . , 2009 ) , making it unlikely that differences in thermal response to MA in mice with different Taar1 genotypes are due to differences in movement after treatment .","In addition to the significant QTL on chromosome 10 for MA intake and MA-induced thermal response , we identified other genomic regions at the suggestive level of significance .","None of these overlapped for the two traits , and the chromosome 4 MA intake QTL that reached the suggestive significance threshold with composite mapping was not identified in our previous studies of the MA drinking lines ( Belknap et al . , 2013 ) .","There may be additional alleles that account for smaller amounts of genetic variance that our analysis did not have the power to detect .","The need for large sample sizes to detect rare variants and alleles with smaller independent effects for complex traits is recognized ( Belknap , 1998; Belknap et al . , 1996; Buchner and Nadeau , 2015; Flint , 2011; Solberg Woods , 2014 ) .","Previous findings indicate that Oprm1 regulation and opioid system activity contribute to MA consumption in mice ( Belknap et al . , 2013; Eastwood et al . , 2018; Eastwood and Phillips , 2014a; Eastwood and Phillips , 2014b ) , and MA dependence/psychosis in humans ( Ide et al . , 2004; Ide et al . , 2006 ) .","Our results in the BXD strains with different combinations of Taar1/Oprm1 genotypes support a potential epistatic interaction in the effects of these genes on MA consumption and on the hypothermic response to MA .","The combined effects of Oprm1 and Taar1 genotype on each trait was non-additive .","Oprm1 genotype impacted MA consumption in Taar1m1J/m1J mice , but not Taar1+/+ mice .","The opposite pattern of effect emerged for MA-induced hypothermia; Oprm1 genotype impacted this trait in Taar1+/+ mice , but not Taar1m1J/m1J mice .","For both traits , the Oprm1D2/D2 mice exhibited the more robust effects , compared to Oprm1B6/B6 mice , and Oprm1 genotype had an impact only in mice with the stronger MA trait .","Thus , Taar1m1J/m1J/Oprm1D2/D2 mice consumed more MA than Taar1m1J/m1J/Oprm1B6/B6 mice , and Taar1+/+/Oprm1D2/D2 mice showed greater MA-induced hypothermia compared to Taar1+/+/Oprm1B6/B6 mice .","Low levels or absence of the MA-related phenotype could preclude the Oprm1 genotype from having an effect .","This interaction between Taar1 and Oprm1 was supported by composite interval QTL mapping for MA-induced body temperature change , in which controlling for Oprm1 genotype resulted in a significantly increased LOD score , specifically for the chromosome 10 QTL .","For MA intake , the LOD score increase did not meet the significance threshold , but the strong statistical trend deserves further consideration , which could be accomplished by testing more BXD strains of appropriate genotypes , as they become available , to increase power .","When we assessed the independent associations of each genotype with these MA-related traits , significant correlations were found only with Taar1 genotype .","That Oprm1 genotype alone did not correspond with amount of MA consumed is consistent with our previous findings , indicating that Oprm1 genotype is not a risk factor for MA intake , and its interactive effect with Taar1 may be associated with the regulation of Oprm1 by a significant MA intake risk gene network ( Belknap et al . , 2013 ) .","The interactive effect of Oprm1 with Taar1 , in the absence of an independent effect of Oprm1 , is consistent with other studies demonstrating epistatic interactions involving polymorphisms that do not have significant independent associations with phenotypes ( Lehner , 2011 ) .","Epistasis has been proposed as a reason for variability in disease occurrence among individuals with disease risk mutations .","However , like the many intermolecular epistatic interactions with unknown mechanisms ( Lehner , 2011 ) , the mechanism ( s ) underlying the impact of Oprm1 genotype on these Taar1-associated traits is not yet known .","One possible mechanism to explore relates to differential Oprm1 expression .","A polymorphism in the promoter region of Oprm1 was related to promoter activity , in which D2 mice had greater Oprm1 promoter activity compared to B6 mice ( Doyle et al . , 2006 ) .","It was hypothesized that this might alter MOP-r expression .","Doyle et al . ( 2014 ) found greater Oprm1 expression in the frontal cortex of D2 , compared to B6 mice .","It is possible that greater expression of Oprm1 could result in greater MA intake in Taar1m1J/m1J/Oprm1D2/D2 mice , and greater MA-induced hypothermia in Taar1+/+/Oprm1D2/D2 mice .","However , Eastwood et al . ( 2018 ) examined MOP-r protein density in D2 vs . B6 mice in the prefrontal cortex and did not find a difference .","On the other hand , our low line mice , which consumes little MA , is sensitive to MA-induced hypothermia and is largely of the Taar1+/+ genotype , had both greater Oprm1 expression and MOP-r density exclusively in the prefrontal cortex , compared to our high line mice , which has the opposite MA-related phenotypes and Taar1m1J/m1J genotype ( Belknap et al . , 2013; Eastwood et al . , 2018 ) .","Thus , the hypothesis that greater Oprm1 expression results in greater MA-induced hypothermia in Taar1+/+ genotype mice is consistent with these findings , but the hypothesis that greater Oprm1 expression results in greater MA intake in Taar1m1J/m1J genotype mice is not consistent with the existing data .","In fact , greater MA intake is associated with reduced prefrontal cortex Oprm1 and MOP-r expression in the high line , and peripheral application of drugs that increase MOP-r activity reduced MA consumption in high line mice ( Eastwood et al . , 2018; Eastwood and Phillips , 2014a ) .","We are currently creating Taar1+/+ knock-in mice on a D2 strain background and Taar1m1J/m1J knock-in mice on a B6 background .","Epistatic behavioral and molecular effects may be more approachable for study on these homogeneous backgrounds .","We observed genotype-dependent effects on total volume consumed from the MA and water tubes , but conclude that genotypic differences in MA consumption were not an artifact of overall fluid intake differences .","The total volume consumption difference between Taar1m1J/m1J and Taar1+/+ mice was small , compared to their robust difference for MA intake .","Furthermore , the interactive effect of Taar1 and Oprm1 was specific to MA intake .","We previously reported greater total fluid intake in high line mice , compared to low line mice , of about the same magnitude ( ~0 . 5 ml ) in some , but not all , replicate sets of lines ( Hitzemann et al . , 2019; Shabani et al . , 2011; Shabani et al . , 2019; Wheeler et al . , 2009 ) .","Locomotor behavior has been found to be positively associated with MA intake in the low and high lines ( Shabani et al . , 2012a ) , and larger intake volumes in mice consuming more MA could be related to stimulant effects that impact fluid needs .","But , because a difference in total fluid intake has not always been found , another interpretation is that , due to greater reinforcing effects of MA in mice of the Taar1m1J/m1J genotype , such as the high line mice ( Shabani et al . , 2012a ) , avidity for MA is greater , leading to larger intake volumes .","We observed some differences in body temperature between the knock-in and control mice that were unrelated to MA treatment .","However , these baseline differences did not appear to account for the genotype-dependent MA response .","Although the knock-in mice had higher baseline temperatures and therefore , a greater opportunity to experience a reduction in body temperature when treated with MA , the difference at baseline was of about 0 . 5°C , whereas the difference at 30 min post MA treatment was over 1°C .","This was , in part , because the MA responses were qualitatively opposite , with the knock-in mice exhibiting decreases in body temperature , similar to MALDR mice , and the controls exhibiting a time-dependent increase , similar to MAHDR mice ( Harkness et al . , 2015 ) .","In our previous studies , sex differences for MA consumption and the thermal response to MA were inconsistently found , and some are summarized in Reed et al . ( 2018 ) .","Generally , when there have been differences , females have consumed about 15% or 0 . 5 mg/kg more MA than males , but in no case were there differences between the MAHDR and MALDR lines or between mice with the different Taar1 genotypes in only one sex ( Eastwood and Phillips , 2014a; Harkness et al . , 2015; Reed et al . , 2018; Shabani et al . , 2011; Shabani et al . , 2016; Wheeler et al . , 2009 ) .","Likewise , sex did not have a significant impact on genotype-dependent differences in MA consumption in the present studies .","Similarly , there were some sex effects in the body temperature studies , but sex did not determine the pattern of response to saline vs . MA in each genotype across time .","Thus , these sex effects did not impact overall interpretations of data for either trait .","One consideration regarding Taar1 involvement in MA consumption and MA thermal response is the potential for differential potency of agonists interacting with the two TAAR1 isoforms .","However , our analyses indicate that the receptor expressed by Taar1m1J is non-functional , rather than sub-functional .","Thus , the D2-like isoform of TAAR1 , expressed by Taar1m1J , exhibits no cAMP response to a wide range of MA concentrations , or to TAAR1-specific agonists ( Harkness et al . , 2015; Shi et al . , 2016 ) .","If the Taar1 mutation reduces potency of agonists , we would expect a shift in the agonist dose-response curve for cAMP accumulation .","Further , at doses of MA up to 16 mg/kg , high line mice fail to exhibit MA-induced hypothermia , whereas low line mice exhibit hypothermia at doses as low as 1 mg/kg that reverses toward hyperthermia at higher doses ( Harkness et al . , 2015 ) .","Thus , the hypothermic effect of MA via TAAR1 may compete with the expression of MA-induced hyperthermia occurring via a non-TAAR1 mechanism in low mice .","The gRNA used to produce the MAHDR-Taar1+/+ knock-in mice was a perfect match for the sequence on either side of the chromosome 10 Taar1 SNP , with imperfect mapping to some additional chromosomes .","In our original QTL analysis for MA drinking and in the current analysis ( Figure 7 ) , there were no significant or suggestive QTLs mapped to those additional chromosomes ( Belknap et al . , 2013; and data herein ) .","This suggests that if off-target deletions or insertions occurred at these locations , they would be unlikely to have the profound impact on the MA-related traits that we have observed .","The chromosome 10 QTL accounts for 60% of the genetic variance in MA intake , and Taar1 genotype-phenotype correlations range from r = 0 . 81 to 0 . 96 for 40 mg/L MA intake , and r = 0 . 71 to 0 . 82 for 2 mg/kg MA-induced hypothermia ( Reed et al . , 2018; and data herein ) .","Therefore , the large effects we observed would be expected for replacement of Taar1m1J with Taar1+ .","The high line mice possess a genetically heterogeneous background; thus , sequencing the CRISPR knock-in and control mice would not provide specific information about off-target effects , because the existing genetic variation would not be separable from such effects .","Further , there appears to be some consensus that off-target effects of CRISPR-Cas9 are rare and indistinguishable from the background rate of de novo mutation ( e . g . , Anderson et al . , 2018; Ayabe et al . , 2019; Iyer et al . , 2018; Mianné et al . , 2016; Nakajima et al . , 2016; Willi et al . , 2018 ) .","The knock-in and control mice used in our studies were produced by independent breeding pairs .","As noted in Materials and methods , this interbreeding of like-Taar1-genotype individuals is a consistent feature for all other existing populations in which the Taar1 SNP exists .","The only exception has been for the B6 x D2 F2 mice that were produced to create the high and low selected lines , which existed as multi-Taar1-genotype littermates .","An equally strong association of Taar1 genotype with MA intake was found in these mice ( Reed et al . , 2018 ) .","In summary , these data demonstrate that Taar1 is a major contributor to MA intake and MA-induced hypothermia , and provide evidence for an interaction between Taar1 and Oprm1 in their effects on these MA traits .","Genetic variation in the human Oprm1 gene has been associated with MA dependence/psychosis ( Ide et al . , 2004; Ide et al . , 2006 ) , but additional research results in this area have not appeared in the literature .","The potential impact of human TAAR1 genetic variation on risk for MA use or on the magnitude of MA-related phenotypes is not yet known .","There are over 200 non-synonymous SNPs with the potential for missense variants in human TAAR1 ( Rutigliano et al . , 2017 ) Initial examination of some of these human TAAR1 variants indicates that TAAR1 proteins with variable levels of function are expressed ( Shi et al . , 2016 ) .","The success of TAAR1 agonists in treatment is dependent upon at least partial receptor function that can be enhanced .","Whether human TAAR1 variants are relevant to MA use and addiction in humans , and whether TAAR1 agonists are a viable treatment option , are important research questions to pursue .","Further , research into potential interactive effects between human TAAR1 and OPRM1 variants for their impact on MA use risk and response would be valuable ."],["All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals ( National Research Council , 2011 ) and were approved by the Institutional Animal Care and Use Committee of the VA Portland Health Care System ( VAPORHCS ) .","Male and female mice were group housed ( 2–5 per cage ) in filtered polycarbonate shoebox cages ( 28 . 5 × 17 . 5×12 cm ) that were lined with Bed-o’Cobs bedding ( The Andersons , Inc , Maumee , OH ) and fitted with wire tops , except during MA drinking studies .","For these studies , mice were individually housed in the same type of caging and provided with a cotton fiber nestlet for enrichment .","All mice were maintained on a 12:12h light:dark schedule ( lights on at 0600h ) , and had ad libitum access to laboratory rodent block food ( PicoLab Laboratory Rodent Diet 5LOD , 4 . 5% fat content; Animal Specialties , Woodburn , OR ) and tap water .","The Taar1m1J mouse SNP , rs33645709 , encodes a non-synonymous proline to threonine mutation at amino acid position 77 that originally occurred in D2 mice in 2001–2003 as a spontaneous mutation ( Reed et al . , 2018 ) .","Proline is often highly conserved , because it is essential for conservation of a particular protein fold .","In fact , this mutation renders TAAR1 non-functional , and the SNP is fixed ( homozygous ) in high line mice ( Harkness et al . , 2015; Reed et al . , 2018; Shi et al . , 2016 ) .","CRISPR-Cas9 technology ( Jinek et al . , 2012; Zhang , 2012 ) was used to replace the mutant Taar1m1J allele with the reference Taar1+ allele in high line mice to generate a homozygous Taar1+/+ knock-in on the high MA drinking line background at the Oregon Health & Science University Transgenic Mouse Models Shared Resource .","CRISPR in vitro transcribed guide RNAs ( gRNAs ) , targeting the specified region of Taar1 , and donor ( 100b ) oligo single strand DNA ( ossDNA ) for incorporation of the Taar1+ sequence were designed and synthesized by ThermoFisher Scientific ( Carlsbad , CA , USA ) ( Figure 8a ) .","During the development of the gRNA , in silico analysis with the basic local alignment search tool or BLAST was used to assure specificity of the sequence .","The gRNA chosen for use was a perfect match for Taar1 , with no mismatches , except at the SNP location .","The gRNA sequence had similarity to nine additional regions on chromosomes 1 , 3 , 5 , 9 , 11 and 19 , but with 2–3 mismatches in each case .","This gRNA had the sequence tctgataatgAcctgcagcatgg .","The location of the SNP is indicated by the capital A , encoding the mutant genotype present in the high line .","The location of the SNP within the Taar1 ossDNA sequence , which is the reference genotype , is indicated below by the capital C , with the gRNA sequence underlined .","The gene editing replaces A with C: cFFctggctccttcactccatggccattgtcgactttctgctgggctgtctgataatgCcctgcagcatggtgagaactgttgagcgctgttggtatZZt .","The ossDNA was designed without a silent mutation and to be asymmetric to provide increased efficiency of the gRNA .","Cas9 mRNA ( catalog number L-7606 ) was purchased from TriLink ( San Diego , CA , USA ) .","High line mice were embryo donors .","Three-four week old , female mice were super-ovulated , mated with high line males , and 0 . 5 day old eggs were isolated as previously described ( Hogan et al . , 1994 ) .","A mixture of Cas9 mRNA , Taar1 gRNA , and donor ossDNA for Taar1+ ( at concentrations of 100 ng/µl , 50 ng/µl , and 100 ng/µl , respectively ) was injected into the pronuclei of one-cell fertilized eggs .","The injected eggs were transferred into the oviducts of pseudo pregnant recipient CD1/NCrl foster dams ( Charles River , San Diego , CA , USA ) , and the surviving offspring were sequenced ( see Genotyping and sequencing ) to determine if they had retained the Taar1m1J allele or if an alteration resulting in insertion of Taar1+ had occurred .","Control mice ( MAHDR-Taar1m1J/m1J ) were derived at the same time , from those mice in which the Taar1m1J allele was not successfully altered .","These offspring were transported to the VAPORHCS vivarium and bred to produce the MAHDR-Taar1+/+ knock-in and MAHDR-Taar1m1J/m1J control mice that participated in our experiments .","Knock-in and control mice were maintained as within-line breeding pairs in a shared colony room .","This breeding of individuals with the same Taar1 genotype is consistent with the maintenance of all other populations in which the Taar1 SNP exists ( e . g . , MAHDR vs . MALDR; BXD strains; D2 mice from The Jackson Laboratory vs . D2 mice from other suppliers; Reed et al . , 2018 ) .","Representative chromatographs for the Taar1m1J ( control ) and Taar1+ ( knock-in ) sequences are displayed in Figure 8b and c , respectively .","Separate cohorts of knock-in and control mice were tested for two-bottle choice MA intake or MA-induced change in body temperature , as detailed below and in our previous publications ( Harkness et al . , 2015; Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .","For MA intake , 20 knock-in and 20 control mice were tested in a single cohort ( n = 10/genotype/sex ) , and the mice were 87–90 days old at the start of testing .","For body temperature , 48 knock-in and 50 control mice were tested in 3 cohorts of 25–46 mice ( final n = 12–13/genotype/dose/sex ) , and the mice were 79–94 days old on the day of testing .","Breeding pairs that produced the BXD mice tested in these studies were obtained from RWW ( University of Tennessee Health Science Center , Memphis , TN ) , and established within the VAPORHCS vivarium .","Specific strains were chosen according to Taar1 and Oprm1 genotypes to allow identification of potential independent and interactive effects on MA-related phenotypes .","Separate cohorts of offspring were tested for two-bottle choice MA intake or MA-induced body temperature change .","Mice were homozygous for either the reference Taar1+ or mutant Taar1m1J allele , as well as for the B6 or D2 Oprm1 allele , in all possible combinations ( Table 1 ) .","A total of 233 BXD mice ( 134 female and 99 male ) , ranging from 53 to 114 days of age , were tested for MA intake in 4 cohorts of 27–97 mice .","A total of 325 BXD mice ( 171 female and 154 male ) , ranging from 49 to 124 days of age , were tested for MA-induced body temperature change in 16 cohorts of 8–36 mice .","Numbers of BXD mice with the four potential combined Taar1/Oprm1 genotypes ( Table","1 ) were n = 22–45/genotype/sex for MA intake , and n = 14–25/genotype/sex/MA dose for the body temperature study .","We previously established and described genotyping methods for the Taar1 SNP ( Harkness et al . , 2015; Reed et al . , 2018 ) .","Genomic DNA was extracted from ear punch or tail snip samples , using QuickExtract DNA Extraction Solution ( Lucigen , Middleton , WI , USA ) .","For samples from the BXD mice and the original mice created by the CRISPR-Cas9 procedure , the Taar1 region was PCR amplified using a Hotstart DNA polymerase kit ( Qiagen , Valencia , CA , USA ) , with sequence-specific primers surrounding the region of interest ( see Harkness et al . , 2015 for details ) .","PCR products were sequenced at the Oregon Health & Science University Sequencing Core and aligned with the mouse Taar1 sequence ( NM_053205 . 1 ) to identify which allele ( s ) was present based on the rs33645709 SNP .","All knock-in and control offspring of the original mice were genotyped with an updated protocol that uses a standard Taqman ( ThermoFisher Scientific ) with fluorescent probes to detect and differentiate the Taar1 alleles ( Reed et al . , 2018 ) .","We performed Oprm1 genotyping for BXD mice as previously described ( Ferraro et al . , 2005 ) .","Sequence-specific primers for exon 10 of Oprm1 were used as the forward primer ( 5'-ggttatgcctctctggattag-3' ) and reverse primer ( 5'-tccatcgcttacatcttacca-3' ) .","A SNP in exon 10 of the B6 Oprm1 gene ( Oprm1B6 ) creates a DdeI restriction site ( Ferraro et al . , 2005 ) , so that two bands ( 235 and 168 bp ) are created when amplified DNA from mice with Oprm1B6 is digested with the DdeI restriction enzyme; DdeI does not excise the D2 Oprm1 gene ( Oprm1D2 ) , resulting in one band .","After amplification , PCR products were digested with DdeI , run on a 4% agarose gel , and visualized using ethidium bromide staining .","A representative gel is displayed in Figure 8d to indicate differentiation of homozygous ( B6/B6 , D2/D2 ) and heterozygous ( B6/D2 ) Oprm1 genotypes .","However , all BXD mice used in these studies were homozygous .","( + ) MA hydrochloride was purchased from Sigma ( St . Louis , MO , USA ) and dissolved in tap water for drinking or in sterile 0 . 9% saline ( Baxter Healthcare Corp . , Deerfield , IL , USA ) for injection .","For the body temperature studies , saline and MA were administered by intraperitoneal injection at a volume of 10 ml/kg body weight .","Methods were consistent with those we used to measure two-bottle choice MA intake during the production of the MA drinking lines ( Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .","During the initial two days of the study , mice had access to two tubes filled with tap water to familiarize them with the novel drinking apparatus .","Starting on the third day , one water tube was replaced with a tube containing MA dissolved in tap water to which mice had access for 18h/day , beginning 3h before the lights turned off .","The MA tube was removed for the remaining 6h of each day .","Unpublished data ( Phillips ) indicate that the 6h forced abstinence periods between periods of 18h MA access are important for higher MA intake levels , compared to 24h MA access periods .","The MA concentration was 20 mg/l for 4 days , and was increased to 40 mg/l for an additional 4 days .","The MA and water tube positions were alternated every other day to account for potential side bias .","MA consumption was indexed as the average intake on the second and fourth days of access to each MA concentration , as these days represent the day after the tube positions were switched , when mice should be familiar with the relative locations of the water and MA tubes .","MA consumption was measured in ml ( accuracy = 0 . 2 ml ) , and then converted to mg/kg , based on body weight measured every two days .","Total volume consumed ( ml ) from the water and MA tubes during the 18h MA access periods was also measured .","Methods for determining MA-induced change in body temperature were consistent with those we established previously ( Harkness et al . , 2015 ) .","Mice were tested after injection of saline or 2 mg/kg MA at an ambient temperature of 21 ± 2°C , for 120 min beginning at 0800h , two hours after lights on .","The MA dose was chosen from previous results demonstrating that mice with functional TAAR1 exhibit a robust hypothermic response to 2 mg/kg MA 30 min after administration that is absent in mice with non-functional TAAR1 ( Harkness et al . , 2015; Reed et al . , 2018 ) .","Treatment groups ( saline or MA ) were assigned so that males and females were equally represented , and mice from each family were distributed equally between the groups .","Mice were weighed and then placed into individual perforated acrylic plastic cubicles ( 8 × 19×8 cm in W x H x L ) that served to prevent huddling-associated effects on body temperature ( Crabbe et al . , 1987; Crabbe et al . , 1989 ) .","Mice were undisturbed for 1h before a baseline rectal temperature was obtained at time 0 ( T0 ) by inserting a glycerin-coated , 5 mm probe attached to a Thermalert model TH-8 digital thermometer ( Sensortek , Clifton , NJ , USA ) 2 . 5 cm into the rectum for 5 s .","Saline or MA was then administered , and mice were returned to their cubicles .","Rectal temperature was again measured at 30 , 60 , 90 , and 120 min post-injection ( T30-T120 ) .","QTL analyses using BXD strain means were conducted using the WebQTL mapping module of GeneNetwork ( www . genenetwork . org; RRID:SCR_002388 ) .","The traits mapped were:","1 ) MA intake when the 40 mg/l MA concentration was offered , which is the phenotype used for selective breeding ( Shabani et al . , 2011; Wheeler et al . , 2009 ) , and","2 ) MA-induced body temperature change at 30 min post-treatment .","The 30 min time point is when the hypothermic effect of the 2 mg/kg dose of MA is largest ( Harkness et al . , 2015; Reed et al . , 2018 ) .","The change score was calculated by subtracting temperatures at 30 min from baseline temperatures taken immediately before treatment .","QTL mapping was initially performed using the interval mapping function and then composite interval mapping was applied , with Oprm1 genotype at rs29351111 as a co-factor , to assess the potential interaction of Oprm1 and Taar1 .","QTL significance was assessed using the LOD score obtained after 1000 or 2000 permutations , for interval or composite interval mapping , respectively , and was considered significant if the genome-wide p-value was <0 . 05 , and considered suggestive if the genome-wide p-value was <0 . 63 , which yields one false positive per genome scan on average .","These are the standard settings used for GeneNetwork QTL mapping .","R version 3 . 6 . 0 ( The R Foundation for Statistical Computing , https://www . r-project . org/foundation/; RRID:SCR_001905 ) was used to analyze changes in LOD scores computed from interval vs . composite interval QTL mapping with a Chi-squared test comparing additive vs . interactive models for the effects of Taar1 and Oprm1 genotype .","MA consumption and MA-induced body temperature change means for the BXD strains are available in GeneNetwork .","For additional information about using GeneNetwork for QTL mapping , see Mulligan et al . ( 2017 ) .","Statistica 13 . 3 ( TIBCO Software , Inc , Palo Alto , CA , USA ) was used for statistical analyses other than QTL mapping .","For MA drinking studies , MA consumption ( mg/kg ) and total volume consumed ( ml ) data were analyzed by repeated measures ANOVA , with MA concentration as the repeated measure , and genotype ( or strain ) and sex as possible independent variables .","Body temperature data were analyzed by repeated measures ANOVA , with time as the repeated measure , and genotype ( or strain ) , sex , and MA dose as possible independent variables .","For BXD MA drinking and body temperature data , initial analyses characterizing the strains did not include sex as a factor , due to group sizes that were too small .","However , analyses examining associations between Taar1/Oprm1 genotypes and MA-related phenotypes did examine the potential impact of sex .","For these analyses , to examine potential interaction effects on the measured phenotypes , data for the entire BXD population were analyzed with sex , Taar1 genotype , and Oprm1 genotype coded as separate independent variables ( Reed et al . , 2018 ) .","Effects were considered statistically significant at p<0 . 05 .","Complex interactions were simplified by ANOVAs at levels of a particular factor .","Two-way interactions were further interpreted using simple main effects analysis , with Bonferroni correction .","To compare body temperature data at post-injection time points ( T30-120 ) to T0 within a particular genotype , Dunnett’s post hoc test was used .","Pearson’s r was used to calculate correlations between phenotypes and Taar1 or Oprm1 genotype .","Sample sizes for these studies were based on our previous MA drinking and body temperature studies ( e . g . , Harkness et al . , 2015; Reed et al . , 2018 ) ."]],"string":"[\n [\n \"Amphetamine-like stimulants , including methamphetamine ( MA ) , remain the second most common class of illicit drugs used worldwide , behind cannabis-containing drugs ( United Nations Office on Drugs and Crime , 2016 ) .\",\n \"MA is the most widely abused psychostimulant ( Chomchai and Chomchai , 2015 ) , and the consequences of MA addiction pose major societal and health concerns ( National Institute on Drug Abuse , 2019 ) .\",\n \"MA-associated deaths in the United States were relatively stable between 2005 and 2013 , but have since been on an increasing trajectory , rising from about 3600 individuals in 2013 to almost 11 , 000 in 2017 ( National Institute on Drug Abuse , 2018 ) .\",\n \"Discovering and understanding genetic factors that contribute to MA addiction risk , and uncovering the linked molecular and cellular processes , may help to curb this trend and improve interventions and long-term treatment success .\",\n \"Whereas use of large human cohorts is effective at mapping loci and nominating candidate genes , precise experimental control over genetic composition , development , and drug exposure history is practical only in animal models .\",\n \"Evidence that mouse models continue to contribute critical information to our understanding of pathological conditions was recently reviewed ( Nadeau and Auwerx , 2019 ) .\",\n \"Selected lines of rodents have been derived to model genetic risk factors for drug use .\",\n \"These include , particularly , those bred for various drug intake phenotypes , such as our mice bred for different amounts of voluntary MA intake ( Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .\",\n \"In previous studies , we demonstrated a strong genetic contribution to level of voluntary MA consumption using multiple replicate sets of mice that we bidirectionally , selectively bred for MA high drinking ( MAHDR ) and MA low drinking ( MALDR ) , which for simplicity are also referred to here as the ‘high’ and ‘low’ lines .\",\n \"Multiple lines of evidence support the involvement of the trace amine-associated receptor 1 gene , Taar1 , in differences in MA intake , including: quantitative trait locus ( QTL ) mapping in the high and low lines ( Belknap et al . , 2013 ) ; the direct agonist activity of MA at the trace amine-associated receptor 1 ( TAAR1 ) ( Bunzow et al . , 2001; Wolinsky et al . , 2007 ) ; the presence of a single nucleotide polymorphism ( SNP ) in Taar1 ( Taar1m1J ) that renders the receptor it expresses ( TAAR1 ) non-functional in response to MA and to other direct agonists ( Harkness et al . , 2015; Shi et al . , 2016 ) ; fixation of the Taar1m1J allele in high line mice of all five replicates , and retention of the wildtype , Taar1+ allele in all low line replicates ( Harkness et al . , 2015; Reed et al . , 2018 ) ; greater level of MA intake in Taar1 knockout mice compared to wildtype mice ( Harkness et al . , 2015 ) ; strong association of Taar1 genotype with MA intake in multiple genetic models ( Reed et al . , 2018 ) ; and strong association of Taar1 genotype with additional MA-related traits that correspond with level of MA intake , including level of sensitivity to MA-induced hypothermia and conditioned taste aversion ( Harkness et al . , 2015; Reed et al . , 2018 ) .\",\n \"In the current studies , to demonstrate a causal role for Taar1 , we used CRISPR-Cas9 technology to replace the Taar1m1J allele found in high line mice with the Taar1+ allele , creating a knock-in with functional TAAR1 .\",\n \"These mice were then tested for MA consumption in comparison to an unaltered high line control , with the prediction that knock-in of Taar1+ would reduce MA intake .\",\n \"In addition , we tested knock-in and control mice for MA-induced change in body temperature to determine if this gene has a pleiotropic effect on this MA trait .\",\n \"We predicted that insertion of the Taar1+ allele would increase sensitivity to MA-induced hypothermia in the knock-in mice .\",\n \"Our existing data indicate that Taar1 genotype has a major impact on MA consumption , and gene mapping results demonstrate that a chromosome 10 QTL , at the location of Taar1 , accounts for as much as 60% of the genetic variance in risk for MA intake ( Belknap et al . , 2013 ) .\",\n \"Other undefined variants must account for the remaining genetic variance .\",\n \"We previously examined the µ-opioid receptor 1 gene , Oprm1 , as a quantitative trait gene ( QTG ) residing at the proximal end of mouse chromosome 10 , but it was eliminated as a risk factor for MA intake ( Eastwood et al . , 2018 ) .\",\n \"Although Oprm1 variants do not predict risk , Oprm1 is involved in MA consumption via regulation by a top-ranked transcription factor network of genes that is differentially expressed between the MA drinking lines .\",\n \"We found that Oprm1 and its product , the µ-opioid receptor ( MOP-r ) , are more highly expressed in the prefrontal cortex of low line mice , relative to high line mice ( Belknap et al . , 2013; Eastwood et al . , 2018 ) .\",\n \"In addition , when MOP-r agonists were given to high line mice to test the hypothesis that increased MOP-r-regulated activity reduces MA intake , the acquisition of MA intake was attenuated , simulating the voluntary intake phenotype of the low line ( Eastwood et al . , 2018; Eastwood and Phillips , 2014a ) .\",\n \"High line mice possess the same mutant Taar1m1J/m1J genotype as their progenitor DBA/2J ( D2 ) strain and are more likely to also possess the Oprm1 variant from the D2 strain , due to the relatively close proximity of Taar1 and Oprm1 on chromosome 10 ( 17 Mb apart ) .\",\n \"Due to this linkage , we have been unable to explore independent and interactive effects of the Taar1 and Oprm1 genotypes on MA intake or other MA-related traits in the MA drinking lines .\",\n \"Instead , we explored this in the present studies , using a family of related recombinant inbred mouse strains derived from a C57BL/6J ( B6 ) x D2 inbred strain intercross ( collectively the BXD strains ) in which the linkage is disrupted .\",\n \"We examined the independent and interactive effects of these genes on MA intake and sensitivity to MA-induced change in body temperature , and performed QTL mapping for both traits using the BXD strain data .\"\n ],\n [\n \"We predicted that MA intake would be reduced in high line mice in which the Taar1m1J allele was removed and replaced with the Taar1+ allele .\",\n \"In the initial repeated measures analyses of variance ( ANOVAs ) , there were no interactions involving sex for either mg/kg MA consumption or total volume consumed , so we performed additional analyses with data for the sexes combined .\",\n \"For MA consumption ( Figure 1a; Figure 1—source data 1 ) , there was a significant MA concentration x genotype interaction ( F[1 , 36]=66 . 8 , p<0 . 0001 ) .\",\n \"Replacement of the Taar1m1J allele with the Taar1+ allele converted high MA intake to low MA intake for both MA concentrations .\",\n \"Control mice , which are homozygous Taar1m1J , consumed significantly more MA at the 40 mg/l concentration , compared to the 20 mg/l concentration , whereas low and comparable levels of MA consumption were found for both MA concentrations in knock-in mice .\",\n \"For total volume consumed from the water and MA tubes , during the time that MA was available ( Figure 1b; Figure 1—source data 1 ) , there was only a significant main effect of MA concentration ( F[1 , 36]=21 . 4 , p<0 . 0001 ) .\",\n \"Total volume consumed was greater when mice were offered 40 mg/l MA , compared to 20 mg/l MA .\",\n \"Thermal response to MA is a genetically-correlated response to selection for level of MA intake; thus , low MA drinking line mice display MA-induced hypothermia , whereas high line mice do not ( Harkness et al . , 2015 ) .\",\n \"We tested the hypothesis that Taar1 genotype has a causal role .\",\n \"Knock-in of Taar1+ in high line mice restored a hypothermic response to MA that did not occur in control mice ( Figure 2; Figure 2—source data 1 ) .\",\n \"In the initial repeated measures ANOVA there were significant independent interactions of sex with genotype ( F[1 , 90]=7 . 1 , p=0 . 009 ) , MA dose ( F[1 , 90]=4 . 9 , p=0 . 03 ) and time ( F[4 , 360]=2 . 9 , p=0 . 02 ) .\",\n \"However , sex did not determine the pattern of response to saline vs . MA in each genotype across time .\",\n \"There was a significant 3-way genotype x MA dose x time interaction ( F[4 , 360]=10 . 2 , p<0 . 0001 ) that did not interact with sex .\",\n \"We next examined the effects of genotype and time within each dose group .\",\n \"In the saline-treated mice ( Figure 2a ) , there were significant main effects of time ( F[4 , 188]=35 . 9 , p<0 . 0001 ) and genotype ( F[1 , 47]=4 . 7 , p=0 . 04 ) .\",\n \"Temperature declined by 0 . 3°C over time , regardless of genotype , and knock-in mice had 0 . 25°C higher body temperature than control mice , regardless of time .\",\n \"In the MA-treated mice ( Figure 2b ) , the genotype x time interaction was significant ( F[4 , 188]=13 . 0 , p<0 . 0001 ) .\",\n \"Knock-in mice had a significant hypothermic response to MA at T30 compared to T0 , whereas control mice were resistant to the hypothermic effects of MA , instead exhibiting significant MA-induced increases in body temperature at all post-MA treatment time points , compared to T0 .\",\n \"Body temperatures were significantly higher in knock-in than control mice at baseline , by about 0 . 6°C , but significantly lower by about 1 . 2°C , at T30 .\",\n \"Initial analyses characterized 18 BXD strains for MA consumption ( Figure 3; Figure 3—source data 1 ) .\",\n \"There was a significant MA concentration x strain interaction ( F[17 , 215]=17 . 2 , p<0 . 0001 ) .\",\n \"MA intake differed among the strains for both the 20 ( Figure 3a; p<0 . 0001 ) and 40 ( Figure 3b; p<0 . 0001 ) mg/l MA concentrations , and all Taar1m1J/m1J strains , but not Taar1+/+ strains , exhibited higher levels of MA intake from the 40 mg/l MA concentration than from the 20 mg/l concentration ( p=0 . 0006 to<0 . 0001 ) .\",\n \"For total volume consumed when mice were offered water vs . MA , there was a main effect of strain ( F[17 , 215]=27 . 7 , p<0 . 0001 ) and a main effect of MA concentration ( F[1 , 215]=58 . 6 , p<0 . 0001 ) .\",\n \"Total volume consumed differed among the strains by as much as 4 ml ( Figure 3c ) , but the strain distribution pattern for total volume did not correspond with the distributions for MA intake at either MA concentration .\",\n \"Total volume consumed was significantly less when water and 20 mg/l MA were available , compared to when water and 40 mg/l MA were available ( mean ± SEM = 5 . 3±0 . 1 and 5 . 7 ± 0 . 1 for 20 and 40 mg/l , respectively ) .\",\n \"We next examined the data from the BXD mice with regard to Taar1 and Oprm1 genotype to detect potential independent and epistatic effects on MA consumption .\",\n \"First , to identify potential independent effects , we calculated Pearson’s correlations of each genotype with individual 20 and 40 mg/l MA intake amounts .\",\n \"MA intake was significantly associated with Taar1 genotype ( r = 0 . 74 and 0 . 81 , ps <0 . 0001 , for 20 and 40 mg/l MA , respectively ) , but not Oprm1 genotype ( r = 0 . 06 and 0 . 10 , p=0 . 36 and 0 . 13 , for 20 and 40 mg/l MA , respectively ) .\",\n \"Next , we considered main and interaction effects by repeated measures ANOVA with MA concentration , Oprm1 genotype , Taar1 genotype , and sex as factors .\",\n \"There were no significant interactions involving sex , so further analyses were performed with data for the sexes combined .\",\n \"Data are summarized in Figure 4 ( Figure 4—source data 1 ) , with mice grouped according to their four possible Taar1/Oprm1 genotypes ( Table 1 ) .\",\n \"For MA consumption ( Figure 4a ) , there was a significant Oprm1 genotype x Taar1 genotype x MA concentration interaction ( F[1 , 225]=17 . 6 , p<0 . 0001 ) .\",\n \"For consumption of MA from the 20 mg/l MA concentration , main effects of the Oprm1 ( F[1 , 229]=8 . 6 , p=0 . 004 ) and Taar1 ( F[1 , 229]=301 . 3 , p<0 . 0001 ) genotypes indicated that both genes influenced MA intake , with greater MA intake in Taar1m1J/m1J and Oprm1D2/D2 mice , compared to Taar1+/+ and Oprm1B6/B6 mice , respectively .\",\n \"However , there was no significant Oprm1 genotype x Taar1 genotype interaction .\",\n \"In contrast , for MA consumption from the 40 mg/l MA concentration , there was a significant Oprm1 genotype x Taar1 genotype interaction ( F[1 , 229]=13 . 9 , p=0 . 0002 ) .\",\n \"MA intake was significantly higher in Taar1m1J/m1J mice , compared to Taar1+/+ mice , regardless of Oprm1 genotype ( ps <0 . 0001 ) .\",\n \"However , Taar1m1J/m1J/Oprm1D2/D2 mice consumed significantly more MA than Taar1m1J/m1J/Oprm1B6/B6 mice .\",\n \"Oprm1 genotype did not significantly impact MA intake in Taar1+/+ mice .\",\n \"For total volume consumed ( Figure 4b ) , there were significant main effects of Oprm1 genotype ( F[1 , 225]=24 . 8 , p<0 . 0001 ) , Taar1 genotype ( F[1 , 225]=4 . 7 , p=0 . 03 ) , and MA concentration ( F[1 , 225]=75 . 7 , p<0 . 0001 ) , but no significant interactions .\",\n \"Oprm1D2/D2 mice consumed more total volume than Oprm1B6/B6 mice , and Taar1m1J/m1J mice consumed more than Taar1+/+ mice .\",\n \"In addition , total volume consumed was greater when mice were offered 40 mg/l MA , compared to 20 mg/l MA .\",\n \"We used a similar approach to analyze body temperature data that were collected after saline or MA treatment in 15 BXD strains ( Figure 5; Figure 5—source data 1 ) .\",\n \"For ease of viewing , the data in Figure 5 are separated by saline/MA treatment and Taar1 genotype for strain responses across time .\",\n \"A repeated measures ANOVA detected a significant strain x MA dose x time interaction ( F[56 , 1172]=10 . 0 , p<0 . 0001 ) .\",\n \"In saline-treated mice ( Figure 5a , b ) , the BXD strains differed at each time point ( ps = 0 . 04 to<0 . 0001 ) , except T0 , and body temperature differed across time in strains 161 ( Taar1+/+/Oprm1D2/D2 ) , 171 , 194 ( both Taar1m1J/m1J/Oprm1B6/B6 ) , 186 , 210 ( both Taar1m1J/m1J/Oprm1D2/D2 ) , and 218 ( Taar1+/+/Oprm1B6/B6 ) ( ps = 0 . 004 to<0 . 0001 ) .\",\n \"In MA-treated mice ( Figure 5c , d ) , the BXD strains differed at each time point ( ps <0 . 0001 ) , except T0 , and body temperature differed across time in all strains ( ps = 0 . 007 to<0 . 0001 ) , except 160 , 171 , 194 ( all Taar1m1J/m1J/Oprm1B6/B6 ) , 178 , 186 , and 210 ( all Taar1m1J/m1J/Oprm1D2/D2 ) .\",\n \"The maximum mean drop in body temperature in saline-treated mice was 0 . 9°C ( Figure 5a , b ) , whereas there was a maximum mean drop of 4°C in MA-treated Taar1+/+ mice ( Figure 5c ) , and a maximum increase of 1°C in MA-treated Taar1m1J/m1J mice ( Figure 5d ) .\",\n \"We next examined the BXD data with regard to Taar1 and Oprm1 genotype to detect potential independent and epistatic effects on the hypothermic effect of MA .\",\n \"Again , to identify potential independent effects , we calculated Pearson’s correlations of each genotype with MA-induced change in body temperature from T0 to T30 .\",\n \"There was a significant correlation of body temperature change with Taar1 genotype ( r = 0 . 71 , p<0 . 0001 ) , but not with Oprm1 genotype ( r = 0 . 11 , p=0 . 16 ) .\",\n \"Next , we considered main and interaction effects by repeated measures ANOVA with time , Oprm1 genotype , Taar1 genotype , sex and MA dose as factors .\",\n \"There were significant independent interactions of sex with MA dose ( F[1 , 307]=8 . 0 , p=0 . 006 ) and time ( F[4 , 1228]=5 . 0 , p=0 . 0007 ) .\",\n \"However , sex did not play a significant role in the pattern of response of each genotype to each dose across time .\",\n \"There was a significant Oprm1 genotype x Taar1 genotype x MA dose x time interaction ( F[4 , 1228]=8 . 0 , p<0 . 0001 ) that did not interact with sex .\",\n \"We next examined the effects of genotype and time within each dose group ( Figure 6; Figure 6—source data 1 ) .\",\n \"In the saline-treated mice ( Figure 6a ) , the Oprm1 genotype x time interaction was significant ( F[4 , 596]=2 . 6 , p=0 . 04 ) .\",\n \"Both Oprm1B6/B6 and Oprm1D2/D2 mice exhibited reductions in body temperatures at T60-120 ( ps <0 . 001 ) , compared to T0 .\",\n \"However , these differences in body temperature were of no more than 0 . 3°C on average .\",\n \"In the MA-treated mice ( Figure 6b ) , there was a significant 3-way interaction of Oprm1 genotype x Taar1 genotype x time ( F[4 , 664]=11 . 4 , p<0 . 0001 ) .\",\n \"Taar1+/+/Oprm1D2/D2 mice had significantly lower body temperatures at all post-MA treatment time points , compared to Taar1+/+/Oprm1B6/B6 mice .\",\n \"Taar1+/+/Oprm1B6/B6 mice exhibited a significant hypothermic response to MA at T30-90 , compared to T0 , and recovered to baseline ( T0 ) temperatures by T120 , whereas Taar1+/+/Oprm1D2/D2 mice exhibited a significant hypothermic response to MA at T30-120 , and remained below baseline for the duration of testing .\",\n \"However , the rate of increase after the initial decrease at T30 was similar for the two genotypes .\",\n \"Regardless of Oprm1 genotype , Taar1m1J/m1J mice were resistant to the hypothermic effects of MA , exhibiting increases in body temperature at all post-MA treatment time points , compared to T0 .\",\n \"The QTL results for both traits are displayed in Figure 7 ( Figure 7—source data 1 ) .\",\n \"A significant QTL in the same region of chromosome 10 emerged for both MA consumption ( Figure 7a ) and body temperature response to MA ( Figure 7b ) .\",\n \"This QTL is at the location of the Taar1 SNP ( 23 . 9 Mb ) .\",\n \"The correlations between the strain means and the Taar1 SNP were r = 0 . 94 , p<0 . 0001 for MA consumption and r = 0 . 82 , p=0 . 02 for temperature response to MA .\",\n \"In addition , suggestive QTLs were detected on chromosomes 17 and 18 for MA effect on body temperature .\",\n \"There was a strong statistical trend for an increase in the chromosome 10 logarithm of the odds ( LOD ) score for MA intake ( from 8 . 2 to 8 . 8; p=0 . 07 ) , and a significant increase for the chromosome 10 LOD score for temperature response ( from 3 . 7 to 4 . 2; p=0 . 01 ) , when results were compared for interval mapping and composite interval mapping .\",\n \"Composite mapping identified a suggestive QTL on chromosome four for MA intake that had not reached the suggestive significance threshold in initial mapping; however , the LOD change from 2 . 5 to 2 . 7 was not statistically significant ( Figure 7c ) .\",\n \"The LOD scores for the suggestive QTLs on chromosomes 17 and 18 for temperature response were comparable for initial and composite mapping ( Figure 7d ) .\"\n ],\n [\n \"We leveraged CRISPR-Cas9 technology to replace the mutant Taar1m1J allele with the common Taar1+ allele in selectively bred high line mice .\",\n \"The patterns of MA consumption in mice with the different Taar1 genotypes mimicked the patterns previously observed in the MA drinking lines ( Harkness et al . , 2015; Shabani et al . , 2011; Wheeler et al . , 2009 ) .\",\n \"Thus , control mice escalated their MA consumption with increasing MA concentration , similar to previous findings in high line mice , whereas MA consumption was low and not concentration-dependent in the knock-in mice , similar to previous findings in the selectively bred low line mice .\",\n \"Although oral is not the most common route of administration , some MA users do take MA orally , and when taken via this route , MA has a half-life similar to that for other routes of administration ( Cruickshank and Dyer , 2009 ) .\",\n \"Furthermore , oral consumption , like other routes of administration , can lead to dependence ( Galloway et al . , 2010 ) .\",\n \"On average , mice with absent TAAR1 function consume about 6 mg/kg/18h from a 40 mg/l MA solution ( Harkness et al . , 2015; Hitzemann et al . , 2019; Reed et al . , 2018; Shabani et al . , 2011; Wheeler et al . , 2009 ) .\",\n \"High line mice will consume an average of as much as 14 mg/kg/18h , when MA is offered as a 140 mg/l concentration ( Shabani et al . , 2016 ) .\",\n \"Daily MA use in humans ranges from 300 to 800 mg/day on average ( Cho et al . , 2001; Simon et al . , 2002 ) , which translates to 3 . 9 to 10 . 4 mg/kg/day in a 77 kg individual .\",\n \"Whereas the half-life of MA is shorter in rodents compared to humans ( Cho et al . , 2001 ) , relevant levels of MA are attained in our mouse model , with locomotor stimulant effects observed in the high line mice at consumed doses as low as 0 . 4 mg/kg in 1h ( Shabani et al . , 2012a ) .\",\n \"In rats with functional TAAR1 , TAAR1 agonists delivered systemically or to specific subregions of the mesocorticolimbic system , decreased MA seeking and cocaine seeking ( Liu et al . , 2017; Pei et al . , 2017 ) , suggesting that treatments that increase TAAR1 activity could be beneficial therapeutics .\",\n \"However , when TAAR1 is non-functional , receptor agonists are not an option , leaving us to consider downstream mechanisms of TAAR1 activation .\",\n \"Details of the mechanisms enlisted by TAAR1 activation remain elusive .\",\n \"Expressing TAAR1 using a heterologous expression system to identify its messenger system ( s ) has proved difficult , potentially due to the predominant intracellular localization of the receptor .\",\n \"Studying the function of TAAR1 is also complicated by the lack of good quality antibodies and selective antagonists ( Liu and Li , 2018; Rutigliano et al . , 2017 ) .\",\n \"There is one available TAAR1 antagonist , EPPTB ( Bradaia et al . , 2009 ) , but it has a high rate of clearance ( Rutigliano et al . , 2017; Stalder et al . , 2011 ) and poor solubility , which limit in vivo use .\",\n \"Our attempts to develop better antagonists with chemist collaborators have not yet met with success , and other investigators in the field have had a similar experience ( Lam et al . , 2018 ) .\",\n \"One strategy that could be considered for reducing MA consumption is to target mechanisms that are impacted by the absence of TAAR1 function .\",\n \"For example , TAAR1 modulates monoaminergic neurotransmission ( Espinoza et al . , 2015a; Leo et al . , 2014; Lindemann et al . , 2008; Revel et al . , 2011; Xie and Miller , 2008 ) , and recently has been implicated in glutamatergic neurotransmission ( Espinoza et al . , 2018; Espinoza et al . , 2015b ) .\",\n \"High line mice are hyperglutamatergic at baseline , compared to low line mice , in both the nucleus accumbens ( NAc ) and medial prefrontal cortex ( mPFC ) .\",\n \"They also exhibit a larger glutamate response to MA in the NAc , but not mPFC ( Lominac et al . , 2016; Szumlinski et al . , 2017 ) .\",\n \"In addition , compared to low line mice , high line mice have blunted dopamine levels in both the NAc and mPFC , and exhibit a heightened dopaminergic response to MA in the mPFC , but not NAc ( Lominac et al . , 2014 ) .\",\n \"Whether these differences are related to Taar1 genotype is unknown , but they suggest certain manipulations to be examined for their role in MA intake .\",\n \"Our previous findings in the MA drinking lines demonstrated that the thermal response to MA is genetically-correlated with level of MA consumption .\",\n \"Thus , high line mice are resistant to the hypothermic effect of MA , whereas low line mice are highly sensitive to this effect of MA ( Harkness et al . , 2015 ) .\",\n \"Here , we performed QTL analysis using data from the BXD strains and identified a significant QTL on chromosome 10 for the effect of MA on body temperature .\",\n \"This QTL is in the SNP-poor region of chromosome 10 ( Shi et al . , 2016 ) , where we mapped the QTL for MA consumption in the MA drinking lines and BXD strains .\",\n \"QTL mapping was previously performed in BXD strains for the effects of 4 , 8 and 16 mg/kg MA on body temperature recorded 48 min after administration .\",\n \"Although a QTL was mapped on chromosome 10 for the 4 mg/kg dose , it was considerably distal to the current QTL ( Grisel et al . , 1997 ) , and not likely to be the same one for several reasons .\",\n \"First , the MA doses and time after administration were considerably different from those in our studies .\",\n \"But , more importantly , the Grisel et al . ( 1997 ) paper was published well before the Taar1m1J mutation arose in D2 mice ( Reed et al . , 2018 ) ; therefore , all of the BXD strains in that study shared the common Taar1+/+ genotype .\",\n \"We have confirmed the Taar1+/+ genotype of many of the BXD strains that were derived prior to when the mutation appeared ( Reed et al . , 2018; Shi et al . , 2016 ) .\",\n \"Sensitivity to MA-induced hypothermia corresponds with low levels of MA consumption in MAHDR-Taar1+/+ knock-in mice , MALDR line mice , the wildtype littermates of Taar1 knockout mice , and BXD strains with the Taar1+/+ genotype .\",\n \"These data could indicate that Taar1 has independent pleiotropic effects on the two traits .\",\n \"Another possibility is that the subjective experience of MA-induced hypothermia in mice with functional TAAR1 limits MA consumption , thereby playing a protective role .\",\n \"Not known is whether MA-induced hypothermia is subjectively unpleasant .\",\n \"However , hypothermia prolonged the associative period during which aversion could be conditioned in a conditioned taste aversion ( CTA ) procedure ( Misanin et al . , 1998; Misanin et al . , 2002 ) , and mice with functional TAAR1 form MA-induced CTA , whereas those without functional TAAR1 do not ( Harkness et al . , 2015; Shabani et al . , 2012b; Wheeler et al . , 2009 ) .\",\n \"Low line and high line mice voluntarily consume similar amounts of MA on the first day that MA is offered , but low line mice then decrease consumption on the subsequent day , and remain at low intake levels from then on ( Eastwood et al . , 2014; Shabani et al . , 2012a ) .\",\n \"It is possible that initial MA consumption induces hypothermia in low line mice , so that initial MA consumption is associated with this potentially unpleasant physiological effect , and reduces the desire to consume more MA .\",\n \"Additional research is needed to determine if MA drinking produces changes in body temperature , as occurs in response to an IP injection of MA .\",\n \"Regardless of the chicken-egg question , the Taar1+ allele replacement in high line mice produced MA intake levels like those found in low line mice , indicating that this gene has a major role in determining level of MA intake .\",\n \"Similarly , both the pattern and magnitude of hypothermic response to MA in the knock-in mice resembled those of low line mice .\",\n \"Low line mice had a maximal response of 1–2°C ( depending upon replicate line ) after treatment with 2 mg/kg MA ( Harkness et al . , 2015 ) , and the response in the knock-in mice was also maximal at 30 min and of about 1 . 2°C .\",\n \"Therefore , Taar1 appears also to have a primary role in the hypothermic response to MA .\",\n \"However , Taar1 genotype does not impact basal body temperature , nor the hypothermic response to ethanol ( Harkness et al . , 2015 ) .\",\n \"Further , the Taar1 mutation does not appear to affect locomotor activity at baseline or in response to the 2 mg/kg dose of MA used here ( Shabani et al . , 2011; Wheeler et al . , 2009 ) , making it unlikely that differences in thermal response to MA in mice with different Taar1 genotypes are due to differences in movement after treatment .\",\n \"In addition to the significant QTL on chromosome 10 for MA intake and MA-induced thermal response , we identified other genomic regions at the suggestive level of significance .\",\n \"None of these overlapped for the two traits , and the chromosome 4 MA intake QTL that reached the suggestive significance threshold with composite mapping was not identified in our previous studies of the MA drinking lines ( Belknap et al . , 2013 ) .\",\n \"There may be additional alleles that account for smaller amounts of genetic variance that our analysis did not have the power to detect .\",\n \"The need for large sample sizes to detect rare variants and alleles with smaller independent effects for complex traits is recognized ( Belknap , 1998; Belknap et al . , 1996; Buchner and Nadeau , 2015; Flint , 2011; Solberg Woods , 2014 ) .\",\n \"Previous findings indicate that Oprm1 regulation and opioid system activity contribute to MA consumption in mice ( Belknap et al . , 2013; Eastwood et al . , 2018; Eastwood and Phillips , 2014a; Eastwood and Phillips , 2014b ) , and MA dependence/psychosis in humans ( Ide et al . , 2004; Ide et al . , 2006 ) .\",\n \"Our results in the BXD strains with different combinations of Taar1/Oprm1 genotypes support a potential epistatic interaction in the effects of these genes on MA consumption and on the hypothermic response to MA .\",\n \"The combined effects of Oprm1 and Taar1 genotype on each trait was non-additive .\",\n \"Oprm1 genotype impacted MA consumption in Taar1m1J/m1J mice , but not Taar1+/+ mice .\",\n \"The opposite pattern of effect emerged for MA-induced hypothermia; Oprm1 genotype impacted this trait in Taar1+/+ mice , but not Taar1m1J/m1J mice .\",\n \"For both traits , the Oprm1D2/D2 mice exhibited the more robust effects , compared to Oprm1B6/B6 mice , and Oprm1 genotype had an impact only in mice with the stronger MA trait .\",\n \"Thus , Taar1m1J/m1J/Oprm1D2/D2 mice consumed more MA than Taar1m1J/m1J/Oprm1B6/B6 mice , and Taar1+/+/Oprm1D2/D2 mice showed greater MA-induced hypothermia compared to Taar1+/+/Oprm1B6/B6 mice .\",\n \"Low levels or absence of the MA-related phenotype could preclude the Oprm1 genotype from having an effect .\",\n \"This interaction between Taar1 and Oprm1 was supported by composite interval QTL mapping for MA-induced body temperature change , in which controlling for Oprm1 genotype resulted in a significantly increased LOD score , specifically for the chromosome 10 QTL .\",\n \"For MA intake , the LOD score increase did not meet the significance threshold , but the strong statistical trend deserves further consideration , which could be accomplished by testing more BXD strains of appropriate genotypes , as they become available , to increase power .\",\n \"When we assessed the independent associations of each genotype with these MA-related traits , significant correlations were found only with Taar1 genotype .\",\n \"That Oprm1 genotype alone did not correspond with amount of MA consumed is consistent with our previous findings , indicating that Oprm1 genotype is not a risk factor for MA intake , and its interactive effect with Taar1 may be associated with the regulation of Oprm1 by a significant MA intake risk gene network ( Belknap et al . , 2013 ) .\",\n \"The interactive effect of Oprm1 with Taar1 , in the absence of an independent effect of Oprm1 , is consistent with other studies demonstrating epistatic interactions involving polymorphisms that do not have significant independent associations with phenotypes ( Lehner , 2011 ) .\",\n \"Epistasis has been proposed as a reason for variability in disease occurrence among individuals with disease risk mutations .\",\n \"However , like the many intermolecular epistatic interactions with unknown mechanisms ( Lehner , 2011 ) , the mechanism ( s ) underlying the impact of Oprm1 genotype on these Taar1-associated traits is not yet known .\",\n \"One possible mechanism to explore relates to differential Oprm1 expression .\",\n \"A polymorphism in the promoter region of Oprm1 was related to promoter activity , in which D2 mice had greater Oprm1 promoter activity compared to B6 mice ( Doyle et al . , 2006 ) .\",\n \"It was hypothesized that this might alter MOP-r expression .\",\n \"Doyle et al . ( 2014 ) found greater Oprm1 expression in the frontal cortex of D2 , compared to B6 mice .\",\n \"It is possible that greater expression of Oprm1 could result in greater MA intake in Taar1m1J/m1J/Oprm1D2/D2 mice , and greater MA-induced hypothermia in Taar1+/+/Oprm1D2/D2 mice .\",\n \"However , Eastwood et al . ( 2018 ) examined MOP-r protein density in D2 vs . B6 mice in the prefrontal cortex and did not find a difference .\",\n \"On the other hand , our low line mice , which consumes little MA , is sensitive to MA-induced hypothermia and is largely of the Taar1+/+ genotype , had both greater Oprm1 expression and MOP-r density exclusively in the prefrontal cortex , compared to our high line mice , which has the opposite MA-related phenotypes and Taar1m1J/m1J genotype ( Belknap et al . , 2013; Eastwood et al . , 2018 ) .\",\n \"Thus , the hypothesis that greater Oprm1 expression results in greater MA-induced hypothermia in Taar1+/+ genotype mice is consistent with these findings , but the hypothesis that greater Oprm1 expression results in greater MA intake in Taar1m1J/m1J genotype mice is not consistent with the existing data .\",\n \"In fact , greater MA intake is associated with reduced prefrontal cortex Oprm1 and MOP-r expression in the high line , and peripheral application of drugs that increase MOP-r activity reduced MA consumption in high line mice ( Eastwood et al . , 2018; Eastwood and Phillips , 2014a ) .\",\n \"We are currently creating Taar1+/+ knock-in mice on a D2 strain background and Taar1m1J/m1J knock-in mice on a B6 background .\",\n \"Epistatic behavioral and molecular effects may be more approachable for study on these homogeneous backgrounds .\",\n \"We observed genotype-dependent effects on total volume consumed from the MA and water tubes , but conclude that genotypic differences in MA consumption were not an artifact of overall fluid intake differences .\",\n \"The total volume consumption difference between Taar1m1J/m1J and Taar1+/+ mice was small , compared to their robust difference for MA intake .\",\n \"Furthermore , the interactive effect of Taar1 and Oprm1 was specific to MA intake .\",\n \"We previously reported greater total fluid intake in high line mice , compared to low line mice , of about the same magnitude ( ~0 . 5 ml ) in some , but not all , replicate sets of lines ( Hitzemann et al . , 2019; Shabani et al . , 2011; Shabani et al . , 2019; Wheeler et al . , 2009 ) .\",\n \"Locomotor behavior has been found to be positively associated with MA intake in the low and high lines ( Shabani et al . , 2012a ) , and larger intake volumes in mice consuming more MA could be related to stimulant effects that impact fluid needs .\",\n \"But , because a difference in total fluid intake has not always been found , another interpretation is that , due to greater reinforcing effects of MA in mice of the Taar1m1J/m1J genotype , such as the high line mice ( Shabani et al . , 2012a ) , avidity for MA is greater , leading to larger intake volumes .\",\n \"We observed some differences in body temperature between the knock-in and control mice that were unrelated to MA treatment .\",\n \"However , these baseline differences did not appear to account for the genotype-dependent MA response .\",\n \"Although the knock-in mice had higher baseline temperatures and therefore , a greater opportunity to experience a reduction in body temperature when treated with MA , the difference at baseline was of about 0 . 5°C , whereas the difference at 30 min post MA treatment was over 1°C .\",\n \"This was , in part , because the MA responses were qualitatively opposite , with the knock-in mice exhibiting decreases in body temperature , similar to MALDR mice , and the controls exhibiting a time-dependent increase , similar to MAHDR mice ( Harkness et al . , 2015 ) .\",\n \"In our previous studies , sex differences for MA consumption and the thermal response to MA were inconsistently found , and some are summarized in Reed et al . ( 2018 ) .\",\n \"Generally , when there have been differences , females have consumed about 15% or 0 . 5 mg/kg more MA than males , but in no case were there differences between the MAHDR and MALDR lines or between mice with the different Taar1 genotypes in only one sex ( Eastwood and Phillips , 2014a; Harkness et al . , 2015; Reed et al . , 2018; Shabani et al . , 2011; Shabani et al . , 2016; Wheeler et al . , 2009 ) .\",\n \"Likewise , sex did not have a significant impact on genotype-dependent differences in MA consumption in the present studies .\",\n \"Similarly , there were some sex effects in the body temperature studies , but sex did not determine the pattern of response to saline vs . MA in each genotype across time .\",\n \"Thus , these sex effects did not impact overall interpretations of data for either trait .\",\n \"One consideration regarding Taar1 involvement in MA consumption and MA thermal response is the potential for differential potency of agonists interacting with the two TAAR1 isoforms .\",\n \"However , our analyses indicate that the receptor expressed by Taar1m1J is non-functional , rather than sub-functional .\",\n \"Thus , the D2-like isoform of TAAR1 , expressed by Taar1m1J , exhibits no cAMP response to a wide range of MA concentrations , or to TAAR1-specific agonists ( Harkness et al . , 2015; Shi et al . , 2016 ) .\",\n \"If the Taar1 mutation reduces potency of agonists , we would expect a shift in the agonist dose-response curve for cAMP accumulation .\",\n \"Further , at doses of MA up to 16 mg/kg , high line mice fail to exhibit MA-induced hypothermia , whereas low line mice exhibit hypothermia at doses as low as 1 mg/kg that reverses toward hyperthermia at higher doses ( Harkness et al . , 2015 ) .\",\n \"Thus , the hypothermic effect of MA via TAAR1 may compete with the expression of MA-induced hyperthermia occurring via a non-TAAR1 mechanism in low mice .\",\n \"The gRNA used to produce the MAHDR-Taar1+/+ knock-in mice was a perfect match for the sequence on either side of the chromosome 10 Taar1 SNP , with imperfect mapping to some additional chromosomes .\",\n \"In our original QTL analysis for MA drinking and in the current analysis ( Figure 7 ) , there were no significant or suggestive QTLs mapped to those additional chromosomes ( Belknap et al . , 2013; and data herein ) .\",\n \"This suggests that if off-target deletions or insertions occurred at these locations , they would be unlikely to have the profound impact on the MA-related traits that we have observed .\",\n \"The chromosome 10 QTL accounts for 60% of the genetic variance in MA intake , and Taar1 genotype-phenotype correlations range from r = 0 . 81 to 0 . 96 for 40 mg/L MA intake , and r = 0 . 71 to 0 . 82 for 2 mg/kg MA-induced hypothermia ( Reed et al . , 2018; and data herein ) .\",\n \"Therefore , the large effects we observed would be expected for replacement of Taar1m1J with Taar1+ .\",\n \"The high line mice possess a genetically heterogeneous background; thus , sequencing the CRISPR knock-in and control mice would not provide specific information about off-target effects , because the existing genetic variation would not be separable from such effects .\",\n \"Further , there appears to be some consensus that off-target effects of CRISPR-Cas9 are rare and indistinguishable from the background rate of de novo mutation ( e . g . , Anderson et al . , 2018; Ayabe et al . , 2019; Iyer et al . , 2018; Mianné et al . , 2016; Nakajima et al . , 2016; Willi et al . , 2018 ) .\",\n \"The knock-in and control mice used in our studies were produced by independent breeding pairs .\",\n \"As noted in Materials and methods , this interbreeding of like-Taar1-genotype individuals is a consistent feature for all other existing populations in which the Taar1 SNP exists .\",\n \"The only exception has been for the B6 x D2 F2 mice that were produced to create the high and low selected lines , which existed as multi-Taar1-genotype littermates .\",\n \"An equally strong association of Taar1 genotype with MA intake was found in these mice ( Reed et al . , 2018 ) .\",\n \"In summary , these data demonstrate that Taar1 is a major contributor to MA intake and MA-induced hypothermia , and provide evidence for an interaction between Taar1 and Oprm1 in their effects on these MA traits .\",\n \"Genetic variation in the human Oprm1 gene has been associated with MA dependence/psychosis ( Ide et al . , 2004; Ide et al . , 2006 ) , but additional research results in this area have not appeared in the literature .\",\n \"The potential impact of human TAAR1 genetic variation on risk for MA use or on the magnitude of MA-related phenotypes is not yet known .\",\n \"There are over 200 non-synonymous SNPs with the potential for missense variants in human TAAR1 ( Rutigliano et al . , 2017 ) Initial examination of some of these human TAAR1 variants indicates that TAAR1 proteins with variable levels of function are expressed ( Shi et al . , 2016 ) .\",\n \"The success of TAAR1 agonists in treatment is dependent upon at least partial receptor function that can be enhanced .\",\n \"Whether human TAAR1 variants are relevant to MA use and addiction in humans , and whether TAAR1 agonists are a viable treatment option , are important research questions to pursue .\",\n \"Further , research into potential interactive effects between human TAAR1 and OPRM1 variants for their impact on MA use risk and response would be valuable .\"\n ],\n [\n \"All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals ( National Research Council , 2011 ) and were approved by the Institutional Animal Care and Use Committee of the VA Portland Health Care System ( VAPORHCS ) .\",\n \"Male and female mice were group housed ( 2–5 per cage ) in filtered polycarbonate shoebox cages ( 28 . 5 × 17 . 5×12 cm ) that were lined with Bed-o’Cobs bedding ( The Andersons , Inc , Maumee , OH ) and fitted with wire tops , except during MA drinking studies .\",\n \"For these studies , mice were individually housed in the same type of caging and provided with a cotton fiber nestlet for enrichment .\",\n \"All mice were maintained on a 12:12h light:dark schedule ( lights on at 0600h ) , and had ad libitum access to laboratory rodent block food ( PicoLab Laboratory Rodent Diet 5LOD , 4 . 5% fat content; Animal Specialties , Woodburn , OR ) and tap water .\",\n \"The Taar1m1J mouse SNP , rs33645709 , encodes a non-synonymous proline to threonine mutation at amino acid position 77 that originally occurred in D2 mice in 2001–2003 as a spontaneous mutation ( Reed et al . , 2018 ) .\",\n \"Proline is often highly conserved , because it is essential for conservation of a particular protein fold .\",\n \"In fact , this mutation renders TAAR1 non-functional , and the SNP is fixed ( homozygous ) in high line mice ( Harkness et al . , 2015; Reed et al . , 2018; Shi et al . , 2016 ) .\",\n \"CRISPR-Cas9 technology ( Jinek et al . , 2012; Zhang , 2012 ) was used to replace the mutant Taar1m1J allele with the reference Taar1+ allele in high line mice to generate a homozygous Taar1+/+ knock-in on the high MA drinking line background at the Oregon Health & Science University Transgenic Mouse Models Shared Resource .\",\n \"CRISPR in vitro transcribed guide RNAs ( gRNAs ) , targeting the specified region of Taar1 , and donor ( 100b ) oligo single strand DNA ( ossDNA ) for incorporation of the Taar1+ sequence were designed and synthesized by ThermoFisher Scientific ( Carlsbad , CA , USA ) ( Figure 8a ) .\",\n \"During the development of the gRNA , in silico analysis with the basic local alignment search tool or BLAST was used to assure specificity of the sequence .\",\n \"The gRNA chosen for use was a perfect match for Taar1 , with no mismatches , except at the SNP location .\",\n \"The gRNA sequence had similarity to nine additional regions on chromosomes 1 , 3 , 5 , 9 , 11 and 19 , but with 2–3 mismatches in each case .\",\n \"This gRNA had the sequence tctgataatgAcctgcagcatgg .\",\n \"The location of the SNP is indicated by the capital A , encoding the mutant genotype present in the high line .\",\n \"The location of the SNP within the Taar1 ossDNA sequence , which is the reference genotype , is indicated below by the capital C , with the gRNA sequence underlined .\",\n \"The gene editing replaces A with C: cFFctggctccttcactccatggccattgtcgactttctgctgggctgtctgataatgCcctgcagcatggtgagaactgttgagcgctgttggtatZZt .\",\n \"The ossDNA was designed without a silent mutation and to be asymmetric to provide increased efficiency of the gRNA .\",\n \"Cas9 mRNA ( catalog number L-7606 ) was purchased from TriLink ( San Diego , CA , USA ) .\",\n \"High line mice were embryo donors .\",\n \"Three-four week old , female mice were super-ovulated , mated with high line males , and 0 . 5 day old eggs were isolated as previously described ( Hogan et al . , 1994 ) .\",\n \"A mixture of Cas9 mRNA , Taar1 gRNA , and donor ossDNA for Taar1+ ( at concentrations of 100 ng/µl , 50 ng/µl , and 100 ng/µl , respectively ) was injected into the pronuclei of one-cell fertilized eggs .\",\n \"The injected eggs were transferred into the oviducts of pseudo pregnant recipient CD1/NCrl foster dams ( Charles River , San Diego , CA , USA ) , and the surviving offspring were sequenced ( see Genotyping and sequencing ) to determine if they had retained the Taar1m1J allele or if an alteration resulting in insertion of Taar1+ had occurred .\",\n \"Control mice ( MAHDR-Taar1m1J/m1J ) were derived at the same time , from those mice in which the Taar1m1J allele was not successfully altered .\",\n \"These offspring were transported to the VAPORHCS vivarium and bred to produce the MAHDR-Taar1+/+ knock-in and MAHDR-Taar1m1J/m1J control mice that participated in our experiments .\",\n \"Knock-in and control mice were maintained as within-line breeding pairs in a shared colony room .\",\n \"This breeding of individuals with the same Taar1 genotype is consistent with the maintenance of all other populations in which the Taar1 SNP exists ( e . g . , MAHDR vs . MALDR; BXD strains; D2 mice from The Jackson Laboratory vs . D2 mice from other suppliers; Reed et al . , 2018 ) .\",\n \"Representative chromatographs for the Taar1m1J ( control ) and Taar1+ ( knock-in ) sequences are displayed in Figure 8b and c , respectively .\",\n \"Separate cohorts of knock-in and control mice were tested for two-bottle choice MA intake or MA-induced change in body temperature , as detailed below and in our previous publications ( Harkness et al . , 2015; Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .\",\n \"For MA intake , 20 knock-in and 20 control mice were tested in a single cohort ( n = 10/genotype/sex ) , and the mice were 87–90 days old at the start of testing .\",\n \"For body temperature , 48 knock-in and 50 control mice were tested in 3 cohorts of 25–46 mice ( final n = 12–13/genotype/dose/sex ) , and the mice were 79–94 days old on the day of testing .\",\n \"Breeding pairs that produced the BXD mice tested in these studies were obtained from RWW ( University of Tennessee Health Science Center , Memphis , TN ) , and established within the VAPORHCS vivarium .\",\n \"Specific strains were chosen according to Taar1 and Oprm1 genotypes to allow identification of potential independent and interactive effects on MA-related phenotypes .\",\n \"Separate cohorts of offspring were tested for two-bottle choice MA intake or MA-induced body temperature change .\",\n \"Mice were homozygous for either the reference Taar1+ or mutant Taar1m1J allele , as well as for the B6 or D2 Oprm1 allele , in all possible combinations ( Table 1 ) .\",\n \"A total of 233 BXD mice ( 134 female and 99 male ) , ranging from 53 to 114 days of age , were tested for MA intake in 4 cohorts of 27–97 mice .\",\n \"A total of 325 BXD mice ( 171 female and 154 male ) , ranging from 49 to 124 days of age , were tested for MA-induced body temperature change in 16 cohorts of 8–36 mice .\",\n \"Numbers of BXD mice with the four potential combined Taar1/Oprm1 genotypes ( Table\",\n \"1 ) were n = 22–45/genotype/sex for MA intake , and n = 14–25/genotype/sex/MA dose for the body temperature study .\",\n \"We previously established and described genotyping methods for the Taar1 SNP ( Harkness et al . , 2015; Reed et al . , 2018 ) .\",\n \"Genomic DNA was extracted from ear punch or tail snip samples , using QuickExtract DNA Extraction Solution ( Lucigen , Middleton , WI , USA ) .\",\n \"For samples from the BXD mice and the original mice created by the CRISPR-Cas9 procedure , the Taar1 region was PCR amplified using a Hotstart DNA polymerase kit ( Qiagen , Valencia , CA , USA ) , with sequence-specific primers surrounding the region of interest ( see Harkness et al . , 2015 for details ) .\",\n \"PCR products were sequenced at the Oregon Health & Science University Sequencing Core and aligned with the mouse Taar1 sequence ( NM_053205 . 1 ) to identify which allele ( s ) was present based on the rs33645709 SNP .\",\n \"All knock-in and control offspring of the original mice were genotyped with an updated protocol that uses a standard Taqman ( ThermoFisher Scientific ) with fluorescent probes to detect and differentiate the Taar1 alleles ( Reed et al . , 2018 ) .\",\n \"We performed Oprm1 genotyping for BXD mice as previously described ( Ferraro et al . , 2005 ) .\",\n \"Sequence-specific primers for exon 10 of Oprm1 were used as the forward primer ( 5'-ggttatgcctctctggattag-3' ) and reverse primer ( 5'-tccatcgcttacatcttacca-3' ) .\",\n \"A SNP in exon 10 of the B6 Oprm1 gene ( Oprm1B6 ) creates a DdeI restriction site ( Ferraro et al . , 2005 ) , so that two bands ( 235 and 168 bp ) are created when amplified DNA from mice with Oprm1B6 is digested with the DdeI restriction enzyme; DdeI does not excise the D2 Oprm1 gene ( Oprm1D2 ) , resulting in one band .\",\n \"After amplification , PCR products were digested with DdeI , run on a 4% agarose gel , and visualized using ethidium bromide staining .\",\n \"A representative gel is displayed in Figure 8d to indicate differentiation of homozygous ( B6/B6 , D2/D2 ) and heterozygous ( B6/D2 ) Oprm1 genotypes .\",\n \"However , all BXD mice used in these studies were homozygous .\",\n \"( + ) MA hydrochloride was purchased from Sigma ( St . Louis , MO , USA ) and dissolved in tap water for drinking or in sterile 0 . 9% saline ( Baxter Healthcare Corp . , Deerfield , IL , USA ) for injection .\",\n \"For the body temperature studies , saline and MA were administered by intraperitoneal injection at a volume of 10 ml/kg body weight .\",\n \"Methods were consistent with those we used to measure two-bottle choice MA intake during the production of the MA drinking lines ( Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .\",\n \"During the initial two days of the study , mice had access to two tubes filled with tap water to familiarize them with the novel drinking apparatus .\",\n \"Starting on the third day , one water tube was replaced with a tube containing MA dissolved in tap water to which mice had access for 18h/day , beginning 3h before the lights turned off .\",\n \"The MA tube was removed for the remaining 6h of each day .\",\n \"Unpublished data ( Phillips ) indicate that the 6h forced abstinence periods between periods of 18h MA access are important for higher MA intake levels , compared to 24h MA access periods .\",\n \"The MA concentration was 20 mg/l for 4 days , and was increased to 40 mg/l for an additional 4 days .\",\n \"The MA and water tube positions were alternated every other day to account for potential side bias .\",\n \"MA consumption was indexed as the average intake on the second and fourth days of access to each MA concentration , as these days represent the day after the tube positions were switched , when mice should be familiar with the relative locations of the water and MA tubes .\",\n \"MA consumption was measured in ml ( accuracy = 0 . 2 ml ) , and then converted to mg/kg , based on body weight measured every two days .\",\n \"Total volume consumed ( ml ) from the water and MA tubes during the 18h MA access periods was also measured .\",\n \"Methods for determining MA-induced change in body temperature were consistent with those we established previously ( Harkness et al . , 2015 ) .\",\n \"Mice were tested after injection of saline or 2 mg/kg MA at an ambient temperature of 21 ± 2°C , for 120 min beginning at 0800h , two hours after lights on .\",\n \"The MA dose was chosen from previous results demonstrating that mice with functional TAAR1 exhibit a robust hypothermic response to 2 mg/kg MA 30 min after administration that is absent in mice with non-functional TAAR1 ( Harkness et al . , 2015; Reed et al . , 2018 ) .\",\n \"Treatment groups ( saline or MA ) were assigned so that males and females were equally represented , and mice from each family were distributed equally between the groups .\",\n \"Mice were weighed and then placed into individual perforated acrylic plastic cubicles ( 8 × 19×8 cm in W x H x L ) that served to prevent huddling-associated effects on body temperature ( Crabbe et al . , 1987; Crabbe et al . , 1989 ) .\",\n \"Mice were undisturbed for 1h before a baseline rectal temperature was obtained at time 0 ( T0 ) by inserting a glycerin-coated , 5 mm probe attached to a Thermalert model TH-8 digital thermometer ( Sensortek , Clifton , NJ , USA ) 2 . 5 cm into the rectum for 5 s .\",\n \"Saline or MA was then administered , and mice were returned to their cubicles .\",\n \"Rectal temperature was again measured at 30 , 60 , 90 , and 120 min post-injection ( T30-T120 ) .\",\n \"QTL analyses using BXD strain means were conducted using the WebQTL mapping module of GeneNetwork ( www . genenetwork . org; RRID:SCR_002388 ) .\",\n \"The traits mapped were:\",\n \"1 ) MA intake when the 40 mg/l MA concentration was offered , which is the phenotype used for selective breeding ( Shabani et al . , 2011; Wheeler et al . , 2009 ) , and\",\n \"2 ) MA-induced body temperature change at 30 min post-treatment .\",\n \"The 30 min time point is when the hypothermic effect of the 2 mg/kg dose of MA is largest ( Harkness et al . , 2015; Reed et al . , 2018 ) .\",\n \"The change score was calculated by subtracting temperatures at 30 min from baseline temperatures taken immediately before treatment .\",\n \"QTL mapping was initially performed using the interval mapping function and then composite interval mapping was applied , with Oprm1 genotype at rs29351111 as a co-factor , to assess the potential interaction of Oprm1 and Taar1 .\",\n \"QTL significance was assessed using the LOD score obtained after 1000 or 2000 permutations , for interval or composite interval mapping , respectively , and was considered significant if the genome-wide p-value was <0 . 05 , and considered suggestive if the genome-wide p-value was <0 . 63 , which yields one false positive per genome scan on average .\",\n \"These are the standard settings used for GeneNetwork QTL mapping .\",\n \"R version 3 . 6 . 0 ( The R Foundation for Statistical Computing , https://www . r-project . org/foundation/; RRID:SCR_001905 ) was used to analyze changes in LOD scores computed from interval vs . composite interval QTL mapping with a Chi-squared test comparing additive vs . interactive models for the effects of Taar1 and Oprm1 genotype .\",\n \"MA consumption and MA-induced body temperature change means for the BXD strains are available in GeneNetwork .\",\n \"For additional information about using GeneNetwork for QTL mapping , see Mulligan et al . ( 2017 ) .\",\n \"Statistica 13 . 3 ( TIBCO Software , Inc , Palo Alto , CA , USA ) was used for statistical analyses other than QTL mapping .\",\n \"For MA drinking studies , MA consumption ( mg/kg ) and total volume consumed ( ml ) data were analyzed by repeated measures ANOVA , with MA concentration as the repeated measure , and genotype ( or strain ) and sex as possible independent variables .\",\n \"Body temperature data were analyzed by repeated measures ANOVA , with time as the repeated measure , and genotype ( or strain ) , sex , and MA dose as possible independent variables .\",\n \"For BXD MA drinking and body temperature data , initial analyses characterizing the strains did not include sex as a factor , due to group sizes that were too small .\",\n \"However , analyses examining associations between Taar1/Oprm1 genotypes and MA-related phenotypes did examine the potential impact of sex .\",\n \"For these analyses , to examine potential interaction effects on the measured phenotypes , data for the entire BXD population were analyzed with sex , Taar1 genotype , and Oprm1 genotype coded as separate independent variables ( Reed et al . , 2018 ) .\",\n \"Effects were considered statistically significant at p<0 . 05 .\",\n \"Complex interactions were simplified by ANOVAs at levels of a particular factor .\",\n \"Two-way interactions were further interpreted using simple main effects analysis , with Bonferroni correction .\",\n \"To compare body temperature data at post-injection time points ( T30-120 ) to T0 within a particular genotype , Dunnett’s post hoc test was used .\",\n \"Pearson’s r was used to calculate correlations between phenotypes and Taar1 or Oprm1 genotype .\",\n \"Sample sizes for these studies were based on our previous MA drinking and body temperature studies ( e . g . , Harkness et al . , 2015; Reed et al . , 2018 ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["We identified a locus on mouse chromosome 10 that accounts for 60% of the genetic variance in methamphetamine intake in mice selectively bred for high versus low methamphetamine consumption .","We nominated the trace amine-associated receptor 1 gene , Taar1 , as the strongest candidate and identified regulation of the mu-opioid receptor 1 gene , Oprm1 , as another contributor .","This study exploited CRISPR-Cas9 to test the causal role of Taar1 in methamphetamine intake and a genetically-associated thermal response to methamphetamine .","The methamphetamine-related traits were rescued , converting them to levels found in methamphetamine-avoiding animals .","We used a family of recombinant inbred mouse strains for interval mapping and to examine independent and epistatic effects of Taar1 and Oprm1 .","Both methamphetamine intake and the thermal response mapped to Taar1 and the independent effect of Taar1 was dependent on genotype at Oprm1 .","Our findings encourage investigation of the contribution of Taar1 and Oprm1 variants to human methamphetamine addiction ."],"string":"[\n \"We identified a locus on mouse chromosome 10 that accounts for 60% of the genetic variance in methamphetamine intake in mice selectively bred for high versus low methamphetamine consumption .\",\n \"We nominated the trace amine-associated receptor 1 gene , Taar1 , as the strongest candidate and identified regulation of the mu-opioid receptor 1 gene , Oprm1 , as another contributor .\",\n \"This study exploited CRISPR-Cas9 to test the causal role of Taar1 in methamphetamine intake and a genetically-associated thermal response to methamphetamine .\",\n \"The methamphetamine-related traits were rescued , converting them to levels found in methamphetamine-avoiding animals .\",\n \"We used a family of recombinant inbred mouse strains for interval mapping and to examine independent and epistatic effects of Taar1 and Oprm1 .\",\n \"Both methamphetamine intake and the thermal response mapped to Taar1 and the independent effect of Taar1 was dependent on genotype at Oprm1 .\",\n \"Our findings encourage investigation of the contribution of Taar1 and Oprm1 variants to human methamphetamine addiction .\"\n]"},"summary":{"kind":"list like","value":["People who misuse drugs often do so partly in response to the environment they find themselves in , and partly because of their genetics .","The genetic component of someone’s risk is influenced by many different genes , and most research has found that each gene has a small individual effect .","A method called quantitative trait locus ( QTL ) analysis can help find parts of the genome that influence someone’s risk of misusing drugs .","In 2013 , researchers found one region on chromosome 10 in mice has a particularly large influence on how much methamphetamine an individual mouse will ingest if the drug was available in one of its two water bottles .","A gene called Taar1 was particularly important in this region and another gene , called Oprm1 , may also play a significant role .","When the Taar1 gene is switched off , mice consume larger amounts of methamphetamine , have a heightened reward response from the drug , and are insensitive to the adverse effects – such as hypothermia .","But whether Taar1 directly caused these effects , and whether Taar1 and Oprm1 interact , had not yet been determined .","If these genes played a causal role , they could be useful targets for treatment of methamphetamine-use disorder .","Stafford , Reed et al . – who include several of the researchers involved in the 2013 work – now report that when a particular variant of Taar1 was present in mice they consumed large amounts of methamphetamine .","The variant codes for a faulty version of a receptor protein .","When this variant was replaced with a working version using gene editing , the mice consumed less methamphetamine and also became sensitive to hypothermia induced by the drug .","This confirms that this gene does play a causal role in methamphetamine consumption and hypothermia .","Next , Stafford , Reed et al . tested mice with different combinations of variants of Oprm1 and Taar1 to see how the genes interacted .","The results showed that the effects of Taar1 on both consumption of the drug and hypothermia depended on the Oprm1 variant present .","The findings suggest that variants of these two genes in humans could influence an individual’s risk of addiction to methamphetamine .","It is possible that in future the disorder could be treated by drugs that modify the brain activity impacted by these receptors .","But first , it will be important to find out if these genes play a similar role in humans as they do in mice ."],"string":"[\n \"People who misuse drugs often do so partly in response to the environment they find themselves in , and partly because of their genetics .\",\n \"The genetic component of someone’s risk is influenced by many different genes , and most research has found that each gene has a small individual effect .\",\n \"A method called quantitative trait locus ( QTL ) analysis can help find parts of the genome that influence someone’s risk of misusing drugs .\",\n \"In 2013 , researchers found one region on chromosome 10 in mice has a particularly large influence on how much methamphetamine an individual mouse will ingest if the drug was available in one of its two water bottles .\",\n \"A gene called Taar1 was particularly important in this region and another gene , called Oprm1 , may also play a significant role .\",\n \"When the Taar1 gene is switched off , mice consume larger amounts of methamphetamine , have a heightened reward response from the drug , and are insensitive to the adverse effects – such as hypothermia .\",\n \"But whether Taar1 directly caused these effects , and whether Taar1 and Oprm1 interact , had not yet been determined .\",\n \"If these genes played a causal role , they could be useful targets for treatment of methamphetamine-use disorder .\",\n \"Stafford , Reed et al . – who include several of the researchers involved in the 2013 work – now report that when a particular variant of Taar1 was present in mice they consumed large amounts of methamphetamine .\",\n \"The variant codes for a faulty version of a receptor protein .\",\n \"When this variant was replaced with a working version using gene editing , the mice consumed less methamphetamine and also became sensitive to hypothermia induced by the drug .\",\n \"This confirms that this gene does play a causal role in methamphetamine consumption and hypothermia .\",\n \"Next , Stafford , Reed et al . tested mice with different combinations of variants of Oprm1 and Taar1 to see how the genes interacted .\",\n \"The results showed that the effects of Taar1 on both consumption of the drug and hypothermia depended on the Oprm1 variant present .\",\n \"The findings suggest that variants of these two genes in humans could influence an individual’s risk of addiction to methamphetamine .\",\n \"It is possible that in future the disorder could be treated by drugs that modify the brain activity impacted by these receptors .\",\n \"But first , it will be important to find out if these genes play a similar role in humans as they do in mice .\"\n]"},"year":{"kind":"string","value":"2019"}}},{"rowIdx":4184,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["epidemiology and global health"],"string":"[\n \"epidemiology and global health\"\n]"},"title":{"kind":"string","value":"A user-friendly, open-source tool to project impact and cost of diagnostic tests for tuberculosis"},"id":{"kind":"string","value":"elife-02565-v2"},"sections":{"kind":"list like","value":[["Infectious disease transmission models are important tools for translating the best current knowledge of the natural history and epidemiology of infectious diseases into projections of epidemiological impact ( e . g . , incidence , mortality ) and costs under alternative strategies for disease control ( Garnett et al . , 2011 ) .","Currently , most published transmission models are either loosely calibrated to reflect global/regional outcomes or more tightly fit to specific epidemiological settings; in either case , model results may be difficult for local decision-makers in the majority of public health settings to utilize .","Simplified models designed for in-country use by decision-makers , most notably the Spectrum suite of models supported by the Futures Institute ( Stover et al . , 2010 ) , have been used to inform decision-making in the fields of reproductive health and human immunodeficiency virus ( HIV ) for over a decade ( Stover , 2004 ) .","Estimates from the Spectrum models are now routinely incorporated into official global and country-level estimates of HIV disease burden ( Brown et al . , 2010 ) and intervention impact ( Farnham et al . , 2013 ) .","Other simplified models are readily available for impact projections related to non-infectious diseases , where transmission assumptions are less important ( Betz Brown et al . , 2000; Walker et al . , 2013 ) .","However , to date , simple , user-friendly transmission models have not been widely used for decision-making related to many infectious diseases other than HIV .","Diagnosis of active tuberculosis ( TB ) is an example of a public health intervention for which transmission models may provide guidance on both global ( Dowdy et al . , 2006; Abu-Raddad et al . , 2009 ) and country-specific levels ( Menzies et al . , 2012 ) .","Specifically , an unprecedented number of new diagnostic strategies for active TB are now recommended by the World Health Organization ( WHO ) , including same-day microscopy ( World Health Organization , 2011 ) , microcolony-based culture techniques ( Leung et al . , 2012 ) such as the microscopic-observation drug-susceptibility ( MODS ) assay ( Moore et al . , 2006 ) , line-probe assays for drug susceptibility testing ( Bwanga et al . , 2009 ) , and Xpert MTB/RIF ( ‘Xpert’ ) , a molecular assay capable of providing results ( including rifampin resistance ) in 90 min with minimal human resource requirements ( Boehme et al . , 2010 , 2011 ) .","TB program decision-makers must repeatedly determine when to invest in scaling up a novel diagnostic test , which test ( s ) to promote , and whether the implementation strategy should differ by epidemiological situation ( Cobelens et al . , 2012 ) .","Without transmission models to provide locally relevant estimates of cost and impact under alternative implementation strategies , such decisions will be made without systematically considering the implications of available scientific evidence .","To aid in this decision-making process , we created a flexible , simple modeling tool that allows non-expert users to define their local situation according to three key epidemiological parameters ( TB incidence , proportion of new TB cases that are multidrug-resistant [MDR] , and adult human immunodeficiency virus [HIV] prevalence ) and local unit costs of TB diagnosis and treatment .","This tool then incorporates those estimates into a combined decision analysis-transmission framework to generate 5-year projections of TB incidence , mortality , and control costs for nine diagnostic strategies ( Figures 1 and 2 ) .","These strategies are:‘Baseline’: Sputum smear microscopy for each diagnostic attempt , with liquid-media TB culture only to evaluate smear-positive cases with a history of previous TB treatment for drug resistance .","( Cultures in all scenarios trigger drug-susceptibility testing if positive . ) ‘TB culture if previously treated’: Sputum smear microscopy used for patients without a history of TB treatment; smear plus liquid-media culture used to diagnose TB in any previously treated individual with symptoms ( regardless of smear status ) .","‘Xpert if HIV-positive’: Xpert MTB/RIF for HIV-infected patients only , with a positive test for rifampin resistance triggering treatment for MDR-TB .","Xpert is assumed to be deployed at the district level , such that results cannot generally be provided during the same clinical encounter ( Lawn et al . , 2012 ) .","This strategy is conceived as a ‘best-case’ scenario for HIV-targeted TB testing: if individuals unaware of their HIV status are not tested with Xpert , this strategy will overestimate effectiveness , and if those unaware of their status are tested , it will underestimate costs .","‘Xpert if Smear-Positive’: Xpert MTB/RIF for smear-positive patients only ( i . e . , for rapid DST ) , with a positive test for rifampin resistance triggering treatment for MDR-TB .","‘Xpert for All’: Xpert MTB/RIF for all patients .","‘Xpert with Culture DST Confirmation’: Xpert MTB/RIF for all patients , but treatment for MDR-TB only initiated if rifampin resistance is confirmed by culture .","‘MODS/TLA’: Sputum smear , plus microcolony-based TB culture ( e . g . , MODS or thin-layer agar , TLA ) for all patients .","‘Same-Day Microscopy’: Double the per-test cost of sputum smear microscopy , in exchange for the ability to provide results to patients in the same clinical encounter ( e . g . , with peripheral , unbatched reading of sputum smears ) .","‘Same-Day Xpert’: Double the per-test cost of Xpert MTB/RIF , in exchange for the ability to provide results to patients in the same clinical encounter ( e . g . , peripheral deployment , with greater costs reflecting lower volume per machine [Vassall et al . , 2011] ) . 10 . 7554/eLife . 02565 . 003Figure 1 . Overview of user-friendly model . Users are asked , via open-source computer script or Web interface , to select one of the nine diagnostic strategies and to provide unit costs and three basic epidemiological parameters ( TB incidence , MDR-TB prevalence among new cases , and adult HIV prevalence ) .","The selected diagnostic strategy is used to populate a decision tree that calculates","( a ) the probability of missed diagnosis , unsuccessful treatment , and successful treatment ,","( b ) costs , and","( c ) diagnostic delays .","These outputs depend on patients' TB ( yes/no , and drug susceptibility status ) , HIV , and TB treatment history status .","The selected epidemiological parameters are then used to populate a dynamic transmission model , creating a steady-state population that reflects local TB epidemiology .","The decision tree—which inputs user-defined unit costs—is then incorporated into the transmission model to project outcomes under the selected diagnostic scenario .","Users can sequentially select multiple diagnostic scenarios for comparison , and the computer script ( though not the Web interface ) allows users to manipulate input parameters at their discretion . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 00310 . 7554/eLife . 02565 . 004Figure 2 . Transmission model of TB diagnosis . Boxes represent sub-populations in the model , and arrows represent rates of movement between those sub-populations .","Parallel structures exist for:","( a ) HIV-infected vs HIV-uninfected;","( b ) never-treated vs previously treated ( for TB ) ; and","( c ) among TB-infected individuals , drug-susceptible vs isoniazid-monoresistant vs rifampin-resistant ( including MDR ) .","‘Pre-diagnostic’ TB refers to individuals who are infectious but have not yet begun to seek care .","Mortality occurs from all sub-populations ( not shown ) , and at a higher rate among those with HIV and active TB . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 004 For purposes of illustration , we evaluated each of these diagnostic strategies in four emblematic epidemiological settings , defined by TB incidence , MDR-TB prevalence among new cases , and adult HIV prevalence:‘Reference/High-Incidence Setting’ ( e . g . , Southeast Asia ) : TB incidence 250 per 100 , 000/year ( twice the global incidence [World Health Organization , 2012] ) , MDR-TB prevalence of 3 . 7% in new TB cases ( the estimated global prevalence [World Health Organization , 2012] ) , adult HIV prevalence of 0·83% ( the estimated global prevalence [UNAIDS , 2012] ) ;‘Low-Incidence Setting’ ( e . g . , United States , Western Europe ) : TB incidence at entry of 8 . 9 per 100 , 000/year and declining , with similar MDR-TB ( as a proportion of new cases ) and HIV prevalence as above;‘High MDR Setting’ ( e . g . , former Soviet Union ) : TB incidence 100 per 100 , 000/year , MDR-TB prevalence of 10% in new TB cases , adult HIV prevalence of 0 . 83%; and‘High HIV Setting’ ( e . g . , sub-Saharan Africa ) : Adult HIV prevalence of 20% , TB incidence 500 per 100 , 000 , and MDR-TB prevalence among new cases of 3 . 7% .","In each setting , we used a uniform set of costs for purposes of comparison ( Table 1 ) .","Although these four settings form the basis of the results presented here , decision-makers can use the open-source model program ( included as Supplementary file 1 , written in the open-source programming language Python , Version 2 . 7 , www . python . org ) to re-define any parameter in Table 1 according to their best local knowledge; a manual for doing so is also included as Supplementary file 2 .","Capacity to create non-equilibrium settings ( e . g . , declining TB incidence , increasing MDR-TB ) is included .","We also provide a web-based interface ( flexdx . modeltb . org ) that allows users to input local TB incidence , MDR-TB prevalence , HIV prevalence , and unit costs; this interface provides customized results without the requirement to manipulate programming code .","The program corresponding to the web version is also available on a public repository ( https://github . com/JJPennington/FlexDx-TB-Web-Django ) . 10 . 7554/eLife . 02565 . 005Table 1 . Model input parameters*DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 005ParameterValueReference ( s ) /RationaleTB and HIV TransmissionTransmission rate , per smear-positive/highly infectious person-yearCalibrated to user-defined TB incidence†Proportional reduction in per-case transmission rate , MDR-TBCalibrated to user-defined MDR-TB prevalence†Proportional reduction in fitness , isoniazid-monoresistant TB25% of MDR-TB reductionAssumptionHIV incidence rate , per yearCalibrated to user-defined HIV prevalence†Relative transmission rate from smear-negative/less infectious TB0 . 22 ( Behr et al . , 1999 ) Proportion of pulmonary TB that is smear-positive/highly infectious HIV-negative0 . 63 ( Steingart et al . , 2006a; Steingart et al . , 2006b ) HIV-infected0 . 50 ( Getahun et al . , 2007 ) TB ProgressionEndogenous reactivation rate HIV-negative0 . 0005/year ( Horsburgh et al . , 2010 ) HIV-infected0 . 05/year ( Antonucci et al . , 1995 ) Proportion of recent infections resulting in rapid progression HIV-negative0 . 14 ( Vynnycky and Fine , 1997; Dye et al . , 1998 ) HIV-infected0 . 470 . 75 without ART , ( Daley et al . , 1992 ) 75% reduction if on ART , ( Williams et al . , 2010 ) 50% ART coverageReduction in TB rapid progression probability due to latent TB infection ( HIV-negative only ) 0 . 79 ( Andrews et al . , 2012 ) TB Mortality and ResolutionLife expectancy at age 1555 years ( World Bank , 2012 ) Annual mortality from HIV0 . 05/year ( UNAIDS , 2012 ) Annual mortality from TB HIV-negative , smear-positive/highly infectious0 . 23/year ( Tiemersma et al . , 2011 ) HIV-negative , smear-negative/less infectious0 . 07/year ( Tiemersma et al . , 2011 ) HIV-infected1 . 0/year ( Corbett et al . , 2003; Corbett et al . , 2007; Wood et al . , 2007 ) Rate of spontaneous TB resolution ( HIV-negative only ) Smear-positive/highly infectious0 . 1/year ( Tiemersma et al . , 2011 ) Smear-negative/less infectious0 . 27/year ( Tiemersma et al . , 2011 ) TB Treatment Outcomes and Emergence of Drug ResistanceProbability of failure or relapse ( within 1 year ) Drug-susceptible0 . 04 ( World Health Organization , 2012 ) INH-monoresistant , first-line therapy0 . 21 ( Menzies et al . , 2009b ) INH-monoresistant , retreatment or 2nd-line0 . 16 ( Menzies et al . , 2009b ) MDR-TB , first-line or retreatment0 . 50 ( Espinal et al . , 2000 ) MDR-TB , second-line therapy0 . 30 ( World Health Organization , 2010 ) Proportion of one-year recurrence due to failure Drug-susceptible0 . 14 ( Lew et al . , 2008 ) INH-monoresistant0 . 33 MDR-TB0 . 56Probability of acquired drug resistance ( per treatment course ) Susceptible becoming INH-monoresistant0 . 001 ( Menzies et al . , 2009a; Menzies et al . , 2009b ) Susceptible becoming MDR-TB0 . 002 INH-monoresistant becoming MDR-TB0 . 045 If treated with 2 effective drugs for >6 mos0 . 017Behavioral ParametersInfectious months before starting to seek care HIV-negative9 months ( Dowdy et al . , 2013 ) HIV-infected1 month ( Corbett et al . , 2004 ) Diagnostic frequency while seeking care5 . 0/year ( Storla et al . , 2008; Sreeramareddy et al . , 2009 ) Probability of treatment in a TB patient whose microbiological test is negative0 . 25 ( Wilkinson et al . , 2000; Dowdy et al . , 2008 ) Loss to follow-up between diagnostic presentation and treatment initiation Sputum smear or GXP ( not same-day ) 0 . 15 ( MacPherson et al . , 2014 ) Culture ( microcolony or commercial liquid ) 0 . 25 ( Dowdy et al . , 2008 ) Same-day diagnosis0AssumptionDiagnostic AccuracySensitivity for smear-negative/less-infectious TB Sputum smear microscopy0 Xpert MTB/RIF0 . 72 ( Brownell et al . , 2012 ) Culture ( microcolony or commercial liquid ) 0 . 85 ( Cruciani et al . , 2004; Leung et al . , 2012 ) Specificity for TB ( Steingart et al . , 2006; Boehme et al . , 2011; Leung et al . , 2012 ) Sputum smear microscopy0 . 98 Xpert MTB/RIF0 . 98 Microcolony culture0 . 98Sensitivity for drug resistance ( if TB detected ) Microcolony culture ( rifampin and isoniazid ) 0 . 98 ( Minion et al . , 2010 ) Xpert MTB/RIF ( rifampin only ) 0 . 94 ( Boehme et al . , 2011 ) Specificity for drug resistance ( if TB detected ) Microcolony culture ( isoniazid ) 0 . 96 ( Minion et al . , 2010 ) Microcolony culture ( rifampin ) 0 . 99 ( Minion et al . , 2010 ) Xpert MTB/RIF ( rifampin ) 0 . 98 ( Boehme et al . , 2011 ) Diagnostic Delay and non-TB Care-SeekingDays from presentation to treatment initiation Sputum smear or Xpert MTB/RIF7 daysAssume 1 week Microcolony or commercial liquid culture30 days ( Boehme et al . , 2011 ) Months of therapy before a failing regimen will be changed , or before default and recurrence6 monthsAssumptionAnnual rate of diagnostic evaluation for TB , among people who do not have active TB0 . 01/year10% of suspects have TB , high-incidence settingCost Parameters ( user-defined; values below for comparison purposes only ) Per-patient cost of TB therapy First-lineUS$500User-defined RetreatmentUS$1000 ( Vassall et al . , 2011 ) Second-line/MDRUS$5000 ( Vassall et al . , 2011 ) Outpatient visit ( diagnosis or follow-up ) US$10 ( Vassall et al . , 2011 ) Per-test cost: Sputum smearUS$2 ( Vassall et al . , 2011 ) Same-day sputum smearUS$10Assumption Xpert MTB/RIFUS$15 ( Vassall et al . , 2011 ) Same-day Xpert MTB/RIFUS$30Assumption Microcolony culture ( with DST ) US$5 ( Solari et al . , 2011 ) Commercial liquid-media cultureUS$20 ( Vassall et al . , 2011 ) Commercial liquid-media culture + DSTUS$40 ( Vassall et al . , 2011 ) *In the actual model program ( Supplementary file 1 ) , users can change any parameter based on local values .","†For reference , the transmission rate ( in infections per person-year during diagnosis-seeking active TB ) is 36 . 9 in the reference scenario , 14 . 0 in the low-incidence scenario , 25 . 4 in the high MDR scenario , and 12 . 9 in the high HIV scenario .","Corresponding proportional reductions in MDR-TB transmission rate are 0 . 23 , 0 . 23 , 0 . 21 , and 0 . 19; and HIV incidence estimates ( per 1000 adult person-years ) are 0 . 7 , 0 . 6 , 0 . 6 , and 18 . 9 ."],["To validate the model , we compared selected model outcomes to published global estimates .","In the high-incidence setting , our model estimated TB mortality at 14% of incidence ( 95% uncertainty range: 7–20% , WHO global estimate 14% [World Health Organization , 2012] ) , HIV-associated TB at 13% of all incident TB ( 95% uncertainty range: 4–14% , global estimate 13% [World Health Organization , 2012] ) , previously treated cases at 13% of all incident cases ( 95% uncertainty range: 9–34% , global estimate 14% [World Health Organization , 2012] ) , and duration of TB disease at 1 . 2 years ( 95% uncertainty range 0 . 8–2 . 1 , global estimate 1 . 4 years [World Health Organization , 2012] ) .","Our model estimated that MDR-TB prevalence in previously treated cases was 15 . 4% ( WHO estimate 20% [World Health Organization , 2012] ) , but unlike our model , WHO notifications often count failure and recurrence after default ( in the same person ) as two separate cases .","At steady-state in the model , 80% of incident TB was due to recent infection rather than reactivation .","Figure 3 shows the projected incremental 5-year cost and impact of each of nine selected diagnostic strategies ( described in greater detail in the ‘Materials and methods’ section ) , in the high-incidence scenario .","In general , both the cost and impact of targeted strategies ( culture for retreatment , Xpert for HIV-positive , Xpert for smear-positive ) on incidence were small relative to broader diagnostic strategies ( Xpert for all , MODS/TLA , same-day Xpert ) .","The incremental cost-effectiveness , in terms of cost per case averted ( i . e . , slope of the line from origin to each point in Figure 3 ) , was similar across all strategies with the exception of same-day smear , which was cost-saving and had greater effectiveness than the baseline .","Same-day microscopy remained cost-saving by year 5 as long as same-day results could be provided at less than five times the per-smear cost of routine results ( i . e . , <$10/test ) .","Among the targeted strategies , Xpert for smear-positives was the most expensive but had the greatest impact on MDR-TB cases averted , whereas Xpert for HIV-infected individuals and culture for retreatment cases offered smaller gains at lower cost .","Among the broad strategies , culture confirmation of rifampin-resistant tests on Xpert saved costs with little reduction in effectiveness .","There was little difference between culture-confirmed Xpert and MODS/TLA , and same-day Xpert was the most expensive and most effective strategy . 10 . 7554/eLife . 02565 . 006Figure 3 . Incremental 5-year cost and impact of TB diagnostic strategies , high-incidence setting . Shown are cumulative projected 5-year costs and impact ( averted TB cases [panel A] or MDR-TB cases [panel B] ) of each diagnostic strategy described in the Introduction , incremental to the baseline strategy , per 100 , 000 population .","Strategies with greater impact appear to the right on the x-axis; more costly strategies appear higher on the y-axis .","The same-day smear strategy is cost-saving but shown at an incremental cost of $0 for simplicity . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 006 The projected impacts and costs of the nine diagnostic strategies relative to the baseline strategy , in each of four selected epidemiological settings , are shown in Figure","4 . In all four settings , the ranking of diagnostic strategies remained similar for all outcomes , although the cost of broader diagnostic strategies relative to targeted strategies fell substantially over 5 years in higher-incidence settings as the broader strategies generated declines in TB incidence .","In the low-incidence setting , where a higher proportion of TB treatments are false-positive and more incident TB is also due to reactivation ( 60% of all new cases ) , the relative cost of improved TB diagnosis was the highest , while the relative impact was the least .","In the high HIV setting , the impact of diagnostic interventions on TB incidence was diminished relative to the high-incidence setting , though the impact on TB mortality was similar .","Additionally in this setting , the Xpert for HIV-positive strategy was substantially more costly , but also more effective , than in other settings . 10 . 7554/eLife . 02565 . 007Figure 4 . Relative impact of diagnostic strategies in emblematic settings . Shown are projected changes in TB incidence , MDR-TB incidence , TB mortality , and costs ( in Year 1 and Year 5 after immediate implementation ) , relative to baseline ( Strategy 1 ) after implementing each of the diagnostic strategies described in the text .","Epidemiological outcomes are measured at the end of Year","5 . Panel A ( high incidence ) shows a setting with TB incidence of 250 per 100 , 000/year , stable MDR-TB prevalence of 3 . 7% among new cases , adult HIV prevalence of 0 . 83% , and cost of $500 to treat one case of TB with first-line therapy .","In panel B ( low incidence ) , the TB incidence is reduced to 8 . 3 per 100 , 000/year ( implemented by gradual decline in incidence over 50 years ) .","In panel C ( high MDR ) , MDR-TB prevalence among new cases is set at 3 . 7% in the beginning of year 1 , increasing to 10 . 7% by the end of year","5 . In panel D ( high HIV ) , adult HIV prevalence is set to 20% and TB incidence is set to 500 per 100 , 000/year . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 007 The impact of the ‘Xpert for all’ strategy on TB incidence ( selected a priori as the primary outcome for sensitivity analysis ) was most sensitive to three parameters , both in terms of absolute effects on impact estimates and partial rank correlation coefficients .","These three parameters were: ( 1 ) the proportion of TB patients who would be empirically treated even if microbiologic testing yielded a negative result ( ‘empiric treatment proportion’ ) , ( 2 ) duration of infectiousness before seeking care ( ‘pre-diagnostic delay’ ) , and ( 3 ) rate of reactivation from latent infection to active disease ( ‘reactivation rate’ ) .","For this latter parameter , in-depth investigation revealed that the key determinant was not the reactivation rate per se , but rather the proportion of active TB representing recent vs remote infection .","If the empiric treatment proportion was increased from 25% to 37 . 5% , the projected reduction in TB incidence fell from 20% to 13% .","By contrast , when only 12 . 5% of false-negative patients were started on therapy , ‘Xpert for all’ achieved a 31% reduction in incidence .","Corresponding reductions in incidence with ‘Xpert for all’ ( 20% at baseline ) included: 27% if pre-diagnostic delay was shortened from 9 months to 4 . 5 months , 14% if pre-diagnostic delay was lengthened to 13 . 5 months , 15% if the reactivation rate was doubled from 0 . 05% to 0 . 1% per year , and 23% if reactivation was halved to 0 . 025% per year .","The projected 5-year reduction in incidence for this strategy did not fall outside the range of 12–25% under variation of any other model parameter , up to 50% of that parameter's baseline value ( Table 1 ) .","Both costs and incremental costs were more sensitive to the cost of TB treatment than the cost of diagnostics .","For example , doubling the cost of first-line therapy ( from $500 to $1000 per person ) augmented the incremental year 1 costs for ‘Xpert for all’ from a 57% increase over baseline to a 77% increase , and doubling the cost of MDR therapy ( from $5000 to $10 , 000 per person ) generated an 86% increase in incremental year 1 costs , whereas doubling the unit cost of Xpert ( from $15 to $30 ) only resulted in a 72% increase .","Unit costs other than those for first-line treatment , MDR treatment , and the diagnostic modality under study in each scenario were not important determinants of incremental costs ."],["To date , most transmission models of infectious disease control interventions present results that are not directly usable by decision makers because they are not customizable to local conditions .","We present a flexible , user-friendly model of TB diagnosis and transmission that allows users without modeling expertise to define an epidemiologic setting ( according to TB incidence , MDR-TB prevalence , and HIV prevalence ) and unit costs , evaluating various diagnostic strategies in that setting in terms of population-level costs and impact .","While this model cannot precisely replicate the epidemiological situation in any given location , it applies a standardized methodology across a wide range of settings , thereby illustrating important interactions between epidemiological parameters and projected impact .","We provide both a web interface for rapid calculations and full model code whereby users can change any model parameter for ‘personalized’ sensitivity analysis .","Our model results suggest that the rank-ordering of diagnostic strategies may be relatively stable across epidemiological settings , but that the actual population-level costs and impact differ dramatically .","This model also serves as an example of how epidemiologists can provide decision-makers across a wide variety of local settings with rapid access to customizable ‘first-pass’ projections of cost and impact from transmission models without the need to construct tightly fitted models to represent all epidemiological settings .","Our results provide important guidance to TB decision-makers .","Specifically , in settings where little additional up-front investment is possible ( <25% increase in TB control budget ) , same-day microscopy has the greatest impact on TB incidence with no increase in overall cost at 5 years , provided that same-day microscopy can be feasibly delivered for less than $10 per patient .","For settings in which containing MDR-TB is the most important consideration , Xpert for smear-positives has the greatest effectiveness for this outcome , but at substantial price and very little impact on overall TB incidence and mortality .","Xpert for HIV-positives and culture for retreatment cases both offer meaningful , albeit small , gains; the cost and impact of these strategies are the highest in HIV-endemic settings .","In settings where more initial investment is possible ( about 50% increase in TB control budget ) , broader scale-up of either Xpert or MODS/TLA for all TB diagnosis can offer substantially greater benefits , both in terms of reduced incidence ( often 10 times more TB cases averted than with the more narrowly-targeted strategies above ) and long-term costs ( in that the incremental cost of these strategies declines greatly by Year 5 ) .","In general , culture confirmation of positive Xpert results before committing to a course of second-line therapy is preferred .","Finally , where the greatest impact is sought , combination of Xpert for all plus infrastructure for same-day diagnosis achieves this aim in all settings , but at the highest cost .","Although this model represents a highly simplified framework , it compares well to other global estimates to which it was not fit ( e . g . , WHO estimates of TB mortality , previously treated TB , HIV-associated TB , and MDR-TB prevalence among previously treated cases ) .","Its results are also similar to those of other mathematical models of TB diagnosis that are fit to specific locations .","For example , Menzies et al . ( 2012 ) modeled scale-up of Xpert for people living with HIV in southern Africa , estimating a 6% reduction in incidence , 21% reduction in TB mortality , 25% reduction in MDR-TB incidence that declines to <10% over a longer time frame , and 40% increase in costs ( not including costs of antiretroviral therapy ) .","Our corresponding estimates for the Xpert for HIV-positive strategy in the high-HIV setting are 5% reduction in incidence , 25% reduction in TB mortality , 7% reduction in MDR-TB incidence , and increase in costs from 41% ( year 1 ) to 28% ( year 5 ) .","For purposes of deciding between alternative strategies of scaling up TB diagnostics , the estimates from these two models are likely to provide similar guidance .","For any such simplified transmission model to have an impact on decision-making , it is important to consider who the eventual users of such a model might be , and to develop the model in consultation with those groups .","In this case , we identified a number of potential end-user organizations including technical assistance agencies ( e . g . , KNCV Tuberculosis Foundation ) , funding bodies ( e . g . , Global Fund for AIDS , Tuberculosis , and Malaria ) , non-governmental organizations ( e . g . , Medecins Sans Frontiers ) , National Tuberculosis Programs , and regional offices of the World Health Organization .","We then invited representatives of these organizations to attend a 1-day workshop ( April 2014 , The Hague , The Netherlands ) describing methods and challenges related to modeling of TB diagnostics in general , including this transmission model in particular .","We developed ‘hands-on’ exercises for participants to better learn the model and solicited specific feedback , which is being incorporated into the model structure and web interface in ongoing fashion .","We next intend to develop a series of informal ‘case studies’ whereby the use of this model for in-country decision-making can be demonstrated and disseminated .","As with any modeling analysis , this research has important limitations .","In order to provide sufficient flexibility and generalizability , we make a number of strong assumptions that include a constant population , homogeneous mixing , no change in parameter values over time , and simplistic incorporation of HIV and drug resistance .","Without making such simplifying assumptions , it is impossible to deliver a flexible modeling framework that can generate transparent , customizable , rapid results ( i . e . , without complex statistical fitting to each individual epidemiological scenario ) .","This model can replicate user-defined TB incidence , MDR-TB prevalence , and HIV incidence but does not describe the breakdown of these values in key subpopulations ( e . g . , congregate settings or geographical ‘hotspots’ ) , nor does it incorporate operational aspects of the TB diagnostic system in any one setting .","Thus , this model does not ameliorate the need for more detailed models in settings where precise estimates are needed; rather it provides access to ‘first-pass’ estimates in settings ( i . e . , the vast majority of local decision-makers ) where such tightly calibrated model projections are not available .","This model focuses on transmission dynamics and thus does not include pediatric TB and extrapulmonary TB; these largely non-infectious disease manifestations remain very important components of the TB epidemic that are not captured here .","We validate our model against global estimates and other models; ideally , further validation would be performed over time using field data across a wide variety of settings .","Our model allows users to define three key epidemiological parameters ( as well as other model assumptions within the program ) , but data to inform even these three parameters—as well as unit costs—are unlikely to be available on sub-national levels .","As a result , users wishing to adapt the model to a smaller geographic scale will need to perform additional data-gathering exercises to inform these estimates if they wish to maximize the utility of the model .","Nevertheless , even if high-quality data are not available at the local level , this model allows decision-makers to estimate epidemiological and economic values according to reasonable assumptions ( e . g . , comparison of TB notification rates or budget line-items to those in the published literature or at the national level ) , vary those assumptions in real-time , and obtain corresponding projections of comparative impact that incorporate the best available current data on epidemiological or natural history parameters ( e . g . , TB progression and reactivation ) .","Future efforts might also provide flexibility to specify operational characteristics ( e . g . , health system capacity ) as well .","A final concern is that , by providing users the ability to specify TB incidence , MDR-TB prevalence , and adult HIV prevalence , a number of scenarios can be created ( e . g . , low TB incidence and very high adult HIV prevalence ) that are not epidemiologically realistic .","Although there is danger in allowing uninformed users to make projections for such scenarios ( and the model will reject or alert users to highly implausible values ) , we believe that this risk is outweighed by the benefit of providing full flexibility to model epidemiological scenarios ( e . g . , sub-district level data ) that will never be captured by a limited number of closely-calibrated TB transmission models .","In summary , we have created a flexible modeling framework that allows users without modeling expertise to generate simulated populations with locally relevant values for TB incidence , MDR-TB prevalence , adult HIV prevalence , and TB treatment costs .","By comparing an array of diagnostic options across emblematic epidemiological scenarios , we provide guidance to decision-makers who seek to ascertain the optimal diagnostic strategy to achieve their selected disease control targets , and to do so using a standardized methodology .","Success in the fight against infectious disease generally , and TB specifically , depends on our ability to place global knowledge in the hands of local decision-makers , enabling them to choose those interventions that are likely to have the greatest impact , given existing resources and local epidemiological realities .","This flexible modeling framework of diagnostic interventions takes an important step in that direction ."],["Using previously published models of TB diagnostics as a guide ( Dye et al . , 1998; Abu-Raddad et al . , 2009; Menzies et al . , 2012; Dowdy et al . , 2013 ) , we constructed a transmission model of TB using ordinary differential equations .","This model categorizes patients according to HIV status ( positive or negative ) , TB treatment status ( never treated or previously treated ) , TB disease status ( as shown in Figure 2 ) , and among those who are infected with TB , drug resistance status ( susceptible , isoniazid-monoresistant , and rifampin-resistant including MDR ) , and level of infectiousness ( smear-negative/less-infectious and smear-positive/highly infectious ) .","Individuals enter the model at age 15 , and TB with no pulmonary component ( i . e . , not infectious ) is not included .","For purposes of transparent communication , we chose a population size of 100 , 000 ( to match standard reporting of TB outcomes ) and assumed a constant population with no net population growth or immigration/emigration .","After constructing the transmission framework , we used decision analysis to estimate","( a ) the probability of each diagnostic outcome;","( b ) the diagnostic delay; and","( c ) the cost of TB diagnosis and treatment under each of the nine diagnostic strategies , assuming immediate implementation at the beginning of a given year ( ‘Year 1’ ) .","Two separate authors ( DWD and PJD ) independently coded the model; these models gave comparable results .","After setting probabilities and costs under each diagnostic strategy , we then created a flexible modeling structure capable of generating epidemiological scenarios as a function of three variables , which the user specifies: HIV prevalence ( assuming a global mean level of antiretroviral therapy coverage ) , TB incidence , and MDR-TB prevalence among new TB cases .","We accomplished this by allowing three key parameters to vary across model scenarios: annual HIV incidence , rate of TB transmission per smear-positive/highly infectious person-year , and relative per-case transmission rate of rifampin-resistant TB .","We also allow users to specify all relevant unit costs for TB diagnosis and treatment; other model parameters were estimated from existing literature ( Table 1 ) .","We then created a program that numerically generates a unique steady-state population meeting the user-defined values; this population serves as the baseline strategy ( Strategy 1 above ) at the beginning of the time frame under evaluation .","The program has flexibility to create its steady-state population 50 years in the past , allowing it to replicate the protracted , slow declines in TB incidence as seen in many lower-incidence settings; this is done automatically for any scenario with a target TB incidence less than 50 per 100 , 000/year .","The mathematical model consists of the following TB compartments:Uhp , UninfectedLhdp , Latently infectedEhdp , Early active ( infectious status i = 0 ) Ahdip , Late activePhdip , Active ‘pre-treatment’: diagnosis in progress , will lead to appropriate therapyIhdip , Active ‘inappropriate treatment’: receiving therapy that ends in default or failure In these compartments , the subscript h refers to HIV status ( h = 0 if HIV-uninfected , 1 if HIV-infected ) , d refers to drug resistance status ( d = 0 if drug-susceptible , 1 if isoniazid [INH]-monoresistant , and 2 if multidrug-resistant [MDR] ) , i refers to infectious status ( i = 0 if smear-negative/less infectious and 1 if smear-positive/highly infectious ) , and p refers to previous treatment status ( p=0 if never treated , 1 if previously treated ) .","Infectious status can be conceptualized as an individual's sputum smear status , if two smears were to be performed in a quality-assured laboratory at any given point in time .","The model assumes an adult population of stable size with no immigration or emigration: the number of individuals entering the uninfected compartment U is set as equal to the number who die ( whether from TB or other causes ) from all other compartments .","Pediatric and purely extrapulmonary TB are not explicitly considered because the diagnostic considerations for these manifestations are different .","In the short-term , however , to the extent that these forms of TB are non-infectious and equally fatal as adult pulmonary TB , their effects on TB incidence and mortality may be approximated by dividing the model's projected incidence and mortality by ( 1 − proportion of TB that is not adult pulmonary ) , to obtain a new incidence/mortality estimate .","Thus , if 20% of all TB in a given location is extrapulmonary or paediatric , the rough projected total TB incidence would be ( projected TB incidence ) / ( 0 . 8 ) .","In this model , we consider latent TB infection to be asymptomatic and non-infectious , with a constant rate of reactivation and ongoing risk of exogenous reinfection leading to active TB; individuals successfully treated for TB are assumed to return to this compartment upon initiation of effective therapy ( i . e . , therapy that will result in completion , with no relapse for 2 years ) .","Upon developing active TB , individuals enter a ‘pre-diagnostic’ phase that is characterized by a low level of infectiousness and mortality ( equivalent to smear-negative TB ) and during which individuals do not actively seek diagnosis .","The duration of this phase ( 9 months ) was selected a priori based on an existing model ( Dowdy et al . , 2013 ) in which the total duration of disease after incorporating this phase reflected the global ratio of prevalence to incidence , as estimated by the World Health Organization .","We compared the total duration of disease to this ratio as part of our model validation and assumed that this ‘pre-diagnostic’ phase is much shorter for HIV-infected vs HIV-uninfected individuals .","Upon completing this ‘pre-diagnostic’ phase , individuals progress to a diagnosis-seeking phase of active disease , which is characterized by separation into highly infectious ( ‘smear-positive’ ) and less infectious ( ‘smear-negative’ ) compartments .","Among HIV-uninfected individuals , the highly infectious compartment also carries higher mortality risk .","Diagnosis-seeking active TB implies active seeking of diagnosis at a defined rate; the probability that any single diagnostic attempt will result in effective therapy is calculated as a function of diagnostic sensitivity , probability of empiric therapy ( i . e . , without bacteriological confirmation ) , prior treatment status , and losses to follow-up , as described below .","Each diagnostic attempt , if successful , leads either to effective therapy ( which is initiated after a defined diagnostic delay ) or to ineffective therapy ( defined as leading to failure or default ) .","In order to focus on differences between the nine selected strategies above in a tractable framework , we subsumed all other diagnostic tests and procedures ( e . g . , chest X-ray , antibiotic trials ) as a probability of non-microbiologic diagnosis , without attempting to specify the associated cost or diagnostic delay .","Once effective therapy is initiated , it is assumed to immediately render the individual non-infectious , with no residual risk of mortality .","Upon initiation of ineffective therapy , individuals are assumed to remain infectious ( at the ‘smear-negative’/less infectious level ) for a defined period before either failing ( followed by another round of therapy , which can be either appropriate/curative or ineffective ) or default .","Reasoning that default will occur , on average , at the midpoint between receipt of fully-ineffective and fully-effective therapy , half of defaulters are presumed to develop recurrent TB ( which is assumed to occur immediately ) , while the other half return to the latent TB compartment ( from which reactivation or reinfection remains possible ) .","All individuals who relapse within 1 year are included as failures; thus , no specific parameter for relapse is incorporated .","Individuals who are effectively treated , or whose disease is contained without therapy , return to the latent compartment following the convention of other TB models ( Dye et al . , 1998; Abu-Raddad et al . , 2009 ) .","As the goal of this model is to focus on TB-related interventions , HIV is modeled as occurring at a defined annual incidence , calibrated to achieve a given user-defined prevalence at baseline .","We do not explicitly model HIV infection in dynamic fashion ( i . e . , the HIV incidence rate does not depend on the number of HIV-infected individuals in the model ) .","HIV infection is assumed to affect all parameters related to TB disease , including the level of immune protection afforded by latent infection ( assumed zero if HIV-infected ) , mortality rate ( increased ) , rate of reactivation from latent TB ( increased ) , duration of ‘pre-diagnostic’ TB ( decreased ) , and risk of ‘primary’ progression to active disease upon infection ( increased ) .","For purposes of maintaining a simple model structure , we do not explicitly model CD4 counts or antiretroviral therapy ( ART ) , but instead assume the global average of ART coverage , as estimated by UNAIDS ( 2012 ) .","We weight all HIV-related parameters according to this estimated probability of ART receipt; this probability can be modified by users .","We assume infection with TB strains of three different drug resistance levels: fully susceptible , INH-monoresistant , and MDR .","Dual infection with multiple strains is not considered in this model , but superinfection ( i . e . , reinfection of a latently infected individual with a different strain , resulting in primary progression to disease with the reinfecting strain ) is allowed , as is acquisition of resistance ( i . e . , change of strain from more susceptible to less susceptible ) as a result of therapy .","A key consideration with any TB diagnostic strategy is the role of the diagnostic test as applied to individuals who do not have TB ( i . e . , specificity ) .","We included this element by considering that a small proportion of the population without TB would present with TB-like symptoms each year .","This proportion ( selected such that 10% of individuals being evaluated in the high-incidence setting actually have underlying TB ) remains constant across all scenarios , such that the pre-test probability of TB is higher in settings of high TB incidence , and declines over time as successful TB control strategies are employed .","Individuals without TB who are ( inappropriately ) treated for TB incur costs of TB therapy and are also marked as ‘previously treated’ for purposes of diagnostic evaluation in the future .","Economic parameters are estimated using a unit-costing approach , whereby the unit cost of a TB diagnostic attempt and a TB treatment course ( separately for first-line , category two , and second-line therapy ) is enumerated under each scenario , and this cost is multiplied by the number of diagnostic attempts and treatment courses performed .","For simplicity , we adopt the perspective of a TB control program for our costing; additional costs of HIV care and general health services ( e . g . , hospitalization ) are not included .","The decision tree below is used to estimate the cost per diagnostic attempt or treatment course , conditional on an individual's HIV , drug resistance , and previous treatment status .","Individuals who default or die on therapy are assumed to incur half the cost of a treatment course .","Given the short time horizon ( 5 years ) , the focus on costs and outcomes ( rather than cost-effectiveness per se ) , and the desire to compare costs in year 1 and at the end of year 5 in equivalent terms , we did not discount future costs or outcomes for this analysis .","All costs are reported in US dollars , assuming the year of costs that is specified by the user .","Under each scenario , we use decision analysis to ascertain the following quantities related to each diagnostic attempt:Probability of receiving successful treatment . Probability of receiving ineffective treatment ( resulting in failure or default , including the probability of acquired resistance ) .","Cost of treatment ( conditional on whether treatment is successful or ineffective ) .","Cost of diagnosis . Diagnostic delay incurred before treatment initiated .","These quantities are calculated conditional on each patient's infectious ( smear ) status , drug resistance status , HIV status , and prior treatment status .","These probabilities are calculated for each diagnostic attempt , with the result fed back into the transmission model for purposes of appropriately allocating flows between compartments .","Inputs into the decision model include the probabilities of failure/recurrence , probability of empiric therapy , loss to follow-up before treatment , diagnostic accuracy , diagnostic delay , and economic parameters as shown in Table 1 .","Outputs from the decision tree appear as parameters in the model , as described in Table 2 and the following equations . 10 . 7554/eLife . 02565 . 008Table 2 . Model parameters and symbolic representationsDOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 008ParameterRepresentationBaseline value ( see Table 1 ) Transmission rate ( transmission events per highly infectious person-year ) βCalibrated to TB incidenceProportional reduction in per-case transmission rate Drug-susceptible TBφ01 . 0 Isoniazid-monoresistant TBφ125%* of φ2 MDR-TBφ2CalibratedHIV incidence rate , per yearθCalibrated to HIV prevalenceRelative transmission rate from smear-negative/less infectious TBζ0 . 22Proportion of pulmonary TB that is smear-positive/highly infectious HIV-negativeψ00 . 63 HIV-infectedψ10 . 50Endogenous reactivation rate , per year HIV-negativeε00 . 005 HIV-infectedε10 . 05Proportion of recent infections resulting in rapid progression HIV-negativeπ00 . 14 HIV-infectedπ10 . 47Reduction in TB rapid progression probability due to latent TB infection HIV-negativeι0 . 79 HIV-infectedNot included0Baseline mortality rate , per yearμbl1/55 = 0 . 018Additional HIV-related mortality rate , per yearμh0 . 05Additional untreated TB-related mortality rate , per year HIV-negative , smear-positive/highly infectiousμt10 . 23 HIV-negative , smear-negative/less infectiousμt00 . 07 HIV-infectedμth1 . 0Rate of spontaneous TB resolution , per year Smear-positive/highly infectiousν10 . 1 Smear-negative/less infectiousν00 . 27 HIV-infectedNot included0Rate of starting diagnosis-seeking in active TB , per year HIV-negativeδe01 . 33 ( 9 months ) HIV-infectedδe112 ( 1/month ) Rate of progression: ineffective therapy to repeat therapy ( failure ) or active TB ( relapse ) , per yearδf6/12 = 0 . 5Rate of diagnostic evaluation for TB , per year Late active TBInput into decision tree5 . 0 No active TBτ00 . 01Decision tree outputs ( in addition to unit costs ) :Vary by intervention Successful diagnosis rate of late active TB , per yearσhdip Rate of movement from successful diagnosis to treatment ( 1/diagnostic delay ) , per yearρhdip Ineffective diagnosis rate of late active TB , per yearκhdip Rate of diagnosis and treatment leading to new resistance , per year susceptible to INH-monoresistantαsihip susceptible to MDRαsmhip INH-monoresistant to MDRαimhip*Calculated such that ( 1−φ1 ) = 0 . 25* ( 1−φ2 ) .","Our primary outcomes under each scenario were TB incidence , TB mortality , MDR-TB incidence , and incremental TB diagnostic and treatment costs ( during year 1 and at the end of the 5-year period ) relative to the baseline strategy .","By giving flexibility to change model inputs , we provide users the ability to conduct any sensitivity analysis desired .","However , for illustrative purposes , we also conducted a series of one-way sensitivity analyses in which each model parameter in Table 1 was varied by ±50% of its listed value ( for proportions , 50% of the difference between the value and either zero or one ) .","Our primary outcome for sensitivity analysis was the change in TB incidence , comparing the ‘Xpert for all’ strategy to baseline in the high incidence setting .","We also conducted multivariable uncertainty analyses by calculating partial rank correlation coefficients ( Kendall , 1942 ) between each natural history parameter and the outcomes of TB incidence , TB mortality , and 5-year costs .","In addition , we constructed 95% uncertainty intervals around our estimates of outcomes in each individual country by simultaneously varying each model parameter by ±10% over a uniform distribution and each target value ( i . e . , TB incidence , HIV prevalence , and MDR-TB prevalence ) over its reported uncertainty range .","In this fashion , we constructed 10 , 000 simulations using Latin Hypercube Sampling ( McKay et al . , 1979 ) and took 95% uncertainty ranges as the 2 . 5th and 97 . 5th percentiles of outcomes in these simulations; these ranges are provided in the web-based version of the model for each country .","Rates of flow between compartments are governed by the system of ordinary differential equations listed in Equations 1–6 .","We first define the forces of infection ( according to resistance status ) and total mortality for simplicity .","( 1 ) dUhp ( t ) dt=Ih=0 , p=0*μtot ( t ) −[λ0 ( t ) +λ1 ( t ) +λ2 ( t ) +μbl ( t ) ]*Uhp ( t ) −Ih=0*[θ*U0p ( t ) ]+Ih=1*[θ*U0p ( t ) −μh*U1p ( t ) ]−Ip=0*[τ0* ( 1−sh ) *Uh0 ( t ) ]+Ip=1*[τ0* ( 1−sh ) *Uh0 ( t ) ]where μtot is the sum of all mortality ( to maintain a constant population ) , λd is the force of infection for a given drug resistance strain , μbl is the baseline mortality rate , μh is the HIV-specific mortality rate , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , θ is the HIV incidence rate , τ0 is the rate of seeking diagnosis among people without TB , and sh is the specificity of the diagnostic test .","Thus , uninfected individuals exit through infection and death , acquire HIV according to the HIV incidence rate , and become previously treated for TB ( inappropriately ) according to the specificity of the test .","The HIV-uninfected , not previously treated compartment is replenished at a rate that matches total mortality and thereby maintains a constant population .","( 2 ) dLhdp ( t ) dt=λd ( t ) * ( 1−πh ) *{Uhp ( t ) +∑d[Lhdp ( t ) * ( 1−Ih=0*ι ) ]}+Ip=1*∑i , p[ρhdip*Phdip ( t ) ]+Ih=0*{ν0*E0dp+∑i[νi* ( A0dip+P0dip+I0dip ) ]}−{[ ( λ0 ( t ) +λ1 ( t ) +λ2 ( t ) ) * ( 1−Ih=0*ι ) +εh+μbl+Ih=1*μh]*Lhdp ( t ) }−Ih=0*[θ*L0dp ( t ) ]+Ih=1*[θ*L0dp ( t ) ]where λd is the strain-specific force of infection , πh is the proportion of recent infections that progress rapidly to active TB , ι is the relative reduction in rapid progression after infection among people with latent TB , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , ρhdip is the rate of treatment after successful diagnosis is initiated , νi is the spontaneous recovery rate , εh is the endogenous reactivation rate , μbl is the baseline mortality rate , μh is the HIV-specific mortality rate , and θ is the HIV incidence rate .","Thus , individuals enter the latently infected compartment through initial TB infection ( without rapid progression ) , reinfection ( without rapid progression , and accounting for immune protection ) , initiation of successful treatment , or spontaneous resolution .","Individuals completing treatment only enter the previously treated compartment ( p=1 ) .","Individuals exit this compartment through TB reinfection , endogenous reactivation , and death , and they acquire HIV infection at a constant rate .","( 3 ) dEhdp ( t ) dt=λd ( t ) *πh*{Uhp ( t ) +∑d[Lhdp ( t ) * ( 1−Ih=0*ι ) ]}+Lhdp ( t ) *εh−{[μbl+Ih=0* ( μt0+ν0+δe0 ) +Ih=1* ( μh+μth+δe1 ) ]*Ehdp ( t ) }−Ih=0*[θ*E0dp ( t ) ]+Ih=1*[θ*E0dp ( t ) ]where λd is the force of infection , πh is the proportion of recent infections that progress rapidly to active TB , ι is the relative reduction in rapid progression after infection among people with latent TB , Ih=0 is an indicator function of HIV status ( = 1 if h = 0 , 0 otherwise ) , εh is the endogenous reactivation rate , μbl is the baseline mortality rate , μt0 is the TB-specific mortality rate for less-infectious TB , ν0 is the spontaneous recovery rate for less-infectious TB , δeh is the HIV-specific rate of progression to late active TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , and θ is the HIV incidence rate .","Thus , individuals enter this compartment through rapid progression of recent infection or endogenous reactivation of latent infection .","They exit through progression to late active disease , spontaneous cure ( if HIV-uninfected ) , or death , and they acquire HIV infection at a constant rate .","( 4 ) dAhdip ( t ) dt=δeh*[Ii=1*ψh+Ii=0* ( 1−ψh ) ]*Ehdp ( t ) +δf*Ihdip ( t ) −Ahdip ( t ) *[μbl+Ih=0* ( μti+νi+σ0dip+κ0dip+Id=0*αsi0ip+Id=0*αsm0ip+Id=1*αim0ip ) +Ih=1* ( μh+μth+σ1dip+κ1dip+Id=0*αsi1ip+Id=0*αsm1ip+Id=1*αim1ip ) ]−Ih=0*[θ*A0dip ( t ) ]+Ih=1*[θ*A0dip ( t ) ]where δeh is the rate of progression from early active TB , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , ψh is the proportion of active TB that is highly infectious ( smear-positive ) , δf is the rate ( 1/duration ) of ineffective therapy , μbl is the baseline mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , σhdip is the rate of diagnosis ultimately leading to successful treatment , κhdip is the rate of placing individuals on ineffective treatment that does not generate acquired resistance , αsihip is the rate of placing individuals on ineffective treatment that generates INH monoresistance , αsmhip and αimhip are the rates of placing individuals on ineffective treatment that generates MDR-TB , and θ is the HIV incidence rate .","Thus , individuals enter this compartment through progression from early active disease or relapse/failure after ineffective treatment .","They exit through spontaneous recovery , diagnosis leading to successful treatment , initiation of ineffective treatment ( which can , in turn , generate acquired drug resistance ) , or death , and they acquire HIV infection at a constant rate .","This compartment consists of individuals who have initiated a diagnostic attempt that will lead to successful treatment , yet remain infectious while awaiting initiation of treatment .","Inclusion of this compartment is designed to capture the effects of diagnostic delays .","( 5 ) dPhdip ( t ) dt=σhdip*Ahdip ( t ) −Phdip ( t ) *[μbl+Ih=0* ( μti+νi+ρ0dip ) +Ih=1* ( μh+μth+ρ1dip ) ]−Ih=0*[θ*P0dip ( t ) ]+Ih=1*[θ*P0dip ( t ) ]where σhdip is the rate of initiating successful diagnostic attempts , Ih=0 is an indicator function of HIV status ( = 1 if h = 0 , 0 otherwise ) , μbl is the non-TB mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , ρhdip is the rate of starting therapy after initiating a successful diagnostic attempt ( 1/diagnostic delay ) , and θ is the HIV incidence rate .","Thus , individuals enter this compartment by initiation of successful diagnostic attempts and exit through initiation of treatment , spontaneous cure , or death .","They acquire HIV infection at a constant rate .","This compartment contains all individuals being treated whose treatment course ends in default or failure .","Unlike the previous ( successful treatment ) compartment , diagnostic delay is not explicitly incorporated into this compartment , as to do so would prolong the duration of time until individuals who default re-enter the ‘late active’ compartment .","Inclusion of a diagnostic delay before this compartment does not materially affect results .","Individuals exit this compartment either in default after partial therapy—which has a defined probability of achieving cure despite not being completed—or failure .","Failure leads immediately to another course of treatment , which can either be successful ( i . e . , transition to the latent compartment ) or unsuccessful ( i . e . , remain in the inappropriate treatment compartment—which results in transition to the ‘previously treated’ compartment , p=1 , not shown below ) .","( 6 ) dIhdip ( t ) dt=κhdip*Ahdip ( t ) +Id=1*αsihdip*Ah0ip ( t ) +Id=2*[αsmhip*Ah0ip ( t ) +αimhip*Ah1ip ( t ) ]−Ihdip ( t ) *[μbl+Ih=0* ( μt0+νi+δf ) +Ih=1* ( μh+μth+δf ) ]−Ih=0*[θ*I0dip ( t ) ]+Ih=1*[θ*I0dip ( t ) ]where κhdip is the rate of placing individuals on ineffective treatment that does not generate acquired resistance , αsihip is the rate of placing individuals on ineffective treatment that generates INH monoresistance from drug-susceptible TB , αsmhip is the rate of placing individuals on ineffective treatment that generates MDR-TB from drug-susceptible TB , αimhip is the rate of placing individuals on ineffective treatment that generates MDR-TB from INH-monoresistant TB , μbl is the non-TB mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , δf is the rate ( 1/duration ) of ineffective therapy , and θ is the HIV incidence rate .","Thus , individuals enter this compartment through ineffective treatment from the late active compartment ( conditional on whether that treatment also generates new drug resistance ) and exit through completion of a course of ineffective therapy , spontaneous resolution , or death .","They acquire HIV infection at a constant rate .","The equations above comprise a series of 100 ordinary differential equations .","In order to generate an equilibrium population according to user specifications of TB incidence , MDR-TB prevalence , and HIV prevalence , it is necessary to solve for the roots of a system of these 100 equations , plus three additional equations to account for the user inputs .","We accomplish this using the ‘fsolve’ routine in SciPy ( www . scipy . org ) .","To solve for these three additional equations , we first match each user input to a single parameter: TB incidence to the transmission rate β , MDR-TB prevalence to the relative reduction in transmission for MDR-TB φ2 , and HIV prevalence to the HIV incidence rate θ .","We then constrain the system of equations such that the total population remains constant at 100 , 000 , and we add the following three equations to the system:dβ/dt= ( calculated TB incidence ) − ( user-defined TB incidence ) dφ2/dt= ( calculated MDR-TB prevalence ) − ( user-defined MDR-TB prevalence ) dθ/dt= ( calculated HIV prevalence ) − ( user-defined HIV prevalence ) By solving for the roots at which this system of equations equals zero , we generate an equilibrium ( steady-state ) population that also defines β , φ2 , and θ such that the user-defined targets are also met .","Additional complexity is added to account for non-equilibrium in TB incidence and MDR-TB prevalence .","We accomplish this by fitting an equilibrium defined by the user-specified target of HIV prevalence , and the user-specified ‘initial’ values of TB incidence and MDR-TB prevalence .","This equilibrium is set to be 50 years in the past; at this time , the system of equations is solved as above .","We then allow for the parameters β and φ2 to be altered from their original values ( corresponding to the equilibrium condition ) such that a second set of targets are attained after 50 years ( start of the analysis ) .","If these calculated values at the end of 50 years do not match the user-defined targets , the parameter values are changed accordingly , and the model is re-run from equilibrium until the appropriate parameter value is identified that generates the user-defined TB incidence and MDR-TB prevalence , within a relative tolerance of 0 . 05 in each variable .","These parameters may then be further modified such that they change over the analysis frame of five years ( e . g . , to describe an epidemic with increasing MDR-TB prevalence over time ) .","In the primary version of the model , this is only automated for low-incidence scenarios in which the user inputs a TB incidence of less than 50 per 100 , 000/year .","In such cases , the model generates an equilibrium population 50 years in the past with a TB incidence of 50 per 100 , 000/year and reduces the transmission parameter β until the user-defined TB incidence is achieved 50 years later ( i . e . , the start of the analytic period ) .","This accounts for the fact that most low-incidence settings have substantially more latent TB infection than would be estimated by an equilibrium population with a very low TB incidence—thereby more appropriately reflecting a higher proportion of TB due to reactivation rather than recent infection .","However , we include code ( described in the user manual below ) that allows users to define high incidence scenarios that are likewise not at equilibrium , as well as ‘emerging MDR’ scenarios in which the prevalence of MDR-TB is increasing rather than stable ."]],"string":"[\n [\n \"Infectious disease transmission models are important tools for translating the best current knowledge of the natural history and epidemiology of infectious diseases into projections of epidemiological impact ( e . g . , incidence , mortality ) and costs under alternative strategies for disease control ( Garnett et al . , 2011 ) .\",\n \"Currently , most published transmission models are either loosely calibrated to reflect global/regional outcomes or more tightly fit to specific epidemiological settings; in either case , model results may be difficult for local decision-makers in the majority of public health settings to utilize .\",\n \"Simplified models designed for in-country use by decision-makers , most notably the Spectrum suite of models supported by the Futures Institute ( Stover et al . , 2010 ) , have been used to inform decision-making in the fields of reproductive health and human immunodeficiency virus ( HIV ) for over a decade ( Stover , 2004 ) .\",\n \"Estimates from the Spectrum models are now routinely incorporated into official global and country-level estimates of HIV disease burden ( Brown et al . , 2010 ) and intervention impact ( Farnham et al . , 2013 ) .\",\n \"Other simplified models are readily available for impact projections related to non-infectious diseases , where transmission assumptions are less important ( Betz Brown et al . , 2000; Walker et al . , 2013 ) .\",\n \"However , to date , simple , user-friendly transmission models have not been widely used for decision-making related to many infectious diseases other than HIV .\",\n \"Diagnosis of active tuberculosis ( TB ) is an example of a public health intervention for which transmission models may provide guidance on both global ( Dowdy et al . , 2006; Abu-Raddad et al . , 2009 ) and country-specific levels ( Menzies et al . , 2012 ) .\",\n \"Specifically , an unprecedented number of new diagnostic strategies for active TB are now recommended by the World Health Organization ( WHO ) , including same-day microscopy ( World Health Organization , 2011 ) , microcolony-based culture techniques ( Leung et al . , 2012 ) such as the microscopic-observation drug-susceptibility ( MODS ) assay ( Moore et al . , 2006 ) , line-probe assays for drug susceptibility testing ( Bwanga et al . , 2009 ) , and Xpert MTB/RIF ( ‘Xpert’ ) , a molecular assay capable of providing results ( including rifampin resistance ) in 90 min with minimal human resource requirements ( Boehme et al . , 2010 , 2011 ) .\",\n \"TB program decision-makers must repeatedly determine when to invest in scaling up a novel diagnostic test , which test ( s ) to promote , and whether the implementation strategy should differ by epidemiological situation ( Cobelens et al . , 2012 ) .\",\n \"Without transmission models to provide locally relevant estimates of cost and impact under alternative implementation strategies , such decisions will be made without systematically considering the implications of available scientific evidence .\",\n \"To aid in this decision-making process , we created a flexible , simple modeling tool that allows non-expert users to define their local situation according to three key epidemiological parameters ( TB incidence , proportion of new TB cases that are multidrug-resistant [MDR] , and adult human immunodeficiency virus [HIV] prevalence ) and local unit costs of TB diagnosis and treatment .\",\n \"This tool then incorporates those estimates into a combined decision analysis-transmission framework to generate 5-year projections of TB incidence , mortality , and control costs for nine diagnostic strategies ( Figures 1 and 2 ) .\",\n \"These strategies are:‘Baseline’: Sputum smear microscopy for each diagnostic attempt , with liquid-media TB culture only to evaluate smear-positive cases with a history of previous TB treatment for drug resistance .\",\n \"( Cultures in all scenarios trigger drug-susceptibility testing if positive . ) ‘TB culture if previously treated’: Sputum smear microscopy used for patients without a history of TB treatment; smear plus liquid-media culture used to diagnose TB in any previously treated individual with symptoms ( regardless of smear status ) .\",\n \"‘Xpert if HIV-positive’: Xpert MTB/RIF for HIV-infected patients only , with a positive test for rifampin resistance triggering treatment for MDR-TB .\",\n \"Xpert is assumed to be deployed at the district level , such that results cannot generally be provided during the same clinical encounter ( Lawn et al . , 2012 ) .\",\n \"This strategy is conceived as a ‘best-case’ scenario for HIV-targeted TB testing: if individuals unaware of their HIV status are not tested with Xpert , this strategy will overestimate effectiveness , and if those unaware of their status are tested , it will underestimate costs .\",\n \"‘Xpert if Smear-Positive’: Xpert MTB/RIF for smear-positive patients only ( i . e . , for rapid DST ) , with a positive test for rifampin resistance triggering treatment for MDR-TB .\",\n \"‘Xpert for All’: Xpert MTB/RIF for all patients .\",\n \"‘Xpert with Culture DST Confirmation’: Xpert MTB/RIF for all patients , but treatment for MDR-TB only initiated if rifampin resistance is confirmed by culture .\",\n \"‘MODS/TLA’: Sputum smear , plus microcolony-based TB culture ( e . g . , MODS or thin-layer agar , TLA ) for all patients .\",\n \"‘Same-Day Microscopy’: Double the per-test cost of sputum smear microscopy , in exchange for the ability to provide results to patients in the same clinical encounter ( e . g . , with peripheral , unbatched reading of sputum smears ) .\",\n \"‘Same-Day Xpert’: Double the per-test cost of Xpert MTB/RIF , in exchange for the ability to provide results to patients in the same clinical encounter ( e . g . , peripheral deployment , with greater costs reflecting lower volume per machine [Vassall et al . , 2011] ) . 10 . 7554/eLife . 02565 . 003Figure 1 . Overview of user-friendly model . Users are asked , via open-source computer script or Web interface , to select one of the nine diagnostic strategies and to provide unit costs and three basic epidemiological parameters ( TB incidence , MDR-TB prevalence among new cases , and adult HIV prevalence ) .\",\n \"The selected diagnostic strategy is used to populate a decision tree that calculates\",\n \"( a ) the probability of missed diagnosis , unsuccessful treatment , and successful treatment ,\",\n \"( b ) costs , and\",\n \"( c ) diagnostic delays .\",\n \"These outputs depend on patients' TB ( yes/no , and drug susceptibility status ) , HIV , and TB treatment history status .\",\n \"The selected epidemiological parameters are then used to populate a dynamic transmission model , creating a steady-state population that reflects local TB epidemiology .\",\n \"The decision tree—which inputs user-defined unit costs—is then incorporated into the transmission model to project outcomes under the selected diagnostic scenario .\",\n \"Users can sequentially select multiple diagnostic scenarios for comparison , and the computer script ( though not the Web interface ) allows users to manipulate input parameters at their discretion . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 00310 . 7554/eLife . 02565 . 004Figure 2 . Transmission model of TB diagnosis . Boxes represent sub-populations in the model , and arrows represent rates of movement between those sub-populations .\",\n \"Parallel structures exist for:\",\n \"( a ) HIV-infected vs HIV-uninfected;\",\n \"( b ) never-treated vs previously treated ( for TB ) ; and\",\n \"( c ) among TB-infected individuals , drug-susceptible vs isoniazid-monoresistant vs rifampin-resistant ( including MDR ) .\",\n \"‘Pre-diagnostic’ TB refers to individuals who are infectious but have not yet begun to seek care .\",\n \"Mortality occurs from all sub-populations ( not shown ) , and at a higher rate among those with HIV and active TB . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 004 For purposes of illustration , we evaluated each of these diagnostic strategies in four emblematic epidemiological settings , defined by TB incidence , MDR-TB prevalence among new cases , and adult HIV prevalence:‘Reference/High-Incidence Setting’ ( e . g . , Southeast Asia ) : TB incidence 250 per 100 , 000/year ( twice the global incidence [World Health Organization , 2012] ) , MDR-TB prevalence of 3 . 7% in new TB cases ( the estimated global prevalence [World Health Organization , 2012] ) , adult HIV prevalence of 0·83% ( the estimated global prevalence [UNAIDS , 2012] ) ;‘Low-Incidence Setting’ ( e . g . , United States , Western Europe ) : TB incidence at entry of 8 . 9 per 100 , 000/year and declining , with similar MDR-TB ( as a proportion of new cases ) and HIV prevalence as above;‘High MDR Setting’ ( e . g . , former Soviet Union ) : TB incidence 100 per 100 , 000/year , MDR-TB prevalence of 10% in new TB cases , adult HIV prevalence of 0 . 83%; and‘High HIV Setting’ ( e . g . , sub-Saharan Africa ) : Adult HIV prevalence of 20% , TB incidence 500 per 100 , 000 , and MDR-TB prevalence among new cases of 3 . 7% .\",\n \"In each setting , we used a uniform set of costs for purposes of comparison ( Table 1 ) .\",\n \"Although these four settings form the basis of the results presented here , decision-makers can use the open-source model program ( included as Supplementary file 1 , written in the open-source programming language Python , Version 2 . 7 , www . python . org ) to re-define any parameter in Table 1 according to their best local knowledge; a manual for doing so is also included as Supplementary file 2 .\",\n \"Capacity to create non-equilibrium settings ( e . g . , declining TB incidence , increasing MDR-TB ) is included .\",\n \"We also provide a web-based interface ( flexdx . modeltb . org ) that allows users to input local TB incidence , MDR-TB prevalence , HIV prevalence , and unit costs; this interface provides customized results without the requirement to manipulate programming code .\",\n \"The program corresponding to the web version is also available on a public repository ( https://github . com/JJPennington/FlexDx-TB-Web-Django ) . 10 . 7554/eLife . 02565 . 005Table 1 . Model input parameters*DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 005ParameterValueReference ( s ) /RationaleTB and HIV TransmissionTransmission rate , per smear-positive/highly infectious person-yearCalibrated to user-defined TB incidence†Proportional reduction in per-case transmission rate , MDR-TBCalibrated to user-defined MDR-TB prevalence†Proportional reduction in fitness , isoniazid-monoresistant TB25% of MDR-TB reductionAssumptionHIV incidence rate , per yearCalibrated to user-defined HIV prevalence†Relative transmission rate from smear-negative/less infectious TB0 . 22 ( Behr et al . , 1999 ) Proportion of pulmonary TB that is smear-positive/highly infectious HIV-negative0 . 63 ( Steingart et al . , 2006a; Steingart et al . , 2006b ) HIV-infected0 . 50 ( Getahun et al . , 2007 ) TB ProgressionEndogenous reactivation rate HIV-negative0 . 0005/year ( Horsburgh et al . , 2010 ) HIV-infected0 . 05/year ( Antonucci et al . , 1995 ) Proportion of recent infections resulting in rapid progression HIV-negative0 . 14 ( Vynnycky and Fine , 1997; Dye et al . , 1998 ) HIV-infected0 . 470 . 75 without ART , ( Daley et al . , 1992 ) 75% reduction if on ART , ( Williams et al . , 2010 ) 50% ART coverageReduction in TB rapid progression probability due to latent TB infection ( HIV-negative only ) 0 . 79 ( Andrews et al . , 2012 ) TB Mortality and ResolutionLife expectancy at age 1555 years ( World Bank , 2012 ) Annual mortality from HIV0 . 05/year ( UNAIDS , 2012 ) Annual mortality from TB HIV-negative , smear-positive/highly infectious0 . 23/year ( Tiemersma et al . , 2011 ) HIV-negative , smear-negative/less infectious0 . 07/year ( Tiemersma et al . , 2011 ) HIV-infected1 . 0/year ( Corbett et al . , 2003; Corbett et al . , 2007; Wood et al . , 2007 ) Rate of spontaneous TB resolution ( HIV-negative only ) Smear-positive/highly infectious0 . 1/year ( Tiemersma et al . , 2011 ) Smear-negative/less infectious0 . 27/year ( Tiemersma et al . , 2011 ) TB Treatment Outcomes and Emergence of Drug ResistanceProbability of failure or relapse ( within 1 year ) Drug-susceptible0 . 04 ( World Health Organization , 2012 ) INH-monoresistant , first-line therapy0 . 21 ( Menzies et al . , 2009b ) INH-monoresistant , retreatment or 2nd-line0 . 16 ( Menzies et al . , 2009b ) MDR-TB , first-line or retreatment0 . 50 ( Espinal et al . , 2000 ) MDR-TB , second-line therapy0 . 30 ( World Health Organization , 2010 ) Proportion of one-year recurrence due to failure Drug-susceptible0 . 14 ( Lew et al . , 2008 ) INH-monoresistant0 . 33 MDR-TB0 . 56Probability of acquired drug resistance ( per treatment course ) Susceptible becoming INH-monoresistant0 . 001 ( Menzies et al . , 2009a; Menzies et al . , 2009b ) Susceptible becoming MDR-TB0 . 002 INH-monoresistant becoming MDR-TB0 . 045 If treated with 2 effective drugs for >6 mos0 . 017Behavioral ParametersInfectious months before starting to seek care HIV-negative9 months ( Dowdy et al . , 2013 ) HIV-infected1 month ( Corbett et al . , 2004 ) Diagnostic frequency while seeking care5 . 0/year ( Storla et al . , 2008; Sreeramareddy et al . , 2009 ) Probability of treatment in a TB patient whose microbiological test is negative0 . 25 ( Wilkinson et al . , 2000; Dowdy et al . , 2008 ) Loss to follow-up between diagnostic presentation and treatment initiation Sputum smear or GXP ( not same-day ) 0 . 15 ( MacPherson et al . , 2014 ) Culture ( microcolony or commercial liquid ) 0 . 25 ( Dowdy et al . , 2008 ) Same-day diagnosis0AssumptionDiagnostic AccuracySensitivity for smear-negative/less-infectious TB Sputum smear microscopy0 Xpert MTB/RIF0 . 72 ( Brownell et al . , 2012 ) Culture ( microcolony or commercial liquid ) 0 . 85 ( Cruciani et al . , 2004; Leung et al . , 2012 ) Specificity for TB ( Steingart et al . , 2006; Boehme et al . , 2011; Leung et al . , 2012 ) Sputum smear microscopy0 . 98 Xpert MTB/RIF0 . 98 Microcolony culture0 . 98Sensitivity for drug resistance ( if TB detected ) Microcolony culture ( rifampin and isoniazid ) 0 . 98 ( Minion et al . , 2010 ) Xpert MTB/RIF ( rifampin only ) 0 . 94 ( Boehme et al . , 2011 ) Specificity for drug resistance ( if TB detected ) Microcolony culture ( isoniazid ) 0 . 96 ( Minion et al . , 2010 ) Microcolony culture ( rifampin ) 0 . 99 ( Minion et al . , 2010 ) Xpert MTB/RIF ( rifampin ) 0 . 98 ( Boehme et al . , 2011 ) Diagnostic Delay and non-TB Care-SeekingDays from presentation to treatment initiation Sputum smear or Xpert MTB/RIF7 daysAssume 1 week Microcolony or commercial liquid culture30 days ( Boehme et al . , 2011 ) Months of therapy before a failing regimen will be changed , or before default and recurrence6 monthsAssumptionAnnual rate of diagnostic evaluation for TB , among people who do not have active TB0 . 01/year10% of suspects have TB , high-incidence settingCost Parameters ( user-defined; values below for comparison purposes only ) Per-patient cost of TB therapy First-lineUS$500User-defined RetreatmentUS$1000 ( Vassall et al . , 2011 ) Second-line/MDRUS$5000 ( Vassall et al . , 2011 ) Outpatient visit ( diagnosis or follow-up ) US$10 ( Vassall et al . , 2011 ) Per-test cost: Sputum smearUS$2 ( Vassall et al . , 2011 ) Same-day sputum smearUS$10Assumption Xpert MTB/RIFUS$15 ( Vassall et al . , 2011 ) Same-day Xpert MTB/RIFUS$30Assumption Microcolony culture ( with DST ) US$5 ( Solari et al . , 2011 ) Commercial liquid-media cultureUS$20 ( Vassall et al . , 2011 ) Commercial liquid-media culture + DSTUS$40 ( Vassall et al . , 2011 ) *In the actual model program ( Supplementary file 1 ) , users can change any parameter based on local values .\",\n \"†For reference , the transmission rate ( in infections per person-year during diagnosis-seeking active TB ) is 36 . 9 in the reference scenario , 14 . 0 in the low-incidence scenario , 25 . 4 in the high MDR scenario , and 12 . 9 in the high HIV scenario .\",\n \"Corresponding proportional reductions in MDR-TB transmission rate are 0 . 23 , 0 . 23 , 0 . 21 , and 0 . 19; and HIV incidence estimates ( per 1000 adult person-years ) are 0 . 7 , 0 . 6 , 0 . 6 , and 18 . 9 .\"\n ],\n [\n \"To validate the model , we compared selected model outcomes to published global estimates .\",\n \"In the high-incidence setting , our model estimated TB mortality at 14% of incidence ( 95% uncertainty range: 7–20% , WHO global estimate 14% [World Health Organization , 2012] ) , HIV-associated TB at 13% of all incident TB ( 95% uncertainty range: 4–14% , global estimate 13% [World Health Organization , 2012] ) , previously treated cases at 13% of all incident cases ( 95% uncertainty range: 9–34% , global estimate 14% [World Health Organization , 2012] ) , and duration of TB disease at 1 . 2 years ( 95% uncertainty range 0 . 8–2 . 1 , global estimate 1 . 4 years [World Health Organization , 2012] ) .\",\n \"Our model estimated that MDR-TB prevalence in previously treated cases was 15 . 4% ( WHO estimate 20% [World Health Organization , 2012] ) , but unlike our model , WHO notifications often count failure and recurrence after default ( in the same person ) as two separate cases .\",\n \"At steady-state in the model , 80% of incident TB was due to recent infection rather than reactivation .\",\n \"Figure 3 shows the projected incremental 5-year cost and impact of each of nine selected diagnostic strategies ( described in greater detail in the ‘Materials and methods’ section ) , in the high-incidence scenario .\",\n \"In general , both the cost and impact of targeted strategies ( culture for retreatment , Xpert for HIV-positive , Xpert for smear-positive ) on incidence were small relative to broader diagnostic strategies ( Xpert for all , MODS/TLA , same-day Xpert ) .\",\n \"The incremental cost-effectiveness , in terms of cost per case averted ( i . e . , slope of the line from origin to each point in Figure 3 ) , was similar across all strategies with the exception of same-day smear , which was cost-saving and had greater effectiveness than the baseline .\",\n \"Same-day microscopy remained cost-saving by year 5 as long as same-day results could be provided at less than five times the per-smear cost of routine results ( i . e . , <$10/test ) .\",\n \"Among the targeted strategies , Xpert for smear-positives was the most expensive but had the greatest impact on MDR-TB cases averted , whereas Xpert for HIV-infected individuals and culture for retreatment cases offered smaller gains at lower cost .\",\n \"Among the broad strategies , culture confirmation of rifampin-resistant tests on Xpert saved costs with little reduction in effectiveness .\",\n \"There was little difference between culture-confirmed Xpert and MODS/TLA , and same-day Xpert was the most expensive and most effective strategy . 10 . 7554/eLife . 02565 . 006Figure 3 . Incremental 5-year cost and impact of TB diagnostic strategies , high-incidence setting . Shown are cumulative projected 5-year costs and impact ( averted TB cases [panel A] or MDR-TB cases [panel B] ) of each diagnostic strategy described in the Introduction , incremental to the baseline strategy , per 100 , 000 population .\",\n \"Strategies with greater impact appear to the right on the x-axis; more costly strategies appear higher on the y-axis .\",\n \"The same-day smear strategy is cost-saving but shown at an incremental cost of $0 for simplicity . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 006 The projected impacts and costs of the nine diagnostic strategies relative to the baseline strategy , in each of four selected epidemiological settings , are shown in Figure\",\n \"4 . In all four settings , the ranking of diagnostic strategies remained similar for all outcomes , although the cost of broader diagnostic strategies relative to targeted strategies fell substantially over 5 years in higher-incidence settings as the broader strategies generated declines in TB incidence .\",\n \"In the low-incidence setting , where a higher proportion of TB treatments are false-positive and more incident TB is also due to reactivation ( 60% of all new cases ) , the relative cost of improved TB diagnosis was the highest , while the relative impact was the least .\",\n \"In the high HIV setting , the impact of diagnostic interventions on TB incidence was diminished relative to the high-incidence setting , though the impact on TB mortality was similar .\",\n \"Additionally in this setting , the Xpert for HIV-positive strategy was substantially more costly , but also more effective , than in other settings . 10 . 7554/eLife . 02565 . 007Figure 4 . Relative impact of diagnostic strategies in emblematic settings . Shown are projected changes in TB incidence , MDR-TB incidence , TB mortality , and costs ( in Year 1 and Year 5 after immediate implementation ) , relative to baseline ( Strategy 1 ) after implementing each of the diagnostic strategies described in the text .\",\n \"Epidemiological outcomes are measured at the end of Year\",\n \"5 . Panel A ( high incidence ) shows a setting with TB incidence of 250 per 100 , 000/year , stable MDR-TB prevalence of 3 . 7% among new cases , adult HIV prevalence of 0 . 83% , and cost of $500 to treat one case of TB with first-line therapy .\",\n \"In panel B ( low incidence ) , the TB incidence is reduced to 8 . 3 per 100 , 000/year ( implemented by gradual decline in incidence over 50 years ) .\",\n \"In panel C ( high MDR ) , MDR-TB prevalence among new cases is set at 3 . 7% in the beginning of year 1 , increasing to 10 . 7% by the end of year\",\n \"5 . In panel D ( high HIV ) , adult HIV prevalence is set to 20% and TB incidence is set to 500 per 100 , 000/year . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 007 The impact of the ‘Xpert for all’ strategy on TB incidence ( selected a priori as the primary outcome for sensitivity analysis ) was most sensitive to three parameters , both in terms of absolute effects on impact estimates and partial rank correlation coefficients .\",\n \"These three parameters were: ( 1 ) the proportion of TB patients who would be empirically treated even if microbiologic testing yielded a negative result ( ‘empiric treatment proportion’ ) , ( 2 ) duration of infectiousness before seeking care ( ‘pre-diagnostic delay’ ) , and ( 3 ) rate of reactivation from latent infection to active disease ( ‘reactivation rate’ ) .\",\n \"For this latter parameter , in-depth investigation revealed that the key determinant was not the reactivation rate per se , but rather the proportion of active TB representing recent vs remote infection .\",\n \"If the empiric treatment proportion was increased from 25% to 37 . 5% , the projected reduction in TB incidence fell from 20% to 13% .\",\n \"By contrast , when only 12 . 5% of false-negative patients were started on therapy , ‘Xpert for all’ achieved a 31% reduction in incidence .\",\n \"Corresponding reductions in incidence with ‘Xpert for all’ ( 20% at baseline ) included: 27% if pre-diagnostic delay was shortened from 9 months to 4 . 5 months , 14% if pre-diagnostic delay was lengthened to 13 . 5 months , 15% if the reactivation rate was doubled from 0 . 05% to 0 . 1% per year , and 23% if reactivation was halved to 0 . 025% per year .\",\n \"The projected 5-year reduction in incidence for this strategy did not fall outside the range of 12–25% under variation of any other model parameter , up to 50% of that parameter's baseline value ( Table 1 ) .\",\n \"Both costs and incremental costs were more sensitive to the cost of TB treatment than the cost of diagnostics .\",\n \"For example , doubling the cost of first-line therapy ( from $500 to $1000 per person ) augmented the incremental year 1 costs for ‘Xpert for all’ from a 57% increase over baseline to a 77% increase , and doubling the cost of MDR therapy ( from $5000 to $10 , 000 per person ) generated an 86% increase in incremental year 1 costs , whereas doubling the unit cost of Xpert ( from $15 to $30 ) only resulted in a 72% increase .\",\n \"Unit costs other than those for first-line treatment , MDR treatment , and the diagnostic modality under study in each scenario were not important determinants of incremental costs .\"\n ],\n [\n \"To date , most transmission models of infectious disease control interventions present results that are not directly usable by decision makers because they are not customizable to local conditions .\",\n \"We present a flexible , user-friendly model of TB diagnosis and transmission that allows users without modeling expertise to define an epidemiologic setting ( according to TB incidence , MDR-TB prevalence , and HIV prevalence ) and unit costs , evaluating various diagnostic strategies in that setting in terms of population-level costs and impact .\",\n \"While this model cannot precisely replicate the epidemiological situation in any given location , it applies a standardized methodology across a wide range of settings , thereby illustrating important interactions between epidemiological parameters and projected impact .\",\n \"We provide both a web interface for rapid calculations and full model code whereby users can change any model parameter for ‘personalized’ sensitivity analysis .\",\n \"Our model results suggest that the rank-ordering of diagnostic strategies may be relatively stable across epidemiological settings , but that the actual population-level costs and impact differ dramatically .\",\n \"This model also serves as an example of how epidemiologists can provide decision-makers across a wide variety of local settings with rapid access to customizable ‘first-pass’ projections of cost and impact from transmission models without the need to construct tightly fitted models to represent all epidemiological settings .\",\n \"Our results provide important guidance to TB decision-makers .\",\n \"Specifically , in settings where little additional up-front investment is possible ( <25% increase in TB control budget ) , same-day microscopy has the greatest impact on TB incidence with no increase in overall cost at 5 years , provided that same-day microscopy can be feasibly delivered for less than $10 per patient .\",\n \"For settings in which containing MDR-TB is the most important consideration , Xpert for smear-positives has the greatest effectiveness for this outcome , but at substantial price and very little impact on overall TB incidence and mortality .\",\n \"Xpert for HIV-positives and culture for retreatment cases both offer meaningful , albeit small , gains; the cost and impact of these strategies are the highest in HIV-endemic settings .\",\n \"In settings where more initial investment is possible ( about 50% increase in TB control budget ) , broader scale-up of either Xpert or MODS/TLA for all TB diagnosis can offer substantially greater benefits , both in terms of reduced incidence ( often 10 times more TB cases averted than with the more narrowly-targeted strategies above ) and long-term costs ( in that the incremental cost of these strategies declines greatly by Year 5 ) .\",\n \"In general , culture confirmation of positive Xpert results before committing to a course of second-line therapy is preferred .\",\n \"Finally , where the greatest impact is sought , combination of Xpert for all plus infrastructure for same-day diagnosis achieves this aim in all settings , but at the highest cost .\",\n \"Although this model represents a highly simplified framework , it compares well to other global estimates to which it was not fit ( e . g . , WHO estimates of TB mortality , previously treated TB , HIV-associated TB , and MDR-TB prevalence among previously treated cases ) .\",\n \"Its results are also similar to those of other mathematical models of TB diagnosis that are fit to specific locations .\",\n \"For example , Menzies et al . ( 2012 ) modeled scale-up of Xpert for people living with HIV in southern Africa , estimating a 6% reduction in incidence , 21% reduction in TB mortality , 25% reduction in MDR-TB incidence that declines to <10% over a longer time frame , and 40% increase in costs ( not including costs of antiretroviral therapy ) .\",\n \"Our corresponding estimates for the Xpert for HIV-positive strategy in the high-HIV setting are 5% reduction in incidence , 25% reduction in TB mortality , 7% reduction in MDR-TB incidence , and increase in costs from 41% ( year 1 ) to 28% ( year 5 ) .\",\n \"For purposes of deciding between alternative strategies of scaling up TB diagnostics , the estimates from these two models are likely to provide similar guidance .\",\n \"For any such simplified transmission model to have an impact on decision-making , it is important to consider who the eventual users of such a model might be , and to develop the model in consultation with those groups .\",\n \"In this case , we identified a number of potential end-user organizations including technical assistance agencies ( e . g . , KNCV Tuberculosis Foundation ) , funding bodies ( e . g . , Global Fund for AIDS , Tuberculosis , and Malaria ) , non-governmental organizations ( e . g . , Medecins Sans Frontiers ) , National Tuberculosis Programs , and regional offices of the World Health Organization .\",\n \"We then invited representatives of these organizations to attend a 1-day workshop ( April 2014 , The Hague , The Netherlands ) describing methods and challenges related to modeling of TB diagnostics in general , including this transmission model in particular .\",\n \"We developed ‘hands-on’ exercises for participants to better learn the model and solicited specific feedback , which is being incorporated into the model structure and web interface in ongoing fashion .\",\n \"We next intend to develop a series of informal ‘case studies’ whereby the use of this model for in-country decision-making can be demonstrated and disseminated .\",\n \"As with any modeling analysis , this research has important limitations .\",\n \"In order to provide sufficient flexibility and generalizability , we make a number of strong assumptions that include a constant population , homogeneous mixing , no change in parameter values over time , and simplistic incorporation of HIV and drug resistance .\",\n \"Without making such simplifying assumptions , it is impossible to deliver a flexible modeling framework that can generate transparent , customizable , rapid results ( i . e . , without complex statistical fitting to each individual epidemiological scenario ) .\",\n \"This model can replicate user-defined TB incidence , MDR-TB prevalence , and HIV incidence but does not describe the breakdown of these values in key subpopulations ( e . g . , congregate settings or geographical ‘hotspots’ ) , nor does it incorporate operational aspects of the TB diagnostic system in any one setting .\",\n \"Thus , this model does not ameliorate the need for more detailed models in settings where precise estimates are needed; rather it provides access to ‘first-pass’ estimates in settings ( i . e . , the vast majority of local decision-makers ) where such tightly calibrated model projections are not available .\",\n \"This model focuses on transmission dynamics and thus does not include pediatric TB and extrapulmonary TB; these largely non-infectious disease manifestations remain very important components of the TB epidemic that are not captured here .\",\n \"We validate our model against global estimates and other models; ideally , further validation would be performed over time using field data across a wide variety of settings .\",\n \"Our model allows users to define three key epidemiological parameters ( as well as other model assumptions within the program ) , but data to inform even these three parameters—as well as unit costs—are unlikely to be available on sub-national levels .\",\n \"As a result , users wishing to adapt the model to a smaller geographic scale will need to perform additional data-gathering exercises to inform these estimates if they wish to maximize the utility of the model .\",\n \"Nevertheless , even if high-quality data are not available at the local level , this model allows decision-makers to estimate epidemiological and economic values according to reasonable assumptions ( e . g . , comparison of TB notification rates or budget line-items to those in the published literature or at the national level ) , vary those assumptions in real-time , and obtain corresponding projections of comparative impact that incorporate the best available current data on epidemiological or natural history parameters ( e . g . , TB progression and reactivation ) .\",\n \"Future efforts might also provide flexibility to specify operational characteristics ( e . g . , health system capacity ) as well .\",\n \"A final concern is that , by providing users the ability to specify TB incidence , MDR-TB prevalence , and adult HIV prevalence , a number of scenarios can be created ( e . g . , low TB incidence and very high adult HIV prevalence ) that are not epidemiologically realistic .\",\n \"Although there is danger in allowing uninformed users to make projections for such scenarios ( and the model will reject or alert users to highly implausible values ) , we believe that this risk is outweighed by the benefit of providing full flexibility to model epidemiological scenarios ( e . g . , sub-district level data ) that will never be captured by a limited number of closely-calibrated TB transmission models .\",\n \"In summary , we have created a flexible modeling framework that allows users without modeling expertise to generate simulated populations with locally relevant values for TB incidence , MDR-TB prevalence , adult HIV prevalence , and TB treatment costs .\",\n \"By comparing an array of diagnostic options across emblematic epidemiological scenarios , we provide guidance to decision-makers who seek to ascertain the optimal diagnostic strategy to achieve their selected disease control targets , and to do so using a standardized methodology .\",\n \"Success in the fight against infectious disease generally , and TB specifically , depends on our ability to place global knowledge in the hands of local decision-makers , enabling them to choose those interventions that are likely to have the greatest impact , given existing resources and local epidemiological realities .\",\n \"This flexible modeling framework of diagnostic interventions takes an important step in that direction .\"\n ],\n [\n \"Using previously published models of TB diagnostics as a guide ( Dye et al . , 1998; Abu-Raddad et al . , 2009; Menzies et al . , 2012; Dowdy et al . , 2013 ) , we constructed a transmission model of TB using ordinary differential equations .\",\n \"This model categorizes patients according to HIV status ( positive or negative ) , TB treatment status ( never treated or previously treated ) , TB disease status ( as shown in Figure 2 ) , and among those who are infected with TB , drug resistance status ( susceptible , isoniazid-monoresistant , and rifampin-resistant including MDR ) , and level of infectiousness ( smear-negative/less-infectious and smear-positive/highly infectious ) .\",\n \"Individuals enter the model at age 15 , and TB with no pulmonary component ( i . e . , not infectious ) is not included .\",\n \"For purposes of transparent communication , we chose a population size of 100 , 000 ( to match standard reporting of TB outcomes ) and assumed a constant population with no net population growth or immigration/emigration .\",\n \"After constructing the transmission framework , we used decision analysis to estimate\",\n \"( a ) the probability of each diagnostic outcome;\",\n \"( b ) the diagnostic delay; and\",\n \"( c ) the cost of TB diagnosis and treatment under each of the nine diagnostic strategies , assuming immediate implementation at the beginning of a given year ( ‘Year 1’ ) .\",\n \"Two separate authors ( DWD and PJD ) independently coded the model; these models gave comparable results .\",\n \"After setting probabilities and costs under each diagnostic strategy , we then created a flexible modeling structure capable of generating epidemiological scenarios as a function of three variables , which the user specifies: HIV prevalence ( assuming a global mean level of antiretroviral therapy coverage ) , TB incidence , and MDR-TB prevalence among new TB cases .\",\n \"We accomplished this by allowing three key parameters to vary across model scenarios: annual HIV incidence , rate of TB transmission per smear-positive/highly infectious person-year , and relative per-case transmission rate of rifampin-resistant TB .\",\n \"We also allow users to specify all relevant unit costs for TB diagnosis and treatment; other model parameters were estimated from existing literature ( Table 1 ) .\",\n \"We then created a program that numerically generates a unique steady-state population meeting the user-defined values; this population serves as the baseline strategy ( Strategy 1 above ) at the beginning of the time frame under evaluation .\",\n \"The program has flexibility to create its steady-state population 50 years in the past , allowing it to replicate the protracted , slow declines in TB incidence as seen in many lower-incidence settings; this is done automatically for any scenario with a target TB incidence less than 50 per 100 , 000/year .\",\n \"The mathematical model consists of the following TB compartments:Uhp , UninfectedLhdp , Latently infectedEhdp , Early active ( infectious status i = 0 ) Ahdip , Late activePhdip , Active ‘pre-treatment’: diagnosis in progress , will lead to appropriate therapyIhdip , Active ‘inappropriate treatment’: receiving therapy that ends in default or failure In these compartments , the subscript h refers to HIV status ( h = 0 if HIV-uninfected , 1 if HIV-infected ) , d refers to drug resistance status ( d = 0 if drug-susceptible , 1 if isoniazid [INH]-monoresistant , and 2 if multidrug-resistant [MDR] ) , i refers to infectious status ( i = 0 if smear-negative/less infectious and 1 if smear-positive/highly infectious ) , and p refers to previous treatment status ( p=0 if never treated , 1 if previously treated ) .\",\n \"Infectious status can be conceptualized as an individual's sputum smear status , if two smears were to be performed in a quality-assured laboratory at any given point in time .\",\n \"The model assumes an adult population of stable size with no immigration or emigration: the number of individuals entering the uninfected compartment U is set as equal to the number who die ( whether from TB or other causes ) from all other compartments .\",\n \"Pediatric and purely extrapulmonary TB are not explicitly considered because the diagnostic considerations for these manifestations are different .\",\n \"In the short-term , however , to the extent that these forms of TB are non-infectious and equally fatal as adult pulmonary TB , their effects on TB incidence and mortality may be approximated by dividing the model's projected incidence and mortality by ( 1 − proportion of TB that is not adult pulmonary ) , to obtain a new incidence/mortality estimate .\",\n \"Thus , if 20% of all TB in a given location is extrapulmonary or paediatric , the rough projected total TB incidence would be ( projected TB incidence ) / ( 0 . 8 ) .\",\n \"In this model , we consider latent TB infection to be asymptomatic and non-infectious , with a constant rate of reactivation and ongoing risk of exogenous reinfection leading to active TB; individuals successfully treated for TB are assumed to return to this compartment upon initiation of effective therapy ( i . e . , therapy that will result in completion , with no relapse for 2 years ) .\",\n \"Upon developing active TB , individuals enter a ‘pre-diagnostic’ phase that is characterized by a low level of infectiousness and mortality ( equivalent to smear-negative TB ) and during which individuals do not actively seek diagnosis .\",\n \"The duration of this phase ( 9 months ) was selected a priori based on an existing model ( Dowdy et al . , 2013 ) in which the total duration of disease after incorporating this phase reflected the global ratio of prevalence to incidence , as estimated by the World Health Organization .\",\n \"We compared the total duration of disease to this ratio as part of our model validation and assumed that this ‘pre-diagnostic’ phase is much shorter for HIV-infected vs HIV-uninfected individuals .\",\n \"Upon completing this ‘pre-diagnostic’ phase , individuals progress to a diagnosis-seeking phase of active disease , which is characterized by separation into highly infectious ( ‘smear-positive’ ) and less infectious ( ‘smear-negative’ ) compartments .\",\n \"Among HIV-uninfected individuals , the highly infectious compartment also carries higher mortality risk .\",\n \"Diagnosis-seeking active TB implies active seeking of diagnosis at a defined rate; the probability that any single diagnostic attempt will result in effective therapy is calculated as a function of diagnostic sensitivity , probability of empiric therapy ( i . e . , without bacteriological confirmation ) , prior treatment status , and losses to follow-up , as described below .\",\n \"Each diagnostic attempt , if successful , leads either to effective therapy ( which is initiated after a defined diagnostic delay ) or to ineffective therapy ( defined as leading to failure or default ) .\",\n \"In order to focus on differences between the nine selected strategies above in a tractable framework , we subsumed all other diagnostic tests and procedures ( e . g . , chest X-ray , antibiotic trials ) as a probability of non-microbiologic diagnosis , without attempting to specify the associated cost or diagnostic delay .\",\n \"Once effective therapy is initiated , it is assumed to immediately render the individual non-infectious , with no residual risk of mortality .\",\n \"Upon initiation of ineffective therapy , individuals are assumed to remain infectious ( at the ‘smear-negative’/less infectious level ) for a defined period before either failing ( followed by another round of therapy , which can be either appropriate/curative or ineffective ) or default .\",\n \"Reasoning that default will occur , on average , at the midpoint between receipt of fully-ineffective and fully-effective therapy , half of defaulters are presumed to develop recurrent TB ( which is assumed to occur immediately ) , while the other half return to the latent TB compartment ( from which reactivation or reinfection remains possible ) .\",\n \"All individuals who relapse within 1 year are included as failures; thus , no specific parameter for relapse is incorporated .\",\n \"Individuals who are effectively treated , or whose disease is contained without therapy , return to the latent compartment following the convention of other TB models ( Dye et al . , 1998; Abu-Raddad et al . , 2009 ) .\",\n \"As the goal of this model is to focus on TB-related interventions , HIV is modeled as occurring at a defined annual incidence , calibrated to achieve a given user-defined prevalence at baseline .\",\n \"We do not explicitly model HIV infection in dynamic fashion ( i . e . , the HIV incidence rate does not depend on the number of HIV-infected individuals in the model ) .\",\n \"HIV infection is assumed to affect all parameters related to TB disease , including the level of immune protection afforded by latent infection ( assumed zero if HIV-infected ) , mortality rate ( increased ) , rate of reactivation from latent TB ( increased ) , duration of ‘pre-diagnostic’ TB ( decreased ) , and risk of ‘primary’ progression to active disease upon infection ( increased ) .\",\n \"For purposes of maintaining a simple model structure , we do not explicitly model CD4 counts or antiretroviral therapy ( ART ) , but instead assume the global average of ART coverage , as estimated by UNAIDS ( 2012 ) .\",\n \"We weight all HIV-related parameters according to this estimated probability of ART receipt; this probability can be modified by users .\",\n \"We assume infection with TB strains of three different drug resistance levels: fully susceptible , INH-monoresistant , and MDR .\",\n \"Dual infection with multiple strains is not considered in this model , but superinfection ( i . e . , reinfection of a latently infected individual with a different strain , resulting in primary progression to disease with the reinfecting strain ) is allowed , as is acquisition of resistance ( i . e . , change of strain from more susceptible to less susceptible ) as a result of therapy .\",\n \"A key consideration with any TB diagnostic strategy is the role of the diagnostic test as applied to individuals who do not have TB ( i . e . , specificity ) .\",\n \"We included this element by considering that a small proportion of the population without TB would present with TB-like symptoms each year .\",\n \"This proportion ( selected such that 10% of individuals being evaluated in the high-incidence setting actually have underlying TB ) remains constant across all scenarios , such that the pre-test probability of TB is higher in settings of high TB incidence , and declines over time as successful TB control strategies are employed .\",\n \"Individuals without TB who are ( inappropriately ) treated for TB incur costs of TB therapy and are also marked as ‘previously treated’ for purposes of diagnostic evaluation in the future .\",\n \"Economic parameters are estimated using a unit-costing approach , whereby the unit cost of a TB diagnostic attempt and a TB treatment course ( separately for first-line , category two , and second-line therapy ) is enumerated under each scenario , and this cost is multiplied by the number of diagnostic attempts and treatment courses performed .\",\n \"For simplicity , we adopt the perspective of a TB control program for our costing; additional costs of HIV care and general health services ( e . g . , hospitalization ) are not included .\",\n \"The decision tree below is used to estimate the cost per diagnostic attempt or treatment course , conditional on an individual's HIV , drug resistance , and previous treatment status .\",\n \"Individuals who default or die on therapy are assumed to incur half the cost of a treatment course .\",\n \"Given the short time horizon ( 5 years ) , the focus on costs and outcomes ( rather than cost-effectiveness per se ) , and the desire to compare costs in year 1 and at the end of year 5 in equivalent terms , we did not discount future costs or outcomes for this analysis .\",\n \"All costs are reported in US dollars , assuming the year of costs that is specified by the user .\",\n \"Under each scenario , we use decision analysis to ascertain the following quantities related to each diagnostic attempt:Probability of receiving successful treatment . Probability of receiving ineffective treatment ( resulting in failure or default , including the probability of acquired resistance ) .\",\n \"Cost of treatment ( conditional on whether treatment is successful or ineffective ) .\",\n \"Cost of diagnosis . Diagnostic delay incurred before treatment initiated .\",\n \"These quantities are calculated conditional on each patient's infectious ( smear ) status , drug resistance status , HIV status , and prior treatment status .\",\n \"These probabilities are calculated for each diagnostic attempt , with the result fed back into the transmission model for purposes of appropriately allocating flows between compartments .\",\n \"Inputs into the decision model include the probabilities of failure/recurrence , probability of empiric therapy , loss to follow-up before treatment , diagnostic accuracy , diagnostic delay , and economic parameters as shown in Table 1 .\",\n \"Outputs from the decision tree appear as parameters in the model , as described in Table 2 and the following equations . 10 . 7554/eLife . 02565 . 008Table 2 . Model parameters and symbolic representationsDOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 008ParameterRepresentationBaseline value ( see Table 1 ) Transmission rate ( transmission events per highly infectious person-year ) βCalibrated to TB incidenceProportional reduction in per-case transmission rate Drug-susceptible TBφ01 . 0 Isoniazid-monoresistant TBφ125%* of φ2 MDR-TBφ2CalibratedHIV incidence rate , per yearθCalibrated to HIV prevalenceRelative transmission rate from smear-negative/less infectious TBζ0 . 22Proportion of pulmonary TB that is smear-positive/highly infectious HIV-negativeψ00 . 63 HIV-infectedψ10 . 50Endogenous reactivation rate , per year HIV-negativeε00 . 005 HIV-infectedε10 . 05Proportion of recent infections resulting in rapid progression HIV-negativeπ00 . 14 HIV-infectedπ10 . 47Reduction in TB rapid progression probability due to latent TB infection HIV-negativeι0 . 79 HIV-infectedNot included0Baseline mortality rate , per yearμbl1/55 = 0 . 018Additional HIV-related mortality rate , per yearμh0 . 05Additional untreated TB-related mortality rate , per year HIV-negative , smear-positive/highly infectiousμt10 . 23 HIV-negative , smear-negative/less infectiousμt00 . 07 HIV-infectedμth1 . 0Rate of spontaneous TB resolution , per year Smear-positive/highly infectiousν10 . 1 Smear-negative/less infectiousν00 . 27 HIV-infectedNot included0Rate of starting diagnosis-seeking in active TB , per year HIV-negativeδe01 . 33 ( 9 months ) HIV-infectedδe112 ( 1/month ) Rate of progression: ineffective therapy to repeat therapy ( failure ) or active TB ( relapse ) , per yearδf6/12 = 0 . 5Rate of diagnostic evaluation for TB , per year Late active TBInput into decision tree5 . 0 No active TBτ00 . 01Decision tree outputs ( in addition to unit costs ) :Vary by intervention Successful diagnosis rate of late active TB , per yearσhdip Rate of movement from successful diagnosis to treatment ( 1/diagnostic delay ) , per yearρhdip Ineffective diagnosis rate of late active TB , per yearκhdip Rate of diagnosis and treatment leading to new resistance , per year susceptible to INH-monoresistantαsihip susceptible to MDRαsmhip INH-monoresistant to MDRαimhip*Calculated such that ( 1−φ1 ) = 0 . 25* ( 1−φ2 ) .\",\n \"Our primary outcomes under each scenario were TB incidence , TB mortality , MDR-TB incidence , and incremental TB diagnostic and treatment costs ( during year 1 and at the end of the 5-year period ) relative to the baseline strategy .\",\n \"By giving flexibility to change model inputs , we provide users the ability to conduct any sensitivity analysis desired .\",\n \"However , for illustrative purposes , we also conducted a series of one-way sensitivity analyses in which each model parameter in Table 1 was varied by ±50% of its listed value ( for proportions , 50% of the difference between the value and either zero or one ) .\",\n \"Our primary outcome for sensitivity analysis was the change in TB incidence , comparing the ‘Xpert for all’ strategy to baseline in the high incidence setting .\",\n \"We also conducted multivariable uncertainty analyses by calculating partial rank correlation coefficients ( Kendall , 1942 ) between each natural history parameter and the outcomes of TB incidence , TB mortality , and 5-year costs .\",\n \"In addition , we constructed 95% uncertainty intervals around our estimates of outcomes in each individual country by simultaneously varying each model parameter by ±10% over a uniform distribution and each target value ( i . e . , TB incidence , HIV prevalence , and MDR-TB prevalence ) over its reported uncertainty range .\",\n \"In this fashion , we constructed 10 , 000 simulations using Latin Hypercube Sampling ( McKay et al . , 1979 ) and took 95% uncertainty ranges as the 2 . 5th and 97 . 5th percentiles of outcomes in these simulations; these ranges are provided in the web-based version of the model for each country .\",\n \"Rates of flow between compartments are governed by the system of ordinary differential equations listed in Equations 1–6 .\",\n \"We first define the forces of infection ( according to resistance status ) and total mortality for simplicity .\",\n \"( 1 ) dUhp ( t ) dt=Ih=0 , p=0*μtot ( t ) −[λ0 ( t ) +λ1 ( t ) +λ2 ( t ) +μbl ( t ) ]*Uhp ( t ) −Ih=0*[θ*U0p ( t ) ]+Ih=1*[θ*U0p ( t ) −μh*U1p ( t ) ]−Ip=0*[τ0* ( 1−sh ) *Uh0 ( t ) ]+Ip=1*[τ0* ( 1−sh ) *Uh0 ( t ) ]where μtot is the sum of all mortality ( to maintain a constant population ) , λd is the force of infection for a given drug resistance strain , μbl is the baseline mortality rate , μh is the HIV-specific mortality rate , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , θ is the HIV incidence rate , τ0 is the rate of seeking diagnosis among people without TB , and sh is the specificity of the diagnostic test .\",\n \"Thus , uninfected individuals exit through infection and death , acquire HIV according to the HIV incidence rate , and become previously treated for TB ( inappropriately ) according to the specificity of the test .\",\n \"The HIV-uninfected , not previously treated compartment is replenished at a rate that matches total mortality and thereby maintains a constant population .\",\n \"( 2 ) dLhdp ( t ) dt=λd ( t ) * ( 1−πh ) *{Uhp ( t ) +∑d[Lhdp ( t ) * ( 1−Ih=0*ι ) ]}+Ip=1*∑i , p[ρhdip*Phdip ( t ) ]+Ih=0*{ν0*E0dp+∑i[νi* ( A0dip+P0dip+I0dip ) ]}−{[ ( λ0 ( t ) +λ1 ( t ) +λ2 ( t ) ) * ( 1−Ih=0*ι ) +εh+μbl+Ih=1*μh]*Lhdp ( t ) }−Ih=0*[θ*L0dp ( t ) ]+Ih=1*[θ*L0dp ( t ) ]where λd is the strain-specific force of infection , πh is the proportion of recent infections that progress rapidly to active TB , ι is the relative reduction in rapid progression after infection among people with latent TB , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , ρhdip is the rate of treatment after successful diagnosis is initiated , νi is the spontaneous recovery rate , εh is the endogenous reactivation rate , μbl is the baseline mortality rate , μh is the HIV-specific mortality rate , and θ is the HIV incidence rate .\",\n \"Thus , individuals enter the latently infected compartment through initial TB infection ( without rapid progression ) , reinfection ( without rapid progression , and accounting for immune protection ) , initiation of successful treatment , or spontaneous resolution .\",\n \"Individuals completing treatment only enter the previously treated compartment ( p=1 ) .\",\n \"Individuals exit this compartment through TB reinfection , endogenous reactivation , and death , and they acquire HIV infection at a constant rate .\",\n \"( 3 ) dEhdp ( t ) dt=λd ( t ) *πh*{Uhp ( t ) +∑d[Lhdp ( t ) * ( 1−Ih=0*ι ) ]}+Lhdp ( t ) *εh−{[μbl+Ih=0* ( μt0+ν0+δe0 ) +Ih=1* ( μh+μth+δe1 ) ]*Ehdp ( t ) }−Ih=0*[θ*E0dp ( t ) ]+Ih=1*[θ*E0dp ( t ) ]where λd is the force of infection , πh is the proportion of recent infections that progress rapidly to active TB , ι is the relative reduction in rapid progression after infection among people with latent TB , Ih=0 is an indicator function of HIV status ( = 1 if h = 0 , 0 otherwise ) , εh is the endogenous reactivation rate , μbl is the baseline mortality rate , μt0 is the TB-specific mortality rate for less-infectious TB , ν0 is the spontaneous recovery rate for less-infectious TB , δeh is the HIV-specific rate of progression to late active TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , and θ is the HIV incidence rate .\",\n \"Thus , individuals enter this compartment through rapid progression of recent infection or endogenous reactivation of latent infection .\",\n \"They exit through progression to late active disease , spontaneous cure ( if HIV-uninfected ) , or death , and they acquire HIV infection at a constant rate .\",\n \"( 4 ) dAhdip ( t ) dt=δeh*[Ii=1*ψh+Ii=0* ( 1−ψh ) ]*Ehdp ( t ) +δf*Ihdip ( t ) −Ahdip ( t ) *[μbl+Ih=0* ( μti+νi+σ0dip+κ0dip+Id=0*αsi0ip+Id=0*αsm0ip+Id=1*αim0ip ) +Ih=1* ( μh+μth+σ1dip+κ1dip+Id=0*αsi1ip+Id=0*αsm1ip+Id=1*αim1ip ) ]−Ih=0*[θ*A0dip ( t ) ]+Ih=1*[θ*A0dip ( t ) ]where δeh is the rate of progression from early active TB , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , ψh is the proportion of active TB that is highly infectious ( smear-positive ) , δf is the rate ( 1/duration ) of ineffective therapy , μbl is the baseline mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , σhdip is the rate of diagnosis ultimately leading to successful treatment , κhdip is the rate of placing individuals on ineffective treatment that does not generate acquired resistance , αsihip is the rate of placing individuals on ineffective treatment that generates INH monoresistance , αsmhip and αimhip are the rates of placing individuals on ineffective treatment that generates MDR-TB , and θ is the HIV incidence rate .\",\n \"Thus , individuals enter this compartment through progression from early active disease or relapse/failure after ineffective treatment .\",\n \"They exit through spontaneous recovery , diagnosis leading to successful treatment , initiation of ineffective treatment ( which can , in turn , generate acquired drug resistance ) , or death , and they acquire HIV infection at a constant rate .\",\n \"This compartment consists of individuals who have initiated a diagnostic attempt that will lead to successful treatment , yet remain infectious while awaiting initiation of treatment .\",\n \"Inclusion of this compartment is designed to capture the effects of diagnostic delays .\",\n \"( 5 ) dPhdip ( t ) dt=σhdip*Ahdip ( t ) −Phdip ( t ) *[μbl+Ih=0* ( μti+νi+ρ0dip ) +Ih=1* ( μh+μth+ρ1dip ) ]−Ih=0*[θ*P0dip ( t ) ]+Ih=1*[θ*P0dip ( t ) ]where σhdip is the rate of initiating successful diagnostic attempts , Ih=0 is an indicator function of HIV status ( = 1 if h = 0 , 0 otherwise ) , μbl is the non-TB mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , ρhdip is the rate of starting therapy after initiating a successful diagnostic attempt ( 1/diagnostic delay ) , and θ is the HIV incidence rate .\",\n \"Thus , individuals enter this compartment by initiation of successful diagnostic attempts and exit through initiation of treatment , spontaneous cure , or death .\",\n \"They acquire HIV infection at a constant rate .\",\n \"This compartment contains all individuals being treated whose treatment course ends in default or failure .\",\n \"Unlike the previous ( successful treatment ) compartment , diagnostic delay is not explicitly incorporated into this compartment , as to do so would prolong the duration of time until individuals who default re-enter the ‘late active’ compartment .\",\n \"Inclusion of a diagnostic delay before this compartment does not materially affect results .\",\n \"Individuals exit this compartment either in default after partial therapy—which has a defined probability of achieving cure despite not being completed—or failure .\",\n \"Failure leads immediately to another course of treatment , which can either be successful ( i . e . , transition to the latent compartment ) or unsuccessful ( i . e . , remain in the inappropriate treatment compartment—which results in transition to the ‘previously treated’ compartment , p=1 , not shown below ) .\",\n \"( 6 ) dIhdip ( t ) dt=κhdip*Ahdip ( t ) +Id=1*αsihdip*Ah0ip ( t ) +Id=2*[αsmhip*Ah0ip ( t ) +αimhip*Ah1ip ( t ) ]−Ihdip ( t ) *[μbl+Ih=0* ( μt0+νi+δf ) +Ih=1* ( μh+μth+δf ) ]−Ih=0*[θ*I0dip ( t ) ]+Ih=1*[θ*I0dip ( t ) ]where κhdip is the rate of placing individuals on ineffective treatment that does not generate acquired resistance , αsihip is the rate of placing individuals on ineffective treatment that generates INH monoresistance from drug-susceptible TB , αsmhip is the rate of placing individuals on ineffective treatment that generates MDR-TB from drug-susceptible TB , αimhip is the rate of placing individuals on ineffective treatment that generates MDR-TB from INH-monoresistant TB , μbl is the non-TB mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , δf is the rate ( 1/duration ) of ineffective therapy , and θ is the HIV incidence rate .\",\n \"Thus , individuals enter this compartment through ineffective treatment from the late active compartment ( conditional on whether that treatment also generates new drug resistance ) and exit through completion of a course of ineffective therapy , spontaneous resolution , or death .\",\n \"They acquire HIV infection at a constant rate .\",\n \"The equations above comprise a series of 100 ordinary differential equations .\",\n \"In order to generate an equilibrium population according to user specifications of TB incidence , MDR-TB prevalence , and HIV prevalence , it is necessary to solve for the roots of a system of these 100 equations , plus three additional equations to account for the user inputs .\",\n \"We accomplish this using the ‘fsolve’ routine in SciPy ( www . scipy . org ) .\",\n \"To solve for these three additional equations , we first match each user input to a single parameter: TB incidence to the transmission rate β , MDR-TB prevalence to the relative reduction in transmission for MDR-TB φ2 , and HIV prevalence to the HIV incidence rate θ .\",\n \"We then constrain the system of equations such that the total population remains constant at 100 , 000 , and we add the following three equations to the system:dβ/dt= ( calculated TB incidence ) − ( user-defined TB incidence ) dφ2/dt= ( calculated MDR-TB prevalence ) − ( user-defined MDR-TB prevalence ) dθ/dt= ( calculated HIV prevalence ) − ( user-defined HIV prevalence ) By solving for the roots at which this system of equations equals zero , we generate an equilibrium ( steady-state ) population that also defines β , φ2 , and θ such that the user-defined targets are also met .\",\n \"Additional complexity is added to account for non-equilibrium in TB incidence and MDR-TB prevalence .\",\n \"We accomplish this by fitting an equilibrium defined by the user-specified target of HIV prevalence , and the user-specified ‘initial’ values of TB incidence and MDR-TB prevalence .\",\n \"This equilibrium is set to be 50 years in the past; at this time , the system of equations is solved as above .\",\n \"We then allow for the parameters β and φ2 to be altered from their original values ( corresponding to the equilibrium condition ) such that a second set of targets are attained after 50 years ( start of the analysis ) .\",\n \"If these calculated values at the end of 50 years do not match the user-defined targets , the parameter values are changed accordingly , and the model is re-run from equilibrium until the appropriate parameter value is identified that generates the user-defined TB incidence and MDR-TB prevalence , within a relative tolerance of 0 . 05 in each variable .\",\n \"These parameters may then be further modified such that they change over the analysis frame of five years ( e . g . , to describe an epidemic with increasing MDR-TB prevalence over time ) .\",\n \"In the primary version of the model , this is only automated for low-incidence scenarios in which the user inputs a TB incidence of less than 50 per 100 , 000/year .\",\n \"In such cases , the model generates an equilibrium population 50 years in the past with a TB incidence of 50 per 100 , 000/year and reduces the transmission parameter β until the user-defined TB incidence is achieved 50 years later ( i . e . , the start of the analytic period ) .\",\n \"This accounts for the fact that most low-incidence settings have substantially more latent TB infection than would be estimated by an equilibrium population with a very low TB incidence—thereby more appropriately reflecting a higher proportion of TB due to reactivation rather than recent infection .\",\n \"However , we include code ( described in the user manual below ) that allows users to define high incidence scenarios that are likewise not at equilibrium , as well as ‘emerging MDR’ scenarios in which the prevalence of MDR-TB is increasing rather than stable .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Most models of infectious diseases , including tuberculosis ( TB ) , do not provide results customized to local conditions .","We created a dynamic transmission model to project TB incidence , TB mortality , multidrug-resistant ( MDR ) TB prevalence , and incremental costs over 5 years after scale-up of nine alternative diagnostic strategies .","A corresponding web-based interface allows users to specify local costs and epidemiology .","In settings with little capacity for up-front investment , same-day microscopy had the greatest impact on TB incidence and became cost-saving within 5 years if delivered at $10/test .","With greater initial investment , population-level scale-up of Xpert MTB/RIF or microcolony-based culture often averted 10 times more TB cases than narrowly-targeted strategies , at minimal incremental long-term cost .","Xpert for smear-positive TB had reasonable impact on MDR-TB incidence , but at substantial price and little impact on overall TB incidence and mortality .","This user-friendly modeling framework improves decision-makers' ability to evaluate the local impact of TB diagnostic strategies ."],"string":"[\n \"Most models of infectious diseases , including tuberculosis ( TB ) , do not provide results customized to local conditions .\",\n \"We created a dynamic transmission model to project TB incidence , TB mortality , multidrug-resistant ( MDR ) TB prevalence , and incremental costs over 5 years after scale-up of nine alternative diagnostic strategies .\",\n \"A corresponding web-based interface allows users to specify local costs and epidemiology .\",\n \"In settings with little capacity for up-front investment , same-day microscopy had the greatest impact on TB incidence and became cost-saving within 5 years if delivered at $10/test .\",\n \"With greater initial investment , population-level scale-up of Xpert MTB/RIF or microcolony-based culture often averted 10 times more TB cases than narrowly-targeted strategies , at minimal incremental long-term cost .\",\n \"Xpert for smear-positive TB had reasonable impact on MDR-TB incidence , but at substantial price and little impact on overall TB incidence and mortality .\",\n \"This user-friendly modeling framework improves decision-makers' ability to evaluate the local impact of TB diagnostic strategies .\"\n]"},"summary":{"kind":"list like","value":["Tuberculosis is an infectious bacterial disease caused predominantly by the microorganism Mycobacterium tuberculosis .","Although the number of deaths from tuberculosis has been falling in recent years , the disease still kills more than 1 million people every year , mainly in developing countries .","Tuberculosis can be treated with antibiotics , but the emergence of bacteria that are resistant to existing drugs is threatening efforts to eradicate the disease .","Preventing the spread of tuberculosis is heavily dependent on accurate diagnosis of individuals with the disease .","This is challenging because the initial symptoms are often mild , usually just a cough , which means that someone can spread the disease to many others over a period of several months before the symptoms become worse—fever , night sweats , and weight loss—and they realize that they are sick .","Multiple diagnostic strategies are available , from the relatively low-tech—examining sputum samples under a microscope to detect tuberculosis bacteria—to more sophisticated tests that can detect bacterial DNA and determine whether the bacteria are drug-resistant in less than 2 hr .","Choosing which diagnostic strategy to adopt can be challenging because the optimal solution in a region will depend on the specific local conditions .","To overcome this problem , Dowdy et al . have developed a computer program that enables decision-makers to input four key parameters that describe the tuberculosis situation in their region , and to obtain 5-year projections of the rate of new infections , mortality , and total costs likely to result from adopting any of nine different diagnostic strategies .","The four parameters are the number of new cases of tuberculosis each year ( incidence ) , the proportion of new cases that are multi-drug resistant , the proportion of the adult population that has HIV , and the local costs of various diagnostic techniques and treatments .","Since the entire computer program is written in a freely available open-source programming language ( Python ) , any user can tweak these parameters to provide a more precise fit to their own region .","Alternatively , the standard version of the program can be run directly from a website without any need to interact with computer code .","This model is the first to enable local decision-makers to evaluate the impact of different diagnostic strategies for tuberculosis under the conditions specific to their region .","The model predicts , for example , that in areas where there is little money available for up-front investment , same-day microscopy analysis of sputum samples and starting patients on treatment is the most cost-effective strategy for reducing the rate of new infections .","Given the wide variation in conditions within even small geographical areas , this more flexible approach should lead to the more efficient use of resources and may , ultimately , help to reduce the spread of tuberculosis ."],"string":"[\n \"Tuberculosis is an infectious bacterial disease caused predominantly by the microorganism Mycobacterium tuberculosis .\",\n \"Although the number of deaths from tuberculosis has been falling in recent years , the disease still kills more than 1 million people every year , mainly in developing countries .\",\n \"Tuberculosis can be treated with antibiotics , but the emergence of bacteria that are resistant to existing drugs is threatening efforts to eradicate the disease .\",\n \"Preventing the spread of tuberculosis is heavily dependent on accurate diagnosis of individuals with the disease .\",\n \"This is challenging because the initial symptoms are often mild , usually just a cough , which means that someone can spread the disease to many others over a period of several months before the symptoms become worse—fever , night sweats , and weight loss—and they realize that they are sick .\",\n \"Multiple diagnostic strategies are available , from the relatively low-tech—examining sputum samples under a microscope to detect tuberculosis bacteria—to more sophisticated tests that can detect bacterial DNA and determine whether the bacteria are drug-resistant in less than 2 hr .\",\n \"Choosing which diagnostic strategy to adopt can be challenging because the optimal solution in a region will depend on the specific local conditions .\",\n \"To overcome this problem , Dowdy et al . have developed a computer program that enables decision-makers to input four key parameters that describe the tuberculosis situation in their region , and to obtain 5-year projections of the rate of new infections , mortality , and total costs likely to result from adopting any of nine different diagnostic strategies .\",\n \"The four parameters are the number of new cases of tuberculosis each year ( incidence ) , the proportion of new cases that are multi-drug resistant , the proportion of the adult population that has HIV , and the local costs of various diagnostic techniques and treatments .\",\n \"Since the entire computer program is written in a freely available open-source programming language ( Python ) , any user can tweak these parameters to provide a more precise fit to their own region .\",\n \"Alternatively , the standard version of the program can be run directly from a website without any need to interact with computer code .\",\n \"This model is the first to enable local decision-makers to evaluate the impact of different diagnostic strategies for tuberculosis under the conditions specific to their region .\",\n \"The model predicts , for example , that in areas where there is little money available for up-front investment , same-day microscopy analysis of sputum samples and starting patients on treatment is the most cost-effective strategy for reducing the rate of new infections .\",\n \"Given the wide variation in conditions within even small geographical areas , this more flexible approach should lead to the more efficient use of resources and may , ultimately , help to reduce the spread of tuberculosis .\"\n]"},"year":{"kind":"string","value":"2014"}}},{"rowIdx":4185,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["computational and systems biology","tools and resources"],"string":"[\n \"computational and systems biology\",\n \"tools and resources\"\n]"},"title":{"kind":"string","value":"DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics"},"id":{"kind":"string","value":"elife-64940-v3"},"sections":{"kind":"list like","value":[["The 23 intervertebral discs ( IVDs ) in the human spine provide stability , mobility , and flexibility .","IVD degeneration ( IDD ) , most common in the lumbar region ( Saleem et al . , 2013; Teraguchi et al . , 2014 ) , is associated with a decline in function and a major cause of back pain , affecting up to 80% of the world’s population at some point in life ( Rubin , 2007 ) , presenting significant socioeconomic burdens .","Multiple interacting factors such as genetics , influenced by ageing , mechanical and other stress factors , contribute to the pathobiology , onset , severity , and progression of IDD ( Munir et al . , 2018 ) .","IVDs are large , avascular , extracellular matrix ( ECM ) -rich structures comprising three compartments: a hydrated nucleus pulposus ( NP ) at the centre , surrounded by a tough annulus fibrosus ( AF ) at the periphery , and cartilaginous endplates of the adjoining vertebral bodies ( Humzah and Soames , 1988 ) .","The early adolescent NP is populated with vacuolated notochordal-like cells , which are gradually replaced by small chondrocyte-like cells ( Risbud et al . , 2015 ) .","Blood vessels terminate at the endplates , nourishing and oxygenating the NP via diffusion , whose limited capacity mean that NP cells are constantly subject to hypoxic , metabolic , and mechanical stresses ( Urban et al . , 2004 ) .","With ageing and degeneration , there is an overall decline in cell ‘health’ and numbers ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) , disrupting homeostasis of the disc proteome .","The ECM has key roles in biomechanical function and disc hydration .","Indeed , a hallmark of IDD is reduced hydration in the NP , diminishing the disc’s capacity to dissipate mechanical loads .","Clinically , T-2 weighted MRI is the gold standard for assessing IDD , that uses disc hydration and structural features such as bulging or annular tears to measure severity ( Pfirrmann et al . , 2001; Schneiderman et al . , 1987 ) .","The hydration and mechanical properties of the IVD are dictated by the ECM composition , which is produced and maintained by the IVD cells .","To meet specific biomechanical needs , cells in the IVD compartments synthesise different compositions of ECM proteins .","Defined as the ‘matrisome’ ( Naba et al . , 2012 ) , the ECM houses the cells and facilitates their inter-communication by regulation of the availability and presentation of signalling molecules ( Taha and Naba , 2019 ) .","With ageing or degeneration , the NP becomes more fibrotic and less compliant ( Yee et al . , 2016 ) , ultimately affecting disc biomechanics ( Newell et al . , 2017 ) .","Changes in matrix stiffness can have a profound impact on cell-matrix interactions and downstream transcriptional regulation , signalling activity , and cell fate ( Park et al . , 2011 ) .","The resulting alterations in the matrisome lead to vicious feedback cycles that reinforce cellular degeneration and ECM changes .","Notably , many of the associated IDD genetic risk factors , such as COL9A1 ( Jim et al . , 2005 ) , ASPN ( Song et al . , 2008 ) , and CHST3 ( Song et al . , 2013 ) , are variants in genes encoding matrisome proteins , highlighting their importance for disc function .","Therefore , knowledge of the cellular and extracellular proteome and their spatial distribution in the IVD is crucial to understanding the mechanisms underlying the onset and progression of IDD ( Feng et al . , 2006 ) .","Current knowledge of IVD biology is inferred from a limited number of transcriptomic studies on human ( Minogue et al . , 2010; Riester et al . , 2018; Rutges et al . , 2010 ) and animal ( Veras et al . , 2020 ) discs .","Studies showed that cells in young healthy NP express markers including CD24 , KRT8 , KRT19 , and T ( Fujita et al . , 2005; Minogue et al . , 2010; Rutges et al . , 2010 ) , whereas NP cells in aged or degenerated discs have different and variable molecular signatures ( Chen et al . , 2006; Rodrigues-Pinto et al . , 2016 ) , such as genes involved in TGFβ signalling ( TGFA , INHA , INHBA , BMP2/6 ) .","The healthy AF expresses genes including collagens ( COL1A1 and COL12A1 ) ( van den Akker et al . , 2017 ) , growth factors ( PDGFB , FGF9 , VEGFC ) , and signalling molecules ( NOTCH and WNT ) ( Riester et al . , 2018 ) .","Although transcriptomic data provides valuable cellular information , it does not faithfully reflect the molecular composition .","Cells represent only a small fraction of the disc volume , transcriptome-proteome discordance does not enable accurate predictions of protein levels from mRNA ( Fortelny et al . , 2017 ) , and the disc matrisome accumulates and remodels over time .","Proteomic studies on animal models of IDD , including murine ( McCann et al . , 2015 ) , canine ( Erwin et al . , 2015 ) , and bovine ( Caldeira et al . , 2017 ) , have been reported .","Nevertheless , human-animal differences in cellular phenotypes and mechanical loading physiologies mean that these findings might not translate to the human scenario .","So far , human proteomic studies have compared IVDs with other cartilaginous tissues ( Önnerfjord et al . , 2012 ) and have shown increases in fibrotic changes in aging and degeneration ( Yee et al . , 2016 ) , a role for inflammation in degenerated discs ( Rajasekaran et al . , 2020 ) , the presence of haemoglobins and immunoglobulins in discs with spondylolisthesis and herniation ( Maseda et al . , 2016 ) , and changes in proteins related to cell adhesion and migration in IDD ( Sarath Babu et al . , 2016 ) .","The reported human disc proteomes were limited in the numbers of proteins identified and finer compartmentalisation within the IVD , and disc levels along the lumbar spine have yet to be studied .","Nor have the proteome dynamics in term of ECM remodelling ( synthesis and degradation ) in young human IVDs and changes in ageing and degeneration been described .","In this study , we presented DIPPER ( the Big Dipper are point-reference stars for guiding nautical voyages ) , a comprehensive disc proteomic resource , comprising static spatial proteome , dynamic proteome and transcriptome , and a methodological flow , for studying the human IVD in youth , ageing , and degeneration .","First , we established a high-resolution point-reference map of static spatial proteomes along the lateral and anteroposterior directions of IVDs at three lumbar levels , contributed by a young ( 16M ) and an aged ( 59M ) cadavers with no reported scoliosis or degeneration .","We evaluated variations among the disc compartments and levels by principal component analysis ( PCA ) , analysis of variance ( ANOVA ) , and identification of differentially expressed proteins ( DEPs ) .","We discovered modules containing specific sets of proteins that describe the directional trends of a young IVD , and the deconstruction of these modules with ageing and degeneration .","Using a LASSO regression model , we identified proteins ( the hydration matrisome ) predictive of tissue hydration as indicated by high-resolution MRI of the aged discs .","Finally , we showed how the point-reference proteomes can be utilised , to integrate with other independent transcriptome and dynamic proteome ( SILAC and degradome ) datasets from 15 additional clinical disc specimens , elevating the robustness of the proteomic findings .","An explorable web interface hosting the data and findings is presented , serving as a useful resource for the scientific community ."],["DIPPER comprises 94 genome-wide measurements from lumbar disc components of 17 individuals ( Figure 1A; Table 1 ) , with data types ranging from label-free proteomics , transcriptomics , SILAC to degradome ( López-Otín and Overall , 2002 ) .","High-resolution static spatial proteomes were generated from multiple intact disc segments of young trauma-induced ( 16 M ) and aged ( 57 M ) cadaveric spines .","T1- and T2-weighted MRI ( 3T ) showed the young discs ( L3/4 , L4/5 , L5/S1 ) were non-degenerated , with a Schneiderman score of 1 from T2 images ( Figure 1B ) .","The NP of young IVD were well hydrated ( white ) with no disc bulging , endplate changes , or observable inter-level variations ( Figure 1B; Figure 1—figure supplement 1A ) , consistent with healthy discs and were deemed fit to serve as a benchmarking point-reference .","To investigate structural changes associated with ageing , high-resolution ( 7T ) MRI was taken for the aged discs ( Figure 1C ) .","All discs had irregular endplates , and annular tears were present ( green arrowheads ) adjacent to the lower endplate and extending towards the posterior region at L3/4 and L4/5 ( Figure 1C ) .","The NP exhibited regional variations in hydration in both sagittal and transverse images ( Figure 1C ) .","Morphologically , the aged discs were less hydrated and the NP and AF structures less distinct , consistent with gross observations ( Figure 1—figure supplement 1A ) .","Scoliosis was not detected in these two individuals .","Information of the disc samples used in the generation of other profiling data are described in Materials and methods and Table","1 . They are clinical samples taken from patients undergoing surgery .","The disc levels and intactness varied , and thus are more suitable for cross-validation purposes and some are directly relevant to IDD .","The intact discs from the two cadaveric spines enabled us to derive spatial proteomes for young and aged human IVDs .","We subdivided each lumbar disc into 11 key regions ( Figure 1D ) , spanning the outer-most ( outer AF; OAF ) to the central-most ( NP ) region of the disc , traversing both anteroposterior and lateral axes , adding valuable spatial information to our proteomic dataset .","Since the disc is an oval shape , an inner AF ( IAF ) region was assigned in the lateral directions .","A ‘mixed’ compartment between the NP and IAF with undefined boundary was designated as the NP/IAF in all four ( anteroposterior and lateral ) directions .","In all , this amounted to 66 specimens with different compartments , ages , directions and levels , which then underwent LC-MS/MS profiling ( Supplementary file 1 ) .","Systematic analyses of the 66 profiles are depicted in a flowchart ( Figure 1A ) .","A median of 654 proteins per profile were identified for the young samples and 829 proteins for the aged samples , with a median of 742 proteins per profile for young and aged combined .","The proteome-wide distributions were on similar scales across the profiles ( Figure 1—figure supplement 1B ) .","Of the 3100 proteins detected in total , 418 were matrisome proteins ( 40 . 7% of all known matrisome proteins ) and 2682 non-matrisome proteins ( ~14% of genome-wide non-matrisome genes ) ( Figure 1E; Figure 1—figure supplement 1C ) , and 983 were common to all four major compartments , namely the OAF , IAF , NP/IAF , and NP ( Figure 1E , upper panel ) .","A total of 1883 proteins were identified in young discs , of which 690 ( 36% ) were common to all regions .","Additionally , 45 proteins ( 2 . 4% ) were unique to NP , whilst NP/IAF , IAF , and OAF had 86 ( 4 . 6% ) , 54 ( 2 . 9% ) , and 536 ( 28% ) unique proteins , respectively ( Figure 1E , middle panel ) .","For the aged discs , 2791 proteins were identified , of which 803 ( 28 . 8% ) were common to all regions .","NP , NP/IAF , and IAF had 34 ( 12% ) , 80 ( 28 . 7% ) , and 44 ( 15% ) unique proteins , respectively , with the OAF accounting for the highest proportion of 1314 unique proteins ( 47% ) ( Figure 1E , lower panel ) .","The aged OAF had the highest number of detected proteins with an average of 1156 , followed by the young OAF with an average of 818 ( Figure 1F ) .","The quantity and spectrum of protein categories identified suggest sufficient proteins had been extracted and the data are of high quality .","We divided the detected matrisome proteins into core matrisome ( ECM proteins , encompassing collagens , proteoglycans , and glycoproteins ) and non-core matrisome ( ECM regulators , ECM affiliated , and secreted factors ) , according to a matrisome classification database ( Naba et al . , 2012 ) ( matrisomeproject . mit . edu ) ( Figure 1F ) .","Despite the large range of total numbers of proteins detected ( 419 to 1920 ) across the 66 profiles ( Figure 1F ) , all six subcategories of the matrisome contained similar numbers of ECM proteins ( Figure 1F and G; Figure 1—figure supplement 2A ) .","The non-core matrisome proteins were significantly more abundant in aged than in young discs ( Figure 1—figure supplement 2A ) .","On average , 19 collagens , 18 proteoglycans , 68 glycoproteins , 52 ECM regulators , 22 ECM affiliated proteins , and 29 secreted factors of ECM were detected per profile .","The majority of the proteins in these matrisome categories were detected in all disc compartments , and in both age groups .","A summary of all the comparisons are presented in Figure 1—figure supplement 1C–E , and the commonly expressed matrisome proteins are listed in Table","2 . Even though there are approximately three times more non-matrisome than matrisome proteins per profile on average ( Figure 1F ) , their expression levels in terms of label-free quantification ( LFQ ) values are markedly lower ( Figure 1—figure supplement 2B ) .","Specifically , the expression levels of core-matrisome were the highest , with an average log2 ( LFQ ) of 30 . 65 , followed by non-core matrisome at 28 . 56 , and then non-matrisome at 27 . 28 ( Figure 1—figure supplement 2B ) .","Within the core-matrisome , the expression was higher ( p=6 . 4 × 10−21 ) in young ( median 30 . 74 ) than aged ( median 29 . 72 ) discs ( Figure 1—figure supplement 2C & D ) .","This difference between young and aged discs is consistent within the subcategories of core and non-core matrisome , with the exception of the ECM regulator category ( Figure 1H ) .","The non-core matrisome and non-matrisome , however , exhibited smaller cross-compartment and cross-age differences in terms of expression levels ( Figure 1—figure supplement 2E–H ) .","That is , the levels of ECM proteins in each compartment of the disc declines with ageing and possibly changes in the relative composition , while the numbers of proteins detected per matrisome subcategory remain similar .","This agrees with the concept that with ageing , ECM synthesis is not sufficient to counterbalance degradation , as exemplified in a proteoglycan study ( Silagi et al . , 2018 ) .","Although 86 . 5% ( 2682 ) of the detected proteins were non-matrisome , their expression levels were considerably lower than matrisome proteins across all sample profiles ( Figure 1F ) .","A functional categorisation according to the Human Genome Nomenclature Committee gene family annotations ( Yates et al . , 2017 ) showed many categories containing information for cellular components and activities , with the top 30 listed in Figure 1I .","These included transcriptional and translational machineries , post-translational modifications , mitochondrial function , protein turnover and importantly , transcriptional factors , cell surface markers , and inflammatory proteins that can inform gene regulation , cell identity , and response in the context of IVD homeostasis , ageing , and degeneration .","These functional overviews highlighted 77 DNA-binding proteins and/or transcription factors , 83 cell surface markers , and 175 inflammatory-related proteins , with their clustering data presented as heatmaps ( Figure 1—figure supplement 3 ) .","Transcription factors and cell surface markers are detected in some profiles ( Figure 1—figure supplement 3A and B ) .","The heatmap of the inflammatory-related proteins showed that more than half of the proteins are detected in the majority of samples , with four major clusters distinguished by age and expression levels ( Figure 1—figure supplement 3C ) .","For example , one of the clusters in the aged samples showed enrichment for complement and coagulation cascades ( False Discovery Rate , FDR q = 1 . 62 × 10−21 ) and clotting factors ( FDR q = 6 . 05 × 10−9 ) , indicating potential infiltration of blood vessels .","Lastly , there are 371 proteins involved in signalling pathways , and their detection frequency in the different compartments and heat map expression levels are illustrated in Figure 1—figure supplement 3D .","Cellularity within the IVD , especially the NP , decreases with age and degeneration ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) .","We assessed whether cellularity of the different compartments could be inferred from the proteomic data .","Quantitation of histones can reflect the relative cellular content of tissues ( Wiśniewski et al . , 2014 ) .","We detected 10 histones , including subunits of histone 1 ( HIST1H1B/C/D/E , HIST1H2BL , HIST1H3A , HIST1H4A ) and histone 2 ( HIST2H2AC , HIST2H2BE , HIST2H3A ) , with four subunits identified in over 60 sample profiles that are mutually co-expressed ( Figure 1—figure supplement 4A ) .","Interestingly , histone concentrations , and thus cellularity , increased from the inner to the outer compartments of the disc , and showed a highly significant decrease in aged discs compared to young discs across all compartments ( Figure 1J , Wilcoxon p=5 . 6 × 10−4 ; Figure 1—figure supplement 4B ) .","GAPDH and ACTA2 are two commonly used reference proteins , involved in the metabolic process and the cytoskeletal organisation of cells , respectively .","They are expected to be relatively constant between cells and are used to quantify the relative cellular content of tissues ( Barber et al . , 2005 ) .","They were detected in all 66 profiles .","GAPDH and ACTA2 amounts were significantly correlated with a Pearson correlation coefficient ( PCC ) of 0 . 794 ( p=9 . 3 × 10−9 ) ( Figure 1—figure supplement 4C ) , and they were both significantly co-expressed with the detected histone subunits , with PCCs of 0 . 785 and 0 . 636 , respectively ( Figure 1K; Figure 1—figure supplement 4D ) .","As expected , expression of the histones , GAPDH , and ACTA2 was not correlated with two core-matrisome proteins , ACAN and COL2A1 ( Figure 1—figure supplement 4E ) , whereas ACAN and COL2A1 were significantly co-expressed ( Figure 1—figure supplement 4F ) , as expected due to their related regulation of expression and tissue function .","Thus , cellularity information can be obtained from proteomic information , and the histone quantification showing reduced cellularity in the aged IVD is consistent with the reported changes ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) .","To gain a global overview of the data , we performed PCA on a set of 507 proteins selected computationally , allowing maximal capture of valid values , while incurring minimal missing values , followed by imputations ( Figure 2—figure supplement 1; Materials and methods ) .","The first two principal components ( PCs ) explained a combined 65 . 5% of the variance , with 39 . 9% and 25 . 6% for the first and second PCs , respectively ( Figure 2A and B ) .","A support vector machine with polynomial kernel was trained to predict the boundaries: it showed PC1 to be most informative to predict age , with a clear demarcation between the two age groups ( Figure 2A , vertical boundary ) , whereas PC2 distinguished disc sample localities , separating the inner compartments ( NP , NP/IAF , and IAF ) from the OAF ( Figure 2A , horizontal boundary ) .","PC3 captured only 5 . 0% of the variance ( Figure 2C & D ) , but it distinguished disc level , separating the lowest level ( L5/S1 ) from the rest of the lumbar discs ( L3/4 and L4/5 ) ( Figure 2D , horizontal boundary ) .","Samples in the upper level ( L3/4 and L4/5 ) appeared to be more divergent , with the aged disc samples deviating from the young ones ( Figure 2D ) .","To extract the most informative features of the PCA , we performed proteome-wide associations with each of the top three PCs , which accounted for over 70% of total variance , and presented the top 100 most positively and top 100 most negatively correlated proteins for each of the PCs ( Figure 2E–G ) .","As expected , the correlation coefficients in absolute values were in the order of PC1 > PC2 > PC3 ( Figure 2E–G ) .","The protein content is presented as non-matrisome proteins ( grey colour ) and matrisome proteins ( coloured ) that are subcategorised as previously .","For the negatively correlated proteins , the matrisome proteins contributed to PC1 in distinguishing young disc samples , as well as to PC2 for sample location within the disc , but less so for disc level in PC3 .","Further , the relative composition of the core and non-core matrisome proteins varied between the three PCs , depicting the dynamic ECM requirement and its relevance in aging ( PC1 ) , tissue composition within the disc ( PC2 ) and mechanical loading ( PC3 ) .","PC1 of young discs identified known chondrocyte markers , CLEC3A ( Lau et al . , 2018 ) and LECT1/2 ( Zhu et al . , 2019 ) , hedgehog signalling proteins , HHIPL2 , HHIP , and SCUBE1 ( Johnson et al . , 2012 ) , and xylosyltransferase-1 ( XYLT1 ) , a key enzyme for initiating the attachment of glycosaminoglycan side chains to proteoglycan core proteins ( Silagi et al . , 2018; Figure 2E ) .","Most of the proteins that were positively correlated in PC1 were coagulation factors or coagulation related , suggesting enhanced blood infiltration in aged discs .","PC2 implicated key changes in molecular signalling proteins ( hedgehog , WNT and Nodal ) in the differences between the inner and outer disc regions ( Figure 2F ) .","Notably , PC2 contains heat shock proteins ( HSPA1B , HSPA8 , HSP90AA1 , HSPB1 ) which are more strongly expressed in the OAF than in inner disc , indicating the OAF is under stress ( Takao and Iwaki , 2002 ) .","Although the correlations in PC3 were much weaker , proteins such as CILP/CILP2 , DCN , and LUM were associated with lower disc level .","To investigate how age , disc compartment , level , and direction affect the protein profiles , we carried out ANOVA for each of these phenotypic factors for the categories of matrisome and non-matrisome proteins ( Figure 2—figure supplement 1H–J ) .","In the young discs , the dominant phenotype explaining the variances for all protein categories was disc compartment .","It is crucial that each disc compartment ( NP , IAF , and OAF ) has the appropriate protein composition to function correctly ( Figure 2—figure supplement 1I ) .","This also fits the understanding that young healthy discs are axially symmetric and do not vary across disc levels .","In aged discs , compartment is still relevant for non-matrisome proteins and collagen , but disc level and directions become influential for other protein categories , which is consistent with variations in mechanical loading occurring in the discs with ageing and degeneration ( Figure 2—figure supplement 1J ) .","In the combined ( young and aged ) disc samples , age was the dominant phenotype across major matrisome categories , while compartment best explained the variance in non-matrisome , reflecting the expected changes in cellular ( non-matrisome ) and structural ( matrisome ) functions of the discs ( Figure 2—figure supplement 1H ) .","This guided us to analyse the young and aged profiles separately , before performing cross-age comparisons .","PCA of the 33 young profiles showed a distinctive separation of the OAF from the inner disc regions on PC1 ( upper panel of Figure 3A ) .","In PC2 , the lower level L5/S1 could generally be distinguished from the upper lumbar levels ( lower panel of Figure 3A ) .","The detected proteome of the young discs ( Figure 3B ) accounts for 9 . 2% of the human proteome ( or 1883 out of the 20 , 368 on UniProt ) .","We performed multiple levels of pairwise comparisons ( summarised in Figure 3—figure supplement 1A ) to detect proteins associated with individual phenotypes , using three approaches ( see Materials and methods ) : statistical tests; proteins detected in one group only; or proteins using a fold-change threshold .","We detected a set of 671 DEPs ( Supplementary file 2 ) ( termed the ‘variable set’ ) , containing both matrisome and non-matrisome proteins ( Figure 3D ) , and visualised in a heatmap ( Figure 3—figure supplement 2 ) , with identification of four modules ( Y1-Y4 ) .","To investigate how the modules are associated with disc components , we compared their protein expression profiles along the lateral and anteroposterior axes .","The original log2 ( LFQ ) values were transformed to z-scores to be on the same scale .","Proteins of the respective modules were superimposed on the same charts , disc levels combined or separated ( Figure 3E–H; Figure 3—figure supplement 3 ) .","Module Y1 is functionally relevant to NP , containing previously reported NP and novel markers KRT19 , CD109 , KRT8 and CHRD ( Anderson et al . , 2002 ) , CHRDL2 , SCUBE1 ( Johnson et al . , 2012 ) , and CLEC3B .","Proteins levels in Y1 are lower on one side of the OAF , increases towards the central NP , before declining towards the opposing side , forming a concave pattern in both lateral and anteroposterior directions , with a shoulder drop occurring between IAF and OAF ( Figure 3E ) .","Module Y2 was enriched in proteins for ECM organisation ( FDR q = 8 . 8 × 10−24 ) ; Y3 was enriched for proteins involved in smooth muscle contraction processes ( FDR q = 8 . 96 × 10−19 ) ; and Y4 was enriched for proteins of the innate immune system ( FDR q = 2 . 93 × 10−20 ) .","Interestingly , these modules all showed a tendency for convex patterns with upward expression toward the OAF regions , in both lateral and anteroposterior directions , in all levels , combined or separated ( Figure 3F–H; Figure 3—figure supplement 3B–D ) .","The higher proportions of ECM , muscle contraction , and immune system proteins in the OAF are consistent with the contractile function of the AF ( Nakai et al . , 2016 ) , and with the NP being avascular and ‘immune-privileged’ in a young homeostatic environment ( Sun et al . , 2020 ) .","The most distinctive pattern on the PCA is the separation of the OAF from the inner disc , with 99 proteins expressed higher in the OAF and 55 expressed higher in the inner disc ( Figure 3I , J ) .","Notably , OAF and inner disc contained different types of ECM proteins .","The inner disc regions were enriched in collagens ( COL3A1 , COL5A1 , COL5A2 , and COL11A2 ) , matrillin ( MATN3 ) , and proteins associated with ECM synthesis ( PCOLCE ) and matrix remodelling ( MXRA5 ) .","We also identified in the inner disc previously reported NP markers ( KRT19 , KRT8 ) ( Risbud et al . , 2015 ) , in addition to inhibitors of WNT ( FRZB , DKK3 ) and BMP ( CHRD , CHRDL2 ) signalling ( Figure 3I , J ) .","Of note , FRZB and CHRDL2 were recently shown to have potential protective characteristics in osteoarthritis ( Ji et al . , 2019 ) .","The TGFβ pathway appears to be suppressed in the inner disc where antagonist CD109 ( Bizet et al . , 2011; Li et al . , 2016 ) is highly expressed , and the TGFβ activity indicator TGFBI is expressed higher in the OAF than in the inner disc .","The OAF is enriched with various collagens ( COL1A1 , COL6A1/2/3 , COL12A1 and COL14A1 ) , basement membrane ( BM ) proteins ( LAMA4 , LAMB2 and LAMC1 ) , small leucine-rich proteoglycans ( SLRP ) ( BGN , DCN , FMOD , OGN , PRELP ) , and BM-anchoring protein ( PRELP ) ( Figure 3I , J ) .","Tendon-related markers such as thrombospondins ( THBS1/2/4 ) ( Subramanian and Schilling , 2014 ) and cartilage intermediate layer proteins ( CILP/CILP2 ) are also expressed higher in the OAF .","Tenomodulin ( TNMD ) was exclusively expressed in 9 of the 12 young OAF profiles , and not in any other compartments ( Supplementary file 2 ) .","This fits a current understanding of the AF as a tendon/ligament-like structure ( Nakamichi et al . , 2018 ) .","In addition , the OAF was enriched in actin-myosin ( Figure 3I ) , suggesting a role of contractile function in the OAF , and in heat-shock proteins ( HSPA1B , HSPA8 , HSPB1 , HSP90B1 , HSP90AA1 ) , suggesting a stress response to fluctuating mechanical loads .","We sought to identify transitions in proteomic signatures between adjacent compartments .","The NP and NP/IAF protein profiles were highly similar ( Figure 3—figure supplement 1B ) .","Likewise , NP/IAF and IAF showed few DEPs , except COMP which was expressed higher in IAF ( Figure 3—figure supplement 1C ) .","OAF and NP ( Figure 3—figure supplement 1R ) and OAF and NP/IAF ( Figure 3—figure supplement 1O ) showed overlapping DEPs , consistent with NP and NP/IAF having highly similar protein profiles , despite some differences in the anteroposterior direction ( Figure 3—figure supplement 1P & Q ) .","The clearest boundary within the IVD , between IAF and OAF , was marked by a set of DEPs , of which COL5A1 , SERPINA5 , MXRA5 were enriched in the IAF , whereas LAMB2 , THBS1 , CTSD typified the OAF ( Figure 3—figure supplement 1D ) .","These findings agreed with the modular patterns ( Figure 3E–H ) .","Of the 1 , 880 proteins detected , 1 , 204 proteins were not found to vary with respect to the phenotypic factors .","The majority of these proteins were detected in few profiles ( Figure 3B ) and were not used in the comparisons .","We set a cutoff for a detection in >1/2 of the profiles to prioritise a set of 245 proteins , hereby referred to as the ‘constant set’ ( Figure 3C ) .","Both the variable and the constant sets contained high proportions of ECM proteins ( Figure 3D ) .","Amongst the proteins in the constant set that were detected in all 33 young profiles were known protein markers defining a young NP or disc , including COL2A1 , ACAN , and A2M ( Risbud et al . , 2015 ) .","Other key proteins in the constant set included CHAD , HAPLN1 , VCAN , HTRA1 , CRTAC1 , and CLU .","Collectively , these proteins showed the common characteristics shared by compartments of young discs , and they , alongside the variable set , form the architectural landscape of the young disc .","PCA was used to identify compartmental , directional , and level patterns for the aged discs ( Figure 4A ) .","Albeit less clear than for the young discs ( Figure 3A ) , the OAF could be distinguished from the inner disc regions on PC1 , explaining 46 . 7% of the total variance ( Figure 4A ) .","PC2 showed a more distinct separation of signatures from lumbar disc levels L5/S1 to the upper disc levels ( L4/L5 and L3/4 ) , accounting for 21 . 8% of the total variance ( Figure 4A ) .","As with the young discs , we performed a series of comparative analyses ( Figure 4C; Figure 4—figure supplement 1A–H; Supplementary file 3 ) .","Detection of DEPs between the OAF and the inner disc ( Figure 4B ) , showed that 100 proteins were expressed higher in the OAF , similar to young discs ( Figure 3C ) .","However , in the inner regions , only nine proteins were significantly expressed higher , in marked contrast to the situation in young discs .","Fifty-five of the 100 DEPs in the OAF region overlapped in the same region in the young discs , but only 3 of the 9 DEPs in the inner region were identified in the young disc; indicating changes in both regions but more dramatic in the inner region .","This suggests that ageing and associated changes may have initiated at the centre of the disc .","The typical NP markers ( KRT8/19 , CD109 , CHRD , CHRDL2 ) were not detectable as DEPs in the aged disc; but CHI3L2 , A2M and SERPING1 ( Figure 4B ) , which have known roles in tissue fibrosis and wound healing ( Lee et al . , 2011; Naveau et al . , 1994; Wang et al . , 2019a ) , were detected uniquely in the aged discs .","A comparative analysis of the protein profiles indicated that the aged OAF retained 55% of the proteins of a young OAF .","These changes are primarily reflected in the class of SLRPs ( BGN , DCN , FMOD , OGN , and PRELP ) , and glycoproteins such as CILP , CILP2 , COMP , FGA/B , and FGG .","From a cellular perspective , 45 proteins enriched in the aged OAF could be classified under ‘responses to stress’ ( FDR q = 1 . 86 × 10−7; contributed by CAT , PRDX6 , HSP90AB1 , EEF1A1 , TUBB4B , P4HB , PRDX1 , HSPA5 , CRYAB , HIST1H1C ) , suggesting OAF ECM and cells are responding to a changing environment such as mechanical loading and other stress factors .","To map the relative changes between inner and outer regions of the aged discs , we performed a systematic comparison between compartments ( Figure 4—figure supplement 1A–D ) .","The most significant observation was a weakening of the distinction between IAF and OAF that was seen in young discs , with only 17 DEPs expressed higher in the OAF of the aged disc ( Figure 4—figure supplement 1C ) .","More differences were seen when we included the NP in the comparison with OAF ( Figure 4—figure supplement 1D ) , indicating some differences remain between inner and outer regions of the aged discs .","While the protein profiles of the NP and IAF were similar , with no detectable DEPs ( Figure 4—figure supplement 1A & B ) , their compositions shared more resemblance with the OAF .","These progressive changes of the protein profiles and DEPs between inner and outer compartments suggest the protein composition of the inner disc compartments becomes more similar to the OAF with ageing , with the greatest changes in the inner regions .","This further supports a change initiating from the inner region of the discs with ageing .","We investigated protein composition in the young disc modules ( Y1-Y4 ) across the lateral and anteroposterior axes in the aged discs ( Figure 4C–F ) .","For module Y1 that consists of proteins defining the NP region , the distinctive concave pattern has flattened along both axes , but more so for the anteroposterior direction where the clear interface between IAF and OAF was lost ( Figure 4C; Figure 4—figure supplement 1I ) .","Similarly for modules Y2 and Y3 , which consist of proteins defining the AF region , the trends between inner and outer regions of the disc have changed such that the patterns become more convex , with a change that is a continuum from inner to outer regions ( Figure 4D , E; Figure 4—figure supplement 1J , K ) .","These changes in modules Y1-3 in the aged disc further illustrate the convergence of the inner and outer regions , with the NP/IAF becoming more OAF-like .","For Y4 , the patterns along the lateral and anteroposterior axes were completely disrupted ( Figure 4F; Figure 4—figure supplement 1L ) .","As Y4 contains proteins involved in vascularity and inflammatory processes ( Supplementary file 5 ) , these changes indicate disruption of cellular homeostasis in the NP .","The protein profiles of the aged disc levels , consistent with the PCA findings , showed similarity between L3/4 and L4/5 ( Figure 4—figure supplement 1E ) , but , in contrast to young discs , differences between L5/S1 and L4/5 ( Figure 4—figure supplement 1F ) , and more marked differences between L5/S1 and L3/4 ( Figure 4—figure supplement 1G , H ) .","Overall , the findings from PCA , protein profiles ( Figure 2B–D ) , and MRI ( Figure 1C ) agree .","As compared to young discs , the more divergent differences across the aged disc levels potentially reflect progressive transmission from the initiating disc to the adjacent discs with ageing .","To further investigate the aetiologies underlying IDD , cross-age comparisons are needed .","Next , we performed extensive pair-wise comparisons between the young and aged samples under a defined scheme ( Figure 5—figure supplement 1A; Supplementary file 4 ) .","First , we compared all 33 young samples with all 33 aged samples , which identified 169 DEPs with 104 expressed higher in the young and 65 expressed higher in the aged discs ( Figure 5A ) .","A simple gene ontology ( GO ) term analysis showed that the most important biological property for a young disc is structural integrity , which is lost in aged discs ( Figure 5B; Supplementary file 5 ) .","The protein classes most enriched in the young discs were related to cartilage synthesis , chondrocyte development , and ECM organisation ( Figure 5B ) .","The major changes in the aged discs relative to young ones , were proteins involved in cellular responses to an ageing environment , including inflammatory and cellular stress signals , progressive remodelling of disc compartments , and diminishing metabolic activities ( Figure 5C; Supplementary file 5 ) .","For young versus old discs , we compared DEPs of the whole disc ( Figure 5A ) with those from the inner regions ( Figure 5D ) and OAF ( Figure 5E ) only .","Seventy-five percent ( 78/104 ) of the downregulated DEPs ( Figure 5F ) were attributed to the inner regions , and only 17% ( 18/104 ) were attributed to the OAF ( Figure 5F ) .","Similarly , 65% ( 42/65 ) of the upregulated DEPs were solely contributed by inner disc regions , and only 18 . 4% ( 12/65 ) by the OAF .","Only 5 DEPs were higher in the OAF , while 49 were uniquely upregulated in the inner discs ( Figure 5G ) .","The key biological processes in each of the compartments are highlighted in Figure 5F and G . Expression of known NP markers was reduced in aged discs , especially proteins involved in the ECM and its remodelling , where many of the core matrisome proteins essential for the structural function of the NP were less abundant or absent .","While this is consistent with previous observations ( Feng et al . , 2006 ) , of interest was the presence of a set of protein changes that were also seen in the OAF , which was also rich in ECM and matrix remodelling proteins ( HTRA1 , SERPINA1 , SERPINA3 , SERPINC1 , SERPINF1 , and TIMP3 ) and proteins involved in fibrotic events ( FN1 , POSTN , APOA1 , APOB ) , suggesting these changes are occurring in the ageing IVDs ( Figure 5F , G ) .","Proteins associated with cellular stress are decreased in the aged inner disc , with functions ranging from molecular chaperones needed for protein folding ( HSPB1 , HSPA1B and HSPA9 ) to modulation of oxidative stress ( SOD1 ) ( Figure 5F ) .","SOD1 has been shown to become less abundant in the aged IVD ( Hou et al . , 2014 ) and in osteoarthritis ( Scott et al . , 2010 ) .","HSPB1 is cytoprotective and a deficiency is associated with inflammation reported in degenerative discs ( Wuertz et al . , 2012 ) .","We found an increased concentration of clusterin ( CLU ) ( Figure 5G ) , an extracellular chaperone that aids the solubilisation of misfolded protein complexes by binding directly and preventing protein aggregation ( Trougakos , 2013; Wyatt et al . , 2009 ) , and also has a role in suppressing fibrosis ( Peix et al . , 2018 ) .","Inhibitors of WNT ( DKK3 and FRZB ) , and antagonists of BMP/TGFβ ( CD109 , CHRDL2 , DCN FMOD , INHBA and THBS1 ) signalling were decreased or absent in the aged inner region ( Figure 5F , G ) , consistent with the reported upregulation of these pathways in IDD ( Hiyama et al . , 2010 ) and its closely related condition osteoarthritis ( Leijten et al . , 2013 ) .","Targets of hedgehog signalling ( HHIPL2 and SCUBE1 ) were also reduced ( Figure 5F ) , consistent with SHH’s key roles in IVD development and maintenance ( Rajesh and Dahia , 2018 ) .","TGFβ signalling is a well-known pathway associated with fibrotic outcomes .","WNT is known to induce chondrocyte hypertrophy ( Dong et al . , 2006 ) , that can be enhanced by a reduction in S100A1 ( Figure 5F ) , a known inhibitor of chondrocyte hypertrophy ( Saito et al . , 2007 ) .","To gain an overview of the disc compartment variations between young and aged discs , we followed the same strategy as in Figure 3—figure supplement 2 to aggregate three categories of DEPs in all 23 comparisons ( Figure 5—figure supplement 1A ) and created a heatmap from the resulting 719 DEPs .","This allowed us to identify six major protein modules ( Figure 5H ) .","A striking feature is module 6 ( M6 ) , which is enriched for proteins involved in the complement pathway ( GSEA Hallmark FDR q = 4 . 9 × 10−14 ) and angiogenesis ( q = 2 . 3 × 10−3 ) .","This module contains proteins that are all highly expressed in the inner regions of the aged disc , suggesting the presence of blood .","M6 also contains the macrophage marker CD14 , which supports this notion .","We visualised the relationship between the young ( Y1-4 ) and young/aged ( M1-6 ) modules using an alluvial chart ( Figure 5I ) .","Y1 corresponds primarily to M1b that is enriched with fibrosis , angiogenesis , apoptosis , and EMT ( epithelial to mesenchymal transition ) proteins .","Y2 seems to have been deconstructed into three M modules ( M2b > M3 > M4 ) .","M2b and M3 contain proteins linked to heterogeneous functions , while proteins in M4 are associated with myogenesis and cellular metabolism , but also linked to fibrosis and angiogenesis .","Y3 primarily links to M2a with a strong link to myogenesis , and mildly connects with M3 and M4 .","Y4 has the strongest connection with M6a , which is linked to coagulation .","Both the variable and the constant sets of the young disc were also changed in ageing .","In the constant set , there is a higher tendency for a decrease in ECM-related proteins , and an increase in blood and immune related proteins with ageing , that may reflect an erosion of the foundational proteome , and infiltration of immune cells ( Figure 5—figure supplement 1L ) .","In all , the IVD proteome showed that with ageing , activities of the SHH pathways were decreased , while those of the WNT and BMP/TGFβ pathways , EMT , angiogenesis , fibrosis , cellular stresses , and chondrocyte hypertrophy-like events were increased .","The proteome reflects both current and past transcriptional activities .","To investigate upstream cellular and regulatory activities , we obtained transcriptome profiles from two IVD compartments ( NP and AF ) and two sample states ( young , scoliotic but non-degenerated , YND; aged individuals with clinically diagnosed IDD , AGD ) ( Table 1 ) .","The transcriptome profiles of YND and AGD are similar to the young and aged disc proteome samples , respectively .","After normalisation ( Figure 6—figure supplement 1A ) and hierarchical clustering , we found patterns reflecting relationships among IVD compartments and ages/states ( Figure 6—figure supplement 1B–D ) .","PCA of the transcriptome profiles showed that PC1 captured age/state variations ( Figure 6A ) and PC2 captured the compartment ( AF or NP ) differences , with a high degree of similarity to the proteomic PCA that explained 65 . 0% of all data variance ( Figure 2A ) .","We compared the transcriptome profiles of different compartments and age/state-groups ( Figure 6—figure supplement 1E–H ) .","We detected 88 DEGs ( Materials and methods ) between young AF and young NP samples ( Figure 6—figure supplement 1E ) , in which 39 were more abundant in young NP ( including known NP markers CD24 , KRT19 ) and 49 were more abundant in young AF , including the AF markers , COL1A1 , and THBS1 .","In the AGD samples , 11 genes differed between AF and NP ( Figure 6—figure supplement 1F ) , comparable to the proteome profiles ( Figure 4C ) .","Between the YND and AGD AF , there were 45 DEGs , with COL1A1 and MMP1 more abundant in YND and COL10A1 , WNT signalling ( WIF1 , WNT16 ) , inflammatory ( TNFAIP6 , CXCL14 , IL11 ) , and fibrosis-associated ( FN1 , CXCL14 ) genes more abundant in AGD ( Figure 6—figure supplement 1G ) .","The greatest difference was between YND and AGD NP , with 216 DEGs ( Figure 6—figure supplement 1H ) , with a marked loss of NP markers ( KRT19 , CD24 ) , and gain of AF ( THBS1 , DCN ) , proteolytic ( ADAMTS5 ) , and EMT ( COL1A1 , COL3A1 , PDPN , NT5E , LTBP1 ) markers with age .","Again , consistent with the proteomic findings , the most marked changes are in the NP , with the transcriptome profiles becoming AF-like .","We partitioned the DEGs between the YND and AGD into DEGs for individual compartments ( Figure 6B ) .","The transcriptomic ( Figure 6B ) Venn diagram was very similar to the proteomic one ( Figure 5F–G ) .","For example , WNT/TGFβ antagonists and ECM genes were all downregulated with ageing/degeneration , while genes associated with stress and ECM remodelling were more common .","When we directly compared the transcriptomic DEGs and proteomic DEPs across age/states and compartments ( Figure 6C–F ) , we observed strong concordance between the two types of datasets for a series of markers .","In the young discs , concordant markers included KRT19 and KRT8 , CHRDL2 , FRZB , and DKK3 in the NP , and COL1A1 , SERPINF1 , COL14A1 , and THBS4 in the AF ( Figure 6C ) .","In the AGD discs , concordant markers included CHI3L2 , A2M and ANGPTL4 in the NP and MYH9 , HSP90AB1 , HBA1 , and ACTA2 in the AF ( Figure 6D ) .","A high degree of concordance was also observed when we compared across age/states for the AF ( Figure 6E ) and NP ( Figure 6F ) .","Despite the transcriptomic samples having diagnoses ( scoliosis for YND and IDD for AGD ) , whereas the proteome samples were cadaver samples with no reported diagnosis of IDD , the changes detected in the transcriptome profiles substantially support the proteomic findings .","A surprising indication from the transcriptome was the increased levels of COL10A1 ( Lu et al . , 2014 ) , BMP2 ( Grimsrud et al . , 2001 ) , IBSP , defensin beta-1 ( DEFB1 ) , ADAMTS5 , pro-inflammatory ( TNFAIP6 , CXCL ) and proliferation ( CCND1 , IGFBP ) genes in the AGD NP ( Figure 6—figure supplement 1E–H ) , reaffirming the involvement of hypertrophic-like events ( Melas et al . , 2014 ) in the aged and degenerated NP .","The genome-wide transcriptomic data included over 20 times more genes per profile than the proteomic data , providing additional biological information about the disc , particularly low abundance proteins , such as transcription factors and surface markers .","For example , additional WNT antagonists were WIF1 ( Wnt inhibitory factor ) and GREM1 ( Figure 6B Leijten et al . , 2013 ) .","Comparing the YND NP against YND AF or AGD NP ( Figure 6—figure supplement 1E , H ) , we identified higher expression of three transcription factors , T ( brachyury ) , HOPX ( homeodomain-only protein homeobox ) , and ZNF385B in the YND NP .","Brachyury is a well-known marker for the NP ( Risbud et al . , 2015 ) , and HOPX is differentially expressed in mouse NP as compared to AF ( Veras et al . , 2020 ) , and expressed in mouse notochordal NP cells ( Lam , 2013 ) .","Overall , transcriptomic data confirmed the proteomic findings and revealed additional markers .","The proteomic data up to this point is a static form of measurement ( static proteome ) and represents the accumulation and turnover of all proteins up to the time of harvest .","The transcriptome indicates genes that are actively transcribed , but does not necessarily correlate to translation or protein turnover .","Thus , we studied changes in the IVD proteome ( dynamic proteome ) that would reflect newly synthesised proteins and proteins cleaved by proteases ( degradome ) , and how they relate to the static proteomic and transcriptomic findings reported above .","We performed ex vivo labelling of newly synthesised proteins using the SILAC protocol ( Ong et al . , 2002; Figure 7A; Materials and methods ) on AF and NP samples from four YND individuals and one AGD individual ( Table 1 ) .","In the SILAC profiles , light isotope-containing signals correspond to the pre-existing unlabelled proteome , and heavy isotope-containing signals to newly synthesised proteins ( Figure 7B ) .","The ECM compositions in the light isotope-containing profiles ( Figure 7B , middle panel ) are similar to the static proteome samples of the corresponding age groups described above ( Figure 1F ) .","Although for NP_YND152 , the numbers of identified proteins in the heavy profiles are considerably less than NP_YND151 due to a technical issue during sample preparation , it is overall still more similar to NP_YND152 than to other samples ( Figure 7—figure supplement 1B ) , indicating that its biological information is still representative of a young NP and the respective AF samples are similar .","In contrast , the heavy isotope-containing profiles contained fewer proteins in the AGD than in the YND samples ( Figure 7B , left panel ) and showed variable heavy to light ratio profiles ( Figure 7B , right panel ) .","To facilitate comparisons , we averaged the abundance of the proteins detected in the NP or AF for which we had more than one sample , then ranked the abundance of the heavy isotope-containing ( Figure 7C ) , light isotope-containing ( Figure 7D ) proteins .","The number of proteins newly synthesised in the AGD samples was about half that in the YND samples ( Figure 7C ) .","This is unlikely to be a technical artefact as the total number of light isotope-containing proteins detected in the AGD samples is comparable to the YND , in both AF and NP ( Figure 7D ) , and the difference is again well illustrated in the heavy to light ratios ( Figure 7—figure supplement 1A ) .","Reduced synthesis of non-matrisome proteins was found for the AGD samples ( GAPDH ) as a reference point ( dotted red lines ) ( Figure 7C and D; Figure 7C and E , grey portions ) .","Of the 68 high abundant non-matrisome proteins in the YND NP compartment that were not present in the AGD NP , 28 are ribosomal proteins ( Figure 7—figure supplement 1C ) , suggesting reduced translational activities .","This agrees with our earlier findings of cellularity , as represented by histones , in the static proteome ( Figure 1J , K ) .","Changes in protein synthesis in response to the cell microenvironment affects the architecture of the disc proteome .","To understand how the cells may contribute and respond to the accumulated matrisome in the young and aged disc , we compared the newly synthesised matrisome proteins of AGD and YND samples rearranged in order of abundance ( Figure 7E ) .","More matrisome proteins were synthesised in YND samples across all classes .","In YND AF , collagens were synthesised in higher proportions than in AGD AF with the exception of fibril-associated COL12A1 ( Figure 7E , top panel ) .","Similarly , higher proportions in YND AF were observed for proteoglycans ( except FMOD ) , glycoproteins ( except TNC , FBN1 , FGG and FGA ) , ECM affiliated proteins ( except C1QB ) , ECM regulators ( except SERPINF2 , SERPIND1 , A2M , ITIH2 , PLG ) , and secreted factors ( except ANGPTL2 ) .","Notably , regulators that were exclusively synthesised in young AF are involved in collagen synthesis ( P4HA1/2 , LOXL2 , LOX , PLOD1/2 ) and matrix turnover ( MMP3 ) , with enrichment of protease HTRA1 and protease inhibitors TIMP3 and ITIH in YND AF compared to AGD AF .","In AGD NP , overall collagen synthesis was less than in YND NP ( Figure 7E , lower panel ) ; however , there was more synthesis of COL6A1/2/3 and COL12A1 .","Furthermore , AGD NP synthesised more LUM , FMOD , DCN , PRG4 , and PRELP proteoglycans than YND NP .","Notably , there was less synthesis of ECM-affiliated proteins ( except C1QC and SEMA3A ) and regulators – particularly those involved in collagen synthesis ( P4HA1/2 , LOXL2 , LOX ) – but an increase in protease inhibitors .","A number of newly synthesised proteins in AGD NP were similarly represented in the transcriptome data , including POSTN , ITIH2 , SERPINC1 , IGFBP3 , and PLG .","Some genes were simultaneously underrepresented in the AGD NP transcriptome and newly synthesised proteins , including hypertrophy inhibitor GREM1 ( Leijten et al . , 2013 ) .","The degradome reflects protein turnover by identifying cleaved proteins in a sample ( López-Otín and Overall , 2002 ) .","When combined with relative quantification of proteins through the use of isotopic and isobaric tagging and enrichment for cleaved neo amine ( N ) -termini of proteins before labelled samples are quantified by mass-spectrometry , degradomics is a powerful approach to identify the actual status of protein cleavage in vivo .","We employed the well-validated and sensitive terminal amine isotopic labelling of substrates ( TAILS ) method ( Kleifeld et al . , 2010; Rauniyar and Yates , 2014 ) to analyse and compare six discs from six individuals ( two young and non-degenerated , YND; and four aged and/or degenerated , AGD ) ( Table 1; Figure 7F; Kleifeld et al . , 2010; Rauniyar and Yates , 2014 ) using the 6-plex tandem mass tag ( TMT ) -TAILS ( labelling six independent samples and analysed together on the mass spectrometer ) ( Figure 7F ) .","Although shotgun proteomics is intended to identify the proteome components , N-terminome data is designed to identify the exact cleavage site in proteins that also evidence stable cleavage products in vivo .","Here , TAILS identified 123 and 84 cleaved proteins in the AF and NP disc samples , respectively .","Performing hierarchical clustering on the data we found that the two YND samples ( 136 and 141; Table 1 ) tend to cluster together in both AF and NP ( Figure 7G , H; Figure 7—figure supplement 2A , B ) .","Interestingly , the trauma sample AGD143 ( 53 year male ) , who has no known IDD diagnosis , tend to cluster with other clinically diagnosed AGD samples , in both AF and NP .","This might be because AGD143 has unreported degeneration or ageing is a dominant factor in degradome signals .","We identified two protein/peptide modules in the AF ( Figure 7G ) , corresponding to more degradation/cleaving in YND AF ( magenta ) and AGD AF ( blue ) , respectively .","There are only 13 unique proteins for proteins/peptides more degraded in the YND AF , the most common of which is COL1A1/2 , followed by COL2A1 .","In comparison , the module corresponding to more degradation in AGD AF recorded 24 unique proteins , 7 ( CILP , CILP2 , COL1A1 , COMP , HBA1 , HBB , PRELP ) of which are in strong overlap ( χ2 p=2 . 0 × 10−71 ) with the 99 proteins higher in outer AF in the spatial proteome ( Figure 3I ) .","This indicates that key proteins defining a young outer AF is experiencing faster degradation in aged and degenerated samples .","Similarly , we identified two modules in the NP ( Figure 7H ) , whereby one ( magenta ) corresponds to more degradation in the YND , and the other ( blue ) corresponds to more degradation in the AGD .","Only 10 unique proteins were recorded in the magenta module ( for YND ) , with COL2A1 being the most dominant ( 928 peptides ) ; whereas 32 were recorded for the blue module ( for AGD ) .","Overall , there are more unique proteins involved in faster degradation in AGD AF and NP .","We tested for a correlation between MRI signal intensity and proteome composition .","In conventional 3T MRI of young discs , the NP is brightest reflecting its high hydration state while the AF is darker , thus less hydrated ( Figure 1B; Figure 8—figure supplement 1B ) .","Since aged discs present with more MRI phenotypes , we used higher resolution MRI ( 7T ) on them ( Figure 1C; Figure 8A ) , which showed less contrast between NP and AF than in the young discs .","To enhance robustness , we obtained three transverse stacks per disc level for the aged discs ( Figure 8B; Figure 8—figure supplement 1D ) , and averaged the pixel intensities for the different compartments showing that overall , the inner regions were still brighter than the outer ( Figure 8C ) .","Next , we performed a level-compartment bi-clustering on the pixel intensities of the aged disc MRIs , which was bound by disc level and compartment ( Figure 8D ) .","The findings resembled the proteomic PCA ( Figure 2 ) and clustering ( Figure 3—figure supplement 1V–W ) patterns .","We performed a pixel intensity averaging of the disc compartments from the 3T images ( Figure 8—figure supplement 1D ) , and a level-compartment bi-clustering on the pixel intensities ( Figure 8—figure supplement 1C ) .","While the clustering can clearly partition the inner from the outer disc compartments , the information value from each of the compartments is less due to the lower resolution of the MRI .","In all , these results indicate a link between regional MRI landscapes and proteome profiles , prompting us to investigate their potential connections .","The MRI and the static proteome were done on the same specimens in both individuals , so we could perform proteome-wide associations with the MRI intensities .","We detected 85 significantly correlated ECM proteins , hereby referred to as the hydration matrisome ( Figure 8E ) .","We found no collagen to be positively correlated with brighter MRI , which fits current understanding as collagens contribute to fibrosis and dehydration .","Other classes of matrisome proteins were either positively or negatively correlated , with differential components for each class ( Figure 8E ) .","Positively correlated proteoglycans included EPYC , PRG4 and VCAN , consistent with their normal expression in a young disc and hydration properties .","Negatively correlated proteins included OAF ( TNC , SLRPs ) and fibrotic ( POSTN ) markers ( Figure 8E ) .","Given this MRI-proteome link and the greater dynamic ranges of MRI in the aged discs enabled by the higher resolution 7T MRIs ( Figure 8D ) , we hypothesised that the hydration matrisome might be used to provide information about MRI intensities and thus disc hydration .","To test this , we trained a LASSO regression model ( Tibshirani , 1996 ) of the aged MRIs using the hydration matrisome ( 85 proteins ) , and applied the model to predict the intensity of the MRIs of the young discs , based on the young proteome of the same 85 proteins .","Remarkably , we obtained a PCC of 0 . 689 ( p=8 . 9 × 10−6; Spearman = 0 . 776 ) between the actual and predicted MRI ( Figure 8—figure supplement 1E ) .","The predicted MRI intensities of the young disc exhibited a smooth monotonic decrease from the NP towards IAF , then dropped suddenly towards the OAF ( Figure 8F , right panel ) , with an ROC AUC ( receiver operating characteristics , area under the curve ) of 0 . 996 between IAF and OAF ( Figure 8—figure supplement 1F ) .","In comparison , actual MRIs exhibited a linear decrease from NP to OAF ( Figure 8F , left panel ) .","On reviewing these two patterns , we argue that the predicted intensities may be a more faithful representation of the young discs’ water contents than the actual MRI , as it reflects the gross images ( Figure 1—figure supplement 1A ) , PCA ( Figure 3A ) and Y1 modular trend in the young discs ( Figure 3E ) .","This exercise not only revealed the inherent connections between regional MRI and regional proteome , but also identified a set of ECM components that is predictive of MRI relating to disc hydration , which may be valuable for future clinical applications ."],["Here , we present DIPPER – a human IVD proteomic resource comprising point-reference genome-wide profiles .","The discovery dataset was established from intact lumbar discs of a young cadaver with no history of skeletal abnormalities ( e . g . scoliosis ) , and an aged cadaver with reduced IVD MRI intensity and annular tears .","Although these two individuals may not be representative of their respective age-groups , this is the first known attempt to achieve high spatial resolution profiles in the discs , adding a critical and much needed dimension to the current available IVD proteomic datasets .","We showed that our spatiotemporal proteomes integrate well with the dynamic proteome and transcriptome of clinical samples , demonstrating their application values with other datasets .","In creating the point-references , we use a well-established protein extraction protocol ( Önnerfjord et al . , 2012 ) , and chromatographic fractionation of the peptides prior to mass spectrometry , we produced a dataset of the human IVD comprising 3 , 100 proteins , encompassing ~400 matrisome and 2 , 700 non-matrisome proteins , with 1 , 769 proteins detected in three or more profiles , considerably higher than recent studies ( Maseda et al . , 2016; Rajasekaran et al . , 2020; Ranjani et al . , 2016; Sarath Babu et al . , 2016 ) .","The high quality of our data enabled the application of unbiased approaches including PCA and ANOVA to reveal the relative importance of the phenotypic factors .","Particularly , age was found to be the dominant factor influencing proteome profiles .","Comparisons between different compartments of the young disc produced a reference landscape containing known ( KRT8/19 for the NP; COL1A1 , CILP and COMP for the AF ) and novel ( FRZB , CHRD , CHRDL2 for the NP; TNMD , SLRPs , and SOD1 for the AF ) markers .","The young healthy discs were enriched for matrisome components consistent with a healthy functional young IVD .","Despite morphological differences between NP and IAF , the inner disc compartments ( NP , NP/IAF , and IAF ) display high similarities , in contrast to the large differences between IAF and OAF , which was consistent for discs from all lumbar disc levels .","This morphological-molecular discrepancy might be accounted for by subtle differences in the ECM organisation , such as differences in GAG moieties on proteoglycans , or levels of glycosylation or other modifications of ECM that diversify function ( Silagi et al . , 2018 ) .","Nonetheless , we partitioned the detected proteins into a variable set that captures the diversity , and a constant set that lays the common foundation of all young compartments , which work in synergy to achieve disc function .","Clustering analysis of the 671 DEPs of the variable set identified four key modules ( Y1-Y4 ) .","Visually , Y1 and Y2 mapped across the lateral and anteroposterior axes with opposing trends .","Molecularly , module Y1 ( NP ) contained proteins promoting regulation of matrix remodelling , such as matrix degradation inhibitor MATN3 ( Jayasuriya et al . , 2012 ) and MMP inhibitor TIMP1 .","Inhibitors of WNT and BMP signalling were also present .","Module Y2 ( OAF ) included COL1A1 , THBS1/2/3 , CILP1/CILP2 , and TNMD , consistent with the OAF’s tendon-like features ( Nakamichi et al . , 2018 ) .","It also included a set of SLRPs that might play roles in regulation of collagen assembly ( Robinson et al . , 2017; Taye et al . , 2020 ) , fibril alignment ( Robinson et al . , 2017 ) , maturation and crosslinking ( Kalamajski et al . , 2016 ) , while others are known to inhibit or promote TGFβ signalling ( Markmann et al . , 2000 ) .","Notably , the composition of the IAF appeared to be a transition zone between NP and OAF rather than an independent compartment , as few proteins can distinguish it from adjacent compartments .","Classes of proteins in both Y3 ( smooth muscle feature ) and Y4 ( immune and blood ) resemble Y2 , which reflects the contractile property of the AF ( Nakai et al . , 2016 ) and the capillaries infiltrating or present at the superficial outer surface of the IVD .","In the aged disc , the DEPs between the inner and outer regions of the discs suggests extensive changes in the inner compartment ( s ) .","Mapping the aged data onto modules Y1-Y4 allowed a visualisation of the changes .","The flattening of the Y1 and Y2 modules along both the lateral and anteroposterior axes indicated a convergence of the inner and outer disc .","This is supported by the observed rapid decline of NP proteins and increase of AF proteins in the inner region .","Fewer changes were seen in the aged OAF , which concurs with the notion that degenerative changes originate from the NP and radiate outwards; however , infringement of IAF into the NP cannot be excluded .","The most marked change was seen in module Y4 ( blood ) , where the pattern was inverted , characterised by high expression in the NP but low in OAF .","While contamination cannot be excluded and there are reports that capillaries do not infiltrate the NP even in degeneration ( Nerlich et al . , 2007 ) , our finding is consistent with other proteomic studies showing enrichment of blood proteins in pathological NP ( Maseda et al . , 2016 ) .","The route of infiltration can be from the fissured AF or cartilage endplates .","Calcified endplates are more susceptible to microfractures , which can lead to blood infiltration into the NP ( Sun et al . , 2020 ) .","Of interest is the involvement of an immune response within the inner disc .","This corroborates reports of inflammatory processes in ageing and degenerative discs , with the upregulation of pro-inflammatory cytokines and presence of inflammatory cells ( Molinos et al . , 2015; Wuertz et al . , 2012 ) .","The SILAC and degradome studies provided important insights into age-related differences in the biosynthetic and turnover activity in the IVD .","The SILAC data indicated that protein synthesis is significantly impaired in aged degenerated discs .","These findings correlate with reports of reduced cellularity in ageing ( Rodriguez et al . , 2011 ) , which we have also ascertained by leveraging the relationship of histones and housekeeping genes with cell numbers .","From the TAILS degradome analysis , we observed more cleaved protein fragments in aged compartments , particularly for structural proteins important for tissue integrity such as COMP and those involved in cell-matrix interactions such as FN1 , which was coupled with the enrichment of the proteolytic process GO terms in the aged static proteome .","Collectively , this reveals a systematic modification and replacement of the primary proteomic architecture of the young IVD with age that is associated with diminished or failure in functional properties in ageing or degeneration .","Despite known transcriptome-proteome discordance ( Fortelny et al . , 2017 ) , our identification of their concordance allows insights into active changes in the young and aged discs .","For example , inhibitors of the WNT pathway and antagonists of BMP/TGFβ signalling ( Leijten et al . , 2013 ) were down-regulated in the aged discs in both the proteome and transcriptome .","Interestingly , the activation of these pathways is known to promote chondrocyte hypertrophy ( Dong et al . , 2006 ) , and hypertrophy has been noted in IDD ( Rutges et al . , 2010 ) .","This suggests a model whereby the regulatory environment suppressing cellular hypertrophy changes with ageing or degeneration , resulting in conditions such as cellular senescence and tissue mineralisation that are part of the pathological process .","In support , S100A1 , a known inhibitor of chondrocyte hypertrophy ( Saito et al . , 2007 ) is downregulated , while chondrocyte hypertrophy markers COL10A1 and IBSP ( Komori , 2010 ) are upregulated in the aged disc .","Similar changes have been observed in ageing mouse NP ( Veras et al . , 2020 ) as well as in osteoarthritis ( Zhu et al . , 2009 ) where chondrocyte hypertrophy is thought to be involved in its aetiology ( Ji et al . , 2019; van der Kraan and van den Berg , 2012 ) .","Given that WNT inhibitors are already in clinical trials for osteoarthritis ( Wang et al . , 2019b ) , this may point to a prospective therapeutic strategy for IDD .","A key finding of our study is the direct demonstration , within a single individual , of association between the hydration status of the disc as revealed by MRI , and the matrisome composition of the disc proteome .","The remarkable correlation between predicted hydration states inferred from the spatial proteomic data and the high-definition phenotyping of the aged disc afforded by 7T MRI has enormous potential for understanding the molecular processes underlying IDD .","In conclusion , we have generated point-reference datasets of the young and aged disc proteome , at a significantly higher spatial resolution than previous works .","By means of a methodological framework , we revealed compartmentalised information on the ECM composition and cellular activities , and their changes with ageing .","Integration of this point-reference with additional age- and protein-specific information of synthesis/degradation helps gain insights into the underlying molecular pathology of degeneration ( Figure 8G and H ) .","The richness of information in DIPPER makes it a valuable resource for cross referencing with human , animal and in vitro studies to evaluate clinical relevance and guide the development of therapeutics for human IDD ."],["Two human lumbar spines were obtained through approved regulations and governing bodies , with one young ( 16M ) provided by L . H . ( McGill University ) and one aged ( 59M ) from Articular Engineering , LLC ( IL , USA ) .","The young lumbar spine was received frozen as an intact whole lumbar spine .","The aged lumbar spine was received frozen , dissected into bone-disc-bone segments .","The cadaveric samples were stored at −80⁰C until use .","Clinical specimens were obtained with approval by the Institutional Review Board ( references UW 13–576 and EC 1516–00 11/01/2001 ) and with informed consent in accordance with the Helsinki Declaration of 1975 ( revision 1983 ) from another 15 patients undergoing surgery for IDD , trauma or adolescent idiopathic scoliosis at Queen Mary Hospital ( Hong Kong ) , and Duchess of Kent Children’s Hospital ( Hong Kong ) .","Information of both the cadaveric and clinical samples are summarised in Table 1 .","The discs were thawed overnight at 4°C , and then pre-equalised for scanning at room temperature .","For the young IVD , these were imaged together as the lumbar spine was kept intact .","T2-weighted and T1-weighted sagittal and axial MRI , T1-rho MRI and Ultrashort-time-to-echo MRI images were obtained using a 3T Philips Achieva 3 . 0 system at the Department of Diagnostic Radiology , The University of Hong Kong .","For the aged discs , the IVD were imaged separately as bone-disc-bone segments , at the Department of Electrical and Electronic Engineering , The University of Hong Kong .","The MRS and CEST imaging were performed .","The FOV for the CEST imaging was adjusted to 76 . 8 × 76 . 8 mm2 to accommodate the size of human lumbar discs ( matrix size = 64 × 64 , slice thickness = 2 mm ) .","All MRI experiments were performed at room temperature using a 7 T pre-clinical scanner ( 70/16 Pharmascan , Bruker BioSpin GmbH , Germany ) equipped with a 370 mT/m gradient system along each axis .","Single-channel volume RF coils with different diameters were used for the samples based on size ( 60 mm for GAG phantoms and human cadaveric discs ) .","The MRI images in the transverse view were then assessed for intensity of the image ( brighter signifying more water content ) .","Three transverse MRI images per IVD were overlaid with a grid representing the areas that were cut for mass-spectrometry measurements as outlined previously .","For each region , the ‘intensity’ was represented by the average of the pixel intensities , which were graphically visualised and used for correlative studies .","The endplates were carefully cut off with a scalpel , exposing the surface of the IVD , which were then cut into small segments spanning seven segments in the central left-right lateral axis , and five segments in the central anteroposterior axis ( Figure 1C ) .","In all , this corresponds to a total of 11 locations per IVD .","Among them , four are from the OAF , two from the IAF ( but only in the lateral axis ) , one from the central NP , and four from a transition zone between IAF and the NP ( designated the ‘NP/IAF’ ) .","Samples were stored frozen at −80⁰C until use .","NP and AF disc tissues from spine surgeries ( Table 1 ) were cultured in custom-made Arg- and Lys-free α-MEM ( AthenaES ) as per formulation of Gibco α-MEM ( Cat #11900–024 ) , supplemented with 10% dialysed FBS ( 10 , 000 MWCO , Biowest , Cat# S181D ) , penicillin/streptomycin , 2 . 2 g/L sodium bicarbonate ( Sigma ) , 30 mg/L L-methionine ( Sigma ) , 21 mg/L ‘heavy’ isotope-labelled 13C6L-arginine ( Arg6 , Cambridge Isotopes , Cat # CLM-2265-H ) , 146 mg/L ‘heavy’ isotope-labelled 4 , 4 , 5 , 5-D4 L-Lysine ( Lys4 , Cambridge Isotopes , Cat # DLM-2640 ) .","Tissue explants were cultured for 7 days in hypoxia ( 1% O2 and 5% CO2 in air ) at 37⁰C before being washed with PBS and frozen until use .","The frozen samples were pulverised using a freezer mill ( Spex ) under liquid nitrogen .","Samples were extracted using 15 volumes ( w/v ) of extraction buffer ( 4M guanidine hydrochloride ( GuHCl ) , 50 mM sodium acetate , 100 mM 6-aminocaproic acid , and HALT protease inhibitor cocktail ( Thermo Fischer Scientific ) , pH 5 . 8 ) .","Samples were mechanically dissociated with 10 freeze-thaw cycles and sonicated in a cold water bath , before extraction with gentle agitation at 4°C for 48 hr .","Samples were centrifuged at 15 , 000 g for 30 min at 4°C and the supernatant was ethanol precipitated at a ratio of 1:9 for 16 hr at −20°C .","The ethanol step was repeated and samples were centrifuged at 5000 g for 45 min at 4°C , and the protein pellets were air dried for 30 min .","Protein pellets were re-suspended in fresh 4M urea in 50 mM ammonium bicarbonate , pH 8 , using water bath sonication to aid in the re-solubilisation of the samples .","Samples underwent reduction with TCEP ( 5 mM final concentration ) at 60°C for 1 hr , and alkylation with iodoacetamide ( 500 mM final concentration ) for 20 min at RT .","Protein concentration was measured using the BCA assay ( Biorad ) according to manufacturer’s instructions .","A total of 200 μg of protein was then buffer exchanged with 50 mM ammonium bicarbonate with centricon filters ( Millipore , 30 kDa cutoff ) according to manufacturer’s instructions .","Samples were digested with mass spec grade Trypsin/LysC ( Promega ) as per manufacturer’s instructions .","For SILAC-labelled samples , formic acid was added to a final concentration of 1% , and centrifuged and the supernatant then desalted prior to LC-MS/MS measurements .","For the cadaveric samples , the digested peptides were then acidified with TFA ( 0 . 1% final concentration ) and quantified using the peptide quantitative colorimetric peptide assay kit ( Pierce , catalogue 23275 ) before undergoing fractionation using the High pH reversed phase peptide fractionation kit ( Pierce , catalogue number 84868 ) into four fractions .","Desalted peptides were dried , re-suspended in 0 . 1% formic acid prior to LC-MS/MS measurements .","Samples were loaded onto the Dionex UltiMate 3000 RSLC nano Liquid Chromatography coupled to the Orbitrap Fusion Lumos Tribid Mass Spectrometer .","Peptides were separated on a commercial Acclaim C18 column ( 75 μm internal diameter ×50 cm length , 1 . 9 μm particle size; Thermo ) .","Separation was attained using a linear gradient of increasing buffer B ( 80% ACN and 0 . 1% formic acid ) and declining buffer A ( 0 . 1% formic acid ) at 300 nL/min .","Buffer B was increased to 30% B in 210 min and ramped to 40% B in 10 min followed by a quick ramp to 95% B , where it was held for 5 min before a quick ramp back to 5% B , where it was held and the column was re-equilibrated .","Mass spectrometer was operated in positive polarity mode with capillary temperature of 300°C .","Full survey scan resolution was set to 120 , 000 with an automatic gain control ( AGC ) target value of 2 × 106 , maximum ion injection time of 30 ms , and for a scan range of 400–1500 m/z .","Data acquisition was in DDA mode to automatically isolate and fragment topN multiply charged precursors according to their intensities .","Spectra were obtained at 30 , 000 MS2 resolution with AGC target of 1 × 105 and maximum ion injection time of 100 ms , 1 . 6 m/z isolation width , and normalised collisional energy of 31 .","Preceding precursor ions targeted for HCD were dynamically excluded of 50 s .","Raw data were analysed using MaxQuant ( v . 1 . 6 . 3 . 3 , Germany ) .","Briefly , raw files were searched using Andromeda search engine against human UniProt protein database ( 20 , 395 entries , Oct 2018 ) , supplemented with sequences of contaminant proteins .","Andromeda search parameters for protein identification were set to a tolerance of 6 ppm for the parental peptide , and 20 ppm for fragmentation spectra and trypsin specificity allowing up to two miscleaved sites .","Oxidation of methionine , carboxyamidomethylation of cysteines was specified as a fixed modification .","Minimal required peptide length was specified at seven amino acids .","Peptides and proteins detected by at least two LFQ ion counts for each peptide in one of the samples were accepted , with a false discovery rate ( FDR ) of 1% .","Proteins were quantified by normalised summed peptide intensities computed in MaxQuant with the LFQ option enabled .","A total of 66 profiles were obtained: 11 locations × 3 disc levels × 2 individuals; with a median of 665 proteins ( minimum 419 , maximum 1920 ) per profile .","The high-resolution , high mass accuracy mass spectrometry ( MS ) data obtained were processed using Proteome Discoverer ( Ver 2 . 1 ) , wherein data were searched using Sequest algorithm against Human UniProt database ( 29 , 900 entries , May 2016 ) , supplemented with sequences of contaminant proteins , using the following search parameters settings: oxidised methionine ( M ) , acetylation ( Protein N-term ) , heavy Arginine ( R6 ) and Lysine ( K4 ) were selected as dynamic modifications , carboxyamidomethylation of cysteines was specified as a fixed modification , minimum peptide length of 7 amino acids was enabled , tolerance of 10 ppm for the parental peptide , and 20 ppm for fragmentation spectra , and trypsin specificity allowing up to two miscleaved sites .","Confident proteins were identified using a target-decoy approach with a reversed database , strict FDR 1% at peptide and PSM level .","Newly synthesised proteins were heavy labelled with Arg6- and Lys4 , and the data was expressed as the normalised protein abundance obtained from heavy ( labelled ) /light ( un-labelled ) ratio .","Degradome analyses was performed on NP and AF from three non-degenerated and three degenerated individuals ( Table 1 ) .","Frozen tissues were pulverised as described above and prepared for TAILS as previously reported ( Kleifeld et al . , 2010 ) .","After extraction with SDS buffer ( 1% SDS , 100 mM dithiothreitol , 1X protease inhibitor in deionised water ) and sonication ( three cycles , 15 s/cycle ) , the supernatant ( soluble fraction ) underwent reduction at 37°C and alkylation with a final concentration of 15 mM iodoacetamide for 30 min at RT .","Samples were precipitated using chloroform/methanol , and the protein pellet air dried .","Samples were re-suspended in 1M NaOH , quantified by nanodrop , diluted to 100 mM HEPES and 4M GnHCl and pH adjusted ( pH6 . 5–7 . 5 ) prior to 6-plex TMT labelling as per manufacturer’s instructions ( Sixplex TMT , Cat# 90061 , ThermoFisher Scientific ) .","Equal ratios of TMT-labelled samples were pooled and methanol/chloroform precipitated .","Protein pellets were air-dried and re-suspended in 200 mM HEPES ( pH8 ) , and digested with trypsin ( 1:100 ratio ) for 16 hr at 37°C , pH 6 . 5 and a sample was taken for pre-TAILS .","High-molecular-weight dendritic polyglycerol aldehyde polymer ( ratio of 5:1 w/w polymer to sample ) and NaBH3CN ( to a final concentration of 80 mM ) was added , incubated at 37°C for 16 hr , followed by quenching with 100 mM ethanolamine ( 30 min at 37°C ) and underwent ultrafiltration ( MWCO of 10 , 000 ) .","Collected samples were desalted , acidified to 0 . 1% formic acid and dried , prior to MS analysis .","Samples were analysed on a Thermo Scientific Easy nLC-1000 coupled online to a Bruker Daltonics Impact II UHR QTOF .","Briefly , peptides were loaded onto a 20 cm x 75 µm I . D . analytical column packed with 1 . 8 µm C18 material ( Dr . Maisch GmbH , Germany ) in 100% buffer A ( 99 . 9% H2O , 0 . 1% formic acid ) at 800 bar followed by a linear gradient elution in buffer B ( 99 . 9% acetonitrile , 0 . 1% formic acid ) to a final 30% buffer B for a total 180 min including washing with 95% buffer B . Eluted peptides were ionised by ESI and peptide ions were subjected to tandem MS analysis using a data-dependent acquisition method .","A top17 method was employed , where the top 17 most intense multiply charged precursor ions were isolated for MS/MS using collision-induced-dissociation , and actively excluded for 30 s .","MGF files were extracted and searched using Mascot against the UniProt Homo sapiens database , with semi-ArgC specificity , TMT6plex quantification , variable oxidation of methionine , variable acetylation of N termini , 20 ppm MS1 error tolerance , 0 . 05 Da MS2 error tolerance and two missed cleavages .","Mascot .","dat files were imported into Scaffold Q+S v4 . 4 . 3 for peptide identification processing to a final FDR of 1% .","Quantitative values were calculated through Scaffold and used for subsequent analyses .","AF and NP tissues from four individuals were cut into approximately 0 . 5 cm3 pieces , and put into the Dulbecco's modified Eagle's medium ( DMEM ) ( Gibco ) supplemented with 20 mM HEPES ( USB ) , 1% penicillin-streptomycin ( Gibco ) and 0 . 4% fungizone ( Gibco ) .","The tissues were digested with 0 . 2% pronase ( Roche ) for 1 hr , and centrifuged at 200 g for 5 min to remove supernatant .","AF and NP were then digested by 0 . 1% type II collagenase ( Worthington Biochemical ) for 14 hr and 0 . 05% type II collagenase for 8 hr , respectively .","Cell suspension was filtered through a 70 µm cell strainer ( BD Falcon ) and centrifuged at 200 g for 5 min .","The cell pellet was washed with phosphate buffered saline ( PBS ) and centrifuged again to remove the supernatant .","RNA was then extracted from the isolated disc cells using Absolutely RNA Nanoprep Kit ( Stratagene ) , following manufacturer’s protocol , and stored at −80°C until further processing .","The quality and quantity of total RNA were assessed on the Bioanalyzer ( Agilent ) using the RNA 6000 Nano total RNA assay .","cDNA was generated using Affymetrix GeneChip Two-Cycle cDNA Synthesis Kit , followed by in vitro transcription to produce biotin-labelled cRNA .","The sample was then hybridised onto the Affymetrix GeneChip Human Genome U133 Plus 2 . 0 Array .","The array image , CEL file and other related files were generated using Affymetrix GeneChip Command Console .","The experiment was conducted as a service at the Centre for PanorOmic Sciences of the University of Hong Kong .","CEL and other files were loaded into GeneSpring GX 10 ( Agilent ) software .","The RMA algorithm was used for probe summation .","Data were normalised with baseline transformed to median of all samples .","A loose filtering based on the raw intensity values was then applied to remove background noise .","Consequently , transcriptomic data with a total of 54 , 675 probes ( corresponding to 20 , 887 genes ) and eight profiles were obtained .","The detected proteins were compared against the transcription factor ( TF ) database ( Vaquerizas et al . , 2009 ) and the human genome nomenclature consortium database for cell surface markers ( CDs ) ( Braschi et al . , 2019 ) , where 77 TFs and 83 CDs were detected ( Figure 1—figure supplement 3A ) .","Excluding missing values , the LFQ levels among the data-points range from 15 . 6 to 41 . 1 , with a Gaussian-like empirical distribution ( Figure 2—figure supplement 1A ) .","The numbers of valid values per protein were found to decline rapidly when they were sorted in descending order ( Figure 2—figure supplement 1B , upper panel ) .","To perform PCAs , only a subset of genes with sufficiently large numbers of valid values ( i . e . non-missing values ) were used .","The cutoff for this was chosen based on a point corresponding to the steepest slope of descending order of valid protein numbers ( Figure 2—figure supplement 1B , second panel ) , such that the increase of valid values is slower than the increase of missing values beyond that point .","Subsequently , the top 507 genes were picked representing 59 . 8% of all valid values .","This new subset includes 12 . 4% of all missing values .","Since the subset of data still contains some missing values , an imputation strategy was adopted employing the Multiple Imputation by Chained Equations ( MICE ) method and package ( Buuren and Groothuis-Oudshoorn , 2011 ) , with a max iteration set at 50 and the default PMM method ( predictive mean matching ) .","To further ensure normality , Winsorisation was applied such that genes whose average is below 5% or above 95% of all genes were also excluded from PCA .","The data was then profile-wise standardised ( zero-mean and one standard deviation ) before PCA was applied on the R platform ( R Development Core Team , 2013 ) .","To assess the impact of the spatiotemporal factors on the proteomic profiles , we performed Analysis of Variance ( ANOVA ) , correlating each protein to the age , compartments , level , and directionality .","To draw the soft boundaries on the PCA plot between groups of samples , support vector machines with polynomial ( degree of 2 ) kernel were applied using the LIBSVM package ( Chang and Lin , 2011 ) and the PCA coordinates as inputs for training .","A meshed grid covering the whole PCA field was created to make prediction and draw probability contours for −0 . 5 , 0 , and 0 . 5 from the fitted model .","Hierarchical clustering was performed with ( 1- correlation coefficient ) as the distance metrics unless otherwise specified .","To address the problem of ‘dropout’ effects while avoiding extra inter-dependency introduced due to imputations , we adopted three strategies in calculating the DEPs , namely , by statistical testing , exclusively detected , and fold-change cutoff approaches .","First , for the proteins that have over half valid values in both groups under comparison , we performed t-testing with p-values adjusted for multiple testing by the false discovery rate ( FDR ) .","Those with FDR below 0 . 05 were considered statistical DEPs .","Second , for the proteins where one group has some valid values while the other group is completely not detected , we considered the ones with over half valid values in one group to be exclusive DEPs .","For those proteins that were expressed in <50% in both groups , the ones with fold-change greater than two were also considered to be DEPs .","To fit the lateral and anteroposterior trends for the modules of genes identified in the young samples , a Gaussian Process Estimation ( GPE ) model was trained using the GauPro package in R Development Core Team , 2013 .","Pathway analyses was conducted on the GSEA ( Subramanian et al . , 2005 ) .","Signalling proteins was compiled based on 25 Signal transduction pathways listed on KEGG ( Kanehisa et al . , 2019 ) .","For transcriptomic data , we used a thresholding approach to detect DEGs ( differentially expressed genes ) , whereby a gene was considered a DEG if the log2 ( fold-change ) is greater than three and the average expression ( logarithmic scale ) is greater than 10 ( Figure 6—figure supplement 1E–H ) .","The LASSO model between MRI and proteome was trained using the R package ‘glmnet’ , wherein the 85 ECMs were first imputed for missing values in them using MICE .","Nine ECMs were not imputed for too many missing values , leaving 76 for training and testing .","The best value for λ was determined by cross-validations .","A model was then trained on the aged MRIs ( dependent variable ) and aged proteome of the 76 genes ( independent variable ) .","The fitted model was then applied to the young proteome to predict MRIs of the young discs .","The custom scripts for processing and analysing the data were housed at github . com/hkudclab/DIPPER; Tam , 2021; copy archived at swh:1:rev:49e4da79786de1dfa496d7c7472343f3b865696c .","An interactive web interface for the data is available at http://www . sbms . hku . hk/dclab/DIPPER/ ."]],"string":"[\n [\n \"The 23 intervertebral discs ( IVDs ) in the human spine provide stability , mobility , and flexibility .\",\n \"IVD degeneration ( IDD ) , most common in the lumbar region ( Saleem et al . , 2013; Teraguchi et al . , 2014 ) , is associated with a decline in function and a major cause of back pain , affecting up to 80% of the world’s population at some point in life ( Rubin , 2007 ) , presenting significant socioeconomic burdens .\",\n \"Multiple interacting factors such as genetics , influenced by ageing , mechanical and other stress factors , contribute to the pathobiology , onset , severity , and progression of IDD ( Munir et al . , 2018 ) .\",\n \"IVDs are large , avascular , extracellular matrix ( ECM ) -rich structures comprising three compartments: a hydrated nucleus pulposus ( NP ) at the centre , surrounded by a tough annulus fibrosus ( AF ) at the periphery , and cartilaginous endplates of the adjoining vertebral bodies ( Humzah and Soames , 1988 ) .\",\n \"The early adolescent NP is populated with vacuolated notochordal-like cells , which are gradually replaced by small chondrocyte-like cells ( Risbud et al . , 2015 ) .\",\n \"Blood vessels terminate at the endplates , nourishing and oxygenating the NP via diffusion , whose limited capacity mean that NP cells are constantly subject to hypoxic , metabolic , and mechanical stresses ( Urban et al . , 2004 ) .\",\n \"With ageing and degeneration , there is an overall decline in cell ‘health’ and numbers ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) , disrupting homeostasis of the disc proteome .\",\n \"The ECM has key roles in biomechanical function and disc hydration .\",\n \"Indeed , a hallmark of IDD is reduced hydration in the NP , diminishing the disc’s capacity to dissipate mechanical loads .\",\n \"Clinically , T-2 weighted MRI is the gold standard for assessing IDD , that uses disc hydration and structural features such as bulging or annular tears to measure severity ( Pfirrmann et al . , 2001; Schneiderman et al . , 1987 ) .\",\n \"The hydration and mechanical properties of the IVD are dictated by the ECM composition , which is produced and maintained by the IVD cells .\",\n \"To meet specific biomechanical needs , cells in the IVD compartments synthesise different compositions of ECM proteins .\",\n \"Defined as the ‘matrisome’ ( Naba et al . , 2012 ) , the ECM houses the cells and facilitates their inter-communication by regulation of the availability and presentation of signalling molecules ( Taha and Naba , 2019 ) .\",\n \"With ageing or degeneration , the NP becomes more fibrotic and less compliant ( Yee et al . , 2016 ) , ultimately affecting disc biomechanics ( Newell et al . , 2017 ) .\",\n \"Changes in matrix stiffness can have a profound impact on cell-matrix interactions and downstream transcriptional regulation , signalling activity , and cell fate ( Park et al . , 2011 ) .\",\n \"The resulting alterations in the matrisome lead to vicious feedback cycles that reinforce cellular degeneration and ECM changes .\",\n \"Notably , many of the associated IDD genetic risk factors , such as COL9A1 ( Jim et al . , 2005 ) , ASPN ( Song et al . , 2008 ) , and CHST3 ( Song et al . , 2013 ) , are variants in genes encoding matrisome proteins , highlighting their importance for disc function .\",\n \"Therefore , knowledge of the cellular and extracellular proteome and their spatial distribution in the IVD is crucial to understanding the mechanisms underlying the onset and progression of IDD ( Feng et al . , 2006 ) .\",\n \"Current knowledge of IVD biology is inferred from a limited number of transcriptomic studies on human ( Minogue et al . , 2010; Riester et al . , 2018; Rutges et al . , 2010 ) and animal ( Veras et al . , 2020 ) discs .\",\n \"Studies showed that cells in young healthy NP express markers including CD24 , KRT8 , KRT19 , and T ( Fujita et al . , 2005; Minogue et al . , 2010; Rutges et al . , 2010 ) , whereas NP cells in aged or degenerated discs have different and variable molecular signatures ( Chen et al . , 2006; Rodrigues-Pinto et al . , 2016 ) , such as genes involved in TGFβ signalling ( TGFA , INHA , INHBA , BMP2/6 ) .\",\n \"The healthy AF expresses genes including collagens ( COL1A1 and COL12A1 ) ( van den Akker et al . , 2017 ) , growth factors ( PDGFB , FGF9 , VEGFC ) , and signalling molecules ( NOTCH and WNT ) ( Riester et al . , 2018 ) .\",\n \"Although transcriptomic data provides valuable cellular information , it does not faithfully reflect the molecular composition .\",\n \"Cells represent only a small fraction of the disc volume , transcriptome-proteome discordance does not enable accurate predictions of protein levels from mRNA ( Fortelny et al . , 2017 ) , and the disc matrisome accumulates and remodels over time .\",\n \"Proteomic studies on animal models of IDD , including murine ( McCann et al . , 2015 ) , canine ( Erwin et al . , 2015 ) , and bovine ( Caldeira et al . , 2017 ) , have been reported .\",\n \"Nevertheless , human-animal differences in cellular phenotypes and mechanical loading physiologies mean that these findings might not translate to the human scenario .\",\n \"So far , human proteomic studies have compared IVDs with other cartilaginous tissues ( Önnerfjord et al . , 2012 ) and have shown increases in fibrotic changes in aging and degeneration ( Yee et al . , 2016 ) , a role for inflammation in degenerated discs ( Rajasekaran et al . , 2020 ) , the presence of haemoglobins and immunoglobulins in discs with spondylolisthesis and herniation ( Maseda et al . , 2016 ) , and changes in proteins related to cell adhesion and migration in IDD ( Sarath Babu et al . , 2016 ) .\",\n \"The reported human disc proteomes were limited in the numbers of proteins identified and finer compartmentalisation within the IVD , and disc levels along the lumbar spine have yet to be studied .\",\n \"Nor have the proteome dynamics in term of ECM remodelling ( synthesis and degradation ) in young human IVDs and changes in ageing and degeneration been described .\",\n \"In this study , we presented DIPPER ( the Big Dipper are point-reference stars for guiding nautical voyages ) , a comprehensive disc proteomic resource , comprising static spatial proteome , dynamic proteome and transcriptome , and a methodological flow , for studying the human IVD in youth , ageing , and degeneration .\",\n \"First , we established a high-resolution point-reference map of static spatial proteomes along the lateral and anteroposterior directions of IVDs at three lumbar levels , contributed by a young ( 16M ) and an aged ( 59M ) cadavers with no reported scoliosis or degeneration .\",\n \"We evaluated variations among the disc compartments and levels by principal component analysis ( PCA ) , analysis of variance ( ANOVA ) , and identification of differentially expressed proteins ( DEPs ) .\",\n \"We discovered modules containing specific sets of proteins that describe the directional trends of a young IVD , and the deconstruction of these modules with ageing and degeneration .\",\n \"Using a LASSO regression model , we identified proteins ( the hydration matrisome ) predictive of tissue hydration as indicated by high-resolution MRI of the aged discs .\",\n \"Finally , we showed how the point-reference proteomes can be utilised , to integrate with other independent transcriptome and dynamic proteome ( SILAC and degradome ) datasets from 15 additional clinical disc specimens , elevating the robustness of the proteomic findings .\",\n \"An explorable web interface hosting the data and findings is presented , serving as a useful resource for the scientific community .\"\n ],\n [\n \"DIPPER comprises 94 genome-wide measurements from lumbar disc components of 17 individuals ( Figure 1A; Table 1 ) , with data types ranging from label-free proteomics , transcriptomics , SILAC to degradome ( López-Otín and Overall , 2002 ) .\",\n \"High-resolution static spatial proteomes were generated from multiple intact disc segments of young trauma-induced ( 16 M ) and aged ( 57 M ) cadaveric spines .\",\n \"T1- and T2-weighted MRI ( 3T ) showed the young discs ( L3/4 , L4/5 , L5/S1 ) were non-degenerated , with a Schneiderman score of 1 from T2 images ( Figure 1B ) .\",\n \"The NP of young IVD were well hydrated ( white ) with no disc bulging , endplate changes , or observable inter-level variations ( Figure 1B; Figure 1—figure supplement 1A ) , consistent with healthy discs and were deemed fit to serve as a benchmarking point-reference .\",\n \"To investigate structural changes associated with ageing , high-resolution ( 7T ) MRI was taken for the aged discs ( Figure 1C ) .\",\n \"All discs had irregular endplates , and annular tears were present ( green arrowheads ) adjacent to the lower endplate and extending towards the posterior region at L3/4 and L4/5 ( Figure 1C ) .\",\n \"The NP exhibited regional variations in hydration in both sagittal and transverse images ( Figure 1C ) .\",\n \"Morphologically , the aged discs were less hydrated and the NP and AF structures less distinct , consistent with gross observations ( Figure 1—figure supplement 1A ) .\",\n \"Scoliosis was not detected in these two individuals .\",\n \"Information of the disc samples used in the generation of other profiling data are described in Materials and methods and Table\",\n \"1 . They are clinical samples taken from patients undergoing surgery .\",\n \"The disc levels and intactness varied , and thus are more suitable for cross-validation purposes and some are directly relevant to IDD .\",\n \"The intact discs from the two cadaveric spines enabled us to derive spatial proteomes for young and aged human IVDs .\",\n \"We subdivided each lumbar disc into 11 key regions ( Figure 1D ) , spanning the outer-most ( outer AF; OAF ) to the central-most ( NP ) region of the disc , traversing both anteroposterior and lateral axes , adding valuable spatial information to our proteomic dataset .\",\n \"Since the disc is an oval shape , an inner AF ( IAF ) region was assigned in the lateral directions .\",\n \"A ‘mixed’ compartment between the NP and IAF with undefined boundary was designated as the NP/IAF in all four ( anteroposterior and lateral ) directions .\",\n \"In all , this amounted to 66 specimens with different compartments , ages , directions and levels , which then underwent LC-MS/MS profiling ( Supplementary file 1 ) .\",\n \"Systematic analyses of the 66 profiles are depicted in a flowchart ( Figure 1A ) .\",\n \"A median of 654 proteins per profile were identified for the young samples and 829 proteins for the aged samples , with a median of 742 proteins per profile for young and aged combined .\",\n \"The proteome-wide distributions were on similar scales across the profiles ( Figure 1—figure supplement 1B ) .\",\n \"Of the 3100 proteins detected in total , 418 were matrisome proteins ( 40 . 7% of all known matrisome proteins ) and 2682 non-matrisome proteins ( ~14% of genome-wide non-matrisome genes ) ( Figure 1E; Figure 1—figure supplement 1C ) , and 983 were common to all four major compartments , namely the OAF , IAF , NP/IAF , and NP ( Figure 1E , upper panel ) .\",\n \"A total of 1883 proteins were identified in young discs , of which 690 ( 36% ) were common to all regions .\",\n \"Additionally , 45 proteins ( 2 . 4% ) were unique to NP , whilst NP/IAF , IAF , and OAF had 86 ( 4 . 6% ) , 54 ( 2 . 9% ) , and 536 ( 28% ) unique proteins , respectively ( Figure 1E , middle panel ) .\",\n \"For the aged discs , 2791 proteins were identified , of which 803 ( 28 . 8% ) were common to all regions .\",\n \"NP , NP/IAF , and IAF had 34 ( 12% ) , 80 ( 28 . 7% ) , and 44 ( 15% ) unique proteins , respectively , with the OAF accounting for the highest proportion of 1314 unique proteins ( 47% ) ( Figure 1E , lower panel ) .\",\n \"The aged OAF had the highest number of detected proteins with an average of 1156 , followed by the young OAF with an average of 818 ( Figure 1F ) .\",\n \"The quantity and spectrum of protein categories identified suggest sufficient proteins had been extracted and the data are of high quality .\",\n \"We divided the detected matrisome proteins into core matrisome ( ECM proteins , encompassing collagens , proteoglycans , and glycoproteins ) and non-core matrisome ( ECM regulators , ECM affiliated , and secreted factors ) , according to a matrisome classification database ( Naba et al . , 2012 ) ( matrisomeproject . mit . edu ) ( Figure 1F ) .\",\n \"Despite the large range of total numbers of proteins detected ( 419 to 1920 ) across the 66 profiles ( Figure 1F ) , all six subcategories of the matrisome contained similar numbers of ECM proteins ( Figure 1F and G; Figure 1—figure supplement 2A ) .\",\n \"The non-core matrisome proteins were significantly more abundant in aged than in young discs ( Figure 1—figure supplement 2A ) .\",\n \"On average , 19 collagens , 18 proteoglycans , 68 glycoproteins , 52 ECM regulators , 22 ECM affiliated proteins , and 29 secreted factors of ECM were detected per profile .\",\n \"The majority of the proteins in these matrisome categories were detected in all disc compartments , and in both age groups .\",\n \"A summary of all the comparisons are presented in Figure 1—figure supplement 1C–E , and the commonly expressed matrisome proteins are listed in Table\",\n \"2 . Even though there are approximately three times more non-matrisome than matrisome proteins per profile on average ( Figure 1F ) , their expression levels in terms of label-free quantification ( LFQ ) values are markedly lower ( Figure 1—figure supplement 2B ) .\",\n \"Specifically , the expression levels of core-matrisome were the highest , with an average log2 ( LFQ ) of 30 . 65 , followed by non-core matrisome at 28 . 56 , and then non-matrisome at 27 . 28 ( Figure 1—figure supplement 2B ) .\",\n \"Within the core-matrisome , the expression was higher ( p=6 . 4 × 10−21 ) in young ( median 30 . 74 ) than aged ( median 29 . 72 ) discs ( Figure 1—figure supplement 2C & D ) .\",\n \"This difference between young and aged discs is consistent within the subcategories of core and non-core matrisome , with the exception of the ECM regulator category ( Figure 1H ) .\",\n \"The non-core matrisome and non-matrisome , however , exhibited smaller cross-compartment and cross-age differences in terms of expression levels ( Figure 1—figure supplement 2E–H ) .\",\n \"That is , the levels of ECM proteins in each compartment of the disc declines with ageing and possibly changes in the relative composition , while the numbers of proteins detected per matrisome subcategory remain similar .\",\n \"This agrees with the concept that with ageing , ECM synthesis is not sufficient to counterbalance degradation , as exemplified in a proteoglycan study ( Silagi et al . , 2018 ) .\",\n \"Although 86 . 5% ( 2682 ) of the detected proteins were non-matrisome , their expression levels were considerably lower than matrisome proteins across all sample profiles ( Figure 1F ) .\",\n \"A functional categorisation according to the Human Genome Nomenclature Committee gene family annotations ( Yates et al . , 2017 ) showed many categories containing information for cellular components and activities , with the top 30 listed in Figure 1I .\",\n \"These included transcriptional and translational machineries , post-translational modifications , mitochondrial function , protein turnover and importantly , transcriptional factors , cell surface markers , and inflammatory proteins that can inform gene regulation , cell identity , and response in the context of IVD homeostasis , ageing , and degeneration .\",\n \"These functional overviews highlighted 77 DNA-binding proteins and/or transcription factors , 83 cell surface markers , and 175 inflammatory-related proteins , with their clustering data presented as heatmaps ( Figure 1—figure supplement 3 ) .\",\n \"Transcription factors and cell surface markers are detected in some profiles ( Figure 1—figure supplement 3A and B ) .\",\n \"The heatmap of the inflammatory-related proteins showed that more than half of the proteins are detected in the majority of samples , with four major clusters distinguished by age and expression levels ( Figure 1—figure supplement 3C ) .\",\n \"For example , one of the clusters in the aged samples showed enrichment for complement and coagulation cascades ( False Discovery Rate , FDR q = 1 . 62 × 10−21 ) and clotting factors ( FDR q = 6 . 05 × 10−9 ) , indicating potential infiltration of blood vessels .\",\n \"Lastly , there are 371 proteins involved in signalling pathways , and their detection frequency in the different compartments and heat map expression levels are illustrated in Figure 1—figure supplement 3D .\",\n \"Cellularity within the IVD , especially the NP , decreases with age and degeneration ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) .\",\n \"We assessed whether cellularity of the different compartments could be inferred from the proteomic data .\",\n \"Quantitation of histones can reflect the relative cellular content of tissues ( Wiśniewski et al . , 2014 ) .\",\n \"We detected 10 histones , including subunits of histone 1 ( HIST1H1B/C/D/E , HIST1H2BL , HIST1H3A , HIST1H4A ) and histone 2 ( HIST2H2AC , HIST2H2BE , HIST2H3A ) , with four subunits identified in over 60 sample profiles that are mutually co-expressed ( Figure 1—figure supplement 4A ) .\",\n \"Interestingly , histone concentrations , and thus cellularity , increased from the inner to the outer compartments of the disc , and showed a highly significant decrease in aged discs compared to young discs across all compartments ( Figure 1J , Wilcoxon p=5 . 6 × 10−4 ; Figure 1—figure supplement 4B ) .\",\n \"GAPDH and ACTA2 are two commonly used reference proteins , involved in the metabolic process and the cytoskeletal organisation of cells , respectively .\",\n \"They are expected to be relatively constant between cells and are used to quantify the relative cellular content of tissues ( Barber et al . , 2005 ) .\",\n \"They were detected in all 66 profiles .\",\n \"GAPDH and ACTA2 amounts were significantly correlated with a Pearson correlation coefficient ( PCC ) of 0 . 794 ( p=9 . 3 × 10−9 ) ( Figure 1—figure supplement 4C ) , and they were both significantly co-expressed with the detected histone subunits , with PCCs of 0 . 785 and 0 . 636 , respectively ( Figure 1K; Figure 1—figure supplement 4D ) .\",\n \"As expected , expression of the histones , GAPDH , and ACTA2 was not correlated with two core-matrisome proteins , ACAN and COL2A1 ( Figure 1—figure supplement 4E ) , whereas ACAN and COL2A1 were significantly co-expressed ( Figure 1—figure supplement 4F ) , as expected due to their related regulation of expression and tissue function .\",\n \"Thus , cellularity information can be obtained from proteomic information , and the histone quantification showing reduced cellularity in the aged IVD is consistent with the reported changes ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) .\",\n \"To gain a global overview of the data , we performed PCA on a set of 507 proteins selected computationally , allowing maximal capture of valid values , while incurring minimal missing values , followed by imputations ( Figure 2—figure supplement 1; Materials and methods ) .\",\n \"The first two principal components ( PCs ) explained a combined 65 . 5% of the variance , with 39 . 9% and 25 . 6% for the first and second PCs , respectively ( Figure 2A and B ) .\",\n \"A support vector machine with polynomial kernel was trained to predict the boundaries: it showed PC1 to be most informative to predict age , with a clear demarcation between the two age groups ( Figure 2A , vertical boundary ) , whereas PC2 distinguished disc sample localities , separating the inner compartments ( NP , NP/IAF , and IAF ) from the OAF ( Figure 2A , horizontal boundary ) .\",\n \"PC3 captured only 5 . 0% of the variance ( Figure 2C & D ) , but it distinguished disc level , separating the lowest level ( L5/S1 ) from the rest of the lumbar discs ( L3/4 and L4/5 ) ( Figure 2D , horizontal boundary ) .\",\n \"Samples in the upper level ( L3/4 and L4/5 ) appeared to be more divergent , with the aged disc samples deviating from the young ones ( Figure 2D ) .\",\n \"To extract the most informative features of the PCA , we performed proteome-wide associations with each of the top three PCs , which accounted for over 70% of total variance , and presented the top 100 most positively and top 100 most negatively correlated proteins for each of the PCs ( Figure 2E–G ) .\",\n \"As expected , the correlation coefficients in absolute values were in the order of PC1 > PC2 > PC3 ( Figure 2E–G ) .\",\n \"The protein content is presented as non-matrisome proteins ( grey colour ) and matrisome proteins ( coloured ) that are subcategorised as previously .\",\n \"For the negatively correlated proteins , the matrisome proteins contributed to PC1 in distinguishing young disc samples , as well as to PC2 for sample location within the disc , but less so for disc level in PC3 .\",\n \"Further , the relative composition of the core and non-core matrisome proteins varied between the three PCs , depicting the dynamic ECM requirement and its relevance in aging ( PC1 ) , tissue composition within the disc ( PC2 ) and mechanical loading ( PC3 ) .\",\n \"PC1 of young discs identified known chondrocyte markers , CLEC3A ( Lau et al . , 2018 ) and LECT1/2 ( Zhu et al . , 2019 ) , hedgehog signalling proteins , HHIPL2 , HHIP , and SCUBE1 ( Johnson et al . , 2012 ) , and xylosyltransferase-1 ( XYLT1 ) , a key enzyme for initiating the attachment of glycosaminoglycan side chains to proteoglycan core proteins ( Silagi et al . , 2018; Figure 2E ) .\",\n \"Most of the proteins that were positively correlated in PC1 were coagulation factors or coagulation related , suggesting enhanced blood infiltration in aged discs .\",\n \"PC2 implicated key changes in molecular signalling proteins ( hedgehog , WNT and Nodal ) in the differences between the inner and outer disc regions ( Figure 2F ) .\",\n \"Notably , PC2 contains heat shock proteins ( HSPA1B , HSPA8 , HSP90AA1 , HSPB1 ) which are more strongly expressed in the OAF than in inner disc , indicating the OAF is under stress ( Takao and Iwaki , 2002 ) .\",\n \"Although the correlations in PC3 were much weaker , proteins such as CILP/CILP2 , DCN , and LUM were associated with lower disc level .\",\n \"To investigate how age , disc compartment , level , and direction affect the protein profiles , we carried out ANOVA for each of these phenotypic factors for the categories of matrisome and non-matrisome proteins ( Figure 2—figure supplement 1H–J ) .\",\n \"In the young discs , the dominant phenotype explaining the variances for all protein categories was disc compartment .\",\n \"It is crucial that each disc compartment ( NP , IAF , and OAF ) has the appropriate protein composition to function correctly ( Figure 2—figure supplement 1I ) .\",\n \"This also fits the understanding that young healthy discs are axially symmetric and do not vary across disc levels .\",\n \"In aged discs , compartment is still relevant for non-matrisome proteins and collagen , but disc level and directions become influential for other protein categories , which is consistent with variations in mechanical loading occurring in the discs with ageing and degeneration ( Figure 2—figure supplement 1J ) .\",\n \"In the combined ( young and aged ) disc samples , age was the dominant phenotype across major matrisome categories , while compartment best explained the variance in non-matrisome , reflecting the expected changes in cellular ( non-matrisome ) and structural ( matrisome ) functions of the discs ( Figure 2—figure supplement 1H ) .\",\n \"This guided us to analyse the young and aged profiles separately , before performing cross-age comparisons .\",\n \"PCA of the 33 young profiles showed a distinctive separation of the OAF from the inner disc regions on PC1 ( upper panel of Figure 3A ) .\",\n \"In PC2 , the lower level L5/S1 could generally be distinguished from the upper lumbar levels ( lower panel of Figure 3A ) .\",\n \"The detected proteome of the young discs ( Figure 3B ) accounts for 9 . 2% of the human proteome ( or 1883 out of the 20 , 368 on UniProt ) .\",\n \"We performed multiple levels of pairwise comparisons ( summarised in Figure 3—figure supplement 1A ) to detect proteins associated with individual phenotypes , using three approaches ( see Materials and methods ) : statistical tests; proteins detected in one group only; or proteins using a fold-change threshold .\",\n \"We detected a set of 671 DEPs ( Supplementary file 2 ) ( termed the ‘variable set’ ) , containing both matrisome and non-matrisome proteins ( Figure 3D ) , and visualised in a heatmap ( Figure 3—figure supplement 2 ) , with identification of four modules ( Y1-Y4 ) .\",\n \"To investigate how the modules are associated with disc components , we compared their protein expression profiles along the lateral and anteroposterior axes .\",\n \"The original log2 ( LFQ ) values were transformed to z-scores to be on the same scale .\",\n \"Proteins of the respective modules were superimposed on the same charts , disc levels combined or separated ( Figure 3E–H; Figure 3—figure supplement 3 ) .\",\n \"Module Y1 is functionally relevant to NP , containing previously reported NP and novel markers KRT19 , CD109 , KRT8 and CHRD ( Anderson et al . , 2002 ) , CHRDL2 , SCUBE1 ( Johnson et al . , 2012 ) , and CLEC3B .\",\n \"Proteins levels in Y1 are lower on one side of the OAF , increases towards the central NP , before declining towards the opposing side , forming a concave pattern in both lateral and anteroposterior directions , with a shoulder drop occurring between IAF and OAF ( Figure 3E ) .\",\n \"Module Y2 was enriched in proteins for ECM organisation ( FDR q = 8 . 8 × 10−24 ) ; Y3 was enriched for proteins involved in smooth muscle contraction processes ( FDR q = 8 . 96 × 10−19 ) ; and Y4 was enriched for proteins of the innate immune system ( FDR q = 2 . 93 × 10−20 ) .\",\n \"Interestingly , these modules all showed a tendency for convex patterns with upward expression toward the OAF regions , in both lateral and anteroposterior directions , in all levels , combined or separated ( Figure 3F–H; Figure 3—figure supplement 3B–D ) .\",\n \"The higher proportions of ECM , muscle contraction , and immune system proteins in the OAF are consistent with the contractile function of the AF ( Nakai et al . , 2016 ) , and with the NP being avascular and ‘immune-privileged’ in a young homeostatic environment ( Sun et al . , 2020 ) .\",\n \"The most distinctive pattern on the PCA is the separation of the OAF from the inner disc , with 99 proteins expressed higher in the OAF and 55 expressed higher in the inner disc ( Figure 3I , J ) .\",\n \"Notably , OAF and inner disc contained different types of ECM proteins .\",\n \"The inner disc regions were enriched in collagens ( COL3A1 , COL5A1 , COL5A2 , and COL11A2 ) , matrillin ( MATN3 ) , and proteins associated with ECM synthesis ( PCOLCE ) and matrix remodelling ( MXRA5 ) .\",\n \"We also identified in the inner disc previously reported NP markers ( KRT19 , KRT8 ) ( Risbud et al . , 2015 ) , in addition to inhibitors of WNT ( FRZB , DKK3 ) and BMP ( CHRD , CHRDL2 ) signalling ( Figure 3I , J ) .\",\n \"Of note , FRZB and CHRDL2 were recently shown to have potential protective characteristics in osteoarthritis ( Ji et al . , 2019 ) .\",\n \"The TGFβ pathway appears to be suppressed in the inner disc where antagonist CD109 ( Bizet et al . , 2011; Li et al . , 2016 ) is highly expressed , and the TGFβ activity indicator TGFBI is expressed higher in the OAF than in the inner disc .\",\n \"The OAF is enriched with various collagens ( COL1A1 , COL6A1/2/3 , COL12A1 and COL14A1 ) , basement membrane ( BM ) proteins ( LAMA4 , LAMB2 and LAMC1 ) , small leucine-rich proteoglycans ( SLRP ) ( BGN , DCN , FMOD , OGN , PRELP ) , and BM-anchoring protein ( PRELP ) ( Figure 3I , J ) .\",\n \"Tendon-related markers such as thrombospondins ( THBS1/2/4 ) ( Subramanian and Schilling , 2014 ) and cartilage intermediate layer proteins ( CILP/CILP2 ) are also expressed higher in the OAF .\",\n \"Tenomodulin ( TNMD ) was exclusively expressed in 9 of the 12 young OAF profiles , and not in any other compartments ( Supplementary file 2 ) .\",\n \"This fits a current understanding of the AF as a tendon/ligament-like structure ( Nakamichi et al . , 2018 ) .\",\n \"In addition , the OAF was enriched in actin-myosin ( Figure 3I ) , suggesting a role of contractile function in the OAF , and in heat-shock proteins ( HSPA1B , HSPA8 , HSPB1 , HSP90B1 , HSP90AA1 ) , suggesting a stress response to fluctuating mechanical loads .\",\n \"We sought to identify transitions in proteomic signatures between adjacent compartments .\",\n \"The NP and NP/IAF protein profiles were highly similar ( Figure 3—figure supplement 1B ) .\",\n \"Likewise , NP/IAF and IAF showed few DEPs , except COMP which was expressed higher in IAF ( Figure 3—figure supplement 1C ) .\",\n \"OAF and NP ( Figure 3—figure supplement 1R ) and OAF and NP/IAF ( Figure 3—figure supplement 1O ) showed overlapping DEPs , consistent with NP and NP/IAF having highly similar protein profiles , despite some differences in the anteroposterior direction ( Figure 3—figure supplement 1P & Q ) .\",\n \"The clearest boundary within the IVD , between IAF and OAF , was marked by a set of DEPs , of which COL5A1 , SERPINA5 , MXRA5 were enriched in the IAF , whereas LAMB2 , THBS1 , CTSD typified the OAF ( Figure 3—figure supplement 1D ) .\",\n \"These findings agreed with the modular patterns ( Figure 3E–H ) .\",\n \"Of the 1 , 880 proteins detected , 1 , 204 proteins were not found to vary with respect to the phenotypic factors .\",\n \"The majority of these proteins were detected in few profiles ( Figure 3B ) and were not used in the comparisons .\",\n \"We set a cutoff for a detection in >1/2 of the profiles to prioritise a set of 245 proteins , hereby referred to as the ‘constant set’ ( Figure 3C ) .\",\n \"Both the variable and the constant sets contained high proportions of ECM proteins ( Figure 3D ) .\",\n \"Amongst the proteins in the constant set that were detected in all 33 young profiles were known protein markers defining a young NP or disc , including COL2A1 , ACAN , and A2M ( Risbud et al . , 2015 ) .\",\n \"Other key proteins in the constant set included CHAD , HAPLN1 , VCAN , HTRA1 , CRTAC1 , and CLU .\",\n \"Collectively , these proteins showed the common characteristics shared by compartments of young discs , and they , alongside the variable set , form the architectural landscape of the young disc .\",\n \"PCA was used to identify compartmental , directional , and level patterns for the aged discs ( Figure 4A ) .\",\n \"Albeit less clear than for the young discs ( Figure 3A ) , the OAF could be distinguished from the inner disc regions on PC1 , explaining 46 . 7% of the total variance ( Figure 4A ) .\",\n \"PC2 showed a more distinct separation of signatures from lumbar disc levels L5/S1 to the upper disc levels ( L4/L5 and L3/4 ) , accounting for 21 . 8% of the total variance ( Figure 4A ) .\",\n \"As with the young discs , we performed a series of comparative analyses ( Figure 4C; Figure 4—figure supplement 1A–H; Supplementary file 3 ) .\",\n \"Detection of DEPs between the OAF and the inner disc ( Figure 4B ) , showed that 100 proteins were expressed higher in the OAF , similar to young discs ( Figure 3C ) .\",\n \"However , in the inner regions , only nine proteins were significantly expressed higher , in marked contrast to the situation in young discs .\",\n \"Fifty-five of the 100 DEPs in the OAF region overlapped in the same region in the young discs , but only 3 of the 9 DEPs in the inner region were identified in the young disc; indicating changes in both regions but more dramatic in the inner region .\",\n \"This suggests that ageing and associated changes may have initiated at the centre of the disc .\",\n \"The typical NP markers ( KRT8/19 , CD109 , CHRD , CHRDL2 ) were not detectable as DEPs in the aged disc; but CHI3L2 , A2M and SERPING1 ( Figure 4B ) , which have known roles in tissue fibrosis and wound healing ( Lee et al . , 2011; Naveau et al . , 1994; Wang et al . , 2019a ) , were detected uniquely in the aged discs .\",\n \"A comparative analysis of the protein profiles indicated that the aged OAF retained 55% of the proteins of a young OAF .\",\n \"These changes are primarily reflected in the class of SLRPs ( BGN , DCN , FMOD , OGN , and PRELP ) , and glycoproteins such as CILP , CILP2 , COMP , FGA/B , and FGG .\",\n \"From a cellular perspective , 45 proteins enriched in the aged OAF could be classified under ‘responses to stress’ ( FDR q = 1 . 86 × 10−7; contributed by CAT , PRDX6 , HSP90AB1 , EEF1A1 , TUBB4B , P4HB , PRDX1 , HSPA5 , CRYAB , HIST1H1C ) , suggesting OAF ECM and cells are responding to a changing environment such as mechanical loading and other stress factors .\",\n \"To map the relative changes between inner and outer regions of the aged discs , we performed a systematic comparison between compartments ( Figure 4—figure supplement 1A–D ) .\",\n \"The most significant observation was a weakening of the distinction between IAF and OAF that was seen in young discs , with only 17 DEPs expressed higher in the OAF of the aged disc ( Figure 4—figure supplement 1C ) .\",\n \"More differences were seen when we included the NP in the comparison with OAF ( Figure 4—figure supplement 1D ) , indicating some differences remain between inner and outer regions of the aged discs .\",\n \"While the protein profiles of the NP and IAF were similar , with no detectable DEPs ( Figure 4—figure supplement 1A & B ) , their compositions shared more resemblance with the OAF .\",\n \"These progressive changes of the protein profiles and DEPs between inner and outer compartments suggest the protein composition of the inner disc compartments becomes more similar to the OAF with ageing , with the greatest changes in the inner regions .\",\n \"This further supports a change initiating from the inner region of the discs with ageing .\",\n \"We investigated protein composition in the young disc modules ( Y1-Y4 ) across the lateral and anteroposterior axes in the aged discs ( Figure 4C–F ) .\",\n \"For module Y1 that consists of proteins defining the NP region , the distinctive concave pattern has flattened along both axes , but more so for the anteroposterior direction where the clear interface between IAF and OAF was lost ( Figure 4C; Figure 4—figure supplement 1I ) .\",\n \"Similarly for modules Y2 and Y3 , which consist of proteins defining the AF region , the trends between inner and outer regions of the disc have changed such that the patterns become more convex , with a change that is a continuum from inner to outer regions ( Figure 4D , E; Figure 4—figure supplement 1J , K ) .\",\n \"These changes in modules Y1-3 in the aged disc further illustrate the convergence of the inner and outer regions , with the NP/IAF becoming more OAF-like .\",\n \"For Y4 , the patterns along the lateral and anteroposterior axes were completely disrupted ( Figure 4F; Figure 4—figure supplement 1L ) .\",\n \"As Y4 contains proteins involved in vascularity and inflammatory processes ( Supplementary file 5 ) , these changes indicate disruption of cellular homeostasis in the NP .\",\n \"The protein profiles of the aged disc levels , consistent with the PCA findings , showed similarity between L3/4 and L4/5 ( Figure 4—figure supplement 1E ) , but , in contrast to young discs , differences between L5/S1 and L4/5 ( Figure 4—figure supplement 1F ) , and more marked differences between L5/S1 and L3/4 ( Figure 4—figure supplement 1G , H ) .\",\n \"Overall , the findings from PCA , protein profiles ( Figure 2B–D ) , and MRI ( Figure 1C ) agree .\",\n \"As compared to young discs , the more divergent differences across the aged disc levels potentially reflect progressive transmission from the initiating disc to the adjacent discs with ageing .\",\n \"To further investigate the aetiologies underlying IDD , cross-age comparisons are needed .\",\n \"Next , we performed extensive pair-wise comparisons between the young and aged samples under a defined scheme ( Figure 5—figure supplement 1A; Supplementary file 4 ) .\",\n \"First , we compared all 33 young samples with all 33 aged samples , which identified 169 DEPs with 104 expressed higher in the young and 65 expressed higher in the aged discs ( Figure 5A ) .\",\n \"A simple gene ontology ( GO ) term analysis showed that the most important biological property for a young disc is structural integrity , which is lost in aged discs ( Figure 5B; Supplementary file 5 ) .\",\n \"The protein classes most enriched in the young discs were related to cartilage synthesis , chondrocyte development , and ECM organisation ( Figure 5B ) .\",\n \"The major changes in the aged discs relative to young ones , were proteins involved in cellular responses to an ageing environment , including inflammatory and cellular stress signals , progressive remodelling of disc compartments , and diminishing metabolic activities ( Figure 5C; Supplementary file 5 ) .\",\n \"For young versus old discs , we compared DEPs of the whole disc ( Figure 5A ) with those from the inner regions ( Figure 5D ) and OAF ( Figure 5E ) only .\",\n \"Seventy-five percent ( 78/104 ) of the downregulated DEPs ( Figure 5F ) were attributed to the inner regions , and only 17% ( 18/104 ) were attributed to the OAF ( Figure 5F ) .\",\n \"Similarly , 65% ( 42/65 ) of the upregulated DEPs were solely contributed by inner disc regions , and only 18 . 4% ( 12/65 ) by the OAF .\",\n \"Only 5 DEPs were higher in the OAF , while 49 were uniquely upregulated in the inner discs ( Figure 5G ) .\",\n \"The key biological processes in each of the compartments are highlighted in Figure 5F and G . Expression of known NP markers was reduced in aged discs , especially proteins involved in the ECM and its remodelling , where many of the core matrisome proteins essential for the structural function of the NP were less abundant or absent .\",\n \"While this is consistent with previous observations ( Feng et al . , 2006 ) , of interest was the presence of a set of protein changes that were also seen in the OAF , which was also rich in ECM and matrix remodelling proteins ( HTRA1 , SERPINA1 , SERPINA3 , SERPINC1 , SERPINF1 , and TIMP3 ) and proteins involved in fibrotic events ( FN1 , POSTN , APOA1 , APOB ) , suggesting these changes are occurring in the ageing IVDs ( Figure 5F , G ) .\",\n \"Proteins associated with cellular stress are decreased in the aged inner disc , with functions ranging from molecular chaperones needed for protein folding ( HSPB1 , HSPA1B and HSPA9 ) to modulation of oxidative stress ( SOD1 ) ( Figure 5F ) .\",\n \"SOD1 has been shown to become less abundant in the aged IVD ( Hou et al . , 2014 ) and in osteoarthritis ( Scott et al . , 2010 ) .\",\n \"HSPB1 is cytoprotective and a deficiency is associated with inflammation reported in degenerative discs ( Wuertz et al . , 2012 ) .\",\n \"We found an increased concentration of clusterin ( CLU ) ( Figure 5G ) , an extracellular chaperone that aids the solubilisation of misfolded protein complexes by binding directly and preventing protein aggregation ( Trougakos , 2013; Wyatt et al . , 2009 ) , and also has a role in suppressing fibrosis ( Peix et al . , 2018 ) .\",\n \"Inhibitors of WNT ( DKK3 and FRZB ) , and antagonists of BMP/TGFβ ( CD109 , CHRDL2 , DCN FMOD , INHBA and THBS1 ) signalling were decreased or absent in the aged inner region ( Figure 5F , G ) , consistent with the reported upregulation of these pathways in IDD ( Hiyama et al . , 2010 ) and its closely related condition osteoarthritis ( Leijten et al . , 2013 ) .\",\n \"Targets of hedgehog signalling ( HHIPL2 and SCUBE1 ) were also reduced ( Figure 5F ) , consistent with SHH’s key roles in IVD development and maintenance ( Rajesh and Dahia , 2018 ) .\",\n \"TGFβ signalling is a well-known pathway associated with fibrotic outcomes .\",\n \"WNT is known to induce chondrocyte hypertrophy ( Dong et al . , 2006 ) , that can be enhanced by a reduction in S100A1 ( Figure 5F ) , a known inhibitor of chondrocyte hypertrophy ( Saito et al . , 2007 ) .\",\n \"To gain an overview of the disc compartment variations between young and aged discs , we followed the same strategy as in Figure 3—figure supplement 2 to aggregate three categories of DEPs in all 23 comparisons ( Figure 5—figure supplement 1A ) and created a heatmap from the resulting 719 DEPs .\",\n \"This allowed us to identify six major protein modules ( Figure 5H ) .\",\n \"A striking feature is module 6 ( M6 ) , which is enriched for proteins involved in the complement pathway ( GSEA Hallmark FDR q = 4 . 9 × 10−14 ) and angiogenesis ( q = 2 . 3 × 10−3 ) .\",\n \"This module contains proteins that are all highly expressed in the inner regions of the aged disc , suggesting the presence of blood .\",\n \"M6 also contains the macrophage marker CD14 , which supports this notion .\",\n \"We visualised the relationship between the young ( Y1-4 ) and young/aged ( M1-6 ) modules using an alluvial chart ( Figure 5I ) .\",\n \"Y1 corresponds primarily to M1b that is enriched with fibrosis , angiogenesis , apoptosis , and EMT ( epithelial to mesenchymal transition ) proteins .\",\n \"Y2 seems to have been deconstructed into three M modules ( M2b > M3 > M4 ) .\",\n \"M2b and M3 contain proteins linked to heterogeneous functions , while proteins in M4 are associated with myogenesis and cellular metabolism , but also linked to fibrosis and angiogenesis .\",\n \"Y3 primarily links to M2a with a strong link to myogenesis , and mildly connects with M3 and M4 .\",\n \"Y4 has the strongest connection with M6a , which is linked to coagulation .\",\n \"Both the variable and the constant sets of the young disc were also changed in ageing .\",\n \"In the constant set , there is a higher tendency for a decrease in ECM-related proteins , and an increase in blood and immune related proteins with ageing , that may reflect an erosion of the foundational proteome , and infiltration of immune cells ( Figure 5—figure supplement 1L ) .\",\n \"In all , the IVD proteome showed that with ageing , activities of the SHH pathways were decreased , while those of the WNT and BMP/TGFβ pathways , EMT , angiogenesis , fibrosis , cellular stresses , and chondrocyte hypertrophy-like events were increased .\",\n \"The proteome reflects both current and past transcriptional activities .\",\n \"To investigate upstream cellular and regulatory activities , we obtained transcriptome profiles from two IVD compartments ( NP and AF ) and two sample states ( young , scoliotic but non-degenerated , YND; aged individuals with clinically diagnosed IDD , AGD ) ( Table 1 ) .\",\n \"The transcriptome profiles of YND and AGD are similar to the young and aged disc proteome samples , respectively .\",\n \"After normalisation ( Figure 6—figure supplement 1A ) and hierarchical clustering , we found patterns reflecting relationships among IVD compartments and ages/states ( Figure 6—figure supplement 1B–D ) .\",\n \"PCA of the transcriptome profiles showed that PC1 captured age/state variations ( Figure 6A ) and PC2 captured the compartment ( AF or NP ) differences , with a high degree of similarity to the proteomic PCA that explained 65 . 0% of all data variance ( Figure 2A ) .\",\n \"We compared the transcriptome profiles of different compartments and age/state-groups ( Figure 6—figure supplement 1E–H ) .\",\n \"We detected 88 DEGs ( Materials and methods ) between young AF and young NP samples ( Figure 6—figure supplement 1E ) , in which 39 were more abundant in young NP ( including known NP markers CD24 , KRT19 ) and 49 were more abundant in young AF , including the AF markers , COL1A1 , and THBS1 .\",\n \"In the AGD samples , 11 genes differed between AF and NP ( Figure 6—figure supplement 1F ) , comparable to the proteome profiles ( Figure 4C ) .\",\n \"Between the YND and AGD AF , there were 45 DEGs , with COL1A1 and MMP1 more abundant in YND and COL10A1 , WNT signalling ( WIF1 , WNT16 ) , inflammatory ( TNFAIP6 , CXCL14 , IL11 ) , and fibrosis-associated ( FN1 , CXCL14 ) genes more abundant in AGD ( Figure 6—figure supplement 1G ) .\",\n \"The greatest difference was between YND and AGD NP , with 216 DEGs ( Figure 6—figure supplement 1H ) , with a marked loss of NP markers ( KRT19 , CD24 ) , and gain of AF ( THBS1 , DCN ) , proteolytic ( ADAMTS5 ) , and EMT ( COL1A1 , COL3A1 , PDPN , NT5E , LTBP1 ) markers with age .\",\n \"Again , consistent with the proteomic findings , the most marked changes are in the NP , with the transcriptome profiles becoming AF-like .\",\n \"We partitioned the DEGs between the YND and AGD into DEGs for individual compartments ( Figure 6B ) .\",\n \"The transcriptomic ( Figure 6B ) Venn diagram was very similar to the proteomic one ( Figure 5F–G ) .\",\n \"For example , WNT/TGFβ antagonists and ECM genes were all downregulated with ageing/degeneration , while genes associated with stress and ECM remodelling were more common .\",\n \"When we directly compared the transcriptomic DEGs and proteomic DEPs across age/states and compartments ( Figure 6C–F ) , we observed strong concordance between the two types of datasets for a series of markers .\",\n \"In the young discs , concordant markers included KRT19 and KRT8 , CHRDL2 , FRZB , and DKK3 in the NP , and COL1A1 , SERPINF1 , COL14A1 , and THBS4 in the AF ( Figure 6C ) .\",\n \"In the AGD discs , concordant markers included CHI3L2 , A2M and ANGPTL4 in the NP and MYH9 , HSP90AB1 , HBA1 , and ACTA2 in the AF ( Figure 6D ) .\",\n \"A high degree of concordance was also observed when we compared across age/states for the AF ( Figure 6E ) and NP ( Figure 6F ) .\",\n \"Despite the transcriptomic samples having diagnoses ( scoliosis for YND and IDD for AGD ) , whereas the proteome samples were cadaver samples with no reported diagnosis of IDD , the changes detected in the transcriptome profiles substantially support the proteomic findings .\",\n \"A surprising indication from the transcriptome was the increased levels of COL10A1 ( Lu et al . , 2014 ) , BMP2 ( Grimsrud et al . , 2001 ) , IBSP , defensin beta-1 ( DEFB1 ) , ADAMTS5 , pro-inflammatory ( TNFAIP6 , CXCL ) and proliferation ( CCND1 , IGFBP ) genes in the AGD NP ( Figure 6—figure supplement 1E–H ) , reaffirming the involvement of hypertrophic-like events ( Melas et al . , 2014 ) in the aged and degenerated NP .\",\n \"The genome-wide transcriptomic data included over 20 times more genes per profile than the proteomic data , providing additional biological information about the disc , particularly low abundance proteins , such as transcription factors and surface markers .\",\n \"For example , additional WNT antagonists were WIF1 ( Wnt inhibitory factor ) and GREM1 ( Figure 6B Leijten et al . , 2013 ) .\",\n \"Comparing the YND NP against YND AF or AGD NP ( Figure 6—figure supplement 1E , H ) , we identified higher expression of three transcription factors , T ( brachyury ) , HOPX ( homeodomain-only protein homeobox ) , and ZNF385B in the YND NP .\",\n \"Brachyury is a well-known marker for the NP ( Risbud et al . , 2015 ) , and HOPX is differentially expressed in mouse NP as compared to AF ( Veras et al . , 2020 ) , and expressed in mouse notochordal NP cells ( Lam , 2013 ) .\",\n \"Overall , transcriptomic data confirmed the proteomic findings and revealed additional markers .\",\n \"The proteomic data up to this point is a static form of measurement ( static proteome ) and represents the accumulation and turnover of all proteins up to the time of harvest .\",\n \"The transcriptome indicates genes that are actively transcribed , but does not necessarily correlate to translation or protein turnover .\",\n \"Thus , we studied changes in the IVD proteome ( dynamic proteome ) that would reflect newly synthesised proteins and proteins cleaved by proteases ( degradome ) , and how they relate to the static proteomic and transcriptomic findings reported above .\",\n \"We performed ex vivo labelling of newly synthesised proteins using the SILAC protocol ( Ong et al . , 2002; Figure 7A; Materials and methods ) on AF and NP samples from four YND individuals and one AGD individual ( Table 1 ) .\",\n \"In the SILAC profiles , light isotope-containing signals correspond to the pre-existing unlabelled proteome , and heavy isotope-containing signals to newly synthesised proteins ( Figure 7B ) .\",\n \"The ECM compositions in the light isotope-containing profiles ( Figure 7B , middle panel ) are similar to the static proteome samples of the corresponding age groups described above ( Figure 1F ) .\",\n \"Although for NP_YND152 , the numbers of identified proteins in the heavy profiles are considerably less than NP_YND151 due to a technical issue during sample preparation , it is overall still more similar to NP_YND152 than to other samples ( Figure 7—figure supplement 1B ) , indicating that its biological information is still representative of a young NP and the respective AF samples are similar .\",\n \"In contrast , the heavy isotope-containing profiles contained fewer proteins in the AGD than in the YND samples ( Figure 7B , left panel ) and showed variable heavy to light ratio profiles ( Figure 7B , right panel ) .\",\n \"To facilitate comparisons , we averaged the abundance of the proteins detected in the NP or AF for which we had more than one sample , then ranked the abundance of the heavy isotope-containing ( Figure 7C ) , light isotope-containing ( Figure 7D ) proteins .\",\n \"The number of proteins newly synthesised in the AGD samples was about half that in the YND samples ( Figure 7C ) .\",\n \"This is unlikely to be a technical artefact as the total number of light isotope-containing proteins detected in the AGD samples is comparable to the YND , in both AF and NP ( Figure 7D ) , and the difference is again well illustrated in the heavy to light ratios ( Figure 7—figure supplement 1A ) .\",\n \"Reduced synthesis of non-matrisome proteins was found for the AGD samples ( GAPDH ) as a reference point ( dotted red lines ) ( Figure 7C and D; Figure 7C and E , grey portions ) .\",\n \"Of the 68 high abundant non-matrisome proteins in the YND NP compartment that were not present in the AGD NP , 28 are ribosomal proteins ( Figure 7—figure supplement 1C ) , suggesting reduced translational activities .\",\n \"This agrees with our earlier findings of cellularity , as represented by histones , in the static proteome ( Figure 1J , K ) .\",\n \"Changes in protein synthesis in response to the cell microenvironment affects the architecture of the disc proteome .\",\n \"To understand how the cells may contribute and respond to the accumulated matrisome in the young and aged disc , we compared the newly synthesised matrisome proteins of AGD and YND samples rearranged in order of abundance ( Figure 7E ) .\",\n \"More matrisome proteins were synthesised in YND samples across all classes .\",\n \"In YND AF , collagens were synthesised in higher proportions than in AGD AF with the exception of fibril-associated COL12A1 ( Figure 7E , top panel ) .\",\n \"Similarly , higher proportions in YND AF were observed for proteoglycans ( except FMOD ) , glycoproteins ( except TNC , FBN1 , FGG and FGA ) , ECM affiliated proteins ( except C1QB ) , ECM regulators ( except SERPINF2 , SERPIND1 , A2M , ITIH2 , PLG ) , and secreted factors ( except ANGPTL2 ) .\",\n \"Notably , regulators that were exclusively synthesised in young AF are involved in collagen synthesis ( P4HA1/2 , LOXL2 , LOX , PLOD1/2 ) and matrix turnover ( MMP3 ) , with enrichment of protease HTRA1 and protease inhibitors TIMP3 and ITIH in YND AF compared to AGD AF .\",\n \"In AGD NP , overall collagen synthesis was less than in YND NP ( Figure 7E , lower panel ) ; however , there was more synthesis of COL6A1/2/3 and COL12A1 .\",\n \"Furthermore , AGD NP synthesised more LUM , FMOD , DCN , PRG4 , and PRELP proteoglycans than YND NP .\",\n \"Notably , there was less synthesis of ECM-affiliated proteins ( except C1QC and SEMA3A ) and regulators – particularly those involved in collagen synthesis ( P4HA1/2 , LOXL2 , LOX ) – but an increase in protease inhibitors .\",\n \"A number of newly synthesised proteins in AGD NP were similarly represented in the transcriptome data , including POSTN , ITIH2 , SERPINC1 , IGFBP3 , and PLG .\",\n \"Some genes were simultaneously underrepresented in the AGD NP transcriptome and newly synthesised proteins , including hypertrophy inhibitor GREM1 ( Leijten et al . , 2013 ) .\",\n \"The degradome reflects protein turnover by identifying cleaved proteins in a sample ( López-Otín and Overall , 2002 ) .\",\n \"When combined with relative quantification of proteins through the use of isotopic and isobaric tagging and enrichment for cleaved neo amine ( N ) -termini of proteins before labelled samples are quantified by mass-spectrometry , degradomics is a powerful approach to identify the actual status of protein cleavage in vivo .\",\n \"We employed the well-validated and sensitive terminal amine isotopic labelling of substrates ( TAILS ) method ( Kleifeld et al . , 2010; Rauniyar and Yates , 2014 ) to analyse and compare six discs from six individuals ( two young and non-degenerated , YND; and four aged and/or degenerated , AGD ) ( Table 1; Figure 7F; Kleifeld et al . , 2010; Rauniyar and Yates , 2014 ) using the 6-plex tandem mass tag ( TMT ) -TAILS ( labelling six independent samples and analysed together on the mass spectrometer ) ( Figure 7F ) .\",\n \"Although shotgun proteomics is intended to identify the proteome components , N-terminome data is designed to identify the exact cleavage site in proteins that also evidence stable cleavage products in vivo .\",\n \"Here , TAILS identified 123 and 84 cleaved proteins in the AF and NP disc samples , respectively .\",\n \"Performing hierarchical clustering on the data we found that the two YND samples ( 136 and 141; Table 1 ) tend to cluster together in both AF and NP ( Figure 7G , H; Figure 7—figure supplement 2A , B ) .\",\n \"Interestingly , the trauma sample AGD143 ( 53 year male ) , who has no known IDD diagnosis , tend to cluster with other clinically diagnosed AGD samples , in both AF and NP .\",\n \"This might be because AGD143 has unreported degeneration or ageing is a dominant factor in degradome signals .\",\n \"We identified two protein/peptide modules in the AF ( Figure 7G ) , corresponding to more degradation/cleaving in YND AF ( magenta ) and AGD AF ( blue ) , respectively .\",\n \"There are only 13 unique proteins for proteins/peptides more degraded in the YND AF , the most common of which is COL1A1/2 , followed by COL2A1 .\",\n \"In comparison , the module corresponding to more degradation in AGD AF recorded 24 unique proteins , 7 ( CILP , CILP2 , COL1A1 , COMP , HBA1 , HBB , PRELP ) of which are in strong overlap ( χ2 p=2 . 0 × 10−71 ) with the 99 proteins higher in outer AF in the spatial proteome ( Figure 3I ) .\",\n \"This indicates that key proteins defining a young outer AF is experiencing faster degradation in aged and degenerated samples .\",\n \"Similarly , we identified two modules in the NP ( Figure 7H ) , whereby one ( magenta ) corresponds to more degradation in the YND , and the other ( blue ) corresponds to more degradation in the AGD .\",\n \"Only 10 unique proteins were recorded in the magenta module ( for YND ) , with COL2A1 being the most dominant ( 928 peptides ) ; whereas 32 were recorded for the blue module ( for AGD ) .\",\n \"Overall , there are more unique proteins involved in faster degradation in AGD AF and NP .\",\n \"We tested for a correlation between MRI signal intensity and proteome composition .\",\n \"In conventional 3T MRI of young discs , the NP is brightest reflecting its high hydration state while the AF is darker , thus less hydrated ( Figure 1B; Figure 8—figure supplement 1B ) .\",\n \"Since aged discs present with more MRI phenotypes , we used higher resolution MRI ( 7T ) on them ( Figure 1C; Figure 8A ) , which showed less contrast between NP and AF than in the young discs .\",\n \"To enhance robustness , we obtained three transverse stacks per disc level for the aged discs ( Figure 8B; Figure 8—figure supplement 1D ) , and averaged the pixel intensities for the different compartments showing that overall , the inner regions were still brighter than the outer ( Figure 8C ) .\",\n \"Next , we performed a level-compartment bi-clustering on the pixel intensities of the aged disc MRIs , which was bound by disc level and compartment ( Figure 8D ) .\",\n \"The findings resembled the proteomic PCA ( Figure 2 ) and clustering ( Figure 3—figure supplement 1V–W ) patterns .\",\n \"We performed a pixel intensity averaging of the disc compartments from the 3T images ( Figure 8—figure supplement 1D ) , and a level-compartment bi-clustering on the pixel intensities ( Figure 8—figure supplement 1C ) .\",\n \"While the clustering can clearly partition the inner from the outer disc compartments , the information value from each of the compartments is less due to the lower resolution of the MRI .\",\n \"In all , these results indicate a link between regional MRI landscapes and proteome profiles , prompting us to investigate their potential connections .\",\n \"The MRI and the static proteome were done on the same specimens in both individuals , so we could perform proteome-wide associations with the MRI intensities .\",\n \"We detected 85 significantly correlated ECM proteins , hereby referred to as the hydration matrisome ( Figure 8E ) .\",\n \"We found no collagen to be positively correlated with brighter MRI , which fits current understanding as collagens contribute to fibrosis and dehydration .\",\n \"Other classes of matrisome proteins were either positively or negatively correlated , with differential components for each class ( Figure 8E ) .\",\n \"Positively correlated proteoglycans included EPYC , PRG4 and VCAN , consistent with their normal expression in a young disc and hydration properties .\",\n \"Negatively correlated proteins included OAF ( TNC , SLRPs ) and fibrotic ( POSTN ) markers ( Figure 8E ) .\",\n \"Given this MRI-proteome link and the greater dynamic ranges of MRI in the aged discs enabled by the higher resolution 7T MRIs ( Figure 8D ) , we hypothesised that the hydration matrisome might be used to provide information about MRI intensities and thus disc hydration .\",\n \"To test this , we trained a LASSO regression model ( Tibshirani , 1996 ) of the aged MRIs using the hydration matrisome ( 85 proteins ) , and applied the model to predict the intensity of the MRIs of the young discs , based on the young proteome of the same 85 proteins .\",\n \"Remarkably , we obtained a PCC of 0 . 689 ( p=8 . 9 × 10−6; Spearman = 0 . 776 ) between the actual and predicted MRI ( Figure 8—figure supplement 1E ) .\",\n \"The predicted MRI intensities of the young disc exhibited a smooth monotonic decrease from the NP towards IAF , then dropped suddenly towards the OAF ( Figure 8F , right panel ) , with an ROC AUC ( receiver operating characteristics , area under the curve ) of 0 . 996 between IAF and OAF ( Figure 8—figure supplement 1F ) .\",\n \"In comparison , actual MRIs exhibited a linear decrease from NP to OAF ( Figure 8F , left panel ) .\",\n \"On reviewing these two patterns , we argue that the predicted intensities may be a more faithful representation of the young discs’ water contents than the actual MRI , as it reflects the gross images ( Figure 1—figure supplement 1A ) , PCA ( Figure 3A ) and Y1 modular trend in the young discs ( Figure 3E ) .\",\n \"This exercise not only revealed the inherent connections between regional MRI and regional proteome , but also identified a set of ECM components that is predictive of MRI relating to disc hydration , which may be valuable for future clinical applications .\"\n ],\n [\n \"Here , we present DIPPER – a human IVD proteomic resource comprising point-reference genome-wide profiles .\",\n \"The discovery dataset was established from intact lumbar discs of a young cadaver with no history of skeletal abnormalities ( e . g . scoliosis ) , and an aged cadaver with reduced IVD MRI intensity and annular tears .\",\n \"Although these two individuals may not be representative of their respective age-groups , this is the first known attempt to achieve high spatial resolution profiles in the discs , adding a critical and much needed dimension to the current available IVD proteomic datasets .\",\n \"We showed that our spatiotemporal proteomes integrate well with the dynamic proteome and transcriptome of clinical samples , demonstrating their application values with other datasets .\",\n \"In creating the point-references , we use a well-established protein extraction protocol ( Önnerfjord et al . , 2012 ) , and chromatographic fractionation of the peptides prior to mass spectrometry , we produced a dataset of the human IVD comprising 3 , 100 proteins , encompassing ~400 matrisome and 2 , 700 non-matrisome proteins , with 1 , 769 proteins detected in three or more profiles , considerably higher than recent studies ( Maseda et al . , 2016; Rajasekaran et al . , 2020; Ranjani et al . , 2016; Sarath Babu et al . , 2016 ) .\",\n \"The high quality of our data enabled the application of unbiased approaches including PCA and ANOVA to reveal the relative importance of the phenotypic factors .\",\n \"Particularly , age was found to be the dominant factor influencing proteome profiles .\",\n \"Comparisons between different compartments of the young disc produced a reference landscape containing known ( KRT8/19 for the NP; COL1A1 , CILP and COMP for the AF ) and novel ( FRZB , CHRD , CHRDL2 for the NP; TNMD , SLRPs , and SOD1 for the AF ) markers .\",\n \"The young healthy discs were enriched for matrisome components consistent with a healthy functional young IVD .\",\n \"Despite morphological differences between NP and IAF , the inner disc compartments ( NP , NP/IAF , and IAF ) display high similarities , in contrast to the large differences between IAF and OAF , which was consistent for discs from all lumbar disc levels .\",\n \"This morphological-molecular discrepancy might be accounted for by subtle differences in the ECM organisation , such as differences in GAG moieties on proteoglycans , or levels of glycosylation or other modifications of ECM that diversify function ( Silagi et al . , 2018 ) .\",\n \"Nonetheless , we partitioned the detected proteins into a variable set that captures the diversity , and a constant set that lays the common foundation of all young compartments , which work in synergy to achieve disc function .\",\n \"Clustering analysis of the 671 DEPs of the variable set identified four key modules ( Y1-Y4 ) .\",\n \"Visually , Y1 and Y2 mapped across the lateral and anteroposterior axes with opposing trends .\",\n \"Molecularly , module Y1 ( NP ) contained proteins promoting regulation of matrix remodelling , such as matrix degradation inhibitor MATN3 ( Jayasuriya et al . , 2012 ) and MMP inhibitor TIMP1 .\",\n \"Inhibitors of WNT and BMP signalling were also present .\",\n \"Module Y2 ( OAF ) included COL1A1 , THBS1/2/3 , CILP1/CILP2 , and TNMD , consistent with the OAF’s tendon-like features ( Nakamichi et al . , 2018 ) .\",\n \"It also included a set of SLRPs that might play roles in regulation of collagen assembly ( Robinson et al . , 2017; Taye et al . , 2020 ) , fibril alignment ( Robinson et al . , 2017 ) , maturation and crosslinking ( Kalamajski et al . , 2016 ) , while others are known to inhibit or promote TGFβ signalling ( Markmann et al . , 2000 ) .\",\n \"Notably , the composition of the IAF appeared to be a transition zone between NP and OAF rather than an independent compartment , as few proteins can distinguish it from adjacent compartments .\",\n \"Classes of proteins in both Y3 ( smooth muscle feature ) and Y4 ( immune and blood ) resemble Y2 , which reflects the contractile property of the AF ( Nakai et al . , 2016 ) and the capillaries infiltrating or present at the superficial outer surface of the IVD .\",\n \"In the aged disc , the DEPs between the inner and outer regions of the discs suggests extensive changes in the inner compartment ( s ) .\",\n \"Mapping the aged data onto modules Y1-Y4 allowed a visualisation of the changes .\",\n \"The flattening of the Y1 and Y2 modules along both the lateral and anteroposterior axes indicated a convergence of the inner and outer disc .\",\n \"This is supported by the observed rapid decline of NP proteins and increase of AF proteins in the inner region .\",\n \"Fewer changes were seen in the aged OAF , which concurs with the notion that degenerative changes originate from the NP and radiate outwards; however , infringement of IAF into the NP cannot be excluded .\",\n \"The most marked change was seen in module Y4 ( blood ) , where the pattern was inverted , characterised by high expression in the NP but low in OAF .\",\n \"While contamination cannot be excluded and there are reports that capillaries do not infiltrate the NP even in degeneration ( Nerlich et al . , 2007 ) , our finding is consistent with other proteomic studies showing enrichment of blood proteins in pathological NP ( Maseda et al . , 2016 ) .\",\n \"The route of infiltration can be from the fissured AF or cartilage endplates .\",\n \"Calcified endplates are more susceptible to microfractures , which can lead to blood infiltration into the NP ( Sun et al . , 2020 ) .\",\n \"Of interest is the involvement of an immune response within the inner disc .\",\n \"This corroborates reports of inflammatory processes in ageing and degenerative discs , with the upregulation of pro-inflammatory cytokines and presence of inflammatory cells ( Molinos et al . , 2015; Wuertz et al . , 2012 ) .\",\n \"The SILAC and degradome studies provided important insights into age-related differences in the biosynthetic and turnover activity in the IVD .\",\n \"The SILAC data indicated that protein synthesis is significantly impaired in aged degenerated discs .\",\n \"These findings correlate with reports of reduced cellularity in ageing ( Rodriguez et al . , 2011 ) , which we have also ascertained by leveraging the relationship of histones and housekeeping genes with cell numbers .\",\n \"From the TAILS degradome analysis , we observed more cleaved protein fragments in aged compartments , particularly for structural proteins important for tissue integrity such as COMP and those involved in cell-matrix interactions such as FN1 , which was coupled with the enrichment of the proteolytic process GO terms in the aged static proteome .\",\n \"Collectively , this reveals a systematic modification and replacement of the primary proteomic architecture of the young IVD with age that is associated with diminished or failure in functional properties in ageing or degeneration .\",\n \"Despite known transcriptome-proteome discordance ( Fortelny et al . , 2017 ) , our identification of their concordance allows insights into active changes in the young and aged discs .\",\n \"For example , inhibitors of the WNT pathway and antagonists of BMP/TGFβ signalling ( Leijten et al . , 2013 ) were down-regulated in the aged discs in both the proteome and transcriptome .\",\n \"Interestingly , the activation of these pathways is known to promote chondrocyte hypertrophy ( Dong et al . , 2006 ) , and hypertrophy has been noted in IDD ( Rutges et al . , 2010 ) .\",\n \"This suggests a model whereby the regulatory environment suppressing cellular hypertrophy changes with ageing or degeneration , resulting in conditions such as cellular senescence and tissue mineralisation that are part of the pathological process .\",\n \"In support , S100A1 , a known inhibitor of chondrocyte hypertrophy ( Saito et al . , 2007 ) is downregulated , while chondrocyte hypertrophy markers COL10A1 and IBSP ( Komori , 2010 ) are upregulated in the aged disc .\",\n \"Similar changes have been observed in ageing mouse NP ( Veras et al . , 2020 ) as well as in osteoarthritis ( Zhu et al . , 2009 ) where chondrocyte hypertrophy is thought to be involved in its aetiology ( Ji et al . , 2019; van der Kraan and van den Berg , 2012 ) .\",\n \"Given that WNT inhibitors are already in clinical trials for osteoarthritis ( Wang et al . , 2019b ) , this may point to a prospective therapeutic strategy for IDD .\",\n \"A key finding of our study is the direct demonstration , within a single individual , of association between the hydration status of the disc as revealed by MRI , and the matrisome composition of the disc proteome .\",\n \"The remarkable correlation between predicted hydration states inferred from the spatial proteomic data and the high-definition phenotyping of the aged disc afforded by 7T MRI has enormous potential for understanding the molecular processes underlying IDD .\",\n \"In conclusion , we have generated point-reference datasets of the young and aged disc proteome , at a significantly higher spatial resolution than previous works .\",\n \"By means of a methodological framework , we revealed compartmentalised information on the ECM composition and cellular activities , and their changes with ageing .\",\n \"Integration of this point-reference with additional age- and protein-specific information of synthesis/degradation helps gain insights into the underlying molecular pathology of degeneration ( Figure 8G and H ) .\",\n \"The richness of information in DIPPER makes it a valuable resource for cross referencing with human , animal and in vitro studies to evaluate clinical relevance and guide the development of therapeutics for human IDD .\"\n ],\n [\n \"Two human lumbar spines were obtained through approved regulations and governing bodies , with one young ( 16M ) provided by L . H . ( McGill University ) and one aged ( 59M ) from Articular Engineering , LLC ( IL , USA ) .\",\n \"The young lumbar spine was received frozen as an intact whole lumbar spine .\",\n \"The aged lumbar spine was received frozen , dissected into bone-disc-bone segments .\",\n \"The cadaveric samples were stored at −80⁰C until use .\",\n \"Clinical specimens were obtained with approval by the Institutional Review Board ( references UW 13–576 and EC 1516–00 11/01/2001 ) and with informed consent in accordance with the Helsinki Declaration of 1975 ( revision 1983 ) from another 15 patients undergoing surgery for IDD , trauma or adolescent idiopathic scoliosis at Queen Mary Hospital ( Hong Kong ) , and Duchess of Kent Children’s Hospital ( Hong Kong ) .\",\n \"Information of both the cadaveric and clinical samples are summarised in Table 1 .\",\n \"The discs were thawed overnight at 4°C , and then pre-equalised for scanning at room temperature .\",\n \"For the young IVD , these were imaged together as the lumbar spine was kept intact .\",\n \"T2-weighted and T1-weighted sagittal and axial MRI , T1-rho MRI and Ultrashort-time-to-echo MRI images were obtained using a 3T Philips Achieva 3 . 0 system at the Department of Diagnostic Radiology , The University of Hong Kong .\",\n \"For the aged discs , the IVD were imaged separately as bone-disc-bone segments , at the Department of Electrical and Electronic Engineering , The University of Hong Kong .\",\n \"The MRS and CEST imaging were performed .\",\n \"The FOV for the CEST imaging was adjusted to 76 . 8 × 76 . 8 mm2 to accommodate the size of human lumbar discs ( matrix size = 64 × 64 , slice thickness = 2 mm ) .\",\n \"All MRI experiments were performed at room temperature using a 7 T pre-clinical scanner ( 70/16 Pharmascan , Bruker BioSpin GmbH , Germany ) equipped with a 370 mT/m gradient system along each axis .\",\n \"Single-channel volume RF coils with different diameters were used for the samples based on size ( 60 mm for GAG phantoms and human cadaveric discs ) .\",\n \"The MRI images in the transverse view were then assessed for intensity of the image ( brighter signifying more water content ) .\",\n \"Three transverse MRI images per IVD were overlaid with a grid representing the areas that were cut for mass-spectrometry measurements as outlined previously .\",\n \"For each region , the ‘intensity’ was represented by the average of the pixel intensities , which were graphically visualised and used for correlative studies .\",\n \"The endplates were carefully cut off with a scalpel , exposing the surface of the IVD , which were then cut into small segments spanning seven segments in the central left-right lateral axis , and five segments in the central anteroposterior axis ( Figure 1C ) .\",\n \"In all , this corresponds to a total of 11 locations per IVD .\",\n \"Among them , four are from the OAF , two from the IAF ( but only in the lateral axis ) , one from the central NP , and four from a transition zone between IAF and the NP ( designated the ‘NP/IAF’ ) .\",\n \"Samples were stored frozen at −80⁰C until use .\",\n \"NP and AF disc tissues from spine surgeries ( Table 1 ) were cultured in custom-made Arg- and Lys-free α-MEM ( AthenaES ) as per formulation of Gibco α-MEM ( Cat #11900–024 ) , supplemented with 10% dialysed FBS ( 10 , 000 MWCO , Biowest , Cat# S181D ) , penicillin/streptomycin , 2 . 2 g/L sodium bicarbonate ( Sigma ) , 30 mg/L L-methionine ( Sigma ) , 21 mg/L ‘heavy’ isotope-labelled 13C6L-arginine ( Arg6 , Cambridge Isotopes , Cat # CLM-2265-H ) , 146 mg/L ‘heavy’ isotope-labelled 4 , 4 , 5 , 5-D4 L-Lysine ( Lys4 , Cambridge Isotopes , Cat # DLM-2640 ) .\",\n \"Tissue explants were cultured for 7 days in hypoxia ( 1% O2 and 5% CO2 in air ) at 37⁰C before being washed with PBS and frozen until use .\",\n \"The frozen samples were pulverised using a freezer mill ( Spex ) under liquid nitrogen .\",\n \"Samples were extracted using 15 volumes ( w/v ) of extraction buffer ( 4M guanidine hydrochloride ( GuHCl ) , 50 mM sodium acetate , 100 mM 6-aminocaproic acid , and HALT protease inhibitor cocktail ( Thermo Fischer Scientific ) , pH 5 . 8 ) .\",\n \"Samples were mechanically dissociated with 10 freeze-thaw cycles and sonicated in a cold water bath , before extraction with gentle agitation at 4°C for 48 hr .\",\n \"Samples were centrifuged at 15 , 000 g for 30 min at 4°C and the supernatant was ethanol precipitated at a ratio of 1:9 for 16 hr at −20°C .\",\n \"The ethanol step was repeated and samples were centrifuged at 5000 g for 45 min at 4°C , and the protein pellets were air dried for 30 min .\",\n \"Protein pellets were re-suspended in fresh 4M urea in 50 mM ammonium bicarbonate , pH 8 , using water bath sonication to aid in the re-solubilisation of the samples .\",\n \"Samples underwent reduction with TCEP ( 5 mM final concentration ) at 60°C for 1 hr , and alkylation with iodoacetamide ( 500 mM final concentration ) for 20 min at RT .\",\n \"Protein concentration was measured using the BCA assay ( Biorad ) according to manufacturer’s instructions .\",\n \"A total of 200 μg of protein was then buffer exchanged with 50 mM ammonium bicarbonate with centricon filters ( Millipore , 30 kDa cutoff ) according to manufacturer’s instructions .\",\n \"Samples were digested with mass spec grade Trypsin/LysC ( Promega ) as per manufacturer’s instructions .\",\n \"For SILAC-labelled samples , formic acid was added to a final concentration of 1% , and centrifuged and the supernatant then desalted prior to LC-MS/MS measurements .\",\n \"For the cadaveric samples , the digested peptides were then acidified with TFA ( 0 . 1% final concentration ) and quantified using the peptide quantitative colorimetric peptide assay kit ( Pierce , catalogue 23275 ) before undergoing fractionation using the High pH reversed phase peptide fractionation kit ( Pierce , catalogue number 84868 ) into four fractions .\",\n \"Desalted peptides were dried , re-suspended in 0 . 1% formic acid prior to LC-MS/MS measurements .\",\n \"Samples were loaded onto the Dionex UltiMate 3000 RSLC nano Liquid Chromatography coupled to the Orbitrap Fusion Lumos Tribid Mass Spectrometer .\",\n \"Peptides were separated on a commercial Acclaim C18 column ( 75 μm internal diameter ×50 cm length , 1 . 9 μm particle size; Thermo ) .\",\n \"Separation was attained using a linear gradient of increasing buffer B ( 80% ACN and 0 . 1% formic acid ) and declining buffer A ( 0 . 1% formic acid ) at 300 nL/min .\",\n \"Buffer B was increased to 30% B in 210 min and ramped to 40% B in 10 min followed by a quick ramp to 95% B , where it was held for 5 min before a quick ramp back to 5% B , where it was held and the column was re-equilibrated .\",\n \"Mass spectrometer was operated in positive polarity mode with capillary temperature of 300°C .\",\n \"Full survey scan resolution was set to 120 , 000 with an automatic gain control ( AGC ) target value of 2 × 106 , maximum ion injection time of 30 ms , and for a scan range of 400–1500 m/z .\",\n \"Data acquisition was in DDA mode to automatically isolate and fragment topN multiply charged precursors according to their intensities .\",\n \"Spectra were obtained at 30 , 000 MS2 resolution with AGC target of 1 × 105 and maximum ion injection time of 100 ms , 1 . 6 m/z isolation width , and normalised collisional energy of 31 .\",\n \"Preceding precursor ions targeted for HCD were dynamically excluded of 50 s .\",\n \"Raw data were analysed using MaxQuant ( v . 1 . 6 . 3 . 3 , Germany ) .\",\n \"Briefly , raw files were searched using Andromeda search engine against human UniProt protein database ( 20 , 395 entries , Oct 2018 ) , supplemented with sequences of contaminant proteins .\",\n \"Andromeda search parameters for protein identification were set to a tolerance of 6 ppm for the parental peptide , and 20 ppm for fragmentation spectra and trypsin specificity allowing up to two miscleaved sites .\",\n \"Oxidation of methionine , carboxyamidomethylation of cysteines was specified as a fixed modification .\",\n \"Minimal required peptide length was specified at seven amino acids .\",\n \"Peptides and proteins detected by at least two LFQ ion counts for each peptide in one of the samples were accepted , with a false discovery rate ( FDR ) of 1% .\",\n \"Proteins were quantified by normalised summed peptide intensities computed in MaxQuant with the LFQ option enabled .\",\n \"A total of 66 profiles were obtained: 11 locations × 3 disc levels × 2 individuals; with a median of 665 proteins ( minimum 419 , maximum 1920 ) per profile .\",\n \"The high-resolution , high mass accuracy mass spectrometry ( MS ) data obtained were processed using Proteome Discoverer ( Ver 2 . 1 ) , wherein data were searched using Sequest algorithm against Human UniProt database ( 29 , 900 entries , May 2016 ) , supplemented with sequences of contaminant proteins , using the following search parameters settings: oxidised methionine ( M ) , acetylation ( Protein N-term ) , heavy Arginine ( R6 ) and Lysine ( K4 ) were selected as dynamic modifications , carboxyamidomethylation of cysteines was specified as a fixed modification , minimum peptide length of 7 amino acids was enabled , tolerance of 10 ppm for the parental peptide , and 20 ppm for fragmentation spectra , and trypsin specificity allowing up to two miscleaved sites .\",\n \"Confident proteins were identified using a target-decoy approach with a reversed database , strict FDR 1% at peptide and PSM level .\",\n \"Newly synthesised proteins were heavy labelled with Arg6- and Lys4 , and the data was expressed as the normalised protein abundance obtained from heavy ( labelled ) /light ( un-labelled ) ratio .\",\n \"Degradome analyses was performed on NP and AF from three non-degenerated and three degenerated individuals ( Table 1 ) .\",\n \"Frozen tissues were pulverised as described above and prepared for TAILS as previously reported ( Kleifeld et al . , 2010 ) .\",\n \"After extraction with SDS buffer ( 1% SDS , 100 mM dithiothreitol , 1X protease inhibitor in deionised water ) and sonication ( three cycles , 15 s/cycle ) , the supernatant ( soluble fraction ) underwent reduction at 37°C and alkylation with a final concentration of 15 mM iodoacetamide for 30 min at RT .\",\n \"Samples were precipitated using chloroform/methanol , and the protein pellet air dried .\",\n \"Samples were re-suspended in 1M NaOH , quantified by nanodrop , diluted to 100 mM HEPES and 4M GnHCl and pH adjusted ( pH6 . 5–7 . 5 ) prior to 6-plex TMT labelling as per manufacturer’s instructions ( Sixplex TMT , Cat# 90061 , ThermoFisher Scientific ) .\",\n \"Equal ratios of TMT-labelled samples were pooled and methanol/chloroform precipitated .\",\n \"Protein pellets were air-dried and re-suspended in 200 mM HEPES ( pH8 ) , and digested with trypsin ( 1:100 ratio ) for 16 hr at 37°C , pH 6 . 5 and a sample was taken for pre-TAILS .\",\n \"High-molecular-weight dendritic polyglycerol aldehyde polymer ( ratio of 5:1 w/w polymer to sample ) and NaBH3CN ( to a final concentration of 80 mM ) was added , incubated at 37°C for 16 hr , followed by quenching with 100 mM ethanolamine ( 30 min at 37°C ) and underwent ultrafiltration ( MWCO of 10 , 000 ) .\",\n \"Collected samples were desalted , acidified to 0 . 1% formic acid and dried , prior to MS analysis .\",\n \"Samples were analysed on a Thermo Scientific Easy nLC-1000 coupled online to a Bruker Daltonics Impact II UHR QTOF .\",\n \"Briefly , peptides were loaded onto a 20 cm x 75 µm I . D . analytical column packed with 1 . 8 µm C18 material ( Dr . Maisch GmbH , Germany ) in 100% buffer A ( 99 . 9% H2O , 0 . 1% formic acid ) at 800 bar followed by a linear gradient elution in buffer B ( 99 . 9% acetonitrile , 0 . 1% formic acid ) to a final 30% buffer B for a total 180 min including washing with 95% buffer B . Eluted peptides were ionised by ESI and peptide ions were subjected to tandem MS analysis using a data-dependent acquisition method .\",\n \"A top17 method was employed , where the top 17 most intense multiply charged precursor ions were isolated for MS/MS using collision-induced-dissociation , and actively excluded for 30 s .\",\n \"MGF files were extracted and searched using Mascot against the UniProt Homo sapiens database , with semi-ArgC specificity , TMT6plex quantification , variable oxidation of methionine , variable acetylation of N termini , 20 ppm MS1 error tolerance , 0 . 05 Da MS2 error tolerance and two missed cleavages .\",\n \"Mascot .\",\n \"dat files were imported into Scaffold Q+S v4 . 4 . 3 for peptide identification processing to a final FDR of 1% .\",\n \"Quantitative values were calculated through Scaffold and used for subsequent analyses .\",\n \"AF and NP tissues from four individuals were cut into approximately 0 . 5 cm3 pieces , and put into the Dulbecco's modified Eagle's medium ( DMEM ) ( Gibco ) supplemented with 20 mM HEPES ( USB ) , 1% penicillin-streptomycin ( Gibco ) and 0 . 4% fungizone ( Gibco ) .\",\n \"The tissues were digested with 0 . 2% pronase ( Roche ) for 1 hr , and centrifuged at 200 g for 5 min to remove supernatant .\",\n \"AF and NP were then digested by 0 . 1% type II collagenase ( Worthington Biochemical ) for 14 hr and 0 . 05% type II collagenase for 8 hr , respectively .\",\n \"Cell suspension was filtered through a 70 µm cell strainer ( BD Falcon ) and centrifuged at 200 g for 5 min .\",\n \"The cell pellet was washed with phosphate buffered saline ( PBS ) and centrifuged again to remove the supernatant .\",\n \"RNA was then extracted from the isolated disc cells using Absolutely RNA Nanoprep Kit ( Stratagene ) , following manufacturer’s protocol , and stored at −80°C until further processing .\",\n \"The quality and quantity of total RNA were assessed on the Bioanalyzer ( Agilent ) using the RNA 6000 Nano total RNA assay .\",\n \"cDNA was generated using Affymetrix GeneChip Two-Cycle cDNA Synthesis Kit , followed by in vitro transcription to produce biotin-labelled cRNA .\",\n \"The sample was then hybridised onto the Affymetrix GeneChip Human Genome U133 Plus 2 . 0 Array .\",\n \"The array image , CEL file and other related files were generated using Affymetrix GeneChip Command Console .\",\n \"The experiment was conducted as a service at the Centre for PanorOmic Sciences of the University of Hong Kong .\",\n \"CEL and other files were loaded into GeneSpring GX 10 ( Agilent ) software .\",\n \"The RMA algorithm was used for probe summation .\",\n \"Data were normalised with baseline transformed to median of all samples .\",\n \"A loose filtering based on the raw intensity values was then applied to remove background noise .\",\n \"Consequently , transcriptomic data with a total of 54 , 675 probes ( corresponding to 20 , 887 genes ) and eight profiles were obtained .\",\n \"The detected proteins were compared against the transcription factor ( TF ) database ( Vaquerizas et al . , 2009 ) and the human genome nomenclature consortium database for cell surface markers ( CDs ) ( Braschi et al . , 2019 ) , where 77 TFs and 83 CDs were detected ( Figure 1—figure supplement 3A ) .\",\n \"Excluding missing values , the LFQ levels among the data-points range from 15 . 6 to 41 . 1 , with a Gaussian-like empirical distribution ( Figure 2—figure supplement 1A ) .\",\n \"The numbers of valid values per protein were found to decline rapidly when they were sorted in descending order ( Figure 2—figure supplement 1B , upper panel ) .\",\n \"To perform PCAs , only a subset of genes with sufficiently large numbers of valid values ( i . e . non-missing values ) were used .\",\n \"The cutoff for this was chosen based on a point corresponding to the steepest slope of descending order of valid protein numbers ( Figure 2—figure supplement 1B , second panel ) , such that the increase of valid values is slower than the increase of missing values beyond that point .\",\n \"Subsequently , the top 507 genes were picked representing 59 . 8% of all valid values .\",\n \"This new subset includes 12 . 4% of all missing values .\",\n \"Since the subset of data still contains some missing values , an imputation strategy was adopted employing the Multiple Imputation by Chained Equations ( MICE ) method and package ( Buuren and Groothuis-Oudshoorn , 2011 ) , with a max iteration set at 50 and the default PMM method ( predictive mean matching ) .\",\n \"To further ensure normality , Winsorisation was applied such that genes whose average is below 5% or above 95% of all genes were also excluded from PCA .\",\n \"The data was then profile-wise standardised ( zero-mean and one standard deviation ) before PCA was applied on the R platform ( R Development Core Team , 2013 ) .\",\n \"To assess the impact of the spatiotemporal factors on the proteomic profiles , we performed Analysis of Variance ( ANOVA ) , correlating each protein to the age , compartments , level , and directionality .\",\n \"To draw the soft boundaries on the PCA plot between groups of samples , support vector machines with polynomial ( degree of 2 ) kernel were applied using the LIBSVM package ( Chang and Lin , 2011 ) and the PCA coordinates as inputs for training .\",\n \"A meshed grid covering the whole PCA field was created to make prediction and draw probability contours for −0 . 5 , 0 , and 0 . 5 from the fitted model .\",\n \"Hierarchical clustering was performed with ( 1- correlation coefficient ) as the distance metrics unless otherwise specified .\",\n \"To address the problem of ‘dropout’ effects while avoiding extra inter-dependency introduced due to imputations , we adopted three strategies in calculating the DEPs , namely , by statistical testing , exclusively detected , and fold-change cutoff approaches .\",\n \"First , for the proteins that have over half valid values in both groups under comparison , we performed t-testing with p-values adjusted for multiple testing by the false discovery rate ( FDR ) .\",\n \"Those with FDR below 0 . 05 were considered statistical DEPs .\",\n \"Second , for the proteins where one group has some valid values while the other group is completely not detected , we considered the ones with over half valid values in one group to be exclusive DEPs .\",\n \"For those proteins that were expressed in <50% in both groups , the ones with fold-change greater than two were also considered to be DEPs .\",\n \"To fit the lateral and anteroposterior trends for the modules of genes identified in the young samples , a Gaussian Process Estimation ( GPE ) model was trained using the GauPro package in R Development Core Team , 2013 .\",\n \"Pathway analyses was conducted on the GSEA ( Subramanian et al . , 2005 ) .\",\n \"Signalling proteins was compiled based on 25 Signal transduction pathways listed on KEGG ( Kanehisa et al . , 2019 ) .\",\n \"For transcriptomic data , we used a thresholding approach to detect DEGs ( differentially expressed genes ) , whereby a gene was considered a DEG if the log2 ( fold-change ) is greater than three and the average expression ( logarithmic scale ) is greater than 10 ( Figure 6—figure supplement 1E–H ) .\",\n \"The LASSO model between MRI and proteome was trained using the R package ‘glmnet’ , wherein the 85 ECMs were first imputed for missing values in them using MICE .\",\n \"Nine ECMs were not imputed for too many missing values , leaving 76 for training and testing .\",\n \"The best value for λ was determined by cross-validations .\",\n \"A model was then trained on the aged MRIs ( dependent variable ) and aged proteome of the 76 genes ( independent variable ) .\",\n \"The fitted model was then applied to the young proteome to predict MRIs of the young discs .\",\n \"The custom scripts for processing and analysing the data were housed at github . com/hkudclab/DIPPER; Tam , 2021; copy archived at swh:1:rev:49e4da79786de1dfa496d7c7472343f3b865696c .\",\n \"An interactive web interface for the data is available at http://www . sbms . hku . hk/dclab/DIPPER/ .\"\n ]\n]"},"abstract":{"kind":"list like","value":["The spatiotemporal proteome of the intervertebral disc ( IVD ) underpins its integrity and function .","We present DIPPER , a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals .","To begin with , protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs .","They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs .","Machine learning methods predicted a ‘hydration matrisome’ that connects extracellular matrix with MRI intensity .","Importantly , the static proteome used as point-references can be integrated with dynamic proteome ( SILAC/degradome ) and transcriptome data from multiple clinical samples , enhancing robustness and clinical relevance .","The data , findings , and methodology , available on a web interface ( http://www . sbms . hku . hk/dclab/DIPPER/ ) , will be valuable references in the field of IVD biology and proteomic analytics ."],"string":"[\n \"The spatiotemporal proteome of the intervertebral disc ( IVD ) underpins its integrity and function .\",\n \"We present DIPPER , a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals .\",\n \"To begin with , protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs .\",\n \"They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs .\",\n \"Machine learning methods predicted a ‘hydration matrisome’ that connects extracellular matrix with MRI intensity .\",\n \"Importantly , the static proteome used as point-references can be integrated with dynamic proteome ( SILAC/degradome ) and transcriptome data from multiple clinical samples , enhancing robustness and clinical relevance .\",\n \"The data , findings , and methodology , available on a web interface ( http://www . sbms . hku . hk/dclab/DIPPER/ ) , will be valuable references in the field of IVD biology and proteomic analytics .\"\n]"},"summary":{"kind":"list like","value":["The backbone of vertebrate animals consists of a series of bones called vertebrae that are joined together by disc-like structures that allow the back to move and distribute forces to protect it during daily activities .","It is common for these intervertebral discs to degenerate with age , resulting in back pain and severely reducing quality of life .","The mechanical features of intervertebral discs are the result of their proteins .","These include extracellular matrix proteins , which form the external scaffolding that binds cells together in a tissue , and signaling proteins , which allow cells to communicate .","However , how the levels of different proteins in each region of the disc vary with time has not been fully examined .","To establish how protein composition changes with age , Tam , Chen et al . quantified the protein levels and gene activity ( which leads to protein production ) of intervertebral discs from young and old deceased individuals .","They found that the position of different mixtures of proteins in the intervertebral disc changes with age , and that young people have high levels of extracellular matrix proteins and signaling proteins .","Levels of these proteins decreased as people got older , as did the amount of proteins produced .","To determine which region of the intervertebral disc different proteins were in , Tam , Chen et al . also performed magnetic resonance imaging ( MRI ) of the samples to correlate image intensity ( which represents water content ) with the corresponding protein signature .","The data obtained provides a high-quality map of how the location of different proteins changes with age , and is available online under the name DIPPER .","This database is an informative resource for research into skeletal biology , and it will likely advance the understanding of intervertebral disc degeneration in humans and animals , potentially leading to the development of new treatment strategies for this condition ."],"string":"[\n \"The backbone of vertebrate animals consists of a series of bones called vertebrae that are joined together by disc-like structures that allow the back to move and distribute forces to protect it during daily activities .\",\n \"It is common for these intervertebral discs to degenerate with age , resulting in back pain and severely reducing quality of life .\",\n \"The mechanical features of intervertebral discs are the result of their proteins .\",\n \"These include extracellular matrix proteins , which form the external scaffolding that binds cells together in a tissue , and signaling proteins , which allow cells to communicate .\",\n \"However , how the levels of different proteins in each region of the disc vary with time has not been fully examined .\",\n \"To establish how protein composition changes with age , Tam , Chen et al . quantified the protein levels and gene activity ( which leads to protein production ) of intervertebral discs from young and old deceased individuals .\",\n \"They found that the position of different mixtures of proteins in the intervertebral disc changes with age , and that young people have high levels of extracellular matrix proteins and signaling proteins .\",\n \"Levels of these proteins decreased as people got older , as did the amount of proteins produced .\",\n \"To determine which region of the intervertebral disc different proteins were in , Tam , Chen et al . also performed magnetic resonance imaging ( MRI ) of the samples to correlate image intensity ( which represents water content ) with the corresponding protein signature .\",\n \"The data obtained provides a high-quality map of how the location of different proteins changes with age , and is available online under the name DIPPER .\",\n \"This database is an informative resource for research into skeletal biology , and it will likely advance the understanding of intervertebral disc degeneration in humans and animals , potentially leading to the development of new treatment strategies for this condition .\"\n]"},"year":{"kind":"string","value":"2020"}}},{"rowIdx":4186,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"SIR-2.1 integrates metabolic homeostasis with the reproductive neuromuscular excitability in early aging male Caenorhabditis elegans"},"id":{"kind":"string","value":"elife-01730-v1"},"sections":{"kind":"list like","value":[["Although lifespan is well studied in the model organism Caenorhabditis elegans ( Kenyon , 2010 ) , the aging process per se is still under intense research ( Jin , 2010 ) .","For example , behavioral and/or mental ability starts to decline during early aging , prior to any dramatic structural or morphological dysfunction ( Salthouse , 2004; Guo et al . , 2012 ) .","The decline could be due to physiological alterations toward a non-optimal state , which leads to the failure of proper behavioral execution .","Therefore , it is urgent to uncover the molecular mechanism ( s ) underlying the physiological changes , so that certain actions could prevent or postpone the non-optimal modifications that occur during aging .","Our previous studies used C . elegans males to establish a behavioral model for studying physiological alterations that occur during early aging ( Guo et al . , 2012 ) .","The spicule intromission motor step , which occurs during C . elegans male copulation , is sensitive to changes that occur during early aging ( Garcia et al . , 2001; Garcia and Sternberg , 2003 ) .","Through pharmacological , optogenetics , and genetic analyses , we found a correlation between the increased excitability of the spicule intromission circuit and the decline of mating at early adulthood ( Guo et al . , 2012 ) .","However , the molecular mechanisms underlying this correlation were unknown .","Caloric restriction is an effective way to extend lifespan ( Hursting et al . , 2003 ) .","In C . elegans , caloric deprivation such as transient starvation for 3 to 18 hr during early adulthood can reduce excitability in the spicule intromission circuitry and prolong the mating potency of wild type ( LeBoeuf et al . , 2011 ) .","We showed that the effect of transient starvation is partially mediated by UNC-43/CaMKII and up-regulation of the unc-103 and egl-2-encoded ERG-like K+ channels ( LeBoeuf et al . , 2011; Guo et al . , 2012 ) .","Although males with mutations in both K+ channels abrogate most of the beneficial effects of transient starvation , we still observed a small increase in the mating ability of aged double-mutant males , suggesting that other mechanisms are applied by transient starvation to improve mating ( Guo et al . , 2012 ) .","One possibility is that metabolic alteration , induced by transient starvation , compensates for the physiological changes that occur during aging .","The connection between metabolic change and physiological status might be the generation of reactive oxygen species ( ROS ) .","First , metabolic status determines the oxidative stress burden: ROS is the byproduct of oxidative phosphorylation ( Murphy , 2009 ) .","Second , emerging evidence shows that ROS modulates the excitability of neuromuscular system through modification of ion channels ( Taglialatela et al . , 1997; Cai and Sesti , 2009; Aggarwal and Makielski , 2013 ) .","However , there are few comprehensive in vivo studies , which link ROS-mediated physiological changes to complex behavior alteration .","To address this , we investigated how a mutation in the C . elegans metabolism regulator , SIR-2 . 1 , alters behavior .","SIR-2 . 1 is an ortholog of yeast SIR2 ( Tissenbaum and Guarente , 2001 ) .","Yeast SIR2 , a NAD+-dependent histone deacetylase , with a role in chromatin regulation , can silence ribosome DNA expression and recombination ( Gottlieb and Esposito , 1989 ) .","When overexpressed , sir2 extends yeast lifespan , whereas deletion of the gene shortens the lifespan by 50% ( Kaeberlein et al . , 1999 ) .","Although there is controversy on whether overexpression of invertebrate sir2 ortholog extends lifespan ( Burnett et al . , 2011; Viswanathan and Guarente , 2011 ) , sirtuin family proteins have been shown to regulate glucose and fat metabolism ( Houtkooper et al . , 2012 ) .","For example , the C . elegans SIR-2 . 1 inhibits lipid synthesis during fasting ( Walker et al . , 2010 ) .","In addition , sirtuin proteins mediate an oxidative stress response by regulating antioxidant gene expression through transcriptional factors such as FOXO in a 14-3-3-dependent manner ( Berdichevsky et al . , 2006; Webster et al . , 2012; Merksamer et al . , 2013 ) .","Overall , sirtuin proteins could be involved in age-related diseases , such as type II diabetes and neurodegenerative diseases ( Satoh et al . , 2011; Houtkooper et al . , 2012 ) .","Taken together , it is possible that sirtuin proteins regulate the cell’s metabolic status and physiology , which further alters the coordination of specific behaviors .","In this study , we used C . elegans male mating behavior to study the molecular and physiological alterations underlying the behavioral decline that occur during early aging .","We found that wild-type males require SIR-2 . 1 to maintain mating potency , and sir-2 . 1 mutant males show a premature decline in copulation behavior , consistent with oxidative stress-induced hyperexcitability of their mating circuit .","We propose that in sir-2 . 1 ( 0 ) males , enhanced glycolysis/fatty acid oxidation , coupled with a compromised anti-stress system , contribute to premature mating decline .","However , in mutant and aged wild-type males , pyruvate carboxylase and phosphenolpyruvate carboxykinase are up-regulated , likely as a compensatory mechanism to shunt excess pyruvate from glycolysis and the TCA cycle to lipid biosynthesis , gluconeogenesis , and glyceroneogenesis ."],["Previously , we reported that C . elegans male mating behavior deteriorates during early aging .","N2 and him-5 ( e1490 ) C . elegans ( henceforth , will be referred to as wild type ) males’ mating capability begins to decline at day 3 of their adulthood , although their median lifespan is 11–12 days ( Guo et al . , 2012 ) .","We demonstrated that transient starvation of young males can extend their mating span , partially through up-regulation of ether-a-go-go K+ channels ( LeBoeuf et al . , 2011 ) ; however , our data also suggested that transient starvation can improve mating through additional mechanisms ( Guo et al . , 2012 ) .","Considering that metabolism is altered in food-deprived males ( Tan et al . , 2011 ) , we tested whether perturbing the histone deacetylase metabolic regulator , sir-2 . 1 , affects the functional span of copulation behavior in fed and transiently starved/re-fed males .","In adult hermaphrodites , animals that lack sir-2 . 1 have increased intestinal lipids ( Walker et al . , 2010 ) , a phenotype opposite of starved animals .","Likewise , we found that 1-day-old sir-2 . 1 ( ok434 ) null ( 0 ) males also contain more lipids than wild type ( Figure 1A ) .","In addition , we observed that 2-day-old wild-type males have more fat ( Figure 1A ) .","Thus , we asked if sir-2 . 1 ( 0 ) males might have altered mating due to metabolic dysregulation .","Allowing the males to mate for 5 hr , we found that well-fed aging sir-2 . 1 ( 0 ) males’ mating ability drops prematurely , compared to wild-type males ( Figure 1B","( i ) ) .","Even under unlimited mating conditions , the mating potency of 2-day-old sir-2 . 1 ( 0 ) drops to 42% ( p<0 . 0001 , n = 47 ) ( Figure 1B","( ii ) ) . 10 . 7554/eLife . 01730 . 003Figure 1 . sir-2 . 1 ( 0 ) males have altered lipid content and their mating ability deteriorates prematurely .","( A ) 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild-type males have more lipid content than 1-day-old wild type .","Left: quantification of fat staining , mean ± SEM , unpaired t-test .","Right: representative images of fat staining .","( B ) Mating potency of wild-type and sir-2 . 1 ( 0 ) males .","Copulations were allowed to occur for 5 hr","( i ) or for an unlimited time","( ii ) .","The number of males in each assay is listed at the bottom of each bar .","The numerical percentage of wild-type males that mated on day 1 was normalized to 100% .","The normalization factor was then applied to the other experimental conditions .","The normalized percentages for each day are listed on the top .","Fisher’s exact test was used to compare the mating potency prior to normalization .","( C ) Transient starvation reduces sir-2 . 1 ( 0 ) mating deficiency .","( D ) Mating potency of sir-2 . 1 ( 0 ) and rescued strain sir-2 . 1 ( 0 ) ; rgEX399 [Psir-2 . 1:sir-2 . 1::yfp] .","ns , not significant .","Asterisks * , ** and *** indicate the p<0 . 05 , 0 . 01 , and 0 . 0001 in this paper , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00310 . 7554/eLife . 01730 . 004Figure 1—figure supplement 1 . ( A ) Tissue specific expression of sir-2 . 1 does not rescue the reduced mating potency of sir-2 . 1 ( 0 ) males at day 2 . The lev-11 promoter expresses sir-2 . 1 in all body wall and sex muscles .","The ges-1 promoter expresses sir-2 . 1 in the intestine .","The aex-3 promoter expresses sir-2 . 1 in all neurons .","( B ) Adult lifespan of wild-type ( circles ) and sir-2 . 1 ( 0 ) ( squares ) males ( n = 50 for wild-type males; n = 76 for sir-2 . 1 ( 0 ) males ) ( Log-rank [Mantel-Cox] Test ) .","( C ) Muscle fiber organization in the genital muscles of 1-day-old and 2-day-old sir-2 . 1 ( 0 ) males .","Asterisks indicate the diagonal muscles , arrow head indicates the oblique muscles .","( D ) In vitro sperm activation assays of 2-day-old wild-type and sir-2 . 1 ( 0 ) males .","Representative images are shown on the top of percentage bars .","Arrow indicates the activated sperm with pseudopod , and solid arrow head indicate the inactivated sperm .","No significant differences were observed between wild type and sir-2 . 1 ( 0 ) males ( unpaired t-test , N = 5 trials ) .","In each trial , 50–60 sperm cells were analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 004 We then asked if transient starvation can suppress the mating defect in sir-2 . 1 ( 0 ) .","To do so , we starved sir-2 . 1 ( 0 ) males for ∼20 hr from L4 , and conducted a 5 hr mating potency assay .","Transient starvation improved mating of 2-day-old sir-2 . 1 ( 0 ) males from 13% to 75% ( p<0 . 0001 , Figure 1C ) , but at day 3 , the mating potency of transiently starved sir-2 . 1 ( 0 ) males was still lower than wild type .","Thus , similar to wild type , the metabolic alteration and/or up-regulated EAG K+ channel functions caused by starvation alleviate some of the dysfunction caused by the sir-2 . 1 deletion .","However , the mutant’s reduced mating potencies between day 1 and 3 under both conditions indicate that SIR-2 . 1 contributes to the functionality of the mating circuits during this period .","To confirm that premature mating deterioration in sir-2 . 1 ( 0 ) males is caused by the ok434 allele , we introduced into sir-2 . 1 ( 0 ) animals a rescuing transgene containing the sir-2 . 1 endogenous promoter driving the sir-2 . 1 genomic sequence fused to yfp .","The extrachromosomal expression of sir-2 . 1 significantly improved the mating potency of 2-day-old sir-2 . 1 ( 0 ) males from 26% to 75% ( p<0 . 0001 , Figure 1D ) .","sir-2 . 1 is expressed broadly in C . elegans ( Bamps et al . , 2009 ) , thus we further conducted tissue-specific rescue assays , but found that none of the tissue-specific promoters driving the expression of sir-2 . 1 , including neuronal , muscle , and intestinal promoters can rescue the premature mating decline ( Figure1—figure supplement 1A ) , suggesting that sir-2 . 1 is required in multiple tissues to maintain male mating .","To exclude the possibility that sir-2 . 1 ( 0 ) mating deficiency at day 2 is due to a shorter lifespan , we conducted a lifespan assay and found that sir-2 . 1 ( 0 ) males lived as long as wild type ( Figure 1—figure supplement 1B ) .","Another possibility for the mating deterioration is morphological deformities of the sex musculature .","However , in 2-day-old sir-2 . 1 ( 0 ) males expressing a functional yfp:act-1 transgene ( Figure 1—figure supplement 1C ) , we did not observe any obvious muscle fiber disorganization , which normally occurs in 8-day-old wild-type males ( Guo et al . , 2012 ) .","Although we did not inspect neural morphology , published studies showed that neural morphology does not change in C . elegans during aging ( Herndon et al . , 2002 ) .","Another potential explanation for the lower mating efficiency is that sperm activity in 2-day-old sir-2 . 1 ( 0 ) males is defective .","C . elegans male sperm are stored in the seminal vesicle as a non-activated form and become activated after transfer into a hermaphrodite .","Regulated by proteases , individual sperm goes through a morphological change to form a pseudopod .","This pseudopod provides mobility for the sperm to fertilize the hermaphrodite oocyte ( Smith and Stanfield , 2011 ) .","To test whether the low mating potency of 2-day-old sir-2 . 1 ( 0 ) males is due to failure in sperm activation , we did an in vitro sperm activation assay and found that similar to wild type , 92 . 0 ± 4 . 3% of sperms from 2-day-old sir-2 . 1 ( 0 ) can be artificially activated by pronase ( Figure 1—figure supplement 1D ) .","Taken together , we speculate that the premature mating decline in sir-2 . 1 ( 0 ) is due to physiological changes , rather than the structural degeneration of either neuromuscular circuits or sperm .","We showed that wild-type mating deterioration at day 3 is correlated with an increased excitability in the mating circuitry ( Guo et al . , 2012 ) .","Hence , we hypothesized that sir-2 . 1 ( 0 ) mating decline might also be due to a premature increase in the cellular excitability .","To test this , we used two acetylcholine ( ACh ) agonists , levemisole ( LEV ) and arecoline ( ARE ) to determine the responses of wild-type and sir-2 . 1 ( 0 ) males at multiple ages .","In the male spicule intromission circuit , LEV binds to ionotropic ACh receptors , whereas ARE is a nonselective ACh agonist ( Liu et al . , 2007; Correa et al . , 2012 ) .","Activation of ACh receptors depolarizes the male’s neurons and muscles , and ultimately causes sex muscle contractions; as a result , males protrude their copulatory spicules .","We found that at day 1 , sir-2 . 1 ( 0 ) and wild-type males had similar response to a sub-threshold effective concentration of ARE ( 50 μM ) ( Figure 2A","( i ) ) .","However , 2-day-old sir-2 . 1 ( 0 ) males were more sensitive to agonist stimulation and required significantly less time to respond ( Figure 2A","( ii ) ) .","Additionally , 2-day-old sir-2 . 1 ( 0 ) males were more sensitive to sub-threshold LEV stimulation .","58% sir-2 . 1 ( 0 ) compared to 35% of wild type protracted their spicules in 500 nM LEV ( p<0 . 05 , n > 30 ) ( Figure 2B","( ii ) ) .","These results indicate that the loss of sir-2 . 1 in males alters the spicule intromission circuit’s excitability during early aging . 10 . 7554/eLife . 01730 . 005Figure 2 . sir-2 . 1 ( 0 ) males’ sex circuitry becomes more excitable during aging , and those males display ejaculation defects .","( A ) 1-day-old wild-type and sir-2 . 1 ( 0 ) males ( n = 30 ) have similar response to the ACh agonist arecoline ( ARE ) .","The time required for those males to protrude their spicules out in 50 μM ARE solution are not significantly different","( i ) ( unpaired t-test ) , whereas 2-day-old sir-2 . 1 ( 0 ) males require significantly less time to respond to ARE","( ii ) ( unpaired t-test ) .","Mean and SEM are indicated .","( B ) 1-day-old wild-type and sir-2 . 1 ( 0 ) males ( n = 30 ) have similar response to the ACh agonist levamisole ( LEV )","( i ) .","However , 2-day-old sir-2 . 1 ( 0 ) males are more sensitive to LEV","( ii ) .","( Fisher’s exact test ) .","( C , D ) 2-day-old sir-2 . 1 ( 0 ) males have an ejaculation defect .","( C ) The percentages of 2-day-old wild-type and sir-2 . 1 ( 0 ) males that ejaculated during copulation .","( Fisher’s exact test ) .","( D ) The numbers of cross progeny produced by individual 2-day-old wild-type and sir-2 . 1 ( 0 ) with unc-64 ( e240 ) hermaphrodites .","Mean and SEM are indicated ( unpaired t-test ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00510 . 7554/eLife . 01730 . 006Figure 2—figure supplement 1 . ( A ) A cartoon illustration of C . elegans male mating behavior .","( B ) 2-day-old sir-2 . 1 ( 0 ) males have no defect in turning behavior , ( C ) sensing the vulva of the hermaphrodite and ( D ) staying at the vulva . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 006 To address how hyperexcitability disrupts copulation , we observed the mating behavior of 2-day-old sir-2 . 1 ( 0 ) and wild-type males .","We found that 2-day-old sir-2 . 1 ( 0 ) males performed most of the mating steps similarly to the wild-type control ( Figure 2— figures supplement 1A , B , C , D ) .","Although 2-day-old sir-2 . 1 ( 0 ) males can effectively insert their spicules , a significant number of them failed to transfer sperm into the hermaphrodite ( Figure 2C ) .","Upon spicule insertion , sperm moved out from the seminal vesicle and traveled through the vas deferens; however , they remained stuck in the vas deferens and did not drain through the cloacal opening ( Videos 1 , 2 and 3 ) .","Even the exceptional sir-2 . 1 ( 0 ) males that successfully ejaculated , transferred less sperm and produced fewer progeny ( Figure 2D ) . 10 . 7554/eLife . 01730 . 007Video 1 . Wild-type male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00710 . 7554/eLife . 01730 . 008Video 2 . 2-day-old sir-2 . 1 ( 0 ) male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00810 . 7554/eLife . 01730 . 009Video 3 . 2-day-old sir-2 . 1 ( 0 ) male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 009 The male copulatory spicules are attached to three sets of sex muscles: the retractor , protractor , and anal depressor muscles .","Contraction of the protractor muscles leads to spicules insertion into the vulva ( Garcia et al . , 2001 ) .","During the normal ejaculation step of mating behavior , after spicule penetration , the posterior gubernaculum erector and retractor muscles contract , presumably to pull the proctodeum posteriorly , so that sperm can drain from the vas deferens ( Figure 3—figure supplement 1 ) ( Liu et al . , 2007 ) .","In sir-2 . 1 ( 0 ) males , we speculated that after spicule insertion , the abnormal increased cell excitability causes the spicule protractor and anal depressor muscles to hypercontract , which during sperm transfer , would pinch close the vas deferens opening .","To test this , we imaged the Ca2+ in the spicule-associated dorsal protractor and anal depressor muscles ( region-of-interest , ROI , indicated in Figure 3A , B ) , by expressing G-CaMP3 in these sex muscles of both sir-2 . 1 ( 0 ) and wild-type males ( Tian et al . , 2009; Guo et al . , 2012 ) .","During the mating behavior of 2-day-old wild-type males , the G-CaMP3 ΔF/F0 increased to 129 . 0 ± 32 . 5% ( n = 5 ) at the time of spicule insertion , and the Ca2+ signal started to decline to 86 . 7 ± 30 . 8% during the 10-s period after spicule insertion ( Figure 3A and Video 4 ) .","This indicates that the spicule protractor muscles partially relax after spicule insertion .","However , in 2-day-old sir-2 . 1 ( 0 ) males , ΔF/F0 increased up to 204 . 3 ± 97 . 5% ( n = 5 ) upon spicule insertion .","Unlike wild type , Ca2+ transients did not decrease as much , and the ΔF/F0 fluctuated at about 129 . 8 ± 33 . 0% ( Figure 3B and Video 5 ) .","The sustained higher Ca2+ levels in 2-day-old sir-2 . 1 ( 0 ) males suggest that spicule protractor and anal depressor muscles are hypercontracted and pinch the vas deferens opening , thus blocking sperm release . 10 . 7554/eLife . 01730 . 010Figure 3 . Ca2+ imaging of spicule-associated muscles in mating males . Pseudo-colored images of Ca2+ in the spicule muscles of 2-day-old wild-type and sir-2 . 1 ( 0 ) males during mating ( A ) and ( B ) are representative frames to show the Ca2+ levels of the spicule-associated muscles during spicule insertion attempts , penetration and the start of sperm transfer ( ∼10 s after insertion for wild type ) or 19 s after insertion ( for sir-2 . 1 ( 0 ) males ) .","The asterisks indicate the hermaphrodite vulva .","Below the images , the Ca2+ transients in the protractor and anal depressor muscles ( indicated by the black rectangle in A and B ) are plotted for 5 individual wild-type ( A ) and sir-2 . 1 ( 0 ) males ( B ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01010 . 7554/eLife . 01730 . 011Figure 3—figure supplement 1 . A cartoon illustrating the contractile changes of the spicule-associated muscles during intromission and ejaculation behaviors of a 2-day-old wild-type and sir-2 . 1 ( 0 ) male , respectively . Muscles displaying warmer colors signify the increasing intensity of the contractile events .","The square box refers to the region of interest used for calcium imaging measurements shown in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01110 . 7554/eLife . 01730 . 012Video 4 . Ca2+ transient in a 2-day-old wild-type male . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01210 . 7554/eLife . 01730 . 013Video 5 . Ca2+ transient in a 2-day-old sir-2 . 1 ( 0 ) male . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 013 C . elegans hermaphrodite studies showed that SIR-2 . 1 promotes the expression of antioxidant genes through its association with the FOXO/DAF-16 transcription factor ( Berdichevsky et al . , 2006 ) .","sir-2 . 1 ( 0 ) hermaphrodites are more sensitive to stresses such as reactive oxygen species ( ROS ) ( Rizki et al . , 2011 ) .","Therefore , we asked if ROS-induced damage contributes to the premature mating deterioration .","We confirmed that similar to hermaphrodites , sir-2 . 1 ( 0 ) males are also more sensitive to paraquat , a ROS-generator .","Mutant males are less viable in 10 mM paraquat after 24 hr; 89% of sir-2 . 1 ( 0 ) males survived , compared to 99% of wild type ( p<0 . 01 , n > 100 ) ( Figure 4A ) .","When exposure time reached 48 hr , the difference between two strains became more obvious , 39% of wild-type males survived , compared to 4% of sir-2 . 1 ( 0 ) ( p<0 . 001 ) ( Figure 4A ) . 10 . 7554/eLife . 01730 . 014Figure 4 . ROS contributes to the mating deterioration .","( A ) Survival rates of wild-type and sir-2 . 1 ( 0 ) males on NGM containing 10 mM paraquat at 24 hr and 48 hr post paraquat exposure .","( B ) Mating potency of 1-day-old wild-type males exposed to 0 . 01 , 0 . 1 , and 1 mM paraquat .","( C ) The percentages of males with their spicules protruding out ( SpOUT ) in response to 1 μM levamisole ( LEV ) after treatment with 1 mM paraquat .","( D–G )","Exposing males to N-acetyl-cystine ( NAC ) improves mating .","The percentages of 3-day-old wild-type ( D ) and 2-day-old sir-2 . 1 ( 0 ) ( E ) males that protrude their spicules out in response to 100 nM LEV after NAC exposure .","Mating potency of 3-day-old wild-type ( F ) and 2-day-old sir-2 . 1 ( 0 ) ( G ) males after NAC exposure ( Fisher’s exact test ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 014 Since sir-2 . 1 ( 0 ) males are more sensitive to oxidative stress , we hypothesized that during aging , accumulated ROS from metabolism might contribute to the decreased mating efficiency and to the increased excitability of the spicule intromission circuit .","To test this , we grew wild-type males on plates containing 1 mM paraquat from late L4 to adult and assayed their mating ability and genital muscle excitability .","After exposure to paraquat for 24 hr , males showed significant decline in mating potency ( Figure 4B ) .","Additionally , these males also displayed increased genital muscle sensitivity to the day 1 EC50 concentration ( 1 μM ) of LEV .","56% of wild type protracted their spicules; however , 83% males exposed to paraquat responded to the ACh agonist ( p<0 . 05 , n = 54 ) ( Figure 4C ) .","To further test if ROS contributes to the copulation decline , we supplemented the males’ media with the antioxidant N-acetyl-cysteine ( NAC ) ( Schulz et al . , 2007 ) , and asked if NAC can delay genital muscle excitability changes and improve fertility .","Indeed , when we exposed wild-type and sir-2 . 1 ( 0 ) males to NAC from L4 to adulthood day 3 and adulthood day 2 , respectively , the antioxidant decreased the males’ sensitivity to 100 nM LEV ( the EC50 concentration for older males [Guo et al . , 2012] ) ( Figure 4D , E ) and increased their mating potency ( Figure 4F , G ) .","These results are consistent with the idea that ROS contributes to the behavioral decline .","Next , we asked why 2-day-old sir-2 . 1 ( 0 ) males are more sensitive to ROS .","Metabolism as a major source of endogenous ROS stress might contribute to behavioral decline .","SIR-2 . 1’s role in regulating metabolic processes has not been well described in C . elegans hermaphrodites , and scarcely in males .","Therefore , we compared the expression levels of 55 genes involved in multiple metabolic processes including: glycolysis , gluconeogenesis/glyceroneogenesis , citrate acid cycle , glyoxylate cycle , fatty acid metabolism and electron transport chain ( ETC ) /oxidative phosphorylation ( OXPHOS ) ( Castelein et al . , 2008 ) between age-matched sir-2 . 1 ( 0 ) and wild-type males .","Out of the 55 genes we surveyed , 17 showed statistically significant changes ( Figure 5 ) ; the information for all the genes is tabulated in the Supplementary file 1 . 10 . 7554/eLife . 01730 . 015Figure 5 . sir-2 . 1 ( 0 ) males have altered expression of metabolic genes . Relative mRNA expression level of genes involved in metabolic processes such as glycolysis ( A ) , TCA cycle ( B ) , fatty acid oxidation ( C ) , Gluconeogenesis/glyceroneogenesis/lipid synthesis ( D ) , Glyoxylate cycle ( E ) , and ETC ( F ) in 2-day-old wild type , 1-day-old , and 2-day-old sir-2 . 1 ( 0 ) males relative to 1-day-old wild type .","d1 WT refers to day1 wild type; d2 WT refers to day 2 wild type; d1 s2 refers to day1 sir-2 . 1 ( 0 ) ; d2 s2 refers to day 2 sir-2 . 1 ( 0 ) ( unpaired t-test compared to 1-day-old wild type ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 015 Through real-time PCR analyses , we found that mRNAs encoding key enzymes involved in the initiation of glycolysis ( hexokinase [F14B4 . 2] and glucose-6-phosphate isomerase [Y87G2A . 8] ) and fatty acid oxidation ( fatty acid acyl-CoA synthetase [C46F4 . 2] ) were up-regulated in 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild-type males , relative to 1-day-old wild type ( Figure 5A , B ) .","This is consistent with the published observation that hexokinase is also up-regulated in the whole body of conditional sirt1 knock-out mice ( Gomes et al . , 2013 ) .","Most enzymes involved in the TCA cycle did not change in their levels ( Supplementary file 1 ) .","In contrast , expression of ETC/OXPHOS components ( cco-1 , W09C5 . 8 ) was reduced in sir-2 . 1 ( 0 ) ( Figure 5F ) .","Other genes that were significantly up-regulated include key anabolic enzymes like fatty acid desaturase ( fat-5 , 6 , 7 ) , pyruvate carboxylase ( PC ) ( pyc-1 ) and phosphoenolpyruvate carboxykinase ( PEPCK ) ( pck-1 and pck-2 ) , isocytrate lysase ( icl-1 ) and aconitase-cytosol ( aco-1 ) , which are important for fatty acid biosynthesis , gluconeogenesis , glyceroneogensis , and glyoxylate cycle ( Figure 5D , E ) ( Yang et al . , 2009 ) .","Consistent with this , hepatic cells without sirt1 also have an up-regualtion of PEPCK gene expression ( Wang et al . , 2011 ) .","Fatty acid desaturase plays a critical role in lipid/triglyceride biosynthesis ( Van Gilst et al . , 2005; Flowers and Ntambi , 2008 ) .","PC catalyzes the carboxylation of pyruvate to oxaloacetate ( OAA ) , the first step that shunts pyruvate from glycolysis to gluconeogenesis/glyceroneogenesis .","Alternatively , OAA , an intermediate of TCA , can be converted to phosphoenolpyruvate ( PEP ) by PEPCK directly inside the mitochondrion or transported and converted to PEP by PEPCK in cytosol ( Figure 6A ) .","The up-regulation of fatty acid desaturase is consistent with the increased lipid staining in sir-2 . 1 ( 0 ) males ( Figure 1A ) . 10 . 7554/eLife . 01730 . 016Figure 6 . sir-2 . 1 ( 0 ) males might have enhanced metabolism .","( A ) Schematic illustration of main metabolic enzymes which have altered expression in sir-2 . 1 ( 0 ) males .","Red arrows indicate catabolic pathways .","Blue arrows indicate anabolic pathways .","( B ) ATP content measured in 1 , 2 and 3-day-old wild-type and sir-2 . 1 ( 0 ) males .","( C ) Glycogen staining in 1-day-old wild type and sir-2 . 1 ( 0 ) .","The glycogen staining level is quantified by measuring the mean gray level of the ROI indicated on the top right corner .","The mean gray level is inversely correlated with the iodine stain .","( D ) Oil Red O staining of wild type and mutant C . elegans males .","( E ) sir-2 . 1 ( + ) and sir-2 . 1 ( 0 ) need pck-2 to maintain their mating at day 2 and day 1 respectively .","All percentages of mating potency are normalized to that of 1-day-old wild-type male .","( F ) sir-2 . 1 ( 0 ) requires pck-1 to maintain their mating at day 1 and day 2 , while sir-2 . 1 ( + ) males do not need pck-2 to maintain their mating at either day 1 or day 2 .","( G ) 2% glucose reduces 1-day-old sir-2 . 1 ( 0 ) mating potency , but not 2-day-old wild-type males ( Fisher’s exact test ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01610 . 7554/eLife . 01730 . 017Figure 6—figure supplement 1 . The level of glucose content is similar between 1-day-old sir-2 . 1 ( 0 ) and wild-type males . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 017 To confirm if the changes in mRNA levels of those metabolic enzymes reflect functional alterations in the metabolic processes , we also measured ATP , glucose and glycogen accumulation in wild-type and sir-2 . 1 ( 0 ) males .","Consistent with increased expression levels of glycolysis and fatty acid oxidation genes , sir-2 . 1 ( 0 ) males produced significantly more ATP at day 1 .","At day 2 , wild-type ATP levels increased to match the level of sir-2 . 1 ( 0 ) males .","But at day 3 , sir-2 . 1 ( 0 ) males again accumulated more ATP ( Figure 6B ) .","These data suggest that sir-2 . 1 ( 0 ) and older wild-type males might have an enhanced catabolism , consistent with the potential to generate more ROS .","Based on metabolic roles of PEPCK ( Yang et al . , 2009 ) , up-regulation of pck genes could lead to excess glucose/glycogen in sir-2 . 1 ( 0 ) males .","Although similar amounts of glucose were detected in sir-2 . 1 ( 0 ) and wild-type males ( Figure 6—figure supplement 1 ) , more glycogen was synthesized in the mutant ( Figure 6C ) .","In addition to gluconeogenesis , PEPCK catalysis of OAA to PEP is also a key step for the synthesis of glycerol-3-phosphate , which is used in triglyceride biosynthesis ( Figure 6A ) ( Nye et al . , 2008 ) .","Indeed , males lacking functional pck-2 , but not pck-1 have less lipid content ( Figure 6D ) .","Additionally , sir-2 . 1 ( 0 ) males that lack pck-2 , but not pck-1 , showed reduced lipid staining ( Figure 6D ) , indicating that pck-2 is necessary for the up-regulation of fat synthesis in sir-2 . 1 ( 0 ) .","Taking together the real-time PCR results , accumulation of metabolic products and hypersensitivity to paraquat , we propose that in sir-2 . 1 ( 0 ) males , glycolysis and fatty acid oxidation are up-regulated to provide excessive NADH to the electron transport chain .","Since we also measured reduced expression of ETC components cytochrome c oxidase , more ROS might be generated via electron leak ( Lee et al . , 2010 ) .","However , we hypothesized that the enhanced expression of enzymes involved in anabolic processes might be a suboptimal self-compensatory mechanism to shunt excess pyruvate from being oxidized in the TCA cycle .","To test if the up-regulation of pck genes in sir-2 . 1 ( 0 ) is a compensatory response , we assayed the mating potency of pck-1 ( 0 ) and pck-2 ( 0 ) single mutants and sir-2 . 1 ( 0 ) ; pck-1 ( 0 ) and pck-2 ( 0 ) ; sir-2 . 1 ( 0 ) double mutants .","At day 1 , sir-2 . 1 ( 0 ) and pck-2 ( 0 ) males mated comparable to wild type ( Figure 6E ) .","However at day 2 , the potency of pck-2 ( 0 ) males started to decline similarly to sir-2 . 1 ( 0 ) .","In contrast , for males containing both sir-2 . 1 ( 0 ) and pck-2 ( 0 ) , their mating potency dropped at day 1 ( Figure 6E ) .","This indicates that without pck-2 , males that contain or lack sir-2 . 1 display accelerated behavioral decline .","Similar to the requirement for functional pck-2 , males that lack sir-2 . 1 also needed pck-1 to maintain their mating potency at day 1; however , pck-1 was not required for sir-2 . 1 ( + ) males to mate efficiently at day 2 ( Figure 6F ) .","Next , we reasoned that if excessive glycolysis contributes to the behavioral deterioration , artificially adding extra glucose to the males’ media could accelerate their mating decline .","To test this , we grew males on UV-killed-OP50 NGM plates supplemented with 2% glucose , from hatched larvae up to the adult age prior to behavioral decline , which is day 1 or day 2 , for sir 2 . 1 ( 0 ) and wild-type males , respectively .","We found that the glucose reduced mating potency of 1-day-old sir-2 . 1 ( 0 ) , but not 2-day-old wild type ( Figure 6G ) , indicating that wild type can cope with the extra glucose better than sir-2 . 1 ( 0 ) .","We hypothesized that unlike wild type , sir-2 . 1 ( 0 ) males cannot efficiently respond to the oxidative stress generated by the enhanced catabolism .","To test this , we used qRT-PCR to measure the mRNA levels of antioxidant genes: superoxide dismutase ( sod-1 , 2 , 3 , 4 and 5 ) , catalase ( ctl-1 , 2 ) , and glutathione transferase ( gst-10 and gsto-1 ) relative to 1-day-old wild-type males ( Figure 7 ) .","As expected , the expression of sod-1 , sod-5 , gst-10 and gsto-1 was reduced in 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild type ( Figure 7 ) .","For sod-2 , day 1 expression was also reduced in sir-2 . 1 ( 0 ) , but this gene’s expression increased in both wild type and sir-2 . 1 ( 0 ) at day 2 , possibly a stress response .","For sod-3 and ctl-2 , their day 1 expression was similar in both strains; however at day 2 , sod-3 expression became higher and ctl-2 expression became lower in mutants .","Finally , sir-2 . 1 ( 0 ) males displayed an increased ctl-1 expression at day 1 , which is also reported in antioxidant-compromised daf-16 ( 0 ) mutant .","The enhanced expression of ctl-1 is considered as an adaptive response ( Yanase et al . , 2002 ) .","These results indicate that in addition to a potentially altered metabolism , which could generate excessive ROS , sir-2 . 1 ( 0 ) males might also have a comprised antioxidant response , which is consistent with their hypersensitivity to excessive glucose intake ( Figure 6G ) and to the ROS generator ( Figure 4A ) . 10 . 7554/eLife . 01730 . 018Figure 7 . sir-2 . 1 ( 0 ) males have compromised expression of anti-oxidant genes . Relative mRNA expression level of anti-oxidant genes superoxide dismutase ( A ) , catalase ( B ) , and glutathione transferase ( C ) in 1 , 2-day-old wild type and sir-2 . 1 ( 0 ) males ( unpaired t-test ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 018 Based on the above results , one could hypothesize that increasing SIR-2 . 1 expression or activity might delay mating deterioration during aging .","However , we found that transgenic overexpression of sir-2 . 1 does not improve the mating potency of 3-day-old wild type ( Figure 8A ) .","It is unlikely that the fusion with YFP disrupts SIR-2 . 1 function , because the same transgene can rescue the sir-2 . 1 ( 0 ) phenotype .","Thus , we speculate that up to a point , the expression level of sir-2 . 1 is not rate limiting for SIR-2 . 1 activity during early aging .","However , one could also speculate that the normal endogenous levels of NAD+ limit the function of SIR-2 . 1 .","To test this , we grew males in the presence of the NAD+ precursor nicotinamide ( NAM ) at 200 μM concentration ( Houtkooper et al . , 2010 ) , and then conducted the mating potency .","Indeed , NAM exposure significantly improves 3-day-old wild-type mating potency , but not 2-day-old sir-2 . 1 ( 0 ) males ( Figure 8B , C ) .","This result is consistent with the idea that excess NAD+ might stimulate SIR-2 . 1 activity .","But additionally , excess NAD+ might also reduce ROS production by relaxing the demand of oxidizing NADH back to NAD+; as a corollary to this possibility , the lack of excess NAM to positively affect the sir-2 . 1 ( 0 ) male’s behavior might be aggravated by the abnormally high expression of catabolic enzymes in the mutant males .","To further test if overexpressing SIR-2 . 1 activity can promote mating behavior in older males , we exposed 3-day-old transgenic SIR-2 . 1 over-expressed males with exogenous NAM , but found that excess SIR-2 . 1 does not amplify the positive effect of NAM ( Figure 8D ) .","Thus , we cannot exclude the possibility that NAM or possibly NAD+ additionally promotes behavioral extension through mechanisms parallel to SIR-2 . 1 activity . 10 . 7554/eLife . 01730 . 019Figure 8 . Exogenous nicotinamide improves mating during aging . sir-2 . 1 overexpression cannot increase mating potency of 3-day-old wild type ( A ) .","However , feeding with a NAD+ precursor nicotinamide ( NAM ) significantly improve the mating potency of 3-day-old wild type ( B ) but not 2-day-old sir-2 . 1 ( 0 ) males ( C ) .","Overexpression of sir-2 . 1 cannot further promote the effect of exogenous NAM ( D ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 019"],["Behavioral decline at advanced age can be attributed to morphological degeneration , such as neuronal death and muscle sarcopenia ( Herndon et al . , 2002; Glenn et al . , 2004 ) .","However , during early aging , prior to any significant cellular decay , physiological changes that affect neuronal and muscular functionality can contribute to the reduction of behavioral coordination ( Salthouse , 2004 ) .","Male mating in C . elegans is a complex behavior that requires coordination of multiple motor systems to impregnate the hermaphrodite ( Liu et al . , 2011; Correa et al . , 2012 ) .","Previously , we showed that male mating behavior declines significantly during early aging .","Wild type cannot mate well at adulthood day 3 , and this decline is correlated with an increased excitability in the sex circuitry ( Guo et al . , 2012 ) .","Similar to aging males , a recent study using hermaphrodites showed that synaptic transmission in the locomotion circuit is enhanced after 5 days of adulthood ( Mulcahy et al . , 2013 ) .","Thus , it is possible that during early aging , the neuromuscular systems of both sexes become hyperexcitable .","However , the faster decline in mating suggests that the male reproductive circuitry is more sensitive to age-related physiological changes .","We found that SIR-2 . 1 is a modulator of behavior and is required to maintain mating in aging males .","1-day-old sir-2 . 1 ( 0 ) males can mate similarly to wild type , suggesting that sir-2 . 1 is not essential for mating .","However , unlike wild-type males , the mating ability of sir-2 . 1 ( 0 ) prematurely drops at day","2 . This is due to hyperexcitability of the reproductive circuitry that coordinates spicule intromission and ejaculation .","The hyperexcitability of the spicule muscles causes the male proctodeum to block the connection between the vas deferens and the cloacal opening , which indirectly obstructs the transfer of sperm .","The mutant phenotype resembles the behavioral , physiological , and pharmacological changes that occur in older wild-type males .","This indicates that in wild type , SIR-2 . 1 maintains the functional excitability of the intromission and ejaculation circuit , possibly by slowing down the deteriorative events that accumulate during aging .","We suggest that as the male ages , SIR-2 . 1 regulates the amounts of catabolic , anabolic , and free radical scavenging enzymes to balance the energy demands needed for rapid reproductive motor responses , with the generation of damaging metabolic by-products , such as ROS .","Our data indicate that the male’s cellular physiology is correlated with his ability to mate .","We showed previously and in this study that perturbation of the male’s physiology through genetic mutation or through dietary alterations during early adulthood will affect his ability to mate later in life .","The physiology of wild-type males is likely changing from day 1 to day 2 , as determined by the level of mRNAs encoding metabolic enzymes and the amount of terminal metabolic products .","These physiological changes can ultimately lead to excessive carbon flow into the TCA cycle , and consequently , more NADH to be oxidized by ETC complexes ( Figures 6A and 9 ) .","This promotes ROS generation via electron leak ( Federico et al . , 2012 ) , which can be reflected by behavioral decay at day","3 . Analysis of sir-2 . 1 ( 0 ) males allowed us to extrapolate how this protein deacetylase regulates male physiology .","Lack of SIR-2 . 1 will induce these deleterious changes to occur sooner , and degenerative behavioral responses in the mutant males are measured at day","2 . Under standard laboratory conditions , wild-type males are raised constitutively on abundant E . coli until senescence .","We speculate that since SIR-2 . 1 uses NAD+ as a cofactor to deacetylate proteins , the physiological changes that occur after 2 days of constitutive feeding in wild type adults might be due to reduction in SIR-2 . 1 function , via the lower ratio of NAD+ to NADH in wild type .","The phenomenon of altering NAD+ to NADH levels in vertebrate cells is shown to reduce the activity of SIRT1 ( Braidy et al . , 2011 ) .","Decreased activity of SIR-2 . 1 will not only aggravate a bias towards catabolism , but will also decrease the levels of ROS scavengers .","This is consistent with a hermaphrodite study , which showed that sir-2 . 1 overexpression can protect the organism from ROS , possibly via HCF-1 and FOXO/DAF-16 , to regulate the expression of stress response genes ( Rizki et al . , 2011 ) . 10 . 7554/eLife . 01730 . 020Figure 9 . A cartoon of the metabolism and behavior that occurs in wild-type and sir-2 . 1 ( 0 ) males during early aging . For successful reproductive behavior , SIR-2 . 1 is required to maintain proper carbon flow to meet the male’s energy demands and balance the generation of ROS .","In 1-day-old old sir-2 . 1 ( 0 ) males , catabolism such as glycolysis and fatty acid oxidation is enhanced , and consequently , oxidative phosphorylation and generation of ROS are also increased .","Without SIR-2 . 1 , ROS accumulation by day 2 of adulthood can lead to hyperexcitability of the male’s genital neuromuscular circuitry .","This results in blocked ejaculation and impotency .","It is possible that in 2- to 3-day-old wild-type males , the NAD+-dependent SIR-2 . 1 activity declines due to a lower ratio of NAD to NADH; thus older wild-type males might have a similar physiology as 1-day-old sir-2 . 1 ( 0 ) males . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 020 Although our qPCR analyses suggest that enhanced catabolism might be occurring in 1-day-old of sir-2 . 1 ( 0 ) and 2-day-old wild-type males , their mating ability could be facilitated via anabolic compensatory mechanisms .","In addition to enhanced expression of catabolic genes , mRNAs encoding enzymes such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase ( PEPCK ) appeared to be also up-regulated .","This could be a likely reason for why the males contain more measurable lipids and glycogen .","We propose that the up-regulation of anabolic process is a self-compensatory mechanism to divert carbon from the TCA cycle ( Figure 9 ) .","sir-2 . 1 ( 0 ) males that are mutant for PEPCK genes , lose their ability to generate fat and fail to mate efficiently on day 1 .","Likewise sir-2 . 1 ( + ) males with a mutation in PEPCK genes also display premature mating decline .","We hypothesize that anabolic pathways could act as a homeostatic mechanism to reduce ROS production .","This idea raises the possibility that obesity as a phenotype might be a compensatory mechanism to alleviate the effects of other underlying metabolic dysfunctions .","Indeed , by switching of glycolysis to gluconeogenesis has been shown to be a potential strategy to cure hepatocarcinoma ( Ma et al . , 2013 ) .","A recent study showed that diabetic patients , who are not obese , have higher mortality than overweight ones ( Carnethon et al . , 2012 ) .","Another study showed that a lifestyle intervention focusing on weight loss did not reduce the rate of cardiovascular events in obese adults with type II diabetes ( Look AHEAD Research Group , 2013 ) , challenging the traditional viewpoint that obesity is a major contributor to metabolic disorders .","The free radical aging theory states that aging is the result of free radical-induced molecular damages ( Harman , 1956 ) .","Although this notion is challenged by the hormesis theory , which posits that moderate amounts of physiological or environmental insults can reinforce cellular processes that reduce stress-induced damage ( Afanas’ev , 2010; Schulz et al . , 2007 ) , artificially applied ROS is reported to change the excitability of cultured neurons and muscles through chemically damaging ion channels ( Danson and Paterson , 2006 ) .","Therefore , aspects of the free radical theory of aging might still apply to physiological changes in neural muscular systems and behavioral decay .","Consistent with this , we showed here that genetic and environmental conditions , which can lead to oxidative stress , caused increased excitability of the male mating circuitry and behavioral decay during early aging .","There is not a strict correlation between oxidative stress and changes in the electrical properties of neurons and muscles .","Oxidation of different types of ion channels changes their conductance and alters the cell’s excitability ( Annunziato et al . , 2002 ) .","One C . elegans study indicates that oxidative stress reduces cell excitability by increasing the conductance of K+ channels ( Sesti et al . , 2010 ) .","Another mammalian study showed that oxidative stress hyperpolarizes the resting potential , but extends the duration of the action potential in cardiac ganglion ( Whyte et al . , 2009 ) .","Mitochondria ROS has been shown to trigger Ca2+ increases in the pulmonary arterial myocytes ( Waypa et al . , 2002 , 2006 ) .","In a study using glia , L-type voltage-gated Ca2+ channels ( L-VGCC ) were found to be a target of ROS .","After modification by ROS , their conductance of Ca2+ was increased ( Bond and Greenfield , 2007 ) .","Different from vertebrate skeletal muscles , L-VGCC in C . elegans propagates action potentials , and the entry of external Ca2+ directly promotes excitation–contraction coupling ( Lee et al . , 1997; Maryon et al . , 1998 ) .","Previous work showed that L-VGCC EGL-19 in C . elegans is required for sustained tonic contraction of the copulatory spicule muscles ( Garcia et al . , 2001 ) .","Therefore , oxidation of L-VGCC in C . elegans might contribute to the increased excitability of mating circuits .","Other major targets of ROS are voltage-gated K+ channels .","In C . elegans , oxidation of KVS-1 slows down its inactivation , leading to hyperpolarization and sensory function loss ( Cai and Sesti , 2009 ) .","The ERG-like K+ channel UNC-103 is a major excitability regulator of the sex circuit ( Reiner et al . , 2006 ) .","Although there is no report of oxidative modification on UNC-103 , human encoded H-ERG channels can be activated by oxidative stress , so that cells become hyperpolarized and less excitable ( Cui and Zhang , 2013 ) .","Considering that ROS increases the excitability of the mating circuit , it is possible that L-VGCC is more prone to be oxidized than K+ channels in male reproductive cells ."],["Worms were grown at 20°C on nematode growth media ( NGM ) plates seeded with E . coli strain OP50 , except for strains containing the pha-1 ( e2123 ) , which were maintained at 15°C .","The alleles used in this work included: lite-1 ( ce314 ) on LGX; pck-2 ( ok2586 ) on LGI; pck-1 ( ok2098 ) , pha-1 ( e2123 ) ( Schnabel and Schnabel , 1990 ) , unc-64 ( e240 ) ( Brenner , 1974 ) on LGIII; sir-2 . 1 ( ok434 ) on LGIV; and him-5 ( e1490 ) ( Hodgkin et al . , 1979 ) on LGV .","The males were generated by the him-5 ( e1490 ) mutation .","Males containing only this mutation are referred to as wild-type; him-5 ( e1490 ) males have been shown to mate efficiently as wild type ( Hodgkin , 1983 ) .","pck-2 ( ok2586 ) , pck-1 ( ok2098 ) , and sir-2 . 1 ( ok434 ) were generated by the C . elegans Gene Knockout Consortium .","sir-2 . 1 ( ok434 ) animals were out-crossed 4 times with the him-5 ( e1490 ) strain .","The deletion in sir-2 . 1 ( 0 ) was detected through PCR using primers listed in the Supplementary file 2 .","Altered mediums used here included NGM medium containing glucose ( 2% ) , paraquat , N-acetyl-cystine ( NAC ) ( Sigma , St . Louis , MO ) , and nicotinamide ( NAM ) ( Sigma ) respectively .","The latter three were added at the indicated concentration just before pouring the plates .","OP50 used for the special medium containing glucose and NAC was UV-killed and concentrated to make sure the worms were not food deprived .","We assume that the animals ingest these compounds as they feed on E . coli or absorb them through their cuticle .","DNA primers are listed in the Supplementary file 2 .","The sir-2 . 1 genomic sequence , plus 2 kb upstream of its ATG , was PCR-amplified from N2 DNA .","The PCR product was digested and ligated between the SphI and SalI sites of pSX322YFP to obtain the plasmid pXG5 .","To obtain promoters for driving sir-2 . 1 expression , the sir-2 . 1 endogenous promoter was removed via PCR-mutagenesis from pXG5 to construct pXG6 .","The Gateway ATTR cassette frame A was inserted in front of the sir-2 . 1 genomic sequence to make the destination clone pXG7 .","Plasmids ( pXG8 , pXG9 , and pXG11 ) that promote neuronal , muscular , and intestinal expression of sir-2 . 1 were obtained through Gateway LR reactions between pXG7 and pLR35 ( Paex-3 ) ( LeBoeuf et al . , 2007 ) , pLR22 ( Plev-11 ) ( Gruninger et al . , 2008 ) and pBL50 ( Pges-1 ) ( Urano et al . , 2002 ) , respectively .","pXG5 ( 25 ng/μl ) , pXG8 ( 10 ng/μl ) , pXG9 ( 1 ng/μl ) or pXG11 ( 50 ng/μl ) were injected to sir-2 . 1 ( 0 ) hermaphrodites and transgenic animals were selected via YFP fluorescence .","pXG5 ( 50 ng/μl ) was injected into wild-type hermaphrodites to obtain strains with overexpression of sir-2 . 1 ( referred as sir-2 . 1 ( OE ) ) .","The mating potency assay was performed as described in Guo et al . ( 2012 ) .","Briefly , L4 males were isolated from hermaphrodites .","One male and one pha-1 ( e2123 ) hermaphrodite were co-transferred to a 5-mm-diameter OP50 mating lawn at 20°C , which is a restrictive temperature for pha-1 ( e2123 ) to sire viable self-progeny .","This allowed us to score the mating potency of males , as only cross-progenies would develop to adulthood .","To quantify different parameters of mating , we observed , up to 5 min , copulations between males and paralyzed unc-64 ( e240 ) hermaphrodites .","The ability of males to sense the vulva was calculated by counting the number of times he stopped at the vulva divided by the total number of times he stopped and/or passed by the vulva .","The turning quality was calculated as: the number of smooth turns ( defined as the male tail keeping contact with hermaphrodite and turning without hesitation ) divided by the total number of turns .","Ejaculation assays were conducted in two ways .","We directly observed sperm transfer after spicule insertion , and determined if sperm drained into the unc-64 ( e240 ) hermaphrodite’s uterus .","Additionally , cross-progeny were counted 1–2 days after spicules insertion .","Lifespan assay was conducted as described in Guo et al . ( 2012 ) .","L4 males were isolated and raised 20 per plate .","The males that can respond to gentle touch with a platinum wire were counted and transferred to new plates every day .","Males that dried on the wall of the Petri plate were censored from the assay on the day they died .","To assess the sensitivity to paraquat , L4 males were transferred to plates containing 10 mM paraquat and scored at 24 and 48 hr .","Sperm activation assays were done according to L’Hernault and Roberts ( 1995 ) ; Smith and Stanfield ( 2011 ) .","Briefly , three 2-day-old males , isolated at L4 stage , were cut at the posterior portion with a needle in 20 μl sperm media ( 50 mM Hepes , pH7 . 0 , 45 mM NaCl , 25 mM KCl , 1 mM MgSO4 , and 5 mM CaCl2 ) freshly supplemented with polyvinylprolidone ( PVP ) 40 , 000 molecular weight ( Sigma ) and the activator pronase ( Roche , Indianapolis , IN ) at the final concentration of 10 mg/ml and 500 μg/ml on a slide .","A coverslip with a thin layer of Vaseline applied around the edge was put on the top of the sperm media to form a chamber over the sperm .","After 5-min incubation , activated sperm with a pseudopod and inactive ones were counted using a compound scope fitted with a 100X objective .","Approximately , 50–60 sperm cells were counted in each sample section .","Arecoline ( ARE ) and levamisole ( LEV ) were dissolved in water at the concentration of 100 mM , and then diluted accordingly , as described in Liu et al . ( 2007 ) .","Briefly , 50 μM of ARE , 1 μM and 500 nM of LEV for 1 , 2-day-old males , and 100 nM LEV for 3-day-old males were used .","Males were introduced to 1 ml drug baths and scored if they protruded their spicules for more than 5 s within 5 min of drug exposure .","Ca2+ imaging was conducted and analyzed as described in Guo et al . ( 2012 ) .","50 ng/μl pLR289[Punc-103E:G-CaMP3::sl2:::DsRed] ( Correa et al . , 2012 ) and 50 ng/μl pBX1 ( Granato et al . , 1994 ) were co-injected into pha-1 ( e2123 ) ; him-5 ( e1490 ) ; lite-1 ( ce314 ) hermaphrodites to generate the extrachromosomal array rgEx566[Punc-103E:G-CaMP3::sl2:::DsRed] .","rgEx566 was crossed into pha-1 ( e2123 ) ; him-5 ( e1490 ) ; sir-2 . 1 ( 0 ) ; lite-1 ( ce314 ) hermaphrodites .","Fluorescence signals from G-CaMP3 and DsRed were recorded simultaneously during the copulations of 2-day-old wild-type and sir-2 . 1 ( 0 ) males with paralyzed hermaphrodites containing the transgene rgEx431[Phsp-16:egl-2 ( gf ) ; Punc-103E:DsRed] .","300 day 1 and day 2 adult males were accumulated over a period of time .","RNA was extracted by Trizol , and cDNA was synthesized by SuperScript II ( Life technology , Grand Island , NY ) using around 2 μg total RNA , as described in LeBoeuf and Garcia ( 2012 ) .","The RT-qPCR reactions were performed using BIO-RAD CFX96 real-time system and SsoFast EvaGreen supermix .","11 candidate reference genes were tested to see whether their expression changed from day 1 and day 2 in both wild-type and sir-2 . 1 males ( Hoogewijs et al . , 2008 ) ; from our analyses , act-1 and gpd-2 were selected as the reference to normalize the expression of the metabolic genes .","Many of the primers used to detect the expression of metabolic enzymes are described in Castelein et al . ( 2008 ) .","Other primers for additional metabolic and antioxidant stress genes are listed in the Supplementary file 2 .","Three replicates were conducted on the same RNA samples .","We used the t-test to determine which mRNA transcripts in sir-2 . 1 ( 0 ) males were significantly different from their cognate wild-type transcripts .","To measure ATP and glucose , 100 males were collected at different ages , frozen and thawed three times .","The worms were homogenized , and the supernatant was collected and measured using an ATP determination Kit ( Life technology ) and the Glucose Oxidase Assay Kit ( Life technology ) .","The ATP and glucose were normalized to the amount of dsDNA quantified by picoGreen ( Life technology ) .","To stain glycogen , 1-day-old sir-2 . 1 ( 0 ) and wild-type virgin males were transferred to 2% agar pads .","The pads containing both genotypes were then placed over a bottle of iodine crystals for 30 s ( Frazier and Roth , 2009 ) .","The pictures were taken by a Leica compound scope mounted with OLYMPUS DP70 camera .","The RGB images were then converted to 16-bit gray scale , and the mean gray levels of the isthmus regions were measured using the SimplePCI image quantification software ( Hamamatsu , Janpan ) .","The mean gray level was reversely correlated with the red signal .","Oil Red O staining was done according to O’Rourke et al . ( 2009 ) .","Briefly , males were collected and washed with PBS , and then fixed with Modified Ruvkuns witches brew ( MRWB ) buffer containing 1% paraformaldehyde ( PFA ) for 1 hr .","Worms were then washed with PBS and suspended in 60% isopropanol for 15 min at room temperature .","The 60% isopropanol was removed and worms were bathed in 60% Red Oil O staining solution .","The RGB images were taken by a Leica compound scope mounted with OLYMPUS DP70 camera .","The images were quantified by ImageJ according to Mehlem et al . ( 2013 ) ."]],"string":"[\n [\n \"Although lifespan is well studied in the model organism Caenorhabditis elegans ( Kenyon , 2010 ) , the aging process per se is still under intense research ( Jin , 2010 ) .\",\n \"For example , behavioral and/or mental ability starts to decline during early aging , prior to any dramatic structural or morphological dysfunction ( Salthouse , 2004; Guo et al . , 2012 ) .\",\n \"The decline could be due to physiological alterations toward a non-optimal state , which leads to the failure of proper behavioral execution .\",\n \"Therefore , it is urgent to uncover the molecular mechanism ( s ) underlying the physiological changes , so that certain actions could prevent or postpone the non-optimal modifications that occur during aging .\",\n \"Our previous studies used C . elegans males to establish a behavioral model for studying physiological alterations that occur during early aging ( Guo et al . , 2012 ) .\",\n \"The spicule intromission motor step , which occurs during C . elegans male copulation , is sensitive to changes that occur during early aging ( Garcia et al . , 2001; Garcia and Sternberg , 2003 ) .\",\n \"Through pharmacological , optogenetics , and genetic analyses , we found a correlation between the increased excitability of the spicule intromission circuit and the decline of mating at early adulthood ( Guo et al . , 2012 ) .\",\n \"However , the molecular mechanisms underlying this correlation were unknown .\",\n \"Caloric restriction is an effective way to extend lifespan ( Hursting et al . , 2003 ) .\",\n \"In C . elegans , caloric deprivation such as transient starvation for 3 to 18 hr during early adulthood can reduce excitability in the spicule intromission circuitry and prolong the mating potency of wild type ( LeBoeuf et al . , 2011 ) .\",\n \"We showed that the effect of transient starvation is partially mediated by UNC-43/CaMKII and up-regulation of the unc-103 and egl-2-encoded ERG-like K+ channels ( LeBoeuf et al . , 2011; Guo et al . , 2012 ) .\",\n \"Although males with mutations in both K+ channels abrogate most of the beneficial effects of transient starvation , we still observed a small increase in the mating ability of aged double-mutant males , suggesting that other mechanisms are applied by transient starvation to improve mating ( Guo et al . , 2012 ) .\",\n \"One possibility is that metabolic alteration , induced by transient starvation , compensates for the physiological changes that occur during aging .\",\n \"The connection between metabolic change and physiological status might be the generation of reactive oxygen species ( ROS ) .\",\n \"First , metabolic status determines the oxidative stress burden: ROS is the byproduct of oxidative phosphorylation ( Murphy , 2009 ) .\",\n \"Second , emerging evidence shows that ROS modulates the excitability of neuromuscular system through modification of ion channels ( Taglialatela et al . , 1997; Cai and Sesti , 2009; Aggarwal and Makielski , 2013 ) .\",\n \"However , there are few comprehensive in vivo studies , which link ROS-mediated physiological changes to complex behavior alteration .\",\n \"To address this , we investigated how a mutation in the C . elegans metabolism regulator , SIR-2 . 1 , alters behavior .\",\n \"SIR-2 . 1 is an ortholog of yeast SIR2 ( Tissenbaum and Guarente , 2001 ) .\",\n \"Yeast SIR2 , a NAD+-dependent histone deacetylase , with a role in chromatin regulation , can silence ribosome DNA expression and recombination ( Gottlieb and Esposito , 1989 ) .\",\n \"When overexpressed , sir2 extends yeast lifespan , whereas deletion of the gene shortens the lifespan by 50% ( Kaeberlein et al . , 1999 ) .\",\n \"Although there is controversy on whether overexpression of invertebrate sir2 ortholog extends lifespan ( Burnett et al . , 2011; Viswanathan and Guarente , 2011 ) , sirtuin family proteins have been shown to regulate glucose and fat metabolism ( Houtkooper et al . , 2012 ) .\",\n \"For example , the C . elegans SIR-2 . 1 inhibits lipid synthesis during fasting ( Walker et al . , 2010 ) .\",\n \"In addition , sirtuin proteins mediate an oxidative stress response by regulating antioxidant gene expression through transcriptional factors such as FOXO in a 14-3-3-dependent manner ( Berdichevsky et al . , 2006; Webster et al . , 2012; Merksamer et al . , 2013 ) .\",\n \"Overall , sirtuin proteins could be involved in age-related diseases , such as type II diabetes and neurodegenerative diseases ( Satoh et al . , 2011; Houtkooper et al . , 2012 ) .\",\n \"Taken together , it is possible that sirtuin proteins regulate the cell’s metabolic status and physiology , which further alters the coordination of specific behaviors .\",\n \"In this study , we used C . elegans male mating behavior to study the molecular and physiological alterations underlying the behavioral decline that occur during early aging .\",\n \"We found that wild-type males require SIR-2 . 1 to maintain mating potency , and sir-2 . 1 mutant males show a premature decline in copulation behavior , consistent with oxidative stress-induced hyperexcitability of their mating circuit .\",\n \"We propose that in sir-2 . 1 ( 0 ) males , enhanced glycolysis/fatty acid oxidation , coupled with a compromised anti-stress system , contribute to premature mating decline .\",\n \"However , in mutant and aged wild-type males , pyruvate carboxylase and phosphenolpyruvate carboxykinase are up-regulated , likely as a compensatory mechanism to shunt excess pyruvate from glycolysis and the TCA cycle to lipid biosynthesis , gluconeogenesis , and glyceroneogenesis .\"\n ],\n [\n \"Previously , we reported that C . elegans male mating behavior deteriorates during early aging .\",\n \"N2 and him-5 ( e1490 ) C . elegans ( henceforth , will be referred to as wild type ) males’ mating capability begins to decline at day 3 of their adulthood , although their median lifespan is 11–12 days ( Guo et al . , 2012 ) .\",\n \"We demonstrated that transient starvation of young males can extend their mating span , partially through up-regulation of ether-a-go-go K+ channels ( LeBoeuf et al . , 2011 ) ; however , our data also suggested that transient starvation can improve mating through additional mechanisms ( Guo et al . , 2012 ) .\",\n \"Considering that metabolism is altered in food-deprived males ( Tan et al . , 2011 ) , we tested whether perturbing the histone deacetylase metabolic regulator , sir-2 . 1 , affects the functional span of copulation behavior in fed and transiently starved/re-fed males .\",\n \"In adult hermaphrodites , animals that lack sir-2 . 1 have increased intestinal lipids ( Walker et al . , 2010 ) , a phenotype opposite of starved animals .\",\n \"Likewise , we found that 1-day-old sir-2 . 1 ( ok434 ) null ( 0 ) males also contain more lipids than wild type ( Figure 1A ) .\",\n \"In addition , we observed that 2-day-old wild-type males have more fat ( Figure 1A ) .\",\n \"Thus , we asked if sir-2 . 1 ( 0 ) males might have altered mating due to metabolic dysregulation .\",\n \"Allowing the males to mate for 5 hr , we found that well-fed aging sir-2 . 1 ( 0 ) males’ mating ability drops prematurely , compared to wild-type males ( Figure 1B\",\n \"( i ) ) .\",\n \"Even under unlimited mating conditions , the mating potency of 2-day-old sir-2 . 1 ( 0 ) drops to 42% ( p<0 . 0001 , n = 47 ) ( Figure 1B\",\n \"( ii ) ) . 10 . 7554/eLife . 01730 . 003Figure 1 . sir-2 . 1 ( 0 ) males have altered lipid content and their mating ability deteriorates prematurely .\",\n \"( A ) 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild-type males have more lipid content than 1-day-old wild type .\",\n \"Left: quantification of fat staining , mean ± SEM , unpaired t-test .\",\n \"Right: representative images of fat staining .\",\n \"( B ) Mating potency of wild-type and sir-2 . 1 ( 0 ) males .\",\n \"Copulations were allowed to occur for 5 hr\",\n \"( i ) or for an unlimited time\",\n \"( ii ) .\",\n \"The number of males in each assay is listed at the bottom of each bar .\",\n \"The numerical percentage of wild-type males that mated on day 1 was normalized to 100% .\",\n \"The normalization factor was then applied to the other experimental conditions .\",\n \"The normalized percentages for each day are listed on the top .\",\n \"Fisher’s exact test was used to compare the mating potency prior to normalization .\",\n \"( C ) Transient starvation reduces sir-2 . 1 ( 0 ) mating deficiency .\",\n \"( D ) Mating potency of sir-2 . 1 ( 0 ) and rescued strain sir-2 . 1 ( 0 ) ; rgEX399 [Psir-2 . 1:sir-2 . 1::yfp] .\",\n \"ns , not significant .\",\n \"Asterisks * , ** and *** indicate the p<0 . 05 , 0 . 01 , and 0 . 0001 in this paper , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00310 . 7554/eLife . 01730 . 004Figure 1—figure supplement 1 . ( A ) Tissue specific expression of sir-2 . 1 does not rescue the reduced mating potency of sir-2 . 1 ( 0 ) males at day 2 . The lev-11 promoter expresses sir-2 . 1 in all body wall and sex muscles .\",\n \"The ges-1 promoter expresses sir-2 . 1 in the intestine .\",\n \"The aex-3 promoter expresses sir-2 . 1 in all neurons .\",\n \"( B ) Adult lifespan of wild-type ( circles ) and sir-2 . 1 ( 0 ) ( squares ) males ( n = 50 for wild-type males; n = 76 for sir-2 . 1 ( 0 ) males ) ( Log-rank [Mantel-Cox] Test ) .\",\n \"( C ) Muscle fiber organization in the genital muscles of 1-day-old and 2-day-old sir-2 . 1 ( 0 ) males .\",\n \"Asterisks indicate the diagonal muscles , arrow head indicates the oblique muscles .\",\n \"( D ) In vitro sperm activation assays of 2-day-old wild-type and sir-2 . 1 ( 0 ) males .\",\n \"Representative images are shown on the top of percentage bars .\",\n \"Arrow indicates the activated sperm with pseudopod , and solid arrow head indicate the inactivated sperm .\",\n \"No significant differences were observed between wild type and sir-2 . 1 ( 0 ) males ( unpaired t-test , N = 5 trials ) .\",\n \"In each trial , 50–60 sperm cells were analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 004 We then asked if transient starvation can suppress the mating defect in sir-2 . 1 ( 0 ) .\",\n \"To do so , we starved sir-2 . 1 ( 0 ) males for ∼20 hr from L4 , and conducted a 5 hr mating potency assay .\",\n \"Transient starvation improved mating of 2-day-old sir-2 . 1 ( 0 ) males from 13% to 75% ( p<0 . 0001 , Figure 1C ) , but at day 3 , the mating potency of transiently starved sir-2 . 1 ( 0 ) males was still lower than wild type .\",\n \"Thus , similar to wild type , the metabolic alteration and/or up-regulated EAG K+ channel functions caused by starvation alleviate some of the dysfunction caused by the sir-2 . 1 deletion .\",\n \"However , the mutant’s reduced mating potencies between day 1 and 3 under both conditions indicate that SIR-2 . 1 contributes to the functionality of the mating circuits during this period .\",\n \"To confirm that premature mating deterioration in sir-2 . 1 ( 0 ) males is caused by the ok434 allele , we introduced into sir-2 . 1 ( 0 ) animals a rescuing transgene containing the sir-2 . 1 endogenous promoter driving the sir-2 . 1 genomic sequence fused to yfp .\",\n \"The extrachromosomal expression of sir-2 . 1 significantly improved the mating potency of 2-day-old sir-2 . 1 ( 0 ) males from 26% to 75% ( p<0 . 0001 , Figure 1D ) .\",\n \"sir-2 . 1 is expressed broadly in C . elegans ( Bamps et al . , 2009 ) , thus we further conducted tissue-specific rescue assays , but found that none of the tissue-specific promoters driving the expression of sir-2 . 1 , including neuronal , muscle , and intestinal promoters can rescue the premature mating decline ( Figure1—figure supplement 1A ) , suggesting that sir-2 . 1 is required in multiple tissues to maintain male mating .\",\n \"To exclude the possibility that sir-2 . 1 ( 0 ) mating deficiency at day 2 is due to a shorter lifespan , we conducted a lifespan assay and found that sir-2 . 1 ( 0 ) males lived as long as wild type ( Figure 1—figure supplement 1B ) .\",\n \"Another possibility for the mating deterioration is morphological deformities of the sex musculature .\",\n \"However , in 2-day-old sir-2 . 1 ( 0 ) males expressing a functional yfp:act-1 transgene ( Figure 1—figure supplement 1C ) , we did not observe any obvious muscle fiber disorganization , which normally occurs in 8-day-old wild-type males ( Guo et al . , 2012 ) .\",\n \"Although we did not inspect neural morphology , published studies showed that neural morphology does not change in C . elegans during aging ( Herndon et al . , 2002 ) .\",\n \"Another potential explanation for the lower mating efficiency is that sperm activity in 2-day-old sir-2 . 1 ( 0 ) males is defective .\",\n \"C . elegans male sperm are stored in the seminal vesicle as a non-activated form and become activated after transfer into a hermaphrodite .\",\n \"Regulated by proteases , individual sperm goes through a morphological change to form a pseudopod .\",\n \"This pseudopod provides mobility for the sperm to fertilize the hermaphrodite oocyte ( Smith and Stanfield , 2011 ) .\",\n \"To test whether the low mating potency of 2-day-old sir-2 . 1 ( 0 ) males is due to failure in sperm activation , we did an in vitro sperm activation assay and found that similar to wild type , 92 . 0 ± 4 . 3% of sperms from 2-day-old sir-2 . 1 ( 0 ) can be artificially activated by pronase ( Figure 1—figure supplement 1D ) .\",\n \"Taken together , we speculate that the premature mating decline in sir-2 . 1 ( 0 ) is due to physiological changes , rather than the structural degeneration of either neuromuscular circuits or sperm .\",\n \"We showed that wild-type mating deterioration at day 3 is correlated with an increased excitability in the mating circuitry ( Guo et al . , 2012 ) .\",\n \"Hence , we hypothesized that sir-2 . 1 ( 0 ) mating decline might also be due to a premature increase in the cellular excitability .\",\n \"To test this , we used two acetylcholine ( ACh ) agonists , levemisole ( LEV ) and arecoline ( ARE ) to determine the responses of wild-type and sir-2 . 1 ( 0 ) males at multiple ages .\",\n \"In the male spicule intromission circuit , LEV binds to ionotropic ACh receptors , whereas ARE is a nonselective ACh agonist ( Liu et al . , 2007; Correa et al . , 2012 ) .\",\n \"Activation of ACh receptors depolarizes the male’s neurons and muscles , and ultimately causes sex muscle contractions; as a result , males protrude their copulatory spicules .\",\n \"We found that at day 1 , sir-2 . 1 ( 0 ) and wild-type males had similar response to a sub-threshold effective concentration of ARE ( 50 μM ) ( Figure 2A\",\n \"( i ) ) .\",\n \"However , 2-day-old sir-2 . 1 ( 0 ) males were more sensitive to agonist stimulation and required significantly less time to respond ( Figure 2A\",\n \"( ii ) ) .\",\n \"Additionally , 2-day-old sir-2 . 1 ( 0 ) males were more sensitive to sub-threshold LEV stimulation .\",\n \"58% sir-2 . 1 ( 0 ) compared to 35% of wild type protracted their spicules in 500 nM LEV ( p<0 . 05 , n > 30 ) ( Figure 2B\",\n \"( ii ) ) .\",\n \"These results indicate that the loss of sir-2 . 1 in males alters the spicule intromission circuit’s excitability during early aging . 10 . 7554/eLife . 01730 . 005Figure 2 . sir-2 . 1 ( 0 ) males’ sex circuitry becomes more excitable during aging , and those males display ejaculation defects .\",\n \"( A ) 1-day-old wild-type and sir-2 . 1 ( 0 ) males ( n = 30 ) have similar response to the ACh agonist arecoline ( ARE ) .\",\n \"The time required for those males to protrude their spicules out in 50 μM ARE solution are not significantly different\",\n \"( i ) ( unpaired t-test ) , whereas 2-day-old sir-2 . 1 ( 0 ) males require significantly less time to respond to ARE\",\n \"( ii ) ( unpaired t-test ) .\",\n \"Mean and SEM are indicated .\",\n \"( B ) 1-day-old wild-type and sir-2 . 1 ( 0 ) males ( n = 30 ) have similar response to the ACh agonist levamisole ( LEV )\",\n \"( i ) .\",\n \"However , 2-day-old sir-2 . 1 ( 0 ) males are more sensitive to LEV\",\n \"( ii ) .\",\n \"( Fisher’s exact test ) .\",\n \"( C , D ) 2-day-old sir-2 . 1 ( 0 ) males have an ejaculation defect .\",\n \"( C ) The percentages of 2-day-old wild-type and sir-2 . 1 ( 0 ) males that ejaculated during copulation .\",\n \"( Fisher’s exact test ) .\",\n \"( D ) The numbers of cross progeny produced by individual 2-day-old wild-type and sir-2 . 1 ( 0 ) with unc-64 ( e240 ) hermaphrodites .\",\n \"Mean and SEM are indicated ( unpaired t-test ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00510 . 7554/eLife . 01730 . 006Figure 2—figure supplement 1 . ( A ) A cartoon illustration of C . elegans male mating behavior .\",\n \"( B ) 2-day-old sir-2 . 1 ( 0 ) males have no defect in turning behavior , ( C ) sensing the vulva of the hermaphrodite and ( D ) staying at the vulva . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 006 To address how hyperexcitability disrupts copulation , we observed the mating behavior of 2-day-old sir-2 . 1 ( 0 ) and wild-type males .\",\n \"We found that 2-day-old sir-2 . 1 ( 0 ) males performed most of the mating steps similarly to the wild-type control ( Figure 2— figures supplement 1A , B , C , D ) .\",\n \"Although 2-day-old sir-2 . 1 ( 0 ) males can effectively insert their spicules , a significant number of them failed to transfer sperm into the hermaphrodite ( Figure 2C ) .\",\n \"Upon spicule insertion , sperm moved out from the seminal vesicle and traveled through the vas deferens; however , they remained stuck in the vas deferens and did not drain through the cloacal opening ( Videos 1 , 2 and 3 ) .\",\n \"Even the exceptional sir-2 . 1 ( 0 ) males that successfully ejaculated , transferred less sperm and produced fewer progeny ( Figure 2D ) . 10 . 7554/eLife . 01730 . 007Video 1 . Wild-type male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00710 . 7554/eLife . 01730 . 008Video 2 . 2-day-old sir-2 . 1 ( 0 ) male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00810 . 7554/eLife . 01730 . 009Video 3 . 2-day-old sir-2 . 1 ( 0 ) male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 009 The male copulatory spicules are attached to three sets of sex muscles: the retractor , protractor , and anal depressor muscles .\",\n \"Contraction of the protractor muscles leads to spicules insertion into the vulva ( Garcia et al . , 2001 ) .\",\n \"During the normal ejaculation step of mating behavior , after spicule penetration , the posterior gubernaculum erector and retractor muscles contract , presumably to pull the proctodeum posteriorly , so that sperm can drain from the vas deferens ( Figure 3—figure supplement 1 ) ( Liu et al . , 2007 ) .\",\n \"In sir-2 . 1 ( 0 ) males , we speculated that after spicule insertion , the abnormal increased cell excitability causes the spicule protractor and anal depressor muscles to hypercontract , which during sperm transfer , would pinch close the vas deferens opening .\",\n \"To test this , we imaged the Ca2+ in the spicule-associated dorsal protractor and anal depressor muscles ( region-of-interest , ROI , indicated in Figure 3A , B ) , by expressing G-CaMP3 in these sex muscles of both sir-2 . 1 ( 0 ) and wild-type males ( Tian et al . , 2009; Guo et al . , 2012 ) .\",\n \"During the mating behavior of 2-day-old wild-type males , the G-CaMP3 ΔF/F0 increased to 129 . 0 ± 32 . 5% ( n = 5 ) at the time of spicule insertion , and the Ca2+ signal started to decline to 86 . 7 ± 30 . 8% during the 10-s period after spicule insertion ( Figure 3A and Video 4 ) .\",\n \"This indicates that the spicule protractor muscles partially relax after spicule insertion .\",\n \"However , in 2-day-old sir-2 . 1 ( 0 ) males , ΔF/F0 increased up to 204 . 3 ± 97 . 5% ( n = 5 ) upon spicule insertion .\",\n \"Unlike wild type , Ca2+ transients did not decrease as much , and the ΔF/F0 fluctuated at about 129 . 8 ± 33 . 0% ( Figure 3B and Video 5 ) .\",\n \"The sustained higher Ca2+ levels in 2-day-old sir-2 . 1 ( 0 ) males suggest that spicule protractor and anal depressor muscles are hypercontracted and pinch the vas deferens opening , thus blocking sperm release . 10 . 7554/eLife . 01730 . 010Figure 3 . Ca2+ imaging of spicule-associated muscles in mating males . Pseudo-colored images of Ca2+ in the spicule muscles of 2-day-old wild-type and sir-2 . 1 ( 0 ) males during mating ( A ) and ( B ) are representative frames to show the Ca2+ levels of the spicule-associated muscles during spicule insertion attempts , penetration and the start of sperm transfer ( ∼10 s after insertion for wild type ) or 19 s after insertion ( for sir-2 . 1 ( 0 ) males ) .\",\n \"The asterisks indicate the hermaphrodite vulva .\",\n \"Below the images , the Ca2+ transients in the protractor and anal depressor muscles ( indicated by the black rectangle in A and B ) are plotted for 5 individual wild-type ( A ) and sir-2 . 1 ( 0 ) males ( B ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01010 . 7554/eLife . 01730 . 011Figure 3—figure supplement 1 . A cartoon illustrating the contractile changes of the spicule-associated muscles during intromission and ejaculation behaviors of a 2-day-old wild-type and sir-2 . 1 ( 0 ) male , respectively . Muscles displaying warmer colors signify the increasing intensity of the contractile events .\",\n \"The square box refers to the region of interest used for calcium imaging measurements shown in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01110 . 7554/eLife . 01730 . 012Video 4 . Ca2+ transient in a 2-day-old wild-type male . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01210 . 7554/eLife . 01730 . 013Video 5 . Ca2+ transient in a 2-day-old sir-2 . 1 ( 0 ) male . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 013 C . elegans hermaphrodite studies showed that SIR-2 . 1 promotes the expression of antioxidant genes through its association with the FOXO/DAF-16 transcription factor ( Berdichevsky et al . , 2006 ) .\",\n \"sir-2 . 1 ( 0 ) hermaphrodites are more sensitive to stresses such as reactive oxygen species ( ROS ) ( Rizki et al . , 2011 ) .\",\n \"Therefore , we asked if ROS-induced damage contributes to the premature mating deterioration .\",\n \"We confirmed that similar to hermaphrodites , sir-2 . 1 ( 0 ) males are also more sensitive to paraquat , a ROS-generator .\",\n \"Mutant males are less viable in 10 mM paraquat after 24 hr; 89% of sir-2 . 1 ( 0 ) males survived , compared to 99% of wild type ( p<0 . 01 , n > 100 ) ( Figure 4A ) .\",\n \"When exposure time reached 48 hr , the difference between two strains became more obvious , 39% of wild-type males survived , compared to 4% of sir-2 . 1 ( 0 ) ( p<0 . 001 ) ( Figure 4A ) . 10 . 7554/eLife . 01730 . 014Figure 4 . ROS contributes to the mating deterioration .\",\n \"( A ) Survival rates of wild-type and sir-2 . 1 ( 0 ) males on NGM containing 10 mM paraquat at 24 hr and 48 hr post paraquat exposure .\",\n \"( B ) Mating potency of 1-day-old wild-type males exposed to 0 . 01 , 0 . 1 , and 1 mM paraquat .\",\n \"( C ) The percentages of males with their spicules protruding out ( SpOUT ) in response to 1 μM levamisole ( LEV ) after treatment with 1 mM paraquat .\",\n \"( D–G )\",\n \"Exposing males to N-acetyl-cystine ( NAC ) improves mating .\",\n \"The percentages of 3-day-old wild-type ( D ) and 2-day-old sir-2 . 1 ( 0 ) ( E ) males that protrude their spicules out in response to 100 nM LEV after NAC exposure .\",\n \"Mating potency of 3-day-old wild-type ( F ) and 2-day-old sir-2 . 1 ( 0 ) ( G ) males after NAC exposure ( Fisher’s exact test ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 014 Since sir-2 . 1 ( 0 ) males are more sensitive to oxidative stress , we hypothesized that during aging , accumulated ROS from metabolism might contribute to the decreased mating efficiency and to the increased excitability of the spicule intromission circuit .\",\n \"To test this , we grew wild-type males on plates containing 1 mM paraquat from late L4 to adult and assayed their mating ability and genital muscle excitability .\",\n \"After exposure to paraquat for 24 hr , males showed significant decline in mating potency ( Figure 4B ) .\",\n \"Additionally , these males also displayed increased genital muscle sensitivity to the day 1 EC50 concentration ( 1 μM ) of LEV .\",\n \"56% of wild type protracted their spicules; however , 83% males exposed to paraquat responded to the ACh agonist ( p<0 . 05 , n = 54 ) ( Figure 4C ) .\",\n \"To further test if ROS contributes to the copulation decline , we supplemented the males’ media with the antioxidant N-acetyl-cysteine ( NAC ) ( Schulz et al . , 2007 ) , and asked if NAC can delay genital muscle excitability changes and improve fertility .\",\n \"Indeed , when we exposed wild-type and sir-2 . 1 ( 0 ) males to NAC from L4 to adulthood day 3 and adulthood day 2 , respectively , the antioxidant decreased the males’ sensitivity to 100 nM LEV ( the EC50 concentration for older males [Guo et al . , 2012] ) ( Figure 4D , E ) and increased their mating potency ( Figure 4F , G ) .\",\n \"These results are consistent with the idea that ROS contributes to the behavioral decline .\",\n \"Next , we asked why 2-day-old sir-2 . 1 ( 0 ) males are more sensitive to ROS .\",\n \"Metabolism as a major source of endogenous ROS stress might contribute to behavioral decline .\",\n \"SIR-2 . 1’s role in regulating metabolic processes has not been well described in C . elegans hermaphrodites , and scarcely in males .\",\n \"Therefore , we compared the expression levels of 55 genes involved in multiple metabolic processes including: glycolysis , gluconeogenesis/glyceroneogenesis , citrate acid cycle , glyoxylate cycle , fatty acid metabolism and electron transport chain ( ETC ) /oxidative phosphorylation ( OXPHOS ) ( Castelein et al . , 2008 ) between age-matched sir-2 . 1 ( 0 ) and wild-type males .\",\n \"Out of the 55 genes we surveyed , 17 showed statistically significant changes ( Figure 5 ) ; the information for all the genes is tabulated in the Supplementary file 1 . 10 . 7554/eLife . 01730 . 015Figure 5 . sir-2 . 1 ( 0 ) males have altered expression of metabolic genes . Relative mRNA expression level of genes involved in metabolic processes such as glycolysis ( A ) , TCA cycle ( B ) , fatty acid oxidation ( C ) , Gluconeogenesis/glyceroneogenesis/lipid synthesis ( D ) , Glyoxylate cycle ( E ) , and ETC ( F ) in 2-day-old wild type , 1-day-old , and 2-day-old sir-2 . 1 ( 0 ) males relative to 1-day-old wild type .\",\n \"d1 WT refers to day1 wild type; d2 WT refers to day 2 wild type; d1 s2 refers to day1 sir-2 . 1 ( 0 ) ; d2 s2 refers to day 2 sir-2 . 1 ( 0 ) ( unpaired t-test compared to 1-day-old wild type ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 015 Through real-time PCR analyses , we found that mRNAs encoding key enzymes involved in the initiation of glycolysis ( hexokinase [F14B4 . 2] and glucose-6-phosphate isomerase [Y87G2A . 8] ) and fatty acid oxidation ( fatty acid acyl-CoA synthetase [C46F4 . 2] ) were up-regulated in 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild-type males , relative to 1-day-old wild type ( Figure 5A , B ) .\",\n \"This is consistent with the published observation that hexokinase is also up-regulated in the whole body of conditional sirt1 knock-out mice ( Gomes et al . , 2013 ) .\",\n \"Most enzymes involved in the TCA cycle did not change in their levels ( Supplementary file 1 ) .\",\n \"In contrast , expression of ETC/OXPHOS components ( cco-1 , W09C5 . 8 ) was reduced in sir-2 . 1 ( 0 ) ( Figure 5F ) .\",\n \"Other genes that were significantly up-regulated include key anabolic enzymes like fatty acid desaturase ( fat-5 , 6 , 7 ) , pyruvate carboxylase ( PC ) ( pyc-1 ) and phosphoenolpyruvate carboxykinase ( PEPCK ) ( pck-1 and pck-2 ) , isocytrate lysase ( icl-1 ) and aconitase-cytosol ( aco-1 ) , which are important for fatty acid biosynthesis , gluconeogenesis , glyceroneogensis , and glyoxylate cycle ( Figure 5D , E ) ( Yang et al . , 2009 ) .\",\n \"Consistent with this , hepatic cells without sirt1 also have an up-regualtion of PEPCK gene expression ( Wang et al . , 2011 ) .\",\n \"Fatty acid desaturase plays a critical role in lipid/triglyceride biosynthesis ( Van Gilst et al . , 2005; Flowers and Ntambi , 2008 ) .\",\n \"PC catalyzes the carboxylation of pyruvate to oxaloacetate ( OAA ) , the first step that shunts pyruvate from glycolysis to gluconeogenesis/glyceroneogenesis .\",\n \"Alternatively , OAA , an intermediate of TCA , can be converted to phosphoenolpyruvate ( PEP ) by PEPCK directly inside the mitochondrion or transported and converted to PEP by PEPCK in cytosol ( Figure 6A ) .\",\n \"The up-regulation of fatty acid desaturase is consistent with the increased lipid staining in sir-2 . 1 ( 0 ) males ( Figure 1A ) . 10 . 7554/eLife . 01730 . 016Figure 6 . sir-2 . 1 ( 0 ) males might have enhanced metabolism .\",\n \"( A ) Schematic illustration of main metabolic enzymes which have altered expression in sir-2 . 1 ( 0 ) males .\",\n \"Red arrows indicate catabolic pathways .\",\n \"Blue arrows indicate anabolic pathways .\",\n \"( B ) ATP content measured in 1 , 2 and 3-day-old wild-type and sir-2 . 1 ( 0 ) males .\",\n \"( C ) Glycogen staining in 1-day-old wild type and sir-2 . 1 ( 0 ) .\",\n \"The glycogen staining level is quantified by measuring the mean gray level of the ROI indicated on the top right corner .\",\n \"The mean gray level is inversely correlated with the iodine stain .\",\n \"( D ) Oil Red O staining of wild type and mutant C . elegans males .\",\n \"( E ) sir-2 . 1 ( + ) and sir-2 . 1 ( 0 ) need pck-2 to maintain their mating at day 2 and day 1 respectively .\",\n \"All percentages of mating potency are normalized to that of 1-day-old wild-type male .\",\n \"( F ) sir-2 . 1 ( 0 ) requires pck-1 to maintain their mating at day 1 and day 2 , while sir-2 . 1 ( + ) males do not need pck-2 to maintain their mating at either day 1 or day 2 .\",\n \"( G ) 2% glucose reduces 1-day-old sir-2 . 1 ( 0 ) mating potency , but not 2-day-old wild-type males ( Fisher’s exact test ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01610 . 7554/eLife . 01730 . 017Figure 6—figure supplement 1 . The level of glucose content is similar between 1-day-old sir-2 . 1 ( 0 ) and wild-type males . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 017 To confirm if the changes in mRNA levels of those metabolic enzymes reflect functional alterations in the metabolic processes , we also measured ATP , glucose and glycogen accumulation in wild-type and sir-2 . 1 ( 0 ) males .\",\n \"Consistent with increased expression levels of glycolysis and fatty acid oxidation genes , sir-2 . 1 ( 0 ) males produced significantly more ATP at day 1 .\",\n \"At day 2 , wild-type ATP levels increased to match the level of sir-2 . 1 ( 0 ) males .\",\n \"But at day 3 , sir-2 . 1 ( 0 ) males again accumulated more ATP ( Figure 6B ) .\",\n \"These data suggest that sir-2 . 1 ( 0 ) and older wild-type males might have an enhanced catabolism , consistent with the potential to generate more ROS .\",\n \"Based on metabolic roles of PEPCK ( Yang et al . , 2009 ) , up-regulation of pck genes could lead to excess glucose/glycogen in sir-2 . 1 ( 0 ) males .\",\n \"Although similar amounts of glucose were detected in sir-2 . 1 ( 0 ) and wild-type males ( Figure 6—figure supplement 1 ) , more glycogen was synthesized in the mutant ( Figure 6C ) .\",\n \"In addition to gluconeogenesis , PEPCK catalysis of OAA to PEP is also a key step for the synthesis of glycerol-3-phosphate , which is used in triglyceride biosynthesis ( Figure 6A ) ( Nye et al . , 2008 ) .\",\n \"Indeed , males lacking functional pck-2 , but not pck-1 have less lipid content ( Figure 6D ) .\",\n \"Additionally , sir-2 . 1 ( 0 ) males that lack pck-2 , but not pck-1 , showed reduced lipid staining ( Figure 6D ) , indicating that pck-2 is necessary for the up-regulation of fat synthesis in sir-2 . 1 ( 0 ) .\",\n \"Taking together the real-time PCR results , accumulation of metabolic products and hypersensitivity to paraquat , we propose that in sir-2 . 1 ( 0 ) males , glycolysis and fatty acid oxidation are up-regulated to provide excessive NADH to the electron transport chain .\",\n \"Since we also measured reduced expression of ETC components cytochrome c oxidase , more ROS might be generated via electron leak ( Lee et al . , 2010 ) .\",\n \"However , we hypothesized that the enhanced expression of enzymes involved in anabolic processes might be a suboptimal self-compensatory mechanism to shunt excess pyruvate from being oxidized in the TCA cycle .\",\n \"To test if the up-regulation of pck genes in sir-2 . 1 ( 0 ) is a compensatory response , we assayed the mating potency of pck-1 ( 0 ) and pck-2 ( 0 ) single mutants and sir-2 . 1 ( 0 ) ; pck-1 ( 0 ) and pck-2 ( 0 ) ; sir-2 . 1 ( 0 ) double mutants .\",\n \"At day 1 , sir-2 . 1 ( 0 ) and pck-2 ( 0 ) males mated comparable to wild type ( Figure 6E ) .\",\n \"However at day 2 , the potency of pck-2 ( 0 ) males started to decline similarly to sir-2 . 1 ( 0 ) .\",\n \"In contrast , for males containing both sir-2 . 1 ( 0 ) and pck-2 ( 0 ) , their mating potency dropped at day 1 ( Figure 6E ) .\",\n \"This indicates that without pck-2 , males that contain or lack sir-2 . 1 display accelerated behavioral decline .\",\n \"Similar to the requirement for functional pck-2 , males that lack sir-2 . 1 also needed pck-1 to maintain their mating potency at day 1; however , pck-1 was not required for sir-2 . 1 ( + ) males to mate efficiently at day 2 ( Figure 6F ) .\",\n \"Next , we reasoned that if excessive glycolysis contributes to the behavioral deterioration , artificially adding extra glucose to the males’ media could accelerate their mating decline .\",\n \"To test this , we grew males on UV-killed-OP50 NGM plates supplemented with 2% glucose , from hatched larvae up to the adult age prior to behavioral decline , which is day 1 or day 2 , for sir 2 . 1 ( 0 ) and wild-type males , respectively .\",\n \"We found that the glucose reduced mating potency of 1-day-old sir-2 . 1 ( 0 ) , but not 2-day-old wild type ( Figure 6G ) , indicating that wild type can cope with the extra glucose better than sir-2 . 1 ( 0 ) .\",\n \"We hypothesized that unlike wild type , sir-2 . 1 ( 0 ) males cannot efficiently respond to the oxidative stress generated by the enhanced catabolism .\",\n \"To test this , we used qRT-PCR to measure the mRNA levels of antioxidant genes: superoxide dismutase ( sod-1 , 2 , 3 , 4 and 5 ) , catalase ( ctl-1 , 2 ) , and glutathione transferase ( gst-10 and gsto-1 ) relative to 1-day-old wild-type males ( Figure 7 ) .\",\n \"As expected , the expression of sod-1 , sod-5 , gst-10 and gsto-1 was reduced in 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild type ( Figure 7 ) .\",\n \"For sod-2 , day 1 expression was also reduced in sir-2 . 1 ( 0 ) , but this gene’s expression increased in both wild type and sir-2 . 1 ( 0 ) at day 2 , possibly a stress response .\",\n \"For sod-3 and ctl-2 , their day 1 expression was similar in both strains; however at day 2 , sod-3 expression became higher and ctl-2 expression became lower in mutants .\",\n \"Finally , sir-2 . 1 ( 0 ) males displayed an increased ctl-1 expression at day 1 , which is also reported in antioxidant-compromised daf-16 ( 0 ) mutant .\",\n \"The enhanced expression of ctl-1 is considered as an adaptive response ( Yanase et al . , 2002 ) .\",\n \"These results indicate that in addition to a potentially altered metabolism , which could generate excessive ROS , sir-2 . 1 ( 0 ) males might also have a comprised antioxidant response , which is consistent with their hypersensitivity to excessive glucose intake ( Figure 6G ) and to the ROS generator ( Figure 4A ) . 10 . 7554/eLife . 01730 . 018Figure 7 . sir-2 . 1 ( 0 ) males have compromised expression of anti-oxidant genes . Relative mRNA expression level of anti-oxidant genes superoxide dismutase ( A ) , catalase ( B ) , and glutathione transferase ( C ) in 1 , 2-day-old wild type and sir-2 . 1 ( 0 ) males ( unpaired t-test ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 018 Based on the above results , one could hypothesize that increasing SIR-2 . 1 expression or activity might delay mating deterioration during aging .\",\n \"However , we found that transgenic overexpression of sir-2 . 1 does not improve the mating potency of 3-day-old wild type ( Figure 8A ) .\",\n \"It is unlikely that the fusion with YFP disrupts SIR-2 . 1 function , because the same transgene can rescue the sir-2 . 1 ( 0 ) phenotype .\",\n \"Thus , we speculate that up to a point , the expression level of sir-2 . 1 is not rate limiting for SIR-2 . 1 activity during early aging .\",\n \"However , one could also speculate that the normal endogenous levels of NAD+ limit the function of SIR-2 . 1 .\",\n \"To test this , we grew males in the presence of the NAD+ precursor nicotinamide ( NAM ) at 200 μM concentration ( Houtkooper et al . , 2010 ) , and then conducted the mating potency .\",\n \"Indeed , NAM exposure significantly improves 3-day-old wild-type mating potency , but not 2-day-old sir-2 . 1 ( 0 ) males ( Figure 8B , C ) .\",\n \"This result is consistent with the idea that excess NAD+ might stimulate SIR-2 . 1 activity .\",\n \"But additionally , excess NAD+ might also reduce ROS production by relaxing the demand of oxidizing NADH back to NAD+; as a corollary to this possibility , the lack of excess NAM to positively affect the sir-2 . 1 ( 0 ) male’s behavior might be aggravated by the abnormally high expression of catabolic enzymes in the mutant males .\",\n \"To further test if overexpressing SIR-2 . 1 activity can promote mating behavior in older males , we exposed 3-day-old transgenic SIR-2 . 1 over-expressed males with exogenous NAM , but found that excess SIR-2 . 1 does not amplify the positive effect of NAM ( Figure 8D ) .\",\n \"Thus , we cannot exclude the possibility that NAM or possibly NAD+ additionally promotes behavioral extension through mechanisms parallel to SIR-2 . 1 activity . 10 . 7554/eLife . 01730 . 019Figure 8 . Exogenous nicotinamide improves mating during aging . sir-2 . 1 overexpression cannot increase mating potency of 3-day-old wild type ( A ) .\",\n \"However , feeding with a NAD+ precursor nicotinamide ( NAM ) significantly improve the mating potency of 3-day-old wild type ( B ) but not 2-day-old sir-2 . 1 ( 0 ) males ( C ) .\",\n \"Overexpression of sir-2 . 1 cannot further promote the effect of exogenous NAM ( D ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 019\"\n ],\n [\n \"Behavioral decline at advanced age can be attributed to morphological degeneration , such as neuronal death and muscle sarcopenia ( Herndon et al . , 2002; Glenn et al . , 2004 ) .\",\n \"However , during early aging , prior to any significant cellular decay , physiological changes that affect neuronal and muscular functionality can contribute to the reduction of behavioral coordination ( Salthouse , 2004 ) .\",\n \"Male mating in C . elegans is a complex behavior that requires coordination of multiple motor systems to impregnate the hermaphrodite ( Liu et al . , 2011; Correa et al . , 2012 ) .\",\n \"Previously , we showed that male mating behavior declines significantly during early aging .\",\n \"Wild type cannot mate well at adulthood day 3 , and this decline is correlated with an increased excitability in the sex circuitry ( Guo et al . , 2012 ) .\",\n \"Similar to aging males , a recent study using hermaphrodites showed that synaptic transmission in the locomotion circuit is enhanced after 5 days of adulthood ( Mulcahy et al . , 2013 ) .\",\n \"Thus , it is possible that during early aging , the neuromuscular systems of both sexes become hyperexcitable .\",\n \"However , the faster decline in mating suggests that the male reproductive circuitry is more sensitive to age-related physiological changes .\",\n \"We found that SIR-2 . 1 is a modulator of behavior and is required to maintain mating in aging males .\",\n \"1-day-old sir-2 . 1 ( 0 ) males can mate similarly to wild type , suggesting that sir-2 . 1 is not essential for mating .\",\n \"However , unlike wild-type males , the mating ability of sir-2 . 1 ( 0 ) prematurely drops at day\",\n \"2 . This is due to hyperexcitability of the reproductive circuitry that coordinates spicule intromission and ejaculation .\",\n \"The hyperexcitability of the spicule muscles causes the male proctodeum to block the connection between the vas deferens and the cloacal opening , which indirectly obstructs the transfer of sperm .\",\n \"The mutant phenotype resembles the behavioral , physiological , and pharmacological changes that occur in older wild-type males .\",\n \"This indicates that in wild type , SIR-2 . 1 maintains the functional excitability of the intromission and ejaculation circuit , possibly by slowing down the deteriorative events that accumulate during aging .\",\n \"We suggest that as the male ages , SIR-2 . 1 regulates the amounts of catabolic , anabolic , and free radical scavenging enzymes to balance the energy demands needed for rapid reproductive motor responses , with the generation of damaging metabolic by-products , such as ROS .\",\n \"Our data indicate that the male’s cellular physiology is correlated with his ability to mate .\",\n \"We showed previously and in this study that perturbation of the male’s physiology through genetic mutation or through dietary alterations during early adulthood will affect his ability to mate later in life .\",\n \"The physiology of wild-type males is likely changing from day 1 to day 2 , as determined by the level of mRNAs encoding metabolic enzymes and the amount of terminal metabolic products .\",\n \"These physiological changes can ultimately lead to excessive carbon flow into the TCA cycle , and consequently , more NADH to be oxidized by ETC complexes ( Figures 6A and 9 ) .\",\n \"This promotes ROS generation via electron leak ( Federico et al . , 2012 ) , which can be reflected by behavioral decay at day\",\n \"3 . Analysis of sir-2 . 1 ( 0 ) males allowed us to extrapolate how this protein deacetylase regulates male physiology .\",\n \"Lack of SIR-2 . 1 will induce these deleterious changes to occur sooner , and degenerative behavioral responses in the mutant males are measured at day\",\n \"2 . Under standard laboratory conditions , wild-type males are raised constitutively on abundant E . coli until senescence .\",\n \"We speculate that since SIR-2 . 1 uses NAD+ as a cofactor to deacetylate proteins , the physiological changes that occur after 2 days of constitutive feeding in wild type adults might be due to reduction in SIR-2 . 1 function , via the lower ratio of NAD+ to NADH in wild type .\",\n \"The phenomenon of altering NAD+ to NADH levels in vertebrate cells is shown to reduce the activity of SIRT1 ( Braidy et al . , 2011 ) .\",\n \"Decreased activity of SIR-2 . 1 will not only aggravate a bias towards catabolism , but will also decrease the levels of ROS scavengers .\",\n \"This is consistent with a hermaphrodite study , which showed that sir-2 . 1 overexpression can protect the organism from ROS , possibly via HCF-1 and FOXO/DAF-16 , to regulate the expression of stress response genes ( Rizki et al . , 2011 ) . 10 . 7554/eLife . 01730 . 020Figure 9 . A cartoon of the metabolism and behavior that occurs in wild-type and sir-2 . 1 ( 0 ) males during early aging . For successful reproductive behavior , SIR-2 . 1 is required to maintain proper carbon flow to meet the male’s energy demands and balance the generation of ROS .\",\n \"In 1-day-old old sir-2 . 1 ( 0 ) males , catabolism such as glycolysis and fatty acid oxidation is enhanced , and consequently , oxidative phosphorylation and generation of ROS are also increased .\",\n \"Without SIR-2 . 1 , ROS accumulation by day 2 of adulthood can lead to hyperexcitability of the male’s genital neuromuscular circuitry .\",\n \"This results in blocked ejaculation and impotency .\",\n \"It is possible that in 2- to 3-day-old wild-type males , the NAD+-dependent SIR-2 . 1 activity declines due to a lower ratio of NAD to NADH; thus older wild-type males might have a similar physiology as 1-day-old sir-2 . 1 ( 0 ) males . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 020 Although our qPCR analyses suggest that enhanced catabolism might be occurring in 1-day-old of sir-2 . 1 ( 0 ) and 2-day-old wild-type males , their mating ability could be facilitated via anabolic compensatory mechanisms .\",\n \"In addition to enhanced expression of catabolic genes , mRNAs encoding enzymes such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase ( PEPCK ) appeared to be also up-regulated .\",\n \"This could be a likely reason for why the males contain more measurable lipids and glycogen .\",\n \"We propose that the up-regulation of anabolic process is a self-compensatory mechanism to divert carbon from the TCA cycle ( Figure 9 ) .\",\n \"sir-2 . 1 ( 0 ) males that are mutant for PEPCK genes , lose their ability to generate fat and fail to mate efficiently on day 1 .\",\n \"Likewise sir-2 . 1 ( + ) males with a mutation in PEPCK genes also display premature mating decline .\",\n \"We hypothesize that anabolic pathways could act as a homeostatic mechanism to reduce ROS production .\",\n \"This idea raises the possibility that obesity as a phenotype might be a compensatory mechanism to alleviate the effects of other underlying metabolic dysfunctions .\",\n \"Indeed , by switching of glycolysis to gluconeogenesis has been shown to be a potential strategy to cure hepatocarcinoma ( Ma et al . , 2013 ) .\",\n \"A recent study showed that diabetic patients , who are not obese , have higher mortality than overweight ones ( Carnethon et al . , 2012 ) .\",\n \"Another study showed that a lifestyle intervention focusing on weight loss did not reduce the rate of cardiovascular events in obese adults with type II diabetes ( Look AHEAD Research Group , 2013 ) , challenging the traditional viewpoint that obesity is a major contributor to metabolic disorders .\",\n \"The free radical aging theory states that aging is the result of free radical-induced molecular damages ( Harman , 1956 ) .\",\n \"Although this notion is challenged by the hormesis theory , which posits that moderate amounts of physiological or environmental insults can reinforce cellular processes that reduce stress-induced damage ( Afanas’ev , 2010; Schulz et al . , 2007 ) , artificially applied ROS is reported to change the excitability of cultured neurons and muscles through chemically damaging ion channels ( Danson and Paterson , 2006 ) .\",\n \"Therefore , aspects of the free radical theory of aging might still apply to physiological changes in neural muscular systems and behavioral decay .\",\n \"Consistent with this , we showed here that genetic and environmental conditions , which can lead to oxidative stress , caused increased excitability of the male mating circuitry and behavioral decay during early aging .\",\n \"There is not a strict correlation between oxidative stress and changes in the electrical properties of neurons and muscles .\",\n \"Oxidation of different types of ion channels changes their conductance and alters the cell’s excitability ( Annunziato et al . , 2002 ) .\",\n \"One C . elegans study indicates that oxidative stress reduces cell excitability by increasing the conductance of K+ channels ( Sesti et al . , 2010 ) .\",\n \"Another mammalian study showed that oxidative stress hyperpolarizes the resting potential , but extends the duration of the action potential in cardiac ganglion ( Whyte et al . , 2009 ) .\",\n \"Mitochondria ROS has been shown to trigger Ca2+ increases in the pulmonary arterial myocytes ( Waypa et al . , 2002 , 2006 ) .\",\n \"In a study using glia , L-type voltage-gated Ca2+ channels ( L-VGCC ) were found to be a target of ROS .\",\n \"After modification by ROS , their conductance of Ca2+ was increased ( Bond and Greenfield , 2007 ) .\",\n \"Different from vertebrate skeletal muscles , L-VGCC in C . elegans propagates action potentials , and the entry of external Ca2+ directly promotes excitation–contraction coupling ( Lee et al . , 1997; Maryon et al . , 1998 ) .\",\n \"Previous work showed that L-VGCC EGL-19 in C . elegans is required for sustained tonic contraction of the copulatory spicule muscles ( Garcia et al . , 2001 ) .\",\n \"Therefore , oxidation of L-VGCC in C . elegans might contribute to the increased excitability of mating circuits .\",\n \"Other major targets of ROS are voltage-gated K+ channels .\",\n \"In C . elegans , oxidation of KVS-1 slows down its inactivation , leading to hyperpolarization and sensory function loss ( Cai and Sesti , 2009 ) .\",\n \"The ERG-like K+ channel UNC-103 is a major excitability regulator of the sex circuit ( Reiner et al . , 2006 ) .\",\n \"Although there is no report of oxidative modification on UNC-103 , human encoded H-ERG channels can be activated by oxidative stress , so that cells become hyperpolarized and less excitable ( Cui and Zhang , 2013 ) .\",\n \"Considering that ROS increases the excitability of the mating circuit , it is possible that L-VGCC is more prone to be oxidized than K+ channels in male reproductive cells .\"\n ],\n [\n \"Worms were grown at 20°C on nematode growth media ( NGM ) plates seeded with E . coli strain OP50 , except for strains containing the pha-1 ( e2123 ) , which were maintained at 15°C .\",\n \"The alleles used in this work included: lite-1 ( ce314 ) on LGX; pck-2 ( ok2586 ) on LGI; pck-1 ( ok2098 ) , pha-1 ( e2123 ) ( Schnabel and Schnabel , 1990 ) , unc-64 ( e240 ) ( Brenner , 1974 ) on LGIII; sir-2 . 1 ( ok434 ) on LGIV; and him-5 ( e1490 ) ( Hodgkin et al . , 1979 ) on LGV .\",\n \"The males were generated by the him-5 ( e1490 ) mutation .\",\n \"Males containing only this mutation are referred to as wild-type; him-5 ( e1490 ) males have been shown to mate efficiently as wild type ( Hodgkin , 1983 ) .\",\n \"pck-2 ( ok2586 ) , pck-1 ( ok2098 ) , and sir-2 . 1 ( ok434 ) were generated by the C . elegans Gene Knockout Consortium .\",\n \"sir-2 . 1 ( ok434 ) animals were out-crossed 4 times with the him-5 ( e1490 ) strain .\",\n \"The deletion in sir-2 . 1 ( 0 ) was detected through PCR using primers listed in the Supplementary file 2 .\",\n \"Altered mediums used here included NGM medium containing glucose ( 2% ) , paraquat , N-acetyl-cystine ( NAC ) ( Sigma , St . Louis , MO ) , and nicotinamide ( NAM ) ( Sigma ) respectively .\",\n \"The latter three were added at the indicated concentration just before pouring the plates .\",\n \"OP50 used for the special medium containing glucose and NAC was UV-killed and concentrated to make sure the worms were not food deprived .\",\n \"We assume that the animals ingest these compounds as they feed on E . coli or absorb them through their cuticle .\",\n \"DNA primers are listed in the Supplementary file 2 .\",\n \"The sir-2 . 1 genomic sequence , plus 2 kb upstream of its ATG , was PCR-amplified from N2 DNA .\",\n \"The PCR product was digested and ligated between the SphI and SalI sites of pSX322YFP to obtain the plasmid pXG5 .\",\n \"To obtain promoters for driving sir-2 . 1 expression , the sir-2 . 1 endogenous promoter was removed via PCR-mutagenesis from pXG5 to construct pXG6 .\",\n \"The Gateway ATTR cassette frame A was inserted in front of the sir-2 . 1 genomic sequence to make the destination clone pXG7 .\",\n \"Plasmids ( pXG8 , pXG9 , and pXG11 ) that promote neuronal , muscular , and intestinal expression of sir-2 . 1 were obtained through Gateway LR reactions between pXG7 and pLR35 ( Paex-3 ) ( LeBoeuf et al . , 2007 ) , pLR22 ( Plev-11 ) ( Gruninger et al . , 2008 ) and pBL50 ( Pges-1 ) ( Urano et al . , 2002 ) , respectively .\",\n \"pXG5 ( 25 ng/μl ) , pXG8 ( 10 ng/μl ) , pXG9 ( 1 ng/μl ) or pXG11 ( 50 ng/μl ) were injected to sir-2 . 1 ( 0 ) hermaphrodites and transgenic animals were selected via YFP fluorescence .\",\n \"pXG5 ( 50 ng/μl ) was injected into wild-type hermaphrodites to obtain strains with overexpression of sir-2 . 1 ( referred as sir-2 . 1 ( OE ) ) .\",\n \"The mating potency assay was performed as described in Guo et al . ( 2012 ) .\",\n \"Briefly , L4 males were isolated from hermaphrodites .\",\n \"One male and one pha-1 ( e2123 ) hermaphrodite were co-transferred to a 5-mm-diameter OP50 mating lawn at 20°C , which is a restrictive temperature for pha-1 ( e2123 ) to sire viable self-progeny .\",\n \"This allowed us to score the mating potency of males , as only cross-progenies would develop to adulthood .\",\n \"To quantify different parameters of mating , we observed , up to 5 min , copulations between males and paralyzed unc-64 ( e240 ) hermaphrodites .\",\n \"The ability of males to sense the vulva was calculated by counting the number of times he stopped at the vulva divided by the total number of times he stopped and/or passed by the vulva .\",\n \"The turning quality was calculated as: the number of smooth turns ( defined as the male tail keeping contact with hermaphrodite and turning without hesitation ) divided by the total number of turns .\",\n \"Ejaculation assays were conducted in two ways .\",\n \"We directly observed sperm transfer after spicule insertion , and determined if sperm drained into the unc-64 ( e240 ) hermaphrodite’s uterus .\",\n \"Additionally , cross-progeny were counted 1–2 days after spicules insertion .\",\n \"Lifespan assay was conducted as described in Guo et al . ( 2012 ) .\",\n \"L4 males were isolated and raised 20 per plate .\",\n \"The males that can respond to gentle touch with a platinum wire were counted and transferred to new plates every day .\",\n \"Males that dried on the wall of the Petri plate were censored from the assay on the day they died .\",\n \"To assess the sensitivity to paraquat , L4 males were transferred to plates containing 10 mM paraquat and scored at 24 and 48 hr .\",\n \"Sperm activation assays were done according to L’Hernault and Roberts ( 1995 ) ; Smith and Stanfield ( 2011 ) .\",\n \"Briefly , three 2-day-old males , isolated at L4 stage , were cut at the posterior portion with a needle in 20 μl sperm media ( 50 mM Hepes , pH7 . 0 , 45 mM NaCl , 25 mM KCl , 1 mM MgSO4 , and 5 mM CaCl2 ) freshly supplemented with polyvinylprolidone ( PVP ) 40 , 000 molecular weight ( Sigma ) and the activator pronase ( Roche , Indianapolis , IN ) at the final concentration of 10 mg/ml and 500 μg/ml on a slide .\",\n \"A coverslip with a thin layer of Vaseline applied around the edge was put on the top of the sperm media to form a chamber over the sperm .\",\n \"After 5-min incubation , activated sperm with a pseudopod and inactive ones were counted using a compound scope fitted with a 100X objective .\",\n \"Approximately , 50–60 sperm cells were counted in each sample section .\",\n \"Arecoline ( ARE ) and levamisole ( LEV ) were dissolved in water at the concentration of 100 mM , and then diluted accordingly , as described in Liu et al . ( 2007 ) .\",\n \"Briefly , 50 μM of ARE , 1 μM and 500 nM of LEV for 1 , 2-day-old males , and 100 nM LEV for 3-day-old males were used .\",\n \"Males were introduced to 1 ml drug baths and scored if they protruded their spicules for more than 5 s within 5 min of drug exposure .\",\n \"Ca2+ imaging was conducted and analyzed as described in Guo et al . ( 2012 ) .\",\n \"50 ng/μl pLR289[Punc-103E:G-CaMP3::sl2:::DsRed] ( Correa et al . , 2012 ) and 50 ng/μl pBX1 ( Granato et al . , 1994 ) were co-injected into pha-1 ( e2123 ) ; him-5 ( e1490 ) ; lite-1 ( ce314 ) hermaphrodites to generate the extrachromosomal array rgEx566[Punc-103E:G-CaMP3::sl2:::DsRed] .\",\n \"rgEx566 was crossed into pha-1 ( e2123 ) ; him-5 ( e1490 ) ; sir-2 . 1 ( 0 ) ; lite-1 ( ce314 ) hermaphrodites .\",\n \"Fluorescence signals from G-CaMP3 and DsRed were recorded simultaneously during the copulations of 2-day-old wild-type and sir-2 . 1 ( 0 ) males with paralyzed hermaphrodites containing the transgene rgEx431[Phsp-16:egl-2 ( gf ) ; Punc-103E:DsRed] .\",\n \"300 day 1 and day 2 adult males were accumulated over a period of time .\",\n \"RNA was extracted by Trizol , and cDNA was synthesized by SuperScript II ( Life technology , Grand Island , NY ) using around 2 μg total RNA , as described in LeBoeuf and Garcia ( 2012 ) .\",\n \"The RT-qPCR reactions were performed using BIO-RAD CFX96 real-time system and SsoFast EvaGreen supermix .\",\n \"11 candidate reference genes were tested to see whether their expression changed from day 1 and day 2 in both wild-type and sir-2 . 1 males ( Hoogewijs et al . , 2008 ) ; from our analyses , act-1 and gpd-2 were selected as the reference to normalize the expression of the metabolic genes .\",\n \"Many of the primers used to detect the expression of metabolic enzymes are described in Castelein et al . ( 2008 ) .\",\n \"Other primers for additional metabolic and antioxidant stress genes are listed in the Supplementary file 2 .\",\n \"Three replicates were conducted on the same RNA samples .\",\n \"We used the t-test to determine which mRNA transcripts in sir-2 . 1 ( 0 ) males were significantly different from their cognate wild-type transcripts .\",\n \"To measure ATP and glucose , 100 males were collected at different ages , frozen and thawed three times .\",\n \"The worms were homogenized , and the supernatant was collected and measured using an ATP determination Kit ( Life technology ) and the Glucose Oxidase Assay Kit ( Life technology ) .\",\n \"The ATP and glucose were normalized to the amount of dsDNA quantified by picoGreen ( Life technology ) .\",\n \"To stain glycogen , 1-day-old sir-2 . 1 ( 0 ) and wild-type virgin males were transferred to 2% agar pads .\",\n \"The pads containing both genotypes were then placed over a bottle of iodine crystals for 30 s ( Frazier and Roth , 2009 ) .\",\n \"The pictures were taken by a Leica compound scope mounted with OLYMPUS DP70 camera .\",\n \"The RGB images were then converted to 16-bit gray scale , and the mean gray levels of the isthmus regions were measured using the SimplePCI image quantification software ( Hamamatsu , Janpan ) .\",\n \"The mean gray level was reversely correlated with the red signal .\",\n \"Oil Red O staining was done according to O’Rourke et al . ( 2009 ) .\",\n \"Briefly , males were collected and washed with PBS , and then fixed with Modified Ruvkuns witches brew ( MRWB ) buffer containing 1% paraformaldehyde ( PFA ) for 1 hr .\",\n \"Worms were then washed with PBS and suspended in 60% isopropanol for 15 min at room temperature .\",\n \"The 60% isopropanol was removed and worms were bathed in 60% Red Oil O staining solution .\",\n \"The RGB images were taken by a Leica compound scope mounted with OLYMPUS DP70 camera .\",\n \"The images were quantified by ImageJ according to Mehlem et al . ( 2013 ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["The decline of aging C . elegans male’s mating behavior is correlated with the increased excitability of the cholinergic circuitry that executes copulation .","In this study , we show that the mating circuits’ functional durability depends on the metabolic regulator SIR-2 . 1 , a NAD+-dependent histone deacetylase .","Aging sir-2 . 1 ( 0 ) males display accelerated mating behavior decline due to premature hyperexcitability of cholinergic circuits used for intromission and ejaculation .","In sir-2 . 1 ( 0 ) males , the hypercontraction of the spicule-associated muscles pinch the vas deferens opening , thus blocking sperm release .","The hyperexcitability is aggravated by reactive oxygen species ( ROS ) .","Our genetic , pharmacological , and behavioral analyses suggest that in sir-2 . 1 ( 0 ) and older wild-type males , enhanced catabolic enzymes expression , coupled with the reduced expression of ROS-scavengers contribute to the behavioral decline .","However , as a compensatory response to reduce altered catabolism/ROS production , anabolic enzymes expression levels are also increased , resulting in higher gluconeogenesis and lipid synthesis ."],"string":"[\n \"The decline of aging C . elegans male’s mating behavior is correlated with the increased excitability of the cholinergic circuitry that executes copulation .\",\n \"In this study , we show that the mating circuits’ functional durability depends on the metabolic regulator SIR-2 . 1 , a NAD+-dependent histone deacetylase .\",\n \"Aging sir-2 . 1 ( 0 ) males display accelerated mating behavior decline due to premature hyperexcitability of cholinergic circuits used for intromission and ejaculation .\",\n \"In sir-2 . 1 ( 0 ) males , the hypercontraction of the spicule-associated muscles pinch the vas deferens opening , thus blocking sperm release .\",\n \"The hyperexcitability is aggravated by reactive oxygen species ( ROS ) .\",\n \"Our genetic , pharmacological , and behavioral analyses suggest that in sir-2 . 1 ( 0 ) and older wild-type males , enhanced catabolic enzymes expression , coupled with the reduced expression of ROS-scavengers contribute to the behavioral decline .\",\n \"However , as a compensatory response to reduce altered catabolism/ROS production , anabolic enzymes expression levels are also increased , resulting in higher gluconeogenesis and lipid synthesis .\"\n]"},"summary":{"kind":"list like","value":["Although the signs of aging are clear to us all , precisely why we age is less well understood .","One possibility is that as cells use oxygen to fuel the breakdown of large molecules into smaller ones to release energy , they also generate by-products called reactive oxygen species that can damage DNA .","As we get older , this damage gets worse .","Consistent with this idea , it has been shown that a reduced calorie intake can reduce oxidative damage in certain species , in addition to extending lifespan .","Many experiments on aging have been performed on worms belonging to the species C . elegans .","Male worms of this species live for an average of 11–12 days , but begin to show signs of aging—for example , a reduced ability to mate—as early as day 3 of their adult lives .","Now , Guo and García have revealed that a protein called SIR-2 . 1 , which regulates metabolism in worms , also helps to protect the animals from the effects of aging .","Male worms in which the gene for this protein has been ‘knocked out’ have a normal lifespan , but show signs of aging earlier than normal males .","They are also more susceptible to the damaging effects of reactive oxygen species , suggesting that SIR-2 . 1 may offer protection against oxidative damage .","Indeed , levels of ATP—the molecule used to move energy around inside cells—are increased in knockout worms .","This suggests that certain metabolic processes and the production of reactive oxygen species , are increased in the knockout worms , which speeds up the aging process .","While the link between metabolism and aging is well known , the work of Guo and García offers insights into some of the molecular mechanisms that may form the basis of this relationship ."],"string":"[\n \"Although the signs of aging are clear to us all , precisely why we age is less well understood .\",\n \"One possibility is that as cells use oxygen to fuel the breakdown of large molecules into smaller ones to release energy , they also generate by-products called reactive oxygen species that can damage DNA .\",\n \"As we get older , this damage gets worse .\",\n \"Consistent with this idea , it has been shown that a reduced calorie intake can reduce oxidative damage in certain species , in addition to extending lifespan .\",\n \"Many experiments on aging have been performed on worms belonging to the species C . elegans .\",\n \"Male worms of this species live for an average of 11–12 days , but begin to show signs of aging—for example , a reduced ability to mate—as early as day 3 of their adult lives .\",\n \"Now , Guo and García have revealed that a protein called SIR-2 . 1 , which regulates metabolism in worms , also helps to protect the animals from the effects of aging .\",\n \"Male worms in which the gene for this protein has been ‘knocked out’ have a normal lifespan , but show signs of aging earlier than normal males .\",\n \"They are also more susceptible to the damaging effects of reactive oxygen species , suggesting that SIR-2 . 1 may offer protection against oxidative damage .\",\n \"Indeed , levels of ATP—the molecule used to move energy around inside cells—are increased in knockout worms .\",\n \"This suggests that certain metabolic processes and the production of reactive oxygen species , are increased in the knockout worms , which speeds up the aging process .\",\n \"While the link between metabolism and aging is well known , the work of Guo and García offers insights into some of the molecular mechanisms that may form the basis of this relationship .\"\n]"},"year":{"kind":"string","value":"2014"}}},{"rowIdx":4187,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["cell biology"],"string":"[\n \"cell biology\"\n]"},"title":{"kind":"string","value":"Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery"},"id":{"kind":"string","value":"elife-01641-v1"},"sections":{"kind":"list like","value":[["When mitotic chromosomes are examined by whole mount microscopy the surface chromatin is obscured by a layer of proteins and RNA derived from the dense fibrillar component ( DFC ) and granular component ( GC ) of the nucleolus ( Moyne and Garrido , 1976; Harrison et al . , 1982; Adolph and Kreisman , 1983; Sumner , 1991; Gautier et al . , 1992b; Wanner et al . , 2005 ) .","This perichromosomal layer includes pre-rRNA , U3 snoRNAs , and over 20 ribosomal proteins , including nucleolin and Nopp140 , NPM/B23 , Bop1 , Nop52 , PM-Scl 100 , and Ki-67 ( Gautier et al . , 1992a , 1994; Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005; Fomproix et al . , 1998; Angelier et al . , 2005 ) .","The perichromosomal layer represents 1 . 4% of the chromosome proteome ( Ohta et al . , 2010 ) .","At present , its functional significance remains unstudied ( Van Hooser et al . , 2005 ) .","Ki-67 is one of the earliest proteins to bind the perichromosomal layer in mitosis .","Ki-67 is an antigen recognised by a monoclonal antibody generated by immunizing mice with nuclei of Hodgkin lymphoma cells ( Gerdes et al . , 1983 ) .","Because Ki-67 is nuclear only in proliferating cells , it is widely used as a marker to assess cell proliferation .","For example , immuno-histochemical assessment of the proportion of cells staining for nuclear Ki-67 is used to predict the responsiveness or resistance of tumours to therapy ( Dowsett et al . , 2011 ) .","Ki-67 protein is predominantly localised in the cortex and dense fibrillar components of the nucleolus during interphase ( Kill , 1996; MacCallum and Hall , 2000b ) .","During mitosis it relocates to the periphery of the condensed chromosomes ( Verheijen et al . , 1989b; Isola et al . , 1990 ) .","It has been reported that Ki-67 is associated with the nuclear matrix ( Verheijen et al . , 1989b ) , preribosomes ( Isola et al . , 1990 ) , satellite DNA in G1 ( Bridger et al . , 1998 ) , and with the chromosome scaffold of mitotic cells ( Verheijen et al . , 1989a ) .","Approximately 40% of the cellular pool of protein is present on isolated mitotic chromosomes ( Ohta et al . , 2010 ) .","Previous studies suggested that Ki-67 is regulated by phosphorylation .","Hyperphosphorylated Ki-67 does not bind DNA during mitosis ( Endl and Gerdes , 2000; MacCallum and Hall , 1999 ) and is characterized by increased mobility , on mitotic chromosomes measured by FRAP .","In interphase , non-phosphorylated Ki-67 binds DNA and does not recover after FRAP ( Saiwaki et al . , 2005 ) .","Its localisation and cell-cycle behaviour suggest that Ki-67 could be involved in the organisation of nucleolar chromatin during interphase in proliferating cells .","Recently , it was reported that Ki-67 also functions in mitosis in stabilisation and maintenance of the mitotic spindle by recruiting Hklp2 to mitotic chromosomes ( Vanneste et al . , 2009 ) .","In this study , we have identified Ki-67 as ancestral to the PP1 targeting subunit Repo-Man ( Trinkle-Mulcahy et al . , 2006 ) and have shown that Ki-67 is a PIP ( PP1 Interacting Protein ) that contributes to the phospho-regulation of nucleophosmin/B23 by CKII .","Ki-67 is also a major organiser of the perichromosomal layer , possibly acting as an interaction platform .","Remarkably , Ki-67 depletion prevents all nucleolar proteins tested from accumulating around the chromosomes in mitosis .","Thus , chromosomes in cells depleted of Ki-67 appear to lack most or all of their perichromosomal layer .","This enabled us to gain insights into the function of this enigmatic chromosomal compartment .","Interestingly , loss of the perichromosomal layer does not detectibly compromise the condensed morphology or intrinsic structure of mitotic chromosomes but does result in significant changes in nucleolar morphology and nuclear organization in the following interphase ."],["We used a phylogenetic approach to identify putative functional regions within the sequence of Repo-Man , a targeting protein that binds PP1 in a cell-cycle specific manner regulated by a phospho-switch ( Trinkle-Mulcahy et al . , 2006; Qian et al . , 2011; Vagnarelli et al . , 2011 ) .","A BLAST search revealed significant ( E = 5 × 10−4 ) similarity between a small region ( amino acids 388–420 ) of human Repo-Man and Ki-67 ( Figure 1A1 , 2 ) , a very large protein that exhibits strong links to cell proliferation ( Gerdes et al . , 1983 ) .","The region conserved between Repo-Man and Ki-67 contains the PP1 binding motif ( RVTF ) of Repo-Man , which is conserved as RVSF in human Ki-67 ( Figure 1C3 ) . 10 . 7554/eLife . 01641 . 003Figure 1 . Ki-67 is evolutionary related to Repo-Man but shows distinct behaviour during mitosis .","( A1 )","Schematic representations of evolutionarily conserved regions in human Repo-Man and Ki-67 proteins ( shown approximately to scale ) .","( A2 )","E-values corresponding to global profile-to-sequence ( HMMer3 ) comparisons between the PP1 binding conserved regions ( blue oval ) in Repo-Man and Ki-67 families .","Arrows indicate the profile search direction .","The Repo-Man profile identified Ki-67 proteins as homologous with a highly significant E-value of 4 . 9 × 10−11 .","Conversely , the profile of Ki-67 homologous sequences in animals identified Repo-Man proteins with a significant E-value of 9 . 9 × 10−13 .","( A3 )","Representative multiple sequence alignment of conserved regions from Repo-Man and Ki-67 families .","Important mitotic phospho-residues in Repo-Man ( T412 and T419 ) are indicated in blue .","The RVTF motif is indicated with an orange box .","The most parsimonious explanation of Repo-Man and Ki-67 evolution is shown to the left of the alignment .","Vertebrate branches are coloured in blue .","Sequences are named according to: Repo_Human , NCBI:NP_689775 , Homo sapiens; Repo_Frog , NCBI:ACR33033 , Xenopus laevis; Repo_Danre , UniProt:A2CEF0 , Danio rerio; Ki_Human , UniProt:P46013 , Homo sapiens; Ki_Frog , UniProt:Q0VA85 , Xenopus laevis; Ki_Fugu , UniProt:UPI00016EA029 , Takifugu rubripes; Ki_Cioin , UniProt:UPI000180CFDA , Ciona intestinalis; Ki_Sacko , Baylor College of Medicine genome and FGENESH+ , Saccoglossus kowalevskii; Ki_Hydra , UniProt:UPI0001926DD5 , Hydra magnipapillata; and , Ki_Triad , UniProt:B3SB24 , Trichoplax adhaerens .","The amino acid colouring scheme indicates average BLOSUM62 scores ( which are correlated with amino acid conservation ) for each alignment column: red ( greater than 3 . 5 ) , violet ( between 3 . 5 and 2 . 5 ) , and light yellow ( between 2 . 5 and 0 . 5 ) .","( B–C )","Ki-67 and PP1γ interact in vivo .","Ki-67301–700 fused to Lac repressor:GFP ( Lac repressor:Ki-67PP1BD_wt ) was transfected together with RFP:PP1γ into a DT40 cell line containing a LacO array integrated on a single chromosome .","Ki-67 was enriched at the LacO site ( panels 2 , 2′ ) where it recruited PP1γ ( 2 , 2″ ) .","However , neither Lac repressor:GFP ( panels 1–1″ ) or the Ki-67 PP1-non-binding mutant ( Lac repressor:Ki-67PP1BD_RASA ) ( panels 3–3″ ) caused PP1 accumulation at the LacO site .","( D ) Ki-67 recruits PP1γ more efficiently than PP1beta and more efficiently in interphase than in mitosis .","The experimental set up was as in ( B ) .","The enrichment of PP1 signal at the Lac repressor spot was compared to the background nuclear ( interphase ) or cytoplasmic ( mitosis ) signal within the same cell .","Scale bar 10 μm .","( E ) Direct and indirect interactors of Ki-67 .","( F ) The B23S125 phosphorylation level remains high in Ki-67 depleted mitotic cells .","Immunoblots of whole cell extracts of cycling ( interphase ) of Nocodazole-arrested ( mitotic ) HeLa cells transfected with Ki-67 RNAi oligo 5 or control oligos , were probed for Ki-67 , tubulin , B23T199ph , and B23S125ph .","Two exposures of the B23S125ph blot are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00310 . 7554/eLife . 01641 . 004Figure 1—figure supplement 1 . Characterisation of Ki-67 RNAi . Two siRNAs against Ki-67 deplete the protein efficiently in HeLa cells .","Top and middle panels show two different exposures of the same blot . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00410 . 7554/eLife . 01641 . 005Figure 1—figure supplement 2 . Distribution of PP1gamma in mitosis after the Ki-67 siRNA . In cells depleted of Ki-67 , PP1 is still recruited to the nucleolus in interphase ( panels 4–4′ ) and to kinetochores in metaphase ( panels 5–5′ ) but its levels are lower on anaphase chromosomes ( panels 6–6′ ) .","HeLa cells were transfected with RFP:PP1γ ( green ) and with oligo 5 ( panels 4–6 ) or control oligo ( panels 1–3 ) .","Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 005 The similarity between Repo-Man and Ki-67 within the PP1 binding domain suggested that Ki-67 could be a PP1-interacting protein ( PIP ) .","Indeed , a possible connection between Ki-67 and PP1 was previously identified in two large scale screens for PP1 interactors .","In one , in silico-screening based on a stringent definition of the RVxF motif ( the PP1 binding motif ) identified Ki-67 as putative interactor , however no in vivo interaction was demonstrated ( Hendrickx et al . , 2009 ) .","Another study identified Ki-67 in a displacement affinity chromatography analysis of PP1 nuclear interactors ( Moorhead et al . , 2008 ) .","To analyse if Ki-67 was a cell-cycle regulated PIP in vivo , we performed a tethering/recruitment experiment ( Vagnarelli et al . , 2011 ) .","Ki-67301–700 wild type ( wt ) and a PP1 non-binding RASA mutant were fused to GFP:Lac repressor ( Figure 1B ) and co-expressed with RFP:PP1γ or β in DT40 cells carrying a lacO array integrated on a single chromosome ( Vagnarelli et al . , 2011 ) .","GFP:Lac repressor on its own was used as a control in this experiment .","All GFP:Lac repressor fusion constructs accumulated at the LacO integration site ( Figure 1C1′–3′ ) .","Furthermore , the Ki-67 wt construct recruited PP1 to the same spot ( Figure 1C2″ ) .","Quantification of the PP1 signal intensity at the GFP:Lac repressor spot compared to the general nuclear distribution ( excluding nucleoli ) revealed that Ki-67 recruitment of PP1 is more efficient in interphase than in mitosis and that PP1γ is recruited more efficiently than PP1β during interphase ( Figure 1D ) .","These data suggest that Ki-67 is a PIP in vivo and the interaction with PP1γ is cell-cycle regulated .","In order to determine whether Ki-67 is required to target PP1γ to any of its known locations in vivo , we used RNAi to deplete Ki-67 in human cells and determined the effects that this had on the distribution of PP1γ .","A previous report described the successful depletion of Ki-67 ( Vanneste et al . , 2009 ) .","However , the target sequence recognised by the published siRNA lies within a repeated stretch and rescue experiments to validate the phenotype were not reported .","We therefore identified two new siRNAs that could deplete the protein as efficiently as the published siRNA ( Figure 1 , Figure 1—figure supplement 1 ) .","Both new siRNAs yielded comparable phenotypes and we used siRNA 5 ( Ki5 ) for the depletion experiments presented below .","This siRNA was validated in a rescue experiment that is discussed in a later section ( Figure 2B ) . 10 . 7554/eLife . 01641 . 006Figure 2 . Ki-67 is required for targeting of nucleolar proteins to the chromosome periphery in mitosis .","( A ) Localisation of endogenous nucleolin is aberrant in mitotic cells after Ki-67 depletion ( panels 2 , 3 , 5 ) .","HeLa cells were transfected with Ki-67 RNAi oligo 5 ( panels 2 , 3 ,","5 ) or control oligos ( panels 1 ,","4 ) and stained for nucleolin ( green ) .","Quantification of the phenotypes is indicated in the graph above the corresponding representative images .","Scale bar 5 μm .","( B ) RNAi rescue experiment .","HeLa cells depleted of Ki-67 were transfected with either mCherry:Ki-67wt , or mCherry:Ki-67RASA , together with Ki-67 RNAi oligo 5 or control oligo and stained for nucleolin .","The localisation of nucleolin in mitotic cells was quantified in the different experimental conditions .","See Figure 2—figure supplement 1 for representative images .","Scale bar 10 μm .","( C ) The mitotic chromosome peripheral localisation of NIFK is disrupted upon Ki-67 RNAi ( panels 5–6 ) .","HeLa cells were transfected with GFP:NIFK ( green ) and oligo 5 ( panels 5–8 ) or control oligo ( panels 1–4 ) .","Scale bar 10 μm .","( D ) All novel cPERPs tested failed to accumulate on the chromosome periphery in mitosis .","HeLa cells were co-transfected with GFP:cPERPs identified in an earlier study ( Ohta et al . , 2010 ) ( green ) and oligo 5 ( panels 3–4 , 7–8 , 11–12 , 15–16 ) or control oligo ( panels 1–2 , 5–6 , 9–10 , 13–14 ) : GFP:cPERP-B ( panels 1–4 ) , GFP:cPERP-C ( panels 5–8 ) , GFP:cPERP-D ( panels 9–12 ) , GFP:cPERP-F ( panels 13–16 ) .","Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00610 . 7554/eLife . 01641 . 007Figure 2—figure supplement 1 . Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides with and without rescue by Ki-67 cDNA . RNAi rescue experiment .","Representative microscopy images for quantifications shown in Figure 2B .","HeLa cells depleted of Ki-67 were transfected with either mCherry:Ki-67wt ( panels 2 ,","5 ) or mCherry:Ki-67RASA ( panels 3 ,","6 ) ( red ) together with Ki-67 RNAi oligo 5 ( panels 4 , 5 ,","6 ) or control oligo ( panels 1 , 2 ,","3 ) and stained for nucleolin ( green ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00710 . 7554/eLife . 01641 . 008Figure 2—figure supplement 2 . Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides . HeLa cells were transfected with Ki-67 RNAi oligo 1 , 2 or 5 or control oligos and stained for nucleolin .","Nucleolin localisation was classified as for Figure 2B ( diffuse , aberrant , and big foci ) and the graph represents the quantification of the phenotypes .","Scale bar 5 μm .","The three different oligos produce the same phenotype . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00810 . 7554/eLife . 01641 . 009Figure 2—figure supplement 3 . Distribution of NIFK in mitosis following Ki-67 depletion . NIFK T234 phosphorylation is regulated normally in the presence and absence of Ki-67 .","Hela cells were transfected with Ki-67 RNAi oligo 5 ( panels 3 ,","4 ) or control oligos ( panels 1 ,","2 ) and stained with NIFK234ph antibody ( green ) .","Scale bar 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 009 Ki-67 depletion in a HeLa cell line has no effect on the accumulation of RFP:PP1γ in the nucleolus ( Figure 1 , Figure 1—figure supplement 2[1 , 4] ) .","Indeed , the targeting subunit for PP1 nucleolar localisation has been recently reported to be RRP1B ( Chamousset et al . , 2010 ) .","In early mitosis , PP1γ localised normally on the spindle and at kinetochores in both control and Ki-67 depleted cells ( Figure 1 , Figure 1—figure supplement 2[2 , 5] ) .","However , we observed a significant decrease in PP1 levels on anaphase chromatin in Ki-67 depleted cells ( Figure 1 , Figure 1—figure supplement 2[3′ , 6′] ) .","Previous reports identified Repo-Man and Sds22 as responsible for targeting PP1 to anaphase chromatin ( Trinkle-Mulcahy et al . , 2006; Wurzenberger et al . , 2013 ) .","Thus , Ki-67 is one of the several factors contributing to the accumulation of PP1γ on chromatin during mitotic exit .","Analysis of the phosphorylation status of several known direct and indirect Ki-67 interacting proteins ( Figure 1E ) in interphase and mitosis revealed that nucleophosmin/B23 phospho-regulation was dependent on Ki-67 .","B23 is phosphorylated both in interphase and in mitosis by several kinases ( Pfaff and Anderer , 1988; Jiang et al . , 2000; Louvet et al . , 2006; Krause and Hoffmann , 2010; Ramos-Echazabal et al . , 2012; Reboutier et al . , 2012 ) , including CyclinB/CDK1 at T199 ( Tokuyama et al . , 2001 ) in mitosis and by casein kinase II ( CKII ) on S125 during interphase ( Szebeni et al . , 2003 ) .","Use of phospho-specific antibodies revealed a reproducible difference in nucleophosmin/B23 phosphorylation on S125 in the presence and absence of Ki-67 exponential cultures and in prometaphase cells ( Figure 1F ) .","In both cases , the levels of S125ph were significantly increased following Ki-67 depletion .","This was particularly evident in prometaphase-arrested cells .","In contrast , we observed no significant difference in the phosphorylation status of B23 at T199 in the presence or absence of Ki-67 ( data not shown ) .","These experiments support the notion that Ki-67 is a functional PP1-targeting subunit in vivo .","Several aspects of mitotic chromosome structure remain relatively poorly understood , but amongst these , the perichromosomal compartment ( also known as the chromosome periphery ) stands out as a structure about which almost nothing is known .","This is remarkable , as an ever-increasing list of chromosome periphery proteins has been compiled over the years ( Chaly et al . , 1984; McKeon et al . , 1984; Gautier et al . , 1992b; Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005; Gassmann et al . , 2005; Ohta et al . , 2010 ) .","Some of these are among the most abundant proteins associated with mitotic chromosomes ( Ohta et al . , 2010 ) .","Despite their abundance , the mechanism of their localisation and the role that they play on the chromosomes , if any , is still not understood ( Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005 ) .","In a recent proteomic study , we identified a number of novel chromosome periphery proteins , which we termed cPERPs ( Ohta et al . , 2010 ) .","Ki-67 is one of many nucleolar proteins that are recruited to the chromosome periphery at the transition from prophase to prometaphase .","As Ki-67 is recruited to the perichromosomal compartment relatively early , we decided to examine whether its depletion affected the localisation of other known chromosome periphery proteins .","We first examined the localisation of the endogenous nucleolin using a specific antibody .","Indeed , nucleolin failed to accumulate at the chromosome periphery in the absence of Ki-67 ( Figure 2A ) .","The phenotype was observed using all of the siRNAs that we have tested and was rescued by an siRNA-resistant cDNA ( Figure 2B , Figure 2—figure supplements 1 , 2 ) .","To examine whether Ki-67 depletion affected the behaviour of other chromosome periphery proteins , we next examined the behaviour of NIFK , a known KI-67 interactor ( Takagi et al . , 2001 ) .","During normal mitotic exit , NIFK concentrates around the segregating chromosomes before associating with nucleolar-derived foci ( NDF–Dundr et al . , 1996; Figure 2C3 ) .","NDF formation varies between cell lines ( Dundr et al . , 2000 ) , but in the HeLa cells that we have used for RNAi studies , prominent NDFs are seldom observed during unperturbed mitosis ( Figure 2C1–4 ) .","To follow NIFK behaviour , we transiently expressed GFP-tagged hNIFK in HeLa cells after control or Ki-67 RNAi .","The nucleolar localisation of GFP-NIFK in interphase was unaltered after Ki-67 depletion ( Figure 2C8 ) , but in early mitosis the protein failed to properly accumulate around the mitotic chromosomes ( Figure 2C5–7 ) .","Instead , in metaphase cells , it accumulated in large cytoplasmic aggregates , one of which was frequently found to ‘cap’ one end of the cluster of chromosomes on the metaphase plate ( Figure 2C5 ) .","This extraordinary behaviour was also seen for endogenous NIFK phosphorylated on Thr234 using a phospho-specific antibody ( Figure 2—figure supplement 3[3′] ) .","During mitotic exit , in the absence of Ki-67 , GFP-NIFK accumulated in NDF-like cytoplasmic aggregates that persisted until the reformation of the nuclear membrane ( Figure 2C5–7 ) .","In view of the striking similarity of the behaviour of nucleolin and NIFK in the absence of Ki-67 , we tested the generality of this effect by localizing four novel cPERPs identified in our proteomics studies ( Ohta et al . , 2010 ) .","Remarkably , all were mislocalised in Ki-67-depleted cells , and all were frequently observed to ‘cap’ one end of the metaphase plate ( Figure 2D3 , 7 , 11 , 15 ) .","As in the case of nucleolin and NIFK , these aggregates dispersed into clusters of smaller cytoplasmic foci during mitotic exit ( Figure 2D4 , 8 , 12 , 16 ) .","We considered two hypotheses to explain the observed failure of nucleolar proteins to associate with the chromosome periphery in mitosis of Ki-67-depleted cells .","First , Ki-67 might be required for the complete disassembly of the nucleolus during mitotic entry .","In this case , the larger cytoplasmic aggregates observed during metaphase might be remnants of incompletely disassembled nucleoli .","Correlative light-microscopy/Electron microscopy ( CLEM ) analysis of cells transfected with both GFP-cPERP-C and the indicated siRNA oligos eliminated this hypothesis .","The GFP-containing aggregates observed in mitosis in Ki-67-depleted cells did not correspond to nucleolar remnants or any other recognisable electron-dense structures such as NDFs ( Dundr et al . , 2000; Figure 3A , Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 01641 . 010Figure 3 . Ki-67 organizes the mitotic chromosome periphery .","( A ) CLEM of HeLa cells transfected with GFP-PerP-C and control oligos ( top panels ) or Ki-67 oligos ( bottom panels ) .","Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates ( arrow ) were identified and processed for CLEM using an adapted protocol ( Booth et al . , 2011 ) .","Appropriate light and electron micrographs , from the matching z position , were overlaid using Adobe Photoshop Elements .","Scale bar 5 μm .","( B ) EM of mitotic cells from Control ( top panels ) and Ki-67 RNAi ( bottom panels ) .","At higher magnification ( zoom","2 ) it is possible to note that the amorphous material surrounding the mitotic chromosomes in control cells ( arrow ) is substantially reduced around Ki-67 depleted chromosomes .","Scale bars ( left to right ) 4 , 2 , and 1 μm .","Far right panels show regions of interest for pixel density analysis ( Figure 3C ) .","( C ) Line scans of the peripheral regions of mitotic chromosomes in control ( blue line ) and Ki-67 depleted cells ( red line ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01010 . 7554/eLife . 01641 . 011Figure 3—figure supplement 1 . Depletion of Ki-67 does not leave electron-dense nucleolar remnants in the cytoplasm . CLEM of HeLa cells transfected with PERP-C and control oligos ( left panels ) or Ki-67 oligonucleotides ( right panels ) .","Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates ( green arrow ) were identified and processed for CLEM using an adapted protocol ( Booth et al . , 2011 ) .","Panels from top to bottom show electron micrographs of consecutive serial sections , together with the appropriate fluorescence micrograph of the same z position .","No obvious electron-dense material can be seen colocalising with the fluorescent aggregates in the cytoplasm .","The white arrow shows regions of the metaphase plate where chromosomes appear abnormally closely clustered together after Ki-67 depletion .","Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 011 A second hypothesis suggested that Ki-67 might function as a scaffolding platform for organising the perichromosomal compartment .","Indeed , all candidate proteins that we have tested to date have failed to localise to the chromosomal periphery in Ki-67-depleted cells .","Although analyses of individual proteins can provide part of the story , it is difficult or impossible to make conclusions about the compartment as a whole since the chromosome periphery has a complex and as-yet undefined protein and RNA composition .","Nonetheless , further analysis of our CLEM images strongly suggests that depletion of Ki-67 causes a loss of most or all of the perichromosomal compartment .","Examination of electron micrographs of control and Ki-67-depleted cells at higher magnification revealed a subtle difference in the appearance of the edge of the chromosomes .","In control cells there was a ‘fuzzy’ transition zone between the electron-dense chromatin and the cytoplasm ( Figure 3B ) .","This transition appeared to be more abrupt in Ki-67-depleted chromosomes .","This was confirmed by line scans across the chromosome-to-cytoplasm boundary , which showed that there is normally a gradual decrease in density at the edge of mitotic chromosomes .","This transition was significantly more abrupt in the absence of Ki-67 ( Figure 3C ) .","These experiments suggest that Ki-67 directs the binding of nucleolar components to the chromosome periphery , perhaps by acting as a scaffold .","Alternatively , the macromolecular network at the chromosomal periphery could be delicately balanced , such that removal of any single component causes the entire structure to fail .","However , depletion of cPERP-B , -D , or -E using RNAi ( Figure 4—figure supplement","1 ) failed to alter the striking perichromosomal localization of Ki-67 ( Figure 4 ) .","This is consistent with Ki-67 being upstream of the other components in the assembly of the perichromosomal compartment . 10 . 7554/eLife . 01641 . 012Figure 4 . Ki-67 localisation is not dependant on other c-PERPS tested .","( A ) Ki-67 localisation was analysed following the RNAi depletion of several novel c-PERPs ( PERP B , D , and E ) .","Following a 48 hr knock-down period , cells were fixed and labelled with anti-Ki67 ( green ) antibody and Dapi ( blue ) .","Examples shown include metaphase and anaphase cells .","Scale bar 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01210 . 7554/eLife . 01641 . 013Figure 4—figure supplement 1 . Depletion of cPERPs by RNAi . As no antibodies are available yet targeting these novel cPERPs , HeLa cells were transfected with GFP:PERP cDNA with or without the co-transfection of siRNA .","Cells were harvested after 48 hr and prepared for Western analysis .","Anti-GFP probing shows decreased levels of GFP-PERP expression in samples co-transfected with the appropriate siRNA . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 013 The observations presented above suggest two possible hypotheses for how Ki-67 could function in the assembly of the chromosome periphery .","First , the protein might comprise part of a PP1 holoenzyme whose removal of phosphates from chromosomal proteins , could be required for those proteins to assemble at the chromosome periphery .","Alternatively , Ki-67 , which is a very large protein with multiple repeated domains , might function as a scaffold linking together key components at the chromosome periphery .","To begin to distinguish between these two hypotheses , we generated constructs expressing mRFP coupled to either wild-type Ki-67 or a Ki-67 RASA mutant that is incapable of binding PP1 at the conserved site shared with Repo-Man ( Figure 1A3 ) .","Both constructs encoded mRNAs that were engineered to be resistant to siRNA-5 .","Both mRFP:Ki-67-wt and mRFP:Ki-67-RASA proteins rescued the localisation of endogenous nucleolin throughout mitosis in cells transfected with Ki-67 siRNAs .","In transfected cells , nucleolin was once again concentrated at the chromosome periphery from prometaphase through telophase ( Figure 2B , Figure 2—figure supplement 1 ) .","When combined with our previous observations these data support the hypothesis that Ki-67 acts as a scaffold for formation of the perichromosomal compartment .","Importantly , this rescue experiment also confirmed that the chromosome periphery phenotypes observed following Ki-67 depletion by siRNA-5 are not due to off-target effects .","Because depletion of Ki-67 appears to result in either a dramatic reduction , or even complete loss , of the perichromosomal compartment , these experiments offer a unique opportunity to investigate the function ( s ) of this mysterious structure .","If the perichromosomal compartment is a kind of pellicle or ‘skin’ on the surface of the chromosomes ( Schrader , 1944 ) , then two obvious potential functions come to mind .","First , the perichromosomal layer could protect the chromosomal DNA from damage once it is released into a potentially hostile cytoplasmic environment following nuclear envelope breakdown .","Immunostaining for 53BP1 , a protein that associates with DNA damage foci revealed that levels of intrinsic DNA damage are low in mitotic cells under our culture conditions ( Figure 5A , B ) .","These levels were not substantially increased in chromosomes lacking Ki-67 .","Thus , this hypothesis appears unlikely . 10 . 7554/eLife . 01641 . 014Figure 5 . Ki-67 is does not function to protect chromosomes from DNA damage or provide structural maintenance .","( A ) Representative overview images of HeLa cells transfected with control or Ki-67 specific siRNA oligos probed with anti-53bp antibodies to assess levels of DNA damage .","Scale bar 10 μm .","( B ) A bar graph showing quantification of the percentage of mitotic cells found with DNA damage , marked using an anti-53bp antibody .","( C ) Chromosome spreads from control RNAi ( panels 1–1″ ) and Ki-67 oligo 5 RNAi ( panels 2–2″ ) were stained for the chromosome scaffold protein KIF4A ( green ) and for the kinetochore with ACA ( red ) .","Ki-67 depleted chromosomes still maintain a proper localisation of the chromosome scaffold and kinetochore proteins .","Scale bar 2 μm .","( D ) IMS Assay ( Intrinsic Metaphase Structure Assay ) .","Chromosomes from HeLa cells after Ki-67 depletion ( panels 2 , 4 ,","6 ) and control depletion ( panels 1 , 3 ,","5 ) at the beginning of the assay ( panels 1 , 2 ) , after the first TEEN treatment ( panels 3 , 4 ) , and after the second RSB addition ( panels 5 , 6 ) .","Scale bar 10 μm .","( E ) Quantification of the experiment in ( D ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 014 An alternative hypothesis is that by forming a layer coating each chromosome , the perichromosomal compartment could promote the formation of physically separated chromosomes that is characteristic of mitosis .","Our prior studies revealed that condensin , the chromokinesin Kif4 and DNA topoisomerase II all contribute to shaping mitotic chromosomes ( Hudson et al . , 2003; Green et al . , 2012; Samejima et al . , 2012 ) .","Furthermore , proteomic analysis revealed a substantial reduction in levels of Ki-67 associated with isolated mitotic chromosomes depleted of KIF4A ( Samejima et al . , 2012 ) .","The converse is not true , however , as neither the localisation nor abundance ( as determined by immunostaining ) of KIF4A was altered in the absence of Ki-67 ( Figure 5C ) .","Thus , Ki-67 depletion does not affect the known mitotic chromosome structural proteins .","Mitotic chromosomes have an intrinsic metaphase structure ( IMS ) that can be probed using a specialized assay , in which chromosomes are induced to unfold down to the level of 10 nm fibres by removal of divalent cations , and then induced to re-fold by re-addition of Mg2+ ( Earnshaw and Laemmli , 1983; Hudson et al . , 2003 ) .","Even though chromosomes lacking condensin or KIF4 appear morphologically normal when cells are processed under optimal conditions , those chromosomes are severely impaired in the IMS assay—failing to re-adopt a normal appearance after restoration of divalent cations ( Hudson et al . , 2003; Samejima et al . , 2012 ) .","Other abundant chromosomal proteins , including , cohesin and DNA topoisomerase II are not required for this aspect of mitotic chromosome structure ( Vagnarelli et al . , 2004; Johnson et al . , 2009 ) .","We transfected cells with either control or Ki-67 siRNAs and then performed the IMS assay .","This experiment showed clearly that chromosomes depleted of Ki-67 efficiently regain their normal morphology after two rounds of unfolding and refolding .","Thus , the perichromosomal layer is not required for maintenance of the intrinsic structure of mitotic chromosomes ( Figure 5D , E ) .","In summary , we could find no evidence for a role of Ki-67—and by extension much or all of the perichromosomal compartment—in mitotic chromosome structure or integrity under these conditions .","Given the abnormal localization of nucleolar components during mitosis , it is no surprise that the segregation of these components is perturbed in mitosis .","The detailed behaviour of the complex population of nucleolar components following abolishment of the perichromosomal compartment is a subject for a follow-up study , but here as an example , we have examined the segregation of the abundant component nucleophosmin/B23 .","We followed cell division in living HeLa cells expressing GFP-B23 after either control or Ki-67 siRNA .","In Ki-67-depleted cells , B23 was never observed to associate with the chromosomes during mitosis .","Instead , it started forming cytoplasmic aggregates just after anaphase onset ( Figure 6A , 45' ) .","We then analysed the distribution of endogenous nucleolin between daughter cells in cytokinesis .","While in control cells there was a relatively even distribution of chromatin-associated nucleolin between daughter cells , in Ki-67-depleted cells the nucleolin aggregates were often not chromatin bound and their distribution between daughter cells tended to be more asymmetric ( Figure 6B , C ) . 10 . 7554/eLife . 01641 . 015Figure 6 . Segregation of nucleolin .","( A ) In cells depleted of Ki-67 the nucleolus never disassembles completely during mitosis and B23 never accumulates around the mitotic chromosomes .","Time-lapse imaging of GFP:B23 in HeLa cells after control or Ki-67 RNAi .","Scale bar 10 μm .","( B ) Nucleolin localisation was analysed in cells in cytokinesis after control or Ki67 RNAi .","Nucleolin distribution between the two daughter cells is uneven following Ki-67 RNAi compared to the control and the protein is predominantly not associated with the chromatin .","( C ) Quantification of the experiment in ( B ) .","The graphs represent the ratio of the total nucleolin intensity between daughter cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01510 . 7554/eLife . 01641 . 016Figure 6—figure supplement 1 . Electron micrographs of interphase nuclei from Ki-67 RNAi cells . Three major components of the nucleolus are recognised: Fibrillar centres ( FC ) , dense fibrillar component ( DFC ) , and granular component ( GC ) .","Scale bar 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 016 Despite this perturbation of the behaviour of major nucleolar components during mitosis , nucleoli did indeed re-form during mitotic exit .","Furthermore , the ultrastructure of the reformed nucleoli appeared to be normal , with fibrillar centres , dense fibrillar component , and granular component all readily observed ( Figure 6—figure supplement 1 ) .","Thus , Ki-67 is apparently not required for the structural organisation within interphase nucleoli .","Human cells contain five chromosomes with ribosomal gene clusters that can form NORs .","Thus , human cells could in theory have 10 nucleoli .","This is almost never seen , presumably due to natural clustering of the NORs and the failure of all NORs to become activated .","In our line of HeLa cells , we observed 3 . 5 ± 0 . 3 nucleoli per nucleus ( Figure 7D , E ) .","This changed dramatically following Ki-67 depletion , when the number of nucleoli observed dropped to 1 . 6 ± 0 . 3 .","This strongly suggests that chromosomes lacking a perichromosomal layer might associate with one another more closely than normal , thus promoting NOR fusion during reactivation . 10 . 7554/eLife . 01641 . 017Figure 7 . The nucleoli of Ki-67 depleted cells are fewer , smaller , and functionally repressed . Normal cell , nucleus , and nucleolus size is compromised following depletion of Ki-67 .","( A ) Representative images showing a reduced cell size and nucleolar size phenotype in Ki-67 depleted cells , using Rhodamine Phalloidin and DAPI as markers .","Scale bar 5 μm .","( B and C )","Quantification of cell area and nuclear area in control ( white bar ) and Ki-67 depleted ( black bar ) cells .","Bars show mean ± SEM .","ncell = 100 .","( D ) Representative images showing the small nuclei phenotype , with single nucleoli following depletion of Ki-67 .","Scale bar 5 μm .","( E and F )","Quantification of nucleolar number and area in control ( white bar ) and Ki-67 depleted ( black bar ) cells .","Bars show mean ± SEM .","ncell = 50 .","( G ) A 2D scatter plot showing combined nucleolar area ( per cell ) on the Y axis , vs nuclear area on the X axis , for control ( green ) and Ki-67 depleted ( red ) cells .","Each individual translucent dot represents one cell .","Black squares represent the means .","( H ) Northern blot of RNA samples prepared from control ( C ) or Ki-67 depleted cells , for 24 , 48 , and 72 hr of Ki-67 knock-down .","Blot shows decreased signal for 47S bands and a time course dependent decrease for 26S signal ( red stars ) .","No clear change was seen with bands for 18SE ( black star ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01710 . 7554/eLife . 01641 . 018Figure 7—figure supplement 1 . Ki-67 depletion influences cell size , nuclei size , and nucleoli size . A 2D scatter plot showing combined nuclei area on the Y axis , vs cell area on the X axis , for control ( green ) and Ki-67 depleted ( red ) cells .","Each individual translucent dot represents one cell .","Black squares represent the means . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01810 . 7554/eLife . 01641 . 019Figure 7—figure supplement 2 . Ki-67 depletion has only minor effects on the cell cycle detected by FACS .","( top )","HeLa cells transfected with control or Ki-67 siRNA oligos were incubated for 48 , 72 , or 96 hr , before cell-cycle analysis using FACS .","Gated cells were manually categorised into cell-cycle stages using FACS histograms .","( bottom ) bar graph representation of the histogram data in the top panel . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01910 . 7554/eLife . 01641 . 020Figure 7—figure supplement 3 . Ki-67 depletion influences nucleolar size . A bar graph showing the mean cross-sectional area measurements of nucleoli from control and Ki-67 depleted cells .","Nucleoli measurements are on a per cell basis and placed in ascending order , from the largest nucleolus , to the smallest . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 020 A second reason why Ki-67-depleted cells might have single large nucleoli could be due to a decreased efficiency of NOR reactivation during mitotic exit .","Ki-67-depleted cells tended to be smaller and to have smaller nuclei ( Figure 7A–C , Figure 7—figure supplement 1 ) .","This was not due to an accumulation of the cells in G1 , as shown by FACs analysis ( Figure 7—figure supplement 2 ) , but could potentially be due to ribosomal insufficiency .","We have made several observations that are consistent with this .","First , if we calculate the total nucleolar area in optical sections of Ki-67-depleted nuclei , we find that these nuclei tend to have a smaller aggregate nucleolar area ( Figure 7F , G , Figure 7—figure supplement 3 ) .","If we assume total nucleolar area to be a proxy for the number of genes involved in rDNA transcription , this suggests that reactivation of ribosomal clusters following mitotic exit may be less efficient in Ki-67-depleted cells .","This is consistent with the results of a Northern analysis of total rRNA in control and Ki-67-depleted cells , which revealed decreased levels of pre-rRNA species , consistent with lower levels of rRNA transcription in the depleted cells ( Figure 7H ) .","To further examine NOR reactivation following mitotic exit , we exploited the fact that chromosomes bearing re-activated NORs are associated with nucleoli .","We used a HT1080 cell line carrying a LacO array integrated on chromosome 13p next to the NOR and expressing GFP fused to Lac repressor ( Chubb et al . , 2002 ) .","In this cell line the 13p locus ( visualized as a GFP spot ) tends to localise in the nuclear interior ( Figure 8A1 ) where it is usually closely associated with a nucleolus ( Figure 8B1 ) .","A nuclear erosion script developed by Bickmore and Perry ( Croft et al . , 1999 ) was used to divide the nucleus into 5 concentric rings of equal area in order to score the position of the locus within the nucleus ( Figure 8A2 ) . 10 . 7554/eLife . 01641 . 021Figure 8 . Ki-67 depletion affects nuclear architecture .","( A ) The position of a chromosome 13p ( marked by a LacO array:LaciGFP ) in HT1080 cells ( 1 ) was assessed in control and Ki67 RNAi experiments .","150 nuclei from three independent experiments were imaged and analysed with an erosion script software to locate the position of the locus ( 2 ) .","The locus repositions from the interior toward the periphery after Ki-67 RNAi ( 3 ) .","( B ) The cells described in A were stained for nucleolin after control or Ki-67 RNAi .","The association with the nucleolus was recorded as proximal ( 1–1′ ) or distal ( 2–2′ ) .","( 3 ) Quantification of the analyses .","( C ) Model for Ki-67 function in mitosis; ( blue: chromatin; green perichromosomal proteins; grey: microtubules ) .","See text for details . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 021 Depletion of Ki-67 had a strong effect on the nuclear localization of chromosome 13p .","In cells depleted of Ki-67 , the tagged 13p locus tended to move from the nuclear interior towards the nuclear periphery ( Figure 8A3 ) .","This reorganization of chromosome 13p was correlated with a disengagement from the nucleolus ( Figure 8B2 , 3 ) .","Both phenotypes are consistent with a decreased efficiency of reactivation of the 13p NOR .","Thus , although Ki-67 depletion and loss of much or all of the perichromosomal compartment has little obvious effect on chromosome structure or segregation in the first mitosis after depletion , after mitotic exit the nucleus undergoes fundamental changes consistent with decreased activity of the ribosomal gene clusters and with altered interactions between chromosomes .","More detailed examination of the link between Ki-67 , the perichromosomal compartment , and nucleolar organizer region ( NOR ) reactivation will be an important subject for future study ."],["Our studies have revealed for the first time that Ki-67 functions as a structural/scaffolding protein required for assembly of the perichromosomal compartment on condensed mitotic chromosomes .","Thus , Ki-67 has an important role in determining the behaviour of many nucleolar components during mitosis .","Interestingly , this method of segregating nucleolar components means that nucleolar reassembly can begin immediately after CDK activity declines and before the re-establishment of nuclear-cytoplasmic transport during mitotic exit .","Ki-67 is a PP1-interacting protein , however the biological role of this activity remains elusive and indeed the protein could execute this function during another phase of the cell cycle rather than mitosis .","How the various activities of Ki-67 combine to determine the nucleolar morphology in proliferating cells and whether the perichromosomal layer has a subtle role in chromosome dynamics during mitosis or nuclear reformation remain to be determined in future studies ."],["HeLa Kyoto and MRC5 cells were maintained in DMEM supplemented with 10% and 15% FBS respectively .","DT40 cells carrying a single integration of the LacO array ( Vagnarelli et al . , 2006 ) were cultured in RPMI1640 supplemented with 10% FBS and 1% chicken serum .","For RNAi treatments HeLa cells in exponential growth were seeded in six-well plates with or without polylysine-coated glass coverslips and grown overnight .","Transfections were performed using Polyplus jetPRIME ( PEQLAB , Southampton , UK ) with the indicated siRNA oligos and analysed 48 hr later as previously described ( Vagnarelli et al . , 2006 ) .","For the rescue experiments HeLa cells at 50% confluence were transfected with 400 ng of plasmid DNA and 50 nM of siRNA oligonucleotides and analysed 48 hr post-transfection .","The siRNA oligonucleotides against Ki67 are as follows: Ki-1 as published by Vanneste et al . ( 2009 ) ; Ki-2 :5′AAGCACCAGAGACCCUGUATT3′; Ki-5:5′GCAUUUAAGCAACCUGCAA3′; a 21-mer oligonucleotide ( CGUACGCGGAAUACUUCGAdTdT ) was used as a control ( Elbashir et al . , 2001 ) .","To test for successful depletion of cPerP proteins , HeLa cells were co-transfected with specific siRNA oligos together with the appropriate GFP-cPerP cDNA constructs .","Control samples were prepared in parallel via cells transfected with GFP-PerP cDNA only .","Following a 48 hr expression/knock-down period cells were harvested and processed routinely for Western analysis .","Knockdown was considered successful if Western analysis revealed decreased GFP expression levels in siRNA treated samples ( rabbit polyclonal anti-GFP , Invitrogen , Paisley , UK ) .","The primary antibodies were used as follows: Ki-67 ( mouse monoclonal BD Transduction laboratory , Oxford , UK ) 1:100; nucleolin ( rabbit polyclonal; Abcam ) 1:300; NIFK T234ph ( Rabbit polyclonal; Abcam , Cambridge , UK ) 1:100; Repo-Man ( Vagnarelli et al . , 2011 ) ; anti-alpha-tubulin antibody ( B512; SIGMA , Gillingham , UK ) , anti-B23 S125ph , ( Abcam ) ; anti-B23T199ph ( Abcam ) ; anti-nucleolin ( Abcam ) .","For immunofluorescence , cells were fixed in 4% PFA and processed as previously described ( Vagnarelli et al . , 2011 ) .","Fluorescence-labelled secondary antibodies were applied at 1:200 ( Jackson ImmunoResearch ) .","3D data sets were acquired using a cooled CCD camera ( CH350; Photometrics ) on a wide-field microscope ( DeltaVision Spectris; Applied Precision ) with a NA 1 . 4 Plan Apochromat lens .","The data sets were deconvolved with softWoRx ( Applied Precision ) .","3D data sets were converted to Quick Projections in softWoRx , exported as TIFF files , and imported into Adobe Photoshop for final presentation .","Live cell imaging was performed with a DeltaVision microscope as previously described ( Vagnarelli et al . , 2011 ) .","For quantification of PP1 binding in vivo , images of prometaphase and interphase transfected cells were acquired and the intensity of PP1 staining at the GFP spot was calculated relative to the average nuclear intensity .","The 3D data sets obtained at the same exposure were projected as mean intensities .","A 12 × 12 pixel area containing the GFP spot was used to measure the total intensity of the signal .","An area of the same size was used to identify the background signal in each cell , and this value was subtracted from the measurement of the nuclear and spot area .","The IMS assay was conducted as previously described ( Hudson et al . , 2003 ) .","For immunoblotting , whole cell lysates were loaded onto polyacrylamide gels .","SDS-PAGE and immunoblotting was performed following standard procedures .","Ki-67 ( aa 161–659 ) was obtained by RT-PCR from HeLa cDNA using the following primers: GGATCCGGCGCCACGTTTCCTCTC and CTCGAGTTTTACTACATCTGC and CLONED PGEX4T3 BamHi/XhoI .","The PP1-non binding mutant version was generated from this vector using a QuikChange Site-Directed Mutagenesis Kit ( Agilent Technologies , Edinburgh , UK ) .","The Lac repressor fusion constructs were obtained by cloning Ki-67 ( aa 161–659 ) into pEGFP:Lac repressor .","hPP1γ was cloned into PET28 EcoRI/HindIII .","hNIFK was cloned by RT PCR from HeLa cDNA using the primers CTCGAGGGATGGCGACTTTTTCTGGC and GAATTCTCACTGATTGCTGCTTCT and cloned into the XhoI/EcoR1 sites of pEGFPC1 .","The cPERPs were cloned into the gateway system by PCR as previously described ( Ohta et al . , 2010 ) .","Accession numbers for cPERPs are as follows: cPERP-A: ( C1orf131 ) –NM_152379 . 2 , cPERP-B: ( CCDC137 ) –NM_199287 . 2 , cPERP-C: ( KIAA0020 ) –NM_014878 . 4 , cPERP-D: ( DDX18 ) –NM_006773 . 3 , cPERP-E: ( CIRH1A ) –NM_032830 . 2 , cPERP-F: ( DDX27 ) –XM_006723815 . 1 .","The CLEM processing method was an adapted version of a previously established protocol ( Booth et al . , 2011 , Booth et al . , 2013 ) .","Cells were seeded onto glass-bottomed , gridded dishes ( MatTek Corporation , USA ) and co-transfected with GFP-cPerpC together with control or Ki-67 specific siRNA oligos .","Following a 48 hr expression period , cells of interest were identified using a wide-field epifluorescence microscope ( DeltaVision RT; Applied Precision ) .","GFP-expressing mitotic cells were located and their position mapped using transmitted light to visualise reference coordinates .","Cells were then fixed for 1 hr ( 3% glutaraldehyde , 0 . 5% paraformaldehyde in 0 . 2 M sodium cacodylate buffer containing 5 µg/ml Hoechst ) and washed in PBS ( 3 × 5 min ) .","Cells of interest were then re-imaged to acquire micrographs of Hoechst stained chromosomes .","Next , cells were osmicated ( 1% osmium tetroxide in PBS ) for 1 hr , washed with PBS ( 3 × 5 min ) , ddH2O ( 2 × 20 min ) , and then 30% ethanol ( 1 × 10 min ) before contrast staining with uranyl acetate ( 0 . 5% in 30% ethanol ) for 1 hr .","Cells were then dehydrated using a graded series of ethanol washes culminating in 2 × 10 min incubations with 100% ethanol , followed by infiltration with ethanol:resin mixtures ( at 2:1 and then 1:1 ) .","Finally , cells were embedded in 100% resin , with a gelatin capsule of resin covering the cells of interest , before curing at 60°C for 3 days .","Ultra-small resin blocks ( 50 µm2 ) were fine trimmed and serial sections ( 85 nm thickness ) taken at areas corresponding to previously chosen coordinate positions , before post-staining in Reynold's lead citrate and uranyl acetate ( 5% in 50% ethanol ) for 10 and 5 min , respectively .","Cells were visualised with a Phillips CM120 BioTwin transmission electron microscope ( FEI ) and micrographs acquired using a Gatan Orius CCD camera ( Gatan ) .","The appropriate Z position correlative light/EM images of a cell were concatenated using ImageJ and then analysed and overlaid using Photoshop Elements 6 ( Adobe ) .","Electron micrographs containing metaphase chromosomes from control or Ki-67 depleted cells were used for chromosome periphery analysis .","The pixel density of a 1 μm region of interest was measured using the raw data from the ‘plot profile’ function of imageJ .","For unbiased consistency , the 1 μm region of interest always started within the chromosome body and finished in the cytoplasm , with the halfway point lying at the expected origin of the chromosome periphery .","Approximately 200 individual pixel density measurements were taken within each 1 μm region .","The data were plotted as a line scan profile , with each data point representing the mean of 6 × 1 μm regions of interest per chromosome .","HeLa cells were seeded onto coverslips and transfected with control or Ki-67 specific siRNA oligonucleotides .","Following a 48 hr knock-down period , cells were fixed using 4% paraformaldehyde , blocked with 3% BSA , and either directly mounted onto slides using hard-setting Vectashield ( containing DAPI ) or first stained with Rhodamine Phalloidin ( Biotium , Inc . , Cambridge , UK ) , before mounting .","All measurements were scored using imageJ and area measurement tools .","Total cell cross-sectional area was measured using Phalloidin to reveal the cytoplasm and by extension a guide for cell periphery .","The area of individual cells or clustered groups of cells were traced and scored using freehand measurement tools .","Nuclei and nucleoli cross-sectional area was measured and nucleolar numbers scored using DAPI as a marker .","A contrast threshold was applied to micrographs allowing the semi-automated , unbiased measurement of nucleolar area and number using the ImageJ wand ( tracing ) tool .","Control or Ki-67 depleted HeLa cells were subjected to cell-cycle analysis by FACS .","Briefly , 1 × 106 cells , per condition , were fixed with cold ethanol ( 70% ) for 1 hr , centrifuged , and resuspended in PBS containing RNase A ( 0 . 2 mg/ml ) and Propidium Iodide ( 10 µg/ml ) .","Following a 20-min incubation , cells were analysed using FACS .","The channel FL2 was used to analyse 20 , 000 events per condition .","Gated cells were manually categorised into cell-cycle stages .","Cells were analysed following knock-down periods of 24 , 48 , 72 , and 96 hr .","HT1080 cell line carrying a LacO integration on chromosome 13p and expressing a Laci:GFP ( kindly provided by W Bickmore ) was used for depleting Ki-67 in an RNAi experiment as described for HeLa above .","At 48 hr the cells were fixed and processed for immunostaining with a nucleolin antibody as described before .","150 nuclei from three independent experiments were imaged and analysed for with the nuclear erosion scrip ( Croft et al . , 1999 ) to assign the position of the 13p locus .","Control or Ki-67 depleted HeLa cells were cultured in 10 cm dishes and processed for Northern analysis .","RNA was extracted using fresh TRIzol ( Ambion ) according to manufacturer’s guidelines .","3 µg of RNA per sample was separated using a standard 1 . 2% TBE agarose gel and transferred to Hybon N+ membrane overnight , by capillary action .","Membranes were hybridized in 6X SSPE , 5X Denhardts , 5X SDS at 37°C before washing with 2XSSPE .","Probes ( see below ) were 5′ labelled with y32P-ATP and T4PNK .","Probe_d_human AGACGAGAACGCCTGACACGCACGGCAC 5'ETS probe .","Probe_e_human CCTCGCCCTCCGGGCTCCGTTAATGATC 5′ end of ITS1 ( 21S and 18SE ) ."]],"string":"[\n [\n \"When mitotic chromosomes are examined by whole mount microscopy the surface chromatin is obscured by a layer of proteins and RNA derived from the dense fibrillar component ( DFC ) and granular component ( GC ) of the nucleolus ( Moyne and Garrido , 1976; Harrison et al . , 1982; Adolph and Kreisman , 1983; Sumner , 1991; Gautier et al . , 1992b; Wanner et al . , 2005 ) .\",\n \"This perichromosomal layer includes pre-rRNA , U3 snoRNAs , and over 20 ribosomal proteins , including nucleolin and Nopp140 , NPM/B23 , Bop1 , Nop52 , PM-Scl 100 , and Ki-67 ( Gautier et al . , 1992a , 1994; Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005; Fomproix et al . , 1998; Angelier et al . , 2005 ) .\",\n \"The perichromosomal layer represents 1 . 4% of the chromosome proteome ( Ohta et al . , 2010 ) .\",\n \"At present , its functional significance remains unstudied ( Van Hooser et al . , 2005 ) .\",\n \"Ki-67 is one of the earliest proteins to bind the perichromosomal layer in mitosis .\",\n \"Ki-67 is an antigen recognised by a monoclonal antibody generated by immunizing mice with nuclei of Hodgkin lymphoma cells ( Gerdes et al . , 1983 ) .\",\n \"Because Ki-67 is nuclear only in proliferating cells , it is widely used as a marker to assess cell proliferation .\",\n \"For example , immuno-histochemical assessment of the proportion of cells staining for nuclear Ki-67 is used to predict the responsiveness or resistance of tumours to therapy ( Dowsett et al . , 2011 ) .\",\n \"Ki-67 protein is predominantly localised in the cortex and dense fibrillar components of the nucleolus during interphase ( Kill , 1996; MacCallum and Hall , 2000b ) .\",\n \"During mitosis it relocates to the periphery of the condensed chromosomes ( Verheijen et al . , 1989b; Isola et al . , 1990 ) .\",\n \"It has been reported that Ki-67 is associated with the nuclear matrix ( Verheijen et al . , 1989b ) , preribosomes ( Isola et al . , 1990 ) , satellite DNA in G1 ( Bridger et al . , 1998 ) , and with the chromosome scaffold of mitotic cells ( Verheijen et al . , 1989a ) .\",\n \"Approximately 40% of the cellular pool of protein is present on isolated mitotic chromosomes ( Ohta et al . , 2010 ) .\",\n \"Previous studies suggested that Ki-67 is regulated by phosphorylation .\",\n \"Hyperphosphorylated Ki-67 does not bind DNA during mitosis ( Endl and Gerdes , 2000; MacCallum and Hall , 1999 ) and is characterized by increased mobility , on mitotic chromosomes measured by FRAP .\",\n \"In interphase , non-phosphorylated Ki-67 binds DNA and does not recover after FRAP ( Saiwaki et al . , 2005 ) .\",\n \"Its localisation and cell-cycle behaviour suggest that Ki-67 could be involved in the organisation of nucleolar chromatin during interphase in proliferating cells .\",\n \"Recently , it was reported that Ki-67 also functions in mitosis in stabilisation and maintenance of the mitotic spindle by recruiting Hklp2 to mitotic chromosomes ( Vanneste et al . , 2009 ) .\",\n \"In this study , we have identified Ki-67 as ancestral to the PP1 targeting subunit Repo-Man ( Trinkle-Mulcahy et al . , 2006 ) and have shown that Ki-67 is a PIP ( PP1 Interacting Protein ) that contributes to the phospho-regulation of nucleophosmin/B23 by CKII .\",\n \"Ki-67 is also a major organiser of the perichromosomal layer , possibly acting as an interaction platform .\",\n \"Remarkably , Ki-67 depletion prevents all nucleolar proteins tested from accumulating around the chromosomes in mitosis .\",\n \"Thus , chromosomes in cells depleted of Ki-67 appear to lack most or all of their perichromosomal layer .\",\n \"This enabled us to gain insights into the function of this enigmatic chromosomal compartment .\",\n \"Interestingly , loss of the perichromosomal layer does not detectibly compromise the condensed morphology or intrinsic structure of mitotic chromosomes but does result in significant changes in nucleolar morphology and nuclear organization in the following interphase .\"\n ],\n [\n \"We used a phylogenetic approach to identify putative functional regions within the sequence of Repo-Man , a targeting protein that binds PP1 in a cell-cycle specific manner regulated by a phospho-switch ( Trinkle-Mulcahy et al . , 2006; Qian et al . , 2011; Vagnarelli et al . , 2011 ) .\",\n \"A BLAST search revealed significant ( E = 5 × 10−4 ) similarity between a small region ( amino acids 388–420 ) of human Repo-Man and Ki-67 ( Figure 1A1 , 2 ) , a very large protein that exhibits strong links to cell proliferation ( Gerdes et al . , 1983 ) .\",\n \"The region conserved between Repo-Man and Ki-67 contains the PP1 binding motif ( RVTF ) of Repo-Man , which is conserved as RVSF in human Ki-67 ( Figure 1C3 ) . 10 . 7554/eLife . 01641 . 003Figure 1 . Ki-67 is evolutionary related to Repo-Man but shows distinct behaviour during mitosis .\",\n \"( A1 )\",\n \"Schematic representations of evolutionarily conserved regions in human Repo-Man and Ki-67 proteins ( shown approximately to scale ) .\",\n \"( A2 )\",\n \"E-values corresponding to global profile-to-sequence ( HMMer3 ) comparisons between the PP1 binding conserved regions ( blue oval ) in Repo-Man and Ki-67 families .\",\n \"Arrows indicate the profile search direction .\",\n \"The Repo-Man profile identified Ki-67 proteins as homologous with a highly significant E-value of 4 . 9 × 10−11 .\",\n \"Conversely , the profile of Ki-67 homologous sequences in animals identified Repo-Man proteins with a significant E-value of 9 . 9 × 10−13 .\",\n \"( A3 )\",\n \"Representative multiple sequence alignment of conserved regions from Repo-Man and Ki-67 families .\",\n \"Important mitotic phospho-residues in Repo-Man ( T412 and T419 ) are indicated in blue .\",\n \"The RVTF motif is indicated with an orange box .\",\n \"The most parsimonious explanation of Repo-Man and Ki-67 evolution is shown to the left of the alignment .\",\n \"Vertebrate branches are coloured in blue .\",\n \"Sequences are named according to: Repo_Human , NCBI:NP_689775 , Homo sapiens; Repo_Frog , NCBI:ACR33033 , Xenopus laevis; Repo_Danre , UniProt:A2CEF0 , Danio rerio; Ki_Human , UniProt:P46013 , Homo sapiens; Ki_Frog , UniProt:Q0VA85 , Xenopus laevis; Ki_Fugu , UniProt:UPI00016EA029 , Takifugu rubripes; Ki_Cioin , UniProt:UPI000180CFDA , Ciona intestinalis; Ki_Sacko , Baylor College of Medicine genome and FGENESH+ , Saccoglossus kowalevskii; Ki_Hydra , UniProt:UPI0001926DD5 , Hydra magnipapillata; and , Ki_Triad , UniProt:B3SB24 , Trichoplax adhaerens .\",\n \"The amino acid colouring scheme indicates average BLOSUM62 scores ( which are correlated with amino acid conservation ) for each alignment column: red ( greater than 3 . 5 ) , violet ( between 3 . 5 and 2 . 5 ) , and light yellow ( between 2 . 5 and 0 . 5 ) .\",\n \"( B–C )\",\n \"Ki-67 and PP1γ interact in vivo .\",\n \"Ki-67301–700 fused to Lac repressor:GFP ( Lac repressor:Ki-67PP1BD_wt ) was transfected together with RFP:PP1γ into a DT40 cell line containing a LacO array integrated on a single chromosome .\",\n \"Ki-67 was enriched at the LacO site ( panels 2 , 2′ ) where it recruited PP1γ ( 2 , 2″ ) .\",\n \"However , neither Lac repressor:GFP ( panels 1–1″ ) or the Ki-67 PP1-non-binding mutant ( Lac repressor:Ki-67PP1BD_RASA ) ( panels 3–3″ ) caused PP1 accumulation at the LacO site .\",\n \"( D ) Ki-67 recruits PP1γ more efficiently than PP1beta and more efficiently in interphase than in mitosis .\",\n \"The experimental set up was as in ( B ) .\",\n \"The enrichment of PP1 signal at the Lac repressor spot was compared to the background nuclear ( interphase ) or cytoplasmic ( mitosis ) signal within the same cell .\",\n \"Scale bar 10 μm .\",\n \"( E ) Direct and indirect interactors of Ki-67 .\",\n \"( F ) The B23S125 phosphorylation level remains high in Ki-67 depleted mitotic cells .\",\n \"Immunoblots of whole cell extracts of cycling ( interphase ) of Nocodazole-arrested ( mitotic ) HeLa cells transfected with Ki-67 RNAi oligo 5 or control oligos , were probed for Ki-67 , tubulin , B23T199ph , and B23S125ph .\",\n \"Two exposures of the B23S125ph blot are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00310 . 7554/eLife . 01641 . 004Figure 1—figure supplement 1 . Characterisation of Ki-67 RNAi . Two siRNAs against Ki-67 deplete the protein efficiently in HeLa cells .\",\n \"Top and middle panels show two different exposures of the same blot . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00410 . 7554/eLife . 01641 . 005Figure 1—figure supplement 2 . Distribution of PP1gamma in mitosis after the Ki-67 siRNA . In cells depleted of Ki-67 , PP1 is still recruited to the nucleolus in interphase ( panels 4–4′ ) and to kinetochores in metaphase ( panels 5–5′ ) but its levels are lower on anaphase chromosomes ( panels 6–6′ ) .\",\n \"HeLa cells were transfected with RFP:PP1γ ( green ) and with oligo 5 ( panels 4–6 ) or control oligo ( panels 1–3 ) .\",\n \"Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 005 The similarity between Repo-Man and Ki-67 within the PP1 binding domain suggested that Ki-67 could be a PP1-interacting protein ( PIP ) .\",\n \"Indeed , a possible connection between Ki-67 and PP1 was previously identified in two large scale screens for PP1 interactors .\",\n \"In one , in silico-screening based on a stringent definition of the RVxF motif ( the PP1 binding motif ) identified Ki-67 as putative interactor , however no in vivo interaction was demonstrated ( Hendrickx et al . , 2009 ) .\",\n \"Another study identified Ki-67 in a displacement affinity chromatography analysis of PP1 nuclear interactors ( Moorhead et al . , 2008 ) .\",\n \"To analyse if Ki-67 was a cell-cycle regulated PIP in vivo , we performed a tethering/recruitment experiment ( Vagnarelli et al . , 2011 ) .\",\n \"Ki-67301–700 wild type ( wt ) and a PP1 non-binding RASA mutant were fused to GFP:Lac repressor ( Figure 1B ) and co-expressed with RFP:PP1γ or β in DT40 cells carrying a lacO array integrated on a single chromosome ( Vagnarelli et al . , 2011 ) .\",\n \"GFP:Lac repressor on its own was used as a control in this experiment .\",\n \"All GFP:Lac repressor fusion constructs accumulated at the LacO integration site ( Figure 1C1′–3′ ) .\",\n \"Furthermore , the Ki-67 wt construct recruited PP1 to the same spot ( Figure 1C2″ ) .\",\n \"Quantification of the PP1 signal intensity at the GFP:Lac repressor spot compared to the general nuclear distribution ( excluding nucleoli ) revealed that Ki-67 recruitment of PP1 is more efficient in interphase than in mitosis and that PP1γ is recruited more efficiently than PP1β during interphase ( Figure 1D ) .\",\n \"These data suggest that Ki-67 is a PIP in vivo and the interaction with PP1γ is cell-cycle regulated .\",\n \"In order to determine whether Ki-67 is required to target PP1γ to any of its known locations in vivo , we used RNAi to deplete Ki-67 in human cells and determined the effects that this had on the distribution of PP1γ .\",\n \"A previous report described the successful depletion of Ki-67 ( Vanneste et al . , 2009 ) .\",\n \"However , the target sequence recognised by the published siRNA lies within a repeated stretch and rescue experiments to validate the phenotype were not reported .\",\n \"We therefore identified two new siRNAs that could deplete the protein as efficiently as the published siRNA ( Figure 1 , Figure 1—figure supplement 1 ) .\",\n \"Both new siRNAs yielded comparable phenotypes and we used siRNA 5 ( Ki5 ) for the depletion experiments presented below .\",\n \"This siRNA was validated in a rescue experiment that is discussed in a later section ( Figure 2B ) . 10 . 7554/eLife . 01641 . 006Figure 2 . Ki-67 is required for targeting of nucleolar proteins to the chromosome periphery in mitosis .\",\n \"( A ) Localisation of endogenous nucleolin is aberrant in mitotic cells after Ki-67 depletion ( panels 2 , 3 , 5 ) .\",\n \"HeLa cells were transfected with Ki-67 RNAi oligo 5 ( panels 2 , 3 ,\",\n \"5 ) or control oligos ( panels 1 ,\",\n \"4 ) and stained for nucleolin ( green ) .\",\n \"Quantification of the phenotypes is indicated in the graph above the corresponding representative images .\",\n \"Scale bar 5 μm .\",\n \"( B ) RNAi rescue experiment .\",\n \"HeLa cells depleted of Ki-67 were transfected with either mCherry:Ki-67wt , or mCherry:Ki-67RASA , together with Ki-67 RNAi oligo 5 or control oligo and stained for nucleolin .\",\n \"The localisation of nucleolin in mitotic cells was quantified in the different experimental conditions .\",\n \"See Figure 2—figure supplement 1 for representative images .\",\n \"Scale bar 10 μm .\",\n \"( C ) The mitotic chromosome peripheral localisation of NIFK is disrupted upon Ki-67 RNAi ( panels 5–6 ) .\",\n \"HeLa cells were transfected with GFP:NIFK ( green ) and oligo 5 ( panels 5–8 ) or control oligo ( panels 1–4 ) .\",\n \"Scale bar 10 μm .\",\n \"( D ) All novel cPERPs tested failed to accumulate on the chromosome periphery in mitosis .\",\n \"HeLa cells were co-transfected with GFP:cPERPs identified in an earlier study ( Ohta et al . , 2010 ) ( green ) and oligo 5 ( panels 3–4 , 7–8 , 11–12 , 15–16 ) or control oligo ( panels 1–2 , 5–6 , 9–10 , 13–14 ) : GFP:cPERP-B ( panels 1–4 ) , GFP:cPERP-C ( panels 5–8 ) , GFP:cPERP-D ( panels 9–12 ) , GFP:cPERP-F ( panels 13–16 ) .\",\n \"Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00610 . 7554/eLife . 01641 . 007Figure 2—figure supplement 1 . Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides with and without rescue by Ki-67 cDNA . RNAi rescue experiment .\",\n \"Representative microscopy images for quantifications shown in Figure 2B .\",\n \"HeLa cells depleted of Ki-67 were transfected with either mCherry:Ki-67wt ( panels 2 ,\",\n \"5 ) or mCherry:Ki-67RASA ( panels 3 ,\",\n \"6 ) ( red ) together with Ki-67 RNAi oligo 5 ( panels 4 , 5 ,\",\n \"6 ) or control oligo ( panels 1 , 2 ,\",\n \"3 ) and stained for nucleolin ( green ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00710 . 7554/eLife . 01641 . 008Figure 2—figure supplement 2 . Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides . HeLa cells were transfected with Ki-67 RNAi oligo 1 , 2 or 5 or control oligos and stained for nucleolin .\",\n \"Nucleolin localisation was classified as for Figure 2B ( diffuse , aberrant , and big foci ) and the graph represents the quantification of the phenotypes .\",\n \"Scale bar 5 μm .\",\n \"The three different oligos produce the same phenotype . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00810 . 7554/eLife . 01641 . 009Figure 2—figure supplement 3 . Distribution of NIFK in mitosis following Ki-67 depletion . NIFK T234 phosphorylation is regulated normally in the presence and absence of Ki-67 .\",\n \"Hela cells were transfected with Ki-67 RNAi oligo 5 ( panels 3 ,\",\n \"4 ) or control oligos ( panels 1 ,\",\n \"2 ) and stained with NIFK234ph antibody ( green ) .\",\n \"Scale bar 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 009 Ki-67 depletion in a HeLa cell line has no effect on the accumulation of RFP:PP1γ in the nucleolus ( Figure 1 , Figure 1—figure supplement 2[1 , 4] ) .\",\n \"Indeed , the targeting subunit for PP1 nucleolar localisation has been recently reported to be RRP1B ( Chamousset et al . , 2010 ) .\",\n \"In early mitosis , PP1γ localised normally on the spindle and at kinetochores in both control and Ki-67 depleted cells ( Figure 1 , Figure 1—figure supplement 2[2 , 5] ) .\",\n \"However , we observed a significant decrease in PP1 levels on anaphase chromatin in Ki-67 depleted cells ( Figure 1 , Figure 1—figure supplement 2[3′ , 6′] ) .\",\n \"Previous reports identified Repo-Man and Sds22 as responsible for targeting PP1 to anaphase chromatin ( Trinkle-Mulcahy et al . , 2006; Wurzenberger et al . , 2013 ) .\",\n \"Thus , Ki-67 is one of the several factors contributing to the accumulation of PP1γ on chromatin during mitotic exit .\",\n \"Analysis of the phosphorylation status of several known direct and indirect Ki-67 interacting proteins ( Figure 1E ) in interphase and mitosis revealed that nucleophosmin/B23 phospho-regulation was dependent on Ki-67 .\",\n \"B23 is phosphorylated both in interphase and in mitosis by several kinases ( Pfaff and Anderer , 1988; Jiang et al . , 2000; Louvet et al . , 2006; Krause and Hoffmann , 2010; Ramos-Echazabal et al . , 2012; Reboutier et al . , 2012 ) , including CyclinB/CDK1 at T199 ( Tokuyama et al . , 2001 ) in mitosis and by casein kinase II ( CKII ) on S125 during interphase ( Szebeni et al . , 2003 ) .\",\n \"Use of phospho-specific antibodies revealed a reproducible difference in nucleophosmin/B23 phosphorylation on S125 in the presence and absence of Ki-67 exponential cultures and in prometaphase cells ( Figure 1F ) .\",\n \"In both cases , the levels of S125ph were significantly increased following Ki-67 depletion .\",\n \"This was particularly evident in prometaphase-arrested cells .\",\n \"In contrast , we observed no significant difference in the phosphorylation status of B23 at T199 in the presence or absence of Ki-67 ( data not shown ) .\",\n \"These experiments support the notion that Ki-67 is a functional PP1-targeting subunit in vivo .\",\n \"Several aspects of mitotic chromosome structure remain relatively poorly understood , but amongst these , the perichromosomal compartment ( also known as the chromosome periphery ) stands out as a structure about which almost nothing is known .\",\n \"This is remarkable , as an ever-increasing list of chromosome periphery proteins has been compiled over the years ( Chaly et al . , 1984; McKeon et al . , 1984; Gautier et al . , 1992b; Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005; Gassmann et al . , 2005; Ohta et al . , 2010 ) .\",\n \"Some of these are among the most abundant proteins associated with mitotic chromosomes ( Ohta et al . , 2010 ) .\",\n \"Despite their abundance , the mechanism of their localisation and the role that they play on the chromosomes , if any , is still not understood ( Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005 ) .\",\n \"In a recent proteomic study , we identified a number of novel chromosome periphery proteins , which we termed cPERPs ( Ohta et al . , 2010 ) .\",\n \"Ki-67 is one of many nucleolar proteins that are recruited to the chromosome periphery at the transition from prophase to prometaphase .\",\n \"As Ki-67 is recruited to the perichromosomal compartment relatively early , we decided to examine whether its depletion affected the localisation of other known chromosome periphery proteins .\",\n \"We first examined the localisation of the endogenous nucleolin using a specific antibody .\",\n \"Indeed , nucleolin failed to accumulate at the chromosome periphery in the absence of Ki-67 ( Figure 2A ) .\",\n \"The phenotype was observed using all of the siRNAs that we have tested and was rescued by an siRNA-resistant cDNA ( Figure 2B , Figure 2—figure supplements 1 , 2 ) .\",\n \"To examine whether Ki-67 depletion affected the behaviour of other chromosome periphery proteins , we next examined the behaviour of NIFK , a known KI-67 interactor ( Takagi et al . , 2001 ) .\",\n \"During normal mitotic exit , NIFK concentrates around the segregating chromosomes before associating with nucleolar-derived foci ( NDF–Dundr et al . , 1996; Figure 2C3 ) .\",\n \"NDF formation varies between cell lines ( Dundr et al . , 2000 ) , but in the HeLa cells that we have used for RNAi studies , prominent NDFs are seldom observed during unperturbed mitosis ( Figure 2C1–4 ) .\",\n \"To follow NIFK behaviour , we transiently expressed GFP-tagged hNIFK in HeLa cells after control or Ki-67 RNAi .\",\n \"The nucleolar localisation of GFP-NIFK in interphase was unaltered after Ki-67 depletion ( Figure 2C8 ) , but in early mitosis the protein failed to properly accumulate around the mitotic chromosomes ( Figure 2C5–7 ) .\",\n \"Instead , in metaphase cells , it accumulated in large cytoplasmic aggregates , one of which was frequently found to ‘cap’ one end of the cluster of chromosomes on the metaphase plate ( Figure 2C5 ) .\",\n \"This extraordinary behaviour was also seen for endogenous NIFK phosphorylated on Thr234 using a phospho-specific antibody ( Figure 2—figure supplement 3[3′] ) .\",\n \"During mitotic exit , in the absence of Ki-67 , GFP-NIFK accumulated in NDF-like cytoplasmic aggregates that persisted until the reformation of the nuclear membrane ( Figure 2C5–7 ) .\",\n \"In view of the striking similarity of the behaviour of nucleolin and NIFK in the absence of Ki-67 , we tested the generality of this effect by localizing four novel cPERPs identified in our proteomics studies ( Ohta et al . , 2010 ) .\",\n \"Remarkably , all were mislocalised in Ki-67-depleted cells , and all were frequently observed to ‘cap’ one end of the metaphase plate ( Figure 2D3 , 7 , 11 , 15 ) .\",\n \"As in the case of nucleolin and NIFK , these aggregates dispersed into clusters of smaller cytoplasmic foci during mitotic exit ( Figure 2D4 , 8 , 12 , 16 ) .\",\n \"We considered two hypotheses to explain the observed failure of nucleolar proteins to associate with the chromosome periphery in mitosis of Ki-67-depleted cells .\",\n \"First , Ki-67 might be required for the complete disassembly of the nucleolus during mitotic entry .\",\n \"In this case , the larger cytoplasmic aggregates observed during metaphase might be remnants of incompletely disassembled nucleoli .\",\n \"Correlative light-microscopy/Electron microscopy ( CLEM ) analysis of cells transfected with both GFP-cPERP-C and the indicated siRNA oligos eliminated this hypothesis .\",\n \"The GFP-containing aggregates observed in mitosis in Ki-67-depleted cells did not correspond to nucleolar remnants or any other recognisable electron-dense structures such as NDFs ( Dundr et al . , 2000; Figure 3A , Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 01641 . 010Figure 3 . Ki-67 organizes the mitotic chromosome periphery .\",\n \"( A ) CLEM of HeLa cells transfected with GFP-PerP-C and control oligos ( top panels ) or Ki-67 oligos ( bottom panels ) .\",\n \"Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates ( arrow ) were identified and processed for CLEM using an adapted protocol ( Booth et al . , 2011 ) .\",\n \"Appropriate light and electron micrographs , from the matching z position , were overlaid using Adobe Photoshop Elements .\",\n \"Scale bar 5 μm .\",\n \"( B ) EM of mitotic cells from Control ( top panels ) and Ki-67 RNAi ( bottom panels ) .\",\n \"At higher magnification ( zoom\",\n \"2 ) it is possible to note that the amorphous material surrounding the mitotic chromosomes in control cells ( arrow ) is substantially reduced around Ki-67 depleted chromosomes .\",\n \"Scale bars ( left to right ) 4 , 2 , and 1 μm .\",\n \"Far right panels show regions of interest for pixel density analysis ( Figure 3C ) .\",\n \"( C ) Line scans of the peripheral regions of mitotic chromosomes in control ( blue line ) and Ki-67 depleted cells ( red line ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01010 . 7554/eLife . 01641 . 011Figure 3—figure supplement 1 . Depletion of Ki-67 does not leave electron-dense nucleolar remnants in the cytoplasm . CLEM of HeLa cells transfected with PERP-C and control oligos ( left panels ) or Ki-67 oligonucleotides ( right panels ) .\",\n \"Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates ( green arrow ) were identified and processed for CLEM using an adapted protocol ( Booth et al . , 2011 ) .\",\n \"Panels from top to bottom show electron micrographs of consecutive serial sections , together with the appropriate fluorescence micrograph of the same z position .\",\n \"No obvious electron-dense material can be seen colocalising with the fluorescent aggregates in the cytoplasm .\",\n \"The white arrow shows regions of the metaphase plate where chromosomes appear abnormally closely clustered together after Ki-67 depletion .\",\n \"Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 011 A second hypothesis suggested that Ki-67 might function as a scaffolding platform for organising the perichromosomal compartment .\",\n \"Indeed , all candidate proteins that we have tested to date have failed to localise to the chromosomal periphery in Ki-67-depleted cells .\",\n \"Although analyses of individual proteins can provide part of the story , it is difficult or impossible to make conclusions about the compartment as a whole since the chromosome periphery has a complex and as-yet undefined protein and RNA composition .\",\n \"Nonetheless , further analysis of our CLEM images strongly suggests that depletion of Ki-67 causes a loss of most or all of the perichromosomal compartment .\",\n \"Examination of electron micrographs of control and Ki-67-depleted cells at higher magnification revealed a subtle difference in the appearance of the edge of the chromosomes .\",\n \"In control cells there was a ‘fuzzy’ transition zone between the electron-dense chromatin and the cytoplasm ( Figure 3B ) .\",\n \"This transition appeared to be more abrupt in Ki-67-depleted chromosomes .\",\n \"This was confirmed by line scans across the chromosome-to-cytoplasm boundary , which showed that there is normally a gradual decrease in density at the edge of mitotic chromosomes .\",\n \"This transition was significantly more abrupt in the absence of Ki-67 ( Figure 3C ) .\",\n \"These experiments suggest that Ki-67 directs the binding of nucleolar components to the chromosome periphery , perhaps by acting as a scaffold .\",\n \"Alternatively , the macromolecular network at the chromosomal periphery could be delicately balanced , such that removal of any single component causes the entire structure to fail .\",\n \"However , depletion of cPERP-B , -D , or -E using RNAi ( Figure 4—figure supplement\",\n \"1 ) failed to alter the striking perichromosomal localization of Ki-67 ( Figure 4 ) .\",\n \"This is consistent with Ki-67 being upstream of the other components in the assembly of the perichromosomal compartment . 10 . 7554/eLife . 01641 . 012Figure 4 . Ki-67 localisation is not dependant on other c-PERPS tested .\",\n \"( A ) Ki-67 localisation was analysed following the RNAi depletion of several novel c-PERPs ( PERP B , D , and E ) .\",\n \"Following a 48 hr knock-down period , cells were fixed and labelled with anti-Ki67 ( green ) antibody and Dapi ( blue ) .\",\n \"Examples shown include metaphase and anaphase cells .\",\n \"Scale bar 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01210 . 7554/eLife . 01641 . 013Figure 4—figure supplement 1 . Depletion of cPERPs by RNAi . As no antibodies are available yet targeting these novel cPERPs , HeLa cells were transfected with GFP:PERP cDNA with or without the co-transfection of siRNA .\",\n \"Cells were harvested after 48 hr and prepared for Western analysis .\",\n \"Anti-GFP probing shows decreased levels of GFP-PERP expression in samples co-transfected with the appropriate siRNA . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 013 The observations presented above suggest two possible hypotheses for how Ki-67 could function in the assembly of the chromosome periphery .\",\n \"First , the protein might comprise part of a PP1 holoenzyme whose removal of phosphates from chromosomal proteins , could be required for those proteins to assemble at the chromosome periphery .\",\n \"Alternatively , Ki-67 , which is a very large protein with multiple repeated domains , might function as a scaffold linking together key components at the chromosome periphery .\",\n \"To begin to distinguish between these two hypotheses , we generated constructs expressing mRFP coupled to either wild-type Ki-67 or a Ki-67 RASA mutant that is incapable of binding PP1 at the conserved site shared with Repo-Man ( Figure 1A3 ) .\",\n \"Both constructs encoded mRNAs that were engineered to be resistant to siRNA-5 .\",\n \"Both mRFP:Ki-67-wt and mRFP:Ki-67-RASA proteins rescued the localisation of endogenous nucleolin throughout mitosis in cells transfected with Ki-67 siRNAs .\",\n \"In transfected cells , nucleolin was once again concentrated at the chromosome periphery from prometaphase through telophase ( Figure 2B , Figure 2—figure supplement 1 ) .\",\n \"When combined with our previous observations these data support the hypothesis that Ki-67 acts as a scaffold for formation of the perichromosomal compartment .\",\n \"Importantly , this rescue experiment also confirmed that the chromosome periphery phenotypes observed following Ki-67 depletion by siRNA-5 are not due to off-target effects .\",\n \"Because depletion of Ki-67 appears to result in either a dramatic reduction , or even complete loss , of the perichromosomal compartment , these experiments offer a unique opportunity to investigate the function ( s ) of this mysterious structure .\",\n \"If the perichromosomal compartment is a kind of pellicle or ‘skin’ on the surface of the chromosomes ( Schrader , 1944 ) , then two obvious potential functions come to mind .\",\n \"First , the perichromosomal layer could protect the chromosomal DNA from damage once it is released into a potentially hostile cytoplasmic environment following nuclear envelope breakdown .\",\n \"Immunostaining for 53BP1 , a protein that associates with DNA damage foci revealed that levels of intrinsic DNA damage are low in mitotic cells under our culture conditions ( Figure 5A , B ) .\",\n \"These levels were not substantially increased in chromosomes lacking Ki-67 .\",\n \"Thus , this hypothesis appears unlikely . 10 . 7554/eLife . 01641 . 014Figure 5 . Ki-67 is does not function to protect chromosomes from DNA damage or provide structural maintenance .\",\n \"( A ) Representative overview images of HeLa cells transfected with control or Ki-67 specific siRNA oligos probed with anti-53bp antibodies to assess levels of DNA damage .\",\n \"Scale bar 10 μm .\",\n \"( B ) A bar graph showing quantification of the percentage of mitotic cells found with DNA damage , marked using an anti-53bp antibody .\",\n \"( C ) Chromosome spreads from control RNAi ( panels 1–1″ ) and Ki-67 oligo 5 RNAi ( panels 2–2″ ) were stained for the chromosome scaffold protein KIF4A ( green ) and for the kinetochore with ACA ( red ) .\",\n \"Ki-67 depleted chromosomes still maintain a proper localisation of the chromosome scaffold and kinetochore proteins .\",\n \"Scale bar 2 μm .\",\n \"( D ) IMS Assay ( Intrinsic Metaphase Structure Assay ) .\",\n \"Chromosomes from HeLa cells after Ki-67 depletion ( panels 2 , 4 ,\",\n \"6 ) and control depletion ( panels 1 , 3 ,\",\n \"5 ) at the beginning of the assay ( panels 1 , 2 ) , after the first TEEN treatment ( panels 3 , 4 ) , and after the second RSB addition ( panels 5 , 6 ) .\",\n \"Scale bar 10 μm .\",\n \"( E ) Quantification of the experiment in ( D ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 014 An alternative hypothesis is that by forming a layer coating each chromosome , the perichromosomal compartment could promote the formation of physically separated chromosomes that is characteristic of mitosis .\",\n \"Our prior studies revealed that condensin , the chromokinesin Kif4 and DNA topoisomerase II all contribute to shaping mitotic chromosomes ( Hudson et al . , 2003; Green et al . , 2012; Samejima et al . , 2012 ) .\",\n \"Furthermore , proteomic analysis revealed a substantial reduction in levels of Ki-67 associated with isolated mitotic chromosomes depleted of KIF4A ( Samejima et al . , 2012 ) .\",\n \"The converse is not true , however , as neither the localisation nor abundance ( as determined by immunostaining ) of KIF4A was altered in the absence of Ki-67 ( Figure 5C ) .\",\n \"Thus , Ki-67 depletion does not affect the known mitotic chromosome structural proteins .\",\n \"Mitotic chromosomes have an intrinsic metaphase structure ( IMS ) that can be probed using a specialized assay , in which chromosomes are induced to unfold down to the level of 10 nm fibres by removal of divalent cations , and then induced to re-fold by re-addition of Mg2+ ( Earnshaw and Laemmli , 1983; Hudson et al . , 2003 ) .\",\n \"Even though chromosomes lacking condensin or KIF4 appear morphologically normal when cells are processed under optimal conditions , those chromosomes are severely impaired in the IMS assay—failing to re-adopt a normal appearance after restoration of divalent cations ( Hudson et al . , 2003; Samejima et al . , 2012 ) .\",\n \"Other abundant chromosomal proteins , including , cohesin and DNA topoisomerase II are not required for this aspect of mitotic chromosome structure ( Vagnarelli et al . , 2004; Johnson et al . , 2009 ) .\",\n \"We transfected cells with either control or Ki-67 siRNAs and then performed the IMS assay .\",\n \"This experiment showed clearly that chromosomes depleted of Ki-67 efficiently regain their normal morphology after two rounds of unfolding and refolding .\",\n \"Thus , the perichromosomal layer is not required for maintenance of the intrinsic structure of mitotic chromosomes ( Figure 5D , E ) .\",\n \"In summary , we could find no evidence for a role of Ki-67—and by extension much or all of the perichromosomal compartment—in mitotic chromosome structure or integrity under these conditions .\",\n \"Given the abnormal localization of nucleolar components during mitosis , it is no surprise that the segregation of these components is perturbed in mitosis .\",\n \"The detailed behaviour of the complex population of nucleolar components following abolishment of the perichromosomal compartment is a subject for a follow-up study , but here as an example , we have examined the segregation of the abundant component nucleophosmin/B23 .\",\n \"We followed cell division in living HeLa cells expressing GFP-B23 after either control or Ki-67 siRNA .\",\n \"In Ki-67-depleted cells , B23 was never observed to associate with the chromosomes during mitosis .\",\n \"Instead , it started forming cytoplasmic aggregates just after anaphase onset ( Figure 6A , 45' ) .\",\n \"We then analysed the distribution of endogenous nucleolin between daughter cells in cytokinesis .\",\n \"While in control cells there was a relatively even distribution of chromatin-associated nucleolin between daughter cells , in Ki-67-depleted cells the nucleolin aggregates were often not chromatin bound and their distribution between daughter cells tended to be more asymmetric ( Figure 6B , C ) . 10 . 7554/eLife . 01641 . 015Figure 6 . Segregation of nucleolin .\",\n \"( A ) In cells depleted of Ki-67 the nucleolus never disassembles completely during mitosis and B23 never accumulates around the mitotic chromosomes .\",\n \"Time-lapse imaging of GFP:B23 in HeLa cells after control or Ki-67 RNAi .\",\n \"Scale bar 10 μm .\",\n \"( B ) Nucleolin localisation was analysed in cells in cytokinesis after control or Ki67 RNAi .\",\n \"Nucleolin distribution between the two daughter cells is uneven following Ki-67 RNAi compared to the control and the protein is predominantly not associated with the chromatin .\",\n \"( C ) Quantification of the experiment in ( B ) .\",\n \"The graphs represent the ratio of the total nucleolin intensity between daughter cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01510 . 7554/eLife . 01641 . 016Figure 6—figure supplement 1 . Electron micrographs of interphase nuclei from Ki-67 RNAi cells . Three major components of the nucleolus are recognised: Fibrillar centres ( FC ) , dense fibrillar component ( DFC ) , and granular component ( GC ) .\",\n \"Scale bar 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 016 Despite this perturbation of the behaviour of major nucleolar components during mitosis , nucleoli did indeed re-form during mitotic exit .\",\n \"Furthermore , the ultrastructure of the reformed nucleoli appeared to be normal , with fibrillar centres , dense fibrillar component , and granular component all readily observed ( Figure 6—figure supplement 1 ) .\",\n \"Thus , Ki-67 is apparently not required for the structural organisation within interphase nucleoli .\",\n \"Human cells contain five chromosomes with ribosomal gene clusters that can form NORs .\",\n \"Thus , human cells could in theory have 10 nucleoli .\",\n \"This is almost never seen , presumably due to natural clustering of the NORs and the failure of all NORs to become activated .\",\n \"In our line of HeLa cells , we observed 3 . 5 ± 0 . 3 nucleoli per nucleus ( Figure 7D , E ) .\",\n \"This changed dramatically following Ki-67 depletion , when the number of nucleoli observed dropped to 1 . 6 ± 0 . 3 .\",\n \"This strongly suggests that chromosomes lacking a perichromosomal layer might associate with one another more closely than normal , thus promoting NOR fusion during reactivation . 10 . 7554/eLife . 01641 . 017Figure 7 . The nucleoli of Ki-67 depleted cells are fewer , smaller , and functionally repressed . Normal cell , nucleus , and nucleolus size is compromised following depletion of Ki-67 .\",\n \"( A ) Representative images showing a reduced cell size and nucleolar size phenotype in Ki-67 depleted cells , using Rhodamine Phalloidin and DAPI as markers .\",\n \"Scale bar 5 μm .\",\n \"( B and C )\",\n \"Quantification of cell area and nuclear area in control ( white bar ) and Ki-67 depleted ( black bar ) cells .\",\n \"Bars show mean ± SEM .\",\n \"ncell = 100 .\",\n \"( D ) Representative images showing the small nuclei phenotype , with single nucleoli following depletion of Ki-67 .\",\n \"Scale bar 5 μm .\",\n \"( E and F )\",\n \"Quantification of nucleolar number and area in control ( white bar ) and Ki-67 depleted ( black bar ) cells .\",\n \"Bars show mean ± SEM .\",\n \"ncell = 50 .\",\n \"( G ) A 2D scatter plot showing combined nucleolar area ( per cell ) on the Y axis , vs nuclear area on the X axis , for control ( green ) and Ki-67 depleted ( red ) cells .\",\n \"Each individual translucent dot represents one cell .\",\n \"Black squares represent the means .\",\n \"( H ) Northern blot of RNA samples prepared from control ( C ) or Ki-67 depleted cells , for 24 , 48 , and 72 hr of Ki-67 knock-down .\",\n \"Blot shows decreased signal for 47S bands and a time course dependent decrease for 26S signal ( red stars ) .\",\n \"No clear change was seen with bands for 18SE ( black star ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01710 . 7554/eLife . 01641 . 018Figure 7—figure supplement 1 . Ki-67 depletion influences cell size , nuclei size , and nucleoli size . A 2D scatter plot showing combined nuclei area on the Y axis , vs cell area on the X axis , for control ( green ) and Ki-67 depleted ( red ) cells .\",\n \"Each individual translucent dot represents one cell .\",\n \"Black squares represent the means . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01810 . 7554/eLife . 01641 . 019Figure 7—figure supplement 2 . Ki-67 depletion has only minor effects on the cell cycle detected by FACS .\",\n \"( top )\",\n \"HeLa cells transfected with control or Ki-67 siRNA oligos were incubated for 48 , 72 , or 96 hr , before cell-cycle analysis using FACS .\",\n \"Gated cells were manually categorised into cell-cycle stages using FACS histograms .\",\n \"( bottom ) bar graph representation of the histogram data in the top panel . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01910 . 7554/eLife . 01641 . 020Figure 7—figure supplement 3 . Ki-67 depletion influences nucleolar size . A bar graph showing the mean cross-sectional area measurements of nucleoli from control and Ki-67 depleted cells .\",\n \"Nucleoli measurements are on a per cell basis and placed in ascending order , from the largest nucleolus , to the smallest . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 020 A second reason why Ki-67-depleted cells might have single large nucleoli could be due to a decreased efficiency of NOR reactivation during mitotic exit .\",\n \"Ki-67-depleted cells tended to be smaller and to have smaller nuclei ( Figure 7A–C , Figure 7—figure supplement 1 ) .\",\n \"This was not due to an accumulation of the cells in G1 , as shown by FACs analysis ( Figure 7—figure supplement 2 ) , but could potentially be due to ribosomal insufficiency .\",\n \"We have made several observations that are consistent with this .\",\n \"First , if we calculate the total nucleolar area in optical sections of Ki-67-depleted nuclei , we find that these nuclei tend to have a smaller aggregate nucleolar area ( Figure 7F , G , Figure 7—figure supplement 3 ) .\",\n \"If we assume total nucleolar area to be a proxy for the number of genes involved in rDNA transcription , this suggests that reactivation of ribosomal clusters following mitotic exit may be less efficient in Ki-67-depleted cells .\",\n \"This is consistent with the results of a Northern analysis of total rRNA in control and Ki-67-depleted cells , which revealed decreased levels of pre-rRNA species , consistent with lower levels of rRNA transcription in the depleted cells ( Figure 7H ) .\",\n \"To further examine NOR reactivation following mitotic exit , we exploited the fact that chromosomes bearing re-activated NORs are associated with nucleoli .\",\n \"We used a HT1080 cell line carrying a LacO array integrated on chromosome 13p next to the NOR and expressing GFP fused to Lac repressor ( Chubb et al . , 2002 ) .\",\n \"In this cell line the 13p locus ( visualized as a GFP spot ) tends to localise in the nuclear interior ( Figure 8A1 ) where it is usually closely associated with a nucleolus ( Figure 8B1 ) .\",\n \"A nuclear erosion script developed by Bickmore and Perry ( Croft et al . , 1999 ) was used to divide the nucleus into 5 concentric rings of equal area in order to score the position of the locus within the nucleus ( Figure 8A2 ) . 10 . 7554/eLife . 01641 . 021Figure 8 . Ki-67 depletion affects nuclear architecture .\",\n \"( A ) The position of a chromosome 13p ( marked by a LacO array:LaciGFP ) in HT1080 cells ( 1 ) was assessed in control and Ki67 RNAi experiments .\",\n \"150 nuclei from three independent experiments were imaged and analysed with an erosion script software to locate the position of the locus ( 2 ) .\",\n \"The locus repositions from the interior toward the periphery after Ki-67 RNAi ( 3 ) .\",\n \"( B ) The cells described in A were stained for nucleolin after control or Ki-67 RNAi .\",\n \"The association with the nucleolus was recorded as proximal ( 1–1′ ) or distal ( 2–2′ ) .\",\n \"( 3 ) Quantification of the analyses .\",\n \"( C ) Model for Ki-67 function in mitosis; ( blue: chromatin; green perichromosomal proteins; grey: microtubules ) .\",\n \"See text for details . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 021 Depletion of Ki-67 had a strong effect on the nuclear localization of chromosome 13p .\",\n \"In cells depleted of Ki-67 , the tagged 13p locus tended to move from the nuclear interior towards the nuclear periphery ( Figure 8A3 ) .\",\n \"This reorganization of chromosome 13p was correlated with a disengagement from the nucleolus ( Figure 8B2 , 3 ) .\",\n \"Both phenotypes are consistent with a decreased efficiency of reactivation of the 13p NOR .\",\n \"Thus , although Ki-67 depletion and loss of much or all of the perichromosomal compartment has little obvious effect on chromosome structure or segregation in the first mitosis after depletion , after mitotic exit the nucleus undergoes fundamental changes consistent with decreased activity of the ribosomal gene clusters and with altered interactions between chromosomes .\",\n \"More detailed examination of the link between Ki-67 , the perichromosomal compartment , and nucleolar organizer region ( NOR ) reactivation will be an important subject for future study .\"\n ],\n [\n \"Our studies have revealed for the first time that Ki-67 functions as a structural/scaffolding protein required for assembly of the perichromosomal compartment on condensed mitotic chromosomes .\",\n \"Thus , Ki-67 has an important role in determining the behaviour of many nucleolar components during mitosis .\",\n \"Interestingly , this method of segregating nucleolar components means that nucleolar reassembly can begin immediately after CDK activity declines and before the re-establishment of nuclear-cytoplasmic transport during mitotic exit .\",\n \"Ki-67 is a PP1-interacting protein , however the biological role of this activity remains elusive and indeed the protein could execute this function during another phase of the cell cycle rather than mitosis .\",\n \"How the various activities of Ki-67 combine to determine the nucleolar morphology in proliferating cells and whether the perichromosomal layer has a subtle role in chromosome dynamics during mitosis or nuclear reformation remain to be determined in future studies .\"\n ],\n [\n \"HeLa Kyoto and MRC5 cells were maintained in DMEM supplemented with 10% and 15% FBS respectively .\",\n \"DT40 cells carrying a single integration of the LacO array ( Vagnarelli et al . , 2006 ) were cultured in RPMI1640 supplemented with 10% FBS and 1% chicken serum .\",\n \"For RNAi treatments HeLa cells in exponential growth were seeded in six-well plates with or without polylysine-coated glass coverslips and grown overnight .\",\n \"Transfections were performed using Polyplus jetPRIME ( PEQLAB , Southampton , UK ) with the indicated siRNA oligos and analysed 48 hr later as previously described ( Vagnarelli et al . , 2006 ) .\",\n \"For the rescue experiments HeLa cells at 50% confluence were transfected with 400 ng of plasmid DNA and 50 nM of siRNA oligonucleotides and analysed 48 hr post-transfection .\",\n \"The siRNA oligonucleotides against Ki67 are as follows: Ki-1 as published by Vanneste et al . ( 2009 ) ; Ki-2 :5′AAGCACCAGAGACCCUGUATT3′; Ki-5:5′GCAUUUAAGCAACCUGCAA3′; a 21-mer oligonucleotide ( CGUACGCGGAAUACUUCGAdTdT ) was used as a control ( Elbashir et al . , 2001 ) .\",\n \"To test for successful depletion of cPerP proteins , HeLa cells were co-transfected with specific siRNA oligos together with the appropriate GFP-cPerP cDNA constructs .\",\n \"Control samples were prepared in parallel via cells transfected with GFP-PerP cDNA only .\",\n \"Following a 48 hr expression/knock-down period cells were harvested and processed routinely for Western analysis .\",\n \"Knockdown was considered successful if Western analysis revealed decreased GFP expression levels in siRNA treated samples ( rabbit polyclonal anti-GFP , Invitrogen , Paisley , UK ) .\",\n \"The primary antibodies were used as follows: Ki-67 ( mouse monoclonal BD Transduction laboratory , Oxford , UK ) 1:100; nucleolin ( rabbit polyclonal; Abcam ) 1:300; NIFK T234ph ( Rabbit polyclonal; Abcam , Cambridge , UK ) 1:100; Repo-Man ( Vagnarelli et al . , 2011 ) ; anti-alpha-tubulin antibody ( B512; SIGMA , Gillingham , UK ) , anti-B23 S125ph , ( Abcam ) ; anti-B23T199ph ( Abcam ) ; anti-nucleolin ( Abcam ) .\",\n \"For immunofluorescence , cells were fixed in 4% PFA and processed as previously described ( Vagnarelli et al . , 2011 ) .\",\n \"Fluorescence-labelled secondary antibodies were applied at 1:200 ( Jackson ImmunoResearch ) .\",\n \"3D data sets were acquired using a cooled CCD camera ( CH350; Photometrics ) on a wide-field microscope ( DeltaVision Spectris; Applied Precision ) with a NA 1 . 4 Plan Apochromat lens .\",\n \"The data sets were deconvolved with softWoRx ( Applied Precision ) .\",\n \"3D data sets were converted to Quick Projections in softWoRx , exported as TIFF files , and imported into Adobe Photoshop for final presentation .\",\n \"Live cell imaging was performed with a DeltaVision microscope as previously described ( Vagnarelli et al . , 2011 ) .\",\n \"For quantification of PP1 binding in vivo , images of prometaphase and interphase transfected cells were acquired and the intensity of PP1 staining at the GFP spot was calculated relative to the average nuclear intensity .\",\n \"The 3D data sets obtained at the same exposure were projected as mean intensities .\",\n \"A 12 × 12 pixel area containing the GFP spot was used to measure the total intensity of the signal .\",\n \"An area of the same size was used to identify the background signal in each cell , and this value was subtracted from the measurement of the nuclear and spot area .\",\n \"The IMS assay was conducted as previously described ( Hudson et al . , 2003 ) .\",\n \"For immunoblotting , whole cell lysates were loaded onto polyacrylamide gels .\",\n \"SDS-PAGE and immunoblotting was performed following standard procedures .\",\n \"Ki-67 ( aa 161–659 ) was obtained by RT-PCR from HeLa cDNA using the following primers: GGATCCGGCGCCACGTTTCCTCTC and CTCGAGTTTTACTACATCTGC and CLONED PGEX4T3 BamHi/XhoI .\",\n \"The PP1-non binding mutant version was generated from this vector using a QuikChange Site-Directed Mutagenesis Kit ( Agilent Technologies , Edinburgh , UK ) .\",\n \"The Lac repressor fusion constructs were obtained by cloning Ki-67 ( aa 161–659 ) into pEGFP:Lac repressor .\",\n \"hPP1γ was cloned into PET28 EcoRI/HindIII .\",\n \"hNIFK was cloned by RT PCR from HeLa cDNA using the primers CTCGAGGGATGGCGACTTTTTCTGGC and GAATTCTCACTGATTGCTGCTTCT and cloned into the XhoI/EcoR1 sites of pEGFPC1 .\",\n \"The cPERPs were cloned into the gateway system by PCR as previously described ( Ohta et al . , 2010 ) .\",\n \"Accession numbers for cPERPs are as follows: cPERP-A: ( C1orf131 ) –NM_152379 . 2 , cPERP-B: ( CCDC137 ) –NM_199287 . 2 , cPERP-C: ( KIAA0020 ) –NM_014878 . 4 , cPERP-D: ( DDX18 ) –NM_006773 . 3 , cPERP-E: ( CIRH1A ) –NM_032830 . 2 , cPERP-F: ( DDX27 ) –XM_006723815 . 1 .\",\n \"The CLEM processing method was an adapted version of a previously established protocol ( Booth et al . , 2011 , Booth et al . , 2013 ) .\",\n \"Cells were seeded onto glass-bottomed , gridded dishes ( MatTek Corporation , USA ) and co-transfected with GFP-cPerpC together with control or Ki-67 specific siRNA oligos .\",\n \"Following a 48 hr expression period , cells of interest were identified using a wide-field epifluorescence microscope ( DeltaVision RT; Applied Precision ) .\",\n \"GFP-expressing mitotic cells were located and their position mapped using transmitted light to visualise reference coordinates .\",\n \"Cells were then fixed for 1 hr ( 3% glutaraldehyde , 0 . 5% paraformaldehyde in 0 . 2 M sodium cacodylate buffer containing 5 µg/ml Hoechst ) and washed in PBS ( 3 × 5 min ) .\",\n \"Cells of interest were then re-imaged to acquire micrographs of Hoechst stained chromosomes .\",\n \"Next , cells were osmicated ( 1% osmium tetroxide in PBS ) for 1 hr , washed with PBS ( 3 × 5 min ) , ddH2O ( 2 × 20 min ) , and then 30% ethanol ( 1 × 10 min ) before contrast staining with uranyl acetate ( 0 . 5% in 30% ethanol ) for 1 hr .\",\n \"Cells were then dehydrated using a graded series of ethanol washes culminating in 2 × 10 min incubations with 100% ethanol , followed by infiltration with ethanol:resin mixtures ( at 2:1 and then 1:1 ) .\",\n \"Finally , cells were embedded in 100% resin , with a gelatin capsule of resin covering the cells of interest , before curing at 60°C for 3 days .\",\n \"Ultra-small resin blocks ( 50 µm2 ) were fine trimmed and serial sections ( 85 nm thickness ) taken at areas corresponding to previously chosen coordinate positions , before post-staining in Reynold's lead citrate and uranyl acetate ( 5% in 50% ethanol ) for 10 and 5 min , respectively .\",\n \"Cells were visualised with a Phillips CM120 BioTwin transmission electron microscope ( FEI ) and micrographs acquired using a Gatan Orius CCD camera ( Gatan ) .\",\n \"The appropriate Z position correlative light/EM images of a cell were concatenated using ImageJ and then analysed and overlaid using Photoshop Elements 6 ( Adobe ) .\",\n \"Electron micrographs containing metaphase chromosomes from control or Ki-67 depleted cells were used for chromosome periphery analysis .\",\n \"The pixel density of a 1 μm region of interest was measured using the raw data from the ‘plot profile’ function of imageJ .\",\n \"For unbiased consistency , the 1 μm region of interest always started within the chromosome body and finished in the cytoplasm , with the halfway point lying at the expected origin of the chromosome periphery .\",\n \"Approximately 200 individual pixel density measurements were taken within each 1 μm region .\",\n \"The data were plotted as a line scan profile , with each data point representing the mean of 6 × 1 μm regions of interest per chromosome .\",\n \"HeLa cells were seeded onto coverslips and transfected with control or Ki-67 specific siRNA oligonucleotides .\",\n \"Following a 48 hr knock-down period , cells were fixed using 4% paraformaldehyde , blocked with 3% BSA , and either directly mounted onto slides using hard-setting Vectashield ( containing DAPI ) or first stained with Rhodamine Phalloidin ( Biotium , Inc . , Cambridge , UK ) , before mounting .\",\n \"All measurements were scored using imageJ and area measurement tools .\",\n \"Total cell cross-sectional area was measured using Phalloidin to reveal the cytoplasm and by extension a guide for cell periphery .\",\n \"The area of individual cells or clustered groups of cells were traced and scored using freehand measurement tools .\",\n \"Nuclei and nucleoli cross-sectional area was measured and nucleolar numbers scored using DAPI as a marker .\",\n \"A contrast threshold was applied to micrographs allowing the semi-automated , unbiased measurement of nucleolar area and number using the ImageJ wand ( tracing ) tool .\",\n \"Control or Ki-67 depleted HeLa cells were subjected to cell-cycle analysis by FACS .\",\n \"Briefly , 1 × 106 cells , per condition , were fixed with cold ethanol ( 70% ) for 1 hr , centrifuged , and resuspended in PBS containing RNase A ( 0 . 2 mg/ml ) and Propidium Iodide ( 10 µg/ml ) .\",\n \"Following a 20-min incubation , cells were analysed using FACS .\",\n \"The channel FL2 was used to analyse 20 , 000 events per condition .\",\n \"Gated cells were manually categorised into cell-cycle stages .\",\n \"Cells were analysed following knock-down periods of 24 , 48 , 72 , and 96 hr .\",\n \"HT1080 cell line carrying a LacO integration on chromosome 13p and expressing a Laci:GFP ( kindly provided by W Bickmore ) was used for depleting Ki-67 in an RNAi experiment as described for HeLa above .\",\n \"At 48 hr the cells were fixed and processed for immunostaining with a nucleolin antibody as described before .\",\n \"150 nuclei from three independent experiments were imaged and analysed for with the nuclear erosion scrip ( Croft et al . , 1999 ) to assign the position of the 13p locus .\",\n \"Control or Ki-67 depleted HeLa cells were cultured in 10 cm dishes and processed for Northern analysis .\",\n \"RNA was extracted using fresh TRIzol ( Ambion ) according to manufacturer’s guidelines .\",\n \"3 µg of RNA per sample was separated using a standard 1 . 2% TBE agarose gel and transferred to Hybon N+ membrane overnight , by capillary action .\",\n \"Membranes were hybridized in 6X SSPE , 5X Denhardts , 5X SDS at 37°C before washing with 2XSSPE .\",\n \"Probes ( see below ) were 5′ labelled with y32P-ATP and T4PNK .\",\n \"Probe_d_human AGACGAGAACGCCTGACACGCACGGCAC 5'ETS probe .\",\n \"Probe_e_human CCTCGCCCTCCGGGCTCCGTTAATGATC 5′ end of ITS1 ( 21S and 18SE ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["When the nucleolus disassembles during open mitosis , many nucleolar proteins and RNAs associate with chromosomes , establishing a perichromosomal compartment coating the chromosome periphery .","At present nothing is known about the function of this poorly characterised compartment .","In this study , we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells .","Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin .","Following siRNA depletion of Ki-67 , NIFK , B23 , nucleolin , and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes .","Correlative light and electron microscopy ( CLEM ) images suggest a near-complete loss of the entire perichromosomal compartment .","Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells ."],"string":"[\n \"When the nucleolus disassembles during open mitosis , many nucleolar proteins and RNAs associate with chromosomes , establishing a perichromosomal compartment coating the chromosome periphery .\",\n \"At present nothing is known about the function of this poorly characterised compartment .\",\n \"In this study , we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells .\",\n \"Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin .\",\n \"Following siRNA depletion of Ki-67 , NIFK , B23 , nucleolin , and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes .\",\n \"Correlative light and electron microscopy ( CLEM ) images suggest a near-complete loss of the entire perichromosomal compartment .\",\n \"Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells .\"\n]"},"summary":{"kind":"list like","value":["The genetic information of an organism is found in the nucleus of each cell in the form of DNA organised into chromosomes .","The exact structure of those chromosomes changes as the cell moves through the different stages of the cell division cycle .","During the stage called mitosis , where the DNA of a cell ( which has previously been duplicated ) is shared into two daughter cells , the chromosomes become tightly packed structures that can be readily moved through the cytoplasm .","Since the late nineteenth century , it has been known that a layer of proteins , called the perichromosomal layer , coats the condensed chromosomes .","However , virtually nothing was known about the role this layer performs .","One of the first proteins to join the perichromosomal layer after mitosis begins is called Ki-67 .","This is only found in the cell nucleus when a cell is actively growing and dividing , and so is widely used as a marker in experiments investigating these processes: for example , Ki-67 is used to detect growing tumour cells amongst the normal cells in tissues of the body , and to measure the effectiveness of drugs designed to stop the growth of tumours .","Again , however , little is known about what Ki-67 actually does .","Booth et al . now reveal that when Ki-67 is not present in a cell , chromosomes do not have a perichromosomal layer—or at best , have a small remnant of one .","This allowed Booth et al . to investigate the role of the perichromosomal layer as well .","When the chromosomes first go through mitosis without a perichromosomal layer , no changes to the shape or the behaviour of the chromosomes are seen .","However , the new nuclei are smaller than normal and their contents are arranged differently .","This causes problems with the ability of daughter cells to synthesise protein building blocks and leads to an increased rate of spontaneous cell death when daughter cells try to undergo the next mitosis .","Further research is needed to understand why this happens ."],"string":"[\n \"The genetic information of an organism is found in the nucleus of each cell in the form of DNA organised into chromosomes .\",\n \"The exact structure of those chromosomes changes as the cell moves through the different stages of the cell division cycle .\",\n \"During the stage called mitosis , where the DNA of a cell ( which has previously been duplicated ) is shared into two daughter cells , the chromosomes become tightly packed structures that can be readily moved through the cytoplasm .\",\n \"Since the late nineteenth century , it has been known that a layer of proteins , called the perichromosomal layer , coats the condensed chromosomes .\",\n \"However , virtually nothing was known about the role this layer performs .\",\n \"One of the first proteins to join the perichromosomal layer after mitosis begins is called Ki-67 .\",\n \"This is only found in the cell nucleus when a cell is actively growing and dividing , and so is widely used as a marker in experiments investigating these processes: for example , Ki-67 is used to detect growing tumour cells amongst the normal cells in tissues of the body , and to measure the effectiveness of drugs designed to stop the growth of tumours .\",\n \"Again , however , little is known about what Ki-67 actually does .\",\n \"Booth et al . now reveal that when Ki-67 is not present in a cell , chromosomes do not have a perichromosomal layer—or at best , have a small remnant of one .\",\n \"This allowed Booth et al . to investigate the role of the perichromosomal layer as well .\",\n \"When the chromosomes first go through mitosis without a perichromosomal layer , no changes to the shape or the behaviour of the chromosomes are seen .\",\n \"However , the new nuclei are smaller than normal and their contents are arranged differently .\",\n \"This causes problems with the ability of daughter cells to synthesise protein building blocks and leads to an increased rate of spontaneous cell death when daughter cells try to undergo the next mitosis .\",\n \"Further research is needed to understand why this happens .\"\n]"},"year":{"kind":"string","value":"2014"}}},{"rowIdx":4188,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"Contrasting roles for parvalbumin-expressing inhibitory neurons in two forms of adult visual cortical plasticity"},"id":{"kind":"string","value":"elife-11450-v1"},"sections":{"kind":"list like","value":[["Understanding how brain synapses , cells , and circuits are persistently modified by experience to store information represents one of the great challenges in neuroscience .","Mouse visual cortex has proven to be an excellent model system in which to examine experience-dependent neural response modification .","One robust type of visual response plasticity is reliably elicited in adult ( > P60 ) mice by simply closing one eyelid .","Over the course of 5–7 days of monocular deprivation ( MD ) , the responses in visual cortex evoked by stimulation of the non-deprived eye progressively increase ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .","This deprivation-enabled response potentiation is driven by visual experience through the non-deprived eye , as only the responses through one eye are potentiated ( i . e . , it is input specific ) and it fails to occur if both eyelids are closed or if the animals are kept in a dark room ( Blais et al . , 2008 ) .","There is evidence that the response potentiation is mediated in part by “Hebbian” strengthening of excitatory synaptic transmission in visual cortex , as induction requires cortical NMDA receptor ( NMDAR ) activation ( Sawtell et al . , 2003 ) ( Sato and Stryker , 2008 ) and α-calcium/calmodulin-dependent protein kinase II ( αCAMKII ) expression in principal cells ( Ranson et al . , 2012 ) .","This form of ocular dominance ( OD ) plasticity is likely responsible for the increase in visual acuity that occurs through the non-deprived eye following adult monocular deprivation ( Iny et al . , 2006 ) , and is of particular interest in the context of recovery of brain function after deprivation , disease , or damage ( Cho and Bear , 2010 ) .","Another robust form of visual response plasticity is induced by exposure of awake mice to oriented visual grating stimuli .","Brief daily presentation of a phase-reversing grating of a single orientation causes a large and persistent increase in the peak cortical response to this orientation , as measured by visual evoked potentials ( VEPs ) or unit recordings ( Frenkel et al . , 2006; Cooke et al . , 2015 ) .","This phenomenon is termed stimulus-selective response potentiation ( SRP ) because only responses to the experienced orientation are increased .","Abundant evidence suggests that SRP is also mediated by “Hebbian” mechanisms , particularly those revealed by the study of long-term synaptic potentiation ( Cooke and Bear , 2010; 2014 ) .","The mechanisms utilized for SRP within V1 have also been shown to mediate a fundamental form of long-term visual recognition memory , manifested behaviorally as orientation-selective habituation ( OSH ) ( Cooke and Bear , 2015; Cooke et al . , 2015 ) .","Both monocular deprivation and selective visual experience trigger input-specific increases in the short latency VEP measured in layer 4 of mouse visual cortex and , as reviewed above , both OD plasticity and SRP share some molecular requirements ( e . g . , NMDAR activation ) .","Thus , it came as a surprise that response potentiation after monocular deprivation and SRP do not occlude one another ( Frenkel and Bear , 2004 ) , suggesting that they employ different mechanisms or are expressed by different synapses .","We became interested in the possibility of differential involvement of cortical inhibition mediated by the parvalbumin-expressing ( PV+ ) fast spiking neurons .","PV+ fast-spiking inhibitory neurons comprise the most numerous sub-class of GABAergic cortical neurons ( Xu et al . , 2010 ) and receive substantial feed-forward glutamatergic input from the thalamus ( Cruikshank et al . , 2007 ) .","These interneurons synapse preferentially on the somata and initial axon segments of principal cells , and therefore are in a position to strongly and precisely modulate even short latency visually evoked responses ( Cristo et al . , 2004; Markram et al . , 2004; Kepecs and Fishell , 2014 ) .","Moreover , previous studies have suggested that fast-spiking interneurons participate in the expression of cortical eye dominance and OD plasticity in juvenile mice ( Yazaki-Sugiyama et al . , 2009; Smith and Bear , 2010 ) .","The contribution of this class of neuron to cortical plasticity is also of particular interest given the evidence for cortical PV+ neuron dysfunction in various psychiatric disorders characterized by impaired cognitive function ( Gogolla et al . , 2009 ) .","In the current study we used a variety of approaches to understand the contribution of PV+ neurons to adult OD plasticity and SRP .","We find that neither the pharmacogenetic silencing of PV+ cells nor the specific deletion of NMDARs within them affect the relative eye dominance or the expression of deprivation-enabled potentiation of VEPs in adult mice .","In contrast , and quite unexpectedly , we discovered that the expression of SRP is dependent on PV+ cell activity , and that perturbations of PV+ neuron function , most notably cell-type specific ablation of NMDARs , disrupt the expression of both SRP and its behavioral correlate , familiarity recognition ."],["In order to understand the role of PV+ neurons in the expression of experience-dependent visual cortical plasticity , we selectively inactivated these cells using a Dreadd ( Designer Receptors Exclusively Activated by Designer Drugs ) pharmacogenetic system: Specifically , we expressed a re-engineered G-protein coupled receptor , hM4D ( Gi ) , which is activated exclusively by an otherwise inert small molecule , clozapine-N-oxide ( CNO ) ( Nichols and Roth , 2009 ) .","The binding of CNO to the hM4D ( Gi ) receptor activates intracellular Gi-mediated signaling and subsequent hyperpolarization of the cell in which the receptors are expressed .","We locally expressed hM4D ( Gi ) receptors in PV+ cells of binocular V1 using an adeno-associated viral vector containing a construct for Cre-selective expression ( AAV9-hSyn-DIO-HA-hM4D ( Gi ) -IRES-mCitrine ) in mice that express Cre recombinase only in PV+ cells ( B6;129P2-Pvalbtm1 ( cre ) Arbr/J , PV-Cre ) .","We confirmed the selective expression of hM4D ( Gi ) receptors in PV+ neurons in binocular V1 using immunohistochemistry ( Figure 1A–E ) .","In order to ensure that PV+ neurons could be inactivated by this method , we took slices of V1 for ex vivo intracellular recordings .","Bath application of CNO drastically inhibited current evoked action potential firing of PV+ neurons in Layer 4 ( 2-way repeated measures ANOVA , interaction of CNO x current injection , F ( 8 , 72 ) = 6 . 227 , P < 0 . 001 , n = 10 cells , significant at data points above 100pA: q ( 8 ) = 3 . 716 , P = 0 . 014 ) but had no effect on neighboring non-expressing cells ( Figure 1F–G ) .","Layer 4 recordings in vivo demonstrated that systemic administration of CNO resulted in the elevation of visually-evoked potential ( VEP ) magnitude , and an increase in the firing rate of excitatory single units ( Figure 1H–I ) , which are expected consequences of inactivating PV+ inhibitory neurons in binocular V1 . 10 . 7554/eLife . 11450 . 003Figure 1 . The hM4D ( Gi ) DREADD system locally inactivates parvalbumin+ neurons in binocular primary visual cortex ( V1 ) .","( A ) An example of V1 expression of hM4D ( Gi ) in a parvalbumin ( PV ) -Cre recombinase ( Cre ) mouse infected locally in binocular V1 with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine .","( B ) A DAPI stain for cell nuclei is shown in blue .","( C ) Infected cells expressing hM4D ( Gi ) are labeled in green .","( D ) Immuno-labeled PV+ cells are shown in red .","( E ) The merged image reveals that hM4D ( Gi ) -expressing cells are also PV+ .","( F ) Intracellular current clamp recordings of hM4D ( Gi ) -infected PV+ layer 4 neurons in ex vivo slices of V1 reveal that green-labeled infected cells exhibit a non-adapting fast-spiking phenotype typical of fast-spiking inhibitory neurons ( black ) .","These cells do not fire action potentials in the presence of CNO ( red ) , the exogenous ligand for hM4D ( Gi ) receptors , despite depolarizing current injection .","In contrast , neighboring cells that are not mCitrine+ show no impact of CNO application .","( G ) HM4D ( Gi ) -mediated inactivation of putative fast-spiking PV+ inhibitory neurons is here summarized as the number of action potentials resulting from a given current injection before ( black ) and after CNO application ( red ) .","( H ) The effects of hM4D ( Gi ) -mediated inactivation of putative PV+ fast-spiking inhibitory neurons in vivo are apparent from electrophysiological recordings from V1 of awake , head-fixed mice viewing phase-reversing sinusoidal grating stimuli .","Averaged Visually Evoked Potential ( VEP ) recordings recorded in layer 4 reveal increased VEP magnitude in the presence of CNO ( red ) , relative to pre-CNO ( baseline ) recordings ( black ) , indicative of reduced inhibition .","( I ) Phase reversal-evoked action potentials recorded from neurons in layer 4 also exhibit a similar effect with elevated firing rates in the presence of CNO ( red ) relative to the baseline recording ( black ) .","Labeled scale bars are presented throughout .","Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00310 . 7554/eLife . 11450 . 004Figure 1—source data 1 . Action potential number for current injection . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 004 Mouse binocular V1 is ~2–3 times more strongly activated by the contralateral ( contra ) eye than the ipsilateral ( ipsi ) eye , an inherent bias known as ocular dominance ( OD ) .","We probed the involvement of PV+ neuron activity in maintaining baseline OD .","PV-Cre mice were infected with AAV virus to deliver hM4D ( Gi ) Dreadd receptors to PV+ neurons in binocular V1 ( Figure 2A ) .","Electrodes were simultaneously implanted into layer 4 of V1 for VEP recordings .","VEPs elicited through just contralateral or ipsilateral eyes were acquired consecutively before and after CNO administration ( Figure 2A–B ) .","After CNO injection , VEPs driven through each eye increased significantly in magnitude ( 2-way repeated measures ANOVA , effect of CNO , F ( 7 ) = 75 . 986 , P < 0 . 001; contralateral eye: 222 . 18 ± 14 . 79 µV baseline vs . 679 . 81 ± 64 . 12 µV CNO , Student-Newman-Keuls ( SNK ) post hoc test , q ( 7 ) = 13 . 925 , n = 8 mice , P < 0 . 001; ipsilateral eye: 105 . 56 ± 6 . 78 µV baseline vs . 358 . 63 ± 35 . 79 µV CNO , SNK post hoc test , q ( 7 ) = 7 . 711 , n = 8 mice , P < 0 . 001 , ( Figure 2C ) , consistent with the occurrence of cortical disinhibition .","However , the OD of visual responses in V1 ( contra/ipsi ratio ) was not significantly altered by CNO injection ( 2 . 13 ± 0 . 12 baseline vs . 1 . 96 ± 0 . 18 CNO , student’s paired two-tailed t-test , t ( 7 ) = 0 . 859 , n = 8 mice , P = 0 . 42 , Figure 2D ) .","This result suggests that the inherent OD of binocular visual cortex is not dependent on PV+ neuron activity . 10 . 7554/eLife . 11450 . 005Figure 2 . Inactivation of parvalbumin+ neurons has no impact on expression of ocular dominance ( OD ) or the ocular dominance shift as a result of monocular deprivation ( MD ) in the adult mouse .","( A ) For experiments described in this figure mice were infected across all cortical depths bilaterally in binocular V1 ( green ) .","During the same surgical implantation procedure VEP recording electrodes were also positioned in layer 4 and chronically fixed .","( B ) Mice ( P45-60 ) were infected and implanted with electrodes and then left for 3 weeks for the AAV9 viral vector to reach maximal expression , after which they underwent habituation to head-fixation and a gray screen for two consecutive days .","Following this , on experimental day 0 , mice were presented with an Xo phase-reversing sinusoidal grating stimulus separately to the left and right eye .","VEPs were recorded from each hemisphere in order to determine ocular dominance in binocular V1 .","Mice were then removed from the recording apparatus and , CNO was delivered systemically half an hour prior to undertaking the same recording procedure , this time using an orthogonal X + 90° visual stimulus .","( C ) As is well documented , responses in V1 to stimuli viewed through the contralateral eye ( blue ) were greater in magnitude than those elicited through the ipsilateral eye ( yellow ) , as measured here by VEP magnitude .","After application of CNO ( red outlines ) , VEP magnitude dramatically increased .","( D ) This increase was scaled such that the ratio of Contralateral:Ipsilateral VEP magnitude was maintained before ( white ) and after CNO ( red ) .","( E ) In a second group of mice , a similar experimental protocol was observed prior to measuring ocular dominance by recording VEPs in each hemisphere elicited by an Xo stimulus through each eye .","One hemisphere was then selected and the contralateral eye was sutured closed .","After 7 days of monocular deprivation , the eye was opened and VEPs driven through either eye were again recorded , this time elicited by an X + 60° stimulus .","Mice were then systemically injected with CNO and , half an hour later , VEPs driven through either eye were recorded , this time elicited by an X - 60° stimulus .","( F ) After the adult mice underwent 7 days of MD , there was a significant potentiation of the V1 response to visual input through the ipsilateral eye ( yellow ) .","After application of CNO ( red outlines ) , VEPs driven through each eye were elevated in magnitude , but again the increase in VEP magnitude was scaled .","( G ) As a result of open eye potentiation after MD , the OD ratio shifted dramatically from the contralateral bias of pre MD ( white ) to an almost equal cortical response through contralateral and ipsilateral eyes ( black ) .","This shifted ratio was unaffected by hM4Di-mediated inactivation of putative fast-spiking inhibitory neurons during CNO application ( red ) , indicating that the expression of OD and its shift as a result of MD in adult mice do not require fast-spiking inhibition .","Significant comparisons are labeled with an asterisk and non-significant comparisons with n . s . throughout .","Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00510 . 7554/eLife . 11450 . 006Figure 2—source data 1 . Ocular dominance and deprivation effects are maintained after PV neuron inactivation . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 006 The normal OD ratio is altered in the adult mouse by MD of the contralateral eye over 7 days , resulting in an ocular dominance shift that features potentiation of response through the open ipsilateral eye ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .","In a new group of mice we assayed the effect of PV+ neuron inactivation on the expression of the shift in OD caused by MD .","We recorded VEPs elicited through each eye from PV-Cre mice expressing hM4D ( Gi ) receptors in binocular V1 ( Figure 2E ) .","After baseline recordings the mice underwent contralateral eyelid suture and 7 days of monocular deprivation .","Subsequently , the contralateral ( deprived ) eye was opened and VEPs were re-recorded in order to reveal the expression of an OD shift .","There was a significant potentiation of VEP magnitude driven through the ipsilateral eye following 7 days of MD ( 69 . 5 ± 5 . 81 µV pre MD vs . 139 . 5 ± 9 . 33 µV post MD , n = 10 mice , student’s paired one-tailed t-test , t ( 9 ) = -10 . 184 , P < 0 . 001 , Figure 2F ) .","This potentiation of response through the ipsilateral ( non-deprived ) eye resulted in a significant shift in the OD ratio ( contra/ipsi ratio , 3 . 08 ± 0 . 26 pre MD vs . 1 . 33 ± 0 . 10 post MD , 1-way repeated measures ANOVA , SNK post hoc test , q ( 2 ) = 11 . 77 , P < 0 . 001 , Figure 2G ) .","Mice were then injected with CNO and re-recorded to assess whether the OD shift would persist in the absence of PV+ neuron activity .","CNO injection resulted in an increase in VEP magnitudes ( Figure 2F ) , but importantly the shift in the contra/ipsi ratio was maintained ( 1 . 33 ± 0 . 10 post MD vs . 1 . 35 ± 0 . 18 post MD CNO , SNK post hoc test , q ( 2 ) = 0 . 127 , P = 0 . 93 , Figure 2G ) .","Therefore , the expression of the adult OD shift induced by 7 days of MD persists after a strong reduction in PV+ neuron inhibition .","We next assayed the role of PV+ neurons in the expression mechanism underlying stimulus-selective response potentiation ( SRP ) , a second form of experience-dependent visual cortical plasticity that also manifests as an increase in V1 responses ( Frenkel et al . , 2006; Cooke and Bear , 2010 ) .","Binocular VEPs were recorded from awake , head-fixed adult mice viewing phase-reversing gratings of a particular orientation ( Xo stimulus ) on each of 6 consecutive days ( Figure 3A and B ) .","On the 7th day mice viewed blocks of the now familiar visual stimulus interleaved with blocks of a novel oriented stimulus ( X +/- 60° ) .","To address whether PV+ neuron activity is required for the expression of SRP , familiar and novel oriented grating stimuli were presented before and after mice received CNO ( Figure 3A ) . 10 . 7554/eLife . 11450 . 007Figure 3 . The expression of Stimulus-selective Response Potentiation ( SRP ) requires activity in PV+ neurons in V1 . ( A ) Mice expressing hM4D ( Gi ) receptors in PV+ cells underwent a standard SRP induction protocol .","On day 7 , mice viewed a novel oriented stimulus in addition to the familiar stimulus; before and after CNO injection .","( B ) We acquired binocular VEPs from awake , head-fixed mice elicited by the same full-field oriented sinusoidal grating stimulus over several days .","( C ) As a result of multiple days of experience cortical response was dramatically potentiated such that the familiar stimulus evoked VEPs of significantly greater magnitude than the novel stimulus ( black bars ) .","( D ) After application of CNO , VEPs underwent a notable increase in magnitude as a result of disinhibition ( red bars ) .","Most notably , CNO rendered response to familiar and novel stimuli equivalent in magnitude .","( E ) This lost discrimination of familiar and novel stimuli is reflected as a drop in the ratio of response to familiar and novel stimuli from approximately 2:1 ( black ) to approximately 1:1 ( red ) .","Significant comparisons are labeled with an asterisk and non-significant comparisons with n . s . throughout .","Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00710 . 7554/eLife . 11450 . 008Figure 3—source data 1 . PV Dreadds SRP induction and expression . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00810 . 7554/eLife . 11450 . 009Figure 3—figure supplement 1 . CNO has no impact on SRP expression in WT mice .","( A ) C57BL/6 WT mice were bilaterally infected with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine in binocular V1 .","Because these mice did not express Cre recombinase there was no subsequent expression of hM4D ( Gi ) DREADDS receptors .","After 4 weeks , during which hM4D ( Gi ) was expressed at high levels in PV-Cre mice , WT mice were taken through a standard SRP protocol of electrode implantation , habituation and visual experience of a single oriented grating .","This progressed as usual , resulting in a significant increase in the magnitude of the VEP .","( B ) Expression of SRP was then tested on day 7 by interleaving presentations of familiar and novel orientations and VEPs elicited by the familiar orientation were of significantly greater magnitude than the novel .","After delivery of CNO , VEPs were again tested in response to the familiar orientation and a second novel orientation .","The drug had no impact and a similar degree of significant stimulus selectivity was present .","Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00910 . 7554/eLife . 11450 . 010Figure 3—figure supplement 1—source data 1 . CNO has no effect on SRP expression in WT mice . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 010 As is characteristic of SRP , there was a significant increase in the magnitude of the average VEP evoked by the familiar oriented grating stimulus over days ( Friedman 1-way repeated measures ANOVA on ranks , n = 10 mice , X2 ( 5 ) = 40 . 40 , P < 0 . 001 , Figure 3C ) .","This potentiation was evident by day 2 ( 263 . 3 ± 19 . 96 µV ) in comparison with day 1 ( 184 ± 17 . 13 µV , SNK post hoc test , q ( 9 ) = 4 . 472 , P < 0 . 05 ) .","The stimulus selectivity of VEP magnitude potentiation was apparent on day 7 and significantly affected by inactivating PV+ inhibitory neurons in V1 ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F = 78 . 927 ( 1 , 9 ) , P < 0 . 001 , Figure 3D ) : Prior to delivery of CNO the average VEP magnitude driven by the familiar stimulus ( 324 . 7 ± 22 . 18 µV ) was significantly greater than that driven by a novel oriented stimulus ( 162 . 9 ± 16 . 31 µV , SNK post hoc test , q ( 9 ) = 10 . 709 , P < 0 . 001 ) .","Following injection of CNO to inactivate hM4D ( Gi ) -infected neurons in V1 , there was no longer a significant difference in the magnitude of VEPs driven by the familiar stimulus ( 537 . 2 ± 66 . 69 µV ) compared with a novel stimulus ( 525 . 1 ± 68 . 30 µV , SNK post hoc test , q ( 9 ) = 0 . 804 , P = 0 . 58 ) .","The selectivity of the potentiation to a familiar stimulus can be summarized by plotting the ratio of VEP magnitudes driven by familiar and novel stimuli ( Figure 3E ) .","Mice expressed a significantly larger familiar/novel ratio before CNO injection ( 2 . 11 ± 0 . 17 ) , corresponding to a greater response to the familiar stimulus , compared to after CNO injection ( 1 . 03 ± 0 . 04 , Mann Whitney rank sum test , U = 0 . 00 , P < 0 . 001 ) .","CNO injection did not affect the stimulus selectivity of SRP in wild type animals infected with virus ( Figure 3—figure supplement 1A–B ) .","These animals underwent SRP ( Figure 3—figure supplement 1A ) and on test day showed significantly larger VEPs to the familiar stimulus , both prior to CNO injection ( familiar VEP: 361 . 06 ± 27 . 97 µV , novel VEP: 219 . 25 ± 17 . 9 µV , 2-way repeated measures ANOVA , n = 8 mice , SNK post hoc test , q ( 7 ) = 13 . 618 , P < 0 . 001 ) and following CNO injection ( familiar VEP: 369 . 00 ± 31 . 16 µV , novel VEP: 256 . 75 ± 19 . 59 µV , SNK post hoc test , q ( 7 ) = 10 . 779 , P < 0 . 001 , Figure 3—figure supplement 1B ) .","These results show that the inactivation of PV+ neurons in binocular V1 disrupts the expression of SRP .","Cortical neurons respond within a dynamic range , and it is possible that discrimination of familiar and novel stimuli would be lost as responses approach saturation .","Our observation that OD is maintained after PV+ neuron inactivation ( Figure 2 ) indicates that V1 can still respond selectively to a strong ( contralateral eye ) and weak ( ipsilateral eye ) input .","However , to address the possibility that a “ceiling effect” contributes to the disruption of SRP expression during PV+ cell inactivation , we conducted an additional experiment in which mice viewed sinusoidal grating stimuli across a range of contrast values ( 5 , 10 , 25 , 50 , 100% ) .","VEPs were progressively greater in magnitude the greater the contrast of the viewed stimulus ( 2-way repeated measures ANOVA , n = 4 mice , effect of contrast , F ( 4 , 12 ) = 25 . 908 , P < 0 . 001 ) both before and during PV+ neuronal activation using the hM4D ( Gi ) system ( SNK post hoc test , 5 vs . 100 percent contrast , baseline; q ( 4 ) = 5 . 178 , P = 0 . 013; CNO; q ( 4 ) = 17 . 595 , P < 0 . 001 , Figure 4A ) .","Preservation of the approximately linear relationship of contrast and response during PV+ neuron inactivation indicates that responses have not exceeded their dynamic range . 10 . 7554/eLife . 11450 . 011Figure 4 . Expression of SRP to two separate contrast values is blocked by hM4D ( Gi ) -mediated PV+ neuron inactivation , but differential response to contrast is maintained .","( A ) CNO delivery to PV-Cre mice that had been infected with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine impacted VEP magnitude ( red ) significantly across a range of contrast compared to baseline ( black ) .","( B ) SRP was induced to two differently oriented stimuli , each at different contrast values: 50% ( gray ) and 100% ( black ) .","Modest but significant SRP was expressed at 50% contrast prior to CNO application .","After CNO application ( red outlines ) , VEPs increased significantly in magnitude but were no longer significantly different for familiar and a second novel orientation .","( C ) SRP was also expressed for a different orientation at 100% contrast and , again , VEP magnitude was increased and SRP blocked by delivery of CNO .","( D ) The blockade of SRP expression by CNO at 50% contrast was apparent in the reduction in the familiar/novel ratio for VEP magnitude .","( E ) The blockade of SRP expression at 100% contrast was also observed as a significant drop in familiar/novel ratio of VEP magnitude after CNO delivery .","Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01110 . 7554/eLife . 11450 . 012Figure 4—source data 1 . PV-neuron inactivation does not produce ceiling effect . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 012 We then induced SRP to different orientations in a different set of mice , one stimulus at 50% contrast and the other stimulus at 100% contrast .","After 6 days of SRP at 50% , there was a modest but significant difference in VEP magnitude for familiar ( 188 . 81 ± 11 . 80 µV ) and novel orientations ( 145 . 06 ± 8 . 11 µV , 2-way repeated measures ANOVA , n = 8 mice , SNK post hoc test , q ( 7 ) = 3 . 608 , P = 0 . 023 , Figure 4B ) , just as there was for the familiar ( 259 . 81 ± 17 . 66 µV ) and novel orientations ( 192 . 81 ± 17 . 27 µV , SNK post hoc test , q ( 7 ) = 3 . 793 , P = 0 . 018 , Figure 4C ) at 100% contrast .","SRP expression was abolished by PV+ neuronal inactivation using the hM4D ( Gi ) system at 50% contrast as VEPs elicited by familiar ( 404 . 69 ± 59 . 17 µV ) and novel orientations ( 376 . 94 ± 56 . 79 µV ) were no longer significantly different ( SNK post hoc test , q ( 7 ) = 2 . 289 , P = 0 . 128 , Figure 4B ) .","The same was true at 100% contrast as VEPs evoked by the familiar ( 532 . 44 ± 63 . 82 µV ) and novel orientations ( 514 . 88 ± 40 . 38 µV ) were also no longer significantly different ( SNK post hoc test , q ( 7 ) = 0 . 994 , P = 0 . 494 , Figure 4C ) .","Again , the blockade of SRP expression was also clearly observed in the familiar/novel ratio , which dropped significantly from ( 1 . 31 ± 0 . 05 ) to ( 1 . 08 ± 0 . 08 ) with CNO application at 50% contrast ( student’s paired two-tailed t-test , t ( 7 ) = 2 . 983 , P = 0 . 02 , Figure 4D ) and from ( 1 . 40 ± 0 . 10 ) to ( 1 . 02 ± 0 . 05 ) with CNO application at 100% contrast ( student’s paired two-tailed t-test , t ( 7 ) = 2 . 955 , P = 0 . 021 , Figure 4E ) .","Thus , SRP could still be abolished through selective loss of PV+ neuron activity even at reduced contrasts eliciting submaximal responses .","The disruption of SRP expression by inactivation of PV+ neurons is not a trivial consequence of a “ceiling effect” .","PV+ neurons have been implicated in the sharpening of orientation selectivity in V1 ( Runyan et al . , 2010; Adesnik et al . , 2012; Atallah et al . , 2012; Lee et al . , 2012; Wilson et al . , 2012 ) .","If orientation selectivity were completely abolished by inactivation of PV+ neurons then the loss of SRP expression could be attributed to a failure of orientation discrimination rather than familiarity .","A previous experiment has indicated that activation of PV+ neurons in V1 actually mildly enhances orientation-selectivity and visual discrimination ( Lee et al . , 2012 ) .","Therefore , we used optogenetics to activate PV+ neurons while mice were presented with familiar and novel stimuli to test whether SRP expression would be enhanced , unaffected or disrupted .","PV+ neurons in binocular V1 of PV-Cre mice expressed Channel-rhodopsin 2 ( ChR2 ) as a result of infection with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP .","During the same surgery VEP recording electrodes were implanted and optic fibers chronically implanted to deliver light to the recording site ( Figure 5A ) .","After stable expression 4 weeks after infection , mice were habituated to head-fixation on each of 2 days before undergoing a standard SRP experiment over 6 days ( Figure 5B , C ) .","Mice showed a significant increase in the magnitude of the average VEP evoked by the familiar oriented grating stimulus over days ( Friedman repeated measures ANOVA on ranks , n = 11 mice , X2 ( 5 ) = 24 . 818 , P < 0 . 001 , Figure 5C ) .","This potentiation was evident by day 2 ( 237 . 55 ± 29 . 01 µV ) in comparison with day 1 ( 182 . 59 ± 21 . 97 µV , SNK post hoc test , q ( 10 ) = 7 . 675 , P < 0 . 05 ) .","On day 7 , mice were presented with interleaved blocks of familiar and novel stimuli .","Also interleaved were blocks of familiar and novel stimuli in which blue light ( 473 nm ) was continuously delivered via the optic fiber to the recording site in binocular V1 .","VEPs elicited by the novel X + 90° stimulus were significantly lower in magnitude ( 221 . 34 ± 33 . 55 µV ) than those elicited by the familiar stimulus prior to optogenetic activation of PV+ neurons ( 310 . 70 ± 36 . 70 µV , 2-way repeated measures ANOVA , n = 11 mice , SNK post hoc test , q ( 10 ) = 11 . 799 , P < 0 . 001 , Figure 5D ) .","Optogenetic activation of PV+ neurons significantly diminished the selectivity of SRP , as VEPs elicited by the familiar stimulus ( 181 . 23 ± 26 . 43 µV ) and novel stimulus ( 157 . 80 ± 21 . 05 µV ) were more similar in magnitude ( interaction of stimulus x laser , F ( 1 , 10 ) = 46 . 606 , P < 0 . 001; familiar vs . novel SNK post hoc test , q ( 10 ) = 3 . 094 , P = 0 . 045 , Figure 5D ) .","The reduction of SRP selectivity is most clearly observed in the significant difference in the familiar/novel ratio without ( 1 . 55 ± 0 . 14 ) and with optogenetic stimulation ( 1 . 16 ± 0 . 07 , student’s paired two-tailed t-test , t ( 10 ) = 4 . 423 , P = 0 . 001 , Figure 5E ) .","Laser stimulation did not affect the stimulus selectivity of SRP in wild type animals infected with virus ( Figure 5—figure supplement 1 ) , as there was no significant interaction between stimulus and laser ( 2-way repeated measures ANOVA , F ( 1 , 8 ) = 0 . 677 , P = 0 . 434 ) .","These mice underwent SRP ( Figure 5—figure supplement 1A ) and on test day showed significantly larger VEPs to the familiar stimulus , both with the laser off ( familiar VEP: 273 . 69 ± 28 . 25 µV , novel VEP: 200 . 19 ± 19 . 53 µV , 2-way repeated measures ANOVA , n = 9 mice , SNK post hoc test , q ( 8 ) = 5 . 234 , P = 0 . 005 ) and the laser on ( familiar VEP: 277 . 08 ± 29 . 80 µV , novel VEP: 194 ± 18 . 39 µV , q ( 8 ) = 5 . 916 , P = 0 . 002 , Figure 5—figure supplement 1B ) .","Thus , SRP expression was disrupted with activation of PV+ neurons , just as it was with inactivation ( Figure 3D–E ) .","This result implies that PV+ neurons play a specific role in SRP expression beyond any role in enhancing orientation tuning . 10 . 7554/eLife . 11450 . 013Figure 5 . Optogenetic stimulation of PV+ inhibitory neurons prevents SRP expression .","( A ) Blue light was delivered locally into V1 via optic fibers chronically implanted at a 45° angle to target the VEP recording site in layer 4 of binocular V1 of PV-Cre mice infected with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP .","( B ) Experimental timeline showing that after viral infection , electrode implantation , and ChR2 expression; mice were accustomed to head-fixation and gray screen viewing .","Subsequently , they underwent a standard SRP induction protocol over 6 days .","On day 7 , mice viewed a novel oriented stimulus in addition to the familiar stimulus and , on 50% of presentations of each stimulus , blue light ( 473 nm ) was delivered to cortex to optogenetically activate PV+ cells .","( C ) Significant SRP was induced over 6 days as VEPs underwent a typical potentiation .","( D ) On day 7 , SRP was expressed through significantly larger VEP magnitude in response to the familiar Xo orientation than a novel X + 90° stimulus when blue light was not delivered ( Black bars ) .","In the presence of blue light ( blue bars ) , VEPs were suppressed , and there was a significant reduction in the differential magnitude of VEPs driven by familiar and novel stimuli .","( E ) The ratio of VEP magnitude elicited by familiar/novel stimuli was significantly reduced by optogenetic activation of PV+ neurons , reflecting a decrement in SRP expression .","Significant comparisons are marked with an asterisk and post hoc test p values are reported in D to emphasize the impact of laser stimulation on SRP selectivity .","Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01310 . 7554/eLife . 11450 . 014Figure 5—source data 1 . PV-neuronal activation . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01410 . 7554/eLife . 11450 . 015Figure 5—figure supplement 1 . Blue light has no impact on SRP expression .","( A ) To check that light itself had no effect on cortical physiology in the optogenetic experiments , WT mice were infected bilaterally with AAV5-EF1α -DIO-hChR2 ( H134R ) -eYFP in binocular V1 .","After 4 weeks , during which ChR2 was expressed at high levels in PV-Cre mice , WT mice were taken through the standard SRP protocol described above .","Again , significant SRP occurred .","( B ) On test day , during interleaved presentations of a familiar and novel oriented stimulus together with either blue light ( 473 nm ) or no light stimulation delivery to V1 , VEPs were significantly greater in magnitude for the familiar than the novel stimulus , regardless of the presence of blue light .","Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01510 . 7554/eLife . 11450 . 016Figure 5—figure supplement 1—source data 1 . Laser does not effect VEPs in WT animals . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 016 Multiple lines of evidence indicate that SRP is dependent upon the NMDA class of glutamate receptors ( Frenkel et al . , 2006; Cooke et al . , 2015 ) .","Given the additional clear requirement for normal PV+ inhibitory cell function in SRP ( Figure 3D , E ) we selectively ablated the NMDAR from just PV+ cells by crossing the PV-Cre line of mice with a line in which the mandatory GluN1 subunit of NMDAR is excised by Cre recombinase activity ( B6 . 129S4-Grin1tm2Stl/J , GluN1 fl/fl ) .","The progeny of this cross , in which both alleles of Grin1 ( the gene encoding GluN1 ) were floxed , are henceforth described as either PV-GluN1 KO or Wildtype ( WT ) -GluN1 fl/fl depending on whether , respectively , Cre was expressed or not .","We implanted PV-GluN1 KO ( n = 14 ) and littermate WT-GluN1 fl/fl mice ( n = 17 ) with VEP recording electrodes in layer 4 , binocular V1 .","After recovery and 2 daily sessions of habituation we recorded VEPs elicited by an Xo oriented grating stimulus .","Immediately apparent was the significantly greater basal magnitude of VEPs recorded in the PV-GluN1 mice ( 242 . 45 ± 20 . 20 µV ) relative to their littermate controls ( 130 . 88 ± 9 . 69 µV , student’s two-tailed t-test , t ( 29 ) = -5 . 269 , P < 0 . 001 ) , consistent with the occurrence of disinhibition as a result of reduced glutamatergic drive on cortical PV+ inhibitory cells ( Figure 6A ) .","We then presented the same stimulus to these mice over several consecutive days and observed a significant difference in SRP across genotypes ( 2-way repeated measures ANOVA , interaction of genotype x day , F ( 4 , 116 ) = 3 . 835 , P = 0 . 006 , Figure 6A ) .","Beyond day 3 , VEP magnitudes were no longer significantly different between the PV-GluN1 KO mice ( 289 . 15 ± 24 . 57 µV ) and WT-GluN1 fl/fl littermates ( 239 . 94 ± 20 . 72 µV , SNK post hoc test , q ( 29 ) = 2 . 242 , P = 0 . 119 ) , suggesting an occlusion of SRP by the already elevated basal VEP magnitude in the PV-GluN1 KO mice .","The significant deficit in SRP is clearly apparent when the data are normalized to day 1 values ( 2-way repeated measures ANOVA , interaction of genotype x day , F ( 4 , 116 ) = 12 . 326 , P < 0 . 001 , Figure 6B ) .","Again , a significant deficit in SRP emerged by day 3 in the PV-GluN1 KO mice ( 119 . 26 ± 10 . 13% day 1 ) compared with WT-GluN1 fl/fl littermates ( 183 . 33 ± 15 . 83% day 1 , SNK post hoc test , q ( 29 ) = 4 . 727 , P = 0 . 002 ) , demonstrating that SRP is compromised by a loss of NMDAR expressed in PV+ neurons . 10 . 7554/eLife . 11450 . 017Figure 6 . Loss of NMDA receptors selectively from parvalbumin+ cells impacts SRP but not adult OD plasticity .","( A ) VEPs recorded from mice in which the mandatory GluN1 subunit of the NMDA receptor was genetically ablated from PV+ cells using Cre recombinase technology ( PV GluN1 KO , gray ) were significantly greater in magnitude than those recorded from WT littermates ( black ) , suggesting disinhibition of the visual response .","In these same PV GluN1 KO mice , SRP was also significantly impacted as there is was significantly less gain in magnitude over days of repeated presentation of an X° stimulus than observed in WT littermates .","( B ) This significant reduction in the magnitude of SRP was most clearly observed if VEP magnitude was normalized to the magnitude on day 1 .","( C ) After SRP , both PV GluN1 KO mice and their WT littermates exhibited a significantly greater VEP magnitude elicited by the now familiar stimulus than interleaved presentations of a novel oriented stimulus .","However , consistent with the observed difference in magnitude on day 1 , VEPs elicited by a novel X + 90° stimulus in PV GluN1 KO mice were significantly greater in magnitude than those in WT littermate mice .","No significant difference was observed for VEPs elicited by the familiar stimulus .","( D ) A significant difference in the ratio of VEP magnitude elicited by the familiar and novel stimuli reveals a deficit in SRP expression in PV GluN1 KO mice .","( E ) In contrast , PV GluN1 KO mice exhibited a normal adult OD shift after 7 days of MD , resulting from open eye potentiation ( yellow outlines ) .","( F ) Wild-type ( WT ) littermates exhibited the same significant open eye potentiation ( yellow bars ) after 7 days of MD . ( G ) A comparison of the degree of OD shift as a result of 7 days of MD reveals a significant shift in OD ratio in both genotypes but no difference between genotypes , indicating that NMDA receptors in PV+ cells are not required for induction or expression of the OD shift .","( H ) To confirm genetic ablation of NMDARs selectively from parvalbumin ( PV+ ) neurons in the PV-GluN1 KO; mice were injected with an AAV5 vector to express GFP in a Cre-dependent fashion in PV+ cells only .","After 1 month , fluorescence-guided intracellular recordings were performed from ex vivo slices of visual cortex .","NMDAR-mediated synaptic transmission was normal in excitatory control cells but abolished in PV+ cells .","This is expressed here as the NMDAR EPSC/AMPAR EPSC ratio in excitatory cells ( black outline ) and PV+ cells ( green outline ) .","Sample EPSC traces mediated by the AMPAR ( downward ) and NMDAR ( upward ) are shown at the top of the panel .","Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01710 . 7554/eLife . 11450 . 018Figure 6—source data 1 . PV-GluN1 KODOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 018 We also tested for the stimulus-selectivity of SRP expression by presenting both groups of animals with interleaved blocks of the familiar Xo stimulus and a novel X + 90° stimulus ( Figure 6C ) .","Significant stimulus selectivity was present in both genotypes ( 2-way repeated measures ANOVA , stimulus , F ( 1 ) = 67 . 397 , P < 0 . 001 , interaction of genotype x stimulus , F ( 1 , 29 ) = 2 . 359 , P = 0 . 135 , Figure 6C ) : Although PV-GluN1 KO mice showed significant differences in VEP magnitude for familiar ( 340 . 24 ± 27 . 96 µV ) and novel orientations ( 242 . 54 ± 24 . 40 µV , SNK post hoc test , q ( 13 ) = 6 . 372 , P < 0 . 001 ) the difference was more pronounced in the WT-GluN1 fl/fl mice ( SNK post hoc test , q ( 16 ) = 10 . 254 , P < 0 . 001 ) , in which the familiar stimulus elicited VEPs 318 . 74 ± 25 . 19 µV in magnitude and the novel stimulus elicited VEPs 176 . 06 ± 11 . 55 µV in magnitude .","The familiar stimulus evoked VEPs that were not significantly different in the WT-GluN1 fl/fl mice ( 318 . 74 ± 25 . 19 µV ) and the PV-GluN1 KO mice ( 340 . 24 ± 27 . 96 µV , SNK post hoc test , q ( 29 ) = 0 . 947 , P = 0 . 507 ) but those evoked in the WT-GluN1 fl/fl by the novel stimulus ( 176 . 06 ± 11 . 55 µV ) were significantly lower in magnitude than in the PV-GluN1 KO mice ( 242 . 54 ± 24 . 40 µV , SNK post hoc test , q ( 29 ) = 2 . 926 , P = 0 . 045 ) .","The significant deficit in SRP expression in the PV-GluN1 KO mice is most apparent when the familiar/novel ratio of VEP magnitude ( 1 . 48 ± 0 . 13 ) is compared with the WT-GluN1 fl/fl littermates ( 1 . 83 ± 0 . 13 , Mann Whitney rank sum test , U = 60 . 000 , P = 0 . 020 , Figure 6D ) .","Thus , loss of NMDAR function selectively within PV+ neurons impairs the full expression of SRP .","We also examined if loss of NMDAR from PV+ neurons has an effect on OD plasticity .","We implanted VEP recording electrodes in a second cohort of 11 PV–GluN1 KO and 7 WT-GluN1 fl/fl mice .","After recovery and habituation over 2 days , monocular VEPs were acquired through each eye .","After 7 days of MD , PV-GluN1 KO mice exhibited a significant potentiation of response through the open ipsilateral eye ( 187 . 91 ± 25 . 52 µV ) compared with day 0 ( 90 . 64 ± 10 . 69 µV , 2-way repeated measures ANOVA , SNK post hoc test , q ( 16 ) = 8 . 849 , P < 0 . 001 , Figure 6E ) .","Similarly , WT-GluN1 fl/fl mice also showed a significant potentiation of the open ipsilateral eye-response ( 154 . 57 ± 7 . 60 µV ) after 7 days of MD compared with responses measured on day 0 ( 97 . 57 ± 15 . 85 µV , SNK post hoc test , q ( 16 ) = 4 . 137 , P = 0 . 01 , Figure 6F ) .","Comparisons of OD ratios prior to MD in the adult PV-GluN1 KO ( 3 . 40 ± 0 . 29 ) and WT-GluN1 fl/fl mice ( 2 . 57 ± 0 . 33 ) did not reveal any significant difference ( 2-way repeated measures ANOVA , effect of genotype , F ( 1 ) = 3 . 424 , P = 0 . 083 ) .","Significant shifts in the OD ratio occurred as a result of 7 days of MD in the adult PV-GluN1 KO ( 1 . 28 ± 0 . 33 , SNK post hoc test , q ( 16 ) = 10 . 6 , P < 0 . 001 ) and WT-GluN1 fl/fl mice ( 1 . 19 ± 0 . 09 , SNK post hoc test , q ( 16 ) = 5 . 517 , P = 0 . 001 ) .","The shifted OD ratio also did not differ significantly across genotype ( 2-way repeated measures ANOVA , interaction of genotype and MD , F ( 1 , 16 ) = 2 . 638 , P = 0 . 124 , Figure 6G ) .","Thus , the loss of NMDAR function from PV+ neurons did not have a significant impact on either the induction or the expression of adult OD plasticity , in contrast to SRP .","In order to confirm the removal of NMDARs selectively from PV+ cells , the PV-GluN1 KO mice were injected with an AAV5 vector to express GFP in a Cre-dependent fashion in PV+ cells only .","After 1 month , fluorescence-guided intracellular recordings were performed from ex vivo slices of visual cortex .","NMDAR-mediated synaptic transmission was normal in excitatory control cells but abolished in PV+ neurons ( Figure 6H ) .","This is expressed as significantly reduced NMDAR EPSC/AMPAR EPSC ratio in PV+ cells ( 0 . 040 ± 0 . 011 , n = 8 cells from 5 mice ) compared to neighboring non-fluorescent excitatory control cells ( 0 . 345 ± 0 . 039 , n = 10 cells from 4 mice , student’s one-tailed t-test , t ( 16 ) = -6 . 819 , P < 0 . 001 ) .","A group of non-competitive , open-channel NMDAR blockers , including ketamine , PCP and MK801 are known to have the paradoxical impact of increasing net neuronal activity in the brain .","It is thought that this apparent disinhibition arises from the preferential impact of these molecules on fast-spiking neurons , due to the tonic activation and the increased open-time of NMDAR expressed within these cells ( Homayoun and Moghaddam , 2007; Seamans , 2008 ) .","Interestingly , these compounds are also psychotomimetic and can reproduce , at high sub-anesthetic doses , most of the symptoms of schizophrenia ( Krystal et al . , 1994 ) .","Here we tested the possibility that a single acute dose of one of these substances , ketamine , would have an impact on the expression of SRP due to its action on NMDAR expressed in PV+ fast spiking inhibitory neurons .","We implanted VEP recording electrodes in layer 4 of binocular V1 in a group of 10 C57BL/6 mice .","After recovery and a standard SRP protocol we recorded VEP magnitudes driven by familiar and novel stimuli before , during and 2 days after recovery from systemic injection ( i . p . ) of a high but sub-anesthetic dose of ketamine ( 50 mg/kg ) ( Figure 7A ) .","Ketamine had a significant effect on SRP expression ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F ( 2 , 18 ) = 29 . 479 , P < 0 . 001 , Figure 7B ) .","After SRP but prior to ketamine delivery the familiar Xo stimulus elicited VEPs of significantly greater magnitude ( 245 . 47 ± 21 . 00 µV ) than a novel X + 60° stimulus ( 138 . 86 ± 9 . 32 µV , SNK post hoc test , q ( 9 ) = 9 . 228 , P < 0 . 001 ) in these mice .","After an hour of respite from head-fixation , mice were injected with ketamine and , 15 min later , they were returned to head-fixation and VEP magnitudes were again recorded .","Under the influence of ketamine , the familiar Xo stimulus elicited VEPs of significantly increased magnitude ( 381 . 90 ± 37 . 25 µV ) relative to pre ketamine ( 245 . 47 ± 21 . 00 µV , SNK post hoc test , q ( 9 ) = 7 . 223 , P < 0 . 001 ) .","However , VEPs elicited by a second novel X - 60° stimulus were increased relative to pre-ketamine by an even greater extent ( 397 . 53 ± 37 . 62 µV ) relative to pre ketamine ( 138 . 86 ± 9 . 32 µV , SNK post hoc test , q ( 9 ) = 13 . 695 , P < 0 . 001 ) , such that VEPs were no longer significantly different in magnitude in response to familiar and novel stimuli ( SNK post hoc test , q ( 9 ) = 1 . 353 , P = 0 . 349 ) .","After 2 day’s rest , allowing for complete recovery from drug effects , the mice were returned to the head-fixation apparatus and exposed to the familiar Xo stimulus and a third novel stimulus , X + 90° .","Just as observed prior to the ketamine injection , the Xo stimulus evoked VEPs of significantly greater magnitude ( 267 . 46 ± 26 . 13 µV ) than the novel X + 90° stimulus ( 149 . 40 ± 9 . 85 µV , q ( 9 ) = 10 . 219 , P < 0 . 001 ) ( Figure 7A and B ) . 10 . 7554/eLife . 11450 . 019Figure 7 . Ketamine prevents expression of SRP through blockade of NMDA receptors expressed in parvalbumin+ neurons but does not impact expression of the adult OD shift .","( A ) Mice were bilaterally implanted with VEP recording electrodes in layer 4 of binocular V1 .","After habituation to head-fixation and a gray screen for 2 days , SRP was induced over 4 days by repeatedly presenting sessions of an X° stimulus .","On day 5 , SRP expression was tested by presenting interleaved blocks of the familiar X° stimulus and a novel X + 60° stimulus .","In order to test the acute impact of blocking NMDA receptors on SRP expression , 50 mg/kg of ketamine was then delivered systemically 15 min before re-acquiring VEPs elicited by the familiar X° stimulus and interleaved presentations of a second novel X - 60° stimulus .","Mice were then allowed 2 days recovery and a complete washout of ketamine before re-testing SRP expression by again testing VEP magnitude in response to the familiar X° stimulus and a third novel X + 90° stimulus on day 7 .","( B ) Significant SRP was expressed on experimental day 5 prior to ketamine delivery as the familiar X° stimulus elicited VEPs of greater magnitude than the novel stimulus .","Delivery of 50 mg/kg ketamine ( purple ) had two notable impacts on the VEP: First , the overall magnitude of the VEP increased .","Second , and most importantly , the significant difference in magnitude of VEPs elicited by familiar and novel stimuli was no longer present .","This effect was acute , as SRP expression was again significantly apparent 2 days later .","( C ) The ratio of VEP magnitude elicited by the familiar stimulus over the novel .","This ratio was close to 2 and not significantly different prior to or after recovery from ketamine administration but dropped significantly to approximately 1 during ketamine exposure .","( D ) We tested whether ketamine had a differential impact on VEP magnitude in PV GluN1 KO mice and WT littermate mice .","( E ) In the WT littermate mice ( white bars ) 50 mg/kg ketamine had a significant potentiating effect on VEP magnitude , consistent with our previous observation .","In contrast , ketamine had no significant impact on VEP magnitude in the PV GluN1 KO mice ( gray bars ) .","( F ) The selectivity of ketamine’s impact on the WT mice is observed by comparing the ratio of VEP magnitude during ketamine over baseline , which was significantly greater for WT mice than the PV GluN1 KO mice , in which the ratio was approximately 1 .","( G ) In a separate group of mice , a similar protocol was then used to determine whether the OD ratio is affected by ketamine .","( H ) Ketamine impacted both the VEPs driven through the contralateral eye ( blue ) and ipsilateral eye ( yellow ) equally .","( I ) This scaled effect is demonstrated by a lack of significant difference between OD ratios prior to ( white ) and during 50 mg/kg ketamine ( purple ) .","( J ) We next tested whether ketamine has any impact on the expression of adult OD plasticity by recording VEP magnitudes through either eye in a new group of adult mice before taking them through a standard 7 day MD protocol .","( K ) As anticipated , 7 days of contralateral eye MD induced a significant ipsilateral eye potentiation ( yellow ) and ketamine then further potentiated VEPs elicited through both contralateral ( blue ) and ipsilateral eyes .","( L ) The OD ratio shifts significantly from a ratio heavily biased towards the contralateral eye , to a less biased ratio .","Ketamine administration did not significantly affect the magnitude of the OD shift .","Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01910 . 7554/eLife . 11450 . 020Figure 7—source data 1 . Ketamine impact on cortical plasticity . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 020 This significant effect of ketamine on the discrimination of familiar and novel stimuli is summarized by the ratio of VEP magnitude driven by the familiar/novel stimulus ( 1-way repeated measures ANOVA , F ( 2 , 18 ) = 24 . 683 , P < 0 . 001 , Figure 7C ) .","This familiar/novel ratio dropped significantly from 1 . 76 ± 0 . 10 prior to ketamine to 0 . 96 ± 0 . 02 after ketamine ( SNK post hoc test , q ( 9 ) = 8 . 443 , P < 0 . 001 ) .","Upon recovery , the familiar/novel ratio significantly recovered ( 1 . 79 ± 0 . 16 , SNK post hoc test , q ( 9 ) = 8 . 759 , P < 0 . 001 ) and was not significantly different from the first test after SRP but prior to ketamine injection ( SNK post hoc test , q ( 9 ) = 0 . 316 , P = 0 . 826 ) .","Thus , the psychotomimetic non-competitive NMDAR antagonist ketamine disrupts SRP .","The fact that this is an acute effect on already established SRP that recovers after drug washout indicates that the role for NMDAR in PV+ fast-spiking GABAergic neurons may be in memory retrieval rather than learning .","Ketamine blocks NMDAR expressed in all cell types throughout the CNS and is also known to have targets other than the NMDAR ( Chen et al . , 2009 ) .","In order to determine if ketamine has its effect on V1 responses specifically through NMDAR expressed in PV+ cells , we tested if there was a differential effect of ketamine on VEP magnitude in PV-GluN1 KO mice and WT-GluN1 fl/fl mice .","After a typical implantation and habituation protocol ( Figure 7D ) we tested VEP magnitudes elicited by a novel oriented Xo stimulus in WT-GluN1 fl/fl mice ( 178 . 11 ± 38 . 41 µV , n = 8 ) and PV-GluN1 KO mice ( 260 . 79 ± 50 . 75 µV , n = 8 ) ( Figure 7E ) .","We then removed mice from head-fixation and allowed them to recover in their home-cage before delivering 50 mg/kg ketamine ( i . p . ) .","After 15 min , the mice were returned to head-fixation and we observed the impact of ketamine on VEPs elicited by a novel X + 90° oriented stimulus , which was significantly different in its effect on the two genotypes ( 2-way repeated measures ANOVA , interaction of genotype x treatment , F ( 1 , 14 ) = 18 . 454 , P < 0 . 001 ) .","In the control WT-GluN1 fl/fl mice there was a significant potentiation of VEP magnitude as a result of ketamine application ( 386 . 65 ± 70 . 75 µV ) in comparison to pre treatment ( 178 . 11 ± 38 . 41 µV , SNK post hoc test , q ( 7 ) = 8 . 505 , P < 0 . 001 , Figure 7E ) , replicating our previous finding ( Figure 7B ) .","However , in the PV-GluN1 KO mice , ketamine had no ostensible significant impact ( 258 . 67 ± 44 . 86 µV ) in comparison to pre treatment ( 260 . 79 ± 50 . 75 µV , SNK post hoc test , q ( 7 ) = 0 . 087 , P = 0 . 952 ) .","A ratio of VEP magnitudes pre and post ketamine treatment reveals the significant difference between ketamine’s action on WT-GluN1 fl/fl mice ( 2 . 40 ± 0 . 25 ) and PV-GluN1 KO mice ( 1 . 12 ± 0 . 13 , student’s two-tailed t-test , t ( 14 ) = 4 . 610 , P < 0 . 001 ) ( Figure 7F ) .","Thus , while ketamine has a wide range of effects in the CNS , it exerts itself on the response of V1 to visual input selectively through NMDAR expressed in PV+ cells .","We then tested whether or not ketamine would disrupt either the OD ratio or the shift induced by 7 days of MD in the adult mouse .","Again , C57BL/6 mice ( n = 8 ) were implanted with VEP electrodes and taken through a standard surgery recovery and habituation protocol before measuring the OD ratio prior to 50 mg/kg ketamine and 15 min after ketamine delivery ( Figure 7G and H ) .","The normal contra/ipsi OD ratio exhibiting contralateral eye dominance prior to ketamine ( 2 . 84 ± 0 . 18 ) was not significantly altered by ketamine ( 2 . 61 ± 0 . 19 , student’s paired two-tailed t-test , t ( 7 ) = 0 . 871 , P = 0 . 413 ) ( Figure 7I ) .","A separate group of mice ( n = 11 ) then underwent a standard 7 day MD protocol .","VEPs elicited by an Xo oriented stimulus were recorded at baseline , followed by the deprivation period .","After eye opening VEP magnitudes were re-tested with a novel X + 60°stimulus ( the standard protocol to avoid contamination of the OD shift by SRP [Frenkel and Bear , 2004] ) .","These mice were then tested again with a second novel X - 60°stimulus after 1 hr rest and an additional 15 min after systemic 50 mg/kg ketamine administration ( Figure 7J ) .","As expected , VEPs elicited through the open ipsilateral eye ( 93 . 73 ± 12 . 85 µV ) were significantly potentiated by 7 days of MD in the adult mouse ( 155 . 73 ± 15 . 86 µV , 2-way repeated measures ANOVA , SNK post hoc test , n = 11 mice , q ( 10 ) = 7 . 102 , P < 0 . 001 ) , reflecting the well-documented OD shift .","These same ipsilateral VEPs were then further potentiated by ketamine administration ( 232 . 03 ± 22 . 19 µV , SNK post hoc test , q ( 10 ) = 8 . 741 , P < 0 . 001 ) .","However , ketamine had a similar significant potentiating effect on the contralateral VEP ( 279 . 07 ± 14 . 53 µV ) , relative to pre-ketamine ( 180 . 14 ± 11 . 22 µV , SNK post hoc test , q ( 10 ) = 11 . 333 , P < 0 . 001 , Figure 7K ) , suggesting a uniform scaling of response through the two eyes .","This observation is confirmed by the fact that the OD ratio , significantly shifted from ( 2 . 84 ± 0 . 29 ) to ( 1 . 23 ± 0 . 11 , Friedman repeated measures ANOVA on ranks , n = 11 mice , X2 ( 2 ) = 16 . 545 , p<0 . 001 , SNK post hoc test , q ( 10 ) = 5 . 126 , P < 0 . 05 ) by 7 days of MD , was not further significantly affected by delivery of ketamine ( 1 . 28 ± 0 . 09 , SNK post hoc test , q ( 10 ) = 0 . 426 , P > 0 . 05 , Figure 7L ) .","Thus , while ketamine prevents SRP expression through action on NMDAR expressed in PV+ cells , it does not significantly affect the adult OD shift after 7 days of MD , consistent once again with PV+ cells contributing to the expression of SRP but not adult OD plasticity .","Given the clear involvement of PV+ neurons in the expression of SRP we wanted to determine if loss of PV+ neuronal function local to V1 would have any behavioral impact .","Head-restrained mice viewing a phase reversing visual grating stimulus are known to exhibit a stereotyped motor response called a visually-induced fidget , or vidget ( Cooke et al . , 2015 ) .","Vidgets can be measured via a piezoelectric sensor located beneath the forepaws of the mouse ( Figure 8A ) .","Importantly , it has been shown that the magnitude of the vidget response is inversely correlated to the familiarity of the visual stimulus .","That is , a visual stimulus that is very familiar to the animal will on average evoke a relatively weak vidget behavioral response .","In contrast , the presentation of a novel stimulus will on average evoke a vidget of significantly greater magnitude .","Therefore , this behavioral response reflects the ability of the animal to discriminate and respond to a novel visual stimulus in its environment .","Importantly , genetic and pharmacological manipulations local to V1 that inhibit SRP also disrupt the behavioral discrimination of familiar and novel stimuli ( Cooke et al . , 2015 ) , demonstrating that this differential vidget response to familiar and novel stimuli is dependent on the plasticity in visual cortex .","Since PV+ neuron inactivation disrupted the expression of SRP , we tested whether this manipulation would likewise disrupt visual novelty detection . 10 . 7554/eLife . 11450 . 021Figure 8 . Discrimination of familiar and novel oriented stimuli involves PV+ neurons in V1 and NMDAR expressed within PV+ neurons .","( A ) Using the same protocol as described in Figure 3B , mice were progressively familiarized with a specific oriented stimulus .","On the test day , as mice viewed familiar and novel stimuli , vidget behavioral responses were measured via a piezoelectric sensor located beneath the forepaws of the head-fixed mouse .","( B ) After a standard SRP protocol , mice expressing hM4D ( Gi ) receptors selectively within PV+ cells of binocular V1 were exposed to both familiar and novel stimuli .","Prior to application of CNO , mice exhibited significant behavioral evidence of discriminating this familiar orientation from interleaved presentations of a novel oriented stimulus ( black bars ) .","After application of CNO , there was no longer successful discrimination of familiar and novel stimuli ( red bars ) .","Averaged vidget responses are displayed at the top of this panel .","( C ) A significant difference was observed in the ratio of response to familiar and novel stimuli from pre CNO ( black ) to post CNO ( red ) .","( D ) A deficit in OSH was also apparent in PV GluN1 KO mice as vidget recordings demonstrated a failure to significantly discriminate familiar from novel orientations ( gray bars ) .","WT littermates exhibited significantly greater vidget magnitudes for novel than familiar stimuli , indicating unimpaired discrimination of familiarity from novelty ( white bars ) .","Averaged behavioral responses are displayed above with accompanying scale bars .","( E ) The significant deficit of PV GluN1 KO mice in discriminating familiar from novel stimuli is apparent in the ratio of behavior elicited by the familiar over the novel stimulus in comparison to WT littermates .","Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02110 . 7554/eLife . 11450 . 022Figure 8—source data 1 . Novelty detection after PV+ neuronal disruption . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02210 . 7554/eLife . 11450 . 023Figure 8—figure supplement 1 . Cumulative distributions of average vidget behavioral response to familiar and novel stimuli for each individual animal included in analysis presented in Figure 8D and Figure 8E .","( A ) Prior to CNO administration , average behavioral response to the familiar stimulus ( light gray ) and the novel stimulus ( black ) of each individual animal bilaterally expressing hM4D ( Gi ) in PV+ neurons of binocular V1 .","Dotted line represents behavior no greater than pre stimulus baseline .","( B ) Average behavioral response of the same animals to the familiar and a new novel stimulus during PV+ neuron inactivation via CNO delivery , revealing consequent deficit in discrimination of familiar and novel stimuli .","( C ) Cumulative distributions of average vidget behavioral response to the familiar stimulus ( light gray ) and the novel stimulus ( black ) of each individual WT GluN1 fl/fl mouse .","( D ) Average vidget behavioral response of each PV-GluN1 KO mouse to the familiar and a new novel stimulus , revealing deficit in discrimination of familiar and novel stimuli .","Dotted line represents behavior no greater than pre stimulus baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02310 . 7554/eLife . 11450 . 024Figure 8—figure supplement 1—source data 1 . Per animal plots of Novelty detection after PV+ neuronal disruption . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 024 A group of PV-Cre mice expressing hM4D ( Gi ) receptors in PV+ cells ( n = 19 ) , underwent a SRP protocol similar to the previous experiment ( Figure 3A ) in which mice viewed phase-reversing gratings of a particular orientation ( Xo stimulus ) each day for 6 days .","On the 7th day mice viewed blocks of the now familiar visual stimulus interleaved with blocks of a novel oriented stimulus ( X + 60° ) .","On day 7 , vidget behavioral responses were acquired via a piezoelectric device situated underneath the forepaws of the mice ( Figure 8A ) , in order to measure the animal’s discrimination of familiar and novel stimuli .","On day 8 , PV+ cells in V1 were then inactivated by systemic delivery of CNO ( i . p . ) and vidget responses were acquired to the familiar Xo stimulus and a second X - 60° novel stimulus .","Inactivation of PV+ neurons in V1 significantly affected stimulus discrimination ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F ( 1 , 18 ) = 13 . 644 , P = 0 . 002 , Figure 8B ) : Prior to CNO , mice exhibited significantly larger vidget responses to the novel visual stimulus ( 3 . 64 ± 0 . 32 a . u . ) than the familiar stimulus ( 1 . 95 ± 0 . 26 a . u . , SNK post-hoc test , q ( 18 ) = 9 . 237 , P < 0 . 001 ) .","During PV+ cell inactivation in V1 , behavioral responses to the familiar ( 2 . 01 ± 0 . 16 a . u . ) and novel visual stimuli ( 2 . 46 ± 0 . 22 a . u . ) were no longer significantly different ( SNK post-hoc test , q ( 18 ) = 2 . 503 , P = 0 . 086 ) .","The deleterious effect of PV+ cell inactivation in V1 on the animal’s ability to discriminate familiar and novel stimuli is most obvious when a ratio of response to familiar/novel visual stimuli is calculated .","Prior to CNO delivery this ratio was significantly lower ( 0 . 54 ± 0 . 04 ) than during CNO treatment ( 1 . 00 ± 0 . 15 , Wilcoxon signed rank test , W = 138 . 000 , Z = 2 . 777 , P = 0 . 004 , Figure 8C ) .","These findings indicate that PV+ neuron activity is required not only for the expression of SRP , but also for visual novelty detection .","Finally , given the observation that the discrimination of familiar and novel visual stimuli is diminished by inactivation of PV+ neurons in V1 , we tested whether a similar failure would occur in the PV-GluN1 KO mice ( n = 17 ) compared with WT-GluN1 fl/fl littermates ( n = 15 , 2-way repeated measures ANOVA , stimulus , F ( 1 ) = 14 . 632 , P < 0 . 001 , Figure 8D ) .","In the PV-GluN1 KO mice , the familiar stimulus produced less behavioral response ( 2 . 75 ± 0 . 42 a . u . ) than a novel stimulus ( 3 . 67 ± 0 . 79 a . u . ) .","However , the difference was not significant ( SNK post hoc test , q ( 16 ) = 2 . 570 , P = 0 . 079 ) .","In comparison , the concurrently tested WT-GluN1 fl/fl littermate mice showed a suppression of behavioral response to the familiar stimulus ( 1 . 76 ± 0 . 26 a . u . ) relative to the novel stimulus ( 4 . 08 ± 0 . 51 a . u . ) that was significant ( SNK post hoc test , q ( 14 ) = 5 . 008 , P = 0 . 001 ) .","This observation was reinforced by a comparison of the ratio of behavior produced by familiar and novel stimuli ( Figure 8E ) , in which there was a significant difference between the PV-GluN1 KO mice ( 0 . 59 ± 0 . 11 ) and their WT-GluN1 fl/fl littermates ( 0 . 94 ± 0 . 14 , student’s one-tailed t-test , t ( 30 ) = -1 . 992 , P = 0 . 028 ) , reflective of the deficit in discrimination of familiar and novel stimuli as a result of lost NMDAR function in PV+ cells .","Individual animals’ average vidget responses to familiar and novel stimuli ( Figure 8—figure supplement 1 ) reveal significantly decreased stimulus selectivity subsequent to CNO administration ( Figure 8—figure supplement 1A–B ) , and in the PV GluN1 KO mice as compared to WT-GluN1 fl/fl littermate controls ( Figure 8—figure supplement 1C–D ) .","Thus , not only do NMDAR in PV+ cells contribute to SRP expression but they are also involved in its behavioral correlate ."],["There has been a long-standing interest in the possible roles of PV+ neurons in juvenile OD plasticity .","These include modulation of the sensitivity of visual cortex to the effects of MD ( Fagiolini et al . , 1994; Hanover et al . , 1999; Huang et al . , 1999; Chattopadhyaya et al . , 2004 ) and the functional expression of the OD shift ( Maffei et al . , 2006; Yazaki-Sugiyama et al . , 2009; Smith and Bear , 2010 ) .","Our findings indicate that expression of OD plasticity in adult mice does not require participation of the PV+ neurons .","In interpreting the current findings , it is important to recognize how juvenile and adult OD plasticity differ .","In juvenile mice ( < ~P35 ) , a rapid and reliable consequence of MD is depression in cortex of responses mediated by the deprived eye .","This is followed by a progressive compensatory increase in responses through the non-deprived eye ( Frenkel and Bear , 2004 ) .","In adult mice maintained under standard laboratory conditions , depression of deprived-eye responses can be a weak and variable consequence of MD , but potentiation of the non-deprived eye still occurs reliably ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .","This deprivation-enabled response potentiation is not mediated by homeostatic synaptic scaling because it requires visual experience through the non-deprived eye ( Blais et al . , 2008 ) , activation of cortical NMDA receptors ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) , and is unaffected ( in adults ) by genetic deletion of TNFα that abolishes scaling ( Ranson et al . , 2012 ) .","These features are consistent with an alternative hypothesis that the adult OD shift occurs because deprivation of the dominant contralateral eye causes a metaplastic shift in the LTP threshold , enabling visual experience through the weaker ipsilateral eye to drive “Hebbian” synaptic strengthening ( Cooper and Bear , 2012 ) .","This interpretation is supported by evidence that light deprivation promotes LTP in visual cortex ( Kirkwood et al . , 1996; Philpot et al . , 2001; 2003 ) and that αCaMKII mutants that lack LTP also lack adult OD plasticity ( Ranson et al . , 2012 ) , and it is compatible with our finding that expression of the adult OD shift is not dependent on the activity of PV+ inhibitory neurons .","Saiepour and colleagues also recently observed that ocular dominance index ( ODI ) values of V1 neurons are not altered by PV+ interneuron suppression in monocularly deprived adult mice ( Saiepour et al . , 2015 ) .","Using single unit recordings they first showed that monocular deprivation caused a shift in ODI values towards the non-deprived eye , as expected .","They then tested the dependence of this shift on PV+ cell activity by measuring ODI values of V1 neurons during optogenetic suppression of PV+ cells .","This optogenetic-suppression did not alter the deprivation-induced shift in ODI values .","Our results using VEP recordings and PV+ suppression via hM4Di Dreadd receptors are in agreement with these results from Saiepour and colleagues , demonstrating that suppression of PV+ cell activity after deprivation does not interfere with the expression of the adult OD shift .","Although the findings that PV+ neurons are not necessary for expression of OD plasticity do not rule out a modulatory role in the induction mechanisms , we note that the adult OD shift still occurred in mutant mice in which PV+ neurons lack NMDARs .","In contrast to the adult OD shift , we found that the expression of SRP relies on PV+ neuron circuitry .","This finding came as a surprise , as SRP reflects the potentiation of short-latency synaptic currents and increased spiking activity in layer 4 ( Cooke et al . , 2015 ) , and previous experiments had strongly indicated that SRP might be considered a naturally occurring form of LTP .","For example , induction of SRP is input-specific , dependent upon NMDA receptor activation and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ( AMPAR ) insertion in principal cells ( Frenkel et al . , 2006 ) , and reversed by infusion of the zeta-inhibitory peptide ( ZIP ) that also erases LTP ( Cooke and Bear , 2010 ) .","Furthermore , LTP elicited by theta-burst stimulation of the thalamus occludes and is occluded by SRP ( Cooke and Bear , 2010 ) .","Altogether , these findings suggest a modification of feed-forward excitatory synaptic transmission .","Before assigning a specific role to inhibition in the expression of SRP , two relatively trivial explanations must be addressed .","The first is that the loss of discrimination of familiar and novel orientations could be accounted for simply by a failure of orientation selectivity and not by a failure of memory .","Throughout the experiments presented here we have used orientations that are at least 60° and often 90° different , meaning that orientation discrimination should be as unchallenging as possible .","Given the modest impact on orientation tuning of suppressing PV+ interneuron activity in V1 ( Runyan et al . , 2010; Adesnik et al . , 2012; Wilson et al . , 2012 ) we believe that this explanation is improbable .","However , we took a direct approach to testing this possibility by stimulating activity in PV+ cells using channelrhodopsin-2 ( ChR2 ) while mice are viewing familiar and novel oriented stimuli .","Stimulation of PV+ inhibition has been shown to actually mildly enhance orientation-selectivity ( Lee et al . , 2012 ) and yet our experiments reveal that the difference between visual cortical response to familiar and novel orientations is significantly diminished by activation PV+ neurons .","This result casts PV+ neurons in a very specific role in the recognition of familiar stimuli in addition to modest participation in orientation-selectivity .","A second concern is that , after the suppression of PV+ inhibition , V1 may be operating outside the dynamic range where discrimination is possible .","However , we show that the cortex responds to input from the contralateral eye with a response of approximately double the magnitude of that elicited by input through the ipsilateral eye even after silencing the PV+ cells , suggesting that the response of the cortex is not saturated .","We also find that PV+ neuron silencing affects SRP even when it is induced using lower contrast stimuli that evoke submaximal responses .","Moreover , it is also worth noting that less specific pharmacological approaches to suppressing inhibition in V1 ( Khibnik et al . , 2010 ) result in much greater magnitude of response to visual input than we observe here for PV+ neuron inactivation , PV-GluN1 KO or ketamine application , indicating that our manipulations do not encroach on a physiological ceiling .","One relatively simple explanation for the current findings is that SRP results from lasting homosynaptic ( i . e . input-specific ) long-term depression ( LTD ) of glutamatergic input to PV+ inhibitory neurons .","Thereafter , presentation of a familiar stimulus evokes a greater response in V1 due to selective disinhibition of cortical circuitry .","This hypothesis is consistent with our observation that SRP is deficient if NMDARs are no longer expressed on PV+ cells .","However , because we used a non-reversible genetic modification in our initial PV-GluN1 KO experiment , we were initially unable to show if these receptors were important for the storage of information or the retrieval of information already stored .","Ketamine , which among other actions serves as a non-competitive NMDAR antagonist , has been shown to preferentially antagonize NMDARs on PV+ cells ( Homayoun and Moghaddam , 2007; Seamans , 2008 ) .","By using ketamine to induce a temporary blockade of these receptors , which we confirmed by showing that there was no impact of ketamine on V1 responses in the PV-GluN1 KO mice , we were able to demonstrate that interference with NMDARs on PV+ cells disrupts expression of SRP even after it has been induced and saturated .","Importantly , acute application of ketamine did not affect the maintenance or stability of stored information , because a strong bias for the familiar stimulus returned after ketamine washout .","If SRP is indeed accounted for by homosynaptic depression of excitatory synapses carrying information about the familiar stimulus orientation , this LTD would need to be expressed by a synapse-specific reduction in NMDAR-mediated responses in order to account for these findings .","However , this simple model is difficult to reconcile with the findings reviewed above that SRP depends on LTP-like mechanisms , e . g . , NMDAR-dependent AMPAR delivery in principal cells .","Considered together , the findings to date indicate that induction and maintenance of SRP occur by modification of excitatory synapses onto excitatory neurons , but that expression of SRP is mediated by stimulus-selective modulation of PV-cell activity in V1 .","Obviously a polysynaptic circuit is required to yield these properties , and dissecting this essential cortical circuitry will require much more work .","However , it is interesting to note a recent study by Makino and Komiyama who showed , in addition to a stimulus-selective reduction in PV+ neuron activity in V1 as mice were repeatedly exposed to a specific visual stimulus , an experience-dependent increase in the responsiveness of somatostatin-positive interneurons ( Makino and Komiyama , 2015 ) .","This result is particularly interesting as it is known that many interneuron types innervate one another and , therefore , that the activity of these cell types are interdependent .","It is not difficult to sketch circuits in which stimulus-selective , feed-forward polysynaptic inhibition of PV+ cells accounts for SRP expression .","Clearly , experiments aimed at teasing apart the involvement of other interneuron types in SRP should now be a high priority .","We note that other studies have suggested that PV+ neurons may be important for plasticity following repeated exposure to sensory stimuli .","For instance , Meyer and colleagues , studying infero-temporal cortex ( IT ) in monkeys , showed that familiarity resulted in a sharpening of cortical responses to dynamic visual stimuli , which was likely orchestrated by local fast-spiking putative PV+ cells ( Meyer et al . , 2014 ) .","Additionally , mice constitutively lacking neuronal activity-regulated pentraxin ( NARP ) , a synaptic protein specifically enriched at excitatory synapses on PV+ cells , displayed a reduction in excitatory synapses onto PV+ cells and impairment in SRP-like effects ( Chang et al . , 2010; Gu et al . , 2013 ) .","Intriguingly , however , the NARP KO mice also failed to exhibit both juvenile and adult OD plasticity .","In juvenile animals , it is well established that reducing inhibition can impair induction of OD plasticity ( Ramoa et al . , 1988 ) and delay the developmental decline in the mechanism of deprived-eye depression ( Hanover et al . , 1999; Huang et al . , 1999 ) .","We speculate that the loss of OD plasticity in the adult NARP KO may be related to disruption of the developmental switch from juvenile to adult-type OD plasticity ( Heimel et al . , 2011 ) .","Our findings indicate that although open-eye potentiation as a result of MD in the adult and SRP are superficially similar , the expression mechanisms of these two forms of plasticity are quite distinct .","Based on evidence provided here as well as by others ( Sawtell et al . , 2003; Ranson et al . , 2012; Saiepour et al . , 2015 ) , the deprivation enabled potentiation of the open eye is consistent with a potentiation of synapses between excitatory cells and is independent of PV+ cell inhibition .","However , further work will be necessary to confirm that open eye potentiation in the adult animal is expressed by synaptic alterations between excitatory cells , and to determine whether these processes are present at the level of thalamocortical synapses or intracortical synapses .","SRP correlates with OSH ( Cooke et al . , 2015 ) , a form of visual learning which is dependent upon NMDARs in V1 , and which enables the animal to make the critically important discrimination between familiarity and novelty .","We have shown here that modulation of PV+ neuron activity in V1 is required for this selective recognition of familiarity and consequent novelty detection .","Loss of NMDAR function in these same cells also impairs familiarity recognition .","The cognitive symptoms of schizophrenia , and other psychiatric disorders , are characterized by deficits in habituation and familiarity ( Braff et al . , 1995; Ramaswami , 2014 ) and individuals with schizophrenia display impairment in visual cortical plasticity ( Çavuş et al . , 2012 ) .","Dysfunction of PV+ inhibitory neurons and the NMDARs expressed on PV+ cells have also been implicated in these same psychiatric disorders through a range of approaches ( Lewis et al . , 2005; Gogolla et al . , 2009; Lewis et al . , 2011 ) .","This notably includes observations of the profound psychotomimetic effect in humans of non-competitive NMDAR antagonists such as ketamine ( Krystal et al . , 1994; Krystal , 2015 ) , a substance that , as we show here , prevents SRP expression .","We suggest that understanding the cortical physiology underlying the processes of familiarity recognition and novelty detection may yield great insight into dysfunction underlying some symptoms of these disorders and that , because these processes are as important to mice as they are to humans , they are likely to be experimentally tractable ."],["All procedures adhered to the guidelines of the National Institutes of Health and were approved by the Committee on Animal Care at MIT , Cambridge , MA , USA .","For all experiments mice were male , aged between P60-90 and on a C57BL/6 background ( Charles River laboratory international , Wilmington , MA ) .","They were housed in groups of 2–5 with food and water available ad libitum and maintained on a 12 hr light-dark cycle .","For hM4D ( Gi ) experiments , mice were Parvalbumin-Cre recombinase knock-in mice ( B6;129P2-Pvalbtm1 ( cre ) Arbr/J , PV-Cre ) on a C57BL/6 background .","For GluN1 knockdown experiments , these PV-Cre mice were bred with homozygous mice in which the Grin1 gene , which encodes the GluN1 sub-unit was flanked by LoxP sites ( B6 . 129S4-Grin1tm2Stl/J , GluN1 fl/fl ) , enabling Cre-dependent ablation of this mandatory subunit of the NMDA receptor .","Multiple generations were required to set up crosses yielding offspring that were homozygous GluN1 fl/fl and Cre-expressing .","Of these approximately 50% expressed Cre and 50% served as wild-type littermates on the GluN1 fl/fl background .","All experiments were conducted blind to genotype and treatment .","Mice were first injected with 0 . 1 mg/kg Buprenex sub-cutaneously ( s . c . ) to provide analgesia .","They were then anesthetized with an intraperitoneal ( i . p . ) injection of 50 mg/kg ketamine and 10 mg/kg xylazine .","Prior to surgical incision , 1% lidocaine hydrochloride anesthetic was injected under the mouse’s scalp .","The skull was then cleaned with iodine and 70% ethanol .","A steel head post was affixed to the skull anterior to bregma using cyanoacrylate glue .","Burr holes ( < 0 . 5 mm ) were then drilled in the skull over binocular V1 ( 3 . 1 mm lateral of lambda ) .","Tapered tungsten recording electrodes ( FHC , Bowdoinham , ME , US ) , 75 μm in diameter at their widest point , were implanted in each hemisphere , 450 μm below cortical surface .","Silver wire ( A-M systems , Sequim , WA , US ) reference electrodes were placed over prefrontal cortex .","Mice were allowed to recover for at least 24 hr prior to head-fixation .","For hM4D ( Gi ) experiments , we infected V1 of P30-60 PV-Cre mice or wild-type littermates with an AAV9-hSyn-DIO-HA-hM4D ( Gi ) -IRES-mCitrine virus ( UNC viral core – generated by Dr . Brian Roth’s laboratory ) and for optogenetic experiments we infected mice from the same line with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP ( UNC viral core – generated by Dr . Karl Deisseroth’s laboratory ) .","Using a glass pipette and nanoject system ( Drummond scientific , Broomall , PA , US ) , we delivered 81 nl of virus at each of 3 cortical depths: 600 , 450 , and 300 µm from the cortical surface , and allowed 5 min between re-positioning for depth .","Mice were allowed 3–4 weeks recovery for virus expression to peak before experiments were initiated .","Clozapine-N-oxide ( CNO , Enzo Life Sciences ) was diluted in saline and injected i . p . at a dosage of 5 mg/kg 30 min prior to stimulus delivery .","Ketamine hydrochloride ( Vedco ) was diluted in water and delivered i . p . at 50 mg/kg 15 min prior to stimulus delivery .","Visual stimuli consisted of full-field , 100% contrast , sinusoidal gratings that were presented on a computer monitor .","Visual stimuli were generated using custom software ( http://bearlab-s1 . mit . edu/supp6/PlxStimOne_Opto . zip ) written in either C++ for interaction with a VSG2/2 card ( Cambridge Research systems , Kent , U . K . ) or Matlab ( MathWorks , Natick , MA , U . S . ) using the PsychToolbox extension ( http://psychtoolbox . org ) to control stimulus drawing and timing .","The display was positioned 20 cm in front of the mouse and centered , thereby occupying 92° × 66° of the visual field .","Visual stimuli consisted of full-field sinusoidal grating stimuli phase reversing at a frequency of 2 Hz .","Mean stimulus luminance was 27 cd/m2 .","Grating stimuli spanned the full range of monitor display values between black and white , with gamma-correction to ensure constant total luminance in both gray-screen and patterned stimulus conditions .","For data described in Figures 1–3 and 6 , experiments were fully automated and each stimulus block consisted of 200 phase reversals with 30-s intervals between each stimulus presentation , during which the screen was gray but of equivalent luminance .","For experiments described in Figures 4 and 5 stimulus blocks were also 200 phase reversals in length but each was triggered directly by the experimenter , meaning that the interval was approximately 30-s .","Throughout , stimulus orientation varied such that a novel orientation was always a minimum of 25° different from any experienced previously by the individual mouse and was never within 20° of horizontal because these orientations are known to elicit VEPs of greater magnitude than vertical or oblique stimuli .","If more than 1 orientation was shown within a session , stimuli were pseudo-randomly interleaved such that 3 consecutive presentations of the same stimulus never occurred .","For monocular presentation an occluding paddle was positioned in front of one eye to limit stimulus presentation to the opposite eye .","VEP recordings were conducted in awake , head-restrained mice .","Prior to recording , mice were habituated to the restraint apparatus by sitting in situ in front of a gray screen for a 30-minute session on each of two consecutive days .","For experiments in which monocular VEPs were subsequently acquired , mice were also habituated to the occluding paddle positioned in front of each eye .","On stimulus presentation days , mice were presented with 5 blocks of 200 phase reversals of each oriented stimulus separated by gray screen presentation for ~30 s .","For monocular presentations , recordings were conducted in sequence for each eye .","VEP magnitude was then quantified by measuring trough-peak response magnitude averaged over all phase reversals .","Recordings presented in Figures 1–3 and 6 were amplified and digitized using the Recorder-64 system ( Plexon Inc . , Dallas , TX ) .","Two recording channels were dedicated to recording EEG/VEPs from V1 in each implanted hemisphere and a third recording channel was reserved for the Piezo-electrical input carrying the behavioral information .","Recordings presented in Figures 4 and 5 were amplified using DAM80 amplifiers ( World Precision Instruments , Florida , U . S . ) and digitized using a custom National Instruments system ( National Instruments , Texas , U . S . ) ) .","Local field potential was recorded from V1 with 1 kHz sampling and a 500 Hz low-pass filter .","Data was extracted from the binary storage files and analyzed using custom software ( http://bearlab-s1 . mit . edu/supp6/VEP_Analysis . zip ) written in C++ , Matlab and Labview .","VEPs were averaged across all phase reversals within a block and trough-peak difference measured during a 200-millisecond period from phase reversal .","For experiments concerning Dreadd receptor expression in PV+ cells presented in Figure 1: Three weeks after infection with AAV9-hSyn-DIO- HA-hM4D ( Gi ) -IRES-mCitrine virus , visual cortical slices were prepared from the injected mice as previously described ( Philpot et al . , 2001 ) .","After dissection 350-µm-thick coronal slices recovered for 30 min at 32°C and then for an additional 2 hr at room temperature , in a holding chamber filled with warmed artificial cerebrospinal fluid ( ACSF ) , which contained: 124 mM NaCl , 3 . 5 mM KCl , 1 . 25 mM Na2PO4 , 26 mM NaHCO3 , 1 . 2 mM MgCl2 , 2 mM CaCl2 , and 10 mM dextrose , saturated with 95% O2 , 5% CO2 .","Whole cell patch clamp recordings were performed at 30°C from the parvalbumin-positive interneurons in the layer 4 , identified by the ECFP fluorescence .","Pipette tip resistances were 3–5 MΩ .","Internal solution contained: 20 mM KCl , 100 mM Na-gluconate , 10 Hepes , 4 mM MgATP , 0 . 3 mM Na2GTP , 7 mM phosphocreatine-Tris , 0 . 2% biocytin with pH adjusted to 7 . 2 and osmolarity adjusted to 300 mOsm .","All recordings were made using Axopatch 200B ( Molecular Devices , Sunnyvale , CA ) at 10 kHz sampling rate .","Cells with the series resistance <30 MΩ were included for analysis .","Data analysis was done using pClamp ( Molecular Devices ) and custom-written python scripts .","For experiments concerning NMDAR EPSCs and AMPAR EPSCs in PV-GluN1 KO mice as presented in Figure 6H: 4 PV-GluN1 KO mice were infected with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP , to allow for fluorescence-guided intracellular recordings of PV+ cells .","Four weeks after infection , visual cortical slices were prepared as describe above .","For fluorescently labeled PV+ cells and unlabeled neighboring excitatory cells: EPSCs were recorded at -70 mV and +40 mV with pipettes filled with balanced intracellular solutions ( in mM ) : 115 cesium methane-sulfonate ( CsMeSO3 ) , 2 . 8 NaCl , 0 . 4 EGTA , 4 ATP-Mg2+ , 10 Na+-phosphocreatine , 0 . 5 Na+-GTP , 5 TEA-Cl- , 5 QX-314 Br- buffered with 20 HEPES , pH 7 . 25 , osmolarity 290 mOsm .","Synaptic events were evoked by stimulation of the white mater ( 150 μs , 0 . 1 Hz , glass electrode , and stimulator A365 from WPI ) .","AMPA component was measured from EPSCs at -70 mV in the presence of PTX ( 100 μM ) and glycine ( 1 μM ) and NMDA component was measured from EPSCs at +40 mV in the presence of DNQX ( 20 μM ) .","All behavioral experiments were performed during the mouse subject’s light cycle .","A piezo-electrical recording device ( C . B . Gitty , Barrington , NH , USA ) was placed under the forepaws of head-restrained mice during all sessions .","Mice became accustomed to the apparatus by sitting in situ in front of a gray screen for a 30-min session on each of 2 days .","Before stimulus presentation on each day mice also underwent 5 min of gray screen presentation after the experimenter had left the room .","A continuous voltage signal was recorded from the piezo device for the entire session .","Movements were detected as a shift in the voltage signal .","The recording system was automated so that no one was ever present in the closed room for any of the recording period and white noise was played at 67 dB in order to mask outside noise .","For vidget scoring , the continuous voltage signal was down-sampled to 100 Hz .","The period of interest in the experiments described here lasted from 2 s prior to stimulus onset until 5 s after stimulus onset ( which was the first 10 phase reversals in a block ) .","Quantification of movement driven by the onset of the stimulus ( the vidget ) was calculated by taking the Root Mean Square ( SQRT ( X2 ) ) of the voltage signal .","Post-stimulus signal was then normalized to the average magnitude during the 2-s period prior to stimulus onset .","The average normalized magnitude across the 5-s period subsequent to stimulus presentation was then used to quantify the degree of stimulus-driven movement and this is described throughout in arbitrary units ( a . u . ) .","After viral infection mice were also bilaterally implanted with VEP recording electrodes positioned in layer 4 .","Ready-made optic fibres ( 200 µm girth ) mounted in stainless steel ferrules ( 1 . 25 mm diameter , 2 mm fibre projection , Thor labs , Newton , NJ , US ) were then implanted positioned lateral ( 3 . 5 mm lateral to lambda ) to the recording site and at a 45° angle to the recording electrode , 0 . 1 mm below surface in each hemisphere .","1 month later , after peak of viral expression , mice were habituated to the head-fixation apparatus over 2 days before conducting optogenetic experiments .","We delivered 31-s long continuous pulses of blue light ( 473 nm , 150 mW ) into V1 using a laser ( Optoengine LLC , Midvale , UT , US ) .","These light pulses were delivered simultaneous to 50% of the 30-s long visual stimulus presentations , commencing 30 ms prior to visual stimulus onset and ending 30 ms after offset .","Animals were sacrificed and perfused within a week after this experiment for histological analysis .","Mice were deeply anaesthetized with fatal plus ( pentobarbital ) and perfused with saline followed by 4% paraformaldehyde in 0 . 1 M phosphate buffer .","The brain was removed and post-fixed for 24 hr at room temperature .","After fixation , the brain was sectioned into 60 µm coronal slices using a vibratome .","Slices were incubated with blocking solution ( 10% fetal bovine serum in PBS with 0 . 2% Triton X-100 ) for 1 hr at room temperature and then with anti-Parvalbumin mouse primary antibody ( MAB1572 , Millipore , Billerica , MA; 1:1000 ) and anti-GFP antibody ( Ab290 , Abcam , Cambridge , United Kingdom; 1:7000 ) details diluted in blocking solution overnight at 4 degrees Celsius .","Slices were then washed three times with PBS and incubated with the secondary antibody for 1 hr at room temperature ( Alexa488-conjugated anti-rabbit IgG , Invitrogen , Carlsbad , CA; 1:500 , Alexa594-conjugated anti-mouse IgG , Invitrogen; 1:500 ) .","Slices were washed three times with PBS and mounted with 49 , 6-diamidino-2 phenylindole ( DAPI ) -containing Vectashield ( Vector Laboratories , Burlingame , CA ) .","Fluorescence images were taken with a confocal fluorescence microscope ( Olympus , Tokyo , Japan ) .","In the results section , all data is expressed as a mean ± standard error of the mean ( S . E . M ) .","Sigmaplot was used for statistical analysis .","Normality of distribution and homogeneity of variation was tested and parametric ANOVAs ( for multiple groups ) or t-tests ( for 2 groups ) were performed when data passed these tests .","Otherwise , non-parametric ANOVAs or t-tests on ranks were used .","If ANOVAs yielded significance , Student-Newman-Keuls post-hoc tests with adjustment for multiple comparisons were applied for individual comparisons .","Repeated measures ANOVAs or paired t-tests were applied for all within subject comparisons .","For other comparisons unpaired ANOVAs or t-tests were used .","Individual tests used are described in the results .","P < 0 . 05 is used as a threshold for significance throughout but exact P values are given for all comparisons for which the P value is above 0 . 001 .","Post-hoc power analyses were used to determine that comparisons reached a power of > 0 . 8 for an alpha value of 0 . 05 .","Technical replicates were only used as N for the ex vivo electrophysiological test of hM4D ( Gi ) efficacy , when 10 cells were recorded from 7 mice , and the test of NMDAR functional loss in PV-GluN1 KO mice , when 8 cells were recorded from 5 mice .","Throughout the remainder of the study we report the N is an individual animal ( biological replicate ) .","Technical replicates ( secondary samples within each animal ) were not undertaken for in vivo electrophysiology .","Although both hemispheres were implanted initially , a decision was made as to the best hemisphere , based on response magnitude , morphology and binocularity after the very first recording ( prior to any measure of plasticity ) .","Technical replicates were undertaken for the behavior , with 10 , 6 ( for PV-HM4D ( Gi ) experiments ) or 5 stimulus onsets ( for PV-GluN1 KO experiment ) being delivered per day , although we only report the daily average ( biological replicate ) .","There variations in protocol were due experiments being conducted by different experimenters at different times .","However , protocols were completely consistent across treatments/genotypes .","The individual technical replicates are available in the uploaded source data files ."]],"string":"[\n [\n \"Understanding how brain synapses , cells , and circuits are persistently modified by experience to store information represents one of the great challenges in neuroscience .\",\n \"Mouse visual cortex has proven to be an excellent model system in which to examine experience-dependent neural response modification .\",\n \"One robust type of visual response plasticity is reliably elicited in adult ( > P60 ) mice by simply closing one eyelid .\",\n \"Over the course of 5–7 days of monocular deprivation ( MD ) , the responses in visual cortex evoked by stimulation of the non-deprived eye progressively increase ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .\",\n \"This deprivation-enabled response potentiation is driven by visual experience through the non-deprived eye , as only the responses through one eye are potentiated ( i . e . , it is input specific ) and it fails to occur if both eyelids are closed or if the animals are kept in a dark room ( Blais et al . , 2008 ) .\",\n \"There is evidence that the response potentiation is mediated in part by “Hebbian” strengthening of excitatory synaptic transmission in visual cortex , as induction requires cortical NMDA receptor ( NMDAR ) activation ( Sawtell et al . , 2003 ) ( Sato and Stryker , 2008 ) and α-calcium/calmodulin-dependent protein kinase II ( αCAMKII ) expression in principal cells ( Ranson et al . , 2012 ) .\",\n \"This form of ocular dominance ( OD ) plasticity is likely responsible for the increase in visual acuity that occurs through the non-deprived eye following adult monocular deprivation ( Iny et al . , 2006 ) , and is of particular interest in the context of recovery of brain function after deprivation , disease , or damage ( Cho and Bear , 2010 ) .\",\n \"Another robust form of visual response plasticity is induced by exposure of awake mice to oriented visual grating stimuli .\",\n \"Brief daily presentation of a phase-reversing grating of a single orientation causes a large and persistent increase in the peak cortical response to this orientation , as measured by visual evoked potentials ( VEPs ) or unit recordings ( Frenkel et al . , 2006; Cooke et al . , 2015 ) .\",\n \"This phenomenon is termed stimulus-selective response potentiation ( SRP ) because only responses to the experienced orientation are increased .\",\n \"Abundant evidence suggests that SRP is also mediated by “Hebbian” mechanisms , particularly those revealed by the study of long-term synaptic potentiation ( Cooke and Bear , 2010; 2014 ) .\",\n \"The mechanisms utilized for SRP within V1 have also been shown to mediate a fundamental form of long-term visual recognition memory , manifested behaviorally as orientation-selective habituation ( OSH ) ( Cooke and Bear , 2015; Cooke et al . , 2015 ) .\",\n \"Both monocular deprivation and selective visual experience trigger input-specific increases in the short latency VEP measured in layer 4 of mouse visual cortex and , as reviewed above , both OD plasticity and SRP share some molecular requirements ( e . g . , NMDAR activation ) .\",\n \"Thus , it came as a surprise that response potentiation after monocular deprivation and SRP do not occlude one another ( Frenkel and Bear , 2004 ) , suggesting that they employ different mechanisms or are expressed by different synapses .\",\n \"We became interested in the possibility of differential involvement of cortical inhibition mediated by the parvalbumin-expressing ( PV+ ) fast spiking neurons .\",\n \"PV+ fast-spiking inhibitory neurons comprise the most numerous sub-class of GABAergic cortical neurons ( Xu et al . , 2010 ) and receive substantial feed-forward glutamatergic input from the thalamus ( Cruikshank et al . , 2007 ) .\",\n \"These interneurons synapse preferentially on the somata and initial axon segments of principal cells , and therefore are in a position to strongly and precisely modulate even short latency visually evoked responses ( Cristo et al . , 2004; Markram et al . , 2004; Kepecs and Fishell , 2014 ) .\",\n \"Moreover , previous studies have suggested that fast-spiking interneurons participate in the expression of cortical eye dominance and OD plasticity in juvenile mice ( Yazaki-Sugiyama et al . , 2009; Smith and Bear , 2010 ) .\",\n \"The contribution of this class of neuron to cortical plasticity is also of particular interest given the evidence for cortical PV+ neuron dysfunction in various psychiatric disorders characterized by impaired cognitive function ( Gogolla et al . , 2009 ) .\",\n \"In the current study we used a variety of approaches to understand the contribution of PV+ neurons to adult OD plasticity and SRP .\",\n \"We find that neither the pharmacogenetic silencing of PV+ cells nor the specific deletion of NMDARs within them affect the relative eye dominance or the expression of deprivation-enabled potentiation of VEPs in adult mice .\",\n \"In contrast , and quite unexpectedly , we discovered that the expression of SRP is dependent on PV+ cell activity , and that perturbations of PV+ neuron function , most notably cell-type specific ablation of NMDARs , disrupt the expression of both SRP and its behavioral correlate , familiarity recognition .\"\n ],\n [\n \"In order to understand the role of PV+ neurons in the expression of experience-dependent visual cortical plasticity , we selectively inactivated these cells using a Dreadd ( Designer Receptors Exclusively Activated by Designer Drugs ) pharmacogenetic system: Specifically , we expressed a re-engineered G-protein coupled receptor , hM4D ( Gi ) , which is activated exclusively by an otherwise inert small molecule , clozapine-N-oxide ( CNO ) ( Nichols and Roth , 2009 ) .\",\n \"The binding of CNO to the hM4D ( Gi ) receptor activates intracellular Gi-mediated signaling and subsequent hyperpolarization of the cell in which the receptors are expressed .\",\n \"We locally expressed hM4D ( Gi ) receptors in PV+ cells of binocular V1 using an adeno-associated viral vector containing a construct for Cre-selective expression ( AAV9-hSyn-DIO-HA-hM4D ( Gi ) -IRES-mCitrine ) in mice that express Cre recombinase only in PV+ cells ( B6;129P2-Pvalbtm1 ( cre ) Arbr/J , PV-Cre ) .\",\n \"We confirmed the selective expression of hM4D ( Gi ) receptors in PV+ neurons in binocular V1 using immunohistochemistry ( Figure 1A–E ) .\",\n \"In order to ensure that PV+ neurons could be inactivated by this method , we took slices of V1 for ex vivo intracellular recordings .\",\n \"Bath application of CNO drastically inhibited current evoked action potential firing of PV+ neurons in Layer 4 ( 2-way repeated measures ANOVA , interaction of CNO x current injection , F ( 8 , 72 ) = 6 . 227 , P < 0 . 001 , n = 10 cells , significant at data points above 100pA: q ( 8 ) = 3 . 716 , P = 0 . 014 ) but had no effect on neighboring non-expressing cells ( Figure 1F–G ) .\",\n \"Layer 4 recordings in vivo demonstrated that systemic administration of CNO resulted in the elevation of visually-evoked potential ( VEP ) magnitude , and an increase in the firing rate of excitatory single units ( Figure 1H–I ) , which are expected consequences of inactivating PV+ inhibitory neurons in binocular V1 . 10 . 7554/eLife . 11450 . 003Figure 1 . The hM4D ( Gi ) DREADD system locally inactivates parvalbumin+ neurons in binocular primary visual cortex ( V1 ) .\",\n \"( A ) An example of V1 expression of hM4D ( Gi ) in a parvalbumin ( PV ) -Cre recombinase ( Cre ) mouse infected locally in binocular V1 with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine .\",\n \"( B ) A DAPI stain for cell nuclei is shown in blue .\",\n \"( C ) Infected cells expressing hM4D ( Gi ) are labeled in green .\",\n \"( D ) Immuno-labeled PV+ cells are shown in red .\",\n \"( E ) The merged image reveals that hM4D ( Gi ) -expressing cells are also PV+ .\",\n \"( F ) Intracellular current clamp recordings of hM4D ( Gi ) -infected PV+ layer 4 neurons in ex vivo slices of V1 reveal that green-labeled infected cells exhibit a non-adapting fast-spiking phenotype typical of fast-spiking inhibitory neurons ( black ) .\",\n \"These cells do not fire action potentials in the presence of CNO ( red ) , the exogenous ligand for hM4D ( Gi ) receptors , despite depolarizing current injection .\",\n \"In contrast , neighboring cells that are not mCitrine+ show no impact of CNO application .\",\n \"( G ) HM4D ( Gi ) -mediated inactivation of putative fast-spiking PV+ inhibitory neurons is here summarized as the number of action potentials resulting from a given current injection before ( black ) and after CNO application ( red ) .\",\n \"( H ) The effects of hM4D ( Gi ) -mediated inactivation of putative PV+ fast-spiking inhibitory neurons in vivo are apparent from electrophysiological recordings from V1 of awake , head-fixed mice viewing phase-reversing sinusoidal grating stimuli .\",\n \"Averaged Visually Evoked Potential ( VEP ) recordings recorded in layer 4 reveal increased VEP magnitude in the presence of CNO ( red ) , relative to pre-CNO ( baseline ) recordings ( black ) , indicative of reduced inhibition .\",\n \"( I ) Phase reversal-evoked action potentials recorded from neurons in layer 4 also exhibit a similar effect with elevated firing rates in the presence of CNO ( red ) relative to the baseline recording ( black ) .\",\n \"Labeled scale bars are presented throughout .\",\n \"Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00310 . 7554/eLife . 11450 . 004Figure 1—source data 1 . Action potential number for current injection . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 004 Mouse binocular V1 is ~2–3 times more strongly activated by the contralateral ( contra ) eye than the ipsilateral ( ipsi ) eye , an inherent bias known as ocular dominance ( OD ) .\",\n \"We probed the involvement of PV+ neuron activity in maintaining baseline OD .\",\n \"PV-Cre mice were infected with AAV virus to deliver hM4D ( Gi ) Dreadd receptors to PV+ neurons in binocular V1 ( Figure 2A ) .\",\n \"Electrodes were simultaneously implanted into layer 4 of V1 for VEP recordings .\",\n \"VEPs elicited through just contralateral or ipsilateral eyes were acquired consecutively before and after CNO administration ( Figure 2A–B ) .\",\n \"After CNO injection , VEPs driven through each eye increased significantly in magnitude ( 2-way repeated measures ANOVA , effect of CNO , F ( 7 ) = 75 . 986 , P < 0 . 001; contralateral eye: 222 . 18 ± 14 . 79 µV baseline vs . 679 . 81 ± 64 . 12 µV CNO , Student-Newman-Keuls ( SNK ) post hoc test , q ( 7 ) = 13 . 925 , n = 8 mice , P < 0 . 001; ipsilateral eye: 105 . 56 ± 6 . 78 µV baseline vs . 358 . 63 ± 35 . 79 µV CNO , SNK post hoc test , q ( 7 ) = 7 . 711 , n = 8 mice , P < 0 . 001 , ( Figure 2C ) , consistent with the occurrence of cortical disinhibition .\",\n \"However , the OD of visual responses in V1 ( contra/ipsi ratio ) was not significantly altered by CNO injection ( 2 . 13 ± 0 . 12 baseline vs . 1 . 96 ± 0 . 18 CNO , student’s paired two-tailed t-test , t ( 7 ) = 0 . 859 , n = 8 mice , P = 0 . 42 , Figure 2D ) .\",\n \"This result suggests that the inherent OD of binocular visual cortex is not dependent on PV+ neuron activity . 10 . 7554/eLife . 11450 . 005Figure 2 . Inactivation of parvalbumin+ neurons has no impact on expression of ocular dominance ( OD ) or the ocular dominance shift as a result of monocular deprivation ( MD ) in the adult mouse .\",\n \"( A ) For experiments described in this figure mice were infected across all cortical depths bilaterally in binocular V1 ( green ) .\",\n \"During the same surgical implantation procedure VEP recording electrodes were also positioned in layer 4 and chronically fixed .\",\n \"( B ) Mice ( P45-60 ) were infected and implanted with electrodes and then left for 3 weeks for the AAV9 viral vector to reach maximal expression , after which they underwent habituation to head-fixation and a gray screen for two consecutive days .\",\n \"Following this , on experimental day 0 , mice were presented with an Xo phase-reversing sinusoidal grating stimulus separately to the left and right eye .\",\n \"VEPs were recorded from each hemisphere in order to determine ocular dominance in binocular V1 .\",\n \"Mice were then removed from the recording apparatus and , CNO was delivered systemically half an hour prior to undertaking the same recording procedure , this time using an orthogonal X + 90° visual stimulus .\",\n \"( C ) As is well documented , responses in V1 to stimuli viewed through the contralateral eye ( blue ) were greater in magnitude than those elicited through the ipsilateral eye ( yellow ) , as measured here by VEP magnitude .\",\n \"After application of CNO ( red outlines ) , VEP magnitude dramatically increased .\",\n \"( D ) This increase was scaled such that the ratio of Contralateral:Ipsilateral VEP magnitude was maintained before ( white ) and after CNO ( red ) .\",\n \"( E ) In a second group of mice , a similar experimental protocol was observed prior to measuring ocular dominance by recording VEPs in each hemisphere elicited by an Xo stimulus through each eye .\",\n \"One hemisphere was then selected and the contralateral eye was sutured closed .\",\n \"After 7 days of monocular deprivation , the eye was opened and VEPs driven through either eye were again recorded , this time elicited by an X + 60° stimulus .\",\n \"Mice were then systemically injected with CNO and , half an hour later , VEPs driven through either eye were recorded , this time elicited by an X - 60° stimulus .\",\n \"( F ) After the adult mice underwent 7 days of MD , there was a significant potentiation of the V1 response to visual input through the ipsilateral eye ( yellow ) .\",\n \"After application of CNO ( red outlines ) , VEPs driven through each eye were elevated in magnitude , but again the increase in VEP magnitude was scaled .\",\n \"( G ) As a result of open eye potentiation after MD , the OD ratio shifted dramatically from the contralateral bias of pre MD ( white ) to an almost equal cortical response through contralateral and ipsilateral eyes ( black ) .\",\n \"This shifted ratio was unaffected by hM4Di-mediated inactivation of putative fast-spiking inhibitory neurons during CNO application ( red ) , indicating that the expression of OD and its shift as a result of MD in adult mice do not require fast-spiking inhibition .\",\n \"Significant comparisons are labeled with an asterisk and non-significant comparisons with n . s . throughout .\",\n \"Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00510 . 7554/eLife . 11450 . 006Figure 2—source data 1 . Ocular dominance and deprivation effects are maintained after PV neuron inactivation . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 006 The normal OD ratio is altered in the adult mouse by MD of the contralateral eye over 7 days , resulting in an ocular dominance shift that features potentiation of response through the open ipsilateral eye ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .\",\n \"In a new group of mice we assayed the effect of PV+ neuron inactivation on the expression of the shift in OD caused by MD .\",\n \"We recorded VEPs elicited through each eye from PV-Cre mice expressing hM4D ( Gi ) receptors in binocular V1 ( Figure 2E ) .\",\n \"After baseline recordings the mice underwent contralateral eyelid suture and 7 days of monocular deprivation .\",\n \"Subsequently , the contralateral ( deprived ) eye was opened and VEPs were re-recorded in order to reveal the expression of an OD shift .\",\n \"There was a significant potentiation of VEP magnitude driven through the ipsilateral eye following 7 days of MD ( 69 . 5 ± 5 . 81 µV pre MD vs . 139 . 5 ± 9 . 33 µV post MD , n = 10 mice , student’s paired one-tailed t-test , t ( 9 ) = -10 . 184 , P < 0 . 001 , Figure 2F ) .\",\n \"This potentiation of response through the ipsilateral ( non-deprived ) eye resulted in a significant shift in the OD ratio ( contra/ipsi ratio , 3 . 08 ± 0 . 26 pre MD vs . 1 . 33 ± 0 . 10 post MD , 1-way repeated measures ANOVA , SNK post hoc test , q ( 2 ) = 11 . 77 , P < 0 . 001 , Figure 2G ) .\",\n \"Mice were then injected with CNO and re-recorded to assess whether the OD shift would persist in the absence of PV+ neuron activity .\",\n \"CNO injection resulted in an increase in VEP magnitudes ( Figure 2F ) , but importantly the shift in the contra/ipsi ratio was maintained ( 1 . 33 ± 0 . 10 post MD vs . 1 . 35 ± 0 . 18 post MD CNO , SNK post hoc test , q ( 2 ) = 0 . 127 , P = 0 . 93 , Figure 2G ) .\",\n \"Therefore , the expression of the adult OD shift induced by 7 days of MD persists after a strong reduction in PV+ neuron inhibition .\",\n \"We next assayed the role of PV+ neurons in the expression mechanism underlying stimulus-selective response potentiation ( SRP ) , a second form of experience-dependent visual cortical plasticity that also manifests as an increase in V1 responses ( Frenkel et al . , 2006; Cooke and Bear , 2010 ) .\",\n \"Binocular VEPs were recorded from awake , head-fixed adult mice viewing phase-reversing gratings of a particular orientation ( Xo stimulus ) on each of 6 consecutive days ( Figure 3A and B ) .\",\n \"On the 7th day mice viewed blocks of the now familiar visual stimulus interleaved with blocks of a novel oriented stimulus ( X +/- 60° ) .\",\n \"To address whether PV+ neuron activity is required for the expression of SRP , familiar and novel oriented grating stimuli were presented before and after mice received CNO ( Figure 3A ) . 10 . 7554/eLife . 11450 . 007Figure 3 . The expression of Stimulus-selective Response Potentiation ( SRP ) requires activity in PV+ neurons in V1 . ( A ) Mice expressing hM4D ( Gi ) receptors in PV+ cells underwent a standard SRP induction protocol .\",\n \"On day 7 , mice viewed a novel oriented stimulus in addition to the familiar stimulus; before and after CNO injection .\",\n \"( B ) We acquired binocular VEPs from awake , head-fixed mice elicited by the same full-field oriented sinusoidal grating stimulus over several days .\",\n \"( C ) As a result of multiple days of experience cortical response was dramatically potentiated such that the familiar stimulus evoked VEPs of significantly greater magnitude than the novel stimulus ( black bars ) .\",\n \"( D ) After application of CNO , VEPs underwent a notable increase in magnitude as a result of disinhibition ( red bars ) .\",\n \"Most notably , CNO rendered response to familiar and novel stimuli equivalent in magnitude .\",\n \"( E ) This lost discrimination of familiar and novel stimuli is reflected as a drop in the ratio of response to familiar and novel stimuli from approximately 2:1 ( black ) to approximately 1:1 ( red ) .\",\n \"Significant comparisons are labeled with an asterisk and non-significant comparisons with n . s . throughout .\",\n \"Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00710 . 7554/eLife . 11450 . 008Figure 3—source data 1 . PV Dreadds SRP induction and expression . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00810 . 7554/eLife . 11450 . 009Figure 3—figure supplement 1 . CNO has no impact on SRP expression in WT mice .\",\n \"( A ) C57BL/6 WT mice were bilaterally infected with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine in binocular V1 .\",\n \"Because these mice did not express Cre recombinase there was no subsequent expression of hM4D ( Gi ) DREADDS receptors .\",\n \"After 4 weeks , during which hM4D ( Gi ) was expressed at high levels in PV-Cre mice , WT mice were taken through a standard SRP protocol of electrode implantation , habituation and visual experience of a single oriented grating .\",\n \"This progressed as usual , resulting in a significant increase in the magnitude of the VEP .\",\n \"( B ) Expression of SRP was then tested on day 7 by interleaving presentations of familiar and novel orientations and VEPs elicited by the familiar orientation were of significantly greater magnitude than the novel .\",\n \"After delivery of CNO , VEPs were again tested in response to the familiar orientation and a second novel orientation .\",\n \"The drug had no impact and a similar degree of significant stimulus selectivity was present .\",\n \"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00910 . 7554/eLife . 11450 . 010Figure 3—figure supplement 1—source data 1 . CNO has no effect on SRP expression in WT mice . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 010 As is characteristic of SRP , there was a significant increase in the magnitude of the average VEP evoked by the familiar oriented grating stimulus over days ( Friedman 1-way repeated measures ANOVA on ranks , n = 10 mice , X2 ( 5 ) = 40 . 40 , P < 0 . 001 , Figure 3C ) .\",\n \"This potentiation was evident by day 2 ( 263 . 3 ± 19 . 96 µV ) in comparison with day 1 ( 184 ± 17 . 13 µV , SNK post hoc test , q ( 9 ) = 4 . 472 , P < 0 . 05 ) .\",\n \"The stimulus selectivity of VEP magnitude potentiation was apparent on day 7 and significantly affected by inactivating PV+ inhibitory neurons in V1 ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F = 78 . 927 ( 1 , 9 ) , P < 0 . 001 , Figure 3D ) : Prior to delivery of CNO the average VEP magnitude driven by the familiar stimulus ( 324 . 7 ± 22 . 18 µV ) was significantly greater than that driven by a novel oriented stimulus ( 162 . 9 ± 16 . 31 µV , SNK post hoc test , q ( 9 ) = 10 . 709 , P < 0 . 001 ) .\",\n \"Following injection of CNO to inactivate hM4D ( Gi ) -infected neurons in V1 , there was no longer a significant difference in the magnitude of VEPs driven by the familiar stimulus ( 537 . 2 ± 66 . 69 µV ) compared with a novel stimulus ( 525 . 1 ± 68 . 30 µV , SNK post hoc test , q ( 9 ) = 0 . 804 , P = 0 . 58 ) .\",\n \"The selectivity of the potentiation to a familiar stimulus can be summarized by plotting the ratio of VEP magnitudes driven by familiar and novel stimuli ( Figure 3E ) .\",\n \"Mice expressed a significantly larger familiar/novel ratio before CNO injection ( 2 . 11 ± 0 . 17 ) , corresponding to a greater response to the familiar stimulus , compared to after CNO injection ( 1 . 03 ± 0 . 04 , Mann Whitney rank sum test , U = 0 . 00 , P < 0 . 001 ) .\",\n \"CNO injection did not affect the stimulus selectivity of SRP in wild type animals infected with virus ( Figure 3—figure supplement 1A–B ) .\",\n \"These animals underwent SRP ( Figure 3—figure supplement 1A ) and on test day showed significantly larger VEPs to the familiar stimulus , both prior to CNO injection ( familiar VEP: 361 . 06 ± 27 . 97 µV , novel VEP: 219 . 25 ± 17 . 9 µV , 2-way repeated measures ANOVA , n = 8 mice , SNK post hoc test , q ( 7 ) = 13 . 618 , P < 0 . 001 ) and following CNO injection ( familiar VEP: 369 . 00 ± 31 . 16 µV , novel VEP: 256 . 75 ± 19 . 59 µV , SNK post hoc test , q ( 7 ) = 10 . 779 , P < 0 . 001 , Figure 3—figure supplement 1B ) .\",\n \"These results show that the inactivation of PV+ neurons in binocular V1 disrupts the expression of SRP .\",\n \"Cortical neurons respond within a dynamic range , and it is possible that discrimination of familiar and novel stimuli would be lost as responses approach saturation .\",\n \"Our observation that OD is maintained after PV+ neuron inactivation ( Figure 2 ) indicates that V1 can still respond selectively to a strong ( contralateral eye ) and weak ( ipsilateral eye ) input .\",\n \"However , to address the possibility that a “ceiling effect” contributes to the disruption of SRP expression during PV+ cell inactivation , we conducted an additional experiment in which mice viewed sinusoidal grating stimuli across a range of contrast values ( 5 , 10 , 25 , 50 , 100% ) .\",\n \"VEPs were progressively greater in magnitude the greater the contrast of the viewed stimulus ( 2-way repeated measures ANOVA , n = 4 mice , effect of contrast , F ( 4 , 12 ) = 25 . 908 , P < 0 . 001 ) both before and during PV+ neuronal activation using the hM4D ( Gi ) system ( SNK post hoc test , 5 vs . 100 percent contrast , baseline; q ( 4 ) = 5 . 178 , P = 0 . 013; CNO; q ( 4 ) = 17 . 595 , P < 0 . 001 , Figure 4A ) .\",\n \"Preservation of the approximately linear relationship of contrast and response during PV+ neuron inactivation indicates that responses have not exceeded their dynamic range . 10 . 7554/eLife . 11450 . 011Figure 4 . Expression of SRP to two separate contrast values is blocked by hM4D ( Gi ) -mediated PV+ neuron inactivation , but differential response to contrast is maintained .\",\n \"( A ) CNO delivery to PV-Cre mice that had been infected with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine impacted VEP magnitude ( red ) significantly across a range of contrast compared to baseline ( black ) .\",\n \"( B ) SRP was induced to two differently oriented stimuli , each at different contrast values: 50% ( gray ) and 100% ( black ) .\",\n \"Modest but significant SRP was expressed at 50% contrast prior to CNO application .\",\n \"After CNO application ( red outlines ) , VEPs increased significantly in magnitude but were no longer significantly different for familiar and a second novel orientation .\",\n \"( C ) SRP was also expressed for a different orientation at 100% contrast and , again , VEP magnitude was increased and SRP blocked by delivery of CNO .\",\n \"( D ) The blockade of SRP expression by CNO at 50% contrast was apparent in the reduction in the familiar/novel ratio for VEP magnitude .\",\n \"( E ) The blockade of SRP expression at 100% contrast was also observed as a significant drop in familiar/novel ratio of VEP magnitude after CNO delivery .\",\n \"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01110 . 7554/eLife . 11450 . 012Figure 4—source data 1 . PV-neuron inactivation does not produce ceiling effect . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 012 We then induced SRP to different orientations in a different set of mice , one stimulus at 50% contrast and the other stimulus at 100% contrast .\",\n \"After 6 days of SRP at 50% , there was a modest but significant difference in VEP magnitude for familiar ( 188 . 81 ± 11 . 80 µV ) and novel orientations ( 145 . 06 ± 8 . 11 µV , 2-way repeated measures ANOVA , n = 8 mice , SNK post hoc test , q ( 7 ) = 3 . 608 , P = 0 . 023 , Figure 4B ) , just as there was for the familiar ( 259 . 81 ± 17 . 66 µV ) and novel orientations ( 192 . 81 ± 17 . 27 µV , SNK post hoc test , q ( 7 ) = 3 . 793 , P = 0 . 018 , Figure 4C ) at 100% contrast .\",\n \"SRP expression was abolished by PV+ neuronal inactivation using the hM4D ( Gi ) system at 50% contrast as VEPs elicited by familiar ( 404 . 69 ± 59 . 17 µV ) and novel orientations ( 376 . 94 ± 56 . 79 µV ) were no longer significantly different ( SNK post hoc test , q ( 7 ) = 2 . 289 , P = 0 . 128 , Figure 4B ) .\",\n \"The same was true at 100% contrast as VEPs evoked by the familiar ( 532 . 44 ± 63 . 82 µV ) and novel orientations ( 514 . 88 ± 40 . 38 µV ) were also no longer significantly different ( SNK post hoc test , q ( 7 ) = 0 . 994 , P = 0 . 494 , Figure 4C ) .\",\n \"Again , the blockade of SRP expression was also clearly observed in the familiar/novel ratio , which dropped significantly from ( 1 . 31 ± 0 . 05 ) to ( 1 . 08 ± 0 . 08 ) with CNO application at 50% contrast ( student’s paired two-tailed t-test , t ( 7 ) = 2 . 983 , P = 0 . 02 , Figure 4D ) and from ( 1 . 40 ± 0 . 10 ) to ( 1 . 02 ± 0 . 05 ) with CNO application at 100% contrast ( student’s paired two-tailed t-test , t ( 7 ) = 2 . 955 , P = 0 . 021 , Figure 4E ) .\",\n \"Thus , SRP could still be abolished through selective loss of PV+ neuron activity even at reduced contrasts eliciting submaximal responses .\",\n \"The disruption of SRP expression by inactivation of PV+ neurons is not a trivial consequence of a “ceiling effect” .\",\n \"PV+ neurons have been implicated in the sharpening of orientation selectivity in V1 ( Runyan et al . , 2010; Adesnik et al . , 2012; Atallah et al . , 2012; Lee et al . , 2012; Wilson et al . , 2012 ) .\",\n \"If orientation selectivity were completely abolished by inactivation of PV+ neurons then the loss of SRP expression could be attributed to a failure of orientation discrimination rather than familiarity .\",\n \"A previous experiment has indicated that activation of PV+ neurons in V1 actually mildly enhances orientation-selectivity and visual discrimination ( Lee et al . , 2012 ) .\",\n \"Therefore , we used optogenetics to activate PV+ neurons while mice were presented with familiar and novel stimuli to test whether SRP expression would be enhanced , unaffected or disrupted .\",\n \"PV+ neurons in binocular V1 of PV-Cre mice expressed Channel-rhodopsin 2 ( ChR2 ) as a result of infection with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP .\",\n \"During the same surgery VEP recording electrodes were implanted and optic fibers chronically implanted to deliver light to the recording site ( Figure 5A ) .\",\n \"After stable expression 4 weeks after infection , mice were habituated to head-fixation on each of 2 days before undergoing a standard SRP experiment over 6 days ( Figure 5B , C ) .\",\n \"Mice showed a significant increase in the magnitude of the average VEP evoked by the familiar oriented grating stimulus over days ( Friedman repeated measures ANOVA on ranks , n = 11 mice , X2 ( 5 ) = 24 . 818 , P < 0 . 001 , Figure 5C ) .\",\n \"This potentiation was evident by day 2 ( 237 . 55 ± 29 . 01 µV ) in comparison with day 1 ( 182 . 59 ± 21 . 97 µV , SNK post hoc test , q ( 10 ) = 7 . 675 , P < 0 . 05 ) .\",\n \"On day 7 , mice were presented with interleaved blocks of familiar and novel stimuli .\",\n \"Also interleaved were blocks of familiar and novel stimuli in which blue light ( 473 nm ) was continuously delivered via the optic fiber to the recording site in binocular V1 .\",\n \"VEPs elicited by the novel X + 90° stimulus were significantly lower in magnitude ( 221 . 34 ± 33 . 55 µV ) than those elicited by the familiar stimulus prior to optogenetic activation of PV+ neurons ( 310 . 70 ± 36 . 70 µV , 2-way repeated measures ANOVA , n = 11 mice , SNK post hoc test , q ( 10 ) = 11 . 799 , P < 0 . 001 , Figure 5D ) .\",\n \"Optogenetic activation of PV+ neurons significantly diminished the selectivity of SRP , as VEPs elicited by the familiar stimulus ( 181 . 23 ± 26 . 43 µV ) and novel stimulus ( 157 . 80 ± 21 . 05 µV ) were more similar in magnitude ( interaction of stimulus x laser , F ( 1 , 10 ) = 46 . 606 , P < 0 . 001; familiar vs . novel SNK post hoc test , q ( 10 ) = 3 . 094 , P = 0 . 045 , Figure 5D ) .\",\n \"The reduction of SRP selectivity is most clearly observed in the significant difference in the familiar/novel ratio without ( 1 . 55 ± 0 . 14 ) and with optogenetic stimulation ( 1 . 16 ± 0 . 07 , student’s paired two-tailed t-test , t ( 10 ) = 4 . 423 , P = 0 . 001 , Figure 5E ) .\",\n \"Laser stimulation did not affect the stimulus selectivity of SRP in wild type animals infected with virus ( Figure 5—figure supplement 1 ) , as there was no significant interaction between stimulus and laser ( 2-way repeated measures ANOVA , F ( 1 , 8 ) = 0 . 677 , P = 0 . 434 ) .\",\n \"These mice underwent SRP ( Figure 5—figure supplement 1A ) and on test day showed significantly larger VEPs to the familiar stimulus , both with the laser off ( familiar VEP: 273 . 69 ± 28 . 25 µV , novel VEP: 200 . 19 ± 19 . 53 µV , 2-way repeated measures ANOVA , n = 9 mice , SNK post hoc test , q ( 8 ) = 5 . 234 , P = 0 . 005 ) and the laser on ( familiar VEP: 277 . 08 ± 29 . 80 µV , novel VEP: 194 ± 18 . 39 µV , q ( 8 ) = 5 . 916 , P = 0 . 002 , Figure 5—figure supplement 1B ) .\",\n \"Thus , SRP expression was disrupted with activation of PV+ neurons , just as it was with inactivation ( Figure 3D–E ) .\",\n \"This result implies that PV+ neurons play a specific role in SRP expression beyond any role in enhancing orientation tuning . 10 . 7554/eLife . 11450 . 013Figure 5 . Optogenetic stimulation of PV+ inhibitory neurons prevents SRP expression .\",\n \"( A ) Blue light was delivered locally into V1 via optic fibers chronically implanted at a 45° angle to target the VEP recording site in layer 4 of binocular V1 of PV-Cre mice infected with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP .\",\n \"( B ) Experimental timeline showing that after viral infection , electrode implantation , and ChR2 expression; mice were accustomed to head-fixation and gray screen viewing .\",\n \"Subsequently , they underwent a standard SRP induction protocol over 6 days .\",\n \"On day 7 , mice viewed a novel oriented stimulus in addition to the familiar stimulus and , on 50% of presentations of each stimulus , blue light ( 473 nm ) was delivered to cortex to optogenetically activate PV+ cells .\",\n \"( C ) Significant SRP was induced over 6 days as VEPs underwent a typical potentiation .\",\n \"( D ) On day 7 , SRP was expressed through significantly larger VEP magnitude in response to the familiar Xo orientation than a novel X + 90° stimulus when blue light was not delivered ( Black bars ) .\",\n \"In the presence of blue light ( blue bars ) , VEPs were suppressed , and there was a significant reduction in the differential magnitude of VEPs driven by familiar and novel stimuli .\",\n \"( E ) The ratio of VEP magnitude elicited by familiar/novel stimuli was significantly reduced by optogenetic activation of PV+ neurons , reflecting a decrement in SRP expression .\",\n \"Significant comparisons are marked with an asterisk and post hoc test p values are reported in D to emphasize the impact of laser stimulation on SRP selectivity .\",\n \"Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01310 . 7554/eLife . 11450 . 014Figure 5—source data 1 . PV-neuronal activation . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01410 . 7554/eLife . 11450 . 015Figure 5—figure supplement 1 . Blue light has no impact on SRP expression .\",\n \"( A ) To check that light itself had no effect on cortical physiology in the optogenetic experiments , WT mice were infected bilaterally with AAV5-EF1α -DIO-hChR2 ( H134R ) -eYFP in binocular V1 .\",\n \"After 4 weeks , during which ChR2 was expressed at high levels in PV-Cre mice , WT mice were taken through the standard SRP protocol described above .\",\n \"Again , significant SRP occurred .\",\n \"( B ) On test day , during interleaved presentations of a familiar and novel oriented stimulus together with either blue light ( 473 nm ) or no light stimulation delivery to V1 , VEPs were significantly greater in magnitude for the familiar than the novel stimulus , regardless of the presence of blue light .\",\n \"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01510 . 7554/eLife . 11450 . 016Figure 5—figure supplement 1—source data 1 . Laser does not effect VEPs in WT animals . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 016 Multiple lines of evidence indicate that SRP is dependent upon the NMDA class of glutamate receptors ( Frenkel et al . , 2006; Cooke et al . , 2015 ) .\",\n \"Given the additional clear requirement for normal PV+ inhibitory cell function in SRP ( Figure 3D , E ) we selectively ablated the NMDAR from just PV+ cells by crossing the PV-Cre line of mice with a line in which the mandatory GluN1 subunit of NMDAR is excised by Cre recombinase activity ( B6 . 129S4-Grin1tm2Stl/J , GluN1 fl/fl ) .\",\n \"The progeny of this cross , in which both alleles of Grin1 ( the gene encoding GluN1 ) were floxed , are henceforth described as either PV-GluN1 KO or Wildtype ( WT ) -GluN1 fl/fl depending on whether , respectively , Cre was expressed or not .\",\n \"We implanted PV-GluN1 KO ( n = 14 ) and littermate WT-GluN1 fl/fl mice ( n = 17 ) with VEP recording electrodes in layer 4 , binocular V1 .\",\n \"After recovery and 2 daily sessions of habituation we recorded VEPs elicited by an Xo oriented grating stimulus .\",\n \"Immediately apparent was the significantly greater basal magnitude of VEPs recorded in the PV-GluN1 mice ( 242 . 45 ± 20 . 20 µV ) relative to their littermate controls ( 130 . 88 ± 9 . 69 µV , student’s two-tailed t-test , t ( 29 ) = -5 . 269 , P < 0 . 001 ) , consistent with the occurrence of disinhibition as a result of reduced glutamatergic drive on cortical PV+ inhibitory cells ( Figure 6A ) .\",\n \"We then presented the same stimulus to these mice over several consecutive days and observed a significant difference in SRP across genotypes ( 2-way repeated measures ANOVA , interaction of genotype x day , F ( 4 , 116 ) = 3 . 835 , P = 0 . 006 , Figure 6A ) .\",\n \"Beyond day 3 , VEP magnitudes were no longer significantly different between the PV-GluN1 KO mice ( 289 . 15 ± 24 . 57 µV ) and WT-GluN1 fl/fl littermates ( 239 . 94 ± 20 . 72 µV , SNK post hoc test , q ( 29 ) = 2 . 242 , P = 0 . 119 ) , suggesting an occlusion of SRP by the already elevated basal VEP magnitude in the PV-GluN1 KO mice .\",\n \"The significant deficit in SRP is clearly apparent when the data are normalized to day 1 values ( 2-way repeated measures ANOVA , interaction of genotype x day , F ( 4 , 116 ) = 12 . 326 , P < 0 . 001 , Figure 6B ) .\",\n \"Again , a significant deficit in SRP emerged by day 3 in the PV-GluN1 KO mice ( 119 . 26 ± 10 . 13% day 1 ) compared with WT-GluN1 fl/fl littermates ( 183 . 33 ± 15 . 83% day 1 , SNK post hoc test , q ( 29 ) = 4 . 727 , P = 0 . 002 ) , demonstrating that SRP is compromised by a loss of NMDAR expressed in PV+ neurons . 10 . 7554/eLife . 11450 . 017Figure 6 . Loss of NMDA receptors selectively from parvalbumin+ cells impacts SRP but not adult OD plasticity .\",\n \"( A ) VEPs recorded from mice in which the mandatory GluN1 subunit of the NMDA receptor was genetically ablated from PV+ cells using Cre recombinase technology ( PV GluN1 KO , gray ) were significantly greater in magnitude than those recorded from WT littermates ( black ) , suggesting disinhibition of the visual response .\",\n \"In these same PV GluN1 KO mice , SRP was also significantly impacted as there is was significantly less gain in magnitude over days of repeated presentation of an X° stimulus than observed in WT littermates .\",\n \"( B ) This significant reduction in the magnitude of SRP was most clearly observed if VEP magnitude was normalized to the magnitude on day 1 .\",\n \"( C ) After SRP , both PV GluN1 KO mice and their WT littermates exhibited a significantly greater VEP magnitude elicited by the now familiar stimulus than interleaved presentations of a novel oriented stimulus .\",\n \"However , consistent with the observed difference in magnitude on day 1 , VEPs elicited by a novel X + 90° stimulus in PV GluN1 KO mice were significantly greater in magnitude than those in WT littermate mice .\",\n \"No significant difference was observed for VEPs elicited by the familiar stimulus .\",\n \"( D ) A significant difference in the ratio of VEP magnitude elicited by the familiar and novel stimuli reveals a deficit in SRP expression in PV GluN1 KO mice .\",\n \"( E ) In contrast , PV GluN1 KO mice exhibited a normal adult OD shift after 7 days of MD , resulting from open eye potentiation ( yellow outlines ) .\",\n \"( F ) Wild-type ( WT ) littermates exhibited the same significant open eye potentiation ( yellow bars ) after 7 days of MD . ( G ) A comparison of the degree of OD shift as a result of 7 days of MD reveals a significant shift in OD ratio in both genotypes but no difference between genotypes , indicating that NMDA receptors in PV+ cells are not required for induction or expression of the OD shift .\",\n \"( H ) To confirm genetic ablation of NMDARs selectively from parvalbumin ( PV+ ) neurons in the PV-GluN1 KO; mice were injected with an AAV5 vector to express GFP in a Cre-dependent fashion in PV+ cells only .\",\n \"After 1 month , fluorescence-guided intracellular recordings were performed from ex vivo slices of visual cortex .\",\n \"NMDAR-mediated synaptic transmission was normal in excitatory control cells but abolished in PV+ cells .\",\n \"This is expressed here as the NMDAR EPSC/AMPAR EPSC ratio in excitatory cells ( black outline ) and PV+ cells ( green outline ) .\",\n \"Sample EPSC traces mediated by the AMPAR ( downward ) and NMDAR ( upward ) are shown at the top of the panel .\",\n \"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01710 . 7554/eLife . 11450 . 018Figure 6—source data 1 . PV-GluN1 KODOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 018 We also tested for the stimulus-selectivity of SRP expression by presenting both groups of animals with interleaved blocks of the familiar Xo stimulus and a novel X + 90° stimulus ( Figure 6C ) .\",\n \"Significant stimulus selectivity was present in both genotypes ( 2-way repeated measures ANOVA , stimulus , F ( 1 ) = 67 . 397 , P < 0 . 001 , interaction of genotype x stimulus , F ( 1 , 29 ) = 2 . 359 , P = 0 . 135 , Figure 6C ) : Although PV-GluN1 KO mice showed significant differences in VEP magnitude for familiar ( 340 . 24 ± 27 . 96 µV ) and novel orientations ( 242 . 54 ± 24 . 40 µV , SNK post hoc test , q ( 13 ) = 6 . 372 , P < 0 . 001 ) the difference was more pronounced in the WT-GluN1 fl/fl mice ( SNK post hoc test , q ( 16 ) = 10 . 254 , P < 0 . 001 ) , in which the familiar stimulus elicited VEPs 318 . 74 ± 25 . 19 µV in magnitude and the novel stimulus elicited VEPs 176 . 06 ± 11 . 55 µV in magnitude .\",\n \"The familiar stimulus evoked VEPs that were not significantly different in the WT-GluN1 fl/fl mice ( 318 . 74 ± 25 . 19 µV ) and the PV-GluN1 KO mice ( 340 . 24 ± 27 . 96 µV , SNK post hoc test , q ( 29 ) = 0 . 947 , P = 0 . 507 ) but those evoked in the WT-GluN1 fl/fl by the novel stimulus ( 176 . 06 ± 11 . 55 µV ) were significantly lower in magnitude than in the PV-GluN1 KO mice ( 242 . 54 ± 24 . 40 µV , SNK post hoc test , q ( 29 ) = 2 . 926 , P = 0 . 045 ) .\",\n \"The significant deficit in SRP expression in the PV-GluN1 KO mice is most apparent when the familiar/novel ratio of VEP magnitude ( 1 . 48 ± 0 . 13 ) is compared with the WT-GluN1 fl/fl littermates ( 1 . 83 ± 0 . 13 , Mann Whitney rank sum test , U = 60 . 000 , P = 0 . 020 , Figure 6D ) .\",\n \"Thus , loss of NMDAR function selectively within PV+ neurons impairs the full expression of SRP .\",\n \"We also examined if loss of NMDAR from PV+ neurons has an effect on OD plasticity .\",\n \"We implanted VEP recording electrodes in a second cohort of 11 PV–GluN1 KO and 7 WT-GluN1 fl/fl mice .\",\n \"After recovery and habituation over 2 days , monocular VEPs were acquired through each eye .\",\n \"After 7 days of MD , PV-GluN1 KO mice exhibited a significant potentiation of response through the open ipsilateral eye ( 187 . 91 ± 25 . 52 µV ) compared with day 0 ( 90 . 64 ± 10 . 69 µV , 2-way repeated measures ANOVA , SNK post hoc test , q ( 16 ) = 8 . 849 , P < 0 . 001 , Figure 6E ) .\",\n \"Similarly , WT-GluN1 fl/fl mice also showed a significant potentiation of the open ipsilateral eye-response ( 154 . 57 ± 7 . 60 µV ) after 7 days of MD compared with responses measured on day 0 ( 97 . 57 ± 15 . 85 µV , SNK post hoc test , q ( 16 ) = 4 . 137 , P = 0 . 01 , Figure 6F ) .\",\n \"Comparisons of OD ratios prior to MD in the adult PV-GluN1 KO ( 3 . 40 ± 0 . 29 ) and WT-GluN1 fl/fl mice ( 2 . 57 ± 0 . 33 ) did not reveal any significant difference ( 2-way repeated measures ANOVA , effect of genotype , F ( 1 ) = 3 . 424 , P = 0 . 083 ) .\",\n \"Significant shifts in the OD ratio occurred as a result of 7 days of MD in the adult PV-GluN1 KO ( 1 . 28 ± 0 . 33 , SNK post hoc test , q ( 16 ) = 10 . 6 , P < 0 . 001 ) and WT-GluN1 fl/fl mice ( 1 . 19 ± 0 . 09 , SNK post hoc test , q ( 16 ) = 5 . 517 , P = 0 . 001 ) .\",\n \"The shifted OD ratio also did not differ significantly across genotype ( 2-way repeated measures ANOVA , interaction of genotype and MD , F ( 1 , 16 ) = 2 . 638 , P = 0 . 124 , Figure 6G ) .\",\n \"Thus , the loss of NMDAR function from PV+ neurons did not have a significant impact on either the induction or the expression of adult OD plasticity , in contrast to SRP .\",\n \"In order to confirm the removal of NMDARs selectively from PV+ cells , the PV-GluN1 KO mice were injected with an AAV5 vector to express GFP in a Cre-dependent fashion in PV+ cells only .\",\n \"After 1 month , fluorescence-guided intracellular recordings were performed from ex vivo slices of visual cortex .\",\n \"NMDAR-mediated synaptic transmission was normal in excitatory control cells but abolished in PV+ neurons ( Figure 6H ) .\",\n \"This is expressed as significantly reduced NMDAR EPSC/AMPAR EPSC ratio in PV+ cells ( 0 . 040 ± 0 . 011 , n = 8 cells from 5 mice ) compared to neighboring non-fluorescent excitatory control cells ( 0 . 345 ± 0 . 039 , n = 10 cells from 4 mice , student’s one-tailed t-test , t ( 16 ) = -6 . 819 , P < 0 . 001 ) .\",\n \"A group of non-competitive , open-channel NMDAR blockers , including ketamine , PCP and MK801 are known to have the paradoxical impact of increasing net neuronal activity in the brain .\",\n \"It is thought that this apparent disinhibition arises from the preferential impact of these molecules on fast-spiking neurons , due to the tonic activation and the increased open-time of NMDAR expressed within these cells ( Homayoun and Moghaddam , 2007; Seamans , 2008 ) .\",\n \"Interestingly , these compounds are also psychotomimetic and can reproduce , at high sub-anesthetic doses , most of the symptoms of schizophrenia ( Krystal et al . , 1994 ) .\",\n \"Here we tested the possibility that a single acute dose of one of these substances , ketamine , would have an impact on the expression of SRP due to its action on NMDAR expressed in PV+ fast spiking inhibitory neurons .\",\n \"We implanted VEP recording electrodes in layer 4 of binocular V1 in a group of 10 C57BL/6 mice .\",\n \"After recovery and a standard SRP protocol we recorded VEP magnitudes driven by familiar and novel stimuli before , during and 2 days after recovery from systemic injection ( i . p . ) of a high but sub-anesthetic dose of ketamine ( 50 mg/kg ) ( Figure 7A ) .\",\n \"Ketamine had a significant effect on SRP expression ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F ( 2 , 18 ) = 29 . 479 , P < 0 . 001 , Figure 7B ) .\",\n \"After SRP but prior to ketamine delivery the familiar Xo stimulus elicited VEPs of significantly greater magnitude ( 245 . 47 ± 21 . 00 µV ) than a novel X + 60° stimulus ( 138 . 86 ± 9 . 32 µV , SNK post hoc test , q ( 9 ) = 9 . 228 , P < 0 . 001 ) in these mice .\",\n \"After an hour of respite from head-fixation , mice were injected with ketamine and , 15 min later , they were returned to head-fixation and VEP magnitudes were again recorded .\",\n \"Under the influence of ketamine , the familiar Xo stimulus elicited VEPs of significantly increased magnitude ( 381 . 90 ± 37 . 25 µV ) relative to pre ketamine ( 245 . 47 ± 21 . 00 µV , SNK post hoc test , q ( 9 ) = 7 . 223 , P < 0 . 001 ) .\",\n \"However , VEPs elicited by a second novel X - 60° stimulus were increased relative to pre-ketamine by an even greater extent ( 397 . 53 ± 37 . 62 µV ) relative to pre ketamine ( 138 . 86 ± 9 . 32 µV , SNK post hoc test , q ( 9 ) = 13 . 695 , P < 0 . 001 ) , such that VEPs were no longer significantly different in magnitude in response to familiar and novel stimuli ( SNK post hoc test , q ( 9 ) = 1 . 353 , P = 0 . 349 ) .\",\n \"After 2 day’s rest , allowing for complete recovery from drug effects , the mice were returned to the head-fixation apparatus and exposed to the familiar Xo stimulus and a third novel stimulus , X + 90° .\",\n \"Just as observed prior to the ketamine injection , the Xo stimulus evoked VEPs of significantly greater magnitude ( 267 . 46 ± 26 . 13 µV ) than the novel X + 90° stimulus ( 149 . 40 ± 9 . 85 µV , q ( 9 ) = 10 . 219 , P < 0 . 001 ) ( Figure 7A and B ) . 10 . 7554/eLife . 11450 . 019Figure 7 . Ketamine prevents expression of SRP through blockade of NMDA receptors expressed in parvalbumin+ neurons but does not impact expression of the adult OD shift .\",\n \"( A ) Mice were bilaterally implanted with VEP recording electrodes in layer 4 of binocular V1 .\",\n \"After habituation to head-fixation and a gray screen for 2 days , SRP was induced over 4 days by repeatedly presenting sessions of an X° stimulus .\",\n \"On day 5 , SRP expression was tested by presenting interleaved blocks of the familiar X° stimulus and a novel X + 60° stimulus .\",\n \"In order to test the acute impact of blocking NMDA receptors on SRP expression , 50 mg/kg of ketamine was then delivered systemically 15 min before re-acquiring VEPs elicited by the familiar X° stimulus and interleaved presentations of a second novel X - 60° stimulus .\",\n \"Mice were then allowed 2 days recovery and a complete washout of ketamine before re-testing SRP expression by again testing VEP magnitude in response to the familiar X° stimulus and a third novel X + 90° stimulus on day 7 .\",\n \"( B ) Significant SRP was expressed on experimental day 5 prior to ketamine delivery as the familiar X° stimulus elicited VEPs of greater magnitude than the novel stimulus .\",\n \"Delivery of 50 mg/kg ketamine ( purple ) had two notable impacts on the VEP: First , the overall magnitude of the VEP increased .\",\n \"Second , and most importantly , the significant difference in magnitude of VEPs elicited by familiar and novel stimuli was no longer present .\",\n \"This effect was acute , as SRP expression was again significantly apparent 2 days later .\",\n \"( C ) The ratio of VEP magnitude elicited by the familiar stimulus over the novel .\",\n \"This ratio was close to 2 and not significantly different prior to or after recovery from ketamine administration but dropped significantly to approximately 1 during ketamine exposure .\",\n \"( D ) We tested whether ketamine had a differential impact on VEP magnitude in PV GluN1 KO mice and WT littermate mice .\",\n \"( E ) In the WT littermate mice ( white bars ) 50 mg/kg ketamine had a significant potentiating effect on VEP magnitude , consistent with our previous observation .\",\n \"In contrast , ketamine had no significant impact on VEP magnitude in the PV GluN1 KO mice ( gray bars ) .\",\n \"( F ) The selectivity of ketamine’s impact on the WT mice is observed by comparing the ratio of VEP magnitude during ketamine over baseline , which was significantly greater for WT mice than the PV GluN1 KO mice , in which the ratio was approximately 1 .\",\n \"( G ) In a separate group of mice , a similar protocol was then used to determine whether the OD ratio is affected by ketamine .\",\n \"( H ) Ketamine impacted both the VEPs driven through the contralateral eye ( blue ) and ipsilateral eye ( yellow ) equally .\",\n \"( I ) This scaled effect is demonstrated by a lack of significant difference between OD ratios prior to ( white ) and during 50 mg/kg ketamine ( purple ) .\",\n \"( J ) We next tested whether ketamine has any impact on the expression of adult OD plasticity by recording VEP magnitudes through either eye in a new group of adult mice before taking them through a standard 7 day MD protocol .\",\n \"( K ) As anticipated , 7 days of contralateral eye MD induced a significant ipsilateral eye potentiation ( yellow ) and ketamine then further potentiated VEPs elicited through both contralateral ( blue ) and ipsilateral eyes .\",\n \"( L ) The OD ratio shifts significantly from a ratio heavily biased towards the contralateral eye , to a less biased ratio .\",\n \"Ketamine administration did not significantly affect the magnitude of the OD shift .\",\n \"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01910 . 7554/eLife . 11450 . 020Figure 7—source data 1 . Ketamine impact on cortical plasticity . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 020 This significant effect of ketamine on the discrimination of familiar and novel stimuli is summarized by the ratio of VEP magnitude driven by the familiar/novel stimulus ( 1-way repeated measures ANOVA , F ( 2 , 18 ) = 24 . 683 , P < 0 . 001 , Figure 7C ) .\",\n \"This familiar/novel ratio dropped significantly from 1 . 76 ± 0 . 10 prior to ketamine to 0 . 96 ± 0 . 02 after ketamine ( SNK post hoc test , q ( 9 ) = 8 . 443 , P < 0 . 001 ) .\",\n \"Upon recovery , the familiar/novel ratio significantly recovered ( 1 . 79 ± 0 . 16 , SNK post hoc test , q ( 9 ) = 8 . 759 , P < 0 . 001 ) and was not significantly different from the first test after SRP but prior to ketamine injection ( SNK post hoc test , q ( 9 ) = 0 . 316 , P = 0 . 826 ) .\",\n \"Thus , the psychotomimetic non-competitive NMDAR antagonist ketamine disrupts SRP .\",\n \"The fact that this is an acute effect on already established SRP that recovers after drug washout indicates that the role for NMDAR in PV+ fast-spiking GABAergic neurons may be in memory retrieval rather than learning .\",\n \"Ketamine blocks NMDAR expressed in all cell types throughout the CNS and is also known to have targets other than the NMDAR ( Chen et al . , 2009 ) .\",\n \"In order to determine if ketamine has its effect on V1 responses specifically through NMDAR expressed in PV+ cells , we tested if there was a differential effect of ketamine on VEP magnitude in PV-GluN1 KO mice and WT-GluN1 fl/fl mice .\",\n \"After a typical implantation and habituation protocol ( Figure 7D ) we tested VEP magnitudes elicited by a novel oriented Xo stimulus in WT-GluN1 fl/fl mice ( 178 . 11 ± 38 . 41 µV , n = 8 ) and PV-GluN1 KO mice ( 260 . 79 ± 50 . 75 µV , n = 8 ) ( Figure 7E ) .\",\n \"We then removed mice from head-fixation and allowed them to recover in their home-cage before delivering 50 mg/kg ketamine ( i . p . ) .\",\n \"After 15 min , the mice were returned to head-fixation and we observed the impact of ketamine on VEPs elicited by a novel X + 90° oriented stimulus , which was significantly different in its effect on the two genotypes ( 2-way repeated measures ANOVA , interaction of genotype x treatment , F ( 1 , 14 ) = 18 . 454 , P < 0 . 001 ) .\",\n \"In the control WT-GluN1 fl/fl mice there was a significant potentiation of VEP magnitude as a result of ketamine application ( 386 . 65 ± 70 . 75 µV ) in comparison to pre treatment ( 178 . 11 ± 38 . 41 µV , SNK post hoc test , q ( 7 ) = 8 . 505 , P < 0 . 001 , Figure 7E ) , replicating our previous finding ( Figure 7B ) .\",\n \"However , in the PV-GluN1 KO mice , ketamine had no ostensible significant impact ( 258 . 67 ± 44 . 86 µV ) in comparison to pre treatment ( 260 . 79 ± 50 . 75 µV , SNK post hoc test , q ( 7 ) = 0 . 087 , P = 0 . 952 ) .\",\n \"A ratio of VEP magnitudes pre and post ketamine treatment reveals the significant difference between ketamine’s action on WT-GluN1 fl/fl mice ( 2 . 40 ± 0 . 25 ) and PV-GluN1 KO mice ( 1 . 12 ± 0 . 13 , student’s two-tailed t-test , t ( 14 ) = 4 . 610 , P < 0 . 001 ) ( Figure 7F ) .\",\n \"Thus , while ketamine has a wide range of effects in the CNS , it exerts itself on the response of V1 to visual input selectively through NMDAR expressed in PV+ cells .\",\n \"We then tested whether or not ketamine would disrupt either the OD ratio or the shift induced by 7 days of MD in the adult mouse .\",\n \"Again , C57BL/6 mice ( n = 8 ) were implanted with VEP electrodes and taken through a standard surgery recovery and habituation protocol before measuring the OD ratio prior to 50 mg/kg ketamine and 15 min after ketamine delivery ( Figure 7G and H ) .\",\n \"The normal contra/ipsi OD ratio exhibiting contralateral eye dominance prior to ketamine ( 2 . 84 ± 0 . 18 ) was not significantly altered by ketamine ( 2 . 61 ± 0 . 19 , student’s paired two-tailed t-test , t ( 7 ) = 0 . 871 , P = 0 . 413 ) ( Figure 7I ) .\",\n \"A separate group of mice ( n = 11 ) then underwent a standard 7 day MD protocol .\",\n \"VEPs elicited by an Xo oriented stimulus were recorded at baseline , followed by the deprivation period .\",\n \"After eye opening VEP magnitudes were re-tested with a novel X + 60°stimulus ( the standard protocol to avoid contamination of the OD shift by SRP [Frenkel and Bear , 2004] ) .\",\n \"These mice were then tested again with a second novel X - 60°stimulus after 1 hr rest and an additional 15 min after systemic 50 mg/kg ketamine administration ( Figure 7J ) .\",\n \"As expected , VEPs elicited through the open ipsilateral eye ( 93 . 73 ± 12 . 85 µV ) were significantly potentiated by 7 days of MD in the adult mouse ( 155 . 73 ± 15 . 86 µV , 2-way repeated measures ANOVA , SNK post hoc test , n = 11 mice , q ( 10 ) = 7 . 102 , P < 0 . 001 ) , reflecting the well-documented OD shift .\",\n \"These same ipsilateral VEPs were then further potentiated by ketamine administration ( 232 . 03 ± 22 . 19 µV , SNK post hoc test , q ( 10 ) = 8 . 741 , P < 0 . 001 ) .\",\n \"However , ketamine had a similar significant potentiating effect on the contralateral VEP ( 279 . 07 ± 14 . 53 µV ) , relative to pre-ketamine ( 180 . 14 ± 11 . 22 µV , SNK post hoc test , q ( 10 ) = 11 . 333 , P < 0 . 001 , Figure 7K ) , suggesting a uniform scaling of response through the two eyes .\",\n \"This observation is confirmed by the fact that the OD ratio , significantly shifted from ( 2 . 84 ± 0 . 29 ) to ( 1 . 23 ± 0 . 11 , Friedman repeated measures ANOVA on ranks , n = 11 mice , X2 ( 2 ) = 16 . 545 , p<0 . 001 , SNK post hoc test , q ( 10 ) = 5 . 126 , P < 0 . 05 ) by 7 days of MD , was not further significantly affected by delivery of ketamine ( 1 . 28 ± 0 . 09 , SNK post hoc test , q ( 10 ) = 0 . 426 , P > 0 . 05 , Figure 7L ) .\",\n \"Thus , while ketamine prevents SRP expression through action on NMDAR expressed in PV+ cells , it does not significantly affect the adult OD shift after 7 days of MD , consistent once again with PV+ cells contributing to the expression of SRP but not adult OD plasticity .\",\n \"Given the clear involvement of PV+ neurons in the expression of SRP we wanted to determine if loss of PV+ neuronal function local to V1 would have any behavioral impact .\",\n \"Head-restrained mice viewing a phase reversing visual grating stimulus are known to exhibit a stereotyped motor response called a visually-induced fidget , or vidget ( Cooke et al . , 2015 ) .\",\n \"Vidgets can be measured via a piezoelectric sensor located beneath the forepaws of the mouse ( Figure 8A ) .\",\n \"Importantly , it has been shown that the magnitude of the vidget response is inversely correlated to the familiarity of the visual stimulus .\",\n \"That is , a visual stimulus that is very familiar to the animal will on average evoke a relatively weak vidget behavioral response .\",\n \"In contrast , the presentation of a novel stimulus will on average evoke a vidget of significantly greater magnitude .\",\n \"Therefore , this behavioral response reflects the ability of the animal to discriminate and respond to a novel visual stimulus in its environment .\",\n \"Importantly , genetic and pharmacological manipulations local to V1 that inhibit SRP also disrupt the behavioral discrimination of familiar and novel stimuli ( Cooke et al . , 2015 ) , demonstrating that this differential vidget response to familiar and novel stimuli is dependent on the plasticity in visual cortex .\",\n \"Since PV+ neuron inactivation disrupted the expression of SRP , we tested whether this manipulation would likewise disrupt visual novelty detection . 10 . 7554/eLife . 11450 . 021Figure 8 . Discrimination of familiar and novel oriented stimuli involves PV+ neurons in V1 and NMDAR expressed within PV+ neurons .\",\n \"( A ) Using the same protocol as described in Figure 3B , mice were progressively familiarized with a specific oriented stimulus .\",\n \"On the test day , as mice viewed familiar and novel stimuli , vidget behavioral responses were measured via a piezoelectric sensor located beneath the forepaws of the head-fixed mouse .\",\n \"( B ) After a standard SRP protocol , mice expressing hM4D ( Gi ) receptors selectively within PV+ cells of binocular V1 were exposed to both familiar and novel stimuli .\",\n \"Prior to application of CNO , mice exhibited significant behavioral evidence of discriminating this familiar orientation from interleaved presentations of a novel oriented stimulus ( black bars ) .\",\n \"After application of CNO , there was no longer successful discrimination of familiar and novel stimuli ( red bars ) .\",\n \"Averaged vidget responses are displayed at the top of this panel .\",\n \"( C ) A significant difference was observed in the ratio of response to familiar and novel stimuli from pre CNO ( black ) to post CNO ( red ) .\",\n \"( D ) A deficit in OSH was also apparent in PV GluN1 KO mice as vidget recordings demonstrated a failure to significantly discriminate familiar from novel orientations ( gray bars ) .\",\n \"WT littermates exhibited significantly greater vidget magnitudes for novel than familiar stimuli , indicating unimpaired discrimination of familiarity from novelty ( white bars ) .\",\n \"Averaged behavioral responses are displayed above with accompanying scale bars .\",\n \"( E ) The significant deficit of PV GluN1 KO mice in discriminating familiar from novel stimuli is apparent in the ratio of behavior elicited by the familiar over the novel stimulus in comparison to WT littermates .\",\n \"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02110 . 7554/eLife . 11450 . 022Figure 8—source data 1 . Novelty detection after PV+ neuronal disruption . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02210 . 7554/eLife . 11450 . 023Figure 8—figure supplement 1 . Cumulative distributions of average vidget behavioral response to familiar and novel stimuli for each individual animal included in analysis presented in Figure 8D and Figure 8E .\",\n \"( A ) Prior to CNO administration , average behavioral response to the familiar stimulus ( light gray ) and the novel stimulus ( black ) of each individual animal bilaterally expressing hM4D ( Gi ) in PV+ neurons of binocular V1 .\",\n \"Dotted line represents behavior no greater than pre stimulus baseline .\",\n \"( B ) Average behavioral response of the same animals to the familiar and a new novel stimulus during PV+ neuron inactivation via CNO delivery , revealing consequent deficit in discrimination of familiar and novel stimuli .\",\n \"( C ) Cumulative distributions of average vidget behavioral response to the familiar stimulus ( light gray ) and the novel stimulus ( black ) of each individual WT GluN1 fl/fl mouse .\",\n \"( D ) Average vidget behavioral response of each PV-GluN1 KO mouse to the familiar and a new novel stimulus , revealing deficit in discrimination of familiar and novel stimuli .\",\n \"Dotted line represents behavior no greater than pre stimulus baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02310 . 7554/eLife . 11450 . 024Figure 8—figure supplement 1—source data 1 . Per animal plots of Novelty detection after PV+ neuronal disruption . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 024 A group of PV-Cre mice expressing hM4D ( Gi ) receptors in PV+ cells ( n = 19 ) , underwent a SRP protocol similar to the previous experiment ( Figure 3A ) in which mice viewed phase-reversing gratings of a particular orientation ( Xo stimulus ) each day for 6 days .\",\n \"On the 7th day mice viewed blocks of the now familiar visual stimulus interleaved with blocks of a novel oriented stimulus ( X + 60° ) .\",\n \"On day 7 , vidget behavioral responses were acquired via a piezoelectric device situated underneath the forepaws of the mice ( Figure 8A ) , in order to measure the animal’s discrimination of familiar and novel stimuli .\",\n \"On day 8 , PV+ cells in V1 were then inactivated by systemic delivery of CNO ( i . p . ) and vidget responses were acquired to the familiar Xo stimulus and a second X - 60° novel stimulus .\",\n \"Inactivation of PV+ neurons in V1 significantly affected stimulus discrimination ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F ( 1 , 18 ) = 13 . 644 , P = 0 . 002 , Figure 8B ) : Prior to CNO , mice exhibited significantly larger vidget responses to the novel visual stimulus ( 3 . 64 ± 0 . 32 a . u . ) than the familiar stimulus ( 1 . 95 ± 0 . 26 a . u . , SNK post-hoc test , q ( 18 ) = 9 . 237 , P < 0 . 001 ) .\",\n \"During PV+ cell inactivation in V1 , behavioral responses to the familiar ( 2 . 01 ± 0 . 16 a . u . ) and novel visual stimuli ( 2 . 46 ± 0 . 22 a . u . ) were no longer significantly different ( SNK post-hoc test , q ( 18 ) = 2 . 503 , P = 0 . 086 ) .\",\n \"The deleterious effect of PV+ cell inactivation in V1 on the animal’s ability to discriminate familiar and novel stimuli is most obvious when a ratio of response to familiar/novel visual stimuli is calculated .\",\n \"Prior to CNO delivery this ratio was significantly lower ( 0 . 54 ± 0 . 04 ) than during CNO treatment ( 1 . 00 ± 0 . 15 , Wilcoxon signed rank test , W = 138 . 000 , Z = 2 . 777 , P = 0 . 004 , Figure 8C ) .\",\n \"These findings indicate that PV+ neuron activity is required not only for the expression of SRP , but also for visual novelty detection .\",\n \"Finally , given the observation that the discrimination of familiar and novel visual stimuli is diminished by inactivation of PV+ neurons in V1 , we tested whether a similar failure would occur in the PV-GluN1 KO mice ( n = 17 ) compared with WT-GluN1 fl/fl littermates ( n = 15 , 2-way repeated measures ANOVA , stimulus , F ( 1 ) = 14 . 632 , P < 0 . 001 , Figure 8D ) .\",\n \"In the PV-GluN1 KO mice , the familiar stimulus produced less behavioral response ( 2 . 75 ± 0 . 42 a . u . ) than a novel stimulus ( 3 . 67 ± 0 . 79 a . u . ) .\",\n \"However , the difference was not significant ( SNK post hoc test , q ( 16 ) = 2 . 570 , P = 0 . 079 ) .\",\n \"In comparison , the concurrently tested WT-GluN1 fl/fl littermate mice showed a suppression of behavioral response to the familiar stimulus ( 1 . 76 ± 0 . 26 a . u . ) relative to the novel stimulus ( 4 . 08 ± 0 . 51 a . u . ) that was significant ( SNK post hoc test , q ( 14 ) = 5 . 008 , P = 0 . 001 ) .\",\n \"This observation was reinforced by a comparison of the ratio of behavior produced by familiar and novel stimuli ( Figure 8E ) , in which there was a significant difference between the PV-GluN1 KO mice ( 0 . 59 ± 0 . 11 ) and their WT-GluN1 fl/fl littermates ( 0 . 94 ± 0 . 14 , student’s one-tailed t-test , t ( 30 ) = -1 . 992 , P = 0 . 028 ) , reflective of the deficit in discrimination of familiar and novel stimuli as a result of lost NMDAR function in PV+ cells .\",\n \"Individual animals’ average vidget responses to familiar and novel stimuli ( Figure 8—figure supplement 1 ) reveal significantly decreased stimulus selectivity subsequent to CNO administration ( Figure 8—figure supplement 1A–B ) , and in the PV GluN1 KO mice as compared to WT-GluN1 fl/fl littermate controls ( Figure 8—figure supplement 1C–D ) .\",\n \"Thus , not only do NMDAR in PV+ cells contribute to SRP expression but they are also involved in its behavioral correlate .\"\n ],\n [\n \"There has been a long-standing interest in the possible roles of PV+ neurons in juvenile OD plasticity .\",\n \"These include modulation of the sensitivity of visual cortex to the effects of MD ( Fagiolini et al . , 1994; Hanover et al . , 1999; Huang et al . , 1999; Chattopadhyaya et al . , 2004 ) and the functional expression of the OD shift ( Maffei et al . , 2006; Yazaki-Sugiyama et al . , 2009; Smith and Bear , 2010 ) .\",\n \"Our findings indicate that expression of OD plasticity in adult mice does not require participation of the PV+ neurons .\",\n \"In interpreting the current findings , it is important to recognize how juvenile and adult OD plasticity differ .\",\n \"In juvenile mice ( < ~P35 ) , a rapid and reliable consequence of MD is depression in cortex of responses mediated by the deprived eye .\",\n \"This is followed by a progressive compensatory increase in responses through the non-deprived eye ( Frenkel and Bear , 2004 ) .\",\n \"In adult mice maintained under standard laboratory conditions , depression of deprived-eye responses can be a weak and variable consequence of MD , but potentiation of the non-deprived eye still occurs reliably ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .\",\n \"This deprivation-enabled response potentiation is not mediated by homeostatic synaptic scaling because it requires visual experience through the non-deprived eye ( Blais et al . , 2008 ) , activation of cortical NMDA receptors ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) , and is unaffected ( in adults ) by genetic deletion of TNFα that abolishes scaling ( Ranson et al . , 2012 ) .\",\n \"These features are consistent with an alternative hypothesis that the adult OD shift occurs because deprivation of the dominant contralateral eye causes a metaplastic shift in the LTP threshold , enabling visual experience through the weaker ipsilateral eye to drive “Hebbian” synaptic strengthening ( Cooper and Bear , 2012 ) .\",\n \"This interpretation is supported by evidence that light deprivation promotes LTP in visual cortex ( Kirkwood et al . , 1996; Philpot et al . , 2001; 2003 ) and that αCaMKII mutants that lack LTP also lack adult OD plasticity ( Ranson et al . , 2012 ) , and it is compatible with our finding that expression of the adult OD shift is not dependent on the activity of PV+ inhibitory neurons .\",\n \"Saiepour and colleagues also recently observed that ocular dominance index ( ODI ) values of V1 neurons are not altered by PV+ interneuron suppression in monocularly deprived adult mice ( Saiepour et al . , 2015 ) .\",\n \"Using single unit recordings they first showed that monocular deprivation caused a shift in ODI values towards the non-deprived eye , as expected .\",\n \"They then tested the dependence of this shift on PV+ cell activity by measuring ODI values of V1 neurons during optogenetic suppression of PV+ cells .\",\n \"This optogenetic-suppression did not alter the deprivation-induced shift in ODI values .\",\n \"Our results using VEP recordings and PV+ suppression via hM4Di Dreadd receptors are in agreement with these results from Saiepour and colleagues , demonstrating that suppression of PV+ cell activity after deprivation does not interfere with the expression of the adult OD shift .\",\n \"Although the findings that PV+ neurons are not necessary for expression of OD plasticity do not rule out a modulatory role in the induction mechanisms , we note that the adult OD shift still occurred in mutant mice in which PV+ neurons lack NMDARs .\",\n \"In contrast to the adult OD shift , we found that the expression of SRP relies on PV+ neuron circuitry .\",\n \"This finding came as a surprise , as SRP reflects the potentiation of short-latency synaptic currents and increased spiking activity in layer 4 ( Cooke et al . , 2015 ) , and previous experiments had strongly indicated that SRP might be considered a naturally occurring form of LTP .\",\n \"For example , induction of SRP is input-specific , dependent upon NMDA receptor activation and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ( AMPAR ) insertion in principal cells ( Frenkel et al . , 2006 ) , and reversed by infusion of the zeta-inhibitory peptide ( ZIP ) that also erases LTP ( Cooke and Bear , 2010 ) .\",\n \"Furthermore , LTP elicited by theta-burst stimulation of the thalamus occludes and is occluded by SRP ( Cooke and Bear , 2010 ) .\",\n \"Altogether , these findings suggest a modification of feed-forward excitatory synaptic transmission .\",\n \"Before assigning a specific role to inhibition in the expression of SRP , two relatively trivial explanations must be addressed .\",\n \"The first is that the loss of discrimination of familiar and novel orientations could be accounted for simply by a failure of orientation selectivity and not by a failure of memory .\",\n \"Throughout the experiments presented here we have used orientations that are at least 60° and often 90° different , meaning that orientation discrimination should be as unchallenging as possible .\",\n \"Given the modest impact on orientation tuning of suppressing PV+ interneuron activity in V1 ( Runyan et al . , 2010; Adesnik et al . , 2012; Wilson et al . , 2012 ) we believe that this explanation is improbable .\",\n \"However , we took a direct approach to testing this possibility by stimulating activity in PV+ cells using channelrhodopsin-2 ( ChR2 ) while mice are viewing familiar and novel oriented stimuli .\",\n \"Stimulation of PV+ inhibition has been shown to actually mildly enhance orientation-selectivity ( Lee et al . , 2012 ) and yet our experiments reveal that the difference between visual cortical response to familiar and novel orientations is significantly diminished by activation PV+ neurons .\",\n \"This result casts PV+ neurons in a very specific role in the recognition of familiar stimuli in addition to modest participation in orientation-selectivity .\",\n \"A second concern is that , after the suppression of PV+ inhibition , V1 may be operating outside the dynamic range where discrimination is possible .\",\n \"However , we show that the cortex responds to input from the contralateral eye with a response of approximately double the magnitude of that elicited by input through the ipsilateral eye even after silencing the PV+ cells , suggesting that the response of the cortex is not saturated .\",\n \"We also find that PV+ neuron silencing affects SRP even when it is induced using lower contrast stimuli that evoke submaximal responses .\",\n \"Moreover , it is also worth noting that less specific pharmacological approaches to suppressing inhibition in V1 ( Khibnik et al . , 2010 ) result in much greater magnitude of response to visual input than we observe here for PV+ neuron inactivation , PV-GluN1 KO or ketamine application , indicating that our manipulations do not encroach on a physiological ceiling .\",\n \"One relatively simple explanation for the current findings is that SRP results from lasting homosynaptic ( i . e . input-specific ) long-term depression ( LTD ) of glutamatergic input to PV+ inhibitory neurons .\",\n \"Thereafter , presentation of a familiar stimulus evokes a greater response in V1 due to selective disinhibition of cortical circuitry .\",\n \"This hypothesis is consistent with our observation that SRP is deficient if NMDARs are no longer expressed on PV+ cells .\",\n \"However , because we used a non-reversible genetic modification in our initial PV-GluN1 KO experiment , we were initially unable to show if these receptors were important for the storage of information or the retrieval of information already stored .\",\n \"Ketamine , which among other actions serves as a non-competitive NMDAR antagonist , has been shown to preferentially antagonize NMDARs on PV+ cells ( Homayoun and Moghaddam , 2007; Seamans , 2008 ) .\",\n \"By using ketamine to induce a temporary blockade of these receptors , which we confirmed by showing that there was no impact of ketamine on V1 responses in the PV-GluN1 KO mice , we were able to demonstrate that interference with NMDARs on PV+ cells disrupts expression of SRP even after it has been induced and saturated .\",\n \"Importantly , acute application of ketamine did not affect the maintenance or stability of stored information , because a strong bias for the familiar stimulus returned after ketamine washout .\",\n \"If SRP is indeed accounted for by homosynaptic depression of excitatory synapses carrying information about the familiar stimulus orientation , this LTD would need to be expressed by a synapse-specific reduction in NMDAR-mediated responses in order to account for these findings .\",\n \"However , this simple model is difficult to reconcile with the findings reviewed above that SRP depends on LTP-like mechanisms , e . g . , NMDAR-dependent AMPAR delivery in principal cells .\",\n \"Considered together , the findings to date indicate that induction and maintenance of SRP occur by modification of excitatory synapses onto excitatory neurons , but that expression of SRP is mediated by stimulus-selective modulation of PV-cell activity in V1 .\",\n \"Obviously a polysynaptic circuit is required to yield these properties , and dissecting this essential cortical circuitry will require much more work .\",\n \"However , it is interesting to note a recent study by Makino and Komiyama who showed , in addition to a stimulus-selective reduction in PV+ neuron activity in V1 as mice were repeatedly exposed to a specific visual stimulus , an experience-dependent increase in the responsiveness of somatostatin-positive interneurons ( Makino and Komiyama , 2015 ) .\",\n \"This result is particularly interesting as it is known that many interneuron types innervate one another and , therefore , that the activity of these cell types are interdependent .\",\n \"It is not difficult to sketch circuits in which stimulus-selective , feed-forward polysynaptic inhibition of PV+ cells accounts for SRP expression .\",\n \"Clearly , experiments aimed at teasing apart the involvement of other interneuron types in SRP should now be a high priority .\",\n \"We note that other studies have suggested that PV+ neurons may be important for plasticity following repeated exposure to sensory stimuli .\",\n \"For instance , Meyer and colleagues , studying infero-temporal cortex ( IT ) in monkeys , showed that familiarity resulted in a sharpening of cortical responses to dynamic visual stimuli , which was likely orchestrated by local fast-spiking putative PV+ cells ( Meyer et al . , 2014 ) .\",\n \"Additionally , mice constitutively lacking neuronal activity-regulated pentraxin ( NARP ) , a synaptic protein specifically enriched at excitatory synapses on PV+ cells , displayed a reduction in excitatory synapses onto PV+ cells and impairment in SRP-like effects ( Chang et al . , 2010; Gu et al . , 2013 ) .\",\n \"Intriguingly , however , the NARP KO mice also failed to exhibit both juvenile and adult OD plasticity .\",\n \"In juvenile animals , it is well established that reducing inhibition can impair induction of OD plasticity ( Ramoa et al . , 1988 ) and delay the developmental decline in the mechanism of deprived-eye depression ( Hanover et al . , 1999; Huang et al . , 1999 ) .\",\n \"We speculate that the loss of OD plasticity in the adult NARP KO may be related to disruption of the developmental switch from juvenile to adult-type OD plasticity ( Heimel et al . , 2011 ) .\",\n \"Our findings indicate that although open-eye potentiation as a result of MD in the adult and SRP are superficially similar , the expression mechanisms of these two forms of plasticity are quite distinct .\",\n \"Based on evidence provided here as well as by others ( Sawtell et al . , 2003; Ranson et al . , 2012; Saiepour et al . , 2015 ) , the deprivation enabled potentiation of the open eye is consistent with a potentiation of synapses between excitatory cells and is independent of PV+ cell inhibition .\",\n \"However , further work will be necessary to confirm that open eye potentiation in the adult animal is expressed by synaptic alterations between excitatory cells , and to determine whether these processes are present at the level of thalamocortical synapses or intracortical synapses .\",\n \"SRP correlates with OSH ( Cooke et al . , 2015 ) , a form of visual learning which is dependent upon NMDARs in V1 , and which enables the animal to make the critically important discrimination between familiarity and novelty .\",\n \"We have shown here that modulation of PV+ neuron activity in V1 is required for this selective recognition of familiarity and consequent novelty detection .\",\n \"Loss of NMDAR function in these same cells also impairs familiarity recognition .\",\n \"The cognitive symptoms of schizophrenia , and other psychiatric disorders , are characterized by deficits in habituation and familiarity ( Braff et al . , 1995; Ramaswami , 2014 ) and individuals with schizophrenia display impairment in visual cortical plasticity ( Çavuş et al . , 2012 ) .\",\n \"Dysfunction of PV+ inhibitory neurons and the NMDARs expressed on PV+ cells have also been implicated in these same psychiatric disorders through a range of approaches ( Lewis et al . , 2005; Gogolla et al . , 2009; Lewis et al . , 2011 ) .\",\n \"This notably includes observations of the profound psychotomimetic effect in humans of non-competitive NMDAR antagonists such as ketamine ( Krystal et al . , 1994; Krystal , 2015 ) , a substance that , as we show here , prevents SRP expression .\",\n \"We suggest that understanding the cortical physiology underlying the processes of familiarity recognition and novelty detection may yield great insight into dysfunction underlying some symptoms of these disorders and that , because these processes are as important to mice as they are to humans , they are likely to be experimentally tractable .\"\n ],\n [\n \"All procedures adhered to the guidelines of the National Institutes of Health and were approved by the Committee on Animal Care at MIT , Cambridge , MA , USA .\",\n \"For all experiments mice were male , aged between P60-90 and on a C57BL/6 background ( Charles River laboratory international , Wilmington , MA ) .\",\n \"They were housed in groups of 2–5 with food and water available ad libitum and maintained on a 12 hr light-dark cycle .\",\n \"For hM4D ( Gi ) experiments , mice were Parvalbumin-Cre recombinase knock-in mice ( B6;129P2-Pvalbtm1 ( cre ) Arbr/J , PV-Cre ) on a C57BL/6 background .\",\n \"For GluN1 knockdown experiments , these PV-Cre mice were bred with homozygous mice in which the Grin1 gene , which encodes the GluN1 sub-unit was flanked by LoxP sites ( B6 . 129S4-Grin1tm2Stl/J , GluN1 fl/fl ) , enabling Cre-dependent ablation of this mandatory subunit of the NMDA receptor .\",\n \"Multiple generations were required to set up crosses yielding offspring that were homozygous GluN1 fl/fl and Cre-expressing .\",\n \"Of these approximately 50% expressed Cre and 50% served as wild-type littermates on the GluN1 fl/fl background .\",\n \"All experiments were conducted blind to genotype and treatment .\",\n \"Mice were first injected with 0 . 1 mg/kg Buprenex sub-cutaneously ( s . c . ) to provide analgesia .\",\n \"They were then anesthetized with an intraperitoneal ( i . p . ) injection of 50 mg/kg ketamine and 10 mg/kg xylazine .\",\n \"Prior to surgical incision , 1% lidocaine hydrochloride anesthetic was injected under the mouse’s scalp .\",\n \"The skull was then cleaned with iodine and 70% ethanol .\",\n \"A steel head post was affixed to the skull anterior to bregma using cyanoacrylate glue .\",\n \"Burr holes ( < 0 . 5 mm ) were then drilled in the skull over binocular V1 ( 3 . 1 mm lateral of lambda ) .\",\n \"Tapered tungsten recording electrodes ( FHC , Bowdoinham , ME , US ) , 75 μm in diameter at their widest point , were implanted in each hemisphere , 450 μm below cortical surface .\",\n \"Silver wire ( A-M systems , Sequim , WA , US ) reference electrodes were placed over prefrontal cortex .\",\n \"Mice were allowed to recover for at least 24 hr prior to head-fixation .\",\n \"For hM4D ( Gi ) experiments , we infected V1 of P30-60 PV-Cre mice or wild-type littermates with an AAV9-hSyn-DIO-HA-hM4D ( Gi ) -IRES-mCitrine virus ( UNC viral core – generated by Dr . Brian Roth’s laboratory ) and for optogenetic experiments we infected mice from the same line with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP ( UNC viral core – generated by Dr . Karl Deisseroth’s laboratory ) .\",\n \"Using a glass pipette and nanoject system ( Drummond scientific , Broomall , PA , US ) , we delivered 81 nl of virus at each of 3 cortical depths: 600 , 450 , and 300 µm from the cortical surface , and allowed 5 min between re-positioning for depth .\",\n \"Mice were allowed 3–4 weeks recovery for virus expression to peak before experiments were initiated .\",\n \"Clozapine-N-oxide ( CNO , Enzo Life Sciences ) was diluted in saline and injected i . p . at a dosage of 5 mg/kg 30 min prior to stimulus delivery .\",\n \"Ketamine hydrochloride ( Vedco ) was diluted in water and delivered i . p . at 50 mg/kg 15 min prior to stimulus delivery .\",\n \"Visual stimuli consisted of full-field , 100% contrast , sinusoidal gratings that were presented on a computer monitor .\",\n \"Visual stimuli were generated using custom software ( http://bearlab-s1 . mit . edu/supp6/PlxStimOne_Opto . zip ) written in either C++ for interaction with a VSG2/2 card ( Cambridge Research systems , Kent , U . K . ) or Matlab ( MathWorks , Natick , MA , U . S . ) using the PsychToolbox extension ( http://psychtoolbox . org ) to control stimulus drawing and timing .\",\n \"The display was positioned 20 cm in front of the mouse and centered , thereby occupying 92° × 66° of the visual field .\",\n \"Visual stimuli consisted of full-field sinusoidal grating stimuli phase reversing at a frequency of 2 Hz .\",\n \"Mean stimulus luminance was 27 cd/m2 .\",\n \"Grating stimuli spanned the full range of monitor display values between black and white , with gamma-correction to ensure constant total luminance in both gray-screen and patterned stimulus conditions .\",\n \"For data described in Figures 1–3 and 6 , experiments were fully automated and each stimulus block consisted of 200 phase reversals with 30-s intervals between each stimulus presentation , during which the screen was gray but of equivalent luminance .\",\n \"For experiments described in Figures 4 and 5 stimulus blocks were also 200 phase reversals in length but each was triggered directly by the experimenter , meaning that the interval was approximately 30-s .\",\n \"Throughout , stimulus orientation varied such that a novel orientation was always a minimum of 25° different from any experienced previously by the individual mouse and was never within 20° of horizontal because these orientations are known to elicit VEPs of greater magnitude than vertical or oblique stimuli .\",\n \"If more than 1 orientation was shown within a session , stimuli were pseudo-randomly interleaved such that 3 consecutive presentations of the same stimulus never occurred .\",\n \"For monocular presentation an occluding paddle was positioned in front of one eye to limit stimulus presentation to the opposite eye .\",\n \"VEP recordings were conducted in awake , head-restrained mice .\",\n \"Prior to recording , mice were habituated to the restraint apparatus by sitting in situ in front of a gray screen for a 30-minute session on each of two consecutive days .\",\n \"For experiments in which monocular VEPs were subsequently acquired , mice were also habituated to the occluding paddle positioned in front of each eye .\",\n \"On stimulus presentation days , mice were presented with 5 blocks of 200 phase reversals of each oriented stimulus separated by gray screen presentation for ~30 s .\",\n \"For monocular presentations , recordings were conducted in sequence for each eye .\",\n \"VEP magnitude was then quantified by measuring trough-peak response magnitude averaged over all phase reversals .\",\n \"Recordings presented in Figures 1–3 and 6 were amplified and digitized using the Recorder-64 system ( Plexon Inc . , Dallas , TX ) .\",\n \"Two recording channels were dedicated to recording EEG/VEPs from V1 in each implanted hemisphere and a third recording channel was reserved for the Piezo-electrical input carrying the behavioral information .\",\n \"Recordings presented in Figures 4 and 5 were amplified using DAM80 amplifiers ( World Precision Instruments , Florida , U . S . ) and digitized using a custom National Instruments system ( National Instruments , Texas , U . S . ) ) .\",\n \"Local field potential was recorded from V1 with 1 kHz sampling and a 500 Hz low-pass filter .\",\n \"Data was extracted from the binary storage files and analyzed using custom software ( http://bearlab-s1 . mit . edu/supp6/VEP_Analysis . zip ) written in C++ , Matlab and Labview .\",\n \"VEPs were averaged across all phase reversals within a block and trough-peak difference measured during a 200-millisecond period from phase reversal .\",\n \"For experiments concerning Dreadd receptor expression in PV+ cells presented in Figure 1: Three weeks after infection with AAV9-hSyn-DIO- HA-hM4D ( Gi ) -IRES-mCitrine virus , visual cortical slices were prepared from the injected mice as previously described ( Philpot et al . , 2001 ) .\",\n \"After dissection 350-µm-thick coronal slices recovered for 30 min at 32°C and then for an additional 2 hr at room temperature , in a holding chamber filled with warmed artificial cerebrospinal fluid ( ACSF ) , which contained: 124 mM NaCl , 3 . 5 mM KCl , 1 . 25 mM Na2PO4 , 26 mM NaHCO3 , 1 . 2 mM MgCl2 , 2 mM CaCl2 , and 10 mM dextrose , saturated with 95% O2 , 5% CO2 .\",\n \"Whole cell patch clamp recordings were performed at 30°C from the parvalbumin-positive interneurons in the layer 4 , identified by the ECFP fluorescence .\",\n \"Pipette tip resistances were 3–5 MΩ .\",\n \"Internal solution contained: 20 mM KCl , 100 mM Na-gluconate , 10 Hepes , 4 mM MgATP , 0 . 3 mM Na2GTP , 7 mM phosphocreatine-Tris , 0 . 2% biocytin with pH adjusted to 7 . 2 and osmolarity adjusted to 300 mOsm .\",\n \"All recordings were made using Axopatch 200B ( Molecular Devices , Sunnyvale , CA ) at 10 kHz sampling rate .\",\n \"Cells with the series resistance <30 MΩ were included for analysis .\",\n \"Data analysis was done using pClamp ( Molecular Devices ) and custom-written python scripts .\",\n \"For experiments concerning NMDAR EPSCs and AMPAR EPSCs in PV-GluN1 KO mice as presented in Figure 6H: 4 PV-GluN1 KO mice were infected with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP , to allow for fluorescence-guided intracellular recordings of PV+ cells .\",\n \"Four weeks after infection , visual cortical slices were prepared as describe above .\",\n \"For fluorescently labeled PV+ cells and unlabeled neighboring excitatory cells: EPSCs were recorded at -70 mV and +40 mV with pipettes filled with balanced intracellular solutions ( in mM ) : 115 cesium methane-sulfonate ( CsMeSO3 ) , 2 . 8 NaCl , 0 . 4 EGTA , 4 ATP-Mg2+ , 10 Na+-phosphocreatine , 0 . 5 Na+-GTP , 5 TEA-Cl- , 5 QX-314 Br- buffered with 20 HEPES , pH 7 . 25 , osmolarity 290 mOsm .\",\n \"Synaptic events were evoked by stimulation of the white mater ( 150 μs , 0 . 1 Hz , glass electrode , and stimulator A365 from WPI ) .\",\n \"AMPA component was measured from EPSCs at -70 mV in the presence of PTX ( 100 μM ) and glycine ( 1 μM ) and NMDA component was measured from EPSCs at +40 mV in the presence of DNQX ( 20 μM ) .\",\n \"All behavioral experiments were performed during the mouse subject’s light cycle .\",\n \"A piezo-electrical recording device ( C . B . Gitty , Barrington , NH , USA ) was placed under the forepaws of head-restrained mice during all sessions .\",\n \"Mice became accustomed to the apparatus by sitting in situ in front of a gray screen for a 30-min session on each of 2 days .\",\n \"Before stimulus presentation on each day mice also underwent 5 min of gray screen presentation after the experimenter had left the room .\",\n \"A continuous voltage signal was recorded from the piezo device for the entire session .\",\n \"Movements were detected as a shift in the voltage signal .\",\n \"The recording system was automated so that no one was ever present in the closed room for any of the recording period and white noise was played at 67 dB in order to mask outside noise .\",\n \"For vidget scoring , the continuous voltage signal was down-sampled to 100 Hz .\",\n \"The period of interest in the experiments described here lasted from 2 s prior to stimulus onset until 5 s after stimulus onset ( which was the first 10 phase reversals in a block ) .\",\n \"Quantification of movement driven by the onset of the stimulus ( the vidget ) was calculated by taking the Root Mean Square ( SQRT ( X2 ) ) of the voltage signal .\",\n \"Post-stimulus signal was then normalized to the average magnitude during the 2-s period prior to stimulus onset .\",\n \"The average normalized magnitude across the 5-s period subsequent to stimulus presentation was then used to quantify the degree of stimulus-driven movement and this is described throughout in arbitrary units ( a . u . ) .\",\n \"After viral infection mice were also bilaterally implanted with VEP recording electrodes positioned in layer 4 .\",\n \"Ready-made optic fibres ( 200 µm girth ) mounted in stainless steel ferrules ( 1 . 25 mm diameter , 2 mm fibre projection , Thor labs , Newton , NJ , US ) were then implanted positioned lateral ( 3 . 5 mm lateral to lambda ) to the recording site and at a 45° angle to the recording electrode , 0 . 1 mm below surface in each hemisphere .\",\n \"1 month later , after peak of viral expression , mice were habituated to the head-fixation apparatus over 2 days before conducting optogenetic experiments .\",\n \"We delivered 31-s long continuous pulses of blue light ( 473 nm , 150 mW ) into V1 using a laser ( Optoengine LLC , Midvale , UT , US ) .\",\n \"These light pulses were delivered simultaneous to 50% of the 30-s long visual stimulus presentations , commencing 30 ms prior to visual stimulus onset and ending 30 ms after offset .\",\n \"Animals were sacrificed and perfused within a week after this experiment for histological analysis .\",\n \"Mice were deeply anaesthetized with fatal plus ( pentobarbital ) and perfused with saline followed by 4% paraformaldehyde in 0 . 1 M phosphate buffer .\",\n \"The brain was removed and post-fixed for 24 hr at room temperature .\",\n \"After fixation , the brain was sectioned into 60 µm coronal slices using a vibratome .\",\n \"Slices were incubated with blocking solution ( 10% fetal bovine serum in PBS with 0 . 2% Triton X-100 ) for 1 hr at room temperature and then with anti-Parvalbumin mouse primary antibody ( MAB1572 , Millipore , Billerica , MA; 1:1000 ) and anti-GFP antibody ( Ab290 , Abcam , Cambridge , United Kingdom; 1:7000 ) details diluted in blocking solution overnight at 4 degrees Celsius .\",\n \"Slices were then washed three times with PBS and incubated with the secondary antibody for 1 hr at room temperature ( Alexa488-conjugated anti-rabbit IgG , Invitrogen , Carlsbad , CA; 1:500 , Alexa594-conjugated anti-mouse IgG , Invitrogen; 1:500 ) .\",\n \"Slices were washed three times with PBS and mounted with 49 , 6-diamidino-2 phenylindole ( DAPI ) -containing Vectashield ( Vector Laboratories , Burlingame , CA ) .\",\n \"Fluorescence images were taken with a confocal fluorescence microscope ( Olympus , Tokyo , Japan ) .\",\n \"In the results section , all data is expressed as a mean ± standard error of the mean ( S . E . M ) .\",\n \"Sigmaplot was used for statistical analysis .\",\n \"Normality of distribution and homogeneity of variation was tested and parametric ANOVAs ( for multiple groups ) or t-tests ( for 2 groups ) were performed when data passed these tests .\",\n \"Otherwise , non-parametric ANOVAs or t-tests on ranks were used .\",\n \"If ANOVAs yielded significance , Student-Newman-Keuls post-hoc tests with adjustment for multiple comparisons were applied for individual comparisons .\",\n \"Repeated measures ANOVAs or paired t-tests were applied for all within subject comparisons .\",\n \"For other comparisons unpaired ANOVAs or t-tests were used .\",\n \"Individual tests used are described in the results .\",\n \"P < 0 . 05 is used as a threshold for significance throughout but exact P values are given for all comparisons for which the P value is above 0 . 001 .\",\n \"Post-hoc power analyses were used to determine that comparisons reached a power of > 0 . 8 for an alpha value of 0 . 05 .\",\n \"Technical replicates were only used as N for the ex vivo electrophysiological test of hM4D ( Gi ) efficacy , when 10 cells were recorded from 7 mice , and the test of NMDAR functional loss in PV-GluN1 KO mice , when 8 cells were recorded from 5 mice .\",\n \"Throughout the remainder of the study we report the N is an individual animal ( biological replicate ) .\",\n \"Technical replicates ( secondary samples within each animal ) were not undertaken for in vivo electrophysiology .\",\n \"Although both hemispheres were implanted initially , a decision was made as to the best hemisphere , based on response magnitude , morphology and binocularity after the very first recording ( prior to any measure of plasticity ) .\",\n \"Technical replicates were undertaken for the behavior , with 10 , 6 ( for PV-HM4D ( Gi ) experiments ) or 5 stimulus onsets ( for PV-GluN1 KO experiment ) being delivered per day , although we only report the daily average ( biological replicate ) .\",\n \"There variations in protocol were due experiments being conducted by different experimenters at different times .\",\n \"However , protocols were completely consistent across treatments/genotypes .\",\n \"The individual technical replicates are available in the uploaded source data files .\"\n ]\n]"},"abstract":{"kind":"list like","value":["The roles played by cortical inhibitory neurons in experience-dependent plasticity are not well understood .","Here we evaluate the participation of parvalbumin-expressing ( PV+ ) GABAergic neurons in two forms of experience-dependent modification of primary visual cortex ( V1 ) in adult mice: ocular dominance ( OD ) plasticity resulting from monocular deprivation and stimulus-selective response potentiation ( SRP ) resulting from enriched visual experience .","These two forms of plasticity are triggered by different events but lead to a similar increase in visual cortical response .","Both also require the NMDA class of glutamate receptor ( NMDAR ) .","However , we find that PV+ inhibitory neurons in V1 play a critical role in the expression of SRP and its behavioral correlate of familiarity recognition , but not in the expression of OD plasticity .","Furthermore , NMDARs expressed within PV+ cells , reversibly inhibited by the psychotomimetic drug ketamine , play a critical role in SRP , but not in the induction or expression of adult OD plasticity ."],"string":"[\n \"The roles played by cortical inhibitory neurons in experience-dependent plasticity are not well understood .\",\n \"Here we evaluate the participation of parvalbumin-expressing ( PV+ ) GABAergic neurons in two forms of experience-dependent modification of primary visual cortex ( V1 ) in adult mice: ocular dominance ( OD ) plasticity resulting from monocular deprivation and stimulus-selective response potentiation ( SRP ) resulting from enriched visual experience .\",\n \"These two forms of plasticity are triggered by different events but lead to a similar increase in visual cortical response .\",\n \"Both also require the NMDA class of glutamate receptor ( NMDAR ) .\",\n \"However , we find that PV+ inhibitory neurons in V1 play a critical role in the expression of SRP and its behavioral correlate of familiarity recognition , but not in the expression of OD plasticity .\",\n \"Furthermore , NMDARs expressed within PV+ cells , reversibly inhibited by the psychotomimetic drug ketamine , play a critical role in SRP , but not in the induction or expression of adult OD plasticity .\"\n]"},"summary":{"kind":"list like","value":["What we see or fail to see through our eyes leaves a lasting impression by changing the strength of connections between neurons in a part of the brain called the visual cortex .","These changes are referred to as synaptic plasticity .","One example of synaptic plasticity results in the visual cortex becoming more responsive to the stimulation of one eye when the other eye is patched for about a week .","This phenomenon is known as “ocular dominance plasticity” .","Another example is the increase in responsiveness that occurs in the visual cortex when animals repeatedly view stripes of a single orientation .","This phenomenon is known as “stimulus-selective response potentiation” .","Some previous studies had suggested that both kinds of plasticity might be induced in the same way .","However , both forms of plasticity can happen at the same time , suggesting that distinct mechanisms may be involved .","To tease out how these two kinds of plasticity work , Kaplan , Cooke et al . inactivated one particular type of neuron that is thought to be involved in triggering ocular dominance plasticity and is found in the visual cortex .","These inhibitory neurons produce a molecular marker called parvalbumin and are therefore referred to as “parvalbumin-expressing neurons” .","The experiments showed that ocular dominance plasticity could still be seen in adult mice when these parvalbumin-expressing neurons were inactivated .","However , when these same neurons were inactivated , the visual cortex no longer responded differently to lines with familiar or new orientations .","This was the case even when the mice had seen lines in a given orientation for long periods of time .","Similarly , observations of the behavior of the mice also showed that their ability to distinguish new from familiar stimuli was lost when the parvalbumin-expressing neurons were inactivated locally within part of the visual cortex .","The connections between neurons that bring information from the eyes to the visual cortex release a chemical neurotransmitter called glutamate .","One important protein that detects glutamate , called an NMDA receptor , is required for ocular dominance plasticity .","Further experiments showed that NMDA receptors within the parvalbumin-expressing neurons are needed for stimulus-selective response potentiation , but not for ocular dominance plasticity .","This indicates that although the two forms of plasticity may share molecular requirements , different connections and cells types are likely involved .","Changes in parvalbumin-expressing neurons and NMDA receptors have been implicated in disorders such as autism and schizophrenia .","To improve treatments for these disorders , it is crucially important to better understand the links between these neurons and the retrieval of memories ."],"string":"[\n \"What we see or fail to see through our eyes leaves a lasting impression by changing the strength of connections between neurons in a part of the brain called the visual cortex .\",\n \"These changes are referred to as synaptic plasticity .\",\n \"One example of synaptic plasticity results in the visual cortex becoming more responsive to the stimulation of one eye when the other eye is patched for about a week .\",\n \"This phenomenon is known as “ocular dominance plasticity” .\",\n \"Another example is the increase in responsiveness that occurs in the visual cortex when animals repeatedly view stripes of a single orientation .\",\n \"This phenomenon is known as “stimulus-selective response potentiation” .\",\n \"Some previous studies had suggested that both kinds of plasticity might be induced in the same way .\",\n \"However , both forms of plasticity can happen at the same time , suggesting that distinct mechanisms may be involved .\",\n \"To tease out how these two kinds of plasticity work , Kaplan , Cooke et al . inactivated one particular type of neuron that is thought to be involved in triggering ocular dominance plasticity and is found in the visual cortex .\",\n \"These inhibitory neurons produce a molecular marker called parvalbumin and are therefore referred to as “parvalbumin-expressing neurons” .\",\n \"The experiments showed that ocular dominance plasticity could still be seen in adult mice when these parvalbumin-expressing neurons were inactivated .\",\n \"However , when these same neurons were inactivated , the visual cortex no longer responded differently to lines with familiar or new orientations .\",\n \"This was the case even when the mice had seen lines in a given orientation for long periods of time .\",\n \"Similarly , observations of the behavior of the mice also showed that their ability to distinguish new from familiar stimuli was lost when the parvalbumin-expressing neurons were inactivated locally within part of the visual cortex .\",\n \"The connections between neurons that bring information from the eyes to the visual cortex release a chemical neurotransmitter called glutamate .\",\n \"One important protein that detects glutamate , called an NMDA receptor , is required for ocular dominance plasticity .\",\n \"Further experiments showed that NMDA receptors within the parvalbumin-expressing neurons are needed for stimulus-selective response potentiation , but not for ocular dominance plasticity .\",\n \"This indicates that although the two forms of plasticity may share molecular requirements , different connections and cells types are likely involved .\",\n \"Changes in parvalbumin-expressing neurons and NMDA receptors have been implicated in disorders such as autism and schizophrenia .\",\n \"To improve treatments for these disorders , it is crucially important to better understand the links between these neurons and the retrieval of memories .\"\n]"},"year":{"kind":"string","value":"2016"}}},{"rowIdx":4189,"cells":{"headings":{"kind":"list like","value":["Introduction","Materials and methods"],"string":"[\n \"Introduction\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["short report","cell biology"],"string":"[\n \"short report\",\n \"cell biology\"\n]"},"title":{"kind":"string","value":"Degradation of Gadd45 mRNA by nonsense-mediated decay is essential for viability"},"id":{"kind":"string","value":"elife-12876-v2"},"sections":{"kind":"list like","value":[["Maintaining proper gene expression is critical for normal development and physiology .","In addition to de novo transcription , mRNA stability substantially contributes to forming the landscape of expression in a cell .","The nonsense-mediated mRNA decay ( NMD ) pathway is a trans-acting mechanism that destabilizes mRNAs , and is best known for its well-described role as a quality control system , degrading abnormal mRNAs containing premature termination codons ( PTCs ) ( Celik et al . , 2015 ) .","NMD also degrades many wild-type endogenous mRNAs and thus is an important aspect of their post-transcriptional ( Peccarelli and Kebaara , 2014 ) .","Loss of either of the core NMD genes Upf1 ( Rent1 ) or Upf2 causes lethality in most eukaryotes ( Kerényi et al . , 2008; Medghalchi et al . , 2001; Metzstein and Krasnow , 2006; Weischenfeldt et al . , 2008; Wittkopp et al . , 2009 ) , indicating regulation of mRNA stability by NMD is critical for viability .","However , the relative contributions to lethality from ectopic stabilization of PTC-containing mRNAs or endogenous NMD targets in NMD mutants remains unclear ( Hwang and Maquat , 2011 ) .","To identify which ectopically stabilized mRNAs are responsible for inducing lethality in NMD mutants , we performed an unbiased genetic suppressor screen seeking to restore viability in a Drosophila NMD mutant .","To detect subtle increases in survival , we screened to suppress the lethality of animals mutant for the partially viable , hypomorphic Upf225G allele , of which 10% survive to adulthood ( Chapin et al . , 2014; Metzstein and Krasnow , 2006 ) .","We crossed this allele to heterozygous deficiencies to simultaneously reduce the mRNA abundance of several loci ( Figure 1A ) .","Of the 376 deficiencies tested , covering more than half the genome , ~10% suppressed NMD mutant lethality ( Figure 1B , Figure 1—figure supplement 1A ) .","The suppression effect could not be explained by a reduction in overall mRNA load , as there was only a weak correlation between the increase in mRNAs expressed from a genomic region upon loss of NMD function and the strength of suppression when that region was removed by a deficiency ( Figure 1—figure supplement 1B ) .","Rather , deficiencies that suppressed NMD-mutant lethality clustered in three genomic regions ( Figure 1—figure supplement 1A ) .","These findings suggest that NMD mutant lethality is not the result of a global excess of nonspecific mRNAs , but rather is mediated by specific genes residing within the few identified regions . 10 . 7554/eLife . 12876 . 003Figure 1 . Drosophila suppressor screen identifies the Gadd45 pathway as the inducer of NMD-mutant lethality .","( A ) Scheme to screen deficiencies for the suppression of Upf225G partial lethality .","The Deficiency Suppression Score ( DSS ) represents the relative difference in Upf225G viability when crossed to a heterozygous deficiency ( Df ) compared to when crossed to a balancer ( Bal ) ( See Methods ) .","( B ) DSS from 376 screened deficiencies ranked by score .","A DSS greater than 0 . 1 ( dotted line ) indicates that deficiency suppresses Upf225G lethality .","( C and E )","Candidate suppressing regions uncovering Gadd45 ( C ) and Mekk1 ( E ) .","DSSs are shown in parenthesis .","Dotted lines denote extent of regions deleted by suppressing deficiencies but not non-suppressing deficiencies .","Filled blocks on chromosomes indicate predicted gene spans , Gadd45 pathway genes are indicated in red; suppressing deficiencies indicated in green , sple-J1 has undefined breakpoints located within hashed regions .","( D and F )","NMD mutant adult viability in combination with Gadd45F17 ( D ) or Mekk1Ur36 ( F ) mutants .","Upf126A and Upf27-5A are null alleles ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .","p-value compared to controls determined by the test of equal or given proportions indicated .","Error bars represent 95% confidence interval of the binomial distribution .","n equals total number of animals scored in each cross . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00310 . 7554/eLife . 12876 . 004Figure 1—figure supplement 1 . Reduced expression of specific loci , not overall mRNA abundance , produces NMD mutant suppression by deficiencies .","( A ) Map of 376 autosomal DrosDel deficiencies with an isogenic background and molecularly defined breakpoints ( Ryder et al . , 2007 ) and eight other deficiencies used to further test candidate suppressing regions without overlapping DrosDel deficiencies .","39 total deficiencies suppress Upf225G lethality , shown in green .","Regions deleted by any non-suppressing deficiencies were eliminated as candidate suppressing regions , removing false positives and reducing the size of the candidate intervals .","The three candidate suppressing regions that are deleted only by suppressing deficiencies are indicated in black and labeled 1–3 .","( B ) Each deficiency’s Deficiency Suppression Score ( DSS ) compared to percent increase in RNA abundance in Upf225G compared to wild-type from loci removed by that deficiency according to RNA-seq from Chapin et al . ( 2014 ) .","Trend line in red; statistics calculated using Pearson correlation test . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00410 . 7554/eLife . 12876 . 005Figure 1—figure supplement 2 . Drosophila Gadd45 is an endogenous direct NMD target .","( A ) Gadd45 mRNA expression in adults of the given genotypes measured by qRT-PCR .","Gadd45 mRNA expression is increased 16 . 7-fold in Upf225G mutants , and is eliminated by Gadd45F17 mutants .","p-values display one-sided Student’s t-test of indicated condition compared to control .","Error bars represent 2 SEM .","( B ) Fluorescence of GFP transgenes with SV40 , Act5C , or Gadd45 3’ UTRs expressed by UAS driven by Actin:GAL4 in Upf2+ or Upf225G third instar larvae .","SV40 and Gadd45 3’ UTR constructs show significantly increased fluorescence in Upf225G animals compared to Upf2+ , indicating NMD-dependent post-transcriptional degradation of mRNAs containing these UTRs .","The Act5C 3’ UTR construct has similar fluorescence in both backgrounds , indicating NMD does not regulate the post-transcriptional stability of this UTR .","Micrographs show dorsal views with anterior at top .","( C ) MAL-A2 , traL , and Gadd45 5’ and 3’ fragment mRNA expression measured by qRT-PCR in pcm14 null mutants ( Waldron et al . , 2015 ) normalized to controls .","Transcript structures of a non-NMD-target , maltase A2 ( MAL-A2 ) ; a known NMD-targeted transcript , the non-sex specific isoform of transformer ( traL ) ( Rehwinkel , 2005; Metzstein and Krasnow , 2006 ) ; and the Gadd45 transcript ( note Gadd45 has no introns ) .","Open boxes indicate UTRs; grey boxes indicate coding regions .","NMD targeting initiates endonucleolytic cleavage near the stop codon ( Gatfield and Izaurralde , 2004 ) , producing 5’ and 3’ fragments with unprotected ends , which are then subjected to degradation by cytoplasmic 3’-to-5’ and 5’-to-3’ exonucleases , respectively .","qRT-PCR primer pairs 5’ ( red ) and 3’ ( blue ) to the cleavage site can be used to differentially measure the quantity of these fragments .","The Drosophila 5’-to-3’ exonuclease is encoded by the XRN1 homologue pacman ( pcm ) , and fragments 3’ to an endonucleolytic NMD cleavage accumulate in Drosophila cells with reduced XRN1 activity ( Gatfield and Izaurralde , 2004 ) .","The MAL-A2 3’ primers show no difference in relative expression in pcm14 mutants compared to the 5’ primers , while tra and Gadd45 have relatively increased levels of a 3’ fragment in pcm14 mutants , revealing endonucleolytic cleavage has occurred between the primer pairs , probably near the stop codon , indicative of NMD-initiated degradation .","p-value between indicated samples using a two-sided Student’s t-test are displayed .","ns indicates a p-value greater than 0 . 05 .","Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00510 . 7554/eLife . 12876 . 006Figure 1—figure supplement 3 . F17 is a null allele of Gadd45 . ( A ) Gadd45F17 is an imprecise excision of the P-element P{EPg}HP20647 , deleting a 894 bp region that includes the entire Gadd45 coding region .","Gadd45E8 is a precise excision of the same P-element , leaving Gadd45 intact .","Coding region in grey; untranslated regions in white .","Arrowhead indicates direction of transcription .","( B ) Adult viability of control and Gadd45F17mutants .","p = 0 . 4463 between Gadd45F17 and controls , using the test of equal or given proportions .","Error bars represent 95% confidence interval of the binomial distribution .","n equals total number of animals scored in each cross . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00610 . 7554/eLife . 12876 . 007Figure 1—figure supplement 4 . Loss of Gadd45 does not restore NMD activity in NMD mutants .","( A ) Expression of the endogenous NMD target transcript traL ( Rehwinkel , 2005; Metzstein and Krasnow , 2006 ) in control male , Upf225G/Y , and Upf225G/Y; Gadd45F17animals , measured by qRT-PCR .","There is no significant difference in traL expression between Upf225G/Y and Upf225G/Y; Gadd45F17animals .","p-values determined by one-sided Student’s t-test between indicated conditions are displayed .","ns indicates a p-value greater than 0 . 05 .","( B ) Relative abundance of PTC-containing Adhn4 ( Chia et al . , 1987 ) and dHR783 ( Fisk and Thummel , 1998 ) allele mRNAs compared to wild-type allele mRNA abundance in animals heterozygous for Adhn4or dHR783in each indicated genotype ( stabilization of dHR783 was not determined in Upf225G/Y; Gadd45F17 animals ) .","Neither reduction nor elimination of Gadd45 restored destabilization of these alleles .","p-values determined by two-sided Student’s t-test between indicated conditions are displayed .","ns indicates a p-value greater than 0 . 05 .","Error represents 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 007 We expected that any specific genes mediating NMD-mutant lethality would have increased expression levels in an NMD mutant and be a direct NMD target .","The only gene located within the suppressing regions to fit these criteria is Gadd45 ( Figure 1C , Figure 1—figure supplement 2A–C ) ( Chapin et al . , 2014 ) .","To determine if NMD targeting of Gadd45 mRNA is critical for viability , we generated a Gadd45 null allele , F17 , which completely removes the Gadd45 coding region ( Figure 1—figure supplement 3A ) and eliminates Gadd45 mRNA expression ( Figure 1—figure supplement 2A ) .","As a heterozygote , Gadd45F17 suppressed Upf225Glethality as strongly as the corresponding deficiency identified by our screen ( Figure 1D ) .","We found that Gadd45F17 homozygous mutants are fully viable ( Figure 1—figure supplement 3B ) , allowing us to test complete loss of Gadd45 for the suppression of NMD-mutant lethality .","Homozygous Gadd45F17 restored full viability to Upf225Gmutants , and remarkably even partially suppressed the complete lethality observed in null Upf1 and Upf2 mutants ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) ( Figure 1D ) .","Importantly , neither reducing nor eliminating Gadd45 restored NMD function to Upf225G mutants , as measured by the expression of both an endogenous NMD target ( Figure 1—figure supplement 4A ) and PTC-containing mRNAs ( Figure 1—figure supplement 4B ) .","In mammals , GADD45 activates the MTK1/MEKK4 kinase in a well-defined stress response pathway ( Takekawa and Saito , 1998 ) .","Strikingly , the Drosophila MTK1 orthologue , Mekk1 , resides within another Upf225G suppressing region ( Figure 1E ) .","Similar to Gadd45 , we found that Mekk1 null mutants ( Inoue et al . , 2001 ) suppressed Upf1 and Upf2 mutant lethality ( Figure 1F ) .","This suppression was not as strong as that caused by a loss of Gadd45 , revealing that although MEKK1 mediates NMD mutant lethality , it is likely that GADD45 has additional downstream effectors that influence viability .","Overall , our findings reveal that increased Gadd45 mRNA stability is the major factor inducing NMD mutant lethality , primarily via increased MEKK1 activity .","Activation of MTK1 in mammals triggers a MAPK signaling cascade that promotes apoptosis ( Takekawa and Saito , 1998 ) .","Over-expression of Gadd45 in Drosophila also induces apoptosis ( Peretz et al . , 2007 ) .","Interestingly , Drosophila cells lacking NMD function show excess cell death in a variety of tissues ( Avery et al . , 2011; Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .","To test if increased Gadd45 contributes to this excess death , we used TUNEL staining to examine cell death in wing imaginal discs from Upf225G mutant third instar larvae .","This analysis revealed elevated levels of cell death compared to controls ( Figure 2A , B , E ) , and this defect was completely suppressed by Gadd45F17 ( Figure 2C–E ) .","To confirm that , this effect was not specific to the Upf2 gene or 25G allele , we examined the wing discs in mutants of another essential NMD gene , Smg5 .","We found that Smg5 discs also showed elevated TUNEL signal , which was eliminated by loss of Gadd45 ( Figure 2—figure supplement 1A–E ) .","These results demonstrate that excess Gadd45 accounts for ectopic cell death in NMD mutant tissues . 10 . 7554/eLife . 12876 . 008Figure 2 . Loss of Gadd45 suppresses NMD-mutant cell death .","( A to D )","DAPI ( blue ) and ( A’to D’ ) TUNEL ( red ) staining in late 3rd instar larval wing discs from control ( A ) ; Upf225G ( B ) ; Gadd45F17 ( C ) ; and Upf225G; Gadd45F17 ( D ) animals .","( A’’ to D’’ ) are 4x view of outlined section at the base of the blade of the wing disc from A’-D’ , respectively .","Scale bar represents 100 μm .","( E ) Relative TUNEL signal in control and mutant wing discs , normalized to control .","p-value between indicated samples using a two-sided Student’s t-test are displayed .","ns indicates a p-value greater than 0 . 05 .","Error bars represent 2 SEM .","n equals total number of discs scored .","( F to I ) w- eye clones in Gadd45+ and Gadd45F17 backgrounds .","Dashed lines indicate clone boundaries .","( J ) Quantification of the fraction of the eye composed of w- cells in control and mutant eyes .","p-values indicate differences between Gadd45 mutant and control in the same NMD background ( indicated by horizontal bars ) or NMD mutant and control in the same Gadd45 background ( indicated by value above each individual bar ) , using a two-sided Student’s t-test .","ns indicates a p-value greater than 0 . 05 .","Error bars represent 2 SEM .","n = 20 eyes for all conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00810 . 7554/eLife . 12876 . 009Figure 2—figure supplement 1 . Loss of Gadd45 suppresses ectopic cell death in Smg5 mutant wing discs .","( A to D )","DAPI ( blue ) and ( A’ to D’ ) TUNEL ( red ) staining in late 3rd instar larval wing discs from Smg5G115/+ ( A ) ; Smg5G115/C391 ( B ) ; Smg5G115/+ Gadd45F17 ( C ) ; and Smg5G115/C391 Gadd45F17 ( D ) animals .","Smg5G115 and Smg5C391 are null Smg5 alleles ( J . O . N . and M . M . M . , unpublished ) .","Scale bar represents 100 μm .","( E ) Relative TUNEL signal in wing discs , normalized to Smg5G115/+ .","p-values determined by two-sided Student’s t-test between indicated conditions are displayed .","ns indicates a p-value greater than 0 . 05 .","Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 009 To test if Gadd45-induced cell death is the only cellular defect in NMD mutants , we examined NMD function in the developing eye .","NMD is required for proper development of eye cells , as clonal patches of NMD mutant cells in eyes are reduced in size ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .","We found that Gadd45 is partially responsible for this defect , as the size of eye-cell clones lacking NMD activity in a Gadd45F17 background was increased , although not fully restored ( Figure 2F–J ) .","These results indicate that some , but not all , defects associated with loss of NMD are dependent on Gadd45 .","Gadd45 is one of the few genes that is directly regulated by NMD in both flies and mammals ( Huang et al . , 2011; Tani et al . , 2012; Viegas et al . , 2007 ) , raising the possibility that excess Gadd45 abundance may also contribute to the NMD-mutant lethality observed in mammalian cells ( Azzalin and Lingner , 2006; Li et al . , 2015; Medghalchi et al . , 2001; Weischenfeldt et al . , 2008 ) .","To test this hypothesis , we analyzed the effects of Gadd45 and Upf1 depletion in mouse NIH-3T3 cells .","Gadd45b mRNA ( also known as MyD118 ) , which is expressed at least 10-fold higher than any other Gadd45 paralogue in these cells ( Yue et al . , 2014 ) , was degraded rapidly in a partially Upf1-dependent manner after transcription was blocked with actinomycin D ( Figure 3A ) , and had increased expression during Upf1 knockdown ( Figure 3D ) , confirming it is sensitive to NMD .","We found that transfection of 3T3 cells with siRNAs targeting Upf1 resulted in significant reduction in cell counts after 48 hr ( Figure 3B ) , but co-transfection with siRNAs targeting both Upf1 and Gadd45b largely reversed this effect ( Figure 3B ) .","The reduction in cell counts was primarily due to increased cell death , as we found that ~25% of cells transfected with Upf1 siRNA were undergoing apoptosis ( Figure 3C ) .","Co-transfection of siRNA targeting Gadd45b almost entirely eliminated this increase ( Figure 3C ) , indicating the excess apoptosis observed in Upf1-knockdown cells was mostly due to increased Gadd45 activity .","However , while Gadd45b knockdown very greatly suppresses this excess death , it does not as fully rescue cell numbers , suggesting loss of NMD may lead to both Gadd45b-dependent cell death as well as a Gadd45b-independent effect on proliferation .","This mirrors the conclusions we made about the partial suppression of cell number defects in the Drosophila eye .","Importantly , Upf1 mRNA expression was equivalently reduced and the expression of the mammalian endogenous NMD targets Rassf1 and CRCP ( Tani et al . , 2012 ) was equivalently increased in both the single and double knockdown experiments ( Figure 3D ) , indicating that the restoration of viability was not due to a recovery of NMD pathway activity . 10 . 7554/eLife . 12876 . 010Figure 3 . Gadd45b mediates cell lethality in Upf1 siRNA knockdown 3T3 mouse embryonic fibroblasts .","( A ) Relative Gadd45b mRNA expression measured by qRT-PCR in NIH-3T3 cells after 48 hr of control ( black ) or Upf1 ( red ) siRNA treatment and 0 to 2 hr of actinomycin D treatment , normalized to expression prior to actinomycin treatment .","The half-life calculated for each decay curve is indicated .","( B ) Relative viable cell count of Upf1 and Gadd45b single and double siRNA treatment normalized to control siRNA .","p-values display two-sided Student’s t-test between indicated conditions .","( C ) Quantification of apoptosis as measured by annexin V staining .","p-values display two-sided Student’s t-test between indicated conditions .","( D ) Relative mRNA expression of Upf1 , Gadd45b , and two mammalian endogenous NMD targets , Rassf1 and CRCP ( Tani et al . , 2012 ) measured by qRT-PCR in Gadd45b and Upf1 single and double siRNA knockdown cells , normalized to expression in the control siRNA condition .","p-values display one-sided Student’s t-test for each condition compared to control .","Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 01010 . 7554/eLife . 12876 . 011Figure 3—figure supplement 1 . GADD45A mediates cell lethality in Upf1 knockdown HEK293 cells .","( A ) Relative GADD45A mRNA expression measured by qRT-PCR in UPF1 and GADD45A single and double siRNA knockdown 72 hr after siRNA transfection in HEK293 cells , normalized to expression in the control siRNA condition .","p-values display one-sided Student’s t-test for each condition compared to control .","( B ) Viable cell count of UPF1 and GADD45A single and double siRNA-treated cells normalized to control siRNA -treated cells .","p-values display two-sided Student’s t-test between indicated conditions .","( C ) Relative UPF1 mRNA expression measured by qRT-PCR in UPF1 and GADD45A single and double siRNA knockdown , normalized to expression in control siRNA condition .","p-values display one-sided Student’s t-test for each condition compared to control .","Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 011 To extend our analysis to other mammalian cells , we analyzed the role of Gadd45 mediating the effects of loss of NMD in HEK293 cells .","We found , similarly to 3T3 cells , that siRNA knockdown of UPF1 in HEK293 cells led to increased GADD45A expression and reduced cell numbers compared to control siRNA ( Figure 3—figure supplement 1A , B ) .","Although transfection of siRNA targeting GADD45A alone slightly reduced HEK293 cell numbers , co-transfection with UPF1 siRNA did not further reduce cell count ( Figure 3—figure supplement 1B ) , and UPF1 expression was equivalently reduced in the single and double knockdown conditions ( Figure 3—figure supplement 1C ) .","These results suggest that UPF1 knockdown is no longer detrimental to HEK293 cell viability in the absence of GADD45A expression .","We conclude that increased expression of mammalian Gadd45 genes contributes to lethality in NMD-deficient mouse and human cells , as Gadd45 does in Drosophila .","Deconvoluting the contributions to organismal viability of the PTC-surveillance versus gene-regulatory functions of NMD has been historically difficult ( Hwang and Maquat , 2011 ) .","Here , we show that viability can be restored to Drosophila lacking core NMD factors when a single endogenous NMD target , Gadd45 , is eliminated , and that the requirement for the regulation of Gadd45 by NMD is evolutionarily conserved from flies to mammals .","Although our data suggest that up-regulation of Gadd45 is a major factor contributing to lethality when NMD activity is lost , it is likely that other NMD targets also contribute to the observed lethality .","In particular , viability is not restored to 100% in null Upf1; or Upf2; Gadd45 double mutants .","In addition , loss of Gadd45 suppresses programmed cell death caused by defects in NMD , but not additional cell cycle defects , as implied by the incomplete suppression in the Drosophila eye and mammalian cell culture .","Such defects in the cell cycle may be particularly pronounced during the development of certain tissue , or specific developmental stages .","Indeed , NMD has been reported to have differing stage and tissue- specific activities ( Bao et al . , 2015; Bruno et al . , 2011; Colak et al . , 2013; Li et al . , 2015 ) .","Whether this is due to a role in surveillance or another specific target remains unclear , but examination of the effects of loss of NMD in Gadd45 mutants should allow exploration of these possibilities .","The benefit for such a mechanism regulating Gadd45 expression may lie in a function of NMD in restricting viral growth ( Balistreri et al . , 2014 ) .","Because viruses encode trans-acting factors to inhibit NMD ( Mocquet et al . , 2012 ) , the resulting accumulation of GADD45 in infected cells may act as a “molecular tripwire” that rapidly elicits a stress response and cell death .","This outcome suggests that regulating responses to infection may underlie a conserved essential function of NMD .","Intriguingly , restriction of pathogens via NMD extends to plants ( Garcia et al . , 2014 ) , where NMD mutant lethality in A . thaliana , which do not encode Gadd45 orthologues , may be caused by the overexpression of a subset of immune-related intracellular nucleotide-binding leucine-rich repeat receptors , some of which are endogenous NMD targets ( Gloggnitzer et al . , 2014 ) .","In contrast , eukaryotes that do not rely on the activation of programmed cell death to protect against viruses , such as S . cerevisiae , S . pombe , and C . elegans , do not require NMD for viability ( Hodgkin et al . , 1989; Leeds et al . , 1991; Mendell et al . , 2000 ) .","Together these observations suggest a potential novel role for NMD and Gadd45 in immune responses , triggering the death of infected cells during pathogenic challenges .","Restoring the expression of PTC-containing alleles via NMD inhibition has been proposed as a promising therapy for a wide range of recessive genetic diseases ( Keeling et al . , 2014 ) .","Translation of stable PTC-containing mRNAs would produce truncated proteins that may be partially functional and alleviate disease symptoms normally caused by complete loss of the protein .","However , the essential function for NMD in viability has raised the concern that these therapies may have prohibitive side effects .","Our findings reveal a molecular basis for dealing with this obstacle by suggesting that inhibiting both the NMD and Gadd45 pathways ( Tornatore et al . , 2014 ) in combination could provide an effective and safe treatment for patients with debilitating genetic disorders ."],["Drosophila melanogaster stocks were raised on standard cornmeal/dextrose food at 25° .","The NMD mutant alleles Upf225G , Upf27-5A , and Upf126A ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) are on y w FRT19A chromosomes .","These alleles were balanced over FM7i , P{ActGFP}JMR3 ( Reichhart and Ferrandon , 1998 ) .","Smg5G115 and Smg5C391 are null alleles of Smg5 ( J . O . N . , D . Förster , S . Luschnig , and M . M . M . , unpublished ) and will be described in detail later .","The Smg5 alleles are balanced over CyO , P{Dfd:eYFP w+} ( Le et al . , 2006 ) .","Other alleles used were P{w[+mC]=EPg}HP20647 ( Staudt et al . , 2005 ) , Mekk1Ur36 ( Inoue et al . , 2001 ) recombined on FRT82B by D . Ryoo , ey-FLP ( Newsome et al . , 2000 ) , pcm14 ( Waldron et al . , 2015 ) , Adhn4 ( Chia et al . , 1987 ) and DHR783 ( Fisk and Thummel , 1998 ) .","Control chromosomes were y w FRT19A ( for Upf1 and Upf2 ) and FRT82B ( for Mekk1 ) ( Xu and Rubin , 1993 ) .","For all experiments using Gadd45F17 we used the Gadd45E8 precise excision as a control .","For viability assays , we mated flies for 3 days and collected all progeny each day for 10 days , starting 10 days after the cross was initiated .","The total numbers of F1 mutant and balancer males were scored , and the ratio of mutant males to balancer males was used to determine mutant animal viability .","To control for balancer viability within each experiment , we normalized the ratio of mutant to balancer animals to a ratio of the appropriate control chromosome to balancer animals produced from a parallel cross .","We screened autosomal deficiencies from the DrosDel collection ( Ryder et al . , 2007 ) .","All deficiencies scored can be found in Supplementary file","1 . Deficiencies on chromosome 2 were balanced over CyO , and deficiencies on chromosome 3 were balanced over TM6C .","We mated males from each deficiency stock to y w Upf225G FRT19A/FM7i , P{ActGFP}JMR3 females and scored all F1 males for the presence or absence of each balancer .","For any given deficiency tested , the percentage of Deficiency / + males that are Upf225G mutants , less the percentage of Balancer / + males that are Upf225G mutants was calculated , producing a Deficiency Suppression Score ( DSS ) , which represents the effect of an individual deficiency on the increase or decrease in Upf225G viability , while controlling for each deficiency’s general influence on viability .","A DSS greater than 0 . 1 indicates suppression of lethality .","Supplemental deficiencies used were from the Exelixis collection ( Parks et al . , 2004 ) and Df ( 2R ) sple-J1 ( Heitzler et al . , 1993 ) .","Deficiency mapping to the Drosophila genome was performed using the 5 . 1 genome release .","RNA-seq data sets were acquired from Chapin et al . ( 2014 ) ( archives SRR896609 , SRR896616 , SRR503415 , and SRR503416 ) and aligned using Bowtie and TopHat alignment with standard remapping parameters to the 5 . 1 Drosophila genome release .","SAMtools accessory scripts were used to retrieve read counts for deficiency and control regions .","All read counts were normalized to reads per million within each data set .","Average normalized reads in Upf225G samples were normalized to the relative reads of 74 ribosomal proteins in Upf225G samples compared to control samples .","Total normalized reads within the regions removed by each deficiency were averaged between biological replicates , and the difference between the Upf225Gand control samples was divided by one million to determine percent increase in genomic load across each deficiency region .","We produced P-element excision lines from the P{w[+mC]=EPg}HP20647 P-element insertion line crossed to a Δ2–3 transposase stock .","We mated F1 males containing the P-element and transposase on a CyO balancer to w; Tft / CyO females .","Cy+ Tft white-eyed F2 males were then individually mated to w; Tft / CyO females .","We then collected Tft+ , Cy males and females to create an isogenic stock from each individually mated F2 male .","To identify precise excisions we used the primers Gadd45_F1 / Gadd45_R1 flanking the P-element insert site to amplify a region across the excised P-element .","Lines that failed to amplify with these primers were candidate imprecise excisions , which we then tested with Gadd45_F1 / Gadd45_R3 primers for deletions .","Any detected deletions were subsequently sequenced using these same primers .","Primer sequences are found in Supplementary file","2 . We generated eye clones with the FLP/FRT system using the ey-FLP driver ( Newsome et al . , 2000 ) to induce recombination .","We imaged eyes on a Leica MZ125 stereo microscope with a Retiga-2000R camera ( QImaging , Canada ) with QCapture 3 . 1 . 2 software ( QImaging ) .","We focused images using the ImageJ stack focuser plugin and quantified relative eye clone size using the ImageJ analyzer tools .","A total of 20 eyes from 20 individual animals were scored for each condition .","For TUNEL assays , third instar larval wing discs were dissected as described in Sullivan et al . ( Sullivan et al . , 2000 ) .","TUNEL staining was performed using the Apoptag Red in situ Apoptosis Detection Kit ( Chimicon International Inc . , Billerica , MA ) according to Chakraborty et al . ( Chakraborty et al . , 2015 ) .","We DAPI stained wing discs ( 1:5000 ) for 5 min prior to mounting .","Confocal images were acquired using a Zeiss LSM710 laser scanning confocal microscope ( Carl Zeiss AG , Germany ) .","3-dimensional datasets were acquired with a Plan-Apochromat 20X/0 . 8 lens , 1 . 34 μm z-step , using the Zeiss ZEN software .","To measure TUNEL signal intensity z-projections images were summed with ImageJ .","Background signal was removed by using the ImageJ MaxEntropy auto-threshold .","Relative total TUNEL signal intensity was calculated using the ImageJ analyzer tools to measure the total pixel intensity within the wing discs of TUNEL images and normalized to the average intensity in control conditions .","For annexin V staining , we collected media ( including floating cells ) from siRNA treated cells .","We spun down media at 950g for 4 min to pellet cells , and then aspirated remaining media .","Concurrently , we trypsinized siRNA-treated cells still on plates and added them to the same respective tube as previously spun-down media .","Following the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit ( Abcam , UK ) protocol , we stained for apoptotic cells .","We visualized cells on an Olympus IX51 microscope ( Olympus , Japan ) with 20X objective .","We collected bright field as well as fluorescent images using a FITC filter with a QImaging QICam Fast1394 camera and QCaptureP software ( QImaging ) .","We analyzed cells by counting all cells within a bright field image as well as the annexin V positive cells from the same image .","The number of annexin V positive cells was divided by total cell number to generate the fraction of apoptotic cells for each treatment .",">3000 total cells were counted across three biological replicates for each treatment .","We cultured mouse NIH-3T3 cells ( ATCC ) or HEK293 cells ( ATCC ) in DMEM ( Thermo-Fisher , Waltham , MA ) supplemented with 10% fetal bovine serum and glutamine .","For siRNA experiments , we transfected cells using RNAiMax and 24 pmol of negative control siRNA ( Qiagen , Netherlands ) , Upf1 siRNA ( Qiagen ) , or Gadd45b siRNA ( Sigma-Aldrich ) for 3T3 cell experiments , or negative control siRNA ( Qiagen ) , UPF1 siRNA ( Qiagen ) , or GADD45A siRNA ( Sigma-Aldrich , St . Louis , MO ) for HEK293 experiments .","For double siRNA-treated cells , we used 24 pmol of each Upf1 and Gadd45b siRNA for 3T3 experiments or UPF1 and GADD45A siRNA for HEK293 experiments .","For actinomycin experiments , we incubated cells with siRNA for 48 hr before changing the media and then incubated with 2 μg/mL actinomycin ( Sigma-Aldrich ) for 1 or 2 hr . mRNA half-life was determined by fitting an exponential decay curve to the relative expression at each time point ( Tani et al . , 2012 ) .","t1/2 was calculated based on the average expression at each time point , and the mean t1/2 for each condition is represented .","For cell counting experiments , we trypsinized cells , incubated a small aliquot with Trypan Blue at a final concentration of 0 . 04% in complete media , and counted Trypan Blue negative cells .","RNA was collected from the remaining cells , and relative mRNA levels were measured as described below .","For Drosophila qRT-PCR analyses , we collected 5–10 adult animals frozen in liquid nitrogen .","We isolated total RNA using TRIzol reagent ( Invitrogen ) and phase-lock tubes ( 5-Prime ) , and the RNeasy mini kit ( Qiagen ) .","We used on-column RNase-free DNase treatment ( Qiagen ) to reduce genomic contamination .","We determined RNA concentration by spectrophotometer and normalized concentration for reverse transcription .","For reverse transcription , we used random decamers and MMLV8 reverse transcriptase ( Retroscript Kit , Thermo-FIsher ) .","We performed qRT-PCR analysis using the SYBR Green qPCR Supermix ( Bio-Rad , Hercules , CA ) and the Bio-Rad iCycler thermocycler .","All experimental reactions were performed using three technical replicates and a minimum of three biological replicates per condition , and the expression level of all experimental assays was normalized to RpL32 mRNA expression .","For cell culture qRT-PCR analyses , we collected RNA following the Zymo Research Quick RNA MiniPrep kits protocol , and synthesized cDNA using MMLV reverse transcriptase ( NEB , Ipswich , MA ) with a template of 1 µg of total RNA and priming with a T18 oligo .","We measured relative mRNA levels by qRT-PCR using the Masterplex ep realplex ( Eppendorf , Germany ) with SYBR green fluorescent dye .","Each sample was measured with technical triplicates and three biological replicates , and target mRNA levels were normalized to those of ribosomal protein 19 ( Rpl19 ) mRNA .","For all qRT-PCR analyses we also measured samples that had been made without reverse transcriptase to ensure that signal was not due to genomic DNA .","Primer sequences can be found in Supplementary file","2 . We cloned the UAS-GFP::Gadd45 3’ UTR and control UAS-GFP::Act5C 3’ UTR constructs using the primers G45_3U_X1_F / G45_3U_S1_R or Act5C_X1_F / Act5C_S1_R ( Supplementary file 2 ) to amplify the Gadd45 and Act5C 3’ UTRs , respectively , from genomic DNA .","PCR fragments were inserted into the Zero Blunt TOPO vector ( Thermo-Fisher ) , sequenced to assure fidelity , and digested and cloned into a pUAST-attB GFP vector using standard cloning procedures to replace the SV40 3’ UTR .","Plasmids were injected by BestGene ( Chino Hills , CA ) into a stock containing the VK00027 attP site ( Venken et al . , 2006 ) for phiC31 directed integration .","We used previously described UAS-GFP::SV40 3’ UTR animals ( Metzstein and Krasnow , 2006 ) .","For imaging , wandering late L3 larvae were collected and examined using a Leica MZ 16F microscope and the Leica DFC340 FX camera with the Leica Application Suite v3 . 3 . 0 software .","We collected adult F1 Upf2+; Gadd45E8/+ , Upf225G; Gadd45E8/+ , and Upf225G; Gadd45F17/+males that were also heterozygous for either the dHR783 or Adhn4 .","The Adhn4allele is a PTC-containing allele and has been demonstrated to be a direct NMD target based on cleavage by Smg6 ( Gatfield and Izaurralde , 2004 ) .","The dHR783 allele is also a PTC-containing allele and thus is presumably degraded by NMD ( Fisk and Thummel , 1998 ) .","At least three biological replicates were collected for each condition .","We isolated RNA and generated cDNA as described in methods above and used this cDNA as a template for PCR amplification of the dHR78 transcript with the DRH78_F3 / DHR78_R3 primers and the Adh transcript with the Adh_F and Adh_R primers ( Supplementary file 2 ) , which flank the nonsense mutation in the respective transcripts .","To compare the relative abundance of the dHR783 allele to the wild-type allele , PCR products were Sanger sequenced , and the relative peak intensity for a T ( dHR783 allele ) compared to a C ( wild-type allele ) at nucleotide 1063 was compared .","To compare the relative abundance of the Adhn4 allele to the wild-type allele , PCR products were digested with PvuII ( a site disrupted by the n4 mutation ) , separated on a 1% agarose gel and stained with ethidium bromide .","The relative intensity of the cut and uncut bands was determined using ImageJ and normalized for fragment length .","All samples were ran on the same gel and compared under identical conditions .","All ratios were normalized to the ratio in the Upf225G; Gadd45E8/+condition .","All figures displaying viability assays represent a proportion of animals of the indicated genotypes that survive to adulthood; error bars for these figures represent the 95% confidence interval of the binomial distribution , and the Test of qual or Given Proportions was used to determine significance difference in these proportions between genotypes .","All other figures represent the mean value of multiple replicates have error bars depicting ± 2 SEM , which is a close approximation of the 95% confidence interval ( Krzywinski and Altman , 2013 ) .","For tests between two variable measures , a two-sided paired Student’s t-test was used to determine significance difference between mean value data .","For most qPCR experiments , data was compared to a normalized control , set to a constant of 1 , so these tests were performed with a one-sided Student’s t-test ."]],"string":"[\n [\n \"Maintaining proper gene expression is critical for normal development and physiology .\",\n \"In addition to de novo transcription , mRNA stability substantially contributes to forming the landscape of expression in a cell .\",\n \"The nonsense-mediated mRNA decay ( NMD ) pathway is a trans-acting mechanism that destabilizes mRNAs , and is best known for its well-described role as a quality control system , degrading abnormal mRNAs containing premature termination codons ( PTCs ) ( Celik et al . , 2015 ) .\",\n \"NMD also degrades many wild-type endogenous mRNAs and thus is an important aspect of their post-transcriptional ( Peccarelli and Kebaara , 2014 ) .\",\n \"Loss of either of the core NMD genes Upf1 ( Rent1 ) or Upf2 causes lethality in most eukaryotes ( Kerényi et al . , 2008; Medghalchi et al . , 2001; Metzstein and Krasnow , 2006; Weischenfeldt et al . , 2008; Wittkopp et al . , 2009 ) , indicating regulation of mRNA stability by NMD is critical for viability .\",\n \"However , the relative contributions to lethality from ectopic stabilization of PTC-containing mRNAs or endogenous NMD targets in NMD mutants remains unclear ( Hwang and Maquat , 2011 ) .\",\n \"To identify which ectopically stabilized mRNAs are responsible for inducing lethality in NMD mutants , we performed an unbiased genetic suppressor screen seeking to restore viability in a Drosophila NMD mutant .\",\n \"To detect subtle increases in survival , we screened to suppress the lethality of animals mutant for the partially viable , hypomorphic Upf225G allele , of which 10% survive to adulthood ( Chapin et al . , 2014; Metzstein and Krasnow , 2006 ) .\",\n \"We crossed this allele to heterozygous deficiencies to simultaneously reduce the mRNA abundance of several loci ( Figure 1A ) .\",\n \"Of the 376 deficiencies tested , covering more than half the genome , ~10% suppressed NMD mutant lethality ( Figure 1B , Figure 1—figure supplement 1A ) .\",\n \"The suppression effect could not be explained by a reduction in overall mRNA load , as there was only a weak correlation between the increase in mRNAs expressed from a genomic region upon loss of NMD function and the strength of suppression when that region was removed by a deficiency ( Figure 1—figure supplement 1B ) .\",\n \"Rather , deficiencies that suppressed NMD-mutant lethality clustered in three genomic regions ( Figure 1—figure supplement 1A ) .\",\n \"These findings suggest that NMD mutant lethality is not the result of a global excess of nonspecific mRNAs , but rather is mediated by specific genes residing within the few identified regions . 10 . 7554/eLife . 12876 . 003Figure 1 . Drosophila suppressor screen identifies the Gadd45 pathway as the inducer of NMD-mutant lethality .\",\n \"( A ) Scheme to screen deficiencies for the suppression of Upf225G partial lethality .\",\n \"The Deficiency Suppression Score ( DSS ) represents the relative difference in Upf225G viability when crossed to a heterozygous deficiency ( Df ) compared to when crossed to a balancer ( Bal ) ( See Methods ) .\",\n \"( B ) DSS from 376 screened deficiencies ranked by score .\",\n \"A DSS greater than 0 . 1 ( dotted line ) indicates that deficiency suppresses Upf225G lethality .\",\n \"( C and E )\",\n \"Candidate suppressing regions uncovering Gadd45 ( C ) and Mekk1 ( E ) .\",\n \"DSSs are shown in parenthesis .\",\n \"Dotted lines denote extent of regions deleted by suppressing deficiencies but not non-suppressing deficiencies .\",\n \"Filled blocks on chromosomes indicate predicted gene spans , Gadd45 pathway genes are indicated in red; suppressing deficiencies indicated in green , sple-J1 has undefined breakpoints located within hashed regions .\",\n \"( D and F )\",\n \"NMD mutant adult viability in combination with Gadd45F17 ( D ) or Mekk1Ur36 ( F ) mutants .\",\n \"Upf126A and Upf27-5A are null alleles ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .\",\n \"p-value compared to controls determined by the test of equal or given proportions indicated .\",\n \"Error bars represent 95% confidence interval of the binomial distribution .\",\n \"n equals total number of animals scored in each cross . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00310 . 7554/eLife . 12876 . 004Figure 1—figure supplement 1 . Reduced expression of specific loci , not overall mRNA abundance , produces NMD mutant suppression by deficiencies .\",\n \"( A ) Map of 376 autosomal DrosDel deficiencies with an isogenic background and molecularly defined breakpoints ( Ryder et al . , 2007 ) and eight other deficiencies used to further test candidate suppressing regions without overlapping DrosDel deficiencies .\",\n \"39 total deficiencies suppress Upf225G lethality , shown in green .\",\n \"Regions deleted by any non-suppressing deficiencies were eliminated as candidate suppressing regions , removing false positives and reducing the size of the candidate intervals .\",\n \"The three candidate suppressing regions that are deleted only by suppressing deficiencies are indicated in black and labeled 1–3 .\",\n \"( B ) Each deficiency’s Deficiency Suppression Score ( DSS ) compared to percent increase in RNA abundance in Upf225G compared to wild-type from loci removed by that deficiency according to RNA-seq from Chapin et al . ( 2014 ) .\",\n \"Trend line in red; statistics calculated using Pearson correlation test . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00410 . 7554/eLife . 12876 . 005Figure 1—figure supplement 2 . Drosophila Gadd45 is an endogenous direct NMD target .\",\n \"( A ) Gadd45 mRNA expression in adults of the given genotypes measured by qRT-PCR .\",\n \"Gadd45 mRNA expression is increased 16 . 7-fold in Upf225G mutants , and is eliminated by Gadd45F17 mutants .\",\n \"p-values display one-sided Student’s t-test of indicated condition compared to control .\",\n \"Error bars represent 2 SEM .\",\n \"( B ) Fluorescence of GFP transgenes with SV40 , Act5C , or Gadd45 3’ UTRs expressed by UAS driven by Actin:GAL4 in Upf2+ or Upf225G third instar larvae .\",\n \"SV40 and Gadd45 3’ UTR constructs show significantly increased fluorescence in Upf225G animals compared to Upf2+ , indicating NMD-dependent post-transcriptional degradation of mRNAs containing these UTRs .\",\n \"The Act5C 3’ UTR construct has similar fluorescence in both backgrounds , indicating NMD does not regulate the post-transcriptional stability of this UTR .\",\n \"Micrographs show dorsal views with anterior at top .\",\n \"( C ) MAL-A2 , traL , and Gadd45 5’ and 3’ fragment mRNA expression measured by qRT-PCR in pcm14 null mutants ( Waldron et al . , 2015 ) normalized to controls .\",\n \"Transcript structures of a non-NMD-target , maltase A2 ( MAL-A2 ) ; a known NMD-targeted transcript , the non-sex specific isoform of transformer ( traL ) ( Rehwinkel , 2005; Metzstein and Krasnow , 2006 ) ; and the Gadd45 transcript ( note Gadd45 has no introns ) .\",\n \"Open boxes indicate UTRs; grey boxes indicate coding regions .\",\n \"NMD targeting initiates endonucleolytic cleavage near the stop codon ( Gatfield and Izaurralde , 2004 ) , producing 5’ and 3’ fragments with unprotected ends , which are then subjected to degradation by cytoplasmic 3’-to-5’ and 5’-to-3’ exonucleases , respectively .\",\n \"qRT-PCR primer pairs 5’ ( red ) and 3’ ( blue ) to the cleavage site can be used to differentially measure the quantity of these fragments .\",\n \"The Drosophila 5’-to-3’ exonuclease is encoded by the XRN1 homologue pacman ( pcm ) , and fragments 3’ to an endonucleolytic NMD cleavage accumulate in Drosophila cells with reduced XRN1 activity ( Gatfield and Izaurralde , 2004 ) .\",\n \"The MAL-A2 3’ primers show no difference in relative expression in pcm14 mutants compared to the 5’ primers , while tra and Gadd45 have relatively increased levels of a 3’ fragment in pcm14 mutants , revealing endonucleolytic cleavage has occurred between the primer pairs , probably near the stop codon , indicative of NMD-initiated degradation .\",\n \"p-value between indicated samples using a two-sided Student’s t-test are displayed .\",\n \"ns indicates a p-value greater than 0 . 05 .\",\n \"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00510 . 7554/eLife . 12876 . 006Figure 1—figure supplement 3 . F17 is a null allele of Gadd45 . ( A ) Gadd45F17 is an imprecise excision of the P-element P{EPg}HP20647 , deleting a 894 bp region that includes the entire Gadd45 coding region .\",\n \"Gadd45E8 is a precise excision of the same P-element , leaving Gadd45 intact .\",\n \"Coding region in grey; untranslated regions in white .\",\n \"Arrowhead indicates direction of transcription .\",\n \"( B ) Adult viability of control and Gadd45F17mutants .\",\n \"p = 0 . 4463 between Gadd45F17 and controls , using the test of equal or given proportions .\",\n \"Error bars represent 95% confidence interval of the binomial distribution .\",\n \"n equals total number of animals scored in each cross . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00610 . 7554/eLife . 12876 . 007Figure 1—figure supplement 4 . Loss of Gadd45 does not restore NMD activity in NMD mutants .\",\n \"( A ) Expression of the endogenous NMD target transcript traL ( Rehwinkel , 2005; Metzstein and Krasnow , 2006 ) in control male , Upf225G/Y , and Upf225G/Y; Gadd45F17animals , measured by qRT-PCR .\",\n \"There is no significant difference in traL expression between Upf225G/Y and Upf225G/Y; Gadd45F17animals .\",\n \"p-values determined by one-sided Student’s t-test between indicated conditions are displayed .\",\n \"ns indicates a p-value greater than 0 . 05 .\",\n \"( B ) Relative abundance of PTC-containing Adhn4 ( Chia et al . , 1987 ) and dHR783 ( Fisk and Thummel , 1998 ) allele mRNAs compared to wild-type allele mRNA abundance in animals heterozygous for Adhn4or dHR783in each indicated genotype ( stabilization of dHR783 was not determined in Upf225G/Y; Gadd45F17 animals ) .\",\n \"Neither reduction nor elimination of Gadd45 restored destabilization of these alleles .\",\n \"p-values determined by two-sided Student’s t-test between indicated conditions are displayed .\",\n \"ns indicates a p-value greater than 0 . 05 .\",\n \"Error represents 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 007 We expected that any specific genes mediating NMD-mutant lethality would have increased expression levels in an NMD mutant and be a direct NMD target .\",\n \"The only gene located within the suppressing regions to fit these criteria is Gadd45 ( Figure 1C , Figure 1—figure supplement 2A–C ) ( Chapin et al . , 2014 ) .\",\n \"To determine if NMD targeting of Gadd45 mRNA is critical for viability , we generated a Gadd45 null allele , F17 , which completely removes the Gadd45 coding region ( Figure 1—figure supplement 3A ) and eliminates Gadd45 mRNA expression ( Figure 1—figure supplement 2A ) .\",\n \"As a heterozygote , Gadd45F17 suppressed Upf225Glethality as strongly as the corresponding deficiency identified by our screen ( Figure 1D ) .\",\n \"We found that Gadd45F17 homozygous mutants are fully viable ( Figure 1—figure supplement 3B ) , allowing us to test complete loss of Gadd45 for the suppression of NMD-mutant lethality .\",\n \"Homozygous Gadd45F17 restored full viability to Upf225Gmutants , and remarkably even partially suppressed the complete lethality observed in null Upf1 and Upf2 mutants ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) ( Figure 1D ) .\",\n \"Importantly , neither reducing nor eliminating Gadd45 restored NMD function to Upf225G mutants , as measured by the expression of both an endogenous NMD target ( Figure 1—figure supplement 4A ) and PTC-containing mRNAs ( Figure 1—figure supplement 4B ) .\",\n \"In mammals , GADD45 activates the MTK1/MEKK4 kinase in a well-defined stress response pathway ( Takekawa and Saito , 1998 ) .\",\n \"Strikingly , the Drosophila MTK1 orthologue , Mekk1 , resides within another Upf225G suppressing region ( Figure 1E ) .\",\n \"Similar to Gadd45 , we found that Mekk1 null mutants ( Inoue et al . , 2001 ) suppressed Upf1 and Upf2 mutant lethality ( Figure 1F ) .\",\n \"This suppression was not as strong as that caused by a loss of Gadd45 , revealing that although MEKK1 mediates NMD mutant lethality , it is likely that GADD45 has additional downstream effectors that influence viability .\",\n \"Overall , our findings reveal that increased Gadd45 mRNA stability is the major factor inducing NMD mutant lethality , primarily via increased MEKK1 activity .\",\n \"Activation of MTK1 in mammals triggers a MAPK signaling cascade that promotes apoptosis ( Takekawa and Saito , 1998 ) .\",\n \"Over-expression of Gadd45 in Drosophila also induces apoptosis ( Peretz et al . , 2007 ) .\",\n \"Interestingly , Drosophila cells lacking NMD function show excess cell death in a variety of tissues ( Avery et al . , 2011; Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .\",\n \"To test if increased Gadd45 contributes to this excess death , we used TUNEL staining to examine cell death in wing imaginal discs from Upf225G mutant third instar larvae .\",\n \"This analysis revealed elevated levels of cell death compared to controls ( Figure 2A , B , E ) , and this defect was completely suppressed by Gadd45F17 ( Figure 2C–E ) .\",\n \"To confirm that , this effect was not specific to the Upf2 gene or 25G allele , we examined the wing discs in mutants of another essential NMD gene , Smg5 .\",\n \"We found that Smg5 discs also showed elevated TUNEL signal , which was eliminated by loss of Gadd45 ( Figure 2—figure supplement 1A–E ) .\",\n \"These results demonstrate that excess Gadd45 accounts for ectopic cell death in NMD mutant tissues . 10 . 7554/eLife . 12876 . 008Figure 2 . Loss of Gadd45 suppresses NMD-mutant cell death .\",\n \"( A to D )\",\n \"DAPI ( blue ) and ( A’to D’ ) TUNEL ( red ) staining in late 3rd instar larval wing discs from control ( A ) ; Upf225G ( B ) ; Gadd45F17 ( C ) ; and Upf225G; Gadd45F17 ( D ) animals .\",\n \"( A’’ to D’’ ) are 4x view of outlined section at the base of the blade of the wing disc from A’-D’ , respectively .\",\n \"Scale bar represents 100 μm .\",\n \"( E ) Relative TUNEL signal in control and mutant wing discs , normalized to control .\",\n \"p-value between indicated samples using a two-sided Student’s t-test are displayed .\",\n \"ns indicates a p-value greater than 0 . 05 .\",\n \"Error bars represent 2 SEM .\",\n \"n equals total number of discs scored .\",\n \"( F to I ) w- eye clones in Gadd45+ and Gadd45F17 backgrounds .\",\n \"Dashed lines indicate clone boundaries .\",\n \"( J ) Quantification of the fraction of the eye composed of w- cells in control and mutant eyes .\",\n \"p-values indicate differences between Gadd45 mutant and control in the same NMD background ( indicated by horizontal bars ) or NMD mutant and control in the same Gadd45 background ( indicated by value above each individual bar ) , using a two-sided Student’s t-test .\",\n \"ns indicates a p-value greater than 0 . 05 .\",\n \"Error bars represent 2 SEM .\",\n \"n = 20 eyes for all conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00810 . 7554/eLife . 12876 . 009Figure 2—figure supplement 1 . Loss of Gadd45 suppresses ectopic cell death in Smg5 mutant wing discs .\",\n \"( A to D )\",\n \"DAPI ( blue ) and ( A’ to D’ ) TUNEL ( red ) staining in late 3rd instar larval wing discs from Smg5G115/+ ( A ) ; Smg5G115/C391 ( B ) ; Smg5G115/+ Gadd45F17 ( C ) ; and Smg5G115/C391 Gadd45F17 ( D ) animals .\",\n \"Smg5G115 and Smg5C391 are null Smg5 alleles ( J . O . N . and M . M . M . , unpublished ) .\",\n \"Scale bar represents 100 μm .\",\n \"( E ) Relative TUNEL signal in wing discs , normalized to Smg5G115/+ .\",\n \"p-values determined by two-sided Student’s t-test between indicated conditions are displayed .\",\n \"ns indicates a p-value greater than 0 . 05 .\",\n \"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 009 To test if Gadd45-induced cell death is the only cellular defect in NMD mutants , we examined NMD function in the developing eye .\",\n \"NMD is required for proper development of eye cells , as clonal patches of NMD mutant cells in eyes are reduced in size ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .\",\n \"We found that Gadd45 is partially responsible for this defect , as the size of eye-cell clones lacking NMD activity in a Gadd45F17 background was increased , although not fully restored ( Figure 2F–J ) .\",\n \"These results indicate that some , but not all , defects associated with loss of NMD are dependent on Gadd45 .\",\n \"Gadd45 is one of the few genes that is directly regulated by NMD in both flies and mammals ( Huang et al . , 2011; Tani et al . , 2012; Viegas et al . , 2007 ) , raising the possibility that excess Gadd45 abundance may also contribute to the NMD-mutant lethality observed in mammalian cells ( Azzalin and Lingner , 2006; Li et al . , 2015; Medghalchi et al . , 2001; Weischenfeldt et al . , 2008 ) .\",\n \"To test this hypothesis , we analyzed the effects of Gadd45 and Upf1 depletion in mouse NIH-3T3 cells .\",\n \"Gadd45b mRNA ( also known as MyD118 ) , which is expressed at least 10-fold higher than any other Gadd45 paralogue in these cells ( Yue et al . , 2014 ) , was degraded rapidly in a partially Upf1-dependent manner after transcription was blocked with actinomycin D ( Figure 3A ) , and had increased expression during Upf1 knockdown ( Figure 3D ) , confirming it is sensitive to NMD .\",\n \"We found that transfection of 3T3 cells with siRNAs targeting Upf1 resulted in significant reduction in cell counts after 48 hr ( Figure 3B ) , but co-transfection with siRNAs targeting both Upf1 and Gadd45b largely reversed this effect ( Figure 3B ) .\",\n \"The reduction in cell counts was primarily due to increased cell death , as we found that ~25% of cells transfected with Upf1 siRNA were undergoing apoptosis ( Figure 3C ) .\",\n \"Co-transfection of siRNA targeting Gadd45b almost entirely eliminated this increase ( Figure 3C ) , indicating the excess apoptosis observed in Upf1-knockdown cells was mostly due to increased Gadd45 activity .\",\n \"However , while Gadd45b knockdown very greatly suppresses this excess death , it does not as fully rescue cell numbers , suggesting loss of NMD may lead to both Gadd45b-dependent cell death as well as a Gadd45b-independent effect on proliferation .\",\n \"This mirrors the conclusions we made about the partial suppression of cell number defects in the Drosophila eye .\",\n \"Importantly , Upf1 mRNA expression was equivalently reduced and the expression of the mammalian endogenous NMD targets Rassf1 and CRCP ( Tani et al . , 2012 ) was equivalently increased in both the single and double knockdown experiments ( Figure 3D ) , indicating that the restoration of viability was not due to a recovery of NMD pathway activity . 10 . 7554/eLife . 12876 . 010Figure 3 . Gadd45b mediates cell lethality in Upf1 siRNA knockdown 3T3 mouse embryonic fibroblasts .\",\n \"( A ) Relative Gadd45b mRNA expression measured by qRT-PCR in NIH-3T3 cells after 48 hr of control ( black ) or Upf1 ( red ) siRNA treatment and 0 to 2 hr of actinomycin D treatment , normalized to expression prior to actinomycin treatment .\",\n \"The half-life calculated for each decay curve is indicated .\",\n \"( B ) Relative viable cell count of Upf1 and Gadd45b single and double siRNA treatment normalized to control siRNA .\",\n \"p-values display two-sided Student’s t-test between indicated conditions .\",\n \"( C ) Quantification of apoptosis as measured by annexin V staining .\",\n \"p-values display two-sided Student’s t-test between indicated conditions .\",\n \"( D ) Relative mRNA expression of Upf1 , Gadd45b , and two mammalian endogenous NMD targets , Rassf1 and CRCP ( Tani et al . , 2012 ) measured by qRT-PCR in Gadd45b and Upf1 single and double siRNA knockdown cells , normalized to expression in the control siRNA condition .\",\n \"p-values display one-sided Student’s t-test for each condition compared to control .\",\n \"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 01010 . 7554/eLife . 12876 . 011Figure 3—figure supplement 1 . GADD45A mediates cell lethality in Upf1 knockdown HEK293 cells .\",\n \"( A ) Relative GADD45A mRNA expression measured by qRT-PCR in UPF1 and GADD45A single and double siRNA knockdown 72 hr after siRNA transfection in HEK293 cells , normalized to expression in the control siRNA condition .\",\n \"p-values display one-sided Student’s t-test for each condition compared to control .\",\n \"( B ) Viable cell count of UPF1 and GADD45A single and double siRNA-treated cells normalized to control siRNA -treated cells .\",\n \"p-values display two-sided Student’s t-test between indicated conditions .\",\n \"( C ) Relative UPF1 mRNA expression measured by qRT-PCR in UPF1 and GADD45A single and double siRNA knockdown , normalized to expression in control siRNA condition .\",\n \"p-values display one-sided Student’s t-test for each condition compared to control .\",\n \"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 011 To extend our analysis to other mammalian cells , we analyzed the role of Gadd45 mediating the effects of loss of NMD in HEK293 cells .\",\n \"We found , similarly to 3T3 cells , that siRNA knockdown of UPF1 in HEK293 cells led to increased GADD45A expression and reduced cell numbers compared to control siRNA ( Figure 3—figure supplement 1A , B ) .\",\n \"Although transfection of siRNA targeting GADD45A alone slightly reduced HEK293 cell numbers , co-transfection with UPF1 siRNA did not further reduce cell count ( Figure 3—figure supplement 1B ) , and UPF1 expression was equivalently reduced in the single and double knockdown conditions ( Figure 3—figure supplement 1C ) .\",\n \"These results suggest that UPF1 knockdown is no longer detrimental to HEK293 cell viability in the absence of GADD45A expression .\",\n \"We conclude that increased expression of mammalian Gadd45 genes contributes to lethality in NMD-deficient mouse and human cells , as Gadd45 does in Drosophila .\",\n \"Deconvoluting the contributions to organismal viability of the PTC-surveillance versus gene-regulatory functions of NMD has been historically difficult ( Hwang and Maquat , 2011 ) .\",\n \"Here , we show that viability can be restored to Drosophila lacking core NMD factors when a single endogenous NMD target , Gadd45 , is eliminated , and that the requirement for the regulation of Gadd45 by NMD is evolutionarily conserved from flies to mammals .\",\n \"Although our data suggest that up-regulation of Gadd45 is a major factor contributing to lethality when NMD activity is lost , it is likely that other NMD targets also contribute to the observed lethality .\",\n \"In particular , viability is not restored to 100% in null Upf1; or Upf2; Gadd45 double mutants .\",\n \"In addition , loss of Gadd45 suppresses programmed cell death caused by defects in NMD , but not additional cell cycle defects , as implied by the incomplete suppression in the Drosophila eye and mammalian cell culture .\",\n \"Such defects in the cell cycle may be particularly pronounced during the development of certain tissue , or specific developmental stages .\",\n \"Indeed , NMD has been reported to have differing stage and tissue- specific activities ( Bao et al . , 2015; Bruno et al . , 2011; Colak et al . , 2013; Li et al . , 2015 ) .\",\n \"Whether this is due to a role in surveillance or another specific target remains unclear , but examination of the effects of loss of NMD in Gadd45 mutants should allow exploration of these possibilities .\",\n \"The benefit for such a mechanism regulating Gadd45 expression may lie in a function of NMD in restricting viral growth ( Balistreri et al . , 2014 ) .\",\n \"Because viruses encode trans-acting factors to inhibit NMD ( Mocquet et al . , 2012 ) , the resulting accumulation of GADD45 in infected cells may act as a “molecular tripwire” that rapidly elicits a stress response and cell death .\",\n \"This outcome suggests that regulating responses to infection may underlie a conserved essential function of NMD .\",\n \"Intriguingly , restriction of pathogens via NMD extends to plants ( Garcia et al . , 2014 ) , where NMD mutant lethality in A . thaliana , which do not encode Gadd45 orthologues , may be caused by the overexpression of a subset of immune-related intracellular nucleotide-binding leucine-rich repeat receptors , some of which are endogenous NMD targets ( Gloggnitzer et al . , 2014 ) .\",\n \"In contrast , eukaryotes that do not rely on the activation of programmed cell death to protect against viruses , such as S . cerevisiae , S . pombe , and C . elegans , do not require NMD for viability ( Hodgkin et al . , 1989; Leeds et al . , 1991; Mendell et al . , 2000 ) .\",\n \"Together these observations suggest a potential novel role for NMD and Gadd45 in immune responses , triggering the death of infected cells during pathogenic challenges .\",\n \"Restoring the expression of PTC-containing alleles via NMD inhibition has been proposed as a promising therapy for a wide range of recessive genetic diseases ( Keeling et al . , 2014 ) .\",\n \"Translation of stable PTC-containing mRNAs would produce truncated proteins that may be partially functional and alleviate disease symptoms normally caused by complete loss of the protein .\",\n \"However , the essential function for NMD in viability has raised the concern that these therapies may have prohibitive side effects .\",\n \"Our findings reveal a molecular basis for dealing with this obstacle by suggesting that inhibiting both the NMD and Gadd45 pathways ( Tornatore et al . , 2014 ) in combination could provide an effective and safe treatment for patients with debilitating genetic disorders .\"\n ],\n [\n \"Drosophila melanogaster stocks were raised on standard cornmeal/dextrose food at 25° .\",\n \"The NMD mutant alleles Upf225G , Upf27-5A , and Upf126A ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) are on y w FRT19A chromosomes .\",\n \"These alleles were balanced over FM7i , P{ActGFP}JMR3 ( Reichhart and Ferrandon , 1998 ) .\",\n \"Smg5G115 and Smg5C391 are null alleles of Smg5 ( J . O . N . , D . Förster , S . Luschnig , and M . M . M . , unpublished ) and will be described in detail later .\",\n \"The Smg5 alleles are balanced over CyO , P{Dfd:eYFP w+} ( Le et al . , 2006 ) .\",\n \"Other alleles used were P{w[+mC]=EPg}HP20647 ( Staudt et al . , 2005 ) , Mekk1Ur36 ( Inoue et al . , 2001 ) recombined on FRT82B by D . Ryoo , ey-FLP ( Newsome et al . , 2000 ) , pcm14 ( Waldron et al . , 2015 ) , Adhn4 ( Chia et al . , 1987 ) and DHR783 ( Fisk and Thummel , 1998 ) .\",\n \"Control chromosomes were y w FRT19A ( for Upf1 and Upf2 ) and FRT82B ( for Mekk1 ) ( Xu and Rubin , 1993 ) .\",\n \"For all experiments using Gadd45F17 we used the Gadd45E8 precise excision as a control .\",\n \"For viability assays , we mated flies for 3 days and collected all progeny each day for 10 days , starting 10 days after the cross was initiated .\",\n \"The total numbers of F1 mutant and balancer males were scored , and the ratio of mutant males to balancer males was used to determine mutant animal viability .\",\n \"To control for balancer viability within each experiment , we normalized the ratio of mutant to balancer animals to a ratio of the appropriate control chromosome to balancer animals produced from a parallel cross .\",\n \"We screened autosomal deficiencies from the DrosDel collection ( Ryder et al . , 2007 ) .\",\n \"All deficiencies scored can be found in Supplementary file\",\n \"1 . Deficiencies on chromosome 2 were balanced over CyO , and deficiencies on chromosome 3 were balanced over TM6C .\",\n \"We mated males from each deficiency stock to y w Upf225G FRT19A/FM7i , P{ActGFP}JMR3 females and scored all F1 males for the presence or absence of each balancer .\",\n \"For any given deficiency tested , the percentage of Deficiency / + males that are Upf225G mutants , less the percentage of Balancer / + males that are Upf225G mutants was calculated , producing a Deficiency Suppression Score ( DSS ) , which represents the effect of an individual deficiency on the increase or decrease in Upf225G viability , while controlling for each deficiency’s general influence on viability .\",\n \"A DSS greater than 0 . 1 indicates suppression of lethality .\",\n \"Supplemental deficiencies used were from the Exelixis collection ( Parks et al . , 2004 ) and Df ( 2R ) sple-J1 ( Heitzler et al . , 1993 ) .\",\n \"Deficiency mapping to the Drosophila genome was performed using the 5 . 1 genome release .\",\n \"RNA-seq data sets were acquired from Chapin et al . ( 2014 ) ( archives SRR896609 , SRR896616 , SRR503415 , and SRR503416 ) and aligned using Bowtie and TopHat alignment with standard remapping parameters to the 5 . 1 Drosophila genome release .\",\n \"SAMtools accessory scripts were used to retrieve read counts for deficiency and control regions .\",\n \"All read counts were normalized to reads per million within each data set .\",\n \"Average normalized reads in Upf225G samples were normalized to the relative reads of 74 ribosomal proteins in Upf225G samples compared to control samples .\",\n \"Total normalized reads within the regions removed by each deficiency were averaged between biological replicates , and the difference between the Upf225Gand control samples was divided by one million to determine percent increase in genomic load across each deficiency region .\",\n \"We produced P-element excision lines from the P{w[+mC]=EPg}HP20647 P-element insertion line crossed to a Δ2–3 transposase stock .\",\n \"We mated F1 males containing the P-element and transposase on a CyO balancer to w; Tft / CyO females .\",\n \"Cy+ Tft white-eyed F2 males were then individually mated to w; Tft / CyO females .\",\n \"We then collected Tft+ , Cy males and females to create an isogenic stock from each individually mated F2 male .\",\n \"To identify precise excisions we used the primers Gadd45_F1 / Gadd45_R1 flanking the P-element insert site to amplify a region across the excised P-element .\",\n \"Lines that failed to amplify with these primers were candidate imprecise excisions , which we then tested with Gadd45_F1 / Gadd45_R3 primers for deletions .\",\n \"Any detected deletions were subsequently sequenced using these same primers .\",\n \"Primer sequences are found in Supplementary file\",\n \"2 . We generated eye clones with the FLP/FRT system using the ey-FLP driver ( Newsome et al . , 2000 ) to induce recombination .\",\n \"We imaged eyes on a Leica MZ125 stereo microscope with a Retiga-2000R camera ( QImaging , Canada ) with QCapture 3 . 1 . 2 software ( QImaging ) .\",\n \"We focused images using the ImageJ stack focuser plugin and quantified relative eye clone size using the ImageJ analyzer tools .\",\n \"A total of 20 eyes from 20 individual animals were scored for each condition .\",\n \"For TUNEL assays , third instar larval wing discs were dissected as described in Sullivan et al . ( Sullivan et al . , 2000 ) .\",\n \"TUNEL staining was performed using the Apoptag Red in situ Apoptosis Detection Kit ( Chimicon International Inc . , Billerica , MA ) according to Chakraborty et al . ( Chakraborty et al . , 2015 ) .\",\n \"We DAPI stained wing discs ( 1:5000 ) for 5 min prior to mounting .\",\n \"Confocal images were acquired using a Zeiss LSM710 laser scanning confocal microscope ( Carl Zeiss AG , Germany ) .\",\n \"3-dimensional datasets were acquired with a Plan-Apochromat 20X/0 . 8 lens , 1 . 34 μm z-step , using the Zeiss ZEN software .\",\n \"To measure TUNEL signal intensity z-projections images were summed with ImageJ .\",\n \"Background signal was removed by using the ImageJ MaxEntropy auto-threshold .\",\n \"Relative total TUNEL signal intensity was calculated using the ImageJ analyzer tools to measure the total pixel intensity within the wing discs of TUNEL images and normalized to the average intensity in control conditions .\",\n \"For annexin V staining , we collected media ( including floating cells ) from siRNA treated cells .\",\n \"We spun down media at 950g for 4 min to pellet cells , and then aspirated remaining media .\",\n \"Concurrently , we trypsinized siRNA-treated cells still on plates and added them to the same respective tube as previously spun-down media .\",\n \"Following the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit ( Abcam , UK ) protocol , we stained for apoptotic cells .\",\n \"We visualized cells on an Olympus IX51 microscope ( Olympus , Japan ) with 20X objective .\",\n \"We collected bright field as well as fluorescent images using a FITC filter with a QImaging QICam Fast1394 camera and QCaptureP software ( QImaging ) .\",\n \"We analyzed cells by counting all cells within a bright field image as well as the annexin V positive cells from the same image .\",\n \"The number of annexin V positive cells was divided by total cell number to generate the fraction of apoptotic cells for each treatment .\",\n \">3000 total cells were counted across three biological replicates for each treatment .\",\n \"We cultured mouse NIH-3T3 cells ( ATCC ) or HEK293 cells ( ATCC ) in DMEM ( Thermo-Fisher , Waltham , MA ) supplemented with 10% fetal bovine serum and glutamine .\",\n \"For siRNA experiments , we transfected cells using RNAiMax and 24 pmol of negative control siRNA ( Qiagen , Netherlands ) , Upf1 siRNA ( Qiagen ) , or Gadd45b siRNA ( Sigma-Aldrich ) for 3T3 cell experiments , or negative control siRNA ( Qiagen ) , UPF1 siRNA ( Qiagen ) , or GADD45A siRNA ( Sigma-Aldrich , St . Louis , MO ) for HEK293 experiments .\",\n \"For double siRNA-treated cells , we used 24 pmol of each Upf1 and Gadd45b siRNA for 3T3 experiments or UPF1 and GADD45A siRNA for HEK293 experiments .\",\n \"For actinomycin experiments , we incubated cells with siRNA for 48 hr before changing the media and then incubated with 2 μg/mL actinomycin ( Sigma-Aldrich ) for 1 or 2 hr . mRNA half-life was determined by fitting an exponential decay curve to the relative expression at each time point ( Tani et al . , 2012 ) .\",\n \"t1/2 was calculated based on the average expression at each time point , and the mean t1/2 for each condition is represented .\",\n \"For cell counting experiments , we trypsinized cells , incubated a small aliquot with Trypan Blue at a final concentration of 0 . 04% in complete media , and counted Trypan Blue negative cells .\",\n \"RNA was collected from the remaining cells , and relative mRNA levels were measured as described below .\",\n \"For Drosophila qRT-PCR analyses , we collected 5–10 adult animals frozen in liquid nitrogen .\",\n \"We isolated total RNA using TRIzol reagent ( Invitrogen ) and phase-lock tubes ( 5-Prime ) , and the RNeasy mini kit ( Qiagen ) .\",\n \"We used on-column RNase-free DNase treatment ( Qiagen ) to reduce genomic contamination .\",\n \"We determined RNA concentration by spectrophotometer and normalized concentration for reverse transcription .\",\n \"For reverse transcription , we used random decamers and MMLV8 reverse transcriptase ( Retroscript Kit , Thermo-FIsher ) .\",\n \"We performed qRT-PCR analysis using the SYBR Green qPCR Supermix ( Bio-Rad , Hercules , CA ) and the Bio-Rad iCycler thermocycler .\",\n \"All experimental reactions were performed using three technical replicates and a minimum of three biological replicates per condition , and the expression level of all experimental assays was normalized to RpL32 mRNA expression .\",\n \"For cell culture qRT-PCR analyses , we collected RNA following the Zymo Research Quick RNA MiniPrep kits protocol , and synthesized cDNA using MMLV reverse transcriptase ( NEB , Ipswich , MA ) with a template of 1 µg of total RNA and priming with a T18 oligo .\",\n \"We measured relative mRNA levels by qRT-PCR using the Masterplex ep realplex ( Eppendorf , Germany ) with SYBR green fluorescent dye .\",\n \"Each sample was measured with technical triplicates and three biological replicates , and target mRNA levels were normalized to those of ribosomal protein 19 ( Rpl19 ) mRNA .\",\n \"For all qRT-PCR analyses we also measured samples that had been made without reverse transcriptase to ensure that signal was not due to genomic DNA .\",\n \"Primer sequences can be found in Supplementary file\",\n \"2 . We cloned the UAS-GFP::Gadd45 3’ UTR and control UAS-GFP::Act5C 3’ UTR constructs using the primers G45_3U_X1_F / G45_3U_S1_R or Act5C_X1_F / Act5C_S1_R ( Supplementary file 2 ) to amplify the Gadd45 and Act5C 3’ UTRs , respectively , from genomic DNA .\",\n \"PCR fragments were inserted into the Zero Blunt TOPO vector ( Thermo-Fisher ) , sequenced to assure fidelity , and digested and cloned into a pUAST-attB GFP vector using standard cloning procedures to replace the SV40 3’ UTR .\",\n \"Plasmids were injected by BestGene ( Chino Hills , CA ) into a stock containing the VK00027 attP site ( Venken et al . , 2006 ) for phiC31 directed integration .\",\n \"We used previously described UAS-GFP::SV40 3’ UTR animals ( Metzstein and Krasnow , 2006 ) .\",\n \"For imaging , wandering late L3 larvae were collected and examined using a Leica MZ 16F microscope and the Leica DFC340 FX camera with the Leica Application Suite v3 . 3 . 0 software .\",\n \"We collected adult F1 Upf2+; Gadd45E8/+ , Upf225G; Gadd45E8/+ , and Upf225G; Gadd45F17/+males that were also heterozygous for either the dHR783 or Adhn4 .\",\n \"The Adhn4allele is a PTC-containing allele and has been demonstrated to be a direct NMD target based on cleavage by Smg6 ( Gatfield and Izaurralde , 2004 ) .\",\n \"The dHR783 allele is also a PTC-containing allele and thus is presumably degraded by NMD ( Fisk and Thummel , 1998 ) .\",\n \"At least three biological replicates were collected for each condition .\",\n \"We isolated RNA and generated cDNA as described in methods above and used this cDNA as a template for PCR amplification of the dHR78 transcript with the DRH78_F3 / DHR78_R3 primers and the Adh transcript with the Adh_F and Adh_R primers ( Supplementary file 2 ) , which flank the nonsense mutation in the respective transcripts .\",\n \"To compare the relative abundance of the dHR783 allele to the wild-type allele , PCR products were Sanger sequenced , and the relative peak intensity for a T ( dHR783 allele ) compared to a C ( wild-type allele ) at nucleotide 1063 was compared .\",\n \"To compare the relative abundance of the Adhn4 allele to the wild-type allele , PCR products were digested with PvuII ( a site disrupted by the n4 mutation ) , separated on a 1% agarose gel and stained with ethidium bromide .\",\n \"The relative intensity of the cut and uncut bands was determined using ImageJ and normalized for fragment length .\",\n \"All samples were ran on the same gel and compared under identical conditions .\",\n \"All ratios were normalized to the ratio in the Upf225G; Gadd45E8/+condition .\",\n \"All figures displaying viability assays represent a proportion of animals of the indicated genotypes that survive to adulthood; error bars for these figures represent the 95% confidence interval of the binomial distribution , and the Test of qual or Given Proportions was used to determine significance difference in these proportions between genotypes .\",\n \"All other figures represent the mean value of multiple replicates have error bars depicting ± 2 SEM , which is a close approximation of the 95% confidence interval ( Krzywinski and Altman , 2013 ) .\",\n \"For tests between two variable measures , a two-sided paired Student’s t-test was used to determine significance difference between mean value data .\",\n \"For most qPCR experiments , data was compared to a normalized control , set to a constant of 1 , so these tests were performed with a one-sided Student’s t-test .\"\n ]\n]"},"abstract":{"kind":"list like","value":["The nonsense-mediated mRNA decay ( NMD ) pathway functions to degrade both abnormal and wild-type mRNAs .","NMD is essential for viability in most organisms , but the molecular basis for this requirement is unknown .","Here we show that a single , conserved NMD target , the mRNA coding for the stress response factor growth arrest and DNA-damage inducible 45 ( GADD45 ) can account for lethality in Drosophila lacking core NMD genes .","Moreover , depletion of Gadd45 in mammalian cells rescues the cell survival defects associated with NMD knockdown .","Our findings demonstrate that degradation of Gadd45 mRNA is the essential NMD function and , surprisingly , that the surveillance of abnormal mRNAs by this pathway is not necessarily required for viability ."],"string":"[\n \"The nonsense-mediated mRNA decay ( NMD ) pathway functions to degrade both abnormal and wild-type mRNAs .\",\n \"NMD is essential for viability in most organisms , but the molecular basis for this requirement is unknown .\",\n \"Here we show that a single , conserved NMD target , the mRNA coding for the stress response factor growth arrest and DNA-damage inducible 45 ( GADD45 ) can account for lethality in Drosophila lacking core NMD genes .\",\n \"Moreover , depletion of Gadd45 in mammalian cells rescues the cell survival defects associated with NMD knockdown .\",\n \"Our findings demonstrate that degradation of Gadd45 mRNA is the essential NMD function and , surprisingly , that the surveillance of abnormal mRNAs by this pathway is not necessarily required for viability .\"\n]"},"summary":{"kind":"list like","value":["Messenger RNA ( mRNA ) molecules act as the templates from which proteins are made , and so control the amount of protein in a cell .","Having too much of certain proteins can harm cells .","Additionally , some mRNAs contain errors , and so can create faulty proteins that may also harm the cell .","Cells have therefore developed ways to destroy excess or error-ridden mRNAs to avoid a deadly build up of proteins .","One such quality control mechanism is called nonsense-mediated decay ( NMD ) .","This mechanism is so important that cells that cannot perform nonsense-mediated decay die , although it is not clear exactly what kills the cells .","Now , Nelson et al . have found that fruit flies whose cells are unable to perform nonsense-mediated decay die because a harmful protein called Gadd45 builds up in the cells .","In normal cells , nonsense-mediated decay destroys the mRNA that relays the instructions for making Gadd45 , which keeps the amount of the Gadd45 protein in the cell low .","Further experiments show that removing Gadd45 from cells that lack nonsense-mediated decay saves the flies .","Removing Gadd45 from human and mouse cells that are unable to perform nonsense-mediated decay also allows these cells to survive .","These findings imply that the only nonsense-mediated decay function needed for cells to live is the destruction of Gadd45 mRNA .","This further implies that most faulty and normal mRNAs that are normally destroyed by nonsense-mediated decay do not cause the cells to die when nonsense-mediated decay is lost .","Learning that creating faulty proteins when nonsense-mediated decay is lost is not necessarily harmful to cells opens new possibilities to treating numerous genetic diseases .","In some diseases , cells can only produce faulty forms of a particular protein .","Nonsense-mediated decay normally destroys all of these mutant proteins , but it may sometimes be better to have faulty versions of a protein than to have none of it .","Safely getting rid of nonsense-mediated decay by also eliminating Gadd45 from cells may therefore be a treatment strategy worth exploring ."],"string":"[\n \"Messenger RNA ( mRNA ) molecules act as the templates from which proteins are made , and so control the amount of protein in a cell .\",\n \"Having too much of certain proteins can harm cells .\",\n \"Additionally , some mRNAs contain errors , and so can create faulty proteins that may also harm the cell .\",\n \"Cells have therefore developed ways to destroy excess or error-ridden mRNAs to avoid a deadly build up of proteins .\",\n \"One such quality control mechanism is called nonsense-mediated decay ( NMD ) .\",\n \"This mechanism is so important that cells that cannot perform nonsense-mediated decay die , although it is not clear exactly what kills the cells .\",\n \"Now , Nelson et al . have found that fruit flies whose cells are unable to perform nonsense-mediated decay die because a harmful protein called Gadd45 builds up in the cells .\",\n \"In normal cells , nonsense-mediated decay destroys the mRNA that relays the instructions for making Gadd45 , which keeps the amount of the Gadd45 protein in the cell low .\",\n \"Further experiments show that removing Gadd45 from cells that lack nonsense-mediated decay saves the flies .\",\n \"Removing Gadd45 from human and mouse cells that are unable to perform nonsense-mediated decay also allows these cells to survive .\",\n \"These findings imply that the only nonsense-mediated decay function needed for cells to live is the destruction of Gadd45 mRNA .\",\n \"This further implies that most faulty and normal mRNAs that are normally destroyed by nonsense-mediated decay do not cause the cells to die when nonsense-mediated decay is lost .\",\n \"Learning that creating faulty proteins when nonsense-mediated decay is lost is not necessarily harmful to cells opens new possibilities to treating numerous genetic diseases .\",\n \"In some diseases , cells can only produce faulty forms of a particular protein .\",\n \"Nonsense-mediated decay normally destroys all of these mutant proteins , but it may sometimes be better to have faulty versions of a protein than to have none of it .\",\n \"Safely getting rid of nonsense-mediated decay by also eliminating Gadd45 from cells may therefore be a treatment strategy worth exploring .\"\n]"},"year":{"kind":"string","value":"2016"}}},{"rowIdx":4190,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration"},"id":{"kind":"string","value":"elife-16043-v2"},"sections":{"kind":"list like","value":[["FRDA , an inherited recessive ataxia , is caused by mutations in FXN ( Campuzano et al . , 1996 ) .","During childhood or early adulthood , FRDA patients show a progressive neurodegeneration of dorsal root ganglia , sensory peripheral nerves , corticospinal tracts , and dentate nuclei of the cerebellum ( Koeppen , 2011 ) .","FXN is evolutionarily conserved and the homologs have been identified in most phyla ( Bencze et al . , 2006; Campuzano et al . , 1996 ) .","It encodes a mitochondrial protein that is required for iron-sulfur cluster assembly ( Layer et al . , 2006; Lill , 2009; Muhlenhoff et al . , 2002; Rotig et al . , 1997; Yoon and Cowan , 2003 ) .","Once synthesized , iron-sulfur clusters are incorporated into a variety of metalloproteins , including proteins of the mitochondrial electron transport chain ( ETC ) complexes and aconitase , where they function as electron carriers , enzyme catalysts , or regulators of gene expression ( Lill , 2009 ) .","It has been proposed that loss of FXN leads to impaired ETC complex , which in turn triggers ROS production that directly contributes to cellular toxicity ( Al-Mahdawi et al . , 2006; Anderson et al . , 2008; Calabrese et al . , 2005; Schulz et al . , 2000 ) .","However , the ROS hypothesis has been questioned in several studies .","For example , loss of FXN only leads to a modest hypersensitivity to oxidative stress ( Macevilly and Muller , 1997; Seznec et al . , 2005; Shidara and Hollenbeck , 2010 ) .","In addition , several clinical trails based on antioxidant therapy in FRDA patients have shown no or limited benefits ( Lynch et al . , 2010; Parkinson et al . , 2013; Santhera Pharmaceuticals , 2010 ) .","Loss of yeast frataxin homolog results in iron accumulation ( Babcock et al . , 1997 ) , and this phenotype has also been reported in cardiac muscles of a Fxn deficiency mouse and FRDA patients ( Koeppen , 2011; Lamarche et al . , 1980; Michael et al . , 2006; Puccio et al . , 2001 ) .","However , whether iron accumulates in the nervous system upon loss of FXN remains controversial .","Furthermore , whether iron deposits contribute to the pathogenesis is not clear .","It has been reported that elevated iron levels were observed in the dentate nuclei or in glia cells of FRDA patients ( Boddaert et al . , 2007; Koeppen et al . , 2012 ) .","Contrary to these results , others suggested that there is no increase of iron in the nervous system of Fxn deficiency mice and FRDA patients ( Koeppen et al . , 2007; Puccio et al . , 2001; Solbach et al . , 2014 ) .","Taken together , current data provide insufficient evidence to establish that iron dysregulation contributes to neurodegeneration .","In addition , the mechanism underlying iron toxicity is still unclear .","In summary , the pathological interplay of mitochondrial dysfunction , ROS , and iron accumulation remains to be established .","We identified the first mutant allele of fh in an unbiased forward genetic screen aimed at isolating mutations that cause neurodegenerative phenotypes .","We show that loss of fh causes an age dependent neurodegeneration in photoreceptors and affects mitochondrial function .","Unlike other mitochondrial mutants with impaired ETC activity , we do not observe an increase in ROS .","However , loss of fh causes an iron accumulation in the nervous system , induces an up-regulation of sphingolipid synthesis , and activation of Pdk1 and Mef2 .","Reducing iron toxicity or inhibiting the sphingolipid/Pdk1/Mef2 pathway significantly suppresses neurodegeneration in fh mutants .","To our knowledge , this is the first evidence that sphingolipids , Pdk1 , and the transcription factor Mef2 are shown to play a primary role in FXN-induced neurodegeneration ."],["We identified the first EMS ( ethyl methanesulfonate ) induced missense mutation in fh , the Drosophila homolog of FXN , through a forward genetic screen on the X chromosome ( Figure 1—figure supplement 1A ) ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) .","The molecular lesion , S136R , is in a highly conserved region , which is required for the FXN binding to the iron-sulfur cluster assembly complex ( Tsai et al . , 2011 ) .","In addition , six point mutations adjacent to S136 ( S157 in human ) have been reported in FRDA patients ( Figure 1—figure supplement 1B ) ( Santos et al . , 2010 ) .","Hemizygous fh mutants are L3 to pupal lethal , and the lethality is not enhanced in transheterozygous mutants that carry a deficiency ( fh/Df ( 1 ) BSC537 ) , suggesting that S136R is a severe loss-of-function ( Figure 1—figure supplement 1C ) .","The lethality of fh can be rescued by a genomic fh construct ( gfh ) , or by ubiquitous expression ( da-GAL4 driver ) of fh cDNA using the UAS/GAL4 system ( Brand and Perrimon , 1993 ) ( Figure 1—figure supplement 1C ) .","The lethality cannot be rescued with tissue specific drivers , including neuronal ( nSyb-GAL4 ) , muscular ( Mef2-GAL4 ) , or glial ( repo-GAL4 ) drivers , suggesting a requirement for fh in multiple tissues ( Figure 1—figure supplement 1C ) .","In addition , fh mutants exhibit a smaller body size and prolonged larval stages ( 10 to 12 days instead of 5 days for wild type animals ) , similar to other mitochondrial mutants ( Sandoval et al . , 2014; Zhang et al . , 2013 ) .","Interestingly , removal of maternal wild type fh mRNA or protein in the egg exacerbates the lethal phase from pupal to embryonic lethality ( Figure 1—figure supplement 1C ) .","In essence , the residual maternally deposited wild type Fh not only extends the lifespan but also creates a partial loss of Fh condition in the fh mutants .","To bypass pupal lethality of fh mutants , mosaic mutant clones of adult photoreceptor neurons were generated with the eyeless-FLP/FRT system .","As a functional readout , we recorded and compared electroretinograms ( ERGs ) in young and aged flies .","In response to a light stimulus , the ERG amplitudes of newly eclosed fh mutants ( Day 1 ) are ~60% of control animals ( y , w , FRT19Aiso , the isogenized flies used in the screen ) ( Figure 1A ) .","The ERG amplitudes rapidly decline over a six day period ( Figure 1A ) .","Morphologically , the fh mutant clones contain a normal number of photoreceptors with a typical trapezoidal organization at Day 1 ( Figure 1B ) .","However , by using transmission electron microscopy ( TEM ) , we found that Day 1 fh mutant photoreceptors exhibit an expansion of the endoplasmic reticulum ( ER ) and a dramatic accumulation of lipid droplets in glial cells that are not observed in controls ( Figure 1—figure supplement 2A and B ) .","At Day 3 , photoreceptors in mutant clones exhibit aberrant elongated rhabdomeres ( Figure 1B ) .","By Day 6 , many of the photoreceptors are missing in the mutant clones , whereas control retinas exhibit intact morphology ( Figure 1B and Figure 1—figure supplement 2C ) .","These data show that the mutant photoreceptors undergo a rapid and severe functional as well as morphological degeneration . 10 . 7554/eLife . 16043 . 003Figure 1 . Loss of fh results in age dependent neurodegeneration in photoreceptors .","( A ) ERG of control ( y w FRT19A ) and fh ( y w fh FRT19A ) mosaic eyes .","The ERG amplitudes from Day 1 to Day 6 are indicated by red double arrows .","Quantification of the ERG amplitudes of control ( y w FRT19A ) and fh ( y w fh FRT19A ) is on the right .","( B ) Retina morphology of control ( y w FRT19A ) and fh mosaic eyes .","Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .","Neuronal nuclei are labeled by anti-Elav antibody ( blue ) .","Scale bar: 10 µm .","( C ) ERG of control ( y w FRT19A ) , fh mutants , fh mutants carrying a genomic fh construct ( fh; gfh ) , and fh mutants that express the fh cDNA ( fh; Rh1>fh ) or human FXN cDNA ( fh; Rh1>hFXN ) in photoreceptors using a Rh1-GAL4 driver .","The ERG amplitudes were recorded at Day 6 .","Data are presented as mean ± SEM .","**p<0 . 01 , ***p<0 . 001 , Student’s t-test .","L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00310 . 7554/eLife . 16043 . 004Figure 1—figure supplement 1 . Mutation and lethal phase analysis of fh mutants .","( A ) Molecular lesion of fh mutants .","A 3 . 4 kb genomic rescue construct is shown below the gene .","( B ) FXN sequences alignment between fly , human , and mouse .","Mutation S136R identified in fh mutants is indicated by a box .","Black arrow heads indicate the point mutations identified in the FRDA patients .","( C ) Lethal phase analysis of fh mutants .","The lethality can be rescued by a genomic fh construct , or expressing fh cDNA by the ubiquitous driver daughterless-GAL4 .","Overexpression of fh cDNA in a tissue specific manner cannot rescue lethality .","Finally , removing maternal Fh proteins in the female germ line by using OvoD ( Perrimon and Gans , 1983 ) leads to embryonic to early L1 lethality . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00410 . 7554/eLife . 16043 . 005Figure 1—figure supplement 2 . Ultrastructure of adult eyes exhibits morphological defects in fh mutant clones .","( A ) Transmission electron microscopy ( TEM ) images of retina morphology in control ( y w FRT19A ) and fh mutant clones at Day 1 .","Lipid droplets are indicated by red arrow .","Scale bar: 2 µm .","( B ) Quantification of lipid droplet in Day 1 retina of control ( y w FRT19A ) and fh mutants .","n = 6 .","( C ) Retina morphology in control ( y w FRT19A ) and fh mutant clones at Day 6 .","Scale bar: 2 µm .","( D ) Retina morphology of fh mutants ( fh; Rh1-Gal4 ) , fh mutants carrying a genomic fh construct ( fh; gfh ) , and fh mutants that express the fh cDNA ( fh; Rh1>fh ) or human FXN cDNA ( fh; Rh1>hFXN ) .","Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .","The red eye marker ( w+ ) of Rh1-GAL4 creates autofluorescence background that makes imaging difficult .","Scale bar: 10 µm .","Data are presented as mean ± SEM .","***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 005 The neurodegeneration can be fully rescued by a genomic fh construct or by expressing fh cDNA in photoreceptors ( Figure 1C and Figure 1—figure supplement 2D ) , suggesting that photoreceptor degeneration is cell-autonomous .","Moreover , expression of the human FXN cDNA rescues neurodegeneration in fh mutants ( Figure 1C and Figure 1—figure supplement 2D ) , showing that fly and human FXN have conserved molecular function , and that our studies are relevant to the biological role of human FXN .","FXN has been reported to be localized to mitochondria ( Koutnikova et al . , 1997 ) .","Indeed , Fh colocalizes with mitoGFP in larval motor neuron , and removing the predicted mitochondrial targeting sequence ( MTS ) of fh cDNA ( Fh-∆MTS-V5 ) leads to a diffused signal in the cytosol ( Figure 2—figure supplement 1A ) .","Removal of the MTS leads to a dysfunctional protein as expression of Fh-∆MTS-V5 in the fh mutant clone is not able to rescue the ERG defect ( Figure 2—figure supplement 1B ) .","Loss of fh causes a highly aberrant mitochondrial morphology in one day old adult photoreceptors .","The mitochondria in fh mutants are larger and exhibit aberrant cristae and altered inner membrane morphology ( Figure 2A and Figure 2—figure supplement 1C ) .","Loss of FXN has been reported to cause various ETC and respiratory defects in different animal models , including yeast , fly , mouse , as well as FRDA patients ( Al-Mahdawi et al . , 2006; Anderson et al . , 2005; Carletti et al . , 2014; Koutnikova et al . , 1997; Puccio et al . , 2001; Rotig et al . , 1997; Wilson and Roof , 1997 ) .","We find that ETC Complex I ( CI ) activity is severely reduced in fh mutants when compared to control and genomic rescued animals ( Figure 2B ) .","Consistent with this data , the ADP/ATP ratio is increased in fh mutants ( Figure 2C ) , showing that energy production is impaired . 10 . 7554/eLife . 16043 . 006Figure 2 . Loss of fh causes impaired mitochondria , and reducing ROS levels fails to suppress degeneration in fh mutants .","( A ) TEM images of mitochondria in photoreceptors of control ( y w FRT19A ) and fh mutant clones .","The insets show a single mitochondrion ( red box ) .","Scale bar: 500 nm .","( B ) ETC complex activities in control ( y w FRT19A ) , fh mutant , and genomic rescued animal ( fh; gfh ) .","Complex activities are normalized to citrate synthase ( CS ) , which act as a control of mitochondrial mass . n = 3 . ( C ) ADP/ATP ratio in control ( y w FRT19A ) , fh mutant , and rescued animal ( fh; gfh ) .","n = 3 . ( D ) ROS levels are measured by DHE in the larval ventral nerve cord .","Neuronal down-regulation of ND42 ( nSyb>ND42 RNAi ) , a subunit of CI , acts as a positive control .","Quantification of DHE fluorescence is on the right .","n = 8 .","Scale bar: 50 µm .","( E ) Day 3 ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which ROS scavengers are overexpressed in photoreceptors .","Data are presented as mean ± SEM .","**p<0 . 01 , ***p<0 . 001 , Student’s t-test .","ND , no significant difference .","L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00610 . 7554/eLife . 16043 . 007Figure 2—figure supplement 1 . Mitochondrial phenotype of fh mutants .","( A ) The immunostaining of larval motor neuron cell body with expression of fh or fh without the mitochondrial targeting sequence ( MTS ) ( Fh-∆MTS ) .","Fh and Fh-∆MTS are labeled by V5 ( red ) , and mitochondria are labeled by mitoGFP ( green ) .","The nuclei are marked by DAPI ( blue ) .","Scale bar: 5 µm .","( B ) Quantification of ERG amplitudes of fh mutants with different cDNA constructs at Day","3 . ( C ) Quantification of abnormal mitochondria in photoreceptor in TEM image .","n = 6 .","( D ) Day 6 ERG of fh mutants ( fh; Rh1-GAL4 ) in which yeast Ndi1p are overexpressed in photoreceptors .","( E ) Quantification of ERG amplitudes of fh mutants at Day","3 . Treating fh mutants with an antioxidant drug AD4 with 40 μg/ml concentration does not suppress degeneration .","Data are presented as mean ± SEM .","***p<0 . 001 , Student’s t-test .","L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00710 . 7554/eLife . 16043 . 008Figure 2—figure supplement 2 . ROS/JNK/SREBP pathway does not contribute to degeneration in fh mutants .","( A ) Nile red staining of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants that overexpress lipases .","Quantification of ERG amplitudes at Day 3 is on the right .","Scale bar: 5 µm .","( B ) ROS/JNK/SREBP pathway causes accumulation of lipid droplets and degeneration in other mitochondrial mutants ( Liu et al . , 2015 ) .","( C ) The levels of puc-lacZ ( green ) in the larval ventral nerve cord of control ( y w FRT19A ) and fh mutants .","Neuronal membrane is marked by anti-DLG antibody ( blue ) .","Scale bar: 40 µm .","Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 008 It has been reported that expression of yeast Ndi1p , which is a single subunit of NADH dehydrogenase , can mitigate the deleterious effects of an ETC CI deficit in the fly ( Vilain et al . , 2012 ) .","However , overexpression of Ndi1p in fh mutant clones does not suppress neurodegeneration ( Figure 2—figure supplement 1D ) .","Impaired ETC CI activity has been linked to increased ROS production that in turn causes cellular stress or toxicity ( Sugioka et al . , 1988; Turrens and Boveris , 1980 ) .","We therefore evaluated ROS production in vivo with dihydroethidium ( DHE ) , a dye that emits fluorescence upon oxidation by ROS .","Neuronal down-regulation of ND42 ( nSyb>ND42 RNAi ) , a subunit of CI , was used as a positive control ( Owusu-Ansah et al . , 2008 ) .","As shown in Figure 2D , loss of ND42 exhibits strong DHE fluorescence in the larval ventral nerve cord , indicating elevated levels of ROS .","However , DHE levels are not obviously increased in fh mutants , suggesting no or a very mild increase in ROS ( Figure 2D ) .","Elevated ROS levels are proposed to be a major contributor to the pathogenesis in FRDA .","To further investigate the role of ROS in photoreceptor degeneration of fh mutants , we overexpressed several ROS scavengers , including Catalase , Super Oxide Dismutase 1 ( SOD1 ) , and SOD2 , which have all been shown to effectively reduce ROS in flies ( Orr and Sohal , 1992; Parkes et al . , 1998; Sun et al . , 2002 ) .","Consistent with the DHE data , overexpression of these ROS scavengers does not suppress neurodegeneration in fh mutants ( Figure 2E ) .","Furthermore , feeding fh mutants with AD4 , a potent antioxidant that dampens oxidative stress ( Amer et al . , 2008; Liu et al . , 2015 ) , does not suppress neurodegeneration ( Figure 2—figure supplement 1E ) .","Taken together , our findings argue that ROS does not contribute to the demise of neurons in fh mutants .","It has been proposed that lipid droplets play a role in the pathogenesis of FRDA as knockdown of fh in flies causes lipid droplet accumulation in glia ( Navarro et al . , 2010 ) .","From TEM and nile red staining , we observed a strong lipid droplet accumulation in glial cells of fh mutant clones ( Figure 1—figure supplement 2A and Figure 2—figure supplement 2A ) .","We recently reported that similar phenotype is observed in several mitochondrial mutants and promotes neurodegeneration via a ROS/JNK/SREBP pathway ( Liu et al . , 2015 ) ( Figure 2—figure supplement 2B ) .","However , we do not observe elevated ROS levels ( Figure 2D ) , and the level of a JNK pathway reporter , puc-lacZ , is not altered in fh mutants ( Figure 2—figure supplement 2C ) .","Finally , overexpression of brummer lipase ( Bmm ) or lipase 4 ( lip4 ) , homologs of human Adipose Triglyceride Lipase ( ATGL ) or acid lipase ( Gronke et al . , 2005 ) respectively , effectively reduces the number of lipid droplet but does not delay neurodegeneration of fh mutants ( Figure 2—figure supplement 2A ) , indicating that these lipid droplets do not contribute to neurodegeneration .","We recently discovered that some mutants that are required for mitochondrial function display an activity dependent degeneration of photoreceptors ( Jaiswal et al . , 2015 ) .","These mitochondrial mutants share several common phenotypes , including reduced ATP production , ERG rundown upon repetitive stimulation , and Rhodopsin1 ( Rh1 ) accumulation in the photoreceptor cytoplasm upon light exposure ( Jaiswal et al . , 2015 ) .","In these mutants , dysfunctional mitochondria cause reduced ATP production and decreased calcium influx , which not only leads to reduced photoreceptor depolarization , but also triggers an internalization of Rh1 leading to an overload of the endolysosomal system upon light stimulation ( Jaiswal et al . , 2015 ) .","The accumulation of Rh1 results in activity dependent degeneration , as the photoreceptors only degenerate when flies are raised in light ( light-induced neuronal activity ) but not in dark ( no neuronal activity ) ( Alloway et al . , 2000; Chinchore et al . , 2009; Jaiswal et al . , 2015 ) .","Since loss of fh leads to decreased CI activity and reduced energy production ( Figure 2B and C ) , we hypothesized that a similar activity dependent degeneration mechanism contributes to neurodegeneration in fh mutants .","To test this hypothesis , we first examined whether ERG traces rundown upon repetitive light stimulation in fh mutant photoreceptors .","As shown in Figure 3A , the ERG amplitudes of the first and last traces show a similar size in control animals .","In contrast , the ERG amplitude rapidly declines during repetitive light stimulation in fh mutants ( Figure 3A ) , similar to other mitochondrial mutants ( Jaiswal et al . , 2015 ) .","Therefore , we assessed whether Rh1 internalization is affected in fh mutants .","Wild type flies exposed to 12 hr of constant light typically exhibit low levels of Rh1 in photoreceptors ( Figure 3B ) .","However , in fh mutants , Rh1 accumulates in the cytoplasm of photoreceptors ( Figure 3B ) .","Since fh mutant photoreceptors share several features with other mitochondrial mutants , we hypothesized that reducing photoreceptor activity by raising fh mutants in the dark would suppress degeneration .","However , fh mutant flies that are raised in the dark still display severe photoreceptor degeneration ( Figure 3B ) .","Hence , we propose that in addition to the Rh1 accumulation , another pathogenic mechanism must contribute to neurodegeneration and mask the rescue effect when flies are raised in dark . 10 . 7554/eLife . 16043 . 009Figure 3 . Loss of fh causes Rh1 accumulation and iron deposit .","( A ) ERG traces upon repetitive light stimulation in both control ( y w FRT19A ) and fh mutants .","The double red arrows indicate ERG amplitudes of the first and last trace during the stimulation .","Quantification of the ratio of last/first ERG amplitude is on the right .","( B ) Rh1 distribution in control ( y w FRT19A ) and fh mutant clones after a 12 hr light exposure .","Rh1 is labeled in red , and rhabdomeres are labeled in green .","Scale bar: 5 µm .","Quantification of ERG amplitudes of control ( y w FRT19A ) and fh mutant clones in different light conditions is on the right .","L/D , flies are raised in 12 hr light and dark cycle .","D , flies are raised in dark .","( C ) Fe2+ levels are assessed by RPA in larval muscle and NMJ in fh mutants and rescued animals ( fh; gfh ) .","RPA fluorescence is represented as a heat map .","The NMJ boutons are indicated by white arrows .","Quantification of normalized RPA intensity is on the right .","n = 8 .","Scale bar: 20 µm .","( D ) Larval brain Perls’/DAB staining of control ( y w FRT19A ) , fh mutants , and rescued animals ( fh; gfh ) with regular food or low iron food conditions .","Scale bar: 100 µm .","( E ) Percent of animals that develop into pupae in control ( y w FRT19A ) and fh mutants when animals are raised by low iron food with different iron concentration .","n = 3 . Data are presented as mean ± SEM .","***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00910 . 7554/eLife . 16043 . 010Figure 3—figure supplement 1 . Fe2+ staining control . RPAC intensity in larva muscle and NMJ of fh mutants and rescued animals ( fh; gfh ) .","Scale bar: 25 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 010 Whether iron accumulates in the nervous system and contributes to the pathogenesis in mice and FRDA patients has remained a topic of debate ( Boddaert et al . , 2007; Puccio et al . , 2001; Solbach et al . , 2014 ) .","We therefore investigated whether iron is involved in neurodegeneration of fh mutants .","To assess if iron homeostasis is affected in fh mutants , we first examined ferrous iron ( Fe2+ ) levels by Rhodamine B- ( ( 1 , 10-phenanthrolin-5-yl ) -aminocarbonyl ) benzyl ester ( RPA ) .","RPA is a cell-permeable fluorescent dye that selectively accumulates in mitochondria , and its fluorescence is stoichiometrically quenched by Fe2+ ( Petrat et al . , 2002 ) .","As RPA poorly crosses the blood brain barrier ( data not shown ) , we performed RPA staining of larval muscles and neuromuscular junctions ( NMJ ) .","In fh mutants rescued with the genomic fh , which serves as a negative control , the RPA signal exhibits strong fluorescence ( Figure 3C ) .","In contrast , the RPA intensity in fh mutants is dramatically reduced , in both muscle and NMJ boutons , suggesting a strong Fe2+ accumulation in mitochondria ( Figure 3C ) .","RPAC ( Rhodamine B- ( ( phenanthren-9-yl ) -aminocarbonyl ) benzyl ester ) , a dye that is structurally very similar to RPA , is also taken up by mitochondria but is insensitive to Fe2+ mediated fluorescence quenching .","Indeed , RPAC fluorescence is not quenched in rescued animals ( fh; gfh ) and fh mutants ( Figure 3—figure supplement 1 ) , suggesting that the decreased fluorescence of RPA in fh mutants is not due to impaired dye uptake .","To investigate whether ferric iron ( Fe3+ ) levels are increased in fh mutants , we performed Perls’ staining and used 3 , 3'-Diaminobenzidine ( DAB ) to enhance the signal ( Meguro et al . , 2007 ) .","In control and rescued animals , the DAB signal is most obvious in the neuropil of the ventral nerve cord ( Figure 3D ) , suggesting that Fe3+ is unevenly distributed in the nervous system .","In fh mutants , however , the DAB signal is dramatically increased ( Figure 3D ) , indicating that loss of fh leads to a severe accumulation of Fe3+ .","Increased Fe3+ levels are not only found in the larval brain , but can also be observed in fat body and gut ( data not shown ) .","Together , these results indicate that loss of fh leads to both Fe2+ and Fe3+ accumulation in multiple tissues , including the nervous system .","To determine if the iron accumulation contributes to neurodegeneration in fh mutant clones , we reduced iron levels in the fly food .","Our regular food contains iron-rich molasses .","To reduce iron levels , we replaced molasses with sucrose as the main carbohydrate source and refer to this food as 'low iron food' .","As shown in Figure 3D , feeding larvae with low iron food reduces iron levels in the brains of both wild type control and fh mutants .","In addition , the lethal phase of fh mutants is susceptible to iron levels in the food .","In low iron food , 80% of fh mutants develop into pupae , but addition of 10 mM Fe3+ to the low iron food reduces pupation to about 10% , whereas the pupation rate of controls is not affected ( Figure 3E ) .","These data indicate that fh mutants are highly sensitive to iron levels .","To determine if iron toxicity is underlying the observed neurodegeneration , we fed low iron food to fh mutants and examined photoreceptor degeneration .","Treating fh mutants with low iron food improves the ERG defects when compared to regular food condition ( Figure 4A ) , suggesting that iron mediated toxicity contributes to neurodegeneration in fh mutants . 10 . 7554/eLife . 16043 . 011Figure 4 . Neuronal activity and iron interact synergistically to contribute to photoreceptor degeneration in fh mutants .","( A ) Quantification of ERG amplitudes of control ( y w FRT19A ) and fh mutants under different light and iron conditions .","( B ) Retina morphology of control ( y w FRT19A ) and fh mutants under different food conditions at Day 6 .","All animals are raised in dark .","Regular , flies are raised in regular food .","Low iron , flies are raised in low iron food .","Scale bar: 5 µm .","( C ) Quantification of ERG amplitudes of fh mutants in low iron food with different iron concentrations .","( D ) ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which ferritins are overexpressed .","Fer1* , enzymatic dead form of Fer1HCH .","Data are presented as mean ± SEM .","***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01110 . 7554/eLife . 16043 . 012Figure 4—figure supplement 1 . Control of low iron food treatment . Quantification of ERG amplitudes of control animals ( y w FRT19A ) in different food and iron conditions .","Regular , flies are raised in regular food .","Low iron , flies are raised in low iron food .","Data are presented as mean ± SEM .","L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 012 We next assessed if excess neuronal activity and iron accumulations act synergistically to promote neurodegeneration in fh mutants .","Interestingly , reducing photoreceptor activity ( raising flies in dark ) and iron ( low iron food ) leads to a synergistic effect in restoring both photoreceptor function and morphology ( Figure 4A and B ) .","To exclude the possibility that changes in other compounds than iron in low iron food suppresses neurodegeneration in fh mutants , we reintroduced iron into the low iron food ( low iron/10 mM iron ) and found that this treatment again induces rapid degeneration in fh mutant clones even when animals are raised in dark ( Figure 4B and C ) .","The ERG amplitudes of the wild type controls are not affected by different iron concentrations in the food ( Figure 4—figure supplement 1 ) .","These data suggest that iron toxicity contributes to the activity independent degeneration , as photoreceptors degenerate in the absence of neuronal activity .","We also tested if feeding 100 mM iron to wild type controls is sufficient to trigger neurodegeneration .","This concentration of iron is toxic as most animals die as embryos or young larval instars , and the Fe3+ levels were not increased in the brains ( data not shown ) .","We next explored if genetic manipulations can suppress iron toxicity and neurodegeneration in fh mutants .","We expressed Ferritins , the iron storage proteins that chelate and sequester irons .","In Drosophila , Ferritin 1 heavy chain homologue ( Fer1HCH ) and Ferritin 2 light chain homologue ( Fer2LCH ) form a heteropolymeric complex , whereas Ferritin 3 heavy chain homologue ( Fer3HCH ) forms a homopolymeric complex to store irons ( Missirlis et al . , 2006 , 2007 ) .","In fh mutant clones , co-expression of Fer1HCH and Fer2LCH ( fh; Rh1>Fer1 , Fer2 ) or expression of Fer3HCH alone ( fh; Rh1>Fer3 ) significantly suppresses degeneration when flies are raised in dark ( Figure 4D ) .","Co-expression of a ferroxidase inactive form of Fer1HCH along with wild type Fer2LCH in fh mutants ( fh; Rh1>Fer1* , Fer2 ) did not rescue degeneration ( Figure 4D ) .","These results further confirm that iron toxicity contributes to neurodegeneration in fh mutant clones .","In summary , we conclude that excess neuronal activity ( activity dependent ) and accumulated iron ( activity independent ) synergize to promote photoreceptor degeneration in fh mutants .","Given that iron accumulates in fh mutants , we investigated the mechanism downstream of iron toxicity .","Previous studies proposed that highly active iron interacts with hydrogen peroxide to generate free radicals , the Fenton reaction .","However , we did not observe an increase in ROS ( Figure 2D ) , and expression of different ROS scavengers did not suppress degeneration in fh mutants ( Figure 2E ) .","It has been previously documented that wild type yeast grow in medium with high iron levels increases sphingolipid synthesis ( Lee et al . , 2012 ) .","However , this phenotype was not documented in the yeast frataxin homolog mutants or FXN mutants in other organisms .","As we observed a strong iron accumulation in fh mutants , we hypothesized that iron toxicity induces sphingolipid synthesis and causes neurodegeneration .","To test our hypothesis , we first assessed the de novo synthesis of sphingolipids by mass spectrometry .","Consistent to our hypothesis , several upstream metabolic intermediates of sphingolipids are increased in fh mutants ( Figure 5A ) .","Strikingly , dihydrosphingosine ( dhSph ) is increased more than 10 fold in fh mutants .","Other sphingolipids , such as dihydroceramide ( dhCer ) and sphingosine ( Sph ) , are also increased to different levels ( Figure 5A ) .","This data suggest that the de novo synthesis of sphingolipids is increased in fh mutants . 10 . 7554/eLife . 16043 . 013Figure 5 . Increased sphingolipids contribute to degeneration of fh mutants .","( A ) Mass spectrometry analysis of sphingolipids in control ( y w FRT19A ) , fh mutants , and rescued animals ( fh; gfh ) .","dhsph , dihydrosphingosine; dhCer , dihydroceramide; Cer , ceramide; Sph , sphingosine .","n = 2 . ( B ) ERG of control ( y w FRT19A; Rh1-GAL4 ) , and fh mutants in which an RNAi against lace is expressed .","( C ) ERG amplitude and retinal morphology of fh mutants treated with 100 μM myriocin .","Scale bar: 5 µm .","( D ) Quantification of Day 3 ERG amplitudes of fh mutants with low iron food and myriocin treatments .","Data are presented as mean ± SEM .","The error bars represent the range of data in ( A ) .","**p<0 . 01 , ***p<0 . 001 , Student’s t-test .","D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01310 . 7554/eLife . 16043 . 014Figure 5—figure supplement 1 . Control of myriocin treatment . Quantification of Day 6 ERG amplitudes of control animals ( y w FRT19A ) with myriocin treatment .","Data are presented as mean ± SEM .","D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 014 To determine if increased sphingolipid synthesis contributes to neurodegeneration in fh mutants , we performed RNAi experiments against the first and rate-limiting enzyme in the de novo synthesis pathway , lace , the fly homolog of Serine palmitoyltransferase .","This significantly improved the ERG defects of fh mutants ( Figure 5B ) .","In addition , feeding fh mutants Myriocin , a serine palmitoyltransferase inhibitor , suppressed both the functional and morphological photoreceptor defects of fh mutant clones ( Figure 5C ) .","Feeding myriocin to control animals does not adversely affect photoreceptor function ( Figure 5—figure supplement 1 ) .","To assess if iron accumulation and increased sphingolipid synthesis act in the same or a different pathway , we treated fh mutants with low iron food as well as myriocin .","As shown in Figure 5D , we did not observe an additive or synergistic effect .","These results suggest that iron toxicity induces sphingolipid synthesis which in turn causes neurodegeneration in fh mutants .","Increased sphingolipids in yeast have been shown to activate a kinase pathway ( Pkh1/Ypk1/Smp1 ) that converges on a transcription factor Smp1p ( Lee et al . , 2012 ) .","However , this pathway has not yet been studied in higher eukaryotic cells , or FXN mutant animal models .","The corresponding proteins in mammals are PDK1 , SGK ( serum- and glucocorticoid regulated kinase ) , and Mef2 transcription factor ( Casamayor et al . , 1999; Dodou and Treisman , 1997 ) .","The Drosophila homolog of SGK has not been identified , but both Pdk1 and Mef2 are present in the fly .","Mef2 is a well known transcription factor required for muscle differentiation , however , it is also expressed in certain neurons and photoreceptors ( Blanchard et al . , 2010 ) .","As we observed an increase in sphingolipids , we assessed if increased levels of sphingolipids activate the Pdk1/Mef2 pathway and underlie the neurodegenerative phenotype in fh mutants .","We first tested if Pdk1/Mef2 is activated in fh mutants .","As a proxy for Mef2 activation , we examined the mRNA levels of known downstream targets of Mef2 .","Seven Mef2 targets , which have been validated through in situ hybridization and qRT-PCR , are all ectopically expressed in response to ectopic expression of Mef2 in vivo ( Elgar et al . , 2008 ) .","We therefore examined these seven Mef2 targets in the nervous system of fh mutants .","As shown in Figure 6A , all of these Mef2 targets are up-regulated .","Several negative control genes , the downstream targets of other transcription factors , including Enhancer of split ( E ( Spl ) ) , senseless ( sens ) , and patched ( ptc ) , as well as several housekeeping genes that are not Mef2 targets ( Sandmann et al . , 2006 ) , are not changed in fh mutants ( Figure 6—figure supplement 1A ) .","These data indicate that Mef2 is abnomally activated in fh mutants . 10 . 7554/eLife . 16043 . 015Figure 6 . Activated Pdk1/Mef2 pathway causes degeneration in fh mutants .","( A ) mRNA levels of Mef2 downstream targets in fh mutants and rescued animals ( fh; gfh ) .","n = 3 . ( B ) Day 3 ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which an RNAi against Pdk1 or Mef2 is expressed .","( C ) ERG of flies with Mef2 overexpression in the eye .","( D ) Mef2 luciferase reporter assay in Neuro-2A cells with different concentration of dhSph treatment .","GSK 2334470 , a PDK1 inhibitor .","n = 4 . Data are presented as mean ± SEM .","*p<0 . 05 .","**p<0 . 01 .","***p<0 . 001 , Student’s t-test .","D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01510 . 7554/eLife . 16043 . 016Figure 6—figure supplement 1 . Control of qRT-PCR .","( A ) mRNA levels of downstream targets of Notch , Wingless , and Hedgehog pathway as well as several ribosomal genes are not changed in fh mutants .","( B ) Retina morphology of fh mutants , and fh mutants that express Pdk1 or Mef2 RNAi .","Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .","Scale bar: 10 µm .","Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 016 Next , we tested if the activated Pdk1/Mef2 pathway contributes to neurodegeneration of fh mutants .","We reasoned that if Pdk1 and Mef2 are activated and toxic , their down-regulation may suppress degeneration .","Consistent with our hypothesis , RNAi mediated knockdown of Pdk1 suppresses the ERG and morphological defects in fh mutants ( Figure 6B and Figure 6—figure supplement 1B ) .","Furthermore , knock down of Mef2 by two independent RNAi lines suppresses neurodegeneration of fh mutants ( Figure 6B and Figure 6—figure supplement 1B ) , suggesting that activated Pdk1 and Mef2 are toxic and contribute to the degeneration .","To determine if up-regulation of Mef2 in wild type photoreceptors is sufficient to induce neurodegeneration , we ectopically expressed Mef2 in the adult eye using Rh1-Gal4 .","Overexpression of Mef2 is sufficient to cause photoreceptor degeneration ( Figure 6C ) .","Furthermore , flies with neuronal expression of Mef2 ( nSyb>Mef2 ) die within a week , instead of 70–80 days of the wild type flies ( data not shown ) .","These data suggest that abnormal activation of Mef2 in neuron is sufficient to trigger its demise .","To assess if a similar pathway is affected in vertebrate cells , we tested if sphingolipids can induce Mef2 activation in Neuro-2A cells .","dhSph , the sphingolipid that is increased more than 10 fold in fly fh mutants ( Figure 5A ) , induces luciferase signals of a Mef2 reporter in a concentration dependent manner ( Figure 6D ) .","In addition , the increased luciferase signal is suppressed by adding the PDK1 inhibitor GSK2334470 ( Figure 6D ) .","This data indicate that dhsph activates Mef2 through PDK1 , consistent with what we observed in the fly .","Collectively , these data suggest that sphingolipids act as signaling molecules to activate the Pdk1/Mef2 pathway and that this pathway contributes to photoreceptor degeneration in fh mutants ."],["Here , we report the isolation of the first severe loss of function allele of fh in Drosophila .","This allowed us to identify a novel molecular pathway that contributes to neurodegeneration .","Loss of fh in Drosophila causes an age dependent degeneration of photoreceptors in mutant clones .","Mutant photoreceptors display abnormal mitochondrial cristae morphology , reduced ETC CI activity , and impaired ATP production .","However , we do not observe an increase in ROS .","Our data indicate that Rh1 trafficking defects caused by mitochondria dysfunction lead to an activity dependent degeneration in fh mutants .","However , the accumulation of Fe2+ and/or Fe3+ stimulates sphingolipid synthesis which in turn activates the Pdk1/Mef2 pathway to cause an activity independent degeneration .","Hence , mitochondrial dysfunction , and iron toxicity through activation of the sphingolipid/Pdk1/Mef2 pathway , synergistically contribute to the demise of neurons in fh mutants .","The data indicate that ectopic activation of the transcription factor Mef2 induced by loss of FXN may play a role in FRDA .","Elevated ROS levels , either through impaired ETC CI or CIII , or iron associated Fenton reaction , have been proposed to be the major driving force of disease pathogenesis in FRDA ( Bayot et al . , 2011; Santos et al . , 2010 ) .","However , we do not observe increased ROS levels in fh mutants ( Figure 2D ) .","In addition , overexpression of ROS scavengers or feeding antioxidant AD4 does not suppress degeneration ( Figure 2E and Figure 2—figure supplement 1E ) .","The lack of increased ROS is surprising as mitochondrial mutants in Drosophila that exhibit a severely reduced CI activity typically show elevated ROS levels ( Owusu-Ansah et al . , 2008; Zhang et al . , 2013 ) .","Numerous studies using different animal models or cell lines have established that a partial or complete loss of FXN leads to different degrees of defects in the ETC .","Importantly , several reports have also documented that loss of FXN leads to an hypersensitivity to ROS , rather than elevated ROS levels ( Babcock et al . , 1997; Llorens et al . , 2007; Macevilly and Muller , 1997; Seznec et al . , 2005; Shidara and Hollenbeck , 2010 ) .","Consistent with our data , overexpression of ROS scavengers in an RNAi mediated fh knock down did not improve viability or cardiac function , and no ROS could be detected ( Anderson et al . , 2005; Shidara and Hollenbeck , 2010; Tricoire et al . , 2014 ) .","Similarly , loss of Fxn in mice does not lead to an increase in ROS ( Puccio et al . , 2001; Seznec et al . , 2005 ) , although elevated ROS levels in another mouse model have been documented ( Al-Mahdawi et al . , 2006 ) .","Combined , these data suggest that ROS is not a prominent player in model organisms and a clinical trial designed to suppress ROS have not provided compelling data ( Santhera Pharmaceuticals , 2010 ) .","Iron accumulation in mitochondria was first described in yeast frataxin homolog mutants in yeast ( Babcock et al . , 1997 ) .","Although iron deposits have been reported in cardiac muscles of Fxn deficiency mice and FRDA patients , iron accumulation in the nervous system has been reported by some ( Boddaert et al . , 2007; Koeppen et al . , 2009 ) but not by others ( Puccio et al . , 2001; Simon et al . , 2004; Solbach et al . , 2014 ) .","More importantly , the role of iron in the pathophysiology of FRDA has not yet been determined , and mitochondrial defects are thought to presage iron accumulation in mammals ( Puccio et al . , 2001 ) .","It has been argued that Fe3+ accumulations are a non-reactive , intra-mitochondrial precipitates ( Seguin et al . , 2010; Whitnall et al . , 2012 ) .","Finally , others proposed that iron accumulation is toxic as it induces the Fenton’s reaction and promotes ROS production .","We find that in Drosophila fh mutants , both Fe2+ and Fe3+ accumulate in the nervous system ( Figure 3C and D ) .","As we observed no increased ROS , we argue that Fe2+ and Fe3+ accumulation is toxic because:","1 ) the lethality is enhanced when iron is increased;","2 ) reduction of iron in food suppresses the progression of photoreceptor degeneration; and","3 ) overexpression of Ferritin delays the demise of neurons .","Taken together , we argue that iron accumulation in neurons is toxic and plays a primary role in the pathogenesis in fh mutants .","Our data indicate that iron accumulation enhances the synthesis of sphingolipid , and that the excess amount of sphingolipids is toxic and promotes neurodegeneration in fh mutants .","How iron accumulation leads to an increase in sphingolipid synthesis is unknown .","Although we show that Fe2+ accumulates in mitochondria of the larval muscle and at the NMJ in fh mutants , it is still not clear whether iron accumulates in other cellular organelles .","The de novo synthesis of sphingolipids occurs in the ER , and mitochondria have close contacts with ER at the MAMs ( mitochondria-associated endoplasmic reticulum membrane ) .","Mitochondrial or ER localized iron may promote the enzymatic function of Lace or other proteins in the ER to enhance sphingolipid synthesis .","However , treatment with Myriocin which targets Lace to decrease sphingolipid synthesis or down-regulation of lace via RNAi mediated knockdown suppresses degeneration in fh mutants ( Figure 5B and C ) , suggesting that increased sphingolipid metabolites contribute to toxicity .","These data are also in agreement with the observation that overexpression of Sk2 ( sphingosine kinase","2 ) or feeding dhSph-1P to wild type flies promotes the degeneration of photoreceptors ( Yonamine et al . , 2011 ) .","An increase in sphingolipids may affect various processes , including membrane integrity , lipid metabolism , and signal transduction ( Brown and London , 2000; Hannun and Obeid , 2008; Worgall , 2008 ) .","We found that down-regulation of Pdk1 or Mef2 in mutant photoreceptor delays neurodegeneration in fh mutants , while overexpression of Mef2 in photoreceptor promotes the demise of neurons ( Figure 6B and C ) .","It has been reported that Sph or dhSph , two sphingolipids that are elevated in fh mutants , directly induce autophosphorylation of PDK1 or SGK in an in vitro kinase assay ( Caohuy et al . , 2014; King et al . , 2000 ) .","Although there is no evidence that PDK1 or SGK can directly activate Mef2 , our data clearly show that Mef2 activity is up-regulated by elevating dhSph in the medium of cultured Neuro-2A cells , and this increased activity is suppressed by a PDK1 inhibitor ( Figure 6D ) , indicating that Mef2 can be activated by PDK1 or a downstream substrate of PDK1 ( Mora et al . , 2004 ) .","The MEF2 family includes four vertebrate genes ( MEF2A–D ) that play a key role in muscle differentiation ( Molkentin et al . , 1995 ) .","However , the MEFs are also expressed in neurons where they are involved in neuronal development ( Akhtar et al . , 2012; Leifer et al . , 1994; Lyons et al . , 1995; Mao et al . , 1999 ) .","The MEFs are known to regulate the expression of numerous target genes , including genes required for muscle differentiation , immediate-early genes , neuronal-activity-regulated genes , as well as genes involved in energy storage and immune response ( Clark et al . , 2013; Dietrich , 2013 ) .","In flies , ectopic expression of Mef2 in ectoderm is sufficient to induce nearly half of its predicted downstream targets that are typically expressed in muscles ( Sandmann et al . , 2006 ) .","Hence , its unusual activity may trigger neurodegeneration via the activation of some of its downstream targets .","Importantly , it has been shown that the up-regulation of Mef2 is implicated in the cardiac hypertrophy and dilation ( Molkentin and Markham , 1993; Xu et al . , 2006 ) , which is the major cause of death in FRDA patients .","In sum , our data indicate that reducing the levels of sphingolipid synthesis with Myriocin , or antisense strategy against enzymes involved in sphingolipid synthesis , or reducing the levels of Pdk1or Mef2 , are options that should be explored in FRDA models ."],["Flies were obtained from the Bloomington Drosophila Stock Center at Indiana University ( BDSC ) unless otherwise noted .","The stocks were routinely maintained at room temperature .","For genetic interaction experiments , flies were raised at 28°C to enhance the GAL4 activity .","For all the larval experiments , flies were allowed to lay eggs for 48 hr on grape juice plates with yeast paste .","Hemizygous mutant larvae and wild type controls were isolated by GFP selection at the first instar phase and transferred to standard fly food for the duration of their development .","To create mosaic mutant clones in the adult eye , y w FRT19A and y w fh FRT19A/FM7c , Kr>GFP females were crossed with y w GMR-hid FRT19A; ey-GAL4 UAS-FLP or y w GMR-hid l ( 1 ) cl FRT19A/FM7c , Kr>GFP; eyFLP Rh1-GAL4/CyO flies .","The following strains were used to generate fly stocks in this study: y w FRT19A ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) y w fh1 FRT19A/FM7c , Kr>GFP ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) y w fh1 FRT19A/FM7c , Kr>GFP; genomic-fh ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) Df ( 1 ) BSC537 , w1118/FM7h/Dp ( 2;Y ) G , P{hs-hid}Y y w GMR-hid FRT19A; ey-GAL4 UAS-FLP y w GMR-hid l ( 1 ) cl FRT19A/FM7c , Kr>GFP; eyFLP Rh1-GAL4/CyO ( Jaiswal et al . , 2015 ) w1; P{UAS-fh . A}1 ( Anderson et al . , 2005 ) w1; P{UAS-Cat . A}2 ( Anderson et al . , 2005 ) w1; P{UAS-Sod . A}B37 ( Anderson et al . , 2005 ) w1; P{UAS-Sod2 . M}UM83 ( Anderson et al . , 2005 ) P{rh1-GAL4}3 , ry506 P{Gal4-da . G32} UH1 y1 w*; P{nSyb-GAL4 . S}3 w; repo-GAL4/TM3 , Sb w*; P{w+mC bmmScer\\UAS = UAS-bmm} ( Gronke et al . , 2005 ) ( gift from Ronald Kühnlein ) w*; UAS-Lip4/CyO ( Liu et al . , 2015 ) P{UAS-Mef2-HA} ( Sandmann et al . , 2006 ) ( gift from Eileen Furlong ) P{UAS-Fer1HCH} P{UAS-Fer2LCH} ( Missirlis et al . , 2006 , 2007 ) ( gift from Hermann Steller ) P{UAS-Fer1HCH . mut} P{UAS-Fer2LCH} ( Missirlis et al . , 2006 , 2007 ) ( gift from Hermann Steller ) P{UAS-Fer3HCH} ( Missirlis et al . , 2006 ) ( gift from Patrik Verstreken ) w1118; P{GawB}D42 , P{UAS-mito-HA-GFP . AP}3 e1/TM6B , Tb1 ( Horiuchi et al . , 2005 ) ( gift from Bill Saxton ) w*; cno3 P{A92}pucE69/TM6B , abdA-LacZ ( Ring and Martinez Arias , 1993 ) ( Drosophila Genetic Resource Center ) ND42 RNAi: y1 sc* v1; P{TRiP . HMS00798}attP2 ( Owusu-Ansah et al . , 2008 ) lace RNAi: P{KK102282}VIE-260B ( Ghosh et al . , 2013 ) ( VDRC ) Pdk1 RNAi: P{KK108363}VIE-260B ( Tokusumi et al . , 2012 ) ( VDRC ) Mef2 RNAi: w1118; P{GD5039}v15549/TM3 and w1118; P{GD5039}v15550 ( Clark et al . , 2013 ) ( VDRC ) ERG recordings were performed as described ( Verstreken et al . , 2003 ) .","Briefly , flies were glued on a glass slide .","A recording electrode filled with 3M NaCl was placed on the eye , and another reference electrode was inserted into the fly head .","Before the recording , photoreceptors were allowed to recover by keeping flies in the dark for 3 min .","During the recording , a 1 s pulse of light stimulation was given , and the ERG traces of five to ten flies were recorded and analyzed by AXON -pCLAMP 8 software .","To detect Fe2+ levels , L3 larvae were dissected in ice-cold 0 . 25 mM Ca2+ HL3 ( Stewart et al . , 1994 ) ( 70 mM NaCl , 5 mM KCl , 20 mM MgCl2 , 10 mM NaHCO3 , 5 mM trehalose , 5 mM HEPES , 115 mM sucrose , pH 7 . 2 ) and rinsed in HL3 with 1 mM Ca2+ .","The animals were then incubated with 10 µM RPA or RPAC ( Squarix Biotechnology , Germany ) in 1 mM Ca2+ HL3 for 20 min at room temperature in the dark .","The animals were washed subsequently three times with ice-cold 1 mM Ca2+ HL3 and then incubated in 1 mM Ca2+ HL3 with 7 mM L-glutamic acid for 15 min at room temperature in the dark .","The RPA or RPAC fluorescence was obtained with a Zeiss LSM 510 confocal microscope ( Carl Zeiss ) and quantified by Image J . To evaluate Fe3+ levels , L3 larval brains were dissected and fixed in 3 . 7% formaldehyde for 20 min .","Samples were quickly washed three times with 0 . 4% Triton-PBS and incubated with Perls solution ( 1% K4Fe ( CN ) 6 and 1% HCl in PBT ) for 5 min .","After 3 quick washes in PBT , samples were incubated with DAB solution ( 10 mg DAB with 0 . 07% H2O2 in 0 . 4% Triton-PBS ) to enhance the signal .","The brains were then quickly washed three times with 0 . 4% Triton-PBS and mounted .","The brain images were obtained with a Leica MZ16 stereomicroscope equipped with Optronics MicroFire Camera .","L3 larvae were dissected in ice-cold 0 . 25 mM Ca2+ HL3 and rinsed with 1 mM Ca2+ HL3 .","The animals were then incubated with 10 µM dihydroethidium ( DHE , Sigma-Aldrich ) in 1 mM Ca2+ HL3 for 20 min at room temperature in the dark .","The samples were quickly washed three times with 1 mM Ca2+ HL3 with 7 mM L-glutamic acid , and the DHE fluorescence in larval brains were live imaged by Zeiss 510 confocal microscope ( Carl Zeiss ) and quantified by Image J . Low iron food was made using 10% dry yeast , 10% sucrose , and 0 . 6% agar in w/v in water .","The solution was microwaved and dried at room temperature , divided into vials , and stored at 4°C for further use .","To increase iron levels in the food , ammonium iron ( Ш ) citrate ( Sigma-Aldrich ) was added to a final concentration of 1 mM or 10 mM in the low iron food .","To test if inhibition of sphingolipid synthesis suppresses neurodegeneration in fh mutants , myriocin from Mycelia sterilia ( Sigma-Aldrich ) was dissolved in regular fly food ( molasses based ) or low iron food , and vials were then stored immediately at −20°C for future use .","The flies were transfer to fresh food vials every two days .","For the fh genomic rescue construct , a DNA fragment containing Drosophila X: 9 , 147 , 070 . . 9 , 150 , 371 was retrieved from a 20 kb P[acman] construct that covers the entire fh genomic locus ( clone CH322-14E18 from BACPAC Resources Center ) ( Venken et al . , 2009 ) .","The fh genomic sequence was then subcloned into p{w+}attB using KpnI and NotI sites .","The construct was then injected into y w ΦC31;VK33 embryos , and transgenic flies were selected .","To generate pUAST-attB-UAS-hFXN construct , the primers 5’- TCCGAATTCGCCACCATGTGGACTCTCGGGCGCCGCGC-3’ and 5’-GCGGTACCTCAAGCATCTTTTCCGGAATAGGC-3’ were used to clone the full length FXN cDNA from pcDNA-hFXN-HA , a gift from Gino Cortopassi ( Shan et al . , 2007 ) .","The PCR product was then subcloned into the pUAST-attB vector .","The construct was then injected into y w ΦC31;VK33 or y w ΦC31;VK37 embryos , and the transgenic flies were selected .","For whole mount eye staining , we dissected fly heads in cold PBS and fixed with 4% paraformaldehyde at 4°C overnight .","The retinas were then dissected and fixed for an additional 30 min .","For larval ventral nerve cord staining , L3 instar larvae were dissected and fixed with 3 . 7% formaldehyde for 20 min .","The antibodies were used at the following concentrations: mouse anti-Na/K ATPase ( α5 , Developmental Studies Hybridoma Bank ( DSHB ) ) , 1:200; rat anti-Elav ( 7E8A10 , DSHB ) , 1:500; mouse anti-Rh1 ( 4C5 , DSHB ) , 1:100; mouse anti-V5 ( R96025 , Invitrogen ) , 1:1000; rabbit anti-HRP ( 323-005-021 , Jackson ImmunoResearch ) , 1:1000; chicken anti-GFP ( ab13970 , abcam ) , 1:1000; rabbit anti-β-Galactosidase ( Cappel ) , 1:1000; mouse anti-DLG ( 4F3 , DSHB ) , 1:200; Alexa 488-conjugated phalloidin ( Invitrogen ) , 1:100; Alexa 405- , Alexa 488- , Cy3- , or Cy5-conjugated secondary antibodies ( Jackson ImmunoResearch ) , 1:250 .","Samples were then mounted in Vectashield ( Vector Laboratories ) before being analyzed under a confocal microscope .","All the confocal scans were acquired with a confocal microscope ( model LSM 510 or LSM 710; Carl Zeiss ) and processed using LSM Image Browser ( Carl Zeiss ) and Photoshop ( Adobe ) .","The Nile red staining was performed as described ( Liu et al . , 2015 ) .","Briefly , the retina was dissected and fixed in 3 . 7%formaldehyde .","The samples were then incubated for 10 min at 1:1 , 000 dilution of PBS with 1 mg/ml Nile Red ( Sigma ) .","The retina was then washed with PBS and mounted in Vectashield ( Vector Laboratories ) .","Images were obtained with a Zeiss LSM 510 confocal microscope .","Mitochondria were extracted as previously described ( Zhang et al . , 2013 ) .","Mitochondrial ETC enzymatic activity assay and aconitase activity assay were performed as previously described ( Zhang et al . , 2013 ) .","To determine ADP/ATP ratio , L3 larvae were collected and the ratio was determined with an ADP/ATP Ratio Assay Kit ( ab65313 , abcam ) .","Briefly , 5–10 L3 instar larvae were collected and frozen with liquid nitrogen .","The samples were then homogenized and the ADP/ATP ratio was measured following manufacturer’s protocol .","The luminescence was measured by Synergy 2 Microplate Reader ( BioTek ) .","Drosophila retina ultrastructure was imaged following standard electron microscopy procedures using a Ted Pella Bio Wave processing microwave with vacuum attachments .","Briefly , fly heads were dissected and fixed at 4°C in fixative ( 2% paraformaldehyde , 2 . 5% glutaraldehyde , 0 . 1 M sodium cacodylate , and 0 . 005% CaCl2 , pH 7 . 2 ) overnight .","The samples were then postfixed in 1% OsO4 , dehydrated in ethanol and propylene oxide , and then embedded in Embed-812 resin ( Electron Microscopy Sciences ) under vacuum .","Photoreceptors were then sectioned and stained in 1% uranyl acetate and saturated lead nitrate .","TEM images of photoreceptor sections were taken using a JEOL JEM 1010 transmission electron microscope at 80 kV with an AMT XR-16 mid-mount 16 mega-pixel digital camera .","ESI/MS/MS analysis of endogenous sphingosine bases and ceramide species ( C14- and C16-Sphingoid Base ) were performed on a Thermo_Fisher TSQ Quantum triple quadrupole mass spectrometer , operating in a Multiple Reaction Monitoring ( MRM ) positive ionization mode , using modified version ( Bielawski et al . , 2009 ) .","Briefly , whole larvae were fortified with the internal standards ( ISs: C17 base D-erythro-sphingosine ( 17CSph ) , C17 sphingosine-1-phosphate ( 17CSph-1P ) , N-palmitoyl-D-erythro-C13 sphingosine ( 13C16-Cer ) and heptadecanoyl-D-erythro-sphingosine ( C17-Cer ) ) , and extracted with ethyl acetate/iso-propanol/water ( 60/30/10 v/v ) solvent system .","After evaporation and reconstitution in 100 μl of methanol samples were injected on the HP1100/TSQ Quantum LC/MS system and gradient eluted from the BDS Hypersil C8 , 150 × 3 . 2 mm , 3 μm particle size column , with 1 . 0 mM methanolic ammonium formate / 2 mM aqueous ammonium formate mobile phase system .","Peaks corresponding to the target analytes and internal standards were collected and processed using the Xcalibur software system .","Quantitative analysis was based on the calibration curves generated by spiking an artificial matrix with the known amounts of the target analyte synthetic standards and an equal amount of the internal standards ( ISs ) .","The target analyte/IS peak areas ratios were plotted against anlyte concentration .","The target analyte/IS peak area ratios from the samples were similarly normalized to their respective ISs and compared to the calibration curves , using a linear regression model .","Applying consistent mass spectral conditions of Collision Assistant Dessociation ( CAD ) ; 35 eV and Electron Spray Ionization ( ESI ) all sphingoid bases and related ceramides undergo uniform transition from initial molecular ion ( M+1 ) to the respective sphingoid backbone secondary ions .","Consequently , calibration curves , generated from authentic standards of the typical , 18C-sphingosine and ceramides , can be used for quantitation of other , e . g . 20C- counterparts .","The levels of sphingolipid species are normalized to the phosphate amount of the samples .","Although absolute concentrations determined for compounds without authentic standards ( 20C-LCB derivatives ) may not be precise , due to possible differences in instrument response for 18C- and 20C-LCB related compounds , for comparative study , where changes in sphingolipids level rather than absolute concentration , are most important this indirect methodology provide reliable results .","Total RNA from the fly larval brains were extracted by Trizol RNA Isolation Reagents ( Thermo Fisher Scientific ) follow the manufacturer’s instructions .","The cDNA was synthesized by High-Capacity cDNA Reverse Transcription Kit ( Applied Biosystems ) .","Real-time PCR was carried out using iQ SYBR Green Supermix ( Bio-Rad ) and performed in a thermal cycler ( iCycler; Bio-Rad Laboratories ) .","The data were collected and analyzed using the optical module ( iQ5; Bio-Rad Laboratories ) .","The following primer pairs are used ( 5’to 3’ ) : RP49 forward ( control primer ) , TCCTACCAGCTTCAAGATGAC; RP49 reverse ( control primer ) , CACGTTGTGCACCAGGAACT; Act57B forward , TTGAGACCTTCAACTCGCCC; Act58B reverse , CCATCACCGGAGTCCAGAAC; mib2 forward , CGCCAGAAAACACTGTCGTG; mib2 reverse , GACGAACTCCAACTTGAGCATTA; CG5080 forward , CGCCCTCTCCAATTAGTTCTCC; CG5080 reverse , CAGCGACTGGATAGTTCCGC; Mlc2 forward , TCGGGTCCGATCAACTTCAC; Mlc2 reverse , ATTTCGCGGAATTTGTCACCG; Mlc1 forward , CCGAGGATGATGAAGGATTT; Mlc1 reverse , CTGGTCTGTCACACATTCTGG; CG6972 forward , AGGGATCACACACTGATGAACT; CG6972 reverse , CAACAGCCATTCGGAGGGAC; Mhc forward , ATCAATCCTTACAAGCGTTACCC; Mhc reverse , CCGTCAGAGATGGCGAAAATATG; E ( Spl ) forward , ATGGAATACACCACCAAGACCC; E ( Spl ) reverse , GGCGACAAGTGTTTTCAGGTT; sens forward , TACTGTGGCCCCAATTTTTGT; sens reverse , AAGGCAAAGTCACGATCCCG; ptc forward , GGATCTTTACATACGCACCAGC; ptc reverse , GGACTGGAATACTGATCGCAG; Neuro-2A cells were seed on 12 well plates , and each well was transfected with 3XMef2-luc reporter ( Addgene ) and Renilla control pRL-TK vector ( gift from Huda Zoghbi ) .","After two days , DMSO , DL-Dihydrosphingosine ( Sigma ) , or GSK2334470 ( gift from Huda Zoghbi ) was added into the medium .","Cells were cultured one more day with drug treatment .","The luciferase assay was then performed by Dual-Luciferase Reporter Assay System ( Promega ) and followed manufacturer instruction ."]],"string":"[\n [\n \"FRDA , an inherited recessive ataxia , is caused by mutations in FXN ( Campuzano et al . , 1996 ) .\",\n \"During childhood or early adulthood , FRDA patients show a progressive neurodegeneration of dorsal root ganglia , sensory peripheral nerves , corticospinal tracts , and dentate nuclei of the cerebellum ( Koeppen , 2011 ) .\",\n \"FXN is evolutionarily conserved and the homologs have been identified in most phyla ( Bencze et al . , 2006; Campuzano et al . , 1996 ) .\",\n \"It encodes a mitochondrial protein that is required for iron-sulfur cluster assembly ( Layer et al . , 2006; Lill , 2009; Muhlenhoff et al . , 2002; Rotig et al . , 1997; Yoon and Cowan , 2003 ) .\",\n \"Once synthesized , iron-sulfur clusters are incorporated into a variety of metalloproteins , including proteins of the mitochondrial electron transport chain ( ETC ) complexes and aconitase , where they function as electron carriers , enzyme catalysts , or regulators of gene expression ( Lill , 2009 ) .\",\n \"It has been proposed that loss of FXN leads to impaired ETC complex , which in turn triggers ROS production that directly contributes to cellular toxicity ( Al-Mahdawi et al . , 2006; Anderson et al . , 2008; Calabrese et al . , 2005; Schulz et al . , 2000 ) .\",\n \"However , the ROS hypothesis has been questioned in several studies .\",\n \"For example , loss of FXN only leads to a modest hypersensitivity to oxidative stress ( Macevilly and Muller , 1997; Seznec et al . , 2005; Shidara and Hollenbeck , 2010 ) .\",\n \"In addition , several clinical trails based on antioxidant therapy in FRDA patients have shown no or limited benefits ( Lynch et al . , 2010; Parkinson et al . , 2013; Santhera Pharmaceuticals , 2010 ) .\",\n \"Loss of yeast frataxin homolog results in iron accumulation ( Babcock et al . , 1997 ) , and this phenotype has also been reported in cardiac muscles of a Fxn deficiency mouse and FRDA patients ( Koeppen , 2011; Lamarche et al . , 1980; Michael et al . , 2006; Puccio et al . , 2001 ) .\",\n \"However , whether iron accumulates in the nervous system upon loss of FXN remains controversial .\",\n \"Furthermore , whether iron deposits contribute to the pathogenesis is not clear .\",\n \"It has been reported that elevated iron levels were observed in the dentate nuclei or in glia cells of FRDA patients ( Boddaert et al . , 2007; Koeppen et al . , 2012 ) .\",\n \"Contrary to these results , others suggested that there is no increase of iron in the nervous system of Fxn deficiency mice and FRDA patients ( Koeppen et al . , 2007; Puccio et al . , 2001; Solbach et al . , 2014 ) .\",\n \"Taken together , current data provide insufficient evidence to establish that iron dysregulation contributes to neurodegeneration .\",\n \"In addition , the mechanism underlying iron toxicity is still unclear .\",\n \"In summary , the pathological interplay of mitochondrial dysfunction , ROS , and iron accumulation remains to be established .\",\n \"We identified the first mutant allele of fh in an unbiased forward genetic screen aimed at isolating mutations that cause neurodegenerative phenotypes .\",\n \"We show that loss of fh causes an age dependent neurodegeneration in photoreceptors and affects mitochondrial function .\",\n \"Unlike other mitochondrial mutants with impaired ETC activity , we do not observe an increase in ROS .\",\n \"However , loss of fh causes an iron accumulation in the nervous system , induces an up-regulation of sphingolipid synthesis , and activation of Pdk1 and Mef2 .\",\n \"Reducing iron toxicity or inhibiting the sphingolipid/Pdk1/Mef2 pathway significantly suppresses neurodegeneration in fh mutants .\",\n \"To our knowledge , this is the first evidence that sphingolipids , Pdk1 , and the transcription factor Mef2 are shown to play a primary role in FXN-induced neurodegeneration .\"\n ],\n [\n \"We identified the first EMS ( ethyl methanesulfonate ) induced missense mutation in fh , the Drosophila homolog of FXN , through a forward genetic screen on the X chromosome ( Figure 1—figure supplement 1A ) ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) .\",\n \"The molecular lesion , S136R , is in a highly conserved region , which is required for the FXN binding to the iron-sulfur cluster assembly complex ( Tsai et al . , 2011 ) .\",\n \"In addition , six point mutations adjacent to S136 ( S157 in human ) have been reported in FRDA patients ( Figure 1—figure supplement 1B ) ( Santos et al . , 2010 ) .\",\n \"Hemizygous fh mutants are L3 to pupal lethal , and the lethality is not enhanced in transheterozygous mutants that carry a deficiency ( fh/Df ( 1 ) BSC537 ) , suggesting that S136R is a severe loss-of-function ( Figure 1—figure supplement 1C ) .\",\n \"The lethality of fh can be rescued by a genomic fh construct ( gfh ) , or by ubiquitous expression ( da-GAL4 driver ) of fh cDNA using the UAS/GAL4 system ( Brand and Perrimon , 1993 ) ( Figure 1—figure supplement 1C ) .\",\n \"The lethality cannot be rescued with tissue specific drivers , including neuronal ( nSyb-GAL4 ) , muscular ( Mef2-GAL4 ) , or glial ( repo-GAL4 ) drivers , suggesting a requirement for fh in multiple tissues ( Figure 1—figure supplement 1C ) .\",\n \"In addition , fh mutants exhibit a smaller body size and prolonged larval stages ( 10 to 12 days instead of 5 days for wild type animals ) , similar to other mitochondrial mutants ( Sandoval et al . , 2014; Zhang et al . , 2013 ) .\",\n \"Interestingly , removal of maternal wild type fh mRNA or protein in the egg exacerbates the lethal phase from pupal to embryonic lethality ( Figure 1—figure supplement 1C ) .\",\n \"In essence , the residual maternally deposited wild type Fh not only extends the lifespan but also creates a partial loss of Fh condition in the fh mutants .\",\n \"To bypass pupal lethality of fh mutants , mosaic mutant clones of adult photoreceptor neurons were generated with the eyeless-FLP/FRT system .\",\n \"As a functional readout , we recorded and compared electroretinograms ( ERGs ) in young and aged flies .\",\n \"In response to a light stimulus , the ERG amplitudes of newly eclosed fh mutants ( Day 1 ) are ~60% of control animals ( y , w , FRT19Aiso , the isogenized flies used in the screen ) ( Figure 1A ) .\",\n \"The ERG amplitudes rapidly decline over a six day period ( Figure 1A ) .\",\n \"Morphologically , the fh mutant clones contain a normal number of photoreceptors with a typical trapezoidal organization at Day 1 ( Figure 1B ) .\",\n \"However , by using transmission electron microscopy ( TEM ) , we found that Day 1 fh mutant photoreceptors exhibit an expansion of the endoplasmic reticulum ( ER ) and a dramatic accumulation of lipid droplets in glial cells that are not observed in controls ( Figure 1—figure supplement 2A and B ) .\",\n \"At Day 3 , photoreceptors in mutant clones exhibit aberrant elongated rhabdomeres ( Figure 1B ) .\",\n \"By Day 6 , many of the photoreceptors are missing in the mutant clones , whereas control retinas exhibit intact morphology ( Figure 1B and Figure 1—figure supplement 2C ) .\",\n \"These data show that the mutant photoreceptors undergo a rapid and severe functional as well as morphological degeneration . 10 . 7554/eLife . 16043 . 003Figure 1 . Loss of fh results in age dependent neurodegeneration in photoreceptors .\",\n \"( A ) ERG of control ( y w FRT19A ) and fh ( y w fh FRT19A ) mosaic eyes .\",\n \"The ERG amplitudes from Day 1 to Day 6 are indicated by red double arrows .\",\n \"Quantification of the ERG amplitudes of control ( y w FRT19A ) and fh ( y w fh FRT19A ) is on the right .\",\n \"( B ) Retina morphology of control ( y w FRT19A ) and fh mosaic eyes .\",\n \"Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .\",\n \"Neuronal nuclei are labeled by anti-Elav antibody ( blue ) .\",\n \"Scale bar: 10 µm .\",\n \"( C ) ERG of control ( y w FRT19A ) , fh mutants , fh mutants carrying a genomic fh construct ( fh; gfh ) , and fh mutants that express the fh cDNA ( fh; Rh1>fh ) or human FXN cDNA ( fh; Rh1>hFXN ) in photoreceptors using a Rh1-GAL4 driver .\",\n \"The ERG amplitudes were recorded at Day 6 .\",\n \"Data are presented as mean ± SEM .\",\n \"**p<0 . 01 , ***p<0 . 001 , Student’s t-test .\",\n \"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00310 . 7554/eLife . 16043 . 004Figure 1—figure supplement 1 . Mutation and lethal phase analysis of fh mutants .\",\n \"( A ) Molecular lesion of fh mutants .\",\n \"A 3 . 4 kb genomic rescue construct is shown below the gene .\",\n \"( B ) FXN sequences alignment between fly , human , and mouse .\",\n \"Mutation S136R identified in fh mutants is indicated by a box .\",\n \"Black arrow heads indicate the point mutations identified in the FRDA patients .\",\n \"( C ) Lethal phase analysis of fh mutants .\",\n \"The lethality can be rescued by a genomic fh construct , or expressing fh cDNA by the ubiquitous driver daughterless-GAL4 .\",\n \"Overexpression of fh cDNA in a tissue specific manner cannot rescue lethality .\",\n \"Finally , removing maternal Fh proteins in the female germ line by using OvoD ( Perrimon and Gans , 1983 ) leads to embryonic to early L1 lethality . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00410 . 7554/eLife . 16043 . 005Figure 1—figure supplement 2 . Ultrastructure of adult eyes exhibits morphological defects in fh mutant clones .\",\n \"( A ) Transmission electron microscopy ( TEM ) images of retina morphology in control ( y w FRT19A ) and fh mutant clones at Day 1 .\",\n \"Lipid droplets are indicated by red arrow .\",\n \"Scale bar: 2 µm .\",\n \"( B ) Quantification of lipid droplet in Day 1 retina of control ( y w FRT19A ) and fh mutants .\",\n \"n = 6 .\",\n \"( C ) Retina morphology in control ( y w FRT19A ) and fh mutant clones at Day 6 .\",\n \"Scale bar: 2 µm .\",\n \"( D ) Retina morphology of fh mutants ( fh; Rh1-Gal4 ) , fh mutants carrying a genomic fh construct ( fh; gfh ) , and fh mutants that express the fh cDNA ( fh; Rh1>fh ) or human FXN cDNA ( fh; Rh1>hFXN ) .\",\n \"Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .\",\n \"The red eye marker ( w+ ) of Rh1-GAL4 creates autofluorescence background that makes imaging difficult .\",\n \"Scale bar: 10 µm .\",\n \"Data are presented as mean ± SEM .\",\n \"***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 005 The neurodegeneration can be fully rescued by a genomic fh construct or by expressing fh cDNA in photoreceptors ( Figure 1C and Figure 1—figure supplement 2D ) , suggesting that photoreceptor degeneration is cell-autonomous .\",\n \"Moreover , expression of the human FXN cDNA rescues neurodegeneration in fh mutants ( Figure 1C and Figure 1—figure supplement 2D ) , showing that fly and human FXN have conserved molecular function , and that our studies are relevant to the biological role of human FXN .\",\n \"FXN has been reported to be localized to mitochondria ( Koutnikova et al . , 1997 ) .\",\n \"Indeed , Fh colocalizes with mitoGFP in larval motor neuron , and removing the predicted mitochondrial targeting sequence ( MTS ) of fh cDNA ( Fh-∆MTS-V5 ) leads to a diffused signal in the cytosol ( Figure 2—figure supplement 1A ) .\",\n \"Removal of the MTS leads to a dysfunctional protein as expression of Fh-∆MTS-V5 in the fh mutant clone is not able to rescue the ERG defect ( Figure 2—figure supplement 1B ) .\",\n \"Loss of fh causes a highly aberrant mitochondrial morphology in one day old adult photoreceptors .\",\n \"The mitochondria in fh mutants are larger and exhibit aberrant cristae and altered inner membrane morphology ( Figure 2A and Figure 2—figure supplement 1C ) .\",\n \"Loss of FXN has been reported to cause various ETC and respiratory defects in different animal models , including yeast , fly , mouse , as well as FRDA patients ( Al-Mahdawi et al . , 2006; Anderson et al . , 2005; Carletti et al . , 2014; Koutnikova et al . , 1997; Puccio et al . , 2001; Rotig et al . , 1997; Wilson and Roof , 1997 ) .\",\n \"We find that ETC Complex I ( CI ) activity is severely reduced in fh mutants when compared to control and genomic rescued animals ( Figure 2B ) .\",\n \"Consistent with this data , the ADP/ATP ratio is increased in fh mutants ( Figure 2C ) , showing that energy production is impaired . 10 . 7554/eLife . 16043 . 006Figure 2 . Loss of fh causes impaired mitochondria , and reducing ROS levels fails to suppress degeneration in fh mutants .\",\n \"( A ) TEM images of mitochondria in photoreceptors of control ( y w FRT19A ) and fh mutant clones .\",\n \"The insets show a single mitochondrion ( red box ) .\",\n \"Scale bar: 500 nm .\",\n \"( B ) ETC complex activities in control ( y w FRT19A ) , fh mutant , and genomic rescued animal ( fh; gfh ) .\",\n \"Complex activities are normalized to citrate synthase ( CS ) , which act as a control of mitochondrial mass . n = 3 . ( C ) ADP/ATP ratio in control ( y w FRT19A ) , fh mutant , and rescued animal ( fh; gfh ) .\",\n \"n = 3 . ( D ) ROS levels are measured by DHE in the larval ventral nerve cord .\",\n \"Neuronal down-regulation of ND42 ( nSyb>ND42 RNAi ) , a subunit of CI , acts as a positive control .\",\n \"Quantification of DHE fluorescence is on the right .\",\n \"n = 8 .\",\n \"Scale bar: 50 µm .\",\n \"( E ) Day 3 ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which ROS scavengers are overexpressed in photoreceptors .\",\n \"Data are presented as mean ± SEM .\",\n \"**p<0 . 01 , ***p<0 . 001 , Student’s t-test .\",\n \"ND , no significant difference .\",\n \"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00610 . 7554/eLife . 16043 . 007Figure 2—figure supplement 1 . Mitochondrial phenotype of fh mutants .\",\n \"( A ) The immunostaining of larval motor neuron cell body with expression of fh or fh without the mitochondrial targeting sequence ( MTS ) ( Fh-∆MTS ) .\",\n \"Fh and Fh-∆MTS are labeled by V5 ( red ) , and mitochondria are labeled by mitoGFP ( green ) .\",\n \"The nuclei are marked by DAPI ( blue ) .\",\n \"Scale bar: 5 µm .\",\n \"( B ) Quantification of ERG amplitudes of fh mutants with different cDNA constructs at Day\",\n \"3 . ( C ) Quantification of abnormal mitochondria in photoreceptor in TEM image .\",\n \"n = 6 .\",\n \"( D ) Day 6 ERG of fh mutants ( fh; Rh1-GAL4 ) in which yeast Ndi1p are overexpressed in photoreceptors .\",\n \"( E ) Quantification of ERG amplitudes of fh mutants at Day\",\n \"3 . Treating fh mutants with an antioxidant drug AD4 with 40 μg/ml concentration does not suppress degeneration .\",\n \"Data are presented as mean ± SEM .\",\n \"***p<0 . 001 , Student’s t-test .\",\n \"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00710 . 7554/eLife . 16043 . 008Figure 2—figure supplement 2 . ROS/JNK/SREBP pathway does not contribute to degeneration in fh mutants .\",\n \"( A ) Nile red staining of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants that overexpress lipases .\",\n \"Quantification of ERG amplitudes at Day 3 is on the right .\",\n \"Scale bar: 5 µm .\",\n \"( B ) ROS/JNK/SREBP pathway causes accumulation of lipid droplets and degeneration in other mitochondrial mutants ( Liu et al . , 2015 ) .\",\n \"( C ) The levels of puc-lacZ ( green ) in the larval ventral nerve cord of control ( y w FRT19A ) and fh mutants .\",\n \"Neuronal membrane is marked by anti-DLG antibody ( blue ) .\",\n \"Scale bar: 40 µm .\",\n \"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 008 It has been reported that expression of yeast Ndi1p , which is a single subunit of NADH dehydrogenase , can mitigate the deleterious effects of an ETC CI deficit in the fly ( Vilain et al . , 2012 ) .\",\n \"However , overexpression of Ndi1p in fh mutant clones does not suppress neurodegeneration ( Figure 2—figure supplement 1D ) .\",\n \"Impaired ETC CI activity has been linked to increased ROS production that in turn causes cellular stress or toxicity ( Sugioka et al . , 1988; Turrens and Boveris , 1980 ) .\",\n \"We therefore evaluated ROS production in vivo with dihydroethidium ( DHE ) , a dye that emits fluorescence upon oxidation by ROS .\",\n \"Neuronal down-regulation of ND42 ( nSyb>ND42 RNAi ) , a subunit of CI , was used as a positive control ( Owusu-Ansah et al . , 2008 ) .\",\n \"As shown in Figure 2D , loss of ND42 exhibits strong DHE fluorescence in the larval ventral nerve cord , indicating elevated levels of ROS .\",\n \"However , DHE levels are not obviously increased in fh mutants , suggesting no or a very mild increase in ROS ( Figure 2D ) .\",\n \"Elevated ROS levels are proposed to be a major contributor to the pathogenesis in FRDA .\",\n \"To further investigate the role of ROS in photoreceptor degeneration of fh mutants , we overexpressed several ROS scavengers , including Catalase , Super Oxide Dismutase 1 ( SOD1 ) , and SOD2 , which have all been shown to effectively reduce ROS in flies ( Orr and Sohal , 1992; Parkes et al . , 1998; Sun et al . , 2002 ) .\",\n \"Consistent with the DHE data , overexpression of these ROS scavengers does not suppress neurodegeneration in fh mutants ( Figure 2E ) .\",\n \"Furthermore , feeding fh mutants with AD4 , a potent antioxidant that dampens oxidative stress ( Amer et al . , 2008; Liu et al . , 2015 ) , does not suppress neurodegeneration ( Figure 2—figure supplement 1E ) .\",\n \"Taken together , our findings argue that ROS does not contribute to the demise of neurons in fh mutants .\",\n \"It has been proposed that lipid droplets play a role in the pathogenesis of FRDA as knockdown of fh in flies causes lipid droplet accumulation in glia ( Navarro et al . , 2010 ) .\",\n \"From TEM and nile red staining , we observed a strong lipid droplet accumulation in glial cells of fh mutant clones ( Figure 1—figure supplement 2A and Figure 2—figure supplement 2A ) .\",\n \"We recently reported that similar phenotype is observed in several mitochondrial mutants and promotes neurodegeneration via a ROS/JNK/SREBP pathway ( Liu et al . , 2015 ) ( Figure 2—figure supplement 2B ) .\",\n \"However , we do not observe elevated ROS levels ( Figure 2D ) , and the level of a JNK pathway reporter , puc-lacZ , is not altered in fh mutants ( Figure 2—figure supplement 2C ) .\",\n \"Finally , overexpression of brummer lipase ( Bmm ) or lipase 4 ( lip4 ) , homologs of human Adipose Triglyceride Lipase ( ATGL ) or acid lipase ( Gronke et al . , 2005 ) respectively , effectively reduces the number of lipid droplet but does not delay neurodegeneration of fh mutants ( Figure 2—figure supplement 2A ) , indicating that these lipid droplets do not contribute to neurodegeneration .\",\n \"We recently discovered that some mutants that are required for mitochondrial function display an activity dependent degeneration of photoreceptors ( Jaiswal et al . , 2015 ) .\",\n \"These mitochondrial mutants share several common phenotypes , including reduced ATP production , ERG rundown upon repetitive stimulation , and Rhodopsin1 ( Rh1 ) accumulation in the photoreceptor cytoplasm upon light exposure ( Jaiswal et al . , 2015 ) .\",\n \"In these mutants , dysfunctional mitochondria cause reduced ATP production and decreased calcium influx , which not only leads to reduced photoreceptor depolarization , but also triggers an internalization of Rh1 leading to an overload of the endolysosomal system upon light stimulation ( Jaiswal et al . , 2015 ) .\",\n \"The accumulation of Rh1 results in activity dependent degeneration , as the photoreceptors only degenerate when flies are raised in light ( light-induced neuronal activity ) but not in dark ( no neuronal activity ) ( Alloway et al . , 2000; Chinchore et al . , 2009; Jaiswal et al . , 2015 ) .\",\n \"Since loss of fh leads to decreased CI activity and reduced energy production ( Figure 2B and C ) , we hypothesized that a similar activity dependent degeneration mechanism contributes to neurodegeneration in fh mutants .\",\n \"To test this hypothesis , we first examined whether ERG traces rundown upon repetitive light stimulation in fh mutant photoreceptors .\",\n \"As shown in Figure 3A , the ERG amplitudes of the first and last traces show a similar size in control animals .\",\n \"In contrast , the ERG amplitude rapidly declines during repetitive light stimulation in fh mutants ( Figure 3A ) , similar to other mitochondrial mutants ( Jaiswal et al . , 2015 ) .\",\n \"Therefore , we assessed whether Rh1 internalization is affected in fh mutants .\",\n \"Wild type flies exposed to 12 hr of constant light typically exhibit low levels of Rh1 in photoreceptors ( Figure 3B ) .\",\n \"However , in fh mutants , Rh1 accumulates in the cytoplasm of photoreceptors ( Figure 3B ) .\",\n \"Since fh mutant photoreceptors share several features with other mitochondrial mutants , we hypothesized that reducing photoreceptor activity by raising fh mutants in the dark would suppress degeneration .\",\n \"However , fh mutant flies that are raised in the dark still display severe photoreceptor degeneration ( Figure 3B ) .\",\n \"Hence , we propose that in addition to the Rh1 accumulation , another pathogenic mechanism must contribute to neurodegeneration and mask the rescue effect when flies are raised in dark . 10 . 7554/eLife . 16043 . 009Figure 3 . Loss of fh causes Rh1 accumulation and iron deposit .\",\n \"( A ) ERG traces upon repetitive light stimulation in both control ( y w FRT19A ) and fh mutants .\",\n \"The double red arrows indicate ERG amplitudes of the first and last trace during the stimulation .\",\n \"Quantification of the ratio of last/first ERG amplitude is on the right .\",\n \"( B ) Rh1 distribution in control ( y w FRT19A ) and fh mutant clones after a 12 hr light exposure .\",\n \"Rh1 is labeled in red , and rhabdomeres are labeled in green .\",\n \"Scale bar: 5 µm .\",\n \"Quantification of ERG amplitudes of control ( y w FRT19A ) and fh mutant clones in different light conditions is on the right .\",\n \"L/D , flies are raised in 12 hr light and dark cycle .\",\n \"D , flies are raised in dark .\",\n \"( C ) Fe2+ levels are assessed by RPA in larval muscle and NMJ in fh mutants and rescued animals ( fh; gfh ) .\",\n \"RPA fluorescence is represented as a heat map .\",\n \"The NMJ boutons are indicated by white arrows .\",\n \"Quantification of normalized RPA intensity is on the right .\",\n \"n = 8 .\",\n \"Scale bar: 20 µm .\",\n \"( D ) Larval brain Perls’/DAB staining of control ( y w FRT19A ) , fh mutants , and rescued animals ( fh; gfh ) with regular food or low iron food conditions .\",\n \"Scale bar: 100 µm .\",\n \"( E ) Percent of animals that develop into pupae in control ( y w FRT19A ) and fh mutants when animals are raised by low iron food with different iron concentration .\",\n \"n = 3 . Data are presented as mean ± SEM .\",\n \"***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00910 . 7554/eLife . 16043 . 010Figure 3—figure supplement 1 . Fe2+ staining control . RPAC intensity in larva muscle and NMJ of fh mutants and rescued animals ( fh; gfh ) .\",\n \"Scale bar: 25 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 010 Whether iron accumulates in the nervous system and contributes to the pathogenesis in mice and FRDA patients has remained a topic of debate ( Boddaert et al . , 2007; Puccio et al . , 2001; Solbach et al . , 2014 ) .\",\n \"We therefore investigated whether iron is involved in neurodegeneration of fh mutants .\",\n \"To assess if iron homeostasis is affected in fh mutants , we first examined ferrous iron ( Fe2+ ) levels by Rhodamine B- ( ( 1 , 10-phenanthrolin-5-yl ) -aminocarbonyl ) benzyl ester ( RPA ) .\",\n \"RPA is a cell-permeable fluorescent dye that selectively accumulates in mitochondria , and its fluorescence is stoichiometrically quenched by Fe2+ ( Petrat et al . , 2002 ) .\",\n \"As RPA poorly crosses the blood brain barrier ( data not shown ) , we performed RPA staining of larval muscles and neuromuscular junctions ( NMJ ) .\",\n \"In fh mutants rescued with the genomic fh , which serves as a negative control , the RPA signal exhibits strong fluorescence ( Figure 3C ) .\",\n \"In contrast , the RPA intensity in fh mutants is dramatically reduced , in both muscle and NMJ boutons , suggesting a strong Fe2+ accumulation in mitochondria ( Figure 3C ) .\",\n \"RPAC ( Rhodamine B- ( ( phenanthren-9-yl ) -aminocarbonyl ) benzyl ester ) , a dye that is structurally very similar to RPA , is also taken up by mitochondria but is insensitive to Fe2+ mediated fluorescence quenching .\",\n \"Indeed , RPAC fluorescence is not quenched in rescued animals ( fh; gfh ) and fh mutants ( Figure 3—figure supplement 1 ) , suggesting that the decreased fluorescence of RPA in fh mutants is not due to impaired dye uptake .\",\n \"To investigate whether ferric iron ( Fe3+ ) levels are increased in fh mutants , we performed Perls’ staining and used 3 , 3'-Diaminobenzidine ( DAB ) to enhance the signal ( Meguro et al . , 2007 ) .\",\n \"In control and rescued animals , the DAB signal is most obvious in the neuropil of the ventral nerve cord ( Figure 3D ) , suggesting that Fe3+ is unevenly distributed in the nervous system .\",\n \"In fh mutants , however , the DAB signal is dramatically increased ( Figure 3D ) , indicating that loss of fh leads to a severe accumulation of Fe3+ .\",\n \"Increased Fe3+ levels are not only found in the larval brain , but can also be observed in fat body and gut ( data not shown ) .\",\n \"Together , these results indicate that loss of fh leads to both Fe2+ and Fe3+ accumulation in multiple tissues , including the nervous system .\",\n \"To determine if the iron accumulation contributes to neurodegeneration in fh mutant clones , we reduced iron levels in the fly food .\",\n \"Our regular food contains iron-rich molasses .\",\n \"To reduce iron levels , we replaced molasses with sucrose as the main carbohydrate source and refer to this food as 'low iron food' .\",\n \"As shown in Figure 3D , feeding larvae with low iron food reduces iron levels in the brains of both wild type control and fh mutants .\",\n \"In addition , the lethal phase of fh mutants is susceptible to iron levels in the food .\",\n \"In low iron food , 80% of fh mutants develop into pupae , but addition of 10 mM Fe3+ to the low iron food reduces pupation to about 10% , whereas the pupation rate of controls is not affected ( Figure 3E ) .\",\n \"These data indicate that fh mutants are highly sensitive to iron levels .\",\n \"To determine if iron toxicity is underlying the observed neurodegeneration , we fed low iron food to fh mutants and examined photoreceptor degeneration .\",\n \"Treating fh mutants with low iron food improves the ERG defects when compared to regular food condition ( Figure 4A ) , suggesting that iron mediated toxicity contributes to neurodegeneration in fh mutants . 10 . 7554/eLife . 16043 . 011Figure 4 . Neuronal activity and iron interact synergistically to contribute to photoreceptor degeneration in fh mutants .\",\n \"( A ) Quantification of ERG amplitudes of control ( y w FRT19A ) and fh mutants under different light and iron conditions .\",\n \"( B ) Retina morphology of control ( y w FRT19A ) and fh mutants under different food conditions at Day 6 .\",\n \"All animals are raised in dark .\",\n \"Regular , flies are raised in regular food .\",\n \"Low iron , flies are raised in low iron food .\",\n \"Scale bar: 5 µm .\",\n \"( C ) Quantification of ERG amplitudes of fh mutants in low iron food with different iron concentrations .\",\n \"( D ) ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which ferritins are overexpressed .\",\n \"Fer1* , enzymatic dead form of Fer1HCH .\",\n \"Data are presented as mean ± SEM .\",\n \"***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01110 . 7554/eLife . 16043 . 012Figure 4—figure supplement 1 . Control of low iron food treatment . Quantification of ERG amplitudes of control animals ( y w FRT19A ) in different food and iron conditions .\",\n \"Regular , flies are raised in regular food .\",\n \"Low iron , flies are raised in low iron food .\",\n \"Data are presented as mean ± SEM .\",\n \"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 012 We next assessed if excess neuronal activity and iron accumulations act synergistically to promote neurodegeneration in fh mutants .\",\n \"Interestingly , reducing photoreceptor activity ( raising flies in dark ) and iron ( low iron food ) leads to a synergistic effect in restoring both photoreceptor function and morphology ( Figure 4A and B ) .\",\n \"To exclude the possibility that changes in other compounds than iron in low iron food suppresses neurodegeneration in fh mutants , we reintroduced iron into the low iron food ( low iron/10 mM iron ) and found that this treatment again induces rapid degeneration in fh mutant clones even when animals are raised in dark ( Figure 4B and C ) .\",\n \"The ERG amplitudes of the wild type controls are not affected by different iron concentrations in the food ( Figure 4—figure supplement 1 ) .\",\n \"These data suggest that iron toxicity contributes to the activity independent degeneration , as photoreceptors degenerate in the absence of neuronal activity .\",\n \"We also tested if feeding 100 mM iron to wild type controls is sufficient to trigger neurodegeneration .\",\n \"This concentration of iron is toxic as most animals die as embryos or young larval instars , and the Fe3+ levels were not increased in the brains ( data not shown ) .\",\n \"We next explored if genetic manipulations can suppress iron toxicity and neurodegeneration in fh mutants .\",\n \"We expressed Ferritins , the iron storage proteins that chelate and sequester irons .\",\n \"In Drosophila , Ferritin 1 heavy chain homologue ( Fer1HCH ) and Ferritin 2 light chain homologue ( Fer2LCH ) form a heteropolymeric complex , whereas Ferritin 3 heavy chain homologue ( Fer3HCH ) forms a homopolymeric complex to store irons ( Missirlis et al . , 2006 , 2007 ) .\",\n \"In fh mutant clones , co-expression of Fer1HCH and Fer2LCH ( fh; Rh1>Fer1 , Fer2 ) or expression of Fer3HCH alone ( fh; Rh1>Fer3 ) significantly suppresses degeneration when flies are raised in dark ( Figure 4D ) .\",\n \"Co-expression of a ferroxidase inactive form of Fer1HCH along with wild type Fer2LCH in fh mutants ( fh; Rh1>Fer1* , Fer2 ) did not rescue degeneration ( Figure 4D ) .\",\n \"These results further confirm that iron toxicity contributes to neurodegeneration in fh mutant clones .\",\n \"In summary , we conclude that excess neuronal activity ( activity dependent ) and accumulated iron ( activity independent ) synergize to promote photoreceptor degeneration in fh mutants .\",\n \"Given that iron accumulates in fh mutants , we investigated the mechanism downstream of iron toxicity .\",\n \"Previous studies proposed that highly active iron interacts with hydrogen peroxide to generate free radicals , the Fenton reaction .\",\n \"However , we did not observe an increase in ROS ( Figure 2D ) , and expression of different ROS scavengers did not suppress degeneration in fh mutants ( Figure 2E ) .\",\n \"It has been previously documented that wild type yeast grow in medium with high iron levels increases sphingolipid synthesis ( Lee et al . , 2012 ) .\",\n \"However , this phenotype was not documented in the yeast frataxin homolog mutants or FXN mutants in other organisms .\",\n \"As we observed a strong iron accumulation in fh mutants , we hypothesized that iron toxicity induces sphingolipid synthesis and causes neurodegeneration .\",\n \"To test our hypothesis , we first assessed the de novo synthesis of sphingolipids by mass spectrometry .\",\n \"Consistent to our hypothesis , several upstream metabolic intermediates of sphingolipids are increased in fh mutants ( Figure 5A ) .\",\n \"Strikingly , dihydrosphingosine ( dhSph ) is increased more than 10 fold in fh mutants .\",\n \"Other sphingolipids , such as dihydroceramide ( dhCer ) and sphingosine ( Sph ) , are also increased to different levels ( Figure 5A ) .\",\n \"This data suggest that the de novo synthesis of sphingolipids is increased in fh mutants . 10 . 7554/eLife . 16043 . 013Figure 5 . Increased sphingolipids contribute to degeneration of fh mutants .\",\n \"( A ) Mass spectrometry analysis of sphingolipids in control ( y w FRT19A ) , fh mutants , and rescued animals ( fh; gfh ) .\",\n \"dhsph , dihydrosphingosine; dhCer , dihydroceramide; Cer , ceramide; Sph , sphingosine .\",\n \"n = 2 . ( B ) ERG of control ( y w FRT19A; Rh1-GAL4 ) , and fh mutants in which an RNAi against lace is expressed .\",\n \"( C ) ERG amplitude and retinal morphology of fh mutants treated with 100 μM myriocin .\",\n \"Scale bar: 5 µm .\",\n \"( D ) Quantification of Day 3 ERG amplitudes of fh mutants with low iron food and myriocin treatments .\",\n \"Data are presented as mean ± SEM .\",\n \"The error bars represent the range of data in ( A ) .\",\n \"**p<0 . 01 , ***p<0 . 001 , Student’s t-test .\",\n \"D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01310 . 7554/eLife . 16043 . 014Figure 5—figure supplement 1 . Control of myriocin treatment . Quantification of Day 6 ERG amplitudes of control animals ( y w FRT19A ) with myriocin treatment .\",\n \"Data are presented as mean ± SEM .\",\n \"D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 014 To determine if increased sphingolipid synthesis contributes to neurodegeneration in fh mutants , we performed RNAi experiments against the first and rate-limiting enzyme in the de novo synthesis pathway , lace , the fly homolog of Serine palmitoyltransferase .\",\n \"This significantly improved the ERG defects of fh mutants ( Figure 5B ) .\",\n \"In addition , feeding fh mutants Myriocin , a serine palmitoyltransferase inhibitor , suppressed both the functional and morphological photoreceptor defects of fh mutant clones ( Figure 5C ) .\",\n \"Feeding myriocin to control animals does not adversely affect photoreceptor function ( Figure 5—figure supplement 1 ) .\",\n \"To assess if iron accumulation and increased sphingolipid synthesis act in the same or a different pathway , we treated fh mutants with low iron food as well as myriocin .\",\n \"As shown in Figure 5D , we did not observe an additive or synergistic effect .\",\n \"These results suggest that iron toxicity induces sphingolipid synthesis which in turn causes neurodegeneration in fh mutants .\",\n \"Increased sphingolipids in yeast have been shown to activate a kinase pathway ( Pkh1/Ypk1/Smp1 ) that converges on a transcription factor Smp1p ( Lee et al . , 2012 ) .\",\n \"However , this pathway has not yet been studied in higher eukaryotic cells , or FXN mutant animal models .\",\n \"The corresponding proteins in mammals are PDK1 , SGK ( serum- and glucocorticoid regulated kinase ) , and Mef2 transcription factor ( Casamayor et al . , 1999; Dodou and Treisman , 1997 ) .\",\n \"The Drosophila homolog of SGK has not been identified , but both Pdk1 and Mef2 are present in the fly .\",\n \"Mef2 is a well known transcription factor required for muscle differentiation , however , it is also expressed in certain neurons and photoreceptors ( Blanchard et al . , 2010 ) .\",\n \"As we observed an increase in sphingolipids , we assessed if increased levels of sphingolipids activate the Pdk1/Mef2 pathway and underlie the neurodegenerative phenotype in fh mutants .\",\n \"We first tested if Pdk1/Mef2 is activated in fh mutants .\",\n \"As a proxy for Mef2 activation , we examined the mRNA levels of known downstream targets of Mef2 .\",\n \"Seven Mef2 targets , which have been validated through in situ hybridization and qRT-PCR , are all ectopically expressed in response to ectopic expression of Mef2 in vivo ( Elgar et al . , 2008 ) .\",\n \"We therefore examined these seven Mef2 targets in the nervous system of fh mutants .\",\n \"As shown in Figure 6A , all of these Mef2 targets are up-regulated .\",\n \"Several negative control genes , the downstream targets of other transcription factors , including Enhancer of split ( E ( Spl ) ) , senseless ( sens ) , and patched ( ptc ) , as well as several housekeeping genes that are not Mef2 targets ( Sandmann et al . , 2006 ) , are not changed in fh mutants ( Figure 6—figure supplement 1A ) .\",\n \"These data indicate that Mef2 is abnomally activated in fh mutants . 10 . 7554/eLife . 16043 . 015Figure 6 . Activated Pdk1/Mef2 pathway causes degeneration in fh mutants .\",\n \"( A ) mRNA levels of Mef2 downstream targets in fh mutants and rescued animals ( fh; gfh ) .\",\n \"n = 3 . ( B ) Day 3 ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which an RNAi against Pdk1 or Mef2 is expressed .\",\n \"( C ) ERG of flies with Mef2 overexpression in the eye .\",\n \"( D ) Mef2 luciferase reporter assay in Neuro-2A cells with different concentration of dhSph treatment .\",\n \"GSK 2334470 , a PDK1 inhibitor .\",\n \"n = 4 . Data are presented as mean ± SEM .\",\n \"*p<0 . 05 .\",\n \"**p<0 . 01 .\",\n \"***p<0 . 001 , Student’s t-test .\",\n \"D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01510 . 7554/eLife . 16043 . 016Figure 6—figure supplement 1 . Control of qRT-PCR .\",\n \"( A ) mRNA levels of downstream targets of Notch , Wingless , and Hedgehog pathway as well as several ribosomal genes are not changed in fh mutants .\",\n \"( B ) Retina morphology of fh mutants , and fh mutants that express Pdk1 or Mef2 RNAi .\",\n \"Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .\",\n \"Scale bar: 10 µm .\",\n \"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 016 Next , we tested if the activated Pdk1/Mef2 pathway contributes to neurodegeneration of fh mutants .\",\n \"We reasoned that if Pdk1 and Mef2 are activated and toxic , their down-regulation may suppress degeneration .\",\n \"Consistent with our hypothesis , RNAi mediated knockdown of Pdk1 suppresses the ERG and morphological defects in fh mutants ( Figure 6B and Figure 6—figure supplement 1B ) .\",\n \"Furthermore , knock down of Mef2 by two independent RNAi lines suppresses neurodegeneration of fh mutants ( Figure 6B and Figure 6—figure supplement 1B ) , suggesting that activated Pdk1 and Mef2 are toxic and contribute to the degeneration .\",\n \"To determine if up-regulation of Mef2 in wild type photoreceptors is sufficient to induce neurodegeneration , we ectopically expressed Mef2 in the adult eye using Rh1-Gal4 .\",\n \"Overexpression of Mef2 is sufficient to cause photoreceptor degeneration ( Figure 6C ) .\",\n \"Furthermore , flies with neuronal expression of Mef2 ( nSyb>Mef2 ) die within a week , instead of 70–80 days of the wild type flies ( data not shown ) .\",\n \"These data suggest that abnormal activation of Mef2 in neuron is sufficient to trigger its demise .\",\n \"To assess if a similar pathway is affected in vertebrate cells , we tested if sphingolipids can induce Mef2 activation in Neuro-2A cells .\",\n \"dhSph , the sphingolipid that is increased more than 10 fold in fly fh mutants ( Figure 5A ) , induces luciferase signals of a Mef2 reporter in a concentration dependent manner ( Figure 6D ) .\",\n \"In addition , the increased luciferase signal is suppressed by adding the PDK1 inhibitor GSK2334470 ( Figure 6D ) .\",\n \"This data indicate that dhsph activates Mef2 through PDK1 , consistent with what we observed in the fly .\",\n \"Collectively , these data suggest that sphingolipids act as signaling molecules to activate the Pdk1/Mef2 pathway and that this pathway contributes to photoreceptor degeneration in fh mutants .\"\n ],\n [\n \"Here , we report the isolation of the first severe loss of function allele of fh in Drosophila .\",\n \"This allowed us to identify a novel molecular pathway that contributes to neurodegeneration .\",\n \"Loss of fh in Drosophila causes an age dependent degeneration of photoreceptors in mutant clones .\",\n \"Mutant photoreceptors display abnormal mitochondrial cristae morphology , reduced ETC CI activity , and impaired ATP production .\",\n \"However , we do not observe an increase in ROS .\",\n \"Our data indicate that Rh1 trafficking defects caused by mitochondria dysfunction lead to an activity dependent degeneration in fh mutants .\",\n \"However , the accumulation of Fe2+ and/or Fe3+ stimulates sphingolipid synthesis which in turn activates the Pdk1/Mef2 pathway to cause an activity independent degeneration .\",\n \"Hence , mitochondrial dysfunction , and iron toxicity through activation of the sphingolipid/Pdk1/Mef2 pathway , synergistically contribute to the demise of neurons in fh mutants .\",\n \"The data indicate that ectopic activation of the transcription factor Mef2 induced by loss of FXN may play a role in FRDA .\",\n \"Elevated ROS levels , either through impaired ETC CI or CIII , or iron associated Fenton reaction , have been proposed to be the major driving force of disease pathogenesis in FRDA ( Bayot et al . , 2011; Santos et al . , 2010 ) .\",\n \"However , we do not observe increased ROS levels in fh mutants ( Figure 2D ) .\",\n \"In addition , overexpression of ROS scavengers or feeding antioxidant AD4 does not suppress degeneration ( Figure 2E and Figure 2—figure supplement 1E ) .\",\n \"The lack of increased ROS is surprising as mitochondrial mutants in Drosophila that exhibit a severely reduced CI activity typically show elevated ROS levels ( Owusu-Ansah et al . , 2008; Zhang et al . , 2013 ) .\",\n \"Numerous studies using different animal models or cell lines have established that a partial or complete loss of FXN leads to different degrees of defects in the ETC .\",\n \"Importantly , several reports have also documented that loss of FXN leads to an hypersensitivity to ROS , rather than elevated ROS levels ( Babcock et al . , 1997; Llorens et al . , 2007; Macevilly and Muller , 1997; Seznec et al . , 2005; Shidara and Hollenbeck , 2010 ) .\",\n \"Consistent with our data , overexpression of ROS scavengers in an RNAi mediated fh knock down did not improve viability or cardiac function , and no ROS could be detected ( Anderson et al . , 2005; Shidara and Hollenbeck , 2010; Tricoire et al . , 2014 ) .\",\n \"Similarly , loss of Fxn in mice does not lead to an increase in ROS ( Puccio et al . , 2001; Seznec et al . , 2005 ) , although elevated ROS levels in another mouse model have been documented ( Al-Mahdawi et al . , 2006 ) .\",\n \"Combined , these data suggest that ROS is not a prominent player in model organisms and a clinical trial designed to suppress ROS have not provided compelling data ( Santhera Pharmaceuticals , 2010 ) .\",\n \"Iron accumulation in mitochondria was first described in yeast frataxin homolog mutants in yeast ( Babcock et al . , 1997 ) .\",\n \"Although iron deposits have been reported in cardiac muscles of Fxn deficiency mice and FRDA patients , iron accumulation in the nervous system has been reported by some ( Boddaert et al . , 2007; Koeppen et al . , 2009 ) but not by others ( Puccio et al . , 2001; Simon et al . , 2004; Solbach et al . , 2014 ) .\",\n \"More importantly , the role of iron in the pathophysiology of FRDA has not yet been determined , and mitochondrial defects are thought to presage iron accumulation in mammals ( Puccio et al . , 2001 ) .\",\n \"It has been argued that Fe3+ accumulations are a non-reactive , intra-mitochondrial precipitates ( Seguin et al . , 2010; Whitnall et al . , 2012 ) .\",\n \"Finally , others proposed that iron accumulation is toxic as it induces the Fenton’s reaction and promotes ROS production .\",\n \"We find that in Drosophila fh mutants , both Fe2+ and Fe3+ accumulate in the nervous system ( Figure 3C and D ) .\",\n \"As we observed no increased ROS , we argue that Fe2+ and Fe3+ accumulation is toxic because:\",\n \"1 ) the lethality is enhanced when iron is increased;\",\n \"2 ) reduction of iron in food suppresses the progression of photoreceptor degeneration; and\",\n \"3 ) overexpression of Ferritin delays the demise of neurons .\",\n \"Taken together , we argue that iron accumulation in neurons is toxic and plays a primary role in the pathogenesis in fh mutants .\",\n \"Our data indicate that iron accumulation enhances the synthesis of sphingolipid , and that the excess amount of sphingolipids is toxic and promotes neurodegeneration in fh mutants .\",\n \"How iron accumulation leads to an increase in sphingolipid synthesis is unknown .\",\n \"Although we show that Fe2+ accumulates in mitochondria of the larval muscle and at the NMJ in fh mutants , it is still not clear whether iron accumulates in other cellular organelles .\",\n \"The de novo synthesis of sphingolipids occurs in the ER , and mitochondria have close contacts with ER at the MAMs ( mitochondria-associated endoplasmic reticulum membrane ) .\",\n \"Mitochondrial or ER localized iron may promote the enzymatic function of Lace or other proteins in the ER to enhance sphingolipid synthesis .\",\n \"However , treatment with Myriocin which targets Lace to decrease sphingolipid synthesis or down-regulation of lace via RNAi mediated knockdown suppresses degeneration in fh mutants ( Figure 5B and C ) , suggesting that increased sphingolipid metabolites contribute to toxicity .\",\n \"These data are also in agreement with the observation that overexpression of Sk2 ( sphingosine kinase\",\n \"2 ) or feeding dhSph-1P to wild type flies promotes the degeneration of photoreceptors ( Yonamine et al . , 2011 ) .\",\n \"An increase in sphingolipids may affect various processes , including membrane integrity , lipid metabolism , and signal transduction ( Brown and London , 2000; Hannun and Obeid , 2008; Worgall , 2008 ) .\",\n \"We found that down-regulation of Pdk1 or Mef2 in mutant photoreceptor delays neurodegeneration in fh mutants , while overexpression of Mef2 in photoreceptor promotes the demise of neurons ( Figure 6B and C ) .\",\n \"It has been reported that Sph or dhSph , two sphingolipids that are elevated in fh mutants , directly induce autophosphorylation of PDK1 or SGK in an in vitro kinase assay ( Caohuy et al . , 2014; King et al . , 2000 ) .\",\n \"Although there is no evidence that PDK1 or SGK can directly activate Mef2 , our data clearly show that Mef2 activity is up-regulated by elevating dhSph in the medium of cultured Neuro-2A cells , and this increased activity is suppressed by a PDK1 inhibitor ( Figure 6D ) , indicating that Mef2 can be activated by PDK1 or a downstream substrate of PDK1 ( Mora et al . , 2004 ) .\",\n \"The MEF2 family includes four vertebrate genes ( MEF2A–D ) that play a key role in muscle differentiation ( Molkentin et al . , 1995 ) .\",\n \"However , the MEFs are also expressed in neurons where they are involved in neuronal development ( Akhtar et al . , 2012; Leifer et al . , 1994; Lyons et al . , 1995; Mao et al . , 1999 ) .\",\n \"The MEFs are known to regulate the expression of numerous target genes , including genes required for muscle differentiation , immediate-early genes , neuronal-activity-regulated genes , as well as genes involved in energy storage and immune response ( Clark et al . , 2013; Dietrich , 2013 ) .\",\n \"In flies , ectopic expression of Mef2 in ectoderm is sufficient to induce nearly half of its predicted downstream targets that are typically expressed in muscles ( Sandmann et al . , 2006 ) .\",\n \"Hence , its unusual activity may trigger neurodegeneration via the activation of some of its downstream targets .\",\n \"Importantly , it has been shown that the up-regulation of Mef2 is implicated in the cardiac hypertrophy and dilation ( Molkentin and Markham , 1993; Xu et al . , 2006 ) , which is the major cause of death in FRDA patients .\",\n \"In sum , our data indicate that reducing the levels of sphingolipid synthesis with Myriocin , or antisense strategy against enzymes involved in sphingolipid synthesis , or reducing the levels of Pdk1or Mef2 , are options that should be explored in FRDA models .\"\n ],\n [\n \"Flies were obtained from the Bloomington Drosophila Stock Center at Indiana University ( BDSC ) unless otherwise noted .\",\n \"The stocks were routinely maintained at room temperature .\",\n \"For genetic interaction experiments , flies were raised at 28°C to enhance the GAL4 activity .\",\n \"For all the larval experiments , flies were allowed to lay eggs for 48 hr on grape juice plates with yeast paste .\",\n \"Hemizygous mutant larvae and wild type controls were isolated by GFP selection at the first instar phase and transferred to standard fly food for the duration of their development .\",\n \"To create mosaic mutant clones in the adult eye , y w FRT19A and y w fh FRT19A/FM7c , Kr>GFP females were crossed with y w GMR-hid FRT19A; ey-GAL4 UAS-FLP or y w GMR-hid l ( 1 ) cl FRT19A/FM7c , Kr>GFP; eyFLP Rh1-GAL4/CyO flies .\",\n \"The following strains were used to generate fly stocks in this study: y w FRT19A ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) y w fh1 FRT19A/FM7c , Kr>GFP ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) y w fh1 FRT19A/FM7c , Kr>GFP; genomic-fh ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) Df ( 1 ) BSC537 , w1118/FM7h/Dp ( 2;Y ) G , P{hs-hid}Y y w GMR-hid FRT19A; ey-GAL4 UAS-FLP y w GMR-hid l ( 1 ) cl FRT19A/FM7c , Kr>GFP; eyFLP Rh1-GAL4/CyO ( Jaiswal et al . , 2015 ) w1; P{UAS-fh . A}1 ( Anderson et al . , 2005 ) w1; P{UAS-Cat . A}2 ( Anderson et al . , 2005 ) w1; P{UAS-Sod . A}B37 ( Anderson et al . , 2005 ) w1; P{UAS-Sod2 . M}UM83 ( Anderson et al . , 2005 ) P{rh1-GAL4}3 , ry506 P{Gal4-da . G32} UH1 y1 w*; P{nSyb-GAL4 . S}3 w; repo-GAL4/TM3 , Sb w*; P{w+mC bmmScer\\\\UAS = UAS-bmm} ( Gronke et al . , 2005 ) ( gift from Ronald Kühnlein ) w*; UAS-Lip4/CyO ( Liu et al . , 2015 ) P{UAS-Mef2-HA} ( Sandmann et al . , 2006 ) ( gift from Eileen Furlong ) P{UAS-Fer1HCH} P{UAS-Fer2LCH} ( Missirlis et al . , 2006 , 2007 ) ( gift from Hermann Steller ) P{UAS-Fer1HCH . mut} P{UAS-Fer2LCH} ( Missirlis et al . , 2006 , 2007 ) ( gift from Hermann Steller ) P{UAS-Fer3HCH} ( Missirlis et al . , 2006 ) ( gift from Patrik Verstreken ) w1118; P{GawB}D42 , P{UAS-mito-HA-GFP . AP}3 e1/TM6B , Tb1 ( Horiuchi et al . , 2005 ) ( gift from Bill Saxton ) w*; cno3 P{A92}pucE69/TM6B , abdA-LacZ ( Ring and Martinez Arias , 1993 ) ( Drosophila Genetic Resource Center ) ND42 RNAi: y1 sc* v1; P{TRiP . HMS00798}attP2 ( Owusu-Ansah et al . , 2008 ) lace RNAi: P{KK102282}VIE-260B ( Ghosh et al . , 2013 ) ( VDRC ) Pdk1 RNAi: P{KK108363}VIE-260B ( Tokusumi et al . , 2012 ) ( VDRC ) Mef2 RNAi: w1118; P{GD5039}v15549/TM3 and w1118; P{GD5039}v15550 ( Clark et al . , 2013 ) ( VDRC ) ERG recordings were performed as described ( Verstreken et al . , 2003 ) .\",\n \"Briefly , flies were glued on a glass slide .\",\n \"A recording electrode filled with 3M NaCl was placed on the eye , and another reference electrode was inserted into the fly head .\",\n \"Before the recording , photoreceptors were allowed to recover by keeping flies in the dark for 3 min .\",\n \"During the recording , a 1 s pulse of light stimulation was given , and the ERG traces of five to ten flies were recorded and analyzed by AXON -pCLAMP 8 software .\",\n \"To detect Fe2+ levels , L3 larvae were dissected in ice-cold 0 . 25 mM Ca2+ HL3 ( Stewart et al . , 1994 ) ( 70 mM NaCl , 5 mM KCl , 20 mM MgCl2 , 10 mM NaHCO3 , 5 mM trehalose , 5 mM HEPES , 115 mM sucrose , pH 7 . 2 ) and rinsed in HL3 with 1 mM Ca2+ .\",\n \"The animals were then incubated with 10 µM RPA or RPAC ( Squarix Biotechnology , Germany ) in 1 mM Ca2+ HL3 for 20 min at room temperature in the dark .\",\n \"The animals were washed subsequently three times with ice-cold 1 mM Ca2+ HL3 and then incubated in 1 mM Ca2+ HL3 with 7 mM L-glutamic acid for 15 min at room temperature in the dark .\",\n \"The RPA or RPAC fluorescence was obtained with a Zeiss LSM 510 confocal microscope ( Carl Zeiss ) and quantified by Image J . To evaluate Fe3+ levels , L3 larval brains were dissected and fixed in 3 . 7% formaldehyde for 20 min .\",\n \"Samples were quickly washed three times with 0 . 4% Triton-PBS and incubated with Perls solution ( 1% K4Fe ( CN ) 6 and 1% HCl in PBT ) for 5 min .\",\n \"After 3 quick washes in PBT , samples were incubated with DAB solution ( 10 mg DAB with 0 . 07% H2O2 in 0 . 4% Triton-PBS ) to enhance the signal .\",\n \"The brains were then quickly washed three times with 0 . 4% Triton-PBS and mounted .\",\n \"The brain images were obtained with a Leica MZ16 stereomicroscope equipped with Optronics MicroFire Camera .\",\n \"L3 larvae were dissected in ice-cold 0 . 25 mM Ca2+ HL3 and rinsed with 1 mM Ca2+ HL3 .\",\n \"The animals were then incubated with 10 µM dihydroethidium ( DHE , Sigma-Aldrich ) in 1 mM Ca2+ HL3 for 20 min at room temperature in the dark .\",\n \"The samples were quickly washed three times with 1 mM Ca2+ HL3 with 7 mM L-glutamic acid , and the DHE fluorescence in larval brains were live imaged by Zeiss 510 confocal microscope ( Carl Zeiss ) and quantified by Image J . Low iron food was made using 10% dry yeast , 10% sucrose , and 0 . 6% agar in w/v in water .\",\n \"The solution was microwaved and dried at room temperature , divided into vials , and stored at 4°C for further use .\",\n \"To increase iron levels in the food , ammonium iron ( Ш ) citrate ( Sigma-Aldrich ) was added to a final concentration of 1 mM or 10 mM in the low iron food .\",\n \"To test if inhibition of sphingolipid synthesis suppresses neurodegeneration in fh mutants , myriocin from Mycelia sterilia ( Sigma-Aldrich ) was dissolved in regular fly food ( molasses based ) or low iron food , and vials were then stored immediately at −20°C for future use .\",\n \"The flies were transfer to fresh food vials every two days .\",\n \"For the fh genomic rescue construct , a DNA fragment containing Drosophila X: 9 , 147 , 070 . . 9 , 150 , 371 was retrieved from a 20 kb P[acman] construct that covers the entire fh genomic locus ( clone CH322-14E18 from BACPAC Resources Center ) ( Venken et al . , 2009 ) .\",\n \"The fh genomic sequence was then subcloned into p{w+}attB using KpnI and NotI sites .\",\n \"The construct was then injected into y w ΦC31;VK33 embryos , and transgenic flies were selected .\",\n \"To generate pUAST-attB-UAS-hFXN construct , the primers 5’- TCCGAATTCGCCACCATGTGGACTCTCGGGCGCCGCGC-3’ and 5’-GCGGTACCTCAAGCATCTTTTCCGGAATAGGC-3’ were used to clone the full length FXN cDNA from pcDNA-hFXN-HA , a gift from Gino Cortopassi ( Shan et al . , 2007 ) .\",\n \"The PCR product was then subcloned into the pUAST-attB vector .\",\n \"The construct was then injected into y w ΦC31;VK33 or y w ΦC31;VK37 embryos , and the transgenic flies were selected .\",\n \"For whole mount eye staining , we dissected fly heads in cold PBS and fixed with 4% paraformaldehyde at 4°C overnight .\",\n \"The retinas were then dissected and fixed for an additional 30 min .\",\n \"For larval ventral nerve cord staining , L3 instar larvae were dissected and fixed with 3 . 7% formaldehyde for 20 min .\",\n \"The antibodies were used at the following concentrations: mouse anti-Na/K ATPase ( α5 , Developmental Studies Hybridoma Bank ( DSHB ) ) , 1:200; rat anti-Elav ( 7E8A10 , DSHB ) , 1:500; mouse anti-Rh1 ( 4C5 , DSHB ) , 1:100; mouse anti-V5 ( R96025 , Invitrogen ) , 1:1000; rabbit anti-HRP ( 323-005-021 , Jackson ImmunoResearch ) , 1:1000; chicken anti-GFP ( ab13970 , abcam ) , 1:1000; rabbit anti-β-Galactosidase ( Cappel ) , 1:1000; mouse anti-DLG ( 4F3 , DSHB ) , 1:200; Alexa 488-conjugated phalloidin ( Invitrogen ) , 1:100; Alexa 405- , Alexa 488- , Cy3- , or Cy5-conjugated secondary antibodies ( Jackson ImmunoResearch ) , 1:250 .\",\n \"Samples were then mounted in Vectashield ( Vector Laboratories ) before being analyzed under a confocal microscope .\",\n \"All the confocal scans were acquired with a confocal microscope ( model LSM 510 or LSM 710; Carl Zeiss ) and processed using LSM Image Browser ( Carl Zeiss ) and Photoshop ( Adobe ) .\",\n \"The Nile red staining was performed as described ( Liu et al . , 2015 ) .\",\n \"Briefly , the retina was dissected and fixed in 3 . 7%formaldehyde .\",\n \"The samples were then incubated for 10 min at 1:1 , 000 dilution of PBS with 1 mg/ml Nile Red ( Sigma ) .\",\n \"The retina was then washed with PBS and mounted in Vectashield ( Vector Laboratories ) .\",\n \"Images were obtained with a Zeiss LSM 510 confocal microscope .\",\n \"Mitochondria were extracted as previously described ( Zhang et al . , 2013 ) .\",\n \"Mitochondrial ETC enzymatic activity assay and aconitase activity assay were performed as previously described ( Zhang et al . , 2013 ) .\",\n \"To determine ADP/ATP ratio , L3 larvae were collected and the ratio was determined with an ADP/ATP Ratio Assay Kit ( ab65313 , abcam ) .\",\n \"Briefly , 5–10 L3 instar larvae were collected and frozen with liquid nitrogen .\",\n \"The samples were then homogenized and the ADP/ATP ratio was measured following manufacturer’s protocol .\",\n \"The luminescence was measured by Synergy 2 Microplate Reader ( BioTek ) .\",\n \"Drosophila retina ultrastructure was imaged following standard electron microscopy procedures using a Ted Pella Bio Wave processing microwave with vacuum attachments .\",\n \"Briefly , fly heads were dissected and fixed at 4°C in fixative ( 2% paraformaldehyde , 2 . 5% glutaraldehyde , 0 . 1 M sodium cacodylate , and 0 . 005% CaCl2 , pH 7 . 2 ) overnight .\",\n \"The samples were then postfixed in 1% OsO4 , dehydrated in ethanol and propylene oxide , and then embedded in Embed-812 resin ( Electron Microscopy Sciences ) under vacuum .\",\n \"Photoreceptors were then sectioned and stained in 1% uranyl acetate and saturated lead nitrate .\",\n \"TEM images of photoreceptor sections were taken using a JEOL JEM 1010 transmission electron microscope at 80 kV with an AMT XR-16 mid-mount 16 mega-pixel digital camera .\",\n \"ESI/MS/MS analysis of endogenous sphingosine bases and ceramide species ( C14- and C16-Sphingoid Base ) were performed on a Thermo_Fisher TSQ Quantum triple quadrupole mass spectrometer , operating in a Multiple Reaction Monitoring ( MRM ) positive ionization mode , using modified version ( Bielawski et al . , 2009 ) .\",\n \"Briefly , whole larvae were fortified with the internal standards ( ISs: C17 base D-erythro-sphingosine ( 17CSph ) , C17 sphingosine-1-phosphate ( 17CSph-1P ) , N-palmitoyl-D-erythro-C13 sphingosine ( 13C16-Cer ) and heptadecanoyl-D-erythro-sphingosine ( C17-Cer ) ) , and extracted with ethyl acetate/iso-propanol/water ( 60/30/10 v/v ) solvent system .\",\n \"After evaporation and reconstitution in 100 μl of methanol samples were injected on the HP1100/TSQ Quantum LC/MS system and gradient eluted from the BDS Hypersil C8 , 150 × 3 . 2 mm , 3 μm particle size column , with 1 . 0 mM methanolic ammonium formate / 2 mM aqueous ammonium formate mobile phase system .\",\n \"Peaks corresponding to the target analytes and internal standards were collected and processed using the Xcalibur software system .\",\n \"Quantitative analysis was based on the calibration curves generated by spiking an artificial matrix with the known amounts of the target analyte synthetic standards and an equal amount of the internal standards ( ISs ) .\",\n \"The target analyte/IS peak areas ratios were plotted against anlyte concentration .\",\n \"The target analyte/IS peak area ratios from the samples were similarly normalized to their respective ISs and compared to the calibration curves , using a linear regression model .\",\n \"Applying consistent mass spectral conditions of Collision Assistant Dessociation ( CAD ) ; 35 eV and Electron Spray Ionization ( ESI ) all sphingoid bases and related ceramides undergo uniform transition from initial molecular ion ( M+1 ) to the respective sphingoid backbone secondary ions .\",\n \"Consequently , calibration curves , generated from authentic standards of the typical , 18C-sphingosine and ceramides , can be used for quantitation of other , e . g . 20C- counterparts .\",\n \"The levels of sphingolipid species are normalized to the phosphate amount of the samples .\",\n \"Although absolute concentrations determined for compounds without authentic standards ( 20C-LCB derivatives ) may not be precise , due to possible differences in instrument response for 18C- and 20C-LCB related compounds , for comparative study , where changes in sphingolipids level rather than absolute concentration , are most important this indirect methodology provide reliable results .\",\n \"Total RNA from the fly larval brains were extracted by Trizol RNA Isolation Reagents ( Thermo Fisher Scientific ) follow the manufacturer’s instructions .\",\n \"The cDNA was synthesized by High-Capacity cDNA Reverse Transcription Kit ( Applied Biosystems ) .\",\n \"Real-time PCR was carried out using iQ SYBR Green Supermix ( Bio-Rad ) and performed in a thermal cycler ( iCycler; Bio-Rad Laboratories ) .\",\n \"The data were collected and analyzed using the optical module ( iQ5; Bio-Rad Laboratories ) .\",\n \"The following primer pairs are used ( 5’to 3’ ) : RP49 forward ( control primer ) , TCCTACCAGCTTCAAGATGAC; RP49 reverse ( control primer ) , CACGTTGTGCACCAGGAACT; Act57B forward , TTGAGACCTTCAACTCGCCC; Act58B reverse , CCATCACCGGAGTCCAGAAC; mib2 forward , CGCCAGAAAACACTGTCGTG; mib2 reverse , GACGAACTCCAACTTGAGCATTA; CG5080 forward , CGCCCTCTCCAATTAGTTCTCC; CG5080 reverse , CAGCGACTGGATAGTTCCGC; Mlc2 forward , TCGGGTCCGATCAACTTCAC; Mlc2 reverse , ATTTCGCGGAATTTGTCACCG; Mlc1 forward , CCGAGGATGATGAAGGATTT; Mlc1 reverse , CTGGTCTGTCACACATTCTGG; CG6972 forward , AGGGATCACACACTGATGAACT; CG6972 reverse , CAACAGCCATTCGGAGGGAC; Mhc forward , ATCAATCCTTACAAGCGTTACCC; Mhc reverse , CCGTCAGAGATGGCGAAAATATG; E ( Spl ) forward , ATGGAATACACCACCAAGACCC; E ( Spl ) reverse , GGCGACAAGTGTTTTCAGGTT; sens forward , TACTGTGGCCCCAATTTTTGT; sens reverse , AAGGCAAAGTCACGATCCCG; ptc forward , GGATCTTTACATACGCACCAGC; ptc reverse , GGACTGGAATACTGATCGCAG; Neuro-2A cells were seed on 12 well plates , and each well was transfected with 3XMef2-luc reporter ( Addgene ) and Renilla control pRL-TK vector ( gift from Huda Zoghbi ) .\",\n \"After two days , DMSO , DL-Dihydrosphingosine ( Sigma ) , or GSK2334470 ( gift from Huda Zoghbi ) was added into the medium .\",\n \"Cells were cultured one more day with drug treatment .\",\n \"The luciferase assay was then performed by Dual-Luciferase Reporter Assay System ( Promega ) and followed manufacturer instruction .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Mutations in Frataxin ( FXN ) cause Friedreich’s ataxia ( FRDA ) , a recessive neurodegenerative disorder .","Previous studies have proposed that loss of FXN causes mitochondrial dysfunction , which triggers elevated reactive oxygen species ( ROS ) and leads to the demise of neurons .","Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants .","We show that loss of frataxin homolog ( fh ) in Drosophila leads to iron toxicity , which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 ( Pdk1 ) and myocyte enhancer factor-2 ( Mef2 ) .","Dampening iron toxicity , inhibiting sphingolipid synthesis by Myriocin , or reducing Pdk1 or Mef2 levels , all effectively suppress neurodegeneration in fh mutants .","Moreover , increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved .","Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA ."],"string":"[\n \"Mutations in Frataxin ( FXN ) cause Friedreich’s ataxia ( FRDA ) , a recessive neurodegenerative disorder .\",\n \"Previous studies have proposed that loss of FXN causes mitochondrial dysfunction , which triggers elevated reactive oxygen species ( ROS ) and leads to the demise of neurons .\",\n \"Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants .\",\n \"We show that loss of frataxin homolog ( fh ) in Drosophila leads to iron toxicity , which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 ( Pdk1 ) and myocyte enhancer factor-2 ( Mef2 ) .\",\n \"Dampening iron toxicity , inhibiting sphingolipid synthesis by Myriocin , or reducing Pdk1 or Mef2 levels , all effectively suppress neurodegeneration in fh mutants .\",\n \"Moreover , increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved .\",\n \"Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA .\"\n]"},"summary":{"kind":"list like","value":["Friedreich’s ataxia is a disorder in which nerve cells in the spinal cord , cerebellum and dorsal root ganglia progressively die as a person ages .","People with this disorder often have difficulties with walking and can eventually develop other problems such as heart disease and diabetes .","Mutations in a gene called Frataxin , known as FXN for short , are the primary cause of the disorder .","The FXN gene encodes a protein normally found in mitochondria – the structures that are best known for providing energy inside cells .","Previous studies suggest that mutations in the FXN gene prevent mitochondria from working normally , which triggers the production of toxic chemicals called reactive oxygen species .","However , therapies based on antioxidants ( which combat reactive oxygen species ) only have limited benefits in patients with Friedreich’s ataxia; this suggests that other mechanisms contribute to the progression of the disease .","Mutations in the FXN gene also cause iron to accumulate inside cells , which can be toxic too .","However , it remains hotly debated whether or not iron toxicity contributes to Friedreich’s ataxia .","Chen et al . set out to identify other mechanisms that can explain the loss of nerve cells seen in Friedreich’s ataxia using fruit flies as an experimental system .","Flies without the equivalent of FXN gene accumulated iron in their nervous systems and other tissues , but did not produce more reactive oxygen species .","The experiments also revealed that this build-up of iron increased the production of fatty molecules ( called sphingolipids ) , which in turn triggered the activation of two proteins ( called Pdk1 and Mef2 ) .","Chen et al . then showed that blocking any of these effects could effectively delay the death of nerve cells in the mutant flies .","Further experiments showed that boosting the levels of the Mef2 protein in the nerve cells of otherwise normal flies was enough to cause these cells to die .","The next step is to see whether the pathway also operates in mice and humans .","Future studies could also see if dampening down this pathway could provide new treatments for Friedreich’s ataxia ."],"string":"[\n \"Friedreich’s ataxia is a disorder in which nerve cells in the spinal cord , cerebellum and dorsal root ganglia progressively die as a person ages .\",\n \"People with this disorder often have difficulties with walking and can eventually develop other problems such as heart disease and diabetes .\",\n \"Mutations in a gene called Frataxin , known as FXN for short , are the primary cause of the disorder .\",\n \"The FXN gene encodes a protein normally found in mitochondria – the structures that are best known for providing energy inside cells .\",\n \"Previous studies suggest that mutations in the FXN gene prevent mitochondria from working normally , which triggers the production of toxic chemicals called reactive oxygen species .\",\n \"However , therapies based on antioxidants ( which combat reactive oxygen species ) only have limited benefits in patients with Friedreich’s ataxia; this suggests that other mechanisms contribute to the progression of the disease .\",\n \"Mutations in the FXN gene also cause iron to accumulate inside cells , which can be toxic too .\",\n \"However , it remains hotly debated whether or not iron toxicity contributes to Friedreich’s ataxia .\",\n \"Chen et al . set out to identify other mechanisms that can explain the loss of nerve cells seen in Friedreich’s ataxia using fruit flies as an experimental system .\",\n \"Flies without the equivalent of FXN gene accumulated iron in their nervous systems and other tissues , but did not produce more reactive oxygen species .\",\n \"The experiments also revealed that this build-up of iron increased the production of fatty molecules ( called sphingolipids ) , which in turn triggered the activation of two proteins ( called Pdk1 and Mef2 ) .\",\n \"Chen et al . then showed that blocking any of these effects could effectively delay the death of nerve cells in the mutant flies .\",\n \"Further experiments showed that boosting the levels of the Mef2 protein in the nerve cells of otherwise normal flies was enough to cause these cells to die .\",\n \"The next step is to see whether the pathway also operates in mice and humans .\",\n \"Future studies could also see if dampening down this pathway could provide new treatments for Friedreich’s ataxia .\"\n]"},"year":{"kind":"string","value":"2016"}}},{"rowIdx":4191,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Material and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Material and methods\"\n]"},"keywords":{"kind":"list like","value":["cell biology","neuroscience"],"string":"[\n \"cell biology\",\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"A novel synaptic plasticity rule explains homeostasis of neuromuscular transmission"},"id":{"kind":"string","value":"elife-12190-v1"},"sections":{"kind":"list like","value":[["In the nervous system , presynaptic neurotransmitter release , postsynaptic receptors , and postsynaptic excitability can be modulated to bi-directionally and durably change synaptic efficacy .","There is a large diversity of plasticity processes , which together shape the neuronal network ( Changeux and Danchin , 1976; Goda and Davis , 2003; Munz et al . , 2014 ) and tune its properties ( Nelson and Turrigiano , 2008 ) .","In the brain , concomitantly to various forms of associative plasticity which sustain memory and learning , homeostatic plasticity processes are proposed to restrain the mean level of neuronal activity within a physiological regime , and to maintain the stability of recurrent network activity that can be challenged by associative plasticity ( Turrigiano and Nelson , 2004; Macleod and Zinsmaier , 2006; Marder and Goaillard , 2006; Turrigiano , 2007; Nelson and Turrigiano , 2008 ) .","Homeostatic plasticity has been extensively studied at the neuromuscular synapse , in particular at the glutamatergic neuromuscular junction ( NMJ ) in Drosophila ( Frank , 2014 ) , due to the robustness of the homeostatic control of synaptic transmission and the great accessibility of the experimental model to genetic manipulations .","In vertebrates , each skeletal muscle fiber is mono-innervated ( Sanes and Lichtman , 1999 ) , and each single presynaptic action potential ( AP ) induces one postsynaptic AP , corresponding to a unity synaptic gain ( ratio between the numbers of post- and presynaptic APs equal to 1 ) ( Wood and Slater , 2001 ) .","The stability of the gain implies that presynaptic neurotransmitter release and/or postsynaptic receptors adapt the effective synaptic strength to the excitability of the muscle fiber , which depends on the fiber characteristics , and presumably decreases with growth and exercise ( Turrigiano , 2007 ) .","In adult vertebrate skeletal muscles , cholinergic nicotinic receptors are clustered in the synaptic region .","Expression and location of nicotinic receptors have been shown to depend not only on agrin ( McMahan , 1990; Hall and Sanes , 1993; Gautam et al . , 1995; 1996; Sandrock et al . , 1997 ) but also on activity ( Lømo , 2003 ) , suggesting their possible role as adjustment variables in the control of synaptic strength .","In the recent years however , thorough studies of the glutamatergic NMJ in Drosophila revealed that presynaptic regulation of neurotransmitter release is certainly a major adjustment variable for synaptic homeostasis ( Davis and Müller , 2015 ) .","In such 'presynaptic homeostasis' , increase in neurotransmitter release counterbalances a genetically-induced decrease of the postsynaptic sensitivity to glutamate ( Petersen et al . , 1997; Davis et al . , 1998; DiAntonio et al . , 1999 ) , a genetically-induced increase of postsynaptic input conductance ( Paradis et al . , 2001 ) or a pharmacological blockade of postsynaptic receptors ( Frank et al . , 2006 ) , thus resulting in the strict maintenance of the level of evoked postsynaptic depolarization .","Presynaptic compensation of postsynaptic excitability changes implies the existence of a retrograde feedback process from the innervated muscle fiber to the motor neuron .","Particular attention was paid to the determination of the molecular targets of the homeostatic retrograde signaling and of the presynaptic cell signaling involved .","It appeared that both the readily releasable vesicle pool ( Müller et al . , 2012; 2015 ) and the presynaptic voltage-gated Ca2+ channels ( Müller and Davis , 2012 ) are targeted to promote glutamate release when postsynaptic excitability is reduced .","The abundance of voltage-gated Ca2+ channels can also be decreased in response to a vesicular content increase ( Gaviño et al . , 2015 ) , confirming the bi-directionality of the process .","In Drosophila , endostatin is a candidate trans-synaptic factor for the retrograde feedback targeting presynaptic neurotransmitter release ( Wang et al . , 2014 ) .","Upstream from the retrograde factors , the postsynaptic kinases , CAMKII ( Haghighi et al . , 2003 ) and TOR ( Penney et al . , 2012 ) , pathways are necessary for a functional homeostatic control at the Drosophila neuromuscular transmission .","An abundant literature is thus progressively assembling pieces of the signaling puzzle underlying the interactions between the motor neuron and the muscle cell for the control of synaptic strength .","However , besides the nature of the retrograde feedback and its presynaptic targets , a mechanism for the evaluation of the synaptic strength should be present at the postsynaptic level .","The nature and dynamics of these mechanisms , primarily sensing synaptic strength in the muscle cell and initiating retrograde feedback , are key issues of homeostatic plasticity that still remain enigmatic in both invertebrates and vertebrates .","Here we investigate this issue in vertebrate ( Xenopus and mouse ) cholinergic transmission .","Motor neurons and muscle cells of Xenopus laevis embryos establish functional neuromuscular synapses ( Tabti et al . , 1998 ) in primary co-culture .","Standard intracellular recording from muscle cells is possible in Xenopus culture owing to the small size of the cells ( Figure 1A , perforated patch-clamp ) , while it is not possible in muscle cells from adult mice , due to macroscopic movements during contraction .","To circumvent this issue and maintain intracellular recordings in moving tissue , we adapted a ‘floating electrode’ device from previous methods invented for muscles ( Woodbury and Brady , 1956 ) or neurons ( Kunze , 1998 ) .","We applied this method to mouse ex vivo nerve-skeletal muscle preparations ( Figure 1B , Methods , and Figure 1—figure supplement 1 ) .","This approach preserved the physiological conditions , necessary to reveal the control exercised by the muscle cell over the neuromuscular synaptic transmission , a role that might have been previously overlooked in vertebrates due to commonly used high concentrations of the nicotinic receptor antagonist curare to prevent contraction . 10 . 7554/eLife . 12190 . 003Figure 1 . Synaptic transmission is homeostatically regulated .","( A ) Perforated patch-clamp on Xenopus neuron ( N ) and muscle cell ( M ) in primary culture .","Presynaptic APs were triggered with current steps .","Postsynaptic APs were recorded under current-clamp and nicotinic synaptic currents under voltage-clamp ( -80 mV ) .","( B ) Intracellular recording in soleus muscle fibers from an adult mouse using a floating sharp electrode ( see Materials and Methods and Figure 1—figure supplement 1 ) .","( C ) Nicotinic conductance calculated from averaged ePSCs ( n = 30 ePSCs for each dot ) in different Xenopus muscle cells as a function of their input conductance .","The black line shows the linear regression .","( D ) In mice , membrane potential reached by the ePSP in individual FDB muscle fibers after treatment with µ-conotoxin GIIIB , in absence of burst stimulation of the nerve ( black dots , n= 88 fibers , 2 muscles , 2 mice ) , and in test preparations ( grey dots , n = 108 fibers , 2 muscles , 2 mice ) for which the nerve was burst stimulated prior to conotoxin treatment ( 15 bursts in 10 min , each of 120 events at 30 Hz ) .","( E ) Mean synaptic gain at Xenopus synapses ( dots , n = 5 synaptic connections ) and in mouse neuromuscular junctions ( squares , n = 4 muscle fibers from different mice ) during chronic bursts of presynaptic stimulation ( bursts of 80 to 120 pulses , 30 Hz for 30 min ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00310 . 7554/eLife . 12190 . 004Figure 1—figure supplement 1 . Floating electrode . In conventional electrophysiology , an intracellular electrode made from pulled glass is rigidly fixed to an amplifier headstage and/or to the micromanipulator by a holder preventing free movements .","To allow intracellular recordings in contracting mouse muscle , we cut off the tip of the pipette and used this as an electrode connected to the amplifier headstage by a loose , 5–10 cm length 50 µm diameter silver wire ( see Materials and methods ) .","The lower trace shows the force developed by the muscle during a train of nerve stimulations with a frequency close to the tetanus .","The contraction force was measured with a FT03 force transducer from Grass Technologies ( Astro-Med Inc . , West Warwick ) and expressed in Newton .","The middle trace shows a single muscle cell recording of the membrane potential with the floating electrode technique .","The upper trace shows a detail view of the ePSPs and action potentials . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00410 . 7554/eLife . 12190 . 005Figure 1—figure supplement 2 . Mouse muscles fibers have a wide range of input conductances . Input conductances ( G ) of muscle fibers were calculated from the depolarization induced by injection of a positive current and application of Ohm’s law .","The histogram represents the normalized count distribution of the input conductances for 33 fibers in an Extensor Digitorum Longus muscle , for 34 fibers in a Flexor Digitorum Brevis muscle , and for 50 fibers in a Soleus muscle .","Data were binned with a step increment of 0 . 2 µS . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00510 . 7554/eLife . 12190 . 006Figure 1—figure supplement 3 . Characterization of the K+ conductances that determine the Xenopus muscle cell input conductance .","( A ) A K+ inward rectifying ( Kir ) conductance dominates the input conductance of Xenopus muscle cells .","The upper panel shows membrane currents recorded under voltage-clamp during ramp potentials ( 125 mV/s ) , in control conditions ( grey trace ) and after addition of external Ba2+ 300 µM ( black trace ) .","The Kir component , sensitive to external Ba2+ , was obtained by the difference between the two traces .","The lower panel shows the isolated Kir conductance ( gray trace ) and the remaining conductances in presence of Ba2+ ( black trace ) , composed of a passive K+ leak conductance and of the voltage-activated K+ ( Kv ) conductances .","The Kir conductance dominates the input conductance around the resting potential , decreases with depolarization and becomes null at the excitability threshold .","( B ) A linear correlation between the Kir current and the membrane capacitance ( an estimation of the surface ) of the cells reveals the strong homogeneity of the membrane conductance density in the cells culture , and emphasizes the wide range of input conductances and excitabilities found in muscle cells ( i . e . an increase of surface with constant leak density implies an increase in the input conductance , and a decrease in excitability ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 006"],["Synaptic efficacy is linked to the ability of evoked postsynaptic potentials ( ePSP ) to reach firing threshold .","In vertebrate muscle cells , the amplitude of PSPs depends on the ratio between the nicotinic conductance activated by acetylcholine ( ACh ) and the muscle input conductance , which is largely due to resting K+ leak currents .","Fibers in flexor digitorum brevis ( FDB ) , extensor digitorum longus and soleus mouse muscles have a wide distribution of input conductances ( Figure 1—figure supplement 2 ) .","A similar distribution in Xenopus muscle cells ( Figure 1C , X axis ) is linked to the diversity of membrane surfaces with homogenous conductance density ( 196 ± 14 pS/pF , n=19 , Figure 1—figure supplement 3 ) .","Despite these differences in muscle cell excitability , a single presynaptic AP triggered a single postsynaptic AP , and a contraction , in all tested cells from Xenopus and mouse ( Figure 1 ) .","In Xenopus cultures , we found that the nicotinic conductance activated in muscle cells by spike-evoked ACh release varied linearly with the postsynaptic input conductances measured in a population of synaptic neuron/muscle cell pairs ( Figure 1C ) , and resulted in comparable ePSP ( evoked PSP ) voltage amplitudes .","Similarly , in mouse muscles , we assessed ePSP characteristics after exogenous application of µ-conotoxin GIIIB which selectively blocks Na+v1 . 4 channels involved in the muscle cell AP ( Cruz et al . , 1985; Li et al . , 2003 ) .","This manipulation does not affect nerve APs , since this Na+ channel subtype is not critical for spiking in motor neurons .","Single-spike-evoked PSPs exhibited a narrow range of voltage magnitudes ( Figure 1D , in FDB muscle ) .","The comparable amplitude of ePSPs , despite widely ranging resting conductances in the postsynaptic muscle cells , indicates that the neuromuscular synapse exhibits robust plasticity that regulates its functional strength within a narrow range . 10 . 7554/eLife . 12190 . 007Figure 2 . Calcium signaling in Xenopus muscle cell and synaptic homeostasis . The middle scheme depicts our model of homeostatic control of the synaptic strength .","The nicotinic Ca2+ influx elicits a positive retrograde feedback signal ( red arrow ) on the presynaptic neurotransmitter release .","The positive feedback is balanced by a negative retrograde feedback signal ( blue arrow ) triggered by the muscle AP-induced DICR .","( DICR = depolarization-induced calcium release , AP = action potential , RyR = ryanodine receptor , SR = sarcoplasmic reticulum , R nico = nicotinic receptors ) .","( A ) Independence of the DICR signal from external Ca2+ .","AP ( black trace , induced with a brief postsynaptic current step ) , Ca2+ dye ( Fluo4 ) relative fluorescence in standard external medium ( dark blue trace ) and in Ca2+ free medium ( light blue trace ) , and qualitative measure of the cell contraction ( green trace , see Materials and methods ) .","( B ) Voltage-dependence of the DICR signal .","The Fluo4 fluorescence was measured at the steady-state of the Ca2+ signal during a classical voltage-step protocol from a holding potential of -80 mV and averaged ( n = 3 cells ) .","( C ) Blockade of the DICR signal by loading the muscle cell with 100 µM ryanodine .","( D ) Representative example of the DICR signal ( blue trace ) during repetitive muscle APs ( black trace ) .","( E ) Ca2+ build-up upon nicotinic receptor activation .","Fluo4 signal ( upper traces ) in control and in Ca2+ free medium , during iontophoretic ACh applications ( 5 pulses ) under postsynaptic voltage-clamp ( -80 mV ) .","The lower trace shows the nicotinic currents .","( F ) Dependence of the nicotinic Ca2+ build-up on the nicotinic conductance with increasing iontophoretic ACh applications at a constant membrane potential ( holding potential –80 mV ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00710 . 7554/eLife . 12190 . 008Figure 2—figure supplement 1 . Ionic permeability of the Xenopus nicotinic receptor-channel . To determine the ionic selectivity of the Xenopus nicotinic receptor in cultured cells , the reversal potential of the nicotinic current induced by ionophoretic acetylcholine application was measured under voltage-clamp in solutions of various ionic compositions , with an intra-pipette medium of the following composition ( in mM ) : 110 KCl , 3 NaCl , 2 MgCl2 , 0 . 5 EGTA and 10 HEPES ( pH 7 . 2 ) .","( A ) Current-voltage relationship of the nicotinic current induced by ionophoretic application of acetylcholine , in a classical physiological medium .","Inset shows the current traces recorded at the different holding potentials .","( B ) Table summarizing the external media compositions and the corresponding reversal potentials ( Vr ) .","Concentrations are expressed in mM , and the mean ± SEM reversal potentials in mV .","Fitting the measured reversal potentials to the GHK voltage equation ( see Materials and methods ) gave: PK/PNa = 1 . 07 , PCa/PNa = 0 . 23 , and PMg/PNa = 0 . 31 , used to calculate the theoretical reversal potentials t-Vr . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 008 How does the neuromuscular synapse adjust its strength depending upon the excitability of the postsynaptic muscle fiber ?","Previous research reported that in Xenopus cell cultures , burst activation of presynaptic input results in marked enhancement of ACh release , in both voltage-clamp and current-clamp conditions ( Wan and Poo , 1999 ) .","We reproduced this result in the specific condition of post-synaptic voltage-clamp ( not shown ) but not in that of current-clamp where we found instead absence of potentiation .","To observe such absence in current-clamp , we believe that it is essential that all or most of the ePSPs evoked by the conditioning burst , reach threshold for spike initiation in the postsynaptic cell .","This was not the case in the only example of current-clamp presented by Wan and Poo 1999 ( see their Figure 1A ( i ) ) .","Homeostatic plasticity rules suggest in fact that this form of plasticity should not occur in the presence of synaptic transmission that is effective in generating a muscle AP .","To test this hypothesis , we initiated burst stimulation of motor neurons in both Xenopus and mice –during current-clamp recordings– in order to allow voltage responses and APs in the muscle cells .","In these physiological conditions and despite the chronic burst activity , no significant change in the synaptic gain was visible .","The probability for a synaptic event to induce a postsynaptic AP was close to unity and kept constant ( Figure 1E ) .","In Xenopus however , test evoked postsynaptic currents ( ePSCs ) were recorded under brief periods of postsynaptic voltage-clamp before and after the 30 min of chronic activity under current-clamp , and showed a slight relative decrease of 0 . 2 in the averaged ePSCs amplitude ( see first bar in Figure 3C ) .","In mice , the average ePSPs were not changed by high-frequency conditioning stimulations ( Figure 1D and 3F ) .","Altogether , these observations suggest that synapses able to trigger postsynaptic APs do not exhibit strong plasticity , and that the occurrence of muscle APs may be involved in the homeostatic mechanisms adjusting and maintaining the synaptic strength in concordance with the postsynaptic input conductance . 10 . 7554/eLife . 12190 . 009Figure 3 . Nicotinic receptor activity induces a positive feedback on ACh release .","( A ) In order to obtain the nicotinic calcium signal in absence of DICR in Xenopus , synaptic events were transiently kept subthreshold either with curare ( decreased nicotinic conductance , Gnico ) or by dynamic-clamp injection of gK+ leaks ( increased input conductance , Gin ) , or left suprathreshold while DICR was blocked by ryanodine .","( B ) Effect of presynaptic burst stimulation and curare on spontaneous ( upper trace , sPSC ) and evoked synaptic currents ( ePSC ) .","ePSCs recorded under voltage-clamp were evoked at low rate ( 0 . 03 Hz ) and averaged by 30–40 events ( lower traces ) .","During conditioning presynaptic stimulations ( 3 bursts of 5 events at 30 Hz ) , the postsynaptic potential was released from clamp and curare transiently applied ( middle trace ) .","Upper trace: for clarity , ePSCs were removed from the continuous trace in order to display the sPSCs only .","( C ) , In Xenopus , mean ePSC relative change 30 min after control chronic bursting activity ( 'chronic activity' , n = 5 , illustrated in Figure 1E ) , 45 min after subthreshold synaptic activity ( 'Curare' , n = 9 , illustrated in B; 'Dynamic-clamp' , n = 5 , illustrated in Figure 3—figure supplement 2A ) , transient curare application in absence of stimulation ( 'curare No Burst' , n = 3 ) , sub- ( 'curare-ryanodine' , n = 3 ) and suprathreshold synaptic activity ( 'ryanodine' , n = 6 , illustrated in Figure 3— figure supplement 3 ) in muscle cells preloaded with ryanodine .","( D ) , Relative change in amplitude and frequency of sPSCs after potentiation in Xenopus .","( E ) , In FDB mice muscles , voltage reached by ePSPs in ryanodine treated ( black dots , n = 60 fibers , 2 mice ) , and in ryanodine treated and burst stimulated preparations ( red dots , n = 70 fibers , 2 mice ) .","( F ) Mean ePSP amplitude in control ( 'no stim' ) and high frequency nerve stimulation ( 'Pre stim' ) shown in Figure 1D , in non-stimulated ( 'Ryanodine' ) and in high frequency stimulated ( 'Pre stim Rya' ) ryanodine treated preparations shown in E . * , p<0 . 05; ** , p<0 . 01; *** , p<0 . 001; t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00910 . 7554/eLife . 12190 . 010Figure 3—figure supplement 1 . Decreasing muscle cell excitability by injection of artificial conductances .","( A ) We used the K+ currents data of Figure 1—figure supplement 3 to establish and inject models of leak conductances into the muscle cell with the dynamic-clamp technique ( see Materials and methods ) .","Trace is a negative of the current output of the Kir conductance model when a ramp potential ( 125 mV/s ) was used as input .","( B ) Time course of sPSPs in the presence of a simulated Kir conductance with dynamic-clamp ( Kir DC ) .","Artificial increase of the postsynaptic input conductance decreased the averaged sPSP amplitude . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01010 . 7554/eLife . 12190 . 011Figure 3—figure supplement 2 . LTP induction with dynamic-clamp or postsynaptic ryanodine .","( A ) Potentiating effect of maintaining synaptic efficacy subthreshold by dynamic-clamp injection of gK+ leak ( see Materials and methods ) .","Middle trace: subthreshold nicotinic ePSPs in presence of the dynamic-clamp current ( trace below ) .","( B ) .","Potentiating effect of intact synaptic activity generating muscle cell AP firing with DICR blocked ( 100 µM ryanodine ) .","The muscle cell was pre-loaded with ryanodine using the classical whole-cell configuration of the patch-clamp technique .","After removing the patch pipette , a perforated patch was performed for electrophysiological recordings . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 011 How might the strength of synaptic transmission depend upon the occurrence of an AP in the postsynaptic muscle cell ?","We reasoned that there may be two distinct postsynaptic Ca2+ signaling pathways involved in this homeostatic regulation: a postsynaptic reporter of the presynaptic activity that signals the occurrence of a synaptic event , and a postsynaptic reporter that signals the occurrence of a postsynaptic AP .","The postsynaptic Ca2+ build up ( Figure 2E–F ) due to a high Ca2+ permeability of the nicotinic receptor-channel ( Fucile et al . , 2006 ) ( PCa2+/PNa+=0 . 23 in Xenopus , Figure 2—figure supplement 1 ) is a good candidate for signaling the occurrence of synaptic events .","Furthermore , in vertebrates , the skeletal muscle cells exhibit a specific Ca2+ signal linked to the AP and involved in the excitation-contraction coupling , i . e . 'the depolarization-induced Ca2+ release' ( DICR ) .","The DICR signal ( Figure 2A–D ) is a Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors , triggered by plasma membrane depolarizations above –40 mV , and thus triggered by AP in physiological conditions .","Depolarization is detected by a voltage-sensor ( the dihydropyridine receptor ) located in the plasma membrane .","This voltage-sensor derives from an L-Type Ca2+ channel; it is voltage-dependent but not ion permeable ( Almers et al . , 1981; Melzer et al . , 1995 ) .","Functionally linked to the ryanodine receptor at the level of the T-tubule , its activation triggers the opening of the ryanodine receptor ( Rios and Brum , 1987; Catterall , 1991; Franzini-Armstrong and Protasi , 1997 ) .","The resulting Ca2+ release is independent of the extra-cellular Ca2+ ( Figure 2A ) , purely voltage-dependent , and its voltage-dependency follows the typical pattern of the activation curve of a 'High Voltage-Activated' Ca2+ channel ( Figure 2B ) .","Ryanodine fully blocks this Ca2+ signal ( Figure 2C ) .","We hypothesized that these two distinct Ca2+ signals , nicotinic Ca2+ and DICR , are used by the muscle cell to detect synaptic activity and its efficiency to elicit an AP , respectively , leading to retrograde control of the presynaptic ACh release ( Figure 2 scheme ) .","To validate this hypothesis , we examined each of these Ca2+ dependent mechanisms in isolation and their respective impact on synaptic efficacy .","We first examined the specific role of nicotinic Ca2+ influx in the regulation of synaptic transmission .","To trigger the nicotinic Ca2+ influx but not the DICR in Xenopus muscle cells , we transiently reduced synaptic strength below AP threshold during the conditioning presynaptic burst stimulations performed in postsynaptic current-clamp .","We monitored subsequent synaptic conductance changes under postsynaptic voltage-clamp during low-rate single-pulse intracellular stimulation of the neuron .","We reduced the synaptic efficacy either by decreasing the synaptic conductance with low-doses of the reversible nicotinic antagonist curare ( Figure 3A–C ) , or by artificially increasing the muscle cell input conductance using dynamic-clamp ( Sharp et al . , 1993; Robinson and Kawai , 1993; Prinz et al . , 2004 ) ( Figure 3A , C , and Figure 3—figures supplements 1 , 2A ) .","The lowered synaptic conductance mimicked the effects of low ACh release in developing synapses .","Simulation and dynamic-clamp injection of a combination of a linear and an inwardly rectifying K+ leak conductances ( Materials and methods and Figure 3—figure supplement 1 ) mimicked the increased endogenous input conductance associated with muscle cell growth .","In both cases , presynaptic burst-stimulation ( 3 bursts of 5 pulses at 30 Hz ) induced a strong , fast ( within minutes ) and long-term potentiation ( LTP ) of ePSCs ( Figure 3B , C , and Figure 3—figure supplement 2A ) , while the transient application of curare in absence of presynaptic stimulation did not induce potentiation ( Figure 3C , 'curare No Burst' bar ) .","This form of LTP induced by subthreshold synaptic events depends solely on nicotinic receptor activity and therefore was insensitive to DICR blockade obtained by pre-loading the muscle cell with ryanodine ( Figure 3C ) .","Furthermore , when ePSPs were left intact but in presence of postsynaptic ryanodine , presynaptic burst-stimulation produced postsynaptic APs and nonetheless a strong LTP of the ePSCs ( Figure 3C , and Figure 3—figure supplement 2B ) , while no potentiation occurred when DICR was functional ( Figure 1E and Figure 3C ) .","The LTP induced in our postsynaptic current-clamp protocols was accompanied with a strong increase of the frequency , but not amplitude , of spontaneous postsynaptic currents ( sPSCs ) ( Figure 3B , D ) .","As previously observed in voltage-clamp protocols ( Wan and Poo , 1999 ) , this indicates that LTP is due to an evoked ACh release increase and not to a postsynaptic receptor modulation .","We found similar results in ryanodine-treated mouse muscles ( Figure 3E , F ) : it is only when nicotinic channels were activated following conditioning bursts of nerve stimulations and ACh release —in the absence of DICR and contraction— that subsequent single test pulses revealed a strong LTP of the ePSPs .","In contrast , as shown above , when DICR was functional and the nerve was stimulated , nicotinic ePSPs did not change and remained identical to those in the absence of nerve stimulation ( Figure 1D and Figure 3F ) .","Therefore the simultaneous occurrence of postsynaptic nicotinic receptor activation and DICR is essential to the stability of functional synaptic gain .","Because the regulation targets ACh release , this implies the existence of retrograde feedback onto the presynaptic compartment .","In order to understand the specific role of DICR , we examined its effect on synaptic efficacy in isolation from the effect of nicotinic receptor activation .","Direct stimulation of the Xenopus muscle cell induces postsynaptic APs and DICR , with no ACh release and no involvement of nicotinic receptors ( Figure 4A ) .","Conditioning bursts of suprathreshold current steps injected in the muscle cell induced a strong , fast and long-term depression ( LTD ) of ePSCs ( Figure 4B , C ) .","The average amplitude of sPSCs did not change , confirming the presynaptic locus of gain control , for negative modulations of synaptic strength just like for the positive modulations described above ( Figure 4D ) .","LTD induced by repetitive postsynaptic depolarizations has been previously reported ( Lo et al . , 1994; Dan et al . , 1995 ) .","Here we show that this form of LTD relies strictly on DICR: it was insensitive to removal of external Ca2+ ( Figure 4C and Figure 4—figure supplement 1A ) , and blocked by postsynaptic pre-loading with ryanodine ( Figure 4C and Figure 4—figure supplement 1B ) .","In adult mouse nerve-muscle preparations , external electrical stimulation elicits an AP in both the nerve and the muscle fibers , a situation in which both DICR and nicotinic Ca2+ influx occur .","As expected , in the presence of external Ca2+ , the external burst stimulation did not change the average ePSP ( Figure 4E , F ) compared to nerve stimulation only ( Figure 1D and Figure 4F ) .","When the external Ca2+ was transiently removed , the external stimulation still elicited APs in both the nerve and the muscle , but neither the ACh release nor its associated postsynaptic nicotinic Ca2+ influx .","In these conditions , the external stimulation induced DICR alone and resulted in a strong LTD of the ePSPs ( Figure 4E , F ) .","This form of depression is reversible , since subsequent burst stimulation of the nerve induced a rapid potentiation of the ePSPs ( Figure 4—figure supplement 2 ) . 10 . 7554/eLife . 12190 . 012Figure 4 . Muscle DICR induces a negative feedback on ACh release .","( A ) In order to trigger the DICR signal in absence of nicotinic Ca2+ influx in Xenopus , postsynaptic APs were directly induced in the muscle cell with brief current steps , without presynaptic stimulation , in presence or absence of external Ca2+ ( upper right scheme ) and with ryanodine that blocks the DICR ( bottom right scheme ) .","( B ) Effect of selective postsynaptic firing on synaptic currents .","Same layout as in Figure 3B .","The middle trace shows the muscle APs firing in response to positive current steps injection .","( C ) In Xenopus , mean ePSC relative change 45 min after control chronic bursting synaptic activity ( 'chronic activity' , n = 5 , see 1E ) , direct triggering of the muscle APs shown in B ( 'Direct AP' , n = 5 ) , APs triggered in a Ca2+-free medium ( 'Direct AP Ca2+-free' , n = 6 , illustrated in Figure 4—figure supplement 1 ) , APs triggered in muscle cells loaded with ryanodine ( 'Direct AP Ryanodine' , n = 3 , illustrated in Figure 4—figure supplement 2 ) .","( D ) , Relative change in sPSCs amplitude and frequency in Xenopus after potentiation ( red ) or depression ( green ) .","( E ) , In FDB mouse muscles , voltage reached by ePSPs in externally stimulated preparations ( 15 bursts of 120 events at 30 Hz , 10 min ) in presence ( black dots , n = 75 fibers , 2 mice ) and in absence of external Ca2+ ( green dots , n = 120 fibers , 2 mice ) .","( F ) , Mean ePSP amplitude in control ( 'no stim' ) and high frequency nerve stimulation ( 'Pre stim' ) shown in Figure 1D , in externally stimulated preparations shown in E in presence ( 'External stim-Ca2+' ) and absence of external Ca2+ ( 'External stim-0Ca2+' ) .","*** , p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01210 . 7554/eLife . 12190 . 013Figure 4—figure supplement 1 . External calcium-independent LTD and blockade by postsynaptic ryanodine .","( A ) LTD induction in a Ca2+ free medium .","For the conditioning bursts , postsynaptic APs were triggered by postsynaptic injection of positive current steps , without presynaptic stimulation .","( B ) .","ePSC depression via muscle firing is prevented when DICR is blocked with 100 µM ryanodine pre-loaded into the muscle cell . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01310 . 7554/eLife . 12190 . 014Figure 4—figure supplement 2 . Recovery from LTD . In adult mice preparations , external stimulation in a Ca2+ free medium is a situation comparable to direct stimulation of the Xenopus muscle cells , and induces a depression of the ePSPs .","Here , after a depression induced by 3 bursts of external stimulations in a Ca2+ free medium , subsequent bursts of nerve-stimulations ( arrow ) induced a partial recovery of the ePSPs amplitude .","We transiently increased further the external Ca2+ concentration to 8mM ( which increases the nicotinic Ca2+ influx ) , and this resulted in an increase of the potentiating effect of the bursting nerve stimulations ( arrow ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 014 In the previous experiments , we used the extreme cases of either fully subthreshold synaptic events or of postsynaptic APs in the absence of presynaptic neuron activity , to emphasize the roles of the nicotinic calcium and the DICR signals respectively .","However , during normal synaptic activity the two calcium signals are combined and a unique plasticity orientation rule , reflecting the interaction between the potentiating and depressing synaptic processes , should be possible to define in terms of synaptic efficacy .","Patch-clamp on Xenopus cells in culture allows the examining of changes in the synaptic strength in individual neuromuscular synapses , and the assessing of synaptic conductance .","In Figure 1E , we observed in non-treated Xenopus synapses that 30 min of chronic burst stimulation of the motor neuron did not drastically change the synaptic gain , but nevertheless induced a slight depression of the averaged ePSCs .","Next , we set to closely examine these small synaptic changes in individual synapses .","Given the variety of average synaptic conductance among neuromuscular synapses ( Figure 1C , Y axis ) , and its strong correlation with the muscle input conductance ( Figure 1C ) , the range of the ratio between the synaptic and the input conductances 'Gsyn/Gin' is much tighter than the synaptic conductance range .","We reasoned that because this ratio , and not the synaptic conductance alone , determines the synaptic efficacy ( ePSP amplitude ) , it should be the appropriate synaptic parameter targeted by the homeostatic process .","Therefore , we recorded the ePSCs under brief test periods of postsynaptic voltage-clamp , before and after 20–30 min of chronic burst stimulation of the motor-neuron under postsynaptic current-clamp , and normalized these averaged ePSCs by the input conductance of the muscle cell .","Figure 5A ( black dots ) shows that the slight depression was not homogenous among neuromuscular synapses .","The degree of depression seems to depend on the distance of the initial Gsyn/Gin ratio from the mean ratio obtained after chronic activity ( Figure 5A , dashed line ) .","Consequently , standard deviation from the mean is reduced after chronic activity ( Figure 5A , inset ) .","This synaptic depression behavior can be interpreted as the convergence of the Gsyn/Gin ratios towards the set point of the homeostasis . 10 . 7554/eLife . 12190 . 015Figure 5 . Homeostatic control of the synaptic efficacy .","( A ) In Xenopus , ratios between averaged synaptic conductance ( 'Gsyn' , calculated from 30–40 ePSCs ) and muscle cell input conductance ( 'Gin' ) before and after 20–30 min of chronic burst stimulation of the motor neuron ( burst of 20–60 events at a 20–30 Hz frequency , every 30–40 s ) under postsynaptic current-clamp in non-treated ( black dots ) and low curare-treated ( red dots ) synapses .","Green dots represent the Gsyn/Gin ratio before and after 1–3 bursts of 5 presynaptic stimulations at 30 Hz ( green dots ) in ryanodine loaded muscle cells ( same data than in Figure 3C , ryanodine bar ) .","Inset shows the mean ± standard deviation of the Gsyn/Gin ratios in the three conditions .","The dotted lines show the averaged Gsyn/Gin ratio after chronic activity in non-treated synapses .","( B ) Degree of plasticity ( relative change in Gsyn/Gin ratio ) shown in A expressed as a function of the difference between the initial individual Gsyn/Gin ratio and the averaged ratio after chronic burst activity ( 'Distance to the set point' ) , in non-treated ( black dots ) and curare-treated ( red dots ) synapses .","The solid line shows the theoretical homeostatic relationship between plasticity and the distance to a set point of 2 . 36 , calculated as the mean Gsyn/Gin ratio after chronic activity in non-treated synapses .","( C ) Apparent averaged Gsyn/Gin ratios calculated in mouse FDB muscles from the data of Figure 3E , in non-stimulated and non-treated synapses ( no burst , black dot ) , in burst-stimulated and non-treated synapses ( after chronic bursts , black dot ) , in non-stimulated and ryanodine-treated synapses ( no burst , green dot ) and in burst-stimulated and ryanodine-treated synapses ( after chronic bursts , green dot ) .","Dots represent the mean ± Standard Deviation .","( D ) Relative change of contraction force during 2s-30Hz bursts of nerve stimulations in mouse soleus muscles ( n=4 ) , before and during exposure to a low dose ( 0 . 1 µM ) of curare . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 015 In order to test whether synapses also converge if the initial Gsyn/Gin ratio is below the set point , we applied the same chronic activity in the continuous presence of low doses of curare ( 0 . 1–2 µM ) .","At these concentrations , the bursts of evoked synaptic events are still composed of a majority of suprathreshold events , combining both potentiating and depressing processes .","Figure 5A ( red dots ) shows that , again , the degree of plasticity ( potentiation in this case ) depends on the distance of the initial Gsyn/Gin ratio to the set point .","The plasticity orientation rule may then be expressed in a way showing its homeostatic character .","We plotted the degrees of plasticity ( relative change in the Gsyn/Gin ratio ) in non-treated ( Figure 5B , black dots ) and curare treated ( Figure 5B , red dots ) synapses shown in Figure 5A , as a function of the difference between the initial Gsyn/Gin ratios and the mean Gsyn/Gin ratio after chronic activity ( distance to the set point ) .","In a perfect homeostatic process , with an attractor set point , the degree of plasticity can be expressed as a function of the distance to the set point: ( GsynGin ) after ( GsynGin ) before=1−11+set point ( GsynGin ) before−set point The solid line in Figure 5B shows this theoretical relationship with a set point of 2 . 36 , equal to the mean Gsyn/Gin ratio obtained after chronic activity .","The overlap between the data points and the theoretical relationship shows the homeostatic function of the neuromuscular plasticity .","In order to place the potentiation we obtained with ryanodine ( Figure 3C , 'ryanodine' bar ) in regard of this homeostatic rule , we normalized the averaged synaptic conductances by the input conductance and added the data to the Figure 5A ( green dots ) .","The ryanodine-treated synapses did not converge towards a set point .","The averaged Gsyn/Gin ratios after burst activity were above the set point , and the standard deviations from the mean were increased .","These data suggest that the ryanodine receptors-dependent calcium signal participates to the stabilization of the synaptic efficacy at the set point .","In adult mouse neuromuscular synapses , an apparent Gsyn/Gin ratio can be extrapolated from the ePSPs .","If we assume a linear passive leak , the Gsyn/Gin ratio can be expressed as: GsynGin=−80−VpVp with Vp the peak membrane potential reached by the ePSP and 0 and -80 mV the reversal potentials of the synaptic and the leak currents respectively .","These ratios are only apparent and underestimated given the effect of distance between the recording site and the neuromuscular junction .","Figure 5C shows the apparent Gsyn/Gin ratio in non-stimulated and in burst-stimulated preparations , both for non-treated ( black dots ) and ryanodine-treated ( green dots ) preparations ( same data as in Figure 1D and Figure 3D–E ) .","These data in mouse ( Figure 5C ) confirmed the results seen in Xenopus ( Figure 5A ) .","Finally , in mouse , we applied a continuous low dose of curare ( 0 . 1 µM ) on soleus muscles , together with chronic burst stimulation of the nerve , in order to show that the homeostatic processes are also able to compensate for a decrease in the postsynaptic sensitivity to the neurotransmitter .","At this curare concentration , the synaptic activity is still composed of a majority of suprathreshold events , combining both nicotinic and DICR calcium signals .","We took advantage of the fact that the muscle contraction force integrates the synaptic efficacy over all the fibers of the muscle and all the synaptic events of the bursts , and as such is an indicator of even small changes in the synaptic gain .","Figure 5D shows the 26% decrease in the contraction force due to low curare , followed by progressive recovery of the force back to a normal level in response to subsequent nerve burst stimulations .","We could thus directly observe the functional effect of the synaptic homeostasis mechanism we describe ."],["When selectively triggered , the nicotinic Ca2+ influx and the DICR signal induce a form of LTP and LTD respectively .","The causal links between the nicotinic Ca2+ influx and the synaptic events , and between the DICR signal and the muscle APs , allow the establishment of an orientation rule for neuromuscular synaptic plasticity: synaptic events-induced LTP versus muscle AP-induced LTD .","This orientation rule finds its equilibrium in the suprathreshold range of the synaptic strength and provides the specific 'homeostatic' function to this plasticity .","This neuromuscular plasticity differs from the other forms of long-term plasticity , and in particular should not be confounded with anti-Hebbian plasticity ( Kullmann and Lamsa , 2008; Roberts and Leen , 2010 ) .","Anti-Hebbian plasticity underlies synaptic strength modulations while the present homeostatic mechanism maintains the stability of synaptic efficacy .","Modulation of synaptic transmission has been extensively studied in vitro at the Xenopus neuromuscular synapse by Poo group ( Fu and Poo , 1991; Lo and Poo , 1991; 1994; Lohof et al . , 1993; Lo et al . , 1994; Dan et al . , 1995; Cash et al . , 1996; Wang and Poo , 1997; Wan and Poo , 1999 ) .","Our work , partly done on the same experimental model , suggests that a number of results obtained by Poo group might be interpreted in the more specific framework of homeostatic plasticity .","In vertebrates , neurotrophic factors such as brain-derived neurotrophic factor , neurotrophin 3 and neurotrophin 4 , are trans-synaptic factors considered candidates to mediate presynaptic release modulations in many forms of synaptic plasticity ( Schinder and Poo , 2000; Poo , 2001 ) .","Skeletal muscle synthesize and release neurotrophins in an activity-dependent manner ( Funakoshi et al . , 1995; Wang and Poo , 1997; Xie et al . , 1997 ) , and motor nerve terminals contain the receptors tyrosine kinase TrkB and C ( Henderson et al . , 1993; Koliatsos et al . , 1993; Wong et al . , 1993; Yan et al . , 1993 ) .","At the Xenopus neuromuscular synapse in vitro , Poo group has shown that the exogenous application ( Lohof et al . , 1993; Wang and Poo , 1997 ) of these factors reproduces the synaptic LTP induced in the present work with subthreshold synaptic events or postsynaptic ryanodine treatment .","Finally , upregulation of the ACh evoked release in α-bungarotoxin treated rat was markedly reduced by inhibition of the tyrosin kinase receptors of the neurotrophins ( Plomp and Molenaar , 1996 ) .","Therefore , neurotrophic factors should be considered for future investigations to determine whether they can be retrograde factors mediating homeostatic plasticity at the neuromuscular synapse .","The diversity of the effects of the calcium signaling comes from the variety of its temporal patterns , amplitude , and location .","The two antagonist calcium signals that we demonstrated here to be involved in the orientation of the neuromuscular plasticity exhibit different temporal patterns and amplitudes .","The AP-associated DICR signal exhibits fast transient large calcium concentration elevations for each AP .","The kinetics of the nicotinic calcium build-up is much slower and does not contain transients during repetitive nicotinic receptor stimulations .","Its amplitude under voltage-clamp is low compare to the DICR signal ( Figure 2F ) , and even lower under current-clamp given the reduced driving-force for the calcium ion due to the postsynaptic depolarization .","This dichotomy between fast large transient and slow low calcium build-up is known to trigger selectively , via calmodulin , the CAMKII kinase and the calcineurin phosphatase respectively .","This mechanism is considered in the central nervous system to implement Hebbian plasticity ( Malenka et al . , 1989; Malinow et al . , 1989; Mulkey et al . , 1993; 1994 ) by modulating the conductance and number of the postsynaptic receptors ( Barria et al . , 1997; Mammen et al . , 1997; Beattie et al . , 2000 ) .","The CAMKII signaling pathway was also shown to be required for normal 'presynaptic homeostasis' in Drosophila ( Haghighi et al . , 2003 ) .","Furthermore , Wan and Poo 1999 showed at the Xenopus synapse in vitro that induction of LTP and LTD can be blocked by pre-loading the muscle cell with peptide inhibitors of calcineurin and CAMKII respectively .","As noted by Wan and Poo 1999 , this situation is opposite to the central synapses , where calcineurin is associated to depression and CAMKII to potentiation .","Our results suggest that the low-calcium nicotinic signal and the calcineurin activation have a possible causal link with the detection of the synaptic events and the synaptic potentiation , while the high-calcium DICR signal and the CAMKII activation are associated with the detection of the postsynaptic APs and the synaptic depression .","The convergence of the synaptic efficacy towards a set point ( Figure 5 ) suggests that the potentiating and depressing synaptic processes balance each other in the suprathreshold range of the synaptic strength .","These observations strongly suggest that the set point of the homeostatic plasticity is sustained by a push-pull mechanism .","Our results , however , do not determine the stages where this push-pull mechanism could operate in the causal chain linking the evaluation of synaptic efficacy to the presynaptic modulation .","In particular , our findings do not necessarily imply that two independent antagonist trans-synaptic retrograde factors balance their effects at the presynaptic level .","The coexistence of potentiating and depressing trans-synaptic factors is a possibility among others .","The increased and decreased secretion of a single positive retrograde factor —like for example endostatin in Drosophila ( Wang et al . , 2014 ) and neurotrophins in vertebrates— could also bi-directionally regulate neurotransmitter release .","In this case , it could be hypothesized that a push-pull mechanism operates downstream of the calcium signaling , for example at the level of the calcineurin/CAMKII balance , ultimately determining the secretion level of a single factor .","Therefore , the use of blue and red arrows in Figure 2 is intended as a schematic representation of the retrograde mechanisms that can bi-directionally change neurotransmitter release , without presuming the existence of two distinct retrograde factors .","Most of what we know about synaptic homeostasis stems from experimental studies at the Drosophila larva neuromuscular junction .","Given the numerous differences between Drosophila and vertebrates the comparison between these systems is not straightforward .","Action potentials and the associated DICR signal in vertebrates being absent in Drosophila muscle cells ( Hong and Ganetzky , 1994 ) , the homeostatic mechanism we propose here cannot be directly transposed to Drosophila .","Since no orthologue of the tyrosine kinase receptors are found in Drosophila ( Frank , 2014 ) , the neurotrophin hypothesis in vertebrates cannot either be applied to Drosophila .","However , different actors could play similar roles in both systems .","The calcium permeability of glutamate receptors in Drosophila could have a similar role than the calcium permeability of nicotinic receptors in vertebrates .","The low-voltage activated calcium channels activated by the ePSPs and the 'calcium-induced calcium release' signal responsible for the excitation-contraction coupling in Drosophila ( Peron et al . , 2009 ) could be the orthologue of the DICR signal in vertebrates .","Finally , endostatin in Drosophila ( Wang et al . , 2014 ) may play the role of neurotrophins in vertebrates .","In Drosophila , Frank et al . ( 2006 ) found that spontaneous glutamate release was sufficient to induce a rapid potentiation in the absence of nerve activity , while we had to use a 30Hz motor command to induce rapid potentiation in vertebrates .","In order to establish the Figure 5A , cells were incubated in curare in absence of neurons spikes during 30–60 min before recording ( red dots ) .","Therefore , the spontaneous ACh release did not restore the normal Gsyn/Gin ratio ( found in non-treated synapses ) before chronic bursts were applied .","A possible explanation of this difference between the two systems is the high frequency of miniatures in Drosophila ( 10–20 Hz ) ( Frank et al . , 2006 ) , while the average frequency of spontaneous release in the Xenopus cells culture was 100 fold lower .","In vertebrates , evoked activity responsible for muscular tonus and voluntary motions represents most of the synaptic activity , and the 20–30 Hz frequencies we used in our experiments are in the normal range for a motor command ( Gorassini et al . , 2000 ) .","Therefore , the evoked activity in vertebrate is more likely able to rapidly mobilize the homeostatic machinery than the spontaneous miniatures .","In the central nervous system , homeostatic forms of synaptic plasticity have been proposed to restrain the mean level of neuronal activity within a physiological regime , and to maintain the stability of recurrent network activity challenged by associative plasticity .","Because of the robustness of its synaptic transmission , the neuromuscular junction is considered as a model of homeostasis .","However , the neuromuscular junction being the end effector of the motor network , its main function is the faithful translation of the motor command into muscle activity .","The mean level of muscle activity is not involved in any recurrent network that requires stability , and therefore does not need to be controlled .","While the synaptic strength is the adjustment variable for the control of the mean level of neuronal activity in the CNS , at the NMJ it is the efficacy of synaptic transmission itself that must be maintained constant for reliable relay of the motor command .","Therefore , homeostatic plasticity could be regarded as a functionally different phenomenon at the NMJ than in the CNS .","Nevertheless , the plasticity orientation rule described in the present work matches a synaptic 'relay' function .","Despite differences in the nature of the activity sensors , other 'relay' synapses in the CNS , such as those involved in sensory inputs to the thalamus ( Guido , 2008 ) , might share with the NMJ a comparable homeostatic control based on pre- and post- synaptic activity detection ."],["Myotomal and spinal tissues from 1-day-old Xenopus laevis embryos ( stages 23 to 25 ) were mechanically dissociated using a Ca2+- and Mg2+-free medium of the following composition ( in mM ) : 115 NaCl , 2 . 6 KCl , 0 . 4 EDTA , 10 HEPES ( pH = 7 . 6 ) .","Cells were directly plated in a plastic recording chamber , and grown at 19°C for 12 hr prior to the experiments .","The culture medium consisted of 50% Leibovitz L-15 medium ( Gibco , Invitrogen Corp . , Cergy-Pontoise , France ) , 1% fetal calf serum , 1% antibiotic mixture ( ibid . , final concentration: 100 units/mL penicillin G and 100 µg/mL streptomycin ) , and 48% physiological solution of the following composition ( in mM ) : 113 NaCl , 2 KCl , 0 . 7 CaCl2 , 5 HEPES ( pH=7 . 8 ) .","Perforated patch-clamp recordings , to preserve the cell integrity , were performed at room temperature ( 20–22°C ) .","Pipettes were made from borosilicate glass ( Clark Electromedical Instruments , Reading , England ) and pulled on a P-1000 puller ( Sutter Instrument Company , Novato , CA , U . S . A . ) .","Patch electrodes had a resistance of 2–3 MΩ when filled with internal physiological solution .","Membrane currents and potential were recorded using Axopatch200B patch-clamp amplifiers ( Axon Instruments , Union City , CA ) .","Access resistances were compensated at 80% .","Myocyte membrane currents were filtered with an integrated low-pass Bessel filter at 2 kHz .","The filtered signals were digitized by a 12 bit A/D converter ( Digidata 1200B , ibid . ) and stored using pCLAMP 8 software ( ibid . ) .","Recordings were analyzed using the Origin 7 software ( OriginLab Corp . , Northampton , MA ) .","Motor neurons were current-clamped through an amphotericin-perforated membrane patch ( 400 pA , 3 ms current step to induce the presynaptic AP ) ; the intra-pipette solution had the following composition ( in mM ) : 1 NaCl , 140 K-gluconate , 1 MgCl2 , 10 HEPES ( pH=7 . 2 ) , and amphotericin-B at 300 µg/ml .","Myocytes were either voltage- or current-clamped , through an amphotericin-perforated membrane patch , using the following intra-pipette composition ( in mM ) : 1 NaCl , 20 KCl , 125 K-gluconate , 1 MgCl2 , 10 HEPES ( pH=7 . 2 ) , and amphotericin-B at 300 µg/ml .","The external solution had the following composition ( in mM ) : 140 NaCl , 3 KCl , 1 MgCl2 , 2 CaCl2 , 10 HEPES ( pH=7 . 4 ) .","Pipettes made with borosilicate glass had a resistance of 80–120 MOhm when filled with 0 . 5 to 1 M ACh-Cl .","ACh+ efflux was induced by positive current steps , using a home-made constant current generator .","Xenopus muscle cells were loaded with the calcium indicator in whole-cell patch-clamp configuration for 10 min with an intra-pipette medium of the following composition ( in mM ) : 110 KCl , 1 NaCl , 2 Mg2+ , 10 HEPES ( pH 7 . 2 ) , and 0 . 2 Fluo4-pentapotassium salt .","The patch pipette was then removed and a perforated patch was performed as described above .","Fluorescence intensity was quantified with an Olympus photomultiplier ( forming part of the OSP system , Olympus , Japan ) , and the tension signal was digitized with the Digidata converter .","After background fluorescence subtraction , signals were normalized according to the baseline fluorescence .","The reversal potential of the nicotinic current induced by ionophoretic acetylcholine application was measured under voltage-clamp in solutions of various ionic compositions ( Figure 2—figure supplement 1 ) .","The following equation , derived from the Goldman-Hodgkin-Katz flux equation ( Goldman , 1943; Hodgkin and Katz , 1949 ) , was used to calculate the permeability ratios:Vrev=RTF[0 . 75×PK[K]o+0 . 75×PNa[Na]o+0 . 25×4PCa[Ca]o+0 . 25×4PMg[Mg]o0 . 75×PK[K]i+0 . 75×PNa×[Na]i+0 . 25×Mg[Mg]i] In this equation , the terms in square brackets are ion concentrations , PS is the permeability of the S ion species , T is the absolute temperature , R and F are the gas and Faraday constants , and i and o are the intra- and extracellular compartments , respectively .","Factors represent the ionic activity coefficients .","The dynamic-clamp technique ( Sharp et al . , 1993; Robinson and Kawai , 1993 ) was used to inject computer-generated conductances in Xenopus muscle cells .","Dynamic-clamp experiments were run using the hybrid RT-NEURON environment ( Le Franc et al . , 2001; Sadoc et al . , 2009 ) , a modified version of NEURON ( Hines and Carnevale , 1997 ) running under the Windows operating system ( Microsoft Corp . , Redmond , Washington ) , augmented with the capacity of simulating models in real time , synchronized with the intracellular recording .","A PCI DSP board with 16 bit A/D-D/A converters ( Innovative Integration , SimiValley ) was used to input the membrane potential into the equations of the model , and to output the current to be injected into the cell with a time resolution of 0 . 1 ms . Passive K+ leak and K+ inward rectifying ( Kir ) conductances were injected into the muscle cell to simulate an increase in the input conductance ( Figure 3—figure supplement 1 ) .","The passive K+ leak was expressed in the form: Ileak = gleak x ( V-Eleak ) .","Eleak was set to -80 mV , and gleak to 5–15 nS .","The Kir conductance was expressed in the form: IKir =gmax x m x ( V – EK ) where the maximal conductance gmax was set to 20–50 nS , and changes with time of the gating variable m were calculated by solving the differential equation:dmdt=m∞−mτmm ( t ) =m∞− ( m∞−m0 ) ×⟮−tτm⟯ where fitting voltage-clamp recordings of the real isolated Kir current of Xenopus muscle cells gave and m∞ ( v ) =11+exp ( −0 . 074× ( EK−V ) ) andτm=0 . 2ms The 'contraction' trace shown in Figure 2A represents a qualitative measurement of the contraction of a muscle cell in culture .","In addition to the membrane potential recording pipette , a second patch pipette vertically approached the membrane cell until a slight increase in the pipette electrical resistance became visible .","The pipette was then held in that position , and increase in the cell thickness accompanying contraction was monitored through further variations of the pipette resistance .","3- to 5-month-old Swiss mice were anesthetized with isoflurane , and cervical dislocated .","Dissections were performed within 15 min in an oxygenated Ringer solution of the following composition ( in mM ) : 145 NaCl , 3 KCl , 2 CaCl2 , 1 MgCl2 , 10 HEPES ( pH 7 . 4 ) and 11 glucose .","Intracellular recordings ( Figure 1—figure supplement 1 ) were performed at 34°C , in the oxygenated Ringer solution defined above .","Sharp pipettes were made from borosilicate glass ( Clark Electromedical Instruments ) , pulled on a P-1000 puller ( Sutter Instrument Company ) , and had a resistance of 40–60 MΩ when filled with a KCl 3 M solution .","The filled pipette tip was cut at the limit of the pulled zone , and used as electrode .","The chlorided end of a 10 cm long , 50 µm diameter , silver wire was introduced inside this electrode , and plugged by its opposite end to the headstage of an Axoclamp 2A amplifier ( Axon Instruments ) , where it hung loosely above the muscle .","A drop of mineral oil was added at the back of the intra-pipette medium to avoid evaporation .","The pipette pendulum was vertically manipulated to enter the muscle cells , and its flexibility allowed stable membrane potential recordings in contracting muscles .","Nerve APs were induced with 6 V , 30 µs voltage steps in a suction pipette .","2 µM of µ-conotoxin GIIIB ( 10 min ) was used to isolate the ePSPs .","Data acquisition and analysis were made as above .","Under µ-conotoxin the amplitudes of the recorded ePSPs depend on the distance between the recording site and the synaptic region .","With the floating electrode method , the placement of the electrode is not precisely controlled .","We limited the distance effect by using the FDB mouse muscle , where cells are shorter ( 300 µm ) than the muscle length ( 1 cm ) .","Therefore , blind placement of the electrode can be performed on this muscle , with a maximal 150 µm distance between the recording site and the end plate .","The specific cells size and organization in FDB limit the distance effect in our recordings , but this effect is presumably responsible for an underestimation of the ePSPs amplitudes and for most of the variability between the recorded cells .","Despite the underestimation of the ePSPs amplitudes , the recorded amplitudes were above the -63 mV AP threshold determined in muscle cells of rat fast and slow muscles ( Wood and Slater , 1995 ) .","The salts composing the media used for electrophysiological recordings , EGTA used to obtain Ca2+-free media , and Amphotericin B used for perforated patch-clamp were obtained from Sigma-Aldrich ( Sigma-Aldrich , Saint Quentin Fallavier , France ) .","Ryanodine used in some experiments was 'Ryanodine fractions' from Latoxan ( Latoxan , Valence , France ) .","The µ-conotoxin GIIIB used to block mouse muscle action-potentials was obtained from Alomone Labs ( Alomone Labs , Jerusalem , Israel ) .","The Ca2+ dye 'Fluo4-pentapotassium salt' was obtained from Molecular Probes ( Molecular Probes , Eugene , Oregon ) ."]],"string":"[\n [\n \"In the nervous system , presynaptic neurotransmitter release , postsynaptic receptors , and postsynaptic excitability can be modulated to bi-directionally and durably change synaptic efficacy .\",\n \"There is a large diversity of plasticity processes , which together shape the neuronal network ( Changeux and Danchin , 1976; Goda and Davis , 2003; Munz et al . , 2014 ) and tune its properties ( Nelson and Turrigiano , 2008 ) .\",\n \"In the brain , concomitantly to various forms of associative plasticity which sustain memory and learning , homeostatic plasticity processes are proposed to restrain the mean level of neuronal activity within a physiological regime , and to maintain the stability of recurrent network activity that can be challenged by associative plasticity ( Turrigiano and Nelson , 2004; Macleod and Zinsmaier , 2006; Marder and Goaillard , 2006; Turrigiano , 2007; Nelson and Turrigiano , 2008 ) .\",\n \"Homeostatic plasticity has been extensively studied at the neuromuscular synapse , in particular at the glutamatergic neuromuscular junction ( NMJ ) in Drosophila ( Frank , 2014 ) , due to the robustness of the homeostatic control of synaptic transmission and the great accessibility of the experimental model to genetic manipulations .\",\n \"In vertebrates , each skeletal muscle fiber is mono-innervated ( Sanes and Lichtman , 1999 ) , and each single presynaptic action potential ( AP ) induces one postsynaptic AP , corresponding to a unity synaptic gain ( ratio between the numbers of post- and presynaptic APs equal to 1 ) ( Wood and Slater , 2001 ) .\",\n \"The stability of the gain implies that presynaptic neurotransmitter release and/or postsynaptic receptors adapt the effective synaptic strength to the excitability of the muscle fiber , which depends on the fiber characteristics , and presumably decreases with growth and exercise ( Turrigiano , 2007 ) .\",\n \"In adult vertebrate skeletal muscles , cholinergic nicotinic receptors are clustered in the synaptic region .\",\n \"Expression and location of nicotinic receptors have been shown to depend not only on agrin ( McMahan , 1990; Hall and Sanes , 1993; Gautam et al . , 1995; 1996; Sandrock et al . , 1997 ) but also on activity ( Lømo , 2003 ) , suggesting their possible role as adjustment variables in the control of synaptic strength .\",\n \"In the recent years however , thorough studies of the glutamatergic NMJ in Drosophila revealed that presynaptic regulation of neurotransmitter release is certainly a major adjustment variable for synaptic homeostasis ( Davis and Müller , 2015 ) .\",\n \"In such 'presynaptic homeostasis' , increase in neurotransmitter release counterbalances a genetically-induced decrease of the postsynaptic sensitivity to glutamate ( Petersen et al . , 1997; Davis et al . , 1998; DiAntonio et al . , 1999 ) , a genetically-induced increase of postsynaptic input conductance ( Paradis et al . , 2001 ) or a pharmacological blockade of postsynaptic receptors ( Frank et al . , 2006 ) , thus resulting in the strict maintenance of the level of evoked postsynaptic depolarization .\",\n \"Presynaptic compensation of postsynaptic excitability changes implies the existence of a retrograde feedback process from the innervated muscle fiber to the motor neuron .\",\n \"Particular attention was paid to the determination of the molecular targets of the homeostatic retrograde signaling and of the presynaptic cell signaling involved .\",\n \"It appeared that both the readily releasable vesicle pool ( Müller et al . , 2012; 2015 ) and the presynaptic voltage-gated Ca2+ channels ( Müller and Davis , 2012 ) are targeted to promote glutamate release when postsynaptic excitability is reduced .\",\n \"The abundance of voltage-gated Ca2+ channels can also be decreased in response to a vesicular content increase ( Gaviño et al . , 2015 ) , confirming the bi-directionality of the process .\",\n \"In Drosophila , endostatin is a candidate trans-synaptic factor for the retrograde feedback targeting presynaptic neurotransmitter release ( Wang et al . , 2014 ) .\",\n \"Upstream from the retrograde factors , the postsynaptic kinases , CAMKII ( Haghighi et al . , 2003 ) and TOR ( Penney et al . , 2012 ) , pathways are necessary for a functional homeostatic control at the Drosophila neuromuscular transmission .\",\n \"An abundant literature is thus progressively assembling pieces of the signaling puzzle underlying the interactions between the motor neuron and the muscle cell for the control of synaptic strength .\",\n \"However , besides the nature of the retrograde feedback and its presynaptic targets , a mechanism for the evaluation of the synaptic strength should be present at the postsynaptic level .\",\n \"The nature and dynamics of these mechanisms , primarily sensing synaptic strength in the muscle cell and initiating retrograde feedback , are key issues of homeostatic plasticity that still remain enigmatic in both invertebrates and vertebrates .\",\n \"Here we investigate this issue in vertebrate ( Xenopus and mouse ) cholinergic transmission .\",\n \"Motor neurons and muscle cells of Xenopus laevis embryos establish functional neuromuscular synapses ( Tabti et al . , 1998 ) in primary co-culture .\",\n \"Standard intracellular recording from muscle cells is possible in Xenopus culture owing to the small size of the cells ( Figure 1A , perforated patch-clamp ) , while it is not possible in muscle cells from adult mice , due to macroscopic movements during contraction .\",\n \"To circumvent this issue and maintain intracellular recordings in moving tissue , we adapted a ‘floating electrode’ device from previous methods invented for muscles ( Woodbury and Brady , 1956 ) or neurons ( Kunze , 1998 ) .\",\n \"We applied this method to mouse ex vivo nerve-skeletal muscle preparations ( Figure 1B , Methods , and Figure 1—figure supplement 1 ) .\",\n \"This approach preserved the physiological conditions , necessary to reveal the control exercised by the muscle cell over the neuromuscular synaptic transmission , a role that might have been previously overlooked in vertebrates due to commonly used high concentrations of the nicotinic receptor antagonist curare to prevent contraction . 10 . 7554/eLife . 12190 . 003Figure 1 . Synaptic transmission is homeostatically regulated .\",\n \"( A ) Perforated patch-clamp on Xenopus neuron ( N ) and muscle cell ( M ) in primary culture .\",\n \"Presynaptic APs were triggered with current steps .\",\n \"Postsynaptic APs were recorded under current-clamp and nicotinic synaptic currents under voltage-clamp ( -80 mV ) .\",\n \"( B ) Intracellular recording in soleus muscle fibers from an adult mouse using a floating sharp electrode ( see Materials and Methods and Figure 1—figure supplement 1 ) .\",\n \"( C ) Nicotinic conductance calculated from averaged ePSCs ( n = 30 ePSCs for each dot ) in different Xenopus muscle cells as a function of their input conductance .\",\n \"The black line shows the linear regression .\",\n \"( D ) In mice , membrane potential reached by the ePSP in individual FDB muscle fibers after treatment with µ-conotoxin GIIIB , in absence of burst stimulation of the nerve ( black dots , n= 88 fibers , 2 muscles , 2 mice ) , and in test preparations ( grey dots , n = 108 fibers , 2 muscles , 2 mice ) for which the nerve was burst stimulated prior to conotoxin treatment ( 15 bursts in 10 min , each of 120 events at 30 Hz ) .\",\n \"( E ) Mean synaptic gain at Xenopus synapses ( dots , n = 5 synaptic connections ) and in mouse neuromuscular junctions ( squares , n = 4 muscle fibers from different mice ) during chronic bursts of presynaptic stimulation ( bursts of 80 to 120 pulses , 30 Hz for 30 min ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00310 . 7554/eLife . 12190 . 004Figure 1—figure supplement 1 . Floating electrode . In conventional electrophysiology , an intracellular electrode made from pulled glass is rigidly fixed to an amplifier headstage and/or to the micromanipulator by a holder preventing free movements .\",\n \"To allow intracellular recordings in contracting mouse muscle , we cut off the tip of the pipette and used this as an electrode connected to the amplifier headstage by a loose , 5–10 cm length 50 µm diameter silver wire ( see Materials and methods ) .\",\n \"The lower trace shows the force developed by the muscle during a train of nerve stimulations with a frequency close to the tetanus .\",\n \"The contraction force was measured with a FT03 force transducer from Grass Technologies ( Astro-Med Inc . , West Warwick ) and expressed in Newton .\",\n \"The middle trace shows a single muscle cell recording of the membrane potential with the floating electrode technique .\",\n \"The upper trace shows a detail view of the ePSPs and action potentials . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00410 . 7554/eLife . 12190 . 005Figure 1—figure supplement 2 . Mouse muscles fibers have a wide range of input conductances . Input conductances ( G ) of muscle fibers were calculated from the depolarization induced by injection of a positive current and application of Ohm’s law .\",\n \"The histogram represents the normalized count distribution of the input conductances for 33 fibers in an Extensor Digitorum Longus muscle , for 34 fibers in a Flexor Digitorum Brevis muscle , and for 50 fibers in a Soleus muscle .\",\n \"Data were binned with a step increment of 0 . 2 µS . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00510 . 7554/eLife . 12190 . 006Figure 1—figure supplement 3 . Characterization of the K+ conductances that determine the Xenopus muscle cell input conductance .\",\n \"( A ) A K+ inward rectifying ( Kir ) conductance dominates the input conductance of Xenopus muscle cells .\",\n \"The upper panel shows membrane currents recorded under voltage-clamp during ramp potentials ( 125 mV/s ) , in control conditions ( grey trace ) and after addition of external Ba2+ 300 µM ( black trace ) .\",\n \"The Kir component , sensitive to external Ba2+ , was obtained by the difference between the two traces .\",\n \"The lower panel shows the isolated Kir conductance ( gray trace ) and the remaining conductances in presence of Ba2+ ( black trace ) , composed of a passive K+ leak conductance and of the voltage-activated K+ ( Kv ) conductances .\",\n \"The Kir conductance dominates the input conductance around the resting potential , decreases with depolarization and becomes null at the excitability threshold .\",\n \"( B ) A linear correlation between the Kir current and the membrane capacitance ( an estimation of the surface ) of the cells reveals the strong homogeneity of the membrane conductance density in the cells culture , and emphasizes the wide range of input conductances and excitabilities found in muscle cells ( i . e . an increase of surface with constant leak density implies an increase in the input conductance , and a decrease in excitability ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 006\"\n ],\n [\n \"Synaptic efficacy is linked to the ability of evoked postsynaptic potentials ( ePSP ) to reach firing threshold .\",\n \"In vertebrate muscle cells , the amplitude of PSPs depends on the ratio between the nicotinic conductance activated by acetylcholine ( ACh ) and the muscle input conductance , which is largely due to resting K+ leak currents .\",\n \"Fibers in flexor digitorum brevis ( FDB ) , extensor digitorum longus and soleus mouse muscles have a wide distribution of input conductances ( Figure 1—figure supplement 2 ) .\",\n \"A similar distribution in Xenopus muscle cells ( Figure 1C , X axis ) is linked to the diversity of membrane surfaces with homogenous conductance density ( 196 ± 14 pS/pF , n=19 , Figure 1—figure supplement 3 ) .\",\n \"Despite these differences in muscle cell excitability , a single presynaptic AP triggered a single postsynaptic AP , and a contraction , in all tested cells from Xenopus and mouse ( Figure 1 ) .\",\n \"In Xenopus cultures , we found that the nicotinic conductance activated in muscle cells by spike-evoked ACh release varied linearly with the postsynaptic input conductances measured in a population of synaptic neuron/muscle cell pairs ( Figure 1C ) , and resulted in comparable ePSP ( evoked PSP ) voltage amplitudes .\",\n \"Similarly , in mouse muscles , we assessed ePSP characteristics after exogenous application of µ-conotoxin GIIIB which selectively blocks Na+v1 . 4 channels involved in the muscle cell AP ( Cruz et al . , 1985; Li et al . , 2003 ) .\",\n \"This manipulation does not affect nerve APs , since this Na+ channel subtype is not critical for spiking in motor neurons .\",\n \"Single-spike-evoked PSPs exhibited a narrow range of voltage magnitudes ( Figure 1D , in FDB muscle ) .\",\n \"The comparable amplitude of ePSPs , despite widely ranging resting conductances in the postsynaptic muscle cells , indicates that the neuromuscular synapse exhibits robust plasticity that regulates its functional strength within a narrow range . 10 . 7554/eLife . 12190 . 007Figure 2 . Calcium signaling in Xenopus muscle cell and synaptic homeostasis . The middle scheme depicts our model of homeostatic control of the synaptic strength .\",\n \"The nicotinic Ca2+ influx elicits a positive retrograde feedback signal ( red arrow ) on the presynaptic neurotransmitter release .\",\n \"The positive feedback is balanced by a negative retrograde feedback signal ( blue arrow ) triggered by the muscle AP-induced DICR .\",\n \"( DICR = depolarization-induced calcium release , AP = action potential , RyR = ryanodine receptor , SR = sarcoplasmic reticulum , R nico = nicotinic receptors ) .\",\n \"( A ) Independence of the DICR signal from external Ca2+ .\",\n \"AP ( black trace , induced with a brief postsynaptic current step ) , Ca2+ dye ( Fluo4 ) relative fluorescence in standard external medium ( dark blue trace ) and in Ca2+ free medium ( light blue trace ) , and qualitative measure of the cell contraction ( green trace , see Materials and methods ) .\",\n \"( B ) Voltage-dependence of the DICR signal .\",\n \"The Fluo4 fluorescence was measured at the steady-state of the Ca2+ signal during a classical voltage-step protocol from a holding potential of -80 mV and averaged ( n = 3 cells ) .\",\n \"( C ) Blockade of the DICR signal by loading the muscle cell with 100 µM ryanodine .\",\n \"( D ) Representative example of the DICR signal ( blue trace ) during repetitive muscle APs ( black trace ) .\",\n \"( E ) Ca2+ build-up upon nicotinic receptor activation .\",\n \"Fluo4 signal ( upper traces ) in control and in Ca2+ free medium , during iontophoretic ACh applications ( 5 pulses ) under postsynaptic voltage-clamp ( -80 mV ) .\",\n \"The lower trace shows the nicotinic currents .\",\n \"( F ) Dependence of the nicotinic Ca2+ build-up on the nicotinic conductance with increasing iontophoretic ACh applications at a constant membrane potential ( holding potential –80 mV ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00710 . 7554/eLife . 12190 . 008Figure 2—figure supplement 1 . Ionic permeability of the Xenopus nicotinic receptor-channel . To determine the ionic selectivity of the Xenopus nicotinic receptor in cultured cells , the reversal potential of the nicotinic current induced by ionophoretic acetylcholine application was measured under voltage-clamp in solutions of various ionic compositions , with an intra-pipette medium of the following composition ( in mM ) : 110 KCl , 3 NaCl , 2 MgCl2 , 0 . 5 EGTA and 10 HEPES ( pH 7 . 2 ) .\",\n \"( A ) Current-voltage relationship of the nicotinic current induced by ionophoretic application of acetylcholine , in a classical physiological medium .\",\n \"Inset shows the current traces recorded at the different holding potentials .\",\n \"( B ) Table summarizing the external media compositions and the corresponding reversal potentials ( Vr ) .\",\n \"Concentrations are expressed in mM , and the mean ± SEM reversal potentials in mV .\",\n \"Fitting the measured reversal potentials to the GHK voltage equation ( see Materials and methods ) gave: PK/PNa = 1 . 07 , PCa/PNa = 0 . 23 , and PMg/PNa = 0 . 31 , used to calculate the theoretical reversal potentials t-Vr . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 008 How does the neuromuscular synapse adjust its strength depending upon the excitability of the postsynaptic muscle fiber ?\",\n \"Previous research reported that in Xenopus cell cultures , burst activation of presynaptic input results in marked enhancement of ACh release , in both voltage-clamp and current-clamp conditions ( Wan and Poo , 1999 ) .\",\n \"We reproduced this result in the specific condition of post-synaptic voltage-clamp ( not shown ) but not in that of current-clamp where we found instead absence of potentiation .\",\n \"To observe such absence in current-clamp , we believe that it is essential that all or most of the ePSPs evoked by the conditioning burst , reach threshold for spike initiation in the postsynaptic cell .\",\n \"This was not the case in the only example of current-clamp presented by Wan and Poo 1999 ( see their Figure 1A ( i ) ) .\",\n \"Homeostatic plasticity rules suggest in fact that this form of plasticity should not occur in the presence of synaptic transmission that is effective in generating a muscle AP .\",\n \"To test this hypothesis , we initiated burst stimulation of motor neurons in both Xenopus and mice –during current-clamp recordings– in order to allow voltage responses and APs in the muscle cells .\",\n \"In these physiological conditions and despite the chronic burst activity , no significant change in the synaptic gain was visible .\",\n \"The probability for a synaptic event to induce a postsynaptic AP was close to unity and kept constant ( Figure 1E ) .\",\n \"In Xenopus however , test evoked postsynaptic currents ( ePSCs ) were recorded under brief periods of postsynaptic voltage-clamp before and after the 30 min of chronic activity under current-clamp , and showed a slight relative decrease of 0 . 2 in the averaged ePSCs amplitude ( see first bar in Figure 3C ) .\",\n \"In mice , the average ePSPs were not changed by high-frequency conditioning stimulations ( Figure 1D and 3F ) .\",\n \"Altogether , these observations suggest that synapses able to trigger postsynaptic APs do not exhibit strong plasticity , and that the occurrence of muscle APs may be involved in the homeostatic mechanisms adjusting and maintaining the synaptic strength in concordance with the postsynaptic input conductance . 10 . 7554/eLife . 12190 . 009Figure 3 . Nicotinic receptor activity induces a positive feedback on ACh release .\",\n \"( A ) In order to obtain the nicotinic calcium signal in absence of DICR in Xenopus , synaptic events were transiently kept subthreshold either with curare ( decreased nicotinic conductance , Gnico ) or by dynamic-clamp injection of gK+ leaks ( increased input conductance , Gin ) , or left suprathreshold while DICR was blocked by ryanodine .\",\n \"( B ) Effect of presynaptic burst stimulation and curare on spontaneous ( upper trace , sPSC ) and evoked synaptic currents ( ePSC ) .\",\n \"ePSCs recorded under voltage-clamp were evoked at low rate ( 0 . 03 Hz ) and averaged by 30–40 events ( lower traces ) .\",\n \"During conditioning presynaptic stimulations ( 3 bursts of 5 events at 30 Hz ) , the postsynaptic potential was released from clamp and curare transiently applied ( middle trace ) .\",\n \"Upper trace: for clarity , ePSCs were removed from the continuous trace in order to display the sPSCs only .\",\n \"( C ) , In Xenopus , mean ePSC relative change 30 min after control chronic bursting activity ( 'chronic activity' , n = 5 , illustrated in Figure 1E ) , 45 min after subthreshold synaptic activity ( 'Curare' , n = 9 , illustrated in B; 'Dynamic-clamp' , n = 5 , illustrated in Figure 3—figure supplement 2A ) , transient curare application in absence of stimulation ( 'curare No Burst' , n = 3 ) , sub- ( 'curare-ryanodine' , n = 3 ) and suprathreshold synaptic activity ( 'ryanodine' , n = 6 , illustrated in Figure 3— figure supplement 3 ) in muscle cells preloaded with ryanodine .\",\n \"( D ) , Relative change in amplitude and frequency of sPSCs after potentiation in Xenopus .\",\n \"( E ) , In FDB mice muscles , voltage reached by ePSPs in ryanodine treated ( black dots , n = 60 fibers , 2 mice ) , and in ryanodine treated and burst stimulated preparations ( red dots , n = 70 fibers , 2 mice ) .\",\n \"( F ) Mean ePSP amplitude in control ( 'no stim' ) and high frequency nerve stimulation ( 'Pre stim' ) shown in Figure 1D , in non-stimulated ( 'Ryanodine' ) and in high frequency stimulated ( 'Pre stim Rya' ) ryanodine treated preparations shown in E . * , p<0 . 05; ** , p<0 . 01; *** , p<0 . 001; t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00910 . 7554/eLife . 12190 . 010Figure 3—figure supplement 1 . Decreasing muscle cell excitability by injection of artificial conductances .\",\n \"( A ) We used the K+ currents data of Figure 1—figure supplement 3 to establish and inject models of leak conductances into the muscle cell with the dynamic-clamp technique ( see Materials and methods ) .\",\n \"Trace is a negative of the current output of the Kir conductance model when a ramp potential ( 125 mV/s ) was used as input .\",\n \"( B ) Time course of sPSPs in the presence of a simulated Kir conductance with dynamic-clamp ( Kir DC ) .\",\n \"Artificial increase of the postsynaptic input conductance decreased the averaged sPSP amplitude . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01010 . 7554/eLife . 12190 . 011Figure 3—figure supplement 2 . LTP induction with dynamic-clamp or postsynaptic ryanodine .\",\n \"( A ) Potentiating effect of maintaining synaptic efficacy subthreshold by dynamic-clamp injection of gK+ leak ( see Materials and methods ) .\",\n \"Middle trace: subthreshold nicotinic ePSPs in presence of the dynamic-clamp current ( trace below ) .\",\n \"( B ) .\",\n \"Potentiating effect of intact synaptic activity generating muscle cell AP firing with DICR blocked ( 100 µM ryanodine ) .\",\n \"The muscle cell was pre-loaded with ryanodine using the classical whole-cell configuration of the patch-clamp technique .\",\n \"After removing the patch pipette , a perforated patch was performed for electrophysiological recordings . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 011 How might the strength of synaptic transmission depend upon the occurrence of an AP in the postsynaptic muscle cell ?\",\n \"We reasoned that there may be two distinct postsynaptic Ca2+ signaling pathways involved in this homeostatic regulation: a postsynaptic reporter of the presynaptic activity that signals the occurrence of a synaptic event , and a postsynaptic reporter that signals the occurrence of a postsynaptic AP .\",\n \"The postsynaptic Ca2+ build up ( Figure 2E–F ) due to a high Ca2+ permeability of the nicotinic receptor-channel ( Fucile et al . , 2006 ) ( PCa2+/PNa+=0 . 23 in Xenopus , Figure 2—figure supplement 1 ) is a good candidate for signaling the occurrence of synaptic events .\",\n \"Furthermore , in vertebrates , the skeletal muscle cells exhibit a specific Ca2+ signal linked to the AP and involved in the excitation-contraction coupling , i . e . 'the depolarization-induced Ca2+ release' ( DICR ) .\",\n \"The DICR signal ( Figure 2A–D ) is a Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors , triggered by plasma membrane depolarizations above –40 mV , and thus triggered by AP in physiological conditions .\",\n \"Depolarization is detected by a voltage-sensor ( the dihydropyridine receptor ) located in the plasma membrane .\",\n \"This voltage-sensor derives from an L-Type Ca2+ channel; it is voltage-dependent but not ion permeable ( Almers et al . , 1981; Melzer et al . , 1995 ) .\",\n \"Functionally linked to the ryanodine receptor at the level of the T-tubule , its activation triggers the opening of the ryanodine receptor ( Rios and Brum , 1987; Catterall , 1991; Franzini-Armstrong and Protasi , 1997 ) .\",\n \"The resulting Ca2+ release is independent of the extra-cellular Ca2+ ( Figure 2A ) , purely voltage-dependent , and its voltage-dependency follows the typical pattern of the activation curve of a 'High Voltage-Activated' Ca2+ channel ( Figure 2B ) .\",\n \"Ryanodine fully blocks this Ca2+ signal ( Figure 2C ) .\",\n \"We hypothesized that these two distinct Ca2+ signals , nicotinic Ca2+ and DICR , are used by the muscle cell to detect synaptic activity and its efficiency to elicit an AP , respectively , leading to retrograde control of the presynaptic ACh release ( Figure 2 scheme ) .\",\n \"To validate this hypothesis , we examined each of these Ca2+ dependent mechanisms in isolation and their respective impact on synaptic efficacy .\",\n \"We first examined the specific role of nicotinic Ca2+ influx in the regulation of synaptic transmission .\",\n \"To trigger the nicotinic Ca2+ influx but not the DICR in Xenopus muscle cells , we transiently reduced synaptic strength below AP threshold during the conditioning presynaptic burst stimulations performed in postsynaptic current-clamp .\",\n \"We monitored subsequent synaptic conductance changes under postsynaptic voltage-clamp during low-rate single-pulse intracellular stimulation of the neuron .\",\n \"We reduced the synaptic efficacy either by decreasing the synaptic conductance with low-doses of the reversible nicotinic antagonist curare ( Figure 3A–C ) , or by artificially increasing the muscle cell input conductance using dynamic-clamp ( Sharp et al . , 1993; Robinson and Kawai , 1993; Prinz et al . , 2004 ) ( Figure 3A , C , and Figure 3—figures supplements 1 , 2A ) .\",\n \"The lowered synaptic conductance mimicked the effects of low ACh release in developing synapses .\",\n \"Simulation and dynamic-clamp injection of a combination of a linear and an inwardly rectifying K+ leak conductances ( Materials and methods and Figure 3—figure supplement 1 ) mimicked the increased endogenous input conductance associated with muscle cell growth .\",\n \"In both cases , presynaptic burst-stimulation ( 3 bursts of 5 pulses at 30 Hz ) induced a strong , fast ( within minutes ) and long-term potentiation ( LTP ) of ePSCs ( Figure 3B , C , and Figure 3—figure supplement 2A ) , while the transient application of curare in absence of presynaptic stimulation did not induce potentiation ( Figure 3C , 'curare No Burst' bar ) .\",\n \"This form of LTP induced by subthreshold synaptic events depends solely on nicotinic receptor activity and therefore was insensitive to DICR blockade obtained by pre-loading the muscle cell with ryanodine ( Figure 3C ) .\",\n \"Furthermore , when ePSPs were left intact but in presence of postsynaptic ryanodine , presynaptic burst-stimulation produced postsynaptic APs and nonetheless a strong LTP of the ePSCs ( Figure 3C , and Figure 3—figure supplement 2B ) , while no potentiation occurred when DICR was functional ( Figure 1E and Figure 3C ) .\",\n \"The LTP induced in our postsynaptic current-clamp protocols was accompanied with a strong increase of the frequency , but not amplitude , of spontaneous postsynaptic currents ( sPSCs ) ( Figure 3B , D ) .\",\n \"As previously observed in voltage-clamp protocols ( Wan and Poo , 1999 ) , this indicates that LTP is due to an evoked ACh release increase and not to a postsynaptic receptor modulation .\",\n \"We found similar results in ryanodine-treated mouse muscles ( Figure 3E , F ) : it is only when nicotinic channels were activated following conditioning bursts of nerve stimulations and ACh release —in the absence of DICR and contraction— that subsequent single test pulses revealed a strong LTP of the ePSPs .\",\n \"In contrast , as shown above , when DICR was functional and the nerve was stimulated , nicotinic ePSPs did not change and remained identical to those in the absence of nerve stimulation ( Figure 1D and Figure 3F ) .\",\n \"Therefore the simultaneous occurrence of postsynaptic nicotinic receptor activation and DICR is essential to the stability of functional synaptic gain .\",\n \"Because the regulation targets ACh release , this implies the existence of retrograde feedback onto the presynaptic compartment .\",\n \"In order to understand the specific role of DICR , we examined its effect on synaptic efficacy in isolation from the effect of nicotinic receptor activation .\",\n \"Direct stimulation of the Xenopus muscle cell induces postsynaptic APs and DICR , with no ACh release and no involvement of nicotinic receptors ( Figure 4A ) .\",\n \"Conditioning bursts of suprathreshold current steps injected in the muscle cell induced a strong , fast and long-term depression ( LTD ) of ePSCs ( Figure 4B , C ) .\",\n \"The average amplitude of sPSCs did not change , confirming the presynaptic locus of gain control , for negative modulations of synaptic strength just like for the positive modulations described above ( Figure 4D ) .\",\n \"LTD induced by repetitive postsynaptic depolarizations has been previously reported ( Lo et al . , 1994; Dan et al . , 1995 ) .\",\n \"Here we show that this form of LTD relies strictly on DICR: it was insensitive to removal of external Ca2+ ( Figure 4C and Figure 4—figure supplement 1A ) , and blocked by postsynaptic pre-loading with ryanodine ( Figure 4C and Figure 4—figure supplement 1B ) .\",\n \"In adult mouse nerve-muscle preparations , external electrical stimulation elicits an AP in both the nerve and the muscle fibers , a situation in which both DICR and nicotinic Ca2+ influx occur .\",\n \"As expected , in the presence of external Ca2+ , the external burst stimulation did not change the average ePSP ( Figure 4E , F ) compared to nerve stimulation only ( Figure 1D and Figure 4F ) .\",\n \"When the external Ca2+ was transiently removed , the external stimulation still elicited APs in both the nerve and the muscle , but neither the ACh release nor its associated postsynaptic nicotinic Ca2+ influx .\",\n \"In these conditions , the external stimulation induced DICR alone and resulted in a strong LTD of the ePSPs ( Figure 4E , F ) .\",\n \"This form of depression is reversible , since subsequent burst stimulation of the nerve induced a rapid potentiation of the ePSPs ( Figure 4—figure supplement 2 ) . 10 . 7554/eLife . 12190 . 012Figure 4 . Muscle DICR induces a negative feedback on ACh release .\",\n \"( A ) In order to trigger the DICR signal in absence of nicotinic Ca2+ influx in Xenopus , postsynaptic APs were directly induced in the muscle cell with brief current steps , without presynaptic stimulation , in presence or absence of external Ca2+ ( upper right scheme ) and with ryanodine that blocks the DICR ( bottom right scheme ) .\",\n \"( B ) Effect of selective postsynaptic firing on synaptic currents .\",\n \"Same layout as in Figure 3B .\",\n \"The middle trace shows the muscle APs firing in response to positive current steps injection .\",\n \"( C ) In Xenopus , mean ePSC relative change 45 min after control chronic bursting synaptic activity ( 'chronic activity' , n = 5 , see 1E ) , direct triggering of the muscle APs shown in B ( 'Direct AP' , n = 5 ) , APs triggered in a Ca2+-free medium ( 'Direct AP Ca2+-free' , n = 6 , illustrated in Figure 4—figure supplement 1 ) , APs triggered in muscle cells loaded with ryanodine ( 'Direct AP Ryanodine' , n = 3 , illustrated in Figure 4—figure supplement 2 ) .\",\n \"( D ) , Relative change in sPSCs amplitude and frequency in Xenopus after potentiation ( red ) or depression ( green ) .\",\n \"( E ) , In FDB mouse muscles , voltage reached by ePSPs in externally stimulated preparations ( 15 bursts of 120 events at 30 Hz , 10 min ) in presence ( black dots , n = 75 fibers , 2 mice ) and in absence of external Ca2+ ( green dots , n = 120 fibers , 2 mice ) .\",\n \"( F ) , Mean ePSP amplitude in control ( 'no stim' ) and high frequency nerve stimulation ( 'Pre stim' ) shown in Figure 1D , in externally stimulated preparations shown in E in presence ( 'External stim-Ca2+' ) and absence of external Ca2+ ( 'External stim-0Ca2+' ) .\",\n \"*** , p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01210 . 7554/eLife . 12190 . 013Figure 4—figure supplement 1 . External calcium-independent LTD and blockade by postsynaptic ryanodine .\",\n \"( A ) LTD induction in a Ca2+ free medium .\",\n \"For the conditioning bursts , postsynaptic APs were triggered by postsynaptic injection of positive current steps , without presynaptic stimulation .\",\n \"( B ) .\",\n \"ePSC depression via muscle firing is prevented when DICR is blocked with 100 µM ryanodine pre-loaded into the muscle cell . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01310 . 7554/eLife . 12190 . 014Figure 4—figure supplement 2 . Recovery from LTD . In adult mice preparations , external stimulation in a Ca2+ free medium is a situation comparable to direct stimulation of the Xenopus muscle cells , and induces a depression of the ePSPs .\",\n \"Here , after a depression induced by 3 bursts of external stimulations in a Ca2+ free medium , subsequent bursts of nerve-stimulations ( arrow ) induced a partial recovery of the ePSPs amplitude .\",\n \"We transiently increased further the external Ca2+ concentration to 8mM ( which increases the nicotinic Ca2+ influx ) , and this resulted in an increase of the potentiating effect of the bursting nerve stimulations ( arrow ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 014 In the previous experiments , we used the extreme cases of either fully subthreshold synaptic events or of postsynaptic APs in the absence of presynaptic neuron activity , to emphasize the roles of the nicotinic calcium and the DICR signals respectively .\",\n \"However , during normal synaptic activity the two calcium signals are combined and a unique plasticity orientation rule , reflecting the interaction between the potentiating and depressing synaptic processes , should be possible to define in terms of synaptic efficacy .\",\n \"Patch-clamp on Xenopus cells in culture allows the examining of changes in the synaptic strength in individual neuromuscular synapses , and the assessing of synaptic conductance .\",\n \"In Figure 1E , we observed in non-treated Xenopus synapses that 30 min of chronic burst stimulation of the motor neuron did not drastically change the synaptic gain , but nevertheless induced a slight depression of the averaged ePSCs .\",\n \"Next , we set to closely examine these small synaptic changes in individual synapses .\",\n \"Given the variety of average synaptic conductance among neuromuscular synapses ( Figure 1C , Y axis ) , and its strong correlation with the muscle input conductance ( Figure 1C ) , the range of the ratio between the synaptic and the input conductances 'Gsyn/Gin' is much tighter than the synaptic conductance range .\",\n \"We reasoned that because this ratio , and not the synaptic conductance alone , determines the synaptic efficacy ( ePSP amplitude ) , it should be the appropriate synaptic parameter targeted by the homeostatic process .\",\n \"Therefore , we recorded the ePSCs under brief test periods of postsynaptic voltage-clamp , before and after 20–30 min of chronic burst stimulation of the motor-neuron under postsynaptic current-clamp , and normalized these averaged ePSCs by the input conductance of the muscle cell .\",\n \"Figure 5A ( black dots ) shows that the slight depression was not homogenous among neuromuscular synapses .\",\n \"The degree of depression seems to depend on the distance of the initial Gsyn/Gin ratio from the mean ratio obtained after chronic activity ( Figure 5A , dashed line ) .\",\n \"Consequently , standard deviation from the mean is reduced after chronic activity ( Figure 5A , inset ) .\",\n \"This synaptic depression behavior can be interpreted as the convergence of the Gsyn/Gin ratios towards the set point of the homeostasis . 10 . 7554/eLife . 12190 . 015Figure 5 . Homeostatic control of the synaptic efficacy .\",\n \"( A ) In Xenopus , ratios between averaged synaptic conductance ( 'Gsyn' , calculated from 30–40 ePSCs ) and muscle cell input conductance ( 'Gin' ) before and after 20–30 min of chronic burst stimulation of the motor neuron ( burst of 20–60 events at a 20–30 Hz frequency , every 30–40 s ) under postsynaptic current-clamp in non-treated ( black dots ) and low curare-treated ( red dots ) synapses .\",\n \"Green dots represent the Gsyn/Gin ratio before and after 1–3 bursts of 5 presynaptic stimulations at 30 Hz ( green dots ) in ryanodine loaded muscle cells ( same data than in Figure 3C , ryanodine bar ) .\",\n \"Inset shows the mean ± standard deviation of the Gsyn/Gin ratios in the three conditions .\",\n \"The dotted lines show the averaged Gsyn/Gin ratio after chronic activity in non-treated synapses .\",\n \"( B ) Degree of plasticity ( relative change in Gsyn/Gin ratio ) shown in A expressed as a function of the difference between the initial individual Gsyn/Gin ratio and the averaged ratio after chronic burst activity ( 'Distance to the set point' ) , in non-treated ( black dots ) and curare-treated ( red dots ) synapses .\",\n \"The solid line shows the theoretical homeostatic relationship between plasticity and the distance to a set point of 2 . 36 , calculated as the mean Gsyn/Gin ratio after chronic activity in non-treated synapses .\",\n \"( C ) Apparent averaged Gsyn/Gin ratios calculated in mouse FDB muscles from the data of Figure 3E , in non-stimulated and non-treated synapses ( no burst , black dot ) , in burst-stimulated and non-treated synapses ( after chronic bursts , black dot ) , in non-stimulated and ryanodine-treated synapses ( no burst , green dot ) and in burst-stimulated and ryanodine-treated synapses ( after chronic bursts , green dot ) .\",\n \"Dots represent the mean ± Standard Deviation .\",\n \"( D ) Relative change of contraction force during 2s-30Hz bursts of nerve stimulations in mouse soleus muscles ( n=4 ) , before and during exposure to a low dose ( 0 . 1 µM ) of curare . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 015 In order to test whether synapses also converge if the initial Gsyn/Gin ratio is below the set point , we applied the same chronic activity in the continuous presence of low doses of curare ( 0 . 1–2 µM ) .\",\n \"At these concentrations , the bursts of evoked synaptic events are still composed of a majority of suprathreshold events , combining both potentiating and depressing processes .\",\n \"Figure 5A ( red dots ) shows that , again , the degree of plasticity ( potentiation in this case ) depends on the distance of the initial Gsyn/Gin ratio to the set point .\",\n \"The plasticity orientation rule may then be expressed in a way showing its homeostatic character .\",\n \"We plotted the degrees of plasticity ( relative change in the Gsyn/Gin ratio ) in non-treated ( Figure 5B , black dots ) and curare treated ( Figure 5B , red dots ) synapses shown in Figure 5A , as a function of the difference between the initial Gsyn/Gin ratios and the mean Gsyn/Gin ratio after chronic activity ( distance to the set point ) .\",\n \"In a perfect homeostatic process , with an attractor set point , the degree of plasticity can be expressed as a function of the distance to the set point: ( GsynGin ) after ( GsynGin ) before=1−11+set point ( GsynGin ) before−set point The solid line in Figure 5B shows this theoretical relationship with a set point of 2 . 36 , equal to the mean Gsyn/Gin ratio obtained after chronic activity .\",\n \"The overlap between the data points and the theoretical relationship shows the homeostatic function of the neuromuscular plasticity .\",\n \"In order to place the potentiation we obtained with ryanodine ( Figure 3C , 'ryanodine' bar ) in regard of this homeostatic rule , we normalized the averaged synaptic conductances by the input conductance and added the data to the Figure 5A ( green dots ) .\",\n \"The ryanodine-treated synapses did not converge towards a set point .\",\n \"The averaged Gsyn/Gin ratios after burst activity were above the set point , and the standard deviations from the mean were increased .\",\n \"These data suggest that the ryanodine receptors-dependent calcium signal participates to the stabilization of the synaptic efficacy at the set point .\",\n \"In adult mouse neuromuscular synapses , an apparent Gsyn/Gin ratio can be extrapolated from the ePSPs .\",\n \"If we assume a linear passive leak , the Gsyn/Gin ratio can be expressed as: GsynGin=−80−VpVp with Vp the peak membrane potential reached by the ePSP and 0 and -80 mV the reversal potentials of the synaptic and the leak currents respectively .\",\n \"These ratios are only apparent and underestimated given the effect of distance between the recording site and the neuromuscular junction .\",\n \"Figure 5C shows the apparent Gsyn/Gin ratio in non-stimulated and in burst-stimulated preparations , both for non-treated ( black dots ) and ryanodine-treated ( green dots ) preparations ( same data as in Figure 1D and Figure 3D–E ) .\",\n \"These data in mouse ( Figure 5C ) confirmed the results seen in Xenopus ( Figure 5A ) .\",\n \"Finally , in mouse , we applied a continuous low dose of curare ( 0 . 1 µM ) on soleus muscles , together with chronic burst stimulation of the nerve , in order to show that the homeostatic processes are also able to compensate for a decrease in the postsynaptic sensitivity to the neurotransmitter .\",\n \"At this curare concentration , the synaptic activity is still composed of a majority of suprathreshold events , combining both nicotinic and DICR calcium signals .\",\n \"We took advantage of the fact that the muscle contraction force integrates the synaptic efficacy over all the fibers of the muscle and all the synaptic events of the bursts , and as such is an indicator of even small changes in the synaptic gain .\",\n \"Figure 5D shows the 26% decrease in the contraction force due to low curare , followed by progressive recovery of the force back to a normal level in response to subsequent nerve burst stimulations .\",\n \"We could thus directly observe the functional effect of the synaptic homeostasis mechanism we describe .\"\n ],\n [\n \"When selectively triggered , the nicotinic Ca2+ influx and the DICR signal induce a form of LTP and LTD respectively .\",\n \"The causal links between the nicotinic Ca2+ influx and the synaptic events , and between the DICR signal and the muscle APs , allow the establishment of an orientation rule for neuromuscular synaptic plasticity: synaptic events-induced LTP versus muscle AP-induced LTD .\",\n \"This orientation rule finds its equilibrium in the suprathreshold range of the synaptic strength and provides the specific 'homeostatic' function to this plasticity .\",\n \"This neuromuscular plasticity differs from the other forms of long-term plasticity , and in particular should not be confounded with anti-Hebbian plasticity ( Kullmann and Lamsa , 2008; Roberts and Leen , 2010 ) .\",\n \"Anti-Hebbian plasticity underlies synaptic strength modulations while the present homeostatic mechanism maintains the stability of synaptic efficacy .\",\n \"Modulation of synaptic transmission has been extensively studied in vitro at the Xenopus neuromuscular synapse by Poo group ( Fu and Poo , 1991; Lo and Poo , 1991; 1994; Lohof et al . , 1993; Lo et al . , 1994; Dan et al . , 1995; Cash et al . , 1996; Wang and Poo , 1997; Wan and Poo , 1999 ) .\",\n \"Our work , partly done on the same experimental model , suggests that a number of results obtained by Poo group might be interpreted in the more specific framework of homeostatic plasticity .\",\n \"In vertebrates , neurotrophic factors such as brain-derived neurotrophic factor , neurotrophin 3 and neurotrophin 4 , are trans-synaptic factors considered candidates to mediate presynaptic release modulations in many forms of synaptic plasticity ( Schinder and Poo , 2000; Poo , 2001 ) .\",\n \"Skeletal muscle synthesize and release neurotrophins in an activity-dependent manner ( Funakoshi et al . , 1995; Wang and Poo , 1997; Xie et al . , 1997 ) , and motor nerve terminals contain the receptors tyrosine kinase TrkB and C ( Henderson et al . , 1993; Koliatsos et al . , 1993; Wong et al . , 1993; Yan et al . , 1993 ) .\",\n \"At the Xenopus neuromuscular synapse in vitro , Poo group has shown that the exogenous application ( Lohof et al . , 1993; Wang and Poo , 1997 ) of these factors reproduces the synaptic LTP induced in the present work with subthreshold synaptic events or postsynaptic ryanodine treatment .\",\n \"Finally , upregulation of the ACh evoked release in α-bungarotoxin treated rat was markedly reduced by inhibition of the tyrosin kinase receptors of the neurotrophins ( Plomp and Molenaar , 1996 ) .\",\n \"Therefore , neurotrophic factors should be considered for future investigations to determine whether they can be retrograde factors mediating homeostatic plasticity at the neuromuscular synapse .\",\n \"The diversity of the effects of the calcium signaling comes from the variety of its temporal patterns , amplitude , and location .\",\n \"The two antagonist calcium signals that we demonstrated here to be involved in the orientation of the neuromuscular plasticity exhibit different temporal patterns and amplitudes .\",\n \"The AP-associated DICR signal exhibits fast transient large calcium concentration elevations for each AP .\",\n \"The kinetics of the nicotinic calcium build-up is much slower and does not contain transients during repetitive nicotinic receptor stimulations .\",\n \"Its amplitude under voltage-clamp is low compare to the DICR signal ( Figure 2F ) , and even lower under current-clamp given the reduced driving-force for the calcium ion due to the postsynaptic depolarization .\",\n \"This dichotomy between fast large transient and slow low calcium build-up is known to trigger selectively , via calmodulin , the CAMKII kinase and the calcineurin phosphatase respectively .\",\n \"This mechanism is considered in the central nervous system to implement Hebbian plasticity ( Malenka et al . , 1989; Malinow et al . , 1989; Mulkey et al . , 1993; 1994 ) by modulating the conductance and number of the postsynaptic receptors ( Barria et al . , 1997; Mammen et al . , 1997; Beattie et al . , 2000 ) .\",\n \"The CAMKII signaling pathway was also shown to be required for normal 'presynaptic homeostasis' in Drosophila ( Haghighi et al . , 2003 ) .\",\n \"Furthermore , Wan and Poo 1999 showed at the Xenopus synapse in vitro that induction of LTP and LTD can be blocked by pre-loading the muscle cell with peptide inhibitors of calcineurin and CAMKII respectively .\",\n \"As noted by Wan and Poo 1999 , this situation is opposite to the central synapses , where calcineurin is associated to depression and CAMKII to potentiation .\",\n \"Our results suggest that the low-calcium nicotinic signal and the calcineurin activation have a possible causal link with the detection of the synaptic events and the synaptic potentiation , while the high-calcium DICR signal and the CAMKII activation are associated with the detection of the postsynaptic APs and the synaptic depression .\",\n \"The convergence of the synaptic efficacy towards a set point ( Figure 5 ) suggests that the potentiating and depressing synaptic processes balance each other in the suprathreshold range of the synaptic strength .\",\n \"These observations strongly suggest that the set point of the homeostatic plasticity is sustained by a push-pull mechanism .\",\n \"Our results , however , do not determine the stages where this push-pull mechanism could operate in the causal chain linking the evaluation of synaptic efficacy to the presynaptic modulation .\",\n \"In particular , our findings do not necessarily imply that two independent antagonist trans-synaptic retrograde factors balance their effects at the presynaptic level .\",\n \"The coexistence of potentiating and depressing trans-synaptic factors is a possibility among others .\",\n \"The increased and decreased secretion of a single positive retrograde factor —like for example endostatin in Drosophila ( Wang et al . , 2014 ) and neurotrophins in vertebrates— could also bi-directionally regulate neurotransmitter release .\",\n \"In this case , it could be hypothesized that a push-pull mechanism operates downstream of the calcium signaling , for example at the level of the calcineurin/CAMKII balance , ultimately determining the secretion level of a single factor .\",\n \"Therefore , the use of blue and red arrows in Figure 2 is intended as a schematic representation of the retrograde mechanisms that can bi-directionally change neurotransmitter release , without presuming the existence of two distinct retrograde factors .\",\n \"Most of what we know about synaptic homeostasis stems from experimental studies at the Drosophila larva neuromuscular junction .\",\n \"Given the numerous differences between Drosophila and vertebrates the comparison between these systems is not straightforward .\",\n \"Action potentials and the associated DICR signal in vertebrates being absent in Drosophila muscle cells ( Hong and Ganetzky , 1994 ) , the homeostatic mechanism we propose here cannot be directly transposed to Drosophila .\",\n \"Since no orthologue of the tyrosine kinase receptors are found in Drosophila ( Frank , 2014 ) , the neurotrophin hypothesis in vertebrates cannot either be applied to Drosophila .\",\n \"However , different actors could play similar roles in both systems .\",\n \"The calcium permeability of glutamate receptors in Drosophila could have a similar role than the calcium permeability of nicotinic receptors in vertebrates .\",\n \"The low-voltage activated calcium channels activated by the ePSPs and the 'calcium-induced calcium release' signal responsible for the excitation-contraction coupling in Drosophila ( Peron et al . , 2009 ) could be the orthologue of the DICR signal in vertebrates .\",\n \"Finally , endostatin in Drosophila ( Wang et al . , 2014 ) may play the role of neurotrophins in vertebrates .\",\n \"In Drosophila , Frank et al . ( 2006 ) found that spontaneous glutamate release was sufficient to induce a rapid potentiation in the absence of nerve activity , while we had to use a 30Hz motor command to induce rapid potentiation in vertebrates .\",\n \"In order to establish the Figure 5A , cells were incubated in curare in absence of neurons spikes during 30–60 min before recording ( red dots ) .\",\n \"Therefore , the spontaneous ACh release did not restore the normal Gsyn/Gin ratio ( found in non-treated synapses ) before chronic bursts were applied .\",\n \"A possible explanation of this difference between the two systems is the high frequency of miniatures in Drosophila ( 10–20 Hz ) ( Frank et al . , 2006 ) , while the average frequency of spontaneous release in the Xenopus cells culture was 100 fold lower .\",\n \"In vertebrates , evoked activity responsible for muscular tonus and voluntary motions represents most of the synaptic activity , and the 20–30 Hz frequencies we used in our experiments are in the normal range for a motor command ( Gorassini et al . , 2000 ) .\",\n \"Therefore , the evoked activity in vertebrate is more likely able to rapidly mobilize the homeostatic machinery than the spontaneous miniatures .\",\n \"In the central nervous system , homeostatic forms of synaptic plasticity have been proposed to restrain the mean level of neuronal activity within a physiological regime , and to maintain the stability of recurrent network activity challenged by associative plasticity .\",\n \"Because of the robustness of its synaptic transmission , the neuromuscular junction is considered as a model of homeostasis .\",\n \"However , the neuromuscular junction being the end effector of the motor network , its main function is the faithful translation of the motor command into muscle activity .\",\n \"The mean level of muscle activity is not involved in any recurrent network that requires stability , and therefore does not need to be controlled .\",\n \"While the synaptic strength is the adjustment variable for the control of the mean level of neuronal activity in the CNS , at the NMJ it is the efficacy of synaptic transmission itself that must be maintained constant for reliable relay of the motor command .\",\n \"Therefore , homeostatic plasticity could be regarded as a functionally different phenomenon at the NMJ than in the CNS .\",\n \"Nevertheless , the plasticity orientation rule described in the present work matches a synaptic 'relay' function .\",\n \"Despite differences in the nature of the activity sensors , other 'relay' synapses in the CNS , such as those involved in sensory inputs to the thalamus ( Guido , 2008 ) , might share with the NMJ a comparable homeostatic control based on pre- and post- synaptic activity detection .\"\n ],\n [\n \"Myotomal and spinal tissues from 1-day-old Xenopus laevis embryos ( stages 23 to 25 ) were mechanically dissociated using a Ca2+- and Mg2+-free medium of the following composition ( in mM ) : 115 NaCl , 2 . 6 KCl , 0 . 4 EDTA , 10 HEPES ( pH = 7 . 6 ) .\",\n \"Cells were directly plated in a plastic recording chamber , and grown at 19°C for 12 hr prior to the experiments .\",\n \"The culture medium consisted of 50% Leibovitz L-15 medium ( Gibco , Invitrogen Corp . , Cergy-Pontoise , France ) , 1% fetal calf serum , 1% antibiotic mixture ( ibid . , final concentration: 100 units/mL penicillin G and 100 µg/mL streptomycin ) , and 48% physiological solution of the following composition ( in mM ) : 113 NaCl , 2 KCl , 0 . 7 CaCl2 , 5 HEPES ( pH=7 . 8 ) .\",\n \"Perforated patch-clamp recordings , to preserve the cell integrity , were performed at room temperature ( 20–22°C ) .\",\n \"Pipettes were made from borosilicate glass ( Clark Electromedical Instruments , Reading , England ) and pulled on a P-1000 puller ( Sutter Instrument Company , Novato , CA , U . S . A . ) .\",\n \"Patch electrodes had a resistance of 2–3 MΩ when filled with internal physiological solution .\",\n \"Membrane currents and potential were recorded using Axopatch200B patch-clamp amplifiers ( Axon Instruments , Union City , CA ) .\",\n \"Access resistances were compensated at 80% .\",\n \"Myocyte membrane currents were filtered with an integrated low-pass Bessel filter at 2 kHz .\",\n \"The filtered signals were digitized by a 12 bit A/D converter ( Digidata 1200B , ibid . ) and stored using pCLAMP 8 software ( ibid . ) .\",\n \"Recordings were analyzed using the Origin 7 software ( OriginLab Corp . , Northampton , MA ) .\",\n \"Motor neurons were current-clamped through an amphotericin-perforated membrane patch ( 400 pA , 3 ms current step to induce the presynaptic AP ) ; the intra-pipette solution had the following composition ( in mM ) : 1 NaCl , 140 K-gluconate , 1 MgCl2 , 10 HEPES ( pH=7 . 2 ) , and amphotericin-B at 300 µg/ml .\",\n \"Myocytes were either voltage- or current-clamped , through an amphotericin-perforated membrane patch , using the following intra-pipette composition ( in mM ) : 1 NaCl , 20 KCl , 125 K-gluconate , 1 MgCl2 , 10 HEPES ( pH=7 . 2 ) , and amphotericin-B at 300 µg/ml .\",\n \"The external solution had the following composition ( in mM ) : 140 NaCl , 3 KCl , 1 MgCl2 , 2 CaCl2 , 10 HEPES ( pH=7 . 4 ) .\",\n \"Pipettes made with borosilicate glass had a resistance of 80–120 MOhm when filled with 0 . 5 to 1 M ACh-Cl .\",\n \"ACh+ efflux was induced by positive current steps , using a home-made constant current generator .\",\n \"Xenopus muscle cells were loaded with the calcium indicator in whole-cell patch-clamp configuration for 10 min with an intra-pipette medium of the following composition ( in mM ) : 110 KCl , 1 NaCl , 2 Mg2+ , 10 HEPES ( pH 7 . 2 ) , and 0 . 2 Fluo4-pentapotassium salt .\",\n \"The patch pipette was then removed and a perforated patch was performed as described above .\",\n \"Fluorescence intensity was quantified with an Olympus photomultiplier ( forming part of the OSP system , Olympus , Japan ) , and the tension signal was digitized with the Digidata converter .\",\n \"After background fluorescence subtraction , signals were normalized according to the baseline fluorescence .\",\n \"The reversal potential of the nicotinic current induced by ionophoretic acetylcholine application was measured under voltage-clamp in solutions of various ionic compositions ( Figure 2—figure supplement 1 ) .\",\n \"The following equation , derived from the Goldman-Hodgkin-Katz flux equation ( Goldman , 1943; Hodgkin and Katz , 1949 ) , was used to calculate the permeability ratios:Vrev=RTF[0 . 75×PK[K]o+0 . 75×PNa[Na]o+0 . 25×4PCa[Ca]o+0 . 25×4PMg[Mg]o0 . 75×PK[K]i+0 . 75×PNa×[Na]i+0 . 25×Mg[Mg]i] In this equation , the terms in square brackets are ion concentrations , PS is the permeability of the S ion species , T is the absolute temperature , R and F are the gas and Faraday constants , and i and o are the intra- and extracellular compartments , respectively .\",\n \"Factors represent the ionic activity coefficients .\",\n \"The dynamic-clamp technique ( Sharp et al . , 1993; Robinson and Kawai , 1993 ) was used to inject computer-generated conductances in Xenopus muscle cells .\",\n \"Dynamic-clamp experiments were run using the hybrid RT-NEURON environment ( Le Franc et al . , 2001; Sadoc et al . , 2009 ) , a modified version of NEURON ( Hines and Carnevale , 1997 ) running under the Windows operating system ( Microsoft Corp . , Redmond , Washington ) , augmented with the capacity of simulating models in real time , synchronized with the intracellular recording .\",\n \"A PCI DSP board with 16 bit A/D-D/A converters ( Innovative Integration , SimiValley ) was used to input the membrane potential into the equations of the model , and to output the current to be injected into the cell with a time resolution of 0 . 1 ms . Passive K+ leak and K+ inward rectifying ( Kir ) conductances were injected into the muscle cell to simulate an increase in the input conductance ( Figure 3—figure supplement 1 ) .\",\n \"The passive K+ leak was expressed in the form: Ileak = gleak x ( V-Eleak ) .\",\n \"Eleak was set to -80 mV , and gleak to 5–15 nS .\",\n \"The Kir conductance was expressed in the form: IKir =gmax x m x ( V – EK ) where the maximal conductance gmax was set to 20–50 nS , and changes with time of the gating variable m were calculated by solving the differential equation:dmdt=m∞−mτmm ( t ) =m∞− ( m∞−m0 ) ×⟮−tτm⟯ where fitting voltage-clamp recordings of the real isolated Kir current of Xenopus muscle cells gave and m∞ ( v ) =11+exp ( −0 . 074× ( EK−V ) ) andτm=0 . 2ms The 'contraction' trace shown in Figure 2A represents a qualitative measurement of the contraction of a muscle cell in culture .\",\n \"In addition to the membrane potential recording pipette , a second patch pipette vertically approached the membrane cell until a slight increase in the pipette electrical resistance became visible .\",\n \"The pipette was then held in that position , and increase in the cell thickness accompanying contraction was monitored through further variations of the pipette resistance .\",\n \"3- to 5-month-old Swiss mice were anesthetized with isoflurane , and cervical dislocated .\",\n \"Dissections were performed within 15 min in an oxygenated Ringer solution of the following composition ( in mM ) : 145 NaCl , 3 KCl , 2 CaCl2 , 1 MgCl2 , 10 HEPES ( pH 7 . 4 ) and 11 glucose .\",\n \"Intracellular recordings ( Figure 1—figure supplement 1 ) were performed at 34°C , in the oxygenated Ringer solution defined above .\",\n \"Sharp pipettes were made from borosilicate glass ( Clark Electromedical Instruments ) , pulled on a P-1000 puller ( Sutter Instrument Company ) , and had a resistance of 40–60 MΩ when filled with a KCl 3 M solution .\",\n \"The filled pipette tip was cut at the limit of the pulled zone , and used as electrode .\",\n \"The chlorided end of a 10 cm long , 50 µm diameter , silver wire was introduced inside this electrode , and plugged by its opposite end to the headstage of an Axoclamp 2A amplifier ( Axon Instruments ) , where it hung loosely above the muscle .\",\n \"A drop of mineral oil was added at the back of the intra-pipette medium to avoid evaporation .\",\n \"The pipette pendulum was vertically manipulated to enter the muscle cells , and its flexibility allowed stable membrane potential recordings in contracting muscles .\",\n \"Nerve APs were induced with 6 V , 30 µs voltage steps in a suction pipette .\",\n \"2 µM of µ-conotoxin GIIIB ( 10 min ) was used to isolate the ePSPs .\",\n \"Data acquisition and analysis were made as above .\",\n \"Under µ-conotoxin the amplitudes of the recorded ePSPs depend on the distance between the recording site and the synaptic region .\",\n \"With the floating electrode method , the placement of the electrode is not precisely controlled .\",\n \"We limited the distance effect by using the FDB mouse muscle , where cells are shorter ( 300 µm ) than the muscle length ( 1 cm ) .\",\n \"Therefore , blind placement of the electrode can be performed on this muscle , with a maximal 150 µm distance between the recording site and the end plate .\",\n \"The specific cells size and organization in FDB limit the distance effect in our recordings , but this effect is presumably responsible for an underestimation of the ePSPs amplitudes and for most of the variability between the recorded cells .\",\n \"Despite the underestimation of the ePSPs amplitudes , the recorded amplitudes were above the -63 mV AP threshold determined in muscle cells of rat fast and slow muscles ( Wood and Slater , 1995 ) .\",\n \"The salts composing the media used for electrophysiological recordings , EGTA used to obtain Ca2+-free media , and Amphotericin B used for perforated patch-clamp were obtained from Sigma-Aldrich ( Sigma-Aldrich , Saint Quentin Fallavier , France ) .\",\n \"Ryanodine used in some experiments was 'Ryanodine fractions' from Latoxan ( Latoxan , Valence , France ) .\",\n \"The µ-conotoxin GIIIB used to block mouse muscle action-potentials was obtained from Alomone Labs ( Alomone Labs , Jerusalem , Israel ) .\",\n \"The Ca2+ dye 'Fluo4-pentapotassium salt' was obtained from Molecular Probes ( Molecular Probes , Eugene , Oregon ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Excitability differs among muscle fibers and undergoes continuous changes during development and growth , yet the neuromuscular synapse maintains a remarkable fidelity of execution .","Here we show in two evolutionarily distant vertebrates ( Xenopus laevis cell culture and mouse nerve-muscle ex-vivo ) that the skeletal muscle cell constantly senses , through two identified calcium signals , synaptic events and their efficacy in eliciting spikes .","These sensors trigger retrograde signal ( s ) that control presynaptic neurotransmitter release , resulting in synaptic potentiation or depression .","In the absence of spikes , synaptic events trigger potentiation .","Once the synapse is sufficiently strong to initiate spiking , the occurrence of these spikes activates a negative retrograde feedback .","These opposing signals dynamically balance the synapse in order to continuously adjust neurotransmitter release to a level matching current muscle cell excitability ."],"string":"[\n \"Excitability differs among muscle fibers and undergoes continuous changes during development and growth , yet the neuromuscular synapse maintains a remarkable fidelity of execution .\",\n \"Here we show in two evolutionarily distant vertebrates ( Xenopus laevis cell culture and mouse nerve-muscle ex-vivo ) that the skeletal muscle cell constantly senses , through two identified calcium signals , synaptic events and their efficacy in eliciting spikes .\",\n \"These sensors trigger retrograde signal ( s ) that control presynaptic neurotransmitter release , resulting in synaptic potentiation or depression .\",\n \"In the absence of spikes , synaptic events trigger potentiation .\",\n \"Once the synapse is sufficiently strong to initiate spiking , the occurrence of these spikes activates a negative retrograde feedback .\",\n \"These opposing signals dynamically balance the synapse in order to continuously adjust neurotransmitter release to a level matching current muscle cell excitability .\"\n]"},"summary":{"kind":"list like","value":["Nerve cells communicate with each other , and with targets such as muscle cells , at junctions called synapses .","The nerve cell before the synapses releases a chemical called a neurotransmitter , which binds to receptors on the cell after the synapses .","However , the first cell cannot determine by itself whether it is releasing the correct amount of neurotransmitter to activate its partner .","For this , it requires feedback from the second cell .","This feedback is particularly important at synapses between nerve cells and muscle cells , which are known as neuromuscular junctions .","The likelihood that a given amount of transmitter will activate a muscle cell can vary with age and after exercise .","Muscle cells must therefore be able to instruct their nerve cell partners to increase or decrease neurotransmitter release to accommodate these changes .","Ouanounou et al . have now identified the mechanism by which muscle cells determine whether nerve cells are releasing an appropriate amount of neurotransmitter .","Experiments in two distantly related animals – mice and embryos from a frog called Xenopus – revealed that muscle cells use two calcium-based signals .","The first is the flow of calcium ions into the muscle cell in response to binding of neurotransmitter to receptors at the synapses: this tells the muscle cell how active the nerve cell is .","The second is the release of calcium ions from internal stores inside the muscle cell: this occurs whenever neurotransmitter release is sufficient to activate the muscle cell .","In response to the first calcium signal , the muscle cell sends positive feedback to the neuron , telling it to increase neurotransmitter release further .","In response to the second signal , the muscle cell sends negative feedback to reduce neurotransmitter release .","Thus , when neurotransmitter release is not enough to activate the muscle , positive feedback dominates and neurotransmitter release increases .","However , when the muscle is activated , the two types of feedback act in balance to maintain efficient communication across the synapse .","The next steps are to identify the cell signaling cascades that are mobilized by the two calcium signals , including the specific molecule ( or molecules ) that regulate neurotransmitter release ."],"string":"[\n \"Nerve cells communicate with each other , and with targets such as muscle cells , at junctions called synapses .\",\n \"The nerve cell before the synapses releases a chemical called a neurotransmitter , which binds to receptors on the cell after the synapses .\",\n \"However , the first cell cannot determine by itself whether it is releasing the correct amount of neurotransmitter to activate its partner .\",\n \"For this , it requires feedback from the second cell .\",\n \"This feedback is particularly important at synapses between nerve cells and muscle cells , which are known as neuromuscular junctions .\",\n \"The likelihood that a given amount of transmitter will activate a muscle cell can vary with age and after exercise .\",\n \"Muscle cells must therefore be able to instruct their nerve cell partners to increase or decrease neurotransmitter release to accommodate these changes .\",\n \"Ouanounou et al . have now identified the mechanism by which muscle cells determine whether nerve cells are releasing an appropriate amount of neurotransmitter .\",\n \"Experiments in two distantly related animals – mice and embryos from a frog called Xenopus – revealed that muscle cells use two calcium-based signals .\",\n \"The first is the flow of calcium ions into the muscle cell in response to binding of neurotransmitter to receptors at the synapses: this tells the muscle cell how active the nerve cell is .\",\n \"The second is the release of calcium ions from internal stores inside the muscle cell: this occurs whenever neurotransmitter release is sufficient to activate the muscle cell .\",\n \"In response to the first calcium signal , the muscle cell sends positive feedback to the neuron , telling it to increase neurotransmitter release further .\",\n \"In response to the second signal , the muscle cell sends negative feedback to reduce neurotransmitter release .\",\n \"Thus , when neurotransmitter release is not enough to activate the muscle , positive feedback dominates and neurotransmitter release increases .\",\n \"However , when the muscle is activated , the two types of feedback act in balance to maintain efficient communication across the synapse .\",\n \"The next steps are to identify the cell signaling cascades that are mobilized by the two calcium signals , including the specific molecule ( or molecules ) that regulate neurotransmitter release .\"\n]"},"year":{"kind":"string","value":"2016"}}},{"rowIdx":4192,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["developmental biology","neuroscience"],"string":"[\n \"developmental biology\",\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"Electrical synaptic transmission requires a postsynaptic scaffolding protein"},"id":{"kind":"string","value":"elife-66898-v1"},"sections":{"kind":"list like","value":[["Synapses are specialized cellular adhesions between neurons that rapidly transfer information to facilitate neural network function .","There are two modalities of fast synaptic transmission , chemical and electrical , both found throughout animal nervous systems including in mammals ( Moroz and Kohn , 2016; Ryan and Grant , 2009 ) .","Chemical synapses are inherently asymmetric structures , derived from presynaptic specializations that regulate the release of neurotransmitters and postsynaptic specializations that contain neurotransmitter receptors and the machinery required to propagate signal transmission .","Both specializations require hundreds to thousands of proteins , which together tightly control the structure , function , and modulation of synaptic communication ( Ackermann et al . , 2015; Grant , 2019; Siddiqui and Craig , 2011 ) .","For example , intracellular scaffolding proteins of the Post Synaptic Density ( PSD ) at chemical synapses regulate the number and functional state of AMPA and NMDA receptors , which are ligand-gated ion channels , at glutamatergic synapses ( Zhu et al . , 2016 ) .","By contrast , electrical synapses are often perceived as simple aggregates of intercellular channels known as gap junctions ( GJs ) ( Goodenough and Paul , 2009 ) .","Intercellular GJ channels are formed by the docking of two hemichannels , composed of Connexin proteins in vertebrates and Innexins in invertebrates ( Bhattacharya et al . , 2019; Phelan , 2005; Shruti et al . , 2014; Söhl et al . , 2005 ) .","Each neuron contributes a hemichannel from each side of the synapse , which form a channel and support communication by allowing the spread of electrical currents and small metabolites between adjacent ‘coupled’ neurons .","While multiple Connexins and Innexins can contribute to individual electrical synapses ( Bhattacharya et al . , 2019; Miller et al . , 2017; Phelan et al . , 2008; Rash et al . , 2013 ) , the complexity of neuronal GJ cellular biology ( Lynn et al . , 2012; Martin et al . , 2020; Meyer et al . , 2014; Sigulinsky et al . , 2020 ) and the variety of mechanisms regulating their synaptic strength ( Arroyo et al . , 2016; Bloomfield and Völgyi , 2009; Marder , 1998; O'Brien and Bloomfield , 2018; Pereda , 2014 ) suggest they require complex multimolecular structures to support function .","Several Connexin-associated proteins have been identified ( Lynn et al . , 2012; Miller and Pereda , 2017 ) ; however , it remains undetermined whether such associated proteins are ancillary to the channels or requisite for electrical synapse function .","Perhaps the best characterized Connexin-associated protein is Zonula Occludens 1 ( ZO1 ) ( Bauer et al . , 2010; Willott et al . , 1993 ) , which is an intracellular scaffolding protein and a member of the membrane-associated guanylate kinase ( MAGUK ) family of proteins .","MAGUKs constitute a large family of multifunctional adaptor proteins that play key roles in scaffolding membrane channels and receptors to intracellular signaling complexes and the cytoskeleton ( González-Mariscal et al . , 2000 ) .","MAGUK proteins , including ZO1 , contain PSD95/Dlg/ZO1 ( PDZ ) protein-protein interaction domains , which bind to PDZ-binding motifs often located at the carboxy terminus of partner proteins , including Connexins ( Zhu et al . , 2016 ) .","The best studied ZO1/Connexin interaction is with Connexin 43 ( Cx43 ) , a widely expressed , non-neuronal , GJ-channel forming protein ( Giepmans and Moolenaar , 1998 ) .","The ZO1/Cx43 interaction is thought to play important functional roles in GJ regulation by facilitating the docking of newly inserted hemichannels , which promotes the formation of intercellular channels ( Hunter et al . , 2005 ) .","Moreover , the ZO1/Cx43 interaction is critical for channels to remain conductive prior to removal during channel turnover at epithelial GJs ( Hervé et al . , 2014; Thévenin et al . , 2017 ) .","While ZO1 is an important regulator of Cx43-contaning GJs , less is known about its role at neuronal GJs , which are primarily formed by the Cx36-family of proteins and mediate electrical synaptic transmission in vertebrate nervous systems ( Connors and Long , 2004; Miller et al . , 2017; Rash et al . , 2013; Söhl and Willecke , 2004 ) .","In neurons , ZO1 immunostaining correlates with synapses containing Cx36 and its fish homologs ( Flores et al . , 2008; Li et al . , 2004; Marsh et al . , 2017; Yao et al . , 2014 ) , and its presence at synapses may play regulatory roles ( Flores et al . , 2008 ) , the nature of its contributions to electrical transmission remains unknown .","Despite mounting evidence for the widespread dynamic functional contributions of electrical synapses to neural circuit function , the perception of the simplicity of their molecular organization remains .","We hypothesized that scaffolding molecules form part of a multimolecular structure that is required for channel function akin to that found at chemical synapses .","Here , we explore the functional role of ZO1 in zebrafish by examining identifiable synaptic contacts of the Mauthner cell ( Bartelmez , 1915; Bodian , 1937; Hildebrand et al . , 2017; Kimmel , 1982; Robertson et al . , 1963 ) , which forms stereotyped electrical synapses accessible to genetic , biochemical , cell biological , electrophysiological , and behavioral analyses .","We show that the presence ZO1 protein is critically required for the structure and function of the intercellular channels .","Moreover , we find that the localization of ZO1 is compartmentalized postsynaptically where it functions in the formation of neuronal GJs .","Our results stand in contrast with current views on electrical synapse organization centered solely on the proteins forming GJ channels .","Thus , our findings provide strong support to the notion that electrical synapses constitute complex and asymmetric synaptic structures at which intercellular channels are governed by multimolecular structures with features that parallel the molecular and functional organization of the PSD at chemical synapses ."],["We sought to examine the relationship between the intracellular scaffold ZO1 and neuronal Connexins ( Cxs ) by utilizing the stereotyped synapses of the zebrafish Mauthner cell .","This circuit drives a fast escape response to threatening stimuli using both electrical and chemical connections ( Eaton et al . , 1977; Jacoby and Kimmel , 1982; Liu and Fetcho , 1999; Wolman et al . , 2015 ) .","Each animal has two Mauthner cells that receive multimodal sensory input that relay information to the spinal cord to coordinate circuits to elicit fast turns .","We focus on two populations of stereotyped electrical synapses made by Mauthner cells: ( 1 ) ‘club ending’ ( CE ) synapses ( Bartelmez and Hoerr , 1933; Pereda et al . , 2004; Yao et al . , 2014 ) , which are mixed electrical/glutamatergic chemical synaptic contacts formed between auditory afferents of the eighth cranial nerve and the Mauthner cell's lateral dendrite ( Figure 1A , B ) and ( 2 ) en passant electrical synapses formed between the Mauthner axon and Commissural Local ( CoLo ) interneurons found in each spinal-cord segment ( Figure 1A , M/CoLo synapses ) ( Satou et al . , 2009 ) .","Neuronal GJs at both CEs and M/CoLo synapses are made of heterotypic channels formed by Cx35 . 5 , encoded by the gene gap junction delta 2a ( gjd2a ) , and Cx34 . 1 , encoded by gjd1a , both homologous to mammalian Cx36 ( gjd2 ) .","We previously found that Cx35 . 5 and Cx34 . 1 are localized asymmetrically to the pre- and postsynaptic sides at CE and M/CoLo synaptic contacts ( Figure 1B; Miller et al . , 2017 ) .","Throughout we use the terms pre- and postsynaptic to reference the neuronal , cell-biological compartment in which a Connexin is localized .","At CE synapses , the auditory afferent axons are presynaptic to the postsynaptic Mauthner lateral dendrite; while at M/CoLo synapses , the Mauthner axon is presynaptic to the postsynaptic CoLo .","We first determined the localization of ZO1 and the Connexin proteins at Mauthner electrical synapses using immunofluorescence and confocal imaging .","We stained 5 day post fertilization ( dpf ) larvae , a time at which the Mauthner circuit elicits a mature startle response , with antibodies against the human ZO1 protein and those that distinguish the zebrafish Cx35 . 5 and Cx34 . 1 ( Figure 1C–L; Miller et al . , 2017 ) .","We observed extensive colocalized signal for these three proteins at CE and M/Colo electrical synapses , with each protein apparent in the stereotyped shape and position of the neuronal gap junctions ( GJs ) at these contacts .","We identified CEs unambiguously as large ( 1 . 5–2 µm ) oval areas localized in the distal portion of the lateral dendrite of the Mauthner cell ( Figure 1C; Yao et al . , 2014 ) .","M/Colo synapses were identified by their regularly spaced sites of contact in the spinal cord ( Figure 1K ) .","Next , we examined the role of ZO1 at electrical synapses using CRISPR/Cas9-induced mutations to knock out gene function .","Mammalian ZO1 is encoded by the gene tight junction protein 1 ( tjp1 ) , while zebrafish have two homologous genes , tjp1a and tjp1b .","Using a CRISPR-based screen , we found that mutations in tjp1b/ZO1b , but not tjp1a/ZO1a , caused a failure of Connexin localization at M/CoLo synapses ( Figure 1; Figure 1—figure supplement 1; Marsh et al . , 2017; Shah et al . , 2015 ) .","We examined the effect of these mutations on CEs and found that tjp1b/ZO1b-/- mutants lack most of the detectable fluorescent staining for both Cx35 . 5 and Cx34 . 1 , as well as ZO1 , at the stereotyped synaptic contact sites ( Figure 1D , L ) .","Quantitation of Cx35 . 5 , Cx34 . 1 , and ZO1 fluorescence at CEs and M/CoLo contacts confirmed that staining for all three antibodies was greatly diminished in tjp1b/ZO1b-/- mutants ( Figure 1M , N ) .","By contrast , homozygous tjp1a/ZO1a -/- mutants had extensive Connexin and ZO1 staining at these contacts , while tjp1a-/-; tjp1b-/- double mutants were indistinguishable from tjp1b-/- single mutants ( Figure 1—figure supplement 1A–G ) .","We conclude that ZO1b protein , encoded by the tjp1b gene , is localized to electrical synapses and required for the robust localization of both Cx35 . 5 and Cx34 . 1 at synaptic contacts .","While tjp1b/ZO1b-/- mutants showed significantly diminished levels of ZO1 and Connexin staining at presumptive synaptic locations , we wondered whether neurons were still attempting to assemble GJs .","Indeed , we detected both Cx35 . 5 and Cx34 . 1 by western blot from brain homogenates of tjp1b/ZO1b-/- mutant animals ( Figure 1—figure supplement 1H ) .","We therefore examined CEs using higher contrast and magnification to assess GJ structure as detectable by immunolabeling and individually stained for ZO1 or Connexin to avoid confounding the image analysis due to bleed through of signal amongst stained proteins .","We found that in tjp1b/ZO1b-/- mutants , each of the three antibodies revealed structures located at the stereotyped position of CE contacts and had morphologies reminiscent of wild-type animals , albeit with much dimmer fluorescence intensity ( Figure 1E–J; note that image contrast was increased in mutants ( H-J ) ) .","While we observed the stereotypical oval-shaped CE structures in mutants , the staining for each protein was weak and irregular in its distribution , suggesting the residual staining in mutants might represent incomplete , abortive synaptic structures ( see electrophysiology below ) .","Consistent with a reduced presence of GJ proteins , we also observed a reduced number of CEs detected by immunolabeling in tjp1b/ZO1b-/- mutants ( Figure 1—figure supplement 1I ) .","We found similar results at M/CoLo synapses , although their smaller size precluded an analogous detailed analysis ( Figure 1K , L , N ) .","In contrast to tjp1b/ZO1b-/- , the staining of tjp1a/ZO1a-/- mutants was indistinguishable from wildtype ( Figure 1—figure supplement 1E–G , I ) .","These observations suggest that neurons of the Mauthner cell network in tjp1b/ZO1b-/- mutants persist in attempting to create electrical synapses , despite their inability to robustly localize neuronal Connexins at synaptic contacts .","We conclude that ZO1 is localized to electrical synapses where it plays a critical role in neuronal GJ formation .","Given that Connexin localization was dependent on ZO1b , we sought to determine if the converse was true – did ZO1 localization require Connexins ?","Using previously generated mutations in gjd2a/Cx35 . 5 and gjd1a/Cx34 . 1 ( Miller et al . , 2017 ) , we examined the localization of Connexin and ZO1 proteins in mutants by immunolabeling ( Figure 2A–L ) .","First , we found that gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant animals revealed a complete loss of detectable staining for the mutated protein .","In addition , there was a failure of the non-mutated Connexin protein to robustly localize to the electrical synapse although low levels of staining were present .","In line with these observations , by using brain homogenates and western blots , we found a complete loss of the Connexin affected by each mutation , but no effect on the non-mutated Connexin protein ( Figure 1—figure supplement 1H ) .","By contrast , ZO1 staining in the Connexin mutants was robust at the synaptic contact sites with the stereotyped appearance , distribution , and position clearly evident ( Figure 2A–L ) .","By comparing the relative ZO1 fluorescence between wild-type and Connexin mutant animals , we found that ZO1 was present at synaptic contacts at approximately half the normal level ( Figure 2M , N ) .","We examined gjd2a-/-; gjd1a-/- double mutants and found that ZO1 still robustly localized to CE and M/CoLo contact sites ( Figure 2M , N; Figure 2—figure supplement 1A–F ) .","These results reveal two critical organizational principles about electrical synapses in the Mauthner cell: ( 1 ) ZO1b localizes to putative electrical synaptic sites largely independent of Connexin proteins and ( 2 ) each Connexin requires the other for robust localization to the synapse .","Based on these data , we conclude that ZO1 can localize to neuronal GJs independent of the presence of channel-forming proteins , yet ZO1 is absolutely essential for proper Connexin localization .","To further examine the electrical synapse structure of Connexin mutants , we assessed CEs at higher contrast ( Figure 2D–I ) .","We found that: ( 1 ) immunofluorescence for the mutated Connexin is completely lost at the synapse , ( 2 ) the non-mutated Connexin is detectable but with weak and irregular labeling , suggestive of incomplete , abortive structures , and ( 3 ) ZO1 labeling resembles wildtype with a distribution and morphology that appears normal across the expanse of the putative synaptic contact .","Consistent with these findings , the number of CEs detected by ZO1 labeling in Connexin mutants was indistinguishable from that observed in wildtype , while those detected by antibodies for the mutated Connexin were significantly reduced ( Figure 1—figure supplement 1I ) .","In addition , the zebrafish genome contains two additional homologous Connexin genes , gjd2b/Cx35 . 1 and gjd1b/Cx34 . 7 .","We found that animals that were homozygous mutant for these two genes had normal Connexin and ZO1 labeling at CEs ( Figure 2—figure supplement 1G–L ) .","Similarly , we previously found there was no effect on M/CoLo synapses in the gjd2b/Cx35 . 1 and gjd1b/Cx34 . 7 mutants ( Miller et al . , 2017 ) .","Together , these results support a hierarchical relationship in the formation of neuronal GJs , in which ZO1 localizes to electrical synaptic contact sites where it is essential to robustly localize neuronal Connexins .","We next sought to examine the functional consequences of ZO1 and Connexin mutants , so we explored the properties of synaptic transmission at CEs during whole-cell recordings of the Mauthner cell .","In wildtype zebrafish , extracellular stimulation of CE afferents near the posterior macula where they contact hair cells ( Figure 3A ) evoked a mixed synaptic response in the Mauthner cell composed of an early and large GJ-mediated electrical component followed by a delayed and smaller glutamatergic chemical response ( Figure 3B; Yao et al . , 2014 ) .","We first aimed to establish the functional consequences of removing the Connexins on this mixed synaptic response .","Consistent with the presence of Cx35 . 5 and Cx34 . 1 at CEs , synaptic responses in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant zebrafish lacked a detectable electrical component , while exhibiting a response with the same delay as the chemical component of the mixed synaptic potential of wildtypes ( Figure 3C–E ) .","By contrast , electrical transmission was unaffected in gjd2b/Cx35 . 1-/- and gjd1b/Cx34 . 7-/- mutant zebrafish ( Figure 3E; Figure 3—figure supplement 1A-B ) , as expected given our immunolabeling results ( Figure 2—figure supplement 1G-L ) .","The apparent chemical response in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants was blocked by extracellular application of a combination of the AMPA and NMDA glutamate receptor ( GluR ) antagonists cyanquixaline ( CNQX ) and D-2-Amino-5-phosphonovaleric acid ( DAP5 ) ( gray traces in Figure 3C , D; Figure 3—figure supplement 1C ) .","No change in membrane potential was observed after application of the blockers .","To confirm that the chemical synaptic potential observed in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants arises from the stimulation of CEs lacking electrical transmission , we examined if blocking GJs with meclofenamic acid ( MA ) could reproduce the observed synaptic phenotype .","We found that application of MA to wildtype recapitulated the gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant phenotype , as the characteristic mixed synaptic response was replaced by a larger chemical synaptic response that was sensitive to GluR antagonists ( Figure 3F; Figure 3—figure supplement 1C ) .","We also observed a small hyperpolarization of the Mauthner cell ( from −80 . 2 ± 0 . 7 mV in control to −84 ± 1 mV in MA; p=0 . 04 , n = 5 ) , likely resulting from the action of MA on other membrane channels .","We conclude that together Cx35 . 5 and Cx34 . 1 generate functional neuronal GJs at CEs .","We next examined the properties of synaptic transmission in ZO1 mutants .","Strikingly , and consistent with the requirement for Connexin localization at contact sites ( Figure 1 ) , tjp1b/ZO1b-/- mutants exhibited a failure in electrical transmission .","The synaptic phenotype was indistinguishable from gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants , and similarly consisted of a single , delayed response that was blocked by GluRs antagonists ( Figure 3G; Figure 3—figure supplement 1D ) .","This functional deficit was specific for ZO1b , as tjp1a/ZO1a-/- mutant fish exhibited normal mixed synaptic responses ( Figure 3H , I ) .","These findings indicate the specificity of the mutants to electrical synapses as glutamatergic transmission at CEs remained intact .","Accordingly , neurotransmitter release properties ( Figure 3—figure supplement 1E ) and the localization of GluR2/3 receptors at these terminals were not affected in mutant fish ( Figure 3—figure supplement 2 ) .","We conclude that the presence of the ZO1b scaffold protein is critical for electrical transmission at CEs , even though its absence does not prevent the formation of glutamatergic synapses coexisting within the same contact .","To investigate the extent of the deficit on electrical transmission within the brainstem network , we investigated whether Connexin and ZO1 mutations affected other synaptic contacts onto the Mauthner cell .","The Mauthner cell receives mixed synaptic inputs from a variety of descending and ascending sensory modalities , including visual information from the optic tectum and somatic information from the spinal cord ( Dunn et al . , 2016; Faber and Pereda , 2011; Kimmel et al . , 1981; Korn and Faber , 2005 ) .","For this purpose , we recorded spontaneous synaptic responses that represent the activity of most , if not all , synaptic inputs received by this cell .","Electrically and chemically mediated spontaneous responses are easily differentiated due to their dramatically different duration ( Figure 4A ) .","Thus , for automated detection purposes , we defined fast spontaneous events ( <1 . 1 ms ) , which represent the electrical coupling of presynaptic spikes , as electrical responses and slow spontaneous events ( >1 . 1 ms ) as chemical responses .","Fast events were nearly absent in tjp1b/ZO1b-/- , gjd2a/Cx35 . 5-/- , and gjd1a/Cx34 . 1-/- mutants ( Figure 4B , C ) , and were dramatically reduced in wild-type fish by application of MA ( Figure 4C ) , confirming that they represent electrical transmission .","Slow events that remained after losing the electrical component were dramatically reduced by application of GluRs antagonists ( Figure 4D ) , confirming that they represent chemical responses .","We conclude that the deficit in electrical transmission observed in mutant zebrafish is likely to be widespread within hindbrain and spinal cord circuits .","Given the primary role of the Mauthner cell in triggering escape responses , we also investigated possible changes in cellular excitability in mutant fish .","Interestingly , the frequency of slow , chemical responses was increased in mutants and MA-treated animals ( Figure 4D ) , even though we observed no changes on presynaptic neurotransmitter release properties ( Figure 3—figure supplement 1E ) .","This suggests that the lack of electrical transmission could have enhanced the detection of smaller amplitude chemical responses .","We posit this could arise from electrical coupling influencing neuronal excitability , as the loss of neuronal GJs in mutants would increase the input resistance of the Mauthner cell ( Alcamí and Pereda , 2019 ) .","Thus , during whole cell recordings , we determined the input resistance ( Rin ) , rheobase , resting potential ( Vrest ) , and firing threshold ( Vthreshold ) of the Mauthner cells from wildtype and mutant zebrafish ( Table 1 ) .","The input resistance , the main determinant of neuronal excitability , was increased in tjp1b/ZO1b-/- , gjd2a/Cx35 . 5-/- , and gjd1a/Cx34 . 1-/- mutants .","Accordingly , the rheobase , a parameter negatively correlated with neuronal excitability , was decreased in these mutant animals ( Table 1 ) .","We found that the change in Rin correlated with the lack of electrical transmission and was not observed in fish that retained electrical transmission ( Figure 4E , F; note however that Rin in tjp1a/ZO1a-/- was found to slightly increase ) .","Differences in magnitude of the effects observed on Rin could be due to distinct compensatory mechanisms in each case or to the mutations affecting channels contributing to leak conductance in the Mauthner cell .","Thus , the lack of electrical transmission in tjp1b/ZO1b-/- fish rendered synaptic transmission exclusively mediated by a relatively delayed glutamatergic response and more excitable Mauthner cells .","Both deficits likely influence the behavioral responses generated by the Mauthner cell and its associated network .","Since tjp1b/ZO1b-/- mutants had functional deficits in electrical transmission , but not chemical , we assessed the consequences on the behavioral output of the Mauthner cell network .","Animals were placed into individual chambers of a multi-well testing stage and presented with acoustic-vibrational stimuli to elicit Mauthner-dependent startle responses ( Wolman et al . , 2015 ) .","Movements were captured with a high-speed camera ( 1000 frames per second ) and analyzed with FLOTE software to automatically track and measure the kinematics ( body movements ) of responses ( Burgess and Granato , 2007 ) .","In this paradigm , wild-type fish exhibit two types of escape responses: ( 1 ) Mauthner-cell-dependent short-latency C-bends ( SLCs , hereafter referred to as ‘startles’ ) and ( 2 ) Mauthner-cell-independent long-latency C-bends ( LLCs ) .","These two behavioral responses were automatically distinguished using well-established kinematic parameters ( Burgess and Granato , 2007 ) .","Larvae generated from crossing tjp1b/ZO1b+/- heterozygous animals were tested and analyzed blind to genotype with subsequent post-hoc identification .","We found that tjp1b/ZO1b-/- mutants startled to strong acoustic stimuli ( 25 . 9 dB ) as frequently as their wildtype siblings ( Figure 5A ) .","While these turns were classified as startles , we found that the tjp1b/ZO1b-/- mutants initiated their responses ~ 2 ms slower than their wildtype siblings ( Figure 5B ) .","A delayed behavioral response in consistent with our electrophysiological findings indicating that the mutant escape network operates with longer synaptic delays due to the lack of electrical transmission ( Figure 3 ) .","In mutant animals , we found that the escapes were often performed with the normal startle kinematic parameters , particularly the maximum angle of the turn and the maximum angular velocity of the response , albeit occurring later than in wild-type siblings ( Figure 5C–H ) .","However , we found a subset of mutant responses ( ~15% ) that showed abnormally shallow and slow turns ( Figure 5C , D red arrows ) .","These abnormal responses suggested deficits in performing the stereotyped C-bend elicited by the Mauthner-cell network , and so we reanalyzed the video data from these responses .","In these startles , we observed that the mutants displayed abnormal postures where the body would bend slightly to one side , creating ‘kinked’ or ‘S-shaped’ postures ( Figure 5I–L ) .","We note that the phenotypes observed in the tjp1b/ZO1b-/- mutants are strikingly similar to those we previously observed in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants ( Miller et al . , 2017 ) .","Such kinked body shapes are reminiscent of startles following CoLo neuron ablation ( Satou et al . , 2009 ) .","Based on these data , we conclude that electrical synapses are essential for generating the speed and coordination of the Mauthner-induced startle response .","The electrophysiological analysis of tjp1b/ZO1b-/- mutants also revealed that Mauthner cells showed increased excitability ( Figure 4 ) , suggesting animals may be hypersensitive with a lower threshold of response for environmental stimuli .","To examine this possibility , we presented larvae with 60 pseudo-randomized stimuli , 10 at six different intensities with a 20 s inter-stimulus interval to eliminate habituation ( Wolman et al . , 2015 ) .","We then assessed the frequency with which animals responded to the stimuli with turns ( Figure 5M–O ) .","As stimulus intensity increased , both wildtype siblings and tjp1b/ZO1b-/- mutants increased the likelihood of performing a startle response .","However , across the mid-range of stimulus intensities , tjp1b/ZO1b-/- mutants were more likely to respond with a startle than their wild-type siblings ( Figure 5M , O ) .","The increased tendency of mutants to perform Mauthner-dependent startles came at the expense of Mauthner-independent LLC responses , which were nearly absent in mutants ( Figure 5N ) .","We conclude that electrical synapses alter the sensitivity of Mauthner cells to environmental stimuli and contribute to the innate startle threshold , which alters the probability of eliciting a startle or LLC response .","These results lend additional evidence for the critical role of ZO1b for creating functional electrical synapses , ultimately contributing to appropriately balanced neural network function and behavior .","Mutant zebrafish revealed a hierarchical relationship between ZO1b and neuronal Connexins ( Figures 1 and 2 ) , so we next investigated the mechanisms underlying this relationship .","ZO1 is a membrane-associated guanylate kinase ( MAGUK ) scaffold protein and contains PSD95/Dlg/ZO1 ( PDZ ) protein-protein interaction domains that bind to PDZ binding motifs ( PBMs ) ( Zhu et al . , 2016 ) .","Previous work demonstrated that the C-terminal four amino acids of mouse Cx36 and perch Cx35 compose PBMs that are essential for interacting with ZO1 ( Flores et al . , 2008; Li et al . , 2004 ) .","Given that the PBM sequence is conserved in zebrafish Cx35 . 5 and Cx34 . 1 proteins ( Figure 6A ) , we tested whether these Connexins could mediate binding to zebrafish ZO1b .","We cloned full-length sequences of tjp1b/ZO1b , gjd2a/Cx35 . 5 , and gjd1a/Cx34 . 1 and used heterologous expression to test for interactions between the scaffold and Connexins .","HEK293T cells were co-transfected with mVenus-ZO1b and full-length Cx35 . 5 or Cx34 . 1 .","Using western blot analysis with antibodies specific to each Connexin , we found that both Cx34 . 1 and Cx35 . 5 were individually detected in mVenus-ZO1b immune complexes ( Figure 6B , lanes 1 , 3 ) compared to control immunoprecipitates ( Figure 6—figure supplement 1A , lanes 1 , 2 , 5 , 6 ) .","We further found that removing the presumptive PBMs , by deleting the C-terminal four amino acids from both Connexins , resulted in a loss of co-purifying Connexins from mVenus-ZO1b immunoprecipitates ( Figure 6B , lanes 2 , 4; Figure 6—figure supplement 1A , lanes 4 , 8 ) .","Control blots demonstrated the ability of the Connexin antibodies to equally recognize both full-length and ∆PBM versions of the proteins ( Figure 6B; Figure 6—figure supplement 1A , bottom input panels ) .","We conclude that zebrafish Cx35 . 5 and Cx34 . 1 can interact with ZO1b in a PBM-dependent manner .","The ZO1b scaffold has three PDZ domains that could mediate the interaction with neuronal Connexins ( Figure 6A ) .","Previous studies testing the three mammalian ZO1 PDZ domains demonstrated that Cx35/36 PBMs exclusively bound to ZO1-PDZ1 , while PDZ2 and PDZ3 did not interact ( Flores et al . , 2008; Li et al . , 2004 ) .","Given the high degree of conservation of the zebrafish ZO1 PDZ1 domain , including the amino acids of the putative PBM binding pocket ( Figure 6A ) , we tested whether zebrafish ZO1b-PDZ1 could directly interact with zebrafish neuronal Connexins .","To examine this question , we isolated the minimal domains of each zebrafish protein , produced them in bacteria , and performed in vitro binding studies .","We found that purified ZO1b-PDZ1 could be pulled down with a GST-Cx34 . 1 or a GST-Cx35 . 5 C-terminal intracellular-tail ( Figure 6C , lanes 2 , 4 ) , but not with control GST protein ( Figure 6C , lane1 ) .","Further , this interaction was significantly decreased when the predicted PBM in the Connexin tails were removed ( Figure 6C , lanes 3 , 5 ) .","We next used an overlay assay to compare the ability of ZO1b-PDZ1 and ZO1b-PDZ2 to bind to immobilized GST-Connexin tails .","Similar to the binding assays , significant amounts of ZO1b-PDZ1 bound to GST-Cx34 . 1 and GST-Cx35 . 5 tails in a PBM-dependent manner ( Figure 6—figure supplement 1B , left panels ) .","By contrast , little ZO1b-PDZ2 bound to the GST-Cx34 . 1 or GST-Cx35 . 5 tails , particularly when compared to the non-neuronal Cx43 C-terminal tail ( Figure 6—figure supplement 1B , right panels ) , which is known to bind PDZ2 ( Flores et al . , 2008; Li et al . , 2004 ) .","We conclude that ZO1b utilizes the PDZ1 domain to directly interact with the neuronal Connexin PBMs .","Since ZO1b and the neuronal Connexins colocalized at electrical synapses ( Figure 1 ) , we next investigated whether these proteins interacted in vivo .","We utilized adult zebrafish brains that maintain widespread electrical synapses , including those in Mauthner cell ( Kimmel et al . , 1981 ) , and provide an abundant source to derive ZO1b immunoprecipitates .","Homogenates derived from wild-type fish brains were immunoprecipitated with anti-ZO1 and control antibodies ( mIgG ) .","Immunoprecipitates demonstrated that Cx34 . 1 copurified with ZO1 , whereas Cx35 . 5 did not copurify with the anti-ZO1 antibody ( Figure 6D , lanes 1 , 2 ) .","To confirm that copurification of Cx34 . 1 was dependent upon ZO1b , we replicated the experiment using homogenates from tjp1b/ZO1b-/- mutants and found that Cx34 . 1 was lost in these immmunocomplexes ( Figure 6D , lane4 ) .","We conclude that ZO1 preferentially interacts with Cx34 . 1 in vivo .","We observed multiple ZO1 bands upon Western analysis of the immunoprecipitates , several of which were lost in the tjp1b/ZO1b-/- mutants ( Figure 6D , lanes 2 , 4 ) .","Since the ZO1 antibody was made against human protein , we reasoned it might be detecting the ZO1b protein and the highly similar ZO1a protein , so we sought evidence to confirm that ZO1b was the primary scaffold for Cx34 . 1 in vivo .","Upon examining ZO1 immunocomplexes from wildtype , tjp1b/ZO1b-/- , and tjp1a/ZO1a-/- brains , we found that only ZO1b deficiency resulted in concomitant loss of Cx34 . 1 ( Figure 6—figure supplement 1C ) .","Taken together , we conclude that ZO1b preferentially interacts with Cx34 . 1 in vivo , despite the fact that the scaffold can interact with either neuronal Connexin .","Next , we determined the functional relevance for a preferential ZO1b/Cx34 . 1 interaction at zebrafish electrical synapses .","We previously observed that Cx35 . 5 localizes presynaptically in axons , while Cx34 . 1 localizes postsynaptically in dendrites ( Miller et al . , 2017 ) .","Since ZO1b preferentially interacts with Cx34 . 1 in vivo ( Figure 6 ) , we speculated that ZO1b might share a similar dendritically compartmentalized localization .","To directly examine this , we visualized the localization of ZO1b protein after inserting a V5 epitope at the N-terminus of the endogenous tjp1b locus ( Figure 7—figure supplement 1A ) .","We found that in transgenicV5-tjp1b larvae , V5 antibody staining colocalized with both Cx34 . 1 and Cx35 . 5 at CEs and M/CoLo synaptic contacts ( Figure 7—figure supplement 1B–E ) .","Moreover , we found no effect on the stereotyped patterns of Connexin staining at Mauthner electrical synaptic contact sites in homozygous V5-tjp1b/V5-tjp1b animals ( Figure 7—figure supplement 1D , E ) , suggesting that V5-ZO1b is functional .","While V5-tjp1b larvae permitted ZO1b visualization at electrical synapses , we could not discriminate whether it was localized asymmetrically at synaptic contacts due to the small size of these structures and the resolution limits of light microscopy .","To overcome this , we utilized an alternate approach to address ZO1b compartmentalization , exploiting the fact that the Mauthner cell is both the postsynaptic partner at CEs and the presynaptic partner at the M/CoLo synapses ( Figure 7A ) .","We generated chimeric embryos by blastula transplantation ( Kemp et al . , 2009 ) , extracted GFP and V5-ZO1b expressing transgenic cells from donor embryos , and transferred them to wild-type hosts ( Figure 7B ) , allowing us to address V5-ZO1b localization within the Mauthner cell ( Figure 7C–E ) .","In animals containing only a donor-derived , V5-ZO1b-expressing Mauthner cell , we found that V5 staining was present at the CEs with Connexin staining , demonstrating that ZO1b was within the dendrite of the Mauthner cell and localized postsynaptically at electrical synapses ( Figure 7D ) .","Conversely , when we examined the M/CoLo synapses of these same embryos , we found that V5 staining was not present at these synaptic contacts despite the Mauthner cell expressing V5-ZO1b protein ( Figure 7E ) .","We note that in the non-chimeric , V5-ZO1b transgenic animals , V5 staining was observed at the M/CoLo synapses ( Figure 7—figure supplement 1C , E ) , suggesting that ZO1b at these synapses derives from the postsynaptic CoLo .","Our transplant experiments produce chimeric larvae in which only Mauthner , only CoLo , or both cells are derived from the transgenic donor embryos ( Figure 7—figure supplement 1F–H ) .","In larvae in which only CoLo expresses V5-ZO1b , we found that V5 staining is present at M/CoLo synapses ( Figure 7—figure supplement 1G ) , confirming ZO1b’s postsynaptic localization .","We conclude that ZO1b is robustly compartmentalized within the somato-dendritic compartment of the neuron and asymmetrically localized on the postsynaptic side of the electrical synapse .","We then addressed whether ZO1b functions postsynaptically to facilitate Connexin localization at the electrical synapse .","We utilized chimeric animals and again took advantage of Mauthner cell morphology .","However , in these experiments , we transplanted GFP-expressing cells from tjp1b/ZO1b-/- mutant donors into wildtype hosts , producing animals in which ZO1b was specifically removed from the Mauthner cell ( Figure 7F–I ) .","At CEs , the removal of ZO1b exclusively from the Mauthner cell resulted in the loss of staining for ZO1 , Cx34 . 1 , and Cx35 . 5 ( Figure 7H; Figure 7—figure supplement 1I ) .","The data indicates that ZO1b is required postsynaptically for the cell autonomous localization of Cx34 . 1 and non-autonomously for presynaptic Cx35 . 5 localization .","By contrast , when we examined the same chimeric animals with ZO1b removed from the Mauthner cell but focused on the M/CoLo synapses , in which Mauthner is presynaptic , there was no effect on either Connexin or ZO1 staining ( Figure 7I; Figure 7—figure supplement 1J ) , indicating that ZO1b is dispensable presynaptically .","As further support for this compartmentalized scaffold function , we reasoned that resupplying ZO1b to the Mauthner cell in an otherwise tjp1b/ZO1b-/- mutant animal would be sufficient to rescue Connexin localization at CEs , but not at M/CoLo synapses .","To test this prediction , we transplanted GFP-expressing cells from wildtype donor embryos into tjp1b/ZO1b-/- mutant hosts and identified animals with donor-derived Mauthner cells .","In such chimeras , where the tjp1b/ZO1b gene is functional only in the Mauthner cell , CEs had normal staining for both postsynaptic ZO1 and Cx34 . 1 and also for presynaptic Cx35 . 5 .","By contrast , there was no rescue of staining for these proteins at M/CoLo synaptic contacts ( Figure 7J , K; Figure 7—figure supplement 1J ) .","We conclude that ZO1b is both necessary and sufficient postsynaptically for building the structure of the neuronal GJ channels .","Taken together , we find that ZO1b is exclusively localized to the postsynaptic compartment where it functions both cell-autonomously and non-autonomously to localize Connexins and build functional neuronal gap junctions ."],["Despite the continuous nature of electrical transmission , the thousands of channels that make up GJ plaques found at electrical synapses are maintained by the active turnover of Connexin proteins ( Flores et al . , 2012 ) , similar to neurotransmitter receptors at chemical synapses ( Carroll and Zukin , 2002; Chen et al . , 2000; Ehlers , 2000; Lüscher et al . , 1999 ) .","Our findings show that ZO1b is required for the robust localization of Connexins , suggesting it functions to stabilize Connexin hemichannels at the synaptic site .","Additionally , ZO1 can localize to electrical synapses in the absence of the Connexins , although its apparent concentration was diminished .","This suggests that building robust neuronal GJ structures involves a reciprocal interaction between ZO1 and the Connexins .","Strikingly , our results reveal that ZO1b is compartmentalized to the dendrite and functions asymmetrically at postsynaptic sites of the Mauthner cell circuit .","This localization is consistent with ZO1b’s preferential in vivo interaction with Cx34 . 1 , as this Connexin is also localized and required postsynaptically at Mauthner cell electrical synapses ( Miller et al . , 2017 ) .","Despite the apparent autonomous ZO1b/Cx34 . 1 postsynaptic interaction at the GJ hemiplaque , tjp1b/ZO1b-/- mutants revealed that presynaptic Cx35 . 5 localization is also affected non-autonomously in the neighboring cell .","This trans-synaptic interaction likely occurs via the Connexins themselves , as mutations to the postsynaptic Cx34 . 1 prevented the robust localization of the presynaptic Cx35 . 5 .","Our results are complementary to recent analysis of the mouse rod/cone network , where removing Cx36 from one neuron of a coupled pair results in the failure of Connexin localization in the adjacent neuron ( Jin et al . , 2020 ) .","Taken together , our results reveal that ZO1b acts as a postsynaptic molecular scaffold that localizes Cx34 . 1 to the GJ hemiplaque , which in turn ensures Cx35 . 5 stabilization at presynaptic hemiplaques ( Figure 7C ) .","Whether presynaptic Cx35 . 5 lacks an in vivo scaffold , or utilizes another unidentified presynaptic scaffolding protein , remains unresolved .","If the molecular function and organization of ZO1 revealed here applies to all electrical synapses , including those formed by homotypic channels between various homologous cellular processes ( dendro-dendritic , somato-somatic , or axo-axonic ) , remains to be determined in future studies .","Never-the-less , we posit that the molecular organization of the electrical synapse can be asymmetrically compartmentalized , thereby enabling preferential biochemical interactions at each side of the junction .","Our results support the prediction that ZO1 likely plays distinct functional roles at GJs in different tissue types .","ZO1 was first described at epithelial tight junctions and later shown to interact with various Connexins , notably with Cx43 , a widespread Connexin expressed in many non-neuronal cell types ( Giepmans , 2004 ) .","Evidence from cell expression systems suggested that ZO1 played critical functions on the periphery of Cx43-contaning GJ plaques forming part of the ‘perinexus’ to facilitate newly inserted hemichannels at each side of the junction ( Rhett and Gourdie , 2012 ) .","Further , the ZO1/Cx43 interaction was critical for GJ communication before the channel ‘ages’ and is subsequently removed during channel turnover , a process governed by Cx43 phosphorylation ( Laird , 1996; Laird , 2006; Márquez-Rosado et al . , 2012; Solan and Lampe , 2016; Thévenin et al . , 2017 ) .","However , preventing the ZO1/Cx43 interaction does not prevent GJ formation ( Hunter and Gourdie , 2008; Hunter et al . , 2003; Hunter et al . , 2005 ) .","By contrast , our results demonstrated that ZO1b’s presence was asymmetric and required for robust Connexin localization and synaptic function .","Beyond our observations here , ZO1 is likely to have homeostatic functions in the modulation of Connexin usage at electrical synapses .","For example , analysis of the interactions between ZO1 and Cx36 ( and its fish homologs ) revealed the interactions occurs via a different PDZ domain than the interaction with Cx43 and the interaction with Cx36 has lower affinity and faster kinetics than that of Cx43 ( Flores et al . , 2008; Li et al . , 2004 ) .","This suggests ZO1 has a more dynamic interaction with neuronal Connexins , which may serve the plastic , activity-dependent regulation of electrical transmission observed in fish and mammalian electrical synapses ( Haas et al . , 2011; Landisman and Connors , 2005; Mathy et al . , 2014; Pereda and Faber , 1996; Pereda et al . , 1998; Turecek et al . , 2014; Yang et al . , 1990 ) .","Thus , our findings suggest that ZO1’s function at electrical synapses differs from its role at Cx43-containing GJs , perhaps serving a specialized function in synaptic communication .","Our results highlight that neural circuit function requires functional electrical and chemical synapses to create an appropriate behavioral response .","Our data indicates that the lack of electrical transmission in ZO1b and Connexin mutants did not prevent the formation of co-existing glutamatergic synapses at CEs on the Mauthner cell .","The remaining chemical transmission supported a behavioral response organized by the Mauthner-cell network , although importantly , the response showed deficits in performance and altered sensitivity .","Given that the startle behavior mediates predator avoidance , these defects would likely be detrimental to survival ( Hecker et al . , 2020 ) .","The lack of effect on glutamatergic transmission contrasts a wealth of data supporting a strong , interdependent relationship between the formation of electrical and chemical synapses during development in both invertebrate and vertebrate nervous systems ( Jabeen and Thirumalai , 2018 ) .","For example , in the leech , knockdown of Innexins at a developmental stage where synaptic contacts are solely electrically coupled prevents the formation of later-forming chemical transmission ( Todd et al . , 2010 ) .","Similarly , in the developing mouse neocortex , dominant negative constructs of Cx26 prevent the initial electrical coupling and subsequent chemical synapse formation amongst sister excitatory neurons within ontogenetic columns ( Yu et al . , 2012 ) .","Our findings indicate that this deficit does not occur at zebrafish CEs when Cx35 . 5 , Cx34 . 1 , and ZO1b are removed .","Whether this is due to the differences in the GJ proteins used in the Mauthner cell or instead due to mechanisms that specify CE formation , remains unknown .","One intriguing possibility is that other proteins may act as a common synaptic control mechanism that is independent of GJ-forming proteins .","Indeed , mutations in the scaffold Neurobeachin caused parallel defects in the formation of both electrical and chemical synapses of the Mauthner circuit ( Miller et al . , 2015 ) , yet the mechanism by which this coordination occurs remains to be elucidated .","Alternatively , rather than functional ( conductive ) GJ channels , other structural components of the electrical synapse may be sufficient to trigger chemical synapse formation via protein-protein interactions .","We posit such interactions would apply to mammalian electrical synapses , which can co-exist with neighboring glutamatergic synapses at distances comparable to those found in fish mixed synapses ( Nagy et al . , 2018 ) and may mediate similar functional interactions .","Identifying the functional relevance of an intracellular scaffolding protein as a critical part of the electrical synapse draws parallels with our understanding of chemical transmission , where neurotransmitter receptors are clustered and modified by a rich network of postsynaptic proteins that dynamically shape synaptic structure and function .","This chemical synapse protein network is known as the ‘postsynaptic density’ ( PSD ) , a term resulting from its structural identification by EM ( Cohen , 2013; Palay , 1956 ) and is composed of hundreds of unique proteins ( Grant , 2019 ) .","Mirroring those findings , EM images of electrical synapses in mammals ( Llinas et al . , 1974 ) and fish ( Brightman and Reese , 1969 ) revealed the presence of clearly identifiable electrodense structures , first described as ‘semi-dense material’ by Sotelo and Korn , 1978 .","These electrodense structures are localized intracellularly and form an undercoating band at neuronal GJs .","The identified structures are presumably formed by a proteinaceous ‘organelle’ that resembles that found at PSDs ( Feng et al . , 2019 ) .","As evidence grows for an array of proteins localized to electrical synapses , we propose that ZO1 is member of such an organelle .","Our data provide enticing hints that the molecular framework of the electrical synapse extends beyond the ZO/Connexin interaction .","In particular , the fact that ZO1 can localize to sites of synaptic contact independent of the Connexins , and that there remains immunofluorescent staining at Mauthner cell synapses in tjp1b/ZO1b-/- mutants , both imply the existence of additional proteins that contribute to this synaptic structure .","We presume the remaining ZO1 staining in tjp1b/ZO1b-/- mutants comes from either the paralogous ZO1a protein ( tjp1a ) or from the related ZO2 ( tjp2a , tjp2b ) and ZO3 ( tjp3 ) proteins .","However , our previous analysis of ZO2/ZO3 mutants in zebrafish did not reveal overt defects in Connexin localization ( Marsh et al . , 2017 ) , yet both proteins are localized to mammalian electrical synapses ( Li et al . , 2009 ) .","Whether these related scaffolds have functional roles at electrical synapses that were undetected in our initial screen remains to be determined .","Beyond the ZO-family , other molecules can directly interact with Cx36 and/or localize at mammalian electrical synapses , such as cell adhesion molecules , cytoskeletal interacting proteins , and molecules that regulate Connexin post-translational modifications ( Lynn et al . , 2012; Martin et al . , 2020; O'Brien and Bloomfield , 2018 ) .","Yet , the molecular roles of these proteins at the electrical synapse remain poorly defined .","Therefore , as opposed to simple aggregates of intercellular channels , we propose that electrical synapses are complex synaptic structures at which communicating pre- and postsynaptic Connexin hemichannels are governed by a yet to be determined mechanism that builds an asymmetric molecular scaffold .","Based on its analogy to the known functions of glutamatergic PSDs , we propose to name this organizational organelle the ‘electrical synapse density’ , or ‘ESD’ ( Lynn et al . , 2012; Miller and Pereda , 2017; Figure 7C ) .","Chemical synapses are organized to match their specific functional requirements by combining presynaptic release properties with unique combinations of postsynaptic receptors .","Electrical synapses also provide a variety of specific synaptic functions , yet we know little about the source of their diversity .","Work in C . elegans exposed the large variety of Innexin expression in neurons contributing to neural circuits , with dozens of potentially unique cellular combinations that are altered during development and following environmental stress ( Bhattacharya et al . , 2019 ) .","These observations have greatly increased the appreciation of the incidence and potential functional diversity of electrical transmission in invertebrates .","Our results indicate that the complexity of electrical synaptic transmission must also include their molecular scaffolds .","Scaffold diversity may be more relevant for the function of vertebrate electrical synapses , which in contrast to C . elegans , are formed by a smaller number of GJ-forming proteins , with most electrical communication being reliant on Cx36-related proteins investigated here .","By promoting and regulating channel trafficking and regulatory molecules , ZO1 , and other scaffolding proteins , could support a variety of functions by governing channel function and the local regulatory environment .","Thus , future investigations will further expose the molecular composition and functional roles of ZO1 and the ESD .","Unraveling the functional complexity of the ESD will lead to a deeper understanding of the diversity of the molecular organization underlying electrical transmission and its contributions to brain function ."],["Fish were maintained in the University of Oregon’s and the Albert Einstein College of Medicine fish facilities with approval from Institutional Animal Care and Use Committees of each institution .","Zebrafish , Danio rerio , were bred and maintained at 28°C on a 14 hr on and 10 hr off light cycle .","Animals were housed in groups , generally of 25 animals per tank .","Development time points were assigned via standard developmental staging ( Kimmel et al . , 1995 ) .","All fish used for this project were maintained in the ABC background developed at the University of Oregon .","Most fish had the enhancer trap transgene zf206Et ( M/CoLo:GFP ) in the background ( Satou et al . , 2009 ) , unless otherwise noted .","Mutant lines were genotyped for all experiments .","All immunohistochemistry , electrophysiological , and behavioral experiments were performed at 5 dpf .","At this stage of development , zebrafish sex is not yet determined ( Wilson et al . , 2014 ) .","Protein extractions were performed from both male and female adult brains and combined .","HEK293T/17 verified cells were purchased from ATCC ( CRL-11268; STR profile , amelogenin: X ) .","Cells were expanded and maintained in Dulbecco's Modified Eagle's Medium ( DMEM , ATCC ) plus 10% fetal bovine serum ( FBS , Gibco ) at 37°C in a humidified incubator in the presence of 5% CO2 .","Low passage aliquots were cryopreserved and stored according to manufacturer’s instructions .","Cells from each thawed cryovial were monitored for mycoplasma contamination using the Universal Mycoplasma Detection Kit ( ATCC , 30–1012K ) .","Anesthetized , 5–6 days post fertilization ( dpf ) larvae were fixed for 3 hr in 2% trichloroacetic acid in PBS .","Fixed tissue was washed in PBS + 0 . 5% Triton X-100 , followed by standard blocking and antibody incubations .","Primary antibody mixes included combinations of the following: rabbit anti-Cx35 . 5 ( Fred Hutch Antibody Technology Facility , clone 12H5 , 1:800 ) , mouse IgG1 anti-Cx35 . 5 ( Fred Hutch Antibody Technology Facility , clone 4B12 , 1:250 ) , rabbit anti-Cx34 . 1 ( Fred Hutch Antibody Technology Facility , clone 3A4 , 1:250 ) , mouse IgG2A anti-Cx34 . 1 ( Fred Hutch Antibody Technology Facility , clone 5C10A , 1:350 ) , mouse IgG1 anti-ZO1 ( Invitrogen , 33–9100 , 1:350 ) , mouse IgG2a anti-V5 peptide ( Invitrogen , R960-25 , 1:50 ) , and chicken anti-GFP ( abcam , ab13970 , 1:350- 1:500 ) .","All secondary antibodies were raised in goat ( Invitrogen , conjugated with Alexa-405 , –488 , −555 , or −633 fluorophores , 1:500 ) .","Tissue was then cleared stepwise in a 25% , 50% , 75% glycerol series , dissected , and mounted in ProLong Gold antifade reagent ( ThermoFisher , P36930 ) .","Images were acquired on a Leica SP8 Confocal using a 405-diode laser and a white light laser set to 499 , 553/554/557 ( consistent within experiments ) , and 631 nm , depending on the fluorescent dye imaged .","Each laser line’s data was collected sequentially using custom detection filters based on the dye .","Quantitative images of the Club Endings ( CEs ) were collected using a 63x , 1 . 40 numerical aperture ( NA ) , oil immersion lens , and images of M/Colo synapses were collected using a 40x , 1 . 20 NA , water immersion lens .","For each set of images , the optimal optical section thickness was used as calculated by the Leica software based on the pinhole , emission wavelengths , and NA of the lens .","Within each experiment where fluorescence intensity was to be quantified , all animals ( including 3–5 wildtype controls ) were stained together with the same antibody mix , processed at the same time , and all confocal settings ( laser power , scan speed , gain , offset , objective , and zoom ) were identical .","Multiple animals per genotype were analyzed to account for biological variation .","To account for technical variation , fluorescence intensity values for each region of each animal were an average across multiple synapses .","For high-contrast imaging of the CEs , fixed samples were washed three times with PBS , incubated at 4°C overnight , and hindbrains were dissected out .","Dissected hindbrains were washed , blocked , and stained as above with the following primary antibodies: rabbit anti-Cx35 . 5 ( 12H5 , 1:500 ) , mouse anti-Cx35/36 ( EMD Millipore , MAB3045 , 1:250 ) , mouse IgG2A anti-Cx34 . 1 ( 5C10A , 1:200 ) , mouse IgG1 anti-ZO1 ( 33–9100 , 1:200 ) , rabbit anti-GFP ( Invitrogen , G10362 , 1:200 ) , and chicken anti-GFP ( Invitrogen , A10262 , 1:200 ) .","Secondary antibodies were raised in goat ( Invitrogen , conjugated with Alexa-405 , –488 , −546 , –555 , −633 , or −647 fluorophores , 1:200 ) .","Samples were then transferred onto a slide in the dark and mounted with Fluoromount-G ( Southern Biotech , 0100–01 ) , covered using the standard ‘bridge’ procedure , and sealed with nail polish .","Samples were imaged on LSM 710 and LSM 880 Zeiss microscopes using appropriate laser wavelengths and detection filters .","Image stacks of roughly 20 µm were collected using a 63x , 1 . 40 numerical aperture ( NA ) , oil immersion lens .","For each Mauthner , laser strengths and gains were adjusted to achieve maximum visualization of CE staining .","Electrophysiological responses were obtained during whole-cell recordings of Mauthner cells ( M-cells ) in wt , Cx and ZO-1 mutant zebrafish larvae ( 5–7 dpf ) .","For this purpose , fish were first anesthetized with a 0 . 03% solution of MS222 ( pH adjusted to 7 . 4 with NaHCO3 ) and later transferred to external solution containing d-tubocurarine ( 10 μM , Sigma ) .","The external solution ( in mM ) : 134 NaCl , 2 . 9 KCl , 2 . 1 CaCl2 , 1 . 2 MgCl2 , 10 HEPES , 10 Glucose , pH adjusted to 7 . 8 with NaOH ( Yao et al . , 2014 ) .","Zebrafish larvae were put on their backs onto a Sylgard-coated small culture dish ( FluoroDish , WPI ) and kept in place using fine tungsten pins .","The hindbrain was then exposed ventrally following the dissection approach previously described ( Koyama et al . , 2011 ) .","Following this procedure , the larvae were placed on an Axio Examiner upright microscope ( Carl Zeiss AG ) equipped with a recording set-up and superfused with external solution during the entire recording session .","The M-cells were identified by GFP expression and/or far-red DIC optics .","Patch pipettes ( 3–4 MΩ ) were filled with internal solution ( in mM ) : 105 K-Methanesulfonate , 10 HEPES , 10 EGTA , 2 MgCl2 , 2 CaCl2 , 4 Na2ATP , 0 . 4 Tris-GTP , 10 K2-Phosphocreatine , 25 mannitol , pH adjusted to 7 . 2 with KOH .","Whole-cell recordings under the current-clamp configuration were performed with a Multiclamp 700B amplifier and a Digidata 1440A ( Molecular Devices ) digitizer .","The liquid-liquid junction potential was estimated in −16 mV using Clampex 10 . 6 ( Molecular Devices ) and was subtracted from the measured values .","The electrode’s resistance was compensated using the bridge balance feature of the amplifier .","To activate the auditory afferents terminating as CEs on the M-cell , a septated ( theta ) glass pipette was filled with external solution and positioned near the posterior macula of the ear , where the dendritic processes of auditory afferents contact the hair cells ( Yao et al . , 2014 ) .","The maximal amplitude of the electrical and chemical components was estimated by applying shocks of increasing intensity until the amplitude of the electrical component did not further increase and before additional responses with longer latency were evoked .","To estimate the Paired-Pulse Ratio ( PPR ) of the chemical component one , a stimulating-pulse was applied to record 10–20 traces in basal conditions .","Then two-stimulating pulses ( 2 ms apart ) were applied to record ( 10–20 traces ) facilitation of the chemical component .","Traces that clearly showed a chemical component were averaged for basal and facilitated conditions .","The trace in basal conditions was subtracted from the facilitated trace .","The PPR was then calculated using the amplitude of the chemical component of the facilitated trace divided by the amplitude of the chemical component in basal conditions .","Spontaneous electrical and chemical synaptic responses were assessed during offline analysis of continuous ( 10 s long ) recordings using Clampfit ( Axon instruments ) to automatically identify events based on their duration ( <1 . 1 ms for electrical spontaneous events and >1 . 1 ms for chemical spontaneous events ) .","Potential erroneous identification of spontaneous events by the software was monitored manually by verifying the duration of the events .","The assignment of electrical vs . chemical nature of spontaneous synaptic events by their duration was confirmed pharmacologically .","For synaptic transmission blockade , CNQX was first dissolved in DMSO to have a stock solution of 10 mM .","The pharmacological agents used to block synaptic transmission were added to the external solution: Meclofenamic Acid ( 200 μM , Sigma ) , CNQX and DAP5 ( 20 μM , Tocris Biosciences ) .","The M-cell input resistance was estimated by applying a hyperpolarizing-current step of −1 nA and 20 ms in duration and measuring the voltage deflection caused , followed by derivation of resistance with Ohm’s law .","The rheobase , defined as the minimum depolarizing current of infinite duration necessary to evoke an action potential , was determined by delivering a 20 ms current pulse of increasing intensity .","Voltage threshold for action potential generation was determined by applying a depolarizing-current step .","Startle behavior of 5dpf larvae was analyzed as described previously ( Marsden et al . , 2018 ) .","Briefly , larvae were adapted to the testing temperature and lighting conditions for 30 min and then transferred to individual wells of a custom , laser-cut acrylic multi-well testing arena , illuminated from below with an infrared ( IR ) LED array and from above with a white light LED bulb to simulate daylight conditions .","A total of 60 acoustic stimuli , 10 at each of 6 intensities , were delivered pseudorandomly using an acoustic-vibrational shaker ( Bruel and Kjaer ) with an inter-stimulus interval of 20 s to eliminate habituation to repeated stimulation ( Wolman et al . , 2015 ) .","The intensity of each stimulus was calibrated using a PCB Piezotronics accelerometer ( model #355B04 ) and signal conditioner ( model #482A21 ) , and voltage outputs were converted to dB using the formula dB = 20 log ( V/0 . 775 ) .","Behavioral responses were captured at 1000 frames per second with an IR-sensitive Photron mini-UX50 high-speed camera .","After testing , larvae were fixed in methanol for subsequent genotyping , thus all testing and analysis was performed blind to genotype .","Behavioral responses were tracked and analyzed using FLOTE software ( Burgess and Granato , 2007 ) , with short and long latency C-bend responses ( SLCs and LLCs , respectively ) automatically defined based on the kinematic parameters of the response .","Startle sensitivity index was calculated by measuring the area under the curve of stimulus intensity versus SLC frequency for each larva .","Cell lines were obtained from ATCC , identity ensured by using exclusively low-passage cells , and were confirmed to be mycoplama free .","Full-length Cx34 . 1 and full-length Cx35 . 5 were cloned into the pCMV expression vector .","Full-length ZO1b was cloned into the pCMV expression vector with an NH2-terminal mVenus tag and a COOH-terminal 8xHIS tag .","Low passage HEK293T/17 cells were seeded 24 hr prior to transfection ( 1 × 106 cells/well of a six-well dish ) , and the indicated plasmids were co-transfected using Lipofectamine 3000 ( Invitrogen ) following the manufacturer’s instructions .","Cells were collected 36–48 hr post-transfection and lysed in 0 . 25 ml solubilization buffer ( 50 mM Tris [pH7 . 4] , 100 mM NaCl , 5 mM EDTA , 1 . 5 mM MgCl2 , 1 mM DTT and 1% Triton X-100 ) plus a protease inhibitor cocktail ( Pierce ) .","Lysates were centrifuged at 20 , 000 x g for 30 min at 4°C , and equal amounts of extract were immunoprecipitated with 0 . 5 ug rabbit anti-GFP ( Abcam , Ab290 ) overnight with rocking at 4°C .","Immunocomplexes were captured with 25 µl prewashed Protein A/G agarose beads for 1 hr with rocking at 4°C .","Beads were washed three times with lysis buffer , and bound proteins were boiled for 3 min in the presence of LDS-PAGE loading dye containing 200 mM DTT .","Samples were resolved by SDS-PAGE using a 4–15% gradient gel and analyzed by Western blot using the following primary antibodies: rabbit anti-GFP ( Abcam Ab290 ) , rabbit anti-Cx34 . 1 3A4-conjugated-680LT , and mouse anti-Cx35 . 5 4B12 .","Compatible near-infrared secondary antibodies were used for visualization with the Odyssey system ( LI-COR ) .","Brains from wildtype , tjp1a/ZO1a-/- , or tjp1b/ZO1b-/- euthanized adult fish ( 4–15 months old ) were removed , snap frozen in liquid nitrogen and stored at −80C until use .","Brains were homogenized in 1 ml of HSE buffer ( 20 mM Hepes [pH7 . 5] , 150 mM NaCl , 5 mM EDTA , 5 mM EGTA , and 1 mM DTT ) plus a protease inhibitor cocktail using a glass homogenizer .","Detergent was added to the homogenate ( final 2% octyl ß-D-glucopyranoside , Anatrace ) and solubilized overnight with rocking at 4°C .","Solubilized homogenate was cleared by centrifugation at 20 , 000 x g for 30 min at 4°C , then pre-cleared for 1 hr at 4°C with Protein A/G beads before immunoprecipitation .","The protein concentration for each homogenate was measured by Bradford assay .","Pre-cleared homogenates ( 2 mg/IP ) were immunoprecipitated with 0 . 5 µg mouse anti-ZO1 ( Life Technologies , 33–9100 ) , control mouse IgG ( Jackson ImmunoResearch ) , mouse anti-Cx34 . 1 5C10 , or mouse anti-Cx35 . 5 4B12 antibody overnight with rocking at 4°C .","Immunocomplexes were captured with 25 ul prewashed Protein A/G agarose beads for 1 hr with rocking at 4°C .","Beads were washed three times with HSE buffer , and bound proteins were boiled for 3 min in the presence of LDS-PAGE loading dye containing 200 mM DTT .","Immune complexes were examined by western analysis using the following primary antibodies: mouse anti-ZO1 , rabbit anti-Cx34 . 1 3A4-conjugated-680LT , rabbit anti-Cx35 . 5 12H5-conjugated-680LT , or mouse anti-Cx35 . 5 4B12 .","Compatible near-infrared secondary antibodies were used for visualization .","The Cx34 . 1-tail ( aa256-299 ) , Cx34 . 1-tail ∆PBM ( aa256-295 ) , Cx35 . 5-tail ( aa267-309 ) , and Cx35 . 5-tail ∆PBM ( aa267-305 ) were cloned into the pGEX expression vector allowing for an NH2-terminal GST tag .","ZO1b-PDZ1 ( aa105-207 ) and ZO1b-PDZ2 ( aa298-387 ) were cloned into a modified pET expression vector ( pBH ) to allow for an NH2-terminal 6xHis tag followed by a TEV cleavage site ( vectors kindly provided by Ken Prehoda ) .","Plasmids were transformed in E . coli BL21 ( DE3 ) cells and plated on selective LB plates .","Single colonies were picked to inoculate 2 ml starter cultures and grown overnight .","Overnight cultures were inoculated into 250 ml selective LB and grown for ~3 hr at 37°C with shaking until OD600 reached 0 . 8–1 followed by 4 hr induction with 0 . 4 mM IPTG .","Cell pellets were collected by centrifugation at 6000 RPM for 5 min at 4°C and frozen at −20°C until test samples confirmed expression .","Pellets were resuspended in sonication buffer ( 50 mM NaPO4 [pH7 . 4] , 300 mM NaCl , and 1 mM PMSF ) .","After adding a dash of lysozyme , the mixture was incubated on ice for 30 min .","Resuspended bacteria were sonicated on ice at 50% amplitude , 1 s/1 s pulse on/off , four times for 20 s .","Debris was cleared by centrifugation at 16 , 000 x g for 30 min at 4°C .","For GST fusions , supernatant was added to 200 ul pre-washed glutathione agarose resin and incubated overnight with rocking at 4°C .","Beads were washed three times with sonication buffer and stored at 4°C .","Purity and amount loaded onto resin was determined by SDS-PAGE followed by Coomassie stain .","For 6xHIS fusions , supernatant was brought to a final concentration of 20 mM imidazole and incubated with pre-washed His60 resin overnight with rocking at 4°C .","Resin was washed with sonication buffer containing 20 mM imidazole .","Protein was eluted from the resin with sonication buffer containing 250 mM imidazole .","The protein was concentrated and exchanged into imidazole-free buffer using an Amicon centrifugal filter unit ( 10K MWCO ) and stored at 4°C on ice .","Protein concentration was estimated by A205 ( https://spin . niddk . nih . gov/clore/ ) ( Anthis and Clore , 2013 ) , and purity was determined by SDS-PAGE followed by Coomassie stain .","Equal amounts of GST fusions ( 10 µl bed of resin ) were aliquoted and the storage buffer was removed .","To each sample 15 µl of 6xHIS-ZO1b-PDZ1 ( 7 mg/ml ) was added , gently mixed and incubated at room temperature for 15 min .","Resin was washed three times with cold wash buffer ( 50 mM NaPO4 [pH7 . 4] , 300 mM NaCl ) .","After the last wash , all buffer was removed and resin was resuspended in 10 µl LDS-PAGE dye with 200 mM DTT .","Samples were boiled for 3 min and resolved by SDS-PAGE using a 4–20% gradient gel .","Samples were analyzed by Western blot using rabbit anti-TEV cleavage site primary antibody ( ThermoFisher , PA1-119 ) and visualized with a compatible near-infrared secondary antibody .","A portion of the GST fusion resin was analyzed by Coomassie stain to demonstrate equal loading .","Equal amounts of GST fusion were resuspended in LDS-PAGE dye plus 200 mM DTT , boiled for 3 min , resolved by SDS-PAGE on a 4–15% gradient gel , transferred to nitrocellulose , and blocked with 5% milk in TBS ( 20 mM Tris [pH7 . 4] , 150 mM NaCl ) .","Blocked membranes were incubated with TBS-T buffer ( TBS + 0 . 1% Tween-20 ) alone ( mock overlay ) , or TBS-T containing 1 µM 6xHIS-ZO1b-PDZ1 or 1 uM 6xHIS-ZO1b-PDZ2 overnight with shaking at 4°C .","Membranes were processed for western analysis using rabbit anti-TEV cleavage site primary antibody and a compatible near-infrared secondary antibody to detect bound PDZ protein .","Equal loading of GST fusions was determined by Stain-Free imaging technology .","A single guide RNA ( sgRNA ) targeting the 5’ region of the endogenous tjp1b coding sequence ( sequence in Key Resources Table ) was designed using the CRISPRscan algorithm ( Moreno-Mateos et al . , 2015 ) and synthesized as previously described ( Shah et al . , 2015 ) .","The sgRNA was generated using the T7 megascript kit ( ThermoFisher , AMB13345 ) .","The V5-tjp1b single stranded donor oligo ( ssODN ) was designed to repair into the endogenous tjp1b locus and was synthesized complimentary to the coding strand .","The ssODN contained 40 bp homology arms which flanked an XbaI restriction site , V5 sequence , and a 5x glycine linker , respectively ( sequence in Key Resources Table ) .","Upon correct repair , the inserted sequence was designed to disrupt the endogenous sgRNA recognition site to prevent further double stranded breaks after repair .","Injection mixes were prepared in a pH 7 . 5 buffer solution of 300 mM KCl and 4 mM HEPES and contained a final concentration of 200 pg/nL ssODN , 200 pg/nL gRNA , and 1600 pg/nL Cas9 protein ( IDT , 1081058 ) .","Injection mixes were incubated at 37 °C for 5 min immediately prior to injection to promote formation of the Cas9 and sgRNA complex .","Finally , 1 nL of solution was injected into embryos at the one-cell stage .","Injected embryos were raised to adulthood and outcrossed to wild-type animals .","Animals carrying insertions were identified and verified using PCR and Sanger sequencing .","Cell transplantation was performed at the high stage approximately 3 . 3 hr into zebrafish development using standard techniques ( Kemp et al . , 2009 ) .","Embryos were chemically de-chorionated with protease ( Sigma Aldrich , 9036-06-0 ) prior to transplantation .","Cells were transplanted using a 50 mm wide glass capillary needle attached to an oil hydraulic .","For ‘V5-tjp1b+/- into wildtype’ transplants ( Figures 7C , D and Figure 7—figure supplement 1B-H ) cells from animals heterozygous for V5-tjp1b in the M/CoLo:GFP background were transplanted into non-transgenic wildtype hosts .","For ‘tjp1b-/- into wildtype’ transplants ( Figure 7G and H ) , genotyped animals homozygous for the tjp1bΔ16bp mutation in the M/CoLo:GFP background were crossed and progeny were transplanted into non-transgenic wildtype hosts .","For 'wildtype into tjp1b-/-' transplants ( Figure 7I and J ) , transgenic M/CoLo:GFP wildtype animals were crossed to use as donors , and non-transgenic , homozygous tjp1bΔ16bp animals were crossed to produce hosts .","Approximately 20 cells were deposited ∼10–15 cell diameters away from the margin , with a single donor embryo supplying cells to 3–5 hosts .","At 5 dpf , larvae were fixed in TCA and processed for immunohistochemistry .","For fluorescence intensity quantitation , confocal images were processed and analyzed ( in part or in full ) using FiJi ( Schindelin et al . , 2012 ) software .","To quantify staining at M/Colo synapses , a standard region of interest ( ROI ) surrounding each M/CoLo site of contact was drawn , and the mean fluorescence intensity was measured .","For the quantification of staining at the club endings , confocal z-stacks of the Mauthner soma and lateral dendrite were cropped to 36 . 08 µm x 36 . 08 µm centered around the lateral dendritic bifurcation .","Using the SciPy ( Virtanen et al . , 2020 ) and scikit-image ( van der Walt et al . , 2014 ) computing packages , the cropped stack was then cleared outside of the Mauthner cell , a 33 median filter was applied to reduce noise , and a standard threshold was set within each experiment to remove background staining .","The image was then transformed into a max intensity projection and the integrated density of each stain within the Mauthner cell was extracted .","Where counts were used to quantify antibody labeling of CEs in high-contrast images , CEs were identified as large fluorescently labeled oval areas of approximately 1 . 5–2 microns on the distal portion and fork of the Mauthner cell’s lateral dendrite , which were thoroughly examined with the confocal microscope .","For Figure 7—figure supplement 1I , J , the presence or absence of electrical synapses on Mauthner was quantified as counts of stereotyped electrical synapse structures dually labeled for Cx34 . 1 and Cx35 . 5 .","Standard deviations and errors were computed using Prism ( GraphPad ) or Excel ( Microsoft ) software .","Figure images were created using FiJi , Photoshop ( Adobe ) , and Illustrator ( Adobe ) .","Statistical analyses were performed using Prism ( GraphPad ) and either an unpaired t-test with Welch’s correction or a one-way analysis of variance with Bonferrroni’s multiple comparison test was performed .","For all experiments , values were normalized to wildtype control animals , and n represented the number of fish used .","Fish were excluded from analysis if Mauthner morphology/GFP staining was abnormal .","Electrophysiology traces of spontaneous synaptic events were analyzed using Clampfit 10 . 7 software .","Electrophysiology traces were transferred to Canvas for illustration and to OriginPro software for quantification and statistical analyses .","For the different parameters measured using electrophysiological recordings , means and standard error of the mean ( SEM ) were illustrated using Prism ( GraphPad ) and computed using OriginPro software .","For statistical analyses comparing electrophysiological recordings in wildtype and mutant zebrafish , Mann-Whitney and Kruskal-Wallis non-parametric tests were performed using OriginPro software ."]],"string":"[\n [\n \"Synapses are specialized cellular adhesions between neurons that rapidly transfer information to facilitate neural network function .\",\n \"There are two modalities of fast synaptic transmission , chemical and electrical , both found throughout animal nervous systems including in mammals ( Moroz and Kohn , 2016; Ryan and Grant , 2009 ) .\",\n \"Chemical synapses are inherently asymmetric structures , derived from presynaptic specializations that regulate the release of neurotransmitters and postsynaptic specializations that contain neurotransmitter receptors and the machinery required to propagate signal transmission .\",\n \"Both specializations require hundreds to thousands of proteins , which together tightly control the structure , function , and modulation of synaptic communication ( Ackermann et al . , 2015; Grant , 2019; Siddiqui and Craig , 2011 ) .\",\n \"For example , intracellular scaffolding proteins of the Post Synaptic Density ( PSD ) at chemical synapses regulate the number and functional state of AMPA and NMDA receptors , which are ligand-gated ion channels , at glutamatergic synapses ( Zhu et al . , 2016 ) .\",\n \"By contrast , electrical synapses are often perceived as simple aggregates of intercellular channels known as gap junctions ( GJs ) ( Goodenough and Paul , 2009 ) .\",\n \"Intercellular GJ channels are formed by the docking of two hemichannels , composed of Connexin proteins in vertebrates and Innexins in invertebrates ( Bhattacharya et al . , 2019; Phelan , 2005; Shruti et al . , 2014; Söhl et al . , 2005 ) .\",\n \"Each neuron contributes a hemichannel from each side of the synapse , which form a channel and support communication by allowing the spread of electrical currents and small metabolites between adjacent ‘coupled’ neurons .\",\n \"While multiple Connexins and Innexins can contribute to individual electrical synapses ( Bhattacharya et al . , 2019; Miller et al . , 2017; Phelan et al . , 2008; Rash et al . , 2013 ) , the complexity of neuronal GJ cellular biology ( Lynn et al . , 2012; Martin et al . , 2020; Meyer et al . , 2014; Sigulinsky et al . , 2020 ) and the variety of mechanisms regulating their synaptic strength ( Arroyo et al . , 2016; Bloomfield and Völgyi , 2009; Marder , 1998; O'Brien and Bloomfield , 2018; Pereda , 2014 ) suggest they require complex multimolecular structures to support function .\",\n \"Several Connexin-associated proteins have been identified ( Lynn et al . , 2012; Miller and Pereda , 2017 ) ; however , it remains undetermined whether such associated proteins are ancillary to the channels or requisite for electrical synapse function .\",\n \"Perhaps the best characterized Connexin-associated protein is Zonula Occludens 1 ( ZO1 ) ( Bauer et al . , 2010; Willott et al . , 1993 ) , which is an intracellular scaffolding protein and a member of the membrane-associated guanylate kinase ( MAGUK ) family of proteins .\",\n \"MAGUKs constitute a large family of multifunctional adaptor proteins that play key roles in scaffolding membrane channels and receptors to intracellular signaling complexes and the cytoskeleton ( González-Mariscal et al . , 2000 ) .\",\n \"MAGUK proteins , including ZO1 , contain PSD95/Dlg/ZO1 ( PDZ ) protein-protein interaction domains , which bind to PDZ-binding motifs often located at the carboxy terminus of partner proteins , including Connexins ( Zhu et al . , 2016 ) .\",\n \"The best studied ZO1/Connexin interaction is with Connexin 43 ( Cx43 ) , a widely expressed , non-neuronal , GJ-channel forming protein ( Giepmans and Moolenaar , 1998 ) .\",\n \"The ZO1/Cx43 interaction is thought to play important functional roles in GJ regulation by facilitating the docking of newly inserted hemichannels , which promotes the formation of intercellular channels ( Hunter et al . , 2005 ) .\",\n \"Moreover , the ZO1/Cx43 interaction is critical for channels to remain conductive prior to removal during channel turnover at epithelial GJs ( Hervé et al . , 2014; Thévenin et al . , 2017 ) .\",\n \"While ZO1 is an important regulator of Cx43-contaning GJs , less is known about its role at neuronal GJs , which are primarily formed by the Cx36-family of proteins and mediate electrical synaptic transmission in vertebrate nervous systems ( Connors and Long , 2004; Miller et al . , 2017; Rash et al . , 2013; Söhl and Willecke , 2004 ) .\",\n \"In neurons , ZO1 immunostaining correlates with synapses containing Cx36 and its fish homologs ( Flores et al . , 2008; Li et al . , 2004; Marsh et al . , 2017; Yao et al . , 2014 ) , and its presence at synapses may play regulatory roles ( Flores et al . , 2008 ) , the nature of its contributions to electrical transmission remains unknown .\",\n \"Despite mounting evidence for the widespread dynamic functional contributions of electrical synapses to neural circuit function , the perception of the simplicity of their molecular organization remains .\",\n \"We hypothesized that scaffolding molecules form part of a multimolecular structure that is required for channel function akin to that found at chemical synapses .\",\n \"Here , we explore the functional role of ZO1 in zebrafish by examining identifiable synaptic contacts of the Mauthner cell ( Bartelmez , 1915; Bodian , 1937; Hildebrand et al . , 2017; Kimmel , 1982; Robertson et al . , 1963 ) , which forms stereotyped electrical synapses accessible to genetic , biochemical , cell biological , electrophysiological , and behavioral analyses .\",\n \"We show that the presence ZO1 protein is critically required for the structure and function of the intercellular channels .\",\n \"Moreover , we find that the localization of ZO1 is compartmentalized postsynaptically where it functions in the formation of neuronal GJs .\",\n \"Our results stand in contrast with current views on electrical synapse organization centered solely on the proteins forming GJ channels .\",\n \"Thus , our findings provide strong support to the notion that electrical synapses constitute complex and asymmetric synaptic structures at which intercellular channels are governed by multimolecular structures with features that parallel the molecular and functional organization of the PSD at chemical synapses .\"\n ],\n [\n \"We sought to examine the relationship between the intracellular scaffold ZO1 and neuronal Connexins ( Cxs ) by utilizing the stereotyped synapses of the zebrafish Mauthner cell .\",\n \"This circuit drives a fast escape response to threatening stimuli using both electrical and chemical connections ( Eaton et al . , 1977; Jacoby and Kimmel , 1982; Liu and Fetcho , 1999; Wolman et al . , 2015 ) .\",\n \"Each animal has two Mauthner cells that receive multimodal sensory input that relay information to the spinal cord to coordinate circuits to elicit fast turns .\",\n \"We focus on two populations of stereotyped electrical synapses made by Mauthner cells: ( 1 ) ‘club ending’ ( CE ) synapses ( Bartelmez and Hoerr , 1933; Pereda et al . , 2004; Yao et al . , 2014 ) , which are mixed electrical/glutamatergic chemical synaptic contacts formed between auditory afferents of the eighth cranial nerve and the Mauthner cell's lateral dendrite ( Figure 1A , B ) and ( 2 ) en passant electrical synapses formed between the Mauthner axon and Commissural Local ( CoLo ) interneurons found in each spinal-cord segment ( Figure 1A , M/CoLo synapses ) ( Satou et al . , 2009 ) .\",\n \"Neuronal GJs at both CEs and M/CoLo synapses are made of heterotypic channels formed by Cx35 . 5 , encoded by the gene gap junction delta 2a ( gjd2a ) , and Cx34 . 1 , encoded by gjd1a , both homologous to mammalian Cx36 ( gjd2 ) .\",\n \"We previously found that Cx35 . 5 and Cx34 . 1 are localized asymmetrically to the pre- and postsynaptic sides at CE and M/CoLo synaptic contacts ( Figure 1B; Miller et al . , 2017 ) .\",\n \"Throughout we use the terms pre- and postsynaptic to reference the neuronal , cell-biological compartment in which a Connexin is localized .\",\n \"At CE synapses , the auditory afferent axons are presynaptic to the postsynaptic Mauthner lateral dendrite; while at M/CoLo synapses , the Mauthner axon is presynaptic to the postsynaptic CoLo .\",\n \"We first determined the localization of ZO1 and the Connexin proteins at Mauthner electrical synapses using immunofluorescence and confocal imaging .\",\n \"We stained 5 day post fertilization ( dpf ) larvae , a time at which the Mauthner circuit elicits a mature startle response , with antibodies against the human ZO1 protein and those that distinguish the zebrafish Cx35 . 5 and Cx34 . 1 ( Figure 1C–L; Miller et al . , 2017 ) .\",\n \"We observed extensive colocalized signal for these three proteins at CE and M/Colo electrical synapses , with each protein apparent in the stereotyped shape and position of the neuronal gap junctions ( GJs ) at these contacts .\",\n \"We identified CEs unambiguously as large ( 1 . 5–2 µm ) oval areas localized in the distal portion of the lateral dendrite of the Mauthner cell ( Figure 1C; Yao et al . , 2014 ) .\",\n \"M/Colo synapses were identified by their regularly spaced sites of contact in the spinal cord ( Figure 1K ) .\",\n \"Next , we examined the role of ZO1 at electrical synapses using CRISPR/Cas9-induced mutations to knock out gene function .\",\n \"Mammalian ZO1 is encoded by the gene tight junction protein 1 ( tjp1 ) , while zebrafish have two homologous genes , tjp1a and tjp1b .\",\n \"Using a CRISPR-based screen , we found that mutations in tjp1b/ZO1b , but not tjp1a/ZO1a , caused a failure of Connexin localization at M/CoLo synapses ( Figure 1; Figure 1—figure supplement 1; Marsh et al . , 2017; Shah et al . , 2015 ) .\",\n \"We examined the effect of these mutations on CEs and found that tjp1b/ZO1b-/- mutants lack most of the detectable fluorescent staining for both Cx35 . 5 and Cx34 . 1 , as well as ZO1 , at the stereotyped synaptic contact sites ( Figure 1D , L ) .\",\n \"Quantitation of Cx35 . 5 , Cx34 . 1 , and ZO1 fluorescence at CEs and M/CoLo contacts confirmed that staining for all three antibodies was greatly diminished in tjp1b/ZO1b-/- mutants ( Figure 1M , N ) .\",\n \"By contrast , homozygous tjp1a/ZO1a -/- mutants had extensive Connexin and ZO1 staining at these contacts , while tjp1a-/-; tjp1b-/- double mutants were indistinguishable from tjp1b-/- single mutants ( Figure 1—figure supplement 1A–G ) .\",\n \"We conclude that ZO1b protein , encoded by the tjp1b gene , is localized to electrical synapses and required for the robust localization of both Cx35 . 5 and Cx34 . 1 at synaptic contacts .\",\n \"While tjp1b/ZO1b-/- mutants showed significantly diminished levels of ZO1 and Connexin staining at presumptive synaptic locations , we wondered whether neurons were still attempting to assemble GJs .\",\n \"Indeed , we detected both Cx35 . 5 and Cx34 . 1 by western blot from brain homogenates of tjp1b/ZO1b-/- mutant animals ( Figure 1—figure supplement 1H ) .\",\n \"We therefore examined CEs using higher contrast and magnification to assess GJ structure as detectable by immunolabeling and individually stained for ZO1 or Connexin to avoid confounding the image analysis due to bleed through of signal amongst stained proteins .\",\n \"We found that in tjp1b/ZO1b-/- mutants , each of the three antibodies revealed structures located at the stereotyped position of CE contacts and had morphologies reminiscent of wild-type animals , albeit with much dimmer fluorescence intensity ( Figure 1E–J; note that image contrast was increased in mutants ( H-J ) ) .\",\n \"While we observed the stereotypical oval-shaped CE structures in mutants , the staining for each protein was weak and irregular in its distribution , suggesting the residual staining in mutants might represent incomplete , abortive synaptic structures ( see electrophysiology below ) .\",\n \"Consistent with a reduced presence of GJ proteins , we also observed a reduced number of CEs detected by immunolabeling in tjp1b/ZO1b-/- mutants ( Figure 1—figure supplement 1I ) .\",\n \"We found similar results at M/CoLo synapses , although their smaller size precluded an analogous detailed analysis ( Figure 1K , L , N ) .\",\n \"In contrast to tjp1b/ZO1b-/- , the staining of tjp1a/ZO1a-/- mutants was indistinguishable from wildtype ( Figure 1—figure supplement 1E–G , I ) .\",\n \"These observations suggest that neurons of the Mauthner cell network in tjp1b/ZO1b-/- mutants persist in attempting to create electrical synapses , despite their inability to robustly localize neuronal Connexins at synaptic contacts .\",\n \"We conclude that ZO1 is localized to electrical synapses where it plays a critical role in neuronal GJ formation .\",\n \"Given that Connexin localization was dependent on ZO1b , we sought to determine if the converse was true – did ZO1 localization require Connexins ?\",\n \"Using previously generated mutations in gjd2a/Cx35 . 5 and gjd1a/Cx34 . 1 ( Miller et al . , 2017 ) , we examined the localization of Connexin and ZO1 proteins in mutants by immunolabeling ( Figure 2A–L ) .\",\n \"First , we found that gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant animals revealed a complete loss of detectable staining for the mutated protein .\",\n \"In addition , there was a failure of the non-mutated Connexin protein to robustly localize to the electrical synapse although low levels of staining were present .\",\n \"In line with these observations , by using brain homogenates and western blots , we found a complete loss of the Connexin affected by each mutation , but no effect on the non-mutated Connexin protein ( Figure 1—figure supplement 1H ) .\",\n \"By contrast , ZO1 staining in the Connexin mutants was robust at the synaptic contact sites with the stereotyped appearance , distribution , and position clearly evident ( Figure 2A–L ) .\",\n \"By comparing the relative ZO1 fluorescence between wild-type and Connexin mutant animals , we found that ZO1 was present at synaptic contacts at approximately half the normal level ( Figure 2M , N ) .\",\n \"We examined gjd2a-/-; gjd1a-/- double mutants and found that ZO1 still robustly localized to CE and M/CoLo contact sites ( Figure 2M , N; Figure 2—figure supplement 1A–F ) .\",\n \"These results reveal two critical organizational principles about electrical synapses in the Mauthner cell: ( 1 ) ZO1b localizes to putative electrical synaptic sites largely independent of Connexin proteins and ( 2 ) each Connexin requires the other for robust localization to the synapse .\",\n \"Based on these data , we conclude that ZO1 can localize to neuronal GJs independent of the presence of channel-forming proteins , yet ZO1 is absolutely essential for proper Connexin localization .\",\n \"To further examine the electrical synapse structure of Connexin mutants , we assessed CEs at higher contrast ( Figure 2D–I ) .\",\n \"We found that: ( 1 ) immunofluorescence for the mutated Connexin is completely lost at the synapse , ( 2 ) the non-mutated Connexin is detectable but with weak and irregular labeling , suggestive of incomplete , abortive structures , and ( 3 ) ZO1 labeling resembles wildtype with a distribution and morphology that appears normal across the expanse of the putative synaptic contact .\",\n \"Consistent with these findings , the number of CEs detected by ZO1 labeling in Connexin mutants was indistinguishable from that observed in wildtype , while those detected by antibodies for the mutated Connexin were significantly reduced ( Figure 1—figure supplement 1I ) .\",\n \"In addition , the zebrafish genome contains two additional homologous Connexin genes , gjd2b/Cx35 . 1 and gjd1b/Cx34 . 7 .\",\n \"We found that animals that were homozygous mutant for these two genes had normal Connexin and ZO1 labeling at CEs ( Figure 2—figure supplement 1G–L ) .\",\n \"Similarly , we previously found there was no effect on M/CoLo synapses in the gjd2b/Cx35 . 1 and gjd1b/Cx34 . 7 mutants ( Miller et al . , 2017 ) .\",\n \"Together , these results support a hierarchical relationship in the formation of neuronal GJs , in which ZO1 localizes to electrical synaptic contact sites where it is essential to robustly localize neuronal Connexins .\",\n \"We next sought to examine the functional consequences of ZO1 and Connexin mutants , so we explored the properties of synaptic transmission at CEs during whole-cell recordings of the Mauthner cell .\",\n \"In wildtype zebrafish , extracellular stimulation of CE afferents near the posterior macula where they contact hair cells ( Figure 3A ) evoked a mixed synaptic response in the Mauthner cell composed of an early and large GJ-mediated electrical component followed by a delayed and smaller glutamatergic chemical response ( Figure 3B; Yao et al . , 2014 ) .\",\n \"We first aimed to establish the functional consequences of removing the Connexins on this mixed synaptic response .\",\n \"Consistent with the presence of Cx35 . 5 and Cx34 . 1 at CEs , synaptic responses in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant zebrafish lacked a detectable electrical component , while exhibiting a response with the same delay as the chemical component of the mixed synaptic potential of wildtypes ( Figure 3C–E ) .\",\n \"By contrast , electrical transmission was unaffected in gjd2b/Cx35 . 1-/- and gjd1b/Cx34 . 7-/- mutant zebrafish ( Figure 3E; Figure 3—figure supplement 1A-B ) , as expected given our immunolabeling results ( Figure 2—figure supplement 1G-L ) .\",\n \"The apparent chemical response in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants was blocked by extracellular application of a combination of the AMPA and NMDA glutamate receptor ( GluR ) antagonists cyanquixaline ( CNQX ) and D-2-Amino-5-phosphonovaleric acid ( DAP5 ) ( gray traces in Figure 3C , D; Figure 3—figure supplement 1C ) .\",\n \"No change in membrane potential was observed after application of the blockers .\",\n \"To confirm that the chemical synaptic potential observed in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants arises from the stimulation of CEs lacking electrical transmission , we examined if blocking GJs with meclofenamic acid ( MA ) could reproduce the observed synaptic phenotype .\",\n \"We found that application of MA to wildtype recapitulated the gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant phenotype , as the characteristic mixed synaptic response was replaced by a larger chemical synaptic response that was sensitive to GluR antagonists ( Figure 3F; Figure 3—figure supplement 1C ) .\",\n \"We also observed a small hyperpolarization of the Mauthner cell ( from −80 . 2 ± 0 . 7 mV in control to −84 ± 1 mV in MA; p=0 . 04 , n = 5 ) , likely resulting from the action of MA on other membrane channels .\",\n \"We conclude that together Cx35 . 5 and Cx34 . 1 generate functional neuronal GJs at CEs .\",\n \"We next examined the properties of synaptic transmission in ZO1 mutants .\",\n \"Strikingly , and consistent with the requirement for Connexin localization at contact sites ( Figure 1 ) , tjp1b/ZO1b-/- mutants exhibited a failure in electrical transmission .\",\n \"The synaptic phenotype was indistinguishable from gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants , and similarly consisted of a single , delayed response that was blocked by GluRs antagonists ( Figure 3G; Figure 3—figure supplement 1D ) .\",\n \"This functional deficit was specific for ZO1b , as tjp1a/ZO1a-/- mutant fish exhibited normal mixed synaptic responses ( Figure 3H , I ) .\",\n \"These findings indicate the specificity of the mutants to electrical synapses as glutamatergic transmission at CEs remained intact .\",\n \"Accordingly , neurotransmitter release properties ( Figure 3—figure supplement 1E ) and the localization of GluR2/3 receptors at these terminals were not affected in mutant fish ( Figure 3—figure supplement 2 ) .\",\n \"We conclude that the presence of the ZO1b scaffold protein is critical for electrical transmission at CEs , even though its absence does not prevent the formation of glutamatergic synapses coexisting within the same contact .\",\n \"To investigate the extent of the deficit on electrical transmission within the brainstem network , we investigated whether Connexin and ZO1 mutations affected other synaptic contacts onto the Mauthner cell .\",\n \"The Mauthner cell receives mixed synaptic inputs from a variety of descending and ascending sensory modalities , including visual information from the optic tectum and somatic information from the spinal cord ( Dunn et al . , 2016; Faber and Pereda , 2011; Kimmel et al . , 1981; Korn and Faber , 2005 ) .\",\n \"For this purpose , we recorded spontaneous synaptic responses that represent the activity of most , if not all , synaptic inputs received by this cell .\",\n \"Electrically and chemically mediated spontaneous responses are easily differentiated due to their dramatically different duration ( Figure 4A ) .\",\n \"Thus , for automated detection purposes , we defined fast spontaneous events ( <1 . 1 ms ) , which represent the electrical coupling of presynaptic spikes , as electrical responses and slow spontaneous events ( >1 . 1 ms ) as chemical responses .\",\n \"Fast events were nearly absent in tjp1b/ZO1b-/- , gjd2a/Cx35 . 5-/- , and gjd1a/Cx34 . 1-/- mutants ( Figure 4B , C ) , and were dramatically reduced in wild-type fish by application of MA ( Figure 4C ) , confirming that they represent electrical transmission .\",\n \"Slow events that remained after losing the electrical component were dramatically reduced by application of GluRs antagonists ( Figure 4D ) , confirming that they represent chemical responses .\",\n \"We conclude that the deficit in electrical transmission observed in mutant zebrafish is likely to be widespread within hindbrain and spinal cord circuits .\",\n \"Given the primary role of the Mauthner cell in triggering escape responses , we also investigated possible changes in cellular excitability in mutant fish .\",\n \"Interestingly , the frequency of slow , chemical responses was increased in mutants and MA-treated animals ( Figure 4D ) , even though we observed no changes on presynaptic neurotransmitter release properties ( Figure 3—figure supplement 1E ) .\",\n \"This suggests that the lack of electrical transmission could have enhanced the detection of smaller amplitude chemical responses .\",\n \"We posit this could arise from electrical coupling influencing neuronal excitability , as the loss of neuronal GJs in mutants would increase the input resistance of the Mauthner cell ( Alcamí and Pereda , 2019 ) .\",\n \"Thus , during whole cell recordings , we determined the input resistance ( Rin ) , rheobase , resting potential ( Vrest ) , and firing threshold ( Vthreshold ) of the Mauthner cells from wildtype and mutant zebrafish ( Table 1 ) .\",\n \"The input resistance , the main determinant of neuronal excitability , was increased in tjp1b/ZO1b-/- , gjd2a/Cx35 . 5-/- , and gjd1a/Cx34 . 1-/- mutants .\",\n \"Accordingly , the rheobase , a parameter negatively correlated with neuronal excitability , was decreased in these mutant animals ( Table 1 ) .\",\n \"We found that the change in Rin correlated with the lack of electrical transmission and was not observed in fish that retained electrical transmission ( Figure 4E , F; note however that Rin in tjp1a/ZO1a-/- was found to slightly increase ) .\",\n \"Differences in magnitude of the effects observed on Rin could be due to distinct compensatory mechanisms in each case or to the mutations affecting channels contributing to leak conductance in the Mauthner cell .\",\n \"Thus , the lack of electrical transmission in tjp1b/ZO1b-/- fish rendered synaptic transmission exclusively mediated by a relatively delayed glutamatergic response and more excitable Mauthner cells .\",\n \"Both deficits likely influence the behavioral responses generated by the Mauthner cell and its associated network .\",\n \"Since tjp1b/ZO1b-/- mutants had functional deficits in electrical transmission , but not chemical , we assessed the consequences on the behavioral output of the Mauthner cell network .\",\n \"Animals were placed into individual chambers of a multi-well testing stage and presented with acoustic-vibrational stimuli to elicit Mauthner-dependent startle responses ( Wolman et al . , 2015 ) .\",\n \"Movements were captured with a high-speed camera ( 1000 frames per second ) and analyzed with FLOTE software to automatically track and measure the kinematics ( body movements ) of responses ( Burgess and Granato , 2007 ) .\",\n \"In this paradigm , wild-type fish exhibit two types of escape responses: ( 1 ) Mauthner-cell-dependent short-latency C-bends ( SLCs , hereafter referred to as ‘startles’ ) and ( 2 ) Mauthner-cell-independent long-latency C-bends ( LLCs ) .\",\n \"These two behavioral responses were automatically distinguished using well-established kinematic parameters ( Burgess and Granato , 2007 ) .\",\n \"Larvae generated from crossing tjp1b/ZO1b+/- heterozygous animals were tested and analyzed blind to genotype with subsequent post-hoc identification .\",\n \"We found that tjp1b/ZO1b-/- mutants startled to strong acoustic stimuli ( 25 . 9 dB ) as frequently as their wildtype siblings ( Figure 5A ) .\",\n \"While these turns were classified as startles , we found that the tjp1b/ZO1b-/- mutants initiated their responses ~ 2 ms slower than their wildtype siblings ( Figure 5B ) .\",\n \"A delayed behavioral response in consistent with our electrophysiological findings indicating that the mutant escape network operates with longer synaptic delays due to the lack of electrical transmission ( Figure 3 ) .\",\n \"In mutant animals , we found that the escapes were often performed with the normal startle kinematic parameters , particularly the maximum angle of the turn and the maximum angular velocity of the response , albeit occurring later than in wild-type siblings ( Figure 5C–H ) .\",\n \"However , we found a subset of mutant responses ( ~15% ) that showed abnormally shallow and slow turns ( Figure 5C , D red arrows ) .\",\n \"These abnormal responses suggested deficits in performing the stereotyped C-bend elicited by the Mauthner-cell network , and so we reanalyzed the video data from these responses .\",\n \"In these startles , we observed that the mutants displayed abnormal postures where the body would bend slightly to one side , creating ‘kinked’ or ‘S-shaped’ postures ( Figure 5I–L ) .\",\n \"We note that the phenotypes observed in the tjp1b/ZO1b-/- mutants are strikingly similar to those we previously observed in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants ( Miller et al . , 2017 ) .\",\n \"Such kinked body shapes are reminiscent of startles following CoLo neuron ablation ( Satou et al . , 2009 ) .\",\n \"Based on these data , we conclude that electrical synapses are essential for generating the speed and coordination of the Mauthner-induced startle response .\",\n \"The electrophysiological analysis of tjp1b/ZO1b-/- mutants also revealed that Mauthner cells showed increased excitability ( Figure 4 ) , suggesting animals may be hypersensitive with a lower threshold of response for environmental stimuli .\",\n \"To examine this possibility , we presented larvae with 60 pseudo-randomized stimuli , 10 at six different intensities with a 20 s inter-stimulus interval to eliminate habituation ( Wolman et al . , 2015 ) .\",\n \"We then assessed the frequency with which animals responded to the stimuli with turns ( Figure 5M–O ) .\",\n \"As stimulus intensity increased , both wildtype siblings and tjp1b/ZO1b-/- mutants increased the likelihood of performing a startle response .\",\n \"However , across the mid-range of stimulus intensities , tjp1b/ZO1b-/- mutants were more likely to respond with a startle than their wild-type siblings ( Figure 5M , O ) .\",\n \"The increased tendency of mutants to perform Mauthner-dependent startles came at the expense of Mauthner-independent LLC responses , which were nearly absent in mutants ( Figure 5N ) .\",\n \"We conclude that electrical synapses alter the sensitivity of Mauthner cells to environmental stimuli and contribute to the innate startle threshold , which alters the probability of eliciting a startle or LLC response .\",\n \"These results lend additional evidence for the critical role of ZO1b for creating functional electrical synapses , ultimately contributing to appropriately balanced neural network function and behavior .\",\n \"Mutant zebrafish revealed a hierarchical relationship between ZO1b and neuronal Connexins ( Figures 1 and 2 ) , so we next investigated the mechanisms underlying this relationship .\",\n \"ZO1 is a membrane-associated guanylate kinase ( MAGUK ) scaffold protein and contains PSD95/Dlg/ZO1 ( PDZ ) protein-protein interaction domains that bind to PDZ binding motifs ( PBMs ) ( Zhu et al . , 2016 ) .\",\n \"Previous work demonstrated that the C-terminal four amino acids of mouse Cx36 and perch Cx35 compose PBMs that are essential for interacting with ZO1 ( Flores et al . , 2008; Li et al . , 2004 ) .\",\n \"Given that the PBM sequence is conserved in zebrafish Cx35 . 5 and Cx34 . 1 proteins ( Figure 6A ) , we tested whether these Connexins could mediate binding to zebrafish ZO1b .\",\n \"We cloned full-length sequences of tjp1b/ZO1b , gjd2a/Cx35 . 5 , and gjd1a/Cx34 . 1 and used heterologous expression to test for interactions between the scaffold and Connexins .\",\n \"HEK293T cells were co-transfected with mVenus-ZO1b and full-length Cx35 . 5 or Cx34 . 1 .\",\n \"Using western blot analysis with antibodies specific to each Connexin , we found that both Cx34 . 1 and Cx35 . 5 were individually detected in mVenus-ZO1b immune complexes ( Figure 6B , lanes 1 , 3 ) compared to control immunoprecipitates ( Figure 6—figure supplement 1A , lanes 1 , 2 , 5 , 6 ) .\",\n \"We further found that removing the presumptive PBMs , by deleting the C-terminal four amino acids from both Connexins , resulted in a loss of co-purifying Connexins from mVenus-ZO1b immunoprecipitates ( Figure 6B , lanes 2 , 4; Figure 6—figure supplement 1A , lanes 4 , 8 ) .\",\n \"Control blots demonstrated the ability of the Connexin antibodies to equally recognize both full-length and ∆PBM versions of the proteins ( Figure 6B; Figure 6—figure supplement 1A , bottom input panels ) .\",\n \"We conclude that zebrafish Cx35 . 5 and Cx34 . 1 can interact with ZO1b in a PBM-dependent manner .\",\n \"The ZO1b scaffold has three PDZ domains that could mediate the interaction with neuronal Connexins ( Figure 6A ) .\",\n \"Previous studies testing the three mammalian ZO1 PDZ domains demonstrated that Cx35/36 PBMs exclusively bound to ZO1-PDZ1 , while PDZ2 and PDZ3 did not interact ( Flores et al . , 2008; Li et al . , 2004 ) .\",\n \"Given the high degree of conservation of the zebrafish ZO1 PDZ1 domain , including the amino acids of the putative PBM binding pocket ( Figure 6A ) , we tested whether zebrafish ZO1b-PDZ1 could directly interact with zebrafish neuronal Connexins .\",\n \"To examine this question , we isolated the minimal domains of each zebrafish protein , produced them in bacteria , and performed in vitro binding studies .\",\n \"We found that purified ZO1b-PDZ1 could be pulled down with a GST-Cx34 . 1 or a GST-Cx35 . 5 C-terminal intracellular-tail ( Figure 6C , lanes 2 , 4 ) , but not with control GST protein ( Figure 6C , lane1 ) .\",\n \"Further , this interaction was significantly decreased when the predicted PBM in the Connexin tails were removed ( Figure 6C , lanes 3 , 5 ) .\",\n \"We next used an overlay assay to compare the ability of ZO1b-PDZ1 and ZO1b-PDZ2 to bind to immobilized GST-Connexin tails .\",\n \"Similar to the binding assays , significant amounts of ZO1b-PDZ1 bound to GST-Cx34 . 1 and GST-Cx35 . 5 tails in a PBM-dependent manner ( Figure 6—figure supplement 1B , left panels ) .\",\n \"By contrast , little ZO1b-PDZ2 bound to the GST-Cx34 . 1 or GST-Cx35 . 5 tails , particularly when compared to the non-neuronal Cx43 C-terminal tail ( Figure 6—figure supplement 1B , right panels ) , which is known to bind PDZ2 ( Flores et al . , 2008; Li et al . , 2004 ) .\",\n \"We conclude that ZO1b utilizes the PDZ1 domain to directly interact with the neuronal Connexin PBMs .\",\n \"Since ZO1b and the neuronal Connexins colocalized at electrical synapses ( Figure 1 ) , we next investigated whether these proteins interacted in vivo .\",\n \"We utilized adult zebrafish brains that maintain widespread electrical synapses , including those in Mauthner cell ( Kimmel et al . , 1981 ) , and provide an abundant source to derive ZO1b immunoprecipitates .\",\n \"Homogenates derived from wild-type fish brains were immunoprecipitated with anti-ZO1 and control antibodies ( mIgG ) .\",\n \"Immunoprecipitates demonstrated that Cx34 . 1 copurified with ZO1 , whereas Cx35 . 5 did not copurify with the anti-ZO1 antibody ( Figure 6D , lanes 1 , 2 ) .\",\n \"To confirm that copurification of Cx34 . 1 was dependent upon ZO1b , we replicated the experiment using homogenates from tjp1b/ZO1b-/- mutants and found that Cx34 . 1 was lost in these immmunocomplexes ( Figure 6D , lane4 ) .\",\n \"We conclude that ZO1 preferentially interacts with Cx34 . 1 in vivo .\",\n \"We observed multiple ZO1 bands upon Western analysis of the immunoprecipitates , several of which were lost in the tjp1b/ZO1b-/- mutants ( Figure 6D , lanes 2 , 4 ) .\",\n \"Since the ZO1 antibody was made against human protein , we reasoned it might be detecting the ZO1b protein and the highly similar ZO1a protein , so we sought evidence to confirm that ZO1b was the primary scaffold for Cx34 . 1 in vivo .\",\n \"Upon examining ZO1 immunocomplexes from wildtype , tjp1b/ZO1b-/- , and tjp1a/ZO1a-/- brains , we found that only ZO1b deficiency resulted in concomitant loss of Cx34 . 1 ( Figure 6—figure supplement 1C ) .\",\n \"Taken together , we conclude that ZO1b preferentially interacts with Cx34 . 1 in vivo , despite the fact that the scaffold can interact with either neuronal Connexin .\",\n \"Next , we determined the functional relevance for a preferential ZO1b/Cx34 . 1 interaction at zebrafish electrical synapses .\",\n \"We previously observed that Cx35 . 5 localizes presynaptically in axons , while Cx34 . 1 localizes postsynaptically in dendrites ( Miller et al . , 2017 ) .\",\n \"Since ZO1b preferentially interacts with Cx34 . 1 in vivo ( Figure 6 ) , we speculated that ZO1b might share a similar dendritically compartmentalized localization .\",\n \"To directly examine this , we visualized the localization of ZO1b protein after inserting a V5 epitope at the N-terminus of the endogenous tjp1b locus ( Figure 7—figure supplement 1A ) .\",\n \"We found that in transgenicV5-tjp1b larvae , V5 antibody staining colocalized with both Cx34 . 1 and Cx35 . 5 at CEs and M/CoLo synaptic contacts ( Figure 7—figure supplement 1B–E ) .\",\n \"Moreover , we found no effect on the stereotyped patterns of Connexin staining at Mauthner electrical synaptic contact sites in homozygous V5-tjp1b/V5-tjp1b animals ( Figure 7—figure supplement 1D , E ) , suggesting that V5-ZO1b is functional .\",\n \"While V5-tjp1b larvae permitted ZO1b visualization at electrical synapses , we could not discriminate whether it was localized asymmetrically at synaptic contacts due to the small size of these structures and the resolution limits of light microscopy .\",\n \"To overcome this , we utilized an alternate approach to address ZO1b compartmentalization , exploiting the fact that the Mauthner cell is both the postsynaptic partner at CEs and the presynaptic partner at the M/CoLo synapses ( Figure 7A ) .\",\n \"We generated chimeric embryos by blastula transplantation ( Kemp et al . , 2009 ) , extracted GFP and V5-ZO1b expressing transgenic cells from donor embryos , and transferred them to wild-type hosts ( Figure 7B ) , allowing us to address V5-ZO1b localization within the Mauthner cell ( Figure 7C–E ) .\",\n \"In animals containing only a donor-derived , V5-ZO1b-expressing Mauthner cell , we found that V5 staining was present at the CEs with Connexin staining , demonstrating that ZO1b was within the dendrite of the Mauthner cell and localized postsynaptically at electrical synapses ( Figure 7D ) .\",\n \"Conversely , when we examined the M/CoLo synapses of these same embryos , we found that V5 staining was not present at these synaptic contacts despite the Mauthner cell expressing V5-ZO1b protein ( Figure 7E ) .\",\n \"We note that in the non-chimeric , V5-ZO1b transgenic animals , V5 staining was observed at the M/CoLo synapses ( Figure 7—figure supplement 1C , E ) , suggesting that ZO1b at these synapses derives from the postsynaptic CoLo .\",\n \"Our transplant experiments produce chimeric larvae in which only Mauthner , only CoLo , or both cells are derived from the transgenic donor embryos ( Figure 7—figure supplement 1F–H ) .\",\n \"In larvae in which only CoLo expresses V5-ZO1b , we found that V5 staining is present at M/CoLo synapses ( Figure 7—figure supplement 1G ) , confirming ZO1b’s postsynaptic localization .\",\n \"We conclude that ZO1b is robustly compartmentalized within the somato-dendritic compartment of the neuron and asymmetrically localized on the postsynaptic side of the electrical synapse .\",\n \"We then addressed whether ZO1b functions postsynaptically to facilitate Connexin localization at the electrical synapse .\",\n \"We utilized chimeric animals and again took advantage of Mauthner cell morphology .\",\n \"However , in these experiments , we transplanted GFP-expressing cells from tjp1b/ZO1b-/- mutant donors into wildtype hosts , producing animals in which ZO1b was specifically removed from the Mauthner cell ( Figure 7F–I ) .\",\n \"At CEs , the removal of ZO1b exclusively from the Mauthner cell resulted in the loss of staining for ZO1 , Cx34 . 1 , and Cx35 . 5 ( Figure 7H; Figure 7—figure supplement 1I ) .\",\n \"The data indicates that ZO1b is required postsynaptically for the cell autonomous localization of Cx34 . 1 and non-autonomously for presynaptic Cx35 . 5 localization .\",\n \"By contrast , when we examined the same chimeric animals with ZO1b removed from the Mauthner cell but focused on the M/CoLo synapses , in which Mauthner is presynaptic , there was no effect on either Connexin or ZO1 staining ( Figure 7I; Figure 7—figure supplement 1J ) , indicating that ZO1b is dispensable presynaptically .\",\n \"As further support for this compartmentalized scaffold function , we reasoned that resupplying ZO1b to the Mauthner cell in an otherwise tjp1b/ZO1b-/- mutant animal would be sufficient to rescue Connexin localization at CEs , but not at M/CoLo synapses .\",\n \"To test this prediction , we transplanted GFP-expressing cells from wildtype donor embryos into tjp1b/ZO1b-/- mutant hosts and identified animals with donor-derived Mauthner cells .\",\n \"In such chimeras , where the tjp1b/ZO1b gene is functional only in the Mauthner cell , CEs had normal staining for both postsynaptic ZO1 and Cx34 . 1 and also for presynaptic Cx35 . 5 .\",\n \"By contrast , there was no rescue of staining for these proteins at M/CoLo synaptic contacts ( Figure 7J , K; Figure 7—figure supplement 1J ) .\",\n \"We conclude that ZO1b is both necessary and sufficient postsynaptically for building the structure of the neuronal GJ channels .\",\n \"Taken together , we find that ZO1b is exclusively localized to the postsynaptic compartment where it functions both cell-autonomously and non-autonomously to localize Connexins and build functional neuronal gap junctions .\"\n ],\n [\n \"Despite the continuous nature of electrical transmission , the thousands of channels that make up GJ plaques found at electrical synapses are maintained by the active turnover of Connexin proteins ( Flores et al . , 2012 ) , similar to neurotransmitter receptors at chemical synapses ( Carroll and Zukin , 2002; Chen et al . , 2000; Ehlers , 2000; Lüscher et al . , 1999 ) .\",\n \"Our findings show that ZO1b is required for the robust localization of Connexins , suggesting it functions to stabilize Connexin hemichannels at the synaptic site .\",\n \"Additionally , ZO1 can localize to electrical synapses in the absence of the Connexins , although its apparent concentration was diminished .\",\n \"This suggests that building robust neuronal GJ structures involves a reciprocal interaction between ZO1 and the Connexins .\",\n \"Strikingly , our results reveal that ZO1b is compartmentalized to the dendrite and functions asymmetrically at postsynaptic sites of the Mauthner cell circuit .\",\n \"This localization is consistent with ZO1b’s preferential in vivo interaction with Cx34 . 1 , as this Connexin is also localized and required postsynaptically at Mauthner cell electrical synapses ( Miller et al . , 2017 ) .\",\n \"Despite the apparent autonomous ZO1b/Cx34 . 1 postsynaptic interaction at the GJ hemiplaque , tjp1b/ZO1b-/- mutants revealed that presynaptic Cx35 . 5 localization is also affected non-autonomously in the neighboring cell .\",\n \"This trans-synaptic interaction likely occurs via the Connexins themselves , as mutations to the postsynaptic Cx34 . 1 prevented the robust localization of the presynaptic Cx35 . 5 .\",\n \"Our results are complementary to recent analysis of the mouse rod/cone network , where removing Cx36 from one neuron of a coupled pair results in the failure of Connexin localization in the adjacent neuron ( Jin et al . , 2020 ) .\",\n \"Taken together , our results reveal that ZO1b acts as a postsynaptic molecular scaffold that localizes Cx34 . 1 to the GJ hemiplaque , which in turn ensures Cx35 . 5 stabilization at presynaptic hemiplaques ( Figure 7C ) .\",\n \"Whether presynaptic Cx35 . 5 lacks an in vivo scaffold , or utilizes another unidentified presynaptic scaffolding protein , remains unresolved .\",\n \"If the molecular function and organization of ZO1 revealed here applies to all electrical synapses , including those formed by homotypic channels between various homologous cellular processes ( dendro-dendritic , somato-somatic , or axo-axonic ) , remains to be determined in future studies .\",\n \"Never-the-less , we posit that the molecular organization of the electrical synapse can be asymmetrically compartmentalized , thereby enabling preferential biochemical interactions at each side of the junction .\",\n \"Our results support the prediction that ZO1 likely plays distinct functional roles at GJs in different tissue types .\",\n \"ZO1 was first described at epithelial tight junctions and later shown to interact with various Connexins , notably with Cx43 , a widespread Connexin expressed in many non-neuronal cell types ( Giepmans , 2004 ) .\",\n \"Evidence from cell expression systems suggested that ZO1 played critical functions on the periphery of Cx43-contaning GJ plaques forming part of the ‘perinexus’ to facilitate newly inserted hemichannels at each side of the junction ( Rhett and Gourdie , 2012 ) .\",\n \"Further , the ZO1/Cx43 interaction was critical for GJ communication before the channel ‘ages’ and is subsequently removed during channel turnover , a process governed by Cx43 phosphorylation ( Laird , 1996; Laird , 2006; Márquez-Rosado et al . , 2012; Solan and Lampe , 2016; Thévenin et al . , 2017 ) .\",\n \"However , preventing the ZO1/Cx43 interaction does not prevent GJ formation ( Hunter and Gourdie , 2008; Hunter et al . , 2003; Hunter et al . , 2005 ) .\",\n \"By contrast , our results demonstrated that ZO1b’s presence was asymmetric and required for robust Connexin localization and synaptic function .\",\n \"Beyond our observations here , ZO1 is likely to have homeostatic functions in the modulation of Connexin usage at electrical synapses .\",\n \"For example , analysis of the interactions between ZO1 and Cx36 ( and its fish homologs ) revealed the interactions occurs via a different PDZ domain than the interaction with Cx43 and the interaction with Cx36 has lower affinity and faster kinetics than that of Cx43 ( Flores et al . , 2008; Li et al . , 2004 ) .\",\n \"This suggests ZO1 has a more dynamic interaction with neuronal Connexins , which may serve the plastic , activity-dependent regulation of electrical transmission observed in fish and mammalian electrical synapses ( Haas et al . , 2011; Landisman and Connors , 2005; Mathy et al . , 2014; Pereda and Faber , 1996; Pereda et al . , 1998; Turecek et al . , 2014; Yang et al . , 1990 ) .\",\n \"Thus , our findings suggest that ZO1’s function at electrical synapses differs from its role at Cx43-containing GJs , perhaps serving a specialized function in synaptic communication .\",\n \"Our results highlight that neural circuit function requires functional electrical and chemical synapses to create an appropriate behavioral response .\",\n \"Our data indicates that the lack of electrical transmission in ZO1b and Connexin mutants did not prevent the formation of co-existing glutamatergic synapses at CEs on the Mauthner cell .\",\n \"The remaining chemical transmission supported a behavioral response organized by the Mauthner-cell network , although importantly , the response showed deficits in performance and altered sensitivity .\",\n \"Given that the startle behavior mediates predator avoidance , these defects would likely be detrimental to survival ( Hecker et al . , 2020 ) .\",\n \"The lack of effect on glutamatergic transmission contrasts a wealth of data supporting a strong , interdependent relationship between the formation of electrical and chemical synapses during development in both invertebrate and vertebrate nervous systems ( Jabeen and Thirumalai , 2018 ) .\",\n \"For example , in the leech , knockdown of Innexins at a developmental stage where synaptic contacts are solely electrically coupled prevents the formation of later-forming chemical transmission ( Todd et al . , 2010 ) .\",\n \"Similarly , in the developing mouse neocortex , dominant negative constructs of Cx26 prevent the initial electrical coupling and subsequent chemical synapse formation amongst sister excitatory neurons within ontogenetic columns ( Yu et al . , 2012 ) .\",\n \"Our findings indicate that this deficit does not occur at zebrafish CEs when Cx35 . 5 , Cx34 . 1 , and ZO1b are removed .\",\n \"Whether this is due to the differences in the GJ proteins used in the Mauthner cell or instead due to mechanisms that specify CE formation , remains unknown .\",\n \"One intriguing possibility is that other proteins may act as a common synaptic control mechanism that is independent of GJ-forming proteins .\",\n \"Indeed , mutations in the scaffold Neurobeachin caused parallel defects in the formation of both electrical and chemical synapses of the Mauthner circuit ( Miller et al . , 2015 ) , yet the mechanism by which this coordination occurs remains to be elucidated .\",\n \"Alternatively , rather than functional ( conductive ) GJ channels , other structural components of the electrical synapse may be sufficient to trigger chemical synapse formation via protein-protein interactions .\",\n \"We posit such interactions would apply to mammalian electrical synapses , which can co-exist with neighboring glutamatergic synapses at distances comparable to those found in fish mixed synapses ( Nagy et al . , 2018 ) and may mediate similar functional interactions .\",\n \"Identifying the functional relevance of an intracellular scaffolding protein as a critical part of the electrical synapse draws parallels with our understanding of chemical transmission , where neurotransmitter receptors are clustered and modified by a rich network of postsynaptic proteins that dynamically shape synaptic structure and function .\",\n \"This chemical synapse protein network is known as the ‘postsynaptic density’ ( PSD ) , a term resulting from its structural identification by EM ( Cohen , 2013; Palay , 1956 ) and is composed of hundreds of unique proteins ( Grant , 2019 ) .\",\n \"Mirroring those findings , EM images of electrical synapses in mammals ( Llinas et al . , 1974 ) and fish ( Brightman and Reese , 1969 ) revealed the presence of clearly identifiable electrodense structures , first described as ‘semi-dense material’ by Sotelo and Korn , 1978 .\",\n \"These electrodense structures are localized intracellularly and form an undercoating band at neuronal GJs .\",\n \"The identified structures are presumably formed by a proteinaceous ‘organelle’ that resembles that found at PSDs ( Feng et al . , 2019 ) .\",\n \"As evidence grows for an array of proteins localized to electrical synapses , we propose that ZO1 is member of such an organelle .\",\n \"Our data provide enticing hints that the molecular framework of the electrical synapse extends beyond the ZO/Connexin interaction .\",\n \"In particular , the fact that ZO1 can localize to sites of synaptic contact independent of the Connexins , and that there remains immunofluorescent staining at Mauthner cell synapses in tjp1b/ZO1b-/- mutants , both imply the existence of additional proteins that contribute to this synaptic structure .\",\n \"We presume the remaining ZO1 staining in tjp1b/ZO1b-/- mutants comes from either the paralogous ZO1a protein ( tjp1a ) or from the related ZO2 ( tjp2a , tjp2b ) and ZO3 ( tjp3 ) proteins .\",\n \"However , our previous analysis of ZO2/ZO3 mutants in zebrafish did not reveal overt defects in Connexin localization ( Marsh et al . , 2017 ) , yet both proteins are localized to mammalian electrical synapses ( Li et al . , 2009 ) .\",\n \"Whether these related scaffolds have functional roles at electrical synapses that were undetected in our initial screen remains to be determined .\",\n \"Beyond the ZO-family , other molecules can directly interact with Cx36 and/or localize at mammalian electrical synapses , such as cell adhesion molecules , cytoskeletal interacting proteins , and molecules that regulate Connexin post-translational modifications ( Lynn et al . , 2012; Martin et al . , 2020; O'Brien and Bloomfield , 2018 ) .\",\n \"Yet , the molecular roles of these proteins at the electrical synapse remain poorly defined .\",\n \"Therefore , as opposed to simple aggregates of intercellular channels , we propose that electrical synapses are complex synaptic structures at which communicating pre- and postsynaptic Connexin hemichannels are governed by a yet to be determined mechanism that builds an asymmetric molecular scaffold .\",\n \"Based on its analogy to the known functions of glutamatergic PSDs , we propose to name this organizational organelle the ‘electrical synapse density’ , or ‘ESD’ ( Lynn et al . , 2012; Miller and Pereda , 2017; Figure 7C ) .\",\n \"Chemical synapses are organized to match their specific functional requirements by combining presynaptic release properties with unique combinations of postsynaptic receptors .\",\n \"Electrical synapses also provide a variety of specific synaptic functions , yet we know little about the source of their diversity .\",\n \"Work in C . elegans exposed the large variety of Innexin expression in neurons contributing to neural circuits , with dozens of potentially unique cellular combinations that are altered during development and following environmental stress ( Bhattacharya et al . , 2019 ) .\",\n \"These observations have greatly increased the appreciation of the incidence and potential functional diversity of electrical transmission in invertebrates .\",\n \"Our results indicate that the complexity of electrical synaptic transmission must also include their molecular scaffolds .\",\n \"Scaffold diversity may be more relevant for the function of vertebrate electrical synapses , which in contrast to C . elegans , are formed by a smaller number of GJ-forming proteins , with most electrical communication being reliant on Cx36-related proteins investigated here .\",\n \"By promoting and regulating channel trafficking and regulatory molecules , ZO1 , and other scaffolding proteins , could support a variety of functions by governing channel function and the local regulatory environment .\",\n \"Thus , future investigations will further expose the molecular composition and functional roles of ZO1 and the ESD .\",\n \"Unraveling the functional complexity of the ESD will lead to a deeper understanding of the diversity of the molecular organization underlying electrical transmission and its contributions to brain function .\"\n ],\n [\n \"Fish were maintained in the University of Oregon’s and the Albert Einstein College of Medicine fish facilities with approval from Institutional Animal Care and Use Committees of each institution .\",\n \"Zebrafish , Danio rerio , were bred and maintained at 28°C on a 14 hr on and 10 hr off light cycle .\",\n \"Animals were housed in groups , generally of 25 animals per tank .\",\n \"Development time points were assigned via standard developmental staging ( Kimmel et al . , 1995 ) .\",\n \"All fish used for this project were maintained in the ABC background developed at the University of Oregon .\",\n \"Most fish had the enhancer trap transgene zf206Et ( M/CoLo:GFP ) in the background ( Satou et al . , 2009 ) , unless otherwise noted .\",\n \"Mutant lines were genotyped for all experiments .\",\n \"All immunohistochemistry , electrophysiological , and behavioral experiments were performed at 5 dpf .\",\n \"At this stage of development , zebrafish sex is not yet determined ( Wilson et al . , 2014 ) .\",\n \"Protein extractions were performed from both male and female adult brains and combined .\",\n \"HEK293T/17 verified cells were purchased from ATCC ( CRL-11268; STR profile , amelogenin: X ) .\",\n \"Cells were expanded and maintained in Dulbecco's Modified Eagle's Medium ( DMEM , ATCC ) plus 10% fetal bovine serum ( FBS , Gibco ) at 37°C in a humidified incubator in the presence of 5% CO2 .\",\n \"Low passage aliquots were cryopreserved and stored according to manufacturer’s instructions .\",\n \"Cells from each thawed cryovial were monitored for mycoplasma contamination using the Universal Mycoplasma Detection Kit ( ATCC , 30–1012K ) .\",\n \"Anesthetized , 5–6 days post fertilization ( dpf ) larvae were fixed for 3 hr in 2% trichloroacetic acid in PBS .\",\n \"Fixed tissue was washed in PBS + 0 . 5% Triton X-100 , followed by standard blocking and antibody incubations .\",\n \"Primary antibody mixes included combinations of the following: rabbit anti-Cx35 . 5 ( Fred Hutch Antibody Technology Facility , clone 12H5 , 1:800 ) , mouse IgG1 anti-Cx35 . 5 ( Fred Hutch Antibody Technology Facility , clone 4B12 , 1:250 ) , rabbit anti-Cx34 . 1 ( Fred Hutch Antibody Technology Facility , clone 3A4 , 1:250 ) , mouse IgG2A anti-Cx34 . 1 ( Fred Hutch Antibody Technology Facility , clone 5C10A , 1:350 ) , mouse IgG1 anti-ZO1 ( Invitrogen , 33–9100 , 1:350 ) , mouse IgG2a anti-V5 peptide ( Invitrogen , R960-25 , 1:50 ) , and chicken anti-GFP ( abcam , ab13970 , 1:350- 1:500 ) .\",\n \"All secondary antibodies were raised in goat ( Invitrogen , conjugated with Alexa-405 , –488 , −555 , or −633 fluorophores , 1:500 ) .\",\n \"Tissue was then cleared stepwise in a 25% , 50% , 75% glycerol series , dissected , and mounted in ProLong Gold antifade reagent ( ThermoFisher , P36930 ) .\",\n \"Images were acquired on a Leica SP8 Confocal using a 405-diode laser and a white light laser set to 499 , 553/554/557 ( consistent within experiments ) , and 631 nm , depending on the fluorescent dye imaged .\",\n \"Each laser line’s data was collected sequentially using custom detection filters based on the dye .\",\n \"Quantitative images of the Club Endings ( CEs ) were collected using a 63x , 1 . 40 numerical aperture ( NA ) , oil immersion lens , and images of M/Colo synapses were collected using a 40x , 1 . 20 NA , water immersion lens .\",\n \"For each set of images , the optimal optical section thickness was used as calculated by the Leica software based on the pinhole , emission wavelengths , and NA of the lens .\",\n \"Within each experiment where fluorescence intensity was to be quantified , all animals ( including 3–5 wildtype controls ) were stained together with the same antibody mix , processed at the same time , and all confocal settings ( laser power , scan speed , gain , offset , objective , and zoom ) were identical .\",\n \"Multiple animals per genotype were analyzed to account for biological variation .\",\n \"To account for technical variation , fluorescence intensity values for each region of each animal were an average across multiple synapses .\",\n \"For high-contrast imaging of the CEs , fixed samples were washed three times with PBS , incubated at 4°C overnight , and hindbrains were dissected out .\",\n \"Dissected hindbrains were washed , blocked , and stained as above with the following primary antibodies: rabbit anti-Cx35 . 5 ( 12H5 , 1:500 ) , mouse anti-Cx35/36 ( EMD Millipore , MAB3045 , 1:250 ) , mouse IgG2A anti-Cx34 . 1 ( 5C10A , 1:200 ) , mouse IgG1 anti-ZO1 ( 33–9100 , 1:200 ) , rabbit anti-GFP ( Invitrogen , G10362 , 1:200 ) , and chicken anti-GFP ( Invitrogen , A10262 , 1:200 ) .\",\n \"Secondary antibodies were raised in goat ( Invitrogen , conjugated with Alexa-405 , –488 , −546 , –555 , −633 , or −647 fluorophores , 1:200 ) .\",\n \"Samples were then transferred onto a slide in the dark and mounted with Fluoromount-G ( Southern Biotech , 0100–01 ) , covered using the standard ‘bridge’ procedure , and sealed with nail polish .\",\n \"Samples were imaged on LSM 710 and LSM 880 Zeiss microscopes using appropriate laser wavelengths and detection filters .\",\n \"Image stacks of roughly 20 µm were collected using a 63x , 1 . 40 numerical aperture ( NA ) , oil immersion lens .\",\n \"For each Mauthner , laser strengths and gains were adjusted to achieve maximum visualization of CE staining .\",\n \"Electrophysiological responses were obtained during whole-cell recordings of Mauthner cells ( M-cells ) in wt , Cx and ZO-1 mutant zebrafish larvae ( 5–7 dpf ) .\",\n \"For this purpose , fish were first anesthetized with a 0 . 03% solution of MS222 ( pH adjusted to 7 . 4 with NaHCO3 ) and later transferred to external solution containing d-tubocurarine ( 10 μM , Sigma ) .\",\n \"The external solution ( in mM ) : 134 NaCl , 2 . 9 KCl , 2 . 1 CaCl2 , 1 . 2 MgCl2 , 10 HEPES , 10 Glucose , pH adjusted to 7 . 8 with NaOH ( Yao et al . , 2014 ) .\",\n \"Zebrafish larvae were put on their backs onto a Sylgard-coated small culture dish ( FluoroDish , WPI ) and kept in place using fine tungsten pins .\",\n \"The hindbrain was then exposed ventrally following the dissection approach previously described ( Koyama et al . , 2011 ) .\",\n \"Following this procedure , the larvae were placed on an Axio Examiner upright microscope ( Carl Zeiss AG ) equipped with a recording set-up and superfused with external solution during the entire recording session .\",\n \"The M-cells were identified by GFP expression and/or far-red DIC optics .\",\n \"Patch pipettes ( 3–4 MΩ ) were filled with internal solution ( in mM ) : 105 K-Methanesulfonate , 10 HEPES , 10 EGTA , 2 MgCl2 , 2 CaCl2 , 4 Na2ATP , 0 . 4 Tris-GTP , 10 K2-Phosphocreatine , 25 mannitol , pH adjusted to 7 . 2 with KOH .\",\n \"Whole-cell recordings under the current-clamp configuration were performed with a Multiclamp 700B amplifier and a Digidata 1440A ( Molecular Devices ) digitizer .\",\n \"The liquid-liquid junction potential was estimated in −16 mV using Clampex 10 . 6 ( Molecular Devices ) and was subtracted from the measured values .\",\n \"The electrode’s resistance was compensated using the bridge balance feature of the amplifier .\",\n \"To activate the auditory afferents terminating as CEs on the M-cell , a septated ( theta ) glass pipette was filled with external solution and positioned near the posterior macula of the ear , where the dendritic processes of auditory afferents contact the hair cells ( Yao et al . , 2014 ) .\",\n \"The maximal amplitude of the electrical and chemical components was estimated by applying shocks of increasing intensity until the amplitude of the electrical component did not further increase and before additional responses with longer latency were evoked .\",\n \"To estimate the Paired-Pulse Ratio ( PPR ) of the chemical component one , a stimulating-pulse was applied to record 10–20 traces in basal conditions .\",\n \"Then two-stimulating pulses ( 2 ms apart ) were applied to record ( 10–20 traces ) facilitation of the chemical component .\",\n \"Traces that clearly showed a chemical component were averaged for basal and facilitated conditions .\",\n \"The trace in basal conditions was subtracted from the facilitated trace .\",\n \"The PPR was then calculated using the amplitude of the chemical component of the facilitated trace divided by the amplitude of the chemical component in basal conditions .\",\n \"Spontaneous electrical and chemical synaptic responses were assessed during offline analysis of continuous ( 10 s long ) recordings using Clampfit ( Axon instruments ) to automatically identify events based on their duration ( <1 . 1 ms for electrical spontaneous events and >1 . 1 ms for chemical spontaneous events ) .\",\n \"Potential erroneous identification of spontaneous events by the software was monitored manually by verifying the duration of the events .\",\n \"The assignment of electrical vs . chemical nature of spontaneous synaptic events by their duration was confirmed pharmacologically .\",\n \"For synaptic transmission blockade , CNQX was first dissolved in DMSO to have a stock solution of 10 mM .\",\n \"The pharmacological agents used to block synaptic transmission were added to the external solution: Meclofenamic Acid ( 200 μM , Sigma ) , CNQX and DAP5 ( 20 μM , Tocris Biosciences ) .\",\n \"The M-cell input resistance was estimated by applying a hyperpolarizing-current step of −1 nA and 20 ms in duration and measuring the voltage deflection caused , followed by derivation of resistance with Ohm’s law .\",\n \"The rheobase , defined as the minimum depolarizing current of infinite duration necessary to evoke an action potential , was determined by delivering a 20 ms current pulse of increasing intensity .\",\n \"Voltage threshold for action potential generation was determined by applying a depolarizing-current step .\",\n \"Startle behavior of 5dpf larvae was analyzed as described previously ( Marsden et al . , 2018 ) .\",\n \"Briefly , larvae were adapted to the testing temperature and lighting conditions for 30 min and then transferred to individual wells of a custom , laser-cut acrylic multi-well testing arena , illuminated from below with an infrared ( IR ) LED array and from above with a white light LED bulb to simulate daylight conditions .\",\n \"A total of 60 acoustic stimuli , 10 at each of 6 intensities , were delivered pseudorandomly using an acoustic-vibrational shaker ( Bruel and Kjaer ) with an inter-stimulus interval of 20 s to eliminate habituation to repeated stimulation ( Wolman et al . , 2015 ) .\",\n \"The intensity of each stimulus was calibrated using a PCB Piezotronics accelerometer ( model #355B04 ) and signal conditioner ( model #482A21 ) , and voltage outputs were converted to dB using the formula dB = 20 log ( V/0 . 775 ) .\",\n \"Behavioral responses were captured at 1000 frames per second with an IR-sensitive Photron mini-UX50 high-speed camera .\",\n \"After testing , larvae were fixed in methanol for subsequent genotyping , thus all testing and analysis was performed blind to genotype .\",\n \"Behavioral responses were tracked and analyzed using FLOTE software ( Burgess and Granato , 2007 ) , with short and long latency C-bend responses ( SLCs and LLCs , respectively ) automatically defined based on the kinematic parameters of the response .\",\n \"Startle sensitivity index was calculated by measuring the area under the curve of stimulus intensity versus SLC frequency for each larva .\",\n \"Cell lines were obtained from ATCC , identity ensured by using exclusively low-passage cells , and were confirmed to be mycoplama free .\",\n \"Full-length Cx34 . 1 and full-length Cx35 . 5 were cloned into the pCMV expression vector .\",\n \"Full-length ZO1b was cloned into the pCMV expression vector with an NH2-terminal mVenus tag and a COOH-terminal 8xHIS tag .\",\n \"Low passage HEK293T/17 cells were seeded 24 hr prior to transfection ( 1 × 106 cells/well of a six-well dish ) , and the indicated plasmids were co-transfected using Lipofectamine 3000 ( Invitrogen ) following the manufacturer’s instructions .\",\n \"Cells were collected 36–48 hr post-transfection and lysed in 0 . 25 ml solubilization buffer ( 50 mM Tris [pH7 . 4] , 100 mM NaCl , 5 mM EDTA , 1 . 5 mM MgCl2 , 1 mM DTT and 1% Triton X-100 ) plus a protease inhibitor cocktail ( Pierce ) .\",\n \"Lysates were centrifuged at 20 , 000 x g for 30 min at 4°C , and equal amounts of extract were immunoprecipitated with 0 . 5 ug rabbit anti-GFP ( Abcam , Ab290 ) overnight with rocking at 4°C .\",\n \"Immunocomplexes were captured with 25 µl prewashed Protein A/G agarose beads for 1 hr with rocking at 4°C .\",\n \"Beads were washed three times with lysis buffer , and bound proteins were boiled for 3 min in the presence of LDS-PAGE loading dye containing 200 mM DTT .\",\n \"Samples were resolved by SDS-PAGE using a 4–15% gradient gel and analyzed by Western blot using the following primary antibodies: rabbit anti-GFP ( Abcam Ab290 ) , rabbit anti-Cx34 . 1 3A4-conjugated-680LT , and mouse anti-Cx35 . 5 4B12 .\",\n \"Compatible near-infrared secondary antibodies were used for visualization with the Odyssey system ( LI-COR ) .\",\n \"Brains from wildtype , tjp1a/ZO1a-/- , or tjp1b/ZO1b-/- euthanized adult fish ( 4–15 months old ) were removed , snap frozen in liquid nitrogen and stored at −80C until use .\",\n \"Brains were homogenized in 1 ml of HSE buffer ( 20 mM Hepes [pH7 . 5] , 150 mM NaCl , 5 mM EDTA , 5 mM EGTA , and 1 mM DTT ) plus a protease inhibitor cocktail using a glass homogenizer .\",\n \"Detergent was added to the homogenate ( final 2% octyl ß-D-glucopyranoside , Anatrace ) and solubilized overnight with rocking at 4°C .\",\n \"Solubilized homogenate was cleared by centrifugation at 20 , 000 x g for 30 min at 4°C , then pre-cleared for 1 hr at 4°C with Protein A/G beads before immunoprecipitation .\",\n \"The protein concentration for each homogenate was measured by Bradford assay .\",\n \"Pre-cleared homogenates ( 2 mg/IP ) were immunoprecipitated with 0 . 5 µg mouse anti-ZO1 ( Life Technologies , 33–9100 ) , control mouse IgG ( Jackson ImmunoResearch ) , mouse anti-Cx34 . 1 5C10 , or mouse anti-Cx35 . 5 4B12 antibody overnight with rocking at 4°C .\",\n \"Immunocomplexes were captured with 25 ul prewashed Protein A/G agarose beads for 1 hr with rocking at 4°C .\",\n \"Beads were washed three times with HSE buffer , and bound proteins were boiled for 3 min in the presence of LDS-PAGE loading dye containing 200 mM DTT .\",\n \"Immune complexes were examined by western analysis using the following primary antibodies: mouse anti-ZO1 , rabbit anti-Cx34 . 1 3A4-conjugated-680LT , rabbit anti-Cx35 . 5 12H5-conjugated-680LT , or mouse anti-Cx35 . 5 4B12 .\",\n \"Compatible near-infrared secondary antibodies were used for visualization .\",\n \"The Cx34 . 1-tail ( aa256-299 ) , Cx34 . 1-tail ∆PBM ( aa256-295 ) , Cx35 . 5-tail ( aa267-309 ) , and Cx35 . 5-tail ∆PBM ( aa267-305 ) were cloned into the pGEX expression vector allowing for an NH2-terminal GST tag .\",\n \"ZO1b-PDZ1 ( aa105-207 ) and ZO1b-PDZ2 ( aa298-387 ) were cloned into a modified pET expression vector ( pBH ) to allow for an NH2-terminal 6xHis tag followed by a TEV cleavage site ( vectors kindly provided by Ken Prehoda ) .\",\n \"Plasmids were transformed in E . coli BL21 ( DE3 ) cells and plated on selective LB plates .\",\n \"Single colonies were picked to inoculate 2 ml starter cultures and grown overnight .\",\n \"Overnight cultures were inoculated into 250 ml selective LB and grown for ~3 hr at 37°C with shaking until OD600 reached 0 . 8–1 followed by 4 hr induction with 0 . 4 mM IPTG .\",\n \"Cell pellets were collected by centrifugation at 6000 RPM for 5 min at 4°C and frozen at −20°C until test samples confirmed expression .\",\n \"Pellets were resuspended in sonication buffer ( 50 mM NaPO4 [pH7 . 4] , 300 mM NaCl , and 1 mM PMSF ) .\",\n \"After adding a dash of lysozyme , the mixture was incubated on ice for 30 min .\",\n \"Resuspended bacteria were sonicated on ice at 50% amplitude , 1 s/1 s pulse on/off , four times for 20 s .\",\n \"Debris was cleared by centrifugation at 16 , 000 x g for 30 min at 4°C .\",\n \"For GST fusions , supernatant was added to 200 ul pre-washed glutathione agarose resin and incubated overnight with rocking at 4°C .\",\n \"Beads were washed three times with sonication buffer and stored at 4°C .\",\n \"Purity and amount loaded onto resin was determined by SDS-PAGE followed by Coomassie stain .\",\n \"For 6xHIS fusions , supernatant was brought to a final concentration of 20 mM imidazole and incubated with pre-washed His60 resin overnight with rocking at 4°C .\",\n \"Resin was washed with sonication buffer containing 20 mM imidazole .\",\n \"Protein was eluted from the resin with sonication buffer containing 250 mM imidazole .\",\n \"The protein was concentrated and exchanged into imidazole-free buffer using an Amicon centrifugal filter unit ( 10K MWCO ) and stored at 4°C on ice .\",\n \"Protein concentration was estimated by A205 ( https://spin . niddk . nih . gov/clore/ ) ( Anthis and Clore , 2013 ) , and purity was determined by SDS-PAGE followed by Coomassie stain .\",\n \"Equal amounts of GST fusions ( 10 µl bed of resin ) were aliquoted and the storage buffer was removed .\",\n \"To each sample 15 µl of 6xHIS-ZO1b-PDZ1 ( 7 mg/ml ) was added , gently mixed and incubated at room temperature for 15 min .\",\n \"Resin was washed three times with cold wash buffer ( 50 mM NaPO4 [pH7 . 4] , 300 mM NaCl ) .\",\n \"After the last wash , all buffer was removed and resin was resuspended in 10 µl LDS-PAGE dye with 200 mM DTT .\",\n \"Samples were boiled for 3 min and resolved by SDS-PAGE using a 4–20% gradient gel .\",\n \"Samples were analyzed by Western blot using rabbit anti-TEV cleavage site primary antibody ( ThermoFisher , PA1-119 ) and visualized with a compatible near-infrared secondary antibody .\",\n \"A portion of the GST fusion resin was analyzed by Coomassie stain to demonstrate equal loading .\",\n \"Equal amounts of GST fusion were resuspended in LDS-PAGE dye plus 200 mM DTT , boiled for 3 min , resolved by SDS-PAGE on a 4–15% gradient gel , transferred to nitrocellulose , and blocked with 5% milk in TBS ( 20 mM Tris [pH7 . 4] , 150 mM NaCl ) .\",\n \"Blocked membranes were incubated with TBS-T buffer ( TBS + 0 . 1% Tween-20 ) alone ( mock overlay ) , or TBS-T containing 1 µM 6xHIS-ZO1b-PDZ1 or 1 uM 6xHIS-ZO1b-PDZ2 overnight with shaking at 4°C .\",\n \"Membranes were processed for western analysis using rabbit anti-TEV cleavage site primary antibody and a compatible near-infrared secondary antibody to detect bound PDZ protein .\",\n \"Equal loading of GST fusions was determined by Stain-Free imaging technology .\",\n \"A single guide RNA ( sgRNA ) targeting the 5’ region of the endogenous tjp1b coding sequence ( sequence in Key Resources Table ) was designed using the CRISPRscan algorithm ( Moreno-Mateos et al . , 2015 ) and synthesized as previously described ( Shah et al . , 2015 ) .\",\n \"The sgRNA was generated using the T7 megascript kit ( ThermoFisher , AMB13345 ) .\",\n \"The V5-tjp1b single stranded donor oligo ( ssODN ) was designed to repair into the endogenous tjp1b locus and was synthesized complimentary to the coding strand .\",\n \"The ssODN contained 40 bp homology arms which flanked an XbaI restriction site , V5 sequence , and a 5x glycine linker , respectively ( sequence in Key Resources Table ) .\",\n \"Upon correct repair , the inserted sequence was designed to disrupt the endogenous sgRNA recognition site to prevent further double stranded breaks after repair .\",\n \"Injection mixes were prepared in a pH 7 . 5 buffer solution of 300 mM KCl and 4 mM HEPES and contained a final concentration of 200 pg/nL ssODN , 200 pg/nL gRNA , and 1600 pg/nL Cas9 protein ( IDT , 1081058 ) .\",\n \"Injection mixes were incubated at 37 °C for 5 min immediately prior to injection to promote formation of the Cas9 and sgRNA complex .\",\n \"Finally , 1 nL of solution was injected into embryos at the one-cell stage .\",\n \"Injected embryos were raised to adulthood and outcrossed to wild-type animals .\",\n \"Animals carrying insertions were identified and verified using PCR and Sanger sequencing .\",\n \"Cell transplantation was performed at the high stage approximately 3 . 3 hr into zebrafish development using standard techniques ( Kemp et al . , 2009 ) .\",\n \"Embryos were chemically de-chorionated with protease ( Sigma Aldrich , 9036-06-0 ) prior to transplantation .\",\n \"Cells were transplanted using a 50 mm wide glass capillary needle attached to an oil hydraulic .\",\n \"For ‘V5-tjp1b+/- into wildtype’ transplants ( Figures 7C , D and Figure 7—figure supplement 1B-H ) cells from animals heterozygous for V5-tjp1b in the M/CoLo:GFP background were transplanted into non-transgenic wildtype hosts .\",\n \"For ‘tjp1b-/- into wildtype’ transplants ( Figure 7G and H ) , genotyped animals homozygous for the tjp1bΔ16bp mutation in the M/CoLo:GFP background were crossed and progeny were transplanted into non-transgenic wildtype hosts .\",\n \"For 'wildtype into tjp1b-/-' transplants ( Figure 7I and J ) , transgenic M/CoLo:GFP wildtype animals were crossed to use as donors , and non-transgenic , homozygous tjp1bΔ16bp animals were crossed to produce hosts .\",\n \"Approximately 20 cells were deposited ∼10–15 cell diameters away from the margin , with a single donor embryo supplying cells to 3–5 hosts .\",\n \"At 5 dpf , larvae were fixed in TCA and processed for immunohistochemistry .\",\n \"For fluorescence intensity quantitation , confocal images were processed and analyzed ( in part or in full ) using FiJi ( Schindelin et al . , 2012 ) software .\",\n \"To quantify staining at M/Colo synapses , a standard region of interest ( ROI ) surrounding each M/CoLo site of contact was drawn , and the mean fluorescence intensity was measured .\",\n \"For the quantification of staining at the club endings , confocal z-stacks of the Mauthner soma and lateral dendrite were cropped to 36 . 08 µm x 36 . 08 µm centered around the lateral dendritic bifurcation .\",\n \"Using the SciPy ( Virtanen et al . , 2020 ) and scikit-image ( van der Walt et al . , 2014 ) computing packages , the cropped stack was then cleared outside of the Mauthner cell , a 33 median filter was applied to reduce noise , and a standard threshold was set within each experiment to remove background staining .\",\n \"The image was then transformed into a max intensity projection and the integrated density of each stain within the Mauthner cell was extracted .\",\n \"Where counts were used to quantify antibody labeling of CEs in high-contrast images , CEs were identified as large fluorescently labeled oval areas of approximately 1 . 5–2 microns on the distal portion and fork of the Mauthner cell’s lateral dendrite , which were thoroughly examined with the confocal microscope .\",\n \"For Figure 7—figure supplement 1I , J , the presence or absence of electrical synapses on Mauthner was quantified as counts of stereotyped electrical synapse structures dually labeled for Cx34 . 1 and Cx35 . 5 .\",\n \"Standard deviations and errors were computed using Prism ( GraphPad ) or Excel ( Microsoft ) software .\",\n \"Figure images were created using FiJi , Photoshop ( Adobe ) , and Illustrator ( Adobe ) .\",\n \"Statistical analyses were performed using Prism ( GraphPad ) and either an unpaired t-test with Welch’s correction or a one-way analysis of variance with Bonferrroni’s multiple comparison test was performed .\",\n \"For all experiments , values were normalized to wildtype control animals , and n represented the number of fish used .\",\n \"Fish were excluded from analysis if Mauthner morphology/GFP staining was abnormal .\",\n \"Electrophysiology traces of spontaneous synaptic events were analyzed using Clampfit 10 . 7 software .\",\n \"Electrophysiology traces were transferred to Canvas for illustration and to OriginPro software for quantification and statistical analyses .\",\n \"For the different parameters measured using electrophysiological recordings , means and standard error of the mean ( SEM ) were illustrated using Prism ( GraphPad ) and computed using OriginPro software .\",\n \"For statistical analyses comparing electrophysiological recordings in wildtype and mutant zebrafish , Mann-Whitney and Kruskal-Wallis non-parametric tests were performed using OriginPro software .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins .","While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins , it is unknown if channels at electrical synapses require similar scaffold support .","Here , we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 ( ZO1 ) , an intracellular scaffolding protein localized to electrical synapses .","Using model electrical synapses in zebrafish Mauthner cells , we demonstrated that ZO1 is required for robust synaptic Connexin localization , but Connexins are dispensable for ZO1 localization .","Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses .","We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels .","Thus , forming functional neuronal gap junctions requires a postsynaptic scaffolding protein .","The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses ."],"string":"[\n \"Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins .\",\n \"While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins , it is unknown if channels at electrical synapses require similar scaffold support .\",\n \"Here , we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 ( ZO1 ) , an intracellular scaffolding protein localized to electrical synapses .\",\n \"Using model electrical synapses in zebrafish Mauthner cells , we demonstrated that ZO1 is required for robust synaptic Connexin localization , but Connexins are dispensable for ZO1 localization .\",\n \"Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses .\",\n \"We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels .\",\n \"Thus , forming functional neuronal gap junctions requires a postsynaptic scaffolding protein .\",\n \"The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses .\"\n]"},"summary":{"kind":"list like","value":["Neurons ‘talk’ with each another at junctions called synapses , which can either be chemical or electrical .","Communication across a chemical synapse involves a ‘sending’ neuron releasing chemicals that diffuse between the cells and subsequently bind to specialized receptors on the receiving neuron .","These complex junctions involve a large number of well-studied molecular actors .","Electrical synapses , on the other hand , are believed to be simpler .","There , neurons are physically connected via channels formed of ‘connexin’ proteins , which allow electrically charged ions to flow between the cells .","However , it is likely that other proteins help to create these structures .","In particular , recent evidence shows that without a structurally supporting ‘scaffolding’ protein called ZO1 , electrical synapses cannot form in the brain of a tiny freshwater fish known as zebrafish .","As their name implies , scaffolding proteins help cells organize their internal structure , for example by anchoring other molecules to the cell membrane .","By studying electrical synapses in zebrafish , Lasseigne , Echeverry , Ijaz , Michel et al . now show that these structures are more complex than previously assumed .","In particular , the experiments reveal that ZO1 proteins are only present on one side of electrical synapses; despite their deceptively symmetrical anatomical organization , these junctions can be asymmetric , like their chemical cousins .","The results also show that ZO1 must be present for connexins to gather at electrical synapses , whereas the converse is not true .","This suggests that when a new electrical synapse forms , ZO1 moves into position first: it then recruits or stabilizes connexins to form the channels connecting the two cells .","In many animals with a spine , electrical synapses account for about 20% of all neural junctions .","Understanding how these structures form and work could help to find new treatments for disorders linked to impaired electrical synapses , such as epilepsy ."],"string":"[\n \"Neurons ‘talk’ with each another at junctions called synapses , which can either be chemical or electrical .\",\n \"Communication across a chemical synapse involves a ‘sending’ neuron releasing chemicals that diffuse between the cells and subsequently bind to specialized receptors on the receiving neuron .\",\n \"These complex junctions involve a large number of well-studied molecular actors .\",\n \"Electrical synapses , on the other hand , are believed to be simpler .\",\n \"There , neurons are physically connected via channels formed of ‘connexin’ proteins , which allow electrically charged ions to flow between the cells .\",\n \"However , it is likely that other proteins help to create these structures .\",\n \"In particular , recent evidence shows that without a structurally supporting ‘scaffolding’ protein called ZO1 , electrical synapses cannot form in the brain of a tiny freshwater fish known as zebrafish .\",\n \"As their name implies , scaffolding proteins help cells organize their internal structure , for example by anchoring other molecules to the cell membrane .\",\n \"By studying electrical synapses in zebrafish , Lasseigne , Echeverry , Ijaz , Michel et al . now show that these structures are more complex than previously assumed .\",\n \"In particular , the experiments reveal that ZO1 proteins are only present on one side of electrical synapses; despite their deceptively symmetrical anatomical organization , these junctions can be asymmetric , like their chemical cousins .\",\n \"The results also show that ZO1 must be present for connexins to gather at electrical synapses , whereas the converse is not true .\",\n \"This suggests that when a new electrical synapse forms , ZO1 moves into position first: it then recruits or stabilizes connexins to form the channels connecting the two cells .\",\n \"In many animals with a spine , electrical synapses account for about 20% of all neural junctions .\",\n \"Understanding how these structures form and work could help to find new treatments for disorders linked to impaired electrical synapses , such as epilepsy .\"\n]"},"year":{"kind":"string","value":"2021"}}},{"rowIdx":4193,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["plant biology","genetics and genomics"],"string":"[\n \"plant biology\",\n \"genetics and genomics\"\n]"},"title":{"kind":"string","value":"Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2"},"id":{"kind":"string","value":"elife-72375-v2"},"sections":{"kind":"list like","value":[["N6-methyladenosine ( m6A ) is the most abundant modified nucleotide in eukaryotic mRNA bodies .","It is required for embryonic development and stem cell differentiation in several animals and plants ( Zhong et al . , 2008; Batista et al . , 2014; Ping et al . , 2014; Geula et al . , 2015; Zhang et al . , 2017 ) and for the control of the meiotic program in yeast ( Shah and Clancy , 1992; Clancy et al . , 2002; Agarwala et al . , 2012 ) .","Most N6-adenosine methylation of mRNA is catalyzed in the nucleus ( Salditt-Georgieff et al . , 1976; Ke et al . , 2017; Huang et al . , 2019 ) by a highly conserved , multimeric methylase ( the m6A ‘writer’; Balacco and Soller , 2019 ) whose catalytic core consists of the heterodimer METTL3/METTL14 ( MTA/MTB in plants; Bokar et al . , 1997; Zhong et al . , 2008; Liu et al . , 2014 ) .","In addition , a number of highly conserved proteins is required for N6-methylation in vivo ( Balacco and Soller , 2019 ) .","The strong conservation of these core factors suggests that the biochemical basis of N6-adenosine methylation is common in eukaryotes , and indeed , m6A occurs in the consensus site RR ( m6A ) CH ( R = G/A , H = A/C/U ) , primarily in 3′-UTRs in vertebrates , plants , and fungi that possess the canonical METTL3/METTL14 methyltransferase ( Bodi et al . , 2012; Dominissini et al . , 2012; Meyer et al . , 2012; Schwartz et al . , 2013; Luo et al . , 2014a; Zhao et al . , 2017; Miao et al . , 2020; Parker et al . , 2020 ) .","Conversely , the characteristic motif and gene body location is not detected in organisms that lack METTL3/METTL14 homologs , such as the nematode Caenorhabditis elegans ( Sendinc et al . , 2020 ) and bacteria ( Deng et al . , 2015 ) .","m6A may impact mRNA function by different mechanisms , including the creation of binding sites for reader proteins that specifically recognize m6A in mRNA ( Dominissini et al . , 2012; Fu et al . , 2014; Meyer and Jaffrey , 2014 ) .","The best understood class of readers contains a so-called YT521-B homology ( YTH ) domain ( Stoilov et al . , 2002 ) of which two phylogenetic groups , YTHDF and YTHDC , have been defined ( Patil et al . , 2018; Balacco and Soller , 2019 ) .","The YTH domain harbors a hydrophobic methyl-binding pocket that increases the affinity of m6A-containing RNA by more than 10-fold compared to unmethylated RNA ( Li et al . , 2014b; Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014; Zhu et al . , 2014 ) .","Apart from interactions with the methylated adenosine and the purine at the –1 position , YTH domain-RNA interactions mostly involve the sugar-phosphate backbone of the RNA ( Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014 ) .","That is consistent with only mild reductions in the binding affinity of the YTH domain of human YTHDC1 upon substitution of nucleotides −2 , +1 , and +3 that abrogate the canonical RR ( m6A ) CH motif ( Xu et al . , 2014 ) , and poor sequence specificity of RNA binding by isolated YTH domains of human YTHDF1 , YTHDF2 , and YTHDC1 ( Arguello et al . , 2019 ) .","Thus , the methyltransferase complex gives the sequence specificity , while YTH domain proteins may bind to m6A-containing RNA regardless of the identity of the immediately adjacent nucleotides .","YTHDF proteins are typically cytoplasmic and consist of a long N-terminal intrinsically disordered region ( IDR ) followed by the globular YTH domain ( Patil et al . , 2018 ) .","Because the affinity of isolated YTH domains for m6A-containing RNA is modest , typically with dissociation constants on the order of 0 . 1–1 μM ( Li et al . , 2014b; Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014; Zhu et al . , 2014 ) , it has been suggested that the IDR may participate in RNA binding ( Patil et al . , 2018 ) .","Nonetheless , the clearest evidence for functions of the IDRs in YTHDF proteins reported thus far includes direct interactions with effectors such as the CCR4-NOT complex in mammalian cells ( Du et al . , 2016 ) , and the ability to cause liquid-liquid phase transition when sufficiently high local concentrations are reached ( Arribas-Hernández et al . , 2018; Gao et al . , 2019; Ries et al . , 2019; Fu and Zhuang , 2020; Wang et al . , 2020 ) .","The YTHDF family comprises 11 proteins in Arabidopsis that are referred to as EVOLUTIONARILY CONSERVED C-TERMINAL REGION1-11 ( ECT1-11 ) ( Li et al . , 2014c; Scutenaire et al . , 2018 ) .","ECT2 , ECT3 , and ECT4 are expressed in rapidly dividing cells of root , leaf , and flower primordia , and genetic analyses have revealed their general importance in organogenesis ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .","Importantly , the biological functions of ECT2/ECT3/ECT4 described thus far are shared with those of m6A writer components and , where tested , have been shown to depend on intact m6A-binding pockets , strongly suggesting that the basis for the observed phenotypes in ect2/ect3/ect4 mutants is defective regulation of m6A-modified mRNA targets ( Bodi et al . , 2012; Shen et al . , 2016; Ružička et al . , 2017; Arribas-Hernández et al . , 2018; Scutenaire et al . , 2018; Wei et al . , 2018; Arribas-Hernández et al . , 2020 ) .","Despite the progress in identifying biological functions of plant m6A-YTHDF axes , a number of fundamental questions regarding their molecular basis remains unanswered .","For example , it is unclear whether sequence determinants in addition to m6A are important for mRNA target association of ECT proteins in vivo , the mRNA targets of ECT2/ECT3/ECT4 responsible for the developmental delay of ect2/ect3/ ( ect4 ) mutants have not been identified , and it is not clear what the effects of ECT2/ECT3/ECT4 binding to them may be ( Arribas-Hernández and Brodersen , 2020 ) .","Clearly , robust identification of the mRNA targets directly bound by ECT proteins is key to obtain satisfactory answers to all of these questions .","Towards that goal , formaldehyde crosslinking and immunoprecipitation ( FA-CLIP ) was used to identify mRNA targets of ECT2 ( Wei et al . , 2018 ) .","Nonetheless , because formaldehyde , in contrast to UV illumination , generates both protein-protein and protein-RNA crosslinks , it is not an ideal choice for identification of mRNAs bound directly by a protein of interest ( see Arribas-Hernández and Brodersen , 2020 for a discussion ) .","In particular , this problem concerns the unexpected conclusion that ECT2 binds to the ‘plant-specific consensus motif’ URU ( m6A ) Y ( Y = U/C ) , not RR ( m6A ) CH ( Wei et al . , 2018 ) .","Thus , the field of gene regulation via m6A-YTHDF modules in plants is in a state of confusion: on the one hand , m6A mapping ( Luo et al . , 2014a; Wan et al . , 2015; Shen et al . , 2016; Duan et al . , 2017; Anderson et al . , 2018; Miao et al . , 2020; Parker et al . , 2020 ) and phenotypes of mutants defective in m6A writing ( Bodi et al . , 2012; Shen et al . , 2016; Ružička et al . , 2017 ) or m6A binding of ECT2/ECT3/ECT4 ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) suggest that these YTHDF proteins should act via recognition of m6A in the RRACH context .","On the other hand , the only attempt at a mechanistic understanding of ECT2 function via mRNA target identification concluded that ECT2 binds to a sequence element different from RRACH ( Wei et al . , 2018 ) .","To complicate matters further , a number of motifs including not only URUAY , but also UGUAMM ( M = A/C ) , UGWAMH ( W = A/U ) , UGUAWA , and GGAU have been reported to be enriched around m6A sites in Arabidopsis and other plant species ( Li et al . , 2014a; Anderson et al . , 2018; Miao et al . , 2020; Zhang et al . , 2019; Zhou et al . , 2019 ) , but it remains unclear whether the adenosines in such motifs are methylated in vivo .","Alternatively , these sequence contexts may play a role in guiding m6A deposition or ECT recognition nearby , either directly by ECT interaction or indirectly via additional RNA-binding proteins assisting or competing with ECT binding .","To clarify principles underlying mRNA recognition by ECT2 , we undertook rigorous analysis of its mRNA-binding sites using two orthogonal methods , the proximity-labeling method HyperTRIBE ( targets of RNA-binding proteins identified by editing ) ( McMahon Aoife et al . , 2016; Xu et al . , 2018 ) and iCLIP ( individual nucleotide resolution crosslinking and immunoprecipitation ) ( König et al . , 2010 ) .","This resulted in identification of high-quality target sets as judged by mutual overlaps and by overlaps with previously reported m6A maps from plants at a similar developmental stage ( Shen et al . , 2016; Parker et al . , 2020 ) .","Relying on this high-quality target set , we used the position information inherent to iCLIP and a single-nucleotide resolution m6A dataset ( Parker et al . , 2020 ) to establish six properties of m6A-containing mRNA and mRNA targeting by ECT2 .","( 1 ) RRACH and its variant DRACH ( D = R/U ) are unequivocally the most highly enriched motifs at m6A sites in Arabidopsis .","( 2 ) ECT2 binds to m6A sites in the canonical RRACH context as ECT2 crosslinking sites are preferentially found immediately 5′ to m6A sites , and RRACH is enriched immediately 3′ to ECT2 crosslinking sites .","( 3 ) GGAU is a minor m6A consensus site in plants .","( 4 ) U- and U/C-rich motifs are enriched around , but not at , m6A sites , and , together with RRACH and GGAU , constitute core elements that distinguish m6A-containing 3′-UTRs from non-m6A-containing 3′-UTRs in plants .","( 5 ) The IDR of ECT2 participates in RNA binding as it crosslinks to target mRNAs at U-rich elements highly abundant upstream of m6A sites .","( 6 ) Although URUAY , URURU , and similar motifs may crosslink to ECT2 , their presence in m6A-containing mRNA disfavors ECT2 binding , consistent with those motifs acting predominantly as sites of interaction for RNA-binding proteins that may compete with ECT2 ."],["HyperTRIBE uses fusion of RNA-binding proteins to the hyperactive E488Q mutant of the catalytic domain of the Drosophila melanogaster adenosine deaminase acting on RNA ( DmADARE488Qcd ) ( Kuttan and Bass , 2012 ) to achieve proximity labeling in vivo ( McMahon Aoife et al . , 2016; Xu et al . , 2018 ) .","Targets are identified as those mRNAs that contain adenosine-inosine sites significantly more highly edited than background controls , measured as A-G changes upon reverse transcription and sequencing .","To develop material suitable for ECT2 HyperTRIBE , we expressed AtECT2pro:AtECT2-FLAG-DmADARE488Qcd-AtECT2ter ( henceforth ‘ECT2-FLAG-ADAR’ ) in the single ect2-1 and triple ect2-1/ect3-1/ect4-2 ( te234 ) knockout backgrounds ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .","We identified lines exhibiting nearly complete rescue of te234 mutant seedling phenotypes , indicating that the fusion protein was functional ( Figure 1A ) .","We then used the expression level in complementing lines as a criterion to select lines in the ect2-1 single mutant background , for which no easily scorable phenotype has been described ( Figure 1—figure supplement 1A ) .","Lines expressing free DmADARE488Qcd under the control of the endogenous ECT2 promoter ( AtECT2pro:FLAG-DmADARE488Qcd-AtECT2ter; henceforth FLAG-ADAR ) at levels similar to or higher than those of the fusion lines ( Figure 1—figure supplement 1A , B ) were used to control for background editing after verification that FLAG-ADAR expression did not result in phenotypic abnormalities in Col-0 WT plants ( Figure 1A ) .","To identify ECT2 HyperTRIBE targets ( HT-targets ) , we sequenced mRNA from dissected root tips and shoot apices of 10-day-old seedlings of ect2-1/ECT2-FLAG-ADAR and FLAG-ADAR transgenic lines using five independent lines of each type as biological replicates to prevent line-specific artifacts .","Next , we generated nucleotide base counts for all positions with at least one mismatch across the full set of samples of mapped reads ( Figure 1B ) , resulting in a raw list of potential editing positions .","This revealed that the amount of editing was clearly higher in the lines expressing the ECT2-FLAG-ADAR fusion protein than in the negative control lines ( Figure 1C , Figure 1—figure supplement 1C ) .","To identify positions with significantly higher editing rates in ECT2-FLAG-ADAR lines compared to controls , we developed a new approach to detect differential editing ( Figure 1B ) described in detail by Rennie et al . , 2021 .","Briefly , the hyperTRIBER method of detecting differential editing exploits the powerful statistical capabilities of a method originally designed to detect differential exon usage ( Anders et al . , 2012 ) .","It efficiently takes replicates and possible differences in expression into account , resulting in high power to detect sites despite the generally low editing proportions that we found in our data ( Figure 1D ) .","As expected , the tendency towards higher editing proportions in fusion lines compared to controls was even more pronounced after filtering nonsignificantly edited sites ( Figure 1C , Figure 1—figure supplement 1C ) .","Three additional properties of the resulting editing sites indicate that they are the result of ADARcd activity guided by its fusion to ECT2 .","First , the vast majority of significant hits corresponded to A-to-G transitions ( Figure 1—figure supplement 1D ) .","Second , the consensus motif at the edited sites matched the sequence preference of DmADARE488Qcd ( 5′ and 3′ nearest-neighbor preference of U>A>C>G and G>C>A~U , respectively [Xu et al . , 2018; Figure 1E , Figure 1—figure supplement 1E] ) , with highly edited sites more closely matching the optimal sequence context than lowly edited ones ( Figure 1—figure supplement 1F ) .","Third , principal component analysis of editing proportions at significant sites over the different lines clearly separated the ECT2-FLAG-ADAR fusion lines from the control lines ( Figure 1F , Figure 1—figure supplement 1G ) .","Application of subsequent filtering steps , including removal of non- ( A-to-G ) mismatches and of potential line-specific single-nucleotide variants ( see Materials and methods ) , resulted in a final list of 16 , 176 edited sites for aerial tissues and 19 , 242 for roots , corresponding to 4864 and 5052 genes ( ECT2 HT-targets ) , respectively ( Figure 1B , Supplementary file 1 ) .","In both cases , this represents 27% of all expressed genes .","We note that the editing proportions were generally low ( Figure 1D ) compared to previous work in Drosophila ( Xu et al . , 2018 ) , perhaps in part due to the limited number of cells that express ECT2 ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .","Indeed , the ADAR expression level ( TPMs ) correlated strongly with editing proportions among ECT2-FLAG-ADAR lines ( Figure 1G , Figure 1—figure supplement 1H ) , and editing proportions were higher for target mRNAs that are coexpressed with ECT2 in a large percentage of cells according to single-cell RNA-seq ( Denyer et al . , 2019; Figure 1H ) , lending further support to the conclusion that the observed editing is ADAR-specific and driven to target mRNAs by ECT2 .","Hence , HyperTRIBE can be used to identify targets of RNA-binding proteins in planta .","To evaluate the properties of ECT2 HT-targets , we first noted that most of them were common between root and aerial tissues ( Figure 2A ) , as expected given the recurrent function of ECT2 in stimulating cell division in all organ primordia ( Arribas-Hernández et al . , 2020 ) .","In agreement with this result , most of the targets specific to root or aerial tissues were simply preferentially expressed in either tissue ( Figure 2B ) .","Moreover , the significant editing sites in roots and aerial tissues had a considerable overlap ( Figure 2A ) , and their editing proportions were similar in the two tissues ( Figure 2C ) .","Of most importance , we observed a large overlap between the ECT2 HT-targets and m6A-containing transcripts mapped by different methods in seedlings ( Shen et al . , 2016; Parker et al . , 2020 ) as more than 76% of ECT2 HT-targets had m6A support by either study ( Figure 2D ) .","These results validate our HyperTRIBE experimental setup and data analysis , and confirm that ECT2 binds predominantly to m6A-containing transcripts in vivo .","We next moved on to independent target and binding site identification via iCLIP ( Figure 3A ) .","We used transgenic lines expressing functional ECT2-mCherry under the control of the endogenous ECT2 promoter in the ect2-1 knockout background ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) to co-purify mRNAs crosslinked to ECT2 for iCLIP .","Lines expressing the ECT2W464A-mCherry variant were used as negative controls because this Trp-to-Ala mutation in the hydrophobic methyl-binding pocket of the YTH domain abrogates the increased affinity for m6A-RNA ( Li et al . , 2014b; Xu et al . , 2014; Zhu et al . , 2014 ) .","Accordingly , the point mutant behaves like a null allele in plants despite its wild-type-like expression pattern and level ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .","To test the feasibility of iCLIP , we first assessed the specificity of RNA co-purified with ECT2-mCherry after UV illumination of whole seedlings by 5′-radiolabeling of the immunoprecipitated RNP complexes followed by SDS-PAGE .","These tests showed that substantially more RNA co-purifies with wild-type ECT2 than with ECT2W464A upon UV-crosslinking , and that no RNA is detected without UV irradiation or from irradiated plants of non-transgenic backgrounds ( Figure 3B , Figure 3—figure supplement 1A ) .","RNAse and DNAse treatments also established that the co-purified nucleic acid is RNA ( Figure 3—figure supplement 1B ) .","Thus , UV crosslinking of intact Arabidopsis seedlings followed by immunopurification successfully captures ECT2-RNA complexes that exist in vivo .","Curiously , although the pattern of ECT2-RNA complexes with bands migrating at ~110 and 55kDa is highly reproducible , it does not correspond to the majority of the purified ECT2-mCherry protein , which runs at ~125kDa in SDS-PAGE ( Figure 3B and C ) .","A variety of control experiments ( Figure 3—figure supplement 1C-E ) , most importantly the disappearance of additional bands with use of an N-terminal rather than a C-terminal tag ( Figure 3C and D ) , indicate that the band pattern arises as a consequence of proteolytic cleavage of the N-terminal IDR in the lysis buffer , such that fragments purified using the C-terminal mCherry tag include the YTH domain with portions of the IDR of variable lengths ( Figure 3—figure supplement 2 ) .","Comparative analysis of RNA in 55-kDa and 110–125-kDa complexes may , therefore , provide insight into the possible role of the N-terminal IDR of ECT2 in mRNA binding ( Figure 3E ) , an idea consistent with the comparatively low polynucleotide kinase labeling efficiency of full-length ECT2-mCherry-mRNA complexes ( ~125kDa ) ( Figure 3B , Figure 3—figure supplement 2 ) .","Thus , we prepared separate iCLIP libraries from RNA crosslinked to ECT2-mCherry/ECT2W464A-mCherry that migrates at ~110–280kDa ( ‘110-kDa band’ ) and at ~55–75kDa ( ’55-kDa band’ ) ( Figure 3—figure supplement","3 ) to investigate the possible existence of IDR-dependent crosslink sites , and thereby gain deeper insights into the mode of YTHDF binding to mRNA in vivo .","We identified a total of 15 , 960 iCLIP ‘peaks’ or crosslink sites ( i . e . , single-nucleotide positions called by PureCLIP from mapped iCLIP reads [Krakau et al . , 2017] ) in 2281 genes from the 110-kDa band of wild-type ECT2-mCherry ( henceforth referred to as ECT2 iCLIP peaks and targets , respectively ) .","In the corresponding 55-kDa band , 4549 crosslink sites in 1127 genes were called , 93% of them contained in the 110-kDa target set ( Figure 3F and G , Figure 3—figure supplement 4 , Supplementary file 2 ) .","We note that these numbers perfectly agree with the idea of the 55-kDa band containing only YTH domain crosslink sites , while the full length may also include IDR crosslink sites .","Importantly , for both libraries , the majority of crosslink sites mapped to the 3′-UTRs of mRNAs ( Figure 3H , see Figure 4A , and Figure 4—figure supplement 1 for more examples ) , coincident with the main location of m6A ( Figure 4B; Parker et al . , 2020 ) .","Accordingly , the 3′-UTR specificity was largely lost in RNA isolated from 55-kDa ECT2W464A ( Figure 3H ) , for which neither YTH domain nor IDR binding to RNA can be expected .","Finally , iCLIP targets in full-length ( 110-kDa band ) ECT2 WT and ECT2W464A overlapped only marginally ( Figure 3G ) , providing molecular proof of the dependence of m6A-binding activity for ECT2 function .","Nonetheless , the bias towards occurrence in the 3′-UTR was only reduced , not abolished , for crosslinks to the full-length ECT2W464A protein , providing another indication that the IDR itself is able to associate with RNA-elements in 3′-UTRs ( Figure 3H ) .","We elaborate further on this important point by analysis of IDR-specific crosslinks to wild-type ECT2 after in-depth validation of sets of ECT2 target mRNAs and determination of the sequence motifs enriched around m6A and ECT2 crosslink sites .","To evaluate the congruence of the results obtained by iCLIP and HyperTRIBE , we investigated the cumulative number of iCLIP sites as a function of distance to the nearest editing site determined by HyperTRIBE .","This analysis showed a clear tendency for iCLIP peaks called with ECT2WT-mCherry , but not for ECT2W464A-mCherry , to be in the vicinity of editing sites ( Figure 4C ) , indicating that the majority of called iCLIP peaks identify genuine ECT2-binding sites on mRNAs .","Similar tendencies of proximity between iCLIP peaks and HyperTRIBE editing sites were previously observed for a Drosophila hnRNP protein ( Xu et al . , 2018 ) .","Although manual inspection of individual target genes confirmed these tendencies , it also revealed that ADAR-edited sites are too dispersed around iCLIP peaks to give precise information on the actual ECT2-binding sites ( Figure 4A , Figure 4—figure supplement 1 ) .","Therefore , we used both HyperTRIBE and iCLIP for gene target identification , but relied on iCLIP peaks for motif analyses .","To examine the quality of our target identification in further detail , we analyzed the overlap between ECT2 targets identified by iCLIP and HyperTRIBE .","This analysis also included m6A mapping data obtained with either m6A-seq ( Shen et al . , 2016 ) or the single-nucleotide resolution methods miCLIP and Nanopore sequencing ( Parker et al . , 2020 ) as young seedlings were used in all cases .","ECT2 targets identified by iCLIP and HyperTRIBE showed clear overlaps , both with each other and with m6A-containing transcripts , further supporting the robustness of ECT2 target identification via combined iCLIP and HyperTRIBE approaches ( Figure 4D , upper panel , Figure 4—figure supplement 2 ) .","Importantly , although some m6A targets are expected not to be bound by ECT2 because of the presence of MTA in cells that do not express ECT2 ( Arribas-Hernández et al . , 2020 ) , only 18% of the high-confident set of m6A-containing genes ( with support from miCLIP and Nanopore ) did not overlap with either ECT2 iCLIP or HT target sets ( Figure 4—figure supplement 2 , arrow ) .","We also observed that HyperTRIBE identifies approximately three times more ECT2 targets than iCLIP , possibly because of the bias towards high abundance inherent to purification-based methods like iCLIP ( Wheeler et al . , 2018 ) .","To test this idea , we compared the distribution of target mRNAs identified by the different techniques across nine expression bins .","As expected , a bias towards highly abundant transcripts was evident for iCLIP-identified targets compared to HyperTRIBE ( Figure 4E ) .","We also observed a similar bias for m6A-containing transcripts detected by miCLIP , another purification-based method , and in the Nanopore dataset ( Figure 4E ) , probably explained by its relatively low sequencing depth ( Parker et al . , 2020 ) .","These observations also suggest that the higher sensitivity of HyperTRIBE ( analyzed in detail in Figure 4—figure supplement","3 ) explains the lack of m6A support ( by Nanopore or miCLIP ) for 28% of ECT2 HT-targets ( 1689 ) compared to only 4% ( 83 ) of ECT2 iCLIP targets ( Figure 4D , Figure 4—figure supplement 2 , upper row ) since HT-targets may simply include genes that escape detection by m6A mapping methods due to low expression .","Indeed , ECT2-HT targets without any m6A support were distributed in lower-expression bins compared to those with m6A support ( Figure 4F ) .","Intriguingly , ECT2 FA-CLIP targets ( Wei et al . , 2018 ) did not show a bias towards highly expressed genes as their distribution over expression bins largely reflected that of the total number of genes ( Figure 4E ) , and as many as 37% of FA-CLIP targets did not have m6A support ( Figure 4D , Figure 4—figure supplement 2 , upper row ) .","In summary , these analyses show that ECT2 iCLIP and HT target sets are in excellent agreement with each other and with independently generated m6A maps , and that HyperTRIBE identifies targets below the detection limit of other techniques .","To characterize the sequence composition and exact positions of ECT2-binding sites relative to m6A , we first used the high resolution of iCLIP data to examine the position of ECT2 crosslink sites relative to m6A sites , determined at single-nucleotide resolution ( Parker et al . , 2020 ) .","This analysis showed that ECT2 crosslinks in the immediate vicinity , but preferentially upstream ( ~11 nt ) of Nanopore-determined m6A sites , with a mild depletion at the exact m6A site ( Figure 5A , upper panel ) .","Furthermore , while m6A-miCLIP sites corresponded to m6A-Nanopore sites overall , a subset of m6A-miCLIP sites were located upstream of m6A-Nanopore sites and coincided well with ECT2-iCLIP peaks ( Figure 5A ) .","This pattern is probably explained by the fact that the UV illumination used in both iCLIP and miCLIP preferentially generates RNA-protein crosslinks involving uridine ( Hafner et al . , 2021 ) , also detectable in the datasets analyzed here ( Figure 5B and C ) .","Thus , the depletion of ECT2-iCLIP sites at Nanopore- , but enrichment at miCLIP-determined m6A sites ( Figure 5A ) , might be explained by the absence of uridine within the RRAC core of the m6A consensus motif , and perhaps also to some extent by reduced photoreactivity of the m6A base stacking with indole side chains of the YTH domain .","Furthermore , the fact that nucleotides at −2 , +1 , and +2 positions are only expected to contribute sugar-phosphate backbone interactions with the YTH domain ( Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014 ) may also contribute to the absence of direct crosslinks at the m6A site relative to the adjacent bases .","The 5′ shift observed for iCLIP and miCLIP sites relative to Nanopore sites might be explained by a higher occurrence of uridines upstream of m6A sites , a particularly interesting possibility given the numerous reports of U-rich motifs enriched around m6A sites in plants ( Li et al . , 2014a; Anderson et al . , 2018; Miao et al . , 2020; Zhang et al . , 2019; Zhou et al . , 2019; Luo et al . , 2020 ) and animals ( Patil et al . , 2016 ) .","To investigate the sequence composition around m6A and ECT2 sites , we first performed exhaustive unbiased de novo motif searches using Homer ( Heinz et al . , 2010; Figure 5—figure supplement","1 ) and extracted all candidate motifs , including the m6A consensus motif RRACH , as well as GGAU ( Anderson et al . , 2018 ) , URUAY ( Wei et al . , 2018 ) , and several other U-rich sequences .","Combined with manually derived candidate motifs ( Figure 5—figure supplement 1B ) , we then calculated position weight matrices ( PWMs ) for a final set of 48 motifs and scanned for their occurrences genome-wide using FIMO ( Grant et al . , 2011; Figure 5—figure supplements 1 and 2 ) .","This allowed us to determine three key properties .","First , the global enrichment of the motifs at locations across the gene body .","Second , the total count of occurrences of each motif at m6A sites and ECT2-iCLIP crosslink sites compared to a set of sites in non-target mRNAs matching the location within gene bodies of m6A/ECT2-iCLIP sites ( expected background ) .","Third , the distribution of the motifs relative to m6A and ECT2 iCLIP sites .","The results of this systematic analysis ( Supplementary file","3 ) were used to select those motifs with a more prominent enrichment at or around m6A and ECT2 sites ( Figure 5D ) .","This approach defined two major categories of motifs of outstanding interest , RRACH-like and GGAU on the one side , and a variety of U/Y-rich motifs on the other .","Figure 5D shows a minimal selection of such motifs , while a more comprehensive compilation is displayed in Figure 5—figure supplements 3 and 4 .","Not surprisingly , RRACH-like motifs were the most highly enriched at m6A sites and showed a clear enrichment immediately downstream of ECT2 crosslink sites in our analyses , with the degenerate variant DRACH being the most frequently observed ( Figure 5D , Figure 5—figure supplement 3 ) .","Motifs containing GGAU behaved similarly to DRACH , with a sharp enrichment exactly at m6A sites and mild enrichment downstream of ECT2 peaks ( Figure 5D ) , supporting a previous suggestion of GGAU as an alternative methylation site ( Anderson et al . , 2018 ) .","The possible roles of the U/Y-rich motifs in m6A deposition and ECT2 binding are analyzed in the following sections .","We first noticed that several motifs retrieved around ECT2 crosslink sites by Homer constituted extended versions of DRACH/GGAU with Us upstream ( e . g . , UGAAC/UGGAU ) or remnants of DRACH with U/Cs ( Ys ) downstream ( e . g . , ACUCU ) .","To test whether these motifs are indeed located adjacent to m6A , we examined their distribution and enrichment around ECT2 and m6A sites .","The distributions showed a clear enrichment at m6A positions with a shift in the direction of the U/Y-extension ( see Figure 5D for ACUCU and Figure 5—figure supplement 4 for others ) .","An enrichment over location-matched background sites close to ECT2-iCLIP sites was also apparent ( see Figure 5D for ACUCU and Figure 5—figure supplement 4 for others ) , further supporting that ECT2 preferentially crosslinks to uridines located in the immediate vicinity of DRACH ( /GGAU ) .","Thus , several enriched motifs around ECT2 crosslink sites are DRACH/GGAU-derived , and their detection in unbiased searches simply reflects a tendency of methylated DRACH/GGAU sites to be flanked by U/Ys .","U/R-rich motifs without traces of adjacent DRACH ( e . g . , YUGUM , URUAY , URURU ) showed a characteristic enrichment around , but depletion at , m6A sites .","For some motifs , the enrichment was more pronounced 5′ than 3′ to m6A sites ( see Figure 5D for URUAY and Figure 5—figure supplement 4 for others ) .","The distance between the site of maximal motif occurrence and the m6A site roughly coincided with the shift observed in ECT2 crosslink sites relative to m6A ( Figure 5A , upper panel ) .","Accordingly , these motifs were enriched exactly at ECT2 crosslink sites ( see Figure 5D for URUAY and Figure 5—figure supplement 4 for others ) , suggesting that they may constitute additional m6A-independent sites of interaction with ECT2 .","We also observed that the 3′ enrichment of YYYYY was asymmetric and closer to m6A than that of UUUUU/URURU/URUAY ( Figure 5—figure supplement 4 , second row from the top ) , indicating a preference for hetero-oligopyrimidine tracts immediately downstream the m6A site , as suggested by the 3′-enrichment of DRACUCU-type motifs as described above .","Taken together , these results suggest that N6-adenosine methylation preferentially occurs in DRACH/GGAU sequences surrounded by stretches of pyrimidines , with a preference for YYYYY ( e . g . , CUCU ) immediately downstream , URURU ( including URUAY ) immediately upstream , and UUUUU/UNUNU slightly further away in both directions .","The enrichment of ECT2 crosslink sites at these motifs , and the fact that the m6A-binding-deficient mutant of ECT2 ( W464A ) crosslinks preferentially to 3′-UTRs through its N-terminal IDR , indicates IDR-mediated binding to U/R- and Y-rich motifs around m6A .","Since our analysis thus far uncovered several motifs of potential importance for m6A deposition and ECT2 binding , we employed machine learning to distinguish m6A and ECT2 iCLIP sites from random location-matched background sites using motif-based features .","Importantly , the underlying classification model includes all motif features within the same model , allowing an evaluation of the importance of the motifs relative to each other .","We used as features the number of matches to each of the 48 motifs ( Figure 5—figure supplement","2 ) in three distinct regions relative to the methylated site according to Nanopore sequencing ( Parker et al . , 2020 ) , defined as position 0: ‘at’ [–10 nt; +10 nt] , ‘down’ [–50 nt; –10 nt] , or ‘up’ [+ 10 nt; +50 nt] ( Figure 6A ) .","The model involving all motifs could successfully distinguish the methylated sites from the background as indicated by an area under the receiver operating characteristic ( ROC ) curve ( true positive rate versus false positive rate , area under the curve [AUC] ) of 0 . 93 , and even a reduced model incorporating only the top 10 features from the full model classified sites largely correctly ( AUC = 0 . 86; Figure 6—figure supplement 1 ) .","The top 16 features ordered by importance from the full model confirmed that RRAC/DRACH or GGAU at the site was indicative of the presence of m6A ( Figure 6B ) .","Interestingly , U/Y-rich sequences ( UNUNU and YYYYY in particular ) flanking the site were also strongly indicative ( Figure 6B ) .","Some motifs showed a skew in their feature importance score , with UNUNU and YUGUM showing a preference to be upstream , and YYYYY downstream ( Figure 6B ) , thus corroborating our previous observations ( Figure 6C ) .","We used a similar modeling approach to identify non-m6A determinants of ECT2 binding , in this case comparing m6A sites within 10 nt distance of ECT2-iCLIP sites to m6A sites without ECT2-iCLIP sites nearby ( AUC = 0 . 94 , and AUC = 0 . 84 using only the top 10 features , Figure 6—figure supplement 1 ) .","In agreement with previous observations , this model showed flanking U/Y-rich sequences as the main determinants for ECT2 crosslinking ( Figure 6D ) .","To investigate the idea of URURU-like motifs as additional sites of ECT2 binding upstream of the m6A-YTH interaction site , we split Nanopore-m6A sites according to two criteria: ( 1 ) whether they occur in ECT2-target transcripts ( both permissive and stringent sets analyzed separately ) , and ( 2 ) for ECT2 targets , whether there is an ECT2 crosslink site within 25 nt of the m6A site ( ‘near’ ) or not ( ‘far’ ) .","Although there was no obvious differences between these categories for most of the motifs ( Supplementary file 3 , page 2 ) , some U-rich sequences displayed distinctive features ( Figure 7A , Figure 7—figure supplement","1 ) that can be summarized as follows .","If a transcript has m6A and ECT2 sites in close proximity , it is ( 1 ) more likely to have UNUNU/UUUUU/YYYYY sequences upstream of the m6A site than targets with distantly located ECT2-binding sites or than non-ECT2 targets; ( 2 ) less likely to have UUUUU/URURU sequences downstream of the m6A site , possibly because ECT2 prefers CUCU-like sequences downstream; and ( 3 ) less likely to have URURU/URUAY-like motifs upstream of the m6A site .","The latter observation is striking because for the specific subset of ECT2-bound m6A sites with URURU/URUAY upstream of m6A , these sequences tend to crosslink to ECT2 , as seen by the enrichment spike at ECT2 crosslink sites ( Figure 5D , Figure 7—figure supplement 1 , bottom panels ) .","Although these two results seem contradictory at first glance , they may be reconciled by a model in which a URURU/URUAY-binding protein would compete with ECT2 for binding adjacent to m6A .","If that protein is absent , ECT2 may bind to the site , potentially via its IDR , to stabilize the low-affinity YTH-m6A interaction and crosslink efficiently due to the U-content .","Conversely , if occupied by the alternative interacting protein , the site might repel ECT2 ( see Discussion and Figure 7B ) .","We reasoned that insights into contacts between ECT2 and mRNA may be gained by analysis of the iCLIP libraries prepared with the ‘YTH-mCherry’ truncation devoid of the N-terminal IDR ( ‘55-kDa band’ ) compared to the full-length ECT2-mCherry ( ‘110-kDa band’ ) ( Figure 3 , Figure 3—figure supplements 2–4 ) .","Initial inspection of the distribution of ECT2 peaks relative to Nanopore-m6A sites showed that the 5′–3′ asymmetry observed with full-length ECT2 was largely reduced with the truncated protein ( Figure 7C ) , as was the bias towards uridines ( Figure 7—figure supplement 2A ) .","These observations suggest that the IDR indeed is implicated in binding to U-rich regions upstream of m6A .","We next split the full-length ECT2 iCLIP peaks according to whether they are present in libraries from both full-length and truncated forms ( ‘IDR-independent’ ) or exclusively in the full-length ( ‘IDR-dependent’ ) ( distance >10 nt ) and plotted the enrichment of the studied motifs relative to the crosslink site ( Figure 7D , Figure 7—figure supplement 2B; Supplementary file 3 , page 2 ) .","UUUUUU/UNUNU-like motifs were more enriched at and immediately upstream of IDR-dependent crosslink sites relative to the IDR-independent ones , supporting preferential crosslinking of the IDR to Us in this region .","Remarkably , the exact opposite was true for URURU/URUAY motifs that showed modest depletion 5′ to IDR-dependent crosslink sites relative to their IDR-independent counterparts ( Figure 7D ) .","These observations are consistent with a model of an RNA-binding protein competing with the ECT2 IDR for interaction with upstream URURU/URUAY motifs ( Figure 7B ) ."],["Our work establishes experimental and computational approaches to implement HyperTRIBE for unbiased and sensitive mapping of direct targets of RNA-binding proteins in plants .","Two points are particularly relevant in this regard .","First , the examples studied here show that stable transgenic expression of DmADARcd does not lead to detrimental phenotypes , perhaps because of the generally low editing proportions obtained in vivo .","Second , the rigorous statistical approach developed to call editing sites makes HyperTRIBE powerful , despite the low editing proportions observed .","We also note that ECT2 is well suited to verify that HyperTRIBE mostly recovers directly bound target RNAs because of the possibility to cross-reference the data with independently obtained m6A maps ( Parker et al . , 2020 ) .","The combination of iCLIP and HyperTRIBE for unbiased mapping of targets proved particularly attractive for at least two reasons .","First , the convergence on overlapping target sets by orthogonal methods strengthens the confidence that the identified targets are biologically meaningful .","Second , HyperTRIBE , especially with the novel computational approach for calling of editing sites ( Rennie et al . , 2021 ) , offers higher sensitivity than iCLIP , while iCLIP is unmatched in providing information on binding sites within target RNAs .","It is possible that better positional information on binding sites may be obtained from HyperTRIBE data using maximal editing proportions rather than statistical significance as the parameter to call editing sites .","Indeed , recent work on the use of HyperTRIBE to identify targets of the RNA-binding protein MUSASHI-2 ( MSI-2 ) in leukemic stem cells recovered the known MSI-2-binding site as enriched around editing sites in targets ( Nguyen et al . , 2020 ) .","Nonetheless , our data shows that highly edited sites match the ADAR substrate consensus site better than lowly edited sites , suggesting that site proximity to ADAR is not the only determinant of editing proportions .","Finally , our work also clearly indicates that FA-CLIP , now used in at least two studies involving YTH domain proteins ( Wei et al . , 2018; Song et al . , 2021 ) , is not a recommendable technique as it recovers many false positives and fails to include many genuine targets .","Thus , with the possible exception of cases in which evidence for indirect association is specifically in demand , such as the recent study in human cells of mixed tailing of viral RNA by the cellular terminal nucleotidyl transferase TENT4 ( Kim et al . , 2020 ) , FA-CLIP should not be used for identification of RNAs associating with a particular RNA-binding protein of interest .","Our analyses of motif enrichments around m6A and ECT2 crosslink sites clarify roles of previously reported motifs and uncover new motifs of importance in m6A writing and ECT2 binding .","Since m6A is a prerequisite for ECT2 binding , any analysis of determinants of ECT2 binding must consider determinants of N6-adenosine methylation separately .","Three conclusions stand out from our analysis in this regard .","First , the major N6-adenosine methylation site is DRACH , consistent with conclusions from multiple other studies .","Second , GGAU is a minor N6-adenosine methylation site , as seen by its enrichment directly at m6A sites .","Third , m6A occurs in DRACH/GGAU islands embedded in U-rich regions .","Such U-rich regions around m6A sites emerged from sorting of methylated from non-methylated transcripts by machine learning as being of similar importance for recognition of m6A-containing transcripts from sequence features as DRACH and GGAU at m6A sites , suggesting their implication in MTA/MTB-catalyzed adenosine methylation ( Figure 6C ) .","This , in turn , may also explain the pronounced 3′-UTR bias of m6A occurrence as extensive poly-U and poly-pyrimidine tracts are rare in coding regions ( Figure 5D , second row on the right-most column; Supplementary file 3 , page 1 ) .","As a special case in this context , our analyses suggest a simple explanation for the tendency of m6A to occur at stop codons .","UAA and UGA correspond to DRA , increasing the frequency of occurrence of DRACH directly at stop codons ( Figure 5D , second row on the left-most column ) , many of which have adjacent U-rich elements in the 3′-UTRs .","We note that the observed pattern is in agreement with a role of the poly ( U ) -interacting proteins RBM15A/B associated with the mammalian methyltransferase complex in guiding methylation ( Patil et al . , 2016 ) .","Whether a similar mechanism operates in plants , potentially via the distant RBM15A/B homologue FPA ( Arribas-Hernández and Brodersen , 2020 ) , remains to be investigated .","It is a major conclusion of the present work that ECT2 binds to m6A predominantly in the DR ( m6A ) CH sequence context in vivo , consistent with reading of m6A written by the conserved nuclear MTA/MTB methyltransferase .","This key conclusion refutes the claim by Wei et al . , 2018 that ECT2 binds to the supposedly plant-specific m6A-containing sequence motif URU ( m6A ) Y , and it thereby reconciles knowledge on m6A-YTHDF axes in plants specifically and in eukaryotes more broadly .","The phenotypic similarity of plants defective in MTA/MTB writer and ECT2/ECT3/ECT4 reader function is now coherent with the locations of MTA/MTB-written m6A and ECT2-binding sites transcriptome-wide , and it is now clear that plants do not constitute an exception to the general biochemical framework for eukaryotic m6A-YTHDF function in which YTHDF proteins read the m6A signal written by the MTA/MTB methyltransferase .","The pronounced protease sensitivity of IDRs , leading to limited proteolysis of ECT2 upon cell lysis after in vivo crosslinking , allowed us to extract information on the mode of ECT2-RNA binding from different observations , all converging on the conclusion that the IDR of ECT2 participates in RNA binding .","First , RNA complexes with YTH-mCherry were 5′-labeled by polynucleotide kinase much more efficiently than RNA complexes with full-length ECT2-mCherry , indicating that the IDR limits accessibility to the 5′ of bound mRNAs .","Second , in contrast to the m6A-binding-deficient YTHW464A-mCherry truncation , the full-length ECT2W464A-mCherry mutant retained an enrichment of crosslink sites in 3′-UTRs .","Third , crosslinks specific to the IDR ( i . e . , observed only with full-length ECT2-mCherry-RNA complexes , but not with YTH-Cherry-RNA complexes ) , could be assigned and have two notable properties .","They are mainly 5′ to m6A sites , and thereby cause a conspicuously asymmetric distribution of ECT2 crosslink sites around m6A sites , not seen with crosslinks to the YTH-mCherry fragment .","In addition , the IDR-specific crosslinks are specifically enriched in U-rich elements of the type UUUUUU and UNUNU immediately upstream .","Taken together , these observations suggest that the IDR of ECT2 participates in locating ECT2 to 3′-UTRs by association with U-rich elements .","Thus , ECT2 , and perhaps YTHDF proteins more generally given their highly similar YTH domains , appears to bind RNA through multivalent interactions among which the YTH domain is responsible for m6A binding , and the IDR is responsible for interaction with adjacent elements .","We note that the notion of RNA interaction by IDRs has precedent ( Corley et al . , 2020 ) , is consistent with the modest affinity of isolated YTHDF domains for m6A-containing oligonucleotides ( Patil et al . , 2018 ) , and is reminiscent of the recent demonstration that transcription factors use their globular DNA-binding domains to recognize core sequence elements of promoters , and their IDRs to provide additional DNA contacts , contributing to specificity ( Brodsky et al . , 2020 ) .","Similarly , it is possible that diverging IDRs among YTHDF paralogs could confer target specificity via binding to distinct motifs in the vicinity of m6A sites , such that specific YTHDF-target mRNA repertoires could exist even for YTHDF proteins coexpressed in the same cells .","Finally , we stress that although our data point to an important role of the IDR in RNA binding , it does not in any way suggest that this is the only function of the IDR , and protein-protein interactions involving the IDR are likely to be key to understanding YTHDF function molecularly .","Despite the conclusions that URUAY does not contain m6A in Arabidopsis , and that ECT2 binds to DR ( m6A ) CH , our detailed analysis of sequence motifs enriched around m6A and ECT2 iCLIP crosslink sites shows that additional motifs , including URUAY , are likely to be implicated in m6A reading by ECT2 , even if not directly .","In contrast to other m6A-proximal , pyrimidine-rich sequences ( e . g . , UNUNU , YYYYY ) that may be of importance for both m6A writing and ECT2 binding , URUAY appears to have ties more specifically to ECT2 binding thanks to three properties .","( 1 ) When present 5′ to m6A sites , it crosslinks to ECT2 , suggesting that some part of the protein can be in contact with URUAY .","( 2 ) URUAY is more enriched close to m6A sites for which there is no evidence of ECT2 binding , suggesting that it weakens ECT2 binding .","This latter point is also consistent with the distinction of ECT2-bound from non-ECT2-bound m6A sites by machine learning that did not find URUAY to be of importance for ECT2-bound sites .","( 3 ) The URUAY enrichment 5′ to ECT2 crosslink sites is observed only when crosslinks to both full-length protein and the YTH-mCherry fragment are considered ( IDR-independent ) , but disappears when crosslinks specific to the full-length protein ( IDR-dependent ) are analyzed .","Although these observations may be explained by multiple scenarios , we find a simple , yet at present speculative , model attractive: URUAY may be a site of competition between the IDR of ECT2 and another , as yet unknown , RNA-binding protein .","Such a competing factor could in theory be another YTHDF protein using higher-affinity IDR-URUAY contacts than ECT2 to achieve competitive binding .","Many other possibilities exist , however .","For example , it is intriguing that URUAY resembles part of a Pumilio-binding site ( Hafner et al . , 2010; Huh et al . , 2013 ) as it raises the tantalizing possibility of functional interaction between YTHDF and Pumilio proteins .","In any event , the functional dissection of the URUAY element in m6A reading now constitutes a subject of major importance , emphasized by the broad conservation of its enrichment around m6A sites across multiple plant species , including rice ( Li et al . , 2014a; Zhang et al . , 2019 ) , maize ( Luo et al . , 2020; Miao et al . , 2020 ) , tomato ( Zhou et al . , 2019 ) , and Arabidopsis ( Miao et al . , 2020 ) ."],["We use the term ‘biological replicate’ in the following way: plants were grown at the same time , under the same conditions , but in separate plates .","Each sample replicate contains pools of seedlings prepared in such a way that no two replicates contain seedlings grown on the same plates .","This sampling ensures that plate-to-plate variation in growth conditions , if any , will have an effect on measurements of gene expression within a single genotype , and hence minimize the risk that any differences due to such variation are called as significant in comparisons between genotypes .","‘Technical replicates’ are understood to be independently conducted measurements using the same technique on the same biological material ( e . g . , on one biological replicate as defined above ) .","Technical replicates were not carried out in this study , and the term ‘replicate’ refers to biological replicate as defined above .","In our definition , an ‘experiment’ results in generation and comparison of measurements arising from multiple biological replicates of different biological entities , in the present case often Arabidopsis seedlings differing in genotype with respect to the genes ECT2 , ECT3 , and ECT4 .","Thus , repetition of an experiment in our definition entails generation and analysis of the required biological replicates at different points in time .","All lines used in this study are in the Arabidopsis thaliana Col-0 ecotype .","The mutant alleles or their combinations – ect2-1 ( SALK_002225 ) ( Arribas-Hernández et al . , 2018; Scutenaire et al . , 2018; Wei et al . , 2018 ) , ect3-1 ( SALKseq_63401 ) , ect4-2 ( GK_241H02 ) , and ect2-1/ect3-1/ect4-2 ( te234 ) ( Arribas-Hernández et al . , 2018 ) – have been previously described .","The transgenic lines expressing ECT2pro:ECT2-mCherry-ECT2ter , ECT2pro:ECT2W464A-mCherry-ECT2ter , ECT2pro:3xHA-ECT2-ECT2ter , or ECT2pro:3xHA-ECT2W464A-ECT2ter in the ect2-1 background have also been described or generated by floral dip in additional mutant backgrounds using the same plasmids and methodology ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .","Seeds were surface-sterilized by 2 min incubation in 70% EtOH plus 10 min in sterilizing solution ( 1 . 5% NaOCl , 0 . 05% Tween-20 ) and 2 H2O washes .","After 2–5 days of stratification at 4°C in darkness , seeds were germinated and grown on plates containing Murashige and Skoog ( MS ) -agar medium ( 4 . 4 g/L MS , 10 g/L sucrose , 10 g/L agar ) pH 5 . 7 at 20°C , receiving ~70 μmol m–2 s–1 of light in a 16 hr light/8 hr dark cycle as default .","For HyperTRIBE and iCLIP experiments , the plates were placed vertically to facilitate root harvesting .","MS-agar media for HyperTRIBE T2 seedlings was supplemented with 7 . 5 mg/L of glufosinate ammonium ( Sigma ) to select plants expressing the ADAR-containing transgenes .","To assess phenotypes of adult plants , ~8-day-old seedlings were transferred from horizontal MS plates ( 4 . 4 g/L MS , 10 g/L sucrose , 8 g/L agar; pH 5 . 7 ) to soil and maintained in Percival incubators under 16 hr light/8 hr dark cycles , 21°C day/18°C night temperature , and ~100 μmol m–2 s–1 light intensity .","We used Philips fluorescent tubes TL-D 90 De Luxe 36 W as light source .","We employed USER cloning ( Bitinaite and Nichols , 2009 ) to generate ECT2pro:ECT2-FLAG- DmADARE488Qcd-ECT2ter and ECT2pro:FLAG-DmADARE488Qcd-ECT2ter constructs in pCAMBIA3300U ( pCAMBIA3300 with a double PacI USER cassette inserted between the PstI-XmaI sites at the multiple cloning site; Nour-Eldin et al . , 2006 ) .","Fragments containing ECT2 gDNA sequences were amplified by PCR ( KAPA HiFi Hotstart Uracil + ReadyMix , Roche ) from plasmids previously generated in our lab ( Arribas-Hernández et al . , 2018 ) .","The FLAG-DmADARE488Qcd fragment was produced in the same way using a pGEM-T Easy ( Promega ) plasmid containing FLAG-DmADARE488Qcd as template , previously subcloned to introduce the E488Q hyperactive mutation by site-directed mutagenesis ( QuickChange , Agilent Technologies ) with primers LA729-LA730 ( Phusion HF DNA Polymerase , NEB ) .","The E488Q mutation was detected by NlaIII ( ThermoFisher ) digestion of the PCR reaction ( DreamTaq , ThermoFisher ) obtained with primers LA660-LA735 .","Of note , the FLAG and DmADARcd sequences had been previously glued together by USER cloning to produce AGO1pro:FLAG-DmADARcd-AGO1ter in pCAMBIA3300U for unrelated purposes ( unpublished work ) , and subsequently amplified by PCR with primers LA696-615 for introduction into pGEM-T Easy .","To build AGO1pro:FLAG-DmADARcd-AGO1ter in the first place , the catalytic domain of the ADAR deaminase isoform N ( Y268-E669 ) was amplified from cDNA of D . melanogaster Canton-S wild-type flies and larvae with USER primers MVUSER12-22 .","The rest of the fragments were amplified from pCAMBIA3300U AGO1pro:FLAG-AGO1-AGO1ter ( Arribas-Hernández et al . , 2016 ) with primers MVUSER1-11 and MVUSER23-6 .","USER primers to amplify all fragments were designed to create overhangs compatible with either the PacI USER cassette present in the pCAMBIA3300U plasmid or the flanking sequences of the neighboring fragments .","All primer sequences , their combinations to produce PCR fragments , and the arrangement of the fragments for USER cloning can be found in Appendix 1 .","Kanamycin-resistant colonies of Escherichia coli DH5α ( NEB ) transformed with the constructs were analyzed by restriction digestion and sequencing prior introduction of the plasmids in Agrobacterium tumefaciens GV3101 ( Koncz and Schell , 1986 ) for plant transformation .","Arabidopsis stable transgenic lines were generated by floral dip transformation ( Clough and Bent , 1998 ) of Col-0 WT , ect2-1 , or te234 , and selection of primary transformants ( T1 ) was done on MS-agar plates supplemented with glufosinate-ammonium ( Sigma ) ( 10 mg/L ) .","We selected five independent lines of each type based on segregation studies ( to isolate single T-DNA insertions ) , phenotypic complementation ( in the te234 background ) , and transgene expression levels assessed by FLAG western blot .","Protein extraction from 10-day-old seedlings and western blotting with FLAG , HA , and mCherry antibodies were done as previously described ( Arribas-Hernández et al . , 2018 ) .","Loading was documented by amido black , Coomassie , or Ponceau staining of the total protein on the membrane .","We extracted total RNA from manually dissected root tips and apices ( removing cotyledons ) of five independent lines ( 10-day-old T2 seedlings ) of each of the lines used for ECT2-HT to use as biological replicates .","The tissue was flash-frozen in liquid nitrogen and ground into a fine powder using liquid nitrogen-cooled adaptors in a tissue homogenizer .","For RNA extraction , we added 1 mL of TRI Reagent ( Sigma ) to the frozen tissue ( <100 mg ) , mixed quickly by vortexing , added 0 . 2 mL of chloroform , and separated the two resulting phases by vigorous shaking and 10 min centrifugation at 4°C .","The RNA was then precipitated from the aqueous phase for 30 min at room temperature with 1 volume of isopropanol .","RNA pellets were solubilized in 300 μL of H2O to remove polysaccharides through a mild precipitation by addition of 1/10 vol .","99% EtOH and 1/30 vol .","of 3 M NaOAc ( pH 5 . 2 ) and incubation on ice for 30 min .","After 15 min of full-speed centrifugation at 4°C to pellet polysaccharides , we re-precipitated the RNA from the supernatant with 2 , 5× vol .","99% EtOH and 1/10 vol .","of 3 M NaOAc ( pH 5 . 2 ) , washed the pellet two times with 70% EtOH , and resuspended in 20–40 μL of H2O .","This highly pure total RNA was then used to produce mRNA libraries through enrichment of mRNA with oligo ( dT ) beads ( 18-mers ) , random fragmentation , cDNA synthesis with random hexamers , custom second-strand synthesis ( Illumina ) , terminal repair , A-ligation and sequencing adaptor ligation , size selection ( 250–300 bp insert ) , and PCR enrichment .","The libraries were prepared and sequenced ( Illumina PE150 , Q30 ≥ 80% ) as a service from Novogene .","The entire HyperTRIBE experiment was done once .","Significant differentially edited sites between ECT2-FLAG-ADAR ( fusion ) and FLAG-ADAR ( control ) samples for ECT2 HyperTRIBE ( ECT2-HT ) were called according to the hyperTRIBER pipeline ( Rennie et al . , 2021 ) .","First , reads were trimmed using trimmomatic ( Bolger et al . , 2014 ) and mapped to the Arabidopsis genome ( TAIR10 ) using STAR ( Dobin et al . , 2013 ) , according to parameters suggested in a previous HyperTRIBE analysis ( Xu et al . , 2018 ) .","All HyperTRIBE samples were also quantified using Salmon ( Patro et al . , 2017 ) , with appropriate settings for pair-end sequencing and non-stranded library setup and based on the transcriptome for Araport11 ( Cheng et al . , 2017 ) with manual addition of the FLAG-ADAR sequence .","A custom Perl script based on SAMtools mpileup ( Li et al . , 2009 ) returned base counts for all positions where there is a mismatch from the reference in at least one sample .","For running the hyperTRIBER analysis pipeline , we specified that any tested position must have a putative edit in at least four of the five replicates in the ECT2-FLAG-ADAR samples ( three of four in the case of roots since one of these samples , ‘L3 , ’ was deemed as low quality and subsequently removed from the significance calling pipeline ) .","Significant hits ( adjusted p-value<0 . 01 and log2FC > 1 ) were further filtered as follows: ( 1 ) hits that did not correspond to an A-to-G change ( or a T-to-C change for the negative strand ) , ( 2 ) hits that were likely SNPs arising specifically in either the ECT2-FLAG-ADAR or FLAG-ADAR line manifesting in an editing proportion at or close to 1 , and ( 3 ) hits where the coverage of tags at the edit base over the ECT2-FLAG-ADAR were fewer than 10 reads .","Specific scripts for the analysis of ECT2-HT data can be found at https://github . com/sarah-ku/targets_arabidopsis .","Editing proportions were calculated as G/ ( A + G ) ( alternatively C/ ( U + C ) for the negative strand ) for all significant sites , averaged over all samples , separately for the ECT2-FLAG-ADAR and FLAG-ADAR samples .","Significant sites were annotated to genes from Araport11 , prioritizing the gene with the highest expression ( annotated TPMs are based on Salmon quantifications of FLAG-ADAR control samples only ) in the given tissue in the case of multiple possibilities .","Possible transcripts were subsequently ordered by expression , along with gene body location along the transcript ( 5′-UTR , CDS , 3′-UTR ) .","Principal component analysis was carried out on the raw editing proportions per sample for all sites with significant evidence of editing .","For the comparison of sites between aerial tissues and roots , genes defined as commonly expressed in both types of tissues were considered in all gene-based comparisons .","For significant editing site-based comparison , we directly compared sites that were common and significant to both .","To calculate correlations between editing proportions and FLAG-ADAR expression levels among lines , transcripts per million ( TPM ) mapping to FLAG-ADAR were extracted from quantifications from Salmon ( Patro et al . , 2017 ) and correlated with the raw editing proportions per sample , separately for the fusion and control samples .","Background correlation estimates were calculated by first scrambling the order of the FLAG-ADAR TPM vector .","For motif identification at significant ECT2-HT sites , all sequences for bases at and -/+ 2 nt of the significant editing positions were derived from TAIR10 using the R package rtracklayer ( Lawrence et al . , 2009 ) in either aerial tissues or roots .","A matrix of nt frequencies ( A , C , G , or U ) was generated , and the R package ggseqlogo ( Wagih , 2017 ) was used to generate the final motif .","For the calculation of editing proportions as a function of the proportion of cells coexpressing ECT2 , we first downloaded the expression matrix based on a total of 4727 individual cells from scRNA-seq in roots from Denyer et al . , 2019 .","To estimate the relationship between coexpression of target genes with ECT2 and their average editing proportions , the expression matrix was used to calculate coexpression for each target gene as follows: ( # cells expressing ECT2 AND target gene ) / ( # cells expressing target gene ) .","These proportions were then split into groups and plotted against the maximum editing proportions from HyperTRIBE in the containing genes .","For expression binning , log2 ( TPM +1 ) values for all expressed genes in either aerial tissues , roots , or combined were split into nine bins of increasing expression , using the cut ( ) function from the R package Hmisc version 4 . 5-0 ( https://github . com/harrelfe/Hmisc/ ) .","For the proportion of target genes in every expression bin , we calculated the proportion of genes in each set ( ECT2 HT/iCLIP-targets or nontargets , ECT2 FA-CLIP; Wei et al . , 2018 ) or m6A sets ( Parker et al . , 2020 ) falling into each expression bin out of the total number of genes in that bin .","To demonstrate expression biases in unsupported ECT2-HT target genes , the genes were further split according to whether or not they had support from m6A ( Nanopore , miCLIP [Parker et al . , 2020] and m6A-seq [Shen et al . , 2016] ) .","In vivo UV crosslinking of 12-day-old seedlings and construction of iCLIP libraries were optimized for ECT2-mCherry from the method previously employed for Arabidopsis GRP7-GFP ( Meyer et al . , 2017; Köster and Staiger , 2020 ) as follows .","Crosslinked plant tissues ( see details below ) were finely ground in liquid nitrogen with mortar and pestle , homogenized in iCLIP buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 4 mM MgCl2 , 5 mM DTT , 1% SDS , 0 . 25% sodium deoxycholate , 0 . 25% Igepal ) supplemented with protease inhibitors ( 4 mM PMSF , 1 tablet/10 mL of Complete Protease Inhibitor Cocktail [Roche] , and 1/30 vol . of Protease Inhibitor Optimized for Plant Extracts [Sigma P9599] ) , and cleared by centrifugation and filtration ( 0 . 45 μm pore ) of the supernatant .","RNP-complexes were then immunopurified with beads coupled to anti-RFP nanobodies ( ChromoTek RFP-Trap in our case ) for 1 hr at 4°C under constant rotation .","In particular , we used 20 μL of beads for 4 g of tissue in 6 mL of iCLIP buffer for every replicate .","After thorough washes with RIP-Wash Buffer ( 2 M urea , 50 mM Tris-HCl pH 7 . 5 , 500 mM NaCl , 4 mM MgCl2 , 2 mM DTT , 1% SDS , 0 . 5% sodium deoxycholate , 0 . 5% Igepal ) , RNP-complexes attached to the beads were subjected to treatment with DNase ( Turbo DNase [Ambion] , 4 U/100 μL ) and RNase I ( Ambion , 1 U/mL ) at 37°C for 10 min , dephosphorylation of RNA 3′ ends ( PNK [ThermoFisher] in pH 6 . 5 buffer ) , and 3′ RNA linker ligation ( L3-App linker [Huppertz et al . , 2014] and NEB HC RNA Ligase ) at 16°C overnight .","RNA was radioactively labeled at the 5′ end by PNK-mediated phosphorylation using γ-32P-ATP ( 20 min at 37°C ) .","The labeled RNP complexes were subjected to SDS-PAGE and blotting on a nitrocellulose membrane ( Protran BA-85 ) .","Pieces of membrane containing a size range of RNA species bound to the protein ( a smear above the expected molecular weight localized by autoradiography ) were excised and subjected to proteolysis ( 200 μg of Proteinase K [Roche] in 200 μL of PK buffer [100 mM Tris-HCl pH 7 . 4 , 50 mM NaCl , 10 mM EDTA] for 20 min at 37°C ) to release RNA bound to small peptides .","The RNA was then purified with TRI-Reagent ( Sigma ) and used to prepare sequencing libraries through the following steps: reverse transcription ( Superscript III , Invitrogen ) using a two-part cleavable DNA adapter complementary to the 3′ RNA linker as primer , gel purification and size selection of cDNA ( high , 120–200 nt; medium , 85–120 nt; low , 70–85 nt ) , circularization ( CircLigase II Epicentre ) , relinearization ( BamHI ) , and PCR amplification ( AccuPrime Supermix I , Invitrogen ) .","All steps were performed as described by Huppertz et al . , 2014 , and the amount of cycles in the final PCR was optimized to the amount of cDNA in each sample .","Notice that we introduced a few modifications in the original protocol ( Köster and Staiger , 2020 ) to account for ( 1 ) low abundance of ECT2 compared to AtGRP7 .","To obtain enough RNA , we increased the crosslinking energy and irradiated 12-day-old seedlings with 2000 mJ/cm2 of 254 nm UV light , harvesting roots and shoots ( 4 g of tissue per replicate ) to maximize the amount of purified ECT2-mCherry .","( 2 ) ECT2 sensitivity to proteolysis .","We did not pre-clear the lysates to reduce the incubation time , and we used high amounts of protease inhibitors during immunoprecipitation .","( 3 ) High molecular weight of ECT2-mCherry .","Due to the size of the protein , we required longer electrophoresis time and cooling ( 3 hr at 180 V with the tank on ice ) .","( 4 ) Different RNA-binding capacity of ECT2 .","Based on trials , we decided to adjust the RNase I treatment to 1 U/mL , incubating for 10 min at 37°C ( 5 μL of RNase I [Ambion , 100 U/μL pre-diluted 1:5000] in 100 μL ) .","Of note , the conditions indicated here were specifically used for library preparation .","Although we used the same conditions as default for CLIP experiments to assess ECT2 RNA-binding capacity , ECT2 sensitivity to proteolysis and ECT2-bound RNA sensitivity to RNase treatment , variations in buffer composition , incubation time , concentration of protease inhibitors , and/or RNase I are specified in the corresponding figure legends where necessary .","The entire iCLIP-seq experiment including three replicates of each group was done once .","Sequenced reads from all samples were investigated after each processing step with fastqc 0 . 11 . 5 ( https://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) .","Adapters at the 3′ end were trimmed using cutadapt version 1 . 16 ( Martin , 2011 ) .","The demultiplexing of the samples was performed using flexbar 3 . 4 . 0 with the -bk parameter to conserve the barcode information for further steps ( Roehr et al . , 2017 ) .","Reads with a length below 24 nucleotides were discarded .","Barcodes were trimmed and saved to the read_id field .","Processed reads were mapped to the TAIR10 genome with STAR version 2 . 6 . 0a allowing a maximum of two mismatches and soft clipping only at 3′ end ( Dobin et al . , 2013 ) .","PCR duplicates were removed by grouping the reads by their mapping start position .","Reads with the identical start position and random barcode were removed from the samples ( Python3 and pybedtools ) .","The peak calling of uniquely mapped reads was done using PureCLIP 1 . 0 . 4 , choosing the second peak-shape option to allow more broader peaks to be called ( Krakau et al . , 2017 ) .","For consistency with the ECT2-HT datasets , the ECT2-iCLIP datasets were annotated using the hyperTRIBER annotation ( Rennie et al . , 2021 ) , using quantifications based on the average of roots and aerial tissues from FLAG-ADAR samples in ECT2-HT ( to reflect that the ECT2-iCLIP data is based on whole seedlings ) .","To calculate the proportion of sites falling at each nucleotide , nucleotide sequences from the reference genome were first obtained from site coordinates for ECT2 iCLIP/m6A-Nanopore/ m6A-miCLIP using the R packages GenomicRanges ( Lawrence et al . , 2013 ) and rtracklayer ( Lawrence et al . , 2009 ) .","Nucleotide proportions were plotted using ggplot2 ( https://ggplot2 . tidyverse . org ) .","Single-cell expression data and marker genes associated with 15 clusters annotated to cell types in roots were downloaded from Denyer et al . , 2019 .","Single-nucleotide resolution locations of m6A sites ( defined according to Nanopore or miCLIP ) were downloaded from Parker et al . , 2020 .","Intervals defining m6A locations based on m6A-seq were downloaded from Shen et al . , 2016 , and intervals defining locations of ECT2-bound sites as determined by FA-CLIP were downloaded from Wei et al . , 2018 .","For consistency with HyperTRIBE and ECT2-iCLIP , all sets of m6A or ECT2-bound sites were gene annotated using the hyperTRIBER pipeline , based on genes and transcripts from Araport11 .","To remove redundancy after ECT2-iCLIP peak calling , directly adjacent peaks ( crosslink sites ) were grouped together and only the peak with the highest pureCLIP score ( dominant ) was kept .","The called peak position ( 1 nt resolution ) was extended by 4 nt up- and downstream to define a ‘collapsed crosslink site’ ( CSS ) with length 9 nt .","The center position marks the dominant called peak .","The extension of the peak positions was computed using bedtools version 2 . 27 . 1 ( Quinlan and Hall , 2010; Dale et al . , 2011 ) .","The collapsed ECT2-iCLIP crosslink sites and m6A-Nanopore sites ( Parker et al . , 2020 ) were used to find motifs significantly enriched by Homer ( Heinz et al . , 2010 ) using a variety of window sizes , settings , and backgrounds .","Motifs resulting from Homer searches were collated manually , and a range of variants of the consensus motif RRACH ( e . g . , RACH , DRAY , DRACH , URACH , DRACG ) were also added to the list , as well as various combinations of U-rich sequences ( e . g . , UUUUU , UNUNU , etc . ) , specific motifs found to be of interest in scientific literature ( e . g . , URUAY [Wei et al . , 2018] , GGAU [Anderson et al . , 2018] ) , and extra motifs that appeared of potential interest from manually browsing with IGV ( Robinson et al . , 2011 ) the sequence in the vicinity of iCLIP peaks ( e . g . , YYYYY , DRACUCU ) .","This resulted in a final list of 48 motifs for further analysis .","For each of the 48 motifs compiled from multiple sources , a custom PWM was generated based on local sequence frequencies around ECT2-iCLIP peaks and used as input to FIMO 5 . 1 . 1 ( Grant et al . , 2011 ) to detect genome-wide occurrences .","To generate PWMs , we used the formula PWMb , j = log2 [p ( b , j ) /p ( b ) ] , where p ( b ) is the background frequency of each nucleotide ( see further down ) , and p ( b , i ) is the frequency of the nucleotides in each position j .","We also included an extra small frequency count in the calculation to account for potential uncertainty in redundancies .","In order to account for location-specific sequence contexts ( typically 3′-UTR ) , each site from iCLIP or m6A ( Parker et al . , 2020 ) sets was assigned a random ‘matched background’ site , in a non-target gene , at the same relative location along the annotated genomic feature of the site ( 5′-UTR , CDS or 3′-UTR ) , according to a resolution of 10 bins per feature .","Logos for all motifs were generated using the R packages ggplot2 ( https://ggplot2 . tidyverse . org ) and ggseqlogo ( Wagih , 2017 ) .","To run the calculated PWMs through FIMO , we specified background letter frequencies ( A: 0 . 273 , C: 0 . 165 , G: 0 . 173 , U: 0 . 389 ) , a threshold of 0 . 05 , and scanning across the full TAIR10 Arabidopsis genome .","Sites were further filtered downstream to have a score of at least 4 – in the vast majority of cases corresponding to an exact match the ( short ) motif .","Distributions of motifs per 1000 sites over distance , centering on ECT2-iCLIP or m6A sites and the respective matched backgrounds , were generated using a custom R-script ( https://github . com/sarah-ku/targets_arabidopsis ) based on overlaps using GenomicRanges ( Lawrence et al . , 2013 ) .","At any given shift from the peak set , the raw number of overlaps of the motif ( at any point ) was calculated and normalized to give a motif count per 1000 peaks .","To adjust for the potential for downstream regions overshooting the end of the 3′-UTR , at each given distance only sites that continue to overlap an annotated gene ( Araport11 ) are counted .","For some analyses , peak sets were further split according to IDR-dependency or target status as indicated .","To calculate motif enrichment over the gene body , motifs were first annotated a value according to their relative position within the gene body regions: 5′-UTR , CDS or 3′-UTR .","In order to account for over-representation of counts within the CDS , due to greater sequence coverage within transcript annotations , a random background set of 10 million positions were generated from the transcript annotation file and annotated in the same way as the motif locations to obtain an expected distribution of all positions over the gene body regions .","This Expected distribution was used to normalize the Observed distribution of each motif , and O/E values were plotted as a metagene plot over the gene .","An enrichment of 1 suggests that the motif is neither over- or under-represented at that location .","Called positions from either Nanopore m6A data ( Parker et al . , 2020 ) or ECT2 iCLIP were first reduced to remove redundant regions of multiple peaks within the same window , then paired with matched background sets ( described above ) .","Windows representing ‘at’ ( ±10 nt ) the motif together with adjacent upstream ‘up’ and downstream ‘down’ windows of length 50 nt ( resulting in total window sizes of 120 nt ) around each position were annotated according to the number of each of the motifs overlapping ( truncated at 10 ) , and the final data set normalized .","To create a held-out set , 1/5th of the peaks were removed from the set , and the other 4/5th were used to build a random forest model using gradient boosting ( R package gbm version 2 . 1 . 8; https://github . com/gbm-developers/gbm ) , with settings specifying a shrinkage of 0 . 05 , an interaction . depth of 6 , cv . folds = 5 , and n . trees = 2000 .","For each model ( m6A Nanopore-based or ECT2 iCLIP-based ) , importance scores were extracted from the model and the top features were selected .","The held-out data was further used to estimate the predictive score of the model by calculating the AUC ( R package pROC; Robin et al . , 2011 ) .","Two further models were run – one involving the top 10 features from the full feature model , and ( only for the m6A-Nanopore set-up ) one involving features from only DRACH and UNUNU ( equating to six features in total ) , and AUC values were calculated and compared to that of the full feature model ."]],"string":"[\n [\n \"N6-methyladenosine ( m6A ) is the most abundant modified nucleotide in eukaryotic mRNA bodies .\",\n \"It is required for embryonic development and stem cell differentiation in several animals and plants ( Zhong et al . , 2008; Batista et al . , 2014; Ping et al . , 2014; Geula et al . , 2015; Zhang et al . , 2017 ) and for the control of the meiotic program in yeast ( Shah and Clancy , 1992; Clancy et al . , 2002; Agarwala et al . , 2012 ) .\",\n \"Most N6-adenosine methylation of mRNA is catalyzed in the nucleus ( Salditt-Georgieff et al . , 1976; Ke et al . , 2017; Huang et al . , 2019 ) by a highly conserved , multimeric methylase ( the m6A ‘writer’; Balacco and Soller , 2019 ) whose catalytic core consists of the heterodimer METTL3/METTL14 ( MTA/MTB in plants; Bokar et al . , 1997; Zhong et al . , 2008; Liu et al . , 2014 ) .\",\n \"In addition , a number of highly conserved proteins is required for N6-methylation in vivo ( Balacco and Soller , 2019 ) .\",\n \"The strong conservation of these core factors suggests that the biochemical basis of N6-adenosine methylation is common in eukaryotes , and indeed , m6A occurs in the consensus site RR ( m6A ) CH ( R = G/A , H = A/C/U ) , primarily in 3′-UTRs in vertebrates , plants , and fungi that possess the canonical METTL3/METTL14 methyltransferase ( Bodi et al . , 2012; Dominissini et al . , 2012; Meyer et al . , 2012; Schwartz et al . , 2013; Luo et al . , 2014a; Zhao et al . , 2017; Miao et al . , 2020; Parker et al . , 2020 ) .\",\n \"Conversely , the characteristic motif and gene body location is not detected in organisms that lack METTL3/METTL14 homologs , such as the nematode Caenorhabditis elegans ( Sendinc et al . , 2020 ) and bacteria ( Deng et al . , 2015 ) .\",\n \"m6A may impact mRNA function by different mechanisms , including the creation of binding sites for reader proteins that specifically recognize m6A in mRNA ( Dominissini et al . , 2012; Fu et al . , 2014; Meyer and Jaffrey , 2014 ) .\",\n \"The best understood class of readers contains a so-called YT521-B homology ( YTH ) domain ( Stoilov et al . , 2002 ) of which two phylogenetic groups , YTHDF and YTHDC , have been defined ( Patil et al . , 2018; Balacco and Soller , 2019 ) .\",\n \"The YTH domain harbors a hydrophobic methyl-binding pocket that increases the affinity of m6A-containing RNA by more than 10-fold compared to unmethylated RNA ( Li et al . , 2014b; Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014; Zhu et al . , 2014 ) .\",\n \"Apart from interactions with the methylated adenosine and the purine at the –1 position , YTH domain-RNA interactions mostly involve the sugar-phosphate backbone of the RNA ( Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014 ) .\",\n \"That is consistent with only mild reductions in the binding affinity of the YTH domain of human YTHDC1 upon substitution of nucleotides −2 , +1 , and +3 that abrogate the canonical RR ( m6A ) CH motif ( Xu et al . , 2014 ) , and poor sequence specificity of RNA binding by isolated YTH domains of human YTHDF1 , YTHDF2 , and YTHDC1 ( Arguello et al . , 2019 ) .\",\n \"Thus , the methyltransferase complex gives the sequence specificity , while YTH domain proteins may bind to m6A-containing RNA regardless of the identity of the immediately adjacent nucleotides .\",\n \"YTHDF proteins are typically cytoplasmic and consist of a long N-terminal intrinsically disordered region ( IDR ) followed by the globular YTH domain ( Patil et al . , 2018 ) .\",\n \"Because the affinity of isolated YTH domains for m6A-containing RNA is modest , typically with dissociation constants on the order of 0 . 1–1 μM ( Li et al . , 2014b; Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014; Zhu et al . , 2014 ) , it has been suggested that the IDR may participate in RNA binding ( Patil et al . , 2018 ) .\",\n \"Nonetheless , the clearest evidence for functions of the IDRs in YTHDF proteins reported thus far includes direct interactions with effectors such as the CCR4-NOT complex in mammalian cells ( Du et al . , 2016 ) , and the ability to cause liquid-liquid phase transition when sufficiently high local concentrations are reached ( Arribas-Hernández et al . , 2018; Gao et al . , 2019; Ries et al . , 2019; Fu and Zhuang , 2020; Wang et al . , 2020 ) .\",\n \"The YTHDF family comprises 11 proteins in Arabidopsis that are referred to as EVOLUTIONARILY CONSERVED C-TERMINAL REGION1-11 ( ECT1-11 ) ( Li et al . , 2014c; Scutenaire et al . , 2018 ) .\",\n \"ECT2 , ECT3 , and ECT4 are expressed in rapidly dividing cells of root , leaf , and flower primordia , and genetic analyses have revealed their general importance in organogenesis ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .\",\n \"Importantly , the biological functions of ECT2/ECT3/ECT4 described thus far are shared with those of m6A writer components and , where tested , have been shown to depend on intact m6A-binding pockets , strongly suggesting that the basis for the observed phenotypes in ect2/ect3/ect4 mutants is defective regulation of m6A-modified mRNA targets ( Bodi et al . , 2012; Shen et al . , 2016; Ružička et al . , 2017; Arribas-Hernández et al . , 2018; Scutenaire et al . , 2018; Wei et al . , 2018; Arribas-Hernández et al . , 2020 ) .\",\n \"Despite the progress in identifying biological functions of plant m6A-YTHDF axes , a number of fundamental questions regarding their molecular basis remains unanswered .\",\n \"For example , it is unclear whether sequence determinants in addition to m6A are important for mRNA target association of ECT proteins in vivo , the mRNA targets of ECT2/ECT3/ECT4 responsible for the developmental delay of ect2/ect3/ ( ect4 ) mutants have not been identified , and it is not clear what the effects of ECT2/ECT3/ECT4 binding to them may be ( Arribas-Hernández and Brodersen , 2020 ) .\",\n \"Clearly , robust identification of the mRNA targets directly bound by ECT proteins is key to obtain satisfactory answers to all of these questions .\",\n \"Towards that goal , formaldehyde crosslinking and immunoprecipitation ( FA-CLIP ) was used to identify mRNA targets of ECT2 ( Wei et al . , 2018 ) .\",\n \"Nonetheless , because formaldehyde , in contrast to UV illumination , generates both protein-protein and protein-RNA crosslinks , it is not an ideal choice for identification of mRNAs bound directly by a protein of interest ( see Arribas-Hernández and Brodersen , 2020 for a discussion ) .\",\n \"In particular , this problem concerns the unexpected conclusion that ECT2 binds to the ‘plant-specific consensus motif’ URU ( m6A ) Y ( Y = U/C ) , not RR ( m6A ) CH ( Wei et al . , 2018 ) .\",\n \"Thus , the field of gene regulation via m6A-YTHDF modules in plants is in a state of confusion: on the one hand , m6A mapping ( Luo et al . , 2014a; Wan et al . , 2015; Shen et al . , 2016; Duan et al . , 2017; Anderson et al . , 2018; Miao et al . , 2020; Parker et al . , 2020 ) and phenotypes of mutants defective in m6A writing ( Bodi et al . , 2012; Shen et al . , 2016; Ružička et al . , 2017 ) or m6A binding of ECT2/ECT3/ECT4 ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) suggest that these YTHDF proteins should act via recognition of m6A in the RRACH context .\",\n \"On the other hand , the only attempt at a mechanistic understanding of ECT2 function via mRNA target identification concluded that ECT2 binds to a sequence element different from RRACH ( Wei et al . , 2018 ) .\",\n \"To complicate matters further , a number of motifs including not only URUAY , but also UGUAMM ( M = A/C ) , UGWAMH ( W = A/U ) , UGUAWA , and GGAU have been reported to be enriched around m6A sites in Arabidopsis and other plant species ( Li et al . , 2014a; Anderson et al . , 2018; Miao et al . , 2020; Zhang et al . , 2019; Zhou et al . , 2019 ) , but it remains unclear whether the adenosines in such motifs are methylated in vivo .\",\n \"Alternatively , these sequence contexts may play a role in guiding m6A deposition or ECT recognition nearby , either directly by ECT interaction or indirectly via additional RNA-binding proteins assisting or competing with ECT binding .\",\n \"To clarify principles underlying mRNA recognition by ECT2 , we undertook rigorous analysis of its mRNA-binding sites using two orthogonal methods , the proximity-labeling method HyperTRIBE ( targets of RNA-binding proteins identified by editing ) ( McMahon Aoife et al . , 2016; Xu et al . , 2018 ) and iCLIP ( individual nucleotide resolution crosslinking and immunoprecipitation ) ( König et al . , 2010 ) .\",\n \"This resulted in identification of high-quality target sets as judged by mutual overlaps and by overlaps with previously reported m6A maps from plants at a similar developmental stage ( Shen et al . , 2016; Parker et al . , 2020 ) .\",\n \"Relying on this high-quality target set , we used the position information inherent to iCLIP and a single-nucleotide resolution m6A dataset ( Parker et al . , 2020 ) to establish six properties of m6A-containing mRNA and mRNA targeting by ECT2 .\",\n \"( 1 ) RRACH and its variant DRACH ( D = R/U ) are unequivocally the most highly enriched motifs at m6A sites in Arabidopsis .\",\n \"( 2 ) ECT2 binds to m6A sites in the canonical RRACH context as ECT2 crosslinking sites are preferentially found immediately 5′ to m6A sites , and RRACH is enriched immediately 3′ to ECT2 crosslinking sites .\",\n \"( 3 ) GGAU is a minor m6A consensus site in plants .\",\n \"( 4 ) U- and U/C-rich motifs are enriched around , but not at , m6A sites , and , together with RRACH and GGAU , constitute core elements that distinguish m6A-containing 3′-UTRs from non-m6A-containing 3′-UTRs in plants .\",\n \"( 5 ) The IDR of ECT2 participates in RNA binding as it crosslinks to target mRNAs at U-rich elements highly abundant upstream of m6A sites .\",\n \"( 6 ) Although URUAY , URURU , and similar motifs may crosslink to ECT2 , their presence in m6A-containing mRNA disfavors ECT2 binding , consistent with those motifs acting predominantly as sites of interaction for RNA-binding proteins that may compete with ECT2 .\"\n ],\n [\n \"HyperTRIBE uses fusion of RNA-binding proteins to the hyperactive E488Q mutant of the catalytic domain of the Drosophila melanogaster adenosine deaminase acting on RNA ( DmADARE488Qcd ) ( Kuttan and Bass , 2012 ) to achieve proximity labeling in vivo ( McMahon Aoife et al . , 2016; Xu et al . , 2018 ) .\",\n \"Targets are identified as those mRNAs that contain adenosine-inosine sites significantly more highly edited than background controls , measured as A-G changes upon reverse transcription and sequencing .\",\n \"To develop material suitable for ECT2 HyperTRIBE , we expressed AtECT2pro:AtECT2-FLAG-DmADARE488Qcd-AtECT2ter ( henceforth ‘ECT2-FLAG-ADAR’ ) in the single ect2-1 and triple ect2-1/ect3-1/ect4-2 ( te234 ) knockout backgrounds ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .\",\n \"We identified lines exhibiting nearly complete rescue of te234 mutant seedling phenotypes , indicating that the fusion protein was functional ( Figure 1A ) .\",\n \"We then used the expression level in complementing lines as a criterion to select lines in the ect2-1 single mutant background , for which no easily scorable phenotype has been described ( Figure 1—figure supplement 1A ) .\",\n \"Lines expressing free DmADARE488Qcd under the control of the endogenous ECT2 promoter ( AtECT2pro:FLAG-DmADARE488Qcd-AtECT2ter; henceforth FLAG-ADAR ) at levels similar to or higher than those of the fusion lines ( Figure 1—figure supplement 1A , B ) were used to control for background editing after verification that FLAG-ADAR expression did not result in phenotypic abnormalities in Col-0 WT plants ( Figure 1A ) .\",\n \"To identify ECT2 HyperTRIBE targets ( HT-targets ) , we sequenced mRNA from dissected root tips and shoot apices of 10-day-old seedlings of ect2-1/ECT2-FLAG-ADAR and FLAG-ADAR transgenic lines using five independent lines of each type as biological replicates to prevent line-specific artifacts .\",\n \"Next , we generated nucleotide base counts for all positions with at least one mismatch across the full set of samples of mapped reads ( Figure 1B ) , resulting in a raw list of potential editing positions .\",\n \"This revealed that the amount of editing was clearly higher in the lines expressing the ECT2-FLAG-ADAR fusion protein than in the negative control lines ( Figure 1C , Figure 1—figure supplement 1C ) .\",\n \"To identify positions with significantly higher editing rates in ECT2-FLAG-ADAR lines compared to controls , we developed a new approach to detect differential editing ( Figure 1B ) described in detail by Rennie et al . , 2021 .\",\n \"Briefly , the hyperTRIBER method of detecting differential editing exploits the powerful statistical capabilities of a method originally designed to detect differential exon usage ( Anders et al . , 2012 ) .\",\n \"It efficiently takes replicates and possible differences in expression into account , resulting in high power to detect sites despite the generally low editing proportions that we found in our data ( Figure 1D ) .\",\n \"As expected , the tendency towards higher editing proportions in fusion lines compared to controls was even more pronounced after filtering nonsignificantly edited sites ( Figure 1C , Figure 1—figure supplement 1C ) .\",\n \"Three additional properties of the resulting editing sites indicate that they are the result of ADARcd activity guided by its fusion to ECT2 .\",\n \"First , the vast majority of significant hits corresponded to A-to-G transitions ( Figure 1—figure supplement 1D ) .\",\n \"Second , the consensus motif at the edited sites matched the sequence preference of DmADARE488Qcd ( 5′ and 3′ nearest-neighbor preference of U>A>C>G and G>C>A~U , respectively [Xu et al . , 2018; Figure 1E , Figure 1—figure supplement 1E] ) , with highly edited sites more closely matching the optimal sequence context than lowly edited ones ( Figure 1—figure supplement 1F ) .\",\n \"Third , principal component analysis of editing proportions at significant sites over the different lines clearly separated the ECT2-FLAG-ADAR fusion lines from the control lines ( Figure 1F , Figure 1—figure supplement 1G ) .\",\n \"Application of subsequent filtering steps , including removal of non- ( A-to-G ) mismatches and of potential line-specific single-nucleotide variants ( see Materials and methods ) , resulted in a final list of 16 , 176 edited sites for aerial tissues and 19 , 242 for roots , corresponding to 4864 and 5052 genes ( ECT2 HT-targets ) , respectively ( Figure 1B , Supplementary file 1 ) .\",\n \"In both cases , this represents 27% of all expressed genes .\",\n \"We note that the editing proportions were generally low ( Figure 1D ) compared to previous work in Drosophila ( Xu et al . , 2018 ) , perhaps in part due to the limited number of cells that express ECT2 ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .\",\n \"Indeed , the ADAR expression level ( TPMs ) correlated strongly with editing proportions among ECT2-FLAG-ADAR lines ( Figure 1G , Figure 1—figure supplement 1H ) , and editing proportions were higher for target mRNAs that are coexpressed with ECT2 in a large percentage of cells according to single-cell RNA-seq ( Denyer et al . , 2019; Figure 1H ) , lending further support to the conclusion that the observed editing is ADAR-specific and driven to target mRNAs by ECT2 .\",\n \"Hence , HyperTRIBE can be used to identify targets of RNA-binding proteins in planta .\",\n \"To evaluate the properties of ECT2 HT-targets , we first noted that most of them were common between root and aerial tissues ( Figure 2A ) , as expected given the recurrent function of ECT2 in stimulating cell division in all organ primordia ( Arribas-Hernández et al . , 2020 ) .\",\n \"In agreement with this result , most of the targets specific to root or aerial tissues were simply preferentially expressed in either tissue ( Figure 2B ) .\",\n \"Moreover , the significant editing sites in roots and aerial tissues had a considerable overlap ( Figure 2A ) , and their editing proportions were similar in the two tissues ( Figure 2C ) .\",\n \"Of most importance , we observed a large overlap between the ECT2 HT-targets and m6A-containing transcripts mapped by different methods in seedlings ( Shen et al . , 2016; Parker et al . , 2020 ) as more than 76% of ECT2 HT-targets had m6A support by either study ( Figure 2D ) .\",\n \"These results validate our HyperTRIBE experimental setup and data analysis , and confirm that ECT2 binds predominantly to m6A-containing transcripts in vivo .\",\n \"We next moved on to independent target and binding site identification via iCLIP ( Figure 3A ) .\",\n \"We used transgenic lines expressing functional ECT2-mCherry under the control of the endogenous ECT2 promoter in the ect2-1 knockout background ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) to co-purify mRNAs crosslinked to ECT2 for iCLIP .\",\n \"Lines expressing the ECT2W464A-mCherry variant were used as negative controls because this Trp-to-Ala mutation in the hydrophobic methyl-binding pocket of the YTH domain abrogates the increased affinity for m6A-RNA ( Li et al . , 2014b; Xu et al . , 2014; Zhu et al . , 2014 ) .\",\n \"Accordingly , the point mutant behaves like a null allele in plants despite its wild-type-like expression pattern and level ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .\",\n \"To test the feasibility of iCLIP , we first assessed the specificity of RNA co-purified with ECT2-mCherry after UV illumination of whole seedlings by 5′-radiolabeling of the immunoprecipitated RNP complexes followed by SDS-PAGE .\",\n \"These tests showed that substantially more RNA co-purifies with wild-type ECT2 than with ECT2W464A upon UV-crosslinking , and that no RNA is detected without UV irradiation or from irradiated plants of non-transgenic backgrounds ( Figure 3B , Figure 3—figure supplement 1A ) .\",\n \"RNAse and DNAse treatments also established that the co-purified nucleic acid is RNA ( Figure 3—figure supplement 1B ) .\",\n \"Thus , UV crosslinking of intact Arabidopsis seedlings followed by immunopurification successfully captures ECT2-RNA complexes that exist in vivo .\",\n \"Curiously , although the pattern of ECT2-RNA complexes with bands migrating at ~110 and 55kDa is highly reproducible , it does not correspond to the majority of the purified ECT2-mCherry protein , which runs at ~125kDa in SDS-PAGE ( Figure 3B and C ) .\",\n \"A variety of control experiments ( Figure 3—figure supplement 1C-E ) , most importantly the disappearance of additional bands with use of an N-terminal rather than a C-terminal tag ( Figure 3C and D ) , indicate that the band pattern arises as a consequence of proteolytic cleavage of the N-terminal IDR in the lysis buffer , such that fragments purified using the C-terminal mCherry tag include the YTH domain with portions of the IDR of variable lengths ( Figure 3—figure supplement 2 ) .\",\n \"Comparative analysis of RNA in 55-kDa and 110–125-kDa complexes may , therefore , provide insight into the possible role of the N-terminal IDR of ECT2 in mRNA binding ( Figure 3E ) , an idea consistent with the comparatively low polynucleotide kinase labeling efficiency of full-length ECT2-mCherry-mRNA complexes ( ~125kDa ) ( Figure 3B , Figure 3—figure supplement 2 ) .\",\n \"Thus , we prepared separate iCLIP libraries from RNA crosslinked to ECT2-mCherry/ECT2W464A-mCherry that migrates at ~110–280kDa ( ‘110-kDa band’ ) and at ~55–75kDa ( ’55-kDa band’ ) ( Figure 3—figure supplement\",\n \"3 ) to investigate the possible existence of IDR-dependent crosslink sites , and thereby gain deeper insights into the mode of YTHDF binding to mRNA in vivo .\",\n \"We identified a total of 15 , 960 iCLIP ‘peaks’ or crosslink sites ( i . e . , single-nucleotide positions called by PureCLIP from mapped iCLIP reads [Krakau et al . , 2017] ) in 2281 genes from the 110-kDa band of wild-type ECT2-mCherry ( henceforth referred to as ECT2 iCLIP peaks and targets , respectively ) .\",\n \"In the corresponding 55-kDa band , 4549 crosslink sites in 1127 genes were called , 93% of them contained in the 110-kDa target set ( Figure 3F and G , Figure 3—figure supplement 4 , Supplementary file 2 ) .\",\n \"We note that these numbers perfectly agree with the idea of the 55-kDa band containing only YTH domain crosslink sites , while the full length may also include IDR crosslink sites .\",\n \"Importantly , for both libraries , the majority of crosslink sites mapped to the 3′-UTRs of mRNAs ( Figure 3H , see Figure 4A , and Figure 4—figure supplement 1 for more examples ) , coincident with the main location of m6A ( Figure 4B; Parker et al . , 2020 ) .\",\n \"Accordingly , the 3′-UTR specificity was largely lost in RNA isolated from 55-kDa ECT2W464A ( Figure 3H ) , for which neither YTH domain nor IDR binding to RNA can be expected .\",\n \"Finally , iCLIP targets in full-length ( 110-kDa band ) ECT2 WT and ECT2W464A overlapped only marginally ( Figure 3G ) , providing molecular proof of the dependence of m6A-binding activity for ECT2 function .\",\n \"Nonetheless , the bias towards occurrence in the 3′-UTR was only reduced , not abolished , for crosslinks to the full-length ECT2W464A protein , providing another indication that the IDR itself is able to associate with RNA-elements in 3′-UTRs ( Figure 3H ) .\",\n \"We elaborate further on this important point by analysis of IDR-specific crosslinks to wild-type ECT2 after in-depth validation of sets of ECT2 target mRNAs and determination of the sequence motifs enriched around m6A and ECT2 crosslink sites .\",\n \"To evaluate the congruence of the results obtained by iCLIP and HyperTRIBE , we investigated the cumulative number of iCLIP sites as a function of distance to the nearest editing site determined by HyperTRIBE .\",\n \"This analysis showed a clear tendency for iCLIP peaks called with ECT2WT-mCherry , but not for ECT2W464A-mCherry , to be in the vicinity of editing sites ( Figure 4C ) , indicating that the majority of called iCLIP peaks identify genuine ECT2-binding sites on mRNAs .\",\n \"Similar tendencies of proximity between iCLIP peaks and HyperTRIBE editing sites were previously observed for a Drosophila hnRNP protein ( Xu et al . , 2018 ) .\",\n \"Although manual inspection of individual target genes confirmed these tendencies , it also revealed that ADAR-edited sites are too dispersed around iCLIP peaks to give precise information on the actual ECT2-binding sites ( Figure 4A , Figure 4—figure supplement 1 ) .\",\n \"Therefore , we used both HyperTRIBE and iCLIP for gene target identification , but relied on iCLIP peaks for motif analyses .\",\n \"To examine the quality of our target identification in further detail , we analyzed the overlap between ECT2 targets identified by iCLIP and HyperTRIBE .\",\n \"This analysis also included m6A mapping data obtained with either m6A-seq ( Shen et al . , 2016 ) or the single-nucleotide resolution methods miCLIP and Nanopore sequencing ( Parker et al . , 2020 ) as young seedlings were used in all cases .\",\n \"ECT2 targets identified by iCLIP and HyperTRIBE showed clear overlaps , both with each other and with m6A-containing transcripts , further supporting the robustness of ECT2 target identification via combined iCLIP and HyperTRIBE approaches ( Figure 4D , upper panel , Figure 4—figure supplement 2 ) .\",\n \"Importantly , although some m6A targets are expected not to be bound by ECT2 because of the presence of MTA in cells that do not express ECT2 ( Arribas-Hernández et al . , 2020 ) , only 18% of the high-confident set of m6A-containing genes ( with support from miCLIP and Nanopore ) did not overlap with either ECT2 iCLIP or HT target sets ( Figure 4—figure supplement 2 , arrow ) .\",\n \"We also observed that HyperTRIBE identifies approximately three times more ECT2 targets than iCLIP , possibly because of the bias towards high abundance inherent to purification-based methods like iCLIP ( Wheeler et al . , 2018 ) .\",\n \"To test this idea , we compared the distribution of target mRNAs identified by the different techniques across nine expression bins .\",\n \"As expected , a bias towards highly abundant transcripts was evident for iCLIP-identified targets compared to HyperTRIBE ( Figure 4E ) .\",\n \"We also observed a similar bias for m6A-containing transcripts detected by miCLIP , another purification-based method , and in the Nanopore dataset ( Figure 4E ) , probably explained by its relatively low sequencing depth ( Parker et al . , 2020 ) .\",\n \"These observations also suggest that the higher sensitivity of HyperTRIBE ( analyzed in detail in Figure 4—figure supplement\",\n \"3 ) explains the lack of m6A support ( by Nanopore or miCLIP ) for 28% of ECT2 HT-targets ( 1689 ) compared to only 4% ( 83 ) of ECT2 iCLIP targets ( Figure 4D , Figure 4—figure supplement 2 , upper row ) since HT-targets may simply include genes that escape detection by m6A mapping methods due to low expression .\",\n \"Indeed , ECT2-HT targets without any m6A support were distributed in lower-expression bins compared to those with m6A support ( Figure 4F ) .\",\n \"Intriguingly , ECT2 FA-CLIP targets ( Wei et al . , 2018 ) did not show a bias towards highly expressed genes as their distribution over expression bins largely reflected that of the total number of genes ( Figure 4E ) , and as many as 37% of FA-CLIP targets did not have m6A support ( Figure 4D , Figure 4—figure supplement 2 , upper row ) .\",\n \"In summary , these analyses show that ECT2 iCLIP and HT target sets are in excellent agreement with each other and with independently generated m6A maps , and that HyperTRIBE identifies targets below the detection limit of other techniques .\",\n \"To characterize the sequence composition and exact positions of ECT2-binding sites relative to m6A , we first used the high resolution of iCLIP data to examine the position of ECT2 crosslink sites relative to m6A sites , determined at single-nucleotide resolution ( Parker et al . , 2020 ) .\",\n \"This analysis showed that ECT2 crosslinks in the immediate vicinity , but preferentially upstream ( ~11 nt ) of Nanopore-determined m6A sites , with a mild depletion at the exact m6A site ( Figure 5A , upper panel ) .\",\n \"Furthermore , while m6A-miCLIP sites corresponded to m6A-Nanopore sites overall , a subset of m6A-miCLIP sites were located upstream of m6A-Nanopore sites and coincided well with ECT2-iCLIP peaks ( Figure 5A ) .\",\n \"This pattern is probably explained by the fact that the UV illumination used in both iCLIP and miCLIP preferentially generates RNA-protein crosslinks involving uridine ( Hafner et al . , 2021 ) , also detectable in the datasets analyzed here ( Figure 5B and C ) .\",\n \"Thus , the depletion of ECT2-iCLIP sites at Nanopore- , but enrichment at miCLIP-determined m6A sites ( Figure 5A ) , might be explained by the absence of uridine within the RRAC core of the m6A consensus motif , and perhaps also to some extent by reduced photoreactivity of the m6A base stacking with indole side chains of the YTH domain .\",\n \"Furthermore , the fact that nucleotides at −2 , +1 , and +2 positions are only expected to contribute sugar-phosphate backbone interactions with the YTH domain ( Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014 ) may also contribute to the absence of direct crosslinks at the m6A site relative to the adjacent bases .\",\n \"The 5′ shift observed for iCLIP and miCLIP sites relative to Nanopore sites might be explained by a higher occurrence of uridines upstream of m6A sites , a particularly interesting possibility given the numerous reports of U-rich motifs enriched around m6A sites in plants ( Li et al . , 2014a; Anderson et al . , 2018; Miao et al . , 2020; Zhang et al . , 2019; Zhou et al . , 2019; Luo et al . , 2020 ) and animals ( Patil et al . , 2016 ) .\",\n \"To investigate the sequence composition around m6A and ECT2 sites , we first performed exhaustive unbiased de novo motif searches using Homer ( Heinz et al . , 2010; Figure 5—figure supplement\",\n \"1 ) and extracted all candidate motifs , including the m6A consensus motif RRACH , as well as GGAU ( Anderson et al . , 2018 ) , URUAY ( Wei et al . , 2018 ) , and several other U-rich sequences .\",\n \"Combined with manually derived candidate motifs ( Figure 5—figure supplement 1B ) , we then calculated position weight matrices ( PWMs ) for a final set of 48 motifs and scanned for their occurrences genome-wide using FIMO ( Grant et al . , 2011; Figure 5—figure supplements 1 and 2 ) .\",\n \"This allowed us to determine three key properties .\",\n \"First , the global enrichment of the motifs at locations across the gene body .\",\n \"Second , the total count of occurrences of each motif at m6A sites and ECT2-iCLIP crosslink sites compared to a set of sites in non-target mRNAs matching the location within gene bodies of m6A/ECT2-iCLIP sites ( expected background ) .\",\n \"Third , the distribution of the motifs relative to m6A and ECT2 iCLIP sites .\",\n \"The results of this systematic analysis ( Supplementary file\",\n \"3 ) were used to select those motifs with a more prominent enrichment at or around m6A and ECT2 sites ( Figure 5D ) .\",\n \"This approach defined two major categories of motifs of outstanding interest , RRACH-like and GGAU on the one side , and a variety of U/Y-rich motifs on the other .\",\n \"Figure 5D shows a minimal selection of such motifs , while a more comprehensive compilation is displayed in Figure 5—figure supplements 3 and 4 .\",\n \"Not surprisingly , RRACH-like motifs were the most highly enriched at m6A sites and showed a clear enrichment immediately downstream of ECT2 crosslink sites in our analyses , with the degenerate variant DRACH being the most frequently observed ( Figure 5D , Figure 5—figure supplement 3 ) .\",\n \"Motifs containing GGAU behaved similarly to DRACH , with a sharp enrichment exactly at m6A sites and mild enrichment downstream of ECT2 peaks ( Figure 5D ) , supporting a previous suggestion of GGAU as an alternative methylation site ( Anderson et al . , 2018 ) .\",\n \"The possible roles of the U/Y-rich motifs in m6A deposition and ECT2 binding are analyzed in the following sections .\",\n \"We first noticed that several motifs retrieved around ECT2 crosslink sites by Homer constituted extended versions of DRACH/GGAU with Us upstream ( e . g . , UGAAC/UGGAU ) or remnants of DRACH with U/Cs ( Ys ) downstream ( e . g . , ACUCU ) .\",\n \"To test whether these motifs are indeed located adjacent to m6A , we examined their distribution and enrichment around ECT2 and m6A sites .\",\n \"The distributions showed a clear enrichment at m6A positions with a shift in the direction of the U/Y-extension ( see Figure 5D for ACUCU and Figure 5—figure supplement 4 for others ) .\",\n \"An enrichment over location-matched background sites close to ECT2-iCLIP sites was also apparent ( see Figure 5D for ACUCU and Figure 5—figure supplement 4 for others ) , further supporting that ECT2 preferentially crosslinks to uridines located in the immediate vicinity of DRACH ( /GGAU ) .\",\n \"Thus , several enriched motifs around ECT2 crosslink sites are DRACH/GGAU-derived , and their detection in unbiased searches simply reflects a tendency of methylated DRACH/GGAU sites to be flanked by U/Ys .\",\n \"U/R-rich motifs without traces of adjacent DRACH ( e . g . , YUGUM , URUAY , URURU ) showed a characteristic enrichment around , but depletion at , m6A sites .\",\n \"For some motifs , the enrichment was more pronounced 5′ than 3′ to m6A sites ( see Figure 5D for URUAY and Figure 5—figure supplement 4 for others ) .\",\n \"The distance between the site of maximal motif occurrence and the m6A site roughly coincided with the shift observed in ECT2 crosslink sites relative to m6A ( Figure 5A , upper panel ) .\",\n \"Accordingly , these motifs were enriched exactly at ECT2 crosslink sites ( see Figure 5D for URUAY and Figure 5—figure supplement 4 for others ) , suggesting that they may constitute additional m6A-independent sites of interaction with ECT2 .\",\n \"We also observed that the 3′ enrichment of YYYYY was asymmetric and closer to m6A than that of UUUUU/URURU/URUAY ( Figure 5—figure supplement 4 , second row from the top ) , indicating a preference for hetero-oligopyrimidine tracts immediately downstream the m6A site , as suggested by the 3′-enrichment of DRACUCU-type motifs as described above .\",\n \"Taken together , these results suggest that N6-adenosine methylation preferentially occurs in DRACH/GGAU sequences surrounded by stretches of pyrimidines , with a preference for YYYYY ( e . g . , CUCU ) immediately downstream , URURU ( including URUAY ) immediately upstream , and UUUUU/UNUNU slightly further away in both directions .\",\n \"The enrichment of ECT2 crosslink sites at these motifs , and the fact that the m6A-binding-deficient mutant of ECT2 ( W464A ) crosslinks preferentially to 3′-UTRs through its N-terminal IDR , indicates IDR-mediated binding to U/R- and Y-rich motifs around m6A .\",\n \"Since our analysis thus far uncovered several motifs of potential importance for m6A deposition and ECT2 binding , we employed machine learning to distinguish m6A and ECT2 iCLIP sites from random location-matched background sites using motif-based features .\",\n \"Importantly , the underlying classification model includes all motif features within the same model , allowing an evaluation of the importance of the motifs relative to each other .\",\n \"We used as features the number of matches to each of the 48 motifs ( Figure 5—figure supplement\",\n \"2 ) in three distinct regions relative to the methylated site according to Nanopore sequencing ( Parker et al . , 2020 ) , defined as position 0: ‘at’ [–10 nt; +10 nt] , ‘down’ [–50 nt; –10 nt] , or ‘up’ [+ 10 nt; +50 nt] ( Figure 6A ) .\",\n \"The model involving all motifs could successfully distinguish the methylated sites from the background as indicated by an area under the receiver operating characteristic ( ROC ) curve ( true positive rate versus false positive rate , area under the curve [AUC] ) of 0 . 93 , and even a reduced model incorporating only the top 10 features from the full model classified sites largely correctly ( AUC = 0 . 86; Figure 6—figure supplement 1 ) .\",\n \"The top 16 features ordered by importance from the full model confirmed that RRAC/DRACH or GGAU at the site was indicative of the presence of m6A ( Figure 6B ) .\",\n \"Interestingly , U/Y-rich sequences ( UNUNU and YYYYY in particular ) flanking the site were also strongly indicative ( Figure 6B ) .\",\n \"Some motifs showed a skew in their feature importance score , with UNUNU and YUGUM showing a preference to be upstream , and YYYYY downstream ( Figure 6B ) , thus corroborating our previous observations ( Figure 6C ) .\",\n \"We used a similar modeling approach to identify non-m6A determinants of ECT2 binding , in this case comparing m6A sites within 10 nt distance of ECT2-iCLIP sites to m6A sites without ECT2-iCLIP sites nearby ( AUC = 0 . 94 , and AUC = 0 . 84 using only the top 10 features , Figure 6—figure supplement 1 ) .\",\n \"In agreement with previous observations , this model showed flanking U/Y-rich sequences as the main determinants for ECT2 crosslinking ( Figure 6D ) .\",\n \"To investigate the idea of URURU-like motifs as additional sites of ECT2 binding upstream of the m6A-YTH interaction site , we split Nanopore-m6A sites according to two criteria: ( 1 ) whether they occur in ECT2-target transcripts ( both permissive and stringent sets analyzed separately ) , and ( 2 ) for ECT2 targets , whether there is an ECT2 crosslink site within 25 nt of the m6A site ( ‘near’ ) or not ( ‘far’ ) .\",\n \"Although there was no obvious differences between these categories for most of the motifs ( Supplementary file 3 , page 2 ) , some U-rich sequences displayed distinctive features ( Figure 7A , Figure 7—figure supplement\",\n \"1 ) that can be summarized as follows .\",\n \"If a transcript has m6A and ECT2 sites in close proximity , it is ( 1 ) more likely to have UNUNU/UUUUU/YYYYY sequences upstream of the m6A site than targets with distantly located ECT2-binding sites or than non-ECT2 targets; ( 2 ) less likely to have UUUUU/URURU sequences downstream of the m6A site , possibly because ECT2 prefers CUCU-like sequences downstream; and ( 3 ) less likely to have URURU/URUAY-like motifs upstream of the m6A site .\",\n \"The latter observation is striking because for the specific subset of ECT2-bound m6A sites with URURU/URUAY upstream of m6A , these sequences tend to crosslink to ECT2 , as seen by the enrichment spike at ECT2 crosslink sites ( Figure 5D , Figure 7—figure supplement 1 , bottom panels ) .\",\n \"Although these two results seem contradictory at first glance , they may be reconciled by a model in which a URURU/URUAY-binding protein would compete with ECT2 for binding adjacent to m6A .\",\n \"If that protein is absent , ECT2 may bind to the site , potentially via its IDR , to stabilize the low-affinity YTH-m6A interaction and crosslink efficiently due to the U-content .\",\n \"Conversely , if occupied by the alternative interacting protein , the site might repel ECT2 ( see Discussion and Figure 7B ) .\",\n \"We reasoned that insights into contacts between ECT2 and mRNA may be gained by analysis of the iCLIP libraries prepared with the ‘YTH-mCherry’ truncation devoid of the N-terminal IDR ( ‘55-kDa band’ ) compared to the full-length ECT2-mCherry ( ‘110-kDa band’ ) ( Figure 3 , Figure 3—figure supplements 2–4 ) .\",\n \"Initial inspection of the distribution of ECT2 peaks relative to Nanopore-m6A sites showed that the 5′–3′ asymmetry observed with full-length ECT2 was largely reduced with the truncated protein ( Figure 7C ) , as was the bias towards uridines ( Figure 7—figure supplement 2A ) .\",\n \"These observations suggest that the IDR indeed is implicated in binding to U-rich regions upstream of m6A .\",\n \"We next split the full-length ECT2 iCLIP peaks according to whether they are present in libraries from both full-length and truncated forms ( ‘IDR-independent’ ) or exclusively in the full-length ( ‘IDR-dependent’ ) ( distance >10 nt ) and plotted the enrichment of the studied motifs relative to the crosslink site ( Figure 7D , Figure 7—figure supplement 2B; Supplementary file 3 , page 2 ) .\",\n \"UUUUUU/UNUNU-like motifs were more enriched at and immediately upstream of IDR-dependent crosslink sites relative to the IDR-independent ones , supporting preferential crosslinking of the IDR to Us in this region .\",\n \"Remarkably , the exact opposite was true for URURU/URUAY motifs that showed modest depletion 5′ to IDR-dependent crosslink sites relative to their IDR-independent counterparts ( Figure 7D ) .\",\n \"These observations are consistent with a model of an RNA-binding protein competing with the ECT2 IDR for interaction with upstream URURU/URUAY motifs ( Figure 7B ) .\"\n ],\n [\n \"Our work establishes experimental and computational approaches to implement HyperTRIBE for unbiased and sensitive mapping of direct targets of RNA-binding proteins in plants .\",\n \"Two points are particularly relevant in this regard .\",\n \"First , the examples studied here show that stable transgenic expression of DmADARcd does not lead to detrimental phenotypes , perhaps because of the generally low editing proportions obtained in vivo .\",\n \"Second , the rigorous statistical approach developed to call editing sites makes HyperTRIBE powerful , despite the low editing proportions observed .\",\n \"We also note that ECT2 is well suited to verify that HyperTRIBE mostly recovers directly bound target RNAs because of the possibility to cross-reference the data with independently obtained m6A maps ( Parker et al . , 2020 ) .\",\n \"The combination of iCLIP and HyperTRIBE for unbiased mapping of targets proved particularly attractive for at least two reasons .\",\n \"First , the convergence on overlapping target sets by orthogonal methods strengthens the confidence that the identified targets are biologically meaningful .\",\n \"Second , HyperTRIBE , especially with the novel computational approach for calling of editing sites ( Rennie et al . , 2021 ) , offers higher sensitivity than iCLIP , while iCLIP is unmatched in providing information on binding sites within target RNAs .\",\n \"It is possible that better positional information on binding sites may be obtained from HyperTRIBE data using maximal editing proportions rather than statistical significance as the parameter to call editing sites .\",\n \"Indeed , recent work on the use of HyperTRIBE to identify targets of the RNA-binding protein MUSASHI-2 ( MSI-2 ) in leukemic stem cells recovered the known MSI-2-binding site as enriched around editing sites in targets ( Nguyen et al . , 2020 ) .\",\n \"Nonetheless , our data shows that highly edited sites match the ADAR substrate consensus site better than lowly edited sites , suggesting that site proximity to ADAR is not the only determinant of editing proportions .\",\n \"Finally , our work also clearly indicates that FA-CLIP , now used in at least two studies involving YTH domain proteins ( Wei et al . , 2018; Song et al . , 2021 ) , is not a recommendable technique as it recovers many false positives and fails to include many genuine targets .\",\n \"Thus , with the possible exception of cases in which evidence for indirect association is specifically in demand , such as the recent study in human cells of mixed tailing of viral RNA by the cellular terminal nucleotidyl transferase TENT4 ( Kim et al . , 2020 ) , FA-CLIP should not be used for identification of RNAs associating with a particular RNA-binding protein of interest .\",\n \"Our analyses of motif enrichments around m6A and ECT2 crosslink sites clarify roles of previously reported motifs and uncover new motifs of importance in m6A writing and ECT2 binding .\",\n \"Since m6A is a prerequisite for ECT2 binding , any analysis of determinants of ECT2 binding must consider determinants of N6-adenosine methylation separately .\",\n \"Three conclusions stand out from our analysis in this regard .\",\n \"First , the major N6-adenosine methylation site is DRACH , consistent with conclusions from multiple other studies .\",\n \"Second , GGAU is a minor N6-adenosine methylation site , as seen by its enrichment directly at m6A sites .\",\n \"Third , m6A occurs in DRACH/GGAU islands embedded in U-rich regions .\",\n \"Such U-rich regions around m6A sites emerged from sorting of methylated from non-methylated transcripts by machine learning as being of similar importance for recognition of m6A-containing transcripts from sequence features as DRACH and GGAU at m6A sites , suggesting their implication in MTA/MTB-catalyzed adenosine methylation ( Figure 6C ) .\",\n \"This , in turn , may also explain the pronounced 3′-UTR bias of m6A occurrence as extensive poly-U and poly-pyrimidine tracts are rare in coding regions ( Figure 5D , second row on the right-most column; Supplementary file 3 , page 1 ) .\",\n \"As a special case in this context , our analyses suggest a simple explanation for the tendency of m6A to occur at stop codons .\",\n \"UAA and UGA correspond to DRA , increasing the frequency of occurrence of DRACH directly at stop codons ( Figure 5D , second row on the left-most column ) , many of which have adjacent U-rich elements in the 3′-UTRs .\",\n \"We note that the observed pattern is in agreement with a role of the poly ( U ) -interacting proteins RBM15A/B associated with the mammalian methyltransferase complex in guiding methylation ( Patil et al . , 2016 ) .\",\n \"Whether a similar mechanism operates in plants , potentially via the distant RBM15A/B homologue FPA ( Arribas-Hernández and Brodersen , 2020 ) , remains to be investigated .\",\n \"It is a major conclusion of the present work that ECT2 binds to m6A predominantly in the DR ( m6A ) CH sequence context in vivo , consistent with reading of m6A written by the conserved nuclear MTA/MTB methyltransferase .\",\n \"This key conclusion refutes the claim by Wei et al . , 2018 that ECT2 binds to the supposedly plant-specific m6A-containing sequence motif URU ( m6A ) Y , and it thereby reconciles knowledge on m6A-YTHDF axes in plants specifically and in eukaryotes more broadly .\",\n \"The phenotypic similarity of plants defective in MTA/MTB writer and ECT2/ECT3/ECT4 reader function is now coherent with the locations of MTA/MTB-written m6A and ECT2-binding sites transcriptome-wide , and it is now clear that plants do not constitute an exception to the general biochemical framework for eukaryotic m6A-YTHDF function in which YTHDF proteins read the m6A signal written by the MTA/MTB methyltransferase .\",\n \"The pronounced protease sensitivity of IDRs , leading to limited proteolysis of ECT2 upon cell lysis after in vivo crosslinking , allowed us to extract information on the mode of ECT2-RNA binding from different observations , all converging on the conclusion that the IDR of ECT2 participates in RNA binding .\",\n \"First , RNA complexes with YTH-mCherry were 5′-labeled by polynucleotide kinase much more efficiently than RNA complexes with full-length ECT2-mCherry , indicating that the IDR limits accessibility to the 5′ of bound mRNAs .\",\n \"Second , in contrast to the m6A-binding-deficient YTHW464A-mCherry truncation , the full-length ECT2W464A-mCherry mutant retained an enrichment of crosslink sites in 3′-UTRs .\",\n \"Third , crosslinks specific to the IDR ( i . e . , observed only with full-length ECT2-mCherry-RNA complexes , but not with YTH-Cherry-RNA complexes ) , could be assigned and have two notable properties .\",\n \"They are mainly 5′ to m6A sites , and thereby cause a conspicuously asymmetric distribution of ECT2 crosslink sites around m6A sites , not seen with crosslinks to the YTH-mCherry fragment .\",\n \"In addition , the IDR-specific crosslinks are specifically enriched in U-rich elements of the type UUUUUU and UNUNU immediately upstream .\",\n \"Taken together , these observations suggest that the IDR of ECT2 participates in locating ECT2 to 3′-UTRs by association with U-rich elements .\",\n \"Thus , ECT2 , and perhaps YTHDF proteins more generally given their highly similar YTH domains , appears to bind RNA through multivalent interactions among which the YTH domain is responsible for m6A binding , and the IDR is responsible for interaction with adjacent elements .\",\n \"We note that the notion of RNA interaction by IDRs has precedent ( Corley et al . , 2020 ) , is consistent with the modest affinity of isolated YTHDF domains for m6A-containing oligonucleotides ( Patil et al . , 2018 ) , and is reminiscent of the recent demonstration that transcription factors use their globular DNA-binding domains to recognize core sequence elements of promoters , and their IDRs to provide additional DNA contacts , contributing to specificity ( Brodsky et al . , 2020 ) .\",\n \"Similarly , it is possible that diverging IDRs among YTHDF paralogs could confer target specificity via binding to distinct motifs in the vicinity of m6A sites , such that specific YTHDF-target mRNA repertoires could exist even for YTHDF proteins coexpressed in the same cells .\",\n \"Finally , we stress that although our data point to an important role of the IDR in RNA binding , it does not in any way suggest that this is the only function of the IDR , and protein-protein interactions involving the IDR are likely to be key to understanding YTHDF function molecularly .\",\n \"Despite the conclusions that URUAY does not contain m6A in Arabidopsis , and that ECT2 binds to DR ( m6A ) CH , our detailed analysis of sequence motifs enriched around m6A and ECT2 iCLIP crosslink sites shows that additional motifs , including URUAY , are likely to be implicated in m6A reading by ECT2 , even if not directly .\",\n \"In contrast to other m6A-proximal , pyrimidine-rich sequences ( e . g . , UNUNU , YYYYY ) that may be of importance for both m6A writing and ECT2 binding , URUAY appears to have ties more specifically to ECT2 binding thanks to three properties .\",\n \"( 1 ) When present 5′ to m6A sites , it crosslinks to ECT2 , suggesting that some part of the protein can be in contact with URUAY .\",\n \"( 2 ) URUAY is more enriched close to m6A sites for which there is no evidence of ECT2 binding , suggesting that it weakens ECT2 binding .\",\n \"This latter point is also consistent with the distinction of ECT2-bound from non-ECT2-bound m6A sites by machine learning that did not find URUAY to be of importance for ECT2-bound sites .\",\n \"( 3 ) The URUAY enrichment 5′ to ECT2 crosslink sites is observed only when crosslinks to both full-length protein and the YTH-mCherry fragment are considered ( IDR-independent ) , but disappears when crosslinks specific to the full-length protein ( IDR-dependent ) are analyzed .\",\n \"Although these observations may be explained by multiple scenarios , we find a simple , yet at present speculative , model attractive: URUAY may be a site of competition between the IDR of ECT2 and another , as yet unknown , RNA-binding protein .\",\n \"Such a competing factor could in theory be another YTHDF protein using higher-affinity IDR-URUAY contacts than ECT2 to achieve competitive binding .\",\n \"Many other possibilities exist , however .\",\n \"For example , it is intriguing that URUAY resembles part of a Pumilio-binding site ( Hafner et al . , 2010; Huh et al . , 2013 ) as it raises the tantalizing possibility of functional interaction between YTHDF and Pumilio proteins .\",\n \"In any event , the functional dissection of the URUAY element in m6A reading now constitutes a subject of major importance , emphasized by the broad conservation of its enrichment around m6A sites across multiple plant species , including rice ( Li et al . , 2014a; Zhang et al . , 2019 ) , maize ( Luo et al . , 2020; Miao et al . , 2020 ) , tomato ( Zhou et al . , 2019 ) , and Arabidopsis ( Miao et al . , 2020 ) .\"\n ],\n [\n \"We use the term ‘biological replicate’ in the following way: plants were grown at the same time , under the same conditions , but in separate plates .\",\n \"Each sample replicate contains pools of seedlings prepared in such a way that no two replicates contain seedlings grown on the same plates .\",\n \"This sampling ensures that plate-to-plate variation in growth conditions , if any , will have an effect on measurements of gene expression within a single genotype , and hence minimize the risk that any differences due to such variation are called as significant in comparisons between genotypes .\",\n \"‘Technical replicates’ are understood to be independently conducted measurements using the same technique on the same biological material ( e . g . , on one biological replicate as defined above ) .\",\n \"Technical replicates were not carried out in this study , and the term ‘replicate’ refers to biological replicate as defined above .\",\n \"In our definition , an ‘experiment’ results in generation and comparison of measurements arising from multiple biological replicates of different biological entities , in the present case often Arabidopsis seedlings differing in genotype with respect to the genes ECT2 , ECT3 , and ECT4 .\",\n \"Thus , repetition of an experiment in our definition entails generation and analysis of the required biological replicates at different points in time .\",\n \"All lines used in this study are in the Arabidopsis thaliana Col-0 ecotype .\",\n \"The mutant alleles or their combinations – ect2-1 ( SALK_002225 ) ( Arribas-Hernández et al . , 2018; Scutenaire et al . , 2018; Wei et al . , 2018 ) , ect3-1 ( SALKseq_63401 ) , ect4-2 ( GK_241H02 ) , and ect2-1/ect3-1/ect4-2 ( te234 ) ( Arribas-Hernández et al . , 2018 ) – have been previously described .\",\n \"The transgenic lines expressing ECT2pro:ECT2-mCherry-ECT2ter , ECT2pro:ECT2W464A-mCherry-ECT2ter , ECT2pro:3xHA-ECT2-ECT2ter , or ECT2pro:3xHA-ECT2W464A-ECT2ter in the ect2-1 background have also been described or generated by floral dip in additional mutant backgrounds using the same plasmids and methodology ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .\",\n \"Seeds were surface-sterilized by 2 min incubation in 70% EtOH plus 10 min in sterilizing solution ( 1 . 5% NaOCl , 0 . 05% Tween-20 ) and 2 H2O washes .\",\n \"After 2–5 days of stratification at 4°C in darkness , seeds were germinated and grown on plates containing Murashige and Skoog ( MS ) -agar medium ( 4 . 4 g/L MS , 10 g/L sucrose , 10 g/L agar ) pH 5 . 7 at 20°C , receiving ~70 μmol m–2 s–1 of light in a 16 hr light/8 hr dark cycle as default .\",\n \"For HyperTRIBE and iCLIP experiments , the plates were placed vertically to facilitate root harvesting .\",\n \"MS-agar media for HyperTRIBE T2 seedlings was supplemented with 7 . 5 mg/L of glufosinate ammonium ( Sigma ) to select plants expressing the ADAR-containing transgenes .\",\n \"To assess phenotypes of adult plants , ~8-day-old seedlings were transferred from horizontal MS plates ( 4 . 4 g/L MS , 10 g/L sucrose , 8 g/L agar; pH 5 . 7 ) to soil and maintained in Percival incubators under 16 hr light/8 hr dark cycles , 21°C day/18°C night temperature , and ~100 μmol m–2 s–1 light intensity .\",\n \"We used Philips fluorescent tubes TL-D 90 De Luxe 36 W as light source .\",\n \"We employed USER cloning ( Bitinaite and Nichols , 2009 ) to generate ECT2pro:ECT2-FLAG- DmADARE488Qcd-ECT2ter and ECT2pro:FLAG-DmADARE488Qcd-ECT2ter constructs in pCAMBIA3300U ( pCAMBIA3300 with a double PacI USER cassette inserted between the PstI-XmaI sites at the multiple cloning site; Nour-Eldin et al . , 2006 ) .\",\n \"Fragments containing ECT2 gDNA sequences were amplified by PCR ( KAPA HiFi Hotstart Uracil + ReadyMix , Roche ) from plasmids previously generated in our lab ( Arribas-Hernández et al . , 2018 ) .\",\n \"The FLAG-DmADARE488Qcd fragment was produced in the same way using a pGEM-T Easy ( Promega ) plasmid containing FLAG-DmADARE488Qcd as template , previously subcloned to introduce the E488Q hyperactive mutation by site-directed mutagenesis ( QuickChange , Agilent Technologies ) with primers LA729-LA730 ( Phusion HF DNA Polymerase , NEB ) .\",\n \"The E488Q mutation was detected by NlaIII ( ThermoFisher ) digestion of the PCR reaction ( DreamTaq , ThermoFisher ) obtained with primers LA660-LA735 .\",\n \"Of note , the FLAG and DmADARcd sequences had been previously glued together by USER cloning to produce AGO1pro:FLAG-DmADARcd-AGO1ter in pCAMBIA3300U for unrelated purposes ( unpublished work ) , and subsequently amplified by PCR with primers LA696-615 for introduction into pGEM-T Easy .\",\n \"To build AGO1pro:FLAG-DmADARcd-AGO1ter in the first place , the catalytic domain of the ADAR deaminase isoform N ( Y268-E669 ) was amplified from cDNA of D . melanogaster Canton-S wild-type flies and larvae with USER primers MVUSER12-22 .\",\n \"The rest of the fragments were amplified from pCAMBIA3300U AGO1pro:FLAG-AGO1-AGO1ter ( Arribas-Hernández et al . , 2016 ) with primers MVUSER1-11 and MVUSER23-6 .\",\n \"USER primers to amplify all fragments were designed to create overhangs compatible with either the PacI USER cassette present in the pCAMBIA3300U plasmid or the flanking sequences of the neighboring fragments .\",\n \"All primer sequences , their combinations to produce PCR fragments , and the arrangement of the fragments for USER cloning can be found in Appendix 1 .\",\n \"Kanamycin-resistant colonies of Escherichia coli DH5α ( NEB ) transformed with the constructs were analyzed by restriction digestion and sequencing prior introduction of the plasmids in Agrobacterium tumefaciens GV3101 ( Koncz and Schell , 1986 ) for plant transformation .\",\n \"Arabidopsis stable transgenic lines were generated by floral dip transformation ( Clough and Bent , 1998 ) of Col-0 WT , ect2-1 , or te234 , and selection of primary transformants ( T1 ) was done on MS-agar plates supplemented with glufosinate-ammonium ( Sigma ) ( 10 mg/L ) .\",\n \"We selected five independent lines of each type based on segregation studies ( to isolate single T-DNA insertions ) , phenotypic complementation ( in the te234 background ) , and transgene expression levels assessed by FLAG western blot .\",\n \"Protein extraction from 10-day-old seedlings and western blotting with FLAG , HA , and mCherry antibodies were done as previously described ( Arribas-Hernández et al . , 2018 ) .\",\n \"Loading was documented by amido black , Coomassie , or Ponceau staining of the total protein on the membrane .\",\n \"We extracted total RNA from manually dissected root tips and apices ( removing cotyledons ) of five independent lines ( 10-day-old T2 seedlings ) of each of the lines used for ECT2-HT to use as biological replicates .\",\n \"The tissue was flash-frozen in liquid nitrogen and ground into a fine powder using liquid nitrogen-cooled adaptors in a tissue homogenizer .\",\n \"For RNA extraction , we added 1 mL of TRI Reagent ( Sigma ) to the frozen tissue ( <100 mg ) , mixed quickly by vortexing , added 0 . 2 mL of chloroform , and separated the two resulting phases by vigorous shaking and 10 min centrifugation at 4°C .\",\n \"The RNA was then precipitated from the aqueous phase for 30 min at room temperature with 1 volume of isopropanol .\",\n \"RNA pellets were solubilized in 300 μL of H2O to remove polysaccharides through a mild precipitation by addition of 1/10 vol .\",\n \"99% EtOH and 1/30 vol .\",\n \"of 3 M NaOAc ( pH 5 . 2 ) and incubation on ice for 30 min .\",\n \"After 15 min of full-speed centrifugation at 4°C to pellet polysaccharides , we re-precipitated the RNA from the supernatant with 2 , 5× vol .\",\n \"99% EtOH and 1/10 vol .\",\n \"of 3 M NaOAc ( pH 5 . 2 ) , washed the pellet two times with 70% EtOH , and resuspended in 20–40 μL of H2O .\",\n \"This highly pure total RNA was then used to produce mRNA libraries through enrichment of mRNA with oligo ( dT ) beads ( 18-mers ) , random fragmentation , cDNA synthesis with random hexamers , custom second-strand synthesis ( Illumina ) , terminal repair , A-ligation and sequencing adaptor ligation , size selection ( 250–300 bp insert ) , and PCR enrichment .\",\n \"The libraries were prepared and sequenced ( Illumina PE150 , Q30 ≥ 80% ) as a service from Novogene .\",\n \"The entire HyperTRIBE experiment was done once .\",\n \"Significant differentially edited sites between ECT2-FLAG-ADAR ( fusion ) and FLAG-ADAR ( control ) samples for ECT2 HyperTRIBE ( ECT2-HT ) were called according to the hyperTRIBER pipeline ( Rennie et al . , 2021 ) .\",\n \"First , reads were trimmed using trimmomatic ( Bolger et al . , 2014 ) and mapped to the Arabidopsis genome ( TAIR10 ) using STAR ( Dobin et al . , 2013 ) , according to parameters suggested in a previous HyperTRIBE analysis ( Xu et al . , 2018 ) .\",\n \"All HyperTRIBE samples were also quantified using Salmon ( Patro et al . , 2017 ) , with appropriate settings for pair-end sequencing and non-stranded library setup and based on the transcriptome for Araport11 ( Cheng et al . , 2017 ) with manual addition of the FLAG-ADAR sequence .\",\n \"A custom Perl script based on SAMtools mpileup ( Li et al . , 2009 ) returned base counts for all positions where there is a mismatch from the reference in at least one sample .\",\n \"For running the hyperTRIBER analysis pipeline , we specified that any tested position must have a putative edit in at least four of the five replicates in the ECT2-FLAG-ADAR samples ( three of four in the case of roots since one of these samples , ‘L3 , ’ was deemed as low quality and subsequently removed from the significance calling pipeline ) .\",\n \"Significant hits ( adjusted p-value<0 . 01 and log2FC > 1 ) were further filtered as follows: ( 1 ) hits that did not correspond to an A-to-G change ( or a T-to-C change for the negative strand ) , ( 2 ) hits that were likely SNPs arising specifically in either the ECT2-FLAG-ADAR or FLAG-ADAR line manifesting in an editing proportion at or close to 1 , and ( 3 ) hits where the coverage of tags at the edit base over the ECT2-FLAG-ADAR were fewer than 10 reads .\",\n \"Specific scripts for the analysis of ECT2-HT data can be found at https://github . com/sarah-ku/targets_arabidopsis .\",\n \"Editing proportions were calculated as G/ ( A + G ) ( alternatively C/ ( U + C ) for the negative strand ) for all significant sites , averaged over all samples , separately for the ECT2-FLAG-ADAR and FLAG-ADAR samples .\",\n \"Significant sites were annotated to genes from Araport11 , prioritizing the gene with the highest expression ( annotated TPMs are based on Salmon quantifications of FLAG-ADAR control samples only ) in the given tissue in the case of multiple possibilities .\",\n \"Possible transcripts were subsequently ordered by expression , along with gene body location along the transcript ( 5′-UTR , CDS , 3′-UTR ) .\",\n \"Principal component analysis was carried out on the raw editing proportions per sample for all sites with significant evidence of editing .\",\n \"For the comparison of sites between aerial tissues and roots , genes defined as commonly expressed in both types of tissues were considered in all gene-based comparisons .\",\n \"For significant editing site-based comparison , we directly compared sites that were common and significant to both .\",\n \"To calculate correlations between editing proportions and FLAG-ADAR expression levels among lines , transcripts per million ( TPM ) mapping to FLAG-ADAR were extracted from quantifications from Salmon ( Patro et al . , 2017 ) and correlated with the raw editing proportions per sample , separately for the fusion and control samples .\",\n \"Background correlation estimates were calculated by first scrambling the order of the FLAG-ADAR TPM vector .\",\n \"For motif identification at significant ECT2-HT sites , all sequences for bases at and -/+ 2 nt of the significant editing positions were derived from TAIR10 using the R package rtracklayer ( Lawrence et al . , 2009 ) in either aerial tissues or roots .\",\n \"A matrix of nt frequencies ( A , C , G , or U ) was generated , and the R package ggseqlogo ( Wagih , 2017 ) was used to generate the final motif .\",\n \"For the calculation of editing proportions as a function of the proportion of cells coexpressing ECT2 , we first downloaded the expression matrix based on a total of 4727 individual cells from scRNA-seq in roots from Denyer et al . , 2019 .\",\n \"To estimate the relationship between coexpression of target genes with ECT2 and their average editing proportions , the expression matrix was used to calculate coexpression for each target gene as follows: ( # cells expressing ECT2 AND target gene ) / ( # cells expressing target gene ) .\",\n \"These proportions were then split into groups and plotted against the maximum editing proportions from HyperTRIBE in the containing genes .\",\n \"For expression binning , log2 ( TPM +1 ) values for all expressed genes in either aerial tissues , roots , or combined were split into nine bins of increasing expression , using the cut ( ) function from the R package Hmisc version 4 . 5-0 ( https://github . com/harrelfe/Hmisc/ ) .\",\n \"For the proportion of target genes in every expression bin , we calculated the proportion of genes in each set ( ECT2 HT/iCLIP-targets or nontargets , ECT2 FA-CLIP; Wei et al . , 2018 ) or m6A sets ( Parker et al . , 2020 ) falling into each expression bin out of the total number of genes in that bin .\",\n \"To demonstrate expression biases in unsupported ECT2-HT target genes , the genes were further split according to whether or not they had support from m6A ( Nanopore , miCLIP [Parker et al . , 2020] and m6A-seq [Shen et al . , 2016] ) .\",\n \"In vivo UV crosslinking of 12-day-old seedlings and construction of iCLIP libraries were optimized for ECT2-mCherry from the method previously employed for Arabidopsis GRP7-GFP ( Meyer et al . , 2017; Köster and Staiger , 2020 ) as follows .\",\n \"Crosslinked plant tissues ( see details below ) were finely ground in liquid nitrogen with mortar and pestle , homogenized in iCLIP buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 4 mM MgCl2 , 5 mM DTT , 1% SDS , 0 . 25% sodium deoxycholate , 0 . 25% Igepal ) supplemented with protease inhibitors ( 4 mM PMSF , 1 tablet/10 mL of Complete Protease Inhibitor Cocktail [Roche] , and 1/30 vol . of Protease Inhibitor Optimized for Plant Extracts [Sigma P9599] ) , and cleared by centrifugation and filtration ( 0 . 45 μm pore ) of the supernatant .\",\n \"RNP-complexes were then immunopurified with beads coupled to anti-RFP nanobodies ( ChromoTek RFP-Trap in our case ) for 1 hr at 4°C under constant rotation .\",\n \"In particular , we used 20 μL of beads for 4 g of tissue in 6 mL of iCLIP buffer for every replicate .\",\n \"After thorough washes with RIP-Wash Buffer ( 2 M urea , 50 mM Tris-HCl pH 7 . 5 , 500 mM NaCl , 4 mM MgCl2 , 2 mM DTT , 1% SDS , 0 . 5% sodium deoxycholate , 0 . 5% Igepal ) , RNP-complexes attached to the beads were subjected to treatment with DNase ( Turbo DNase [Ambion] , 4 U/100 μL ) and RNase I ( Ambion , 1 U/mL ) at 37°C for 10 min , dephosphorylation of RNA 3′ ends ( PNK [ThermoFisher] in pH 6 . 5 buffer ) , and 3′ RNA linker ligation ( L3-App linker [Huppertz et al . , 2014] and NEB HC RNA Ligase ) at 16°C overnight .\",\n \"RNA was radioactively labeled at the 5′ end by PNK-mediated phosphorylation using γ-32P-ATP ( 20 min at 37°C ) .\",\n \"The labeled RNP complexes were subjected to SDS-PAGE and blotting on a nitrocellulose membrane ( Protran BA-85 ) .\",\n \"Pieces of membrane containing a size range of RNA species bound to the protein ( a smear above the expected molecular weight localized by autoradiography ) were excised and subjected to proteolysis ( 200 μg of Proteinase K [Roche] in 200 μL of PK buffer [100 mM Tris-HCl pH 7 . 4 , 50 mM NaCl , 10 mM EDTA] for 20 min at 37°C ) to release RNA bound to small peptides .\",\n \"The RNA was then purified with TRI-Reagent ( Sigma ) and used to prepare sequencing libraries through the following steps: reverse transcription ( Superscript III , Invitrogen ) using a two-part cleavable DNA adapter complementary to the 3′ RNA linker as primer , gel purification and size selection of cDNA ( high , 120–200 nt; medium , 85–120 nt; low , 70–85 nt ) , circularization ( CircLigase II Epicentre ) , relinearization ( BamHI ) , and PCR amplification ( AccuPrime Supermix I , Invitrogen ) .\",\n \"All steps were performed as described by Huppertz et al . , 2014 , and the amount of cycles in the final PCR was optimized to the amount of cDNA in each sample .\",\n \"Notice that we introduced a few modifications in the original protocol ( Köster and Staiger , 2020 ) to account for ( 1 ) low abundance of ECT2 compared to AtGRP7 .\",\n \"To obtain enough RNA , we increased the crosslinking energy and irradiated 12-day-old seedlings with 2000 mJ/cm2 of 254 nm UV light , harvesting roots and shoots ( 4 g of tissue per replicate ) to maximize the amount of purified ECT2-mCherry .\",\n \"( 2 ) ECT2 sensitivity to proteolysis .\",\n \"We did not pre-clear the lysates to reduce the incubation time , and we used high amounts of protease inhibitors during immunoprecipitation .\",\n \"( 3 ) High molecular weight of ECT2-mCherry .\",\n \"Due to the size of the protein , we required longer electrophoresis time and cooling ( 3 hr at 180 V with the tank on ice ) .\",\n \"( 4 ) Different RNA-binding capacity of ECT2 .\",\n \"Based on trials , we decided to adjust the RNase I treatment to 1 U/mL , incubating for 10 min at 37°C ( 5 μL of RNase I [Ambion , 100 U/μL pre-diluted 1:5000] in 100 μL ) .\",\n \"Of note , the conditions indicated here were specifically used for library preparation .\",\n \"Although we used the same conditions as default for CLIP experiments to assess ECT2 RNA-binding capacity , ECT2 sensitivity to proteolysis and ECT2-bound RNA sensitivity to RNase treatment , variations in buffer composition , incubation time , concentration of protease inhibitors , and/or RNase I are specified in the corresponding figure legends where necessary .\",\n \"The entire iCLIP-seq experiment including three replicates of each group was done once .\",\n \"Sequenced reads from all samples were investigated after each processing step with fastqc 0 . 11 . 5 ( https://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) .\",\n \"Adapters at the 3′ end were trimmed using cutadapt version 1 . 16 ( Martin , 2011 ) .\",\n \"The demultiplexing of the samples was performed using flexbar 3 . 4 . 0 with the -bk parameter to conserve the barcode information for further steps ( Roehr et al . , 2017 ) .\",\n \"Reads with a length below 24 nucleotides were discarded .\",\n \"Barcodes were trimmed and saved to the read_id field .\",\n \"Processed reads were mapped to the TAIR10 genome with STAR version 2 . 6 . 0a allowing a maximum of two mismatches and soft clipping only at 3′ end ( Dobin et al . , 2013 ) .\",\n \"PCR duplicates were removed by grouping the reads by their mapping start position .\",\n \"Reads with the identical start position and random barcode were removed from the samples ( Python3 and pybedtools ) .\",\n \"The peak calling of uniquely mapped reads was done using PureCLIP 1 . 0 . 4 , choosing the second peak-shape option to allow more broader peaks to be called ( Krakau et al . , 2017 ) .\",\n \"For consistency with the ECT2-HT datasets , the ECT2-iCLIP datasets were annotated using the hyperTRIBER annotation ( Rennie et al . , 2021 ) , using quantifications based on the average of roots and aerial tissues from FLAG-ADAR samples in ECT2-HT ( to reflect that the ECT2-iCLIP data is based on whole seedlings ) .\",\n \"To calculate the proportion of sites falling at each nucleotide , nucleotide sequences from the reference genome were first obtained from site coordinates for ECT2 iCLIP/m6A-Nanopore/ m6A-miCLIP using the R packages GenomicRanges ( Lawrence et al . , 2013 ) and rtracklayer ( Lawrence et al . , 2009 ) .\",\n \"Nucleotide proportions were plotted using ggplot2 ( https://ggplot2 . tidyverse . org ) .\",\n \"Single-cell expression data and marker genes associated with 15 clusters annotated to cell types in roots were downloaded from Denyer et al . , 2019 .\",\n \"Single-nucleotide resolution locations of m6A sites ( defined according to Nanopore or miCLIP ) were downloaded from Parker et al . , 2020 .\",\n \"Intervals defining m6A locations based on m6A-seq were downloaded from Shen et al . , 2016 , and intervals defining locations of ECT2-bound sites as determined by FA-CLIP were downloaded from Wei et al . , 2018 .\",\n \"For consistency with HyperTRIBE and ECT2-iCLIP , all sets of m6A or ECT2-bound sites were gene annotated using the hyperTRIBER pipeline , based on genes and transcripts from Araport11 .\",\n \"To remove redundancy after ECT2-iCLIP peak calling , directly adjacent peaks ( crosslink sites ) were grouped together and only the peak with the highest pureCLIP score ( dominant ) was kept .\",\n \"The called peak position ( 1 nt resolution ) was extended by 4 nt up- and downstream to define a ‘collapsed crosslink site’ ( CSS ) with length 9 nt .\",\n \"The center position marks the dominant called peak .\",\n \"The extension of the peak positions was computed using bedtools version 2 . 27 . 1 ( Quinlan and Hall , 2010; Dale et al . , 2011 ) .\",\n \"The collapsed ECT2-iCLIP crosslink sites and m6A-Nanopore sites ( Parker et al . , 2020 ) were used to find motifs significantly enriched by Homer ( Heinz et al . , 2010 ) using a variety of window sizes , settings , and backgrounds .\",\n \"Motifs resulting from Homer searches were collated manually , and a range of variants of the consensus motif RRACH ( e . g . , RACH , DRAY , DRACH , URACH , DRACG ) were also added to the list , as well as various combinations of U-rich sequences ( e . g . , UUUUU , UNUNU , etc . ) , specific motifs found to be of interest in scientific literature ( e . g . , URUAY [Wei et al . , 2018] , GGAU [Anderson et al . , 2018] ) , and extra motifs that appeared of potential interest from manually browsing with IGV ( Robinson et al . , 2011 ) the sequence in the vicinity of iCLIP peaks ( e . g . , YYYYY , DRACUCU ) .\",\n \"This resulted in a final list of 48 motifs for further analysis .\",\n \"For each of the 48 motifs compiled from multiple sources , a custom PWM was generated based on local sequence frequencies around ECT2-iCLIP peaks and used as input to FIMO 5 . 1 . 1 ( Grant et al . , 2011 ) to detect genome-wide occurrences .\",\n \"To generate PWMs , we used the formula PWMb , j = log2 [p ( b , j ) /p ( b ) ] , where p ( b ) is the background frequency of each nucleotide ( see further down ) , and p ( b , i ) is the frequency of the nucleotides in each position j .\",\n \"We also included an extra small frequency count in the calculation to account for potential uncertainty in redundancies .\",\n \"In order to account for location-specific sequence contexts ( typically 3′-UTR ) , each site from iCLIP or m6A ( Parker et al . , 2020 ) sets was assigned a random ‘matched background’ site , in a non-target gene , at the same relative location along the annotated genomic feature of the site ( 5′-UTR , CDS or 3′-UTR ) , according to a resolution of 10 bins per feature .\",\n \"Logos for all motifs were generated using the R packages ggplot2 ( https://ggplot2 . tidyverse . org ) and ggseqlogo ( Wagih , 2017 ) .\",\n \"To run the calculated PWMs through FIMO , we specified background letter frequencies ( A: 0 . 273 , C: 0 . 165 , G: 0 . 173 , U: 0 . 389 ) , a threshold of 0 . 05 , and scanning across the full TAIR10 Arabidopsis genome .\",\n \"Sites were further filtered downstream to have a score of at least 4 – in the vast majority of cases corresponding to an exact match the ( short ) motif .\",\n \"Distributions of motifs per 1000 sites over distance , centering on ECT2-iCLIP or m6A sites and the respective matched backgrounds , were generated using a custom R-script ( https://github . com/sarah-ku/targets_arabidopsis ) based on overlaps using GenomicRanges ( Lawrence et al . , 2013 ) .\",\n \"At any given shift from the peak set , the raw number of overlaps of the motif ( at any point ) was calculated and normalized to give a motif count per 1000 peaks .\",\n \"To adjust for the potential for downstream regions overshooting the end of the 3′-UTR , at each given distance only sites that continue to overlap an annotated gene ( Araport11 ) are counted .\",\n \"For some analyses , peak sets were further split according to IDR-dependency or target status as indicated .\",\n \"To calculate motif enrichment over the gene body , motifs were first annotated a value according to their relative position within the gene body regions: 5′-UTR , CDS or 3′-UTR .\",\n \"In order to account for over-representation of counts within the CDS , due to greater sequence coverage within transcript annotations , a random background set of 10 million positions were generated from the transcript annotation file and annotated in the same way as the motif locations to obtain an expected distribution of all positions over the gene body regions .\",\n \"This Expected distribution was used to normalize the Observed distribution of each motif , and O/E values were plotted as a metagene plot over the gene .\",\n \"An enrichment of 1 suggests that the motif is neither over- or under-represented at that location .\",\n \"Called positions from either Nanopore m6A data ( Parker et al . , 2020 ) or ECT2 iCLIP were first reduced to remove redundant regions of multiple peaks within the same window , then paired with matched background sets ( described above ) .\",\n \"Windows representing ‘at’ ( ±10 nt ) the motif together with adjacent upstream ‘up’ and downstream ‘down’ windows of length 50 nt ( resulting in total window sizes of 120 nt ) around each position were annotated according to the number of each of the motifs overlapping ( truncated at 10 ) , and the final data set normalized .\",\n \"To create a held-out set , 1/5th of the peaks were removed from the set , and the other 4/5th were used to build a random forest model using gradient boosting ( R package gbm version 2 . 1 . 8; https://github . com/gbm-developers/gbm ) , with settings specifying a shrinkage of 0 . 05 , an interaction . depth of 6 , cv . folds = 5 , and n . trees = 2000 .\",\n \"For each model ( m6A Nanopore-based or ECT2 iCLIP-based ) , importance scores were extracted from the model and the top features were selected .\",\n \"The held-out data was further used to estimate the predictive score of the model by calculating the AUC ( R package pROC; Robin et al . , 2011 ) .\",\n \"Two further models were run – one involving the top 10 features from the full feature model , and ( only for the m6A-Nanopore set-up ) one involving features from only DRACH and UNUNU ( equating to six features in total ) , and AUC values were calculated and compared to that of the full feature model .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Specific recognition of N6-methyladenosine ( m6A ) in mRNA by RNA-binding proteins containing a YT521-B homology ( YTH ) domain is important in eukaryotic gene regulation .","The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU ( m6A ) Y sites , yet RR ( m6A ) CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity .","Here , we apply iCLIP ( individual nucleotide resolution crosslinking and immunoprecipitation ) and HyperTRIBE ( targets of RNA-binding proteins identified by editing ) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites .","Our analyses show that ECT2 does in fact bind to RR ( m6A ) CH .","Pyrimidine-rich motifs are enriched around , but not at m6A sites , reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions .","Such motifs , particularly oligo-U and UNUNU upstream of m6A sites , are also implicated in ECT2 binding via its intrinsically disordered region ( IDR ) .","Finally , URUAY-type motifs are enriched at ECT2 crosslink sites , but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins .","Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins ."],"string":"[\n \"Specific recognition of N6-methyladenosine ( m6A ) in mRNA by RNA-binding proteins containing a YT521-B homology ( YTH ) domain is important in eukaryotic gene regulation .\",\n \"The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU ( m6A ) Y sites , yet RR ( m6A ) CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity .\",\n \"Here , we apply iCLIP ( individual nucleotide resolution crosslinking and immunoprecipitation ) and HyperTRIBE ( targets of RNA-binding proteins identified by editing ) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites .\",\n \"Our analyses show that ECT2 does in fact bind to RR ( m6A ) CH .\",\n \"Pyrimidine-rich motifs are enriched around , but not at m6A sites , reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions .\",\n \"Such motifs , particularly oligo-U and UNUNU upstream of m6A sites , are also implicated in ECT2 binding via its intrinsically disordered region ( IDR ) .\",\n \"Finally , URUAY-type motifs are enriched at ECT2 crosslink sites , but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins .\",\n \"Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins .\"\n]"},"summary":{"kind":"list like","value":["Genes are strings of genetic code that contain instructions for producing a cell’s proteins .","Active genes are copied from DNA into molecules called mRNAs , and mRNA molecules are subsequently translated to create new proteins .","However , the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA .","Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production .","One of these methods involves adding a chemical tag , a methyl group , onto mRNA while it is being created .","These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation .","Most eukaryotic species – which include animals , plants and fungi – use the same system to add methyl tags to mRNA molecules .","One methyl tag in particular , known as m6A , is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants .","However , in the flowering plant Arabidopsis thaliana , ECT2 was thought to bind to an mRNA sequence different from the one normally carrying the chemical tag , creating obvious confusion about how the system works in plants .","Arribas-Hernández , Rennie et al . investigated this question using advanced large-scale biochemical techniques , and discovered that conventional m6A methyl tags are indeed used by ECT2 in Arabidopsis thaliana .","The confusion likely arose because the sequence ECT2 was thought bind is often located in close proximity to the m6A tags , possibly acting as docking stations for proteins that can influence the ability of ECT2 to bind mRNA .","Arribas-Hernández , Rennie et al . also uncovered additional mRNA sequences that directly interact with parts of ECT2 previously unknown to participate in mRNA binding .","These findings provide new insights into how chemical labels in mRNA control gene activity .","They have broad implications that extend beyond plants into other eukaryotic species , including humans .","Since this chemical labelling system has a major role in controlling plant growth , these findings could be leveraged in biotechnology applications to improve crop yields and enhance plant-based food production ."],"string":"[\n \"Genes are strings of genetic code that contain instructions for producing a cell’s proteins .\",\n \"Active genes are copied from DNA into molecules called mRNAs , and mRNA molecules are subsequently translated to create new proteins .\",\n \"However , the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA .\",\n \"Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production .\",\n \"One of these methods involves adding a chemical tag , a methyl group , onto mRNA while it is being created .\",\n \"These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation .\",\n \"Most eukaryotic species – which include animals , plants and fungi – use the same system to add methyl tags to mRNA molecules .\",\n \"One methyl tag in particular , known as m6A , is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants .\",\n \"However , in the flowering plant Arabidopsis thaliana , ECT2 was thought to bind to an mRNA sequence different from the one normally carrying the chemical tag , creating obvious confusion about how the system works in plants .\",\n \"Arribas-Hernández , Rennie et al . investigated this question using advanced large-scale biochemical techniques , and discovered that conventional m6A methyl tags are indeed used by ECT2 in Arabidopsis thaliana .\",\n \"The confusion likely arose because the sequence ECT2 was thought bind is often located in close proximity to the m6A tags , possibly acting as docking stations for proteins that can influence the ability of ECT2 to bind mRNA .\",\n \"Arribas-Hernández , Rennie et al . also uncovered additional mRNA sequences that directly interact with parts of ECT2 previously unknown to participate in mRNA binding .\",\n \"These findings provide new insights into how chemical labels in mRNA control gene activity .\",\n \"They have broad implications that extend beyond plants into other eukaryotic species , including humans .\",\n \"Since this chemical labelling system has a major role in controlling plant growth , these findings could be leveraged in biotechnology applications to improve crop yields and enhance plant-based food production .\"\n]"},"year":{"kind":"string","value":"2021"}}},{"rowIdx":4194,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["chromosomes and gene expression","cell biology"],"string":"[\n \"chromosomes and gene expression\",\n \"cell biology\"\n]"},"title":{"kind":"string","value":"A conserved function for pericentromeric satellite DNA"},"id":{"kind":"string","value":"elife-34122-v2"},"sections":{"kind":"list like","value":[["Satellite DNA is AT-rich , non-coding , repetitive DNA that is abundant in centromeric and pericentromeric heterochromatin .","Unlike the satellite DNAs that comprise the vast majority of natural centromeres ( Willard , 1990; Sun et al . , 1997 , 2003 ) , the role of pericentromeric satellite DNA remains obscure: although function for a few satellite DNA repeats has been implied in certain cellular processes such as meiotic segregation of achiasmatic chromosomes , X chromosome dosage compensation and formation of lampbrush-like loops on the Y chromosome during male meiosis ( Yunis and Yasmineh , 1971; Bonaccorsi et al . , 1990; Dernburg et al . , 1996; Menon et al . , 2014 ) , a unifying theme for pericentromeric satellite DNA function remains elusive .","Moreover , highly divergent satellite DNA sequences even among closely related species has led to the idea that satellite DNA does not serve a conserved function and is mostly a selfish element or junk ( Doolittle and Sapienza , 1980; Walker , 1971 ) .","Pericentromeric satellite DNA repeats are proposed to be sources of genomic instability , as their misexpression is associated with the formation of genotoxic R-loops and DNA damage ( Zhu et al . , 2011; Zeller et al . , 2016; Zeller and Gasser , 2017 ) .","Most studies on pericentromeric heterochromatin have focused on the mechanisms to repress satellite DNA transcription , and accordingly , a clear rationale for the existence of most pericentromeric satellite DNA is still lacking .","Cytologically , it is well documented that pericentromeric satellite DNA from multiple chromosomes is clustered into chromocenters in interphase nuclei in diverse eukaryotes including Drosophila , mouse and plants ( Figure 1A ) ( Jones , 1970; Pardue and Gall , 1970; Gall et al . , 1971; Fransz et al . , 2002 ) .","While multiple factors such as epigenetic modifications and transcription of repetitive DNA from pericentromeric DNA sequences are known to be required for chromocenter formation ( Peters et al . , 2001; Probst et al . , 2010; Bulut-Karslioglu et al . , 2012; Pinheiro et al . , 2012; Hahn et al . , 2013 ) , the ultimate consequences of disrupted chromocenter formation has never been addressed , leaving the function of chromocenters unknown .","In this study , we explored the role of pericentromeric satellite DNA/chromocenters by studying multi-AT-hook proteins , D1 from Drosophila melanogaster and HMGA1 from mouse .","D1 and HMGA1 are known to bind specific pericentromeric satellite DNA , and we show that these proteins are required for chromocenter formation .","When chromocenters are disrupted in the absence of these proteins , cells exhibited a high frequency of micronuclei formation , leading to DNA breakage and cell death .","We show that micronuclei are formed during interphase by budding from the nucleus .","We further show that D1 binding to the target DNA sequence is sufficient to bring it to the chromocenter .","High-resolution imaging revealed chromatin threads positive for D1/HMGA proteins and satellite DNA that connect heterologous chromosomes .","Taken together , we propose that chromocenter formation via bundling of satellite DNA from heterologous chromosomes functions as a mechanism to encapsulate the full complement of the genome into a single nucleus .","We suggest that satellite DNA function as a critical constituent of chromosomes and may serve an evolutionarily conserved role across eukaryotic species ."],["D1 in Drosophila melanogaster and HMGA1 in mouse are multi-AT-hook proteins; D1 contains 10 AT-hooks , HMGA1 contains three AT-hooks and both proteins contain C-terminal acidic domains ( Aulner et al . , 2002 ) .","D1 and HMGA1 are known to bind the Drosophila {AATAT}n satellite DNA ( ~8% of the Drosophila male diploid genome ) and mouse major satellite DNA ( ~6% of the mouse genome ) , respectively ( Goodwin et al . , 1973; Rodriguez Alfageme et al . , 1980; Levinger and Varshavsky , 1982b; Levinger and Varshavsky , 1982a; Lund et al . , 1983 ) .","The {AATAT}n satellite is distributed across 11 loci on multiple chromosomes as visualized by DNA fluorescence in situ hybridization ( FISH ) on mitotic chromosome spreads ( Figure 1B ) ( Lohe et al . , 1993; Jagannathan et al . , 2017 ) .","However , it is typically clustered into a few foci in Drosophila interphase nuclei , colocalizing with the D1 protein ( Figure 1C ) .","The D1/{AATAT}n foci stained positively for H3K9me2 in interphase nuclei ( Figure 1C ) , a well-established characteristic of constitutive heterochromatin/chromocenters ( Guenatri et al . , 2004 ) .","Consistently , D1 localized near the centromere ( marked by Drosophila CENP-A , Cid ) on mitotic chromosome spreads ( marked by phospho-H3 S10 ) ( Figure 1D ) .","These results suggest that D1 is a chromocenter-localizing protein , via its binding to the {AATAT}n satellite DNA .","The mouse HMGA1 protein was originally identified as an abundant non-histone component of mammalian chromatin ( Goodwin et al . , 1973; Lund et al . , 1983 ) with subsequent studies demonstrating its binding to satellite DNA ( Strauss and Varshavsky , 1984; Radic et al . , 1992 ) .","Mouse major satellite , which is present in pericentromeric regions of all chromosomes ( Figure 1E ) ( Lyon and Searle , 1989 ) , clustered into DAPI-dense chromocenters positive for HMGA1 protein ( Figure 1F , Figure 1—figure supplement 1A , B ) , revealing an analogous relationship to D1/{AATAT}n satellite in Drosophila .","Interestingly , we found that Drosophila D1 protein localizes to major satellite/chromocenters when ectopically expressed in multiple mouse cell lines ( Figure 1G , Figure 1—figure supplement 1C , D ) , suggesting that D1 and HMGA1 may possess an orthologous and conserved function as satellite DNA/chromocenter-binding proteins .","We next examined the effects of D1 mutation and siRNA-mediated knockdown of HMGA1 on chromocenters .","We used two D1 alleles , D1LL03310 and D1EY05004 , which we show to be protein null alleles , evidenced by near-complete loss of anti-D1 antibody staining ( Figure 1—figure supplement 2A–C ) .","When these alleles were combined with the D1 deficiency allele , Df ( 3R ) BSC666 , it led to severe declustering of {AATAT}n satellite DNA ( Figure 1H–J , Figure 1—figure supplement 2D–E ) , suggesting that D1 is required for clustering of pericentromeric satellite DNA into chromocenters .","We observed D1’s requirement for chromocenter formation in multiple cell types ( Figure 1—figure supplement 2F–I ) , but we largely focused on spermatogonial cells , where the phenotypes ( such as cell death ) were most penetrant and severe .","We also examined the requirement for HMGA1 in mouse chromocenter formation .","Following siRNA-mediated knockdown of HMGA1 , which led to near complete loss of HMGA1 protein ( see Figure 2D , E and Figure 2—figure supplement 1A–B , D–E for efficiencies of HMGA1 knockdown ) , we observed chromocenter disruption in multiple mouse cell lines ( Figure 1K–M , Figure 1—figure supplement 2J–L ) .","These results suggest that D1 and HMGA1 have an orthologous function to organize pericentromeric satellite DNA into chromocenters .","To explore the function of chromocenters and satellite DNA , we examined the effects of D1 mutation/HMGA1 knockdown , which showed strikingly similar phenotypes .","We found that D1 mutation as well as siRNA-mediated HMGA1 knockdown in multiple mouse cell lines resulted in a significant increase in micronuclei formation ( Figure 2A–F , Figure 2—figure supplement 1A–F ) .","Micronuclei are known to have compromised nuclear envelope integrity , leading to DNA damage and catastrophic chromosomal rearrangement therein ( Crasta et al . , 2012; Hatch et al . , 2013 ) .","Therefore , we first examined a possible defect in nuclear envelope integrity in D1 mutant .","We found that loss of D1 led to breaching of the nuclear envelope both in major and micronuclei , visualized by the cytoplasmic leakage of nuclear GFP ( nlsGFP ) ( Figure 2G–I ) , suggesting that nuclear envelope integrity might be generally compromised .","Consistently , we observed mislocalization of nuclear envelope proteins in D1 mutant spermatogonia .","We frequently observed that lamin surrounded the nucleus incompletely in D1 mutant ( 1 . 9% in control ( n = 52 ) and 68 . 9% in D1 mutant ( n = 58 ) ) ( Figure 2J , K , arrows indicate lamin-negative regions on the nuclear membrane ) .","We also observed cytoplasmic ‘holes’ , which resemble the nucleus in that they exclude cytoplasmic proteins such as Vasa ( Figure 2K , arrowhead ) , but are devoid of nuclear lamin ( Figure 2K , arrowhead ) .","These ‘holes’ were often surrounded by an ER marker , which normally surrounds the nuclear envelope ( Figure 2J ) ( Dorn et al . , 2011 ) .","Similarly , Otefin , an inner nuclear membrane LEM-domain protein ( Barton et al . , 2014 ) , also showed perturbed localization ( 2 . 7% in control ( n = 109 ) and 24 . 5% in D1 mutant ( n = 106 ) ) ( Figure 2L , M , arrows indicate lamin/Otefin negative regions on the nuclear envelope while the arrowhead indicates Otefin-positive micronuclei ) .","Taken together , these results show that D1 mutant cells exhibit compromised nuclear envelope integrity , which is associated with micronuclei formation .","It has been shown that defects in nuclear envelope integrity can lead to extensive DNA damage in the major nucleus and micronuclei ( Crasta et al . , 2012; Hatch et al . , 2013; Zhang et al . , 2015; Denais et al . , 2016; Raab et al . , 2016 ) .","Nuclear envelope defects and extensive DNA damages therein lead to catastrophic chromosomal breaks/rearrangements termed chromothripsis ( Crasta et al . , 2012; Hatch et al . , 2013 ) .","Such catastrophic DNA breaks/rearrangements are speculated to lead to tumorigenesis ( Hatch and Hetzer , 2015 ) .","Consistent with defective nuclear envelope integrity , we observed extensive DNA damage ( revealed by γ-H2Av ) in both major and micronuclei ( Figure 3A–F , arrows point to damaged DNA in micronuclei in B and D ) .","Likely as a result of DNA damage and defective nuclear envelope integrity , D1 mutant testes rapidly degenerated ( Figure 3—figure supplement 1A , B ) .","When Omi , a gene required to promote germ cell death ( Yacobi-Sharon et al . , 2013 ) , was knocked down in D1 mutant testes , it restored the cellularity in D1 mutant testis ( Figure 3—figure supplement 1C–D ) , but the surviving cells showed a dramatic increase in DNA damage ( Figure 3—figure supplement 1E–F ) .","Under these conditions , we observed that surviving germ cells in D1 mutant testes showed a high frequency of chromosome breaks compared to control , revealed by FISH on metaphase chromosome spreads from spermatocytes ( 3 . 7% in control ( n = 27 ) vs . 15 . 8% in D1 mutant ( n = 57 ) ) ( Figure 3G , H , arrowheads indicate sites of chromosome breaks ) .","These results show that loss of D1/HMGA1 results in compromised nuclear envelope integrity , leading to extensive DNA damage and chromosomal breaks .","It has been shown that micronuclei form by lagging chromosomes ( Crasta et al . , 2012 ) .","Thus , we examined whether D1 mutation/HMGA1 knockdown resulted in mitotic chromosome segregation errors , causing micronuclei formation .","However , we did not observe an increase in lagging chromosomes in D1 mutant spermatogonia or HMGA1-depleted mouse cells ( Figure 4—figure supplement 1A–G ) , suggesting an alternative route for micronuclei formation .","Instead , time-lapse live observation showed that micronuclei formed by budding from the interphase nucleus both in Drosophila spermatogonia and mouse cells ( Figure 4A–D ) .","In Drosophila spematogonia , nuclear contents were visualized by a GFP-tagged nuclear protein , Df31 , and RFP-tagged histone H2Av .","Control cells stably maintained nuclear contents for a prolonged time period ( only 1 event of nuclear blebbing without concurrent micronuclei formation ( as detected by H2Av-RFP ) over 1552 min of live imaging ) ( Figure 4A ) .","In contrast , D1 mutant cells showed budding off of nuclear contents and micronuclei formation in interphase ( 15 nuclear breaches with eight micronuclei formed over 3427 min of live imaging with a total budding duration of 172 min ) ( Figure 4B ) .","Similarly , live imaging in mouse cells using the Hoechst DNA dye revealed that HMGA1 knockdown also resulted in micronuclei formation during interphase ( siControl – no micronuclei formation over 253 min of live observation , siHMGA1 – three micronuclei formed by budding over 5962 min of live imaging with a total budding duration of 310 min ) ( Figure 4C , D ) .","These results show that micronuclei in D1 mutant/HMGA1-knockdown cells are generated during interphase , via budding from the nucleus .","Based on the above results , we postulated that chromocenter formation , that is clustering of satellite DNA from multiple chromosomes , might be a mechanism to bundle heterologous chromosomes together to prevent individual chromosomes from floating out of the nucleus .","In this manner , the full set of chromosomes may be retained within a single nucleus .","In the absence of chromocenter formation , individual chromosomes may bud off the nucleus , leading to micronuclei formation .","Previous in vitro experiments indicated that HMGA1 is capable of crosslinking multiple DNA strands with individual AT-hooks binding AT-rich DNA strands ( Vogel et al . , 2011 ) .","Bundling of DNA in this manner by D1/HMGA1 could explain how pericentromeric satellite DNA from multiple chromosomes may be clustered to form chromocenters .","A few lines of evidence support this idea .","When Drosophila D1 was expressed in mouse cells , it localized to the chromocenter as described above ( Figure 1G ) , and its overexpression enhanced chromocenter formation in a dose-dependent manner ( Figure 5A–C ) : the higher the amount of D1 that was expressed in mouse cells , the fewer chromocenters per cell was observed ( i . e . more clustering ) .","These results suggest that D1 is sufficient to bundle its binding target , tethering it to chromocenter .","Consistent with this idea , we found that artificial tethering of D1 protein to euchromatic LacO repeat DNA sequences was sufficient to bring LacO repeats to the chromocenter .","D1 protein or D1-LacI fusion protein was expressed in a Drosophila strain in which LacO repeats are inserted in the distal regions of the 2nd chromosome ( Figure 5D , arrows ) .","In control spermatogonial cells expressing wild type D1 , LacO repeats were observed far away from the {AATAT}n satellite foci/chromocenters ( Figure 5E , G , arrow indicates site of LacO repeats in interphase nucleus ) .","However , in cells expressing the LacI-D1 chimeric protein , we observed recruitment of the LacO repeats close to {AATAT}n/chromocenters ( Figure 5F , G , arrow indicates site of LacO repeats recruited to the chromocenter ) , demonstrating that D1’s binding to a DNA sequence is sufficient to incorporate the target sequence into chromocenters .","Although it cannot be visualized how DNA strands from multiple chromosomes might be bundled in these interphase chromocenters , deconvolution microscopy of D1/HMGA1 proteins on early mitotic chromosomes revealed proteinaceous threads between chromatin in the process of condensation ( Figure 6A , B , arrows indicate D1/HMGA1 threads ) , which we speculate contributed to bundling of chromosomes in the previous interphase .","These threads were also detectable by DNA FISH against {AATAT}n and the mouse major satellite ( Figure 6C , D , dotted lines are alongside the satellite DNA threads ) , suggesting that satellite DNA bound by D1/HMGA1 can form threads .","These threads likely connect heterologous chromosomes , as we see threads between chromosomes that are clearly distinct in their morphology ( e . g . Figure 6C ) .","These D1/HMGA1 threads are reminiscent of ‘DNA fibers’ , which were observed among mitotic chromosomes , although their function has never been appreciated ( Takayama , 1975; Burdick , 1976; Kuznetsova et al . , 2007 ) .","Taken together , these results support a model , in which D1/HMGA1 bind their target sequences ( satellite DNA ) on multiple chromosomes and bundle them into chromocenters , likely via their multivalent DNA-binding domains ( multiple AT-hooks ) ( Figure 6E ) ."],["The function of chromocenters , as well as that of satellite DNA , has remained enigmatic , even though cytological association of pericentromeric satellite DNA into chromocenters was identified almost 50 years ago ( Jones , 1970; Pardue and Gall , 1970 ) .","Pericentromeric heterochromatin has most often been studied and discussed in the context of how to maintain its heterochromatic , repressed nature ( Nishibuchi and Déjardin , 2017 ) , based on the assumption that the underlying sequences are mostly selfish , which have negative phenotypic consequences when derepressed in cells ( Zeller and Gasser , 2017 ) .","Although satellite DNA’s function has been speculated and implicated in several examples ( Yunis and Yasmineh , 1971; Bonaccorsi et al . , 1990; Dernburg et al . , 1996; Menon et al . , 2014 ) , the non-coding nature and lack of conservation in repeat sequence among closely related species led to the idea that they are mostly junk DNA , serving no essential function ( Walker , 1971; Doolittle and Sapienza , 1980 ) .","Instead , we propose that satellite DNA is a critical constituent of eukaryotic chromosomes to ensure encapsulation of all chromosomes in interphase nucleus .","Our results may also explain why the sequences of pericentromeric satellite DNA are so divergent among closely related species , a contributing factor that led to their dismissal as junk .","Based on our model that pericentromeric satellite DNA serves as a platform for generating heterologous chromosome association to form chromocenters , the essential feature of satellite DNA is that they are bound by protein ( s ) capable of bundling multiple DNA strands .","If so , the underlying sequence does not have to be conserved .","Instead , the binding of satellite DNA by a chromocenter bundling protein may be a critical feature of pericentromeric satellite DNAs .","Based on this idea , chromocenter bundling proteins and pericentromeric satellite DNA may be co-evolving .","We observed perturbation of nuclear envelope integrity upon chromocenter disruption .","Understanding the mechanisms underlying perturbation of nuclear envelope integrity in D1 mutant awaits future investigation .","Previous studies have documented that cytoskeletal forces are transmitted to chromatin through nuclear envelope and external mechanical forces can cause temporary nuclear envelope breaches ( King et al . , 2008; Denais et al . , 2016; Hatch and Hetzer , 2016; Raab et al . , 2016 ) .","Therefore , we speculate that chromosome bundling in the form of chromocenter may help prevent cytoskeletal forces from shearing chromosomes and nuclear envelope: when chromosomes are not bundled , cytoskeletal forces may be transmitted to individual chromosomes and associated nuclear envelope , resulting in shearing of nuclear envelope , disrupting its integrity .","In summary , our study provides the first evidence for a conserved function of pericentromeric satellite DNA and chromocenters .","Our data suggest that the multi-AT-hook proteins , D1 and HMGA1 , play an evolutionarily conserved role in the formation of chromocenters , likely via their ability to bind and bundle satellite DNA from heterologous chromosomes .","Heterologous chromosome association , mediated by chromocenter-binding proteins , may represent a third mode of chromosomal ‘gluing’ after meiotic homologous pairing and sister chromatid cohesion .","Through heterologous association , the chromocenter plays a fundamental role in maintaining the full complement of the genome , which is divided into multiple chromosomes , into a single nucleus .","This function of the chromocenter may be conserved in eukaryotic species that contain pericentromeric satellite DNA , thereby bringing about a signature characteristic of eukaryotic cells ."],["All fly stocks were raised on standard Bloomington medium at 25°C .","The following fly stocks were used: D1EY05004 ( BDSC17340 ) , Df ( 3R ) BSC666 ( BDSC26518 ) , UAS-OmiRNAi ( BDSC55165 ) , UAS-GFP-nls ( BDSC4776 ) and UAS-GFP-ER-SR ( BDSC59042 ) were obtained from the Bloomington Drosophila stock center .","D1LL03310 ( DGRC140754 ) and Df31-GFP ( DGRC110806 ) were obtained from the Kyoto stock center .","nos-gal4 ( Van Doren et al . , 1998 ) , hs-flp;nos-FRT-stop-FRT-gal4 , UAS-GFP ( Salzmann et al . , 2013 ) , UAS-H2A-YFP ( Bellaïche et al . , 2001 ) and B1 LacO ( Vazquez et al . , 2002 ) have been previously described .","A stock containing B chromosomes , mtrm126 +B ( Bauerly et al . , 2014 ) , was a kind gift from Scott Hawley .","Chromocenter disruption was scored in Drosophila testes by assessing {AATAT}n morphology in GFP +cells that were generated as follows in control ( hs-flp; nos-FRT-stop-FRT-gal4 , UAS-GFP ) and D1 mutant ( hs-flp;nos-FRT-stop-FRT-gal4 , UAS-GFP;D1LL03310/Df ) flies .","Testes were dissected 24 hr following a 20 min heat shock at 37°C .","Chromocenters were considered disrupted in Drosophila and mouse when satellite DNA adopted thread-like morphology in interphase nuclei .","Micronuclei were scored in 0-3 d testes where early germ cell chromosomes were labeled with H2A-YFP .","The genotypes used were , control – nos > H2 A-YFP and D1 mutant – nos > H2 A-YFP; D1LL03310/Df .","For construction of UAS-GFP-D1 , the D1 ORF was PCR-amplified from cDNA using the following primer pair , 5’-GATCAGATCTATGGAGGAAGTTGCGGTAAAG-3’ and 5’-GATCCTCGAGTTAGGCAGCTACCGATTCGG-3’ .","The amplified fragment was subcloned into the BglII and XhoI sites of pUASt-EGFP-attB ( Salzmann et al . , 2013 ) resulting in UAS-GFP-D1 .","For UAS-GFP-LacI-D1 , the LacI ORF ( lacking 11 C-terminal residues ) ( Straight et al . , 1996 ) was synthesized using GeneArt ( Thermofisher ) and inserted into the BglII site of UAS-GFP-D1 resulting in UAS-GFP-LacI-D1 .","Transgenic flies were generated by PhiC31 integrase-mediated transgenesis into the attP40 site ( BestGene ) .","For expression of GFP and GFP-D1 in mouse cells , GFP and GFP-D1 was subcloned from pUASt-EGFP-attB into pCDNA3 ( gift from Cheng-Yu Lee ) using EcoRI and XhoI sites .","Mouse MOVAS cells were obtained from Daniel Eitzman .","Mouse C2C12 cells were obtained from David Bridges .","Mouse RAW264 . 7 cells were obtained from Dr . Harry Mobley .","Mouse C3H10T1/2 cells were obtained from Stephen Weiss .","MOVAS , C2C12 and RAW264 . 7 cells were maintained in Dulbecco's minimal essential medium ( DMEM ) ( Gibco ) supplemented with 10% fetal bovine serum ( FBS ) .","C3H10T1/2 cell line was maintained in alpha minimal essential media ( Gibco ) supplemented with 10% fetal bovine serum .","All cell lines used were authenticated as mouse cells by the presence of mouse-specific satellite DNA as is shown throughout the manuscript .","Two major cell lines used in this study , C2C12 and MOVAS cells , were treated with Plasmocin ( Invivogen ) prior to use as a precaution for mycoplasma infection .","RNA interference ( RNAi ) against HMGA1 was performed using ON-TARGET plus Mouse HMGA1 siRNA SMARTpool ( Dharmacon , L-049293–01 ) consisting of the following target sequences , CCAUUUAGCCGCAGCCCGA , AGGCAAACGGGCACCAACA , GGGCGCAGCAGACUGGUUA , GUUCAUUCUUAGAUACCCA .","ON-TARGET plus Non-targeting pool ( Dharmacon , D-001810–10 ) consisting of the following sequences , UGGUUUACAUGUCGACUAA , UGGUUUACAUGUUGUGUGA , UGGUUUACAUGUUUUCUGA , UGGUUUACAUGUUUUCCUA , was used as a negative control .","siRNA transfections were performed using DharmaFECT four reagent ( Dharmacon , Lafayette , CO ) according to the manufacturer's protocol .","25 nM of siControl and siHMGA1 were transfected using DharmaFECT 4 ( Dharmacon ) according to the manufacturer’s protocol .","Cells were fixed for immunostaining/in situ hybridization 6 days post-transfection .","Transient transfection of GFP and GFP-D1 was performed using Fugene HD ( Roche ) reagent according to the manufacturer’s protocol .","For Drosophila tissues , immunofluorescence staining was performed as described previously ( Cheng et al . , 2008 ) .","Briefly , tissues were dissected in PBS , transferred to 4% formaldehyde in PBS and fixed for 30 min .","Testes were then washed in PBS-T ( PBS containing 0 . 1% Triton-X ) for at least 60 min , followed by incubation with primary antibody in 3% bovine serum albumin ( BSA ) in PBS-T at 4°C overnight .","Samples were washed for 60 min ( three 20 min washes ) in PBS-T , incubated with secondary antibody in 3% BSA in PBS-T at 4°C overnight , washed as above , and mounted in VECTASHIELD with DAPI ( Vector Labs ) .","The following primary antibodies were used: rabbit anti-vasa ( 1:200; d-26; Santa Cruz Biotechnology ) , rabbit anti-H3K9 dimethyl ( 1:200; Abcam , ab32521 ) , guinea pig anti-Otefin ( gift from Georg Krohne , 1:400 ) , chicken anti-Cid ( 1:500 , generated using the synthetic peptide CDGENDANDGYVSDNYNDSESVAA ( Covance ) ) , mouse anti-LaminDm0 ( ADL84 . 12 , 1:200 , Developmental Studies Hybridoma Bank ) , mouse anti-γ−H2Av ( UNC93-5 . 2 . 1 , 1:400 , Developmental Studies Hybridoma Bank ) , Phalloidin-Alexa546 ( ThermoFisher , a22283 , 1:200 ) .","Adherent mouse cells were fixed in 4% formaldehyde in PBS for 20 min at room temperature on coverslips .","Cells were permeabilized in PBS-T for 5 min , rinsed three times with PBS , blocked using 3% BSA in PBS-T for 30 min at room temperature and incubated with primary antibody diluted in 3% BSA in PBS-T overnight at 4°C .","Cells were then washed for 30 min ( three 10 min washes ) , incubated with secondary antibody in 3% BSA in PBS-T for 2 hr at room temperature , washed as above and mounted in VECTASHIELD with DAPI ( Vector Labs ) .","For nucleoplasmic extraction , cells were incubated with CSK buffer ( 10 mM PIPES pH7 , 100 mM NaCl , 300 mM sucrose , 3 mM MgCl2 , 0 . 5% Triton X-100 , 1 mM PMSF ) for 10 min at room temperature .","After CSK extraction , cells were washed with PBS and fixed and immunostained as above .","The following antibodies were used: rabbit anti-HMGA1 ( 1:400 , Abcam , ab129153 ) , goat anti-LaminB ( C-20 ) ( 1:20 , Santa Cruz Biotechnology , sc-2616 ) , mouse anti-α-tubulin ( 4 . 3 , 1:100 , Developmental Studies Hybridoma Bank ) and γ−H2Ax S139 ( 2577 , 1:200 , Cell Signaling Technologies ) .","Images were taken using a Leica TCS SP8 confocal microscope with 63x oil-immersion objectives ( NA = 1 . 4 ) .","Deconvolution was performed when indicated using the Hyvolution package from Leica .","Images were processed using Adobe Photoshop software .","Testes from newly eclosed flies were dissected into Schneider’s Drosophila medium containing 10% fetal bovine serum .","The testis tips were placed inside a sterile glass-bottom chamber and were mounted on a three-axis computer-controlled piezoelectric stage .","An inverted Leica TCS SP8 confocal microscope with a 63 × oil immersion objective ( NA = 1 . 4 ) was used for imaging .","For mouse live cell imaging , transfected cells were seeded onto a sterile glass-bottom chamber coated with poly-lysine .","Cells were incubated with Hoechst 33342 for 10 min , rinsed with PBS and fresh medium was added to the chamber .","Cells were imaged using a stage-top Tokai-Hit incubator that was mounted on an inverted TCS SP5 confocal microscope with a 63x oil immersion objective ( NA = 1 . 4 ) .","All images were processed using Adobe Photoshop software .","Metrics used for quantification of live imaging were total imaging duration ( defined as number of cells x imaging duration ) , total budding duration ( defined as number of cells with micronuclei that formed by budding x time with budded micronuclei ) .","Whole mount Drosophila testes were prepared as described above , and optional immunofluorescence staining protocol was carried out first .","Subsequently , samples were post-fixed with 4% formaldehyde for 10 min and washed in PBS-T for 30 min .","Fixed samples were incubated with 2 mg/ml RNase A solution at 37°C for 10 min , then washed with PBS-T +1 mM EDTA .","Samples were washed in 2xSSC-T ( 2xSSC containing 0 . 1% Tween-20 ) with increasing formamide concentrations ( 20% , 40% and 50% ) for 15 min each followed by a final 30 min wash in 50% formamide .","Hybridization buffer ( 50% formamide , 10% dextran sulfate , 2x SSC , 1 mM EDTA , 1 μM probe ) was added to washed samples .","Samples were denatured at 91°C for 2 min , then incubated overnight at 37°C .","For mitotic chromosome spreads , testes and larval 3rd instar brains were squashed according to previously described methods ( Larracuente and Ferree , 2015 ) .","Briefly , tissue was dissected into 0 . 5% sodium citrate for 5–10 min and fixed in 45% acetic acid/2 . 2% formaldehyde for 4–5 min .","Fixed tissues were firmly squashed with a cover slip and slides were submerged in liquid nitrogen until bubbling ceased .","Coverslips were then removed with a razor blade and slides were dehydrated in 100% ethanol for at least 5 min .","After drying , hybridization mix ( 50% formamide , 2x SSC , 10% dextran sulfate , 100 ng of each probe ) was applied directly to the slide , samples were heat denatured at 95°C for 5 min and allowed to hybridize overnight at room temperature .","Following hybridization , slides were washed thrice for 15 min in 0 . 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .","For the in situ experiment described in Figure 4j–m , testes were dissected into PBS and fixed in 4% formaldehyde for 4 min .","Tips of fixed testes were firmly squashed with a cover slip and slides were submerged in liquid nitrogen until bubbling ceased .","Coverslips were removed with a razor blade and slides were subjected to 5 min washes in 2XSSC and 2XSSC with 0 . 1% Tween-20 .","The samples were denatured in freshly made 0 . 07N NaOH for 5 min , rinsed in 2X SSC .","Hybridization mix ( 50% formamide , 2x SSC , 10% dextran sulfate , 100 ng of each probe ) was added directly to the slide and allowed to hybridize overnight at room temperature .","Following hybridization , slides were washed three times for 15 min in 0 . 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .","The following probes were used for Drosophila in situ hybridization: {AATAT}6 , {AAGAG}6 , IGS and have been previously described ( Jagannathan et al . , 2017 ) .","LacO probe - 5’-Cy5-CCACAAATTGTTATCCGCTCACAATTCCAC-3’ .","For interphase mouse cells , optional immunostaining was carried out as above .","Subsequently , samples were post-fixed with 4% formaldehyde in PBS for 10 min and rinsed three times in PBS .","Post-fixed cells were incubated with 0 . 1 mg/ml RNase A solution at 37°C for 1 hr , rinsed three times in PBS and denatured in 1 . 9M HCl for 30 min .","After three rinses in ice-cold PBS , hybridization mix ( 2X SSC , 60% formamide , 5 µg/ml salmon sperm DNA and 500 nM probe ) was added to the samples and incubated overnight at room temperature .","Following hybridization , coverslips were washed three times for 15 min in 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .","For mouse mitotic cells , chromosomes were spread on slides as previously described .","Subsequently , chromosomes were denatured in 70% formamide in 2XSSC for 1 . 5 min at 70°C , dehydrated in 100% ethanol and hybridization mix ( 2X SSC , 60% formamide , 5 µg/ml salmon sperm DNA and 500 nM probe ) was added directly to the slide and incubated overnight at room temperature .","Following hybridization , slides were washed three times for 15 min in 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .","The following probe was used: Major satellite - 5’-Cy3-GGAAAATTTAGAAATGTCCACTG-3’ ."]],"string":"[\n [\n \"Satellite DNA is AT-rich , non-coding , repetitive DNA that is abundant in centromeric and pericentromeric heterochromatin .\",\n \"Unlike the satellite DNAs that comprise the vast majority of natural centromeres ( Willard , 1990; Sun et al . , 1997 , 2003 ) , the role of pericentromeric satellite DNA remains obscure: although function for a few satellite DNA repeats has been implied in certain cellular processes such as meiotic segregation of achiasmatic chromosomes , X chromosome dosage compensation and formation of lampbrush-like loops on the Y chromosome during male meiosis ( Yunis and Yasmineh , 1971; Bonaccorsi et al . , 1990; Dernburg et al . , 1996; Menon et al . , 2014 ) , a unifying theme for pericentromeric satellite DNA function remains elusive .\",\n \"Moreover , highly divergent satellite DNA sequences even among closely related species has led to the idea that satellite DNA does not serve a conserved function and is mostly a selfish element or junk ( Doolittle and Sapienza , 1980; Walker , 1971 ) .\",\n \"Pericentromeric satellite DNA repeats are proposed to be sources of genomic instability , as their misexpression is associated with the formation of genotoxic R-loops and DNA damage ( Zhu et al . , 2011; Zeller et al . , 2016; Zeller and Gasser , 2017 ) .\",\n \"Most studies on pericentromeric heterochromatin have focused on the mechanisms to repress satellite DNA transcription , and accordingly , a clear rationale for the existence of most pericentromeric satellite DNA is still lacking .\",\n \"Cytologically , it is well documented that pericentromeric satellite DNA from multiple chromosomes is clustered into chromocenters in interphase nuclei in diverse eukaryotes including Drosophila , mouse and plants ( Figure 1A ) ( Jones , 1970; Pardue and Gall , 1970; Gall et al . , 1971; Fransz et al . , 2002 ) .\",\n \"While multiple factors such as epigenetic modifications and transcription of repetitive DNA from pericentromeric DNA sequences are known to be required for chromocenter formation ( Peters et al . , 2001; Probst et al . , 2010; Bulut-Karslioglu et al . , 2012; Pinheiro et al . , 2012; Hahn et al . , 2013 ) , the ultimate consequences of disrupted chromocenter formation has never been addressed , leaving the function of chromocenters unknown .\",\n \"In this study , we explored the role of pericentromeric satellite DNA/chromocenters by studying multi-AT-hook proteins , D1 from Drosophila melanogaster and HMGA1 from mouse .\",\n \"D1 and HMGA1 are known to bind specific pericentromeric satellite DNA , and we show that these proteins are required for chromocenter formation .\",\n \"When chromocenters are disrupted in the absence of these proteins , cells exhibited a high frequency of micronuclei formation , leading to DNA breakage and cell death .\",\n \"We show that micronuclei are formed during interphase by budding from the nucleus .\",\n \"We further show that D1 binding to the target DNA sequence is sufficient to bring it to the chromocenter .\",\n \"High-resolution imaging revealed chromatin threads positive for D1/HMGA proteins and satellite DNA that connect heterologous chromosomes .\",\n \"Taken together , we propose that chromocenter formation via bundling of satellite DNA from heterologous chromosomes functions as a mechanism to encapsulate the full complement of the genome into a single nucleus .\",\n \"We suggest that satellite DNA function as a critical constituent of chromosomes and may serve an evolutionarily conserved role across eukaryotic species .\"\n ],\n [\n \"D1 in Drosophila melanogaster and HMGA1 in mouse are multi-AT-hook proteins; D1 contains 10 AT-hooks , HMGA1 contains three AT-hooks and both proteins contain C-terminal acidic domains ( Aulner et al . , 2002 ) .\",\n \"D1 and HMGA1 are known to bind the Drosophila {AATAT}n satellite DNA ( ~8% of the Drosophila male diploid genome ) and mouse major satellite DNA ( ~6% of the mouse genome ) , respectively ( Goodwin et al . , 1973; Rodriguez Alfageme et al . , 1980; Levinger and Varshavsky , 1982b; Levinger and Varshavsky , 1982a; Lund et al . , 1983 ) .\",\n \"The {AATAT}n satellite is distributed across 11 loci on multiple chromosomes as visualized by DNA fluorescence in situ hybridization ( FISH ) on mitotic chromosome spreads ( Figure 1B ) ( Lohe et al . , 1993; Jagannathan et al . , 2017 ) .\",\n \"However , it is typically clustered into a few foci in Drosophila interphase nuclei , colocalizing with the D1 protein ( Figure 1C ) .\",\n \"The D1/{AATAT}n foci stained positively for H3K9me2 in interphase nuclei ( Figure 1C ) , a well-established characteristic of constitutive heterochromatin/chromocenters ( Guenatri et al . , 2004 ) .\",\n \"Consistently , D1 localized near the centromere ( marked by Drosophila CENP-A , Cid ) on mitotic chromosome spreads ( marked by phospho-H3 S10 ) ( Figure 1D ) .\",\n \"These results suggest that D1 is a chromocenter-localizing protein , via its binding to the {AATAT}n satellite DNA .\",\n \"The mouse HMGA1 protein was originally identified as an abundant non-histone component of mammalian chromatin ( Goodwin et al . , 1973; Lund et al . , 1983 ) with subsequent studies demonstrating its binding to satellite DNA ( Strauss and Varshavsky , 1984; Radic et al . , 1992 ) .\",\n \"Mouse major satellite , which is present in pericentromeric regions of all chromosomes ( Figure 1E ) ( Lyon and Searle , 1989 ) , clustered into DAPI-dense chromocenters positive for HMGA1 protein ( Figure 1F , Figure 1—figure supplement 1A , B ) , revealing an analogous relationship to D1/{AATAT}n satellite in Drosophila .\",\n \"Interestingly , we found that Drosophila D1 protein localizes to major satellite/chromocenters when ectopically expressed in multiple mouse cell lines ( Figure 1G , Figure 1—figure supplement 1C , D ) , suggesting that D1 and HMGA1 may possess an orthologous and conserved function as satellite DNA/chromocenter-binding proteins .\",\n \"We next examined the effects of D1 mutation and siRNA-mediated knockdown of HMGA1 on chromocenters .\",\n \"We used two D1 alleles , D1LL03310 and D1EY05004 , which we show to be protein null alleles , evidenced by near-complete loss of anti-D1 antibody staining ( Figure 1—figure supplement 2A–C ) .\",\n \"When these alleles were combined with the D1 deficiency allele , Df ( 3R ) BSC666 , it led to severe declustering of {AATAT}n satellite DNA ( Figure 1H–J , Figure 1—figure supplement 2D–E ) , suggesting that D1 is required for clustering of pericentromeric satellite DNA into chromocenters .\",\n \"We observed D1’s requirement for chromocenter formation in multiple cell types ( Figure 1—figure supplement 2F–I ) , but we largely focused on spermatogonial cells , where the phenotypes ( such as cell death ) were most penetrant and severe .\",\n \"We also examined the requirement for HMGA1 in mouse chromocenter formation .\",\n \"Following siRNA-mediated knockdown of HMGA1 , which led to near complete loss of HMGA1 protein ( see Figure 2D , E and Figure 2—figure supplement 1A–B , D–E for efficiencies of HMGA1 knockdown ) , we observed chromocenter disruption in multiple mouse cell lines ( Figure 1K–M , Figure 1—figure supplement 2J–L ) .\",\n \"These results suggest that D1 and HMGA1 have an orthologous function to organize pericentromeric satellite DNA into chromocenters .\",\n \"To explore the function of chromocenters and satellite DNA , we examined the effects of D1 mutation/HMGA1 knockdown , which showed strikingly similar phenotypes .\",\n \"We found that D1 mutation as well as siRNA-mediated HMGA1 knockdown in multiple mouse cell lines resulted in a significant increase in micronuclei formation ( Figure 2A–F , Figure 2—figure supplement 1A–F ) .\",\n \"Micronuclei are known to have compromised nuclear envelope integrity , leading to DNA damage and catastrophic chromosomal rearrangement therein ( Crasta et al . , 2012; Hatch et al . , 2013 ) .\",\n \"Therefore , we first examined a possible defect in nuclear envelope integrity in D1 mutant .\",\n \"We found that loss of D1 led to breaching of the nuclear envelope both in major and micronuclei , visualized by the cytoplasmic leakage of nuclear GFP ( nlsGFP ) ( Figure 2G–I ) , suggesting that nuclear envelope integrity might be generally compromised .\",\n \"Consistently , we observed mislocalization of nuclear envelope proteins in D1 mutant spermatogonia .\",\n \"We frequently observed that lamin surrounded the nucleus incompletely in D1 mutant ( 1 . 9% in control ( n = 52 ) and 68 . 9% in D1 mutant ( n = 58 ) ) ( Figure 2J , K , arrows indicate lamin-negative regions on the nuclear membrane ) .\",\n \"We also observed cytoplasmic ‘holes’ , which resemble the nucleus in that they exclude cytoplasmic proteins such as Vasa ( Figure 2K , arrowhead ) , but are devoid of nuclear lamin ( Figure 2K , arrowhead ) .\",\n \"These ‘holes’ were often surrounded by an ER marker , which normally surrounds the nuclear envelope ( Figure 2J ) ( Dorn et al . , 2011 ) .\",\n \"Similarly , Otefin , an inner nuclear membrane LEM-domain protein ( Barton et al . , 2014 ) , also showed perturbed localization ( 2 . 7% in control ( n = 109 ) and 24 . 5% in D1 mutant ( n = 106 ) ) ( Figure 2L , M , arrows indicate lamin/Otefin negative regions on the nuclear envelope while the arrowhead indicates Otefin-positive micronuclei ) .\",\n \"Taken together , these results show that D1 mutant cells exhibit compromised nuclear envelope integrity , which is associated with micronuclei formation .\",\n \"It has been shown that defects in nuclear envelope integrity can lead to extensive DNA damage in the major nucleus and micronuclei ( Crasta et al . , 2012; Hatch et al . , 2013; Zhang et al . , 2015; Denais et al . , 2016; Raab et al . , 2016 ) .\",\n \"Nuclear envelope defects and extensive DNA damages therein lead to catastrophic chromosomal breaks/rearrangements termed chromothripsis ( Crasta et al . , 2012; Hatch et al . , 2013 ) .\",\n \"Such catastrophic DNA breaks/rearrangements are speculated to lead to tumorigenesis ( Hatch and Hetzer , 2015 ) .\",\n \"Consistent with defective nuclear envelope integrity , we observed extensive DNA damage ( revealed by γ-H2Av ) in both major and micronuclei ( Figure 3A–F , arrows point to damaged DNA in micronuclei in B and D ) .\",\n \"Likely as a result of DNA damage and defective nuclear envelope integrity , D1 mutant testes rapidly degenerated ( Figure 3—figure supplement 1A , B ) .\",\n \"When Omi , a gene required to promote germ cell death ( Yacobi-Sharon et al . , 2013 ) , was knocked down in D1 mutant testes , it restored the cellularity in D1 mutant testis ( Figure 3—figure supplement 1C–D ) , but the surviving cells showed a dramatic increase in DNA damage ( Figure 3—figure supplement 1E–F ) .\",\n \"Under these conditions , we observed that surviving germ cells in D1 mutant testes showed a high frequency of chromosome breaks compared to control , revealed by FISH on metaphase chromosome spreads from spermatocytes ( 3 . 7% in control ( n = 27 ) vs . 15 . 8% in D1 mutant ( n = 57 ) ) ( Figure 3G , H , arrowheads indicate sites of chromosome breaks ) .\",\n \"These results show that loss of D1/HMGA1 results in compromised nuclear envelope integrity , leading to extensive DNA damage and chromosomal breaks .\",\n \"It has been shown that micronuclei form by lagging chromosomes ( Crasta et al . , 2012 ) .\",\n \"Thus , we examined whether D1 mutation/HMGA1 knockdown resulted in mitotic chromosome segregation errors , causing micronuclei formation .\",\n \"However , we did not observe an increase in lagging chromosomes in D1 mutant spermatogonia or HMGA1-depleted mouse cells ( Figure 4—figure supplement 1A–G ) , suggesting an alternative route for micronuclei formation .\",\n \"Instead , time-lapse live observation showed that micronuclei formed by budding from the interphase nucleus both in Drosophila spermatogonia and mouse cells ( Figure 4A–D ) .\",\n \"In Drosophila spematogonia , nuclear contents were visualized by a GFP-tagged nuclear protein , Df31 , and RFP-tagged histone H2Av .\",\n \"Control cells stably maintained nuclear contents for a prolonged time period ( only 1 event of nuclear blebbing without concurrent micronuclei formation ( as detected by H2Av-RFP ) over 1552 min of live imaging ) ( Figure 4A ) .\",\n \"In contrast , D1 mutant cells showed budding off of nuclear contents and micronuclei formation in interphase ( 15 nuclear breaches with eight micronuclei formed over 3427 min of live imaging with a total budding duration of 172 min ) ( Figure 4B ) .\",\n \"Similarly , live imaging in mouse cells using the Hoechst DNA dye revealed that HMGA1 knockdown also resulted in micronuclei formation during interphase ( siControl – no micronuclei formation over 253 min of live observation , siHMGA1 – three micronuclei formed by budding over 5962 min of live imaging with a total budding duration of 310 min ) ( Figure 4C , D ) .\",\n \"These results show that micronuclei in D1 mutant/HMGA1-knockdown cells are generated during interphase , via budding from the nucleus .\",\n \"Based on the above results , we postulated that chromocenter formation , that is clustering of satellite DNA from multiple chromosomes , might be a mechanism to bundle heterologous chromosomes together to prevent individual chromosomes from floating out of the nucleus .\",\n \"In this manner , the full set of chromosomes may be retained within a single nucleus .\",\n \"In the absence of chromocenter formation , individual chromosomes may bud off the nucleus , leading to micronuclei formation .\",\n \"Previous in vitro experiments indicated that HMGA1 is capable of crosslinking multiple DNA strands with individual AT-hooks binding AT-rich DNA strands ( Vogel et al . , 2011 ) .\",\n \"Bundling of DNA in this manner by D1/HMGA1 could explain how pericentromeric satellite DNA from multiple chromosomes may be clustered to form chromocenters .\",\n \"A few lines of evidence support this idea .\",\n \"When Drosophila D1 was expressed in mouse cells , it localized to the chromocenter as described above ( Figure 1G ) , and its overexpression enhanced chromocenter formation in a dose-dependent manner ( Figure 5A–C ) : the higher the amount of D1 that was expressed in mouse cells , the fewer chromocenters per cell was observed ( i . e . more clustering ) .\",\n \"These results suggest that D1 is sufficient to bundle its binding target , tethering it to chromocenter .\",\n \"Consistent with this idea , we found that artificial tethering of D1 protein to euchromatic LacO repeat DNA sequences was sufficient to bring LacO repeats to the chromocenter .\",\n \"D1 protein or D1-LacI fusion protein was expressed in a Drosophila strain in which LacO repeats are inserted in the distal regions of the 2nd chromosome ( Figure 5D , arrows ) .\",\n \"In control spermatogonial cells expressing wild type D1 , LacO repeats were observed far away from the {AATAT}n satellite foci/chromocenters ( Figure 5E , G , arrow indicates site of LacO repeats in interphase nucleus ) .\",\n \"However , in cells expressing the LacI-D1 chimeric protein , we observed recruitment of the LacO repeats close to {AATAT}n/chromocenters ( Figure 5F , G , arrow indicates site of LacO repeats recruited to the chromocenter ) , demonstrating that D1’s binding to a DNA sequence is sufficient to incorporate the target sequence into chromocenters .\",\n \"Although it cannot be visualized how DNA strands from multiple chromosomes might be bundled in these interphase chromocenters , deconvolution microscopy of D1/HMGA1 proteins on early mitotic chromosomes revealed proteinaceous threads between chromatin in the process of condensation ( Figure 6A , B , arrows indicate D1/HMGA1 threads ) , which we speculate contributed to bundling of chromosomes in the previous interphase .\",\n \"These threads were also detectable by DNA FISH against {AATAT}n and the mouse major satellite ( Figure 6C , D , dotted lines are alongside the satellite DNA threads ) , suggesting that satellite DNA bound by D1/HMGA1 can form threads .\",\n \"These threads likely connect heterologous chromosomes , as we see threads between chromosomes that are clearly distinct in their morphology ( e . g . Figure 6C ) .\",\n \"These D1/HMGA1 threads are reminiscent of ‘DNA fibers’ , which were observed among mitotic chromosomes , although their function has never been appreciated ( Takayama , 1975; Burdick , 1976; Kuznetsova et al . , 2007 ) .\",\n \"Taken together , these results support a model , in which D1/HMGA1 bind their target sequences ( satellite DNA ) on multiple chromosomes and bundle them into chromocenters , likely via their multivalent DNA-binding domains ( multiple AT-hooks ) ( Figure 6E ) .\"\n ],\n [\n \"The function of chromocenters , as well as that of satellite DNA , has remained enigmatic , even though cytological association of pericentromeric satellite DNA into chromocenters was identified almost 50 years ago ( Jones , 1970; Pardue and Gall , 1970 ) .\",\n \"Pericentromeric heterochromatin has most often been studied and discussed in the context of how to maintain its heterochromatic , repressed nature ( Nishibuchi and Déjardin , 2017 ) , based on the assumption that the underlying sequences are mostly selfish , which have negative phenotypic consequences when derepressed in cells ( Zeller and Gasser , 2017 ) .\",\n \"Although satellite DNA’s function has been speculated and implicated in several examples ( Yunis and Yasmineh , 1971; Bonaccorsi et al . , 1990; Dernburg et al . , 1996; Menon et al . , 2014 ) , the non-coding nature and lack of conservation in repeat sequence among closely related species led to the idea that they are mostly junk DNA , serving no essential function ( Walker , 1971; Doolittle and Sapienza , 1980 ) .\",\n \"Instead , we propose that satellite DNA is a critical constituent of eukaryotic chromosomes to ensure encapsulation of all chromosomes in interphase nucleus .\",\n \"Our results may also explain why the sequences of pericentromeric satellite DNA are so divergent among closely related species , a contributing factor that led to their dismissal as junk .\",\n \"Based on our model that pericentromeric satellite DNA serves as a platform for generating heterologous chromosome association to form chromocenters , the essential feature of satellite DNA is that they are bound by protein ( s ) capable of bundling multiple DNA strands .\",\n \"If so , the underlying sequence does not have to be conserved .\",\n \"Instead , the binding of satellite DNA by a chromocenter bundling protein may be a critical feature of pericentromeric satellite DNAs .\",\n \"Based on this idea , chromocenter bundling proteins and pericentromeric satellite DNA may be co-evolving .\",\n \"We observed perturbation of nuclear envelope integrity upon chromocenter disruption .\",\n \"Understanding the mechanisms underlying perturbation of nuclear envelope integrity in D1 mutant awaits future investigation .\",\n \"Previous studies have documented that cytoskeletal forces are transmitted to chromatin through nuclear envelope and external mechanical forces can cause temporary nuclear envelope breaches ( King et al . , 2008; Denais et al . , 2016; Hatch and Hetzer , 2016; Raab et al . , 2016 ) .\",\n \"Therefore , we speculate that chromosome bundling in the form of chromocenter may help prevent cytoskeletal forces from shearing chromosomes and nuclear envelope: when chromosomes are not bundled , cytoskeletal forces may be transmitted to individual chromosomes and associated nuclear envelope , resulting in shearing of nuclear envelope , disrupting its integrity .\",\n \"In summary , our study provides the first evidence for a conserved function of pericentromeric satellite DNA and chromocenters .\",\n \"Our data suggest that the multi-AT-hook proteins , D1 and HMGA1 , play an evolutionarily conserved role in the formation of chromocenters , likely via their ability to bind and bundle satellite DNA from heterologous chromosomes .\",\n \"Heterologous chromosome association , mediated by chromocenter-binding proteins , may represent a third mode of chromosomal ‘gluing’ after meiotic homologous pairing and sister chromatid cohesion .\",\n \"Through heterologous association , the chromocenter plays a fundamental role in maintaining the full complement of the genome , which is divided into multiple chromosomes , into a single nucleus .\",\n \"This function of the chromocenter may be conserved in eukaryotic species that contain pericentromeric satellite DNA , thereby bringing about a signature characteristic of eukaryotic cells .\"\n ],\n [\n \"All fly stocks were raised on standard Bloomington medium at 25°C .\",\n \"The following fly stocks were used: D1EY05004 ( BDSC17340 ) , Df ( 3R ) BSC666 ( BDSC26518 ) , UAS-OmiRNAi ( BDSC55165 ) , UAS-GFP-nls ( BDSC4776 ) and UAS-GFP-ER-SR ( BDSC59042 ) were obtained from the Bloomington Drosophila stock center .\",\n \"D1LL03310 ( DGRC140754 ) and Df31-GFP ( DGRC110806 ) were obtained from the Kyoto stock center .\",\n \"nos-gal4 ( Van Doren et al . , 1998 ) , hs-flp;nos-FRT-stop-FRT-gal4 , UAS-GFP ( Salzmann et al . , 2013 ) , UAS-H2A-YFP ( Bellaïche et al . , 2001 ) and B1 LacO ( Vazquez et al . , 2002 ) have been previously described .\",\n \"A stock containing B chromosomes , mtrm126 +B ( Bauerly et al . , 2014 ) , was a kind gift from Scott Hawley .\",\n \"Chromocenter disruption was scored in Drosophila testes by assessing {AATAT}n morphology in GFP +cells that were generated as follows in control ( hs-flp; nos-FRT-stop-FRT-gal4 , UAS-GFP ) and D1 mutant ( hs-flp;nos-FRT-stop-FRT-gal4 , UAS-GFP;D1LL03310/Df ) flies .\",\n \"Testes were dissected 24 hr following a 20 min heat shock at 37°C .\",\n \"Chromocenters were considered disrupted in Drosophila and mouse when satellite DNA adopted thread-like morphology in interphase nuclei .\",\n \"Micronuclei were scored in 0-3 d testes where early germ cell chromosomes were labeled with H2A-YFP .\",\n \"The genotypes used were , control – nos > H2 A-YFP and D1 mutant – nos > H2 A-YFP; D1LL03310/Df .\",\n \"For construction of UAS-GFP-D1 , the D1 ORF was PCR-amplified from cDNA using the following primer pair , 5’-GATCAGATCTATGGAGGAAGTTGCGGTAAAG-3’ and 5’-GATCCTCGAGTTAGGCAGCTACCGATTCGG-3’ .\",\n \"The amplified fragment was subcloned into the BglII and XhoI sites of pUASt-EGFP-attB ( Salzmann et al . , 2013 ) resulting in UAS-GFP-D1 .\",\n \"For UAS-GFP-LacI-D1 , the LacI ORF ( lacking 11 C-terminal residues ) ( Straight et al . , 1996 ) was synthesized using GeneArt ( Thermofisher ) and inserted into the BglII site of UAS-GFP-D1 resulting in UAS-GFP-LacI-D1 .\",\n \"Transgenic flies were generated by PhiC31 integrase-mediated transgenesis into the attP40 site ( BestGene ) .\",\n \"For expression of GFP and GFP-D1 in mouse cells , GFP and GFP-D1 was subcloned from pUASt-EGFP-attB into pCDNA3 ( gift from Cheng-Yu Lee ) using EcoRI and XhoI sites .\",\n \"Mouse MOVAS cells were obtained from Daniel Eitzman .\",\n \"Mouse C2C12 cells were obtained from David Bridges .\",\n \"Mouse RAW264 . 7 cells were obtained from Dr . Harry Mobley .\",\n \"Mouse C3H10T1/2 cells were obtained from Stephen Weiss .\",\n \"MOVAS , C2C12 and RAW264 . 7 cells were maintained in Dulbecco's minimal essential medium ( DMEM ) ( Gibco ) supplemented with 10% fetal bovine serum ( FBS ) .\",\n \"C3H10T1/2 cell line was maintained in alpha minimal essential media ( Gibco ) supplemented with 10% fetal bovine serum .\",\n \"All cell lines used were authenticated as mouse cells by the presence of mouse-specific satellite DNA as is shown throughout the manuscript .\",\n \"Two major cell lines used in this study , C2C12 and MOVAS cells , were treated with Plasmocin ( Invivogen ) prior to use as a precaution for mycoplasma infection .\",\n \"RNA interference ( RNAi ) against HMGA1 was performed using ON-TARGET plus Mouse HMGA1 siRNA SMARTpool ( Dharmacon , L-049293–01 ) consisting of the following target sequences , CCAUUUAGCCGCAGCCCGA , AGGCAAACGGGCACCAACA , GGGCGCAGCAGACUGGUUA , GUUCAUUCUUAGAUACCCA .\",\n \"ON-TARGET plus Non-targeting pool ( Dharmacon , D-001810–10 ) consisting of the following sequences , UGGUUUACAUGUCGACUAA , UGGUUUACAUGUUGUGUGA , UGGUUUACAUGUUUUCUGA , UGGUUUACAUGUUUUCCUA , was used as a negative control .\",\n \"siRNA transfections were performed using DharmaFECT four reagent ( Dharmacon , Lafayette , CO ) according to the manufacturer's protocol .\",\n \"25 nM of siControl and siHMGA1 were transfected using DharmaFECT 4 ( Dharmacon ) according to the manufacturer’s protocol .\",\n \"Cells were fixed for immunostaining/in situ hybridization 6 days post-transfection .\",\n \"Transient transfection of GFP and GFP-D1 was performed using Fugene HD ( Roche ) reagent according to the manufacturer’s protocol .\",\n \"For Drosophila tissues , immunofluorescence staining was performed as described previously ( Cheng et al . , 2008 ) .\",\n \"Briefly , tissues were dissected in PBS , transferred to 4% formaldehyde in PBS and fixed for 30 min .\",\n \"Testes were then washed in PBS-T ( PBS containing 0 . 1% Triton-X ) for at least 60 min , followed by incubation with primary antibody in 3% bovine serum albumin ( BSA ) in PBS-T at 4°C overnight .\",\n \"Samples were washed for 60 min ( three 20 min washes ) in PBS-T , incubated with secondary antibody in 3% BSA in PBS-T at 4°C overnight , washed as above , and mounted in VECTASHIELD with DAPI ( Vector Labs ) .\",\n \"The following primary antibodies were used: rabbit anti-vasa ( 1:200; d-26; Santa Cruz Biotechnology ) , rabbit anti-H3K9 dimethyl ( 1:200; Abcam , ab32521 ) , guinea pig anti-Otefin ( gift from Georg Krohne , 1:400 ) , chicken anti-Cid ( 1:500 , generated using the synthetic peptide CDGENDANDGYVSDNYNDSESVAA ( Covance ) ) , mouse anti-LaminDm0 ( ADL84 . 12 , 1:200 , Developmental Studies Hybridoma Bank ) , mouse anti-γ−H2Av ( UNC93-5 . 2 . 1 , 1:400 , Developmental Studies Hybridoma Bank ) , Phalloidin-Alexa546 ( ThermoFisher , a22283 , 1:200 ) .\",\n \"Adherent mouse cells were fixed in 4% formaldehyde in PBS for 20 min at room temperature on coverslips .\",\n \"Cells were permeabilized in PBS-T for 5 min , rinsed three times with PBS , blocked using 3% BSA in PBS-T for 30 min at room temperature and incubated with primary antibody diluted in 3% BSA in PBS-T overnight at 4°C .\",\n \"Cells were then washed for 30 min ( three 10 min washes ) , incubated with secondary antibody in 3% BSA in PBS-T for 2 hr at room temperature , washed as above and mounted in VECTASHIELD with DAPI ( Vector Labs ) .\",\n \"For nucleoplasmic extraction , cells were incubated with CSK buffer ( 10 mM PIPES pH7 , 100 mM NaCl , 300 mM sucrose , 3 mM MgCl2 , 0 . 5% Triton X-100 , 1 mM PMSF ) for 10 min at room temperature .\",\n \"After CSK extraction , cells were washed with PBS and fixed and immunostained as above .\",\n \"The following antibodies were used: rabbit anti-HMGA1 ( 1:400 , Abcam , ab129153 ) , goat anti-LaminB ( C-20 ) ( 1:20 , Santa Cruz Biotechnology , sc-2616 ) , mouse anti-α-tubulin ( 4 . 3 , 1:100 , Developmental Studies Hybridoma Bank ) and γ−H2Ax S139 ( 2577 , 1:200 , Cell Signaling Technologies ) .\",\n \"Images were taken using a Leica TCS SP8 confocal microscope with 63x oil-immersion objectives ( NA = 1 . 4 ) .\",\n \"Deconvolution was performed when indicated using the Hyvolution package from Leica .\",\n \"Images were processed using Adobe Photoshop software .\",\n \"Testes from newly eclosed flies were dissected into Schneider’s Drosophila medium containing 10% fetal bovine serum .\",\n \"The testis tips were placed inside a sterile glass-bottom chamber and were mounted on a three-axis computer-controlled piezoelectric stage .\",\n \"An inverted Leica TCS SP8 confocal microscope with a 63 × oil immersion objective ( NA = 1 . 4 ) was used for imaging .\",\n \"For mouse live cell imaging , transfected cells were seeded onto a sterile glass-bottom chamber coated with poly-lysine .\",\n \"Cells were incubated with Hoechst 33342 for 10 min , rinsed with PBS and fresh medium was added to the chamber .\",\n \"Cells were imaged using a stage-top Tokai-Hit incubator that was mounted on an inverted TCS SP5 confocal microscope with a 63x oil immersion objective ( NA = 1 . 4 ) .\",\n \"All images were processed using Adobe Photoshop software .\",\n \"Metrics used for quantification of live imaging were total imaging duration ( defined as number of cells x imaging duration ) , total budding duration ( defined as number of cells with micronuclei that formed by budding x time with budded micronuclei ) .\",\n \"Whole mount Drosophila testes were prepared as described above , and optional immunofluorescence staining protocol was carried out first .\",\n \"Subsequently , samples were post-fixed with 4% formaldehyde for 10 min and washed in PBS-T for 30 min .\",\n \"Fixed samples were incubated with 2 mg/ml RNase A solution at 37°C for 10 min , then washed with PBS-T +1 mM EDTA .\",\n \"Samples were washed in 2xSSC-T ( 2xSSC containing 0 . 1% Tween-20 ) with increasing formamide concentrations ( 20% , 40% and 50% ) for 15 min each followed by a final 30 min wash in 50% formamide .\",\n \"Hybridization buffer ( 50% formamide , 10% dextran sulfate , 2x SSC , 1 mM EDTA , 1 μM probe ) was added to washed samples .\",\n \"Samples were denatured at 91°C for 2 min , then incubated overnight at 37°C .\",\n \"For mitotic chromosome spreads , testes and larval 3rd instar brains were squashed according to previously described methods ( Larracuente and Ferree , 2015 ) .\",\n \"Briefly , tissue was dissected into 0 . 5% sodium citrate for 5–10 min and fixed in 45% acetic acid/2 . 2% formaldehyde for 4–5 min .\",\n \"Fixed tissues were firmly squashed with a cover slip and slides were submerged in liquid nitrogen until bubbling ceased .\",\n \"Coverslips were then removed with a razor blade and slides were dehydrated in 100% ethanol for at least 5 min .\",\n \"After drying , hybridization mix ( 50% formamide , 2x SSC , 10% dextran sulfate , 100 ng of each probe ) was applied directly to the slide , samples were heat denatured at 95°C for 5 min and allowed to hybridize overnight at room temperature .\",\n \"Following hybridization , slides were washed thrice for 15 min in 0 . 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .\",\n \"For the in situ experiment described in Figure 4j–m , testes were dissected into PBS and fixed in 4% formaldehyde for 4 min .\",\n \"Tips of fixed testes were firmly squashed with a cover slip and slides were submerged in liquid nitrogen until bubbling ceased .\",\n \"Coverslips were removed with a razor blade and slides were subjected to 5 min washes in 2XSSC and 2XSSC with 0 . 1% Tween-20 .\",\n \"The samples were denatured in freshly made 0 . 07N NaOH for 5 min , rinsed in 2X SSC .\",\n \"Hybridization mix ( 50% formamide , 2x SSC , 10% dextran sulfate , 100 ng of each probe ) was added directly to the slide and allowed to hybridize overnight at room temperature .\",\n \"Following hybridization , slides were washed three times for 15 min in 0 . 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .\",\n \"The following probes were used for Drosophila in situ hybridization: {AATAT}6 , {AAGAG}6 , IGS and have been previously described ( Jagannathan et al . , 2017 ) .\",\n \"LacO probe - 5’-Cy5-CCACAAATTGTTATCCGCTCACAATTCCAC-3’ .\",\n \"For interphase mouse cells , optional immunostaining was carried out as above .\",\n \"Subsequently , samples were post-fixed with 4% formaldehyde in PBS for 10 min and rinsed three times in PBS .\",\n \"Post-fixed cells were incubated with 0 . 1 mg/ml RNase A solution at 37°C for 1 hr , rinsed three times in PBS and denatured in 1 . 9M HCl for 30 min .\",\n \"After three rinses in ice-cold PBS , hybridization mix ( 2X SSC , 60% formamide , 5 µg/ml salmon sperm DNA and 500 nM probe ) was added to the samples and incubated overnight at room temperature .\",\n \"Following hybridization , coverslips were washed three times for 15 min in 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .\",\n \"For mouse mitotic cells , chromosomes were spread on slides as previously described .\",\n \"Subsequently , chromosomes were denatured in 70% formamide in 2XSSC for 1 . 5 min at 70°C , dehydrated in 100% ethanol and hybridization mix ( 2X SSC , 60% formamide , 5 µg/ml salmon sperm DNA and 500 nM probe ) was added directly to the slide and incubated overnight at room temperature .\",\n \"Following hybridization , slides were washed three times for 15 min in 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .\",\n \"The following probe was used: Major satellite - 5’-Cy3-GGAAAATTTAGAAATGTCCACTG-3’ .\"\n ]\n]"},"abstract":{"kind":"list like","value":["A universal and unquestioned characteristic of eukaryotic cells is that the genome is divided into multiple chromosomes and encapsulated in a single nucleus .","However , the underlying mechanism to ensure such a configuration is unknown .","Here , we provide evidence that pericentromeric satellite DNA , which is often regarded as junk , is a critical constituent of the chromosome , allowing the packaging of all chromosomes into a single nucleus .","We show that the multi-AT-hook satellite DNA-binding proteins , Drosophila melanogaster D1 and mouse HMGA1 , play an evolutionarily conserved role in bundling pericentromeric satellite DNA from heterologous chromosomes into ‘chromocenters’ , a cytological association of pericentromeric heterochromatin .","Defective chromocenter formation leads to micronuclei formation due to budding from the interphase nucleus , DNA damage and cell death .","We propose that chromocenter and satellite DNA serve a fundamental role in encapsulating the full complement of the genome within a single nucleus , the universal characteristic of eukaryotic cells ."],"string":"[\n \"A universal and unquestioned characteristic of eukaryotic cells is that the genome is divided into multiple chromosomes and encapsulated in a single nucleus .\",\n \"However , the underlying mechanism to ensure such a configuration is unknown .\",\n \"Here , we provide evidence that pericentromeric satellite DNA , which is often regarded as junk , is a critical constituent of the chromosome , allowing the packaging of all chromosomes into a single nucleus .\",\n \"We show that the multi-AT-hook satellite DNA-binding proteins , Drosophila melanogaster D1 and mouse HMGA1 , play an evolutionarily conserved role in bundling pericentromeric satellite DNA from heterologous chromosomes into ‘chromocenters’ , a cytological association of pericentromeric heterochromatin .\",\n \"Defective chromocenter formation leads to micronuclei formation due to budding from the interphase nucleus , DNA damage and cell death .\",\n \"We propose that chromocenter and satellite DNA serve a fundamental role in encapsulating the full complement of the genome within a single nucleus , the universal characteristic of eukaryotic cells .\"\n]"},"summary":{"kind":"list like","value":["On Earth , life is divided into three domains .","The smallest of these domains includes all the creatures , from sunflowers to yeasts to humans , that have the genetic information within their cells encased in a structure known as the nucleus .","The genomes of these organisms are formed of long pieces of DNA , called chromosomes , which are packaged tightly and then unpackaged every time the cell divides .","When a cell is not dividing , the chromosomes in the nucleus are loosely bundled up together .","It is well known that DNA is the blueprint for the building blocks of life , but actually most of the genetic information in a cell codes for nothing , and has unknown roles .","An example of such ‘junk DNA’ is pericentrometric satellite DNA , small repetitive sequences found on all chromosomes .","However , new experiments by Jagannathan et al . show that , in the nucleus of animal cells , certain DNA binding proteins make chromosomes huddle together by attaching to multiple pericentrometric satellite DNA sequences on different chromosomes .","In fact , if these proteins are removed from mice and fruit flies cells grown in the laboratory , the chromosomes cannot be clustered together .","Instead , they ‘float away’ , and the membranes of the nucleus get damaged , possibly buckling under the pressure of the unorganized DNA .","These events damage the genetic information , which can lead to the cell dying or forming tumors .","‘Junk DNA’ therefore appears to participate in fundamental cellular processes across species , a result that opens up several new lines of research ."],"string":"[\n \"On Earth , life is divided into three domains .\",\n \"The smallest of these domains includes all the creatures , from sunflowers to yeasts to humans , that have the genetic information within their cells encased in a structure known as the nucleus .\",\n \"The genomes of these organisms are formed of long pieces of DNA , called chromosomes , which are packaged tightly and then unpackaged every time the cell divides .\",\n \"When a cell is not dividing , the chromosomes in the nucleus are loosely bundled up together .\",\n \"It is well known that DNA is the blueprint for the building blocks of life , but actually most of the genetic information in a cell codes for nothing , and has unknown roles .\",\n \"An example of such ‘junk DNA’ is pericentrometric satellite DNA , small repetitive sequences found on all chromosomes .\",\n \"However , new experiments by Jagannathan et al . show that , in the nucleus of animal cells , certain DNA binding proteins make chromosomes huddle together by attaching to multiple pericentrometric satellite DNA sequences on different chromosomes .\",\n \"In fact , if these proteins are removed from mice and fruit flies cells grown in the laboratory , the chromosomes cannot be clustered together .\",\n \"Instead , they ‘float away’ , and the membranes of the nucleus get damaged , possibly buckling under the pressure of the unorganized DNA .\",\n \"These events damage the genetic information , which can lead to the cell dying or forming tumors .\",\n \"‘Junk DNA’ therefore appears to participate in fundamental cellular processes across species , a result that opens up several new lines of research .\"\n]"},"year":{"kind":"string","value":"2018"}}},{"rowIdx":4195,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["structural biology and molecular biophysics"],"string":"[\n \"structural biology and molecular biophysics\"\n]"},"title":{"kind":"string","value":"Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival"},"id":{"kind":"string","value":"elife-20914-v2"},"sections":{"kind":"list like","value":[["Mg2+ is the most abundant intracellular divalent cation and is essential for the regulation of a broad spectrum of metabolic and signalling pathways ( de Baaij et al . , 2015 ) .","In addition , direct association with Mg2+ fosters the structural integrity of key metabolites ( such as ATP ) , proteins , lipid membranes and nucleic acids ( de Baaij et al . , 2015 ) implying that organismal Mg2+ deficiency , a surprisingly common condition in humans ( King et al . , 2005; Rosanoff et al . , 2012 ) , may be especially harmful during prenatal development and early postnatal life , when the production of and the demand for Mg2+-bound metabolites is particularly high .","There is growing evidence to suggest that Mg2+ deprivation is accompanied by different types of metabolic , immune , cardiovascular and neurological disorders ( de Baaij et al . , 2015 ) .","However , mainly due to the lack of adequate mammalian genetic models , it still remains unclear whether an imbalance in Mg2+ metabolism is merely associated with or can directly trigger the latter pathophysiological processes .","Furthermore , it has recently been shown that cellular Mg2+ fluxes regulate the circadian rhythm and energy balance ( Feeney et al . , 2016 ) , CGRP-mediated osteogenic differentiation ( Zhang et al . , 2016 ) and synaptic plasticity ( Palacios-Prado et al . , 2014 ) , and that changes in the composition of brain interstitial Mg2+ concentrations participate in the control of the sleep-wake cycle ( Ding et al . , 2016 ) .","The remarkable recent progress in our understanding of the critical role of Mg2+ in health and disease contrasts with the dearth of knowledge about the mechanisms governing cellular and organismal Mg2+ balance .","Approximately 10 plasma membrane Mg2+ channels have been proposed ( Quamme , 2010 ) indicating a high degree of redundancy .","However , quite some controversy surrounds the biological role of many of these proteins , and the question whether there is a central gatekeeper responsible for organismal Mg2+ balance has not yet been answered .","The kinase-coupled ion channel TRPM7 has been proposed as a ubiquitous , indispensable cellular Mg2+ entry pathway ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010; Stritt et al . , 2016 ) .","However , studies with Trpm7 gene-deficient mice failed to confirm a corresponding in vivo role of Trpm7 .","Thus , constitutive inactivation of Trpm7 in mice entailed early embryonic lethality for as yet unknown reasons ( Jin et al . , 2008 ) .","Furthermore , tissue-specific deletions of Trpm7 in mouse embryos affected morphogenesis of internal organs apparently in a Mg2+-independent manner ( Jin et al . , 2008 , 2012; Sah et al . , 2013 ) .","More recently , it was suggested that the Mg2+ transporter MagT1 rather than TRPM7 might play a critical role for Mg2+ homeostasis in T lymphocytes ( Li et al . , 2011 ) and probably also in the whole embryo ( Zhou and Clapham , 2009 ) .","Hence , the biological role of TRPM7 requires further clarification .","In the present work , we focussed on the closest TRPM7 relative , TRPM6 , because loss-of-function mutations in TRPM6 cause hypomagnesemia ( low Mg2+ blood levels ) in human infants thought to mainly result from renal Mg2+ wasting ( Schlingmann et al . , 2002; Walder et al . , 2002; Voets et al . , 2004 ) .","However , deletion of Trpm6 in mice has resulted in neural tube closure defects and embryonic death ( Walder et al . , 2009 ) indicating a direct role of TRPM6 in developmental processes and calling into question the simplistic view on the human TRPM6 phenotype .","By integrating systematic phenotyping of Trpm6 gene-modified mice with biochemical analysis , gene expression , metabolomics , and cell biological approaches , we decipher the molecular and organismal roles of TRPM6 in prenatal development and postnatal survival ."],["To understand the role of Trpm6 in prenatal development , we determined the onset of embryonic lethality in Trpm6 null embryos and investigated the expression pattern of Trpm6 at this stage .","Using a mouse strain carrying a gene-trap mutation in Trpm6 ( Trpm6βgeo ) ( Table 1 ) , we found that Trpm6βgeo/βgeo embryos were present at embryonic days ( e ) 8 . 5–10 . 5 ( Figure 1A ) .","However , only a few mutants were found between e11 . 5–12 . 5 and no Trpm6βgeo/βgeo individuals were viable after e14 . 5 ( Figure 1A ) .","Compared to e9 . 5 C-shaped Trpm6+/+ individuals , all Trpm6βgeo/βgeo embryos isolated had not turned ( S-shaped ) and were smaller indicating a developmental retardation after e8 . 5 ( Figure 1B ) .","Consequently , we investigated the expression pattern of Trpm6 in e8 . 5 fetuses by in situ hybridization ( ISH ) and found that Trpm6 was specifically expressed in the visceral yolk sac endoderm and extraembryonic chorion ( Figure 1C ) and that Trpm6 was not detectable in the neural tube ( Figure 1—figure supplement 1 ) .","Within the placental labyrinth a network of maternal sinusoids are intertwined with fetal blood capillaries , separated by two layers of transporting trophoblast cells , syncytiotrophoblasts I ( SynT-I ) and II ( SynT-II ) ( Simmons and Cross , 2005; Simmons et al . , 2008 ) .","At e8 . 5 , morphogenesis of the labyrinth is in the initial stages and SynT-I/SynT-II cell layers are distinguishable ( Simmons and Cross , 2005; Simmons et al . , 2008 ) .","We observed that Trpm6 expression was restricted to SynT-I cells ( Figure 1D ) .","In the fully maturated labyrinth at e14 . 5 Trpm6 mRNA was detected in syncytiotrophoblasts as well ( Figure 1E ) . 10 . 7554/eLife . 20914 . 003Figure 1 . Assessment of Trpm6 function in extraembryonic tissues .","( A ) Survival of Trpm6βgeo/βgeo embryos obtained from Trpm6βgeo/+ intercrosses .","( B ) Representative images of e9 . 5 Trpm6+/+ ( +/+ , n = 13 ) and Trpm6βgeo/βgeo ( βgeo/βgeo , n = 5 ) embryos from dataset in ( A ) .","Dashed lines underline C-shaped versus S-shaped morphology of Trpm6+/+ and Trpm6βgeo/βgeo embryos , respectively .","( C ) ISH on serial paraffin sections obtained from wildtype n = 5 e8 . 5 fetus using antisense ( left ) and sense ( right ) probes for Trpm6 .","Boxes indicate the positions of the magnified images of the chorion ( ch ) and yolk sac ( yc ) .","Arrows indicate Trpm6-positive cells in the developing labyrinth ( chorion ) and the endoderm layer in the visceral yolk sac .","( D ) ISH on serial paraffin sections of wildtype e8 . 5 placenta using DIG-labelled probes for Trpm6 ( left ) , SynA ( middle ) and Gcm1 ( right ) , respectively .","Note: Trpm6 expression was restricted to cells positive for SynA , a marker of SynT-I , and absent in cells expressing Gcm1 , a marker of SynT-II .","Representative images of n = 2 independent tissues are shown .","( E ) ISH of WT e14 . 5 placenta with the antisense Trpm6 probe .","The box indicates the position of the magnified image .","The Trpm6 signal is restricted to the labyrinth ( lab ) and not detectable in the decidua ( dec ) and trophoblast giant cells ( GT ) .","Representative images of n = 8 independent placentas are shown .","( F ) Mg2+ levels in e9 . 5 Trpm6+/+ ( n = 4 ) and Trpm6βgeo/βgeo ( n = 3 ) embryos .","Distal segments of the embryos were used for genotyping , and the remaining parts were analysed by ICP-MS .","Elementary magnesium ( Mg ) contents were normalized to phosphorus ( P ) and sulfur ( S ) levels represented as mean±SEM .","*-p≤0 . 05 ( Student’s t-test ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00310 . 7554/eLife . 20914 . 004Figure 1—figure supplement 1 . ISH on serial paraffin sections obtained from wildtype e8 . 5 fetus using antisense ( left ) and sense ( right ) probes for Trpm6 . Arrows indicate Trpm6-positive cells in the chorion ( blue arrows ) and the endoderm layer in the visceral yolk sac ( black arrows ) stained only by the antisense probe .","Note: Trpm6 was not detectable in embryonic tissues including the neural tube ( red arrows ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00410 . 7554/eLife . 20914 . 005Table 1 . Postnatal survival of the mice with global and tissue-restricted deletions of Trpm6 . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 005Targeted tissueBreeding strategyExpected F1 outcome* Survival of the mutantConstitutive mutagenesisWhole fetus♂Trpm6βgeo/+ x ♀Trpm6βgeo/+ 25%Trpm6βgeo/βgeo 50% Trpm6βgeo/+ 25% Trpm6+/+ noWhole fetus♂Trpm6Δ17/+ x ♀Trpm6 Δ17/+ 25% Trpm6 Δ17/Δ17 50% Trpm6 Δ17/+ 25% Trpm6+/+ noWhole fetus♂Trpm6Δ17/+ x ♀Trpm6βgeo/+ 25% Trpm6βgeo/Δ17 25% Trpm6βgeo/+ 25% Trpm6Δ17/+ 25% Trpm6+/+ noConditional mutagenesis using Cre/LoxP systemEpiblast♂Trpm6Δ17/+;Sox2-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/Δ17;Sox2-Cre 25% Trpm6Δ17/fl 25% Trpm6Δ17/+;Sox2-Cre 25% Trpm6fl/+ yesIntestine♂Trpm6Δ17/+;Villin1-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/fl;Villin1-Cre† 25% Trpm6Δ17/fl 25% Trpm6fl/+;Villin1-Cre 25% Trpm6fl/+ yesKidney♂Trpm6Δ17/+;Ksp-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/fl;Ksp-Cre† 25% Trpm6Δ17/fl 25% Trpm6fl/+;Ksp-Cre 25% Trpm6fl/+ yes*Genotypes were determined using genomic DNA extracted from tail fragments .","†Individuals were homozygous for Trpm6Δ17 allele in the targeted cells .","Syncytiotrophoblasts and endoderm cells of the yolk sac exchange metabolites between the maternal and fetal blood ( Simmons and Cross , 2005 ) .","To clarify whether TRPM6 is required for Mg2+ supply by extraembryonic tissues , we used inductively coupled plasma mass spectrometry ( ICP-MS ) and found that relative magnesium ( Mg2+ ) levels were reduced in the whole e9 . 5 Trpm6βgeo/βgeo embryos ( Figure 1F ) .","Thus , Trpm6 is specifically expressed in the placental labyrinth and the yolk sac at the stage when the Mg2+ deficiency and growth delay of Trpm6-deficient embryos become apparent .","To investigate whether TRPM6 activity in extraembryonic cells underlies the lethality of Trpm6 null embryos , we characterized a mouse strain with a ‘floxed’ ( Trpm6fl ) allele ( Table 1 ) .","Cre-mediated excision engendered viable mice heterozygous for the constitutive deletion mutation in Trpm6 ( Trpm6Δ17/+ ) .","However , we were unable to produce live Trpm6βgeo/Δ17 or Trpm6Δ17/Δ17 offspring , indicating that Trpm6∆17 is a true null mutation ( Table 1 ) .","The paternally inherited Sox2-Cre transgene drives recombination only in epiblast cells , but not in extraembryonic tissues ( Hayashi et al . , 2003 ) .","Notably , intercrosses of Trpm6Δ17/+;Sox2-Cre males and Trpm6fl/fl females resulted in viable Trpm6Δ17/Δ17 pups at the expected ratio ( Table 1 ) .","Therefore , the embryonic mortality of Trpm6-deficient mice appears to be caused by the loss of TRPM6 in extraembryonic tissues .","We next studied the impact of a global deletion of Trpm6 postnatally .","Examination of Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre ) mice at weaning did not reveal conspicuous abnormalities .","However , during the follow-up period , we observed the gradual development of pathologies .","Thus , Trpm6-deficient mice had a lifespan of no longer than 16 weeks ( Figure 2A ) .","Mutants were growth-delayed , and displayed a lighter fur colour and low night-time activity ( Figure 2B , C ) .","Weight gain and lean body mass of Trpm6-deficient mice were reduced ( Figure 2D , E ) , as was the muscle fibre area of the gastrocnemius muscle of 12–13 week-old Trpm6-deficient mice indicative of sarcopenia ( Figure 2F ) .","Mutant mice displayed kyphosis ( Figure 2G ) and completely lacked abdominal and subcutaneous fat depots ( Figure 2H , I ) indicative of catabolic metabolism .","However , the total amount of faeces ( Figure 2—figure supplement 1A ) and the calorimetrically determined faecal energy content , as a measure of energy excretion ( Figure 2—figure supplement 1B ) , were not altered in Trpm6 mutants , ruling out insufficient food intake .","Histological analysis of internal organs ( Figure","3 ) showed that Trpm6-deficient mice developed lung emphysema and degeneration of lymphoid organs .","Thus , the thymus of mutant mice was rudimentary and the cortex region was not distinguishable .","In the spleen of Trpm6-deficient mice , the red pulp was substantially reduced .","Hepatocytes of Trpm6-deficient mice were depleted of glycogen granules ( Figure 3 ) , corroborating catabolic metabolism .","It has been suggested that low serum Mg2+ and TRPM6 function are associated with atherosclerosis in humans ( Maier , 2012; Tin et al . , 2015 ) .","Therefore , we investigated whether such a phenotype would develop in our mouse model as well .","However , examination of thoracal aorta showed no signs of atherosclerosis development in mutant mice ( Figure 2—figure supplement 2 ) . 10 . 7554/eLife . 20914 . 006Figure 2 . Pathophysiological changes displayed by Trpm6-deficient adult mice . Unless stated otherwise , 10–12 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were studied .","( A–E )","Mice were examined for survival rate ( A ) , overall physical appearance ( B ) , day/night activity of 8 week-old individuals ( C ) , growth rate ( D ) and lean mass ( E ) .","( F ) Fibre size of the gastrocnemius muscle after hematoxylin-eosin staining .","( G ) X-ray images of mice .","The red arrow indicates the characteristic skeletal deformation ( kyphosis ) observed in Trpm6-deficient mice .","( H ) Assessment of abdominal fat .","Arrows indicate fat deposits observed only in control mice .","( I ) H and E staining of paraffin skin sections .","Arrows indicate a layer of fat cells present only in control mice .","Histological analysis was performed with three animals per group resulting in similar observations .","( J ) The levels of main elements in the serum of 8 week-old mice assessed by ICP-MS .","( K ) The survival rate of mice maintained on high Mg2+ ( 0 . 75% ) and regular ( 0 . 22% ) chows .","Data are represented as mean±SEM .","***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00610 . 7554/eLife . 20914 . 007Figure 2—figure supplement 1 . Examination of energy balance in Trpm6-deficient mice . Eight week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were evaluated for ( A ) chow intake as assessed by feces production , ( B ) energy content of feces studied by bomb calorimetry and ( C ) serum levels of β-hydroxybutyrate .","Glucose ( D ) and insulin levels ( E ) in the serum of mice subjected to a glucose tolerance test .","Note: mutant mice had lower peripheral glucose concentrations than controls despite of a similar amount of insulin released , thus reflecting increased insulin sensitivity .","Data are represented as mean±SEM .","***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00710 . 7554/eLife . 20914 . 008Figure 2—figure supplement 2 . Evaluation of atherosclerosis development in Trpm6-deficient mice . An assessment of 8 week-old control ( Control , n = 3 ) and Trpm6-deficient ( KO , n = 3 ) with ApoE-/- mice ( as a positive control , n = 1 ) using en-face thoracal aorta preparation and Oil-Red O staining ( A ) and hematoxylin-eosin staining ( B ) , Mac2-staining ( green ) ( C ) of aortic arches .","Representative images are shown .","Arrows indicate atherosclerosis lesions observed only in ApoE-/- mice .","( D ) Plasma cholesterol levels ( mean±SEM ) of control and Trpm6-deficient mice .","n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00810 . 7554/eLife . 20914 . 009Figure 3 . Histology of internal organs of Trpm6-deficient mice . Hematoxylin-eosin staining of paraffin embedded tissue sections of 12–13 week-old control ( Control ) and Trpm6-deficient ( KO ) mice maintained either on regular ( 0 . 22% Mg2+ ) or Mg2+ supplemented ( 0 . 75% Mg2+ ) chows .","Trpm6-deficient mice maintained on the regular diet showed marked airspace enlargement ( indicated by stars ) mimicking lung emphysema , distortion of splenic red pulp ( rp ) / white pulp ( wp ) microarchitecture , thymic atrophy , and reduction of intracellular glycogen in hepatocytes ( indicated by arrows ) .","Histological analysis was performed with three animals per group resulting in similar observations . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 009 Next , we asked whether the phenotype of Trpm6-deficient mice is caused by Mg2+ deficiency .","We employed ICP-MS to compare the concentrations of main elements in serum from controls and Trpm6-deficient littermates .","We found that , similar to humans with mutations in the TRPM6 gene ( Schlingmann et al . , 2002; Walder et al . , 2002 ) , Trpm6-deficient mice developed hypomagnesemia ( Figure 2J ) .","Serum Mg2+ levels of mutant mice were only 0 . 58 mM ( 36% of control value , 1 . 58 mM ) , whereas concentrations of other elements were not changed ( Figure 2J ) .","A Mg2+-enriched diet is an efficient way to alleviate hypomagnesemia in humans lacking TRPM6 ( Schlingmann et al . , 2002; Walder et al . , 2002 ) .","Therefore , we asked whether the phenotypes of Trpm6-deficient mice were caused by Mg2+ deprivation and could be rescued by dietary supplementation .","To this end , we changed the regular chow ( 0 . 22% Mg2+ ) of 4 week-old mutant mice and control littermates for a Mg2+ enriched diet ( 0 . 75% Mg2+ ) .","Notably , none of the Trpm6-deficient mice died during the following 12 weeks of Mg2+ supplementation ( Figure 2K ) .","However , returning to regular chow resulted in 100% mortality of mutant mice within the following 13 weeks ( Figure 2K ) .","Mg2+ supplemented mutants neither exhibited kyphosis nor lipodystrophy ( data not shown ) .","Furthermore , the morphology of the lung , spleen , and thymus of Mg2+ supplemented mutants closely resembled that of control mice ( Figure 3 ) .","We asked whether dietary Mg2+ supplementation of Trpm6βgeo/+ parents would benefit the survival of Trpm6-deficient offspring .","However , similar to a previous study ( Walder et al . , 2009 ) , we found that this treatment was inefficient .","Trpm6-deficient adult mice phenocopy salient pathologies reported for a set of mouse strains advocated as genetic models of ‘accelerated’ or ‘premature’ aging ( Kuro-o et al . , 1997; Trifunovic et al . , 2004; Kujoth et al . , 2005; Varela et al . , 2005; Mostoslavsky et al . , 2006; Niedernhofer et al . , 2006; van der Pluijm et al . , 2007; López-Otín et al . , 2013 ) .","Similar to Trpm6-deficient mice , the latter mutants display short lifespan , growth failure , low physical activity , kyphosis , lung emphysema , sarcopenia , lipodystrophy and degeneration of lymphoid organs .","A characteristic feature of these mouse strains is suppression of the somatotropic axis accompanied by induction of xenobiotic detoxification gene networks in the liver ( Niedernhofer et al . , 2006; van de Ven et al . , 2006; van der Pluijm et al . , 2007; Schumacher et al . , 2008; Garinis et al . , 2009; Mariño et al . , 2010 ) , interpreted as a defensive organismal response , slowing down growth and metabolism in favor of somatic preservation ( López-Otín et al . , 2013 ) .","We asked whether Trpm6-deficient mice would also display such protective metabolic responses .","In fact , we found that serum IGF1 concentrations were reduced in Trpm6-deficient mice as well ( Figure 4A ) .","Mutant mice had a lower core body temperature ( Figure 4B ) and a profoundly reduced urinary content of major urinary proteins ( MUPs ) ( Figure 4C ) , two known features of supressed IGF1 signalling ( Mariño et al . , 2010; Bartke et al . , 2013 ) .","Even though mutant mice showed signs of overall energy shortage , circulating levels of ketone bodies ( ß-hydroxybutyrate ) were not elevated ( Figure 2—figure supplement 1C ) .","When subjected to an oral glucose tolerance test , mutant mice displayed lower peripheral glucose concentrations than controls despite of a similar amount of insulin released , thus reflecting increased insulin sensitivity ( Figure 2—figure supplement 1D , E ) , another hallmark of suppressed IGF1 signalling ( Bartke et al . , 2013 ) .","Notably , body weight and IGF1 serum levels were indistinguishable in Mg2+ supplemented mutant and control mice suggesting that the variations observed in mice maintained on a regular diet were induced by Mg2+ deficiency ( Figure 2—figure supplement 1F , G ) . 10 . 7554/eLife . 20914 . 010Figure 4 . Assessment of metabolic profiles of Trpm6-deficient mice .","( A–C ) 8 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were evaluated for ( A ) serum IGF1 , ( B ) body temperature , ( C ) urinary MUPs content in individual mice .","Data are represented as mean±SEM .","***-p≤0 . 001; *-p≤0 . 05 ( Student’s t-test ) ; n – number of mice examined .","( D ) IPA analysis of genome-wide hepatic transcriptome profiling of Trpm6-deficient ( n = 3 ) vs control ( n = 4 ) littermates .","The diagram shows the top 5 of IPA Canonical Pathways significantly changed in mutant mice ( Supplementary file 2 ) .","Numbers of the commonly changed transcripts are indicated close to the lines connecting the pathways .","( E ) Venn diagram for sets of metabolites significantly changed ( FDR p≤0 . 05 ) in serum , liver and gastrocnemius muscle Trpm6-deficient ( n = 6 ) vs control ( n = 8 ) littermates ( Supplementary file 3 ) .","Commonly changed metabolites are listed in different colours as outlined in the Venn diagram .","( F–J )","Levels of AC C18:1 ( F–H ) and AC C18 ( I–J ) examined in the serum ( F , I ) , liver ( G ) and gastrocnemius muscle ( H , J ) of Trpm6-deficient and control mice .","Data are represented as mean±SEM .","***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different to control group maintained on a regular Mg2+ diet ( one-way ANOVA ) ; n – number of mice examined .","( K ) ATP production by mitochondria isolated from the liver of wildtype C57BL/6 mice with succinate , palmitoylcarnitine or octanoylcarnitine as energy sources .","ATP levels were determined after 30 min incubation of untreated ( - ) or treated ( + ) mitochondria by EDTA with or without Mg2+ .","Data are represented as mean±SEM of 4–5 independent isolations ( N ) .","##-p≤0 . 01; #-p≤0 . 05 significantly different to the control group; ***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05 significantly different to the EDTA treated group ( Student’s t-test ) .","n . s . – not significantly different . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01010 . 7554/eLife . 20914 . 011Figure 4—figure supplement 1 . Gene expression profiling of Trpm6-deficient and control mice . Heatmap diagram of differently expressed genes with FDR p≤0 . 1 in the liver of 12–13 week-old control ( Trpm6fl/+ , n = 4 ) vs Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 3 ) male littermates .","n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01110 . 7554/eLife . 20914 . 012Figure 4—figure supplement 2 . Metabolomic profiling of Trpm6-deficient and control mice . Profiling of metabolites in the serum , liver and gastrocnemius muscle samples from 8–10 week-old control ( Trpm6fl/+ , n = 8 ) and KO ( Trpm6Δ17/Δ17;Sox2-Cre , n = 6 ) male littermates were studied for a panel of 237 metabolites outlined in Supplementary file 3 .","The heatmap diagram shows concentration levels scaled to zero mean and unit standard deviation of significantly changed metabolites for control and KO genotypes in a colour-coded way .","n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01210 . 7554/eLife . 20914 . 013Figure 4—figure supplement 3 . Assessment of the membrane potential ( Δψm ) in isolated mitochondria .","( A ) Mitochondria isolated from the liver of wildtype C57BL/6 mice were incubated ( 30 min ) in the presence of succinate/rotenone , octanoylcarnitine/malate or palmitoylcarnitine/malate as energy source .","The left panel shows representative measurements of Δψm using Rh123 probe .","As a positive control , carbonyl cyanide 4- ( trifluoromethoxy ) phenylhydrazone ( FCCP ) was added at the end of recording to induce breakdown of Δψm .","The right panel shows the calculated start- and endpoints of Δψm elicited by the application of 3 mM EDTA in the absence or presence of 1–5 mM Mg2+ for traces shown in left panel .","Data are represented as mean±SD .","N – independent mitochondria isolations; n – independent measurements .","***-p≤0 . 001 , **-p≤0 . 01 , *-p≤0 . 05 significant to the untreated ( control ) group; ###-p≤0 . 001 , ##-p≤0 . 01 , #-p≤0 . 05 significant to 3 mM EDTA + 0 mM Mg2+ group; ‡‡‡-p≤0 . 001 , ‡‡-p≤0 . 01 , ‡-p≤0 . 05 significant to 3 mM EDTA + 1 mM Mg2+ group ( Student’s t-test ) .","( B ) Representative measurements of ψm in the presence of 3 mM EDTA with/without 3–5 mM Ca2+ or Zn2+ performed analogously to ( A ) .","Three independent experiments showed similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 013 Finally , we performed whole-genome profiling of the liver transcriptome ( Supplementary file 1 ) .","Applying a cut-off value of p≤0 . 1 for the false discovery rate ( FDR ) , we identified 46 genes up- or down-regulated in the livers of Trpm6-deficient mice ( Figure 4—figure supplement 1 , Supplementary file 1 ) .","The majority of affected transcripts code for solute carrier transporters , cytochrome P450 metabolising enzymes , glutathione S-transferases and proteins metabolizing steroids .","As expected , Ingenuity Pathway Analysis ( IPA ) revealed that the inactivation of Trpm6 is associated with an induction of interconnected gene networks controlling toxicity responses and xenobiotic metabolism governed by nuclear receptors such as retinoid X receptors ( RXR ) , liver X receptor ( LXR ) and farnesoid X receptor ( FXR ) ( Figure 4D , Supplementary file 2 ) .","Hence , Trpm6-deficient mice were characterized by suppression of the somatotropic axis and induction of xenobiotic detoxification responses .","To gain mechanistic insight into the altered energy metabolism of mutant mice , we quantified serum , liver and skeletal muscle levels of 237 metabolites ( Supplementary file 3 , Figure 4—figure supplement 2 ) .","Unexpectedly , alterations in Trpm6-deficient mice ( FDR p≤0 . 1 ) were restricted to only several metabolites representing mainly long-chain acylcarnitines ( AC ) , phosphatidylcholines ( PC ) , and sphingomyelins ( SM ) ( Figure 4E ) .","These findings , as well as the results of gene array profiling ( Figure 4D ) suggest that sustained Mg2+ deficiency triggers a specific metabolic response rather than widespread unsystematic changes .","In line with this idea , we noted that AC C18:1 and the related AC C18 were consistently increased in tissues of Trpm6-deficient mice ( Figure 4F–J ) , whereas carnitine levels were not changed ( Supplementary file 3 ) .","In conjunction with lowered concentrations of glucose and unchanged ketogenesis ( Figure 2—figure supplement 1C ) , this constellation is a metabolic ‘signature’ of a frequent inherited human disorder characterized by inefficient β-oxidation of fatty acids due to mitochondrial carnitine palmitoyltransferase II deficiency ( Gempel et al . , 2002; Bonnefont et al . , 2004 ) .","AC C18:1 and C18 levels were normalized in serum and tissues obtained from Mg2+ supplemented mutants ( Figure 4F–J ) , implying that metabolic changes of AC were caused by Mg2+ deficiency .","Consequently , we asked whether Mg2+ would specifically affect mitochondrial ATP production ( Figure 4K ) and maintenance of the mitochondrial membrane potential ( MMP ) ( Figure 4—figure supplement 3A ) .","To address this question , wildtype liver mitochondria were incubated in a buffer containing 3 mM EDTA with or without 1–5 mM Mg2+ .","Such manipulations had no negative effect on mitochondrial respiration when succinate was offered as an energy source ( Figure 4K , Figure 4—figure supplement 3A ) .","In contrast , mitochondria failed to utilize octanoylcarnitine or palmitylcarnitine for ATP production ( Figure 4K ) and to maintain MMP ( Figure 4—figure supplement 3A ) in the presence of EDTA .","Importantly , ATP production and MMP could be fully rescued by the administration of 3–5 mM Mg2+ ( Figure 4K , Figure 4—figure supplement 3A ) , but not Zn2+ or Ca2+ ( Figure 4—figure supplement 3B ) .","Thus , sustained Mg2+ deprivation impairs energy homeostasis resulting in a catabolic metabolism in Trpm6-deficient mice , at least partially due to insufficient mitochondrial utilization of AC .","Next , we investigated the etiology of hypomagnesemia in Trpm6-deficient mice .","~50% of body Mg2+ content is stored in bones , ~30% in muscle tissues and only ~1% in the serum ( de Baaij et al . , 2015 ) .","Using ICP-MS we studied the Mg2+ content in bones ( right tibia ) and gastrocnemius muscle , and observed that Mg2+ levels in bones of Trpm6 null mice were only 24% of control values ( Figure 5A ) .","Furthermore , the Mg2+ content of muscle was also significantly reduced in Trpm6-deficient mice ( Figure 5B ) .","Hence , Trpm6-deficient mice develop a severe systemic Mg2+ deficit . 10 . 7554/eLife . 20914 . 014Figure 5 . Examining of Mg2+ balance in Trpm6-deficient adult mice .","( A–F )","Assessment of 8 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermate males .","( A ) Mg2+ levels in bones and ( B ) gastrocnemius muscle assessed by ICP-MS .","( C ) Immunostaining of kidney cryosections using a TRPM6-specific antibody .","Representative images are shown ( n = 2 tissues per genotype ) .","The blue square indicates the position of the confocal and differential interference contrast magnified images acquired from control tissue .","Arrows indicate labelling of the apical surface of renal tubules .","( D ) 24 hr urinary and ( E ) fecal Mg2+ excretion rates .","( F ) ISH on paraffin sections obtained from the colon of control and Trpm6-deficient mice ( n = 2 tissues per genotype ) .","( G–J )","Examination of 6 month-old Trpm6fl/+ ( Control ) and Trpm6Δ17/fl;Ksp-Cre ( Kidney KO ) littermate males .","( G ) Immunostaining of TRPM6 in kidney cryosections .","Arrows indicate labelling of renal tubules .","( H–I )","Determination of Mg2+ in serum ( H ) and bones ( I ) .","( J ) 24 hr urinary Mg2+ excretion rate .","( K–N )","Assessment of 6 month-old Trpm6fl/+ ( Control ) and Trpm6Δ17/fl;Villin1-Cre ( Intestine KO ) littermate males .","( K ) ISH on paraffin sections of the colon using a Trpm6-specific probe ( n = 2 tissues per genotype ) .","( L , M )","Mg2+ levels in the serum ( L ) and bones ( M ) .","( N ) 24 hr urinary Mg2+ excretion rate .","Data are represented as mean±SEM .","***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined .","Histological analysis in ( F ) and ( K ) was performed with n = 3 animals per group resulting in similar observations . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01410 . 7554/eLife . 20914 . 015Figure 5—figure supplement 1 . Expression pattern of Trpm6 in the intestine . ISH on serial paraffin sections obtained from wildtype intestine using sense ( left ) and antisense ( right ) DIG-labelled probes for Trpm6 .","Boxes indicate the positions of the magnified images of the proximal and distal colon .","Representative images of n = 3 tissues are shown .","Arrows indicate Trpm6-positive cells in the colon .","Note: Trpm6 transcripts were not detectable in the duodenum and ileum and specifically present in the absorptive epithelium cells of the proximal and distal colon . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 015 It is generally assumed that the distal convoluted tubule ( DCT ) of the kidney is critical for whole-body Mg2+ balance ( de Baaij et al . , 2015 ) .","Accordingly , immunostaining of control kidney cryosections with a TRPM6-specific antibody labelled nephron segments resembling DCT ( Figure 5C ) .","TRPM6 was not detectable in the kidneys of Trpm6-deficient mice .","Surprisingly , mutant mice exhibited substantially reduced urinary Mg2+ excretion ( only 14% of control values , Figure 5D ) , whereas fecal Mg2+ loss was significantly increased ( 166% , Figure 5E ) , indicating that mice lacking TRPM6 develop Mg2+ deficiency primarily due to impaired intestinal Mg2+ uptake .","Therefore , we studied the expression pattern of Trpm6 in the intestine .","Because the TRPM6-specific antibody did not efficiently and specifically detect TRPM6 protein in the intestine , we resorted to ISH ( Figure 5—figure supplement 1 ) .","Trpm6 transcripts were not detectable in the small intestine , but Trpm6-specific signal was observed in absorptive epithelial cells of the colon ( Figure 5—figure supplement 1 ) .","The colon of mutant mice was not stained by a Trpm6-specific ISH probe ( Figure 5F ) , consistent with the notion that systemic Mg2+ deficit in Trpm6-deficient mice was primarily caused by a defect of Mg2+ uptake in the colon .","To directly assess the contribution of the kidney versus intestine to the Trpm6 null phenotype , we employed Ksp-Cre ( Shao et al . , 2002 ) and Villin1-Cre ( Madison et al . , 2002 ) transgenic mice to specifically ablate floxed Trpm6 alleles in renal and intestinal epithelial cells , respectively ( Table 1 ) .","Surprisingly , conditional Trpm6 inactivation in the kidney neither impacted serum Mg2+ concentration nor urinary Mg2+ excretion , and bone Mg2+ content was only slightly reduced ( Figure 5G–J ) .","In contrast , disruption of Trpm6 in the intestine resulted in hypomagnesemia , reduced bone Mg2+ content and lowered urinary Mg2+ excretion ( Figure 5K–N ) , indicating that wildtype kidneys are not able to compensate for the ablation of intestinal TRPM6 .","Hence , in contrast to current thinking , our findings support the new concept that Trpm6-dependent Mg2+ uptake in the intestine plays an indispensable role for systemic Mg2+ balance .","TRPM6 is invariably co-expressed with the TRPM7 channel and the question as to why TRPM6 function is non-redundant remains central to a mechanistic understanding of the Trpm6 null phenotype .","Because Trpm6 expression levels in the epithelial cells of the colon ( Figure 5—figure supplement","1 ) and the kidney ( Figure 5C ) are highly heterogeneous , we searched for an alternative native cell model to dissect the functional interplay of TRPM6 and TRPM7 .","Trophoblast stem ( TS ) cells are widely used to study the transport function of placental trophoblasts ( Tanaka et al . , 1998; Simmons and Cross , 2005 ) and , as shown before ( Figure 1 ) , this cell type is crucial for the fetal Trpm6 phenotype .","Therefore , we derived Trpm6+/+ and Trpm6-deficient ( Trpm6βgeo/βgeo ) TS cells from e3 . 5 blastocysts isolated from Trpm6βgeo/+ parents ( Figure 6—figure supplement 1A ) .","We also produced Trpm7+/+ and Trpm7-deficient ( Trpm7Δ17/Δ17 ) TS cells ( Figure 6—figure supplement 2A ) using Trpm7Δ17/+ mice ( Jin et al . , 2008 ) .","As expected , RT-PCR analysis revealed that wildtype TS cells expressed TRPM6 and TRPM7 ( Figure 6—figure supplement 1B , Figure 6—figure supplement 2B ) .","Trpm6βgeo/βgeo TS cells were maintained in culture for >40 passages .","Furthermore , analysis of DNA content showed that the proportion of polyploidy was similar in Trpm6+/+ and Trpm6βgeo/βgeo TS cells ( Figure 6—figure supplement 1C , D ) , suggesting that inactivation of Trpm6 did not affect the self-renewal of Trpm6βgeo/βgeo stem cells .","In contrast , Trpm7Δ17/Δ17 TS cells did not proliferate , unless the cell culture medium was supplemented with additional Mg2+ ( Figure 6—figure supplement 2C ) , supporting the concept that TRPM7 plays a pivotal role in cellular Mg2+ uptake that cannot be maintained by TRPM6 alone ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010 ) .","TRPM6 and TRPM7 have been suggested as molecular correlates of MgATP- and Mg2+-regulated cation currents responsible for the cellular uptake of divalent cations including Mg2+ ( Aarts et al . , 2003; Nadler et al . , 2001; Schmitz et al . , 2003; Chubanov et al . , 2004; Voets et al . , 2004; Zhang et al . , 2014 ) .","Patch-clamp analysis showed that Trpm6βgeo/βgeo TS cells display substantially reduced TRPM6/M7-like outward currents at +80 mV ( Figure 6A ) .","Due to permeation block by extracellular divalent cations ( Nadler et al . , 2001; Fleig and Chubanov , 2014 ) , inward currents at physiological membrane potentials were very small ( Figure 6A; p≤0 . 001 , t-test ) .","However , exposure of TS cells to a divalent cation free ( DVF ) solution resulted in large monovalent cation currents ( Figure 6B ) .","Under these conditions , mutant TS cells exhibited a comparable reduction of inward ( p≤0 . 01 , t-test ) as well as outward ( p≤0 . 05 , t-test ) monovalent currents ( Figure 6B ) . 10 . 7554/eLife . 20914 . 016Figure 6 . Characterization of TRPM6/M7-like currents in Trpm6- and Trpm7-deficient TS cells .","( A ) Left panel: Whole-cell currents measured at −80 mV and +80 mV over time in Trpm6+/+ ( n = 22 ) and Trpm6βgeo/βgeo ( n = 22 ) TS cells .","Middle panel: Representative current-voltage relationships obtained at 12 s and 400 s .","Right panel: Bar graphs of current amplitudes at +80 mV ( 400 s ) .","( B ) Measurements were performed in control ( n = 16 ) and Trpm6-deficient ( n = 14 ) TS cells analogous to ( A ) except that the external saline ( containing 2 mM Mg2+ and 1 mM Ca2+ ) was exchanged with divalent-free ( DVF ) solution ( black bar ) .","Right panel shows currents measured before ( filled bars ) and after application of DVF solution ( open bars ) at 400 s and 600 s , respectively .","( C , D )","Dose-dependent inhibition of currents ( +80 mV , 400 s ) by [MgATP]i and [Mg2+]i , respectively ( n = 4–18 cells per concentration ) .","( E , F )","Whole-cell currents of Trpm7+/+ ( n = 15 ) and Trpm7Δ17/Δ17 ( n = 10 ) TS cells studied similar to ( A , B ) .","Data are represented as mean±SEM .","***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) .","n – number of cells examined .","( G ) A suggested model for the molecular role of TRPM6 in epithelial cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01610 . 7554/eLife . 20914 . 017Figure 6—figure supplement 1 . Characterization of Trpm6-deficient TS cells .","( A ) Phase-contrast images of Trpm6+/+ ( +/+ ) and Trpm6βgeo/βgeo ( βgeo/βgeo ) TS cells cultured in a regular cell culture medium .","( B ) Expression of Trpm6 and Trpm7 in TS cells assessed by RT-PCR .","( C , D )","Assessment of self-renewal of Trpm6+/+ and Trpm6βgeo/βgeo cells .","( C ) Diploid ( 2N ) , tetraploid ( 4N ) , and polyploid ( 8–16N ) DNA content was analysed by flow cytometry of Trpm6+/+ and Trpm6βgeo/βgeo TS cells stained with propidium iodide ( PI ) .","( D ) Bar graphs showing DNA contents ( mean+/-SEM ) calculated from three independent experiments outlined in ( A ) .","n . s . – not significantly different ( Student’s t-test ) .","Note: 2N DNA content ( diploid cells in G1 ) , 4N ( diploid cells in G2 or tetraploid cells in G1 ) and 8–16N ( spontaneously differentiated polyploid trophoblasts ) were not significantly altered in Trpm6-deficient TS cells indicating that self-renewal of Trpm6βgeo/βgeo cells was not affected . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01710 . 7554/eLife . 20914 . 018Figure 6—figure supplement 2 . Examination of TS cells deficient in Trpm7 . ( A ) Phase-contrast images of Trpm7+/+ ( +/+ ) and Trpm7Δ17/Δ17 ( Δ17/Δ17 ) TS cells cultured in a cell culture medium supplemented by 10 mM Mg2+ .","( B ) RT-PCR analysis of Trpm7 and Trpm6 in TS cells and mouse intestine ( positive control ) .","( C ) Proliferation rate of Trpm7+/+ ( +/+ ) and Trpm7Δ17/Δ17 ( Δ17/Δ17 ) TS cells in standard and Mg2+ ( 10 mM ) supplemented medium .","The experiment was repeated three times .","Student’s t-test was applied for comparison of Trpm7+/+versus Trpm7Δ17/Δ17 datasets . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01810 . 7554/eLife . 20914 . 019Figure 6—figure supplement 3 . Evaluation of human haploid leukaemia ( HAP1 ) cells deficient in TRPM7 . ( A ) Phase-contrast images of parental ( WT ) and TRPM7-deficient ( KO ) HAP1 cells cultured in a cell culture medium supplemented with 10 mM Mg2+ .","( B ) Western-blot analysis of TRPM7 in WT and KO HAP1 cells .","( C ) Whole-cell currents in WT and KO HAP1 cells ( determined as described in Figure 6 ) .","Left panel: currents measured at −80 mV and +80 mV over time in WT ( n = 9 ) and KO ( n = 4 ) HAP1 cells .","Data are represented as mean±SEM .","Right panel: Representative current-voltage relationships obtained at 300 s .","( D ) Proliferation rate of WT and KO HAP1 cells either in standard or in Mg2+ ( 10 mM ) supplemented medium .","The experiment was repeated three times ( n = 3 ) .","Data are represented as mean±SEM .","Student’s t-test was applied for comparison of the growth rates of WT versus KO cells cultured in standard medium ( ***-p≤0 . 001 ) .","( E ) Determination of total Mg content in WT and KO HAP1 cells .","Dried cell pellets ( n = 4 for each genotype ) were obtained from WT and KO HAP1 cells cultured for 24 hr in standard medium and analysed by ICP-MS .","Elementary magnesium ( Mg ) content was normalized to sulfur ( S ) levels and represented as mean±SEM .","***-p≤0 . 001 ( Student’s t-test ) .","( F ) Assessment of total ATP levels in WT and KO HAP1 cells cultured for 24 hr in standard medium .","ATP-induced luminescent of luciferase ( CellTiter-Glo2 . 0 reagent ) was normalized to a number of viable cells ( Cell Counting Kit-8 ) .","The normalized luminescent signal in WT HAP1 cells was designated 100% .","The experiment was repeated six times ( n = 6 ) .","**-p≤0 . 01 ( Student’s t-test ) .","( G–H )","Routine respiration rate ( G ) and maximal respiration rate ( H ) analysed by Oxygraph-2k in WT and KO HAP1 cells cultured for 24 hr in standard cell culture medium ( n – number of independent experiments ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 019 Cytosolic levels of free Mg2+ ( [Mg2+]i ) and MgATP ( [MgATP]i ) have been suggested to exert a negative feedback mechanism on TRPM6/M7 channel activity ( Fleig and Chubanov , 2014 ) .","We observed ( Figure 6C ) that currents in Trpm6βgeo/βgeo TS cells were more susceptible to concentration-dependent inhibition by cytosolic MgATP ( p≤0 . 0001 , F-test ) .","The calculated IC50 value for Trpm6+/+ was 3 . 17 mM ( Hill ( h ) slope = −1 . 81 ) .","Currents in Trpm6βgeo/βgeo cells were inhibited by [MgATP]i with an IC50 value of 1 . 45 mM ( h = −1 . 95; p≤0 . 0015 , F-test ) .","These results suggest that physiological concentrations of [MgATP]i varying between 2–7 mM in mammalian cells ( Günther , 2006; Romani , 2011 ) will affect currents in Trpm6+/+ and Trpm6βgeo/βgeo cells differently .","In contrast , we observed no significant differences in [Mg2+]i concentration-response curves for Trpm6+/+ and Trpm6βgeo/βgeo currents ( p=0 . 62 , F-test ) ( Figure 6D ) .","The obtained IC50 value for Trpm6+/+ currents was 0 . 60 mM ( h = −1 . 14 ) and was not significantly altered in Trpm6βgeo/βgeo TS cells ( 0 . 72 mM , h = −1 . 18; p=0 . 22 , F-test ) , suggesting that physiological concentrations ( 0 . 3–1 mM , ( Günther , 2006; Romani , 2011 ) ) of [Mg2+]i will exert similar effects on ion currents in Trpm6+/+ and Trpm6βgeo/βgeo cells .","Next , we asked whether TRPM6 channel activity would be detectable in the absence of TRPM7 .","Remarkably , we observed that Trpm7Δ17/Δ17 TS cells completely lacked TRPM6/M7-like currents ( Figure 6E , F ) .","These findings cogently support our model ( Chubanov et al . , 2004 ) that native TRPM6 primarily functions in close cooperation with TRPM7 .","TRPM6 facilitates Mg2+ uptake by increasing the amplitude of TRPM7-like currents and relieving TRPM7 from the negative feedback by MgATP ( Figure 6G ) .","Finally , we studied whether Mg2+ deprivation may affect energy metabolism at the cellular level .","We chose the genetically tractable human haploid leukaemia cell line ( HAP1 cells ) ( Essletzbichler et al . , 2014; Blomen et al . , 2015; Wang et al . , 2015 ) as a new model system .","CRISPR/Cas9-mediated ablation of the TRPM7 protein in HAP1 cells ( Figure 6—figure supplement 3A , B ) completely abolished TRPM7-like currents ( Figure 6—figure supplement 3C ) .","When cultured in standard medium for 24 hr , TRPM7-deficient cells were characterized by a reduced total cellular Mg2+ content and a Mg2+-dependent proliferation defect ( Figure 6—figure supplement 3D , E ) .","In addition , TRPM7-deficient HAP1 cells had reduced intracellular ATP levels ( Figure 6—figure supplement 3F ) .","Finally , the respiration rate of TRPM7-deficient HAP1 cells was significantly lower when compared to control cells ( Figure 6—figure supplement 3G , H ) .","Our studies with Trpm6-deficient mice clearly demonstrated that a sustained disruption of Mg2+ homeostasis is detrimental for overall health and eventually reduces the lifespan of affected animals .","Conversely , we asked whether a Mg2+-enriched diet might exert a beneficial effect on the lifespan of wildtype mice .","Since there are no prior reports on lifespan extension of mice subjected to life-long dietary Mg2+ supplementation ( or any other mineral ) , we performed proof-of-principle experiments to investigate , if such an effect can be observed .","To this end , we used only the long-lived B6C3F1 hybrid mouse strain to avoid genotype-specific effects on disease susceptibility observed in longevity experiments with inbred strains ( Lipman et al . , 1999; Turturro et al . , 1999; Mitchell et al . , 2016 ) .","At this stage , we studied only females because of a larger number of early losses of males due to fighting ( Miller et al . , 2007 ) .","Finally , we studied animals only under pathogen-free conditions , since Mg2+ may elicit a protective effect via the immune system ( Brandao et al . , 2013; Chaigne-Delalande et al . , 2013 ) .","Consistent with published reports ( Turturro et al . , 1999 ) , the mean lifespan of B6C3F1 mice ( 886 days regarded as 100% ) was significantly extended ( 1100 days , 125% ) by dietary restriction ( DR ) ( Figure 7A , B , Table 2 ) .","Remarkably , supplementation with three Mg2+ salts ( Mg ( CH3COO ) 2 , Mg ( OH ) 2 and MgCl2 ) increased the mean lifespan of mice by approximately 10% ( 976 , 976 and 961 days , respectively ) .","The nutritional CaCl2 administration was without any significant effect ( 828 days ) .","In contrast to DR , animals supplemented with Mg2+ had a normal or even increased body weight ( Figure 7C ) , ruling out the possibility that high dietary Mg2+ affected the lifespan of mice due to reduced food intake .","Hence , opposite to Trpm6-dependent Mg2+ deprivation , dietary Mg2+ supplementation may have a beneficial effect on the lifespan of mice suggestive future large-scale longevity studies with varied conditions and species . 10 . 7554/eLife . 20914 . 020Figure 7 . Effects of whole-life Mg2+ dietary treatments on B6C3F1 mouse strain .","( A ) Mean survival ages of B6C3F1 mice maintained at a control diet ( Control , n = 335 ) , under dietary restriction ( DR , n = 60 ) , or supplemented by Mg ( CH3COO ) 2 ( MgAc , n = 15 ) , Mg ( OH ) 2 ( n = 15 ) , MgCl2 ( n = 15 ) and CaCl2 ( n = 15 ) in drinking water as outlined in Table 2 .","Pooled Mg shows results for all Mg2+ supplemented mice pooled within a common group ( n = 45 ) .","The obtained survival distributions were analysed by the MATLAB computing environment to calculate mean lifespans and corresponding P-values: ***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different .","Alternatively , survival data of control mice versus individually treated groups were assessed by log-rank test: ###-p≤0 . 001; ##-p≤0 . 01; #-p≤0 . 05; n . s . – not significantly different .","( B ) Kaplan-Meier survival distributions of B6C3F1 mice maintained on control diet ( Control ) , Mg2+ supplemented groups ( MgAc , MgCl2 , Mg ( OH ) 2 ) or mice under dietary restriction ( DR ) .","( C ) Body weights ( mean+/-SEM ) of control and nutritionally fortified mice studied in ( A ) .","***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( one-way ANOVA ) .","n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 02010 . 7554/eLife . 20914 . 021Table 2 . Dietary regimes used to maintain B6C3F1 mice . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 021Experimental groupChow 5 K54 Drinking water ( ad libitum ) Number of miceControlad libitumRegular water* 335Dietary restriction60% of ad libitumRegular water60Magnesium acetate supplementedad libitum1 g/l Mg ( CH3COO ) 2 . ( H2O ) 4 in regular water15Magnesium chloride supplementedad libitum1 g/l MgCl2 in regular water15Magnesium hydroxide supplementedad libitum14 mg/l Mg ( OH ) 2 in regular water† 15Calcium chloride supplementedad libitum0 . 8 g/l CaCl2 in regular water15*Regular water contained 0 . 44 mg/l Mg2+ .","†Mg ( OH ) 2 concentration was lowered to prevent overall alkalization of the diet ."],["Here , we present a new mechanistic model of the regulation of Mg2+ homeostasis during development and postnatal life of mice .","Against current thinking we show in vivo that TRPM6 is not required for embryonic development per se , but primarily operates in placenta and intestine to regulate Mg2+ levels by transcellular transport , while TRPM6 function in the kidney – commonly thought to be essential – is expendable for organismal Mg2+ balance .","We demonstrate that ablation of TRPM6 in adult mice leads to reduced lifespan , growth defects and profoundly impaired health of mutant mice due to defective energy metabolism .","We also show that dietary Mg2+ supplementation is not only sufficient to prevent all Trpm6 null pathologies , but Mg2+ is the only mineral known so far able to extend the lifespan of wildtype mice .","It has been reported that constitutive Trpm6 inactivation leads to embryonic lethality , resulting in the generally accepted tenet that Trpm6 is required for the development of the embryo proper ( Walder et al . , 2009 ) .","On the contrary , we provide genetic evidence that the embryonic mortality of Trpm6-deficient mice is caused by the loss of TRPM6 activity in placental SynT-I and yolk sac endoderm cells , and that Trpm6-deficient embryos are depleted of Mg2+ .","Altogether , we postulate that Trpm6 controls the maternal Mg2+ supply to the fetus , and that growth failure and death are secondary phenotypes induced by Mg2+ deprivation .","Differences in the role of TRPM6 for embryonic survival of humans and mice may be attributable to different morphologies of the placental exchange interfaces , fetal growth rate , litter size and dietary preferences ( Simmons and Cross , 2005 ) .","However , at present we cannot exclude that loss-of-function mutations in TRPM6 might be associated with prenatal mortality in humans as well .","Pioneering positional cloning studies ( Schlingmann et al . , 2002; Walder et al . , 2002 ) and follow-up case reports ( Schlingmann et al . , 2005; Konrad and Schlingmann , 2014 ) focused on hospitalized human infants selected by the criterion of deleterious Mg2+ deficiency .","Hence , additional TRPM6 phenotypes , such as infertility or embryonic death , might have been overlooked .","Accordingly , it has recently been shown that single nucleotide polymorphisms in human TRPM6 are associated with a neural tube closure defect , i . e . meningomyelocele ( Saraç et al . , 2016 ) .","Of note , dietary Mg2+ supplementation is common practice of pregnant women ( Durlach et al . , 2004 ) .","Our work provides first mechanistic insight as to how this essential mineral is delivered to the fetus .","There is growing evidence to suggest that Mg2+ deprivation is involved in the development of metabolic , immune , cardiovascular and neurological disorders ( de Baaij et al . , 2015 ) .","However , due to the lack of adequate mammalian genetic models , it is still unclear whether impaired Mg2+ homeostasis can be regarded as the cause or the consequence of the latter pathophysiological processes .","Therefore , we studied the impact of TRPM6 deletion on postnatal mice .","Trpm6-deficient mice displayed shorter lifespans , failure to thrive , low physical activity , kyphosis , lung emphysema , sarcopenia and degeneration of lymphoid organs .","In addition , Trpm6-deficient animals developed signs of catabolic metabolism such as lipodystrophy , increased insulin sensitivity and hypothermia , and showed suppression of the somatotropic axis accompanied by induction of xenobiotic detoxification gene networks in the liver .","Altogether , we noted that the complex phenotype of Trpm6-deficient mice mirrors many phenotypic hallmarks of mutant mouse strains that are generally considered genetic models of ‘accelerated aging’ in the scientific literature ( López-Otín et al . , 2013 ) .","Furthermore , our unbiased screen for metabolic pathways dysregulated in Trpm6-deficient mice revealed an error in energy metabolism reminiscent of humans with mutations in the gene coding for mitochondrial carnitine palmitoyltransferase II , the most common inherited disorder of lipid metabolism in adult humans ( Bonnefont et al . , 2004; Gempel et al . , 2002 ) .","Interestingly , an early biochemical study revealed that Mg2+ and MgATP could regulate activity of mitochondrial carnitine acyltransferase activity ( Saggerson , 1982 ) .","Accordingly , in proof-of-concept experiments , we demonstrated that Mg2+ is specifically required for the utilization of acyl carnitines ( AC ) as an energy source in liver mitochondria .","Hence , these results suggest that insufficient mitochondrial utilization of AC represents a plausible mechanism contributing to the pathologies developed by Trpm6-deficient mice .","The exact role of Mg2+ in AC metabolism as well as the molecular pathophysiology of carnitine palmitoyltransferase II deficiency is still not completely understood and , being beyond of the scope of the current manuscript , has to be addressed in future studies .","Notably , if Trpm6-deficient mice were fed with a high Mg2+ diet , they were viable and displayed normal physical activity , morphology of internal organs and tissue levels of AC , indicating that the phenotypes in Trpm6-deficient mice were triggered by Mg2+ deficiency .","Therefore , we studied in more detail how mutant mice develop organismal Mg2+ deprivation .","According to current thinking , the active transport of Mg2+ in the kidney , in particular in DCT , determines the final urinary Mg2+ concentration and is central to whole-body Mg2+ balance ( de Baaij et al . , 2015 ) .","Contrary to this model , we demonstrate that global inactivation of Trpm6 leads to Mg2+ deficiency due to a defect in intestinal Mg2+ uptake .","To interrogate the renal role of TRPM6 , we conditionally inactivated Trpm6 in the kidney and , surprisingly , did not observe any changes in serum Mg2+ levels .","In contrast , intestine-specific disruption of Trpm6 resulted in hypomagnesemia indicating that wildtype kidneys were not able to counterbalance the ablation of TRPM6 in the intestine .","Taken together , our findings lend strong support to the new concept that Trpm6-dependent Mg2+ uptake by the colon plays an indispensable role for systemic Mg2+ balance in mice .","However , we assume that renal TRPM6 activity may have an important role under conditions of insufficient dietary Mg2+ intake , for instance , during prolonged fasting periods .","Currently there are two suggested mechanistic models of hypomagnesemia in humans carrying mutations in TRPM6 .","A pioneering study of patients with congenital hypomagnesemia anticipated that Mg2+ malabsorption in the intestine plays a key role in organismal Mg2+ deprivation ( Friedman et al . , 1967; Milla et al . , 1979 ) .","More recently , it was reported that in affected humans renal Mg2+ loss plays a key role ( Schlingmann et al . , 2002; Walder et al . , 2002 ) .","The results obtained with Trpm6-deficient mice are well compatible with the former model .","However , we cannot exclude physiological differences between humans and mice , an important issue to address in future studies .","In conclusion , our findings support the idea that TRPM6 is a central gatekeeper of organismal Mg2+ balance in mammals and that this role cannot be compensated for by any other Mg2+ channel and transporter .","What is the molecular mechanism underlying insufficient Mg2+ intake in Trpm6-deficient mice ?","TRPM6 and its close homolog TRPM7 have been invoked as molecular correlates of cation currents responsible for Mg2+ entry into cells .","In order to circumvent the limitations and pitfalls imposed by overexpression of recombinant proteins , we employed TS cells to compare the roles of native TRPM6 and TRPM7 .","We found that stem cells express both TRPM6 and TRPM7 , thus mimicking the in vivo situation .","Inactivation of Trpm6 did not affect the self-renewal of TS cells .","In contrast , Trpm7-deficient cells were not able to proliferate unless the cell culture medium was supplemented with additional Mg2+ , supporting the idea that TRPM7 plays a non-redundant role in cellular Mg2+ uptake ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010 ) .","We further showed that wildtype TS cells exhibited [Mg2+]i- and [MgATP]i-sensitive divalent cation currents , and that inactivation of Trpm6 reduced current amplitudes .","In contrast , deletion of TRPM7 caused complete ablation of these currents .","Remarkably , ion currents in Trpm6 deficient TS cells still expressing TRPM7 were substantially more sensitive to intracellular MgATP when compared to TRPM6/M7 co-expressing cells .","Thus , in a native environment the presence of TRPM6 reduces the sensitivity of TRPM6/M7-like currents to inhibition by intracellular MgATP .","It has recently been reported that the TRPM6/M7 heteromer is completely insensitive to MgATP after heterologous expression in HEK 293 cells ( Zhang et al . , 2014 ) .","Thus , native TRPM6/M7 currents do not fully recapitulate the latter findings obtained in a heterologous expression system and further studies are required to clarify these discrepancies .","Nevertheless , our results are concordant with our model ( Chubanov et al . , 2004 ) that native TRPM6 functions primarily as a subunit of heteromeric TRPM6/M7 complexes by increasing current amplitudes and relieving TRPM7 from inhibition by [MgATP]i ( Figure 6G ) .","Such facilitated Mg2+ entry is most probably not required for any cell autonomous function , but is necessary for epithelial Mg2+ transport and the maintenance of serum Mg2+ levels .","Inadequate nutritional Mg2+ intake is commonplace in Western societies ( in up to 68% of the US population [King et al . , 2005] ) .","In addition , a growing percentage of the population is exposed to drug-induced forms of hypomagnesemia ( de Baaij et al . , 2015 ) .","Consequently , we asked whether wildtype mice would benefit from extra Mg2+ supplementation .","Our proof-of-concept experiments suggest that life-long Mg2+ supplementation may extend the lifespan of mice .","This idea is concordant with the recent observation that Mg2+acting alone or in conjunction with dietary calorie restriction counteracts lifespan-shortening effects of RNA-DNA hybrid ( R loop ) accumulation in yeasts and human HeLa cells ( Abraham et al . , 2016 ) .","Hence , large-scale investigations of nutritional Mg2+ adjustments and their impact on health- and lifespan of different species should be enlightening in this regard ."],["Experiments involving animals were done in accordance with the EU Animal Welfare Act and were approved by the local councils on animal care ( permit No 55 . 2-1-54-2532-134-13 from the Government of Oberbayern , Germany , and permit No 2347-15-2014 from the State Ministry of Brandenburg , Germany ) .","Microarray data were deposited in NCBI Gene Expression Omnibus ( GEO ) ( GSE70457 ) .","Liver tissues were collected from 12–13 week-old Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 3 ) and control ( Trpm6+/fl , n = 4 ) male littermates , snap-frozen in liquid nitrogen and stored at −80°C .","Total RNA was extracted using the GenElute mammalian total RNA purification kit ( Sigma-Aldrich ) .","Whole genome profiling was performed using a GeneChip Mouse Gene 1 . 0 ST Array ( Affymetrix ) at Source Bioscience ( Berlin , Germany ) .","Biotinylated single-stranded DNA was prepared according to the standard Affymetrix protocol ( Whole Transcript Expression arrays ) from 100 ng total RNA using the WT terminal labelling kit .","2 . 5 µg of fragmented and labelled ssDNA were hybridized for 16–18 hr at 45°C .","GeneChips were washed and stained in an Affymetrix Fluidics Station 450 .","GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 .","Processing of the array data , including quality assessment , background correction , normalization and summarization was performed with the Affymetrix Expression Console ( version 1 . 4 . 0 ) .","All statistical analyses were carried out with the statistical computing environment R ( version 3 . 1 . 2 , www . R-project . org ) .","Differential expression analysis was performed with the R package limma ( version 3 . 22 . 4 ) ( Ritchie et al . , 2015 ) .","p-values were adjusted for multiple testing with the Benjamini-Hochberg method for controlling the false discovery rate ( FDR ) .","A heatmap was generated for a group of 46 transcripts differentially expressed at a level of FDR p≤0 . 1 .","Analysis of the affected pathways and causal transcriptional regulators was performed by Ingenuity pathway analysis ( IPA ) environment ( www . ingenuity . com , RRID:SCR_008653 ) using a set of 2443 transcripts changed at p≤0 . 05 confidence level ( t-test ) .","Serum , liver and gastrocnemius muscle samples were collected from 8–10 week-old control ( Trpm6+/fl , n = 8 ) and Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 6 ) male littermates , flash frozen in liquid nitrogen and stored at −80°C .","Metabolomic analyses were performed at Biocrates Life Sciences AG ( Innsbruck , Austria ) .","Measurements comprised the quantification of 237 metabolites including 41 amino acids/biogenic amines , 40 acylcarnitines ( AC ) , 22 bile acids ( BA ) , 14 lysophosphatidylcholines ( LysPC ) , 77 phosphatidylcholines ( PC ) , 15 sphingomyelins ( SM ) , 17 eicosanoids/prostaglandins and 11 energy metabolism intermediates as outlined in Supplementary file 3 .","To extract metabolites , tissue samples were treated with corresponding extraction buffers and incubated in a chilled ultrasonic bath for 5 min .","Afterwards samples were centrifuged and the supernatant was used for analysis .","FIA- and LC-MS/MS measurement techniques were applied as described in detail previously ( Pena et al . , 2014 ) .","All statistical analyses have been applied by using the statistical computing environment R ( www . r-project . org ) .","Metabolite measurements containing more than 75% missing values or more than 75% of values below the limit of detection ( LOD ) across all samples per matrix were removed from analysis .","Measured concentrations of metabolites were log2-transformed for moderated statistical tests .","Measurements were scaled to µ = 0 mean and unit standard deviation for each biological matrix separately for heatmap visualization .","Changes in average metabolite levels between control and mutant individuals were tested using a linear model framework implemented in the R package Limma ( Smyth , 2004 ) .","Resulting P-values for moderated t-test were corrected for multiple testing by Benjamini and Hochberg approach .","A p-value threshold of 0 . 05 was considered as significant .","A Heatmap diagram for metabolites with significant changes was calculated with the R-package Heatmap using ward clustering and Euclidean distance measure and the R-package VennDiagram was applied to calculate a Venn diagram of significantly changed metabolites across sample matrices .","Mouse liver mitochondria were isolated by differential centrifugation from freshly prepared homogenates as previously described ( Springer et al . , 2002; Schulz et al . , 2015 ) .","Liver mitochondria were further purified by Percoll density gradient centrifugation ( Schulz et al . , 2013 ) .","Isolated mitochondria were subjected to quantification by the Bradford assay and kept on ice until use .","Assessment of the mitochondrial membrane potential Δψm ( MMP ) was followed by Rh123 fluorescence quenching ( Ex . 485 nm , Em . 528 nm ) in a 96-well plate reader ( BioTek ) and quantitatively evaluated by curve analysis as previously described ( Schulz et al . , 2013 ) ( set threshold slopes were ≥0 . 67 for start points and ≤0 . 67 for end points , respectively ) .","In order to exclude fluctuations at measurement start , slope calculations were started after 6 min measurement time with slope values ≤ 1 . 5 .","A kit-based assay ( ATP Bioluminescence Assay Kit , Roche ) was used to analyze the ATP content from cleared lysates after 30 min mitochondrial ATP synthesis at RT , initiated by the addition of 160 µM ADP and stopped at 95°C for 5 min .","For both analyses assay buffer composition was 0 . 2 M sucrose , 10 mM MOPS-Tris , 1 mM Pi and 10 µM EGTA .","Respiratory substrates were either succinate ( 25 mM ) /rotenone ( 2 µM ) , or DL-octanoylcarnitine ( 10 µM ) /malate ( 12 . 5 mM ) , or DL-palmitoylcarnitine ( 10 µM ) /malate ( 12 . 5 mM ) .","Buffers and solutions were essentially Mg²+-free , as determined by ICP-OES ( Ciros Vision , SPECTRO Analytical Instruments GmbH ) after wet ashing with 65% nitric acid ( Zischka et al . , 2011 ) .","EDTA , Mg2+ , Ca2+ , or Zn2+ was added at the concentrations indicated in the respective figures .","Mice were raised in a specific pathogen-free facility at Jackson Laboratory Sacramento ( Sacramento , CA , USA ) .","The long-lived B6C3F1 hybrid strain ( Lipman et al . , 1999; Turturro et al . , 1999 ) was used .","Specifically , F1 females were derived by crossing C57BL/6J females with C3H/HeJ males .","The produced B6C3F1 females were weaned at 3 weeks of age , and afterwards kept in individually ventilated polycarbonate cages ( Thoren Caging Systems ) on multigrain chow 5 K54 ( Purina ) containing 0 . 22% Mg2+ and drinking water containing 0 . 44 mg/l Mg2+ .","Five mice were housed per cage and cages were changed every two weeks .","The housing rooms were with artificial lighting and 12 hr light/dark cycle ( 6 am to 6 pm ) .","Temperature and relative humidity in animal rooms were 22 ± 4°C and 50 ± 15% , respectively .","At 5 months of age , mice were assigned to control , dietary restricted ( DR ) or supplemented cohorts as outlined in Table 2 .","For supplementation experiments , corresponding salts ( Sigma-Aldrich ) were diluted in drinking water that was administered ad libitum throughout the lifespan of all groups ( Table 2 ) .","To reduce bias , the supplemented and control groups were examined simultaneously and the study was performed as a blinded trial .","Mice were inspected daily .","Necropsies of randomly selected dead mice revealed that 58 of 88 control females ( 66% ) developed tumours .","8 of 11 females ( 73% ) from the three Mg2+ supplemented groups also had tumours suggesting that high dietary Mg2+ had no substantial effect on tumor rate at death .","Kaplan-Meier survival distributions were computed to illustrate survival times .","For statistical analysis , we used MATLAB computing environment and programming language ( MathWorks , www . de . mathworks . com ) , in particular its built-in fast convolution function that allows to find the distribution of the sum of independent random variables , given the distributions of individual variables .","To enable statistical comparisons between control and dosed groups , the distribution of the mean lifespan of 15 control mice was found as a convolution of the original distribution of lifespans of all control mice .","To calculate P-values of the mean lifespan from the dosed groups relative to the control group , we calculated the probability of the average of 15 control mice showing the same or more extreme ( away from control mean ) lifespan than the experimentally determined mean lifespan of the dosed mice .","A similar technique was used to find P-values for pooled Mg and dietary restriction groups relative to controls .","The MATLAB code is available from the Dryad Digital Repository ( Chubanov and Gudermann , 2016 ) .","In addition , the survival data of control mice versus individual treated groups were assessed by log-rank test using GraphPad Prism software .","P-values for both approaches are indicated in Figure 7A ."]],"string":"[\n [\n \"Mg2+ is the most abundant intracellular divalent cation and is essential for the regulation of a broad spectrum of metabolic and signalling pathways ( de Baaij et al . , 2015 ) .\",\n \"In addition , direct association with Mg2+ fosters the structural integrity of key metabolites ( such as ATP ) , proteins , lipid membranes and nucleic acids ( de Baaij et al . , 2015 ) implying that organismal Mg2+ deficiency , a surprisingly common condition in humans ( King et al . , 2005; Rosanoff et al . , 2012 ) , may be especially harmful during prenatal development and early postnatal life , when the production of and the demand for Mg2+-bound metabolites is particularly high .\",\n \"There is growing evidence to suggest that Mg2+ deprivation is accompanied by different types of metabolic , immune , cardiovascular and neurological disorders ( de Baaij et al . , 2015 ) .\",\n \"However , mainly due to the lack of adequate mammalian genetic models , it still remains unclear whether an imbalance in Mg2+ metabolism is merely associated with or can directly trigger the latter pathophysiological processes .\",\n \"Furthermore , it has recently been shown that cellular Mg2+ fluxes regulate the circadian rhythm and energy balance ( Feeney et al . , 2016 ) , CGRP-mediated osteogenic differentiation ( Zhang et al . , 2016 ) and synaptic plasticity ( Palacios-Prado et al . , 2014 ) , and that changes in the composition of brain interstitial Mg2+ concentrations participate in the control of the sleep-wake cycle ( Ding et al . , 2016 ) .\",\n \"The remarkable recent progress in our understanding of the critical role of Mg2+ in health and disease contrasts with the dearth of knowledge about the mechanisms governing cellular and organismal Mg2+ balance .\",\n \"Approximately 10 plasma membrane Mg2+ channels have been proposed ( Quamme , 2010 ) indicating a high degree of redundancy .\",\n \"However , quite some controversy surrounds the biological role of many of these proteins , and the question whether there is a central gatekeeper responsible for organismal Mg2+ balance has not yet been answered .\",\n \"The kinase-coupled ion channel TRPM7 has been proposed as a ubiquitous , indispensable cellular Mg2+ entry pathway ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010; Stritt et al . , 2016 ) .\",\n \"However , studies with Trpm7 gene-deficient mice failed to confirm a corresponding in vivo role of Trpm7 .\",\n \"Thus , constitutive inactivation of Trpm7 in mice entailed early embryonic lethality for as yet unknown reasons ( Jin et al . , 2008 ) .\",\n \"Furthermore , tissue-specific deletions of Trpm7 in mouse embryos affected morphogenesis of internal organs apparently in a Mg2+-independent manner ( Jin et al . , 2008 , 2012; Sah et al . , 2013 ) .\",\n \"More recently , it was suggested that the Mg2+ transporter MagT1 rather than TRPM7 might play a critical role for Mg2+ homeostasis in T lymphocytes ( Li et al . , 2011 ) and probably also in the whole embryo ( Zhou and Clapham , 2009 ) .\",\n \"Hence , the biological role of TRPM7 requires further clarification .\",\n \"In the present work , we focussed on the closest TRPM7 relative , TRPM6 , because loss-of-function mutations in TRPM6 cause hypomagnesemia ( low Mg2+ blood levels ) in human infants thought to mainly result from renal Mg2+ wasting ( Schlingmann et al . , 2002; Walder et al . , 2002; Voets et al . , 2004 ) .\",\n \"However , deletion of Trpm6 in mice has resulted in neural tube closure defects and embryonic death ( Walder et al . , 2009 ) indicating a direct role of TRPM6 in developmental processes and calling into question the simplistic view on the human TRPM6 phenotype .\",\n \"By integrating systematic phenotyping of Trpm6 gene-modified mice with biochemical analysis , gene expression , metabolomics , and cell biological approaches , we decipher the molecular and organismal roles of TRPM6 in prenatal development and postnatal survival .\"\n ],\n [\n \"To understand the role of Trpm6 in prenatal development , we determined the onset of embryonic lethality in Trpm6 null embryos and investigated the expression pattern of Trpm6 at this stage .\",\n \"Using a mouse strain carrying a gene-trap mutation in Trpm6 ( Trpm6βgeo ) ( Table 1 ) , we found that Trpm6βgeo/βgeo embryos were present at embryonic days ( e ) 8 . 5–10 . 5 ( Figure 1A ) .\",\n \"However , only a few mutants were found between e11 . 5–12 . 5 and no Trpm6βgeo/βgeo individuals were viable after e14 . 5 ( Figure 1A ) .\",\n \"Compared to e9 . 5 C-shaped Trpm6+/+ individuals , all Trpm6βgeo/βgeo embryos isolated had not turned ( S-shaped ) and were smaller indicating a developmental retardation after e8 . 5 ( Figure 1B ) .\",\n \"Consequently , we investigated the expression pattern of Trpm6 in e8 . 5 fetuses by in situ hybridization ( ISH ) and found that Trpm6 was specifically expressed in the visceral yolk sac endoderm and extraembryonic chorion ( Figure 1C ) and that Trpm6 was not detectable in the neural tube ( Figure 1—figure supplement 1 ) .\",\n \"Within the placental labyrinth a network of maternal sinusoids are intertwined with fetal blood capillaries , separated by two layers of transporting trophoblast cells , syncytiotrophoblasts I ( SynT-I ) and II ( SynT-II ) ( Simmons and Cross , 2005; Simmons et al . , 2008 ) .\",\n \"At e8 . 5 , morphogenesis of the labyrinth is in the initial stages and SynT-I/SynT-II cell layers are distinguishable ( Simmons and Cross , 2005; Simmons et al . , 2008 ) .\",\n \"We observed that Trpm6 expression was restricted to SynT-I cells ( Figure 1D ) .\",\n \"In the fully maturated labyrinth at e14 . 5 Trpm6 mRNA was detected in syncytiotrophoblasts as well ( Figure 1E ) . 10 . 7554/eLife . 20914 . 003Figure 1 . Assessment of Trpm6 function in extraembryonic tissues .\",\n \"( A ) Survival of Trpm6βgeo/βgeo embryos obtained from Trpm6βgeo/+ intercrosses .\",\n \"( B ) Representative images of e9 . 5 Trpm6+/+ ( +/+ , n = 13 ) and Trpm6βgeo/βgeo ( βgeo/βgeo , n = 5 ) embryos from dataset in ( A ) .\",\n \"Dashed lines underline C-shaped versus S-shaped morphology of Trpm6+/+ and Trpm6βgeo/βgeo embryos , respectively .\",\n \"( C ) ISH on serial paraffin sections obtained from wildtype n = 5 e8 . 5 fetus using antisense ( left ) and sense ( right ) probes for Trpm6 .\",\n \"Boxes indicate the positions of the magnified images of the chorion ( ch ) and yolk sac ( yc ) .\",\n \"Arrows indicate Trpm6-positive cells in the developing labyrinth ( chorion ) and the endoderm layer in the visceral yolk sac .\",\n \"( D ) ISH on serial paraffin sections of wildtype e8 . 5 placenta using DIG-labelled probes for Trpm6 ( left ) , SynA ( middle ) and Gcm1 ( right ) , respectively .\",\n \"Note: Trpm6 expression was restricted to cells positive for SynA , a marker of SynT-I , and absent in cells expressing Gcm1 , a marker of SynT-II .\",\n \"Representative images of n = 2 independent tissues are shown .\",\n \"( E ) ISH of WT e14 . 5 placenta with the antisense Trpm6 probe .\",\n \"The box indicates the position of the magnified image .\",\n \"The Trpm6 signal is restricted to the labyrinth ( lab ) and not detectable in the decidua ( dec ) and trophoblast giant cells ( GT ) .\",\n \"Representative images of n = 8 independent placentas are shown .\",\n \"( F ) Mg2+ levels in e9 . 5 Trpm6+/+ ( n = 4 ) and Trpm6βgeo/βgeo ( n = 3 ) embryos .\",\n \"Distal segments of the embryos were used for genotyping , and the remaining parts were analysed by ICP-MS .\",\n \"Elementary magnesium ( Mg ) contents were normalized to phosphorus ( P ) and sulfur ( S ) levels represented as mean±SEM .\",\n \"*-p≤0 . 05 ( Student’s t-test ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00310 . 7554/eLife . 20914 . 004Figure 1—figure supplement 1 . ISH on serial paraffin sections obtained from wildtype e8 . 5 fetus using antisense ( left ) and sense ( right ) probes for Trpm6 . Arrows indicate Trpm6-positive cells in the chorion ( blue arrows ) and the endoderm layer in the visceral yolk sac ( black arrows ) stained only by the antisense probe .\",\n \"Note: Trpm6 was not detectable in embryonic tissues including the neural tube ( red arrows ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00410 . 7554/eLife . 20914 . 005Table 1 . Postnatal survival of the mice with global and tissue-restricted deletions of Trpm6 . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 005Targeted tissueBreeding strategyExpected F1 outcome* Survival of the mutantConstitutive mutagenesisWhole fetus♂Trpm6βgeo/+ x ♀Trpm6βgeo/+ 25%Trpm6βgeo/βgeo 50% Trpm6βgeo/+ 25% Trpm6+/+ noWhole fetus♂Trpm6Δ17/+ x ♀Trpm6 Δ17/+ 25% Trpm6 Δ17/Δ17 50% Trpm6 Δ17/+ 25% Trpm6+/+ noWhole fetus♂Trpm6Δ17/+ x ♀Trpm6βgeo/+ 25% Trpm6βgeo/Δ17 25% Trpm6βgeo/+ 25% Trpm6Δ17/+ 25% Trpm6+/+ noConditional mutagenesis using Cre/LoxP systemEpiblast♂Trpm6Δ17/+;Sox2-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/Δ17;Sox2-Cre 25% Trpm6Δ17/fl 25% Trpm6Δ17/+;Sox2-Cre 25% Trpm6fl/+ yesIntestine♂Trpm6Δ17/+;Villin1-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/fl;Villin1-Cre† 25% Trpm6Δ17/fl 25% Trpm6fl/+;Villin1-Cre 25% Trpm6fl/+ yesKidney♂Trpm6Δ17/+;Ksp-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/fl;Ksp-Cre† 25% Trpm6Δ17/fl 25% Trpm6fl/+;Ksp-Cre 25% Trpm6fl/+ yes*Genotypes were determined using genomic DNA extracted from tail fragments .\",\n \"†Individuals were homozygous for Trpm6Δ17 allele in the targeted cells .\",\n \"Syncytiotrophoblasts and endoderm cells of the yolk sac exchange metabolites between the maternal and fetal blood ( Simmons and Cross , 2005 ) .\",\n \"To clarify whether TRPM6 is required for Mg2+ supply by extraembryonic tissues , we used inductively coupled plasma mass spectrometry ( ICP-MS ) and found that relative magnesium ( Mg2+ ) levels were reduced in the whole e9 . 5 Trpm6βgeo/βgeo embryos ( Figure 1F ) .\",\n \"Thus , Trpm6 is specifically expressed in the placental labyrinth and the yolk sac at the stage when the Mg2+ deficiency and growth delay of Trpm6-deficient embryos become apparent .\",\n \"To investigate whether TRPM6 activity in extraembryonic cells underlies the lethality of Trpm6 null embryos , we characterized a mouse strain with a ‘floxed’ ( Trpm6fl ) allele ( Table 1 ) .\",\n \"Cre-mediated excision engendered viable mice heterozygous for the constitutive deletion mutation in Trpm6 ( Trpm6Δ17/+ ) .\",\n \"However , we were unable to produce live Trpm6βgeo/Δ17 or Trpm6Δ17/Δ17 offspring , indicating that Trpm6∆17 is a true null mutation ( Table 1 ) .\",\n \"The paternally inherited Sox2-Cre transgene drives recombination only in epiblast cells , but not in extraembryonic tissues ( Hayashi et al . , 2003 ) .\",\n \"Notably , intercrosses of Trpm6Δ17/+;Sox2-Cre males and Trpm6fl/fl females resulted in viable Trpm6Δ17/Δ17 pups at the expected ratio ( Table 1 ) .\",\n \"Therefore , the embryonic mortality of Trpm6-deficient mice appears to be caused by the loss of TRPM6 in extraembryonic tissues .\",\n \"We next studied the impact of a global deletion of Trpm6 postnatally .\",\n \"Examination of Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre ) mice at weaning did not reveal conspicuous abnormalities .\",\n \"However , during the follow-up period , we observed the gradual development of pathologies .\",\n \"Thus , Trpm6-deficient mice had a lifespan of no longer than 16 weeks ( Figure 2A ) .\",\n \"Mutants were growth-delayed , and displayed a lighter fur colour and low night-time activity ( Figure 2B , C ) .\",\n \"Weight gain and lean body mass of Trpm6-deficient mice were reduced ( Figure 2D , E ) , as was the muscle fibre area of the gastrocnemius muscle of 12–13 week-old Trpm6-deficient mice indicative of sarcopenia ( Figure 2F ) .\",\n \"Mutant mice displayed kyphosis ( Figure 2G ) and completely lacked abdominal and subcutaneous fat depots ( Figure 2H , I ) indicative of catabolic metabolism .\",\n \"However , the total amount of faeces ( Figure 2—figure supplement 1A ) and the calorimetrically determined faecal energy content , as a measure of energy excretion ( Figure 2—figure supplement 1B ) , were not altered in Trpm6 mutants , ruling out insufficient food intake .\",\n \"Histological analysis of internal organs ( Figure\",\n \"3 ) showed that Trpm6-deficient mice developed lung emphysema and degeneration of lymphoid organs .\",\n \"Thus , the thymus of mutant mice was rudimentary and the cortex region was not distinguishable .\",\n \"In the spleen of Trpm6-deficient mice , the red pulp was substantially reduced .\",\n \"Hepatocytes of Trpm6-deficient mice were depleted of glycogen granules ( Figure 3 ) , corroborating catabolic metabolism .\",\n \"It has been suggested that low serum Mg2+ and TRPM6 function are associated with atherosclerosis in humans ( Maier , 2012; Tin et al . , 2015 ) .\",\n \"Therefore , we investigated whether such a phenotype would develop in our mouse model as well .\",\n \"However , examination of thoracal aorta showed no signs of atherosclerosis development in mutant mice ( Figure 2—figure supplement 2 ) . 10 . 7554/eLife . 20914 . 006Figure 2 . Pathophysiological changes displayed by Trpm6-deficient adult mice . Unless stated otherwise , 10–12 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were studied .\",\n \"( A–E )\",\n \"Mice were examined for survival rate ( A ) , overall physical appearance ( B ) , day/night activity of 8 week-old individuals ( C ) , growth rate ( D ) and lean mass ( E ) .\",\n \"( F ) Fibre size of the gastrocnemius muscle after hematoxylin-eosin staining .\",\n \"( G ) X-ray images of mice .\",\n \"The red arrow indicates the characteristic skeletal deformation ( kyphosis ) observed in Trpm6-deficient mice .\",\n \"( H ) Assessment of abdominal fat .\",\n \"Arrows indicate fat deposits observed only in control mice .\",\n \"( I ) H and E staining of paraffin skin sections .\",\n \"Arrows indicate a layer of fat cells present only in control mice .\",\n \"Histological analysis was performed with three animals per group resulting in similar observations .\",\n \"( J ) The levels of main elements in the serum of 8 week-old mice assessed by ICP-MS .\",\n \"( K ) The survival rate of mice maintained on high Mg2+ ( 0 . 75% ) and regular ( 0 . 22% ) chows .\",\n \"Data are represented as mean±SEM .\",\n \"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00610 . 7554/eLife . 20914 . 007Figure 2—figure supplement 1 . Examination of energy balance in Trpm6-deficient mice . Eight week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were evaluated for ( A ) chow intake as assessed by feces production , ( B ) energy content of feces studied by bomb calorimetry and ( C ) serum levels of β-hydroxybutyrate .\",\n \"Glucose ( D ) and insulin levels ( E ) in the serum of mice subjected to a glucose tolerance test .\",\n \"Note: mutant mice had lower peripheral glucose concentrations than controls despite of a similar amount of insulin released , thus reflecting increased insulin sensitivity .\",\n \"Data are represented as mean±SEM .\",\n \"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00710 . 7554/eLife . 20914 . 008Figure 2—figure supplement 2 . Evaluation of atherosclerosis development in Trpm6-deficient mice . An assessment of 8 week-old control ( Control , n = 3 ) and Trpm6-deficient ( KO , n = 3 ) with ApoE-/- mice ( as a positive control , n = 1 ) using en-face thoracal aorta preparation and Oil-Red O staining ( A ) and hematoxylin-eosin staining ( B ) , Mac2-staining ( green ) ( C ) of aortic arches .\",\n \"Representative images are shown .\",\n \"Arrows indicate atherosclerosis lesions observed only in ApoE-/- mice .\",\n \"( D ) Plasma cholesterol levels ( mean±SEM ) of control and Trpm6-deficient mice .\",\n \"n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00810 . 7554/eLife . 20914 . 009Figure 3 . Histology of internal organs of Trpm6-deficient mice . Hematoxylin-eosin staining of paraffin embedded tissue sections of 12–13 week-old control ( Control ) and Trpm6-deficient ( KO ) mice maintained either on regular ( 0 . 22% Mg2+ ) or Mg2+ supplemented ( 0 . 75% Mg2+ ) chows .\",\n \"Trpm6-deficient mice maintained on the regular diet showed marked airspace enlargement ( indicated by stars ) mimicking lung emphysema , distortion of splenic red pulp ( rp ) / white pulp ( wp ) microarchitecture , thymic atrophy , and reduction of intracellular glycogen in hepatocytes ( indicated by arrows ) .\",\n \"Histological analysis was performed with three animals per group resulting in similar observations . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 009 Next , we asked whether the phenotype of Trpm6-deficient mice is caused by Mg2+ deficiency .\",\n \"We employed ICP-MS to compare the concentrations of main elements in serum from controls and Trpm6-deficient littermates .\",\n \"We found that , similar to humans with mutations in the TRPM6 gene ( Schlingmann et al . , 2002; Walder et al . , 2002 ) , Trpm6-deficient mice developed hypomagnesemia ( Figure 2J ) .\",\n \"Serum Mg2+ levels of mutant mice were only 0 . 58 mM ( 36% of control value , 1 . 58 mM ) , whereas concentrations of other elements were not changed ( Figure 2J ) .\",\n \"A Mg2+-enriched diet is an efficient way to alleviate hypomagnesemia in humans lacking TRPM6 ( Schlingmann et al . , 2002; Walder et al . , 2002 ) .\",\n \"Therefore , we asked whether the phenotypes of Trpm6-deficient mice were caused by Mg2+ deprivation and could be rescued by dietary supplementation .\",\n \"To this end , we changed the regular chow ( 0 . 22% Mg2+ ) of 4 week-old mutant mice and control littermates for a Mg2+ enriched diet ( 0 . 75% Mg2+ ) .\",\n \"Notably , none of the Trpm6-deficient mice died during the following 12 weeks of Mg2+ supplementation ( Figure 2K ) .\",\n \"However , returning to regular chow resulted in 100% mortality of mutant mice within the following 13 weeks ( Figure 2K ) .\",\n \"Mg2+ supplemented mutants neither exhibited kyphosis nor lipodystrophy ( data not shown ) .\",\n \"Furthermore , the morphology of the lung , spleen , and thymus of Mg2+ supplemented mutants closely resembled that of control mice ( Figure 3 ) .\",\n \"We asked whether dietary Mg2+ supplementation of Trpm6βgeo/+ parents would benefit the survival of Trpm6-deficient offspring .\",\n \"However , similar to a previous study ( Walder et al . , 2009 ) , we found that this treatment was inefficient .\",\n \"Trpm6-deficient adult mice phenocopy salient pathologies reported for a set of mouse strains advocated as genetic models of ‘accelerated’ or ‘premature’ aging ( Kuro-o et al . , 1997; Trifunovic et al . , 2004; Kujoth et al . , 2005; Varela et al . , 2005; Mostoslavsky et al . , 2006; Niedernhofer et al . , 2006; van der Pluijm et al . , 2007; López-Otín et al . , 2013 ) .\",\n \"Similar to Trpm6-deficient mice , the latter mutants display short lifespan , growth failure , low physical activity , kyphosis , lung emphysema , sarcopenia , lipodystrophy and degeneration of lymphoid organs .\",\n \"A characteristic feature of these mouse strains is suppression of the somatotropic axis accompanied by induction of xenobiotic detoxification gene networks in the liver ( Niedernhofer et al . , 2006; van de Ven et al . , 2006; van der Pluijm et al . , 2007; Schumacher et al . , 2008; Garinis et al . , 2009; Mariño et al . , 2010 ) , interpreted as a defensive organismal response , slowing down growth and metabolism in favor of somatic preservation ( López-Otín et al . , 2013 ) .\",\n \"We asked whether Trpm6-deficient mice would also display such protective metabolic responses .\",\n \"In fact , we found that serum IGF1 concentrations were reduced in Trpm6-deficient mice as well ( Figure 4A ) .\",\n \"Mutant mice had a lower core body temperature ( Figure 4B ) and a profoundly reduced urinary content of major urinary proteins ( MUPs ) ( Figure 4C ) , two known features of supressed IGF1 signalling ( Mariño et al . , 2010; Bartke et al . , 2013 ) .\",\n \"Even though mutant mice showed signs of overall energy shortage , circulating levels of ketone bodies ( ß-hydroxybutyrate ) were not elevated ( Figure 2—figure supplement 1C ) .\",\n \"When subjected to an oral glucose tolerance test , mutant mice displayed lower peripheral glucose concentrations than controls despite of a similar amount of insulin released , thus reflecting increased insulin sensitivity ( Figure 2—figure supplement 1D , E ) , another hallmark of suppressed IGF1 signalling ( Bartke et al . , 2013 ) .\",\n \"Notably , body weight and IGF1 serum levels were indistinguishable in Mg2+ supplemented mutant and control mice suggesting that the variations observed in mice maintained on a regular diet were induced by Mg2+ deficiency ( Figure 2—figure supplement 1F , G ) . 10 . 7554/eLife . 20914 . 010Figure 4 . Assessment of metabolic profiles of Trpm6-deficient mice .\",\n \"( A–C ) 8 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were evaluated for ( A ) serum IGF1 , ( B ) body temperature , ( C ) urinary MUPs content in individual mice .\",\n \"Data are represented as mean±SEM .\",\n \"***-p≤0 . 001; *-p≤0 . 05 ( Student’s t-test ) ; n – number of mice examined .\",\n \"( D ) IPA analysis of genome-wide hepatic transcriptome profiling of Trpm6-deficient ( n = 3 ) vs control ( n = 4 ) littermates .\",\n \"The diagram shows the top 5 of IPA Canonical Pathways significantly changed in mutant mice ( Supplementary file 2 ) .\",\n \"Numbers of the commonly changed transcripts are indicated close to the lines connecting the pathways .\",\n \"( E ) Venn diagram for sets of metabolites significantly changed ( FDR p≤0 . 05 ) in serum , liver and gastrocnemius muscle Trpm6-deficient ( n = 6 ) vs control ( n = 8 ) littermates ( Supplementary file 3 ) .\",\n \"Commonly changed metabolites are listed in different colours as outlined in the Venn diagram .\",\n \"( F–J )\",\n \"Levels of AC C18:1 ( F–H ) and AC C18 ( I–J ) examined in the serum ( F , I ) , liver ( G ) and gastrocnemius muscle ( H , J ) of Trpm6-deficient and control mice .\",\n \"Data are represented as mean±SEM .\",\n \"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different to control group maintained on a regular Mg2+ diet ( one-way ANOVA ) ; n – number of mice examined .\",\n \"( K ) ATP production by mitochondria isolated from the liver of wildtype C57BL/6 mice with succinate , palmitoylcarnitine or octanoylcarnitine as energy sources .\",\n \"ATP levels were determined after 30 min incubation of untreated ( - ) or treated ( + ) mitochondria by EDTA with or without Mg2+ .\",\n \"Data are represented as mean±SEM of 4–5 independent isolations ( N ) .\",\n \"##-p≤0 . 01; #-p≤0 . 05 significantly different to the control group; ***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05 significantly different to the EDTA treated group ( Student’s t-test ) .\",\n \"n . s . – not significantly different . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01010 . 7554/eLife . 20914 . 011Figure 4—figure supplement 1 . Gene expression profiling of Trpm6-deficient and control mice . Heatmap diagram of differently expressed genes with FDR p≤0 . 1 in the liver of 12–13 week-old control ( Trpm6fl/+ , n = 4 ) vs Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 3 ) male littermates .\",\n \"n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01110 . 7554/eLife . 20914 . 012Figure 4—figure supplement 2 . Metabolomic profiling of Trpm6-deficient and control mice . Profiling of metabolites in the serum , liver and gastrocnemius muscle samples from 8–10 week-old control ( Trpm6fl/+ , n = 8 ) and KO ( Trpm6Δ17/Δ17;Sox2-Cre , n = 6 ) male littermates were studied for a panel of 237 metabolites outlined in Supplementary file 3 .\",\n \"The heatmap diagram shows concentration levels scaled to zero mean and unit standard deviation of significantly changed metabolites for control and KO genotypes in a colour-coded way .\",\n \"n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01210 . 7554/eLife . 20914 . 013Figure 4—figure supplement 3 . Assessment of the membrane potential ( Δψm ) in isolated mitochondria .\",\n \"( A ) Mitochondria isolated from the liver of wildtype C57BL/6 mice were incubated ( 30 min ) in the presence of succinate/rotenone , octanoylcarnitine/malate or palmitoylcarnitine/malate as energy source .\",\n \"The left panel shows representative measurements of Δψm using Rh123 probe .\",\n \"As a positive control , carbonyl cyanide 4- ( trifluoromethoxy ) phenylhydrazone ( FCCP ) was added at the end of recording to induce breakdown of Δψm .\",\n \"The right panel shows the calculated start- and endpoints of Δψm elicited by the application of 3 mM EDTA in the absence or presence of 1–5 mM Mg2+ for traces shown in left panel .\",\n \"Data are represented as mean±SD .\",\n \"N – independent mitochondria isolations; n – independent measurements .\",\n \"***-p≤0 . 001 , **-p≤0 . 01 , *-p≤0 . 05 significant to the untreated ( control ) group; ###-p≤0 . 001 , ##-p≤0 . 01 , #-p≤0 . 05 significant to 3 mM EDTA + 0 mM Mg2+ group; ‡‡‡-p≤0 . 001 , ‡‡-p≤0 . 01 , ‡-p≤0 . 05 significant to 3 mM EDTA + 1 mM Mg2+ group ( Student’s t-test ) .\",\n \"( B ) Representative measurements of ψm in the presence of 3 mM EDTA with/without 3–5 mM Ca2+ or Zn2+ performed analogously to ( A ) .\",\n \"Three independent experiments showed similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 013 Finally , we performed whole-genome profiling of the liver transcriptome ( Supplementary file 1 ) .\",\n \"Applying a cut-off value of p≤0 . 1 for the false discovery rate ( FDR ) , we identified 46 genes up- or down-regulated in the livers of Trpm6-deficient mice ( Figure 4—figure supplement 1 , Supplementary file 1 ) .\",\n \"The majority of affected transcripts code for solute carrier transporters , cytochrome P450 metabolising enzymes , glutathione S-transferases and proteins metabolizing steroids .\",\n \"As expected , Ingenuity Pathway Analysis ( IPA ) revealed that the inactivation of Trpm6 is associated with an induction of interconnected gene networks controlling toxicity responses and xenobiotic metabolism governed by nuclear receptors such as retinoid X receptors ( RXR ) , liver X receptor ( LXR ) and farnesoid X receptor ( FXR ) ( Figure 4D , Supplementary file 2 ) .\",\n \"Hence , Trpm6-deficient mice were characterized by suppression of the somatotropic axis and induction of xenobiotic detoxification responses .\",\n \"To gain mechanistic insight into the altered energy metabolism of mutant mice , we quantified serum , liver and skeletal muscle levels of 237 metabolites ( Supplementary file 3 , Figure 4—figure supplement 2 ) .\",\n \"Unexpectedly , alterations in Trpm6-deficient mice ( FDR p≤0 . 1 ) were restricted to only several metabolites representing mainly long-chain acylcarnitines ( AC ) , phosphatidylcholines ( PC ) , and sphingomyelins ( SM ) ( Figure 4E ) .\",\n \"These findings , as well as the results of gene array profiling ( Figure 4D ) suggest that sustained Mg2+ deficiency triggers a specific metabolic response rather than widespread unsystematic changes .\",\n \"In line with this idea , we noted that AC C18:1 and the related AC C18 were consistently increased in tissues of Trpm6-deficient mice ( Figure 4F–J ) , whereas carnitine levels were not changed ( Supplementary file 3 ) .\",\n \"In conjunction with lowered concentrations of glucose and unchanged ketogenesis ( Figure 2—figure supplement 1C ) , this constellation is a metabolic ‘signature’ of a frequent inherited human disorder characterized by inefficient β-oxidation of fatty acids due to mitochondrial carnitine palmitoyltransferase II deficiency ( Gempel et al . , 2002; Bonnefont et al . , 2004 ) .\",\n \"AC C18:1 and C18 levels were normalized in serum and tissues obtained from Mg2+ supplemented mutants ( Figure 4F–J ) , implying that metabolic changes of AC were caused by Mg2+ deficiency .\",\n \"Consequently , we asked whether Mg2+ would specifically affect mitochondrial ATP production ( Figure 4K ) and maintenance of the mitochondrial membrane potential ( MMP ) ( Figure 4—figure supplement 3A ) .\",\n \"To address this question , wildtype liver mitochondria were incubated in a buffer containing 3 mM EDTA with or without 1–5 mM Mg2+ .\",\n \"Such manipulations had no negative effect on mitochondrial respiration when succinate was offered as an energy source ( Figure 4K , Figure 4—figure supplement 3A ) .\",\n \"In contrast , mitochondria failed to utilize octanoylcarnitine or palmitylcarnitine for ATP production ( Figure 4K ) and to maintain MMP ( Figure 4—figure supplement 3A ) in the presence of EDTA .\",\n \"Importantly , ATP production and MMP could be fully rescued by the administration of 3–5 mM Mg2+ ( Figure 4K , Figure 4—figure supplement 3A ) , but not Zn2+ or Ca2+ ( Figure 4—figure supplement 3B ) .\",\n \"Thus , sustained Mg2+ deprivation impairs energy homeostasis resulting in a catabolic metabolism in Trpm6-deficient mice , at least partially due to insufficient mitochondrial utilization of AC .\",\n \"Next , we investigated the etiology of hypomagnesemia in Trpm6-deficient mice .\",\n \"~50% of body Mg2+ content is stored in bones , ~30% in muscle tissues and only ~1% in the serum ( de Baaij et al . , 2015 ) .\",\n \"Using ICP-MS we studied the Mg2+ content in bones ( right tibia ) and gastrocnemius muscle , and observed that Mg2+ levels in bones of Trpm6 null mice were only 24% of control values ( Figure 5A ) .\",\n \"Furthermore , the Mg2+ content of muscle was also significantly reduced in Trpm6-deficient mice ( Figure 5B ) .\",\n \"Hence , Trpm6-deficient mice develop a severe systemic Mg2+ deficit . 10 . 7554/eLife . 20914 . 014Figure 5 . Examining of Mg2+ balance in Trpm6-deficient adult mice .\",\n \"( A–F )\",\n \"Assessment of 8 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermate males .\",\n \"( A ) Mg2+ levels in bones and ( B ) gastrocnemius muscle assessed by ICP-MS .\",\n \"( C ) Immunostaining of kidney cryosections using a TRPM6-specific antibody .\",\n \"Representative images are shown ( n = 2 tissues per genotype ) .\",\n \"The blue square indicates the position of the confocal and differential interference contrast magnified images acquired from control tissue .\",\n \"Arrows indicate labelling of the apical surface of renal tubules .\",\n \"( D ) 24 hr urinary and ( E ) fecal Mg2+ excretion rates .\",\n \"( F ) ISH on paraffin sections obtained from the colon of control and Trpm6-deficient mice ( n = 2 tissues per genotype ) .\",\n \"( G–J )\",\n \"Examination of 6 month-old Trpm6fl/+ ( Control ) and Trpm6Δ17/fl;Ksp-Cre ( Kidney KO ) littermate males .\",\n \"( G ) Immunostaining of TRPM6 in kidney cryosections .\",\n \"Arrows indicate labelling of renal tubules .\",\n \"( H–I )\",\n \"Determination of Mg2+ in serum ( H ) and bones ( I ) .\",\n \"( J ) 24 hr urinary Mg2+ excretion rate .\",\n \"( K–N )\",\n \"Assessment of 6 month-old Trpm6fl/+ ( Control ) and Trpm6Δ17/fl;Villin1-Cre ( Intestine KO ) littermate males .\",\n \"( K ) ISH on paraffin sections of the colon using a Trpm6-specific probe ( n = 2 tissues per genotype ) .\",\n \"( L , M )\",\n \"Mg2+ levels in the serum ( L ) and bones ( M ) .\",\n \"( N ) 24 hr urinary Mg2+ excretion rate .\",\n \"Data are represented as mean±SEM .\",\n \"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined .\",\n \"Histological analysis in ( F ) and ( K ) was performed with n = 3 animals per group resulting in similar observations . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01410 . 7554/eLife . 20914 . 015Figure 5—figure supplement 1 . Expression pattern of Trpm6 in the intestine . ISH on serial paraffin sections obtained from wildtype intestine using sense ( left ) and antisense ( right ) DIG-labelled probes for Trpm6 .\",\n \"Boxes indicate the positions of the magnified images of the proximal and distal colon .\",\n \"Representative images of n = 3 tissues are shown .\",\n \"Arrows indicate Trpm6-positive cells in the colon .\",\n \"Note: Trpm6 transcripts were not detectable in the duodenum and ileum and specifically present in the absorptive epithelium cells of the proximal and distal colon . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 015 It is generally assumed that the distal convoluted tubule ( DCT ) of the kidney is critical for whole-body Mg2+ balance ( de Baaij et al . , 2015 ) .\",\n \"Accordingly , immunostaining of control kidney cryosections with a TRPM6-specific antibody labelled nephron segments resembling DCT ( Figure 5C ) .\",\n \"TRPM6 was not detectable in the kidneys of Trpm6-deficient mice .\",\n \"Surprisingly , mutant mice exhibited substantially reduced urinary Mg2+ excretion ( only 14% of control values , Figure 5D ) , whereas fecal Mg2+ loss was significantly increased ( 166% , Figure 5E ) , indicating that mice lacking TRPM6 develop Mg2+ deficiency primarily due to impaired intestinal Mg2+ uptake .\",\n \"Therefore , we studied the expression pattern of Trpm6 in the intestine .\",\n \"Because the TRPM6-specific antibody did not efficiently and specifically detect TRPM6 protein in the intestine , we resorted to ISH ( Figure 5—figure supplement 1 ) .\",\n \"Trpm6 transcripts were not detectable in the small intestine , but Trpm6-specific signal was observed in absorptive epithelial cells of the colon ( Figure 5—figure supplement 1 ) .\",\n \"The colon of mutant mice was not stained by a Trpm6-specific ISH probe ( Figure 5F ) , consistent with the notion that systemic Mg2+ deficit in Trpm6-deficient mice was primarily caused by a defect of Mg2+ uptake in the colon .\",\n \"To directly assess the contribution of the kidney versus intestine to the Trpm6 null phenotype , we employed Ksp-Cre ( Shao et al . , 2002 ) and Villin1-Cre ( Madison et al . , 2002 ) transgenic mice to specifically ablate floxed Trpm6 alleles in renal and intestinal epithelial cells , respectively ( Table 1 ) .\",\n \"Surprisingly , conditional Trpm6 inactivation in the kidney neither impacted serum Mg2+ concentration nor urinary Mg2+ excretion , and bone Mg2+ content was only slightly reduced ( Figure 5G–J ) .\",\n \"In contrast , disruption of Trpm6 in the intestine resulted in hypomagnesemia , reduced bone Mg2+ content and lowered urinary Mg2+ excretion ( Figure 5K–N ) , indicating that wildtype kidneys are not able to compensate for the ablation of intestinal TRPM6 .\",\n \"Hence , in contrast to current thinking , our findings support the new concept that Trpm6-dependent Mg2+ uptake in the intestine plays an indispensable role for systemic Mg2+ balance .\",\n \"TRPM6 is invariably co-expressed with the TRPM7 channel and the question as to why TRPM6 function is non-redundant remains central to a mechanistic understanding of the Trpm6 null phenotype .\",\n \"Because Trpm6 expression levels in the epithelial cells of the colon ( Figure 5—figure supplement\",\n \"1 ) and the kidney ( Figure 5C ) are highly heterogeneous , we searched for an alternative native cell model to dissect the functional interplay of TRPM6 and TRPM7 .\",\n \"Trophoblast stem ( TS ) cells are widely used to study the transport function of placental trophoblasts ( Tanaka et al . , 1998; Simmons and Cross , 2005 ) and , as shown before ( Figure 1 ) , this cell type is crucial for the fetal Trpm6 phenotype .\",\n \"Therefore , we derived Trpm6+/+ and Trpm6-deficient ( Trpm6βgeo/βgeo ) TS cells from e3 . 5 blastocysts isolated from Trpm6βgeo/+ parents ( Figure 6—figure supplement 1A ) .\",\n \"We also produced Trpm7+/+ and Trpm7-deficient ( Trpm7Δ17/Δ17 ) TS cells ( Figure 6—figure supplement 2A ) using Trpm7Δ17/+ mice ( Jin et al . , 2008 ) .\",\n \"As expected , RT-PCR analysis revealed that wildtype TS cells expressed TRPM6 and TRPM7 ( Figure 6—figure supplement 1B , Figure 6—figure supplement 2B ) .\",\n \"Trpm6βgeo/βgeo TS cells were maintained in culture for >40 passages .\",\n \"Furthermore , analysis of DNA content showed that the proportion of polyploidy was similar in Trpm6+/+ and Trpm6βgeo/βgeo TS cells ( Figure 6—figure supplement 1C , D ) , suggesting that inactivation of Trpm6 did not affect the self-renewal of Trpm6βgeo/βgeo stem cells .\",\n \"In contrast , Trpm7Δ17/Δ17 TS cells did not proliferate , unless the cell culture medium was supplemented with additional Mg2+ ( Figure 6—figure supplement 2C ) , supporting the concept that TRPM7 plays a pivotal role in cellular Mg2+ uptake that cannot be maintained by TRPM6 alone ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010 ) .\",\n \"TRPM6 and TRPM7 have been suggested as molecular correlates of MgATP- and Mg2+-regulated cation currents responsible for the cellular uptake of divalent cations including Mg2+ ( Aarts et al . , 2003; Nadler et al . , 2001; Schmitz et al . , 2003; Chubanov et al . , 2004; Voets et al . , 2004; Zhang et al . , 2014 ) .\",\n \"Patch-clamp analysis showed that Trpm6βgeo/βgeo TS cells display substantially reduced TRPM6/M7-like outward currents at +80 mV ( Figure 6A ) .\",\n \"Due to permeation block by extracellular divalent cations ( Nadler et al . , 2001; Fleig and Chubanov , 2014 ) , inward currents at physiological membrane potentials were very small ( Figure 6A; p≤0 . 001 , t-test ) .\",\n \"However , exposure of TS cells to a divalent cation free ( DVF ) solution resulted in large monovalent cation currents ( Figure 6B ) .\",\n \"Under these conditions , mutant TS cells exhibited a comparable reduction of inward ( p≤0 . 01 , t-test ) as well as outward ( p≤0 . 05 , t-test ) monovalent currents ( Figure 6B ) . 10 . 7554/eLife . 20914 . 016Figure 6 . Characterization of TRPM6/M7-like currents in Trpm6- and Trpm7-deficient TS cells .\",\n \"( A ) Left panel: Whole-cell currents measured at −80 mV and +80 mV over time in Trpm6+/+ ( n = 22 ) and Trpm6βgeo/βgeo ( n = 22 ) TS cells .\",\n \"Middle panel: Representative current-voltage relationships obtained at 12 s and 400 s .\",\n \"Right panel: Bar graphs of current amplitudes at +80 mV ( 400 s ) .\",\n \"( B ) Measurements were performed in control ( n = 16 ) and Trpm6-deficient ( n = 14 ) TS cells analogous to ( A ) except that the external saline ( containing 2 mM Mg2+ and 1 mM Ca2+ ) was exchanged with divalent-free ( DVF ) solution ( black bar ) .\",\n \"Right panel shows currents measured before ( filled bars ) and after application of DVF solution ( open bars ) at 400 s and 600 s , respectively .\",\n \"( C , D )\",\n \"Dose-dependent inhibition of currents ( +80 mV , 400 s ) by [MgATP]i and [Mg2+]i , respectively ( n = 4–18 cells per concentration ) .\",\n \"( E , F )\",\n \"Whole-cell currents of Trpm7+/+ ( n = 15 ) and Trpm7Δ17/Δ17 ( n = 10 ) TS cells studied similar to ( A , B ) .\",\n \"Data are represented as mean±SEM .\",\n \"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) .\",\n \"n – number of cells examined .\",\n \"( G ) A suggested model for the molecular role of TRPM6 in epithelial cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01610 . 7554/eLife . 20914 . 017Figure 6—figure supplement 1 . Characterization of Trpm6-deficient TS cells .\",\n \"( A ) Phase-contrast images of Trpm6+/+ ( +/+ ) and Trpm6βgeo/βgeo ( βgeo/βgeo ) TS cells cultured in a regular cell culture medium .\",\n \"( B ) Expression of Trpm6 and Trpm7 in TS cells assessed by RT-PCR .\",\n \"( C , D )\",\n \"Assessment of self-renewal of Trpm6+/+ and Trpm6βgeo/βgeo cells .\",\n \"( C ) Diploid ( 2N ) , tetraploid ( 4N ) , and polyploid ( 8–16N ) DNA content was analysed by flow cytometry of Trpm6+/+ and Trpm6βgeo/βgeo TS cells stained with propidium iodide ( PI ) .\",\n \"( D ) Bar graphs showing DNA contents ( mean+/-SEM ) calculated from three independent experiments outlined in ( A ) .\",\n \"n . s . – not significantly different ( Student’s t-test ) .\",\n \"Note: 2N DNA content ( diploid cells in G1 ) , 4N ( diploid cells in G2 or tetraploid cells in G1 ) and 8–16N ( spontaneously differentiated polyploid trophoblasts ) were not significantly altered in Trpm6-deficient TS cells indicating that self-renewal of Trpm6βgeo/βgeo cells was not affected . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01710 . 7554/eLife . 20914 . 018Figure 6—figure supplement 2 . Examination of TS cells deficient in Trpm7 . ( A ) Phase-contrast images of Trpm7+/+ ( +/+ ) and Trpm7Δ17/Δ17 ( Δ17/Δ17 ) TS cells cultured in a cell culture medium supplemented by 10 mM Mg2+ .\",\n \"( B ) RT-PCR analysis of Trpm7 and Trpm6 in TS cells and mouse intestine ( positive control ) .\",\n \"( C ) Proliferation rate of Trpm7+/+ ( +/+ ) and Trpm7Δ17/Δ17 ( Δ17/Δ17 ) TS cells in standard and Mg2+ ( 10 mM ) supplemented medium .\",\n \"The experiment was repeated three times .\",\n \"Student’s t-test was applied for comparison of Trpm7+/+versus Trpm7Δ17/Δ17 datasets . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01810 . 7554/eLife . 20914 . 019Figure 6—figure supplement 3 . Evaluation of human haploid leukaemia ( HAP1 ) cells deficient in TRPM7 . ( A ) Phase-contrast images of parental ( WT ) and TRPM7-deficient ( KO ) HAP1 cells cultured in a cell culture medium supplemented with 10 mM Mg2+ .\",\n \"( B ) Western-blot analysis of TRPM7 in WT and KO HAP1 cells .\",\n \"( C ) Whole-cell currents in WT and KO HAP1 cells ( determined as described in Figure 6 ) .\",\n \"Left panel: currents measured at −80 mV and +80 mV over time in WT ( n = 9 ) and KO ( n = 4 ) HAP1 cells .\",\n \"Data are represented as mean±SEM .\",\n \"Right panel: Representative current-voltage relationships obtained at 300 s .\",\n \"( D ) Proliferation rate of WT and KO HAP1 cells either in standard or in Mg2+ ( 10 mM ) supplemented medium .\",\n \"The experiment was repeated three times ( n = 3 ) .\",\n \"Data are represented as mean±SEM .\",\n \"Student’s t-test was applied for comparison of the growth rates of WT versus KO cells cultured in standard medium ( ***-p≤0 . 001 ) .\",\n \"( E ) Determination of total Mg content in WT and KO HAP1 cells .\",\n \"Dried cell pellets ( n = 4 for each genotype ) were obtained from WT and KO HAP1 cells cultured for 24 hr in standard medium and analysed by ICP-MS .\",\n \"Elementary magnesium ( Mg ) content was normalized to sulfur ( S ) levels and represented as mean±SEM .\",\n \"***-p≤0 . 001 ( Student’s t-test ) .\",\n \"( F ) Assessment of total ATP levels in WT and KO HAP1 cells cultured for 24 hr in standard medium .\",\n \"ATP-induced luminescent of luciferase ( CellTiter-Glo2 . 0 reagent ) was normalized to a number of viable cells ( Cell Counting Kit-8 ) .\",\n \"The normalized luminescent signal in WT HAP1 cells was designated 100% .\",\n \"The experiment was repeated six times ( n = 6 ) .\",\n \"**-p≤0 . 01 ( Student’s t-test ) .\",\n \"( G–H )\",\n \"Routine respiration rate ( G ) and maximal respiration rate ( H ) analysed by Oxygraph-2k in WT and KO HAP1 cells cultured for 24 hr in standard cell culture medium ( n – number of independent experiments ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 019 Cytosolic levels of free Mg2+ ( [Mg2+]i ) and MgATP ( [MgATP]i ) have been suggested to exert a negative feedback mechanism on TRPM6/M7 channel activity ( Fleig and Chubanov , 2014 ) .\",\n \"We observed ( Figure 6C ) that currents in Trpm6βgeo/βgeo TS cells were more susceptible to concentration-dependent inhibition by cytosolic MgATP ( p≤0 . 0001 , F-test ) .\",\n \"The calculated IC50 value for Trpm6+/+ was 3 . 17 mM ( Hill ( h ) slope = −1 . 81 ) .\",\n \"Currents in Trpm6βgeo/βgeo cells were inhibited by [MgATP]i with an IC50 value of 1 . 45 mM ( h = −1 . 95; p≤0 . 0015 , F-test ) .\",\n \"These results suggest that physiological concentrations of [MgATP]i varying between 2–7 mM in mammalian cells ( Günther , 2006; Romani , 2011 ) will affect currents in Trpm6+/+ and Trpm6βgeo/βgeo cells differently .\",\n \"In contrast , we observed no significant differences in [Mg2+]i concentration-response curves for Trpm6+/+ and Trpm6βgeo/βgeo currents ( p=0 . 62 , F-test ) ( Figure 6D ) .\",\n \"The obtained IC50 value for Trpm6+/+ currents was 0 . 60 mM ( h = −1 . 14 ) and was not significantly altered in Trpm6βgeo/βgeo TS cells ( 0 . 72 mM , h = −1 . 18; p=0 . 22 , F-test ) , suggesting that physiological concentrations ( 0 . 3–1 mM , ( Günther , 2006; Romani , 2011 ) ) of [Mg2+]i will exert similar effects on ion currents in Trpm6+/+ and Trpm6βgeo/βgeo cells .\",\n \"Next , we asked whether TRPM6 channel activity would be detectable in the absence of TRPM7 .\",\n \"Remarkably , we observed that Trpm7Δ17/Δ17 TS cells completely lacked TRPM6/M7-like currents ( Figure 6E , F ) .\",\n \"These findings cogently support our model ( Chubanov et al . , 2004 ) that native TRPM6 primarily functions in close cooperation with TRPM7 .\",\n \"TRPM6 facilitates Mg2+ uptake by increasing the amplitude of TRPM7-like currents and relieving TRPM7 from the negative feedback by MgATP ( Figure 6G ) .\",\n \"Finally , we studied whether Mg2+ deprivation may affect energy metabolism at the cellular level .\",\n \"We chose the genetically tractable human haploid leukaemia cell line ( HAP1 cells ) ( Essletzbichler et al . , 2014; Blomen et al . , 2015; Wang et al . , 2015 ) as a new model system .\",\n \"CRISPR/Cas9-mediated ablation of the TRPM7 protein in HAP1 cells ( Figure 6—figure supplement 3A , B ) completely abolished TRPM7-like currents ( Figure 6—figure supplement 3C ) .\",\n \"When cultured in standard medium for 24 hr , TRPM7-deficient cells were characterized by a reduced total cellular Mg2+ content and a Mg2+-dependent proliferation defect ( Figure 6—figure supplement 3D , E ) .\",\n \"In addition , TRPM7-deficient HAP1 cells had reduced intracellular ATP levels ( Figure 6—figure supplement 3F ) .\",\n \"Finally , the respiration rate of TRPM7-deficient HAP1 cells was significantly lower when compared to control cells ( Figure 6—figure supplement 3G , H ) .\",\n \"Our studies with Trpm6-deficient mice clearly demonstrated that a sustained disruption of Mg2+ homeostasis is detrimental for overall health and eventually reduces the lifespan of affected animals .\",\n \"Conversely , we asked whether a Mg2+-enriched diet might exert a beneficial effect on the lifespan of wildtype mice .\",\n \"Since there are no prior reports on lifespan extension of mice subjected to life-long dietary Mg2+ supplementation ( or any other mineral ) , we performed proof-of-principle experiments to investigate , if such an effect can be observed .\",\n \"To this end , we used only the long-lived B6C3F1 hybrid mouse strain to avoid genotype-specific effects on disease susceptibility observed in longevity experiments with inbred strains ( Lipman et al . , 1999; Turturro et al . , 1999; Mitchell et al . , 2016 ) .\",\n \"At this stage , we studied only females because of a larger number of early losses of males due to fighting ( Miller et al . , 2007 ) .\",\n \"Finally , we studied animals only under pathogen-free conditions , since Mg2+ may elicit a protective effect via the immune system ( Brandao et al . , 2013; Chaigne-Delalande et al . , 2013 ) .\",\n \"Consistent with published reports ( Turturro et al . , 1999 ) , the mean lifespan of B6C3F1 mice ( 886 days regarded as 100% ) was significantly extended ( 1100 days , 125% ) by dietary restriction ( DR ) ( Figure 7A , B , Table 2 ) .\",\n \"Remarkably , supplementation with three Mg2+ salts ( Mg ( CH3COO ) 2 , Mg ( OH ) 2 and MgCl2 ) increased the mean lifespan of mice by approximately 10% ( 976 , 976 and 961 days , respectively ) .\",\n \"The nutritional CaCl2 administration was without any significant effect ( 828 days ) .\",\n \"In contrast to DR , animals supplemented with Mg2+ had a normal or even increased body weight ( Figure 7C ) , ruling out the possibility that high dietary Mg2+ affected the lifespan of mice due to reduced food intake .\",\n \"Hence , opposite to Trpm6-dependent Mg2+ deprivation , dietary Mg2+ supplementation may have a beneficial effect on the lifespan of mice suggestive future large-scale longevity studies with varied conditions and species . 10 . 7554/eLife . 20914 . 020Figure 7 . Effects of whole-life Mg2+ dietary treatments on B6C3F1 mouse strain .\",\n \"( A ) Mean survival ages of B6C3F1 mice maintained at a control diet ( Control , n = 335 ) , under dietary restriction ( DR , n = 60 ) , or supplemented by Mg ( CH3COO ) 2 ( MgAc , n = 15 ) , Mg ( OH ) 2 ( n = 15 ) , MgCl2 ( n = 15 ) and CaCl2 ( n = 15 ) in drinking water as outlined in Table 2 .\",\n \"Pooled Mg shows results for all Mg2+ supplemented mice pooled within a common group ( n = 45 ) .\",\n \"The obtained survival distributions were analysed by the MATLAB computing environment to calculate mean lifespans and corresponding P-values: ***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different .\",\n \"Alternatively , survival data of control mice versus individually treated groups were assessed by log-rank test: ###-p≤0 . 001; ##-p≤0 . 01; #-p≤0 . 05; n . s . – not significantly different .\",\n \"( B ) Kaplan-Meier survival distributions of B6C3F1 mice maintained on control diet ( Control ) , Mg2+ supplemented groups ( MgAc , MgCl2 , Mg ( OH ) 2 ) or mice under dietary restriction ( DR ) .\",\n \"( C ) Body weights ( mean+/-SEM ) of control and nutritionally fortified mice studied in ( A ) .\",\n \"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( one-way ANOVA ) .\",\n \"n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 02010 . 7554/eLife . 20914 . 021Table 2 . Dietary regimes used to maintain B6C3F1 mice . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 021Experimental groupChow 5 K54 Drinking water ( ad libitum ) Number of miceControlad libitumRegular water* 335Dietary restriction60% of ad libitumRegular water60Magnesium acetate supplementedad libitum1 g/l Mg ( CH3COO ) 2 . ( H2O ) 4 in regular water15Magnesium chloride supplementedad libitum1 g/l MgCl2 in regular water15Magnesium hydroxide supplementedad libitum14 mg/l Mg ( OH ) 2 in regular water† 15Calcium chloride supplementedad libitum0 . 8 g/l CaCl2 in regular water15*Regular water contained 0 . 44 mg/l Mg2+ .\",\n \"†Mg ( OH ) 2 concentration was lowered to prevent overall alkalization of the diet .\"\n ],\n [\n \"Here , we present a new mechanistic model of the regulation of Mg2+ homeostasis during development and postnatal life of mice .\",\n \"Against current thinking we show in vivo that TRPM6 is not required for embryonic development per se , but primarily operates in placenta and intestine to regulate Mg2+ levels by transcellular transport , while TRPM6 function in the kidney – commonly thought to be essential – is expendable for organismal Mg2+ balance .\",\n \"We demonstrate that ablation of TRPM6 in adult mice leads to reduced lifespan , growth defects and profoundly impaired health of mutant mice due to defective energy metabolism .\",\n \"We also show that dietary Mg2+ supplementation is not only sufficient to prevent all Trpm6 null pathologies , but Mg2+ is the only mineral known so far able to extend the lifespan of wildtype mice .\",\n \"It has been reported that constitutive Trpm6 inactivation leads to embryonic lethality , resulting in the generally accepted tenet that Trpm6 is required for the development of the embryo proper ( Walder et al . , 2009 ) .\",\n \"On the contrary , we provide genetic evidence that the embryonic mortality of Trpm6-deficient mice is caused by the loss of TRPM6 activity in placental SynT-I and yolk sac endoderm cells , and that Trpm6-deficient embryos are depleted of Mg2+ .\",\n \"Altogether , we postulate that Trpm6 controls the maternal Mg2+ supply to the fetus , and that growth failure and death are secondary phenotypes induced by Mg2+ deprivation .\",\n \"Differences in the role of TRPM6 for embryonic survival of humans and mice may be attributable to different morphologies of the placental exchange interfaces , fetal growth rate , litter size and dietary preferences ( Simmons and Cross , 2005 ) .\",\n \"However , at present we cannot exclude that loss-of-function mutations in TRPM6 might be associated with prenatal mortality in humans as well .\",\n \"Pioneering positional cloning studies ( Schlingmann et al . , 2002; Walder et al . , 2002 ) and follow-up case reports ( Schlingmann et al . , 2005; Konrad and Schlingmann , 2014 ) focused on hospitalized human infants selected by the criterion of deleterious Mg2+ deficiency .\",\n \"Hence , additional TRPM6 phenotypes , such as infertility or embryonic death , might have been overlooked .\",\n \"Accordingly , it has recently been shown that single nucleotide polymorphisms in human TRPM6 are associated with a neural tube closure defect , i . e . meningomyelocele ( Saraç et al . , 2016 ) .\",\n \"Of note , dietary Mg2+ supplementation is common practice of pregnant women ( Durlach et al . , 2004 ) .\",\n \"Our work provides first mechanistic insight as to how this essential mineral is delivered to the fetus .\",\n \"There is growing evidence to suggest that Mg2+ deprivation is involved in the development of metabolic , immune , cardiovascular and neurological disorders ( de Baaij et al . , 2015 ) .\",\n \"However , due to the lack of adequate mammalian genetic models , it is still unclear whether impaired Mg2+ homeostasis can be regarded as the cause or the consequence of the latter pathophysiological processes .\",\n \"Therefore , we studied the impact of TRPM6 deletion on postnatal mice .\",\n \"Trpm6-deficient mice displayed shorter lifespans , failure to thrive , low physical activity , kyphosis , lung emphysema , sarcopenia and degeneration of lymphoid organs .\",\n \"In addition , Trpm6-deficient animals developed signs of catabolic metabolism such as lipodystrophy , increased insulin sensitivity and hypothermia , and showed suppression of the somatotropic axis accompanied by induction of xenobiotic detoxification gene networks in the liver .\",\n \"Altogether , we noted that the complex phenotype of Trpm6-deficient mice mirrors many phenotypic hallmarks of mutant mouse strains that are generally considered genetic models of ‘accelerated aging’ in the scientific literature ( López-Otín et al . , 2013 ) .\",\n \"Furthermore , our unbiased screen for metabolic pathways dysregulated in Trpm6-deficient mice revealed an error in energy metabolism reminiscent of humans with mutations in the gene coding for mitochondrial carnitine palmitoyltransferase II , the most common inherited disorder of lipid metabolism in adult humans ( Bonnefont et al . , 2004; Gempel et al . , 2002 ) .\",\n \"Interestingly , an early biochemical study revealed that Mg2+ and MgATP could regulate activity of mitochondrial carnitine acyltransferase activity ( Saggerson , 1982 ) .\",\n \"Accordingly , in proof-of-concept experiments , we demonstrated that Mg2+ is specifically required for the utilization of acyl carnitines ( AC ) as an energy source in liver mitochondria .\",\n \"Hence , these results suggest that insufficient mitochondrial utilization of AC represents a plausible mechanism contributing to the pathologies developed by Trpm6-deficient mice .\",\n \"The exact role of Mg2+ in AC metabolism as well as the molecular pathophysiology of carnitine palmitoyltransferase II deficiency is still not completely understood and , being beyond of the scope of the current manuscript , has to be addressed in future studies .\",\n \"Notably , if Trpm6-deficient mice were fed with a high Mg2+ diet , they were viable and displayed normal physical activity , morphology of internal organs and tissue levels of AC , indicating that the phenotypes in Trpm6-deficient mice were triggered by Mg2+ deficiency .\",\n \"Therefore , we studied in more detail how mutant mice develop organismal Mg2+ deprivation .\",\n \"According to current thinking , the active transport of Mg2+ in the kidney , in particular in DCT , determines the final urinary Mg2+ concentration and is central to whole-body Mg2+ balance ( de Baaij et al . , 2015 ) .\",\n \"Contrary to this model , we demonstrate that global inactivation of Trpm6 leads to Mg2+ deficiency due to a defect in intestinal Mg2+ uptake .\",\n \"To interrogate the renal role of TRPM6 , we conditionally inactivated Trpm6 in the kidney and , surprisingly , did not observe any changes in serum Mg2+ levels .\",\n \"In contrast , intestine-specific disruption of Trpm6 resulted in hypomagnesemia indicating that wildtype kidneys were not able to counterbalance the ablation of TRPM6 in the intestine .\",\n \"Taken together , our findings lend strong support to the new concept that Trpm6-dependent Mg2+ uptake by the colon plays an indispensable role for systemic Mg2+ balance in mice .\",\n \"However , we assume that renal TRPM6 activity may have an important role under conditions of insufficient dietary Mg2+ intake , for instance , during prolonged fasting periods .\",\n \"Currently there are two suggested mechanistic models of hypomagnesemia in humans carrying mutations in TRPM6 .\",\n \"A pioneering study of patients with congenital hypomagnesemia anticipated that Mg2+ malabsorption in the intestine plays a key role in organismal Mg2+ deprivation ( Friedman et al . , 1967; Milla et al . , 1979 ) .\",\n \"More recently , it was reported that in affected humans renal Mg2+ loss plays a key role ( Schlingmann et al . , 2002; Walder et al . , 2002 ) .\",\n \"The results obtained with Trpm6-deficient mice are well compatible with the former model .\",\n \"However , we cannot exclude physiological differences between humans and mice , an important issue to address in future studies .\",\n \"In conclusion , our findings support the idea that TRPM6 is a central gatekeeper of organismal Mg2+ balance in mammals and that this role cannot be compensated for by any other Mg2+ channel and transporter .\",\n \"What is the molecular mechanism underlying insufficient Mg2+ intake in Trpm6-deficient mice ?\",\n \"TRPM6 and its close homolog TRPM7 have been invoked as molecular correlates of cation currents responsible for Mg2+ entry into cells .\",\n \"In order to circumvent the limitations and pitfalls imposed by overexpression of recombinant proteins , we employed TS cells to compare the roles of native TRPM6 and TRPM7 .\",\n \"We found that stem cells express both TRPM6 and TRPM7 , thus mimicking the in vivo situation .\",\n \"Inactivation of Trpm6 did not affect the self-renewal of TS cells .\",\n \"In contrast , Trpm7-deficient cells were not able to proliferate unless the cell culture medium was supplemented with additional Mg2+ , supporting the idea that TRPM7 plays a non-redundant role in cellular Mg2+ uptake ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010 ) .\",\n \"We further showed that wildtype TS cells exhibited [Mg2+]i- and [MgATP]i-sensitive divalent cation currents , and that inactivation of Trpm6 reduced current amplitudes .\",\n \"In contrast , deletion of TRPM7 caused complete ablation of these currents .\",\n \"Remarkably , ion currents in Trpm6 deficient TS cells still expressing TRPM7 were substantially more sensitive to intracellular MgATP when compared to TRPM6/M7 co-expressing cells .\",\n \"Thus , in a native environment the presence of TRPM6 reduces the sensitivity of TRPM6/M7-like currents to inhibition by intracellular MgATP .\",\n \"It has recently been reported that the TRPM6/M7 heteromer is completely insensitive to MgATP after heterologous expression in HEK 293 cells ( Zhang et al . , 2014 ) .\",\n \"Thus , native TRPM6/M7 currents do not fully recapitulate the latter findings obtained in a heterologous expression system and further studies are required to clarify these discrepancies .\",\n \"Nevertheless , our results are concordant with our model ( Chubanov et al . , 2004 ) that native TRPM6 functions primarily as a subunit of heteromeric TRPM6/M7 complexes by increasing current amplitudes and relieving TRPM7 from inhibition by [MgATP]i ( Figure 6G ) .\",\n \"Such facilitated Mg2+ entry is most probably not required for any cell autonomous function , but is necessary for epithelial Mg2+ transport and the maintenance of serum Mg2+ levels .\",\n \"Inadequate nutritional Mg2+ intake is commonplace in Western societies ( in up to 68% of the US population [King et al . , 2005] ) .\",\n \"In addition , a growing percentage of the population is exposed to drug-induced forms of hypomagnesemia ( de Baaij et al . , 2015 ) .\",\n \"Consequently , we asked whether wildtype mice would benefit from extra Mg2+ supplementation .\",\n \"Our proof-of-concept experiments suggest that life-long Mg2+ supplementation may extend the lifespan of mice .\",\n \"This idea is concordant with the recent observation that Mg2+acting alone or in conjunction with dietary calorie restriction counteracts lifespan-shortening effects of RNA-DNA hybrid ( R loop ) accumulation in yeasts and human HeLa cells ( Abraham et al . , 2016 ) .\",\n \"Hence , large-scale investigations of nutritional Mg2+ adjustments and their impact on health- and lifespan of different species should be enlightening in this regard .\"\n ],\n [\n \"Experiments involving animals were done in accordance with the EU Animal Welfare Act and were approved by the local councils on animal care ( permit No 55 . 2-1-54-2532-134-13 from the Government of Oberbayern , Germany , and permit No 2347-15-2014 from the State Ministry of Brandenburg , Germany ) .\",\n \"Microarray data were deposited in NCBI Gene Expression Omnibus ( GEO ) ( GSE70457 ) .\",\n \"Liver tissues were collected from 12–13 week-old Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 3 ) and control ( Trpm6+/fl , n = 4 ) male littermates , snap-frozen in liquid nitrogen and stored at −80°C .\",\n \"Total RNA was extracted using the GenElute mammalian total RNA purification kit ( Sigma-Aldrich ) .\",\n \"Whole genome profiling was performed using a GeneChip Mouse Gene 1 . 0 ST Array ( Affymetrix ) at Source Bioscience ( Berlin , Germany ) .\",\n \"Biotinylated single-stranded DNA was prepared according to the standard Affymetrix protocol ( Whole Transcript Expression arrays ) from 100 ng total RNA using the WT terminal labelling kit .\",\n \"2 . 5 µg of fragmented and labelled ssDNA were hybridized for 16–18 hr at 45°C .\",\n \"GeneChips were washed and stained in an Affymetrix Fluidics Station 450 .\",\n \"GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 .\",\n \"Processing of the array data , including quality assessment , background correction , normalization and summarization was performed with the Affymetrix Expression Console ( version 1 . 4 . 0 ) .\",\n \"All statistical analyses were carried out with the statistical computing environment R ( version 3 . 1 . 2 , www . R-project . org ) .\",\n \"Differential expression analysis was performed with the R package limma ( version 3 . 22 . 4 ) ( Ritchie et al . , 2015 ) .\",\n \"p-values were adjusted for multiple testing with the Benjamini-Hochberg method for controlling the false discovery rate ( FDR ) .\",\n \"A heatmap was generated for a group of 46 transcripts differentially expressed at a level of FDR p≤0 . 1 .\",\n \"Analysis of the affected pathways and causal transcriptional regulators was performed by Ingenuity pathway analysis ( IPA ) environment ( www . ingenuity . com , RRID:SCR_008653 ) using a set of 2443 transcripts changed at p≤0 . 05 confidence level ( t-test ) .\",\n \"Serum , liver and gastrocnemius muscle samples were collected from 8–10 week-old control ( Trpm6+/fl , n = 8 ) and Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 6 ) male littermates , flash frozen in liquid nitrogen and stored at −80°C .\",\n \"Metabolomic analyses were performed at Biocrates Life Sciences AG ( Innsbruck , Austria ) .\",\n \"Measurements comprised the quantification of 237 metabolites including 41 amino acids/biogenic amines , 40 acylcarnitines ( AC ) , 22 bile acids ( BA ) , 14 lysophosphatidylcholines ( LysPC ) , 77 phosphatidylcholines ( PC ) , 15 sphingomyelins ( SM ) , 17 eicosanoids/prostaglandins and 11 energy metabolism intermediates as outlined in Supplementary file 3 .\",\n \"To extract metabolites , tissue samples were treated with corresponding extraction buffers and incubated in a chilled ultrasonic bath for 5 min .\",\n \"Afterwards samples were centrifuged and the supernatant was used for analysis .\",\n \"FIA- and LC-MS/MS measurement techniques were applied as described in detail previously ( Pena et al . , 2014 ) .\",\n \"All statistical analyses have been applied by using the statistical computing environment R ( www . r-project . org ) .\",\n \"Metabolite measurements containing more than 75% missing values or more than 75% of values below the limit of detection ( LOD ) across all samples per matrix were removed from analysis .\",\n \"Measured concentrations of metabolites were log2-transformed for moderated statistical tests .\",\n \"Measurements were scaled to µ = 0 mean and unit standard deviation for each biological matrix separately for heatmap visualization .\",\n \"Changes in average metabolite levels between control and mutant individuals were tested using a linear model framework implemented in the R package Limma ( Smyth , 2004 ) .\",\n \"Resulting P-values for moderated t-test were corrected for multiple testing by Benjamini and Hochberg approach .\",\n \"A p-value threshold of 0 . 05 was considered as significant .\",\n \"A Heatmap diagram for metabolites with significant changes was calculated with the R-package Heatmap using ward clustering and Euclidean distance measure and the R-package VennDiagram was applied to calculate a Venn diagram of significantly changed metabolites across sample matrices .\",\n \"Mouse liver mitochondria were isolated by differential centrifugation from freshly prepared homogenates as previously described ( Springer et al . , 2002; Schulz et al . , 2015 ) .\",\n \"Liver mitochondria were further purified by Percoll density gradient centrifugation ( Schulz et al . , 2013 ) .\",\n \"Isolated mitochondria were subjected to quantification by the Bradford assay and kept on ice until use .\",\n \"Assessment of the mitochondrial membrane potential Δψm ( MMP ) was followed by Rh123 fluorescence quenching ( Ex . 485 nm , Em . 528 nm ) in a 96-well plate reader ( BioTek ) and quantitatively evaluated by curve analysis as previously described ( Schulz et al . , 2013 ) ( set threshold slopes were ≥0 . 67 for start points and ≤0 . 67 for end points , respectively ) .\",\n \"In order to exclude fluctuations at measurement start , slope calculations were started after 6 min measurement time with slope values ≤ 1 . 5 .\",\n \"A kit-based assay ( ATP Bioluminescence Assay Kit , Roche ) was used to analyze the ATP content from cleared lysates after 30 min mitochondrial ATP synthesis at RT , initiated by the addition of 160 µM ADP and stopped at 95°C for 5 min .\",\n \"For both analyses assay buffer composition was 0 . 2 M sucrose , 10 mM MOPS-Tris , 1 mM Pi and 10 µM EGTA .\",\n \"Respiratory substrates were either succinate ( 25 mM ) /rotenone ( 2 µM ) , or DL-octanoylcarnitine ( 10 µM ) /malate ( 12 . 5 mM ) , or DL-palmitoylcarnitine ( 10 µM ) /malate ( 12 . 5 mM ) .\",\n \"Buffers and solutions were essentially Mg²+-free , as determined by ICP-OES ( Ciros Vision , SPECTRO Analytical Instruments GmbH ) after wet ashing with 65% nitric acid ( Zischka et al . , 2011 ) .\",\n \"EDTA , Mg2+ , Ca2+ , or Zn2+ was added at the concentrations indicated in the respective figures .\",\n \"Mice were raised in a specific pathogen-free facility at Jackson Laboratory Sacramento ( Sacramento , CA , USA ) .\",\n \"The long-lived B6C3F1 hybrid strain ( Lipman et al . , 1999; Turturro et al . , 1999 ) was used .\",\n \"Specifically , F1 females were derived by crossing C57BL/6J females with C3H/HeJ males .\",\n \"The produced B6C3F1 females were weaned at 3 weeks of age , and afterwards kept in individually ventilated polycarbonate cages ( Thoren Caging Systems ) on multigrain chow 5 K54 ( Purina ) containing 0 . 22% Mg2+ and drinking water containing 0 . 44 mg/l Mg2+ .\",\n \"Five mice were housed per cage and cages were changed every two weeks .\",\n \"The housing rooms were with artificial lighting and 12 hr light/dark cycle ( 6 am to 6 pm ) .\",\n \"Temperature and relative humidity in animal rooms were 22 ± 4°C and 50 ± 15% , respectively .\",\n \"At 5 months of age , mice were assigned to control , dietary restricted ( DR ) or supplemented cohorts as outlined in Table 2 .\",\n \"For supplementation experiments , corresponding salts ( Sigma-Aldrich ) were diluted in drinking water that was administered ad libitum throughout the lifespan of all groups ( Table 2 ) .\",\n \"To reduce bias , the supplemented and control groups were examined simultaneously and the study was performed as a blinded trial .\",\n \"Mice were inspected daily .\",\n \"Necropsies of randomly selected dead mice revealed that 58 of 88 control females ( 66% ) developed tumours .\",\n \"8 of 11 females ( 73% ) from the three Mg2+ supplemented groups also had tumours suggesting that high dietary Mg2+ had no substantial effect on tumor rate at death .\",\n \"Kaplan-Meier survival distributions were computed to illustrate survival times .\",\n \"For statistical analysis , we used MATLAB computing environment and programming language ( MathWorks , www . de . mathworks . com ) , in particular its built-in fast convolution function that allows to find the distribution of the sum of independent random variables , given the distributions of individual variables .\",\n \"To enable statistical comparisons between control and dosed groups , the distribution of the mean lifespan of 15 control mice was found as a convolution of the original distribution of lifespans of all control mice .\",\n \"To calculate P-values of the mean lifespan from the dosed groups relative to the control group , we calculated the probability of the average of 15 control mice showing the same or more extreme ( away from control mean ) lifespan than the experimentally determined mean lifespan of the dosed mice .\",\n \"A similar technique was used to find P-values for pooled Mg and dietary restriction groups relative to controls .\",\n \"The MATLAB code is available from the Dryad Digital Repository ( Chubanov and Gudermann , 2016 ) .\",\n \"In addition , the survival data of control mice versus individual treated groups were assessed by log-rank test using GraphPad Prism software .\",\n \"P-values for both approaches are indicated in Figure 7A .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Mg2+ regulates many physiological processes and signalling pathways .","However , little is known about the mechanisms underlying the organismal balance of Mg2+ .","Capitalizing on a set of newly generated mouse models , we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6 .","We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development .","In adult mice , TRPM6 is required in the intestine to maintain organismal Mg2+ balance , but is dispensable in the kidney .","Trpm6 inactivation in adult mice leads to a shortened lifespan , growth deficit and metabolic alterations indicative of impaired energy balance .","Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice , but also may extend the lifespan of wildtype mice .","Hence , maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood ."],"string":"[\n \"Mg2+ regulates many physiological processes and signalling pathways .\",\n \"However , little is known about the mechanisms underlying the organismal balance of Mg2+ .\",\n \"Capitalizing on a set of newly generated mouse models , we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6 .\",\n \"We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development .\",\n \"In adult mice , TRPM6 is required in the intestine to maintain organismal Mg2+ balance , but is dispensable in the kidney .\",\n \"Trpm6 inactivation in adult mice leads to a shortened lifespan , growth deficit and metabolic alterations indicative of impaired energy balance .\",\n \"Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice , but also may extend the lifespan of wildtype mice .\",\n \"Hence , maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood .\"\n]"},"summary":{"kind":"list like","value":["A balanced diet contains a variety of minerals such as magnesium ions , which are required for many chemical reactions in our body .","A shortage of magnesium ions is linked to many diseases and is thought to be especially harmful to babies in the womb and shortly after birth .","Magnesium ion deficiency is widespread in human populations and in the US is thought to affect up to 68% of people .","Despite its prominent role in human health , our understanding of how the body maintains the right balance of magnesium ions remains extremely vague .","Magnesium ions can enter and leave a cell by passing through specific types of proteins that form channels in the membrane surrounding the cell .","There are thought to be around ten types of these magnesium ion channels in human cells , but we do not know what roles any of them perform in the body .","One such channel called TRPM6 may be particularly important because mutations in the gene that encodes this channel can cause magnesium ion deficiency in human infants .","However , the loss of TRPM6 in mice disrupts how mouse embryos develop , suggesting that our current view on the role that TRPM6 plays in regulating the magnesium ion balance in humans may be too simplistic .","To address this question , Chubanov et al . studied mice with mutations that disrupted the production of TRPM6 in specific tissues only .","The experiments show that TRPM6 primarily operates in the placenta and intestine to regulate the balance of magnesium ions in the body .","Further experiments show that the loss of TRPM6 in adult mice leads to reduced lifespan , growth defects and poor health by disrupting important biochemical reactions .","Supplying the mutant mice with magnesium ion supplements improved their health and could extend lifespans of normal animals .","The findings of Chubanov et al . demonstrate that TRPM6 plays a crucial role in regulating the levels of magnesium ions in mice before birth and into adulthood .","The next step is to carry out large-scale experiments to investigate the effects of altering the levels of magnesium ions in human diets ."],"string":"[\n \"A balanced diet contains a variety of minerals such as magnesium ions , which are required for many chemical reactions in our body .\",\n \"A shortage of magnesium ions is linked to many diseases and is thought to be especially harmful to babies in the womb and shortly after birth .\",\n \"Magnesium ion deficiency is widespread in human populations and in the US is thought to affect up to 68% of people .\",\n \"Despite its prominent role in human health , our understanding of how the body maintains the right balance of magnesium ions remains extremely vague .\",\n \"Magnesium ions can enter and leave a cell by passing through specific types of proteins that form channels in the membrane surrounding the cell .\",\n \"There are thought to be around ten types of these magnesium ion channels in human cells , but we do not know what roles any of them perform in the body .\",\n \"One such channel called TRPM6 may be particularly important because mutations in the gene that encodes this channel can cause magnesium ion deficiency in human infants .\",\n \"However , the loss of TRPM6 in mice disrupts how mouse embryos develop , suggesting that our current view on the role that TRPM6 plays in regulating the magnesium ion balance in humans may be too simplistic .\",\n \"To address this question , Chubanov et al . studied mice with mutations that disrupted the production of TRPM6 in specific tissues only .\",\n \"The experiments show that TRPM6 primarily operates in the placenta and intestine to regulate the balance of magnesium ions in the body .\",\n \"Further experiments show that the loss of TRPM6 in adult mice leads to reduced lifespan , growth defects and poor health by disrupting important biochemical reactions .\",\n \"Supplying the mutant mice with magnesium ion supplements improved their health and could extend lifespans of normal animals .\",\n \"The findings of Chubanov et al . demonstrate that TRPM6 plays a crucial role in regulating the levels of magnesium ions in mice before birth and into adulthood .\",\n \"The next step is to carry out large-scale experiments to investigate the effects of altering the levels of magnesium ions in human diets .\"\n]"},"year":{"kind":"string","value":"2016"}}},{"rowIdx":4196,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"Principles of operation of a cerebellar learning circuit"},"id":{"kind":"string","value":"elife-55217-v2"},"sections":{"kind":"list like","value":[["A hallmark of brain function is the ability to learn and remember .","We can learn and successfully recall faces , events , language , concepts , places , facts , things that were frightening or rewarding , and the motor commands required to skillfully move our motor effectors .","The basic currency of learning and memory is ‘plasticity’ , changes in either the strength of synapses or the intrinsic excitability of a neuron’s membrane .","While decades of research have identified the basic rules that govern plasticity and , for some memory systems , have even defined the neural sites that undergo plasticity , behavioral learning is not solely a property of synapses , neurons , or individual brain sites .","Rather , it is the emergent property of a complete learning neural circuit in which the sites and plasticity mechanisms of learning are embedded .","Only when we incorporate our current knowledge about the specific sites of learning and the rules of plasticity with an understanding of circuit-level interactions can we truly understand learning and memory .","Here , our goal is to define the requisite set of circuit-level computational principles that operate during cerebellar-dependent motor learning .","Arguably , motor learning is the domain that affords the best chance of understanding the principles of learning and memory , due to the exquisite relationship between sensory stimuli and adaptive motor behavior , combined with the tight link from known neural circuits to the output motoneurons .","Across a wide range of movement modalities , motor learning depends crucially on the cerebellum ( Gilbert and Thach , 1977; Golla et al . , 2008; Herzfeld et al . , 2014a; Martin et al . , 1996; Medina and Lisberger , 2008; Smith and Shadmehr , 2005; Robinson , 1974 ) , providing a well-defined neural substrate to investigate how the fundamental principles of learning are implemented in a specific neural circuit .","In the motor learning paradigm we study , direction learning in smooth pursuit eye movements , neurophysiological data has demonstrated that short-term motor learning , on the order of a single learning trial , occurs at or upstream of Purkinje cells in the floccular complex of the cerebellum ( Medina and Lisberger , 2008; Yang and Lisberger , 2013; Yang and Lisberger , 2014 ) .","Single-trial learning is tightly linked to climbing fiber inputs , in agreement with the predictions of the classical theory of cerebellar learning ( Marr , 1969; Albus , 1971; Ito , 1984 ) .","Crucially , Purkinje cells are only two synapses away from the motoneurons that drive the adaptive motor response we measure , significantly constraining the circuit architecture that implements the transformation from sensory inputs to adapted behavior across short timescales .","However , recent behavioral results have suggested that longer term pursuit direction learning , on the order of hundreds to thousands of trials , may be mediated by multiple sites and/or learning mechanisms ( Hall et al . , 2018; Yang and Lisberger , 2010 ) .","The existence of multiple components in pursuit learning is consistent with behavioral and computational evidence from other cerebellar-dependent motor learning behaviors ( Boyden et al . , 2004; Ethier et al . , 2008; Kojima et al . , 2004; Kording et al . , 2007; Lee and Schweighofer , 2009; Medina et al . , 2000; Smith et al . , 2006; Kassardjian et al . , 2005; Shutoh et al . , 2006 ) .","While plasticity is possible across many synapses in the cerebellar circuit ( Carey , 2011; D'Angelo and De Zeeuw , 2009; Hansel et al . , 2001; McElvain et al . , 2010; Mittmann and Häusser , 2007 ) , a prime candidate for long-term learning is the deep cerebellar nucleus , one synapse downstream of the Purkinje cells .","Convergent neurophysiological and behavioral evidence across multiple learning paradigms lends credence to role of the deep cerebellar nucleus for long-term storage of cerebellar-dependent memories ( Raymond and Medina , 2018; Kassardjian et al . , 2005; Shutoh et al . , 2006; Lee et al . , 2015; Raymond and Lisberger , 1998; Popa et al . , 2016; Broussard and Kassardjian , 2004; Lisberger , 1994 ) .","Taken together , these results highlight the idea that multiple neurophysiological mechanisms within the cerebellar circuit may underlie behavioral motor learning across longer timescales .","Our goal in the present paper is to elucidate the operation of the full and well-characterized cerebellar learning circuit .","We identify the properties of the signals that guide acquisition and expression of motor learning across timescales through strategic manipulations of the experimental learning conditions coupled with quantitative measures of behavior .","Our results suggest four circuit-level principles that define pursuit direction learning .","First , rapid acquisition of learning occurs at the parallel fiber to Purkinje cell synapse , driven by climbing fiber responses , with limited retention .","Second , learned responses in Purkinje cells guide the slow acquisition of learning in the deep cerebellar nucleus , which learns slowly but has excellent retention .","Third , the functional properties of the inputs that are subject to learning are different in the cerebellar cortex and deep cerebellar nuclei .","Finally , the extent of learning is limited by negative feedback from the deep nucleus to the inferior olive , reducing the teaching signal that drives learning in the cerebellar cortex ."],["Given the substantial neurophysiological evidence suggesting the primary role of complex-spike-linked plasticity at the parallel fiber to Purkinje cell synapse for single-trial motor learning , our first objective was to fully characterize the properties underlying the acquisition of a motor memory following a single movement error .","To isolate the properties of short-term motor learning , we developed a ‘dual-trial’ experimental paradigm that is a small , but important , modification of our previous methods for studying single-trial learning ( see Materials and methods ) .","In each pair of trials , the first trial is defined as the ‘learning’ trial .","In the learning trial , the monkey begins to pursue a smoothly moving target in a randomly chosen ‘pursuit direction . ’ After 250 ms of target motion in the pursuit direction , the target abruptly changes direction due to the addition of a velocity component in an orthogonal ‘learning direction’ , either +90 or −90 degrees relative to the pursuit direction ( Figure 2A ) .","The discrepancy between animal’s smooth eye movement in the original pursuit direction and target’s net direction due to the addition of orthogonal target motion creates a movement error that serves as an instruction for motor learning ( Figure 2B ) .","We therefore refer to the addition of the target velocity component orthogonal to the original pursuit direction as the ‘instruction' . We measure motor learning in the subsequent ‘probe’ trial , where the target moves in the same pursuit direction as the learning trial but without any instruction .","In probe trials , the animal exhibits a ‘learned response’ due to the error induced by the instruction in the preceding learning trial .","The learned response ( Figure 2C , arrowhead ) starts about 200 ms after the onset of target motion in the pursuit direction , anticipating the change in target direction imposed in the preceding learning trial .","We measure learning in the 50 ms interval surrounding the time of the instruction , from 225 to 275 ms after the onset of pursuit target motion .","The measurement interval is chosen strategically to capture the anticipatory response of the pursuit system at the time of the instruction .","Later in the paper , it also allows us to measure the learned response even in learning trials , because the measurement interval precedes any visually driven eye movement resulting from retinal image motion caused by the instruction ( Hall et al . , 2018; Yang and Lisberger , 2017; Medina and Lisberger , 2008 ) .","We first characterized the dependence of single-trial learning on the magnitude of the error imposed in the learning trial .","To ensure that we were measuring only the learned response due to the occurrence of a single movement error , we prevented any long-term learning by choosing the pursuit direction for each learning-probe pair randomly from the cardinal axes ( see Materials and methods ) .","In each learning trial , we also chose the speed of the instruction randomly to be 0 , 5 , 10 , 15 , 20 , 25 , or 30 deg/s ( Figure 3A ) , and measured the effect of varying instruction speed on the learned response in the subsequent probe trial .","Single-trial learning using randomized pursuit directions creates a situation where the error magnitude experienced in the learning trial is identical to the imposed instruction magnitude , because the average eye speed in the learning direction during the learning trial is zero at the time of the instruction .","This allowed us to control the size of the error that drives learning and to measure directly the shape of the relationship between learning and error .","In both monkeys , learned responses in the probe trial increased as a function of the error magnitude experienced in the learning trial ( Figure 3B ) .","Quantitative analysis revealed that the learned response did not increase linearly as a function of error magnitude in the learning trial , but rather saturated as error increased ( Figure 3C ) .","We quantified the effect of error magnitude on single-trial learning as a sigmoid of the form: ( 1 ) Y2E1=a1+e-τE1-a2where E1 is the magnitude of the error imposed on the learning trial and Y2 is the measured learned response in the subsequent probe trial .","The parameters a and τ were obtained by fitting Equation 1 to the data for each monkey in Figure 3C ( dashed traces versus connected , filled , symbols ) .","Equation 1 fitted the data well for both monkeys ( Monkey RE: a = 1 . 35 , τ = 0 . 21 , R2 = 0 . 99; Monkey YO: a = 1 . 25 , τ = 0 . 118 , R2 = 0 . 98 ) .","The sigmoidal model of single-trial learning was superior to an alternative linear model ( Akaike’s information criterion ( AIC ) ( Akaike , 1974 ) , Monkey RE: -53 . 3 ( sigmoid ) versus -14 . 5 ( linear ) ; Monkey YO: -29 . 2 ( sigmoid ) versus -20 . 5 ( linear ) ) .","Our next step was the characterize the stability of short-term pursuit learning across trials that did not include a movement error .","To do so , we used a modified experimental design where the first 20 trials were standard learning trials with an instruction magnitude of 30 deg/s .","The subsequent 10 trials were ‘error-clamp’ trials ( Figure 3D ) that we used to estimate retention in the absence of movement error ( after Scheidt et al . , 2000 ) .","Error-clamp trials used the same pursuit target motion as in the instruction trials , but the motion of the target in the learning direction was yoked to the animal’s eye movement in that direction , to ensure that the animal experienced no visual error signals in the learning direction .","Thus , any trial-to-trial change in the animal’s learned response over the course of 10 error-clamp trials was due solely to forgetting of the previously acquired short-term motor memory .","We normalized the learned response in the 10 error-clamp trials by the magnitude of the animal’s learned response in the final learning trial of the 20-trial learning block .","Both monkeys demonstrated an exponential decay in the magnitude of the learned response across error-clamp trials ( Figure 3E ) .","Fitting an exponential model to each monkey’s retention data ( Figure 3E , black lines ) suggested a retention constant of approximately 0 . 85 ( 95% confidence intervals based on four experiments per monkey , Monkey RE: 0 . 80–0 . 86 , Monkey YO: 0 . 85–0 . 91 ) .","Therefore , in the absence of any error to drive learning , approximately 15% of the short-term learned response is forgotten on each trial .","We performed this experiment only for instruction speeds of 30 deg/s because the larger amplitude of learning provided more reliable estimates of retention .","We next sought to use generalization of learning to constrain the functional properties of the site of plasticity that causes single-trial motor learning .","Our strategy is based on the following line of reasoning .","Imagine , for example , that the input signals that are subject to single-trial learning are linearly related to eye speed in the pursuit direction ( ve ) and that plasticity operates as a scalar gain ( g ) .","Then , the learned change in firing of the relevant post-synaptic cells should be proportional to gve .","As a result , the learned eye speed should generalize linearly as a function of pursuit speed in the probe trial .","We can invert this logic to use the measured characteristics of generalization of learning to reveal the functional properties of signals that undergo plasticity .","Under the assumption that the site of plasticity for single-trial learning acts as a scalar gain , if learned eye speed generalizes linearly with pursuit eye speed in the probe trial then the population response of neurons upstream from the site of plasticity should be linearly related to eye speed in the pursuit direction .","We again employed our dual-trial paradigm with a randomized pursuit direction for each pair of learning and probe trials .","Here , the learning trials always had a pursuit speed of 20 deg/s and an orthogonal instruction speed of 30 deg/s .","The pursuit speed of the target in the probe trial was selected randomly from 5 , 10 , 15 , or 20 deg/s ( Figure 4A ) .","We found that average eye speed in the learning direction scaled with the speed of the probe target for both monkeys RE and YO , especially in the interval from 225 to 275 ms after the onset of target motion ( Figure 4B , shaded area ) .","We note that scaling is less clear earlier in the probe trial for Monkey YO , partly because the animal’s eye speed in the pursuit direction scales poorly with target speed during this earlier interval .","A RM-ANOVA showed a main effect of eye speed in the pursuit direction in the probe trial on the amount of measured behavioral learning for both monkeys ( Monkey RE: F ( 3 , 6 ) =24 . 3 , p<10−3 , Monkey YO: F ( 3 , 15 ) =13 . 7 , p<0 . 001 ) .","For both monkeys , the effect of eye speed in the pursuit direction on the learned response from 225 to 275 ms was highly linear ( Figure 4C , Monkey RE: R2 = 0 . 99 , Monkey YO: R2 = 0 . 97 ) .","These results suggest that signals related to the speed of eye motion in the pursuit direction may be part of the substrate that is subject to plasticity .","The linear generalization of single-trial learning with pursuit direction eye speed is consistent with prior observations that the eye speed at the time of the instruction linearly modulates the expression of behavioral learning ( Hall et al . , 2018 ) .","In contrast , target/eye speed in the pursuit direction during the learning trial did not affect the expression of single-trial learning on the subsequent probe trial .","Here , we again used the dual-trial paradigm with pursuit speed randomly chosen to be 5 , 10 , 15 , or 20 deg/s in the learning trials but always 20 deg/s in the probe trials .","The instruction occurred 250 ms after the onset of target motion and had a magnitude of 30 deg/s .","The direction of the instruction was chosen randomly to be either +90° or −90° relative to the pursuit direction .","Both Monkeys RE and YO ( Figure 4D ) showed no effect of pursuit speed in the learning trial on the magnitude of the learned response measured in the probe trials .","A RM-ANOVA failed to show a significant effect at 250 ms in the probe trial ( Figure 4E , Monkey RE: F ( 3 , 15 ) =0 . 56 , p=0 . 65 , Monkey YO: F ( 3 , 15 ) =0 . 39 , p=0 . 76 ) .","Thus , we can conclude that target/eye speed in the pursuit direction has distinct and very different effects on the acquisition of a motor memory during the learning trial versus on the expression of learning in the probe trial .","Previous neurophysiological results suggest that complex-spike-linked plasticity at the parallel fiber to Purkinje cell synapse in the cerebellar cortex is likely the primary driver of single-trial pursuit learning ( Medina and Lisberger , 2008; Yang and Lisberger , 2014 ) .","Combining these neurophysiological observations with our behavioral results thus far allows us to begin to constrain the parameters of a cerebellar model of motor learning .","A model of short-term pursuit learning appears in Figure 5A ( Source code 1 ) .","Here , we chose to model the activity of parallel fibers as linearly related to eye speed in the pursuit direction , to account for the generalization of single-trial learning ( Figure 4B–D ) .","We use only two parallel fibers with opposite preferred directions ( more parallel fibers are trivially possible but unnecessary ) with an arbitrary scaling parameter , r , that converts eye velocity to spikes/s: ( 2 ) PF1∝rE˙PF2∝−rE˙ In the absence of learning , the parallel fiber inputs cancel perfectly , ensuring that the firing of the post-synaptic Purkinje cell does not modulate in relation to eye movement in the pursuit direction , consistent with experimental data ( Medina and Lisberger , 2008 ) .","We can model Purkinje cell firing on the nth trial , PCn , as the weighted contributions of the pre-synaptic parallel fibers and a background response , PC0: ( 3 ) PCn=wn1PFn1+wn2PFn2+PC0 The synaptic weight for each parallel fiber , wi , is adaptable via the complex spikes caused by climbing fiber inputs .","The plasticity rule includes two terms: ( 1 ) a decay term , αPF , that allows the relaxation of any learned weight changes back to their baseline value in the absence of error and ( 2 ) an update term that depresses weights by a fractional amount ( β ) depending on the positive firing on the parallel fiber in question and the probability of a complex spike given the neural representation of the image motion from the instruction , PCSEn: ( 4 ) wn+1i={wni− ( 1−αPF ) ( wni−w0i ) −βP ( CS|En ) if PFi > 0wni− ( 1−αPF ) ( wni−w0i ) if PFi ≤ 0 When αPF=1 , the weights are maintained completely from one trial to the next and learning is allowed to perfectly accumulate; when αPF<1 , the weights decay back to their baseline levels , w0i , in the absence of any climbing fiber input .","We model only learning for instructions in the direction that causes complex spikes so that we do not need to explicitly simulate synaptic potentiation beyond the relaxation back toward the baseline weights provided by αPF .","Our results in Figures 3 and 4 suggest appropriate values for the unknown parameters of this simplified cerebellar model of learning .","First , we know that , in the absence of error , short-term motor learning decays across trials .","The time constant analysis in Figure 3E suggests that αPF≈0 . 85 for both monkeys .","In addition , Equation 1 describes how the parallel fiber to Purkinje cell weights are modified as a function of error during single-trial learning: Y2E1∝βPCSE1 .","Previous neurophysiological data has demonstrated that the probability of observing a complex spike following a 30 deg/s instruction is approximately 30% ( Yang and Lisberger , 2014; Yang and Lisberger , 2017 ) .","Given these observations , we can derive a rule linking the probability of observing a complex spike to a given error magnitude using a sigmoid function with the same form as Equation 1: ( 5 ) PCSEn=0 . 61+e-τEn-0 . 3 Here , the probability of observing a complex spike saturates at 30% for large error magnitudes .","The value of τ matches the values we obtained for each monkey in Equation 1 .","Purkinje cells inhibit the floccular target neurons ( FTNs ) in the vestibular nucleus which , in turn , modulate eye movements via their projections to motoneurons .","In the absence of other inputs to FTNs that are modulated by learning , the change in the firing of FTNs from their baseline activity is FTNn=PC0-PCn .","The resulting learned response , Yn , measured via generated eye movements is then Yn= cFTNn , where c is an arbitrary constant that scales FTN activity to deg/s .","Equations 2-5 describe a 'single-learning-process' model .","The constants , r , β , w0i and c are arbitrary and affect only the scaling of firing rates to eye movements .","The values we chose for these parameters are shown in Table 1 , but any number of parameter combinations would give qualitatively similar behavioral results .","The single-learning-process model appropriately estimated our behavioral observations for two different stimulus conditions , one that was more-or-less built into the model and one that matches our data as an emergent property of the model .","First , the trial-to-trial change in output following a range of imposed error magnitudes closely matched the learned behavior of both animals ( Figure 5B ) , because this relationship was built into the model based on the data in Figure 3C .","Second , the model reproduced the results of an experiment that was a superset of the conditions used in Figure 4A–C .","Here , we presented a series of dual-trial stimuli where the instruction trial used a randomly chosen pursuit speed of 0 , 10 , 15 , or 20 deg/s and delivered an instruction at 30 deg/s .","The probe trial also delivered a pursuit speed that was randomly chosen from 0 , 10 , 15 or 20 deg/s .","For each probe trial , we computed the ‘learning expression ratio’ as the magnitude of the learned response expressed in each probe trial normalized by the average learning expressed in probe trials with the same pursuit speed as the preceding learning trial .","When the pursuit speed in probe trial matched the pursuit speed in the learning trial , the learning expression ratio is ~1 . 0 .","We then plotted the learning expression ratio as a function of the ratio of the pursuit speeds of the probe trial and the preceding learning trial to assess the generalization function of learning across any combination of pursuit speeds in the learning and probe trials .","The model reproduces the results of this experiment on both monkeys ( Figure 5C ) , demonstrating strong linear generalization across probe pursuit speeds .","In summary , a cerebellar model with a single learning site where plasticity operates on signals linearly related to pursuit eye speed predicts all of our behavioral data for single-trial learning .","In the more natural situation where the same instruction for learning occurs on movement after movement and is corrected gradually , learning could be simply due to an accumulation of single-trial learning plus forgetting at the rate of 15% per trial .","At some point , the forgetting and learning on each trial would balance and the learning curve would reach an asymptote .","To provide a dataset to ask whether the ‘single-learning-process’ model of single-trial cerebellar learning with forgetting ( Equations 2-5 ) can explain the accumulation of behavioral learning across multiple learning trials , monkeys completed 100 consecutive learning trials .","Each learning trial used identical pursuit and learning directions and speeds .","Within each learning block of 100 trials , the instruction magnitude was either 0 , 5 , 10 , 15 , 20 , 25 , or 30 deg/s .","Note that during 100-trial learning blocks , we control the magnitude of the instruction , but the magnitude of the error on any given trial is the difference between the instruction and the monkey’s expression of behavioral learning on that trial .","When the same learning trial is repeated , acquisition of behavioral learning leads to a gradual reduction in the magnitude of the error for a given magnitude of instruction .","Therefore , we use the terms ‘instruction magnitude’ and ‘error magnitude’ carefully to convey this distinction .","For each instruction magnitude , the trial-course of learning revealed dual-exponential learning curves that largely saturated by the end of 100 learning trials ( Figure 6A ) .","Yet , even after 100 consecutive learning trials , the magnitude of the animal’s learned response was always much smaller than the speed of the imposed instruction: neither animal ever exhibited full compensation for the imposed instruction ( Figure 6B ) .","It is especially noteworthy that the system is able to achieve a learned response of 5 deg/s after 100 learning trials for an instruction at 30 deg/s , but not for an instruction at 5 deg/s .","The effect of instruction magnitude on the learned response was much more linear after 100 consecutive learning trials than after a single learning trial .","The learned eye speed in the last 25 trials of the learning epoch was linearly related to instruction magnitude for both monkeys ( Monkey RE: R2 = 0 . 99 , t-test on the slope of the regression fit: t ( 5 ) =21 . 9 , p<10−4; Monkey YO: R2 = 0 . 99 , t ( 5 ) =18 . 9 , p<10−4 ) .","After 100 learning trials , a linear model was preferred when compared against the best-fit saturating model from Equation 1 ( AIC , Monkey RE: 7 . 7 ( sigmoid ) versus 1 . 5 ( linear ) ; Monkey YO: 0 . 6 ( sigmoid ) versus 0 . 02 ( linear ) , Figure 6C ) .","The difference in the effect of instruction magnitude on learning after 1 versus 100 learning trials hinted that multiple plasticity mechanisms and/or sites within the cerebellar circuit could be driving learning across the different trial-courses .","Using the single-learning-process model , we simulated blocks of 100 consecutive learning trials .","In each trial , we drove learning with an error that was the difference between model output and In , the imposed instruction: En≡In-cFTN , simulating the gradual reduction in the error signal for a fixed instruction magnitude due to the acquisition of learning across trials .","The single-learning-process model failed to predict two crucial features of the true trial-course of learning .","First , it produced single exponential trial-courses that learned too quickly and reached asymptote within the first 20 learning trials ( Figure 7A ) .","Second , it systematically over-estimated the extent of asymptotic learning for instruction magnitudes between 5 and 20 deg/s ( Figure 7B , arrows ) and underestimated learning for instruction magnitudes of 30 deg/s .","Indeed , the model predicted a sigmoidal relationship between asymptotic learning and instruction magnitude ( Figure 7B ) , inherited from the relationship between single-trial learning and instruction magnitude , in contrast to the highly linear relationship in the data ( Figures 6C and 7B ) .","We note that we did not fit the model to the data .","Instead , we used the parameters derived from our behavioral experiments that allowed Equations 2-5 to predict the details of single-trial learning .","We therefore conclude that pursuit learning cannot be mediated solely by the accumulation of single-trial learning with forgetting ( Yang and Lisberger , 2010; Hall et al . , 2018 ) .","Our next step was to identify the minimal principles of a cerebellar circuit model that can explain behavioral learning across multiple timescales .","The features of behavioral learning in Figures 3 and 4 mandate several properties of a more complete cerebellar model: ( 1 ) acquisition of single-trial learning saturates as a function of the magnitude of the error according to a sigmoidal relationship dictated by the data in Figure 3C; ( 2 ) at least across the 100 learning trials we tested in Figure 6 but also after 2000 trials ( Hall et al . , 2018 ) , the asymptotic response for a consistent instruction magnitude falls short of completely correcting the movement error created by the imposed instruction; ( 3 ) the trial-course of learning across long bouts of consecutive learning trials ( Hall et al . , 2018 ) and the different relationship between learned response and error magnitude after 1 versus 100 trials suggest the presence of at least two learning processes ( Hall et al . , 2018; Kojima et al . , 2004; Lee and Schweighofer , 2009; Smith et al . , 2006 ) .","To test our conclusion about multiple learning processes contributing to cerebellar learning across longer learning sessions , we next asked whether the retention of the motor memory changed across long bouts of learning trials .","We used the same error-clamp strategy as before , but now to measure the retention constant after blocks of >1000 identical learning trials .","At the end of long blocks of learning trials , we alternated short blocks of 10 error-clamp trials with runs of 20 learning trials to ‘top up’ the asymptotic learning .","We found significantly better retention after 1000 trials than after 20 trials ( Figure 8A ) : the measured retention constant after 1000 trials was approximately 0 . 95 ( 95% confidence intervals , Monkey RE: 0 . 95–0 . 97 , Monkey YO: 0 . 94–0 . 96 ) ) .","These data suggested that the motor memory gradually transitions , at least conceptually , from a relatively labile , low-retention learning process early in learning to a second learning process with high retention ( Smith et al . , 2006; Herzfeld et al . , 2014a ) .","Armed with knowledge that learning appears to transition from a low-retention learning process to a higher retention process after extended learning , we expanded our circuit model of cerebellar learning to include a secondary site of plasticity ( Figure 8B , Source code 2 ) .","Given neurophysiological results from other cerebellar-dependent learning tasks ( Kassardjian et al . , 2005; Shutoh et al . , 2006; Lee et al . , 2015; Raymond and Lisberger , 1998; Lisberger , 1994 ) as well as the proximity of the cerebellar circuit to motoneurons , we view the floccular target neurons , or FTNs , in the vestibular nucleus as the most likely candidate for long-term storage of learning .","FTNs are immediately downstream of the Purkinje cells , the presumed site of rapid learning , and synapse onto output motoneurons .","We modeled the learned responses of FTNs on trial n as the sum of the firing of input axons , INni , weighted by the scalar synaptic weight , vni , minus the baseline-subtracted firing of the Purkinje cell: ( 6 ) FTNn=∑ivniINni- ( PCn-PC0 ) To account for a secondary site of plasticity within the learning circuit , we implemented Hebbian-style plasticity at the synapse from the eye movement inputs to FTNs .","Plasticity at the inputs to FTNs was driven by the learned responses of presynaptic Purkinje cells: ( 7 ) vn+1i=vni+η ( PC0-PCn ) INni Here , η determines the strength of the Purkinje cell’s teaching signal and the rate of plasticity in the inputs to FTNs .","The value of η was set to be small so that the FTNs represented the site of a slow-learning process , in contrast to the rapid learning at the parallel fiber to Purkinje cell synapse .","Based on our behavioral results in Figure 8A , plasticity in the slow-learning-process is retained almost perfectly , so that Equation 7 lacks the forgetting factor provided by αPF in Equation","4 . Together , Equations 6 and 7 assume that the learning in the Purkinje cell responses acts as a teacher for long-term plasticity at the inputs to the FTNs .","Simulation of Equations 2-7 for a variety of instruction magnitudes ( Figure 8C ) correctly demonstrates one crucial feature of our long-term learning data .","The simulated learning is dual-exponential , with initial fast acquisition and a second , slower , timescale of acquisition .","However , the simulations do not predict a crucial aspect of our behavioral data .","Due to the stability of the long-term memory at the FTN inputs , the asymptotic response of the model is much larger than is found in our data .","Also , Figure 8C shows that the slow-learning process produces a linear relationship between asymptotic learning and instruction magnitude , but only after 2000 trials and not after 100 trials ( vertical dashed line ) as seen in the data .","The excessive learning in the model of Figure 8B suggests the existence of inhibition within the learning circuit .","Multiple deficiencies of the model in Equations 2-7 are corrected when recurrent inhibition from the cerebellum to the inferior olive modulates the error signals used to drive learning at the parallel fiber to Purkinje cell synapse .","We modeled inhibition by reducing the magnitude of the error signal that drives climbing fiber responses in proportion to the size of the learned response: ( 8 ) En=In−cFTNn−γcFTNnE˙ Here , In is the speed of the instruction on the nth trial and cFTNn is the FTN firing converted to units of learned eye velocity .","The term ( In-cFTNn ) in Equation 8 represents the physical error that results from the difference between the instruction magnitude and the response of FTNs .","This formula makes the distinction between the physical error , ( In-cFTNn ) , and the learning system’s internal representation of this error used to drive learning in the cerebellar cortex ( En ) .","The last term in Equation 8 implements modulation of the internal representation of the physical image motion in proportion to γ and the learned response , cFTNn .","There is no recurrent control when the value of γ is zero .","The modulation is normalized by eye speed in the pursuit direction ( E˙ ) so that inhibition of the climbing fiber input occurs in proportion to the change in weight of the parallel fiber to Purkinje cell synapse .","The combination of the Purkinje cell modulation as a teacher ( Equation 7 ) as well as the presence of recurrent inhibition ( Equation 8 ) transfers the motor memory from the cerebellar cortex to the vestibular nucleus across long blocks of learning trials .","We note that the probability of complex spikes decreases considerably across 100 repetitions of the same learning trial ( Yang and Lisberger , 2017 ) , compatible with the mechanism implemented by Equation 8 .","We adjusted the strength of the teaching signal from the Purkinje cells to the FTN inputs ( η ) and the strength of recurrent inhibition of the instructive signal ( γ ) to allow the model in Figure 8B and Equations 2-8 to reproduce both the two-component trial-course of learning ( Figure 8D ) and the correct linear relationship between the asymptotic learning and instruction magnitude after 100 learning trials ( Figure 8E ) .","The best-fit model ( two unknowns , η and γ ) agrees well with the data for both monkeys ( Figure 8E , black curves , Monkey RE: R2 = 0 . 99; Monkey YO: R2 = 0 . 97 ) .","In addition , both monkeys had similar values for both estimated parameters ( 95% confidence intervals , Monkey RE: η = 5 . 1×10−4–9 . 2 × 10−4 , γ = 55 . 9–68 . 5 , Monkey YO: η = 4 . 5×10−4–9 . 4 × 10−4 , γ = 38 . 8–65 . 0 ) .","Linearity emerges after 100 trials because recurrent feedback switches the mechanism driving asymptotic behavior from a tradeoff between learning and forgetting to an emergent property of the operation of a dynamic feedback loop .","The successful model in Figure 8B has three crucial features: ( 1 ) a site of rapid learning with limited retention at the parallel fiber to Purkinje cell synapse , ( 2 ) gradual transfer of learning from the cerebellar cortex to a slower-learning process with excellent retention at the inputs to the FTNs , and ( 3 ) recurrent inhibition from FTNs to the inferior olive that limits learning and linearizes the stimulus-response function of learning by controlling the error signals available to drive learning at the parallel fiber to Purkinje cell synapse .","We next provide additional behavioral evidence for separate sites of learning within the cerebellar circuit .","By probing the expression of learning as a function of pursuit speed across the trial-course of learning , we show that the linear pattern of generalization to pursuit speed we observed following a single learning trial ( Figure 4 ) changes gradually but qualitatively across 1000 trials .","We suggest that the site and/or mechanism of learning changes across a long trial-course .","We again deployed the dual-trial paradigm , but with the modification that the instruction trial in each pair of trials repeated the same fixed parameters of pursuit ( 5 deg/s or 20 deg/s ) , instruction magnitude ( 30 deg/s ) , instruction direction , and pursuit direction for the entire experimental session .","We studied generalization across the gradual accumulation of learning by varying target speed in the pursuit direction randomly in the probe trial to be 5 , 10 , 15 or 20 deg/s , while keeping the pursuit direction the same as in the learning trials .","An example experiment demonstrates how generalization changed over a long trial-course when pursuit speed was 5 deg/s in the learning trials and was always equal or faster in the probe trials .","Averages of the trajectory of the learned eye velocity as a function of time during the first 250 trials confirmed that the learned response scaled with pursuit speed in the initial stages of learning ( Figure 9A , left ) , in agreement with the data for single-trial learning in Figure","4 . However , at the end of the same learning session , the expression of learning no longer scaled with pursuit speed in the probe trials ( Figure 9A , right ) .","The change in generalization across the session could not be explained by any systematic differences in the eye speed in the pursuit direction during the first versus last 250 trials ( Figure 9B , continuous versus dashed traces ) .","The change in the generalization of the expression of learning in the probe trials occurred gradually ( Figure 10A ) .","Across 2250 trials ( 1125 dual-trial stimuli ) in Monkey RE , the expression of learning declined for probes with pursuit speeds of 20 deg/s and remained constant for probes with pursuit speeds of 5 deg/s .","By 2250 trials , the learned response did not depend on the target speed in the probe , in agreement with the right graph of Figure 9A .","The observations were similar in Monkey YO , although he did not work for as many trials as Monkey RE ( note the shorter x-axis ) .","These results suggest that something about the inputs that are subject to plasticity or the mechanism of plasticity at the site of single-trial , short-term , learning may differ from those to the site of long-term pursuit learning .","We observed a qualitatively different pattern of generalization across learning blocks when pursuit speed was 20 deg/s in the learning trials and was always equal or slower in the probe trials .","Now , the expression of learning increased slightly for probe trials with a pursuit speed of 20 deg/s and decreased as a function of trial when the pursuit speed in the probe trial was 5 deg/s ( Figure 10B ) .","Together , the results of learning with pursuit target speeds in the learning trials of 5 and 20 deg/s show a general rule: learning generalizes approximately linearly for pursuit speed across all timescales of learning when the pursuit speed in the probe trial is slower than the learning trial; when pursuit speed in the probe trial is faster than that in the learning trial , linear generalization early in learning is replaced gradually with a learned response that appears largely independent of pursuit speed after long-term learning .","To verify the statistical veracity of the effects in Figure 10A and B , we performed a regression analysis on the trial-course of generalization of each experiment ( lines in Figure 10A and B ) .","For each experimental condition , we measured the rate of change of the learned response across trials ( Figure 10C ) .","Here , a negative slope indicated that the learned response for a particular probe pursuit speed decayed over the experiment ( e . g . 20 deg/s probe trials when the pursuit speed of the learning trial was 5 deg/s ) .","Positive slopes indicated that the learned response measured in the probe trials tended to increase across the experiment , as was the case when the pursuit speed in both the learning trial and the probe trial was 20 deg/s ( Figure 10B , red curves ) .","A two-way ANOVA confirmed the differential effect of pursuit speed in the learning trials on the generalization properties of the motor memory .","We tested for main effects of probe pursuit speed ( 5 , 10 , 15 or 20 deg/s ) , pursuit speed in the learning trials ( 5 deg/s or 20 deg/s ) , and their interaction .","For both monkeys , we observed a main effect of pursuit speed in the learning trials ( RE: F ( 1 , 32 ) =46 . 8 , p<10−7; YO: F ( 1 , 20 ) =31 . 6 , p<10−4 ) , no main effect of probe speed ( RE: F ( 3 , 32 ) =1 . 39 , p=0 . 26; YO: F ( 3 , 20 ) =0 . 33; p=0 . 81 ) , and crucially a significant learning pursuit speed by probe pursuit speed interaction ( RE: F ( 3 , 32 ) =14 . 2 , p<10−5 , YO: F ( 3 , 20 ) =9 . 0 , p<10−3 ) .","Thus , the relationship between pursuit speeds of the target in the learning and probe trials significantly affected the shape of generalization across trials .","To further validate the interaction between pursuit speed in the learning and probe trials , we conducted additional experiments in Monkey RE that tested other pursuit speeds in the learning trial ( not shown ) .","Here , different experiments used one of either 5 , 10 or 20 deg/s as the consistent pursuit speed in the learning trials and probed with pursuit speeds selected randomly from the same three speeds .","After 1000 learning trials , the expression of learning generalized approximately linearly as a function of slower pursuit speeds in the probe trial compared to the learning trial , but not for probe speeds faster than used in the learning trial , in agreement with Figure 10A and B . Thus , the rules for generalization depend not on whether the probe speed is the same or different from the pursuit speed in the instruction trials , but rather on whether it is faster or slower .","These preliminary experiments also served as a control to ensure that the change in generalization across many trials was not a result of the alternation of learning and probe trials , because probe trials with the other two pursuit speeds were interspersed randomly among the learning trials with a ratio of 10 learning trials to every probe .","In the previous section , we showed that learning generalizes differently in relation to pursuit target speed early versus late in a long learning block .","Next , we analyze our data in a way that quantifies the shape of the pursuit speed generalization function at different stages of learning .","We used the data from the long blocks of learning/probe pairs in Figure 10 , where the pursuit and instruction speed were fixed in the learning trial but pursuit speed in each interleaving probe trial was selected randomly among 5 , 10 , 15 , or 20 deg/s .","Here , we computed the ‘learning expression ratio’ for each pair of trials as the magnitude of the learned response in the probe trial divided by the learning expressed in the preceding learning trial .","The learning expression ratio is a single-trial metric of generalization , allowing us to probe the shape of generalization across long bouts of learning while controlling for the gradual accumulation of behavioral learning across the session .","We then binned the resulting learning expression ratio by the ratio between the target pursuit speed in each probe trial and learning trial .","When the probe trial and the preceding learning trial have the same pursuit speed ( probe speed to pursuit speed ratio of 1 . 0 ) , we would expect nearly complete generalization between the learning and probe trials ( a learning expression ratio ~1 . 0 ) .","We refer to the relationship between the learning expression ratio and the ratio of the probe and learning trial pursuit speeds as the ‘generalization function' .","In the first 250 trials of a long learning block ( blue curve , Figure 11A ) , both monkeys demonstrated an approximately linear generalization function , paralleling our finding of single-trial generalization in Figure","5 . Thus , early in learning , both monkeys expressed greater ( less ) learning in the probe trial than in the learning trial when pursuit speed in the probe trial was faster ( slower ) than in the preceding learning trial .","As learning progressed through a long block of trials ( Figure 11A , pink and purple traces ) , the shape of the generalization function changed gradually .","After 1000 trials , probe trials with pursuit target speed faster than in the learning trial ( probe speed to pursuit speed ratio >1 . 0 ) no longer showed enhancement of the expression of learning ( learning expression ratio ~1 . 0 ) .","The changing shape of the generalization function was further accentuated in Monkey RE after 2000 trials when probe trials expressed less behavioral learning ( learning expression ratio <1 . 0 ) if the pursuit speed was faster in the probe than in the learning trial .","That the generalization function changes across 2000 trials indicates either that the properties of a single site of learning change or that learning transfers between sites with different functional properties .","Cognizant that factors such as non-linear synapses and recurrent inhibition can control the properties of the signals at a site of learning , the simplest way to model generalization is in terms of the signals conveyed by the afferent fibers whose synapses are subject to plasticity .","Figure 11 suggests that we can model learning under the assumption that the motor memory gradually transitions from an early site of learning where the inputs are linearly related to pursuit speed to a secondary site/synapse where the inputs are unimodally tuned for pursuit speed .","We completed our model of cerebellar learning ( Figure 12A ) by defining the functional properties of non-Purkinje cell inputs to FTNs .","We simulated 50 inputs to the FTNs , each with a preferred direction either equal to or opposite the pursuit direction and a Gaussian tuned response for pursuit speed: ( 9 ) INni=cos ( θne−θi ) exp ( − ( E˙−si ) 22σ2 ) Here , for the ith input axon , θi is either 0 or 180 deg , si is the preferred eye speed ranging from 0 to 50 deg/s , and σ is the standard deviation of the Gaussian function .","The eye movement vector on the nth trial is defined as ( θne , E˙n ) .","Note that any unimodal function ( e . g . cosine ) would yield comparable results to the Gaussian function .","The circuit-level learning system defined by the schematic in Figure 12A and Equations 2-9 reproduces our data on both the trial-course of learning and the trial-course of the generalization of learning to pursuit speed .","The learned response , like the real data , shows a saturating relationship to instruction magnitude for single-trial learning ( Figure 12B ) and a linear relationship after 100 trials ( Figure 12C ) .","For learning with pursuit target motion at 5 deg/s , the expression of the learned response in the model generalizes differently early versus late in a 1000 trial learning block ( Figure 12D ) .","Early in the learning block , the learned response generalizes linearly to pursuit speeds regardless of the pursuit speed in the learning trials .","Late in the learning block , the learned response is largely identical across probe speeds .","For learning with pursuit target motion at 20 deg/s , the learned response generalizes linearly across probe speeds lower than 20 deg/s for the entire duration of the learning block ( Figure 12E ) .","The model also makes predictions .","It suggests that adaptive changes in the Purkinje cell responses should diminish over the course of thousands of trials ( Figure 12F , G ) as learning transfers to the FTN inputs ( Figure 12H , I ) .","We considered a number of other model forms besides our final cerebellar model ( Figure 12A ) .","The criteria for a plausible model were imposed by our data for single-trial learning ( Figures 3 and 4 ) and repeated presentation of the same learning trial ( Figure 6 ) .","Single-trial learning always showed a saturating relationship as a function of the error imposed by the instruction .","Therefore , all candidate models were constrained by the fits in Figure 3C , ensuring that the measured learned behavior on trial 2 as a function of the error magnitude experienced on the first trial , Y2e1 , was approximately equal to Equation 1 .","We then asked whether each candidate model could account for the nearly linear asymptotic learned response as a function of instruction magnitude after a block of 100 learning trials ( Figure 6C ) .","We began by assuming the simplest model capable of reaching an asymptotic learned response that was less than the imposed instruction .","This model featured a single learning process , x , with a retention parameter that allowed trial-to-trial forgetting: ( 10 ) xn+1=αxn+ΔYn ( en ) Yn=xn Here , α is the retention factor where α=1 indicates complete retention ( no forgetting ) and Yn is the measured behavioral response on trial n .","The change in the measured amount of learning on each trial , ∆Ynen , is given by extending Equation 1 from the case of single trial learning ( trials 1 and 2 ) to trial-over-trial learning at any stage ( trials n and n+1 ) .","The asymptotic response of this single learning process occurs when Yn+1≈Yn , which is given by: ( 11 ) Y∞=∆Y∞e∞1-α The form of the asymptotic response is a scaled version of the measured single-trial learning from Figure 3C and therefore cannot , by itself , reproduce the linear relationship between instruction magnitude and asymptotic learning that we measured after 100 consecutive learning trials .","Any single-learning-process model that ( 1 ) has a static relationship between learning and error and ( 2 ) predicts saturating single-trial learning in Figure 3 would fail to predict the linear asymptotic learning after 100 repeated learning trials in Figure 6 .","Pursuit learning might align with other models of motor learning that also have posited two ( Lee and Schweighofer , 2009; Smith et al . , 2006 ) or more ( Kording et al . , 2007 ) learning processes .","For our system , learning could be the net sum of two learning processes that run independently and in parallel: a fast-learning process , xf , with rapid learning from error but limited retention and a slow-learning process , xs , with strong retention: ( 12 ) xn+1f=αfxnf+ΔYn ( en ) xn+1s=αsxns+ηsenYn=xnf+ xns A central requirement is that the retention of the slower-learning process is greater than the retention of the faster process , namely αs≫αf , and the rate of learning is greater in the fast-learning process versus the slow-learning process , ∆Ynen≫ηsen .","As long as the fast-learning process shows a saturating relationship between error and single-trial learning , the model will reproduce the single-trial learning data in Figure 3C .","The two-independent process model in Equation 12 fails to predict a linear relationship between the asymptotic response after extended learning and instruction magnitude .","Given the retention values , we measured experimentally ( αf≈0 . 85 and αs>0 . 95 ) , the value of ηs must be very small to ensure that learning saturates at a level significantly below the imposed instruction after extended learning ( Figure 6A ) .","With this constraint ( ηs≈0 ) , we can derive the asymptotic response of the two-learning-process model from Equation 12: ( 13 ) Y∞=x∞f+x∞s=ΔYn ( en ) ( 1−αs ) ( 1−αf ) ( 1−αs+η ) + ηI ( 1−αf ) ( 1−αs+η ) ≈Δxf ( e∞ ) 1−αf Equation 13 represents a scaled version of the single-trial response shown in Figure 3 and is largely identical to the asymptotic response predicted by a single-learning-process in Equation 11 .","We expressly set out to model our data in a way that was driven by the known architecture of the cerebellar learning circuit .","Adherence to the known circuit and the ability to reproduce a wide set of behavioral data with relatively simple assumptions are virtues of the model in Figure 12A .","At the same time , we doubt that our successful model is unique and other strategies might ( or might not ) be able to reproduce our data as well .","We give two examples .","( 1 ) A single-site model might work if the rate of learning is not constant but rather changes from trial-to-trial .","It would be possible to account for some of our data in a single-state model where complex-spike-mediated depression of the parallel-fiber to Purkinje cell synapse intrinsically weakened or saturated across repeated identical learning trials .","( 2 ) Some models modulate learning based on Bayesian integration of noisy sensory feedback with internal estimates of the perturbation ( i . e . the Kalman filter ) , experienced errors ( Herzfeld et al . , 2014b ) , or environmental consistency ( Gonzalez Castro et al . , 2014 ) .","Each of these models predicts that learning rate should increase for repeated presentations of the same learning stimulus versus single-trial learning , whereas we observed that the learning rate decreases over trials .","However , one might contrive a model based on these principles where learning rate is lower when the stimulus regime is highly regular and consistent .","In addition to their other failings , none of these approaches have the advantage of working seamlessly within the constraint of the architecture of the cerebellar learning circuit ."],["Our data add to the evidence for a fast-learning process with poor retention based on climbing-fiber mediated plasticity at the parallel to Purkinje cell synapse in the cerebellar cortex ( Medina and Lisberger , 2008; Yang and Lisberger , 2014; Nguyen-Vu et al . , 2013; Kimpo et al . , 2014 ) .","Long-term depression at this particular synapse has been a staple of cerebellar learning for many years ( Ito , 2001; Marr , 1969; Albus , 1971 ) , and short-term forms of plasticity also occur at the same site ( Brenowitz and Regehr , 2005; Suvrathan et al . , 2016 ) .","Still , the tight link between the occurrence of a complex spike and neural learning in Purkinje cell simple-spike responses ( and behavior ) does not prove that the parallel fiber to Purkinje cell synapse is the mechanism of the fast-learning process and we need to remain open to the possibility that the actual site of plasticity may be upstream of the parallel fiber to Purkinje cell synapse .","We are able to measure the properties of the fast-learning process largely independent of other , slower , components of the recurrent learning circuit using the analysis of single-trial learning .","Thus , we effectively measured the ‘open-loop’ features of the fast-learning process and discovered that the fast-learning process features a non-linear , saturating relationship between the magnitude of learning and the size of the movement error .","We suspect that the non-linear relationship reflects the effects of the retinal slip on the probability and/or duration of the complex spikes that occur in Purkinje cells ( Mathy et al . , 2009; Najafi and Medina , 2013; Yang and Lisberger , 2014 ) .","However , we cannot exclude two alternatives .","The asymptote of single-trial learning for the highest instruction magnitude could reflect the influence of inhibitory neurons on the learning signal ( Rowan et al . , 2018 ) and/or a simple limit on the maximum amount of plasticity that can be induced by a single climbing fiber input to most Purkinje cells .","The saturating dependence of single-trial adaptation in our system on instruction magnitude or stimulus strength parallels findings of single-trial learning in other cerebellar-dependent adaptive behaviors ( Fine and Thoroughman , 2006; Herzfeld et al . , 2014b; Marko et al . , 2012; Hutter and Taylor , 2018 ) .","Our finding of qualitatively different generalization functions early versus late in learning provides experimental support for two distinct sites of learning , one that learns quickly and predominates early versus a second process that learns slowly and therefore predominates late in a 1000-trial learning block .","Evidence that learning transfers from the fast- to the slow-learning site , rather than proceeding independently in parallel , comes from our computational and theoretical analysis .","The cerebellar circuit model with recurrent inhibition of the error input to the cerebellar cortex can explain linearization of the relationship between the size of learning and instruction magnitude after 100 learning trials , whereas a standard model with independent fast and slow-learning processes would require considerable modification to do so ( see Equations 12 and 13 ) .","Thus , we think that the fast-learning process for pursuit learning occurs in the floccular complex of the cerebellum ( Medina and Lisberger , 2008 ) and that the floccular target neurons in the vestibular nucleus ( Lisberger et al . , 1994 ) are likely the site of the slow-learning process and long-term storage .","At this time , we cannot rule out the possible role of additional downstream areas .","There are multiple reasons to think that learning could transfer from the cerebellar cortex to the deep cerebellar nuclei , even though there is currently no direct neurophysiological evidence .","Mechanisms of plasticity are abundant in the deep cerebellar nuclei and optogenetic modulation of the activity of Purkinje cells can adapt the vestibulo-ocular reflex , presumably through Purkinje-cell induced plasticity in the deep cerebellar nuclei ( Jang et al . , 2020; Nguyen-Vu et al . , 2013 ) .","Recordings from Purkinje cells and deep cerebellar nucleus neurons following long-term vestibulo-ocular gain adaptation lead to the conclusion of neural learning in both sites ( Lisberger , 1994 ) and are entirely compatible with the suggestion that the learned responses in Purkinje cells instruct learning in the deep cerebellar nucleus ( Miles and Lisberger , 1981 ) .","For both eye movements ( Pastor , Pastor et al . , 1994 ) and classical conditioning of the eyelid response ( Garcia et al . , 1999 ) , inactivation or lesions of the cerebellar cortex prevents adaptation but does not fully abolish learned responses from previous adaptation ( McCormick and Thompson , 1984; Perrett et al . , 1993 ) , suggesting that the residual long-term learning resides outside of the cerebellar cortex .","Finally , for learning in the optokinetic response , lesions of the flocculus abolish the learned response completely after short-term learning but only partially after long-term learning ( Shutoh et al . , 2006 ) .","We think that the accessible neural substrate of pursuit learning affords the opportunity to study the neural mechanisms of memory transfer , a ubiquitous characteristic across memory systems .","For example , different sites of early acquisition versus long-term storage exist for fear memories ( Rogan et al . , 1997 ) , ( Do-Monte et al . , 2015 ) , and in bird song ( Bottjer et al . , 1984; Nordeen and Nordeen , 1993; Warren et al . , 2011 ) .","Indeed , the acquisition of a memory in one structure and the long-term storage of this memory in separate structures is clear from amnesic subjects , such as HM ( Zola-Morgan and Squire , 1990 ) , as well as from many more modern observations ( Tonegawa et al . , 2018 ) .","In our data , the early learned response generalizes linearly as a function of all pursuit speeds in the probe trials , even when the probe speed is faster than the pursuit speed in the learning trial .","After 1000 trials of learning , the learned response scales only for probe speeds slower than pursuit speed in the learning trial .","The literature contains other evidence of changes in the generalization of the expression of learning across timescales .","In birdsong , learned changes in the pitch of one syllable generalizes differently early versus late in learning ( Warren et al . , 2011 ) , suggesting that early learning in LMAN changes signals that are related to the overall song , while the late learning in the motor structure RA operates on signals related to the motor act of producing the syllable .","In the vestibulo-ocular reflex , Kimpo et al . , 2005 showed different generalization for stimulus frequency for gain-up versus gain-down learning , suggesting different sites of learning based on modification of different vestibular signals .","After Shadmehr , 2004 , we interpret the change in generalization of learning as possible evidence for a change in the properties of the neural signals that are subjected to learning .","Our circuit model accounts for the change in generalization by assuming that a different set of eye speed signals is learned at Purkinje cells versus FTNs .","The use in the model of different inputs to the sites of plasticity in the floccular complex and at the floccular target neurons suggests two features of the neural system that run counter to current doctrine .","First , the general rule is that Purkinje cells and their targets in the deep cerebellar nuclei receive identical inputs from mossy fibers .","In the floccular complex , however , this does not seem to be the case: brainstem areas that send afferent mossy fibers to the ventral paraflocculus region of the floccular complex send only sparse projections to the location of FTNs in the vestibular nucleus ( Osanai et al . , 1999; Nagao et al . , 1997 ) .","Second , the general rule in the oculomotor system is the firing rate is linearly related to eye position and velocity , and this is true for mossy fibers in the floccular complex ( Miles et al . , 1980; Lisberger and Fuchs , 1978 ) .","Thus , there is no obvious precedent for neural responses that are unimodally tuned for eye velocity .","Limited recordings from FTNs during pursuit learning Joshua et al . , 2013 demonstrate a diversity of FTN velocity-dependent responses to a 30 deg/s pursuit stimulus , perhaps consistent with a distribution of preferred speeds across the neural population .","We realize that the assumptions in the model may not reflect the actual implementation of the system in the brain , and that the differences in generalization at different sites could reflect properties of synapses or plasticity mechanisms rather than of the input signals that are subject to plasticity .","We have always been troubled by the fact that pursuit learning , like other forms of motor learning ( Krakauer et al . , 2000; Tseng et al . , 2007; Vaswani et al . , 2015 ) , never reaches an amplitude that completely eliminates the error created by the imposed instruction .","Even after more than 1000 learning trials , the adapted pursuit eye movements shows residual errors than are in excess of 50% of the imposed instruction ( Hall et al . , 2018 ) .","We suggest that motor learning never fully adjusts for the instruction because of active inhibition of the instruction for learning Kenyon et al . , 1998 ) .","Via the known double-inhibitory pathway from Purkinje cells to the inferior olive via the deep cerebellar nucleus ( Houck and Person , 2014; Najac and Raman , 2015; de Zeeuw et al . , 1988 ) , learned depression of Purkinje cell simple-spike firing could reduce the effectiveness of a given movement error by attenuating the probability and/or duration of climbing fiber inputs to the cerebellar cortex .","Previous neurophysiological observations over 100 trials of pursuit learning demonstrate a reduction in the probability of complex spikes , hinting at possible inhibition of the olive ( Medina and Lisberger , 2008; Yang and Lisberger , 2017 ) .","Our data and modeling render unlikely the alternate explanation that incomplete adaptation is the result of a competition between the learning signals and a restoring force due to forgetting ( Smith et al . , 2006; Albert et al . , 2019 ) .","During error-clamp trials , we measured the retention parameter after 1000 trials to be close to 1 . 0 , corresponding to almost complete retention .","We imagine that recurrent inhibition of the error inputs to the fast-learning process promotes slow , conservative learning that does not respond hastily to noisy inputs .","We do not understand fully why it is advantageous for the fast-learning process to remain inhibited after learning has been transferred from the cerebellar cortex to FTNs .","Perhaps the goal is to force other parts of the pursuit circuit to take over some of the learning under conditions where permanent changes seem desirable , by transferring some of the learning away even from the FTNs .","Alternatively , our somewhat rarified stimulus conditions may not be engaging the neural learning circuit fully so that we are seeing only a part of the full learning capability of the system .","The cerebellar learning circuit is stereotyped , well-known , accessible for analyses at the level of the activity of single neurons , and engaged in quantitatively-defined learning behavior .","We see it as an exemplar where we have the greatest potential to determine not just the sites and mechanisms of plasticity , but also how the essential neural circuit exploits the local mechanisms of plasticity and converts them into the global deliverable of an adaptive change in behavior .","Similar analysis and thinking are going to be essential in all other learning systems and neural circuits before we can say that we ‘understand’ any form of learning and memory .","The particular behavioral system we study affords the huge advantage that we can constrain models by both the architecture of the essential circuit and data on the operation of the system .","Our data and computational thinking assemble a statement of the principles of operation of this learning neural circuit .","Once additional behavioral and neurophysiological evidence further refine these principles of operation , we will have a strong statement of how one learning circuit works ."],["Monkeys were positioned with their heads fixed 30 cm in front of a CRT monitor ( resolution: 2304 × 1440 , framerate: 80 Hz ) .","The monitor subtended 58 × 46° of the monkey’s visual field .","All experiments took place while the monkey was in a dimly lit room .","The visual target for all experiments was a 0 . 5° diameter black dot presented on a light grey background ( approximate luminance 32 . 9 cd/m2 ) .","The motion of the target at each frame was controlled by our laboratory’s custom ‘Maestro’ software .","Separate voltage signals from the scleral coil system , corresponding to the monkey’s horizontal and vertical eye position , were digitized at 1 kHz .","We also digitized eye velocity signals obtained by passing the eye position voltages through an analog differentiator circuit with a low-pass filter that reduced the amplitude of signals above 25 Hz ( −20 dB/decade ) .","All signals were stored for later offline processing and analysis .","At the start of each trial , the monkey fixated within ±3° of a target at the center of the screen for a random interval , chosen from a uniform distribution of 400–800 ms . After the fixation period , the target instantaneously jumped eccentrically by 0 . 15 vt degrees , and then began moving at a constant speed , vt , in the opposite direction , termed the ‘pursuit direction’ ( step-ramp paradigm of Rashbass , 1961 ) .","The eccentric target jump minimizes the number of catch-up saccades during the initial tracking of the target .","During baseline , probe , and washout trials , the target proceeded at the constant ‘pursuit speed’ for 850 ms before stopping .","The monkey then fixated the target at its final position for an additional 200 ms . In exchange for appropriate tracking of the target as well as fixating the target within ±3° at the end of the trial , the monkey received a small fluid reward .","If the monkey broke fixation during the initial fixation period or failed to adequately track the moving target and fixate its final position , the trial was aborted immediately and the monkey received no reward .","Aborted trials were not included in the data analysis .","To induce cerebellar-dependent motor learning , we used a direction learning paradigm ( Medina et al . , 2005 ) .","In ‘learning trials’ ( Figure 2A , dotted line , Figure 2B , D ) , the target proceeded in the original pursuit direction for 250 ms , before the addition of an orthogonal velocity component , termed the ‘instruction . ’ We call the direction of the orthogonal instruction the ‘learning direction , ’ since the instruction induces visual motion that serves as an error and drives cerebellar-dependent motor learning .","During the instruction , we suspended the eye position requirements for liquid reward .","In some experiments , we varied the speed of the instruction .","In all experiments , the instruction had a duration of 400 ms , so that it was followed by 200 ms of target motion in the original pursuit direction and then 200 ms of fixation .","Reward was not contingent upon generation of a learned response .","Monkeys typically worked for 1500 to 3000 successful trials per experimental session .","To measure the amount of trial-to-trial forgetting in the absence of error , we used ‘error-clamp’ trials .","During error-clamp trials , the target proceeded in the original pursuit direction , as in a probe trial .","However , the location of the target in the direction orthogonal to the pursuit direction ( the learning direction ) was ‘clamped’ to the animal’s eye position , such that the position of the target on each of the monitor’s frames matched the animal’s current eye position in the learning direction ( Figure 3D ) .","Target stabilization in the learning direction removes any errors that could drive unlearning and allowed us to measure the intrinsic retention/decay of motor memories without any stimulus for learning .","To separately investigate the characteristics of the signals that drive learning of a motor memory from those that affect generalization on the timescale of a single-trial , we developed a ‘dual-trial’ experimental paradigm as a variant of our previously described paradigm for single-trial learning ( Yang and Lisberger , 2010 ) .","Our modified paradigm uses pairs of trials , where the first trial , n , is the learning trial that provides a direction-change instruction ( Figure 2A , dotted line , Figure 2B ) and the second trial , n+1 , is a probe trial without an instruction ( Figure 2A , solid line , Figure 2C ) .","Both the learning and probe trial in each dual-trial pair used the same pursuit direction , chosen randomly for each pair of trials from the cardinal axes ( 0° , 90° , 180° , or 270° ) .","For some experiments , the speed of the target in the pursuit axis ( i . e . , the pursuit speed ) was identical in both the learning and probe trials .","In other experiments , we strategically altered the pursuit speed in either the learning trial or the probe trial .","The direction of the instruction in the learning trial was chosen randomly to be either +90° or −90° relative to the pursuit direction .","Due to the random directions of the initial pursuit and the instruction within a daily experiment , learning did not accumulate across trials .","We explicitly prohibited the consecutive occurrence of pairs of trials with the same pursuit and instruction directions .","To quantify the behavioral learning expressed in the second trial of each pair , we measured the ‘learned response’ from the eye velocity in the direction of the instruction in the preceding learning trial ( i . e . , the learning direction ) .","Monkeys typically performed 750 to 1500 pairs of trials in a given experimental session .","We extended our dual-trial paradigm to investigate changes in the generalization of learning to pursuit speed across long blocks of trials .","Now , we allowed the learning to accumulate by using a consistent pursuit direction in both the learning and probe trials across the entire experimental session .","The pursuit direction for both the learning and probe trial was chosen randomly from the cardinal axes ( 0° , 90° , 180° , or 270° ) for each experimental session .","Within an experimental session , the pursuit speed during the learning trial was either 5°/s or 20°/s .","To measure the generalization of learning to pursuit speed , the speed of each probe trial was chosen randomly ( 5 , 10 , 15 , or 20 deg/s ) .","For all experiments that used the dual-trial paradigm , we report statistics for each monkey across days/experimental sessions .","To measure changes in the acquisition of a motor memory across timescales longer than a single-trial , we used blocks of trials with consistent instruction statistics .","For each ‘learning block , ’ we chose a single direction of pursuit for all trials from one of the cardinal axes .","Monkeys performed a series of baseline trials without an instruction , followed by a series of learning trials with an imposed instruction in a consistent learning direction that was orthogonal to the pursuit direction .","The behavioral learning was subsequently extinguished by presenting washout trials that did not feature an instruction .","The number of washout trials was always equal to or larger than the number of the preceding learning trials .","Because the direction of the instruction was consistent across the learning trials , we define the behavioral learning on each trial as the speed of the eye movement in the direction of the instruction experienced during the learning trials .","After a complete learning block , including washout , a new pursuit direction and instruction direction were chosen randomly for the subsequent block .","Monkeys typically performed multiple repetitions of repeated-instruction learning paradigms in a given experimental session .","No two consecutive learning blocks were allowed to have the same pursuit and instruction directions .","For all experiments in a repeated-instruction design , we report statistics for each monkey across repetitions of the learning paradigm .","To ensure that saccades did not contaminate our estimate of the learned pursuit response , we identified and removed saccades from eye velocity traces .","We identified saccades using a combined speed and acceleration threshold: any instance when the speed of the eye exceeded 20 deg/s and the eye acceleration exceeded 1250 deg/s/s was labeled as a saccade .","Time points were marked as a saccade from 10 ms before the first time point that exceeded joint velocity and acceleration thresholds to 10 ms after the final time point that exceeded both thresholds .","We eliminated these intervals from data analysis by treating them as missing data .","Because the target motions were designed to minimize their occurrence , saccades were typically infrequent in the analysis intervals and occurred at variable times .","To summarize the magnitude of behavioral learning on a single-trial , we averaged the speed of the eye in the learning direction from 25 ms before to 25 ms after the time of the instruction , 225–275 ms after the onset of pursuit-direction target motion ( grey shading in Figure 2B , C ) .","Even during trials that included an instruction ( e . g . during block-wise learning ) , this measurement provides an estimate of the learned response because the effects of visual feedback and online corrections in pursuit movements do not appear until at least 75 ms after the onset of the instruction .","This measurement of motor learning is consistent with previous pursuit direction learning experiments ( Hall et al . , 2018; Yang and Lisberger , 2010; Yang and Lisberger , 2017 ) .","We used two tailed t-tests to compare sample means for conditions that were not sampled within the same experimental session , with the significance level set at p=0 . 05 .","Remaining tests with conditions that were simultaneously sampled within an experimental session were performed using a repeated measures analysis of variance ( RM-ANOVA ) ."]],"string":"[\n [\n \"A hallmark of brain function is the ability to learn and remember .\",\n \"We can learn and successfully recall faces , events , language , concepts , places , facts , things that were frightening or rewarding , and the motor commands required to skillfully move our motor effectors .\",\n \"The basic currency of learning and memory is ‘plasticity’ , changes in either the strength of synapses or the intrinsic excitability of a neuron’s membrane .\",\n \"While decades of research have identified the basic rules that govern plasticity and , for some memory systems , have even defined the neural sites that undergo plasticity , behavioral learning is not solely a property of synapses , neurons , or individual brain sites .\",\n \"Rather , it is the emergent property of a complete learning neural circuit in which the sites and plasticity mechanisms of learning are embedded .\",\n \"Only when we incorporate our current knowledge about the specific sites of learning and the rules of plasticity with an understanding of circuit-level interactions can we truly understand learning and memory .\",\n \"Here , our goal is to define the requisite set of circuit-level computational principles that operate during cerebellar-dependent motor learning .\",\n \"Arguably , motor learning is the domain that affords the best chance of understanding the principles of learning and memory , due to the exquisite relationship between sensory stimuli and adaptive motor behavior , combined with the tight link from known neural circuits to the output motoneurons .\",\n \"Across a wide range of movement modalities , motor learning depends crucially on the cerebellum ( Gilbert and Thach , 1977; Golla et al . , 2008; Herzfeld et al . , 2014a; Martin et al . , 1996; Medina and Lisberger , 2008; Smith and Shadmehr , 2005; Robinson , 1974 ) , providing a well-defined neural substrate to investigate how the fundamental principles of learning are implemented in a specific neural circuit .\",\n \"In the motor learning paradigm we study , direction learning in smooth pursuit eye movements , neurophysiological data has demonstrated that short-term motor learning , on the order of a single learning trial , occurs at or upstream of Purkinje cells in the floccular complex of the cerebellum ( Medina and Lisberger , 2008; Yang and Lisberger , 2013; Yang and Lisberger , 2014 ) .\",\n \"Single-trial learning is tightly linked to climbing fiber inputs , in agreement with the predictions of the classical theory of cerebellar learning ( Marr , 1969; Albus , 1971; Ito , 1984 ) .\",\n \"Crucially , Purkinje cells are only two synapses away from the motoneurons that drive the adaptive motor response we measure , significantly constraining the circuit architecture that implements the transformation from sensory inputs to adapted behavior across short timescales .\",\n \"However , recent behavioral results have suggested that longer term pursuit direction learning , on the order of hundreds to thousands of trials , may be mediated by multiple sites and/or learning mechanisms ( Hall et al . , 2018; Yang and Lisberger , 2010 ) .\",\n \"The existence of multiple components in pursuit learning is consistent with behavioral and computational evidence from other cerebellar-dependent motor learning behaviors ( Boyden et al . , 2004; Ethier et al . , 2008; Kojima et al . , 2004; Kording et al . , 2007; Lee and Schweighofer , 2009; Medina et al . , 2000; Smith et al . , 2006; Kassardjian et al . , 2005; Shutoh et al . , 2006 ) .\",\n \"While plasticity is possible across many synapses in the cerebellar circuit ( Carey , 2011; D'Angelo and De Zeeuw , 2009; Hansel et al . , 2001; McElvain et al . , 2010; Mittmann and Häusser , 2007 ) , a prime candidate for long-term learning is the deep cerebellar nucleus , one synapse downstream of the Purkinje cells .\",\n \"Convergent neurophysiological and behavioral evidence across multiple learning paradigms lends credence to role of the deep cerebellar nucleus for long-term storage of cerebellar-dependent memories ( Raymond and Medina , 2018; Kassardjian et al . , 2005; Shutoh et al . , 2006; Lee et al . , 2015; Raymond and Lisberger , 1998; Popa et al . , 2016; Broussard and Kassardjian , 2004; Lisberger , 1994 ) .\",\n \"Taken together , these results highlight the idea that multiple neurophysiological mechanisms within the cerebellar circuit may underlie behavioral motor learning across longer timescales .\",\n \"Our goal in the present paper is to elucidate the operation of the full and well-characterized cerebellar learning circuit .\",\n \"We identify the properties of the signals that guide acquisition and expression of motor learning across timescales through strategic manipulations of the experimental learning conditions coupled with quantitative measures of behavior .\",\n \"Our results suggest four circuit-level principles that define pursuit direction learning .\",\n \"First , rapid acquisition of learning occurs at the parallel fiber to Purkinje cell synapse , driven by climbing fiber responses , with limited retention .\",\n \"Second , learned responses in Purkinje cells guide the slow acquisition of learning in the deep cerebellar nucleus , which learns slowly but has excellent retention .\",\n \"Third , the functional properties of the inputs that are subject to learning are different in the cerebellar cortex and deep cerebellar nuclei .\",\n \"Finally , the extent of learning is limited by negative feedback from the deep nucleus to the inferior olive , reducing the teaching signal that drives learning in the cerebellar cortex .\"\n ],\n [\n \"Given the substantial neurophysiological evidence suggesting the primary role of complex-spike-linked plasticity at the parallel fiber to Purkinje cell synapse for single-trial motor learning , our first objective was to fully characterize the properties underlying the acquisition of a motor memory following a single movement error .\",\n \"To isolate the properties of short-term motor learning , we developed a ‘dual-trial’ experimental paradigm that is a small , but important , modification of our previous methods for studying single-trial learning ( see Materials and methods ) .\",\n \"In each pair of trials , the first trial is defined as the ‘learning’ trial .\",\n \"In the learning trial , the monkey begins to pursue a smoothly moving target in a randomly chosen ‘pursuit direction . ’ After 250 ms of target motion in the pursuit direction , the target abruptly changes direction due to the addition of a velocity component in an orthogonal ‘learning direction’ , either +90 or −90 degrees relative to the pursuit direction ( Figure 2A ) .\",\n \"The discrepancy between animal’s smooth eye movement in the original pursuit direction and target’s net direction due to the addition of orthogonal target motion creates a movement error that serves as an instruction for motor learning ( Figure 2B ) .\",\n \"We therefore refer to the addition of the target velocity component orthogonal to the original pursuit direction as the ‘instruction' . We measure motor learning in the subsequent ‘probe’ trial , where the target moves in the same pursuit direction as the learning trial but without any instruction .\",\n \"In probe trials , the animal exhibits a ‘learned response’ due to the error induced by the instruction in the preceding learning trial .\",\n \"The learned response ( Figure 2C , arrowhead ) starts about 200 ms after the onset of target motion in the pursuit direction , anticipating the change in target direction imposed in the preceding learning trial .\",\n \"We measure learning in the 50 ms interval surrounding the time of the instruction , from 225 to 275 ms after the onset of pursuit target motion .\",\n \"The measurement interval is chosen strategically to capture the anticipatory response of the pursuit system at the time of the instruction .\",\n \"Later in the paper , it also allows us to measure the learned response even in learning trials , because the measurement interval precedes any visually driven eye movement resulting from retinal image motion caused by the instruction ( Hall et al . , 2018; Yang and Lisberger , 2017; Medina and Lisberger , 2008 ) .\",\n \"We first characterized the dependence of single-trial learning on the magnitude of the error imposed in the learning trial .\",\n \"To ensure that we were measuring only the learned response due to the occurrence of a single movement error , we prevented any long-term learning by choosing the pursuit direction for each learning-probe pair randomly from the cardinal axes ( see Materials and methods ) .\",\n \"In each learning trial , we also chose the speed of the instruction randomly to be 0 , 5 , 10 , 15 , 20 , 25 , or 30 deg/s ( Figure 3A ) , and measured the effect of varying instruction speed on the learned response in the subsequent probe trial .\",\n \"Single-trial learning using randomized pursuit directions creates a situation where the error magnitude experienced in the learning trial is identical to the imposed instruction magnitude , because the average eye speed in the learning direction during the learning trial is zero at the time of the instruction .\",\n \"This allowed us to control the size of the error that drives learning and to measure directly the shape of the relationship between learning and error .\",\n \"In both monkeys , learned responses in the probe trial increased as a function of the error magnitude experienced in the learning trial ( Figure 3B ) .\",\n \"Quantitative analysis revealed that the learned response did not increase linearly as a function of error magnitude in the learning trial , but rather saturated as error increased ( Figure 3C ) .\",\n \"We quantified the effect of error magnitude on single-trial learning as a sigmoid of the form: ( 1 ) Y2E1=a1+e-τE1-a2where E1 is the magnitude of the error imposed on the learning trial and Y2 is the measured learned response in the subsequent probe trial .\",\n \"The parameters a and τ were obtained by fitting Equation 1 to the data for each monkey in Figure 3C ( dashed traces versus connected , filled , symbols ) .\",\n \"Equation 1 fitted the data well for both monkeys ( Monkey RE: a = 1 . 35 , τ = 0 . 21 , R2 = 0 . 99; Monkey YO: a = 1 . 25 , τ = 0 . 118 , R2 = 0 . 98 ) .\",\n \"The sigmoidal model of single-trial learning was superior to an alternative linear model ( Akaike’s information criterion ( AIC ) ( Akaike , 1974 ) , Monkey RE: -53 . 3 ( sigmoid ) versus -14 . 5 ( linear ) ; Monkey YO: -29 . 2 ( sigmoid ) versus -20 . 5 ( linear ) ) .\",\n \"Our next step was the characterize the stability of short-term pursuit learning across trials that did not include a movement error .\",\n \"To do so , we used a modified experimental design where the first 20 trials were standard learning trials with an instruction magnitude of 30 deg/s .\",\n \"The subsequent 10 trials were ‘error-clamp’ trials ( Figure 3D ) that we used to estimate retention in the absence of movement error ( after Scheidt et al . , 2000 ) .\",\n \"Error-clamp trials used the same pursuit target motion as in the instruction trials , but the motion of the target in the learning direction was yoked to the animal’s eye movement in that direction , to ensure that the animal experienced no visual error signals in the learning direction .\",\n \"Thus , any trial-to-trial change in the animal’s learned response over the course of 10 error-clamp trials was due solely to forgetting of the previously acquired short-term motor memory .\",\n \"We normalized the learned response in the 10 error-clamp trials by the magnitude of the animal’s learned response in the final learning trial of the 20-trial learning block .\",\n \"Both monkeys demonstrated an exponential decay in the magnitude of the learned response across error-clamp trials ( Figure 3E ) .\",\n \"Fitting an exponential model to each monkey’s retention data ( Figure 3E , black lines ) suggested a retention constant of approximately 0 . 85 ( 95% confidence intervals based on four experiments per monkey , Monkey RE: 0 . 80–0 . 86 , Monkey YO: 0 . 85–0 . 91 ) .\",\n \"Therefore , in the absence of any error to drive learning , approximately 15% of the short-term learned response is forgotten on each trial .\",\n \"We performed this experiment only for instruction speeds of 30 deg/s because the larger amplitude of learning provided more reliable estimates of retention .\",\n \"We next sought to use generalization of learning to constrain the functional properties of the site of plasticity that causes single-trial motor learning .\",\n \"Our strategy is based on the following line of reasoning .\",\n \"Imagine , for example , that the input signals that are subject to single-trial learning are linearly related to eye speed in the pursuit direction ( ve ) and that plasticity operates as a scalar gain ( g ) .\",\n \"Then , the learned change in firing of the relevant post-synaptic cells should be proportional to gve .\",\n \"As a result , the learned eye speed should generalize linearly as a function of pursuit speed in the probe trial .\",\n \"We can invert this logic to use the measured characteristics of generalization of learning to reveal the functional properties of signals that undergo plasticity .\",\n \"Under the assumption that the site of plasticity for single-trial learning acts as a scalar gain , if learned eye speed generalizes linearly with pursuit eye speed in the probe trial then the population response of neurons upstream from the site of plasticity should be linearly related to eye speed in the pursuit direction .\",\n \"We again employed our dual-trial paradigm with a randomized pursuit direction for each pair of learning and probe trials .\",\n \"Here , the learning trials always had a pursuit speed of 20 deg/s and an orthogonal instruction speed of 30 deg/s .\",\n \"The pursuit speed of the target in the probe trial was selected randomly from 5 , 10 , 15 , or 20 deg/s ( Figure 4A ) .\",\n \"We found that average eye speed in the learning direction scaled with the speed of the probe target for both monkeys RE and YO , especially in the interval from 225 to 275 ms after the onset of target motion ( Figure 4B , shaded area ) .\",\n \"We note that scaling is less clear earlier in the probe trial for Monkey YO , partly because the animal’s eye speed in the pursuit direction scales poorly with target speed during this earlier interval .\",\n \"A RM-ANOVA showed a main effect of eye speed in the pursuit direction in the probe trial on the amount of measured behavioral learning for both monkeys ( Monkey RE: F ( 3 , 6 ) =24 . 3 , p<10−3 , Monkey YO: F ( 3 , 15 ) =13 . 7 , p<0 . 001 ) .\",\n \"For both monkeys , the effect of eye speed in the pursuit direction on the learned response from 225 to 275 ms was highly linear ( Figure 4C , Monkey RE: R2 = 0 . 99 , Monkey YO: R2 = 0 . 97 ) .\",\n \"These results suggest that signals related to the speed of eye motion in the pursuit direction may be part of the substrate that is subject to plasticity .\",\n \"The linear generalization of single-trial learning with pursuit direction eye speed is consistent with prior observations that the eye speed at the time of the instruction linearly modulates the expression of behavioral learning ( Hall et al . , 2018 ) .\",\n \"In contrast , target/eye speed in the pursuit direction during the learning trial did not affect the expression of single-trial learning on the subsequent probe trial .\",\n \"Here , we again used the dual-trial paradigm with pursuit speed randomly chosen to be 5 , 10 , 15 , or 20 deg/s in the learning trials but always 20 deg/s in the probe trials .\",\n \"The instruction occurred 250 ms after the onset of target motion and had a magnitude of 30 deg/s .\",\n \"The direction of the instruction was chosen randomly to be either +90° or −90° relative to the pursuit direction .\",\n \"Both Monkeys RE and YO ( Figure 4D ) showed no effect of pursuit speed in the learning trial on the magnitude of the learned response measured in the probe trials .\",\n \"A RM-ANOVA failed to show a significant effect at 250 ms in the probe trial ( Figure 4E , Monkey RE: F ( 3 , 15 ) =0 . 56 , p=0 . 65 , Monkey YO: F ( 3 , 15 ) =0 . 39 , p=0 . 76 ) .\",\n \"Thus , we can conclude that target/eye speed in the pursuit direction has distinct and very different effects on the acquisition of a motor memory during the learning trial versus on the expression of learning in the probe trial .\",\n \"Previous neurophysiological results suggest that complex-spike-linked plasticity at the parallel fiber to Purkinje cell synapse in the cerebellar cortex is likely the primary driver of single-trial pursuit learning ( Medina and Lisberger , 2008; Yang and Lisberger , 2014 ) .\",\n \"Combining these neurophysiological observations with our behavioral results thus far allows us to begin to constrain the parameters of a cerebellar model of motor learning .\",\n \"A model of short-term pursuit learning appears in Figure 5A ( Source code 1 ) .\",\n \"Here , we chose to model the activity of parallel fibers as linearly related to eye speed in the pursuit direction , to account for the generalization of single-trial learning ( Figure 4B–D ) .\",\n \"We use only two parallel fibers with opposite preferred directions ( more parallel fibers are trivially possible but unnecessary ) with an arbitrary scaling parameter , r , that converts eye velocity to spikes/s: ( 2 ) PF1∝rE˙PF2∝−rE˙ In the absence of learning , the parallel fiber inputs cancel perfectly , ensuring that the firing of the post-synaptic Purkinje cell does not modulate in relation to eye movement in the pursuit direction , consistent with experimental data ( Medina and Lisberger , 2008 ) .\",\n \"We can model Purkinje cell firing on the nth trial , PCn , as the weighted contributions of the pre-synaptic parallel fibers and a background response , PC0: ( 3 ) PCn=wn1PFn1+wn2PFn2+PC0 The synaptic weight for each parallel fiber , wi , is adaptable via the complex spikes caused by climbing fiber inputs .\",\n \"The plasticity rule includes two terms: ( 1 ) a decay term , αPF , that allows the relaxation of any learned weight changes back to their baseline value in the absence of error and ( 2 ) an update term that depresses weights by a fractional amount ( β ) depending on the positive firing on the parallel fiber in question and the probability of a complex spike given the neural representation of the image motion from the instruction , PCSEn: ( 4 ) wn+1i={wni− ( 1−αPF ) ( wni−w0i ) −βP ( CS|En ) if PFi > 0wni− ( 1−αPF ) ( wni−w0i ) if PFi ≤ 0 When αPF=1 , the weights are maintained completely from one trial to the next and learning is allowed to perfectly accumulate; when αPF<1 , the weights decay back to their baseline levels , w0i , in the absence of any climbing fiber input .\",\n \"We model only learning for instructions in the direction that causes complex spikes so that we do not need to explicitly simulate synaptic potentiation beyond the relaxation back toward the baseline weights provided by αPF .\",\n \"Our results in Figures 3 and 4 suggest appropriate values for the unknown parameters of this simplified cerebellar model of learning .\",\n \"First , we know that , in the absence of error , short-term motor learning decays across trials .\",\n \"The time constant analysis in Figure 3E suggests that αPF≈0 . 85 for both monkeys .\",\n \"In addition , Equation 1 describes how the parallel fiber to Purkinje cell weights are modified as a function of error during single-trial learning: Y2E1∝βPCSE1 .\",\n \"Previous neurophysiological data has demonstrated that the probability of observing a complex spike following a 30 deg/s instruction is approximately 30% ( Yang and Lisberger , 2014; Yang and Lisberger , 2017 ) .\",\n \"Given these observations , we can derive a rule linking the probability of observing a complex spike to a given error magnitude using a sigmoid function with the same form as Equation 1: ( 5 ) PCSEn=0 . 61+e-τEn-0 . 3 Here , the probability of observing a complex spike saturates at 30% for large error magnitudes .\",\n \"The value of τ matches the values we obtained for each monkey in Equation 1 .\",\n \"Purkinje cells inhibit the floccular target neurons ( FTNs ) in the vestibular nucleus which , in turn , modulate eye movements via their projections to motoneurons .\",\n \"In the absence of other inputs to FTNs that are modulated by learning , the change in the firing of FTNs from their baseline activity is FTNn=PC0-PCn .\",\n \"The resulting learned response , Yn , measured via generated eye movements is then Yn= cFTNn , where c is an arbitrary constant that scales FTN activity to deg/s .\",\n \"Equations 2-5 describe a 'single-learning-process' model .\",\n \"The constants , r , β , w0i and c are arbitrary and affect only the scaling of firing rates to eye movements .\",\n \"The values we chose for these parameters are shown in Table 1 , but any number of parameter combinations would give qualitatively similar behavioral results .\",\n \"The single-learning-process model appropriately estimated our behavioral observations for two different stimulus conditions , one that was more-or-less built into the model and one that matches our data as an emergent property of the model .\",\n \"First , the trial-to-trial change in output following a range of imposed error magnitudes closely matched the learned behavior of both animals ( Figure 5B ) , because this relationship was built into the model based on the data in Figure 3C .\",\n \"Second , the model reproduced the results of an experiment that was a superset of the conditions used in Figure 4A–C .\",\n \"Here , we presented a series of dual-trial stimuli where the instruction trial used a randomly chosen pursuit speed of 0 , 10 , 15 , or 20 deg/s and delivered an instruction at 30 deg/s .\",\n \"The probe trial also delivered a pursuit speed that was randomly chosen from 0 , 10 , 15 or 20 deg/s .\",\n \"For each probe trial , we computed the ‘learning expression ratio’ as the magnitude of the learned response expressed in each probe trial normalized by the average learning expressed in probe trials with the same pursuit speed as the preceding learning trial .\",\n \"When the pursuit speed in probe trial matched the pursuit speed in the learning trial , the learning expression ratio is ~1 . 0 .\",\n \"We then plotted the learning expression ratio as a function of the ratio of the pursuit speeds of the probe trial and the preceding learning trial to assess the generalization function of learning across any combination of pursuit speeds in the learning and probe trials .\",\n \"The model reproduces the results of this experiment on both monkeys ( Figure 5C ) , demonstrating strong linear generalization across probe pursuit speeds .\",\n \"In summary , a cerebellar model with a single learning site where plasticity operates on signals linearly related to pursuit eye speed predicts all of our behavioral data for single-trial learning .\",\n \"In the more natural situation where the same instruction for learning occurs on movement after movement and is corrected gradually , learning could be simply due to an accumulation of single-trial learning plus forgetting at the rate of 15% per trial .\",\n \"At some point , the forgetting and learning on each trial would balance and the learning curve would reach an asymptote .\",\n \"To provide a dataset to ask whether the ‘single-learning-process’ model of single-trial cerebellar learning with forgetting ( Equations 2-5 ) can explain the accumulation of behavioral learning across multiple learning trials , monkeys completed 100 consecutive learning trials .\",\n \"Each learning trial used identical pursuit and learning directions and speeds .\",\n \"Within each learning block of 100 trials , the instruction magnitude was either 0 , 5 , 10 , 15 , 20 , 25 , or 30 deg/s .\",\n \"Note that during 100-trial learning blocks , we control the magnitude of the instruction , but the magnitude of the error on any given trial is the difference between the instruction and the monkey’s expression of behavioral learning on that trial .\",\n \"When the same learning trial is repeated , acquisition of behavioral learning leads to a gradual reduction in the magnitude of the error for a given magnitude of instruction .\",\n \"Therefore , we use the terms ‘instruction magnitude’ and ‘error magnitude’ carefully to convey this distinction .\",\n \"For each instruction magnitude , the trial-course of learning revealed dual-exponential learning curves that largely saturated by the end of 100 learning trials ( Figure 6A ) .\",\n \"Yet , even after 100 consecutive learning trials , the magnitude of the animal’s learned response was always much smaller than the speed of the imposed instruction: neither animal ever exhibited full compensation for the imposed instruction ( Figure 6B ) .\",\n \"It is especially noteworthy that the system is able to achieve a learned response of 5 deg/s after 100 learning trials for an instruction at 30 deg/s , but not for an instruction at 5 deg/s .\",\n \"The effect of instruction magnitude on the learned response was much more linear after 100 consecutive learning trials than after a single learning trial .\",\n \"The learned eye speed in the last 25 trials of the learning epoch was linearly related to instruction magnitude for both monkeys ( Monkey RE: R2 = 0 . 99 , t-test on the slope of the regression fit: t ( 5 ) =21 . 9 , p<10−4; Monkey YO: R2 = 0 . 99 , t ( 5 ) =18 . 9 , p<10−4 ) .\",\n \"After 100 learning trials , a linear model was preferred when compared against the best-fit saturating model from Equation 1 ( AIC , Monkey RE: 7 . 7 ( sigmoid ) versus 1 . 5 ( linear ) ; Monkey YO: 0 . 6 ( sigmoid ) versus 0 . 02 ( linear ) , Figure 6C ) .\",\n \"The difference in the effect of instruction magnitude on learning after 1 versus 100 learning trials hinted that multiple plasticity mechanisms and/or sites within the cerebellar circuit could be driving learning across the different trial-courses .\",\n \"Using the single-learning-process model , we simulated blocks of 100 consecutive learning trials .\",\n \"In each trial , we drove learning with an error that was the difference between model output and In , the imposed instruction: En≡In-cFTN , simulating the gradual reduction in the error signal for a fixed instruction magnitude due to the acquisition of learning across trials .\",\n \"The single-learning-process model failed to predict two crucial features of the true trial-course of learning .\",\n \"First , it produced single exponential trial-courses that learned too quickly and reached asymptote within the first 20 learning trials ( Figure 7A ) .\",\n \"Second , it systematically over-estimated the extent of asymptotic learning for instruction magnitudes between 5 and 20 deg/s ( Figure 7B , arrows ) and underestimated learning for instruction magnitudes of 30 deg/s .\",\n \"Indeed , the model predicted a sigmoidal relationship between asymptotic learning and instruction magnitude ( Figure 7B ) , inherited from the relationship between single-trial learning and instruction magnitude , in contrast to the highly linear relationship in the data ( Figures 6C and 7B ) .\",\n \"We note that we did not fit the model to the data .\",\n \"Instead , we used the parameters derived from our behavioral experiments that allowed Equations 2-5 to predict the details of single-trial learning .\",\n \"We therefore conclude that pursuit learning cannot be mediated solely by the accumulation of single-trial learning with forgetting ( Yang and Lisberger , 2010; Hall et al . , 2018 ) .\",\n \"Our next step was to identify the minimal principles of a cerebellar circuit model that can explain behavioral learning across multiple timescales .\",\n \"The features of behavioral learning in Figures 3 and 4 mandate several properties of a more complete cerebellar model: ( 1 ) acquisition of single-trial learning saturates as a function of the magnitude of the error according to a sigmoidal relationship dictated by the data in Figure 3C; ( 2 ) at least across the 100 learning trials we tested in Figure 6 but also after 2000 trials ( Hall et al . , 2018 ) , the asymptotic response for a consistent instruction magnitude falls short of completely correcting the movement error created by the imposed instruction; ( 3 ) the trial-course of learning across long bouts of consecutive learning trials ( Hall et al . , 2018 ) and the different relationship between learned response and error magnitude after 1 versus 100 trials suggest the presence of at least two learning processes ( Hall et al . , 2018; Kojima et al . , 2004; Lee and Schweighofer , 2009; Smith et al . , 2006 ) .\",\n \"To test our conclusion about multiple learning processes contributing to cerebellar learning across longer learning sessions , we next asked whether the retention of the motor memory changed across long bouts of learning trials .\",\n \"We used the same error-clamp strategy as before , but now to measure the retention constant after blocks of >1000 identical learning trials .\",\n \"At the end of long blocks of learning trials , we alternated short blocks of 10 error-clamp trials with runs of 20 learning trials to ‘top up’ the asymptotic learning .\",\n \"We found significantly better retention after 1000 trials than after 20 trials ( Figure 8A ) : the measured retention constant after 1000 trials was approximately 0 . 95 ( 95% confidence intervals , Monkey RE: 0 . 95–0 . 97 , Monkey YO: 0 . 94–0 . 96 ) ) .\",\n \"These data suggested that the motor memory gradually transitions , at least conceptually , from a relatively labile , low-retention learning process early in learning to a second learning process with high retention ( Smith et al . , 2006; Herzfeld et al . , 2014a ) .\",\n \"Armed with knowledge that learning appears to transition from a low-retention learning process to a higher retention process after extended learning , we expanded our circuit model of cerebellar learning to include a secondary site of plasticity ( Figure 8B , Source code 2 ) .\",\n \"Given neurophysiological results from other cerebellar-dependent learning tasks ( Kassardjian et al . , 2005; Shutoh et al . , 2006; Lee et al . , 2015; Raymond and Lisberger , 1998; Lisberger , 1994 ) as well as the proximity of the cerebellar circuit to motoneurons , we view the floccular target neurons , or FTNs , in the vestibular nucleus as the most likely candidate for long-term storage of learning .\",\n \"FTNs are immediately downstream of the Purkinje cells , the presumed site of rapid learning , and synapse onto output motoneurons .\",\n \"We modeled the learned responses of FTNs on trial n as the sum of the firing of input axons , INni , weighted by the scalar synaptic weight , vni , minus the baseline-subtracted firing of the Purkinje cell: ( 6 ) FTNn=∑ivniINni- ( PCn-PC0 ) To account for a secondary site of plasticity within the learning circuit , we implemented Hebbian-style plasticity at the synapse from the eye movement inputs to FTNs .\",\n \"Plasticity at the inputs to FTNs was driven by the learned responses of presynaptic Purkinje cells: ( 7 ) vn+1i=vni+η ( PC0-PCn ) INni Here , η determines the strength of the Purkinje cell’s teaching signal and the rate of plasticity in the inputs to FTNs .\",\n \"The value of η was set to be small so that the FTNs represented the site of a slow-learning process , in contrast to the rapid learning at the parallel fiber to Purkinje cell synapse .\",\n \"Based on our behavioral results in Figure 8A , plasticity in the slow-learning-process is retained almost perfectly , so that Equation 7 lacks the forgetting factor provided by αPF in Equation\",\n \"4 . Together , Equations 6 and 7 assume that the learning in the Purkinje cell responses acts as a teacher for long-term plasticity at the inputs to the FTNs .\",\n \"Simulation of Equations 2-7 for a variety of instruction magnitudes ( Figure 8C ) correctly demonstrates one crucial feature of our long-term learning data .\",\n \"The simulated learning is dual-exponential , with initial fast acquisition and a second , slower , timescale of acquisition .\",\n \"However , the simulations do not predict a crucial aspect of our behavioral data .\",\n \"Due to the stability of the long-term memory at the FTN inputs , the asymptotic response of the model is much larger than is found in our data .\",\n \"Also , Figure 8C shows that the slow-learning process produces a linear relationship between asymptotic learning and instruction magnitude , but only after 2000 trials and not after 100 trials ( vertical dashed line ) as seen in the data .\",\n \"The excessive learning in the model of Figure 8B suggests the existence of inhibition within the learning circuit .\",\n \"Multiple deficiencies of the model in Equations 2-7 are corrected when recurrent inhibition from the cerebellum to the inferior olive modulates the error signals used to drive learning at the parallel fiber to Purkinje cell synapse .\",\n \"We modeled inhibition by reducing the magnitude of the error signal that drives climbing fiber responses in proportion to the size of the learned response: ( 8 ) En=In−cFTNn−γcFTNnE˙ Here , In is the speed of the instruction on the nth trial and cFTNn is the FTN firing converted to units of learned eye velocity .\",\n \"The term ( In-cFTNn ) in Equation 8 represents the physical error that results from the difference between the instruction magnitude and the response of FTNs .\",\n \"This formula makes the distinction between the physical error , ( In-cFTNn ) , and the learning system’s internal representation of this error used to drive learning in the cerebellar cortex ( En ) .\",\n \"The last term in Equation 8 implements modulation of the internal representation of the physical image motion in proportion to γ and the learned response , cFTNn .\",\n \"There is no recurrent control when the value of γ is zero .\",\n \"The modulation is normalized by eye speed in the pursuit direction ( E˙ ) so that inhibition of the climbing fiber input occurs in proportion to the change in weight of the parallel fiber to Purkinje cell synapse .\",\n \"The combination of the Purkinje cell modulation as a teacher ( Equation 7 ) as well as the presence of recurrent inhibition ( Equation 8 ) transfers the motor memory from the cerebellar cortex to the vestibular nucleus across long blocks of learning trials .\",\n \"We note that the probability of complex spikes decreases considerably across 100 repetitions of the same learning trial ( Yang and Lisberger , 2017 ) , compatible with the mechanism implemented by Equation 8 .\",\n \"We adjusted the strength of the teaching signal from the Purkinje cells to the FTN inputs ( η ) and the strength of recurrent inhibition of the instructive signal ( γ ) to allow the model in Figure 8B and Equations 2-8 to reproduce both the two-component trial-course of learning ( Figure 8D ) and the correct linear relationship between the asymptotic learning and instruction magnitude after 100 learning trials ( Figure 8E ) .\",\n \"The best-fit model ( two unknowns , η and γ ) agrees well with the data for both monkeys ( Figure 8E , black curves , Monkey RE: R2 = 0 . 99; Monkey YO: R2 = 0 . 97 ) .\",\n \"In addition , both monkeys had similar values for both estimated parameters ( 95% confidence intervals , Monkey RE: η = 5 . 1×10−4–9 . 2 × 10−4 , γ = 55 . 9–68 . 5 , Monkey YO: η = 4 . 5×10−4–9 . 4 × 10−4 , γ = 38 . 8–65 . 0 ) .\",\n \"Linearity emerges after 100 trials because recurrent feedback switches the mechanism driving asymptotic behavior from a tradeoff between learning and forgetting to an emergent property of the operation of a dynamic feedback loop .\",\n \"The successful model in Figure 8B has three crucial features: ( 1 ) a site of rapid learning with limited retention at the parallel fiber to Purkinje cell synapse , ( 2 ) gradual transfer of learning from the cerebellar cortex to a slower-learning process with excellent retention at the inputs to the FTNs , and ( 3 ) recurrent inhibition from FTNs to the inferior olive that limits learning and linearizes the stimulus-response function of learning by controlling the error signals available to drive learning at the parallel fiber to Purkinje cell synapse .\",\n \"We next provide additional behavioral evidence for separate sites of learning within the cerebellar circuit .\",\n \"By probing the expression of learning as a function of pursuit speed across the trial-course of learning , we show that the linear pattern of generalization to pursuit speed we observed following a single learning trial ( Figure 4 ) changes gradually but qualitatively across 1000 trials .\",\n \"We suggest that the site and/or mechanism of learning changes across a long trial-course .\",\n \"We again deployed the dual-trial paradigm , but with the modification that the instruction trial in each pair of trials repeated the same fixed parameters of pursuit ( 5 deg/s or 20 deg/s ) , instruction magnitude ( 30 deg/s ) , instruction direction , and pursuit direction for the entire experimental session .\",\n \"We studied generalization across the gradual accumulation of learning by varying target speed in the pursuit direction randomly in the probe trial to be 5 , 10 , 15 or 20 deg/s , while keeping the pursuit direction the same as in the learning trials .\",\n \"An example experiment demonstrates how generalization changed over a long trial-course when pursuit speed was 5 deg/s in the learning trials and was always equal or faster in the probe trials .\",\n \"Averages of the trajectory of the learned eye velocity as a function of time during the first 250 trials confirmed that the learned response scaled with pursuit speed in the initial stages of learning ( Figure 9A , left ) , in agreement with the data for single-trial learning in Figure\",\n \"4 . However , at the end of the same learning session , the expression of learning no longer scaled with pursuit speed in the probe trials ( Figure 9A , right ) .\",\n \"The change in generalization across the session could not be explained by any systematic differences in the eye speed in the pursuit direction during the first versus last 250 trials ( Figure 9B , continuous versus dashed traces ) .\",\n \"The change in the generalization of the expression of learning in the probe trials occurred gradually ( Figure 10A ) .\",\n \"Across 2250 trials ( 1125 dual-trial stimuli ) in Monkey RE , the expression of learning declined for probes with pursuit speeds of 20 deg/s and remained constant for probes with pursuit speeds of 5 deg/s .\",\n \"By 2250 trials , the learned response did not depend on the target speed in the probe , in agreement with the right graph of Figure 9A .\",\n \"The observations were similar in Monkey YO , although he did not work for as many trials as Monkey RE ( note the shorter x-axis ) .\",\n \"These results suggest that something about the inputs that are subject to plasticity or the mechanism of plasticity at the site of single-trial , short-term , learning may differ from those to the site of long-term pursuit learning .\",\n \"We observed a qualitatively different pattern of generalization across learning blocks when pursuit speed was 20 deg/s in the learning trials and was always equal or slower in the probe trials .\",\n \"Now , the expression of learning increased slightly for probe trials with a pursuit speed of 20 deg/s and decreased as a function of trial when the pursuit speed in the probe trial was 5 deg/s ( Figure 10B ) .\",\n \"Together , the results of learning with pursuit target speeds in the learning trials of 5 and 20 deg/s show a general rule: learning generalizes approximately linearly for pursuit speed across all timescales of learning when the pursuit speed in the probe trial is slower than the learning trial; when pursuit speed in the probe trial is faster than that in the learning trial , linear generalization early in learning is replaced gradually with a learned response that appears largely independent of pursuit speed after long-term learning .\",\n \"To verify the statistical veracity of the effects in Figure 10A and B , we performed a regression analysis on the trial-course of generalization of each experiment ( lines in Figure 10A and B ) .\",\n \"For each experimental condition , we measured the rate of change of the learned response across trials ( Figure 10C ) .\",\n \"Here , a negative slope indicated that the learned response for a particular probe pursuit speed decayed over the experiment ( e . g . 20 deg/s probe trials when the pursuit speed of the learning trial was 5 deg/s ) .\",\n \"Positive slopes indicated that the learned response measured in the probe trials tended to increase across the experiment , as was the case when the pursuit speed in both the learning trial and the probe trial was 20 deg/s ( Figure 10B , red curves ) .\",\n \"A two-way ANOVA confirmed the differential effect of pursuit speed in the learning trials on the generalization properties of the motor memory .\",\n \"We tested for main effects of probe pursuit speed ( 5 , 10 , 15 or 20 deg/s ) , pursuit speed in the learning trials ( 5 deg/s or 20 deg/s ) , and their interaction .\",\n \"For both monkeys , we observed a main effect of pursuit speed in the learning trials ( RE: F ( 1 , 32 ) =46 . 8 , p<10−7; YO: F ( 1 , 20 ) =31 . 6 , p<10−4 ) , no main effect of probe speed ( RE: F ( 3 , 32 ) =1 . 39 , p=0 . 26; YO: F ( 3 , 20 ) =0 . 33; p=0 . 81 ) , and crucially a significant learning pursuit speed by probe pursuit speed interaction ( RE: F ( 3 , 32 ) =14 . 2 , p<10−5 , YO: F ( 3 , 20 ) =9 . 0 , p<10−3 ) .\",\n \"Thus , the relationship between pursuit speeds of the target in the learning and probe trials significantly affected the shape of generalization across trials .\",\n \"To further validate the interaction between pursuit speed in the learning and probe trials , we conducted additional experiments in Monkey RE that tested other pursuit speeds in the learning trial ( not shown ) .\",\n \"Here , different experiments used one of either 5 , 10 or 20 deg/s as the consistent pursuit speed in the learning trials and probed with pursuit speeds selected randomly from the same three speeds .\",\n \"After 1000 learning trials , the expression of learning generalized approximately linearly as a function of slower pursuit speeds in the probe trial compared to the learning trial , but not for probe speeds faster than used in the learning trial , in agreement with Figure 10A and B . Thus , the rules for generalization depend not on whether the probe speed is the same or different from the pursuit speed in the instruction trials , but rather on whether it is faster or slower .\",\n \"These preliminary experiments also served as a control to ensure that the change in generalization across many trials was not a result of the alternation of learning and probe trials , because probe trials with the other two pursuit speeds were interspersed randomly among the learning trials with a ratio of 10 learning trials to every probe .\",\n \"In the previous section , we showed that learning generalizes differently in relation to pursuit target speed early versus late in a long learning block .\",\n \"Next , we analyze our data in a way that quantifies the shape of the pursuit speed generalization function at different stages of learning .\",\n \"We used the data from the long blocks of learning/probe pairs in Figure 10 , where the pursuit and instruction speed were fixed in the learning trial but pursuit speed in each interleaving probe trial was selected randomly among 5 , 10 , 15 , or 20 deg/s .\",\n \"Here , we computed the ‘learning expression ratio’ for each pair of trials as the magnitude of the learned response in the probe trial divided by the learning expressed in the preceding learning trial .\",\n \"The learning expression ratio is a single-trial metric of generalization , allowing us to probe the shape of generalization across long bouts of learning while controlling for the gradual accumulation of behavioral learning across the session .\",\n \"We then binned the resulting learning expression ratio by the ratio between the target pursuit speed in each probe trial and learning trial .\",\n \"When the probe trial and the preceding learning trial have the same pursuit speed ( probe speed to pursuit speed ratio of 1 . 0 ) , we would expect nearly complete generalization between the learning and probe trials ( a learning expression ratio ~1 . 0 ) .\",\n \"We refer to the relationship between the learning expression ratio and the ratio of the probe and learning trial pursuit speeds as the ‘generalization function' .\",\n \"In the first 250 trials of a long learning block ( blue curve , Figure 11A ) , both monkeys demonstrated an approximately linear generalization function , paralleling our finding of single-trial generalization in Figure\",\n \"5 . Thus , early in learning , both monkeys expressed greater ( less ) learning in the probe trial than in the learning trial when pursuit speed in the probe trial was faster ( slower ) than in the preceding learning trial .\",\n \"As learning progressed through a long block of trials ( Figure 11A , pink and purple traces ) , the shape of the generalization function changed gradually .\",\n \"After 1000 trials , probe trials with pursuit target speed faster than in the learning trial ( probe speed to pursuit speed ratio >1 . 0 ) no longer showed enhancement of the expression of learning ( learning expression ratio ~1 . 0 ) .\",\n \"The changing shape of the generalization function was further accentuated in Monkey RE after 2000 trials when probe trials expressed less behavioral learning ( learning expression ratio <1 . 0 ) if the pursuit speed was faster in the probe than in the learning trial .\",\n \"That the generalization function changes across 2000 trials indicates either that the properties of a single site of learning change or that learning transfers between sites with different functional properties .\",\n \"Cognizant that factors such as non-linear synapses and recurrent inhibition can control the properties of the signals at a site of learning , the simplest way to model generalization is in terms of the signals conveyed by the afferent fibers whose synapses are subject to plasticity .\",\n \"Figure 11 suggests that we can model learning under the assumption that the motor memory gradually transitions from an early site of learning where the inputs are linearly related to pursuit speed to a secondary site/synapse where the inputs are unimodally tuned for pursuit speed .\",\n \"We completed our model of cerebellar learning ( Figure 12A ) by defining the functional properties of non-Purkinje cell inputs to FTNs .\",\n \"We simulated 50 inputs to the FTNs , each with a preferred direction either equal to or opposite the pursuit direction and a Gaussian tuned response for pursuit speed: ( 9 ) INni=cos ( θne−θi ) exp ( − ( E˙−si ) 22σ2 ) Here , for the ith input axon , θi is either 0 or 180 deg , si is the preferred eye speed ranging from 0 to 50 deg/s , and σ is the standard deviation of the Gaussian function .\",\n \"The eye movement vector on the nth trial is defined as ( θne , E˙n ) .\",\n \"Note that any unimodal function ( e . g . cosine ) would yield comparable results to the Gaussian function .\",\n \"The circuit-level learning system defined by the schematic in Figure 12A and Equations 2-9 reproduces our data on both the trial-course of learning and the trial-course of the generalization of learning to pursuit speed .\",\n \"The learned response , like the real data , shows a saturating relationship to instruction magnitude for single-trial learning ( Figure 12B ) and a linear relationship after 100 trials ( Figure 12C ) .\",\n \"For learning with pursuit target motion at 5 deg/s , the expression of the learned response in the model generalizes differently early versus late in a 1000 trial learning block ( Figure 12D ) .\",\n \"Early in the learning block , the learned response generalizes linearly to pursuit speeds regardless of the pursuit speed in the learning trials .\",\n \"Late in the learning block , the learned response is largely identical across probe speeds .\",\n \"For learning with pursuit target motion at 20 deg/s , the learned response generalizes linearly across probe speeds lower than 20 deg/s for the entire duration of the learning block ( Figure 12E ) .\",\n \"The model also makes predictions .\",\n \"It suggests that adaptive changes in the Purkinje cell responses should diminish over the course of thousands of trials ( Figure 12F , G ) as learning transfers to the FTN inputs ( Figure 12H , I ) .\",\n \"We considered a number of other model forms besides our final cerebellar model ( Figure 12A ) .\",\n \"The criteria for a plausible model were imposed by our data for single-trial learning ( Figures 3 and 4 ) and repeated presentation of the same learning trial ( Figure 6 ) .\",\n \"Single-trial learning always showed a saturating relationship as a function of the error imposed by the instruction .\",\n \"Therefore , all candidate models were constrained by the fits in Figure 3C , ensuring that the measured learned behavior on trial 2 as a function of the error magnitude experienced on the first trial , Y2e1 , was approximately equal to Equation 1 .\",\n \"We then asked whether each candidate model could account for the nearly linear asymptotic learned response as a function of instruction magnitude after a block of 100 learning trials ( Figure 6C ) .\",\n \"We began by assuming the simplest model capable of reaching an asymptotic learned response that was less than the imposed instruction .\",\n \"This model featured a single learning process , x , with a retention parameter that allowed trial-to-trial forgetting: ( 10 ) xn+1=αxn+ΔYn ( en ) Yn=xn Here , α is the retention factor where α=1 indicates complete retention ( no forgetting ) and Yn is the measured behavioral response on trial n .\",\n \"The change in the measured amount of learning on each trial , ∆Ynen , is given by extending Equation 1 from the case of single trial learning ( trials 1 and 2 ) to trial-over-trial learning at any stage ( trials n and n+1 ) .\",\n \"The asymptotic response of this single learning process occurs when Yn+1≈Yn , which is given by: ( 11 ) Y∞=∆Y∞e∞1-α The form of the asymptotic response is a scaled version of the measured single-trial learning from Figure 3C and therefore cannot , by itself , reproduce the linear relationship between instruction magnitude and asymptotic learning that we measured after 100 consecutive learning trials .\",\n \"Any single-learning-process model that ( 1 ) has a static relationship between learning and error and ( 2 ) predicts saturating single-trial learning in Figure 3 would fail to predict the linear asymptotic learning after 100 repeated learning trials in Figure 6 .\",\n \"Pursuit learning might align with other models of motor learning that also have posited two ( Lee and Schweighofer , 2009; Smith et al . , 2006 ) or more ( Kording et al . , 2007 ) learning processes .\",\n \"For our system , learning could be the net sum of two learning processes that run independently and in parallel: a fast-learning process , xf , with rapid learning from error but limited retention and a slow-learning process , xs , with strong retention: ( 12 ) xn+1f=αfxnf+ΔYn ( en ) xn+1s=αsxns+ηsenYn=xnf+ xns A central requirement is that the retention of the slower-learning process is greater than the retention of the faster process , namely αs≫αf , and the rate of learning is greater in the fast-learning process versus the slow-learning process , ∆Ynen≫ηsen .\",\n \"As long as the fast-learning process shows a saturating relationship between error and single-trial learning , the model will reproduce the single-trial learning data in Figure 3C .\",\n \"The two-independent process model in Equation 12 fails to predict a linear relationship between the asymptotic response after extended learning and instruction magnitude .\",\n \"Given the retention values , we measured experimentally ( αf≈0 . 85 and αs>0 . 95 ) , the value of ηs must be very small to ensure that learning saturates at a level significantly below the imposed instruction after extended learning ( Figure 6A ) .\",\n \"With this constraint ( ηs≈0 ) , we can derive the asymptotic response of the two-learning-process model from Equation 12: ( 13 ) Y∞=x∞f+x∞s=ΔYn ( en ) ( 1−αs ) ( 1−αf ) ( 1−αs+η ) + ηI ( 1−αf ) ( 1−αs+η ) ≈Δxf ( e∞ ) 1−αf Equation 13 represents a scaled version of the single-trial response shown in Figure 3 and is largely identical to the asymptotic response predicted by a single-learning-process in Equation 11 .\",\n \"We expressly set out to model our data in a way that was driven by the known architecture of the cerebellar learning circuit .\",\n \"Adherence to the known circuit and the ability to reproduce a wide set of behavioral data with relatively simple assumptions are virtues of the model in Figure 12A .\",\n \"At the same time , we doubt that our successful model is unique and other strategies might ( or might not ) be able to reproduce our data as well .\",\n \"We give two examples .\",\n \"( 1 ) A single-site model might work if the rate of learning is not constant but rather changes from trial-to-trial .\",\n \"It would be possible to account for some of our data in a single-state model where complex-spike-mediated depression of the parallel-fiber to Purkinje cell synapse intrinsically weakened or saturated across repeated identical learning trials .\",\n \"( 2 ) Some models modulate learning based on Bayesian integration of noisy sensory feedback with internal estimates of the perturbation ( i . e . the Kalman filter ) , experienced errors ( Herzfeld et al . , 2014b ) , or environmental consistency ( Gonzalez Castro et al . , 2014 ) .\",\n \"Each of these models predicts that learning rate should increase for repeated presentations of the same learning stimulus versus single-trial learning , whereas we observed that the learning rate decreases over trials .\",\n \"However , one might contrive a model based on these principles where learning rate is lower when the stimulus regime is highly regular and consistent .\",\n \"In addition to their other failings , none of these approaches have the advantage of working seamlessly within the constraint of the architecture of the cerebellar learning circuit .\"\n ],\n [\n \"Our data add to the evidence for a fast-learning process with poor retention based on climbing-fiber mediated plasticity at the parallel to Purkinje cell synapse in the cerebellar cortex ( Medina and Lisberger , 2008; Yang and Lisberger , 2014; Nguyen-Vu et al . , 2013; Kimpo et al . , 2014 ) .\",\n \"Long-term depression at this particular synapse has been a staple of cerebellar learning for many years ( Ito , 2001; Marr , 1969; Albus , 1971 ) , and short-term forms of plasticity also occur at the same site ( Brenowitz and Regehr , 2005; Suvrathan et al . , 2016 ) .\",\n \"Still , the tight link between the occurrence of a complex spike and neural learning in Purkinje cell simple-spike responses ( and behavior ) does not prove that the parallel fiber to Purkinje cell synapse is the mechanism of the fast-learning process and we need to remain open to the possibility that the actual site of plasticity may be upstream of the parallel fiber to Purkinje cell synapse .\",\n \"We are able to measure the properties of the fast-learning process largely independent of other , slower , components of the recurrent learning circuit using the analysis of single-trial learning .\",\n \"Thus , we effectively measured the ‘open-loop’ features of the fast-learning process and discovered that the fast-learning process features a non-linear , saturating relationship between the magnitude of learning and the size of the movement error .\",\n \"We suspect that the non-linear relationship reflects the effects of the retinal slip on the probability and/or duration of the complex spikes that occur in Purkinje cells ( Mathy et al . , 2009; Najafi and Medina , 2013; Yang and Lisberger , 2014 ) .\",\n \"However , we cannot exclude two alternatives .\",\n \"The asymptote of single-trial learning for the highest instruction magnitude could reflect the influence of inhibitory neurons on the learning signal ( Rowan et al . , 2018 ) and/or a simple limit on the maximum amount of plasticity that can be induced by a single climbing fiber input to most Purkinje cells .\",\n \"The saturating dependence of single-trial adaptation in our system on instruction magnitude or stimulus strength parallels findings of single-trial learning in other cerebellar-dependent adaptive behaviors ( Fine and Thoroughman , 2006; Herzfeld et al . , 2014b; Marko et al . , 2012; Hutter and Taylor , 2018 ) .\",\n \"Our finding of qualitatively different generalization functions early versus late in learning provides experimental support for two distinct sites of learning , one that learns quickly and predominates early versus a second process that learns slowly and therefore predominates late in a 1000-trial learning block .\",\n \"Evidence that learning transfers from the fast- to the slow-learning site , rather than proceeding independently in parallel , comes from our computational and theoretical analysis .\",\n \"The cerebellar circuit model with recurrent inhibition of the error input to the cerebellar cortex can explain linearization of the relationship between the size of learning and instruction magnitude after 100 learning trials , whereas a standard model with independent fast and slow-learning processes would require considerable modification to do so ( see Equations 12 and 13 ) .\",\n \"Thus , we think that the fast-learning process for pursuit learning occurs in the floccular complex of the cerebellum ( Medina and Lisberger , 2008 ) and that the floccular target neurons in the vestibular nucleus ( Lisberger et al . , 1994 ) are likely the site of the slow-learning process and long-term storage .\",\n \"At this time , we cannot rule out the possible role of additional downstream areas .\",\n \"There are multiple reasons to think that learning could transfer from the cerebellar cortex to the deep cerebellar nuclei , even though there is currently no direct neurophysiological evidence .\",\n \"Mechanisms of plasticity are abundant in the deep cerebellar nuclei and optogenetic modulation of the activity of Purkinje cells can adapt the vestibulo-ocular reflex , presumably through Purkinje-cell induced plasticity in the deep cerebellar nuclei ( Jang et al . , 2020; Nguyen-Vu et al . , 2013 ) .\",\n \"Recordings from Purkinje cells and deep cerebellar nucleus neurons following long-term vestibulo-ocular gain adaptation lead to the conclusion of neural learning in both sites ( Lisberger , 1994 ) and are entirely compatible with the suggestion that the learned responses in Purkinje cells instruct learning in the deep cerebellar nucleus ( Miles and Lisberger , 1981 ) .\",\n \"For both eye movements ( Pastor , Pastor et al . , 1994 ) and classical conditioning of the eyelid response ( Garcia et al . , 1999 ) , inactivation or lesions of the cerebellar cortex prevents adaptation but does not fully abolish learned responses from previous adaptation ( McCormick and Thompson , 1984; Perrett et al . , 1993 ) , suggesting that the residual long-term learning resides outside of the cerebellar cortex .\",\n \"Finally , for learning in the optokinetic response , lesions of the flocculus abolish the learned response completely after short-term learning but only partially after long-term learning ( Shutoh et al . , 2006 ) .\",\n \"We think that the accessible neural substrate of pursuit learning affords the opportunity to study the neural mechanisms of memory transfer , a ubiquitous characteristic across memory systems .\",\n \"For example , different sites of early acquisition versus long-term storage exist for fear memories ( Rogan et al . , 1997 ) , ( Do-Monte et al . , 2015 ) , and in bird song ( Bottjer et al . , 1984; Nordeen and Nordeen , 1993; Warren et al . , 2011 ) .\",\n \"Indeed , the acquisition of a memory in one structure and the long-term storage of this memory in separate structures is clear from amnesic subjects , such as HM ( Zola-Morgan and Squire , 1990 ) , as well as from many more modern observations ( Tonegawa et al . , 2018 ) .\",\n \"In our data , the early learned response generalizes linearly as a function of all pursuit speeds in the probe trials , even when the probe speed is faster than the pursuit speed in the learning trial .\",\n \"After 1000 trials of learning , the learned response scales only for probe speeds slower than pursuit speed in the learning trial .\",\n \"The literature contains other evidence of changes in the generalization of the expression of learning across timescales .\",\n \"In birdsong , learned changes in the pitch of one syllable generalizes differently early versus late in learning ( Warren et al . , 2011 ) , suggesting that early learning in LMAN changes signals that are related to the overall song , while the late learning in the motor structure RA operates on signals related to the motor act of producing the syllable .\",\n \"In the vestibulo-ocular reflex , Kimpo et al . , 2005 showed different generalization for stimulus frequency for gain-up versus gain-down learning , suggesting different sites of learning based on modification of different vestibular signals .\",\n \"After Shadmehr , 2004 , we interpret the change in generalization of learning as possible evidence for a change in the properties of the neural signals that are subjected to learning .\",\n \"Our circuit model accounts for the change in generalization by assuming that a different set of eye speed signals is learned at Purkinje cells versus FTNs .\",\n \"The use in the model of different inputs to the sites of plasticity in the floccular complex and at the floccular target neurons suggests two features of the neural system that run counter to current doctrine .\",\n \"First , the general rule is that Purkinje cells and their targets in the deep cerebellar nuclei receive identical inputs from mossy fibers .\",\n \"In the floccular complex , however , this does not seem to be the case: brainstem areas that send afferent mossy fibers to the ventral paraflocculus region of the floccular complex send only sparse projections to the location of FTNs in the vestibular nucleus ( Osanai et al . , 1999; Nagao et al . , 1997 ) .\",\n \"Second , the general rule in the oculomotor system is the firing rate is linearly related to eye position and velocity , and this is true for mossy fibers in the floccular complex ( Miles et al . , 1980; Lisberger and Fuchs , 1978 ) .\",\n \"Thus , there is no obvious precedent for neural responses that are unimodally tuned for eye velocity .\",\n \"Limited recordings from FTNs during pursuit learning Joshua et al . , 2013 demonstrate a diversity of FTN velocity-dependent responses to a 30 deg/s pursuit stimulus , perhaps consistent with a distribution of preferred speeds across the neural population .\",\n \"We realize that the assumptions in the model may not reflect the actual implementation of the system in the brain , and that the differences in generalization at different sites could reflect properties of synapses or plasticity mechanisms rather than of the input signals that are subject to plasticity .\",\n \"We have always been troubled by the fact that pursuit learning , like other forms of motor learning ( Krakauer et al . , 2000; Tseng et al . , 2007; Vaswani et al . , 2015 ) , never reaches an amplitude that completely eliminates the error created by the imposed instruction .\",\n \"Even after more than 1000 learning trials , the adapted pursuit eye movements shows residual errors than are in excess of 50% of the imposed instruction ( Hall et al . , 2018 ) .\",\n \"We suggest that motor learning never fully adjusts for the instruction because of active inhibition of the instruction for learning Kenyon et al . , 1998 ) .\",\n \"Via the known double-inhibitory pathway from Purkinje cells to the inferior olive via the deep cerebellar nucleus ( Houck and Person , 2014; Najac and Raman , 2015; de Zeeuw et al . , 1988 ) , learned depression of Purkinje cell simple-spike firing could reduce the effectiveness of a given movement error by attenuating the probability and/or duration of climbing fiber inputs to the cerebellar cortex .\",\n \"Previous neurophysiological observations over 100 trials of pursuit learning demonstrate a reduction in the probability of complex spikes , hinting at possible inhibition of the olive ( Medina and Lisberger , 2008; Yang and Lisberger , 2017 ) .\",\n \"Our data and modeling render unlikely the alternate explanation that incomplete adaptation is the result of a competition between the learning signals and a restoring force due to forgetting ( Smith et al . , 2006; Albert et al . , 2019 ) .\",\n \"During error-clamp trials , we measured the retention parameter after 1000 trials to be close to 1 . 0 , corresponding to almost complete retention .\",\n \"We imagine that recurrent inhibition of the error inputs to the fast-learning process promotes slow , conservative learning that does not respond hastily to noisy inputs .\",\n \"We do not understand fully why it is advantageous for the fast-learning process to remain inhibited after learning has been transferred from the cerebellar cortex to FTNs .\",\n \"Perhaps the goal is to force other parts of the pursuit circuit to take over some of the learning under conditions where permanent changes seem desirable , by transferring some of the learning away even from the FTNs .\",\n \"Alternatively , our somewhat rarified stimulus conditions may not be engaging the neural learning circuit fully so that we are seeing only a part of the full learning capability of the system .\",\n \"The cerebellar learning circuit is stereotyped , well-known , accessible for analyses at the level of the activity of single neurons , and engaged in quantitatively-defined learning behavior .\",\n \"We see it as an exemplar where we have the greatest potential to determine not just the sites and mechanisms of plasticity , but also how the essential neural circuit exploits the local mechanisms of plasticity and converts them into the global deliverable of an adaptive change in behavior .\",\n \"Similar analysis and thinking are going to be essential in all other learning systems and neural circuits before we can say that we ‘understand’ any form of learning and memory .\",\n \"The particular behavioral system we study affords the huge advantage that we can constrain models by both the architecture of the essential circuit and data on the operation of the system .\",\n \"Our data and computational thinking assemble a statement of the principles of operation of this learning neural circuit .\",\n \"Once additional behavioral and neurophysiological evidence further refine these principles of operation , we will have a strong statement of how one learning circuit works .\"\n ],\n [\n \"Monkeys were positioned with their heads fixed 30 cm in front of a CRT monitor ( resolution: 2304 × 1440 , framerate: 80 Hz ) .\",\n \"The monitor subtended 58 × 46° of the monkey’s visual field .\",\n \"All experiments took place while the monkey was in a dimly lit room .\",\n \"The visual target for all experiments was a 0 . 5° diameter black dot presented on a light grey background ( approximate luminance 32 . 9 cd/m2 ) .\",\n \"The motion of the target at each frame was controlled by our laboratory’s custom ‘Maestro’ software .\",\n \"Separate voltage signals from the scleral coil system , corresponding to the monkey’s horizontal and vertical eye position , were digitized at 1 kHz .\",\n \"We also digitized eye velocity signals obtained by passing the eye position voltages through an analog differentiator circuit with a low-pass filter that reduced the amplitude of signals above 25 Hz ( −20 dB/decade ) .\",\n \"All signals were stored for later offline processing and analysis .\",\n \"At the start of each trial , the monkey fixated within ±3° of a target at the center of the screen for a random interval , chosen from a uniform distribution of 400–800 ms . After the fixation period , the target instantaneously jumped eccentrically by 0 . 15 vt degrees , and then began moving at a constant speed , vt , in the opposite direction , termed the ‘pursuit direction’ ( step-ramp paradigm of Rashbass , 1961 ) .\",\n \"The eccentric target jump minimizes the number of catch-up saccades during the initial tracking of the target .\",\n \"During baseline , probe , and washout trials , the target proceeded at the constant ‘pursuit speed’ for 850 ms before stopping .\",\n \"The monkey then fixated the target at its final position for an additional 200 ms . In exchange for appropriate tracking of the target as well as fixating the target within ±3° at the end of the trial , the monkey received a small fluid reward .\",\n \"If the monkey broke fixation during the initial fixation period or failed to adequately track the moving target and fixate its final position , the trial was aborted immediately and the monkey received no reward .\",\n \"Aborted trials were not included in the data analysis .\",\n \"To induce cerebellar-dependent motor learning , we used a direction learning paradigm ( Medina et al . , 2005 ) .\",\n \"In ‘learning trials’ ( Figure 2A , dotted line , Figure 2B , D ) , the target proceeded in the original pursuit direction for 250 ms , before the addition of an orthogonal velocity component , termed the ‘instruction . ’ We call the direction of the orthogonal instruction the ‘learning direction , ’ since the instruction induces visual motion that serves as an error and drives cerebellar-dependent motor learning .\",\n \"During the instruction , we suspended the eye position requirements for liquid reward .\",\n \"In some experiments , we varied the speed of the instruction .\",\n \"In all experiments , the instruction had a duration of 400 ms , so that it was followed by 200 ms of target motion in the original pursuit direction and then 200 ms of fixation .\",\n \"Reward was not contingent upon generation of a learned response .\",\n \"Monkeys typically worked for 1500 to 3000 successful trials per experimental session .\",\n \"To measure the amount of trial-to-trial forgetting in the absence of error , we used ‘error-clamp’ trials .\",\n \"During error-clamp trials , the target proceeded in the original pursuit direction , as in a probe trial .\",\n \"However , the location of the target in the direction orthogonal to the pursuit direction ( the learning direction ) was ‘clamped’ to the animal’s eye position , such that the position of the target on each of the monitor’s frames matched the animal’s current eye position in the learning direction ( Figure 3D ) .\",\n \"Target stabilization in the learning direction removes any errors that could drive unlearning and allowed us to measure the intrinsic retention/decay of motor memories without any stimulus for learning .\",\n \"To separately investigate the characteristics of the signals that drive learning of a motor memory from those that affect generalization on the timescale of a single-trial , we developed a ‘dual-trial’ experimental paradigm as a variant of our previously described paradigm for single-trial learning ( Yang and Lisberger , 2010 ) .\",\n \"Our modified paradigm uses pairs of trials , where the first trial , n , is the learning trial that provides a direction-change instruction ( Figure 2A , dotted line , Figure 2B ) and the second trial , n+1 , is a probe trial without an instruction ( Figure 2A , solid line , Figure 2C ) .\",\n \"Both the learning and probe trial in each dual-trial pair used the same pursuit direction , chosen randomly for each pair of trials from the cardinal axes ( 0° , 90° , 180° , or 270° ) .\",\n \"For some experiments , the speed of the target in the pursuit axis ( i . e . , the pursuit speed ) was identical in both the learning and probe trials .\",\n \"In other experiments , we strategically altered the pursuit speed in either the learning trial or the probe trial .\",\n \"The direction of the instruction in the learning trial was chosen randomly to be either +90° or −90° relative to the pursuit direction .\",\n \"Due to the random directions of the initial pursuit and the instruction within a daily experiment , learning did not accumulate across trials .\",\n \"We explicitly prohibited the consecutive occurrence of pairs of trials with the same pursuit and instruction directions .\",\n \"To quantify the behavioral learning expressed in the second trial of each pair , we measured the ‘learned response’ from the eye velocity in the direction of the instruction in the preceding learning trial ( i . e . , the learning direction ) .\",\n \"Monkeys typically performed 750 to 1500 pairs of trials in a given experimental session .\",\n \"We extended our dual-trial paradigm to investigate changes in the generalization of learning to pursuit speed across long blocks of trials .\",\n \"Now , we allowed the learning to accumulate by using a consistent pursuit direction in both the learning and probe trials across the entire experimental session .\",\n \"The pursuit direction for both the learning and probe trial was chosen randomly from the cardinal axes ( 0° , 90° , 180° , or 270° ) for each experimental session .\",\n \"Within an experimental session , the pursuit speed during the learning trial was either 5°/s or 20°/s .\",\n \"To measure the generalization of learning to pursuit speed , the speed of each probe trial was chosen randomly ( 5 , 10 , 15 , or 20 deg/s ) .\",\n \"For all experiments that used the dual-trial paradigm , we report statistics for each monkey across days/experimental sessions .\",\n \"To measure changes in the acquisition of a motor memory across timescales longer than a single-trial , we used blocks of trials with consistent instruction statistics .\",\n \"For each ‘learning block , ’ we chose a single direction of pursuit for all trials from one of the cardinal axes .\",\n \"Monkeys performed a series of baseline trials without an instruction , followed by a series of learning trials with an imposed instruction in a consistent learning direction that was orthogonal to the pursuit direction .\",\n \"The behavioral learning was subsequently extinguished by presenting washout trials that did not feature an instruction .\",\n \"The number of washout trials was always equal to or larger than the number of the preceding learning trials .\",\n \"Because the direction of the instruction was consistent across the learning trials , we define the behavioral learning on each trial as the speed of the eye movement in the direction of the instruction experienced during the learning trials .\",\n \"After a complete learning block , including washout , a new pursuit direction and instruction direction were chosen randomly for the subsequent block .\",\n \"Monkeys typically performed multiple repetitions of repeated-instruction learning paradigms in a given experimental session .\",\n \"No two consecutive learning blocks were allowed to have the same pursuit and instruction directions .\",\n \"For all experiments in a repeated-instruction design , we report statistics for each monkey across repetitions of the learning paradigm .\",\n \"To ensure that saccades did not contaminate our estimate of the learned pursuit response , we identified and removed saccades from eye velocity traces .\",\n \"We identified saccades using a combined speed and acceleration threshold: any instance when the speed of the eye exceeded 20 deg/s and the eye acceleration exceeded 1250 deg/s/s was labeled as a saccade .\",\n \"Time points were marked as a saccade from 10 ms before the first time point that exceeded joint velocity and acceleration thresholds to 10 ms after the final time point that exceeded both thresholds .\",\n \"We eliminated these intervals from data analysis by treating them as missing data .\",\n \"Because the target motions were designed to minimize their occurrence , saccades were typically infrequent in the analysis intervals and occurred at variable times .\",\n \"To summarize the magnitude of behavioral learning on a single-trial , we averaged the speed of the eye in the learning direction from 25 ms before to 25 ms after the time of the instruction , 225–275 ms after the onset of pursuit-direction target motion ( grey shading in Figure 2B , C ) .\",\n \"Even during trials that included an instruction ( e . g . during block-wise learning ) , this measurement provides an estimate of the learned response because the effects of visual feedback and online corrections in pursuit movements do not appear until at least 75 ms after the onset of the instruction .\",\n \"This measurement of motor learning is consistent with previous pursuit direction learning experiments ( Hall et al . , 2018; Yang and Lisberger , 2010; Yang and Lisberger , 2017 ) .\",\n \"We used two tailed t-tests to compare sample means for conditions that were not sampled within the same experimental session , with the significance level set at p=0 . 05 .\",\n \"Remaining tests with conditions that were simultaneously sampled within an experimental session were performed using a repeated measures analysis of variance ( RM-ANOVA ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["We provide behavioral evidence using monkey smooth pursuit eye movements for four principles of cerebellar learning .","Using a circuit-level model of the cerebellum , we link behavioral data to learning’s neural implementation .","The four principles are: ( 1 ) early , fast , acquisition driven by climbing fiber inputs to the cerebellar cortex , with poor retention; ( 2 ) learned responses of Purkinje cells guide transfer of learning from the cerebellar cortex to the deep cerebellar nucleus , with excellent retention; ( 3 ) functionally different neural signals are subject to learning in the cerebellar cortex versus the deep cerebellar nuclei; and ( 4 ) negative feedback from the cerebellum to the inferior olive reduces the magnitude of the teaching signal in climbing fibers and limits learning .","Our circuit-level model , based on these four principles , explains behavioral data obtained by strategically manipulating the signals responsible for acquisition and recall of direction learning in smooth pursuit eye movements across multiple timescales ."],"string":"[\n \"We provide behavioral evidence using monkey smooth pursuit eye movements for four principles of cerebellar learning .\",\n \"Using a circuit-level model of the cerebellum , we link behavioral data to learning’s neural implementation .\",\n \"The four principles are: ( 1 ) early , fast , acquisition driven by climbing fiber inputs to the cerebellar cortex , with poor retention; ( 2 ) learned responses of Purkinje cells guide transfer of learning from the cerebellar cortex to the deep cerebellar nucleus , with excellent retention; ( 3 ) functionally different neural signals are subject to learning in the cerebellar cortex versus the deep cerebellar nuclei; and ( 4 ) negative feedback from the cerebellum to the inferior olive reduces the magnitude of the teaching signal in climbing fibers and limits learning .\",\n \"Our circuit-level model , based on these four principles , explains behavioral data obtained by strategically manipulating the signals responsible for acquisition and recall of direction learning in smooth pursuit eye movements across multiple timescales .\"\n]"},"summary":{"kind":"list like","value":["The human brain can do many things , from reading and remembering the words written on a page to adapting and improving movements .","When a movement misses its goal , the strength of the connections between cells in a part of the brain known as the cerebellum changes .","The cerebellum is important for coordinating movements , including eye movements .","When the connections between the cells in the cerebellum – known as neurons – strengthen or weaken , the cerebellum changes how it will respond in the future , leading to more accurate movements .","However , the speed of the changes in the connections and how the connections between different neurons evolve and coordinate were unknown .","Herzfeld et al . have now combined eye-tracking studies in monkeys with computer modeling based on what is known about the neural circuits in the cerebellum to learn more about the changes in these connections .","Monkeys watched a moving target that would abruptly change direction .","In the next movement , the eye-tracking equipment monitored how well the monkey’s eyes anticipated the unexpected change in the target’s direction – a form of motor learning .","Using the experimental data , Herzfeld et al . produced a model that outlines general principles of how the cerebellum might manage this process .","The model suggested that neurons in one region in the cerebellum , known as Purkinje cells , learn from mistakes quickly , but have poor long-term retention .","If the movement is repeated , Purkinje cells teach another area of the cerebellum , the cerebellar nucleus , which takes longer to learn but has much better retention .","Although these findings are based on a simple motor learning task , they are the first step to understanding how the brain forms memories and how we might learn more complex behaviors ."],"string":"[\n \"The human brain can do many things , from reading and remembering the words written on a page to adapting and improving movements .\",\n \"When a movement misses its goal , the strength of the connections between cells in a part of the brain known as the cerebellum changes .\",\n \"The cerebellum is important for coordinating movements , including eye movements .\",\n \"When the connections between the cells in the cerebellum – known as neurons – strengthen or weaken , the cerebellum changes how it will respond in the future , leading to more accurate movements .\",\n \"However , the speed of the changes in the connections and how the connections between different neurons evolve and coordinate were unknown .\",\n \"Herzfeld et al . have now combined eye-tracking studies in monkeys with computer modeling based on what is known about the neural circuits in the cerebellum to learn more about the changes in these connections .\",\n \"Monkeys watched a moving target that would abruptly change direction .\",\n \"In the next movement , the eye-tracking equipment monitored how well the monkey’s eyes anticipated the unexpected change in the target’s direction – a form of motor learning .\",\n \"Using the experimental data , Herzfeld et al . produced a model that outlines general principles of how the cerebellum might manage this process .\",\n \"The model suggested that neurons in one region in the cerebellum , known as Purkinje cells , learn from mistakes quickly , but have poor long-term retention .\",\n \"If the movement is repeated , Purkinje cells teach another area of the cerebellum , the cerebellar nucleus , which takes longer to learn but has much better retention .\",\n \"Although these findings are based on a simple motor learning task , they are the first step to understanding how the brain forms memories and how we might learn more complex behaviors .\"\n]"},"year":{"kind":"string","value":"2020"}}},{"rowIdx":4197,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["cell biology"],"string":"[\n \"cell biology\"\n]"},"title":{"kind":"string","value":"Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy"},"id":{"kind":"string","value":"elife-38064-v2"},"sections":{"kind":"list like","value":[["Heritable hypertrophic cardiomyopathy ( HCM ) is a leading cause of death among young adults , particularly competitive athletes .","This heterogeneous and complex disease typically involves asymmetric growth of the heart , interventricular septum thickening , disorganized cellular architecture , diastolic dysfunction , arrhythmias , and an increased risk of sudden cardiac death ( Davis et al . , 2016; Maron , 2002; Maron and Maron , 2013; Masarone et al . , 2018 ) .","Diastolic dysfunction is characterized by impaired left ventricular relaxation , chamber stiffening , elevated filling pressures , and , consequently , reduced stroke volumes and cardiac output .","Enhanced cardiomyocyte contractile activity is a leading hypothesis for the underlying cause of HCM .","Increased Ca2+-sensitivity of the contractile apparatus as well as heightened function of the myosin molecular motor are potential molecular mechanisms .","The over-active contractile apparatus can prolong mechanical tension , which apparently initiates hypertrophy via activation of specific signaling cascades that regulate heart growth ( Davis et al . , 2016 ) .","Although therapies exist to treat problematic HCM abnormalities , there is no cure .","Thus , it is critical to better understand both the disease-causing mutations and the mechanism by which they produce HCM .","Over 1400 point mutations in sarcomeric proteins cause HCM ( Maron and Maron , 2013 ) , with the largest number ( >300 ) found in the β-cardiac myosin heavy chain .","This myosin II molecule forms sarcomere thick filaments and serves as a molecular motor to drive ATP-dependent movement along actin-containing thin filaments .","Mapping the locations of the mutations onto a three-dimensional structure of myosin reveals several hot spots .","These include the converter domain ( Colegrave and Peckham , 2014 ) and the recently described ‘myosin mesa’ ( Homburger et al . , 2016 ) .","The converter guides the myosin lever arm in its rotation during the power stroke that generates force and motion ( Mesentean et al . , 2007 ) .","The myosin mesa has been implicated in enabling formation of an interacting head motif ( Trivedi et al . , 2018 ) , wherein the heads of the dimeric molecule fold together to dampen motor function ( Woodhead et al . , 2005 ) .","Several molecular and physiological studies suggest that enhanced ATPase , in vitro actin sliding speed and muscle contractile force and/or velocity correlate with many , but not all , of the HCM-causing myosin mutations ( Adhikari et al . , 2016; Moore et al . , 2012; Sommese et al . , 2013; Spudich , 2014 ) .","For the domains cited above , this could occur via activation of the lever arm through enhanced converter function or stimulation of myosin cross-bridge initiation through inhibiting the formation of the interacting head motif .","The mechanism by which increased contractility results in development of HCM phenotypes remains an active area of investigation ( Davis et al . , 2016; Garfinkel et al . , 2018 ) .","In this communication , we model a human myosin HCM mutation in Drosophila melanogaster , which provides a highly integrative approach that yields unique insights into the disease .","The Drosophila heart , although a relatively simple tube-shaped structure , shares numerous gene expression patterns , as well as developmental and functional properties , with the human heart ( Bier and Bodmer , 2004 ) .","Further , the fly system allows evaluation of the effects of a mutation upon isolated myosin ( Swank et al . , 2000 ) , as well as on the myofibrillar structure and physiological function of both skeletal ( Maughan and Vigoreaux , 1999 ) and cardiac muscles ( Ocorr et al . , 2014 ) .","A major advantage of Drosophila is its simplified genetics , in that one Mhc gene gives rise to all myosin isoforms through alternative RNA splicing ( George et al . , 1989 ) .","Hence compensation by myosin multigene family members that can mask mutant protein effects does not occur .","Further , myosin expression levels and tissue-specificity can be controlled by combining Mhc transgenes with lines containing Mhc alleles that specifically knock out myosin heavy chain expression in the indirect flight muscles ( Collier et al . , 1990 ) or in all muscle types ( O'Donnell and Bernstein , 1988 ) .","Previously , using this approach , we showed that a mutant myosin with enhanced ATPase activity yielded disrupted skeletal muscle structure and function and restricted the Drosophila cardiac tube to reduce its output ( Cammarato et al . , 2008 ) .","This indicates that Drosophila models could yield insights into the basis of HCM through assessing human mutations that cause overactive myosin .","For the current study , we produced transgenic flies expressing the Drosophila equivalent of the K146N human HCM myosin mutation .","This mutation was identified in the family of an adult patient with increased left ventricular wall thickness in the absence of hypertension ( Ingles et al . , 2005 ) and as a spontaneous mutation in a child who displayed left ventricular hypertrophy ( Morita et al . , 2008 ) .","Residue 146 is particularly interesting in that it does not map to a well-studied HCM hot spot .","Instead , it maps near the N-terminus of the protein , which has few HCM-causing mutations ( Colegrave and Peckham , 2014 ) .","However , mutational analysis in Dictyostelium ( Ruppel et al . , 1994 ) and a domain swap study in Drosophila ( Swank et al . , 2003 ) suggest that effects on the mechanochemical cycle of myosin might be expected from alterations to the N-terminal region of myosin II molecules .","Our molecular modeling predicts that mutation of residue 146 of muscle myosin reduces its interaction with the myosin lever arm during the pre-power stroke state .","The myosin S1 head domain is in a cocked position during this state , so that it is prepared to drive the power stroke and muscle contraction upon actin binding during the mechanochemical cycle .","We found that the primary result of the 146N mutation is that myosin spends increased time in strongly bound states of the cross-bridge cycle .","This prolonged binding of myosin to actin increases force generation , but slows actin motility , decreases cyclical muscle power generation and reduces flight ability .","Higher force production and stiffness also cause progressive skeletal and cardiac muscle structural degeneration , and a restricted cardiac phenotype with diastolic dysfunction .","Some of our observations , such as the prolonged periods of systolic tension as well as increased ATPase rates , indicate this HCM mutation causes hyperdynamic contractile properties .","However , our use of the integrative approach in the Drosophila system shows that the mutant phenotype is complex , with reduced functions for a number of other parameters .","This emphasizes the need for a comprehensive analysis , from the molecular to organismal levels , which has allowed us to provide key insights into defining the molecular mechanism behind myosin-based HCM ."],["We determined the location and interactions for the amino acid residue corresponding to human K146 , which in its mutant form ( K146N ) causes HCM .","The Drosophila indirect flight muscle ( IFM ) myosin heavy chain sequence was modeled onto the scallop muscle myosin II crystal structure during the pre-power stroke state ( PDB 1QVI ) and the actin-detached post-power stroke state ( PDB 1KK8 ) ( Figure 1A and B ) .","Both scallop and Drosophila substitute an identically charged arginine residue for lysine at residue 146 , as do some human muscle myosins ( Rossi et al . , 2010 ) ( for clarity of comparison , the human β-cardiac myosin numbering system is used throughout ) .","The location of the modeled Drosophila residue , which is at the surface of the motor domain of the molecule , is shown in magenta .","In the pre-power stroke state , this residue is in close proximity to the lever arm ( Figure 1A ) , a domain that rotates to generate movement and force during muscle contraction .","As a result of configuration changes during the mechanochemical cycle , the N-terminus and R146 are distant from the lever arm in the post-power stroke state ( Figure 1B ) .","The Drosophila , scallop and human β-cardiac myosins show strong sequence similarity in the R146 region and also in the lever arm interacting region ( Figure 1B inset ) .","Analysis of the pre-power stroke model reveals specific charge interactions with residue 146 ( Figure 1C ) .","There is a 3 . 2 Å contact distance between positively charged R146 and negatively charged E774 , as well as a 3 . 0 Å contact distance between R146 and E775 .","These close contact distances permit the formation of salt bridges with E774 and E775 residues in the lever arm of the molecule .","In a model with HCM mutant residue R146N , the close contact and salt bridges with E774 and E775 are eliminated , with distances increased to 4 . 5 and 5 . 7 Å , respectively ( Figure 1D ) .","This could decrease the stability of the pre-power stroke state and alter rates of conformational changes required for the actin-myosin mechanochemical cycle to proceed normally .","A similar disruption may occur for the human β-cardiac mutant myosin as well ( Figure 1—figure supplement 1 ) .","To study the effects of the R146N mutation within myosin and Drosophila muscle , we constructed a mutant myosin transgene and obtained 29 transgenic lines by P element-mediated transformation .","Three independent transgenic inserts that mapped to the third chromosome ( PwMhcR146N-11 , PwMhcR146N-15 , PwMhcR146N-28; abbreviated hereafter with PwMhc deleted ) were crossed into the Mhc10 background , which is null for myosin heavy chain in IFM ( Collier et al . , 1990 ) .","RT-PCR of RNA isolated from dissected IFM verified that each transgenic line expresses only the mutant myosin in the IFM and that the normal alternative exon splicing pattern is not disrupted ( see Materials and methods ) .","SDS-PAGE analysis confirmed that upper thoraces of mutant 2-day-old adult female flies from lines 11 , 15 and 28 express normalized ratios of myosin to actin ( 1 . 06 ± 0 . 06 , 0 . 99 ± 0 . 02 , 0 . 98 ± 0 . 01 , respectively ) that are essentially identical to the wild-type control transgenic line PwMhc2 ( 1 . 00 ± 0 . 04 ) .","To explore the impact of the R146N mutation on the mechanochemical cycle of myosin , we assessed the steady-state ATPase parameters of myosin isolated from the IFM of R146N homozygotes ( Figure 2A–E ) .","Both Ca-ATPase ( 16 . 64 ± 1 . 87 vs . 9 . 51 ± 1 . 08 sec−1 ) and basal Mg-ATPase ( 0 . 62 ± 0 . 14 vs . 0 . 25 ± 0 . 04 sec−1 ) rates of R146N myosin increased significantly , about two-fold compared to wild-type myosin ( Figure 2A , B ) .","While Ca2+ typically yields higher in vitro ATPase activities , Mg2+ is the physiologically relevant cation that is employed for actin stimulation .","We found that actin-stimulated Mg-ATPase activity ( Vmax ) ( Figure 2C ) was not significantly different from the control ( 1 . 57 ± 0 . 27 vs . 1 . 62 ± 0 . 14 sec−1 ) .","The Km of actin concentration relative to ATPase activity was significantly increased , indicating that a 35% higher actin concentration is required to reach 50% Vmax ( 0 . 42 ± 0 . 06 vs . 0 . 31 ± 0 . 04 µM ) ( Figure 2D ) .","Further , the mutant’s catalytic efficiency ( defined as Vmax/Km ) was significantly lower than that of the control ( 3 . 80 ± 0 . 60 vs . 5 . 34 ± 1 . 07 sec−1/µM ) ( Figure 2E ) .","Analysis of in vitro actin-sliding velocity data showed that the mutant myosin possesses an ~two fold reduction in its ability to drive actin filament sliding compared to myosin isolated from control IFM ( 3 . 32 ± 0 . 21 vs . 6 . 71 ± 0 . 17 µm/s ) ( Figure 2F ) .","Thus the major effects of the R146N mutation on ATPase and in vitro motility are an enhancement of basal Mg- and Ca-ATPase rates and a decrease in actin filament sliding velocity .","To determine if the R146N mutation affects muscle structure and stability , we employed transmission electron microscopy to examine IFMs of homozygous female flies at late-pupal stage as well as fibers from 2-hr-old , 2-day-old and 1-week-old adults .","Transverse and longitudinal sections of R146N late-stage pupae and 2-hr-old adults ( Figure 3E , F ) showed normal sarcomeres containing double hexagonal arrays of thick and thin filaments , which are indistinguishable from those of the PwMhc2 homozygous control line at the same developmental stages ( Figure 3A , B ) .","Transverse and longitudinal sections of R146N 2-day-old adults showed minor disruptions of thick and thin filament packing ( Figure 3G ) compared to the control ( Figure 3C ) , with even more modest disruption seen in R146N/+ heterozygotes ( Figure 3—figure supplement 1 ) .","Transverse and longitudinal sections of R146N 7-day-old adults showed more disorder in myofibril morphology , with gaps in the hexagonal packing of thick and thin filaments and disruption in the Z- and M-lines ( Figure 3H ) compared to the control ( Figure 3D ) .","Hence R146N mutant myosin is capable of contributing to the formation of normal myofibrillar structures that begin to deteriorate at 2 days into adulthood , presumably as a result of mechanical stress during use .","We minimized any influence of structural changes in R146N adult IFMs on our muscle mechanical assays by employing fibers from 2-hr-old flies for these assays .","We also assessed heterozygous organisms ( R146N/+ ) to determine whether the dominant nature of the mutation in humans is mirrored in Drosophila .","We assessed the effects of R146N myosin on IFM function by testing flight ability .","Line 15 R146N homozygotes exhibited ~50% and ~70% decreases in flight index at 15°C ( temperature employed for flight muscle mechanics ) and 22°C ( ambient temperature ) , respectively , compared to the control line ( Table 4 ) .","The flight index of R146N/+ heterozygotes was significantly higher than that of homozygotes at both temperatures , with a statistically significant 15% reduction compared to the control line at 22°C and no significant difference with the control line at 15°C ( Table 4 ) .","The flight ability of the other two lines of homozygous R146N mutants also was severely diminished ( Supplementary file 2 ) .","Impairment for all lines increased with age in a statistically significant manner , as flight index at 7 days decreased to about half that at 2 days ( Supplementary file 2 ) .","In contrast , the flight index of control organisms showed a significant decrease of only 11% during the same timeframe ( Supplementary file 2 ) .","We also assessed wing beat frequency ( WBF ) at 2 days of age and found a significant reduction in the homozygous R146N mutants , with 10% ( 15°C ) and 7% ( 22°C ) decreases compared to controls ( Table 4 ) .","The R146N/+ heterozygous mutant showed no significant decrease in WBF at either temperature .","However , there was a significant difference between the WBFs of the heterozygous and homozygous mutants at both temperatures .","Thus , R146N myosin detrimentally affects WBF and flight ability .","Homozygotes display more severe phenotypes than heterozygotes .","We next assessed the structure of adult hearts of R146N/+ heterozygous flies by transmission electron microcopy to determine the effects of the dominant myosin mutation on cardiac ultrastructure .","Two distinct layers are observed in transverse sections taken between the second and third abdominal segments ( Figure 6 ) .","The lumen-facing layer is composed of contractile cardiomyocytes that contain a circumferential array of myofibrils oriented perpendicularly to the anterior-posterior axis of the heart .","A layer of structurally supportive ventral-longitudinal skeletal muscle is located ventral to the cardiomyocyte layer ( Figure 6 , VL ) .","In heterozygous controls ( PwMhc2/+ ) , both young 1-week-old and aged 3-week-old flies showed intact myofibrils with characteristic discontinuous Z-lines ( Achal et al . , 2016 ) ( Figure 6 left ) .","In contrast , R146N-15/+ heterozygotes displayed areas of myofibrillar discontinuity , where filamentous fields appear to have been pulled apart ( Figure 6 center , arrows ) and these defects worsened with aging .","Although R146N-28/+ hearts showed essentially normal myofibrillar integrity and organization in young flies , they displayed myofibrillar discontinuities in aged flies ( Figure 6 right , arrows ) .","This suggests that defects in older mutant flies are not due to gross assembly defects but occur during the aging process .","To assess whether concentric cardiac hypertrophy ( addition of myofibrils in parallel ) occurs in R146N/+ heterozygotes , we measured the average cardiomyocyte thickness in ventral and dorsal areas and compared values between young and aged flies for mutant lines and controls .","Cardiomyocyte thickness in both young and aged mutant heterozygotes showed no statistically significant differences compared to controls ( Supplementary file 3 ) .","Further , we observed no evidence of eccentric hypertrophy ( addition of sarcomeres in series ) , given that our cardiac physiological analysis showed no increases in chamber dimensions in young or old mutant hearts under basal conditions ( Figure 7A–C ) , and a dilated phenotype was not observed after complete relaxation with EGTA+ blebbistatin ( Figure 8A–C ) .","Hence the R146N myosin mutation caused progressive defects in myofibrillar continuity , but did not yield hypertrophy in the Drosophila heart .","To investigate the pathophysiological consequences of the dominant R146N HCM mutation on the adult Drosophila heart , we surgically exposed , imaged , and assessed wall movement in semi-intact R146N/+ heterozygous flies .","The effects of mutant myosin expression on cardiac dimensions and contractile performance , at 1- and 3-weeks of age , were quantified and compared to those resulting from wild-type transgenic myosin expression in age-matched PwMhc2/+ controls ( Figure 7A–C ) .","The diastolic and systolic diameters across the heart wall of 1-week-old control ( PwMhc2/+ ) Drosophila were 65 . 68 ± 0 . 73 and 39 . 60 ± 0 . 46 µm , respectively , and were 67 . 58 ± 0 . 80 and 43 . 49 ± 0 . 67 µm in 3-week-old animals ( Supplementary file 4 ) .","R146N myosin expression triggered a significant reduction in dimensions at both ages .","The diastolic and systolic diameters of R146N-15/+at 1 week were 52 . 13 ± 0 . 53 and 32 . 83 ± 0 . 45 µm , respectively , and were 48 . 62 ± 0 . 64 and 34 . 76 ± 0 . 53 µm at 3 weeks .","The greater mutational effect on diastolic vs . systolic diameter resulted in 7 . 5% and 22% decreases in fractional shortening at 1 and 3 weeks of age relative to controls ( Figure 7C ) .","Similar results were found for R146N-28/+ flies ( Supplementary file 4 ) .","The age-related decrease in fractional shortening suggests progressive remodeling in the mutant , which correlates with the decreasing myofibrillar continuity we observed .","The heart period , which is the time required for a complete cardiac cycle consisting of a diastolic and subsequent systolic phase , was not significantly different between mutant and control flies at either age tested ( Figure 7C ) .","The systolic interval , however , was 0 . 19 ± 0 . 01 and 0 . 21 ± 0 . 01 s for 1- and 3-week-old control flies and 0 . 21 ± 0 . 01 and 0 . 23 ± 0 . 01 s for R146N-15/+ heterozygotes .","Therefore , the proportion of time during the cardiac cycle that was spent generating tension , calculated by determining the ratio of systolic interval to heart period ( SI/HP ) , was significantly greater for the mutant heart tubes relative to controls at all ages studied ( Figure 7C ) .","Together , these findings illustrate that the R146N/+ hearts exhibit reduced cardiac diameters and potentially elevated tone during diastole , as well as prolonged periods of systolic tension generation , which are indicative of restrictive physiology , diastolic dysfunction , and impaired relaxation ( Cammarato et al . , 2008; Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .","To further investigate the molecular basis of diastolic restriction and potentially altered resting tone in the R146N/+ heterozygote hearts , we compared the responses of three-week-old control and mutant cardiac tubes to treatments with EGTA-AM , a cell permeable chelator of intracellular Ca2+ ( Johnson et al . , 1997 ) and blebbistatin , a small molecule myosin inhibitor ( Fedorov et al . , 2007; Limouze et al . , 2004 ) .","Upon incubation with artificial hemolymph supplemented with 10 mM EGTA and 100 µM EGTA-AM , which removes extra- and intracellular Ca2+ , the hearts ceased beating and ‘relaxed’ , as manifest by an increase in diameter across the wall of both control and R146N/+ hearts relative to diastole ( Figure 8A ) .","Interestingly , there was a significantly amplified relaxation response in R146N/+hearts ( Δ = 4 . 50 ± 0 . 13 µm ) relative to controls ( Δ = 3 . 77 ± 0 . 19 µm ) ( Figure 8B ) .","We previously demonstrated in vivo , that a small population of residual cross-bridges actively cycles and generates force , and helps establish basal mechanical tone in wild-type Drosophila myocardium during diastole ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .","In this regard , subsequent treatment of control and mutant hearts with artificial hemolymph containing 10 mM EGTA , 100 µM EGTA-AM , and 100 µM blebbistatin generated a second ‘relaxation’ response ( Figure 8A ) .","Comparing the cardiac diameters after extra- and intra-cellular Ca2+ chelation and following blebbistatin treatment revealed a significant increase of roughly 2 . 8% ( Δ = 2 . 34 ± 0 . 17 µm ) in control hearts ( Figure 8C ) .","Notably , there was a strikingly augmented response to blebbistatin treatment in the R146N/+ heterozygote hearts that led to a ~ 10% ( Δ = 6 . 14 ± 0 . 35 µm ) increase in heart tube diameter ( Figure 8C ) .","Comparing the change in diameters between the two genotypes revealed that the response to blebbistatin is significantly greater in the R146N/+ mutant hearts vs . that for controls ( Figure 8C ) .","Our observations suggest that , in addition to elevated diastolic Ca2+ and Ca2+-activated cross-bridge cycling , excessive Ca2+-independent cross-bridges contribute to diastolic dysfunction and inadequate relaxation in the R146N/+ mutant hearts .","Overall , we utilized an integrative approach in the Drosophila system to determine the effects of a myosin HCM mutation on myosin function , skeletal muscle structure , muscle mechanics and cardiac physiology .","We found that altering modeled interactions between the mutant R146N residue and the lever arm leads to increased basal ATPase activity and changes to myosin cross-bridge kinetics that disrupt actin motility , myofibril power output , myofibril stability , flight ability and cardiac structure and function .","Our analyses provide insight into how a myosin mutation translates into mutant phenotypes , thereby elucidating underlying mechanisms of HCM ."],["We leveraged the strengths of the Drosophila myosin system to determine new phenotypic and mechanistic insights into HCM , a common disease of young adults .","The Drosophila model is an outstanding tool for integrative analysis of contractile protein mutations and for dissecting the mechanistic basis of disease , allowing an understanding from the molecular through the whole organism level .","In our HCM model , the Drosophila heart tube shows a restricted phenotype , which , like abnormalities in the mutated IFM , develops from prolonged cross-bridge binding to actin .","The restricted cardiac tube and diastolic dysfunction in this model of human HCM is congruent with phenotypes observed when expressing myosin with enhanced function ( Cammarato et al . , 2008 ) or tropomyosin that is inefficient at blocking the myosin binding site on actin ( Viswanathan et al . , 2014 ) .","This suggests that Drosophila is an excellent analytical system for screening mutations to assess their potential for causing human HCM , which are typically expected to enhance contractility ( Adhikari et al . , 2016; Nag et al . , 2017; Spudich , 2014 ) .","Integrating the results from our ATPase , actin motility and muscle mechanical studies reveals that there are alterations to the myosin cross-bridge cycle caused by the R146N mutation .","Muscle mechanics measurements showed an increase in apparent rate constant 2πb , which , according to the modeling of Kawai and Brandt ( 1980 ) is influenced by rates associated with myosin attachment to actin , including binding rate , phosphate release and the power stroke ( Figure 5—figure supplement 1 ) .","The enhanced basal myosin ATPase rates suggest that phosphate release rate has been increased , since this parameter is rate limiting for basal ATPase rate .","This would yield an increased speed of weak to strong actin binding .","Our modeling of molecular interactions within the myosin molecule suggests a destabilization of the pre-power stroke state , which could account for the increased affinity of the R146N myosin ADP . Pi state for actin and faster phosphate release rate .","Drosophila myosin residue R146 normally has charge-based interactions with E774 and E775 of the myosin lever arm at the pre-power stroke state ( Figure 1C ) .","This interaction is abolished by the R146N mutation ( Figure 1D ) , which would reduce the stability of the pre-power stroke molecule .","This could drive the cycle forward by enhancing ATP hydrolysis and the production of cross-bridges , possibly contributing to prolonged actin binding .","A similar mechanism may occur for human β-cardiac myosin as well , since the analogous K146 residue is also predicted to interact with E775 in the pre-power stroke state ( Figure 1—figure supplement 1A ) and this contact is destroyed by the K146N mutation ( Figure 1—figure supplement 1B ) .","Interestingly , analysis of another HCM mutation suggests a similar mode of action as for R146N ( Guhathakurta et al . , 2017 ) .","Time-resolved fluorescence resonance energy transfer analysis of myosin containing the E56G mutation in the lever-arm-binding essential light chain showed a reduction in pre-power stroke state molecules and enhanced levels of those in the post-power stroke state in the presence of ATP .","As with R146N , this yielded greater actin attachment and an increased duty ratio ( Guhathakurta et al . , 2017 ) .","Recent studies of omecamtiv mecarbil , a compound that enhances cardiac myosin power output ( Liu et al . , 2015; Planelles-Herrero et al . , 2017; Shen et al . , 2010 ) , are also relevant to understanding the molecular basis of the R146N mutant phenotypes .","As shown in Figure 1—figure supplement 1C , the compound binds in a pocket at the nexus of the N-terminus ( including K146 ) , the relay helix and the converter domain/lever arm ( including residue E774 ) ( Planelles-Herrero et al . , 2017 ) .","Omecamtiv mecarbil enhances the actin attachment rate and the duty ratio and slows in vitro motility ( Liu et al . , 2015 ) , similar to the effects that we observed with R146N myosin .","In contrast to our findings , ATPase activity in the absence of actin is inhibited by the compound ( Liu et al . , 2015 ) .","However , the rate of phosphate release is increased upon actin binding .","While the R146N mutation enhances basal activity , which may arise from destabilization of the pre-power stroke state , omecamtiv mecarbil appears to stabilize this state prior to actin interaction , including facilitation of a weak charge-based interaction between K146 and E774 ( Figure 1—figure supplement 1C ) .","Further clinical evidence supports the importance of these interactions in relationship to HCM , in that mutation of E774 to V774 also yields HCM ( Moric et al . , 2003 ) .","More rapid cross-bridge formation due to the R146N or E774V mutations destabilizing the pre-power stroke state coupled with decreased detachment rates ( see below ) stabilize actin-myosin interaction , leading to enhanced force production and decreased actin filament in vitro motility .","A similar outcome could arise from omecamtiv mecarbil increasing the number of molecules in the pre-power stroke state , which would increase the number of attached cross-bridges as the myosin molecules move through the mechanochemical cycle .","Although myosin attachment ( Brizendine et al . , 2017 ) and detachment rates ( Harris and Warshaw , 1993 ) both influence the duty ratio ( the fraction of time myosin is strongly bound to actin during a mechanochemical cycle ) , the latter is thought to have the major influence on in vitro motility at high myosin concentrations .","Thus , the ~50% decrease in the rate of actin movement in vitro for R146N myosin ( Figure 2F ) is likely facilitated by the decreased actin detachment rate suggested by our fiber mechanics measurements .","We observed a decrease in apparent rate constant 2πc ( Table 3 ) , a measure of rates associated with myosin detachment from actin , which includes detachment induced by ADP release and ATP binding ( Kawai and Brandt , 1980 ) ( Figure 5—figure supplement 1 ) .","Further , our observed increase in ATP Km in the fiber studies ( Figure 5A ) suggests decreased ATP affinity , which would slow the ATP-induced detachment rate of myosin from actin .","This is supported by the trends showing decreases in fmax with increasing phosphate concentration for the mutant ( Figure 5B ) , which does not occur in control myosin .","fmax is most likely being slowed by phosphate competing with ATP for the rigor state ( Pate and Cooke , 1989 ) .","Normally , increasing phosphate either has no effect ( as seen in the control ) or causes fmax and 2πb to increase ( as seen in many slow vertebrate muscle types [Galler et al . , 2005; Kawai et al . , 1993; Siemankowski et al . , 1985] ) .","Overall , the increase in attachment and decrease in detachment rate , in concert with no change in actin-activated ATPase rate arising from the mutation , indicate an increase in duty ratio .","In addition to slowed in vitro motility , an increase in duty ratio would cause the increased fiber tension generation and stiffness that we observed from analysis of elastic modulus values ( Figure 5C , D ) .","The increased duty ratio is also expected to negatively influence mutant IFM power generation ( Figure 4A ) .","The major cause of power loss is likely decreased net work production , given that there was relatively little change in optimal frequency for muscle power generation ( fmax ) .","Lack of change in fmax may be due to increased actin attachment and phosphate release being balanced by reduced ATP-induced detachment rate .","Work production , however , was decreased proportionally to power for homozygous fibers examined via sinusoidal analysis , and for both homozygous and heterozygous fibers when measured using the work loop technique ( Figure 4B , Table 2 ) .","When amplitude and length change conditions were optimized for power generation , the ratio of work absorption to work generation was increased in the mutants compared to control fibers .","Tellingly , decreasing optimal muscle length change amplitude allowed the mutant fibers to generate more power ( compare optimal power work loops with work loops performed under the identical length change and frequency conditions ) .","This indicates that less muscle stretching was beneficial because it decreased work absorbed , resulting in more net work and hence more power .","It appears that increased time of cross-bridge attachment causes cross-bridge elements to act more like brakes or shock absorbers during cyclical power generation .","These abnormally high stresses on the myofilaments likely result in the disruption of sarcomere integrity that develops over time in the IFM expressing the mutant myosin ( Figure 3 ) , as well as the reduced flight ability and WBF ( Table 4 ) .","Our analysis of the heart tube in R146N/+ heterozygotes produced results that support the thesis that an increased myosin duty ratio yields significant mutant phenotypes .","While the hearts in the mutants did not show a change in heart period ( suggesting no overall change in kinetics , in agreement with no change for fmax in IFM and for actin-activated ATPase ) , the heart spent a greater portion of each heart period in systole compared to the control ( Figure 7 ) .","This is consistent with prolonged contractions arising from increased myosin attachment kinetics and a decreased detachment rate .","An enhanced attachment rate would also increase the rate of force generation , which may partially account for the restricted cardiac phenotype .","The reduced fractional shortening observed for the R146N/+ heart may correspond to its optimal pumping mode , mimicking the improved IFM fiber mechanical performance at shorter muscle length changes .","At the ultrastructural level , the observed myofibrillar discontinuity in cardiomyocytes ( Figure 6 ) could be caused by the same mechanism as IFM degeneration , that is , excessive tension generation disrupting the integrity of the sarcomeric ultrastructure .","We did not detect hypertrophy of the cardiomyocytes in R146N/+ heterozygotes , although it has been shown that hypertrophy can occur in the conical chamber of the Drosophila heart as a result of abnormal receptor tyrosine kinase pathway signaling ( Yu et al . , 2013 ) .","It is important to note , however , that there is significant variability in the presence and degree of hypertrophy detected in patients carrying the 146N mutation ( personal communication from Jodie Ingles , Sydney Medical School ) , suggesting that interacting genes or environment may play a role in hypertrophy .","Interestingly , our data suggest that cross-bridges in the Drosophila heart bind to thin filaments via Ca2+-dependent and Ca2+-independent mechanisms during diastole .","This contributes to impaired relaxation .","Our results imply that small amounts of diastolic Ca2+ promote contraction and yield slightly shortened cardiomyocytes at rest in both mutant and control lines .","However , based upon the greater diameter increase ( greater tension drop ) in R146N/+ hearts compared to control hearts upon extra- and intracellular Ca2+ chelation with EGTA/EGTA , AM ( Figure 8B ) , disruption of Ca2+-handling in the mutant appears likely , which may promote the restricted cardiac tube phenotype .","Therefore , it is possible that perturbation of Ca2+-handling in R146N/+ mutant fly hearts , as is frequently observed in human cardiomyopathies ( Kranias and Bers , 2007 ) , results in excessive diastolic Ca2+ levels , yielding enhancement of cell shortening and elevated basal tone .","Additionally , the higher duty ratio of R146N myosin could indirectly enhance the Ca2+ sensitivity of the cross-bridges through thin filament activation .","Full thin filament activation requires both Ca2+ binding to troponin and strong binding of cross-bridges .","Thus , a higher duty-ratio myosin will have a greater number of myosin molecules strongly bound at a lower Ca2+ concentration than a lower-duty-ratio myosin .","We experimentally demonstrated this when we expressed the high-duty-ratio EMB myosin in the jump muscle , which caused enhanced Ca2+ sensitivity for tension development compared to jump muscle myosin ( Eldred et al . , 2010 ) .","Thus , the same concentration of Ca2+ in the R146N/+ mutant heart would generate higher force than in the control , yielding decreased cardiac diameters .","Our analysis with blebbistatin , which impedes acto-myosin cycling ( Kovács et al . , 2004 ) , indicates that a portion of the cross-bridges responsible for diastolic tone is calcium-independent in both control and R146N/+ hearts .","Treatment with the drug ( Figure 8C ) shows that there is a greater diameter increase in R146N/+ hearts than in control hearts , suggesting an enhanced number of Ca2+-independent cross-bridges .","This enhancement likely arises from the higher on-rate ( actin affinity ) , which contributes to the restricted phenotype and impaired relaxation observed in R146N/+ mutant hearts .","Increased cross-bridge availability could also result from disruption of the super-relaxed state that has been documented in both skeletal and cardiac muscles ( Hooijman et al . , 2011; Stewart et al . , 2010 ) .","This state correlates with the presence of the interacting head motif , wherein the dimeric heads of myosin molecules interact to reduce their actin binding and ATPase activity ( Trivedi et al . , 2018; Woodhead et al . , 2005 ) .","Disruption of this motif by mutation is linked to myosin activation that correlates with HCM ( Alamo et al . , 2017b; Nag et al . , 2017 ) .","Interestingly , myosin is found in its pre-power stroke configuration in the interacting head motif ( Alamo et al . , 2017a; Alamo et al . , 2016 ) and disturbance of this state of filament packing by the R146N mutation could reduce the number of molecules in the super-relaxed state .","This would enhance myosin activity within the myofibril , leading to the phenotypes we observed .","Drosophila myosins are indeed capable of entering the interacting head motif ( Lee et al . , 2018 ) , but the presence of the super-relaxed state in thick filaments or muscle fibers is yet to be documented .","In summary , our integrative analysis of the Drosophila analogue of the K146N human HCM mutation revealed increases in several , but not all , contractile parameters , as posited for a general mechanism of action for HCM mutations in humans ( Spudich , 2014 ) .","Notably , enhancements in basal ATPase rate , length of systole and cross-bridge binding are concordant with these expectations .","Further , the restricted phenotype and reduced fractional shortening observed in the Drosophila heart mimic the diastolic dysfunction/impaired relaxation and reduced ejection fraction that can accompany HCM ( Davis et al . , 2016; Maron , 2002; Maron and Maron , 2013; Masarone et al . , 2018 ) .","The restricted phenotype observed in our model of an HCM mutant , as well as the restricted phenotypes arising from a hyperactive myosin ( Cammarato et al . , 2008 ) or mutant troponin T or actin that enhance myosin binding ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) , suggest that restriction is a common phenotype produced in the Drosophila cardiac tube due to over-active cross-bridges .","If all or most HCM mutations have increased force production as their basis , the restricted cardiac tube phenotype in Drosophila could serve as a useful screening tool for determining whether specific human mutations are causative of HCM .","The fly might also serve to illuminate conserved signaling pathways that lead from contractile protein mutations to HCM .","Further , the amelioration of the mutant phenotype through genetic or drug screens in Drosophila may yield insights into potential treatments for human HCM ."],["Scallop muscle myosin II crystal structures in the pre-power stroke state ( PDB 1QVI ) ( Gourinath et al . , 2003 ) and the actin-detached post-power stroke state ( PDB 1KK8 ) were used as templates to predict Drosophila and human myosin structures ( Himmel et al . , 2002 ) .","Myosin S1 amino acid sequences were modeled using the SWISS-MODEL homology modeling server ( http://swissmodel . expasy . org/ ) ( Arnold et al . , 2006 ) .","Structures were viewed using PyMOL ( http://www . pymol . org; DeLano Scientific , Palo Alto , CA ) .","A P element-containing Mhc transgene with the R146N mutation was constructed using standard cloning techniques along with site directed mutagenesis .","The wild-type genomic construct PwMhc2 ( Swank et al . , 2000 ) was digested with Eag I to produced two subclones , PwMhc-5’ and pMhc3’ .","The PwMhc-5’ subclone contains an 11 . 3 kb Eag I Mhc fragment cloned into the pCaSpeR vector ( Thummel and Pirrotta , 1992 ) .","The PMhc-3’ subclone contains a 12 . 5 kb Eag I Mhc fragment cloned into pBluescriptKS Eag I site ( Stratagene , La Jolla , CA ) .","The PwMhc-5’ subclone was further digested with Xho I and Avr II .","A 6 . 8 kb Xho I-Avr II digested fragment from PwMhc-5’ was gel isolated and ligated into an Xho I-Avr II site in pLitmus 28I vector ( New England Biolabs , Ipswich , MA ) to produce pMhc-5’-XA .","The PwMhc-5’-XA subclone was further digested with Pst I and Avr II .","A 4 . 3 kb Pst I-Avr II digested fragment from PwMhc-5’-XA was gel isolated and ligated into an Pst I-Avr II site in pLitmus 28I vector , to produce pMhc-5’-PA .","The PwMhc-5’-PA subclone was further digested with Pst I and Age I .","A 1 . 6 kb Pst I-Age I digested fragment from PwMhc-5’-PA was gel isolated and ligated into an Pst I-Age I site in pLitmus 28I vector , to produce pMhc-5’-PA1 . 6 .","The pMhc-5’-PA1 . 6 subclone was subjected to site-directed mutagenesis using the QuickChange II kit ( Stratagene ) and exon specific-primer 5’-CCGTGGCAAGAACCGTAATGAGG-3’ containing the R146N nucleotide coding change ( bold ) to yield , pR146N-5’PA-1 . 6 .","Upon sequence confirmation of the R146N site-directed mutagenesis product , the pR146N-5’-PA-1 . 6 subclone was digested with Pst I and Age I .","The 1 . 6 kb R146N fragment from this digest was used to replace the wild-type Pst I-Age I fragment of pMhc-5’PA .","The resulting clone was digested with Pst I-Avr II and the Pst I-Avr II fragment was used to replace the wild-type Pst I-Avr II fragment of pMhc-5’-XA .","The resulting clone was digested with Xho I-Avr II and the isolated Xho I-Avr II fragment was used to replace the wild-type Xho I-Avr II fragment of PwMhc-5’ .","The resulting clone , PwMhcR146N-5’ , was digested with Eag I .","The wild-type 3’ end subclone pMhc-3’ also was digested with Eag I .","The 12 . 5 kb Eag I fragment was gel isolated and ligated into the Eag I of PwMhcR146N-5’ , to yield PwMhcR146N .","The entire coding region and all ligation sites of the final PwMhcR146N plasmid were confirmed by DNA sequencing ( Eton Bioscience , San Diego , CA ) prior to P element transformation .","Transgenic lines for PwMhcR146N were generated by P element-mediated germline transformation ( Rubin and Spradling , 1982 ) by BestGene , Inc . ( Chino Hills , CA ) .","BestGene injected 1200 embryos with the PwMhcR146N transgene , and 29 transgenic lines were obtained .","Each insert was mapped to its chromosomal location using balancer chromosomes and standard genetic crosses .","Ten inserts mapped to the second chromosome , 3 to the X chromosome and 16 to the third chromosome .","Three independent lines that mapped to the third chromosome were crossed into Mhc10 background , which is null for myosin heavy chain in IFM and TDT muscle due to mutation of the endogenous Mhc gene , which is located on the second chromosome ( Collier et al . , 1990 ) .","RT-PCR was employed to verify that Mhc transcripts from the PwMhcR146N transgenic lines contained the appropriate site-directed nucleotide changes in exon four and that flanking alternative exons 3 and 7 were spliced correctly .","RNA was prepared by LiCl2 extraction ( Becker et al . , 1992 ) from 2-day-old adult female transgenic flies .","cDNAs were generated for each line using the Protoscript cDNA synthesis kit ( New England Biolabs ) .","A reverse primer specific to exon 8 ( 5’-GTTCGTCACCCAGGGCCGTA-3’ ) and a forward primer specific to exon 2 ( 5’-TGGATCCCCGACGAGAAGGA-3’ ) were used to generate cDNA .","Briefly , 3 μmol of reverse specific primer were mixed with 0 . 5 μg of total RNA from each transgenic line .","PCR was performed using 1 μl of cDNA and 3 μmol of forward and reverse primers under the following conditions: 60 s at 94° C then 30 cycles of: 30 s at 94° C , 30 s at 55° C and 2 min at 68° C . RT-PCR products were sequenced by Eton Bioscience .","To determine myosin protein expression levels for each transgene in an Mhc10 background , SDS polyacrylamide gel electrophoresis was utilized as previously described ( O'Donnell et al . , 1989 ) .","Upper thoraces from six 2-day-old homozygous female flies were homogenized in 60 µl SDS gel buffer .","Six µl of sample were loaded on a 9% polyacrylamide gel .","This was repeated five different times each time using a freshly prepared sample .","Protein accumulation was determined using Coomassie blue stained gels that were digitally scanned and analyzed on NIH Image J software .","Values are expressed relative to the control transgene ±S . E . M . As previously reported , actin was prepared from chicken skeletal muscle after multiple steps of polymerization–depolymerization ( Kronert et al . , 2014; Kronert et al . , 2015 ) .","Myosin was isolated from dissected dorsolongitudinal IFMs of ~250 transgenic flies and its concentration was determined by spectrophotometry ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .","A final concentration of 2 . 0 µg/µl myosin was used for ATPase assays and 0 . 5 µg/µl was used for in vitro motility assays .","Steady-state calcium , magnesium and actin-stimulated magnesium ATPase activities of myosin were obtained using [γ-32P]-ATP as previously described ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .","Basal Mg-ATPase activities obtained in the absence of actin and actin-stimulated Mg-ATPase was determined using increasing concentrations of filamentous actin .","Basal Mg-ATPase values were subtracted from all data points , which were then fit with the Michaelis–Menten equation to determine actin-stimulated ATPase ( Vmax ) and actin affinity relative to ATPase ( Km ) .","Catalytic efficiency ( defined as Vmax/Km ) was determined as previously reported ( Kronert et al . , 2014; Kronert et al . , 2015 ) .","In vitro motility assays were carried out by adding ATP and filamentous actin labeled with fluorescent phalloidin to nitrocellulose treated cover slips coated with wild-type or mutant myosin ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .","Video sequences captured under fluorescence optics were analyzed computationally to determine actin-sliding velocity .","Mean values from five independent experiments ( two assays for each ) for the mutant and wild-type ATPase or from three independent motility assays ( at least 30 filaments/assay ) were compared for statistically significant differences ( p<0 . 05 ) by Student’s t-tests .","To determine the effects of transgene expression on IFM structure in an Mhc10 null background , transmission electron microscopy was performed as previously described ( O'Donnell and Bernstein , 1988 ) .","We examined four different stages of development; late stage pupae , 2-hr-old adults , 2-day-old adults and 1-week-old adults .","Cross-sections and longitudinal sections were obtained from females for each homozygous transgenic line , with at least three different samples examined for each of the three transgenic lines .","One myofibril is shown in each panel and is representative of the entire population at the given stage of development .","Images were obtained using a FEI Tecnai 12 transmission electron microscope with a TVIPS 214 high-resolution camera .","For cardiac electron microscopy , heart samples at 1 or 3 weeks of age ( three per age per genotype ) were surgically exposed in an oxygenated artificial hemolymph solution and fixed using a modified procedure as described previously ( Achal et al . , 2016 ) .","Rhythmic beating was observed to confirm structural integrity of samples .","Hearts were relaxed in 10 mM EGTA , fixed in primary fixative ( 3% formaldehyde , 3% glutaraldehyde , 0 . 1 M cacodylate buffer pH 7 . 4 ) , washed 6x in 0 . 1 M cacodylate buffer pH 7 . 4 , then bathed in secondary fixative ( 1% OsO4 , 10 mM MgCl2 , 100 mM phosphate buffer pH 7 . 4 ) and washed 3x in 0 . 1 M cacodylate buffer pH 7 . 4 .","The samples were dehydrated in an acetone series , oriented and embedded in Epon-filled BEEM capsules , and polymerized at 60°C under vacuum .","Thin sections ( ≤50 nm ) were obtained using a Leica ultramicrotome , picked up on Formvar-coated grids , and stained with 4% uranyl acetate for 10 min .","Images were obtained at 120 kV on a FEI Tecnai 12 transmission electron microscope .","Transverse sections of the heart were utilized to determine average cardiac thickness between the second and third abdominal segments .","For each measurement , a roughly rectangular area of cardiomyocyte tissue ( defined as myofibrillar and mitochondrial material ) of 10 µm in length within the inner and outer edges of the cardiac tube was highlighted using Adobe Photoshop .","The image was imported into ImageJ to calculate the area of the highlighted region .","Dividing the resulting cardiomyocyte area by 10 µm yielded the average cardiac thickness for that highlighted section .","At least three images each for dorsal areas and for ventral areas per heart section were analyzed .","Three or more transverse sections along the anterior-poster axis at least 10 µm apart were assessed in the same way .","Cardiac thickness measurements from dorsal-side or ventral-side images were averaged per section and the data were then averaged for each biological replicate .","Data from at least three hearts were averaged per line to yield the final values , which are presented as mean ± SEM .","A one-way ANOVA with the Bonferroni correction determined statistical significance ( p<0 . 05 ) .","Newly eclosed female PwMhcR146N-15 homozygous or heterozygous flies were collected every half hour and allowed to mature for 2 hr .","Control flies were taken from the PwMhc2 stock .","Individual IFM fibers were isolated by dissection as previously described by Swank ( Swank , 2012 ) .","Briefly , the head , abdomen and wings were removed and the thorax was placed into dissection solution ( Swank , 2012 ) .","The thorax was split in half , and the IFM fiber bundle was removed .","The fibers were skinned in the dissection solution for 1 hr at 6°C before being transferred to storage solution ( same as the dissection solution but without Triton X-100 ) .","Individual fibers were then separated from the bundle and split in half with a long , thinly etched tungsten probe .","The split fiber was then fitted with a pair of aluminum foil t-clips .","The clipped fiber was mounted in relaxing solution ( pCa 8 . 0 , 12 mM MgATP , 30 mM creatine phosphate , 600 U/ml creatine phosphokinase , 1 mM free Mg2+ , 5 mM EGTA , 20 mM pH 7 . 0 BES , 200 mM ionic strength , adjusted with Na methane sulfonate , 1 mM DTT ) onto a mechanics apparatus by settling it onto two hooks attached to a motor arm and a force transducer .","The muscle was lengthened to be just taught , then stretched 5% beyond this length .","The resulting length , width and height were measured to obtain cross-sectional area .","Passive tension ( Po ) was recorded before adding activating solution ( the same as relaxing solution except that pCa was adjusted to 5 . 0 ) .","The maximum power of the muscle was determined by performing sinusoidal analysis ( see below ) , under low amplitude conditions ( 0 . 125% muscle length ) .","After every run , the muscle was stretched by 2% muscle length ( ML ) until the power did not increase by more than 3% .","The active tension ( Ao ) at the optimal muscle length was measured and the net tension ( Fo ) was calculated by Ao-Po .","Two- and 7-day-old homozygotes for each of the three transgenic lines and controls were tested for flight ability in an Mhc10 background at 22°C .","Flight was determined by the ability to fly up ( U ) , horizontal ( H ) , down ( D ) or not at all ( N ) ( Drummond et al . , 1991 ) .","As previously described ( Tohtong et al . , 1995 ) flight index is defined as: 6 U/T+ 4 H/T+ 2 D/T+ 0 N/T where T is the total number of flies tested .","Flight abilities and wing beat frequencies of two-day-old PwMhcR146N-15 homozygotes and heterozygotes were examined at 15°C and 22°C .","An optical tachometer was used to measure the wing beat frequency of tethered flies ( Tohtong et al . , 1995 ) .","All values represent mean ± SEM .","Significance was assessed by a Student’s t-test at p<0 . 05 .","Cardiac tubes of 1- and 3-week-old female adult flies were surgically exposed under oxygenated artificial hemolymph as described by Vogler and Ocorr ( 2009 ) .","High-speed videos of contracting hearts were taken at ~120 frames per second using a Hamamatsu Orca Flash 2 . 8 CMOS camera on a Leica DM5000B TL microscope with a 10x ( 0 . 30 NA ) immersion lens .","Physiological parameters were assessed from individual videos using the semiautomatic optical heartbeat analysis ( SOHA v . 3 ) program as previously outlined ( Cammarato et al . , 2015; Fink et al . , 2009; Viswanathan et al . , 2017 ) .","M-mode kymograms were generated to provide an edge trace documenting the movement of heart wall in the y-axis over time in the x-axis .","Values represent mean ± S . E . M . Two-way ANOVAs were employed to test if the effects of genotype and age ( or if an interaction exists ) were significant for each cardiac parameter investigated .","Significance was assessed at p<0 . 05 .","Beating hearts from 3-week-old heterozygous female PwMhc2/+ and PwMhcR146N-15/+ flies were imaged as described above using a 20x ( 0 . 50 NA ) immersion objective lens and analyzed as previously detailed ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .","The effects of EGTA/EGTA-AM and blebbistatin treatment on cardiac diameters were evaluated using paired t-tests of the matched groups .","Unpaired t-tests were used to distinguish significant differences in the cardiac responses to EGTA , EGTA . AM and blebbistatin between PwMhc2 and mutant MHC expressing hearts .","Significance was assessed at p<0 . 05 ."]],"string":"[\n [\n \"Heritable hypertrophic cardiomyopathy ( HCM ) is a leading cause of death among young adults , particularly competitive athletes .\",\n \"This heterogeneous and complex disease typically involves asymmetric growth of the heart , interventricular septum thickening , disorganized cellular architecture , diastolic dysfunction , arrhythmias , and an increased risk of sudden cardiac death ( Davis et al . , 2016; Maron , 2002; Maron and Maron , 2013; Masarone et al . , 2018 ) .\",\n \"Diastolic dysfunction is characterized by impaired left ventricular relaxation , chamber stiffening , elevated filling pressures , and , consequently , reduced stroke volumes and cardiac output .\",\n \"Enhanced cardiomyocyte contractile activity is a leading hypothesis for the underlying cause of HCM .\",\n \"Increased Ca2+-sensitivity of the contractile apparatus as well as heightened function of the myosin molecular motor are potential molecular mechanisms .\",\n \"The over-active contractile apparatus can prolong mechanical tension , which apparently initiates hypertrophy via activation of specific signaling cascades that regulate heart growth ( Davis et al . , 2016 ) .\",\n \"Although therapies exist to treat problematic HCM abnormalities , there is no cure .\",\n \"Thus , it is critical to better understand both the disease-causing mutations and the mechanism by which they produce HCM .\",\n \"Over 1400 point mutations in sarcomeric proteins cause HCM ( Maron and Maron , 2013 ) , with the largest number ( >300 ) found in the β-cardiac myosin heavy chain .\",\n \"This myosin II molecule forms sarcomere thick filaments and serves as a molecular motor to drive ATP-dependent movement along actin-containing thin filaments .\",\n \"Mapping the locations of the mutations onto a three-dimensional structure of myosin reveals several hot spots .\",\n \"These include the converter domain ( Colegrave and Peckham , 2014 ) and the recently described ‘myosin mesa’ ( Homburger et al . , 2016 ) .\",\n \"The converter guides the myosin lever arm in its rotation during the power stroke that generates force and motion ( Mesentean et al . , 2007 ) .\",\n \"The myosin mesa has been implicated in enabling formation of an interacting head motif ( Trivedi et al . , 2018 ) , wherein the heads of the dimeric molecule fold together to dampen motor function ( Woodhead et al . , 2005 ) .\",\n \"Several molecular and physiological studies suggest that enhanced ATPase , in vitro actin sliding speed and muscle contractile force and/or velocity correlate with many , but not all , of the HCM-causing myosin mutations ( Adhikari et al . , 2016; Moore et al . , 2012; Sommese et al . , 2013; Spudich , 2014 ) .\",\n \"For the domains cited above , this could occur via activation of the lever arm through enhanced converter function or stimulation of myosin cross-bridge initiation through inhibiting the formation of the interacting head motif .\",\n \"The mechanism by which increased contractility results in development of HCM phenotypes remains an active area of investigation ( Davis et al . , 2016; Garfinkel et al . , 2018 ) .\",\n \"In this communication , we model a human myosin HCM mutation in Drosophila melanogaster , which provides a highly integrative approach that yields unique insights into the disease .\",\n \"The Drosophila heart , although a relatively simple tube-shaped structure , shares numerous gene expression patterns , as well as developmental and functional properties , with the human heart ( Bier and Bodmer , 2004 ) .\",\n \"Further , the fly system allows evaluation of the effects of a mutation upon isolated myosin ( Swank et al . , 2000 ) , as well as on the myofibrillar structure and physiological function of both skeletal ( Maughan and Vigoreaux , 1999 ) and cardiac muscles ( Ocorr et al . , 2014 ) .\",\n \"A major advantage of Drosophila is its simplified genetics , in that one Mhc gene gives rise to all myosin isoforms through alternative RNA splicing ( George et al . , 1989 ) .\",\n \"Hence compensation by myosin multigene family members that can mask mutant protein effects does not occur .\",\n \"Further , myosin expression levels and tissue-specificity can be controlled by combining Mhc transgenes with lines containing Mhc alleles that specifically knock out myosin heavy chain expression in the indirect flight muscles ( Collier et al . , 1990 ) or in all muscle types ( O'Donnell and Bernstein , 1988 ) .\",\n \"Previously , using this approach , we showed that a mutant myosin with enhanced ATPase activity yielded disrupted skeletal muscle structure and function and restricted the Drosophila cardiac tube to reduce its output ( Cammarato et al . , 2008 ) .\",\n \"This indicates that Drosophila models could yield insights into the basis of HCM through assessing human mutations that cause overactive myosin .\",\n \"For the current study , we produced transgenic flies expressing the Drosophila equivalent of the K146N human HCM myosin mutation .\",\n \"This mutation was identified in the family of an adult patient with increased left ventricular wall thickness in the absence of hypertension ( Ingles et al . , 2005 ) and as a spontaneous mutation in a child who displayed left ventricular hypertrophy ( Morita et al . , 2008 ) .\",\n \"Residue 146 is particularly interesting in that it does not map to a well-studied HCM hot spot .\",\n \"Instead , it maps near the N-terminus of the protein , which has few HCM-causing mutations ( Colegrave and Peckham , 2014 ) .\",\n \"However , mutational analysis in Dictyostelium ( Ruppel et al . , 1994 ) and a domain swap study in Drosophila ( Swank et al . , 2003 ) suggest that effects on the mechanochemical cycle of myosin might be expected from alterations to the N-terminal region of myosin II molecules .\",\n \"Our molecular modeling predicts that mutation of residue 146 of muscle myosin reduces its interaction with the myosin lever arm during the pre-power stroke state .\",\n \"The myosin S1 head domain is in a cocked position during this state , so that it is prepared to drive the power stroke and muscle contraction upon actin binding during the mechanochemical cycle .\",\n \"We found that the primary result of the 146N mutation is that myosin spends increased time in strongly bound states of the cross-bridge cycle .\",\n \"This prolonged binding of myosin to actin increases force generation , but slows actin motility , decreases cyclical muscle power generation and reduces flight ability .\",\n \"Higher force production and stiffness also cause progressive skeletal and cardiac muscle structural degeneration , and a restricted cardiac phenotype with diastolic dysfunction .\",\n \"Some of our observations , such as the prolonged periods of systolic tension as well as increased ATPase rates , indicate this HCM mutation causes hyperdynamic contractile properties .\",\n \"However , our use of the integrative approach in the Drosophila system shows that the mutant phenotype is complex , with reduced functions for a number of other parameters .\",\n \"This emphasizes the need for a comprehensive analysis , from the molecular to organismal levels , which has allowed us to provide key insights into defining the molecular mechanism behind myosin-based HCM .\"\n ],\n [\n \"We determined the location and interactions for the amino acid residue corresponding to human K146 , which in its mutant form ( K146N ) causes HCM .\",\n \"The Drosophila indirect flight muscle ( IFM ) myosin heavy chain sequence was modeled onto the scallop muscle myosin II crystal structure during the pre-power stroke state ( PDB 1QVI ) and the actin-detached post-power stroke state ( PDB 1KK8 ) ( Figure 1A and B ) .\",\n \"Both scallop and Drosophila substitute an identically charged arginine residue for lysine at residue 146 , as do some human muscle myosins ( Rossi et al . , 2010 ) ( for clarity of comparison , the human β-cardiac myosin numbering system is used throughout ) .\",\n \"The location of the modeled Drosophila residue , which is at the surface of the motor domain of the molecule , is shown in magenta .\",\n \"In the pre-power stroke state , this residue is in close proximity to the lever arm ( Figure 1A ) , a domain that rotates to generate movement and force during muscle contraction .\",\n \"As a result of configuration changes during the mechanochemical cycle , the N-terminus and R146 are distant from the lever arm in the post-power stroke state ( Figure 1B ) .\",\n \"The Drosophila , scallop and human β-cardiac myosins show strong sequence similarity in the R146 region and also in the lever arm interacting region ( Figure 1B inset ) .\",\n \"Analysis of the pre-power stroke model reveals specific charge interactions with residue 146 ( Figure 1C ) .\",\n \"There is a 3 . 2 Å contact distance between positively charged R146 and negatively charged E774 , as well as a 3 . 0 Å contact distance between R146 and E775 .\",\n \"These close contact distances permit the formation of salt bridges with E774 and E775 residues in the lever arm of the molecule .\",\n \"In a model with HCM mutant residue R146N , the close contact and salt bridges with E774 and E775 are eliminated , with distances increased to 4 . 5 and 5 . 7 Å , respectively ( Figure 1D ) .\",\n \"This could decrease the stability of the pre-power stroke state and alter rates of conformational changes required for the actin-myosin mechanochemical cycle to proceed normally .\",\n \"A similar disruption may occur for the human β-cardiac mutant myosin as well ( Figure 1—figure supplement 1 ) .\",\n \"To study the effects of the R146N mutation within myosin and Drosophila muscle , we constructed a mutant myosin transgene and obtained 29 transgenic lines by P element-mediated transformation .\",\n \"Three independent transgenic inserts that mapped to the third chromosome ( PwMhcR146N-11 , PwMhcR146N-15 , PwMhcR146N-28; abbreviated hereafter with PwMhc deleted ) were crossed into the Mhc10 background , which is null for myosin heavy chain in IFM ( Collier et al . , 1990 ) .\",\n \"RT-PCR of RNA isolated from dissected IFM verified that each transgenic line expresses only the mutant myosin in the IFM and that the normal alternative exon splicing pattern is not disrupted ( see Materials and methods ) .\",\n \"SDS-PAGE analysis confirmed that upper thoraces of mutant 2-day-old adult female flies from lines 11 , 15 and 28 express normalized ratios of myosin to actin ( 1 . 06 ± 0 . 06 , 0 . 99 ± 0 . 02 , 0 . 98 ± 0 . 01 , respectively ) that are essentially identical to the wild-type control transgenic line PwMhc2 ( 1 . 00 ± 0 . 04 ) .\",\n \"To explore the impact of the R146N mutation on the mechanochemical cycle of myosin , we assessed the steady-state ATPase parameters of myosin isolated from the IFM of R146N homozygotes ( Figure 2A–E ) .\",\n \"Both Ca-ATPase ( 16 . 64 ± 1 . 87 vs . 9 . 51 ± 1 . 08 sec−1 ) and basal Mg-ATPase ( 0 . 62 ± 0 . 14 vs . 0 . 25 ± 0 . 04 sec−1 ) rates of R146N myosin increased significantly , about two-fold compared to wild-type myosin ( Figure 2A , B ) .\",\n \"While Ca2+ typically yields higher in vitro ATPase activities , Mg2+ is the physiologically relevant cation that is employed for actin stimulation .\",\n \"We found that actin-stimulated Mg-ATPase activity ( Vmax ) ( Figure 2C ) was not significantly different from the control ( 1 . 57 ± 0 . 27 vs . 1 . 62 ± 0 . 14 sec−1 ) .\",\n \"The Km of actin concentration relative to ATPase activity was significantly increased , indicating that a 35% higher actin concentration is required to reach 50% Vmax ( 0 . 42 ± 0 . 06 vs . 0 . 31 ± 0 . 04 µM ) ( Figure 2D ) .\",\n \"Further , the mutant’s catalytic efficiency ( defined as Vmax/Km ) was significantly lower than that of the control ( 3 . 80 ± 0 . 60 vs . 5 . 34 ± 1 . 07 sec−1/µM ) ( Figure 2E ) .\",\n \"Analysis of in vitro actin-sliding velocity data showed that the mutant myosin possesses an ~two fold reduction in its ability to drive actin filament sliding compared to myosin isolated from control IFM ( 3 . 32 ± 0 . 21 vs . 6 . 71 ± 0 . 17 µm/s ) ( Figure 2F ) .\",\n \"Thus the major effects of the R146N mutation on ATPase and in vitro motility are an enhancement of basal Mg- and Ca-ATPase rates and a decrease in actin filament sliding velocity .\",\n \"To determine if the R146N mutation affects muscle structure and stability , we employed transmission electron microscopy to examine IFMs of homozygous female flies at late-pupal stage as well as fibers from 2-hr-old , 2-day-old and 1-week-old adults .\",\n \"Transverse and longitudinal sections of R146N late-stage pupae and 2-hr-old adults ( Figure 3E , F ) showed normal sarcomeres containing double hexagonal arrays of thick and thin filaments , which are indistinguishable from those of the PwMhc2 homozygous control line at the same developmental stages ( Figure 3A , B ) .\",\n \"Transverse and longitudinal sections of R146N 2-day-old adults showed minor disruptions of thick and thin filament packing ( Figure 3G ) compared to the control ( Figure 3C ) , with even more modest disruption seen in R146N/+ heterozygotes ( Figure 3—figure supplement 1 ) .\",\n \"Transverse and longitudinal sections of R146N 7-day-old adults showed more disorder in myofibril morphology , with gaps in the hexagonal packing of thick and thin filaments and disruption in the Z- and M-lines ( Figure 3H ) compared to the control ( Figure 3D ) .\",\n \"Hence R146N mutant myosin is capable of contributing to the formation of normal myofibrillar structures that begin to deteriorate at 2 days into adulthood , presumably as a result of mechanical stress during use .\",\n \"We minimized any influence of structural changes in R146N adult IFMs on our muscle mechanical assays by employing fibers from 2-hr-old flies for these assays .\",\n \"We also assessed heterozygous organisms ( R146N/+ ) to determine whether the dominant nature of the mutation in humans is mirrored in Drosophila .\",\n \"We assessed the effects of R146N myosin on IFM function by testing flight ability .\",\n \"Line 15 R146N homozygotes exhibited ~50% and ~70% decreases in flight index at 15°C ( temperature employed for flight muscle mechanics ) and 22°C ( ambient temperature ) , respectively , compared to the control line ( Table 4 ) .\",\n \"The flight index of R146N/+ heterozygotes was significantly higher than that of homozygotes at both temperatures , with a statistically significant 15% reduction compared to the control line at 22°C and no significant difference with the control line at 15°C ( Table 4 ) .\",\n \"The flight ability of the other two lines of homozygous R146N mutants also was severely diminished ( Supplementary file 2 ) .\",\n \"Impairment for all lines increased with age in a statistically significant manner , as flight index at 7 days decreased to about half that at 2 days ( Supplementary file 2 ) .\",\n \"In contrast , the flight index of control organisms showed a significant decrease of only 11% during the same timeframe ( Supplementary file 2 ) .\",\n \"We also assessed wing beat frequency ( WBF ) at 2 days of age and found a significant reduction in the homozygous R146N mutants , with 10% ( 15°C ) and 7% ( 22°C ) decreases compared to controls ( Table 4 ) .\",\n \"The R146N/+ heterozygous mutant showed no significant decrease in WBF at either temperature .\",\n \"However , there was a significant difference between the WBFs of the heterozygous and homozygous mutants at both temperatures .\",\n \"Thus , R146N myosin detrimentally affects WBF and flight ability .\",\n \"Homozygotes display more severe phenotypes than heterozygotes .\",\n \"We next assessed the structure of adult hearts of R146N/+ heterozygous flies by transmission electron microcopy to determine the effects of the dominant myosin mutation on cardiac ultrastructure .\",\n \"Two distinct layers are observed in transverse sections taken between the second and third abdominal segments ( Figure 6 ) .\",\n \"The lumen-facing layer is composed of contractile cardiomyocytes that contain a circumferential array of myofibrils oriented perpendicularly to the anterior-posterior axis of the heart .\",\n \"A layer of structurally supportive ventral-longitudinal skeletal muscle is located ventral to the cardiomyocyte layer ( Figure 6 , VL ) .\",\n \"In heterozygous controls ( PwMhc2/+ ) , both young 1-week-old and aged 3-week-old flies showed intact myofibrils with characteristic discontinuous Z-lines ( Achal et al . , 2016 ) ( Figure 6 left ) .\",\n \"In contrast , R146N-15/+ heterozygotes displayed areas of myofibrillar discontinuity , where filamentous fields appear to have been pulled apart ( Figure 6 center , arrows ) and these defects worsened with aging .\",\n \"Although R146N-28/+ hearts showed essentially normal myofibrillar integrity and organization in young flies , they displayed myofibrillar discontinuities in aged flies ( Figure 6 right , arrows ) .\",\n \"This suggests that defects in older mutant flies are not due to gross assembly defects but occur during the aging process .\",\n \"To assess whether concentric cardiac hypertrophy ( addition of myofibrils in parallel ) occurs in R146N/+ heterozygotes , we measured the average cardiomyocyte thickness in ventral and dorsal areas and compared values between young and aged flies for mutant lines and controls .\",\n \"Cardiomyocyte thickness in both young and aged mutant heterozygotes showed no statistically significant differences compared to controls ( Supplementary file 3 ) .\",\n \"Further , we observed no evidence of eccentric hypertrophy ( addition of sarcomeres in series ) , given that our cardiac physiological analysis showed no increases in chamber dimensions in young or old mutant hearts under basal conditions ( Figure 7A–C ) , and a dilated phenotype was not observed after complete relaxation with EGTA+ blebbistatin ( Figure 8A–C ) .\",\n \"Hence the R146N myosin mutation caused progressive defects in myofibrillar continuity , but did not yield hypertrophy in the Drosophila heart .\",\n \"To investigate the pathophysiological consequences of the dominant R146N HCM mutation on the adult Drosophila heart , we surgically exposed , imaged , and assessed wall movement in semi-intact R146N/+ heterozygous flies .\",\n \"The effects of mutant myosin expression on cardiac dimensions and contractile performance , at 1- and 3-weeks of age , were quantified and compared to those resulting from wild-type transgenic myosin expression in age-matched PwMhc2/+ controls ( Figure 7A–C ) .\",\n \"The diastolic and systolic diameters across the heart wall of 1-week-old control ( PwMhc2/+ ) Drosophila were 65 . 68 ± 0 . 73 and 39 . 60 ± 0 . 46 µm , respectively , and were 67 . 58 ± 0 . 80 and 43 . 49 ± 0 . 67 µm in 3-week-old animals ( Supplementary file 4 ) .\",\n \"R146N myosin expression triggered a significant reduction in dimensions at both ages .\",\n \"The diastolic and systolic diameters of R146N-15/+at 1 week were 52 . 13 ± 0 . 53 and 32 . 83 ± 0 . 45 µm , respectively , and were 48 . 62 ± 0 . 64 and 34 . 76 ± 0 . 53 µm at 3 weeks .\",\n \"The greater mutational effect on diastolic vs . systolic diameter resulted in 7 . 5% and 22% decreases in fractional shortening at 1 and 3 weeks of age relative to controls ( Figure 7C ) .\",\n \"Similar results were found for R146N-28/+ flies ( Supplementary file 4 ) .\",\n \"The age-related decrease in fractional shortening suggests progressive remodeling in the mutant , which correlates with the decreasing myofibrillar continuity we observed .\",\n \"The heart period , which is the time required for a complete cardiac cycle consisting of a diastolic and subsequent systolic phase , was not significantly different between mutant and control flies at either age tested ( Figure 7C ) .\",\n \"The systolic interval , however , was 0 . 19 ± 0 . 01 and 0 . 21 ± 0 . 01 s for 1- and 3-week-old control flies and 0 . 21 ± 0 . 01 and 0 . 23 ± 0 . 01 s for R146N-15/+ heterozygotes .\",\n \"Therefore , the proportion of time during the cardiac cycle that was spent generating tension , calculated by determining the ratio of systolic interval to heart period ( SI/HP ) , was significantly greater for the mutant heart tubes relative to controls at all ages studied ( Figure 7C ) .\",\n \"Together , these findings illustrate that the R146N/+ hearts exhibit reduced cardiac diameters and potentially elevated tone during diastole , as well as prolonged periods of systolic tension generation , which are indicative of restrictive physiology , diastolic dysfunction , and impaired relaxation ( Cammarato et al . , 2008; Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .\",\n \"To further investigate the molecular basis of diastolic restriction and potentially altered resting tone in the R146N/+ heterozygote hearts , we compared the responses of three-week-old control and mutant cardiac tubes to treatments with EGTA-AM , a cell permeable chelator of intracellular Ca2+ ( Johnson et al . , 1997 ) and blebbistatin , a small molecule myosin inhibitor ( Fedorov et al . , 2007; Limouze et al . , 2004 ) .\",\n \"Upon incubation with artificial hemolymph supplemented with 10 mM EGTA and 100 µM EGTA-AM , which removes extra- and intracellular Ca2+ , the hearts ceased beating and ‘relaxed’ , as manifest by an increase in diameter across the wall of both control and R146N/+ hearts relative to diastole ( Figure 8A ) .\",\n \"Interestingly , there was a significantly amplified relaxation response in R146N/+hearts ( Δ = 4 . 50 ± 0 . 13 µm ) relative to controls ( Δ = 3 . 77 ± 0 . 19 µm ) ( Figure 8B ) .\",\n \"We previously demonstrated in vivo , that a small population of residual cross-bridges actively cycles and generates force , and helps establish basal mechanical tone in wild-type Drosophila myocardium during diastole ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .\",\n \"In this regard , subsequent treatment of control and mutant hearts with artificial hemolymph containing 10 mM EGTA , 100 µM EGTA-AM , and 100 µM blebbistatin generated a second ‘relaxation’ response ( Figure 8A ) .\",\n \"Comparing the cardiac diameters after extra- and intra-cellular Ca2+ chelation and following blebbistatin treatment revealed a significant increase of roughly 2 . 8% ( Δ = 2 . 34 ± 0 . 17 µm ) in control hearts ( Figure 8C ) .\",\n \"Notably , there was a strikingly augmented response to blebbistatin treatment in the R146N/+ heterozygote hearts that led to a ~ 10% ( Δ = 6 . 14 ± 0 . 35 µm ) increase in heart tube diameter ( Figure 8C ) .\",\n \"Comparing the change in diameters between the two genotypes revealed that the response to blebbistatin is significantly greater in the R146N/+ mutant hearts vs . that for controls ( Figure 8C ) .\",\n \"Our observations suggest that , in addition to elevated diastolic Ca2+ and Ca2+-activated cross-bridge cycling , excessive Ca2+-independent cross-bridges contribute to diastolic dysfunction and inadequate relaxation in the R146N/+ mutant hearts .\",\n \"Overall , we utilized an integrative approach in the Drosophila system to determine the effects of a myosin HCM mutation on myosin function , skeletal muscle structure , muscle mechanics and cardiac physiology .\",\n \"We found that altering modeled interactions between the mutant R146N residue and the lever arm leads to increased basal ATPase activity and changes to myosin cross-bridge kinetics that disrupt actin motility , myofibril power output , myofibril stability , flight ability and cardiac structure and function .\",\n \"Our analyses provide insight into how a myosin mutation translates into mutant phenotypes , thereby elucidating underlying mechanisms of HCM .\"\n ],\n [\n \"We leveraged the strengths of the Drosophila myosin system to determine new phenotypic and mechanistic insights into HCM , a common disease of young adults .\",\n \"The Drosophila model is an outstanding tool for integrative analysis of contractile protein mutations and for dissecting the mechanistic basis of disease , allowing an understanding from the molecular through the whole organism level .\",\n \"In our HCM model , the Drosophila heart tube shows a restricted phenotype , which , like abnormalities in the mutated IFM , develops from prolonged cross-bridge binding to actin .\",\n \"The restricted cardiac tube and diastolic dysfunction in this model of human HCM is congruent with phenotypes observed when expressing myosin with enhanced function ( Cammarato et al . , 2008 ) or tropomyosin that is inefficient at blocking the myosin binding site on actin ( Viswanathan et al . , 2014 ) .\",\n \"This suggests that Drosophila is an excellent analytical system for screening mutations to assess their potential for causing human HCM , which are typically expected to enhance contractility ( Adhikari et al . , 2016; Nag et al . , 2017; Spudich , 2014 ) .\",\n \"Integrating the results from our ATPase , actin motility and muscle mechanical studies reveals that there are alterations to the myosin cross-bridge cycle caused by the R146N mutation .\",\n \"Muscle mechanics measurements showed an increase in apparent rate constant 2πb , which , according to the modeling of Kawai and Brandt ( 1980 ) is influenced by rates associated with myosin attachment to actin , including binding rate , phosphate release and the power stroke ( Figure 5—figure supplement 1 ) .\",\n \"The enhanced basal myosin ATPase rates suggest that phosphate release rate has been increased , since this parameter is rate limiting for basal ATPase rate .\",\n \"This would yield an increased speed of weak to strong actin binding .\",\n \"Our modeling of molecular interactions within the myosin molecule suggests a destabilization of the pre-power stroke state , which could account for the increased affinity of the R146N myosin ADP . Pi state for actin and faster phosphate release rate .\",\n \"Drosophila myosin residue R146 normally has charge-based interactions with E774 and E775 of the myosin lever arm at the pre-power stroke state ( Figure 1C ) .\",\n \"This interaction is abolished by the R146N mutation ( Figure 1D ) , which would reduce the stability of the pre-power stroke molecule .\",\n \"This could drive the cycle forward by enhancing ATP hydrolysis and the production of cross-bridges , possibly contributing to prolonged actin binding .\",\n \"A similar mechanism may occur for human β-cardiac myosin as well , since the analogous K146 residue is also predicted to interact with E775 in the pre-power stroke state ( Figure 1—figure supplement 1A ) and this contact is destroyed by the K146N mutation ( Figure 1—figure supplement 1B ) .\",\n \"Interestingly , analysis of another HCM mutation suggests a similar mode of action as for R146N ( Guhathakurta et al . , 2017 ) .\",\n \"Time-resolved fluorescence resonance energy transfer analysis of myosin containing the E56G mutation in the lever-arm-binding essential light chain showed a reduction in pre-power stroke state molecules and enhanced levels of those in the post-power stroke state in the presence of ATP .\",\n \"As with R146N , this yielded greater actin attachment and an increased duty ratio ( Guhathakurta et al . , 2017 ) .\",\n \"Recent studies of omecamtiv mecarbil , a compound that enhances cardiac myosin power output ( Liu et al . , 2015; Planelles-Herrero et al . , 2017; Shen et al . , 2010 ) , are also relevant to understanding the molecular basis of the R146N mutant phenotypes .\",\n \"As shown in Figure 1—figure supplement 1C , the compound binds in a pocket at the nexus of the N-terminus ( including K146 ) , the relay helix and the converter domain/lever arm ( including residue E774 ) ( Planelles-Herrero et al . , 2017 ) .\",\n \"Omecamtiv mecarbil enhances the actin attachment rate and the duty ratio and slows in vitro motility ( Liu et al . , 2015 ) , similar to the effects that we observed with R146N myosin .\",\n \"In contrast to our findings , ATPase activity in the absence of actin is inhibited by the compound ( Liu et al . , 2015 ) .\",\n \"However , the rate of phosphate release is increased upon actin binding .\",\n \"While the R146N mutation enhances basal activity , which may arise from destabilization of the pre-power stroke state , omecamtiv mecarbil appears to stabilize this state prior to actin interaction , including facilitation of a weak charge-based interaction between K146 and E774 ( Figure 1—figure supplement 1C ) .\",\n \"Further clinical evidence supports the importance of these interactions in relationship to HCM , in that mutation of E774 to V774 also yields HCM ( Moric et al . , 2003 ) .\",\n \"More rapid cross-bridge formation due to the R146N or E774V mutations destabilizing the pre-power stroke state coupled with decreased detachment rates ( see below ) stabilize actin-myosin interaction , leading to enhanced force production and decreased actin filament in vitro motility .\",\n \"A similar outcome could arise from omecamtiv mecarbil increasing the number of molecules in the pre-power stroke state , which would increase the number of attached cross-bridges as the myosin molecules move through the mechanochemical cycle .\",\n \"Although myosin attachment ( Brizendine et al . , 2017 ) and detachment rates ( Harris and Warshaw , 1993 ) both influence the duty ratio ( the fraction of time myosin is strongly bound to actin during a mechanochemical cycle ) , the latter is thought to have the major influence on in vitro motility at high myosin concentrations .\",\n \"Thus , the ~50% decrease in the rate of actin movement in vitro for R146N myosin ( Figure 2F ) is likely facilitated by the decreased actin detachment rate suggested by our fiber mechanics measurements .\",\n \"We observed a decrease in apparent rate constant 2πc ( Table 3 ) , a measure of rates associated with myosin detachment from actin , which includes detachment induced by ADP release and ATP binding ( Kawai and Brandt , 1980 ) ( Figure 5—figure supplement 1 ) .\",\n \"Further , our observed increase in ATP Km in the fiber studies ( Figure 5A ) suggests decreased ATP affinity , which would slow the ATP-induced detachment rate of myosin from actin .\",\n \"This is supported by the trends showing decreases in fmax with increasing phosphate concentration for the mutant ( Figure 5B ) , which does not occur in control myosin .\",\n \"fmax is most likely being slowed by phosphate competing with ATP for the rigor state ( Pate and Cooke , 1989 ) .\",\n \"Normally , increasing phosphate either has no effect ( as seen in the control ) or causes fmax and 2πb to increase ( as seen in many slow vertebrate muscle types [Galler et al . , 2005; Kawai et al . , 1993; Siemankowski et al . , 1985] ) .\",\n \"Overall , the increase in attachment and decrease in detachment rate , in concert with no change in actin-activated ATPase rate arising from the mutation , indicate an increase in duty ratio .\",\n \"In addition to slowed in vitro motility , an increase in duty ratio would cause the increased fiber tension generation and stiffness that we observed from analysis of elastic modulus values ( Figure 5C , D ) .\",\n \"The increased duty ratio is also expected to negatively influence mutant IFM power generation ( Figure 4A ) .\",\n \"The major cause of power loss is likely decreased net work production , given that there was relatively little change in optimal frequency for muscle power generation ( fmax ) .\",\n \"Lack of change in fmax may be due to increased actin attachment and phosphate release being balanced by reduced ATP-induced detachment rate .\",\n \"Work production , however , was decreased proportionally to power for homozygous fibers examined via sinusoidal analysis , and for both homozygous and heterozygous fibers when measured using the work loop technique ( Figure 4B , Table 2 ) .\",\n \"When amplitude and length change conditions were optimized for power generation , the ratio of work absorption to work generation was increased in the mutants compared to control fibers .\",\n \"Tellingly , decreasing optimal muscle length change amplitude allowed the mutant fibers to generate more power ( compare optimal power work loops with work loops performed under the identical length change and frequency conditions ) .\",\n \"This indicates that less muscle stretching was beneficial because it decreased work absorbed , resulting in more net work and hence more power .\",\n \"It appears that increased time of cross-bridge attachment causes cross-bridge elements to act more like brakes or shock absorbers during cyclical power generation .\",\n \"These abnormally high stresses on the myofilaments likely result in the disruption of sarcomere integrity that develops over time in the IFM expressing the mutant myosin ( Figure 3 ) , as well as the reduced flight ability and WBF ( Table 4 ) .\",\n \"Our analysis of the heart tube in R146N/+ heterozygotes produced results that support the thesis that an increased myosin duty ratio yields significant mutant phenotypes .\",\n \"While the hearts in the mutants did not show a change in heart period ( suggesting no overall change in kinetics , in agreement with no change for fmax in IFM and for actin-activated ATPase ) , the heart spent a greater portion of each heart period in systole compared to the control ( Figure 7 ) .\",\n \"This is consistent with prolonged contractions arising from increased myosin attachment kinetics and a decreased detachment rate .\",\n \"An enhanced attachment rate would also increase the rate of force generation , which may partially account for the restricted cardiac phenotype .\",\n \"The reduced fractional shortening observed for the R146N/+ heart may correspond to its optimal pumping mode , mimicking the improved IFM fiber mechanical performance at shorter muscle length changes .\",\n \"At the ultrastructural level , the observed myofibrillar discontinuity in cardiomyocytes ( Figure 6 ) could be caused by the same mechanism as IFM degeneration , that is , excessive tension generation disrupting the integrity of the sarcomeric ultrastructure .\",\n \"We did not detect hypertrophy of the cardiomyocytes in R146N/+ heterozygotes , although it has been shown that hypertrophy can occur in the conical chamber of the Drosophila heart as a result of abnormal receptor tyrosine kinase pathway signaling ( Yu et al . , 2013 ) .\",\n \"It is important to note , however , that there is significant variability in the presence and degree of hypertrophy detected in patients carrying the 146N mutation ( personal communication from Jodie Ingles , Sydney Medical School ) , suggesting that interacting genes or environment may play a role in hypertrophy .\",\n \"Interestingly , our data suggest that cross-bridges in the Drosophila heart bind to thin filaments via Ca2+-dependent and Ca2+-independent mechanisms during diastole .\",\n \"This contributes to impaired relaxation .\",\n \"Our results imply that small amounts of diastolic Ca2+ promote contraction and yield slightly shortened cardiomyocytes at rest in both mutant and control lines .\",\n \"However , based upon the greater diameter increase ( greater tension drop ) in R146N/+ hearts compared to control hearts upon extra- and intracellular Ca2+ chelation with EGTA/EGTA , AM ( Figure 8B ) , disruption of Ca2+-handling in the mutant appears likely , which may promote the restricted cardiac tube phenotype .\",\n \"Therefore , it is possible that perturbation of Ca2+-handling in R146N/+ mutant fly hearts , as is frequently observed in human cardiomyopathies ( Kranias and Bers , 2007 ) , results in excessive diastolic Ca2+ levels , yielding enhancement of cell shortening and elevated basal tone .\",\n \"Additionally , the higher duty ratio of R146N myosin could indirectly enhance the Ca2+ sensitivity of the cross-bridges through thin filament activation .\",\n \"Full thin filament activation requires both Ca2+ binding to troponin and strong binding of cross-bridges .\",\n \"Thus , a higher duty-ratio myosin will have a greater number of myosin molecules strongly bound at a lower Ca2+ concentration than a lower-duty-ratio myosin .\",\n \"We experimentally demonstrated this when we expressed the high-duty-ratio EMB myosin in the jump muscle , which caused enhanced Ca2+ sensitivity for tension development compared to jump muscle myosin ( Eldred et al . , 2010 ) .\",\n \"Thus , the same concentration of Ca2+ in the R146N/+ mutant heart would generate higher force than in the control , yielding decreased cardiac diameters .\",\n \"Our analysis with blebbistatin , which impedes acto-myosin cycling ( Kovács et al . , 2004 ) , indicates that a portion of the cross-bridges responsible for diastolic tone is calcium-independent in both control and R146N/+ hearts .\",\n \"Treatment with the drug ( Figure 8C ) shows that there is a greater diameter increase in R146N/+ hearts than in control hearts , suggesting an enhanced number of Ca2+-independent cross-bridges .\",\n \"This enhancement likely arises from the higher on-rate ( actin affinity ) , which contributes to the restricted phenotype and impaired relaxation observed in R146N/+ mutant hearts .\",\n \"Increased cross-bridge availability could also result from disruption of the super-relaxed state that has been documented in both skeletal and cardiac muscles ( Hooijman et al . , 2011; Stewart et al . , 2010 ) .\",\n \"This state correlates with the presence of the interacting head motif , wherein the dimeric heads of myosin molecules interact to reduce their actin binding and ATPase activity ( Trivedi et al . , 2018; Woodhead et al . , 2005 ) .\",\n \"Disruption of this motif by mutation is linked to myosin activation that correlates with HCM ( Alamo et al . , 2017b; Nag et al . , 2017 ) .\",\n \"Interestingly , myosin is found in its pre-power stroke configuration in the interacting head motif ( Alamo et al . , 2017a; Alamo et al . , 2016 ) and disturbance of this state of filament packing by the R146N mutation could reduce the number of molecules in the super-relaxed state .\",\n \"This would enhance myosin activity within the myofibril , leading to the phenotypes we observed .\",\n \"Drosophila myosins are indeed capable of entering the interacting head motif ( Lee et al . , 2018 ) , but the presence of the super-relaxed state in thick filaments or muscle fibers is yet to be documented .\",\n \"In summary , our integrative analysis of the Drosophila analogue of the K146N human HCM mutation revealed increases in several , but not all , contractile parameters , as posited for a general mechanism of action for HCM mutations in humans ( Spudich , 2014 ) .\",\n \"Notably , enhancements in basal ATPase rate , length of systole and cross-bridge binding are concordant with these expectations .\",\n \"Further , the restricted phenotype and reduced fractional shortening observed in the Drosophila heart mimic the diastolic dysfunction/impaired relaxation and reduced ejection fraction that can accompany HCM ( Davis et al . , 2016; Maron , 2002; Maron and Maron , 2013; Masarone et al . , 2018 ) .\",\n \"The restricted phenotype observed in our model of an HCM mutant , as well as the restricted phenotypes arising from a hyperactive myosin ( Cammarato et al . , 2008 ) or mutant troponin T or actin that enhance myosin binding ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) , suggest that restriction is a common phenotype produced in the Drosophila cardiac tube due to over-active cross-bridges .\",\n \"If all or most HCM mutations have increased force production as their basis , the restricted cardiac tube phenotype in Drosophila could serve as a useful screening tool for determining whether specific human mutations are causative of HCM .\",\n \"The fly might also serve to illuminate conserved signaling pathways that lead from contractile protein mutations to HCM .\",\n \"Further , the amelioration of the mutant phenotype through genetic or drug screens in Drosophila may yield insights into potential treatments for human HCM .\"\n ],\n [\n \"Scallop muscle myosin II crystal structures in the pre-power stroke state ( PDB 1QVI ) ( Gourinath et al . , 2003 ) and the actin-detached post-power stroke state ( PDB 1KK8 ) were used as templates to predict Drosophila and human myosin structures ( Himmel et al . , 2002 ) .\",\n \"Myosin S1 amino acid sequences were modeled using the SWISS-MODEL homology modeling server ( http://swissmodel . expasy . org/ ) ( Arnold et al . , 2006 ) .\",\n \"Structures were viewed using PyMOL ( http://www . pymol . org; DeLano Scientific , Palo Alto , CA ) .\",\n \"A P element-containing Mhc transgene with the R146N mutation was constructed using standard cloning techniques along with site directed mutagenesis .\",\n \"The wild-type genomic construct PwMhc2 ( Swank et al . , 2000 ) was digested with Eag I to produced two subclones , PwMhc-5’ and pMhc3’ .\",\n \"The PwMhc-5’ subclone contains an 11 . 3 kb Eag I Mhc fragment cloned into the pCaSpeR vector ( Thummel and Pirrotta , 1992 ) .\",\n \"The PMhc-3’ subclone contains a 12 . 5 kb Eag I Mhc fragment cloned into pBluescriptKS Eag I site ( Stratagene , La Jolla , CA ) .\",\n \"The PwMhc-5’ subclone was further digested with Xho I and Avr II .\",\n \"A 6 . 8 kb Xho I-Avr II digested fragment from PwMhc-5’ was gel isolated and ligated into an Xho I-Avr II site in pLitmus 28I vector ( New England Biolabs , Ipswich , MA ) to produce pMhc-5’-XA .\",\n \"The PwMhc-5’-XA subclone was further digested with Pst I and Avr II .\",\n \"A 4 . 3 kb Pst I-Avr II digested fragment from PwMhc-5’-XA was gel isolated and ligated into an Pst I-Avr II site in pLitmus 28I vector , to produce pMhc-5’-PA .\",\n \"The PwMhc-5’-PA subclone was further digested with Pst I and Age I .\",\n \"A 1 . 6 kb Pst I-Age I digested fragment from PwMhc-5’-PA was gel isolated and ligated into an Pst I-Age I site in pLitmus 28I vector , to produce pMhc-5’-PA1 . 6 .\",\n \"The pMhc-5’-PA1 . 6 subclone was subjected to site-directed mutagenesis using the QuickChange II kit ( Stratagene ) and exon specific-primer 5’-CCGTGGCAAGAACCGTAATGAGG-3’ containing the R146N nucleotide coding change ( bold ) to yield , pR146N-5’PA-1 . 6 .\",\n \"Upon sequence confirmation of the R146N site-directed mutagenesis product , the pR146N-5’-PA-1 . 6 subclone was digested with Pst I and Age I .\",\n \"The 1 . 6 kb R146N fragment from this digest was used to replace the wild-type Pst I-Age I fragment of pMhc-5’PA .\",\n \"The resulting clone was digested with Pst I-Avr II and the Pst I-Avr II fragment was used to replace the wild-type Pst I-Avr II fragment of pMhc-5’-XA .\",\n \"The resulting clone was digested with Xho I-Avr II and the isolated Xho I-Avr II fragment was used to replace the wild-type Xho I-Avr II fragment of PwMhc-5’ .\",\n \"The resulting clone , PwMhcR146N-5’ , was digested with Eag I .\",\n \"The wild-type 3’ end subclone pMhc-3’ also was digested with Eag I .\",\n \"The 12 . 5 kb Eag I fragment was gel isolated and ligated into the Eag I of PwMhcR146N-5’ , to yield PwMhcR146N .\",\n \"The entire coding region and all ligation sites of the final PwMhcR146N plasmid were confirmed by DNA sequencing ( Eton Bioscience , San Diego , CA ) prior to P element transformation .\",\n \"Transgenic lines for PwMhcR146N were generated by P element-mediated germline transformation ( Rubin and Spradling , 1982 ) by BestGene , Inc . ( Chino Hills , CA ) .\",\n \"BestGene injected 1200 embryos with the PwMhcR146N transgene , and 29 transgenic lines were obtained .\",\n \"Each insert was mapped to its chromosomal location using balancer chromosomes and standard genetic crosses .\",\n \"Ten inserts mapped to the second chromosome , 3 to the X chromosome and 16 to the third chromosome .\",\n \"Three independent lines that mapped to the third chromosome were crossed into Mhc10 background , which is null for myosin heavy chain in IFM and TDT muscle due to mutation of the endogenous Mhc gene , which is located on the second chromosome ( Collier et al . , 1990 ) .\",\n \"RT-PCR was employed to verify that Mhc transcripts from the PwMhcR146N transgenic lines contained the appropriate site-directed nucleotide changes in exon four and that flanking alternative exons 3 and 7 were spliced correctly .\",\n \"RNA was prepared by LiCl2 extraction ( Becker et al . , 1992 ) from 2-day-old adult female transgenic flies .\",\n \"cDNAs were generated for each line using the Protoscript cDNA synthesis kit ( New England Biolabs ) .\",\n \"A reverse primer specific to exon 8 ( 5’-GTTCGTCACCCAGGGCCGTA-3’ ) and a forward primer specific to exon 2 ( 5’-TGGATCCCCGACGAGAAGGA-3’ ) were used to generate cDNA .\",\n \"Briefly , 3 μmol of reverse specific primer were mixed with 0 . 5 μg of total RNA from each transgenic line .\",\n \"PCR was performed using 1 μl of cDNA and 3 μmol of forward and reverse primers under the following conditions: 60 s at 94° C then 30 cycles of: 30 s at 94° C , 30 s at 55° C and 2 min at 68° C . RT-PCR products were sequenced by Eton Bioscience .\",\n \"To determine myosin protein expression levels for each transgene in an Mhc10 background , SDS polyacrylamide gel electrophoresis was utilized as previously described ( O'Donnell et al . , 1989 ) .\",\n \"Upper thoraces from six 2-day-old homozygous female flies were homogenized in 60 µl SDS gel buffer .\",\n \"Six µl of sample were loaded on a 9% polyacrylamide gel .\",\n \"This was repeated five different times each time using a freshly prepared sample .\",\n \"Protein accumulation was determined using Coomassie blue stained gels that were digitally scanned and analyzed on NIH Image J software .\",\n \"Values are expressed relative to the control transgene ±S . E . M . As previously reported , actin was prepared from chicken skeletal muscle after multiple steps of polymerization–depolymerization ( Kronert et al . , 2014; Kronert et al . , 2015 ) .\",\n \"Myosin was isolated from dissected dorsolongitudinal IFMs of ~250 transgenic flies and its concentration was determined by spectrophotometry ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .\",\n \"A final concentration of 2 . 0 µg/µl myosin was used for ATPase assays and 0 . 5 µg/µl was used for in vitro motility assays .\",\n \"Steady-state calcium , magnesium and actin-stimulated magnesium ATPase activities of myosin were obtained using [γ-32P]-ATP as previously described ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .\",\n \"Basal Mg-ATPase activities obtained in the absence of actin and actin-stimulated Mg-ATPase was determined using increasing concentrations of filamentous actin .\",\n \"Basal Mg-ATPase values were subtracted from all data points , which were then fit with the Michaelis–Menten equation to determine actin-stimulated ATPase ( Vmax ) and actin affinity relative to ATPase ( Km ) .\",\n \"Catalytic efficiency ( defined as Vmax/Km ) was determined as previously reported ( Kronert et al . , 2014; Kronert et al . , 2015 ) .\",\n \"In vitro motility assays were carried out by adding ATP and filamentous actin labeled with fluorescent phalloidin to nitrocellulose treated cover slips coated with wild-type or mutant myosin ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .\",\n \"Video sequences captured under fluorescence optics were analyzed computationally to determine actin-sliding velocity .\",\n \"Mean values from five independent experiments ( two assays for each ) for the mutant and wild-type ATPase or from three independent motility assays ( at least 30 filaments/assay ) were compared for statistically significant differences ( p<0 . 05 ) by Student’s t-tests .\",\n \"To determine the effects of transgene expression on IFM structure in an Mhc10 null background , transmission electron microscopy was performed as previously described ( O'Donnell and Bernstein , 1988 ) .\",\n \"We examined four different stages of development; late stage pupae , 2-hr-old adults , 2-day-old adults and 1-week-old adults .\",\n \"Cross-sections and longitudinal sections were obtained from females for each homozygous transgenic line , with at least three different samples examined for each of the three transgenic lines .\",\n \"One myofibril is shown in each panel and is representative of the entire population at the given stage of development .\",\n \"Images were obtained using a FEI Tecnai 12 transmission electron microscope with a TVIPS 214 high-resolution camera .\",\n \"For cardiac electron microscopy , heart samples at 1 or 3 weeks of age ( three per age per genotype ) were surgically exposed in an oxygenated artificial hemolymph solution and fixed using a modified procedure as described previously ( Achal et al . , 2016 ) .\",\n \"Rhythmic beating was observed to confirm structural integrity of samples .\",\n \"Hearts were relaxed in 10 mM EGTA , fixed in primary fixative ( 3% formaldehyde , 3% glutaraldehyde , 0 . 1 M cacodylate buffer pH 7 . 4 ) , washed 6x in 0 . 1 M cacodylate buffer pH 7 . 4 , then bathed in secondary fixative ( 1% OsO4 , 10 mM MgCl2 , 100 mM phosphate buffer pH 7 . 4 ) and washed 3x in 0 . 1 M cacodylate buffer pH 7 . 4 .\",\n \"The samples were dehydrated in an acetone series , oriented and embedded in Epon-filled BEEM capsules , and polymerized at 60°C under vacuum .\",\n \"Thin sections ( ≤50 nm ) were obtained using a Leica ultramicrotome , picked up on Formvar-coated grids , and stained with 4% uranyl acetate for 10 min .\",\n \"Images were obtained at 120 kV on a FEI Tecnai 12 transmission electron microscope .\",\n \"Transverse sections of the heart were utilized to determine average cardiac thickness between the second and third abdominal segments .\",\n \"For each measurement , a roughly rectangular area of cardiomyocyte tissue ( defined as myofibrillar and mitochondrial material ) of 10 µm in length within the inner and outer edges of the cardiac tube was highlighted using Adobe Photoshop .\",\n \"The image was imported into ImageJ to calculate the area of the highlighted region .\",\n \"Dividing the resulting cardiomyocyte area by 10 µm yielded the average cardiac thickness for that highlighted section .\",\n \"At least three images each for dorsal areas and for ventral areas per heart section were analyzed .\",\n \"Three or more transverse sections along the anterior-poster axis at least 10 µm apart were assessed in the same way .\",\n \"Cardiac thickness measurements from dorsal-side or ventral-side images were averaged per section and the data were then averaged for each biological replicate .\",\n \"Data from at least three hearts were averaged per line to yield the final values , which are presented as mean ± SEM .\",\n \"A one-way ANOVA with the Bonferroni correction determined statistical significance ( p<0 . 05 ) .\",\n \"Newly eclosed female PwMhcR146N-15 homozygous or heterozygous flies were collected every half hour and allowed to mature for 2 hr .\",\n \"Control flies were taken from the PwMhc2 stock .\",\n \"Individual IFM fibers were isolated by dissection as previously described by Swank ( Swank , 2012 ) .\",\n \"Briefly , the head , abdomen and wings were removed and the thorax was placed into dissection solution ( Swank , 2012 ) .\",\n \"The thorax was split in half , and the IFM fiber bundle was removed .\",\n \"The fibers were skinned in the dissection solution for 1 hr at 6°C before being transferred to storage solution ( same as the dissection solution but without Triton X-100 ) .\",\n \"Individual fibers were then separated from the bundle and split in half with a long , thinly etched tungsten probe .\",\n \"The split fiber was then fitted with a pair of aluminum foil t-clips .\",\n \"The clipped fiber was mounted in relaxing solution ( pCa 8 . 0 , 12 mM MgATP , 30 mM creatine phosphate , 600 U/ml creatine phosphokinase , 1 mM free Mg2+ , 5 mM EGTA , 20 mM pH 7 . 0 BES , 200 mM ionic strength , adjusted with Na methane sulfonate , 1 mM DTT ) onto a mechanics apparatus by settling it onto two hooks attached to a motor arm and a force transducer .\",\n \"The muscle was lengthened to be just taught , then stretched 5% beyond this length .\",\n \"The resulting length , width and height were measured to obtain cross-sectional area .\",\n \"Passive tension ( Po ) was recorded before adding activating solution ( the same as relaxing solution except that pCa was adjusted to 5 . 0 ) .\",\n \"The maximum power of the muscle was determined by performing sinusoidal analysis ( see below ) , under low amplitude conditions ( 0 . 125% muscle length ) .\",\n \"After every run , the muscle was stretched by 2% muscle length ( ML ) until the power did not increase by more than 3% .\",\n \"The active tension ( Ao ) at the optimal muscle length was measured and the net tension ( Fo ) was calculated by Ao-Po .\",\n \"Two- and 7-day-old homozygotes for each of the three transgenic lines and controls were tested for flight ability in an Mhc10 background at 22°C .\",\n \"Flight was determined by the ability to fly up ( U ) , horizontal ( H ) , down ( D ) or not at all ( N ) ( Drummond et al . , 1991 ) .\",\n \"As previously described ( Tohtong et al . , 1995 ) flight index is defined as: 6 U/T+ 4 H/T+ 2 D/T+ 0 N/T where T is the total number of flies tested .\",\n \"Flight abilities and wing beat frequencies of two-day-old PwMhcR146N-15 homozygotes and heterozygotes were examined at 15°C and 22°C .\",\n \"An optical tachometer was used to measure the wing beat frequency of tethered flies ( Tohtong et al . , 1995 ) .\",\n \"All values represent mean ± SEM .\",\n \"Significance was assessed by a Student’s t-test at p<0 . 05 .\",\n \"Cardiac tubes of 1- and 3-week-old female adult flies were surgically exposed under oxygenated artificial hemolymph as described by Vogler and Ocorr ( 2009 ) .\",\n \"High-speed videos of contracting hearts were taken at ~120 frames per second using a Hamamatsu Orca Flash 2 . 8 CMOS camera on a Leica DM5000B TL microscope with a 10x ( 0 . 30 NA ) immersion lens .\",\n \"Physiological parameters were assessed from individual videos using the semiautomatic optical heartbeat analysis ( SOHA v . 3 ) program as previously outlined ( Cammarato et al . , 2015; Fink et al . , 2009; Viswanathan et al . , 2017 ) .\",\n \"M-mode kymograms were generated to provide an edge trace documenting the movement of heart wall in the y-axis over time in the x-axis .\",\n \"Values represent mean ± S . E . M . Two-way ANOVAs were employed to test if the effects of genotype and age ( or if an interaction exists ) were significant for each cardiac parameter investigated .\",\n \"Significance was assessed at p<0 . 05 .\",\n \"Beating hearts from 3-week-old heterozygous female PwMhc2/+ and PwMhcR146N-15/+ flies were imaged as described above using a 20x ( 0 . 50 NA ) immersion objective lens and analyzed as previously detailed ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .\",\n \"The effects of EGTA/EGTA-AM and blebbistatin treatment on cardiac diameters were evaluated using paired t-tests of the matched groups .\",\n \"Unpaired t-tests were used to distinguish significant differences in the cardiac responses to EGTA , EGTA . AM and blebbistatin between PwMhc2 and mutant MHC expressing hearts .\",\n \"Significance was assessed at p<0 . 05 .\"\n ]\n]"},"abstract":{"kind":"list like","value":["K146N is a dominant mutation in human β-cardiac myosin heavy chain , which causes hypertrophic cardiomyopathy .","We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical , physiological and mechanical foundations of the disease .","ATPase assays , actin motility , and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment , yielding prolonged binding .","This increases isometric force generation , but also resistive force and work absorption during cyclical contractions , resulting in decreased work , power output , flight ability and degeneration of flight muscle sarcomere morphology .","Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes , 146N/+ hearts are hypercontractile with increased tension generation periods , decreased diastolic/systolic diameters and myofibrillar disarray .","This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments ."],"string":"[\n \"K146N is a dominant mutation in human β-cardiac myosin heavy chain , which causes hypertrophic cardiomyopathy .\",\n \"We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical , physiological and mechanical foundations of the disease .\",\n \"ATPase assays , actin motility , and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment , yielding prolonged binding .\",\n \"This increases isometric force generation , but also resistive force and work absorption during cyclical contractions , resulting in decreased work , power output , flight ability and degeneration of flight muscle sarcomere morphology .\",\n \"Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes , 146N/+ hearts are hypercontractile with increased tension generation periods , decreased diastolic/systolic diameters and myofibrillar disarray .\",\n \"This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments .\"\n]"},"summary":{"kind":"list like","value":["Myosin is a motor protein that drives the contraction of muscles .","Filaments made from myosin molecules slide between filaments of another protein called actin , tugging the edges of the muscle cell inwards .","To achieve this , part of each motor protein – called the 'head' – grabs hold of actin and uses energy to pull on the filaments .","Small genetic mutations in the gene for myosin can change the shape of the protein .","This can change the way that it interacts with actin , altering the molecular machinery that makes muscles contract .","In some cases , gene errors can cause the heart muscle wall to thicken , a condition called hypertrophic cardiomyopathy .","Mapping the locations of known mutations revealed 'hot spots' on the myosin protein where these errors are likely to cause disease .","These include the part of the molecule that swings the myosin heads , and the heads themselves .","It only takes a change to a single letter in the DNA code to thicken the heart wall , but the impact of each possible change is not yet known .","Kronert et al . have now genetically modified fruit flies to give them one of the mutations that causes thickening of the heart wall in humans .","The mutation , known as K146N , does not appear in one of the well-known 'hot spots' .","The experiments revealed that the mutation causes myosin to remain attached to actin for longer than normal .","This increased the amount of force the myosin generated , but slowed down actin movement , causing muscle stiffness .","This resulted in less power for every cycle of muscle movement , and caused the muscles to degenerate over time .","As a result , the flies were less able to use their wings , and their hearts pumped less well .","Hypertrophic cardiomyopathy can cause death in young adults , particularly competitive athletes .","Yet studying the disease in humans is challenging .","Recreating myosin mutations in fruit flies provides a way to study hypertrophic cardiomyopathy in the laboratory .","In the future , extensions to this technique could allow researchers to examine the impact of other mutations .","Models like this one could also allow early testing of new drugs or genetic treatments to repair faulty myosin molecules ."],"string":"[\n \"Myosin is a motor protein that drives the contraction of muscles .\",\n \"Filaments made from myosin molecules slide between filaments of another protein called actin , tugging the edges of the muscle cell inwards .\",\n \"To achieve this , part of each motor protein – called the 'head' – grabs hold of actin and uses energy to pull on the filaments .\",\n \"Small genetic mutations in the gene for myosin can change the shape of the protein .\",\n \"This can change the way that it interacts with actin , altering the molecular machinery that makes muscles contract .\",\n \"In some cases , gene errors can cause the heart muscle wall to thicken , a condition called hypertrophic cardiomyopathy .\",\n \"Mapping the locations of known mutations revealed 'hot spots' on the myosin protein where these errors are likely to cause disease .\",\n \"These include the part of the molecule that swings the myosin heads , and the heads themselves .\",\n \"It only takes a change to a single letter in the DNA code to thicken the heart wall , but the impact of each possible change is not yet known .\",\n \"Kronert et al . have now genetically modified fruit flies to give them one of the mutations that causes thickening of the heart wall in humans .\",\n \"The mutation , known as K146N , does not appear in one of the well-known 'hot spots' .\",\n \"The experiments revealed that the mutation causes myosin to remain attached to actin for longer than normal .\",\n \"This increased the amount of force the myosin generated , but slowed down actin movement , causing muscle stiffness .\",\n \"This resulted in less power for every cycle of muscle movement , and caused the muscles to degenerate over time .\",\n \"As a result , the flies were less able to use their wings , and their hearts pumped less well .\",\n \"Hypertrophic cardiomyopathy can cause death in young adults , particularly competitive athletes .\",\n \"Yet studying the disease in humans is challenging .\",\n \"Recreating myosin mutations in fruit flies provides a way to study hypertrophic cardiomyopathy in the laboratory .\",\n \"In the future , extensions to this technique could allow researchers to examine the impact of other mutations .\",\n \"Models like this one could also allow early testing of new drugs or genetic treatments to repair faulty myosin molecules .\"\n]"},"year":{"kind":"string","value":"2018"}}},{"rowIdx":4198,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["medicine","microbiology and infectious disease"],"string":"[\n \"medicine\",\n \"microbiology and infectious disease\"\n]"},"title":{"kind":"string","value":"Mucosal effects of tenofovir 1% gel"},"id":{"kind":"string","value":"elife-04525-v2"},"sections":{"kind":"list like","value":[["The HIV prevention field has invested considerable resources in testing the phosphonated nucleoside reverse transcriptase inhibitor ( NRTI ) tenofovir as a mucosally applied topical microbicide to prevent sexual HIV transmission .","In a phase 2B trial , CAPRISA 004 , pericoital tenofovir 1% gel was 39% efficacious in preventing vaginal HIV acquisition ( Abdool Karim et al . , 2010 ) .","However , in another phase 2B trial , the VOICE study ( MTN-003 ) , the daily vaginal tenofovir 1% gel arm was discontinued for futility ( Marrazzo et al . , 2015 ) .","Adherence to product use was low in VOICE , likely explaining the differences in findings between the two studies .","Currently , the CAPRISA 004 study is being repeated in a phase 3 trial ( FACTS 001 study ) .","A reduced glycerin formulation of the vaginal tenofovir 1% gel for use as a rectal microbicide appears safe when evaluated by epithelial sloughing , fecal calprotectin , inflammatory cytokine mRNA/protein levels , and cellular immune activation markers ( McGowan et al . , 2013 ) .","However , because topical application of an antiretroviral drug to the mucosa is a novel prevention strategy without clinical precedent , we conducted a comprehensive systems biology assessment of tenofovir gel's effects on the mucosa ."],["We measured mRNA expression changes across the complete human transcriptome by microarrays in rectal biopsies taken at 9 and 15 cm proximal to the anal margin .","Biopsies were obtained before treatment , after a single and after seven consecutive once-daily applications of reduced glycerin tenofovir 1% gel , nonoxynol-9 ( N-9 ) 2% gel , hydroxyethyl cellulose ( HEC ) placebo gel , or no treatment ( eight participants per arm were tested by microarrays ) .","The primary results of the clinical study , MTN-007 , a phase 1 , randomized , double-blind , placebo-controlled trial at three US sites were reported elsewhere ( McGowan et al . , 2013 ) .","Relative to enrollment biopsies , after 7 days of treatment , tenofovir 1% gel suppressed 505 genes and induced 137 genes in the 9 cm biopsies , whereas the detergent N-9 , a transient mucosal toxin , suppressed 56 genes and induced 60 genes ( log2 fold expression change ≥ 0 . 5 for induction or ≤ −0 . 5 for suppression , FDR ≤ 0 . 05 ) ( Figure 1A , B and Supplementary file 2 ) .","15 suppressed and 4 induced genes were common to tenofovir and N-9 ( Figure 1B ) .","In the HEC gel and no treatment arms , 16 and 23 genes changed ( Figure 1B ) .","Tenofovir 1% gel affected more genes after 7 days of treatment than after a single application and more genes in 9 cm than in 15 cm biopsies ( Figure 1B ) , with significant correlations between expression changes at 9 cm and 15 cm ( suppression: Spearman rho = 0 . 4775 [95% CI: 0 . 4051–0 . 5440]; p < 0 . 001 , Figure 1C; induction: Spearman rho = 0 . 427 [95% CI: 0 . 2840–0 . 5514]; p < 0 . 001 , Figure 1—figure supplement 1 ) .","By fold change of the individual genes , tenofovir suppressed genes more strongly than N-9 ( median 0 . 505 vs 0 . 627-fold; p < 0 . 001 ) , but induced genes less strongly ( 1 . 58 vs 1 . 69-fold; p < 0 . 001 ) ( Figure 1—figure supplement 2 ) . 10 . 7554/eLife . 04525 . 003Figure 1 . Tenofovir-induced gene expression changes in the human rectum .","( A ) Heat map of differentially expressed genes in eight participants after a single ( I ) and after seven consecutive once-daily ( VII ) rectal applications of tenofovir 1% gel compared to baseline in biopsies taken at 9 cm and 15 cm proximal to the anal margin .","Red and green bars signify strength of gene induction and suppression , respectively .","642 genes are shown , all of which exhibited an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≥ 0 . 5 ( induction ) or ≤ −0 . 5 ( suppression ) when evaluated jointly for all eight participants at time point VII in the 9 cm biopsies .","( B ) Numbers of significantly suppressed ( green borders and numbers ) and induced ( red borders and numbers ) genes after reduced glycerin tenofovir 1% gel ( TFV ) treatment and their overlap with nonoxynol-9 ( N-9 ) , hydroxyethyl cellulose ( HEC ) and no treatment ( No tx ) .","Circle area symbolizes the number of affected genes , overlap the number of genes independently affected by two or three conditions .","( C ) Correlation of log2 fold gene suppression from baseline to Day VII between 9 and 15 cm biopsies .","All 505 genes significantly suppressed at 9 cm are included .","Genes depicted as blue dots were significantly suppressed in both 9 and 15 cm biopsies ( 15 cm FDR < 0 . 05 ) , genes depicted as black dots were only significant in 9 cm biopsies ( 15 cm FDR ≥ 0 . 05 ) .","Spearman rho correlation between the 9 and 15 cm biopsies expression and the corresponding p-value of a Spearman rank correlation test are indicated on the plot .","Genes tested in Figure 2B by RT-ddPCR are specifically indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00310 . 7554/eLife . 04525 . 004Figure 1—figure supplement 1 . Correlation of log2 fold gene induction by tenofovir 1% gel from baseline to Day VII between 9 and 15 cm biopsies . All 137 genes at 9 cm significantly induced ( FDR < 0 . 05 ) and with log2 fold change > 0 . 5 are included .","Genes depicted as blue dots were significantly induced in both 9 and 15 cm biopsies ( 15 cm FDR < 0 . 05 ) , genes depicted as black dots were only significant in 9 cm biopsies ( 15 cm FDR ≥ 0 . 05 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00410 . 7554/eLife . 04525 . 005Figure 1—figure supplement 2 . Log2 fold gene suppression ( A ) and induction ( B ) from baseline to Day VII caused by N-9 and tenofovir in 9 cm biopsies .","( A ) All genes with an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≤ 0 . 5 are included ( N-9: 56 genes; tenofovir: 505 genes ) .","( B ) All genes with an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≥ 0 . 5 are included ( N-9: 60 genes; tenofovir: 137 genes ) .","Differences in magnitude of gene expression changes were statistically compared between N-9 and tenofovir by unpaired Mann–Whitney tests .","The box plots indicate medians and interquartile ranges and the whiskers indicate 10th–90th percentiles . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 005 To independently confirm the microarray results , we selected nine induced and six suppressed genes and performed reverse transcription digital droplet PCR ( RT-ddPCR ) assays with RNA from the 9 cm biopsies in the remaining seven individuals enrolled in the tenofovir 1% gel arm ( two men and five women ) , whose biopsies had not been analyzed by microarray ( Figure 2 ) .","The mRNA copy numbers of all 15 genes increased or decreased as predicted from the microarray data between baseline and time point VII ( Figure 2A , B ) .","Next , we combined the microarray and RT-ddPCR expression data , normalized fluorescence and copy number values over their respective baselines , and compared the fold change after 7 days of treatment with baseline ( Figure 2C ) .","Expression changes for all 15 genes assessed by microarray and RT-ddPCR were statistically significant ( p < 0 . 01 for all genes except TRIM5 [p = 0 . 02] ) . 10 . 7554/eLife . 04525 . 006Figure 2 . Confirmation of microarray data .","( A and B )","Quantification of mRNA copy numbers measured in 9 cm biopsies by reverse transcription ( RT ) droplet digital PCR ( ddPCR ) relative to the housekeeping gene hemoglobin beta ( HBB ) copy numbers in seven additional study participants .","( A ) Nine selected genes induced in the microarrays: CCL19 , CCL21 , CCL23 , CXCL9 , CCR7 , CD7 , CD19 , matrix metallopeptidase 12 ( MMP12 ) , and serine peptidase inhibitor of the Kazal type 4 ( SPINK4 ) .","Copy numbers at baseline ( 0 ) , after a single tenofovir gel application ( I ) and after seven consecutive once-daily applications ( VII ) are shown .","Line colors signify each of the seven study participants .","( B ) Six selected suppressed genes: p21-activated kinase ( PAK2 ) , nuclear factor of activated T cells 5 ( NFAT5 ) , desmoplakin ( DSP ) , TGF-β receptor associated protein ( TGFBRAP ) , interleukin 10 ( IL-10 ) , and tripartite motif-containing protein 5 ( TRIM5 ) .","( C ) Normalized fold changes of gene expression at Day VII over baseline in all 15 individuals treated with tenofovir 1% gel .","Red dots depict fold changes measured by microarray , blue dots depict fold changes measured by RT-ddPCR .","The boxes indicate median and 25th–75th percentiles and the whiskers indicate 10th–90th percentile .","Asterisks indicate statistical significance level relative to baseline ( one asterisk p < 0 . 05; two asterisks p < 0 . 01 , by one-sided Wilcoxon tests , adjusted for multiple testing ) .","( D ) Immunostaining of formalin-fixed 9 cm rectal biopsies from 10 participants for the proteins CD7 ( immunohistochemistry [IHC] ) , CD3 ( immunofluorescence ) and ubiquitin D ( UBD; IHC ) , predicted to be induced by the microarrays , and for IL-10 ( IHC ) , predicted to be suppressed .","For CD7 and CD3 , tissue sections were evaluated in their entirety and positive cells per mm2 are shown at baseline ( 0 ) and after seven consecutive once-daily applications ( VII ) .","Representative images are shown in Figure 2—figure supplement","1 . For UBD and IL-10 , only columnar epithelial cells were evaluated .","For UBD , the average mean staining intensities ( MSI ) per cell are shown .","Representative images are shown in Figure 2—figure supplement","2 . Colors signify each of the 10 study participants .","The boxes indicate median and 25th–75th percentiles and the whiskers indicate the range .","Paired Wilcoxon signed-rank test p values for differences between 0 and VII are listed . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00610 . 7554/eLife . 04525 . 007Figure 2—figure supplement 1 . Immunohistochemistry for CD7 , and immunofluorescence for CD3 , in rectal biopsies before ( 0 ) and after 7 days ( VII ) of daily tenofovir 1% gel use . Individual study participants are designated by letters .","Blue indicates nuclei , red indicates CD3 or CD7 . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00710 . 7554/eLife . 04525 . 008Figure 2—figure supplement 2 . Immunohistochemistry for IL-10 and ubiquitin D ( UBD ) in rectal biopsies before ( 0 ) and after 7 days ( VII ) of daily tenofovir 1% gel use . Individual study participants are designated by letters .","Blue indicates nuclei , brown indicates IL-10 or UBD . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 008 For additional confirmation , we selected three induced ( CD7 , CD3 and ubiquitin D ) and one suppressed gene ( IL-10 ) for immunohistochemical staining of the respective proteins in 9 cm rectal biopsies from 10 subjects ( Figure 2D and Figure 2—figure supplements 1 , 2 ) .","Consistent with the gene expression studies , infiltrating T lymphocytes increased twofold to fivefold in the mucosa ( mean fold change 5 . 0 , 95% CI 2 . 46–10 . 1 , p < 0 . 001 for CD7+; 2 . 44 , 1 . 17–5 . 11 , p = 0 . 023 for CD3+ ) , whereas IL-10+ columnar epithelial cells decreased by more than half ( 0 . 36 , 0 . 18–0 . 79 , p = 0 . 017 ) , between baseline and following 7 days of tenofovir 1% gel use .","Ubiquitin D was widely expressed in all biopsies , but tenofovir treatment increased the intensity of its expression ( p = 0 . 007 ) , as predicted by the microarrays .","Tenofovir 1% gel was more suppressive than stimulatory , with a ratio of induced to suppressed genes in the 9-cm rectal biopsies of 0 . 116 ( 17 genes up-regulated to 146 down ) for nuclear products .","However , genes encoding secreted proteins were more often induced than suppressed ( Figure 3A ) , with a ratio of 2 . 33 ( 35 genes up-regulated to 15 down; χ2 p < 0 . 001 ) .","Noteworthy among induced genes for secreted products were the chemokines CCL2 , CCL19 , CCL21 , CCL23 , CXCL9 , and CXCL13 ( Figure 3A ) .","Correspondingly , transcripts of a number of leukocyte-specific cell surface markers increased , specifically CD2 , CD3D , CD7 , CD8A , CD19 , CD52 , CD53 , CCR6 , and CCR7 .","The kinetics of gene induction is depicted in Figure 3—figure supplement 1 . 10 . 7554/eLife . 04525 . 009Figure 3 . Expression pattern and functional pathway analysis .","( A ) Ingenuity pathways analysis of tenofovir-induced effects in rectal biopsies , showing cellular localizations of and relationships between individual gene products .","Red symbols indicate induction and green symbols suppression at Day VII relative to baseline in 9 cm biopsies .","The diagram includes all significant genes identified as primarily located in the extracellular space and the cell nucleus .","A few selected significant genes with products localizing to the plasma membrane ( PM ) or cytoplasm ( CP ) are also shown based on their putative functional roles .","Direct ( solid lines ) and indirect ( dashed ) interactions between gene products are indicated .","Line color is arbitrary and meant to indicate relationships between groups of genes .","Yellow-shaded areas indicate zinc finger transcription factors .","( B ) Pathways analysis of tenofovir-induced effects in primary vaginal epithelial cells .","Only genes that were suppressed or induced by tenofovir both in 9 cm rectum in vivo and in vaginal epithelial cells in vitro are shown .","Primary vaginal epithelial cells derived from three healthy women were cultured with 50 or 500 μM tenofovir for 14 days .","Global gene expression microarrays at 4 , 7 , and 14 days of culture were evaluated in comparison to untreated epithelial cells .","Pre-processed microarray expression data were extremely consistent between the three vaginal cell cultures ( mean Pearson correlation coefficient 0 . 9912; Figure 3—source data 1 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00910 . 7554/eLife . 04525 . 010Figure 3—source data 1 . Pearson correlation coefficients of pre-processed microarray probe expression values between the three primary vaginal cell cultures . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 01010 . 7554/eLife . 04525 . 011Figure 3—figure supplement 1 . Average strength of gene induction by tenofovir 1% gel across all 8 microarray study participants . Heat map colors depict fold-change at Day I and Day VII over baseline .","Baseline is depicted as the vertical bar labeled ‘0’ filled with the shade of blue corresponding to a fold-change of","1 . Genes included in the list exhibited ≥1 . 6-fold average induction on Day VII or were ≥1 . 1-fold induced in at least six of eight study participants , and some knowledge about the gene products exists in the literature .","The heat map divisions signify three different kinetic patterns of gene induction: flat at day I and then induced; induced at day I and then flat; or induced at day I and then more strongly induced at day VII . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 01110 . 7554/eLife . 04525 . 012Figure 3—figure supplement 2 . Selected biological processes defined in the InnateDB database with significant enrichment of genes suppressed or induced by tenofovir 1% gel at Day VII in 9 cm biopsies . Green bars depict the percentage of genes identified as suppressed in a particular process out of the total number of genes included by InnateDB in that process .","Red bars depict corresponding percentages for gene induction .","Numbers of suppressed and induced genes are indicated above the bars .","Gene enrichment in each biological process was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .","Not all processes with significant gene enrichment are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 012 Among suppressed genes with products localizing to the cell nucleus , we identified a large number of known or putative transcription factors and their co-factors , including CREB1 ( CAMP responsive element-binding protein 1 ) and CREBBP ( CREB-binding protein ) , both activators of IL-10 transcription ( Woodgett and Ohashi , 2005; Alvarez et al . , 2009 ) , NFAT5 ( nuclear factor of activated T cells 5 ) ( Neuhofer , 2010 ) , and many zinc finger proteins ( Figure 3A ) .","Tenofovir 1% gel also suppressed genes important for regulation of transcription and translation , as well as biological processes involving transforming growth factor beta ( TGF-β ) , epithelial structure organization , regulation of cell proliferation , and apoptosis ( Figure 3—figure supplement 2 ) .","Lastly , tenofovir 1% gel suppressed genes important for mitochondrial function , including PNPT1 ( polyribonucleotide nucleotidyltransferase 1 ) ( Wang et al . , 2010 ) and OPA3 ( optic atrophy 3 ) ( Misaka et al . , 2002 ) .","Among the few genes with nuclear products that were strongly induced , KIAA0101 and ubiquitin D were notable ( Yu et al . , 2001; Lee et al . , 2003; Hosokawa et al . , 2007 ) .","Tenofovir 1% gel is most advanced as a candidate product for vaginal HIV prevention .","We were therefore interested to extend our findings from the rectum to the vaginal mucosa .","We isolated epithelial cells from vaginal tissues donated by three healthy women and tested their response to 50 and 500 μM tenofovir in vitro for up to 14 days of culture using RNA expression analysis .","Preliminary tests showed that these dosages give roughly similar intracellular concentrations of the active drug ( tenofovir diphosphate ) as measured in the vagina after tenofovir 1% gel use ( not shown ) ( Hendrix et al . , 2013 ) .","Pre-processed microarray expression data were extremely consistent between the three vaginal cell cultures ( mean Pearson correlation coefficient 0 . 9912; Figure 3—source data 1 ) .","Tenofovir's effects on the purified vaginal epithelial cells ( Figure 3B ) were similar to its effects on the rectal mucosa ( Figure 3A ) , but , as expected , changes likely driven by leukocytes in the biopsies were not seen in the purified epithelial cells , or were differently regulated .","In the epithelial cells , tenofovir did not significantly change chemokine , chemokine receptor , and cluster of differentiation ( CD ) genes , and it suppressed ISG 15 ( interferon-stimulated gene 15 ) and MX1 ( myxovirus resistance 1 ) , which were induced in the biopsies .","The number of genes affected was initially higher with 500 μM than with 50 μM tenofovir , but equalized after 14 days of culture ( Figure 4A ) .","Next , we confirmed the expression changes of select genes by ddPCR with vaginal epithelial cells from four healthy women: mRNA copies of DSP ( desmoplakin ) and IL-10 significantly decreased , and KIAA0101 significantly increased during 7 days of tenofovir treatment , as seen in the microarray data ( Figure 4B ) .","In fact , IL-10 transcripts were virtually eliminated at 7 days ( p = 0 . 002 and p = 0 . 003 for 50 and 500 μM , respectively ) , and KIAA0101 increased more than 10-fold at 500 μM tenofovir ( p = 0 . 005 ) .","By ELISA of cell lysates , IL-10 protein also decreased significantly ( n = 4 cell lines; p = 0 . 007 and p < 0 . 001 for 50 and 500 μM , respectively ) ( Figure 4C ) . 10 . 7554/eLife . 04525 . 013Figure 4 . Effects of tenofovir on primary vaginal epithelial cells .","( A ) Number of suppressed ( green ) and induced ( red ) genes in response to treatment with 50 μM ( open circles ) or 500 μM ( filled circles ) tenofovir for 1 , 4 , 7 , or 14 days ( n = 3 cell lines from different women ) .","( B ) Quantification of mRNA copy numbers at days 1 and 7 of culture by RT-ddPCR assays for two selected genes identified as suppressed ( DSP and IL-10 ) and one induced ( KIAA0101 ) in the microarray data set ( n = 4 cell lines ) .","( C ) Quantification of IL-10 protein concentrations in vaginal epithelial cells at days 1 and 7 of culture by ELISA .","Mean ( ±standard deviation ) IL-10 concentrations in the untreated cultures were 5 . 65 pg/ml ( ±0 . 25 ) at day 1 and 5 . 86 pg/ml ( ±0 . 32 ) at day 7 of culture .","Boxes and error bars in ( B ) and ( C ) signify means and standard deviations with vaginal epithelial cell cultures derived from four healthy women .","Asterisks indicate statistical significance level relative to untreated ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001 ) .","( D ) Selected biological processes defined in the InnateDB and DAVID databases with significant enrichment of genes suppressed or induced by 50 μM tenofovir in vaginal epithelial cells after 7 days of culture .","Green bars depict the percentage of genes identified as suppressed in a particular process out of the total number of genes included in that process .","Red bars depict gene induction .","Numbers of suppressed and induced genes are indicated above the bars .","Gene enrichment in each biological process was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .","Not all processes with significant gene enrichment are shown .","( E ) Proliferation of vaginal epithelial cells without or with various concentrations of tenofovir ( n = 3 cell lines ) .","Boxes depict mean percent reduction of the alamarBlue reagent in comparison to the maximum reduction .","Error bars signify standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 013 Tenofovir treatment of primary vaginal epithelial cells in vitro mostly impacted the same biological processes as it did in the rectum in vivo ( Figure 4D and Figure 3—figure supplement 2 ) .","Additionally , it suppressed genes important for keratinocyte differentiation and cellular innate immunity , and induced genes involved in DNA damage repair .","Furthermore , tenofovir enhanced vaginal epithelial cell proliferation and/or cell survival in vitro ( p = 0 . 02 ) ( Figure 4E ) .","Our microarray results indicated that tenofovir suppresses PNPT1 ( Figure 1C ) , which has been characterized as a master regulator of RNA import into mitochondria and whose deletion impairs mitochondrial function ( Wang et al . , 2010 ) .","To explore tenofovir's effects on mitochondria , we first confirmed its inhibition of PNPT1 in the 9 cm and 15 cm rectal biopsies of all 15 study participants by RT-ddPCR .","PNPT1 copy numbers decreased more than 10-fold at 9 cm ( mean fold change 0 . 09 , 95% CI 0 . 009–0 . 172 , p < 0 . 001 ) and by half at 15 cm ( 0 . 53 , 0 . 34–0 . 73 , p < 0 . 001 ) after 7 days of treatment ( Figure 5A ) .","To directly assess mitochondrial function , we picked one of the 13 genes encoded by mitochondrial DNA , ATP synthase F0 subunit 6 ( ATP6 ) , a key component of the proton channel ( Houstek et al . , 2006 ) , and measured its transcription by RT-ddPCR in the 9 cm biopsies of all 15 study participants in the tenofovir arm ( Figure 5B ) .","Because mitochondrial genes are not included on the microarray chips , we had no preexisting information on its expression .","ATP6 mRNA copy numbers decreased on average threefold ( p = 0 . 003 ) after a single application and sixfold after 7 days ( p < 0 . 001 ) .","In contrast , ATP6 copy numbers were stable in all 15 study participants treated with 2% N-9 gel ( p = 0 . 491 ) ( Figure 5B ) . 10 . 7554/eLife . 04525 . 014Figure 5 . Quantification of mitochondria-associated parameters .","( A ) PNPT1 mRNA copy numbers measured in 9 and 15 cm biopsies at baseline ( 0 ) , after a single tenofovir gel application ( I ) and after seven consecutive once-daily applications ( VII ) by RT-ddPCR assay .","( B ) Mitochondrial ATP6 mRNA copy numbers measured in 9 cm biopsies after tenofovir or N-9 treatment .","Line colors in ( A ) and ( B ) signify the 15 participants in the tenofovir arm .","Black lines signify the 15 participants in the N-9 arm .","Baseline values were compared between 9 and 15 cm biopsies by paired t-test and between tenofovir and N-9 by unpaired t-test .","Expression changes over time were tested for statistical significance by ANOVA with Bonferroni adjusted post-tests .","( C ) Assessment of mitochondrial density by electron microscopy of 9 cm biopsies in two study participants .","Each dot indicates the mean number of mitochondria per µm2 in a separate 2000× image .","( D ) Assessment of mitochondrial sizes by electron microscopy in the same biopsies .","Each dot depicts the size in µm2 of an individual mitochondrion measured at 5000× .","Dot colors in ( C ) and ( D ) correspond to the line colors of the same two study participants in ( A ) and ( B ) .","Density and size changes were tested for statistical significance by unpaired t-tests .","Horizontal lines and error bars depict means and standard deviations .","( E ) Representative electron microscopy images of normal mitochondria at baseline , and of enlarged and dysmorphic mitochondria at time point VII , in 9 cm biopsies of Subject Y . Fine structural detail is limited due to formalin fixation of biopsies .","( F ) PNPT1 and ATP6 gene expression in vaginal epithelial cell cultures in response to 1 and 7 days of 50 μM ( blue boxes ) or 500 μM ( green boxes ) tenofovir exposure in vitro .","Boxes and error bars signify means and standard deviations across four independent experiments with epithelial cell cultures derived from the vaginal mucosa of four healthy women .","Statistical significance levels in all figure panels are indicated by asterisks ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001; ‘ns’ , not significant ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 014 Next , we evaluated changes in mitochondrial number and size between baseline and after 7 days of treatment in two study participants chosen for exhibiting pronounced PNPT1 suppression by tenofovir .","The number of mitochondria per µm2 decreased by more than half after 7 days of tenofovir treatment ( p < 0 . 001 for both subjects ) ( Figure 5C ) .","In the second participant , mitochondria also increased in size by 1 . 4-fold ( p < 0 . 001 ) ( Figure 5D ) and developed dysmorphic cristae during treatment ( Figure 5E ) .","In parallel , tenofovir also caused statistically significant inhibition of PNPT1 and ATP6 mRNA expression in vaginal epithelial cells ( Figure 5F ) .","Extensive protein studies were not an intended part of MTN-007 and samples were not preserved optimally for this purpose .","However , 483 proteins were detected consistently at baseline and time point VII by mass spectrometry .","382 proteins increased in the tenofovir arm vs 112 in the no treatment arm ( five participants/arm ) .","Among these , significant increases in individual protein expression were only seen in the tenofovir arm ( Figure 6 ) .","The top 100 proteins exhibited an average fold increase of 3 . 8 ( median 3 . 2 , range 1 . 6–128 . 7 ) in the tenofovir arm , listed by p value in Figure 6 .","Enrichment analysis primarily indicated enhancement of cell survival and induction of leukocyte migration by tenofovir ( Figure 6 ) , which is consistent with our other findings . 10 . 7554/eLife . 04525 . 015Figure 6 . Unbiased mass spectrometry proteomics in rectal secretions from five study participants each in the tenofovir and the no treatment arm .","( A ) 483 proteins were consistently detected in all 10 study participants .","Of these , 382 proteins in the tenofovir arm had log2 values > 0 for fold change between baseline and after seven daily gel applications , and 112 had log2 fold change values > 0 in the no treatment arm .","Log2 fold changes ( x axis ) and p values signifying the likelihood of change ( y axis ) for these proteins are shown .","Upper panel , tenofovir arm ( 382 proteins ) ; lower panel , no treatment arm ( 112 proteins ) .","Data for proteins with log2 fold values ≤ 0 were not interpretable and are not shown .","( B ) Log2 fold changes of the top 100 proteins upregulated between baseline and after 7 days of daily gel application in each of the five study participants in the tenofovir arm .","( C ) Selected biofunctional processes defined in the Ingenuity database with significant enrichment of proteins induced in rectal secretions by 7 days of daily tenofovir 1% gel use .","The red bars with arrow heads depict the z scores for these biofunctions , indicating the strength of the directionality of the effect .","Protein enrichment in each biofunction was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .","Numbers of induced proteins are indicated above/below the bars . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 015"],["Our findings indicate that reduced glycerin rectal tenofovir 1% gel affects expression of a different and much broader range of genes than N-9 2% gel , potentially affecting mucosal immune homeostasis , mitochondrial function , and regulation of epithelial cell differentiation and survival .","These results make biological sense given that tenofovir is a DNA chain terminator , with possible off-target effects in human cells ( Lewis et al . , 2003 ) , and that topical application achieves at least 100-fold higher active drug concentrations in the mucosa than oral administration of 300 mg tenofovir disoproxil fumarate ( Anton et al . , 2012; Hendrix et al . , 2013 ) .","Moreover , tenofovir caused similar changes in primary vaginal epithelial cells cultured from several healthy women .","We did not find evidence that tenofovir directly causes inflammation , which is in keeping with our prior report that rectal tenofovir gel did not cause overt histological inflammation or increased mRNA/protein levels of a select panel of pro-inflammatory cytokines ( McGowan et al . , 2013 ) .","Rather , tenofovir dampened anti-inflammatory factors .","Most prominently , it strongly inhibited IL-10 gene and protein expression , likely via blocking of CREB1 and its coactivator CREBBP ( Martin et al . , 2005; Woodgett and Ohashi , 2005; Gee et al . , 2006; Alvarez et al . , 2009 ) .","In addition , it suppressed signaling pathways downstream of TGF-β , a central anti-inflammatory mediator in the gut ( Konkel and Chen , 2011; Surh and Sprent , 2012 ) .","Consequently , a number of chemokines were induced , such as the B lymphocyte chemoattractant CXCL13 ( Ansel et al . , 2002 ) , and CCL19 and CCL21 , both ligands of CCR7 on T lymphocytes and dendritic cells ( Forster et al . , 2008 ) .","Correspondingly , CCR7 , the B cell marker CD19 , and the T cell markers CD2 , CD3D and CD7 increased .","In keeping with this , we observed higher densities of CD3+ and CD7+ T lymphocytes in the rectal mucosa following 7 days of tenofovir 1% gel use .","In concert , these changes suggest that tenofovir creates a state of potential hyper-responsiveness to external inflammatory stimuli but does not itself cause inflammation .","In populations who , unlike our MTN-007 study cohort , have a high incidence of mucosal infections and associated immune activation , this could potentially diminish the anti-viral protective effect of topical tenofovir prophylaxis ( Naranbhai et al . , 2012 ) .","Mitochondrial toxicity of nucleotide/nucleoside reverse transcriptase inhibitors such as tenofovir is well described but the mechanism remains unclear ( Lewis et al . , 2003 ) .","We found that tenofovir consistently inhibited expression of PNPT1 , which encodes polynucleotide phosphorylase ( PNPASE ) .","PNPASE regulates nucleus-encoded RNA import into mitochondria ( Wang et al . , 2010 ) .","In PNPT1 knock-out mice , mitochondrial morphology and respiratory capacity are disrupted in a manner quite similar to the disruption in renal proximal tubular cells in patients with tenofovir-induced nephrotoxicity ( Perazella , 2010; Wang et al . , 2010 ) .","In our study , just 1 week of daily tenofovir 1% gel application lowered transcription of mitochondrial ATP6 by sixfold and caused visible ultrastructural mitochondrial changes .","These findings suggest that tenofovir's suppression of PNPT1 expression may underlie its reported , but heretofore unexplained , mitochondrial toxicity .","A number of changes in rectal biopsies and primary vaginal epithelial cells also suggested that tenofovir can cause increased epithelial proliferation .","Furthermore , tenofovir's negative effect on mitochondrial function could lead to impairment of tumor progenitor cell apoptosis ( Modica-Napolitano et al . , 2007; Ni Chonghaile et al . , 2011 ) , as has been specifically reported for loss-of-function mutations of mtATP6 , a mitochondrial gene strongly suppressed by tenofovir in our study ( Shidara et al . , 2005 ) .","Neoplastic pressure could also arise from the strong induction of KIAA0101 and UBD ( ubiquitin D ) .","KIAA0101 is important for regulation of DNA repair ( Simpson et al . , 2006 ) , is increased in tumor tissues ( Yu et al . , 2001 ) , and enhances cancer cell growth ( Jain et al . , 2011; Hosokawa et al . , 2007 ) .","UBD appears to increase mitotic non-disjunction and chromosome instability ( Ren et al . , 2006 , 2011 ) and is highly up-regulated in gastrointestinal cancers ( Lee et al . , 2003; Ren et al . , 2006 , 2011 ) .","Notably , though , these findings remain circumstantial , as there is no actual clinical evidence for carcinogenicity .","Nevertheless , they raise the question of whether the relatively high concentrations of tenofovir achieved in the mucosa during topical use could potentially lead to neoplastic lesions with continuous and long-term use .","According to Viread's Product Monograph , gastrointestinal tumorigenicity has been observed in mice after high oral dosing of tenofovir disoproxil fumarate .","Vaginal tumorigenicity has been documented for azido-thymidine , an NRTI and DNA chain terminator like tenofovir , which induced vaginal hyperplasia and carcinomas when delivered to mice intravaginally as a 2% solution ( ∼25% carcinoma rate ) ( Ayers et al . , 1996 ) .","This is the first time that a systems biology approach has been applied to a clinical trial of mucosal pre-exposure prophylaxis , and our study shows the value of using these technologies for comprehensive mucosal safety assessment .","Our findings raise concerns regarding the safety of topical tenofovir 1% gel in the rectum with long-term use .","Tenofovir's effects on vaginal epithelial cells suggest similar activities in the vagina , which we are currently verifying in MTN-014 , a phase I clinical trial comparing vaginal and rectal tenofovir 1% gel in a cross-over format .","Further studies are required to gauge whether tenofovir , which has become a valuable cornerstone drug in treating HIV infection , can also be safely and effectively used as a vaginal or rectal microbicide ."],["MTN-007 ( ClinicalTrials . gov registration NCT01232803 ) was a phase 1 , double blind , placebo-controlled trial in which participants were randomized to receive rectal reduced glycerin tenofovir 1% , nonoxynol-9 ( N-9 ) 2% or hydroxyethylcellulose ( HEC ) gels , or no-treatment ( 1:1:1:1 ) , at three clinical research sites ( Pittsburgh , PA; Birmingham , AL; Boston , MA ) .","The study protocol was approved by IRBs at all three sites .","All participants gave written informed consent .","The study details , and general safety and acceptability data , have been published elsewhere and showed that rectal tenofovir 1% gel was well tolerated and appeared safe by established safety parameters ( McGowan et al . , 2013 ) .","Each gel was administered as a single dose and then , after at least a 1-week recovery period , once daily for seven consecutive days .","The first dose of study product was self-administered under supervision by the clinic staff at the Treatment 1 Visit .","Subsequent administrations occurred at home , and study participants were instructed to insert one dose of gel into the rectum once daily throughout the 7-day period in the evening or before the longest period of rest .","A total of 65 study participants were enrolled and randomized in the study , 62 of whom completed it ( tenofovir , n = 15; N-9 , n = 16; HEC , n = 15; and no treatment , n = 16 ) .","43 ( 69% ) were male .","Microarray studies were performed on eight randomly selected male participants in each group , and confirmatory gene expression studies were done on the remaining participants .","The study population consisted of healthy , HIV-uninfected adults aged 18 or older who were required to abstain from receptive anal intercourse during the course of the clinical trial .","Female participants were required to use effective contraception .","Individuals with abnormalities of the colorectal mucosa , significant gastrointestinal symptoms ( such as a history of rectal bleeding or inflammatory bowel disease ) , evidence of anorectal Chlamydia trachomatis or Neisseria gonorrhea infection , hepatitis B infection , or who used anticoagulants were excluded from the study .","Reduced glycerin tenofovir 1% gel and HEC gel , known as the ‘Universal Placebo Gel’ ( Tien et al . , 2005 ) , were supplied by CONRAD ( Arlington , VA , USA ) .","2% N-9 gel was provided as Gynol II ( Johnson & Johnson ) .","All study products were provided in identical opaque HTI polypropylene pre-filled applicators ( HTI Plastics , Lincoln , NE ) containing 4 ml of study product .","Rectal biopsies for the microarray studies were obtained before treatment at enrollment ( time point ‘0’ ) , 30–60 min following application of the single gel dose ( time point ‘I’; to test acute single-dose effects ) , and again on the day following the last dose of the seven once-daily gel applications ( time point ‘VII’; to test multiple-dose effects ) .","Following an enema with Normosol-R pH 7 . 4 , a flexible sigmoidoscope was inserted into the rectum and biopsies were collected at 15 cm from the anal margin .","Following the sigmoidoscopy , a disposable anoscope was inserted into the anal canal for collection of rectal biopsies at 9 cm from the anal margin .","Immediately after harvest , biopsies were immersed in RNA later ( Qiagen , Germany ) , stored at 4°C overnight , and transferred to a −80°C freezer for long-term storage until shipping to Seattle and processing .","Tissues routinely discarded from vaginal repair surgeries were harvested from four otherwise healthy adult women , placed in ice-cooled calcium- and magnesium-free phosphate-buffered saline containing 100 U/ml penicillin , 100 μg/ml streptomycin , and 2 . 5 μg/ml Fungizone ( Thermo Fisher Scientific , Waltham , MA ) , and transported to the laboratory within 1 hr of removal from the donor .","Tissue harvesting and experimental procedures were approved by the Institutional Review Boards of the University of Washington and the Fred Hutchinson Cancer Research Center .","The deep submucosa was removed with surgical scissors and the remaining vaginal mucosa was cut into 5 × 5 mm pieces , which were incubated at 4°C for 18 hr in 5 ml of a 25 U/ml dispase solution ( 354235; BD Biosciences , Franklin Lakes , NJ ) .","The epithelial sheets were dissected off under a stereoscope and incubated for 10–12 min at 37°C in 2 ml 0 . 05% trypsin while gently shaking .","The dispersed cells were poured through a 100-μm cell strainer into a 50-ml tube , pelleted by centrifugation , and resuspended in F medium ( 3:1 [vol/vol] F12 [Ham]-DMEM [Thermo Fisher Scientific] , 5% fetal calf serum [Gemini Bio-Products , Calabasas , CA] , 0 . 4 μg/ml hydrocortisone [H-4001; Sigma-Aldrich , St . Louis , MO] , 5 μg/ml insulin [700-112P; Gemini Bio-Products] , 8 . 4 ng/ml cholera toxin [227036; EMD Millipore , Billerica , MA] , 10 ng/ml epidermal growth factor [PHG0311; Thermo Fisher Scientific] , 24 μg/ml adenine [A-2786; Sigma] , 100 U/ml penicillin , and 100 μg/ml streptomycin [Thermo Fisher Scientific] ) .","The keratinocytes were plated into culture flasks in the presence of ∼12 , 500/cm2 irradiated ( 6000 Rad ) 3T3-J2 feeder fibroblasts ( a kind gift by Cary A Moody ) and 10 μM of Rho kinase inhibitor Y27632 ( 1254; Enzo Life Sciences , Farmingdale , NY ) was added ( Rheinwald and Green , 1975; Chapman et al . , 2010; Liu et al . , 2012 ) .","Keratinocytes were fed every 2–3 days and passaged when around 80% confluent by 1 min treatment with 10 ml versene ( Thermo Fisher Scientific ) to remove the feeder cells , followed by 5 min treatment with trypsin/EDTA ( Thermo Fisher Scientific ) .","Dislodged keratinocytes were washed and re-plated at ∼2500 keratinocytes/cm2 with irradiated 3T3-J2 feeder fibroblasts .","Tenofovir ( CAS 147127-20-6; T018500 , Toronto Research Chemicals , Canada ) was dissolved in phosphate-buffered saline , 7% dimethyl sulfoxide , and 5% 5N sodium hydroxide to result in a 767 mM stock solution , and further diluted in culture media for addition to keratinocyte cultures in concentrations ranging from 0 . 05 to 32 mM .","Based on initial titration experiments in which we measured the intracellular concentration of tenofovir diphosphate , the active cellular metabolite of tenofovir , by liquid chromatography-tandem mass spectrometry ( performed in the laboratory of Dr Craig Hendrix , Johns Hopkins University ) , concentrations in the culture media at the lower end ( 0 . 05–0 . 5 mM range ) were estimated to be equivalent to the active concentrations likely achieved by topical tenofovir 1% gel ( 35 mM ) in mucosal epithelial cells in vivo ( Hendrix et al . , 2013; Louissaint et al . , 2013 ) .","In all experiments , control keratinocytes were cultured in parallel without tenofovir .","Keratinocytes were harvested at pre-determined time points , split into several aliquots , and pelleted .","Pellets were frozen and stored at −80°C either as dry pellets for protein assays or after suspension in RNAprotect Cell Reagent ( Qiagen ) for RNA assays .","Primary vaginal keratinocytes were cultured in six-well plates ( Corning ) with or without various concentrations of tenofovir for up to 14 days .","At day 1 , day 4 , day 7 , or day 14 , the alamarBlue reagent ( BUF012A; AbD Serotec ) was added .","After 5 hr , absorbance was measured at 570 and 600 nm on a Varioskan Flash Multimode reader ( Thermo Fisher Scientific ) .","Percentage reduction of alamarBlue , corresponding to the level of cell proliferation , was calculated as specified in the manufacturer's technical datasheet .","This was converted to percentage of maximal alamarBlue reduction by dividing each well by the well with the highest amount of alamarBlue reduction .","The IL-10 concentration in primary vaginal keratinocytes was measured after lysis of frozen cell pellets in Cell Lysis buffer ( R&D Systems , Minneapolis , MN ) using a commercial human IL-10 immunoassay ( Quantikine HS , HS100C; R&D Systems ) according to the manufacturer's specifications .","RNA was isolated from the biopsies using the RNeasy Fibrous Tissue Mini Kit ( Qiagen ) , and from the vaginal keratinocytes using the Direct-zol RNA MiniPrep kit ( Zymo Research , Irvine , CA ) , according to the manufacturer's instructions , treated with 27 Kunitz units of DNAse ( Qiagen ) to remove genomic DNA contamination , and evaluated for integrity using the Agilent RNA 6000 Nano Kit ( Agilent , Palo Alto , CA ) on an Agilent 2100 Bioanalyzer .","All samples had an RNA Integrity Number of 7 or greater .","500 ng of total RNA was amplified and labeled using the Illumina TotalPrep RNA Amplification kit ( Thermo Fisher Scientific ) .","cRNA from a total of 192 rectal biopsy samples ( eight men per study arm , four study arms , three time points , and two biopsy sites [9 cm and 15 cm] ) , and from a total of 36 primary vaginal keratinocyte cultures ( three tissue donors , three arms , and four time points ) , was hybridized to HumanHT12 v4 Expression BeadChips ( Illumina , San Diego , CA ) according to the manufacturer's protocols .","Each chip contains 47 , 323 probes , corresponding to 30 , 557 genes .","Sponges were obtained on the same visits as the biopsies , prior to the biopsy procedure , and processed as previously described ( Anton et al . , 2011 ) .","Protein concentration of sponge eluates was determined by BCA assay ( EMD Millipore ) .","Equal amounts of total protein from each sample ( 100 μg ) were then denatured in pH 8 . 0 urea exchange buffer ( 8 M Urea [GE HealthCare , United Kingdom] , 50 mM HEPES [Sigma-Aldrich] ) for 20 min at room temperature , and then placed into 10 kDa Nanosep filter cartridges .","After centrifugation , samples were treated with 25 mM dithiothreitol ( Sigma-Aldrich ) for 20 min , then 50 mM iodoacetamide ( Sigma-Aldrich ) for 20 min , followed by a wash with 50 mM HEPES buffer .","Trypsin ( Promega , Fitchburg , WI ) was added ( 2 μg/100 μg protein ) and samples were incubated at 37°C overnight in the cartridge .","Peptides were eluted off the filter with 50 mM HEPES , and the digestion was stopped with 1% formic acid .","Peptides were dried via vacuum centrifugation and cleaned of salts and detergents by reversed-phase liquid chromatography ( high pH RP , Agilent 1200 series micro-flow pump , Water XBridge column ) using a step-function gradient .","The fractions were then dried via vacuum centrifugation .","Equal amounts of peptides were re-suspended in 2% acetonitrile ( Thermo Fisher Scientific ) , 0 . 1% formic acid ( EMD , Canada ) and injected into a nano-flow LC system ( Easy nLC , Thermo Fisher Scientific ) connected in-line to a LTQ Orbitrap Velos mass spectrometer ( Thermo Fisher Scientific ) .","Mass spectrometry instrument settings were the same as described previously ( Burgener et al . , 2013 ) .","A two-step reverse transcription ( RT ) droplet digital PCR ( ddPCR ) was used to confirm microarray transcriptome data for selected genes of interest ( Hindson et al . , 2011; Pinheiro et al . , 2012 ) .","In a ddPCR assay , each sample is partitioned into ∼20 , 000 droplets representing as many individual PCR reactions .","The number of target DNA copies present per sample can be quantified based on Poisson distribution statistics , because each individual droplet is categorized as positive or negative for a given gene .","For rectal biopsies , cDNA was generated using the High Capacity cDNA Reverse Transcription Kit ( Thermo Fisher Scientific ) and the ddPCR was carried out using the 2× ddPCR Supermix for Probes ( BioRad , Hercules , CA ) .","For epithelial cell lines , these steps were combined using the 2× One-Step RT-ddPCR Kit for Probes ( BioRad ) .","ddPCR was performed on the QX100 droplet digital PCR system ( BioRad ) .","Reactions were set up with 20× 6-carboxyfluorescein ( FAM ) -labeled target gene-specific qPCR assay ( Integrated DNA Technologies , Coralville , IA; or Thermo Fisher Scientific ) and 20× VIC-labeled housekeeping hemoglobin B ( HBB ) gene-specific Taqman gene expression assay ( Thermo Fisher Scientific ) .","Each assembled ddPCR reaction mixture was loaded in duplicate into the sample wells of an eight-channel disposable droplet generator cartridge ( BioRad ) and droplet generation oil ( BioRad ) was added .","After droplet generation , the samples were amplified to the endpoint in 96-well PCR plates on a conventional thermal cycler using the following conditions: denaturation/enzyme activation for 10 min at 95°C , 40 cycles of 30 s denaturation at 94°C , and 60 s annealing/amplification at 60°C , followed by a final 10 min incubation step at 98°C .","After PCR , the droplets were read on the QX100 Droplet Reader ( BioRad ) .","Analysis of the ddPCR data was performed with QuantaSoft analysis software version 1 . 3 . 1 . 0 ( BioRad ) .","Four-micron sections were cut on a Leica RM2255 Automated Rotary Microtome ( Leica , Germany ) , mounted on positively charged EP-3000 slides ( Creative Waste Solutions , Tualatin , OR ) , dried for 1 hr at 60°C , and stored until staining at 4°C .","Slides were deparaffinized in xylene and rehydrated in graded dilutions of ethanol in water .","Antigen retrieval was performed by heating the slides in Trilogy Pretreatment Solution ( Sigma-Aldrich ) for 20 min at boiling temperature in a conventional steamer ( Black&Decker , Towson , MD ) .","Slides were then cooled for 20 min , rinsed three times in Tris-buffered 0 . 15 M NaCl solution containing 0 . 05% Tween 20 ( Wash Buffer; Dako , Santa Clara , CA ) , and stained at room temperature in an automated slide-processing system ( Dako Autostainer Plus ) .","Endogenous peroxidase activity was blocked using 3% H2O2 for 8 min , followed by 10 min in Serum-Free Protein Block ( Dako ) .","The slides were then stained for 1 hr with the primary antibodies .","Staining was performed with the following primary antibodies: anti-CD3 ( RM9107S , rabbit clone SP7; Thermo Fisher Scientific ) , anti-ubiquitin D ( NBP2-13498 , rabbit polyclonal anti-FAT10 antibody; Novus Biologicals , Littleton , CO ) , anti-CD7 ( M7255 , mouse clone CBC . 37; Dako ) , and anti-interleukin-10 ( sc-8438 , mouse clone E−10; Santa Cruz Biotechnology , Dallas , TX ) .","Negative control primary immunoglobulins were either whole rabbit IgG ( 011-000-003; Jackson ImmunoResearch Laboratories , West Grove , PA ) or mouse IgG ( I-2000; Vector Laboratories , Burlingame , CA ) .","For CD3 staining , the slides were incubated with anti-CD3 for 60 min at a dilution of 1:25 , washed in Wash Buffer , incubated for 30 min with sheep-anti-rabbit Dylight 649 ( 611-643-122; Rockland Immunochemicals , Limerick , PA ) , washed , and incubated for 30 min with donkey-anti-sheep Dylight 649 ( 613-743-168; Rockland ) .","Sections were counter-stained for 20 min with the nucleic acid-binding dye Sytox Orange ( S11368; Thermo Fisher Scientific ) at a dilution of 1:20 , 000 , and coverslipped in ProLong Gold antifade reagent ( P36930; Thermo Fisher Scientific ) .","For ubiquitin D ( UBD ) , CD7 and interleukin-10 ( IL-10 ) staining , the slides were incubated with 0 . 8 μg/ml ( UBD and CD7 ) or 4 μg/ml ( IL-10 ) of the primary antibody for 60 min .","After washing , the anti-UBD-stained slides were incubated for 30 min with Leica Power Vision HRP rabbit-specific antibody polymer ( PV6119; Leica ) ; and the anti-CD7 and anti-IL-10 stained slides were incubated for 30 min with LeicaPower Vision HRP mouse-specific antibody polymer ( PV6114; Leica ) .","After washing , staining was visualized with 3 , 3′-diaminobenzidine ( Liquid DAB+ Substrate Chromogen System; Dako ) for 7 min , and the sections were counter-stained for 2 min with hematoxylin ( Biocare , Concord , CA ) .","Controls for all antibodies were run with identical procedures but replacing the primary antibodies with either rabbit IgG or mouse IgG , as appropriate , at calculated matching concentrations .","Formalin-fixed paraffin-embedded rectal biopsies were de-paraffinized and fixed overnight in half-strength Karnovsky's fixative .","Staining , embedding , cutting , and viewing on a JEOL 1400 SX transmission electron microscope were performed as previously described ( Hladik et al . , 1999 , 2007 ) .","20 images per sample were acquired at 5 , 000× magnification .","Using ImageJ ( Collins , 2007 ) , the two-dimensional sizes in µm2 of all individual mitochondria with a circularity index of ≥0 . 9 were calculated ( for standardization purposes , only mitochondria cut near perfectly along their minor axis were evaluated ) .","10 images per sample were also acquired at 2 , 000× magnification , always including the epithelial cell brush border .","Using ImageJ , a grid of 1 . 32 µm2 squares of defined size was overlaid onto each image .","All mitochondria in the images were counted , except those in the first row of squares falling on the brush border and in squares along the image rims ( which only partially covered the tissue ) , and the mean numbers of mitochondria per µm2 were calculated for each of the acquired 2 , 000× images ( range of counted squares per image: 23–50 ) .","All p values reported were adjusted for multiple testing as appropriate , except for the exploratory protein mass spectrometry data .","Correlations of log2 fold gene expression changes between 9 cm and 15 cm biopsies in Figure 1C and Figure 1—figure supplement 1 were tested by Spearman's rank correlation coefficient .","Gene and protein expression changes over baseline were compared between study arms by two-tailed Mann–Whitney test ( Figure 1—figure supplement 2 and Figure 6 ) .","The combined expression data from the microarray and RT-ddPCR tests shown in Figure 2C were compared separately for log-fold induction or suppression over baseline using a one-tailed Wilcoxon signed-rank test with Bonferroni adjustment .","Immunohistology measurements in Figure 2D were compared between baseline and after 7 days of treatment by two-tailed paired t tests .","Due to high skewness of the CD7+ , CD3+ , and IL-10+ cell counts , these were tested after log10 transformation .","Ratios of induced to suppressed genes in Figure 3A were tested for a difference between cellular compartments using Chi-square statistics .","DSP , IL-10 , KIAA0101 , PNPT1 , and ATP6 copy numbers or protein concentrations in Figures 4B , C , 5A , B , F were tested for significant change across three time points by repeated measures ANOVA , and exact Sidak's ( Figures 4B , C , 5F ) or Tukey ( Figure 5A , B ) post-tests were run for comparisons between two time points .","Due to high skewness of the KIAA0101 copy numbers , these were tested after log10 transformation .","The effect of increasing tenofovir concentrations on the proliferation of primary vaginal epithelial cells in Figure 4E was assessed for significance by a linear model accounting for days and donors .","Copy numbers between 9 cm and 15 cm biopsies in Figure 5A were compared by two-tailed paired t test .","Copy numbers between baseline tenofovir and N-9 in Figure 5B were compared by a two-tailed unpaired t test .","Mitochondria counts and sizes in Figure 5C , D were compared between baseline and Day VII , separately for the two subjects evaluated by electron microscopy , by two-tailed unpaired t tests with Bonferroni adjustment .","Microarray chip probe expression values were tested for correlation between the three primary vaginal cell cultures by computing pairwise Pearson correlation coefficients ( Figure 3—source data 1 ) .","Statistics packages used were Prism 6 ( Graphpad Software , La Jolla , CA ) and Bioconductor/Lumi in R . Statistical results are summarized in Supplementary file 1 ."]],"string":"[\n [\n \"The HIV prevention field has invested considerable resources in testing the phosphonated nucleoside reverse transcriptase inhibitor ( NRTI ) tenofovir as a mucosally applied topical microbicide to prevent sexual HIV transmission .\",\n \"In a phase 2B trial , CAPRISA 004 , pericoital tenofovir 1% gel was 39% efficacious in preventing vaginal HIV acquisition ( Abdool Karim et al . , 2010 ) .\",\n \"However , in another phase 2B trial , the VOICE study ( MTN-003 ) , the daily vaginal tenofovir 1% gel arm was discontinued for futility ( Marrazzo et al . , 2015 ) .\",\n \"Adherence to product use was low in VOICE , likely explaining the differences in findings between the two studies .\",\n \"Currently , the CAPRISA 004 study is being repeated in a phase 3 trial ( FACTS 001 study ) .\",\n \"A reduced glycerin formulation of the vaginal tenofovir 1% gel for use as a rectal microbicide appears safe when evaluated by epithelial sloughing , fecal calprotectin , inflammatory cytokine mRNA/protein levels , and cellular immune activation markers ( McGowan et al . , 2013 ) .\",\n \"However , because topical application of an antiretroviral drug to the mucosa is a novel prevention strategy without clinical precedent , we conducted a comprehensive systems biology assessment of tenofovir gel's effects on the mucosa .\"\n ],\n [\n \"We measured mRNA expression changes across the complete human transcriptome by microarrays in rectal biopsies taken at 9 and 15 cm proximal to the anal margin .\",\n \"Biopsies were obtained before treatment , after a single and after seven consecutive once-daily applications of reduced glycerin tenofovir 1% gel , nonoxynol-9 ( N-9 ) 2% gel , hydroxyethyl cellulose ( HEC ) placebo gel , or no treatment ( eight participants per arm were tested by microarrays ) .\",\n \"The primary results of the clinical study , MTN-007 , a phase 1 , randomized , double-blind , placebo-controlled trial at three US sites were reported elsewhere ( McGowan et al . , 2013 ) .\",\n \"Relative to enrollment biopsies , after 7 days of treatment , tenofovir 1% gel suppressed 505 genes and induced 137 genes in the 9 cm biopsies , whereas the detergent N-9 , a transient mucosal toxin , suppressed 56 genes and induced 60 genes ( log2 fold expression change ≥ 0 . 5 for induction or ≤ −0 . 5 for suppression , FDR ≤ 0 . 05 ) ( Figure 1A , B and Supplementary file 2 ) .\",\n \"15 suppressed and 4 induced genes were common to tenofovir and N-9 ( Figure 1B ) .\",\n \"In the HEC gel and no treatment arms , 16 and 23 genes changed ( Figure 1B ) .\",\n \"Tenofovir 1% gel affected more genes after 7 days of treatment than after a single application and more genes in 9 cm than in 15 cm biopsies ( Figure 1B ) , with significant correlations between expression changes at 9 cm and 15 cm ( suppression: Spearman rho = 0 . 4775 [95% CI: 0 . 4051–0 . 5440]; p < 0 . 001 , Figure 1C; induction: Spearman rho = 0 . 427 [95% CI: 0 . 2840–0 . 5514]; p < 0 . 001 , Figure 1—figure supplement 1 ) .\",\n \"By fold change of the individual genes , tenofovir suppressed genes more strongly than N-9 ( median 0 . 505 vs 0 . 627-fold; p < 0 . 001 ) , but induced genes less strongly ( 1 . 58 vs 1 . 69-fold; p < 0 . 001 ) ( Figure 1—figure supplement 2 ) . 10 . 7554/eLife . 04525 . 003Figure 1 . Tenofovir-induced gene expression changes in the human rectum .\",\n \"( A ) Heat map of differentially expressed genes in eight participants after a single ( I ) and after seven consecutive once-daily ( VII ) rectal applications of tenofovir 1% gel compared to baseline in biopsies taken at 9 cm and 15 cm proximal to the anal margin .\",\n \"Red and green bars signify strength of gene induction and suppression , respectively .\",\n \"642 genes are shown , all of which exhibited an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≥ 0 . 5 ( induction ) or ≤ −0 . 5 ( suppression ) when evaluated jointly for all eight participants at time point VII in the 9 cm biopsies .\",\n \"( B ) Numbers of significantly suppressed ( green borders and numbers ) and induced ( red borders and numbers ) genes after reduced glycerin tenofovir 1% gel ( TFV ) treatment and their overlap with nonoxynol-9 ( N-9 ) , hydroxyethyl cellulose ( HEC ) and no treatment ( No tx ) .\",\n \"Circle area symbolizes the number of affected genes , overlap the number of genes independently affected by two or three conditions .\",\n \"( C ) Correlation of log2 fold gene suppression from baseline to Day VII between 9 and 15 cm biopsies .\",\n \"All 505 genes significantly suppressed at 9 cm are included .\",\n \"Genes depicted as blue dots were significantly suppressed in both 9 and 15 cm biopsies ( 15 cm FDR < 0 . 05 ) , genes depicted as black dots were only significant in 9 cm biopsies ( 15 cm FDR ≥ 0 . 05 ) .\",\n \"Spearman rho correlation between the 9 and 15 cm biopsies expression and the corresponding p-value of a Spearman rank correlation test are indicated on the plot .\",\n \"Genes tested in Figure 2B by RT-ddPCR are specifically indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00310 . 7554/eLife . 04525 . 004Figure 1—figure supplement 1 . Correlation of log2 fold gene induction by tenofovir 1% gel from baseline to Day VII between 9 and 15 cm biopsies . All 137 genes at 9 cm significantly induced ( FDR < 0 . 05 ) and with log2 fold change > 0 . 5 are included .\",\n \"Genes depicted as blue dots were significantly induced in both 9 and 15 cm biopsies ( 15 cm FDR < 0 . 05 ) , genes depicted as black dots were only significant in 9 cm biopsies ( 15 cm FDR ≥ 0 . 05 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00410 . 7554/eLife . 04525 . 005Figure 1—figure supplement 2 . Log2 fold gene suppression ( A ) and induction ( B ) from baseline to Day VII caused by N-9 and tenofovir in 9 cm biopsies .\",\n \"( A ) All genes with an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≤ 0 . 5 are included ( N-9: 56 genes; tenofovir: 505 genes ) .\",\n \"( B ) All genes with an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≥ 0 . 5 are included ( N-9: 60 genes; tenofovir: 137 genes ) .\",\n \"Differences in magnitude of gene expression changes were statistically compared between N-9 and tenofovir by unpaired Mann–Whitney tests .\",\n \"The box plots indicate medians and interquartile ranges and the whiskers indicate 10th–90th percentiles . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 005 To independently confirm the microarray results , we selected nine induced and six suppressed genes and performed reverse transcription digital droplet PCR ( RT-ddPCR ) assays with RNA from the 9 cm biopsies in the remaining seven individuals enrolled in the tenofovir 1% gel arm ( two men and five women ) , whose biopsies had not been analyzed by microarray ( Figure 2 ) .\",\n \"The mRNA copy numbers of all 15 genes increased or decreased as predicted from the microarray data between baseline and time point VII ( Figure 2A , B ) .\",\n \"Next , we combined the microarray and RT-ddPCR expression data , normalized fluorescence and copy number values over their respective baselines , and compared the fold change after 7 days of treatment with baseline ( Figure 2C ) .\",\n \"Expression changes for all 15 genes assessed by microarray and RT-ddPCR were statistically significant ( p < 0 . 01 for all genes except TRIM5 [p = 0 . 02] ) . 10 . 7554/eLife . 04525 . 006Figure 2 . Confirmation of microarray data .\",\n \"( A and B )\",\n \"Quantification of mRNA copy numbers measured in 9 cm biopsies by reverse transcription ( RT ) droplet digital PCR ( ddPCR ) relative to the housekeeping gene hemoglobin beta ( HBB ) copy numbers in seven additional study participants .\",\n \"( A ) Nine selected genes induced in the microarrays: CCL19 , CCL21 , CCL23 , CXCL9 , CCR7 , CD7 , CD19 , matrix metallopeptidase 12 ( MMP12 ) , and serine peptidase inhibitor of the Kazal type 4 ( SPINK4 ) .\",\n \"Copy numbers at baseline ( 0 ) , after a single tenofovir gel application ( I ) and after seven consecutive once-daily applications ( VII ) are shown .\",\n \"Line colors signify each of the seven study participants .\",\n \"( B ) Six selected suppressed genes: p21-activated kinase ( PAK2 ) , nuclear factor of activated T cells 5 ( NFAT5 ) , desmoplakin ( DSP ) , TGF-β receptor associated protein ( TGFBRAP ) , interleukin 10 ( IL-10 ) , and tripartite motif-containing protein 5 ( TRIM5 ) .\",\n \"( C ) Normalized fold changes of gene expression at Day VII over baseline in all 15 individuals treated with tenofovir 1% gel .\",\n \"Red dots depict fold changes measured by microarray , blue dots depict fold changes measured by RT-ddPCR .\",\n \"The boxes indicate median and 25th–75th percentiles and the whiskers indicate 10th–90th percentile .\",\n \"Asterisks indicate statistical significance level relative to baseline ( one asterisk p < 0 . 05; two asterisks p < 0 . 01 , by one-sided Wilcoxon tests , adjusted for multiple testing ) .\",\n \"( D ) Immunostaining of formalin-fixed 9 cm rectal biopsies from 10 participants for the proteins CD7 ( immunohistochemistry [IHC] ) , CD3 ( immunofluorescence ) and ubiquitin D ( UBD; IHC ) , predicted to be induced by the microarrays , and for IL-10 ( IHC ) , predicted to be suppressed .\",\n \"For CD7 and CD3 , tissue sections were evaluated in their entirety and positive cells per mm2 are shown at baseline ( 0 ) and after seven consecutive once-daily applications ( VII ) .\",\n \"Representative images are shown in Figure 2—figure supplement\",\n \"1 . For UBD and IL-10 , only columnar epithelial cells were evaluated .\",\n \"For UBD , the average mean staining intensities ( MSI ) per cell are shown .\",\n \"Representative images are shown in Figure 2—figure supplement\",\n \"2 . Colors signify each of the 10 study participants .\",\n \"The boxes indicate median and 25th–75th percentiles and the whiskers indicate the range .\",\n \"Paired Wilcoxon signed-rank test p values for differences between 0 and VII are listed . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00610 . 7554/eLife . 04525 . 007Figure 2—figure supplement 1 . Immunohistochemistry for CD7 , and immunofluorescence for CD3 , in rectal biopsies before ( 0 ) and after 7 days ( VII ) of daily tenofovir 1% gel use . Individual study participants are designated by letters .\",\n \"Blue indicates nuclei , red indicates CD3 or CD7 . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00710 . 7554/eLife . 04525 . 008Figure 2—figure supplement 2 . Immunohistochemistry for IL-10 and ubiquitin D ( UBD ) in rectal biopsies before ( 0 ) and after 7 days ( VII ) of daily tenofovir 1% gel use . Individual study participants are designated by letters .\",\n \"Blue indicates nuclei , brown indicates IL-10 or UBD . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 008 For additional confirmation , we selected three induced ( CD7 , CD3 and ubiquitin D ) and one suppressed gene ( IL-10 ) for immunohistochemical staining of the respective proteins in 9 cm rectal biopsies from 10 subjects ( Figure 2D and Figure 2—figure supplements 1 , 2 ) .\",\n \"Consistent with the gene expression studies , infiltrating T lymphocytes increased twofold to fivefold in the mucosa ( mean fold change 5 . 0 , 95% CI 2 . 46–10 . 1 , p < 0 . 001 for CD7+; 2 . 44 , 1 . 17–5 . 11 , p = 0 . 023 for CD3+ ) , whereas IL-10+ columnar epithelial cells decreased by more than half ( 0 . 36 , 0 . 18–0 . 79 , p = 0 . 017 ) , between baseline and following 7 days of tenofovir 1% gel use .\",\n \"Ubiquitin D was widely expressed in all biopsies , but tenofovir treatment increased the intensity of its expression ( p = 0 . 007 ) , as predicted by the microarrays .\",\n \"Tenofovir 1% gel was more suppressive than stimulatory , with a ratio of induced to suppressed genes in the 9-cm rectal biopsies of 0 . 116 ( 17 genes up-regulated to 146 down ) for nuclear products .\",\n \"However , genes encoding secreted proteins were more often induced than suppressed ( Figure 3A ) , with a ratio of 2 . 33 ( 35 genes up-regulated to 15 down; χ2 p < 0 . 001 ) .\",\n \"Noteworthy among induced genes for secreted products were the chemokines CCL2 , CCL19 , CCL21 , CCL23 , CXCL9 , and CXCL13 ( Figure 3A ) .\",\n \"Correspondingly , transcripts of a number of leukocyte-specific cell surface markers increased , specifically CD2 , CD3D , CD7 , CD8A , CD19 , CD52 , CD53 , CCR6 , and CCR7 .\",\n \"The kinetics of gene induction is depicted in Figure 3—figure supplement 1 . 10 . 7554/eLife . 04525 . 009Figure 3 . Expression pattern and functional pathway analysis .\",\n \"( A ) Ingenuity pathways analysis of tenofovir-induced effects in rectal biopsies , showing cellular localizations of and relationships between individual gene products .\",\n \"Red symbols indicate induction and green symbols suppression at Day VII relative to baseline in 9 cm biopsies .\",\n \"The diagram includes all significant genes identified as primarily located in the extracellular space and the cell nucleus .\",\n \"A few selected significant genes with products localizing to the plasma membrane ( PM ) or cytoplasm ( CP ) are also shown based on their putative functional roles .\",\n \"Direct ( solid lines ) and indirect ( dashed ) interactions between gene products are indicated .\",\n \"Line color is arbitrary and meant to indicate relationships between groups of genes .\",\n \"Yellow-shaded areas indicate zinc finger transcription factors .\",\n \"( B ) Pathways analysis of tenofovir-induced effects in primary vaginal epithelial cells .\",\n \"Only genes that were suppressed or induced by tenofovir both in 9 cm rectum in vivo and in vaginal epithelial cells in vitro are shown .\",\n \"Primary vaginal epithelial cells derived from three healthy women were cultured with 50 or 500 μM tenofovir for 14 days .\",\n \"Global gene expression microarrays at 4 , 7 , and 14 days of culture were evaluated in comparison to untreated epithelial cells .\",\n \"Pre-processed microarray expression data were extremely consistent between the three vaginal cell cultures ( mean Pearson correlation coefficient 0 . 9912; Figure 3—source data 1 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00910 . 7554/eLife . 04525 . 010Figure 3—source data 1 . Pearson correlation coefficients of pre-processed microarray probe expression values between the three primary vaginal cell cultures . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 01010 . 7554/eLife . 04525 . 011Figure 3—figure supplement 1 . Average strength of gene induction by tenofovir 1% gel across all 8 microarray study participants . Heat map colors depict fold-change at Day I and Day VII over baseline .\",\n \"Baseline is depicted as the vertical bar labeled ‘0’ filled with the shade of blue corresponding to a fold-change of\",\n \"1 . Genes included in the list exhibited ≥1 . 6-fold average induction on Day VII or were ≥1 . 1-fold induced in at least six of eight study participants , and some knowledge about the gene products exists in the literature .\",\n \"The heat map divisions signify three different kinetic patterns of gene induction: flat at day I and then induced; induced at day I and then flat; or induced at day I and then more strongly induced at day VII . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 01110 . 7554/eLife . 04525 . 012Figure 3—figure supplement 2 . Selected biological processes defined in the InnateDB database with significant enrichment of genes suppressed or induced by tenofovir 1% gel at Day VII in 9 cm biopsies . Green bars depict the percentage of genes identified as suppressed in a particular process out of the total number of genes included by InnateDB in that process .\",\n \"Red bars depict corresponding percentages for gene induction .\",\n \"Numbers of suppressed and induced genes are indicated above the bars .\",\n \"Gene enrichment in each biological process was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .\",\n \"Not all processes with significant gene enrichment are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 012 Among suppressed genes with products localizing to the cell nucleus , we identified a large number of known or putative transcription factors and their co-factors , including CREB1 ( CAMP responsive element-binding protein 1 ) and CREBBP ( CREB-binding protein ) , both activators of IL-10 transcription ( Woodgett and Ohashi , 2005; Alvarez et al . , 2009 ) , NFAT5 ( nuclear factor of activated T cells 5 ) ( Neuhofer , 2010 ) , and many zinc finger proteins ( Figure 3A ) .\",\n \"Tenofovir 1% gel also suppressed genes important for regulation of transcription and translation , as well as biological processes involving transforming growth factor beta ( TGF-β ) , epithelial structure organization , regulation of cell proliferation , and apoptosis ( Figure 3—figure supplement 2 ) .\",\n \"Lastly , tenofovir 1% gel suppressed genes important for mitochondrial function , including PNPT1 ( polyribonucleotide nucleotidyltransferase 1 ) ( Wang et al . , 2010 ) and OPA3 ( optic atrophy 3 ) ( Misaka et al . , 2002 ) .\",\n \"Among the few genes with nuclear products that were strongly induced , KIAA0101 and ubiquitin D were notable ( Yu et al . , 2001; Lee et al . , 2003; Hosokawa et al . , 2007 ) .\",\n \"Tenofovir 1% gel is most advanced as a candidate product for vaginal HIV prevention .\",\n \"We were therefore interested to extend our findings from the rectum to the vaginal mucosa .\",\n \"We isolated epithelial cells from vaginal tissues donated by three healthy women and tested their response to 50 and 500 μM tenofovir in vitro for up to 14 days of culture using RNA expression analysis .\",\n \"Preliminary tests showed that these dosages give roughly similar intracellular concentrations of the active drug ( tenofovir diphosphate ) as measured in the vagina after tenofovir 1% gel use ( not shown ) ( Hendrix et al . , 2013 ) .\",\n \"Pre-processed microarray expression data were extremely consistent between the three vaginal cell cultures ( mean Pearson correlation coefficient 0 . 9912; Figure 3—source data 1 ) .\",\n \"Tenofovir's effects on the purified vaginal epithelial cells ( Figure 3B ) were similar to its effects on the rectal mucosa ( Figure 3A ) , but , as expected , changes likely driven by leukocytes in the biopsies were not seen in the purified epithelial cells , or were differently regulated .\",\n \"In the epithelial cells , tenofovir did not significantly change chemokine , chemokine receptor , and cluster of differentiation ( CD ) genes , and it suppressed ISG 15 ( interferon-stimulated gene 15 ) and MX1 ( myxovirus resistance 1 ) , which were induced in the biopsies .\",\n \"The number of genes affected was initially higher with 500 μM than with 50 μM tenofovir , but equalized after 14 days of culture ( Figure 4A ) .\",\n \"Next , we confirmed the expression changes of select genes by ddPCR with vaginal epithelial cells from four healthy women: mRNA copies of DSP ( desmoplakin ) and IL-10 significantly decreased , and KIAA0101 significantly increased during 7 days of tenofovir treatment , as seen in the microarray data ( Figure 4B ) .\",\n \"In fact , IL-10 transcripts were virtually eliminated at 7 days ( p = 0 . 002 and p = 0 . 003 for 50 and 500 μM , respectively ) , and KIAA0101 increased more than 10-fold at 500 μM tenofovir ( p = 0 . 005 ) .\",\n \"By ELISA of cell lysates , IL-10 protein also decreased significantly ( n = 4 cell lines; p = 0 . 007 and p < 0 . 001 for 50 and 500 μM , respectively ) ( Figure 4C ) . 10 . 7554/eLife . 04525 . 013Figure 4 . Effects of tenofovir on primary vaginal epithelial cells .\",\n \"( A ) Number of suppressed ( green ) and induced ( red ) genes in response to treatment with 50 μM ( open circles ) or 500 μM ( filled circles ) tenofovir for 1 , 4 , 7 , or 14 days ( n = 3 cell lines from different women ) .\",\n \"( B ) Quantification of mRNA copy numbers at days 1 and 7 of culture by RT-ddPCR assays for two selected genes identified as suppressed ( DSP and IL-10 ) and one induced ( KIAA0101 ) in the microarray data set ( n = 4 cell lines ) .\",\n \"( C ) Quantification of IL-10 protein concentrations in vaginal epithelial cells at days 1 and 7 of culture by ELISA .\",\n \"Mean ( ±standard deviation ) IL-10 concentrations in the untreated cultures were 5 . 65 pg/ml ( ±0 . 25 ) at day 1 and 5 . 86 pg/ml ( ±0 . 32 ) at day 7 of culture .\",\n \"Boxes and error bars in ( B ) and ( C ) signify means and standard deviations with vaginal epithelial cell cultures derived from four healthy women .\",\n \"Asterisks indicate statistical significance level relative to untreated ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001 ) .\",\n \"( D ) Selected biological processes defined in the InnateDB and DAVID databases with significant enrichment of genes suppressed or induced by 50 μM tenofovir in vaginal epithelial cells after 7 days of culture .\",\n \"Green bars depict the percentage of genes identified as suppressed in a particular process out of the total number of genes included in that process .\",\n \"Red bars depict gene induction .\",\n \"Numbers of suppressed and induced genes are indicated above the bars .\",\n \"Gene enrichment in each biological process was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .\",\n \"Not all processes with significant gene enrichment are shown .\",\n \"( E ) Proliferation of vaginal epithelial cells without or with various concentrations of tenofovir ( n = 3 cell lines ) .\",\n \"Boxes depict mean percent reduction of the alamarBlue reagent in comparison to the maximum reduction .\",\n \"Error bars signify standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 013 Tenofovir treatment of primary vaginal epithelial cells in vitro mostly impacted the same biological processes as it did in the rectum in vivo ( Figure 4D and Figure 3—figure supplement 2 ) .\",\n \"Additionally , it suppressed genes important for keratinocyte differentiation and cellular innate immunity , and induced genes involved in DNA damage repair .\",\n \"Furthermore , tenofovir enhanced vaginal epithelial cell proliferation and/or cell survival in vitro ( p = 0 . 02 ) ( Figure 4E ) .\",\n \"Our microarray results indicated that tenofovir suppresses PNPT1 ( Figure 1C ) , which has been characterized as a master regulator of RNA import into mitochondria and whose deletion impairs mitochondrial function ( Wang et al . , 2010 ) .\",\n \"To explore tenofovir's effects on mitochondria , we first confirmed its inhibition of PNPT1 in the 9 cm and 15 cm rectal biopsies of all 15 study participants by RT-ddPCR .\",\n \"PNPT1 copy numbers decreased more than 10-fold at 9 cm ( mean fold change 0 . 09 , 95% CI 0 . 009–0 . 172 , p < 0 . 001 ) and by half at 15 cm ( 0 . 53 , 0 . 34–0 . 73 , p < 0 . 001 ) after 7 days of treatment ( Figure 5A ) .\",\n \"To directly assess mitochondrial function , we picked one of the 13 genes encoded by mitochondrial DNA , ATP synthase F0 subunit 6 ( ATP6 ) , a key component of the proton channel ( Houstek et al . , 2006 ) , and measured its transcription by RT-ddPCR in the 9 cm biopsies of all 15 study participants in the tenofovir arm ( Figure 5B ) .\",\n \"Because mitochondrial genes are not included on the microarray chips , we had no preexisting information on its expression .\",\n \"ATP6 mRNA copy numbers decreased on average threefold ( p = 0 . 003 ) after a single application and sixfold after 7 days ( p < 0 . 001 ) .\",\n \"In contrast , ATP6 copy numbers were stable in all 15 study participants treated with 2% N-9 gel ( p = 0 . 491 ) ( Figure 5B ) . 10 . 7554/eLife . 04525 . 014Figure 5 . Quantification of mitochondria-associated parameters .\",\n \"( A ) PNPT1 mRNA copy numbers measured in 9 and 15 cm biopsies at baseline ( 0 ) , after a single tenofovir gel application ( I ) and after seven consecutive once-daily applications ( VII ) by RT-ddPCR assay .\",\n \"( B ) Mitochondrial ATP6 mRNA copy numbers measured in 9 cm biopsies after tenofovir or N-9 treatment .\",\n \"Line colors in ( A ) and ( B ) signify the 15 participants in the tenofovir arm .\",\n \"Black lines signify the 15 participants in the N-9 arm .\",\n \"Baseline values were compared between 9 and 15 cm biopsies by paired t-test and between tenofovir and N-9 by unpaired t-test .\",\n \"Expression changes over time were tested for statistical significance by ANOVA with Bonferroni adjusted post-tests .\",\n \"( C ) Assessment of mitochondrial density by electron microscopy of 9 cm biopsies in two study participants .\",\n \"Each dot indicates the mean number of mitochondria per µm2 in a separate 2000× image .\",\n \"( D ) Assessment of mitochondrial sizes by electron microscopy in the same biopsies .\",\n \"Each dot depicts the size in µm2 of an individual mitochondrion measured at 5000× .\",\n \"Dot colors in ( C ) and ( D ) correspond to the line colors of the same two study participants in ( A ) and ( B ) .\",\n \"Density and size changes were tested for statistical significance by unpaired t-tests .\",\n \"Horizontal lines and error bars depict means and standard deviations .\",\n \"( E ) Representative electron microscopy images of normal mitochondria at baseline , and of enlarged and dysmorphic mitochondria at time point VII , in 9 cm biopsies of Subject Y . Fine structural detail is limited due to formalin fixation of biopsies .\",\n \"( F ) PNPT1 and ATP6 gene expression in vaginal epithelial cell cultures in response to 1 and 7 days of 50 μM ( blue boxes ) or 500 μM ( green boxes ) tenofovir exposure in vitro .\",\n \"Boxes and error bars signify means and standard deviations across four independent experiments with epithelial cell cultures derived from the vaginal mucosa of four healthy women .\",\n \"Statistical significance levels in all figure panels are indicated by asterisks ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001; ‘ns’ , not significant ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 014 Next , we evaluated changes in mitochondrial number and size between baseline and after 7 days of treatment in two study participants chosen for exhibiting pronounced PNPT1 suppression by tenofovir .\",\n \"The number of mitochondria per µm2 decreased by more than half after 7 days of tenofovir treatment ( p < 0 . 001 for both subjects ) ( Figure 5C ) .\",\n \"In the second participant , mitochondria also increased in size by 1 . 4-fold ( p < 0 . 001 ) ( Figure 5D ) and developed dysmorphic cristae during treatment ( Figure 5E ) .\",\n \"In parallel , tenofovir also caused statistically significant inhibition of PNPT1 and ATP6 mRNA expression in vaginal epithelial cells ( Figure 5F ) .\",\n \"Extensive protein studies were not an intended part of MTN-007 and samples were not preserved optimally for this purpose .\",\n \"However , 483 proteins were detected consistently at baseline and time point VII by mass spectrometry .\",\n \"382 proteins increased in the tenofovir arm vs 112 in the no treatment arm ( five participants/arm ) .\",\n \"Among these , significant increases in individual protein expression were only seen in the tenofovir arm ( Figure 6 ) .\",\n \"The top 100 proteins exhibited an average fold increase of 3 . 8 ( median 3 . 2 , range 1 . 6–128 . 7 ) in the tenofovir arm , listed by p value in Figure 6 .\",\n \"Enrichment analysis primarily indicated enhancement of cell survival and induction of leukocyte migration by tenofovir ( Figure 6 ) , which is consistent with our other findings . 10 . 7554/eLife . 04525 . 015Figure 6 . Unbiased mass spectrometry proteomics in rectal secretions from five study participants each in the tenofovir and the no treatment arm .\",\n \"( A ) 483 proteins were consistently detected in all 10 study participants .\",\n \"Of these , 382 proteins in the tenofovir arm had log2 values > 0 for fold change between baseline and after seven daily gel applications , and 112 had log2 fold change values > 0 in the no treatment arm .\",\n \"Log2 fold changes ( x axis ) and p values signifying the likelihood of change ( y axis ) for these proteins are shown .\",\n \"Upper panel , tenofovir arm ( 382 proteins ) ; lower panel , no treatment arm ( 112 proteins ) .\",\n \"Data for proteins with log2 fold values ≤ 0 were not interpretable and are not shown .\",\n \"( B ) Log2 fold changes of the top 100 proteins upregulated between baseline and after 7 days of daily gel application in each of the five study participants in the tenofovir arm .\",\n \"( C ) Selected biofunctional processes defined in the Ingenuity database with significant enrichment of proteins induced in rectal secretions by 7 days of daily tenofovir 1% gel use .\",\n \"The red bars with arrow heads depict the z scores for these biofunctions , indicating the strength of the directionality of the effect .\",\n \"Protein enrichment in each biofunction was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .\",\n \"Numbers of induced proteins are indicated above/below the bars . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 015\"\n ],\n [\n \"Our findings indicate that reduced glycerin rectal tenofovir 1% gel affects expression of a different and much broader range of genes than N-9 2% gel , potentially affecting mucosal immune homeostasis , mitochondrial function , and regulation of epithelial cell differentiation and survival .\",\n \"These results make biological sense given that tenofovir is a DNA chain terminator , with possible off-target effects in human cells ( Lewis et al . , 2003 ) , and that topical application achieves at least 100-fold higher active drug concentrations in the mucosa than oral administration of 300 mg tenofovir disoproxil fumarate ( Anton et al . , 2012; Hendrix et al . , 2013 ) .\",\n \"Moreover , tenofovir caused similar changes in primary vaginal epithelial cells cultured from several healthy women .\",\n \"We did not find evidence that tenofovir directly causes inflammation , which is in keeping with our prior report that rectal tenofovir gel did not cause overt histological inflammation or increased mRNA/protein levels of a select panel of pro-inflammatory cytokines ( McGowan et al . , 2013 ) .\",\n \"Rather , tenofovir dampened anti-inflammatory factors .\",\n \"Most prominently , it strongly inhibited IL-10 gene and protein expression , likely via blocking of CREB1 and its coactivator CREBBP ( Martin et al . , 2005; Woodgett and Ohashi , 2005; Gee et al . , 2006; Alvarez et al . , 2009 ) .\",\n \"In addition , it suppressed signaling pathways downstream of TGF-β , a central anti-inflammatory mediator in the gut ( Konkel and Chen , 2011; Surh and Sprent , 2012 ) .\",\n \"Consequently , a number of chemokines were induced , such as the B lymphocyte chemoattractant CXCL13 ( Ansel et al . , 2002 ) , and CCL19 and CCL21 , both ligands of CCR7 on T lymphocytes and dendritic cells ( Forster et al . , 2008 ) .\",\n \"Correspondingly , CCR7 , the B cell marker CD19 , and the T cell markers CD2 , CD3D and CD7 increased .\",\n \"In keeping with this , we observed higher densities of CD3+ and CD7+ T lymphocytes in the rectal mucosa following 7 days of tenofovir 1% gel use .\",\n \"In concert , these changes suggest that tenofovir creates a state of potential hyper-responsiveness to external inflammatory stimuli but does not itself cause inflammation .\",\n \"In populations who , unlike our MTN-007 study cohort , have a high incidence of mucosal infections and associated immune activation , this could potentially diminish the anti-viral protective effect of topical tenofovir prophylaxis ( Naranbhai et al . , 2012 ) .\",\n \"Mitochondrial toxicity of nucleotide/nucleoside reverse transcriptase inhibitors such as tenofovir is well described but the mechanism remains unclear ( Lewis et al . , 2003 ) .\",\n \"We found that tenofovir consistently inhibited expression of PNPT1 , which encodes polynucleotide phosphorylase ( PNPASE ) .\",\n \"PNPASE regulates nucleus-encoded RNA import into mitochondria ( Wang et al . , 2010 ) .\",\n \"In PNPT1 knock-out mice , mitochondrial morphology and respiratory capacity are disrupted in a manner quite similar to the disruption in renal proximal tubular cells in patients with tenofovir-induced nephrotoxicity ( Perazella , 2010; Wang et al . , 2010 ) .\",\n \"In our study , just 1 week of daily tenofovir 1% gel application lowered transcription of mitochondrial ATP6 by sixfold and caused visible ultrastructural mitochondrial changes .\",\n \"These findings suggest that tenofovir's suppression of PNPT1 expression may underlie its reported , but heretofore unexplained , mitochondrial toxicity .\",\n \"A number of changes in rectal biopsies and primary vaginal epithelial cells also suggested that tenofovir can cause increased epithelial proliferation .\",\n \"Furthermore , tenofovir's negative effect on mitochondrial function could lead to impairment of tumor progenitor cell apoptosis ( Modica-Napolitano et al . , 2007; Ni Chonghaile et al . , 2011 ) , as has been specifically reported for loss-of-function mutations of mtATP6 , a mitochondrial gene strongly suppressed by tenofovir in our study ( Shidara et al . , 2005 ) .\",\n \"Neoplastic pressure could also arise from the strong induction of KIAA0101 and UBD ( ubiquitin D ) .\",\n \"KIAA0101 is important for regulation of DNA repair ( Simpson et al . , 2006 ) , is increased in tumor tissues ( Yu et al . , 2001 ) , and enhances cancer cell growth ( Jain et al . , 2011; Hosokawa et al . , 2007 ) .\",\n \"UBD appears to increase mitotic non-disjunction and chromosome instability ( Ren et al . , 2006 , 2011 ) and is highly up-regulated in gastrointestinal cancers ( Lee et al . , 2003; Ren et al . , 2006 , 2011 ) .\",\n \"Notably , though , these findings remain circumstantial , as there is no actual clinical evidence for carcinogenicity .\",\n \"Nevertheless , they raise the question of whether the relatively high concentrations of tenofovir achieved in the mucosa during topical use could potentially lead to neoplastic lesions with continuous and long-term use .\",\n \"According to Viread's Product Monograph , gastrointestinal tumorigenicity has been observed in mice after high oral dosing of tenofovir disoproxil fumarate .\",\n \"Vaginal tumorigenicity has been documented for azido-thymidine , an NRTI and DNA chain terminator like tenofovir , which induced vaginal hyperplasia and carcinomas when delivered to mice intravaginally as a 2% solution ( ∼25% carcinoma rate ) ( Ayers et al . , 1996 ) .\",\n \"This is the first time that a systems biology approach has been applied to a clinical trial of mucosal pre-exposure prophylaxis , and our study shows the value of using these technologies for comprehensive mucosal safety assessment .\",\n \"Our findings raise concerns regarding the safety of topical tenofovir 1% gel in the rectum with long-term use .\",\n \"Tenofovir's effects on vaginal epithelial cells suggest similar activities in the vagina , which we are currently verifying in MTN-014 , a phase I clinical trial comparing vaginal and rectal tenofovir 1% gel in a cross-over format .\",\n \"Further studies are required to gauge whether tenofovir , which has become a valuable cornerstone drug in treating HIV infection , can also be safely and effectively used as a vaginal or rectal microbicide .\"\n ],\n [\n \"MTN-007 ( ClinicalTrials . gov registration NCT01232803 ) was a phase 1 , double blind , placebo-controlled trial in which participants were randomized to receive rectal reduced glycerin tenofovir 1% , nonoxynol-9 ( N-9 ) 2% or hydroxyethylcellulose ( HEC ) gels , or no-treatment ( 1:1:1:1 ) , at three clinical research sites ( Pittsburgh , PA; Birmingham , AL; Boston , MA ) .\",\n \"The study protocol was approved by IRBs at all three sites .\",\n \"All participants gave written informed consent .\",\n \"The study details , and general safety and acceptability data , have been published elsewhere and showed that rectal tenofovir 1% gel was well tolerated and appeared safe by established safety parameters ( McGowan et al . , 2013 ) .\",\n \"Each gel was administered as a single dose and then , after at least a 1-week recovery period , once daily for seven consecutive days .\",\n \"The first dose of study product was self-administered under supervision by the clinic staff at the Treatment 1 Visit .\",\n \"Subsequent administrations occurred at home , and study participants were instructed to insert one dose of gel into the rectum once daily throughout the 7-day period in the evening or before the longest period of rest .\",\n \"A total of 65 study participants were enrolled and randomized in the study , 62 of whom completed it ( tenofovir , n = 15; N-9 , n = 16; HEC , n = 15; and no treatment , n = 16 ) .\",\n \"43 ( 69% ) were male .\",\n \"Microarray studies were performed on eight randomly selected male participants in each group , and confirmatory gene expression studies were done on the remaining participants .\",\n \"The study population consisted of healthy , HIV-uninfected adults aged 18 or older who were required to abstain from receptive anal intercourse during the course of the clinical trial .\",\n \"Female participants were required to use effective contraception .\",\n \"Individuals with abnormalities of the colorectal mucosa , significant gastrointestinal symptoms ( such as a history of rectal bleeding or inflammatory bowel disease ) , evidence of anorectal Chlamydia trachomatis or Neisseria gonorrhea infection , hepatitis B infection , or who used anticoagulants were excluded from the study .\",\n \"Reduced glycerin tenofovir 1% gel and HEC gel , known as the ‘Universal Placebo Gel’ ( Tien et al . , 2005 ) , were supplied by CONRAD ( Arlington , VA , USA ) .\",\n \"2% N-9 gel was provided as Gynol II ( Johnson & Johnson ) .\",\n \"All study products were provided in identical opaque HTI polypropylene pre-filled applicators ( HTI Plastics , Lincoln , NE ) containing 4 ml of study product .\",\n \"Rectal biopsies for the microarray studies were obtained before treatment at enrollment ( time point ‘0’ ) , 30–60 min following application of the single gel dose ( time point ‘I’; to test acute single-dose effects ) , and again on the day following the last dose of the seven once-daily gel applications ( time point ‘VII’; to test multiple-dose effects ) .\",\n \"Following an enema with Normosol-R pH 7 . 4 , a flexible sigmoidoscope was inserted into the rectum and biopsies were collected at 15 cm from the anal margin .\",\n \"Following the sigmoidoscopy , a disposable anoscope was inserted into the anal canal for collection of rectal biopsies at 9 cm from the anal margin .\",\n \"Immediately after harvest , biopsies were immersed in RNA later ( Qiagen , Germany ) , stored at 4°C overnight , and transferred to a −80°C freezer for long-term storage until shipping to Seattle and processing .\",\n \"Tissues routinely discarded from vaginal repair surgeries were harvested from four otherwise healthy adult women , placed in ice-cooled calcium- and magnesium-free phosphate-buffered saline containing 100 U/ml penicillin , 100 μg/ml streptomycin , and 2 . 5 μg/ml Fungizone ( Thermo Fisher Scientific , Waltham , MA ) , and transported to the laboratory within 1 hr of removal from the donor .\",\n \"Tissue harvesting and experimental procedures were approved by the Institutional Review Boards of the University of Washington and the Fred Hutchinson Cancer Research Center .\",\n \"The deep submucosa was removed with surgical scissors and the remaining vaginal mucosa was cut into 5 × 5 mm pieces , which were incubated at 4°C for 18 hr in 5 ml of a 25 U/ml dispase solution ( 354235; BD Biosciences , Franklin Lakes , NJ ) .\",\n \"The epithelial sheets were dissected off under a stereoscope and incubated for 10–12 min at 37°C in 2 ml 0 . 05% trypsin while gently shaking .\",\n \"The dispersed cells were poured through a 100-μm cell strainer into a 50-ml tube , pelleted by centrifugation , and resuspended in F medium ( 3:1 [vol/vol] F12 [Ham]-DMEM [Thermo Fisher Scientific] , 5% fetal calf serum [Gemini Bio-Products , Calabasas , CA] , 0 . 4 μg/ml hydrocortisone [H-4001; Sigma-Aldrich , St . Louis , MO] , 5 μg/ml insulin [700-112P; Gemini Bio-Products] , 8 . 4 ng/ml cholera toxin [227036; EMD Millipore , Billerica , MA] , 10 ng/ml epidermal growth factor [PHG0311; Thermo Fisher Scientific] , 24 μg/ml adenine [A-2786; Sigma] , 100 U/ml penicillin , and 100 μg/ml streptomycin [Thermo Fisher Scientific] ) .\",\n \"The keratinocytes were plated into culture flasks in the presence of ∼12 , 500/cm2 irradiated ( 6000 Rad ) 3T3-J2 feeder fibroblasts ( a kind gift by Cary A Moody ) and 10 μM of Rho kinase inhibitor Y27632 ( 1254; Enzo Life Sciences , Farmingdale , NY ) was added ( Rheinwald and Green , 1975; Chapman et al . , 2010; Liu et al . , 2012 ) .\",\n \"Keratinocytes were fed every 2–3 days and passaged when around 80% confluent by 1 min treatment with 10 ml versene ( Thermo Fisher Scientific ) to remove the feeder cells , followed by 5 min treatment with trypsin/EDTA ( Thermo Fisher Scientific ) .\",\n \"Dislodged keratinocytes were washed and re-plated at ∼2500 keratinocytes/cm2 with irradiated 3T3-J2 feeder fibroblasts .\",\n \"Tenofovir ( CAS 147127-20-6; T018500 , Toronto Research Chemicals , Canada ) was dissolved in phosphate-buffered saline , 7% dimethyl sulfoxide , and 5% 5N sodium hydroxide to result in a 767 mM stock solution , and further diluted in culture media for addition to keratinocyte cultures in concentrations ranging from 0 . 05 to 32 mM .\",\n \"Based on initial titration experiments in which we measured the intracellular concentration of tenofovir diphosphate , the active cellular metabolite of tenofovir , by liquid chromatography-tandem mass spectrometry ( performed in the laboratory of Dr Craig Hendrix , Johns Hopkins University ) , concentrations in the culture media at the lower end ( 0 . 05–0 . 5 mM range ) were estimated to be equivalent to the active concentrations likely achieved by topical tenofovir 1% gel ( 35 mM ) in mucosal epithelial cells in vivo ( Hendrix et al . , 2013; Louissaint et al . , 2013 ) .\",\n \"In all experiments , control keratinocytes were cultured in parallel without tenofovir .\",\n \"Keratinocytes were harvested at pre-determined time points , split into several aliquots , and pelleted .\",\n \"Pellets were frozen and stored at −80°C either as dry pellets for protein assays or after suspension in RNAprotect Cell Reagent ( Qiagen ) for RNA assays .\",\n \"Primary vaginal keratinocytes were cultured in six-well plates ( Corning ) with or without various concentrations of tenofovir for up to 14 days .\",\n \"At day 1 , day 4 , day 7 , or day 14 , the alamarBlue reagent ( BUF012A; AbD Serotec ) was added .\",\n \"After 5 hr , absorbance was measured at 570 and 600 nm on a Varioskan Flash Multimode reader ( Thermo Fisher Scientific ) .\",\n \"Percentage reduction of alamarBlue , corresponding to the level of cell proliferation , was calculated as specified in the manufacturer's technical datasheet .\",\n \"This was converted to percentage of maximal alamarBlue reduction by dividing each well by the well with the highest amount of alamarBlue reduction .\",\n \"The IL-10 concentration in primary vaginal keratinocytes was measured after lysis of frozen cell pellets in Cell Lysis buffer ( R&D Systems , Minneapolis , MN ) using a commercial human IL-10 immunoassay ( Quantikine HS , HS100C; R&D Systems ) according to the manufacturer's specifications .\",\n \"RNA was isolated from the biopsies using the RNeasy Fibrous Tissue Mini Kit ( Qiagen ) , and from the vaginal keratinocytes using the Direct-zol RNA MiniPrep kit ( Zymo Research , Irvine , CA ) , according to the manufacturer's instructions , treated with 27 Kunitz units of DNAse ( Qiagen ) to remove genomic DNA contamination , and evaluated for integrity using the Agilent RNA 6000 Nano Kit ( Agilent , Palo Alto , CA ) on an Agilent 2100 Bioanalyzer .\",\n \"All samples had an RNA Integrity Number of 7 or greater .\",\n \"500 ng of total RNA was amplified and labeled using the Illumina TotalPrep RNA Amplification kit ( Thermo Fisher Scientific ) .\",\n \"cRNA from a total of 192 rectal biopsy samples ( eight men per study arm , four study arms , three time points , and two biopsy sites [9 cm and 15 cm] ) , and from a total of 36 primary vaginal keratinocyte cultures ( three tissue donors , three arms , and four time points ) , was hybridized to HumanHT12 v4 Expression BeadChips ( Illumina , San Diego , CA ) according to the manufacturer's protocols .\",\n \"Each chip contains 47 , 323 probes , corresponding to 30 , 557 genes .\",\n \"Sponges were obtained on the same visits as the biopsies , prior to the biopsy procedure , and processed as previously described ( Anton et al . , 2011 ) .\",\n \"Protein concentration of sponge eluates was determined by BCA assay ( EMD Millipore ) .\",\n \"Equal amounts of total protein from each sample ( 100 μg ) were then denatured in pH 8 . 0 urea exchange buffer ( 8 M Urea [GE HealthCare , United Kingdom] , 50 mM HEPES [Sigma-Aldrich] ) for 20 min at room temperature , and then placed into 10 kDa Nanosep filter cartridges .\",\n \"After centrifugation , samples were treated with 25 mM dithiothreitol ( Sigma-Aldrich ) for 20 min , then 50 mM iodoacetamide ( Sigma-Aldrich ) for 20 min , followed by a wash with 50 mM HEPES buffer .\",\n \"Trypsin ( Promega , Fitchburg , WI ) was added ( 2 μg/100 μg protein ) and samples were incubated at 37°C overnight in the cartridge .\",\n \"Peptides were eluted off the filter with 50 mM HEPES , and the digestion was stopped with 1% formic acid .\",\n \"Peptides were dried via vacuum centrifugation and cleaned of salts and detergents by reversed-phase liquid chromatography ( high pH RP , Agilent 1200 series micro-flow pump , Water XBridge column ) using a step-function gradient .\",\n \"The fractions were then dried via vacuum centrifugation .\",\n \"Equal amounts of peptides were re-suspended in 2% acetonitrile ( Thermo Fisher Scientific ) , 0 . 1% formic acid ( EMD , Canada ) and injected into a nano-flow LC system ( Easy nLC , Thermo Fisher Scientific ) connected in-line to a LTQ Orbitrap Velos mass spectrometer ( Thermo Fisher Scientific ) .\",\n \"Mass spectrometry instrument settings were the same as described previously ( Burgener et al . , 2013 ) .\",\n \"A two-step reverse transcription ( RT ) droplet digital PCR ( ddPCR ) was used to confirm microarray transcriptome data for selected genes of interest ( Hindson et al . , 2011; Pinheiro et al . , 2012 ) .\",\n \"In a ddPCR assay , each sample is partitioned into ∼20 , 000 droplets representing as many individual PCR reactions .\",\n \"The number of target DNA copies present per sample can be quantified based on Poisson distribution statistics , because each individual droplet is categorized as positive or negative for a given gene .\",\n \"For rectal biopsies , cDNA was generated using the High Capacity cDNA Reverse Transcription Kit ( Thermo Fisher Scientific ) and the ddPCR was carried out using the 2× ddPCR Supermix for Probes ( BioRad , Hercules , CA ) .\",\n \"For epithelial cell lines , these steps were combined using the 2× One-Step RT-ddPCR Kit for Probes ( BioRad ) .\",\n \"ddPCR was performed on the QX100 droplet digital PCR system ( BioRad ) .\",\n \"Reactions were set up with 20× 6-carboxyfluorescein ( FAM ) -labeled target gene-specific qPCR assay ( Integrated DNA Technologies , Coralville , IA; or Thermo Fisher Scientific ) and 20× VIC-labeled housekeeping hemoglobin B ( HBB ) gene-specific Taqman gene expression assay ( Thermo Fisher Scientific ) .\",\n \"Each assembled ddPCR reaction mixture was loaded in duplicate into the sample wells of an eight-channel disposable droplet generator cartridge ( BioRad ) and droplet generation oil ( BioRad ) was added .\",\n \"After droplet generation , the samples were amplified to the endpoint in 96-well PCR plates on a conventional thermal cycler using the following conditions: denaturation/enzyme activation for 10 min at 95°C , 40 cycles of 30 s denaturation at 94°C , and 60 s annealing/amplification at 60°C , followed by a final 10 min incubation step at 98°C .\",\n \"After PCR , the droplets were read on the QX100 Droplet Reader ( BioRad ) .\",\n \"Analysis of the ddPCR data was performed with QuantaSoft analysis software version 1 . 3 . 1 . 0 ( BioRad ) .\",\n \"Four-micron sections were cut on a Leica RM2255 Automated Rotary Microtome ( Leica , Germany ) , mounted on positively charged EP-3000 slides ( Creative Waste Solutions , Tualatin , OR ) , dried for 1 hr at 60°C , and stored until staining at 4°C .\",\n \"Slides were deparaffinized in xylene and rehydrated in graded dilutions of ethanol in water .\",\n \"Antigen retrieval was performed by heating the slides in Trilogy Pretreatment Solution ( Sigma-Aldrich ) for 20 min at boiling temperature in a conventional steamer ( Black&Decker , Towson , MD ) .\",\n \"Slides were then cooled for 20 min , rinsed three times in Tris-buffered 0 . 15 M NaCl solution containing 0 . 05% Tween 20 ( Wash Buffer; Dako , Santa Clara , CA ) , and stained at room temperature in an automated slide-processing system ( Dako Autostainer Plus ) .\",\n \"Endogenous peroxidase activity was blocked using 3% H2O2 for 8 min , followed by 10 min in Serum-Free Protein Block ( Dako ) .\",\n \"The slides were then stained for 1 hr with the primary antibodies .\",\n \"Staining was performed with the following primary antibodies: anti-CD3 ( RM9107S , rabbit clone SP7; Thermo Fisher Scientific ) , anti-ubiquitin D ( NBP2-13498 , rabbit polyclonal anti-FAT10 antibody; Novus Biologicals , Littleton , CO ) , anti-CD7 ( M7255 , mouse clone CBC . 37; Dako ) , and anti-interleukin-10 ( sc-8438 , mouse clone E−10; Santa Cruz Biotechnology , Dallas , TX ) .\",\n \"Negative control primary immunoglobulins were either whole rabbit IgG ( 011-000-003; Jackson ImmunoResearch Laboratories , West Grove , PA ) or mouse IgG ( I-2000; Vector Laboratories , Burlingame , CA ) .\",\n \"For CD3 staining , the slides were incubated with anti-CD3 for 60 min at a dilution of 1:25 , washed in Wash Buffer , incubated for 30 min with sheep-anti-rabbit Dylight 649 ( 611-643-122; Rockland Immunochemicals , Limerick , PA ) , washed , and incubated for 30 min with donkey-anti-sheep Dylight 649 ( 613-743-168; Rockland ) .\",\n \"Sections were counter-stained for 20 min with the nucleic acid-binding dye Sytox Orange ( S11368; Thermo Fisher Scientific ) at a dilution of 1:20 , 000 , and coverslipped in ProLong Gold antifade reagent ( P36930; Thermo Fisher Scientific ) .\",\n \"For ubiquitin D ( UBD ) , CD7 and interleukin-10 ( IL-10 ) staining , the slides were incubated with 0 . 8 μg/ml ( UBD and CD7 ) or 4 μg/ml ( IL-10 ) of the primary antibody for 60 min .\",\n \"After washing , the anti-UBD-stained slides were incubated for 30 min with Leica Power Vision HRP rabbit-specific antibody polymer ( PV6119; Leica ) ; and the anti-CD7 and anti-IL-10 stained slides were incubated for 30 min with LeicaPower Vision HRP mouse-specific antibody polymer ( PV6114; Leica ) .\",\n \"After washing , staining was visualized with 3 , 3′-diaminobenzidine ( Liquid DAB+ Substrate Chromogen System; Dako ) for 7 min , and the sections were counter-stained for 2 min with hematoxylin ( Biocare , Concord , CA ) .\",\n \"Controls for all antibodies were run with identical procedures but replacing the primary antibodies with either rabbit IgG or mouse IgG , as appropriate , at calculated matching concentrations .\",\n \"Formalin-fixed paraffin-embedded rectal biopsies were de-paraffinized and fixed overnight in half-strength Karnovsky's fixative .\",\n \"Staining , embedding , cutting , and viewing on a JEOL 1400 SX transmission electron microscope were performed as previously described ( Hladik et al . , 1999 , 2007 ) .\",\n \"20 images per sample were acquired at 5 , 000× magnification .\",\n \"Using ImageJ ( Collins , 2007 ) , the two-dimensional sizes in µm2 of all individual mitochondria with a circularity index of ≥0 . 9 were calculated ( for standardization purposes , only mitochondria cut near perfectly along their minor axis were evaluated ) .\",\n \"10 images per sample were also acquired at 2 , 000× magnification , always including the epithelial cell brush border .\",\n \"Using ImageJ , a grid of 1 . 32 µm2 squares of defined size was overlaid onto each image .\",\n \"All mitochondria in the images were counted , except those in the first row of squares falling on the brush border and in squares along the image rims ( which only partially covered the tissue ) , and the mean numbers of mitochondria per µm2 were calculated for each of the acquired 2 , 000× images ( range of counted squares per image: 23–50 ) .\",\n \"All p values reported were adjusted for multiple testing as appropriate , except for the exploratory protein mass spectrometry data .\",\n \"Correlations of log2 fold gene expression changes between 9 cm and 15 cm biopsies in Figure 1C and Figure 1—figure supplement 1 were tested by Spearman's rank correlation coefficient .\",\n \"Gene and protein expression changes over baseline were compared between study arms by two-tailed Mann–Whitney test ( Figure 1—figure supplement 2 and Figure 6 ) .\",\n \"The combined expression data from the microarray and RT-ddPCR tests shown in Figure 2C were compared separately for log-fold induction or suppression over baseline using a one-tailed Wilcoxon signed-rank test with Bonferroni adjustment .\",\n \"Immunohistology measurements in Figure 2D were compared between baseline and after 7 days of treatment by two-tailed paired t tests .\",\n \"Due to high skewness of the CD7+ , CD3+ , and IL-10+ cell counts , these were tested after log10 transformation .\",\n \"Ratios of induced to suppressed genes in Figure 3A were tested for a difference between cellular compartments using Chi-square statistics .\",\n \"DSP , IL-10 , KIAA0101 , PNPT1 , and ATP6 copy numbers or protein concentrations in Figures 4B , C , 5A , B , F were tested for significant change across three time points by repeated measures ANOVA , and exact Sidak's ( Figures 4B , C , 5F ) or Tukey ( Figure 5A , B ) post-tests were run for comparisons between two time points .\",\n \"Due to high skewness of the KIAA0101 copy numbers , these were tested after log10 transformation .\",\n \"The effect of increasing tenofovir concentrations on the proliferation of primary vaginal epithelial cells in Figure 4E was assessed for significance by a linear model accounting for days and donors .\",\n \"Copy numbers between 9 cm and 15 cm biopsies in Figure 5A were compared by two-tailed paired t test .\",\n \"Copy numbers between baseline tenofovir and N-9 in Figure 5B were compared by a two-tailed unpaired t test .\",\n \"Mitochondria counts and sizes in Figure 5C , D were compared between baseline and Day VII , separately for the two subjects evaluated by electron microscopy , by two-tailed unpaired t tests with Bonferroni adjustment .\",\n \"Microarray chip probe expression values were tested for correlation between the three primary vaginal cell cultures by computing pairwise Pearson correlation coefficients ( Figure 3—source data 1 ) .\",\n \"Statistics packages used were Prism 6 ( Graphpad Software , La Jolla , CA ) and Bioconductor/Lumi in R . Statistical results are summarized in Supplementary file 1 .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Tenofovir gel is being evaluated for vaginal and rectal pre-exposure prophylaxis against HIV transmission .","Because this is a new prevention strategy , we broadly assessed its effects on the mucosa .","In MTN-007 , a phase-1 , randomized , double-blinded rectal microbicide trial , we used systems genomics/proteomics to determine the effect of tenofovir 1% gel , nonoxynol-9 2% gel , placebo gel or no treatment on rectal biopsies ( 15 subjects/arm ) .","We also treated primary vaginal epithelial cells from four healthy women with tenofovir in vitro .","After seven days of administration , tenofovir 1% gel had broad-ranging effects on the rectal mucosa , which were more pronounced than , but different from , those of the detergent nonoxynol-9 .","Tenofovir suppressed anti-inflammatory mediators , increased T cell densities , caused mitochondrial dysfunction , altered regulatory pathways of cell differentiation and survival , and stimulated epithelial cell proliferation .","The breadth of mucosal changes induced by tenofovir indicates that its safety over longer-term topical use should be carefully monitored .","Clinical trial registration: NCT01232803 ."],"string":"[\n \"Tenofovir gel is being evaluated for vaginal and rectal pre-exposure prophylaxis against HIV transmission .\",\n \"Because this is a new prevention strategy , we broadly assessed its effects on the mucosa .\",\n \"In MTN-007 , a phase-1 , randomized , double-blinded rectal microbicide trial , we used systems genomics/proteomics to determine the effect of tenofovir 1% gel , nonoxynol-9 2% gel , placebo gel or no treatment on rectal biopsies ( 15 subjects/arm ) .\",\n \"We also treated primary vaginal epithelial cells from four healthy women with tenofovir in vitro .\",\n \"After seven days of administration , tenofovir 1% gel had broad-ranging effects on the rectal mucosa , which were more pronounced than , but different from , those of the detergent nonoxynol-9 .\",\n \"Tenofovir suppressed anti-inflammatory mediators , increased T cell densities , caused mitochondrial dysfunction , altered regulatory pathways of cell differentiation and survival , and stimulated epithelial cell proliferation .\",\n \"The breadth of mucosal changes induced by tenofovir indicates that its safety over longer-term topical use should be carefully monitored .\",\n \"Clinical trial registration: NCT01232803 .\"\n]"},"summary":{"kind":"list like","value":["Tenofovir is a drug that can stop some viruses—including HIV—from multiplying .","It is commonly used in multidrug therapies to control HIV infection .","Clinical trials are underway to find out whether using the drug in the form of a gel applied to the vagina or rectum could be an effective way to prevent HIV transmission during sex .","Some of the clinical trials carried out so far have produced promising results .","However , since the use of gels containing anti-viral drugs is a new strategy for HIV prevention , there are limited data available about the safety of these products .","Previous studies have shown that the concentration of tenofovir in the vagina is much higher in individuals using the gel than in those taking the tablet form of the drug .","These high concentrations could lead to unexpected effects on the health of the cells exposed to the gel .","Here , Hladik , Burgener , Ballweber et al . used a systems biology approach to look at the broad effects of tenofovir gel on tissue from the rectum .","Tissue samples taken from the rectums of 15 patients who used tenofovir gel for seven days were compared with tissue samples taken from individuals who used a control gel that did not contain the drug or who did not use any gel .","Genes that regulate inflammation were suppressed in the rectal tissue from patients who used tenofovir , as were genes that help these tissues regenerate and produce energy .","The tissue from these patients also contained more immune cells , suggesting that their local immune systems were more active .","Additionally , Hladik , Burgener , Ballweber et al . observed changes that could potentially lead to the increased growth of cells .","Similar differences were also observed in vaginal cells that had been treated with tenofovir in the laboratory .","These findings suggest that tenofovir delivered directly to the vagina or rectum may have unintentional local side effects .","However , it is important to acknowledge that tenofovir gel has been evaluated in multiple studies that have not observed overt clinical adverse effects .","Therefore , the implication of these findings is currently unclear and warrants further study ."],"string":"[\n \"Tenofovir is a drug that can stop some viruses—including HIV—from multiplying .\",\n \"It is commonly used in multidrug therapies to control HIV infection .\",\n \"Clinical trials are underway to find out whether using the drug in the form of a gel applied to the vagina or rectum could be an effective way to prevent HIV transmission during sex .\",\n \"Some of the clinical trials carried out so far have produced promising results .\",\n \"However , since the use of gels containing anti-viral drugs is a new strategy for HIV prevention , there are limited data available about the safety of these products .\",\n \"Previous studies have shown that the concentration of tenofovir in the vagina is much higher in individuals using the gel than in those taking the tablet form of the drug .\",\n \"These high concentrations could lead to unexpected effects on the health of the cells exposed to the gel .\",\n \"Here , Hladik , Burgener , Ballweber et al . used a systems biology approach to look at the broad effects of tenofovir gel on tissue from the rectum .\",\n \"Tissue samples taken from the rectums of 15 patients who used tenofovir gel for seven days were compared with tissue samples taken from individuals who used a control gel that did not contain the drug or who did not use any gel .\",\n \"Genes that regulate inflammation were suppressed in the rectal tissue from patients who used tenofovir , as were genes that help these tissues regenerate and produce energy .\",\n \"The tissue from these patients also contained more immune cells , suggesting that their local immune systems were more active .\",\n \"Additionally , Hladik , Burgener , Ballweber et al . observed changes that could potentially lead to the increased growth of cells .\",\n \"Similar differences were also observed in vaginal cells that had been treated with tenofovir in the laboratory .\",\n \"These findings suggest that tenofovir delivered directly to the vagina or rectum may have unintentional local side effects .\",\n \"However , it is important to acknowledge that tenofovir gel has been evaluated in multiple studies that have not observed overt clinical adverse effects .\",\n \"Therefore , the implication of these findings is currently unclear and warrants further study .\"\n]"},"year":{"kind":"string","value":"2015"}}},{"rowIdx":4199,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["biochemistry and chemical biology","microbiology and infectious disease"],"string":"[\n \"biochemistry and chemical biology\",\n \"microbiology and infectious disease\"\n]"},"title":{"kind":"string","value":"Angiomotin functions in HIV-1 assembly and budding"},"id":{"kind":"string","value":"elife-03778-v2"},"sections":{"kind":"list like","value":[["As obligate parasites , viruses must harness host cellular pathways to complete many different steps in viral replication .","For example , a large number of enveloped viruses , including HIV-1 , usurp the cellular ESCRT ( Endosomal Sorting Complexes Required for Transport ) pathway to bud from cells ( for recent reviews , see Meng and Lever , 2013; Votteler and Sundquist , 2013; Weissenhorn et al . , 2013 ) .","Viral structural proteins typically use one or more short peptide motifs , termed ‘late assembly domains’ , to bind and recruit early-acting ESCRT factors or ESCRT-associated E3 ubiquitin ligases to sites of viral assembly and budding .","The three best characterized viral late assembly domains are: ( 1 ) the ‘PTAP’ motif ( Pro-Thr/Ser-Ala-Pro ) , which binds the UEV domain of the TSG101 subunit of the ESCRT-I complex , ( 2 ) the ‘YPXnL’ motif ( Tyr-Pro-Xn-Leu , where X can vary in identity and sequence length ) , which binds the V domain of the ESCRT factor ALIX , and ( 3 ) the ‘PPXY’ motif ( Pro-Pro-X-Tyr ) , which binds the WW domains of NEDD4 family E3 ubiquitin ligases .","All three of these viral late assembly domains mimic cellular protein–protein interactions that facilitate ESCRT-dependent vesiculation processes such as multi-vesicular body ( MVB ) biogenesis and ectosome formation .","In addition to the three well-characterized late assembly domains described above , there are indications that enveloped viruses can also connect to the ESCRT pathway via alternative interactions that are not yet well-understood .","For example , efficient release of the model paramyxovirus , PIV5 , requires a non-canonical ‘FPIV’ late assembly domain motif , ubiquitylation of the matrix ( M ) protein , and host Angiomotin-like protein 1 ( AMOTL1 ) ( Schmitt et al . , 2005; Pei et al . , 2010; Harrison et al . , 2012 ) .","There is no evidence that AMOTL1 can bind the FPIV late assembly domain , however , and any relationships between ubiquitin , AMOTL1 and the FPIV late assembly domain activity are not yet understood ( Pei et al . , 2010 ) .","Owing to its medical importance , HIV-1 has become a leading model for understanding ESCRT pathway recruitment and viral budding .","The C-terminal p6 region of the viral Gag structural protein contains canonical PTAP and YPXnL motifs that serve as the major functional late assembly domains in most cell types .","Nevertheless , the release of ‘crippled’ HIV-1 Gag constructs that lack both of these late domains can still be stimulated by overexpression of the HECT E3 ubiquitin ligase , NEDD4L ( also known as NEDD4-2 ) ( Chung et al . , 2008; Usami et al . , 2008 ) .","Humans express nine different NEDD4 protein family members ( reviewed in Ingham et al . , 2004; Bernassola et al . , 2008 ) , but NEDD4L stimulates virus budding more potently than any other NEDD4 family member , implying that HIV-1 Gag preferentially engages this E3 ubiquitin ligase ( Chung et al . , 2008; Usami et al . , 2008 ) .","Previous studies have defined many of the requirements for NEDD4L-stimulated HIV-1 release .","NEDD4L has many different isoforms ( Chen et al . , 2001; Dunn et al . , 2002; Itani et al . , 2005 ) , but isoform 2 ( also called NEDD4-2s , and here termed NEDD4L ) potently stimulates the budding of crippled HIV-1 constructs , and uniquely rescues the Gag processing defects that accompany defective budding ( Chung et al . , 2008; Usami et al . , 2008 ) .","This NEDD4L isoform contains only the final 31 residues of the N-terminal C2 membrane binding domain , a central linker region that contains four WW domains , and a C-terminal HECT E3 ubiquitin ligase domain that forms Lys-63-linked poly-ubiquitin chains ( see Figure 1A ) ( Kim and Huibregtse , 2009; Weiss et al . , 2010 ) .","Stimulation of HIV-1 budding requires NEDD4L E3 ubiquitin ligase activity , indicating that Lys-63-linked poly-ubiquitin chains perform an essential function ( Chung et al . , 2008; Usami et al . , 2008 ) .","The functional target ( s ) of Lys-63 poly-ubiquitylation has not been established definitively , but HIV-1 Gag itself is the leading candidate because functional virus budding correlates with Lys-63-linked poly-ubiquitylation of Gag , and because NEDD4L truncation constructs that include the HECT domain can be functionally rescued by fusion to the HIV-1 Gag-targeting cyclophilin A domain ( Weiss et al . , 2010; Sette et al . , 2013 ) .","HIV-1 Gag lacks identifiable PPXY motifs , however , and there is no evidence that Gag can bind NEDD4L directly . 10 . 7554/eLife . 03778 . 003Figure 1 . AMOT p130 is a NEDD4L binding partner .","( A ) Schematic illustrations of the domain structures and motifs in NEDD4L , AMOT p130 and p80 , AMOTL1 , AMOTL2 and HIV-1 Gag .","Here and throughout , NEDD4L refers to a naturally occurring protein isoform ( isoform","2 ) that contains only the C-terminal 32 residues of the C2 domain ( Genebank accession number AAM46201 . 1 , denoted NEDD4Ls in other publications and NEDD4LΔC2 in our previous publication ( Chung et al . , 2008 ) ) .","To maintain consistency with that publication , the NEDD4L numbering scheme used here corresponds to NEDD4L isoform 1 , which is 121 residues longer at the N-terminus and contains the entire C2 domain ( denoted NEDD4LWT in reference Chung et al . , 2008 ) .","( B ) Affinity co-purification and identification of AMOT p130 as a binding partner of OSF ( One-STrEP-FLAG ) -tagged NEDD4L .","SDS-PAGE/Coomassie-stained gel showing STrEP-Tactin matrix affinity purified proteins from 293T cells transfected with an OSF-NEDD4L expression construct ( lane","2 ) or an empty vector control ( lane 1 ) .","Labels denote OSF-NEDD4L ( bait ) and AMOT p130 ( prey ) .","Trypsin-digested AMOT p130 peptides identified by mass spectrometric analyses are summarized in Figure 1—figure supplement 1 .","A representative image from three independent repetitions is shown .","( C ) NEDD4L WW domains are required for AMOT p130 binding .","Western blots showing co-immunoprecipitations of endogenous 293T cell AMOT proteins ( prey ) with OSF-NEDD4L ( bait ) .","Panel 1: endogenous AMOT proteins ( anti-AMOT blot ) co-immunoprecipitated with OSF-NEDD4L baits from cells that lacked exogenous OSF-NEDD4L ( lane 1 , control ) , expressed wild type OSF-NEDD4L ( lane 2 ) , expressed OSF-NEDD4LΔWW ( lane 3 , construct has inactivating point mutations in all four WW domains ) , expressed OSF-NEDD4LC942A ( lane 4 , construct has an inactivating point mutation in the HECT E3 domain ) , or expressed OSF-NEDD4LC942A/ΔWW ( lane 5 , construct has inactivating point mutations in the WW and HECT E3 domains ) .","Panel 2: same co-immunoprecipitation experiment blotted with anti-FLAG antibodies to detect OSF-NEDD4L proteins .","Panel 3: input levels of endogenous AMOT ( anti-AMOT blot , 10% of total ) .","Panel 4: input levels of exogenous OSF-NEDD4L ( anti-FLAG , 10% of total ) .","Note that endogenous AMOT p80 was present in the input lysate , but did not co-immunoprecipitate with OSF-NEDD4L .","Representative image from three independent repetitions . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00310 . 7554/eLife . 03778 . 004Figure 1—figure supplement 1 . Identification of AMOT p130 as a NEDD4L binding partner . The protein band denoted ‘AMOT p130’ in Figure 1B , lane 2 was excised , digested with trypsin and the eluted peptides were identified by mass spectrometry as described in ‘Materials and methods’ .","This analysis was performed three times and the identified peptides are mapped onto the AMOT p130 primary sequence , with peptides identified in experiments 1–3 color coded in black , blue and red , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00410 . 7554/eLife . 03778 . 005Figure 1—figure supplement 2 . The AMOT p130 PPXY motifs contribute to NEDD4L binding . Western blots show co-immunoprecipitations of OSF-NEDD4L ( bait , captured on STrEP-Tactin affinity matrices ) with endogenous 293T cells AMOT and exogenously expressed HA-AMOT p130 proteins ( prey ) .","Co-immunoprecipitations with OSF-NEDD4L were from cells that lacked exogenous AMOT p130 ( lane 1 ) , expressed wild type HA-AMOT p130 ( lane","2 ) or expressed a mutant HA-AMOT p130 protein carrying PP/AA mutations in the three N-terminal ‘PPXY’ motifs ( Wang et al . , 2012; Yi et al . , 2013 ) .","Panel 1: AMOT protein co-immunoprecipitations ( anti-AMOT blot ) with OSF-NEDD4L .","Note that OSF-NEDD4L co-immunoprecipitated low levels of endogenous AMOT p130 ( lanes 1 and 3 , denoted p130 ) but not endogenous AMOT p80 ( denoted p80 with a dashed line ) .","Panel 2: The same co-immunoprecipitation experiment blotted with anti-HA antibodies to detect exogenously expressed HA-AMOT p130 proteins .","Panel 3: The same co-immunoprecipitation experiment blotted with anti-FLAG antibodies to detect exogenously expressed OSF-NEDD4L .","Panels 4–6 show the input quantities ( 10% ) of endogenous AMOT and HA-AMOT p130 ( panel 4 , anti-AMOT ) , exogenous HA-AMOT p130 ( panel 5 , anti-HA ) , and exogenous OSF-NEDD4L ( panel 6 , anti-FLAG ) .","A representative image from three independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 005 These observations imply that NEDD4L may be recruited to sites of HIV-1 assembly through an alternative mechanism .","We undertook the present study with the goal of identifying co-factors that might help link NEDD4L to HIV-1 Gag and stimulate virus budding .","We identified Angiomotin ( AMOT ) as a protein that binds both NEDD4L and HIV-1 Gag , and functions in HIV-1 assembly and release .","AMOT is required to complete virion assembly and envelopment , and is therefore a new host factor that acts in a previously uncharacterized step of HIV-1 morphogenesis ."],["Affinity purification/mass spectrometry experiments were performed to identify candidate NEDD4L binding partners .","A number of cellular proteins co-purified with OSF ( One-STrEP-FLAG ) -tagged NEDD4L , as visualized by SDS-PAGE ( Figure 1B , compare lanes 1 and 2 ) .","Prominent co-purifying proteins were excised from the gel , digested with trypsin , and identified by mass spectrometry .","The p130 isoform of human Angiomotin was unambiguously identified as a protein that co-purified with OSF-NEDD4L in three independent repetitions of this experiment , ( lane 2 , labeled AMOT p130 , peptide coverage data are summarized in Figure 1—figure supplement 1 ) .","Smaller AMOT p130 fragments were also identified , as was the related AMOT family protein , Angiomotin-like protein 1 ( AMOTL1 , data not shown ) .","Cells express two AMOT isoforms , a longer p130 isoform , and a shorter C-terminal p80 isoform that is expressed from an alternatively spliced message ( Figure 1A ) ( Moreau et al . , 2005 ) .","AMOT p130 binds NEDD4 protein family members via interactions between the four central NEDD4 WW domains and three PPXY motifs located within the N-terminal extension that is unique to the AMOT p130 isoform ( Figure 1A ) ( Wang et al . , 2012 ) .","In good agreement with this report and with our affinity purification/mass spectrometry results , we found that AMOT p130 co-immunoprecipitated with exogenously expressed OSF-NEDD4L ( Figure 1C , panel 1 , lane 2 ) .","In contrast , AMOT p130 did not co-immunoprecipitate with a mutant OSF-NEDD4L protein that contained inactivating Trp to Ala substitutions in the key PPXY-binding ‘W39’ residues from each of the four WW domains ( denoted OSF-NEDD4LΔWW , panel 1 , compare lanes 2 and","3 ) ( Otte et al . , 2003 ) .","A previous study has demonstrated that NEDD4 proteins can ubiquitylate and reduce AMOT p130 levels ( Wang et al . , 2012 ) , and we also observed that OSF-NEDD4L overexpression modestly reduced AMOT p130 levels ( Figure 1C , panel 3 , compare lanes 1 and 2 ) .","We therefore tested whether AMOT p130 also co-precipitated with OSF-NEDD4L proteins that carried inactivating Cys942Ala point mutations in the active site cysteine of the HECT E3 ubiquitin ligase domain ( OSF-NEDD4LC942A ) .","This mutation increased OSF-NEDD4L levels in the lysate and immunoprecipitate ( panels 2 and 4 compare lanes 4 and 5 to lanes 2 and","3 ) and restored normal AMOT protein levels ( panel 3 , compare lanes 4 , 5 and 1 to lane 2 ) .","Once again , AMOT p130 ( and breakdown products ) co-immunoprecipitated with OSF-NEDD4LC942A ( panel 1 , lane 4 ) , but not with a protein that also contained inactivating WW domain mutations ( OSF-NEDD4LΔWW/C942A panel 1 , lane 5 ) .","AMOT p130 co-precipitation levels were higher with OSF-NEDD4LC942A than with wild type OSF-NEDD4L , presumably owing to the higher cellular levels of both OSF-NEDD4L and AMOT p130 ( compare lanes 2 and 4 ) .","Three additional observations confirmed that the NEDD4L WW domains and AMOT p130 PPXY motifs are critical for the NEDD4L-AMOT p130 interaction .","Firstly , mutating the three N-terminal AMOT p130 PPXY motifs strongly inhibited co-immunoprecipitation of HA-AMOT p130 with OSF-NEDD4L ( Figure 1—figure supplement 2 , panel 1 , compare lanes 2 and 3 ) .","Secondly , endogenous AMOT p80 , which lacks the N-terminal PPXY motifs , failed to co-immunoprecipitate with either OSF-NEDD4L or OSF-NEDD4LC942A ( Figure 1C , panel 1 , lanes 2 and 4 ) , even though AMOT p80 was present in the 293T cell extracts ( panel 3 , lanes 1–5 ) .","Thirdly , we observed that exogenously expressed , epitope-tagged NEDD4L and AMOT p130 co-localized well in HeLa-M cells , and this co-localization required both the AMOT PPXY and NEDD4L WW motifs ( data not shown ) , in good agreement with previous reports that motins co-localize with other NEDD4 family members ( Skouloudaki and Walz , 2012; Wang et al . , 2012 ) .","Taken together , these experiments demonstrate that NEDD4L interacts specifically with AMOT p130 in cells and that the interaction requires the WW domains of NEDD4L and the PPXY motifs of AMOT .","Pull-down experiments with purified recombinant proteins were performed to test whether AMOT p130 binds NEDD4L directly , and whether AMOT p130 can also bind HIV-1 Gag and thereby link NEDD4L to the viral Gag protein .","Wild type and mutant NEDD4L proteins and an HIV-1 Gag protein that lacked the flexible p6 domain ( GagΔp6 ) were expressed in Escherichia coli and purified to near homogeneity ( Figure 2A , lanes 1 and 2 , and Figure 2B , lane 1 , respectively ) .","OSF-AMOT p130 was overexpressed in SF-9 insect cells and substantially enriched by binding to STrEP-Tactin affinity resin ( Figure 2A , lane 6 and Figure 2B , lane 7 ) , although we were unable to purify the protein to homogeneity .","As shown in Figure 2A , NEDD4L did not bind an immobilized control OSF-GFP protein , but bound with at least 1:1 stoichiometry to immobilized OSF-AMOT p130 ( compare lanes 4 and 7 ) .","In contrast , binding of the NEDD4LΔWW mutant protein was nearly undetectable ( Figure 2A , compare lanes 7 and 8 , particularly in the western blot in panel 2 ) .","These experiments confirm that NEDD4L and AMOT p130 bind one another directly , with the WW domains of NEDD4L binding the PPXY motifs of AMOT p130 . 10 . 7554/eLife . 03778 . 006Figure 2 . AMOT p130 binds directly and specifically to NEDD4L and HIV-1 Gag∆p6 . ( A ) OSF-AMOT p130 binds NEDD4L .","Recombinant OSF-GFP ( specificity control , lanes 3–5 ) or OSF-AMOT p130 ( lanes 6–8 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 3 and","6 ) or buffers containing 0 . 5 µM wild type NEDD4L ( lanes 4 and","7 ) or NEDD4LΔWW ( lanes 5 and 8 , inactivating point mutations in all four WW domains ) .","Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining ( panel","1 ) or by western blotting ( panel 2 , anti-NEDD4L ) to confirm the identities of the bound NEDD4L and help to distinguish background proteins from low-level binding in panel 1 .","Input levels of NEDD4L ( lane 1 , 1 . 5% of total ) and NEDD4LΔWW ( lane 2 , 1 . 5% of total ) are shown for reference .","A representative image from three independent repetitions is shown .","( B ) OSF-AMOT p130 binds HIV-1 Gag∆p6 .","Recombinant OSF-GFP ( control , lanes 3–6 ) or OSF-AMOT p130 ( lanes 7–10 ) were expressed , captured on STrEP-Tactin affinity matrices and incubated with a buffer control ( lanes 3 and","7 ) or with buffers containing 1 . 0 µM HIV-1 Gag∆p6 ( lanes 3 and 8 ) , 0 . 5 µM wild type NEDD4L ( lanes 5 and 9 ) or both proteins ( lanes 6 and 10 ) .","Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining ( panel","1 ) or by western blotting to confirm the identities of the bound Gag ( panel 2 , anti-MA and anti-CA ) and NEDD4L ( panel 3 , anti-NEDD4L ) proteins and help to distinguish background proteins from low-level binding in panel 1 .","Input Gag∆p6 ( lane 1 , 2% of total ) and NEDD4L ( lane 2 , 1 . 5% of total ) are shown for reference .","A representative image from three independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00610 . 7554/eLife . 03778 . 007Figure 2—figure supplement 1 . AMOT p130 , AMOTL1 and AMOTL2 bind directly and specifically to NEDD4L and HIV-1 Gag∆p6 . ( A ) OSF-AMOT p130 , OSF-AMOTL1 , and OSF-AMOTL2 bind NEDD4L .","Recombinant OSF-GFP ( specificity control , lanes 2 and 6 ) , OSF-AMOT p130 ( lanes 3 and 7 ) , OSF-AMOTL1 ( lanes 4 and 8 ) , and OSF-AMOTL2 ( lanes 5 and 9 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 2–5 ) or with a buffer containing 0 . 5 µM wild type NEDD4L ( lanes 6–9 ) .","Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining .","Input NEDD4L ( lane 1 , 1 . 5% of total ) is shown for reference .","Asterisk denotes an OSF-AMOTL2 breakdown product that co-migrates with NEDD4L .","A representative image from two independent repetitions is shown .","( B ) OSF-AMOT p130 , OSF-AMOTL1 , and OSF-AMOTL2 bind Gag∆p6 .","Recombinant OSF-GFP ( specificity control , lanes 2 and 6 ) , OSF-AMOT p130 ( lanes 3 and 7 ) , OSF-AMOTL1 ( lanes 4 and 8 ) , and OSF-AMOTL2 ( lanes 5 and 9 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 2–5 ) or buffers containing 1 µM Gag∆p6 ( lanes 6–9 ) .","Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining .","Input Gag∆p6 ( lane 1 , 2% of total ) is shown for reference .","A representative image from two independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 007 Analogous pull-down experiments were performed to test whether AMOT binds HIV-1 GagΔp6 .","Removing the p6 region facilitates Gag protein expression and purification , and is not expected to interfere with any biologically relevant interactions because NEDD4L potently stimulates HIV-1 GagΔp6 release from cells ( Chung et al . , 2008; Usami et al . , 2008 ) .","As shown in Figure 2B , HIV-1 GagΔp6 bound immobilized OSF-AMOT p130 , but did not bind an immobilized OSF-GFP control ( compare lanes 4 and 8 ) .","This interaction did not appear to be mediated by nucleic acids because the affinity-purified OSF-AMOT p130 protein was pre-washed with high salt solution to remove any bound nucleic acid ( see ‘Materials and methods’ ) , and because the interaction was maintained , albeit at slightly lower levels , even when immobilized OSF-AMOT p130 was incubated with high concentrations of RNase that were sufficient to eliminate other RNA-mediated Gag-protein interactions ( not shown ) .","GagΔp6 and NEDD4L appeared to bind OSF-AMOT p130 independently because their binding levels did not change significantly when all three proteins were incubated together ( compare lanes 8 and 9 to lane 10 ) .","AMOT is the founding member of the human motin family , which also includes Angiomotin-like protein 1 ( AMOTL1 ) and Angiomotin-like protein 2 ( AMOTL2 ) ( Moleirinho et al . , 2014 ) .","AMOTL1 and AMOTL2 share 52% and 45% pair-wise sequence identity with AMOT p130 , and both contain the N-terminal extension and PPXY motifs that are present in AMOT p130 but absent in AMOT p80 ( Figure 1A ) .","Pull-down experiments demonstrated that AMOTL1 and AMOTL2 also bind both NEDD4L and HIV-1 GagΔp6 ( Figure 2—figure supplement 1 ) .","Thus , AMOT p130 binds directly and specifically to both NEDD4L and HIV-1 Gag , and these binding activities are shared by the other two human motins .","Co-expression experiments were performed to test whether AMOT and NEDD4L can cooperate in stimulating HIV-1 release ( Figure 3 ) .","These experiments employed the crippled HIV-1ΔPTAP , ΔYP viral expression construct , which lacks functional PTAP and YPXnL late domains and is therefore particularly responsive to cellular NEDD4L activity ( Chung et al . , 2008; Usami et al . , 2008 ) .","As expected , HIV-1ΔPTAP , ΔYP was poorly released from control 293T cells , as measured by western blot that detected virion-associated CA and MA proteins ( Figure 3 , upper right panel , lane 1 ) , and low viral titers ( lower right panel , lane 1 ) .","Overexpression of HA-AMOT p130 increased the levels of the full-length protein ( and presumptive breakdown products were also evident , left panel 1 , compare lanes 1 and 2 ) .","AMOT p130 overexpression modestly increased HIV-1ΔPTAP .","ΔYP release and infectivity ( right panels compare lanes 1 and 2 , 1 . 7-fold increases in both release and infectivity ) , without significantly altering the intracellular levels of Gag ( left , panel 4 , compare lanes 1 and","2 ) or a control cellular protein ( GAPDH , left panel 3 , compare lanes 1 and 2 ) .","Thus , AMOT p130 overexpression stimulates HIV-1ΔPTAP , ΔYP release and infectivity , but the effect is modest . 10 . 7554/eLife . 03778 . 008Figure 3 . AMOT p130 stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , FLAG-NEDD4L proteins ( lanes 3–6 , with wild type ( WT ) FLAG-NEDD4L in lanes 3 and 4 and FLAG-NEDD4LΔWW , in lanes 5 and 6 ) , and wild type HA-AMOT p130 ( lanes 2 , 4 and","6 ) or an empty vector control ( lanes 1 , 3 , and 5 ) .","Right panels show corresponding levels of extracellular , virion-associated CAGag and MAGag proteins ( panel 1 , anti-MA and anti-CA ) and viral titers ( panel 2 , IU denotes ‘infectious units’ ) .","Here and in subsequent figures: ( 1 ) error bars denote standard deviations between independent repetitions of the experiment , n = 5 in this case , and ( 2 ) numbers within the blots show integrated intensities of the CA band intensities ( relative to the value in the control experiment , set to 1 . 0 ) .","Here and throughout significance is denoted by: NS , not significant ( p > 0 . 05 ) ; *0 . 05 > p > 0 . 01; **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00810 . 7554/eLife . 03778 . 009Figure 3—figure supplement 1 . Dose-dependent AMOT p130 stimulation of NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–8 ) , FLAG-NEDD4L ( lanes 2–8 ) , and increasing quantities of an AMOT p130 expression construct ( lanes 3–8 , 0 . 25–1 . 5 μg DNA ) .","Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 3 μg DNA total ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00910 . 7554/eLife . 03778 . 010Figure 3—figure supplement 2 . The AMOT p130 isoform specifically stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT , exogenous p80 and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , AMOT p80 ( lanes 2 and 5 ) , AMOT p130 ( lanes 3 and","6 ) and FLAG-NEDD4L ( lanes 4–6 ) .","Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 2 . 5 μg DNA total ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 010 As seen previously , FLAG-NEDD4L overexpression also stimulated HIV-1ΔPTAP , ΔYP release ( Figure 3 , right panel 1 , compare lanes 1 and 3 , 3 . 2-fold stimulation ) and infectivity ( right panel 2 , compare lanes 1 and 3 , 5 . 3-fold stimulation ) , and also largely rescued the HIV-1ΔPTAP , ΔYP Gag processing defects ( left panel 4 , compare lane 3 to lanes 1 and 2 , and note the reduction in p41 and p25 Gag processing intermediates ) ( Chung et al . , 2008; Usami et al . , 2008 ) .","Co-overexpression of both HA-AMOT p130 and FLAG-NEDD4L further increased HIV-1ΔPTAP , ΔYP release ( right panel 1 , lane 4 , sixfold increase over basal levels ) and infectivity ( right panel 2 , lane 4 , 14-fold increase over basal levels ) .","As shown in Figure 3—figure supplement 1 , the degree of hyper-stimulation correlated positively with AMOT p130 expression levels , until an inhibitory level was reached .","In contrast to wild type NEDD4L , the NEDD4LΔWW mutant stimulated HIV-1ΔPTAP , ΔYP release and infectivity only very modestly ( Figure 3 , right panels , compare lanes 5 and","3 ) and did not synergize with AMOT p130 ( right panels , compare lanes 6 and 5 ) .","Similarly , the shorter AMOT p80 isoform , which lacks the PPXY motifs found in the N-terminal domain of AMOT p130 , failed to synergize with NEDD4L in stimulating virus release and infectivity , and instead modestly reduced NEDD4L stimulation , probably by dominantly inhibiting endogenous AMOT p130 activity ( Figure 3—figure supplement 2 , compare lanes 4–6 ) .","These data indicate that AMOT p130 and NEDD4L cooperate in stimulating HIV-1ΔPTAP , ΔYP release and infectivity .","siRNA depletion experiments were performed to determine whether AMOT was also necessary for NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release and infectivity .","We used an siRNA that targeted both AMOT isoforms and reduced cellular AMOT p130 and p80 levels by at least fivefold ( Figure 4 , left panel 1 , compare lanes 1 and 2 and lanes 3 and 4 ) .","AMOT depletion reduced the already low levels of virus release and infectivity even further , to near background levels ( Figure 4 , right panels , compare lanes 1 and 2 , 7 . 5-fold infectivity reduction ) .","Cellular levels of Gag and a GAPDH control were unaffected by this treatment ( left panels 3 and 4 , compare lanes 1 and 2 ) , although AMOT depletion reduced cellular protein levels slightly in some repetitions ( not shown ) . 10 . 7554/eLife . 03778 . 011Figure 4 . AMOT p130 is required for NEDD4L-stimulated release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were co-transfected with a control siRNA ( lanes 1 and","3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–6 ) , with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , and with FLAG-NEDD4L ( lanes 3–6 ) or an empty vector control ( lanes 1 and 2 ) , and with siRNA-resistant expression constructs for HA-AMOT p130 proteins ( with wild type HA-AMOT p130 in lane 5 and an HA-AMOT p130ΔPPXY protein carrying inactivating point mutations in the three PPXY motifs in lane 6 ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01110 . 7554/eLife . 03778 . 012Figure 4—figure supplement 1 . Dose-dependent AMOT p130 rescue of HIV-1 release from cells depleted of endogenous AMOT . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were co-transfected with a control siRNA ( lanes 1 and","3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–10 ) , with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–10 ) , for FLAG-NEDD4L ( lanes 3–10 ) , and with increasing quantities of an siRNA-resistant AMOT p130 construct ( lanes 5–10 , 0 . 25–1 . 5 μg DNA ) .","Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 3 μg DNA total ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01210 . 7554/eLife . 03778 . 013Figure 4—figure supplement 2 . The AMOT p130 isoform specifically stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 or AMOT p80 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were transfected with a control siRNA ( lanes 1 and","3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–7 ) .","Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–7 ) , FLAG-NEDD4L ( lanes 3–7 ) , and siRNA-resistant expression constructs for AMOT p80 ( lane 5 ) , HA-AMOT p130 ( lane","6 ) or both ( lane 7 ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 013 AMOT depletion blocked the ability of NEDD4L to stimulate HIV-1ΔPTAP , ΔYP release and infectivity ( Figure 4 and Figure 4—figure supplements 1 , 2 ) .","In the control case , NEDD4L overexpression stimulated HIV-1ΔPTAP , ΔYP release ( Figure 4 , right panels , compare lanes 1 and 3 , 4 . 1-fold infectivity increase ) .","However , virus release was reduced to the levels seen in untreated control cells when endogenous AMOT p80 and p130 proteins were simultaneously depleted ( compare lanes 2–4 ) .","Similar results were observed for a second siRNA that targeted both AMOT isoforms ( not shown ) .","Thus , AMOT is required for NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release .","Rescue experiments with exogenous siRNA-resistant AMOT expression constructs were performed to confirm the specificity of the AMOT depletion and to test the requirements for AMOT function .","Re-expression of AMOT p130 completely rescued the block to virus release and infectivity imposed by AMOT depletion ( Figure 4 , right panels , compare lanes 5 and 4 ) , restoring viral infectivity to a level that was actually slightly higher than the control ( compare lanes 3 and 5 ) .","The degree of rescue generally correlated positively with AMOT p130 re-expression levels , and peaked at levels that approximated those of the native endogenous protein ( Figure 4—figure supplement 1 , lane 8 ) .","In contrast , an AMOT p130 protein that lacked the three N-terminal PPXY motifs failed to rescue NEDD4L-dependent HIV-1ΔPTAP , ΔYP release ( Figure 4 , AMOT p130ΔPPXY , right panels , compare lanes 6 and 5 ) .","Similarly , re-expression of an siRNA-resistant AMOT p80 construct also failed to rescue virus release and infectivity ( Figure 4—figure supplement 2 , right panels , compare lanes 4 and 5 ) , unless exogenous AMOT p130 was present ( Figure 4—figure supplement 2 , right panels , lane 7 ) .","Hence , NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release and infectivity requires the presence of an AMOT p130 protein that can bind NEDD4L , but is not significantly affected by AMOT proteins that cannot bind NEDD4L , including AMOT p80 .","siRNA depletion and rescue experiments were also performed to test whether AMOT p130 is required for release and infectivity of wild type HIV-1 .","In control experiments , depletion of either of the well-characterized HIV-1 budding factors , ALIX and TSG101 , reduced virus release and infectivity , although the effects were much greater for TSG101 depletion , as reported previously ( Figure 5 , right panels , compare lanes 2 and 3 to lane","1 ) ( Fujii et al . , 2009 ) .","AMOT depletion also significantly reduced both HIV-1 release and infectivity ( Figure 5 , right panels , compare lane 4 to lane 1 , eightfold reduction in infectivity and 2 . 5-fold reduction in release ) .","The magnitudes of these effects were intermediate between those seen for depletion of TSG101 ( 20-fold reduction in infectivity and threefold reduction in virion release ) and ALIX ( 1 . 5-fold reduction in infectivity and insignificant reduction in virion release ) .","Importantly , the detrimental effects of AMOT p130 depletion were almost completely rescued by re-expression of wild type AMOT p130 ( compare lanes 1 , 4 and 5 ) but not by the mutant AMOT p130ΔPPXY protein ( lane 6 ) .","These experiments demonstrate that AMOT p130 is required for efficient release of wild type HIV-1 from 293T cells and mutations that abolish NEDD4L binding block the ability of AMOT p130 to function in virus release . 10 . 7554/eLife . 03778 . 014Figure 5 . AMOT p130 is required for efficient release of wild type HIV-1 . Left panels are western blots showing 293T cellular levels of endogenous ALIX ( panel 1 , anti-ALIX ) , TSG101 ( panel 2 , anti-TSG101 ) , AMOT ( panel 3 , anti-AMOT ) , GAPDH ( panel 4 , anti-GAPDH , loading control ) and levels of HIV-1 Gag proteins ( panel 5 , anti-MA and anti-CA ) .","Cells were co-transfected with a control siRNA ( lane","1 ) or with an siRNA that depleted ALIX ( lane 2 ) , TSG101 ( lane","3 ) or AMOT p130 and p80 ( lanes 4–6 ) , with expression vectors for wild type HIV-1 ( lanes 1–6 ) and with an siRNA-resistant expression constructs for HA-AMOT p130 proteins ( with wild type HA-AMOT p130 in lane 5 and an HA-AMOT p130ΔPPXY protein with inactivating point mutations in all three PPXY motifs in lane 6 ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 014 AMOT mRNA is expressed in 293T cells , as well as in white blood cells and lymphoid tissues that are the natural hosts for HIV-1 infection ( Moleirinho et al . , 2014 ) .","In contrast , HeLa cells reportedly express little or no AMOT mRNA , although they do express AMOTL2 mRNA ( Moleirinho et al . , 2014 ) .","We confirmed that endogenous AMOT protein levels are at least 10-fold lower in HeLa cells than in 293T cells ( Figure 6—figure supplement 1 ) .","This observation begs the question of how HIV-1 can be released from a cell type that lacks AMOT , and this issue is particularly relevant because HeLa cells are commonly used to study HIV-1 assembly .","We therefore tested the effects of AMOT p130 expression on HIV-1 release and infectivity in HeLa cells .","As shown in Figure 6A , AMOT p130 expression increased HIV-1 release from HeLa cells in a dose-dependent fashion , up to 15-fold at the highest levels of AMOT tested ( right panel 1 , compare lanes 1 and 5 ) .","Viral titers similarly increased with AMOT expression in a dose-dependent fashion , although the overall effects were less dramatic ( right panel 2 , compare lanes 1–5 , threefold infectivity increase ) .","Thus , HIV-1 release from wild type HeLa cells is suboptimal , and can be enhanced by expression of AMOT p130 . 10 . 7554/eLife . 03778 . 015Figure 6 . AMOT p130 stimulates release of HIV-1 from HeLa and Jurkat T cells .","( A ) Left panels are western blots showing HeLa cellular levels of endogenous AMOT p130 and exogenous HA-AMOT p130 ( panel 1 , anti-AMOT ) , endogenous GAPDH ( panel 2 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 3 , anti-MA and anti-CA ) .","HeLa cells were co-transfected with expression vectors for HIV-1 ( lanes 1–5 ) , and with increasing concentrations of an expression construct for HA-AMOT p130 ( 0–4 μg DNA in lanes 1–5 ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 .","( B ) Levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) .","Jurkat T cells were co-transfected with expression vectors for HIV-1 ( lanes 1–5 ) , together with a control vector ( lane","1 ) or expression vectors for wild type HA-AMOT p130 ( lanes 2 and 3 , 5 and 10 µg DNA respectively ) or an HA-AMOT p130ΔPPXY protein with inactivating point mutations in the three PPXY motifs ( lanes 4 and 5 , 5 and 10 µg DNA respectively ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01510 . 7554/eLife . 03778 . 016Figure 6—figure supplement 1 . HeLa cells express little or no AMOT . Western blots showing endogenous AMOT p130 and p80 expression levels in 293T cells ( lanes 1–4 ) and in HeLa cells ( lanes 5–8 ) .","Increasing numbers of cell equivalents were added as indicated ( ranging from 1 . 5 × 105 cells in lanes 1 and 5 to 1 . 2 × 106 cells in lanes 4 and 8 ) .","Upper and lower panels show lighter and darker exposures of the same blot .","Image is representative of two independent repetitions of this experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 016 T cells are natural hosts for HIV-1 infection , and AMOT mRNA is present in these cells , including Jurkat T cells ( Yeung et al . , 2009; Sheynkman et al . , 2013 ) .","To determine whether AMOT p130 can function in virus release from Jurkat T cells , we tested whether expressing exogenous AMOT p130 expression stimulated HIV-1 release and infectivity .","As shown in Figure 6B , AMOT p130 expression increased HIV-1 release from Jurkat T cells in a dose-dependent fashion , with up to 16-fold stimulation observed at the highest levels of AMOT p130 tested ( panel 1 , compare lanes 1–3 ) .","Viral titers similarly increased in a dose-dependent fashion ( panel 2 , compare lanes 1–3 , sevenfold infectivity increase ) .","Western blots confirmed that virus expression levels were the same in all cases ( not shown ) , but AMOT p130 expression levels were below our detection limits in this cell type .","We therefore also tested the effects of overexpressing the mutant AMOT p130ΔPPXY protein .","Unlike wild type AMOT p130 , AMOT p130ΔPPXY failed to stimulate virus release , and actually reproducibly decreased viral titers as compared to the untreated control ( compare lanes 4 and 5 to lane 1 ) .","We speculate that this effect again reflects dominant inhibition of endogenous AMOT p130 .","Regardless , our data indicate that AMOT p130 also stimulates virus release and infectivity from T cells , which are natural targets of HIV-1 infection .","Consistent with this conclusion , a global shRNA screen for host cofactors revealed that Jurkat T cells transduced with an shRNA that targeted AMOT were protected against HIV cytotoxicity ( Yeung et al . , 2009 ) .","This observation implies that AMOT contributes to HIV-1 replication in cultured Jurkat T cells , although the role of AMOT in HIV-1 replication was not characterized further in that study .","( Yeung et al . , 2009 ) .","siRNA depletion/rescue experiments were performed to test whether other motin family members can substitute for AMOT p130 in supporting release of wild type HIV-1 ( Figure 7 ) .","In control experiments , HIV-1 release and infectivity were again decreased by depletion of endogenous AMOT from 293T cells ( right panels , compare lanes 1 and 2 , fivefold infectivity reduction ) .","As expected , these effects were largely rescued by expression of exogenous siRNA-resistant AMOT p130 ( right panels , compare lanes 2 and 3 ) .","Expression of either AMOTL1 or AMOTL2 also significantly rescued HIV-1 release and infectivity ( right panels , compare lanes 4 and 5 to lanes 2 and 3 ) .","Hence , both AMOT-like proteins can facilitate HIV-1 release in the absence of endogenous AMOT p130 .","Neither AMOTL1 nor AMOTL2 was quite as effective as AMOT p130 in rescuing HIV-1 infectivity in this assay , possibly because these proteins are intrinsically less active than AMOT p130 and/or because they were expressed at lower levels ( left panel 2 , compare lanes 3–5 ) . 10 . 7554/eLife . 03778 . 017Figure 7 . AMOTL1 and AMOTL2 can substitute for AMOT p130 in HIV-1 release . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 proteins ( panel 2 , anti-HA ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were co-transfected with a control siRNA ( lane","1 ) or with an siRNA that depleted AMOT p130 and AMOT p80 ( lanes 2–5 ) , and with expression vectors for HIV-1 ( lanes 1–5 ) , with siRNA-resistant expression constructs for AMOT p130 ( lane 3 ) , AMOTL1 ( lane 4 ) or AMOTL2 ( lane 5 ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01710 . 7554/eLife . 03778 . 018Figure 7—figure supplement 1 . AMOTL2 can stimulate NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP and rescue HIV-1ΔPTAP , ΔYP release from cells depleted of endogenous AMOT .","( A ) AMOTL2 stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP .","Left panels are western blots showing 293T cellular levels of exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 proteins ( panel 1 , anti-HA ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .","Cells were co-transfected with expression vectors for HIV-1ΔPTAP/ΔYP ( lanes 1–8 ) , FLAG-NEDD4L ( lanes 5–8 ) , and either HA-AMOT p130 ( lanes 2 and 6 ) , HA-AMOTL1 ( lanes 3 and","7 ) or HA-AMOTL2 ( lanes 4 and 8 ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel ) and viral titers ( lower panel ) , n = 3 .","( B ) AMOTL2 can rescue HIV-1ΔPTAP , ΔYP release from 293T cells depleted of endogenous AMOT .","Left panels are western blots showing cellular levels of endogenous AMOT and exogenous HA-AMOT proteins ( panel 1 , anti-AMOT ) , exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 ( panel 2 , anti-HA ) , exogenous FLAG-NEDD4L ( panel 3 , anti-FLAG ) , GAPDH ( panel 4 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 5 , anti-MA and anti-CA ) .","Cells were co-transfected with a control siRNA ( lanes 1 and","3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–7 ) , with expression vectors for HIV-1 ( lanes 1–7 ) , FLAG-NEDD4L ( lanes 3–7 ) , and either an siRNA-resistant HA-AMOT p130 expression construct ( lane 5 ) , or expression constructs for HA-AMOTL1 ( lane 6 ) , or HA-AMOTL2 ( lane 7 ) .","Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 018 We also tested whether the other AMOT family members could cooperate with NEDD4L in stimulating release of the crippled HIV-1ΔPTAP , ΔYP virus ( Figure 7—figure supplement 1A ) and whether AMOTL1 and AMOTL2 could rescue the block to NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release imposed by depletion of endogenous AMOT ( Figure 7—figure supplement 1B ) .","AMOTL2 strongly stimulated/rescued HIV-1ΔPTAP , ΔYP release in both of these assays , albeit slightly less efficiently than AMOT p130 ( Figure 7—figure supplement 1A , compare lane 8 to lanes 5 and 6 and Figure 7—figure supplement 1B , compare lane 7 to lanes 4 and 5 ) .","In contrast , AMOTL1 exhibited little or no activity in either assay ( Figure 7—figure supplement 1A , compare lane 7 to lanes 5 and 6 and Figure 7—figure supplement 1B , compare lane 6 to lanes 4 and 5 ) .","Thus , AMOTL2 could substitute for AMOT p130 in three different virus release assays , whereas AMOTL1 was slightly less active than AMOT in the release of wild type HIV-1 , and failed to stimulate release of the ‘crippled’ HIV-1ΔPTAP , ΔYP construct .","Scanning and transmission electron microscopy ( SEM and TEM , respectively ) were used to visualize the stage at which AMOT depletion inhibited HIV-1 assembly or release ( Figures 8 , 9 ) .","SEM imaging was initially performed because this method can survey the entire cell surface and thereby identify global changes in virion assembly and budding profiles .","SEM analyses were performed on 293T cells that expressed HIV-1 and were treated with either: ( 1 ) a control siRNA , ( 2 ) an siRNA that depleted TSG101 ( positive control ) , or ( 3 ) an siRNA that depleted AMOT .","As expected , viruses budding from control siRNA-treated cells were detectable but rare ( e . g . , Figure 8A–C ) .","In contrast , large numbers of assembling virions were visible on the surfaces of cells that lacked TSG101 ( Figure 8D–F ) or that lacked AMOT ( Figure 8G–I ) .","We also observed that the arrested virions tended to cluster in the center of cells lacking AMOT , whereas they were more evenly distributed across the surfaces of cells lacking TSG101 .","The dramatic increase in the numbers of arrested budding virions implies that AMOT , like TSG101 , must function following the initial stages of Gag assembly , but prior to viral membrane fission . 10 . 7554/eLife . 03778 . 019Figure 8 . AMOT p130 is required for HIV-1 assembly/budding . Scanning electron microscopic ( SEM ) images of HIV virions budding from 293T cells depleted with a control siRNA ( A–C ) , depleted of TSG101 ( D–F ) or depleted of AMOT ( G–I ) .","Successive images across each row show expansions of the adjacent boxed regions , and scale bars are 500 nm in all cases .","Arrows in panel ( C ) highlight budding wild type HIV-1 virions . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01910 . 7554/eLife . 03778 . 020Figure 9 . AMOT p130 is required for an early stage of HIV-1 assembly/budding .","( A ) Transmission electron microscopic ( TEM ) images of HIV virions budding from 293T cells depleted of TSG101 .","Panel 1: wide view .","Panel 2: expanded view .","Scale bars here and in part B are 100 nm .","( B ) TEM images of HIV virions budding from 293T cells depleted of AMOT p130 and p80 .","Panel 1: wide view .","Panel 2: expanded view .","( C ) Quantification of the release and maturation status of HIV-1 virions associated with cells that were treated with a control siRNA ( black bars ) , depleted of TSG101 ( red ) , depleted of AMOT ( teal ) , or depleted of AMOT and re-expressing AMOT p130 from an siRNA-resistant construct ( light blue ) .","This experiment was repeated twice with similar results , and error bars were derived by quantifying three separate sets of >100 virions each from one of the experiments .","( D ) Quantification of the completeness of virions budding from cells treated with a control siRNA ( black triangles ) , depleted of TSG101 ( red squares ) , or depleted of AMOT ( teal diamonds ) .","The extent of the Gag shell arc ( in degrees ) was measured from TEM images of 500 budding virions of each type , the measurements were binned into the intervals shown below the x-axis , and the virion numbers in each bin are shown in the plot . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 020 TEM experiments were performed to characterize how virus assembly/budding arrested in the absence of AMOT .","These experiments again visualized HIV-1 budding from 293T cells treated with a control siRNA , an siRNA that depleted TSG101 ( positive control ) , an siRNA that depleted AMOT , or an siRNA that depleted endogenous AMOT together with an siRNA-resistant construct that re-expressed wild type AMOT p130 ( rescue control ) .","Culture supernatants were removed from the virus producing cells and the adherent cells were fixed while still attached to the culture dish in order to maximize the numbers of cell-associated virions .","Fixed cells were harvested , embedded in resin , thin sectioned , stained , and visualized by TEM .","Cell-associated virions observed in these experiments were scored as being either: ( 1 ) mature ( discernable conical or condensed cores ) , ( 2 ) immature ( discernable immature Gag shells ) , or ( 3 ) budding ( contiguous cellular and viral membranes ) .","HIV-1 virions associated with wild type cells were usually mature ( 76 ± 5% , see Figure 9C ) , but immature and budding virions were also observed ( 9 ± 3% and 15 ± 5% , respectively ) .","As expected , TSG101 depletion induced a strong virus budding defect , with the majority of detected virions arrested at the budding stage ( 67 ± 7% , shown in Figure 9A and quantified in Figure 9C ) .","In addition , the ratio of mature ( 10 ± 6% ) to immature ( 23 ± 2% ) virions ( 1:2 . 3 ) was much lower than in the control case ( 1:0 . 1 ) , consistent with previous reports that TSG101 is also required for efficient Gag processing and maturation ( Göttlinger et al . , 1991; Garrus et al . , 2001; Martin-Serrano et al . , 2003 ) .","AMOT depletion also inhibited virus release , as reflected in a large increase in the percentage of virions detected at the budding stage ( 72 ± 6% , shown in Figure 9B and quantified in Figure 9C ) .","In this case , however , the virions that were released exhibited a normal ratio of mature:immature virions ( 25 ± 5% vs 3 ± 2%; 1:0 . 1 ) .","Importantly , the budding defect seen in cells lacking endogenous AMOT was almost completely rescued by re-expression of the siRNA-resistant AMOT p130 ( Figure 9C ) , so that the majority of virions were now mature ( 70 ± 5% vs 76 ± 5% in the control ) , and budding virion levels were nearly normal ( 26 ± 9% vs 15 ± 5% in the control ) .","Thus , AMOT depletion blocks virus release at the assembly/budding stage , but does not inhibit Gag processing or virion maturation .","It was also striking that AMOT depletion caused virion assembly/budding to arrest at an earlier stage than TSG101 depletion .","This difference can be seen in the EM images shown in Figure 9A , B , where most of the virions in TSG101-depleted cells exhibit the characteristic ‘lollipop’ morphology associated with a late assembly/membrane fission defect ( Figure 9A ) , whereas most of the virions budding from AMOT-depleted cells exhibit a ‘half-moon’ morphology in which the nascent viral membrane and Gag shell are incomplete ( Figure 9B ) .","This observation was quantified by imaging 500 budding HIV-1 virions from: ( 1 ) wild type cells ( control ) , ( 2 ) cells depleted of TSG101 , and ( 3 ) cells depleted of AMOT .","The arc formed by the Gag shell was measured manually for each budding virion , and the 500 measurements for each condition were then binned in 15° intervals and displayed graphically ( Figure 9D ) .","The Gag arc distribution of control virions budding from wild type cells exhibited two similarly sized peaks; one in which the Gag shells covered an arc of ∼155° , and another in which the Gag shells covered an arc of ∼320° .","These measurements indicate that virion assembly intermediates ( or possibly dead-end products ) appreciably accumulate at two different stages , suggesting that either stage can be rate limiting .","In cells lacking TSG101 , budding-arrested virions were much more prevalent and they typically arrested at very late stages of assembly , with the maximal population exhibiting a Gag shell arc of ∼320° .","In cells lacking AMOT , arrested virions were again prevalent but they arrested at a much earlier stage , with the maximal population exhibiting a Gag shell arc of ∼165° .","The dramatic increases in numbers of budding virions induced by depletion of TSG101 or AMOT can explain how the assembly phenotypes could distribute as single peaks in these cases , whereas virions budding from wild type cells ( which were much rarer ) distributed into two peaks .","Our experiments reveal that HIV budding arrests at an earlier stage in the absence of AMOT than in the absence of TSG101 , implying that AMOT acts at an earlier stage of virion morphogenesis ."],["We have identified Angiomotin ( AMOT ) as a host protein required for efficient HIV-1 release .","In the absence of AMOT , most virions arrested at a ‘half-moon’ stage in which a hemisphere of assembled Gag molecules distended the membrane , but did not form a spherical particle ( Figure 9 ) .","The AMOT depletion phenotype was distinct from the defect induced by the absence of ESCRT factors , where virus budding arrested at a ‘lollipop’ stage in which nearly complete immature virions remained tethered to the host cell via thin membrane stalks .","Thus , cellular factors facilitate ( at least ) two distinct steps in virion morphogenesis , with AMOT required to complete assembly/envelopment and the ESCRT machinery required for membrane fission .","HIV-1 virions budding from control cells accumulated at both stages , suggesting that both steps can be kinetic bottlenecks ( see Figure 9D and reference Ku et al . , 2013 ) .","The Gag lattice organizes the virion and undoubtedly helps to drive membrane envelopment because pure recombinant Gag proteins can form spherical particles in vitro ( Campbell et al . , 2001 ) .","In cells , however , Gag appears to require additional activities to bend the membrane , extrude the particle , and sever the bud neck .","The well-studied process of endocytic vesicle formation provides precedence for these additional requirements because clathrin alone can form spherical assemblies but nevertheless requires assistance from BAR domain-containing proteins to help bend membranes , and assistance from actin and dynamin to release the vesicle ( Weinberg and Drubin , 2012 ) .","It is intriguing that AMOT p130 also contains a candidate BAR-like domain ( Heller et al . , 2010 ) and can associate with F-actin ( Ernkvist et al . , 2006 , 2008; Chan et al . , 2013 ) .","The AMOT BAR-like domain can remodel membranes in vitro ( Heller et al . , 2010 ) , although it is not yet clear whether it functions as a classical or an inverse BAR domain .","This distinction is mechanistically significant because classical BAR domains stabilize positive membrane curvature whereas inverse BAR domains stabilize negative curvature ( Mim and Unger , 2012 ) , both of which are generated during enveloped virus assembly .","In principle , the BAR domain could mediate AMOT p130 recruitment at the half-moon stage , once the nascent viral membrane has reached the proper degree of curvature .","The BAR domain could also help drive the additional membrane curvature required to proceed to the lollipop stage .","Similarly , F-actin recruitment and polymerization could help ‘push’ the virion toward the lollipop stage , although several recent studies argue strongly against an essential role for F-actin in HIV-1 assembly ( Bharat et al . , 2014; Rahman et al . , 2014 ) .","Our experiments indicate that another important AMOT p130 function is to recruit NEDD4L to sites of virus budding .","In support of this idea , we have shown that: ( 1 ) the two proteins bind one another directly through interactions between the NEDD4L WW domains and PPXY elements present in the unique N-terminal region of the AMOT p130 isoform ( Figures 1 , 2 and references Wang et al . , 2012; Moleirinho et al . , 2014 ) , ( 2 ) AMOT p130 can recruit NEDD4L to cellular membranes , and this activity requires the NEDD4L WW-AMOT p130 PPXY interactions ( [Heller et al . , 2010] and our data not shown ) , ( 3 ) NEDD4L cannot stimulate release of ‘crippled’ HIV-1 constructs in the absence of AMOT ( Figure 4 ) , ( 4 ) NEDD4L mutants that cannot bind AMOT are also impaired in their ability to stimulate virus budding ( Figure 5 ) , ( 5 ) AMOT p80 and AMOT p130 PPXY mutants that cannot bind NEDD4L do not support virus budding ( Figures 3–6 ) and ( 6 ) recombinant AMOT p130 and HIV-1 Gag proteins can bind one another directly , albeit weakly in vitro ( Figure 2B ) , and could bind more robustly in cells owing to avidity and membrane topology effects .","Previous studies indicate that other retroviruses also employ distinct host factors to promote membrane envelopment and fission .","The clearest example is the D-type Betaretrovirus Mason-Pfizer Monkey Virus ( M-PMV ) , which forms spherical particles in the cytoplasm and then obtains its outer membrane and exits the cell by crossing the plasma membrane .","M-PMV Gag contains two distinct late assembly domains that function non-redundantly: a PPPY motif that binds NEDD4 ( and perhaps other NEDD4 family members ) , and a PSAP motif that binds TSG101/ESCRT-I ( Gottwein et al . , 2003 ) .","PPPY and PSAP mutations induce strikingly different phenotypes: mutation of the PPPY motif causes virions to stack up against the plasma membrane where they fail to initiate membrane envelopment whereas virions lacking the PSAP motif acquire an envelope , but accumulate at the lollipop stage or are released as chains of membrane-tethered particles .","These observations imply that Gag PPPY-NEDD4 complexes are required for early stages of membrane bending and envelopment , whereas Gag PSAP-ESCRT-I complexes recruit the ESCRT machinery to mediate membrane fission .","Like other family members , NEDD4 binds AMOT ( Moleirinho et al . , 2014 ) , and we therefore speculate that M-PMV may also recruit AMOT to facilitate membrane bending and particle envelopment .","This would imply that viruses can recruit NEDD4-motin complexes by binding either member of the complex .","The deltaretrovirus HTLV-I is another case where a PPXY motif appears to be required for an early step in particle envelopment and a PTAP motif may function at a later membrane fission step ( [Le Blanc et al . , 2002; Blot et al . , 2004; Heidecker et al . , 2004] , but also see reference [Wang et al . , 2004] ) .","Distinct roles for PPXY and PTAP motifs have not been delineated in other well-studied viruses that use both motifs , such as the gammaretrovirus Murine Leukemia Virus ( Yuan et al . , 2000 ) or the filovirus Ebola Virus ( Timmins et al . , 2003 ) .","Although many details of HIV-1 assembly and budding remain to be elucidated , our data are consistent with a stepwise pathway in which: ( 1 ) AMOT p130 is recruited at the half-moon stage of HIV-1 assembly , perhaps by binding cooperatively to Gag and to appropriately curved membranes , ( 2 ) AMOT p130 promotes further membrane curvature and recruits NEDD4L via PPXY-WW domain interactions , ( 3 ) NEDD4L builds Lys-63-linked ubiquitin chains on the viral Gag protein ( and/or other nearby substrates ) , ( 4 ) the juxtaposition of Gag late assembly domains and Lys-63-linked Ub chains creates high affinity binding sites for the early-acting ESCRT factors , TSG101 and ALIX , and ( 5 ) these factors then recruit the downstream ESCRT-III and VPS4 components required to effect membrane scission .","This model is attractive because: ( 1 ) our work shows that AMOT functions upstream of the ESCRT pathway , ( 2 ) AMOT appears to function as a NEDD4L adaptor ( see above ) , ( 3 ) ubiquitin transfer is required for efficient HIV-1 release ( Martin-Serrano , 2007 ) , ( 4 ) NEDD4L builds Lys-63-linked ubiquitin chains and can use HIV-1 Gag as a substrate ( Weiss et al . , 2010; Sette et al . , 2013 ) , and ( 5 ) both TSG101/ESCRT-I and ALIX have ubiquitin binding activities .","The ALIX activity is specific for Lys-63-linked ubiquitin chains ( Dowlatshahi et al . , 2012; Keren-Kaplan et al . , 2013; Pashkova et al . , 2013 ) , and ESCRT-I complexes also have multiple ubiquitin binding sites , although linkage specificity has not been observed ( Stefani et al . , 2011; Agromayor et al . , 2012; McCullough et al . , 2013 ) .","Cooperative binding to the late domains and ubiquitin could explain how ALIX and TSG101/ESCRT-I can be efficiently recruited , despite making intrinsically weak interactions with the p6Gag late assembly domains .","The proposed sequence of events could also provide a ‘timing’ mechanism for recruiting ( or activating ) these early-acting ESCRT proteins to function at a late stage of virion assembly , although the precise timing of early-acting ESCRT recruitment remains to be clarified because published reports differ on this issue ( Jouvenet et al . , 2011; Ku et al . , 2014 ) .","All enveloped viruses must bend and remodel membranes as they bud , and AMOT requirements may therefore be quite general .","Indeed , AMOTL1 has been shown to bind the M protein of PIV5 and to be required for the efficient , ubiquitin-dependent release of this virus ( Pei et al . , 2010 ) .","Motin family members therefore contribute to the assembly of at least two highly diverged enveloped viruses; the paramyxovirus PIV5 and the lentivirus HIV-1 .","This suggests that AMOT proteins , like the ESCRT machinery , may provide general activities required for the assembly and release of a variety of different enveloped viruses ."],["Expression constructs , siRNA sequences , and antibodies are described in detail in Supplementary file 1 .","Constructs for expressing wild type and mutant NEDD4L and AMOT proteins in E . coli , insect SF-9 cells , and human 293T cells were created by standard cloning and mutagenesis methods , with detailed methods available upon request .","All of the new expression constructs used in our studies have been deposited in the public DNASU plasmid repository ( http://dnasu . org/DNASU/Home . jsp ) .","For the experiment shown in Figure 1B , an empty vector control or a pCAG-OSF-NEDD4L expression vector ( Morita et al . , 2007 ) expressing NEDD4L with an N-terminal One-STrEP-FLAG ( OSF ) affinity tag was transfected into 293T cells ( calcium phosphate method , 2 × 10 cm plates each , 25 μg DNA/plate ) .","After 48 hr , cells from each sample were collected by scraping , pooled , washed three times with PBS ( 1 . 5 mM KH2PO4 , 155 mM NaCl , 2 . 7 mM Na2HPO4 , pH 7 . 4 ) , and lysed with 500 μl Triton lysis buffer ( 1% Triton X-100 , 50 mM Tris pH 8 . 0 , 150 mM NaCl , 150 μg/ml PMSF ) .","This , and all subsequent preparation steps , were performed at 4°C .","Soluble extracts were pre-incubated for 30 min with 5 μl Protein-A resin slurry ( Millipore , Temecula , CA ) to remove non-specific binding proteins .","Supernatants were collected and incubated for 2 hr with 10 μl STrEP-Tactin resin ( IBA-Lifesciences , Göttingen , Germany ) .","The resins were washed three times with 500 μl washing buffer ( 0 . 1% Triton-X100 , 50 mM Tris , 150 mM NaCl , pH 8 . 0 ) , resuspended in 35 μl SDS-PAGE loading buffer ( 2% SDS , 10% glycerol , 2% 2-mercaptoethanol ( BME ) , 0 . 002% bromophenol blue and 62 . 5 mM Tris , pH 6 . 8 ) , and boiled to release the bound proteins .","12 μl samples of each solution were electrophoresed ( 10% SDS-PAGE ) and proteins were visualized by staining with Coomassie brilliant blue .","This procedure was repeated in three independent experiments , and in each case the protein band corresponding to AMOT p130 was visible in the Coomassie-stained gel ( Figure 1B ) .","This band was excised ( and the equivalent region from the control lane was also excised in one of the repetitions ) , de-stained in 50% methanol with 50 mM ammonium bicarbonate ( 2 × 1 ml , 1 hr , 23°C , gentle vortexing ) , re-hydrated in 50 mM ammonium bicarbonate ( 1 ml , 30 min , 23°C ) , and cut into several pieces .","Each piece was dehydrated in acetonitrile ( 1 ml , 30 min , 23°C , gentle shaking ) , and the excess acetonitrile was removed .","Proteins were in-gel digested using 10–20 μl sequence-grade modified trypsin ( Promega , Madison , WI , 20 ng/μl ) in 50 mM ammonium bicarbonate ( 16 hr , 37°C ) , and the reaction was quenched by the addition of 20 μl 1% formic acid .","The solution was removed and the gel washed twice with 50% acetonitrile in 1% formic acid ( 20 μl , 20 min , 37°C , with sonication ) and once with 100% acetonitrile ( 20 min , 37°C , with sonication ) .","The wash solutions were combined , dried , and reconstituted in 100 μl 5% acetonitrile with 0 . 1% formic acid for LC/MS/MS analysis .","Peptides were analyzed using a nano-LC/MS/MS system comprising a nano-LC pump ( 2D-ultra , Eksigent ) and an LTQ-FT mass spectrometer ( ThermoElectron Corporation , San Jose , CA ) , equipped with a nanospray ion source ( ThermoElectron Corporation ) .","5–20 fmoles of each tryptic digest were injected onto a homemade C18 nanobore LC column ( C18 [Atlantis , Waters Corporation , Milford , MA] ) ; 3 μm particle; column ( 75 μm ID × 100 mm length ) , and eluted using a linear gradient of 5–70% solvent B in 78 min ( solvent B: 80% acetonitrile with 0 . 1% formic acid; solvent A: 5% acetonitrile with 0 . 1% formic acid , 350 nl/min ) .","The LTQ-FT mass spectrometer was operated in the data-dependent acquisition mode ( Xcalibur 1 . 4 software ) with the 10 most intense peaks in each FT primary scan determined on-the-fly and trapped for MS/MS analysis and CID peptide fragmentation/sequencing in the LTQ linear ion trap .","Spectra in the FT-ICR were acquired from m/z 350 to 1400 at 50 , 000 resolution ( ∼2 ppm mass accuracy ) with the following LTQ linear ion trap parameters: precursor activation time 30 ms and activation Q at 0 . 25; collision energy at 35%; dynamic exclusion width at 0 . 1 Da–2 . 1 Da , with one repeat count and 10 s duration .","Raw LTQ FT MS data files were converted to peak lists with BioworksBrowser 3 . 2 software ( ThermoElectron Corporation ) using the following processing parameters: precursor mass 351–5500 Da; grouping enabled , 5 intermediate MS/MS scans; precursor mass tolerance 5 ppm , minimum ion count in MS/MS = 15 , and minimum group count = 1 .","Resulting DTA files were merged , and searched to identify peptides/proteins against NCBInr using the MASCOT search engine ( Matrix Science Ltd . ; version 2 . 2 . 1 , Boston , MA ) .","Searches were done with tryptic specificity , allowing two missed cleavages , or ‘non-specific cleavage’ and a mass error tolerance of 5 ppm in MS spectra ( i . e . , FT-ICR data ) and 0 . 5 Da for MS/MS ions ( i . e . , LTQ linear ion trap ) .","Identified peptides were accepted when the MASCOT ion score value exceeded 20 .","In all three repetitions , at least two peptides corresponding to AMOT p130 were identified in the tryptic digest , and the maximal coverage was 33% ( see Figure 1—figure supplement 1 ) .","To assay OSF-NEDD4L protein binding to endogenous AMOT p130 ( Figure 1C ) , 293T cells ( 2 × 106 cells per 10 cm plate ) were transfected using Lipofectamine 2000 ( Invitrogen/Life Technologies , Carlsbad , CA ) with either 3 . 5 μg of an empty pCAG-OSF expression vector ( control ) or with pCAG-OSF vectors that expressed the designated OSF-NEDD4L proteins , using 3 . 5 μg of expression constructs for wild type NEDD4L or NEDD4LΔWW , or 2 μg each of expression constructs for NEDD4LC942A or NEDD4LΔWW/C942A .","In this case , and in all other experiments , empty vector was added to keep total DNA levels constant ( i . e . , 1 . 5 μg empty pCAG-OSF vector in the case of NEDD4LC942A or NEDD4LΔWW/C942A ) .","48 hr post transfection , cells were washed in PBS buffer and lysed ( 25 mM Tris , 150 mM NaCl , 1 mM EDTA , 0 . 5% CHAPS , pH 7 . 5 , 10 min , 4°C ) .","Soluble lysates were collected by centrifugation ( 5000×g , 5 min , 4°C ) and incubated with 50 μl STrEP-Tactin resin ( IBA-Lifesciences , Göttingen , Germany , 3 hr , 4°C ) equilibrated with lysis buffer .","The resin was washed ( 25 mM Tris-HCl , 150 mM NaCl , 1 mM EDTA , 0 . 1% CHAPS , pH 7 . 5 , 3 × 1 ml ) , resuspended in 50 μl 2× SDS-PAGE loading buffer , boiled , and the released proteins were analyzed by SDS-PAGE and western blotting .","Separated proteins were transferred to PVDF membranes , blocked with 5% non-fat dry milk for 45 min at RT , and incubated overnight at 4°C with primary antibodies ( see Supplementary file 1 ) .","Secondary antibodies were anti-rabbit IgG or anti-mouse IgG conjugated to IRdye800 or IRdye700 , respectively ( 1:10 , 000 and 1:20 , 000 respectively , Rockland Immunochemicals Inc . , Gilbertsville , PA ) .","All western blots were visualized using an Odyssey scanner ( Li-Cor Biosciences , Lincoln , NB ) .","To assay OSF-NEDD4L protein binding to exogenous HA-AMOT p130 proteins ( Figure 1—figure supplement 2 ) , 293T cells ( 2 × 106 cells per 10 cm plate ) were co-transfected using Lipofectamine 2000 ( Invitrogen/Life Technologies ) with 3 . 5 μg of a pCAG-OSF vector expressing wild type OSF-NEDD4L and 3 . 5 μg of an empty pCAG vector ( control ) , or pcDNA vectors expressing HA-AMOT p130 or HA-AMOT p130ΔPPXY .","Thereafter , the co-immunoprecipitation protocol was identical to the one described above .","Proteins were expressed in the Rosetta-pLysS bacterial strain grown in auto-induction media ZYP-5052 ( Studier , 2005 ) .","For the experiments described in Figure 2 and Figure 2—figure supplement 1 , SF9 cells ( 2 l ) were infected at an MOI of 5 with Bac-to-Bac baculoviruses expressing OSF-tagged GFP ( control ) , AMOT p130 , AMOTL1 or AMOTL2 .","Pellets from 200 ml of culture were lysed in 10 ml of lysis buffer ( 1% NP-40 , 150 mM NaCl , 20 mM Tris pH 8 . 0 , 0 . 5 mM EDTA supplemented with protease inhibitors ) .","All steps were performed at 4°C .","Supernatants were collected by centrifugation for 30 min at 13 , 000 RPM in a microcentrifuge and 0 . 5 ml of each lysate was incubated with 30 μl of a slurry of STrEP-Tactin resin ( 1 hr ) .","Resins were washed with high salt wash buffer ( 20 mM Tris , 1 M LiCl , 0 . 5 mM EDTA , 0 . 1% NP-40 , pH 8 . 0 ) , followed by low salt buffer ( 20 mM Tris , 150 mM NaCl , 0 . 5 mM EDTA , 0 . 1% NP-40 , pH 8 . 0 ) .","For NEDD4L binding experiments , the OSF-GFP or OSF-AMOT-bound resins were washed with binding buffer ( 150 mM NaCl , 50 mM Tris , 1 mM TCEP , 10 μM ZnCl2 , 0 . 1% NP-40 , pH 7 . 5 ) .","Pure recombinant NEDD4L or NEDD4LΔWW proteins ( 0 . 5 μM in 500 μl binding buffer ) were pre-incubated with washed resins for 1 hr , and then incubated for 1 hr with GFP- or AMOT-bound resins .","Resins were washed three times with 250 μl binding buffer , resuspended in 30 μl SDS-PAGE loading buffer , boiled , separated by SDS-PAGE , and visualized by Coomassie staining or by Western blotting ( with 1 μl samples used for Coomassie staining , and 1 μl samples of a 50-fold dilution used for Western blotting ) .","For GagΔp6 binding experiments , the OSF-GFP ( control ) - or OSF-AMOT-bound resins were washed with binding buffer .","Pure recombinant HIV-1 GagΔp6 ( 1 μM ) in 500 μl binding buffer was incubated with the washed resins for 1 hr .","Resins were washed with binding buffer , and processed as described above .","NEDD4L and AMOT protein family co-overexpression experiments shown in Figure 3 and Figure 3—figure supplement 1 , Figure 3—figure supplement 2 , and Figure 7—figure supplement 1A were performed following the protocol: 293T cells ( 4 × 105 cells/well in a 6-well plate ) were co-transfected ( Lipofectamine , 2000; Invitrogen/Life Technologies ) with 1 μg of an R9-based expression vector for the NL4-3 strain of HIV-1ΔPTAP , ΔYP ( Swingler et al . , 1997 ) , 0 . 5 μg of pCI-FLAG NEDD4L or NEDD4LΔWW ( Chung et al . , 2008 ) or pCI-FLAG empty vector in combination with either 0 . 5 μg of pCMV AMOT p80 ( Figure 3—figure supplement 2 ) or 1 μg pcDNA3 HA-AMOT p130 ( Figure 3 and Figure 3—figure supplement 2 ) or 0 , 0 . 25 , 0 . 5 , 0 . 75 , 1 , 1 . 25 or 1 . 5 μg pcDNA3 HA-AMOT p130 , ( Figure 3—figure supplement","1 ) or 1 μg pcDNA3 HA AMOTL1 or 1 μg pcDNA3 HA AMOTL2 ( Figure 7—figure supplement 1A ) .","48 hr post-transfection , media was harvested for titer measurements and virus purification , and cells were washed off the plate in PBS .","Viral titers were assayed in HeLa-TZM indicator cells using a β-galactosidase assay , and following the manufacturer's instructions ( Promega , Madison , WI ) .","Briefly , HeLa-TZM cells ( 5000 cells per well , 96-well plate ) were infected with four different dilutions of virus-containing culture media , harvested after 48 hr , washed once ( PBS , 4°C ) , and lysed in 25 mM Tris phosphate ( pH 7 . 8 ) , 2 mM DTT , 2 mM 1 , 2-diaminocyclohexane N , N , N′ , N′-tetraacetic acid ( DCTA ) , 10% glycerol and 1% Triton X-100 ( 10 min , 23°C ) .","Cell lysates were incubated in 200 mM sodium phosphate buffer ( pH 7 . 3 ) , 2 mM MgCl2 , 100 mM BME and 1 . 33 mg/ml ortho-Nitrophenyl-β-galactoside for 60 min at 37°C .","Reactions were terminated by addition of 200 μl 1 M Tris base and absorbance at 420 nm was read in a plate reader .","For western blot analyses , virions were pelleted by centrifugation through a 20% sucrose cushion ( 90 min , 15 , 000×g , 4°C ) and resuspended in SDS-PAGE loading buffer .","Cells were washed in PBS , lysed in RIPA buffer ( 10 mM Tris , 1 mM EDTA , 0 . 5 mM EGTA , 1% Triton X-100 , 0 . 1% sodium deoxycholate , 0 . 1% SDS , 140 mM NaCl , pH 8 . 0 ) supplemented with protease inhibitors ( cOmplete , Roche ) ( 10 min , 4°C ) , and the insoluble material was removed by centrifugation ( 10 min , 15 , 000×g , 4°C ) .","Primary antibodies and dilutions used for western blots are given in Supplementary file 1 .","Depletion/re-expression experiments ( Figure 4 , Figure 4—figure supplement 1 , Figure 4—figure supplement 2 , Figure 5 , Figure 7 , and Figure 7—figure supplement","1 ) were performed following the protocol: t = 0: 293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were transfected with 10 nM siRNA using 7 . 5 μl Lipofectamine RNAiMax ( Invitrogen/Life Technologies ) ; t = 24 hr: media changed and second siRNA co-transfection with 10 nM siRNA and 0 . 5 μg wild type HIV-1 R9 expression vector or 1 μg HIV-1ΔPTAP , ΔYPXL expression vector and 0 . 5 μg of pCI-FLAG NEDD4L ( 10 μl Lipofectamine , 2000; Invitrogen/Life Technologies ) ; t = 72 hr: media and cells harvested for titer measurements and western blot analyses as described above .","For the rescue experiments , siRNA-resistant expression constructs expressing AMOT p80 , AMOT p130 , AMOT p130ΔPPXY , AMOTL1 or AMOTL2 were co-transfected at t = 24 hr .","AMOT mRNA is expressed in 293T cells and in white blood cells and lymphoid tissues , but cannot be detected in HeLa cells ( Moleirinho et al . , 2014 ) .","Western blots were performed to compare AMOT protein expression levels in 293T vs HeLa cells ( Figure 6—figure supplement 1 ) .","293T ( 2 x 106 cells/10 cm plate ) and HeLa M ( 1 . 2 x 106 cells/10 cm plate ) cells were seeded , harvested 72 hr later by trypsin treatment , washed twice in PBS , counted , lysed in RIPA buffer ( 1 . 2 × 105 cells/μl ) for 15 min at 4°C , clarified by centrifugation ( 15 min , 15 , 000×g , 4°C ) and diluted 1:1 with 2× SDS loading buffer .","2 . 5 , 5 , 10 and 20 μl of each cell lysate mixture was loaded on a 4–15% gradient gel and AMOT proteins were detected by western blotting .","Experiments to test whether AMOT overexpression can stimulate HIV-1 release from HeLa cells ( Figure 6A ) were performed as follows: HeLa M cells ( 2 . 5 × 105 cells/well in a 6-well plate ) were co-transfected with 0 . 5 μg of wild-type R9 HIV-1 and 0 . 5 , 1 , 2 or 4 μg of pcDNA3-HA AMOT p130 expression vectors .","Cells and viruses were harvested 48 hr post-transfection .","To test whether AMOT could stimulate virus release from physiologically relevant T cells , AMOT was overexpressed in Jurkat T cells as follows: t = 0 , cells were harvested , washed in PBS and resuspended in Neon resuspension buffer R ( Invitrogen/Life Technologies ) .","5 × 106 cells were combined with 3 μg of the wild type R9 HIV-1 expression vector and 0 , 5 or 10 μg of pcDNA3-HA AMOT p130 or AMOT p130ΔPPXY expression vectors .","Cells were then electroporated in a 100 µl tip with the Neon Transfection System using three 10 ms pulses of 1325 V . Following electroporation , each sample was immediately transferred to a 10 cm plate containing 10 ml pre-warmed RPMI ( supplemented with 10% FBS and 2 mM glutamine ) .","t = 96 hr: media and cells were harvested .","Protocols for analyzing protein expression and viral titers were identical to those described for 293T cells experiments .","293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were seeded onto glass cover slips ( 24 hr prior transfection ) and co-transfected with the designated siRNAs and the R9 HIV-1 expression construct as described above .","48 hr post-transfection , the media was removed and cells were fixed in 1 ml fixation buffer ( 2 . 5% glutaraldehyde/1% paraformaldehyde in sodium cacodylate buffer; 50 mM sodium cacodylate , 18 mM sucrose , 2 mM CaCl2 , pH 7 . 4 , 10 min ) .","This and all subsequent steps were performed at 23°C .","Fixed cells were washed three times in sodium cacodylate buffer ( 5 min ) , and stained with 2% OsO4 for 1 hr .","Each sample was then dehydrated in a graded ethanol series , dried in HDMS ( hexamethyldisilazane ) , and sputter coated with platinum .","SEM images were collected on a FEI Quanta 600 FEG scanning electron microscope at a beam energy of 10 kV , a spot size of three , and magnifications of 10 , 000 , 30 , 000 or 65 , 000 X . 293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were co-transfected with the designated siRNAs and the R9 HIV-1 expression construct as described above .","48 hr post-transfection , the media was removed and cells were fixed in 1 ml fixation buffer ( 2 . 5% glutaraldehyde/1% paraformaldehyde in sodium cacodylate buffer [50 mM sodium cacodylate , 18 mM sucrose , 2 mM CaCl2 , pH 7 . 4 , 10 min] ) .","This and all subsequent steps were performed at 23°C .","Fixed cells were harvested , washed three times in sodium cacodylate buffer ( 5 min ) , and stained with 2% OsO4 for 1 hr .","The pellet was rinsed once in sodium cacodylate buffer and twice in water ( 5 min ) , followed by incubation in 100 μl of a 4% uranyl acetate solution ( 30 min ) .","Stained cells were dehydrated in a graded ethanol series followed by acetone , and embedded in epoxy resin EMBed-812 ( Electron Microscopy Sciences , Hatfield , PA ) .","Thin sections ( 80–100 nm ) were cut , post-stained with saturated uranyl acetate ( 20 min ) , rinsed with water , dried , stained with Reynolds' lead citrate ( 10 min ) and dried again .","TEM images were collected on a Hitachi H-7100 transmission electron microscope at an accelerating voltage of 75 kV , equipped with a Gatan Orius sc1000 camera .","Imaged virions were scored as ‘mature’ if they lacked an observable cellular tether and a condensed core was evident , as ‘immature’ if they were untethered but lacked a condensed core , and as ‘budding’ if a Gag layer was clearly evident and the nascent virion was assembling or was clearly tethered to the cell .","The degree of assembly was assessed by rendering a ( hypothetical ) spherical particle and measuring the arc angle of the nascent Gag layer .","To image 500 virions budding from wild type cells , it was necessary to screen about eight-times as many thin slices of wild type control cells ( ∼2500 cells ) vs TSG101- or AMOT-depleted cells ( 275 and 300 , respesctively ) ."]],"string":"[\n [\n \"As obligate parasites , viruses must harness host cellular pathways to complete many different steps in viral replication .\",\n \"For example , a large number of enveloped viruses , including HIV-1 , usurp the cellular ESCRT ( Endosomal Sorting Complexes Required for Transport ) pathway to bud from cells ( for recent reviews , see Meng and Lever , 2013; Votteler and Sundquist , 2013; Weissenhorn et al . , 2013 ) .\",\n \"Viral structural proteins typically use one or more short peptide motifs , termed ‘late assembly domains’ , to bind and recruit early-acting ESCRT factors or ESCRT-associated E3 ubiquitin ligases to sites of viral assembly and budding .\",\n \"The three best characterized viral late assembly domains are: ( 1 ) the ‘PTAP’ motif ( Pro-Thr/Ser-Ala-Pro ) , which binds the UEV domain of the TSG101 subunit of the ESCRT-I complex , ( 2 ) the ‘YPXnL’ motif ( Tyr-Pro-Xn-Leu , where X can vary in identity and sequence length ) , which binds the V domain of the ESCRT factor ALIX , and ( 3 ) the ‘PPXY’ motif ( Pro-Pro-X-Tyr ) , which binds the WW domains of NEDD4 family E3 ubiquitin ligases .\",\n \"All three of these viral late assembly domains mimic cellular protein–protein interactions that facilitate ESCRT-dependent vesiculation processes such as multi-vesicular body ( MVB ) biogenesis and ectosome formation .\",\n \"In addition to the three well-characterized late assembly domains described above , there are indications that enveloped viruses can also connect to the ESCRT pathway via alternative interactions that are not yet well-understood .\",\n \"For example , efficient release of the model paramyxovirus , PIV5 , requires a non-canonical ‘FPIV’ late assembly domain motif , ubiquitylation of the matrix ( M ) protein , and host Angiomotin-like protein 1 ( AMOTL1 ) ( Schmitt et al . , 2005; Pei et al . , 2010; Harrison et al . , 2012 ) .\",\n \"There is no evidence that AMOTL1 can bind the FPIV late assembly domain , however , and any relationships between ubiquitin , AMOTL1 and the FPIV late assembly domain activity are not yet understood ( Pei et al . , 2010 ) .\",\n \"Owing to its medical importance , HIV-1 has become a leading model for understanding ESCRT pathway recruitment and viral budding .\",\n \"The C-terminal p6 region of the viral Gag structural protein contains canonical PTAP and YPXnL motifs that serve as the major functional late assembly domains in most cell types .\",\n \"Nevertheless , the release of ‘crippled’ HIV-1 Gag constructs that lack both of these late domains can still be stimulated by overexpression of the HECT E3 ubiquitin ligase , NEDD4L ( also known as NEDD4-2 ) ( Chung et al . , 2008; Usami et al . , 2008 ) .\",\n \"Humans express nine different NEDD4 protein family members ( reviewed in Ingham et al . , 2004; Bernassola et al . , 2008 ) , but NEDD4L stimulates virus budding more potently than any other NEDD4 family member , implying that HIV-1 Gag preferentially engages this E3 ubiquitin ligase ( Chung et al . , 2008; Usami et al . , 2008 ) .\",\n \"Previous studies have defined many of the requirements for NEDD4L-stimulated HIV-1 release .\",\n \"NEDD4L has many different isoforms ( Chen et al . , 2001; Dunn et al . , 2002; Itani et al . , 2005 ) , but isoform 2 ( also called NEDD4-2s , and here termed NEDD4L ) potently stimulates the budding of crippled HIV-1 constructs , and uniquely rescues the Gag processing defects that accompany defective budding ( Chung et al . , 2008; Usami et al . , 2008 ) .\",\n \"This NEDD4L isoform contains only the final 31 residues of the N-terminal C2 membrane binding domain , a central linker region that contains four WW domains , and a C-terminal HECT E3 ubiquitin ligase domain that forms Lys-63-linked poly-ubiquitin chains ( see Figure 1A ) ( Kim and Huibregtse , 2009; Weiss et al . , 2010 ) .\",\n \"Stimulation of HIV-1 budding requires NEDD4L E3 ubiquitin ligase activity , indicating that Lys-63-linked poly-ubiquitin chains perform an essential function ( Chung et al . , 2008; Usami et al . , 2008 ) .\",\n \"The functional target ( s ) of Lys-63 poly-ubiquitylation has not been established definitively , but HIV-1 Gag itself is the leading candidate because functional virus budding correlates with Lys-63-linked poly-ubiquitylation of Gag , and because NEDD4L truncation constructs that include the HECT domain can be functionally rescued by fusion to the HIV-1 Gag-targeting cyclophilin A domain ( Weiss et al . , 2010; Sette et al . , 2013 ) .\",\n \"HIV-1 Gag lacks identifiable PPXY motifs , however , and there is no evidence that Gag can bind NEDD4L directly . 10 . 7554/eLife . 03778 . 003Figure 1 . AMOT p130 is a NEDD4L binding partner .\",\n \"( A ) Schematic illustrations of the domain structures and motifs in NEDD4L , AMOT p130 and p80 , AMOTL1 , AMOTL2 and HIV-1 Gag .\",\n \"Here and throughout , NEDD4L refers to a naturally occurring protein isoform ( isoform\",\n \"2 ) that contains only the C-terminal 32 residues of the C2 domain ( Genebank accession number AAM46201 . 1 , denoted NEDD4Ls in other publications and NEDD4LΔC2 in our previous publication ( Chung et al . , 2008 ) ) .\",\n \"To maintain consistency with that publication , the NEDD4L numbering scheme used here corresponds to NEDD4L isoform 1 , which is 121 residues longer at the N-terminus and contains the entire C2 domain ( denoted NEDD4LWT in reference Chung et al . , 2008 ) .\",\n \"( B ) Affinity co-purification and identification of AMOT p130 as a binding partner of OSF ( One-STrEP-FLAG ) -tagged NEDD4L .\",\n \"SDS-PAGE/Coomassie-stained gel showing STrEP-Tactin matrix affinity purified proteins from 293T cells transfected with an OSF-NEDD4L expression construct ( lane\",\n \"2 ) or an empty vector control ( lane 1 ) .\",\n \"Labels denote OSF-NEDD4L ( bait ) and AMOT p130 ( prey ) .\",\n \"Trypsin-digested AMOT p130 peptides identified by mass spectrometric analyses are summarized in Figure 1—figure supplement 1 .\",\n \"A representative image from three independent repetitions is shown .\",\n \"( C ) NEDD4L WW domains are required for AMOT p130 binding .\",\n \"Western blots showing co-immunoprecipitations of endogenous 293T cell AMOT proteins ( prey ) with OSF-NEDD4L ( bait ) .\",\n \"Panel 1: endogenous AMOT proteins ( anti-AMOT blot ) co-immunoprecipitated with OSF-NEDD4L baits from cells that lacked exogenous OSF-NEDD4L ( lane 1 , control ) , expressed wild type OSF-NEDD4L ( lane 2 ) , expressed OSF-NEDD4LΔWW ( lane 3 , construct has inactivating point mutations in all four WW domains ) , expressed OSF-NEDD4LC942A ( lane 4 , construct has an inactivating point mutation in the HECT E3 domain ) , or expressed OSF-NEDD4LC942A/ΔWW ( lane 5 , construct has inactivating point mutations in the WW and HECT E3 domains ) .\",\n \"Panel 2: same co-immunoprecipitation experiment blotted with anti-FLAG antibodies to detect OSF-NEDD4L proteins .\",\n \"Panel 3: input levels of endogenous AMOT ( anti-AMOT blot , 10% of total ) .\",\n \"Panel 4: input levels of exogenous OSF-NEDD4L ( anti-FLAG , 10% of total ) .\",\n \"Note that endogenous AMOT p80 was present in the input lysate , but did not co-immunoprecipitate with OSF-NEDD4L .\",\n \"Representative image from three independent repetitions . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00310 . 7554/eLife . 03778 . 004Figure 1—figure supplement 1 . Identification of AMOT p130 as a NEDD4L binding partner . The protein band denoted ‘AMOT p130’ in Figure 1B , lane 2 was excised , digested with trypsin and the eluted peptides were identified by mass spectrometry as described in ‘Materials and methods’ .\",\n \"This analysis was performed three times and the identified peptides are mapped onto the AMOT p130 primary sequence , with peptides identified in experiments 1–3 color coded in black , blue and red , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00410 . 7554/eLife . 03778 . 005Figure 1—figure supplement 2 . The AMOT p130 PPXY motifs contribute to NEDD4L binding . Western blots show co-immunoprecipitations of OSF-NEDD4L ( bait , captured on STrEP-Tactin affinity matrices ) with endogenous 293T cells AMOT and exogenously expressed HA-AMOT p130 proteins ( prey ) .\",\n \"Co-immunoprecipitations with OSF-NEDD4L were from cells that lacked exogenous AMOT p130 ( lane 1 ) , expressed wild type HA-AMOT p130 ( lane\",\n \"2 ) or expressed a mutant HA-AMOT p130 protein carrying PP/AA mutations in the three N-terminal ‘PPXY’ motifs ( Wang et al . , 2012; Yi et al . , 2013 ) .\",\n \"Panel 1: AMOT protein co-immunoprecipitations ( anti-AMOT blot ) with OSF-NEDD4L .\",\n \"Note that OSF-NEDD4L co-immunoprecipitated low levels of endogenous AMOT p130 ( lanes 1 and 3 , denoted p130 ) but not endogenous AMOT p80 ( denoted p80 with a dashed line ) .\",\n \"Panel 2: The same co-immunoprecipitation experiment blotted with anti-HA antibodies to detect exogenously expressed HA-AMOT p130 proteins .\",\n \"Panel 3: The same co-immunoprecipitation experiment blotted with anti-FLAG antibodies to detect exogenously expressed OSF-NEDD4L .\",\n \"Panels 4–6 show the input quantities ( 10% ) of endogenous AMOT and HA-AMOT p130 ( panel 4 , anti-AMOT ) , exogenous HA-AMOT p130 ( panel 5 , anti-HA ) , and exogenous OSF-NEDD4L ( panel 6 , anti-FLAG ) .\",\n \"A representative image from three independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 005 These observations imply that NEDD4L may be recruited to sites of HIV-1 assembly through an alternative mechanism .\",\n \"We undertook the present study with the goal of identifying co-factors that might help link NEDD4L to HIV-1 Gag and stimulate virus budding .\",\n \"We identified Angiomotin ( AMOT ) as a protein that binds both NEDD4L and HIV-1 Gag , and functions in HIV-1 assembly and release .\",\n \"AMOT is required to complete virion assembly and envelopment , and is therefore a new host factor that acts in a previously uncharacterized step of HIV-1 morphogenesis .\"\n ],\n [\n \"Affinity purification/mass spectrometry experiments were performed to identify candidate NEDD4L binding partners .\",\n \"A number of cellular proteins co-purified with OSF ( One-STrEP-FLAG ) -tagged NEDD4L , as visualized by SDS-PAGE ( Figure 1B , compare lanes 1 and 2 ) .\",\n \"Prominent co-purifying proteins were excised from the gel , digested with trypsin , and identified by mass spectrometry .\",\n \"The p130 isoform of human Angiomotin was unambiguously identified as a protein that co-purified with OSF-NEDD4L in three independent repetitions of this experiment , ( lane 2 , labeled AMOT p130 , peptide coverage data are summarized in Figure 1—figure supplement 1 ) .\",\n \"Smaller AMOT p130 fragments were also identified , as was the related AMOT family protein , Angiomotin-like protein 1 ( AMOTL1 , data not shown ) .\",\n \"Cells express two AMOT isoforms , a longer p130 isoform , and a shorter C-terminal p80 isoform that is expressed from an alternatively spliced message ( Figure 1A ) ( Moreau et al . , 2005 ) .\",\n \"AMOT p130 binds NEDD4 protein family members via interactions between the four central NEDD4 WW domains and three PPXY motifs located within the N-terminal extension that is unique to the AMOT p130 isoform ( Figure 1A ) ( Wang et al . , 2012 ) .\",\n \"In good agreement with this report and with our affinity purification/mass spectrometry results , we found that AMOT p130 co-immunoprecipitated with exogenously expressed OSF-NEDD4L ( Figure 1C , panel 1 , lane 2 ) .\",\n \"In contrast , AMOT p130 did not co-immunoprecipitate with a mutant OSF-NEDD4L protein that contained inactivating Trp to Ala substitutions in the key PPXY-binding ‘W39’ residues from each of the four WW domains ( denoted OSF-NEDD4LΔWW , panel 1 , compare lanes 2 and\",\n \"3 ) ( Otte et al . , 2003 ) .\",\n \"A previous study has demonstrated that NEDD4 proteins can ubiquitylate and reduce AMOT p130 levels ( Wang et al . , 2012 ) , and we also observed that OSF-NEDD4L overexpression modestly reduced AMOT p130 levels ( Figure 1C , panel 3 , compare lanes 1 and 2 ) .\",\n \"We therefore tested whether AMOT p130 also co-precipitated with OSF-NEDD4L proteins that carried inactivating Cys942Ala point mutations in the active site cysteine of the HECT E3 ubiquitin ligase domain ( OSF-NEDD4LC942A ) .\",\n \"This mutation increased OSF-NEDD4L levels in the lysate and immunoprecipitate ( panels 2 and 4 compare lanes 4 and 5 to lanes 2 and\",\n \"3 ) and restored normal AMOT protein levels ( panel 3 , compare lanes 4 , 5 and 1 to lane 2 ) .\",\n \"Once again , AMOT p130 ( and breakdown products ) co-immunoprecipitated with OSF-NEDD4LC942A ( panel 1 , lane 4 ) , but not with a protein that also contained inactivating WW domain mutations ( OSF-NEDD4LΔWW/C942A panel 1 , lane 5 ) .\",\n \"AMOT p130 co-precipitation levels were higher with OSF-NEDD4LC942A than with wild type OSF-NEDD4L , presumably owing to the higher cellular levels of both OSF-NEDD4L and AMOT p130 ( compare lanes 2 and 4 ) .\",\n \"Three additional observations confirmed that the NEDD4L WW domains and AMOT p130 PPXY motifs are critical for the NEDD4L-AMOT p130 interaction .\",\n \"Firstly , mutating the three N-terminal AMOT p130 PPXY motifs strongly inhibited co-immunoprecipitation of HA-AMOT p130 with OSF-NEDD4L ( Figure 1—figure supplement 2 , panel 1 , compare lanes 2 and 3 ) .\",\n \"Secondly , endogenous AMOT p80 , which lacks the N-terminal PPXY motifs , failed to co-immunoprecipitate with either OSF-NEDD4L or OSF-NEDD4LC942A ( Figure 1C , panel 1 , lanes 2 and 4 ) , even though AMOT p80 was present in the 293T cell extracts ( panel 3 , lanes 1–5 ) .\",\n \"Thirdly , we observed that exogenously expressed , epitope-tagged NEDD4L and AMOT p130 co-localized well in HeLa-M cells , and this co-localization required both the AMOT PPXY and NEDD4L WW motifs ( data not shown ) , in good agreement with previous reports that motins co-localize with other NEDD4 family members ( Skouloudaki and Walz , 2012; Wang et al . , 2012 ) .\",\n \"Taken together , these experiments demonstrate that NEDD4L interacts specifically with AMOT p130 in cells and that the interaction requires the WW domains of NEDD4L and the PPXY motifs of AMOT .\",\n \"Pull-down experiments with purified recombinant proteins were performed to test whether AMOT p130 binds NEDD4L directly , and whether AMOT p130 can also bind HIV-1 Gag and thereby link NEDD4L to the viral Gag protein .\",\n \"Wild type and mutant NEDD4L proteins and an HIV-1 Gag protein that lacked the flexible p6 domain ( GagΔp6 ) were expressed in Escherichia coli and purified to near homogeneity ( Figure 2A , lanes 1 and 2 , and Figure 2B , lane 1 , respectively ) .\",\n \"OSF-AMOT p130 was overexpressed in SF-9 insect cells and substantially enriched by binding to STrEP-Tactin affinity resin ( Figure 2A , lane 6 and Figure 2B , lane 7 ) , although we were unable to purify the protein to homogeneity .\",\n \"As shown in Figure 2A , NEDD4L did not bind an immobilized control OSF-GFP protein , but bound with at least 1:1 stoichiometry to immobilized OSF-AMOT p130 ( compare lanes 4 and 7 ) .\",\n \"In contrast , binding of the NEDD4LΔWW mutant protein was nearly undetectable ( Figure 2A , compare lanes 7 and 8 , particularly in the western blot in panel 2 ) .\",\n \"These experiments confirm that NEDD4L and AMOT p130 bind one another directly , with the WW domains of NEDD4L binding the PPXY motifs of AMOT p130 . 10 . 7554/eLife . 03778 . 006Figure 2 . AMOT p130 binds directly and specifically to NEDD4L and HIV-1 Gag∆p6 . ( A ) OSF-AMOT p130 binds NEDD4L .\",\n \"Recombinant OSF-GFP ( specificity control , lanes 3–5 ) or OSF-AMOT p130 ( lanes 6–8 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 3 and\",\n \"6 ) or buffers containing 0 . 5 µM wild type NEDD4L ( lanes 4 and\",\n \"7 ) or NEDD4LΔWW ( lanes 5 and 8 , inactivating point mutations in all four WW domains ) .\",\n \"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining ( panel\",\n \"1 ) or by western blotting ( panel 2 , anti-NEDD4L ) to confirm the identities of the bound NEDD4L and help to distinguish background proteins from low-level binding in panel 1 .\",\n \"Input levels of NEDD4L ( lane 1 , 1 . 5% of total ) and NEDD4LΔWW ( lane 2 , 1 . 5% of total ) are shown for reference .\",\n \"A representative image from three independent repetitions is shown .\",\n \"( B ) OSF-AMOT p130 binds HIV-1 Gag∆p6 .\",\n \"Recombinant OSF-GFP ( control , lanes 3–6 ) or OSF-AMOT p130 ( lanes 7–10 ) were expressed , captured on STrEP-Tactin affinity matrices and incubated with a buffer control ( lanes 3 and\",\n \"7 ) or with buffers containing 1 . 0 µM HIV-1 Gag∆p6 ( lanes 3 and 8 ) , 0 . 5 µM wild type NEDD4L ( lanes 5 and 9 ) or both proteins ( lanes 6 and 10 ) .\",\n \"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining ( panel\",\n \"1 ) or by western blotting to confirm the identities of the bound Gag ( panel 2 , anti-MA and anti-CA ) and NEDD4L ( panel 3 , anti-NEDD4L ) proteins and help to distinguish background proteins from low-level binding in panel 1 .\",\n \"Input Gag∆p6 ( lane 1 , 2% of total ) and NEDD4L ( lane 2 , 1 . 5% of total ) are shown for reference .\",\n \"A representative image from three independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00610 . 7554/eLife . 03778 . 007Figure 2—figure supplement 1 . AMOT p130 , AMOTL1 and AMOTL2 bind directly and specifically to NEDD4L and HIV-1 Gag∆p6 . ( A ) OSF-AMOT p130 , OSF-AMOTL1 , and OSF-AMOTL2 bind NEDD4L .\",\n \"Recombinant OSF-GFP ( specificity control , lanes 2 and 6 ) , OSF-AMOT p130 ( lanes 3 and 7 ) , OSF-AMOTL1 ( lanes 4 and 8 ) , and OSF-AMOTL2 ( lanes 5 and 9 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 2–5 ) or with a buffer containing 0 . 5 µM wild type NEDD4L ( lanes 6–9 ) .\",\n \"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining .\",\n \"Input NEDD4L ( lane 1 , 1 . 5% of total ) is shown for reference .\",\n \"Asterisk denotes an OSF-AMOTL2 breakdown product that co-migrates with NEDD4L .\",\n \"A representative image from two independent repetitions is shown .\",\n \"( B ) OSF-AMOT p130 , OSF-AMOTL1 , and OSF-AMOTL2 bind Gag∆p6 .\",\n \"Recombinant OSF-GFP ( specificity control , lanes 2 and 6 ) , OSF-AMOT p130 ( lanes 3 and 7 ) , OSF-AMOTL1 ( lanes 4 and 8 ) , and OSF-AMOTL2 ( lanes 5 and 9 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 2–5 ) or buffers containing 1 µM Gag∆p6 ( lanes 6–9 ) .\",\n \"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining .\",\n \"Input Gag∆p6 ( lane 1 , 2% of total ) is shown for reference .\",\n \"A representative image from two independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 007 Analogous pull-down experiments were performed to test whether AMOT binds HIV-1 GagΔp6 .\",\n \"Removing the p6 region facilitates Gag protein expression and purification , and is not expected to interfere with any biologically relevant interactions because NEDD4L potently stimulates HIV-1 GagΔp6 release from cells ( Chung et al . , 2008; Usami et al . , 2008 ) .\",\n \"As shown in Figure 2B , HIV-1 GagΔp6 bound immobilized OSF-AMOT p130 , but did not bind an immobilized OSF-GFP control ( compare lanes 4 and 8 ) .\",\n \"This interaction did not appear to be mediated by nucleic acids because the affinity-purified OSF-AMOT p130 protein was pre-washed with high salt solution to remove any bound nucleic acid ( see ‘Materials and methods’ ) , and because the interaction was maintained , albeit at slightly lower levels , even when immobilized OSF-AMOT p130 was incubated with high concentrations of RNase that were sufficient to eliminate other RNA-mediated Gag-protein interactions ( not shown ) .\",\n \"GagΔp6 and NEDD4L appeared to bind OSF-AMOT p130 independently because their binding levels did not change significantly when all three proteins were incubated together ( compare lanes 8 and 9 to lane 10 ) .\",\n \"AMOT is the founding member of the human motin family , which also includes Angiomotin-like protein 1 ( AMOTL1 ) and Angiomotin-like protein 2 ( AMOTL2 ) ( Moleirinho et al . , 2014 ) .\",\n \"AMOTL1 and AMOTL2 share 52% and 45% pair-wise sequence identity with AMOT p130 , and both contain the N-terminal extension and PPXY motifs that are present in AMOT p130 but absent in AMOT p80 ( Figure 1A ) .\",\n \"Pull-down experiments demonstrated that AMOTL1 and AMOTL2 also bind both NEDD4L and HIV-1 GagΔp6 ( Figure 2—figure supplement 1 ) .\",\n \"Thus , AMOT p130 binds directly and specifically to both NEDD4L and HIV-1 Gag , and these binding activities are shared by the other two human motins .\",\n \"Co-expression experiments were performed to test whether AMOT and NEDD4L can cooperate in stimulating HIV-1 release ( Figure 3 ) .\",\n \"These experiments employed the crippled HIV-1ΔPTAP , ΔYP viral expression construct , which lacks functional PTAP and YPXnL late domains and is therefore particularly responsive to cellular NEDD4L activity ( Chung et al . , 2008; Usami et al . , 2008 ) .\",\n \"As expected , HIV-1ΔPTAP , ΔYP was poorly released from control 293T cells , as measured by western blot that detected virion-associated CA and MA proteins ( Figure 3 , upper right panel , lane 1 ) , and low viral titers ( lower right panel , lane 1 ) .\",\n \"Overexpression of HA-AMOT p130 increased the levels of the full-length protein ( and presumptive breakdown products were also evident , left panel 1 , compare lanes 1 and 2 ) .\",\n \"AMOT p130 overexpression modestly increased HIV-1ΔPTAP .\",\n \"ΔYP release and infectivity ( right panels compare lanes 1 and 2 , 1 . 7-fold increases in both release and infectivity ) , without significantly altering the intracellular levels of Gag ( left , panel 4 , compare lanes 1 and\",\n \"2 ) or a control cellular protein ( GAPDH , left panel 3 , compare lanes 1 and 2 ) .\",\n \"Thus , AMOT p130 overexpression stimulates HIV-1ΔPTAP , ΔYP release and infectivity , but the effect is modest . 10 . 7554/eLife . 03778 . 008Figure 3 . AMOT p130 stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , FLAG-NEDD4L proteins ( lanes 3–6 , with wild type ( WT ) FLAG-NEDD4L in lanes 3 and 4 and FLAG-NEDD4LΔWW , in lanes 5 and 6 ) , and wild type HA-AMOT p130 ( lanes 2 , 4 and\",\n \"6 ) or an empty vector control ( lanes 1 , 3 , and 5 ) .\",\n \"Right panels show corresponding levels of extracellular , virion-associated CAGag and MAGag proteins ( panel 1 , anti-MA and anti-CA ) and viral titers ( panel 2 , IU denotes ‘infectious units’ ) .\",\n \"Here and in subsequent figures: ( 1 ) error bars denote standard deviations between independent repetitions of the experiment , n = 5 in this case , and ( 2 ) numbers within the blots show integrated intensities of the CA band intensities ( relative to the value in the control experiment , set to 1 . 0 ) .\",\n \"Here and throughout significance is denoted by: NS , not significant ( p > 0 . 05 ) ; *0 . 05 > p > 0 . 01; **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00810 . 7554/eLife . 03778 . 009Figure 3—figure supplement 1 . Dose-dependent AMOT p130 stimulation of NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–8 ) , FLAG-NEDD4L ( lanes 2–8 ) , and increasing quantities of an AMOT p130 expression construct ( lanes 3–8 , 0 . 25–1 . 5 μg DNA ) .\",\n \"Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 3 μg DNA total ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00910 . 7554/eLife . 03778 . 010Figure 3—figure supplement 2 . The AMOT p130 isoform specifically stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT , exogenous p80 and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , AMOT p80 ( lanes 2 and 5 ) , AMOT p130 ( lanes 3 and\",\n \"6 ) and FLAG-NEDD4L ( lanes 4–6 ) .\",\n \"Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 2 . 5 μg DNA total ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 010 As seen previously , FLAG-NEDD4L overexpression also stimulated HIV-1ΔPTAP , ΔYP release ( Figure 3 , right panel 1 , compare lanes 1 and 3 , 3 . 2-fold stimulation ) and infectivity ( right panel 2 , compare lanes 1 and 3 , 5 . 3-fold stimulation ) , and also largely rescued the HIV-1ΔPTAP , ΔYP Gag processing defects ( left panel 4 , compare lane 3 to lanes 1 and 2 , and note the reduction in p41 and p25 Gag processing intermediates ) ( Chung et al . , 2008; Usami et al . , 2008 ) .\",\n \"Co-overexpression of both HA-AMOT p130 and FLAG-NEDD4L further increased HIV-1ΔPTAP , ΔYP release ( right panel 1 , lane 4 , sixfold increase over basal levels ) and infectivity ( right panel 2 , lane 4 , 14-fold increase over basal levels ) .\",\n \"As shown in Figure 3—figure supplement 1 , the degree of hyper-stimulation correlated positively with AMOT p130 expression levels , until an inhibitory level was reached .\",\n \"In contrast to wild type NEDD4L , the NEDD4LΔWW mutant stimulated HIV-1ΔPTAP , ΔYP release and infectivity only very modestly ( Figure 3 , right panels , compare lanes 5 and\",\n \"3 ) and did not synergize with AMOT p130 ( right panels , compare lanes 6 and 5 ) .\",\n \"Similarly , the shorter AMOT p80 isoform , which lacks the PPXY motifs found in the N-terminal domain of AMOT p130 , failed to synergize with NEDD4L in stimulating virus release and infectivity , and instead modestly reduced NEDD4L stimulation , probably by dominantly inhibiting endogenous AMOT p130 activity ( Figure 3—figure supplement 2 , compare lanes 4–6 ) .\",\n \"These data indicate that AMOT p130 and NEDD4L cooperate in stimulating HIV-1ΔPTAP , ΔYP release and infectivity .\",\n \"siRNA depletion experiments were performed to determine whether AMOT was also necessary for NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release and infectivity .\",\n \"We used an siRNA that targeted both AMOT isoforms and reduced cellular AMOT p130 and p80 levels by at least fivefold ( Figure 4 , left panel 1 , compare lanes 1 and 2 and lanes 3 and 4 ) .\",\n \"AMOT depletion reduced the already low levels of virus release and infectivity even further , to near background levels ( Figure 4 , right panels , compare lanes 1 and 2 , 7 . 5-fold infectivity reduction ) .\",\n \"Cellular levels of Gag and a GAPDH control were unaffected by this treatment ( left panels 3 and 4 , compare lanes 1 and 2 ) , although AMOT depletion reduced cellular protein levels slightly in some repetitions ( not shown ) . 10 . 7554/eLife . 03778 . 011Figure 4 . AMOT p130 is required for NEDD4L-stimulated release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with a control siRNA ( lanes 1 and\",\n \"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–6 ) , with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , and with FLAG-NEDD4L ( lanes 3–6 ) or an empty vector control ( lanes 1 and 2 ) , and with siRNA-resistant expression constructs for HA-AMOT p130 proteins ( with wild type HA-AMOT p130 in lane 5 and an HA-AMOT p130ΔPPXY protein carrying inactivating point mutations in the three PPXY motifs in lane 6 ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01110 . 7554/eLife . 03778 . 012Figure 4—figure supplement 1 . Dose-dependent AMOT p130 rescue of HIV-1 release from cells depleted of endogenous AMOT . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with a control siRNA ( lanes 1 and\",\n \"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–10 ) , with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–10 ) , for FLAG-NEDD4L ( lanes 3–10 ) , and with increasing quantities of an siRNA-resistant AMOT p130 construct ( lanes 5–10 , 0 . 25–1 . 5 μg DNA ) .\",\n \"Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 3 μg DNA total ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01210 . 7554/eLife . 03778 . 013Figure 4—figure supplement 2 . The AMOT p130 isoform specifically stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 or AMOT p80 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were transfected with a control siRNA ( lanes 1 and\",\n \"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–7 ) .\",\n \"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–7 ) , FLAG-NEDD4L ( lanes 3–7 ) , and siRNA-resistant expression constructs for AMOT p80 ( lane 5 ) , HA-AMOT p130 ( lane\",\n \"6 ) or both ( lane 7 ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 013 AMOT depletion blocked the ability of NEDD4L to stimulate HIV-1ΔPTAP , ΔYP release and infectivity ( Figure 4 and Figure 4—figure supplements 1 , 2 ) .\",\n \"In the control case , NEDD4L overexpression stimulated HIV-1ΔPTAP , ΔYP release ( Figure 4 , right panels , compare lanes 1 and 3 , 4 . 1-fold infectivity increase ) .\",\n \"However , virus release was reduced to the levels seen in untreated control cells when endogenous AMOT p80 and p130 proteins were simultaneously depleted ( compare lanes 2–4 ) .\",\n \"Similar results were observed for a second siRNA that targeted both AMOT isoforms ( not shown ) .\",\n \"Thus , AMOT is required for NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release .\",\n \"Rescue experiments with exogenous siRNA-resistant AMOT expression constructs were performed to confirm the specificity of the AMOT depletion and to test the requirements for AMOT function .\",\n \"Re-expression of AMOT p130 completely rescued the block to virus release and infectivity imposed by AMOT depletion ( Figure 4 , right panels , compare lanes 5 and 4 ) , restoring viral infectivity to a level that was actually slightly higher than the control ( compare lanes 3 and 5 ) .\",\n \"The degree of rescue generally correlated positively with AMOT p130 re-expression levels , and peaked at levels that approximated those of the native endogenous protein ( Figure 4—figure supplement 1 , lane 8 ) .\",\n \"In contrast , an AMOT p130 protein that lacked the three N-terminal PPXY motifs failed to rescue NEDD4L-dependent HIV-1ΔPTAP , ΔYP release ( Figure 4 , AMOT p130ΔPPXY , right panels , compare lanes 6 and 5 ) .\",\n \"Similarly , re-expression of an siRNA-resistant AMOT p80 construct also failed to rescue virus release and infectivity ( Figure 4—figure supplement 2 , right panels , compare lanes 4 and 5 ) , unless exogenous AMOT p130 was present ( Figure 4—figure supplement 2 , right panels , lane 7 ) .\",\n \"Hence , NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release and infectivity requires the presence of an AMOT p130 protein that can bind NEDD4L , but is not significantly affected by AMOT proteins that cannot bind NEDD4L , including AMOT p80 .\",\n \"siRNA depletion and rescue experiments were also performed to test whether AMOT p130 is required for release and infectivity of wild type HIV-1 .\",\n \"In control experiments , depletion of either of the well-characterized HIV-1 budding factors , ALIX and TSG101 , reduced virus release and infectivity , although the effects were much greater for TSG101 depletion , as reported previously ( Figure 5 , right panels , compare lanes 2 and 3 to lane\",\n \"1 ) ( Fujii et al . , 2009 ) .\",\n \"AMOT depletion also significantly reduced both HIV-1 release and infectivity ( Figure 5 , right panels , compare lane 4 to lane 1 , eightfold reduction in infectivity and 2 . 5-fold reduction in release ) .\",\n \"The magnitudes of these effects were intermediate between those seen for depletion of TSG101 ( 20-fold reduction in infectivity and threefold reduction in virion release ) and ALIX ( 1 . 5-fold reduction in infectivity and insignificant reduction in virion release ) .\",\n \"Importantly , the detrimental effects of AMOT p130 depletion were almost completely rescued by re-expression of wild type AMOT p130 ( compare lanes 1 , 4 and 5 ) but not by the mutant AMOT p130ΔPPXY protein ( lane 6 ) .\",\n \"These experiments demonstrate that AMOT p130 is required for efficient release of wild type HIV-1 from 293T cells and mutations that abolish NEDD4L binding block the ability of AMOT p130 to function in virus release . 10 . 7554/eLife . 03778 . 014Figure 5 . AMOT p130 is required for efficient release of wild type HIV-1 . Left panels are western blots showing 293T cellular levels of endogenous ALIX ( panel 1 , anti-ALIX ) , TSG101 ( panel 2 , anti-TSG101 ) , AMOT ( panel 3 , anti-AMOT ) , GAPDH ( panel 4 , anti-GAPDH , loading control ) and levels of HIV-1 Gag proteins ( panel 5 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with a control siRNA ( lane\",\n \"1 ) or with an siRNA that depleted ALIX ( lane 2 ) , TSG101 ( lane\",\n \"3 ) or AMOT p130 and p80 ( lanes 4–6 ) , with expression vectors for wild type HIV-1 ( lanes 1–6 ) and with an siRNA-resistant expression constructs for HA-AMOT p130 proteins ( with wild type HA-AMOT p130 in lane 5 and an HA-AMOT p130ΔPPXY protein with inactivating point mutations in all three PPXY motifs in lane 6 ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 014 AMOT mRNA is expressed in 293T cells , as well as in white blood cells and lymphoid tissues that are the natural hosts for HIV-1 infection ( Moleirinho et al . , 2014 ) .\",\n \"In contrast , HeLa cells reportedly express little or no AMOT mRNA , although they do express AMOTL2 mRNA ( Moleirinho et al . , 2014 ) .\",\n \"We confirmed that endogenous AMOT protein levels are at least 10-fold lower in HeLa cells than in 293T cells ( Figure 6—figure supplement 1 ) .\",\n \"This observation begs the question of how HIV-1 can be released from a cell type that lacks AMOT , and this issue is particularly relevant because HeLa cells are commonly used to study HIV-1 assembly .\",\n \"We therefore tested the effects of AMOT p130 expression on HIV-1 release and infectivity in HeLa cells .\",\n \"As shown in Figure 6A , AMOT p130 expression increased HIV-1 release from HeLa cells in a dose-dependent fashion , up to 15-fold at the highest levels of AMOT tested ( right panel 1 , compare lanes 1 and 5 ) .\",\n \"Viral titers similarly increased with AMOT expression in a dose-dependent fashion , although the overall effects were less dramatic ( right panel 2 , compare lanes 1–5 , threefold infectivity increase ) .\",\n \"Thus , HIV-1 release from wild type HeLa cells is suboptimal , and can be enhanced by expression of AMOT p130 . 10 . 7554/eLife . 03778 . 015Figure 6 . AMOT p130 stimulates release of HIV-1 from HeLa and Jurkat T cells .\",\n \"( A ) Left panels are western blots showing HeLa cellular levels of endogenous AMOT p130 and exogenous HA-AMOT p130 ( panel 1 , anti-AMOT ) , endogenous GAPDH ( panel 2 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 3 , anti-MA and anti-CA ) .\",\n \"HeLa cells were co-transfected with expression vectors for HIV-1 ( lanes 1–5 ) , and with increasing concentrations of an expression construct for HA-AMOT p130 ( 0–4 μg DNA in lanes 1–5 ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 .\",\n \"( B ) Levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) .\",\n \"Jurkat T cells were co-transfected with expression vectors for HIV-1 ( lanes 1–5 ) , together with a control vector ( lane\",\n \"1 ) or expression vectors for wild type HA-AMOT p130 ( lanes 2 and 3 , 5 and 10 µg DNA respectively ) or an HA-AMOT p130ΔPPXY protein with inactivating point mutations in the three PPXY motifs ( lanes 4 and 5 , 5 and 10 µg DNA respectively ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01510 . 7554/eLife . 03778 . 016Figure 6—figure supplement 1 . HeLa cells express little or no AMOT . Western blots showing endogenous AMOT p130 and p80 expression levels in 293T cells ( lanes 1–4 ) and in HeLa cells ( lanes 5–8 ) .\",\n \"Increasing numbers of cell equivalents were added as indicated ( ranging from 1 . 5 × 105 cells in lanes 1 and 5 to 1 . 2 × 106 cells in lanes 4 and 8 ) .\",\n \"Upper and lower panels show lighter and darker exposures of the same blot .\",\n \"Image is representative of two independent repetitions of this experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 016 T cells are natural hosts for HIV-1 infection , and AMOT mRNA is present in these cells , including Jurkat T cells ( Yeung et al . , 2009; Sheynkman et al . , 2013 ) .\",\n \"To determine whether AMOT p130 can function in virus release from Jurkat T cells , we tested whether expressing exogenous AMOT p130 expression stimulated HIV-1 release and infectivity .\",\n \"As shown in Figure 6B , AMOT p130 expression increased HIV-1 release from Jurkat T cells in a dose-dependent fashion , with up to 16-fold stimulation observed at the highest levels of AMOT p130 tested ( panel 1 , compare lanes 1–3 ) .\",\n \"Viral titers similarly increased in a dose-dependent fashion ( panel 2 , compare lanes 1–3 , sevenfold infectivity increase ) .\",\n \"Western blots confirmed that virus expression levels were the same in all cases ( not shown ) , but AMOT p130 expression levels were below our detection limits in this cell type .\",\n \"We therefore also tested the effects of overexpressing the mutant AMOT p130ΔPPXY protein .\",\n \"Unlike wild type AMOT p130 , AMOT p130ΔPPXY failed to stimulate virus release , and actually reproducibly decreased viral titers as compared to the untreated control ( compare lanes 4 and 5 to lane 1 ) .\",\n \"We speculate that this effect again reflects dominant inhibition of endogenous AMOT p130 .\",\n \"Regardless , our data indicate that AMOT p130 also stimulates virus release and infectivity from T cells , which are natural targets of HIV-1 infection .\",\n \"Consistent with this conclusion , a global shRNA screen for host cofactors revealed that Jurkat T cells transduced with an shRNA that targeted AMOT were protected against HIV cytotoxicity ( Yeung et al . , 2009 ) .\",\n \"This observation implies that AMOT contributes to HIV-1 replication in cultured Jurkat T cells , although the role of AMOT in HIV-1 replication was not characterized further in that study .\",\n \"( Yeung et al . , 2009 ) .\",\n \"siRNA depletion/rescue experiments were performed to test whether other motin family members can substitute for AMOT p130 in supporting release of wild type HIV-1 ( Figure 7 ) .\",\n \"In control experiments , HIV-1 release and infectivity were again decreased by depletion of endogenous AMOT from 293T cells ( right panels , compare lanes 1 and 2 , fivefold infectivity reduction ) .\",\n \"As expected , these effects were largely rescued by expression of exogenous siRNA-resistant AMOT p130 ( right panels , compare lanes 2 and 3 ) .\",\n \"Expression of either AMOTL1 or AMOTL2 also significantly rescued HIV-1 release and infectivity ( right panels , compare lanes 4 and 5 to lanes 2 and 3 ) .\",\n \"Hence , both AMOT-like proteins can facilitate HIV-1 release in the absence of endogenous AMOT p130 .\",\n \"Neither AMOTL1 nor AMOTL2 was quite as effective as AMOT p130 in rescuing HIV-1 infectivity in this assay , possibly because these proteins are intrinsically less active than AMOT p130 and/or because they were expressed at lower levels ( left panel 2 , compare lanes 3–5 ) . 10 . 7554/eLife . 03778 . 017Figure 7 . AMOTL1 and AMOTL2 can substitute for AMOT p130 in HIV-1 release . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 proteins ( panel 2 , anti-HA ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with a control siRNA ( lane\",\n \"1 ) or with an siRNA that depleted AMOT p130 and AMOT p80 ( lanes 2–5 ) , and with expression vectors for HIV-1 ( lanes 1–5 ) , with siRNA-resistant expression constructs for AMOT p130 ( lane 3 ) , AMOTL1 ( lane 4 ) or AMOTL2 ( lane 5 ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01710 . 7554/eLife . 03778 . 018Figure 7—figure supplement 1 . AMOTL2 can stimulate NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP and rescue HIV-1ΔPTAP , ΔYP release from cells depleted of endogenous AMOT .\",\n \"( A ) AMOTL2 stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP .\",\n \"Left panels are western blots showing 293T cellular levels of exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 proteins ( panel 1 , anti-HA ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with expression vectors for HIV-1ΔPTAP/ΔYP ( lanes 1–8 ) , FLAG-NEDD4L ( lanes 5–8 ) , and either HA-AMOT p130 ( lanes 2 and 6 ) , HA-AMOTL1 ( lanes 3 and\",\n \"7 ) or HA-AMOTL2 ( lanes 4 and 8 ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel ) and viral titers ( lower panel ) , n = 3 .\",\n \"( B ) AMOTL2 can rescue HIV-1ΔPTAP , ΔYP release from 293T cells depleted of endogenous AMOT .\",\n \"Left panels are western blots showing cellular levels of endogenous AMOT and exogenous HA-AMOT proteins ( panel 1 , anti-AMOT ) , exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 ( panel 2 , anti-HA ) , exogenous FLAG-NEDD4L ( panel 3 , anti-FLAG ) , GAPDH ( panel 4 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 5 , anti-MA and anti-CA ) .\",\n \"Cells were co-transfected with a control siRNA ( lanes 1 and\",\n \"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–7 ) , with expression vectors for HIV-1 ( lanes 1–7 ) , FLAG-NEDD4L ( lanes 3–7 ) , and either an siRNA-resistant HA-AMOT p130 expression construct ( lane 5 ) , or expression constructs for HA-AMOTL1 ( lane 6 ) , or HA-AMOTL2 ( lane 7 ) .\",\n \"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 018 We also tested whether the other AMOT family members could cooperate with NEDD4L in stimulating release of the crippled HIV-1ΔPTAP , ΔYP virus ( Figure 7—figure supplement 1A ) and whether AMOTL1 and AMOTL2 could rescue the block to NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release imposed by depletion of endogenous AMOT ( Figure 7—figure supplement 1B ) .\",\n \"AMOTL2 strongly stimulated/rescued HIV-1ΔPTAP , ΔYP release in both of these assays , albeit slightly less efficiently than AMOT p130 ( Figure 7—figure supplement 1A , compare lane 8 to lanes 5 and 6 and Figure 7—figure supplement 1B , compare lane 7 to lanes 4 and 5 ) .\",\n \"In contrast , AMOTL1 exhibited little or no activity in either assay ( Figure 7—figure supplement 1A , compare lane 7 to lanes 5 and 6 and Figure 7—figure supplement 1B , compare lane 6 to lanes 4 and 5 ) .\",\n \"Thus , AMOTL2 could substitute for AMOT p130 in three different virus release assays , whereas AMOTL1 was slightly less active than AMOT in the release of wild type HIV-1 , and failed to stimulate release of the ‘crippled’ HIV-1ΔPTAP , ΔYP construct .\",\n \"Scanning and transmission electron microscopy ( SEM and TEM , respectively ) were used to visualize the stage at which AMOT depletion inhibited HIV-1 assembly or release ( Figures 8 , 9 ) .\",\n \"SEM imaging was initially performed because this method can survey the entire cell surface and thereby identify global changes in virion assembly and budding profiles .\",\n \"SEM analyses were performed on 293T cells that expressed HIV-1 and were treated with either: ( 1 ) a control siRNA , ( 2 ) an siRNA that depleted TSG101 ( positive control ) , or ( 3 ) an siRNA that depleted AMOT .\",\n \"As expected , viruses budding from control siRNA-treated cells were detectable but rare ( e . g . , Figure 8A–C ) .\",\n \"In contrast , large numbers of assembling virions were visible on the surfaces of cells that lacked TSG101 ( Figure 8D–F ) or that lacked AMOT ( Figure 8G–I ) .\",\n \"We also observed that the arrested virions tended to cluster in the center of cells lacking AMOT , whereas they were more evenly distributed across the surfaces of cells lacking TSG101 .\",\n \"The dramatic increase in the numbers of arrested budding virions implies that AMOT , like TSG101 , must function following the initial stages of Gag assembly , but prior to viral membrane fission . 10 . 7554/eLife . 03778 . 019Figure 8 . AMOT p130 is required for HIV-1 assembly/budding . Scanning electron microscopic ( SEM ) images of HIV virions budding from 293T cells depleted with a control siRNA ( A–C ) , depleted of TSG101 ( D–F ) or depleted of AMOT ( G–I ) .\",\n \"Successive images across each row show expansions of the adjacent boxed regions , and scale bars are 500 nm in all cases .\",\n \"Arrows in panel ( C ) highlight budding wild type HIV-1 virions . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01910 . 7554/eLife . 03778 . 020Figure 9 . AMOT p130 is required for an early stage of HIV-1 assembly/budding .\",\n \"( A ) Transmission electron microscopic ( TEM ) images of HIV virions budding from 293T cells depleted of TSG101 .\",\n \"Panel 1: wide view .\",\n \"Panel 2: expanded view .\",\n \"Scale bars here and in part B are 100 nm .\",\n \"( B ) TEM images of HIV virions budding from 293T cells depleted of AMOT p130 and p80 .\",\n \"Panel 1: wide view .\",\n \"Panel 2: expanded view .\",\n \"( C ) Quantification of the release and maturation status of HIV-1 virions associated with cells that were treated with a control siRNA ( black bars ) , depleted of TSG101 ( red ) , depleted of AMOT ( teal ) , or depleted of AMOT and re-expressing AMOT p130 from an siRNA-resistant construct ( light blue ) .\",\n \"This experiment was repeated twice with similar results , and error bars were derived by quantifying three separate sets of >100 virions each from one of the experiments .\",\n \"( D ) Quantification of the completeness of virions budding from cells treated with a control siRNA ( black triangles ) , depleted of TSG101 ( red squares ) , or depleted of AMOT ( teal diamonds ) .\",\n \"The extent of the Gag shell arc ( in degrees ) was measured from TEM images of 500 budding virions of each type , the measurements were binned into the intervals shown below the x-axis , and the virion numbers in each bin are shown in the plot . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 020 TEM experiments were performed to characterize how virus assembly/budding arrested in the absence of AMOT .\",\n \"These experiments again visualized HIV-1 budding from 293T cells treated with a control siRNA , an siRNA that depleted TSG101 ( positive control ) , an siRNA that depleted AMOT , or an siRNA that depleted endogenous AMOT together with an siRNA-resistant construct that re-expressed wild type AMOT p130 ( rescue control ) .\",\n \"Culture supernatants were removed from the virus producing cells and the adherent cells were fixed while still attached to the culture dish in order to maximize the numbers of cell-associated virions .\",\n \"Fixed cells were harvested , embedded in resin , thin sectioned , stained , and visualized by TEM .\",\n \"Cell-associated virions observed in these experiments were scored as being either: ( 1 ) mature ( discernable conical or condensed cores ) , ( 2 ) immature ( discernable immature Gag shells ) , or ( 3 ) budding ( contiguous cellular and viral membranes ) .\",\n \"HIV-1 virions associated with wild type cells were usually mature ( 76 ± 5% , see Figure 9C ) , but immature and budding virions were also observed ( 9 ± 3% and 15 ± 5% , respectively ) .\",\n \"As expected , TSG101 depletion induced a strong virus budding defect , with the majority of detected virions arrested at the budding stage ( 67 ± 7% , shown in Figure 9A and quantified in Figure 9C ) .\",\n \"In addition , the ratio of mature ( 10 ± 6% ) to immature ( 23 ± 2% ) virions ( 1:2 . 3 ) was much lower than in the control case ( 1:0 . 1 ) , consistent with previous reports that TSG101 is also required for efficient Gag processing and maturation ( Göttlinger et al . , 1991; Garrus et al . , 2001; Martin-Serrano et al . , 2003 ) .\",\n \"AMOT depletion also inhibited virus release , as reflected in a large increase in the percentage of virions detected at the budding stage ( 72 ± 6% , shown in Figure 9B and quantified in Figure 9C ) .\",\n \"In this case , however , the virions that were released exhibited a normal ratio of mature:immature virions ( 25 ± 5% vs 3 ± 2%; 1:0 . 1 ) .\",\n \"Importantly , the budding defect seen in cells lacking endogenous AMOT was almost completely rescued by re-expression of the siRNA-resistant AMOT p130 ( Figure 9C ) , so that the majority of virions were now mature ( 70 ± 5% vs 76 ± 5% in the control ) , and budding virion levels were nearly normal ( 26 ± 9% vs 15 ± 5% in the control ) .\",\n \"Thus , AMOT depletion blocks virus release at the assembly/budding stage , but does not inhibit Gag processing or virion maturation .\",\n \"It was also striking that AMOT depletion caused virion assembly/budding to arrest at an earlier stage than TSG101 depletion .\",\n \"This difference can be seen in the EM images shown in Figure 9A , B , where most of the virions in TSG101-depleted cells exhibit the characteristic ‘lollipop’ morphology associated with a late assembly/membrane fission defect ( Figure 9A ) , whereas most of the virions budding from AMOT-depleted cells exhibit a ‘half-moon’ morphology in which the nascent viral membrane and Gag shell are incomplete ( Figure 9B ) .\",\n \"This observation was quantified by imaging 500 budding HIV-1 virions from: ( 1 ) wild type cells ( control ) , ( 2 ) cells depleted of TSG101 , and ( 3 ) cells depleted of AMOT .\",\n \"The arc formed by the Gag shell was measured manually for each budding virion , and the 500 measurements for each condition were then binned in 15° intervals and displayed graphically ( Figure 9D ) .\",\n \"The Gag arc distribution of control virions budding from wild type cells exhibited two similarly sized peaks; one in which the Gag shells covered an arc of ∼155° , and another in which the Gag shells covered an arc of ∼320° .\",\n \"These measurements indicate that virion assembly intermediates ( or possibly dead-end products ) appreciably accumulate at two different stages , suggesting that either stage can be rate limiting .\",\n \"In cells lacking TSG101 , budding-arrested virions were much more prevalent and they typically arrested at very late stages of assembly , with the maximal population exhibiting a Gag shell arc of ∼320° .\",\n \"In cells lacking AMOT , arrested virions were again prevalent but they arrested at a much earlier stage , with the maximal population exhibiting a Gag shell arc of ∼165° .\",\n \"The dramatic increases in numbers of budding virions induced by depletion of TSG101 or AMOT can explain how the assembly phenotypes could distribute as single peaks in these cases , whereas virions budding from wild type cells ( which were much rarer ) distributed into two peaks .\",\n \"Our experiments reveal that HIV budding arrests at an earlier stage in the absence of AMOT than in the absence of TSG101 , implying that AMOT acts at an earlier stage of virion morphogenesis .\"\n ],\n [\n \"We have identified Angiomotin ( AMOT ) as a host protein required for efficient HIV-1 release .\",\n \"In the absence of AMOT , most virions arrested at a ‘half-moon’ stage in which a hemisphere of assembled Gag molecules distended the membrane , but did not form a spherical particle ( Figure 9 ) .\",\n \"The AMOT depletion phenotype was distinct from the defect induced by the absence of ESCRT factors , where virus budding arrested at a ‘lollipop’ stage in which nearly complete immature virions remained tethered to the host cell via thin membrane stalks .\",\n \"Thus , cellular factors facilitate ( at least ) two distinct steps in virion morphogenesis , with AMOT required to complete assembly/envelopment and the ESCRT machinery required for membrane fission .\",\n \"HIV-1 virions budding from control cells accumulated at both stages , suggesting that both steps can be kinetic bottlenecks ( see Figure 9D and reference Ku et al . , 2013 ) .\",\n \"The Gag lattice organizes the virion and undoubtedly helps to drive membrane envelopment because pure recombinant Gag proteins can form spherical particles in vitro ( Campbell et al . , 2001 ) .\",\n \"In cells , however , Gag appears to require additional activities to bend the membrane , extrude the particle , and sever the bud neck .\",\n \"The well-studied process of endocytic vesicle formation provides precedence for these additional requirements because clathrin alone can form spherical assemblies but nevertheless requires assistance from BAR domain-containing proteins to help bend membranes , and assistance from actin and dynamin to release the vesicle ( Weinberg and Drubin , 2012 ) .\",\n \"It is intriguing that AMOT p130 also contains a candidate BAR-like domain ( Heller et al . , 2010 ) and can associate with F-actin ( Ernkvist et al . , 2006 , 2008; Chan et al . , 2013 ) .\",\n \"The AMOT BAR-like domain can remodel membranes in vitro ( Heller et al . , 2010 ) , although it is not yet clear whether it functions as a classical or an inverse BAR domain .\",\n \"This distinction is mechanistically significant because classical BAR domains stabilize positive membrane curvature whereas inverse BAR domains stabilize negative curvature ( Mim and Unger , 2012 ) , both of which are generated during enveloped virus assembly .\",\n \"In principle , the BAR domain could mediate AMOT p130 recruitment at the half-moon stage , once the nascent viral membrane has reached the proper degree of curvature .\",\n \"The BAR domain could also help drive the additional membrane curvature required to proceed to the lollipop stage .\",\n \"Similarly , F-actin recruitment and polymerization could help ‘push’ the virion toward the lollipop stage , although several recent studies argue strongly against an essential role for F-actin in HIV-1 assembly ( Bharat et al . , 2014; Rahman et al . , 2014 ) .\",\n \"Our experiments indicate that another important AMOT p130 function is to recruit NEDD4L to sites of virus budding .\",\n \"In support of this idea , we have shown that: ( 1 ) the two proteins bind one another directly through interactions between the NEDD4L WW domains and PPXY elements present in the unique N-terminal region of the AMOT p130 isoform ( Figures 1 , 2 and references Wang et al . , 2012; Moleirinho et al . , 2014 ) , ( 2 ) AMOT p130 can recruit NEDD4L to cellular membranes , and this activity requires the NEDD4L WW-AMOT p130 PPXY interactions ( [Heller et al . , 2010] and our data not shown ) , ( 3 ) NEDD4L cannot stimulate release of ‘crippled’ HIV-1 constructs in the absence of AMOT ( Figure 4 ) , ( 4 ) NEDD4L mutants that cannot bind AMOT are also impaired in their ability to stimulate virus budding ( Figure 5 ) , ( 5 ) AMOT p80 and AMOT p130 PPXY mutants that cannot bind NEDD4L do not support virus budding ( Figures 3–6 ) and ( 6 ) recombinant AMOT p130 and HIV-1 Gag proteins can bind one another directly , albeit weakly in vitro ( Figure 2B ) , and could bind more robustly in cells owing to avidity and membrane topology effects .\",\n \"Previous studies indicate that other retroviruses also employ distinct host factors to promote membrane envelopment and fission .\",\n \"The clearest example is the D-type Betaretrovirus Mason-Pfizer Monkey Virus ( M-PMV ) , which forms spherical particles in the cytoplasm and then obtains its outer membrane and exits the cell by crossing the plasma membrane .\",\n \"M-PMV Gag contains two distinct late assembly domains that function non-redundantly: a PPPY motif that binds NEDD4 ( and perhaps other NEDD4 family members ) , and a PSAP motif that binds TSG101/ESCRT-I ( Gottwein et al . , 2003 ) .\",\n \"PPPY and PSAP mutations induce strikingly different phenotypes: mutation of the PPPY motif causes virions to stack up against the plasma membrane where they fail to initiate membrane envelopment whereas virions lacking the PSAP motif acquire an envelope , but accumulate at the lollipop stage or are released as chains of membrane-tethered particles .\",\n \"These observations imply that Gag PPPY-NEDD4 complexes are required for early stages of membrane bending and envelopment , whereas Gag PSAP-ESCRT-I complexes recruit the ESCRT machinery to mediate membrane fission .\",\n \"Like other family members , NEDD4 binds AMOT ( Moleirinho et al . , 2014 ) , and we therefore speculate that M-PMV may also recruit AMOT to facilitate membrane bending and particle envelopment .\",\n \"This would imply that viruses can recruit NEDD4-motin complexes by binding either member of the complex .\",\n \"The deltaretrovirus HTLV-I is another case where a PPXY motif appears to be required for an early step in particle envelopment and a PTAP motif may function at a later membrane fission step ( [Le Blanc et al . , 2002; Blot et al . , 2004; Heidecker et al . , 2004] , but also see reference [Wang et al . , 2004] ) .\",\n \"Distinct roles for PPXY and PTAP motifs have not been delineated in other well-studied viruses that use both motifs , such as the gammaretrovirus Murine Leukemia Virus ( Yuan et al . , 2000 ) or the filovirus Ebola Virus ( Timmins et al . , 2003 ) .\",\n \"Although many details of HIV-1 assembly and budding remain to be elucidated , our data are consistent with a stepwise pathway in which: ( 1 ) AMOT p130 is recruited at the half-moon stage of HIV-1 assembly , perhaps by binding cooperatively to Gag and to appropriately curved membranes , ( 2 ) AMOT p130 promotes further membrane curvature and recruits NEDD4L via PPXY-WW domain interactions , ( 3 ) NEDD4L builds Lys-63-linked ubiquitin chains on the viral Gag protein ( and/or other nearby substrates ) , ( 4 ) the juxtaposition of Gag late assembly domains and Lys-63-linked Ub chains creates high affinity binding sites for the early-acting ESCRT factors , TSG101 and ALIX , and ( 5 ) these factors then recruit the downstream ESCRT-III and VPS4 components required to effect membrane scission .\",\n \"This model is attractive because: ( 1 ) our work shows that AMOT functions upstream of the ESCRT pathway , ( 2 ) AMOT appears to function as a NEDD4L adaptor ( see above ) , ( 3 ) ubiquitin transfer is required for efficient HIV-1 release ( Martin-Serrano , 2007 ) , ( 4 ) NEDD4L builds Lys-63-linked ubiquitin chains and can use HIV-1 Gag as a substrate ( Weiss et al . , 2010; Sette et al . , 2013 ) , and ( 5 ) both TSG101/ESCRT-I and ALIX have ubiquitin binding activities .\",\n \"The ALIX activity is specific for Lys-63-linked ubiquitin chains ( Dowlatshahi et al . , 2012; Keren-Kaplan et al . , 2013; Pashkova et al . , 2013 ) , and ESCRT-I complexes also have multiple ubiquitin binding sites , although linkage specificity has not been observed ( Stefani et al . , 2011; Agromayor et al . , 2012; McCullough et al . , 2013 ) .\",\n \"Cooperative binding to the late domains and ubiquitin could explain how ALIX and TSG101/ESCRT-I can be efficiently recruited , despite making intrinsically weak interactions with the p6Gag late assembly domains .\",\n \"The proposed sequence of events could also provide a ‘timing’ mechanism for recruiting ( or activating ) these early-acting ESCRT proteins to function at a late stage of virion assembly , although the precise timing of early-acting ESCRT recruitment remains to be clarified because published reports differ on this issue ( Jouvenet et al . , 2011; Ku et al . , 2014 ) .\",\n \"All enveloped viruses must bend and remodel membranes as they bud , and AMOT requirements may therefore be quite general .\",\n \"Indeed , AMOTL1 has been shown to bind the M protein of PIV5 and to be required for the efficient , ubiquitin-dependent release of this virus ( Pei et al . , 2010 ) .\",\n \"Motin family members therefore contribute to the assembly of at least two highly diverged enveloped viruses; the paramyxovirus PIV5 and the lentivirus HIV-1 .\",\n \"This suggests that AMOT proteins , like the ESCRT machinery , may provide general activities required for the assembly and release of a variety of different enveloped viruses .\"\n ],\n [\n \"Expression constructs , siRNA sequences , and antibodies are described in detail in Supplementary file 1 .\",\n \"Constructs for expressing wild type and mutant NEDD4L and AMOT proteins in E . coli , insect SF-9 cells , and human 293T cells were created by standard cloning and mutagenesis methods , with detailed methods available upon request .\",\n \"All of the new expression constructs used in our studies have been deposited in the public DNASU plasmid repository ( http://dnasu . org/DNASU/Home . jsp ) .\",\n \"For the experiment shown in Figure 1B , an empty vector control or a pCAG-OSF-NEDD4L expression vector ( Morita et al . , 2007 ) expressing NEDD4L with an N-terminal One-STrEP-FLAG ( OSF ) affinity tag was transfected into 293T cells ( calcium phosphate method , 2 × 10 cm plates each , 25 μg DNA/plate ) .\",\n \"After 48 hr , cells from each sample were collected by scraping , pooled , washed three times with PBS ( 1 . 5 mM KH2PO4 , 155 mM NaCl , 2 . 7 mM Na2HPO4 , pH 7 . 4 ) , and lysed with 500 μl Triton lysis buffer ( 1% Triton X-100 , 50 mM Tris pH 8 . 0 , 150 mM NaCl , 150 μg/ml PMSF ) .\",\n \"This , and all subsequent preparation steps , were performed at 4°C .\",\n \"Soluble extracts were pre-incubated for 30 min with 5 μl Protein-A resin slurry ( Millipore , Temecula , CA ) to remove non-specific binding proteins .\",\n \"Supernatants were collected and incubated for 2 hr with 10 μl STrEP-Tactin resin ( IBA-Lifesciences , Göttingen , Germany ) .\",\n \"The resins were washed three times with 500 μl washing buffer ( 0 . 1% Triton-X100 , 50 mM Tris , 150 mM NaCl , pH 8 . 0 ) , resuspended in 35 μl SDS-PAGE loading buffer ( 2% SDS , 10% glycerol , 2% 2-mercaptoethanol ( BME ) , 0 . 002% bromophenol blue and 62 . 5 mM Tris , pH 6 . 8 ) , and boiled to release the bound proteins .\",\n \"12 μl samples of each solution were electrophoresed ( 10% SDS-PAGE ) and proteins were visualized by staining with Coomassie brilliant blue .\",\n \"This procedure was repeated in three independent experiments , and in each case the protein band corresponding to AMOT p130 was visible in the Coomassie-stained gel ( Figure 1B ) .\",\n \"This band was excised ( and the equivalent region from the control lane was also excised in one of the repetitions ) , de-stained in 50% methanol with 50 mM ammonium bicarbonate ( 2 × 1 ml , 1 hr , 23°C , gentle vortexing ) , re-hydrated in 50 mM ammonium bicarbonate ( 1 ml , 30 min , 23°C ) , and cut into several pieces .\",\n \"Each piece was dehydrated in acetonitrile ( 1 ml , 30 min , 23°C , gentle shaking ) , and the excess acetonitrile was removed .\",\n \"Proteins were in-gel digested using 10–20 μl sequence-grade modified trypsin ( Promega , Madison , WI , 20 ng/μl ) in 50 mM ammonium bicarbonate ( 16 hr , 37°C ) , and the reaction was quenched by the addition of 20 μl 1% formic acid .\",\n \"The solution was removed and the gel washed twice with 50% acetonitrile in 1% formic acid ( 20 μl , 20 min , 37°C , with sonication ) and once with 100% acetonitrile ( 20 min , 37°C , with sonication ) .\",\n \"The wash solutions were combined , dried , and reconstituted in 100 μl 5% acetonitrile with 0 . 1% formic acid for LC/MS/MS analysis .\",\n \"Peptides were analyzed using a nano-LC/MS/MS system comprising a nano-LC pump ( 2D-ultra , Eksigent ) and an LTQ-FT mass spectrometer ( ThermoElectron Corporation , San Jose , CA ) , equipped with a nanospray ion source ( ThermoElectron Corporation ) .\",\n \"5–20 fmoles of each tryptic digest were injected onto a homemade C18 nanobore LC column ( C18 [Atlantis , Waters Corporation , Milford , MA] ) ; 3 μm particle; column ( 75 μm ID × 100 mm length ) , and eluted using a linear gradient of 5–70% solvent B in 78 min ( solvent B: 80% acetonitrile with 0 . 1% formic acid; solvent A: 5% acetonitrile with 0 . 1% formic acid , 350 nl/min ) .\",\n \"The LTQ-FT mass spectrometer was operated in the data-dependent acquisition mode ( Xcalibur 1 . 4 software ) with the 10 most intense peaks in each FT primary scan determined on-the-fly and trapped for MS/MS analysis and CID peptide fragmentation/sequencing in the LTQ linear ion trap .\",\n \"Spectra in the FT-ICR were acquired from m/z 350 to 1400 at 50 , 000 resolution ( ∼2 ppm mass accuracy ) with the following LTQ linear ion trap parameters: precursor activation time 30 ms and activation Q at 0 . 25; collision energy at 35%; dynamic exclusion width at 0 . 1 Da–2 . 1 Da , with one repeat count and 10 s duration .\",\n \"Raw LTQ FT MS data files were converted to peak lists with BioworksBrowser 3 . 2 software ( ThermoElectron Corporation ) using the following processing parameters: precursor mass 351–5500 Da; grouping enabled , 5 intermediate MS/MS scans; precursor mass tolerance 5 ppm , minimum ion count in MS/MS = 15 , and minimum group count = 1 .\",\n \"Resulting DTA files were merged , and searched to identify peptides/proteins against NCBInr using the MASCOT search engine ( Matrix Science Ltd . ; version 2 . 2 . 1 , Boston , MA ) .\",\n \"Searches were done with tryptic specificity , allowing two missed cleavages , or ‘non-specific cleavage’ and a mass error tolerance of 5 ppm in MS spectra ( i . e . , FT-ICR data ) and 0 . 5 Da for MS/MS ions ( i . e . , LTQ linear ion trap ) .\",\n \"Identified peptides were accepted when the MASCOT ion score value exceeded 20 .\",\n \"In all three repetitions , at least two peptides corresponding to AMOT p130 were identified in the tryptic digest , and the maximal coverage was 33% ( see Figure 1—figure supplement 1 ) .\",\n \"To assay OSF-NEDD4L protein binding to endogenous AMOT p130 ( Figure 1C ) , 293T cells ( 2 × 106 cells per 10 cm plate ) were transfected using Lipofectamine 2000 ( Invitrogen/Life Technologies , Carlsbad , CA ) with either 3 . 5 μg of an empty pCAG-OSF expression vector ( control ) or with pCAG-OSF vectors that expressed the designated OSF-NEDD4L proteins , using 3 . 5 μg of expression constructs for wild type NEDD4L or NEDD4LΔWW , or 2 μg each of expression constructs for NEDD4LC942A or NEDD4LΔWW/C942A .\",\n \"In this case , and in all other experiments , empty vector was added to keep total DNA levels constant ( i . e . , 1 . 5 μg empty pCAG-OSF vector in the case of NEDD4LC942A or NEDD4LΔWW/C942A ) .\",\n \"48 hr post transfection , cells were washed in PBS buffer and lysed ( 25 mM Tris , 150 mM NaCl , 1 mM EDTA , 0 . 5% CHAPS , pH 7 . 5 , 10 min , 4°C ) .\",\n \"Soluble lysates were collected by centrifugation ( 5000×g , 5 min , 4°C ) and incubated with 50 μl STrEP-Tactin resin ( IBA-Lifesciences , Göttingen , Germany , 3 hr , 4°C ) equilibrated with lysis buffer .\",\n \"The resin was washed ( 25 mM Tris-HCl , 150 mM NaCl , 1 mM EDTA , 0 . 1% CHAPS , pH 7 . 5 , 3 × 1 ml ) , resuspended in 50 μl 2× SDS-PAGE loading buffer , boiled , and the released proteins were analyzed by SDS-PAGE and western blotting .\",\n \"Separated proteins were transferred to PVDF membranes , blocked with 5% non-fat dry milk for 45 min at RT , and incubated overnight at 4°C with primary antibodies ( see Supplementary file 1 ) .\",\n \"Secondary antibodies were anti-rabbit IgG or anti-mouse IgG conjugated to IRdye800 or IRdye700 , respectively ( 1:10 , 000 and 1:20 , 000 respectively , Rockland Immunochemicals Inc . , Gilbertsville , PA ) .\",\n \"All western blots were visualized using an Odyssey scanner ( Li-Cor Biosciences , Lincoln , NB ) .\",\n \"To assay OSF-NEDD4L protein binding to exogenous HA-AMOT p130 proteins ( Figure 1—figure supplement 2 ) , 293T cells ( 2 × 106 cells per 10 cm plate ) were co-transfected using Lipofectamine 2000 ( Invitrogen/Life Technologies ) with 3 . 5 μg of a pCAG-OSF vector expressing wild type OSF-NEDD4L and 3 . 5 μg of an empty pCAG vector ( control ) , or pcDNA vectors expressing HA-AMOT p130 or HA-AMOT p130ΔPPXY .\",\n \"Thereafter , the co-immunoprecipitation protocol was identical to the one described above .\",\n \"Proteins were expressed in the Rosetta-pLysS bacterial strain grown in auto-induction media ZYP-5052 ( Studier , 2005 ) .\",\n \"For the experiments described in Figure 2 and Figure 2—figure supplement 1 , SF9 cells ( 2 l ) were infected at an MOI of 5 with Bac-to-Bac baculoviruses expressing OSF-tagged GFP ( control ) , AMOT p130 , AMOTL1 or AMOTL2 .\",\n \"Pellets from 200 ml of culture were lysed in 10 ml of lysis buffer ( 1% NP-40 , 150 mM NaCl , 20 mM Tris pH 8 . 0 , 0 . 5 mM EDTA supplemented with protease inhibitors ) .\",\n \"All steps were performed at 4°C .\",\n \"Supernatants were collected by centrifugation for 30 min at 13 , 000 RPM in a microcentrifuge and 0 . 5 ml of each lysate was incubated with 30 μl of a slurry of STrEP-Tactin resin ( 1 hr ) .\",\n \"Resins were washed with high salt wash buffer ( 20 mM Tris , 1 M LiCl , 0 . 5 mM EDTA , 0 . 1% NP-40 , pH 8 . 0 ) , followed by low salt buffer ( 20 mM Tris , 150 mM NaCl , 0 . 5 mM EDTA , 0 . 1% NP-40 , pH 8 . 0 ) .\",\n \"For NEDD4L binding experiments , the OSF-GFP or OSF-AMOT-bound resins were washed with binding buffer ( 150 mM NaCl , 50 mM Tris , 1 mM TCEP , 10 μM ZnCl2 , 0 . 1% NP-40 , pH 7 . 5 ) .\",\n \"Pure recombinant NEDD4L or NEDD4LΔWW proteins ( 0 . 5 μM in 500 μl binding buffer ) were pre-incubated with washed resins for 1 hr , and then incubated for 1 hr with GFP- or AMOT-bound resins .\",\n \"Resins were washed three times with 250 μl binding buffer , resuspended in 30 μl SDS-PAGE loading buffer , boiled , separated by SDS-PAGE , and visualized by Coomassie staining or by Western blotting ( with 1 μl samples used for Coomassie staining , and 1 μl samples of a 50-fold dilution used for Western blotting ) .\",\n \"For GagΔp6 binding experiments , the OSF-GFP ( control ) - or OSF-AMOT-bound resins were washed with binding buffer .\",\n \"Pure recombinant HIV-1 GagΔp6 ( 1 μM ) in 500 μl binding buffer was incubated with the washed resins for 1 hr .\",\n \"Resins were washed with binding buffer , and processed as described above .\",\n \"NEDD4L and AMOT protein family co-overexpression experiments shown in Figure 3 and Figure 3—figure supplement 1 , Figure 3—figure supplement 2 , and Figure 7—figure supplement 1A were performed following the protocol: 293T cells ( 4 × 105 cells/well in a 6-well plate ) were co-transfected ( Lipofectamine , 2000; Invitrogen/Life Technologies ) with 1 μg of an R9-based expression vector for the NL4-3 strain of HIV-1ΔPTAP , ΔYP ( Swingler et al . , 1997 ) , 0 . 5 μg of pCI-FLAG NEDD4L or NEDD4LΔWW ( Chung et al . , 2008 ) or pCI-FLAG empty vector in combination with either 0 . 5 μg of pCMV AMOT p80 ( Figure 3—figure supplement 2 ) or 1 μg pcDNA3 HA-AMOT p130 ( Figure 3 and Figure 3—figure supplement 2 ) or 0 , 0 . 25 , 0 . 5 , 0 . 75 , 1 , 1 . 25 or 1 . 5 μg pcDNA3 HA-AMOT p130 , ( Figure 3—figure supplement\",\n \"1 ) or 1 μg pcDNA3 HA AMOTL1 or 1 μg pcDNA3 HA AMOTL2 ( Figure 7—figure supplement 1A ) .\",\n \"48 hr post-transfection , media was harvested for titer measurements and virus purification , and cells were washed off the plate in PBS .\",\n \"Viral titers were assayed in HeLa-TZM indicator cells using a β-galactosidase assay , and following the manufacturer's instructions ( Promega , Madison , WI ) .\",\n \"Briefly , HeLa-TZM cells ( 5000 cells per well , 96-well plate ) were infected with four different dilutions of virus-containing culture media , harvested after 48 hr , washed once ( PBS , 4°C ) , and lysed in 25 mM Tris phosphate ( pH 7 . 8 ) , 2 mM DTT , 2 mM 1 , 2-diaminocyclohexane N , N , N′ , N′-tetraacetic acid ( DCTA ) , 10% glycerol and 1% Triton X-100 ( 10 min , 23°C ) .\",\n \"Cell lysates were incubated in 200 mM sodium phosphate buffer ( pH 7 . 3 ) , 2 mM MgCl2 , 100 mM BME and 1 . 33 mg/ml ortho-Nitrophenyl-β-galactoside for 60 min at 37°C .\",\n \"Reactions were terminated by addition of 200 μl 1 M Tris base and absorbance at 420 nm was read in a plate reader .\",\n \"For western blot analyses , virions were pelleted by centrifugation through a 20% sucrose cushion ( 90 min , 15 , 000×g , 4°C ) and resuspended in SDS-PAGE loading buffer .\",\n \"Cells were washed in PBS , lysed in RIPA buffer ( 10 mM Tris , 1 mM EDTA , 0 . 5 mM EGTA , 1% Triton X-100 , 0 . 1% sodium deoxycholate , 0 . 1% SDS , 140 mM NaCl , pH 8 . 0 ) supplemented with protease inhibitors ( cOmplete , Roche ) ( 10 min , 4°C ) , and the insoluble material was removed by centrifugation ( 10 min , 15 , 000×g , 4°C ) .\",\n \"Primary antibodies and dilutions used for western blots are given in Supplementary file 1 .\",\n \"Depletion/re-expression experiments ( Figure 4 , Figure 4—figure supplement 1 , Figure 4—figure supplement 2 , Figure 5 , Figure 7 , and Figure 7—figure supplement\",\n \"1 ) were performed following the protocol: t = 0: 293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were transfected with 10 nM siRNA using 7 . 5 μl Lipofectamine RNAiMax ( Invitrogen/Life Technologies ) ; t = 24 hr: media changed and second siRNA co-transfection with 10 nM siRNA and 0 . 5 μg wild type HIV-1 R9 expression vector or 1 μg HIV-1ΔPTAP , ΔYPXL expression vector and 0 . 5 μg of pCI-FLAG NEDD4L ( 10 μl Lipofectamine , 2000; Invitrogen/Life Technologies ) ; t = 72 hr: media and cells harvested for titer measurements and western blot analyses as described above .\",\n \"For the rescue experiments , siRNA-resistant expression constructs expressing AMOT p80 , AMOT p130 , AMOT p130ΔPPXY , AMOTL1 or AMOTL2 were co-transfected at t = 24 hr .\",\n \"AMOT mRNA is expressed in 293T cells and in white blood cells and lymphoid tissues , but cannot be detected in HeLa cells ( Moleirinho et al . , 2014 ) .\",\n \"Western blots were performed to compare AMOT protein expression levels in 293T vs HeLa cells ( Figure 6—figure supplement 1 ) .\",\n \"293T ( 2 x 106 cells/10 cm plate ) and HeLa M ( 1 . 2 x 106 cells/10 cm plate ) cells were seeded , harvested 72 hr later by trypsin treatment , washed twice in PBS , counted , lysed in RIPA buffer ( 1 . 2 × 105 cells/μl ) for 15 min at 4°C , clarified by centrifugation ( 15 min , 15 , 000×g , 4°C ) and diluted 1:1 with 2× SDS loading buffer .\",\n \"2 . 5 , 5 , 10 and 20 μl of each cell lysate mixture was loaded on a 4–15% gradient gel and AMOT proteins were detected by western blotting .\",\n \"Experiments to test whether AMOT overexpression can stimulate HIV-1 release from HeLa cells ( Figure 6A ) were performed as follows: HeLa M cells ( 2 . 5 × 105 cells/well in a 6-well plate ) were co-transfected with 0 . 5 μg of wild-type R9 HIV-1 and 0 . 5 , 1 , 2 or 4 μg of pcDNA3-HA AMOT p130 expression vectors .\",\n \"Cells and viruses were harvested 48 hr post-transfection .\",\n \"To test whether AMOT could stimulate virus release from physiologically relevant T cells , AMOT was overexpressed in Jurkat T cells as follows: t = 0 , cells were harvested , washed in PBS and resuspended in Neon resuspension buffer R ( Invitrogen/Life Technologies ) .\",\n \"5 × 106 cells were combined with 3 μg of the wild type R9 HIV-1 expression vector and 0 , 5 or 10 μg of pcDNA3-HA AMOT p130 or AMOT p130ΔPPXY expression vectors .\",\n \"Cells were then electroporated in a 100 µl tip with the Neon Transfection System using three 10 ms pulses of 1325 V . Following electroporation , each sample was immediately transferred to a 10 cm plate containing 10 ml pre-warmed RPMI ( supplemented with 10% FBS and 2 mM glutamine ) .\",\n \"t = 96 hr: media and cells were harvested .\",\n \"Protocols for analyzing protein expression and viral titers were identical to those described for 293T cells experiments .\",\n \"293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were seeded onto glass cover slips ( 24 hr prior transfection ) and co-transfected with the designated siRNAs and the R9 HIV-1 expression construct as described above .\",\n \"48 hr post-transfection , the media was removed and cells were fixed in 1 ml fixation buffer ( 2 . 5% glutaraldehyde/1% paraformaldehyde in sodium cacodylate buffer; 50 mM sodium cacodylate , 18 mM sucrose , 2 mM CaCl2 , pH 7 . 4 , 10 min ) .\",\n \"This and all subsequent steps were performed at 23°C .\",\n \"Fixed cells were washed three times in sodium cacodylate buffer ( 5 min ) , and stained with 2% OsO4 for 1 hr .\",\n \"Each sample was then dehydrated in a graded ethanol series , dried in HDMS ( hexamethyldisilazane ) , and sputter coated with platinum .\",\n \"SEM images were collected on a FEI Quanta 600 FEG scanning electron microscope at a beam energy of 10 kV , a spot size of three , and magnifications of 10 , 000 , 30 , 000 or 65 , 000 X . 293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were co-transfected with the designated siRNAs and the R9 HIV-1 expression construct as described above .\",\n \"48 hr post-transfection , the media was removed and cells were fixed in 1 ml fixation buffer ( 2 . 5% glutaraldehyde/1% paraformaldehyde in sodium cacodylate buffer [50 mM sodium cacodylate , 18 mM sucrose , 2 mM CaCl2 , pH 7 . 4 , 10 min] ) .\",\n \"This and all subsequent steps were performed at 23°C .\",\n \"Fixed cells were harvested , washed three times in sodium cacodylate buffer ( 5 min ) , and stained with 2% OsO4 for 1 hr .\",\n \"The pellet was rinsed once in sodium cacodylate buffer and twice in water ( 5 min ) , followed by incubation in 100 μl of a 4% uranyl acetate solution ( 30 min ) .\",\n \"Stained cells were dehydrated in a graded ethanol series followed by acetone , and embedded in epoxy resin EMBed-812 ( Electron Microscopy Sciences , Hatfield , PA ) .\",\n \"Thin sections ( 80–100 nm ) were cut , post-stained with saturated uranyl acetate ( 20 min ) , rinsed with water , dried , stained with Reynolds' lead citrate ( 10 min ) and dried again .\",\n \"TEM images were collected on a Hitachi H-7100 transmission electron microscope at an accelerating voltage of 75 kV , equipped with a Gatan Orius sc1000 camera .\",\n \"Imaged virions were scored as ‘mature’ if they lacked an observable cellular tether and a condensed core was evident , as ‘immature’ if they were untethered but lacked a condensed core , and as ‘budding’ if a Gag layer was clearly evident and the nascent virion was assembling or was clearly tethered to the cell .\",\n \"The degree of assembly was assessed by rendering a ( hypothetical ) spherical particle and measuring the arc angle of the nascent Gag layer .\",\n \"To image 500 virions budding from wild type cells , it was necessary to screen about eight-times as many thin slices of wild type control cells ( ∼2500 cells ) vs TSG101- or AMOT-depleted cells ( 275 and 300 , respesctively ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 E3 ubiquitin ligase family to facilitate virus release .","Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif , suggesting that NEDD4L may function through an adaptor protein .","Here , we demonstrate that the cellular protein Angiomotin ( AMOT ) can bind both NEDD4L and HIV-1 Gag .","HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT depletion , whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release .","Electron microscopic analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle .","Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding ."],"string":"[\n \"Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 E3 ubiquitin ligase family to facilitate virus release .\",\n \"Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif , suggesting that NEDD4L may function through an adaptor protein .\",\n \"Here , we demonstrate that the cellular protein Angiomotin ( AMOT ) can bind both NEDD4L and HIV-1 Gag .\",\n \"HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT depletion , whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release .\",\n \"Electron microscopic analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle .\",\n \"Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding .\"\n]"},"summary":{"kind":"list like","value":["To multiply and spread infections , viruses must enter and exit cells .","Once inside a cell , many viruses conscript the cell's machinery to produce new viral particles and release them into the surroundings .","Some viruses—like HIV-1—exit the cell in a way that leads to them being wrapped ( or ‘enveloped’ ) in membrane from the host cell .","A virus protein called Gag is required for the release of HIV-1 and other enveloped viruses .","In some cases , Gag proteins bind directly to members of the NEDD4 protein family to enable the viruses to be released .","However , the Gag protein from HIV-1 does not appear to be able to interact directly with NEDD4 proteins , so it was not clear how Gag works in this case .","Mercenne et al . studied how HIV-1 is released from human cells grown in the laboratory .","The experiments show that members of a human protein family called the Angiomotins bind to both Gag and NEDD4L ( a member of the NEDD4 family ) and are required for the efficient release of viruses .","Using a technique called electron microscopy , Mercenne et al . observed that when Angiomotins are present , Gag proteins assemble in spheres at the cell membrane and viruses are able to exit the cell .","However , when Angiomotins are depleted or absent , incomplete spheres of Gag proteins accumulate on the inner membrane surface and viruses are not released .","These findings show that Angiomotins act as a link between Gag and NEDD4L to promote the release of HIV-1 from human cells .","The next step will be to learn precisely how this works .","There are indications that the Angiomotins may also be involved in the release of other enveloped viruses , so the findings may be useful for the development of treatments for a variety of viral infections ."],"string":"[\n \"To multiply and spread infections , viruses must enter and exit cells .\",\n \"Once inside a cell , many viruses conscript the cell's machinery to produce new viral particles and release them into the surroundings .\",\n \"Some viruses—like HIV-1—exit the cell in a way that leads to them being wrapped ( or ‘enveloped’ ) in membrane from the host cell .\",\n \"A virus protein called Gag is required for the release of HIV-1 and other enveloped viruses .\",\n \"In some cases , Gag proteins bind directly to members of the NEDD4 protein family to enable the viruses to be released .\",\n \"However , the Gag protein from HIV-1 does not appear to be able to interact directly with NEDD4 proteins , so it was not clear how Gag works in this case .\",\n \"Mercenne et al . studied how HIV-1 is released from human cells grown in the laboratory .\",\n \"The experiments show that members of a human protein family called the Angiomotins bind to both Gag and NEDD4L ( a member of the NEDD4 family ) and are required for the efficient release of viruses .\",\n \"Using a technique called electron microscopy , Mercenne et al . observed that when Angiomotins are present , Gag proteins assemble in spheres at the cell membrane and viruses are able to exit the cell .\",\n \"However , when Angiomotins are depleted or absent , incomplete spheres of Gag proteins accumulate on the inner membrane surface and viruses are not released .\",\n \"These findings show that Angiomotins act as a link between Gag and NEDD4L to promote the release of HIV-1 from human cells .\",\n \"The next step will be to learn precisely how this works .\",\n \"There are indications that the Angiomotins may also be involved in the release of other enveloped viruses , so the findings may be useful for the development of treatments for a variety of viral infections .\"\n]"},"year":{"kind":"string","value":"2015"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":41,"numItemsPerPage":100,"numTotalItems":4346,"offset":4100,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc0MzczNzQ1NSwic3ViIjoiL2RhdGFzZXRzL3NhbmR5MzcvZWxpZmUiLCJleHAiOjE3NDM3NDEwNTUsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.sn-segyVy5eYpAfxRs1r9F95km7SzFfqoA80rufG3YOnFA6cztx2dR4SMDDX-poHCyHHEJEl3Zz0o_zJpdLjAg","displayUrls":true},"discussionsStats":{"closed":0,"open":1,"total":1},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false}">
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[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease"
] | Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants | elife-18296-v3 | [
[
"Viruses hijack host proteins and processes to optimize the cellular environment for viral replication and/or persistence .",
"Manipulation by viruses signposts critical pathways in viral pathogenesis and cell biology , and evolutionary pressure has led to conflict between cellular restriction factors ( limiting viral replication ) and viral countermeasures ( overcoming restriction in vivo ) .",
"We previously used multiplex whole cell proteomic analysis of Human Cytomegalovirus ( HCMV ) -infected fibroblasts to define expression time courses of viral and cellular proteins and identify novel proteins involved in the host-HCMV interaction , a technique we termed Quantitative Temporal Viromics ( QTV ) ( Weekes et al . , 2014 ) .",
"Here , we provide a comprehensive temporal proteomic analysis of HIV infection .",
"The HIV-1 'accessory proteins' Vif , Vpr , Nef and Vpu share a common ability to target cellular proteins for degradation ( Simon et al . , 2015; Sugden et al . , 2016 ) .",
"Whilst dispensible for viral replication in vitro , they are essential for pathogenesis in vivo .",
"Nef and Vpu are multifunctional adaptors which co-opt endolysosomal and proteasomal machinery to downregulate numerous plasma membrane proteins , including their canonical substrates CD4 , tetherin and MHC class I .",
"In contrast , although Vif and Vpr are known to target cytoplasmic and nuclear proteins for proteasomal degradation , relatively few cellular substrates have been reported .",
"The only known Vif targets are members of the APOBEC family of cytosine deaminases , which are otherwise incorporated into viral particles and act as dominant restriction factors causing hyper-mutation of the HIV genome ( Desimmie et al . , 2014; Malim , 2009 ) .",
"Whilst Nef , Vpr and Vpu are found exclusively in primate lentiviruses , Vif is found in four of the five extant lentiviral lineages , infecting primate , feline , bovine and small ruminant hosts ( Gifford , 2012 ) , and the ability to target cognate host APOBEC proteins is conserved across Vif variants from all these diverse lineages ( Larue et al . , 2010 ) .",
"Cellular proteins regulated by HIV have generally been identified using non-systematic , candidate approaches .",
"We recently used a different , unbiased plasma membrane proteomic approach to reveal >100 previously unsuspected cell surface proteins depleted by HIV-1 , including novel Nef ( SERINC3/5 ) and Vpu ( SNAT1 ) targets ( Matheson et al . , 2015 ) .",
"Whole cell proteomic studies of HIV-infected cells have been variably hampered by limited proteome coverage , asynchronous infections and confounding by the presence of bystander ( uninfected ) cells ( Supplementary file 1 ) .",
"Consequently , it has been difficult to attribute changes in protein levels to expression of specific viral genes , and intracellular proteins targeted by HIV accessory proteins have not been discovered in this fashion .",
"In this study , we extend our tandem mass tag ( TMT ) -based temporal proteomic approach to describe global changes in HIV-infected T cells , comprising expression time courses of >6500 proteins .",
"We cluster proteins according to their patterns of temporal expression , and identify >100 cellular proteins regulated by HIV , including candidate resistance/restriction factors and HIV accessory protein targets .",
"To test the utility of our approach , we focus on proteins depleted with similar kinetics to APOBEC3C , and confirm the B56 family of regulatory subunits of the key cellular phosphatase PP2A ( PPP2R5A-E ) to be novel Vif targets .",
"We use large-scale quantitative phosphoproteomics to demonstrate Vif-dependent remodelling of the cellular phosphoproteome during HIV infection , and show that , along with APOBEC proteins , antagonism of PP2A-B56 is an ancient and conserved Vif function ."
],
[
"To gain a comprehensive , unbiased overview of viral and cellular protein dynamics during HIV infection , we analysed total proteomes of CEM-T4 T cells infected with HIV .",
"As previously described ( Matheson et al . , 2015 ) , cells were spinoculated with Env-deficient , VSVg-pseudotyped virus at an MOI sufficient to achieve a synchronous single round infection with <10% uninfected bystander cells .",
"We exploited 6-plex TMT labelling to quantitate 6538 proteins in whole cell lysates of uninfected cells ( 0 hr ) , at four timepoints following HIV-1 infection ( 6 , 24 , 48 , and 72 hr ) , and in cells infected for 72 hr in the presence of reverse transcriptase inhibitors ( RTi ) ( Figure 1A ) .",
"The complete dataset has been deposited to the ProteomeXchange consortium with the dataset identifier PXD004187 ( accessible at http://proteomecentral . proteomexchange . org ) and is summarised in an interactive spreadsheet ( Figure 1—source data 1 ) , which allows generation of temporal profiles for any quantitated genes of interest . 10 . 7554/eLife . 18296 . 003Figure 1 . TMT-based proteomic time course analysis of HIV-infected cells .",
"( A ) Workflow of 6-plex TMT-based whole cell proteomic time course experiment .",
"CEM-T4 T cells were infected with NL4-3-dE-EGFP HIV at an MOI of 10 .",
"In subsequent figures timepoints 1–5 show protein abundance 0 , 6 , 24 , 48 and 72 hr after HIV infection ( where 0 hr = uninfected cells ) and timepoint 6 shows protein abundance 72 hr after HIV infection in the presence of reverse transcriptase inhibitors ( RTi ) .",
"( B ) Comparison of temporal profiles of Env-GFP obtained by proteomic ( TMT ) versus flow cytometric quantitation .",
"Cells from ( A ) were analysed by flow cytometry .",
"Relative abundance is expressed as a fraction of maximum TMT reporter ion or fluorescence intensity .",
"For linear regression , log2 ( fold change in protein abundance compared with uninfected cells ) is shown .",
"( C–D )",
"Temporal profiles of viral proteins ( C ) and previously reported HIV targets ( D ) .",
"GAPDH and β-actin are included as controls .",
"Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 00310 . 7554/eLife . 18296 . 004Figure 1—source data 1 . Interactive spreadsheet of TMT time course data . Interactive spreadsheet enabling generation of temporal profiles of protein abundance for any quantitated genes of interest ( 'Gene search and plots' worksheet ) .",
"Detailed instructions are incorporated into the spreadsheet .",
"The complete ( unfiltered ) TMT-based proteomic time course dataset ( 'Complete TMT time course data' worksheet ) and a database of gene name aliases ( 'Gene name aliases' worksheet ) are also included .",
"Protein abundance is depicted on a colour scale ( red = downregulated; green = upregulated ) .",
"The number of unique peptides , peptides and peptide spectral matches are specified for each protein , along with ratio counts and variability for each TMT condition . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 00410 . 7554/eLife . 18296 . 005Figure 1—figure supplement 1 . Additional temporal profiles and comparison with plasma membrane profiling .",
"( A ) Comparison of temporal profiles of cell surface Nef- ( CD4 and HLA-A ) and Vpu- ( CD4 , tetherin and SNAT1 ) targets obtained by whole cell or plasma membrane proteomics .",
"Expression levels from our whole cell proteomic analysis ( WCP , red ) and our previous plasma membrane profiling analysis ( [Matheson et al . , 2015]; PMP , blue ) are shown .",
"Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity .",
"( B ) Dynamic range of protein regulation observed by whole cell or plasma membrane proteomics .",
"Histograms show the frequencies of log2 ( fold change compared with mock/uninfected cells ) values for proteins quantitated in our whole cell proteomic analysis ( WCP , red ) and our previous PMP ( [Matheson et al . , 2015]; blue ) at 24 , 48 and 72 hr .",
"All proteins identified by >1 unique peptide in both WCP and PMP experiments are shown .",
"( C–G )",
"Temporal profiles of selected control proteins ( C–E ) , actin regulatory proteins ( F ) and mitotic kinases ( G ) .",
"Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity .",
"( H ) Quantitation of plasma membrane proteins by whole cell or plasma membrane proteomics .",
"The pie chart shows overlap of proteins with Gene Ontology Cellular Compartment annotations indicative of plasma membrane localisation quantitated in our whole cell proteomic analysis ( WCP , red ) and our previous PMP analysis ( [Matheson et al . , 2015]; blue ) .",
"Protein numbers are indicated .",
"The bar chart details the glycosylation status of proteins quantitated in WCP ( red ) , PMP ( blue ) or both ( tan ) experiments .",
"Glycosylation sites were identified from the UniProt Knowledgebase ( accessed on 4/12/15 at http://www . uniprot . org ) .",
"Protein numbers ( glycosylated/total ) are indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 00510 . 7554/eLife . 18296 . 006Figure 1—figure supplement 2 . Gene set enrichment analysis of HIV infection .",
"( A ) Pathways and processes upregulated by HIV infection .",
"KEGG Pathway and Gene Ontology Biological Process gene sets enriched in infected cells at the indicated timepoints compared with uninfected cells were determined using GSEA .",
"Indicative false discovery rate ( FDR ) thresholds for gene set enrichment are shown and gene sets related to lipid metabolism are highlighted ( green ) .",
"( B ) Pathways and processes downregulated by HIV infection .",
"KEGG Pathway and Gene Ontology Biological Process gene sets enriched in uninfected cells compared with infected cells at the indicated timepoints were determined using GSEA .",
"Indicative FDR thresholds for gene set enrichment are shown and gene sets related to RNA processing are highlighted ( green ) .",
"( C–D )",
"Expression levels of all quantitated proteins in Lipid_Metabolic_Process ( C ) and RNA_Processing ( D ) gene sets .",
"Log2 ( fold change in protein abundance compared with uninfected cells ) is shown at the indicated timepoints . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 006 We observed a tight correlation between levels of Env-GFP expression determined by mass spectrometry and flow cytometry ( r2 = 0 . 97 ) ( Figure 1B ) .",
"As expected , the well characterised HIV cell surface targets downregulated in our plasma membrane proteomic analysis were also depleted in our whole cell proteomic analysis ( Figure 1—figure supplement 1A ) .",
"The magnitude of effect was generally greater in the plasma membrane proteomic analysis ( Figure 1—figure supplement 1A–B ) , suggesting that regulation of cell surface proteins by redistribution or sequestration is an important feature of this system .",
"We detected gene products from 7/9 HIV-1 open reading frames ( ORFs; Figure 1B–C ) .",
"As previously reported , expression of regulatory proteins ( Tat and Rev ) from Rev-independent completely spliced mRNA transcripts occurred earlier in viral replication than expression of structural proteins from Rev-dependent unspliced ( Gag and Gagpol ) and partially spliced ( Env ) mRNA transcripts ( Karn and Stoltzfus , 2012; Pollard and Malim , 1998 ) , with Rev expression lagging Tat in our experiment .",
"Vif and Nef showed intermediate temporal profiles ( Figure 1C ) , with progressively increasing Nef expression from 24–48 hr inversely correlating with downregulation of CD4 and HLA-A ( Figure 1—figure supplement 1A ) .",
"Finally , we saw an increase in plasma membrane VSVg levels immediately after infection ( reflecting fusion of incoming virions ) , followed by a rapid decline ( Figure 1—figure supplement 1C ) .",
"Compared with numerous cell surface targets ( Haller et al . , 2014; Matheson et al . , 2015 ) , relatively few intracellular proteins depleted by HIV accessory proteins have been described .",
"Nonetheless , we confirmed downregulation of the Vif target APOBEC3C ( Smith and Pathak , 2010 ) and the Vpr target UNG ( Schrofelbauer et al . , 2005 ) ( Figure 1D ) .",
"The temporal pattern of UNG depletion was distinct from that of other accessory protein substrates , including APOBEC3C , with degradation seen as early as 6 hr post-infection , and preserved in the presence of reverse transcriptase inhibitors .",
"This is likely to reflect the high abundance of Vpr packaged within incoming viral particles ( Lu et al . , 1993; Paxton et al . , 1993 ) , abrogating the need for de novo protein synthesis .",
"As well as recruiting substrates for degradation , Vpu increases β-catenin levels by sequestering the ß-TrCP substrate-recognition unit of the SCFß-TrCP E3 ubiquitin ligase complex ( Besnard-Guerin et al . , 2004 ) .",
"In addition , HIV infection causes cell cycle arrest at G2/M ( Jowett et al . , 1995 ) , a point in the cell cycle associated with upregulation of cyclin B1 ( Norbury and Nurse , 1992 ) .",
"Accordingly , we observed progressive accumulation of both β-catenin and cyclin B1 ( Figure 1D ) .",
"Gene Set Enrichment Analysis ( GSEA ) revealed time-dependent perturbation of multiple cellular processes and pathways during HIV infection ( Figure 1—figure supplement 2A–B ) , with protein-level changes generally supporting earlier transcriptome-level data .",
"For example , genes associated with lipid metabolism ( Figure 1—figure supplement 2C ) are induced by expression of Nef ( Shrivastava et al . , 2016; van 't Wout et al . , 2005 ) , whereas genes associated with RNA processing ( Figure 1—figure supplement 2D ) are suppressed in HIV-infected cells ( Chang et al . , 2011; Sherrill-Mix et al . , 2015 ) . To facilitate data mining and identify specific host factors regulated by HIV infection , we classified cellular proteins according to their patterns of temporal expression ( Figure 2A ) . We observed 4 main clusters: ( #1 ) 29 proteins downregulated late in infection , rescued in the presence of reverse transcriptase inhibitors; ( #2 ) 59 proteins downregulated earlier in infection , incompletely rescued in the presence of reverse transcriptase inhibitors; ( #3 ) 29 proteins progressively upregulated during infection , abolished in the presence of reverse transcriptase inhibitors; and ( #4 ) 49 proteins progressively upregulated during infection , even in the presence of reverse transcriptase inhibitors ( Figure 2B ) . We validated protein downregulation ( clusters #1 and #2 ) and upregulation ( clusters #3 and #4 ) in an independent infection time course experiment , using stable isotope labelling with amino acids in cell culture ( SILAC ) as an alternative quantitative proteomic approach ( Figure 2C and Figure 2—figure supplement 1A ) . Details of all proteins in clusters #1–4 , including validation time course data , are available in Figure 2—source data 1 . 10 . 7554/eLife . 18296 . 007Figure 2 . Identification and SILAC-based proteomic validation of novel HIV targets . ( A ) Hierarchical cluster analysis of temporal profiles of proteins regulated by HIV . The heatmap shows log2 ( fold change in protein abundance compared with uninfected cells ) and clusters #1–4 are indicated . ( B ) Average temporal profiles of proteins in clusters #1–4 . Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity . ( C ) SILAC-based validation of novel HIV targets . CEM-T4 T cells pre-labelled with heavy amino acids were infected with NL4-3-dE-EGFP HIV at an MOI of 10 , and control cells pre-labelled with medium amino acids were mock-infected without virus . Aliquots of HIV-infected ( heavy; H ) and mock ( medium; M ) cells were harvested sequentially at the indicated timepoints and subjected to SILAC-based whole cell proteomic analysis ( Figure 2—figure supplement 1A ) . Log2 ( H/M protein abundance ) at 24 , 48 and 72 hr is shown for proteins from clusters #1–4 . ***p value<0 . 001 . ( D ) Temporal profiles of novel HIV-1 targets FMR1 and TFAP4 . Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity . ( E ) Antagonism of HIV production by FMR1 and TFAP4 . 293T cells were co-transfected with pNL4-3-dE-EGFP/pMD . G and either mCherry or the indicated cellular protein . 48 hr culture supernatants were assayed for infectious virus by infection of HeLa cells . Well-characterised restriction factors APOBEC3G and tetherin were included as controls , and infectious virus release normalised compared with mCherry . Mean values and standard errors are shown from at least 4 replicates . All four proteins significantly reduced viral release compared with mCherry in an ANOVA analysis with Bonferroni post-test , p values<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 00710 . 7554/eLife . 18296 . 008Figure 2—source data 1 . Clusters #1–4 summary proteomic data . Spreadsheet of all proteomic data for proteins in clusters #1–4 . Each cluster is represented by a single worksheet , with proteins ranked according to the hierarchical cluster analysis shown ( Figure 2A ) and protein abundance depicted on a colour scale ( red = downregulated; green = upregulated ) . As well as data from the TMT-based proteomic time course experiment ( Figure 1 and Figure 1—source data 1 ) , additional data from the SILAC-based proteomic validation ( Figure 2C and Figure 2—figure supplement 1A ) and TMT-based single timepoint experiment ( Figure 4A and Figure 4—figure supplement 1A ) are also included . The number of unique peptides is shown for each protein ( TMT experiments ) and each timepoint ( SILAC experiment ) . q values for the TMT-based single timepoint experiment were determined using Limma with Benjamini-Hochberg adjustment for multiple testing , with q values < 0 . 01 highlighted in gold . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 00810 . 7554/eLife . 18296 . 009Figure 2—figure supplement 1 . Workflow of SILAC-based proteomic time course experiment , functional analysis of clusters #1–4 and prediction of novel Vpr targets . ( A ) Workflow of SILAC-based proteomic validation time course experiment . CEM-T4 T cells pre-labelled with heavy amino acids were infected with NL4-3-dE-EGFP HIV at an MOI of 10 , and control cells pre-labelled with medium amino acids were mock-infected without virus . Aliquots of HIV-infected ( heavy ) and mock ( medium ) cells were harvested sequentially at the indicated timepoints and subjected to SILAC-based whole cell proteomic analysis . ( B ) Functional annotation clusters enriched amongst proteins from clusters #1–4 . Enrichment of Gene Ontology Molecular Function and Biological Process terms against a background of all quantitated proteins was determined using DAVID . Functional annotation clusters with enrichment scores > 1 . 3 ( equivalent to a geometric mean of all included enrichment p values<0 . 05 ) were considered significant . Representative Gene Ontology terms are indicated . ( C ) Temporal profiles of Vpr targets UNG and HLTF . Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity . ( D ) Enlargement of part of cluster #2 from hierarchical cluster analysis ( Figure 2A ) . The heatmap shows log2 ( fold change in protein abundance compared with uninfected cells ) . Previously characterised Vpr targets UNG and HLTF are highlighted ( red ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 009 Distinct patterns of temporal regulation imply different mechanisms and biological significance . The Nef , Vpu and Vif accessory protein targets CD4 , SNAT1 , APOBEC3C are found in cluster #1 , where progressive downregulation and rescue by reverse transcriptase inhibitors suggest dependence on de novo viral protein synthesis ( compare Figure 2B with Figure 1D , top left panel , and Figure 1—figure supplement 1A ) . Upregulation of proteins in cluster #3 is also likely to require de novo viral protein synthesis , and the indirect Vpu target β-catenin is found in this cluster ( compare Figure 2B with Figure 1D , middle left panel ) . Conversely , reverse transcriptase inhibitor-independent regulation of proteins in clusters #2 and #4 implies a cellular response to HIV infection , or a direct effect of viral proteins in incoming virions , and the Vpr target UNG is found in cluster #2 ( compare Figure 2B with Figure 1D , top right panel ) . As well as mechanistic differences , analysis using the Database for Annotation , Visualisation and Integrated Discovery ( DAVID ) revealed that clusters #1–4 contained proteins associated with distinct biological functions and processes ( Figure 2—figure supplement 1B ) . Whilst accumulation of some proteins in cluster #3 may be secondary to G2/M cell cycle arrest , other changes are unlikely to reflect the interferon ( IFN ) or unfolded protein responses , because we did not see accumulation of either the highly IFN-inducible protein ISG15 ( Figure 1—figure supplement 1D ) or proteins associated with ER stress ( Figure 1—figure supplement 1E ) . Restriction factors are cellular proteins whose primary biological activity is antiviral , and which are induced by IFN or viral infection , antagonized by viral proteins , and show genetic evidence of positive selection ( Duggal and Emerman , 2012 ) . Proteins which reduce permissivity for viral infection , but fail to meet strict criteria for restriction factors , may be classified as resistance factors ( Goujon et al . , 2013 ) . Restriction and resistance factors are characteristically increased ( cellular response ) or decreased ( viral antagonism ) during viral infection . Our temporal proteomic approach therefore identifies unsuspected viral regulation of known resistance/restriction factors , and provides a strategy for the discovery of novel host antiviral factors . Accordingly , we found the actin regulatory proteins gelsolin and CAPG ( both cluster #4 ) to be strongly induced during HIV infection ( Figure 1—figure supplement 1F ) . Actin cytoskeletal remodeling is required for virological synapse formation and cell-cell transmission of HIV ( Jolly et al . , 2004 ) , and gelsolin levels have been reported to control early HIV infection in macrophages ( Garcia-Exposito et al . , 2013 ) . In contrast , we discovered marked depletion of FMR1 ( cluster #1 , Figure 2D , upper panel ) and TFAP4 ( cluster #2 , Figure 2D , lower panel ) during HIV infection . Both these proteins reduce production of infectious HIV virus ( Figure 2E ) ( Imai and Okamoto , 2006; Pan et al . , 2009 ) , but their distinct patterns of temporal expression suggest different mechanisms of viral regulation . We predict that other regulated proteins in clusters #1–4 , without known roles in HIV infection , will also represent novel cellular resistance/restriction factors . To test the utility of our approach , we focussed on proteins in cluster #1 highlighted in our functional analysis ( Figure 3A–B and Figure 2—figure supplement 1B ) . First , three members of the deoxynucleotide triphosphate ( dNTP ) biosynthetic pathway were depleted during HIV infection: thymidylate synthetase ( TYMS ) , which catalyses the methylation of deoxyuridylate ( dUMP ) to deoxythymidylate ( dTMP ) ; and two subunits of ribonucleotide reductase ( RNR ) , RRM1 and RRM2 , which catalyses the formation of deoxyribonucleotides from ribonucleotides ( Figure 3B , left panels ) . HIV replication is tightly regulated by dNTP availability , and SAMHD1 ( which tends to oppose the effects of RNR ) is a well-described HIV restriction factor ( Ayinde et al . , 2012; Baldauf et al . , 2012; Hrecka et al . , 2011; Laguette et al . , 2011; Lahouassa et al . , 2012; Taylor et al . , 2015 ) . Second , and most strikingly , all detected members of the B56 family of protein phosphatase 2 A ( PP2A ) regulatory subunits ( PPP2R5A , C , D and E ) were profoundly depleted by HIV ( Figure 3B , right panels ) . These subunits determine the specificity and localisation of PP2A holoenzymes ( PP2A-B56 ) , a ubiquitous family of heterotrimeric serine-threonine phosphatases with critical roles in many aspects of cellular physiology ( McCright et al . , 1996 ) . We confirmed downregulation of RRM2 , PPP2R5A and PPP2R5D by immunoblot ( Figure 3—figure supplement 1 ) . Because cluster #1 also contained APOBEC3C , and intracellular proteins in this cluster ( including PPP2R5 subunits ) show near-identical patterns of temporal expression , we hypothesised that some or all of these proteins might be novel Vif targets . 10 . 7554/eLife . 18296 . 010Figure 3 . Cellular proteins progressively downregulated by HIV infection . ( A ) Enlargement of cluster #1 from hierarchical cluster analysis ( Figure 2A ) . The heatmap shows log2 ( fold change in protein abundance compared with uninfected cells ) . Enzymes associated with deoxynucleotide metabolism ( blue ) and B56 family regulatory subunits of serine/threonine protein phosphatase PP2A ( red ) are highlighted , along with known Vif target APOBEC3C ( boxed ) and other proteins of interest ( bold ) . ( B ) Temporal profiles of enzymes associated with deoxynucleotide metabolism ( blue ) and B56 family regulatory subunits of serine/threonine protein phosphatase PP2A ( red ) . Relative abundance is expressed as a fraction of maximum TMT reporter ion intensity , and the temporal profile of APOBEC3C is shown for comparison . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 01010 . 7554/eLife . 18296 . 011Figure 3—figure supplement 1 . Immunoblot validation of novel HIV targets . Depletion of proteins in cluster #1 by HIV-1 infection . CEM-T4s were infected with NL4-3-dE-EGFP HIV at an MOI of 10 and analysed by immunoblot ( IB ) 48 hr post-infection . Depletion of positive control ( CD4 and APOBEC3G ) and novel ( RRM2 and PPP2R5A/D ) HIV targets was confirmed . ImageJ ( Schneider et al . , 2012 ) was used to determine band intensities , which were normalised to calreticulin intensity and compared with TMT-based proteomic quantitation at 48 hr . p24 ( capsid ) and Vif are included as additional controls . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 011 To examine this hypothesis and systematically identify novel Vif targets , we performed a 3-way proteomic comparison of mock-infected cells and cells infected with wildtype ( WT ) or Vif-deficient ( ∆Vif ) HIV viruses ( Figure 4—figure supplement 1A ) . To complement our high MOI time course experiment , we used an MOI of 1 . 5 , resulting in approximately 75% productive infection and ( typically ) one or two copies of the viral genome per cell ( Figure 4—figure supplement 1B ) . We exploited 10-plex TMT labelling to analyse samples in triplicate at a single timepoint 48 hr post-infection , with the resulting statistical power compensating for the reduced magnitude of changes due to the presence of bystander ( uninfected ) cells . As expected , Vif protein was only detected in WT infection , but levels of other viral proteins were equivalent ( Figure 4A ) . 10 . 7554/eLife . 18296 . 012Figure 4 . Vif-mediated depletion of PP2A-B56 family members PPP2R5A-E . ( A ) Proteomic analysis of CEM-T4 T cells infected with WT and ΔVif HIV . Cells were infected with NL4-3-ΔE-EGFP viruses at an MOI of 1 . 5 , and harvested 48 hr post-infection . Scatterplots display pairwise comparisons between WT , ΔVif and mock-infected cells . Each point represents a single protein , plotted by its log2 ( fold change in abundance ) versus the statistical significance of that change . q values were determined using Limma with Benjamini-Hochberg adjustment for multiple testing , with increasing −log2 ( q value ) indicating increasing significance . Points above the dotted line change with a q value < 0 . 01 . HIV proteins and host proteins of interest are highlighted with different symbols ( see key ) . ( B ) Depletion of PPP2R5A and PPP2R5D during HIV infection . CEM-T4 T-cells were infected with NL4-3-dE-EGFP WT and ΔVif viruses at an MOI of 1 or 10 and analysed by immunoblot ( IB ) 48 hr post-infection . p24 ( capsid ) , Vif and calreticulin are included as controls . ( C ) Depletion of exogenous PPP2R5A by Vif . 293T cells stably expressing HA-PPP2R5A were co-transfected with GFP plus empty vector , NL4-3 Vif or NL4-3 Vif with a single amino acid mutation C114S and analysed by intracellular flow cytometry for HA 36 hr post-transfection . Histograms show GFP positive ( transfected , red shading ) and negative ( untransfected , blue line ) cells . Median fluorescence intensity ( MFI ) values are shown for GFP positive ( red ) and negative ( blue ) cells . ( D ) Depletion of PPP2R5A-E family members by Vif . 293T cells stably expressing HA-tagged PPP2R5A-E or APOBEC3G were co-transfected with GFP plus NL4-3 Vif expression vectors , and intracellular HA staining quantitated by flow cytometry 36 hr post transfection . Histograms show GFP positive ( transfected , red shading ) and negative ( untransfected , blue line ) cells . MFI values are shown for GFP positive ( red ) and negative ( blue ) cells . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 01210 . 7554/eLife . 18296 . 013Figure 4—figure supplement 1 . Workflow and controls for single timepoint proteomic/phosphoproteomic experiment . ( A ) Workflow of TMT-based single timepoint whole cell proteomic and phosphoproteomic experiment . CEM-T4 T cells were mock-infected or infected with NL4-3-dE-EGFP WT or ΔVif HIV at an MOI of 1 . 5 . Cells were harvested for proteomic analysis 48 hr post-infection . Samples were subjected to ( 1 ) whole cell proteome and ( 2 ) phosphopeptide analysis . Each condition was carried out in triplicate . ( B ) Quantitation of infected cells from experiment described in ( A ) . Cells were analysed by flow cytometry for CD4 and GFP expression , with infected cells losing CD4 and gaining GFP . Example flow cytometric analysis of one replicate for each condition is shown , demonstrating the % infected cells . Across all replicates , cells infected with WT or ΔVif viruses were 74–78 % infected . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 01310 . 7554/eLife . 18296 . 014Figure 4—figure supplement 2 . Depletion of endogenous PPP2R5D during HIV infection of primary cells . ( A–B ) CEM-T4 T cells ( A ) or activated primary human CD4+ T cells ( B ) were infected with NL4-3-dE-EGFP WT and ΔVif viruses at an MOI of 1 and analysed by intracellular flow cytometry for PPP2R5D 48 hr post-infection . ICAM3 is included as a control . MFI values are shown for ΔVif ( blue ) and WT ( red ) viruses . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 014 Vif-independent HIV targets such as tetherin ( Vpu substrate ) , SNAT1 ( Vpu substrate ) , CD4 ( Nef/Vpu substrate ) and UNG ( Vpr substrate ) were depleted in cells infected with both WT ( Figure 4A , left panel ) and ∆Vif ( Figure 4A , middle panel ) viruses , with no difference in abundance in the presence or absence of Vif ( Figure 4A , right panel ) . Conversely , known Vif targets APOBEC3C , APOBEC3G and APOBEC3D were all decreased by WT but not ΔVif HIV infection ( Figure 4A ) , and APOBEC3B , which is resistant to Vif ( Doehle et al . , 2005 ) , was unchanged across all conditions . In addition to APOBEC family members , all five PP2A-B56 regulatory subunits PPP2R5A-E were depleted in the presence of Vif ( Figure 4A ) . Vif-dependent degradation of PPP2R5 subunits was confirmed by immunoblot of HIV-infected CEM-T4 T cells ( PPP2R5A and PPP2R5D; Figure 4B ) and intracellular flow cytometry of HIV-infected CEM-T4 and primary human CD4+ T cells ( PPP2R5D; Figure 4—figure supplement 2A–B ) . To test whether degradation of PPP2R5 subunits by Vif was post-translational , we expressed HA-tagged PPP2R5A in 293T cells . Transfection of Vif caused a marked loss of intracellular HA staining ( Figure 4C , middle panels ) , and the same effect was also seen in cells expressing all other PPP2R5 subunits or APOBEC3G ( Figure 4D ) . Degradation of APOBEC family members by Vif is mediated by recruitment of a cullin-5 ( CUL5 ) E3 ubiquitin ligase complex , resulting in substrate-specific ubiquitination and proteasomal degradation ( Malim and Bieniasz , 2012 ) . We therefore predicted that depletion of PPP2R5 subunits would exploit the same pathway . Accordingly , we found that the Vif C114S mutant , which is unable to recruit CUL5 ( Bergeron et al . , 2010 ) , was defective for PPP2R5A degradation ( Figure 4C , right panels ) , and PPP2R5A degradation by wildtype Vif was rescued in the presence of the proteasome inhibitor bortezomib ( Figure 5A ) . A similar rescue of PPP2R5B was seen when Vif was co-transfected with dominant negative , but not wildtype , CUL5 ( Figure 5B ) , and knockdown of other cellular components of the CUL5 E3 ligase complex recruited by Vif ( EloB , EloC and CBFβ ) ( Jager et al . , 2012; Malim and Bieniasz , 2012 ) rescued both PPP2R5B and APOBEC3G from degradation , with the magnitude of rescue similar for both substrates ( Figure 5C ) . 10 . 7554/eLife . 18296 . 015Figure 5 . Mechanism of Vif-mediated degradation of PPP2R5A-E subunits . ( A ) Proteasomal degradation . 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO ( control ) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA . ( B ) CUL5-dependent degradation . 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector , wildtype cullin-5 ( CUL5 WT ) or a dominant negative cullin-5 mutant ( CUL5 DN ) and analysed by flow cytometry for GFP . ( C ) CUL5 complex-dependent degradation . 293T cells stably expressing HA-PPP2R5B ( upper panels ) or HA-APOBEC3G ( lower panels ) were transduced with the indicated shRNA . Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA . Green/red shading shows Vif-transfected cells in the indicated shRNA background . Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference . In all experiments , cells were analysed 36 hr post-transfection , and transfected cells determined by co-transfection with GFP ( A and C ) or mCherry ( B ) . MFI values are shown for transfected ( red/green ) and untransfected ( blue ) cells . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 01510 . 7554/eLife . 18296 . 016Figure 5—figure supplement 1 . Co-immunoprecipitation of Vif and PPP2R5D . ( A ) Co-immunoprecipitation in 293Ts . WT 293T cells or 293T cells stably expressing HA-tagged PPP2R5D were transfected with FLAG-tagged NL4-3 Vif , pre-treated with bortezomib ( 10 nM ) for 16 hr , and analysed by immunoblot ( IB ) for HA-PPP2R5D and FLAG-Vif 48 hr post-infection ( left panels ) . Lysates were subjected to immunoprecipitation ( IP ) with anti-HA ( middle panels ) or anti-PPP2R5D ( right panels , WT 293Ts only ) and re-analysed by immunoblot . 293T cells transfected with empty vector or FLAG-tagged K5 protein of Kaposi's sarcoma-associated herpesvirus ( KSHV ) were included as controls .",
"( B ) Co-immunoprecipitation during HIV infection of T cells .",
"CEM-T4 T cells were infected with NL4-3-dE-EGFP HIV at an MOI of 1 . 5 , pre-treated with bortezomib ( 10 nM ) for 16 hr , and analysed by immunoblot ( IB ) for PPP2R5D and Vif 48 hr post-infection ( left panels ) .",
"Lysates were subjected to immunoprecipitation ( IP ) with anti-PPP2R5D ( right panels ) and re-analysed by immunoblot .",
"Uninfected CEM-T4 T cells and infected CEM-T4 T cells without bortezomib pre-treatment were included as controls . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 01610 . 7554/eLife . 18296 . 017Figure 5—figure supplement 2 . Time course analysis of endogenous PPP2R5D during HIV infection of T cells .",
"( A–B )",
"Rescue by proteasome inhibition .",
"CEM-T4 T cells were infected with NL4-3-dE-EGFP HIV at an MOI of 1 . 5 and analysed by flow cytometry ( A ) at the indicated timepoints in the presence ( bottom panels ) or absence ( top panels ) of bortezomib ( BZB ) added 24 hr post-infection ( +0 hr ) .",
"MFI values ( PPP2R5D staining ) for GFP positive ( infected ) cells are shown , normalized to values at +0 hr ( B ) .",
"( C–D )",
"Cycloheximide chase .",
"CEM-T4 T cells were infected with NL4-3-dE-EGFP WT ( top panels ) and ΔVif ( bottom panels ) viruses at an MOI of 1 . 5 and analysed by flow cytometry ( C ) at the indicated timepoints in the presence of cycloheximide ( CHX ) added 24 hr post-infection ( +0 hr ) .",
"MFI values ( PPP2R5D staining ) for GFP positive ( infected ) cells are shown , normalized to values at +0 hr ( D ) .",
"As predicted , the presence of CHX inhibited the production of new Env-EGFP protein . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 01710 . 7554/eLife . 18296 . 018Figure 5—figure supplement 3 . Pulse-chase analysis of endogenous PPP2R5D during HIV infection of T cells . CEM-T4 T cells were infected with NL4-3-dE-EGFP WT or ΔVif viruses at an MOI of 1 .",
"5 and pulsed with [35S]methionine/[35S]cysteine 48 hr post-infection .",
"Cells were chased until the indicated timepoints , subjected to immunoprecipitation ( IP ) with anti-PPP2R5D and analysed by autoradiography . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 018 Consistent with a protein-level interaction between Vif and PPP2R5 subunits , we observed co-immunoprecipitation of FLAG-tagged Vif with HA-tagged and endogenous PPP2R5D in 293T cells ( Figure 5—figure supplement 1A ) , and co-immunoprecipitation of untagged Vif with endogenous PPP2R5D in CEM-T4 T cells infected with HIV ( Figure 5—figure supplement 1B ) .",
"As in 293T cells transfected with Vif , PPP2R5 subunit depletion in CEM-T4 T cells infected with HIV was abolished in the presence of bortezomib ( Figure 5—figure supplement 2A–B ) .",
"Finally , we confirmed using cycloheximide chase ( Figure 5—figure supplement 2C–D ) and [35S]methionine/[35S]cysteine metabolic labelling/pulse-chase ( Figure 5—figure supplement",
"3 ) analyses that degradation of PPP2R5D was accelerated in the presence of Vif in HIV-infected CEM-T4 T cells .",
"Vif is therefore both necessary and sufficient for degradation of PPP2R5 subunits , and employs the same cellular machinery required for degradation of APOBEC family members .",
"The substrate specificity of the PP2A phosphatase holoenzyme is determined by binding of its regulatory subunits ( Yang and Phiel , 2010 ) .",
"To identify the phenotypic consequences of Vif-mediated PPP2R5A-E subunit depletion , we used titanium dioxide-based phosphopeptide enrichment and 10-plex TMT labelling to analyse total phosphoproteomes of the mock- , WT and ∆Vif virus-infected cells described in Figure 4A and Figure 4—figure supplement 1A–B .",
"In total , we quantitated 8631 phosphopeptides from 2767 proteins ( Figure 6—source data 1 ) .",
"Phosphopeptide abundance was normalized to total protein abundance determined from the whole cell proteomic analysis , allowing differential phosphorylation to be distinguished from altered protein expression ( Wu et al . , 2011 ) .",
"HIV infection resulted in marked remodelling of the cellular phosphoproteome ( Figure 6—figure supplement 1A ) , and analysis using the Database for Annotation , Visualisation and Integrated Discovery ( DAVID ) revealed enhanced phosphorylation of proteins associated with cell cycle regulation and activation of the DNA damage response ( Figure 6—figure supplement 1B ) .",
"To isolate those changes which specifically resulted from Vif-mediated PPP2R5A-E subunit depletion , we focused on differences between cells infected with WT and ∆Vif viruses .",
"Remarkably , compared with the small number of protein-level changes in the presence or absence of Vif ( specifically , APOBEC and PPP2R5 family members; Figure 4A , right panel and Figure 6A , left panel ) , we saw striking Vif-dependent changes in the phosphoproteome ( Figure 6A , right panel ) .",
"Furthermore , as predicted for antagonism of a phosphatase , almost all changes represented increases in phosphopeptide abundance , indicating increased protein phosphorylation ( with a total of 238 peptides from 192 proteins showing abundance changes of >2 fold with a q value of <0 . 01 ) .",
"To confirm that the observed changes resulted from PP2A antagonism , we compared our Vif-dependent changes in protein phosphorylation with published alterations to the phosphoproteome of HeLa cells following treatment with the PP2A inhibitor okadaic acid ( Kauko et al . , 2015 ) .",
"Despite the different cell types and treatments , a highly significant correlation was found between our observed Vif-dependent changes in HIV-infected cells and the published changes resulting from okadaic acid treatment ( Figure 6B and Figure 6—figure supplement 1D ) . 10 . 7554/eLife . 18296 . 019Figure 6 . Global phosphoproteomic analysis of cells infected with WT or ∆Vif HIV .",
"( A ) Vif-dependent changes in peptide and phosphopeptide abundance .",
"CEM-T4 T cells from Figure 4A and Figure 4—figure supplement 1A were subjected to TMT-based phosphoproteomic analysis .",
"Scatterplots display differences in protein ( left panel , as in Figure 4A , right panel ) and phosphopeptide abundance ( right panel ) between WT and ΔVif-infected cells .",
"Each point represents a single protein or phosphopeptide , plotted by its log2 ( fold change in abundance ) versus the statistical significance of that change .",
"q values were determined using Limma with Benjamini-Hochberg adjustment for multiple testing , with increasing −log2 ( q value ) indicating increasing significance .",
"Proteins and phosphopeptides downregulated ( red ) or upregulated ( green ) with a fold change > 2 and q value < 0 . 01 are highlighted .",
"( B ) Comparison of changes in phosphopeptide abundance between WT and ΔVif-infected CEM-T4 T cells with previously published data for okadaic acid-treated HeLa cells ( Kauko et al . , 2015 ) .",
"Lines show linear correlation with associated 95% confidence areas , r2 values and p values of a non-zero correlation .",
"( C ) Analysis of changes in phosphopeptide abundance between WT and ΔVif-infected cells CEM-T4 T cells using the PhosphoSitePlus kinase-substrate database .",
"Bars show log2 ( fold change in phosphopeptide abundance ) for peptides spanning known kinase substrate sites .",
"Error bars show the standard error of the mean .",
"( D ) Comparison of changes in phosphopeptide abundance between WT and ΔVif-infected CEM-T4 T cells with previously published data for kinase inhibitor-treated HeLa cells ( Kettenbach et al . , 2011 ) .",
"At low concentrations , MLN8054 is a selective AURKA inhibitor , but at 5 μM ( as shown ) reduced activity of AURKB and PLK1 is also observed .",
"Lines show linear correlation with associated 95% confidence areas , r2 values and p values of a non-zero correlation .",
"( E ) Vif-specific hyperphosphorylation of aurora kinase substrates .",
"Protein abundances of PLK1 , AURKA and AURKB were compared with normalised abundances of manually curated phosphopeptides targeted by the respective kinases .",
"Abundances of kinase proteins were compared using Limma with Benjamini-Hochberg adjustment for multiple testing .",
"Abundances of target phosphopeptides were compared by Repeated Measures ANOVA with Bonferroni post-test .",
"N . S . , p value>0 . 05; *p value<0 . 05; **p value<0 . 01; ***p value<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 01910 . 7554/eLife . 18296 . 020Figure 6—source data 1 . Single timepoint phosphoproteomic data . Spreadsheet of cellular phosphopeptides identified in mock , WT and ΔVif HIV-infected cells in the TMT-based single timepoint phosphoproteomic experiment ( Figure 6 , Figure 4—figure supplement 1A and Figure 6—figure supplement 1 ) .",
"Peptide sequence , details of the cognate protein and the position of the peptide within the protein are shown .",
"The column 'Phosphosite Probabilities' indicates the probability that each serine , threonine or tyrosine within the peptide is phosphorylated .",
"The amino acid is stated ( S , serine; T , threonine; Y , tyrosine ) with the position in the peptide in parentheses , followed by the probability ( % ) .",
"Each potential phosphosite is separated by a semicolon .",
"Phosphosites with a probability of over 75% are listed in the column 'Modifications in Master Proteins' , which shows a summary of the phosphorylated amino acids identified and their position in the protein .",
"Log2 ( fold change ) compares phosphopeptide abundance normalized to total protein abundance , with abundance depicted on a colour scale ( red = downregulated; green = upregulated ) .",
"q values were determined using Limma with Benjamini-Hochberg adjustment for multiple testing , with q values < 0 . 01 highlighted in gold . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 02010 . 7554/eLife . 18296 . 021Figure 6—source data 2 . Previously reported AURKA , AURKB and PLK1 targets . AURKA , AURKB and PLK1 targets were manually curated from the literature .",
"Peptides listed overlap reported sites of phosphorylation by AURKA , AURAKB or PLK1 , or are the only phosphopeptide identified from a protein known to be phosphorylated by one of these kinases , but at an unknown site .",
"Peptides where the identified phosphorylation site explicitly matches the one reported are plotted in Figure 6E ( blue shading; excluded peptides highlighted in red text ) .",
"Studies cited: ( Asano et al . , 2013; Dephoure et al . , 2008; Hengeveld et al . , 2012; Kettenbach et al . , 2011; Santamaria et al . , 2011; Welburn et al . , 2010; Yu et al . , 2005 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 02110 . 7554/eLife . 18296 . 022Figure 6—figure supplement 1 . Further phosphoproteomic analysis .",
"( A ) Remodelling of the cellular phosphoproteome by HIV infection .",
"CEM-T4 T cells from Figure 4A and Figure 4—figure supplement 1A were subjected to TMT-based phosphoproteomic analysis .",
"The scatterplot displays differences in phosphopeptide abundance between WT HIV and mock-infected cells .",
"Each point represents a single phosphopeptide , plotted by its log2 ( fold change in abundance ) versus the statistical significance of that change .",
"q values were determined using Limma with Benjamini-Hochberg adjustment for multiple testing , with increasing -log2 ( q value ) indicating increasing significance .",
"Phosphopeptides downregulated ( red ) or upregulated ( green ) with a fold change > 2 and q value < 0 . 01 are highlighted .",
"( B ) Functional annotation clusters enriched amongst proteins hyperphosphorylated in the presence of HIV infection .",
"Proteins containing phosphopeptides significantly upregulated ( q values < 0 . 01 ) in cells infected with WT HIV compared with mock-infected cells were analysed .",
"Enrichment of Gene Ontology Molecular Function and Biological Process terms against a background of all identified phosphoproteins was determined using DAVID .",
"Functional annotation clusters with enrichment scores > 1 . 3 ( equivalent to a geometric mean of all included enrichment p values<0 . 05 ) were considered significant .",
"Representative Gene Ontology terms are indicated .",
"( C ) PhosFate analysis of kinase activity in HIV-infected versus mock-infected cells .",
"Data are shown for WT HIV ( upper panel ) and ΔVif HIV ( lower panel ) .",
"A positive activity score indicates enhanced phosphorylation of kinase-specific phosphosites in infected cells .",
"Aurora kinases A and B ( AurA/AurB; red ) and other control mitotic/checkpoint kinases ( PLK1 , ATR and ATM; blue ) are highlighted .",
"Kinases represented in the dataset by a single target phosphosite were excluded .",
"( D–E )",
"Comparison of phosphoproteomic dataset with previously published data for ( D ) okadaic acid-treated ( Kauko et al . , 2015 ) and ( E ) kinase inhibitor-treated ( Kettenbach et al . , 2011 ) HeLa cells .",
"Each row shows a different pairwise comparison: top row , HIV WT versus mock; middle row , HIV ΔVif vs mock; bottom row , HIV WT vs HIV ΔVif .",
"Each column shows a different inhibitor treatment .",
"At low concentrations , MLN8054 is a selective AURKA inhibitor , but at 5 μM ( as shown ) reduced activity of AURKB and PLK1 is also observed .",
"AZDZM indicates a combined analysis of selective AURKB inhibitors AZD1152 and ZM447439 .",
"BI2536 is a selective PLK1-3 inhibitor .",
"Each scatterplot compares log2 ( fold change ) in WT/ ΔVif/mock-infected cells ( y axis ) with log2 ( fold change ) in inhibitor treated cells ( x axis ) .",
"Lines indicate linear correlation with 95% confidence areas , r2 values and p values of a non-zero correlation .",
"For each column , the most significant correlation is highlighted ( red ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 022 To identify individual kinases with enhanced activity in the presence of Vif-dependent PPP2R5A-E subunit depletion , we interrogated our data using PhosFate ( http://phosfate . com/ ) , which infers kinase activity from quantitative phosphoproteomic data by examining the coordinated regulation of known phosphosites ( Ochoa et al . , 2016 ) .",
"We found marked activation of aurora kinase A ( AURKA ) and B ( AURKB ) in cells infected with WT but not ∆Vif viruses ( Figure 6—figure supplement 1C ) , and confirmed this observation by comparing phosphorylation of sites listed on the PhosphoSitePlus ( http://www . phosphosite . org/ ) kinase substrate database ( Hornbeck et al . , 2015 ) between WT and ∆Vif virus infections ( Figure 6C ) .",
"Next , we compared Vif-dependent changes in protein phosphorylation with published alterations to the phosphoproteome of HeLa cells following treatment with the aurora kinase inhibitors MLN8054 ( Figure 6D , upper panel , and Figure 6—figure supplement 1E ) and AZD1152/ZM447439 ( AZDZM; Figure 6—figure supplement 1E ) ( Kettenbach et al . , 2011 ) .",
"As expected , we found a significant inverse correlation between our Vif-dependent changes in HIV-infected cells and the published changes resulting from aurora kinase inhibition , whereas no such correlation was seen for control datasets from the same study employing DMSO or the PLK1-3 inhibitor BI2536 ( Figure 6D , lower panel , and Figure 6—figure supplement 1E ) .",
"Finally , to fully characterize the behavior of these kinases in our dataset , we manually curated the literature for substrates of aurora kinases , including PLK1 as a negative control for Vif-specific effects ( Figure 6—source data 2 ) .",
"PLK1 protein abundance was upregulated in both WT and ∆Vif infections , with enhanced phosphorylation of kinase-specific phosphosites , but no difference between WT and ∆Vif viruses ( Figure 6E , left panels , and Figure 1—figure supplement 1G ) .",
"By contrast , whilst the aurora kinases were also upregulated equally in WT and ∆Vif infections , increased phosphorylation of kinase-specific phosphosites was only seen in the presence of Vif ( Figure 6E , middle and right panels , and Figure 1—figure supplement 1G ) .",
"Depletion of PPP2R5A-E subunits by Vif is therefore responsible for the selective amplification of aurora kinase activity in HIV-infected T cells .",
"The vif gene is found in all primate lentiviral lineages , and in most of the extant non-primate lineages .",
"We therefore assembled a panel of vif genes from diverse primate and non-primate lentiviruses ( Figure 7A and Figure 7—figure supplement 1 ) , including 14 vif variants from HIV-1 clades A-F and 6 vif variants from SIVcpz and SIVgor of chimpanzees and gorillas , the most closely related viruses to HIV .",
"Multiple vif variants from two other primate lentiviral lineages were also represented: SIVsmm of sooty mangabeys , and the viruses that resulted from cross species transmission of SIVsmm , HIV-2 and SIVmac; and SIVagm of African green monkeys .",
"Finally , a non-primate lentivirus vif variant was included , from a small ruminant lentivirus ( SRLV , or maedi-visna virus ) isolated from sheep ( Sargan et al . , 1991 ) . 10 . 7554/eLife . 18296 . 023Figure 7 . Phylogenetic conservation of PPP2R5A-E subunit degradation .",
"( A ) Phylogenetic tree based on the amino acid alignment of Vif variants used in this study .",
"( B ) Conservation of PPP2R5B subunit degradation by phylogenetically diverse lentiviral Vif variants .",
"293T cells stably expressing HA-PPP2R5B were transfected with a panel of lentiviral Vif variants and analysed by intracellular flow cytometry for HA 36 hr post-transfection .",
"The median fluorescence intensity of the transfected population is shown as a proportion of median fluorescence intensity of the untransfected population for each condition , normalized to the empty vector control .",
"Datapoints represent mean values for different Vif variants obtained from up to four independent experiments .",
"( C ) Depletion of PPP2R5A-E subunits by small ruminant lentivirus Vif .",
"293T cells stably expressing HA-PPP2R5A-E or HA-APOBEC3G were transfected with NL4-3 ( HIV-1 ) or SRLV Vif variants .",
"Histograms show GFP positive ( transfected , red shading ) and negative ( untransfected , blue line ) cells .",
"MFI values are shown for GFP positive ( red ) and negative ( blue ) cells . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 02310 . 7554/eLife . 18296 . 024Figure 7—figure supplement 1 . Identity/similarity matrix of lentiviral Vif variants . Upper-right half of matrix shows pairwise similarity between Vif variants , lower-right half shows pairwise identity .",
"For pairwise comparisons with NL4-3 Vif , relevant similarity values ( black lines ) and identity values ( white lines ) are highlighted .",
"Matrix is based on a Vif amino acid alignment carried out with the PSI-Coffee variant of the T-Coffee alignment algorithm ( Notredame et al . , 2000 ) and the SIAS tool available at http://imed . med . ucm . es/Tools/sias . html using default settings . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 02410 . 7554/eLife . 18296 . 025Figure 7—figure supplement 2 . Additional data on phylogenetic conservation of PPP2R5A-E subunit degradation .",
"( A ) Conservation of PPP2R5A subunit degradation by HIV-1 Vif variants .",
"293T cells stably expressing HA-PPP2R5A were transfected with the indicated Vif variants and analysed by intracellular flow cytometry for HA 36 hr post-transfection .",
"The median fluorescence intensity of the transfected population is shown as a proportion of median fluorescence intensity of the untransfected population for each condition , normalized to the empty vector control .",
"Mean values and standard errors are shown .",
"( B ) Conservation of PPP2R5A-E subunit degradation by phylogenetically diverse lentiviral Vif variants .",
"293T cells stably expressing different HA-tagged PPP2R5A-E subunits were transfected with the indicated Vif variants and analysed by intracellular flow cytometry for HA 36 hr post-transfection .",
"Median fluorescence intensity of the transfected population is shown as a proportion of median fluorescence intensity of the untransfected population for each condition , normalized to the empty vector control .",
"Each datapoint represents a different Vif variant .",
"( C ) Representative data for primate lentiviral Vif degradation of PPP2R5B .",
"Data shown in Figure 7B was acquired in several experiments , one example is shown here .",
"Histograms show GFP positive ( transfected , red shading ) and negative ( untransfected , blue line ) cells .",
"MFI values are shown for GFP positive ( red ) and negative ( blue ) cells . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 02510 . 7554/eLife . 18296 . 026Figure 7—figure supplement 3 . Mechanism of PPP2R5E degradation by SRLV Vif .",
"( A ) Co-immunoprecipitation of SRLV Vif with PPP2R5E .",
"WT 293T cells or 293T cells stably expressing HA-tagged PPP2R5E were transfected with FLAG-tagged SRLVE Vif , pre-treated with bortezomib ( 10 nM ) for 16 hr , and analysed by immunoblot ( IB ) for HA-PPP2R5E and FLAG-Vif 48 hr post-infection ( left panels ) .",
"Lysates were subjected to immunoprecipitation ( IP ) with anti-HA and re-analysed by immunoblot ( right panels ) .",
"293T cells transfected with empty vector were included as controls .",
"( B ) CBFβ-independent degradation of PPP2R5E by SRLV Vif .",
"293T cells stably expressing HA-PPP2R5E were transduced with the indicated shRNA .",
"Cells were then transfected with NL4-3 Vif ( upper panels ) or SRLV Vif ( lower panels ) and analysed by intracellular flow cytometry for HA .",
"Red/green shading shows Vif-transfected cells in the indicated shRNA background .",
"Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference .",
"Cells were analysed 36 hr post-transfection , and transfected cells determined by co-transfection with GFP .",
"MFI values are shown for transfected ( red/green ) and untransfected ( blue ) cells . DOI: http://dx . doi . org/10 . 7554/eLife . 18296 . 026 Vif variants were tested by transfection of 293T cells stably expressing HA-tagged PPP2R5 subunits , with the proportion of HA-tagged protein degraded in transfected cells quantitated by intracellular flow cytometry .",
"All HIV-1 variants tested were able to degrade HA-PPP2R5A , but the magnitude of effect was variable ( Figure 7—figure supplement 2A ) .",
"We therefore screened a diverse selection of Vif variants for degradation of different PPP2R5 subunits ( Figure 7—figure supplement 2B ) .",
"The ability to deplete PPP2R5 subunits was conserved across all PPP2R5A-E/Vif combinations , but most marked for PPP2R5B .",
"We therefore tested our entire panel of Vif variants for depletion of PPP2R5B , and found strong and consistent degradation ( Figure 7B and Figure 7—figure supplement 2C ) .",
"Finally , we focused specifically on the distantly related SRLV and NL4-3 ( HIV-1 ) Vif variants .",
"Vif-dependent antagonism of APOBEC proteins shows lineage-specificity , and SRLV Vif is unable to antagonize human APOBEC3G ( Larue et al . , 2010 ) .",
"Nonetheless , despite only sharing 15% amino acid identity with NL4-3 Vif ( Figure 7—figure supplement 1 ) , SRLV Vif was still able to associate with ( Figure 7—figure supplement 3A ) and efficiently degrade human PPP2R5 subunits ( Figure 7C ) .",
"Whilst Vif variants from primate lentiviruses ( including HIV-1 ) require CBFβ to enable proper protein folding , stability and interaction with the CUL5 E3 ligase complex ( Fribourgh et al . , 2014; Kim et al . , 2013; Miyagi et al . , 2014; Salter et al . , 2012 ) and mediate APOBEC depletion ( Hultquist et al . , 2012; Jager et al . , 2012; Zhang et al . , 2012 ) , Vif variants from non-primate lentiviruses ( including SRLV ) neither interact with CBFβ ( Ai et al . , 2014; Kane et al . , 2015; Yoshikawa et al . , 2016; Zhang et al . , 2014 ) nor require CBFβ to antagonize their cognate APOBEC proteins ( Ai et al . , 2014; Kane et al . , 2015 ) .",
"As with APOBEC proteins , we found CBFβ but not EloB to be dispensable for degradation of HA-PPP2R5E by SRLV Vif ( Figure 7—figure supplement 3B ) ."
],
[
"In this study , we provide a comprehensive description of temporal changes in >6500 viral and cellular proteins during HIV infection .",
"Our data confirm known HIV targets , and identify many more proteins regulated by infection .",
"Compared with other studies ( Supplementary file 1 ) , we achieve a step-change in depth of proteomic coverage , and by utilising multiplex TMT-based quantitation , we facilitate high-resolution time-based analysis .",
"To generate a cell surface proteomic map of HIV infection , we previously employed selective aminooxy-biotinylation of sialylated glycoproteins ( Plasma Membrane Profiling; PMP ) to quantitate 804 plasma membrane proteins ( Matheson et al . , 2015 ) .",
"Although 1030 proteins quantitated in our whole cell proteomic analysis also had Gene Ontology Cellular Component annotations suggesting localisation to the plasma membrane , there was limited overlap with our PMP dataset ( Figure 1—figure supplement 1H , upper panel ) .",
"The techniques are therefore non-redundant , and this is likely to reflect differential enrichment of scarce or poorly soluble/aggregate-prone glycoproteins using PMP , and intrinsic transmembrane proteins lacking significant extracellular domains or glycosylation sites using whole cell proteomics ( Figure 1—figure supplement 1H , lower panel ) .",
"In our earlier temporal proteomic study of HCMV infection , we utilised temporal classification of cellular protein expression to predict novel immunoreceptors ( Weekes et al . , 2014 ) .",
"Here , we develop and extend this methodology to predict cellular targets of specific HIV proteins .",
"Whereas HCMV encodes >150 canonical ORFs ( Wilkinson et al . , 2015 ) , HIV-1 encodes only 9 genes and 15 proteins .",
"Amongst these , the accessory proteins Vif , Vpr , Vpu and Nef have distinct patterns of temporal expression , and HIV-1 is therefore ideally suited to this approach .",
"Vpu is translated from the same transcripts as Env ( Schwartz et al . , 1990 ) , and therefore expressed late in the viral replication cycle ( Figure 1B ) .",
"Accordingly , cell surface proteins targeted specifically by Vpu ( tetherin and SNAT1 ) are depleted late in the time course of infection ( Figure 1—figure supplement 1A ) , and intracellular proteins known to be targeted by Vif ( APOBEC3C ) and Vpr ( UNG ) show distinct temporal profiles ( Figure 1D ) .",
"Based on the similarity with the temporal profile of APOBEC3C , we predicted that other proteins in cluster #1 ( Figure 3 ) would be candidate Vif targets .",
"We validated this prediction by comparing changes in protein expression during WT and ΔVif virus infections , and demonstrated that as with APOBEC3C degradation , Vif was necessary for depletion of the PP2A regulatory subunits PPP2R5A-E .",
"Conversely , like known Vpr-target UNG , proteins in cluster #2 are downregulated early in viral infection , in the absence of Vif , and in the presence of reverse transcriptase inhibitors .",
"Vpr is reported to antagonize DNA repair pathways and inhibit innate immune sensing of viral nucleic acids ( Laguette et al . , 2014; Schrofelbauer et al . , 2005 ) .",
"Interestingly , cluster #2 is markedly enriched for nucleic acid binding proteins , including proteins from families with known roles in DNA damage repair and nucleic acid sensing ( Figure 2—figure supplement 1B and Figure 2—source data 1 ) .",
"Whilst this manuscript was in preparation , downregulation of a second protein in cluster #2 , helicase-like transcription factor ( HLTF ) , was also attributed to Vpr in incoming viral particles ( Hrecka et al . , 2016; Lahouassa et al . , 2016 ) .",
"Remarkably , as for Vif targets APOBEC3C and PPP2R5A-E , temporal profiles of Vpr targets UNG and HLTF cluster very tightly ( Figure 2—figure supplement 1C–D ) .",
"Other proteins in cluster #2 with similar temporal profiles are therefore very strong candidates for novel Vpr targets , and downregulation of these proteins by Vpr may antagonize DNA repair pathways or inhibit viral nucleic acid sensing .",
"Reversible serine/threonine phosphorylation is the most commonly observed post-translational modification ( Khoury et al . , 2011 ) , and PP1 and PP2A , are the major cellular serine/threonine phosphatases .",
"The core PP2A enzyme consists of one catalytic subunit encoded by PPP2CA or PPP2CB and one structural subunit encoded by PPP2R1A or PPP2R1B .",
"The specificity of the holoenzyme is determined by the binding of an additional regulatory subunit , encoded by a total of 15 genes , split into four families ( Yang and Phiel , 2010 ) .",
"We found Vif-dependent proteasomal degradation of all five members of the B56 family ( PPP2R5A-E; also known as the B’ , PR61 or PPP2R5 family ) .",
"Since each PP2A holoenzyme contains a single regulatory subunit , it is unlikely that depletion of individual regulatory subunits destabilizes other B56 family members .",
"Given the high sequence similarity between B56 subunits , and the ability of Vif to deplete individual subunits expressed non-stoichiometrically in 293T cells , it is much more likely that degradation is mediated by a conserved Vif interaction domain in all five family members .",
"PP2A makes up 0 . 2–1% of total eukaryotic cellular protein ( Lin et al . , 1998; Ruediger et al . , 1991 ) , and whilst in many cases the relevant regulatory subunits have not been characterized , published targets of PP2A-B56 holoenzymes are nonetheless implicated in a multitude of cellular processes ( Yang and Phiel , 2010 ) .",
"In order to confirm functional PP2A-B56 antagonism and identify relevant PP2A-B56 substrates in HIV-infected cells , we therefore carried out a comprehensive , unbiased analysis of cellular protein phosphorylation during productive HIV infection , and provide a multiplex TMT-based replicated analysis of >8500 cellular phosphopeptides .",
"As expected , we found enhanced phosphorylation of proteins associated with Vpr-mediated activation of the DNA damage response and G2/M cell cycle arrest ( Figure 6—figure supplement 1B ) , reflecting increased activity of the mammalian checkpoint kinases ATR/ATM ( Figure 6—figure supplement 1C ) ( Lai et al . , 2005; Nakai-Murakami et al . , 2007; Roshal et al . , 2003; Vassena et al . , 2013 ) .",
"Conversely , PP2A-B56 antagonism by Vif resulted in hyperphosphorylation of a more limited subset of host phosphoproteins , mirroring previously reported changes seen with PP2A inhibition using okadaic acid .",
"Our unbiased analysis of Vif-dependent kinase pathways in HIV-infected cells identified a striking increase in the activity of the aurora kinases ( Figure 6C and Figure 6—figure supplement 1C ) .",
"Aurora kinase activity and abundance peak in late G2 and mitosis ( Bischoff et al . , 1998; Ly et al . , 2014 ) , and PP2A-B56 holoenzymes antagonize aurora kinase functions in other systems ( Bastos et al . , 2014; Espert et al . , 2014; Kruse et al . , 2013; Xu et al . , 2013 ) .",
"Conversely , aurora kinase activity is typically inhibited by the DNA damage response ( Bensimon et al . , 2011 ) , and reduced activity would therefore be expected in HIV-infected cells .",
"Instead , we propose that Vif-mediated antagonism of PP2A-B56 sustains aurora kinase activity in the presence of the DNA damage response .",
"Interestingly , PLK1 is also inhibited by the DNA damage response in other systems ( Bensimon et al . , 2011 ) , but kinase-active PLK1 is recruited to the SLX4 complex by Vpr in HIV-infected cells ( Laguette et al . , 2014 ) , consistent with the results of this study ( Figure 6E ) .",
"Manipulation of mitotic kinases is therefore a shared feature of the HIV accessory proteins Vpr and Vif , and pharmacological inhibitors targeting these cellular kinases may represent a viable antiviral strategy .",
"Replication of WT and ΔVif viruses in vitro is equivalent in permissive cell lines lacking APOBEC3G expression ( Sheehy et al . , 2002 ) .",
"Aurora kinase activity controls Lck kinase location and phosphorylation at the immunological synapse ( Blas-Rus et al . , 2016 ) , and kinase-active Lck is also recruited to the virological synapse during cell-cell transmission of HIV ( Vasiliver-Shamis et al . , 2009 ) .",
"It is therefore possible that Vif-mediated PP2A-B56 antagonism directly enhances cell-cell spread in vivo or in vitro in primary T cells or macrophages , but it is not currently practicable to compare replication of WT and ΔVif viruses on an APOBEC family-negative background in primary cells .",
"Alternatively , PP2A-B56 antagonism may enhance HIV replication or persistence in vivo indirectly , by modulating T cell activation or macrophage polarization ( Blas-Rus et al . , 2016; Ding et al . , 2015 ) .",
"It is also possible that in other cell types or systems , such as terminally differentiated ( non-cycling ) macrophages , signalling through alternative kinases may be differentially amplified by Vif-mediated PP2A-B56 depletion .",
"Nonetheless , since PP2A-B56 antagonism spans lineages of lentiviruses which are primarily tropic for both lymphocytes ( primate lentiviruses ) and myeloid cells ( non-primate lentiviruses ) , it is likely that modulation of key kinases is conserved across cell types .",
"The significance of host factors targeted by HIV is proven in vivo by evolutionary conservation of antagonism across a range of HIV and SIV viruses , and by the existence of similar mechanisms in other viruses .",
"For example , MHC class I proteins are targeted by Nef variants of all primate lentiviruses ( Specht et al . , 2008 ) , and manipulation of MHC class I is a common attribute of many virus families ( Randow and Lehner , 2009 ) .",
"Here , we show that the degradation of PP2A-B56 subunits is conserved across Vif variants from diverse HIV and SIV lentiviruses of primates , as well as a small ruminant lentivirus of sheep ( SRLV ) .",
"The lentiviral genus is ancient ( Gifford et al . , 2008; Katzourakis et al . , 2007; Keckesova et al . , 2009; Worobey et al . , 2010 ) , and species-specific lineages have developed through virus-host co-evolution .",
"Accordingly , the most recent common ancestor of the primate and small ruminant lentiviruses is likely to have existed in the common ancestor of primates and ruminants , approximately 100 million years ago ( Hedges et al . , 2015 ) .",
"We therefore propose that degradation of PP2A-B56 subunits is a primordial feature of Vif , present in the common ancestor of primate lentiviral and SRLV Vif variants .",
"Alternatively , Vif variants from these lineages may have independently acquired this ability .",
"Either possibility strongly suggests a critical selective advantage for lentiviral replication or persistence in vivo ."
],
[
"CEM-T4 T cells ( AIDS Reagent Program , Division of AIDS , NIAD , NIH: Dr . JP Jacobs ) ( Foley et al . , 1965 ) were cultured in RPMI supplemented with 10% FCS , 100 units/ml penicillin and 0 . 1 mg/ml streptomycin at 37°C in 5% CO2 .",
"HEK 293T cells and HeLa cells ( Lehner laboratory stocks ) ( Matheson et al . , 2015 ) were cultured in DMEM supplemented with 10% FCS , 100 units/ml penicillin and 0 . 1 mg/ml streptomycin at 37°C in 5% CO2 .",
"All cells were confirmed to be mycoplasma negative ( MycoAlert , Lonza , Switzerland ) .",
"Cell line authentication was not undertaken .",
"For SILAC labelling , CEM-T4 T cells were grown for at least 7 cell divisions in SILAC RPMI lacking lysine and arginine ( Thermo Scientific , Thermo Fisher Scientific , UK ) supplemented with 10% dialysed FCS ( Gibco , Thermo Fisher Scientific ) , 100 units/ml penicillin and 0 . 1 mg/ml streptomycin , 280 mg/L proline ( Sigma , UK ) and light ( K0 , R0; Sigma ) , medium ( K4 , R6; Cambridge Isotope Laboratories , Tewksbury , MA ) or heavy ( K8 , R10; Cambridge Isotope Laboratories ) 13C/15N-containing lysine ( K ) and arginine ( R ) at 50 mg/L .",
"Primary human CD4+ T cells were isolated from peripheral blood by density gradient centrifugation over Lympholyte-H ( Cedarlane Laboratories , Canada ) and negative selection using the Dynabeads Untouched Human CD4 T Cells kit ( Invitrogen , Thermo Fisher Scientific ) according to the manufacturer’s instructions .",
"Purity was assessed by flow cytometry for CD3 and CD4 and routinely found to be ≥95% .",
"Cells were activated using Dynabeads Human T-Activator CD3/CD28 beads ( Invitrogen ) according to the manufacturer’s instructions and cultured in RPMI supplemented with 10% FCS , 30 U/ml recombinant human IL-2 ( PeproTech , Rocky Hill , NJ ) , 100 units/ml penicillin and 0 . 1 mg/ml streptomycin at 37°C in 5% CO2 .",
"pNL4-3-dE-EGFP ( derived from the HIV-1 molecular clone pNL4-3 but encoding Enhanced Green Fluorescent Protein ( EGFP ) in the env open reading frame ( ORF ) , rendering Env non-functional ) was obtained through the AIDS Reagent Program , Division of AIDS , NIAD , NIH: Drs Haili Zhang , Yan Zhou , and Robert Siliciano ( Zhang et al . , 2004 ) and the complete sequence verified by Sanger sequencing ( Source BioScience , UK ) .",
"To generate a Vif-deficient clone , overlapping PCR mutagenesis was used to introduce a stop codon early in the Vif ORF , after the final in-frame start codon , as shown below .",
"For lentiviral transgene expression in 293T cells , N-terminal 4xHA tagged PP2R5 genes were subcloned from pCEP-4xHA-PPP2R5A-E ( a kind gift from Dr . David Virshup , Addgene plasmids #14532–14537 [Seeling et al . , 1999] ) into pHRSIN-PGK-puro ( van den Boomen et al . , 2014 ) .",
"APOBEC3G-HA was subcloned from pcDNA3 . 1-APOBEC3G-HA ( AIDS Reagent Program , Division of AIDS , NIAD , NIH: Dr . Warner C Greene [Sheehy et al . , 2002; Stopak et al . , 2003] ) .",
"PcVif , Pc∆Vif , and PcVif C114S expression vectors were a kind gift from Prof . Michael Malim , and have been previously described ( Huthoff and Malim , 2007 ) .",
"pCRV1 Vif expression vectors for HIV-1 and primate lentiviral Vif variants were a kind gift from Prof . Viviana Simon and have been previously described ( Binka et al . , 2012; Letko et al . , 2013 ) .",
"SRLV Vif was subcloned into pCRV1 by PCR from an SRLV EV1 Vif expression cloning vector , a kind gift from Dr . Barbara Blacklaws , University of Cambridge ( Wu et al . , 2008 ) .",
"The dominant negative ( DN ) CUL5 expression plasmid pcDNA3-DN-hCUL5-FLAG was a kind gift from Prof . Wade Harper ( Addgene plasmid #15823 [Jin et al . , 2005] ) .",
"For lentiviral shRNA-mediated knockdown of EloB ( TCEB2 ) , EloC ( TCEB1 ) and CBFβ ( CBFB ) in 293T cells , hairpins were cloned into pHRSIREN-PGK-hygro ( related to pHRSIREN-PGK-SBP-ΔLNGFR-W , but expressing hygromycin resistance ( Matheson et al . , 2014 ) .",
"The following oligonucleotides were inserted using BamHI-EcoRI ( only top oligonucleotides are shown ) .",
"Gene specific target sequences are underlined and the source of the target sequence design shown in parentheses .",
"VSVg-pseudotyped NL4-3-dE-EGFP HIV viral stocks were generated by co-transfection of 293T cells with pNL4-3-dE-EGFP molecular clones and pMD . G at a ratio of 9:1 ( µg ) DNA and a DNA:FuGENE 6 ( Promega , UK ) ratio of 1 µg:6 µl .",
"Media was changed the next day and viral supernatants harvested and filtered ( 0 . 45 µm ) at 48 hr prior to concentration with LentiX Concentrator ( Clontech , Takara Bio Europe , France ) and storage at −80°C .",
"VSVg-pseudotyped pHRSIN and pHRSIREN lentivector stocks were generated by co-transfection of 293T cells with lentivector , p8 . 91 and pMD . G at a ratio of 2:1:1 ( µg ) DNA and a DNA:FuGENE 6 ratio of 1 µg:3 µl .",
"Viral supernatants were harvested , filtered , concentrated if required and stored at −80°C .",
"NL4-3-dE-EGFP HIV viral stocks were titred by infection/transduction of known numbers of relevant target cells under standard experimental conditions followed by flow cytometry for GFP and CD4 at 48–72 hr to identify % infected cells .",
"CEM-T4 T cells were infected with concentrated HIV viral stocks by spinoculation at 800 ×g for 2 hr in a non-refrigerated benchtop centrifuge in complete media supplemented with 10 mM HEPES .",
"In experiments with reverse transcriptase inhibitors , cells were incubated with zidovudine ( 10 μM ) and efavirenz ( 100 nM ) ( AIDS Reagent Program , Division of AIDS , NIAD , NIH ) for 1 hr prior to spinoculation , and inhibitors maintained at these concentrations during subsequent cell culture .",
"Stable 293T cell lines were generated by transduction with pHRSIN-PGK-puro-4xHA-PPP2R5A-E or pHRSIN-PGK-puro-APOBEC3G-HA and selection with puromycin at 1 µg/ml .",
"For knockdown experiments , 293T cells transduced with HA-PPP2R5B or APOEC3G-HA were subsequently transduced with pHRSIREN ( control or gene-specific shRNA expression ) and selected with hygromycin at 200 µg/ml .",
"The following primary antibodies were used for immunoblot ( alphabetical order ) : anti-APOBEC3G ( AIDS Reagent Program , Division of AIDS , NIAID , NIH from Immunodiagnostics , 10069 ) , anti-ß-catenin ( ab32572 , Abcam , UK ) , anti-calreticulin ( PA3-900 , Thermo Scientific ) , anti-FLAG ( M2 , Sigma ) , anti-HA ( 16B12 , Biolegend ) , anti-p24 ( ab9071 , Abcam ) , anti-PPP2R5A ( ab89621 , Abcam ) , anti-PPP2R5D ( ab88075 , Abcam ) , anti-PPP2R5D ( EPR15617 , ab188323 , Abcam ) , anti-RRM2 ( ab57653 , Abcam ) , anti-Vif ( #319 , AIDS Reagent Program , Division of AIDS , NIAID , NIH: Dr . Michael Malim #6459 ( Fouchier et al . , 1996; Simon et al . , 1997 ) .",
"The following primary antibodies were used for flow cytometry: anti-CD4-AF647 ( clone OKT4; BioLegend , UK ) , anti-HA-DyLight 650 ( 16B12 , ab117515 , Abcam ) , anti-PPP2R5D ( EPR15617 , ab188323 , Abcam ) , anti-ICAM3 ( TU41 , 555957 , BD ) .",
"The following primary antibodies were used for immunoprecipitation anti-PPP2R5D ( EPR15617 , ab188323 , Abcam ) .",
"The following secondary antibodies were used: goat anti-mouse-AF647 and donkey anti-rabbit-AF647 ( flow cytometry , Molecular Probes , Thermo Fisher Scientific ) ; goat anti-mouse-HRP and anti-rabbit-HRP ( immunoblot , Jackson ImmunoResearch , West Grove , PA ) .",
"CEM-T4 T cells or 293T cells were typically lysed in TBS/2% SDS supplemented with Benzonase ( Sigma ) to reduce lysate viscosity .",
"Lysates were heated in Laemlli Loading Buffer for 15 min at 95°C , separated by SDS-PAGE and transferred to Immobilon-P membrane ( Millipore , UK ) .",
"Membranes were blocked in PBS/5% non-fat dried milk ( Marvel ) /0 . 2% Tween and probed with the indicated primary antibody overnight at 4°C .",
"Reactive bands were visualised using HRP-conjugated secondary antibodies and SuperSignal West Pico or Dura chemiluminescent substrates ( Thermo Scientific ) .",
"CEM-T4 T cells or 293T cells were lysed in 1 % NP-40 .",
"Lysates were pre-cleared with Protein A-Sepharose ( Sigma ) or IgG-Sepharose ( GE Healthcare , UK ) and incubated for 16 hr at 4°C with anti-HA coupled to agarose beads ( Sigma EZview Red Anti-HA Affinity Gel ) or anti-PPP2R5D/Protein A-Sepharose ( Sigma ) .",
"After washing in 0 . 5% NP-40 , samples were eluted with 0 . 5 mg/ml HA peptide at 37°C for 1 hr ( anti-HA immunoprecipitation ) or in Laemlli Loading Buffer without DTT at 70°C for 10 min ( anti-PPP2R5D immunoprecipitation ) .",
"Samples were separated by SDS-PAGE , and immunoblotted as described .",
"CEM-T4 T cells were starved for 20 min in methionine-free , cysteine-free RPMI/5% dialysed FCS ( Invitrogen ) , labeled with [35S]methionine/[35S]cysteine ( EasyTag EXPRESS , PerkinElmer ) for 15 min , then chased in RPMI/10% FCS at 37°C .",
"Cells were lysed in 1% Triton X-100 at the indicated timepoints , and subjected to immunoprecipitation with anti-PPP2R5D as described .",
"Samples were separated by SDS-PAGE and processed for autoradiography using the Packard Cyclone Storage Phosphor System .",
"pCMV-SPORT6 expression vectors encoding APOBEC3G , tetherin , TFAP4 and FMR1 were obtained from the MGC/IMAGE clone collection ( Dharmacon , GE Healthcare ) with the following identifiers: APOBEC3G ( IMAGE:3905631 ) , BST-2 ( IMAGE:5217945 ) , TFAP4 ( IMAGE:4181538 ) and FMR1 ( IMAGE:30347992 ) .",
"As a control , mCherry was subcloned into pCMV-SPORT6 .",
"293T cells were transfected in 24 well plates using Fugene 6 .",
"Each well received a transfection mix containing 135 ng NL4-3-dE-EGFP and 15 ng pMD . G .",
"Shortly after , each well received a second transfection mix of 150 ng pCMV-SPORT6 mCherry , APOBEC3G , tetherin , TFAP4 or FMR1 .",
"The media was changed 24 hr post-infection , and 48 hr post-infection , cell supernatants were harvested .",
"Contaminating 293T cells in the supernatants were removed by centrifugation , and a small proportion used to infect a fixed number of HeLa cells .",
"After 48 hr , the HeLa cells were analysed by flow cytometry to determine the proportion that had become GFP positive ( infected ) .",
"The MOI in each well was calculated and normalized to the MOI resulting from the supernatants of 293T cells receiving mCherry ."
]
] | [
"Viruses manipulate host factors to enhance their replication and evade cellular restriction .",
"We used multiplex tandem mass tag ( TMT ) -based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection .",
"To enable specific functional predictions , we categorized cellular proteins regulated by HIV according to their patterns of temporal expression .",
"We focussed on proteins depleted with similar kinetics to APOBEC3C , and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A ( PPP2R5A-E ) .",
"Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins , particularly substrates of the aurora kinases .",
"The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages , and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function ."
] | [
"About 100 years since it was first transmitted to humans , the Human Immunodeficiency Virus ( HIV ) infects almost 40 million people worldwide and causes more than a million AIDS-related deaths every year .",
"It is therefore critical to understand how HIV has been able to multiply and spread , and why infection with HIV causes AIDS .",
"In the evolutionary “arms race” between viruses like HIV and the cells they infect , viruses try to enhance their ability to multiply , and cells try to resist .",
"These interactions emphasise the processes that are most important for cells and viruses , and suggest new ways to treat HIV and other viral infections .",
"Proteomics is the large-scale study of molecules known as proteins , the critical building blocks of both cells and viruses .",
"Greenwood , Matheson et al . use proteomics to measure how the abundance of proteins in human cells change during HIV infection , and identify new interactions between the virus and its host .",
"The experiments distinguish more than 6500 proteins , and reveal that an HIV protein called Vif destroys several key components of a cellular protein called PP2A .",
"Previous studies have demonstrated that PP2A plays a critical role in regulating the activities of numerous other proteins and processes in cells .",
"Greenwood , Matheson et al . further show that other HIV-related viruses that infect monkeys , apes and even sheep can also counteract PP2A , suggesting that this interaction has been important during host and virus evolution .",
"The next steps following on from this work are to find out why HIV attacks PP2A , and whether drugs that interfere with this interaction may help to treat HIV infection .",
"A future challenge will be to investigate how HIV interacts with other cellular proteins highlighted by the proteomics approach ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"short report",
"neuroscience"
] | Nostril-specific and structure-based olfactory learning of chiral discrimination in human adults | elife-41296-v1 | [
[
"Asymmetric carbon atoms , those with four chemically distinct substituents , cause chirality in molecules , that is , the possibility of two nonidentical mirror image forms called enantiomers .",
"Whereas the different enantiomers of a chiral molecule have almost identical physical and chemical properties , they usually have different biological properties and can smell differently to the human nose ( Friedman and Miller , 1971; Russell and Hills , 1971 ) .",
"After all , olfactory receptors are proteins that use only one enantiomeric form ( the L form ) of the amino-acid building blocks ( Mombaerts , 1999 ) .",
"The olfactory ability of chiral discrimination has genetic basis ( Polak et al . , 1989; Saito et al . , 2009 ) but can also be acquired through learning by human adults ( Li et al . , 2008 ) , which highlights the plasticity of the adult olfactory system .",
"The mechanism underlying such plasticity is not yet fully understood .",
"In vision , the specificity of perceptual learning has long provided important clues to the locus of cortical plasticity ( Gilbert , 1994 ) .",
"For instance , learning of texture discrimination is specific for retinal input with little interocular transfer ( Karni and Sagi , 1991 ) , and is subserved by dynamic changes of activities in a subregion of the primary visual cortex ( V1 ) corresponding to the trained visual field quadrant ( Yotsumoto et al . , 2008 ) .",
"In olfaction , an ancient sense that resides in the allocortex , the specificity of perceptual learning has not been systematically characterized .",
"An earlier study tested individuals who could not smell androstenone and found that repeatedly exposing one nostril of these individuals to androstenone improved the androstenone-detection accuracy in both the exposed nostril and the unexposed nostril to the same extent ( Mainland et al . , 2002 ) .",
"The consensus seems to be that olfactory learning takes place at the level of cortical synthetic processing that shares information from both nostrils , rather than initial analytical processing of chemical features ( Mainland et al . , 2002; Wilson and Stevenson , 2003 ) .",
"Unlike the detection of androstenone , which could involve perceptual criterion ( Bremner et al . , 2003 ) , the discrimination of odor enantiomers ultimately draws upon the enantioselectivity of olfactory receptors .",
"The specificity/generalization of chiral discrimination learning thus offers a unique window into where the plasticity underlying olfactory learning firstly occurs .",
"In two experiments , we trained participants unirhinally for the discrimination between odor enantiomers ( procedure illustrated in Figure 1a ) and assessed the extent to which the learning effect transferred to the untrained nostril or an untrained enantiomer pair that is structurally distinct from ( Experiments 1 and",
"2 ) or similar to ( Experiment",
"2 ) the enantiomer pair used for training ."
],
[
"The olfactory stimuli in Experiment 1 consisted of two structurally distinct pairs of enantiomers , the enantiomers of α-pinene and those of 2-butanol ( Figure 1b ) .",
"Each served as the training pair for half of the participants and the control pair for the other half .",
"Chiral discrimination was assessed with a triangular unirhinal odor discrimination task .",
"In each trial of the task , participants sampled three bottles ( two containing one odorant , the other containing its chiral counterpart ) , one at a time , by sniffing with one nostril ( the other nostril was pinched firmly shut ) , and then selected the odd one out .",
"At baseline ( pretest ) , discrimination accuracies were at chance ( overall accuracy = 0 . 37 vs . chance = 0 . 33; t11 = 1 . 77 , p = 0 . 10; binomial test , p > 0 . 3 ) irrespective of the enantiomer pair presented ( α-pinene vs . 2-butanol , t11 = 1 . 07 , p = 0 . 31 ) or the nostril of presentation ( left vs . right , t11 = −0 . 64 , p = 0 . 54 ) .",
"Training began the day after pretest and comprised sessions spaced 1 day apart ( Figure 1a ) , where participants completed 12 trials of the unirhinal odor discrimination task and received immediate feedback about the correct choice after each trial .",
"Such reinforcement signals have been shown to guide plasticity and induce perceptual learning even in the absence of awareness of the stimulus presentation ( Roelfsema et al . , 2010 ) .",
"Only the training pair of enantiomers was presented , always to the same nostril ( either the left or the right nostril ) for a given participant .",
"Training concluded when participants reached a criterion of 9 correct choices out of 12 trials ( 75% ) on two consecutive sessions .",
"A posttest was conducted an hour later , which , like the pretest , involved both the training and the control enantiomer pairs presented to each nostril , without feedback .",
"Training lasted 12 . 1 days on average , during which period the participants’ discrimination accuracies showed a continuous improvement from chance level ( Figure 1c ) .",
"It is worth noting that the increments in performance were long-term and referred to learning retained from one daily session to the next .",
"Critically , this improvement did not transfer to the untrained nostril nor to the structurally distinct control enantiomer pair .",
"A direct comparison of the participants’ performances at pretest and posttest ( Figure 1d ) showed that training doubled the chiral discrimination accuracy for the training pair of enantiomers presented to the trained nostril ( from 0 . 36 to 0 . 70 , t11 = 6 . 37 , p < 0 . 0001 , Cohen’s d = 1 . 84 ) , but failed to affect the discrimination accuracy for the training pair presented to the untrained nostril ( t11 = 0 . 52 , p = 0 . 61 ) , or the control pair presented to the trained ( t11 = 0 . 60 , p = 0 . 56 ) or untrained nostril ( t11 = 0 . 84 , p = 0 . 42 ) .",
"Meanwhile , several participants noted at posttest that the trained enantiomers smelled much weaker when presented to the trained as opposed to the untrained nostril .",
"These results presented a sharp contrast to the internarial transfer of learning of androstenone detection ( Mainland et al . , 2002 ) , and pointed to plastic changes at a level in the olfactory system where uninarial information is still retained .",
"Considering that olfactory perception begins with systematic mappings of chemical structural features ( Khan et al . , 2008 ) yet relies heavily on memory ( Wilson and Stevenson , 2003 ) , a natural question that followed was whether such plasticity reflected enhanced differentiation of chirality ( molecular configuration ) or enhanced differentiation of overall odor quality ( odor object ) per se ( Rabin , 1988 ) with respect to the trained enantiomers .",
"In the former but not the latter case , we reasoned that learning would generalize , to a certain extent , to an enantiomer pair that is perceptually different from but structurally similar to the trained pair in terms of the substituents attached to the chiral center .",
"To tease apart the above alternatives , we adopted two structurally similar pairs of enantiomers in Experiment 2 , the enantiomers of carvone and those of limonene ( Figure 2a ) .",
"They share an isopropenyl group at the chiral center and a methyl group at the para-position , that is , at the opposite side of the ring structure relative to the chiral carbon atom bearing the isopropenyl group , and differ only by a carbonyl group .",
"We also included the enantiomers of α-pinene ( Figure 1b ) as a structurally dissimilar control .",
"Carvone , limonene and α-pinene are all found naturally in plants yet smell quite different from one another .",
"Since many volunteers readily distinguished the ( + ) and ( - ) forms of carvone and limonene ( Laska and Teubner , 1999 ) , we screened participants for the inability to do so and were able to recruit 12 non-discriminators ( out of 46 tested , 6 females ) .",
"As a group , they showed chance-level discrimination accuracies at baseline ( pretest , overall accuracy = 0 . 37 vs . chance = 0 . 33; t11 = 1 . 72 , p = 0 . 11; binomial test , p > 0 . 3 ) regardless of the enantiomer pair presented ( carvone vs . limonene vs . α-pinene , F2 , 22 = 1 . 34 , p = 0 . 28 ) or the nostril of presentation ( left vs . right , F1 , 11 = 0 . 37 , p = 0 . 55 ) .",
"Half of them were subsequently trained unirhinally to discriminate the enantiomers of carvone , and the other half the enantiomers of limonene , following identical procedures as in Experiment 1 .",
"Training lasted for 9 . 1 days on average and yielded a similar learning curve to that in Experiment 1 ( Figure 2b ) .",
"The improvement in chiral discrimination was again largely confined to the trained nostril .",
"At posttest relative to pretest , the participants showed a substantially enhanced ability to discriminate the training pair of enantiomers presented to the trained ( from 0 . 37 to 0 . 72 , t11 = 10 . 65 , p < 0 . 0001 , Cohen’s d = 3 . 08 ) , but not the untrained nostril ( t11 = 2 . 11 , p = 0 . 059 ) , with a significant difference between the two nostrils ( t11 = 6 . 05 , p < 0 . 0001 , Cohen’s d = 1 . 75 , Figure 2c ) .",
"In the meantime , they also experienced a reduction in perceived intensity when these enantiomers were presented to the trained ( t11 = −4 . 69 , p = 0 . 001 , Cohen’s d = −1 . 35 ) , but not the untrained ( t11 = 0 , p > 0 . 9 ) , nostril , consistent with the participants’ reports in Experiment 1 ( Figure 2d ) .",
"Importantly , within the trained nostril , a structure-based generalization was evident ( Figure 2c ) : Individuals trained to discriminate the enantiomers of carvone also demonstrated improved discrimination for the enantiomers of limonene ( Wilcoxon signed rank test , p = 0 . 026 ) , but not for the enantiomers of α-pinene ( p = 0 . 32 ) , and vice versa ( carvone: p = 0 . 039; α-pinene: p = 0 . 60 ) .",
"Overall , discrimination accuracies for the untrained but structurally similar enantiomer pair increased by 25 . 9% ( t11 = 7 . 00 , p < 0 . 0001 , Cohen’s d = 2 . 02 ) –– to a lesser extent as compared with the trained pair ( 35 . 2% , t11 = −2 . 59 , p = 0 . 025 , Cohen’s d = −0 . 75 ) –– when it was presented to the trained nostril , and stayed unchanged when it was presented to the untrained nostril ( t11 = 1 . 17 , p = 0 . 27 ) .",
"Those for the structurally dissimilar enantiomer pair ( i . e . the enantiomers of α-pinene ) remained unaltered regardless of the nostril of presentation ( trained: t11 = 0 . 58 , p = 0 . 57; untrained: t11 = 0 . 90 , p = 0 . 39 ) .",
"There was no significant change in perceived intensity for the untrained enantiomers , structurally similar or dissimilar , from pretest to posttest irrespective of which nostril they were presented to ( ps > 0 . 1 , Figure 2d ) .",
"In other words , the acquisition of chiral discrimination was not necessarily accompanied by a change in the perceived saliency ( intensity ) of the enantiomers’ smells .",
"It was also unlikely that overall perceptual quality contributed to the observed generalization , as the participants rated the racemic mixtures of carvone , limonene and α-pinene as smelling distinctively different from one another ( mean similarity rating <20 for any two of the three compounds , on a 100-unit visual analogue scale with 100 denoting extremely similar ) and distinguished them with ceiling-level accuracy ( >0 . 9 for any two of them vs . chance = 0 . 33 , triangular binarial odor discrimination test ) .",
"Taken together , these results suggested that the plasticity underpinning the acquisition of chiral discrimination originates in early olfactory regions that analyze the structural features ( i . e . chirality ) of uninarial olfactory input .",
"We note that they did not fully exclude the possibility that the participants performed the chiral discriminations based on a learned sub-quality of the odors ."
],
[
"Chirality represents a simple chemical feature of odorants .",
"The specificity to nostril of training and to the structure ( rather than overall quality ) of the trained compound in the learning of chiral discrimination points to the involvement of early stages in olfactory processing , where uninarial information is retained and structural features are extracted and mapped .",
"In the olfactory system , inputs from the two nostrils remain largely separated up to the primary olfactory cortex including the piriform cortex , the largest recipient of bulbar projections that is heavily implicated in odor object perception ( Carmichael et al . , 1994; de Olmos et al . , 1978; Gottfried , 2010 ) .",
"But in contrast to the chemotopy seen in the olfactory bulb ( Khan et al . , 2008; but see Soucy et al . , 2009 ) , neurons in the piriform cortex exhibit no correspondence between chemical receptive field and position , show minimal cross-habituation between odorants differing by as few as two carbons , and have been proposed to code synthetic odorant identity and odor quality ( Kadohisa and Wilson , 2006; Stettler and Axel , 2009; Wilson , 2000 ) .",
"Other primary olfactory regions have not been shown to be heavily engaged in odor discrimination .",
"In addition , axonal projections from the olfactory bulb to the anterior olfactory nucleus pars principalis , the amygdala and the olfactory tubercle exhibit only a crude set of topographical relationships ( Friedrich , 2011; Giessel and Datta , 2014; Miyamichi et al . , 2011 ) .",
"We therefore infer that an initial locus for plasticity underlying the learning of chiral discrimination likely resides in or upstream of the olfactory bulb .",
"Experience-dependent modulations of olfactory receptor expressions in the epithelium ( Tan et al . , 2015 ) , odor-induced oscillatory responses in the bulb ( Kay et al . , 2009 ) and/or tuning of the receptive fields of mitral-tufted cells ( Fletcher and Wilson , 2003; Kato et al . , 2012 ) could in turn be utilized by downstream regions like the posterior piriform to facilitate perceptual discrimination between odor enantiomers ( Li et al . , 2008 ) .",
"In mice , adult neurogenesis is found to reinforce functional inhibition in the olfactory bulb and critically mediate olfactory perceptual learning ( Moreno et al . , 2009 ) , but it is highly controversial whether adult neurogenesis exists in human brains ( Bergmann et al . , 2012; Curtis et al . , 2007; Sorrells et al . , 2018 ) .",
"The anterior olfactory nucleus pars externa precisely links mirror-symmetric isofunctional mitral-tufted cells between the olfactory bulbs ( Grobman et al . , 2018; Yan et al . , 2008 ) , which in theory could provide bilateral access to unilaterally stored olfactory associations ( Kucharski and Hall , 1987 ) .",
"However , as only a subset of mitral-tufted cells are interconnected and the connections are weak ( Grobman et al . , 2018 ) , the activities of the mirror-symmetric mitral-tufted cells , if any , are likely insufficient to enable discrimination between the trained enantiomers presented to the untrained nostril , resulting in the observed nostril-specific gain in chiral discrimination .",
"The exact neural substrates subserving human olfactory learning await further investigation .",
"Different theories of perceptual learning have been developed mainly based on studies of visual perceptual learning .",
"They assume that practice improves discrimination by enhancing the signal ( Gold et al . , 1999; Seitz and Dinse , 2007 ) , filtering/reducing external and internal noise ( Dosher and Lu , 1998 ) or inducing a top-down cascade of weight retuning ( Ahissar and Hochstein , 2004 ) .",
"As perceptual training involves exposure to stimuli and consequently adaptation , the impact of adaptation on task performance has been hard to discern ( Ahissar and Hochstein , 2004; but see Harris et al . , 2012 ) .",
"Participants in the current study consistently showed nostril-specific adaptation to the enantiomers used for training as well as nostril-specific learning of chiral discrimination .",
"In other words , training-induced perceptual gain was not accompanied by an overall enhancement of the perceptual salience ( intensity ) of the trained odor enantiomers .",
"Neither did it require perceptual adaptation , as learning generalized in a structure-based manner to an untrained enantiomer pair for which the participants reported no change in perceived intensity ( Experiment 2 ) .",
"As such , our results seem more consistent with the reverse hierarchy theory ( Ahissar and Hochstein , 2004 ) , which asserts that learning is a gradual top-down-guided ( e . g . , feedback about the correct choice ) increase in usability of first high- then lower-level task-relevant information and that this process is subserved by a cascade of top-to-bottom level modifications that enhance task-relevant information ( e . g . , configurations of the substituents attached to the chiral center ) .",
"We note that this framework also nicely reconciles our results with the internarial transfer of learning of androstenone detection observed earlier ( Mainland et al . , 2002 ) .",
"Supposing that learning proceeds as a countercurrent along the olfactory processing hierarchy , the detection of androstenone could be achieved by heightened attention or sensitization to its olfactory quality ( amplification of a weak signal which lowers detection threshold ) and hence possibly engages a higher generalizing level , whereas the learning of chiral discrimination likely relies more on lower-level inputs and is thus more specific .",
"On a separate note , a previous study by researchers in Germany tested the olfactory discrimination ability of 20 participants for 10 pairs of enantiomers ( Laska and Teubner , 1999 ) and found that they significantly discriminated the optical isomers of α-pinene , carvone and limonene ( mean accuracies ≈ 80% for each enantiomer pair vs . chance = 33% ) despite marked individual differences in discrimination performance .",
"In our testing , we also noticed that many individuals readily distinguished the ( + ) and ( - ) forms of carvone and limonene and had to screen participants for the inability to do so in Experiment 2 .",
"However , we did not encounter any individual who could reliably tell apart the enantiomers of α-pinene without training .",
"It is unclear whether this interesting discrepancy was due to genetic or environmental differences between Chinese and German participants .",
"But it indicates that structural properties of a chiral compound cannot fully predict whether the enantiomers smell differently to a human recipient .",
"To summarize , the current study suggests that human olfactory perception can partially be a learned trait .",
"Learning can take place at a very early stage of olfactory processing , where the structural features of odorants are analyzed and extracted .",
"Put differently , early analytical processing of the chemical features of unirhinal input is both plastic ( experience-dependent ) and behaviorally relevant in human adults .",
"Unlike earlier beliefs ( Mainland et al . , 2002 ) , one nostril does not always know what the other learns ."
],
[
"A total of 24 healthy non-smokers completed the training for chiral discrimination , which lasted for 10 to 11 days on average: 12 ( 6 males , mean age ±SD = 24 . 8 ± 2 . 1 years ) in Experiment 1 and another 12 ( 6 males , 21 . 3 ± 2 . 0 years; screened from 46 volunteers ) in Experiment 2 .",
"The sample size was motivated by those used in previous studies ( Karni and Sagi , 1991; Mainland et al . , 2002 ) .",
"All participants reported to have a normal sense of smell and no respiratory allergy or upper respiratory infection at the time of testing .",
"Written informed consent and consent to publish was obtained from participants in accordance with ethical standards of the Declaration of Helsinki ( 1964 ) .",
"The study was approved by the Institutional Review Board at Institute of Psychology , Chinese Academy of Sciences .",
"The olfactory stimuli were presented in identical 280 ml narrow-mouthed glass bottles .",
"They consisted of the enantiomers of α-pinene ( 1% v/v in propylene glycol ) and 2-butanol ( 1% v/v in propylene glycol ) in Experiment 1 ( Figure 1b ) , and the enantiomers of carvone ( 0 . 5% v/v in propylene glycol ) , limonene ( 0 . 5% v/v in propylene glycol ) and α-pinene ( 1% v/v in propylene glycol ) , as well as their racemic mixtures ( 0 . 5% , 0 . 5% and 1% v/v in propylene glycol , respectively ) , in Experiment 2 ( Figure 2a ) .",
"Each bottle contained 10 ml clear liquid and was connected with either one ( for those containing an odor enantiomer ) or two ( for those containing a racemic mixture ) Teflon nosepieces .",
"Participants were instructed to sample the olfactory stimuli by inhaling through the nosepieces and exhaling through the mouth .",
"At the concentrations used , the odor enantiomers were clearly detectable yet did not elicit a significant trigeminal response , as assessed in an independent panel of 20 participants ( 10 males , 22 . 6 ± 1 . 7 years ) , who failed to localize the side of uninarial stimulation for all of them in a lateralization test ( Wysocki et al . , 2003 ) ( 32 trials per participant , mean accuracy for each enantiomer <0 . 52 vs . chance = 0 . 5 , ps >0 . 7 ) .",
"Experiment 1 comprised three phases: pretest , training and posttest ( Figure 1a ) .",
"At pretest ( day",
"0 ) and posttest ( day N ) , the participants performed 36 trials of a triangular unirhinal odor discrimination task .",
"In each trial of the task , they were blindfolded and were instructed to close either the left or the right nostril with the index finger .",
"The open nostril was subsequently presented with three bottles , two containing one odorant , the other containing its chiral counterpart , one at a time in random order , and the participants were asked to select the odd one out .",
"Specifically , the nosepieces connected with the bottles were positioned into the open nostril by the experimenter , one at a time , without the participants holding or touching the bottles .",
"There were 9 trials per enantiomer pair per nostril , with a break of at least 30 s in between two trials .",
"The order of the enantiomer pairs was counterbalanced across participants ( i . e . , half of the participants first performed the task with the enantiomers of α-pinene , followed by the enantiomers of 2-butanol; the other half did the reverse ) .",
"For each participant , the nostril of presentation ( open nostril ) was alternated in consecutive trials .",
"No feedback was provided .",
"Training began the day after pretest ( day",
"1 ) and consisted of sessions spaced 1 day apart ( day 1 to day N ) .",
"In each session , the participants , blindfolded , completed 12 trials of the triangular unirhinal chiral discrimination task and received immediate feedback about the correct choice .",
"Half of the participants were randomly assigned to be trained with the enantiomers of α-pinene and the other half the enantiomers of 2-butanol .",
"The training pair of enantiomers was always presented to the left nostril for 5 of the participants ( randomly assigned , 2 trained with the enantiomers of α-pinene ) and to the right nostril for the other 7 ( 4 trained with the enantiomers of α-pinene ) .",
"We limited the number of trials in each session to reduce olfactory fatigue , as participants in our pilot testing reported that they could only smell a faint odor after about 12 trials , which were at least 36 repetitive exposures to the ( + ) and ( - ) forms of the same chiral compound presented in the same nostril .",
"Training concluded when the participants reached a criterion of 9/12 correct choices on two consecutive sessions ( day N-1 and day N ) .",
"The posttest was conducted an hour later ( day N ) .",
"The same bottles used for the trained nostril were also used for the untrained nostril .",
"During the course of the experiment , the odor solution in each bottle was replaced with a freshly prepared solution of the same compound every two days .",
"Experiment 2 followed similar procedures to those described above , except for the followings: Participants were screened for the inability to distinguish the ( + ) and ( - ) forms of carvone and limonene ( accuracy <0 . 56 for each enantiomer pair vs . chance = 0 . 33 ) .",
"At pretest ( day",
"0 ) and posttest ( day N ) , each participant completed 54 trials of the triangular unirhinal odor discrimination task ( 9 trials per enantiomer pair per nostril , 3 pairs of enantiomers ) .",
"Prior to the odor discrimination task , they rated the intensity of each odor enantiomer , presented monorhinally , on a 7-point Likert scale , with 7 signifying very strong .",
"In the training phase ( day 1 to day N ) , half of the participants were trained with the enantiomers of carvone and the other half the enantiomers of limonene .",
"The training pair of enantiomers was always presented to the left nostril for half of the participants ( 3 trained with the enantiomers of carvone ) and to the right nostril for the other half ( 3 trained with the enantiomers of carvone ) .",
"After the posttest , 10 of the participants sampled the racemic mixtures of carvone , limonene and α-pinene , presented birhinally , and provided ratings for the perceived similarities between every two of them on a 100-unit visual analogue scale , with 100 representing extremely similar .",
"They also completed a triangular odor discrimination task using these three racemic mixtures .",
"In each trial of the task , they were blindfolded and were presented birhinally with three bottles , two containing one racemic mixture , the other containing a different racemic mixture , one at a time in random order , and were asked to select the odd one out .",
"There were 12 trials , 4 trials for every pairwise combination , with a break of at least 30 s in between two trials .",
"No feedback was provided .",
"For each of Experiments 1 and 2 , the participants’ chiral discrimination accuracies at pretest were analyzed in a repeated measures ANOVA , using enantiomer pair ( Experiment 1: α-pinene vs . 2-butanol; Experiment 2: carvone vs . limonene vs . α-pinene ) and nostril of presentation ( left vs . right ) as the within-subject factors .",
"The overall accuracies were subsequently compared against chance ( 1/3 ) in a one-sample t test and a binomial test ( averaged number of correct responses in 36 or 54 trials vs . chance ) .",
"We were primarily interested in whether training-induced perceptual gain would transfer to an untrained pair of odor enantiomers or to the untrained nostril .",
"To this end , we performed a series of paired-samples t tests to compare the chiral discrimination accuracies between posttest and pretest for each pair of odor enantiomers presented to the trained nostril as well as to the untrained nostril .",
"In Experiment 2 , as the participants showed significantly improved chiral discrimination for the untrained enantiomer pair that was structurally similar to the trained pair when it was presented to the trained nostril , we performed follow-up Wilcoxon signed rank tests ( non-parametric , suitable for small sample sizes ) to specifically examine whether those trained with the enantiomers of carvone ( n = 6 ) also demonstrated improved discrimination ( posttest vs . pretest ) for the enantiomers of limonene and α-pinene , and whether those trained with the enantiomers of limonene ( n = 6 ) also demonstrated improved discrimination for the enantiomers of carvone and α-pinene .",
"In addition , we performed paired-samples t tests to compare the increments in chiral discrimination accuracy ( 1 ) between the trained enantiomer pair and the untrained but structurally similar pair presented to the trained nostril and ( 2 ) between the trained enantiomer pair presented to the trained nostril and that presented to the untrained nostril .",
"We also compared in a series of paired-samples t tests the participants’ intensity ratings between posttest and pretest for each pair of odor enantiomers ( ratings averaged between the two enantiomers in each pair ) presented to the trained or the untrained nostril .",
"All statistical tests other than the binomial tests were two-tailed ."
]
] | [
"Practice makes perfect .",
"In human olfaction , such plasticity is generally assumed to occur at the level of cortical synthetic processing that shares information from both nostrils .",
"Here we present findings that challenge this view .",
"In two experiments , we trained human adults unirhinally for the discrimination between odor enantiomers over a course of about 10 to 11 days .",
"Results showed that training-induced perceptual gain was restricted to the trained nostril yet partially generalized to untrained odor enantiomers in a structure- rather than quality- based manner .",
"In other words , learning enhanced the differentiation of chirality ( molecular configuration ) as opposed to overall odor quality ( odor object ) per se .",
"These findings argue that , unlike earlier beliefs , one nostril does not readily know what the other learns .",
"Moreover , the initial analytical processing of the structural features of uninarial olfactory input remains plastic in human adults ."
] | [
"Although we may only become consciously aware of our sense of smell when we encounter something pungent , it can greatly influence our quality of life .",
"Smells are processed by our olfactory system , a collection of receptors and nerve cells in the nose and brain .",
"Odor molecules activate the olfactory system when they bind to receptors in the nostrils .",
"These molecules can have a wide range of chemical and physical properties .",
"However , some odor molecules are mirror images of each other .",
"These variants are known as enantiomers .",
"Some people can naturally smell the difference between enantiomers; others can be taught how to tell them apart .",
"Studying this training process could help us to understand how the olfactory system adapts to new circumstances .",
"Feng and Zhou trained volunteers to distinguish between odor enantiomers – but only using one nostril .",
"After training , the volunteers were better able to tell the difference between different enantiomers – even for certain scents they had not been trained to discriminate – when sniffing through the trained nostril .",
"However , they got no better at distinguishing between enantiomers when they sniffed them using the other nostril .",
"Overall , the results reported by Feng and Zhou confirm that human adults can learn how to process the structural features of odorants .",
"However , in contrast to earlier beliefs , they also suggest that one nostril does not readily know what the other learns .",
"This new understanding of how the olfactory system adapts could ultimately help us to develop therapies to restore a lost sense of smell ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and Methods"
] | [
"immunology and inflammation"
] | Mitochondrial Ca2+ and membrane potential, an alternative pathway for Interleukin 6 to regulate CD4 cell effector function | elife-06376-v2 | [
[
"Interleukin 6 ( IL-6 ) is an inflammatory cytokine that is elevated in several autoimmune and inflammatory disorders , including rheumatoid arthritis ( RA ) ( Kishimoto , 2005 ) .",
"Inhibition of IL-6 signaling by an anti-IL-6R antibody has been shown to be a highly effective therapy in treating patients with RA ( Tanaka and Kishimoto , 2012 ) .",
"IL-6 plays crucial role in regulating CD4 T helper cell differentiation and cytokine production ( Dienz and Rincon , 2009 ) .",
"It enhances Th2 differentiation through an auto-feedback loop by upregulating autocrine IL-4 production ( Rincon et al . , 1997; Diehl et al . , 2002 ) .",
"IL-6 inhibits IFNγ production and Th1 differentiation ( Diehl et al . , 2000 ) .",
"In combination with TGFβ , IL-6 also contributes to the differentiation of Th17 cells ( Bettelli et al . , 2006; Ivanov et al . , 2006; Zhou et al . , 2007 ) .",
"IL-6 inhibits regulatory T cell function and downregulates Foxp3 expression ( Pasare and Medzhitov , 2003; Dienz and Rincon , 2009 ) .",
"In addition , IL-6 alone , without the need of TGFβ , induces IL-21 expression , a mechanism by which it promotes the generation of follicular T helper ( Tfh ) cells ( Nurieva et al . , 2008; Suto et al . , 2008; Dienz et al . , 2009; Diehl et al . , 2012 ) .",
"IL-6 binds to its membrane receptor , which triggers signaling through gp130 , a common transducer that activates Jak/Stat3 and Ras/MAPK pathways in T cells ( Boulanger et al . , 2003; Heinrich et al . , 2003; Kishimoto , 2005 ) .",
"Stat3 is a transcription factor present in cytosol but translocates to the nucleus upon stimulation where it mediates the expression of numerous genes .",
"Stat3 has been previously implicated in the regulation of genes involved in cell survival and proliferation by directly binding to multiple survival genes , including Bcl2 , Fos , Jun , Mcl1 and Fosl2 ( Hirano et al . , 2000; Bourillot et al . , 2009; Durant et al . , 2010; Carpenter and Lo , 2014 ) .",
"Additionally , IL-6-dependent Stat3 activation plays an important role in the expression of several cytokine genes , including Il21 and Il17 ( Mathur et al . , 2007; Zhou et al . , 2007; Dienz et al . , 2009 ) .",
"In addition to its role as a nuclear transcription factor , Stat3 has been found within mitochondria in liver , heart and some cell lines where it enhances the mitochondrial respiratory chain activity ( Gough et al . , 2009; Wegrzyn et al . , 2009 ) .",
"However , no studies have addressed whether IL-6 regulates mitochondrial function through Stat3 .",
"IL-6 has for long been associated with metabolic changes and high levels of IL-6 in serum have been correlated with BMI ( Mohamed-Ali et al . , 1997; Fried et al . , 1998; Vgontzas et al . , 2000 ) .",
"Recent studies indicate that IL-6 is linked to glucose homeostasis in adipose tissue and it participates in the switch from white to brown fat tissue in cancer-induced cachexia ( Stanford et al . , 2013; Petruzzelli et al . , 2014 ) .",
"However , it remains unclear whether IL-6 has a direct effect on the metabolism of cells .",
"But in the context of ischemia-reperfusion injury in cardiomyocytes , IL-6 has been shown to maintain mitochondrial membrane potential ( MMP ) in cardiomyocytes ( Smart et al . , 2006 ) .",
"Despite the known role of IL-6 in the CD4 cell effector function , no studies have addressed whether IL-6 has an effect on mitochondrial function in CD4 cells .",
"Here we show that IL-6 plays an important role in maintaining MMP late during CD4 cell activation in a Stat3-dependent manner .",
"IL-6-mediated mitochondrial hyperpolarization is , however , uncoupled from the oxidative phosphorylation and ATP production .",
"Instead , IL-6 uses the high MMP to raise mitochondrial Ca2+ and , consequently , cytosolic Ca2+ levels to promote cytokine expression late during activation .",
"Thus we have identified a previously undescribed mechanism by which IL-6 regulates CD4 cell effector function ."
],
[
"Although the role of IL-6 in CD4 cell differentiation and cytokine gene expression is well established , little is known about the role of this cytokine in mitochondrial function .",
"An essential function of the mitochondrial electron transport chain ( ETC ) , in addition to the transfer of electrons , is the generation of an electrochemical gradient across the mitochondrial inner membrane by accumulating H+ at the intermembrane space .",
"This electrochemical gradient , known as MMP , is used as a mechanism to generate ATP .",
"Since IL-6 has been associated with maintaining MMP in cardiomyocytes ( Smart et al . , 2006 ) , we examined whether IL-6 regulates the MMP in CD4 cells during activation .",
"Fresh CD4 cells were activated with anti-CD3 and anti-CD28 antibodies ( Abs ) in the presence or absence of IL-6 for different periods of times , stained with TMRE ( an MMP indicator ) , and analyzed by flow cytometry .",
"Most freshly isolated CD4 cells were hyperpolarized as shown by the high TMRE staining ( Figure 1A ) .",
"However , cells activated in the absence of IL-6 depolarized progressively during activation ( Figure 1A ) .",
"Interestingly , the presence of IL-6 prevents mitochondrial depolarization during CD4 cell activation ( Figure 1A ) .",
"After 48hr of activation , most CD4 cells activated in the presence of IL-6 maintained a high MMP ( TMREhigh ) ( Figure 1B ) .",
"In contrast to IL-6 , the presence of exogenous IL-2 , the main growth factor of T cells , did not affect MMP in activated CD4 cells ( Figure 1C ) , supporting a selective role for IL-6 on MMP . 10 . 7554/eLife . 06376 . 003Figure 1 . IL-6 sustains high mitochondrial membrane potential ( MMP ) late during activation .",
"( A ) MMP during activation of CD4 cells with anti-CD3/CD28 Abs over time in the presence or absence of IL-6 , as determined by staining with TMRE and flow cytometry analysis .",
"( B ) Percentage of CD4 cells with TMREhigh ( defined by the gate displayed in ( A ) at 48 hr , after activation as in ( A ) ( n = 3 ) .",
"( C ) MMP during activation of CD4 cells in the absence or presence of IL-2 was determined by staining with TMRE and flow cytometry analysis .",
"( D ) Expression of NDUFA9 , NDUFS3 , COX IV and ACTIN examined by Western blot analysis using whole-cell extracts from CD4 cells activated for 48 hr . ( E ) Percentage of live CD4 cells activated as in ( A ) for 48 hr , determined by flow cytometry .",
"( n = 3 ) .",
"( F ) MMP in OT-II CD4 cells activated by WT or IL-6 KO APCs with OVA peptide in the presence or absence of the supplement of exogenous of IL-6 ( IL-6 ) or blocking anti-IL-6 antibody ( αIL-6 ) for 48 hr . ( n = 3 ) .",
"( G ) Percentage of TMREhigh population in OT-II CD4 cells from ( F ) ( n = 3 ) .",
"( H ) OT-II CD4 cells were adoptively transferred to WT or IL-6 KO recipient mice that were then immunized with ovalbumin ( and Alum ) .",
"After 2 days , cells were harvested to examine for MMP .",
"Percentage of TMREhigh population in activated OT-II T cells from WT or IL-6 KO mice were determined by TMRE staining and flow cytometry analysis .",
"Error bars represent the mean ± SD .",
"*denotes p < 0 . 05 , as determined by Student's t test .",
"Results are representative of 2–3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 003 To examine the effect of IL-6 on mitochondrial mass and levels of ETC complexes , we performed Western blot analysis for subunits of these complexes using whole cell extracts .",
"IL-6 did not affect the overall mitochondrial mass as determined by the levels of COX IV ( Complex IV subunit of ETC ) , NDUFS3 and NDUFA9 ( Complex I subunits ) ( Figure 1D ) .",
"In addition , the frequency of live cells among those activated in the presence of IL-6 was not significantly different from the frequency of live cells in the absence of IL-6 ( Figure 1E ) .",
"Thus , the increase of MMP triggered by IL-6 is not a consequence of survival or change in mitochondrial mass .",
"Antigen presenting cells ( APCs ) are one of the major sources of IL-6 during CD4 cell activation .",
"To examine whether IL-6 was required to maintain the mitochondrial hyperpolarization during antigen activation , naive CD4 cells were obtained from OT-II TCR transgenic mice ( Barnden et al . , 1998 ) and activated with OVA peptide and APCs isolated from WT or IL-6 KO mice .",
"Similar to CD4 cells activated with anti-CD3/CD28 Abs in the presence of IL-6 , a large frequency of OT-II CD4 cells activated with WT APC showed a high MMP ( Figure 1F , G ) .",
"However , a blocking anti-IL-6 Ab drastically decreased the frequency of cells with high MMP ( Figure 1F , G ) .",
"In contrast to WT APCs , very low frequency of activated CD4 cells showed high MMP when APC from IL-6 KO mice were used ( Figure 1F , G ) .",
"Remarkably , addition of exogenous IL-6 to cells activated with IL-6 KO APCs restored high MMP ( Figure 1F , G ) .",
"Thus , these results indicate that IL-6 derived from APC during in vitro activation of CD4 cells is essential to maintain mitochondrial hyperpolarization .",
"To address the role of IL-6 in regulating the MMP in CD4 cells during in vivo activation , we performed adoptive transfer of OT-II CD4 cells into WT or IL-6 KO mice as hosts .",
"Mice were then immunized with ovalbumin , and after two days , cells were harvested to examine their MMP .",
"Similar to in vitro results , the fraction of OT-II cells maintaining a high MMP was significantly greater in WT mice relative to IL-6 KO mice ( Figure 1H ) .",
"Together , these results indicate that IL-6 plays an essential role in maintaining the MMP during activation of CD4 cells .",
"Morphological states of highly pleomorphic inner membrane cristae reflect the different mitochondrial metabolic stages .",
"Mitochondrial cristae shape has been shown to influence the efficiency of the respiratory chain in part by affecting the formation of respiratory chain supercomplexes ( RCS ) ( Hackenbrock , 1966; Gomes et al . , 2011; Cogliati et al . , 2013 ) , formed of Complex I together with Complex III and Complex IV .",
"The function of RCS is to facilitate the transfer of electrons between complexes and increase Complex I activity while reducing the electron leak from ETC and mitigate the production of reactive oxygen species ( ROS ) ( Schägger , 1995; Acín-Pérez et al . , 2008; Althoff et al . , 2011; Winge , 2012 ) .",
"To determine whether IL-6 could affect cristae shape , we examined CD4 cells activated in the presence or absence of IL-6 by transmission electron microscopy ( TEM ) imaging .",
"No obvious differences in mitochondrial integrity or mitochondrial mass were observed in cells activated with or without IL-6 ( Figure 2A ) .",
"Similarly , there was no increase in the number of mitochondria in CD4 cells activated in the presence of IL-6 ( Figure 2—figure supplement 1 ) .",
"However , the morphology of the mitochondrial cristae in cells activated with IL-6 was different from that of cells activated without IL-6 ( Figure 2A ) .",
"The number of mitochondria with expanded and disorganized cristae was greater in CD4 cells activated in the absence of IL-6 compared with CD4 cells activated with IL-6 ( Figure 2B ) .",
"In contrast , the number of mitochondria with tight and organized cristae was higher in cells activated in the presence of IL-6 ( Figure 2C ) .",
"Thus , IL-6 affects mitochondrial cristae shape during activation of CD4 cells . 10 . 7554/eLife . 06376 . 004Figure 2 . IL-6 facilitates the formation of respiratory chain supercomplexes in CD4 cells during activation .",
"( A ) Transmission electron microscopy analysis of mitochondria in CD4 cells activated in the presence or absence of IL-6 .",
"Original magnification , 6 , 000× .",
"Bars represent 500 nm; Blue arrows indicate mitochondria ( B ) Representative image of an ‘expanded cristae’ mitochondrion ( Left ) .",
"Number of mitochondria with expanded cristae in CD4 cells activated in the presence or absence of IL-6 ( right ) .",
"( n = 25 ) .",
"( C ) Representative image of a ‘tight cristae’ mitochondrion ( Left ) .",
"Number of mitochondria with tight cristae in CD4 cells activated in the presence or absence of IL-6 ( right ) .",
"( n = 23 ) .",
"Error bars represent the mean ± SD .",
"*denotes p < 0 . 05 , as determined by Student's t test .",
"Results are representative of 2 experiments .",
"( D ) Digitonin-soluble mitochondrial extracts from CD4 cells were resolved by BN-PAGE and transferred onto a membrane ( Western blot ) and immunoblotted for NDUFA9 and Core1 protein .",
"Immunoreactivity for the two proteins within the supercomplex ( SC ) region is shown .",
"Immunoreactivity for NDUFA9 with monomeric complex I ( Com I ) and Core1 with dimeric complex III ( Com III ) are shown .",
"Lower panels display the densitometry of NDUFA9 ( left ) and Core I ( right ) subunits within the supercomplex region ( SC ) and densitometry at the individual Complex I ( Com I ) and Complex III ( Com III ) respectively .",
"( E ) Mitochondrial ROS during activation of CD4 cells with anti-CD3/28 Abs in the presence or absence of IL-6 for 48 hr was determined by staining with MitoSox and flow cytometry analysis .",
"( F ) Percentage of CD4 cells with mROShigh , defined by the gate displayed in ( E ) at 48 hr , after activation as in ( E ) ( n = 4 ) .",
"Error bars represent the mean ± SD .",
"*denotes p < 0 . 05 , as determined by Student's t-test .",
"Results are representative of 2 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 00410 . 7554/eLife . 06376 . 005Figure 2—figure supplement 1 . IL-6 does not increase the number of mitochondria in CD4 cells during activation . Number of mitochondria in CD4 cells activated in the presence or absence of IL-6 was analyzed by TEM .",
"( n = 25 ) .",
"p > 0 . 5 , as determined by Student's t test . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 005 To determine whether the effect of IL-6 on the mitochondrial cristae morphology could be reflected in an altered formation of RCS as a mechanism to maintain a high MMP , we examined the presence of RCS in activated CD4 cells .",
"We performed blue-native gel electrophoresis ( BN-PAGE ) using mitochondrial extracts generated in the presence of digitonin to preserve the supercomplexes ( SCs ) ( Acín-Pérez et al . , 2008 ) , followed by Western blot analysis .",
"The levels of RCS but not the levels of individual Complex I or Complex III were increased in mitochondria from IL-6-stimulated CD4 cells , as determined by the presence of NDUFA9 ( Complex I ) and Core I ( Complex III ) within the RCS region ( Figure 2D ) .",
"Since the formation of RCS is associated with increased MMP but reduced mitochondrial ROS ( mROS ) ( Schägger , 1995; Acín-Pérez et al . , 2008; Althoff et al . , 2011; Winge , 2012 ) , we examined the production of mROS in CD4 cells activated with or without IL-6 by flow cytometry analysis using MitoSOX , a mitochondrial superoxide indicator .",
"Despite the increased MMP , IL-6 reduced the production of mROS ( Figure 2E , F ) .",
"Thus , the formation of RCS facilitated by IL-6 makes possible for this cytokine to sustain mitochondria hyperpolarization while minimizing the production of mROS during activation of CD4 cells .",
"The energy released from the transport of H+ from the mitochondrial intermembrane space to the mitochondrial matrix through F0F1 ATP synthase , a subunit of Complex V , is coupled to ATP generation .",
"Thus , an increased MMP elicited by IL-6 could potentially lead to an increase in mitochondrial ATP synthesis .",
"We therefore examined ATP production in CD4 cells activated in the presence or absence of IL-6 .",
"Surprisingly , despite of the increased MMP , intracellular ATP levels were not affected by IL-6 ( Figure 3A ) .",
"TCR stimulation has been shown to trigger rapid ATP release from CD4 cells ( Yip et al . , 2009 ) .",
"It was therefore possible that IL-6 increased ATP synthesis but also ATP release .",
"However , analysis of ATP levels in culture supernatants of activated cells showed no difference in the levels of extracellular ATP ( Figure 3B ) .",
"Since most ATP in activated T cells is generated through glycolysis ( Pearce et al . , 2013 ) , increased MMP by IL-6 could enhance mitochondrial oxidative phosphorylation ( OXPHOS ) but have minimal effect on overall ATP levels .",
"To further address the effect of IL-6 on mitochondrial OXPHOS , we examined oxygen consumption rate ( OCR ) using the Seahorse XF24 analyzer ( Wu et al . , 2007 ) .",
"No statistically significant difference in basal mitochondrial OCR or maximal respiratory capacity was detected ( Figure 3C ) .",
"Thus , the effects of IL-6 on the MMP are uncoupled from OXPHOS . 10 . 7554/eLife . 06376 . 006Figure 3 . IL-6-mediated increase in mitochondrial membrane potential in CD4 cells is uncoupled from OXPHOS .",
"( A ) Intracellular ATP levels ( per 104 cells ) in CD4 cells activated in the presence ( IL-6 ) of absence of IL-6 ( Med ) ( n = 5 ) .",
"( B ) Extracellular ATP levels in supernatants of CD4 cells activated for 48 hr ( n = 3 ) .",
"( C ) Oxygen consumption rates in CD4 cells activated with or without IL-6 for 48 hr , under basal conditions and in response to oligomycin ( oligo ) , FCCP or rotenone plus antimycin ( R/A ) .",
"Average of basal level OCR ( n = 3 ) and the average of maximal OCR ( n = 3 ) are shown .",
"( D ) Lactate levels in supernatant of CD4 cells activated for 48 hr ( n = 3 ) .",
"( E ) Extracellular acidification rates ( ECAR ) were measured in activated CD4 cells ( 48 hr ) under basal conditions or in response to glucose , FCCP or 2-deoxyglucose ( 2-DG ) sequentially .",
"Average of basal ECAR levels are graphed on the right ( n = 3 ) .",
"Error bars represent mean ± SD .",
"No statistically significant differences ( p > 0 . 05 ) were found for any of the assays , as determined by Student's t-test or two-way ANOVA .",
"Results are representative of 2–3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 006 We also examined whether the mitochondrial hyperpolarization by IL-6 could compromise anaerobic glycolysis during activation .",
"Culture supernatants of activated CD4 cells with or without IL-6 were assayed for lactate production .",
"Lactate production was not significantly different in cells activated with IL-6 ( Figure 3D ) .",
"The Seahorse XF24 analyzer was also used to measure the extracellular acidification rate ( ECAR ) , another alternative approach to examine the rate of glycolysis .",
"Consistent with the production of lactate , there was no difference in anaerobic glycolysis in the presence of IL-6 during CD4 cell activation ( Figure 3E ) .",
"Thus , although IL-6 maintains high MMP late during the activation of CD4 cells , it does not alter rates of OXPHOS or anaerobic glycolysis .",
"Although the main function of the MMP is to drive the generation of ATP through OXPHOS , MMP also plays an important role in mitochondrial Ca2+ homeostasis ( Rizzuto et al . , 2012 ) .",
"Mitochondria are emerging as the primary subcellular Ca2+ store which buffers cytosolic Ca2+ ( Starkov , 2010 ) .",
"Mitochondrial Ca2+ uptake is modulated by mitochondrial calcium uniporter ( MCU ) and it is dictated by the MMP ( Baughman et al . , 2011; De Stefani et al . , 2011; Mallilankaraman et al . , 2012a , 2012b; Shanmughapriya et al . , 2015 ) , while Ca2+ release from mitochondria is mediated by the mitochondrial Na+/Ca2+ exchanger ( mNCLX ) ( Kirichok et al . , 2004; Palty et al . , 2010; Nita et al . , 2012; Rizzuto et al . , 2012 ) .",
"Upon TCR engaging , it has been reported that the formation of the immunological synapse triggers early store-dependent Ca2+ influx through mitochondrial Ca2+ buffering ( Hoth et al . , 1997; Quintana et al . , 2007 ) .",
"However , little is known about the mitochondrial Ca2+ signaling in activated effector cells and how it may contribute to CD4 cell effector functions .",
"We examined whether an increased MMP regulated by IL-6 could affect mitochondrial Ca2+ homeostasis .",
"CD4 cells activated in the presence or absence of IL-6 for 48 hr were stained with Rhod-2 AM , a selective indicator for mitochondrial Ca2+ ( Hajnoczky et al . , 1995; Brisac et al . , 2010 ) and analyzed by flow cytometry .",
"Consistent with an increased MMP , there was a significantly greater frequency of cells with high levels of mitochondrial Ca2+ ( Rhod-2high ) in the presence of IL-6 ( Figure 4A , C ) .",
"Short treatment of IL-6-activated CD4 cells with the depolarizing agent Carbonyl cyanide m-chlorophenyl hydrazone ( CCCP ) significantly reduced the frequency of cells with high levels of mitochondrial Ca2+ ( Rhod-2high ) ( Figure 4B , C ) , indicating that the increased levels of mitochondrial Ca2+ are dependent on mitochondrial hyperpolarization . 10 . 7554/eLife . 06376 . 007Figure 4 . IL-6-mediated high MMP results in elevated mitochondrial Ca2+ levels .",
"( A ) Mitochondrial Ca2+ in CD4 cells activated in absence or presence of IL-6 for 48 hr was determined by staining with Rhod-2 AM and flow cytometry analysis .",
"( B ) Rhod-2 staining in CD4 cells activated with IL-6 for 46 hr followed by incubation with CCCP or vehicle ( Veh ) for 2 hr . ( C ) Percentage of Rhod-2high population in CD4 cells activated as in ( A ) .",
"Gates are shown in ( A ) and ( B ) ( n = 4 ) .",
"( D ) CD4 cells were activated for 48 hr with or without IL-6 and treated for the last 4 hr with vehicle , rotenone plus antimycin ( R/A ) or CGP-37157 ( CGP ) .",
"Cytoplasmic Ca2+ was measured using Fura-2 AM staining .",
"Fluorometric ratio at 340 nm/380 nm ( F340/380 ) is shown .",
"( n = 3 ) .",
"Error bars represent the mean ± SD .",
"*denotes p < 0 . 05 , as determined by Student's t test and one-way or two-way ANOVA test .",
"Results are representative of 2–3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 00710 . 7554/eLife . 06376 . 008Figure 4—figure supplement 1 . IL-6 maintains elevated cytosolic Ca2+ through its effect on the MMP and mitochondrial Ca2+ .",
"( A ) Cytosolic calcium of CD4 cells activated with anti-CD3/CD28 Abs for 48hr in the absence or presence of IL-6 , as determined by staining with Indo-1 .",
"The ratio of Ca2+-bound Indo-1 fluorescence ( 405 nm ) to unbound indo-1 fluorescence ( 480 nm ) was then determined by flow cytometry analysis .",
"Ionomycin ( 500 ng/mL ) and EGTA ( 50 mM ) were used as positive and negative control for indo-1 measurements .",
"( B ) Mitochondrial Ca2+ determined by Rhod-2 AM staining and flow cytometry analysis in CD4 cells activated in absence or presence of IL-6 for 42 hr followed by treatment with CGP-37157 ( 50 μM ) or vehicle for 6 hr . ( C ) Cytoplasmic Ca2+ determined by Fura-2 AM staining in CD4 cells activated in the presence or absence of IL-6 for 44 hr followed by IL-6 treatment with CCCP ( 2 μM ) or vehicle for 4 hr .",
"Fluorometric ratio at 340 nm/380 nm ( F340/380 ) is shown .",
"n = 3 , *denotes p < 0 . 05 as determined by student's t test .",
"Results are representative of 2–3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 008 Because of their dynamic characteristics and ability to redistribute within the cell , mitochondria play an important role in cytoplasmic Ca2+ homeostasis .",
"Mitochondria uptake Ca2+ through MCU at the cytoplasmic membrane near the extracellular calcium channels , as well as from ER storage , and serve as a delivery vehicle to increase cytosolic Ca2+ ( Rizzuto et al . , 2012; Soboloff et al . , 2012 ) .",
"Thus , early during T cell activation mitochondria have been shown to relocate close to immune synapse and contribute to increase cytosolic Ca2+ ( Quintana et al . , 2007; Schwindling et al . , 2010 ) .",
"To determine whether the increase in mitochondrial Ca2+ elicited by IL-6 could affect the levels of free cytosolic Ca2+ , we examined the basal level of cytosolic Ca2+ in CD4 cells using Fura-2 AM as a calcium indicator .",
"The levels of cytosolic Ca2+ , as determined by fluorometric ratio at 340 nm/380 nm ( F340/380 ) , in cells activated with IL-6 were higher than in cells activated without IL-6 ( Figure 4D ) .",
"It has been previously shown that TCR stimulation fails to induce cytosolic Ca2+ flux in activated CD4 cells , as determined by flow cytometry analysis ( Nagaleekar et al . , 2008 ) .",
"Similarly , no Ca2+ flux was triggered by TCR stimulation in CD4 cells activated in the presence of IL-6 ( data not shown ) .",
"However , similar to the results with Fura-2 staining , analysis of the cytosolic Ca2+ baseline by Indo-1 staining and flow cytometry analysis also revealed higher baseline in CD4 cells activated in the presence of IL-6 relative to cells activated in the absence of IL-6 ( Figure 4—figure supplement 1A ) .",
"Maximum cytosolic Ca2+ levels triggered by the calcium ionophore , ionomycin were comparable between CD4 cells activated in the presence or absence of IL-6 ( Figure 4—figure supplement 1A ) .",
"Thus , the presence of IL-6 during activation maintains increased levels of cytosolic Ca2+ .",
"To demonstrate that this increased cytosolic Ca2+ was dependent on high mitochondrial Ca2+ , we examined the effect of CGP-37157 , a blocker of mitochondrial Ca2+ efflux ( Cox et al . , 1993 ) .",
"As previously demonstrated ( Delmotte et al . , 2012 ) , treatment with CGP-37157 resulted in increased levels of mitochondrial Ca2+ ( Figure 4—figure supplement 1B ) .",
"Importantly , the treatment with CGP-37157 , lowered the cytosolic Ca2+ levels in CD4 cells activated in the presence of IL-6 to the levels found in those without IL-6 ( Figure 4D ) , indicating that this increase was dependent on mitochondrial Ca2+ .",
"In addition , reducing the MMP in IL-6-stimulated cells by treatment with inhibitors of Complex I ( rotenone ) and Complex III ( antimycin ) also lowered the levels of cytosolic Ca2+ ( Figure 4D ) .",
"Similar effects were found by the treatment with CCCP ( Figure 4—figure supplement 1C ) .",
"IL-6 therefore provides a mechanism for CD4 cells to maintain elevated levels of cytosolic Ca2+ through its effect on the MMP and mitochondrial Ca2+ .",
"In addition to its role as a transcription factor , several studies have shown the presence of Stat3 in mitochondria where it regulates the ETC primarily in tissues with high mitochondria content ( Gough et al . , 2009; Wegrzyn et al . , 2009; Heusch et al . , 2011; Lachance et al . , 2013; Zhang et al . , 2013; Erlich et al . , 2014 ) .",
"Although IL-6 is a major activator of Stat3 , no studies have previous address the regulation of Stat3 in mitochondria by this cytokines .",
"However , the maintenance of high MMP late during activation of CD4 cells by IL-6 could possibly be mediated by Stat3 .",
"We first examined whether Stat3 could also be present in mitochondria in activated CD4 cells by Western blot analysis using extracts from different subcellular fractions .",
"As expected , Stat3 was present in both the nucleus and cytosol ( Figure 5A ) .",
"Interestingly , however , high levels of Stat3 were also present in mitochondria ( Figure 5A ) .",
"GAPDH and COX IV were used as cytosolic and mitochondrial fraction markers , respectively ( Figure 5A ) .",
"To examine whether localization of Stat3 in mitochondria was influenced by IL-6 during CD4 cell activation , we performed Western blot analysis using mitochondrial extracts from CD4 cells activated in the presence or absence of IL-6 as well as from freshly isolated CD4 cells .",
"Only low levels of Stat3 were present in the mitochondrial fraction from freshly isolated CD4 cells ( Figure 5B ) .",
"High levels of Stat3 were detected in mitochondria from activated cells , but these levels were further upregulated by IL-6 ( Figure 5B ) .",
"In contrast , as a control , the levels of NDUFA9 were not affected by IL-6 ( Figure 5B ) .",
"IL-6 did not have an effect on the total levels of Stat3 either , as determined by Western-blot using whole cell lysates ( Figure 5C ) .",
"We also examined whether Stat3 in mitochondria was phosphorylated .",
"No phospho-Stat3 was detected in mitochondria from freshly isolated CD4 cells ( Figure 5B ) .",
"Phospho-Stat3 was present in mitochondria of activated CD4 cells , but the levels were substantially higher in the presence of IL-6 ( Figure 5B ) .",
"Thus , IL-6 promotes the accumulation of Stat3 in mitochondria during CD4 cell activation . 10 . 7554/eLife . 06376 . 009Figure 5 . The regulation of the MMP and mitochondrial Ca2+ elicited by IL-6 is Stat3 dependent .",
"( A ) CD4 cells were activated with anti-CD3 and anti-CD28 mAbs .",
"After 48 hr , cytosolic , nuclear and mitochondrial fractions were prepared and used to examine Stat3 by Western blot analysis .",
"GAPDH and COX IV were used as markers for cytosol and mitochondria , respectively .",
"( B ) Mitochondrial fractions from freshly isolated CD4 cells , and CD4 cells activated ( 48 hr ) with ( IL-6 ) or without IL-6 ( Media ) were analyzed for Stat3 , phospho-Stat3 ( p-Stat3 ) and the Complex I subunit NDUFA9 , as mitochondrial loading control .",
"Relative densitometry ratios of p-Stat3 to NDUFA9 and total Stat3 to NDUFA9 in cells activated in the presence and absence of IL-6 are shown .",
"( C ) Total Stat3 levels in CD4 cells activated ( 48 hr ) with or without IL-6 were examined by Western blot analysis using whole cell extracts .",
"GAPDH was used as loading control .",
"( D ) MMP in Stat3+/+ or Stat3−/− CD4 cells activated with anti-CD3/28 Abs in the presence or absence of IL-6 for 48 hr ( n = 3 ) .",
"( E ) Percentage of TMREhigh population in Stat3+/+ or Stat3−/− CD4 cells activated from ( D ) ( n = 3 ) .",
"( F ) Percentage of TMREhigh population in WT or mut-Stat3 CD4 cells activated from ( n = 3 ) .",
"( G ) Mitochondrial Ca2+ ( Rhod-2 AM staining ) in Stat3+/+ or Stat3−/− CD4 cells activated as in ( D ) .",
"( H ) Percentage of Rhod-2high population ( gate shown in panel G ) in Stat3+/+ or Stat3−/− CD4 cells ( n = 3 ) .",
"( I ) Mitochondrial Ca2+ ( Rhod-2 AM staining ) in WT or mut-Stat3 CD4 cells activated as in ( D ) .",
"( J ) Percentage of Rhod-2high population ( gate shown in panel I ) in WT or mut-Stat3 CD4 cells activated as in ( D ) ( n = 3 ) .",
"( K ) Mitochondrial fractions of CD4 cells activated ( 48 hr ) with or without IL-6 were resolved by BN-PAGE .",
"Supercomplexes regions ( SC region ) of BN-PAGE were excised and analyzed for Stat3 , NDUFA9 and NDUFV1 by Western blot analysis .",
"Error bars represent the mean ± SD .",
"*denotes p < 0 . 05 , as determined by Student's t test and one-way or two-way ANOVA test .",
"Results are representative of 2–3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 00910 . 7554/eLife . 06376 . 010Figure 5—figure supplement 1 . Stat3 transcription activity is not required for IL-6 to sustain the MMP .",
"( A and B )",
"Mitochondrial membrane potential determined by TMRE staining and flow cytometry analysis in CD4 cells activated in absence or presence of IL-6 for 42 hr were treated with Stattic ( 10 μM ) or vehicle for 6 hr . ( A ) Representative profiles and ( B ) percentage of CD4 cells with TMREhigh ( defined by the gate displayed in ( A ) ) .",
"n = 3 , denote p < 0 . 05 as significant , Defined n . s . as ‘not significant’ determined by two-way ANOVA .",
"Results are representative of 3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 01010 . 7554/eLife . 06376 . 011Figure 5—figure supplement 2 . Stat3 contributes to elevated cytosolic Ca2+ elicited by IL-6 . Cytoplasmic Ca2+ determined by Fura-2 AM staining in CD4 cells from Stat3+/+ or Stat3−/− mice activated ( 48 hr ) in the presence or absence of IL-6 .",
"Fluorometric ratio at 340 nm/380 nm ( F340/380 ) is shown .",
"n = 3 , *denotes p < 0 . 05 , n . s . denotes ‘not significant’ based on two-way ANOVA test .",
"Results are representative of 2 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 011 We then investigated whether IL-6 increases MMP in activated CD4 cells through Stat3 .",
"CD4 cells from wild-type ( Stat3+/+ ) mice and T-cell conditional Stat3 knockout ( Stat3−/− ) mice ( Takeda et al . , 1998 ) were activated in the absence or presence of IL-6 , and MMP was examined 48 hr later .",
"Interestingly , IL-6 failed to increase MMP in Stat3-deficient CD4 cells during activation ( Figure 5D , E ) , indicating that effect of IL-6 on MMP in CD4 cells is dependent on Stat3 .",
"To address whether this effect of Stat3 dissociates from its activity as a transcription factor , we used CD4 cells from mice expressing a mutant Stat3 ( mut-Stat3 ) carrying a deletion at V463 residue ( Stat3-Δ463 ) that prevents DNA binding but does not affect Stat3 phosphorylation ( Steward-Tharp et al . , 2014 ) .",
"This mutation was found in autosomal dominant hyperimmunoglobulin E syndrome ( Holland et al . , 2007; Minegishi et al . , 2007; Jiao et al . , 2008 ) .",
"Expression of mut-Stat3 in mice has been shown to act as dominant-negative and inhibit Stat3 mediated transcription ( Steward-Tharp et al . , 2014 ) .",
"CD4 cells from WT and mut-Stat3 mice were activated with or without IL-6 and MMP was examined after 48 hr .",
"IL-6 was still able to increase MMP in CD4 cells from mut-Stat3 mice ( Figure 5F ) .",
"In addition , we also tested the effect of Stattic , a well characterized inhibitor of Stat3 that blocks dimerization of Stat3 through phosphor-Tyr705 ( Schust et al . , 2006 ) .",
"The presence of Stattic , even at a relatively high concentration ( Schust et al . , 2006 ) , did not affect the MMP in IL-6-treated CD4 cells ( Figure 5—figure supplement 1A and B ) .",
"Thus , correlating with the accumulation of Stat3 in mitochondria , the increased MMP in CD4 cells activated in the presence of IL-6 requires Stat3 , but it is independent of Stat3-mediated transcription .",
"Although the presence of Stat3 in mitochondria and its role as regulator of ETC activity has now been widely reported in different cell types , no previous studies have addressed the role of Stat3 in mitochondrial Ca2+ .",
"To further determine whether IL-6 increases mitochondrial Ca2+ through Stat3 , we examined mitochondrial Ca2+ in Stat3+/+ and Stat3−/− CD4 cells activated in the presence or absence of IL-6 .",
"Interestingly , in the absence of Stat3 , IL-6 failed to maintain elevated levels of mitochondrial Ca2+ ( Figure 5G , H ) .",
"To show that this effect was not dependent on Stat3 transcriptional activity we also examined mitochondrial Ca2+ in CD4 cells from mut-Stat3 mice .",
"Unlike Stat3 deficient CD4 cells , IL-6 was capable to increase mitochondrial Ca2+ in mut-Stat3 CD4 cells ( Figure 5I , J ) .",
"To further examine whether Stat3 is necessary for the regulation of cytosolic Ca2+ elicited by IL-6 , cytosolic Ca2+ levels were measured in Stat3+/+ or Stat3−/− CD4 cells activated in the presence or absence of IL-6 using the Fura-2 AM assay .",
"Unlike Stat3+/+ CD4 cells , IL-6 failed to increase cytosolic Ca2+ in Stat3−/− CD4 cells ( Figure 5—figure supplement 2 ) .",
"Together , these data show for the first time that Stat3 contributes to mitochondrial Ca2+ in response to IL-6 and , consequently , cytosolic Ca2+ homeostasis .",
"Previous studies have demonstrated the association of Stat3 with Complex I of the ETC through GRIM-19 , a component of Complex I ( Lufei et al . , 2003; Gough et al . , 2009; Wegrzyn et al . , 2009; Tammineni et al . , 2013 ) .",
"No studies have reported whether Stat3 is present in the ETC SCs .",
"Our studies above ( Figure 2D ) indicate that IL-6 facilitates the formation of ETC SCs in CD4 cells .",
"We therefore examined whether mitochondrial Stat3 could also be recruited to the SCs .",
"BN-PAGE was performed using mitochondrial extracts generated with digitonin from CD4 cells activated in the presence or absence of IL-6 .",
"SC region of BN-PAGE was excised and resolved by Western blot analysis for Stat3 .",
"As described above , the levels of SCs were increased in CD4 cells activated in the presence of IL-6 as determined by the levels of NDUFA9 and NDUFV1 subunits of Complex I ( Figure 5K ) .",
"Interestingly , Stat3 was also present in the SC region isolated from IL-6-treated CD4 cells ( Figure 5K ) .",
"Thus , Stat3 is recruited to the ETC SCs , where it can regulate activity of Complex I through interaction with GRIM-19 .",
"IL-6 , in the absence of other cytokines , is the major inducer of IL-21 production by CD4 cells in mouse and human ( Nurieva et al . , 2008; Suto et al . , 2008; Dienz et al . , 2009; Diehl et al . , 2012 ) .",
"Stat3 is considered the main transcription factor that induces Il21 gene expression ( Chen et al . , 2006; Nurieva et al . , 2007; Zhou et al . , 2007; Kaplan et al . , 2011 ) .",
"However , since Stat3 but not its transcriptional activity is required for IL-6 to sustain MMP and Ca2+ during the activation of CD4 cells , this could be an additional mechanism by which IL-6 promotes the production of IL-21 .",
"We therefore examined the ability of IL-6 to induce IL-21 production in CD4 cells from mut-Stat3 mice where Stat3 is present but its transcriptional activity is impaired .",
"Similarly to human CD4 cells from patients with Hyper IgE syndrome expressing mut-Stat3 , CD4 cells from mut-Stat3 mice have been shown to fail to produce IL-17 , another cytokine gene regulated by Stat3 ( Ma et al . , 2008; Milner et al . , 2008; Renner et al . , 2008; de Beaucoudrey et al . , 2008; Minegishi et al . , 2009; Durant et al . , 2010; Steward-Tharp et al . , 2014 ) .",
"Although IL-6 totally failed to induce IL-21 production in Stat3−/− CD4 cells ( Figure 6A ) , it was able to trigger the production of IL-21 in mut-Stat3 CD4 cells ( Figure 6B ) .",
"Thus , correlating with its role on MMP and mitochondrial Ca2+ , Stat3 can contribute to the production of IL-21 in response to IL-6 independently of its function of transcription factor . 10 . 7554/eLife . 06376 . 012Figure 6 . Mitochondrial Ca2+ is essential for IL-6 to sustain the production of IL-21 and IL-4 late during activation of CD4 cells .",
"( A ) CD4 cells from Stat3+/+ or Stat3−/− mice were activated in the presence or absence of IL-6 for 48 hr .",
"IL-21 production was measured by ELISA .",
"( B ) IL-21 production from WT or mut-Stat3 CD4 cells with or without IL-6 during activation was measured as in ( A ) CD4 cells were activated in the presence or absence of IL-6 .",
"After 42 hr , rotenone/antimycin ( R/A ) , CCCP , GCP-37157 or vehicle were added to the cultures .",
"Supernatants were collected 6 hr later .",
"IL-21 ( C ) , IL-4 ( D ) , IL-2 ( E ) production was measured by ELISA .",
"( F ) Relative mRNA levels for IL-21 , IL-4 and IL-2 in activated in CD4 cells ( 48 hr ) were measured by real-time PCR ( RT-PCR ) .",
"( G ) CD4 cells were activated in the presence of IL-6 .",
"After 24 hr , Ru360 or vehicle control ( Veh ) were added to the cultures .",
"Supernatants were collected 24 hr later .",
"IL-21 and IL-2 production was measured by ELISA .",
"Error bars represent the mean ± SD .",
"*denotes p < 0 . 05 , as determined by two-way ANOVA test .",
"Results are representative of 2–3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 01210 . 7554/eLife . 06376 . 013Figure 6—figure supplement 1 . A transcriptionally inactive Stat3 is sufficient for IL-6 to promote IL-21 production through mitochondrial Ca2+ .",
"( A and B )",
"CD4 cells from WT and mut-Stat3 mice were activated in the presence of IL-6 for 42 hr , CGP-17157 ( CGP ) or vehicle ( Veh ) were added and supernatants were harvested 6 hr later .",
"IL-21 ( A ) and IL-2 ( B ) levels in supernatant were measured by ELISA .",
"Cytokine production is shown as fold increase between the levels at 42 hr ( prior to treatment ) and the level at 48 hr ( 6 hr after treatment ) .",
"n = 3 , *denotes p < 0 . 05 as determined by two-way ANOVA . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 01310 . 7554/eLife . 06376 . 014Figure 6—figure supplement 2 . Stat3 contributes to the production of IL-4 in response to IL-6 independently of its function of transcription factor . CD4 cells from WT , mut-Stat3 and Stat3−/−were activated in the presence of IL-6 for 48 hr .",
"IL-4 and IL-2 production in the supernatant was measured by ELISA .",
"The percentage of IL-4 and IL-2 levels in mut-Stat3 and Stat3−/− CD4 cells relative to the levels in WT CD4 cells are provided .",
"n = 3 .",
"*denotes p < 0 . 05 as determined by student's t-test , showing greater reduction in IL-4 production in Stat3−/− CD4 cells than in mut-Stat3 cells relative to WT cells .",
"#denotes p < 0 . 05 as determined by student's t-test , showing greater reduction in IL-2 production in mut-Stat3 cells than in Stat3−/− cells relative to WT cells . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 01410 . 7554/eLife . 06376 . 015Figure 6—figure supplement 3 . Ru360 decreases mitochondrial Ca2+ in CD4 cells in response to IL-6 during activation . Mitochondrial Ca2+ determined by Rhod-2 AM staining and flow cytometry analysis in CD4 cells activated with IL-6 and treated with vehicle ( Veh ) or Ru360 ( 10 μM ) for the last 24 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 015 A recent study has reported that sustained elevated cytosolic Ca2+ levels are associated with the increased expression of Il21 in CD4 cells in vivo ( Shulman et al . , 2014 ) .",
"We therefore investigated whether the sustained high MMP elicited by IL-6 late during the activation of CD4 cells could contribute to the production of IL-21 triggered by this cytokine .",
"CD4 cells were activated in the presence or absence of IL-6 for 42 hr and treated with rotenone and antimycin ( R/A ) or CCCP ( to depolarize mitochondria ) for another 6 hr .",
"IL-21 levels in the supernatants were determined by Enzyme linked immunosorbent assay ( ELISA ) .",
"Although there were already substantial levels of IL-21 at 42 hr in cells activated with IL-6 , these levels steeply rose in the next 6 hr ( Figure 6C ) .",
"However , the increase in IL-21 levels was prevented by the treatment with R/A or CCCP ( Figure 6C ) , indicating that the late production of IL-21 was dependent on the increased MMP caused by IL-6 .",
"To further address whether IL-6-mediated mitochondrial Ca2+ contributes to the late production of IL-21 , CD4 cells were activated in the presence or absence of IL-6 for 42 hr , and treated with CGP-37157 to inhibit mitochondrial Ca2+ export for another 6 hr .",
"The increase in IL-21 production was also prevented by CGP-37157 ( Figure 6C ) , showing that the increased mitochondrial Ca2+ elicited by IL-6 also contributes to the late production of IL-21 .",
"Similarly , CGP-37157 prevented the increase in IL-21 production late during activation in mut-Stat3 CD4 cells , without effecting IL-2 production ( Figure 6—figure supplement 1A and B ) .",
"We and others have shown that IL-6 can also promote the production of IL-4 during activation ( Rincon et al . , 1997; Diehl et al . , 2002; Heijink et al . , 2002 ) .",
"Like IL-21 , sustained elevated cytosolic Ca2+ levels have been associated with the increased expression of Il4 in CD4 cells in vivo ( Shulman et al . , 2014 ) .",
"We therefore examined the effect that interfering with MMP or Ca2+ has on IL-4 production later during activation .",
"Similar to IL-21 , the levels of IL-4 were increased in the last 6 hr in IL-6-stimulated CD4 cells , however R/A , CCCP or CGP-37157 prevented this increase ( Figure 6D ) , indicating that the increased MMP and cytosolic Ca2+ regulated by mitochondrial Ca2+ caused by IL-6 also contributes to the late production of IL-4 .",
"In contrast , IL-6 had no effect on IL-2 production and treatment with R/A , CCCP or CGP-37157 had no effect ( Figure 6E ) .",
"We also examined the relative contribution of transcription-independent function of Stat3 in the regulation of these other cytokines by IL-6 .",
"Similar to IL-21 , IL-4 production was strongly reduced in Stat3−/− CD4 cells , but not in mut-Stat3 CD4 cells ( Figure 6—figure supplement 2 ) .",
"In contrast , IL-2 production was more affected in mut-Stat3 CD4 cells than in Stat3−/− CD4 cells ( Figure 6—figure supplement 2 ) , further supporting a transcription-independent role of Stat3 in the regulation of IL-21 and IL-4 by IL-6 .",
"To address whether mitochondrial Ca2+ could contribute to the IL-6-mediated gene expression of these cytokines , we also examined mRNA levels of Il21 , Il4 and Il2 .",
"CD4 cells were activated in the presence of or absence of IL-6 , and treated with CGP-37157 to inhibit mitochondrial Ca2+ export .",
"The levels of Il21 and Il4 mRNA were significantly increased in cells treated with IL-6 but 6 hr of CGP-37157 treatment was sufficient to reduce these levels ( Figure 6F ) .",
"In contrast , Il2 mRNA levels were not increased by IL-6 , and treatment with CGP-37157 did not have an effect .",
"Thus , mitochondrial Ca2+ regulated by IL-6 is required for sustaining cytokine gene expression induced by IL-6 in CD4 cells late during activation .",
"In addition , we also addressed the relevance of mitochondrial Ca2+ uptake in the regulation of cytokines by IL-6 using the RU360 compound , a specific MCU inhibitor ( Matlib et al . , 1998 ) .",
"We confirmed that the treatment with RU360 lowered the mitochondrial Ca2+ levels in CD4 cells activated in the presence of IL-6 ( Figure 6—figure supplement 3 ) .",
"Importantly , the treatment with RU360 reduced the production of IL-21 in CD4 cells activated with IL-6 ( Figure 6G ) .",
"RU360 however had no effect on IL-2 production ( Figure 6G ) .",
"Thus , both uptake and export of mitochondrial Ca2+ plays a role in the regulation of cytokine production by IL-6 in CD4 cells .",
"Il21 gene expression is regulated by Stat3 , a Ca2+-independent transcription factor , but it is also regulated by the NFAT transcription factor ( Kim et al . , 2005; Durant et al . , 2010 ) .",
"NFAT is also required for Il4 gene expression ( Rao , 1994; Diehl et al . , 2002; Rengarajan et al . , 2002 ) .",
"Nuclear translocation of NFAT is dependent on increased cytosolic Ca2+ and activation of the Ca2+-dependent phosphatase , calcineurin .",
"Mitochondrial Ca2+ has been shown to contribute to NFAT activation in sensory neurons ( Kim and Usachev , 2009 ) .",
"Since we have shown IL-6 promotes NFATc2 nuclear accumulation ( Diehl et al . , 2002 ) , we examined whether this could be dependent on mitochondrial Ca2+ .",
"CD4 cells were activated in the presence of or absence of IL-6 for 42 hr , and treated with CGP-37157 for another 6 hr to inhibit mitochondrial Ca2+ export .",
"The addition of CGP disrupted the nuclear accumulation of NFATc2 in cells treated with IL-6 ( Figure 7A ) .",
"Thus , mitochondrial Ca2+ regulated by IL-6 is required for IL-6 to sustain NFATc2 in the nucleus late during activation . 10 . 7554/eLife . 06376 . 016Figure 7 . Increased mitochondrial Ca2+ by IL-6 is required to sustain nuclear NFAT accumulation late during activation of CD4 cells .",
"( A ) CD4 cells were activated in the presence or absence of IL-6 for 42 hr followed by 6 hr treatment with medium ( Med ) or CGP-37157 ( CGP ) .",
"NFATc2 ( red ) was examined by immunostaining and confocal microscopy .",
"TOPRO was used as nuclear dye .",
"40× Magnification .",
"Bars represent 20 μm .",
"( B ) CD4 cells were activated in the presence or absence of IL-6 for 42 hr .",
"FK506 ( FK ) was added to culture for another 6 hr .",
"Supernatants were collected , and IL-21 and IL-4 levels were measured by ELISA .",
"( C ) Relative mRNA for IL-21 and IL-4 levels in CD4 cells activated as in ( B ) was measured by RT-PCR .",
"Error bars represent the mean ± SD .",
"*denotes p < 0 . 05 , as determined by two-way ANOVA test .",
"Results are representative of 2–3 experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 06376 . 016 To address whether NFAT contributes to the production of IL-21 and IL-4 induced by IL-6 late during activation , CD4 cells were activated in the presence or absence of IL-6 for 42 hr , and treated for another 6 hr with FK506 , a NFAT inhibitor ( FK ) .",
"FK506 blocked the production of IL-21 and IL-4 induced by IL-6 , as determined by ELISA ( Figure 7B ) .",
"In addition , inhibition of NFAT late during activation also reduced Il21 and Il4 mRNA levels in cells exposed to IL-6 ( Figure 7C ) .",
"Therefore , high mitochondrial Ca2+ and nuclear accumulation of NFAT triggered by IL-6 late during activation in CD4 cells is required to sustain expression of Il21 and Il4 ."
],
[
"Most of the functions of IL-6 in CD4 cells have been assigned to a regulatory role on gene expression through Stat3 as a transcription factor .",
"However , in the light of studies indicating that Stat3 localizes in mitochondria where it regulates the mitochondrial respiratory chain through association with Complex I ( Gough et al . , 2009; Wegrzyn et al . , 2009 ) , it was also possible that IL-6 could have an effect on mitochondria in CD4 cells .",
"Our studies here show for the first time that IL-6 maintains mitochondrial hyperpolarization late during activation of CD4 cells and this has an impact in mitochondrial Ca2+ and , thereby cytosolic Ca2+ .",
"We also show that the effect of IL-6 on mitochondrial Ca2+ and baseline cytosolic Ca2+ requires the presence of Stat3 , but it is independent of its role as transcription factor .",
"In recent years , growing interest has been focused on mitochondrial biology in T cells .",
"Bioenergetic profiling of T cells has revealed that T cell metabolism changes dynamically during activation ( Wang and Green , 2012; Pearce et al . , 2013 ) .",
"Naive T cells maintain low rates of glycolysis and predominantly oxidize glucose-derived pyruvate via OXPHOS , or engage fatty acid oxidation ( FAO ) .",
"After activation , they rapidly switch to anabolic growth and biomass accumulation .",
"This adaption to aerobic glycolysis is specifically required for effector functions in T cells ( Chang et al . , 2013 ) .",
"IL-2 and IL-15 have been reported to regulate mitochondrial respiration and the balance between glycolysis and oxidative phosphorylation ( van der Windt et al . , 2012 ) .",
"IL-2 has been shown to support aerobic glycolysis , while IL-15 increases spared respiratory capacity and oxidative metabolism by enhancing mitochondrial biogenesis and FAO in CD8 cells ( Pearce et al . , 2009; van der Windt et al . , 2012 ) .",
"IL-6 has been recently shown to regulate glucose homeostasis in myeloid cells and induce the switch from white adipose tissue to brown fat in cancer induced cachexia ( Mauer et al . , 2014; Petruzzelli et al . , 2014 ) .",
"Here we show that IL-6 enhances the MMP in CD4 cells .",
"However , this is uncoupled from oxidative phosphorylation ( i . e . ATP synthesis ) .",
"In addition , IL-6 does not alter the balance between glycolysis and oxidative glycolysis during activation .",
"Instead , we show that a sustained MMP elicited by IL-6 leads to an effect on mitochondrial Ca2+ .",
"No other studies have linked cytokine effects to mitochondrial Ca2+ in CD4 cells .",
"While endoplasmic reticulum ( ER ) -derived Ca2+ has been extensively studied in T cells , less is known about mitochondrial Ca2+ homeostasis in T cells .",
"Mitochondrial Ca2+ has been previously shown to modulate store-operated calcium signaling early upon T cell activation at the immunological synapse ( Hoth et al . , 1997; Quintana et al . , 2007 ) .",
"Here we show that IL-6 uses MMP to sustain elevated levels of mitochondrial Ca2+ late during activation and , consequently , elevated levels of cytosolic Ca2+ .",
"We have previously shown that the expression of IP3Rs is downregulated during the activation of CD4 cells ( Nagaleekar et al . , 2008 ) .",
"It is therefore possible that the source of Ca2+ in CD4 cells is reprogramed during activation .",
"ER- IP3R is the main source of Ca2+ during early activation of naive CD4 cells at the synapse .",
"However , mitochondrial Ca2+ could be the major source to sustain cytosolic Ca2+ in activated CD4 cells .",
"Our data indicate that IL-6 sustains cytosolic Ca2+ late during activation by increasing the MMP and mitochondrial Ca2+ .",
"This provides a potential mechanism by which Tfh cells have increased free cytosolic calcium levels ( Shulman et al . , 2014 ) .",
"More importantly , we show here for the first time that mitochondrial Ca2+ plays a key role in promoting increased production of cytokine by effector CD4 cells .",
"Although IP3R-mediated Ca2+ release is essential for the initial induction of cytokine gene expression ( Feske , 2007 ) , we have previously shown that IP3R-mediated Ca2+ is not responsible for late production of cytokines by activated CD4 cells ( Nagaleekar et al . , 2008 ) .",
"Thus , the source of Ca2+ for cytokine production is also reprogrammed during activation of CD4 cells .",
"Although we cannot discard the effect of other transcription factors , our study shows that mitochondrial Ca2+ is required for IL-6 to keep NFATc2 in the nucleus , and that NFAT contributes to late expression of Il21 and Il4 .",
"Mitochondrial respiration has been shown to lead to ROS production caused by proton leaks and ROS can lead to oxidative injury .",
"A number of recent studies have shown that mROS can function as signaling intermediates , and the mROS signaling is required for antigen-specific T cell activation and subsequent IL-2 production ( Byun et al . , 2008; Schieke et al . , 2008; Sena et al . , 2013 ) .",
"Although IL-6 increases the MMP , we did not observe an increase in the levels of mROS correlating with the effect of IL-6 facilitating the formation of respiratory SCs .",
"The presence of ETC SCs is emerging as a novel but highly relevant aspect of the mitochondrial function ( Acín-Pérez et al . , 2008; Althoff et al . , 2011 ) .",
"The function of these SCs is to facilitate the transfer of electrons between ETC complexes to minimize the risk of electron leak and , thereby , the risk of producing harmful ROS .",
"Our study demonstrates for the first time the presence of ETC SCs in CD4 cells , and the effect that IL-6 has in promoting the formation of these SCs during activation of CD4 cells .",
"This could be a mechanism by which IL-6 can sustain elevated MMP and Ca2+ while minimizing the production of mROS .",
"Although the association of Stat3 with individual complexes of the ETC has been previous described in heart and cancer cells ( Gough et al . , 2009; Wegrzyn et al . , 2009 ) , here we show for the first time the presence of Stat3 in the ETC SCs in CD4 cells .",
"Stat3 may also be present in mitochondrial SCs in other tissues such as heart .",
"Thus , here we identify a novel mechanism by which IL-6 promotes the production of IL-21 and IL-4 late during the activation of CD4 cells .",
"This new mechanism involves Stat3 but as a factor regulating MMP and Ca2+ instead of its function as mediator of transcription .",
"Our studies also reveal a novel function of mitochondrial respiration in the control of cytokine production through its effect on mitochondrial Ca2+ homeostasis ."
],
[
"C57BL/6J mice were purchased from Jackson Laboratories .",
"Null IL-6 deficient mice ( IL-6 KO ) were previously described ( Poli et al . , 1994 ) .",
"Stat3 conditional knockout ( Stat3−/− ) mice were generated by crossing the homozygous floxed Stat3 mice ( Stat3loxp/loxp ) ( Takeda et al . , 1998 ) with T cell-specific Lck-Cre transgenic [B6 . Cg-Tg ( Lck-cre ) 1Cwi N9] mice ( Lee et al . , 2001 ) .",
"Mutant-Stat3 ( mut-Stat3 ) mice have been previously described ( Steward-Tharp et al . , 2014 ) .",
"OT-II TCR transgenic mice have been previously described ( Barnden et al . , 1998 ) .",
"All mice were housed under sterile conditions at the animal care facility at the University of Vermont .",
"All procedures performed on the mice were approved by the University of Vermont Institutional Animal Care and Use Committee .",
"CD4 cells were isolated from spleen and lymph nodes by negative selection as previously described ( Diehl et al . , 2002 ) .",
"For Stat3+/+ and Stat3−/− mice , CD4 cells were purified by cell sorting ( FACS-Aria; Becton Dickinson ) .",
"CD4 cells were activated with plate-bound anti-CD3 ( 2C11 ) ( 5 μg/ml ) and soluble anti-CD28 ( 1 μg/ml ) ( BD Pharmingen , San Diego , CA ) mAbs in the presence or absence of IL-6 ( 50 ng/mL ) ( Miltenyi Biotec , Auburn , CA ) .",
"Pharmacological inhibitors were added to culture 42 hr after activation and supernatants were harvested 6 hr later .",
"APCs were purified by depleting CD4 and CD8 T cells using positive selection ( Miltenyi ) , and followed by irradiation treatment ( 2000 rad ) .",
"APCs and OT-II CD4 cells were co-cultured at 4:1 ratio in the presence of 5 μM OVA323-339 peptide ( Barnden et al . , 1998 ) with or without IL-6 ( 50 ng /mL ) ( Miltenyi ) or anti-IL-6 ( 2 . 5 μg /mL ) ( BD Pharmingen ) .",
"Pharmacological inhibitors used were CGP-37157 ( Tocris Bioscience , Ellisville , MO ) ( 10 μM ) , CCCP ( 2 μM ) , rotenone ( 2 μM ) , antimycin ( 2 μM ) , Ru360 ( 10 μM ) , FK506 ( InvivoGen , San Diego , CA ) ( 10 nM ) , Stattic ( 10 μM ) .",
"OT-II CD4 cells were purified from OT-II TCR transgenic mice ( Thy1 . 1+ ) by positive selection using anti-CD4 MACS beads ( Miltenyi Biotec ) .",
"2 × 106 naive OT-II TCR Tg T cells in 100 μL Phosphate buffered saline ( PBS ) were transferred i . v . into WT or IL-6 KO hosts ( Thy1 . 2+ ) .",
"After overnight , adoptive hosts were simultaneously immunized i . p . with 200 μL of 50 μg OVA absorbed on alum ( 4 . 5% , w/v ) .",
"After 2 d immunization , spleens from immunized mice were harvested and stained with fluorescent conjugated Abs ( anti-Thy1 . 1 , anti-Vα2 , anti-CD69 , anti-CD4 , anti-CD44 ) and TMRE followed by flow cytometry analysis .",
"For each experiment , three to four hosts were used in each group .",
"MMP analysis was performed by staining CD4 cells with TMRE ( Molecular Probes , Eugene , OR ) as previously described ( Hatle et al . , 2013 ) .",
"Mitochondrial calcium analysis was performed by staining with Rhod-2 AM ( Invitrogen , Carlsbad , CA; 5 or 10 μM ) for 1 hr at 37°C , as previously described ( Brisac et al . , 2010 ) .",
"mROS production was determined by 10 min staining of cells with 5 μM MitoSox Red ( Molecular Probes ) .",
"Live/dead cell viability staining ( Molecular Probes ) was performed as recommended by the manufacturer .",
"All samples were examined by flow cytometry analysis using an LSRII flow cytometer ( BD Biosciences ) and Flowjo software .",
"Whole-cell extracts were prepared in Triton lysis buffer .",
"Mitochondrial , nuclear and cytosolic extracts were purified using the cell fractionation kit-standard ( MitoScience ) for CD4 cells .",
"Western blot analyses were performed as previously described ( Hatle et al . , 2013 ) .",
"Anti-Stat3 , anti-phospho-Stat3 ( Tyr705 ) ( Cell Signaling , Danvers , MA ) , anti-actin , anti-GAPDH , anti-rabbit IgG , and anti-goat IgG ( Santa Cruz Biotechnology , Santa Cruz , CA ) ; anti-mouse IgG ( Jackson Immunologicals , West Grove , PA ) ; anti-CoxIV ( Cell Signaling ) ; anti-NDUFA9 , anti-NDUFS3 ( MitoScience , Eugene , OR ) Abs were used .",
"Cells were suspended in fixative for 60 min at 4°C ( 2% glutaraldehyde , 0 . 05% CaCl2 , 0 . 1% MgCl2 , 22 mM betaine in 0 . 1 M Pipes buffer ) .",
"After rinsing in Pipes buffer , the cell pellets were embedded in 2% SeaPrep agarose , crosslinked with above fixative and postfixed with 1% osmium tetroxide for 1 hr at 4°C .",
"The samples were again rinsed in Pipes buffer , followed by dehydration through graded ethanol , cleared in propylene oxide and embedded in Spurr's epoxy resin .",
"Semithin sections ( 1 μm ) were cut with glass knives on a Reichert ultracut microtome , stained with methylene blue-azure II , and evaluated for areas of interest .",
"Ultrathin sections ( 60–80 nm ) were cut with a diamond knife , retrieved onto 200 mesh thin bar nickel grids , contrasted with uranyl acetate ( 2% in 50% ethanol ) and lead citrate , and examined with a JEOL 1400 TEM ( JOEL USA Inc , Peabody , MA ) operating at 60 kV .",
"Twenty-five digital images were acquired with an AMT XR611 CCD camera by systemic uniform random sampling from each sample .",
"Number of mitochondria and mitochondria with tight or expanded cristae was counted manually .",
"Activated CD4 cells ( 48 hr ) were cytospun and immunostained as previously described ( Diehl et al . , 2002 ) using a specific anti-NFATc2 Ab ( Upstate Biotechnology , Lake Placid , NY ) , followed by Alexa568-conjugated secondary Ab .",
"Nuclei were stained with TOPRO ( Molecular Probes ) .",
"Images were recorded using a Zeiss LSM 510 Meta confocal laser scanning imaging system ( Carl Zeiss Microimaging , Thornwood , NY ) .",
"Purified mitochondria were solubilized in Native PAGE loading buffer ( Invitrogen ) containing 2% digitonin ( Sigma-Aldrich Co . , St Louis , MO ) .",
"Complexes were resolved by native electrophoresis through gradient 4–16% Native PAGE Novex Bis-Tris gels ( Invitrogen ) as previously described ( Hatle et al . , 2013 ) .",
"Proteins were transferred to PVDF membrane for Western blot analysis with anti-NDUFA9 ( MitoScience ) and anti-Core I ( MitoScience ) .",
"SCs regions were also excised from BN-PAGE , eluted in SDS sample buffer and resolved in SDS-PAGE .",
"Proteins were then transferred to PVDF membrane for Western blot analysis with anti-NDUFA9 , anti-NDUFV1 and anti-Stat3 Abs .",
"OCR were measured , as previously described ( van der Windt et al . , 2012 ) under basal conditions and in response to oligomycinv ( 1 μM ) , FCCP ( 1 μM ) , and rotenone + antimycin A ( 1 μM ) with the Seahorse XF-24 Extracellular Flux Analyzer ( Seahorse Bioscience , North Billerica , MA ) using the XF Cell Mito Stress Test Kit .",
"ECAR were measured as recommended by the manufacturer using the XF Glycolysis Stress Test Kit .",
"Total RNA was isolated from CD4 cells using the Qiagen micro RNeasy kit , as recommended by manufacture ( Qiagen , Valencia , CA ) .",
"cDNA synthesis was performed as previously described ( Hatle et al . , 2013 ) .",
"cDNA was used to quantify the relative mRNA levels for mouse Il21 , Il4 and Il2 ( Assays-on-Demand by Applied Biosystems ) by conventional RT-PCR ( Applied Biosystems , San Diego , CA ) using β2-microglobulin as housekeeping gene .",
"The relative values were determined by the comparative CT analysis method .",
"Cytokine levels in cell culture supernatants were determined by ELISA as previously described ( Diehl et al . , 2002; Dienz et al . , 2007 , 2009 ) .",
"ATP was measured on 105 cells and/or 100 μl of culture supernatants by using ATP Lite kit ( Perkin Elmer , Boston , MA ) as recommended by the manufacturer in a TD-20/20 single tube luminometer .",
"Lactate production was examined in CD4 cells ( 2 × 106 ) activated for 48 hr , washed and incubated for 2 hr in media .",
"Measurement of lactate in supernatants was done using the Lactate assay Kit II ( BioVision , Milpitas , CA ) .",
"Cytosolic calcium was measured by staining with Fura-2 AM ( Molecular Probes ) ( 5 μM ) for 30 min , followed by fluorometrically measurement ( 340/380 exication , 510 emission ) in a Synergy H4 plate reader ( Bio-Tek , Winooski , VT ) .",
"F340/380 value was calculated by dividing the fluorescence reading at 340 nm by the fluorescence at 380 nm exication .",
"Cells were also loaded for 45 min at 37°C with 10 μM Indo-1 ( Molecular probes ) ( Grynkiewicz et al . , 1985 ) , harvested , washed and transferred to a standard extracellular solution ( 140 mM NaCl , 4 mM KCl , 1 mM CaCl2 , 2 mM MgCl2 , 1 mM KH2PO4 , 10 mM glucose , 10 mM HEPES [pH 7 . 4] ) .",
"The ratio of Ca2+-bound Indo-1 fluorescence ( 405 nm ) to unbound indo-1 fluorescence ( 480 nm ) was then determined by flow cytometry analysis .",
"Significance of differences between two groups was determined using GraphPad Prism v . 5 . 0 , by standard Student's t-test .",
"Significance of differences among more than 2 groups was determined by one-way or two-way ANOVA .",
"Standard p < 0 . 05 was used as the cutoff for significance .",
"For flow cytometry analysis , percentages of compared samples under the same gate were analyzed by t-test or ANOVA in Prism ."
]
] | [
"IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce .",
"Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function .",
"We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells .",
"Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation .",
"However , it is a mechanism to raise the levels of mitochondrial Ca2+ late during activation of CD4 cells .",
"Increased levels of mitochondrial Ca2+ in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells .",
"Thus , the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca2+ is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases ."
] | [
"Inflammation is a normal part of the body's response to an infection or injury and it helps to start the healing process .",
"However , if left unchecked , inflammation itself can damage tissues , and diseases such as rheumatoid arthritis are the result of uncontrolled inflammation .",
"Certain immune cells release molecules that can either trigger or suppress inflammation .",
"Interleukin 6 is an example of a ‘pro-inflammatory’ molecule , which regulates the activity of groups of immune cells collectively known as ‘CD4 cells’ .",
"People who are overweight or obese have higher levels of interleukin 6 than people of a healthy weight .",
"Obesity and other metabolic conditions have been linked to problems with structures called mitochondria , which make a molecule called ATP that provides cells with the energy they need to survive .",
"But it is not known if interleukin 6 can affect the activity of mitochondria inside CD4 cells .",
"Now , Yang et al . have discovered that interleukin 6 can affect the mitochondria inside CD4 cells and , in doing so , have identified a new way that interleukin 6 can regulate these cells' activity .",
"Experiments involving immune cells from mice revealed that interleukin 6 triggers a cascade of signaling events that aid the formation of so-called ‘mitochondrial respiratory chain supercomplexes’ in CD4 cells .",
"These are groups of proteins that work together in the membranes of mitochondria and are vital for the activity of these structures .",
"The formation of these supercomplexes maintains a large voltage difference across the membrane of the mitochondria that occurs during the later stages of CD4 cell activation .",
"Yang et al . found that this voltage difference was not linked to the production of ATP , but that it did raise the levels of calcium ions inside the mitochondria .",
"Further experiments revealed that these increased levels of calcium ions prolong the production of other pro-inflammatory molecules in the CD4 cells .",
"Following the discovery of a new pathway that regulates the activity of CD4 cells , the next challenge is to see if the parts of this pathway could be targeted with drugs to help treat inflammatory diseases such as rheumatoid arthritis .",
"Moreover , because interleukin 6 plays an active role in other diseases such as cancer , further studies of this new pathway may help explain how this molecule encourages cancers to progress and/or spread around the body ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Neural pattern change during encoding of a narrative predicts retrospective duration estimates | elife-16070-v3 | [
[
"Imagine that you are at the bus stop when you run into a colleague and the two of you become engrossed in a conversation about memory research .",
"After a few minutes , you realize that the bus still has not arrived .",
"Without looking at your watch , you have some sense of how long you have been waiting .",
"Where does this intuition come from ?",
"Estimation of durations lasting a few seconds has been probed in the neuroimaging , neuropsychology and neuropharmacology literatures ( see Wittmann , 2013 , for a review ) .",
"On the other hand , the neural mechanisms underlying time perception on the scale of minutes have remained unexplored .",
"This is particularly true of retrospective judgments , where individuals experience an interval without paying attention to time and must subsequently estimate the interval’s duration .",
"In such cases , individuals must rely on information stored in memory to estimate duration .",
"How is this accomplished ?",
"Memory scholars have long posited that the same contextual cues that help us to retrieve an item from memory can also help us determine its recency .",
"According to extant theories of context and memory ( see Manning et al . , 2014 , for a review ) , mental context refers to aspects of our mental state that tend to persist over a relatively long time scale; this encompasses our representation of slowly-changing aspects of the external world ( e . g . , what room we are in ) as well as other slowly-changing aspects of our internal mental state ( e . g . , our current plans ) .",
"Crucially , these theories posit that slowly-changing contextual features can be episodically associated with more quickly-changing aspects of the world ( e . g . , stimuli that appear at a particular moment in time; Mensink and Raaijmakers , 1988; Howard and Kahana , 2002 ) .",
"Bower ( 1972 ) first proposed that we could determine how long ago an item occurred by comparing our current context with the context associated with the remembered item .",
"The similarity of these two context representations would reflect their temporal distance , with more similar representations associated with events that happened closer together in time .",
"Thus , a slowly varying mental context could serve as a temporal tag ( Polyn and Kahana , 2008 ) .",
"In parallel , researchers in the domain of retrospective time estimation have shown that the degree of context change is a better predictor of duration judgments than alternative explanations , such as the number of items remembered from the interval ( Block and Reed , 1978; Block , 1990 , 1992 ) .",
"Indeed , changes in task processing ( Block and Reed , 1978; Sahakyan and Smith , 2014 ) , environmental context ( Block , 1982 ) , and emotions ( Pollatos et al . , 2014 ) , as well as event boundaries ( Poynter , 1983; Zakay et al . , 1994; Faber and Gennari , 2015 ) , lead to overestimation of past durations .",
"In our study , we set out to obtain neural evidence in support of the hypothesis that mental context change drives duration estimates .",
"Specifically , we hypothesized that , in brain regions representing mental context , the degree of neural pattern change between two events ( operationalized as change in multi-voxel patterns of fMRI activity ) should predict participants’ estimates of how much time passed between those events .",
"Extensive prior work has implicated the medial temporal lobe ( MTL ) and lateral prefrontal cortex ( PFC ) in representing contextual information ( Polyn and Kahana , 2008; for reviews of MTL contributions to representing context , see Eichenbaum et al . , 2007 , and Ritchey and Ranganath , 2012; for related computational modeling work , see Howard and Eichenbaum , 2013 ) .",
"In keeping with our hypothesis , multiple studies have obtained evidence linking neural pattern change in these regions to temporal memory judgments .",
"Manns et al . ( 2007 ) recorded from rat hippocampus during an odor memory task; they found that greater change in hippocampal activity patterns between two stimuli predicted better memory for the order in which the stimuli occurred .",
"In the human neuroimaging literature , Jenkins and Ranganath ( 2010 ) found that the degree to which activity patterns in rostrolateral prefrontal cortex changed during the encoding of a stimulus predicted better memory for the temporal position of that stimulus in the experiment .",
"Jenkins and Ranganath ( 2016 ) also showed that greater pattern distance between two stimuli at encoding in the hippocampus , medial and anterior prefrontal cortex predicted better order memory .",
"Only one study has directly related neural pattern drift to judgments of elapsed time in humans: Ezzyat and Davachi ( 2014 ) found that patterns of fMRI activity in left hippocampus were more similar for pairs of stimuli that were later estimated to have occurred closer together in time , despite equivalent time passage between all pairs ( a little less than a minute ) .",
"While the Ezzyat and Davachi ( 2014 ) study provides support for our hypothesis , it has some limitations .",
"First , in Ezzyat and Davachi ( 2014 ) , participants estimated the temporal distance of stimuli that were linked to their contexts in an artificial way ( by placing pictures of objects or famous faces on unrelated scene backgrounds ) ; it is unclear whether these results will generalize to more naturalistic situations where events are linked through a narrative .",
"Second , since participants performed the temporal memory test after each encoding run , they were not entirely naïve to the manipulation .",
"Knowing that they would have to estimate durations between stimuli could have changed participants’ strategy and enhanced their attention to time ( for evidence that estimating time prospectively engages different mechanisms , see Hicks et al . , 1976 , and Zakay and Block , 2004 ) .",
"In the current study , we sought to address the above issues by eliciting temporal distance judgments for pairs of events that had occurred several minutes apart and that were embedded in the context of a rich naturalistic story; participants listened to the entire story before being informed about the temporal judgment task .",
"Based on the studies reviewed above , we predicted that neural pattern drift in medial temporal and lateral prefrontal regions might support duration estimation .",
"In our study , we examined these regions of interest ( ROIs ) , as well as a broader set of regions that have been implicated in fMRI studies of time estimation , including the inferior parietal cortex , putamen , insula and frontal operculum ( see Box 1 for a review ) .",
"In addition to the ROI analysis , which examined activity patterns in masks that were anatomically defined , we performed a searchlight analysis , which examined activity patterns within small cubes over the whole brain .",
"Participants were scanned while they listened to a 25-minute science fiction radio story .",
"Outside the scanner , they were surprised with a time perception test , in which they had to estimate how much time had passed between pairs of auditory clips from the story .",
"Controlling for objective time , we found that the degree of neural pattern distance between two clips at the time of encoding predicted how much time an individual would later estimate passed between them .",
"The effect was significant in the right entorhinal cortex ROI .",
"Extending the anatomical analysis to all masks in cortex revealed an additional effect in the left caudal anterior cingulate cortex ( ACC ) .",
"Moreover , whole-brain searchlight analyses yielded significant clusters spanning the right anterior temporal lobe .",
"Our results suggest that patterns of neural activity in these regions may carry contextual information that helps us make retrospective time judgments on the order of minutes ."
],
[
"We tested whether BOLD pattern change between two clips correlated with temporal distance estimates , using both ROI and whole-brain searchlight analyses .",
"Each type of analysis was performed both within-participants across intervals and within-intervals across participants .",
"In the within-participant analysis , we correlated each participant’s duration estimates with that participant’s neural pattern distances ( see Within-Participant Correlation between Pattern Change and Duration Estimates and Within-Participant Whole-brain Searchlight ) .",
"In the within-interval analysis , we correlated individual differences in subjective duration for a given interval with individual differences in neural pattern distance for that interval ( see Within-Interval Correlation between Pattern Change and Duration Estimates and Within-Interval Whole-brain Searchlight ) .",
"The two versions of each analysis were performed in order to rule out the possibility that our effects were driven either by participant or interval random effects .",
"In particular , we were concerned that correlations between neural pattern distance and behavior could reflect sensitivity to perceptual or semantic features of the clips ( i . e . , clip pairs with similar perceptual/semantic features might be associated with shorter duration estimates and greater neural similarity , relative to clip pairs with more dissimilar features ) .",
"The within-interval analysis addresses this concern by holding clip identity constant .",
"Next , we fit a mixed-effects model for each ROI ( see Mixed-Effects Model Accounting for Naïve Duration Estimates ) , in which we estimated whether pattern distance in that ROI could predict duration estimates , even when accounting for participant random effects , item ( interval ) random effects , as well as naïve duration estimates ( which are a proxy for the perceptual and semantic similarity between two clips , see Behavioral results ) .",
"Finally , we discuss the brain regions that showed significant effects across all analyses ( see Comparing Results from ROI and Searchlight Analyses ) .",
"As noted in the Materials and methods , the ROI and searchlight analyses were conducted only on high-confidence two-minute intervals .",
"Six-minute intervals were excluded from the fMRI analysis , since we could not successfully dissociate neural pattern change at this timescale from low-frequency scanner noise ( see Methodological challenges with analyzing pattern distance over long time scales in the Materials and methods ) . 10 . 7554/eLife . 16070 . 011Figure 4 . Correlating pattern distance with duration estimates within participants . For each ROI in each participant , the pattern distance between each pair of clips at encoding was correlated with the participant’s retrospective duration estimate ( A–B ) .",
"The top panel ( A ) shows two example intervals .",
"The neural distance ( 1-Pearson’s r ) between clips 2 and 4 ( second interval ) is greater than the neural distance between clips 1 and 3 ( first interval ) , as is the subjective duration estimate .",
"( B ) shows the correlation between neural distance and duration estimates in a hypothetical region and participant .",
"( C ) We used a permutation test to generate 10 , 000 surrogate pattern distance vectors ( see Figure 4—figure supplement 1 ) , which we then used to obtain a distribution of null correlations between neural distances and duration estimates .",
"For each ROI in each participant , we calculated the z-scored correlation value , which reflects the strength of the empirical correlation relative to the distribution of null correlations .",
"For each ROI , we performed a random effects t-test to assess whether the z-score was reliably positive across participants .",
"P-values from this t-test were then subjected to multiple comparisons correction using False Discovery Rate ( FDR ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16070 . 01110 . 7554/eLife . 16070 . 012Figure 4—figure supplement 1 . Permutation test assessing the temporal specificity of correlations between pattern change and behavior . This procedure is described in the Materials and methods ( see Statistical analysis of correlations between pattern change and behavior ) .",
"( A , B )",
"The time course of pattern change is constructed using the distance ( 1 - Pearson’s r ) between each pattern and the pattern 80 TRs ( 2 min ) after it .",
"As in the main analysis , we averaged over the 5 consecutive TRs surrounding each pattern ( for simplicity , this is not shown in the above figure ) .",
"( C ) 10 , 000 surrogate pattern distance time courses are generated by randomizing the phases of the original time course , thus conserving the amplitude of each frequency component .",
"( D ) Surrogate pattern distances are correlated with time estimates , generating 10 , 000 null correlations .",
"A Z-value for each ROI / searchlight in each participant is computed to compare the strength of the empirical correlation with the distribution of null correlations .",
"The p-value for a given ROI is obtained using a right-tailed t-test on the Z-values across participants . DOI: http://dx . doi . org/10 . 7554/eLife . 16070 . 012"
],
[
"After probing human participants’ time perception for intervals from an auditory story they had just heard , we found substantial variability in subjective estimates of the passage of time .",
"This variability was significantly correlated with changes in BOLD activity patterns in the right anterior temporal lobe , particularly the right entorhinal cortex , between the start and end of each interval .",
"Control experiments demonstrated that duration estimates were strongly driven by contextual boundaries and that the relationship between neural distance and behavior still held when we controlled for the perceptual and semantic similarity of the clips .",
"Our findings suggest that patterns of activity in these regions might encode contextual information that participants can later retrieve to infer the durations of intervals on the scale of minutes .",
"Additional work is needed to assess how these regions contribute to representing particular contextual features ( such as physical environment , abstract task states , and emotional states ) and whether changes in each of these features affect retrospective duration estimates differently ."
],
[
"18 participants ( 13 female ) took part in the study .",
"All participants were recruited from the Princeton undergraduate and graduate student population and were between 18 and 31 years of age ( mean = 22 years ) .",
"All participants were screened to ensure no neurological or psychiatric disorders .",
"Written informed consent was obtained for all participants in accordance with the Princeton Institutional Review Board regulations .",
"Participants were compensated $20/hr for the scanning session , and $12/hr for the behavioral session .",
"Given that no previous studies had related neural pattern change during a naturalistic stimulus to subsequent duration estimates for minutes-long intervals , we could not a priori estimate the variance in the pattern change signal , the variance in duration estimates , or the correlation between them .",
"Therefore , rather than performing a power analysis , we chose a sample size that was in the same range as previous fMRI studies that had used naturalistic stimuli to study memory ( Lerner et al . , 2011 , n=11 per condition; Chen et al . , 2015 , n=13 , 14 and 24 per condition; Chen et al . , 2016 , n=22 [5 excluded] ) , as well as fMRI studies that had related neural pattern distance to mnemonic judgments ( Ezzyat and Davachi , 2011 , n=19; Jenkins and Ranganath , 2010 , n=16 ( 1 excluded ) ; Ezzyat and Davachi , 2014 , n=21 ( 3 excluded ) , Jenkins and Ranganath , 2016 , n=17 ) .",
"The experiment consisted of two parts: an approximately 40-min session in the MRI scanner , during which participants listened to the auditory story , followed immediately by a 1-hr behavioral session , during which participants completed a time perception test on the story they had just heard .",
"Figure 1 illustrates the experimental procedure .",
"Participants were scanned in a 3T full-body Skyra MRI scanner ( Siemens , Munich , Germany ) with a 20-channel head coil .",
"Functional images were acquired using a T2*-weighted echo planer imaging ( EPI ) pulse sequence ( repetition time [TR] , 1500 ms; echo time [TE] , 28 ms; flip angle , 64° ) , each volume comprising 27 slices of 4 mm thickness .",
"In-plane resolution was 3×3 mm2 ( field of view [FOV] , 192×192 mm2 ) .",
"Slice acquisition order was interleaved .",
"Anatomical images were acquired using a T1-weighted magnetization-prepared rapid-acquisition gradient echo ( MPRAGE ) pulse sequence ( TR , 2300 ms; TE , 3 . 08 ms; flip angle 9°; 0 . 89 mm3 resolution; FOV , 256 mm2 ) .",
"Participants’ heads were stabilized with foam padding to minimize head movement .",
"Auditory stimuli were presented using the Psychophysics toolbox ( Brainard , 1997; Pelli , 1997 ) .",
"Participants were provided with MRI compatible in-ear mono earbuds ( Model S14 , Sensimetrics Corporation , Malden , MA ) , which provided the same audio input to each ear .",
"MRI-safe passive noise-canceling headphones were placed over the earbuds for additional protection against noise .",
"FMRI data processing was carried out using FEAT ( FMRI Expert Analysis Tool ) Version 5 . 98 , part of FSL ( FMRIB's Software Library , www . fmrib . ox . ac . uk/fsl ) .",
"The following procedure was applied: motion correction using MCFLIRT ( Jenkinson et al . , 2002 ) ; slice-timing correction using Fourier-space time-series phase-shifting; non-brain removal using BET ( Smith , 2002 ) ; spatial smoothing using a Gaussian kernel of FWHM 6 . 0 mm; grand-mean intensity normalization of the entire 4D dataset by a single multiplicative factor; and high-pass temporal filtering ( Gaussian-weighted least-squares straight line fitting , with sigma=240 . 0 s ) .",
"The procedure for selecting the high-pass filter is described below .",
"Preprocessed data were kept in the native functional space for all analyses , except for the within-interval searchlight analysis , which was performed across participants .",
"Preprocessed data were then despiked using the following procedure: for each voxel , data points that deviated from the mean by more than 5 times the inter-quartile range were removed and replaced using cubic interpolation ."
]
] | [
"What mechanisms support our ability to estimate durations on the order of minutes ?",
"Behavioral studies in humans have shown that changes in contextual features lead to overestimation of past durations .",
"Based on evidence that the medial temporal lobes and prefrontal cortex represent contextual features , we related the degree of fMRI pattern change in these regions with people’s subsequent duration estimates .",
"After listening to a radio story in the scanner , participants were asked how much time had elapsed between pairs of clips from the story .",
"Our ROI analyses found that duration estimates were correlated with the neural pattern distance between two clips at encoding in the right entorhinal cortex .",
"Moreover , whole-brain searchlight analyses revealed a cluster spanning the right anterior temporal lobe .",
"Our findings provide convergent support for the hypothesis that retrospective time judgments are driven by 'drift' in contextual representations supported by these regions ."
] | [
"How do humans judge how much time has passed during daily life , such as when waiting for the bus ?",
"Psychology studies have shown that people remember events to have lasted longer when more changes occurred during that time period .",
"These changes can occur either in the environment ( such as changes in location ) or in the individual’s internal state ( such as changes in goals and emotions ) .",
"Brain activity changes from moment to moment .",
"Lositsky et al . hypothesized that when patterns of activity in a person’s brain change a lot across an interval of time , that person will judge that a long time has passed .",
"On the other hand , if brain activity changes less over that interval , individuals will judge that less time has passed .",
"Some regions of the brain are sensitive to information that unfolds over several minutes; many of these regions are vital for forming memories of episodes from our lives .",
"Using a technique called functional magnetic resonance imaging ( fMRI ) , Lositsky et al . specifically looked at the activity of these regions while volunteers listened to a 25-minute radio drama .",
"Afterwards , the volunteers listened to clips from different events in the story and judged how much time passed between those events .",
"Even though each pair of audio clips occurred exactly two minutes apart in the original story , people’s time judgments were strongly influenced by how many scene changes happened in the story between the two clips .",
"In a part of the brain called the right anterior temporal lobe – and especially in a region of it called the entorhinal cortex – Lositsky et al . found that brain activity changed more when audio clips were judged to be further apart in time .",
"Activity in this region fluctuated more slowly overall than in the rest of the brain .",
"This could mean that it combines sensory information ( about images , sounds , smells and so on ) across minutes of time , in order to form a representation of the current situation .",
"Future research could focus on several unanswered questions .",
"Exactly which environmental and internal changes influence our perception of time ?",
"What form does this information take in the entorhinal cortex ?",
"Studies show that the entorhinal cortex contains “grid cells” that track our location in space .",
"Could these cells also help judge the passage of time ?"
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding | elife-05242-v2 | [
[
"The primary cilium provides a unique cellular compartment for interaction with the extracellular milieu .",
"Initiated by discoveries in chemosensation in Caenorhabditis elegans , of ciliary defects in polycystic kidney disease , and of the central role of cilia in the vertebrate hedgehog ( Hh ) signaling pathway , we now know that the organelle is a signaling node for multiple pathways in development , disease , and homeostasis ( reviewed in Gerdes et al . , 2009; Lancaster and Gleeson , 2009; Goetz and Anderson , 2010 ) .",
"Although the signaling functions of the ciliary membrane are crucial for development and homeostasis , we are just beginning to learn the cellular and molecular mechanisms that underlie the dynamic regulation of ciliary membrane protein composition in existing cilia during cilium-generated signaling ( Pazour and Bloodgood , 2008; Nachury et al . , 2010; Hu and Nelson , 2011; Malicki and Avidor-Reiss , 2014 ) .",
"During ciliogenesis , soluble proteins are delivered into the organelle by intraflagellar transport ( IFT ) ( Pedersen and Rosenbaum , 2008 ) .",
"The working model for delivery of ciliary membrane proteins during ciliogenesis is that vesicles derived from the Golgi and containing newly synthesized membrane proteins destined for the cilium traffic via as yet poorly characterized motors to specialized regions of the plasma membrane near the base of the organelle , including the ciliary pocket ( Molla-Herman et al . , 2010; Rohatgi and Snell , 2010 ) , and enter the cilium as the organelle elongates .",
"IFT proteins are required for the presence of some membrane proteins in the cilium , but it remains unknown whether IFT is required for their entry ( Mukhopadhyay et al . , 2010; Wood and Rosenbaum , 2014 ) .",
"Several proteins and protein complexes are involved in trafficking of membrane proteins to the organelle during its formation as well as in establishing the barrier , and their mutational disruption leads to aberrant regulation of constitutively localized ciliary membrane proteins , or to complete absence of cilia and to the ciliopathies ( Garcia-Gonzalo et al . , 2011; Hildebrandt et al . , 2011; Sang et al . , 2011; Williams et al . , 2011; Chih et al . , 2012 ) .",
"After the cilium is fully formed , however , the protein composition of the ciliary membrane does not remain fixed , but is dynamically regulated during cilium-generated signaling .",
"In the well-studied cilium-based Hedgehog ( Hh ) signaling system in vertebrates , the pathway suppressors Ptch and GPR161 are present in the ciliary membrane in the absence of the Hh ligand , and the effector Smoothened ( Smo ) is mostly excluded from the cilium and present on the cell plasma membrane and in internal vesicles .",
"When Hh binds to it receptor Ptch , Smo becomes enriched in the ciliary membrane where it regulates downstream events in the pathway ( Rohatgi et al . , 2007; Milenkovic et al . , 2009; Wang et al . , 2009; Dorn et al . , 2012; Mukhopadhyay et al . , 2013 ) .",
"Concomitantly , both Ptch and GPR161 are depleted from the organelle .",
"Although several models have been proposed to account for the fascinating , dynamic , regulated enrichment and loss of Hh effectors in the ciliary membrane , we still know few mechanistic details .",
"The use of cilia as sensory/signaling organelles is an ancient invention .",
"Interactions between receptors ( agglutinin polypeptides ) on the cilia of plus and minus gametes during fertilization in the green alga Chlamydomonas trigger an anterograde IFT-dependent signaling pathway within the organelles ( Wang and Snell , 2003; Wang et al . , 2006 ) that activates the gametes for cell–cell fusion ( Snell and Goodenough , 2009 ) .",
"The plus agglutinin polypeptide receptor expressed on plus gametes is encoded by the SAG1 gene and the minus agglutinin polypeptide receptor on minus gametes is encoded by the SAD1 gene ( Ferris et al . , 2005 ) .",
"In addition to activating the signaling pathway within each type of gamete , interactions between the SAG1 plus agglutinin and the SAD1 minus agglutinin cause the cilia of the gametes to adhere to each other , thereby bringing the gametes into the close contact required for gamete fusion .",
"Recently , using plus gametes expressing a SAG1-HA transgene , we showed that soon after synthesis of the full-length protein encoded by SAG1 , it is cleaved to yield the N-terminal plus agglutinin polypeptide and a C-terminal , integral membrane polypeptide , SAG1-C65 ( Belzile et al . , 2013 ) .",
"We found that although small amounts of SAG1-C65 were on the cilia of resting plus gametes , most was excluded from the organelles and present at the plasma membrane .",
"When the cilium-generated signaling pathway was activated , however , the C-terminal SAG1-C65 polypeptide was rapidly recruited to the ciliary membrane through a mechanism that did not require the anterograde IFT motor kinesin 2/FLA10 .",
"Moreover , before entering the cilium during signaling , SAG1-C65 became highly polarized , accumulating in the periciliary region as part of a ciliary entry pathway that required cytoplasmic microtubules .",
"Here , we report that during cilium-generated signaling , cells regulate ciliary membrane SAG1-C65 levels by action of the retrograde IFT motor in the cytoplasm and by regulated shedding of SAG1-C65-containing ciliary ectosomes that retain signaling competency and comprise a distinct membrane compartment ."
],
[
"The presence in Chlamydomonas of only a single cytoplasmic dynein , cytoplasmic dynein 1b , and our previous results that cytoplasmic microtubules participated in periciliary accumulation and ciliary entry of SAG1-C65 during signaling raised the possibility that this microtubule minus end-directed IFT motor ( Pazour et al . , 1999 ) might participate in SAG1-C65 redistribution .",
"The benzoyl dihydroquinazolinone , ciliobrevin D , has been shown in metazoans to block cytoplasmic dyneins ( Hyman et al . , 2009; Firestone et al . , 2012; Ye et al . , 2013 ) .",
"And , recently Shih et al . ( 2013 ) showed that ciliobrevin D inhibition of Chlamydomonas cytoplasmic dynein 1b ( DHC1b ) strongly reduced retrograde IFT .",
"We tested for a role of the retrograde IFT motor in SAG1-C65 redistribution during signaling using ciliobrevin D . Early during ciliary adhesion and cilium-generated signaling , activation of a ciliary adenylyl cyclase leads to an ∼15-fold increase in cellular cAMP that activates gametes to prepare for fusion .",
"Thus , it is possible to study cellular events activated by the signaling pathway , such as redistribution of the agglutinin polypeptide , release of cell walls , and upregulation of transcripts for gamete-specific proteins , in gametes of a single mating type by incubating them in the cell-permeable analogue , db-cAMP ( Pijst et al . , 1984b; Pasquale and Goodenough , 1987; Goodenough , 1989; Hunnicutt et al . , 1990; Belzile et al . , 2013; Ning et al . , 2013 ) .",
"We incubated SAG1-HA/sag1-5 gametes ( which express a tagged SAG1-C65 polypeptide , SAG1-C65-HA ) ( Belzile et al . , 2013 ) with and without ciliobrevin D for 20 min , activated them by addition of db-cAMP for 5 min in the continued presence of the inhibitor , and then assessed SAG1-C65-HA localization .",
"As shown previously ( Belzile et al . , 2013 ) , whereas SAG1-C65-HA showed apical localization in only a small portion of resting gametes , the protein became apically localized after gametes were activated by incubation in db-cAMP for 5 min ( Figure 1A , B ) .",
"Incubation of resting cells in ciliobrevin D reduced the already low percentage of cells with apically localized SAG1-C65-HA ( Figure 1B ) , and inhibited SAG1-C65-HA redistribution to the apical ends in cells incubated for 5 min in db-cAMP ( Figure 1A , B ) .",
"Consistent with the results of Shih et al . ( 2013 ) that ciliobrevin did not completely inhibit the motor activity of cytoplasmic dynein-1b , after longer incubation in db-cAMP , SAG1-C65-HA redistribution returned ( not shown ) .",
"These results indicated that the retrograde IFT motor was essential for the rapid redistribution of SAG1-C65-HA to the peri-ciliary region at the cell apex . 10 . 7554/eLife . 05242 . 003Figure 1 . Cytoplasmic dynein 1b is required for the rapid , signaling-induced apical localization and ciliary enrichment of SAG1-C65-HA .",
"( A ) Confocal images of resting gametes in the presence and absence of Cilio-D for 20 min and gametes activated for 5 min in the presence and absence of Cilio-D .",
"( B ) Percent of cells showing apical localization of SAG1-HA ( see ‘Materials and methods’ ) .",
"Data are from three experiments .",
"100 cells were scored for each condition .",
"Error bars indicate ± SD .",
"( C ) Immunoblots of cilia , cell bodies and whole cells ( 8 μg protein/lane ) of SAG1-HA cells treated as indicated and activated ( or not ) with db-cAMP for 5 min . ( D ) Confocal images showing cytoplasmic microtubules in control and Cilio-D treated plus gametes .",
"( E ) Immunoblot for GSP1 of plus cells incubated with or without Cilio-D and with or without db-cAMP .",
"( F ) Confocal images of resting and db-cAMP activated gametes showing high levels of SAG1-C65-HA in cilia of resting fla24 gametes and lack of redistribution upon activation .",
"( G ) Immunoblots of resting and activated whole cells , cell bodies , and cilia ( 2 . 5 μg protein/lane ) of fla24 gametes . DOI: http://dx . doi . org/10 . 7554/eLife . 05242 . 003 Ciliobrevin D inhibition of signaling-induced SAG1-C65-HA apical localization also resulted in a concomitant inhibition of accumulation of the protein in the cilia .",
"Cilia of resting gametes contained little SAG1-C65-HA as assessed by both IF and immunoblotting of isolated cilia , but upon 5-min incubation in db-cAMP , SAG1-C65-HA in the organelles increased in control , but not in the ciliobrevin samples ( Figure 1A , C , upper panels ) .",
"The effects of ciliobrevin D were transient , and cells washed out of the inhibitor rapidly regained the ability to respond to db-cAMP by accumulating SAG1-C65-HA in their cilia ( Figure 1C ) .",
"IF analysis showed that the array of cytoplasmic microtubules was unaffected by ciliobrevin D ( Figure 1D ) .",
"And , the inhibitor had no effect on total cellular SAG1-C65-HA ( Figure 1C , lower panels ) .",
"Interestingly , and consistent with the IF results that ciliobrevin D treatment of resting cells reduced the percent with apical localization of SAG1-C65-HA , the basal level of SAG1-C65-HA in cilia isolated from ciliobrevin D-treated resting gametes was lower than in the control , resting gametes ( Figure 1C ) .",
"We also examined whether the inhibitor blocked signaling per se , by testing its affects on signaling-induced phosphorylation of the homeodomain protein , GSP1 ( Wilson et al . , 1999 ) .",
"As shown in Figure 1E , gametes in ciliobrevin D remained capable of responding to db-cAMP as assessed by the phosphorylation-related change in migration of GSP1 in immunoblots ( Wilson et al . , 1999 ) .",
"Thus , apical localization and ciliary enrichment of SAG1-C65 during signaling were both inhibited by the cytoplasmic dynein inhibitor , ciliobrevin D , indicating that the retrograde IFT motor acts in the cytoplasm to enrich SAG1-C65 in the peri-ciliary region concomitant with entry of the protein into the ciliary membrane .",
"As a further test for a role of the retrograde IFT motor in SAG1-C65 trafficking , we introduced the SAG1-HA transgene into the cytoplasmic dynein mutant strain fla24 , which has an L3242P mutation in dynein heavy chain 1b ( DHC1b ) ( Lin et al . , 2013 ) .",
"fla24 exhibits a temperature-sensitive ciliary phenotype , being ciliated at 21°C , and without cilia after 4–6 hr at 32°C .",
"Even at 21°C , however , DHC1b and its associated light chain protein D1bLIC are reduced dramatically and the level of D1bLIC in fla24 cells is less than 7% of wild type at 21°C ( Lin et al . , 2013 ) .",
"Thus , we tested the effects of reduced retrograde IFT motor function on SAG1-C65-HA apical localization and ciliary entry by using SAG1-HA/fla24 gametes at 21°C .",
"Consistent with the results with ciliobrevin D , at 21°C , SAG1-C65-HA failed to undergo rapid apical redistribution in the DHC1b-depleted fla24 gametes incubated in db-cAMP ( Figure 1F ) and the amount of SAG1-C65-HA in cilia did not increase ( Figure 1G ) .",
"Interestingly , the depleted levels of the retrograde IFT motor in the fla24 cells were also associated with aberrantly large amounts of ciliary SAG1-C65-HA in resting gametes ( Figure 1F , G ) .",
"Thus , the retrograde IFT motor is required for trafficking of SAG1-C65-HA during ciliogenesis of gametes , and it is required for redistribution of SAG1-C65-HA to the peri-ciliary region and into the cilium during cilium-generated signaling .",
"We also followed the fate of SAG1-C65-HA when SAG1-C65-HA plus gametes were mixed with fusion-defective hap2 minus gametes and allowed to undergo sustained ciliary receptor-induced signaling ( Liu et al . , 2008 ) .",
"As expected , cilia isolated after 30 min of signaling possessed substantial amounts of SAG1-C65-HA ( Belzile et al . , 2013 ) , and both the ciliary and cell body SAG1-C65-HA levels remained constant for 3 hr ( Figure 2A , control ) .",
"Previously ( Ning et al . , 2013 ) , we showed that gametes upregulate SAG1 transcripts fourfold to fivefold during gamete activation ( Figure 3B ) , and thus we were surprised that the protein levels failed to increase during the 3-hr experiment .",
"Although protein levels do not necessarily reflect transcript levels , both the transcript ( Ning et al . , 2013 ) and protein amounts of the minus gamete-specific membrane protein HAP2 increased during gamete activation ( Figure 2B , C ) .",
"Therefore , we tested whether SAG1-C65-HA was being synthesized and then turned over by the gametes undergoing sustained , agglutinin receptor-induced ciliary signaling by examining protein amounts in samples in which protein synthesis was blocked .",
"At 30 min after mixing the gametes together in the protein synthesis inhibitor , cycloheximide ( CH ) , the amount of SAG1-C65-HA in the cilia was similar to that of control , signaling gametes as shown previously ( Belzile et al . , 2013 ) .",
"At 1 . 5 hr , however , SAG1-C65-HA in the cilia of the CH-treated samples had decreased , and at 3 hr the amount of SAG1-C65-HA in the cilia had fallen dramatically ( Figure 2A , CH ) . 10 . 7554/eLife . 05242 . 004Figure 2 . SAG1-C65-HA is lost from cells during adhesion and signaling .",
"( A ) Immunoblot analysis of SAG1-C65-HA in isolated cilia and cell bodies ( 8 μg protein/lane ) from cells activated by mixing with hap2 minus gametes .",
"Cells were pretreated ( or not ) for 15 min with 10 mg/ml cycloheximide ( CH ) before mixing .",
"( B ) RNA-Seq data from Ning et al . ( 2013 ) showing transcript abundance as median reads per kilobase per million mapped reads ( RPKM ) values of SAG1 and HAP2 transcripts from vegetative cells , resting gametes , and activated gametes of both mating types .",
"( C ) Immunoblots using HA and tubulin antibodies of HAP2-HA minus gametes ( 1 . 5 × 107 cells/ml ) mixed with an equal number of fus1 plus gametes for the indicated times .",
"( D ) Immunoblot analysis of SAG1-C65-HA in isolated SAG1-HA resting cells , SAG1-HA cells activated with db-cAMP , and SAG1-HA cells activated by undergoing ciliary adhesion with hap2 minus gametes in the presence of the protein kinase inhibitor staurosporine ( 1 μM; st . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05242 . 00410 . 7554/eLife . 05242 . 005Figure 3 . Adhering gametes release SAG1-C65-HA in a membrane-associated form .",
"( A ) Immunoblot analysis of SAG1-C65-HA in cells and medium harvested after undergoing adhesion and signaling with fusion-defective hap2 minus gametes for the indicated times .",
"1 × 106 cell equivalents were loaded in each lane .",
"( B ) Number of cell equivalents of SAG1-HA released into the medium 3 hr after mixing SAG1-C65-HA gametes with hap2 gametes in the presence and absence of CH as assessed by quantitative immunoblotting .",
"Results are averages from the experiment shown in ( A ) and at least one other independent experiment .",
"Error bars show S . D . ( C ) Immunoblot analysis of SAG1-C65-HA in the medium collected from SAG1-HA gametes undergoing adhesion with hap2 minus gametes .",
"Before mixing , cells were activated ( or not ) with db-cAMP and then mixed together in the presence or absence of 1 μM staurosporine ( st . ) for 3 hr . ( D ) Immunoblot analysis of SAG1-HA distribution after differential centrifugation of medium from SAG1-C65-HA and hap2 gametes that had been adhering for 3 hr .",
"S , supernatant; P , pellet .",
"( E ) Distribution of SAG1-C65-HA after 200 , 000×g centrifugation of ectosome samples that had been incubated on ice for 20 min in 0 . 5 M NaCl or 2% NP-40 in HMDEK buffer .",
"S , supernatant; P , pellet . DOI: http://dx . doi . org/10 . 7554/eLife . 05242 . 005 Notably , SAG1-C65-HA was also depleted from the cell body during agglutinin receptor-induced signaling in CH ( Figure 2A , CH ) .",
"This wholescale depletion of cellular SAG1-C65-HA in CH was not simply a reflection of normal turnover , because the amount of SAG1-C65-HA in resting gametes was unchanged after 3 hr in CH ( Figure 2D ) .",
"Moreover , SAG1-C65-HA also remained constant in gametes activated with db-cAMP in the presence of CH ( Figure 2D ) , and no SAG1-C65-HA could be detected in the medium ( not shown ) .",
"And , gametes undergoing sustained agglutinin-induced signaling in CH in the presence of the protein kinase inhibitor staurosporine , which blocks recruitment of SAG1-C65-HA to cilia ( Belzile et al . , 2013 ) , also did not lose SAG1-C65 , indicating that interactions of the agglutinin receptors on plus and minus gametes and signaling were required for loss ( Figure 2D ) .",
"To determine whether the lost SAG1-C65-HA had been shed into the medium , we activated sustained agglutinin receptor interactions by mixing SAG1-C65-HA plus gametes and fusion-defective minus gametes together for 3 hr , harvested the cells , and then determined SAG1-C65-HA amounts in equal portions of the cells and the medium .",
"As shown in Figure 3A ( upper panels ) , cells indeed released SAG1-C65-HA into the medium during receptor-activated signaling .",
"Moreover , the amount recovered in the medium after 3 hr was three times as much as present in the starting cells ( Figure 3A , B ) , indicating that the relatively constant levels of cell body and ciliary SAG1-C65-HA during sustained agglutinin receptor interactions and signaling reflected a dynamic balance between synthesis , ciliary delivery , and release from the cilia .",
"Moreover , when the cells were mixed together in the presence of the protein synthesis inhibitor cycloheximide , nearly 75% of the SAG1-HA in the cells at T = 0 was recovered in the medium after 3 hr of receptor-activated signaling , demonstrating that release was the major fate of the protein ( Figure 3A , lower panels and Figure 3B ) .",
"Consistent with the results above , SAG1-C65-HA was absent from the medium of adhering gametes in which signaling and ciliary recruitment of SAG1-C65-HA were blocked by the addition of staurosporine ( Figure 3C ) .",
"On the other hand , compared to control SAG1-HA gametes undergoing receptor-activated signaling induced by mixing them with hap2 minus gametes , SAG1-HA gametes that had been activated by pre-incubating them with db-cAMP and then mixed with hap2 gametes , released increased amounts of SAG1-C65-HA into the medium , even when staurosporine was also added to the medium ( Figure 3C ) .",
"Thus , once gametes had been activated with db-cAMP , staurosporine no longer blocked release of SAG1-C65-HA during agglutinin receptor interactions .",
"Differential centrifugation showed that the SAG1-C65-HA in the medium was particulate .",
"It was in the supernatant after sedimenting the cells at low speed , remained soluble after centrifugation at intermediate speed ( 20 , 000×g ) , and was sedimented upon centrifugation at 200 , 000×g ( Figure 3D ) .",
"Thus , nearly all of the SAG1-C65-HA that was lost during ciliary adhesion was recovered in the medium in a particulate form .",
"Biochemical fractionation indicated that SAG1-C65-HA harvested from the medium by high-speed centrifugation indeed was an integral membrane protein ( Belzile et al . , 2013 ) , A high salt wash failed to release the protein , but release required detergent ( Figure 3E ) .",
"Taken together , these results indicated that ciliary receptor interactions and signaling triggered release of SAG1-C65-HA from the cilia into the medium in a particulate , membrane-associated form .",
"Several groups have reported that vegetative cells and gametes constitutively release membrane vesicles ( Wiese , 1965; Bergman et al . , 1975; Snell , 1976; Dentler , 2013 ) , and very recently Wood et al . ( 2013 ) demonstrated release of biologically active ciliary ectosomes from vegetative cells during cell division .",
"Moreover , Goodenough and Heuser reported several years ago that cilia on adhering gametes possessed associated membrane vesicles that were absent from non-adhering samples ( Goodenough and Heuser , 1999 ) .",
"Those studies did not determine whether the vesicles seen in the samples of the mixed gametes represented vesicles that were in the medium of each gamete type before the cells were mixed , or if their formation was induced by adhesion .",
"We washed gametes into fresh medium to remove any vesicles that might have been present in the medium , and used negative staining and TEM to examine the cilia of non-adhering and adhering gametes .",
"As expected , the cilia of the non-adhering gametes exhibited a smooth membrane with no evidence of particulate material ( Figure 4A ) .",
"The surfaces of cilia of plus and minus gametes that had been washed into fresh medium and mixed together for 10–15 min , however , were strikingly different ( Figure 4B ) .",
"Adhesion had triggered appearance of vesicles on the ciliary surfaces , some along the shafts of the organelles , and many near the ciliary tips .",
"Some of the vesicles were in clusters , but many were single vesicles in intimate association with the ciliary membrane .",
"Formation of vesicles during ciliary adhesion was unique to cilia , and we did not observe vesicles associated with the cell body surface of the gametes .",
"Importantly , similar to the release of SAG1-C65-HA from cilia detected by biochemical methods , formation of the vesicles required ciliary receptor-induced signaling and vesicles were not detected on cilia of plus gametes alone activated by db-cAMP ( Figure 4D ) .",
"Taken together , the simplest explanation for these results is that ciliary adhesion and signaling triggered shedding of ciliary membranes in the form of ciliary ectosomes . 10 . 7554/eLife . 05242 . 006Figure 4 . Adhering gametes release SAG1-C65-HA as ciliary ectosomes .",
"( A ) Negative stain transmission electron micrographs of resting wild type plus gametes showing the smooth membranes of the cilia .",
"The inset shows a higher magnification view of the tip of a cilium .",
"( The scale bars shown in this and subsequent TEM images are 1000 nm , with the exception that the bar in Figure 4E is 200 nm . ) ( B ) Wild type plus and hap2 minus gametes were washed into fresh N-free , mixed together for 10–15 min , and prepared for TEM .",
"The adhering cilia contained larger numbers of associated vesicles .",
"The inset shows a higher magnification view of the vesicles at the ciliary tips .",
"( C ) High magnification view of adhering cilia of wild type plus and hap2 minus gametes showing vesicles .",
"( D ) Vesicles were absent from the cilia of db-cAMP-activated wild type plus gametes .",
"( E ) TEM of ciliary ectosomes harvested from the medium of adhering wild type plus and hap2 minus gametes .",
"( F ) Immunoblots with the indicated antibodies of equal protein amounts ( 8 μg/lane ) of ciliary ectosomes ( right lane ) and cilia isolated from a mixture of adhering SAG1-C65-HA plus gametes and hap2 minus gametes ( left lane ) .",
"( G ) Relative abundance of SAG1-HA and FMG1 in equal amounts of protein of ciliary ectosomes compared to cilia in a representative experiment .",
"Error bars show S . D . from at least three quantifications of immunoblots . DOI: http://dx . doi . org/10 . 7554/eLife . 05242 . 00610 . 7554/eLife . 05242 . 007Figure 4—figure supplement 1 . Model illustrating unidirectional trafficking of SAG1 during ciliary adhesion and signaling . SAG1* in resting cells is present at the cell periphery , with only small amounts in cilia .",
"Initial ciliary adhesion to minus gametes by SAG1 agglutinin–SAD1** agglutinin interactions triggers gamete activation and redistribution of SAG1 to the peri-ciliary region at the apical ends of the cells followed by ciliary entry .",
"The retrograde IFT motor , cytoplasmic dynein 1b , is required for SAG1 movement along the cytoplasmic microtubules that originate from sites near the bases of the cilia .",
"Although the anterograde motor is not required for ciliary entry of SAG1 , it remains unknown whether it transports SAG1 once it is in the cilia .",
"During sustained ciliary adhesion and signaling , SAG1 is released into the medium in the form of ciliary ectosomes and is replaced by protein synthesis .",
"*Although we lack information on whether the SAG1 plus agglutinin fragment and SAG1-C65 form a complex , for simplicity they are here depicted as a single unit ( orange stars ) .",
"**For the purpose of illustrating ciliary adhesion , the model depicts SAD1 [violet stars] on minus gametes interacting with SAG1 on plus gametes and undergoing similar redistribution .",
"Whether SAD1 behaves similarly to SAG1 is unknown , and thus SAD1 behavior in the model is purely speculative . DOI: http://dx . doi . org/10 . 7554/eLife . 05242 . 007 Negative staining TEM confirmed that the particulate material isolated from the medium of adhering gametes indeed was composed of vesicles similar to those being released from the surface of the adhering cilia ( Figure 4E ) .",
"To our surprise , the protein composition of these ciliary ectosomes was much different than that of isolated cilia .",
"Immunoblotting ( Figure 4F ) showed that isolated cilia contained typical ciliary proteins including IFT81 , IFT139 , alpha tubulin , Bug22 ( an axonemal protein; Meng et al . , 2014 ) , SAG1-C65 , and the major ciliary membrane protein , FMG1 .",
"On the other hand , immunoblot analysis of an equal amount of ciliary ectosome protein showed that SAG1-C65-HA was enriched nearly sixfold in ciliary ectosomes compared to cilia , but most other ciliary proteins were de-enriched ( Figure 4F , G ) .",
"FMG1 was detectable , but strikingly de-enriched ( relative abundance <0 . 1 in ciiary ectosomes compared to cilia; Figure 4F , G ) , and IFT81 , IFT139 , Bug22 , and alpha tubulin typically were not detectable .",
"Thus , SAG1-C65-containing ciliary ectosomes represented a unique ciliary compartment released from cilia during receptor-activated signaling .",
"We examined whether the isolated ectosomes contained biological activity by mixing them with minus gametes and testing for agglutinin receptor interactions and gamete activation .",
"As shown by TEM ( Figure 5A , right panel ) , the vesicles adhered along the lengths of the cilia of the minus gametes , demonstrating that the ectosomes possessed the plus agglutinin polypeptide that bound to the SAD1 agglutinin polypeptide on the cilia of the minus gametes .",
"Vesicles did not adhere to flagella of minus vegetative cells , which do not express the minus agglutinin , demonstrating that binding was specific ( Figure 5A , left panel ) .",
"A direct binding assay confirmed the TEM results .",
"Isolated ectosomes were incubated with minus vegetative cells or minus gametes for 15 min , followed by harvesting of the cells by centrifugation .",
"Analysis of the harvested whole cells by immunoblotting showed that only minus gametes bound the SAG1-C65-HA vesicles .",
"Furthermore , cell fractionation showed that the vesicles bound only to the cilia , and little if any binding was detected in the cell body fraction ( Figure 5B ) . 10 . 7554/eLife . 05242 . 008Figure 5 . Ciliary ectosomes possess agglutinin receptor binding and signaling activity .",
"( A ) TEM of cilia on minus vegetative cells ( left panel ) and gametes ( right panel ) that had been mixed with ciliary ectosomes .",
"Only the cilia on the gametes bound ciliary ectosomes .",
"( B ) Immunoblot analysis of binding of SAG1-C65-HA-containing ectosomes to whole cells , cell bodies , and cilia of minus gametes .",
"The initial sample of cells + ectosomes is also shown ( input ) .",
"The lanes contain equal cell equivalents .",
"( C ) Cilium-generated signaling was activated when ciliary ectosomes were mixed with HAP2-HA minus gametes , as assessed by activation-induced upregulation of expression of the gamete fusion protein , HAP2 . DOI: http://dx . doi . org/10 . 7554/eLife . 05242 . 008 Finally , we tested whether the isolated ectosomes possessed the ability to activate signaling in minus gametes .",
"When minus gametes are activated by mixing with plus gametes , they upregulate synthesis of several genes specifically expressed in minus gametes , including the minus gamete fusion protein HAP2 ( Liu et al . , 2010; Ning et al . , 2013 ) .",
"We tested for signaling activity of the ectosomes by mixing them with minus gametes expressing a tagged form of the gamete fusion protein HAP2 ( HAP2-HA ) and ( as a control ) with minus vegetative cells , and assessed HAP2-HA protein levels .",
"As shown in Figure 5C , addition of the ectosomes indeed led to substantial increase in HAP2-HA .",
"Thus , the isolated ciliary ectosomes were capable of activating cilium-generated signaling ."
],
[
"We report here that membrane protein trafficking through the cilium during cilium-generated signaling in Chlamydomonas is uni-directional and depends on the concerted action of two cellular processes , protein delivery to the ciliary base by the retrograde IFT motor , cytoplasmic dynein 1b , and shedding of a unique ciliary membrane compartment in the form of ciliary ectosomes .",
"Use of both a retrograde IFT motor mutant , fla24 and the cytoplasmic dynein 1b inhibitor , ciliobrevin D , showed that the signaling-triggered rapid polarization of SAG1-C65-HA and its entry into the ciliary membrane were inhibited when motor function was impaired ( Figure 1 ) .",
"Thus , the retrograde IFT motor transports SAG1-C65-HA from distal cellular membrane sites to the periciliary region , either within the plasma membrane or as vesicles within the cytoplasm .",
"The increased amounts of SAG1-C65-HA in the cilia of resting fla24 gametes ( Figure 1F , G ) also indicated that the retrograde motor is important for membrane protein trafficking during the ciliogenesis that accompanies gamete formation .",
"In their studies of a conditional DHC1b mutant , dhc1b-3 , Engel et al . ( 2012 ) found several differences in ciliary protein composition compared to wild-type .",
"Whether the increased amount of SAG1-C65-HA in the fla24 cilia reflects a direct or indirect role for the retrograde motor in regulating ciliary membrane protein composition is unclear ( Kim et al . , 2009; Ocbina et al . , 2011 ) .",
"Although several membrane-associated proteins and protein complexes are strongly implicated in targeting ciliary membrane proteins to the periciliary region in many cell types ( Nachury et al . , 2010; Wood and Rosenbaum , 2014 ) , the motors that carry ciliary-destined membrane proteins to the base of the organelle have been largely unidentified .",
"One notable exception is photoreceptor cells in which cytoplasmic dynein 1 ( not the retrograde IFT motor ) is involved in trafficking of rod outer segment membrane proteins from the golgi to the base of the connecting cilium ( Tai et al . , 1999; Kong et al . , 2013 ) .",
"Kim et al . ( 2009 ) reported that treatment of mammalian cells with vinblastine , an agent that disrupts cytoplasmic microtubules , had no effect on Smoothened accumulation in the primary cilium many hours after addition of the Hh ligand .",
"On the other hand , the parent compound of ciliobrevin D , HPI-4 , inhibited Hh-induced ciliary accumulation of Smoothened by 60–70% 12 hr after addition of the Hh ligand , raising the possibility that a cytoplasmic dynein is involved in trafficking of Smoothened to the cilium in metazoans ( Hyman et al . , 2009 ) .",
"And Ye et al . ( 2013 ) found that ciliobrevin unexpectedly inhibited anterograde IFT after a 30 min incubation , leading them to suggest that a cytoplasmic dynein might be involved in the delivery of IFT complexes in the cytoplasm to the base of the cilium .",
"In addition to this signaling-regulated delivery of pre-existing membrane proteins to an existing organelle , cells also deliver pre-existing and newly synthesized membrane proteins to the cilium during ciliogenesis ( Rosenbaum et al . , 1969; Wood and Rosenbaum , 2014 ) .",
"And , vectorially labeled proteins on the membrane of de-ciliated cells become incorporated into cilia as they regrow ( Hunnicutt et al . , 1990; Dentler , 2013 ) .",
"It will be interesting to determine whether ciliary growth in Chlamydomonas depends on cargo transport along cytoplasmic microtubules by cytoplasmic dynein 1b .",
"Given that growth of new cilia is dependent on anterograde IFT , whereas signaling-induced movement of pre-existing SAG1-C65-HA into existing cilia is independent of anterograde IFT , it may be that cells use one membrane protein trafficking modality for ciliary assembly and a different one for regulated ciliary enrichment of pre-existing membrane proteins .",
"Studies many years ago showed that cilia of Chlamydomonas vegetative cells and gametes undergo constitutive membrane shedding ( Wiese , 1965; Bergman et al . , 1975; Snell , 1976 ) of vesicles similar in protein composition to the ciliary membrane .",
"The recent work of Dentler ( 2013 ) showed that vegetative cells shed over 15% of their ciliary membrane per hr , and that the protein composition of the constitutively shed membrane vesicles was similar to that of the ciliary membrane per se .",
"In our current studies , however , resting gametes did not shed SAG1-C65-containing ciliary ectosomes , but shedding of such ectosomes required receptor interactions and signaling .",
"It is likely that the vesicles Goodenough and Heuser ( 1999 ) observed 25 years ago on adhering cilia corresponded to those we isolated from the medium .",
"Notably , the shed vesicles we isolated were enriched in SAG1-C65-HA and de-enriched in the 350 kDa major membrane protein compared to cilia .",
"And , nearly the entire cellular complement of pre-existing SAG1-C65-HA was shed in ∼3 hr .",
"Thus , receptor signaling-induced shedding of SAG1-C65-containing ciliary ectosomes was much more rapid and selective than the previously reported , constitutive shedding of ciliary membrane vesicles , and likely depends on a mechanism distinct from that used for constitutive shedding .",
"Our discovery that the isolated ciliary ectosomes possessed the ability to bind to the cilia of minus gametes and activate cilium-generated signaling ( Figure 5 ) indicated that the ectosomes also contained the plus receptor agglutinin polypeptide , which likely is an N-terminal fragment of the 340 kDa full length protein encoded by the SAG1 gene ( Belzile et al . , 2013 ) .",
"Several workers have reported that the agglutinin activity is lost during adhesion ( Weise and Wiese , 1978; Snell and Moore , 1980; Adair et al . , 1983; Pijst et al . , 1984a; Hunnicutt et al . , 1990; Hunnicutt and Snell , 1991 ) , and , using a semi-quantitiative bioassay , we reported that over 60% of it could be recovered in an uncharacterized form in the medium ( Hunnicutt et al . , 1990 ) .",
"In future studies , it will be interesting to determine if the integral membrane polypeptide SAG1-C65 is the membrane anchor for the agglutinin polypeptide , which lacks predicted transmembrane domains .",
"These considerations lead to the model that release of ciliary ectosomes during ciliary adhesion and signaling underlies the dynamic nature of ciliary adhesion evidenced by constant cilia adhesion and de-adhesion within mixtures of plus and minus gametes .",
"From another perspective , because the released ectosomes retain biological activity , their formation might help to maximize the probability of zygote formation .",
"As indicated above , recent studies showed that regulated formation of ciliary ectosomes is not unique to the cilia of gametes or possibly not even to Chlamydomonas .",
"Wood et al . ( 2013 ) reported that Chlamydomonas vegetative cells release ciliary ectosomes that contain an enzyme required for release of the extracellular matrix during cell division ( Kubo et al . , 2009 ) .",
"And , several groups have identified biologically active microvesicles from metazoans that contain ciliary proteins and are capable of binding to cilia ( Hogan et al . , 2009; Bakeberg et al . , 2011; Chacon-Heszele et al . , 2014 ) .",
"Regulating the membrane protein composition of the cilium is a central feature of cilium-dependent signaling .",
"Current models posit that regulation of ciliary membrane protein composition is controlled by bidirectional flow of membrane proteins from the cell body to the cilium and back .",
"Direct evidence has been lacking , however , that ciliary membrane proteins lost from the cilium during signaling actually return to the cell , because of the difficulty in determining the fate of ciliary proteins in most systems .",
"Our results demonstrate that cells can maintain a steady-state level of a ciliary membrane protein by unidirectional flow ( Figure 4—figure supplement 1 ) in which proteins move from the cell body to the cilium and then into the extracellular milieu ."
],
[
"Chlamydomonas reinhardtii strains 21gr ( mating type plus; CC-1690 ) , 6145C ( mating type minus; CC-1691 ) , and the fusion-defective minus strain hap2 are available from the Chlamydomonas Genetics Center , University of Minnesota , St . Paul .",
"A SAG1-HA transgene was introduced into the ciliary adhesion mutant mt+/sag1-5 as previously described ( Belzile et al . , 2013 ) .",
"Staurosporine , cycloheximide , dibutyryl-cAMP , and poly-l-lysine were from Sigma–Aldrich ( St . Louis , MO ) and ciliobrevin D was from Calbiochem ( Darmstadt , Germany ) .",
"Dhc1b mutant fla24 plus cells expressing SAG1-HA were obtained from a cross between fla24 minus gametes and SAG1-HA/sag1-5 plus gametes .",
"Cell growth , induction of gametogenesis by transfer of vegetatively growing cells into N-free medium , fractionation of cells into cell bodies and cilia , and storage of samples were as previously described ( Belzile et al . , 2013 ) .",
"Ciliary ectosomes were isolated from the medium as follows .",
"The indicated plus and minus gametes were pre-treated with lysin for 30 min to remove cell walls ( Hunnicutt et al . , 1990 ) .",
"After washing with fresh N-free medium , the plus and minus gametes were mixed with aeration for the times indicated in the figure legends .",
"The samples were centrifuged at 1600×g for 10 min to sediment the cells .",
"The supernatant medium was centrifuged at 20 , 000×g for 30 min and the resulting supernatant was centrifuged at 200 , 000×g for 60 min in a TLA 100 . 3 rotor ( Beckman ) to sediment membrane vesicles , which were resuspended in N-free medium or HMDEK ( 20 mM pH = 7 . 2 HEPES , 5 mM MgCl2 , 1 mM dithiothreitol , 1 mM EDTA , 25 mM KCl ) buffer .",
"Ciliary ectosomes harvested from 1 . 2 × 108 adhering gametes were mixed with freshly washed minus gametes ( 4 × 107 cells ) in 0 . 4 ml N-free medium with gentle agitation .",
"After 15 min , after removing a portion to be used as input , a portion was centrifuged at 20 , 000×g for 2 min to sediment whole cells and any bound vesicles .",
"The remaining portion was de-ciliated , and centrifuged at 600×g for 2 min to sediment the cell bodies .",
"The supernatant was centrifuged at 20 , 000×g for 2 min to sediment the cilia .",
"All the fractions were boiled in 1 × SDS sample buffer for 5 min , and then subjected to SDS/PAGE analysis on 4–20% gradient gels ( GenScript , USA ) .",
"SDS-PAGE and immunoblotting were essentially as described previously ( Cao et al . , 2013 ) with slight modifications ( Belzile et al . , 2013 ) .",
"Cells , cilia , and ciliary ectosome samples were boiled in 1 × SDS sample buffer for 5 min , and then subjected to SDS-PAGE analysis on 4–20% gradient gels ( GenScript , USA ) .",
"The antibodies used for immunoblotting were anti-HA ( 1:1000; Roche ) , anti-α-tubulin ( 1:3000 or 1:200 , 000; Sigma ) , anti-GSP1 ( 1:20 , 000; Wilson et al . , 1999 ) , anti-FMG1 ( 1:100 , 00 ) , anti-IFT139 ( 1:50 , 000 ) , anti-IFT81 ( 1:1000 ) , and anti-BUG22 ( 1:500 , 000 ) .",
"Mouse monoclonal antibody against FMG1 was generously provided by Robert Bloodgood ( University of Virginia ) .",
"Monoclonal antibodies against IFT81 and IFT139 were generously provided by Dennis Deiner and Joel Rosenbaum ( Yale University ) .",
"Rabbit polyclonal antibody against BUG22 was generously provided by Dan Meng and Junmin Pan ( Tsinghua University ) .",
"The amount of SAG1-HA released into the medium during adhesion was determined by use of ImageJ analysis of immunoblots of equal proportions of cells and medium .",
"Determinations were made under conditions in which the amount of sample loaded was linear with the signal obtained .",
"Similar methods were used to determine the relative abundance of FMG1 and SAG1-HA in equal protein amounts of ciliary ectosomes compared to cilia .",
"Immunofluorescence was carried out essentially as described previously ( Snell , 1976; Belzile et al . , 2013 ) .",
"In some cases , the protocol was modified as follows ( Cao et al . , 2013 ) : Cells in N-free medium were collected by centrifugation , resuspended in N-free medium , and fixed for 2 min at room temperature in 4% paraformaldehyde in N-free medium .",
"After removing the fixation buffer , the cells were resuspended in PBS and allowed to adhere to 0 . 1% poly-l-lysine-coated microscope slides for 10 min at room temperature .",
"The slides were immersed in ice-cold 100% methanol for 10 min at −20°C , removed , and allowed to air dry .",
"The primary antibodies were anti–α-tubulin ( 1:400; Sigma ) and rat anti-HA ( 1:100; Roche ) .",
"The secondary antibodies were Alexa Fluor 488 goat anti-rat/mouse IgG ( 1:400; Molecular Probes ) and Texas red goat anti-mouse IgG ( 1:400; Molecular Probes ) .",
"The slides were examined with a Zeiss LSM780 Observer Z1 Confocal Laser Microscope or a Zeiss Axioplan 2E , motorized focus drive with a Hamamatsu monochrome digital camera .",
"Images were acquired and processed by ZEN 2009 Light Edition and Adobe Photoshop software , and assembled in Adobe Illustrator ( Adobe Systems ) .",
"Negative staining and TEM were carried out as described previously ( Snell , 1976 ) with slight modifications .",
"Briefly , cells or ciliary ecotosomes in N-free medium were applied to 200 mesh carbon-formvar coated grids ( Electron Microscopy Sciences , USA ) and allowed to adhere for 30 s .",
"The liquid was removed from the side by wicking with filter paper and the grids were washed twice with distilled water .",
"Uranyl acetate ( 2% ) was then applied for 10 s , and removed with filter paper .",
"The samples were imaged on a FEI Tecnai G2 Spirit BioTWIN Transmission Electron Microscope .",
"Images were processed by Adobe Photoshop software , and assembled in Adobe Illustrator ( Adobe Systems ) ."
]
] | [
"The role of the primary cilium in key signaling pathways depends on dynamic regulation of ciliary membrane protein composition , yet we know little about the motors or membrane events that regulate ciliary membrane protein trafficking in existing organelles .",
"Recently , we showed that cilium-generated signaling in Chlamydomonas induced rapid , anterograde IFT-independent , cytoplasmic microtubule-dependent redistribution of the membrane polypeptide , SAG1-C65 , from the plasma membrane to the periciliary region and the ciliary membrane .",
"Here , we report that the retrograde IFT motor , cytoplasmic dynein 1b , is required in the cytoplasm for this rapid redistribution .",
"Furthermore , signaling-induced trafficking of SAG1-C65 into cilia is unidirectional and the entire complement of cellular SAG1-C65 is shed during signaling and can be recovered in the form of ciliary ectosomes that retain signal-inducing activity .",
"Thus , during signaling , cells regulate ciliary membrane protein composition through cytoplasmic action of the retrograde IFT motor and shedding of ciliary ectosomes ."
] | [
"Nearly every cell in the human body has slender , hair-like structures known as cilia that project outwards from its surface .",
"These structures can sense and respond to light , chemicals and touch , and they are required for normal development .",
"Failure of cilia to form or function in the correct manner can lead to severe diseases—such as kidney disorders , deafness and loss of vision .",
"A major puzzle for researchers who study cilia has been to understand how cells change the composition of these structures as part of their response to a sensory input .",
"Cilia are ancient structures that were present in early single-celled organisms and researchers interested in cilia have often used a single-celled green alga called Chlamydomonas reinhardtii as a model system for their studies .",
"When these algae reproduce sexually , the two types of sex cells sense the presence of each other when their cilia touch and then stick together .",
"This ciliary touching activates signals that are sent into the cells to get them ready to fuse together , much like sperm and egg cells do in animals .",
"Both ciliary touching and signaling depend on a protein called SAG1 , a part of which ( known as SAG1-C65 ) is normally found mostly over the surface membrane of C . reinhardtii .",
"Only very small amounts of SAG1-C65 are normally found on cilia; but , when the sex cells' cilia touch , this protein rapidly moves to the end of the cell nearest the cilia via a previously unknown mechanism .",
"SAG1-C65 then becomes much more enriched in the cilia .",
"Cao , Ning , Hernandez-Lara et al . investigated this process and found that SAG1-C65 movement requires a molecular motor called ‘cytoplasmic dynein’ .",
"This motor protein typically walks along the inside of cilia to transport other molecules away from the tip and towards the cell membrane .",
"However , Cao , Ning , Hernandez-Lara et al . found that this dynein also carries SAG1-C65 from the membrane of the cells towards the base of the cilia in preparation for it to enter into these structures .",
"As part of an effort to understand the fate of the protein after it entered cilia , Cao , Ning , Hernandez-Lara et al . discovered that the SAG1-C65 disappeared from the structures without returning to the cell membrane .",
"Instead , SAG1-C65 was packaged within tiny bubble-like structures near the tips of cilia and these packages were then shed from cilia into the external environment .",
"This discovery challenges a widely held view that proteins are only removed from cilia by returning to the cell .",
"Future work will be required to understand more of the molecular details of these processes , which are likely to be present in most cells with cilia ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"microbiology and infectious disease"
] | Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells | elife-22028-v3 | [
[
"Tuberculosis is characterized by the formation of granulomas , cellular structures which attempt to ‘wall off’ infection by surrounding it with cells of the immune system ( Ramakrishnan , 2012; Russell , 2007; Russell et al . , 2010 ) .",
"Granulomas , which have a defined anatomical structure as well as segregated expression of immune system related proteins within the structure ( Marakalala et al . , 2016 ) , differentiate and mature independent of each other in infected tissues , most often the lung .",
"The process of pulmonary granuloma formation is driven by macrophage phagocytosis of inhaled , viable Mtb , followed by extravasation of monocytes and T cells from the circulation and their accumulation at the site of infection ( Ramakrishnan , 2012; Russell , 2007; Russell et al . , 2010; Barry et al . , 2009 ) .",
"However , granulomas do not always succeed in containing Mtb infection , and different granulomas in the same lung can control the infection or support the growth of the bacilli ( Barry et al . , 2009; Lenaerts et al . , 2015; Lin et al . , 2014; Kaplan et al . , 2003 ) .",
"In the latter case , central necrosis within the granuloma , cavity formation , and breach into the airways , results in release of Mtb into the environment and transmission of the infection to another human host ( Ramakrishnan , 2012; Russell , 2007; Russell et al . , 2010; Barry et al . , 2009 ) .",
"Thus , while Mtb is considered an intracellular pathogen infecting live cells , its proliferation during active pulmonary tuberculosis occurs in an environment containing many dead cells and cell remnants ( Kaplan et al . , 2003; Hunter , 2011; Hunter et al . , 2007; Welsh et al . , 2011; Irwin et al . , 2015 ) .",
"Part of the host response to Mtb infection is the death of the infected phagocyte .",
"Mtb-induced macrophage death has been observed to occur by apoptotic and non-apoptotic mechanisms .",
"Apoptosis was reported to be protective against Mtb ( Fratazzi et al . , 1999; Gan et al . , 2008; Keane et al . , 2000; Molloy et al . , 1994; Oddo et al . , 1998; Behar et al . , 2010 ) .",
"Evidence from mouse macrophages shows that protection is mediated through efferocytosis , the internalization of apoptotic cell fragments by macrophages , leading to the elimination of the Mtb inside the cellular fragments ( Martin et al . , 2012 ) .",
"Mtb was also observed to induce non-apoptotic cell death , differentiated from apoptosis by the lack of caspase activation ( Lee et al . , 2006 ) , lack of DNA fragmentation ( Keane et al . , 2000; Park et al . , 2006 ) , and loss of membrane integrity ( Park et al . , 2006; Dobos et al . , 2000 ) .",
"Mtb was not killed when necrotic cell death was induced by H2O2 ( Molloy et al . , 1994 ) .",
"Mtb is reported to initiate non-apoptotic cell death by the breakdown of mitochondrial membrane integrity ( Duan et al . , 2002 ) , interference with host plasma membrane repair through inhibition of prostaglandin E2 ( Divangahi et al . , 2009; Divangahi et al . , 2010 ) , and secretion of a necrosis inducing toxin which kills macrophages by hydrolyzing NAD ( Sun et al . , 2015 ) .",
"The inflammatory effect of non-apoptotic cell death resulting from the immediate loss of membrane integrity upon death ( Fink and Cookson , 2005 ) may allow for the recruitment of additional host phagocytes to the site of infection which may lead to infection spread ( Clay et al . , 2007 ) .",
"In contrast , control of Mtb through apoptosis and autophagy ( Castillo et al . , 2012; Gutierrez et al . , 2004; Watson et al . , 2012 ) may dampen the inflammatory response .",
"Mtb infection is known to be controlled by the adaptive immune response ( Cooper and Flynn , 1995; Flynn et al . , 1995 , 1992 ) which becomes effective several weeks after exposure ( Wolf et al . , 2008 ) , though a study using intravital imaging observed limited T cell effector function in Mtb granulomas ( Egen et al . , 2011 ) .",
"The adaptive immune response controls Mtb by activating macrophages with factors such as interferon gamma ( IFNγ ) to kill or control the growth of the bacilli , and by targeting infected macrophages with cytotoxic lymphocytes ( Cooper and Flynn , 1995; Flynn et al . , 1995 , 1992 ) .",
"However , given that different infection outcomes can coexist in the same lung , the source for variability ( Russell et al . , 2009 ) in infection outcomes at different foci is not well understood .",
"Substantial variability in other biological systems results from positive feedback ( Kaern et al . , 2005; Sigal et al . , 2006 ) .",
"Positive feedback involves a cascade of events such that once an event occurs , the probability that it will occur again is increased .",
"To understand how an environment conducive to bacterial growth and host cell death is sustained , we investigated virulent Mtb infection dynamics in primary human macrophages in vitro using time-lapse microscopy over approximately 80 hr at 10 min resolution .",
"We observed that macrophage internalization of Mtb aggregates led to the killing of the host cell , with the frequency of host cell death increasing with aggregate size .",
"Uptake of single large aggregates showed a higher frequency of cell death relative to the uptake of a similar number of bacteria as multiple small aggregates , and the time to death was inversely correlated to aggregate size .",
"Once the host cell was killed , Mtb was able to rapidly grow inside the dead infected macrophage regardless of whether the cells were monocyte derived macrophages ( MDM ) or alveolar macrophages , or whether the cells were exposed to IFNγ prior to infection .",
"Growth inside dead cells was significantly faster than in the extracellular environment .",
"Once host cell death occurred , other macrophages internalized the dead infected cells , and this rapidly led to their own death , forming a cell death cascade .",
"These observations of a cycle of host cell death followed by bacterial replication could drive Mtb infection dynamics of necrosis and bacterial proliferation in lung granulomas of patients with active tuberculosis disease ."
],
[
"We infected human MDM with the virulent M . tuberculosis H37Rv strain labelled with red fluorescent protein ( RFP ) or mCherry expressed under the control of a constitutively active promoter ( groEL and smyc’ respectively ) .",
"We used live cell imaging in Biosafety Level 3 containment over a period of approximately 80 hr to follow the outcome of infection in individual macrophages .",
"MDM cell borders were determined by custom written image analysis software ( Video 1 , Materials and methods , Source code 1 ) .",
"Time of cell death was determined to be the time point at which movement of internal cellular structures ceased or the cell detached ( Materials and methods ) .",
"This method of death determination was validated in macrophages with the DNA intercalating dye DRAQ7 , which only enters the cell when membrane integrity has been compromised due to cell death ( Figure 1—figure supplement 1 ) .",
"To examine whether Mtb fluorescence is a valid measure of Mtb number , we tracked the increase in Mtb by fluorescence versus colony forming units ( CFU ) over 3 days of growth .",
"We found a tight correspondence between the two measures ( Figure 1—figure supplement 2 ) , with an incremental increase in fluorescence translating to an incremental increase in the number of bacilli as measured by CFU .",
"Hence , fluorescence measurements reflect Mtb numbers and an increase in Mtb fluorescence reflects Mtb growth . 10 . 7554/eLife . 22028 . 003Video 1 . Macrophage internalization of Mtb aggregates . Mtb infections of human macrophages were imaged by time-lapse microscopy at a resolution of 10 min between image acquisitions .",
"Macrophage borders and locations were tracked using a custom-written , Matlab based image analysis code ( Source code 1 ) .",
"The left panel of the montage is a movie of macrophage infection .",
"The border of the analyzed macrophage is shown as a green outline .",
"RFP-expressing Mtb are shown in red .",
"Mtb internalized by the cell is marked by blue ellipses .",
"Time is hours:minutes .",
"Scale bar is 20 µm .",
"The graph on right shows the fluorescence signal converted to bacterial numbers in the analyzed macrophage .",
"Timing of internalizations of Mtb clumps containing varying numbers of bacilli as detected from the movie are shown as vertical red lines on the graph .",
"Macrophage death is marked by a vertical black line of x’s appearing at the point of cell death . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 003 We asked whether the number of Mtb internalized by MDM , as observed in our time-lapse movies , affected the probability of macrophage death .",
"The aggregation state of Mtb has been hypothesized to impact host-pathogen interactions ( Orme , 2014; Sani et al . , 2010 ) .",
"Mtb naturally aggregate , unless cultured with a nonionic detergent such as Tween 80 ( Dubos and Middlebrook , 1948 ) .",
"To minimize modification of the outer surface of Mtb and its aggregation state ( Sani et al . , 2010 ) , we grew Mtb without detergent for several replication cycles and gently broke up aggregates resulting from such growth into a heterogeneous population of bacterial aggregation states .",
"Hence , MDM in a single experiment were infected by individual bacteria or multibacterial clumps of various sizes ( Video 1 ) .",
"This enabled us to compare the consequences of MDM phagocytosis of different Mtb numbers and aggregation states in the same experiment .",
"We converted Mtb fluorescent signal inside MDM to bacterial numbers by dividing the signal by the mean fluorescence of a single bacterium , with single Mtb obtained by filtering .",
"We then ranked MDM from lowest to highest by the sum of Mtb internalized .",
"This ranking gave the best separation in the frequency of cell death between the top 50% and bottom 50% of ranks ( Figure 1—figure supplement 3 ) .",
"The raw data describing the outcome of the macrophage-Mtb interactions is presented in Figure 1—figure supplement 4 .",
"The full dataset contains the dynamics of macrophage Mtb internalization and fate of 759 MDM , with 720 infected cells and 39 uninfected bystander cells .",
"( Figure 1—figure supplement 4B ) .",
"To compare the death frequencies between cells internalizing different numbers of Mtb over the course of the imaging , we divided the dataset into ten groups of infected cells , where cells internalizing a similar number of Mtb were grouped together and where each group constituted 10% of the total number of infected cells .",
"We also compared death frequency to a group of bystander cells which did not phagocytose any Mtb .",
"We then determined the frequency of cell death in each group , and as well as the mean number of Mtb in the group .",
"Macrophage fate was found to depend on the number of Mtb internalized .",
"As the number of bacteria internalized increased , there was more cell death and it occurred sooner ( Figure 1 ) .",
"Phagocytosis of small numbers of Mtb , less than approximately 10 bacilli , did not induce significantly more macrophage death than observed in uninfected bystander cells ( Figure 1—figure supplement 5 ) .",
"Above 10 Mtb internalized but below approximately 30 , there was a trend toward increased macrophage death relative to bystander cells , but the differences in frequencies were not significant ( see Figure 1—figure supplement 5B for p-values and significance thresholds ) .",
"The probability of death increased significantly relative to bystanders and cells with less than 10 Mtb when more than 30 Mtb were internalized per cell over the course of the movie , and a threshold for Mtb to induce death was clearly visible in Figure 1 at about 50 Mtb internalized , with differences relative to bystander and lightly infected cells becoming highly significant ( Figure 1—figure supplement 5B ) . 10 . 7554/eLife . 22028 . 004Figure 1 . Number of Mtb internalized determines probability of macrophage death . The death frequency for 720 infected MDM was graphed over time as a function of the sum of Mtb internalized ( raw data provided in Figure 1—source data 1 ) .",
"To obtain death frequencies , MDM were divided into 10 groups according to the number of Mtb internalized .",
"The frequency of dead cells was determined at 10 movie frame ( 1 . 7 hr ) intervals for each group ( x-axis ) , and plotted against the mean sum of Mtb internalized for the group ( y-axis ) .",
"Interpolation between points was performed to obtain a surface .",
"The color scale represents the frequency of cell death from low ( blue ) to high ( red ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 00410 . 7554/eLife . 22028 . 005Figure 1—source data 1 . Intracellular Mtb fluorescence through time , Movie frames of Mtb phagocytosis , and MDM frame of death , if it occurs , for IFNγ untreated MDM . Used in Figures 1–6 . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 00510 . 7554/eLife . 22028 . 006Figure 1—figure supplement 1 . Measures of cell death . Time of cell death induced by Mtb and determined by macrophage dynamics was correlated to macrophage death as determined by the uptake of the dead cell stain DRAQ7 .",
"Time of DRAQ7 uptake was correlated with time of macrophage detachment ( A ) or time of cessation of movement of internal cellular structures ( B ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 00610 . 7554/eLife . 22028 . 007Figure 1—figure supplement 2 . Fluorescence as a measure of Mtb numbers . we tracked the increase in Mtb by fluorescence ( blue squares ) versus by colony forming units ( CFU , red circles ) over three days of growth in culture .",
"Means and standard deviations of triplicates .",
"One of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 00710 . 7554/eLife . 22028 . 008Figure 1—figure supplement 3 . MDMs were ranked according to the sum of Mtb internalized ( black ) , number of Mtb in the last aggregate internalized ( red ) , number of Mtb in the first aggregate internalized ( green ) , or randomly ( blue ) .",
"Ranks ( x-axis ) were then plotted against the cumulative number of dead cells present up to each rank ( y-axis ) .",
"Ranking according to the sum of Mtb internalized gave the best separation between the top 50% and the bottom 50% of ranks ( ratio top/bottom = 2 . 6 ) , followed by a number of Mtb in the last clump internalized ( 2 . 4 ) , number of Mtb in the first clump ( 1 . 4 ) , or randomized ranking ( 1 . 0 ) .",
"All values were significant relative to the randomized ranking ( p<0 . 0001 ) , as determined from 10 , 000 randomizations . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 00810 . 7554/eLife . 22028 . 009Figure 1—figure supplement 4 . Outcomes of macrophage-Mtb interactions .",
"( A ) A subset of cells is shown as an example .",
"Macrophages were numbered on the y-axis based on the sum of Mtb internalized , with cells internalizing the largest number at the top of the chart .",
"The fate of each macrophage over time is represented by one line which intersects the y-axis at the rank of the cell .",
"The time of macrophage death , if it occurred , is at the point where the line changes from green to dark blue .",
"The timing and number of bacteria per pickup event is indicated by the x-axis location and shade of the marker , respectively .",
"Pickups of cell-free Mtb are marked as circles , and pickups of dead infected cells as stars .",
"( B ) The full dataset of 759 cells from five independent imaging experiments .",
"For cells which died and detached , the dark blue line was extended from the point of cell death to movie end . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 00910 . 7554/eLife . 22028 . 010Figure 1—figure supplement 5 . Analysis of differences between macrophages grouped by the sum of Mtb internalized .",
"( A ) Fraction of cells which died over time in each of the 10 infected cell groups ( n = 72 , group mean and standard deviation of Mtb per macrophage shown in the legend on the right ) , and the uninfected bystander group ( n = 39 ) .",
"Lines are cubic fits as a guide to the eye .",
"( B ) Table of p-values for differences between groups as determined by bootstrap .",
"The fraction of cells in each group was compared to the fraction of dead cells in infected groups with lower mean Mtb internalized , or to bystanders .",
"Each group was resampled by drawing either 72 cells ( for comparison to infected groups ) or 39 cells ( for comparison to bystanders ) , with replacement , 100 , 000 times .",
"The p-value was calculated as the number of times the resampled fraction of dead cells at the end of the movie was lower than in the comparison group , divided by 100 , 000 ( code provided as Source code 2 ) .",
"As the threshold for statistical significance , we used α/n = 0 . 005 , where α = 0 . 05 for single comparisons was adjusted for n = 10 comparisons by the Bonferroni method .",
"Table shows the groups in the first column and the comparison groups in the first row .",
"p-values which passed the significance threshold are shown in bold . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 010 We examined whether the frequency of cell death was different if a cell internalized a given amount of Mtb in one pickup as opposed to over multiple pickups .",
"In order to control for bacterial number , we limited the comparison to cells which internalized an average of approximately 50 Mtb ( 49 . 6 ± 18 . 9 Mtb for macrophages which phagocytosed multiple smaller aggregates , versus 49 . 4 ± 14 . 3 for macrophages which phagocytosed one large aggregate ) , an infection level where approximately one half of infected cells died by the end of our imaging period of 83 hr ( Figure 1—figure supplement 5A ) .",
"This infection level was chosen so as to increase sensitivity by excluding very highly infected cells , which died with a high frequency , as well as lightly infected cells , which did not die at an appreciably higher frequency relative to bystanders .",
"We obtained a lower frequency of cell death when infection occurred by multiple smaller pickups compared to single large pickups ( Figure 2 ) .",
"While 47% of the cells died when they accumulated Mtb in three or more internalizations , 71% of the cells died when they internalized a similar mean number of Mtb as one pickup .",
"The difference was significant ( p=5×10−4 by bootstrap ) .",
"In addition to the summary of the results ( Figure 2A ) , we also represent the individual cell histories ( Figure 2B for multiple pickups and Figure 2C for single pickups ) .",
"The data are inherently complex as individual macrophages are tracked , and these phagocytose different numbers of Mtb in different states at different times , and may or may not die as a result .",
"We represent each cell as a line , with pickup events ( denoted as circles or stars , where are circles are internalizations of cell-free Mtb , and stars are internalizations of dead infected cells , both color coded according to the number of Mtb internalized ) at a location on the line corresponding to the time of pickup .",
"If macrophage death occurs , line color changes from green to dark blue at the time of death . 10 . 7554/eLife . 22028 . 011Figure 2 . Internalization of single large aggregates is more cytotoxic than several smaller aggregates .",
"( A ) Fraction of dead MDM after phagocytosing one large aggregate of Mtb ( black line ) or multiple small aggregates ( red line ) with a similar cumulative sum of Mtb to the single internalizations , but with all aggregates being smaller than 50 bacilli each .",
"The frequency of cell death was 47% for multiple internalizations ( n = 47 ) and 71% for single internalizations ( n = 62 , p=5×10−4 by bootstrap ) .",
"Individual cell fates are shown in ( B ) for cells internalizing multiple small aggregates , and ( C ) for cells internalizing a similar number of Mtb as single aggregates .",
"The blue circles are internalizations of aggregates of 10 or fewer Mtb , red circles are clumps of 11–49 Mtb , and black circles are 50–81 Mtb .",
"Line color changes from green to dark blue at time of death .",
"Stars indicate internalizations of dead infected cells .",
"An equal number of cells is shown in ( B ) and ( C ) to facilitate comparison .",
"Mean number of Mtb phagocytosed was 49 . 6 ± 18 . 9 for MDM internalizing multiple small Mtb clumps , and 49 . 4 ± 14 . 3 for MDM internalizing a single large clump . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 011 We asked whether time to cell death was dependent on pickup size .",
"We therefore compared macrophages which internalized single clumps of Mtb during the first half of the imaging period , to increase the measurable time to death , if it occurred .",
"In lightly infected cells ( ≤10 Mtb internalized as one aggregate ) cell death was intermittent and not clearly linked to the internalization event .",
"In contrast , the time to cell death appeared to be clearly linked to the time of pickup when the number of Mtb in the clump was large ( Figure 3A ) .",
"The time to cell death after internalization inversely correlated with the number of bacteria internalized in a single pickup ( Figure 3B , R2 = 0 . 45 , p=2×10−6 ) , indicating that uptake of larger clumps led to faster onset of macrophage death . 10 . 7554/eLife . 22028 . 012Figure 3 . Mtb aggregate size determines timing of macrophage death .",
"( A ) MDMs which internalized exactly one clump before the halfway point of the movie were ranked based on the amount of Mtb internalized ( n = 72 cells ) .",
"The blue circles are internalizations of aggregates of 10 or fewer Mtb , red circles are clumps of 11–49 Mtb , and black circles are >50 Mtb .",
"Line color changes from green to dark blue at time of death .",
"Stars indicate internalizations of dead infected cells .",
"( B ) Time differences between internalization and death for cells that died as a function of log transformed bacterial number .",
"R2 = 0 . 56 , p=4×10−8 ( n = 39 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 012 It has been previously reported that high multiplicity infection with Mtb leads to non-apoptotic cell death ( Molloy et al . , 1994; Lee et al . , 2006; Park et al . , 2006; Dobos et al . , 2000; Repasy et al . , 2013 ) .",
"To examine the mode of Mtb aggregate-induced cell death , we compared Mtb-induced cell death to that triggered by cisplatin , a chemotherapeutic agent that induces macrophage apoptosis by DNA damage ( von Knethen et al . , 1998 ) , and the toxin nigericin , reported to induce macrophage cell death by pyroptosis following LPS priming ( Warny and Kelly , 1999 ) .",
"In apoptosis , loss of membrane integrity is delayed to provide an opportunity for the removal of the dead cell ( Fadok et al . , 1992; Vermes et al . , 1995 ) .",
"In pyroptosis , necrosis and other inflammatory cell death types , cell death involves the immediate loss of membrane integrity and spillage of cell contents ( Fink and Cookson , 2005 ) .",
"We used DRAQ7 to track loss of membrane integrity , and cessation of internal movement to define the point of cell death ( Figure 4A–B , Materials and methods ) .",
"Mtb typically induced phagocyte cell death with immediate loss of membrane integrity ( Video 2 , Figure 4 ) , similar to nigericin induced cell death ( Video 3 , Figure 4 ) , and consistent with a previous study using A549 epithelial cells ( Dobos et al . , 2000 ) .",
"In contrast , cisplatin induced cell death was apparent significantly before incorporation of DRAQ7 ( Video 4 , Figure 4 ) . 10 . 7554/eLife . 22028 . 013Figure 4 . Mtb induced macrophage death does not resemble apoptosis .",
"( A ) Cell death was caused by internalization of Mtb clumps ( top row , Mtb in red ) , the pyroptosis inducers Nigericin+LPS ( middle row ) , or the apoptosis inducer cisplatin ( bottom row ) .",
"Time of cell death was determined by the cessation of internal movement , and loss of membrane integrity by entry of the fluorescent dye DRAQ7 ( green ) .",
"( B ) Quantitation of time of cell death and loss of membrane integrity .",
"Black line is the Pearson correlation of within cell image pixels from one time-lapse frame to the next .",
"Cell death as determined by cessation of internal cell movement is indicated by greater than threshold jump in pixel correlation ( red vertical line ) .",
"Loss of membrane integrity as detected by DRAQ7 is shown by the green line , ( normalized to maximum signal ) with blue vertical line representing time DRAQ7 levels cross an experimentally determined threshold .",
"In the case of Mtb-induced cell death , the blue and red lines are superimposed .",
"Each graph represents the cell shown in ( A ) .",
"( C ) Median time difference ( red line , with an interquartile range shown as blue box ) between cell death and loss of membrane integrity for cells killed by Mtb ( n = 119 ) , 20 µM nigericin and 1 µg/ml LPS ( n = 29 ) or 50 µM cisplatin ( n = 76 ) .",
"****p<0 . 001 by Kruskal-Wallis rank sum test corrected for multiple hypotheses . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 01310 . 7554/eLife . 22028 . 014Video 2 . Death of an Mtb-infected macrophage . MDMs were infected with Mtb H37Rv-RFP ( red ) and imaged in the presence of the viability dye DRAQ7 ( green ) .",
"The entry of DRAQ7 corresponds to the time of cell death .",
"Scale bar is 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 01410 . 7554/eLife . 22028 . 015Video 3 . Pyroptotic death of macrophages . Macrophages were sensitized with 1 µg/ml LPS for 3 hr , then treated with 20 µM nigericin to induce pyroptosis .",
"Cells were imaged in the presence of DRAQ7 ( green ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 01510 . 7554/eLife . 22028 . 016Video 4 . Cisplatin-Induced cell death . Macrophages were treated with 50 µM cisplatin and imaged in the presence of DRAQ7 ( green ) .",
"Scale bar is 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 016 We tracked the fate of Mtb in the dead cells .",
"Mtb-induced macrophage death did not lead to the killing of the bacilli , and Mtb fluorescence levels increased in the dead cells ( Figure 5A , Video 5 ) .",
"While RFP or mCherry fluorescence possibly persists after Mtb death , the increase in fluorescent signal is a clear indication of Mtb replication ( Figure 1—figure supplement 2 ) .",
"Figure 5A is typical of the type of bacterial growth we observed: Mtb did not burst out of the cell to spread into the neighboring medium .",
"Instead , the bacilli remained tightly associated with the dead host cell as a growing clump .",
"Out of the 92 dead infected cells we evaluated , 90 showed positive growth rates while two had declining Mtb numbers ( Figure 5B ) .",
"Intracellular Mtb had a median doubling time , excluding the two negative values , of 24 . 7 hr ( interquartile range 19 to 34 hr , Figure 5B inset ) .",
"The extracellular growth rate of Mtb in the same experiments had a median doubling time of 36 . 1 hr ( interquartile range 31 to 44 hr , Figure 5B inset ) , significantly slower than in dead cells ( p=7×10−8 , Wilcoxon rank sum test ) .",
"We confirmed these results by CFU , quantifying the number of Mtb after three days of growth in dead MDM versus extracellular growth in MDM medium .",
"We initiated growth with the same concentrated Mtb culture , which killed approximately 99% of the MDMs after one day , thus allowing Mtb to replicate in the dead macrophages for at least two days ( Figure 5—figure supplement 1A ) .",
"We recovered approximately an order of magnitude more Mtb by CFU after growth in dead cells versus after growth in the MDM extracellular medium ( Figure 5—figure supplement 1B ) . 10 . 7554/eLife . 22028 . 017Figure 5 . Mtb grows robustly inside dead macrophages .",
"( A ) In silico synchronized images of representative macrophages infected with Mtb and imaged before and after death .",
"Each horizontal set of images represents the same macrophage over time , with the cell death event at the center image .",
"On the left of the cell death event are images of the cell at regular intervals before cell death as a percentage of time the cell was imaged alive .",
"On the right are images at regular intervals after death a percentage of time the cell was imaged dead .",
"( B ) Traces of Mtb growth in dead cells .",
"Each trace represents the number of Mtb within the same dead MDM and is normalized to its maximum Mtb number and maximum length of time the dead cell was imaged before detachment or movie end .",
"Minimum time for imaging dead cells was 10 hr .",
"In total , 92 dead macrophages were analyzed , with 90 showing positive exponential slopes .",
"The median doubling time for cells with positive slopes was 24 . 7 hr over five independent experiments .",
"Inset: Doubling times of Mtb in dead cells excluding the two negative values ( red , n = 90 ) and in the extracellular medium ( black , n = 60 ) .",
"The median extracellular doubling time was 36 . 1 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 01710 . 7554/eLife . 22028 . 018Figure 5—figure supplement 1 . Differences in growth measured by CFU between Mtb in dead cells and extracellular Mtb .",
"( A ) A homogeneous population of Mtb in dead cells was produced by infecting at a high multiplicity per cell ( MOI = 30 according to CFU ) .",
"( B ) the same input dose of concentrated Mtb culture was either grown with MDM or cell-free in MDM medium for three days and assayed by CFU .",
"Mean and standard deviation of two replicates .",
"*p=0 . 029 Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 01810 . 7554/eLife . 22028 . 019Video 5 . Mtb grows inside a dead macrophage treated with IFNγ prior to infection . The left panel of the montage is a movie of macrophage infection at a resolution of 10 min between image acquisitions .",
"The border of the analyzed macrophage is shown as a green outline , and the presence of DRAQ7 is shown in blue .",
"RFP-expressing Mtb are in red .",
"Time is hours:minutes .",
"Scale bar is 20 µm .",
"The graph on right shows the fluorescence signal converted to bacterial numbers in the analyzed macrophage .",
"Timing of macrophage death was determined by the lack of internal movement and confirmed by a DRAQ7 entry ( vertical line of x’s ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 019 We next compared the growth of Mtb in dead cells to that in live infected cells .",
"Live macrophages internalized Mtb throughout the imaging period , and hence an increase in Mtb fluorescence could be the result of either new Mtb internalized , or existing Mtb growing in the live cell .",
"Therefore , we marked the time each macrophage internalized Mtb , and used a 10 hr tracking window which could be fitted between internalization events for many of the cells .",
"We calculated the median fold change in Mtb in this interval ( Figure 6A ) .",
"For comparison , we used an interval of the same length in cells after death ( Figure 6B ) .",
"In order to investigate the source of variability and the statistical significance of the data , we fitted the temporal dynamics of Mtb fluorescence of each individual infected MDM with an exponential curve .",
"Variability in Mtb growth was evident in individual slopes in live macrophages , with Mtb growing in some cells and decreasing in others ( Figure 6A inset ) .",
"Mtb growth in dead cells was less variable ( Figure 6B inset ) . 10 . 7554/eLife . 22028 . 020Figure 6 . Mtb growth in macrophages before and after macrophage death . Median ( circle with dot ) and interquartile range ( blue rectangle ) of Mtb signal in live ( A ) and dead cells ( B ) .",
"Insets show exponential fits of Mtb dynamics in the individual live ( n = 101 ) or dead ( n = 92 ) macrophages .",
"X and y-scales on insets same as on main panels .",
"The median doubling time of Mtb was >100 hr in live cells , 21 . 2 hr in dead cells .",
"( C ) MDMs were infected with Mtb-mCherry labelled with pHrodo Green , which fluoresces in low pH . Mtb ( red line ) , pHrodo ( green line ) and DRAQ7 ( dashed blue line ) signals of a representative MDM tracked by time-lapse microscopy is shown .",
"The time of cell death , as determined by DRAQ7 entry , is indicated as a vertical black line ( raw data provided in Figure 6—source data 1 ) .",
"( D ) pHrodo signal was quantified 3 hr before and following death of each cell .",
"Means and standard error of cells ( n = 74 cells ) combined from two independent experiments .",
"The dashed horizontal line represents signal threshold .",
"****p<0 . 0001 , paired t-test of pHrodo signal before and after cell death . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 02010 . 7554/eLife . 22028 . 021Figure 6—source data 1 . pHrodo signal over time . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 021 The median growth constant for Mtb in live cells was 0 . 0012 hour−1 ( interquartile range −0 . 016 to 0 . 022 ) , translating to a doubling time of >100 hr ( Figure 6A ) .",
"The slope was not significantly different from 0 ( p=0 . 3 , right tailed Sign Test ) .",
"In contrast , the median growth constant for Mtb in dead cells was 0 . 033 hour−1 ( interquartile range 0 . 021 to 0 . 045 ) , translating to a doubling time of 21 . 2 hr ( Figure 6B ) .",
"This was faster than growth over the full timescale ( Figure 5B ) , possibly indicating faster growth of Mtb in the first hours after host cell death .",
"Mtb growth in dead cells was positive ( p=5×10−23 , right tailed Sign Test ) , and significantly faster than in live infected cells ( p=8×10−15 , Wilcoxon rank sum test ) .",
"While the Mtb growth inside live cells may be significantly positive if measured over a longer time period , we can conclude that it is much slower than growth in dead cells .",
"To understand the mechanism behind the very slow growth in live MDM versus robust growth in dead MDM , we labelled Mtb with the pH detection dye pHrodo prior to MDM infection .",
"We observed that Mtb were in an acidified compartment in live MDM ( Video 6 and Figure 6C ) , with seemingly variable acidification of different bacilli in the same macrophage .",
"Upon Mtb induced MDM death , pHrodo signal was rapidly reduced to near background , indicating that acidification was lost ( Video 6 , Figure 6D ) .",
"This indicates that live infected MDM sequestered Mtb in a phagosomal compartment , which was at least partially acidified .",
"Upon cell death , this compartmentalization disappeared . 10 . 7554/eLife . 22028 . 022Video 6 . Loss of phagosome acidification upon Mtb induced host cell death . Mtb were stained with the pH detection dye pHrodo at 100 µM and used to infect MDM in the presence of the cell death indicator dye DRAQ7 .",
"pHrodo fluorescence is shown in green , mCherry-expressing Mtb in red , and DRAQ7 fluorescence in blue .",
"Time is hours:minutes .",
"Scale bar is 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 022 We next examined host cell death and Mtb dynamics in several physiological contexts .",
"The immune response to infection activates macrophages to mediate Mtb control ( Russell et al . , 2010; Means et al . , 1999; Mosser , 2003; Nau et al . , 2002 ) .",
"A key macrophage activator is IFNγ , secreted by several immune cell types ( Chackerian et al . , 2001; Flesch and Kaufmann , 1987; Cooper et al . , 1993; Flynn et al . , 1993; Fenton et al . , 1997 ) .",
"We therefore exposed MDM to IFNγ prior to infection and examined their response to internalized aggregates of Mtb .",
"Infection with aggregates led to cell death which , similarly to untreated MDM , resulted in Mtb growth inside the dead infected cells ( Figure 7A ) .",
"The median growth constant in dead MDM treated before death with IFNγ was 0 . 027 hour−1 ( interquartile range 0 . 0076 to 0 . 046 ) , corresponding to a doubling time of 25 . 8 hr .",
"Alveolar macrophages are responsible for the initial macrophage contact with the pathogen in the lung ( Keane et al . , 2000; Fenton et al . , 1997; Nicholson et al . , 1996; Hirsch et al . , 1994; Keane et al . , 1997 ) .",
"Similarly to MDM , infection of these cells with aggregates led to cell death followed by Mtb intracellular growth ( Figure 7B ) .",
"The median growth constant in dead alveolar macrophages was 0 . 028 hour−1 ( interquartile range 0 . 0014 to 0 . 055 ) , corresponding to a doubling time of 24 . 6 hr .",
"We have also quantified the extracellular Mtb growth rate during a ten-hour window ( Figure 7C ) .",
"Here , the median growth constant was 0 . 018 hour−1 ( interquartile range 0 . 0015 to 0 . 034 ) , corresponding to a 39 . 1 hr doubling time . 10 . 7554/eLife . 22028 . 023Figure 7 . Mtb grows in IFNγ treated MDM and alveolar macrophages after cell death .",
"( A ) MDM was exposed to IFNγ for 18 hr , then infected with Mtb and imaged ( raw data provided in Figure 7—source data 1 ) .",
"Shown is the median fold change ( circle with dot ) and interquartile range ( blue rectangle ) of Mtb in dead infected cells .",
"( B ) Median fold change and interquartile range of Mtb in dead infected human alveolar macrophages ( AMφ ) isolated from bronchoalveolar lavage and infected in vitro ( raw data provided in Figure 7—source data 2 ) .",
"( C ) Median fold change in Mtb number in extracellular aggregates of Mtb over a 10 hr timespan ( raw data provided in Figure 7—source data 3 ) .",
"( D ) Combined plot of median values for all Mtb growth conditions .",
"The number of dead cells or extracellular clumps analyzed: n = 49 ( IFNγ treated MDM ) , n = 220 ( alveolar macrophages ) , n = 60 ( extracellular clumps ) .",
"Non-IFNγ treated live and dead MDM numbers are as in Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 02310 . 7554/eLife . 22028 . 024Figure 7—source data 1 . Intracellular Mtb fluorescence through time in dead IFNγ treated MDM . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 02410 . 7554/eLife . 22028 . 025Figure 7—source data 2 . Intracellular Mtb fluorescence through time in dead alveolar macrophages . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 02510 . 7554/eLife . 22028 . 026Figure 7—source data 3 . Extracellular Mtb fluorescence through time . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 026 Interestingly , Mtb seemed to grow somewhat slower in both IFNγ treated MDM and alveolar macrophages relative to untreated MDM ( Figure 7D ) .",
"However , this difference was not statistically significant ( p=0 . 45 for treated versus untreated MDM , and p=0 . 16 for alveolar versus untreated MDM , Kruskal-Wallis rank sum test corrected for multiple hypotheses ) .",
"Hence , the growth of Mtb in dead cells seemed to be relatively independent of cellular activation and tissue source .",
"We next investigated whether internalization of dead infected cells led to macrophage death .",
"We selected from our dataset all live macrophages , which internalized dead infected macrophages before 90% of the video recording elapsed , to avoid cells where we could not capture the death event because it was too close to the end of the movie .",
"While cell-free pickups of Mtb also occurred in some of these cells , internalization of a dead infected cell was the terminal internalization event in all but two of the observed cells .",
"Frequency of cell death was 89% ( Figure 8 ) .",
"There was a strong correlation between the time of internalization and death of the internalizing cell ( R2 = 0 . 93 , p=8×10−23 , Figure 8—figure supplement 1 ) , with a median time to death of 3 . 2 hr ( interquartile range of 1 . 5 to 6 . 0 hr ) .",
"Chains of macrophage internalizations of dead infected cells , followed by cell death of the internalizing macrophage and uptake by the next cell , were also seen ( Video 7 ) .",
"We compared the frequency of death from dead cell pickups to the frequency of death in cells internalizing large cell-free aggregates ( 81–300 bacilli ) , which had a similar median number of bacteria ( dead infected cells contained a median of 114 Mtb , versus 125 Mtb for cell-free aggregates ) .",
"Death frequency was 81% for cells that internalized cell-free aggregates , and the difference between death frequencies was not significant ( p=0 . 16 ) .",
"This indicated that the highly cytotoxic effect of dead infected cell internalization can be mostly explained by the high number of Mtb they contain . 10 . 7554/eLife . 22028 . 027Figure 8 . Internalization of dead infected cells leads to rapid cell death . Cells are ranked according to the time of last pickup , with cells ranked highest internalizing earliest .",
"The frequency of cell death was 89% .",
"The blue circles are internalizations of aggregates of 10 or fewer Mtb , red circles are clumps of 11–49 Mtb , and black circles are >50 Mtb .",
"Line color changes from green to dark blue at time of death .",
"Stars are internalizations of dead infected cells . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 02710 . 7554/eLife . 22028 . 028Figure 8—figure supplement 1 . Correlation of time of last pickup and time to death in the cells internalizing a dead infected cell . R2 = 0 . 93 ( p=8×10−23 ) , with a median time to cell death of 3 . 2 hr ( interquartile range 1 . 5 to 6 hr ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 02810 . 7554/eLife . 22028 . 029Video 7 . Cell death cascade following phagocytosis of a dead infected macrophage . The borders of four macrophages are shown as green , yellow , magenta , or blue outlines .",
"RFP-expressing Mtb are shown in red .",
"Time is hours:minutes .",
"Scale bar is 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 029 If Mtb aggregates kill the phagocyte , remain associated with it , and grow in the dead cell , each new macrophage joining the infection chain should be faced with a greater or equal probability of cell death due to increasing Mtb numbers ( Figure 9A , Video 7 ) .",
"To model the consequences of host cell death associated with bacterial growth , we performed numerical simulations to determine the number of dead host cells through time .",
"Here , we do not claim to capture the exact dynamics of what occurs in vivo in the diseased lung , but rather illustrate the trend ( see Materials and methods for simulation details ) .",
"We compared the number of dead host cells when the Mtb doubling time in dead cells was one day , versus when it was two days ( where two days is within the range of the doubling times observed for extracellular Mtb , which was 22–75 hr for the data presented in the inset of Figure 5B ) .",
"As in the experimental results , host cell death was dependent on the bacterial number internalized , and host cells were restrictive of intracellular bacterial growth when alive and permissive when dead .",
"A one day doubling time yielded substantially higher numbers of dead cells in many of the simulations ( Figure 9B , left panels ) .",
"Strikingly , only the one day doubling time in dead cells resulted in high Mtb growth over the simulation period of 60 days ( Figure 9B , right panels ) .",
"High Mtb growth was very sensitive to the doubling time ( Figure 9—figure supplement 1 ) , and this could not be explained by the differences in doubling time if growth was cell-free ( Figure 9B , white lines in right panels ) , as both growth rates were rapid for the timescale of the simulations .",
"Rather , this simplified simulation shows the presence of positive feedback in the host cell-Mtb interactions: faster Mtb growth in the dead cell leads to larger intracellular clumps upon phagocytosis by another cell , and hence to a higher probability of death of the phagocytosing cell , leading to Mtb growth in the new dead cell and a higher probability to kill the next phagocyte in the infection chain . 10 . 7554/eLife . 22028 . 030Figure 9 . Positive feedback in Mtb infection .",
"( A ) Schematic of the positive feedback loop .",
"Mtb are in red , live cells are in grey and dead cells are outlined by dashed borders .",
"Time is from left to right .",
"Probabilities of death at time points 1 , 2 , and three for macrophages internalizing dead infected cells are represented by p1 , p2 , and p3 , where p3>p2>p1 due to Mtb growth in the dead cells .",
"( B ) Results of numerical simulations of the number of dead host cells over time and the corresponding numbers of accumulated Mtb given a one day ( top panels ) or two day ( bottom panels ) doubling time inside dead infected host cells .",
"Each line represents an independent simulation ( numbered on the x-axis ) , time in days is on the z-axis , and the cumulative number of dead cells or number of Mtb is on the y-axis .",
"Each run was initiated with the internalization of one bacillus inside a dead cell .",
"White line represents Mtb extracellular growth given a one or two day doubling time .",
"The fraction of simulations reaching 1000 or more Mtb within 60 days was 0 . 21 for a one day doubling time and 0 . 0003 for a two day doubling time ( code provided as Source code 3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 03010 . 7554/eLife . 22028 . 031Figure 9—figure supplement 1 . Sensitivity of Mtb expansion to doubling time in dead cells . The stochastic simulation described in Figure 9 was repeated for doubling times ranging from 1 to 3 days , with 10 , 000 iterations per doubling time value .",
"The fraction of iterations where Mtb expanded by more than three orders of magnitude ( from 1 to >1000 bacilli ) was calculated for each doubling time ( y-axis ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22028 . 031"
],
[
"Active Mtb infection and pulmonary disease involve host cell necrosis as well as extensive bacterial proliferation , both necessary for infectious aerosol formation and transmission to the next host ( Russell , 2007; Russell et al . , 2010 ) .",
"While in general Mtb bacilli are hard to find in intact closed granulomas ( Lin et al . , 2014 ) , high Mtb concentrations have been reported in vivo upon examination of the cavity surface in human tuberculous granulomas ( Hunter , 2011; Hunter et al . , 2007; Welsh et al . , 2011 ) as well as cavity containing granulomas in rabbits infected with experimental pulmonary TB ( Kaplan et al . , 2003 ) , and the cavity surface of granulomas with a necrotic core in the granuloma-forming Kramnik mouse model ( Irwin et al . , 2015 ) .",
"Histologic examination has shown clumped bacteria as well as highly infected macrophages at the luminal surface of cavities in areas with extensive cellular necrosis ( Kaplan et al . , 2003; Hunter , 2011; Irwin et al . , 2015 ) .",
"While it can be inferred that high bacillary loads cause host cell death , this view is incomplete , as it lacks insight into the dynamic interactions between host cells and bacilli which cause the buildup of bacterial numbers and host necrosis at a particular site .",
"Furthermore , since Mtb clumps are present in vivo , it is necessary to consider the effect of clumps on the dynamics of the host-pathogen interaction ( Orme , 2014; Sani et al . , 2010 ) .",
"This entails using non-detergent treated Mtb capable of aggregate formation .",
"Here we used time-lapse microscopy of detergent untreated Mtb infection of primary human macrophages to map out the interactions between Mtb and the host macrophages .",
"We quantified host cell-Mtb dynamics over 80 hr of growth at a 10 min resolution between time-lapse frames , necessary to capture several replication cycles of this slow growing pathogen .",
"To our knowledge , this study is the first to use long duration time-lapse microscopy combined with automated image analysis to decipher Mtb infection dynamics of primary human macrophages .",
"We confirmed at the single cell level that the probability of macrophage death increased with multiplicity of infection ( Figure 1 , Video 1 ) , consistent with a previous report of a threshold of pathogens which a macrophage can control in Trypanosoma cruzi infection ( Tanaka et al . , 1982 ) as well as the previously found increase of cell death with increasing Mtb load ( Lee et al . , 2006; Repasy et al . , 2013 ) .",
"The death pathway was non-apoptotic ( Figure 4 ) , consistent with previous reports of host cell death at high multiplicities of Mtb infection ( Lee et al . , 2006; Repasy et al . , 2013 ) .",
"However , in addition to the multiplicity of infection , host cells were found to be sensitive to the aggregation state of the bacilli: pickup of a single large aggregate was more cytotoxic than pickup of several smaller aggregates with a similar total number of Mtb ( Figure 2 ) .",
"After internalization of large clumps , macrophage death was rapid ( Figure 3 , Videos 1 , 5 and 7 ) .",
"The observed rapid breakdown of host cell membranes with Mtb induced cell death ( Videos 2 and 5 , Figure 4 ) is consistent with reports of Mtb being present in the cytoplasm , as opposed to the phagosome ( Russell , 2001 ) , of non-apoptotic cells ( van der Wel et al . , 2007; McDonough et al . , 1993; Simeone et al . , 2012 ) .",
"If breach of the phagolysosomal membrane precedes death in Mtb infection , one reason for the increased cytotoxicity of aggregates may include increased membrane breaching ( Rodríguez-Muela et al . , 2015; Sargeant et al . , 2014; Boya and Kroemer , 2008 ) .",
"In addition , Mtb induced mitochondrial membrane breakdown ( Duan et al . , 2002 ) , interference with host plasma membrane repair ( Divangahi et al . , 2009 , 2010 ) , and toxin secretion ( Sun et al . , 2015 ) would all be expected to scale with Mtb number per macrophage .",
"Host cell death did not eliminate intracellular Mtb ( Figure 5 ) , consistent with previous reports at the cell population level showing that Mtb induced necrosis does not clear the bacilli ( Fratazzi et al . , 1999; Molloy et al . , 1994; Lee et al . , 2006; Park et al . , 2006; Dobos et al . , 2000; Duan et al . , 2002; Divangahi et al . , 2009 , 2010; Sun et al . , 2015; Tobin et al . , 2012 ) , and that upregulation of Ipr1 , which switches the host response to Mtb from necrosis to apoptosis in mice , limits Mtb growth ( Pan et al . , 2005 ) .",
"Pre-stimulation with IFNγ or use of alveolar macrophages instead of MDM did not substantially alter the ability of Mtb to survive host cell death .",
"These observations differ from results showing that efferocytosis leads to the elimination of Mtb ( Martin et al . , 2012 ) .",
"However , the protective effect of efferocytosis may occur when cells die by apoptosis and the number of bacilli inside the dead cell is small while the number of uninfected macrophages is large , enabling the surrounding macrophages to divide up the bacillary load of a single dead infected cell .",
"This was not the case in our experiments .",
"The observation that Mtb growth was slowest in live cells , intermediate in the extracellular environment , and most robust in dead infected cells ( Figures 5–7 ) , suggest that the dead cell may provide Mtb with a favorable growth niche relative to the intact phagosome in live cells or the extracellular environment , while live cells provide a poor growth environment either because of antimicrobial effector mechanisms or reduced access to nutrients in the Mtb compartment .",
"Our results indicating acidification of Mtb ( Figure 6C–D ) are consistent with the presence of antimicrobial effector mechanisms that reduce Mtb growth in Mtb compartmentalized to the phagosome .",
"Mtb can inhibit phagosome-lysosome fusion and complete phagosome acidification ( Vergne et al . , 2003; Rohde et al . , 2007; Via et al . , 1997 ) .",
"However , acidification may occur when macrophages are activated with cytokines such GM-CSF ( Denis and Ghadirian , 1990 ) , as are the MDMs used for this study .",
"In this case , Mtb growth may be impaired or arrested in acidified vacuoles ( Gomes et al . , 1999 ) .",
"Acidification was lost immediately upon cell death ( Video 6 , Figure 6C–D ) .",
"However , loss of antimicrobial effector mechanisms may not be the only factor responsible for robust growth in dead cells , as growth is faster than in the extracellular medium , indicating that Mtb may have access to cytosolic nutrient sources , which promote growth in other intracellular pathogens ( Appelberg , 2006; Ray et al . , 2009 ) .",
"If Mtb growth in the dead cells was solely dependent of leakage of nutrients from the extracellular environment , the intracellular bacilli would not be expected to replicate any faster than extracellular Mtb .",
"This is clearly not what we observe .",
"In these experiments we used a pure macrophage culture to investigate the outcome of macrophage-Mtb interactions .",
"In the granuloma , the presence of other cell types can influence outcomes .",
"Particularly relevant are the T cell subsets of the adaptive cellular immune response ( Cooper and Flynn , 1995; Flynn et al . , 1995 , 1992; Mogues et al . , 2001 ) .",
"T cells control Mtb by activating macrophages to kill Mtb through IFNγ secretion ( Flynn et al . , 1993 ) , and CD8+ cytotoxic T cells target intracellular bacteria by granulysin release ( Stenger et al . , 1998 ) , which has been reported to enable granzymes to enter and kill the bacteria ( Walch et al . , 2014 ) .",
"These mechanisms would counteract the spread of Mtb , but would likely be restricted by the Mtb mediated death of the host cell before recognition and targeting by T cells , limited T cell effector function in the granuloma ( Egen et al . , 2011 ) , lack of T cells in the inner cell layer of granulomas ( Ulrichs et al . , 2004 ) , and impaired expression of perforin and granulysin observed in Mtb granulomas ( Andersson et al . , 2007 ) .",
"The process we observe likely reflects a positive feedback loop for Mtb replication in late stage infection , as illustrated by the outcomes of the simplified model in Figure 9 .",
"At this infection stage , localized expansion of Mtb numbers may occur by the following scenario , captured in Figure 8 and strikingly in Video 7: serial killing is initiated when a human macrophage internalizes a clump of bacilli which causes its death by necrosis .",
"Upon death , Mtb rapidly grows in the dead cell , with a doubling time faster than either in the extracellular environment or in live cells ( where growth is minimal ) .",
"The next macrophage to internalize the Mtb , this time encased in a dead cell , faces a larger Mtb clump and also dies .",
"The new dead infected cell now provides fuel for the next round of bacterial growth and bait for the next macrophage .",
"This illustrates how , once initiated , Mtb replication can be locally stabilized in the active state ."
],
[
"Blood was obtained from adult healthy volunteers after written informed consent ( University of KwaZulu-Natal Institutional Review Board approval BE022/13 ) .",
"Alveolar macrophages were obtained from bronchoalveolar lavage as part of an indicated diagnostic procedure after written informed consent ( University of KwaZulu-Natal Institutional Review Board approval BE037/12 ) .",
"Buffy coats from HIV-negative blood bank donations were obtained from the South African National Blood Service ( Human Research Ethics Committee approved protocol IRB00007553 ) or a cohort of healthy donors ( UKZN Institutional Review Board approval BE022/13 ) .",
"Informed consent was obtained .",
"Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Histopaque 1077 ( Sigma-Aldrich , St Louis , MO ) .",
"CD14+ monocytes were purified under positive selection using anti-CD14 microbeads ( Miltenyi Biotec , San Diego , CA ) .",
"2 ml of 105/ml monocytes were added to 0 . 01% fibronectin ( Sigma-Aldrich ) coated 35 mm glass bottom optical dishes ( Mattek , Ashland , MA ) and differentiated in macrophage growth medium containing 1% each of HEPES , sodium pyruvate , L-glutamine , and non-essential amino acids , 10% human AB serum ( Sigma-Aldrich ) , and 50 ng/ml GM-CSF ( Peprotech , Rocky Hill , NJ ) in RPMI .",
"The cell culture medium was changed one day post plating and half the media was replaced on day 3 and 6 post plating .",
"To isolate alveolar macrophages , bronchoalveolar lavage fluid was centrifuged at 300× g for 10 min , and the cell pellet washed twice with phosphate buffered saline ( PBS ) .",
"The cells were counted , and adjusted to 105 cells/ml using macrophage growth media with 2 . 5 μg/mL amphotericin B to inhibit potential fungal contamination .",
"The cells were plated in glass bottom dishes overnight , followed by three washes with PBS , and the media was replaced .",
"The RFP fluorescent strain of H37Rv Mtb was derived by transforming the parental strain with a plasmid with the TagRFP gene downstream of the constitutive groEL promoter ( gift from D . Russell ) .",
"The mCherry fluorescent strain of H37Rv Mtb was derived by transforming the parental strain with a plasmid with mCherry under the smyc' promoter ( gift from D . Russell ) .",
"Mtb were maintained in Difco Middlebrook 7H9 medium enriched with oleic acid-albumin-dextrose catalase supplement ( BD , Sparks , MD ) .",
"Three days before macrophage infection , Mtb were switched to grow in Tween 80-free media .",
"On the day of infection , exponentially growing bacterial culture was pelleted at 2000 × g for 10 min , washed twice with 10 ml PBS , and large aggregates broken up by shaking with sterilized 2–4 mm glass beads for 30 s .",
"10 ml of PBS was added and large clumps were further excluded by allowing them to settle for 5 min .",
"An inoculum of 1 ml was taken from the center of the suspension for macrophage infections .",
"MDM and alveolar macrophages were inoculated with 5 µl Mtb suspension ( MOI ~5 by CFU ) and either imaged after 2 hr to allow the Mtb to settle , or incubated 18 hr , wash five times to remove extracellular bacteria and then imaged .",
"Macrophages and bacteria were imaged using an Andor integrated ( Andor , Belfast , UK ) Metamorph-controlled ( Molecular Devices , Sunnyvale , CA ) Nikon TiE motorized microscope ( Nikon Corporation , Tokyo , Japan ) with a 20x , 0 . 75 NA phase objective .",
"For Mtb RFP and mCherry fluorescence , excitation source was a 561 laser line and emission was detected through a Semrock Brightline 607 nm filter ( Semrock , Rochester , NY ) .",
"Images were captured using an 888 EMCCD camera ( ( Andor ) .",
"Temperature ( 37°C ) , humidity and CO2 ( 5% ) were controlled using an environmental chamber ( OKO Labs , Naples , Italy ) .",
"Approximately 40 fields of view were captured every 10 min , one phase contrast image and one fluorescent image per field at every time point .",
"Fluorescence readings after death were confirmed by widefield microscopy ( data not shown ) , but fluorescence readings before and particularly at the point of cell death were strongly influenced by cell movement in the z-plane and are not included in the analysis .",
"For imaging data after cell death , the fluorescence signal starting 4 hr after the cell death event was used for analysis .",
"For DRAQ7 ( BioStatus , Leicestershire , UK ) entry , excitation source was a 640 nm laser and emission collected through a Semrock Brightline 685 nm filter For pH dye pHrodo detection , excitation source was a 488 laser while emission was collected through a Semrock 525 nm filter .",
"To determine cell borders , phase contrast images were segmented by a custom code ( Source code",
"1 ) using the Matlab R2014a image analysis toolbox .",
"Cells were identified using edge detection and cell borders between adjacent cells discriminated using the watershed algorithm .",
"Segmentation was manually curated for errors .",
"Cells in each frame in the same field of view were linked to cells in the previous frame by root mean squared closest centroid .",
"To determine the number of Mtb internalized per cell , single Mtb fluorescence was obtained by filtering the culture through a 5 µm filter and validating the resulting product as single cell by microscopy .",
"We converted Mtb fluorescent signal inside cells to bacterial numbers by dividing the signal by mean single Mtb fluorescence at the acquisition settings used for each experiment .",
"Each macrophage internalization event was manually segmented at the time of pickup , and the fluorescence from the internalized bacteria was divided by the movie specific average fluorescence per bacterium to obtain Mtb number per internalization .",
"To confirm that fluorescence measurements by microscopy reflected actual Mtb expansion , we grew fluorescently labeled-Mtb as a suspension in 7H9 media and sampled aliquots of the suspension by imaging or plating on 7H10 agar plates over a three day period .",
"Macrophages were either infected with H37Rv Mtb , treated with 50 µM cisplatin ( Sigma-Aldrich ) , or primed with 1 µg/ml Escherichia coli LPS ( Sigma-Aldrich ) for 3 hr , washed , and then incubated with nigericin ( 20 µM , Sigma-Aldrich ) .",
"Cells were then incubated with 3 µM DRAQ7 and imaged as described above .",
"After seven days of differentiation , MDM was treated with 200 U/ml IFNγ ( Peprotech ) or vehicle control ( 0 . 01% BSA ) in fresh culture media , incubated for 18 hr , then infected with Mtb and imaged in the presence of IFNγ as described above for non-IFNγ treated MDM .",
"Activation of macrophages by IFNγ was confirmed by flow cytometry for the upregulation of HLA-DR and CD86 using anti-HLA-DR conjugated to Alexa Fluor 488 and anti-CD86 conjugated to Alexa Fluor 647 monoclonal antibodies ( Biolegend , San Diego , CA ) .",
"Time of cell death was determined by an image analysis algorithm detecting cell detachment or cessation of internal movement .",
"For the cessation of internal movement , a 20 by 20 pixel square around the centroid of the cell at the last frame was used .",
"The pixels in the square in one frame were compared to the pixels in the same square on the previous frame .",
"Given row r and column c coordinates , for all pixels Xr , c in frame i and all corresponding pixels Yr , c in frame i−1 , the Pearson’s Correlation Coefficient was computed as ∑ ( Xr , c−X¯ ) ( Yr , c−Y¯ ) ∑ ( Xr , c−X¯ ) 2∑ ( Yr , c−Y¯ ) 2 .",
"The correlation between frames in cell interiors in dead cells was high , while internal organelle movement in live cells kept correlation low between frames .",
"The time of transition between a living and dead cell was determined by fitting two horizontal lines to the data .",
"If the mean of the line corresponding to the later time points was greater than 1 . 5-fold the mean of the line for the earlier time-points , the time point at which the transition from the first to second line occurred was the time of death .",
"The fold change threshold was selected to maximize sensitivity and specificity relative to death determined by DRAQ7 .",
"MDMs were infected with Mtb at MOI ~30 , six-fold higher than the MOI used in other infections and sufficient to cause >99% macrophage death after one day .",
"After three days , MDMs were lysed with 0 . 1% Triton X , and the suspensions plated in duplicate on Difco Middlebrook 7H10 agar enriched with oleic acid-albumin-dextrose catalase supplement ( BD ) to quantify Mtb growth in comparison to Mtb grown in parallel in macrophage media without MDMs .",
"Mtb was labelled with 100 µM pHrodo Green STP Ester ( Thermo Fisher , Waltham , MA ) in 100 mM sodium bicarbonate buffer , pH 8 . 5 for 30 min at room temperature , and then washed three times with phosphate buffered saline before proceeding to infection of MDMs .",
"The DNA intercalating dye DRAQ7 was used at 3 µM in the same experiments to detect cell death .",
"Where noted , bootstrap ( Efron and Tibshirani , 1994 ) was used to determine statistical significance .",
"p-values of differences between death frequencies between two samples x and y , with Freq ( death ) x > Freq ( death ) y , were determined using Matlab 2014a by randomly selecting with replacement the same number of cells from x , one at a time , as contained in y .",
"The frequency of death in this randomly selected set of cells was determined , and the procedure repeated 10 , 000 or 100 , 000 times to create vector Frand .",
"The p-value was calculated as the number of elements in Frand smaller than Freq ( death ) y , divided by the number of times the procedure was repeated .",
"In the case of multiple comparisons , the significance threshold was made more stringent by adjusting for the number of comparisons by the Bonferroni method ( Noble , 2009 ) .",
"Statistical analysis not using bootstrap was performed using Graphpad Prism 6 and identified in the figure legends .",
"The simulation determined the total number of dead host cells per unit time ND ( t ) and number of Mtb per unit time m ( t ) as a function of Mtb doubling time ( td ) , where time t is in increments of one day .",
"A doubling time of one day was what was experimentally measured in dead cells .",
"To compare to a slower doubling time , for simplicity we used a value of two days , which was within the physiological range of Mtb growth ( for example , the doubling time range for extracellular Mtb is 22–75 hr for the data shown in the inset of Figure 5B ) .",
"Each run was initiated with the internalization of 1 Mtb .",
"Two approximations were made based on our experimental results: ( 1 ) No Mtb growth occurred inside live cells .",
"( 2 ) Probability of death after internalization of a dead infected cell depended only on the number of Mtb internalized .",
"The probability of cell death PD ( m ) per day after Mtb internalization was determined experimentally from the time-lapse data and binned for simplicity for Mtb numbers m ≤ 10 , 10 < m ≤ 100 , and m > 100 .",
"Death probabilities were determined from the data to be PD ( m ≤ 10 ) =0 . 06 , PD ( 10 < m ≤ 100 ) =0 . 50 , and PD ( m > 100 ) =0 . 93 .",
"To implement the cell death decision , a random number from a uniform distribution was drawn at each time increment .",
"If the random number was less than or equal to PD ( m ) , then ND ( t + 1 ) =ND ( t ) +1 , else ND ( t + 1 ) =ND ( t ) .",
"After cell death at time t , there was a one day delay before the next pickup , and m ( t + 1 ) = m ( t ) er , where r = ln2/td .",
"Simulation script ( Matlab ) is attached as supplementary material .",
"To examine the sensitivity of the number of Mtb after 60 days to doubling time , we ranged td from 1 to 3 days , and calculated the fraction of the simulations in which the number of Mtb expanded by more than three orders of magnitude ( from 1 to >1000 bacilli ) ."
]
] | [
"A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas .",
"These may restrict Mycobacterium tuberculosis ( Mtb ) growth , or progress to central necrosis and cavitation , facilitating pathogen growth .",
"To determine factors leading to Mtb proliferation and host cell death , we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages .",
"Internalization of Mtb aggregates caused macrophage death , and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli .",
"Macrophage death did not result in clearance of Mtb .",
"Rather , it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type .",
"In contrast , bacillary replication was controlled in live phagocytes .",
"Mtb grew as a clump in dead cells , and macrophages which internalized dead infected cells were very likely to die themselves , leading to a cell death cascade .",
"This demonstrates how pathogen virulence can be achieved through numbers and aggregation states ."
] | [
"Every year , around two million people worldwide die from tuberculosis , a disease caused by the bacterium Mycobacterium tuberculosis ( Mtb ) .",
"The bacteria generally infect the lungs .",
"In response , the immune system forms structures called granulomas that attempt to control and isolate the infecting pathogens .",
"Granulomas consist of immune cells known as macrophages , which engulf the M . tuberculosis bacteria and isolate them in a cellular compartment where the bacteria either cannot grow or are killed .",
"However , if a large number of macrophages in a granuloma die , the granuloma’s core liquefies and the structure is coughed up into the airways , from where M . tuberculosis bacteria are transmitted to other people .",
"But how do the bacteria manage to cause the extensive death of the cells that are supposed to control the infection ?",
"By imaging M . tuberculosis in human macrophages using time-lapse microscopy , Mahamed et al . reveal that the bacteria break down macrophage control by serially killing macrophages .",
"M . tuberculosis cells first clump together and ‘gang up’ on a macrophage , which engulfs the clump and dies because the bacteria overwhelm it .",
"This does not kill the bacteria , and they rapidly grow inside the dead macrophage .",
"The dead cell is then cleaned up by another macrophage .",
"However , the increasing number of bacteria inside the dead macrophage means that the new macrophage is even more likely to die than the first one .",
"Hence , the bacteria use dead macrophages as fuel to grow on and as bait to attract the next immune cell .",
"Overall , Mahamed et al . show that once a clump of M . tuberculosis initiates death of a single macrophage , it may lead to serial killing of other macrophages and a loss of control over the infection .",
"An important next step will be to understand how the initial clump of bacteria is allowed to form ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Central neural circuitry mediating courtship song perception in male Drosophila | elife-08477-v1 | [
[
"Sexual behaviors normally involve the exchange of species-specific social cues , including chemical , visual , and auditory information , that elicit a set of sex-specific behaviors ( Dulac and Kimchi , 2007; Manoli et al . , 2013 ) .",
"Here , we are particularly focused on auditory communication , which requires the production and perception of sound signals that are diversified between species and are utilized by both invertebrates and vertebrates to attract female mates during courtship ( Murthy , 2010; Brainard and Doupe , 2013 ) .",
"Drosophila melanogaster males sing a multi-part , species-specific courtship song that is both critical to female receptivity and encourages courtship by nearby males .",
"Although there has been progress in elucidating the circuitry by which these signals are perceived , processed , and integrated with other sensory cues to elicit stereotyped courtship behaviors , there are very significant gaps in our knowledge .",
"The neural circuits underlying sexually dimorphic behavior in Drosophila are specified by the action of the two terminal genes in the sex determination hierarchy , fruitless ( fruM ) and doublesex ( dsx ) .",
"fruM and dsx act together to specify sexual behaviors by altering the number , morphology , and physiology of a small subset of CNS ( Central Nervous System ) neurons ( Manoli et al . , 2013; Pavlou and Goodwin , 2013; Yamamoto and Koganezawa , 2013 ) .",
"dsx is expressed in a limited set of neurons within the CNS , as well as in subsets of non-neuronal cells throughout the body ( Rideout et al . , 2010; Robinett et al . , 2010 ) .",
"In contrast , fruM is expressed only in neurons , including ∼2% of neurons within the CNS , as well as sensory , motor , and higher-order interneurons , all of which might comprise neural circuits dedicated to sexual behaviors ( Manoli et al . , 2005; Stockinger et al . , 2005 ) .",
"This has motivated the proposal that fruM+ neurons might provide a set of genetically labeled pathways that channel courtship-related sensory information through a dedicated circuit ( Manoli et al . , 2005; Stockinger et al . , 2005 ) .",
"Supporting this view , a fruM-labeled pathway has been identified that links olfactory reception of a male-specific pheromone to descending neurons that drive motor output ( Kurtovic et al . , 2007; Ruta et al . , 2010; Kohl et al . , 2013 ) .",
"Despite tantalizing clues , it remains unknown whether Drosophila audition is mediated by such dedicated circuits .",
"When courting a female , the male vibrates his wing to generate a courtship song consisting of two parts: a rhythmic pulse song with an ∼35-ms inter-pulse interval ( IPI ) that strongly elicits male courtship behavior as well as female receptivity , as well as a ∼160-Hz sine song that may play a secondary role ( Shorey , 1962; Bennet-Clark and Ewing , 1967; von Philipsborn et al . , 2011; Arthur et al . , 2013 ) .",
"Drosophila species exhibit significant diversity in the IPI of pulse song , and in D . melanogaster , IPIs around ∼35 ms are critical for enhancing the responses to courtship song of both conspecific males and females ( Ewing and Bennet-Clark , 1968; Bennet-Clark , 1969; Cowling and Burnet , 1981; Yoon et al . , 2013 ) .",
"In females , pulse song induces reduced locomotion ( allowing courting males to come closer ) and increases their receptivity to mating attempts ( Bennet-Clark , 1969; Schilcher , 1976a , 1976b; Rybak et al . , 2002; Shirangi et al . , 2013 ) .",
"In males , pulse song elicits both increased locomotion and exploratory courtship activity directed towards nearby flies ( Schilcher , 1976b; Eberl et al . , 1997; Kowalski et al . , 2004; Kamikouchi et al . , 2009; Vaughan et al . , 2014 ) .",
"Although males and females show very different motor output in response to song , significant sexual dimorphisms in the sensory pathway have not been identified .",
"Drosophila receives sound stimuli by vibration of the arista , a feather-like structure protruding from the second segment of the antenna ( Gopfert and Robert , 2001 ) .",
"Vibration of the arista activates the mechanosensory neurons of the Johnston's organ of the second antennal segment , which project to the antennal mechanosensory and motor center ( AMMC ) of the central brain where they connect with secondary auditory projection neurons ( aPNs ) , local neurons ( aLNs ) , and the giant fiber neurons ( Gopfert et al . , 2006; Kamikouchi et al . , 2009; Yorozu et al . , 2009; Effertz et al . , 2011 , 2012; Tootoonian et al . , 2012; Lehnert et al . , 2013; Pezier et al . , 2014 ) .",
"Among a variety of aPNs , only the aPN1 cell type projecting to the wedge of the ventrolateral protocerebrum ( WED ) is necessary for song responses in either sex ( Kamikouchi et al . , 2009; Lai et al . , 2012; Tootoonian et al . , 2012; Vaughan et al . , 2014 ) .",
"aPN1 is necessary for both female receptivity and the male song-induced locomotion response , and it shows a response to courtship song that is proportional to pulse rate ( Vaughan et al . , 2014 ) .",
"By identifying downstream neurons in this pathway , we hope to identify the sexually dimorphic structure of this critical pathway , as well as elucidate how song is transformed before reaching the central drivers of courtship output .",
"One candidate neuronal cluster for central integration of courtship song and other courtship modalities is the set of dsx+ pC1 neurons that innervate the lateral protocerebral complex ( LPC ) , a region of dense innervation by both fruM+ and dsx+ neurons ( Cachero et al . , 2010; Rideout et al . , 2010; Robinett et al . , 2010; Yu et al . , 2010 ) .",
"These neurons are activated by courtship stimuli in both sexes , but they drive differential behavioral outputs in each sex .",
"Female pC1 neurons are activated by the male-specific pheromone cVA ( Zhou et al . , 2014 ) , while male fruM+/dsx+ P1 neurons ( a subset of the dsx+ pC1 population ) are inhibited by cVA but activated by female pheromones ( Kohatsu et al . , 2011 ) .",
"Female pC1 neurons are also activated by sine and pulse songs ( Zhou et al . , 2014 ) .",
"These observations are consistent with pC1 neurons as a site for multimodal integration of courtship stimuli , but how song information is relayed from aPN1 neurons to the pC1/P1 neurons is unknown .",
"Here , we used a large-scale intersectional screen to identify a labeled line of fru+ neurons that supports courtship hearing in male flies .",
"This approach identified a critical fruM+ interneuron type , which we designate as ventrolateral protocerebrum Projection Neuron 1 ( vPN1 ) , whose neurites lie in close proximity to those of aPN1 in the WED area .",
"We present several lines of evidence suggesting that vPN1 may represent the third-order auditory neurons .",
"First , anatomical registration of aPN1 and vPN1 suggests axon/dendrite overlap in the WED .",
"Second , genetic inactivation of vPN1 recapitulates the attenuation of male song responses observed for aPN1 , while optogenetic activation of vPN1 mimicked pulse song stimuli to induce robust male chaining .",
"Third , GCaMP imaging revealed that both pulse song and sine song elicit strong calcium responses in vPN1 cell bodies and show a preferential response to pulse songs with long IPIs .",
"In addition , we found that vPN1 neurons likely target dsx+ pC1 neurons directly .",
"Anatomically , vPN1 neurons overlap with pC1 neurons in the LPC region .",
"Physiologically , the tuning curve of the pC1 calcium responses to pulse song IPIs closely matches that of behavioral chaining responses .",
"Behaviorally , silencing pC1 neurons reduced male-chaining behavior in response to pulse song , while pC1 activation is sufficient to induce robust chaining responses .",
"Lastly , simultaneous GCaMP imaging and CsChrimson stimulation reveals that the aPN1-vPN1-pC1 pathway is indeed functionally connected .",
"Taken together , we provide anatomical , behavioral , and physiological evidence that the aPN1-vPN1-pC1 pathway provides a labeled line for processing courtship song in Drosophila ."
],
[
"Motivated by the hypothesis that fruM labels neurons that detect courtship-relevant sensory stimuli ( Manoli et al . , 2005; Stockinger et al . , 2005 ) , we performed an anatomical screen aimed at identifying fruM+ neurons in the auditory pathway .",
"Specifically , ∼1000 cis regulatory module ( CRM ) GAL4 lines with relatively sparse neuronal expression patterns ( Jenett et al . , 2012 ) were crossed to LexAop2-FLP; fruLexA , UAS>stop>myr::GFP to restrict expression of GFP to those neurons that express both GAL4 and fruLexA ( Figure 1J ) .",
"These intersectional expression patterns were then registered onto a standard brain for analysis of potentially overlapping projection patterns . 10 . 7554/eLife . 08477 . 003Figure 1 . Intersectional labeling of auditory neurons .",
"( A ) Intersectional labeling of male aPN1 neurons using three independent GAL4 lines shown registered onto a standard brain ( R21B12-GAL4 in green , R22B11-GAL4 in magenta , and R49F09-GAL4 in yellow ) .",
"White region is a result of overlapping between different color channels .",
"( B ) Intersectional labeling of male ventrolateral protocerebrum Projection Neuron 1 ( vPN1 ) neurons using R72E10-GAL4 ( green ) and R46F09-GAL4 ( magenta ) .",
"( C ) Co-registration of male aPN1 neurons ( green ) and vPN1 neurons ( magenta ) onto the standard brain shows significant overlap in WED .",
"aPN1 and vPN1 neurons are labeled by R49F09-GAL4 ∩ fruLexA and R72E10-GAL4 ∩ fruLexA , respectively .",
"( D–F ) aPN1 neurons are present in both male ( D ) and female ( E ) brains of LexAop2-FLP/+; fruLexA , UAS>stop>myr::GFP/49F09-GAL4 flies .",
"( F ) Merge of ( D ) and ( E ) .",
"( G–I ) vPN1 neurons are present in male ( G ) but absent in female ( H ) brains of LexAop2-FLP/+; fruLexA , UAS>stop>myr::GFP/72E10-GAL4 flies .",
"( I ) Merge of ( G ) and ( H ) .",
"( J ) Schematic drawing of GAL4/LexA intersection for labeling subsets of fruM+ neurons .",
"FLP expression induced by fruLexA will remove the stop cassette to allow GFP reporter expression only in neurons expressing both fruLexA and the CRM-GAL4 .",
"Scale bars , 50 μm .",
"All images were aligned and registered onto a standard brain . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 00310 . 7554/eLife . 08477 . 004Figure 1—source data 1 . Quantification of aPN1 or VPN1 neurons labeled by intersectional drivers . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 004 To characterize fruM+ aPN1 cells , we identified GAL4 drivers ( R21B12 , R22B11 , and R49F09 ) that label four to five aPN1 cells per hemisphere when intersected with fruLexA ( Figure 1A , Figure 1—source data 1 ) .",
"These intersectional drivers appear to label the same cell type ( Figure 1A ) , as co-registered expression patterns are morphologically indistinguishable and consistent with the aPN1/AMMC-B1 cell type ( Kamikouchi et al . , 2009; Lai et al . , 2012; Vaughan et al . , 2014 ) .",
"In order to identify putative third-order auditory neurons , we focused on drivers labeling fruM+ neurons that innervate the WED and project to the central brain .",
"Two GAL4 drivers ( R72E10 and R46F09 ) were identified that , when crossed to LexAop2-FLP; fruLexA , UAS>stop>myr::GFP , labeled a subset of the fruM+ aSP-k cell type previously identified via MARCM ( Mosaic analysis with a repressible cell marker ) clones ( Cachero et al . , 2010 ) ( Figure 1B and Figure 1—source data 1 ) .",
"These neurons , termed vPN1 , have cell bodies located laterally in the dorsal anterior brain and neurites that innervate the WED and the LPC ( Figure 1B ) .",
"Co-registration of fruM+ aPN1 and vPN1 neurons revealed substantial overlap in the WED region ( Figure 1C ) , suggesting potential synaptic connectivity between aPN1 and vPN1 neurons .",
"The intersectional labeling of aPN1 and vPN1 was dependent on fruLexA expression .",
"Since many fruM+ neurons have been shown to be sexually dimorphic at the anatomical level , we examined whether the morphologies of either the aPN1 or vPN1 neurons were sexually dimorphic .",
"aPN1 neurons had indistinguishable projection patterns in female and male brains ( Figure 1D–F ) , while vPN1 neurons were only observed in male brains ( Figure 1G ) , with female brains showing only very weak expression in a few seemingly unrelated neurons ( Figure 1H–I ) .",
"However , aSP-k neurons are present in both sexes and extend male-specific processes that innervate the LPC arch ( Cachero et al . , 2010 ) .",
"In addition , we identified a split-GAL4 combination ( R72E10-GAL4AD ∩ VT9665-GAL4DBD , referred to as vPN1 split-GAL4 hereafter ) that labels vPN1 neurons in the male brain ( Figure 2A ) , independent of fruLexA expression .",
"Using this driver , vPN1 neurons remain absent from the female brain , and fruM is expressed in all vPN1 cells of the male ( Figure 2B , C ) .",
"We therefore infer that the vPN1 population constitutes a male-specific subset of the larger aSP-k cell type . 10 . 7554/eLife . 08477 . 005Figure 2 . fru is necessary and sufficient for specifying male-specific vPN1 neurons .",
"( A , B )",
"GFP expression ( green ) in the male ( A ) or female ( B ) brain of R72E10-GAL4AD/UAS-mCD8GFP; VT9665-GAL4DBD/+ flies counter-stained by nc82 antibody ( magenta ) .",
"vPN1 neurons with VLP projections were labeled in the male but not female brain .",
"( C ) vPN1 neurons ( C1 ) in the male brain of R72E10-GAL4AD/UAS-mCD8GFP; VT9665-GAL4DBD/+ flies co-stained with FruM antibody ( C2 ) .",
"( C3 ) is a merge of ( C1 ) and ( C2 ) .",
"( D ) vPN1 neurons are present in the male brain ( D1 ) but not female brain ( D2 ) of fru4–40/+ flies .",
"Genotype is: R72E10-GAL4AD/UAS-mCD8GFP; VT9665-GAL4DBD , fru4–40/+ .",
"( E ) vPN1 neurons are absent in both the male brain ( E1 ) and female brain ( E2 ) of null mutant fru4–40/fruLexA .",
"Genotype is: R72E10-GAL4AD/UAS-mCD8GFP; VT9665-GAL4DBD , fru4–40/fruLexA .",
"( F ) vPN1 neurons are present in both the male brain ( F1 ) and female brain ( F2 ) of fruM mutant flies where FruM is expressed in both males and females .",
"Genotype is: R72E10-GAL4AD/UAS-mCD8GFP; VT9665-GAL4DBD , fruM/+ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 005 We next asked whether fruM is necessary and sufficient for specifying the male-specific vPN1 neurons .",
"We did this by examining vPN1 split-GAL4 expression patterns in various fru mutant backgrounds .",
"The male-specific expression pattern of vPN1 is not affected in heterozygous fru mutants ( fru4–40/+ , Figure 2D ) .",
"However , vPN1 expression is absent in fru null mutant males ( fru4–40/fruLexA , Figure 2E ) , while misexpression of FruM protein in females ( fruM/+ ) leads to an induction of vPN1 expression in the female brain ( Figure 2F ) ( Demir and Dickson , 2005 ) .",
"Thus , fruM plays an instructive role in the specification of sexual dimorphism of vPN1 neurons .",
"Our anatomical results suggest a candidate pathway that may carry auditory information from aPN1 to higher brain regions via male-specific vPN1 projections .",
"We next tested whether these neurons are necessary for male courtship hearing .",
"Male flies respond to courtship song , and pulse song in particular , by increasing locomotion and exploratory courtship of nearby flies ( Schilcher , 1976b; Eberl et al . , 1997; Kowalski et al . , 2004 ) .",
"Ecologically , this response is appropriate for the context of competitive courtship on fruit substrates .",
"Experimentally , stimulation of groups of males with synthetic courtship song can induce both locomotion and chains of males engaging in the initial stages of courtship .",
"This response ( distinct from the unstimulated and dysregulated courtship observed in fru mutant males ) has been used to investigate the genetic and neural mechanisms underlying auditory detection ( Eberl et al . , 1997; Kamikouchi et al . , 2009; Vaughan et al . , 2014 ) .",
"To assay the role of putative auditory interneurons in male courtship hearing , we constructed courtship chambers with sloped side walls for better visualization of song-induced chaining behavior ( Figure 3A and Figure 3—figure supplement",
"1 ) ( Simon and Dickinson , 2010 ) .",
"Upon stimulation by pulse song with a ramping intensity , we indeed observed that male flies displayed robust song-induced chaining behavior ( Figure 3A ) . 10 . 7554/eLife . 08477 . 006Figure 3 . IPI tuning of song-induced male-chaining behavior .",
"( A ) Song-induced chaining assay .",
"Males court each other when exposed to pulse song with a 35-ms inter-pulse interval ( IPI ) and robustly form courtship chains ( bottom ) .",
"( B ) Chaining indices ( CIs ) of wild-type males in response to different IPIs .",
"After 60 s of silence , continuous pulse songs were played back with a ramping sound intensity from 60 dB to 90 dB .",
"The intensity was increased every 30 s .",
"n = 14 for all groups .",
"Error bars represent SEM .",
"( C ) Heat map visualization of chaining responses in ( B ) .",
"Each cell represents the chaining index at a given IPI and song intensity .",
"( D ) CIs of wild-type males in response to intermittent pulse songs with different IPIs at 80 dB .",
"A train of 40 pulses was delivered every five seconds so that the number of pulses was the same for different IPIs .",
"After one minute of silence , two minutes of intermittent pulse song stimuli were presented ( indicated by shadowed box ) , and then followed by two minutes of silence .",
"Chaining persisted and gradually decreased after song presentation .",
"n = 15 for all groups .",
"( E ) CIs during the song presentation period in ( D ) were summed up and plotted as a function of IPIs .",
"Error bars represent SEM .",
"*p < 0 . 05 when chaining responses at 35-ms IPI were compared to those at 15- , 20- , 25- , 75- , 85- , and 95-ms IPIs ( Wilcoxon rank-sum test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 00610 . 7554/eLife . 08477 . 008Figure 3—figure supplement 1 . Song-induced chaining setup .",
"( A–C )",
"Schematic drawing of chaining chamber design .",
"A 30° sloped wall was made to reduce the probability that flies occlude each other .",
"Flies were introduced through the holes in the slipping acrylic cover on the top of the chamber .",
"( D–F )",
"The chaining chamber was assembled and mounted on an acrylic holder , which was placed at the side of an external speaker .",
"Front light was delivered by two regular fluorescent bulbs from the top . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 008 Drosophila pulse songs have two key features: intra-pulse frequency ( IPF ) and IPI .",
"Across different Drosophila species , the antennal receiver is tuned to the conspecific IPF ( Riabinina et al . , 2011 ) , and behavioral responses appear tuned to the conspecific IPI ( Ewing and Bennet-Clark , 1968; Cowling and Burnet , 1981 ) .",
"While mechanical features of the antenna may have evolved to optimally detect conspecific IPFs , it has been hypothesized that the brain is responsible for recognizing conspecific IPIs ( Riabinina et al . , 2011 ) .",
"Therefore , we asked whether song-induced chaining behavior of D . melanogaster males varied as a function of song IPI ( Schilcher , 1976b; Yoon et al . , 2013 ) .",
"Indeed , the chaining response was significantly higher for songs at 35-ms IPI than for all other IPIs , across a wide range of intensities ( p < 0 . 05 , 72 . 5–85 dB , Wilcoxon rank-sum test; Figure 3B , C ) , suggesting that D . melanogaster males to some extent are able to preferentially respond to pulse song of their own species .",
"A similar effect was observed using intermittent song stimuli ( 80 dB; Figure 3D , E ) .",
"We therefore used this assay to test the behavioral role of aPN1 , vPN1 , and pC1 populations .",
"aPN1 neurons had been identified as putative second-order auditory neurons for courtship hearing , based on their anatomical location , physiological response to song , and the behavioral phenotypes produced in response to courtship song in both male and female with aPN1 neurons silenced ( Vaughan et al . , 2014 ) .",
"We asked whether the fruM+ subset of aPN1 is also required for courtship hearing in males .",
"Three independent GAL4 ∩ fruLexA genotypes were used to confirm that silencing fruM+ aPN1 neurons reduced male courtship hearing; indeed , flies expressing tetanus neurotoxin light chain ( TNT ) , which blocks synaptic vesicle release , in fruM+ aPN1 neurons showed significantly reduced chaining compared to controls expressing an inactive form of TNT ( TNTin ) or lacking fruLexA ( Figure 4A–C ) ( Sweeney et al . , 1995 ) .",
"Thus , fruM+ aPN1 neurons are required for song-induced male chaining .",
"We similarly asked whether the vPN1 neurons were required for the male-chaining response to song .",
"Using two GAL4 drivers ( R72E10 , R46F09 ) in an intersectional approach ( GAL4 ∩ fruLexA ) to target TNT expression to vPN1 neurons , we observed that vPN1 silencing reduced song-induced chaining ( Figure 4D , E ) . 10 . 7554/eLife . 08477 . 009Figure 4 . Inactivation of second- and third-order auditory neurons reduced chaining responses to pulse song .",
"( A–C )",
"Silencing aPN1 neurons decreased song-induced chaining responses .",
"aPN1 drivers R21B12-GAL4 ( A ) , R22B11-GAL4 ( B ) , or R49F09-GAL4 ( C ) were crossed to UAS>stop>TNT; fruLexA , LexAop2-FLP ( TNT group ) , UAS>stop>TNTin; fruLexA , LexAop2-FLP ( TNTin group ) , or UAS>stop>TNT; LexAop2-FLP ( no LexA group ) .",
"( D , E )",
"Silencing vPN1 neurons decreased song-induced chaining responses .",
"vPN1 drivers R72E10-GAL4 ( D ) , R46F09-GAL4 ( E ) were crossed to UAS>stop>TNT; fruLexA , LexAop2-FLP ( TNT group ) , UAS>stop>TNTin; fruLexA , LexAop2-FLP ( TNTin group ) , or UAS>stop>TNT; LexAop2-FLP ( no LexA group ) .",
"n = 13–16 for each condition .",
"*p < 0 . 0001 compared to both controls at 80 dB , Wilcoxon rank-sum test .",
"Shown in the left panel is GFP expression of each intersectional driver .",
"In the right panel , a heat map summary shows chaining intensities across the testing time course .",
"Each row corresponds to a group of six flies .",
"Colors represent the number of flies in chain . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 009 As the multiple drivers used for aPN1 and vPN1 share no off-target expression in fruLexA+ neurons ( Figure 4A–E ) , we conclude that both fruM+ aPN1 and fruM+ vPN1 fruM neurons are required for the male-chaining response to pulse song .",
"Courtship song responses in aPN1 have been extensively studied using in vivo calcium imaging and electrophysiology ( Lai et al . , 2012; Tootoonian et al . , 2012; Vaughan et al . , 2014 ) .",
"To investigate the physiological function of downstream vPN1 neurons in auditory perception , we performed in vivo calcium imaging in vPN1 cell bodies during song presentation by expressing UAS-GCaMP6m under the control of R72E10-GAL4 ( Figure 5A–C ) ( Chen et al . , 2013 ) . 10 . 7554/eLife . 08477 . 010Figure 5 . Calcium responses of vPN1 neurons to courtship song .",
"( A ) Diagram of imaging setup in which a speaker was located 20-cm away from the recorded male fly .",
"( B ) Diagram of vPN1 neurons labeled with R72E10-GAL4 driving expression of GCaMP6 .",
"Cell bodies are circled .",
"( C ) Calcium imaging of R72E10-GAL4 driven GCaMP6 expression in vPN1 cell bodies .",
"Heat map of a sample frame shows ∆F/F changes in two vPN1 cell bodies .",
"Scale bar , 10 μm .",
"( D ) Calcium responses of vPN1 neurons to a train of 40 pulses at different IPIs , sine song , and white noise at 80 dB .",
"Black lines represent means .",
"Gray envelopes indicate SEM .",
"Song stimulus durations are indicated as red bars below .",
"( E ) Peak ∆F/F changes of vPN1 neurons stimulated with pulse song ( 35-ms IPI , 40 pulses ) , sine song ( 140 Hz , 1 . 4 s ) , and white noise ( 1 . 4 s ) at 80 dB .",
"*p < 0 . 01 , Wilcoxon rank-sum test .",
"n = 10 for all the groups .",
"( F ) Peak ∆F/F of vPN1 neurons in response to pulse song ( 35-ms IPI , 40 pulses ) at different sound intensities .",
"n = 14 trials for each sound level .",
"( G ) Normalized calcium traces of vPN1 neurons at different IPIs .",
"Each ∆F/F was normalized by the maximum ∆F/F .",
"( H ) Peak ∆F/F of vPN1 neurons in response to different IPIs at 80 dB ( 40 pulses ) .",
"n = 10 for all groups .",
"*p < 0 . 01 , Wilcoxon signed-rank test . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 01010 . 7554/eLife . 08477 . 012Figure 5—figure supplement 1 . Raster plots of vPN1 neurons in individual flies .",
"( A ) Raster plots of ∆F/F during presentation of different song stimuli .",
"Each row represents calcium responses from one male fly .",
"( B ) Raster plots of peak ∆F/F during presentation of different song stimuli .",
"Each row represents calcium responses from one male fly while each column represents a different IPI .",
"All the responses from an individual fly are normalized by the maximum response from that fly . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 012 When auditory stimuli were presented at 80 dB , vPN1 neurons responded to both pulse song and sine song , but not to white noise stimuli ( Figure 5D , E ) .",
"We then looked at the response of vPN1 neurons to pulse songs of different intensities .",
"Strikingly , the threshold of vPN1 activation matched the behavioral threshold for song-induced chaining ( EC50 = 68 . 2 dB for vPN1 responses v . s . EC50 = 70 . 6 dB for chaining responses , half-maximal sigmoid fit; Figure 5F and Figure 3B ) .",
"To further understand how vPN1 neurons process pulse song , we examined their tuning with respect to a variety of IPIs from 15 ms to 95 ms at 80 dB ( Figure 5D , G ) .",
"We found that vPN1 neurons respond strongly to pulse trains with IPIs of 35 ms and above , with significant attenuation at IPIs below 25 ms ( p < 0 . 01 when compared to responses at 35-ms IPI , Wilcoxon signed-rank test; Figure 5H and Figure 5—figure supplement 1 ) .",
"This signal displays low-pass response properties for IPI with a shoulder around 35-ms and is different from the aPN1 response that responds as a function of pulse rate ( Vaughan et al . , 2014 ) .",
"The vPN1 neurons extend projections into the LPC , an area surrounding the mushroom body peduncle that is enlarged in males and densely innervated by both fruM+ and dsx+ neurons ( Figure 1B ) ( Cachero et al . , 2010; Rideout et al . , 2010; Robinett et al . , 2010; Yu et al . , 2010 ) .",
"One of the cell types innervating this region is the male-specific fruM+/dsx+ P1 cell type ( a subset of pC1 ) , which controls the initiation of male courtship ( Kimura et al . , 2008; Kohatsu et al . , 2011; Pan et al . , 2011; von Philipsborn et al . , 2011 ) .",
"Some pC1 neurons are present in both sexes , although their projections and cell number are highly sexually dimorphic ( Rideout et al . , 2010; Robinett et al . , 2010; Zhou et al . , 2014 ) .",
"Female pC1 neurons are sensitive to courtship song and the male-specific pheromone cVA , suggesting a role for female pC1 neurons in integrating multiple courtship-related sensory signals ( Zhou et al . , 2014 ) .",
"We asked whether male pC1 neurons function downstream of vPN1 neurons to process song signals .",
"To evaluate the potential connections between pC1 neurons and vPN1 neurons , we first labeled pC1 neurons via the intersection of R71G01-LexA::p65 with dsxGAL4 ( Pan et al . , 2012 ) and then registered male pC1 neurons and vPN1 neurons onto a standard brain ( Figure 6A ) .",
"The neurites of pC1 and vPN1 neurons extensively overlap in the LPC , including the lateral crescent , the lateral junction , the arch and the ring region ( Yu et al . , 2010 ) , suggesting potential synaptic contacts between these two cell types ( Figure 6B–D ) .",
"To examine the roles of pC1 neurons in song perception , R71G01-LexA::p65 ∩ dsxGAL4 was used to express TNT in dsx+ pC1 neurons .",
"Compared to controls , TNT-mediated inactivation of pC1 neurons almost completely abolished song-induced chaining behavior ( Figure 6E ) . 10 . 7554/eLife . 08477 . 013Figure 6 . Anatomical , behavioral , and physiological characterization of dsx+ pC1 neurons in auditory sensation .",
"( A–D )",
"Co-registration of vPN1 ( green ) and pC1 ( magenta ) neurons onto the standard brain .",
"Genotype for labeling vPN1 neurons: LexAop2-FLP/+; fruLexA , UAS>stop>myr::GFP/R72E10-GAL4 .",
"Genotype for labeling vPN1 neurons: R71G01-LexA/UAS>stop>myr::GFP; dsxGAL4 , LexAop2-FLP/+ .",
"vPN1 processes ( B ) and pC1 ( C ) processes overlap in the region of lateral protocerebral complex ( LPC ) .",
"( D ) Merge of ( B ) and ( C ) .",
"Scale bars , ( A ) 100 μm and ( D ) 20 μm .",
"( E ) Song-induced chaining response was impaired by inactivation of pC1 neurons .",
"Genotypes: UAS>stop>TNTin/71G01-LexA; dsxGAL4 , LexAop2-FLP/+ ( TNTin ) , UAS>stop>TNT/+; dsxGAL4 , LexAop2-FLP/+ ( no LexA ) , UAS>stop>TNT/71G01-LexA; dsxGAL4 , LexAop2-FLP/+ ( TNT ) .",
"n = 16 for all the conditions .",
"*p < 0 . 0001 when comparing TNT group to both controls at 80 dB , Wilcoxon rank-sum test .",
"Bottom , heat map analysis of chaining events for individual groups of flies .",
"( F ) Diagram of pC1 neurons labeled with dsxGAL4 driving expression of GCaMP6 .",
"Neurites innervating the LPC are circled .",
"( G ) Calcium responses of pC1 neurons responding to different IPIs , sine song , and white noise at 80 dB .",
"Pulse song stimuli consist of a train of 40 pulses .",
"Black lines indicate mean values , while gray areas indicate SEM .",
"Song stimulus durations are indicated as red bars .",
"( H ) Peak ∆F/F values of pC1 neurons stimulated with pulse song ( 35-ms IPI , 40 pulses ) , sine song ( 140 Hz , 1 . 4 s ) , and white noise ( 1 . 4 s ) at 80 dB .",
"*p < 0 . 01 , Wilcoxon rank-sum test .",
"No significance was observed for sine vs white noise .",
"n = 12 for all the groups .",
"( I ) Peak ∆F/F values of pC1 neurons stimulated with pulse song ( 35-ms IPI , 40 pulses ) from 60 dB to 90 dB .",
"pC1 neurons are only sensitive to pulse song stimuli above 80 dB .",
"n = 9 trials for each sound level .",
"( J ) Normalized calcium traces of pC1 neurons at different IPIs .",
"Each ∆F/F was normalized by the maximum ∆F/F .",
"( K ) Peak ∆F/F of pC1 neurons stimulated with different IPIs at 80 dB ( 40 pulses ) .",
"n = 12 for all groups .",
"*p < 0 . 01 , Wilcoxon signed-rank test .",
"( L ) Comparison between the calcium response ( peak ∆F/F ) of vPN1 neurons and chaining responses to different IPIs shown in Figure 3E ( R = 0 . 67; p < 0 . 017 , permutation test ) .",
"Both calcium responses and chaining responses are normalized to their respective maximum responses .",
"( M ) Comparison between the calcium response ( peak ∆F/F ) of pC1 neurons and chaining responses to different IPIs shown in Figure 3E ( R = 0 . 89; p < 0 . 001 , permutation test ) .",
"Both calcium responses and chaining responses are normalized to their respective maximum responses .",
"( N ) Comparison between normalized vPN1 responses and pC1 responses .",
"Each colored square represents a different IPI indicated by the heat map .",
"Error bars represent SEM in all panels . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 01310 . 7554/eLife . 08477 . 014Figure 6—figure supplement 1 . Raster plots of pC1 neurons in individual flies .",
"( A ) Raster plots of ∆F/F during presentation of different song stimuli .",
"Each row represents calcium responses from one male fly .",
"( B ) Raster plots of peak ∆F/F during presentation of different song stimuli .",
"Each row represents calcium responses from one male fly while each column represents a different IPI .",
"All the responses from an individual fly are normalized by the maximum response from that fly . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 01410 . 7554/eLife . 08477 . 015Figure 6—figure supplement 2 . Transfer function between vPN1 and pC1 responses to IPIs of the pulse song .",
"( A ) Normalized peak ∆F/Fs for the calcium responses of vPN1 ( green ) and pC1 ( yellow ) neurons to different IPIs .",
"The tuning curve of aPN1 neurons ( gray ) is reported previously ( Vaughan et al . , 2014 ) and superimposed for comparison .",
"*p < 0 . 05 between vPN1 and pC1 responses , One-sided Wilcoxon rank-sum test .",
"( B ) A transfer function was calculated by dividing pC1 responses by vPN1 responses .",
"This transfer function resembles a band-pass filter .",
"The SEM of the transfer function was estimated using standard error propagation . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 01510 . 7554/eLife . 08477 . 016Figure 6—figure supplement 3 . Comparison of synthetic courtship song and natural courtship song with a particle-velocity microphone .",
"( A ) Synthetic pulse song ( top ) and sine song ( bottom ) measured by a particle-velocity microphone .",
"Pulse song train at 80 dB sound pressure level corresponds to particle velocity of ∼2 mm/s , while sine song at 80 dB corresponds to ∼0 . 35 mm/s .",
"( B ) Natural courtship song recorded by a particle-velocity microphone located ∼2 mm above a pair of male and female Canton S flies .",
"Pulse songs from a courting male can reach up to ∼7 . 5 mm/s , and sine songs can reach up to ∼1 mm/s in this setup . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 016 To ask whether male pC1 neurons also respond to song input , we expressed GCaMP6m in dsxGAL4 neurons and recorded the activity of the pC1 neurites in the LPC while presenting song stimuli ( Figure 6F ) .",
"pC1 neurons are only sensitive to pulse song stimuli at 80 dB and above ( EC50 = 79 . 7 dB , sigmoid fit; Figure 6I ) , a higher activation threshold than that observed for vPN1 ( EC50 = 68 . 2 dB; Figure 5F ) .",
"This could be due to intrinsic properties of pC1 neurons , which might require multi-sensory input to become fully activated .",
"As expected , pulse-song stimuli evoked calcium transients in pC1 neurites , while sine song and white noise induced little response ( Figure 6H ) , consistent with the report that sine song and white noise are not capable of inducing chaining behavior ( Eberl et al . , 1997 ) .",
"We next examined the IPI tuning of male pC1 neurons .",
"Unlike the low-pass response of vPN1 , the pC1 neurite response showed a strong response to pulse song with IPIs of 35–65 ms . This response is significantly reduced at both short ( 15–25 ms ) and long ( 85–95 ms ) IPIs ( p < 0 . 01 when compared to responses at 35-ms IPI , Wilcoxon signed-rank test; Figure 6K , Figure 6—figure supplement 1 ) .",
"This response reflects a band-pass sensitivity to pulse song , which is not seen in either aPN1 or vPN1 , and qualitatively matches the IPI sensitivity of the behavioral response in male and female flies ( Bennet-Clark , 1969; Yoon et al . , 2013; Vaughan et al . , 2014 ) .",
"The auditory pathway leading through aPN1 , vPN1 , and pC1 shows a possible transformation of song representation .",
"In particular , while aPN1 responses integrate pulse rate at IPIs longer than 25 ms ( Vaughan et al . , 2014 ) , we found that vPN1 shows a low-pass response and pC1 shows a band-pass response to IPI ( Figure 6—figure supplement 2A ) .",
"We compared vPN1 and pC1 responses and calculated a direct transfer function for them ( Figure 6N and Figure 6—figure supplement 2 ) .",
"This comparison shows significant attenuation of IPIs below ( 25 ms ) and above ( 75–95 ms ) the 35-ms IPI , indicating a possible band-pass filter linking these two responses ( Wilcoxon rank-sum test , one-sided ) .",
"We next examined the relationship between the GCaMP response and song-induced chaining behavior more closely .",
"For vPN1 , we observed only a moderate correlation between this calcium response and the IPI sensitivity of song-induced chaining ( r = 0 . 67; p < 0 . 017 , permutation test , Figure 6L ) .",
"In contrast , the pC1 response closely matches the IPI sensitivity of song-induced chaining ( r = 0 . 89; p < 0 . 001 , permutation test , Figure 6M ) .",
"The correlation with the behavioral response is higher for pC1 than vPN1 ( p < 0 . 037 , Meng's z-test ) .",
"These results suggest that pC1 activity may integrate information from vPN1 and other neurons to determine the level of courtship-chaining behavior .",
"As caveats , however , we note that the slow decay kinetics of GCaMP6m ( Chen et al . , 2013 ) may account for some of the observed low-pass properties observed in our vPN1 or pC1 recordings .",
"In addition , some of the differences between the tuning of vPN1 and pC1 may arise from distinct calcium dynamics in the recording sites for vPN1 ( soma ) and pC1 ( neurites ) , or from cell-type specific differences in calcium dynamics .",
"Indeed , although our results suggest that song representations may be serially transformed in the ascending auditory pathway , a circuit-level understanding of how local and projection neurons in this pathway shape this response remains to be discovered .",
"To investigate whether neurogenetic activation of auditory neurons mimics the effects of courtship song presentation in inducing male-chaining behavior , we expressed the red light-sensitive channelrhodopsin CsChrimson in auditory neurons with intersectional drivers and assessed the effects of red light stimulation on groups of male flies ( Klapoetke et al . , 2014 ) .",
"CsChrimson-mediated activation of aPN1 neurons did not induce male-chaining behavior when we tested two independent GAL4 drivers ( R22B11 and R49F09 ) ( Figure 7A , D ) .",
"This is consistent with the report that dTrpA1-mediated hyperactivation of aPN1 neurons failed to restore receptivity of females toward wingless males ( Vaughan et al . , 2014 ) .",
"In contrast , CsChrimson-mediated activation of putative third-order vPN1 neurons induced robust chaining behavior ( Figure 7B , D and Video 1 ) , which was not observed in the absence of red light stimulation or in control flies lacking GAL4 ( Figure 7D ) .",
"Similarly , activation of pC1 neurons induced robust male-chaining behavior ( Figure 7C , E and Video 2 ) . 10 . 7554/eLife . 08477 . 017Figure 7 . Optogenetic activation of auditory neurons .",
"( A–C )",
"Optogenetic activation of vPN1 neurons ( B ) or pC1 neurons ( C ) induced male chaining , as evidenced by males courting each other on food ( green arrow head ) , while activation of aPN1 neurons did not ( A ) .",
"CsChrimson activation was achieved with constant 655-nm light ( 0 . 06 mW/mm2 ) .",
"R22B11-GAL4 ∩ fruLexA , R72E10-GAL4 ∩ fruLexA , or R71G01-LexA ∩ dsxGAL4 was used to drive CsChrimson expression in aPN1 ( A ) , vPN1 ( B ) , or pC1 ( C ) .",
"( D ) Male-chaining behavior was induced by CsChrimson-mediated activation of vPN1 neurons but not aPN1 neurons .",
"*p < 0 . 0001 ( Student's t-test ) .",
"n = 14–18 for all the genotypes .",
"( E ) Male-chaining behavior was induced by CsChrimson-mediated activation of pC1 neurons .",
"*p < 0 . 0001 ( Student's t-test ) .",
"n = 16–18 for all the genotypes .",
"( F , G )",
"Male-chaining behavior induced by CsChrimson-mediated activation of either vPN1 or pC1 neurons when stimulated with constant red light at 0 . 006 , 0 . 018 , 0 . 03 , 0 . 06 mW/mm2 ( F ) or stimulated with 5-ms light pulses at 10 , 25 , 50 , and 100 Hz ( G ) .",
"vPN1 driver 1 is R72E10-GAL4 ∩ fruLexA; vPN1 driver 2 is R46F09-GAL4 ∩ fruLexA , pC1 driver is R71G01-LexA ∩ dsxGAL4 .",
"n = 16–28 for all the genotypes . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 01710 . 7554/eLife . 08477 . 018Video 1 . CsChrimson activation of vPN1 neurons induced chaining behavior in LexAop2-FLP/+; UAS>dsFRT>CsChrimson-mVenus , fruLexA/72E10-GAL4 male flies . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 01810 . 7554/eLife . 08477 . 019Video 2 . CsChrimson activation of pC1 neurons induced chaining behavior in LexAop2-FLP/71G01-LexA; UAS>dsFRT>CsChrimson-mVenus/dsxGAL4 male flies . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 019 To probe the sensitivity of the chaining response to red light stimulation , we examined chaining behavior when activating either vPN1 or pC1 with various intensities or frequencies of light ( Figure 7F , G ) .",
"While activation of vPN1 induced chaining in an intensity-dependent or frequency-dependent manner , activation of pC1 generated a saturating response in which even the lowest intensity ( 0 . 006 mW/mm2 ) or the lowest frequency ( 10 Hz ) was able to induce robust chaining through >90% of the testing period .",
"Although it is not feasible to control for the expression strength of the aPN1 , vPN1 , and pC1 drivers , we suspect that the different dynamics of chaining behavior induced by activating aPN1 , vPN1 , or pC1 neurons may reflect the intrinsic properties of the cell types and the manner in which courtship song is encoded in these neurons .",
"In particular , we note that the lack of response for aPN1 is consistent with the observation that the stimulus that best activates aPN1 ( 25 ms IPI ) does not induce a strong behavioral response from wild-type flies ( Vaughan et al . , 2014 ) .",
"As song information flows from peripheral to central brain , the higher-order neurons may represent the socially relevant features of courtship in a simpler manner than in lower neurons , and this may account for the gradient of behavior phenotype we observed when we activate aPN1 , vPN1 , or pC1 , respectively .",
"This ( still theoretical ) architecture describes a possible transformation of song representations from a temporal code in aPN1 to a rate code in vPN1—which is ultimately coupled with other sensory signals to encode overall behavioral arousal in PC1 ( Figure 8F and Video 3 ) . 10 . 7554/eLife . 08477 . 020Figure 8 . Directionality and functional connectivity of the auditory pathway .",
"( A–C )",
"Labeling of dendrites and axons by expression of the dendritic marker DenMark ( magenta ) and axonal marker Syt::GFP ( green ) in ( A ) vPN1 neurons ( labeled with split GAL4 driver R72E10-GAL4AD ∩ VT009665-GAL4DBD ) and ( B ) pC1 neurons ( labeled with split GAL4 driver R71G01-GAL4AD ∩ R15A01-GAL4DBD ) .",
"( C ) Co-registration of vPN1 axons ( green ) and pC1 dendrites ( magenta ) onto a standard brain .",
"( D ) Activation of aPN1 neurons induced calcium responses in vPN1 neurons .",
"( D1 )",
"CsChrimson was expressed in aPN1 neurons with 59C10-LexA , while GCaMP6 was expressed in vPN1 neurons with R72E10-GAL4 .",
"Dashed circle indicates the location of the recording site .",
"( D2 ) aPN1 CsChrimson-mediated activation induced calcium responses in vPN1 neurons .",
"Black line indicates mean while gray envelope indicates SEM .",
"n = 46 from 6 flies .",
"( D3 )",
"This effect is suppressed by the acetylcholine receptor antagonist mecamylamine ( **p < 0 . 001 , Student's t-test ) , and partially restored by washing out the antagonist ( *p < 0 . 01 , Student's t-test ) .",
"Dots with the same color represent experiments performed on the same individual .",
"Black lines indicate mean values .",
"n = 9–14 ( depending on the drug condition ) from 3 flies .",
"( E ) Activation of vPN1 neurons induced pC1 calcium responses .",
"( E1 )",
"CsChrimson was expressed in vPN1 neurons with a vPN1 split-GAL4 driver while GCaMP6 was expressed in pC1 neurons with dsxLexA .",
"Dashed circle indicates the location of the recording site .",
"( E2 ) pC1 neurons respond to CsChrimson activation of vPN1 neurons .",
"Black line indicates mean while gray envelope indicates SEM .",
"n = 13 from 3 flies .",
"( E3 )",
"Mecamylamine causes a mild reduction in the pC1 responses ( **p < 0 . 001 , Student's t-test ) .",
"Black lines indicate mean values .",
"n = 4–9 from 2 flies .",
"( F ) Left panel shows co-registration of aPN1 ( magenta ) , vPN1 ( green ) , and pC1 ( yellow ) neurons onto a standard brain .",
"The anatomical overlap between them suggests a potentially interconnected circuit mediating courtship song detection in the male brain .",
"We propose that courtship song is relayed through aPN1 and vPN1 neurons to pC1 neurons , and that the pC1 neurons integrate song signals with other sensory cues to initiate courtship . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 02010 . 7554/eLife . 08477 . 021Figure 8—figure supplement 1 . Characterization of synaptic connections in the aPN1-vPN1-pC1 pathway with GFP reconstitution across synaptic partners ( GRASP ) method .",
"( A–C )",
"GFP Expression ( green ) in aPN1 ( A ) , vPN1 ( B ) , and pC1 ( C ) driven by 59C10-LexA , 72E10-GAL4 and 71G01-LexA and counter-stained by nc82 antibody ( blue ) .",
"Arrow in ( B ) indicates the cell bodies of vPN1 neurons .",
"Scale bars , 100 μm .",
"( D ) Pattern of reconstituted GFP signal ( green , indicated by arrow in D1 ) between aPN1 and vPN1 neurons in the WED region .",
"Neuropil is labeled by nc82 antibody staining ( magenta ) .",
"GRASP signal was not observed in control flies ( D2 ) .",
"Scale bars , 50 μm .",
"( E ) GRASP signal was barely detected between vPN1 neurons and pC1 neurons ( E1 ) .",
"( E2 ) is the control for ( E1 ) .",
"Scale bars , 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 02110 . 7554/eLife . 08477 . 022Video 3 . 3D segmentation and co-registration of aPN1 , vPN1 , and pC1 neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 08477 . 022 It has been reported that the dendrites of aPN1 innervate the AMMC region while the aPN1 axons project to the WED region ( Vaughan et al . , 2014 ) , indicating that the song signal is transmitted from AMMC to WED .",
"To further investigate the directionality of information flow in the aPN1-vPN1-pC1 pathway , we have used DenMark and Syt::GFP for post- and pre-synaptic labeling of vPN1 and pC1 neurons , respectively .",
"The dendrites of vPN1 target the WED region , and vPN1 axonal termini ramify extensively in the LPC region ( Figure 8A ) .",
"Both dendrites and axons of pC1 are observed within the LPC region ( Figure 8B ) , and vPN1 axons overlap with pC1 dendrites when registered in a standard brain ( Figure 8C ) .",
"These results suggest that vPN1 neurons may convey song information from the WED to the LPC region .",
"To assess if the hypothesized pathway is indeed functional , we activated aPN1 or vPN1 with CsChrimson while recording GCaMP6-indicated calcium responses in vPN1 or pC1 , respectively .",
"CsChrimson was expressed in aPN1 by R59C10-LexA::p65 and GCaMP6m was expressed in vPN1 by R72E10-GAL4 .",
"Pre-synaptic activation of aPN1 elicited calcium responses in vPN1 ( Figure 8D ) , suggesting a functional connection between aPN1 and vPN1 .",
"Moreover , to allow simultaneous vPN1 activation/pC1 recording , we generated a dsxLexA knock-in for driving GCaMP6m expression in dsx+ neurons while using R72E10-GAL4AD ∩ VT9665-GAL4DBD for CsChrimson-mediated activation of vPN1 neurons .",
"As anticipated , activation of vPN1 elicited robust calcium responses in pC1 neurites ( Figure 8E ) , supporting the idea that vPN1 neurons form functional synapses with pC1 neurons .",
"In both cases , these effects were reduced by the administration of the acetylcholine receptor antagonist mecamylamine ( Figure 8D , E ) .",
"These data further support the existence of an aPN1-vPN1-pC1 functional pathway that mediates song detection in males ( Figure 8F ) .",
"To further investigate the connectivity in the aPN1-vPN1-pC1 pathway , we first performed the GFP reconstitution across synaptic partners ( GRASP ) analysis to visualize the connections between aPN1 , vPN1 , and pC1 neurons .",
"In this approach , two different populations of neurons were driven by either GAL4 or LexA ( Figure 8—figure supplement 1A–C ) to separately express each half of the GFP molecule ( spGFP11 and spGFP1-10 ) .",
"GFP will be reconstituted in the region where these two groups of cells come into close proximity and form synapses ( Feinberg et al . , 2008; Gordon and Scott , 2009 ) .",
"By expressing spGFP11 in aPN1 neurons and spGFP1-10 in vPN1 neurons , we observed significant GRASP signal in the WED region ( Figure 8—figure supplement 1D ) , suggesting the connections between aPN1 and vPN1 .",
"However , no GRASP signal was detected when we drove spGFP1-10 expression in vPN1 neurons with 72E10-GAL4 and spGFP11 expression in pC1 neurons with 71G01-LexA ( Figure 8—figure supplement 1E ) .",
"One possibility to explain this is that 71G01-LexA only label a subset of pC1 neurons , and given that pC1 neurons are likely to be heterogeneous ( Zhou et al . , 2014 ) , the 71G01-LexA-labeled pC1 neurons may not include the pC1 auditory neurons that synapse with vPN1 neurons .",
"Alternatively , it might be that the neurites of vPN1 and pC1 in the LPC region is too diffuse to allow reliable detection of the GRASP signal .",
"The mono-synaptic nature of this circuit is still awaiting a validation from electron microscopy or eletrophysiology methods ."
],
[
"fru and dsx are two key transcription factors with restricted expression patterns that specify the potential for sexual behaviors in Drosophila ( Manoli et al . , 2013; Pavlou and Goodwin , 2013; Yamamoto and Koganezawa , 2013 ) .",
"fruM expression in primary auditory , tactile , gustatory , and visual neurons as well as the central brain may suggest that there are multiple fruM-labeled pathways conveying and integrating diverse sensory signals related to courtship , and an appealing hypothesis is that fruM labels interconnected neurons in a circuit that is dedicated to courtship ( Manoli et al . , 2005; Stockinger et al . , 2005 ) .",
"For example , the male-specific pheromone cVA is processed by a four-neuron pathway extending from sensory neurons through to the ventral nerve cord ( Ruta et al . , 2010 ) .",
"This circuit appears to function as an olfactory labeled line , in that neurons in this circuit are functionally connected and selectively responsive to cVA .",
"We focused our efforts here on elucidating the auditory pathway underlying courtship song perception .",
"We have demonstrated that the aPN1-vPN1-pC1 pathway is a labeled line for courtship hearing , by fulfilling four criteria: ( 1 ) these neurons are functionally connected; ( 2 ) these neurons respond preferentially to courtship song; ( 3 ) these neurons are necessary for the behavioral response to courtship song in male flies , and ( 4 ) activation of this labeled line provides a fictive stimulus , observable by the chaining response elicited upon CsChrimson activation .",
"Strikingly , this labeled line appears to be specified by the expression of fruM or dsx .",
"The auditory labeled line for courtship hearing begins with fruM-expressing JONs and second-order auditory neurons aPN1/aLN ( al ) in the AMMC ( Manoli et al . , 2005; Stockinger et al . , 2005; Vaughan et al . , 2014 ) .",
"Silencing either fruM JONs or fruM aPN1 neurons reduced male song-induced responses ( Figure",
"4 ) ( Vaughan et al . , 2014 ) .",
"In addition , we have also demonstrated the connectivity , response patterns , necessity , and sufficiency of fruM vPN1 and pC1 neurons in this pathway , thus , delineating a labeled line of fruM neurons leading directly from sensory neuron to multimodal integration .",
"The inclusion of vPN1 in this pathway is supported by three lines of evidence .",
"First , vPN1 projections extensively overlap with projections extended by aPN1 neurons within WED , and we observe functional connectivity between aPN1 and vPN1 .",
"Second , silencing vPN1 reduced pulse song-induced chaining in male flies , while optogenetic activation of vPN1 neurons mimicked a song signal to induce male chaining .",
"Third , GCaMP recordings reveal that vPN1 responds strongly to both pulse song and sine song .",
"We therefore conclude that fruM+ vPN1 neurons are the third-order neurons mediating courtship hearing .",
"vPN1 may provide its output via innervation of the LPC , a region receiving multimodal input that is likely to be a site for multi-sensory integration ( Yu et al . , 2010 ) .",
"This area is heavily innervated by dsx+ pC1 neurons , which include most of the male-specific fruM+ P1 neurons ( Kohatsu et al . , 2011; Zhou et al . , 2014 ) .",
"While the broader pC1 population is important for both male courtship and female receptivity , the P1 neurons play a critical role in the initiation of male courtship and respond to both male and female pheromones ( Kohatsu et al . , 2011; von Philipsborn et al . , 2011; Pan et al . , 2012; Zhou et al . , 2014 ) .",
"These neurons appear to be the downstream targets of vPN1 , based on three lines of evidence .",
"First , the arborizations of pC1 neurons match very closely with the projection of vPN1 neurons in the LPC , and optogenetic activation of vPN1 generates robust activity in pC1 .",
"Second , pC1 neurons show calcium responses to pulse song stimuli , with IPI tuning that matches that of the behavioral response .",
"Third , silencing pC1 neurons in male flies almost completely abolishes song-induced chaining , while activation induces robust chaining in the absence of song .",
"We therefore conclude that vPN1 may carry song stimuli to activate pC1 , where these stimuli are integrated with other sensory modalities such as pheromonal olfactory and gustatory cues to modulate the courtship level in males ( Figure 8F ) .",
"Taken together , the neural circuit we identified suggests that song information flows via a labeled line of fruM neurons from the antenna to AMMC , to WED , and then to LPC , providing a functional explanation of how pulse song induces male courtship behavior .",
"IPI is a key parameter of courtship song that exhibits great variation across Drosophila species ( Cowling and Burnet , 1981 ) .",
"D . melanogaster not only produces song with a specific IPI ( Shorey , 1962; Bennet-Clark and Ewing , 1967; von Philipsborn et al . , 2011 ) , but also behaviorally recognizes song with that conspecific IPI in both males and females ( Bennet-Clark , 1969; Yoon et al . , 2013 ) .",
"We have also shown that song-induced male-chaining behavior is most responsive to a 35-ms IPI , although longer IPIs ( 35–65 ms ) are still able to induce robust chaining behavior .",
"While Drosophila has behavioral preferences toward the conspecific IPI , it has not been clear how IPIs are represented in the nervous system or how the fly discriminates specific IPIs .",
"Our results suggest there is a significant change in pulse song representation across the ascending aPN1-vPN1-pC1 pathway .",
"For aPN1 , the GCaMP ∆F/F responses in female flies reflect an integration of pulse rate at IPIs longer than 25 ms ( Vaughan et al . , 2014 ) .",
"In contrast , vPN1 responses observed here are low-passed and preferentially tuned to longer IPIs .",
"Interestingly , the vPN1 response saturates above ∼35-ms IPI , consistent with the saturating response observed when comparing dendritic and axonal GCaMP signals in aPN1 ( Vaughan et al . , 2014 ) .",
"Notably , however , neither the aPN1 nor vPN1 response corresponds well with the behavioral sensitivity to IPI observed in male or female flies .",
"In contrast , the IPI sensitivity of pC1 reflects a band-pass response to IPI that closely matches the behavioral sensitivity of the chaining response .",
"Indeed , the correlation between pC1 response and chaining behavior is significantly higher than the correlation observed for vPN1 .",
"Thus , while the mechanistic details remain unclear , the IPI sensitivity appropriate for species-appropriate responses is likely to be generated through a multi-stage transformation of song stimuli .",
"Sexual dimorphism at multiple levels in the Drosophila brain may give rise to sex-specific differences in sensory processing and multimodal integration .",
"The central integrators of courtship-related sensory cues in male and female flies , the pC1 neurons , are themselves sexually dimorphic in both cell number and morphology ( Kimura et al . , 2008; Kohatsu et al . , 2011; Zhou et al . , 2014 ) .",
"pC1 neurons arborize within the triangular lateral junction of the LPC in both sexes , where integration of multiple sensory modalities may occur , but they also show male-specific innervation of the LPC arch and male-specific contralateral projections ( Kimura et al . , 2008; Zhou et al . , 2014 ) .",
"For courtship hearing , pC1 neurons are stimulated by pulse song in both sexes , but are also stimulated by sine song in females ( Zhou et al . , 2014 ) .",
"This result is consistent with the behavioral observation that both males and females are responsive to pulse song , while females are also responsive to sine song ( Bennet-Clark , 1969; Schilcher , 1976a , 1976b; Eberl et al . , 1997; Shirangi et al . , 2013 ) .",
"However , the pC1 auditory response cannot be easily explained by the dimorphism of vPN1 , which responds to pulse song in males but is absent in females .",
"Moreover , the absence of vPN1 in females begs the question of how pC1 receives song information in females .",
"One explanation comes from the observation that vPN1 is a subset of the fru+ aSP-k clone ( Cachero et al . , 2010 ) .",
"aSP-k shows arborization in VLP and the LPC ring in both male and females , as well as male-specific innervation of the LPC arch that corresponds with vPN1 morphology .",
"These neurons , including non-fru+ neurons in the same lineage , may compose a parallel pathway for female hearing .",
"More generally , we observe a gradient of sexual dimorphism across the ascending pathway for both olfaction and audition .",
"In both cases , we note only limited sexual dimorphism in second-order neurons ( DA1 and aPN1 , respectively ) , but dramatic changes in third-order neurons ( aSP-f/aSP-g and vPN1 ) and integrative neurons ( pC1 ) , which show significant dimorphisms in cell number and morphology ( Datta et al . , 2008; Ruta et al . , 2010; Kohatsu et al . , 2011; Kohl et al . , 2013; Zhou et al . , 2014 ) .",
"This gradient may reflect a general rule for the flexible assembly of sexually dimorphic circuits on an evolutionary timescale .",
"Our anatomical , behavioral , and physiological analyses here have outlined the architecture of a system supporting species-specific courtship hearing , built upon genetically labeled lines expressing fruM or dsx within the fly .",
"Although it is clear that courtship song representations are systematically transformed along the aPN1-vPN1-pC1 pathway , we await a circuit and synapse-level explanation for how this occurs , as well as an understanding of how pC1 activation gives rise to distinct and appropriate behavioral outputs in each sex ."
],
[
"The fruLexA and dsxGAL4 ( Δ2 ) lines were previously described ( Mellert et al . , 2010; Pan et al . , 2011 ) .",
"CRM-GAL4s , R72E10-GAL4AD , R71G01-GAL4AD , R15A01-GAL4DBD , LexAop2-FLP ( pJFRC79 in attP40 ) , LexAop2-FLP ( pJFRC79 in attP2 ) , UAS>stop>myr::GFP ( pJFRC41 in attP40 ) , UAS>stop>myr::GFP ( pJFRC41 in su ( hw ) attP1 ) , and 20XUAS>dsFRT>CsChrimson-mVenus ( in VK00005 ) were gifts from Gerald Rubin ( Janelia Research Campus ) ( Pfeiffer et al . , 2008; Jenett et al . , 2012 ) .",
"22B11-GAL4 is previously reported ( Vaughan et al . , 2014 ) .",
"R71G01-LexA::p65 ( inserted in attP40 ) was previously described ( Pan et al . , 2012 ) .",
"UAS-GCaMP6m was a gift of Douglas Kim ( Janelia Research Campus ) .",
"fruM , VT9665-GAL4DBD , UAS>stop>TNT , and UAS>stop>TNTin were gifts from Barry Dickson ( Janelia Research Campus ) .",
"LexAop-spGFP11 and UAS-spGFP1-10 were gifts from Kristin Scott ( UC Berkeley ) .",
"UAS-DenMark , UAS-syt::GFP was previously described ( Nicolai et al . , 2010 ) .",
"5- to 7-day-old adult fly CNSs were dissected in Schneider's insect medium ( Sigma , MO , United States , S0146 ) and immediately fixed in 2% paraformaldehyde ( PFA ) in Schneider's insect medium for 55-min at room temperature ( RT ) .",
"After washing three times with PBS ( Phosphate-buffered saline ) containing 0 . 3% Triton X-100 ( PBT ) , the samples were incubated in PBT with 5% normal goat serum ( Vector Laboratories , CA , United States ) for 1 hr at RT .",
"Samples were then incubated at 4°C for 24 hr in primary antibody , washed three times in 0 . 3% PBT at RT , and then incubated at 4°C for 24 hr in secondary antibody .",
"After washing three times with 0 . 3% PBT , samples were fixed in 4% paraformaldehyde ( PFA ) for 3 hr at RT , washed again five times with 0 . 3% PBT , and mounted onto a poly-lysine-coated coverslip .",
"The coverslip was then immersed into 30% , 50% , 75% , 95% , 100% ethanol for dehydration at 5-min intervals .",
"Next , the coverslip was washed with Xylenes ( Fisher Scientific , NJ , United States ) for three times in the hood and mounted in DPX solution ( Electron Microscopy Sciences , PA , United States ) before imaging .",
"Primary antibodies were mouse anti-Bruchpilot nc82 ( Developmental Studies Hybridoma Bank , IA , United States ) used at a 1:50 dilution , FruM antibody used at 1:100 dilution , and rabbit anti-GFP used at a 1:1000 dilution ( Invitrogen ) .",
"Secondary antibodies were Alexa Fluor 546 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG ( Invitrogen , OR , United States ) used at a 1:500 dilution .",
"A standard brain was generated using CMTK software to average six male and female brains stained with nc82 ( anti-Bruchpilot ) that were of good quality ( Rohlfing and Maurer , 2003; Rohlfing , 2012 ) .",
"Confocal stacks were registered into the standard brain by linear registration and non-rigid warping based on the nc82 channel ( Jefferis et al . , 2007; Ostrovsky et al . , 2013 ) .",
"Flies were reared at 22°C and 50% humidity under a 12 hr:12-hrlight/dark cycle .",
"Male flies were collected immediately after eclosion and aged in groups of 8 for 2 days .",
"They were then anesthetized to remove their wings and allowed to recover for 5–7 days before assaying the behavior .",
"Behavioral experiments were performed at 22°C .",
"Briefly , six males were introduced into the chaining chamber by gentle aspiration without anesthesia , and then videotaped by a Stingray camera ( F080B , Allied Vision Technologies , PA , United States ) for 6-min .",
"After 1-min in the chamber , continuous pulse song trains were played back with incremental intensities at 30-s intervals .",
"Two pieces of nylon mesh were inserted at both ends of the chaining chamber to allow the delivery of acoustic stimuli from an external speaker ( HiVi D10G Woofer , Parts Express , Springboro , OH ) located 9-cm from the center of the chamber .",
"Synthetic pulse song was generated by MATLAB as a train of Gaussian-modulated sinusoidal pulses with a 220 Hz fundamental carrier frequency and a defined IPI .",
"A data acquisition device ( USB-6229 BNC , National Instruments ) controlled by a custom-written MATLAB program ( Source code 1 ) was used to relay the song signals to an A500 linear amplifier ( Willich , Germany ) that drives the speaker , triggers the videotaping , and maintains synchrony between the two .",
"To calculate the chaining index , the number of flies engaged in chaining was counted every 3 s and summed up for each 30-s block .",
"The maximum chaining index for one block is 60 if all six flies are chaining throughout the 30-s block .",
"Furthermore , a detailed analysis of chaining behavior for each group of flies was presented in a heat map to reflect the number of chaining flies every 3 s throughout the recording time .",
"R72E10-GAL4/UAS-GCaMP6m and dsxGAL4/UAS-GCaMP6m males were aged in groups of 8–12 for 5–7 days before calcium imaging .",
"Flies were gently anesthetized on ice and then inserted into a rectangular hole ( ∼1 mm × 2 mm ) and stabilized with low-melting wax as described previously ( Zhou et al . , 2014 ) .",
"The dorsal head capsule was facing up to the objective and bathed in insect saline ( 103 mM NaCl , 5 mM HEPES , 8 mM trehalose , 10 mM glucose , 26 mM NaHCO3 , 1 mM NaH2PO4 , 2 mM CaCl2 , 1 . 5 mM MgCl2 , pH = 7 . 3 ) , while the antenna protrudes downward on the other side of the plate to receive acoustic stimulation .",
"Because R72E10-GAL4 labels off-target neurons with projections intermingled with the neurites of vPN1 neurons , it is technically difficult to record calcium responses from the vPN1 neurites .",
"We therefore performed calcium imaging from the somas of vPN1 neurons , which are easily identified nearby the lateral horn .",
"The cuticle at the dorsal region of the head was gently removed with sharp forceps , and calcium signals from the somas of vPN1 neurons were recorded .",
"For imaging the neurites of male pC1 neurons , the procedure was the same as previously described ( Zhou et al . , 2014 ) .",
"At the end of experiments , most flies appeared healthy as they were still exhibiting voluntary abdomen contractions .",
"Calcium-imaging experiments were done with a 488-nm laser on a Zeiss LSM 710 confocal microscope .",
"Images with 128 × 128 pixels resolution at a frame rate of 13 Hz were acquired with a water immersion objective lens ( 40×/1 . 0 DIC VIS-IR , Zeiss ) .",
"A data acquisition device ( USB-6229 BNC , National Instruments ) was used to control a trigger interface box ( 1437-440 , Zeiss ) and an amplifier ( Marantz SR5003 ) to synchronize song stimulus onset and image acquisition .",
"An external speaker located ∼20-cm from fly antenna was used to present the song stimuli .",
"Synthetic song stimuli including pulse song ( 40 pulses ) with a defined IPI , 1 . 4-s sine song ( 140 Hz ) and 1 . 4-s white noise were generated with MATLAB software .",
"The sound intensity was measured with a sound level meter ( NO . 33–2055 , Radioshack ) .",
"We also measured the synthetic song with a particle-velocity microphone ( Microflown Technologies , Arnhem , NL ) to make sure that the song intensity ( 80 dB ) used in calcium imaging is within the range of natural Drosophila courtship song ( Figure 6—figure supplement 3 ) .",
"To reduce the habituation effects , song stimuli were shuffled randomly and presented at 1-min intervals .",
"52 high-power red LEDs ( 655 nm , LXM3-PD01 , Luxeon Rebel ) and 96 blue LEDs ( 468 nm , VAOL-S12SB4 , VCC Optoelectronics , CA , United States ) were mounted onto a heat sink and suspended above the chaining chambers to provide a source of illumination .",
"Blue LEDs provide constant background illumination to allow flies to see each other , as male chaining behavior depends on the ability to visually tracking other flies .",
"Because CsChrimson is also sensitive to blue light , we kept the blue light at a very low level ( 0 . 001 mW/mm2 ) to make sure that no chaining behavior was triggered with only blue light illumination .",
"The intensity and frequency of high-power red LEDs were controlled with a Teensy 2 . 0 microcontroller using MATLAB software .",
"Light intensity was measured by placing an optical power meter ( PM100D , Thorlabs , NJ , United States ) nearby the location of chaining chambers .",
"Fly behavior was recorded by a Stingray camera equipped with a Tokina infrared filter under 880-nm LED illumination ( SL1236 , Advanced Illumination , VT , United States ) to avoid the interference from red and blue LEDs .",
"For all the CsChrimson experiments , crosses were set up on standard fly food with 0 . 2 mM all-trans-retinal .",
"Male flies were collected immediately after eclosion and reared in groups of 8–12 on 0 . 5 mM retinal food for 5–7 days before behavioral test .",
"A group of eight males with the same genotype were gently aspirated into a culture dish ( 430165 , 35 mm × 10 mm , Corning , NY , United States ) containing fly food at the bottom .",
"Videotaping and red light stimulation were triggered simultaneously with a MATLAB interface for a total duration of 5 min .",
"Videos were scored using LifesongX software .",
"The percentage of time when at least three flies are in a courtship chain was calculated to quantify the chaining behavior induced by CsChrimson activation .",
"To genetically label cells that express dsx , we used homologous recombination ( Gong and Golic , 2003 ) to replace the entire coding sequence of dsx exon 2 ( starting after the ATG through the splice donor ) with the coding sequence for LexA::p65 ( Pfeiffer et al . , 2010 ) followed by a stop codon and a transcription stop cassette containing the SV40 poly-A sequence in tandem with the D . melanogaster α-tubulin 84B 3′ UTR ( Stockinger et al . , 2005 ) .",
"For homologous recombination , we generated donor transgenes in which these exogenous sequences were flanked by two homology arms corresponding to the dsx locus: a 2 . 8-kb 5′ homology arm as per ( Robinett et al . , 2010 ) and a 2 . 7-kb 3′ homology arm extending between genomic sequences GCAATATTGGCACTCAGCTATTATC and CACGTTCGATATTGAGTTGGGTGAA in the dsx second intron .",
"The 3′ arm was PCR-amplified from genomic DNA prepared with the DNeasy Tissue Kit ( Qiagen ) .",
"All DNA fragments were generated by PCR using AccuPrime Supermix ( Invitrogen ) and sequence-verified .",
"Using restriction endonuclease sites added to the 5′ ends of the PCR primers , these fragments were cloned in the linear order of dsx 5′ arm-LexA::p65-SV40 poly-A/α-tubulin 84B 3′ UTR-dsx 3′ arm in pBluescript-SK ( Invitrogen ) and then transferred as a unit into pP{WhiteOut2} ( gift of Jeff Sekelsky ) to make pP{WO2-dsx-LexA::p65-stop-2} .",
"pP{WO2-dsx-LexA::p65-stop-2} transgenics were made by P element-mediated germ line transformation using standard methods ( Rainbow Transgenic Flies , Inc . ) , and nine independent , homozygous viable , non-third chromosome transformant lines were isolated to serve as donors of the dsx-LexA::p65-stop-2 DNA fragment for homologous recombination ( Gong and Golic , 2003 ) .",
"Donors were crossed to a line with heat shock-inducible FLP recombinase and I-SceI endonuclease transgenes ( Gong and Golic , 2003 ) , and larvae were heat shocked for 1 hr at 37°C on days 3 and 4 of development .",
"∼7000 female F1 progeny containing the two transgenes and the donor were crossed to lexAop-rCD2::GFP ( Lai and Lee , 2006 ) males and the F2 progeny screened for candidates with a GFP expression pattern matching expression of dsxGAL4 ( 1 ) ( Robinett et al . , 2010 ) .",
"40 independent candidate flies were isolated , but only six were fertile .",
"Of these candidate lines , four produced intersexual progeny when homozygosed or when heterozygous with Df ( 3R ) dsxM+R15 ( Baker et al . , 1991 ) .",
"These four lines were tested by PCR for proper targeting of LexA::p65-stop into the endogenous dsx locus by using the dsx 5′ genomic and LexA::p65 primers , GTGTGTGAGGCTGCCTATGTACTAG and GACACGATTTCAATGACACCCTTGC , respectively , and the dsx 3′ genomic and α-tubulin 84B 3′ UTR primers , GAAAGTCGCAGTTTCCTACTGATAC and CCGTCAAGCATGCGATTGTACATAC , respectively .",
"For each candidate , the predicted 5′ and 3′ PCR products were generated , confirming proper targeting .",
"Male flies 5 to 7 days old were dissected in saline containing: 103 mM NaCl , 3 mM KCl , 5 mM TES , 8 mM trehalose dihydrate , 10 mM glucose , 26 mM NaHCO3 , 1 mM NaH2PO4 , 2 mM CaCl2 , 4 mM MgCl2 , bubbled with carbogen ( 5% CO2/95% O2 ) .",
"The brain and ventral nerve cord were taken out of the fly and laid on a poly-lysine-coated coverslip .",
"The dissection was realized using the minimum level of illumination possible to avoid spurious activation of CsChrimson .",
"Brains were then continuously perfused in the same saline at 60 ml/hr throughout the experiment .",
"Imaging was done on a two-photon scanning microscope ( PrairieTechnologies , Bruker ) .",
"Excitation wavelength was 920 nm .",
"CsChrimson was excited with 50-Hz trains of 2-ms 590-nm light pulses via a LED shining through the objective .",
"Instantaneous powers measured out of the objective ranged between 50 μW/mm2 and 700 μW/mm2 .",
"Experiments usually started with a 50 pulses train at 50 μW/mm2 .",
"If no response could be seen , the power was raised progressively until a response could be seen or the maximum power was reached .",
"For pharmacology experiments , mecamylamine ( 50 μM ) was administered through the perfusion line for times ranging from 3 to 5 min , followed by a wash period where the perfusion was drug-free again .",
"For aPN1 activation/vPN1 recording , R59C10-LexA::p65/LexAop2-CsChrimson; R72E10-GAL4/UAS-GCaMP6m flies were used and the cell soma of vPN1 neurons was recorded , as it was the only place where we could confidently identify the vPN1 neurons .",
"For vPN1 activation/pC1 recording , R72E10-GAL4AD/UAS-CsChrimson; VT9665-GAL4DBD , dsxLexA/LexAop2-GCaMP6m flies were used and the LPC projection of pC1 neurons was imaged .",
"In the cases where cell somas were imaged ( vPN1 imaging ) , fluorescence videos were segmented based on average intensity using k-means clustering , following which individual cell bodies were labeled as different region of interests .",
"In the cases where LPC projection was imaged , fluorescence videos were segmented by k-means ( with 3 clusters ) on the individual pixel traces of every run .",
"In both cases , ROIs were then cleaned up by an erosion/dilation step .",
"ΔF/F were then calculated for each run with F being the average signal before the stimulation .",
"For further analysis , only the most responding ROI was kept .",
"Statistical analysis was performed with MATLAB software .",
"For the song-induced chaining assay , Wilcoxon rank-sum test was used to detect significant differences between TNT and control groups at 80 dB song playback .",
"For GCaMP data , ROI selection and peak ΔF/F calculation were done using custom-written code in MATLAB ( Source code 2 ) .",
"Calcium traces were smoothed with a cubic Savitzky-Golay filter .",
"Peak ΔF/F was calculated from ΔF/F = ( Ft − Fb ) /Fb , where Ft is the maximum value in the 5-s window following stimulus onset and Fb is the averaged value in the 1-s window before the stimulus onset .",
"Wilcoxon signed-rank test was used to compare vPN1/pC1 responses at 35-ms IPI to those at shorter or longer IPIs ( Figures 5H , 6K ) .",
"To normalize responses , each fly's response across IPIs was divided by its maximum response; these responses were subsequently averaged to generate the values presented in Figure 6L–N .",
"To assess the correlation between calcium responses and chaining behavior with respect to IPI tuning , we calculated the Pearson correlation coefficient , r , between GCaMP and behavioral responses normalized by the maximum response value .",
"The significance of this correlation was established by calculating a bootstrap distribution of correlations between samples with randomly permuted IPIs .",
"The vPN1/behavior correlation was compared to the pC1/behavior correlation via Meng's z test ( Meng et al . , 1992 ) .",
"The significance of the tuning curve shown in Figure 6—figure supplement 2A was calculated using a one-sided Wilcoxon rank-sum test ."
]
] | [
"Animals use acoustic signals across a variety of social behaviors , particularly courtship .",
"In Drosophila , song is detected by antennal mechanosensory neurons and further processed by second-order aPN1/aLN ( al ) neurons .",
"However , little is known about the central pathways mediating courtship hearing .",
"In this study , we identified a male-specific pathway for courtship hearing via third-order ventrolateral protocerebrum Projection Neuron 1 ( vPN1 ) neurons and fourth-order pC1 neurons .",
"Genetic inactivation of vPN1 or pC1 disrupts song-induced male-chaining behavior .",
"Calcium imaging reveals that vPN1 responds preferentially to pulse song with long inter-pulse intervals ( IPIs ) , while pC1 responses to pulse song closely match the behavioral chaining responses at different IPIs .",
"Moreover , genetic activation of either vPN1 or pC1 induced courtship chaining , mimicking the behavioral response to song .",
"These results outline the aPN1-vPN1-pC1 pathway as a labeled line for the processing and transformation of courtship song in males ."
] | [
"The seemingly simple fruit fly engages in an intricate courtship ritual before it mates .",
"Male flies use their wings to ‘sing’ a complex song that makes females more willing to mate .",
"The song also encourages nearby males to start courting , and these males may then intervene to compete for the female .",
"Each species of fruit fly has its own song , and it is important for both males and females to detect the right song .",
"The sounds of the courtship song are detected by vibration-sensitive neurons on the flies' antennae .",
"These neurons send signals to the fly's brain .",
"But little is known about how this information is then processed , or how information about the song can be integrated with other courtship cues .",
"Zhou et al . have now identified a pathway of neurons in male flies that is responsible for hearing the courtship song .",
"This pathway stretches from the antennae to neurons deep within the brain .",
"These neural pathways are different in males and females , suggesting that the two sexes use different circuits of neurons for hearing courtship songs .",
"Zhou et al . then used genetic techniques to show that males need every neuron in this pathway to hear courtship songs .",
"Further experiments revealed that stimulating the ‘deep layer’ neurons caused male flies to respond as if they are hearing the courtship song .",
"These neurons are likely to integrate the song with information from other senses and may encode a general signal for arousal .",
"These findings now pave the way to deepen our understanding of how information from different senses—for example , courtship songs , visual cues , and pheromones—can be integrated to drive specific behaviors .",
"The next challenge is to explore how species-specific songs are detected and recognized , a goal that has yet to be achieved in any species ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"neuroscience"
] | Time- and polarity-dependent proteomic changes associated with homeostatic scaling at central synapses | elife-33322-v1 | [
[
"In biological systems , many variables like pH , ion concentration and temperature are under homeostatic control .",
"In the brain , changes in synaptic efficacy underlie at least some forms of information storage , potentially leading to runaway dynamics of synaptic weights and activity .",
"Brain synapses also exhibit homeostasis .",
"Synaptic scaling , a form of homeostatic plasticity , allows neurons to adjust globally the strengths of their synapses up or down to stabilize specific neuronal functions in response to an offset from a setpoint ( Turrigiano et al . , 1998; Davis and Goodman , 1998; Burrone et al . , 2002; Turrigiano , 2012 ) .",
"Synaptic scaling has been observed and studied at central nervous system synapses in the hippocampus and cortex as well as the neuromuscular junction ( see Davis , 2013 and Turrigiano , 2008 for review ) .",
"The cascade of mechanisms that underlie the homeostatic response are not well understood .",
"Mechanistically speaking , there must exist molecules and signaling events that detect the offset from the activity set-point and implement cellular responses to maintain homeostasis .",
"Several studies have suggested that activity-dependent changes in intracellular Ca2+ may serve as an early sensor for the offset from an activity threshold ( Ibata et al . , 2008; Thiagarajan et al . , 2002 , 2005 ) .",
"In homeostatic scaling at central mammalian synapses , there is emerging consensus that one end-point effector mediating both the enhanced and reduced response following a prolonged activity manipulation is the AMPA-type glutamate receptor ( Cingolani et al . , 2008; O'Brien et al . , 1998; Wierenga et al . , 2005; Thiagarajan et al . , 2005; Sutton et al . , 2006; Gainey et al . , 2009 , 2015 ) .",
"At the systems level , homeostatic scaling requires both transcription ( Ibata et al . , 2008 ) and translation ( Schanzenbächer et al . , 2016 ) .",
"Global activity manipulations bring about both transcriptional ( Meadows et al . , 2015; Steinmetz et al . , 2016; Schaukowitch et al . , 2017 ) and translational regulation ( Schanzenbächer et al . , 2016 ) .",
"For example , chronic activity suppression activated a calcium-dependent transcription program of 73 genes that includes AMPA receptors and transcription factors ( SRF , ELK ) and led to synaptic upscaling ( Schaukowitch et al . , 2017 ) .",
"After 24 hr of either enhanced or reduced activity , Schanzenbächer et al . , 2016 detected 307 differentially expressed proteins involved in neuronal functions such as calcium transport , synaptic vesicle trafficking and neurotransmitter release .",
"The earliest reports of homeostatic scaling at mammalian central synapses suggested that relatively long duration activity manipulations were required to elicit the homeostatic response ( O'Brien et al . , 1998; Turrigiano et al . , 1998 ) .",
"However , local manipulations of activity can bring about more rapid scaling ( Sutton et al . , 2004 , 2006 ) and recent studies have also suggested that global scaling can occur within a few hours of an activity manipulation ( Ibata et al . , 2008 ) .",
"Under physiological conditions that elicit synaptic scaling , it is likely that different durations of activity manipulation give rise to different responses that may differ both in the magnitude and nature of regulated proteins .",
"However , the mechanisms that enable neurons to sense the duration and polarity of a stimulus remain poorly understood to date .",
"In order to understand the time-dependent dynamics of the response to an activity manipulation we treated cultured hippocampal cells with either TTX or bicuculline for 2 hr to induce the process of up- or down-scaling ( Turrigiano et al . , 1998 ) , respectively .",
"During the 2 hr activity manipulation we metabolically labeled newly synthesized proteins using BONCAT ( Dieterich et al . , 2006; Schanzenbächer et al . , 2016 ) .",
"We analyzed the newly synthesized proteins and then to obtain a temporal trajectory of the cellular response , we compared the proteins synthesized within 2 hr of activity manipulation to those synthesized during a 24 hr manipulation ( Schanzenbächer et al . , 2016 ) .",
"Surprisingly , there was little overlap in the significantly regulated newly synthesized proteins identified in the early- and integrated late response datasets .",
"Many significantly regulated at 24 hr , however , exhibited trends for intermediate levels of regulation at 2 hr .",
"There was however , overlap in the functional categories that are modulated at the two time points , suggesting that within protein function groups , different choices are made to effect early and late homeostatic responses ."
],
[
"In order to determine the early cellular response to neural activity manipulations , we treated cultured hippocampal cells with either TTX or bicuculline for 2 hr to induce the process of up- or down-scaling ( Turrigiano et al . , 1998 ) respectively , and during this time metabolically labeled newly synthesized proteins using azidohomoalanine ( AHA ) and BONCAT ( Dieterich et al . , 2006; Schanzenbächer et al . , 2016 ) .",
"We compared the newly synthesized proteome in the TTX- and bicuculline-treated samples to each other and to an untreated control group , analyzing five biological replicates .",
"Proteins were identified and quantified using MaxQuant ( Cox and Mann , 2008 ) , with the following criteria:",
"( i ) at least one peptide per protein identified",
"( ii ) protein detected in at least 1/5 ( Figure 1A–B ) or 2/5 ( all subsequent Figures ) experiments and",
"( iii ) protein not detected in the Met control group ( see Materials and methods ) .",
"The methionine content of our AHA-labeled proteins was not different compared to a global proteome ( Rattus norvegicus , UniProtKB database ) as , on average , 2 . 42 and 2 . 40 Met residues are present per 100 amino acids , respectively ( Figure 1—figure supplement 1I ) .",
"As shown in Figure 1A , we identified over 3000 proteins in each treatment group and over 5000 unique proteins altogether ( summing proteins across groups , see Supplementary file 1 ) .",
"The majority of proteins were identified in at least 3/5 replicates and the relative representation of proteins in different functional groups as well as the abundance of proteins was not different between conditions ( Figure 1—figure supplement 1A–C ) .",
"The protein intensities associated with replicates within each treatment group were well-correlated with one another as were the protein intensities for each 2 hr treatment group ( Figure 1—figure supplement 1D–G ) .",
"As observed previously ( Schanzenbächer et al . , 2016 ) the activity manipulations did not result in a significant change in the overall proteome size ( Figure 1A ) .",
"Indeed , the overlap in the proteins identified in the three groups was greater than 90% ( Figure 1B ) .",
"The newly synthesized proteins uniquely identified in each group were 180 , 95 and 182 for TTX- , bicuculline- and the untreated groups , respectively , representing about 3–6% of each total nascent proteome .",
"We analyzed these unique proteins and found that the majority of them were detected in a single replicate ( Figure 1—figure supplement 1H ) .",
"A small number of proteins ( 4 , 2 , and 3 for TTX , bicuc , and untreated , respectively ) were detected in 3/5 replicates and represented cytoskeletal-associated proteins and signaling molecules ( Figure 1—figure supplement 1H ) .",
"Neither the stimulation ( bicuc ) nor inhibition ( TTX ) of activity induced a systematic shift ( e . g . global up- or down-regulation ) of proteins ( Figure 1C ) , but rather induced either up- or down-regulation of different sets of proteins .",
"We focused our analysis on these changes in the level of protein expression , using label-free quantification ( LFQ ) ( Cox et al . , 2014 ) to analyze the intensities of identified peptides in different groups ( Figure 1—figure supplement 1C ) .",
"Altogether , we detected 168 proteins whose expression level was significantly regulated by either enhanced or reduced activity or by both manipulations ( Supplementary file 2 ) .",
"In Figure 1D , proteins significant up- or down-regulated by both enhanced and reduced activity are shown in the upper-right and lower-left quadrant , respectively .",
"Note that the regulation of proteins in these two quadrants does not give information about the direction of the activity manipulation ( e . g . up or down , the ‘polarity’ ) but rather could signal the absolute value of the deviation from an activity set-point .",
"Other proteins exhibited significant increases in expression levels that were specific to either enhanced ( upper-left quadrant ) or reduced ( lower-right quadrant ) activity ( Figure 1D ) .",
"In principle , these classes of regulated proteins could detect or represent both the offset from the setpoint and the sign of the offset .",
"We compared the regulated proteins to a curated list of 35 proteins previously implicated in homeostatic scaling by other groups ( Supplementary file 2; Schanzenbächer et al . , 2016 ) .",
"We found six proteins regulated in our dataset that have been previously implicated in scaling , including two Ca2+ channel subunits ( Cacnb3 and Cacna2d3 ) , two protein phosphatases ( Ppp1r7 and Ppp6r3 ) , and two G-protein family members ( Rheb and Rgs2 ) .",
"Which functional protein classes exhibit significant differential regulation following 2 hr of enhanced or reduced activity ?",
"We conducted a gene annotation ( GO ) analysis of the 168 significantly regulated proteins and found that most ( 152/168 ) regulated proteins had a neuronal function and many were associated with synaptic functions ( Figure 2A , Supplementary file 3 ) , indicating that the majority of the significantly regulated proteins are not simply associated common cellular or metabolic functions .",
"Using the newly synthesized proteins in untreated samples as a background , we examined which particular functional groups were regulated by the activity manipulations ( Figure 2B , Supplementary file 3 ) .",
"Amongst the top 20 terms that showed significant enrichment were many terms associated with the secretory pathway , including Golgi apparatus , ER to Golgi transport vesicle , and Golgi membrane .",
"Within the synapse , we noted the regulation of many proteins including Neuroligin 2 ( Nlgn2 ) , Neurexin 2 ( Nrxn2 ) Agrin ( Agrn ) and Slc32a1 ( Figure 2C ) .",
"Does the up- or downward manipulation of activity result in protein groups which are differentially regulated , reflecting the ‘sign’ of the manipulation ?",
"We found many protein groups that exhibit significant differential regulation between the activity manipulated groups ( Figure 3 , Supplementary file 4 ) .",
"For example , the protein functional groups significantly upregulated following TTX-treatment ( and down-regulated following bicuculline-treatment ) included the following categories: voltage-gated Ca2+ channels , synaptosome , dendritic spine as well as cell adhesion , actin cytoskeleton and proteins associated with phosphorylation and de-phosphorylation .",
"At the other end of the spectrum were several protein groups associated with protein translation , that were negatively regulated by TTX treatment and positively regulated by bicuculline ( Figure 3 ) .",
"Some examples of polarity-dependent regulation of proteins are also shown in Figure 2C , for example , the kinesin Kif3c , the vesicle-associated protein syntaxin 5 ( Stx5 ) , the hyperpolarization-activated channel Hcn1 , and the disease-related protein Teneurin transmembrane protein 4 ( Tenm4 ) .",
"How does the proteomic response to an activity deviation evolve over time ?",
"To address this , we compared the significantly regulated nascent proteins following 2 hr of activity manipulation ( either bicuculline or TTX ) to those regulated during a 24 hr manipulation ( initially described in Schanzenbächer et al . , 2016 ) .",
"In the absence of activity manipulation , the newly synthesized proteomes identified at these two time points were largely ( ~94% ) overlapping ( Figure 4A and Figure 4—figure supplement 1A ) .",
"There were 292 ( 2 hrs ) and 1287 ( 24 hrs ) proteins that were uniquely identified at one time point , but not the other .",
"Using peptide intensities , we analyzed the relative abundance of these unique proteins compared to the total pool and found that they were less abundant ( Figure 4—figure supplement 1A ) , suggesting that the inability to detect them in one group might be due to insufficient sensitivity .",
"We next analyzed the significantly regulated proteins at each time point ( 168 at 2 hr and 307 at 24 hr ) and found a very low level of overlap ( 10 proteins ) ( Figure 4B and Figure 4—figure supplement 1B , Supplementary file 5 ) , not significantly greater than what would be expected by chance .",
"As also noted in Figure 1D , a large fraction of proteins was significantly regulated by both TTX and bicuculline; at 2 hr 52% of the regulated proteins exhibited regulation by both treatments ( Figure 4C ) .",
"We noted with interest that the fraction of proteins that were uniquely regulated by either TTX or bicuculline was enhanced ( Figure 4D ) , suggesting that as the duration of the treatment increases , the distinctiveness of the proteomic response , presumably more focused on the homeostatic effector mechanism , evolves .",
"We next focused our analyses on the translational dynamics of the proteins which showed significant regulation at either the 2 or 24 hr time point .",
"We examined the significantly regulated proteins for each time point and then examined the same protein at the other ( not significantly ) regulated time point .",
"In Figure 4E , the mauve dots represent the proteins regulated at 2 hr , with proteins up- and down-regulated following both enhanced ( bicuculline-treated ) or reduced ( TTX-treated ) activity shown in the upper-right and lower-left quadrants , respectively .",
"The black dots in this plot show the regulation of these same proteins at 24 hr .",
"The clustering of the black dots near the origin indicates that the newly synthesized proteins significantly regulated at 2 hr are largely unregulated at 24 hr .",
"In Figure 4F , the grey dots represent the proteins regulated at 24 hr and the black dots represent these same proteins at 2 hr .",
"Again , the cluster of black dots around the origin thus indicates the absence of significant regulation of these proteins at the 2 hr time point .",
"Although there was little evidence that the same individual proteins were regulated at both 2 and 24 hr following either activity manipulation , we considered the possibility that a functional protein group might be regulated at both time points , with the individual regulated proteins within that group being different at 2 vs 24 hr .",
"Indeed , we found several examples where there was significant regulation of a functional protein group at both time points , although the individual proteins within the group were different at each time point ( Figure 4G and H , Supplementary file 4 ) .",
"For example , the group ‘voltage-gated calcium channels’ was significantly regulated at both 2 and 24 hr , but the individual subunits were different at the time points ( e . g . Cacnb3 and Cacna2d3 at 2 hr and Cacnb4 at 24 hr ) .",
"In Figure 4H , the protein groups that fall on the diagonal line are regulated in the same direction at both the 2 and 24 hr time points , including again , synaptic transmission , dendritic spine , synapse , postsynaptic density , axon and others .",
"Only a few groups , including threonine kinase and Parkinson’s disease 20S proteasome were exclusively regulated at 24 hr ( Figure 4H ) .",
"These data indicate that most of protein groups are hot-spots for regulation at both time points , even if the individual regulated proteins within the groups are different .",
"We next conducted a meta-analysis of all significantly regulated proteins considering , in addition to newly synthesized proteins that are different from untreated samples , proteins that are significantly different between the two time-points and/or the up- and down-scaling conditions .",
"This master set of comparisons resulted in 711 significantly regulated proteins , which were significantly enriched for neuronal , rather than glial markers ( Supplementary file 6 ) .",
"We examined the regulation of each protein at 2 hr and 24 hr for both bicuculline- and TTX-treated samples to discover patterns of co-regulation ( Figures 5 and 6 ) .",
"We used self-organizing maps ( SOM ) to sort and organize the various patterns of regulation of individual proteins into similar groups , calculated the average fold-change for each cluster , and then used hierarchical clustering to examine the relationships between the groups ( Figure 5 ) .",
"In order to extract repeated patterns of regulation , we plotted the individual regulated proteins as a deviation from untreated , including the response at both time points and in both conditions on a single axis ( Figure 6 , Supplementary file 7 ) .",
"We first evaluated the optimal number of clusters to use , optimizing a minimization of within-cluster variance and a maximization of between-cluster variance ( Figure 6—figure supplement 1A ) .",
"Based on these considerations we chose a cluster size of 48 with each cluster containing between 4–53 proteins , ( Figure 6 , Supplementary file 7 ) .",
"The SOMs revealed several distinct patterns of regulation that , in principle , can serve as indicators of either the duration ( early-2hrs or late-24hrs ) or the polarity ( enhanced or reduced ) of the activity manipulation .",
"We also analyzed whether particular regulatory patterns were enriched for particular functional classes of proteins ( Figure 6—figure supplement 1B , Supplementary file 7 ) .",
"We found that most ( 11/12 ) of the regulatory patterns identified by the SOM algorithm were indicators of the time/duration of the manipulation .",
"For example , proteins that were significantly regulated at 2 hr , but not at 24 hr for both up- and down-scaling fall into this category and their regulation pattern is represented by the ‘M’ or the ‘W’ profiles in Figure 6A–D .",
"There were also many examples of ‘sine wave’ shape regulated proteins in which the early ( 2 hr ) and the late ( 24 hr ) time point were regulated in an opposite manner ( Figure 6H ) .",
"We also observed profiles in which there was regulation of proteins exclusively at the 24 hr time point in one or both conditions ( ‘upward or downward trapezoid shapes’ ) ( Figure 6E , F , K ) .",
"The trapezoid patterns were significantly enriched for a variety of channels and transporters ( Figure 6—figure supplement 1B ) .",
"In addition , some patterns of regulation that occurred indicate the polarity ( sign ) of the activity manipulation .",
"Polarity indicators are clusters where regulation of proteins occurs only in response to one ( TTX or bicuc ) of the activity manipulations or where regulation occurs to both activity manipulations but is of an opposite sign ( e . g . protein up regulated following TTX but down-regulated following bicuculline and vice versa ) .",
"We found that many ( 7/12 ) of the regulatory patterns identified by the self-organizing map were indicators of the polarity of the manipulation .",
"Examples of polarity-sensitive patterns include the ‘sun-seeking worm’ ( Figure 6G; enriched for kinases , Figure 6—figure supplement 1B ) , the ‘sine wave’ ( Figure 6H ) or the ‘skewed W’ ( Figure 6I ) , ‘skewed M’ ( Figure 6J ) or ‘flattened trapezoid’ ( Figure 6K ) profiles .",
"We noted with interest that some simple protein regulation patterns were also apparently absent from our data ( Figure 6L , M ) .",
"For example , the sign and magnitude of a single protein’s regulation could convey information about both the time and the direction of the activity manipulation , as shown in the ‘diagonal line’ pattern in Figure 6L .",
"This pattern was not , however , observed , although some sub-features of this pattern can be found in some of the other clusters .",
"Alternatively , time- information , but not polarity information , could be conveyed by the ‘V’ or ‘inverted V’ ( Figure 6M ) ; this pattern was also not observed in our data .",
"Taken together , these data indicate that there are patterns of individual protein regulation that can convey information about either the duration of the activity offset ( time ) or the polarity ( up-scaling or down-scaling ) of the offset .",
"Lastly , to examine whether there is a functional relationship between the large set of regulated proteins we conducted a network analysis ( see Materials and methods ) , including any protein significantly regulated at either 2 or 24 hr following either bicuculline or TTX treatment ( Figure 7 ) , yielding a regulated protein-protein interaction map .",
"This analysis revealed several protein networks , again comprising functions associated with the cytoskeleton and vesicle-mediated transport ( Figure 7A , B ) , neuronal systems ( Figure 7A , C ) , cell-cell communication ( Figure 7A , D ) , the processing and transport of mRNA ( Figure 7A , E ) and mitochondrial translation ( Figure 7F ) .",
"Within these networks signaling molecules , predominantly protein kinases and phosphatases emerged as hubs .",
"Also , two catalytic subunits of the cAMP-dependent Protein Kinase A ( Prkaca and Prkacb ) display highly correlated changes following both Bic and TTX stimulation with a pronounced abundance increase after 24 hr of TTX and decrease after 2 hr of Bic .",
"Prkaca and Prkacb interact with multiple regulated proteins that often exhibit similar changes in abundance and are known to play essential roles in synaptic transmission and plasticity: The voltage-gated calcium channel auxiliary beta subunits 1 , 3 , and 4 ( Cacnb1 , 3 , 4 ) and alpha subunit 2 delta 3 ( Cacna2d3 ) , Calmodulin ( Calm1 ) and the calcium-dependent protein kinase 2 subunit delta ( Camk2d ) .",
"Similarly , Synaptojanin-1 , a protein that affects synaptic transmission and membrane trafficking by regulating membrane levels of phosphatidylinositol-4 , 5-biphosphate , shows an increase in abundance after 24 hr of TTX treatment .",
"We also observed corresponding elevated levels of phosphatidyl-4- and -5-phosphate-kinase subunits ( Pip4k2b and Pip5k1c ) and the related phosphatase ( Inpp4a ) , as well as the AP2 related kinase 1 ( Aak1 ) which phosphorylates AP-2 to trigger clathrin assembly .",
"Some of the patterns of regulation complicate simple linear interpretations of signaling pathways: some regulated proteins that were downstream targets of a regulated kinase/phosphatase were often potential interactors with more than one regulated kinase/phosphatase .",
"For example , the regulated protein Pdpk1 , itself a kinase , interacts with three other regulated kinases ( Lyn , Src and Fyn ) as well as a regulated phosphatase ( Ppp2r5e ) , many of which also interact with one another ( Figure 7A ) .",
"In addition , we observed that the abundance of a group of mitochondrial ribosomal protein subunits increased after both 2 hr of Bic and TTX treatment ( Mrpl9 , 19 , 21 and 42; Mrps26 ) but then displayed different trends after 24 hr ( Figure 7F ) .",
"Lastly , we investigated the regulated proteins that are known to possess the highest number of interaction partners .",
"In this String analysis we acknowledge that we can only capture those interactions that are documented ( e . g . for well-studied proteins ) and thus could miss highly interactive proteins that have not been studied in detail .",
"With this caveat in mind , we used the String database and identified the top ~40 regulated proteins that have the highest number of protein-protein interactions ( Figure 7G ) .",
"This group of proteins exhibits on average , 38 . 3 interactions with other proteins whereas a random sampling of 40 proteins exhibits on average 5 . 6 interactions .",
"This difference is statistically significant ( p<0 . 001 ) .",
"Not surprisingly , several kinases emerge ( e . g . Adrbk1 , Prkaca , Prkacb , Lyn , Kit ) as well as phosphatases ( Ptpn1 , Ptpn11 ) .",
"We also observed several proteins involved in synaptic vesicle cycling ( the syntaxins , Stx1a , Stx5 , Stx6 ) and interestingly , proteins associate with the nuclear pore complex ( Nup98 and Ranbp2 ) .",
"The regulation of proteins that have many interaction partners obviously can exert a multiplicative effect on downstream signaling during synaptic scaling ."
],
[
"Here we examined the proteomic response of neuronal networks to a brief but global manipulation ( 2 hr ) of neuronal activity- either enhanced or reduced- by the addition of TTX or bicuculline .",
"Using BONCAT to metabolically label and identify the nascent proteome , we discovered 168 proteins whose expression levels were significantly regulated by treatments that lead to homeostatic up- or down-scaling .",
"The proteins that we identified are largely associated with neuronal function , including ion channels , adhesion and guidance molecules , synaptic receptors , and synaptic vesicle-associated proteins .",
"We also found that a large number of differentially regulated proteins are involved in post- translational modifications .",
"Adjusting proteome composition and activity by modifying existing protein pools is an energy-efficient and rapid way to alter activity states .",
"Following a 2 hr manipulation of activity , we observed elevated synthesis of several kinases such as Lyn , Aak1 and Src , and phosphatases such as protein tyrosine phosphatases ( Ptprg , see Figure 2 and Figure 3 ) .",
"We also observed the regulation of proteins involved with protein synthesis and degradation including Cullin3 , an essential component of the E3 ubiquitin-protein ligase complexes essential for poly-ubiquitination and protein degradation .",
"The regulation of degradation-related proteins supports observations that protein degradation plays an important role to adjust the neuronal proteome to the desired composition and thus functionality ( e . g . Bingol and Schuman , 2006; Tai and Schuman , 2008 ) .",
"Lastly , while we identified several proteins ( e . g . Rheb , Rgs2 , Ppp1r7 , and Cacnb3 ) that have been implicated in previous studies of homeostatic scaling , we did not observe significant regulation of AMPA receptors , which are known to be end-point effectors for both up- and down-scaling elicited after short-term manipulations of activity ( Ibata et al . , 2008 ) .",
"It is possible that our measurements were not sensitive enough to detect changes in AMPAR after a short ( 2 hr ) metabolic labeling and activity manipulation .",
"We did , however , observe polarized regulation of some AMPAR after 24 hr of labeling and manipulation ( Schanzenbächer et al . , 2016 ) .",
"This indicates that synaptic scaling following short-term activity manipulations may be due to post-translational modifications rather than regulation of new AMPAR synthesis .",
"We compared the regulated nascent proteome after 2 hr of global activity manipulation to a previous data set ( Schanzenbächer et al . , 2016 ) in which the same activity manipulations were applied for 24 hr .",
"Surprisingly , there was very little overlap ( ~10 proteins ) between the two time points .",
"We noted two additional interesting features .",
"First , a prominent feature of both datasets is the regulation of the same protein population by both up- and down-scaling manipulations .",
"In some cases , the regulation of this common protein pool is polarized ( e . g . upregulated in one condition and downregulated in another; Figure 3 ) reflecting the sign of the manipulation .",
"In other cases , the polarity of the regulation is similar for both manipulations .",
"Second , we analyzed if the fraction of overlapping regulated proteins was similar at 2 and 24 hr and found that this overlap was reduced from 52% at 2 hr to 34% at 24 hr .",
"These results suggest that the longer the neuronal system is in a state of offset from a threshold , the more distinct the proteomic response becomes to reflect whether up- or down-scaling must be implemented .",
"From a network perspective , the regulated proteins identified here and in our previous study ( Schanzenbächer et al . , 2016 ) can function as different types of sensors and effectors during homeostatic scaling .",
"The polarity ( positive or negative ) of the offset from an activity setpoint could be coded for by proteins uniquely synthesized in response to TTX or bicuculline treatment .",
"In our experiments , the number of such unique proteins was low as was their expression level .",
"Another means to represent the sign of the offset is the differential regulation ( up- or down ) of a single protein or multiple proteins .",
"We detected several protein examples that fit this profile .",
"Using self-organizing maps , we clustered the patterns of regulation observed over time and with each activity manipulation .",
"Interestingly , we found that many patterns of regulation indicated the duration of the activity offset ( 2 vs 24 hr ) , but not the polarity ( whether activity was enhanced or decreased ) .",
"We also found several regulation patterns that indicate the polarity of the manipulation , showing changes in expression exclusively to one manipulation but not another .",
"The proteins that do not show polarized regulation may represent general offset detectors whereas those that exhibit polarized responses may be effectors for the polarized homeostatic response .",
"Taken together , these data indicate that there are patterns of individual protein regulation that can convey information about either the duration of the activity offset ( time ) or the polarity ( up-scaling or down-scaling ) of the offset , or in some cases , both .",
"Capitalizing on the sensitivity of BONCAT , we were able to monitor the regulation of the neuronal proteome following a relatively brief global activity manipulation .",
"In contrast to other recent publications ( Bowling et al . , 2016 ) , we did not use SILAC-co-incorporation because the stringent biochemical purification method we have developed allows one to directly monitor newly-synthesized proteins .",
"In addition , because of the high number of proteins identified , we did not need to make use of intensity-based thresholds for including proteins also detected at lower levels in control samples .",
"We note , however , that AHA is incorporated into the proteomes of all cells present in the preparation , including glia cells , leading to a dilution of the measured neuronal proteome response analyzed in this study .",
"We recently developed a technique that enables cell-type specific labeling and identification of nascent proteins ( Alvarez-Castelao et al . , 2017 ) .",
"Future studies can make use of this platform to monitor selectively the cell-type specific proteomes both in vitro and in vivo ."
],
[
"Dissociated hippocampal neurons from postnatal day 0–2 rat pups ( strain Sprague-Dawley ) were prepared and maintained as previously described ( Aakalu et al . , 2001 ) .",
"Briefly , hippocampi were dissected and triturated after incubating in L-cystein-papain solution at 37°C for 15 min .",
"Dissociated neurons were plated onto poly-D-Lysine-coated Petri dishes ( MatTek ) and cultured in Neurobasal A medium supplemented with B-27 and Glutamax ( Invitrogen ) at 37°C for 21 days .",
"All experiments were carried out in accordance with the German Animal Welfare Act and supervised by the local government authorities and the Max Planck Society .",
"After a brief ( 30 min ) methionine deprivation , two dishes of cultured neurons ( ~800 k cells in total ) were incubated in each condition with 4 mM AHA ( or methionine as a control ) and treated with 20 µM bicuculline or 1 µM Tetrodotoxin ( Tocris Bioscience ) for 2 hrs .",
"After incubation , the neurons were washed with cold DPBS completed with protease inhibitor cocktail ( cOmplete EDTA-free , Roche ) , harvested , and pelleted at 2000x g for 5 min .",
"The cell pellets were snap-frozen in liquid nitrogen , and stored at −80°C until further use .",
"The pelleted neurons were resuspended in lysis buffer ( 100 µL , 8 M urea , 200 mM Tris [pH 8 . 4] , 4% CHAPS , 1 M NaCl , cOmplete EDTA-free protease inhibitor ) and homogenized using a pestle .",
"The lysates were sonicated with short bursts ( 4 × 30 s ) in a cooled ultrasonic bath , followed by 5 min of benzonase digestion ( 1 µL of a ≥ 250 units/µL stock solution ) , and centrifugation for 5 min at 10000x g .",
"The supernatants ( ~200 µL ) were incubated with freshly prepared catalyst solution ( 250 µL ) and Alkyne-Sepharose slurry ( 50 µL ) according to the manufacturer’s protocol ( #C10416 , Thermo Fisher Scientific ) .",
"The reaction mixture was gently agitated for 19 hr in the dark .",
"After centrifugation ( 2 min , 1000x g ) , the beads were rinsed twice in H2O ( 900 µL each ) and incubated in 250 µL SDS wash buffer ( 100 mM Tris [pH 8] , 1% SDS , 250 mM NaCl , 5 mM EDTA ) containing 10 mM TCEP for 45 min at 55°C under gentle agitation .",
"The mixture was centrifuged for 5 min at 1000x g and the supernatants were discarded .",
"Each sample was incubated with 250 µL SDS wash buffer containing 95 mM iodoacetamide for 30 min at room temperature in the dark under gentle agitation .",
"The samples were transferred to pre-washed ( 400 µL H2O , LC-MS/MS-ChromaSolv; 400 µL SDS wash buffer ) Pierce Spin columns and then washed with 20 mL SDS wash buffer , 20 mL 8 M urea in 100 mM Tris [pH 8] , and 20 mL 20% ( v/v ) acetonitrile/water .",
"The resin was rinsed in digestion buffer ( 250 µL , 100 mM Tris , 2 mM CaCl2 , 10% acetonitrile ) and transferred to an Eppendorf tube .",
"After centrifugation ( 5 min , 1000x g ) , the supernatant was discarded leaving a volume of ~50 µL slurry .",
"EndoLysC ( 0 . 65 µg ) was added to each tube and incubated overnight ( ~22 hr ) at 37°C with constant agitation .",
"For subsequent tryptic digestion , each sample was incubated with 0 . 65 µg trypsin at 37°C overnight ( ~22 hr ) with constant agitation .",
"The samples were rinsed twice with 500 µL H2O ( 0 . 1% TFA ) and centrifuged at 1000x g for 5 min .",
"The collected supernatants were loaded onto C18-SepPak columns ( 50 mg sorbent , Waters Corp . ) conditioned with 2 mL acetonitrile , 1 mL of 50% acetonitrile/water ( 0 . 5% acetic acid ) in 0 . 5% acetic acid , and 2 mL water ( 0 . 1% TFA ) .",
"The samples ( ~1 mL ) were washed with 2 mL of water ( 0 . 1% TFA ) and 200 µL of water ( 0 . 5% acetic acid ) .",
"Desalted peptides were eluted with 0 . 5 mL of 50% acetonitrile/water ( 0 . 5% acetic acid ) , dried using a Speed-Vac ( Eppendorf ) , and stored at −80°C until LC-MS/MS analysis .",
"The dried peptide fractions were dissolved in 5% acetonitrile with 0 . 1% formic acid , and subsequently loaded using a nano-HPLC ( Dionex U3000 RSLCnano ) onto a PepMap100 trapping column ( C18 , particle size 3 µm , L = 20 mm ) .",
"Peptides were separated on a PepMap RSLC analytical column ( C18 , particle size <2 µM , L = 50 cm , Dionex/Thermo Fisher Scientific ) by a gradient of water ( buffer A: water with 5% v/v dimethylsulfoxide and 0 . 1% formic acid ) and acetonitrile ( buffer B: 5% dimethylsulfoxide , 15% water and 80% acetonitrile ( v/v/v ) , and 0 . 08% formic acid ) , running from 4% to 48% B in 178 min at a flowrate of 300 nL/min .",
"All LC-MS-grade solvents were purchased from Fluka .",
"Peptides eluting from the column were ionized online using a Thermo nanoFlex ESI source and analyzed in a ‘Q Exactive Plus’ mass spectrometer ( Thermo Fisher Scientific ) .",
"Mass spectra were acquired over the mass range 350–1 , 400 m/z , and sequence information were acquired by data-dependent automated switching to MS/MS mode using collision energies based on mass and charge state of the candidate ions ( TOP12 , MS resolution 70 k , MS/MS resolution 35 k , injection time: 120 ms , full parameters in Supplementary file 8 ) .",
"All samples were measured in quadruplicate LC-MS/MS runs .",
"MS data were analyzed in MaxQuant ( ver . 1 . 5 . 5 . 1; Cox and Mann , 2008 ) using a customized Andromeda LFQ parameter set ( see Supplementary file 8 ) .",
"In brief , spectra were matched to a Rattus norvegicus database downloaded from uniprot . org ( 35 , 953 entries , reviewed and non-reviewed ) and a contaminant and decoy database .",
"Tryptic peptides with ≥6 amino acids and ≤2 missed cleavages were included .",
"Precursor mass tolerance was set to 4 . 5 ppm , fragment ion tolerance to 20 ppm , with a static modification of Cys residues ( carboxyamidomethylation +57 . 021 ) and variable modifications of Met residues ( oxidation +15 . 995 ) , Lys residues ( acetylation +42 . 011 ) , Asn and Gln residues ( deamidation +0 . 984 ) , and N termini ( carbamylation +43 . 006 ) .",
"Search results were filtered with an FDR of 0 . 01 , and proteins identified by ≥1 unique peptide were included for subsequent analysis .",
"Proteins were quantified in each condition by pair-wise ratio determination using ≥1 common peptide in ≥4 consecutive full scans per run for the label-free quantification ( Cox et al . , 2014 ) .",
"Five independent biological replicates measured in quadruplicates were processed in Perseus software package ( ver . 1 . 5 . 5 . 3; Tyanova et al . , 2016 ) .",
"After combining technical replicates , background proteins detected in vehicle control were subtracted from the other conditions ( untreated , bicuculline-treated , TTX-treated neurons ) .",
"Newly-synthesized proteins identified in each biological replicate were averaged within each condition ( Figure 1A , Supplementary file 1 ) , and counted according to their numbers of biological replicates ( Figure 1—figure supplement 1A ) .",
"Venn diagrams ( e . g . showing the protein overlap in ≥1 biological replicate , Figure 1B ) were generated using Venn Diagram Plotter ( ver . 1 . 5 . 5228 . 29250; PNNL ) .",
"Sub-cellular localization of proteins identified in ≥2 biological replicates ( Figure 1—figure supplement 1B ) was predicted using the LocTree3 database ( ver . 23-08-2016; Goldberg et al . , 2014 ) .",
"Non-normalized protein intensities ( non-LFQ ) were log2 transformed and averaged across replicates to assess differences in overall protein abundances ( Figure 1—figure supplement 1C , Supplementary file 1 ) .",
"LFQ intensities were normalized over experimental conditions using the central tendency adjustment method: protein quantities were divided by a normalization factor representing the median intensity of all proteins measured in ≥2 biological replicates .",
"The normalized log2 intensities were averaged across ≥2 biological replicates and then used for calculating the fold change of treated neurons relative to control ( Figure 1C , Supplementary file 1 ) .",
"After filtering for the presence in ≥2 biological replicates ( 3196 proteins remaining ) and for a coefficient of variation <1 ( 3086 proteins remaining ) , these proteins were annotated using the gene ontology databases ( GOBP , GOMF , GOCC , Ashburner et al . , 2000 ) , the Panther database ( Thomas et al . , 2003 ) and protein keywords retrieved from the UniprotKB database ( The UniProt Consortium , 2017 ) .",
"Differentially regulated groups of annotated proteins were analyzed and extracted using Perseus ( Figure 3 , Supplementary file 4 , 1D annotation enrichment , two-sided Wilcoxon-Mann-Whitney test , Benjamini-Hochberg FDR of 0 . 05 ) , and compared to the protein fold changes after 24 hr ( Figures 4G , 24 hr dataset from Schanzenbächer et al . , 2016 ) .",
"Protein annotations of both time points were then combined and analyzed in a 2D annotation enrichment ( Cox and Mann , 2012 ) using a p value < 0 . 01 ( Figure 4H , Supplementary file 4 ) .",
"Duplicates and annotation groups with more than 100 protein members were discarded .",
"Statistical significance for protein regulation between bicuculline/untreated , TTX/untreated or bicuculline/TTX after 2 hr were measured using an ANOVA ( permutation-based FDR of 0 . 05 , S0 = 0 . 05 , 250 randomizations ) , followed by post-hoc Fisher LSD p<0 . 05 ( in Origin 2015G Sr1 , OriginLab ) , yielding 168 proteins ( Figure 1D , Figure 2C , Supplementary file 2 ) .",
"Significantly regulated proteins were annotated ( Figure 2A , Supplementary file 3 ) using the VarElect interpretation tool ( Stelzer et al . , 2016 ) by LifeMap’s GeneCards suite ( Ben-Ari Fuchs et al . , 2016 ) .",
"Cellular component ontologies enriched in the regulated proteome compared to the 5135 newly synthesized proteins as the background were analyzed in FunRich ( ver . 3; Pathan et al . , 2015 ) using a Benjamini Hochberg corrected p value < 0 . 05 ( Figure 2B , Supplementary file 3 ) .",
"Significantly regulated proteins in early ( 2 hr ) and late response ( 24 hr , Schanzenbächer et al . , 2016 ) were compared ( Figure 4B , Figure 4—figure supplement 1B ) , and the protein overlap regulated in both treatments was calculated for each time point using Fisher LSD p<0 . 05 ( Figure 4C–D , Supplementary file 2 and 5 ) .",
"The ‘degree of regulation’ was calculated for the 168 and 307 regulated proteins ( 2 hr and 24 hr , respectively ) by measuring the distance of each protein to the zero point in Figure 4E–F .",
"Protein fold changes of treated neurons relative to the control were calculated for each biological replicate in both time points .",
"Time- and polarity-dependent regulation was statistically validated by an ANOVA ( permutation-based FDR of 0 . 05 , S0 = 0 . 05 , 250 randomizations ) , yielding 711 differentially regulated proteins ( Supplementary file 6 ) .",
"Cell-type specific markers were extracted from Sharma et al . , 2015 by selecting 290 glial cell and 644 neuronal markers showing an at lest 2-fold higher abundance in astrocytes , oligodendrocytes and microglia cultures compared to neuronal cultures .",
"Statistical significance of neuronal markers enriched in the differentially regulated proteomes were evaluated by comparing the number of regulated neuronal and glial markers .",
"Protein clusters ( Figure 6 , Figure 6—figure supplement 1B , Supplementary file 7 ) were compiled in J-Express pro 2012 ( Dysvik and Jonassen , 2001 ) using a Self-Organizing Map ( SOM ) algorithm ( 49 nodes , theta/momentum = 0 . 998 , phi/momentum = 0 . 998 , Euclidean distance , Gaussian neighbourhood function , ≤4000 iterations ) .",
"SOM clusters were hierarchically clustered in a second layer and assembled in a heatmap ( Figure 5 , Uncentered Pearson Correlation , Weighted Average Linkage , WPGMA ) .",
"Enrichment analysis of cellular component and molecular function ontologies ( Figure 6—figure supplement 1B , Supplementary file 7 ) was conducted in FunRich ( Benjamini Hochberg corrected p value < 0 . 05 ) .",
"Protein networks ( Figure 7A–F ) were analyzed using the String interactome ( v . 10 . 0 , string-db . org , Szklarczyk et al . , 2015 ) with databases only as interaction source and high confidence of interactions ( score >0 . 700 ) .",
"Protein groups ( Figure 7F ) were preprocessed with k-means and hierarchically clustered in Perseus ( Euclidean distance , complete linkage ) .",
"Significantly regulated proteins that are annotated as ‘high interaction hubs’ ( Figure 7G ) were analyzed in NetworkAnalyst ( networkanalyst . ca , Xia et al . , 2015 ) using the String interactome ( confidence score >0 . 900 , experimental evidenced , first-order network ) .",
"The largest network containing 82 regulated proteins , 1178 interaction partners and 1745 protein-protein interactions was plotted in Figure 7G using the force atlas layout algorithm .",
"The proteomics data associated with this manuscript have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository ( Vizcaíno et al . , 2016 ) with the dataset identifier PDX008271 ."
]
] | [
"In homeostatic scaling at central synapses , the depth and breadth of cellular mechanisms that detect the offset from the set-point , detect the duration of the offset and implement a cellular response are not well understood .",
"To understand the time-dependent scaling dynamics we treated cultured rat hippocampal cells with either TTX or bicucculline for 2 hr to induce the process of up- or down-scaling , respectively .",
"During the activity manipulation we metabolically labeled newly synthesized proteins using BONCAT .",
"We identified 168 newly synthesized proteins that exhibited significant changes in expression .",
"To obtain a temporal trajectory of the response , we compared the proteins synthesized within 2 hr or 24 hr of the activity manipulation .",
"Surprisingly , there was little overlap in the significantly regulated newly synthesized proteins identified in the early- and integrated late response datasets .",
"There was , however , overlap in the functional categories that are modulated early and late .",
"These data indicate that within protein function groups , different proteomic choices can be made to effect early and late homeostatic responses that detect the duration and polarity of the activity manipulation ."
] | [
"The brain can store information by changing the strength of connections between neurons , also known as synapses .",
"When two neurons at a synapse are active at the same time , the synapse becomes stronger .",
"This enables the first neuron to activate the second more easily .",
"But it also means that the two neurons will now be active at the same time more often , which will tend to make the synapse even stronger .",
"If this process continues unchecked , the synapse will keep getting stronger until no further changes in strength are possible .",
"This will make it harder for the brain to form new memories .",
"To prevent this from happening , the brain responds to prolonged changes in the activity of neurons by adjusting the strength of synapses in the opposite direction .",
"If neurons are too active for an extended period of time , the brain reduces the strength of synapses .",
"If neurons show too little activity , the brain increases the strength of synapses .",
"This process is known as homeostatic scaling , and the brain achieves it by adjusting the number and/or type of proteins present at synapses .",
"Schanzenbächer et al . now reveal the changes in synaptic proteins that occur in response to a two-hour increase or decrease in neuronal activity .",
"These changes can be tracked in the laboratory by growing cells in a petri dish in the presence of modified amino acids , the building blocks of proteins .",
"Any new proteins the cells produce will contain the modified amino acids , making them easy to spot .",
"Schanzenbächer et al . applied this technique to neurons obtained from the rat hippocampus , a region of the brain involved in learning and memory .",
"Bathing the neurons for two hours in chemicals that either enhanced or reduced their activity , triggered changes in more than 150 proteins .",
"Schanzenbächer et al . compared these results to those of a previous experiment in which neuronal activity had been manipulated for 24 hours .",
"Each set of conditions produced a characteristic profile of protein activity .",
"The profiles indicated whether the activity in neurons had increased or decreased , and whether the changes had lasted for two hours or 24 hours .",
"These findings may provide insights into disease states in which there is too much or too little brain activity ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease",
"immunology and inflammation"
] | Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors | elife-12241-v2 | [
[
"Serotonin ( 5-hydroxytryptamine , 5-HT ) is one of the oldest neurotransmitters/hormones in evolution ( Turlejski , 1996 ) .",
"It regulates or modulates a wide variety of processes in most invertebrates and vertebrates , such as metabolism ( Sze et al . , 2000 ) and locomotion ( Ranganathan et al . , 2000 ) in nematodes; reproduction ( Anstey et al . , 2009 ) , learning and memory ( Sitaraman et al . , 2008 ) in insects; physiologic states and behaviors , including pain , appetite , mood , and sleep ( Mössner and Lesch , 1998 ) in humans .",
"5-HT also plays an important role outside of the central nervous system ( CNS ) in immune signaling .",
"Immune cells can synthesize and sequester 5-HT .",
"For instance , human mast cells express the key peripheral 5-HT synthesizing enzyme , tryptophan hydroxylase 1 ( TPH-1 ) ( Kushnir-Sukhov et al . , 2007 , 2008 ) .",
"Mouse dendritic cells ( DCs ) express the serotonin transporter ( SERT ) , taking up 5-HT from the microenvironment ( O'Connell et al . , 2006 ) .",
"5-HT regulates immune responses and inflammatory cascades via distinct receptors and different immune cells have been shown to express a different composition of 5-HT receptor subtypes ( Baganz and Blakely , 2013 ) .",
"5-HT2A may contribute to chemotaxis of eosinophils ( Boehme et al . , 2008 ) and 5-HT2C receptors on alveolar macrophages can be activated by 5-HT ( Mikulski et al . , 2010 ) .",
"However , most studies assess the in vitro response of immune cells to pharmacological agents .",
"Therefore , the function of 5-HT signaling in vivo in are still unclear .",
"Vertebrate blood cells ( e . g . macrophages ) have evolved a variety of strategies to internalize particles and solutes , including pinocytosis , receptor-mediated endocytosis , and phagocytosis , a highly conserved aspect of innate immunity .",
"Phagocytosis , the uptake of large particles ( >0 . 5 μm ) into cells , allows for rapid engulfment of dying cells and pathogens by specialized phagocytes , such as macrophages and neutrophils in mammals ( Aderem and Underhill , 1999 ) .",
"In insects , hemocyte phagocytosis is an important cellular defense response to pathogens and parasites ( Lavine and Strand , 2002 ) .",
"In lepidopteran insects , the granulocytes and plasmatocytes are the major phagocytes ( Kanost et al . , 2004 ) .",
"Cross-talk between the immune and nervous system may play a role in regulating phagocytosis in insects during infection .",
"However , the roles of serotonin in insect phagocytosis are less well characterized compared with vertebrate counterparts , although there is evidence that 5-HT can enhance phagocytosis ( Baines et al . , 1992; Kim et al . , 2009 ) .",
"Many of the intracellular signaling pathways that drive the insect immune system are very similar to those found in the mammalian innate immune system ( Lemaitre and Hoffmann , 2007 ) , and some of them were first uncovered in insects ( Hoffmann , 2003 ) .",
"Indirect evidence suggests that similarities between insects and mammals extend to the molecular mechanisms involved in the neuroendocrine control of immune function ( Adamo , 2008 ) .",
"Because of the relative simplicity of the insect immune system , examining the basic interactions between serotonin receptor-medicated second messenger systems and immune-related intracellular signaling pathways may be easier in insects .",
"As an initial step toward this goal , we have made a comprehensive study in the caterpillar , Pieris rapae hemocytes to elucidate the function of 5-HT signaling in insect cellular immune responses .",
"We found that hemocyte-derived 5-HT regulates phagocytosis in an autocrine manner through 5-HT1B and 5-HT2B receptors , each of which produces distinct effects .",
"Mortality experiments using Drosophila mutants and hemocyte-specific RNAi-silencing further found that both 5-HT1B and 5-HT2B are necessary for effective resistance to bacterial infections ."
],
[
"After activation with 100 ng/ml of lipopolysaccharide ( LPS ) for 2 hr , we detected 5-HT within hemocytes directly by immunolabeling and fluorescence microscopy .",
"Figure 1A shows that granules of 5-HT labeled with 5-HT antisera ( upper ) are readily visible in the cytosol .",
"As a negative control , 5-HT antisera preabsorbed with 5-HT were invisible ( lower ) .",
"Serotonin synthesis requires two enzymes , tryptophan hydroxylase and aromatic L-amino acid decarboxylase ( AADC ) ( Figure 1B ) .",
"Tryptophan hydroxylase is the rate-limiting enzyme in serotonin biosynthesis encoded by two genes , TPH and TRH in insects , TPH1 and TPH2 in mammals .",
"Both gene products have tryptophan hydroxylase activity in vivo .",
"The TPH/TPH1 gene is expressed in non-neural tissues , and the TRH/TPH2 gene is expressed in neural tissues ( Côté et al . , 2003; Neckameyer et al . , 2007; Watanabe et al . , 2011 ) .",
"The high-affinity SERT is a plasma membrane protein that can take up extracellular 5-HT ( Rudnick , 2006; Torres et al . , 2003 ) .",
"Thus , we performed RT-PCR to characterize TPH , TRH , and SERT expression in hemocytes .",
"Figure 1C shows that hemocytes produced mRNA for TPH , but that transcripts of TRH and SERT were not detected .",
"These results suggest that hemocytes do not selectively sequester 5-HT but can synthesize 5-HT using TPH .",
"Then , we performed ELISA to quantify the amount of 5-HT released into the hemocytes’ culture media .",
"5-HT levels increased significantly above that found in controls in cell supernatants 1 hr after exposure of hemocytes to LPS .",
"5-HT levels reached maximal at 2 hr and decreased at 4 hr ( Figure 1D ) .",
"The real-time quantitative RT-PCR results also show that mRNA expression level of TPH was up-regulated by approximately sixfold relative to controls at 15 min and at 4 hr after LPS stimulation , suggesting a potential negative feedback regulation ( Figure 1E ) .",
"Therefore , it is concluded that hemocytes are able to synthesize and release 5-HT in vitro , and this activity is enhanced following their activation .",
"We hypothesise that 5-HT may play an important role in hemocyte function . 10 . 7554/eLife . 12241 . 003Figure 1 . Activated hemocytes are capable of 5-HT synthesis .",
"( A ) 5-HT was visualized by confocal microscopy in hemocytes activated with 100 ng/ml LPS by labeling with 5-HT antisera ( Alexa Fluor 546; red ) ( upper left ) .",
"5-HT antisera was preabsorbed with 5-HT as the negative control ( lower left ) .",
"Nuclei were counterstained with DAPI ( blue ) .",
"Scale bar represents 10 μM .",
"Data are representatives of two independent experiments .",
"( B ) Serotonin biosynthetic pathway .",
"Tryptophan- phenylalanine hydroxylase ( TRH/TPH ) , aromatic L-amino acid decarboxylase ( AADC ) , 5-hydroxy tryptophan ( 5-HTP ) .",
"( C ) Expression of gene transcripts for TPH , TRH , and SERT were determined by RT-PCR from naive hemocytes and from hemocytes activated with 100 ng/ml LPS for 2 hr , central nervous system ( CNS ) as positive control .",
"Data are representatives of three independent experiments .",
"( D ) 5-HT concentrations in hemocytes supernatants were determined by ELISA .",
"Hemocytes were activated with 100 ng/ml LPS .",
"Naive hemocytes treated with PBS are as control ( n = 4 ) .",
"( E ) Relative expression of TPH was quantified by real-time PCR .",
"Hemocytes were activated with 100 ng/ml LPS .",
"Naive hemocytes treated with PBS is as control ( n = 3 ) .",
"One-way ANOVA followed by Tukey’s multiple comparison test for D and E . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 , *p<0 . 05 and NS means no significant difference . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 003 To examine the physiological role of 5-HT in hemocytes , we tested whether inhibition of 5-HT synthesis would affect hemocyte phagocytosis of Gram-negative E . coli bacteria labeled with pHrodo , a dye that fluoresces in the acidic environment of a mature phagosome upon fusion with lysosomes .",
"α-methyltryptophan ( AMTP ) is a competitive inhibitor of 5-HT synthesis ( Gal and Christiansen , 1975 ) .",
"As shown in Figure 2—figure supplement 1 , 5-HT synthesis was significantly reduced in hemocytes after treatment with 10 μM AMTP , compared with PBS-treated controls .",
"The results show that 10 μM AMTP also significantly impaired hemocyte phagocytic ability .",
"Furthermore , treatment with exogenous 5-HT at 100 nM fully restored hemocyte phagocytosis ( Figure 2A–D ) .",
"Next , we tested siTPH-treated hemocytes ( knock-down effect was approximately 80% , Figure 2E ) for their phagocytosis ability .",
"After incubation with siTPH for 48 hr , hemocyte phagocytosis rate was significantly decreased compared with the negative control .",
"100 nM 5-HT treatment could fully rescue the siTPH induced phenotype ( Figure 2F–I ) .",
"Collectively , these data indicate that hemocyte-derived 5-HT is critical for proper hemocyte phagocytosis upon immune challenge . 10 . 7554/eLife . 12241 . 004Figure 2 . Inhibition of endogenous 5-HT synthesis impairs hemocyte phagocytosis .",
"( A-C )",
"Hemocyte phagocytosis was visualized by florescence microscope .",
"Hemocytes were stained with Cell Tracker Blue CMAC ( blue ) , green represent the phagocytosed pHrodo E . coli .",
"( D ) Quantification of phagocytosis of E . coli by hemocytes ( n = 3 ) .",
"( E ) Confirmation of knock-down effect of TPH by real-time qPCR .",
"The P . rapae18s rRNA gene was used as an internal reference gene ( n = 4 ) .",
"( F-H )",
"Effect of siTPH on hemocyte phagocytosis was visualized by florescence microscope .",
"( I ) Quantification of siTPH effect on hemocyte phagocytosis ( n = 3 ) .",
"One-way ANOVA followed by Tukey’s multiple comparison test for D and I; two-tailed t-test for E . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 and NS means no significant difference . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 00410 . 7554/eLife . 12241 . 005Figure 2—figure supplement 1 . Inhibition of 5-HT synthesis by AMTP . Both PBS treated hemocytes ( control ) and 10 μM AMTP-treated hemocytes were incubated with 100 ng/ml LPS for 2 hr .",
"Error bars indicate ± s . e . m . , n = 3 , the statistical analysis is based on two-tailed t-test , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 005 Since 5-HT is involved in the regulation of hemocyte function , it should exert its effects through corresponding 5-HT receptors .",
"So far , five subtypes of 5-HT receptors including 5-HT1A , 5-HT1B , 5-HT2A , 5-HT2B and 5-HT7 are identified in insects ( Blenau and Thamm , 2011; Gasque et al . , 2013 ) .",
"5-HT1A and 5-HT1B couple with Gi protein and decrease intracellular cAMP levels .",
"5-HT2A and 5-HT2B couple with Gq protein , which can induce increased intracellular Ca2+ .",
"5-HT7 couples with Gs protein and increases cAMP levels ( Blenau and Thamm , 2011; Gasque et al . , 2013 ) .",
"We performed a comprehensive analysis of 5-HT receptor gene expression in hemocytes by RT-PCR after stimulation with 100 ng/ml LPS for 2 hr .",
"The positive control samples were extracted from the CNS of P . rapae .",
"As shown in Figure 3A , naive hemocytes express Pr5-HT1B , Pr5-HT2B and Pr5-HT7 .",
"We used specific antibodies to confirm the expression of Pr5-HT1B ( Figure 3B ) and Pr5-HT2B ( Figure 3C ) on the plasma membranes of hemocytes .",
"We also performed Ca2+ imaging to further confirm functional expression of 5-HT2B in hemocytes .",
"Both plasmatocytes and granulocytes , the two most abundant types of hemocytes , produced intracellular Ca2+ increase in response to 5-HT .",
"5-HT stimulated Ca2+ responses at concentrations ranging from 0 . 01 nM to 10 nM ( maximal response ) , and its EC50 value was estimated at 0 . 15 nM ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 12241 . 006Figure 3 . 5-HT receptor subtypes expressed in naïve and LPS-activated hemocytes .",
"( A ) Hemocytes were negatively purified and activated with 100 ng/ml LPS for 2 hr .",
"The gene expression for 5-HTR subtype was examined by RT-PCR .",
"Data are representatives of three independent experiments .",
"( B ) Gene expression for Pr5-HT1B was examined by immunofluorescence .",
"The scale bar represents 10 μM .",
"Data are representatives of two independent experiments .",
"( C ) Gene expression for Pr5-HT2B was examined by immunofluorescence .",
"PL , plasmatocytes; GR , granulocytes .",
"Data are representatives of two independent experiments .",
"( D-G )",
"The effect of different antagonist on hemocyte phagocytosis was visualized by florescence microscope .",
"SB216641 is an antagonist of 5-HT1B .",
"SB269970 is an antagonist of 5-HT7 and RS127445 is a human 5-HT2B antagonist .",
"( H ) Quantification of different antagonist on hemocyte phagocytosis .",
"Data are from three independent experiments that each consists of cells from ten fifth-instar larvae .",
"One-way ANOVA followed by Tukey’s multiple comparison test for H . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 and NS means no significant difference . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 00610 . 7554/eLife . 12241 . 007Figure 3—figure supplement 1 . Representative Ca2+ responses and dose-response profiles for 5-HT in hemocytes .",
"( A ) Pseudocolored images of hemocytes before and after application of 1 nM 5-HT .",
"Red cells indicate high levels of intracellular Ca2+ measured by fluorescent ratio intensities , and blue cells represent the basal levels .",
"Two major kinds of hemocytes , plasmatocytes and granulocytes , are indicated by arrows .",
"( B ) Increasing concentrations of 5-HT dose-dependently induced changes in the fluorescence ratio of hemocytes .",
"( C ) Dose-response curve for 5-HT in hemocytes , as obtained from Ca2+ imaging .",
"Each point represents the mean ± s . e . m . from three to five replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 00710 . 7554/eLife . 12241 . 008Figure 3—figure supplement 2 . Modulation of intracellular cAMP levels in HEK 293 cells stably expressing the Pr5-HT1B and Pr5-HT2B . To characterize the pharmacological properties of Pr5-HT1B and Pr5-HT7 , We use HEK 293 cell line to stably express Pr5-HT1B and Pr5-HT7 .",
"( A ) Dose-response relationships of the effects of 5-HT on intracellular cAMP levels in HEK 293 cells stably transfected with pcDNA3/Pr5-HT1B ( n = 3 ) .",
"( B ) Effects of 5-HT and the antagonist SB 216641 on intracellular cAMP levels in HEK 293 cells stably transfected with pcDNA3/Pr5-HT1B ( n = 3 ) .",
"( C ) Dose-response relationships of the effects of 5-HT on intracellular cAMP levels in HEK 293 cells stably transfected with pcDNA3/Pr5-HT7 ( n = 3 ) .",
"( D ) Effects of 5-HT and the antagonist SB 269970 on intracellular cAMP levels in HEK 293 cells stably transfected with pcDNA3/Pr5-HT7 ( n = 3 ) .",
"One-way ANOVA followed by Tukey’s multiple comparison test for A and B . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 , and NS means no significant difference . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 008 Since there are multiple 5-HT receptor types on the hemocyte membranes , we next investigated which one was involved in the 5-HT-mediated phagocytosis by pharmacological manipulation .",
"Three selective 5-HT receptor antagonists ( SB 216641 , SB 269970 , and RS 127445 ) were applied at a final concentration of 10 μM to block their corresponding receptors .",
"The inhibition effects of SB 216641 and SB 269970 on Pr5-HT1B and Pr5-HT7 were also confirmed by cAMP assays in heterogeneous expression system ( Figure 3—figure supplement 2 ) .",
"We found that only blockade of Pr5-HT1B significantly reduced the phagocytic ability of hemocytes .",
"Blockade of Pr5-HT7 neither intensified nor reduced hemocyte phagocytosis .",
"Surprisingly , blockade of 5-HT2B significantly increased hemocyte phagocytosis ( Figure 3D–H ) .",
"We further found that the 5-HT1B blocker SB 216641 affected hemocyte phagocytosis in a dose-dependent manner ( Figure 4A ) .",
"To confirm the above results , we performed siRNA-mediated interference to knock-down each receptor in hemocytes .",
"The results showed that siPr5-HT1Bdecreased Pr5-HT1B expression significantly at both mRNA and protein levels ( Figures 4B–C ) after 24 hr and 48 hr , respectively .",
"As expected , the siPr5-HT1B treated hemocytes phagocytose E . coli poorly compared with control ( Figure 4D–F ) .",
"Significantly knock-down of Pr5-HT2B was also observed at both transcript and protein levels ( Figure 4—figure supplement 1A–B ) at 48 hr , which promoted hemocyte phagocytosis ( Figure 4—figure supplement 1C–E ) .",
"However , knock-down of Pr5-HT7have no significant effect on hemocyte phagocytosis ( Figure 4—figure supplement 1F–G ) .",
"The results demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors but that these two receptors act in opposite ways . 10 . 7554/eLife . 12241 . 009Figure 4 . Pr5-HT1B mediates hemocyte phagocytosis .",
"( A ) Dose-response profiles for the effects of Pr5-HT1B blocker SB 216641 on hemocyte phagocytosis .",
"Data are from three independent experiments that each consists of cells from ten fifth-instar larvae .",
"( B ) Confirmation of knock-down effect of Pr5-HT1B by real-time PCR .",
"The P . rapae 18s rRNA gene was used as an internal reference gene ( n = 4 ) .",
"( C ) Western blot analysis of knock-down effect of Pr5-HT1B .",
"β-actin was used to show equal protein loading .",
"( D-E )",
"Effect of siPr5-HT1B on hemocyte phagocytosis was visualized by florescence microscope .",
"( F ) Quantification of siPr5-HT1B effect on hemocyte phagocytosis .",
"Data are from three independent experiments that each consists of cells from ten fifth-instar larvae .",
"One-way ANOVA followed by Tukey’s multiple comparison test for A; two-tailed t-test for B and F . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 00910 . 7554/eLife . 12241 . 010Figure 4—figure supplement 1 . Effect of siPr5-HT2B and siPr5-HT7 on hemocyte phagocytosis .",
"( A ) Confirmation of knock-down effect of Pr5-HT2B by real-time PCR .",
"The P . rapae 18s rRNA gene was used as an internal reference gene ( n = 3 ) .",
"( B ) Western blot analysis of knock-down effect of Pr5-HT2B .",
"β-actin was used to show equal protein loading .",
"( C-D )",
"Effect of siPr5-HT2B on hemocyte phagocytosis was visualized by florescence microscope .",
"( E ) Quantification of siPr5-HT2B effect on hemocyte phagocytosis ( n = 3 ) .",
"( F ) Confirmation of knock-down effect of Pr5-HT7 by real-time PCR .",
"The P . rapae 18s rRNA gene was used as an internal reference gene ( n= 3 ) .",
"( G ) Quantification of siPr5-HT7 effect on hemocyte phagocytosis ( n = 3 ) .",
"Two-tailed t-test for A , E , F and G . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 , *p<0 . 05 , and NS means no significant difference . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 010 Although the above data showed that activation of Pr5-HT1B and Pr5-HT2B produced opposite effects , enhanced hemocyte phagocytosis is the overall effect by hemocytes exposed to released 5-HT .",
"Thus , we tested whether 5-HT receptors were upregulated or downregulated in hemocytes during an immune response .",
"qPCR results showed that the mRNA levels of Pr5-HT1B was significantly increased after LPS stimulation from 15 min to 4 hr ( Figure 5A ) .",
"The protein expression levels also increased at 2 hr and 4 hr ( Figure 5C ) .",
"Interestingly , the mRNA expression level of Pr5-HT2B was significantly decreased after LPS induction at 1 hr and 4 hr ( Figure 5B ) , in accordance with its protein expression levels ( Figure 5D ) . 10 . 7554/eLife . 12241 . 011Figure 5 . Expression analysis of Pr5-HT1B and Pr5-HT2B in naïve and LPS-induced hemocytes .",
"( A–B )",
"Relative expression of Pr5-HT1B and Pr5-HT2B were quantified by q-PCR .",
"The P . rapae 18s rRNA gene was used as an internal reference ( n = 3 ) .",
"( C–D )",
"Western blot analysis of Pr5-HT1B and Pr5-HT2B in naive and LPS-induced hemocytes .",
"β-actin was used to show equal protein loading .",
"One-way ANOVA followed by Tukey’s multiple comparison test for A and B . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 , *p<0 . 05 , and NS means no significant difference . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 011 We hypothesized that 5-HT receptors mediate immunoregulation in other insects and that it is important in host defense in vivo .",
"On the other hand , it is a technical challenge to perform in vivo RNAi in Lepidoptera ( Terenius et al . , 2011 ) .",
"Thus , we chose the model animal Drosophila melanogaster to address the above questions .",
"Both 5-HT1B and 5-HT2B transcripts were detected in wild-type Drosophila hemocytes by RT-PCR .",
"High mRNA levels of Henna , the Drosophila TPH homolog , were also expressed in hemocytes ( Figure 6A ) .",
"We next used a 5-HT1B null allele ( 5-HT1BΔIII-V ) and its corresponding control allele ( Gasque et al . , 2013 ) to examine the ability to phagocytose bacteria as well as susceptibility to infections .",
"A 1344 bp fragment is removed from genomic DNA to disrupt the III-V transmembrane domains of the 5-HT1BΔIII-V allele ( Figure 6B ) . 10 . 7554/eLife . 12241 . 012Figure 6 . 5-HT1B is required for microbial phagocytosis and plays an important role in the Drosophila defense against S . aureus infection .",
"( A ) 5-HT1B , 5-HT2B and TPH are expressed in Drosophila naive hemocytes .",
"RPL11 was used as an internal reference gene .",
"Data are representatives of three independent experiments .",
"( B ) Genomic PCR of 5-HT1B control and 5-HT1BΔIII-V flies .",
"Data are representatives of three independent experiments .",
"( C ) Representative pictures depicting phagocytosis in 5-HT1B control and 5-HT1BΔIII-V flies of fluorescein-labeled E . coli bioparticles .",
"( D ) Quantification of in vivo phagocytosis of E . coli .",
"Approximately 10 flies per genotype were used in each experiment .",
"Data are representatives of three independent experiments .",
"( E ) Representative pictures depicting phagocytosis in 5-HT1B control and 5-HT1BΔIII-V flies of fluorescein-labeled S . aureus bioparticles .",
"( F ) Quantification of in vivo phagocytosis of S . aureus .",
"Approximately eight flies per genotype were used in each experiment .",
"Data are representatives of three independent experiments .",
"( G-H )",
"Representative survival curves of female ( G ) and male ( H ) 5-HT1B control and 5-HT1BΔIII-V flies after injection of S . aureus ( optical density [OD] 0 . 4 ) .",
"n = 20–25 flies .",
"Experiments were performed in triplicate .",
"( I ) Comparison of the S . aureus ( OD 0 . 4 ) recovered in 5-HT1B control and 5-HT1BΔIII-V flies 0 , 6 , and 18 hr post infection .",
"Bacterial load was measured in eight individual female flies per genotype at each time point in each experiment .",
"Two-tailed t-test for D , F and I . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 , *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 012 The phagocytic capacity of flies was measured using an in vivo adult phagocytosis assay ( Kocks et al . , 2005 ) .",
"Flies were injected with fluorescently labeled pHrodo bioparticles .",
"The amount of fluorescence in the dorsal vessel area where sessile phagocytes accumulates were visualized and quantified .",
"In 5-HT1BΔIII-V flies , in vivo phagocytosis of E . coli was strongly impaired when compared to controls ( Figure 6C–D ) .",
"A similar effect was observed with the Gram-positive bacterium S . aureus ( Figure 6E–F ) .",
"After infection with S . aureus , 5-HT1BΔIII-V flies die more quickly than controls , for both male and female flies ( Figure 6G–H ) .",
"To determine whether the increased mortality of 5-HT1BΔIII-V flies following infection was due to defective resistance or decreased tolerance ( Schneider and Ayres , 2008 ) , we also assessed bacterial clearance by comparing colony-forming units ( CFU ) 6 hr and 18 hr after infection .",
"There is an increased bacterial load in 5-HT1B null flies compared to control files ( Figure 6I ) .",
"5-HT1B function in the regulation of hemocyte phagocytosis was further confirmed by RNAi .",
"We used the HmlΔ-Gal4 ( Sinenko et al . , 2004 ) driver to specifically express UAS-1B RNAi ( Yuan et al . 2005 ) and UAS-5-HT1B RNAi25833 respectively , both of which led to decreased phagocytosis of E . coli and S . aureus ( Figure 7A–D ) .",
"To test whether the hemocyte-specific knockdown of 5-HT1B dampened phagocytosis in general , or whether there was a lack of inducibility after immune challenge , we injected flies with PBS or 5-HT and then latex beads .",
"When flies were injected with PBS , all flies showed similar phagocytic capacity .",
"However , when flies were injected with 5-HT , knocking down 5-HT1B failed to enhance latex beads phagocytosis as controls ( Figure 7E ) .",
"After injection with S . aureus , the flies expressing 5-HT1B RNAi in their blood cells were much weaker than the control flies ( Figure 7F–I ) .",
"Our RNAi data indicate that the increased susceptibility to bacteria in 5-HT1B mutants is due to 5-HT1B malfunction in hemocytes . 10 . 7554/eLife . 12241 . 013Figure 7 . Knockdown of 5-HT1B in hemocytes affects Drosophila phagocytosis and survival .",
"( A ) Quantification of in vivo phagocytosis of E . coli in WT/UAS-1B RNAi , hml> UAS-1B RNAi and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice , ( B ) Quantification of in vivo phagocytosis of E . coli in WT/UAS-5-HT1B RNAi25833 , hml>UAS-5-HT1B RNAi25833 , and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice .",
"( C ) Quantification of in vivo phagocytosis of S . aureus in WT/UAS-1B RNAi , hml> UAS-1B RNAi , and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice , ( D ) Quantification of in vivo phagocytosis of S . aureus in WT/UAS-5-HT1B RNAi25833 , hml>UAS-5-HT1B RNAi25833 , and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice .",
"( E ) Quantification of in vivo phagocytosis of red fluorescently labeled latex beads in WT/UAS-1B RNAi , hml>UAS-1B RNAi , and hml/WT flies after a 30 min preinjection of either PBS or 1μg/μl 5-HT .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were done twice , ( F ) Representative survival curves of WT/UAS-1B RNAi , hml>UAS-1B RNAi , and hml/WT male flies after injection of S . aureus .",
"n=19–22 flies .",
"Data are representatives of three independent experiments .",
"Each experiment was performed in triplicate .",
"( G ) Representative survival curves of WT/UAS-5-HT1B RNAi25833 , hml>UAS-5-HT1B RNAi25833 , and hml/WT male flies after injection of S . aureus .",
"n=19–21 flies .",
"Data are representatives of two independent experiments .",
"Each experiment was performed in triplicate .",
"( H ) Comparison of the S . aureus ( OD 0 . 4 ) recovered in WT/UAS-1B RNAi , hml>UAS-1B RNAi and hml/WT flies 0 , and 18 hr post infection .",
"Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment .",
"Experiments were performed in triplicate .",
"( I ) Comparison of the S . aureus ( OD 0 . 4 ) recovered in WT/UAS-5-HT1B RNAi25833 , hml>UAS-5-HT1B RNAi25833 , and hml/WT flies 0 and 18 hr post infection .",
"Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment .",
"Experiments were performed in triplicate .",
"One-way ANOVA followed by Tukey’s multiple comparison test for A , B , C , D , E , H , and I . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 , *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 013 To investigate the role of 5-HT2B in Drosophila hemocyte phagocytosis , we also knocked down 5-HT2B expression in blood cells by RNAi using two independent UAS-5-HT2B RNAi lines .",
"Unlike the results found in P . rapae , the reduced levels of 5-HT2B in Drosophila hemocytes led to a defect in the phagocytosis of E . coli and S . aureus ( Figure 8A–D ) .",
"Moreover , the flies expressing 5-HT2B RNAi in their hemocytes took up significantly fewer latex beads than did control flies after 5-HT injection ( Figure 8E ) .",
"5-HT2B knockdown flies had higher bacterial loads than controls ( Figure 8I ) and increased susceptibility to S . aureus ( Figure 8E–G ) .",
"Taken together , our experiments demonstrate that both 5-HT1B and 5-HT2B play important roles in host defense against bacterial infection . 10 . 7554/eLife . 12241 . 014Figure 8 . Knockdown of 5-HT2B in hemocytes affects Drosophila phagocytosis and survival .",
"( A ) Quantification of in vivo phagocytosis of E . coli in WT/UAS-5-HT2B RNAi25874 , hml>UAS-5-HT2B RNAi25874 , and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice , ( B ) Quantification of in vivo phagocytosis of E . coli in WT/UAS-5-HT2B RNAi60488 , hml>UAS-5-HT2B RNAi60488 , and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice .",
"( C ) Quantification of in vivo phagocytosis of S . aureus in WT/UAS-5-HT2B RNAi25874 , hml>UAS-5-HT2B RNAi25874 , and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice , ( D ) Quantification of in vivo phagocytosis of S . aureus in WT/UAS-5-HT2B RNAi60488 , hml> UAS-5-HT2B RNAi60488 , and hml/WT flies .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were performed twice .",
"( E ) Quantification of in vivo phagocytosis of red fluorescently labeled latex beads in WT/UAS-5-HT2B RNAi25874 , hml> UAS-5-HT2B RNAi25874 , and hml/WT flies after a 30 min preinjection of either PBS or 1μg/μl 5-HT .",
"Approximately six flies per genotype were used in each experiment .",
"Experiments were done twice .",
"( F ) Representative survival curves of WT/UAS-5-HT2B RNAi25874 , hml> UAS-5-HT2B RNAi25874 , and hml/WT male flies after injection of S . aureus .",
"n=20–21 flies .",
"Data are representatives of two independent experiments .",
"Each experiment was performed in triplicate .",
"( G ) Representative survival curves of WT/UAS-5-HT2B RNAi60488 , hml>UAS-5-HT2B RNAi60488 , and hml/WT male flies after injection of S . aureus .",
"n=19–21 flies .",
"Data are representatives of two independent experiments .",
"Each experiment was performed in triplicate .",
"( H ) Comparison of the S . aureus ( OD 0 . 4 ) recovered in WT/UAS-5-HT2B RNAi25874 , hml>UAS-5-HT2B RNAi25874 , and hml/WT flies 0 , and 18 hr post infection .",
"Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment .",
"Experiments were performed in triplicate .",
"( I ) Comparison of the S . aureus ( OD 0 . 4 ) recovered in WT/UAS-5-HT2B RNAi60488 , hml>UAS-5-HT2B RNAi60488 , and hml/WT flies 0 , and 18 hr post infection .",
"Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment .",
"Experiments were performed in triplicate .",
"One-way ANOVA followed by Tukey’s multiple comparison test for A , B , C , D , E , H , and I . Error bars indicate ± s . e . m . , ***p<0 . 001 , **p<0 . 01 , *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 01410 . 7554/eLife . 12241 . 015Figure 9 . A schematic diagram of serotonin signaling on hemocyte phagocytosis . LPS enhances the expression of TPH , which catalyzes tryptophan into 5-HT via 5-HTP .",
"5-HT , which secreted from hemocytes , activates the hemocyte-membrane receptor 5-HT1B and 5-HT2B .",
"The immune responses of P . rapae are labeled in purple: activation of 5-HT1B promotes hemocyte phagocytosis and activation of 5-HT2B lead to opposite effects .",
"LPS increases 5-HT1B expression but decreases that of 5-HT2B .",
"The immune responses of Drosophila are labeled in green arrows: activation of 5-HT1B promotes hemocyte phagocytosis and activation of 5-HT2B lead to the same effects . DOI: http://dx . doi . org/10 . 7554/eLife . 12241 . 015"
],
[
"Our study demonstrates the ubiquity of serotonergic receptor signaling pathway in immune function .",
"This paper is the first to demonstrate that insect hemocytes express TPH and can synthesize 5-HT , like human macrophages ( Nakamura et al . , 2008 ) .",
"The exposure of hemocytes to LPS led to an induction of TPH expression and a release of 5-HT .",
"Moreover , the secreted 5-HT appears to be an important autocrine stimulus .",
"It promotes phagocytosis: inhibition of TPH either by a competitive inhibitor or siRNA silencing resulted in significantly decreased hemocyte phagocytosis .",
"Chemicals that act as neurotransmitters in the nervous system can also modulate immune function ( Meredith et al . , 2005 ) .",
"5-HT is one of these classical neurotransmitters that is also an important immune regulatory molecule in both insects and mammals .",
"Recent research has made progress in determining 5-HT modulated mammalian immune responses , especially regarding the machinery to produce , store , release , and respond to 5-HT in immune cells ( Ahern , 2011 ) .",
"5-HT was also reported to mediate immune responses , such as hemocyte phagocytosis , nodule formation and hemocyte population in insects ( Baines et al . , 1992; Kim et al . , 2009; Kim and Kim , 2010 ) , but the signaling pathway is unclear .",
"Drosophila TPH homolog gene Henna was found to be one hit in a genome-wide RNAi screen for genes that affect phagocytosis of Candida albicans by hemocytes ( Stroschein-Stevenson et al . , 2006 ) .",
"Examining the basic interactions between serotonin receptor-medicated second messenger systems and immune-related intracellular signaling pathways in insects may shed light on their interactions and functions in mammals .",
"Second , our findings show that naive hemocytes express 5-HT1B , 5-HT2B , and 5-HT7 receptors , but only 5-HT1B and 5-HT2B appear to have functional roles .",
"Using selective antagonists and RNAi , we found that inhibition of 5-HT1B decreases hemocyte phagocytosis .",
"However , inhibition of 5-HT2B enhances hemocyte phagocytosis .",
"It seems that activation of these two receptors affects hemocytes in opposite ways .",
"Interestingly , we found that 5-HT1B is dramatically up-regulated following hemocyte activation , but 5-HT2B is significantly down-regulated .",
"Therefore , even though the two receptors have opposite functions upon activation , the overall effects of 5-HT signaling induced by immune challenge are favorable for hemocyte phagocytosis ( Figure 7 ) .",
"Mammalian DCs also express 5-HT1B and 5-HT2B , via which 5-HT induces chemotaxis and in vivo migration of DCs to draining lymph nodes ( Müller et al . , 2009 ) .",
"Even though insect plasmatocytes are macrophage-like cells , 5-HT activates macrophage cells via 5-HT1A ( Nakamura et al . , 2008 ) and 5-HT2C ( Mikulski et al . , 2010 ) , which are different from the receptor sub-types found in insect hemocytes .",
"The 5-HT7 receptor plays a critical role in the immune response in the gut of mice ( Kim et al . , 2013 ) .",
"Although our results indicated that 5-HT7 receptors were not involved in insect hemocyte phagocytosis , they may be critical for other immune activities .",
"Many neurotransmitter receptors are found on mammalian immune cells and regulate innate immune response , however , it is still unclear about their general role at the organismal level because most studies are conducted in vitro ( Sternberg , 2006 ) .",
"However , it is relatively easy to test immunity in insects in vivo ( Lemaitre and Hoffmann , 2007 ) .",
"We found that the 5-HT1B deficiency flies were more vulnerable to bacterial infections due to their poor phagocytic ability .",
"Hemocyte-specific RNAi experiments showed similar results , indicating that the 5-HT1B-mediated hemocyte phagocytosis is important for insects to defend themselves against pathogens .",
"Surprisingly , the flies expressing 5-HT2B RNAi in their hemocytes were more susceptible to bacterial infections , suggesting that activation of 5-HT2B may promote phagocytosis in Drosophila , different from the results in P . rapae .",
"In conclusion , we found that insect hemocytes can synthesis and release 5-HT , which regulates phagocytosis via 5-HT1B and 5-HT2B receptors on the membrane of the hemocyte .",
"We also used the genetic model Drosophila to further confirm the roles of 5-HT1B and 5-HT2B in vivo .",
"These findings suggest that serotonin , an ancient signaling molecule , modifies immune function in animals across phyla .",
"Exploring the interactions between serotonin receptor-mediated pathways and immune-related pathways may be easier to initiate in insects , which have a simpler immune system ."
],
[
"P . rapae larvae were collected primarily from cabbage fields in the experimental farmland of Zhejiang University , Hangzhou , China and the laboratory colony of P . rapae was reared at 25 ± 1°C and 70% relative humidity under a photoperiod of 14 hr light: 10 hr dark in a greenhouse as previously described by Zhang et al . ( 2005 ) .",
"The following fly stocks were from the Bloomington Drosophila Stock Center: HmlΔ-Gal4 ( 30139 ) ; UAS-5-HT1B RNAi ( 27634 , 25833 ) ; UAS-5-HT2B RNAi ( 25874 , 60488 ) .",
"The 5-HT1B control and 5-HT1BΔIII-V flies were generous gifts from Leslie Vosshall ( Gasque et al . , 2013 ) .",
"Serotonin hydrochloride , forskolin , G418 disulfate salt and SB-269970 hydrochloride were obtained from Sigma-Aldrich ( St Louis , MO ) .",
"SB 216641 and RS 127445 were purchased from Tocris Bioscience ( Bristol , UK ) .",
"Fifth-instar larvae of P . rapae were surface sterilized with 70% ethanol .",
"The proleg was cut with a pair of scissors and the hemolymph was collected in Grace’s Insect Medium ( 1:10 , v/v; Invitrogen , Carlsbad , CA ) .",
"The diluted hemolymph was added to 12-well tissue culture plates ( Nunc , Roskilde , Denmark ) at a density of 2× 106 cells per well .",
"Hemocytes were treated with 100 ng/ml LPS ( Escherichia coli 0111:B4; Sigma Aldrich ) for 15 min , 1 hr , 2 hr , and 4 hr ( Ngkelo et al . , 2012; Wu et al . , 2015 ) .",
"The hemocyte culture was collected and centrifugated .",
"The supernatants were collected for 5-HT detection .",
"The hemocytes that adhered to the plate were harvested for RNA isolation .",
"The hemolymph from three first day of fifth instar larvae was collected and mixed .",
"The combined hemolymph ( 30 μl ) was mixed with 170 μl Grace’s Insect Medium ( Invitrogen ) containing 50 μg/ml tetracycline and 2 μl saturated 2-phenylthiourea ( PTU ) .",
"The diluted hemolymph was added to each well of a 8-well chambered coverglasses ( Lab-Tek , Nunc , Thermo Fisher Scientific , Rochester , USA ) and hemocytes were allowed to adhere to the slide for 20 min at 27°C to form monolayers .",
"Then , hemocytes were fixed with 4% paraformaldehyde .",
"5-HT was labeled with 5-HT antisera ( 72 hr , room temperature; S-5545; Sigma ) followed by biotinylated anti- rabbit Ig ( 24 hr , 4°C ) and SA-Alexa Fluor 546 ( 1 hr , room temperature; Invitrogen ) .",
"For use as a negative control , 5-HT antisera was preabsorbed with 5-HT ( 10 mM , 24 hr , 4°C ) .",
"The nuclei of hemocytes were stained with 1 μg/ml of 4′-6-diamidino-2-phenylindole ( DAPI , Beyotime Biotech , Jiangsu , China ) for 5 mins and hemocytes were observed by fluorescent microscope ( Zeiss , Göttingen , Germany ) .",
"We performed ELISA to quantify the amount of 5-HT produced in the hemocyte culture supernatants using the 5-Hydroxytryptamine ( serotonin ) assay kit ( Li et al . , 2014 ) ( Jiancheng , Nanjing , China ) .",
"Total RNA was isolated from P . rapae nerve cord with Trizol reagent ( Invitrogen , Carlsbad , CA , USA ) .",
"Single-strand cDNA , synthesized from the RNA using a ReverTra Ace-α- kit ( Toyobo , Osaka , Japan ) , was used as a template for PCRs .",
"We performed transcriptome sequencing of nerve cord of P . rapae .",
"Through transcriptome sequencing , several putative serotonin receptors were annotated using BlastX ( National Center for Biotechnology Information [NCBI] , Bethesda , MD ) .",
"The full length was obtained using the 5’-Full rapid-amplification of cDNA ends ( RACE ) Kit ( Takara , Dalian , China ) and 3’-Full RACE Kit ( Takara , Dalian , China ) ( Supplementary file 1A ) .",
"To amplify the complete sequence , we use the forward primer located upstream of the putative start codon initiator , and the reverse primer located downstream of the putative stop codon ( Supplementary file 1A ) .",
"Total RNA was extracted from P . rapae hemocytes with high pure RNA isolation kit ( Roche ) in accordance with the manufacturer’s instructions .",
"To collect RNA from larval fly blood cells , approximately 30 larvae were carefully lacerated with tweezers on their anterior end in 100 μl of nuclease-free water .",
"The RNA quantity and quality was measured by using a Nanodrop 2000 spectrophotometer ( Thermo Scientific Inc . , Bremen , Germany ) .",
"Reverse transcription was performed with 1 μg of RNA by using a ReverTra Ace qPCR RT kit ( Toyobo , Osaka , Japan ) .",
"For conventional reverse transcription- polymerase chain reaction ( RT-PCR ) cDNA was amplified using KOD- Plus- ( Toyobo , Osaka , Japan ) .",
"Real-time quantitative PCR was performed on cDNA preparations using the SsoFast Eva Green Supermix with Low Rox ( Bio-Rad , Hercules , CA ) and Applied Biosystems 7500 Real-Time PCR System ( Applied Biosystems by Life Technologies , Carlsbad , CA ) following the manufacturer’s instructions .",
"The quantification of transcript level of different gene was conducted according to the 2−ΔΔCT method ( Livak and Schmittgen , 2001 ) .",
"Comparable quantities of cDNA were ensured by amplification of 18s rRNA , as a stably expressed reference gene ( Wu et al . , 2013 ) in P . rapae .",
"The primers are listed in Supplementary file 1A .",
"To assay E . coli and S . aureus phagocytosis in adults , approximately eight to ten flies with an equal distribution of females and males per genotype were injected with ~0 . 2 μl of 1 mg/mL pHrodo green E . coli or pHrodo red S . aureus ( Invitrogen , Carlsbad , CA ) using a FemtoJet microinjection system ( Eppendorf ) .",
"They were then incubated for 1 hr at room temperature .",
"Fluorescently labeled particles were visualized through the cuticle using a florescence microscope ( Nikon AZ100M , Nikon , Japan ) .",
"We use Image J software to quantify the results .",
"Relative fluorescence calculated as: [fluorescence]dorsal vein area/[fluorescence]adjacent area .",
"To assay phagocytosis of beads , flies were first injected with approximately 36 . 8 nl PBS or 1 μg/μl 5-HT dissolved in PBS using a Drummond Scientific Nanoject II .",
"Flies were incubated at room temperature for 30 min and then injected with approximately 27 nl of 1 . 0 μm Red Fluorescent Carboxylate Modified FluoSpheres diluted 1: 2 ( Invitrogen ) , incubated at room temperature for 10 min , injected with 36 . 8 nl 0 . 4% Trypan blue ( Invitrogen ) , and then mounted and visualized as described above .",
"Small interfering RNA ( siRNA ) molecules were designed from the nucleotide sequence obtained from P . rapae using Invitrogen siRNA design software ( http://rnaidesigner . thermofisher . com/rnaiexpress/ ) ( Supplementary file 1B ) and were synthesized by Invitrogen .",
"siRNA for the negative control was used in transfection experiments as a control .",
"Hemocytes were isolated as described above and were attached to the surface of 96-well tissue culture plates and incubated in Grace’s medium .",
"The monolayers of hemocytes were treated with 2 . 4 ng siRNA and 0 . 7 μl siRNA transfection reagent INTERFERin ( Polyplus-transfection SA , France ) according to the instructions from the manufacturer , and incubated at 27°C .",
"We use the forward primer Pr5-HT1B- BamHI 5′- CGGGATCCCAAACAGCTAGGAAAAGAAT -3′ and the reverse primer Pr5-HT1B- XholI 5′- CCCTCGAGTTATGTCTTTGCCGCTTTCC -3′ to amplify the third intracellular loop cDNA fragment of Pr5-HT1B .",
"Purified PCR products was cloned into vector pET-28a ( + ) and confirmed by DNA sequencing .",
"The expression product was 17 kDa which carried a His-tag .",
"The construct was used to transform Escherichia coli BL21 ( DE3 ) and the cells inoculated into 2 l of Luria–Bertani ( LB ) medium containing kanamycin ( 50 mg/ml ) at 37°C .",
"Until A600 reached 0 . 8 , the cultures were added with 0 . 5 mM isopropyl-β-d-thiogalactopyranoside ( IPTG ) and incubated overnight at 28°C .",
"The cells were collected by centrifugation and disrupted by sonication .",
"The insoluble recombinant protein was purified through Ni-chelating affinity column ( TransGen Biotech , Beijing , China ) under denaturing conditions .",
"To confirm the identity of the recombinant protein , proteins were separated by SDS-PAGE , transferred to PVDF ( Bio-Rad ) and detected with an anti-His polyclonal antibody HRP ( HuaAn Biotechnology , Hangzhou , China ) conjugate by ECL ( Thermo Scientific ) .",
"Then the purified protein was submitted to Hangzhou HuaAn Biotechnology Company as an antigen for immunization in male New Zealand rabbits .",
"The antibody to Pr5-HT1B was purified from antiserum which had been done by the company .",
"Polyclonal antibody against Pr5-HT2B was generated by Hangzhou HuaAn Biotechnology Company using peptide antigen for antibody development .",
"Epitopes are predicted by GenScript Optimum Antigen design tool .",
"Peptide sequence synthesized for Pr5-HT2B antibody development is SAAAKTSKGTNISEC .",
"The amino acid cysteine ( C ) , which locates at the end of the peptide , is used for carrier protein conjugation .",
"Hemocytes were lysed in ice-cold buffer ( 1% Triton X-100 , 150 mM NaCl , 10 mM Tris-HCl pH 7 . 5 , 5 mM EDTA , 1 mM sodiumo-vanadate ) containing protease inhibitors phenylmethyl sulfonylfluoride ( PMSF , Sangon Biotech , P0754 , Shanghai , China ) .",
"For membrane proteins , debris was sedimented by centrifugation ( 15 , 500 g , 10 min , 4°C ) , supernatants were collected and their protein concentration was determined by Bradford reagent ( Sangon Biotech , Shanghai , China ) .",
"Samples were diluted in 4 × Protein SDS PAGE Loading Buffer ( Takara Biotechnology , Japan ) , then boiled for 10 min .",
"Samples were separated in a denaturing polyacrylamide gel and transferred to a PVDF membrane .",
"After blocking ( 5% Tris-buffered saline; pH 7 . 0; containing 0 . 1% Tween 20 ) and washing , membranes were then incubated overnight with primary antibodies against Pr5-HT1B and Pr5-HT2B ( 1: 500 ) .",
"Membranes were then incubated with secondary antibody horseradish peroxidase–conjugated goat anti-rabbit IgG diluted 1:5 , 000 in Tris-buffered saline with Tween-20 .",
"Membranes were rinsed five times with wash buffer and then incubated with the ECL western blotting substrate ( Promega , Wisconsin ) .",
"Membranes were stripped and reprobed with anti-β- actin ( Cell Signaling Technology , Beverly , MA ) for 2 hr at room temperature , followed by incubation with secondary antibody .",
"Immunofluorescence was performed using the same methods as used for 5-HT detection except that the primary antibody was different .",
"An expression plasmid containing the Kozak consensus sequence ( Kozak , 1987 ) was constructed by PCR with specific primers ( Supplementary file 1A ) .",
"The PCR product was double digested .",
"The digested DNA fragments were purified by PCR Clean-Up Kit ( Axygen , Union City , CA ) and then subcloned into pcDNA3 . 0 vector ( Invitrogen ) .",
"The correct insertion was confirmed by DNA sequencing .",
"Human Embryonic Kidney 293 ( HEK 293 ) cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences ( Shanghai , China ) .",
"The cell line identity has been authenticated by short tandem repeat ( STR ) profiling , and the cells were free from the mycoplasma contamination .",
"HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium ( D-MEM ) ( Gibco BRL , Gaithersburg , MD ) supplemented with 10% fetal bovine serum ( FBS ) ( Gibco BRL ) and antibiotics at 37°C and 5% CO2 .",
"After transfection of plasmid into the cells using Lipofectamine 2000 ( Invitrogen ) , the antibiotic G418 ( 0 . 8 mg/mL ) was added to the medium to select for cells that stably expressed the receptors .",
"After 2 weeks of G418 selection , G418- resistant colonies were trypsinized in cloning cylinders and transferred to 12-well plastic plates for expansion .",
"These individual cell lines were analyzed for integration of the receptor DNA by RT-PCR and localization of the protein by immunofluorescence ( data not shown ) .",
"The clonal cell line that most efficiently expressing 5-HT receptor was chosen for this study .",
"Intracellular cAMP concentration ( [cAMP]i ) was determined as previously described ( Huang et al . , 2007 ) .",
"Cells were plated into 12-well tissue culture plates ( Nunc , Roskilde , Denmark ) at a density of 1× 106 cells per well and incubated at 37°C with 10% CO2 in a humidified incubator .",
"The cells were pre-incubated in Dulbecco’s phosphate-buffered saline ( DPBS; Gibco-Invitrogen ) containing 100 uM phosphodiesterase inhibitor IBMX for 20 min at room temperature .",
"After the preincubation , a 50-μl aliquot of D-PBS containing various concentrations of reagents was added .",
"The culture was then incubated for 20 min at room temperature .",
"The reaction was stopped by aspirating the solution and then adding 250 μl ice-cold cell lysis buffer immediately to lysis the cells .",
"The cell lysate was scraped into 1 . 5 ml Eppendorf tubes for collections and stored at –70°C until use .",
"The solution was centrifugated and [cAMP]i in the supernatant was determined using a cAMP ELISA kit ( R&D Systems , Minneapolis , MN ) .",
"Intracellular calcium concentration ( [Ca2+]i ) was estimated and analyzed with a calcium imaging system .",
"Fresh hemocytes from fifth-instar larvae were seeded on the coverslip with Grace’s medium and incubated for 30 min at 27°C .",
"Then , the cells were loaded with the fluorescent probe Fura 2-AM ( Dojindo Laboratories , Kumamoto , Japan ) using 0 . 2% Cremophor EL ( Sigma–Aldrich ) for 30 min at 27°C .",
"The cells were subsequently washed twice with a bathing solution ( 152 mM NaCl , 5 . 4 mM KCl , 5 . 5 mM glucose , 1 . 8 mM CaCl2 , 0 . 8 mM MgCl2 , and 10 mM HEPES , pH 7 . 4 ) .",
"The coverslips were transferred to a microscopic chamber that was constantly perfused with the bathing solution at ≈ 2 ml/min ( Huang et al . , 2012 ) .",
"The fluorescence at 510 nm by excitation at 340 or 380 nm with a xenon lamp was measured with individual cells using an Easy Ratio Pro calcium imaging system ( Photon Technology International , Birmingham , NJ ) .",
"Each experiment was repeated three times or more .",
"EC50 values were estimated by fitting to a dose-dependent curve in Origin Pro ( Origin Lab , Northampton , MA ) .",
"The tetracycline-resistant S . aureus was grown overnight in a shaking incubator at 37ºC shaking at 225 rpm .",
"Cultures were spun down .",
"The resuspended cells were diluted in sterile PBS to achieve an optical density ( OD ) of 0 . 4 .",
"Five to seven days post-eclosion , flies were injected with 32 . 2 nl of the bacterial resuspension using a Drummond Scientific Nanoject II .",
"Flies were kept at 25°C .",
"Flies that died within 6 hr were considered dead and were removed from the count .",
"Flies were transferred every day to new food and death was recorded every 12 hr .",
"The tetracycline-resistant S . aureus was cultured in LB broth overnight at 37ºC shaking at 225 rpm , and subcultured to an OD of 0 . 4 .",
"Approximately 24 female flies per genotype were injected with 23 nl of the bacterial suspension .",
"Eight flies from each group were then immediately homogenized in individual tubes with 100 μl sterile PBS .",
"The material was serially diluted to 1:10 , twice , in sterile PBS , and then plated on LB plates containing tetracycline .",
"After 6 and 18 hr of infection at 25°C , eight additional flies from each genotype per time point were assayed as above with the exception that each sample was serially diluted 1:10 five times .",
"The plates were incubated at 37°C for 8 to 10 hr .",
"Bacterial colonies were counted when the colonies remain small and discrete .",
"Data had a normal distribution and are expressed as means ± standard error ( s . e . m . ) .",
"Data were analyzed using analysis of variance ( ANOVA ) with Tukey-Kramer post-hoc test and Student t tests .",
"Log rank tests were used to determine whether survival curves of male flies were significantly different from one another .",
"Gehan-Breslow-Wilcoxon test were used to determine whether survival curves of female flies were significantly different from one another .",
"All curve fitting and statistical calculations were performed with Origin 8 . 0 ( Origin Lab , Northampton , MA ) and GraphPad Prism 5 . 0 ( San Diego , CA ) ."
]
] | [
"Serotonin ( 5-HT ) modulates both neural and immune responses in vertebrates , but its role in insect immunity remains uncertain .",
"We report that hemocytes in the caterpillar , Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide .",
"The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis .",
"Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors .",
"The blockade of 5-HT1B significantly reduced phagocytic ability; however , the blockade of 5-HT2B increased hemocyte phagocytosis .",
"The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria , due to their decreased phagocytotic ability .",
"Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection .",
"Combined , these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution ."
] | [
"Serotonin is a small molecule found in organisms across the animal kingdom .",
"This molecule plays various roles in the human body and affects many systems including the gut and central nervous system .",
"Over recent decades , serotonin has been found to play a role in the immune system too , and appears to help regulate how immune cells respond to invasion by infectious bacteria or viruses .",
"Various types of immune cells that can engulf foreign particles or microorganisms via a process called phagocytosis have receptors for serotonin on their cell surface and are activated when serotonin is present .",
"Signaling pathways associated with part of the immune system in mammals are often highly similar to pathways found in insects .",
"Serotonin is also known to influence many processes in insects , such as appetite , sleep and reproduction , but its role in the insect’s immune system was not well understood .",
"In particular , insects have phagocytic cells known as hemocytes and it was unknown if serotonin helps to activate these cells .",
"Qi , Huang et al . have now discovered that serotonin does indeed control the activity of insect hemocytes from the caterpillars of the small white butterfly ( Pieris rapae ) and the fruit fly ( Drosophila melanogaster ) .",
"The experiments showed that two distinct receptors on a hemocyte’s cell surface can detect serotonin .",
"One of these receptors increases phagocytic activity in both insects , while the other has the opposite effect in the caterpillar and reduces this activity . Qi , Huang et al . also discovered that phagocytosis depends on which of these receptors is most common on the hemocyte cell surface , and demonstrated that insects exposed to bacteria start to produce more of the serotonin receptors that increase phagocytosis .",
"Further experiments showed that fruit flies in which the gene for a serotonin receptor has been deleted are more vulnerable to bacterial infections due to their poor phagocytic ability .",
"Insects and mammals are separated by about 500 million years of evolution , and so these findings suggest that serotonin is an ancient signaling molecule that can control the immune system across the animal kingdom .",
"The work also supports the idea that studies of the simpler immune systems of insects , including the model organisms such as D . melanogaster , can offer insight into the immune systems of humans and other animals ."
] | 2016 |
[
"Materials and methods"
] | [
"short report",
"computational and systems biology"
] | RNA polymerase errors cause splicing defects and can be regulated by differential expression of RNA polymerase subunits | elife-09945-v2 | [
[
"Much existing RNA-seq data is available as bam files aligned to the human genome .",
"In order to bypass alignment , which is the most computationally expensive step of the pipeline , I developed a method capable of using RNA-seq reads aligned with spliced aligners .",
"First , in order to avoid increased mismatch rates at splice junctions due to alignment problems with both spliced and unspliced reads , I used SAMtools ( Li et al . , 2009 ) and awk to remove all alignments that do not align along the full length of the genome ( e . g . , for 76 bp reads , only reads with a CIGAR flag of 76 M ) .",
"The remaining reads weretrimmed ( bamUtil , trimBam ) to convert the first and last 10 bp of each read to Ns and set the quality strings to ‘ ! ’ .",
"I then used samtools mpileup ( -q30 –C50 –Q30 ) and custom perl code to count the number of reads and number of errors at each position in genome .",
"Positions with too many errors ( e . g . , more than one read of the same nonreference base ) were not counted .",
"I used the University of California Santa Cruz ( UCSC ) table browser ( Karolchik , 2004 ) to download two bed files: hg19 EnsemblGenes introns with -10 bp flanking from each side , and another file with the introns and +10 bp flanking on either side .",
"I then used bedtools ( Quinlan and Hall , 2010 ) ( bedtools flank -b 20 -l 0 and bedtools flank -l 20 -b 0 ) to generate bed files with intervals that contain the splicing donor and acceptor sites , respectively .",
"In addition , I used bedtools getfasta on the +10 bp flanking bed file to keep only introns flanked by GT and AG donor and acceptor sites .",
"The final result is a pair of bam files with intervals centered on the splicing donor or acceptor sites .",
"I used this new bed file to count error rates around each splice junction .",
"The error rate at each position ( e . g . , -10 , -9 , -8 , etc . from the G at the 5’ donor site ) is the sum of all errors at that position , divided by the sum of all reads .",
"Positions are relative to the splicing feature , not to the genome , as error rates at any single genomic position are dominated by sampling bias .",
"Per mono- , di- , and trinucleotide background error rates were-calculated using the same scripts , but without limiting mpileup to the splice junctions .",
"The parental strain DBY12394 ( Mcisaac et al . , 2013 ) ( GAL2 + s288c repaired HAP1 , ura3∆ , leu2∆0::ACT1pr-Z3EV-NatMX ) was transformed with a polymerase chain reaction ( PCR ) product ( KanMX-Z3EVpr ) to generate a genomically integrated inducible RPB9 ( LCY143 ) or DST1 ( LCY142 ) .",
"To induce various levels of expression , strains were re-grown in YPD + 0- , 3- , 6- , 12- , or 25-nM β-estradiol ( Sigma , St . Louis , MO , USA , E4389 ) for more than 12 hr to a final OD600 of 0 . 1 – 0 . 4 .",
"Cellular RNA was extracted using the Epicenter MasterPure RNA Purification Kit , and Illumina sequencing libraries were prepared using the Truseq Stranded mRNA kit , and sequenced on an HiSeq2000 with at least 20 , 000 , 000 50 bp sequencing reads per sample .",
"I used bwa ( Li and Durbin , 2009 ) ( -n 2 , to permit no more than two mismatches in a read ) to align the yeast RNA-seq reads to the reference genome , and trimBam from bamUtil to mask the first and last 10 bp of each read .",
"I used samtools mpileup ( Li et al . , 2009 ) ( -q 30 -d 100000 -C50 –Q39 ) to count the number of reads and mismatches at each position in the genome , discarding low confidence mapping , reads that map to multiple positions , and low quality reads .",
"Duplicate reads can be removed from the fastq file if the coverage is low enough so that all reads that map to identical genome coordinates are expected be PCR duplicates from the same RNA fragment .",
"This is the case for low coverage paired-end reads with a variable insert size , but not for very high coverage datasets or single-ended reads .",
"For the intron retention analysis in human cells , data are from NCBI SRA PRJNA253670 .",
"Data for the elc4 and spt4 analysis are from PRJNA167772 and PRJNA148851 , respectively .",
"For RPB9 correlation , undefined data ( SRA PRJNA30709 ) are all from the Gingeras lab at CSHL ."
]
] | [
"Errors during transcription may play an important role in determining cellular phenotypes: the RNA polymerase error rate is >4 orders of magnitude higher than that of DNA polymerase and errors are amplified >1000-fold due to translation .",
"However , current methods to measure RNA polymerase fidelity are low-throughout , technically challenging , and organism specific .",
"Here I show that changes in RNA polymerase fidelity can be measured using standard RNA sequencing protocols .",
"I find that RNA polymerase is error-prone , and these errors can result in splicing defects .",
"Furthermore , I find that differential expression of RNA polymerase subunits causes changes in RNA polymerase fidelity , and that coding sequences may have evolved to minimize the effect of these errors .",
"These results suggest that errors caused by RNA polymerase may be a major source of stochastic variability at the level of single cells ."
] | [
"Genes encode instructions to make proteins and other molecules .",
"To issue an instruction , a gene is first used as a template to make molecules of ribonucleic acid ( called mRNAs for short ) in a process called transcription .",
"An enzyme called RNA polymerase – which comprises several protein subunits that all work together – is responsible for making the mRNA molecules .",
"Occasionally , this enzyme makes mistakes that lead to small changes in the instruction that is produced .",
"These mistakes are rare , but because cells make thousands of mRNAs , a single human cell can make 10-100 transcription errors per second .",
"It has been difficult to study how often RNA polymerase makes mistakes and what effect these mistakes have on organisms because the techniques available for research are labour-intensive and technically challenging .",
"Here , Lucas Carey demonstrates that it is possible to use a technique called RNA sequencing to study the accuracy of RNA polymerase in human and yeast cells .",
"The experiments show that altering the levels of the different subunits of RNA polymerase in cells can change how many mistakes are made during transcription .",
"This suggests that cells may be able regulate number of mistakes by controlling the production of specific subunits .",
"Carey found that the severity of the mistakes made by RNA polymerase depends on where the mistake is in the mRNA .",
"For example , errors in specific parts of the mRNA can alter how the whole instruction is edited later , while others might make only a tiny change to the protein encoded by the gene .",
"Carey also found evidence that the instructions encoded by genes may have evolved in such a way to minimise the effect of any errors on their roles in cells .",
"RNA sequencing is less labour-intensive than other methods used to study the accuracy of RNA polymerase and is already used to address other research questions on a wide variety of different organisms .",
"Therefore , Carey’s findings will make it easier to study what genes or environmental factors influence the number of errors made during transcription .",
"A major challenge for the future is to find out if the mistakes made by RNA polymerase can lead to cancer and other human diseases ."
] | 2015 |
[
"Introduction",
"Model",
"Results",
"Discussion",
"Methods"
] | [
"structural biology and molecular biophysics"
] | Viral genome structures are optimal for capsid assembly | elife-00632-v1 | [
[
"For many viruses the spontaneous assembly of a protein shell , or capsid , around the viral nucleic acid ( NA ) is an essential step in the viral lifecycle .",
"Identifying the factors which enable capsids to efficiently and selectively assemble around the viral genome could identify targets for new antiviral drugs that block or derail the formation of infectious virions .",
"Conversely , understanding how assembly depends on the NA and protein structure would guide efforts to reengineer capsid proteins and human NAs for gene therapy applications .",
"From a fundamental perspective , high-order complexes that assemble from protein and/or NAs abound in biology .",
"Learning how the properties of viral components determine their co-assembly can shed light on assembly mechanisms of a broad array of structures and the associated selective pressures on their components .",
"In this article , we use GPU computing ( Anderson et al . , 2008; Nguyen et al . , 2011; LeBard et al . , 2012 ) and a simplified , but quantitatively testable , model to elucidate the effects of electrostatics , capsid geometry , and NA tertiary structure on assembly .",
"Assembly around NAs is predominately driven by electrostatic interactions between NA phosphate groups and basic amino acids , often located in flexible tails known as arginine rich motifs ( ARMs ) ( e . g . , Schneemann , 2006 ) .",
"There is a correlation between the net charge of these protein motifs and the genome length for many ssRNA viruses ( Belyi and Muthukumar , 2006; Hu et al . , 2008 ) , with a ‘charge ratio’ of negative charge on NAs to positive charge on proteins typically of order 2:1 ( i . e . , viruses are ‘overcharged’ ) .",
"Electrophoresis measurements confirm that viral particles are negatively charged ( e . g . , [Serwer et al . , 1995; Serwer and Griess , 1999; Porterfield et al . , 2010] ) , though these measurements include contributions from the capsid exteriors ( Bozic et al . , 2012; Zlotnick et al . , 2013 ) .",
"Based on these observations , it has been proposed that viral genome lengths are thermodynamically optimal for assembly , meaning that their lengths minimize the free energy of the assembled nucleocapsids .",
"However , while estimates of optimal lengths have varied ( van der Schoot and Bruinsma , 2005; Angelescu et al . , 2006; Belyi and Muthukumar , 2006; Hu et al . , 2008; Siber and Podgornik , 2008; Ting et al . , 2011; Ni et al . , 2012; Siber et al . , 2012 ) , recent theoretical models based on linear polyelectrolytes ( Siber and Podgornik , 2008; Ting et al . , 2011; Ni et al . , 2012 ) have consistently predicted that optimal NA lengths correspond to ‘undercharging’ ( fewer NA charges than positive capsid charges ) .",
"These results lead to the conclusion that capsid assembly around genomic ( overcharged ) NAs requires an external driving force such as a Donnan potential ( Ting et al . , 2011 ) .",
"Yet , viruses preferentially assemble around genomic length RNAs even in vitro ( Comas-Garcia et al . , 2012 ) , in the absence of such a driving force .",
"The effect of NA structural features other than charge remains unclear .",
"In some cases , genomic NAs are preferentially packaged over others with equivalent charge ( Borodavka et al . , 2012 ) due to virus-specific packaging sequences ( Bunka et al . , 2011; Borodavka et al . , 2012 ) .",
"However , experiments on other viruses have demonstrated a striking lack of virus-specific interactions ( Porterfield et al . , 2010; Comas-Garcia et al . , 2012 ) .",
"For example , cowpea chlorotic mottle virus ( CCMV ) proteins preferentially encapsidate BMV RNA over the genomic CCMV RNA ( Comas-Garcia et al . , 2012 ) .",
"Since the two NAs are of similar length , the authors propose that other structural features , such as NA tertiary structure ( Yoffe et al . , 2008 ) , may drive this preferential encapsidation .",
"However , the relationship between NA structure and assembly has not been explored .",
"To clarify this relationship , we use a computational model to investigate capsid assembly dynamics and thermodynamics as functions of NA and capsid charge , solution ionic strength , capsid geometry , and NA size ( resulting from tertiary structure ) .",
"We first test the proposed link between the thermodynamic optimum length , Leq∗ and assembly , finding that the yield of assembled nucleocapsids at relevant timescales is maximal near Leq∗ .",
"Longer-than-optimal NAs lead to non-functional structures , indicating that the thermodynamic optimum ( Leq∗ ) corresponds to an upper bound for the genome size for capsids which spontaneously assemble and package their genome .",
"We then explore how Leq∗ depends on solution conditions and the structures of capsids and NAs .",
"We find that overcharging occurs spontaneously , requiring no external driving force .",
"When base-pairing is accounted for , predicted optimal NA lengths are consistent with the genome size for a number of viruses , suggesting that electrostatics and NA tertiary structure are important factors in the formation and stability of viral particles .",
"Our predictions can be tested quantitatively in in vitro packaging experiments ( e . g . , [Porterfield et al . , 2010; Cadena-Nava et al . , 2012; Comas-Garcia et al . , 2012] ) ."
],
[
"Our coarse-grained capsid model ( Figure",
"1 ) is motivated by the recent observation ( Kler et al . , 2012 ) that purified simian virus 40 ( SV40 ) capsid proteins assemble around ssRNA molecules in vitro to form capsids comprising 12 homopentamer subunits .",
"We model the capsid as a dodecahedron , composed of 12 pentagonal subunits ( each of which represents a rapidly forming and stable pentameric intermediate , which then more slowly assembles into the complete capsid , as is the case for SV40 [Li et al . , 2002] ) .",
"Our model extends those of Wales ( 2005 ) , Fejer et al . ( 2009 ) , Johnston et al . ( 2010 ) , with subunits attracted to each other via attractive pseudoatoms at the vertices ( type ‘A’ ) and driven toward a preferred subunit–subunit angle by repulsive ‘Top’ pseudoatoms ( type ‘T’ ) and ‘Bottom’ pseudoatoms ( type ‘B’ ) ( see Figure 1 and the ‘Methods’ ) .",
"In contrast to previous models for polyelectrolyte encapsidation ( Angelescu et al . , 2006; Elrad and Hagan , 2010; Kivenson and Hagan , 2010; Mahalik and Muthukumar , 2012 ) , the proteins contain positive charges located in flexible polymeric tails , representing the ARM ( arginine-rich motif ) NA binding domains typical of positive-sense ssRNA virus capsid proteins . 10 . 7554/eLife . 00632 . 003Figure 1 . Schematics and representative images of model systems .",
"( A ) , ( B ) Model schematic for ( A ) a single subunit , and ( B ) two interacting subunits , showing positions of the attractor ( ‘A’ ) , Top ( ‘T’ ) , and Bottom ( ‘B’ ) pseudoatoms , which are defined in the ‘Model’ section and in the ‘Methods’ .",
"( C ) ( left ) The pentameric SV40 capsid protein subunit , which motivates our model .",
"The globular portions of proteins are shown in blue and the beginning of the NA binding motifs ( ARMs ) in yellow , though much of the ARMs are not resolved in the crystal structure ( Stehle et al . , 1996 ) .",
"Space-filling model of the basic subunit model ( middle ) and a pentamer from the PC2 model ( right ) .",
"( D ) A cutaway view of complete CCMV and PC2 capsids ( with respective biological charge ratios of 1 . 8 and 1 . 32 ) .",
"Beads are colored as follows: blue = excluders , green = attractors , yellow = positive ARM bead , gray = neutral ARM bead , red = polyelectrolyte . DOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 003 To investigate the effect of NA properties on assembly we consider two models for the packaged polymer: ( 1 ) a linear flexible polyelectrolyte and ( 2 ) a NA with predefined secondary and tertiary structure ( i . e . , static base-pairs ) that captures the size , shape , and rigidity of NAs .",
"Single-stranded regions are modeled as flexible polymers with one bead per nucleotide ( Zhang and Glotzer , 2004; ElSawy et al . , 2011 ) , with charge −e .",
"Double-stranded regions of NAs comprise two adjoined semiflexible strands with the net persistence length of dsDNA ( ≈50 nm ) , and base-paired nucleotides are connected by harmonic bonds .",
"Electrostatics are modeled using Debye–Huckel interactions to account for screening , except where these are tested against simulations with Coulomb interactions and explicit salt ions ( see Figure 3D below ) .",
"In addition to representing the secondary structures of specific ssRNA genomes , we are able to tune statistical measures of base-pairing , such as the fraction of nucleotides that are base-paired , the relative frequency of hairpins and higher-order junctions ( Figure 6 ) , and the maximum ladder distance ( MLD ) , which measures the extension in graph space of a NA secondary structure ( Yoffe et al . , 2008 ) .",
"As shown in Figure 6 , the radius of gyration RG of the model NAs depends on MLD as 1 . 7×MLD0 . 43 , which has a slightly smaller exponent than a theory in which only base-paired segments were accounted for ( Yoffe et al . , 2008 ) .",
"Further model details and parameters are presented in the ‘Methods’ ."
],
[
"We begin by presenting the results of simulations on our simplest capsid and cargo models .",
"Our model capsid has a dodecahedron inradius ( defined as the distance from the capsid center to a face center ) of Rin=7 . 3 nm , to give an interior volume consistent with that of the smallest icosahedral viruses , and contains 60 ARMs ( i . e . , a T=1 capsid , where T is the triangulation number [Caspar and Klug , 1962] ) each containing five positively charged residues .",
"The cargo is a linear polyelectrolyte .",
"While we systematically alter both the cargo and capsid below to include more biological detail , the simple model demonstrates two important results ( that are consistent with results from more complex models ) : ( 1 ) Viral particles spontaneously overcharge during assembly , and ( 2 ) The thermodynamic optimal polyelectrolyte length closely correlates with the length for which dynamical assembly leads to the highest yield of complete viral particles .",
"To evaluate the significance of the trends identified above for packaging in a biological context , we performed equilibrium calculations in which the structural parameters discussed above ( capsid volume , ARM length , charge , and NA base-pairing ) were based on specific T=1 and T=3 viruses ( whose capsids are assembled from 60 and 180 protein copies respectively ) .",
"For each investigated virus , the capsid radius was fit to protein densities in capsid crystal structures ( Carrillo-Tripp et al . , 2009 ) , the ARM length was determined from the structure , and charges in the ARM and on the capsid inner surface were assigned based on amino acid sequence ( see Table 1 ) .",
"NAs were modeled with 50% base-pairing and MLD/Max MLD≈0 . 5 .",
"Visualizations of T=1 and T=3 viruses ( PC2 and CCMV ) are presented in Figure 1D and further details details are provided in Figure 4—figure supplement 2 . 10 . 7554/eLife . 00632 . 016Table 1 . Details for the models of biological capsids studied in this article .",
"The capsid inradius ( distance from capsid center to face center ) , number of residues in the arginine rich motif ( ARM ) , and net charge of the ARM and inner surface are features used to build these models .",
"The viral genome length is then presented for comparison to the value of Leq∗ predicted for the base-paired model .",
"Finally , the fraction of occupied volume within the capsid is given for the base-paired model at the optimal lengthDOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 016VirusInradius ( nm ) ARM Length/Net chargeGenome lengthLeq∗Occupied volume fractionPaV13 . 048/+13432247660 . 074CCMV11 . 548/+10323331360 . 099BMV11 . 544/+9323330870 . 093PC28 . 043/+22176716720 . 265STNV7 . 728/+16123912420 . 240BBT7 . 527/+12106610580 . 209STMV7 . 219/+1110589220 . 232SPMV6 . 820/+138269180 . 276 The predicted values of Leq∗ for linear polyelectrolytes and base-paired NAs are compared to the actual viral genome lengths in Figure 4 .",
"We see that overcharging ( charge ratios larger than 1 , Figure 4B ) is predicted for all structures .",
"Furthermore , while the values of Leq∗ predicted for linear polyelectrolytes fall short of the viral genome lengths for all investigated structures except for SPMV ( whose virion has an unusually low charge ratio ) , Leq∗ for the NA models are relatively close to the viral genome lengths for most structures .",
"We emphasize that the optimal length is sensitive to all of the control parameters; for example , Leq∗ is correlated not just with the capsid charge , but also with capsid volume and ARM packing fraction ( see Figure 4—figure supplement 2 ) .",
"Recalling that Leq∗ sets an upper bound on length of a polymer that can be efficiently packaged during assembly ( Figure 2B ) , this result suggests that the geometric effects of base-pairing contribute to spontaneous packaging of viral genomes .",
"The largest difference between Leq∗ and genome length occurs for STMV .",
"This discrepancy may reflect the fact that we used a NA base-pairing fraction of fbp=0 . 5 whereas 57% of nucleotides participate in secondary structure elements in the STMV crystal structure ( Larson et al . , 1998; Zeng et al . , 2012 ) ( lower fractions of nucleotides are resolved in other virion structures , suggesting lower values of fbp ) ."
],
[
"Analysis of conformations of encapsulated polymers in our simulations shows that overcharging arises as a consequence of geometry and electrostatic screening .",
"The presence of discrete positive charges located in ARMs ( or on the capsid surface ) combined with nm-scale screening of electrostatics limits the number of direct NA–protein electrostatic interactions; the remaining nucleotides are found in segments which bridge the gaps between positive charges .",
"These interconnecting ( bridging ) segments are the primary origin of overcharging .",
"Earlier approaches which assumed spherical symmetry could not capture these bridging segments and thus did not predict overcharging .",
"The significance of bridging segments to overcharging is clearly revealed by the dependence of optimal length on capsid size under constant ARM length ( Figures 3B ) .",
"For Rin≥12 . 5 nm , the amount of NA interacting with the ARMs is constant , while bridging lengths increase with capsid radius ( Figure 5—figure supplement 3 ) due to the increased typical distance between charges on different ARMs .",
"The increase in Leq∗ with capsid radius in these calculations can be attributed entirely to increased bridge lengths .",
"Although the amounts of bridging segments in the biological capsid models depend on many control parameters ( e . g . , charge , volume , packing fraction , RNA structure ) , we also confirmed the significance of bridging segments to overcharging in these calculations .",
"Figure 5 breaks down the Leq∗ into the number of segments which interact with positive ARM charges and the number of segments which are bridging .",
"If one counts only the NA segments that directly interact with capsid charges , the resulting charge ratio is slightly less than one for each of these capsids .",
"However , when the bridging segments are included , all the capsids are overcharged .",
"Interestingly , more bridging segments are found in the larger T=3 capsids ( 56% bridging ) than in T=1 capsids ( 25% bridging ) , contributing to the larger predicted charge ratios for T=3 capsids ( Figure 4B ) .",
"Though the fraction of nucleotides closely interacting with protein in capsids is difficult to measure experimentally , it might be estimated from the amounts of RNA resolved in crystallographic or EM structures .",
"In a recent summary , Larsson et al . found that for 10 T=3 crystal structures an average of 16% of NA were resolved .",
"For T=1 structures a wider range of values was obtained , where some had a large fraction of NA resolved ( STMV = 62% , STNV = 34% ) , but other ssDNA viruses had resolved fractions similar to T=3 viruses .",
"An additional piece of evidence comes from low resolution neutron diffraction , where 72% of RNA was observed to be in the first layer of density along the inner capsid surface of the T=1 STNV , again suggesting that much of the T=1 viral genome is closely interacting with the protein ( Bentley et al . , 1987 ) .",
"We present additional data describing the conformation of the polymer within the capsid , including the radial and angular densities as Figure 5—figure supplements 1 , 2 , 3 , 4 . 10 . 7554/eLife . 00632 . 017Figure 5 . Bridging in biological capsids .",
"Number of NA segments that directly interact with positively charged ARM segments ( interaction energy ≤−0 . 5kBT , blue squares ) and bridging segments ( interaction energy >−0 . 5kBT , purple circles ) .",
"The numbers are calculated at the optimal length Leq∗ for each capsid shown in Figure 4 using the base-paired model .",
"For visual reference , the dashed line indicates a 1:1 correspondence between capsid charge and number of nucleotides . DOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 01710 . 7554/eLife . 00632 . 018Figure 5—figure supplement 1 . Radial density for linear polymer and ARM segments in the simple capsid ( A ) and CCMV ( B ) .",
"The sharp peak in ARM density is due to the first ARM segment , which is rigidly attached to the capsid shell .",
"In the simple capsid the polymer segments are concentrated within a few nm of the capsid shell , with lower densities in the capsid center .",
"For CCMV , the longer arms result in a more diffuse distribution of positive charges within the capsid interior as compared to the basic capsid model .",
"While there is some co-localization of positively charged ARM and combined neutral and negatively charged polymer segments , their densities peak at slightly different radii .",
"The CCMV ARM sequence contains 48 segments , with 11 positive segments and 1 negative segment .",
"Though the charges are not homogenously distributed throughout the sequence ( 9 occur within a 19 segment stretch ) , the degree of separation observed was unexpected . DOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 01810 . 7554/eLife . 00632 . 019Figure 5—figure supplement 2 . Angular density of linear polymer segments ( heat map ) in the basic capsid model ( A ) and CCMV ( B ) .",
"Green squares indicate the first ARM segment .",
"Segment densities are averaged over radial distances of 5–6 . 25 nm ( A ) and 8 . 75–10 nm ( B ) , as a function of the spherical angles , without angular averaging .",
"For the simple capsid , the polymer more frequently resides in the vertices between subunits ( between the clusters of 3 ARMs ) as well as along the dodecahedral edges , and resides less frequently in the center of the subunit faces .",
"The angular density is heterogeneous in CCMV , though to a lesser extent than found for the simple capsid . DOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 01910 . 7554/eLife . 00632 . 020Figure 5—figure supplement 3 . Capsid radius and polymer bridging . Number of polymer segments interacting with positive capsid charges ( red inverted triangles ) , and number of polymer segments not interacting with positive charges ( bridging segments , blue diamonds ) , using threshold interaction distance of 0 . 74 nm , which corresponds to a screened electrostatic interaction of −0 . 5kBT .",
"The numbers are calculated at the optimal polymer length Leq∗ as a function of capsid inradius Rin for the simple capsid with constant ARM length ( Figure 3B ) .",
"The number of polymer segments strongly interacting with ARM charges is constant for Rin≥12 . 5 nm , while the number of bridging segments increases to span the distances between arms .",
"Hence , for capsids with Rin≥12 . 5 nm , the observed dependence of Leq∗ on capsid size arises entirely due to bridging segments .",
"For smaller capsids , there is a weak increase in the number of interacting segments with size as more conformational space around the ARMs becomes available . DOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 02010 . 7554/eLife . 00632 . 021Figure 5—figure supplement 4 . Number of NA segments that directly interact with positively charged ARM segments and bridging segments , for both the linear and base-paired model . For visual reference , the dashed line indicates a 1:1 correspondence between capsid charge and number of nucleotides .",
"This data shows that while the base-paired polymer increases the charge ratio it does so by increasing both the number of segments which are tightly bound and bridging . DOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 02110 . 7554/eLife . 00632 . 022Figure 6 . Base-paired polymer setup and analysis .",
"( A ) Schematic illustrations of the algorithm we used to obtain a wide range of base-paired structures .",
"From left to right , double-stranded ( ds ) segments are first randomly assigned .",
"These segments are then base-paired together , starting from one end .",
"If base-paired segments are widely separated ( i . e . , lpairis large ) then subsequent nested base-pairs lead to an extended structure .",
"Conversely , if lpair is small , less extended structures form .",
"The right-most panel indicates a psuedoknot , a structural motif we have prevented from occurring in this model , by setting lmax to the last unpaired segment .",
"( B ) Radius of gyration RG for model NAs isolated in solution as a function of maximum ladder distance ( MLD ) normalized by the maximum possible MLD .",
"The nucleic acid has 1000 nt , 50% of which are base-paired .",
"( C ) The frequency of junction numbers can be altered by varying λ in Equation 2 , with large values of λ leading to large values of lpair .",
"The symbols indicate the relative frequency of junction numbers for biological RNAs with indicated lengths , obtained from Ref . ( Gopal et al . , 2012 ) , and the lines are best fits to these distributions generated by varying λ .",
"The inset illustrates several different junction orders .",
"( D ) The thermodynamic optimum length measured for the simple model capsid as a function of the fraction of base-paired nucleotides fbp for a simplified ‘hairpins only’ model ( red squares ) .",
"( E ) Snapshots illustrating assembly around a NA .",
"Beads are colored as follows: blue = excluders , yellow = ARM bead , red = single-stranded NA , cyan = double-stranded NA .",
"‘Top’ , ‘Bottom’ , and ‘Attractor’ beads removed for clarity . DOI: http://dx . doi . org/10 . 7554/eLife . 00632 . 022 We emphasize that our coarse-grained model is designed to incorporate the minimal set of features required to explain the thermodynamic stability of viral particles , and thus neglects some factors that contribute to packaging specific NAs .",
"The in vivo experiments in Ni et al . ( 2012 ) on brome mosaic virus ( BMV ) showed that even charge-conserving mutations to ARM residues could affect the amounts and types of packaged RNA , possibly by interfering with coordination of RNA replication and encapsidation ( Rao , 2006 ) .",
"Similarly , packaging signals , or regions of RNA that have sequence-specific interactions with the capsid protein , are known for some viruses ( e . g . , HIV [D’Souza and Summers , 2005] or MS2 and satellite tobacco necrosis virus [STNV] [Bunka et al . , 2011; Borodavka et al . , 2012] ) .",
"Packaging signals could be added to our model to investigate how they favor selective assembly around the viral genome through kinetic ( Borodavka et al . , 2012 ) or thermodynamic effects .",
"The fact that our model predicts Leq∗ for STNV close to its genome length without accounting for sequence specificity may suggest that packaging signals have only a small effect on the thermodynamic Leq∗ .",
"In conclusion , our results elucidate the connection between structure and assembly for viral capsids .",
"Firstly , our simulations show that ‘overcharged’ capsids are favored thermodynamically and kinetically , even in the absence of cellular factors or other external effects .",
"Secondly , our results delineate how the genome length which is most favorable for assembly depends on virus-specific quantities such as capsid charge , capsid volume , and genomic tertiary structure .",
"While the correlation between predicted optimal lengths and viral genome sizes suggests that our results have biological relevance , the physical foundations of our model can be tested via controlled in vitro experiments .",
"As noted above , several existing experimental observations agree with our results .",
"A positive correlation between protein charge and amounts of packaged RNA has been demonstrated through experiments in which the charge on capsid protein ARMs was altered by mutagenesis ( e . g . , [Dong et al . , 1998; Kaplan et al . , 1998; Venter et al . , 2009] ) .",
"Competition assays ( Porterfield et al . , 2010; Comas-Garcia et al . , 2012 ) , in which two species of NAs or other polymers compete for packaging by a limiting concentration of capsid proteins , offer a quantitative estimate of Leq∗ .",
"For example , our prediction that Leq∗ for CCMV is roughly consistent with the genome length ( Figure 4 ) agrees with the observation that CCMV proteins preferentially package longer RNAs , up to the wildtype genome length , over shorter RNAs in competition assays ( Comas-Garcia et al . , 2012 ) .",
"Now , it is possible to quantitatively test the predictions of our model for the dependence of Leq∗ on protein charge and salt concentration through similar competition assays in which NA length preferences are observed for proteins with charge altered by mutagenesis under different ionic strengths .",
"Similarly , our prediction that base-pairing increases Leq∗ can be evaluated by comparison of assembly experiments on RNA and synthetic polyelectrolytes ( e . g . , polystyrene sulfonate ) or RNA with base-pairing inhibited through chemical modification ( e . g . , etheno-RNA [Dhason et al . , 2012] ) .",
"Our simulations predict that above the optimal length for a linear polyelectrolyte , only base-paired RNA will be packaged in high yields of well-formed capsids ."
],
[
"We have extended a model for empty capsid assembly ( Wales , 2005; Fejer et al . , 2009; Johnston et al . , 2010 ) to describe assembly around NAs .",
"A complete listing of the interaction potentials is provided below; here we present a concise description of our model .",
"The pseudoatoms in the capsid subunit model are illustrated in Figure 1 .",
"Subunit assembly is mediated through an attractive Morse potential between Attractor ( ‘A’ ) pseudoatoms located at each subunit vertex .",
"The Top ( ‘T’ ) pseudoatoms interact with other ‘T’ psuedoatoms through a potential consisting of the repulsive term of the LJ potential , the radius of which is chosen to favor a subunit-subunit angle consistent with a dodecahedron ( 116° ) .",
"The Bottom ( ‘B’ ) pseudoatom has a repulsive LJ interaction with ‘T’ pseudoatoms , intended to prevent ‘upside-down’ assembly .",
"The ‘T’ , ‘B’ , and ‘A’ pseudoatoms form a rigid body ( Wales , 2005; Fejer et al . , 2009; Johnston et al . , 2010 ) .",
"See Schwartz et al . ( 1998 ) , Rapaport et al . ( 1999 ) , Rapaport ( 2004 , 2008 ) , Hagan and Chandler ( 2006 ) , Hicks and Henley ( 2006 ) , Nguyen et al . ( 2007 ) , Wilber et al . , ( 2007 , 2009a , 2009b ) , Hagan ( 2008 ) , Nguyen and Brooks ( 2008 ) , Nguyen et al . ( 2009 ) , Elrad and Hagan ( 2010 ) , Johnston et al . ( 2010 ) , Hagan et al . ( 2011 ) , Mahalik and Muthukumar ( 2012 ) , Hagan ( 2013 ) for related models .",
"To model electrostatic interaction with a negatively charged NA or polyelectrolyte we extend the model as follows .",
"Firstly , to better represent the capsid shell we add a layer of ‘Excluder’ pseudoatoms which have a repulsive LJ interaction with the polyelectrolyte and the ARMs .",
"Each ARM is modeled as a bead–spring polymer , with one bead per amino acid .",
"The ‘Excluders’ and first ARM segment are part of the subunit rigid body .",
"ARM beads interact through repulsive Lennard–Jones interactions and , if charged , electrostatic interactions modeled by a Debye–Huckel potential .",
"Comparison to Coulomb interactions with explicit counterions is shown in Figure 3D .",
"We also show that the results do not change significantly when experimentally relevant concentrations of divalent cations are added to the system ( Figure 3D ) .",
"The ability of the Debye–Huckel model to provide a reasonable representation of electrostatics in the system can be understood based on the relatively low packing fractions ( see Table 1 ) within the assembled capsids and the fact that the relevant experimental and physiological conditions correspond to moderate to high salt concentrations .",
"Simulations were performed with the Brownian Dynamics algorithm of HOOMD , which uses the Langevin equation to evolve positions and rigid body orientations in time ( Anderson et al . , 2008; Nguyen et al . , 2011; LeBard et al . , 2012 ) .",
"Simulations were run using a set of fundamental units .",
"The fundamental energy unit is selected to be Eu≡1kBT .",
"The unit of length Du is set to the circumradius of a pentagonal subunit , which is taken to be 1Du≡5 nm so that the dodecahedron inradius of 1 . 46Du=7 . 3 nm gives an interior volume consistent with that of the smallest T=1 capsids .",
"Assembly simulations were run at least 10 times for each set of parameters , each of which were concluded at 2×108 time steps .",
"The following parameters values were used in all of our dynamical assembly simulations: λD=1 nm , box size = 200 × 200 × 200 nm , subunit concentration = 12μM .",
"During calculation of the thermodynamic optimal polymer length Leq∗ , calculations were run at least 1×107 timesteps , with equilibrium assessed after convergence .",
"Standard error was obtained based on averages of multiple ( ≥3 ) independent simulations .",
"Separate calculations of Leq∗ were also performed using using the Widom test-particle method ( Widom , 1963 ) as extended to calculate polymer residual chemical potentials ( Kumar et al . , 1991; Elrad and Hagan , 2010 ) ( described in more detail below ) .",
"Snapshots from simulations were visualized using VMD ( Humphrey et al . , 1996 ) .",
"To obtain base-paired polymers with a wide and tunable range of structures ( i . e . , maximum ladder distances ) , we implement the following strategy .",
"Firstly , the polymer contour length LC , length of the base-paired segments Lbp , and fraction of nucleotides in base-pairs fbp are free parameters which we specify ( typical values are LC=1000 nucleotides , Lbp=5 nucleotides per segment , and fbp=0 . 5 ) .",
"Secondly , we iterate over the linear sequence of the polymer , randomly choosing segments which will undergo base-pairing to form double-stranded ( ds ) segments .",
"Each segment consists of Lbp consecutive nucleotides .",
"Segments are numbered sequentially to facilitate pairing ( i . e . , the first ds segment in the sequence is 1 , the second is 2 , and so on ) .",
"Thirdly , these ds segments are then paired together .",
"In the case of the hairpin model , each ds strand is paired with the next ds segment in the sequence ( i . e . , the first segment with the second , third with fourth , and so on ) .",
"In the general base-pairing model , pairs are assigned stochastically according to an algorithm which allows us to simultaneously tune the distribution of junction orders and the maximum ladder distance ( MLD ) .",
"The algorithm is described in Figure 6A defined as follows: The first step in assigning a base-pair is to obtain a random separation lrandom from an exponential distribution where λ is the inverse of the mean: ( 1 ) ( lrandom ( λ , l ) ) =λe−lλ .",
"To prevent pseudoknots this lrandom is then subtracted from the maximal available separation lmax to yield lpair: ( 2 ) lpair ( lmax , lrandom ) =lmax−lrandom .",
"The obtained lpair defines the number of segments separating the current segment from its base-pairing partner .",
"With this algorithm , the single control parameter parameter λ is used to control both the base-pairing pattern , and thus MLD and the distribution of junction types , that is , the number of double stranded segments emerging from a single stranded intersection ( see Figure 6C ) .",
"When λ is large , we are more likely to obtain small values of lrandom , and thus large values of lpair .",
"Large lpair values lead to more extensive structures ( i . e . , larger MLDs and a larger fraction of two-junctions ) .",
"When λ is lower , we have a broader distribution of lrandom values , and thus obtain smaller values of lpair .",
"If lpair is small , it creates higher-order junctions and regions which are not part of the MLD .",
"To describe the structures of the polymers generated by this algorithm , we make use of two structural parameters: the maximum ladder distance ( MLD ) and radius of gyration ( RG ) .",
"As in ( Yoffe et al . , 2008 ) , we define the MLD as the largest number of base-pairs in any single path across the molecule’s secondary structure .",
"Figure 6B describes the polymer radius of gyration RG as a function of MLD , normalized by the maximal possible MLD ( i . e . , if all base-pairs were along a single path ) , for polymers of length 1000 with fraction base-pairing fbp=0 . 5 .",
"All of the base-paired polymers are compressed relative to the linear polymer ( RG=25 . 5 nm ) , but they differ amongst themselves significantly .",
"We observe RG to vary with MLD as RG∼MLD0 . 43 to yield sizes in the range RG≈8 nm to RG≈20 .",
"Our method of calculating the subunit–subunit binding free energy is similar to that presented in our previous simulations ( Elrad and Hagan , 2010; Hagan et al . , 2011 ) .",
"Briefly , subunits were modified such that only one edge formed attractive bonds , limiting complex formation to dimers .",
"We measured the relative concentration of dimers and monomers for a range of attraction strengths ( ε ) .",
"The free energy of binding along that interface is then gcc=−kBT ln ( css/Kd ) with standard state concentration css=1 M and Kd in molar units .",
"This free energy is well fit by the linear expression gcc−1 . 5ε−Tsb where sb=−9kBT .",
"We can then correct for the multiplicity of dimer conformations , by adding in the additional term T−Δsc=ln ( 25/2 ) , where the five pentagonal edges are assumed to be distinguishable , but complex orientations which differ only through global rotation are not .",
"Our assembly simulations are run at ε=5kBT , for which we observe only transient subunit–subunit associations except in the presence of an anionic polyelectrolyte .",
"Our free energy calculations agree with this observation , suggesting that for this value of ε binding is very weak: Kd=0 . 33M and gcc=−1 . 1kBT .",
"Note that formation of additional bonds in a capsid structure will give rise to substantially more negative binding free energies .",
"As shown in Hagan and Chandler ( 2006 ) much of the binding entropy penalty associated with adding a subunit to a capsid is incurred during the formation of the first bond , with smaller decreases in entropy associated with forming additional bonds .",
"A similar set of calculations for capsids with the ARMs removed decreased the binding free energy to gcc=−1 . 84kBT , indicating that ARM–ARM interactions increase the free energy by about 0 . 74kBT along each interface at Csalt=100 mM .",
"The free energy as a function of encapsidated polymer length was obtained by two different methods .",
"In the first , we placed a very long polymer in or near a preassembled capsid , with one of the capsid subunits made permeable to the polymer .",
"We then performed unbiased Brownian dynamics .",
"Once the amount of packaged polymer reached equilibrium , the thermodynamic optimum length Leq∗ and the distribution of fluctuations around it were measured .",
"In the second approach we used the Widom test-particle method ( Widom , 1963 ) as extended to calculate polymer residual chemical potentials ( Kumar et al . , 1991; Elrad and Hagan , 2010 ) .",
"We performed independent sets of simulations for a free and an encapsidated polymer in which we calculated the residual chemical potential μr according to: ( 3 ) −βμr ( Np ) ≡−β ( μchain ( Np+1 ) −μchain ( Np ) ) =log〈exp ( −βUI ( Np ) ) 〉where Np is the number of segments in the polymer and UI is the interaction energy experienced by a test segment inserted onto either end of the polymer .",
"Importance sampling was used to make the calculation feasible , where the bond length of inserted segments was chosen from a normal distribution matching the equilibrium distribution of bond lengths , truncated at ±3 standard deviations .",
"The effect of using this biased insertion was removed a posteriori according to standard non-Boltzmann sampling .",
"Between incrementing Np , 105 steps of dynamics were run , and 105 insertions were attempted for each value of Np in 100 independent runs .",
"The results of these calculations are presented in Figure 2—figure supplement 2 , and based on the point of intersection between the encapsidated and unencapsidated chemical potentials , we estimate the optimal length Leq∗ to be between 550–575 segments ( or a charge ratio of 1 . 83−1 . 92 ) , which is close agreement with the preassembled dynamics calculations ( 574 segments or a charge ratio of 1 . 91 ) .",
"If we integrate the difference in chemical potential between the encapsidated and unencapsidated polymers between 0 and 575 , we obtain −500kT as an estimate for the free energy of polymer encapsidation due to both polymer–ARM and polymer–polymer interactions in the simple capsid model with ARM length = 5 .",
"Since the primary motivation for the Widom test-particle method calculations was to provide an independent test of optimal lengths calculated using the semipermeable capsid , we only considered the Debye–Huckel model for electrostatics in test-particle method calculations .",
"To further assess the convergence and sampling of both approaches for calculating the Leq∗ , we performed additional replica exchange ( REX ) simulations ( Sugita and Okamoto , 1999 ) .",
"In replica exchange , replicas of the system are simulated in parallel at different temperatures .",
"Periodically , structures are exchanged between temperatures based on the Metropolis Criterion .",
"In our systems , 12 replicas were run , with temperatures distributed exponentially between 1 kT and 1 . 5 kT .",
"This resulted in a satisfactory exchange frequency of 30–40% .",
"We present the results for REX simulations in Figure 2—figure supplement 2 , but in this case and all other cases , the REX results quantitatively agree with the results of our simulations run at a single temperature .",
"In our model , all potentials can be decomposed into pairwise interactions .",
"Potentials involving capsomer subunits further decompose into pairwise interactions between their constituent building blocks—the excluders , attractors , ‘Top’ and ‘Bottom’ , and ARM pseudoatoms .",
"It is convenient to write the total energy of the system as the sum of 6 terms: a capsomer-capsomer Ucc part ( which does not include interactions between ARM pseudoatoms ) , capsomer-polymer Ucp , capsomer-ARM Uca , polymer-polymer Upp , polymer-ARM Upa , and ARM-ARM Uaa parts .",
"Each is summed over all pairs of the appropriate type: ( 4 ) U=∑cap i ∑cap j<iUcc+∑cap i ∑poly jUcp+∑cap i ∑ARM jUca+∑poly i ∑poly j<iUpp+∑poly i ∑ARM jUpa+∑ARM i ∑ARM j<iUaawhere ∑cap i∑cap j<i is the sum over all distinct pairs of capsomers in the system , ∑cap i∑poly j is the sum over all capsomer-polymer pairs , etc .",
"The capsomer-capsomer potential Ucc is the sum of the attractive interactions between complementary attractors , and geometry guiding repulsive interactions between ‘Top’–‘Top’ pairs and ‘Top’–‘Bottom’ pairs .",
"There are no interactions between members of the same rigid body , but ARMs are not rigid and thus there are intra-subunit ARM–ARM interactions .",
"Thus , for notational clarity , we index rigid bodies and non-rigid pseudoatoms in Roman , while the pseudoatoms comprising a particular rigid body are indexed in Greek .",
"For example , for capsomer i we denote its attractor positions as {aiα} with the set comprising all attractors α , its ‘Top’ positions {tiα} , and its ‘Bottom’ positions {biα} .",
"The capsomer–capsomer interaction potential between two capsomers i and j is then defined as: ( 5 ) Ucc ( {aiα} , {tiα} , {biα} , {ajβ , {tjβ} , {bjβ} ) =∑α , βNtεL ( |tiα−tjβ| , σt ) +∑α , βNb , NtεL ( |biα−tjβ| , σb ) +∑α , βNaεM ( |aiα−ajβ| , r0 , ϱ , rcut ) where ε is an adjustable parameter which both sets the strength of the capsomer–capsomer attraction at each attractor site and scales the repulsive interactions which enforce the dodecahedral geometry .",
"Nt , Nb , and Na are the number of ‘Top’ , ‘Bottom’ , and attractor pseudoatoms respectively in one capsomer , σt and σb are the effective diameters of the ‘Top’–‘Top’ interaction and ‘Bottom’–‘Top’ interactions , which are set to 10 . 5 nm and 9 . 0 nm respectively throughout this work , r0 is the minimum energy attractor distance , set to 1 nm , ϱ is a parameter determining the width of the attractive interaction , set to 2 . 5 , and rcut is the cutoff distance for the attractor potential , set to 10 . 0 nm .",
"The function ℒ is defined as the repulsive component of the Lennard–Jones potential shifted to zero at the interaction diameter: ( 6 ) ℒ ( x , σ ) ≡{ ( σx ) 12−1:x<σ0:otherwise The function ℳ is a Morse potential: ( 7 ) ℳ ( x , r0 , ϱ ) ≡{ ( eϱ ( 1−xr0 ) −2 ) eϱ ( 1−xr0 ) :x<rcut0:otherwise The capsomer–polymer interaction is a short-range repulsion that accounts for excluded-volume .",
"For capsomer i with excluder positions {xiα} and polymer subunit j with position Rj , the potential is: ( 8 ) Ucp ( {xiα} , Rj ) =∑αNxℒ ( |xiα−Rj| , σxp ) where Nx is the number of excluders on a capsomer and σxp=0 . 5 ( σx+σp ) is the effective diameter of the excluder–polymer repulsion .",
"The diameter of the polymer bead is σp=0 . 5 nm and the diameter for the excluder beads is σx=3 . 0 nm for the T=1 model and σx=5 . 25 nm for the T=3 model .",
"The capsomer–ARM interaction is a short-range repulsion that accounts for excluded-volume .",
"For capsomer i with excluder positions {xiα} and ARM subunit j with position Rj , the potential is: ( 9 ) UcA ( {xiα} , Rj ) =∑αNxℒ ( |xiα−Rj| , σxA ) with σxA=0 . 5 ( σx+σA ) as the effective diameter of the excluder–ARM repulsion with σA=0 . 5 nm the diameter of an ARM bead ."
]
] | [
"Understanding how virus capsids assemble around their nucleic acid ( NA ) genomes could promote efforts to block viral propagation or to reengineer capsids for gene therapy applications .",
"We develop a coarse-grained model of capsid proteins and NAs with which we investigate assembly dynamics and thermodynamics .",
"In contrast to recent theoretical models , we find that capsids spontaneously ‘overcharge’; that is , the negative charge of the NA exceeds the positive charge on capsid .",
"When applied to specific viruses , the optimal NA lengths closely correspond to the natural genome lengths .",
"Calculations based on linear polyelectrolytes rather than base-paired NAs underpredict the optimal length , demonstrating the importance of NA structure to capsid assembly .",
"These results suggest that electrostatics , excluded volume , and NA tertiary structure are sufficient to predict assembly thermodynamics and that the ability of viruses to selectively encapsidate their genomic NAs can be explained , at least in part , on a thermodynamic basis ."
] | [
"Viruses are infectious agents made up of proteins and a genome made of DNA or RNA .",
"Upon infecting a host cell , viruses hijack the cell’s gene expression machinery and force it to produce copies of the viral genome and proteins , which then assemble into new viruses that can eventually infect other host cells .",
"Because assembly is an essential step in the viral life cycle , understanding how this process occurs could significantly advance the fight against viral diseases .",
"In many viral families , a protein shell called a capsid forms around the viral genome during the assembly process .",
"However , capsids can also assemble around nucleic acids in solution , indicating that a host cell is not required for their formation .",
"Since capsid proteins are positively charged , and nucleic acids are negatively charged , electrostatic interactions between the two are thought to have an important role in capsid assembly .",
"However , it is unclear how structural features of the viral genome affect assembly , and why the negative charge on viral genomes is actually far greater than the positive charge on capsids .",
"These questions are difficult to address experimentally because most of the intermediates that form during virus assembly are too short-lived to be imaged .",
"Here , Perlmutter et al . have used state of the art computational methods and advances in graphical processing units ( GPUs ) to produce the most realistic model of capsid assembly to date .",
"They showed that the stability of the complex formed between the nucleic acid and the capsid depends on the length of the viral genome .",
"Yield was highest for genomes within a certain range of lengths , and capsids that assembled around longer or shorter genomes tended to be malformed .",
"Perlmutter et al . also explored how structural features of the virus—including base-pairing between viral nucleic acids , and the size and charge of the capsid—determine the optimal length of the viral genome .",
"When they included structural data from real viruses in their simulations and predicted the optimal lengths for the viral genome , the results were very similar to those seen in existing viruses .",
"This indicates that the structure of the viral genome has been optimized to promote packaging into capsids .",
"Understanding this relationship between structure and packaging will make it easier to develop antiviral agents that thwart or misdirect virus assembly , and could aid the redesign of viruses for use in gene therapy and drug delivery ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | Stage-specific effects of Notch activation during skeletal myogenesis | elife-17355-v3 | [
[
"Skeletal muscle accounts for approximately 40% of adult human body weight ( Yin et al . , 2013 ) .",
"Besides the motor function that is essential for life , muscle is also a key metabolic organ , responsible for 80–90% of postprandial insulin-stimulated glucose uptake .",
"In addition , muscle intimately interplays with many other organs , and regulates their homeostasis and metabolism ( Baskin et al . , 2015 ) .",
"Therefore , proper muscle function is important for human health and life quality .",
"In adult skeletal muscles , resident stem cells called satellite cells ( SCs ) are indispensable for postnatal muscle growth and regeneration ( Le Grand and Rudnicki , 2007; Lepper et al . , 2011; Murphy et al . , 2011; Pawlikowski et al . , 2015; Relaix and Zammit , 2012; Sambasivan et al . , 2011 ) .",
"SCs are wedged between the plasma membrane of muscle fiber ( i . e . , myofiber ) and the basal lamina that surrounds the myofiber .",
"The juxtaposition of SCs immediately identifies myofiber as an important regulator of SCs .",
"Consistent with this notion , transplantation of myofibers carrying SCs , but not SCs alone , elicited life-long amelioration of aging-related muscle atrophy ( Hall et al . , 2010 ) .",
"Besides myofibers , SCs also actively interact with interstitial fibroblasts , preadipocytes , endothelial cells and macrophages , which together with myofibers constitute the unique microenvironment , or niche that regulates the homeostasis of SCs ( Arnold et al . , 2007; Aurora and Olson , 2014; Christov et al . , 2007; Joe et al . , 2010; Liu et al . , 2012b; Murphy et al . , 2011; Sonnet et al . , 2006 ) .",
"Age-related muscle wasting ( sarcopenia ) and degenerative muscular dystrophy are linked to decline of SCs’ abundance and functionality , as a result of changes in niche function and intrinsic properties of SCs ( Conboy et al . , 2005; Sacco and Puri , 2015 ) .",
"As such , FGF2 , p38 , STAT3 , p16 , canonical Wnt and Notch signaling , and epigenetic status are identified as aberrantly dysregulated factors in aged SCs that account for impaired self-renewal of SCs during aging ( Bernet et al . , 2014; Brack et al . , 2007; Chakkalakal et al . , 2012; Cosgrove et al . , 2014; Liu et al . , 2013; Price et al . , 2014; Sousa-Victor et al . , 2014; Tierney et al . , 2014 ) .",
"Stepwise progression of myogenesis is tightly orchestrated by several key transcriptional factors ( Bentzinger et al . , 2012 ) .",
"Among them , Pax7 ( Paired box 7 ) is the faithful marker for SCs , and also indispensable for proper postnatal muscle regeneration by controlling expansion and differentiation of SCs ( Kuang et al . , 2006; Seale et al . , 2000; von Maltzahn et al . , 2013 ) .",
"Activation of myogenesis relies on the expression of several members of the basic helix-loop-helix domain-containing myogenic regulatory factors: Myf5 , Myod1 ( also known as MyoD ) , Myf6 and myogenin ( Myog ) ( Bentzinger et al . , 2012 ) .",
"When stimulated , SCs break quiescence and express MyoD , which promotes S-phase entry and facilitates SCs activation ( Halevy et al . , 2004; Olguin and Olwin , 2004; Zammit et al . , 2004; Zhang et al . , 2010 ) .",
"In addition , MyoD and Myf5 act as upstream regulators of Myog and Myf6 , which are required for terminal differentiation of myocytes ( Bentzinger et al . , 2012 ) .",
"Notably , certain microRNAs , epigenetics , serum response factor ( SRF ) and members of myocyte enhancer factor 2 ( MEF2 ) are also fundamentally important for proper muscle development ( Cheung et al . , 2012; Haberland et al . , 2009; Li et al . , 2005; Liu et al . , 2014; 2012a; Shenoy and Blelloch , 2014; Williams et al . , 2009 ) , which eventually leads to expression of muscle tissue-specific genes such as muscle creatine kinase ( MCK ) , myosin light chain ( MLC ) and myosin heavy chain ( MHC ) members .",
"The opposite process of differentiation , or dedifferentiation is a fascinating phenomenon that bears great therapeutic potentials by generating stem cell sources for tissue repair .",
"In zebrafish and amphibians , dedifferentiation is an integral part of tissue regeneration ( Jopling et al . , 2010; Morrison et al . , 2006 ) .",
"Specifically , in the newt , fragmentation and dedifferentiation of myofibers generate a pool of proliferating mononucleated cells that give rise to the skeletal muscle of a new limb ( Sandoval-Guzman et al . , 2014 ) .",
"However , in mammals , there is no evidence of dedifferentiation that occurs as a natural part of tissue regeneration .",
"The underlying mechanism that determines such dramatic inter-species difference is largely unknown .",
"Intriguingly , recent studies reported that dedifferentiation can be induced in muscle cells of several genetically engineered mice ( Kubin et al . , 2011; Pajcini et al . , 2010 ) .",
"For instance , concomitant inactivation of two tumor-suppressors Arf and Rb in mononucleated mouse myocytes led to loss of differentiation properties , cell cycle reentry and generation of cells that were capable to redifferentiate into skeletal muscles ( Pajcini et al . , 2010 ) .",
"Undoubtedly , better understanding of mechanisms underlying muscle dedifferentiation will allow the invention of future medicine to treat geriatric muscle disease , where the SC number is limited .",
"Notch signaling is an evolutionarily conserved pathway that plays crucial functions in organ development , tissue homeostasis , stem cell fate choice , metabolism and cancers ( Andersson et al . , 2011; Bi and Kuang , 2015; Koch et al . , 2013; Ranganathan et al . , 2011 ) .",
"Notch signaling transduction is initiated upon binding of a Notch receptor ( Notch1-4 ) with a ligand ( Dll1 , Dll4 , Jag1 , Jag2 ) located on a neighbor cell ( Andersson et al . , 2011 ) .",
"Subsequently , Notch receptors are cleaved by several enzymes including γ–secretase , which releases the Notch intracellular domain ( NICD ) .",
"NICD then translocates to the nucleus , where it binds with Rbpj and other cofactors to activate the transcription of canonical Notch targets , including Hes and Hey family genes ( Kopan , 2012 ) .",
"In postnatal myogenesis , Notch signaling is indispensable for maintaining quiescence and self-renewal of SCs ( Bjornson et al . , 2012; Fukada et al . , 2011; Mourikis et al . , 2012b; Vasyutina et al . , 2007 ) .",
"In addition , during development SCs rely on Notch signaling to acquire 'satellite' position aside of myofiber by controlling the assembly of basal lamina ( Brohl et al . , 2012 ) .",
"Furthermore , Notch is a potent inhibitor of muscle differentiation by inhibiting expression of MyoD ( Delfini et al . , 2000 ) .",
"As such , deletion of Rbpj or Dll1 led to premature differentiation and depletion of SCs , thus a loss of muscle growth and severe muscle hypotrophy ( Schuster-Gossler et al . , 2007; Vasyutina et al . , 2007 ) .",
"Despite of the wealth of knowledge about Notch signaling in SCs , its function in late-stage myogenesis is unknown .",
"Here , we report that activation of Notch signaling dedifferentiates myocytes into Pax7-expressing SCs , leading to defective myogenesis .",
"By contrast , activation of Notch signaling in post-fusion myotubes/myofibers restored the functionality and regenerative capacity of aged and dystrophic muscles ."
],
[
"Myl1Cre ( MLC-Cre ) ( Bothe et al . , 2000 ) and MCK-Cre ( Bruning et al . , 1998 ) mouse strains are widely used tools to study gene function in post-differentiation muscle lineages , by inducing recombination that causes either overexpression or knockout of candidate genes .",
"However , the spatiotemporal activation pattern of MLC-Cre and MCK-Cre has not been characterized .",
"It is conceivable of an earlier expression of the MLC-Cre than MCK-Cre , based on the developmental expression of Myl1 and MCK at day-9 . 5 and day-13 . 0 post conception , respectively ( Lyons et al . , 1991 , 1990; Zammit et al . , 2008 ) .",
"In order to directly visualize and trace the activation of these Cre lines , we generated Td-tomato ( red fluorescence protein ) reporters for MLC-Cre and MCK-Cre , termed MLC-Td and MCK-Td mice , respectively .",
"First , we cultured myoblasts from 1-month old MLC-Td and MCK-Td mice , and found that none of the freshly isolated SCs expressed RFP .",
"Then we expanded the cells and induced myogenic differentiation .",
"Under growth conditions , RFP expression was first detected in a few MLC-Td but not MCK-Td myocytes that were Pax7– , MyoD– , MyoG– , but MF20+ ( Figure 1A and Figure 1—figure supplement 1 ) .",
"Later on , RFP was readily detectable in fusion-competent MLC-Td myocytes immediately after switching to the differentiation medium ( Figure 1B ) .",
"By contrast , RFP was only detected in post-fusion myotubes starting from day-3 after induction of myogenic differentiation in MCK-Td cells ( Figure 1B ) . 10 . 7554/eLife . 17355 . 003Figure 1 . Sequential activation of Myl1Cre ( MLC-Cre ) and MCK-Cre in post-differentiation myocytes and post-fusion myotubes , respectively .",
"( A , B )",
"Immunofluorescence images of cultured myoblasts ( A ) and myocytes ( B ) .",
"Arrow in A points to a weak MyoG+/Td+ cell .",
"Arrowhead in B points to Td– myofibers .",
"( C , D )",
"Immunofluorescence images ( C ) of TA cross sections of postnatal day-12 mice , and myonuclei counting ( D ) .",
"Arrow points to nT myonucleus .",
"( E ) Immunofluorescence images of TA cross sections at different days post CTX injury ( dpi ) : 1 ( MLC-nTnG , 4 dpi ) , 2 ( MCK-nTnG , 4 dpi ) , 3 ( MCK-nTnG , 6 dpi ) , 4 ( MCK-nTnG , 9 dpi ) , 5 ( MLC-nTnG , 21 dpi ) , 6 ( MCK-nTnG , 21 dpi ) .",
"Arrowhead in panel 3 points to the nT/nG myofiber . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 00310 . 7554/eLife . 17355 . 004Figure 1—figure supplement 1 . Expression of myogenic markers in MLC-Td myoblasts . Primary myoblasts were isolated from MLC-Td mice and cultured in growth medium .",
"Cells were fixed and stained with antibodies to detect Pax7 , Myog , Myod and MF20 , and merged with Td fluorescence . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 004 Next , we examined the Cre activation pattern in skeletal muscles in vivo .",
"To unmask potential mosaic Cre activation in multinucleated myofiber that can’t be distinguished by the Td reporter , we utilized the nTnG reporter ( Prigge et al . , 2013 ) , in which nucleus-localized GFP ( nG ) or Td-tomato ( nT ) is expressed in the presence or absence of Cre , respectively .",
"Intriguingly , 96% and 55% myonuclei ( within myofibers outlined by dystrophin ) were nG+ in cross sections of tibialis anterior ( TA ) muscles from postnatal day-12 MLC-nTnG and MCK-nTnG mice , respectively ( Figure 1C and D ) .",
"By 1 . 5-month old , all myonuclei ( 100% ) became nG+ in both MLC-nTnG and MCK-nTnG mice ( data not shown ) , coinciding with the completion of the myonuclei addition in growing muscles ( White et al . , 2010 ) .",
"We further examined the nT and nG expression at different stages of muscle regeneration after cardiotoxin ( CTX ) induced muscle injury .",
"Consistently , we detected massive nG+ myonuclei 4 days post injury ( dpi ) in MLC-nTnG mice ( Figure 1E1 ) .",
"By contrast , MCK-nTnG myofibers didn’t express nG at 4 dpi ( Figure 1E2 ) , but started to express nG at 6 dpi ( Figure 1E3 ) , and peaked at 9 dpi ( Figure 1E4 ) .",
"Eventually , myonuclei in both MCK-nTnG and MLC-nTnG muscles were predominantly nG+ at 21 dpi ( Figure 1E5 and E6 ) , which marks the completion of muscle regeneration .",
"In summary , these in vitro and in vivo genetic mapping results reveal sequential activation of MLC-Cre and MCK-Cre along myogenesis , with earliest MLC-Cre activation detected in mononuclear myocytes and MCK-Cre detected only in multinucleated myotubes ( myofibers ) .",
"To investigate the role of Notch signaling in post-differentiation myocytes , we generated the Myl1Cre/Rosa26 ( Gt ) SorN1ICD ( abbreviated as MLC-N1ICD ) mouse model , in which the expression of constitutively active Notch1-ICD ( N1ICD ) is driven by Myl1Cre ( Figure 2A ) .",
"The MLC-N1ICD mice were born at an expected Mendelian ratio with normal body weight ( data not shown ) .",
"Adult MLC-N1ICD mice expressed higher levels of Notch1ICD and its target genes , including Hes1 , Hey1 and Heyl , in both fast ( gastroceminus , Figure 2B ) and slow ( soleus , Figure 2C ) muscles , confirming activation of Notch signaling .",
"However , MLC-N1ICD mice showed less body weights at weaning ( postnatal day-21 ) , and less body weight gains and muscle mass in adulthood , compared with wildtype ( WT ) mice ( Figure 2D and E ) .",
"By 4-month of age , all MLC-N1ICD mice displayed prominent kyphosis ( Figure 2F ) , which is a sign of severe muscle weakness and a hallmark of aging .",
"As early phase of postnatal muscle growth is normally mediated by myonuclei addition , we quantified the myonucleus numbers of EDL myofibers , and found that MLC-N1ICD had only around half the myonuclei numbers of WT myofibers ( Figure 2G and H ) .",
"Consequently , MLC-N1ICD mice were subjected to premature death starting from 2-month of age , and no MLC-N1ICD mice survived longer than 6 months ( Figure 2I ) . 10 . 7554/eLife . 17355 . 005Figure 2 . Muscle growth and motor-function defects of MLC-N1ICD mice .",
"( A ) Cartoon illustration of Notch activation by Cre in MLC-N1ICD mice .",
"( B , C )",
"Relative gene expression levels in fast ( B , gastrocnemius , n = 3 ) and slow muscles ( C , soleus , n = 4 ) .",
"( D ) Growth curve of MLC-N1ICD mice ( n = 3 ) .",
"( E ) TA muscle weight ( n = 4 ) .",
"( F ) Image of MLC-N1ICD-Td mouse with kyphosis ( arrow ) , note muscles are in red color as labeled by RFP ( Td ) .",
"( G , H )",
"Immunofluorescence images of EDL fiber ( G ) and quantification of myonucleus numbers ( H , n = 3 ) .",
"( I ) Survive curve of MLC-N1ICD mice .",
"( J ) Exhaustive treadmill exercise test results ( n = 4 ) .",
"( K ) Gripping strength measurement result of limbs ( n = 3 ) .",
"*p<0 . 05 , **p<0 . 01 .",
"Bar graphs indicate mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 005 Although 1 . 5-month old MLC-N1ICD mice had smaller muscles , they moved normally in the cage .",
"We utilized treadmill to assess if their exercise performance is impaired .",
"Notably , MLC-N1ICD mice ran significantly more slowly and shorter distance compared with WT mice ( Figure 2J ) .",
"In addition , MLC-N1ICD mice showed lower gripping strength ( Figure 2K ) .",
"Together , these results reveal normal embryonic muscle development but dramatic defects in postnatal muscle growth and function in MLC-N1ICD mice .",
"To evaluate the muscle regeneration potential of MLC-N1ICD mice , we injured TA muscles with CTX and evaluated the regeneration at 7 dpi .",
"In non-CTX injected leg , the MLC-N1ICD myofibers are smaller than those of WT mice ( Figure 3A , left; Figure 2G ) ; with CTX injection , MLC-N1ICD showed drastically compromised muscle regeneration , evidenced by massive infiltration of non-muscle cells into TA ( Figure 3A; right ) .",
"We also performed Pax7 immunostaining to detect satellite cells .",
"Unexpectedly , despite of severe regenerative defects , MLC-N1ICD muscles showed greatly increased number of Pax7+ satellite cells , in both control and injured TA muscles ( Figure 3B ) .",
"Similarly , MLC-N1ICD EDL myofibers showed 10-times more Pax7+ satellite cells than WT myofibers ( Figure 3C ) .",
"Consistently , Pax7 expression was significantly higher at both mRNA and protein levels in MLC-N1ICD muscles ( Figure 3D and E ) .",
"To determine whether these changes are caused by SC proliferation , we co-labeled MLC-N1ICD SCs with Ki67 .",
"However , none of the Pax7+ cells expressed Ki67 ( Figure 3F ) .",
"Similarly , we didn’t detect MyoD and MyoG expression in these cells ( Figure 3—figure supplement 1A ) .",
"As a positive control of staining , the cultured WT myoblasts were Ki67+ and MyoD+ ( Figure 3—figure supplement 2A , B ) .",
"These results indicate that these Pax7+ cells are in a quiescent state .",
"Indeed , we detected significantly higher expression levels of marker genes that are abundantly ( Cdh15 , encodes M-cadherin ) or exclusively ( calcitonin receptor , Calcr; teneurin transmembrane protein 4 , Tenm4 ) expressed by quiescent SCs ( Figure 3G ) ( Fukada et al . , 2007 ) .",
"Similar changes were also observed in soleus muscles of MLC-N1ICD mice ( Figure 3—figure supplement 1B and C ) . 10 . 7554/eLife . 17355 . 006Figure 3 . Muscle regeneration defect , and myocyte dedifferentiation in MLC-N1ICD mice .",
"( A ) H&E staining results of TA muscle cross sections .",
"Right panels , 7 dpi .",
"( B ) Immunofluorescence images of TA muscle cross sections .",
"Arrow points to Pax7+ cell in WT mice .",
"( C ) Immunofluorescence images ( left ) and quantification ( right , n = 3 ) of Pax7+ cells ( arrow ) on EDL fibers .",
"( D ) Western blot results of protein extracts from non-injured muscle .",
"( E , G )",
"Relative expression of Pax7 ( E , n = 3 ) and quiescent SC marker genes ( G , n = 5 ) in gastrocnemius muscle .",
"( F , H )",
"Immunofluorescence images of EDL fibers .",
"Note all Pax7+ cells are Ki67– in F; Note Pax7+/Td+ cells only appeared on MLC-N1ICD-Td but not on MLC-Td fibers in H , arrow points to Pax7+/Td– cells in H . *p<0 . 05 , **p<0 . 01 .",
"Bar graphs indicate mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 00610 . 7554/eLife . 17355 . 007Figure 3—figure supplement 1 . Myocytes dedifferentiation in MLC-N1ICD mice .",
"( A ) Immunostaining result of MyoG on EDL myofibers .",
"( B , C )",
"Relative expression of Pax7 ( B , n = 3 ) and quiescent SC marker genes ( C , n = 4 ) in soleus muscles .",
"( D ) Immunofluorescence result of Pax7 on regenerating MLC-Td EDL fibers , note all Pax7+ cells are Td– .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 00710 . 7554/eLife . 17355 . 008Figure 3—figure supplement 2 . Immunostaining results of myofibers , myoblasts and sections of CTX damaged TA muscles . Lack of Ki67 ( A ) and MyoD ( B ) expression in Pax7+ cells in freshly isolated EDL myofibers from WT and MCK-N1ICD mice .",
"The right panels are control staining for Ki67 and MyoD positive cells in cultured WT myoblasts .",
"( C ) WT and MLC-N1ICD TA muscles at 7 days after CTX injury showing Pax7+ and Ki67+ myoblasts .",
"( D ) Percentage of Pax7+/Ki67+ cells among all Pax7+ cells in WT and MLC-N1ICD muscles as stained in C . n = 182 cells in WT , 660 cells in MLC-N1ICD muscles from two pairs of mice . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 008 As Notch signaling is required for maintaining the quiescence in SCs ( Bjornson et al . , 2012; Mourikis et al . , 2012b ) , we hypothesized that these extra Pax7+ SCs are dedifferentiated from MLC-Cre myocytes , as a consequence of cell-autonomous Notch activation .",
"To test this hypothesis , we generated the Cre-inducible RFP ( Td-tomato ) reporter mice in combination with Notch activation , namely MLC-N1ICD-Td mice .",
"Strikingly , 73% of the Pax7+ SCs in MLC-N1ICD mice were labeled as Td+ ( Figure 3H; second row ) , demonstrating that they were previously differentiated ( Myl1+ ) .",
"As a control , no Pax7+/Td+ SC was detected in resting and CTX-injected muscles of adult MLC-Td mice ( Figure 3 H; first row , Figure 3—figure supplement 1D ) , which is consistent with the earlier tracing result of cultured MLC-Td SCs ( Figure 1A ) .",
"In addition , in the CTX damaged muscles , we detected reduced numbers of Pax7+/Ki67+ cells in MLC-N1ICD versus WT mice ( Figure 3—figure supplement 2C , D ) .",
"In summary , Notch1 activation dedifferentiated MLC-Cre+ myocytes into quiescent SCs .",
"In parallel , to investigate the role of Notch signaling in post-fusion myofibers , we generated the MCK-Cre/Rosa26 ( Gt ) SorN1ICD ( abbreviated as MCK-N1ICD ) mouse model .",
"MCK-N1ICD mice were born at normal Mendelian ratio and expressed higher levels of Notch1 and Notch target genes , including Hes1 , Hes5 , Hey1 and Heyl ( Figure 4—figure supplement 1A ) .",
"Compared to WT littermates , adult MCK-N1ICD mice didn’t show any significant differences in body weight , myosin expression , neuromuscular junction morphology , denervation responses , exercise performance and gripping strength ( Figure 4—figure supplement 1B–H ) .",
"In addition , adult MCK-N1ICD mice displayed normal muscle regeneration after a single episode of CTX injury ( Figure 4—figure supplement 2A; first row ) .",
"In response to multiple rounds of injuries induced by repetitive CTX injections , however , the MCK-N1ICD muscles regenerated much better than the WT muscles , manifested by overall larger muscle volume ( Figure 4—figure supplement 2B ) , appearance of larger regenerating myofibers and homogeneous regenerated area throughout the muscle ( Figure 4—figure supplement 2A; second row ) .",
"As aged muscles ( >1-year old ) expressed reduced levels of Notch receptors and Notch targets than young muscles ( around 1 month old ) ( Figure 4—figure supplement 2C and D ) , we investigated if MCK-N1ICD improves muscle regeneration in aged mice .",
"At 15-month old , MCK-N1ICD muscles regenerated more efficiently than those of WT littermates , evidenced by larger and more regenerating myofibers , reduced adipocyte infiltration ( Figure 4—figure supplement 2A; third row ) , hallmarks of human sarcopenia ( Taaffe et al . , 2009 ) .",
"Moreover , the aged MCK-N1ICD mice achieved a higher maximum speed and longer running distance in the treadmill test ( Figure 4—figure supplement 2E ) .",
"Together , Notch1 activation driven by MCK-Cre improves muscle function and regeneration in aged mice .",
"We next asked if myofiber-specific activation of Notch1 improves muscle pathology in mdx mice , a widely used model for Duchenne Muscular Dystrophy ( DMD ) in humans .",
"To achieve this goal , we generated MCK-N1ICD-mdx mice ( short as N1ICD-mdx ) and detected upregulation of Hes1 expression in the muscles of the N1ICD-mdx mice ( Figure 4A ) , indicating Notch activation .",
"A prominent feature of mdx mice is the continuous cycles of muscle regeneration and degeneration that lead to muscle pseudo-hypertrophy: larger but weaker muscles ( Chamberlain et al . , 2007 ) .",
"Interestingly , compared with littermate mdx mice , adult N1ICD-mdx mice showed 11% less body weight and 27% less muscle mass ( Figure 4B and C ) .",
"Such changes were not observed in 4-week old N1ICD-mdx mice ( before pseudo-hypertrophy ) and adult MCK-N1ICD mice ( Figure 4—figure supplement 1B ) .",
"Therefore , the body weight reduction phenotype of adult N1ICD-mdx mice is specific to the mdx , and coincides with the peak of muscular dystrophy in adult mdx mice ( Faber et al . , 2014 ) .",
"Consistently , H&E and immunostaining revealed the relatively smaller fiber size , but fewer centronuclear and necrotic IgG+ myofibers in N1ICD-mdx mice , compared with mdx mice ( Figure 4D; first row , E and F ) .",
"Given this , we interpreted the reductions of muscle mass as a sign of less compensatory pseudo-hypertrophy and improved muscle function . 10 . 7554/eLife . 17355 . 009Figure 4 . Improved muscle morphology , regeneration and exercise performance of adult MCK-N1ICD-mdx ( short as N1ICD-mdx ) mice .",
"( A ) Western blot results of Notch target gene Hes1 in muscle protein extracts .",
"( B , C )",
"Mice body weights ( B , n = 4 ) and TA muscle weights ( C , n = 3 ) , to show less muscle pseudo-hypertrophy of N1ICD-mdx , versus mdx mice .",
"( D ) H&E staining results of TA muscle sections .",
"( E , F )",
"Quantification of central nuclei fiber ratio ( E , n = 3 ) and IgG+ fiber numbers ( F , n = 7 ) of non-CTX injected mdx and N1ICD-mdx mice .",
"( G ) Results of Evans blue dye ( EBD ) uptake by control ( left leg ) and 7 dpi CTX-injured muscles ( right leg ) .",
"( H ) Immunofluorescence staining results of TA muscle cross sections .",
"( I , J )",
"Exhaustive treadmill exercise test results ( n = 5 ) .",
"( K ) Gripping strength measurement of limbs of adult mice ( n = 15 ) .",
"*p<0 . 05 , **p<0 . 01 .",
"Bar graphs indicate mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 00910 . 7554/eLife . 17355 . 010Figure 4—figure supplement 1 . Normal muscle development , function and denervation response of MCK-N1ICD mice .",
"( A ) Gene expression of Notch1 and its target genes in muscle tissues ( n = 6 ) .",
"( B ) Growth curve of mice ( n = 4 ) .",
"( C , D )",
"Silver staining ( C ) and quantification result ( D ) of gastrocnemius protein extracts separated by SDS-PAGE gel .",
"( E ) α-Bungarotoxin ( green ) staining of EDL muscles .",
"( F ) Weights of control muscle and 2-week post denervation muscles ( n = 4 ) .",
"( G ) Exhaustive treadmill exercise test results of adult mice ( n = 4 WT and 9 MCK-N1ICD mice ) .",
"( H ) Gripping strength measurement result of limbs of adult mice ( n = 5 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 01010 . 7554/eLife . 17355 . 011Figure 4—figure supplement 2 . Improved muscle regeneration and function of aged MCK-N1ICD mice .",
"( A ) H&E staining results of 7 dpi muscle of indicated conditions .",
"Arrows point to adipocytes in aged muscles .",
"( B ) Image of TA muscle sections after 4 serial CTX-injuries , 7 dpi .",
"( C , D )",
"Gene expression of Notch receptors and target genes in WT muscle tissues ( C , n = 5 young and 6 >1-year old muscles ) .",
"( E ) Exhaustive treadmill exercise performance test results of aged mice .",
"*p<0 . 05 , **p<0 . 01 .",
"Bar graphs indicate mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 011 In addition , we performed CTX-induced muscle damage , followed by Evans blue dye ( EBD ) injection to label myofibers with dysfunctional and permeable membranes .",
"Notably , the muscles of N1ICD-mdx mice showed much less EBD uptake than mdx muscles ( Figure 4G ) .",
"Consistently , there were fewer EBD+myofibers in N1ICD-mdx than mdx muscles ( Figure 4H ) .",
"H&E staining revealed better muscle regeneration , characterized by more and larger myofibers , and less inflammatory infiltration in the CTX-injected TA muscles of N1ICD-mdx mice ( Figure 4D; second row ) .",
"Furthermore , adult N1ICD-mdx mice also exhibited better muscle exercise performance and stronger gripping strength than their littermate mdx mice ( Figure 4I–K ) .",
"To systematically evaluate the impact of myofiber-specific Notch1 activation on mdx muscles , we used microarray to survey the transcriptomes of N1ICD-mdx and mdx muscles .",
"Among the total 882 differentially expressed genes ( by ≥1 . 5–fold ) , 505 genes were downregulated and 377 genes were upregulated in N1ICD-mdx , compared with mdx muscles ( Figure 5—source data 1 ) .",
"From the microarray , we identified Gdf11 ( Growth Differentiation Factor 11 ) as the highest upregulated gene in MCK-N1ICD muscles ( Figure 5—figure supplement 1A ) .",
"Consistently , we validated upregulation of Gdf11 expression in all muscles examined in both MCK-N1ICD-mdx and MLC-N1ICD mice , compared with their littermate controls ( Figure 5—figure supplement 1B and C ) .",
"Considering the contrasting phenotypes of MLC-N1ICD and MCK-N1ICD mice , however , it is unlikely that Gdf11 directly mediates the effects of N1ICD overexpression in skeletal muscle function .",
"We performed Gene Set Enrichment Analysis ( GSEA ) ( Subramanian et al . , 2005 ) to interrogate the down- and up-regulated gene sets with a reference human DMD gene expression dataset from NCBI ( GDS3027 ) , which includes 14 healthy human muscle samples and 23 DMD human muscle samples ( Figure 5A ) ( Pescatori et al . , 2007 ) .",
"Commonly regulated genes in these murine and human datasets were assigned with enrichment scores that correlate to the statistical significance and fold changes of the genes’ appearance in the human DMD expression dataset . 10 . 7554/eLife . 17355 . 012Figure 5 . N1ICD-mdx muscle transcriptomes gained gene signatures enriched in healthy versus DMD human muscles .",
"( A ) Cartoon illustration of experiment design for ( B–D ) .",
"( B , C )",
"Gene set enrichment plots from GSEA analysis of the normal human and DMD muscle gene expression database ( GDS3027 ) , interrogated with the down-regulated ( B ) and up-regulated ( C ) gene sets in N1ICD-mdx versus mdx muscles .",
"FC , fold change .",
"( D ) Heatmap results of top 10 commonly up-regulated and down-regulated gene expression in N1ICD-mdx muscles and normal human muscles ( GDS3027 ) .",
"( E ) Ingenuity analysis of 51 genes that are commonly up-regulated in N1ICD-mdx muscles and normal human muscles ( GDS3027 ) .",
"( F ) Heatmap results of gene expression in the indicated pathways .",
"FC , fold change in N1ICD-mdx relative to mdx muscles . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 01210 . 7554/eLife . 17355 . 013Figure 5—source data 1 . Agilent microarray results showing genes up-regulated and down-regulated in N1ICD-mdx versus mdx muscles . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 01310 . 7554/eLife . 17355 . 014Figure 5—figure supplement 1 . Activation of Notch1 signaling upregulates Gdf11 expression in muscles .",
"( A ) Heat-map of gene expression results from microarray analysis , n = 3 .",
"( B , C )",
"Relative expression levels of Gdf11 in different muscles of MCK-N1ICD-mdx and MLC-N1ICD mice , n = 5 for B and 3 for C . *p< 0 . 05 , **p<0 . 01 .",
"Bar graphs indicate mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 014 Strikingly , GSEA predicted significant overall enrichment ( false discovery rate q-value = 0 . 02; familywise-error rate p-value = 0 . 009 ) for the downregulated N1ICD-mdx gene-set with human genes that were upregulated in DMD versus healthy human subjects ( score , –1 . 40 ) ( Figure 5B ) .",
"In other words , the downregulated genes in N1ICD-mdx ( versus mdx ) muscles were expressed at higher levels in DMD ( versus healthy ) human muscles .",
"Similarly , there is a strong trend that N1ICD-mdx upregulated genes were also positively correlated to genes that were expressed at higher levels in healthy human muscles ( score = 1 . 08 , q = 0 . 26 , p = 0 . 13 ) ( Figure 5C ) .",
"Based on the GSEA result , we generated heatmaps for expression of the top-10 commonly down- and up-regulated genes in the murine and human arrays ( Figure 5D ) .",
"Consistent with their reduced degeneration , N1ICD-mdx muscles expressed higher levels of mature muscle specific Myh1 ( MHC-2x ) , and lower levels of regenerating muscle specific Myh8 ( Figure 5D ) .",
"Strikingly , expression of Spp1 , the biomarker of human DMD ( Pegoraro et al . , 2011 ) , is also significantly lower in N1ICD-mdx muscles , than in mdx muscles ( Figure 5D ) .",
"Interestingly , the cell cycle inhibitor p21 ( encoded by gene Cdkn1a ) , a critical modulator of senescence , and the muscle hypertrophy gene Junb ( Raffaello et al . , 2010 ) were significantly downregulated in N1ICD-mdx muscles and normal human muscles , compared with corresponding dystrophic muscles ( Figure 5D ) .",
"Both N1ICD-mdx and healthy human muscle showed upregulation of Mfn2 ( Mitofusin",
"2 ) ( Figure 5D ) , which is involved in mitochondrial fusion , and associated with better muscle glucose metabolism and amelioration of muscle atrophy ( Chen et al . , 2010; Sebastian et al . , 2012 ) .",
"In addition , upregulated in N1ICD-mdx samples was Vegfa ( Figure 5D ) , which stimulates angiogenesis and improves vascular function .",
"Consistently , Ingenuity Pathway Analysis ( IPA ) of commonly up- and down-regulated genes predicted significantly elevated 'vasculogenesis' , and decreased 'fibrosis' in N1ICD-mdx muscles ( Figure 5E ) .",
"We also examined genes that were uniquely changed in our murine microarray .",
"Notch target genes Hey2 , Hes5 and Nrarp were upregulated in N1ICD-mdx muscles ( Figure 5F ) , confirming Notch activation .",
"Interestingly , dysferlin ( dysf ) and α-dystrobrevin ( Dtna ) , two components of dystrophin-glycoprotein complex essential for sarcolemma repair and neuromuscular junction maturation ( Bansal et al . , 2003; Grady et al . , 1999 ) , were significantly elevated in N1ICD-mdx muscles ( Figure 5F ) .",
"Musk ( Muscle-specific kinase receptor ) that regulates the development of the neuromuscular junction ( Perez-Garcia and Burden , 2012 ) was also upregulated in N1ICD-mdx muscles ( Figure 5F ) .",
"Furthermore , a panel of stem cell senescence modulators , p21 ( Cdkn1a ) , p27 ( Cdkn1b ) and Rbl1 ( Cheung and Rando , 2013; Narita et al . , 2003; Sousa-Victor et al . , 2014 ) were all significantly downregulated in N1ICD-mdx muscles ( Figure 5F ) .",
"In summary , these transcriptome analysis results support the phenotypic assays demonstrating the myofiber-specific Notch1 activation improves muscle function in dystrophic skeletal muscles .",
"Sarcopenia and muscle dystrophy are linked to decline of SC abundance and functionality , which is associated with repression of Notch signaling in SCs ( Conboy et al . , 2003; Jiang et al . , 2014 ) .",
"Consistent with improved muscle regeneration , CTX-damaged 17-month old MCK-N1ICD muscles , and adult N1ICD-mdx muscles showed more Pax7+ SCs ( Figure 6A–C ) , and elevated expression of Pax7 than did mdx muscles ( Figure 6D ) .",
"To exclude the possibility that dedifferentiation of MCK-Cre+ myocytes contribute to the increases of SCs , we again performed lineage tracing using MCK-Cre in combination with N1ICD and Td reporter mice .",
"Staining of the EDL myofiber with Pax7 antibody didn’t detect any Pax7+/Td+ cells ( Figure 6E ) .",
"Therefore , unlike the MLC-N1ICD mice , the increase of SCs number in MCK-N1ICD mice is not a result of myocyte dedifferentiation . 10 . 7554/eLife . 17355 . 015Figure 6 . Notch activation in myofiber upregulates Notch ligands’ expression , therefore stimulates Notch-activation and self-renewal of satellite cells niched on MCK-N1ICD myofibers .",
"( A–C )",
"Immunofluorescence images ( A ) and quantification of Pax7+ cell numbers in CTX-damaged ( B , n = 3 ) and dystrophic TA muscles ( C , n = 7 ) .",
"( D ) Western blot results of Pax7 in muscle protein extracts .",
"( E ) Immunofluorescence image of one EDL myofiber to show that Pax7 cells are not from MCK-Cre lineage ( Td – ) of MCK-N1ICD mice .",
"Arrow points to Pax7+/Td– cell , arrow-head points to Pax7–/Td+ myonucleus .",
"( F ) Relative mRNA levels of Notch ligand genes .",
"( G , H )",
"Immunofluorescence images ( G ) and quantification result ( H , n = 3 ) of intact EDL myofibers .",
"Arrow points to Dll4 immuno-signal patch on myofiber .",
"( I , J )",
"Immunofluorescence images ( I ) and quantification ( J , n = 4 ) of GFP percentage in satellite cells ( Pax7 ) on EDL myofibers .",
"Arrow in I points GFP–/Pax7+ satellite cells; black and white images to shown individual channels of the outlined area on left images .",
"*p<0 . 05 , **p<0 . 01 .",
"Bar graphs indicate mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 01510 . 7554/eLife . 17355 . 016Figure 6—figure supplement 1 . Real-time PCR results of Notch-related gene expression in primary myoblasts ( A , n = 3 ) and myofibers ( B , n = 5 ) .",
"*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 . Values are mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 01610 . 7554/eLife . 17355 . 017Figure 6—figure supplement 2 . Expression of Notch pathway genes in myofiber .",
"( A ) Experiment design .",
"( B ) Immunofluorescence images of EDL myofiber from Pax7nGFP mice before and after trypsin stripping .",
"Nuclear GFP labels satellite cells and DAPI labels nuclei .",
"( C ) Gel electrophoresis images of RT-PCR results using mRNA isolated from EDL myofibers .",
"( D ) Immunofluorescence staining of Pax7 and Dll4 in EDL myofibers .",
"( E ) Western blot results of WT myofibers after trypsin-stripping , showing an expression of activated Notch1 ( N1ICD ) and its target Hes1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 017 Considering the juxtaposed position of SCs on their host myofibers , Notch ligands on myofibers are presumably essential triggers of SC-intrinsic Notch signaling that is necessary for the quiescence and maintenance of SCs .",
"Genetic mapping studies showed that the higher Jag1 expression is associated with better muscle phenotypes of dystrophic mice and dogs ( Vieira et al . , 2015 ) .",
"Notably , Jag1 overexpression rescued the muscular dystrophy phenotype of dystrophin-deficient zebrafish ( Vieira et al . , 2015 ) .",
"In skeletal muscle of N1ICD-mdx mice , we detected elevated expression of two Notch ligands , Jag2 and Dll4 , compared with mdx muscles ( Figure 6F ) .",
"Consistently , immunostaining of EDL myofibers revealed stronger Dll4 immunofluorescence in MCK-N1ICD than in WT myofibers ( Figure 6G and H ) .",
"Similar gene expression changes were detected in myofibers of MCK-N1ICD mice ( Figure 6—figure supplement 1A ) .",
"To directly evaluate whether the increased ligand expression in myofibers subsequently activates Notch signaling in SCs , we generated MCK-N1ICD-CpGFP mice by breeding MCK-N1ICD with Notch reporter mice ( CpGFP ) , in which expression of GFP was controlled by a promoter cassette containing the Rbpj-responsive element ( Jiang et al . , 2014; Mizutani et al . , 2007 ) .",
"Immunostaining of EDL myofibers with Pax7 and GFP antibodies revealed that 41% SCs were GFP+/Pax7+ in MCK-N1ICD-CpGFP mice , compared with 26% GFP+/Pax7+ SCs in control CpGFP mice ( Figure 6I and J ) .",
"As a positive control , we observed stronger GFP fluorescence in myofibers of MCK-N1ICD-CpGFP mice , compared with those of CpGFP mice ( Figure 6I and K ) , confirming Notch activation by N1ICD OE .",
"As a negative control , no GFP+ SC was observed on WT myofibers ( Figure 6I; first row ) .",
"Therefore , we hypothesize that MCK-N1ICD upregulates Dll4/Jag2 in myofibers , which in turn activates Notch signaling in juxtaposed SCs to regulate their self-renewal in response to aging and muscular dystrophy .",
"To address whether Notch1 signaling components are expressed in mature myofibers under normal physiological conditions , we examined EDL myofibers free of SCs and interstitial cells after trypsin digestion and extensive PBS washing ( Figure 6—figure supplement 2A ) .",
"We confirmed the complete removal of SCs as evidenced by the absence of Pax7+ cells and Pax7 expression in the trypsin-stripped EDL myofibers ( Figure 6—figure supplement 2B—C ) .",
"Notably , expression of Notch1 and its target gene Hes1 , as well as Notch ligand Dll4 and Jag2 were detected in these trypsin-stripped myofibers , though at lower levels compared to the non-trypsin-stripped myofibers ( Figure 6—figure supplement 2C—E ) .",
"To directly test the hypothesis that Notch ligands on the MCK-N1ICD myofiber mediate SC function , we utilized dibenzazepine ( DBZ ) , a γ-secretase inhibitor to block cleavage of N1ICD ( thus ligand-induced Notch activation ) without affecting the cleavage-independent N1ICD overexpression in myofibers .",
"As myofibers are physically separated from the interstitial cells by basal lamina , Dll/Jag ligands on myofiber surface should only activate Notch receptors in juxtaposed SCs .",
"Therefore , if the improved muscle function in MCK-N1ICD mice is mediated by elevated Notch ligand expression on myofiber , then the effect should be blocked by DBZ treatment .",
"We injected the mice with DBZ or vehicle control ( DMSO ) every two days continuously for 6 times , after which the mice were trained and tested on treadmill , and then examined for their regenerative capacity ( Figure 7A ) .",
"To test the long-term effect of modulating Notch signaling in SCs , we waited 20 days after last DBZ injection before phenotyping these mice .",
"Whereas the N1ICD-mdx mice treated with DMSO ran faster and farther than their littermate mdx mice , after DBZ treatment both genotypes showed similar maximum speed and distance in the treadmill test ( Figure 7B and C ) .",
"In addition , DBZ-treated N1ICD-mdx muscle displayed similar EBD uptake levels to the mdx mice , indicating similar extent of muscle repair ( Figure 7D ) .",
"Indeed , H&E staining revealed better muscle regeneration in N1ICD-mdx than mdx mice in DMSO group , and DBZ administration rendered the regeneration similarly poor in both N1ICD-mdx and mdx mice ( Figure 7E ) .",
"Taken together , these results demonstrate that the amelioration of muscle atrophy and dysfunction in N1ICD-mdx mice is mediated by modulation of Notch signaling in SCs . 10 . 7554/eLife . 17355 . 018Figure 7 . Notch inhibitor DBZ abolished improvements of exercise performance and muscle regeneration of N1ICD-mdx mice .",
"( A ) Experiment design to show the timing of different treatments .",
"EBD , Evans blue dye .",
"( B , C )",
"Exhaustive Treadmill exercise test results ( n = 6 ) .",
"( D ) Image of EBD uptake by TA muscles .",
"( E ) H&E staining results of TA muscle cross sections .",
"*p<0 . 05 .",
"Bar graphs indicate mean SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 01810 . 7554/eLife . 17355 . 019Figure 7—figure supplement 1 . Absence of skeletal muscle development in Myod1Cre-NICD ( MyoD-NICD ) mice .",
"( A ) H&E staining result of hind limb of newborn mice .",
"( B , C )",
"Immunostaining results of hind limb cross-sections for Pax7 and MyoD ( B ) , MyoG ( C ) .",
"Arrow in B points to Pax7+/MyoD+ cell , arrowhead points to Pax7–/MyoD+ cell . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 019 Collectively , our study demonstrates stage-specific functions of Notch during myogenesis .",
"We show that N1ICD overexpression in committed and activated SCs , induced by Myf5Cre and Myod1Cre , respectively , completely blocked the embryonic muscle development ( Figure 7—figure supplement 1A ) ( Mourikis et al . , 2012a ) .",
"Myod1Cre-N1ICD mice failed to survive to birth , and showed total absence of MyoD and MyoG expression ( Figure 7—figure supplement 1B and C ) .",
"However , MLC-N1ICD mice had relatively normal embryonic muscle development evidenced by similar body weight and muscle morphology at birth , but exhibited defective postnatal muscle growth and regeneration ( Figure 8 ) .",
"Furthermore , adult MCK-N1ICD mice had normal muscle growth , and better muscle regeneration and motor function as they age ( Figure 8 ) .",
"More importantly , MCK-N1ICD boosted muscle function and regenerative capacity in mdx mice ( Figure 8 ) .",
"Together , our study revealed the versatile functions of Notch1 in controlling stepwise progression of myogenesis ( Figure 8 ) . 10 . 7554/eLife . 17355 . 020Figure 8 . Summary of stage-dependent effects of Notch1 activation on muscle cell fate choice and myogenesis . Quiescent satellite cells ( SCs ) are marked as Pax7+ .",
"Expression of MyoD activates SCs , which enter into cell cycle .",
"A subpopulation of the replicated SCs downregulate Pax7 to differentiate .",
"After differentiation , MLC starts to express in myocytes and nascent myotubes , while MCK only starts to express in the multi-nucleated myofibers .",
"Activation of Notch1 in MyoDCre lineage ( Timing 1 , T1 ) blocks differentiation , promotes self-renewal of SCs , which causes absence of skeletal musculature and embryonic lethality; Activation of Notch1 in MLCCre lineage ( T2 ) induces dedifferentiation of myocytes , and generates Pax7 quiescent SCs .",
"As a consequence , MLC-N1ICD mice show pronounced defects of muscle growth , motor-function and regeneration; Activation of Notch1 in MCKCre lineage ( T3 ) upregulates expression of Notch ligands on myofiber , which physiological promotes Notch activation in neighboring cells , inducing self-renewal of satellite cells .",
"Thus , it improves muscle regeneration and exercise performance of old and mdx mice . DOI: http://dx . doi . org/10 . 7554/eLife . 17355 . 020"
],
[
"Notch signaling is indispensable for maintaining SC quiescence , and inactivation of Notch diminishes the SC pool ( Bjornson et al . , 2012; Mourikis et al . , 2012b; Wen et al . , 2012 ) .",
"Consistently , Notch signaling activity in the regenerating muscle correlates with quiescent stage of SC ( Bjornson et al . , 2012; Mourikis et al . , 2012b ) .",
"Although SCs can reversibly shift between quiescent and activated states , it’s not clear whether post-differentiation myocyte or myofiber retains the potential to reverse back to the stem cell stage .",
"Our study demonstrates that post-differentiation myocytes can dedifferentiate back into Pax7+ quiescent SCs with activation of Notch1 .",
"Intriguingly , dedifferentiation was only observed in a portion of MLC-N1ICD cells , despite that N1ICD was overexpressed in both pre-fusion myocytes and post-fusion myotubes .",
"By contrast , dedifferentiation was not observed in MCK-N1ICD mice , which specifically overexpress N1ICD in myotubes .",
"Based on these comparisons , we concluded that Notch-induced dedifferentiation is restricted to mononucleated myocytes , which doesn’t require cytokinesis as for multinucleated myofibers .",
"Consistently , although knockout of Rb and Arf promoted cell cycle re-entry of myonuclei in myotubes , cytokinesis was not observed ( Pajcini et al . , 2010 ) .",
"One important future direction is to pinpoint the downstream targets of Notch1 that mediate its dedifferentiation function in myocytes .",
"Activation of Notch1 in myotubes improved motor function and muscle regeneration of both aged and mdx mice .",
"This is accompanied by upregulation of Dll/Jag ligand expression in myofibers , higher Notch activity in juxtaposed SCs , as well as greater regenerative potentials of SCs in MCK-N1ICD mice .",
"Consistently , Notch signaling deficiency in SCs is a prominent feature of aged and dystrophic muscles ( Conboy et al . , 2003; Jiang et al . , 2014 ) .",
"As SCs are the only cells that are directly attached to the myofibers , Dll/Jag ligands on intact myofibers should only activate Notch signaling in the SCs , but not the other cells ( for example blood vessel-associated cells and interstitial cells ) that are prevented from direct physical interaction with the myofiber by the basal lamina .",
"Thus , the normalizing effects of DBZ on the improved muscle function in the MCK-N1ICD mice demonstrates that Notch-dependent enhancement of SCs’ regenerative potential is the underlying molecular and cellular mechanism mediating the effect of N1ICD overexpression in myofibers .",
"Although we detected in MCK-N1ICD muscle a gene expression profile similar to that of healthy human muscles , we didn’t find any significant upregulation of Notch pathway genes in healthy human subjects .",
"Considering the multiple cell types in muscle tissues , expression and function of Notch in individual cell types warrant further investigation .",
"In summary , our results illustrate contrasting stage-dependent effects of Notch1 activation in post-differentiation muscle cells .",
"Most importantly , our study demonstrates that activation of Notch1 improves myotube’s function as a stem cell niche .",
"This finding suggests that one strategy to ameliorate sarcopenia and muscle dystrophy diseases is through modulating Notch signaling in the myofiber ."
],
[
"All procedures involving mice were approved by Purdue University’s Animal Care and Use Committee under protocol # 1112000440 .",
"Myl1Cre mice were generously provided by Steven Burden ( Skirball Institute of Biomolecular Medicine , NYU ) .",
"The other mice were purchased from Jackson lab: Ckmm-Cre , also known as MCK-Cre ( stock# 006475 ) , Rosa26 ( Gt ) SorN1ICD ( stock# 008159 ) , Rosa26 ( Gt ) Sortd-Tomato ( stock# 007914 ) , Rosa26 ( Gt ) SornTnG ( stock# 023035 ) , mdx ( stock# 001801 ) , CpGFP ( stock#005854 ) .",
"Mice were housed in the animal facility with free access to water and standard rodent chow food .",
"Muscle regeneration was induced by injection of cardiotoxin ( CTX; Sigma-Aldrich , St . Louis , MO ) .",
"Briefly , mice were anesthetized using a ketamine-xylazine cocktail , and then 50 μl of 10 μM CTX was injected into the TA muscle .",
"Muscles were then harvested at indicated time post-injection to assess the completion of regeneration and repair .",
"DBZ was purchased from TOCRIS Bioscience ( catalog number 4489 ) , and dissolved in DMSO at a 100 mM concentration .",
"Before use , the stock was suspended at a 1:200 dilution in DMSO .",
"50 µl working solution was injected into each TA muscle of mice .",
"Mice were anesthetized using a ketamine-xylazine cocktail before DBZ injection .",
"MHC isoforms were separated by modified SDS-PAGE ( 8% acrylamide gels containing 3 . 5% glycerol ) , which was run for 22 hr at 4°C .",
"MHC isoforms were detected by silver stain after gel fixation .",
"A detailed protocol for treadmill and grip strength tests of mice are published elsewhere ( Castro and Kuang , 2017 ) .",
"In brief , the mouse was trained on treadmill ( Eco3/6 treadmill; Columbus Instruments , Columbus , OH ) with a fixed 10% slope , at constant 10 m/minute speed for 5 min daily for consecutively 3 days before test .",
"On the exercise testing day , the animals ran on the treadmill at 10 m/min for five minutes and the speed was increased by 2 m/min every two minutes until they were exhausted or a maximal speed of 46 m/min was achieved .",
"The exhaustion was defined as the inability of the animal to run on the treadmill for 10 s despite mechanical prodding .",
"Running time and maximum speed achieved was measured whereas running distance was calculated .",
"A digital grip-strength meter ( Columbus Instruments ) was used to measure four-limb grip strength in mice , which were acclimatized for 10 min before the test .",
"The mouse was allowed to grab the metal pull bar with the paws .",
"Then the mouse tail was gently pulled backward until mice could not hold on the bar .",
"The force at the time of release was recorded as the peak tension .",
"Each mouse was tested three times .",
"The average strength was defined as grip strength .",
"For sciatic nerve denervation , mice were anesthetized with ketamine .",
"The right hind limb was prepared for surgery , a 1-cm incision was made in the skin along with the axis of the femur , and the sciatic nerve was isolated .",
"To prevent reinnervation , 3–5 mm section of the sciatic nerve was cut and removed .",
"Mice were sacrificed by cervical dislocation 2 weeks after denervation .",
"For NMJ staining , muscles were dissected and fixed by 4% PFA and incubated 30% sucrose/PBS for overnight .",
"Muscles were frozen by cold isopentan with OCT-compound and sectioned longitudinally at 50 µm thickness .",
"Sections were incubated with blocking buffer containing 0 . 1% Triton-X 100 and biotinylated α-bungarotoxin ( α-BTX ) to detect NMJs for overnight .",
"NMJs were visualized by FITC-conjugated streptavidin .",
"Primary myoblasts were isolated with collagenase type I and dispase B digestion .",
"Briefly , the hind limb skeletal muscles of mice were collected , minced and digested for around 40 min .",
"The digestions were stopped with F-10 Ham's medium containing 10% FBS and centrifuged at 450 ×g for 5 min .",
"Then the cells were seeded on collagen-coated dishes and cultured in growth medium containing F-10 Ham's medium , with 20% fetal bovine serum ( FBS ) , 4 ng/mL basic fibroblast growth factor , and 1% penicillin–streptomycin at 37°C with 5% CO2 .",
"The medium was changed every 2 days .",
"Single myofiber was isolated from the extensor digitorum longus ( EDL ) muscle by digestion with 0 . 2% collagenase A ( Sigma ) in DMEM for around 45 min .",
"After digestion , EDL muscle was gentally titrated by different pore-sized glass pipettes .",
"Freshly isolated single fiber was quickly fixed in 4% PFA for 10 min , washed in 100 mM glycine three times each 10 min , followed by PBS wash for three times and blocking for 1 hr at room temperature .",
"The primary antibody was incubated at 4°C for overnight .",
"Fluorescence secondary antibody was diluted and incubated at room temperature for 1 hr .",
"Images were taken with a Leica DM6000 microscope with a 20× objective .",
"To strip SCs from myofiber and get SC-free myofibers , freshly isolated EDL myofibers were first washed with PBS for three times .",
"Myofibers were trypsinized with 0 . 25% trypsin for 5 mins to release SCs and other cells , and then washed with PBS for three times .",
"Myofibers without trypsin treatment were used as a control for immunostaining or gene expression analysis .",
"For gene expression analysis , mRNA from all myofibers liberated an EDL muscles was extracted ( ~100 myofibers ) and reverse transcribed , and subjected to PCR analysis .",
"PCR cycle number used for 18 s was 30 , and for Notch1 , Pax7 , Dll4 and Jag2 was 38 .",
"A detailed protocol for muscle histological characterization using H&E staining and myofiber typing using immunofluorescence are published in Bio-Protocol ( Wang et al . , 2017 ) .",
"Briefly , fresh TA muscles were embedded in OCT compound , and frozen in isopentane chilled on dry ice .",
"Then the tissues were cut at 10 µm thickness by Leica CM1850 cryostat .",
"Muscles sections were fixed with fresh 4% PFA for 10 min , washed in 100 mM glycine three times each 10 min , followed by PBS wash for three times and blocking for 1 hr at room temperature .",
"The primary antibody was incubated at 4°C for overnight .",
"Fluorescence secondary antibody was diluted and incubated at room temperature for 1 hr .",
"Images were taken with a Leica DM6000 microscope with a 20× objective .",
"H&E staining images were captured with a Nikon D90 digital camera mounted on a microscope .",
"Protein was isolated from cells or tissue using RIPA buffer contains 50 mM Tris-HCl ( pH 8 . 0 ) , 150 mM NaCl , 1% NP-40 , 0 . 5% sodium deoxycholate and 0 . 1% SDS .",
"Protein concentrations were determined using Pierce BCA Protein Assay Reagent ( Pierce Biotechnology ) , followed by measurement with NanoDrop .",
"Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE ) , transferred to a polyvinylidene fluoride ( PVDF ) membrane ( Millipore Corporation ) , blocked in 5% fat-free milk for one hour at room temperature , and then incubated with primary antibodies diluted in 5% milk overnight at 4°C .",
"The Hes1 ( sc ) , Hey1 ( sc ) , Gapdh ( sc-32233 ) antibodies were from Santa Cruz Biotechnology .",
"The HRP conjugated secondary antibody ( anti-rabbit IgG or anti-mouse IgG , Santa Cruz Biotechnology ) was diluted at 1:5000 .",
"Immunodetection was performed using enhanced chemiluminescence ( ECL ) Western blotting substrate ( Pierce Biotechnology ) and detected with an imaging system ( FluorChem R FR0116 ) .",
"Alternatively , the membrane was incubated with an infrared secondary antibody ( Alexa Fluor 790 goat anti-mouse IgG , A11357; Alexa Fluor 680 goat anti-rabbit IgG , A21109; Life Technologies , USA ) diluted 1:10 , 000 , and the signals were detected by using the Odyssey infrared image scanning system .",
"Total RNA was extracted from cells or tissues using Trizol Reagent according to the manufacturer’s instructions .",
"The purity and concentration of total RNA were determined by a spectrophotometer ( Nanodrop 2000c , Thermo Fisher ) at 260 nm and 280 nm .",
"Ratios of absorption ( 260/280 nm ) of all samples were between 1 . 8 and 2 . 0 .",
"Then 5 μg of total RNA were reverse transcribed using random primers with M-MLV reverse transcriptase ( Invitrogen ) .",
"Real-time PCR was carried out in a Roche Light Cycler 480 PCR System with SYBR Green Master Mix ( Roche ) and gene-specific primers as previously described ( Liu et al . , 2012b ) .",
"The 2−∆∆Ct method was used to analyze the relative changes in each gene’s expression normalized against 18S rRNA expression .",
"RNA was extracted from TA muscles of adult mdx and MCK-N1ICD-mdx mice .",
"Gene expression was analyzed by microarray with Agilent SurePrint G3 Mouse GE 8 X 60 K chip .",
"The list of significantly changed genes with a fold change ≥1 . 5–fold was used for GSEA analysis , as well as analyzed through the QIAGEN’s Ingenuity Pathway Analysis ( IPA , QIAGEN ) .",
"GSEA analysis was performed as instructed by the manual with default settings of software .",
"Trial experiments or experiments done previously were used to determine sample size with adequate statistical power .",
"Measurement values that are beyond the fence as determined by interquartile range were considered as outlier and excluded from following statistical analysis .",
"All analyses were conducted with student t test with two-tail distribution .",
"Comparison with a p value <0 . 05 was considered significant ."
]
] | [
"Skeletal myogenesis involves sequential activation , proliferation , self-renewal/differentiation and fusion of myogenic stem cells ( satellite cells ) .",
"Notch signaling is known to be essential for the maintenance of satellite cells , but its function in late-stage myogenesis , i . e . post-differentiation myocytes and post-fusion myotubes , is unknown .",
"Using stage-specific Cre alleles , we uncovered distinct roles of Notch1 in mononucleated myocytes and multinucleated myotubes .",
"Specifically , constitutive Notch1 activation dedifferentiates myocytes into Pax7 quiescent satellite cells , leading to severe defects in muscle growth and regeneration , and postnatal lethality .",
"By contrast , myotube-specific Notch1 activation improves the regeneration and exercise performance of aged and dystrophic muscles .",
"Mechanistically , Notch1 activation in myotubes upregulates the expression of Notch ligands , which modulate Notch signaling in the adjacent satellite cells to enhance their regenerative capacity .",
"These results highlight context-dependent effects of Notch activation during myogenesis , and demonstrate that Notch1 activity improves myotube’s function as a stem cell niche ."
] | [
"Muscles do much more than enable the body to move; they are also important organs involved in the metabolism .",
"Conditions ranging from muscular dystrophy to insulin resistance result from problems that affect muscle tissue .",
"Hence , understanding the signaling mechanisms that regulate how muscles develop and work will be critical to improving muscle-related health conditions .",
"To form and repair muscles , muscle progenitor cells develop ( or differentiate ) into new muscle cells , which then fuse to form muscle fibers .",
"A signaling pathway involving a protein known as Notch regulates how cells communicate during development , and has been shown to play a key role in muscle progenitor cells .",
"However , it was not known what role Notch signaling plays in the differentiated muscle cells .",
"Bi , Yue et al . have now studied genetically modified mice in which Notch signaling could be manipulated in certain types of cells .",
"In mice with increased Notch signaling in both their newly differentiated muscle cells and muscle fibers , any unfused muscle cells were forced to return to an undifferentiated state , a process called dedifferentiation .",
"This led to the muscles wasting away and resulted in the mice dying young .",
"By contrast , in mice that only experienced activated Notch signaling in their muscle fibers , no dedifferentiation was seen .",
"However , aged and dystrophic muscles in these mice regained the ability to contract and regenerate .",
"Bi , Yue et al . hope that these findings will transform into new strategies to activate or inactivate Notch signaling at different stages of muscle development or regeneration .",
"This could help to repair muscles under various disease conditions ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"immunology and inflammation"
] | IL17 factors are early regulators in the gut epithelium during inflammatory response to Vibrio in the sea urchin larva | elife-23481-v2 | [
[
"Gut epithelial cells deploy an elaborate suite of signals to transmit information about the state of the gut lumen to the wider organism .",
"These communication networks can be difficult to interpret in the context of vertebrate systems , which exhibit complexity at both morphological ( e . g . vertebrate guts are multilayered tissues that interact with many types of peripheral immune cells ) and molecular levels .",
"The gut is an ancient site of intense immune activity , and core aspects of the regulatory circuitry in this tissue are likely to be conserved across phyla .",
"Consequently , invertebrate animals provide alternative models to investigate the fundamental mechanisms that control the connections between the gut lumen and the distributed immune system .",
"Some of these organisms are morphologically and genetically simple , which provides unique experimental advantages , including reduced microbiota diversity , optical transparency and efficient transgenesis .",
"The difficulty in identifying homologs of mammalian cytokines , even within other vertebrate classes ( Secombes et al . , 2011 ) , remains a long-standing barrier to this approach .",
"As central mediators of the immune response , cytokines are key targets for pathogen mimicry or co-option ( Elde and Malik , 2009; Epperson et al . , 2012 ) and are subject to high levels of evolutionary pressure and sequence diversification ( Koyanagi et al . , 2010 ) .",
"An exception to this trend is the IL17 cytokine family .",
"These proteins are characterized by a cysteine-knot fold structure that is formed through interactions among four conserved cysteine residues ( Hymowitz et al . , 2001 ) .",
"This structural constraint provides a means to computationally identify IL17 homologs across phyla .",
"IL17 cytokines have been functionally characterized in jawed ( Kolls and Lindén , 2004 ) and jawless vertebrates ( Smith et al . , 2013; Han et al . , 2015 ) , and orthologs have been identified also in invertebrate deuterostomes ( Huang et al . , 2008; Hibino et al . , 2006 ) molluscs , nematodes and arthropods ( Daphnia ) ( Huang et al . , 2015 ) .",
"In contrast , IL17 factors are absent from available genome sequences of insects and non-bilaterian metazoans .",
"The broad phylogenetic distribution of this signaling system underscores the fundamental role of the IL17 family in animal biology and highlights the opportunity to glean understanding of this system using experimentally accessible invertebrate models .",
"Most mammalian genomes encode six IL17 family members ( IL17A-F ) ( Kolls and Lindén , 2004 ) , of which the most widely studied are the closely related IL17A and IL17F .",
"These two highly expressed cytokines define subsets of effector T cells ( Th17 cells and γδ17 cells ) and innate lymphocyte-like cells ( ILCs ) and induce strong inflammatory responses ( Korn et al . , 2009; Lockhart et al . , 2006; Gladiator et al . , 2013 ) .",
"Importantly , IL17 expression is not restricted to lymphocytes or other mesodermal immune cells .",
"Three members of the IL17 family ( IL17B , IL17C , and IL17E [also known as IL-25] ) are expressed by epithelial cells , including those in the gut ( Song et al . , 2011; Ramirez-Carrozzi et al . , 2011; Reynolds et al . , 2015 ) .",
"In this context , IL17C is a key factor in the early intestinal immune response where it regulates the expression of many innate immune genes .",
"In colonic epithelial cells , IL17E promotes inflammation through the IL17RB receptor , while IL17B competitively binds with IL17RB to interfere with this signal ( Reynolds et al . , 2015 ) .",
"Thus , within humans and mice , the IL17 cytokines are produced from a variety of cellular sources and have wide-ranging functions and downstream transcriptional consequences , some of which are just beginning to be understood .",
"To investigate the role of IL17 in the gut-associated immune response within the context of a morphologically simple organism , the larval stage of the purple sea urchin ( Strongylocentrotus purpuratus ) provides a unique model system ( Ch Ho et al . , 2016 ) .",
"The purple sea urchin genome sequence encodes an expansive set of immune receptors and effectors as well as conserved signaling pathways downstream of pattern recognition receptors and homologs of a suite of transcription factors that have key roles in modulating the immune response and hematopoiesis in vertebrates ( Hibino et al . , 2006; Sodergren et al . , 2006; Rast et al . , 2006; Messier-Solek et al . , 2010; Buckley and Rast , 2012 , 2015; Solek et al . , 2013; Schrankel et al . , 2016 ) .",
"This genetic heritage is shared within the deuterostomes ( e . g . echinoderms , hemichordates and chordates ) , providing a context in which to investigate IL17 function in a simple invertebrate for comparison to mammals .",
"Purple sea urchins undergo indirect development with a bilaterally symmetric , planktonic larval form that metamorphoses into a pentameral adult .",
"Over 5 days , embryos synchronously develop to form free-swimming larvae that feed for 10–12 weeks before settling and metamorphosis ( reviewed in [McClay , 2011] ) .",
"At 10 days post-fertilization ( dpf ) , larvae are 300–400 µM in length and consist of about 4000 cells .",
"Larvae have a tripartite gut composed of an epithelial monolayer separated by two sphincters into a pharynx , midgut and hindgut ( Smith et al . , 2008 ) .",
"Larvae have several types of immune cells ( Ch Ho et al . , 2016 ) including a granular cell population known as ‘pigment cells’ and a heterogeneous suite of several types of ‘blastocoelar cells’ that populate the body cavity ( blastocoel ) ( Solek et al . , 2013; Tamboline and Burke , 1992; Gibson and Burke , 1985 ) .",
"Collectively , these cells mediate the larval immune response through surveillance-like motility , phagocytosis , expression of immune effectors and regulatory cell-cell interactions ( Ch Ho et al . , 2016 ) .",
"The simplicity and optical transparency of the sea urchin larva allows visualization and quantification of the immune response on an organism-wide scale at single-cell resolution .",
"We have developed a model for gut-associated immune response in which larvae are exposed to the Gram-negative bacterium Vibrio diazotrophicus ( Ch Ho et al . , 2016 ) .",
"This marine bacterium was first isolated from the gastrointestinal tract of the congeneric green sea urchin , S . droebachiensis ( Guerinot et al . , 1982 ) .",
"Other Vibrio species have been implicated as causative disease agents in several adult sea urchins species ( Becker et al . , 2008 ) .",
"Upon exposure to V . diazotrophicus , larvae exhibit a synchronous and robust set of cellular responses over a period of 24 hr ( Ch Ho et al . , 2016 ) .",
"The most notable of these is that a subset of pigment cells change shape from a stellate to a rounded form , disengage from their typical positions apposed to the aboral ectoderm , and migrate to the gut epithelium .",
"Some blastocoelar cell types exhibit changes in cell motility and increasingly frequent cell-cell interactions with each other and with the gut epithelium .",
"Additionally , gut morphology is affected: the epithelial wall thickens to constrict the midgut , suggesting that the animals cease feeding .",
"By 24 hr , bacteria are evident within the blastocoel , where they are phagocytosed by filopodial blastocoelar cells .",
"Early changes in gene activity are most evident within the gut epithelium within 2 hr of exposure , which is well before bacteria penetrate the gut lumen .",
"Transcriptional affects are also apparent in peripheral immune cells .",
"These observations suggest that the system-wide response is regulated by recognition of microbial disturbance at the gut epithelium and is mediated in part by signals that transmit the state of the gut lumen to cells distributed throughout the organism .",
"Through comprehensive surveys of larval gene activity during infection , we find that two small subfamilies of IL17 genes emerge as highly regulated factors during the early response .",
"Here , we address the genomic repertoire , expression and function of the IL17 cytokines and receptors in the purple sea urchin immune response .",
"We also present the diversity of IL17 sequences within the purple sea urchin genome with reference to other echinoderms .",
"Expression of the sea urchin IL17 genes is evident only after bacterial exposure and is restricted to the gut epithelium in this infection model , as assessed by both in situ hybridization and transgenic reporters .",
"In the larva , exposure to V . diazatrophicus does not elicit expression of IL17 in mesodermally derived immune cells .",
"In contrast , a third subfamily of IL17 is acutely expressed in the adult by circulating immunocytes in response to immune challenge and injury .",
"The parallel roles of these IL17 subfamilies within the sea urchin immune response mirror the similar division of labor among vertebrate IL17 factors and highlight fundamental aspects of animal immunity .",
"Functional data in the larva indicate that disruption of IL17 signaling leads to decreased expression of several immune regulators and effector genes in the gut epithelium , including some of the IL17 factors .",
"Collectively , these findings indicate that epithelial expression of IL17 family regulators is central to an ancient aspect of gut immunity"
],
[
"Seawater exposure to the marine bacterium V . diazotrophicus ( V . d . ) induces a distinct cellular response in sea urchin larvae that includes the migration of pigment cells to the gut epithelium , changes in cell behavior and altered gut morphology ( Figure 1a , b ) ( Ch Ho et al . , 2016 ) .",
"To investigate the transcriptional underpinnings of this response , whole transcriptome sequencing was performed on mRNA isolated from larval samples collected at 0 , 6 , 12 and 24 hr of exposure to V . d . Given the morphological simplicity of the sea urchin larva and the depth of sequence coverage , these data provide a system-wide picture of biologically relevant transcriptional state changes upon bacterial exposure . 10 . 7554/eLife . 23481 . 003Figure 1 . Sea urchin larvae exhibit changes in cell behavior and gene expression following exposure to specific bacterial strains .",
"( a , b )",
"The larval cellular immune response includes pigment cell migration to the gut epithelium .",
"An uninfected control larva",
"( a ) , and a larva exposed to V . d . for 24 hr",
"( b ) are shown .",
"The red color of pigment cells is a consequence of echinochrome A , a naphthoquinone that is encapsulated in large granules .",
"Under typical laboratory conditions , pigment cells localize to the outer ectoderm .",
"In response to certain bacterial isolates , these cells migrate through the blastocoel to interact with the gut epithelium ( Ch Ho et al . , 2016 ) .",
"( c ) Genes are activated with a variety of kinetics that varies among functional classes .",
"RNA-Seq was performed on larvae collected at 0 , 6 , 12 , and 24 hr of exposure to V . d . Differentially expressed transcripts ( RPKM ≥3 , fold-change in expression ≥2; 3238 total transcripts ) were hierarchically clustered with average linkages to identify similarly temporally regulated genes using Gene Cluster 3 . 0 .",
"High expression is indicated in dark blue; low expression is shown in white .",
"( d ) A subset of IL17 genes is upregulated early in infection .",
"Transcript levels are shown for 16 , 920 genes that are expressed in larvae during the infection time course .",
"Expression levels ( RPKM ) are shown for uninfected larvae ( x-axis ) and larvae exposed to V . d . for 6 hr ( y-axis ) .",
"Most genes were not strongly differentially expressed at these two time points ( gray ) .",
"The 127 genes that exhibit ≥3-fold changes in expression levels between 0 and 6 hr of infection are shown in black .",
"The genes within the SpIL17 subfamilies are indicated ( SpIL17-1 , red; SpIL17-4 , green ) .",
"The dashed box is enlarged in the inset .",
"( e ) Mapped reads were used to identify a novel SpIL17 transcript .",
"RNA-Seq reads were mapped to the S . purpuratus genome .",
"Genomic regions that contained mapped reads but no known genes were investigated for domains that are associated with immunity ( Scaffold1325:145 , 000–160 , 000 from S . purpuratus genome , v3 . 1 is shown ) .",
"The number of reads normalized to library size is plotted for each time point as a stacked bar graph .",
"Reads that map to the positive strand are shown with positive values; negative values indicate reads that map on the negative strand .",
"The exons of the experimentally confirmed SpIL17-4a transcript sequences ( SpIL17-4a and SpIL17-4a´ ) are indicated in black .",
"The location of the IL17 domain encoded by the transcripts is shown in gray . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 003 The sea urchin genome encodes a large complement of genes with homologs involved in immunity in other organisms , including pattern recognition receptors , signaling molecules , immune effector and transcription factors ( Hibino et al . , 2006 ) .",
"Analyses of our RNA-Seq screens indicate that much of this genetic complexity is deployed within the larval immune response ( Figure 1c ) .",
"This includes the expression of homologs of important immune transcription regulators in vertebrates ( e . g . cebpα , cebpγ , atf2 and nf-κB ) , signal mediators ( e . g . nfkbiz , tnfaip3 and traf6 ) , and effector molecules ( e . g . complement factors and the sea urchin-specific immune response gene family 185/333 ) .",
"These data indicate that several cytokines are also transcriptionally regulated in this response , including macrophage inhibitory factors ( mif7 ) ( Ch Ho et al . , 2016 ) , TNF superfamily members , and , notably , homologs of IL17 ( Figure 1c ) .",
"To characterize the events that initiate the larval immune response , early changes in gene expression were analyzed by comparing transcript prevalence in larvae exposed to bacteria for 6 hr relative vs . unexposed controls ( Figure 1d ) .",
"From this analysis , a small group of IL17 genes emerge as the most upregulated genes in the genome ( Figure 1d ) .",
"Notably , these transcripts are completely absent from transcriptomes assembled from unchallenged ( presumably immunoquiescent ) larvae ( Tu et al . , 2012 ) .",
"The acute upregulation of these genes suggests that this group of IL17 genes may play a role in initiating the larval response to perturbation of lumenal bacteria .",
"As a foundation for functional study of these cytokines , we next characterized the purple sea urchin IL17 complement from a genomic perspective .",
"Our surveys of the original S . purpuratus genome assembly ( v2 . 1 ) identified 30 IL17-like factors ( Hibino et al . , 2006 ) .",
"However , because many of these homologs were distantly related to each other and also IL17 sequences in other species , we reanalyzed the current genome assembly ( v4 . 2; www . echinobase . org ) .",
"Using these sequences as queries in BLAST searches , and HMMER analyses to identify IL17 domains ( PF06083 ) in the translated genome sequence , 34 IL17 homologs were identified .",
"Of these , 22 correspond to previously annotated gene models ( gene model numbers and coordinates are shown in Supplementary file 2 ) .",
"In addition to BLAST ( which requires primary sequence similarity ) and HMMER ( which can be complicated by intron sequences ) , we scanned uncharacterized but transcriptionally active regions of the genome to identify divergent IL17 factors .",
"RNA-Seq reads were analyzed as they mapped to the genome without consideration of the established gene or transcript models .",
"Genomic regions that exhibited changing expression levels ( e . g . were expressed in infected larvae but not in uninfected controls ) and lacked any previously described genes were selected .",
"Candidate regions were translated and searched for domains common to immune proteins .",
"One of these expressed , unannotated regions contained a partial IL17 domain .",
"Using the transcriptome data to guide the prediction of coding sequence , a second nearby exon was identified and experimentally confirmed using RT-PCR ( Figure 1e ) .",
"The spliced sequence ( which is the single member of the SpIL17-4 subfamily ) is divergent relative to the other sea urchin IL17 genes and was not identified using BLAST searches .",
"The S . purpuratus genome ( v4 . 2 ) thus contains 35 homologs of IL17 ( hereafter referred to as SpIL17 ) .",
"Five of these sequences appear to be either pseudogenes that contain premature stop codons or frame shifts ( these were verified by reference to raw unassembled trace sequence ) , or are truncated within the genome assembly due to sequence ambiguity .",
"Phylogenetic analysis of the 30 remaining sequences indicates that this family of genes is comprised of 10 subgroups ( designated SpIL17-1-10; Figure 2 ) .",
"As a complementary analysis to define the echinoderm IL17 subfamilies and to provide phylogenetic context for the SpIL17 sequences , we identified IL17 homologs in five additional echinoderm species that represent a range of taxonomic distances ( divergence times of 5–480 million years ago ( Thompson et al . , 2015; Pisani et al . , 2012; Biermann et al . , 2003; Smith et al . , 2006 ) ; Table 1 , Supplementary file 2 ) .",
"The three closely related strongylocentrotid species ( S .",
"purpuratus , S . fragilis [formerly Allocentrotus fragilis ( Kober and Bernardi , 2013 ) ] , and Mesocentrotus franciscanus [formerly Strongylocentrotus franciscanus ( Kober and Bernardi , 2013 ) ] ) are estimated to have similar complements of between 30 and 47 IL17 homologs .",
"In contrast , two other sea urchins ( the euechinoid Lytechinus variegatus and the cidaroid Eucidaris tribuloides ) and the asteroid Patiria miniata each have fewer IL17 genes ( 7-23; Table 1 ) .",
"Phylogenetic analysis of the echinoderm IL17 genes indicates that representatives of the 10 subfamilies are present within each of the euechinoids ( Figure 2—figure supplement 1 ) .",
"The more distant E . tribuloides genome also contains orthologs of most of the SpIL17 subfamilies ( with the exception of groups 3 , 9 , and 10 ) .",
"The conservation of these families over 260 million years may reflect conserved roles within the immune response .",
"Notably , the P . miniata IL17 sequences did not cluster with any of the echinoid sequences , with the exception of PmIL17-1 , which is related to the EtIL17-N4 family .",
"The short , highly divergent IL17 sequences preclude robust phylogenetic analysis beyond the echinoderm lineage .",
"Consequently , orthology cannot be confidently assessed among specific sea urchin IL17 sequences and those from vertebrates . 10 . 7554/eLife . 23481 . 004Figure 2 . Phylogenetic analysis of the sea urchin IL17 sequences defines 10 subfamilies . Predicted amino acid sequences of the 30 IL17 proteins from S . purpuratus ( purple lines ) and 14 IL17 sequences from L . variegatus ( green lines ) were aligned and used in a phylogenetic analysis .",
"The neighbor-joining tree is shown to scale and was constructed in MEGA6 . 0 using Poisson corrected evolutionary distances , a gamma distribution model for rate variation among sites and complete deletion of alignment positions containing gaps ( Tamura et al . , 2013 ) .",
"Asterisks indicate branches with bootstrap values greater than 75 and based on 500 replicates .",
"Each of the 10 major clades was recovered in phylogenetic trees constructed using maximum likelihood and maximum parsimony methods as well as variable model parameters using the neighbor-joining method ( data not shown ) .",
"Groups are indicated by brackets .",
"Subfamilies expressed in the larval immune response are shown in blue; the family that is expressed in adult immune cells is indicated in red . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 00410 . 7554/eLife . 23481 . 005Figure 2—figure supplement 1 . Phylogenetic analysis of the echinoderm IL17 sequences . Predicted IL17 genes were identified from the genome sequences of S . purpuratus ( Sp; purple ) , L . variegatus ( Lv; green ) , E . tribuloides ( Et; brown ) , and P . miniata ( Pm; red ) .",
"Coordinates for each of the genes are shown in Supplementary file 2 .",
"Amino acid sequences were used in a phylogenetic analysis in MEGA6 . 0 .",
"The tree shown was constructed using Neighbor-joining methods using Poisson corrected evolutionary distances and complete deletion of alignment positions that contain gaps .",
"Bootstrap values ( based on 1000 replicates ) are shown for nodes with >50% support .",
"Groups as defined in S . purpuratus are indicated in bold . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 00510 . 7554/eLife . 23481 . 006Table 1 . Numbers of IL17 genes by subfamily in echinoderm species . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 006EchinodermataEchinoideaAsteroideaEuechinoideaCidaroideaStrongylocentrotidaeToxopneustidaeSubfamilyS .",
"purpuratusS .",
"fragilis* ( 5–7 myr ) †M .",
"franciscanus*a ( 20 myr ) †L .",
"variegatus ( 50 myr ) †E .",
"tribuloides ( 268 myr ) †P .",
"miniata ( 480 myr ) †1118 . 610 . 0260210 . 50 . 4110310 . 50 . 9200411 . 90 . 4130523 . 33 . 5220634 . 44 . 4230712 . 97 . 8120811 . 96 . 51209713 . 87 . 11001021 . 05 . 2100Other----412‡Total3038 . 647 . 0152212*Estimates are based on the number of best reciprocal blast hits using the SpIL17 sequences against the unassembled genomic trace sequences ( Buckley and Rast , 2012 ) .",
"†Estimated divergence times shown in million years from S . purpuratus ( Thompson et al . , 2015; Pisani et al . , 2012; Biermann et al . , 2003; Smith et al . , 2006 ) .",
"‡See Figure 2—figure supplement 1 for the phylogenetic analysis of these genes .",
"Within the purple sea urchin genome assembly , the SpIL17 genes are located on nine scaffolds ( Figure 3—figure supplement 1 ) .",
"Each gene is encoded by one to three exons , and all but five are clustered in tandem arrays of two or more genes .",
"The genomic organization of the IL17 genes is largely conserved between S . purpuratus and L . variegatus ( Figure 3—figure supplement 1 ) .",
"To confirm the transcript sequences of the SpIL17-1 and −4 genes , 5´ RACE PCR was carried out on cDNA generated from larvae 6 hr after infection with V . d . Sequences were additionally verified ( including the 3´ untranslated regions ) using the RNA-Seq data .",
"The SpIL17-1 genes have two exons , the first of which encodes a methionine and a single glutamate; the IL17 domain is encoded in the second exon ( Figure 3a ) .",
"SpIL17-4a has two transcripts that initiate at exons with distinct transcription start sites ( TSS ) but share the second and third exons .",
"These alternative first exons result in different N-terminal sequences that modify the predicted secretion signal peptide ( SpIL17-4a encodes five amino acids , whereas the alternative SpIL17-4a´ first exon encodes only a methionine; Figure 3b ) , although the cleavage site is not affected .",
"The functional consequences of this difference are unknown . 10 . 7554/eLife . 23481 . 007Figure 3 . Gene structure and diversity of the SpIL17-1 and -4 genes .",
"( a , b )",
"Coding sequence is shown in the colored boxes; non-coding sequence is in white boxes .",
"Untranslated regions have been verified using RACE PCR and through analysis of the RNA-Seq data .",
"The genomic structure of all the SpIL17 genes is shown in Figure 3—figure supplement 1 .",
"( a ) The SpIL17-1 genes are arrayed in a tandem cluster .",
"The eight SpIL17-1 genes ( light gray ) and the adjacent SpIL17-10a gene ( black ) are located on a single scaffold in a 59 . 8 kb region ( Scaffold1147; Genbank KN912785 ) .",
"The SpIL17-1 genes are encoded in two exons , the first of which includes the methionine and a single amino acid .",
"The entire region is located on BAC clone R3-17F18 , which was used to construct a GFP reporter for gene spIL17-1d .",
"The position of the GFP in this reporter construct is indicated .",
"( b ) The spIL17-4a gene encodes two transcripts that initiate from distinct TSS .",
"The nucleotide and translated amino acid sequences are shown for each of the initial exons .",
"( c ) The sea urchin and human IL17 proteins share key cysteine residues .",
"The amino acid sequences of the IL17 domains of a member of each of the SpIL17 subfamilies as well as the six human IL17 factors are shown .",
"The conserved cysteine residues implicated in forming the cysteine knot are highlighted in dark gray with white text .",
"Positions in which the SpIL17 proteins have a cysteine that corresponds to a conserved serine in vertebrates are shaded light gray .",
"Additional conserved cysteine residues are indicated in bold . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 00710 . 7554/eLife . 23481 . 008Figure 3—figure supplement 1 . Genomic organization of the S . purpuratus and L . variegatus IL17 genes . Coding sequence is shown as colored boxes ( according to the groups defined in Figure 1; the color scheme is indicated below the scaffolds ) .",
"Untranslated regions and predicted pseudogenes are shown as white boxes .",
"Scaffolds and genes are shown to scale except for large intergenic regions , which are abbreviated by brackets ( total size is indicated in kb ) .",
"Scaffolds from S . purpuratus are shown in black; L . variegatus scaffolds are in gray .",
"The red asterisk indicates the location of two additional transcripts: a transcript that encodes a protein with an SGNH_hydrolase domain ( SPU_005467 ) and a predicted non-coding RNA ( ncRNA; Genbank XR_973245 . 1 ) .",
"Analysis of the RNA-Seq data indicates that these two transcripts are expressed at low levels in larvae , but expression levels do not change in the course of infection ( data not shown ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 00810 . 7554/eLife . 23481 . 009Figure 3—figure supplement 2 . The SpIL17 sequences within subfamilies are highly conserved . The average percent identities of the proteins encoded by the SpIL17 genes within ( boxes are outlined in black ) and among subfamilies ( see Figure 2 ) are shown .",
"When only a single sequence is present within a subfamily , the within group identities cannot be calculated ( indicated as blank boxes ) .",
"Protein identities were also calculated against the six human IL17 ( HsIL17 ) proteins ( IL17A-F ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 00910 . 7554/eLife . 23481 . 010Figure 3—figure supplement 3 . Diversity of the IL17 proteins . The proteins encoded by the SpIL17 genes exhibit greater conservation within the IL17 domain .",
"The diversity of each position within the alignment of SpIL17 protein sequences was calculated as a measure of entropy ( Shannon , 1948 ) .",
"The average entropy over a 15 amino acid sliding window is shown ( blue line ) .",
"The position of the IL17 domain is highlight in gray .",
"The average entropy of the N-terminal sequence and the C-terminal IL17 domain is shown as a dashed gray line . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 010 The SpIL17 amino acid sequences contain eight conserved cysteine residues ( Figure 3c ) .",
"In vertebrates , four of these ( highlighted in dark gray in Figure 3c ) form disulfide bonds at the core of stereotypical cysteine-knot structures ( Hymowitz et al . , 2001 ) .",
"The two C-terminal conserved cysteine residues are absent from subfamilies 3 and 9 , which may indicate that these predicted sequences are truncated or that different cysteines are used instead .",
"The vertebrate IL17 sequences also encode two conserved serine residues ( Hymowitz et al . , 2001 ) that are replaced by shared cysteines in the sea urchin sequences ( light gray; Figure 3c ) .",
"Within this framework of conserved amino acids , the SpIL17 sequences exhibit varying levels of diversity within and among the subfamilies .",
"The largest subfamily , SpIL17-1 , consists of 11 very closely related genes ( 94 . 9% average amino acid identity; Figure 3—figure supplement 2a ) .",
"Among subfamilies , these proteins exhibit much higher diversity ( an average of 33 . 3% amino acid identity ) .",
"For comparison , the six human IL17 proteins share an average 35% amino acid identity ( Figure 3—figure supplement 2a ) .",
"Much of this diversity is concentrated within the N-terminus of the SpIL17 sequences , consistent with IL17 cytokine family diversity in other groups Figure 3—figure supplement 2b ( Pappu et al . , 2010 ) . 10 . 7554/eLife . 23481 . 011Figure 4 . Expression of the SpIL17 factors in response to bacterial infection .",
"( a ) .",
"Genes within two SpIL17 subfamilies are quickly upregulated in response to bacteria .",
"Expression of the SpIL17-1 ( red bars ) and SpIL17-4 ( blue bars ) genes was measured by RT-qPCR .",
"Relative expression values are normalized to the level of expression in uninfected larvae ( 0 hr ) .",
"Non-normalized data with error bars are shown in Figure 4—figure supplement 1 .",
"Oligonucleotides used in the RT-qPCR reaction anneal to all the SpIL17-1 genes and both of the SpIL17-4 transcripts ( Supplementary file 1 ) .",
"( b , c ) .",
"The two SpIL17-4a transcripts are both expressed during infection .",
"Transcript levels of the SpIL17-4a ( dark green ) and −4a´ ( light green ) transcripts were measured are shown as expression relative to 18S transcripts",
"( b ) and as the proportion of total SpIL17-4 ( blue ) as measured with primers located in the shared exons 3 and 4 ( c; see gene structure in Figure 2b ) .",
"Only time points with significant SpIL17-4 transcript levels are shown in",
"( c ) .",
"( d ) Activation of the SpIL17 genes precedes transcriptional changes in many other genes .",
"Transcript prevalence was measured using RT-qPCR for genes that are either known to be involved in immune response in either sea urchins or other organisms .",
"Expression values are log transformed and centered on the mean values for each gene . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 01110 . 7554/eLife . 23481 . 012Figure 4—figure supplement 1 . Many genes are transcriptionally regulated in larvae responding to microbial perturbation of the gut . Larvae were collected at 0 , 2 , 4 , 6 , 8 , 12 , and 24 hr of exposure to V . d . RNA isolated from the larvae was used in RT-qPCR assays .",
"Reactions were performed in at least triplicate .",
"Error bars indicate the standard deviation among the replicates .",
"Relative expression is normalized to 18S expression . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 012 The RNA-Seq screens of larvae exposed to V . d for 0 , 6 , 12 , and 24 hr reveal that genes within two SpIL17 subfamilies ( SpIL17-1 and −4 ) are sharply upregulated in response to microbial perturbation in the gut lumen ( Figure 1d ) .",
"To more thoroughly characterize expression of the SpIL17-1 and −4 genes , immune challenged larvae were sampled at higher resolution and gene expression was quantified with RT-qPCR ( Figure 4a ) .",
"In these samples , the SpIL17-1 genes are strongly upregulated within 2 hr of bacterial exposure , peak at 4 hr ( 49-fold higher than at 0 hr ) and are downregulated by 8 hr , although expression remains higher than pre-exposure levels ( Figure 4a ) .",
"The 11 SpIL17-1 genes are 87 . 1–99 . 8% identical at the nucleotide level .",
"This high similarity , as well as the high level of heterozygosity within the sea urchin population precludes determining expression levels for specific genes using either RNA-Seq data or qPCR .",
"We have isolated and sequenced SpIL17-1 transcripts from infected larvae using PCR and find that multiple genes are transcribed .",
"Additionally , analysis of single-nucleotide polymorphisms within the RNA-Seq data indicates that most of the SpIL17-1 genes are biallelically expressed .",
"The single group four gene , SpIL17-4a , is also upregulated in response to bacteria ( Figure 4a–c ) .",
"Like the SpIL17-1 genes , SpIL17-4a expression is undetectable in unexposed larvae , is activated by 2 hr of V . d . exposure , although its expression peaks slightly later ( by 6 hr of exposure ) .",
"In contrast to the SpIL17-1 genes , which are consistently downregulated by 8 hr , SpIL17-4a expression is downregulated more slowly ( Figure 4a ) .",
"While there is some variation in the timing of the downregulation of IL-17-4a , these general expression profiles have been reproduced in these and other independent challenge experiments carried out with larvae generated from different mate pairs ( e . g . RNAseq in Figure 1c and QPCR in Figure 4a ) and are consistent with qualitative findings from independent in situ hybridization time course experiments ( Figure 5a , b ) as well as with from quantitative measurement of GFP reporter transgene expression levels ( Figure 5—figure supplement 1b ) .",
"The relative expression levels of the two SpIL17-4a transcripts were measured using primers that anneal to sequence in the unique first exons for each transcript and the common second exon ( see gene structure in Figure 2b ) and compared to transcript levels measured using primers located in the shared exons ( exons 2 and 3; Figure 4b , c ) .",
"Results indicate that SpIL17-4a is upregulated prior to SpIL17-4a´ ( at 2 and 4 hr , SpIL17-4a transcripts comprise 84% and 80% of the total; Figure 4b , c ) .",
"From 6–12 hr , however , expression levels are comparable for both isoforms . 10 . 7554/eLife . 23481 . 013Figure 5 . The SpIL17-1 and −4 genes are expressed in gut epithelial cells in response to bacterial challenge . Expression of the SpIL17-1 ( a , c , e–g ) and −4 ( b , d , h , i ) genes was assessed using WMISH ( a–d ) and BAC-based GFP reporter constructs ( e–i ) .",
"White numbers shown in ( a ,",
"b ) indicate the number of positive larvae out of the total examined .",
"Hours post-infection ( hpi ) with V . d . are indicated in yellow .",
"Larval morphology is shown in c1 – i1 ( b , blastocoel , yellow; hg , hindgut; mg , midgut; gut , green; skeleton , purple; blastocoelar immune cells , blue ) .",
"White dashed lines shown in c2 – i2 indicate the location of the insets .",
"Black scale bars indicate 50 µM; white bars indicate 20 µM . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 01310 . 7554/eLife . 23481 . 014Figure 5—figure supplement 1 . Transgene reporter constructs recapitulate endogenous SpIL17-1 expression . Larvae transgenic for either the BAC reporter or GFP Construct ( shown in",
"a ) were exposed with V . d . and used for RT-qPCR analysis .",
"Expression values are normalized to the number of transgenes incorporated into the genomic DNA as described in Solek et al . ( 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 014 To put SpIL17 expression in the context of other immune factors , we generated expression profiles for additional genes that are known to be important in animal immunity ( e . g . the echinoid-specific acute immune effector family 185/333 ( Smith , 2012; Figure 4d and Figure 4—figure supplement 1 ) .",
"Analysis of these data reveals that the activation of the SpIL17-1 and −4 genes is one of the first transcriptional events in the larval immune response .",
"Many changes in expression levels are evident by 6 hr of exposure or later ( e . g . tnfaip3 , nfkbiz , and cebpα ) .",
"Of the 23 genes assayed , a similarly early activation was evident only for ets4 ( SPU_008528 , a homolog of the human Prostate-derived Ets transcription factor; PDEF [Rizzo et al . , 2006] ) .",
"The early and rapid activation of these cytokines suggests that they may be involved in the initiation of the immune response .",
"Notably , although de novo assembly of the RNA-Seq reads recovered spliced transcripts from the SpIL17-2 , −5 , –6 , and −9 families , RT-qPCR analysis indicates that these genes are expressed at very low levels and expression is not affected by immune challenge .",
"No expression of genes within the other SpIL17 subfamilies was evident during the larval immune response .",
"Furthermore , the SpIL17-1 and −4 transcripts were not present in unchallenged larvae .",
"IL17 domains are also absent from the extensive sea urchin transcriptome databases , which are generated from immunoquiescent tissues and animals .",
"Expression of SpIL17 genes is therefore tightly regulated and restricted to specific immune challenge conditions .",
"To localize the expression of the SpIL17 genes within the larval immune response , whole mount in situ hybridization ( WMISH ) was used ( Figure 5a , b ) .",
"Data from these analyses are consistent with the temporal kinetics during larval infection described above .",
"No expression of either the SpIL17-1 or −4 genes was evident in WMISH with uninfected larvae ( Figure 5a1 , b1 ) .",
"SpIL17-1 expression was observed in the midgut and hindgut of larvae collected at 6 hr of infection ( 70% of larvae; Figure 5a , c ) .",
"These data suggest that individual larva express the SpIL17-1 genes for a very short period of time , and the overall increase in expression at 6 hr is the average rate of expression over thousands of larvae .",
"Similarly , expression of SpIL17-4 is primarily restricted to the mid- and hindguts of larvae exposed to V . d . for 6 hr , although some expression is evident at 12 hr ( Figure 5b2 , b3 ) .",
"Fluorescent in situ hybridization using probes for both SpIL17-1 and −4 indicate that the two IL17 factors are largely co-expressed within the gut epithelium , however , the two transcripts do not always completely overlap ( Figure 5d ) .",
"Many of the finer details of the larval morphology are lost during the fixation process for WMISH .",
"Thus , to localize expression of the SpIL17 genes in vivo , we generated BAC-based fluorescent reporter constructs to recapitulate endogenous expression ( Figure 5e-i ) .",
"BAC R3-17F18 spans a 140 kb genomic region that encompasses the eight SpIL17-1 genes and SpIL17-10a on Scaffold1147 ( Figure 3—figure supplement 2 ) .",
"Using homologous recombination , the GFP coding sequence was inserted into the coding sequence in exon 1 of the SpIL17-1d gene ( shown in Figure 2a ) .",
"Linearized BACs were injected into fertilized eggs , which were cultured to larval stage ( 10 dpf ) and infected with V . d . No GFP expression was observed in larvae prior to infection ( Figure 5e ) .",
"By 16 hr of infection , however , fluorescent signal was evident within a few cells within the mid- and hindgut epithelium ( Figure 5f , g ) .",
"The lag in visualizing GFP compared to endogenous SpIL17-1 transcription is consistent , at least in part , with the time required to accumulate and fold the GFP protein .",
"RT-qPCR quantitation of GFP transcript levels normalized to incorporated transgene copy number confirms that the kinetics of GFP expression are similar to that of endogenous SpIL17-1 expression , with a sharp increase in expression by 8 hr that is attenuated at 24 hr of infection ( Figure 5—figure supplement 1 ) .",
"Similarly , a BAC reporter construct was generated for the SpIL17-4 transcripts .",
"For this gene , the GFP coding sequence was inserted into the second exon , which is common to both transcripts ( see Figure 2b ) and analyzed as above .",
"This reporter construct also recapitulates endogenous SpIL17-4 expression .",
"No fluorescence was observed in uninfected larvae , but by 24 hr of infection , GFP was evident in midgut epithelial cells ( Figure 4h , i ) , which is delayed relative to SpIL17-1 as is the endogenous SpIL17-4 .",
"Together , these data confirm that the SpIL17-1 and −4 genes are expressed exclusively in the epithelial cells of the larval mid- and hindgut , and that expression of these genes is tightly regulated and dependent upon bacterial challenge .",
"To investigate the role of the SpIL17 gene family in the adult sea urchin immune response , we analyzed RNA-Seq data collected from adult immune cells ( phagocytic coelomocytes ) and gut tissues isolated following immune challenge ( Buckley and Rast , 2012 ) .",
"The tissues used in this experiment were collected from a single animal that was injected intracoelomically with bacteria isolated from the gut lumen of another individual .",
"This complex challenge mimics a gut perforation and generally increases the expression of many genes involved in immunity .",
"Analysis of the RNA-Seq data indicated that while SpIL17 expression was not detected in the gut tissue , genes within a third subfamily , SpIL17-9 , were expressed at low levels in coelomocytes at 12 hr after challenge .",
"To determine whether this low level of expression reflected a larger transcriptional response to immune challenge , adult animals were challenged by intracoelomic injection of either V . d . or sham controls ( seawater injection ) .",
"Coelomocytes were collected at six time points over the course of 24 hr and used for gene expression analysis ( Figure 6 ) .",
"Expression of the sea urchin immune response genes 185/333 were used to assess immune activation .",
"This echinoid-specific family of diverse defense genes is strongly upregulated in response to several types of immune challenge ( Ghosh et al . , 2010 ) .",
"RT-qPCR analysis indicates that the SpIL17-9 genes are strongly upregulated within 3 hr of infection in both animals injected with bacteria ( red bars; Figure 6a ) .",
"Expression peaks at 6 hr , and then returns to lower levels .",
"This timing precedes expression of the 185/333 genes , which are slightly upregulated at 3 hr of infection , but exhibit high levels of expression by 9 hr ( Figure 6b ) .",
"In the animal that received the sham seawater injection , expression of the SpIL17-9 genes also increased , but often more slowly ( expression peaked at 12 hr; Figure 6a ) .",
"This is consistent with a later activation of the 185/333 genes in this animal ( 24 hr; Figure 6b ) .",
"A slower , more attenuated response is typical in sham-injected animals ( Rast et al . , 2000 ) .",
"Additionally , in the experiments described here , the repeated needle sticks during the time course sampling may also elicit an acute injury response even in the absence of injected bacteria .",
"Nonetheless , these experiments demonstrate that adult phagocytic cells can be induced to express the SpIL17-9 subclass and that it is silent in undisturbed animals . 10 . 7554/eLife . 23481 . 015Figure 6 . The SpIL17-9 genes are expressed in adult coelomocytes . Data from two independent experiments are shown ( a , b ) .",
"qPCR was used to measure transcript prevalence in coelomocytes collected from adult animals that were either injected with live V . d . ( animals 1 , 2 , and 4 ) or sham injection controls ( ASW only; animals 3 and 5 ) .",
"The treatment for each animal is indicated below the graphs .",
"Expression levels for the SpIL17-9 genes ( a1 , b1 ) increase strongly by 3 hr of exposure to bacteria , and more slowly in response to injury .",
"Expression of the effector genes 185/333 ( a2 , b2 ) serves as a marker of immune activation ( Ghosh et al . , 2010 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 015 We found no evidence of expression of SpIL17-9 genes in larvae responding to V . d . exposure , and SpIL17-1 or −4 expression was never evident in the adult coelomocytes .",
"Together , these data indicate that genes within at least three of the SpIL17 subfamilies are expressed in the course of the sea urchin immune response and that the subfamilies are deployed in distinct tissues during the different life stages , although it remains to be seen if different modes of challenge will lead to other expression patterns .",
"To characterize the role of IL17 signaling within the immune response , we searched the S . purpuratus genome for homologs of the IL17 receptor ( IL17R ) .",
"Vertebrate IL17 receptors are characterized by a conserved cytoplasmic Sef/IL17 receptor ( SEFIR ) domain ( PF08357 ) that is structurally similar to the Toll/IL-1 receptor ( TIR ) domain ( Novatchkova et al . , 2003 ) but is uniquely associated with IL17 signaling .",
"The SEFIR domain mediates intracellular signaling through interactions with the adaptor molecule Act1 , which also contains a SEFIR domain ( Qian et al . , 2007 ) .",
"Mammalian IL17RA is also characterized by a TIR-like loop ( TILL ) domain , and a loosely defined C/EBPβ activation domain ( CBAD ) that are C-terminal to the SEFIR domain and are required for downstream signaling ( Maitra et al . , 2007 ) .",
"We find that two sea urchin genes encode SEFIR domains ( SpIL17R1 and SpIL17R2; Figure 7; Figure 7—figure supplement 1a , b ) .",
"Each of these genes encodes a signal sequence , a long putative extracellular region ( 535 or 662 amino acids ) , a transmembrane region , and a cytoplasmic SEFIR domain .",
"This structure is consistent with IL17 receptors in other lineages .",
"Additionally , phylogenetic analysis of the sea urchin SEFIR domains supports homology with IL17 receptors in other species ( Figure 7—figure supplement 1 ) .",
"We have confirmed these sequences by amplifying the receptors using PCR and sequencing .",
"A TIR-like loop ( TILL ) sequence is present in SpIL17R1 directly C-terminal to the SEFIR domain but is absent in SpIL17R2 ( Figure 7 ) .",
"SpIL17R1 is encoded in 17 exons ( Figure 7—figure supplement 3a ) .",
"Exon 16 , which encodes the sequence between the transmembrane region and the SEIFR domain is alternatively spliced and is absent from some transcripts .",
"Similarly , SpIL17R2 is expressed in 16 exons; the last exon encodes the SEFIR domain ( Figure 7—figure supplement 3a ) . 10 . 7554/eLife . 23481 . 016Figure 7 . Two IL17 receptors mediate IL17 signaling in the sea urchin .",
"( a ) The sea urchin IL17 receptor sequences have similar domain architectures as those in vertebrates .",
"The sea urchin receptors have conserved SEFIR domains ( red ) .",
"SpIL17R1 also has a TILL domain ( blue ) .",
"The structure of the protein encoded by the SpIL17R1 transcript in the presence of the splice-blocking MASO ( MASOSplice ) is also shown .",
"This MASO interferes with splicing by binding to the donor splice site in exon 15 ( see Figure 7—figure supplement 2 ) .",
"Consequently , a cryptic donor splice site in exon 14 is used , which introduces a frameshift and premature stop codon .",
"The resulting truncated protein does not contain a transmembrane or SEFIR domain ( indicated by white shapes ) .",
"( b ) .",
"Interfering with IL17 signaling affects the expression of downstream genes during immune challenge .",
"Fertilized eggs were injected with the IL17R1 MASOSplice and grown to 10 dpf .",
"Larvae were infected with V . d . and collected for RT-qPCR analysis .",
"Complete data are shown in Figure 7—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 01610 . 7554/eLife . 23481 . 017Figure 7—figure supplement 1 . Phylogeny of SEFIR domain-containing proteins . SEFIR domains from IL17 receptors and Act1/CIKS molecules were collected and used in a phylogenetic analysis .",
"The tree shown was constructed using Neighbor-joining methods using a Poisson substitution model and a Gamma distribution for variation among sites in MEGA6 . 086 .",
"Similar results were obtained using maximum parsimony and maximum likelihood methods ( data not shown ) .",
"Bootstrap values based on 500 replicates are indicated ( * > 50; ** > 75 ) .",
"Act1 sequences are indicated in red; IL17 receptor sequences in shades of blue .",
"Large clusters of sequences are condensed to colored boxes .",
"Species included in these boxes are listed below .",
"The IL17 receptor sequences from sea urchin species are shown in bold .",
"A complete list of the sequences used in the analysis is shown in Supplementary file 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 01710 . 7554/eLife . 23481 . 018Figure 7—figure supplement 2 . The splice-blocking SpIL17R1 MASOS yields a transcript with a frameshift and premature stop codon . Fertilized eggs were injected with either the splice-blocking MASO ( MASOSpl . ; gray sequences ) or a control MASO ( MASOCon . ; black sequences ) .",
"Exons 14 through 17 from SpIL17R1 transcripts were amplified , cloned and sequenced from larvae ( 10 dpf ) .",
"The sequences are shown .",
"The boundaries of exons 14-17 are indicated by black lines .",
"As a consequence of the MASOS , a cryptic donor splice site is used ( indicated in red ) that results in a frameshift and premature stop codon ( shown in green ) that results in a truncated protein . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 01810 . 7554/eLife . 23481 . 019Figure 7—figure supplement 3 . The S . purpuratus genome encodes two IL17 receptors . Gene structures are shown for the SpIL17-R1",
"( a ) and SpIL17-R2",
"( b ) genes .",
"Sequences encoding the predicted transmembrane domains are shown in green; SEFIR domains are shown in red .",
"Genes are shown to scale , except for large introns , which are abbreviated by brackets ( total intron size is shown in kb ) .",
"The binding sites for the SpIL17-R1 MASOs are indicated by red bars .",
"The incorrect splice products that are generated in the presence of the MASOSplice are indicated by the dashed red line .",
"( c ) Expression levels of the SpIL17 receptors are regulated during embryogenesis .",
"qPCR was used to measure the transcript prevalence for the SpIL17-R1 ( blue ) and SpIL17-R2 ( red ) genes .",
"Error bars indicate deviation among replicates .",
"Expression of SpIL17-R2 peaks at 48 hpf , whereas SpIL17-R1 expression increases during development into the larval stage ( 72 hpf ) .",
"( d ) Expression of the SpIL17-R1 transcript varies in response to exposure to V . d .",
"( e ) The SpIL17-R1 transcript is primarily expressed in the larval gut .",
"WMISH was performed on uninfected larvae ( 10 dpf ) .",
"( f ) The SpIL17-R2 transcript is slightly upregulated at 2 hr of exposure to V . d . , but returns to unexposed levels by 4 hr .",
"( g ) The MASOSplice specifically affects the expression of SpIL17-R1 exon 15 .",
"( h ) Perturbation of IL17 signaling does not affect pigment cell migration during immune response . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 01910 . 7554/eLife . 23481 . 020Figure 7—figure supplement 4 . Effects of IL17R1 perturbation on downstream gene expression . RT-qPCR was used to assess expression of genes involved in the larval immune response to bacteria .",
"Larvae were exposed to either control MASO ( gray ) , SpIL17-R1 MASOSplice ( red ) or MASOTranslation ( blue ) .",
"Error bars indicate deviation among replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 23481 . 020 We characterized the temporal expression of the SpIL17 receptor genes in developing embryos and larvae as well as adult tissues .",
"qPCR indicates that SpIL17R1 expression is not evident at 12 hpf , but increases slowly to prism larval stage ( 84 hpf ) .",
"SpIL17R2 is upregulated at 24 hpf , peaks at 48 hpf , and then returns to low levels into the larval stage ( Figure 7—figure supplement 3c ) .",
"In larvae , spIL17R1 is gradually downregulated during infection , whereas SpIL17R2 expression is mostly constant but exhibits a small peak at 2 hr of infection and then again at 24 hr ( Figure 7—figure supplement 3d , f ) .",
"To assess transcript prevalence in adult tissues , we analyzed publicly available RNA-Seq data ( generated from adult coelomocytes , axial organ , gut , radial nerve , ovary and testes [Tu et al . , 2012] ) .",
"Results indicate that low expression of spIL17-R1 is evident in adult gut and testes .",
"The spIL17r2 transcript was not clearly present in any of the adult tissues assayed .",
"Despite extensive searches , we were not able to identify a homolog of Act1 ( the SEFIR domain-containing adaptor molecule for the IL17 receptor in vertebrates [Qian et al . , 2007] ) in any of the echinoderm genomes .",
"Other than the two SpIL17 receptors , we found no evidence of additional sequences that encode SEFIR domains in the purple sea urchin genome ( including all gene models , open reading frames and transcriptome data ) .",
"Similarly , the L . variegatus genome contains two IL17 receptors and lacks an Act1 homolog ( Figure 7—figure supplement 1 ) .",
"Given the close relationship between the SEFIR and TIR domains , we analyzed the seven unique TIR domain-containing molecules within the S . purpuratus genome ( Hibino et al . , 2006 ) , but none of these exhibit sequence or domain similarity to the Act1 proteins .",
"To assess the role of IL17 signaling in the sea urchin larval immune response , we perturbed SpIL17R1 using two Morpholino antisense oligonucleotide ( MASO ) reagents: one that interferes with translation by annealing at the translation start site ( MASOT ) and a splice-blocking MASO that binds to the donor splice site in exon 15 ( MASOS; Figure 7—figure supplement 3 ) .",
"Because these two MASOs resulted in similar effects , we concentrated on the splice-blocking MASO , as the efficiency of this treatment could be directly quantified using qPCR .",
"Translation blocking morpholinos generally do not affect message prevalence of their targets in the absence of regulatory feedbacks ( e . g . [Rast et al . , 2002; Davidson et al . , 2002] ) .",
"To confirm the effect of this splice-blocking MASO , we amplified and sequenced the SpIL17R1 exons 14 through 17 from transcripts expressed in larvae ( 10 dpf ) subjected to MASOS ( Figure 7—figure supplement 2 ) .",
"Analysis of these sequences indicates that treatment with the MASOS produces incorrectly spliced SpIL17R1 transcripts , in which an alternative donor site within exon 14 is spliced directly to exon 16 , resulting in a frame shift and premature stop codon .",
"The subsequently translated protein does not encode a transmembrane domain ( Figure 7a ) .",
"To assess the efficacy of this MASO , qPCR was performed on larvae using oligonucleotides that bind to a series of SpIL17R1 exons ( Figure 7—figure supplement 3g ) .",
"The MASOS specifically affects the transcription of exons 14 and 15 , such that amplification between exons 14/15 , 15/16 and 15/17 is significantly lower than in control larvae , whereas amplification between exons 3/4 and 16/17 is unaffected .",
"The MASOs does not exhibit complete penetrance , however , as low levels of exon 15 transcript amplification are evident in perturbed larvae .",
"This may result in a partial effect on the transcription of downstream genes .",
"Perturbation of SpIL17R2 was developmentally lethal and therefore not pursued in this study ( data not shown ) .",
"This phenotype , however , is consistent with previously described developmental functions for IL17-RD ( Sef ) ( Tsang et al . , 2002; Ron et al . , 2008 ) , Perturbation of IL17 signaling during the larval immune challenge does not significantly affect pigment cell migration ( Figure 7—figure supplement 3h ) ; however , it does affect the message prevalence of several genes that were chosen as candidate response genes from the timing of their upregulation ( Figure 7b , Figure 7—figure supplement 4 ) .",
"Notably , this includes SpIL17-4 , which is upregulated relative to uninfected larvae , but at 2–5-fold lower levels than in larvae injected with control MASO .",
"This decreased expression , in addition to the fact that SpIL17-1 expression consistently lags that of peak SpIL17-4 ( Figure 4d ) suggests that the SpIL17-1 signaling may be involved in upregulating SpIL17-4 .",
"Additionally , transcript levels of tnfaip3 , soul1 , nfkbiz , cebpα , and cebpγ are reduced in larvae exposed to the SpIL17R1 MASOs compared to control MASO ( Figure 7b , Figure 7—figure supplement 4 ) .",
"These data begin to delineate how early expression of SpIL17 factors in response to bacterial challenge mediates the expression of downstream genes in an intact organism ."
],
[
"We present here a characterization of the sea urchin larval immune response to microbial perturbation in the gut from a transcriptional perspective .",
"RNA-Seq screens of larvae exposed to bacteria reveal that two subfamilies of IL17 act as key components of this immune response .",
"The SpIL17-1 genes are acutely upregulated during the earliest phases of immune response .",
"This change in gene expression ( ~90 fold higher at 6 hr of exposure to V . d . relative to unexposed controls that have minimal SpIL17-1 transcript prevalence ) is greater than any other gene at any time point .",
"This early expression pattern may point to a role in activating the downstream immune response and communicating the state of the gut lumen to both other cells of the gut epithelium and to the wider organism .",
"In addition to the IL17 cytokine family , we have characterized two orthologs of IL17 receptors within the S . purpuratus genome .",
"Perturbation of these receptors using antisense reagents results in reduced expression of immune genes and transcription regulators , including the SpIL17-4 genes .",
"These results further support the role of IL17 signaling at the initiation of the sea urchin larval gut-associated immune response .",
"Finally , the purple sea urchin IL17 subfamily structure is conserved throughout a phylogenetic range of representative echinoderm species , suggesting an ancient origin for the sequence diversity of the echinoderm IL17 sequences .",
"The conservation of the IL-17 subfamilies may indicate functional compartmentalization for these genes within the echinoderm immune response .",
"This is supported by the tightly regulated expression of the SpIL17 genes both spatially ( gut epithelium in the larva and immune cells of the adult ) and temporally ( expression is strictly dependent on immune challenge or injury ) .",
"Of the 10 SpIL17 subfamilies , genes within three families ( SpIL17-1 , −4 , and −9 ) are expressed in the context of either the larval or adult immune systems .",
"It is likely that genes with the remaining subfamilies are expressed under different conditions of immune challenge or stress .",
"This is particularly true for subfamilies SpIL17-2 , −5 , and −6 , which can be detected as spliced transcripts from the larval transcriptome assembly , although RT-qPCR indicates that these are present at very low levels .",
"The SpIL17 transcripts are virtually absent under immunoquiescent conditions .",
"We have analyzed available transcriptome data from 16 developmental stages ( egg through juvenile ) and six adult tissues ( Tu et al . , 2012 ) and find no evidence for expression of any of the SpIL17 subfamilies .",
"Furthermore , despite the availability of large EST databases ( there are >350 , 000 echinoderm ESTs from several adult tissues and developmental time points ) , no ESTs have been identified that correspond to the SpIL17 sequences .",
"This lack of SpIL17 expression in non-challenged tissues correlates with our observations and underscores the importance of analyzing transcriptional activity under varied conditions of challenge when targeting immune-related genes .",
"Data presented here indicate that genome sequences from echinoderms ( particularly the strongylocentrotids ) have an unusually large number of genes encoding IL17 homologs .",
"Mammals typically have six IL17 orthologs and the genomes of teleost fish that have been analyzed contain between four and seven IL17 genes ( Secombes et al . , 2011 ) .",
"The presence of 35 IL17 genes within the genome of the purple sea urchin suggests that this gene family has been expanded within this lineage .",
"Although the driving forces behind this expansion remain unknown , it is consistent with other large immune-related gene families within the purple sea urchin genome that have both pathogen recognition and regulatory functions .",
"The purple sea urchin genome contains expanded families of pattern recognition receptors that are 10-fold larger than their vertebrate counterparts ( Rast et al . , 2006; Messier-Solek et al . , 2010 ) .",
"High multiplicity is also apparent among gene families that encode immune effector genes ( e . g . , the 185/333 gene family [Ghosh et al . , 2010] and the perforin-like Macpf family [Hibino et al . , 2006] ) .",
"Most of the echinoid IL17 subfamilies are conserved at least to the last common cidaroid-euechinoid ancestor ( Figure 2—figure supplement 1 ) .",
"Although the L . variegatus genome has fewer IL-17 genes , homologs representative of each of the subfamilies are retained .",
"This phylogenetic analysis indicates that lineage-specific tandem duplications also contribute to the diversity of echinoderm IL17 family ( e . g . the SpIL17-1 homologs; Figure 3—figure supplement 1 ) .",
"Broader phylogenetic comparisons ( e . g . , comparing the echinoderm and chordate IL17 sequences ) are rendered uninformative by the relatively short and divergent sequences .",
"In these analyses , genes tend to cluster within phyla with low confidence .",
"In mammals , studies of the IL17 family are largely focused on expression of IL17A and IL17F in lymphocytes .",
"The Th17 and γδ T cells are major sources of IL17A and IL17F in response to infection ( Littman and Rudensky , 2010; Roark et al . , 2008 ) .",
"Other cell types also produce IL17A and IL17F , including ILCs and myeloid cells ( Roark et al . , 2008; Passos et al . , 2010; Michel et al . , 2007; Takatori et al . , 2009; Zhu et al . , 2008; Li et al . , 2010; Hueber et al . , 2010 ) , and specialized gut epithelial cells known as Paneth cells ( Takahashi et al . , 2008 ) .",
"More recent work , however , has shown the importance of epithelial expression of another IL17 ortholog , IL17C , in directing immune responses in the gut .",
"IL17C is produced by the gut epithelium where it acts in an autocrine manner to activate expression of genes involved in the innate immune response , including proinflammatory cytokines and antimicrobial peptides ( Song et al . , 2011; Ramirez-Carrozzi et al . , 2011 ) .",
"This is observed in dextran sodium sulfate-induced colitis models as well as infection with Citrobacter rodentium .",
"Members of the IL17 family have been implicated in inducing neutrophil migration ( Ye et al . , 2001 ) , regulating tight junction formation ( Kinugasa et al . , 2000; Reynolds et al . , 2012 ) and stimulating mucin production ( Chen et al . , 2003 ) .",
"IL17C plays a role in maintaining intestinal barrier integrity by regulating the expression of occludin , a tight junction protein in colonic epithelial cells ( Reynolds et al . , 2012 ) .",
"IL17C expression in the gut epithelium has also been linked to autoimmunity ( Chang et al . , 2011 ) and tumorigenesis ( Song et al . , 2014 ) .",
"This cytokine is thus a primary mediator in mammalian gut-associated immune response .",
"Data on IL17 function remain limited outside of the jawed vertebrates .",
"In the lamprey , five IL17 homologs are differentially expressed on skin , kidney , intestine and gills , as well as VLRA+ , VLRB+ and VLRC+ lymphocytes ( Han et al . , 2015 ) .",
"Ciona intestinalis upregulates three homologs of IL17 in the pharynx and in immune cells in response to LPS challenge ( Vizzini et al . , 2015 ) .",
"In the oyster , which is the only protostome in which the IL17 response has been characterized , the single IL17 homolog is expressed by circulating hemocytes in response to infection ( Roberts et al . , 2008 ) .",
"In the work presented here , we show that in the sea urchin larva , SpIL17 expression is restricted to gut epithelial cells , with no evidence of expression in mesodermally derived immune cells in response to V . d . challenge .",
"This function is potentially homologous to the mammalian epithelial expression and suggests that the role of IL17 in modulating mucosal immunity is an ancient and fundamental component of immunity .",
"It is notable that , although there is a strong expression of SpIL17-9 in adult coelomic immune cells in response to immune challenge , over the course of many V . d . challenge experiments , we have never observed SpIL17 expression in any larval mesodermal immune cells .",
"This differential expression pattern may reflect the mode of infection with V . d .",
"Ongoing work with different isolated bacteria suggests that other larval cells may be capable of expressing SpIL17-1 and SpIL17-4 in altered infection conditions .",
"Additionally , genes within the other IL17 subfamilies may also be expressed in the larva in response to differential immune challenge .",
"The sea urchin genome encodes two IL17 receptor chains .",
"These contain SEFIR domains with structures that mirror the two types of receptors found in mammals .",
"In situ hybridization indicates that the gut epithelium is a primary site of SpIL17R1 expression ( Figure 7—figure supplement 3e ) .",
"Analysis of cell-specific transcriptome data from larvae indicates that this receptor is also expressed in gcm+ pigment cells ( Barsi et al . , 2014 ) .",
"As in other systems , these receptors may be widely expressed at low levels ( Gaffen , 2009 ) .",
"We were unable to detect an Act1 homolog in any echinoderm genome , although homologs are readily detectable in hemichordates and invertebrate chordates ( Ryzhakov et al . , 2011 ) .",
"This suggests that the sea urchin IL17 receptors signal through an Act1-independent mechanism .",
"One potential mechanism identified in mammals is the direct activation of STAT5 that occurs in vertebrate IL17RB signaling ( Wu et al . , 2015 ) .",
"The recruitment of STAT5 depends on phosphorylation of specific tyrosine residues within the IL17RB cytoplasmic region .",
"Specifically , STAT5 recruitment is mediated by tyrosine residues 444 and 454 .",
"These residues are both conserved in the SpIL17-R1 sequence .",
"There are 13 additional tyrosine residues in the cytoplasmic region of SpIL17R1 and six in SpIL17R2 that may serve similar functions .",
"Alternatively , a novel mechanism may function within the echinoderm SpIL17 system .",
"Several observations are consistent with feedback in the larval gut epithelial system .",
"The IL17R1 is expressed in the gut epithelial cells consistent with the possibility that neighboring cells in the epithelium could communicate using the IL17-1 signal .",
"In addition , the single IL17-4 gene is consistently activated to peak levels several hours after the IL17-1 genes in immune challenge experiments even when these vary in time of initial expression .",
"Reduction of IL17-4 expression in IL17R1 MASO perturbed embryos supports a causal linkage between IL17-1 activation and later IL17-4 expression .",
"Thus , IL17-4 may in some way modify signaling initiated by IL17-1 .",
"These possibilities can be explored in future perturbation experiments .",
"These findings reveal that epithelial IL17 signaling is an ancient and central element of the gut associated immune response .",
"By exploiting the experimental strengths of the morphologically simple sea urchin larva , these findings provide a novel perspective on the regulation and downstream consequences of this highly studied immune signaling factor .",
"Further investigations into the activation and interplay of the IL17 subfamilies within the larval immune response will continue to yield valuable insights that can be applied directly to the more complex mammalian systems ."
],
[
"S . purpuratus animals were obtained from the Point Loma Marine Invertebrate Lab ( Lakeside , CA ) .",
"Animals and larval cultures were maintained and exposed to Vibrio diazotrophicus as previously described ( Ch Ho et al . , 2016 ) .",
"To challenge adult sea urchins , V . diazotrophicus were cultured in LB at 15°C , washed three times with artificial sea water ( Instant Ocean; ASW ) and resuspended in 0 . 2 µM filtered ASW .",
"Animals were injected with 105 bacteria/mL coelomic fluid in a total volume of 500 µL .",
"Sham injected animals were injected with an equal volume of 0 . 2 µM filtered ASW .",
"Coelomocytes were harvested from adult sea urchins by inserting a preloaded syringe ( with a 22-gauge needle ) into the peristomial membrane and extracting coelomic fluid as in Buckley and Smith ( 2007 ) .",
"To prevent clotting , the syringe was preloaded with ice cold calcium/magnesium-free seawater ( 454 mM NaCl , 9 . 4 mM KCl , 48 mM MgSO4 , 6 mM NaHCO3 , pH 7 . 4 ) .",
"Coelomocytes were pelleted and resuspended in Trizol ( Invitrogen ) .",
"Total RNA was isolated with Trizol ( Invitrogen ) .",
"Contaminating genomic DNA was removed using the DNA-free kit ( Ambion ) .",
"First-strand cDNA was synthesized from random hexamers using Superscript III ( Invitrogen ) .",
"Quantitative PCR was carried out as described ( Rast et al . , 2002; Fugmann et al . , 2006 ) .",
"Measurements were made in triplicate on a ViiA7 real-time PCR machine using SYBR green chemistry ( Applied Biosystems ) and expression levels were normalized to parallel 18S rRNA measurements made on samples diluted 1:1000 .",
"Primer sequences are shown in Supplementary file 1 .",
"Whole transcriptome sequence data was generated for larvae exposed to V . diazotrophicus for 0 , 6 , 12 , and 24 hr ( Buckley and Rast , 2012 ) .",
"Data are available at NCBI ( BioProject PRJNA380184 ) .",
"Reads were mapped to the S . purpuratus genome ( v3 . 1; www . echinobase . org ) using Bowtie , version 0 . 12 . 7 ( Langmead et al . , 2009 ) with modified parameters to accommodate both the polymorphic sea urchin genome as well as the large families of highly similar immune genes that are relevant for this analysis ( Buckley and Rast , 2012 ) .",
"To assess expression in adult tissues , RNA-Seq data was downloaded from NCBI for project PRJNA81157 ( Tu et al . , 2012 ) ( axial organ , SRX173268; coelomocytes , SRX173270; gut , SRX173274; radial nerve , SRX173280; ovary , SRX173277; testes , SRX173283 ) .",
"Gene expression in tissues collected from an immune activated adult was also assessed using RNA-Seq methods ( PRJNA381801 ) .",
"Reads were mapped to the S . purpuratus genome ( v3 . 1 ) as above .",
"Expression levels quantification were performed using Cufflinks ( Trapnell et al . , 2012 ) .",
"De novo transcriptome assemblies were done using Trinity ( Haas et al . , 2013 ) .",
"To identify novel genes involved in the immune response , the Bowtie output files from the analysis of the larval RNA-Seq experiments were analyzed directly .",
"Output files ( in SAM format ) were sorted by scaffold and position ( using the Linux sort function ) .",
"Based on the average size of exons within the S . purpuratus genome ( 100–115 nt [Sodergren et al . , 2006] ) , numbers and orientations of reads that mapped within 200 nt regions were tabulated ( using a sliding window with a 100 nt overlap ) .",
"Genomic positions that included known gene or transcript models were excluded from further analysis .",
"Bins that contained at least 20 reads of which at least 90% were in the same orientation were ranked by expression level .",
"These sequences were translated and searched for immunologically relevant domains using HMMER Eddy , 1998 .",
"Primer sequences that were used for cloning the SpIL17 ligands and receptors are located in Supplementary file 1 .",
"Complete cDNA sequences were obtained for the SpIL17 ligands and receptors with RACE PCR using the GeneRacer kit ( Invitrogen ) .",
"Amplified sequences were cloned into pCR-TOPO4 ( Invitrogen ) and sequenced .",
"Infected larvae were washed twice with ASW and fixed overnight in 4% paraformaldehyde , 32 . 5 mM MOPS pH 7 , 32 . 5% ASW , 162 . 5 mM NaCl ( Minokawa et al . , 2004 ) .",
"Larvae were washed five times in MOPS buffer ( 100 mM MOPS pH 7; 500 mM NaCl; 0 . 1% Tween-20 ) , dehydrated and stored in 70% ethanol at −20°C until use .",
"WMISH was performed as described ( colorimetric [Minokawa et al . , 2004; Ransick et al . , 2002]; fluorescent [Croce and McClay , 2010] ) .",
"Reporter constructs were generated using homologous recombination ( Yu et al . , 2000 ) for the SpIL17-1 gene SpIL17-1e using BAC clone R3-17F18 ( GenBank: AC201380 . 1 ) and for the SpIl17-4a gene using BAC clone R3-4009B23 ( GenBank: AC179066 . 1 ) .",
"Primer sequences used to design the recombination arms are shown in Supplementary file 1 .",
"Recombinant BACs were linearized and microinjected into fertilized eggs at 100–200 copies/pL .",
"Injected larvae were cultured to 10 days , infected with V . diazotrophicus as described ( Ch Ho et al . , 2016 ) and imaged to assess fluorescent reporter expression .",
"Fertilized eggs were injected with MASO reagents ( Gene Tools ) at a final concentration of 200 μM as described ( Solek et al . , 2013 ) .",
"The MASO sequences are as follows: SpIL17R1 translation-blocking , 5´-GTGACGACATGTGAACCATGGACAT-3´; SpIL17R1 splice-blocking , 5´- CCATTGTTCCCAAACACCTACCACT-3´; SpIL17R2 translation-blocking 5´-ACACGATTGCGACGGTGGTTAACAT-3´ .",
"Genome sequences for S . purpuratus ( v4 . 2 ) , L . variegatus ( v2 . 2 ) and E . tribuloides ( v1 . 0 ) and unassembled trace sequences from M . franciscanus and A . fragilis were obtained from Echinobase ( www . echinobase . org ) ( Cameron et al . , 2009 ) .",
"The genome sequence from P . miniata ( v1 . 0; Bioproject PRJNA49323 ) was obtained from NCBI .",
"IL17 multiplicity in unassembled genome sequences was estimated by identifying traces with similarity to full-length echinoderm IL17 sequences using BLAST .",
"The number of traces was normalized using the estimated coverage of the genome sequence ( S . fragilis , 2 . 1×; M . franciscanus , 2 . 3× ) .",
"Tools within the EMBOSS suite were used to translate genomic sequence and identify open reading frames ( emboss . sourceforge . net ) .",
"Domain predictions were done using HMMER ( Eddy , 1998 ) using the IL17 PFAM domain ( PFAM accession number PF06083 ) and the SEFIR domain ( PF08357 ) .",
"Signal peptides were predicted with SignalP3 . 0 ( Bendtsen et al . , 2004 ) .",
"Alignments were edited using Bioedit ( Eddy , 1998 ) .",
"Phylogenetic analyses were performed in MEGA , version 6 . 0 ( Tamura et al . , 2013 ) .",
"Genbank accession numbers for the IL17 sequences used in as BLAST queries and in phylogenetic analysis are as follows: human IL17A , AAR23263 . 1; human IL17B , CAG33473 . 1; human IL17C , AAQ88835 . 1; human IL17D , AAQ89471 . 1; human IL17E , AAQ89484 . 1; human IL17F , AAK83350 . 1; zebrafish IL17c , NP_001018624 . 1; zebrafish IL17a/f1 , NP_001018623 . 1; zebrafish IL17d , NP_001018625 . 1; zebrafish IL17a/f2 , NP_001018634 . 1; zebrafish IL17a/f3 , NP_001018626 . 1; oyster IL17 , EW779442 . 1 .",
"Protein structure predictions were performed using Phyre2 ( Kelley and Sternberg , 2009 ) .",
"Protein sequence identities were calculated with Matgat ( Campanella et al . , 2003 ) ."
]
] | [
"IL17 cytokines are central mediators of mammalian immunity .",
"In vertebrates , these factors derive from diverse cellular sources .",
"Sea urchins share a molecular heritage with chordates that includes the IL17 system .",
"Here , we characterize the role of epithelial expression of IL17 in the larval gut-associated immune response .",
"The purple sea urchin genome encodes 10 IL17 subfamilies ( 35 genes ) and 2 IL17 receptors .",
"Most of these subfamilies are conserved throughout echinoderms .",
"Two IL17 subfamilies are sequentially strongly upregulated and attenuated in the gut epithelium in response to bacterial disturbance .",
"IL17R1 signal perturbation results in reduced expression of several response genes including an IL17 subtype , indicating a potential feedback .",
"A third IL17 subfamily is activated in adult immune cells indicating that expression in immune cells and epithelia is divided among families .",
"The larva provides a tractable model to investigate the regulation and consequences of gut epithelial IL17 expression across the organism ."
] | [
"To protect themselves from the constant invasion of harmful microbes , animals have evolved complex immune systems .",
"The gut is one of the most active sites of the immune system and plays a key role in regulating immune responses .",
"In mammals , cells lining the gut wall can sense the presence of harmful bacteria and communicate this information to tissues across the body by producing specialized proteins called Interleukin-17 ( IL-17 ) .",
"IL-17 proteins are important for regulating inflammation and are thought to activate specific immune cells in an infected area .",
"Some aspects of immune systems are similar between different animal species , which can provide clues of how immunity evolved and how it is regulated .",
"For example , sea urchins , which evolved 400-600 million years ago , begin life as simple larvae consisting of a few thousand cells .",
"As oceans harbor a multitude of bacteria and viruses , sea urchin larvae need an efficient immune system to defend themselves .",
"These larvae can respond to specific types of bacteria within a few hours after the microbes have entered their gut by modifying gene expression in distant cells .",
"As these changes occur in cells that are removed from the bacteria , it is thought that the gut cells that initially sense the bacteria , somehow communicate this information .",
"Now , Buckley et al . exposed sea urchin larvae to a marine bacterium and measured the responses of the cells and their gene expression .",
"The infection affected several types of cells , and in the first 24 hours , a subset of immune cells changed shape and started migrating to the gut wall .",
"In addition , IL-17 gene expression changed significantly in gut cells in the early phases of the larval immune response .",
"Buckley et al . identified three types of IL-17 proteins involved in sea urchin immunity: two that are important for the immune response in the gut during the larval stage , and a third that is only present in adults .",
"These findings suggest that IL-17 signaling is an ancient and central element of gut-associated immune response , which even exists in animals that evolved long before humans .",
"These findings demonstrate that the sea urchin larva represents a unique and ideal experimental model to study immune responses in a living organism that is more closely related to mammals than some other models , like fruit flies or worms .",
"By understanding the fundamental mechanisms that mediate gut health , this work may highlight new drug targets to treat conditions like Crohn’s disease and colon cancer ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | The translational landscape of the splicing factor SRSF1 and its role in mitosis | elife-02028-v2 | [
[
"Alternative splicing is a central mechanism for the regulation of gene expression allowing increased proteomic complexity in higher eukaryotes ( Smith and Valcárcel , 2000; Braunschweig et al . , 2013; Kornblihtt et al . , 2013 ) .",
"It is regulated at many different levels , mainly by the binding of protein factors to enhancers and silencers in the pre-mRNA .",
"The importance of chromatin structure and histone modifications in alternative splicing regulation has only begun to emerge recently ( Schwartz et al . , 2009; Tilgner et al . , 2009; Luco et al . , 2010 , 2011; Pradeepa et al . , 2012 ) .",
"The SR proteins are a well-characterized family of splicing factors with a role in both constitutive and alternative splicing ( reviewed by Lin and Fu , 2007 ) .",
"They have a modular structure consisting of one or two N-terminal RNA recognition motifs ( RRMs ) , which determine their RNA-binding specificity and a C-terminal domain rich in arginine and serine residues ( RS domain ) ( Shepard and Hertel , 2009 ) .",
"An extended family of RS domain-containing proteins present in metazoans , termed SR-like or SR-related proteins , are structurally and functionally distinct from canonical SR proteins and have roles not exclusively related to splicing but participate in other cellular functions as well , including transcription and cell cycle progression ( Boucher et al . , 2001 ) .",
"The activity of SR proteins in alternative splicing is defined by the location of their binding sites , generally displaying a stimulatory role in splicing when bound to exons and an inhibitory role when bound to introns ( Han et al . , 2011; Erkelenz et al . , 2013; Zhou and Fu , 2013 ) .",
"Their function in alternative splicing can be antagonized by the activity of hnRNP A/B proteins in a concentration-dependent manner , in such a way that the relative ratios of these antagonists can influence patterns of regulated splicing in a tissue-specific or developmentally regulated manner ( Eperon et al . , 2000; Zhu et al . , 2001 ) .",
"Although initially the RS domain was proposed to solely act as a protein–protein interaction domain , it was later revealed that it also acts to contact the pre-mRNA ( Shen and Green , 2004; Shen et al . , 2004; Hertel and Graveley , 2005 ) .",
"The RS domain has also been shown to determine the localization and nucleo-cytoplasmic shuttling properties of SR proteins ( Cáceres et al . , 1997 , 1998; Allemand et al . , 2001 ) .",
"A role for SR proteins and their natural antagonists , hnRNP proteins , in deregulated alternative splicing during cancer progression has been extensively documented ( reviewed by Venables , 2004 and David and Manley , 2010 ) .",
"For instance , three hnRNP proteins , hnRNP A1 , hnRNP A2 and PTB , control the alternative splicing of pyruvate kinase ( PK-M ) pre-mRNA giving rise to an isoform that is required for aerobic glycolysis used by rapidly growing tumor cells ( Clower et al . , 2010; David et al . , 2010; Chen et al . , 2012 ) .",
"The SR protein SRSF3 ( previously known as SRp20 ) antagonizes the function of the reported hnRNP proteins in PK-M alternative splicing ( Wang et al . , 2012 ) .",
"The antagonistic activities of SRSF1 and hnRNP A1 also control the epithelial-to-mesenchymal transition ( EMT ) and its reversal ( MET ) through production of two different alternatively spliced isoforms of the Ron proto-oncogene ( Ghigna et al . , 2005; Bonomi et al . , 2013 ) .",
"The levels of SRSF1 are regulated during EMT/MET via alternative splicing associated with the nonsense-mediated mRNA decay pathway ( AS-NMD ) , which is regulated by the splicing factor Sam68 ( Valacca et al . , 2010 ) .",
"SRSF1 has been identified as an oncogenic protein with altered expression in several tumors ( Karni et al . , 2007 ) .",
"Its increased expression leads , in cooperation with MYC , to the transformation of mammary epithelial cells ( Anczuków et al . , 2012 ) .",
"SRSF3 has also been proposed to be a proto-oncogene critical for cell proliferation and tumor induction and maintenance ( Jia et al . , 2010 ) , whereas SRSF6 ( SRp55 ) is amplified and is an oncoprotein in lung and colon cancers ( Cohen-Eliav et al . , 2013 ) .",
"Recently , a cellular defense mechanism to deal with the oncogenic potential of increased SRSF1 expression has been described , whereby SRSF1 stabilizes the tumor suppressor protein p53 by blocking its MDM2-dependent proteasomal degradation , which ultimately leads to oncogene-induced senescence ( OIS ) ( Fregoso et al . , 2013 ) .",
"Interestingly , other splicing factors , such as hnRNP A2/B1 , are also overexpressed in some types of cancers , such as glioblastomas , where they are correlated with poor prognosis ( Golan-Gerstl et al . , 2011 ) .",
"A subset of the SR protein family members shuttle from the nucleus to the cytoplasm , including SRSF1 ( SF2/ASF ) , SRSF3 ( SRp20 ) , SRSF4 ( SRp75 ) , SRSF6 ( SRp55 ) , SRSF7 ( 9G8 ) , and SRSF10 ( SRp38 ) ( Cáceres et al . , 1998; Cowper et al . , 2001; Cazalla et al . , 2002; Sapra et al . , 2009 ) .",
"Importantly , shuttling SR proteins have been shown to participate in a wide range of post-splicing activities , including mRNA nuclear export , nonsense-mediated mRNA decay , and mRNA translation ( reviewed by Long and Caceres , 2009 and Twyffels et al . , 2011 ) .",
"As an example , several studies have revealed that three shuttling SR proteins , SRSF1 , SRSF3 , and SRSF7 , can act as mRNA export adaptors via their interaction with the cellular export factor TAP ( Huang and Steitz , 2001; Huang et al . , 2003; Hargous et al . , 2006 ) .",
"Furthermore , increased concentration of SRSF1 promotes nonsense-mediated decay ( NMD ) ( Zhang and Krainer , 2004; Sato et al . , 2008 ) .",
"A number of SR protein family members were found to have a role in translation .",
"We have previously shown that hypophosphorylated SRSF1 protein is associated with polyribosomes in cytoplasmic extracts and enhances translation in HeLa cells both in vitro and in vivo ( Sanford et al . , 2004 , 2005 ) .",
"Furthermore , we also uncovered the molecular mechanism by which SRSF1 promotes translation by showing that it promotes translation initiation of bound mRNAs by suppressing the activity of 4E-BP , a competitive inhibitor of cap-dependent translation .",
"This activity is mediated by interactions of SRSF1 with components of the mTOR signaling pathway ( Michlewski et al . , 2008 ) .",
"In agreement with this , it was also shown that SRSF1 activates the mTORC1 branch of the pathway , as measured by S6K and 4E-BP1 phosphorylation ( Karni et al . , 2008 ) .",
"These findings suggest a model whereby SRSF1 acts as an adaptor protein that recruits the signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs .",
"Another shuttling SR protein , SRSF7 , has also been shown to promote translation of unspliced MMPV retroviral transcripts ( Swartz et al . , 2007 ) , whereas SRSF5 and SRFS6 increase the rate of Gag translation in the HIV virus ( Swanson et al . , 2010 ) .",
"SRSF3 functions as a trans-acting factor for the internal ribosome entry site ( IRES ) -mediated translation of poliovirus , which requires its cytoplasmic relocalization during viral infection ( Bedard et al . , 2007; Fitzgerald and Semler , 2011 , 2013 ) .",
"Despite the presence of growing evidence for a role for shuttling SR proteins in the regulation of mRNA translation , only very few physiological targets have been identified .",
"This raises the issue whether this activity of SR proteins has an important role in gene expression and/or whether it is associated with a particular cellular pathway .",
"Here , we have focused on the identification of the mRNA translational targets of the SRSF1 protein .",
"We carried out high-throughput deep sequencing analysis of polysomal fractions in mammalian cells overexpressing SRSF1 .",
"This resulted in the identification of a large number of mRNAs that are translationally regulated by SRSF1 .",
"These mRNAs encode proteins involved in cell cycle regulation , such as spindle , kinetochore , and M phase proteins , which are essential for accurate chromosome segregation .",
"Interestingly , we also observed that in many cases SRSF1 affects the alternative splicing of a subset of mRNAs and also influences translation of these isoforms , suggesting a role for SRSF1 in the coupling of pre-mRNA splicing and translation .",
"Altogether , the finding that SRSF1 promotes the increased translation of genes associated with cell division could partially explain the oncogenic role of SRSF1 .",
"In summary , these data provide insights on the complex role of SRSF1 in the control of gene expression and its implications in cancer ."
],
[
"In order to identify SRSF1 translational mRNA targets , we performed a polysomal shift analysis to follow mRNAs that move from the subpolysomal fraction to the heavier polysomal fractions in HEK 293T cells upon increased SRSF1 expression .",
"Maintaining proper levels of SRSF1 could be critical for cell function .",
"As such , SRSF1 expression is subjected to negative autoregulation in order to maintain homeostatic levels , which involves multiple layers of post-transcriptional and translational control ( Sun et al . , 2010 ) .",
"Thus , in order to avoid cellular mechanisms that could limit an increased SRSF1 expression , we relied on transient overexpression of an epitope-tagged SR protein cDNA encoding wild-type SRSF1 protein .",
"We used two different concentrations of the SRSF1 expression vector and obtained a maximum of a threefold increase in the levels of transfected SRSF1 protein over endogenous protein in HEK 293T cells that displayed approximately 80–90% transfection efficiency ( Figure 1—figure supplement 1 ) .",
"We chose the highest concentration of transfected SRSF1 protein since this resulted in maximum activation of a luciferase reporter harboring an SRSF1 binding site ( Sanford et al . , 2004 ) ( Figure 1—figure supplement 2 , left panel ) .",
"The translational activation of the luciferase reporter induced by SRSF1 correlated well with a threefold increase in the polysomal/subpolysomal ratio of the reporter RNA ( Figure 1—figure supplement 2 , right panel ) .",
"The expression of SRSF1 varies widely in a tissue-specific manner and differences of up to 20-fold between different tissues have been reported ( Zahler et al . , 1993; Hanamura et al . , 1998 ) .",
"Thus , this level of overexpression is within physiological levels and correlates well with the maximum activation of a translational reporter ( Figure 1—figure supplements 1 and 2 ) .",
"We proceeded to fractionate cell cytoplasm across 10–45% sucrose gradients and isolated RNA from the subpolysomal and heavy polysomal fractions from control cells and from cells transiently overexpressing SRSF1 .",
"Next , we identified by high-throughput sequencing analysis those mRNAs that shifted to the polysomal fractions upon SRSF1 increased expression ( Figure 1A ) .",
"It has been shown that calculating mRNA translation levels as log ratios of actively translated mRNAs divided by the corresponding cytoplasmic mRNA results in a significant number of false positives and false negatives ( Larsson et al . , 2010 ) .",
"Therefore , to precisely identify those mRNAs whose translation is responsive to increased levels of SRSF1 , we used for normalization the log ratios of polysomal mRNAs versus RNAs in subpolysomal plus polysomal fractions .",
"The resulting polysome index measures the proportion/density of each transcript that is present in the polysomal fractions .",
"We compared empty vector-transfected to SRSF1-transfected cells by calculating the distribution of the log2 ratios of their respective polysome index , which we defined as the polysome shift ratio ( PSR ) ( Figure 1B ) .",
"This allowed for the scoring of an increase in translational efficiency independently of the cellular abundance of the corresponding transcript .",
"A cut off of 0 . 889 ( p<0 . 01 ) , corresponding to a 1 . 85-fold increase in the proportion of polysomal-associated transcripts , resulted in the identification of 1576 mRNAs that shifted to heavier polysomal fractions upon SRSF1 overexpression ( Figure 1B and data in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) ) .",
"Gene Ontology analyses showed that a large proportion of those mRNAs identified in the polysomal shift analysis encode for proteins involved in cell cycle regulation , mitosis , transcription , and post-translational protein modification ( based on analysis with DAVID [Dennis et al . , 2003] ) ( Figure 2—figure supplement 1A ) .",
"Among the RNA targets that displayed a polysomal shift are mRNAs encoding proteins related to cancer , such as NRAS , the Ras-related protein R-Ras2 , and those related to cell cycle , such as CDC27 , the retinoblastoma binding protein RBBP8 , and retinoblastoma-like 1 ( RBL1 ) .",
"( A list of all SRSF1 translational targets is provided in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) .",
") The SRSF1 translational targets may represent indirect as well as direct events .",
"In the first scenario , increased expression of SRSF1 could lead to general changes in gene expression , which could result indirectly in the translational upregulation of a subset of mRNAs .",
"Conversely , direct events would represent events whereby SRSF1 binds to its mRNA targets and activates their translation .",
"Interestingly , we observed that 41% of all mRNAs identified in the polysomal shift experiment upon SRSF1 overexpression were previously identified as bona fide RNA targets of this SR protein by CLIP-seq ( Sanford et al . , 2009 ) ( Figure 2—figure supplement 1B ) .",
"This strongly suggests that these are direct mRNA translational targets of SRSF1 .",
"We combined k-mer enrichment analysis ( the enrichment of every 5-mer within the RNA sequences ) with motif discovery to search for over-represented sequences in SRSF1 mRNA translational targets .",
"MEME was used to retrieve a motif logo from mRNA regions containing the over-represented 5-mers ( Bailey and Elkan , 1994 , 1995 ) .",
"This resulted in the identification of a purine-rich motif very similar to the one obtained when identifying genome-wide targets of SRSF1 ( Figure 2A ) ( Sanford et al . , 2008 , 2009 ) .",
"Interestingly , the frequency of this motif showed a clear gradient , being more predominant in CLIP-positive translational targets than in CLIP-negative translational targets and was even more reduced in both the CLIP-positive and CLIP-negative subset of mRNAs that did not shift to polysomes following SRSF1 overexpression ( Figure 2B ) .",
"In fact , statistical analysis showed a significant enrichment of the CLIP-positive mRNAs containing the identified consensus motif ( CM ) in the SRSF1 translational targets ( Fisher’s exact test: OR 1 . 686 , p<2 . 2E-16 ) ( Figure 2C ) .",
"We further refer to these 505 mRNAs as SRSF1 direct translational targets .",
"Next we analyzed whether there was any position bias with respect to the SRSF1 consensus motif in SRSF1 translational targets .",
"We observed that this motif is preferentially located in the coding DNA sequence ( CDS ) and to a lesser extent in the 5′UTR of SRSF1 translational targets , when compared with those mRNAs whose translation is unaffected by increased SRSF1 expression ( referred to as null [PSR∼0] ) ( Figure 2—figure supplement 2 ) .",
"Gene Ontology analysis of direct SRSF1 translational targets revealed an enrichment in mRNAs associated with cell cycle and chromosome organization , as previously seen when analyzing all targets or CLIP-positive targets ( compare Figure 2D and Figure 2—figure supplement 1A , C ) as well as an enrichment in mRNAs linked with transcription and RNA metabolism ( Figure 2D , E , Figure 2—figure supplement 1C ) . 10 . 7554/eLife . 02028 . 003Figure 1 . Identification of SRSF1 mRNA translational targets .",
"( A ) Experimental approach to identify SRSF1 mRNA translational targets .",
"A characteristic fractionation profile of empty vector ( pCG ) and SRSF1 transfected-HEK 293T cells ( Figure 1—figure supplement 1 ) is depicted .",
"Absorbance at 254 nm was monitored .",
"( B ) A plot showing the distribution of mRNAs from RNA-seq analysis according to the polysome shift ratio ( PSR ) .",
"The null distribution ( comparing two control subsamples ) is symmetric and sharply centered at",
"0 . The PSR of SRSF1 versus empty vector shows an enrichment over the null distribution .",
"The mRNAs with a p<0 . 01 ( PSR>0 . 889 ) were considered SRSF1 translational targets ( Supplementary file 1 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 00310 . 7554/eLife . 02028 . 004Figure 1—figure supplement 1 . Optimization of SRSF1 transient transfection for polysomal shift analysis .",
"( A ) Western blot analysis showing the levels of overexpressed T7-epitope tagged SRSF1 protein ( upper band ) compared to the levels of the endogenous SRSF1 protein ( lower band ) .",
"A quantitation of the intensity of the bands is shown below .",
"( B ) Evaluation of transfection efficiency in HEK 293T cells by flow cytometric analysis of GFP expression shows that 84 . 4±1 . 3% of cells transiently transfected with the pmaxGFP plasmid ( right panel ) are GFP positive . DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 00410 . 7554/eLife . 02028 . 005Figure 1—figure supplement 2 . Luciferase reporter containing an SRSF1 binding site . The fold activation of luciferase activity in SRSF1-transfected cells is plotted relative to empty vector ( pCG ) control , whose values were set to",
"1 . Two different concentrations of pCGT7-SRSF1 plasmid were used ( 0 . 016 and 0 . 4 μg/ml ) ( left panel ) .",
"RT-qPCR showing the polysome to subpolysome ratio for the luciferase reporter mRNA and actin mRNA upon SRSF1 overexpresion ( 0 . 4 μg/ml ) .",
"The polysome to subpolysome ratio is relative to cells transfected with empty vector ( pCG ) ( right panel ) .",
"EDA: SRSF1-binding site derived from the fibronectin EDA exonic splicing; LCS: firefly luciferase ORF; SV40: simian virus 40 promoter . DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 00510 . 7554/eLife . 02028 . 006Figure 2 . SRSF1 translational targets .",
"( A ) Motif identified on putative direct translational targets using 5-mers enrichment in combination with MEME algorithm ( width = 10 , sites = 508/508 , E value = 1 . 5E-308 , IC = 7 . 9 bits ) .",
"Over-represented k-mers were obtained by double comparison CLIP+ versus CLIP− translational targets and CLIP+ translational targets versus null CLIP+ .",
"( B ) Box plot showing the density of consensus motif ( translational targets CLIP+ > translational targets CLIP− > null CLIP+ > null CLIP+ ) .",
"( C ) Venn diagram showing the overlap ( 505 mRNAs ) between translational targets ( 1576 mRNAs with p<0 . 01 ) and CLIP-tag mRNAs containing the consensus motif ( CLIP+ CM ) ( 6065 ) ( Fisher’s exact test: OR 1 . 1686; p<2 . 2E-16 .",
"( D ) The most representative classes of Gene Ontology terms enriched in direct translational targets ( with CLIP-tag and consensus motif [CLIP+ CM] ) relative to all the mRNAs detected in HEK 293T by RNA-seq .",
"The number of genes observed in each category is indicated in the pie chart .",
"Modified Fisher’s exact p value , EASE score is given for each category .",
"In all cases , the Benjamini–Hochberg-corrected EASE score was <0 . 1 .",
"( E ) Table giving the gene names of SRSF1 translational targets related to cell cycle and RNA processing pathways . DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 00610 . 7554/eLife . 02028 . 007Figure 2—figure supplement 1 . Analysis of SRSF1 translational targets .",
"( A and C )",
"The most representative classes of Gene Ontology terms enriched in translational targets ( PSR>0 . 889536; p<0 . 01 ) ( A ) and in the subset of CLIP+ translational target mRNAs ( C ) relative to all the mRNAs detected in HEK 293T by RNA-seq .",
"The number of genes observed in each category is indicated in the pie chart .",
"Modified Fisher’s exact p value , EASE score is given for each category .",
"The asterisks indicate a Benjamini–Hochberg-corrected EASE score <0 . 05 .",
"( B ) Venn diagram showing the overlap ( 645 mRNAs ) between translational target mRNAs identified in the polysomal shift analysis ( 1576 ) with mRNAs shown to be bound directly by SRSF1 in a CLIP experiment ( 9094 ) ( Sanford et al . , 2009 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 00710 . 7554/eLife . 02028 . 008Figure 2—figure supplement 2 . The positional bias of the SRSF1 consensus motif in the 5′UTR , protein coding sequence ( CDS ) , and 3′UTR of SRSF1 translational mRNA targets ( red ) was compared to the null population ( blue , PSR∼0 ) .",
"One transcript per gene ( with the longest 5′UTR ) was kept for the analysis .",
"The x axis represents the position of the motif , with each region's length normalized to",
"1 . The y axis represents the density of coverage of motifs over the sequence . DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 008 We proceeded to validate a subset of SRSF1 targets that were identified in the experiment described above .",
"These experiments were performed in polysomal/subpolysomal fractions obtained independently from the samples used in the RNA-seq experiment ( Figure 1 ) .",
"We selected mRNAs encoding for proteins involved in cancer-related pathways such as cell cycle and apoptosis , as well as other targets involved in processes such as transcription , translation , RNA processing , and proteolysis .",
"Moreover , the selected mRNAs covered a wide range of values for PSR , from 0 . 9186 to 2 . 95 ( data in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) ) .",
"As a negative control we used the AVEN and ALAS1 mRNAs , as their distribution along the polysome profile did not change upon SRSF1 overexpression ( PSR = 0 ) and its cellular abundance was comparable to those mRNAs selected for the validation ( data in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) ) .",
"Importantly , we observed a significant increase in the polysomal to subpolysomal ratio for 70% of those mRNAs upon SRSF1 overexpression , as analyzed by RT-qPCR ( Figure 3A ) , confirming a role for SRSF1 in regulating translation of these targets .",
"Notably , we observed the highest fold change in the polysome to subpolysome ratio for mRNAs involved in cell cycle regulation and RNA processing . 10 . 7554/eLife . 02028 . 009Figure 3 . Validation of SRSF1 translational targets .",
"( A ) RT-qPCR validation confirms an increased polysome to subpolysome ratio for selected SRSF1 translational targets upon SRSF1 overexpression ( CLIP+: green; CLIP+ and harboring an SRSF1 consensus motif ( CLIP+ CM+ ) : purple ) .",
"The polysome to subpolysome ratio is relative to cells transfected with empty vector ( pCG ) and normalized to actin .",
"Plotted data are the average of three biological replicates .",
"The asterisks indicate statistical significance ( p<0 . 05 ) Error bars , Gene Ontology terms and cancer relationship are indicated .",
"( B ) RT-qPCR validation of SRSF1 translational targets involved in RNA metabolic processes .",
"The polysome to subpolysome ratio as measured by RT-qPCR in empty vector-transfected cells compared to cells overexpressing SRSF1 is indicated .",
"Two different concentrations of pCGT7-SRSF1 plasmid were used .",
"A mutant version of SRSF1 that is constitutively nuclear and does not activate translation was also included ( SRSF1-NRS ) .",
"( C ) Box plot showing the values of the stable isotope labeling by amino acids in cell culture ( SILAC ) index , defined as log2 SRSF1 ratio/empty vector ratio .",
"‘All’ refers to mRNAs encoding for all the proteins found by SILAC ( 2471 mRNAs ) ; ‘CLIP+’ refers to the SRSF1 translational targets ( PSR>0 . 889536; p<0 . 01 ) harboring CLIP-tag ( 125 mRNAs ) ; ‘CLIP+CM’ is for direct translational targets ( 105 mRNAs ) ; ‘PSR≥1 , CLIP+CM’ refers to direct translational targets with a PSR≥1 ( 72 mRNAs ) ( p = 0 . 002487 ) .",
"( D ) PP242-mediated mTOR inhibition suppresses SRSF1-dependent activation of translation of a subset of mRNA targets .",
"Control and SRSF1-overexpressing cells were treated with PP242 for 90 min .",
"The polysome to subpolysome ratio was measured by RT-qPCR in empty vector-transfected cells compared to cells overexpressing SRSF1 treated with or without PP242 .",
"mTOR inhibition was validated by Western blotting ( data not shown ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 009 We sought to further explore a role for SRSF1 in mediating the translational regulation of proteins involved in different aspects of RNA metabolism , such as splicing , NMD , and translation .",
"As a control we included a chimeric SRSF1 protein harboring a nuclear retention signal ( NRS ) , identified in the non-shuttling protein SRSF2 , which was fused at its C-terminus .",
"This protein , termed SRSF1-NRS , is constitutively retained in the nucleus and does not activate translation ( Cazalla et al . , 2002; Sanford et al . , 2004 ) .",
"In most cases , we confirmed that overexpression of SRSF1 protein , but not of the nuclear-retained SRSF1-NRS variant , results in a shift of the mRNAs encoding for the aforementioned proteins to polysomes ( Figure 3B ) .",
"For instance , we noticed that overexpression of SRSF1 results in increased translation of CWC22 , an essential splicing factor that also has a role in exon junction complex deposition and NMD ( Alexandrov et al . , 2012; Barbosa et al . , 2012; Steckelberg et al . , 2012 ) , as well as of PRPF18 that is required for the second step of pre-mRNA splicing ( Horowitz and Krainer , 1997 ) .",
"SRSF1 translational targets that were validated in this assay also include LSM3 , which is a constituent of the Lsm1-7-Pat1 complex that functions in the 5′-to-3′ mRNA decay pathway ( Sharif and Conti , 2013 ) , as well as proteins involved in the NMD pathway such as UPF2 and PNRC2 ( data in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) and Figure 3B ) ( Nicholson et al . , 2010 ) .",
"In particular , PNRC2 shows a drastic movement to polysomal fractions upon SRSF1 overexpression , but is not responsive to increased expression of the SRSF1-NRS variant ( Figure 3B ) .",
"Together , these observations are consistent with a role for SRSF1 in regulating the translation of mRNAs encoding components of the RNA processing pathway .",
"Of interest , we noticed that SRSF1 also promotes the translation of mRNAs encoding negative regulators of mRNA translation , such as EIF4E3 that recognizes and binds the 7-methylguanosine-containing mRNA cap during an early step in translation initiation ( Osborne et al . , 2013 ) , as well as Paip2 that inhibits translation both in vitro and in vivo by displacing PABP from the poly ( A ) tail ( Khaleghpour et al . , 2001 ) .",
"This could represent a feedback mechanism that becomes activated in response to SRSF1 overexpression to antagonize its role in translation .",
"Alternatively it could suggest a role for SRSF1 in the negative regulation of translation of subsets of mRNAs that are targets of EIF4E3 and/or PAIP2 .",
"Indeed , we observed that 165 mRNAs are translationally repressed by increased expression of SRSF1 ( Figure 1B ) .",
"It is known that protein levels in the cell cannot be always predicted from the mRNA abundance , as other factors , such as post-translational modification and protein stability , contribute to steady-state levels of protein .",
"Thus , we sought to determine whether the SRSF1-induced polysomal shift of target mRNAs correlated with higher protein abundance .",
"Use of stable isotope labeling by amino acids in cell culture ( SILAC ) resulted in the identification of 2157 proteins in the three protein lysates used ( untransfected , empty vector , and SRSF1-transfected HEK 293T cells ) ( data in Supplementary file 2 at Dryad: Maslon et al . ( 2014 ) ) .",
"Following normalization , we calculated for each of those proteins the log2 of the ratio between the levels of each individual protein in cells overexpressing SRSF1 versus control cells ( SILAC index ) .",
"Thus , a positive value of the SILAC index indicates an increase in protein abundance upon SRSF1 overexpression .",
"As expected , we found the higher score for SRSF1 ( data in Supplementary file 2 at Dryad: Maslon et al . ( 2014 ) ) .",
"To establish a correlation with the PSR , the SILAC index for each protein was assigned to the mRNAs encoded by the corresponding gene ( data in Supplementary files 1 and 2 at Dryad: Maslon et al . ( 2014 ) ) .",
"We found a positive correlation showing increased protein levels for SRSF1 direct translational targets ( Figure 3C , third box ) .",
"This correlation was even better for a subset of mRNAs that displayed a high polysome shift ratio ( PSR>1 ) ( Figure 3C , fourth box ) .",
"We previously showed that SRSF1 promotes translation initiation of bound mRNAs by suppressing the activity of 4E-BP , a competitive inhibitor of cap-dependent translation .",
"This activity is mediated by interactions of SRSF1 with components of the mTOR signaling pathway .",
"This suggested a model whereby SRSF1 functions as an adaptor to recruit signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs ( Michlewski et al . , 2008 ) .",
"We sought to determine whether endogenous SRSF1 translational targets responded to the same mechanism of translational activation , as we previously showed using reporter assays .",
"For this , we treated cells transiently expressing SRSF1 ( or control cells ) with a specific inhibitor of the mTOR kinase , PP242 ( Dowling et al . , 2010 ) , and then measured polysomal to subpolysomal ratios of a subset of selected SRSF1 translational targets , including proteins related to RNA processing ( Figure 3B ) .",
"Interestingly , we found that inhibition of mTOR abrogated the stimulatory activity of SRSF1 on the translation of selected targets ( Figure 3D ) .",
"This demonstrates that the activity of SRSF1 in translational activation of endogenous targets requires the mTOR pathway .",
"The function of shuttling SR proteins in both splicing and post-splicing activities raises the possibility that they may act to coordinate nuclear and cytoplasmic events for a subset of pre-mRNAs .",
"Indeed , we previously showed by coupling CLIP with subcellular fractionation that mRNAs found associated with SRSF1 in the nucleus , were also found in the cytoplasm and in the actively translating pool of ribosomes , suggesting that splicing and translation of those mRNAs could be coordinated by SRSF1 ( Sanford et al . , 2008 ) .",
"To assess a global effect of SRSF1 in coupling of pre-mRNA splicing with mRNA translation , we analyzed changes in alternative splicing in cells overexpressing SRSF1 using exon-junction arrays .",
"We identified 382 differentially regulated cassette exons: 209 events associated with skipping of an alternative exon and 173 events where overexpression of SRSF1 resulted in the inclusion of an alternative exon ( data in Supplementary file 3 at Dryad: Maslon et al . ( 2014 ) ) .",
"In order to correlate the polysomal shift of SRSF1 translational targets with the alternative splicing of those mRNAs , the PSR for the isoforms generated by the changes in alternative splicing events upon SRSF1 overexpression were analyzed ( Figure 4A ) .",
"Interestingly , we observed a statistically significant increase in the PSR of the isoforms generated by skipping as well as inclusion of a cassette exon ( Figure 4A ) .",
"This suggests that SRSF1 can influence both the alternative splicing as well as the translational efficiency of subsets of mRNAs .",
"As an example , we focused on the alternative splicing of the SR protein kinase Clk1 pre-mRNA , which is regulated through alternative splicing , giving rise to two isoforms encoding catalytically active and truncated inactive polypeptides ( Clk1Ex4+ and Clk1Ex4− , respectively ) ( Duncan et al . , 1997 ) .",
"Indeed , we could confirm that SRSF1 overexpression caused increased inclusion of the alternatively spliced exon 4 of CLK1 mRNA , giving rise to the active Clk1 isoform ( Figure 4B , left panel ) .",
"Furthermore , an SRSF1 CLIP tag containing a consensus motif mapped to this cassette exon suggesting that it is bound directly by SRSF1 ( data in Supplementary file 3 at Dryad: Maslon et al . ( 2014 ) ) .",
"Interestingly , this isoform was also more translated ( as measured by polysome to subpolysome ratio by RT-qPCR ) upon SRSF1 overexpression ( Figure 4B , right panel ) . 10 . 7554/eLife . 02028 . 010Figure 4 . Coupling of alternative splicing and translational regulation .",
"( A ) Correlation between SRSF1-induced changes in alternative splicing with polysomal distribution of those isoforms .",
"Changes in alternative splicing induced by SRSF1 overexpression were determined by an exon-junction array .",
"PSR: polysome shift ratio .",
"( B ) RT-qPCR analysis of the effect of SRSF1 on CLK1 alternative splicing and preferential polysomal association .",
"The exon-intron structure of both isoforms is indicated ( not to scale ) and the CLK1 isoform that is an SRSF1 direct translational target is underlined .",
"SRSF1-induced changes in CLK1 alternative splicing were determined and normalized to exon 7 ( constitutive exon ) levels ( left panel ) .",
"Two different concentrations of pCGT7-SRSF1 plasmid were used .",
"Polysomal distribution of CLK1 mRNA isoforms upon SRSF1 overexpression normalized to exon 7 ( constitutive exon ) levels ( right panel ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 010 A large number of SRSF1 mRNA translational targets encode for proteins involved in cell cycle regulation ( data in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) ) .",
"This is even more apparent when looking at the functional classification of direct translation targets containing the SRSF1 consensus binding motif ( Figure 2D , E ) that includes several redundant layers such as cell cycle ( p = 3 . 35E-06 ) , chromosome organization ( p = 2 . 79E-07 ) , and M phase ( p = 3 . 61E-05 ) , suggesting that SRSF1-mediated translation may affect proper mitotic progression .",
"In particular , many of these mRNAs encode for proteins with a role in mitotic spindle and kinetochore formation ( data in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) and Figure 5A ) .",
"The aforementioned group of proteins comprises , among others , a group of centrosomal proteins including CEP170 , CEP70 , and CEP57 , proteins involved in kinetochore and spindle function , such as NDC80 and CCDC99 , and proteins forming the condensin complex , including SMC2 and SMC4 .",
"NDC80 , a core protein of the NDC80 complex , is required for stable microtubule binding in the outer plate of kinetochores ( Wei et al . , 2007 ) , whereas CCDC99 , also known as Spindly , is required for the dynein/dynactin localization to kinetochores ( Barisic et al . , 2010 ) .",
"Centrosomal proteins control cell cycle progression and spindle–kinetochore assembly ( Kumar et al . , 2013 ) .",
"In particular , CEP57 is involved in linking central spindle microtubules and is required for spindle integrity ( He et al . , 2013 ) , CEP70 is necessary for the organization and orientation of a bipolar spindle in mitosis , and CEP170 is involved in microtubule organization and associates with spindle microtubules during mitosis ( Shi et al . , 2011 ) .",
"Finally , SMC2 and SMC4 proteins are part of the condensin I and II complexes , which , together with cohesin , restructure chromosomes to promote faithful chromosome segregation during mitosis ( Losada and Hirano , 2005 ) .",
"Importantly , altered translational regulation of any of these proteins could have important implications for faithful chromosome segregation , as this depends on the formation of a bipolar spindle and the correct attachment of kinetochores to spindle microtubules .",
"Notably , another identified SRSF1 translational target , CDK1 , has also been shown to regulate the assembly of mitotic spindles as well as spindle positioning , stability , and elongation ( Enserink and Kolodner , 2010 ) . 10 . 7554/eLife . 02028 . 011Figure 5 . SRSF1 translational targets involved in cell division .",
"( A ) List of cell cycle proteins regulated by SRSF1 at the translational level ( left panel ) .",
"The cartoon depicting their involvement in chromosome segregation during mitosis was adapted from Kitagawa and Hieter ( 2001 ) .",
"APC stands for ( Anaphase-promoting complex ) ( B ) Validation of cell cycle translational targets .",
"The polysome to subpolysome ratio for a subset of cell cycle-related mRNAs was measured by RT-qPCR in empty vector-transfected cells compared to cells overexpressing SRSF1 or depleted of SRSF1 .",
"Two different concentrations of pCGT7-SRSF1 plasmid were used .",
"A mutant version of SRSF1 that is constitutively nuclear and does not activate translation was also included ( SRSF1-NRS ) .",
"The asterisks indicate statistical significance ( p<0 . 05 ) .",
"( C ) Western blot validation of selected cell cycle SRSF1 translational targets in empty vector or SRSF1-transfected cells .",
"β-Actin was used as a loading control .",
"( D ) Western blot validation of selected cell cycle SRSF1 translational targets in control or SRSF1-depleted cells in an asynchronous ( A ) or mitotic population ( M ) .",
"Tubulin was used as a loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 01110 . 7554/eLife . 02028 . 012Figure 5—figure supplement 1 . Cell cycle defects following SRSF1 knockdown .",
"( A ) Cell cycle distributions of asynchronous control and SRSF1-overexpressing cells were monitored by flow cytometry .",
"( B and C )",
"Cell cycle distributions for control and SRSF1-depleted cells were monitored by flow cytometry in asynchronous cells ( B ) and in cell cultures synchronized with a double thymidine block following release for 0–10 hr ( C ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 012 Here , we focused on those SRSF1 translational targets that are related to chromosome segregation during mitosis ( Figure 5A ) .",
"We estimated their polysome to subpolysome ratio in HEK 293T cells overexpressing SRSF1 using RT-qPCR .",
"We observed a shift to the polysomal fraction upon increased SRSF1 expression ( note that both concentrations shown in Figure 1—figure supplement 1 were used here ) .",
"By contrast , increased expression of the translationally inactive SRSF1-NRS variant did not cause a shift to the polysomal fraction in most cases .",
"Also , as expected we observed a decrease in polysomal association of these mRNAs upon siRNA-mediated depletion of SRSF1 ( Figure 5B ) .",
"This analysis validated this subset of mRNAs as bona fide translational targets , suggesting a role for SRSF1 in the translational regulation of proteins that are required for cell cycle progression ( Figure 5B ) .",
"We also performed Western blotting analysis of some of these targets and were able to confirm that SRSF1 overexpression results in increased levels of CEP70 , NDC80 , and SMC4 ( Figure 5C ) .",
"Conversely , we observed decreased protein levels following SRSF1 depletion , in particular of NDC80 , SMC4 , and CEP57 ( Figure 5D ) .",
"Furthermore , SILAC analysis also showed that increased expression of SRSF1 resulted in increased levels of proteins related to cell cycle progression and chromosome segregation ( Figure 3 and data in Supplementary file 2 at Dryad: Maslon et al . ( 2014 ) ) .",
"In particular , CEP170 , CBX3 , and PDS5B protein abundance increased in response to SRSF1 overexpression , further validating the role of SRSF1 in regulating translation of these targets .",
"In agreement with these observations , previous findings from the MitoCheck consortium have revealed that SRSF1 is involved in mitotic progression ( Neumann et al . , 2010 ) .",
"A recent study has revealed a dynamic reprogramming of translation throughout the cell cycle ( Stumpf et al . , 2013 ) .",
"Since our studies were carried out for the most part in asynchronous cell populations , it remains possible that SRSF1 non-translational effects on cell cycle could be the cause of at least some of the observed changes in mRNA translation .",
"Importantly , we observed that increased expression of SRSF1 does not grossly affect the cell cycle profile ( Figure 5—figure supplement 1A ) , strongly suggesting that the observed increase in polysomal to subpolysomal ratios for these targets is primarily linked to SRSF1-mediated translation and not to an indirect effect on the cell cycle .",
"Conversely , SRSF1 depletion led to major cell cycle aberrations ( Figure 5—figure supplement 1B ) , with approximately 50% of SRSF1-depleted cells remaining arrested in the G2/M phase .",
"This could be caused by a loss of SRSF1-dependent translation of targets required for cell cycle progression , as we observed decreased association of these mRNA targets with polysomes in SRSF1-depleted cells ( Figure 5B ) , as well as decreased levels of the corresponding proteins ( Figure 5D ) .",
"Importantly , the levels of SRSF1 itself do not change significantly throughout the cell cycle , albeit there is a 1 . 2-fold increase in SRSF1 protein levels from the G1 to S phase ( Ly et al . , 2014 ) .",
"We cannot rule out , however , that the subcellular localization of SRSF1 is cell cycle regulated , which could potentially affect the translation of SRSF1 targets .",
"We proceeded to further assess the effect of SRSF1 depletion in mitotic progression .",
"The proteasome inhibitor MG132 was added for 2 hr to HeLa cells in culture and the cell cycle profile was evaluated at different times following MG132 release ( Figure 6A ) .",
"As previously demonstrated , proteasome inhibitors induce metaphase arrest ( Wójcik et al . , 1996 ) and indeed following drug treatment we observed accumulation of cells in the prometaphase/metaphase stage of the cell cycle ( Figure 6B ) .",
"Interestingly , while control cells proceeded through the normal stages of mitosis following MG132 withdrawal , HeLa cells that were depleted of SRSF1 remained arrested in metaphase , indicating that SRSF1 was required for normal mitotic progression ( Figure 6B ) .",
"To confirm this , we co-transfected HeLa cells with epitope-tagged GFP-tubulin and mCherry-H2B and compared the cell cycle stage of control cells and SRSF1-depleted cells by time-lapse imaging ( Figure 6C–E ) .",
"The co-transfected proteins were used as markers to follow the cell cycle stage ( for simplicity only the mCherry-H2B is shown ) .",
"This analysis confirmed that SRSF1 is indeed essential for proper cell cycle progression ( Figure 6D , E ) .",
"Specifically , control cells underwent mitosis in around 50 min , whereas cells depleted of SRSF1 remained arrested in metaphase for several hours , and eventually either underwent cell division or apoptosis .",
"We repeated this experiment in HT1080 , a human fibrosarcoma cell line stably expressing GFP-centromere protein A ( CENPA ) .",
"CENPA is homologous to histone H3 and replaces canonical H3 in the nucleosome core of centromeric chromatin .",
"Thus , monitoring GFP-CENPA protein allows progression through mitosis to be followed .",
"Similarly to what was observed in HeLa cells , SRSF1 depletion in HT1080 resulted in a significant , albeit less severe , increase in the time these cells spent in mitosis .",
"Interestingly , the mitotic delay was partially rescued by restoring normal levels of SRSF1 ( Figure 6F ) . 10 . 7554/eLife . 02028 . 013Figure 6 . SRSF1 is required for cell cycle progression .",
"( A ) and ( C ) Schematic representation of the protocols used to assess mitotic progression .",
"( B ) HeLa cells were treated as in ( A ) and the number of cells at different stages of mitosis was determined by classification of images of fixed cells stained for DNA , tubulin , and pericentrin .",
"( D ) HeLa cells were treated as in ( C ) and time-lapse imaging of mCherry-H2B was performed .",
"Images were captured every 15 min over 24 hr at three different positions .",
"Representative images of cells transfected with control siRNA and SRSF1 siRNA are shown .",
"( E ) The graph indicates the elapsed time ( minutes ) from nuclear envelope breakdown ( NEBD ) or chromatin condensation to the onset of anaphase/telophase or to mitotic cell death .",
"( F ) HT1080 cells stably expressing GFP-CENPA were transfected with control or SRSF1-specific siRNA , and 24 hr later cells were retransfected with either empty vector or SRSF1 .",
"The next day cells were seeded in six-well plates and time-lapse imaging of GFP-CENPA was performed as in ( C ) .",
"The graph indicates the elapsed time ( minutes ) from nuclear envelope breakdown ( NEBD ) or chromatin condensation to the onset of anaphase/telophase or to mitotic cell death .",
"Scale bar is 10 μm .",
"The asterisks indicate statistical significance ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 013 The finding that SRSF1 translational targets are enriched for mRNAs implicated in mitotic spindle function could explain the observed mitotic defect .",
"To explore this further , we followed spindle formation in control cells and in cells depleted of SRSF1 .",
"We noticed that upon depletion of SRSF1 , HeLa cells displayed spindle defects , in particular a multipolar spindle phenotype ( Figure 7A–C ) .",
"SRSF1 depletion also resulted in abnormal alignment and chromosome congression problems ( Figure 7—figure supplement 1 ) .",
"Importantly , the multipolar spindle phenotype could be rescued by transient overexpression of wild-type SRSF1 protein .",
"By contrast , transient expression of the non-shuttling SRSF1-NRS did not rescue this phenotype ( Figure 7D ) .",
"This strongly implies that normal levels of SRSF1 protein are required to maintain a bipolar spindle and that the translational function of SRSF1 is necessary for this activity . 10 . 7554/eLife . 02028 . 014Figure 7 . SRSF1 is required for bipolar spindle formation .",
"( A and B )",
"HeLa cells were transfected with control or SRSF1-specific siRNA and 48 hr later stained for DNA ( blue ) , tubulin ( green ) , and pericentrin ( red ) .",
"Representative images for cells transfected with control or SRSF1-siRNAs ( A and B , respectively ) are shown .",
"( B ) Images show the formation of multipolar spindles upon SRSF1 depletion .",
"( C ) Quantitation of the multipolar spindle phenotype observed in ( B ) upon SRSF1 depletion .",
"( D ) HeLa cells were treated as in ( A and B ) and 24 hr later transfected with either wild-type SRSF1 or its nuclear-retained version ( SRSF1-NRS ) .",
"The next day cells were fixed and stained for DNA , tubulin , and pericentrin and the appearance of multipolar spindle was quantified .",
"Scale bar is 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 01410 . 7554/eLife . 02028 . 015Figure 7—figure supplement 1 . SRSF1 is required for appropriate chromosome alignment .",
"( A ) HeLa cells were transfected with a control or an SRSF1-specific siRNA and 48 hr later stained for DNA ( blue ) , tubulin ( green ) , and pericentrin ( red ) .",
"Representative images show abnormal alignment .",
"( B ) Quantitation of the abnormal alignment phenotype upon SRSF1 depletion . DOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 015 In summary , we have identified the translational targets of the shuttling SR protein SRSF1 and have found a particular enrichment in mRNAs that participate in cell cycle regulation and chromosome segregation .",
"In particular , we identified a role for SRSF1 in translating mRNAs encoding for proteins involved in bipolar spindle formation .",
"The translational regulation of SRSF1 targets could contribute partially to the role of this shuttling SR protein in tumorigenesis ."
],
[
"There is extensive coupling among different steps in eukaryotic gene expression , as illustrated by the intimate connection between transcription and pre-mRNA splicing ( Baurén and Wieslander , 1994; Neugebauer , 2002 ) .",
"Recent genome-wide analyses using the CLIP protocol have identified endogenous RNA targets for several SR proteins ( Sanford et al . , 2009; Änkö et al . , 2012; Pandit et al . , 2013 ) .",
"These studies revealed that individual mRNAs bind multiple SR proteins , as was also described in insect cells ( Björk et al . , 2009 ) .",
"Protein synthesis is a tightly regulated process and its misregulation has been linked to the development of cancer ( Blagden and Willis , 2011 ) .",
"We had previously demonstrated that SRSF1 functions as an adaptor protein to recruit the signaling molecules responsible for the regulation of cap-dependent translation of specific mRNAs ( Michlewski et al . , 2008 ) .",
"Here , we present a transcriptome-wide view of the role of SRSF1 in mRNA translation .",
"This analysis revealed that SRSF1 regulates the translation of mRNAs encoding proteins involved in many different cellular processes , including cell cycle progression , RNA processing , and mRNA translation itself ( Figure 3A ) .",
"For instance , SRSF1 promotes the translation of PABP-interacting protein 1 ( PAIP1 ) , a positive regulator of translation that binds to eIF3 and stabilizes the interaction between PABP and eIF4G ( Martineau et al . , 2008 ) .",
"The activity of SRSF1 in promoting the translation of endogenous targets is mTOR dependent; however , we did not find an effect of SRSF1 on the regulation of either 5′TOP mRNAs or pyrimidine rich translational element ( PRTE ) mRNAs , which have been previously shown to be mTOR sensitive ( Hsieh et al . , 2012; Thoreen et al . , 2012 ) .",
"Interestingly , approximately one third of the SRSF1 translational target mRNAs identified here were previously shown to be bona fide RNA targets of this SR protein by CLIP-seq , suggesting that these are direct translational targets ( Sanford et al . , 2009 ) .",
"Recently , an interesting link between alternative splicing and the preferential association of alternative mRNA isoforms to the translational machinery was reported ( Sterne-Weiler et al . , 2013 ) .",
"Here , we have analyzed changes in alternative splicing in response to different levels of SRSF1 protein .",
"Our previous results have suggested that SRSF1 may act to coordinate the nuclear and cytoplasmic steps of post-transcriptional gene expression for a subset of pre-mRNAs ( Sanford et al . , 2008 ) .",
"Here , we observed that mRNAs that display alternative splicing changes upon increased SRSF1 concentration were also translationally regulated by SRSF1 .",
"This suggests that SRSF1 influences several steps of the mRNA life cycle acting to couple nuclear events with mRNA translation ( Figure 4 ) .",
"Interestingly , it was recently shown that SRSF3 regulates the alternative splicing of programmed cell death 4 ( PDCD4 ) mRNA , but also negatively regulates its translational efficiency in the cytoplasm ( Kim et al . , 2014 ) .",
"SRSF1 was previously reported to be required for the maintenance of genomic stability in chicken DT40 cells and its inactivation results in a G2-phase cell cycle arrest and subsequent programmed cell death ( Li and Manley , 2005; Li et al . , 2005 ) .",
"In this study , we show that SRSF1 is required for normal mitotic progression and its depletion results in metaphase arrest ( Figure 6 ) and the formation of a multipolar spindle , which in general is not compatible with cell survival ( Figure 7 ) .",
"Along the same lines , loss of another SR protein , SRSF2 ( SC35 ) in mouse embryonic fibroblasts also results in a G2/M cell cycle arrest and genomic instability ( Xiao et al . , 2007 ) .",
"SRSF1 and SRSF3 associate with interphase chromosomes and post-mitotic chromatin; however , during mitosis they are displaced from chromatin by phosphorylation of histone H3 on serine 10 ( Loomis et al . , 2009 ) .",
"Interestingly , SRSF1 mRNA is deposited onto mitotic microtubules; however , the regulation of its translation during cell cycle is not currently understood ( Blower et al . , 2007 ) .",
"Moreover , SRSF1 has been recently identified as a factor involved in centriole biogenesis , another mechanism essential for mitosis and genomic integrity .",
"These findings suggest a potential role for SRSF1 in regulating chromatin function and cell cycle progression ( Balestra et al . , 2013 ) .",
"The oncogenic potential of SR proteins has so far being described in terms of the nuclear activities of SR proteins , most notably involving differential alternative splicing of pre-mRNAs involved in signaling and/or cellular transformation ( Ghigna et al . , 2005; Karni et al . , 2007 ) .",
"A recent study confirmed that the transformative potential of SRSF1 requires its splicing activity , since deletion of its first RRM ( RRM1 ) , which is required for pre-mRNA splicing , abrogates this activity .",
"Nonetheless , this study also revealed that preventing the nucleo-cytoplasmic shuttling of SRSF1 prevents its oncogenic potential , strongly suggesting a role for the translational activity of SRSF1 in its oncogenic activities ( Shimoni-Sebag et al . , 2013 ) .",
"Interestingly , SRSF1 as well as SRSF9 ( SRp30c ) have been recently shown to promote the translation of β-catenin in an mTOR-dependent manner and this activates Wnt signaling-mediated tumorigenesis .",
"In agreement , SRSF9 displays oncogenic properties and its overexpression has been observed in multiple types of human tumors ( Fu et al . , 2013 ) .",
"Results presented here strongly suggest that overexpressed SRSF1 could contribute to tumorigenesis by influencing the translational rate of key components of the cell cycle machinery .",
"Indeed , many of the SRSF1 translational targets that we have identified here correspond to proteins with roles in chromosome segregation and cell cycle progression ( data in Supplementary file 1 at Dryad: Maslon et al . ( 2014 ) , Figure 2 and Figure 5 ) .",
"In particular , we found that SRSF1 has a role in regulating the translation of proteins that are required for mitotic spindle and kinetochore function .",
"Importantly , altered expression of any of the spindle-associated proteins may contribute to unbalanced chromosome segregation during mitosis .",
"Indeed , increased expression of NDC80 and SMC2 has been observed in cancer cells and an increased requirement for NDC80 at kinetochores of cancer cells has been postulated ( Ferretti et al . , 2010; Dávalos et al . , 2012 ) .",
"Interestingly , some SRSF1 translational targets , including NDC80 and centrosomal proteins , have been previously identified in an siRNA screen looking for proteins involved in centrosome clustering in cancer cells ( Leber et al . , 2010 ) .",
"This suggests that the oncogenic function of SRSF1 could be partially due to its role in promoting the translation of factors that are involved in suppressing multipolar divisions in human tumor cells .",
"Indeed , decreased levels of SRSF1 protein result in multipolar spindle formation and abnormal chromosomal alignment ( Figure 7—figure supplement 1 ) .",
"Importantly , we were able to rescue the multipolar spindle phenotype observed upon SRSF1 depletion only with overexpression of the wild-type SRSF1 protein .",
"By contrast , increased expression of the nuclear-retained SRSF1-NRS did not rescue this phenotype , strongly suggesting a role for SRSF1-regulated translation in this process ( Figure 7 ) .",
"So far , most studies on gene regulation during cell cycle progression have focused on the transcriptional regulation of mRNAs encoding proteins required for this process as well as on the timely proteasome-mediated degradation of checkpoint proteins .",
"Lately however , a crucial role for the translational regulation of hundreds of mRNAs required for cell cycle progression , including most of the mRNAs encoding proteins forming the cohesin and condensin complexes , has been uncovered ( Stumpf et al . , 2013 ) .",
"Interestingly , and despite the fact that our experiments were performed in an unsynchronized cell population , we observed an approximate 15% overlap between SRSF1 direct translational targets identified in this work and those genes that were shown to exhibit translation regulation during cell cycle progression ( Stumpf et al . , 2013 and this study ) , strongly suggesting that SRSF1 may have a central role in this event ( Fisher's exact test: OR 1 . 466548 , p=0 . 02611 ) .",
"Those overlapping targets include the condensin components , SMC2 and SMC4 , the centrosomal protein CEP170 , and the DNA repair protein MRE11A .",
"Altogether , this suggests that SRSF1 could provide a transcript-specific mechanism for translational regulation of the cell cycle .",
"Increased expression of SRSF1 would promote the increased translation of genes associated with cell division and this could partially explain the oncogenic role of SRSF1 .",
"In summary , these data provide insights on the complex role of SRSF1 in the control of gene expression and its implications in cancer ."
],
[
"HEK 293T , HeLa , and HT1080 cell lines were grown in Dulbecco's Modified Eagle's Medium ( Invitrogen ) supplemented with 10% fetal calf serum , and incubated at 37°C in the presence of 5% CO2 .",
"Control pooled siRNA ( D-001810-01 ) , SRSF1 pooled siRNA ( L-018672-01 ) , and SRSF1 UTR-targeting siRNA ( J-018672-12 ) were purchased from Thermo Scientific .",
"Cycloheximide was from Merck Chemicals and used at 50 μg/ml .",
"PP242 was from Cayman Chemicals and was used at 5 μM .",
"Cells were grown to 30% confluency and then incubated with 2 mM thymidine ( VWR International ) for 18 hr , washed with PBS , and released into thymidine-free medium for 9 hr .",
"Thymidine ( 2 mM ) was then added for a further 16 hr .",
"The cells were then washed with PBS , released into thymidine-free medium , and harvested at different time points , as indicated in the figure legend ( Figure 5—figure supplement 1 ) , and analyzed by propidium iodide-flow cytometric analysis .",
"Following the indicated treatment , cells were collected by centrifugation ( 1000 rpm , 4 min ) .",
"After washing with PBS solution , cells were fixed with chilled 70% ethanol at 4°C for 24 hr .",
"The cells were then centrifuged ( 1000 rpm , 4 min ) , washed once with PBS solution , resuspended in PBS , incubated with 5 μl of RNase A ( 0 . 5 μg/ml; Roche ) for 30 min at 37°C , and stained with 50 μg/ml propidium iodide ( Sigma ) for 30 min at room temperature .",
"Cell cycle distribution was then evaluated using flow cytometry .",
"The mammalian expression vector pCGT7-SRSF1 ( previously known as pCG T7-SF2/ASF ) has been previously described ( Cáceres et al . , 1997 ) .",
"Transcription is driven by the cytomegalovirus enhancer-promoter and the coding sequence begins with an N-terminal epitope tag , MASMTGGQQMG; this sequence corresponds to the first 11 residues of the bacteriophage T7 gene 10 capsid protein and is recognized by the T7 . tag monoclonal antibody ( Novagen ) .",
"The mammalian expression vector GFP-α-tubulin and RFP-H2B were provided by Carol-Anne Martin ( MRC HGU ) .",
"Using Lipofectamine 2000 ( Invitrogen ) , 70–90% confluent cells were transfected with the indicated amount of pCG T7 expression vector .",
"The transfection medium was replaced with fresh medium with 10% FCS after 5 hr and following 24 or 48 hr incubation , cells were harvested and lysed or seeded for subsequent analysis .",
"To determine transfection efficiency , HEK 293T cells were transfected in the same conditions with a plasmid encoding green fluorescent protein ( GFP ) .",
"The efficiency of transfection was measured 72 hr later using a FACS cantoII ( BD ) flow cytometer .",
"Using DharmaFECT1 reagent ( Thermo Scientific ) according to manufacturer's protocol , 30–50% confluent cells were transfected with 100 nM siRNA .",
"RNA was isolated using TRIzol LS Reagent ( Invitrogen ) following the manufacturer's protocol .",
"RNA was then treated with Dnase ( Ambion ) and transcribed to cDNA using the First-Strand Synthesis System from Roche .",
"This was followed by a probe detection qPCR assay ( RealTime ready Custom Panel; Roche ) .",
"For splicing validation and luciferase reporter mRNA analysis , the SYBR Green detection system was used ( Lightcycler 2× SYBR Green Mix; Roche ) .",
"The polysomal to monosomal ratio was calculated using the ΔΔCt method and the statistical analyses were performed using the Mann–Whitney U test .",
"Cell pellets were lysed in 50 mM Tris pH 8 . 0 , 150 mM NaCl , and 1% NP-40 buffer containing protease inhibitors .",
"Protein samples either from HEK 293T or HeLa cell extracts were separated by SDS–PAGE and electroblotted onto nitrocellulose membranes ( Whatman ) in 25 mM Tris-base , 40 mM glycine , and 20% methanol in a Genie Blotter unit ( Idea Scientific Company ) , at 12 V for 1 hr or iBlot System ( Invitrogen ) for 6 min .",
"Non-specific binding sites were blocked by incubation of the membrane with 5% non-fat milk in PBS containing 0 . 1% Tween 20 ( PBST ) .",
"Proteins were detected using the following primary antibodies diluted in blocking solution: mouse monoclonal anti-SRSF1 ( clone 96 , 1:1000; Hanamura et al . , 1998 ) , rabbit polyclonal anti-GAPDH ( 1:2000; Abcam ) , mouse monoclonal anti-T7 ( 1:10 , 000; Novagen ) , rabbit polyclonal anti-CEP170 ( 1:1000 , Abcam ) , rabbit polyclonal anti-SMC4 ( 1:1000; Bethyl Laboratories ) , rabbit polyclonal anti-CEP70 ( 1:1000; Abcam ) , rabbit polyclonal anti-CEP57 ( 1:250; Abcam ) , mouse monoclonal anti-NDC80 ( 1:1000; Abcam ) , and mouse anti-β-actin ( 1:5000; Sigma-Aldrich ) .",
"Following washing in PBST , blots were incubated with the appropriate secondary antibodies conjugated to horse-radish peroxidase ( Pierce ) and detected with Super Signal West Pico detection reagent ( Pierce ) .",
"The membranes were stripped using ReBlot Plus Strong Antibody Stripping solution ( Chemicon ) equilibrated in water , blocked in 5% milk in PBST , and reprobed , as described above .",
"Cells were transfected with the indicated constructs , including various pGL3 constructs using Lipofectamine 2000 .",
"Cells were then lysed on the plate using passive lysis buffer ( Promega ) and used for the Dual Luciferase Assay Kit following the manufacturer's guidelines ( Promega ) .",
"Samples were measured on a Monolight 3010 luminometer ( Pharmingen ) .",
"Firefly luciferase activity was normalized to Renilla luciferase expression .",
"HeLa and/or HEK 293T cell were treated with 50 μg/ml cycloheximide for 30 min at 48 hr after transfection .",
"Cells were subsequently washed twice in ice-cold PBS containing cycloheximide .",
"Cytoplasmic extracts were prepared as previously described ( Sanford et al . , 2004 ) .",
"Sucrose gradients ( 10–45% ) containing 20 mM Tris , pH 7 . 5 , 5 mM MgCl2 , and 100 mM KCl were made using the BioComp gradient master .",
"Extracts were loaded onto the gradient and centrifuged for 2 . 5 hr at 41 , 000 rpm in a Sorvall centrifuge with a SW41Ti rotor .",
"Following centrifugation , gradients were fractionated using a BioComp gradient station model 153 ( BioComp Instruments , New Brunswick , Canada ) measuring cytosolic RNA at 254 nm .",
"Fractions 8 to 11 ( polysomal fractions ) and 1 to 7 ( subpolysomal fractions ) were pooled and sucrose concentration was adjusted to 20% w/v .",
"The RNA extraction was performed as described above .",
"All sequence reads were mapped to the RefSeq transcripts ( Pruitt et al . , 2009 ) using GEM ( Marco-Sola et al . , 2012 ) , allowing for up to three mismatches per read and testing for both strands .",
"Unambiguous reads , mapping to unique positions in the reference , and ambiguous reads , mapping to up to 10 multiple positions , were collected ( Table 1 ) . 10 . 7554/eLife . 02028 . 016Table 1 . Number of sequenced and mapped reads from each sampleDOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 016SampleTotal readsMapped readsUnambiguousAmbiguousPolysomal ( mock ) 15 , 303 , 46113 , 225 , 8028 , 629 , 4994 , 596 , 303Polysomal ( SRSF1 ) 19 , 135 , 00815 , 505 , 49310 , 150 , 7755 , 354 , 718Subpolysomal ( mock ) 15 , 553 , 05811 , 676 , 7697 , 489 , 3294 , 187 , 440Subpolysomal ( SRSF1 ) 14 , 417 , 24011 , 025 , 7977 , 106 , 9343 , 918 , 863 Only the reads mapping forward in transcripts were kept for the analysis ( Table 2 ) . 10 . 7554/eLife . 02028 . 017Table 2 . Total number of forward read counts considered in each sampleDOI: http://dx . doi . org/10 . 7554/eLife . 02028 . 017Total forward countsPolysomal ( mock ) 10 , 407 , 102Polysomal ( SRSF1 ) 12 , 219 , 337Subpolysosomal ( mock ) 9 , 188 , 985Subpolyosomal ( SRSF1 ) 8 , 585 , 825 For each mRNA ( a ) , the density of read counts was calculating using the reads per kilobase per million of mapped reads ( RPKM ) in a given sample ( N ) : ( 1 ) d ( a , N ) =109n ( a , N ) N×length ( a ) Using these densities , for each transcript and for each of the two samples , polysomal ( poly ) and subpolysomal ( sub ) , a polysomal index ( P ) was defined: ( 2 ) P ( a ) =d ( a , Npoly ) d ( a , Npoly ) +d ( a , Nsub ) This index measures the proportion of transcript copies that is present in polysomes .",
"Then , in order to determine the mRNAs that shift to polysomes upon SRSF1 overexpression , we defined a polysome shift ratio ( PSR ) , as the log2 ratio of the polysomal index between the SRSF1 overexpressed and mock experiments: ( 3 ) PSR=log2 ( P ( a , SRSF1 ) P ( a , mock ) ) To estimate the cases that change significantly , two non-overlapping subpopulations of read counts from the mock sample were compared to each other .",
"From these two subpopulations , the polysomal index and the ratio between them were calculated .",
"From this comparison , we calculated an empirical p value .",
"The SRSF1 mRNA translational targets were compared with RNAs having tags from the CLIP experiments from Sanford et al . ( 2008 ) and ( 2009 ) .",
"A total of 23 , 633 CLIP tags obtained from a 454 sequencing experiment in Sanford et al . ( 2009 ) were mapped to the RefSeq mRNA set using Exonerate ( Slater and Birney , 2005 ) with an ungapped alignment model .",
"Sequence tags that fully aligned to the mRNA in the forward strand were kept .",
"A total of 9 , 094 different mRNAs from RefSeq were found to contain one or more tags .",
"For the k-mer enrichment analysis , we considered the total count of 5-mers in CLIP-tag regions .",
"The significant differences in relative abundance of 5-mers between the two sets were estimated using the z score statistic ( Fairbrother et al . , 2002 ) : ( 4 ) z=XANA−XBNB ( 1NA−1NB ) g ( 1−g ) where XA and XB are the number of occurrences of a given 5-mer in sets A and B , respectively; NA and NB are the total number of occurrences of all 5-mers in sets A and B , respectively , and g= ( XA + XB ) /NA + NB ) .",
"SRSF1 translational targets were defined as those mRNAs that shifted to polysomes significantly , that is PSR>0 . 889 ( TTR , 1576 mRNAs ) .",
"The set of mRNAs that did not shift were defined as those mRNAs with PSR −0 . 02<p<0 . 02 ( null , 1448 mRNAs ) .",
"If two or more mRNAs from a set had 80% or greater sequence similarity , only the longest one was kept .",
"Accordingly , we were left with 1052 and 1133 sequences , respectively .",
"The 24 most significant 5-mers were selected ( z score≥35 , upper tail ∼2 . 3% ) , including: AAAAG , AAAAT , AAAGA , AAATA , AAATG , AAATT , AATAT , AATTA , AATTT , AGAAA , ATAAA , ATATT , ATTTA , ATTTT , GAAAA , TAAAA , TAAAT , TATTT , TGAAA , TTAAA , TTATT , TTTAA , TTTAT , and TTTTA .",
"In order to select candidates for direct targets of SRSF1 , we considered the tags from a previous CLIP-Seq experiment ( Sanford et al . , 2008 , 2009 ) .",
"First , all mRNAs were separated into those with CLIP-tags ( CLIP+ ) and those without CLIP-tags ( CLIP- ) .",
"Subsequently , a double comparison of 5-mers was considered:5-mers inside CLIP-tags in TTR+ CLIP+ mRNAs versus 5-mers inside CLIP-tags in null CLIP+ mRNAs . 5-mers inside CLIP-tags in TTR+ CLIP+ mRNAs versus 5-mers in TTR+ CLIP+ mRNAs outside the CLIP-tags , that is the rest of the direct targets .",
"The double ranking of z scores was used to select 5-mers associated with direct translational targets .",
"Subsequently , considering positives z scores such that p≤10−5 in both rankings , we found 12 GAA-rich 5-mers associated with direct targets: AAAAG , AAAGA , AAAGG , AAGAA , AAGAT , AGAAA , ATGAA , ATTGG , GAAAA , GAAGA , TGGAA , and TTGGA .",
"In order to infer a consensus motif logo for direct translational targets , we first mapped the selected 5-mers on the mRNA sequences extending 10 nt per flank .",
"The resulting continuous sequences in TTR+ CLIP+ mRNAs were extracted .",
"The background model for MEME was built using a Markov model with the ‘null’ sequences for translational targets ( M1 ) and with the CLIP-tags in null mRNAs and in TTR+ CLIP+ mRNAs outside the CLIP-tags for the direct translational targets ( CM ) .",
"Using these sequences as input , the program MEME ( Bailey and Elkan , 1995 ) was used to recover a motif logo , requiring candidate motifs to appear in at least 90% of the input sequence set .",
"We found only one motif for each pool of sequences that satisfied this criterion .",
"The list of SRSF1 translational targets , including those with CLIP-tags and those estimated to be direct translational targets ( CLIP-tag and consensus motif ) were uploaded as a gene list to the Database for Annotation , Visualization and Integrated Discovery ( DAVID ) v6 . 7 ( http://david . abcc . ncifcrf . gov/home . jsp ) , while all the mRNAs detected by RNA-seq were used as a background ( Dennis et al . , 2003 ) .",
"Then we analyzed the over-represented functional categories in ‘Biological Process’ using the gene functional classification tool containing all the levels of GO terms as described in Huang et al . ( 2009 ) .",
"EASE scores ( modified Fisher's exact p value ) were computed for all categories .",
"The Benjamini–Hochberg correction method was applied to the data in order to identify the most significantly over-represented gene categories .",
"For SILAC , HEK 293T cells were grown for 8 d with two passages in DMEM SILAC media before transfection ( Dundee Cell Products , Dundee , UK ) .",
"The arginine and lysine isotopes were as follows: R0K0 , L-[12C614N4]arginine ( R0 ) , and v-[12C614N2]lysine ( K0 ) ; R6K4 , L-[13C614N4]arginine ( R6 ) , and L-[12C62H414N2]lysine ( K4 ) ; and R10K8 , L-[13C615N4]arginine ( R10 ) , and L-[13C615N2]lysine ( K8 ) .",
"Cells were grown either with R0K0 ( untransfected cells ) , R6K4 ( cells transfected with empty vector ) , or R10K8 ( cell transfected with pCG-T7SRSF1 ) .",
"At 48 hr after transfection , cells were washed twice in ice-cold PBS and scraped into ice-cold RIPA buffer containing a protease inhibitor cocktail ( Roche ) .",
"Total protein extracts were measured by the Bradford assay .",
"Equal amounts of protein from unlabeled and labeled samples were run on SDS–PAGE , and gel lanes were cut into 10 sections , followed by overnight digestion with trypsin at 37°C .",
"Sample processing , mass spectrometry , and data analysis were performed by the Dundee Cell Products service .",
"The protein quantification changes detected by SILAC were compared to the changes in PSR detected with RNA-seq upon SRSF1 overexpression .",
"After applying quantile normalization ( Bolstad et al . , 2003 ) to the enrichment signal of SRSF1 versus untransfected , and to the enrichment signal of the empty vector sample over untransfected , the log2 ratio of the two normalized signals was considered .",
"In order to compare proteins identified by SILAC with mRNAs shifted to polysomes , the IPI identifiers ( Kersey et al . , 2004 ) from the SILAC experiment were mapped to the RefSeq identifiers used for the RNA-seq data .",
"Total RNA was purified and genomic DNA removed using the RNeasy Plus Kit ( 74 , 134; Qiagen ) according to the manufacturer's instructions .",
"The RNA quality was verified by the 2100 Bioanalyzer ( Agilent ) .",
"Each array experiment was performed in triplicate .",
"Array data analysis was performed by GenoSplice ( www . genosplice . com; Paris , France ) .",
"Affymetrix Human JAY arrays were normalized using the probe scaling method and background corrected with ProbeEffect from GeneBase ( Kapur et al . , 2008 ) .",
"The gene expression index was computed from probes that were selected using ProbeSelect from GeneBase ( Kapur et al . , 2008 ) .",
"Gene expression signals were computed using these probes .",
"Genes were considered expressed if mean intensity was ≥200 .",
"Genes were considered regulated if: ( 1 ) they were expressed in at least one condition ( i . e . , SRSF1 and/or empty vector control ) ; ( 2 ) fold-change was ≥1 . 5; and ( 3 ) the unpaired t test p value between gene intensities was ≤0 . 05 .",
"For each probe , a splicing index was computed .",
"Unpaired t tests were performed to test the difference in probe expression between the two samples as described previously ( Shen et al . , 2010 ) .",
"Probe p values in each probeset were then summarized using Fisher's method .",
"Using annotation files , splicing patterns ( cassette exons , 5′/3′ alternative splice sites , and mutually exclusive exons ) were tested for difference between isoforms , selecting those with a minimum number of regulated probesets ( with p≤0 . 01 ) in each competing isoform ( at least one third of ‘exclusion’ probesets have to be significant , and at least one third of ‘inclusion’ probesets have to be significant and show an opposite regulation for the splicing index compared to ‘exclusion’ probesets ) .",
"For example , for a single cassette exon , the exclusion junction and at least one of the three inclusion probesets ( one exon probeset and two inclusion junction probesets ) have to be significant and have to show an opposite regulation for the splicing index .",
"Significantly regulated cassette exons from the array were mapped to RefSeq genes .",
"Each upregulated or downregulated exon was assigned to alternative isoforms that included or excluded the exon , respectively .",
"No isoform with two exons regulated in opposite directions .",
"The values of PSR were compared between the datasets of mRNAs either including or skipping the cassette exons from the array , as well as for all mRNAs detected by RNA-seq belonging to genes with multiple isoforms .",
"Appropriately transfected cells were seeded on coverslips in six-well plates .",
"After 24 hr , cells were rinsed in PBS and fixed with 4% paraformaldehyde for 10 min at room temperature .",
"The fixed cells were washed with PBS and permabilised using PBS + 0 . 2% Triton .",
"After washing , the slides were blocked in 1% BSA in PBS for 1 hr .",
"Various antibodies were used including anti-pericentrin , anti-α-tubulin , and anti-SRSF1 , which were diluted 1:2000 , 1:1000 , and 1:1000 , respectively .",
"Slides were then washed and incubated with the appropriate AlexaFluor 488 and AlexaFluor 594 labeled secondary antibodies at 1:2000 .",
"Final washes were followed by mounting with DAPI Vectashield .",
"A fluorescent upright microscope , Zeiss Axioplan 2 , was used to image the cells at 20× magnification .",
"At least 100 mitotic cells were captured and abnormal mitoses were scored for multipolar spindle and metaphase misalignment .",
"Data are presented as a percentage of abnormal phenotypes versus total number of scored mitotic cells .",
"Transfected cells were seeded in six-well plates .",
"After 24 hr , cells were rinsed twice in PBS and grown in phenol-red free medium .",
"Live imaging for 24 hr at a time lapse of 15 min was performed with a Zeiss Axiovert 200 microscope .",
"Metamorph software was used for image capture and analysis .",
"At least 20 mitotic cells were assessed for the time spent in mitosis .",
"Supplementary files 1–4 are available at Dryad: Maslon et al . ( 2014 ) .",
"Supplementary file",
"1 . List of SRSF1 translational targets .",
"List of 1576 mRNAs enriched in polysomes upon SRSF1 overexpression .",
"Supplementary file",
"2 . SILAC analysis following SRSF1 overexpression .",
"List of proteins identified in SILAC experiment .",
"Supplementary file",
"3 . Alternative splicing analysis .",
"List of the 382 SRSF1-regulated cassette exons determined by exon arrays ( cassette exons ) .",
"List of SRSF1-regulated cassette exons with SRSF1-clip-tags over cassette exon ( clip tag over cassette exon tab ) .",
"Supplementary file",
"4 . Bed file containing positions and sequences of the consensus motif in hg18 assembly of human genome ."
]
] | [
"The shuttling serine/arginine rich ( SR ) protein SRSF1 ( previously known as SF2/ASF ) is a splicing regulator that also activates translation in the cytoplasm .",
"In order to dissect the gene network that is translationally regulated by SRSF1 , we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1 .",
"We identified approximately 1500 mRNAs that are translational targets of SRSF1 .",
"These include mRNAs encoding proteins involved in cell cycle regulation , such as spindle , kinetochore , and M phase proteins , which are essential for accurate chromosome segregation .",
"Indeed , we show that translational activity of SRSF1 is required for normal mitotic progression .",
"Furthermore , we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets , strongly suggesting that SRSF1 couples pre-mRNA splicing and translation .",
"These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer ."
] | [
"Genes contain the instructions to make proteins .",
"These instructions are first transcribed to produce an intermediate molecule called a messenger RNA ( mRNA ) , which is then translated to produce the protein .",
"However , gene sequences are often interrupted by ‘introns’ , sections of DNA that do not code for protein , and these introns must be removed from the mRNA molecules via a process called ‘splicing’ before the protein is produced .",
"Splicing can also be used to ‘mix and match’ sections of gene sequences to produce slightly different versions of the same protein in a process called ‘alternative splicing’ .",
"SRSF1 is one of a family of proteins that control both types of gene splicing but also promotes the translation of specific mRNAs .",
"To date only a few of the genes whose translation is regulated by SRSF1 have been identified .",
"Here , Maslon , Heras et al . have used human cells that artificially produce more SRSF1 protein than normal to identify those genes whose translation is regulated by SRSF1 .",
"Over 1500 ‘target genes’ were found; many of which encoded proteins that are involved in cell division—and cells with less SRSF1 than normal failed to divide properly .",
"Maslon , Heras et al . also found a link between alternative splicing and protein translation: many of the mRNAs that were spliced differently in cells that over-produced SRSF1 were also genes whose translation was affected by SRSF1 .",
"Since uncontrolled cell division , or defects in mRNA splicing or protein synthesis are all often linked to cancer , these discoveries might provide new insights into the mechanisms underlying this disease ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease"
] | Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure | elife-34039-v2 | [
[
"Parasitic diseases have been particularly resistant to the development of effective vaccines and safe and curative therapeutic drugs .",
"The drugs that have been put into use often suffer from one or more of the problems of inefficiency , high toxicity and/or selection of drug resistant mutants .",
"Despite being one of the highest impact infectious diseases in the Americas , T . cruzi infection in humans is relatively rarely treated .",
"Initially this was because of the misconception that Chagas disease had an autoimmune , rather than pathogen persistence etiology ( Tarleton , 2003 ) but more recently the substantial toxic side effects and the limited supply of benznidazole ( BZN ) and nifurtimox ( NFX ) , drugs that have been in use for decades , has also contributed significantly to their low rate of utilization .",
"However , the highly variable outcome of treatment regimens employing these compounds – ranging from 0% to 100% depending on the study - and the difficulty of accurately assessing these outcomes , also keep utilization as low as 1% of those infected ( de Castro et al . , 2006; Guedes et al . , 2011; Jackson et al . , 2010; Navarro et al . , 2012; Yun et al . , 2009 ) .",
"The reason for the high variability in the efficacy of BZN and NFX is not known but has been primarily attributed to the broad genetic diversity of T . cruzi isolates .",
"Importantly , the lack of drug efficacy is almost certainly not a result of selection of genetic resistance by drug use or misuse , as neither BZN nor NFX have been widely or indiscriminately used and the induction of stable resistance to these drugs is difficult to produce in vitro or in vivo .",
"Further , the variable and often low contribution of humans to infection of insect vectors ( many infections in insects originate from feeding on non-human mammals , within which T . cruzi circulates widely and at high levels [Cohen and Gürtler , 2001] ) , offers very few opportunities to drive and spread resistance even if it were to develop .",
"In addition to classical genetic-based drug resistance , it has become increasingly appreciated that in bacterial infections , ‘persister’ cells develop that are transiently unaffected by chemotherapy , due in most cases to metabolic dormancy ( reviewed in [Harms et al . , 2016] ) .",
"Such persisters can arise spontaneously in the presence or absence of stress signals and can reemerge after the termination of environmental challenges – such as antibiotic treatment .",
"In this study , we document the occurrence of dormancy in the protozoan T . cruzi and link this phenomenon to the resistance of this parasite to otherwise highly effective and cytotoxic drugs .",
"We show that both in vitro and in vivo , T . cruzi exhibits a biphasic kill curve , with the rapid death of most parasites but the persistence of transiently non-replicating intracellular parasites that are resistant to high level and prolonged drug exposure ."
],
[
"The anti-T .",
"cruzi drugs BZN and NFX are normally dosed in animals ( including humans ) for 30–60 days , although shorter treatment times can occasionally be curative ( Bustamante et al . , 2014; Viotti et al . , 2011 ) .",
"This general requirement for a long treatment course and the variability in outcome is inconsistent with the documented rapid trypanocidal activity of BNZ for T . cruzi in vitro ( Moraes et al . , 2014 ) , although to our knowledge , kill rates had not previously been determined in vivo .",
"To directly assess the activity of BNZ in vivo , we first established a localized infection of T . cruzi in the footpads of mice and then used a transgenic luciferase reporter to monitor the loss of parasites subsequent to a single oral dose of BNZ .",
"We first determined that T . cruzi trypomastigotes delivered in the footpad or skin , primarily infect host cells at the infection site ( rather than distributing systemically ) and expand as amastigotes within these host cells during the subsequent ~96 hr ( Padilla , et al . , in preparation ) .",
"Administration of a single oral dose of BZN reduced intracellular amastigotes at the initial infection site by ~90% within 48 hr ( Figure 1A–C ) .",
"A similarly rapid reduction in parasite load occurs in mice with established systemic infections within a few days after the initiation of oral BZN treatment ( Figure 1D–F ) .",
"In this later experiment we used multiple T . cruzi isolates , including two ( colombiana and ARC-0704 ) previously determined to be relatively more resistant to BZN in vivo ( Bustamante et al . , 2014 ) .",
"The rate of parasite clearance , approaching the limit of detection by imaging , was similar for all three strains and in the case of the colombiana infection , the 5 days of treatment resulted in a > 3 log10 reduction in systemic parasite load .",
"Thus , consistent with previous in vitro assessments , BZN rapidly and highly effectively kills both ‘resistant’ and ‘susceptible’ T . cruzi strains in vivo .",
"While these experiments demonstrate that BZN treatment rapidly reduces T . cruzi numbers by 10- to 1000-fold over just a few days , we know from previous studies that such short-term treatments , and indeed treatments as long as 60 days , often fail to completely clear T . cruzi infection ( Bustamante et al . , 2014 ) .",
"This disconnect implicates a mechanism by which T . cruzi can resist highly effective trypanocidal drugs for an extended period of time .",
"To investigate whether dormant/non-replicating forms of T . cruzi might be present in infected hosts and possibly contributing to this requirement for long treatment regimens , we used in situ labeling with nucleotide analog 5-ethynyl-2’-deoxyuridine ( EdU ) to determine the replication status of parasites within tissues of infected mice .",
"We first established the in vivo EdU labeling protocol in acutely infected mice with high tissue parasite load , finding that the vast majority of amastigotes incorporated EdU during a 24 hr period after EdU injection ( Figure 2—figure supplement 1 ) .",
"We then applied this protocol to mice with established chronic T . cruzi infections and again found that most intracellular amastigotes incorporated EdU over the 24–72 hr of exposure .",
"However a small fraction of intracellular amastigotes failed to show evidence of replication over the 72 hr after EdU injection ( Figure 2—figure supplement 2 ) .",
"Interestingly , these apparently non-replicating amastigotes were within host cells in which the other amastigotes were clearly replicating , suggesting that there is a heterogeneity of amastigotes in infected cells .",
"Since the low parasite load in chronically infected mice makes it highly unlikely that a single host cell is infected by more than one trypomastigote , this heterogeneity in replication appears to establish within host cells from a single infecting parasite Figure 2 .",
"To examine in greater detail the replication pattern of T . cruzi in host cells , we moved to in vitro systems wherein large numbers of infected cells can be readily monitored longitudinally during infection and the continuous exposure to EdU can be assured .",
"As in the in vivo infection , a small fraction of amastigotes in host cells in vitro consistently failed to take up EdU , even when exposed for up to 72 hr of the ~96 hr intracellular replication phase ( Figure 3 ) .",
"One possible reason for the failure to replicate is that some amastigotes might die within host cells ( perhaps as a result of lethal damage to DNA , defective cytokinesis , or unequal dispersal of chromosomes/organelles during prior replication , among other possible reasons ) .",
"However monitoring for apoptosis of amastigotes in host cells revealed this process to be extremely rare ( Figure 3—figure supplement 1 ) .",
"Incorporation of EdU into DNA can impact DNA replication and thus cell division ( Zhao et al . , 2013 ) .",
"To rule out a role for EdU itself in inhibition of parasite replication , we used the cell division tracker dyes carboxyfluorescein succinimidyl ester ( CFSE ) and CellTrace Violet as a second method to monitor parasite replication in host cells .",
"Both dyes couple to cellular components and are equally distributed to daughter cells during cytokinesis and with vigorous replication , the dyes are eventually diluted beyond the point of detection .",
"T . cruzi trypomastigotes uniformly stain with either dye and upon infection of host cells , replicating amastigotes dilute the dye , while a fraction of amastigotes remain dye positive , indicating again the limited replication of a subset of amastigotes ( Figure 4A and B ) .",
"Amastigotes remaining dye positive after 72–96 hr were also EdU-negative ( Figure 4C ) .",
"By continuing to monitor infected host cells over time , we observed that amastigotes that had failed to incorporate EdU during the final ~72 hr in host cells were fully competent to convert to trypomastigotes and to exit host cells , along with the previously replicating ( EdU+ ) trypomastigotes ( Figure 4D , E ) .",
"The use of the CellTrace Violet to mark parasites that had undergone a limited number or no divisions during the infection cycle in host cells allowed us to further characterize and ultimately isolate this minority population of trypomastigotes from infected cell cultures ( Figure 4F and G ) , and to assess their ability to establish a second round of infection/replication in new host cells .",
"Not only could trypomastigotes from minimally replicating amastigotes infect new host cells , re-convert to amastigotes ( Figure 5A ) , and begin a new round of replication , but progeny of this replication can also stop dividing soon after invasion and retain dye for >6 days while other progeny of this dormant amastigote divided extensively ( Figure 5B ) .",
"Thus , a small proportion of T . cruzi amastigotes halt replication within 24 hr after host cell entry but can convert to trypomastigotes that are infection-competent and able to repeat the processes of replication and arrested replication in new host cells ( Figure 5C ) .",
"The detection of a small subpopulation of T . cruzi amastigotes undergoing minimal replication through 2 rounds of host cell invasion suggests that the cessation of replication can at a minimum happen early after host cell invasion , but did not preclude a more dynamic pattern of transitioning between replication and dormancy during the infection cycle in host cells .",
"In order to better understand the flexibility and heterogeneity of replication within host cells , we monitored the early replication of T . cruzi in host cells by time-lapse video , using violet dye dilution to help mark those parasites undergoing minimal cell division within the same cell where other parasites are actively dividing ( Sup Videos 1 and 2 ) .",
"These videos also indicate other characteristics of non-dividing amastigotes that were apparent in other still images , for example that non-dividing amastigotes are similar in size to recently divided amastigotes and are generally Tdtomato-dim , expressing low levels of the Tdtomato fluorescent protein relative to rapidly dividing amastigotes .",
"These results show conclusively that within host cells infected with T . cruzi , a subset of amastigotes cease replication while others continue rapid division , eventually filling the host cell with amastigotes .",
"The obvious question remaining is ‘Does this cessation of replication play a critical role in the failure of drugs to effectively clear T . cruzi infection ? ’ More specifically , are these replication-arrested parasites resistant to drug treatment ?",
"To directly address these questions , we infected mice with Tdtomato-expressing and CellTrace Violet-stained trypomastigotes and confirmed 2 days post-infection the presence in adipose tissue of parasites displaying both of these markers ( Figure 6A and B ) .",
"As expected based on previous experiments , over the span of the 2 day infection , several strongly violet-positive parasites are obvious in cells along with a larger number of parasites in which the violet dye was faint or not detectable , indicating both slow or non-replicating and actively replicating progeny of the infecting trypomastigotes .",
"On days 2 and 3 , similarly infected mice were treated with BZN or left untreated and on day 4 , examined for the presence of parasites .",
"Remarkably , in the BZN-treated mice , the only parasites detected were brightly violet and present at 1 and occasionally two per infected cell ( Figure 6B and Figure 6—figure supplement 1 ) .",
"Actively replicating parasites ( violet-negative ) were not detected in the BZN-treated mice while a much larger number of violet-negative and less abundant violet-positive parasites were observed in the non-treated mice .",
"A resumption of parasite replication was evident in treated mice 9 days after the last of the 2 BNZ treatment doses , demonstrating that dormant , and therefore drug resistant amastigotes , could also resume division in vivo .",
"To further address the drug resistance and recovery potential of dormant amastigotes in vivo , we repeated these experiments in interferon-gamma deficient mice , which lack the ability to develop immune control of T . cruzi infection .",
"Non-replicating amastigotes developing in these mice survive a minimum of 20 days of BZN treatment ( Figure 6—figure supplement 2A ) and again resume replication upon cessation of BZN treatment ( Figure 6C ) and eventually spread extensively in these animals ( Figure 6—figure supplement 2B , C ) .",
"Tracking very low numbers of persisting parasites in vivo in drug-treated , infected animals is quite difficult , so we returned to in vitro systems to examine the resistance of T . cruzi to drug treatment over longer time periods and the association between drug resistance and dormancy .",
"Here we infected host cells 24 hr previously with Tdtomato-expressing and CellTrace Violet-labeled parasites and then added BZN at 10X the IC50 for up to 30 days of culture before washing out the drug .",
"Parasite recovery following drug treatment/washout was monitored globally by whole-well fluorescence detection of the Tdtomato reporter , and at the individual cell level by microscopy ( Figure 7 ) .",
"In the absence of treatment , parasite numbers peak near the time of the completion of the first round of intracellular replication ( ~day four after infection ) .",
"In all cases , even following 30 days of high dose BZN treatment , parasites can rebound and begin replication after BZN is removed .",
"This rebound can often be observed in the whole well Tdtomato fluorescence readings ( Figure 7A ) , and in all cases , low numbers of parasites with a range of violet dye retention are evident in cultures during drug treatment ( Figure 7B ) , as well as resumption of low level to vigorous replication after drug washout ( Figure 7C ) .",
"The resistance of T . cruzi to drug treatment due to amastigote dormancy was not restricted only to BNZ , as members of a class of newly developed oxaborale compounds currently under preclinical development also failed to overcome dormancy-dependent resistance in vitro ( Figure 7—figure supplement 1 ) .",
"Further , T . cruzi lines rebounding after 30 days of in vitro exposure to BZN had not developed stable resistance to the compound as the IC50 of BZN on this population remained unchanged from that of the pre-exposed population ( Figure 7—figure supplement 2 ) .",
"Thus , T . cruzi amastigotes regularly and spontaneously cease replication and in that dormant state are resistant to otherwise highly effective trypanocidal drugs for extended periods of time .",
"These dormant parasites are capable of re-initiating replication after >30 days of drug exposure and perhaps much longer after the initiation of dormancy ."
],
[
"Although T . cruzi infection is one of the highest impact infectious disease in the Americas , including in the United States , effective prevention , control and treatment methods are virtually non-existent .",
"A rare bright spot in potential interventions for Chagas disease is the increased availability of drugs such as BZN , which have substantial curative efficacy .",
"However , although BZN and NFX have been in use for decades , have relatively clear mechanisms of action and do not appear to readily encourage development of genetic-based drug resistance , their unpredictable treatment outcome coupled with their modest to severe side effects has limited their use primarily to acute , childhood , or immunosuppression-exacerbated infections .",
"This situation leaves largely untreated the millions of individuals with chronic T . cruzi infection , many of whom will eventually develop chagasic cardiomyopathies .",
"Furthermore , human clinical trials of new therapies judged to be promising based upon limited studies in experimental models have been colossal failures ( Molina et al . , 2014; STOP-CHAGAS Investigators et al . , 2017 ) and ClinicalTrials . gov identifiers NCT01489228 , NCT02498782 , and NCT01162967 ) , demonstrating these compounds to be clearly inferior to BNZ and NFX .",
"This study initially sought to determine why compounds like BZN that can , and often do , provide sterile cure in T . cruzi infection , also frequently fail .",
"This and previous studies have largely ruled out several mechanisms as primary reasons for the variable efficacy of BZN .",
"Although there is parasite strain variation with respect to BZN susceptibility in vivo , this variation does not appear to be due to direct resistance to the trypanocidal activity of BZN ( Figure 1 ) .",
"Parasite lines obtained from failed drug treatments are no more resistant to BZN than the pre-treatment parasites ( Sup Figure 7 ) , and although BZN resistance can be induced by drug selection in vitro , such partially resistant lines are crippled with respect to initiating and maintaining an infection ( Mejia et al . , 2012 ) .",
"Thus resistance to BZN treatment is relative , not complete , is not easily selectable , and is not associated with any specific genetic lineage of T . cruzi nor with host genetics .",
"The results of the current study firmly connect drug treatment failure in T . cruzi infection to the presence of a previously unrecognized player in this parasite’s life cycle , the transiently dormant amastigote .",
"We denote these stages as dormant based primarily on their failure to replicate over an extended period of time and their resistance to trypanocidal compounds over >30 days of exposure .",
"These dormant amastigotes appear at a consistently detectable frequency and are present in many infected host cells in vitro and in vivo .",
"Dormancy occurs spontaneously and is often observed soon after host cell infection – as shown by the ability of parasites to retain cell division monitoring dyes through several rounds of host cell invasion and expansion .",
"However dormancy may also occur at other points throughout the 4–5 day replication cycle within host cells , as suggested by the presence of multiple EdU-negative amastigotes in heavily infected cells and supported by time lapse imaging of infected cells .",
"Importantly , these dormant parasites are not permanently arrested , as they resume replication days to weeks after entering dormancy ( Figure 7 ) .",
"These dormant amastigotes also readily respond to the cues that drive conversion to trypomastigote forms within host cells , a process that appears to occur only when the host cell is nearly bursting with amastigotes .",
"Candidates for the inducer of stage conversion would include depleted host cytoplasmic components or byproducts of parasite metabolism that could accumulate inside the host cell .",
"Thus the dormant amastigotes have two potential fates besides continued dormancy: to re-initiate replication like conventional amastigotes – likely to occur when there are none or a limited number of other amastigotes in the cell - or to convert to trypomastigotes in concert with the actively replicating amastigotes undergoing that process within the same cells .",
"An extremely rare outcome for amastigotes in host cells is death , as determined by the paucity of degrading amastigotes detected in host cells ( Sup Figure 3 ) .",
"Dormancy is well-studied in bacteria and plays a critical role in their adaptation to changing environments .",
"This phenomenon had been missed by previous investigations in T . cruzi likely because extended dormancy ( which would be required to survive during weeks of drug treatment ) is relatively rare .",
"In vitro studies often report very high efficiency of drugs such as BZN to kill >99% of T . cruzi amastigotes – and the assumption has been that this was in fact 100% killing ( Moraes et al . , 2014 ) , but this is clearly not the case .",
"To our knowledge , this is the first report of extended dormancy in an otherwise replicating stage of any trypanosomatid .",
"In the related protozoan Leishmania sp , intracellular amastigotes exhibit greatly reduced proliferation and metabolism relative to the extracellular promastigote forms ( Jara et al . , 2017 ) and have been described as ‘semi-quiescent’ but steadily replicating in vivo with a doubling time of 12 days for L . mexicana ( Kloehn et al . , 2015 ) .",
"Studies in L . major by Mandell and Beverley documented slow growing ( ~60 hr doubling time ) and even slower dividing , BrdU-non-incorporating intracellular amastigotes in mouse infections but the technical limitations of the labeling procedure prevented a definitive conclusion of dormancy ( Mandell and Beverley , 2017 ) .",
"The dormancy we describe here for T . cruzi is clearly very distinct from but perhaps most similar to the scantly studied arrested hypnozoite liver stages of Plasmodium vivax .",
"Hypnozoites appear responsible for relapse of vivax malaria even years after the initial infection ( Markus , 2012 ) , although studies in human liver chimeric mice suggest that hypnozoites may actually not be fully metabolically inactive ( Mikolajczak et al . , 2015 ) .",
"Cyst stages of apicomplexans such as Toxoplasma gondii and Cryptosporidium sp exhibit greatly reduced if not fully quiescent metabolism , consistent with a life cycle stage that is resistant to potentially harsh environmental conditions associated with transmission to a new host .",
"For T . cruzi it is much less clear what the evolutionary advantage of dormancy is since transmission depends on putting extracellular parasites into the mammalian host bloodstream for ingestion by its blood feeding reduviid vector .",
"Although we show that dormancy provides a degree of protection from drug-mediated clearance , drug resistance itself cannot be the selective force for this developmental pathway as all the T . cruzi lines we have examined , including ones that had not been previously exposed to these or other drugs , generate dormant amastigotes .",
"If dormancy is programmed and essential for T . cruzi survival , then the property would seem most likely associated with evasion of host immune responses , the major stressor with which T . cruzi amastigotes have to contend .",
"T . cruzi induces a robust and highly effective immune response that greatly reduces parasite transmission to insect vectors .",
"Although it is relatively rare , this immune response can lead to complete resolution of the infection ( Dias et al . , 2008; Tarleton , 2013; Viotti et al . , 2006 ) .",
"Dormancy could facilitate parasite persistence if some trypomastigotes entering host cells immediately went quiescent and as a result were not detected by the immune system .",
"Such persisters could then ‘wake-up’ and begin replication weeks or months later .",
"But those reawakened parasites would likely remain low in numbers as they would have to contend with the now established anti-T .",
"cruzi immune response in order to expand and have an increased chance of being transmitted .",
"Thus the selective pressure for development or retention of a dormancy pathway for this purpose alone seems very low .",
"Alternatively , dormancy might be a consequence of the biology of T . cruzi , rather than an evolutionarily selected process for low-level persistence .",
"T . cruzi very rarely undergoes sexual recombination but instead depends heavily on gene amplification and recombination , as well as horizontal gene transfer , for generating genetic diversity .",
"The resorting of genetic material likely takes place in amastigotes , one of the two replicating stages in the T . cruzi life cycle .",
"Parasites that enter dormancy soon after host cell invasion could be pre-recombination , ‘stem-like’ cells that would be useful should the genetic recombination events in the replicating parasites go awry .",
"Alternatively , parasites might enter dormancy in order to sort out the result of recombination events before proceeding with additional rounds of replication .",
"Whether stochastic or programmed , the mechanisms for entering and exiting the dormant state by T . cruzi is a complete black box .",
"Hints about the process may come from understanding the events involved in transitioning from replicating stages to non-replicating stages in T . cruzi ( e . g . epimastigotes to infective metacyclic trypomastigote and amastigote to blood stage trypomastigote ) or in the related African trypanosome Trypanosoma brucei , which although lacking an intracellular stage , makes several well-characterized transitions from replicating to non-replicating forms .",
"The biggest practical message from the current study is that dormancy in the mammalian infection cycle of T . cruzi is key to the failure of current drug treatments for T . cruzi infection .",
"While other factors , including the differential tissue tropism of parasite strains and tissue distribution of potential drugs , certainly also impact treatment outcomes , only dormancy has been definitively linked .",
"Given this new understanding of dormancy and its impact on drug treatment , simply identifying more compounds that effectively kill metabolically active parasites is not likely on its own to be the solution to achieving more dependable cure in T . cruzi infection .",
"The development of new assays that can screen for compounds capable of overcoming dormancy should also be part of the paradigm for identifying new , more effective compounds .",
"The use of current drugs in extended , less intensive dosing routines ( Álvarez et al . , 2016; Bustamante et al . , 2014 ) that extend beyond the dormancy potential of T . cruzi , in place of the current highly intensive regimens , should also be further explored ."
],
[
"C57BL/6NCr and IFN-γ knockout ( B6 . 129S7-Ifngtm1Ts/J ) mice were purchased from Charles River Laboratories and The Jackson Laboratory respectively .",
"The SKH-1 ‘hairless’ mice backcrossed to C57BL/6 were a gift from Dr . Lisa DeLouise ( University of Rochester ) .",
"All mice were maintained in the University of Georgia Animal Facility under specific pathogen-free conditions .",
"This study was carried out in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and Association for Assessment and Accreditation of Laboratory Animal Care accreditation guidelines .",
"The protocol was approved by the University of Georgia Institutional Animal Care and Use Committee .",
"To generate bioluminescent parasites , CL-3 , ARC-0704 and Colombiana T . cruzi strains were transfected with the pTREX-Luciferase-Neo plasmid , generated by cloning of the firefly luciferase gene from the Luciferase-pcDNA3 plasmid ( gift from William Kaelin; Addgene plasmid # 18964 ) into the multi-cloning site of the pTREX plasmid ( Vazquez and Levin , 1999 ) .",
"Colombiana bioluminescent parasites were transfected with pTREX-Td-tomato plasmid ( Canavaci et al . , 2010 ) to generate parasites co-expressing bioluminescent and fluorescent markers .",
"Transfection and selection conditions were as previously described ( Peng et al . , 2014 ) .",
"For single-cell cloning , parasites were deposited into a 96-well plate to a density of 1 cell/well using a MoFlow XDP ( Beckman Coulter , Hialeah , FL ) cell sorter and cultured in 250 μl LDNT supplemented with 300 mg/ml G418 .",
"Selected clones were screened for luciferase activity as previously described ( Lewis et al . , 2014 ) and a highly luciferase-expressing clone was selected for in vivo experiments .",
"To obtain metacyclic forms , epimastigotes were submitted to stress in triatome artificial urine ( TAU ) medium for 2 hr .",
"Then , parasites were incubated in complemented TAU medium ( TAU3AAG ) ) ( Bourguignon et al . , 1998 ) for 6–7 days , at which time transformation to metacyclic trypomastigote was maximal .",
"Vero cells were obtained from the American Type Culture Collection ( Manassas , VA ) and cultured in RPMI 1640 medium with 10% fetal bovine serum .",
"Human foreskin fibroblast ( HFF ) cells were a gift from Dr . Drew Etheridge ( University of Georgia ) and were maintained in DMEM media supplemented with 10% fetal bovine serum .",
"Cultures were maintained in a humid atmosphere containing 5% CO2 at 37°C and tested periodically for mycoplasma contamination .",
"Luciferase- and Tdtomato-expressing parasites were used to assess in vivo parasite replication and clearance in mice .",
"For bioluminescent detection , mice were injected via an intraperitoneal ( i . p . ) route with D-luciferin ( 150 mg/kg; PerkinElmer , Waltham , MA ) and anesthetized using 2 . 5% ( vol/vol ) gaseous isofluorane in oxygen prior to imaging on an IVIS 100 imager ( Xenogen , Alameda , CA ) as previously described ( Canavaci et al . , 2010 ) .",
"Quantification of bioluminescence and data analysis was performed using Living Image v4 . 3 software ( Xenogen ) .",
"EdU incorporation into amastigote DNA was detected using the Click-iT EdU Imaging Kit ( ThermoFisher Scientific , Waltham , MA ) following manufacturer specifications .",
"Briefly , Vero cells ( 1 × 105 cells ) were plated onto sterile glass-bottom 35 mm petri dishes and incubated overnight ON at 37°C , 5% CO2 , in RPMI-1640 medium plus 10% fresh fetal bovine serum ( FBS ) .",
"Trypomastigotes of the colombiana , CL , Brazil or ARC-0704 strains were used to infect dishes at a ratio of 10 parasites to one host cell .",
"After ON infection , cell cultures were washed with RPMI and incubated with 100 µM EdU for 24 , 48 or 72 hr prior to fixation , staining and mounting in ProLong Diamond anti-fade mounting solution ( ThermoFisher Scientific , Waltham , MA ) containing 4’ , 6-diamidino-2-phenylindole dihydrochloride ( DAPI ) to stain nuclear and kinetoplast DNA .",
"Images were acquired using a laser scanning confocal microscope LSM 710 attached to an EXFO Xcite series 120Q lamp and a digital Zeiss XM10 camera .",
"C57BL/6 mice were infected with 2 . 5 × 105 colombiana T . cruzi strain trypomastigotes co-expressing fluorescent ( Tdtomato ) and luminescent ( luciferase ) proteins .",
"Sixty days post-infection , mice were injected i . p . with 10 mM EdU and sacrificed 12 , 48 or 72 hr later .",
"Before bioluminescence imaging , mice were transcardially perfused with 1x PBS and then with D-luciferin ( 0 . 3 mg/ml ) diluted in 1x PBS .",
"Tissue-specific ex vivo luciferase imaging was performed with tissues soaked in D-luciferin ( 0 . 3 mg/ml ) .",
"Luciferase positive thick tissue sections were excised and consecutive imaging and sectioning were performed to reduce non-luminescent area and increase chances to localize amastigote infected tissues .",
"Selected tissue sections were washed in 1x PBS and fixed with cold 4% PFA .",
"After 4°C ON fixation , tissues were washed with 1x PBS and immersed in CUBIC clarifying solution ( Susaki et al . , 2015 ) ( 25 % N , N , N′ , N′-Tetrakis ( 2-hydroxypropyl ) ethylenediamine; 25% urea; 15% Triton X-100 and distilled water ) with shaking at 37°C during 2 days .",
"After clarification , EdU incorporation on intracellular amastigotes was detected as previously described .",
"For selection of parasite-containing tissue areas for further imaging in non-drug treated mice , luciferase-positive thick tissue sections were excised and consecutive imaging and sectioning were performed to obtain small tissue sections with the brightest luminescent foci .",
"Tissue sections were fixed and clarified as described above , and EdU incorporation on intracellular amastigotes was detected by click chemistry as described above .",
"Tissue sections were placed into sterile glass-bottom 35 mm petri dishes , mounted using ProLong Diamond anti-fade solution and prepared for confocal imaging using a Zeiss LSM 710 attached to an EXFO Xcite series 120Q lamp and a digital Zeiss XM10 camera .",
"In the case of abdominal adipose tissue from drug-treated mice , wherein parasites were extremely scarce , both unfixed and PFA-fixed , non-clarified tissues were scanned for parasites using a 40X objective .",
"DNA fragmentation indicative of parasite death was assessed by TUNEL ( Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling ) assay according to manufacturer's protocol ( Click-iT TUNEL Alexa Fluor 647 imaging assay; ThermoFisher Scientific , Waltham , MA ) .",
"Briefly , Vero cells ( 1 × 105 ) were infected with 1 × 106 colombiana strain trypomastigotes for approximately 12 hr .",
"Seventy-two hours post-infection , cells were washed twice in PBS , fixed with 4% PFA and then washed again in PBS .",
"After permeabilization with 0 . 2% Triton X-100 , the cells were incubated with TdT reaction cocktail and washed twice with 3% BSA .",
"Finally , cells were incubated with click-iT reaction buffer and washed with 3% BSA .",
"Parasites were pre-treated for 10 min at room temperature with 10 IU/mL DNase I prior to the TUNEL for positive control .",
"A negative control was performed in the absence of the terminal transferase .",
"Cells were mounted using ProLong Diamond anti-fade mounting solution containing DAPI for parasite nuclei visualization .",
"Results were quantified counting about 200 cells in duplicate from three independent experiments .",
"Cell suspensions of T . cruzi trypomastigotes were labelled with CellTrace Violet fluorescent dye ( CellTrace Cell Proliferation Kit , ThermoFisher Scientific , Waltham , MA ) and CFSE following manufacturer’s instructions .",
"Briefly , 2 × 106 trypomastigotes were incubated for 20 min at 37°C with 10 μM of CellTrace Violet or for 5 min with 5 μM of CFSE , protected from light .",
"Unbound dye was quenched by the addition of five volumes of 10% FBS-RPMI or one volume of FBS respectively .",
"After washing in fresh media parasites were used for infection of cell cultures or mice .",
"Human foreskin fibroblasts ( HFF ) or Vero cells were seeded in 96 well glass bottom plates ( Corning Life Sciences , NY ) or 8 well 1 µ-slides ( Ibidi , Fitchburg , WI ) and infected with Tdtomato-expressing colombiana or CL strain parasites labeled with CellTrace Violet in ratios of 2:1 to 10:1 ( parasites:host cells ) .",
"For time-lapse video of amastigote replication , extracellular trypomastigotes were removed by washing and plates were placed in humid chamber with CO2 and imaged at 40X magnification every 15 min for 48 hr in a Lionheart FX imager ( BioTek , Winooski , VT ) .",
"Images and time lapse videos were analyzed with the Gene5 software ( BioTek ) .",
"Live cell imaging of cultures at different times post-infection and after drug addition or removal were performed in a Cytation5 cell imager ( BioTek ) or a Delta Vision II Microscope System ( GE Healthcare Biosciences , PA ) .",
"Vero cell cultures were infected with CellTrace Violet-labeled Tdtomato-expressing parasites of colombiana or CL strain ( 5:1 ratio parasites:host cells ) and incubated for 6 days at 37C in 5% CO2 .",
"Free trypomastigotes released to the supernatant were harvested and imaged using an ImageStream Mark II ( MilliporeSigma , MA ) .",
"CellTrace Violet- Tdtomato- positive parasites from those supernatants were sorted using a MoFlo XDP ( Beckman Coulter , FL ) and placed in fresh Vero cell cultures that were periodically monitored and imaged using a Cytation five imager ( BioTek ) .",
"Gamma-irradiated Vero cells at 25 , 000/well were infected with 150 , 000 colombiana or CL strain Tdtomato-luciferase trypomastigotes in each well of a 96 well plate ( Greiner Bio-one ) for 24 hr .",
"After removing non-infecting trypomastigotes , 10 μM BZN ( approx . 10X IC50 ) in complete RPMI media was added .",
"The six replicate wells for each condition were read using BioTek Synergy Hybrid reader and images were taken by BioTek Cytation 5 .",
"At the indicated times post-treatment , BZN was removed , cultures washed and fresh medium without BZN added .",
"Spent medium was removed and fresh medium with or without BZN were added weekly .",
"Additional ( 15 , 000/well ) Vero cells were added biweekly .",
"The relative in vitro resistance to drug treatment by T . cruzi lines was determined as previously described ( Canavaci et al . , 2010 ) , and IC50 was calculated using the GraphPad PRISM 5 . 0 software .",
"The Mann-Whitney U tests and one-way variance analysis ( ANOVA ) of the GraphPad Prism version 5 . 0 software were used .",
"Values are expressed as means ± standard error of mean of at least three separate experiments .",
"P values equal to or minor that 0 . 05 were considered significant ."
]
] | [
"The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection .",
"We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment .",
"Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells .",
"Most significantly , dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure .",
"These results demonstrate a dormancy state in T . cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection .",
"The ability of T . cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches ."
] | [
"Chagas disease is one of the most harmful tropical diseases in the Americas .",
"It affects millions of people , predominantly in Latin America .",
"It is usually spread by kissing bugs infected with Trypanosoma cruzi parasites .",
"It is considered a neglected tropical disease because few effective treatments and preventive methods are routinely used .",
"Several drugs can kill T . cruzi parasites , but they often fail to cure the infection .",
"Many people with Chagas disease go on to have life-long infections and eventually develop heart failure .",
"The reason for the high rate of treatment failure is not known .",
"It does not appear to be the result of the parasites developing resistance to the drugs .",
"One possibility is that the parasites can hide in a dormant state in the body , dodging the toxic drugs and living to reproduce another day .",
"Now , Sánchez-Valdéz et al . identify a dormant form of the T . cruzi parasite that allows the infection to persist after treatment .",
"In the experiments , a non-reproducing form of the so-called amastigote stage of the T . cruzi parasite inside the host cells was observed in infected mice and human cells .",
"While some of the amastigote parasites continue multiplying , a few stop even without drug treatment – but can resume multiplication at a later time .",
"They may also be able to change into the trypomastigote form of the parasite , which can infect new cells .",
"These non-multiplying amastigotes can survive drug treatment for as long as 30 days , whereas the multiplying amastigotes are killed by such drugs .",
"However , the surviving amastigotes then reestablish active infections after treatment has stopped .",
"The experiments explain why treatment so often fails to cure Chagas disease .",
"This suggests new treatment strategies are needed , including using existing drugs for a longer time perhaps with less frequent doses .",
"New therapies that kill the dormant amastigotes may also help .",
"Treatments that overcome the parasite’s ability to hide , could stop the progression of the disease and prevent heart-related deaths in those with persistent T . cruzi infections ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology"
] | Biosynthetic tailoring of existing ascaroside pheromones alters their biological function in C. elegans | elife-33286-v2 | [
[
"The nematode C . elegans secretes ascarosides as pheromones to induce development of the dauer larval stage at high population densities , as well as to control various behaviors , including male attraction to hermaphrodites , hermaphrodite attraction to males , avoidance , foraging behavior , and adult aggregation ( Cassada and Russell , 1975; Golden and Riddle , 1982; Butcher et al . , 2007; Butcher et al . , 2008; Srinivasan et al . , 2008; Butcher et al . , 2009a; Pungaliya et al . , 2009; Izrayelit et al . , 2012; Srinivasan et al . , 2012; Artyukhin et al . , 2013; Greene et al . , 2016 ) .",
"The ascarosides are derivatives of the 3 , 6-dideoxysugar ascarylose that can be named according to their structural features , including the number of carbons ( C# ) in their fatty acid-derived side chains ( Butcher , 2017 ) .",
"These side chains are attached to the ascarylose sugar at their penultimate ( ω−1 ) or terminal ( ω ) carbon and are often unsaturated ( Δ ) at the α-β position .",
"The ascarosides can be modified with various head groups at the 4’-position of the ascarylose sugar , such as the IC , the octopamine succinyl ( OS ) , the 4-hydroxybenzoyl ( HB ) , and the ( E ) -2-methyl-2-butenoyl ( MB ) groups .",
"They can also be modified with various terminus groups at the end of their fatty-acid side chain .",
"These head and terminus modifications can have important consequences for the biological activity of the ascarosides .",
"For example , while unmodified , simple ascarosides are often negative signals that induce avoidance in C . elegans , ascarosides that are modified with an IC head group , such as IC-asc-ΔC9 ( icas#3 ) , are favorable signals that induce aggregation at picomolar concentrations ( Srinivasan et al . , 2012; von Reuss et al . , 2012 ) .",
"However , the consequences of the IC group and other modifications can be quite nuanced .",
"For example , unlike IC-ascarosides with medium-length ( C9 ) side chains , which do not induce dauer , an IC-ascaroside with a short ( C5 ) side chain , IC-asc-C5 ( icas#9 ) , is one of the five primary components of the dauer pheromone and induces the dauer larval stage when it accumulates at low nanomolar concentrations ( Butcher et al . , 2009a ) .",
"Thus , IC-ascarosides have a dual nature to their activity as they are generally favorable signals that induce aggregation on food , but can also indicate unfavorable conditions and induce dauer when they have short side chains and accumulate to higher concentrations .",
"Peroxisomal β-oxidation is central to biosynthesis of the ascarosides .",
"β-oxidation cycles shorten the side chains of long-chain ascaroside precursors by two carbons per cycle to produce short- and medium-chain ascaroside pheromones ( Golden and Riddle , 1985; Wanders and Waterham , 2006; Butcher et al . , 2009b; Joo et al . , 2009; Joo et al . , 2010; von Reuss et al . , 2012; Zhang et al . , 2015; Zhang et al . , 2018 ) .",
"The first step in these β-oxidation cycles is catalyzed by an acyl-CoA oxidase , which installs a double bond at the α-β position of its substrate in a reaction that uses an FAD cofactor and produces H2O2 .",
"Specific acyl-CoA oxidases process ascaroside precursors with specific side-chain lengths and thus help to control which pheromones are produced ( Joo et al . , 2010; von Reuss et al . , 2012; Zhang et al . , 2015; Zhang et al . , 2018 ) .",
"The C . elegans genome encodes seven acyl-CoA oxidases , five of which have been implicated in ascaroside production ( Zhang et al . , 2018 ) .",
"To reflect their similarity to mammalian ACOX-1 , the acyl-CoA oxidases that were previously referred to as ACOX-1 , –2 , −3 , –4 , and −5 ( von Reuss et al . , 2012; Zhang et al . , 2015; 2016 ) have been renamed ACOX-1 . 1 , –1 . 2 , −1 . 3 , –1 . 4 , and −1 . 5 , respectively , and an additional acyl-CoA oxidase ( F59F4 . 1 ) has been named ACOX-1 . 6 .",
"To reflect its similarity to mammalian ACOX-3 , the acyl-CoA oxidase that was previously referred to as ACOX-6 ( Zhang et al . , 2016 ) has been renamed ACOX-3 .",
"The remaining three steps in each β-oxidation cycle are catalyzed by an enoyl-CoA hydratase ( MAOC-1 ) , an ( R ) -3-hydroxyacyl-CoA dehydrogenase ( DHS-28 ) , and a 3-ketoacyl-CoA thiolase ( DAF-22 ) ( Butcher et al . , 2009b; Joo et al . , 2009; Joo et al . , 2010; von Reuss et al . , 2012; Zhang et al . , 2015 ) .",
"Our group has shown that ( ω−1 ) -ascarosides and ω-ascarosides are biosynthesized by two parallel β-oxidation pathways , the former pathway involving ACOX-1 . 1 , ACOX-1 . 3 , ACOX-1 . 4 , and ACOX-3 and the latter pathway involving ACOX-1 . 1 and ACOX-1 . 2 ( Zhang et al . , 2015; Zhang et al . , 2018 ) .",
"In general , extensive β-oxidation of ascaroside precursors by these pathways to make ascarosides with very short side chains generates some of the most potent dauer pheromones , such as asc-ωC3 ( ascr#5 ) .",
"Very little is known about how various head groups , such as the IC , OS , HB , and MB groups , become attached to the ascarosides and how this attachment process is coordinated with the β-oxidation process that shortens the ascaroside side chains .",
"Attachment of the IC head group to the 4’-position of the ascarylose is thought to occur late in the biosynthetic pathway after β-oxidation of the side chain has occurred ( von Reuss et al . , 2012 ) .",
"The exogenous addition of a synthetic ascaroside with a nine-carbon side chain , asc-C9 ( ascr#10 ) , to daf-22 worms ( which are defective in β-oxidation and thus cannot make short- and medium-chain ascarosides , but which presumably can still biosynthesize the IC group and attach it ) leads to production of the corresponding IC-ascaroside , IC-asc-C9 ( icas#10 ) ( von Reuss et al . , 2012 ) .",
"This result suggests that attachment of the IC group occurs after side-chain β-oxidation , at least in the biosynthetic pathway to IC-asc-C9 .",
"The acyl-CoA synthetase gene , acs-7 , has been shown to be required for biosynthesis of the short-chain IC- and OS-modified ascarosides , IC-asc-C5 and OS-asc-C5 ( osas#9 ) , but not for medium-chain IC- and OS-ascarosides , such as IC-asc-C9 and OS-asc-C9 ( osas#10 ) ( Panda et al . , 2017 ) .",
"Panda et al . proposed that ACS-7 activates indole-3-carboxylic acid ( ICA ) as the corresponding CoA-thioester ( IC-CoA ) and is involved in the attachment of the IC group specifically to the short-chain ascaroside asc-C5 ( ascr#9 ) to make IC-asc-C5 ( Figure 1A ) .",
"However , while Panda et al . were able to show that ACS-7 activates ICA as an AMP-ester ( IC-AMP ) , they could not show that ACS-7 catalyzes the formation of IC-CoA or attachment of the IC group to an ascaroside ( Figure 1A ) .",
"Their interpretation of these data was that some other protein or factor was required for the full activity of ACS-7 .",
"Panda et al . showed that mutant worms lacking lysosome-related organelles ( LROs ) did not make 4’-modified ascarosides , and they reported that ACS-7 was expressed in the lysosome ( Panda et al . , 2017 ) .",
"Thus , they concluded that ACS-7 was functioning inside the lysosomes in IC-ascaroside biosynthesis .",
"Here , we show that the role of the acyl-CoA synthetase ACS-7 is not to attach the IC group to ascarosides as proposed by Panda et al . , but to activate the side chains of medium-chain IC-ascarosides for shortening through β-oxidation ( Figure 1B ) .",
"Thus , in contrast to the previously proposed model , we show that β-oxidation of the side chain occurs after attachment of the IC head group in the biosynthesis of the short-chain IC-ascarosides .",
"Our data show that under favorable , growth-promoting conditions , the IC group is preferentially attached to ascarosides with 8–11-carbon side chains to form aggregation pheromones .",
"However , if conditions decline , such as during starvation , the side chains of these IC-ascarosides can be activated by ACS-7 and then shortened through peroxisomal β-oxidation cycles involving ACOX-1 . 1 and ACOX-3 to make the potent dauer pheromone IC-asc-C5 .",
"Consistent with its function in activating ascarosides for peroxisomal β-oxidation , ACS-7 is localized to the peroxisome , rather than the lysosome , as previously reported by Panda et al . Our results uncover the mechanism by which C . elegans responds to declining environmental conditions and converts aggregation-inducing , medium-chain IC-ascarosides to dauer-inducing , short-chain IC-ascarosides .",
"This mechanism is likely also used by C . elegans in the biosynthesis of short-chain OS-ascarosides , which induce nematode dispersal under unfavorable conditions .",
"Using this mechanism , C . elegans can efficiently alter the function of existing ascarosides simply by tailoring the length of the side chain , providing a novel strategy to rapidly modulate chemical signaling in response to environmental conditions ."
],
[
"Previously , it has been shown that daf-22 worms , which cannot make any short- or medium-chain ascarosides , can attach the IC group to exogenously provided synthetic asc-C9 to produce IC-asc-C9 ( structures shown in Figure 1A , B and Figure 2A ) ( von Reuss et al . , 2012 ) .",
"It was also shown that daf-22 worms can attach the IC group to exogenously provided asc-ΔC9 ( ascr#3 ) to produce IC-asc-ΔC9 ( von Reuss et al . , 2012 ) .",
"To determine whether C . elegans could attach the IC group to other ascarosides , we cultured daf-22 worms with ascarosides of various side-chain lengths ( Figure 2A ) .",
"These data suggest that the unknown enzyme that attaches the IC group specifically prefers ascarosides with side chains of 8–11 carbons in length ( Figure 2B ) .",
"Recently , Panda et al . indicated that they were also able to detect the conversion by daf-22 worms of asc-C7 ( ascr#1 ) to IC-asc-C7 ( icas#1 ) ( Panda et al . , 2017 ) .",
"Although we were also able to detect a peak on the LC-MS representing this conversion , the amount of IC-asc-C7 produced was so small relative to background that it could not be quantified .",
"Thus , we conclude that C . elegans does not preferentially attach the IC group to ascarosides with C5 or C7 side chains .",
"On the other hand , C . elegans can make the corresponding IC-ascarosides , IC-asc-C5 and IC-asc-C7 .",
"Therefore , we speculated that these short chain IC-ascarosides could potentially be made from medium-chain IC-ascarosides , such as IC-asc-C9 , which have their side chains shortened through β-oxidation .",
"The acyl-CoA synthetase ACS-7 has been reported to be required for biosynthesis of the short-chain IC-ascaroside , IC-asc-C5 .",
"Panda et al . hypothesized that this enzyme was involved in direct attachment of the IC group to the 4’-position of the ascarylose sugar of asc-C5 ( Figure 1A ) ( Panda et al . , 2017 ) .",
"They were able to show that ACS-7 activates ICA as the AMP-ester , IC-AMP ( Figure 1A ) .",
"However , they were not able to show that ACS-7 promotes conversion of IC-AMP to the corresponding CoA-thioester , IC-CoA , or that it leads to the reaction of IC-CoA with asc-C5 ( Figure 1A ) .",
"Furthermore , the KM of ACS-7 for ICA ( in the one-step reaction to produce IC-AMP ) is 270 ± 90 μM ( Panda et al . , 2017 ) , which is much higher than the KM values of acyl-CoA synthetases from other organisms ( in the two-step reaction to produce an acyl-CoA ) that are generally in the low μM range ( Hall et al . , 2003; Van Horn et al . , 2005 ) .",
"Given our data showing that the IC group is not directly attached to asc-C5 or asc-C7 inside the worm , we considered alternative substrates for ACS-7 .",
"The acs-7 mutant accumulates IC-asc-C9 , while failing to make IC-asc-C5 ( Panda et al . , 2017 ) .",
"Therefore , we speculated that ACS-7 may activate , for example , IC-asc-C9 , as its CoA-thioester , for subsequent β-oxidation of the side chain to make shorter-chain IC-ascarosides .",
"To test this hypothesis , we purified recombinant His-tagged ACS-7 from E . coli and tested its activity against IC-asc-C9 .",
"ACS-7 showed robust activity towards this substrate , rapidly converting it completely to the corresponding CoA-thioester within 10 min ( Figure 3A ) .",
"We considered the possibility that recombinant ACS-7 may copurify with an enzyme from E . coli that might be responsible for this activity .",
"Therefore , we designed as a negative control two ACS-7 catalytic mutants for testing against the IC-asc-C9 substrate .",
"ACS-7 mutants in which the magnesium ion binding site was disrupted were designed through sequence alignment of ACS-7 with long-chain fatty acyl-CoA synthetase from Thermus thermophilus .",
"The ACS-7 ( E339A ) single mutant showed very little activity towards the IC-asc-C9 substrate , and the ACS-7 ( E339A , S186A , S187A ) triple mutant showed no activity towards the IC-asc-C9 substrate , thereby confirming that the activity seen for the wild-type enzyme does not result from any copurifying proteins ( Figure 3A ) .",
"ACS-7 showed very little activity towards ICA , the putative substrate reported by Panda et al . During the reaction of ACS-7 with ICA , we saw accumulation of the AMP-ester intermediate , suggesting that ACS-7 is very sluggish towards the ICA substrate and does not turn it over to the corresponding CoA-thioester ( Figure 3B ) .",
"Although we were able to detect the conversion of ICA to the corresponding CoA-thioester , the reaction required a 2-hour incubation ( Figure 3C ) .",
"Furthermore , based on the UV absorption at 280 nm , more than 60% of the substrate remained as the AMP-ester at this late time point .",
"To determine the substrate specificity of ACS-7 , we tested it against a panel of substrates , including IC-ascarosides ( IC-asc-C5 , IC-asc-C7 , IC-asc-ΔC9 , and IC-asc-C9 ) , ascarosides ( asc-C7 , asc-ΔC9 , and asc-C9 ) , and fatty acids ( fatty acid-C7 and fatty acid-C9 ) .",
"These data show that ACS-7 strongly prefers IC-modified ascarosides over simple ascarosides , demonstrating that it is specifically involved in activation of IC-modified ascarosides for β-oxidation ( Figure 3D ) .",
"ACS-7 activates the longer-chain substrates ( IC-asc-C7 and IC-asc-C9 ) , but not short-chain substrates ( IC-asc-C5 ) , consistent with its role in shortening the longer-chain substrates .",
"ACS-7 can activate fatty acids with medium-length side chains , but not short side chains .",
"Although it is unclear whether the reaction of ACS-7 with fatty acids is physiologically relevant , it could potentially indicate that ACS-7 also plays a role in fatty-acid metabolism , in addition to ascaroside biosynthesis .",
"The KM of ACS-7 for IC-asc-C9 is 14 . 5 ± 3 . 0 μM , the kcat is 0 . 53 ± 0 . 08 s−1 , and the kcat/KM is 36 , 300 ± 9 , 200 M−1 s−1 .",
"Analysis of ascaroside production in acyl-CoA oxidase mutants suggests that β-oxidation does play a role in production of the IC-ascarosides .",
"We have shown that ACOX-1 . 1 and ACOX-3 work together in the β-oxidation cycles that shorten ascarosides with 15-carbon and 13-carbon side chains ( Zhang et al . , 2018 ) .",
"Analysis of the ascarosides produced by acox-1 . 1 and acox-3 mutant worms indicates that these acyl-CoA oxidases also play a role in biosynthesis of short-chain IC-ascarosides .",
"The amounts of the ascarosides and corresponding IC-ascarosides in wild-type , acox-1 . 1 , acox-3 , and acox-1 . 1;acox-3 worms suggest that production of these two groups of ascarosides does not always correlate ( Figure 4 ) .",
"Thus , it is unlikely that the IC group is directly added to short-chain ascarosides to make the corresponding IC-ascarosides .",
"The acox-1 . 1 ( ok2257 ) deletion mutant shows an accumulation of IC-asc-C7 , indicating that ACOX-1 . 1 may function in the β-oxidation cycle that shortens a seven-carbon IC-ascaroside to a five-carbon IC-ascaroside ( Figure 4 ) .",
"The acox-3 ( tm4033 ) deletion mutant shows decreased production of IC-asc-C5 , but increased accumulation of IC-asc-C7 and IC-asc-ΔC9 ( Figure 4 , Figure 4—figure supplement 1 ) .",
"Thus , like ACOX-1 . 1 , ACOX-3 may function in the β-oxidation cycle that shortens a seven-carbon IC-ascaroside to a five-carbon IC-ascaroside .",
"The ascaroside profile of the acox-1 . 1 ( ok2257 ) ;acox-3 ( tm4033 ) double deletion mutant is quite striking in that production of IC-asc-C5 is completely abolished , while the amount of IC-asc-C7 is increased relative to wild type ( Figure 4 ) .",
"The amount of IC-asc-C7 is increased despite the amount of asc-C7 being decreased , indicating that production of the ascarosides and IC-ascarosides are independently regulated .",
"These data thus provide further evidence that ACOX-1 . 1 and ACOX-3 participate in a β-oxidation cycle that shortens IC-asc-C7 to IC-asc-C5 .",
"We used CRISPR-Cas9 to generate an acox-1 . 1 ( reb2[E434A] ) catalytic mutant strain , in which the ACOX-1 . 1 enzyme is mutated in a glutamate in the active site that is important for catalytic activity ( Zhang et al . , 2018 ) .",
"This strain shows an accumulation of IC-asc-C7 , indicating that ACOX-1 . 1’s catalytic activity contributes to the shortening of IC-ascarosides ( Figure 4—figure supplement 1 ) .",
"In addition to ACOX-1 . 1 and ACOX-3 , ACOX-1 . 4 may also contribute to the β-oxidation of IC-ascarosides .",
"An acox-1 . 4 ( reb6[E433A] ) catalytic mutant strain that we generated through CRISPR-Cas9 , as well as an acox-1 . 4 ( tm6415 ) deletion mutant strain and an acox-1 . 4 ( gk892586 ) nonsense mutant strain , did not show defects in IC-ascaroside biosynthesis .",
"However , examination of the acox-1 . 1 ( reb2[E434A] ) ;acox-1 . 4 ( reb6[E433A] ) ;acox-3 ( tm4033 ) triple mutant shows that all three genes contribute to the β-oxidation cycle that shortens IC-asc-C9 to IC-asc-C7 , as well as the β-oxidation cycle that shortens IC-asc-C7 to IC-asc-C5 ( Figure 4—figure supplement 1 ) .",
"To provide direct evidence for the role of ACOX-1 . 1 and ACOX-3 in biosynthesis of the IC-ascarosides , the two enzymes were expressed in E . coli and purified to assay their activity .",
"Unfortunately , ACOX-1 . 4 could not be expressed in E . coli despite repeated attempts .",
"In an LC-MS-based activity assay , the acyl-CoA oxidases , as well as MAOC-1 , DHS-28 , and DAF-22 , were incubated with IC-asc-C9-CoA ( Figure 5A , B; Figure 5—figure supplement 1 ) .",
"ACOX-1 . 1 and ACOX-3 both enabled the conversion of IC-asc-C9-CoA to IC-asc-C5-CoA .",
"On the other hand , ACOX-1 . 2 , which has a small active site and strictly prefers short-chain substrates ( Zhang et al . , 2015; Zhang et al . , 2016 ) , was inactive in this assay ( Figure 5A ) .",
"Under the assay conditions , the CoA-thioester bond was gradually cleaved , and thus , in the control and ACOX-1 . 2 reactions , unreacted IC-asc-C9-CoA substrate was gradually hydrolyzed to IC-asc-C9 .",
"Conversely , in the ACOX-1 . 1 and ACOX-3 reactions , IC-asc-C9-CoA was converted to IC-asc-C5-CoA , which was partially hydrolyzed to IC-asc-C5 ( Figure 5A ) .",
"To further investigate the substrate preferences of the acyl-CoA oxidases , the enzymes were assayed in an enzyme-coupled assay in the presence of peroxidase , which can use the H2O2 produced by the reaction to generate a UV-active product .",
"ACOX-1 . 1 was almost as active towards IC-asc-C7-CoA as it was towards its preferred ascaroside substrate , asc-C9-CoA ( Figure 5C; Figure 5—figure supplement 1 ) .",
"It also showed activity towards IC-asc-C9-CoA , but less than towards IC-asc-C7-CoA .",
"Unfortunately , ACOX-3 showed low activity towards all substrates tested in this assay , including another one of its preferred ascaroside substrates , asc-C13-CoA ( Zhang et al . , 2018 ) .",
"This result may suggest that ACOX-3 requires other β-oxidation enzymes to be fully active ( Figure 5—figure supplement 2 ) .",
"It has been shown previously that ACS-7 is expressed in the lysosome , where it was thought to contribute to IC-ascaroside biosynthesis ( Panda et al . , 2017 ) .",
"Because our data show that ACS-7 activates the side chains of medium-chain IC-ascarosides for peroxisomal β-oxidation , we hypothesized that ACS-7 must be expressed in the peroxisome .",
"Indeed , ACS-7 has a PTS1-type peroxisomal localization signal .",
"Furthermore , when we tested the enzymatic activity of ACS-7 , we found that the enzyme was active at neutral pH , but not at the pH found in the lysosome ( pH 5 . 0 ) .",
"To determine the localization pattern of ACS-7 , we generated translational reporter constructs for ACS-7 , ACOX-3 , as well as ACOX-1 . 1 , which was previously shown to be localized to the peroxisome ( Joo et al . , 2010 ) .",
"Co-injection of these constructs into wild-type C . elegans showed that all three proteins are expressed in a punctate pattern in the intestine ( Figure 6A , B ) .",
"In addition , the intestinal expression pattern of ACS-7 overlaps with that of ACOX-1 . 1 and ACOX-3 ( Figure 6A , B ) .",
"Some GFP signal is occasionally seen in LROs , especially in older worms ( Figure 6—figure supplement 1A , B ) .",
"However , we believe that this GFP signal is caused by autofluorescence of the LROs ( Hermann et al . , 2005 ) because ( 1 ) it only becomes prominent on prolonged bleaching of the worms under the fluorescent microscope , and ( 2 ) it is also observed in wild-type ( N2 ) worms that have not been injected with the Pasc-7::gfp::acs-7 reporter .",
"( Figure 6—figure supplement 1C–E ) .",
"RNAi against prx-13 , which is a component of the peroxisomal import machinery ( Thieringer et al . , 2003 ) , leads to a diffuse pattern of expression for ACOX-1 . 1 and ACS-7 in the intestine , providing further evidence that these enzymes are localized to the peroxisome ( Figure 6C , D ) .",
"Thus , ACS-7 likely participates in biosynthesis of the IC-ascarosides in the peroxisome rather than lysosome ."
],
[
"Our data show that C . elegans can engage in dynamic tailoring of the ascarosides that it has produced .",
"This dynamic tailoring enables the worm to respond rapidly to changing environmental conditions and modulate the nature of its chemical message without having to synthesize new ascarosides de novo .",
"Specifically , we have shown that C . elegans attaches the IC head group to ascarosides with medium-length ( 8–11 ) side chains to generate aggregation pheromones such as IC-asc-ΔC9 ( icas#3 ) ( Srinivasan et al . , 2012 ) .",
"Production of aggregation pheromones likely occurs under favorable environmental conditions such as food-rich conditions as C . elegans tends to aggregate on food ( Srinivasan et al . , 2012 ) .",
"We have shown that C . elegans can activate IC-ascarosides with medium-length side chains for shortening of the side chains through β-oxidation to generate the dauer pheromone IC-asc-C5 ( icas#9 ) ( Figure 1B ) .",
"This β-oxidation process likely occurs when environmental conditions become less favorable , such as during starvation .",
"Indeed , starvation conditions have been shown to lead to an increase in the short-chain IC-ascaroside IC-asc-C5 in L1 larvae ( Artyukhin et al . , 2013 ) .",
"Consistent with this result , bacterial food has been shown to downregulate production of IC-asc-C5 in L4 larvae ( Zhang et al . , 2015 ) .",
"Increased production of short-chain IC-ascarosides by starvation conditions would be consistent with their biological role as IC-asc-C5 is a dauer pheromone ( Butcher et al . , 2009a ) , and C . elegans would likely want to enter dauer under starvation conditions .",
"Our work has thus determined the mechanism by which C . elegans modulates the balance between aggregation-inducing , medium-chain IC-ascarosides and dauer-inducing , short-chain IC-ascarosides in response to changing environmental conditions .",
"Our model for biosynthesis of the short-chain IC-ascarosides , in which β-oxidation of the ascaroside side chain occurs after attachment of the IC head group , contrasts with the model proposed by Panda et al . , in which β-oxidation of the ascaroside side chain occurs before attachment of the IC head group ( Panda et al . , 2017 ) ( Figure 1A , B ) .",
"We have shown that the acyl-CoA synthetase ACS-7 activates the side chains of medium-chain IC-ascarosides , such as IC-asc-C9 , as CoA-thioesters , thereby enabling the side chains to be shortened through β-oxidation ( Figure 1B ) .",
"Previously , Panda et al . proposed that ACS-7 activates ICA as the corresponding CoA-thioester , IC-CoA , and enables attachment of the IC group to asc-C5 to make IC-asc-C5 ( Panda et al . , 2017 ) ( Figure 1A ) .",
"However , ACS-7 could only be shown to convert ICA to IC-AMP ( Panda et al . , 2017 ) .",
"Our data show that IC-asc-C9 is a much better substrate for ACS-7 , which rapidly converts IC-asc-C9 to the corresponding CoA-thioester , IC-asc-C9-CoA ( Figure 1B ) .",
"We have shown that once the side chain of the medium-chain IC-ascarosides is activated , it can be shortened through β-oxidation cycles involving ACOX-1 . 1 , ACOX-3 , and likely ACOX-1 . 4 as well , to make the short-chain IC-ascaroside , IC-asc-C5 .",
"Specifically , we have shown that the acox-1 . 1;acox-3 double deletion mutant fails to make any IC-asc-C5 and accumulates IC-asc-C7 .",
"Furthermore , ACOX-1 . 1 and ACOX-3 are able to process IC-asc-C9-CoA to IC-asc-C5-CoA in vitro with the help of the β-oxidation enzymes MAOC-1 , DHS-28 , and DAF-22 .",
"Consistent with ACS-7 working in the same biosynthetic pathway as ACOX-1 . 1 and ACOX-3 , the three genes appear to be transcriptionally co-regulated ( von Mering et al . , 2005 ) .",
"ACOX-3 has been shown to be induced by dietary restriction ( Palgunow et al . , 2012 ) .",
"ACS-7 and ACOX-3 have also been shown to be co-regulated by the transcription factor SKN-1 , which functions in a variety of stress responses , including detoxification , pathogen defense , the unfolded protein response , and lipid metabolism under starvation conditions ( Blackwell et al . , 2015 ) .",
"However , the significance of this regulation for IC-ascaroside biosynthesis remains to be seen .",
"In addition to the IC-ascarosides , ACS-7 is also necessary for biosynthesis of short-chain OS-ascarosides , as acs-7 mutant worms cannot make the short-chain OS-ascarosides ( Panda et al . , 2017 ) .",
"Thus , it is likely that , in addition to medium-chain IC-ascarosides , ACS-7 also activates medium-chain OS-ascarosides for β-oxidation of their side chains .",
"The β-oxidation of IC- and OS-modified ascarosides may be regulated in a similar fashion .",
"Starvation has been shown to increase the ratio of short-chain ( C5 ) to medium-chain ( C9 ) OS-ascarosides ( Artyukhin et al . , 2013 ) .",
"Production of the short-chain OS-ascaroside OS-asc-C5 ( osas#9 ) under starvation conditions is consistent with its biological role , as it acts as a dispersal signal that encourages the worm population to seek more favorable conditions ( Artyukhin et al . , 2013 ) .",
"In addition to the IC and OS head groups , the 4’-position of the ascarosides can also be modified by the MB and HB head groups ( von Reuss et al . , 2012 ) .",
"However , MB- and HB-ascarosides with fewer than nine-carbons in their side chains have not been detected in C . elegans ( von Reuss et al . , 2012 ) .",
"Thus , unlike medium-chain IC-and OS-ascarosides , medium-chain MB- and HB-ascarosides probably do not undergo activation by an acyl-CoA synthetase for further β-oxidation in the peroxisome .",
"In contrast to previous reports that suggested ACS-7 is localized to the lysosomes in the intestine ( Panda et al . , 2017 ) , we have shown that ACS-7 is localized to the peroxisomes .",
"It is likely that the peroxisome is where ACS-7 contributes to IC-ascaroside biosynthesis , as ACS-7 activates the medium-chain IC-ascarosides as their CoA-thioesters for β-oxidation by the peroxisomal enzymes ACOX-1 . 1/ACOX-3 , MAOC-1 , DHS-28 , and DAF-22 .",
"The lysosome has been shown to be important for biosynthesis of ascarosides modified with a head group on the 4’-position of the ascarylose sugar ( Panda et al . , 2017 ) .",
"Mutant worms that lack LROs , such as glo-1 mutants , do not make any ascarosides modified on the 4’-position with IC , OS , HB , or MB head groups ( Panda et al . , 2017 ) .",
"In a model that accounts for the role of the LROs in ascaroside biosynthesis , long-chain ascarosides could be shortened to medium- and short-chain ascarosides in the peroxisome , and then medium-chain ascarosides , such as asc-C9 , may be transported from the peroxisome to the LROs for attachment of 4’-modifications , such as attachment of the IC head group to make IC-asc-C9 , by unknown enzymes ( Figure 1B ) .",
"In response to changing internal or external conditions , these modified ascarosides could then be transported back to the peroxisome for activation by ACS-7 and further β-oxidation of their side chains ( Figure 1B ) .",
"Trafficking of the ascarosides through multiple cellular organelles , such as the peroxisome and LROs , during biosynthesis may enable C . elegans to induce the production of modified ascarosides with specific side-chain lengths only under certain conditions or only in certain tissues ."
],
[
"The following strains were used: wild-type ( N2 , Bristol ) , RAB1 acox-1 . 1 ( ok2257 ) I , RAB24 acox-1 . 1 ( reb2[E434A] ) I , RAB28 acox-1 . 4 ( reb6[E433A] ) I , RAB21 acox-1 . 4 ( tm6415 ) I , VC40944 acox-1 . 4 ( gk892586 ) I , RAB22 acox-3 ( tm4033 ) IV , RAB30 acox-1 . 1 ( ok2257 ) ;acox-3 ( tm4033 ) , RAB31 acox-1 . 1 ( reb2[E434A] ) ;acox-1 . 4 ( reb6[E433A] ) , RAB32 acox-1 . 1 ( reb2[E434A] ) ;acox-3 ( tm4033 ) , RAB35 acox-1 . 4 ( reb6[E433A] ) ;acox-3 ( tm4033 ) , RAB36 acox-1 . 1 ( reb2[E434A] ) ;acox-1 . 4 ( reb6[E433A] ) ; acox-3 ( tm4033 ) , DR476 daf-22 ( m130 ) II .",
"The acox-1 . 1 ( ok2257 ) , acox-1 . 4 ( tm6415 ) , and acox-3 ( tm4033 ) strains were backcrossed four or six times .",
"The acox-1 . 1 ( reb2[E434A] ) , acox-1 . 4 ( reb6[E433A] ) , and acox-1 . 1 ( reb2[E434A] ) ;acox-1 . 4 ( reb6[E433A] ) mutants were generated using CRISPR-Cas9 and backcrossed two to six times ( Zhang et al . , 2018 ) .",
"All CRISPR-Cas9 mutants were made using the Fire laboratory’s marker-free CRISPR protocol ( Arribere et al . , 2014; Kim et al . , 2014; Cong and Zhang , 2015; Farboud and Meyer , 2015 ) .",
"Cultures were grown similar to a previously published method ( von Reuss et al . , 2012 ) , but with modifications .",
"daf-22 ( m130 ) worms from one 10 cm NGM plate ( seeded with 0 . 75 mL 25X OP50 ) were collected after the bacteria were completely consumed and transferred to a 25 mL pre-culture , feeding with 10 mL 25X OP50 on day 1 and day 3 .",
"On day 4 , 1 . 25 mL of the pre-culture was transferred to 3 . 75 mL fresh S medium to start the ascaroside-supplemented cultures .",
"Each of the 5 mL cultures were supplemented with 10 μL of 3 mM asc-C5 , asc-C6 , asc-C7 , asc-C8 , asc-C9 , asc-C11 , or asc-C13 .",
"10 μL of ethanol was used for control .",
"The supplemented cultures were fed with 1 mL of 25X OP50 each day for 4 days , and the culture medium was collected on the fifth day .",
"1 mL of the culture medium was lyophilized and extracted with 100 μL 50% methanol/water , and 10 μL of the supernatant was analyzed by LC-MS ( Zhang et al . , 2013 ) .",
"Given that detection of IC-ascarosides in culture medium by LC-MS suffers from ion suppression , a calibration curve of pure , synthetic IC-asc-ΔC9 in culture medium extract was generated as follows: 2 . 8 μL 600 μM IC-asc-ΔC9 standard was mixed with 57 . 2 μL daf-22 culture medium extract ( in 50% MeOH/water ) to make a 28 μM stock .",
"A series of 1:2 diluted standards was generated by mixing the stock 1:1 with daf-22 culture medium extract .",
"5 μL of the standards ( each containing 4 . 4 , 8 . 8 , 17 . 5 , 35 , 70 , or 140 pmol IC-asc-ΔC9 , respectively ) were injected into the LC-MS on the same day of sample analysis .",
"Trend lines describing the relationship between peak area and IC-asc-ΔC9 amount were used to analyze the molar amount of all IC-ascarosides present in the supplemented daf-22 cultures .",
"Three independent experiments were performed using worms cultured at three different times .",
"acs-7 was cloned from a C . elegans cDNA library by PCR and ligated into pET-22b using the NdeI and XhoI restriction sites .",
"To engineer an appropriate negative control for enzyme assays , catalytic mutants of ACS-7 were designed through sequence alignment with ttLC-FACS ( Hisanaga et al . , 2004 ) from Thermus thermophiles .",
"pET-22b-acs-7 ( E339A ) was obtained via Q5 Site-Directed Mutagenesis Kit ( New England Biolabs , Ipswich , MA ) using pET-22b-acs-7 as the template , and it was then further modified by the kit to generate pET-22b-acs-7 ( E339A , S186A , S187A ) .",
"Wild-type ACS-7 and the mutants were expressed in BL21 ( DE3 ) cells by inducing with 0 . 3 mM IPTG at 16°C for 42 hr .",
"The enzymes were purified by lysing the cells in lysis buffer ( 50 mM KPO4 pH 7 . 4 , 100 mM KCl ) using a microfluidizer , incubating the lysate with Ni-NTA resin ( Thermo Fisher , Waltham , MA ) , and eluting the resin with lysis buffer containing 500 mM imidazole .",
"The enzymes were further purified through FPLC on a HiLoad 16/600 Superdex 200 column ( GE Healthcare , Chicago , IL ) .",
"Purified ACS-7 , ACS-7 ( E339A ) , and ACS-7 ( E339A , S186A , S187A ) were concentrated to 2 mg/mL .",
"2 μL of the 2 mg/mL enzyme was used in each 50 μL reaction mixture , which gave a 1 . 57 μM final concentration .",
"The reaction mixture contained 100 mM KPO4 pH 7 . 0 , 5 mM ATP , 5 mM MgCl2 , 5 mM CoA , and 100 μM IC-asc-C9 or ICA as substrates .",
"Reaction mixtures were incubated at 25°C for 10 min , 1 hr , or 2 hr . 50 μL of methanol was added to the reaction mixtures to quench them , and 5 μL of the 100 μL 1:1 reaction/methanol mixture was analyzed by LC-MS directly .",
"LC-MS analysis was performed with an Agilent 6130 single quadrupole mass spectrometer , operating in both positive and negative modes , using a method adapted from a previously published one ( Zhang et al . , 2013 ) .",
"The LC conditions were holding for 2 min at 95% solvent A ( water with 10 mM ammonium acetate ) and 5% solvent B ( acetonitrile ) , followed by gradually ramping up to 100% solvent B over 24 min .",
"The MS was operated in full-scan mode ( m/z 150–1500 ) with a fragmentor voltage of 125 V , peak width of 0 . 15 min , and cycle length of 2 . 20 s/cycle .",
"The enzyme kinetics of ACS-7 were determined through an enzyme-coupled spectrophotometric assay that measures the release of AMP ( Tanaka et al . , 1979; Hisanaga et al . , 2004 ) .",
"Each 100 μL assay mixture contained 0 . 1 M Tris-HCl pH 7 . 4 , 5 mM dithiothreitol , 1 . 6 mM Triton X-100 , 10 mM MgCl2 , 7 . 5 mM ATP , 0 . 2 mM phosphoenol pyruvate , 0 . 15 mM NADH , 2 μL of adenylate kinase solution ( Sigma M3003 , prepared according to the manufacturer’s protocol ) , 2 μL of pyruvate kinase and lactate dehydrogenase mixture stock ( Sigma P0294 ) , 4 μg of ACS-7 , and the tested substrates with concentrations from 5 to 250 μM .",
"1 mM final concentration of CoA was added to the assay mixture to start the reaction .",
"The reaction was run at 22°C , and the UV absorbance at 340 nm was measured to determine the activity of ACS-7 .",
"Three independent experiments were performed using protein purified at three different times .",
"Large-scale ( 150 mL ) non-synchronized worm cultures were fed E . coli ( HB101 ) and grown for 9 d , and extracts were generated from the culture medium , as described ( Zhang et al . , 2013 ) .",
"Three independent experiments were performed using worms cultured at three different times .",
"LC-MS/MS analysis of ascarosides from extracts was performed as described ( Zhang et al . , 2015 ) , but with some modifications .",
"A Phenomenex Kinetex 2 . 6 μM C18 100 Å ( 100 × 2 . 1 mm ) column was attached to an Accela UHPLC and a Thermo TSQ Quantum Max mass spectrometer , operating in negative ion , heated ( H ) -ESI , precursor scanning mode ( selecting for a product ion of m/z 73 . 0 ) .",
"Quantitation of ascarosides by LC-MS/MS was done by generating a calibration curve using synthetic standards .",
"All ascarosides were quantified using their corresponding synthetic standard , except for IC-asc-C7 and IC-asc-C9 , which were quantitated using synthetic IC-asc-ΔC9 .",
"For Figure 4—figure supplement 1 , small-scale ( 5 mL ) non-synchronized worm cultures were started with worms from one 6 cm NGM-agar plate , fed E . coli ( HB101 ) , and grown for 7 days .",
"Three independent experiments were performed using worms cultured at three different times .",
"5 mL of culture was centrifuged ( 800 g for 2 min ) , the worms at the bottom were removed , and the supernatant was centrifuged again ( 3500 rpm for 10 min ) .",
"1 mL of this supernatant was lyophilized and resuspended in 100 μL of 50% methanol in water , and the ascarosides were analyzed by LC-MS as described ( Zhang et al . , 2013 ) .",
"LC-MS analysis of ascarosides was performed on a Phenomenex Luna 5 μm C18 2 100 Å ( 100 × 4 . 6 mm ) column attached to an Agilent 1260 infinity binary pump and Agilent 6130 single quad mass spectrometer with API-ES source , operating in dual negative/positive single-ion monitoring mode , as previously described ( Zhang et al . , 2015 ) .",
"In general , all ascarosides were detected by LC-MS using the [M-H]- ion .",
"Ascarosides and IC-ascarosides were synthesized as previously described ( Hollister et al . , 2013 ) , except that for the IC-ascarosides , the final reaction products were purified by Agilent 1200 Series HPLC on a Supelco Discovery 10 μm C18 ( 250 × 10 mm ) column .",
"A water ( with 0 . 1% formic acid ) and acetonitrile ( with 0 . 1% formic acid ) solvent gradient was used , starting from 5% acetonitrile , ramping to 100% acetonitrile over 25 min , and then holding at 100% acetonitrile for 4 min , with a flow rate of 2 mL / min .",
"CoA-thioesters of fatty acids and short- and medium-chain ascarosides were synthesized as previously described with several modifications ( Zhang et al . , 2015 ) .",
"Ascaroside ( 3 μM ) was dissolved in tetrahydrofuran ( 350 μL ) , followed by the addition of carbonyldiimidazole ( 4 . 5 ~ 6 μM ) in tetrahydrofuran ( 90 μL ) .",
"The reaction was allowed to stir at room temperature for 1 hr and then dried under N2 gas or by speedvac .",
"The mixture was then dissolved in tetrahydrofuran ( 400 μL ) followed by adding CoA ( 3 μM ) in water ( 200 μL ) .",
"The reaction was allowed to stir at room temperature for 4 hr .",
"Dried product was purified by Agilent 1260 Infinity HPLC on a Supelco Discovery 10 μm C18 ( 250 × 21 . 2 mm ) column .",
"A water ( with 10 mM ammonium acetate ) and acetonitrile solvent gradient was used , starting from 0% acetonitrile , ramping to 80% acetonitrile over 25 min , and then holding at 100% acetonitrile for 2 min , with a flow rate of 8 mL / min .",
"To synthesize the CoA-thioesters of long-chain ascarosides or IC-ascarosides , a chemoenzymatic approach was taken using the fatty acyl-CoA ligase FadD6 ( Arora et al . , 2005 ) or ACS-7 .",
"The fadD6 gene was cloned from a Mycobacterium tuberculosis H37Ra genomic library ( a gift from Peilan Zhang and Prof . Yousong Ding ) and expressed as described ( Zhang et al . , 2015; Zhang et al . , 2018 ) .",
"FadD6 reaction conditions were similar to previously described conditions ( Arora et al . , 2005 ) with some modifications .",
"For a 200 μL total reaction volume , ~300 μM asc-C13 , IC-asc-C7 , or IC-asc-C9 , 5 mM CoA , 15 mM ATP and 20 μg FadD6 protein were added to reaction buffer ( 100 mM Tris , 8 mM MgCl2 , pH 7 . 5 ) , and the reaction was incubated at 30°C for 1 hr .",
"The asc-C13-CoA , IC-asc-C7-CoA , and IC-asc-C9-CoA were purified by Agilent 1200 Series HPLC on a Phenomenex Luna 5 μm C18 2 100 Å ( 100 × 4 . 6 mm ) column .",
"A water ( with 10 mM ammonium acetate ) and acetonitrile solvent gradient was used , starting from 0% acetonitrile , ramping to 100% acetonitrile over 20 min , and then holding at 100% acetonitrile for 2 min , with a flow rate of 0 . 7 mL / min .",
"Purified asc-C13-CoA , IC-asc-C7-CoA , and IC-asc-C9-CoA were then dried with a speedvac .",
"Although FadD6 was initially used in the chemoenzymatic synthesis of these molecules , once we identified the substrate preferences of ACS-7 , we subsequently used it to make IC-asc-C7-CoA and IC-asc-C9-CoA , as it was much faster and far more consistent than FadD6 at making these compounds .",
"The structures of synthetic IC-asc-C7-CoA , IC-asc-C9-CoA , and asc-C13-CoA were confirmed by LC-MS/MS , using a Nano LCMS Solutions 3 μM 200 Å ( 0 . 3 × 150 mm ) ProtoSIL C18AQ + column attached to an UltiMate 3000 RSLCnano System and a Bruker Impact II QTOF mass spectrometer , operating in positive ion , heated ( H ) -ESI mode .",
"A water ( with 100 mM ammonium acetate ) and acetonitrile ( with 0 . 1% formic acid ) solvent gradient was used for separation , starting from 2% acetonitrile for 7 min , ramping to 98% acetonitrile over 31 min , and then holding at 98% acetonitrile for 10 min , with a flow rate of 5 µL / min .",
"HR-MS/MS analysis was applied to all three samples with collision energy set at 45 eV .",
"Characteristic fragmentation was observed , such as a neutral loss of 507 ( Magnes et al . , 2005 ) ( Figure 5—figure supplement 1 ) .",
"IC-asc-C7-CoA , HR-ESIMS ( m/z ) : [M + H]+ calcd .",
"for C43H64N8O22P3S 1169 . 3068 , found 1169 . 3105 .",
"IC-asc-C9-CoA , HR-ESIMS ( m/z ) : [M + H]+ calcd .",
"for C45H68N8O22P3S 1197 . 3382 , found 1197 . 3390 .",
"asc-C13-CoA , HR-ESIMS ( m/z ) : [M + H]+ calcd .",
"for C40H71N7O21P3S 1110 . 3637 , found 1110 . 3712 .",
"The cloning and expression conditions for the ACOX-1 . 1a ( the longest splice variant of ACOX-1 . 1 ) homodimer were described previously ( Zhang et al . , 2015 ) .",
"Acox-3 was cloned by PCR from a C . elegans ( N2 ) cDNA library and was inserted into a modified pET-16b vector at the NheI/NotI restriction sites ( resulting in a C-terminal His tag ) .",
"The plasmid was transformed into BL21 ( DE3 ) cells , and a culture was grown at 37°C until the OD600 reached 0 . 7 , at which point expression was induced overnight at 25°C using 0 . 6 mM IPTG .",
"The cells were resuspended in buffer ( 25 mM Tris , pH 7 . 5 , 500 mM NaCl , 20 μM FAD ) and lysed using a microfluidizer , and the protein was purified with Ni-NTA resin ( Thermo Fisher ) .",
"After the protein was concentrated with a 10 KDa cut-off centricon ( MilliporeSigma , Burlington , MA ) , it was further purified through FPLC on a HiLoad 16/600 Superdex 200 column ( GE Healthcare ) and concentrated again to 1 mg/mL for assay .",
"maoc-1 , dhs-28 , and daf-22 genes were cloned by PCR from a C . elegans ( N2 ) cDNA library .",
"The maoc-1 gene was inserted into the pACYCDuet-1 vector at the EcoRI/NotI sites ( resulting in an N-terminal His tag ) to generate pACYCDuet-1-maoc-1 plasmid .",
"The dhs-28 gene was inserted into the pACYCDuet-1 vector at the EcoRI/NotI sites ( resulting in an N-terminal His tag ) to generate pACYCDuet-1-dhs-28Δscp-2 .",
"This construct lacks the dhs-28 sequence encoding the SCP-2 ( Sterol Carrier Protein-2 ) domain because of its interference with protein expression and lack of relevance for enzymatic activity .",
"The daf-22 gene was inserted into a modified pET-16b vector at the NcoI/NotI sites ( resulting in a C-terminal His tag ) to generate pET-16b-daf-22 plasmid .",
"MAOC-1 , DHS-28ΔSCP-2 , and DAF-22 were expressed and purified using a similar method as that used for ACOX-3 , except that expression was induced with 0 . 8 mM IPTG and FAD was not included in protein lysis and purification buffer .",
"For the LC-MS-based assay , 40 μM IC-asc-C9-CoA , 20 μM FAD , 20 μM NAD+ , 200 μM CoA , 8 μg of ACOX protein ( ACOX-1 . 1 , ACOX-1 . 2 , or ACOX-3 ) , and 4 μg of other three β-oxidation enzymes ( MAOC-1 , DHS-28 , and DAF-22 ) were added to the reaction buffer ( 100 mM Tris , 8 mM MgCl2 , pH 7 . 5 ) , for a total reaction volume of 50 μL .",
"The control reaction contained all of the above except the ACOX proteins .",
"Reactions were performed at 30°C for 1 hr .",
"Then 50 μL of MeOH was added to quench the reaction .",
"Samples were heated at 95˚C for 5 min and centrifuged at 13000 rpm for 5 min . 10 μL of the supernatant was used for LC-MS analysis .",
"Retention times of the substrate and products were confirmed with synthetic standards IC-asc-C9 , IC-asc-C7 , IC-asc-C5 , IC-asc-C9-CoA , and IC-asc-C7-CoA .",
"The retention time of IC-asc-C5-CoA was predicted using the linear relationship between the retention times of IC-asc-C5-CoA and IC-asc-C7-CoA and IC-asc-C9-CoA .",
"The coupled enzyme assay for acyl-CoA oxidase activity was performed as described previously ( Zhang et al . , 2015 ) , with several modifications .",
"Specifically , reactions were performed at room temperature ( ~23°C ) , and the substrate concentration was 24 µM .",
"Three independent experiments were performed using protein purified at three different times .",
"To generate Pacs-7::gfp::acs-7 , 2 . 2 kb of the acs-7 promoter and the acs-7 gene plus 3’-UTR were inserted into the AscI/NotI and NgoMIV/AatII sites , respectively , of pPD114 . 108 ( from Andy Fire , via Addgene ) .",
"120 ng/µL of Pacs-7::gfp::acs-7 was injected into wild-type worms to give RAB37 rebEx11 ( Pacs-7::gfp::acs-7 ) .",
"To generate Pacox-1 . 1::mcherry::acox-1 . 1 , 1 . 4 kb of the acox-1 . 1 promoter and the acox-1 . 1 gene plus 3’-UTR were inserted into the SalI/NotI and NgoMIV/ApaI sites , respectively , of pPD114 . 108 .",
"The GFP sequence in the vector was replaced with the mCherry sequence amplified from pMC10-mCherry ( gift of Piali Sengupta ) using the AgeI/NheI sites .",
"To generate Pacox-3::gfp::acox-3 , 3 kb of the acox-3 promoter and the acox-3 gene plus 3’-UTR were inserted into the SalI/NotI and NheI/ApaI sites , respectively , of pPD114 . 108 .",
"60 ng/µL of Pacox-1 . 1::mcherry::acox-1 . 1 was co-injected with either 60 ng/µL of Pacs-7::gfp::acs-7 or Pacox-3::gfp::acox-3 into wild-type worms to give RAB38 rebEx12 ( Pacox-1 . 1::mcherry::acox-1 . 1; Pacs-7::gfp::acs-7 ) and RAB39 rebEx13 ( Pacox-1 . 1::mcherry::acox-1 . 1; Pacox-3::gfp::acox-3 ) , respectively .",
"Alternatively , 10 ng/µL of Pacox-1 . 1::mcherry::acox-1 . 1 was co-injected with 120 ng/µL of Pacs-7::gfp::acs-7 into wild-type worms to give RAB40 rebEx14 ( Pacox-1 . 1::mcherry::acox-1 . 1; Pacs-7::gfp::acs-7 ) .",
"Imaging was conducted on a Zeiss Axiovert . A1 microscope equipped with ZEN lite 2012 camera .",
"In the RNAi feeding assay , E . coli strain HT115 ( DE3 ) carrying L4440 ( control ) or L4440-prx-13 ( from Julie Ahringer RNAi strain library ) was cultured in LB medium ( with 150 μg/mL ampicillin ) at 37°C at 225 rpm for 5 hr followed by induction with 4 mM IPTG for 1 hr . 200 μL of the bacterial cultures were then seeded onto individual NGM agar plates ( with 1 mM IPTG , 25 μg/mL carbenicillin ) and the plates were dried overnight .",
"On the next day , two Pacs-7::gfp::acs-7; Pacox-1 . 1::mcherry::acox-1 . 1 worms at the L4 stage were transferred onto each plate .",
"Adults were removed from the plates 2 days later ( on day 3 ) , and on day 5 , the progeny were imaged .",
"To make LysoTracker Red plates , 6 μL of the LysoTracker Deep Red ( Thermo L12492 , 1 mM stock in DMSO ) were added to each 3 cm plate ( containing 3 mL NGM-agar per plate ) to obtain a final concentration of 2 μM .",
"After the agar plates solidified , 50 μL of OP50 ( freshly inoculated culture in LB medium , 37°C , 225 rpm , 6 hr ) were added to the center of each plate .",
"Plates with bacteria were dried in a sterile hood in the dark for about 20 min .",
"Twenty RAB37 rebEx11 ( Pacs-7::gfp::acs-7 ) worms or wild-type worms were transferred to each plate while minimizing exposure to light under the microscope .",
"The plates were kept in the dark for 24 hr before imaging ."
]
] | [
"Caenorhabditis elegans produces ascaroside pheromones to control its development and behavior .",
"Even minor structural differences in the ascarosides have dramatic consequences for their biological activities .",
"Here , we identify a mechanism that enables C . elegans to dynamically tailor the fatty-acid side chains of the indole-3-carbonyl ( IC ) -modified ascarosides it has produced .",
"In response to starvation , C . elegans uses the peroxisomal acyl-CoA synthetase ACS-7 to activate the side chains of medium-chain IC-ascarosides for β-oxidation involving the acyl-CoA oxidases ACOX-1 . 1 and ACOX-3 .",
"This pathway rapidly converts a favorable ascaroside pheromone that induces aggregation to an unfavorable one that induces the stress-resistant dauer larval stage .",
"Thus , the pathway allows the worm to respond to changing environmental conditions and alter its chemical message without having to synthesize new ascarosides de novo .",
"We establish a new model for biosynthesis of the IC-ascarosides in which side-chain β-oxidation is critical for controlling the type of IC-ascarosides produced ."
] | [
"Small roundworms such as Caenorhabditis elegans release chemical signals called ascarosides in order to communicate with other worms of the same species .",
"Using the ascarosides , the worm can tell its friends , for example , how crowded the neighborhood is and whether there is enough food .",
"The ascarosides thus help the worms in the population decide whether the neighborhood is good – meaning they should hang around , eat , and make babies – or whether the neighborhood is bad .",
"If so , the worms should develop into a larval stage specialized for dispersal that will allow them to find a better neighborhood .",
"Roundworms make the ascarosides by attaching a long chemical ‘side chain’ to an ascarylose sugar .",
"Further chemical modifications allow the worms to produce different signals .",
"In general , to signal a good neighborhood , worms attach a structure called an indole group to the ascarosides .",
"To signal a bad neighborhood , worms make the side chain very short .",
"But how does a worm control which ascarosides it makes ?",
"Zhou , Wang et al . now show that C . elegans can change the meaning of its chemical message by modifying the ascarosides that it has already produced instead of making new ones from scratch .",
"Specifically , as their neighborhood runs out of food , C . elegans can use an enzyme called ACS-7 to initiate the shortening of the side chains of indole-ascarosides .",
"The worm can thus change a favorable ascaroside signal that causes the worms to group together into an unfavorable ascaroside signal that causes the worms to enter their dispersal stage .",
"Although Zhou , Wang et al . have focused on chemical communication in C . elegans , the findings could easily apply to the many other species of roundworm that produce ascarosides .",
"Knowing how worms communicate will help us to understand how worms respond to their environment .",
"This knowledge could potentially be used to interfere with the lifecycles and survival of parasitic worm species that harm health and crops ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"cell biology",
"tools and resources"
] | Decoupling global biases and local interactions between cell biological variables | elife-22323-v2 | [
[
"Interpretation of the relations among coupled variables is a classic problem in many areas of cell biology .",
"One example is the spatiotemporal co-localization of molecules – a critical clue to interactions between molecular components; another example is alignment of molecular structures , such as filamentous networks .",
"However , co-localization or alignment may also occur because the observed components are associated with external effectors .",
"For example , the internal components of a polarized cell are organized along the polarization axis , making it difficult to quantify how much of the observed alignment between two filamentous networks is related to common organizational constraints , and how much of it is indeed caused by direct interaction between filaments .",
"Another example is introduced with protein co-localization , where their intensity distributions may be heavily biased to specific levels regulated by the cell state .",
"The combined effects of global bias with local interactions are manifest in the joint distribution of the spatially coupled variables .",
"The contribution of global bias to this joint distribution can be recognized from the deviation of the marginal distributions of each of the two variables from an ( un-biased ) uniform distribution .",
"Although global bias can significantly mislead the interpretation of co-localization and co-orientation measurements , most studies do not account for this effect ( Adler and Parmryd , 2010; Bolte and Cordelières , 2006; Costes et al . , 2004; Das et al . , 2015; Dunn et al . , 2011; Kalaidzidis et al . , 2015; Rizk et al . , 2014; Serra-Picamal et al . , 2012; Tambe et al . , 2011 ) .",
"Previous approaches indirectly assessed spatial correlations ( e . g . , [Drew et al . , 2015; Karlon et al . , 1999] ) , variants of mutual information ( e . g . , [Krishnaswamy et al . , 2014; Reshef et al . , 2011] ) or spatial biases ( Helmuth et al . , 2010 ) but did not explicitly quantify the contribution of the global bias to the observed joint distribution .",
"These methods approach the global bias as a confounding factor ( VanderWeele and Shpitser , 2013 ) that must be eliminated for more accurate assessment of the true local interaction , but ignore the possibility that the global bias contains by-itself valuable mechanistic information to cell behavior .",
"Here , we present DeBias as an algorithm to decouple the global bias ( represented by a global index ) from the bona fide local interaction ( represented by a local index ) in co-localization and co-orientation of two independently-measured spatial variables .",
"The decoupling enables simultaneous investigation of processes that drive global bias and local interactions between spatially-matched variables .",
"Our method is dubbed DeBias because it Decouples the global Bias from local interactions between two variables .",
"To highlight its capabilities , DeBias was applied to data from four different areas in cell biology , ranging in scale from macromolecular to multicellular: ( 1 ) alignment of vimentin fibers and microtubules in the context of polarized cells; ( 2 ) alignment of cell velocity and traction stress during collective migration; ( 3 ) fluorescence resonance energy transfer of Protein Kinase C; and ( 4 ) recruitment of transmembrane receptors to clathrin-coated pits during endocytosis .",
"These examples demonstrate the generalization of the method and underline the potential of extracting global bias as an independent functional measurement in the analysis of multiplex biological variables ."
],
[
"The issue of separating contributions from global bias and local interactions is best illustrated with the alignment of two sets of variables that carry orientational information .",
"Examples of co-orientation include the alignment of two filament networks ( Drew et al . , 2015; Gan et al . , 2016; Nieuwenhuizen et al . , 2015 ) , or the alignment of cell velocity and traction stress , a phenomenon referred to as plithotaxis ( Das et al . , 2015; Tambe et al . , 2011; Trepat and Fredberg , 2011 ) .",
"In these systems , global bias imposes a preferred axis of orientation on the two variables , which is independent of the local interactions between the two variables ( Figure 1A ) . 10 . 7554/eLife . 22323 . 003Figure 1 . Illustration of global bias and local interaction using the alignment of two orientational variables .",
"( A ) The relation between two variables X , Y can be explained from a combination of direct interactions ( orange ) and a common effector .",
"( B ) Simulation .",
"Given two distributions X , Y , pairs of coupled variables are constructed by drawing sample pairs ( xi , yi ) and transforming them to ( xi’ , yi’ ) by a correction parameter ζi = αθi , which represents the effect of a local interaction .",
"α is constant for each of these simulations .",
"( C ) Simulated joint distributions .",
"X , Y truncated normal distributions with mean 0 and σX = σY .",
"Shown are the joint distributions of 4 simulations with reduced global bias ( i . e . , increased standard deviation σX , σY ) and increased local interaction ( left-to-right ) .",
"All scenarios have similar observed mean alignment of ~19° .",
"( D ) Example of 100 draws of coupled orientational variables from the two most extreme scenarios in panel C . Most orientations are aligned with the x-axis when the global bias is high and no local interaction exists ( left ) , while the orientations are less aligned with the x-axis but maintain the mean alignment between ( xi’ , yi’ ) pairs for reduced global bias and increased local interaction ( right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 003 Similar observed alignments may arise from different levels of global bias and local interactions .",
"This is demonstrated by simulation of two independent random variables X and Y , representing orientations ( Figure 1B , left ) , from which pairs of samples xi and yi are drawn to form an alignment angle θi ( Figure 1B , middle ) .",
"Then , a local interaction between the two variables is modeled by co-aligning θi by ζi degrees , resulting in two variables xi’ and yi’ with an observed alignment θi - ζi ( Figure 1B , right ) .",
"We show the joint distribution of X , Y for four simulations ( Figure 1C ) where X and Y are normally distributed with identical means but different standard deviations ( σ ) , truncated to [−90∘ , 90∘] , and different magnitudes of local interactions ( ζ ) .",
"The latter is defined as ζ = αθ ( Figure 1B , α = 1 for perfect alignment ) .",
"Throughout the simulations both σ and α are gradually increased ( Figure 1C , left-to-right ) , implying that the global bias in the orientational variables is reduced while their local interactions increase .",
"As a result , all simulations display similar observed alignments ( mean values , 18 . 9°−19 . 5° ) .",
"Figure 1D visualizes 100 samples from each of the two most distinct scenarios: low σ and no local interaction ( σ = 17° , α = 0 ) leads to tendency of X and Y to align independently to one direction ( left ) ; higher variance together with increased interaction ( σ = 40° , α = 0 . 5 ) leads to more diverse orientations of X and Y ( right ) , while maintaining similar mean alignment .",
"This simple example highlights the possibility of observing similar alignments arising from different mechanisms .",
"While the described properties are well known and many others have used statistical post-processing to eliminate confounding factors for accurate assessment of local interactions ( Drew et al . , 2015; Helmuth et al . , 2010; Karlon et al . , 1999; Krishnaswamy et al . , 2014; Reshef et al . , 2011 ) , we aim at directly quantifying the global bias , with the goal of extracting encapsulated information that is fundamental to the biological question .",
"DeBias models the observed marginal distributions X’ and Y’ as the sum of contributions by a common effector , i . e . , the global bias , and by local interactions that effect the co-alignment of the two variables in every data point ( Figure 2A ) . 10 . 7554/eLife . 22323 . 004Figure 2 . DeBias algorithm .",
"( A ) Underlying assumption: the observed relation between two variables is a cumulative process of a global bias and a local interaction component .",
"( B ) Quantifying local and global indices: sample from the marginal distributions X , Y to construct the resampled distribution .",
"The global index ( GI ) is calculated as the Earth Movers Distance ( EMD ) between the uniform and the resampled distributions .",
"The local index ( LI ) is calculated as the subtraction of the GI from the EMD between the uniform and the observed distribution .",
"( C ) Local and global indices calculated for the examples from Figure 1C .",
"Black circles represent the ( GI , LI ) value for the corresponding example in Figure 1C , bars represent the relative contribution of the local ( green ) and global ( red ) index to the observed alignment .",
"( D-E )",
"Simulation using a constant interaction parameter α = 0 . 2 and varying standard deviations of X , Y , σ = 50° to 5° .",
"( D ) Joint distributions .",
"Correlation between X and Y is ( subjectively ) becoming less obvious for increasing global bias ( decreasing σ ) .",
"( E ) GI and LI are negatively correlated: decreased σ enhances GI and reduces LI .",
"The change in GI is ~4 fold larger compared to the change in LI indicating that the GI has a limited effect on LI values .",
"Inset: stretched LI emphasizes the negative correlation .",
"( F ) Both LI and GI are needed to discriminate between simulations with different interaction parameters .",
"α = 0 . 2 ( red ) or 0 . 25 ( cyan ) , σ is drawn from a normal distribution ( mean = 25° , standard deviation = 4° ) .",
"Number of simulations = 40 , for each parameter setting .",
"Inset: stretched LI emphasizes the discrimination .",
"Number of histogram bins , K = 15 , for all simulations . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 00410 . 7554/eLife . 22323 . 005Figure 2—figure supplement 1 . Simulations demonstrating the negative local interactions induce negative local indices . Simulations were performed as in Figure 1B: X , Y truncated normal distributions with mean 0 and σ = σX = σY; ζ = αθ , but with α < 0 as the negative interaction .",
"Shown are GI and LI values for four simulations with increased global bias ( i . e . , smaller standard deviation σX , σY ) and increased negative local interaction ( left-to-right ) , all scenarios have similar observed mean alignment of ~19° ( corresponding to the simulations in Figure 1C ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 005 In a scenario without any global bias or local interaction between X’ and Y’ , the observed alignment would be uniformly distributed ( denoted uniform ) .",
"Hence any deviation from the uniform distribution would reflect contributions from both the global bias and the local interactions .",
"To extract the contribution of the global bias we constructed a resampled alignment distribution ( denoted resampled ) from independent samples of the marginal distributions X’ and Y’ , which decouples matched pairs ( xi’ , yi’ ) , and thus excludes their local interactions .",
"The global bias is defined as the dissimilarity between the uniform and resampled distributions and accordingly describes to what extent elements of X’ and Y’ are aligned without local interaction ( Figure 2B ) .",
"If a local interaction exists then the distribution of the observed alignment angles will differ from the independently resampled alignment distribution .",
"Hence , the uniform distribution will be less similar to the experimentally observed alignment distribution ( denoted observed ) than to the resampled distribution .",
"Accordingly , the local interaction is defined by the difference of dissimilarity between the observed and uniform distributions and dissimilarity between the resampled and uniform distributions ( Figure 2B ) .",
"The Earth Mover's Distance ( EMD ) ( Peleg et al . , 1989; Rubner et al . , 2000 ) was used to calculate dissimilarities between distributions .",
"The EMD of 1-dimensional distributions is defined as the minimal 'cost' to transform one distribution into the other ( Kantorovich and Rubinstein , 1958 ) .",
"This cost is proportional to the minimal accumulated number of moving observations to adjacent histogram bins needed to complete the transformation .",
"Formally , we calculate EMD ( A , B ) =∑i=1 , … , K|∑j=1 , … , iaj−∑j=1 , … , ibj| , with K - number of histogram bins , aj and bj - fraction of observations in bin j for distributions A and B correspondingly .",
"Introducing the EMD defines scalar values for the dissimilarities and allows us to define the EMD between resampled and uniform alignment distributions as the global index ( GI ) and the local index ( LI ) as the difference of EMD between observed and uniform and the GI ( Figure 2B ) .",
"Figure 2C , demonstrates how the GI and LI recognize the global bias and local interactions between the matched variable pairs ( xi’ , yi’ ) established in Figure 1C .",
"For a scenario with no local interaction ( α = 0 ) DeBias correctly reports LI~0 and GI~3 .",
"For a scenario with gradually wider distributions X , Y , i . e . , less global bias , and gradually stronger local interactions ( α > 0 ) , the LI increases while the GI decreases .",
"Additional simulations showed that similar properties apply for negative local interactions ζ = αθ ( Figure 1B ) were α < 0 ( Figure 2—figure supplement 1 ) .",
"In the previous illustrations , changes in spread of the distributions X and Y were compensated by changes in the local interactions .",
"When leaving the interaction parameter α constant while changing the spread of X and Y , a weak , but intrinsically negative correlation between LI and GI becomes apparent ( Figure 2D–E ) .",
"Thus , while DeBias can correctly distinguish scenarios with substantial shifts from global bias to local interactions , the precise numerical values estimating the contribution of LI varies between scenarios with a low versus high global bias .",
"To address this issue we propose to exploit the variation between experiments for modeling the relation between LI and GI .",
"This is demonstrated by comparing two distinct values of the interaction parameter , α , emulating different experimental settings ( Figure 2F ) .",
"Within experiments variation was obtained by drawing multiple values of σ from a normal distribution .",
"Due to the negative correlation between LI and GI the experimental patterns can only be discriminated by combining LI and GI into a two-dimensional descriptor ( Figure 2F ) .",
"This point will be further demonstrated in one of the following case studies and in the Discussion .",
"To characterize the properties of DeBias we used theoretical statistical reasoning .",
"The first limiting case is set by the scenario in which observations from X and Y are independent .",
"The expected values of the observed and resampled alignments are identical; accordingly , LI converges to 0 for large N ( Appendix 1 , Theorem 1 ) .",
"The second limiting case is set by the scenario in which X and Y are both uniform .",
"The corresponding resampled alignment is also uniform; accordingly , GI converges to 0 for a large N ( Appendix 1 , Theorem 2 ) .",
"The third limiting case occurs with perfect alignment , i . e . , xi = yi for all i .",
"In this case the observed alignment distribution is concentrated in the bin containing θ = 0 .",
"We examine two scenarios of perfect alignment: ( 1 ) When all the locally matched measurements are identical ( xi = yj for all i , j ) , the resampled distribution is also concentrated in the bin θ = 0 implying that LI = 0 and GI assumes the maximal possible value: GI=1K∑i=1 , .",
". , K ( i−1 ) =K−12 , where K is the number of quantization bins ( Appendix 1 , Theorem 3 . I ) .",
"( 2 ) When X , Y are uniform ( and xi = yi for all",
"i ) , the resampled distribution is uniform , thus GI = 0 and LI reaches its maximum value: LI=1K∑i=1 , .",
". , K ( i−1 ) =K−12 , ( Appendix 1 , Theorem 3 . II ) .",
"Generalizing this case , we prove that LI is a lower bound for the actual contribution of the local interaction to the observed alignment ( Appendix 1 , Theorem 4 ) .",
"Complementarily , GI is an upper bound for the contribution of the global bias to the observed alignment .",
"Last , we show that when X and Y are truncated normal distributions , or when the alignment distribution is truncated normal , GI reduces to a limit of 0 as σ → ∞ , when σ is the standard deviation of the normal distribution before truncation ( Appendix 1 , Theorem 5 ) .",
"Simulations complement this result demonstrating that σ and GI are negatively associated , i . e . , GI decreases with increasing σ ( Figure 2E ) .",
"This final property is intuitive , because resampling from more biased distributions ( smaller σ ) tends to generate high agreement between ( xi , yi ) leading to reduced alignment angles and increased GI .",
"The modeling of the observed alignment as the sum of GI and LI allowed us to assess the performance of DeBias from synthetic data .",
"By using a constant local interaction parameter ζ ( ζ = c ) , we were able to retrieve the portion of the observed alignment that is attributed to the local interaction and to compare it with the true predefined ζ ( Appendix 2 , Appendix 2—figure 1 ) .",
"These simulations demonstrated again the need for a GI-dependent interpretation of LI ( first shown in Figure 2E–F ) .",
"Simulations were also performed to assess how the choice of the quantization parameter K ( i . e . , number of histogram bins ) and number of observations N affect GI and LI ( Appendix 2 , Appendix 2—figures 2–3 ) , and this was also verified in our experimental data ( Figure 3—figure supplement 1 ) .",
"In summary , by combining theoretical considerations and simulations we demonstrated the properties and limiting cases of DeBias in decoupling paired matching variables from orientation data .",
"We applied DeBias to investigate the degree of alignment between vimentin intermediate filaments and microtubules in polarized cells .",
"Recent work using genome-edited Retinal Pigment Epithelial ( RPE ) cells with endogenous levels of fluorescently tagged vimentin and α-tubulin showed that vimentin provides a structural template for microtubule growth , and through this maintains cell polarity ( Gan et al . , 2016 ) .",
"The effect was strongest in cells at the wound front where both vimentin and microtubule networks collaboratively align with the direction of migration ( Figure 3A–C ) .",
"An open question remains as to how much of this alignment is caused by the extrinsic directional bias associated with the collective migration of cells into the wound as opposed to a local interaction between the two cytoskeleton systems . 10 . 7554/eLife . 22323 . 006Figure 3 . : Alignment of microtubule and vimentin intermediate filaments in the context of cell polarity .",
"( A ) RPE cells expressing TagRFP α-tubulin ( MT ) and mEmerald-vimentin ( VIM ) at endogenous levels during a wound healing assay .",
"Scale bar 100 μm .",
"( B ) Zooming in on cells in different locations in respect to the wound edge .",
"Right-most column , computer segmented filaments of both cytoskeleton systems .",
"Top row , cells located at the wound edge ( ‘Front’ ) ; Middle row , cells located 2–3 rows away from the wound edge ( ’Back’ ) ; Bottom row , cells located at the wound edge partially with shRNA knock-down of vimentin .",
"Scale bar 10 μm .",
"( C ) Orientation distribution of microtubules ( left column ) and vimentin filaments ( middle columns ) for the cells outlined in B . Vimentin-microtubule alignment distributions ( right column ) .",
"( D ) Scatterplot of GI versus LI derived by DeBias .",
"The GI is significantly higher in WT cells at the wound edge ( ‘Front’ , n = 12 ) compared to cells inside the monolayer ( ‘Back’ , n = 12 , fold change = 4 . 8 , p-value < 0 . 0001 ) ; or compared to vimentin-depleted cells at the wound edge ( ‘VIM KD’ , n = 7 , fold change = 5 . 2 , p-value < 0 . 0001 ) .",
"Statistics based on Wilcoxon rank-sum test .",
"All DeBias analyses performed with K = 15 .",
"( E ) Polarization of RPE cells at the wound edge at different time points after scratching .",
"Scale bar 10 μm .",
"( F ) Representative experiment showing the progression of LI and GI as a function of time after scratching ( see color code ) .",
"Correlation between GI and time ~0 . 90 , p-value < 10−30 ( n time points = 83 ) .",
"N = 5 independent experiments were conducted of which four experiments showed a gradual increase in GI with increased observed polarity .",
"All DeBias analyses performed with K = 15 . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 00610 . 7554/eLife . 22323 . 007Figure 3—figure supplement 1 . LI and GI are independent of the number of observations ( N ) and the number of histogram bins ( K ) – experimental evidence from the data in Figure 3E–F ( time evolution of microtubule-vimentin alignment ) .",
"( A ) Time evolution of N , the number of observations ( paired microtubule and vimentin pixels ) analyzed , N grows linearly in time .",
"Y-axis was normalized to the first time point .",
"( B–C )",
"LI and GI are independent of the number of random resampled observations N = 500–3000 .",
"( B ) LI and GI remain stable .",
"( C ) Deviation from the LI , GI values reported in Figure 3F .",
"Lower N correspond to higher variability .",
"All analyses performed with K = 15 .",
"( D–E )",
"LI and GI patterns are independent of the number of alignment histogram bins K = 5–25 .",
"( D ) GI .",
"( E ) LI . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 007 Analysis of the GI and LI revealed that most of the discrepancy in vimentin-microtubule alignment originated from a shift in the global bias ( Figure 3D ) , suggesting that the local interaction between the two cytoskeletons is unaffected by the cell position or knock-down of vimentin .",
"Instead , the reduced alignment between the two cytoskeletons is caused by a loss of cell polarity in cells away from the wound edge , probably associated with the reduced geometric constraints imposed by the wound edge .",
"In a similar fashion , reduction of vimentin expression relaxes global cell polarity cues that tend to impose alignment .",
"To corroborate our conclusion that the global state of cell polarity is encoded by the GI , we performed a live cell imaging experiment , in which single cells at the edge of a freshly inflicted wound in a RPE monolayer were monitored for 80 min after scratching .",
"DeBias was applied to calculate a time sequence of LI and GI .",
"Cells at the wound edge tended to gradually increase their polarity and started migrating during the imaging time frame ( Figure 3E , Video 1 ) .",
"Accordingly , the GI increased over time ( Figure 3F ) .",
"We also used this data set to verify that the reported shifts in GI are independent of the number of data points in and the binning of the distribution ( Figure 3—figure supplement 1 ) .",
"This demonstrates the capacity of DeBias to distinguish fundamentally different effectors of cytoskeleton alignment . 10 . 7554/eLife . 22323 . 008Video 1 . Polarization of RPE cells at the monolayer edge over time . Please note several occasions ( 44 and 46 min , 65 and 67 min , 73 and 75 min ) of focus drift followed by automated focus correction . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 008 Collective cell migration requires intercellular coordination , achieved by mechanical and chemical information transfer between cells .",
"One mechanism for cell-cell communication is plithotaxis , the tendency of individual cells to align their velocity with the maximum principal stress orientation ( He et al . , 2015; Tambe et al . , 2011; Zaritsky et al . , 2015 ) .",
"As in the previous example of vimentin and microtubule interaction , much of this alignment is associated with a general directionality of velocity and stress field parallel to the axis of collective migration ( Zaritsky et al . , 2015 ) .",
"Using a wound healing assay , Das et al . ( Das et al . , 2015 ) screened 11 tight-junction proteins to identify pathways that promote motion-stress alignment ( Figure 4A ) .",
"Knockdown of Merlin , Claudin1 , Patj and Angiomotin ( Amot ) reduced the alignment of velocity direction and stress orientation ( Das et al . , 2015 ) .",
"Further inspection of these hits showed that the stress orientation remained stable upon depletion of these proteins , but the velocity direction distribution was much less biased towards the wound edge ( Zaritsky et al . , 2015 ) .",
"Here , we further analyze this data to demonstrate the capacity of DeBias to pinpoint tight-junction proteins that alter specifically the global or local components that induce velocity-stress alignment . 10 . 7554/eLife . 22323 . 009Figure 4 . : Alignment of stress orientation and velocity direction during collective cell migration .",
"( A ) Assay illustration .",
"Wound healing assay of MDCK cells .",
"Particle image velocimetry was applied to calculate velocity vectors ( red ) and monolayer stress microscopy to reconstruct stresses ( blue ) .",
"Alignment of velocity direction and stress orientation was assessed .",
"( B ) Mini-screen that includes depletion of 11 tight-junction proteins and Merlin .",
"Data from ( Das et al . , 2015 ) , where effective depletion was demonstrated .",
"Shown are GI and LI values; molecular conditions are sorted by the LI values ( control is ranked sixth , pointed by the black arrow ) .",
"Each dot was calculated from accumulation of 3 independent experiments ( N = 925–1539 for each condition ) .",
"Three groups of tight junction proteins are highlighted by dashed rectangles: red - low LI and GI compared to control , purple – different GI but similar LI , orange – high LI .",
"All DeBias analyses were performed with K = 15 .",
"( C ) Pair-wise statistical significance for LI values .",
"P-values were calculated via a permutation-test on the velocity and stress data ( Materials and methods ) .",
"Red – no significant ( p ≥ 0 . 05 ) change in LI values , blue – highly significant ( < 0 . 01 ) change in LI values .",
"( D ) Highlighted hits: Claudin1 , Claudin2 , Merlin and ZO1 .",
"Top: Distribution of stress orientation ( top ) , velocity direction ( middle ) and motion-stress alignment ( bottom ) .",
"Bottom: table of mean alignment angle , LI and GI .",
"Claudin1 and Claudin2 have similar mechanisms for transforming stress to aligned velocity .",
"ZO1 depletion enhances alignment of velocity by stress . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 009 By distinguishing GI and LI we generated a refined annotation of the functional alteration that depletion of these tight-junction components caused in mechanical coordination of collectively migrating cells ( Figure 4B–C ) .",
"First , we confirmed that the four hits reported by ( Das et al . , 2015 ) massively reduced the GI , consistent with the notion that absence of these proteins diminished the general alignment of velocity to the direction induced by the migrating sheet ( Figure 4B , red dashed rectangle ) .",
"Merlin , Patj and Angiomotin reduced the LI to values close to 0 , suggesting that the local dependency between stress orientation and velocity direction was lost .",
"Depletion of Claudin1 , or of its paralog Claudin2 , which was not reported as a hit in the Das et al . screen , reduced the LI to a lesser extent , similarly for both proteins , but had very different effects on the GI ( Figure 4B , purple dashed rectangle ) .",
"This suggested that the analysis by ( Das et al . , 2015 ) missed effects that do not alter the general alignment of stress or motion , and implied the existence of a local velocity-stress alignment mechanism that does not immediately change the collective aspect of cell migration .",
"When assessing the marginal distributions of stress orientation and velocity direction we observed that depletion of Claudin1 reduced the organization of stress orientations and of velocity direction , while Claudin2 reduced only the latter .",
"The LI values of depletion conditions were similar and lower than control ( Figure 4D ) .",
"Merlin depletion is characterized by an even lower LI and marginal distributions with aligned stress orientation and almost uniform alignment distribution ( Figure 4D ) .",
"Since we think that aligned stress is transformed to aligned motion ( He et al . , 2015; Zaritsky et al . , 2015 ) , we propose that in this data the LI quantifies the effect of local mechanical communication on parallelizing the velocity among neighboring cells .",
"Accordingly , stress-motion transmission mechanism is impaired to a similar extent by reduction of Claudin1 and Claudin2 , albeit less than by reduction of Merlin .",
"Using LI as a discriminative measure also allowed us to identify a group of new hits ( Figure 4C ) .",
"ZO1 , ZO2 , ZO3 , Occludin and ZONAB are all characterized by small reductions in GI but a substantial increase in LI relative to control ( Figure 4B , orange dashed rectangle ) .",
"A quantitative comparison of control and ZO1 depleted cells provides a good example for the type of information DeBias can extract: both conditions yield similar observed alignment distributions with nearly identical means , yet ZO1 depletion has an 83% increase in LI and 8% reduction in GI , i . e . , the mild loss in the marginal alignment of velocity or stress is compensated by enhanced local alignment in ZO1 depleted cells ( Figure 4D ) .",
"This might point to a mechanism , in which stress orientation is reduced by tight-junction depletion , but enhanced by transmission of stress orientation into motion orientation , leading to comparable alignment .",
"Notably , all paralogs , ZO1 , ZO2 and ZO3 fall into the same cluster of elevated LI and slightly reduced GI relative to control experiments .",
"This phenotype is in agreement with the outcomes of a screen that found ZO1 depletion to increase both motility and cell-junctional forces ( Bazellières et al . , 2015 ) .",
"Protein-protein co-localization is another ubiquitous example of correlating spatially matched variables in cell biology .",
"To quantify GI and LI for protein-protein co-localization , we normalized each channel to intensity values between 0 and 1 .",
"The 'alignment' θi of matched observations ( xi , yi ) was replaced by the difference in normalized fluorescent intensities xi - yi ( Materials and methods ) .",
"Simulations demonstrated that stronger interactions in co-localization are translated to larger LI values and validated that the choice of K ( number of histogram bins ) and N ( number of observations ) marginally affect GI and LI ( Appendix 3 , Appendix 3—figure 1 ) .",
"While LI could serve as a measure to assess co-localization , the interpretation of GI is less intuitive .",
"In the following , we present two examples of applying DeBias for protein-protein co-localization , and demonstrate the type of information that can be extracted from the combined GI and LI analysis .",
"To test the potential of DeBias in quantification of pixel-based co-localization , we analyzed the effect of fluorescence resonance energy transfer ( FRET ) in the C kinase activity reporter ( CKAR ) , which reversibly responds to PKC activation and deactivation ( Violin et al . , 2003 ) .",
"Reduced PKC activity leads to energy transfer from CFP to YFPCFP , resulting in reduced FRET ratio ( CFPYFPCFP ) ( Figure 5A ) .",
"Assuming that the CFP signal is dominant ( CFP > YFPCFP ) , this alteration should reduce the difference between the CFP and YFPCFP channels , which would in DeBias yield an increased LI ( Figure 5A , Materials and methods ) . 10 . 7554/eLife . 22323 . 010Figure 5 . : PKC inhibition alters LI and GI .",
"( A ) PKC inhibition is expected to lead to elevated LI for cells with dominant CFP signal ( CFP > YFPCFP ) .",
"Upon FRET , CFP signal is locally transferred to YFPCFP , reducing the difference in normalized intensity between the two channels , which increases LI .",
"( B ) hTERT-RPE-1 cells imaged with the CKAR reporter .",
"A cell before ( top ) and after ( bottom ) PKC inhibition .",
"Region of interest was manually annotated and the ratio CFPYFPCFP was calculated within it .",
"( C ) Pixel distribution of differences in normalized fluorescent intensities CFPnorm - YFPnormCFP before and after PKC inhibition for the cell from panel B . PKC inhibition shifted the average absolute difference from 0 . 054 to 0 . 042 and the LI from 2 . 25 to 2 . 84 .",
"( D–F )",
"PKC inhibition experiment .",
"N = 8 cells .",
"Statistics based on Wilcoxon sign-rank test .",
"( D ) The FRET ratio CFPYFPCFP decreased ( p-value < 0 . 008 ) , ( E ) LI increased ( p-value < 0 . 008 ) , and ( F ) GI decreased ( p-value < 0 . 008 ) after PKC inhibition .",
"( G ) Marginal distribution of CFP and YFPCFP before ( top ) and after ( bottom ) PKC inhibition .",
"( H ) Control experiment .",
"N = 7 cells .",
"hTERT-RPE-1 cells expressing cytoplasmic GFP and mCherry before and after PKC inhibition .",
"No significant change in LI or GI was observed .",
"All DeBias analyses were performed with K = 19 . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 010 To test this we labeled hTERT-RPE-1 cells with CKAR and imaged CFP and YFPCFP channels before and after specific inhibition of PKC with HA-100 dihydrochloride ( Figure 5B , Materials and methods ) , leading to reduced pixel differences in their normalized fluorescent intensities ( Figure 5C ) .",
"As expected , the CFPYFPCFP ratio decreased ( Figure 5D ) , LI values increased ( Figure 5E ) and seemed more sensitive to the FRET .",
"Surprisingly , DeBias indicated a shift in the GI values ( Figure 5F ) , reflected in a more homogeneous marginal distribution of both channels before inhibition ( Figure 5G ) .",
"Control experiments with cytoplasmic GFP and mCherry expression did not show the shifts observed in LI or GI ( Figure 5H ) .",
"Thus , we conclude that PKC inhibition changes the localization of PKC towards a more random spatial distribution .",
"One possible mechanism for this behavior is that deactivation releases the kinase from the substrate .",
"This example illustrates DeBias’ capabilities to simultaneously quantify changes in local interaction and global bias in pixel-based co-localization .",
"Clathrin-mediated endocytosis ( CME ) is the major pathway for entry of cargo receptors into eukaryotic cells .",
"Cargo receptor composition plays an important role in regulating clathrin-coated pit ( CCP ) initiation and maturation ( Liu et al . , 2010; Loerke et al . , 2009 ) .",
"The clustering of transferrin receptors ( TfnR ) , the classic cargo receptor used to study CME , promotes CCP initiation , in concert with clathrin and adaptor proteins ( Liu et al . , 2010 ) .",
"Recent evidence suggests a diversity of mechanisms regulating endocytic trafficking , including cross-talks between signaling receptors and components of the endocytic machinery ( Di Fiore and von Zastrow , 2014 ) .",
"For example , the oncogenic protein kinase Akt has been shown to play an important role in mediating CME in cancer cells ( Liberali et al . , 2014; Reis et al . , 2015 ) , but not in normal epithelial cells ( Reis et al . , 2015 ) .",
"Here we tested how the decoupling by DeBias of global and local contributions to the overall intensity alignment of clathrin and TfnR , can be used to simultaneously investigate co-localization and predict CCP dynamics , using fixed cell fluorescence imaging .",
"We used fluorescence images of fixed non‐small lung cancer cells ( H1299 ) or untransformed human retinal pigment epithelial cells ( ARPE-19 ) expressing clathrin light chain A fused to eGFP ( eGFP-CLCa ) as a CCP marker ( Figure 6A–B ) .",
"Cells were either treated with DMSO or with an AKT inhibitor ( Akt inhibitor X , ‘ten’ ) , and imaged by Total Internal Reflection Fluorescence Microscopy ( TIRFM ) .",
"CCPs were reported in the eGFP-CLCa channel and TfnR was visualized by immunofluorescence in a second channel ( Materials and methods ) .",
"For single cells , the location of fluorescent signals of CLCa and TfnR were recorded and the data were pooled and processed by DeBias ( Materials and methods ) . 10 . 7554/eLife . 22323 . 011Figure 6 . : AKT inhibition differentially alters recruitment of TfnR to CCPs during CME for different cell lines .",
"( A ) H1299 cells expressing CLCa and TfnR ligands .",
"Top row , representative WT cell ( TfnR ligand , GI = 4 . 6 , LI = 1 . 6 ) .",
"Bottom row , representative AKT-inhibited cell ( TfnR ligand , GI = 6 . 0 , LI = 0 . 3 ) .",
"Scale bar 10 μm .",
"( B ) ARPE-19 cells .",
"Top row , representative WT cell ( TfnR ligand , GI = 4 . 3 , LI = 0 . 8 ) .",
"Bottom row , representative AKT-inhibited cell ( TfnR ligand , GI = 4 . 6 , LI = 1 . 6 ) .",
"( C–D )",
"LI and GI of CLCa-TfnR co-localization for Ctrl ( red ) and Aktinh .",
"cells ( cyan ) .",
"Every data point represents the LI and GI values for a single cell .",
"Statistical analyses performed with the Wilcoxon rank-sum test .",
"All DeBias analyses were performed with K = 40 .",
"( C ) H1299: N number of cells Ctrl = 30 , Aktinh .",
"= 30; number of CCPs per cell: Ctrl = 455 . 5 , Aktinh .",
"= 179 . 5 .",
"GI p-value < 0 . 0001 , LI p-value < 0 . 0001 .",
"( D ) ARPE-19: N number of cells Ctrl = 30 , Aktinh .",
"= 20; number of CCPs per cell: Ctrl = 958 . 8 , Aktinh .",
"= 1138 . 2 .",
"GI p-value < 0 . 002 , LI p-value < 0 . 008 .",
"( E–F )",
"Receiver Operating Characteristic ( ROC ) curves showing the true positive rates as a function of false-positive rates for single cell classification , higher curves correspond to enhanced discrimination ( Materials and methods ) .",
"Black – LI , orange – ( GI , LI ) .",
"Statistics via permutation test ( Materials and methods ) .",
"( E ) H1299 AUC: ( GI , LI ) = 0 . 96 versus LI = 0 . 88 , p-value ≤ 0 . 003 .",
"( F ) ARPE-19 AUC: ( GI , LI ) = 0 . 83 versus LI = 0 . 72 , p-value ≤ 0 . 048 .",
"( G–H )",
"Joint distributions of CLCa ( x-axis ) and TfnR ( y-axis ) for H1299 ( G ) and ARPE-19 ( H ) cells .",
"( I–J )",
"Marginal distributions of CLCa ( left ) and TfnR ( right ) for H1299 ( I ) and ARPE-19 ( J ) cells .",
"( K–L )",
"Combined CCP lifetime distribution for 50 Ctrl ( red ) and Aktinh .",
"( cyan ) cells .",
"Statistics with Wilcoxon rank-sum test ( Materials and methods ) .",
"( K ) H1299: p-value < 0 . 006 ( mean EMD: Ctrl = 29 . 3 versus Aktinh . = 43 . 6 ) ; number of cells: 50 ( Ctrl ) , 11 ( Aktinh . ) .",
"( L ) H1299: p-value n . s . ( mean EMD: Ctrl = 36 . 1 versus Aktinh . = 38 . 0 ) ; number of cells: 12 ( Ctrl ) , 12 ( Aktinh . ) .",
"( M–N )",
"Percentage of TfnR uptake: Ctrl versus Aktinh .",
"( whiskers - standard deviation ) .",
"Statistics via two-tailed Student’s t-test .",
"( M ) H1299: p-value < 0 . 005; N = 3 independent experiments .",
"( N ) ARPE-19: p-value n . s . ; N = 3 independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 01110 . 7554/eLife . 22323 . 012Figure 6—figure supplement 1 . : GI encodes information that is distinct from local interactions; Experimental validations of DeBias for co-localization . Data from Figure 6 .",
"( A–F )",
"GI as a complementary measure .",
"( A–B )",
"LI and Pearson’s correlation ( CORR ) are highly associated ( statistical analyses performed using Pearson’s correlation ) .",
"( A ) H1299 cells: rho = 0 . 95 , p-value < 10−29 .",
"( B ) ARPE-19 cells: rho = 0 . 98 , p-value < 10−32 .",
"( C–D )",
"CORR vs . GI .",
"( C ) H1299 .",
"( D ) ARPE-19 .",
"( E–F )",
"ROC curves .",
"Statistics via permutation test ( Materials and methods ) .",
"( E ) H1299 AUC: ( GI , CORR ) = 0 . 96 versus CORR = 0 . 83 , p-value ≤ 0 . 0001 .",
"( F ) ARPE-19 AUC: ( GI , CORR ) = 0 . 84 versus CORR = 0 . 72 , p-value n . s . .",
"( G–H )",
"Similar LI , GI values for different number of observations N = 100 , 200 , 400 , 800 .",
"( G ) H1299 .",
"( H ) ARPE-19 .",
"( I–J )",
"LI and GI patterns are independent of the number of alignment histogram bins K = 10 , 20 , 30 , 40 .",
"( I ) H1299 .",
"( J ) ARPE-19 . DOI: http://dx . doi . org/10 . 7554/eLife . 22323 . 012 LI values , indicative of the co-localization between TfnR and CLCa , were significantly lower in Akt-inhibited H1299 compared to control cells ( Figure 6C ) .",
"In contrast , Akt inhibition increased the LI values in ARPE-19 cells but this effect was less prominent ( Figure 6D ) .",
"Akt inhibition resulted in increased GI values for both cell lines , to a much greater degree in H1299 cells ( Figure 6C–D ) .",
"To test whether GI enhances the ability to distinguish between control and Akt-inhibited cells , we applied Linear Discriminative Analysis ( LDA ) classification to calculate the true positive rate versus the false positive rate for LI alone ( black lines , Figure 6E–F ) or the pair ( GI , LI ) , ( orange lines , Figure 6E–F ) .",
"The area under these curves ( AUC ) provided a direct measure of the ability of each method to accurately classify the experimental condition of single cells .",
"AUC for the ( GI , LI ) representation was superior to using LI alone for both cell lines ( H1299: 0 . 96 versus 0 . 88 , Figure 6E; ARPE-19: 0 . 83 versus 0 . 72 , Figure 6F ) .",
"Similar benefit in discrimination was achieved when using the GI to complement Pearson’s correlation as an alternative to LI for measuring local interaction ( Figure 6—figure supplement 1A–F ) .",
"Such improved discrimination is indicative of distinct molecular processes that were altered upon Akt inhibition .",
"We also used this data set to experimentally validate the independence of GI and LI of the number of observations ( N , Figure 6—figure supplement 1G , H ) and the choice of the number of histogram bins ( K , Figure 6—figure supplement 1I , J ) .",
"To interpret the increased GI values for Akt-inhibited cells , we examined the joint and marginal distributions of CLCa and TfnR .",
"Upon Akt-inhibition , the joint distributions were more biased toward regions of low TfnR intensities ( Figure 6G–H ) .",
"This was clearly observed in the marginal distributions ( Figure 6I–J ) .",
"Hence , although the CLCa distribution appeared not to change upon AKT inhibition , the frequency of CCPs with fewer TfnRs increased .",
"Given the positive relation between TfnR cargo quantities and CCPs maturation ( Loerke et al . , 2009 ) , we wondered whether the increased frequencies of CCPs containing less TfnR might alter CCPs dynamics .",
"Indeed , live-imaging of H1299 cells showed a higher frequency of CCPs with shorter lifetimes upon Akt-inhibition , which was not seen in normal ARPE-19 cells ( Figure 6K–L , Materials and methods ) .",
"It has previously been shown that Akt inhibition reduces the rate of TfnR CME uptake in H1299 cells , but not in ARPE-19 cells ( [Reis et al . , 2015] , see also Figure 6M–N ) ; therefore , these findings indicate that the reduced levels of TfnR in CCPs upon Akt inhibition results in an increase in short-lived , most likely abortive events , and hence a decrease in CME efficiency .",
"Altogether , DeBias could distinguish alterations in the regulation of CME between two cell types .",
"The decoupling to GI and LI indicated that upon Akt inhibition , both untransformed and cancer cells showed a global bias towards CCPs with lowered TfnR intensities .",
"This conclusion could not have been reached by considering only the LI , which increased for normal and decreased for transformed cell lines ."
],
[
"We introduce DeBias as a new method to assess global bias and local interactions between coupled cellular variables .",
"Although the method is generic , we show here specific examples of DeBias analysis in co-orientation and co-localization studies .",
"The source code is available , https://github . com/DanuserLab/DeBias , as well as via a web-based platform , https://debias . biohpc . swmed . edu .",
"The website also provides detailed instructions for the operation of the user interface .",
"DeBias defines a generalizable framework for eliminating confounding factors in the analysis of interacting variables .",
"Our examples demonstrate that the distinction of global and local contributions to the level of variable coupling eliminates much of the global confounder bias in the analysis of more direct interactions and can unearth in the form of global bias mechanisms that are missed by a single parameter analysis ( Figures 1–2 ) .",
"In the example of vimentin-microtubule alignment ( Figure 3 ) , the significant decrease in GI as opposed to the LI upon partial vimentin knock-down indicated that the reduction in alignment between the two cytoskeleton systems is associated with a reduction of cell polarity as the global cue .",
"In the example of stress-velocity alignment ( Figure 4 ) , depletion of some tight junction proteins increased LI , suggestive of enhanced local stress-motion transmission; knock-down of others decreased GI indicating an overall impaired alignment of velocity in the direction of wound closure .",
"In the example of FRET experiments ( Figure 5 ) , PKC inhibition lead to increased LI , validating the FRET response , while a reduced GI was indicative of weaker interactions of the inactivated kinase with its substrates .",
"In the example of Tfn receptor ( TfnR ) co-localization with CCPs during CME ( Figure 6 ) , the increased GI in response to Akt inhibition related to a higher fraction of CCPs containing less TfnR .",
"Moreover , Akt inhibition induced opposite shifts in LI for normal and cancer cells , reflecting differential alterations in co-localization between cell types .",
"Thus , DeBias provided insight into the regulation of cargo-pit association by kinase activity that depended on a proper deconvolution of local and global effects on the interaction of the clathrin and receptor signal .",
"We then validated our conclusions by further analyses of the marginal distributions , live-imaging and uptake assays ( Figure 6 ) .",
"Overall , the four applications shown in this work first emphasize the general need for a confounder analysis when dealing with coupled biological variables and second indicate that the global bias may be linked to mechanistically meaningful properties of the studied system .",
"These properties were either ignored or eliminated by previous methods , and now can be assessed directly by DeBias .",
"Other approaches have been used to address global confounders for assessment of local interactions between biological variables .",
"For the specific example of object-based co-localization , Helmuth et al . simulated the spatial distribution of objects in the absence of local interactions to calibrate co-localization measurement ( Helmuth et al . , 2010 ) .",
"Other methods mostly used second-order spatial statistics on distances between neighbor points to exclude confounders for better co-localization sensitivity ( reviewed in Lagache et al . , 2015 ) .",
"Importantly , we show applications of DeBias on co-localization that do not require initial object detection ( Figure 5 ) .",
"While the phenomenon of confounder bias is independent of object- versus pixel-based co-localization , we distinguish the peculiarities of the two scenarios in Appendix 4 .",
"An important and more general approach to revealing local interactions masked by global biases was recently proposed by ( Krishnaswamy et al . , 2014 ) , using applications to single cell mass cytometry data as examples .",
"The authors developed a measure referred to as conditional-Density Resampled Estimate of Mutual Information ( DREMI ) to quantify the influence of a protein X on protein Y based on the conditional probability P ( Y|X ) .",
"DREMI takes advantage of the abundant mass cytometry data to equally weigh data at different intervals along the range of X values using > 10 , 000 cells per experimental condition .",
"This approach is less reliable when limited data is available , because of the low confidence in the conditional probability of observations with low data abundance .",
"Thus , DREMI is not well suited for image data , which typically has fewer observations .",
"DeBias estimates GI and LI assuming a constant global bias and local interaction for all observations .",
"Moreover , its quantification power is relative .",
"For example , a two-fold increase in the direct interaction of two variables would not necessarily result in a two-fold increase in LI .",
"Another limitation is the absence of complete orthogonality of GI and LI values ( Figure 2E–F , Appendix 3—figure 1 ) , which complicates the interpretation of GI and LI in certain scenarios .",
"These three limitations apply to all current approaches for quantifying interactions between coupled variables .",
"The main conceptual advance DeBias seeks to make relates to the explicit integration of confounding factors in the analysis of coupled variables , which implies an expansion of the coupling metric from a scalar to a two-dimensional score .",
"A forth limitation in the current implementation of DeBias is the linear normalization of multiple intensity variables in co-localization applications .",
"Future versions may include non-linear normalization methods , although such normalization is usually highly specific to a particular data set .",
"Last , the mechanism encoded by the GI is not always obvious .",
"Sometimes it requires additional experiments to unveil the information contained by the GI .",
"For example , we combined fixed cell dual-color imaging with live-imaging and uptake assays to show that shifts in the GI encode a shift in the relative populations of short- and long-lived CCPs between conditions ( Figure 6 ) .",
"Despite some of the discussed complexities , DeBias offers a simple means to quantify and interpret mechanisms that alter confounders in the coupling of two variables and to largely exclude such global biases from the quantification of direct interactions ."
],
[
"The DeBias procedure is depicted in Figure 2A .",
"The marginal distributions X and Y are estimated from the experimental data , ∀i , xi , yi∈[ 0 , 90° ] .",
"The experimentally observed alignment distribution ( denoted observed ) is calculated from the alignment angles θi of matched ( xi , yi ) paired variables , for all i .",
"θi={|xi−yi||xi−yi|≤90180−|xi−yi||xi−yi|>90 The resampled alignment distribution ( denoted resampled ) is constructed by independent sampling from X and Y . N random observations ( where N = |X| is the original sample size ) from X and Y are independently sampled with replacement , arbitrarily matched and their alignment angles calculated to define the resampled alignment .",
"This type of resampling precludes the local dependencies between the originally matched ( xi , yi ) paired variables .",
"The uniform alignment distribution ( denoted uniform ) is used as a baseline for comparison between distributions .",
"This is the expected alignment distribution when neither global bias ( reflected by uniform X , Y distributions ) nor local interactions exist .",
"The Earth Mover's Distance ( EMD ) ( Peleg et al . , 1989; Rubner et al . , 2000 ) was used as a distance metric between alignment distributions .",
"The EMD for two distributions , A and B , is defined as follows: EMD ( A , B ) =∑j=1 , … , K|∑j=1 , … , iaj−∑j=1 , … , ibj| , where aj and bj are the frequencies of observations in bins j of the histograms of distributions A and B , respectively , each containing K bins .",
"The global index ( GI ) is defined as the EMD between the uniform distribution and the resampled alignment: GI = EMD ( uniform , resampled ) The local index is determined by subtraction of the global index from the EMD between the uniform distribution and the experimentally observed alignment distribution: LI = EMD ( uniform , observed ) - global index The following adjustments to this procedure are implemented to allow DeBias to quantify protein-protein co-localization: The number of histogram bins for the alignment distributions ( observed , resampled and uniform ) was K = 15 for orientational data , 19 for PKC and 40 for CME co-localization data .",
"The Freedman-Diaconis rule ( Freedman and Diaconis , 1981 ) was used to automate the selection of histogram bin width: bin size=Q3 ( x ) −Q1 ( x ) n3 , where Qi is the ith quartile of the empirical distribution x and n is the number of data points contained .",
"A function to calculate K is included in our publicly available source code and this functionality was also integrated to the web-server implementation .",
"Importantly , GI and LI across experiments can be compared only when evaluated with the same K value and this is enforced by the web-server .",
"It is the responsibility of the source-code user to validate using the same K values when comparing different experimental conditions .",
"Coupled measurements of velocity direction and stress orientation were taken from the data originally published by Tamal Das et al . ( Das et al . , 2015 ) .",
"Particle image velocimetry ( PIV ) was applied to calculate velocity vectors , and monolayer stress microscopy ( Tambe et al . , 2011 ) was used to extract stress orientations .",
"Velocity and stress measurements were recorded 3 hr after collective migration was induced by lifting off the culture-insert in which the cells have grown to confluence .",
"Validated siRNAs were used for gene screening .",
"Detailed experimental settings can be found in Das et al . , 2015 .",
"hTERT-RPE-1 cells ( ATCC , RRID: CVCL_4388 ) expressing GFP and mCherry ( for the control experiment ) or C kinase activity reporter ( CKAR , for the PKC activation experiment ) ( Violin et al . , 2003 ) were plated with DMEM/F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in fibronectin-coated 35 mm MatTek plates ( P35G-0–10 c ) .",
"The cell line has been tested negative for mycoplasma contamination .",
"Cells were incubated overnight and imaged with a custom-built DeltaVision OMX widefield microscope ( GE healthcare life sciences , Chicago , IL ) equipped with an Olympus PLAN 60 × 1 . 42 N . A . oil objective and CoolSNAP HQ2 interline CCD cameras .",
"FRET experiments were performed with a 445 nm laser , and control experiments were performed with 488 and 561 nm lasers .",
"478/35 , 541/22 , 528/48 and 609/37 emission bandpass filters were used for the CFP , YFP , GFP and mCherry channels , respectively .",
"The output powers of the lasers were set to 10% the maximal output ( 100 mW ) .",
"The exposure time was 200 ms per frame for both channels .",
"10 µM of the PKC inhibitor HA-100 dihydrochloride ( Santa Cruz Biotechnology , Dallas , TX ) was added to the media after the first image was recorded and a second image was recorded 20 min later .",
"Single cells were manually selected for analysis .",
"In particular , cells with higher intensities in the CFP channel were found to provide reproducible changes in their FRET intensity .",
"Each cell was manually annotated and analyzed with the Biosensor Processing Software 2 . 1 to produce the ratio images ( Hodgson et al . , 2010 ) .",
"Briefly , the field of view was corrected for uneven illumination , background was subtracted , the image was masked with the single cell annotation , and the ratio image was calculated as CFP/YFPCFP .",
"Statistics was determined using the non-parametric Wilcoxon signed-rank test .",
"The DeBias code was implemented in Matlab , compiled with Matlab complier SDK and transferred to a web-based platform to allow public access for all users at https://debias . biohpc . swmed . edu .",
"The graphical user interface ( GUI ) was designed to be simple and easy to use .",
"The user uploads one or more datasets to the DeBias webserver and selects the mode of operation ( co-localization/orientation ) .",
"GI and LI values are calculated and the results are displayed and emailed to the user .",
"‘DeBias Analyst’ enables to group experiments into two experimental conditions ( usually control versus treatment ) , visualizes and outputs statistics on the alterations of GI and LI .",
"The software’s flow chart and a detailed user manual are available in the online user manual .",
"Source code is publicly available , https://github . com/DanuserLab/DeBias Danuser , 2017 ( with a copy archived at https://github . com/elifesciences-publications/DeBias ) ."
]
] | [
"Analysis of coupled variables is a core concept of cell biological inference , with co-localization of two molecules as a proxy for protein interaction being a ubiquitous example .",
"However , external effectors may influence the observed co-localization independently from the local interaction of two proteins .",
"Such global bias , although biologically meaningful , is often neglected when interpreting co-localization .",
"Here , we describe DeBias , a computational method to quantify and decouple global bias from local interactions between variables by modeling the observed co-localization as the cumulative contribution of a global and a local component .",
"We showcase four applications of DeBias in different areas of cell biology , and demonstrate that the global bias encapsulates fundamental mechanistic insight into cellular behavior .",
"The DeBias software package is freely accessible online via a web-server at https://debias . biohpc . swmed . edu ."
] | [
"Cell biologists often use microscopes to look closely at cells and see what is happening during an experiment .",
"Cell biology experiments typically involve measuring more than one aspect of the cells , for example , the forces a cell is experiencing and the direction it is moving , or the locations of two different components in the cell .",
"The task is then to decipher the interactions between these independent variables to better understand the inner workings of a living cell .",
"This task , however , can be challenging because other variables can mask the interactions between the pairs of variables being studied .",
"For example , it is difficult to know if two components of cells overlap in microscopy images because they directly interact or simply because the overall organization of the cell makes it more likely they will end up in the same place .",
"Several statistical methods have been developed to estimate and eliminate such confounding effects to reveal specific interactions .",
"However , these confounding effects – termed “global biases” – may themselves contain valuable information about how cells work .",
"Ignoring the possible roles of global biases may limit our understanding of biological processes .",
"Zaritsky et al . have now addressed this issue by developing an algorithm called DeBias that takes global bias into account by decoupling it from true interactions between two variables .",
"DeBias uses global bias as a second measurement to understand how two biological variables interact via confounding variables .",
"Zaritsky et al . then used DeBias with data from four different cell biology experiments to show that the algorithm works .",
"For example , animal cells contain several proteins that form filaments , which give them their shape and help them move .",
"So-called vimentin filaments and microtubules had previously been seen to occur in the same place within cells but it was not clear whether their alignment was due to local interactions or determined by the overall shape of the cell .",
"DeBias was used to analyze cells that were either still or moving in a particular direction .",
"The algorithm could tease apart the effect of this movement and showed that co-alignment of vimentin filaments and microtubules was caused more by the movement and shape of the cell and less by specific interactions .",
"Overall , DeBias is a new mathematical tool for cell biologists that is freely accessible online .",
"Other researchers can now use this tool in future studies to identify local interactions and global biases in a wide range of cell biology experiments and interpret the data in a meaningful way ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | EM connectomics reveals axonal target variation in a sequence-generating network | elife-24364-v2 | [
[
"Neural sequences are central to many models of circuit function ( Diesmann et al . , 1999; Jin et al . , 2007; Gibb et al . , 2009; Fiete et al . , 2010; Mostafa and Indiveri , 2014; Cannon et al . , 2015a; Rajan et al . , 2016 ) , and neurons often fire sequentially during specific behaviors ( Hahnloser et al . , 2002; Peters et al . , 2014; Mello et al . , 2015 ) or cognitive states ( Pastalkova et al . , 2008; Harvey et al . , 2012 ) , but the network properties that underlie such dynamics are poorly understood .",
"Here we explore the synaptic connections within the zebra finch HVC , which is central to generating the neuronal activity necessary to coordinate activation of vocal muscles during the highly reproducible courtship song ( Nottebohm et al . , 1976; Vu et al . , 1994; Aronov et al . , 2008; Long and Fee , 2008 ) .",
"Song progression is paced by HVC ( RA ) neurons , which project to the primary downstream target area , known as the robust nucleus of the arcopallium ( RA ) ( Figure 1a ) .",
"During the song , an HVC ( RA ) neuron is either silent or active in the form of a burst of action potentials that occurs at a single precise and cell-specific time ( Hahnloser et al . , 2002; Kozhevnikov and Fee , 2007; Long et al . , 2010; Vallentin and Long , 2015 ) .",
"At any moment , it is estimated that about 200 of these ‘pacer’ neurons are active and can drive the appropriate motor activity ( Fee et al . , 2004 ) , presumably through a set of specific synaptic connections in RA ( Fee et al . , 2004; Markowitz et al . , 2015; Lynch et al . , 2016; Picardo et al . , 2016 ) . 10 . 7554/eLife . 24364 . 003Figure 1 . Analysis of synaptic inputs onto HVC ( RA ) dendrites .",
"( a ) A schematic of the songbird brain showing HVC and its two main downstream targets , RA and Area X .",
"( b ) A backlabeled HVC ( RA ) neuron ( red ) during juxtacellular filling ( pipette shown in white ) guided by 2-photon imaging of fluoro-Ruby .",
"( c , d )",
"A Neurobiotin-filled cell from",
"( b ) in brightfield LM after histochemical processing",
"( c ) and dendritic reconstruction",
"( d ) .",
"( e ) Normalized count of dendritic path length vs . soma distance for 15 HVC ( RA ) neurons; individual cells ( gray ) and average ( red ) .",
"Bin size: 10 µm .",
"( f ) Cross-section through a SBEM stack showing BDA-labeled HVC ( RA ) somata .",
"( g ) Inhibitory ( blue spheres ) and excitatory ( gold spheres ) synapses onto an HVC ( RA ) dendrite in the SBEM volume .",
"Sphere cross-sectional areas are proportional to the active zone area .",
"( h ) Higher magnification of a dendritic branch from",
"( g ) .",
"( i ) Density of asymmetric and symmetric synapses vs . the distance to the soma .",
"( j ) Two HVC ( RA ) dendrites; red spheres indicate double-labeled synapses , with cross sections through two synapses ( insets ) .",
"Inset , cross sections through the synapses circled in red .",
"( k ) Active zone size distributions of inhibitory ( blue ) , excitatory ( black ) , and double-labeled ( red ) synapses .",
"Scale bars are 10 µm in b and c , 25 µm in f , and 0 . 25 µm in j . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 00310 . 7554/eLife . 24364 . 004Figure 1—figure supplement 1 . Sample preparation for LM and EM .",
"( a , b )",
"For LM , HVC ( RA ) neurons are retrogradely labeled by injecting a fluorescent dextran ( fluoro-Ruby ) into RA",
"( a ) , and a single labeled neuron is targeted and filled with Neurobiotin under the guidance of 2-photon microscopy",
"( b ) .",
"( c ) The tissue is then fixed and 100 µm parasagittal sections are sliced through the entirety of HVC .",
"( d , e )",
"Sections are further processed to stain Neurobiotin-labeled neurites",
"( d ) and then imaged in brightfield with a 100x objective",
"( e ) .",
"( f ) For EM , HVC ( RA ) neurons are retrogradely labeled with biotinylated dextran ( BDA ) .",
"( g , h )",
"A single 200 µm parasagittal section is taken from the center of HVC",
"( g ) and further processed to stain BDA-labeled neurites",
"( h ) .",
"( i , j )",
"A cube of tissue from within the center of HVC is extracted and stained by ROTO ( reduced osmium OTO )",
"( i ) before being imaged via SBEM",
"( j ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 00410 . 7554/eLife . 24364 . 005Figure 1—figure supplement 2 . Synaptic boutons on HVC ( RA ) axon collaterals .",
"( a ) An LM reconstruction of HVC ( RA ) axon collaterals of one neuron with the HVC border indicated by dashed lines .",
"Locations of all synaptic boutons are marked by grey spheres .",
"( b ) Bouton density of collateral branches as a function of their midpoint distances from the soma . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 00510 . 7554/eLife . 24364 . 006Figure 1—figure supplement 3 . Ultrastructural classification of synapses .",
"( a ) An asymmetric synapse onto a BDA-labeled HVC ( RA ) dendrite whose morphology is partially obscured by the label .",
"Red arrows indicate the synaptic cleft .",
"( b to",
"d ) Other synapses made by the same axon onto unlabeled dendrites .",
"The pronounced postsynaptic density ( PSD ) , especially for synapse 4 , confirms the classification of this connection as excitatory .",
"( e ) A symmetric synapse onto a BDA-labeled HVC ( RA ) dendrite .",
"( f to",
"h ) Synapses from the same neuron onto other unlabeled dendrites display a lack of a PSD and a different appearance of synaptic vesicles compared with the asymmetric synapses .",
"Scale bar: 0 . 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 00610 . 7554/eLife . 24364 . 007Figure 1—figure supplement 4 . The BDA label is inefficient and incomplete .",
"( a ) All cells within our SBEM dataset are represented by spheres at the location of the cell body .",
"Known HVC ( RA ) neurons , which were labeled with BDA , were colored red .",
"Also shown are putative HVC ( RA ) neurons , classified by morphological features of the soma and dendrite ( pink ) , other neuron types ( large gray spheres ) , and glia ( small gray spheres ) .",
"Scale bar: 50 µm .",
"( b ) Incomplete labeling of axonal collaterals of HVC ( RA ) neurons inside HVC is demonstrated with a skeleton reconstruction ( labeled axon in black and unlabeled axon in gray ) .",
"The inset electron micrographs correspond to the portions of the axonal field indicated .",
"Scale bars: left: 1 µm , right: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 007 It has been difficult to discriminate between different models of sequence generation in HVC , in part because of the unknown connectivity within that nucleus .",
"One class of models uses a synaptic ( or ‘synfire’ ) chain architecture ( Amari , 1972; Abeles , 1991; Diesmann et al . , 1999 ) , which can deliver highly reliable and precise timing but requires direct connections between the pacer neurons ( Li and Greenside , 2006; Jin et al . , 2007; Long et al . , 2010; Cannon et al . , 2015a ) .",
"Such connections are , however , only rarely seen with paired intracellular recordings , which at the same time showed that HVC ( RA ) neurons are connected with high probability ( >0 . 50 ) to nearby inhibitory interneurons ( Mooney and Prather , 2005; Kosche et al . , 2015 ) .",
"This observation weakened the case for synfire chain-based sequence generation in HVC and sparked the development of alternative hypotheses that do not require direct connections between excitatory cells ( Yildiz and Kiebel , 2011; Hamaguchi and Mooney , 2012; Amador et al . , 2013; Goldin et al . , 2013; Armstrong and Abarbanel , 2016; Hamaguchi et al . , 2016; Rajan et al . , 2016 ) .",
"There are , however , a number of reasons paired recordings may fail to correctly estimate the connection rate between excitatory cells , among them the severing of axons during slice preparation ( Stepanyants et al . , 2009 ) and an oversampling of closely spaced neurons ( Jiang et al . , 2015 ) .",
"To avoid this bias , we used a structural approach combining anatomical reconstructions of complete cells in light microscopy ( LM ) with high-throughput serial block-face electron microscopy ( SBEM ) ( Denk and Horstmann , 2004; Seung , 2009 ) ."
],
[
"We used both LM and EM , because anatomically , synapses can only be identified unambiguously in EM , but currently the size of the volume that can be studied by EM is limited to several hundred microns in one dimension ( Helmstaedter , 2013 ) .",
"This size is too small to explore the full extent of HVC connectivity , given that axon collaterals of HVC neurons ramify widely throughout the nucleus ( e . g . Figure 1—figure supplement 2a ) , which is roughly 2000 × 500 × 500 µm3 in size ( Nixdorf-Bergweiler and Bischof , 2007 ) .",
"We therefore used LM to explore the mesoscale structure of the axonal morphology and EM to analyze synaptic connectivity .",
"To identify HVC ( RA ) cells , we injected markers into RA that are retrogradely transported , fluorescent Tetramethylrhodamine ( TMR , also called fluoro-Ruby ) or biotinylated dextran ( BDA , Figure 1—figure supplement 1 ) , for tissue to be observed in LM or EM , respectively .",
"To enable the LM-based reconstruction of the entire dendrite and of the axonal collaterals within HVC for single HVC ( RA ) cells , we used in vivo two-photon microscopy to target ( Komai et al . , 2006 ) TMR-labeled somata for Neurobiotin labeling ( Figure 1b ) .",
"We eliminated all cells ( 29 of 44 ) where the labeling intensity varied between different parts of the neurite or where no descending axon could be found .",
"The remaining 15 cells were imaged at 92 × 92 × 500 nm3 voxel size using a transmitted light brightfield microscope ( Oberlaender et al . , 2007 ) and reconstructed using Neuromorph ( see Materials and methods ) ( Figure 1c , d , Figure 1—figure supplement 1a–e; Video 1 ) .",
"In agreement with other observations ( Dutar et al . , 1998; Mooney , 2000; Kosche et al . , 2015 ) , we found that HVC ( RA ) dendrites were compact , with 95 . 0 ± 2 . 0% ( SEM ) of the dendritic path found within 100 µm of the soma ( Figure 1e ) .",
"In contrast , the axon collaterals , which were lined with synaptic boutons throughout ( Figure 1—figure supplement 2b ) , ramified across HVC .",
"For each cell ( n = 15 ) , the dendrite was entirely ( 100% ) confined to HVC , while the axon ( with the exception of the branch projecting to RA ) was also largely restricted to the boundaries of HVC ( 97% on average ) . 10 . 7554/eLife . 24364 . 008Video 1 . Video shifting through a z-stack of a sagittal section within HVC , containing a Neurobiotin-filled HVC ( RA ) neuron stained with DAB . Number of z-sections shown is 144 .",
"Voxel size is 92 × 92 × 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 008 To quantify the prevalence of different types of synaptic inputs onto the dendrite of HVC ( RA ) cells , we next acquired a SBEM data set ( 166 × 166 × 77 µm3 overall size , comprising 15104 × 15104×2661 voxels , each 11 × 11 × 29 nm3 in size ) from the central part of HVC ( Figure 1f , Figure 1—figure supplement 1f–j , Videos 2 and 3 ) .",
"All raw data as well as skeletonized reconstructions are available online ( Kornfeld , 2017a ) ( https://github . com/jmrk84/HVC_paper; with a copy archived at https://github . com/elifesciences-publications/HVC_paper ) .",
"Within this volume , 34 somata were positively identified as HVC ( RA ) neurons by the presence of a BDA-derived electron density ( Figure 1f ) .",
"This number is approximately 14% of the expected value of HVC ( RA ) somata ( 240 ± 28 , SEM ) , given that there are about 40 , 000 ± 3800 ( SEM ) HVC ( RA ) cells ( Wang et al . , 2002 ) and the total HVC volume is 0 . 35 ± 0 . 024 mm3 ( n = 14 , SEM ) .",
"For 12 of the 34 labeled HVC ( RA ) neurons , we manually reconstructed ( skeletonized ) ( Helmstaedter et al . , 2011 ) the dendrite as far as possible .",
"These reconstructions ranged in dendritic path length from 642 µm to 1956 µm ( 1290 ± 469 µm , mean ± SD ) compared with complete LM-based reconstructions ( 1438 µm to 4819 µm , mean ± SD: 3187 ± 997 µm ) .",
"Although ~70% ( 174 out of 248 ) of dendritic branches reached the boundary of the EM data set and were thus incomplete , 74 branches were completely reconstructed , including their most distal inputs ( median ± SD of maximum soma distances: 90 . 9 ± 8 . 6 µm and 116 . 7 ± 29 . 4 for EM and LM , respectively ) .",
"Our reconstructions therefore sample the full gamut of input types .",
"While we do not find any variation of the input type with dendritic distance from the soma beyond a distance of 40 microns ( see below ) , it cannot be completely ruled out that a subtle bias exists that lies below our detection threshold but might be discoverable when using larger data volumes . 10 . 7554/eLife . 24364 . 009Video 2 . Video of a subregion of the acquired SBEM dataset , showing the original data resolution ( lossy compression ) .",
"Number of z-sections shown is 100 , translating to 2 . 9 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 00910 . 7554/eLife . 24364 . 010Video 3 . Video of a subregion of the acquired SBEM dataset , showing a larger field of view with a BDA-labeled HVC ( RA ) soma ( lossy compression ) .",
"Number of z-sections shown is 200 , translating to 5 . 8 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 010 We started by classifying for one cell all ( 1 , 003 ) incoming synapses ( Figure 1g–h ) by visually inspecting their ultrastructural details ( Gray , 1959; Colonnier , 1968 ) ( Videos 4 and 5 ) .",
"We found that 396 ( 39 . 5% ) synapses were asymmetric and thus presumably excitatory , and 607 ( 60 . 5% ) were symmetric ( inhibitory ) cases .",
"If it was not possible to classify a synapse based on its inspection directly , additional synapses nearby on the same axon were analyzed , since it can be assumed that they are of the same type ( Eccles , 1976 ) ( Figure 1—figure supplement 3 ) .",
"Our synapse classification is reliable: in 19 out of 20 randomly selected test cases , a second expert independently came to the same conclusion and in another set of test cases ( 8 HVC ( RA ) , 11 HVC ( X ) , and 31 interneuron synapses ) , where the neuron type was known based on somatic and dendritic morphology ( Figure 2d , Figure 2—figure supplement 1 ) , all synapses were correctly classified by an expert unaware of the cell type . 10 . 7554/eLife . 24364 . 011Figure 2 . Classification of postsynaptic targets .",
"( a ) A BDA-labeled axon with four synaptic boutons ( boxes ) .",
"One bouton and its postsynaptic structure labeled in red and blue , respectively: In cross section ( top right ) and as a surface reconstruction ( bottom center ) .",
"( b ) Dendrites from an inhibitory interneuron , an HVC ( RA ) neuron , and an HVC ( X ) neuron ( left to right ) in LM .",
"Spine locations are indicated by grey spheres .",
"( c , d )",
"Spine densities for each of these neuron classes from LM",
"( c ) and EM",
"( d ) reconstructions .",
"Insets show examples with spines indicated by arrowheads .",
"( e ) SBEM-based reconstructions of two HVC ( RA ) somata with their proximal axons .",
"Blue , green , and red spheres mark the location of synapses with inhibitory interneurons , HVC ( RA ) neurons , and HVC ( X ) neurons , respectively .",
"Scale bar is 0 . 25 µm in a . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 01110 . 7554/eLife . 24364 . 012Figure 2—figure supplement 1 . Morphological markers of interneurons .",
"( a ) Ultrastructural and morphological differences of the somata of an HVC interneuron ( left , blue shade ) and an HVC ( RA ) neuron ( right , red shade ) .",
"Compared with HVC ( RA ) neurons , interneurons had large amounts of endoplasmatic reticulum ( ER ) , many mitochondria , and a large cell body .",
"Scale bar: 7 . 5 µm .",
"( b ) An electron micrograph ( cut plane rotated to show spine attached to dendrite ) showing a polysynaptic protrusion ( blue label ) that receives four synapses at its tip , that was previously misclassified as a dendritic spine of an excitatory cell .",
"The four presynaptic axons are colored in yellow , green , purple and red .",
"Scale bar: 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 01210 . 7554/eLife . 24364 . 013Video 4 . Video of a z-stack of 18 consecutive images ( 100 × 100 pixels ) showing a symmetric synapse . Voxel dimensions: 11 × 11 × 29 . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 01310 . 7554/eLife . 24364 . 014Video 5 . Video of a z-stack of 18 consecutive images ( 100 × 100 pixels ) showing an asymmetric synapse . Voxel dimensions: 11 × 11 × 29 . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 014 The dominance of inhibitory synaptic inputs was consistently observed for HVC ( RA ) cells: when we applied our synapse classification procedure to 97 short dendritic stretches ( first and third quartile of stretch length: 13 . 3 µm and 21 . 6 µm ) randomly selected from eight of the other skeletonized HVC ( RA ) cells , we found that across neurons the average ratio between excitatory and inhibitory synapses was statistically indistinguishable ( p=0 . 36 , one-way ANOVA ) from that found in the completely analyzed neuron .",
"Inhibitory synapses were significantly enriched near the soma ( 68 ± 4% of all synapses at most 40 µm from the soma are inhibitory compared to 57 ± 2% , for synapses beyond that distance , mean ± SEM , p<0 . 05 , Wilcoxon rank-sum test , Figure 1i ) , an observation also made in cortical neurons ( Anderson et al . , 1994 ) .",
"To estimate the number of excitatory and inhibitory synapses that a single HVC ( RA ) neuron receives on average , we first calculated dendritic synapse densities for all nine analyzed cells separately for asymmetric ( 0 . 25 ± 0 . 02 µm−1 , mean ± SEM ) and for symmetric synapses ( 0 . 36 ± 0 . 02 µm−1 ) .",
"To get expected counts per cell , we multiplied these with the full dendritic path length ( on average 3 . 2 mm per neuron ) , determined from LM reconstructions .",
"Thus , on average well above half of all synapses onto HVC ( RA ) dendrites are symmetric ( 59% , 1144 ± 429 , mean ± SD ) and only 41% are asymmetric ( 786 ± 311 ) — a surprising dominance of inhibitory inputs that stands in stark contrast to mammalian cortical neurons ( Beaulieu et al . , 1992; Peters , 2002; Kasthuri et al . , 2015 ) , where the inhibitory synapses are typically found to be at most 20% of the total .",
"We next inspected all BDA-labeled dendrites emerging from the 12 aforementioned cells for synapses in which the presynaptic axon was labeled , and thus had to come from other HVC ( RA ) cells ( Figure 1j ) .",
"We found 44 such homotypic synapses between HVC ( RA ) cells ( see Materials and methods ) , but they comprise only about 1% among an estimated total of 3817 ± 926 ( SD ) incoming excitatory synapses .",
"Their median size ( 0 . 21 µm2 ) and size variation ( first and third quartile: 0 . 10 µm2 and 0 . 48 µm2 ) , were statistically indistinguishable from those for all asymmetric synapses ( 0 . 17 µm2; first and third quartile: 0 . 08 µm2 and 0 . 39 µm2 , p>0 . 05 , Wilcoxon rank-sum test , Figure 1k ) .",
"One might be tempted to consider the small number of double-labeled synapses as evidence that HVC ( RA ) -HVC ( RA ) connections are rare .",
"However , BDA labeled only a small fraction ( 1/7th ) of all HVC ( RA ) cells in our data set ( Figure 1—figure supplement 4a ) and even for those , axonal collaterals were often incompletely filled ( Figure 1—figure supplement 4b ) , suggesting the probability that a given stretch of HVC ( RA ) axon is labeled could be quite small .",
"To estimate this probability , we created a 300-member set of 1 µm3 cubes randomly placed throughout the SBEM volume and measured the total labeled axonal path length they contained .",
"The value obtained ( 38 . 6 µm of labeled axon across 300 cubes ) is about 13 times smaller than that expected given an estimate of the combined axonal path length ( 585 . 6 m ) of all 40 , 000 HVC ( RA ) cells .",
"The axonal labeling probability of 7 . 6 ± 1 . 6% ( SEM , see Materials and methods ) in turn implies that the homotypic HVC ( RA ) synapses constitute ~15 ± 4% ( SEM ) of all excitatory synapses onto HVC ( RA ) neurons .",
"We next took a presynaptic perspective to independently estimate the extent of HVC ( RA ) -HVC ( RA ) connectivity and used a transsynaptic tracing scheme ( McGuire et al . , 1991 ) to determine the cell-type of the targets of the outgoing synapses on BDA-labeled axon collaterals ( Figure 2a ) .",
"The three main cell types found in HVC ( Dutar et al . , 1998; Kubota and Taniguchi , 1998; Mooney , 2000 ) are easily distinguished in LM: Inhibitory interneurons have smooth dendrites with a nearly complete lack of spines ( Mooney , 2000; Wild et al . , 2005 ) , and excitatory neurons project to either RA or to the basal ganglia ( Area X ) , with the descending axon clearly recognizable .",
"Even short stretches of dendrite can be reliably ascribed to one of the three types , because the spine density varies widely between but not within them ( Dutar et al . , 1998; Kubota and Taniguchi , 1998; Mooney , 2000 ) ( Figure 2b , c ) .",
"Dendrites were largely aspinous ( 0 . 01 ± 0 . 01 spines/µm , mean ± SD ) for interneurons , densely covered with spines ( 0 . 70 ± 0 . 13 spines/µm ) for HVC ( X ) cells and less so ( 0 . 21 ± 0 . 07 spines/µm ) for HVC ( RA ) neurons .",
"This spine density metric correctly classified 17 out of 18 BDA-labeled HVC ( RA ) dendrites in EM as well as 11 inhibitory neurons that had been classified using other morphological characteristics ( symmetric synapses and a large soma diameter , Figure 2—figure supplement 1a ) .",
"We used this to classify the cell type of postsynaptic dendritic segments ( n = 528 ) transsynaptically traced from nine BDA-labeled axons fully reconstructed in the EM volume .",
"In 41 of 569 cases , the cell type could not be determined .",
"These cases were excluded from further analysis , because the ultrastructure was obstructed by the BDA label ( n = 33 ) or because the recovered dendritic branch was too short ( n = 8 ) , see Materials and methods , Figure 2d .",
"When we examined three BDA-stained axons that each emerged from labeled somata in the SBEM dataset ( path lengths: 1 . 37 , 0 . 88 , and 0 . 72 mm ) , we found that of 121 connections , 115 terminated on dendrites of inhibitory cells but only six onto excitatory cells , four of which being other HVC ( RA ) cells ( e . g . , Figure 2e ) .",
"This agrees with the high connectivity found for closely spaced HVC ( RA ) -interneuron pairs by electrical recordings ( Kosche et al . , 2015 ) as well as with reports using EM connectomics for other cortical tissue ( Bock et al . , 2011 ) .",
"However , at this density , there would only be about 20 homotypic synapses per HVC ( RA ) neuron , which is about six times smaller than our estimate derived from the BDA-labeled inputs onto HVC ( RA ) dendrites .",
"We then examined BDA-labeled axon fragments that were 'orphaned' ( n = 6 , path length: 0 . 56 ± 0 . 27 mm , mean ± SD ) , i . e . , could not be traced back to their soma and were therefore likely farther away from it .",
"Three of the fragments were synaptically connected to one of the labeled dendrites and four were partially myelinated .",
"We discovered that the prevalence of synapses onto excitatory neurons , and onto other HVC ( RA ) cells in particular , was much larger for orphaned fragments than for attached axons; increases were 13-fold ( HVC ( RA ) -E ) , from 5 . 0% ( 6 out of 121 ) to 64 . 6% ( 263 out of 407 ) , and 11-fold ( HVC ( RA ) -HVC ( RA ) ) , from 3 . 3% ( 4 out of 121 ) to 36 . 8% ( 150 out of 407 ) ( Figure 3a ) .",
"HVC ( RA ) dendrites were often connected by more than one synapse to a labeled axon ( 17 doubles , 3 triple , and 1 quintuple among 127 analyzed pairs ) .",
"The much larger ( compared to the proximal outputs ) fraction of excitatory target cells for the orphans implies that the prevalence of the different target types must depend on the distance from the soma .",
"This would also be consistent with the low connection probability of 0 . 7% between HVC ( RA ) cells found in electrophysiological recordings ( Kosche et al . , 2015 ) , where the recorded somata are usually less than 200 µm apart ( Mooney and Prather , 2005; Jiang et al . , 2015 ) , while , as our LM reconstructions show , 56 ± 14% ( SD ) of the axon collaterals' path lies farther than 200 µm from the soma , with some of them ramifying over the extent of HVC ( e . g . , Figure 1—figure supplement 2a ) . 10 . 7554/eLife . 24364 . 015Figure 3 . Spatial variation of postsynaptic cell type .",
"( a ) SBEM-based reconstructions and synaptic targets for two orphaned axon segments .",
"Colored spheres mark the locations and types of synapses .",
"( b ) Axon collaterals ( LM-based reconstruction ) of an HVC ( RA ) neuron with branch nodes ( gold circles ) , the soma ( black circle ) , and the HVC border ( dashed lines ) .",
"( c ) Mean axon length ( black ) and branch node densities ( gold ) vs . soma distance ( n = 15 cells ) .",
"( d ) The ratio of synapses onto inhibitory interneurons vs . estimated distance from the soma ( p<0 . 005 , Pearson's correlation ) .",
"( e , f )",
"The density of synapses onto HVC ( RA )",
"( e ) and HVC ( X )",
"( f ) vs . estimated distance from soma ( p<0 . 002 , Pearson's correlation , combining HVC ( RA ) and HVC ( X ) values ) .",
"( g ) Total synaptic size ( summated active zone area , µm2/mm ) onto excitatory neurons vs . estimated distance of the presynaptic axon from the soma ( p<0 . 05 , Pearson's correlation ) .",
"Vertical error bars: SEM of the Poisson-distribution means estimated from the number of synapses on each axon segment ( e–g ) or the SEM of an assumed underlying binomial count distribution",
"( d ) .",
"Horizontal error bars from quantiles 0 . 16 to 0 . 84 of the distance distribution based on the nearest neighbor sampling approach ( see Materials and methods ) .",
"( h ) Proposed circuit architecture .",
"HVC ( RA ) neurons ( red ) target inhibitory interneurons ( blue ) proximally and other HVC ( RA ) neurons distally . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 01510 . 7554/eLife . 24364 . 016Figure 3—figure supplement 1 . Synaptic properties of HVC ( RA ) axons , using a Bayesian approach to estimate distance from soma .",
"( a ) Total synaptic strength ( summated active zone area/pathlength ) onto excitatory neurons vs . estimated distance of the presynaptic axon from the soma ( p<0 . 05 , Pearson's correlation ) .",
"( b ) The ratio of synapses onto inhibitory interneurons vs . estimated distance from soma ( p<0 . 01 , Pearson's correlation ) .",
"( c , d )",
"The density of synapses onto HVC ( RA )",
"( c ) and HVC ( X )",
"( d ) vs . estimated distance from soma ( p<0 . 002 , Pearson's correlation , combining HVC ( RA ) and HVC ( X ) values ) .",
"Vertical bars represent the SEM of an assumed underlying Poisson distributed synapse counting process ( a , c , d ) or",
"( b ) the SEM of an assumed underlying Binomial count distribution .",
"( a–d )",
"Horizontal error bars correspond to a 0 . 16 and 0 . 84 quantile of the Bayesian posterior distribution , see Materials and methods . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 01610 . 7554/eLife . 24364 . 017Figure 3—figure supplement 2 . A SBEM-based reconstruction and synaptic targets for an orphaned axon with high branch density . Small spheres mark the location of synapses , with the color indicating the target type .",
"Note the higher frequency of inhibitory targets ( blue ) along the length of the reconstruction compared with other orphaned axons with low branch density ( see Figure 3a ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 01710 . 7554/eLife . 24364 . 018Figure 3—figure supplement 3 . A small population of RA neurons project to HVC .",
"( a ) The borders of RA were traced across nine sequential 100 µm thick sagittal sections , and the location of each retrogradely labeled HVC-projecting RA neuron is marked with a dot .",
"( b ) DiI injection in HVC resulted in robust retrograde labeling of upstream motor nuclei Uva and NIf , while only a small percentage ( <1% ) of cells in RA were labeled ( a , c ) .",
"An example confocal image of RA is shown in",
"( c ) , revealing a sparse population of retrogradely labeled neurons in the posterior region of the nucleus . DOI: http://dx . doi . org/10 . 7554/eLife . 24364 . 018 Can we estimate the distance of an orphan segment to its soma based on local information ?",
"It is apparent from our LM reconstructions that branching becomes less frequent as the distance from the soma increases ( Figure 3b , c ) .",
"Consistent with this , HVC ( RA ) axons in the SBEM data set that were connected to a cell body were much more highly branched ( 12 . 4 ± 3 . 7 , mean ± SD , branch points/mm , Figure 2e ) than most orphaned axon fragments , with an average of only 4 . 0 ± 4 . 3 ( mean ± SD ) branch points/mm .",
"To obtain a quantitative estimate of the distance to the soma and its uncertainty based on the number of branch nodes on a branch and its length we used both a nearest neighbor ( Figure 3d–g ) and a Bayesian ( Figure 3—figure supplement 1 ) analysis ( for details see Materials and methods ) .",
"We found that a synapse was much more likely to be connected to another HVC ( RA ) cell or to a HVC ( X ) neuron rather than to an inhibitory neuron if the synapse was farther away from the soma ( Figure 3d ) .",
"The transitions between these regimes may well be gradual: One of the orphaned axons ( Figure 3—figure supplement 2 ) showed an unusually high branch density ( 11 . 4 branch points/mm ) , suggesting a location close to the soma ( 16th to 84th percentile: 35 . 8 to 72 . 3 µm and also made the majority of its connections ( 52 of 83 , 63% ) onto interneurons , twice the fraction seen for the other orphaned axons ( 32 ± 10% , mean ± SD ) .",
"To rule out the possibility that our findings are due to a selection bias , we estimated the fraction of homotypic synapses for the 59 BDA-labeled axon fragments found in the 300-member set ( see above ) , tracing each fragment from the sampling cube until we found two synapses or reached the data set boundary , and determined the postsynaptic cell types .",
"Out of 105 synapses , 65 targeted interneurons , 22 HVC ( X ) neurons , and 18 other HVC ( RA ) neurons .",
"Since there are approximately 1111 ± 513 ( SD ) outgoing synapses inside HVC for each HVC ( RA ) neuron ( given an axon path length of 14 . 7 mm and a total synapse density of 75 . 4 synapses/mm ) , we expect about ~191 ± 88 ( SD ) homotypic synapses per cell ( on average , incoming and outgoing homotypic synapse have to be equal in number ) , comprising about a quarter ( 24 ± 4% , SEM ) of all incoming excitatory synapses , and nearly half of all outgoing excitatory contacts .",
"The discrepancy between the estimates of the homotypic fraction of incoming excitatory synapses from the dendritic ( ~15% ) and axonal perspective ( ~24% ) might be due to the fact that when counting the number of double labeled synapses , we accepted only those where the labeling of the presynaptic terminal was unambiguous .",
"How can we be sure that all or at least most of the orphaned fragments belong to HVC ( RA ) neurons ?",
"Since BDA ( which is transported in the retrograde direction much more efficiently than anterogradely in all tissues tested , including the zebra finch brain ( Reiner et al . , 2000 ) was only injected into RA , any labeled axon has to belong to a cell with an axon that connects HVC and RA , as HVC ( RA ) axons do .",
"If there is indeed a substantial number of cells in RA that project to HVC ( Roberts et al . , 2008 ) , then it is possible that a substantial fraction of the orphaned axons could originate from those cells .",
"To independently confirm the number of RA ( HVC ) cells , we injected the fluorescent tracer DiI ( Invitrogen , Carlsbad , CA ) into HVC , which heavily labeled the upstream nuclei NIf and Uva ( Figure 3—figure supplement 3 ) but yielded only a small number of labeled somata in RA ( 125 , 163 , and 171 , respectively , in three birds ) , approximately one for every 200 HVC ( RA ) neurons on average .",
"To account for the density of labeled axon in our EM volume , each those cells would need a total axon path of ~4 m in HVC , which appears unlikely given that the extensively ramifying HVC ( RA ) axons have a length of only ~0 . 015 m ."
],
[
"We have shown that the synaptic architecture in HVC contains a density of connections between HVC ( RA ) neurons that might be sufficient to support a synaptic-chain model , whereby precisely timed sequences of action potential bursts in HVC ( RA ) neurons are generated by a wave of activity propagating via synaptic connections among these neurons without the need for inhibition-mediated propagation of activity ( Yildiz and Kiebel , 2011 ) or to involve structures outside HVC ( Hamaguchi and Mooney , 2012; Goldin et al . , 2013; Hamaguchi et al . , 2016 ) .",
"While we estimate that 25% of excitatory inputs to HVC ( RA ) neurons are homotypic , the sources of the remaining synapses are unknown .",
"It should be a central goal of future efforts to quantify the relative number of connections from these regions ( e . g . , Uva , NIf , etc . ) at the level of single HVC ( RA ) neurons .",
"That said , many of these connections , such as auditory afferents ( Vallentin and Long , 2015 ) , collaterals from HVC ( X ) neurons ( Scharff et al . , 2000 ) , and descending fibers from NIf are unlikely to play a role in motor patterning , since removal of NIf does not disrupt the song ( Cardin et al . , 2005 ) .",
"The precise role of Uva , a thalamic region also directly projecting to HVC ( Nottebohm et al . , 1982 ) , remains to be determined ( Coleman and Vu , 2005; Hamaguchi et al . , 2016 ) .",
"Somewhat surprisingly , the low rates of pairwise connectivity seen in electrophysiological recordings ( Mooney and Prather , 2005; Kosche et al . , 2015 ) , which previously had been interpreted as evidence against a direct synaptic chain ( Armstrong and Abarbanel , 2016 ) , are not inconsistent with our estimate that each HVC ( RA ) neuron receives a significant amount of its excitatory input from other HVC ( RA ) neurons .",
"The reason is that with the around 200 homotypic inputs per cell , the probability to be connected to any one of around 40 , 000 HVC ( RA ) neurons can be at most 0 . 5% .",
"The question remains what fraction of those inputs are true ‘chain’ synapses in that the presynaptic cell’s activity immediately precedes that of the postsynaptic cell , but our study demonstrates that the anatomical substrate for the chain model exists .",
"An important next step will be to combine functional imaging with volume EM to directly test whether an HVC ( RA ) cell receives more numerous or stronger direct homotypic inputs from cells that fire immediately prior to its own activity .",
"In fact , a recent study describes how calcium activity can be imaged in the singing bird ( Picardo et al . , 2016 ) , a crucial step in that direction .",
"One potential difficulty stems from our finding that HVC ( RA ) neurons preferentially form distal connections , indicating that the timing circuitry in HVC is distributed and therefore requires a large EM volume ( as much as 500 million µm3 , compared to 2 million µm3 in our volume ) for its complete reconstruction .",
"It might take the better part of a year merely to acquire the raw data ( Schalek et al . , 2016 ) .",
"While even a few years ago it seemed impossible to analyze such an amount of data within a reasonable time frame , recent progress in the automation of segmentation are encouraging ( Berning et al . , 2015; Januszewski et al . , 2016; Beier et al . , 2017; Dorkenwald et al . , 2017 ) .",
"Our finding that connections near the soma are often onto inhibitory neurons suggests that inhibition plays an important role in sequence generation , which is further supported by the large overall fraction of inhibitory inputs .",
"One function of those inhibitory connections could be to decorrelate excitatory activity in space and time: Not only are nearby HVC ( RA ) neurons rarely connected and thus unable to drive each other , but even when driven by a common input , only the cell ( s ) with the strongest input ( s ) will continue to fire in the face of the winner-take-all effect due to the strong reciprocal inhibition ( Figure 3h ) .",
"Winner-take-all behavior is normally associated with certain cognitive tasks ( Hopfield and Tank , 1985; Lundqvist et al . , 2016 ) , such as decision making ( Usher and McClelland , 2001 ) .",
"In HVC , it may help to prevent local clusters of activity , which could lead to leakage across different chains passing through adjacent excitatory neurons .",
"An altogether different role for local inhibition may be the improvement of temporal precision by sharpening burst timing through recurrent inhibition ( Hahnloser et al . , 2002; Long et al . , 2010; Cannon et al . , 2015a ) .",
"Inhibition may , furthermore , have a central role in shaping the distance dependency of postsynaptic targets during circuit development without the need for molecular cues ( de Wit and Ghosh , 2016 ) .",
"Instead , the architecture we observed may arise naturally from a pattern that initially follows Peters' rule ( Braitenberg and Schüz , 1998 ) , which predicts synaptic connections between cell types with intermingled axonal and dendritic arbors ( Rees et al . , 2017 ) , but is then refined as the interneurons increasingly prevent the co-activation of nearby excitatory cells , thereby destabilizing connections between them while leaving more distant connections intact .",
"Such a preferentially distal connectivity would also favor more widely distributed synaptic chains , which could have the added benefit of relying more on axonal propagation delays for sequence timing ( Budd et al . , 2010 ) .",
"Overall , the observed synaptic architecture shows some resemblance with local inhibitory/excitatory networks linked by long-range excitatory/excitatory connections ( coupled winner-take-all modules ) that have been shown to make computational models of cortical sequence generation more robust ( Binas et al . , 2014; Mostafa and Indiveri , 2014 ) ."
],
[
"We used adult ( >90 days post hatch ) male zebra finches that were obtained from an outside breeder and maintained in a temperature- and humidity-controlled environment with a 12/12 hr light/dark schedule .",
"All animal maintenance and experimental procedures were performed according to the guidelines established by the Institutional Animal Care and Use Committee at the New York University Langone Medical Center .",
"To label only neurons that projected from HVC to the robust nucleus of the arcopallium ( RA ) , we injected lysine-fixable retrograde dextran tracers ( Invitrogen ) conjugated to either Tetramethylrhodamine ( fluoro-Ruby , mol . Weight: 10 , 000 ) or biotin ( BDA , mol . weight: 3000 ) for preparations to be inspected with light microscopy ( LM ) or electron microscopy ( EM ) , respectively .",
"We injected 200 nL of either Fluoro-Ruby ( 50 mg/mL ) or BDA ( 100 mg/mL ) into RA of anesthetized ( 1–3% isoflurane in oxygen ) zebra finches using an injection system ( Nanoject , Drummond Scientific , Broomall , PA ) outfitted with a glass injection pipette ( tip diameter: 30–40 µm ) .",
"RA was targeted using stereotaxic coordinates ( 2 . 30 mm lateral and 1 . 85 mm posterior from the midsagittal sinus ) and success in finding the RA region was confirmed by observing characteristic spontaneous activity ( Long and Fee , 2008 ) using a carbon-fiber electrode ( Carbostar-1 , Kation Scientific , Minneapolis , MN ) and an extracellular amplifier ( NPI Electronic Instruments , Germany ) .",
"For in vivo imaging and dye loading , we first had to enable optical access to HVC .",
"To accomplish this , a craniotomy ( 1 mm x 1 mm ) was prepared over HVC .",
"The underlying dura was then carefully removed with a flame sharpened tungsten wire ( starting diameter: 0 . 5 mm ) .",
"A small drop of saline buffer was applied to the exposed brain , followed by a 3 mm-diameter round cover glass ( #0 thickness , Warner Instruments , Hamden , CT ) as an optical window , which was first secured to the surrounding skull by applying light-curable acrylic ( Flow-IT ALC; Pentron Clinical Technologies ) around the edges of the glass .",
"Dental acrylic ( Cooralite Dental MFG , Diamond Springs , CA ) and cyanoacrylate were then added to permanently and stably attach the cover glass to the skull .",
"A small metal head plate with two tapped holes was then implanted at the anterior part of the skull using dental acrylic for head fixation .",
"Juxtacellular labeling ( Pinault , 1996; Narayanan et al . , 2015 ) with Neurobiotin ( Vector Labs , Burlingame , CA ) was used to fill individual RA-projecting HVC ( HVC ( RA ) ) neurons out of a population that had been retrogradely labeled from RA with fluoro-Ruby in vivo .",
"After waiting at least 48 hr following the injection of the retrograde tracer into RA , two-photon imaging ( Denk and Webb , 1990 ) was used to identify the target cell and guide the pipette .",
"On the day of single-cell labeling , a small pipette access hole ( ~400–500 µm ) was drilled in the glass coverslip immediately lateral to the target recording region using a carbide bur drill bit ( 1/4 FG-100; Johnson-Promident ) .",
"Glass pipettes were fabricated using a horizontal puller ( P97 , Sutter Instrument Company , Novato , CA ) and had a final resistance of 4–5 MΩ when loaded with internal solution that consisted of 150 mM K-Gluconate ( Sigma-Aldrich , St . Louis , MO ) and 3% Neurobiotin .",
"The microscope ( MOM , Sutter Instrument Company ) was of the moveable objective design ( Euler et al . , 2009 ) and was controlled using ScanImage ( Pologruto et al . , 2003 ) 3 . 8 with a 16x/0 . 8 NA water immersion objective ( Nikon , Japan ) .",
"Pipettes were made fluorescent either by adding 40 µM of Alexa 488 ( Invitrogen ) to the internal solution or by coating the pipette with green fluorescent quantum dots ( Andrásfalvy et al . , 2014 ) .",
"The activity of HVC ( RA ) neurons was recorded ( IR-183 , Cygnus Technology Inc , Delaware Water Gap , PA ) , and cells were filled with Neurobiotin by applying 1000–1500 positive current pulses with an amplitude between 3 and 15 nA and a duration of 200 ms delivered at a frequency of 2 . 5 Hz .",
"Birds were anesthetized with pentobarbital sodium and perfused transcardially with 4% w/v paraformaldehyde ( EMS ) at least one hour after dye loading to permit adequate Neurobiotin diffusion .",
"Brains were removed from the skull using a surgical scoop , immersed in 4% paraformaldehyde for 3–5 days to achieve thorough fixation , and incubated in phosphate buffer for an additional 1–3 days to decrease endogenous peroxidase activity .",
"To prepare sections , the brain was cut across the midline , mounted on the sagittal surface with cyanoacrylate , and stabilized with 3% agarose .",
"Parasagittal sections ( 100 µm thickness ) of HVC were cut using a vibratome ( Leica VT1000S ) .",
"Slices were washed five times with phosphate buffer and treated with 3% H2O2 to further reduce endogenous peroxidase activity .",
"Slices were then immersed overnight at 4°C in a solution containing avidin/biotin complexes and 0 . 5% Triton X-100 in phosphate buffer ( Vector Labs and Sigma-Aldrich , respectively ) to tag the Neurobiotin with peroxidase complex .",
"On the following day , slices were washed five times with phosphate buffer and then immersed in a solution containing 2 . 3 mM diaminobenzidine ( DAB , Sigma-Aldrich ) and 0 . 01% H2O2 in phosphate buffer to label processes containing Neurobiotin .",
"Slices were then washed and mounted on slides with Vectashield ( Vector Labs ) or Mowiol ( Sigma-Aldrich ) mounting medium .",
"To quantify the number of HVC-projecting RA neurons , we injected a retrograde tracer into HVC ( DiI , Invitrogen D3911; 46 nL total injection volume ) that labels neurons with high efficiency in zebra finches ( Scott et al . , 2012 ) .",
"Following a two-day incubation period , animals were perfused with 4% paraformaldehyde , and 100 µm sagittal sections were cut across the entirety of RA , Nucleus Interfacialis ( NIf ) , and nucleus Uvaeformis ( Uva ) .",
"Sections were mounted on slides using Vectashield ( Vector Labs ) and imaged with a confocal microscope ( LSM 800 , Zeiss , Germany; excitation / emission: 551/569 nm ) using a 20x objective ( 0 . 8 NA ) .",
"The z-stacks of retrogradely labeled RA ( HVC ) neurons were captured across the extent of RA , and the position of each cell was manually marked using the landmark function in Amira .",
"Only well-filled HVC ( RA ) neurons were selected for reconstruction , specifically those in which the soma , dendrite , and axon were all labeled ( even if faintly ) without interruptions and with clearly labeled dendritic spines and presynaptic boutons were selected for high resolution LM imaging with a custom-designed high-resolution mosaic/optical-sectioning brightfield microscope system ( Oberlaender et al . , 2007 ) .",
"In brief , a transmitted light brightfield microscope ( Olympus BX51 , Olympus , Japan ) , equipped with a motorized x-y-z stage ( Maerzhaeuser , Germany ) , a narrow bandpass ( 546 ± 5 nm ) illumination filter and a 100x magnification oil-immersion objective ( numerical aperture 1 . 4 ) was used to acquire image stacks from consecutive 100 µm thick brain sections .",
"For each section , a 3D mosaic of images ( e . g . , 10 × 15 fields of view ) covering the entire HVC was acquired at 92 × 92 nm pixel size and in steps of 500 nm mechanical defocus .",
"Next we applied a linear image restoration algorithm ( Tikhonow-Miller ) using the Huygens software package ( Scientific Volume Imaging , Netherlands ) .",
"By inverting the gray values of the brightfield image stacks they could be treated as fluorescent data with an emission wavelength of 546 nm .",
"The deconvolution used a point-spread-function that takes the optical properties of biocytin-labeled brain tissue into account ( Oberlaender et al . , 2009 ) .",
"Deconvolved image stacks were then downsampled by a factor of two in x/y , yielding a final voxel size of 184 × 184 × 500 nm before axonal reconstruction .",
"To quantify the bouton density , subvolumes that contained primarily horizontal ( i . e . within the image plane ) axonal branches were acquired at 200 nm focus increments and used without deconvolution .",
"Neuronal branches ( dendrites and axons ) were reconstructed in 3D using NeuroMorph ( Oberlaender et al . , 2007 ) .",
"Automated tracing results from each histological section were manually proof-edited using FilamentEditor ( Dercksen et al . , 2014 ) , custom-designed based on Amira visualization software ( FEI-VisualizationSciencesGroup ) .",
"In brief , maximum-intensity z-projections of the original image stacks were superimposed onto automatically generated 3D skeleton tracings of all putative neuronal branches contained within the imaged volume and segmented objects that had no correspondence in the projection image were manually deleted ( Dercksen et al . , 2014 ) .",
"Fragmented segments were spliced , and axonal branches were classified as ‘dendrite’ or ‘axon’ based on whether , respectively , spines or boutons were visible in the projection images .",
"Whenever a neuronal branch reached one of the borders of the imaged volume , additional image stack regions were acquired that allowed us to follow the branch further .",
"To account for shrinkage during histological processing , the reconstruction was scaled to match the thickness of 100 µm , as defined by the vibratome .",
"The scaled 3D tracings from all consecutive sections were then combined and manually aligned using the FilamentEditor .",
"The z-coordinate of each point was then replaced by the average of nine points ( the point itself and the four adjacent points in each direction ) and resampled to a point spacing of about 1 µm .",
"Smoothing in z and downsampling make path length measurements comparable to manual tracing results using Neurolucida Software ( Microbrightfield , Williston , VT ) .",
"The NeuroMorph and FilamentEditor tools enable tracings that are independent of the experience of the human operator , with an interuser-variability of approximately 20 µm per 1 mm axonal length ( Dercksen et al . , 2014 ) .",
"The borders of HVC were manually traced in each 100 µm tissue section using Neurolucida .",
"The fraction of dendritic length contained within a certain distance of the soma was determined by conducting a spherical Sholl analysis ( Sholl , 1953 ) in Neurolucida ( Microbrightfield ) .",
"The proportion of axonal pathlength both within HVC and within a 200 µm radius from the soma was computed in Amira for each neuron using the ZIB extension package ( Egger et al . , 2014 ) .",
"Axonal boutons and dendritic spines were annotated manually in Amira using high-resolution LM stacks .",
"The location of each bouton or spine was marked in 3D and aligned in Amira to the corresponding branch reconstruction .",
"Spine-densities were calculated for each branch by dividing its total spine count by its path length .",
"Branch nodes ( points where the axon bifurcates ) were manually located in the reconstructions using Amira .",
"Branch nodes for which one of the daughter branches was <15 µm in length were not included in this analysis .",
"The bird used for the EM experiments was transcardially perfused in a way that preserves the extracellular space and leads to minimal shrinkage ( JK , unpublished observations ) , by using high pressure and the following fixative solution: 0 . 07 M sodium cacodylate ( Serva , Germany ) , 140 M sucrose ( Sigma-Aldrich ) , 2 mM CaCl2 ( Sigma-Aldrich ) with 2% paraformaldehyde and 2% glutaraldehyde ( Serva ) added ( Cragg , 1980 ) .",
"The brain was removed and , using a vibratome ( Leica VT1000S ) , cut into slices each about 200 µm thick .",
"One of the slabs that centrally intersected HVC was selected and post-fixed in the same solution overnight , rinsed several times with cacodylate buffer and permeabilized in a 30% sucrose solution by exposing it to one freeze-thaw cycle in liquid nitrogen .",
"Residual peroxidase activity was suppressed by soaking the sample in 3% H2O2 for 30 min before labeling the sample with an avidin-peroxidase complex and DAB , as described in a previous section .",
"The sample was then rinsed several times in cacodylate buffer .",
"Heavy metal staining was added through a conventional ROTO protocol using the following steps interspersed with rinses in cacodylate buffer ( after first Osmium step ) or H2O ( all others ) : 2% OsO4 ( Serva ) , reduced with 2 . 5% potassium hexacyanoferrate ( II ) ( Sigma-Aldrich ) 2 hr , room temperature; 1% thiocarbohydrazide in H2O , 1 hr , 58°C ( Sigma-Aldrich ) ; 2% OsO4 , 2 hr; 1 . 5% uranyl-acetate in H2O , 53°C ( Serva ) ; 20 mM lead-aspartate , 2 hr , 53°C ( Sigma-Aldrich ) ( Seligman et al . , 1966; Karnovsky , 1971; Walton , 1979 ) .",
"Dehydration was performed using an ethanol series with 10 , 15 , 10 , 10 min at 70% , 100% , 100% , and 100% ethanol ( Electron Microscopy Sciences ) .",
"The sample was infiltrated with epoxy monomer ( epon hard , Serva ) ( Glauert and Lewis , 2014 ) dissolved in propylene oxide ( Sigma-Aldrich ) for 3 hr and for 3 hr with pure monomer before final embedding and curing ( 48 hr at 60°C ) .",
"The sample was then trimmed and glued with epoxy to a custom-made aluminum holder and trimmed into a pyramidal-shape before gold coating for better conductivity .",
"We performed serial block-face electron microscopy ( Denk and Horstmann , 2004 ) at 11 × 11 × 29 nm voxel size using a scanning electron microscope with a field-emission cathode ( UltraPlus , Zeiss , Germany ) equipped with a custom-built in-chamber microtome in high-vacuum ( raw and effective voxel rates were 5 and 2 . 1 MHz respectively ) at a dose of 10 . 3 electrons/nm2 , 2 kV landing energy with a custom back-scatter electron detector and amplification system optimized for fast acquisition speeds .",
"Before each cut , a subregion of the block face was imaged using four overlapping micrographs resulting in an image stack .",
"Images were registered by affine transformations ( https://github . com/billkarsh/Alignment_Projects ) ( Scheffer et al . , 2013; Karsh , 2016 ) and converted to a KNOSSOS ( www . knossostool . org ) data set for reconstruction and browsing with custom Python code ( https://github . com/knossos-project/knossos_python_tools/tree/master/knossos_cuber ) ( Kornfeld , 2017b ) .",
"Copies of the software are archived at https://github . com/elifesciences-publications/Alignment_Projects and https://github . com/elifesciences-publications/knossos_utils ) .",
"Each annotator received at least 10 hr of training and was considered an expert after one year of annotation experience .",
"BDA-labeled neurons , using the soma as a starting place , were skeletonized within the EM stack in KNOSSOS by an expert annotator , and errors were corrected by the same individual in a second pass , which was also used for synapse annotation .",
"All BDA-labeled axons , including orphaned axons , were traced by at least two independent annotators and discrepancies were resolved by an expert that had not participated in the initial annotation .",
"Synapses on each axon were then labeled ( see synapse identification ) and proofread by an expert annotator who excluded cases where the BDA-label obscured the ultrastructure .",
"The remaining synapses were used to seed the tracing of the postsynaptic dendrite segment .",
"Annotators were instructed to reconstruct the postsynaptic dendrite to the end of the branch in one direction and to the next main branch point in the other direction .",
"All dendritic-branch tracings were proofread by an expert and only included if at least a minimum path length of 10 µm could be reconstructed .",
"All EM reconstructions were analyzed and visualized with custom Python code using the Mayavi2 ( Enthought ) library ( Kornfeld , 2017a , Kornfeld , 2017b ) .",
"Synapses were labeled by an expert annotator and classified as symmetric or asymmetric ( Videos 4 and 5 , Figure 1—figure supplement 3 ) .",
"Active-zone’ diameters ‘were quantified by measuring - with KNOSSOS - the cross-sectional length of the synaptic thickening in that plane and principal viewing orientation ( x , y , or z ) in which the contact cross section appeared largest .",
"Diameters were then converted to areas by assuming a circular synaptic contact .",
"To estimate dendritic spine density , a stretch of the postsynaptic dendrite ( >10 µm ) was selected that often included the place where the axon was in contact with the dendrite .",
"We counted as a spine every skeleton branch with a length greater than 1 µm that emerged from the dendritic shaft .",
"Some postsynaptic protrusions found on interneurons contained multiple synapses ( e . g . , Figure 2—figure supplement 1b ) .",
"Therefore , spines were defined as receiving no more than one synapse at their ends by three independent annotators .",
"The resulting spine density Dspine ( in µm−1 ) was used to classify the dendritic stretch as belonging to an interneuron ( Dspine < 0 . 11 ) , HVC ( RA ) ( 0 . 11 < Dspine < 0 . 46 ) , or HVC ( X ) neuron ( 0 . 46 < Dspine ) .",
"To detect dendritic reconstructions that were traced from separate synapses but belonged to the same dendrite , we detected overlap between skeletons using the following criterion: a node was considered to overlap another skeleton if it was less the 400 nm from any edge of all other skeletons .",
"Dendritic reconstructions were defined as belonging to the same neuron when at least 25% of their nodes overlapped .",
"Since the postsynaptic dendritic reconstructions were never complete ( i . e . only parts of the entire neuron could be reconstructed ) , our analysis could only positively identify reconstructions as belonging to the same cell .",
"For dendrites that were found to belong to the same cell ( grouped together after being traced from different synapses ) , spine density was averaged before classification .",
"We used two different ways to estimate the distance between an orphaned branch and its soma from its number of branch nodes inside the EM volume , both based on the LM observation that the density of branch nodes , Db , varies with soma distance ( r ) ( Figure 3c ) .",
"The first way used a Bayesian approach to calculate the probability distribution over r , given a branch of length l and a branch-node count of N ( Figure 3c ) , which can be used to estimate , as needed , mean , median , variance or any quantile for r:P ( r|N , l ) ∝P ( N , l|r ) ∗Pa ( r ) , wherebyP ( N , l|r ) = ( Db ( r ) *l ) N*e−Db ( r ) *lN ! , which assumes that the branch nodes are placed independently from each other and are , therefore , Poisson distributed with a node-count expectation value of λ=Db ( r ) *l .",
"Fitting the LM measurements to an exponential gave Db ( r ) = ( 35 . 448*e−r43 . 5mm+0 . 613 ) mm .",
"The Bayesian prior , Pa ( r ) , i . e . the probability that an axon segment is found at a distance between r and r±� from its soma , was estimated by applying Gaussian kernel density estimation ( Python scipy . stats . gaussian_kde , scott bandwidth selector ) to the LM based axon distribution measurements .",
"The other way to relate r to N and l is to sample the LM data directly: We divided each of the 15 LM stacks into volumes shaped identically to the EM volume and recorded for each volume and for all contained orphaned branches their lengths , distances from the soma , and branch-node counts .",
"Only branches that both entered and left the sampled subvolume were considered ( about 95% of the total ) because all of the reconstructed orphaned branches in the EM volume also had that property .",
"This was repeated with the origin of the division grid shifted in 10 µm increments along all three axes resulting in 17 × 17 × 8 different divisions for each LM stack .",
"For a given orphaned branch in the EM volume , we selected all those sample branches that had the same node count and a length within ± 10% .",
"The distribution of their soma distances was then used in the same way as the probability distribution coming from the Bayesian approach .",
"In order to estimate the homotypic fraction of all excitatory synapses onto HVC ( RA ) cells , we determined the density of homotypic synapses by counting the number of double labeled synapses and correcting it for the axonal labeling efficiency .",
"Labeling efficiency was estimated by comparing the volume density of labeled axon length by inspecting 300 randomly placed 1 µm3 cubes with the density expected for HVC ( RA ) neurons using published estimates for their total number ( Wang et al . , 2002 ) and the average axonal path length from LM reconstructions .",
"To count the number of double labeled synapses , BDA-labeled dendrites were searched by an expert annotator for synapses with labeled axons by following them in KNOSSOS at the full voxel resolution , instructed to annotate also synapses with weak labeling .",
"The found synapses were then scrutinized by JK and the result was confirmed by ML and SB .",
"All error estimates were calculated assuming independence of the errors using the variance formula for error propagation ."
]
] | [
"The sequential activation of neurons has been observed in various areas of the brain , but in no case is the underlying network structure well understood .",
"Here we examined the circuit anatomy of zebra finch HVC , a cortical region that generates sequences underlying the temporal progression of the song .",
"We combined serial block-face electron microscopy with light microscopy to determine the cell types targeted by HVC ( RA ) neurons , which control song timing .",
"Close to their soma , axons almost exclusively targeted inhibitory interneurons , consistent with what had been found with electrical recordings from pairs of cells .",
"Conversely , far from the soma the targets were mostly other excitatory neurons , about half of these being other HVC ( RA ) cells .",
"Both observations are consistent with the notion that the neural sequences that pace the song are generated by global synaptic chains in HVC embedded within local inhibitory networks ."
] | [
"For us to interact with the world around us , our brains must plan and execute our movements and behaviors .",
"For instance , although speaking is often quite effortless , it is also remarkably complex; all of the muscles in our vocal cords have to be activated at just the right moments to create words .",
"It remains poorly understood how exactly the brain generates such precise timing signals that enable these movements .",
"A specific portion of the songbird brain allows the bird to sing its song , a process that has clear parallels with human speech .",
"Previous work had demonstrated that this region in the bird’s brain acts as a ‘clock’ for singing behavior , with individual brain cells active at just a single moment , or ‘tick’ .",
"Little consensus had been reached concerning how this might be achieved .",
"Kornfeld , Benezra et al . have now used new anatomical methods to better understand how the songbird clock works .",
"A technique called 3D electron microscopy allowed the connections between the neurons in the clock brain region to be seen directly .",
"This revealed that these neurons form direct connections with each other , which is consistent with the idea that one ‘tick’ can lead to the next and so on , like a series of falling dominoes .",
"Several mysteries remain to be resolved by future research .",
"First , the connections that Kornfeld , Benezra et al . found are between cells that are quite distant from each other .",
"This arrangement is fundamentally different from many other brain areas where neighboring cells are thought to work together .",
"Second , although these key brain cells form appropriate connections to act as a clock , it is still not clear whether and how the network uses these connections during singing .",
"By resolving these mysteries , we will establish a new framework for understanding how the brain encodes learned motor gestures that may help to spur innovative new approaches for combatting motor-related deficits due to injury or disease ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"plant biology"
] | Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling | elife-17061-v1 | [
[
"Abiotic stress is a major threat to global crop yields and this problem is likely to be exacerbated in the future .",
"A large body of research has focused on the immediate stress responses .",
"However , in nature , stress is frequently chronic or recurring , suggesting that temporal dynamics are an important , but under-researched , component of plant stress responses .",
"Indeed , plants can be primed by a stress exposure such that they respond more efficiently to another stress incident that occurs after a stressless period ( Hilker et al . , 2015 ) .",
"Priming has been described in response to pathogen attack , heat stress ( HS ) , drought , and salt stress ( Charng et al . , 2006; Conrath , 2011; Jaskiewicz et al . , 2011; Ding et al . , 2012; Sani et al . , 2013 ) .",
"Such stress priming and memory may be particularly beneficial to plants due to their sessile life style .",
"Chromatin structure can be modulated by nucleosome positioning , histone variants and posttranslational histone modifications that together control the access of sequence-specific transcription factors and the general transcription machinery to gene loci ( Struhl and Segal , 2013; Zentner and Henikoff , 2013 ) .",
"Chromatin mediates long-term stability of environmentally and developmentally-induced gene expression states ( Gendrel and Heard , 2014; Steffen and Ringrose , 2014; Berry and Dean , 2015 ) .",
"Hence , the modification of chromatin structure has been suggested to mediate the priming and memory of stress-induced gene expression .",
"Indeed , the above mentioned cases of plant stress priming are associated with lasting histone H3 methylation ( Conrath , 2011; Jaskiewicz et al . , 2011; Ding et al . , 2012; Sani et al . , 2013; Lämke et al . , 2016 ) .",
"However , the underlying mechanism and the contribution of other determinants of chromatin structure such as nucleosome positioning and occupancy remain unknown .",
"Moderate HS allows a plant to acquire thermotolerance and subsequently withstand high temperatures that are lethal to a plant in the naïve state ( Mittler et al . , 2012 ) .",
"After returning to non-stress temperatures , acquired thermotolerance is maintained over several days , and this maintenance phase is genetically separable from the acquisition phase ( Charng et al . , 2006 , 2007; Meiri and Breiman , 2009; Stief et al . , 2014 ) .",
"We refer to this maintenance phase as HS memory .",
"The immediate HS response ( acquisition phase ) involves the activation of heat shock transcription factors ( HSFs ) that induce heat shock proteins ( HSPs ) .",
"Their chaperone activity ensures protein homeostasis ( Scharf et al . , 2012 ) .",
"The HS response is conserved in animals , plants and fungi ( Richter et al . , 2010 ) .",
"Among the 21 HSFs in Arabidopsis thaliana ( Scharf et al . , 2012 ) , only HSFA2 is specifically required for HS memory ( Charng et al . , 2007 ) .",
"It activates HEAT-STRESS-ASSOCIATED32 ( HSA32 ) , a gene with chaperone-like activity , although no homology to known chaperone families ( Wu et al . , 2013 ) .",
"Like HSFA2 , HSA32 is critically required for HS memory ( Charng et al . , 2006 ) .",
"HSA32 is induced by HS and this induction is sustained for at least three days .",
"A set of HS memory-related genes was identified based on their similar expression pattern , which is in contrast to that of canonical HS-inducible genes ( HSP70 , HSP101 ) that show upregulation after HS , but not sustained induction ( Stief et al . , 2014 ) .",
"Among the HS memory-related genes are small HSPs ( such as HSP21 , HSP22 . 0 , HSP18 . 2 ) .",
"A subset of these loci show transcriptional memory in the sense that recurring stress causes a more efficient re-activation compared to the first stress incident , even though active transcription has subsided before the second stress ( Lämke et al . , 2016 ) .",
"Sustained induction and transcriptional memory of these genes is associated with hyper-methylation of H3K4 ( H3K4me2 and H3K4me3 ) and requires HSFA2 , which binds directly to these genes ( Lämke et al . , 2016 ) .",
"Interestingly , HSFA2 dissociates from these loci before its requirement becomes apparent at the physiological and gene expression levels , thus implicating the existence of additional factors .",
"Here , we report the identification of FGT1 from an unbiased screen for factors that are required for the sustained induction of HSA32 .",
"FGT1 is required for HS memory at the physiological and gene expression levels .",
"FGT1 is the single A . thaliana orthologue of metazoan Strawberry notch , a highly conserved co-activator of the developmental regulator Notch .",
"We show that FGT1 associates with memory genes in a HS-dependent way .",
"Moreover , FGT1 is widely associated with the transcriptional start site of expressed genes .",
"We further show that FGT1 interacts with highly conserved chromatin remodeling complexes and is required for proper nucleosome dynamics at HS-memory genes .",
"Thus , FGT1 maintains its target loci in an open and transcription-competent state by interacting with remodeler complexes around the transcriptional start site ."
],
[
"In order to identify regulators of HS memory we generated a transgenic HSA32::HSA32-LUCIFERASE ( HSA32::HSA32-LUC ) reporter line .",
"LUC expression in this line was induced by HS and expression remained high for at least 3 d ( Figure 1A ) , thus mimicking expression of the endogenous HSA32 ( Charng et al . , 2006 ) .",
"We mutagenized the HSA32::HSA32-LUC line with ethyl methanesulfonate and screened M2 families for mutants with modified maintenance of LUC activity after HS .",
"To this end , 4 d-old plate-grown seedlings were treated with an acclimatizing heat treatment ( ACC , see Materials and methods ) .",
"LUC-derived bioluminescence was monitored 1 , 2 and 3 d later , and putative mutants were isolated that had normal LUC activity 1 d after ACC and reduced activity 3 d after ACC .",
"Among the recovered mutants with such a LUC expression profile was forgetter1 ( fgt1-1 ) , on which we focused further analyses .",
"LUC expression in fgt1-1 was induced normally , however , it declined precociously , which was most apparent 3 d after ACC . 10 . 7554/eLife . 17061 . 003Figure 1 . FGT1 is required for HS memory and sustained induction of memory genes in A . thaliana .",
"( A ) fgt1-1 displays normal induction but reduced maintenance of pHSA32::HSA32-LUC expression .",
"Bioluminescence of fgt1-1 or the parent assayed 1 , 2 , or 3 d after an acclimatizing HS ( ACC ) .",
"The color scale of relative LUC activity is shown .",
"( B ) fgt1-1 is impaired in HS memory at the physiological level .",
"Seedlings of the indicated genotypes ( cf . fgt1-1#2 in C–D ) were acclimatized 4 d after germination and treated with a tester HS 2 or 3 d later .",
"Pictures were taken after 14 d of recovery .",
"( C–D )",
"Quantification of the data shown in ( B ) .",
"The fgt1-1 lines represent independent backcrosses .",
"Data are averaged over at least two independent assays ( n>36 ) .",
"Fisher’s exact test , *p<0 . 05; **p<0 . 001 .",
"( E ) Transcript levels of HS memory genes after ACC decline prematurely in fgt1-1 .",
"Expression values were normalized to the reference At4g26410 and the corresponding no-HS control ( NHS ) .",
"Data are averages and SE of two biological replicates .",
"*p<0 . 05; **p<0 . 01 ( Student’s t test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 00310 . 7554/eLife . 17061 . 004Figure 1—figure supplement 1 . fgt1-1 is not affected in the acquisition of thermotolerance .",
"( A ) Acquisition of thermotolerance in Col-0 , parent , fgt1-1 and the HS response-deficient hsp101 was assayed by treating 5 d-old seedlings at 37°C for 60 min , recovery for 90 min at 22°C , followed by 44°C for the indicated times .",
"Pictures were taken 13 d after heat treatments .",
"All plants of one treatment were grown on the same plate .",
"One representative of at least three biological replicates is shown .",
"( B ) Quantification of the data shown in ( A ) .",
"Seedlings were categorized 13 d after heat treatments .",
"Fisher’s exact test , **p<0 . 001; n>30 . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 00410 . 7554/eLife . 17061 . 005Figure 1—figure supplement 2 . fgt1-1 has normal basal thermotolerance . Basal thermotolerance in parent , fgt1-1 , and hsp101 was assayed by incubating 4 d-old seedlings for the indicated times at 44°C .",
"Pictures were taken 14 d after HS .",
"All plants of one treatment were grown on the same plate .",
"One representative of at least three biological replicates is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 00510 . 7554/eLife . 17061 . 006Figure 1—figure supplement 3 . Additional qRT-PCR analyses of HS genes . Transcript levels of the indicated genes ( unspliced where indicated ) were determined by qRT-PCR .",
"Expression values were normalized to the reference At4g26410 and the corresponding no-HS control ( NHS ) .",
"Data are averages and SE of two biological replicates .",
"*p<0 . 05; **p<0 . 01 ( Student’s t test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 006 We next investigated whether this correlated with modified HS memory at the physiological level by applying a tester HS 2 or 3 d after ACC .",
"The tester HS is lethal to a naïve plant or a mutant with loss of HS memory .",
"Indeed , fgt1-1 mutants displayed reduced growth and survival under these conditions ( Figure 1B–D ) .",
"To check whether fgt1-1 mutants had a generally impaired HS response , we also tested fgt1-1 seedlings for acquisition of thermotolerance and for basal thermotolerance ,",
"i . e .",
"the amount of heat that can be tolerated without prior acclimation .",
"fgt1-1 mutants behaved very similar to the parental line in these assays ( Figure 1—figure supplements 1 , 2 ) , indicating that the immediate responses to HS were not affected in fgt1-1 .",
"Thus , fgt1-1 is specifically impaired in HS memory .",
"Notably , fgt1-1 did not have any obvious morphological alterations under standard growth conditions .",
"We next examined whether the premature decline of LUC expression mimicked that of the endogenous HSA32 gene by quantitative RT-PCR ( qRT-PCR ) specific for the endogenous HSA32 .",
"HSA32endo and LUC transcripts were induced normally in fgt1-1 during the first day after ACC , but declined faster thereafter ( Figure 1E , Figure 1—figure supplement 3 ) .",
"A similar defect was observed for the HS memory-related genes HSP21 , HSP22 . 0 and HSP18 . 2 , but not the HS-inducible non-memory genes HSP101 and HSP70 .",
"For intron-containing genes , we measured unspliced transcripts as a proxy for transcriptional activity .",
"Unspliced HSA32 transcripts were induced in fgt1-1 similarly as in the parent up to 21 h , but declined faster thereafter .",
"Notably , unspliced transcript levels in the parent were still elevated 20-fold relative to the non-HS control ( NHS ) at 69 h after ACC , indicating continued transcription throughout the memory phase .",
"Similar results were obtained for HSP21 , but not for the non-memory genes .",
"This is in accordance with what was observed previously ( Lämke et al . , 2016 ) .",
"Thus , FGT1 is required to facilitate sustained transcription of HS memory genes after HS .",
"To identify the molecular lesion underlying the fgt1-1 mutant phenotype , we combined recombination breakpoint mapping with Illumina sequencing .",
"We identified a 0 . 77 MB interval at the bottom of chromosome 1 , containing a splice acceptor site mutation in At1g79350 ( Figure 2A ) .",
"This mutation caused retention of intron 19 , resulting in a premature stop codon .",
"At1g79350 was previously tentatively identified as EMB1135 with a reported embryo-defective phenotype ( Meinke et al . , 2008 ) .",
"However , it was not confirmed that the disruption of this gene in the emb1135 allele indeed causes the phenotype and neither fgt1-1 , nor any of several putative loss-of-function T-DNA insertion lines showed any obvious morphological phenotype .",
"Three independent lines of evidence show that FGT1 is At1g79350 .",
"First , we complemented the LUC expression and physiological memory phenotypes by expressing a genomic FGT1 fragment ( SA13 ) in the fgt1-1 background ( Figure 2B–D ) .",
"Second , similar results were obtained for a FGT1-YFP fusion protein that was driven by the constitutive 35S CaMV promoter ( Figure 2—figure supplement 1A , B ) .",
"Finally , fgt1-2 and fgt1-3 , two putative loss-of-function T-DNA alleles displayed reduced HS memory ( Figure 2—figure supplement 1C–E ) . 10 . 7554/eLife . 17061 . 007Figure 2 . FGT1 encodes the A . thaliana orthologue of Drosophila Sno and binds histones .",
"( A ) Gene model of FGT1 ( At1g79350 ) with domains and location of mutations; exons ( grey and colored bars ) ; black line , intron .",
"fgt1-1 has a C to T mutation at the splice acceptor site of intron 19/exon 20 .",
"( B–D )",
"Complementation of fgt1-1 by a genomic FGT1 fragment ( SA13 ) .",
"( B ) pHSA32::HSA32-LUC-derived bioluminescence of indicated genotypes assayed 1 , 2 , or 3 d after ACC .",
"( C , D )",
"Seedlings of the indicated genotypes were acclimatized 5 d after germination and received a tester HS 3 d later .",
"( C ) Quantification; n = 48–49 , Fisher’s exact test , **p<0 . 01 .",
"( D ) Representative picture taken after 14 d recovery .",
"( E ) FGT1 transcript levels increase transiently after ACC .",
"Relative FGT1 transcript levels were determined by qRT-PCR and normalized to At4g26410 .",
"Errors are SE of two biological replicates .",
"( F ) FGT1 is localized to the nucleus in 3 d-old seedling roots .",
"35S::FGT1-YFP transgenic seedlings were imaged for YFP fluorescence .",
"Left , overlay; middle , bright field; right , YFP fluorescence .",
"Scale bar , 40 µm .",
"( G ) FGT1 binds histone H3 in vivo .",
"Nuclear protein extracts of transgenic 35S::FGT1-YFP , 35S::YFP and non-transgenic Col-0 seedlings harvested 28 h after the indicated treatments were immuno-precipitated with anti-GFP antibody .",
"Co-purification of histone H3 was assessed by immunoblotting . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 00710 . 7554/eLife . 17061 . 008Figure 2—figure supplement 1 . Complementation of the fgt1-1 mutant phenotype during HS memory .",
"( A ) pHSA32::HSA32-LUC-derived bioluminescence of the parent , fgt1-1 , fgt1-1 35S::FGT1-YFP ( two independent transgenic lines ) was assayed 1 , 2 , or 3 d after an acclimatizing HS ( ACC ) .",
"Blue indicates low LUC activity , red indicates high activity ( cf . Figure 1A ) .",
"The signal threshold was adjusted to the signal of the parental line .",
"( B ) Complementation of the fgt1-1 mutant by 35S::FGT1-YFP at the physiological level .",
"5 d-old seedlings of the indicated genotypes were acclimatized and received a tester HS of the indicated length 3 d later .",
"Seedlings were categorized after 10 d recovery at normal growth temperatures .",
"Asterisks indicate ( Fisher’s exact test ) *p<0 . 05; **p <0 . 01; n .",
"s .",
", not significant; n = 32–38 .",
"( C ) fgt1-2 and fgt1-3 display reduced HS memory at the physiological level .",
"The assay was performed as described in ( B ) .",
"( D ) fgt1-2 displays reduced pHSA32::HSA32-LUC-derived bioluminescence after ACC .",
"The assay was performed as described in ( A ) .",
"( E ) Characterization of fgt1-2 and fgt1-3 mutants .",
"Top panel: Genotyping using 3-primer PCR confirms mutants are homozygous for the T-DNA insertion .",
"Bottom panel: FGT1 expression as determined by semi-quantitative RT-PCR in the indicated genotypes . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 00810 . 7554/eLife . 17061 . 009Figure 2—figure supplement 2 . FGT1 is highly conserved and encodes the SNO/SBNO orthologue of A . thaliana . Phylogenetic tree generated using the www . phylogeny . fr website with the preset 'one-click' settings .",
"Sno/SBNO/FGT1 sequences were identified using BLAST searches with the full-length FGT1 protein .",
"A . thaliana MDB9 and ATXR6 were used as outgroups as they displayed the highest hits from A . thaliana in a BLAST against full-length FGT1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 00910 . 7554/eLife . 17061 . 010Figure 2—figure supplement 3 . The PHD domain of FGT1 binds to histone H3 in vitro . Recombinant FGT1PHD-GST was immunoprecipitated with Flag-tagged histone peptides and detected by anti-GST immunoblotting .",
"ING1PHD-GST and GST were used as controls . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 010 FGT1 contains an ATP-binding DExD/H-like helicase domain , a Helicase C-like domain , and a PHD finger ( Figure 2A ) .",
"FGT1 is a single copy gene in A . thaliana .",
"Interestingly , it is highly homologous over the whole length of the protein with Sno from Drosophila melanogaster , human SBNO1 and SBNO2 , and Caenorhabditis elegans let-765 ( Figure 2—figure supplement 2 ) ( Majumdar et al . , 1997; Simms and Baillie , 2010; Grill et al . , 2015 ) .",
"Sno genes are required for the expression of Notch and EGFR target genes and it has been hypothesized that they interact with co-activator proteins to spatio-temporally regulate transcription ( Tsuda et al . , 2002 ) , yet no molecular mode of action has been demonstrated .",
"Although DExD helicases have been ascribed a role in RNA processing and translation , roles in gene expression and transcription have been suggested ( Fuller-Pace , 2006 ) .",
"FGT1 is expressed throughout the plant ( Winter et al . , 2007 ) and was slightly induced ( 1 . 7 fold ) at 4 h after HS , but not thereafter ( Figure 2E ) .",
"We next tested the subcellular localization of the complementing FGT1-YFP fusion protein in roots of 3 d-old stably transformed seedlings .",
"FGT1-YFP was localized to the nucleus ( Figure 2F ) .",
"PHD domains have the potential to bind to methylated histone H3 tails ( Musselman and Kutateladze , 2011 ) .",
"We thus tested whether FGT1PHD-GST was precipitated by H3 histone tail peptides that were either unmethylated or mono- , di- , or trimethylated , respectively ( Figure 2—figure supplement 3 ) .",
"We observed comparable binding to H3 aa 1–20 , H3 aa 21–44 or H3 aa 1–20 methylated at K4 or K9 .",
"In contrast , the PHD domain of ING1 ( Lee et al . , 2009 ) bound under the same conditions only to the H3K4me3 peptide ( Figure 2—figure supplement 3 ) .",
"This suggests that FGT1-PHD binds to the N-terminal region of H3 , albeit not in a methylation-specific manner ( at least with respect to methylated K4 and K9 ) .",
"In addition , histone H3 was co-immunoprecipitated with FGT1-YFP , but not YFP alone , from extracts of transgenic A . thaliana seedlings ( Figure 2G ) .",
"In summary , nuclear FGT1 is the A . thaliana orthologue of the DExD helicase Sno and associates with H3 in vivo , consistent with a function as a co-activator .",
"Given its potential function as a co-activator , we next asked whether FGT1 binds directly to its putative target genes during HS memory .",
"To test this , we performed chromatin immunoprecipitation ( ChIP ) followed by qPCR analysis on 35S::FGT1-YFP plants ( Figure 3 ) .",
"FGT1 bound to a broad region around the transcriptional start sites ( TSS ) of HSA32 , HSP18 . 2 , and HSP22 . 0 .",
"The enrichment of FGT1 in the heat-treated samples was highest 4 h and 28 h after ACC compared to the NHS , and was still present at 52 h .",
"Such heat-dependent enrichment was not observed at the ACTIN7 ( ACT7 ) and AtMu1 control loci .",
"The enrichment at the active ACT7 gene was comparable to that of HSA32 before HS , suggesting that FGT1 binds HSA32 and ACT7 already pre-HS .",
"The ChIP-signal in FGT1-YFP plants at bound loci was strongly enhanced compared to non-transgenic control samples ( Figure 3—figure supplement 1A ) .",
"A comparable but overall weaker binding pattern was observed for a FGT1-YFP driven by the endogenous promoter ( Figure 3—figure supplement 1B , C ) , indicating that the observed binding pattern does not result from FGT1 overexpression .",
"Thus , FGT1 binds to memory genes in a region encompassing the TSS and proximal promoter , where it may mediate their sustained expression after HS . 10 . 7554/eLife . 17061 . 011Figure 3 . FGT1 binds memory-associated genes in a HS-dependent manner . FGT1 binds to HSA32 , HSP18 . 2 and HSP22 . 0 .",
"ChIP-qPCR on 35S::FGT1-YFP seedlings was performed 4 , 28 or 52 h after an acclimatizing HS ( ACC ) or no HS ( NHS ) .",
"As controls , the active gene ACT7 or the inactive transposon AtMu1 were used .",
"Schematics show regions analyzed relative to TSS .",
"HSA32 1–4: −175 , −75 , +57 , +194 bp .",
"HSP18 . 2 1–3: −1068 , −367 , +32 bp .",
"HSP22 . 0 1–2: −3000 , −60 bp; ACT7: +55 bp .",
"AtMu1: -175 bp relative to ATG .",
"Amplification values were normalized to input and region 2HSA32 at 28 h NHS ( HSA32 , AtMu1 and ACT7 ) , region 2HSP18 . 2 at 28 h NHS or region 3HSP22 . 0 at 28 h NHS , respectively .",
"Data are averages of at least three biological replicates .",
"Error bars indicate SE . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 01110 . 7554/eLife . 17061 . 012Figure 3—figure supplement 1 . FGT1-YFP expressed from the endogenous promoter binds to HSA32 . ( A ) FGT1-YFP derived from 35S::FGT1-YFP binds the HSP18 . 2 locus already before HS as determined by ChIP-qPCR on 35S::FGT1-YFP or Col-0 seedlings .",
"4 d-old seedlings were subjected to an acclimatizing HS ( ACC ) , or no HS ( NHS ) .",
"ChIP-qPCR was performed at the indicated times after the treatments .",
"Schematic shows positions of regions analyzed .",
"Data show one representative experiment .",
"Error bars are SD of three technical replicates .",
"( B ) FGT1-YFP expressed from the FGT1 promoter binds to HSA32 as determined by ChIP-qPCR .",
"FGT1 enrichment around the TSS of HSA32 ( region 1 ) increases after ACC and remains elevated for at least 28 h .",
"Amplification values were normalized to input and the region 2 at 28 h NHS .",
"Error bars are SE of two biological replicates .",
"( C ) The pFGT1::FGT1-YFP transgene complements the HSA32-LUC phenotype of fgt1-1 .",
"HSA32::LUC-derived bioluminescence of the indicated genotypes was assayed 1 , 2 , or 3 d after an acclimatizing HS .",
"Blue indicates low LUC activity , red indicates high activity ( cf . Figure 1A ) .",
"The signal threshold was adjusted to the signal of the parental line . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 012 Because of the highly conserved nature of FGT1 and the binding to ACT7 , we suspected that FGT1 may have genomic targets beyond the tested candidates .",
"To obtain a global view , we performed ChIP-seq on 35S::FGT1-YFP plants 28 h after ACC or NHS .",
"Peak calling identified 942 ( 60 ) genes with FGT1 enrichment after ACC ( NHS ) , and no binding in the corresponding non-transgenic control samples .",
"While the NHS peaks remained associated with FGT1 after ACC , the ACC peaks were overall less strongly enriched under NHS conditions ( Figure 4—figure supplement 1 ) .",
"Coverage profiling of the FGT1-associated genes indicated that FGT1 bound primarily to the proximal promoter just upstream of the TSS and somewhat more weakly to the region downstream of the transcription termination site ( TTS ) .",
"In contrast , the signal was very low in the transcribed region .",
"For both conditions ( ACC and NHS ) , FGT1-associated genes showed a higher expression in seedlings under normal growth conditions compared to non-target genes ( Figure 4A ) , suggesting that FGT1 binding is positively correlated with transcription . 10 . 7554/eLife . 17061 . 013Figure 4 . FGT1 globally binds expressed genes upstream of the TSS .",
"( A ) FGT1-associated genes ( ChIP-seq peaks ) are more highly expressed than other genes .",
"Violin plot indicates expression levels of FGT1-bound genes compared to all other genes ( Figure 4—figure supplement 1 ) .",
"Expression data were taken from ( Gan et al . , 2011 ) .",
"( B ) Normalized global read coverages of ACC or NHS FGT1-YFP or wild-type control samples as determined by ChIP-seq .",
"Genes were categorized into not expressed genes and equally sized groups of highly , moderately and lowly expressed genes ( Gan et al . , 2011 ) .",
"Coverage profiles include 2 kb up- and downstream of the TSS and TTS , respectively .",
"Genic regions were normalized to a standard length .",
"( C ) Normalized read coverages of HS-responsive genes from ACC or NHS FGT1-YFP .",
"The panels show global analysis of genes in the respective expression class ( cf . B ) according to their expression pattern 4 h after ACC ( Stief et al . , 2014 ) in wild type ( up , down , others ) .",
"( D ) FGT1 is enriched in chromatin state 2 ( Sequeira-Mendes et al . , 2014 ) .",
"Relative enrichment of FGT1-bound sequences or WT control after ACC or NHS in different chromatin states indicated depletion of FGT1 in heterochromatin ( states 8 , 9 ) and enrichment in state 2 ( promoter and intergenic regions , open chromatin ) .",
"Lines denote average of replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 01310 . 7554/eLife . 17061 . 014Figure 4—figure supplement 1 . Coverage profiles of ChIP-seq peaks bound by FGT1 28 h after ACC or NHS . Comparison of normalized coverage profiles of FGT1-bound peak genes identified by peak calling ( 942 genes for FGT1 ACC and 60 genes for FGT1 NHS ) and all other genes in FGT1 ChIP-seq samples from ACC and NHS conditions .",
"Coverage profiles were calculated 2 kb up- and downstream of the TSS and TTS , respectively .",
"The length of the genic regions was normalized . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 014 Given that the identified peaks were wide and flat , we hypothesized that the peak calling may have underestimated the number of targets .",
"Thus , we investigated the global correlation of FGT1 binding and expression under NHS conditions by plotting global coverage profiles grouped according to the relative expression in non-stressed seedlings ( Gan et al . , 2011 ) .",
"This revealed that FGT1 is preferentially associated with expressed genes at a global scale ( Figure 4B ) .",
"FGT1 is most strongly associated with genes that have high or intermediate expression in a pattern similar to that observed for the peak genes ( Figure 4—figure supplement 1 ) .",
"We next asked whether FGT1 shows differential association with genes that are HS-responsive ( Stief et al . , 2014 ) .",
"FGT1 associated most strongly with those expressed genes that are upregulated at 4 h after ACC , irrespective of their expression level before HS ( Figure 4C ) .",
"This is especially true for genes that are lowly expressed without HS; this category contains typical HS-responsive genes .",
"Accordingly , FGT1 associated most strongly with genes that are upregulated at 4 h and/ or 52 h after ACC and this is more pronounced in the ACC samples ( Figure 7C ) .",
"Thus , HS increases binding of FGT1 to HS-responsive genes globally , and these genes are associated with low levels of FGT1 already before HS .",
"The A . thaliana genome was categorized into nine chromatin states based on the differential presence of histone modifications , variants and DNA methylation ( Sequeira-Mendes et al . , 2014 ) .",
"Analyzing the overlap between FGT1-bound sequences with different chromatin states , we found that FGT1 was highly enriched in sequences annotated as chromatin state 2 ( Figure 4D ) .",
"This state is found in poised chromatin , mostly in promoters and intergenic regions ( hence transcript levels are low ) .",
"It is enriched in H3 . 3 , H3K4me2 , H3K4me3 , H2A . Z , H2Bub , H3K27me3 , AT-rich and relatively low in overall nucleosome abundance .",
"Strikingly , state 2 peaks immediately before the TSS , and has a smaller peak just after the TTS , mimicking closely the global coverage profile of FGT1 ( Sequeira-Mendes et al . , 2014 ) .",
"In contrast , FGT1 was depleted from the heterochromatic states 8 and 9 .",
"Thus , FGT1 binding globally associates with the nucleosome-poor regions flanking the transcription units of expressed genes .",
"To elucidate the mechanism of how FGT1 promotes gene expression , we isolated FGT1-interacting proteins .",
"To this end , we purified native FGT1-YFP complexes from 35S::FGT1-YFP seedlings 28 h after ACC or control ( NHS ) treatment .",
"FGT1-YFP and associated proteins were then subjected to LC-MS/MS analysis .",
"As controls , we performed purifications on 35S::YFP and Col-0 plants , respectively .",
"Among the peptides identified specifically in the FGT1-YFP samples were both A . thaliana orthologues of the ISWI chromatin remodeler , CHR11 and CHR17 , and the SWI/SNF chromatin remodeler BRAHMA ( BRM ) , suggesting that FGT1 interacts with chromatin remodeling proteins ( Table 1 ) .",
"Because of the high homology between CHR11 and CHR17 , most of the identified peptides could not be assigned unequivocally to either of the two proteins , however , a few specific peptides were recovered demonstrating the presence of both ISWI proteins ( Table 1 ) .",
"We also identified several known subunits ( SWI3a , b , d , SWP73b ) of the BRM complex .",
"We did not observe differences between the ACC and NHS samples , suggesting that the mode of action of FGT1 is independent of HS .",
"To confirm the interactions between FGT1 and the remodelers we used bimolecular fluorescent complementation in transiently transformed tobacco leaves ( Walter et al . , 2004 ) .",
"We thus confirmed the interaction of FGT1 with CHR11 , CHR17 and BRM in the nucleus ( Figure 5 ) .",
"In summary , FGT1 interacts with chromatin remodeling proteins of the ISWI and SWI/SNF classes . 10 . 7554/eLife . 17061 . 015Table 1 . FGT1 interacts with chromatin remodeling proteins in vivo .",
"FGT1-interacting proteins identified by native co-immunoprecipitation followed by mass spectrometry ( nHPLC-MS/MS ) from 5 d-old 35S::FGT1-YFP seedlings subjected to ACC or NHS 28 h before sampling .",
"Col-0 and 35S::YFP were used as controls .",
"The data represent the number of unique peptides found in the indicated experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 015BackgroundTreatmentExpNumber of peptidesFGT1CHR11/ CHR17Chr11Chr17BRMSWI3aSWI3bSWI3dSWP73b35S::FGT1-YFPACC15841------25612322311334311--2----NHS1332------1252114214--33514-------Col-0ACC1-3---------NHS1-3---------35S::YFPNHS1-3---------10 . 7554/eLife . 17061 . 016Figure 5 . FGT1 interacts in vivo with SWI/SNF ( BRM ) and ISWI ( CHR11 , CHR17 ) chromatin remodeling proteins . Bimolecular Fluorescence Complementation confirms the interaction of FGT1 and CHR11 , CHR17 or BRM in the nucleus of tobacco leaf cells .",
"The indicated constructs were co-transformed and analyzed 2 d later with an LSM710 confocal microscope .",
"YFP , BiFC signal in the YFP spectrum; RFP , signal from co-expressed nuclear RFP-fusion protein .",
"Size bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 016 To determine whether the interaction of FGT1 and the remodelers was functionally relevant during HS memory , we examined whether remodeler mutants displayed normal HS memory .",
"As the loss of BRM causes sterility , we performed the assay on the progeny of a heterozygous brm-1/+ plant and genotyped individual seedlings after phenotyping .",
"Indeed , brm-1 mutants displayed reduced HS memory ( Figure 6A , B ) .",
"The highly similar CHR11 and CHR17 proteins show functional redundancy and the double mutant displays severe developmental defects including dwarfism ( Li et al . , 2012 ) .",
"Thus , we performed the experiment on the progeny of a chr11/chr11 chr17/+ plant .",
"As for brm-1 , we genotyped individual seedlings after the phenotypic analysis was completed .",
"We observed that chr11 single mutants and chr11/chr11 chr17/+ seedlings were defective in the physiological HS memory ( Figure 6A , B ) .",
"Due to their growth defects , we could not analyze chr11 chr17 double mutants .",
"To test whether the remodeler mutants have a generally impaired HS response , we also tested their ability to acquire thermotolerance and their basal thermotolerance .",
"Mutants in the remodelers behaved similar to the wild type or slightly better in these assays ( Figure 6—figure supplements 1 , 2 ) , which indicates that the responses to acute HS were not compromised .",
"Thus , the remodeler mutants under investigation displayed a specific impairment of HS memory and not a general defect in HS responses .",
"We also tested the expression of HS-responsive genes after ACC in wild type , and brm-1/+ or chr11/chr11 chr17/+ -segregating lines , respectively .",
"qRT-PCR analysis revealed that both mutant lines show a premature decline of expression of HSA32 , HSP18 . 2 , HSP21 , HSP22 and HSP101 ( Figure 6C ) .",
"In many cases , transcript levels were already lower at the earliest time point measured ( immediately after the end of ACC ) , suggesting that the remodelers were also necessary for full induction of these genes .",
"Interestingly , this was not correlated with a reduced level of acquired thermotolerance in our assays ( Figure 6—figure supplement 1 ) . 10 . 7554/eLife . 17061 . 017Figure 6 . The ISWI and BRM chromatin remodelers are required for HS memory and BRM interacts genetically with FGT1 . ( A ) chr11/chr11 , chr11/chr11 chr17/+ and brm-1 mutants show reduced HS memory .",
"Seedlings of the indicated genotypes were acclimatized 5 d after germination and treated with a tester HS 3 d later .",
"hsa32 was included as a control .",
"Individual seedlings were phenotypically categorized 14 d after ACC and genotyped by PCR .",
"Data are averaged over at least two independent assays , Fisher’s exact test , *p<0 . 05 , **p<0 . 001; n>28 .",
"( B ) Representative picture of the data shown in ( A ) taken 14 d after ACC .",
"( C ) Transcript levels of HS-inducible genes after ACC are reduced in brm-1/+ and chr11/chr11 chr17/+-segregating lines compared to Col-0 .",
"Transcript levels were normalized to At4g26410 and the corresponding NHS sample .",
"Data are averages and SE of at least three biological replicates .",
"( D , E )",
"The brm-1 fgt1-1 double mutant is delayed in growth and development in long-day conditions .",
"( D ) Flowering time in days to first open flower .",
"Genotypes were isolated from a segregating population .",
"The percentage of flowering plants is plotted against the days of growth .",
"( E ) Representative individuals of the indicated genotypes grown for 51 d ( Col-0 , fgt1-1 ) or 67 d ( brm-1 , brm-1 fgt1-1 ) .",
"Size bar , 2 cm . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 01710 . 7554/eLife . 17061 . 018Figure 6—figure supplement 1 . CHR11 , CHR17 and BRM are not required for the acquisition of thermotolerance . Acquisition of thermotolerance is unaffected in chr11/chr11 chr17/+- and brm-1/+ -segregating families , respectively .",
"Acquisition of thermotolerance in Col-0 , hsp101 , chr11/chr11 chr17/+ and brm-1/+ was assayed by treating seedlings 4 d after germination at 37°C for 60 min , 90 min at 22°C , followed by 44°C for the indicated times .",
"For chr11/chr11 chr17/+ and brm1/+ segregating families were used due to the sterility of the homozygous ( double ) mutants .",
"Pictures were taken 14 d after heat treatment .",
"All plants of one treatment were grown on the same plate .",
"One representative of at least three biological replicates is shown .",
"The quantification is based on n = 25–49 individuals for each genotype .",
"Asterisks indicate ( Fisher’s exact test ) *p<0 . 05; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 01810 . 7554/eLife . 17061 . 019Figure 6—figure supplement 2 . CHR11 , CHR17 and BRM are not required for basal thermotolerance . Basal thermotolerance is unaffected in chr11/chr11 chr17/+ and brm-1/+ .",
"Basal thermotolerance in Col-0 , hsp101 , and segregating families of chr11/chr11 chr17/+ and brm-1/+ , respectively , was assayed by incubating seedlings at 44°C for the indicated times for 4 d after germination .",
"For chr11/chr11 chr17/+ and brm-1/+ segregating families were used due to the sterility of the homozygous mutants .",
"Pictures were taken 14 d after HS .",
"All plants of one treatment were grown on the same plate .",
"One representative of at least three biological replicates is shown .",
"The quantification is based on n=26–55 individuals for each genotype .",
"Asterisks indicate ( Fisher’s exact test ) **p <0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 01910 . 7554/eLife . 17061 . 020Figure 6—figure supplement 3 . Seedling phenotype of the brm-1 fgt1-1 double mutant . The brm-1 fgt1-1 double mutant displays delayed development at the seedling stage . Representative pictures of seedlings of the indicated genotypes grown side-by-side were imaged after the indicated days of growth in long-day conditions .",
"Scale bars: grey , 0 . 25 cm; white , 1 cm .",
"Arrows indicate the shoot meristem region of the seedlings .",
"Growth and development of brm-1 fgt1-1 is delayed compared to brm-1 ( and Col and fgt1-1 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 020 Given the similar HS memory phenotypes and their physical interaction , we asked whether FGT1 interacted with BRM also genetically .",
"Indeed , the brm-1 fgt1-1 double mutants displayed several novel phenotypes compared to the brm-1 single mutant ( Figure 6D , E ) .",
"During seedling development , growth of the brm-1 fgt1-1 double mutant was retarded , resulting in reduced development and delayed leaf initiation ( Figure 6—figure supplement 3 ) .",
"Later on , flowering time ( days to flowering ) was delayed compared to the wild type or either single mutant ( Figure 6D , E ) .",
"As the double mutants have to be isolated from a segregating population due to the sterility of brm-1 , we could not obtain suitable material for testing HS memory .",
"Nevertheless , the additive phenotypes observed in the double mutant clearly suggest that both genes act partially redundantly , in agreement with the idea that they act at least partly on the same targets .",
"The additional phenotypes indicate that FGT1 also plays a role during plant development , which is consistent with the large number of target genes that are not related to HS memory .",
"To compare BRM and FGT1 genomic targets , we took advantage of published BRM ChIP-seq data ( Li et al . , 2016 ) .",
"We observed a highly significant overlap between genes associated with BRM under non-stress conditions and FGT1-associated genes ( NHS and ACC; Figure 7A ) .",
"Globally , BRM was more strongly associated with FGT1 ACC target genes than with the rest of the genome ( Figure 7B ) .",
"Strikingly , BRM showed a very similar coverage profile as FGT1; a pronounced peak at the TSS and a second , weaker peak just downstream the TTS .",
"When comparing the association with genes that are induced at 4 and/or 52 h after ACC , we found BRM to be strongly enriched at memory genes ( up at 4 and 52 h ) and late-inducible genes ( up at 52 h only ) , compared to downregulated or non-responsive genes ( Figure 7C ) .",
"Again , this was very similar to the results observed for FGT1 .",
"Notably , the association of BRM and ( to a lesser extent ) FGT1 with memory genes was established before HS .",
"The overlapping binding pattern of FGT1 and BRM was also apparent from browser screenshots of individual loci ( Figure 7—figure supplement 1 ) .",
"In summary , genome-wide BRM target genes overlap strongly with FGT1 target genes and they are pre-associated with memory genes under non-stress conditions , strongly supporting the interaction of FGT1 and BRM . 10 . 7554/eLife . 17061 . 021Figure 7 . BRM and FGT1 show overlapping genomic targeting at HS-responsive genes .",
"( A ) BRM and FGT1 target genes overlap highly significantly .",
"FGT1 ACC and NHS peak overlapping genes were compared to BRM identified peaks ( Li et al . , 2016 ) .",
"The number of overlapping genes is represented and their significance was estimated by Fisher test ( BRM vs . FGT1 NHS p<10−8 , BRM vs . FGT1 ACC p<10−76 ) .",
"( B ) BRM is enriched at FGT1 target genes and shows a similar coverage profile as FGT1 .",
"Normalized coverage profiles of BRM are displayed for FGT1 ACC peak genes and all other genes .",
"FGT1 panels correspond to those in Figure 4—figure supplement 1 .",
"( C ) Before HS , BRM binds preferentially to HS memory genes and late HS-induced genes in a pattern similar to FGT1 .",
"BRM and FGT1 are strongly enriched at HS memory genes ( 4 h + 52 h up , top panel , blue line ) .",
"Normalized read coverages of BRM , FGT1 ACC and FGT1 NHS of genes with changed ( up , down , other ) expression at 4 and/or 52 h after ACC ( Stief et al . , 2014 ) are displayed . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 02110 . 7554/eLife . 17061 . 022Figure 7—figure supplement 1 . Genome browser views of BRM and FGT1 ChIP-seq reads . Genome browser views of HSA32 , HSP18 . 2 , HSP21 , HSP22 . 0 , HSP101 and ACT7 of read coverages from FGT1 ACC , FGT1 NHS and BRM ChIP-seq experiments .",
"The transcribed region of the indicated genes is marked in grey . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 022 Given the physical and genetic interaction with chromatin remodeling complexes , we next examined the possibility that FGT1 functions by regulating nucleosome dynamics at memory genes .",
"To this end , we determined nucleosome occupancy around the TSS of HSA32 , HSP22 . 0 , HSP18 . 2 and HSP101 during two days after ACC in fgt1-1 by qPCR of Micrococcal Nuclease-digested chromatin ( MNase-qPCR ) .",
"At HSA32 , HSP22 . 0 and HSP18 . 2 , FGT1 was required for the correct positioning and occupancy of the +1 nucleosome already under control conditions ( Figure 8 ) , consistent with the fact that FGT1 was bound to these loci already before HS ( Figure 3 , Figure 7—figure supplement 1 ) .",
"Interestingly , while at HSA32 occupancy at the +1 nucleosome was reduced in fgt1-1 compared to parental seedlings , it was increased at HSP22 . 0 and HSP18 . 2 .",
"In wild type , nucleosome occupancy was strongly reduced at 4 h after ACC for all three genes and recovered slowly over the next two days ( Figure 8—figure supplement 1 ) .",
"At HSA32 and HSP22 . 0 nucleosome occupancy was still not fully recovered after 52 h .",
"At HSP18 . 2 it was fully recovered by 52 h ( but not yet 28 h ) .",
"In fgt1-1 , nucleosome recovery was accelerated ( Figure 8—figure supplement 1 ) .",
"Nucleosome dynamics at HSP101 were not affected in fgt1-1 .",
"Thus , FGT1 is required to maintain low relative nucleosome occupancy at memory genes after HS . 10 . 7554/eLife . 17061 . 023Figure 8 . FGT1 is required for nucleosome occupancy and nucleosome recovery after HS at memory genes . FGT1 is required for proper nucleosome organization around the TSS of memory genes before HS and is required to maintain low nucleosome occupancy during the memory phase .",
"Chromatin dynamics at HSA32 , HSP22 . 0 , HSP18 . 2 , HSP101 and At4g07700 at 4 , 28 , or 52 h after acclimatizing HS ( ACC ) or no HS ( NHS ) in the parent ( blue ) or fgt1-1 ( red ) , respectively .",
"Nucleosome occupancy was determined by MNase-qPCR .",
"Data shown are averages of at least three biological replicates and SE . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 02310 . 7554/eLife . 17061 . 024Figure 8—figure supplement 1 . Nucleosome recovery after ACC is delayed in fgt1-1 . Nucleosomes of memory genes but not HSP101 recover faster in fgt1-1 to pre-HS levels .",
"Nucleosome abundance of ACC-treated samples relative to NHS samples was calculated for the position with the highest signal based on the data shown in Figure 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 02410 . 7554/eLife . 17061 . 025Figure 8—figure supplement 2 . Nucleosome remodeler mutants and fgt1 show similar nucleosome occupancy defects of memory genes but not HSP101 . Nucleosome occupancy was determined by MNase-qPCR at the indicated genes on rosette leaves of 35 d-old Col-0 , brm-1 , chr11 chr17/+ , parent and fgt1-1 .",
"Data shown are averages of three biological replicates and SE . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 025 Because of the sterility and developmental defects of brm-1 and chr11 chr17 mutants we were unable to examine nucleosome dynamics in heat-stressed seedings of the remodeler mutants .",
"Given the pre-association of BRM and FGT1 with the memory loci and the findings for fgt1-1 , we reasoned that nucleosome abundance at these loci may be altered already under non-stress conditions .",
"Indeed , rosette leaves of fgt1-1 , brm-1 and chr11/chr11 chr17/+ showed similarly increased nucleosome abundance under greenhouse conditions for HSA32 , HSP18 . 2 and HSP22 . 0 ( Figure 8—figure supplement 2 ) .",
"In contrast , the nucleosome abundance at HSP101 was not changed ."
],
[
"Here , we have reported the identification of the A . thaliana orthologue of Sno , FGT1 , as a regulator of sustained gene induction after HS .",
"We identified FGT1 from an unbiased mutagenesis screen for factors that are required for the maintenance of high HSA32::LUC expression after HS , but not for its initial induction .",
"Genome-wide determination of FGT1-binding sites and its high phylogenetic conservation suggest that FGT1 has a general function in promoting gene expression .",
"FGT1 interacts with conserved chromatin remodeling complexes , and the catalytic subunits of these complexes are also required for physiological HS memory .",
"Together , our data suggest the following model ( Figure 9 ) ; HS induces memory gene expression through HSF proteins .",
"FGT1 binds to these loci at the nucleosome-depleted region ( NDR ) adjacent to the TSS , where it interacts with nucleosome remodeling complexes .",
"By tuning nucleosome occupancy around the TSS , FGT1 maintains these loci in a memory-competent state .",
"This allows active transcription to be maintained for several days after HS , thus contributing to HS memory . 10 . 7554/eLife . 17061 . 026Figure 9 . FGT1 interacts with chromatin remodelers and affects nucleosome dynamics during transcription and HS memory . Top: Schematic representation of HS memory gene expression in wild type and fgt1 after acclimatizing HS ( ACC ) .",
"Loss of FGT1 causes loss of sustained gene induction .",
"Bottom: FGT1 interacts with ISWI and BRM remodelers near the TSS to maintain low nucleosome occupancy .",
"In the absence of FGT1 , nucleosome recovery is accelerated .",
"Profiles were drawn based on the data obtained for HSA32 .",
"Angled arrows indicate transcriptional activity . DOI: http://dx . doi . org/10 . 7554/eLife . 17061 . 026 We have previously found that sustained induction and transcriptional memory after HS are associated with HSFA2-dependent H3K4 hyper-methylation ( Lämke et al . , 2016 ) .",
"Whether FGT1 interacts with HSFA2 and H3K4 methylation remains a question for future studies .",
"FGT1 and BRM are associated with HS memory loci already before HS , when their expression levels are low .",
"FGT1 binding increases after HS and is maintained at high levels over the course of HS memory .",
"It is unclear whether HS memory is conserved outside the plant kingdom .",
"However , our results strongly indicate that FGT1 has a more generic role in transcription , despite the lack of morphological aberrations under standard growth conditions .",
"This general function of FGT1 is partially redundant with that of the SWI/SNF remodeler BRM ( as apparent in the double mutant ) .",
"It is conceivable that FGT1 is required for regulated gene expression , which is prevalent especially during development and under stress conditions .",
"FGT1 shows a high conservation over the whole protein length with its metazoan orthologues .",
"Sno is a component of the inductive Notch signaling pathway and important for patterning of the Drosophila wing margin ( Majumdar et al . , 1997 ) .",
"It is also required for the activation of downstream genes of the Notch and EGFR pathways ( Majumdar et al . , 1997; Tsuda et al . , 2002 ) .",
"The C . elegans orthologue let-765 was implicated as a positive regulator of lin-3/egf expression in vulval induction ( Simms and Baillie , 2010 ) , and mammalian SBNO may be involved in neuron development ( Grill et al . , 2015 ) .",
"FGT1 contains a DExD helicase domain , which is classically considered as an RNA helicase ( Fuller-Pace , 2006 ) .",
"Interestingly , other DExD/H helicase proteins have been implicated in coordinating transcription and co-transcriptional RNA processing by interacting with co-activator/-repressor proteins ( Fuller-Pace , 2006 ) .",
"While FGT1 binds to active genes , it was not associated with transcribed regions , but rather with the flanking promoter and terminator sequences , supporting the notion of a role in transcriptional regulation rather than RNA processing .",
"However , we currently cannot rule out a role for ( non- ) coding RNAs in FGT1-dependent regulation .",
"FGT1 also contains a Helicase C domain , which is frequently found in chromatin remodeler proteins ( Clapier and Cairns , 2009 ) .",
"In fact , the combination of two helicase domains as present in FGT1 is reminiscent of remodeler proteins .",
"In addition , FGT1 contains a PHD domain .",
"Several PHD domains display very high binding affinities to specific posttranslational modifications of histone H3 ( Musselman and Kutateladze , 2011 ) .",
"Using recombinant FGT1PHD or transgenic FGT1-YFP , we confirmed H3 binding , however , we did not observe preferential binding to any of the tested modifications ( Figure 2G , Figure 2—figure supplement 3 ) , suggesting that other determinants contribute to targeting FGT1 .",
"In agreement with this , the PHD domain of FGT1 could not be assigned to any of the characterized subgroups with high specificity for methylated H3 ( Lee et al . , 2009 ) .",
"Chromatin remodeling complexes use the energy of ATP to move nucleosomes along DNA , and to eject or exchange nucleosomes on DNA ( Clapier and Cairns , 2009; Narlikar et al . , 2013; Struhl and Segal , 2013 ) , thus changing the accessibility of DNA for other proteins .",
"Chromatin remodeling complexes have widespread functions during development and have been linked with several pathologies , including tumorigenesis ( Narlikar et al . , 2013 ) .",
"Four families of chromatin remodelers are conserved in metazoans , plants and yeast; the SWI/SNF , the ISWI , the CHD and the INO80 families ( Clapier and Cairns , 2009 ) .",
"The catalytic subunits of all families contain a Snf2-related helicase domain and form large multi-subunit complexes .",
"Although they have differing functions , there is evidence that remodelers of different families interact on the chromatin ( Clapier and Cairns , 2009; Narlikar et al . , 2013 ) .",
"FGT1 interacts with remodelers of the ISWI and SWI/SNF families .",
"Both ISWI orthologues , BRM and several accessory subunits ( SWI3 , BAF73b ) were identified by co-immunoprecipitation .",
"ISWI is required for the regular spacing of nucleosomes downstream of the +1 border nucleosome ( Clapier and Cairns , 2009; Li et al . , 2014 ) .",
"FGT1 was found previously in a co-immunoprecipitation experiment with CHR17 , thus independently confirming our results ( Smaczniak et al . , 2012 ) .",
"Drosophila BRM was originally identified due to its activating function that antagonizes Polycomb group silencing ( Tamkun et al . , 1992; Clapier and Cairns , 2009 ) .",
"During HS memory , both BRM and ISWI act as positive regulators of the memory , suggesting that both contribute to maintaining the chromatin open and accessible .",
"FGT1 , localized just upstream of the TSS , may bridge both remodelers .",
"This notion is consistent with the localization of BRM and ISWI in other systems ( Gkikopoulos et al . , 2011; Yen et al . , 2012; Narlikar et al . , 2013 ) and with the high overlap in BRM and FGT1 localization ( Figure 7 and Li et al . , 2016 ) .",
"Moreover , remodelers of different families cooperate to regulate dynamic sites ( Morris et al . , 2014 ) .",
"The evidence for interaction with BRM is further strengthened by the double mutant analysis , which demonstrated that FGT1 activity becomes critical in the absence of BRM .",
"FGT1 binds to expressed genes just upstream of the TSS and just downstream of the TTS .",
"Whether this reflects one function or two separable functions , remains to be investigated .",
"The FGT1 peaks that we detected were wide and relatively flat .",
"Consistent with FGT1 interacting with BRM and CHR11/CHR17 , such a binding pattern is characteristic for chromatin remodeling proteins but not sequence-specific transcription factors ( Gkikopoulos et al . , 2011; Yen et al . , 2012; Zentner and Henikoff , 2013; Li et al . , 2016 ) .",
"FGT1 was preferentially associated with medium and highly expressed genes .",
"Among the lowly expressed genes , those that are HS-inducible were more strongly bound than other genes - not only after HS , but also under control conditions .",
"This indicates that despite an overall correlation with expression , FGT1 binding does not simply reflect the transcriptional activity of a locus .",
"It is tempting to speculate that at some loci FGT1 binding indicates a readiness for transcription rather than actual transcription , similar to what was described as a poised state ( Levine et al . , 2014 ) .",
"FGT1 binding overlaps the NDRs present next to the TSS and TTS .",
"Hence , FGT1 may be required to maintain the NDR by interacting with remodeler proteins .",
"Accordingly , we observed changes in the nucleosome occupancy of this region and particularly the adjacent +1 nucleosome in fgt1-1 mutants .",
"The changes in nucleosome occupancy at FGT1 target loci preceded HS , in agreement with the FGT1 binding pattern .",
"In non-stressed adult leaves , similar changes were observed for brm and iswi mutants .",
"Locus-specific and developmental effects indicate the involvement of additional components .",
"Thus , it is likely that BRM and ISWI cooperate with FGT1 to mediate nucleosome occupancy before and after HS .",
"We propose that the molecular function of Sno/FGT1 orthologues is conserved and that they promote transcriptional regulation through interaction with chromatin remodeling complexes .",
"The reported function of Sno as a co-activator is consistent with this mechanism; moreover , the interacting chromatin remodelers are highly conserved in metazoans .",
"In summary , we have uncovered a role for nucleosome occupancy in stress memory that is modulated by the conserved chromatin regulator FGT1 .",
"Sno/FGT1 also functions in a broader context to sustain gene expression during development , stress adaptation and pathologies .",
"Our results identify a mechanism of how environmentally-induced gene expression is sustained after cessation of an external cue and provides a molecular framework for a chromatin memory .",
"This mechanism may be exploited to improve stress tolerance in crop plants ."
],
[
"Arabidopsis thaliana Col-0 seedlings were grown on GM medium ( 1% ( w/v ) glucose ) under a 16 h/ 8 h light/dark cycle at 23°C/21°C .",
"brm-1 , hsa32-1 and hsp101 were previously described ( Charng et al . , 2006; Hurtado et al . , 2006; Stief et al . , 2014 ) .",
"chr11-1-/-chr17-1+/- was obtained from K . Kaufmann ( Li et al . , 2012; Smaczniak et al . , 2012 ) .",
"T-DNA insertion lines in FGT1 ( fgt1-2 , SALKseq_17372; fgt1-3 , SALK_036520 ) and brm-1 were obtained from NASC .",
"Heat treatments were performed on 4 d-old seedlings unless stated otherwise .",
"Seedlings were treated with an acclimatizing HS ( ACC ) of 37°C for 60 min , followed by 90 min at 23°C and 45 min at 44°C starting eight hours after light onset .",
"As tester HS a 44°C treatment for the indicated times was applied .",
"After HS , plants were returned to normal growth conditions .",
"Thermotolerance assays were performed as described ( Stief et al . , 2014 ) .",
"For all assays , all genotypes of one treatment were grown on the same plate .",
"To generate HSA32::HSA32-LUC , a 4 . 6 kb fragment encompassing the complete HSA32 gene and 2 . 2 kb of promoter sequences was amplified ( pHSA32_for_SacI GTGGAGAGCTCAAAGCTGCCATGAATGTGTT , HSA32_rev_PstI AACACTGCAGACAATGCCAAGTTTGATGCCTGA ) from genomic DNA , the Stop codon was mutated and replaced by the LUC reporter gene .",
"The resulting HSA32::HSA32-LUC construct was transformed into Col-0 .",
"For pFGT1::FGT1 ( SA13 ) a 10 . 7 kb genomic fragment encompassing the complete FGT1 gene and including 1 . 5 kb promoter sequences was amplified ( EMBFrag1_FSphI TACTGCATGCCTTTAGCGTTATCGAATCT , EMBFrag4_RBamhI CAAGAGGTTAGGATCCGCTTCCAGACA ) and inserted into pBarMAP ( ML516 ) .",
"To make 35S::FGT1-YFP ( SA1 ) , FGT1 was amplified from cDNA mutating the stop codon ( EMB1135BamHI_F AGGGATCCACAATGACGCAGTCGCCTGTTCAAC , EMBBamHInoStpR A AGG ATC CGC ATC ATC AAT CTC TTG AAC CCA TGC T ) and inserted into pBarM:35S::YFP ( IB30 ) , thus generating pBarM:35S::FGT1-YFP .",
"For pFGT1::FGT1-YFP ( EB19 ) the FGT1 stop codon in SA13 was mutated to a SalI site into which YFP was inserted to generate pFGT1::FGT1-YFP .",
"All constructs were inserted into Agrobacterium tumefaciens strain GV3101 and transformed into A . thaliana using the floral dip method ( Clough and Bent , 1998 ) .",
"LUC activity was detected by spraying seedlings with 2 mM Luciferin ( Promega , Mannheim , Germany ) and imaging with a Nightowl ( Berthold Technologies , Bad Wildbad , Germany ) .",
"Image analysis was performed with IndiGO software ( Berthold ) .",
"The signal threshold was adjusted to the signal of the parental line .",
"YFP and RFP fluorescence was imaged using a Zeiss LSM710 confocal microscope ( Zeiss , Jena , Germany ) .",
"Controls were imaged using the same settings and laser intensities .",
"For BiFC YFPN-FGT1 , YFPC-CHR11 , YFPC-CHR17 and YFPC-BRM constructs were generated using published vectors pE-SPYCE and pE-SPYNE , respectively ( Walter et al . , 2004; Weltmeier et al . , 2006 ) .",
"Combinations of YFPN and YFPC fusion constructs were co-expressed in four to six week-old Nicotiana benthamiana leaves using leaf infiltration of A . tumefaciens GV3101 suspensions containing the tested construct combinations .",
"Fluorescence in the YFP spectrum was analysed 2 d after infiltration .",
"A UBC10::BRU1-RFP construct was co-transformed to image nuclei ( Ohno et al . , 2011 ) .",
"RNA extraction , reverse transcription and qPCR were performed as described previously ( Stief et al . , 2014; Lämke et al . , 2016 ) .",
"At4g26410 was used as a reference gene ( Czechowski et al . , 2005 ) .",
"Primer sequences are listed in Supplementary file",
"1 . The PHD domains of FGT1 ( aa 667–757 ) and ING1 ( Lee et al . , 2009 ) were subcloned into pGEX-4T-1 ( Amersham Biosciences , Pittsburgh , PA ) , expressed in E . coli BL21 ( DE3 ) as GST fusion proteins and purified using Glutathione affinity resins ( Thermo Fisher Scientific , Waltham , MA ) .",
"The histone peptide binding assay was performed as described ( Kabelitz et al . , 2016 ) .",
"In brief , 1 µg of biotinylated histone peptides ( Merck-Millipore , Darmstadt , Germany ) were incubated with 10 µg of GST-fusion protein in binding buffer ( 50 mM Tris-HCl pH 7 . 5 , 300 mM NaCl , 0 . 1% NP-40 , 1 mM PMSF , protease inhibitors ) overnight at 4°C with rotation .",
"After incubation with Streptavidin Dynabeads ( Thermo Fisher Scientific ) and extensive washing with TBST , bound proteins were analyzed by SDS-PAGE and immunoblotting with anti-GST antibodies ( Merck-Millipore ) .",
"Results for GST-ING1 were taken from Kabelitz et al . ( 2016 ) , as the experiments were performed in parallel .",
"ChIP was performed as described ( Lämke et al . , 2016 ) .",
"MNase-qPCR was performed as described ( Liu et al . , 2014 ) .",
"Primer sequences are listed in Supplementary file",
"1 . Curves were created based on a polynomial regression ( HSA32 , HSP22 . 0 , HSP101 , At4g07700 ) or spline interpolation ( HSP18 . 2 ) .",
"The position of the center of each amplicon relative to the TSS is indicated .",
"The +1 nucleosome of At4g07700 was used for normalization ( Kumar and Wigge , 2010 ) .",
"ChIP-seq was done with three biological replicates for two genotypes ( 35S::FGT1-YFP , Col-0 ) and two treatments ( acclimated ( ACC ) and control ( NHS ) ) .",
"5 d-old seedlings were treated with ACC or NHS and harvested 28 h later .",
"ChIP was performed as described above .",
"Library preparation and 100 bp single-end sequencing on an Illumina HiSeq 2500 were performed by ATLAS Biolabs ( Berlin , Germany ) .",
"Data were delivered as de-multiplexed fastq files .",
"Raw data have been deposited at NCBI SRA under accession number SRA GSE79453 .",
"BRM ChIP-seq raw data ( Li et al . , 2016 ) were downloaded from NCBI SRA .",
"All statistical analyses were done using R ( http://www . r-project . org ) .",
"Figures were made using R base plotting or the lattice package .",
"Read adapters were removed using Trimmomatic ( Bolger et al . , 2014 ) .",
"The resulting reads were mapped against the A . thaliana TAIR10 reference genome using bwa mem ( Li , 2013 ) .",
"Mapping files were further transformed into sorted bam files and indexed using samtools ( Li et al . , 2009 ) .",
"Duplicate reads were removed using samtools rmdup .",
"One Col-0 ACC sample was filtered out due to its very low number of mapped reads .",
"Peak calling was done using MACS ( Zhang et al . , 2008 ) for each sample separately , lower mfold was decreased to",
"2 . Only peaks called for all three FGT1 samples with the same treatment and not called for any of the 2 Col-0 control samples were considered as a signal .",
"Distances between the peaks thus identified and annotated genes were calculated using bedtools closestBed ( Quinlan and Hall , 2010 ) .",
"The expression of those genes was estimated based on published RNA-seq data for 11 d-old seedlings ( Gan et al . , 2011 ) .",
"For coverage profiles , base coverages for the whole genome and selected regions were computed using bedtools coverage and normalized by division by the total number of covered bases ( as calculated by multiplying the number of mapped reads by their length ) , chloroplast and mitochondrion sequences were excluded .",
"Coverage profiles around genes were made using 2 kb prior to the TSS , gene regions , and 2 kb after the TTS .",
"Values were averaged over biological replicates .",
"Genic ( transcribed ) region base coverages were scaled to the same length to make them comparable between different genes .",
"Genes were categorized into not expressed genes and equally sized groups of highly , moderately and lowly expressed genes according to their expression under standard conditions as determined by a published RNA-seq experiment ( Gan et al . , 2011 ) .",
"This dataset corresponds to the closest developmental stage and environmental conditions regarding our ChIP-seq experiment with available data at NCBI GEO ( GSM764077 ) .",
"Coverage profiles for each class were calculated by averaging the values for all member genes for each genotype x treatment combination .",
"Another classification was done by grouping genes based on their expression pattern 4h after ACC as determined by ATH1 microarray hybridization ( Stief et al . , 2014 ) .",
"Coverage profiles for those classes were calculated as described above .",
"FGT1 binding enrichment depending on chromatin state was investigated by comparing coverages for chromatin states as described ( Sequeira-Mendes et al . , 2014 ) .",
"Coverages were normalized regarding the total number of covered bases and the length of each chromatin state region .",
"Values for each state were averaged at the sample level .",
"Native FGT1-YFP protein complexes were immunoprecipitated from 35S::FGT1-YFP seedlings and subjected to nHPLC-MS/MS for identification .",
"In detail , 2 . 5 g of 4 d-old seedlings subjected to ACC or NHS were harvested 28 h later and snap-frozen in liquid nitrogen .",
"Nuclei were extracted according to ( Kaufmann et al . , 2010 ) and sonified using a Diagenode Bioruptor ( 3 cycles/ 30 s on/off ) on low intensity settings .",
"Protein extracts were incubated with α-GFP paramagnetic beads for 1 . 5 h at 4°C and native protein complexes recovered using α-GFP isolation kit ( Miltenyi Biotec , Bergisch Gladbach , Germany ) and eluted in 8 M urea ( Sigma-Aldrich , München , Germany ) .",
"Eluates were diluted and digested with trypsin ( Promega ) as described ( Smaczniak et al . , 2012 ) .",
"Peptides were desalted , lyophilized and re-suspended in 30 μL 5% ( v/v ) acetonitrile , 2% ( v/v ) trifluoroacetic acid .",
"Measurements were performed on a Q Exactive Plus orbitrap mass spectrometer coupled with an Easy nLC1000 HPLC ( Thermo Fisher Scientific ) .",
"Spectra were analyzed using MaxQuant software ( Cox and Mann , 2008 ) and the A . thaliana TAIR10 annotations .",
"A decoy database search was used to limit false discovery rates to <1% on the protein level ."
]
] | [
"Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects .",
"As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress .",
"We show that this heat stress memory requires the activity of the FORGETTER1 ( FGT1 ) locus , with fgt1 mutants displaying reduced maintenance of heat-induced gene expression .",
"FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch ( Sno ) , and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion .",
"FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families , which also display reduced heat stress memory .",
"Genomic targets of the BRM remodeler overlap significantly with FGT1 targets .",
"Accordingly , nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1 .",
"Together , our results suggest that by modulating nucleosome occupancy , FGT1 mediates stress-induced chromatin memory ."
] | [
"In nature , plant growth is often limited by unfavourable conditions or disease .",
"Plants have thus evolved sophisticated mechanisms to adapt to such stresses .",
"In fact , brief exposure to stress can prime plants to be better prepared for a future stress following a period without stress .",
"However , the molecular basis of this memory-like phenomenon is poorly understood .",
"Now , Brzezinka , Altmann et al . have used priming by heat stress as a model to dissect the memory of environmental stresses in thale cress , Arabidopsis thaliana .",
"First , a library of mutant plants were tested to identify a gene that is specifically required for heat stress memory but not for the initial responses to heat .",
"Brzezinka , Altmann et al . identified one such gene and termed it FORGETTER1 ( or FGT1 for short ) .",
"Further experiments then revealed that the FGT1 protein binds directly to a specific class of heat-inducible genes that are relevant for heat stress memory .",
"Brzezinka , Altmann et al . propose that the FGT1 protein makes sure that the heat-inducible genes are always accessible and active by modifying the way the DNA containing these genes is packaged .",
"DNA is wrapped around protein complexes called nucleosomes and depending on how tightly the DNA of a gene is wrapped makes it more or less easy to activate the gene .",
"In agreement with this model , FGT1 does interact with proteins that can reposition nucleosomes and leave the DNA more loosely packaged .",
"Also , the fact that plants that lack a working FGT1 gene repackage the DNA of memory-related genes too early after experiencing heat stress provides further support for the model .",
"Together these findings could lead to new approaches for breeding programmes to improve stress tolerance in crop plants .",
"One future challenge will be to find out whether memories involving nucleosomes are also made in response to other stressful conditions , such as attack by pests and disease ."
] | 2016 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"developmental biology",
"short report",
"cell biology"
] | Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc | elife-22319-v3 | [
[
"Morphogens are thought to disperse and form concentration gradients to control tissue patterning and growth ( Rogers and Schier , 2011 ) .",
"The Drosophila wing imaginal disc has served as an excellent model to study how morphogens control patterning and growth .",
"It has been shown that decapentagplegic ( dpp ) , a homologue of vertebrate bone morphogenetic protein 2/4 ( BMP2/4 ) , is expressed in a stripe of cells in the anterior compartment along the anterior-posterior compartmental boundary of the wing imaginal disc .",
"From this source , Dpp protein is thought to spread and form a concentration gradient to control patterning and growth of the wing imaginal disc ( Lecuit et al . , 1996; Nellen et al . , 1996; Matsuda et al . , 2016; Affolter and Basler , 2007; Restrepo et al . , 2014 ) .",
"However , the precise spatial-temporal requirement of dpp remained elusive , mainly because it was not possible to generate an inducible null allele of dpp due to the haploinsufficiency of the locus and the lack of appropriate methods .",
"Recently , two papers addressed the role of the dpp source at the compartment boundary using independent strategies .",
"Akiyama and Gibson used CRISPR-Cas9-mediated genome engineering techniques to insert a FRT ( Flippase Recognition Target ) cassette into the dpp locus , and successfully generated a conditional null allele of dpp using the expression of Flippase ( FLP ) in a spatial-temporal controlled manner ( Akiyama and Gibson , 2015 ) .",
"By genetically removing dpp from the anterior stripe , they showed that the Dpp morphogen gradient derived from this stripe is indeed critical for patterning .",
"However , and rather surprisingly , they also found that dpp from the stripe is largely dispensable for wing disc growth during the third instar larval stage .",
"Instead , growth of the wing disc was compromised by genetically removing dpp from the entire anterior compartment .",
"Based on the constant requirement of dpp derived from the anterior compartment for growth of the wing disc , Akiyama and Gibson proposed that a not-yet identified anterior dpp source outside the stripe of cells is required for wing disc growth ( Akiyama and Gibson , 2015 ) .",
"Harmansa et al . used a membrane-anchored anti-GFP nanobody ( morphotrap ) to trap GFP-Dpp and manipulate GFP-Dpp dispersal ( Harmansa et al . , 2015 ) .",
"Since an endogenously tagged GFP-Dpp strain was not available , dpp disc mutants were rescued by expressing GFP-Dpp in the stripe ( Entchev et al . , 2000; Teleman and Cohen , 2000 ) and morphotrap was concomitantly expressed in the stripe in order to trap GFP-Dpp and block its dispersal .",
"In this setup , the authors confirmed that dpp is required for wing disc patterning , and also found that Dpp morphogen dispersal from the stripe of cells is required for medial but not for lateral wing disc growth in the posterior compartment ( the region they analyzed ) .",
"However , since these experiments were done under rescue conditions , other sources of dpp important for growth in wild type individuals could have been missed .",
"Thus , while both studies confirmed a role of dpp on wing disc patterning , these studies propose different scenarios for the spatial requirement of dpp on wing disc growth , and it remains debated whether the Dpp morphogen gradient derived from the anterior stripe of cells is required for wing disc growth ( Vincent et al . , 2016; Strzyz , 2016 ) .",
"In this study , we first show that the dpp-Gal4 driver line used to remove dpp from the anterior stripe in the previous study ( Akiyama and Gibson , 2015 ) does not faithfully reflect the endogenous dpp expression pattern during third instar larval stages .",
"We therefore genetically removed dpp at different time points using a different Gal4 line ( ptc-Gal4 line ) , which covers the anterior stripe of cells from the early larval stages onward .",
"Using this setup , we demonstrate that dpp from the stripe of cells is indeed critical for growth of the wing disc , even during third instar larval stages .",
"Furthermore , this result indicates that an anterior dpp source outside the stripe of cells , even if it would exist , would not be sufficient to drive growth of the wing disc ."
],
[
"Akiyama and Gibson used a dpp-Gal4 driver line to genetically remove dpp from the anterior stripe .",
"Based on the results they obtained , they proposed that the dpp stripe is not required for wing disc growth ( Akiyama and Gibson , 2015 ) .",
"Although it appears straightforward to use dpp-Gal4 to remove dpp , this setup has intrinsic problems in removing dpp from the early onset of its expression .",
"Since this setup requires the dpp disc enhancer to be activated , endogenous dpp is initially expressed before it can be removed by FLP/FRT recombination .",
"Furthermore , since it takes about 18–24 hr to remove the engineered cassette in the dpp locus from the majority of cells in the anterior stripe and the wing disc grows dramatically during this time ( Akiyama and Gibson , 2015 ) ( Figure 1a ) , this delay may fail to reveal a potential early function of dpp on growth .",
"In addition to this intrinsic problem , we found that dpp-Gal4 expression does not faithfully reflect the spatial-temporal endogenous dpp expression pattern until relatively late third instar stages ( Figure 1a ) .",
"Although dpp has been shown to be expressed in the entire anterior stripe of cells from the early third instar larval stages ( Akiyama and Gibson , 2015 ) , NLS-mCherry expressed under the control of dpp-Gal4 marked only the dorsal stripe of cells at the beginning of third instar larval stages ( 60 hr after egg laying ( AEL ) at 26°C ) ( Figure 1a ) .",
"dpp-Gal4 was expressed in the entire stripe of cells only in relatively late third instar stages ( Figure 1a ) .",
"Most probably , the fragment of the dpp disc enhancer used to drive Gal4 does not cover all the cis-regulatory regions important for proper stripe expression . 10 . 7554/eLife . 22319 . 003Figure 1 . Comparison of dpp-Gal4 and ptc-Gal4 expression pattern with Dpp expression in the Drosophila wing disc .",
"( a ) Temporal expression pattern of dpp-Gal4 ( dppFO/+; dpp-Gal4/UAS-NLS-mCherry )",
"( b ) temporal expression pattern of ptc-Gal4 ( dppFO , ptc-Gal4/+; UAS-NLS-mCherry ) .",
"Single confocal images except 50 . 5–52 . 5 hr AEL by maximum intensity projection .",
"( c ,",
"d ) Comparison of anti-Dpp staining and dpp-Gal4 expression ( NLS-mCherry ) in the early",
"( c ) and late",
"( d ) third instar wing disc of a dppFO/+; dpp-Gal4/UAS-NLS-mCherry larva .",
"( e ,",
"f ) Comparison of anti-Dpp staining and ptc-Gal4 expression ( NLS-mCherry ) in the early",
"( e ) and late",
"( f ) third instar wing disc of a dppFO/+; ptc-Gal4/UAS-NLS-mCherry larva .",
"Average intensity projection from 5 sequential confocal images .",
"Scale bars 50 μm .",
"Anterior is to the left in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 00310 . 7554/eLife . 22319 . 004Figure 1—figure supplement 1 . Comparison of mCherry fluorescent signal and anti-mCherry staining .",
"( a–d )",
"Comparison of mCherry fluorescent signal ( a–d ) and anti-mCherry staining ( a’–d’ ) in the early ( a ,",
"b ) and late ( c ,",
"d ) third instar wing disc of a dppFO/+; dpp-Gal4/UAS-NLS-mCherry larva ( a , b ) and a dppFO/+; ptc-Gal4/UAS-NLS-mCherry larva ( c , d ) .",
"Average intensity projection from 3 sequential confocal images .",
"Scale bars 50 μm .",
"Anterior is to the left in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 004 Therefore , to reinvestigate the role of the dpp stripe on growth , we decided to remove dpp by FLP/FRT recombination using a different Gal4 line expressed in this anterior stripe of cells .",
"Since ptc , a Hedgehog target gene , is expressed in a stripe of cells similar to dpp , we first analyzed the spatial-temporal expression pattern of ptc-Gal4 .",
"We found that NLS-mCherry expressed under the control of ptc-Gal4 marked the entire stripe of cells as early as late second instar ( 50 hr AEL at 26°C ) and that stripe expression continued throughout third instar larval stages ( Figure 1b ) .",
"We then compared the expression pattern of these two Gal4 lines with endogenous Dpp expression using a Dpp antibody that recognizes the prodomain of Dpp ( Akiyama and Gibson , 2015 ) ( Figure 1c–f ) .",
"Consistent with the above observations , dpp-Gal4 expression did not cover the ventral Dpp stripe in the early third instar larval stages and covered the entire stripe only in late third instar larval stages ( Figure 1c , d ) .",
"In contrast , ptc-Gal4 expression continuously covered the Dpp stripe from the early third instar larval stages onward ( Figure 1e , f ) .",
"To ascertain the validity of using NLS-mCherry to mark ptc-Gal4 or dpp-Gal4 expression , which might be somewhat compromised due to the lengthy maturation time for the mCherry protein , we marked Gal4 expression using α-mCherry antibody and found that fluorescent signal and antibody signal overlap well ( Figure 1—figure supplement 1 ) .",
"Taken together , these results show that spatial-temporal ptc-Gal4 expression reflects endogenous dpp expression pattern in the wing disc more precisely than dpp-Gal4 .",
"We then removed dpp using dpp-Gal4 or ptc-Gal4 and compared their effects on wing disc growth ( CRISPR-Cas9-modified flies ( dppFO ) were generously provided by Akiyama and Gibson ) .",
"When dpp was removed using dpp-Gal4 , pMad was not detectable in the wing pouch but the wing disc grew normally , as reported in Akiyama and Gibson ( Figure 2a , b ) ( Akiyama and Gibson , 2015 ) .",
"In sharp contrast , when dpp was removed using ptc-Gal4 , the pMad signal was also lost from the wing pouch ( except for the future alula region ) , and wing disc growth was severely affected ( Figure 2c ) .",
"Wing pouches were very small and often hardly visible , as shown by the lack of internal ring expression of Wg ( Figure 2c ) .",
"We confirmed that Dpp expression was lost from the stripe of anterior cells but remained detectable in the future alular region ( Figure 2d ) , consistent with the pMad signal in this region ( Figure 2c ) .",
"Accordingly , the growth repressor Brk , normally repressed by pMad , was uniformly expressed in the wing pouch ( Figure 2e ) . 10 . 7554/eLife . 22319 . 005Figure 2 . Defects in wing disc growth by removing dpp using ptc-Gal4 . ( a–c ) Anti-pMad and anti-ptc/wg staining in a dppFO/+; UAS-FLP/+ ( control ) late third instar wing disc",
"( a ) , in a dppFO/dppFO; dpp-Gal4/UAS-FLP late third instar wing disc",
"( b ) , and in a dppFO , ptc-Gal4/dppFO; UAS-FLP/+ late third instar wing disc",
"( c ) .",
"( d ) anti-Dpp staining in a dppFO , ptc-Gal4/+; UAS-FLP/+ ( control ) late third instar wing disc ( left ) , and in a dppFO , ptc-Gal4/dppFO; UAS-FLP/+ late third instar wing disc ( right ) .",
"( e ) anti-Brk staining in a dppFO , ptc-Gal4/+; UAS-FLP/+ ( control ) late third instar wing disc ( left ) , and in a dppFO , ptc-Gal4/dppFO; UAS-FLP/+ late third instar wing disc ( right ) .",
"( * ) marks the future alula region .",
"( a–e )",
"Average intensity projection from 5 sequential confocal images .",
"( f , h ) an experimental setup to test the efficiency of FLP/FRT mediated recombination .",
"( g ) anti-β-gal staining in a dppFO , ptc-Ga4/+; UAS-FLP/act5C ( -FRT ) lacZ late third instar wing disc ( control ) .",
"( i ) anti β-gal staining in a dppFO , ptc-Gal4/dppFO;UAS-FLP/act5C ( -FRT ) lacZ late third instar wing disc .",
"( g , i )",
"A single confocal image .",
"Scale bars 50 μm .",
"Anterior is to the left in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 005 The above results show that wing disc growth is severely affected by removing dpp using ptc-gal4 .",
"To test where FLP/FRT recombination was driven by ptc-Gal4 , we then marked cell lineages of ptc-Gal4 ( Figure 2f–i ) .",
"Since there is no lineage separation within the anterior compartment , the temporal Gal4 expression pattern does not necessarily reflect the actual domain , in which FLP/FRT-mediated recombination occurred .",
"For example , cells that leave the stripe in the early stages might not express NLS-mCherry anymore at later stages , but could have excised dpp when they were located in the stripe in earlier stages .",
"Alternatively , Gal4 may be transiently expressed outside the stripe in the early stages and be sufficient to excise dpp there .",
"We found that when one copy of dpp was removed using ptc-Gal4 ( control ) , the entire anterior wing pouch was marked in some wing discs , and only a stripe of anterior cells was marked in other wing discs ( Figure 2f , g ) .",
"Similar cell lineages were also observed when two copies of dpp were removed using ptc-Gal4 ( Figure 2h , i ) .",
"The variations in these ptc-Gal4 lineages observed may be due to slight differences in Gal4 or FLP expression or a degree of randomness in the excision events in each wing disc .",
"Importantly , the wing discs where the ptc-Gal4 lineages were restricted to a stripe of cells showed drastic growth defects ( Figure 2i ) , raising a possibility that dpp derived from the anterior stripe is critical for wing disc growth .",
"However , since ptc-Gal4 is expressed earlier than dpp-Gal4 ( Figure 1 ) , the severe growth defects by ptc-Gal4 may simply reflect an early role of dpp stripe on wing disc growth .",
"Furthermore , since different FRT cassettes can have different sensitivities to FLPase , the FRT cassette in dpp locus and the FRT cassette to follow the ptc-Gal4 lineage could be excised in different regions .",
"For example , the FRT cassette in the dpp locus may be excised outside the anterior stripe , although the excisions of the FRT cassette to follow the ptc-Gal4 lineages are restricted to the anterior stripe .",
"Thus , the growth defect by ptc-Gal4 may be due to elimination of dpp in the early stages , and/or elimination of potential dpp source outside the anterior stripe that may drives wing disc growth .",
"To investigate the temporal requirement of the dpp stripe for wing disc growth , we therefore used the Gal80ts system ( Figure 3a ) .",
"Gal80ts represses Gal4 activity at 17°C and can be inactivated at 29°C .",
"Thus , ptc-Gal4 can be conditionally activated upon a temperature shift .",
"Indeed , we found that at the permissive temperature dppFO , ptc-Gal4 / dppFO; tub-Gal80ts / UAS-FLP adult wing had no obvious wing phenotype , suggesting that Gal80ts effectively suppresses ptc-Gal4 activity at 17°C ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 22319 . 006Figure 3 . dpp stripe is required for wing disc growth during second and third instar larval stages .",
"( a ) A scheme to genetically remove dpp from the second or third instar larval stages using Gal80ts system .",
"At 17°C , Gal4 activity is blocked by Gal80ts .",
"At 29°C , Gal80ts is inactivated and Gal4 starts to induce FLP expression in the anterior stripe of cells .",
"After embryo collection for 2–4 hr at room temperature , the embryos were incubated at 17°C until temperature shift .",
"Temperature was shifted after 4 days ( second instar ) or 6 days ( third instar ) at 17°C , and late third instar wing discs were dissected after 53 hr or 43 hr later respectively .",
"( b–e )",
"Removal of dpp using ptc-Gal4 during second instar larval stages ( b ,",
"c ) or third instar larval stages ( d ,",
"e ) .",
"( b ,",
"d ) anti-pMad staining and anti-β-gal staining ( lineage tracing ) in a control male wing disc ( dppFO , ptc-Gal4/CyO; tub-Gal80ts/UAS-FLP , act5C ( -FRT ) lacZ ) ( left ) , and a male wing disc removing dpp during the specified time point ( dppFO , ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP , act5C ( -FRT ) lacZ ) ( right ) .",
"( c ,",
"e ) anti-Dpp staining in a control male wing disc ( dppFO , ptc-Gal4/+; tub-Gal80ts/ + ) ( left ) , and a male wing disc removing dpp during the specified time point ( dppFO , ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP ) ( right ) .",
"( b’–e’ )",
"Quantification of the wing disc size of ( b–e ) .",
"Mean ± s . d . p<0 . 001 by two sided Student’s t-test .",
"Scale bars 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 00610 . 7554/eLife . 22319 . 007Figure 3—source data 1 . Quantification of wing disc size for Figure 3b–e . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 00710 . 7554/eLife . 22319 . 008Figure 3—figure supplement 1 . A control experiment under permissive temperature ( 17°C ) for Figure 3 .",
"( a ) A control male wing of dppFO , ptc-Gal4/dppFO; tub-Gal80ts/TM3Sb .",
"( b ) a male wing of dppFO , ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 00810 . 7554/eLife . 22319 . 009Figure 3—figure supplement 2 . Visualization of the regions where dpp is removed using ptc-Gal4 in the wing imaginal disc .",
"( a ) A scheme to mark the regions where dpp is removed using ptc-Gal4 .",
"dppFO-GFP contains ubi-GFP within the FRT cassette .",
"Upon FLP/FRT mediated recombination , Ubi-GFP would be removed .",
"Thus the regions where GFP signal is eliminated correspond to the regions where dpp is removed .",
"( b ,",
"c ) a dppFO-GFP/+ late third instar wing disc ( control ) after temperature shift at the beginning of second",
"( b ) or third",
"( c ) instar larval stages .",
"( d ,",
"e ) a ptc-Gal4/dppFO-GFP; UAS-FLP , tub-Gal80ts/+ late third instar wing disc after temperature shift at the beginning of second",
"( d ) and third",
"( e ) instar larval stages .",
"Single confocal images .",
"Scale bars 50 μm .",
"Anterior is to the left in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 00910 . 7554/eLife . 22319 . 010Figure 3—figure supplement 3 . Temporal resolution of dpp removal using ptc-Gal4 . Anti-Dpp staining in dppFO , ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP wing discs at specified time ( 9 , 16 , 20 , 24 hr ) after temperature shift at the beginning of third instar larval stage ( 17°C ) , and in a dppFO , ptc-Gal4/dppFO; tub-Gal80ts/TM6C wing disc ( control ) at 24 hr after temperature shift .",
"Average intensity projection from 3 sequential confocal images .",
"Scale bars 100 μm .",
"Anterior is to the left in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 01010 . 7554/eLife . 22319 . 011Figure 3—figure supplement 4 . Temporal changes in Dpp and Brk expression by removing dpp using dpp-Gal4 . ( a–b ) Anti-Dpp staining",
"( a ) and anti-Brk staining",
"( b ) in a dppFO/+; UAS-FLP/+ ( control ) mid-third instar wing disc ( left ) , and in a dppFO/dppFO; dpp-Gal4/UAS-FLP mid-third instar wing disc ( right ) .",
"( c–d ) anti-Dpp staining",
"( c ) and anti-Brk staining",
"( d ) in a dppFO/+; UAS-FLP/+ ( control ) late third instar wing disc ( left ) , and in a dppFO/dppFO; dpp-Gal4/UAS-FLP late third instar wing disc ( right ) .",
"Average intensity projection from 3 sequential confocal images .",
"Scale bars 50 μm .",
"Anterior is to the left in all figures . DOI: http://dx . doi . org/10 . 7554/eLife . 22319 . 011 We first marked lineages of ptc-Gal4 when FLP/FRT-mediated recombination was temporally activated at the beginning of the second or third instar larval stage using Gal80ts .",
"We found that ptc-Gal4 lineages were strictly restricted to the anterior stripe in control and mutant wing disc ( Figure 3b , d ) .",
"Thus , the more random lineages shown in Figure 2g , i appear to be derived from early expression of the ptc-Gal4 driver .",
"To directly monitor where dpp is removed by conditionally activating ptc-Gal4 , we utilized the dppFO-GFP allele originally generated as an intermediate allele to generate the final dppFO allele ( Akiyama and Gibson , 2015 ) .",
"dppFO-GFP contains the same FRT cassette as dppFO and a ubiquitously expressed GFP construct ( ubi-GFP ) within the cassette ( Figure 3—figure supplement 2 ) .",
"Thus , the regions in which dpp is excised will lack the GFP signal .",
"Using this setup , we found that FLP/FRT-mediated excision in the dpp locus was indeed restricted to the anterior stripe from second and from third instar larval stages although the excision varied within the stripe ( 21/21 and 14/14 wing discs , respectively ) ( Figure 3—figure supplement 2 ) .",
"This result also strongly suggests that the temporal lineages of ptc-Gal4 indeed reflect the actual region where dpp is removed in this setup ( Figure 3b , d ) .",
"By removing dpp during either second or third instar larval stages , we found that wing disc growth was severely affected in both cases , as measured in the late third instar larval stages ( 53 hr or 43 hr later after temperature shift , respectively ) ( Figure 3b’–e’ ) .",
"The majority of Dpp was eliminated around 20 ~ 24 hr after temperature shift as also reported by Akiyama and Gibson ( Akiyama and Gibson , 2015 ) ( Figure 3—figure supplement 3 ) and wing disc appeared to grow at least until 24 hr after temperature shift .",
"Thus , the growth defects resulting from temperature shifting at the beginning of third instar larval stages likely reflect the effects seen from the absence of dpp around mid-third instar larval stages .",
"These results show that the dpp stripe is required for wing disc growth during second and even third instar larval stages .",
"Since we used the same dpp allele ( dppFO ) and UAS-FLP line as Akiyama and Gibson , the different growth defects observed should be due to the differences between the dpp-Gal4 and ptc-Gal4 driver lines .",
"We showed that while ptc-Gal4 is constantly expressed at the anterior stripe during third instar larval stages , dpp-Gal4 is initially expressed only at the dorsal stripe and only later , during third instar larval stages , in the entire stripe ( Figure 1 ) .",
"Thus , the differences in the spatial-temporal expression may be responsible for the different phenotype observed when using dpp-Gal4 or ptc-Gal4 .",
"Interestingly , although both dpp-Gal4 and ptc-Gal4 are expressed in the dorsal stripe in the early third instar larval stages , the dorsal wing disc pouch still grew using dpp-Gal4 with minor growth defects ( Figure 2b ) ( Akiyama and Gibson , 2015 ) .",
"To investigate why the dorsal compartment still grows when using dpp-Gal4 , we analyzed Brk expression , the critical growth repressor repressed by Dpp signaling .",
"( Figure 3—figure supplement 4 ) .",
"We found that at the mid third instar larval stage ( 80 hrAEL at 26°C ) , Brk was repressed in the ventral compartment where dpp was still expressed but was slightly upregulated in the dorsal compartment where the majority of dpp was removed ( Figure 3—figure supplement 4 ) .",
"Interestingly , Brk upregulation in the dorsal compartment was not uniform but graded; lower in ventral and higher in dorsal regions within the dorsal compartment ( Figure 3—figure supplement 4 ) .",
"At the late third instar larval stage , the majority of dpp was eliminated and Brk was upregulated in both dorsal and ventral compartment but again , Brk was not uniformly upregulated ( Figure 3—figure supplement 4 ) .",
"Consistent with our findings , Omb has been shown to be weakly expressed in this setup ( Akiyama and Gibson , 2015 ) .",
"These results suggest that the Dpp signal is not completely removed from the wing imaginal disc when using dpp-Gal4 .",
"The graded Brk expression in the dorsal compartment is consistent with weak Dpp signal derived from the ventral dpp stripe , and this lasting signal can explain the sustained growth there with minor growth defects .",
"In contrast , when dpp was removed by ptc-Gal4 during the third instar larval stages , majority of dpp was removed from the entire anterior stripe ( Figure 3—figure supplement 3 ) and wing disc growth was severely affected ( Figure 3b–d ) .",
"Together , these results suggest that the critical role of the dpp stripe on wing disc growth was missed by Akiyama and Gibson due to imprecise spatial removal of dpp when using dpp-Gal4 during third instar larval stages ( Akiyama and Gibson , 2015 ) .",
"Based on the constant requirement of dpp from the anterior compartment during third instar larval stages for proper wing disc growth , Akiyama and Gibson further proposed that a potential anterior dpp source outside the stripe was critical for wing disc growth ( Akiyama and Gibson , 2015 ) .",
"However , our data show that wing disc growth was severely affected when dpp was removed only from the anterior stripe during third instar larval stages .",
"Thus , the strong growth defects resulting from removing dpp from the entire anterior compartment are most likely due to excision of dpp from the anterior stripe and not due to the excision of the potential dpp outside the stripe .",
"In conclusion , our results establish that the anterior dpp stripe is critical for growth as well as patterning of the wing imaginal disc .",
"Given the slow process of removing dpp by FLP/FRT mediated recombination ( about 20–24 hr ) compared to wing disc growth , it remains an open question whether the requirement of the dpp stripe on wing disc growth changes over time .",
"It would be important to acutely manipulate the endogenous morphogen gradient at the protein level to address the precise temporal requirement of the dpp stripe on wing disc growth ( Matsuda et al . , 2016; Bieli et al . , 2016 ) ."
],
[
"Flies were kept in standard fly vials ( containing polenta and yeast ) in a 26°C incubator .",
"The following fly lines were used: dppFO , dppFO-GFP , dpp-Gal4 , and UAS-FLP ( Matthew Gibson ) , UAS-NLS-mCherry ( Caussinus et al . , 2008 ) , ptc-Gal4 ( w*; P{GawB}ptc559 . 1 ) , P{act5C ( FRT . polyA ) lacZ . nls1}3 , ry506 , tub-Gal80ts ( Bloomington stock center ) .",
"Protocol was described previously ( Harmansa et al . , 2015 ) .",
"Each fly cross was set together with control and >10 wing imaginal discs from each genotype were processed in parallel .",
"If the genotype could be distinguished , experimental and control samples were processed in the same tube .",
"A representative wing disc was shown for all the experiments .",
"Following primary antibodies were used; anti-Dpp ( 1:100; Matthew Gibson ) , anti-phospho-Smad1/5 ( 1:200; Cell Signaling , 9516S ) , anti-Brk ( 1:1000; Gines Morata ) , anti-Wg ( 1:120; DSHB , University of Iowa ) , anti-Ptc ( 1:40; DSHB , University of Iowa ) , anti-β-Galactosidase ( 1:1000; Promega Z378A ) , anti-mCherry ( 1:5000; Nigg lab , University of Basel ) .",
"All the primary antibodies except anti-Dpp antibody were diluted in 5% normal goat serum ( NGS ) ( Sigma ) in PBT ( 0 . 03% Triton X-100/PBS ) .",
"Anti-Dpp antibody was diluted in 5% NGS in Can Get Signal Immunostain Solution B ( TOYOBO ) .",
"All secondary antibodies from the AlexaFluor series were used at 1:500 dilutions .",
"Wing discs were mounted in Vectashield ( H-1000 , Vector Laboratories ) .",
"Images of wing discs were obtained using a Leica TCS SP5 confocal microscope ( section thickness 1 μm ) ."
]
] | [
"The Dpp morphogen gradient derived from the anterior stripe of cells is thought to control growth and patterning of the Drosophila wing disc .",
"However , the spatial-temporal requirement of dpp for growth and patterning remained largely unknown .",
"Recently , two studies re-addressed this question .",
"By generating a conditional null allele , one study proposed that the dpp stripe is critical for patterning but not for growth ( Akiyama and Gibson , 2015 ) .",
"In contrast , using a membrane-anchored nanobody to trap Dpp , the other study proposed that Dpp dispersal from the stripe is required for patterning and also for medial wing disc growth , at least in the posterior compartment ( Harmansa et al . , 2015 ) .",
"Thus , growth control by the Dpp morphogen gradient remains under debate .",
"Here , by removing dpp from the stripe at different time points , we show that the dpp stripe source is indeed required for wing disc growth , also during third instar larval stages ."
] | [
"From the wings of a butterfly to the fingers of a human hand , living tissues often have complex and intricate patterns .",
"Developmental biologists have long been fascinated by the signals – called morphogens – that guide how these kinds of pattern develop .",
"Morphogens are substances that are produced by groups of cells and spread to the rest of the tissue to form a gradient .",
"Depending on where they sit along this gradient , cells in the tissue activate different sets of genes , and the resulting pattern of gene activity ultimately defines the position of the different parts of the tissue .",
"Decades worth of studies into how limbs develop in animals from mice to fruit flies have revealed common principles of morphogen gradients that regulate the development of tissue patterns .",
"Morphogens have been shown to help regulate the growth of tissues in a number of different animals as well .",
"However , how the morphogens regulate tissue size and what role their gradients play in this process remain topics of intense debate in the field of developmental biology .",
"In the developing wing of a fruit fly , a morphogen called Dpp is expressed in a thin stripe located in the center and spreads to the rest of the tissue to form a gradient .",
"Matsuda and Affolter have now characterized where and when the Dpp morphogen must be produced to regulate both the final size of the fly’s wing and the number of cells the wing eventually contains .",
"The experiments involved preventing the production of Dpp in the developing wing in specific cells and at specific stages of development .",
"This approach confirmed that Dpp must be produced in the central stripe for the wing to grow .",
"Bosch , Ziukaite , Alexandre et al . and , independently , Barrio and Milán report the same findings in two related studies , and also conclude that the gradient of Dpp throughout the wing is not required for growth .",
"Further work will be needed to explain how the Dpp signal regulates the growth of the wing .",
"The answer to this question will contribute to a better understanding of the role of morphogens in regulating the size of human organs and how a failure to do so might cause developmental disorders ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and Methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | Proteasome storage granules protect proteasomes from autophagic degradation upon carbon starvation | elife-34532-v2 | [
[
"Protein homeostasis ( proteostasis ) is an essential process by which cells attempt to maintain proteome integrity by regulating protein synthesis , folding , transport and degradation .",
"Key features are mechanisms that control the abundance of regulators necessary for developmental transitions or stress survival; re-cycle the cellular complement of amino acids; and clear mis-folded or dysfunctional proteins and protein complexes ( Hipp et al . , 2014; Vilchez et al . , 2014; Sala et al . , 2017 ) .",
"Importantly , failure to remove aberrant proteins often allows the accumulation of cytotoxic protein aggregates that are frequent hallmarks of aging and an array of degenerative diseases collectively termed aggregation-prone pathologies ( Menzies et al . , 2015; Hjerpe et al . , 2016; Yerbury et al . , 2016 ) .",
"Two major pathways for protein degradation in eukaryotes are the ubiquitin-26S proteasome system ( UPS ) and autophagy .",
"UPS substrates are first tagged with a poly-ubiquitin chain using a highly polymorphic E1-E2-E3 enzymatic cascade , which facilitates their recognition and degradation by the 26S proteasome ( Finley et al . , 2012 ) .",
"This 2 . 5-MDa proteolytic machine is composed of two functionally distinct sub-complexes; the 20S core protease ( CP ) and the 19S regulatory particle ( RP; Lander et al . , 2012; Bhattacharyya et al . , 2014 ) .",
"The CP houses the peptidase active sites responsible for cleaving substrates into short peptides , whereas the RP contains activities for substrate recognition , deubiquitylation , unfolding , and translocation into the CP lumen ( Collins and Goldberg , 2017; Dikic , 2017 ) .",
"While the UPS is exquisitely designed to catabolize proteins individually , it is often not compatible with turnover of larger protein-containing structures .",
"Cells instead employ macroautophagy ( henceforth referred to as autophagy ) , where portions of cytosol are engulfed by a double-membrane-bound structure termed an autophagosome , which is then delivered to the vacuole ( in plants and yeast ) or lysosome ( in animals ) for breakdown ( Reggiori and Klionsky , 2013; Dikic , 2017; Galluzzi et al . , 2017; Marshall and Vierstra , 2018 ) .",
"The delivery of substrates to autophagy is driven by an array of dedicated receptors that recognize appropriate cargo and tether them to the Atg8 ( or LC3 ) protein that coats the enveloping autophagic membrane .",
"In this way , specific proteins , macromolecular complexes , protein aggregates , whole organelles , and even invading pathogens can be selectively eliminated .",
"In addition , less-selective autophagy of cytoplasmic constituents in bulk is often induced upon nutrient starvation as a mechanism to replenish amino acid pools .",
"Besides inducing autophagy , starvation triggers global re-arrangements in cellular transcriptomes , proteomes and metabolomes that ultimately result in cessation of cell growth and entry into quiescence ( Laporte et al . , 2011; Marguerat et al . , 2012; Valcourt et al . , 2012; Honigberg , 2016; Roche et al . , 2017 ) .",
"ATP levels decline during the transition from proliferation to quiescence as glucose depletion restricts glycolysis and thus oxidative phosphorylation .",
"This transition is also accompanied by a drop in intracellular pH and a reduction in protoplasmic fluidity that impacts the dynamics of soluble proteins ( Parry et al . , 2014; Munder et al . , 2016 ) .",
"Widespread re-organization of proteins into membrane-less condensates/granules is also a common phenomenon that might serve to partition , freeze , and/or protect cellular activities until growth resumes ( Laporte et al . , 2008; Narayanaswamy et al . , 2009; O'Connell et al . , 2014; Lee et al . , 2016; Franzmann et al . , 2018; Holehouse and Pappu , 2018 ) .",
"In yeast , one prominent example of cytoplasmic condensates that accumulate as cells enter into stationary phase is the re-localization of proteasomes from the nucleus into cytoplasmic foci known as proteasome storage granules ( PSGs; Laporte et al . , 2008; Chowdhury and Enenkel , 2015; Lee et al . , 2016; Yedidi et al . , 2016; Gu et al . , 2017 ) .",
"A current model proposes that proteasomes first accumulate at the inner face of the nuclear envelope , pass through the nuclear pore , and then gather in an early cytoplasmic intermediate that finally yields mature PSGs ( Peters et al . , 2016 ) .",
"The drop in ATP levels destabilizes the CP-RP interaction ( Bajorek et al . , 2003 ) , and although the CP and RP localize to the same PSGs , they are thought to be targeted by different mechanisms ( Weberruss et al . , 2013 ) .",
"Upon replenishment of the culture medium with a fresh carbon source , ATP levels rapidly increase to trigger the resumption of cell growth , the dissociation of PSGs , and the resorption of proteasomes into the nucleus , all within just a few minutes ( Laporte et al . , 2008 ) .",
"How and why PSGs assemble remains unclear .",
"Factors influencing their formation include intracellular pH , with low pH stimulating PSG formation ( Peters et al . , 2013 ) , the NatB N-terminal acetylation complex ( van Deventer et al . , 2015 ) , the alternative CP capping protein Blm10 ( Weberruss et al . , 2013 ) , and the C-terminal region of Rpn11 , an intrinsic proteasomal deubiquitylase ( DUB; Saunier et al . , 2013 ) .",
"More recently , a high-throughput screen by Gu et al . ( 2017 ) identified 45 genes required for sequestration of the CP into PSGs , 21 of which were also required for sequestration of the RP .",
"Included were factors involved in protein ubiquitylation ( including ubiquitin itself ) and energy regulation .",
"However , with the exception of Blm10 and ubiquitin , none of these proteins accumulated in PSGs , hence their role ( s ) in PSG formation remain largely obscure ( Gu et al . , 2017 ) .",
"In addition to entering PSGs , it was recently reported that proteasomes are rapidly degraded by autophagy via a process termed proteaphagy ( Marshall et al . , 2015; Marshall et al . , 2016; Marshall and Vierstra , 2015; Cohen-Kaplan et al . , 2016; Waite et al . , 2016; Nemec et al . , 2017 ) .",
"Two separate pathways are evident: one that clears inactive proteasomes , and a second that responds to nitrogen deprivation .",
"This former pathway involves the Hsp42-dependent concentration of proteasomes into another cytoplasmic granule termed the insoluble protein deposit ( IPOD ) , extensive ubiquitylation of the particle , and then recognition by the ubiquitin-binding autophagic receptors RPN10 ( in plants ) or Cue5 ( in yeast ) for eventual deposition into autophagosomes ( Marshall et al . , 2015; Marshall et al . , 2016 ) .",
"The machinery underpinning the latter nitrogen-sensitive pathway is less resolved .",
"Besides requiring the core autophagy system , the nutrient-responsive Atg1 kinase complex , and the sorting nexins Snx4/Atg24 , Snx41 and/or Snx42 ( Marshall et al . , 2015; Marshall et al . , 2016; Nemec et al . , 2017 ) , the DUB Ubp3 is needed , implying that the deubiquitylation of an unknown factor is important ( Waite et al . , 2016 ) .",
"Given the contrasting roles of PSGs and proteaphagy in controlling proteasome abundance during carbon and nitrogen starvation , respectively , we hypothesized that the two are inter-related , with the intriguing scenario that proteasomes are specifically recruited to PSGs upon carbon starvation to safeguard them from proteaphagy .",
"Here , we tested this idea by examining a number of conditions and mutants known to impact PSG assembly , and assaying their consequences for proteaphagy .",
"In all cases , PSG assembly and proteaphagy were antagonistic; for example , when PSG formation is blocked , proteaphagy occurs .",
"We confirmed that Blm10 is required for incorporation of the CP into PSGs , and identified the RP-associated protein Spg5 ( Hanna et al . , 2012 ) as integral to the incorporation of the RP into PSGs , thus linking both to proteasome protection .",
"Ubp3 activity is also required for carbon-starvation-induced proteaphagy in the absence of PSG formation , as it is upon nitrogen starvation ( Waite et al . , 2016 ) .",
"Culture growth studies revealed that the ability to form PSGs improves cell fitness , presumably by providing a cache of stored proteasomes that can be rapidly re-mobilized when carbon availability improves .",
"Finally , we demonstrated that Arabidopsis also assembles PSGs upon fixed-carbon starvation via a process requiring the Blm10 ortholog PA200 , making it highly likely that this proteasome protective granule is conserved among eukaryotes ."
],
[
"While yeast proteasomes undergo rapid proteaphagy in response to nitrogen starvation ( Marshall et al . , 2016 ) , recent results from Waite et al . , 2016 suggested that proteasomes are not similarly degraded in response to carbon starvation , even though both conditions activate bulk autophagy ( Takeshige et al . , 1992; Adachi et al . , 2017 ) .",
"To further investigate this possibility , we exploited haploid strains in which the CP subunit Pre10 ( α7 ) or the RP subunit Rpn5 were expressed with C-terminal GFP tags .",
"These reporters allowed us to track proteaphagy by ‘GFP-release’ immunoblot assays that detect the liberation of stable , free GFP from the fusion proteins following their autophagic transport to vacuoles , and by confocal fluorescence microscopy that visualizes the movement of GFP-tagged proteasomes from the nucleus , where over 80% of the particles reside ( Enenkel et al . , 1998; Russell et al . , 1999 ) , to other cellular locations such as the vacuole ( Marshall et al . , 2016; Waite et al . , 2016 ) .",
"Importantly , by measuring the ratio of free GFP to the fusion , and by morphometric analysis of confocal images ( e . g . Figure 1F ) , we could quantitatively assess proteasome fates ( Marshall et al . , 2015; Marshall et al . , 2016 ) .",
"As shown by the GFP-release assays in Figure 1A , proteasomes in wild-type cells undergo rapid proteaphagy upon nitrogen starvation , as evidenced by the accumulation of free GFP from both Pre10-GFP and Rpn5-GFP reporters , which could be seen when total cell lysates were immunoblotted with anti-GFP antibodies .",
"Greater than 90% of both fusions disappeared within 1 day of the onset of starvation , concomitant with the strong accumulation of free GFP .",
"By contrast , loss of the fusions and the release of free GFP were substantially slower upon carbon starvation , which was generated by switching cells from growth on non-fermentable carbon ( i . e . glycerol ) to medium lacking this carbon source ( Takeshige et al . , 1992; Adachi et al . , 2017 ) .",
"Here , free GFP was undetectable within the first 2 days , with only small amounts appearing subsequently ( ~8–12% after 6 days; Figure 1A ) .",
"This relative absence of proteaphagy occurred despite that fact that the carbon starvation regime employed here effectively suppressed culture growth ( Figure 1—figure supplement 1A ) and stimulated bulk autophagy , as judged by the increased activity of the Pho8Δ60 reporter ( Noda and Klionsky , 2008 ) and by the release of free GFP from GFP-Atg8 , which both measure autophagic flux ( Figure 1—figure supplement 1B and C ) .",
"This modest accumulation of free GFP seen from the Pre10-GFP and Rpn5-GFP reporters was autophagy-dependent , as it was absent in mutants eliminating the core autophagy component Atg7 , or the Atg13 regulatory subunit of the Atg1 kinase complex that activates autophagy in response to nutrient deprivation ( Figure 1B ) .",
"Thus , proteaphagy still occurs in yeast upon carbon starvation , but at a substantially slower rate .",
"Surprisingly , when nitrogen and carbon starvation were combined , we found that rapid proteaphagy during nitrogen starvation was suppressed by the simultaneous lack of carbon .",
"Although 8 hr of nitrogen starvation induced rapid clearance of proteasomes , as measured by loss of the Pre10-GFP and Rpn5-GFP reporters together with the appearance of free GFP , little to no clearance was evident in cells starved for both nitrogen and carbon ( Figure 1C ) .",
"This contrasted with other forms of selective autophagy , with the turnover of GFP-tagged substrates related to the cytoplasm-to-vacuole targeting ( CVT ) pathway ( Ape1; Shintani et al . , 2002 ) , pexophagy ( Pex14; Reggiori et al . , 2005 ) , ribophagy ( Rpl25; Kraft et al . , 2008 ) and , to a lesser extent , mitophagy ( Om45; Kanki and Klionsky , 2008 ) being induced by all three starvation regimes ( Figure 1—figure supplement 1D ) .",
"For each substrate , rapid loss of the fusion concomitant with release of free GFP was readily evident upon nitrogen , carbon , or simultaneous nitrogen and carbon starvation .",
"As an aside , we saw slightly slower mitophagy upon carbon starvation versus nitrogen starvation , in agreement with prior observations showing that the rate of mitophagy is dampened in cells exposed to non-fermentable carbon sources , presumably to maintain respiration ( Kanki and Klionsky , 2008 ) .",
"Taken together , it appears that carbon starvation selectively suppresses proteaphagy , despite up-regulating both bulk autophagy and other forms of selective autophagy .",
"Confocal fluorescence microscopy of cells expressing PRE10-GFP or RPN5-GFP confirmed that the rapid transport of proteasomes to the vacuole upon nitrogen starvation was indeed suppressed by simultaneous carbon starvation .",
"Upon switching exponentially growing cells from nitrogen-rich to nitrogen-starvation medium , the GFP signals moved from a mainly nuclear localization to a diffuse vacuolar pattern within 24 hr ( Figure 1E and F ) .",
"Strikingly , this relocation was not evident in cells starved for both nitrogen and carbon .",
"Instead , the Pre10-GFP and Rpn5-GFP signals migrated toward the nuclear periphery and into brightly fluorescent , large ( ~0 . 5 μm ) puncta within the cytoplasm ( Figure 1E and F ) .",
"The appearance of these foci was extremely rapid , being detectable in 50% of the cells within 1 hr of carbon starvation and in 95% of the cells after 4 hr ( Figure 1D and Figure 1—figure supplement 2A and B ) .",
"The time course for entry of Pre10-GFP and Rpn5-GFP into these cytoplasmic puncta ( i . e . within 1 hr ) was faster than the up-regulation of bulk autophagy ( at 2 to 4 hr ) , suggesting that formation of these foci is an early response to carbon deprivation separate from autophagy .",
"In support , the foci were visible in a number of mutants missing factors required for autophagy initiation , including Atg1 , Atg11 , Atg13 and Atg17 that help scaffold the pre-autophagosomal structure ( PAS ) , indicating that they arise independent of autophagy ( Figure 1—figure supplement 2C ) .",
"The appearance of these foci was also rapidly reversible; upon switching starved PRE10-GFP or RPN5-GFP cells to fresh carbon-containing medium , the GFP signals returned to diffuse cytoplasmic and nuclear patterns within 30 min ( Figure 1—figure supplement 2D ) .",
"We previously described the sequestration of proteasomes into cytoplasmic IPODs , which represents an intermediate step in the autophagic clearance of inactive proteasomes ( Marshall et al . , 2016 ) .",
"However , the proteasome-containing puncta emerging after carbon starvation were different , as co-localization studies with Pre10-GFP and the IPOD marker Rnq1-mCherry ( Kaganovich et al . , 2008 ) detected separate cytoplasmic foci in greater than 90% of cells ( Figure 1H ) .",
"Moreover , while the accretion of inactive proteasomes into IPODs requires the Hsp42 chaperone ( Figure 1G; Marshall et al . , 2016 ) , the rapid accumulation of proteasomes into the cytoplasmic puncta seen here upon carbon starvation still occurred in Δhsp42 cells ( Figure 1G; Peters et al . , 2016 ) .",
"These data place the proteasome-containing foci seen upon carbon starvation as different from IPODs .",
"Numerous studies have described the accumulation of PSGs in stationary phase yeast which resemble the proteasome-containing puncta seen here that form during carbon starvation ( Laporte et al . , 2008; Peters et al . , 2013; Saunier et al . , 2013; van Deventer et al . , 2015; Lee et al . , 2016; Gu et al . , 2017; reviewed in Chowdhury and Enenkel , 2015; Yedidi et al . , 2016 ) .",
"Consequently , we hypothesized that these puncta are PSGs , which could protect proteasomes from proteaphagic degradation by sequestering them away from the autophagic machinery .",
"To test this proposed inverse relationship between PSG-type puncta and proteaphagy , we examined the accumulation of these puncta and rates of proteaphagy under several situations previously shown to influence PSG accumulation .",
"One such situation involves the protein acetylase NatB , one of three acetylation complexes in yeast that modify the N-terminus of proteins in a sequence-dependent manner ( Polevoda et al . , 1999 ) .",
"Genetic analysis of both its catalytic ( Nat3 ) and regulatory ( Mdm20 ) subunits recently demonstrated that NatB is essential for PSG assembly ( van Deventer et al . , 2015 ) .",
"Here , we confirmed this observation by showing that both the Pre10-GFP and Rpn5-GFP reporters failed to localize to PSG-type foci in Δnat3 and Δmdm20 cells subjected to carbon starvation ( Figure 2A and Figure 2—figure supplement 1A and B ) .",
"Instead , both reporters accumulated in the vacuole , as expected if proteaphagy became the alternative .",
"Likewise , whereas little free GFP accumulated from both reporters even upon extended carbon starvation of wild-type cells , rapid GFP accumulation was seen in Δnat3 and Δmdm20 cells ( Figure 2B and Figure 2—figure supplement 1C ) .",
"Both the accumulation of PSGs and the stability of the Pre10-GFP and Rpn5-GFP fusions were restored to wild-type levels when Δnat3 cells were rescued with HA-tagged Nat3 , but not with the catalytically defective Nat3 ( C97A ) -HA variant ( Figure 2A and B , and Figure 2—figure supplement 1B ) , demonstrating that an active NatB complex is essential for PSG assembly and proteaphagy suppression .",
"In a similar fashion , we tested a pair of mutants affecting the intrinsic deubiquitylase of the RP , Rpn11 ( termed rpn11-m1 and rpn11-m5; for details see Materials and methods ) , which were previously shown to prevent or delay entry of the RP , but not the CP , into PSGs ( Saunier et al . , 2013 ) .",
"Accordingly , we found that both the rpn11-m1 and rpn11-m5 alleles suppressed formation of PSGs containing Rpn5-GFP upon carbon starvation , and instead allowed concentration of the reporter in vacuoles ( Figure 2C and Figure 2—figure supplement 1B ) .",
"The mutants also promoted the rapid release of free GFP from the Rpn5-GFP reporter but not the Pre10-GFP reporter , indicating that proteaphagy of the RP , but not the CP , was now occurring in these carbon-starved cells ( Figure 2D ) .",
"In both assays , the responses of rpn11-m5 cells were restored to wild type when complemented with an RPN11-FLAG transgene ( Figure 2C and D , and Figure 2—figure supplement 1B ) .",
"Interestingly , small amounts of free GFP accumulated from the Rpn5-GFP reporter in the rpn11-m1 mutant even in the absence of starvation ( Figure 2D ) .",
"This slight accumulation was absent in Δatg7 and Δcue5 backgrounds ( data not shown ) , suggesting that it represents proteaphagy of compromised RPs , as previously observed for the rpn5ΔC mutation that also impairs RP assembly ( Peters et al . , 2015; Marshall et al . , 2016 ) .",
"Intracellular pH also influences yeast PSG abundance , which can be altered by mutating the vacuolar V-ATPase complex or modifying the pH of the growth medium ( Peters et al . , 2013 ) .",
"In the latter situation , growth at low pH , as occurs during quiescence , accelerates the accumulation of PSGs , while growth in high pH medium dampens their accumulation .",
"To test if pH inversely impacts proteaphagy , we grew cells expressing PRE10-GFP or RPN5-GFP in pH 3 . 0 , 6 . 0 and 9 . 0 media buffered to prevent natural acidification of the cultures ( Wasko et al . , 2013 ) , and containing the ionophore carbonyl-cyanide-3-chlorophenylhydrazone ( CCCP ) to suppress effective regulation of internal pH ( Orij et al . , 2009 ) .",
"While PSG accumulation , as assessed by confocal fluorescence microscopy , was more robust at low pH and dampened at high pH ( Figure 3A; Peters et al . , 2013 ) , we found that rates of proteaphagy , as measured by release of free GFP from the Pre10-GFP and Rpn5-GFP reporters , were more robust at high pH and dampened at low pH ( Figure 3B ) .",
"High pH also encouraged transport of the GFP signals to the vacuole , in agreement with increased proteaphagy ( Figure 3A ) .",
"This rapid appearance of free GFP from both reporters in pH 9 . 0 medium was blocked in Δatg1 , Δatg7 and Δatg13 mutants , but allowed in Δcue5 mutants ( Figure 3C ) , indicating that clearance of proteasomes at high pH occurred via the nutrient-responsive proteaphagy pathway , and not by the pathway that clears inactive proteasomes ( Marshall et al . , 2016; Waite et al . , 2016 ) .",
"Certainly , changes in intracellular pH likely have effects on cell growth that could indirectly impact autophagy .",
"Indeed , we found that culture growth was robust at pH 6 . 0 , but substantially slower in pH 3 . 0 or pH 9 . 0 media ( Figure 1—figure supplement 1A ) .",
"However , changes in the growth medium pH only marginally impacted bulk autophagy , based on measurements of autophagic flux using the Pho8Δ60 reporter ( Figure 3D ) .",
"During a screen for factors inhibiting PSG assembly during quiescence , several proteins that regulate energy balance and ATP levels were identified ( Gu et al . , 2017 ) , suggesting that PSG formation accelerates upon energy depletion .",
"To study how reductions in ATP might commensurately impact proteaphagy , we treated PRE10-GFP and RPN5-GFP cells with 2-deoxyglucose ( 2-DG ) , a glycolysis inhibitor that depresses intracellular ATP levels ( Wick et al . , 1957 ) .",
"As predicted , pre-treatment of non-starved , wild-type cells with 5 mM 2-DG rapidly induced the sequestration of proteasomes into PSG-type puncta , as observed by confocal fluorescence microscopy ( Figure 3E ) .",
"Their appearance strongly resembled the puncta observed following carbon starvation , including their rapid reversibility when 2-DG was removed from the culture medium ( Figure 1—figure supplement 2E ) .",
"In fact , PSGs even appeared in nitrogen-starved cells pre-treated with 2-DG , as they do in cells subjected to simultaneous nitrogen and carbon starvation .",
"In contrast , when assayed for proteaphagy by the GFP-release assay of both reporters , we found that 2-DG had the inverse effect; like carbon starvation , 2-DG dampened proteaphagy induced by nitrogen starvation ( Figure 3F ) .",
"Taken together , we found that conditions that suppress PSG formation ( the Δnat3 , Δmdm20 , rpn11-m1 and rpn11-m5 mutations , or growth at high pH ) accentuated proteaphagy , while those that enhanced PSG formation ( low pH and 2-DG ) instead dampened proteaphagy , strongly suggesting that the two processes are inversely related .",
"Blm10 ( known as PA200 in plants and mammals ) is a well-described CP capping factor , where it has been proposed to help assemble α- and β-subunits into the CP barrel , stabilize the complex before RP docking , and/or possibly promote nuclear import of the CP ( Schmidt et al . , 2005; Sadre-Bazzaz et al . , 2010; Dange et al . , 2011; Weberruss et al . , 2013 ) .",
"This 246-kDa protein has also been implicated in PSG assembly , where it appears essential for sequestering the CP specifically ( Weberruss et al . , 2013 ) .",
"Consequently , we hypothesized that absence of Blm10 could lead to proteaphagy of the CP by limiting its incorporation into PSGs .",
"Indeed , we found by confocal fluorescence microscopy that the Pre10-GFP reporter did not localize into PSGs in Δblm10 cells after 24 hr of carbon starvation , but instead appeared in the vacuole ( Figure 4A and B ) , strongly suggesting an absolute requirement for Blm10 in directing the CP to PSGs .",
"By contrast , the Rpn5-GFP reporter behaved normally in carbon-starved Δblm10 cells and rapidly coalesced into PSGs ( Figure 4A and B ) .",
"The appearance of Pre10-GFP in Δblm10 vacuoles upon carbon starvation was blocked in Δatg7 and Δatg13 backgrounds , where the Pre10-GFP reporter instead remained in the cytosol and nucleus , but not in the Δcue5 background ( Figure 4A and B ) , indicating that autophagic transport of the CP proceeds via the nutrient-responsive proteaphagy pathway , and not the pathway that clears inactive proteasomes .",
"Moreover , when we assayed proteasomes in Δblm10 cells by the GFP-release assay , we found that the CP now underwent proteaphagy upon carbon or simultaneous nitrogen and carbon starvation , as evidenced by the rapid accumulation of free GFP from the Pre10-GFP reporter ( Figure 4C and Figure 4—figure supplement 1A ) .",
"Further supporting a nutrient-responsive route , this accumulation of free GFP was blocked in Δatg1 , Δatg7 and Δatg13 cells , but not in Δcue5 cells ( Figure 4D ) .",
"The RP did not encounter the same fate in starved Δblm10 cells , as the release of free GFP from Rpn5-GFP was not similarly accelerated ( Figure 4C ) .",
"Given the stable association of Blm10 with the CP , which can bind to both ends of the CP barrel ( Schmidt et al . , 2005; Sadre-Bazzaz et al . , 2010 ) , it was likely that Blm10 also enters PSGs .",
"To confirm this possibility , we tested for co-localization of Blm10 and the CP by confocal fluorescence microscopy of cells expressing PRE10-GFP and mCherry-BLM10 .",
"The mCherry fusion appeared to retain the activity of non-modified Blm10 , as it could reverse the accelerated turnover of Pre10-GFP in Δblm10 cells ( Figure 4—figure supplement 1B ) .",
"Under carbon-replete conditions , the two reporters had similar intracellular distributions , with a strong enrichment in the nucleus , moderate signal in the cytoplasm , and little to no signal in the vacuole ( Figure 4E ) .",
"Following carbon starvation , mCherry-Blm10 rapidly migrated into PSGs along with Pre10-GFP , strongly suggesting that the CP and Blm10 reside in the same granules ( Figure 4E ) .",
"Similar accretion was seen in cells expressing RPN5-GFP and mCherry-BLM10 ( Figure 4—figure supplement 1C ) , indicating that these PSGs also contain the RP , as previously reported ( Laporte et al . , 2008 ) .",
"This finding corresponds with the recent study by Gu et al . ( 2017 ) , who observed GFP-tagged Blm10 in PSGs upon entry of yeast cells into quiescence .",
"To assess if Blm10 also undergoes autophagy , we examined the Blm10-GFP reporter using the GFP-release assay .",
"Free GFP was evident within hours of nitrogen starvation , indicating that Blm10 is a target of autophagy , possibly through its connection to the CP ( Figure 4F ) .",
"Conversely , free GFP did not accumulate in cells starved for carbon or both nitrogen and carbon ( Figure 4F ) , again strongly implicating PSGs as a mechanism to not only safeguard the CP from proteaphagy , but also Blm10 bound to the CP .",
"Given the possibility that other factor ( s ) help sequester the RP into PSGs upon carbon starvation , as Blm10 does for the CP , we searched for likely candidates among known RP-interacting proteins .",
"One possibility was Ecm29 , which co-purifies with the 26S particle ( Leggett et al . , 2002; Marshall et al . , 2016 ) and appears to have roles in proteasome assembly and quality control ( Lehmann et al . , 2010; Park et al . , 2011; De La Mota-Peynado et al . , 2013; Wang et al . , 2017 ) .",
"However , when the Δecm29 mutation was introduced into PRE10-GFP or RPN5-GFP cells , we found by GFP-release assays that , as in wild type , the autophagic clearance of the CP and RP was slow during carbon starvation ( Figure 4—figure supplement 1D ) , implying RP-containing PSGs still accumulate without Ecm29 .",
"We additionally investigated the roles of Blm10 and Ecm29 in nitrogen starvation- and inhibitor-induced proteaphagy; however , neither Δblm10 nor Δecm29 cells showed any defect in these pathways , as judged by rapid accumulation of free GFP from the Pre10-GFP and Rpn5-GFP reporters after removal of nitrogen from the growth medium or addition of MG132 , respectively ( Figure 4—figure supplement 1E ) .",
"The lack of an effect for Ecm29 in inhibitor-induced proteaphagy was noteworthy , given its proposed role in identifying dysfunctional proteasomes ( Lehmann et al . , 2010 ) .",
"Another intriguing candidate was Spg5 , which was previously shown by Hanna et al . ( 2012 ) to bind the AAA-ATPase ring of the RP but not the complete 26S particle , and to regulate proteasome structure and function in stationary-phase yeast cells .",
"Moreover , evaluation of large-scale transcriptomic studies ( Gasch et al . , 2000; Martinez et al . , 2004 ) revealed that SPG5 is highly expressed following either sudden carbon starvation induced by switching the growth medium , or by more gradual carbon starvation that occurs as cells enter stationary phase , both of which correlate with the timing of PSG formation .",
"In fact , our focused transcript analysis of an assortment of proteasome genes , CUE5 , BLM10 and SPG5 revealed that only the SPG5 mRNA dramatically increases in abundance in carbon-starved cells ( Figure 5—figure supplement 1 ) .",
"As above with Blm10 , we tested the importance of Spg5 to PSG formation and proteaphagy using the confocal fluorescence microscopic and GFP-release assays .",
"For the CP , Δspg5 cells starved for carbon behaved like wild type and rapidly coalesced Pre10-GFP into PSGs within a few hours after the onset of starvation ( Figure 5A and B ) .",
"In contrast , Δspg5 cells failed to similarly sequester Rpn5-GFP into PSGs , with the reporter instead re-localizing to vacuoles ( Figure 5A and B ) .",
"However , unlike the relationship of the CP and Blm10 , the deposition of the RP into PSGs upon carbon starvation was not completely dependent on Spg5 , as a sizable percentage of Δspg5 cells contained PSGs labelled with Rpn5-GFP after prolonged starvation ( Figure 5E; Saunier et al . , 2013 ) , suggesting that absence of Spg5 delays , rather than blocks , deposition of the RP into PSGs .",
"Delivery of Rpn5-GFP to the vacuole in Δspg5 cells was prevented in the Δatg7 and Δatg13 , but not in the Δcue5 backgrounds , again indicating that the vacuolar transport of the RP depended on the nutrient-responsive proteaphagy pathway and not the pathway that clears inactive proteasomes ( Figure 5A and B ) .",
"Accordingly , when we assayed proteasomes by the GFP-release assay , we found that the RP indeed underwent proteaphagy in Δspg5 cells , as evidenced by the rapid accumulation of free GFP from the Rpn5-GFP reporter after 1 day of carbon starvation , a processes again requiring Atg1 , Atg7 and Atg13 , but not Cue5 ( Figure 5C and D ) .",
"However , the CP did not encounter the same fate , as the accumulation of free GFP from Pre10-GFP was not accelerated in carbon-starved Δspg5 cells ( Figure 5C ) .",
"The time course for entry of Rpn5-GFP into vacuoles in Δspg5 cells was noticeably slower than the time taken for Rpn5-GFP to enter into PSGs in wild-type cells , implying that PSG formation is faster than proteaphagy ( Figure 5E ) .",
"Given the possibility that Spg5 binds to the RP and helps shepherd the sub-particle into PSGs , as Blm10 appears to do for the CP , we tested for their co-localization by confocal fluorescence microscopy of cells expressing RPN5-GFP and mCherry-SPG5 .",
"The mCherry fusion appeared to retain the activity of non-modified Spg5 , as its expression could reverse the accelerated turnover of Rpn5-GFP in Δspg5 cells ( Figure 4—figure supplement 1B ) .",
"Under carbon-replete conditions , the two reporters had similar intracellular distributions , with a strong enrichment in the nucleus , moderate signal in the cytoplasm , and little to no signal in the vacuole , similar to that observed with mCherry-Blm10 and Pre10-GFP ( Figure 5F ) .",
"However , unlike with Blm10 , mCherry-Spg5 only rarely co-migrated with Rpn5-GFP into PSGs in carbon-starved cells; puncta containing both Rpn5-GFP and mCherry-Spg5 were visible in just 12% of over 200 cells analysed .",
"Instead , the mCherry reporter mostly retained its nuclear/cytoplasmic pattern , implying that Spg5 does not generally follow the RP into PSGs ( Figure 5F ) .",
"Similarly , mCherry-Spg5 only rarely co-localized with PSGs containing Pre10-GFP ( in just 6% of over 200 cells; Figure 4—figure supplement 1C ) .",
"This lack of association was also confirmed by mass spectrometry of 26S proteasomes; whereas Blm10 was easily detected in proteasomes affinity-purified from carbon-starved cells ( Marshall et al . , 2016 ) , we could not detect Spg5 ( data not shown ) .",
"Previous studies revealed that the CP and RP dissociate upon entry of yeast cultures into stationary phase , presumably because of depleted ATP levels ( Bajorek et al . , 2003 ) , but that they are eventually found together in the same PSGs ( Laporte et al . , 2008 ) .",
"While transport of both sub-particles into PSGs could occur following re-assembly into 26S complexes , results by Weberruss et al . ( 2013 ) and us ( this report ) showing that Blm10 and Spg5 mediate separate delivery of the CP and RP , respectively , implied that the two sub-complexes are sequestered individually via distinct pathways that shield each from autophagy .",
"To address this possibility , we exploited strains in which proteasome subunits ( Pre1 ( β4 ) from the CP and Rpn11 from the RP ) were tagged with Protein A to facilitate their rapid and efficient affinity-purification ( Leggett et al . , 2005 ) , and analyzed the composition of proteasomes purified from wild-type , Δblm10 and Δspg5 cells after 0 , 1 , or 5 days of carbon starvation , in search for differential CP versus RP enrichment .",
"The 26S proteasomes purified from wild-type cells contained the characteristic SDS-PAGE ladder of RP and CP subunits throughout the starvation period , regardless of whether proteasomes were purified via the CP or RP , indicating that stable 26S complexes persist in carbon-starved yeast .",
"Comparisons of core subunits , as detected by silver staining of total protein or by immunoblotting with antibodies against Pre4 ( β7 ) , Rpt1 , Rpn5 and Rpn8 , failed to see changes in relative subunit abundance after 1 and 5 days of carbon starvation versus the non-starved controls ( Figure 6A and B ) .",
"However , when proteasomes were purified via the CP from the Δblm10 and Δspg5 backgrounds , a substantial reduction in the amount of co-purifying RP and its corresponding Rpt1 , Rpn5 and Rpn8 subunits was observed as carbon starvation progressed .",
"Similarly , when proteasomes were purified via the RP in these two backgrounds , less CP and its corresponding Pre4 subunit were co-purified ( Figure 6A and B ) .",
"While other scenarios are possible , the most parsimonious is that CP and RP dissociate upon carbon starvation but can be co-purified if both are deposited into PSGs .",
"If one sub-particle is blocked from entry into PSGs , its enrichment during purifications of the other sub-particle is diminished .",
"For further evidence supporting this dissociation , we measured the proteolytic activity of the CP from whole cell extracts prepared 1 day after carbon starvation , when the levels of RP and CP were unaffected ( see Figure 1A ) , using either a substrate effective for the CP alone ( Suc-LLVY-amc ) or a substrate that requires the RP for import ( Mca-AKVYPYPME-Dpa ( Dnp ) -amide , also known as LFP; Smith et al . , 2005 ) .",
"As a control , we also measured CP activity in the rpn5ΔC mutant , which compromises binding of the RP to the CP ( Peters et al . , 2015 ) .",
"RP-independent CP activity was indistinguishable in cells starved for nitrogen , carbon , or both nitrogen and carbon ( Figure 6C ) , implying that the activity of the CP alone was unaltered by PSG formation .",
"In contrast , RP-dependent CP activity was significantly dampened after carbon and simultaneous nitrogen and carbon starvation , close to that seen for non-starved rpn5ΔC cells , implying that the CP and RP are less associated under these growth conditions ( Figure 6C ) .",
"A similar drop in RP-dependent CP activity was seen for nitrogen-starved cells , in agreement with previous studies showing that the CP and RP separate under this starvation condition as well ( Waite et al . , 2016; Nemec et al . , 2017 ) .",
"In addition to the core autophagy machinery , the deubiquitylase Ubp3 has been connected to proteaphagy in yeast subjected to nitrogen starvation , where it promotes clearance of the CP but not the RP ( Waite et al . , 2016 ) .",
"Ubp3 has also been implicated in both mitophagy and ribophagy ( Kraft et al . , 2008; Müller et al . , 2015 ) , thus raising the possibility that it has a general role in starvation-induced autophagy of organelles and protein complexes .",
"As such , we examined PSG assembly and proteaphagy in carbon-starved Δblm10 , Δnat3 and Δspg5 cells also harboring the Δubp3 mutation by tracking the Pre10-GFP and Rpn5-GFP reporters .",
"As seen above by confocal fluorescence microscopy , delivery of Pre10-GFP into PSGs proceeded normally in wild-type cells and was blocked in both Δblm10 and Δnat3 cells , with the signal instead moving to the vacuole upon carbon starvation ( Figure 7A and Figure 7—figure supplement 1A ) .",
"In Δubp3 cells , the Pre10-GFP signal behaved like wild type and entered PSGs , indicating that Ubp3 is not required for PSG formation .",
"However , when the Δubp3 mutation was combined with either the Δblm10 or Δnat3 mutations , Pre10-GFP failed to enter the vacuole and instead appeared trapped in the nucleus and cytoplasm ( Figure 7A and Figure 7—figure supplement 1A ) .",
"The same pattern was not true for Rpn5-GFP; although this reporter entered PSGs in wild-type cells and vacuoles in Δnat3 and Δspg5 cells upon carbon-starvation , it retained the corresponding responses in Δubp3 , Δnat3 Δubp3 and Δspg5 Δubp3 cells ( Figure 7B and Figure 7—figure supplement 1B ) .",
"When then assayed for proteaphagy by the GFP-release assay , we confirmed that Ubp3 selectively affects the CP .",
"Accumulation of free GFP from the Pre10-GFP reporter was accelerated in carbon-starved Δblm10 or Δnat3 cells , but its release was blocked in Δblm10 Δubp3 or Δnat3 Δubp3 cells , while the release of free GFP from the Rpn5-GFP reporter was equally rapid in Δnat3 and Δspg5 cells with or without the Δubp3 mutation ( Figure 7C and Figure 7—figure supplement 1C and D ) .",
"Ubp3 associates with a co-factor , Bre5 , which promotes its activity ( Cohen et al . , 2003; Kraft et al . , 2008 ) .",
"From analysis of Δbre5 cells , we found that this co-factor is also required for carbon starvation-induced proteaphagy of the CP .",
"When the Pre10-GFP reporter was examined in Δblm10 Δbre5 cells by the GFP-release assay , little free GFP accumulated even after prolonged carbon starvation , while its accumulation was robust after 1 day in Δblm10 cells wild-type for BRE5 ( Figure 7D ) .",
"Complementation studies showed that active Ubp3 and Bre5 are required for proteaphagy of the CP in Δblm10 cells .",
"Whereas UBP3-HA and BRE5-HA transgenes readily restored proteaphagy of the CP in Δblm10 Δubp3 and Δblm10 Δbre5 cells , respectively , similar transgenes expressing alanine substitution mutants of Ubp3 replacing either the catalytic cysteine at residue 469 ( UBP3 ( C469A ) -HA; Cohen et al . , 2003 ) or the Bre5-binding site at residues 208 to 211 ( UBP3 ( LFIN-AAAA ) -HA; Li et al . , 2005 ) were ineffective ( Figure 7D ) .",
"Although Ubp3 appears vital for both nitrogen starvation- and carbon starvation-induced proteaphagy ( this study; Waite et al . , 2016 ) , possible roles for the other 19 yeast DUBs remained unexplored .",
"Consequently , we examined most other ubiquitin-specific DUBs in yeast ( the exceptions being the essential DUB Rpn11 and Yuh1 , which has greater specificity for the ubiquitin relative Rub1 ) .",
"While accumulation of free GFP from Pre10-GFP upon carbon starvation was clearly evident in the Δblm10 mutant and was blocked in the Δblm10 Δubp3 double mutant , deletion of the 17 other DUBs individually had no effect ( Figure 7—figure supplement 1E ) .",
"These data imply that there is a specific role for Ubp3 in proteaphagy , as opposed to deubiquitylation more generally .",
"Because PSGs appear to protect proteasomes from autophagic degradation in response to carbon starvation , we speculated that these granules might be beneficial for cell survival .",
"In particular , the sequestration of proteasomes into PSGs could help cells resume growth as carbon availability improves by providing a rapidly re-mobilizable cache of proteasomes .",
"To test this hypothesis , we examined the growth resumption of yeast cultures in nutrient-rich medium following exposure to carbon and/or nitrogen starvation using mutant backgrounds ( Δblm10 , Δnat3 , Δspg5 and/or Δupb3 ) or culture conditions ( 2-DG ) that impact PSG accumulation and/or proteaphagy ( see above ) .",
"Initially , wild-type yeast cells were subjected to 24 hr of carbon , nitrogen , or simultaneous carbon and nitrogen starvation , before being returned to nutrient-rich medium , at which point their ability to resume growth was monitored by measurement of culture density ( Figure 8—figure supplement 1A ) .",
"While cells not subjected to starvation grew rapidly without lag , reaching an OD600 of more than 8 . 0 after 6 hr of growth , cells subjected to nitrogen starvation suffered a 3 to 4 hr lag before resuming growth , reaching an OD600 of only ~2 . 0 after 6 hr ( Figure 8A and B ) .",
"By contrast , carbon starvation only modestly delayed growth resumption by itself , while remarkably accelerating re-growth of cells also missing nitrogen , indicating that the growth defect caused by nitrogen starvation can be partially overcome by a lack of carbon , in much the same way as carbon starvation protects proteasomes from autophagy even when cells are starved for nitrogen ( Figure 8A and B ) .",
"As a further connection of this growth phenotype to proteasome levels , we exposed nitrogen- and/or carbon-starved cells to the amino acid analogs canavanine and p-fluorophenylalanine; survival under these conditions would be aided by the capacity of proteasomes to clear abnormal proteins incorporating these analogs ( Finley et al . , 2012 ) .",
"Whereas culture growth in the presence of the analogs was dramatically impaired in cells pre-exposed to nitrogen starvation ( ~10% of non-treated cells after 6 hr ) , which would have depleted proteasomes by autophagy , culture growth was better for analog-treated cells starved for either carbon alone or nitrogen and carbon together , and was comparable to non-starved cells ( ~30% of untreated cells ) , all three of which would have avoided autophagic clearance of their proteasomes ( Figure 8C and D ) .",
"As a complementary approach , we examined the resumption of growth for wild-type cells first treated with 2-DG for 6 hr prior to ( and during ) nitrogen starvation , which promotes PSG formation and protects against proteaphagy ( Figure 3E and F ) , and again monitored the ability of these cells to resume growth upon a switch back to carbon- and nitrogen-rich medium lacking 2-DG .",
"As above with simultaneous nitrogen and carbon starvation , we found that cells pre-treated with 2-DG prior to the onset of nitrogen starvation resumed growth more rapidly than cells subjected to nitrogen starvation alone ( Figure 8E , F and G ) .",
"We next investigated the growth resumption of cells harbouring the Δblm10 , Δnat3 and Δspg5 mutations described above .",
"None of the mutants impaired the robust resumption of cell growth in cultures transferred from nutrient-rich medium back into nutrient-rich medium .",
"However , as predicted , Δnat3 , Δblm10 and Δspg5 cells , which block PSG formation and accelerate proteaphagy , showed a substantial delay in growth resumption after exposure to carbon starvation as compared to wild-type cells ( Figure 8H , I , J , K , L , M , N , O and P ) .",
"In agreement with its partial impact on PSG assembly and proteaphagy , the delayed growth response of Δspg5 cells was milder than those of Δnat3 and Δblm10 cells ( Figure 8N and O ) .",
"In all cases , these growth defects could be rescued by expressing the corresponding wild-type transgenes ( mCherry-BLM10 , mCherry-SPG5 or NAT3-HA ) , but not one encoding the catalytically inactive C97A variant of Nat3 ( Figure 8—figure supplement 1B , C , D , G , H , I , J , K and L ) .",
"Based on the observation that the Δubp3 mutation will reverse the effects of the Δblm10 and Δnat3 mutations in allowing proteaphagy in the absence of PSG assembly ( Figure 7A and C , and Figure 7—figure supplement 1A and C; Waite et al . , 2016 ) , we additionally investigated how the growth of carbon-starved Δubp3 , Δblm10 Δupb3 and Δnat3 Δubp3 cells resumed in nutrient-rich medium .",
"Whereas the growth of Δupb3 cells was mostly indistinguishable from wild-type , and both Δblm10 and Δnat3 cells showed a substantial delay in growth resumption following carbon starvation , the growth of Δblm10 Δupb3 and Δnat3 Δubp3 cells was intermediate , indicating that the lack of Ubp3 can partially suppress the lack of Blm10 or Nat3 , as it does for proteaphagy ( Figure 8H , I , J , K , L and M ) .",
"Again the effects of Ubp3 required its DUB activity , as expression of UBP3-HA restored the slow growth phenotype to Δblm10 Δupb3 cells , while the catalytically dead C496A mutant , or the FLIN-AAAA variant that cannot bind Bre5 , were ineffective ( Figure 8—figure supplement 1E and F ) .",
"By contrast , Δspg5 Δubp3 cells did not show improved growth recovery following carbon starvation compared to Δspg5 cells alone ( Figure 8N , O and P ) , in agreement with the lack of a role for Ubp3 in delivering RPs into PSGs ( Figure 7B and C , and Figure 7—figure supplement 1B and D ) .",
"For further support that the autophagic degradation of proteasomes is at least partly responsible for delaying the resumption of culture growth following carbon starvation , we assayed the growth of Δblm10 cells in which the core autophagy component Atg7 was eliminated .",
"The Δatg7 , Δblm10 and Δatg7 Δblm10 cells all grew at similar rates in the absence of starvation , while Δatg7 and Δblm10 cells had moderate and strong delays in growth resumption , respectively , following carbon starvation ( Figure 8—figure supplement 1M , N and O ) .",
"Strikingly , Δatg7 Δblm10 cells also resumed growth more rapidly than Δblm10 cells alone , implying that an active autophagy system plays a role in delaying the growth resumption of Δblm10 cells by clearing proteasomes in the absence of PSG assembly .",
"Taken together , our data are consistent with a model whereby cells that can protect proteasomes from autophagy by sequestering them in PSGs are better able to resume growth when carbon availability and energy status improve .",
"To test if PSGs represent a conserved mechanism to safeguard proteasomes from proteaphagy , we examined PSG dynamics and proteaphagy in Arabidopsis , using previously developed homozygous PAG1 ( α7 ) -GFP and RPN5a-GFP reporters for the CP and RP , respectively ( Marshall et al . , 2015 ) .",
"Here , the GFP-tagged subunits expressed from their native promoters were used to rescue pag1-1 and rpn5a-2 null mutant lines; these transgenic proteins fully rescue the embryo lethality and severe dwarf phenotypes of the corresponding homozygous mutations , and were faithfully integrated into the 26S particle ( Book et al . , 2009; Marshall et al . , 2015 ) .",
"Five-day-old seedlings were examined , which have almost fully completed the transition to photoautotrophic growth , thus rendering them sensitive to light and external supplies of fixed carbon ( Penfield et al . , 2005; Gao et al . , 2015 ) .",
"When we monitored proteaphagy by the GFP-release assay in seedlings grown in nitrogen- and carbon-rich medium , we observed a modest accumulation of free GFP ( Figure 9A ) which likely reflected constitutive proteaphagy , as evidenced by its absence in mutants eliminating the core autophagic machinery ( Marshall et al . , 2015 ) .",
"As described previously , free GFP accumulated and the PAG1-GFP and RPN5a-GFP fusions disappeared as the seedlings were starved for nitrogen , which became obvious by measuring the ratio of free GFP to the corresponding fusions ( Figure 9A and B ) .",
"Conversely , breakdown of the GFP reporters was not evident in seedlings starved for fixed carbon ( achieved by omitting sucrose from the growth medium and placing the plants in the dark; Thompson et al . , 2005 ) and was equally absent in seedlings starved for nitrogen and fixed carbon simultaneously ( Figure 9A and B ) .",
"Bulk autophagy was accelerated under all three conditions , as judged by release of free GFP from the GFP-ATG8a reporter ( Figure 9A and B ) , indicating that proteaphagy in Arabidopsis is selectively suppressed by fixed-carbon starvation , as it is in yeast .",
"To assess accumulation of autophagic vesicles and possible assembly of PSGs , we examined the distribution of the PAG1-GFP and RPN5a-GFP reporters by confocal fluorescence microscopy of root cells treated with concanamycin A ( ConA ) , which stabilizes vacuolar autophagic bodies and thus enhances their visualization ( Thompson et al . , 2005; Marshall et al . , 2015 ) .",
"As shown in Figure 9C , both reporters were concentrated in the nucleus along with a diffuse cytoplasmic signal under nutrient-replete growth conditions , in agreement with the largely nuclear distribution of plant proteasomes ( Book et al . , 2009; Marshall et al . , 2015 ) .",
"This distribution changed substantially upon nitrogen starvation , where the dramatic accumulation of small ( ~1 μm ) autophagic bodies in vacuoles became evident , similar to those seen with the GFP-ATG8a reporter .",
"This re-location was not seen in fixed carbon-starved roots , even though GFP-ATG8a still moved to autophagic bodies .",
"Instead , large , bright puncta ( ~5 μm ) resembling PSGs accumulated in the cytoplasm , concomitant with a substantial loss of nuclear fluorescence ( Figure 9C and D ) .",
"These foci were not similarly decorated with mCherry-ATG8a , implying that they are not phagophores or autophagosomes that sequester cargo prior to their vacuolar deposition ( Figure 9E ) .",
"As with PSGs in yeast , accumulation of these puncta in Arabidopsis was also readily reversible , with the fluorescence signal from the bright PAG1-GFP foci rapidly dispersing back to a diffuse cytosolic and nuclear pattern within 1 to 2 hr following return of the seedlings to sucrose-containing medium and light ( Figure 9F ) .",
"These puncta were almost entirely absent 4 hr after the cessation of starvation ( Figure 9F ) .",
"To help demonstrate that these puncta were PSGs , as well as investigate their ability to suppress proteaphagy , we analysed the fate of the PAG1-GFP and RPN5a-GFP reporters in Arabidopsis mutants missing the plant ortholog of Blm10 , known as PA200 ( Book et al . , 2010 ) .",
"When assayed by the GFP-release assay , fixed-carbon starvation did not accelerate the accumulation of free GFP from the RPN5a-GFP fusion in either wild-type plants or plants homozygous for the null pa200-2 and pa200-3 alleles ( Figure 10A and B; Book et al . , 2010 ) , in agreement with our observations that yeast Δblm10 cells do not accelerate RP autophagy ( Figure 4C ) .",
"However , for the PAG1-GFP reporter , proteaphagy upon fixed-carbon starvation was now evident in the pa200-2 and pa200-3 mutants , as it was for yeast Δblm10 cells , with the accumulation of free GFP and loss of the PAG1-GFP fusion clearly seen ( Figure 10A and B ) .",
"When similarly analysed by confocal fluorescence microscopy , we could easily detect bright cytoplasmic foci reminiscent of PSGs in PAG1-GFP roots , but not in roots also missing PA200 ( Figure 10C ) .",
"Instead , much smaller autophagic bodies containing PAG1-GFP accumulated in pa200-2 and pa200-3 vacuoles .",
"Formation of the bright cytoplasmic foci did not depend on the core autophagic machinery , as their appearance after fixed-carbon starvation was still robust in homozygous atg7-2 seedlings ( Figure 10D ) .",
"They were also clearly visible when the seedlings were starved for fixed carbon in the absence of ConA treatment , indicating that they did not reside in the vacuole ( Figure 10—figure supplement 1 ) .",
"Taken together , our data point to Arabidopsis also generating PSGs during carbon starvation , thus providing a second kingdom that assembles these proteaphagy-protecting condensates ."
],
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"Given the critical roles for the UPS and autophagy in cell regulation , maintaining amino acid supply , and mitigating the toxic effects of aggregation-prone proteins , it is unsurprising that these pathways are highly regulated ( Collins and Goldberg , 2017; Dikic , 2017 ) .",
"The activity and abundance of the proteasome in particular are tightly controlled by a variety of mechanisms , including the autophagic clearance of inactive or excess particles ( Marshall et al . , 2015; Marshall et al . , 2016; Marshall and Vierstra , 2015; Waite et al . , 2016; Cohen-Kaplan et al . , 2016; Nemec et al . , 2017 ) .",
"In this study , we further investigated starvation-induced proteaphagy in yeast and Arabidopsis and surprisingly found that , while proteasomes are rapidly eliminated during nitrogen starvation , they remain stable in response to carbon starvation , even though bulk autophagy is up-regulated .",
"Instead , mature proteasomes exit the nucleus and accumulate in cytoplasmic PSGs , the formation of which has previously been reported to protect yeast cells against stress and confer fitness during aging ( van Deventer et al . , 2015 ) .",
"Although the appearance of PSGs in quiescent yeast cells has long been known ( Laporte et al . , 2008 ) , their function ( s ) have remained obscure .",
"Here , we demonstrated an inverse relationship between PSG accumulation and proteaphagy , where promoting PSG assembly protects proteasomes from autophagy , while blocking delivery into PSGs encourages their degradation .",
"During the PSG assembly process , the CP and RP appear to separately coalesce , such that they accumulate in PSGs even in the absence of the other sub-particle .",
"An array of cell fitness studies in turn demonstrated that a failure to store proteasomes in PSGs directs them to proteaphagy , which substantially delays the resumption of growth when carbon-starved yeast cells are re-fed .",
"The response can even been seen in cells starved for nitrogen and treated with 2-DG , which supresses ATP levels , indicating that PSGs are not solely assembled in the absence of carbon but are more generally tied to the energy status of yeast cells ( this study; Gu et al . , 2017 ) .",
"Taken together , we propose that entry into PSGs shields proteasomes from proteaphagic breakdown , and instead creates a reservoir of stored proteasomes that can be rapidly re-mobilized upon the resumption of cell growth and/or when proteolytic demand rises .",
"While we cannot exclude the remote possibility that PSGs also have alternative functions , and/or that the ability of the various factors studied here to protect proteasomes from autophagy arises from processes unrelated to PSGs , the sum of our results strongly converges to this conclusion .",
"Presumably , the ability to rapidly restore proteasome capacity avoids the need to re-build the proteasome pool de novo , which would be essential for the proper regulation of cell division and other growth-promoting processes .",
"The inverse relationship between PSGs and proteaphagy , and the requirement of Blm10/PA200 for CP aggregation , were also demonstrated in Arabidopsis , indicating that PSGs represent a conserved mechanism for proteasome protection .",
"We confirmed the involvement of several factors previously reported to influence PSG formation , including the NatB N-terminal acetylation complex , the C-terminus of the proteasomal DUB Rpn11 , the proteasome capping factor Blm10/PA200 , intracellular pH , and energy levels ( Peters et al . , 2013; Saunier et al . , 2013; Weberruss et al . , 2013; van Deventer et al . , 2015 ) .",
"How these seemingly unlinked factors work together to condense proteasomes into PSGs remains largely unknown .",
"A number of yeast proteasome subunits are acetylated ( Hirano et al . , 2016 ) , with modification of Pre1 ( β4 ) , Rpt3 and Rpn11 being specifically ascribed to NatB ( Kimura et al . , 2000; Kimura et al . , 2003 ) , although the functions of these modifications are not known .",
"Likewise , while the deubiquitylating activity of Rpn11 is well positioned at the entrance to the substrate channel in the 26S complex to impact ubiquitin recycling ( Collins and Goldberg , 2017 ) , the function ( s ) of the C-terminal amino acids mutated in the rpn11-m5 allele remain ( s ) unclear .",
"The precise role of Blm10/PA200 also remains enigmatic , with various reports proposing that it helps assemble and stabilize the stacked CP barrel prior to RP docking ( Schmidt et al . , 2005; Sadre-Bazzaz et al . , 2010; Dange et al . , 2011 ) .",
"However , Δblm10 strains also display numerous pleiotropic phenotypes associated with genome instability and DNA repair , including reduced cell viability and susceptibility to DNA damaging agents ( Schmidt et al . , 2005 ) .",
"Besides promoting entry of the CP in PSGs , Blm10 bound to PSG-localized CPs could promote the rapid nuclear resorption of the CP or singly capped proteasomes upon restoration of cell growth , based on its ability to facilitate their nuclear import ( Weberruss et al . , 2013 ) .",
"It is also conceivable the Blm10 prevents inadvertent proteolysis by the CP after Blm10-CP particles coalesce into PSGs by covering the substrate entry pore of the CP ( Schmidt et al . , 2005; Sadre-Bazzaz et al . , 2010 ) .",
"Regardless of its activities , we found that Blm10 also becomes a target of autophagy upon nitrogen starvation , presumably because of its association with the CP .",
"In addition , we identified an unanticipated role for Spg5 in delivery of the yeast RP into PSGs .",
"In contrast to the absolute requirement of Blm10 for CP delivery , Spg5 was not essential for the RP , but its absence substantially delayed PSG entry .",
"Spg5 was previous shown to bind free RPs and to be important for cell viability during stationary phase ( Hanna et al . , 2012 ) , likely by safeguarding proteasomes .",
"How Spg5 promotes delivery of RPs into PSGs is unknown .",
"At least with respect to carbon-starved cells , we did not find Spg5 bound to proteasomes by mass spectrometry of purified preparations , nor did we detect consistent co-localization of Spg5 with PSGs by confocal fluorescence microscopy , implying that , unlike Blm10 , Spg5 does not follow proteasomes ( or at least the RP ) into these granules .",
"Given the possibility that orthologs of Spg5 exist beyond yeast ( like Blm10 ) , we search for relatives in other eukaryotes by amino acid sequence similarity; while weak sequence homologs were found in other fungi , they were absent in plants and metazoans , suggesting either that Spg5 is a fungi-specific factor , or that the Spg5 sequence has evolved considerably .",
"Ubp3 was previously shown to be important for proteaphagy upon nitrogen starvation ( Waite et al . , 2016 ) .",
"We confirmed this observation and also showed that Upb3 is critical upon carbon starvation once the transport of proteasomes ( or just the CP ) into PSGs is blocked .",
"Collectively , these data add proteaphagy to the reported roles for Ubp3 during ribophagy in response to nitrogen starvation ( Kraft et al . , 2008 ) and in negatively regulating mitophagy ( Müller et al . , 2015 ) .",
"Ubp3 activity is also required for the efficient formation of stress granules and processing bodies in response to heat stress , sodium azide treatment , or entry into stationary phase ( Nostramo et al . , 2016 ) , but not for PSG assembly ( this study ) , implying that this DUB is differentially required for the formation of various cytoplasmic puncta .",
"While complementation studies confirmed that the catalytic activity of Ubp3 and its interaction with its co-factor Bre5 are important for proteaphagy , the identity of its target ( s ) remains unknown .",
"Based on the observations that:",
"( i ) proteasomes are ubiquitylated ( Besche et al . , 2014; Kim et al . , 2013; Marshall et al . , 2015; Marshall et al . , 2016 ) ;",
"( ii ) Ubp3 interacts directly with proteasomes ( Fehlker et al . , 2003; Mao and Smerdon , 2010 ) ; and",
"( iii ) free ubiquitin has been detected in PSGs and promotes their appearance ( Gu et al . , 2017 ) , it is possible that direct deubiquitylation of one or more proteasome subunit ( s ) is essential for PSG condensation .",
"However , immunoblotting of proteasomes purified before and during nitrogen and carbon starvation did not detect changes in overall ubiquitylation of the particle ( data not shown ) , as has been seen upon proteasome inactivation ( Marshall et al . , 2015; Marshall et al . , 2016 ) .",
"Alternatively , it is possible that deubiquitylation of a hypothetical autophagy receptor permits binding to proteasomes and/or Atg8 upon starvation .",
"Clearly , the involvement of proteasome ubiquitylation in IPOD-mediated proteaphagy of inactive proteasomes , and of Ubp3 in supressing starvation-induced proteaphagy , places ubiquitin as a critical effector of proteasome dynamics , as well as being essential for proteasome substrate recruitment ( Collins and Goldberg , 2017; Dikic , 2017; Gu et al . , 2017 ) .",
"Further quantitative analysis of the ubiquitylation landscape of cells subjected to starvation in the presence and absence of Ubp3 will likely be required to differentiate the above possibilities .",
"How PSGs assemble and are able to shield proteasomes from proteaphagy is unclear .",
"Organisms in natural environments frequently encounter nutrient excess , nutrient deprivation , and rapid shifts between these two extremes , with growth under carbon stress in particular known to trigger the rapid re-organization of the cytoplasm and other compartments to promote cell survival ( Lee et al . , 2016; Saarikangas and Barral , 2016; Kaganovich , 2017 ) .",
"Included is the appearance of large , highly dynamic , membrane-less inclusions that can selectively partition individual proteins , biochemical pathways or cytotoxic protein aggregates away from the cellular milieu ( Narayanaswamy et al . , 2009; O'Connell et al . , 2014; Petrovska et al . , 2014; Shah et al . , 2014; Suresh et al . , 2015; Franzmann et al . , 2018 ) .",
"Besides PSGs , examples include hundreds of yeast proteins that condense into so-called stress granules during heat stress , mRNA and associated RNA-binding proteins that assemble into ribonucleoprotein granules under osmotic stress , and IPODs that concentrate amyloidogenic protein aggregates .",
"As with PSGs , some of these inclusions are thought to serve protective roles ( Saarikangas and Barral , 2016; Kaganovich , 2017; Mateju et al . , 2017; Franzmann et al . , 2018 ) .",
"Furthermore , these inclusions , like PSGs , coalesce rapidly and are often reversible , with possible driving forces being changes in cytoplasmic fluidity , intrinsic physico-chemical properties and folding of the protein ( s ) , changes in the surrounding environment such as the influences of pH seen for PSGs ( Peters et al . , 2013; this report ) , and extrinsic factors such as chaperones and/or post-translational modifications ( Kaganovich , 2017 ) .",
"Condensation is thought to involve phase separation phenomena often caused by reduced protein solubility .",
"In a manner highly reminiscent of PSGs , phase separation was recently reported for the yeast translation termination factor Sup35 upon nutrient starvation in response to changes in intracellular ATP levels and pH , with this accretion helping resumption of cell growth upon exit from starvation ( Franzmann et al . , 2018 ) .",
"Why carbon starvation , but not nitrogen starvation , induces these re-arrangements remains unexplored; for PSGs , this might be caused by alterations in intracellular pH and ATP levels seen upon carbon starvation but not nitrogen starvation ( Narayanaswamy et al . , 2009; Munder et al . , 2016 ) .",
"Similarly , how these condensates are able to evade the autophagic machinery , which is certainly capable of handling large protein aggregates and insoluble deposits , remains unclear , although their unique biophysical properties might be important ( Holehouse and Pappu , 2018 ) .",
"PSGs form independently of the PAS that initiates autophagy , additionally implying a spatial separation between PSGs and sites of autophagy initiation ( Figure 1—figure supplement 2C ) .",
"It is also unclear how proteasomes exit the nucleus prior to PSG formation .",
"A recent study found that nuclear proteasomes likely dissociate into their CP and RP sub-complexes prior to export in response to nitrogen starvation , and identified a role for the exportin Crm1 in this relocation , which was blocked in the temperature-sensitive xpo1-1 mutant ( Nemec et al . , 2017 ) .",
"Proteasomes then seemed to transiently associate with cytosolic IPODs , before forming mature PSGs as separate puncta ( Peters et al . , 2016 ) .",
"We previously showed that inactive proteasomes are triaged into IPODs in an Hsp42-dependent manner prior to Cue5-mediated proteaphagy ( Marshall et al . , 2015 ) , but our finding that PSGs can form even in the absence of Hsp42 implies that the pathway that forms PSGs is different .",
"In conclusion , we identify here an evolutionarily conserved function of PSGs in shielding proteasomes from autophagic degradation during nutrient deprivation and/or entry into quiescence that promotes cell survival when growth conditions improve .",
"An intriguing possibility is that similarly protective aggregation takes place for a variety of other intracellular protein complexes during nutritional and environmental stress .",
"Given the ease with which PSG ( and proteasome-containing IPOD ) assembly can be manipulated through growth conditions , inhibitors , and mutations , proteasome dynamics could provide an excellent paradigm to define the processes underpinning biomolecular condensate formation during stress ."
],
[
"Unless otherwise stated , all manipulations were performed according to standard yeast protocols ( Dunham et al . , 2015; Marshall et al . , 2016 ) .",
"Details of all strains used in this study are given in Supplementary file 1-Table S1 , and all relevant Saccharomyces Genome Database identifiers are given in Supplementary file 1-Table S2 .",
"Cells expressing PRE10-GFP , RPN5-GFP or BLM10-GFP in the BY4741 background ( Brachmann et al . , 1998 ) were obtained from the yeast GFP clone collection ( Thermo Fisher Scientific , Waltham , MA ) and cultured in synthetic complete medium lacking histidine .",
"All deletion strains in the BY4742 background ( Brachmann et al . , 1998 ) were obtained from the yeast gene knockout collection ( GE Healthcare , Chicago , IL ) and cultured in YPDA medium containing 200 µg/ml Geneticin , except for the Δerg6 deletion , which was instead grown in YPDA medium containing 200 µg/ml hygromycin B ( Marshall et al . , 2016 ) .",
"The rpn11-m1 mutation is a frame-shift at position 276 that results in expression of a truncated protein replacing the last C-terminal 31 amino acids with nine non-native residues ( Rinaldi et al . , 2008 ) .",
"The rpn11-m5 mutation is an intragenic suppressor of rpn11-m1 that restored the end of the open-reading frame downstream of residue 282 , but still maintained seven amino acid changes compared to the wild type sequence ( Rinaldi et al . , 2008; Saunier et al . , 2013 ) .",
"Crosses between haploid strains of opposite mating types were selected for on appropriate synthetic dropout media plus antibiotics , with subsequent sporulation and asci dissection performed as previously described ( Marshall et al . , 2016 ) .",
"The identities of the resulting haploid strains were confirmed by PCR genotyping and confocal fluorescence microscopy ( see below ) .",
"All oligonucleotide primers used in this study are listed in Supplementary file 1-Table S3 .",
"For time-course experiments , 15 ml liquid cultures in YPGA medium ( YPDA medium but containing 2% glycerol instead of 2% glucose [Adachi et al . , 2017] ) were grown overnight at 30°C with vigorous shaking , diluted to an OD600 of 0 . 1 in 15 ml , then grown for an additional 2 to 3 hr until an OD600 of approximately 0 . 5 was reached .",
"Cell aliquots corresponding to 1 . 5 OD600 units were taken at the indicated times , pelleted by centrifugation at 5000 x g for 1 min , washed once in sterile distilled H2O , pelleted again , and immediately frozen in liquid nitrogen .",
"For nitrogen starvation , cultures were grown and diluted in YPGA medium as above and , once an OD600 of approximately 0 . 5 was reached , cells were pelleted by centrifugation at 1000 x g for 2 min , washed twice in sterile distilled H2O , re-suspended in synthetic dropout medium lacking nitrogen ( 0 . 17% yeast nitrogen base without amino acids and ammonium sulphate ( Sigma-Aldrich , St . Louis , MO ) , 2% glycerol ) , then incubated at 30°C as above .",
"For carbon starvation , cultures were grown as above , followed by re-suspension in YPGA medium lacking glycerol ( Adachi et al . , 2017 ) .",
"Where indicated , cells were also pre-treated for 6 hr with 5 mM 2-deoxyglucose and 2 mM NaN3 prior to the starvation period , or the medium was adjusted to pH 3 . 0 ( with Na2HPO4/citric acid ) or pH 9 . 0 ( with NaOH ) instead of the usual pH 6 . 0 , in which case cells were simultaneously treated with 100 µM CCCP ( Orij et al . , 2009 ) .",
"For yeast growth assays , cells were grown and treated as above , except a culture volume of 50 ml was used ( Figure 8—figure supplement 1A ) .",
"Following a 24 hr starvation period , cultures were diluted to an OD600 of 0 . 2 in 50 ml YPGA medium , and growth resumption was monitored in the presence or absence of 5 μM canavanine or 25 mM p-fluorophenylalanine ( Sigma-Aldrich ) by measurement of OD600 values , or by growth of cells on solid synthetic complete medium .",
"Susceptibility to canavanine or p-fluorophenylalanine was determined by normalizing the OD600 value of each strain in the presence of the analog to its growth in the absence of the analog .",
"For growth on solid medium , cells were re-suspended in liquid synthetic complete medium to an OD600 of 1 . 0 , subjected to a series of 5-fold dilutions , and 5 μl of each dilution was spotted onto media containing or lacking 5 μM canavanine or 25 mM p-fluorophenylalanine .",
"Cells were then grown for 36 hr at 30°C .",
"For treatment with MG132 ( ( N-benzyloxycarbonyl ) -leucinyl-leucinyl-leucinal; Selleckchem , Houston , TX; Kisselev and Goldberg , 2001 ) , cells containing the Δerg6 deletion were grown in YPGA medium as above and treated with 80 µM MG132 for the indicated times .",
"For the experiment monitoring pexophagy , cells expressing the PEX14-GFP reporter were grown overnight in YPGA medium , then diluted to an OD600 of 0 . 1 in 15 ml SGD medium ( 0 . 67% yeast nitrogen base , 3% glycerol , 0 . 1% glucose ) and grown for an additional 12 hr . 1 . 5 ml of 10X YP medium ( 10% yeast extract , 20% bacto-peptone ) was then added , resulting in final concentrations of 1% yeast extract and 2% bacto-peptone , and the cells were grown for an additional 4 hr .",
"Cultures were then diluted into 15 ml YTO medium ( 0 . 67% yeast nitrogen base , 0 . 1% Tween-20 , 0 . 1% oleic acid ) to an OD600 of 0 . 2 and grown overnight to induce peroxisome proliferation ( Hutchins et al . , 1999 ) .",
"Cells were then subjected to nitrogen or carbon starvation as described above .",
"All other types of selective autophagy were monitored in YPGA medium only .",
"Genetic complementation with the BRE5 , NAT3 , RPN11 , SPG5 , and UBP3 genes used coding sequences amplified from BY4741 cDNA generated at appropriate growth stages , as described below ( see Quantitative real-time PCR ) .",
"The oligonucleotides used for amplification of RPN11 included sequence encoding a C-terminal FLAG tag .",
"Resulting PCR products were recombined first into pDONR221 via the Gateway BP clonase II reaction ( Thermo Fisher Scientific ) , and then into the pAG424GPD-ccdB or pAG424GPD-ccdB-HA vectors ( provided by Susan Lindquist ( Whitehead Institute for Biomedical Research , Massachusetts Institute of Technology ) ) via the Gateway LR clonase II reaction ( Thermo Fisher Scientific ) .",
"Previously described point mutations that abolish Nat3 catalytic activity ( C97A; Polevoda et al . , 2003 ) , Ubp3 catalytic activity ( C469A; Cohen et al . , 2003 ) or Ubp3 binding to its co-factor Bre5 ( L208A F209A V210A N211A; Li et al . , 2005 ) were introduced by the QuikChange method ( Agilent Genomics , Santa Clara , CA ) .",
"The construct encoding mCherry-SPG5 was generated by overlapping fusion PCR , using the mCherry coding region from the pESC::GAL1-RNQ1-mCherry plasmid as the template .",
"The mCherry-BLM10 construct was generated by sequential Gibson assembly of 10 overlapping PCR fragments ( Gibson et al . , 2009 ) .",
"All resulting plasmids were transformed into the indicated yeast strains using the lithium acetate method and subsequently grown in synthetic complete medium lacking tryptophan , in addition to other selective amino acids .",
"Total protein extracts from yeast were obtained by re-suspending harvested cells in 500 µl of yeast extraction buffer ( 0 . 2 N NaOH , 1% 2-mercaptoethanol ) , followed by precipitation of proteins with 50 µl of 50% trichloroacetic acid .",
"Proteins collected by centrifugation at 16 , 000 x g for 5 min at 4°C were washed once with 1 ml of ice-cold acetone , re-suspended into 150 µl SDS-PAGE sample buffer ( 80 mM Tris-HCl ( pH 6 . 8 ) , 10% glycerol , 4% SDS , 4% 2-mercaptoethanol , 0 . 002% bromophenol blue ) , and heated at 95°C for 5 min .",
"Total protein extracts from Arabidopsis were obtained by grinding frozen seedling tissue in 3 volumes of plant extraction buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , 2 mM dithiothreitol ( DTT ) , 1 mM phenylmethylsulphonyl fluoride ( PMSF ) , 50 μM MG132 , 1X protease inhibitor cocktail ( Sigma-Aldrich ) ) , followed by removal of insoluble debris by centrifugation .",
"The supernatant was then made 1X with SDS-PAGE sample buffer ( from a 5X concentrate ) and also heated to 95°C for 5 min .",
"SDS-PAGE gels were then prepared and stained for protein with silver nitrate as previously described ( Marshall et al . , 2017 ) .",
"Alternatively , gels were subjected to immunoblot analysis , where proteins were electrophoretically transferred onto Immobilon-P membrane ( EMD Millipore , Burlington , MA ) at 80 mA for 16 hr , blocked with a 10% non-fat dry milk solution in PBS ( 137 mM NaCl , 2 . 7 mM KCl , 10 mM Na2HPO4 , 1 . 8 mM KH2PO4 ) , then probed with specific antibodies diluted in PBS containing 1% milk .",
"See the Key Resources Table for full details of specific primary and secondary antibodies used .",
"The anti-Rpn5 antibodies were raised against the Arabidopsis protein ( Book et al . , 2009 ) , which has 30% identity and 37% similarity to the yeast version .",
"All blots were developed using the SuperSignal West Pico Plus Chemiluminescent Substrate ( Thermo Fisher Scientific ) .",
"Densitometric quantification of blots was performed using TotalLab Quant software ( Non-linear Dynamics; Newcastle-on-Tyne , UK ) , with at least three different exposures used to ensure the exposure level was within the linear range of the film .",
"The Pho8Δ60 activity assays were performed essentially as previously described ( Noda and Klionsky , 2008 ) , with minor modifications .",
"Strain TN124 was grown in a 250 ml culture , subjected to nitrogen and/or carbon starvation or growth at different pH , and aliquots corresponding to 5 . 0 OD600 units were sampled at the indicated times .",
"Cell pellets were re-suspended in 500 µl lysis buffer ( 20 mM PIPES-KOH ( pH 8 . 5 ) , 50 mM KCl , 100 mM potassium acetate , 10 mM MgSO4 , 10 μM ZnSO4 , 0 . 5% Triton X‐100 , supplemented with 1 mM PMSF immediately before use ) , and lysed by vigorous vortexing in the presence of ~200 μl acid-washed glass beads for a total of 5 min at 4°C ( 10 rounds of vortexing for 30 s , followed by resting on ice for 30 s ) .",
"Remaining non-lysed cells and insoluble debris were pelleted by centrifugation at 16 , 000 x g for 5 min at 4°C , and the supernatant was collected for subsequent analysis .",
"Equal amounts of total protein ( 20 μg , as determined by Pierce BCA protein assay kit ) were then assayed for alkaline phosphatase activity .",
"Protein samples in a volume of 100 μl were mixed with 400 µl of pre-warmed assay buffer ( 250 mM Tris-HCl ( pH 8 . 5 ) , 10 mM MgSO4 , 10 µM ZnSO4 , 1% Triton X-100 ) containing 1 . 5 mM p-nitrophenyl phosphate ( Sigma-Aldrich ) and incubated for 10 min at 37°C .",
"Reactions were stopped by addition of 500 µl of 1 M glycine-KOH ( pH 11 . 0 ) , and the absorbance of p-nitrophenol at 400 nm was measured using a SmartSpec 3000 UV/Vis spectrophotometer ( Bio-Rad , Hercules , CA ) .",
"Following subtraction of the appropriate enzyme and substrate only controls , specific alkaline phosphatase activity was calculated from a p-nitrophenol standard curve .",
"Three technical replicates were performed for each sample , and the data from three independent biological replicates was averaged and normalized to the activity observed at the 0 hr time point .",
"Yeast cells were visualized by confocal laser scanning microscopy using a Nikon A1 microscope with a 100X oil objective ( numerical aperture 1 . 46 ) .",
"Excitation was at 488 or 543 nm , and emission was collected from 500 to 530 nm or 565 to 615 nm , for GFP and mCherry , respectively .",
"To prevent cell movement , all cover slips were first washed with 1 M NaOH , rinsed with sterile distilled H2O , and coated with a 2 mg/ml solution of concanavalin A ( in H2O ) for 10 min .",
"The slips were then air-dried , rinsed with sterile distilled H2O , left to dry again , and stored at room temperature for up to 2 months before use .",
"To avoid auto-fluorescence from the YPGA medium , cells were first pelleted by centrifugation at 1000 x g for 1 min , and then re-suspended in synthetic complete medium lacking appropriate nutrients prior to imaging .",
"For imaging of Arabidopsis roots , seedlings of the indicated genotypes were grown in 5 ml liquid GM medium ( 3 . 2 g/l Gamborg’s B5 basal salts with minimal organics , 1% ( w/v ) sucrose , 0 . 05% ( w/v ) MES ( pH 5 . 7 ) ) at 21°C to 23°C under continuous white light for 5 days with gentle shaking ( 90 rpm ) , before being transferred to fresh medium containing or lacking 1 μM concanamycin A ( Santa Cruz Biotechnology , Dallas , TX ) and being subjected to either nitrogen and/or fixed-carbon starvation as previously described ( Thompson et al . , 2005; Marshall et al . , 2015 ) .",
"Root cells within the lower elongation zone were then visualized as above , using 20X or 40X oil objectives ( numerical apertures 0 . 75 and 1 . 30 , respectively ) .",
"All confocal images were scanned in single-track mode , except for the co-localisation studies , when GFP and mCherry signals were instead detected simultaneously in multi-track mode .",
"Images were processed using Adobe Photoshop CC , before conversion to TIFF files for use in the Figures .",
"Within each Figure , all images were captured using identical microscope settings .",
"Yeast cell cultures ( 15 ml ) grown in YPGA medium were subjected to nitrogen and/or carbon starvation as described above , harvested , and 2 × 107 cells were digested for 1 hr at 30°C with 100 U of lyticase in 100 µl Y1 buffer ( 1 M sorbitol , 100 mM EDTA , 0 . 1% ( v/v ) β-mercaptoethanol ( pH 7 . 4 ) ) .",
"Quantitative real-time PCR was performed exactly as previously described ( Marshall et al . , 2016 ) using a LightCycler 480 in combination with SYBR Green I master mix ( Roche Diagnostics; Basel , Switzerland ) and transcript-specific primers ( see Supplementary file 1-Table S3 ) .",
"Relative transcript abundance was determined by the comparative threshold cycle method ( Pfaffl , 2001 ) , using the ALG9 and TFC1 reference genes as internal controls ( Teste et al . , 2009; Llanos et al . , 2015 ) .",
"All data were normalized to non-starved wild-type cells .",
"26S holo-proteasomes or the CP or RP sub-complexes were affinity purified essentially as previously described ( Leggett et al . , 2005 ) , with minor modifications .",
"Yeast strains in which the Pre1 or Rpn11 subunits had been genetically replaced by variants tagged with Protein A were grown overnight at 30°C in 50 ml YPGA medium , diluted in 500 ml YPGA medium to an OD600 of 0 . 1 , grown for a further 2 to 3 hr until an OD600 of approximately 0 . 5 was reached , then subjected to nitrogen or carbon starvation for the indicated times .",
"Cells were then pelleted by centrifugation at 4000 x g for 20 min at 4°C , washed once in sterile distilled H2O , pelleted again , and immediately frozen in liquid nitrogen until use .",
"Frozen cell pellets were ground to a fine powder at liquid nitrogen temperatures for 15 min each , rehydrated with 1 vol of proteasome lysis buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 5 mM MgCl2 , 1 mM EDTA , 10% ( v/v ) glycerol , with 2 mM ATP , 2 mM PMSF , 10 mM 2-chloroiodoacetamide , 10 mM N-ethylmaleimide , 10 mM sodium metabisulphite , 1 mM benzamidine , 10 µg/ml pepstatin A , 1 µg/ml antipain and 1X protease inhibitor cocktail ( Sigma-Aldrich ) added immediately before use ) , and proteins were extracted on ice for 20 min .",
"Extracts were filtered through two layers of Miracloth ( Calbiochem , San Diego , CA ) , and clarified at 30 , 000 x g for 20 min at 4°C .",
"Equal volumes of supernatant were then incubated with gentle rotation for 2 hr at 4°C with 100 µl of rabbit whole molecule IgG antigen affinity gel ( MP Biomedicals , Santa Ana , CA ) pre-equilibrated in lysis buffer .",
"Samples were then applied to a 12 ml Polyprep chromatography column ( Bio-Rad ) , and the collected beads were washed three times with 2 ml of proteasome wash buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 50 mM NaCl , 5 mM MgCl2 , 1 mM EDTA , 2 mM ATP , 10% ( v/v ) glycerol ) , and twice with 1 ml of tobacco etch virus ( TEV ) protease buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 5 mM MgCl2 , 1 mM EDTA , 2 mM ATP , 1 mM DTT , 10% ( v/v ) glycerol ) .",
"Bound proteins were eluted by incubating the beads for 1 hr at 30°C with 300 µl of TEV protease buffer containing 20 ng/µl recombinant 6His-TEV , then collecting the flow through from the column .",
"The remaining 6His-TEV was removed by addition of 50 µl nickel-nitrilotriacetic acid ( Ni-NTA ) -agarose beads ( Qiagen , Germantown , MD ) , which were pre-equilibrated in TEV protease buffer containing 40 mM imidazole ( resulting in a final concentration of 10 mM ) , and incubating for 1 hr at 4°C with gentle rotation .",
"The beads were pelleted by centrifugation at 5000 x g for 1 min at 4°C , and the supernatant containing purified 26S proteasomes was removed and analyzed by SDS-PAGE followed by silver staining or immunoblotting , as described above .",
"To assay 26S proteasome activity , wild-type or rpn5ΔC cells were grown in a 50 ml culture , subjected to nitrogen and/or carbon starvation treatment as described above , and cell aliquots corresponding to 5 . 0 OD600 units were sampled at the indicated times .",
"Frozen cell pellets were ground to a fine powder at liquid nitrogen temperatures for 5 min each , rehydrated with 1 vol of activity assay lysis buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 5 mM MgCl2 , 1 mM EDTA , 10% ( v/v ) glycerol ) , filtered through two layers of Miracloth ( Calbiochem ) and clarified at 30 , 000 × g for 20 min at 4°C .",
"Supernatants were then made 10% ( w/v ) in PEG 8000 and incubated for 30 min at 4°C with moderate stirring .",
"The resulting precipitate was collected by centrifugation at 12 , 000 × g for 15 min at 4°C and re-suspended in 500 μl of lysis buffer .",
"The total protein concentration of each sample was determined by Pierce BCA protein assay kit ( Thermo Fisher Scientific ) , and equal amounts of protein ( 10 μg ) from each sample were assayed for proteasome activity in the presence or absence of 80 μM MG132 .",
"Protein samples in a volume of 20 μl were incubated for 20 min at 37°C in 1 ml of assay buffer ( 50 mM Tris-HCl ( pH 7 . 0 ) , 2 mM MgCl2 , with 1 mM ATP and 2 mM 2-mercaptoethanol added immediately before use ) containing 100 μM of the fluorogenic substrates N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin ( Suc-LLVY-amc; Sigma-Aldrich ) or ( 7-methoxycoumarin-4-yl ) -acetyl-alanyl-lysyl-valyl-tyrosyl-prolyl-tyrosyl-prolyl-methionyl-glutamyl- ( 2 , 4-dinitrophenyl- ( 2 , 3-diaminopropionic acid ) ) -amide ( Mca-AKVYPYPME-Dpa ( Dnp ) -amide , also known as LFP; GenScript , Piscataway , NJ; Smith et al . , 2005 ) .",
"Reactions were quenched by the addition of 1 ml of 80 mM sodium acetate ( pH 4 . 3 ) , and the resulting fluorescence was monitored using a TKO 100 fluorometer ( Hoefer Scientific Instruments , Holliston , MA ) , with an excitation wavelength of 365 nm and an emission wavelength of 460 nm .",
"Unless otherwise noted , A . thaliana seeds ( ecotype Columbia-0 ) were vapor-phase sterilized , stratified at 4°C for 3 to 4 days , and germinated on solid GM medium ( 3 . 2 g/l Gamborg’s B5 basal salts with minimal organics , 1% ( w/v ) sucrose , 0 . 05% ( w/v ) MES ( pH 5 . 7 ) , 0 . 7% ( w/v ) agar ) at 21°C to 23°C under a long-day photoperiod ( 16 hr light ( 75 to 100 μmol/m2/sec ) /8 hr darkness ) .",
"When required , after 2 to 3 weeks the seedlings were transferred onto soil ( mixed in a 1:1 ratio with organic Coco Coir planting mixture , then supplemented before use with 2 g/l Peters 20-20-20 fertilizer , 80 mg/l Ca ( NO3 ) 2 and 80 mg/l MgSO4 ) and again grown at 21°C to 23°C under a long-day photoperiod until completion of their lifecycle .",
"The pa200-2 , pa200-3 and atg7-2 T-DNA insertion mutants ( SALK_095870 , SALK_070184 and GABI_655_B06 , respectively ) , and the 35S:GFP-ATG8a , PAG1:PAG1-GFP pag1-1 and RPN5a:RPN5a-GFP rpn5a-2 reporter lines , were as previously described ( Thompson et al . , 2005; Chung et al . , 2010; Book et al . , 2010; Marshall et al . , 2015 ) .",
"The T-DNA insertion mutants were confirmed by genomic PCR using 5ʹ and 3ʹ gene-specific primers ( LP and RP , respectively ) in conjunction with appropriate T-DNA left border-specific primers ( BP ) .",
"All oligonucleotide primers used in this study are listed in Supplementary file 1-Table S3 .",
"The PAG1-GFP and RPN5a-GFP reporters were introgressed into the pa200-2 and pa200-3 mutants by standard crossing .",
"For chemical or starvation treatments , seedlings were grown in liquid GM medium at 21°C to 23°C under continuous light with gentle shaking ( 90 rpm ) , with the medium replenished every 3 days where required .",
"To stabilize autophagic bodies in the vacuole , fresh medium was supplemented with 1 μM ConA for 16 hr .",
"For nitrogen starvation , seedlings were transferred to MS medium lacking nitrogen ( MS basal salt micronutrient solution ( Sigma-Aldrich ) supplemented with 3 mM CaCl2 , 1 . 5 mM MgSO4 , 1 . 5 mM KH2PO4 , 5 mM KCl , 1% ( w/v ) sucrose , 0 . 05% ( w/v ) MES ( pH 5 . 7 ) ) for the indicated times .",
"For fixed-carbon starvation , the seedlings were transferred to liquid GM medium lacking sucrose , and incubated in the dark ( to prevent carbon fixation by photosynthesis ) , while simultaneous nitrogen and fixed carbon starvation utilised MS medium lacking nitrogen and sucrose together with incubation in the dark .",
"For all starvation treatments , control and treated seedlings were washed three times in appropriate medium prior to commencing starvation and , following treatment , all tissue was harvested , immediately frozen in liquid nitrogen and stored at −80°C until use .",
"All datasets were statistically analyzed using one-way analysis of variance ( ANOVA ) , followed by Tukey’s post-hoc tests to identify significantly different data points .",
"At least three biological replicates were performed in all cases , unless otherwise indicated in the Figure Legend ."
]
] | [
"26S proteasome abundance is tightly regulated at multiple levels , including the elimination of excess or inactive particles by autophagy .",
"In yeast , this proteaphagy occurs upon nitrogen starvation but not carbon starvation , which instead stimulates the rapid sequestration of proteasomes into cytoplasmic puncta termed proteasome storage granules ( PSGs ) .",
"Here , we show that PSGs help protect proteasomes from autophagic degradation .",
"Both the core protease and regulatory particle sub-complexes are sequestered separately into PSGs via pathways dependent on the accessory proteins Blm10 and Spg5 , respectively .",
"Modulating PSG formation , either by perturbing cellular energy status or pH , or by genetically eliminating factors required for granule assembly , not only influences the rate of proteasome degradation , but also impacts cell viability upon recovery from carbon starvation .",
"PSG formation and concomitant protection against proteaphagy also occurs in Arabidopsis , suggesting that PSGs represent an evolutionarily conserved cache of proteasomes that can be rapidly re-mobilized based on energy availability ."
] | [
"Proteins perform many jobs within an organism , including providing structure and support , and protecting against infection .",
"The levels of the many proteins in a cell need to be carefully controlled so that the correct amounts are present at the right place and time to perform these tasks .",
"This control can be achieved by balancing the production of new proteins with the break down ( or degradation ) of proteins that are no longer required or become dysfunctional .",
"Most cells have two pathways for degrading proteins .",
"One pathway breaks down individual proteins specifically marked for elimination; this causes them to be recognized by a structure called the proteasome , which chops proteins into smaller pieces .",
"Larger protein assemblies – including the proteasome itself – are to big for the proteasome and thus need to be degraded by another pathway called autophagy .",
"This process engulfs and delivers parts of a cell to a membrane-bound compartment called the vacuole , which ‘digests’ and recycles these larger constituents .",
"Proteasomes are degraded by autophagy when they are not working correctly and when nitrogen ( a crucial nutrient ) is in short supply .",
"However , proteasomes are not degraded when cells lack carbon , even though this starvation is known to activate autophagy in the same way that an absence of nitrogen does .",
"So how do proteasomes escape degradation when cells are starved for carbon ?",
"Marshall and Vierstra now show that upon carbon starvation , proteasomes rapidly exit the cell nucleus and cluster together in the main part of the cell ( termed the cytosol ) .",
"These clusters are known as proteasome storage granules ( PSGs ) .",
"In fungi and plants , mutations or conditions inside the cell that make it difficult for PSGs to assemble cause proteasomes to instead be broken down in the vacuole when carbon availability is low .",
"Clustering into PSGs therefore protects proteasomes from autophagy .",
"This clustering appears advantageous to cells; yeast cells that could form PSGs were better able to start growing again when their nutrient supply improved .",
"Protein clustering ( also known as aggregation ) is an important strategy that cells use to survive stressful conditions .",
"However , it can also be harmful when proteins aggregate inappropriately , such as occurs in Alzheimer’s disease .",
"Researchers may be able to use PSG assembly as a convenient model to study the causes and consequences of protein aggregation; this knowledge could ultimately be applied to improve human health and crop productivity ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"epidemiology and global health",
"microbiology and infectious disease"
] | Zika seroprevalence declines and neutralizing antibodies wane in adults following outbreaks in French Polynesia and Fiji | elife-48460-v1 | [
[
"Zika virus ( ZIKV ) , a Flavivirus primarily transmitted to humans by Aedes mosquitoes , was first reported in the Pacific region on Yap island ( Federated States of Micronesia ) in 2007 ( Duffy et al . , 2009 ) .",
"Six years later , there was a large ZIKV outbreak in French Polynesia ( Cao-Lormeau et al . , 2014 ) where an estimated 11 . 5% of the population visited healthcare facilities with clinical symptoms suggestive of ZIKV infection ( Kucharski et al . , 2016 ) .",
"Since then the virus has spread across the Pacific region ( Musso et al . , 2014 ) , including to Fiji where cases of ZIKV infection were first detected in July 2015 ( World Health Organisation , 2015 ) .",
"The same year , cases of ZIKV infection in Latin America were reported for the first time ( Zammarchi et al . , 2015 ) .",
"From February 1 to November 18 , 2016 , due to its rapid spread and association with birth defects , microcephaly in newborns and Guillain-Barré syndrome in adults ( Cao-Lormeau et al . , 2016 ) the WHO declared ZIKV a Public Health Emergency of International Concern ( World Health Organisation , 2016 ) .",
"At the end of 2016 , outbreaks had declined in most of the countries recently affected ( O'Reilly et al . , 2018 ) .",
"However , ZIKV was still circulating in 2018 in several countries , including Fiji and Tonga in the Pacific region ( World Health Organisation , 2019 ) .",
"In countries with known ZIKV outbreaks , the few serological surveys that have been published found a high level of ZIKV seroprevalence following the outbreak .",
"In French Polynesia , a population-representative cross-sectional serological survey at the end of the outbreak in 2014 found a seroprevalence of 49% ( Aubry et al . , 2017 ) .",
"In Martinique , a study of blood donors showed a post-outbreak seroprevalence of 42% in 2015 ( Gallian et al . , 2017 ) .",
"In Salvador , Northeastern Brazil , a serosurvey in 2016 of prospectively sampled individuals including microcephaly and non-microcephaly pregnancies , HIV-infected patients , tuberculosis patients , and university staff , found a post-outbreak seroprevalence of 63% ( Netto et al . , 2017 ) .",
"Another study in Salvador , conducted in a long-term health cohort , also found a post-outbreak seroprevalence of 63% ( Rodriguez-Barraquer et al . , 2019 ) .",
"Finally , in paediatric and household cohort studies in Managua , Nicaragua , ZIKV seroprevalence was estimated to be 46% in households following the outbreak in 2016 ( Zambrana et al . , 2018 ) .",
"It has been suggested that infection with ZIKV confers immunity that lasts several years; if so , the high level of seroprevalence in affected countries may reflect sufficient herd immunity for the current ZIKV epidemic to be over in many locations , with the virus unable to re-emerge for decades to come ( Kucharski et al . , 2016; O'Reilly et al . , 2018; Netto et al . , 2017; Ferguson et al . , 2016 ) .",
"Recent evidence suggests that neutralizing antibodies can distinguish between ZIKV and dengue virus ( DENV ) – a closely related Flavivirus – and that the immune response following ZIKV infection can persist over a year ( Montoya et al . , 2018; Griffin et al . , 2019 ) .",
"It has also been suggested that primary ZIKV infection may confer protective immunity ( Osuna et al . , 2016 ) .",
"However , ZIKV serosurveys conducted at the end of the outbreak in French Polynesia and 18 months later found a drop in seroprevalence in the Society Islands , the archipelago where over 85% of the inhabitants of French Polynesia reside ( Aubry et al . , 2017 ) .",
"Therefore , the long-term antibody response following a ZIKV outbreak remains unclear .",
"Here , we explore short- and long-term seroprevalence against ZIKV as well as neutralizing responses against ZIKV following two ZIKV outbreaks in the Pacific region .",
"We compared results from five serological surveys in the Society Islands , French Polynesia , over a seven-year period , and three serial serological surveys in the same cohort of individuals in Central Division , Fiji , over a four-year period .",
"These surveys span the pre- and post- outbreak period in each country , allowing us to examine temporal changes in antibody responses following a ZIKV outbreak ."
],
[
"In French Polynesia , seroprevalence of IgG antibodies against domain III of the ZIKV envelope glycoprotein in blood donors recruited before October 2013 was <1% ( 0 . 3–2% ) , which confirmed that the virus had not previously circulated in the population ( Table 1 ) .",
"Analysis of samples collected in the general population of the Society Islands of French Polynesia after the emergence of ZIKV showed a decrease in ZIKV seroprevalence from 37% ( 26–47% ) to 22% ( 16–28% ) between February-March 2014 and September-November 2015 ( chi-squared test , p=0 . 03 ) .",
"In Fiji , analysis of the serum samples serially collected from a cohort of participants in the Central Division showed an increase in ZIKV seroprevalence from 6 . 3% ( 3 . 3–11% ) in October-November 2013 to 24% ( 18–31% ) in November 2015 ( chi-squared test , p<0 . 0001 ) , and then a decrease to 12% ( 7 . 9–18% ) by June 2017 ( chi-squared test , p=0 . 005 ) .",
"In this cohort , based on IgG results tested by microsphere immunoassay ( MIA ) , 6 of the 189 participants seroconverted ( from negative to positive ) and 28 seroreverted ( from positive to negative ) to ZIKV between 2015 and 2017 ( McNemar’s test , p=0 . 0003 ) .",
"To investigate possible factors influencing the decline in seroprevalence , we compared the seroprevalence profiles in children ( defined as ≤16 years ) and adults ( >16 years ) in both settings ( Table 1 and Figure 1 ) .",
"In French Polynesia , although ZIKV seroprevalence declined in the general population from the Society Islands over 18 months , there was no evidence of a significant decline in seroprevalence in two serosurveys conducted four years apart in schoolchildren aged 6 to 16 years , with 66% ( 60–71% ) positive in 2014 and 64% ( 58–69% ) in 2018 ( chi-squared test , p=0 . 6 ) ( Table 1 ) .",
"When stratifying the general population from the Society Islands by age ( ≤16 years and >16 years ) , there was a decline in adults in the two consecutive cross-sectional studies conducted in 2014 and 2015 , from 35 . 4% ( 22 . 2–50 . 5% ) to 21 . 3% ( 18 . 2–24 . 5% ) ( Figure 1 ) .",
"A decline in adults was still observed , albeit with larger uncertainty , when the two datasets were standardised according to the age distribution of the population , with age-adjusted seroprevalence decreasing from 32 . 0% ( 16 . 7–62 . 1% ) to 26 . 0% ( 20 . 1–33 . 9% ) ( Table 2 ) .",
"In Fiji , in the subset of individuals who were aged over 16 years ( n = 122 ) , there was a decrease in seroprevalence by MIA from 24% ( 17–33% ) in 2015 to 7 . 3% ( 3 . 4–13% ) 2017 ( Figure 1 ) .",
"There were two seroconversions in the collected samples over this period but 23 seroreversions ( McNemar’s test , p<0 . 0001 ) ( Table 3 ) .",
"In contrast seroprevalence in participants aged 16 and under ( n = 67 ) remained relatively stable over this period ( Figure 1 ) , with four seroconversions and five seroreversions ( McNemar’s test , p=1 ) ( Table 3 ) .",
"In order to assess whether the decline in ZIKV seroprevalence was also observed for other circulating Flaviviruses , the MIA seroprevalence pattern against each of the four DENV serotypes was analyzed in both countries , by age group ( Figure 1—figure supplements 1–4 ) .",
"In Fiji , seroprevalence for DENV-1 , DENV-2 and DENV-4 increased in participants in both age groups between 2013 and 2017 ( Figure 1—figure supplements 1 , 2 and 4 ) .",
"DENV-3 seroprevalence also increased in both age groups between 2013 and 2015 following an outbreak in 2013–14 ( Kucharski et al . , 2018a ) and then declined in 2017 from 44% ( 32–57% ) to 40% ( 28–52% ) in children ( McNemar’s test , p=0 . 6 ) and from 59% ( 50–68% ) to 49% ( 40–58% ) in adults ( McNemar’s test , p=0 . 01 ) ( Figure 1—figure supplement 3 ) .",
"In French Polynesia between 2014 and 2018 , seroprevalence in children aged under 16 years showed no evidence of a change for DENV-1 and DENV-2 ( chi-squared test , p=0 . 1917 and p=1 , respectively ) ( Figure 1—figure supplements 1–2 ) and decreased for DENV-3 and DENV-4 ( chi-squared test , p<0 . 0001 and p=0 . 0085 , respectively ) ( Figure 1—figure supplements 3–4 ) .",
"In adult participants from the general population , seroprevalence for all four DENV serotypes declined between 2014 and 2015 .",
"The age-adjusted values for seroprevalence by MIA for the four DENV serotypes were similar to the raw values ( Table 2 ) , suggesting that the decline in French Polynesia could not be explained by differences in sampling by age .",
"However , a higher proportion of the samples in 2014 tested positive by MIA for all four DENV serotypes ( Table 4 ) , suggesting that the sampling included a group at higher risk for arbovirus infection than those sampled in 2015 .",
"To check that the estimated decline in ZIKV seroprevalence was not an artefact of this sampling bias , we re-estimated seroprevalence for the four DENV serotypes and ZIKV using a bootstrap sample of the 2014 responses , with replacement , weighted by the DENV exposure profile ( excluding the virus of interest ) in the 2015 survey so that the bootstrap sample of the 2014 responses had a similar DENV exposure profile as in the 2015 responses .",
"For example , when generating bootstrap estimates for DENV-1 in 2014 , we resampled participants based on the distribution of number of exposures to DENV-2 , DENV-3 , and DENV-4 in the 2015 data ( Table 5 ) .",
"After adjusting for prior exposure , there was no significant decline in seroprevalence for DENV-1 , DENV-3 , or DENV-4 , which had all circulated in the five years preceding the 2014 data collection , whereas the decline in ZIKV was still present ( chi-squared test , p=0 . 0047 ) .",
"To explore dynamics of antibody waning at the individual level , we performed neutralization assays ( NT ) on a subset of 45 participants from Fiji for whom sufficient sera were available to test against ZIKV from all three collection periods , focusing on those who were seropositive to ZIKV by MIA in 2013 or 2015 .",
"We found that in the 31 individuals who were ZIKV seronegative by NT ( i . e . log titre <2 ) in 2013 and had a rise in log titre ≥2 against ZIKV between 2013 and 2015 , anti-ZIKV antibody responses waned significantly in 2017 , with an average decline in log titre of −1 . 94 ( t-test , p<0 . 0001 ) ( Figure 2A and Table 6 ) .",
"In total , four participants seroreverted between 2015 and 2017; all had a log titre of 4 against ZIKV in 2015 .",
"We observed a similar effect when we analysed all participants who had a rise in log titre of at least 2 between 2013–15 , regardless of serostatus in 2013 ( Figure 2—figure supplement 1 ) .",
"To test whether the dynamics of anti-ZIKV antibody waning were different from the responses to DENV infection , we compared results for ZIKV to the neutralization response following a DENV-3 infection in the same cohort from Fiji .",
"There was a large DENV-3 epidemic during 2013–14 in Fiji ( Osuna et al . , 2016 ) , which meant most seroconversions to DENV-3 occurred between the collection of samples in 2013 and 2015 .",
"In those individuals that seroconverted to DENV-3 ( n = 19 ) or ZIKV ( n = 31 ) between 2013 and 2015 , the initial rise in NT log titres against ZIKV was larger than for DENV-3 , with a mean change of 5 . 0 and 3 . 37 respectively ( Figure 2B and Table 6 ) .",
"All individuals who had seroconverted to DENV-3 remained seropositive to the virus in 2017 , while four individuals who had seroconverted to ZIKV were seronegative in 2017 .",
"Although the NT log titres increased by a mean of 0 . 89 for DENV-3 between 2015 and 2017 ( two-sided t-test , p=0 . 04 ) , log titres against ZIKV declined by a mean of 1 . 94 over the same period ( two-sided t-test , p<0 . 001 ) ( Figure 2A and Table 6 ) .",
"In Fiji , there was a delay of around 18 months between the end of the 2013–14 DENV-3 epidemic and collection of samples in 2015 .",
"As DENV titres can wane following infection , particularly in individuals with a prior DENV exposure ( Clapham et al . , 2016 ) , titres against DENV-3 in Fiji may therefore have had more time to wane and reach a stable persistent level than titres against ZIKV , which may have circulated later than DENV-3 .",
"We therefore analysed changes in titre for participants who were initially seronegative to DENV-1 and DENV-2 , which were circulating at low levels in Fiji between the two serological surveys in 2013 and 2015 ( Figure 1 ) .",
"As with DENV-3 , we found no evidence of a subsequent overall decline during 2015–17 for those participants who seroconverted to DENV-1 or DENV-2 during 2013–15 ( Figure 2—figure supplement 2 ) .",
"Of the 45 participants tested by neutralization assay , nine were initially seropositive to ZIKV by NT in 2013 .",
"Fitting a generalized additive model to these data , we found that higher baseline mean NT log titres against DENV were associated with an increased probability of seropositivity to ZIKV ( Figure 3A ) .",
"In contrast , higher baseline mean DENV titres were not associated with increased seropositivity by MIA in 2013 .",
"There was little difference between the assay results in the 2015 samples ( Figure 3B ) , but we did find evidence of a difference in the 2017 results , with 15/45 participants positive by MIA and 31/45 positive by NT .",
"This difference was associated with participants’ 2013 DENV titres: those with intermediate DENV titres in 2013 had a significantly lower probability of being seropositive in the MIA in 2017 compared to NT ( Figure 3C ) ."
],
[
"Analyzing data from serological surveys conducted in French Polynesia and Fiji at different time points after the first reported autochthonous ZIKV transmission , we found evidence of a decline in ZIKV seroprevalence .",
"The high number of participants from the Fijian cohort that seroreverted between 2015 and 2017 suggested that anti-ZIKV antibody levels waned in these individuals to the point that they were no longer detectable by MIA .",
"Using a neutralization assay to test longitudinal sera collected in Fiji , we found that the mean change in neutralizing antibody titres against ZIKV also decreased significantly between 2015 and 2017 , showing that individual-level antibody titres against ZIKV as well as overall seroprevalence decreased over time .",
"In contrast , over the same period , neutralizing antibody titres against DENV-3 , a closely related Flavivirus which caused a large epidemic in Fiji in 2013–2014 ( Kucharski et al . , 2018a ) , remained stable .",
"In both countries we found seroprevalence against ZIKV in individuals aged over 16 declined over the two-year period following an outbreak , while the overall level of seroprevalence persisted in children .",
"This pattern was unique to ZIKV compared to DENV in both countries .",
"It is possible that this is related to the DENV immunological profile of individuals , given that the older population is likely to have experienced more DENV infections over their lifetime .",
"If an individual has experienced prior DENV infections , high numbers of weakly neutralizing cross-reactive B cells may outcompete naïve B cells for ZIKV antigen ( Midgley et al . , 2011 ) , leading to a short-term boost in antibody response against ZIKV following ZIKV infection ( Robbiani et al . , 2017 ) but not a persistent specific response; a similar phenomenon has been observed for other antigenically variable viruses like influenza ( Kucharski et al . , 2018b ) .",
"In the 2017 samples , more participants remained seropositive in the neutralization assay – which measures the overall ability of sera to neutralize ZIKV – than in the MIA , which tests for IgG antibodies against domain III of the envelope glycoprotein .",
"This difference was greatest for participants who had intermediate baseline titres to DENV in 2013 ( Figure 3C ) , which would support the hypothesis that prior DENV exposure may result in a detectable short-term specific response against ZIKV following ZIKV infection ( as measured by MIA ) , but not a persistent specific response .",
"To our knowledge , the only other study to date that has investigated the long-term persistence of neutralizing antibodies against ZIKV was conducted in 62 residents of Miami ( Florida , USA ) , who had a confirmed ZIKV infection in 2016 ( Griffin et al . , 2019 ) .",
"This cross-sectional study found that all participants had neutralizing antibodies against ZIKV 12–19 months after infection .",
"This study also found that at least 37% of the participants had no evidence of past DENV infection , which is consistent with the hypothesis that anti-ZIKV immune responses may persist longer in populations that have had less exposure to DENV .",
"More data are therefore needed to test hypotheses about the potential impact of pre-existing anti-DENV immune response on anti-ZIKV antibody waning .",
"Although we found evidence of a decline in seroprevalence for antibodies against domain III of the envelope glycoprotein , as well as waning neutralizing antibody responses following two ZIKV outbreaks , the implications for susceptibility to future ZIKV infection remain unclear .",
"Given the antigenic similarity of DENV and ZIKV ( Priyamvada et al . , 2016 ) , it is commonly assumed that the immune response to ZIKV infection will be similar to that following DENV infection .",
"High levels of neutralizing antibodies to DENV have been shown to correlate with protection from symptomatic infection ( Katzelnick et al . , 2016 ) .",
"Moreover , infection with a single DENV serotype can confer lifelong immunity to the infecting serotype as well as a transient period of cross-neutralization against heterologous serotypes ( Wahala and Silva , 2011 ) .",
"However , it is unclear in the context of ZIKV what the relationship is between a specific titre value and susceptibility to further infection .",
"A key aim for future work will be to establish how waning antibody levels as measured by MIA and neutralization assays may impact protective immunity , and hence susceptibility to reinfection in populations that have already experienced transmission of ZIKV .",
"There are some additional limitations to our analysis .",
"First , we did not have reverse transcription polymerase chain reaction ( RT-PCR ) confirmation of ZIKV infection in individuals sampled in this study .",
"We have presented analysis of representative serological surveys in two locations with known , RT-PCR-confirmed ZIKV outbreaks ( Mallet et al . , 2015; World Health Organisation , 2015 ) .",
"However , RT-PCR confirmation for ZIKV at the individual level remains difficult to obtain , in particular from blood samples , and there have been relatively few confirmations globally compared to the number of suspected cases ( Ferguson et al . , 2016 ) , let alone analysis of long-term antibody dynamics in RT-PCR confirmed patients .",
"In French Polynesia , there were approximately 32 , 000 reported clinical cases of ZIKV infection , but only 297 documented RT-PCR-confirmed cases ( Mallet et al . , 2015 ) .",
"As a result , antibody responses in RT-PCR-confirmed cases may not necessarily be representative of immune responses against ZIKV in the wider population , particularly following asymptomatic infection .",
"Although MIA seropositivity in our study was defined using control sera collected over a year after RT-PCR-confirmed infection , our results suggest that this threshold may not detect long-term waning responses in individuals who had unreported , and likely less severe , infections .",
"Our analysis was also limited by study design .",
"In French Polynesia , surveys were cross-sectional , so we were unable to examine temporal antibody dynamics at the individual level .",
"However , both cross-sectional studies of the general population were conducted using population representative cluster sampling ( Aubry et al . , 2017 ) in the same remote island locations with stable population composition , which enabled robust comparisons of overall seroprevalence .",
"We did identify one potential source of sampling bias with different DENV exposure profiles in the two surveys , but our conclusions of declining seroprevalence for ZIKV persisted once we adjusted for this bias .",
"We also used a different serological testing method between the studies in French Polynesia in 2014 and 2015 .",
"However , both used the same recombinant antigens and it has been shown that there was good agreement between ELISA and MIA in the 2014 samples ( see Materials and methods ) .",
"In Fiji , a strength of our study was the collection of longitudinal samples from the same individuals at three time points .",
"However , our sample size was limited given the logistical challenge of recontacting participants twice over a four-year period .",
"These data provided strong evidence that ZIKV seroprevalence declined over the two-year period following first reports of circulation , but our sample size was insufficient to fully explore the potential effect of anti-DENV pre-existing immunity on anti-ZIKV antibody waning once we stratified individuals by previous DENV exposure .",
"Although the outbreaks of DENV-3 in Fiji and ZIKV in French Polynesia were well-documented and occurred over a relatively brief period of time ( Figure 1 ) , it was harder to identify the likely time of infection for other viruses – such as ZIKV in Fiji or DENV in French Polynesia – in our study populations .",
"Several participants in Fiji were seropositive to ZIKV by neutralization assay ( NT ) in 2013 , but this result may be influenced by cross-reaction; participants who had high pre-existing titres to DENV in 2013 were more likely to be seropositive by NT ( Figure 3A ) .",
"In our main analysis of titre dynamics , we therefore focused on the subset of participants who were seronegative by NT in 2013 ( Figure 2 ) .",
"However , we obtained the same conclusion when participants who were initially seropositive were also considered ( Figure 2—figure supplement 1 ) .",
"The global ZIKV epidemic began in the Pacific islands in 2013 before spreading in Central and South America from 2015 .",
"Seroprevalence studies following ZIKV epidemics in Latin America have been reported but data have either been non-representative ( Netto et al . , 2017 ) or not enough time had elapsed since the outbreak to observe long-term dynamics ( Rodriguez-Barraquer et al . , 2019; Zambrana et al . , 2018 ) .",
"To our knowledge , these are the first studies of community seroprevalence over a long-term period following a ZIKV outbreak .",
"Therefore , patterns observed in Pacific islands may be an early indication of what might happen to seroprevalence in Latin America where ZIKV outbreaks began two to three years after the French Polynesia epidemic ( Cao-Lormeau et al . , 2014; Bogoch et al . , 2016 ) .",
"In the short-term , our findings have implications for the design of follow up studies of ZIKV .",
"Our results provide evidence that levels of seroprevalence one to two years following ZIKV circulation may be lower than previously expected and study designs may need to be adapted to reflect this , particularly in settings that exhibit long-term low level circulation of ZIKV as opposed to large sporadic outbreaks ( Ruchusatsawat et al . , 2019 ) .",
"For example , estimates of microcephaly risk may be inflated if derived from long-term seroprevalence data that underestimate the true extent of infection within the population , and results of clinical trials could also be biased if post-outbreak seroprevalence is used an indicator of infection within a population ( Cohen , 2018 ) .",
"In the longer-term , our results demonstrate the value of longitudinal serological studies of flaviviruses , and analysis using multiple serological tests , including neutralization assays ( Clapham et al . , 2016 ) .",
"Such studies will be essential to understand different aspects of the short and long-term immune antibody response against ZIKV , and how prior exposures to DENV may influence these responses ."
],
[
"For data from Fiji , where serial samples were collected from the same individual , changes in seroprevalence between studies were tested using McNemar’s test .",
"In French Polynesia , chi-squared tests were performed to test for evidence of a change in seroprevalence between two cross-sectional surveys .",
"Changes in mean log titre between groups were analyzed using a t-test .",
"To analyse the potentially non-linear relationship between DENV neutralization titres and seroprevalence by MIA and neutralization test ( Figure 3 ) , we used a generalized additive model via the mcgv package in R ( Wood , 2019 ) .",
"The model was of the form g ( E",
"( y ) ) =b + f",
"( x ) , where y was the binary outcome variable , x was the predictor ( i . e . titre ) , g was the link function , b was the intercept , and f was a smooth function represented by a penalized regression spline .",
"Mean DENV titre was calculated as the mean of log titres against the four DENV serotypes for each participant .",
"All data and code used in the analysis are available at: https://github . com/a-henderson91/zika-sero-pacific/ ( Henderson and Kucharski , 2019; copy archived at https://github . com/elifesciences-publications/zika-sero-pacific/settings ) ."
]
] | [
"It has been commonly assumed that Zika virus ( ZIKV ) infection confers long-term protection against reinfection , preventing ZIKV from re-emerging in previously affected areas for several years .",
"However , the long-term immune response to ZIKV following an outbreak remains poorly documented .",
"We compared results from eight serological surveys before and after known ZIKV outbreaks in French Polynesia and Fiji , including cross-sectional and longitudinal studies .",
"We found evidence of a decline in seroprevalence in both countries over a two-year period following first reported ZIKV transmission .",
"This decline was concentrated in adults , while high seroprevalence persisted in children .",
"In the Fiji cohort , there was also a significant decline in neutralizing antibody titres against ZIKV , but not against dengue viruses that circulated during the same period ."
] | [
"Since the Zika virus first emerged in the Pacific Islands in 2007 , it has caused many outbreaks in the Pacific and Latin America .",
"Some scientists thought that after exposure to the virus people would develop long-term immunity to it , reducing the number of outbreaks in the future .",
"Several studies supported this idea .",
"These studies showed that many people recently infected with Zika developed antibodies in their blood that might protect them from becoming ill during future outbreaks .",
"But it was not clear how long this protection would last .",
"To better understand how immunity to the Zika virus changes over time , Henderson , Aubry et al . combined data from eight surveys that collected blood samples at different time points during Zika outbreaks in French Polynesia and Fiji .",
"The analysis showed that the proportion of people with detectable antibodies against the Zika virus increased in both countries after the outbreaks .",
"In children these immune responses persisted for years , but antibody levels declined over time in adults .",
"By contrast , antibodies to the closely related dengue virus did not wane over time in individuals tested for both viruses in Fiji in 2013 , 2015 and 2017 .",
"The data suggest that immunity against the Zika virus may not last as long as previously thought , which could affect the chances of future outbreaks .",
"The findings may also have implications for researchers studying the virus , because the number of people with antibodies against the virus is not a good estimate of how many people were initially infected .",
"More studies are needed to understand immunity to Zika virus over time and how it may affect future outbreaks ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"neuroscience"
] | A receptor and neuron that activate a circuit limiting sucrose consumption | elife-24992-v2 | [
[
"Feeding regulation is a critical problem in animal life ( Morton et al . , 2006 ) .",
"Organisms must consume sufficient levels of nutrients to thrive , but overconsumption can have severe consequences .",
"The initiation of feeding has been studied in the model genetic system Drosophila , whose taste system has many parallels to that of mammals ( Liman et al . , 2014 ) .",
"A variety of molecular and cellular mechanisms must operate for feeding to be initiated .",
"Discrete classes of taste receptors and neurons assess the molecular composition of a potential food source ( Marella et al . , 2006; Thorne et al . , 2004 ) .",
"Receptors sensitive to sugars signal the presence of nutrients ( Dahanukar et al . , 2007; Freeman et al . , 2014; Jiao et al . , 2008; Wisotsky et al . , 2011 ) ; receptors sensitive to bitter-tasting compounds signal the danger of toxins ( Lee et al . , 2009 , 2015; Shim et al . , 2015; Weiss et al . , 2011 ) .",
"If nutrient levels are sufficiently high and toxin levels sufficiently low , the animal begins to feed ( French et al . , 2015 ) .",
"The termination of feeding , once begun , is poorly understood .",
"When nutrients are readily available and toxins are absent , what mechanisms terminate feeding ?",
"Previously described mechanisms that terminate feeding are based in central neural circuits that act downstream from taste neurons ( Hergarden et al . , 2012; Pool et al . , 2014 ) .",
"Some involve internal sensors that monitor post-ingestive nutrient concentrations in the hemolymph ( Dus et al . , 2015; Miyamoto et al . , 2012 ) or mechanical tension in the gut ( Olds and Xu , 2014 ) , while others involve cells in the taste center of the brain that are controlled by the satiety state of the fly ( Marella et al . , 2012; Yapici et al . , 2016; Zhan et al . , 2016 ) .",
"It would seem advantageous for an animal to have an additional means of inhibiting feeding , a mechanism that operates on a fast time-scale and functions directly in the gustatory organs .",
"Such a mechanism could prevent overconsumption at an early stage , before the animal has invested in the ingestion of nutrients that may be not only unnecessary , but detrimental .",
"The gustatory organs of the fly include the legs , the labellum , and the pharyngeal sense organs , which include the labral sense organ ( LSO ) ( Gendre et al . , 2004; Stocker , 1994 ) .",
"Taste reception in these organs is mediated by a large number of receptors , including those of the Gustatory receptor ( Gr ) family ( Clyne et al . , 2000; Scott et al . , 2001 ) , which detect a variety of sugars and bitter compounds ( Liman et al . , 2014 ) .",
"Members of an ancient class of sensory receptors called the ionotropic receptors ( IRs ) were recently found be expressed in taste organs ( Benton et al . , 2009; Croset et al . , 2010 ) .",
"In particular , a large clade of IRs called the IR20a clade were mapped to taste neurons in the legs , labellum , and pharynx ( Koh et al . , 2014; Stewart et al . , 2015 ) .",
"The functions of the IR20a clade genes are virtually unexplored , although analysis of two that are expressed in the male leg has revealed roles in male mating behavior , presumably as pheromone receptors ( Koh et al . , 2014 ) .",
"Among the ~35 members of the IR20a clade , one gene , IR60b , is exceptional in two ways .",
"First , it is remarkably restricted in its expression , as determined by a systematic analysis of IR-GAL4 drivers ( Koh et al . , 2014 ) .",
"Expression was detected in the pharynx , in a single pair of neurons , and nowhere else in the animal .",
"Second , IR60b is unique among the genes of this IR clade in showing the signature of purifying selection ( Figure 1D ) ( Koh et al . , 2014; Mackay et al . , 2012; Stoletzki and Eyre-Walker , 2011 ) , suggesting that IR60b represents a particularly effective solution to a difficult evolutionary problem . 10 . 7554/eLife . 24992 . 003Figure 1 . Expression of IR60b in the LSO .",
"( A ) Drosophila head .",
"The box indicates the region of the proboscis containing the labral sense organ ( LSO , shaded ) of the pharynx .",
"( B ) The pharyngeal region containing the LSO .",
"The position of sensillum 7 is indicated .",
"( C ) IR60b-GAL4; UAS-GFP shows expression in a single pair of neurons that project dendrites ( d ) to the pore of sensillum 7 , whose position is indicated .",
"( cb ) , cell body .",
"To maximize the fidelity of the driver , GAL4 was placed between sequences lying 5’ and 3’ to IR60b .",
"Scale bar = 5 μm .",
"Green color represents UAS-GFP fluorescence , visualized with a 488 nm laser .",
"Magenta color represents cuticular autofluorescence , visualized with a 514 nm laser .",
"( D ) IR60b is the only member of the IR20a clade that shows a significantly negative Direction of Selection ( DoS ) signature .",
"Values were generated by using polymorphism data from the Drosophila Genetic Reference Panel ( Huang et al . , 2014; Mackay et al . , 2012 ) to perform McDonald-Kreitman Tests ( Stoletzki and Eyre-Walker , 2011 ) for the IR20a clade genes .",
"Values were generated using the popDrowser website ( Ràmia et al . , 2012 ) .",
"Briefly , the values are calculated by comparing sequence variation within Drosophila melanogaster to the sequence divergence between Drosophila melanogaster and its sibling species Drosophila simulans .",
"*p<0 . 05; **p<0 . 01 .",
"Figure adapted from data displayed in Figure 6K in Koh et al . ( 2014 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 003 Here , we show that the pair of neurons in which IR60b is expressed mediates a response to sucrose .",
"Strikingly , these IR60b neurons inhibit sucrose consumption , in contrast to other sugar-sensitive taste neurons that promote sucrose consumption .",
"Silencing the IR60b neurons increases sucrose uptake , while activating these neurons reduces sugar uptake .",
"Sucrose detection by these neurons depends on the IR60b receptor , as revealed in behavioral and Ca2+ imaging analysis of an IR60b mutant .",
"The IR60b phenotype shows a high degree of specificity when tested with a broad panel of tastants .",
"An automated analysis of feeding behavior in freely moving flies shows that IR60b acts to limit the duration of feeding bouts .",
"Taken together , this study identifies a novel function for an understudied class of receptor and taste neuron .",
"The analysis reveals a new element in the circuit logic of feeding regulation: sugar-sensing taste neurons that act to prevent overconsumption ."
],
[
"We sought to determine whether IR60b is expressed in neurons that had previously been defined , as opposed to in ‘orphan’ neurons .",
"IR60b is expressed in the LSO in sensillum 7 ( Figure 1A–C ) , which houses eight neurons ( Gendre et al . , 2004 ) .",
"Two of these neurons have been shown to coexpress a set of sugar-sensing Gr-GAL4 or Gr-LexA drivers , including drivers of Gr43a , Gr61a , Gr64a , Gr64e , and Gr64f ( LeDue et al . , 2015 ) .",
"These two neurons promote the consumption of sweet foods .",
"The other six neurons have not been assigned a molecular identity , and their function is unknown .",
"To determine whether IR60b is expressed in an orphan neuron , we performed a systematic double-driver analysis .",
"We first combined IR60b-GAL4 with Gr-GAL4 drivers and used UAS-GFP ( Lee and Luo , 1999 ) to count the number of labeled cells in the LSO .",
"The neurons in this sensillum are tightly grouped , but careful analysis of the confocal z-stacks allowed us to determine the number of labeled neurons .",
"We found that while IR60b-GAL4 labels a single neuron in the LSO ( Figure 2A; the arrowhead indicates a labeled cell body in one of the two bilaterally symmetric neurons ) , and Gr64a-GAL4 labels two neurons ( Figure 2B ) , together the two drivers label three neurons ( Figure 2C ) .",
"The simplest interpretation of these results is that IR60b is expressed in a neuron distinct from those that express Gr64a .",
"Similar analysis with the Gr43a-GAL4 driver confirmed this conclusion ( Figure 2D–F ) . 10 . 7554/eLife . 24992 . 004Figure 2 . Coexpression of IR60b and other receptors .",
"( A–O )",
"Maximum intensity projections of GFP expression in IR60b-GAL4/+; IR60b-GAL4/UAS-mCD8-GFP flies ( A , D , G , J , M ) ; Gr- or IR-GAL4/+; UAS-mCD8-GFP/+ flies ( B , E , H , K , N ) ; IR60b-GAL4/Gr- or IR-GAL4; IR60b-GAL4/UAS-mCD8-GFP flies ( C , F , I , L , O ) .",
"Two copies of IR60b-GAL4 were used to compensate for the low expression level of IR60b-GAL4 .",
"Arrowheads indicate neuronal cell bodies in the LSO .",
"Scale bar = 5 μm .",
"( P ) Mapping of IR60b driver relative to other IR and Gr drivers in the eight gustatory neurons of LSO sensillum 7 .",
"Drivers mapped to neurons indicated in color do not co-express with drivers mapped to neurons indicated in gray , which have not been mapped at single-cell resolution in this analysis .",
"Gr66a is not included in the diagram because the Gr66a-GAL4 driver maps to other sensilla of the LSO . DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 00410 . 7554/eLife . 24992 . 005Figure 2—figure supplement 1 . IR60b-GAL4 expresses in a neuron of the pharynx that does not express several other drivers . Maximum intensity projections of GFP expression in IR60b-GAL4/+; IR60b-GAL4/UAS-CD8-GFP flies ( A , D , G , J , M ) ; Gr- , IR- , or ppk28-GAL4/+; UAS-CD8-GFP/+ flies ( B , E , H , K , N ) ; IR60b-GAL4/Gr- or IR-GAL4; IR60b-GAL4/UAS-CD8-GFP flies ( C , F , I , L ) .",
"( O ) IR60b-GAL4/ppk28-LexA::VP1; IR60b-GAL4/UAS-mCD8-GFP , LexAop-mtdTomato flies .",
"Although ppk28-GAL4 is driving UAS-cD8-GFP in panel ( N ) , the green-channel has been false-colored red to facilitate comparison to the double-labeled IR60b-GAL4/ppk28-LexA::VP1; IR60b-GAL4/UAS-mCD8-GFP sample in ( O ) .",
"Arrowheads indicate distinct neuronal cell bodies in the LSO .",
"In this analysis , some of the labeled cells are less strongly labeled than others , presumably because of differences in the strength of the GAL4 drivers .",
"The interpretations depicted in Figure 2P are supported by examination of the preparations at different focal planes .",
"We note that in an earlier analysis ( Koh et al . , 2014 ) , the IR20a-GAL4 driver could be observed to label two cells in the LSO; however , only one cell body was consistently observed with the IR20a-GAL4 line used in this study .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 005 We next tested other Gr drivers that are expressed in the Drosophila pharynx ( Joseph and Heberlein , 2012; Kwon et al . , 2014 ) .",
"The labeling pattern of IR60b-GAL4 is again distinct from that of Gr2a-GAL4 ( Figure 2G–I ) , and that of Gr66a-GAL4 ( Figure 2—figure supplement 1A–C ) .",
"Although the IR60b driver did not coexpress with any of these Gr drivers , it did coexpress with IR94f-GAL4 ( Figure 2J–L ) and IR94h-GAL4 ( Figure 2M–O ) .",
"We also tested IR67c , IR20a , and IR56a drivers and found that all three are expressed in this sensillum , but in neurons distinct from that expressing IR60b-GAL4 ( Figure 2—figure supplement 1D–L ) .",
"Likewise , a driver representing ppk28 , an osmosensitive ion channel that mediates water consumption ( Cameron et al . , 2010; Thistle et al . , 2012 ) , is expressed in the sensillum but in a different neuron ( Figure 2—figure supplement 1M–O ) .",
"Taken together , this mapping analysis suggests that IR60b is expressed in a previously undefined gustatory neuron of the Drosophila pharynx .",
"The analysis also suggests that IR60b is coexpressed with two other IRs of the IR20a clade ( Figure 2P ) .",
"We asked whether the IR60b neuron acts in the regulation of feeding , given its location in a sensillum of the pharynx .",
"We used a modified pharyngeal pumping paradigm that combines elements of previously described cibarial pumping assays ( Figure 3A , B ) ( Manzo et al . , 2012; Qi et al . , 2015 ) .",
"Our paradigm allows measurement of several parameters of feeding over a short time scale , that is on the order of a minute .",
"The parameters include total feeding time , swallowing rate , and number of swallows .",
"To facilitate analysis of food consumption by direct visual inspection , we added a blue dye to the food samples .",
"Control experiments indicated that the presence of the dye did not affect the results ( Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 24992 . 006Figure 3 . Consumption of sucrose in a modified pharyngeal pumping assay .",
"( A ) Diagram of assay .",
"Figure adapted from Figure 2B of Delventhal et al . ( 2017 ) .",
"( Delventhal et al . , 2017 ) ( B ) Blue dye allows visualization of filling and emptying of the cibarium ( red-dashed circle ) .",
"( C ) Total feeding time of w Canton-S females as a function of sucrose concentration .",
"n = 37–108 .",
"( D ) Swallowing rate of flies as a function of sucrose concentration .",
"Medians do not differ ( one-way ANOVA , Bonferroni post-test , n = 22–34 ) .",
"Lines indicate medians; boxes indicate 25% quartiles above and below the median; whiskers indicate range of values .",
"( E ) Correlation analysis of total number of swallows per fly vs . total feeding time .",
"Data are from all concentrations tested in ( C ) .",
"R2 = 0 . 91 , ***p<0 . 001 , Pearson’s correlation test , n = 140 .",
"( F ) Correlation analysis of calculated volumes of 900 mM sucrose ingested vs . total feeding time ( ***p<0 . 001 , R2 = 0 . 76 , Pearson’s correlation test , n = 20 ) .",
"Flies consumed ~8 nanoliters/s , which is comparable to reported values ( Qi et al . , 2015; Yapici et al . , 2016 ) .",
"Volumes were determined by extracting dye from single flies after feeding , and measuring the absorbance of each sample .",
"Absorbance values were converted into calculated volumes using the slope of a standard absorbance curve for the concentration of the blue dye ( see Materials and methods ) .",
"12 hr starved , mated females were used in this and all other assays . DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 00610 . 7554/eLife . 24992 . 007Figure 3—figure supplement 1 . Addition of erioglaucine blue dye to sucrose food does not affect feeding behaviors .",
"( A ) Total feeding time , ( B ) number of swallows/fly , and ( C ) pumping rate of w Canton-S ( +/+ ) females , in response to increasing sucrose concentrations with or without 0 . 4 ug/μm erioglaucine blue dye .",
"No differences were observed ( two-way ANOVA , Bonferroni post-test , n = 20–34 ) .",
"As with all assays , females were starved for 12 hr before testing .",
"In ( A ) and ( B ) two-way ANOVA revealed only the concentration of sucrose as a significant source of variation ( **p=0 . 0015 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 007 As food samples , we initially used drops of sucrose solution .",
"We verified that flies exhibit robust consumption of sucrose solutions in this paradigm across a wide range of concentrations ( Figure 3C ) .",
"The total time of feeding increased with sucrose concentration and saturated at ~100 mM .",
"The rate of pumping in this paradigm , that is the swallowing rate , was independent of concentration ( Figure 3D ) , consistent with previous results that pumping rate is not impeded by viscosity at sucrose concentrations less than 1M ( Manzo et al . , 2012 ) .",
"To confirm the constant pumping rate , we plotted the number of swallows against total time of feeding for each fly , pooled across all sucrose concentrations and found a strong correlation ( Figure 3E , R2 = 0 . 91; p<0 . 001 ) .",
"To determine whether the time of feeding in turn correlated with the volume of food ingested , we made extracts from individual flies after recording the time of feeding .",
"We measured the light absorbance of each extract to measure the amount of blue dye consumed , and from this value calculated the volume of sucrose solution consumed ( Materials and methods ) .",
"The feeding time was in fact correlated with the volume ingested ( Figure 3F , R2 = 0 . 76; p<0 . 001 ) .",
"Thus , the total feeding time serves as a reasonable measure of the amount of consumption in this paradigm .",
"To test whether the IR60b neuron acts in feeding regulation , we used IR60b-GAL4 to drive UAS-tetanus toxin ( TNT ) , which blocks synaptic signaling from neurons ( Martin et al . , 2002; Sweeney et al . , 1995 ) .",
"We compared feeding time of IR60b-GAL4/UAS-TNT flies to those of parental IR60b-GAL4 and UAS-TNT control flies .",
"Feeding time was scored blind to genotype .",
"As a precaution against genetic background effects , the IR60b-GAL4 and UAS-TNT constructs were each backcrossed for at least five generations against our control genetic background before the experiment .",
"Surprisingly , silencing of IR60b neurons caused a robust increase in feeding time when flies were tested with 300 mM sucrose ( Figure 4A ) .",
"We confirmed and extended this finding by testing across a broad range of sucrose concentrations .",
"At all sucrose concentrations , the silenced flies showed greater feeding times than the parental controls , which were equivalent to each other ( Figure 4B ) .",
"Feeding time was equivalently low among the three genotypes when tested with a control water solution , arguing that the silencing of IR60b neurons did not affect baseline levels of thirst or satiety .",
"The silencing of the IR60b neuron did not affect pumping rate at any concentration ( Figure 4C ) , arguing that increased feeding times were not due to motor defects that caused flies to swallow at a lower rate .",
"The simplest interpretation of these results is that the IR60b neuron acts to limit the intake of the sucrose source . 10 . 7554/eLife . 24992 . 008Figure 4 . Silencing and activating the IR60b neuron alters feeding time .",
"( A ) Initial analysis of total feeding time of IR60b-GAL4/UAS-TNT , IR60b-GAL4/+ , and UAS-TNT/+ flies when 300 mM sucrose was delivered in the pharyngeal pumping assay ( **p<0 . 01 , one-way ANOVA , Bonferroni post-test , n = 16–24 ) .",
"( B ) Total feeding time of silenced flies at a range of sucrose concentrations ( *p<0 . 05 , ***p<0 . 001: two-way ANOVA , Bonferroni post-test , n = 18–30 ) .",
"( C ) Pumping rates of silenced flies at different sucrose concentrations .",
"No differences were observed in pumping rates among different genotypes or doses ( two-way ANOVA , Bonferroni post-test , n = 16–27 ) .",
"( D ) Total feeding time of silenced flies when tested with glucose , fructose , or trehalose .",
"No increases in sugar consumption were observed in IR60b-GAL4/UAS-TNT flies when compared to controls ( one-way ANOVA , Bonferroni post-test , n = 25–38 ) .",
"( E ) UAS-Chrimson ( CHR ) activation: total feeding time with 300 mM trehalose in IR60b-GAL4/UAS-Chrimson , IR60b-GAL4/+ , and UAS-Chrimson/+ flies under low-light conditions , either with or without application of red light .",
"IR60b-GAL4/UAS-Chrimson flies show a decrease in feeding when IR60b neurons are activated with red light ( *p<0 . 05 , two-way ANOVA , Bonferroni post-test , n = 12–24 ) .",
"Under red light , feeding time for IR60b-GAL4/UAS-Chrimson flies is lower than for IR60b-GAL4/+ and UAS-Chrimson/+ controls ( *p<0 . 05 , two-way ANOVA , Bonferroni post-test , n = 12–24 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 00810 . 7554/eLife . 24992 . 009Figure 4—figure supplement 1 . Expression of UAS-TNT driven by IR94f-GAL4 also causes overconsumption .",
"( A ) Total feeding time of 300 mM sucrose for IR94f-GAL4/UAS-TNT , IR94f-GAL4/+ , and UAS-TNT/+ flies in the pharyngeal pumping assay .",
"IR94f-GAL4/UAS-TNT females exhibited increased total feeding time compared to heterozygote controls ( *p<0 . 05 , ***p<0 . 001 , one-way ANOVA , Bonferroni post-test , n = 12–16 ) .",
"These results support the conclusion from mapping data that IR60b and IR94f co-express in the same pharyngeal neuron .",
"The IR94f-GAL4 and UAS-TNT constructs were each backcrossed for at least five generations against our control w Canton S genetic background before the experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 00910 . 7554/eLife . 24992 . 010Figure 4—figure supplement 2 . Optogenetic activation of IR60b neurons does not reduce feeding on sucrose . The feeding time of IR60b-GAL4/UAS-Chrimson flies in red light is not lower than that of parental controls during delivery of 300 mM sucrose ( one-way ANOVA , n = 36–49 ) , suggesting that activation of IR60b neurons by sucrose is sufficiently strong that red light does not produce a demonstrable incremental effect . DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 010 To confirm the role of the IR60b neuron , we used an independent driver , IR94f-GAL4 , which we had found to be coexpressed with IR60b-GAL4 in the same neuron ( Figure 2L ) .",
"We confirmed with this driver that silencing the neuron increased the intake of the sucrose source ( Figure 4—figure supplement 1 ) .",
"Does this neuron affect intake of sugars other than sucrose ?",
"We tested 300 mM concentrations of fructose , trehalose , and glucose , and found no effects of silencing ( Figure 4D ) .",
"This specificity suggests that the neuron is tuned to a relatively narrow subset of taste stimuli , a hypothesis that is considered further below .",
"Since silencing the IR60b neuron increased feeding , we wondered if activating the neuron would decrease feeding .",
"To activate the IR60b neuron , we used UAS-CsChrimson , which encodes a channelrhodopsin that is activated by long-wavelength red light ( Klapoetke et al . , 2014 ) .",
"As an initial test , we asked whether optogenetic activation of the IR60b neuron with red light would decrease sucrose consumption .",
"We found that it did not ( Figure 4—figure supplement 2 ) ; however , it seemed likely that the sucrose stimulus alone may have activated the neuron to such an extent that additional activation by optogenetics would not produce a detectable effect .",
"Further analysis shown below confirmed that sucrose activates the IR60b neuron directly ( Figure 7F , G ) .",
"We therefore wished to test a stimulus that did not activate the IR60b neuron but that elicited a substantial baseline feeding level .",
"Accordingly , we used trehalose , which elicits a substantial level of feeding , and whose consumption was not affected by silencing of the IR60b neuron ( Figure 4D ) ; further analysis shown below confirmed that trehalose does not activate the IR60b neuron ( Figure 7F , G ) .",
"Thus , we asked whether trehalose feeding would be decreased when the IR60b neuron was activated optogenetically .",
"We found that optogenetic activation of the IR60b neuron with red light reduced trehalose feeding of IR60b-GAL4/UAS-Chrimson flies ( Figure 4E ) .",
"As a control , red light did not affect the feeding of either parental genotype .",
"Moreover , in the absence of red light , all genotypes showed the same level of consumption .",
"These results indicate that activation of IR60b neurons is sufficient to reduce trehalose consumption .",
"In summary , the activity of IR60b neurons is necessary to limit sucrose consumption to control levels ( Figure 4A , B ) and is sufficient to limit consumption of another sugar ( Figure 4E ) .",
"Taken together , the silencing and activation results reveal a surprising role for the IR60b neuron , which appears to negatively regulate ingestion in response to sucrose , a nutritive and appetitive sugar that normally promotes feeding .",
"Our results have shown that the activity of the IR60b neuron limits sucrose consumption .",
"Is the activity of the IR60b receptor required for this limitation ?",
"IR60b , IR94f , and IR94h drivers are coexpressed in this neuron , and a priori any of them , or none of them , could act in the regulation of sucrose feeding .",
"To test the hypothesis that the IR60b receptor functions in the limitation of sucrose feeding , we used CRISPR-Cas9 to create a mutant allele that lacks 66% of the IR60b coding sequence ( Gratz et al . , 2014 ) ( Materials and methods ) .",
"The mutation was designed to eliminate all three of the predicted three transmembrane domains of IR60b .",
"We confirmed the presence of the mutation , IR60b1 , by PCR analysis ( Barnes , 1994 ) .",
"The cleavage sites chosen for the construction of the deletion had no predicted off-target sites .",
"We backcrossed the mutation for five generations against our control genetic background prior to testing .",
"The IR60b1 mutant showed a robust increase in feeding time when tested with 300 mM sucrose ( Figure 5A ) .",
"The homozygous IR60b1/IR60b1mutant showed a much greater feeding time than either the parental control or an IR60b1/+ heterozygote .",
"The heterozygote showed the same phenotype as the control , supporting the interpretation that the phenotype is due to a recessive mutation in IR60b .",
"We confirmed and extended these results by testing a broad range of sucrose concentrations ( Figure 5B ) .",
"At all sucrose concentrations , the mutant showed greater feeding times than the controls .",
"When tested with a control water drop , feeding time was equivalently low among all three genotypes , arguing that increased feeding times in the mutant did not result from differences in baseline levels of thirst . 10 . 7554/eLife . 24992 . 011Figure 5 . Mutation of IR60b leads to overconsumption of sucrose .",
"( A ) Initial analysis of feeding time for IR60b1/IR60b1 , IR60b1/+ , and w Canton-S ( +/+ ) flies when 300 mM sucrose was delivered in the pharyngeal pumping assay ( ***p<0 . 001 , one-way ANOVA , Bonferroni post-test , n = 13–20 ) .",
"( B ) Sucrose dose-response curve for IR60b1 ( *p<0 . 05 , ***p<0 . 001 , two-way ANOVA , Bonferroni post-test , n = 12–25 for 0 mM , n = 30 for other doses ) .",
"We note that the feeding times in this experiment are somewhat less than those in Figure 4B , which may reflect differences in genetic background: all flies used in Figure 5B are w1118 , whereas all flies in Figure 4 have a mini-white+ marker in the GAL4 and UAS constructs .",
"( C ) Initial analysis of IR60b2 ( ***p<0 . 001 , one-way ANOVA , Bonferroni post-test , n = 20–21 .",
"( D ) Sucrose dose-response curve for IR60b2 ( **p<0 . 01 , ***p<0 . 001 , two-way ANOVA , Bonferroni post-test , n = 12–25 ) .",
"( E ) The total time feeding and ( F ) calculated volume ingested for IR60b1/IR60b1 and +/+ flies when offered 900 mM sucrose , a concentration selected to elicit robust feeding .",
"( G ) Volume consumed versus total feeding time for individual flies .",
"Volumes ingested were calculated as described in Figure 3F .",
"Data for +/+ shown here are from Figure 3F and are presented for comparison .",
"The volume consumed correlated with total feeding time for both IR60b1/IR60b1 and +/+ flies ( ***p<0 . 001 , R2 = 0 . 94 and 0 . 76 , respectively , Pearson’s correlation test , n = 20 ) .",
"Slopes of regression lines for IR60b1/IR60b1 and +/+ were very similar ( 7 . 8 and 7 . 5 nl/s , respectively ) , showing that both mutant and control flies swallowed the same amount of food per unit time , and arguing that the increased feeding times observed in the mutant translate directly to increased volumes ingested . DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 011 As a further confirmation of the requirement for IR60b , we tested an independent allele , IR60b2 , which was generated and backcrossed using the same protocol as for IR60b1 .",
"This second allele produced an increase in feeding time very similar to that of IR60b1 ( Figure 5C , D ) .",
"The greater feeding time of the mutant could reflect greater food consumption .",
"Alternatively , in principle the mutant could consume the same amount as the controls if the greater feeding time of the mutant were a mechanism of compensating for a decreased feeding rate .",
"To distinguish between these two possibilities , we compared the quantity of food consumed by individual mutant flies and , in parallel , individual control flies .",
"For each fly , we measured feeding time when provided a drop of 900 mM sucrose .",
"We subsequently determined the volume ingested by measuring the amount of blue dye present in extracts of each individual fly .",
"We found that not only was the mean feeding time greater for the mutant than the control ( Figure 5E ) , but the volume ingested was greater as well ( Figure 5F ) .",
"The mutant and control flies showed the same linear relationship between feeding time and volume consumed; the slopes were statistically indistinguishable , consistent with an equivalent feeding rate ( Figure 5G ) .",
"The simplest interpretation of these results is that the IR60b mutant has a defect in the regulation of sucrose consumption , and that the IR60b receptor acts in limiting the intake of the sucrose source .",
"We were interested in the specificity of IR60b: does it act to restrict feeding of many stimuli , or just sucrose ?",
"We initially addressed this question in vivo , via a behavioral analysis .",
"First , we analyzed three classes of sugars and sugar alcohols:",
"( i ) sugars that Drosophila encounters in its natural environment that are highly appetitive in a variety of taste assays , and have high-caloric value ( sucrose , glucose , fructose , trehalose , glycerol , maltose ) ( Amrein and Thorne , 2005; Gordesky-Gold et al . , 2008 ) ;",
"( ii ) a sugar alcohol that is less appetitive but has high-nutritional value ( sorbitol ) ( Stafford et al . , 2012 ) ;",
"( iii ) sugars that are highly appetitive but have limited caloric value ( arabinose and L-fucose ) ( Stafford et al . , 2012 ) .",
"Sugars were offered at 100 , 300 , and 900 mM concentrations .",
"We note that some of these stimuli have viscosities comparable to that of sucrose ( Galmarini et al . , 2011; Nikam et al . , 2000; Sheely , 1932; Swindells et al . , 1958 ) .",
"Only sucrose , among the nine sugars tested , elicited increased feeding in the IR60b1 mutant at concentrations of either 100 mM or 300 mM ( Figure 6A , B ) .",
"At 900 mM concentrations , sucrose and glucose elicited increased feeding ( Figure 6C ) .",
"The mutation had no effect on responses to lower concentrations of glucose , consistent with the lack of an effect on response to 300 mM glucose when silencing the IR60b neuron ( Figure 4D ) .",
"The feeding times of all other sugars were unaffected at all concentrations . 10 . 7554/eLife . 24992 . 012Figure 6 . The IR60b receptor regulates feeding of a highly specific set of taste stimuli .",
"( A–F )",
"Total feeding times of IR60b1/IR60b1and w Canton-S ( +/+ ) females with sugars , bitter compounds , salts , acids , and amino acids .",
"Bitter stimuli , including all doses of caffeine , were delivered in a 300 mM trehalose background to stimulate baseline consumption to a level where decreases in feeding could be observed .",
"All other compounds were dissolved in water .",
"NR indicates flies for 10 mM coumarin and 10 mM lobeline groups did not accept these tastants .",
"Differences between the mutant and +/+ were observed only for 100 , 300 , and 900 mM sucrose , and 900 mM glucose ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , Student’s two-tailed t-test , n = 10–32 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 012 Bitter compounds were also tested .",
"Bitter compounds inhibit the sugar responses elicited by many taste neurons ( French et al . , 2015; Sellier et al . , 2011 ) , although some taste neurons are activated by both classes of tastants ( van Giesen et al . , 2016a ) .",
"We wondered if bitter compounds inhibited sugar response in this system and if so whether the inhibition occurred via IR60b .",
"We tested each of several bitter compounds together with 300 mM trehalose , which was selected because the response to 300 mM trehalose alone is not affected by IR60b1 ( Figure 6B ) .",
"We tested a series of concentrations of three bitter compounds: caffeine , coumarin , and lobeline , and found that in wild type all three reduce mean trehalose feeding time , in a dose-dependent fashion ( Figure 6D ) .",
"The extent of reduction was not affected by the IR60b mutation .",
"Salts and pH were also tested since IR-expressing neurons in Drosophila chemosensory systems have been found to detect these stimuli ( Ai et al . , 2013; Hussain et al . , 2016; Zhang et al . , 2013 ) .",
"We found no effect of IR60b1 on the feeding time elicited by a range of NaCl or KCl concentrations ( Figure 6E ) .",
"Nor was there an effect on feeding at a range of pH values .",
"Finally , we tested several amino acids and found no effect of IR60b1 ( Figure 6F ) .",
"In summary , testing with a broad panel of taste stimuli revealed that IR60b1 affected the feeding time only with sucrose and with a high concentration of glucose .",
"These results suggested the hypothesis that the IR60b receptor responds to sucrose with a high degree of specificity .",
"The behavioral analysis we have performed with flies in which the IR60b neuron is silenced , and with flies in which the IR60b receptor is mutated , together support a model in which the IR60b neuron is a sucrose sensor , and that IR60b is required for the neuron to detect sucrose .",
"To test this model , we used calcium imaging to measure directly the response of the neuron to sucrose .",
"Previous imaging of sugar-sensitive pharyngeal neurons that express Gr43a drivers has focused on their axonal projections in the brain , visualized by cutting a window in the head ( LeDue et al . , 2015 ) .",
"Since IR60b-GAL4 appears to be a weaker driver than these pharyngeal Gr drivers , we found it necessary to devise a new preparation that images IR60b cell bodies directly in the pharynx .",
"Briefly , we removed the head and then excised the labellum from it such that the pharynx remained intact and accessible in the head .",
"We then mounted the head on a slide such that a fluid stimulus could be perfused into the sample and make contact with the pharynx ( Figure 7A ) .",
"We then used UAS-GCaMP6s to image cell bodies ( Chen et al . , 2013 ) .",
"We took special care to avoid misinterpreting fluorescent signals that can arise from shifts in the z-axis during imaging ( see Materials and methods ) . 10 . 7554/eLife . 24992 . 013Figure 7 . The IR60b neuron responds to sucrose and the response depends on IR60b .",
"( A ) The preparation used for imaging of cell bodies of the IR60b neurons .",
"( B–C )",
"Confocal images of merged green fluorescence and DIC channels showing IR60b-GAL4/+; IR60b-GAL4/UAS-GCaMP6s preparations during the delivery of ( B ) 900 mM sucrose or ( C ) water .",
"Inset heat maps show the change in fluorescence at 60 s , after the addition of the stimulus .",
"Yellow dashed boxes outline the regions shown in heat maps .",
"Scale bars = 10 um .",
"( D–E )",
"Representative traces showing changes in fluorescence for ( D ) the 900mM sucrose stimulus or ( E ) water .",
"Blue arrowheads indicate frames excluded because the sample was being re-focused to the appropriate plane of focus .",
"Red arrows indicate time of stimulus application .",
"( F ) Time course of change in fluorescence ( ∆F/F ) for flies that received the indicated stimuli .",
"Values on x-axis represent binned , 30 s intervals , for example , “0” indicates the 0-29 s bin; “30” indicates the 30-59 s bin .",
"The 900mM sucrose response differed from the water response between 0 and 120 s ( p***<0 . 001 for 0-29 s , 30-59 s , and 60-89 s bins; *p<0 . 05 for the 90-119 s bin , two-way ANOVA , Bonferroni post-test , n=7-12 ) .",
"The response to the 900 mM glucose stimulus differed from the water response between 0 and 180 s ( p***<0 . 001 for 0-29 s and 30-59 s bins , and **p<0 . 01 for 60-89 s , 90-119 s , 120-149 s , and 150-179 s bins , n=7-12 ) .",
"( G ) Bar graph of ∆F/F values from the 30-59 s bin in ( F ) , illustrating differences during the bin of maximal fluorescence .",
"( H ) Time course of fluorescence in IR60b1/IR60b1; IR60b-GAL4/UAS-GCaMP6s flies and control flies in response to 900mM sucrose .",
"Values on x-axis are binned in 30 s intervals as in ( F ) .",
"∆F/F values differed between 0 and 60 s ( **p<0 . 01 for 0-29 s and 30-59 s bins , two-way ANOVA , Bonferroni post-test , n=7-12 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 01310 . 7554/eLife . 24992 . 014Figure 7—figure supplement 1 . Response to sucrose in a neuron in which UAS-GCaMP6s is driven by IR94f-GAL4 .",
"Time course of ∆F/F values for binned , 30 s intervals of responses to 900 mM sucrose or water in IR94f-GAL4/UAS-GCaMP6s flies .",
"Values on x-axis represent binned , 30 s intervals , for example , 0 indicates the 0–29 s bin , and 30 indicates the 30–59 s bin , etc .",
"∆F/F values between the groups were different between 0 and 60 s ( p* < 0 . 05 for the 0–29 s bin , ***p<0 . 001 for the 30–59 s bin , two-way ANOVA , Bonferroni post-test , n = 6–9 ) .",
"These data are consistent with mapping results indicating that IR94f and IR60b are co-expressed and further confirm that the IR60b neuron is responsive to sucrose . DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 014 We found that a sucrose stimulus elicited an increase in fluorescence in IR60b neurons , indicating an increase in Ca2+ levels ( Figure 7B ) .",
"A control water stimulus did not elicit such an increase , indicating that the response is not due to mechanosensory stimulation ( Figure 7C ) .",
"Once the stimulus perfuses through the system to the neuron and the Ca2+ response becomes detectable , it takes on the order of 30 s to reach peak amplitude ( Figure 7D , E ) .",
"This rise time is comparable to that observed for some tastants in pharyngeal neurons of the larval taste system ( Apostolopoulou et al . , 2016; Choi et al . , 2016; van Giesen et al . , 2016b ) .",
"However , the rise time of the Gr43a neuron , once it begins to respond to sugar , is faster ( LeDue et al . , 2015 ) , suggesting that the Gr43a neuron activates a circuit that stimulates feeding before the IR60b neuron activates a circuit that inhibits feeding .",
"Our previous behavioral analysis indicated that the IR60b receptor was required for response to sucrose , but not to other stimuli tested , except for 900 mM glucose ( Figure 6A–C ) .",
"Consistent with these results , of five stimuli tested in parallel , the IR60b neuron was activated only by sucrose and glucose , each tested at 900 mM ( Figure 7F , G ) .",
"To independently confirm the response of the IR60b neuron to sucrose , we drove the GCaMP6s reporter with a different driver , IR94f-GAL4 , which is coexpressed in the same neuron ( Figure 2J–L ) .",
"Again , we found an increase in fluorescence , with comparable dynamics ( Figure 7—figure supplement 1 ) .",
"Finally , we asked whether the sucrose response of the IR60b neuron was dependent on IR60b .",
"We used IR60b-GAL4 to express GCaMP6s in an IR60b1 background and , in parallel , in a control background .",
"Both these genotypes contained a single copy of the IR60b-GAL4 driver , whereas the flies in our earlier imaging experiments contained two copies and thus presumably higher levels of GCaMP6s .",
"Nonetheless , sucrose elicited a clear Ca2+ signal in the control background .",
"The signal was essentially eliminated in the IR60b1 mutant background ( Figure 7H ) .",
"The simplest interpretation of these imaging results is that sucrose acts via the IR60b receptor to activate the IR60b neuron , which in turn activates a circuit that inhibits feeding .",
"We have shown that the IR60b receptor limits sucrose consumption in a pharyngeal pumping paradigm in which flies are immobilized .",
"We finally asked whether the receptor also limits sucrose ingestion in freely moving flies .",
"To address this question , we used the Fly Liquid-Food Interaction Counter ( FLIC ) ( Itskov et al . , 2014; Ro et al . , 2014 ) , and we wrote custom software to analyze the data ( Materials and methods ) .",
"In the FLIC system , liquid food is placed in a well surrounded by an aluminum ring ( Figure 8A , B ) .",
"To access the food , a fly must stand on the ring , which acts as an electrode .",
"When the fly makes contact with the liquid , it closes an electrical circuit between the aluminum ring , the liquid food , and an aluminum plate at the base of the well , which also functions as an electrode .",
"Thus , when the fly makes contact with the liquid food , an electrical signal is generated .",
"Previous studies have shown that such electrical signals provide a reliable indication of ingestion ( Itskov et al . , 2014 ) . 10 . 7554/eLife . 24992 . 015Figure 8 . The IR60b receptor limits sucrose consumption in freely moving animals .",
"( A ) The FLIC assay .",
"Diagram has been adapted from the illustration depicted in Figure 1B from Itskov et al . ( 2014 ) .",
"( B ) Fly contacting sucrose solution in a chamber of the FLIC apparatus .",
"( C ) Total contact time , ( D ) number of contacts with liquid food per fly , ( E ) contact time per contact event , for IR60b1/IR60b1 and control w Canton S ( +/+ ) .",
"**p<0 . 01 , *p<0 . 05 , Student’s two-tailed t-test , n = 44–46 .",
"( F , G )",
"Examples of contact events of mutant and control flies .",
"The durations of contact events are longer in the mutant .",
"Mated females in ( A–G ) were starved for 12 hr using the same protocol as in the pharyngeal pumping assays .",
"Results were analyzed using custom software code ( Tam , 2017 ) .",
"( H ) A dual-control model for regulation of short-term sucrose consumption by pharyngeal neurons .",
"After the fly encounters an acceptable food source , the Gr64a/Gr43a neuron would be activated and promote a feeding response .",
"The IR60b neuron would be activated subsequently and activate a circuit that inhibits the Gr64a/Gr43a neuron , thereby limiting feeding .",
"The absence of IR60b would lead to overconsumption ( I ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24992 . 015 Freely moving IR60b1 flies showed hallmarks of overconsumption .",
"We analyzed the response to 300 mM sucrose for 15 min , before post-ingestive effects modulate hunger state and feeding behavior .",
"Strikingly , IR60b1 mutants made contact with sucrose for three times longer than controls during this period ( Figure 8C ) .",
"We note that in the pharyngeal pumping assay , mutants fed for longer than controls by a comparable factor ( three-fold ) .",
"Does the longer total contact time of mutants reflect a greater number of feeding bouts or a greater mean duration of individual feeding bouts ?",
"IR60b1 mutants approached the wells and made contact with the food the same number of times as the control ( Figure 8D ) , as if locomotion and foraging capabilities are unaffected by the mutation .",
"However , the mean duration of individual contact events was longer in the IR60b1 ( Figure 8E–G ) , thereby accounting for the greater overall contact time .",
"The simplest interpretation of these results is that the IR60b receptor is required not only to prevent overconsumption of sucrose in the pharyngeal pumping paradigm but also in freely moving flies under conditions more comparable to natural foraging and feeding .",
"IR60b acts to limit the duration of individual feeding bouts ."
],
[
"This study maps IR60b to a previously undefined pharyngeal neuron .",
"The study identifies a role for both the neuron and the receptor , which belongs to the IR20a clade of IRs .",
"Only two members of this clade have previously been functionally analyzed , and both were implicated in mate-sensing ( Koh et al . , 2014 ) .",
"The current study extends the functions of this clade to include sugar-sensing , which has not previously been observed for any member of the entire IR family .",
"The simplest interpretation of our results is that IR60b is a sucrose receptor or co-receptor .",
"IR60b maps to a pharyngeal neuron that does not express any previously defined Gr sugar receptors such as Gr43a and Gr64a ( LeDue et al . , 2015 ) .",
"Although we have not identified any Gr receptors that map to the IR60b neuron , both IR94f and IR94h co-mapped with IR60b .",
"Further studies will be required to determine if either of these other IRs form a complex with IR60b .",
"We note that of the IR genes of this clade analyzed , IR94h has the next lowest DoS value after IR60b ( Figure 1D ) ( Koh et al . , 2014 ) .",
"Among a wide variety of other stimuli tested , only glucose at the highest tested concentration appears to elicit a response via IR60b .",
"Sucrose is a dissaccharide composed of the monosaccharides glucose and fructose , and it is possible that at high concentrations glucose may bind to a site that has evolved to bind sucrose .",
"We note that the high specificity of the IR60b phenotype argues against a role for the IR60b receptor in signaling osmolarity , viscosity , or mechanosensory stimulation .",
"Fifty years of research on fly taste has enshrined the orthodoxy that sugars act on taste organs to stimulate feeding , whereas bitter compounds act on them to inhibit feeding .",
"In this study , we have identified a peripheral taste neuron that detects sucrose but that inhibits feeding .",
"That is , sucrose assumes a negative rather than a positive valence with respect to this neuron .",
"We note that both loss-of-function and gain-of-function experiments suggest that the IR60b neuron does not alone dictate the level of consumption .",
"In the absence of IR60b , the fly does not feed continuously , and when the IR60b neuron is activated optogenetically the fly does not terminate all feeding .",
"Rather , the IR60b neuron appears to act as a brake , to temper the activity of other neurons that promote feeding .",
"These other neurons include the pharyngeal neurons that coexpress Gr sugar receptors ( Figure 2P ) ( LeDue et al . , 2015 ) .",
"Sucrose has high-caloric value .",
"Why after detecting and locating a source of sucrose would it be advantageous for a fly to have a taste neuron that inhibits its consumption ?",
"First , although sucrose is an energy-rich nutrient , a large increase in sucrose levels may disrupt the nutrient homeostasis in the fly ( Miyamoto et al . , 2012 ) .",
"The IR60b neuron may provide a mechanism for moderating the rate of sucrose intake and thereby helping to maintain homeostasis .",
"There may be special advantages to activating an inhibitory circuit from a peripheral taste receptor .",
"The inhibition can begin quickly , in contrast to the post-ingestive inhibition that occurs only after sugars reach nutrient sensors in the CNS ( Dus et al . , 2015; Miyamoto et al . , 2012 ) .",
"A fast-acting inhibition could prevent a rapid influx of sugar into the system .",
"By tempering the dynamics of sugar intake , the circuit could prevent the system from quickly moving far from equilibrium and requiring major metabolic costs to restore .",
"A similar mechanism could exist in mammals to limit water ingestion: peripheral osmolarity detectors may be responsible in part for the cessation of water drinking before systemic osmolarity levels are affected ( Bourque , 2008 ) .",
"In a more general sense , there are advantages to a system that has circuit mechanisms of both activation and inhibition .",
"Dual control may allow the system to be fine-tuned more precisely .",
"For example , changes in the internal state of the fly could in principle affect feeding by modulation of both positive and negative control mechanisms .",
"Positive control mechanisms in this system have recently been identified ( LeDue et al . , 2015; Yapici et al . , 2016 ) , and now IR60b neurons identify a negative control mechanism; together , these mechanisms may allow for a more precise orchestration of feeding behavior .",
"For example , satiety negatively modulates Gr43a neurons , and it will be interesting to determine whether satiety positively modulates IR60b neurons .",
"Flies encounter many sugars in their environment ( Das et al . , 2016 ) .",
"Why might it be advantageous to have a mechanism that is tuned to sucrose as opposed to other sugars ?",
"As fruits ripen , sucrose levels decline and the levels of other sugars increase in many cases ( Akazawa and Okamoto , 1980; Chareoansiri and Kongkachuichai , 2009; Hardy , 1967; Schwieterman et al . , 2014 ) .",
"Drosophila melanogaster prefers feeding on fruits that are at advanced stages of ripening .",
"Perhaps a circuit that inhibits feeding on sources rich in sucrose might tend to favor feeding on food sources with low sucrose concentrations .",
"Such food sources might be richer in monosaccharides or other nutrients , or in microbial species to which Drosophila melanogaster is well adapted .",
"How is this new circuit element integrated into the overall circuit logic of feeding regulation ?",
"One possibility is that under natural conditions the dynamics of the IR60b response are slower than those of the pharyngeal Gr43a neuron , a neuron that promotes feeding ( LeDue et al . , 2015; Yapici et al . , 2016 ) .",
"According to this model , after the fly encounters an acceptable food source , the Gr43a neuron would be activated and promote a feeding response .",
"The IR60b neuron would be excited subsequently and activate a circuit that inhibits the Gr43a neuron , thereby limiting feeding ( Figure 8H ) .",
"The absence of IR60b would lead to overconsumption ( Figure 8I ) .",
"We note finally that the central projections of the IR60b neuron differ from those of other pharyngeal neurons .",
"GAL4 drivers representing receptors of the IR20a clade label projections in the dorsoanterior subesophageal zone ( Koh et al . , 2014 ) .",
"However , drivers of IR60b and IR94f , which are coexpressed ( Figure 2P ) , differ from the others in that they show no projections toward the midline ( Koh et al . , 2014 ) .",
"In this respect , the IR60b driver also differs from Gr-GAL4 drivers expressed in the pharynx ( Kwon et al . , 2014 ) .",
"A detailed study using GRASP analysis ( Feinberg et al . , 2008 ) to identify synaptic partners of IR60b neurons may help identify higher-order neural circuits that the IR60b neuron connects to , and may determine whether the distinct projections of the IR60b neuron underlie its distinct function .",
"Mutants of the IR60b receptor had a highly specific phenotype when tested with a broad panel of tastants , suggesting that IR60b acts specifically to prevent overconsumption of sucrose .",
"It will be interesting to determine by mutational analysis whether either IR94f or IR94h act in the same neuron to limit overconsumption of other tastants not considered in the present analysis .",
"Likewise , it will be interesting to determine if other taste neurons in the fly play analogous roles , perhaps limiting overconsumption of other taste stimuli .",
"It is striking that sucrose has a negative valence that is mediated via IR60b neurons but a positive valence via the Gr43a neuron .",
"In both flies and mammals , NaCl has a negative valence with some taste neurons ( Amrein and Thorne , 2005; Hiroi et al . , 2004 ) , and a positive valence with others ( Zhang et al . , 2013 ) .",
"However , NaCl acts on these different neurons at different concentrations , whereas sucrose acts on the IR60b and Gr43a neurons at the same concentration .",
"Finally , it will be interesting to determine if there are taste neurons in mammals that detect sugars and limit their consumption by a logic similar to that identified in this study .",
"Overconsumption of sugar is a major cause of a rapidly expanding worldwide obesity epidemic in humans ( WHO , 2011 ) .",
"The identification of neurons that limit sugar consumption could provide targets useful in controlling this massive global health problem ."
],
[
"Flies were reared on standard cornmeal-dextrose agar food at 25°C and 40% humidity .",
"IR-GAL4 drivers are described in Koh et al . ( 2014 ) .",
"Gr-GAL4 drivers are described in Weiss et al . ( 2011 ) .",
"The ppk28-LexA transgene was originally described in Thistle et al . ( 2012 ) .",
"The UAS-mCD8-GFP and LexAop-mtdTomato constructs are described in Koh et al . ( 2014 ) .",
"UAS-tetanus toxin ( TNT ) line was generated from second and third chromosome insertion lines originally described in Sweeney et al . ( 1995 ) .",
"UAS-CsChrimson was a generous gift from Vivek Jayaraman .",
"UAS-GCaMP6s was a gift from Douglas Kim .",
"All lines were backcrossed for at least five generations to w1118 Canton-S before testing in behavioral assays .",
"CRISPR-Cas9 homologous recombination was employed to generate the IR60b1 and IR60b2 mutations .",
"Guide chiRNAs were selected using the CRISPR Optimal Target Finder resource on the flyCRISPR website ( Gratz et al . , 2014 ) .",
"Gibson Assembly was used to generate guide chiRNA using the Gibson Assembly Master Mix ( New England BioLabs , Inc: Ipswich , MA ) .",
"Drosophila embryos were injected with guide chiRNA and donor plasmids by Bestgene , Inc . ( Chino Hills , CA ) .",
"Two non-sibling alleles were isolated by screening DsRed , and were subsequently back-crossed to the w1118 CantonS stock for five generations before experimentation .",
"DNA sequence analysis determined that the mutant allele lacks ~66% of the IR60b coding region .",
"Detailed description of guide chiRNA sequences , verification primers , and mutation strategy is provided in Supplementary file 1 .",
"To perform cell-counting experiments , GAL4 lines were used to express UAS-mCD8::GFP ( Lee and Luo , 1999 ) .",
"Lines containing IR60b-GAL4 had two copies of the transgene to compensate for low IR60b-GAL4 expression levels , while partner IR-GAL4 , Gr-GAL4 , or ppk28-GAL4 transgenes were present as a single copy .",
"UAS-mCD8::GFP was present as a single copy in all tested lines .",
"Flies were aged between 10 and 35 days to ensure sufficient GFP production for visualization .",
"Female flies were decapitated and mounted on a microscope slide in 50% glycerol for immediate imaging on a Zeiss 510 Meta confocal microscope .",
"We systematically mapped the expression of GAL4 drivers by analyzing z-stacks to count the number of cell bodies expressing a UAS-mCD8-GFP reporter in the LSO .",
"For each genotype , n ≥ 10 flies were examined .",
"Tastants from J . T . Baker ( Central Valley , PA ) , American Bio , Inc ( Natick , MA ) , and Sigma-Aldrich ( St . Louis , MA ) were obtained at high purity .",
"Samples were stored as aliquots at −20°C long-term and kept at 4°C during experiments .",
"Thawed aliquots were discarded after one week .",
"We employed a pharyngeal pumping assay , modified from that of Manzo et al . ( 2012 ) .",
"Mated females were aged for 10–14 days to allow for tetanus toxin ( TNT ) or Chrimson ( CHR ) to accumulate via expression from the relatively weak IR60b-GAL4 driver .",
"Flies were also aged for 10–14 days in IR60b1 , IR60b2 , and IR94f-GAL4 experiments .",
"Before testing , flies were placed in a 1000-μl pipette tip .",
"A second 1000-μl pipette tip was inserted into the first tip so as to contain the fly for a starvation period of 12–14 hr .",
"Flies were placed in a 100-mm Petri dish with three Kimwipes wetted with 5 ml water , and then transferred to a humidity-controlled room to prevent dehydration .",
"After starvation , the fly was aspirated into another 1000-μl pipette tip such that it was immobilized with its head and proboscis exposed .",
"Occasionally , ends of the 1000-μl pipette tips were trimmed with a razor blade to widen the opening for the fly’s head .",
"Flies were mounted on a micromanipulator for video recording under a Nikon SMZ800 microscope with an attached Sony HD Camcorder .",
"Before food was delivered , the fly was presented a water droplet to ensure the animal was not overly desiccated from starvation .",
"If it consumed water for longer than 10 s , the animal was discarded .",
"Only ~5% of flies were discarded by this criterion .",
"We added 0 . 4 μg/μl erioglaucine blue dye to the liquid food to facilitate data acquisition .",
"Food was presented with a P20 PipetteMan mounted on a micromanipulator , allowing for fine adjustments during delivery .",
"The fly was offered food for 2 s .",
"Flies that ingested liquid were allowed to continue feeding until they freely terminated feeding .",
"After breaking contact with the drop , flies were given 3 s of rest before a subsequent presentation , in which they were given another 2 s to initiate a second bout .",
"This process was repeated until the fly no longer responded to food .",
"Typically , 2 s is sufficient presentation time to initiate feeding , as longer presentations did not increase the likelihood of initiating a feeding bout .",
"On average , flies engaged in 1–3 bouts .",
"If a fly did not initiate feeding after four attempted presentations , it was discarded as a non-responder .",
"When testing different concentrations and tastants , the fraction of non-responders did not differ significantly between control , mutant , or transgenically manipulated flies , indicating there were no differences in the capacity to initiate feeding between the different genotypes .",
"Typically , the fraction of non-responders was less than ~10% for appetitive sugars like sucrose .",
"Each fly was used for only one experiment to prevent previous experience from influencing its responses .",
"The investigator was blind to the genotypes of the flies .",
"The video was analyzed using QuickTime Media Player .",
"Within each experiment , responses of different genotypes were measured in parallel , at the same time of day , by the same investigator , and within the same 3–5 day time window .",
"Experiments with UAS-CsChrimson were performed as described for the pharyngeal pumping assay , except that flies were kept in the dark prior to testing , including a 24-hr period during which flies were given cornmeal food supplemented with 0 . 5 mM all-trans-retinal , followed by a 12-hr starvation period .",
"After being presented a water droplet , the whole fly was illuminated by high-intensity 626 nm red LEDs at an intensity of ~5 W/m2 for the duration of the assay .",
"The volumes ingested by single flies ( Figures 3F and 5C ) were measured as follows: 10 serial two-fold dilutions of 0 . 4 μg/μl erioglaucine were prepared for spectrophotometer analysis using a NanoDrop 2000c Spectrometer ( Thermo Scientific: Wilmington , DE ) .",
"The average optical density ( OD630 ) was determined for 1 μl of each dilution ( n ≥ 3 for each concentration ) .",
"Average ODs were then used to plot a standard line for absorbance of blue dye ( OD ) versus the dye concentration .",
"The resulting slope was 0 . 14 ± 0 . 001 OD/μl , and was used as a factor for converting OD values of 0 . 4 μg/μl erioglaucine to volume of fluid ingested by the fly .",
"A single mated female was fed 900 mM sucrose with 0 . 4 μg/μl erioglaucine blue dye in the pharyngeal pumping assay .",
"After feeding , the fly was homogenized in water and samples were centrifuged for 1 min at 14 , 000 RPM to pellet the cuticle .",
"1 μl of supernatant from homogenized flies were then assayed with the NanoDrop Spectrometer , and an average OD value was used to determine the amount of blue dye present in each fly ( n ≥ 4 OD values were averaged for each fly ) .",
"The slope of the standard line for 0 . 4 μg/μl erioglaucine was then used to convert average OD values into volumes ingested .",
"To control for endogenous absorbance of the extract , four females were fed 900 mM sucrose without blue dye , and OD values for each fly were obtained .",
"The calculated values for these control flies were averaged , yielding a standard absorbance value in the OD630 range .",
"This standard value was subtracted from each measurement from the flies that were fed 900 mM sucrose with 0 . 4 μg/μl erioglaucine , before determining the final calculated ingested volumes .",
"To image the cell bodies of IR60b neurons directly in the pharynx , we first removed the heads of mated females .",
"To increase access to the esophagus and pharyngeal sensilla , the labellum lobes were carefully excised while leaving the rest of the proboscis and pharynx intact .",
"Heads were mounted in water on a slide with three 18 × 18 mm bridging slips , positioned such that two gaps were left between the bridging slips , so liquid stimulant could be perfused through the sample ( Figure 7A ) .",
"A minimal amount of water was used to minimize dilution of the stimulus during the perfusion process .",
"The head and bridging slips were then secured with a 22 × 40 mm coverslip , positioned with roughly 10 mm of overhang from the microscope slide , which would later allow for liquid stimulant to be delivered to the preparation when the sample was inverted .",
"The three non-overhanging sides of the coverslip were secured with nail polish .",
"UAS-GCaMP6s fluorescence was viewed with an inverted Zeiss 510 Meta confocal microscope .",
"The IR60b neurons were visualized with a 20X objective , with a digital zoom of 2 . 5 .",
"Images were acquired such that a single 512 × 512 resolution frame was scanned each second , with single-line averaging .",
"The pinhole was opened to 3 . 42 Airy Units ( 7 . 3 μm ) , with the 488 nm laser at 15% .",
"Stimulus was perfused into the sample by delivering tastant solution to the overhanging portion of the coverslip , using a P20 PipetteMan .",
"Changes in fluorescent activity were recorded for 5 m after delivery of the stimulus .",
"Scanned images were binned into 30 s intervals , and a maximum change in fluorescence ( ∆F/F ) was calculated for each bin in order to generate a response curve as depicted in Figure 7 .",
"∆F/F for each binned time interval was calculated as the maximum fluorescence change divided by the average baseline fluorescence of the 10 consecutive frames taken immediately before stimulation .",
"Fluorescent intensities were obtained by using open-source Fiji/ImageJ software ( https://fiji . sc ) to draw a region-of-interest ( ROI ) around cell bodies , and measuring an average pixel intensity for the ROI in each frame using the Time-Series Analyzer Plugin written by Balaji , J . ( https://imagej . nih . gov/ij/plugins/time-series . html ) .",
"When imaging single cell bodies , false positives can occur if the preparation shifts out of the initial plane of focus , along the z-axis , thereby causing changes in the baseline fluorescence levels during the assay .",
"To prevent false positives , care was taken to ensure that cell bodies remained in the correct plane of focus .",
"Before the stimulus , we selected focal landmarks in both the DIC and green fluorescent channels , and closely monitored them during the experiment .",
"If the sample shifted , it was refocused to the reference landmarks , and the out-of-focus frames were excluded from the ∆F/F calculations .",
"Typically , refocusing took 5–10 s , which is less than the binned 30 s time segments used in ∆F/F calculations .",
"Thus , we recorded sufficient information to measure the maximum ∆F/F for each 30 s time interval depicted in Figure 7E–G .",
"The FLIC Drosophila Behavior System was from Sable Systems International ( North Las Vegas , NV ) and was described by Ro et al . ( 2014 ) .",
"The FLIC Monitor Software ( version 2 . 1 , downloaded from wikiflic . com ) was used to collect raw data from the Drosophila Feeding Monitor ( DFM ) .",
"Before the assay , mated females were starved in 1000-μl pipette tips using the same protocol as for the pharyngeal pumping assay .",
"After loading 900 mM sucrose into the DFM , the FLIC Monitor Software was initialized .",
"Six mutant and six control flies were then quickly aspirated into the FLIC system ( 12 single-well setup ) .",
"Typically , loading took no longer than 2 min .",
"We analyzed the first 15 m of feeding , a period chosen to reduce the influence of post-ingestive effects that modulate hunger state and feeding behavior .",
"To automate the analysis of raw FLIC data , we wrote custom software in the R programming language ( Tam , 2017 ) .",
"Feeding events were detected as peaks in raw FLIC traces using the open-source numerical analysis R package ‘pracma’ ( available at https://cran . r-project . org/web/packages/pracma/index . html ) .",
"Thresholds and parameters of the software are user-customizable .",
"To assess the accuracy of our software , we selected 10 FLIC traces randomly for manual ground-truth scoring .",
"Our algorithm correctly identified 97 . 6% of the peaks with a false-positive rate of 1 . 8% .",
"Source code for the software can be obtained at https://github . com/edrictam/FLIC-analysis ( with a copy archived at https://github . com/elifesciences-publications/FLIC-Analysis ) .",
"All statistical tests described in the figure legends were performed using GraphPad Prism 7 .",
"D'Agostino-Pearson and Kolmogorov-Smirnov normality tests were employed to confirm that our data approximately followed Gaussian distributions , thereby allowing the use of parametric analysis ."
]
] | [
"The neural control of sugar consumption is critical for normal metabolism .",
"In contrast to sugar-sensing taste neurons that promote consumption , we identify a taste neuron that limits sucrose consumption in Drosophila .",
"Silencing of the neuron increases sucrose feeding; optogenetic activation decreases it .",
"The feeding inhibition depends on the IR60b receptor , as shown by behavioral analysis and Ca2+ imaging of an IR60b mutant .",
"The IR60b phenotype shows a high degree of chemical specificity when tested with a broad panel of tastants .",
"An automated analysis of feeding behavior in freely moving flies shows that IR60b limits the duration of individual feeding bouts .",
"This receptor and neuron provide the molecular and cellular underpinnings of a new element in the circuit logic of feeding regulation .",
"We propose a dynamic model in which sucrose acts via IR60b to activate a circuit that inhibits feeding and prevents overconsumption ."
] | [
"All animals – from the fruit fly to mammals like humans – must control their dietary intake of nutrients to survive and stay healthy .",
"Taste receptors that sense high-calorie sugars are essential to this process .",
"Typically , when food tastes sweet , it signals that the food contains nutrients and promotes consumption .",
"However , eating too much sugar can be detrimental because the animal wastes time and energy eating food that it does not need , and could eventually lead to obesity and other metabolic diseases .",
"This raised the question: are there any taste receptors that , once they detect sugars , cause animals to eat less ?",
"Joseph et al . worked with the fruit fly Drosophila melanogaster and identified one such taste receptor called IR60b .",
"The experiments showed that this taste receptor responds selectively to sucrose ( a high-calorie sugar ) , and that it activates nerve cells that cause fruit flies to eat less food , rather than more .",
"When the receptor was experimentally inactivated , the fruit flies ate for longer and ate too much sucrose .",
"This indicates that the flies need this receptor to control their sugar intake .",
"A next step will be to see if mammals similarly use sweet-sensing taste receptors to limit the amount of food they eat .",
"A better insight into how mammals can control what they eat could provide a deeper understanding of how to tackle major health issues , such as obesity , in humans ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"evolutionary biology"
] | Membranes, energetics, and evolution across the prokaryote-eukaryote divide | elife-20437-v2 | [
[
"The hallmark feature distinguishing eukaryotes from prokaryotes ( bacteria and archaea ) is the universal presence in the former of discrete cellular organelles enveloped within lipid bilayers ( e . g . the nucleus , mitochondria , endoplasmic reticulum , golgi , vacuoles , vesicles , etc . ) .",
"Under a eukaryocentric view of life , these types of cellular features promoted the secondary origin of genomic modifications that ultimately led to the adaptive emergence of fundamentally superior life forms ( Martin and Koonin , 2006; Lane and Martin , 2010 ) .",
"Most notably , it has been proposed that the establishment of the mitochondrion provided an energetic boost that fueled an evolutionary revolution responsible for all things eukaryotic , including novel protein folds , membrane-bound organelles , sexual reproduction , multicellularity , and complex behavior ( Lane , 2002 , 2015 ) .",
"However , despite having more than two billion years to impose their presumed superiority , eukaryotes have not driven prokaryotes extinct .",
"Prokaryotes dominate eukaryotes both on a numerical and biomass basis ( Whitman et al . , 1998; Lynch , 2007 ) , and harbor most of the biosphere’s metabolic diversity .",
"Although there is no logical basis for proclaiming the evolutionary inferiority of prokaryotes , one central issue can be addressed objectively – the degree to which the establishment of eukaryotic-specific morphology altered energetic efficiency at the cellular level .",
"Drawing on observations from biochemistry , physiology , and cell biology , we present a quantitative summary of the relative bioenergetic costs and benefits of the modified architecture of the eukaryotic cell .",
"The data indicate that once cell-size scaling is taken into account , the bioenergetic features of eukaryotic cells are consistent with those in bacteria .",
"This implies that the mitochondrion-host cell consortium that became the primordial eukaryote did not precipitate a bioenergetics revolution ."
],
[
"The starting point is a recap of recent findings on the scaling properties of the lifetime energetic expenditures of single cells .",
"All energy utilized by cells can be partitioned into two basic categories: that employed in cell maintenance and that directly invested in building the physical infrastructure that comprises a daughter cell .",
"Maintenance costs involve a diversity of cellular functions , ranging from turnover of biomolecules , intracellular transport , control of osmotic balance and membrane potential , nutrient uptake , information processing , and motility .",
"Cell growth represents a one-time investment in the production of the minimum set of parts required for a progeny cell , whereas cell maintenance costs scale with cell-division time .",
"The common usage of metabolic rate as a measure of power production is uninformative from an evolutionary perspective , as it fails to distinguish the investment in cellular reproduction from that associated with non-growth-related processes .",
"To make progress in this area , a common currency of energy is required .",
"The number of ATP→ADP turnovers meets this need , as such transformations are universally deployed in most cellular processes of all organisms , and where other cofactors are involved , these can usually be converted into ATP equivalents ( Atkinson , 1970 ) .",
"When cells are grown on a defined medium for which the conversion rate from carbon source to ATP is known ( from principles of biochemistry ) , the two categories of energy allocation can be quantified from the regression of rates of resource consumption per cell on rates of cell division ( Bauchop and Elsden , 1960; Pirt , 1982; Tempest and Neijssel , 1984 ) .",
"A summary of results derived from this method reveals two universal scaling relationships that transcend phylogenetic boundaries ( Lynch and Marinov , 2015 ) .",
"First , basal maintenance costs ( extrapolated to zero-growth rate , in units of 109 molecules of ATP/cell/hour , and normalized to a constant temperature of 20∘C for all species ) scale with cell volume as a power-law relationship ( 1a ) CM=0 . 39V0 . 88 , where cell volume V is in units of μm3 .",
"Second , the growth requirements per cell ( in units of 109 molecules of ATP/cell ) scale as ( 1b ) CG=27V0 . 97 .",
"The total cost of building a cell is ( 1c ) CT=CG+tCM , where t is the cell-division time in hours .",
"Derived from cells ranging over four orders of magnitude in volume , neither of the preceding scaling relationships is significantly different from expectations under isometry ( with exponent 1 . 0 ) , as the standard errors of the exponents in Equations ( 1a , b ) are 0 . 07 and 0 . 04 , respectively .",
"Moreover , as there is no discontinuity in scaling between prokaryotes and eukaryotes , these results suggest that a shift of bioenergetics from the cell membrane in prokaryotes to the mitochondria of eukaryotes conferred no directly favorable energetic effects .",
"In fact , the effect appears to be negative .",
"Taking into account the interspecific relationships between cell-division time and cell volume ( Lynch and Marinov , 2015 ) and using Equation ( 1b ) , one can compute the scaling of the rate of incorporation of energy into biomass , CG/t .",
"For bacteria , cell-division times decline with increasing cell volume as ∼V-0 . 17 , albeit weakly ( the SE of the exponent being 0 . 11 ) , implying that the rate of biomass accumulation scales as ∼V0 . 97+0 . 17=V1 . 14 on a per-cell basis and as ∼V1 . 14-1 . 00=V0 . 14 on a cell volumetric basis ( with the SEs of both exponents being 0 . 12 ) .",
"In contrast , in most eukaryotic groups , cell-division times increase with cell volume , on average scaling as ∼V0 . 13 , implying a scaling of ∼V0 . 84 for the rate of biomass accumulation per cell and ∼V-0 . 16 on a volumetric basis ( with SEs equal to 0 . 06 for the exponents ) .",
"Thus , in terms of biomass production , the bioenergetic efficiency of eukaryotic cells declines with cell volume , whereas that of bacterial cells does not .",
"The pattern observed in bacteria is inconsistent with the view that surface area limits the rate of energy production , as this leads to an expected scaling of ∼V2/3 on a per-cell basis .",
"The argument that mitochondria endow eukaryotic cells with exceptionally high energy provisioning derives from the idea that large internal populations of small mitochondria with high surface area-to-volume ratios provide a dramatic increase in bioenergetic-membrane capacity ( Lane and Martin , 2010 ) .",
"In prokaryotes , the F0F1 ATP synthase ( the molecular machine that transforms ADP to ATP in the process of chemiosmosis ) and the electron transport chain ( ETC ) components ( which create the chemiosmotic proton gradient ) are restricted to the cell membrane , but in eukaryotes , they are confined to inner mitochondrial membranes .",
"A key question is whether the bioenergetic capacity of cells is , in fact , limited by membrane surface area .",
"Although the situation at the time of first colonization of the mitochondrion is unknown , the iconic view of mitochondria being tiny , bean-shaped cellular inclusions is not entirely generalizable .",
"For example , many unicellular eukaryotes harbor just a single mitochondrion or one that developmentally moves among alternative reticulate states ( e . g . Rosen et al . , 1974; Osafune et al . , 1975; Biswas et al . , 2003; Yamaguchi et al . , 2011 ) .",
"Such geometries necessarily have lower total surface areas than a collection of spheroids with similar total volumes .",
"For the range of species that have been examined , many of which do have small individualized mitochondria , the total outer surface area of mitochondria per cell is generally on the order of the total area of the plasma membrane , with no observed ratio exceeding 5:1 , and many being considerably smaller than 1:1 ( Figure 1a ) .",
"It may be argued that the outer surface area of the mitochondrion is of less relevance than that of the inner membrane ( where the ATP synthase complex sits ) , but the ratios of inner ( including the internal cristae ) to outer membrane areas for mitochondria in mammals , the green alga Ochromonas , the plant Rhus toxicodendron , and the ciliate Tetrahymena are 5 . 0 ( SE = 1 . 1 ) , 2 . 4 , 2 . 5 , and 5 . 2 , respectively ( Supplementary material ) .",
"Thus , the data are inconsistent with the idea that the mitochondrion engendered a massive expansion in the surface area of bioenergetic membranes in eukaryotes .",
"Three additional observations raise questions as to whether membrane surface area is a limiting factor in ATP synthesis .",
"First , the localization of mitochondrial ATP synthase complexes is restricted to two rows on the narrow edges of the inner cristae ( Kühlbrandt , 2015 ) .",
"Because this confined region comprises <<10% of the total internal membrane area , the surface area of mitochondrial membranes allocated to ATP synthase appears to be less than the surface area of the cell itself .",
"Second , only a fraction of bacterial membranes appears to be allocated to bioenergetic functions ( Magalon and Alberge , 2016 ) , again shedding doubt on whether membrane area is a limiting factor for energy production .",
"Third , in every bacterial species for which data are available , growth in cell volume is close to exponential , that is , the growth rate of a cell increases as its cell volume increases despite the reduction in the surface area:volume ratio ( Voorn and Koppes , 1998; Godin et al . , 2010; Santi et al . , 2013; Iyer-Biswas et al . , 2014; Osella et al . , 2014; Campos et al . , 2014 ) .",
"Further insight into this issue can be achieved by considering the average packing density of ATP synthase for the few species with proteomic data sufficient for single-cell counts of individual proteins .",
"By accounting for the stoichiometry of the various subunits in the complex , it is possible to obtain several independent estimates of the total number of complexes per cell under the assumption that all the proteins are assembled ( Supplementary material ) .",
"For example , the estimated number of complexes in E . coli is 3018 , and the surface area of the cell is ~15 . 8 μm2 .",
"Based on the largest diameter of the molecule ( the F1 subcomplex ) , a single ATP synthase in this species occupies ~64 nm2 ( Lücken et al . , 1990 ) of surface area , so the total set of complexes occupies ~1 . 8% of the cell membrane .",
"Four other diverse bacterial species for which these analyses can be performed yield occupancies ranging from 0 . 6% to 1 . 5% , for an overall average of 1 . 1% for bacteria .",
"This will be an overestimate if only a fraction of proteins are properly assembled and embedded in the cell membrane .",
"This kind of analysis can be extended to eukaryotes , noting that eukaryotic ATP synthases are slightly larger , with maximum surface area of ~110 nm2 ( Abrahams et al . , 1994; Stock et al . , 1999 ) .",
"Although ATP synthase resides in mitochondria in eukaryotes , it is relevant to evaluate the fractional area that would be occupied were they to be located in the cell membrane .",
"Such hypothetical packing densities are 5 . 0% and 6 . 6% , respectively , for the yeasts S . cerevisiae and S . pombe , and 6 . 6% and 6 . 8% for mouse fibroblasts and human HeLa cells .",
"Although these observations suggest a ~5 fold increase in ATP synthase abundance with cell surface area in eukaryotes , the data conform to a continuous allometric function with no dichotomous break between the bacteria and eukaryotes ( Figure 1b ) .",
"Similar conclusions can be reached regarding the ETCs , although direct comparisons are more difficult due to the diversity of electron transport chain complexes in prokaryotes ( Price and Driessen , 2010 ) .",
"The number of ETC complexes is comparable to that of ATP synthases in both bacteria and eukaryotes ( Supplementary Material ) , and the physical footprint of the ETC is ~5× that of F0F1 ( ~570 nm2; Dudkina et al . , 2011 ) , implying that an average of ~5 . 5% of bacterial cell membranes is dedicated to the ETC and that the corresponding hypothetical packing density for eukaryotes would be ~30% ( if in the cell membrane ) .",
"There are a number of uncertainties in these packing-density estimates , and more direct estimates are desirable .",
"The optimum and maximum-possible packing densities for ATP synthase also remain unclear .",
"Nonetheless , the fact remains that any packing problems that exist for the cell membrane are also relevant to mitochondrial membranes , which have additional protein components ( such as those involved in internal-membrane folding and transport into and out of the mitochondrion ) .",
"Any attempt to determine the implications of membranes for cellular evolution must account for the high biosynthetic costs of lipid molecules .",
"There are two ways to quantify such a cost .",
"First , from an evolutionary perspective , the cost of synthesizing a molecule is taken to be the sum of the direct use of ATP in the biosynthetic pathway plus the indirect loss of ATP resulting from the use of metabolic precursors that would otherwise be converted to ATP and available for alternative cellular functions ( Akashi and Gojobori , 2002; Lynch and Marinov , 2015 ) .",
"Second , to simply quantify the direct contribution to a cell’s total ATP requirement , the costs of diverting metabolic precursors are ignored .",
"By summing the total costs of all molecules underlying a cellular feature and scaling by the lifetime energy expenditure of the cell , one obtains a measure of the relative drain on the cell’s energy budget associated with building and maintaining the trait .",
"This measurement , sc , can then be viewed as the fractional increase in the cell’s energy budget that could be allocated to growth , reproduction , and survival in the absence of such an investment , ignoring the direct fitness benefits of expressing the trait , sa .",
"For selection to be effective , the net selective advantage of the trait , sn=sa-sc , must exceed the power of random genetic drift , 1/Ne in a haploid species and 1/ ( 2Ne ) in a diploid , where Ne is the effective population size .",
"Most cellular membranes are predominantly comprised of glycerophospholipids , which despite containing a variety of head groups ( e . g . glycerol , choline , serine , and inositol ) , all have total biosynthetic costs per molecule ( in units of ATP hydrolyses , and including the cost of diverting intermediate metabolites ) of ( 2a ) cL≃320+[38⋅ ( NL−16 ) ]+ ( 6⋅NU ) , ( 2b ) cL≃340+[40⋅ ( NL−16 ) ]+ ( 6⋅NU ) , in bacteria and eukaryotes , respectively , where NL is the mean fatty-acid chain length , and NU is the mean number of unsaturated carbons per fatty-acid chain ( Supplementary material ) .",
"Although variants on glycerophospholipids are utilized in a variety of species ( Guschina and Harwood , 2006; Geiger et al . , 2010 ) , these are structurally similar enough that the preceding expressions should still provide excellent first-order approximations .",
"The reduced ( direct ) cost , which ignores the loss of ATP-generating potential from the diversion of metabolic precursors , is ( 3a ) cL′≃110+[7⋅ ( NL−16 ) ]+ ( 6⋅NU ) , ( 3b ) cL′≃120+[9⋅ ( NL−16 ) ]+ ( 6⋅NU ) , in bacteria and eukaryotes , respectively .",
"From the standpoint of a cell’s total energy budget , the evolutionary cost of a lipid molecule is cL/CT .",
"For most lipids in biological membranes , 14≤NL≤22 and 0≤NU≤6 , so the cost per lipid molecule is generally in the range of cL≃200 to 600 ATP , although the average over all lipids deployed in species-specific membranes is much narrower ( below ) .",
"Cardiolipin , which rarely constitutes more than 20% of membrane lipids is exceptional , having an evolutionary cost of ~640 ATP/molecule ( and a reduced cost of ~240 ATP ) .",
"To put these expenditures into perspective , the evolutionary biosynthetic costs of each of the four nucleotides is ≈ 50 ATP hydrolyses per molecule ( Lynch and Marinov , 2015 ) , whereas the average cost of an amino acid is ≈30 ATP ( Atkinson , 1970; Akashi and Gojobori , 2002 ) .",
"Application of the preceding expressions to the known membrane compositions of cells indicates that the biosynthetic costs of eukaryotic lipids are higher than those in bacteria ( Supplementary table ) .",
"For example , for a diversity of bacterial species the average direct cost per lipid molecule in the plasma membrane is 123 ( SE = 3 ) ATP , whereas that for eukaryotes is 143 ( 2 ) .",
"The latter estimate is identical to the mean obtained for whole eukaryotic cells , but the cost of mitochondrial lipids is especially high , 155 ( 5 ) .",
"These elevated expenses in eukaryotes are joint effects of the cost of mitochondrial export of oxaloacetate to generate acetyl-CoA and the tendency for eukaryotic lipids to have longer chains containing more desaturated carbons .",
"To understand the total bioenergetic cost associated with membranes , we require information on the numbers of lipid molecules required for membrane formation , which is equivalent to the total surface area of the membrane divided by the number of lipid molecules/unit surface area , and multiplied by two to account for the lipid bilayer .",
"Estimates of the head-group areas of membrane lipids are mostly within 10% of an average value of 6 . 5 × 10−7 μm2 ( Petrache et al . , 2000; Kučerka et al . , 2011 ) , so the cost of a membrane ( in units of ATP , and ignoring lipid turnover and the space occupied by transmembrane proteins ) is ( 4 ) CL≃ ( 3 . 1×106 ) ⋅c¯L⋅A , where A is the membrane surface area in units of μm2 , and c¯L is the average cost of a lipid .",
"Enough information is available on the total investment in mitochondrial membranes that a general statement can be made .",
"Over the eukaryotic domain , the total surface area of mitochondria ( inner plus outer membranes , summed over all mitochondria , in μm2 ) scales with cell volume ( V , in units of μm3 ) as 3 . 0V0 . 99 ( Figure 1c; SEs of intercept and slope on log plots are 0 . 22 and 0 . 08 , respectively ) .",
"Applying this to Equation ( 4 ) , with the average total cost of mitochondrial lipids ( c¯L=440 ATP/ molecule; Appendix 1–table 4 ) , and using the expression for the total growth requirements of a cell , Equation ( 1b ) , the relative cost of mitochondrial membrane lipids is ( 5 ) sc≃0 . 15V0 . 02 , or ∼15% of the total growth budget of a minimum-sized ( 1 μm3 ) eukaryotic cell , and nearly independent of cell size within the range typically found in eukaryotes ( SE of the exponent is 0 . 08 ) .",
"The direct contribution of mitochondrial membrane lipids to a cell’s growth budget is ~36% of this total cost .",
"These costs of mitochondrial membranes represent a baseline price , not incurred by prokaryotes , associated with relocating bioenergetics to the interior of eukaryotic cells , that is , ~5% .",
"Unfortunately , the additional costs of maintenance of mitochondrial lipids is unknown , but for rapidly growing cells , the vast majority of a cell’s energy budget is allocated to growth ( Lynch and Marinov , 2015 ) , so the above costs should still apply as first-order approximations; for slowly growing cells , the costs will be higher or lower depending on whether the cost of mitochondrial-membrane maintenance is above or below that for total cellular maintenance .",
"Proteins do not occupy >50% of membranes , so accounting for this would change the preceding results by a factor <2 .",
"For prokaryotic cells without internal membranes , the relative contribution of the cell membrane to a cell’s total energy budget is expected to decline with increasing cell size , owing to the decline in the surface area to volume ratio .",
"For the tiny cells of Leptospira interrogans and Mycoplasma pneumoniae ( average volumes of 0 . 03 and 0 . 22 μm3 , respectively ) , ~63 and 43% of a cell’s growth budget must be allocated to the plasma membrane , but for the larger Bacillus subtilis and Escherichia coli ( on average , 1 . 4 and 1 . 0 μm3 , respectively ) , these contributions drop to ~14 and 19% , and they would be expected to continue to decline with further increases in cell size , scaling inversely with the linear dimension of the cell .",
"In contrast , owing to the increased investment in internal membranes , the fraction of a eukaryotic cell’s energy budget devoted to membranes does not diminish with increasing cell size .",
"Although there are only a few eukaryotic cell types for which this issue can be evaluated quantitatively ( Table 1 ) , the data span three orders of magnitude in cell volume and uniformly suggest that ~10 to 30% of the total growth budget is allocated to lipid biosynthesis , and that an increasing fraction of such costs is associated with internal membranes in cells of increasing size .",
"The picoplanktonic alga Ostreococcus , which has a cell volume of just 0 . 22 μm3 ( below that of many prokaryotes ) , devotes ~32% of its energy budget to membranes , and 44% of these costs ( ~18% of the total cell budget ) are associated with internal membranes .",
"A moderate-sized mammalian cell devotes a similar ~30% of its energy budget to membranes , but 96% of these costs ( ~29% of the total cell budget ) are associated with internal membranes .",
"Taken together , these observations imply that the use of internal membranes constitutes a major drain on the total energy budgets of eukaryotic cells , much more than would be expected in bacteria of comparable size .",
"Moreover , because the lipids associated with mitochondria alone constitute 20% to 35% of a eukaryotic cell’s investment in membranes ( Table 1 ) , the energetic burden of localizing membrane bioenergetics to mitochondria is substantial .",
"Finally , given that the observations summarized in Figure 1a , b are derived from a diversity of studies , likely with many unique inaccuracies , it is worth considering whether the overall conclusions are consistent with the known capacity of ATP synthase .",
"First , it bears noting that only a fraction of the energy invested in biosynthesis is derived directly from the chemiosmotic activity of ATP synthase .",
"For example , amino-acid biosynthesis involves ~1 . 5 oxidations of NADH and NADPH for every ATP hydrolysis ( Akashi and Gojobori , 2002 ) .",
"Assuming that each of the former is equivalent to ~3 ATP hydrolyses , this implies that only ~18% of the energy invested in amino-acid biosynthesis involves ATP hydrolysis .",
"As noted in the Supplementary text , the ratio of use of NADH/NADPH to ATP is more on the order of 2 . 0 in lipid biosynthesis , reducing the direct investment in ATP to ~14% Thus , as the vast majority of the energetic cost of building a cell is associated with synthesis of the monomeric building blocks of proteins and membranes , only ~15% of biosynthetic energy may be derived from ATP hydrolysis .",
"Given the known energy requirements for the maintenance and growth of a cell , the cell-division time , and the number of ATP synthase complexes per cell , it is possible to estimate the required rate of ADP → ATP conversions per complex .",
"Using the cellular energetic data previously presented ( Lynch and Marinov , 2015 ) and the ATP synthase abundances in Appendix 1–table 2 , after discounting the maximum values by 85% , the estimated required rates of ATP production/complex/sec are: 2109 , 221 , and 19 respectively for the bacteria B . subtilis , E . coli , and M . pneumoniae , and 1440 and 329 for the yeasts S . cerevisiae and S . pombe .",
"Several attempts have been made to estimate the maximum turnover rates ( per sec ) for F0F1 ATP synthase , usually in reconstituted liposomes , and these average 195/s in bacteria ( Etzold et al . , 1997; Slooten and Vandenbranden , 1989; Toei et al . , 2007 ) , 295 in soybean plastids ( Schmidt and Gräbe , 1985; Junesch and Gräber , 1991 ) , 120 in S . cerevisiae ( Förster et al . , 2010 ) , and 440 in bovine heart ( Matsuno-Yagi and Hatefi , 1988 ) .",
"Thus , given that a substantial fraction of complexes are likely to be misassembled in artificial membranes , the energy-budget based estimates of the numbers of ATP turnovers generated per cell appear to be consistent with the known capacity of ATP synthase .",
"The ribosome content of a cell provides a strong indicator of its bioenergetic capacity .",
"Owing to the large number of proteins required to build the complex , ribosomes are energetically costly , and the number per cell appears to be universally correlated with cellular growth rate ( Fraenkel and Neidhardt , 1961; Tempest et al . , 1965; Brown and Rose , 1969; Poyton , 1973; Dennis and Bremer , 1974; Freyssinet and Schiff , 1974; Alberghina et al . , 1975; Boehlke and Friesen , 1975; Waldron and Lacroute , 1975; Scott et al . , 2010 ) .",
"We previously pointed out that the genome-wide total and mean number of transcripts per gene scale with cell volume as V0 . 36 and V0 . 28 respectively , and that the analogous scalings are V0 . 93 and V0 . 66 for proteins , with no dichotomous break between prokaryotes and eukaryotes ( Lynch and Marinov , 2015 ) .",
"As with the transcripts they process and the proteins they produce , the numbers of ribosomes per cell also appear to scale sublinearly with cell volume , in a continuous fashion across bacteria , unicellular eukaryotes , and cells derived from multicellular species ( Figure 2 ) .",
"These observations are inconsistent with the idea that entry into the eukaryotic world resulted in an elevated rate of protein production .",
"Moreover , as noted previously ( Lynch and Marinov , 2015 ) , the absolute costs of producing individual proteins and maintaining the genes associated with them are substantially higher in eukaryotes than in bacteria , owing to the substantial increase in gene lengths , investment in nucleosomes , etc ."
],
[
"Lane ( 2015 ) and Lane and Martin ( 2010 ) have proposed a scenario for how the mitochondrion became established by a series of adaptive steps , arguing that the eukaryotic leap to increased gene number and cellular complexity , and a subsequent adaptive cascade of morphological diversification , ‘was strictly dependent on mitochondrial power' . However , the scaling of the costs of building and maintaining cells is inconsistent with an abrupt shift in volumetric bioenergetic capacity of eukaryotic cells , and although the absolute costs of biosynthesis , maintenance , and operation of individual genes are much greater in eukaryotes , the proportional costs are less ( Lynch and Marinov , 2015 ) . This means that evolutionary additions and modifications of genes are more easily accrued in eukaryotic genomes from a bioenergetics perspective , regardless of their downstream fitness effects . The analyses presented here reveal a number of additional scaling features involving cellular bioenergetic capacity that appear to transcend the substantial morphological differences across the bacterial-eukaryotic divide . There is not a quantum leap in the surface area of bioenergetic membranes exploited in eukaryotes relative to what would be possible on the cell surface alone , nor is the idea that ATP synthesis is limited by total membrane surface area supported . Moreover , the numbers of both ribosomes and ATP synthase complexes per cell , which jointly serve as indicators of a cell’s capacity to convert energy into biomass , scale with cell size in a continuous fashion both within and between bacterial and eukaryotic groups . Although there is considerable room for further comparative analyses in this area , when one additionally considers the substantial cost of building mitochondria , it is difficult to accept the idea that the establishment of the mitochondrion led to a major advance in net bioenergetic capacity . Most discussion of the origin of the mitochondrion by endosymbiosis starts ( and often ends ) with a consideration of the benefits gained by the host cell . This ignores the fact that the eukaryotic consortium consists of two participants . At least initially , the establishment of a stable symbiotic relationship requires that each member of the pair gain as much from the association as is lost by relinquishing independence . Under the scenario painted by Lane and Martin ( 2010 ) , and earlier by Martin and Müller ( 1998 ) , the original mitochondrial-host cell affiliation was one in which the intracellular occupant provided hydrogen by-product to fuel methanogenesis in the host cell , while eventually giving up access to external resources and thereby coming to rely entirely on the host cell for organic substrates . For such a consortium to be evolutionarily stable as a true mutualism , both partners would have to acquire more resources than would be possible by living alone , in which case this novel relationship would be more than the sum of its parts . Although some scenario like this might have existed in the earliest stages of mitochondrial establishment , it is also possible that one member of the original consortium was a parasite rather than a benevolent partner ( made plausible by the fact that many of the α-protobacteria to which mitochondria are most closely related are intracellular parasites ) . Despite its disadvantages , such a system could be rendered stable if one member of the pair ( the primordial mitochondrion ) experienced relocation of just a single self-essential gene to the other member’s genome , while the other lost a key function that was complemented by the presence of the endosymbiont .",
"This scenario certainly applies today , as all mitochondria have relinquished virtually all genes for biosynthesis , replication , and maintenance , and as a consequence depend entirely on their host cells for these essential metabolic functions .",
"In contrast , all eukaryotes have relocated membrane bioenergetics from the cell surface to mitochondrial membranes .",
"Such an outcome represents a possible grand example of the preservation of two ancestral components by complementary degenerative mutations ( Force et al . , 1999 ) .",
"Notably , this process of subfunctionalization is most likely to proceed in relatively small populations because the end state is slightly deleterious from the standpoint of mutational vulnerability , owing to the fact that the original set of tasks becomes reliant on a larger set of genes ( Lynch et al . , 2001 ) .",
"Thus , a plausible scenario is that the full eukaryotic cell plan emerged at least in part by initially nonadaptive processes made possible by a very strong and prolonged population bottleneck ( Lynch , 2007; Koonin , 2015 ) .",
"The origin of the mitochondrion was a singular event , and we may never know with certainty the early mechanisms involved in its establishment , nor the order of prior or subsequent events in the establishment of other eukaryotic cellular features ( Koonin , 2015 ) .",
"However , the preceding observations suggest that if there was an energetic boost associated with the earliest stages of mitochondrial colonization , this has subsequently been offset by the loss of the use of the eukaryotic cell surface for bioenergetics and the resultant increase in costs associated with the construction of internal membranes .",
"Rather than a major bioenergetic revolution being provoked by the origin of the mitochondrion , at best a zero-sum game is implied .",
"Thus , if the establishment of the mitochondrion was a key innovation in the adaptive radiation of eukaryotes , the causal connection does not appear to involve a boost in energy acquisition .",
"Notably , a recent analysis suggests that the origin of the mitochondrion postdated the establishment of many aspects of eukaryotic cellular complexity ( Pittis and Gabaldón , 2016 ) .",
"It is plausible , that phagocytosis was a late-comer in this series of events , made possible only after the movement of membrane bioenergetics to the mitochondrion , which would have eliminated the presumably disruptive effects of ingesting surface membranes containing the ETC and ATP synthase ."
],
[
"The results in this paper are derived from an integration of bioenergetic analyses based on known biochemical pathways and existing morphological observations on a variety of cell-biological features .",
"The sources of this information , as well as the basic approaches employed can be found in the Appendix ( where not mentioned directly in the text ) .",
"The central analyses involve: ( 1 ) estimation of the biosynthetic costs for lipid-molecule production ( in terms of ATP equivalents per molecule produced ) ; ( 2 ) mitochondrial surface areas and cell membrane areas; ( 3 ) investments in lipids at the cell-membrane and organelle levels; and ( 4 ) numbers of ATP synthase complexes , ETC complexes , and ribosomes per cell ."
]
] | [
"The evolution of the eukaryotic cell marked a profound moment in Earth’s history , with most of the visible biota coming to rely on intracellular membrane-bound organelles .",
"It has been suggested that this evolutionary transition was critically dependent on the movement of ATP synthesis from the cell surface to mitochondrial membranes and the resultant boost to the energetic capacity of eukaryotic cells .",
"However , contrary to this hypothesis , numerous lines of evidence suggest that eukaryotes are no more bioenergetically efficient than prokaryotes .",
"Thus , although the origin of the mitochondrion was a key event in evolutionary history , there is no reason to think membrane bioenergetics played a direct , causal role in the transition from prokaryotes to eukaryotes and the subsequent explosive diversification of cellular and organismal complexity ."
] | [
"Over time , life on Earth has evolved into three large groups: archaea , bacteria , and eukaryotes .",
"The most familiar forms of life – such as fungi , plants and animals – all belong to the eukaryotes .",
"Bacteria and archaea are simpler , single-celled organisms and are collectively referred to as prokaryotes .",
"The hallmark feature that distinguishes eukaryotes from prokaryotes is that eukaryotic cells contain compartments called organelles that are surrounded by membranes .",
"Each organelle supports different activities in the cell .",
"Mitochondria , for example , are organelles that provide eukaryotes with most of their energy by producing energy-rich molecules called ATP .",
"Prokaryotes lack mitochondria and instead produce their ATP on their cell surface membrane .",
"Some researchers have suggested that mitochondria might actually be one of the reasons that eukaryotic cells are typically larger than prokaryotes and more varied in their shape and structure .",
"The thinking is that producing ATP on dedicated membranes inside the cell , rather than on the cell surface , boosted the amount of energy available to eukaryotic cells and allowed them to diversify more .",
"However , other researchers are not convinced by this view .",
"Moreover , some recent evidence suggested that eukaryotes are no more efficient in producing energy than prokaryotes .",
"Lynch and Marinov have now used computational and comparative analysis to compare the energy efficiency of different organisms including prokaryotes and eukaryotes grown under defined conditions .",
"To do the comparison , the results were scaled based on cell volume and the total surface area deployed in energy production .",
"From their findings , Lynch and Marinov concluded that mitochondria did not enhance how much energy eukaryotes could produce per unit of cell volume in any substantial way .",
"Although the origin of mitochondria was certainly a key event in evolutionary history , it is unlikely to have been responsible for the diversity and complexity of today’s life forms ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"genetics and genomics"
] | Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism | elife-06315-v1 | [
[
"The steroid hormone aldosterone is normally produced in the adrenal zona glomerulosa in response to either angiotensin II , which is produced in response to volume depletion , or hyperkalemia ( high plasma K+ level ) .",
"Both stimuli cause membrane depolarization , activating voltage-gated Ca2+ channels; increased intracellular Ca2+ provides the signal that triggers aldosterone production ( Spät and Hunyady , 2004 ) .",
"In the setting of volume depletion , aldosterone signaling in renal and intestinal epithelia produces increased salt ( re ) absorption , promoting restoration of intravascular volume; in hyperkalemia , aldosterone promotes increased potassium secretion , restoring electrolyte balance .",
"Pathological secretion of aldosterone in the absence of normal physiological stimuli leads to primary aldosteronism ( PA ) , producing increased salt ( re ) absorption and hypertension .",
"Hypokalemia is a frequently associated finding , resulting from increased renal K+ elimination .",
"PA is found in 10% of patients referred for evaluation of hypertension ( Conn , 1955; Rossi et al . , 2006 ) .",
"About half of these patients have adrenal aldosterone-producing adenomas ( APAs ) .",
"Germline mutations in three genes have been shown to cause rare Mendelian forms of early-onset PA .",
"Gene fusions leading to constitutive expression of aldosterone synthase ( encoded by CYP11B2 ) , a rate-limiting enzyme in aldosterone biosynthesis , cause Glucocorticoid-Remediable Aldosteronism ( GRA ) ( Lifton et al . , 1992 ) .",
"Mutations in and near the selectively filter of the K+ channel encoded by KCNJ5 result in channels that conduct Na+ , leading to adrenal glomerulosa cell depolarization and activation of Ca2+ channels , producing a Mendelian form of aldosteronism ( Choi et al . , 2011 ) .",
"Gain of function mutations in the calcium channel encoded by CACNA1D cause increased Ca2+ channel activity and another form of PA .",
"These latter patients also have seizures , neurodevelopmental and neuromuscular abnormalities owing to gain of function effects of CACNA1D in the nervous system ( Scholl et al . , 2013 ) .",
"Families with GRA often have many affected subjects and were identified by linkage analysis in extended families ( Lifton et al . , 1992 ) .",
"Germline mutations in KCNJ5 are typically de novo or in small nuclear families; similarly , CACNA1D mutations to date are all de novo ( Choi et al . , 2011; Scholl et al . , 2012 , 2013 ) .",
"Germline mutations in KCNJ5 and CACNA1D were found following identification of the same or related somatic mutations as drivers of APAs ( Choi et al . , 2011; Scholl et al . , 2012; Azizan et al . , 2013; Scholl et al . , 2013 ) .",
"The causes of PA in many patients remain undetermined .",
"Although Mendelian inheritance has been suggested by recurrence of PA in some kindreds without mutations in known genes ( Stowasser et al . , 1992; Torpy et al . , 1998; Lafferty et al . , 2000 ) , traditional linkage analysis has failed to identify additional causative genes , likely due to a combination of factors including locus heterogeneity , high frequency of de novo mutations , reduced penetrance and/or variable expressivity .",
"The advent of next-generation sequencing , allowing the search for recurrent mutations or greater burden of rare variants in individual genes than expected by chance , can permit identification of such loci in the absence of classical segregation patterns .",
"Very rare phenotypes , such as childhood PA , are promising candidates for such traits .",
"Using exome sequencing , we here identify five independent occurrences of the identical mutation in CACNA1H among 40 subjects with unexplained PA in childhood .",
"CACNA1H encodes a voltage-gated calcium channel that is expressed in adrenal glomerulosa .",
"Electrophysiology demonstrates that this variant causes reduced inactivation and a shift of activation to more hyperpolarized potentials , effects inferred to produce increased calcium influx and PA ."
],
[
"From a cohort of more than 1500 unrelated subjects referred for evaluation of genetic forms of hypertension , we identified 40 subjects diagnosed with hypertension and PA by age 10 years in whom disease-causing mutations in CYP11B2 , KCNJ5 , and CACNA1D ( Lifton et al . , 1992; Choi et al . , 2011; Scholl et al . , 2013 ) were excluded .",
"Clinical details are shown in Supplementary file 1A .",
"All subjects had hypertension with elevated aldosterone levels despite low plasma renin activity ( PRA ) .",
"None of the subjects studied were the offspring of consanguineous union .",
"DNA from peripheral blood was subjected to exome capture and sequencing; mean coverage was 73 independent reads per targeted base ( Supplementary file 1B ) .",
"Variants were called as described in ‘Materials and methods’ ( Lemaire et al . , 2013 ) .",
"We performed three analyses tailored to the expectation of a rare genetic disease ( ‘Materials and methods’ ) .",
"We sought previously unreported ( absent in dbSNP , NHLBI , 1000Genomes and Yale exome databases ) protein-altering variants that occurred in more than one subject ( Supplementary file 1C ) ; we performed gene burden analyses to search for previously unreported or rare ( minor allele frequency [MAF] < 0 . 01% ) heterozygous variants that collectively occurred in any gene more often than expected by chance ( Supplementary file 1D ) ; we searched for rare ( MAF < 0 . 1% ) homozygous and potential compound heterozygous variants that collectively occurred in any genes more often than expected by chance ( Supplementary file 1E ) .",
"There was only one result that surpassed genome-level significance: we found five apparently unrelated subjects with the identical previously unreported heterozygous A > G variant , resulting in a p . Met1549Val substitution in CACNA1H , which encodes the pore-forming alpha subunit of a T-type , low voltage-activated calcium channel ( aka CaV3 . 2 ) ( Figure 1 , Table 1 , Supplementary file 1F ) ( Perez-Reyes , 2003 ) .",
"This variant is absent among more than 129 , 000 alleles sequenced from diverse populations in the Exome Aggregation Consortium ( Exome Aggregation Consortium ) , and Yale databases .",
"No other CACNA1H alleles with allele frequencies <0 . 01% were found among our cohort .",
"Like other Ca2+ channel alpha subunits , CACNA1H contains four homologous repeats ( I–IV ) , each with six transmembrane segments ( S1–S6 ) .",
"The CACNA1HM1549V variant lies in the S6 segment of repeat III ( Marksteiner et al . , 2001 ) .",
"Sanger sequencing in each case confirmed the heterozygous variant ( Figure 1A ) . 10 . 7554/eLife . 06315 . 003Figure 1 . Kindreds with hypertension and primary aldosteronism ( PA ) with CACNA1HM1549V mutation at conserved position of S6 domain .",
"( A ) Pedigrees of kindreds with CACNA1HM1549V mutation are shown .",
"Studied subjects with early-onset hypertension are shown as black filled symbols , and subjects with early-onset hypertension by family history ( K333 ) or low renin with normal blood pressure ( K1393 ) are shown as grey filled symbols .",
"Genotypes are indicated below each symbol ( +/+ denotes wild type sequence; +/M denotes heterozygosity for CACNA1HM1549V variant ) .",
"Corresponding Sanger sequencing results for selected subjects are depicted to the right .",
"( B ) Transmembrane structure of CaV3 . 2 ( encoded by CACNA1H ) , the pore-forming subunit of a voltage-gated Ca2+ channel , is shown .",
"These channels have four internal homologous repeats ( I–IV ) , each with six transmembrane segments ( S1–S6 ) and a membrane-associated loop between the pore-forming S5 and S6 segments .",
"The p . Met1549Val mutation is located in S6 of repeat III .",
"( C ) Conservation of CACNA1HM1549 in CACNA1H orthologs and paralogs .",
"The amino acid sequences of the S6 segment of domain III of CACNA1H , orthologs and paralogs are shown .",
"The S6 segment , including Met1549 , is virtually completely conserved ( highlighted in yellow ) among orthologs and all paralogs that are activated by small changes in membrane potential ( l , low voltage-activated ) but not those activated by large changes ( h , high voltage-activated ) .",
"M1549 is part of the Met-Phe-Val sequence that is implicated in rapid channel inactivation ( Marksteiner et al . , 2001 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 00310 . 7554/eLife . 06315 . 004Figure 1—source data 1 . Source data corresponding to Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 00410 . 7554/eLife . 06315 . 005Figure 1—figure supplement 1 . Cohort population structure by principal component analysis ( PCA ) .",
"PCA of subjects referred for PA .",
"Individuals in the cohort ( orange crosses ) mostly cluster with HapMap subjects of European and African American subjects .",
"The five individuals with CACNA1H mutation ( filled red circles ) are of African American ( 1390-1 ) , Hispanic ( 1393-1 ) and European ( 1368-1 , 1347-1 and 333-2 ) origin , respectively , by history and PCA .",
"Source files are available in Figure 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 00510 . 7554/eLife . 06315 . 006Table 1 . Clinical features of index cases with CACNA1HM1549VDOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 006Subject IDGenderAge dxBP ( %ile ) Aldo ( ng/dl ) PRA ( ng/ml/hr ) ARR ( ng/dl: ng/ml/hr ) 1347-1M3 yrs160/105 ( >99th ) 20<0 . 1>2001390-1F7 yrs150/90 ( >99th ) 660 . 23301368-1M8 yrs140/90 ( >99th ) 20<0 . 2>100333-2M9 yrs192/144 ( >99th ) 40<0 . 7>571393-1M2 mos170/110 ( >99th ) 87<0 . 6>145M , male; F , female; age dx , age at diagnosis of hypertension; yrs , years; mos , months; BP , blood pressure; ( %ile ) , percentile adjusted for age and gender; Aldo , serum aldosterone; PRA , plasma renin activity; ARR , aldosterone:renin ratio , values >20 with aldosterone level >15 are considered indicative of primary aldosteronism ( PA ) .",
"Three index cases were of European ancestry , one Hispanic , and one African American by self-report and principal component analysis ( Figure 1—figure supplement 1 ) .",
"Members of the extended families were recruited , and sequencing of these subjects demonstrated that CACNA1HM1549V was a de novo mutation ( absent in the biological parents ) in both the index case of kindred 1347 , and in the affected mother of the index case in kindred 1390 ( Figure 1 ) .",
"Analysis of highly polymorphic markers confirmed paternity and maternity in both kindreds ( Supplementary file 1G ) .",
"This establishes independent occurrences of CACNA1HM1549V in these two kindreds .",
"In the remaining three kindreds , the variant was transmitted to the index case from a parent , and samples from grandparents were not available for further analysis of transmission ( Figure 1 ) .",
"Analysis of kinship coefficients using SNP genotypes of affected subjects from Illumina Human 1M-Quad beadchips and the KING algorithm ( Manichaikul et al . , 2010 ) provided no evidence that these three kindreds shared recent common ancestry ( Supplementary file 1H , ‘Materials and methods’ ) .",
"Further , haplotypes flanking the CACNA1H mutation were phased using the BEAGLE program , revealing that the maximum shared haplotype flanking the CACNA1H mutation among these three kindreds was only 53 . 6 kb ( 87 . 0 kb for the two European kindreds , Figure 2 ) .",
"From this data , the maximum likelihood estimate of the number of generations since the last shared common ancestor among these subjects is estimated to be 714 generations 95% CI 290-1268 ( Genin et al . , 2004 ) .",
"A more conservative analysis identifying homozygous discordant SNPs ( eliminating inference of haplotypes by phasing ) still limited the shared haplotype to less than 127 . 1 kb , consistent with the results of phasing using BEAGLE .",
"These findings indicate that the CACNA1HM1549V mutation in these three kindreds has not been inherited from a recent common ancestor , and has either arisen independently or has been inherited from an extremely remote common ancestor .",
"The latter possibility is extremely unlikely given the absence of this mutation in more than 129 , 000 alleles studied to date . 10 . 7554/eLife . 06315 . 007Figure 2 . Shared haplotypes in subjects with inherited CACNA1HM1549V variant . Haplotypes of three affected individuals from kindreds without proven de novo occurrence of CACNA1HM1549V variant were phased using BEAGLE ( ‘Materials and methods’ ) ( Browning and Browning , 2007 ) .",
"This analysis identified a very small maximum interval shared among all three individuals ( ∼53 . 6 kb , green box ) flanked by rs1075789 and rs3760122 .",
"If only homozygous discordant calls ( * ) were considered in the absence of phasing , the maximum interval shared by all three subjects would be 127 . 1 kb and the longest pairwise shared haplotype would be 200 . 0 kb between 1393-1 and 333-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 007 The probability of finding any recurrent protein-altering de novo mutation among 40 kindreds is ∼4 . 0 × 10−6 ( see ‘Materials and methods’ ) .",
"Even with a conservative estimate of the allele frequency of CACNA1HM1549V of 0 . 00001 in the general population ( ‘conservative’ because it has never been seen among >129 , 000 alleles in subjects not selected for early PA ) , the probability of finding three additional instances of this mutation in 38 unrelated subjects is ∼8 . 4 × 10−12 .",
"Combined , the probability of finding these five instances of the identical variant by chance is conservatively estimated to be 3 . 4 × 10−17 , providing extremely strong statistical support for the role of this mutation in PA .",
"If the CACNA1HM1549V mutation causes early-onset PA , CACNA1H ( CaV3 . 2 ) should be expressed in human adrenal glomerulosa .",
"CACNA1H transcripts have previously been reported in human kidney , liver , heart and brain ( Cribbs et al . , 1998 ) , and our prior analysis of gene expression of human adrenal cortex showed that CACNA1H was the second most highly expressed calcium channel alpha subunit , after CACNA1D ( Scholl et al . , 2013 ) .",
"We performed immunohistochemistry with two different antibodies specific for the encoded channel protein ( CaV3 . 2 ) , demonstrating strong staining of human adrenal glomerulosa; this staining was abolished after preincubation with immunogenic peptide ( Figure 3 ) .",
"These results are consistent with prior in situ hybridization and electrophysiological studies of rodent and bovine glomerulosa ( Schrier et al . , 2001; Hu et al . , 2012 ) as well as a recent study of human adrenal gland ( Felizola et al . , 2014 ) . 10 . 7554/eLife . 06315 . 008Figure 3 . Immunohistochemistry of CaV3 . 2 in normal human adrenal gland . Sections of normal human adrenal are shown .",
"C denotes adrenal capsule; G , glomerulosa; F , fasciculata .",
"( A ) Normal adrenal gland stained with hematoxylin and an antibody to CaV3 . 2 ( Alomone ) .",
"( B ) Higher power image of adrenal in panel ( A ) .",
"( C , D )",
"Absence of staining after preincubation of the antibody with the antigenic peptide , demonstrating specificity .",
"( E ) A second normal human adrenal gland stained for CACNA1H as in ( A , B ) .",
"( F ) Gland from ( A–D ) stained with a second α-CACNA1H antibody ( Santa Cruz ) .",
"Scale bars , 100 μm ( A , C ) ; 50 μm ( B , D , E , F ) .",
"The results demonstrate expression of CaV3 . 2 in the normal zona glomerulosa , which is only several cells in depth . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 008 The clinical features of the index cases harboring the CACNA1HM1549V variant were uniform .",
"All presented with hypertension by age 10 and had persistent inappropriate elevation of serum aldosterone with suppressed PRA and high aldosterone:PRA ratio , indicative of autonomous adrenal aldosterone production ( Table 1 ) .",
"Adrenal imaging by computed tomography , magnetic resonance or ultrasound showed no evidence of mass or hyperplasia at the time of presentation .",
"There were no other recurrent or distinctive features of the index cases , specifically no history of seizures , neurologic or neuromuscular disorders as found in patients with CACNA1D mutations ( Scholl et al . , 2013 ) .",
"Additional details are presented in Appendix 1 .",
"By direct Sanger sequencing , we identified five additional CACNA1HM1549V mutation carriers among family members , including four parents and one uncle of an index case ( Figure 1 ) .",
"Of these five , three were diagnosed with early severe hypertension while two were not , and in fact were normotensive as adults ( Appendix 1 and Supplementary file 1I ) .",
"For example , subject 1390-2 was diagnosed with severe hypertension and PA at age 17; her hypertension was difficult to control , leading to unilateral adrenalectomy at age 29 .",
"Her hypertension nonetheless recurred , requiring reinstitution of treatment .",
"Interestingly , the histology of her adrenal gland showed striking microscopic hyperplasia .",
"While the normal adrenal glomerulosa comprises only a few cell layers and is about 70 μm in depth , the glomerulosa of subject 1390-2 was ∼30 cell layers and ∼300 μm in depth ( Figure 4A–C ) .",
"CACNA1H was expressed in the hyperplastic glomerulosa layer ( Figure 4 , Appendix 1 ) .",
"In this kindred , the CACNA1HM1549V variant arose concordantly and segregated precisely with PA and early hypertension ( Table 2 ) .",
"Two mutation carriers ( 1368-2 and 1393-3 ) were normotensive as adults and had not been diagnosed as hypertensive in childhood; in subject 1393-3 , PRA was at the lower limit of normal with normal aldosterone level , while PRA and aldosterone levels were normal in 1368-2 ( Supplementary file 1I ) . 10 . 7554/eLife . 06315 . 009Figure 4 . Glomerulosa hyperplasia in adrenal gland of subject 1390-2 with CACNA1HM1549V mutation . C denotes adrenal capsule; G , glomerulosa; F , fasciculata; R , reticularis; M , medulla .",
"( A ) Low power image stained with hematoxylin and eosin .",
"Scale bar 1000 μm .",
"( B , C )",
"Higher power images of adrenal from panel ( A ) , scale bars 400 μm ( B ) or 100 μm ( C ) .",
"The mutant adrenal shows marked zona glomerulosa hyperplasia , with micronodular invasion of the capsule ( denoted by * ) .",
"( D ) Same adrenal gland stained with hematoxylin and antibody to CaV3 . 2 ( Santa Cruz ) , demonstrating specific staining of zona glomerulosa .",
"Scale bar , 400 μm .",
"( E , F ) , higher power images stained with second antibody to CaV3 . 2 ( Alomone ) .",
"Scale bars 250 μm ( E ) or 100 μm ( F ) .",
"CaV3 . 2 is expressed in the hyperplastic zona glomerulosa . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 00910 . 7554/eLife . 06315 . 010Table 2 . Laboratory values of carriers and non-carriers of CACNA1HM1549V in kindred 1390DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 010Subject IDGenderAge ( yrs ) K+ ( mmol/l ) Aldo ( ng/dl ) PRA ( ng/ml/hr ) ARR ( Aldo/PRA ) Direct renin ( μIU/ml ) Aldo/direct reninCarriers 1390-1F153 . 7370 . 4288 . 1NANA 1390-2F293 . 522NANA37 . 3Non-Carriers 1390-4M174 . 321 . 651 . 2NANA 1390-8M373 . 8<12 . 25<0 . 4NANA 1390-5F624 . 11618 . 920 . 8NANA 1390-6F514 . 041 . 872 . 1NANA 1390-7F464 . 331 . 671 . 8NANAM , male; F , female; Age ( yrs ) , Age in years when sample was obtained; K+ , serum potassium ( reference 3 . 5–5 . 5 mmol/l ) ; Aldo , serum aldosterone; PRA; direct renin <5 is indicative of volume-mediated hypertension; ARR , aldosterone:renin ratio , values >20 ( using PRA ) or values of aldo/direct renin >2 . 4 with aldosterone level greater than 15 are considered indicative of PA .",
"Blood samples were drawn on the same day , and values were determined in the same laboratory ( except for 1390-2 , in whom values are pre-adrenalectomy at age 29 ) .",
"See Figure 1 for relationships .",
"1390-6 and −7 are not included in Figure 1 , and are sisters of 1390-5 .",
"To explore the specificity of this mutation for early-onset PA , we performed targeted Sanger sequencing for the CACNA1HM1549V variant in germline DNA of 1632 additional unrelated subjects , comprising 324 subjects with PA diagnosed after age 10 years , 96 with hypertension and bilateral adrenal hyperplasia , and 1212 referred for potential genetic causes of hypertension without evidence of PA .",
"We also sequenced tumor DNA of 90 APAs , including 40 that did not have mutations in previously implicated genes ( KCNJ5 , CACNA1D , ATP1A1 , ATP2B3 , and CTNNB1 [Choi et al . , 2011; Azizan et al . , 2013; Beuschlein et al . , 2013; Scholl et al . , 2013] ) .",
"No additional CACNA1HM1549V mutations were identified , demonstrating striking specificity for early-onset PA .",
"Members of the CaV3 family are activated by small depolarizing changes in the membrane potential ( activation threshold ∼ −60 mV ) and display very fast voltage-dependent inactivation ( Perez-Reyes , 2003 ) .",
"Methionine at the position corresponding to CACNA1HM1549 is conserved in the S6 helix of repeat three in all identified orthologs , including invertebrates .",
"In addition , methionine occurs at the paralogous position in other calcium channels activated by small depolarizing potential changes ( Figure 1C ) .",
"Prior studies of CaV3 . 1 ( CACNA1G ) have shown that methionine 1549 lies in a methionine-phenylalanine-valine ( MFV ) tripeptide that regulates channel inactivation ( Hering et al . , 1998; Marksteiner et al . , 2001 ) .",
"Mutation of the homologous methionine in CaV3 . 1 to isoleucine or alanine results in delayed channel inactivation ( Marksteiner et al . , 2001 ) , and related calcium channels with isoleucine at the homologous position inactivate more slowly than those with methionine ( Hering et al . , 1997 ) ( Figure 1C ) .",
"To assess the biophysical properties of CACNA1HM1549V , we heterologously expressed either CACNA1HWT or CACNA1HM1549V in HEK293T cells and performed whole-cell patch clamp recordings ( Figure 5A ) .",
"Upon depolarizing voltage steps from −90 mV , CACNA1HWT showed fast activation of calcium currents followed by rapid inactivation , consistent with prior studies ( Cribbs et al . , 1998 ) .",
"In contrast , CACNA1HM1549V exhibited marginally slower activation followed by a dramatically slowed inactivation .",
"While CACNA1HWT is virtually fully inactivated by 400 ms , CACNA1HM1549V shows strong tail currents after returning to the holding potential of −90 mV ( Figure 5A ) , demonstrating loss of normal inactivation , an effect still evident after sustained depolarization for 5 s ( Figure 5B ) . 10 . 7554/eLife . 06315 . 011Figure 5 . CACNA1HM1549V impairs channel inactivation . Whole-cell patch clamp recordings were performed in HEK293T cells transfected with CACNA1HWT or CACNA1HM1549V .",
"( A ) Cells were held at −90 mV , and voltage steps between −90 and +50 mV were applied to elicit calcium currents , followed by a step to −90 mV to evoke tail currents .",
"Representative recordings show rapid activation and inactivation of CACNA1HWT currents and delayed inactivation of CACNA1HM1549V .",
"Tail currents are exclusively present in CACNA1HM1549V and suggest the presence of non-inactivated mutant channels at the end of the depolarizing pulse .",
"( B ) Tail currents are still present after a 5-s pulse to −20 mV .",
"The fraction of non-inactivated channels after 5 s was determined by dividing the peak amplitude at −20 mV before and after 5 s long pulses to voltages between −90 and −20 mV in 5 mV increments ( CACNA1HM1549V: 6 . 7 ± 1% , N = 12; CACNA1HWT: 2 . 4 ± 0 . 5% , N = 9; p = 0 . 004 , protocol not shown in figure ) .",
"( C ) Exponential fits of the current decay between −50 and +30 mV provide inactivation time constants .",
"Data from CACNA1HM1549V are shown in blue circles , CACNA1HWT data are shown in red squares .",
"The mutant channel shows almost 10-fold slower inactivation than wild-type ( N = 9 for CACNA1HWT , N = 7–14 for CACNA1HM1549V , p < 0 . 001 across all voltages studied , Mann–Whitney rank sum test ) .",
"( D ) In contrast , activation time constants at different voltages are only slightly slower in CACNA1HM1549V compared to WT ( cf . ‘Materials and methods’ for details ) .",
"Source files are available in Figure 5—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 01110 . 7554/eLife . 06315 . 012Figure 5—source data 1 . Source data corresponding to Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 01210 . 7554/eLife . 06315 . 013Figure 5—figure supplement 1 . Recovery from inactivation is slightly slower in CACNA1HM1549V .",
"( A ) The recovery from inactivation at −90 mV is decelerated in CACNA1HM1549V .",
"Representative current recordings of CACNA1HWT or CACNA1HM1549V channels .",
"Channels were activated and subsequently inactivated by clamping the membrane potential to −20 mV for 5 s .",
"Afterwards , cells were held at −90 mV for increasing durations followed by short activation at −20 mV .",
"The peak amplitude at the last −20 mV step is dependent on the number of non-inactivated channels that increases upon longer intervals at −90 mV .",
"( B ) Monoexponential fits to the plot of the relative peak amplitudes vs the time spent at −90 mV reveal a slight delay in the recovery from inactivation of CACNA1HM1549V channels ( time constants for CACNA1HWT: 871 . 4 ± 52 . 6 ms , n = 6; CACNA1HM1549V: 1689 . 0 ± 70 . 9 ms , n = 10; p = 1 × 10−6 ) .",
"Source files are available in Figure 5—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 013 We fitted mono-exponential equations to the decay phase of the calcium current between −50 mV and +30 mV .",
"The determined time constants represent the mean time at which the current has decreased to 1/e of its initial amplitude; the results demonstrate ∼10-fold slower inactivation of CACNA1HM1549V compared to CACNA1HWT ( p < 0 . 001 at all voltages studied , Figure 5C ) .",
"In contrast , activation and recovery from inactivation were only marginally slower in mutant channels ( Figure 5D , Figure 5—figure supplement 1 ) .",
"We also observed a significant shift of activation to less depolarizing potentials ( Figure 6 ) .",
"CACNA1HWT showed half-maximal activation ( V1/2 ) at −38 . 9 ± 1 . 1 mV ( N = 11 ) ; in contrast , CACNA1HM1549V showed V1/2 of −44 . 2 ± 1 . 1 mV ( N = 11 , p = 0 . 003 ) , resulting in a lower threshold for activation and increase in size of the ‘window current’ , the area under the intersection of activation and inactivation curves where a fraction of channels are constitutively open .",
"There was no significant effect on single channel conductance ( Figure 6—figure supplement 1 ) . 10 . 7554/eLife . 06315 . 014Figure 6 . CACNA1HM1549V shifts activation to more hyperpolarized potentials .",
"( A ) Current-voltage plots and ( B ) activation curves show a shift of V1/2 for activation of the mutant channel to less depolarizing potentials .",
"The voltage dependence of inactivation is shown as open circles or squares .",
"For CACNA1HM1549V , the area under the intersection of activation and inactivation curves ( where a fraction of channels show continuous activity ) is larger and shifted to more hyperpolarized potentials compared to CACNA1HWT , allowing for increased constitutive Ca2+ influx at potentials close to the resting potential of zona glomerulosa cells ( Hu et al . , 2012 ) .",
"Source files are available in Figure 6—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 01410 . 7554/eLife . 06315 . 015Figure 6—source data 1 . Source data corresponding to Figure 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 01510 . 7554/eLife . 06315 . 016Figure 6—figure supplement 1 . CACNA1HM1549V and CACNA1HWT whole-cell current densities and non-stationary noise analysis .",
"( A ) Whole-cell peak currents were divided by the cell capacitance as determined by the amount of capacitance compensation .",
"CACNA1HM1549V shows a decreased current density compared to CACNA1HWT albeit with large variability .",
"( B , E )",
"Representative mean currents from more than 200 traces recorded from one cell expressing CACNA1HWT ( B ) or CACNA1HM1549V ( E ) channels .",
"( C , F )",
"Analysis of the variance at the −90 mV tail pulse reveals a time dependent decrease .",
"( D , G )",
"A plot of the variance vs the current only allows for a small initial part of the expected parabolic distribution to be visible .",
"Linear fits ( black line ) reveal similar single channel amplitudes ( CACNA1HWT: 273 . 7 ± 3 . 2 fA , n = 3; CACNA1HM1549V: 285 . 0 ± 17 . 3 fA , n = 6; p = 0 . 67 ) , but cannot be used to determine absolute open probabilities .",
"Source files are available in Figure 6—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06315 . 016 Collectively , the changes in inactivation and voltage-dependence of activation cause Ca2+ influx at membrane potentials close to the resting potential and result in channels that remain open longer , allowing increased Ca2+ entry ."
],
[
"These findings define a previously unrecognized form of PA resulting from a recurrent germline gain of function mutation in the Ca2+ channel encoded by CACNA1H .",
"The extremely strong statistical evidence implicating this mutation , its clear gain of function effect , and the known role of Ca2+ signaling on aldosterone production and cell proliferation ( Spät and Hunyady , 2004 ) all strongly support this conclusion .",
"The effects of this mutation phenocopy the adrenal effects of PA-causing mutations in KCNJ5 ( Choi et al . , 2011 ) and CACNA1D ( Scholl et al . , 2013 ) , demonstrating a shared final common pathway by which PA results from increased Ca2+ entry via voltage-gated channels .",
"These results allow a simple genetic test for this specific cause of severe hypertension and suggest that inhibition of mutant CACNA1H activity would ameliorate hypertension in patients with this mutation .",
"While the CACNA1HM1549V mutation explains a significant fraction of the early PA cases , the causes of the remaining cases in our cohort are still unknown .",
"There is striking genotype–phenotype correlation among patients with germline and somatic mutations in KCNJ5 and CACNA1H .",
"Several recurrent germline mutations in KCNJ5 ( e . g . , p . Gly151Arg and p . Thr158Ala ) support robust cell proliferation leading to massive adrenal hyperplasia identifiable on CT scan , leading to adrenalectomy ( Choi et al . , 2011; Scholl et al . , 2012 ) .",
"In contrast , another recurrent mutation in KCNJ5 ( p . Gly151Glu ) shows no or minimal hyperplasia discernable by adrenal imaging ( Mulatero et al . , 2012; Scholl et al . , 2012 ) .",
"While the former mutations are also found as somatic mutations in about 40% of APAs ( Choi et al . , 2011; Mulatero et al . , 2012 ) , the latter have not been found in more than 900 APAs ( Scholl and Lifton , 2013 ) .",
"This phenotypic difference is likely accounted for by different effects on Na+ conductance- the germline mutations that are not associated with APAs or hyperplasia cause markedly greater Na+ conductance , resulting in very high cell lethality , preventing sustained increases in cell mass ( Mulatero et al . , 2012; Scholl et al . , 2012 ) .",
"Similarly , adrenal glands with CACNA1HM1549V show little or no hyperplasia by CT scan and neither this mutation nor other activating mutations in CACNA1H have been seen in APAs .",
"We have not observed high cell lethality in HEK293T cells expressing CACNA1HM1549V .",
"Germline mutations that cause massive hyperplasia and somatic mutations that cause APA formation likely require an optimal Ca2+ signal , one that is high enough to support proliferation but not so high as to cause cell lethality ( Berridge et al . , 2000 ) .",
"While germline mutations in KCNJ5 and CACNA1D were discovered following the initial identification of their somatic mutations in APAs ( Choi et al . , 2011; Scholl et al . , 2013 ) , the discovery of the recurrent CACNA1H mutation relied entirely on brute force sequencing of patients with early severe aldosteronism and hypertension .",
"The occurrence of de novo mutations , the reduced penetrance in adults with the absence of large multiplex families , as well as the absence of distinctive phenotypes that distinguish these patients from others with early hypertension and aldosteronism , all suggest reasons that CACNA1H mutations were not previously linked to PA .",
"The reduced penetrance in adults in particular is interesting—two mutation-carrier parents were normotensive as adults , without clear evidence of PA .",
"Incomplete penetrance among some carriers of mutations that cause aldosteronism ( Stowasser et al . , 1995; Mulatero et al . , 2002; Scholl et al . , 2013 ) has been previously described .",
"The explanations for these effects remain unclear , however age-dependent activity of the renin-angiotensin system and the ability of older individuals to modulate dietary salt intake in response to physiologic demand are potential contributors .",
"This is well described in the case of heterozygous loss of function mutation in the receptor for aldosterone ( the mineralocorticoid receptor , MR ) .",
"These patients have life-threatening salt-wasting and volume depletion in the first years of life due to low signaling through MR , but are asymptomatic as adults .",
"Adult subjects show increased dietary salt intake and increase MR signaling by induction of the renin-angiotensin system , thereby markedly increasing aldosterone levels ( Geller et al . , 1998 ) .",
"Other possible mechanisms for incomplete penetrance include genetic modifiers either in cis or in trans , including the possibility of somatic mosaicism resulting in absence of the gain of function mutation the adrenal gland ( Youssoufian and Pyeritz , 2002 ) .",
"While such mosaicism cannot be excluded , Sanger sequence traces provided no suggestion of mosaicism in circulating white blood cell or saliva DNA ( Figure 1A ) .",
"CACNA1HM1549V shows constitutive activity at membrane potentials close to the resting potential , allowing channels to be activated despite suppression of the renin-angiotensin system and absence of hyperkalemia .",
"CACNA1HM1549V channels also show strikingly delayed inactivation , a finding similar to mechanisms in several other channelopathies ( Cannon et al . , 1991; Lerche et al . , 1993; Scholl et al . , 2013 ) .",
"In glomerulosa , delayed inactivation is inferred to increase the period of membrane potential depolarizations .",
"Interestingly , recent studies in mouse have implicated CACNA1H activity in regular glomerulosa membrane potential oscillations that may amplify small changes in membrane potential to produce significant Ca2+ signals ( Hu et al . , 2012 ) .",
"Thus this regular activation of CACNA1H , together with a shift of activation to less depolarized potentials and prolonged activity , provides a mechanism for increased Ca2+ entry , leading to aldosteronism .",
"While a common variant in CACNA1H has been suggested to be associated with blood pressure in a small genome-wide association study of African American individuals ( Adeyemo et al . , 2009 ) , this result did not pass criteria for genome-wide significance , was only found after exclusion of hypertensive individuals , and was not replicated in larger studies ( International Consortium for Blood Pressure Genome-Wide Association Studies et al . , 2011; Kidambi et al . , 2012 ) .",
"The apparent limitation of the phenotype associated with CACNA1HM1549V to PA with hypertension despite the expression of CACNA1H in other organs including heart and brain ( Cribbs et al . , 1998 ) is notable , and underscores the challenges in predicting human phenotypes from knowledge of underlying mutations .",
"No mutation carrier had a history of seizures or cardiac arrhythmia .",
"While some prior studies have suggested a role of rare gain of function mutations in CACNA1H in epilepsy ( Chen et al . , 2003; Liang et al . , 2006; Heron et al . , 2007 ) , these studies have not approached genome-wide levels of significance , do not appear to confer high risk , and have not been uniformly replicated ( Heron et al . , 2004; Chioza et al . , 2006; Liang et al . , 2006 ) .",
"Our findings are consistent with evidence supporting a normal role for CACNA1H in the regulation of human aldosterone biosynthesis ( Felizola et al . , 2014 ) .",
"Because CACNA1H is activated by small depolarizing changes in glomerulosa membrane potential , it is likely activated in response to small day-to-day changes in serum K+ concentration and angiotensin II levels that require fine adjustments in aldosterone production to maintain volume and electrolyte balance .",
"In contrast , CACNA1D , which is the most highly expressed calcium channel in adrenal cortex , and which shows larger single channel conductance than CACNA1H ( Michels et al . , 2002; Bock et al . , 2011 ) , is only activated by large depolarizations .",
"Activation of this channel likely contributes to the high levels of aldosterone produced in response to marked volume depletion or hyperkalemia .",
"We suggest that CACNA1H and CACNA1D act in series in the regulation of aldosterone , with CACNA1H being activated in response to small , frequent physiologic perturbations and CACNA1D in response to more infrequent large physiologic challenges .",
"These findings also raise the question whether inhibition of wild type CACNA1H would lower blood pressure or aldosterone production .",
"In the general population , loss-of-function variants in CACNA1H are very rare ( cumulative frequency of splice site , frameshift and nonsense variants in the ExAC database of 0 . 06% , resulting in expected compound heterozygosity or homozygosity in about 1 in 2 . 6 million subjects ) , making such studies challenging .",
"It seems plausible that loss of CACNA1H could be compensated by activation of the renin-angiotensin system , leading to greater glomerulosa cell depolarization with consequent activation of CACNA1D , maintaining normal aldosterone production and blood pressure .",
"Consistent with this suggestion , blood pressure was reportedly unchanged in a CACNA1H knockout mouse model , although aldosterone levels were not reported ( Chiang et al . , 2009 ) .",
"Similarly , selective inhibitors of CACNA1H inhibit aldosterone production in vitro ( Rossier et al . , 1998; Perez-Reyes et al . , 2009 ) , but do not apparently reduce aldosterone levels or blood pressure in vivo ( Schmitt et al . , 1992; Ragueneau et al . , 2001 ) .",
"Whether additional non-dihydropyridine compounds will prove to be more effective in lowering aldosterone levels or blood pressure will be interesting to assess ."
],
[
"PA was diagnosed based on elevated ARR ( >20 ng/dl:ng/ml/hr ) , typically with aldosterone >15 ng/dl , or marginally elevated values in the presence of unexplained hypokalemia ( Funder et al . , 2008 ) .",
"Venous blood or saliva samples were obtained from subjects with unexplained early-onset PA and family members .",
"Research protocols were approved by the local institutional review board ( IRB ) , and informed consent was obtained from all research participants .",
"DNA was prepared from venous blood or saliva samples using standard procedures .",
"Exome capture was performed using the 2 . 1M NimbleGen Exome reagent ( Roche NimbleGen , Madison , WI ) , and 75 base paired end sequencing on the Illumina ( San Diego , CA ) platform was performed as previously described ( Lemaire et al . , 2013 ) .",
"Coverage statistics are provided in Supplementary file 1B .",
"Direct bidirectional Sanger sequencing of CACNA1HP1523-R1584 from genomic DNA of indicated subjects was performed following PCR amplification using primers CACNA1H_25F ( 5′-GACCCACCGCCTCTGTG-3′ ) and CACNA1H_25R ( 5′-AGCGCCTTACTCCTGCG-3′ ) .",
"Parent-offspring trios were genotyped as previously described , except for locus D7S820 in kindred 1390 ( primers [5′-ATGTTGGTCAGGCTGACTATG-3′] and [5′-GATTCCACATTTATCCTCATTGAC-3′] ) ( Scholl et al . , 2013 ) .",
"Alleles without known frequencies in the population were omitted from the analysis .",
"Normal human adrenal tissue was obtained from the Yale Pathology archive , and adrenal tissue from subject 1390-2 from Pathology Services of Beaufort/Charleston ( South Carolina , USA ) .",
"Immunohistochemistry was performed as previously described ( Scholl et al . , 2013 ) .",
"Primary antibodies were α-CaV3 . 2 ( #ACC-025 , Alomone , Jerusalem , Israel ) or T-type Ca++ CP α1H ( SC-25691 , Santa Cruz Biotechnology , Santa Cruz , CA ) , both at dilutions of 1:100; secondary antibody was donkey α-rabbit ( #035-152 , 1:500 , Jackson , Bar Harbor , ME ) .",
"For the Alomone antibody , preincubation with the antigenic peptide ( 1:1 , wt/wt in 10% FBS ) was performed for 1 hr at RT .",
"Both antibodies were tested on two independent glands .",
"H&E staining was performed at Yale Research Histology using routine procedures .",
"Myc-DDK-tagged CACNA1H in pCMV6-Entry was obtained from Origene ( Rockville , MD ) ( RC212772 , NM_021098 . 2 ) .",
"Site-directed mutagenesis ( QuikChange , Agilent Technologies , Santa Clara , CA ) was performed to introduce the p . Met1549Val mutation according to the manufacturer's instruction .",
"Each construct was validated by sequencing of the entire coding region .",
"Culturing of HEK293T cells was performed as described ( Scholl et al . , 2013 ) .",
"Cells were transfected with 3 µg of CACNA1HWT or CACNA1HM1549V expression plasmids .",
"For each construct , two clones were functionally tested .",
"Whole cell patch clamp recordings were performed on a HEKA EPC10 amplifier ( HEKA Elektronik , Ludwigshafen , Germany ) as described previously ( Scholl et al . , 2013 ) .",
"The extracellular solution contained: 5 mM CaCl2 , 125 mM TEA-Cl , 10 mM HEPES , 15 mM Mannitol , pH 7 . 4 .",
"Pipette solution contained: 100 mM CsCl , 5 mM TEA-Cl , 3 . 6 mM PCr-Na2 , 10 mM EGTA , 5 mM Mg-ATP , 0 . 2 mM Na-GTP , 10 mM HEPES , pH 7 . 4 ( titration with CsOH ) .",
"Voltage dependences of activation were determined from the peak current–voltage relation and fit by a Boltzmann function as described ( Marcantoni et al . , 2010; Scholl et al . , 2013 ) .",
"The fraction of non-inactivated channels was determined by dividing the peak amplitude at −20 mV before and after 5 s long pulses to voltages between −90 and −20 mV .",
"Time courses of activation or inactivation were analyzed by fitting a mono-exponential function ( Scholl et al . , 2013 ) .",
"The recovery from inactivation was measured using envelope protocols consisting of an inactivation of channels during a 5 s pulse to −20 mV followed by holding the membrane potential at −90 for increasing durations ( Coulter et al . , 1989 ) .",
"Afterwards , peak currents at −20 mV were measured and divided by the previous peak current .",
"A plot of these ratios vs the duration of the pulse to −90 mV was fit with a mono-exponential function to obtain time constants for the recovery from inactivation .",
"Non-stationary noise analysis was performed as described ( Hebeisen and Fahlke , 2005 ) using a voltage protocol that activates channels at −20 mV followed by the analysis of the decay of currents and variance at −90 mV .",
"The initial variance at the holding potential of −90 mV before activation was regarded as background variance and subtracted from the recordings .",
"The Lorentzian noise produced by channel opening and closing depends on the unitary current amplitude ( i ) , the number of channels ( N ) , and the absolute open probability ( P ) : ( 1 ) σ2=N·i2·p· ( 1−p ) .",
"Since the macroscopic current amplitude is given by ( 2 ) I=N·p·i , the variance-current relationship results in a quadratic distribution: ( 3 ) σ2=i·〈I〉− ( 〈I〉2N ) .",
"The single channel amplitude ( i ) was derived from the initial slope of a plot of the variance against the mean isochronal current results .",
"Due to a low open probability ( p < 0 . 5 ) at 5 mM of external Ca2+ , the recorded data points only described a small part of the usual parabola and did not allow for determination of the number of channels and open probabilities .",
"Data were analyzed in FitMaster ( HEKA Elektronik ) , SigmaPlot ( Jandel Scientific , San Rafael , CA ) and Python .",
"Statistical comparisons were performed using Student's t-test or Mann–Whitney rank sum test .",
"Proteins encoded by orthologs or close paralogs of CACNA1H in vertebrate and invertebrate species were identified by a BLAST search .",
"GenBank accessions included NP_066921 . 2 ( Homo sapiens ) , O88427 . 3 ( Mus musculus ) , XP_414830 . 4 ( Gallus gallus ) , XP_002932520 . 2 ( Silurana tropicalis ) , XP_002122425 . 1 ( Ciona intestinalis ) and NP_001024496 . 1 ( Caenorhabditis elegans ) .",
"Human α1 subunit paralogs were as previously described ( Scholl et al . , 2013 ) .",
"Principal component analysis was performed as previously described ( Lemaire et al . , 2013 ) .",
"For analysis of close relatedness , genomic DNA from subjects 333-1 , 1347-1 , 1368-1 , 1390-1 , and 1393-1 was genotyped on Illumina Human 1M-Quad beadchips according to the manufacturer's instructions .",
"Data were analyzed using a combination of GenomeStudio ( Illumina ) and PLINK v1 . 07 softwares ( Purcell et al . , 2007 ) .",
"Mean call rate was 95 . 7% .",
"Kinship coefficients were calculated by using the robust algorithm in KING 1 . 4 ( Manichaikul et al . , 2010 ) .",
"For 1393-1 , 1368-1 and 333-1 , PLINK format was converted to BEAGLE format using Mega2 ( Mukhopadhyay et al . , 2005 ) .",
"Haplotypes flanking the CACNA1HM1549V mutation were phased by observed transmission in kindred 1393 and by maximum likelihood in kindreds 1368 and 333 using BEAGLE v . 3 . 3 . 2 ( Browning and Browning , 2007 ) and a reference panel ( phase 1 1000Genomes project ) .",
"Only SNPs called in at least two samples were used for imputation , and only called SNPs were used for determination of the shared interval .",
"Four additional heterozygous variants in close proximity to CACNA1HM1549V were identified from the 1393-1 exome .",
"For 1368-1 and 333-1 , the inferred haplotype producing the largest shared interval was chosen for further analysis .",
"Mutation age was determined from haplotypes including flanking 41 markers using ESTIAGE ( Genin et al . , 2004 ) .",
"Recombination fractions were calculated from marker distances and average recombination rate across the interval ( 2 . 9 cM/Mb , deCODE ) .",
"Shared allele frequencies were from EUR population ( 1000 Genomes project ) , and mutation rate was set to 2 × 10−8 .",
"For statistical analysis , a de novo mutation rate of 1 . 4 × 10−8 was assumed .",
"The binomial probability of observing two or more de novo mutations at a specified position in a set of 41 cases ( including one affected parent ) was calculated and corrected for the target size of the human exome ( 24 . 75 Mb ) .",
"The likelihood of observing three additional independent mutations at the identical position in 38 patients was calculated as a binomial probability from the assumed allele frequency .",
"The mutation burden per gene in the cohort of patients with PA was compared to that in a control cohort comprising 724 unaffected parents of patients with congenital heart disease sequenced to similar depth of coverage on the same exome platform ( Zaidi et al . , 2013 ) using Fisher's exact test ."
]
] | [
"Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations , incomplete penetrance , and/or variable expressivity .",
"Genome-level sequencing can overcome these complications .",
"Extreme childhood phenotypes are promising candidates for new Mendelian traits .",
"One example is early onset hypertension , a rare form of a global cause of morbidity and mortality .",
"We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10 .",
"Five subjects ( 12 . 5% ) shared the identical , previously unidentified , heterozygous CACNA1HM1549V mutation .",
"Two mutations were demonstrated to be de novo events , and all mutations occurred independently .",
"CACNA1H encodes a voltage-gated calcium channel ( CaV3 . 2 ) expressed in adrenal glomerulosa .",
"CACNA1HM1549V showed drastically impaired channel inactivation and activation at more hyperpolarized potentials , producing increased intracellular Ca2+ , the signal for aldosterone production .",
"This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension ."
] | [
"The consequence of mutations to the large majority of human genes is unknown .",
"Most mutations that are currently known were discovered by tracing their effects through families .",
"This allows the locations of mutations to be pinpointed on chromosomes—the structures that genetic material is packaged into .",
"Other mutations are harder to trace because individuals with these mutations may develop very different signs and symptoms , or not develop clinical abnormalities at all .",
"Alternatively , a trait may appear sporadically in a family because the mutation arises anew in the affected subject .",
"Recently developed technologies that allow scientists to rapidly sequence all the gene-encoding regions of an individual's DNA—their genome—offer a new way to identify harmful genetic variants .",
"Comparing the genomes of individuals with rare disorders can reveal if the individuals share any genetic mutations in common that could cause their symptoms .",
"Scholl et al . used this strategy to sequence the genomes of 40 individuals with a rare type of hypertension—a condition that causes high blood pressure , and increases the risk of strokes , kidney failure and heart attacks—that develops early in childhood .",
"In this form of the disease , high blood pressure is caused by the adrenal glands above the kidneys producing too much of a hormone called aldosterone .",
"Some genetic causes of this form of the disease have already been identified .",
"Now , Scholl et al . have found a new genetic mutation present in five families with this condition .",
"Two of the individuals were the first in their families to develop this mutation , while three others inherited it .",
"Some of the family members with this mutation had hypertension and some did not .",
"The mutation is in a gene that encodes a type of calcium channel—a protein found in the membrane that surrounds cells , and which can open and close to control the amount of calcium in the cell .",
"This particular calcium channel is abundant in the cells of the adrenal gland .",
"Scholl et al . found that the mutation causes the calcium channels to be more likely to open and take longer to close .",
"This increases the number of calcium ions that move into the cell , which causes the adrenal gland to produce more aldosterone .",
"These new insights have provided a new way of diagnosing early-onset hypertension , and suggest that targeting calcium channels could help to develop new treatments for this disease ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Rab5 and Alsin regulate stress-activated cytoprotective signaling on mitochondria | elife-32282-v3 | [
[
"Mitochondria , the organelle for cellular metabolism and ATP production , play an essential role in a number of other cellular processes such as calcium signaling , lipid synthesis and trafficking , metabolite transport , apoptosis , and reactive oxygen species ( ROS ) production in the cell ( Mesmin , 2016; Ott et al . , 2007; Rizzuto et al . , 2012 ) .",
"Many of these processes necessitate communication with other cellular compartments .",
"For example , membrane contact sites ( MCS ) between endoplasmic reticulum ( ER ) and mitochondria are important for Ca2+ and lipid transfer ( de Brito and Scorrano , 2008 ) , mitochondria fission ( Friedman et al . , 2011 ) , and regulation of apoptosis ( Prudent et al . , 2015 ) .",
"Lipid droplets and peroxisomes interact with mitochondria to regulate fatty acid oxidation ( Cohen et al . , 2014; Pu et al . , 2011 ) .",
"Oxidized and damaged proteins can be selectively delivered to peroxisomes and lysosomes via mitochondrial-derived vesicles ( Sugiura et al . , 2014 ) .",
"These examples demonstrate an extensive functional interplay between organelles , either directly via MCS and/or indirectly via vesicular intermediates .",
"However , the underlying molecular mechanisms remain poorly understood and , in particular , the functional relationship between mitochondria and the endocytic system is largely unexplored .",
"The endocytic pathway is responsible for maintaining cellular homeostasis by internalizing , sorting , recycling and/or degrading distinct types of cargo molecules ( Huotari and Helenius , 2011 ) .",
"Rab GTPases serve as molecular signatures on endosomes , regulating their biogenesis and functions ( Pfeffer , 2017; Zerial and McBride , 2001; Zhen and Stenmark , 2015 ) .",
"Ligand-receptor complexes at the plasma membrane ( PM ) are internalized into early endosomes ( EE ) marked by small GTPase Rab5 , followed by either recycling to the PM via Rab4 and Rab11-positive recycling endosomes ( RE ) , or routed to late endosomes ( LE ) and lysosomes for degradation .",
"The latter process occurs via the conversion of Rab5-positive EE into Rab7-positive LE ( de Renzis et al . , 2002; Rink et al . , 2005; Sönnichsen et al . , 2000; Ullrich et al . , 1996 ) .",
"Rab proteins can thus dynamically associate with the membranes , conferring functional plasticity to organelles .",
"On these endosomal membranes , Rab proteins recruit a plethora of effectors for membrane tethering and fusion , cargo sorting and signaling ( Sorkin and von Zastrow , 2009; Stenmark , 2009; Zerial and McBride , 2001 ) .",
"For example , EEA1 is a dimeric coiled-coiled Rab5 effector protein that tethers two vesicles to allow efficient fusion between Rab5-harbouring membranes ( Murray et al . , 2016; Ohya et al . , 2009 ) .",
"Other Rab5 effectors such as APPL1 are involved in regulating metabolic and inflammatory responses ( Schenck et al . , 2008; Wen et al . , 2010 ) .",
"Rab activation , and thus stabilization after recruitment , on the membrane requires guanine nucleotide exchange factors ( GEFs ) ( Blümer et al . , 2013 ) .",
"Rab5 GEFs constitute a family of VPS9 domain-containing proteins , including Rabex-5 ( Horiuchi et al . , 1997 ) , RME-6 ( Sato et al . , 2005 ) , amyotrophic lateral sclerosis protein 2 ( ALS2/Alsin ) ( Otomo et al . , 2003 ) , and mammalian Ras and Rab interactor 1 , 2 , 3 ( Rin1-3 ) ( Hu et al . , 2005 ) .",
"The rationale behind this complexity is that Rab5 must be specifically regulated by different GEFs in space and time .",
"In this respect , the function of many Rab5 GEFs remains unclear .",
"Physical interactions between the endosomal machinery and mitochondria serve important functions in cell homeostasis , repair and apoptosis .",
"For example , transfer of iron ( Das et al . , 2016; Sheftel et al . , 2007 ) and cholesterol ( Charman et al . , 2010 ) from endosomes to mitochondria is mediated by physical interactions between the two organelles .",
"Another classical example of mitochondria-endolysosome interactions is autophagy .",
"Autophagy is a clearance mechanism whereby cells identify defective organelles following damage or stress , and eliminate them via the formation of an autophagosome and subsequent fusion with lysosomes ( Mizushima and Levine , 2010 ) .",
"The mechanism of degrading mitochondria has been termed macroautophagy or mitophagy .",
"Intriguingly , the expression of pro-apoptotic factors such as canonical BH3-only proteins drive Rab5- and Rab7-positive endolysosomes into inner mitochondrial compartments via a pathway that appears to differ from autophagy/mitophagy ( Hamacher-Brady et al . , 2014 ) .",
"Interestingly , our previous conducted genome-wide RNAi screen of endocytosis ( Collinet et al . , 2010 ) revealed that ~8% of the hit genes had mitochondrial-related functions , pointing at hitherto unexplored molecular connections between the endosomal system and mitochondria .",
"This led us to hypothesize that other mitochondrial functions may be regulated by endocytic components .",
"Here by exploring the interactions between EE and mitochondria , we unexpectedly found that upon laser- or chemically induced oxidative stress in mammalian cells , mitochondria outer membrane permeabilization ( MOMP ) releases cytochrome c , and concomitantly , triggers the assembly of the Rab5 machinery on mitochondria , in a process which is reversible and independent of mitophagy .",
"Remarkably , we found that the Rab5 GEF responsible for Rab5 activation is ALS2/Alsin , which is necessary for efficient Rab5 recruitment to mitochondria .",
"Our findings suggest that the Rab endocytic machineries interact with mitochondria during oxidative stress as a cytoprotective mechanism , with important implications for amyotrophic lateral sclerosis ( ALS ) and other neurodegenerative diseases ."
],
[
"We first explored the potential physical link between the endosomal system and mitochondria at steady state .",
"HeLa cells stably expressing TagRFP-MTS ( mitochondria targeting sequence ) ( Takeuchi et al . , 2013 ) were incubated with two types of endocytic cargo , Alexa-488-conjugated transferrin ( Tfn ) or epidermal growth factor ( EGF ) for 10 min at 37°C , to visualize the endocytic/recycling and degradative pathway , respectively .",
"Cell were then fixed and imaged via confocal microscopy .",
"We observed a subset of endosomes that appeared to partially overlap with , or were in close proximity to , mitochondria ( Figure 1A , B ) .",
"To avoid potential morphological changes induced by the TagRFP-MTS overexpression , we also performed quantitative measurements in cells stained for mitochondria with MitoTracker-Red CMXRos ( Mito-Red ) and labeled with Tfn-488 and EGF-488 for 10 and 60 min at 37°C .",
"All acquired images were subjected to chromatic shift correction , deconvolution , and localization analysis ( MotionTracking ) based on subtraction of random colocalization ( Kalaidzidis and Zerial , 2015 ) .",
"Tfn and EGF were efficiently internalized at 10 min , and further sorted to distinct perinuclear compartments representing RE ( Maxfield and McGraw , 2004 ) and LE/lysosomes , respectively , at 60 min ( Figure 1C ) .",
"In both time points , Tfn-containing endosomes consistently exhibited higher colocalization with Mito-Red ( Figure 1D ) compared to EGF-structures , despite both having similar signal intensities ( Figure 1E ) , suggesting that the interactions with mitochondria may preferentially involve early/recycling endosomal structures .",
"Given that early endosomes are motile and omnipresent in the cytoplasm , we next asked whether the observed physical proximity with mitochondria may reflect real MCS or be simply due to random chance ( Kalaidzidis and Zerial , 2015 ) .",
"To further assess the interactions , we monitored the dynamics of organelle contacts during early endocytic events by live-cell imaging .",
"Cells were incubated with Tfn-488 for 1 min to label EE and immediately imaged using a spinning disk confocal microscope .",
"A number of Tfn-containing endosomes and mitochondria labeled by TagRFP-MTS were observed in close proximity , suggesting possible interactions ( Video 1 ) .",
"Some endosomal vesicles remained in close contact with mitochondria for 3–5 min and , interestingly , we could also observe interactions that were followed by fission-like events ( Video 2 ) .",
"Our data suggest that the occurrence of physical contacts between EE and mitochondria observed in fixed and live cells may reflect bona fide , albeit transient interactions , as suggested previously ( Das et al . , 2016; Sheftel et al . , 2007 ) .",
"Given the key role of mitochondria in sensing and responding to oxidative stress , we asked whether acute perturbation on mitochondria may affect endosomes-mitochondria interactions .",
"For this , we used HeLa cells stably expressing GFP-Rab5 under its endogenous promoter with a bacterial artificial chromosome ( BAC ) transgene ( BAC GFP-Rab5 ) ( Villaseñor et al . , 2015 ) .",
"These cells were validated based on the lack of detectable alterations on endocytic trafficking .",
"Live-cell imaging of BAC GFP-Rab5 expressing RFP-MTS also confirmed the occurrence of Rab5-positive EE ( >200 nm ) in close contact with mitochondria ( Video 3 ) .",
"To validate the results with the ectopically expressed mitochondrial marker , we tested mitochondrial-selective dyes in our live-cell imaging .",
"Unexpectedly , we found that photoirradiation in cells labeled with Mito-Red not only robustly induced alterations in mitochondrial morphology but also the appearance of GFP-Rab5 signals around mitochondria ( Figure 2A ) .",
"This is consistent with the fact that certain rosamines and rhodamine-derived dyes used to assay mitochondrial functions possess photosensitizing properties ( Hsieh et al . , 2015 ) .",
"Specifically , Mito-Red has been used to perturb mitochondrial function ( Minamikawa et al . , 1999 ) .",
"Therefore , we hypothesized that the change in Rab5 localization may be a consequence of alterations in mitochondrial function .",
"Consistent with previous reports , low exposure of Mito-Red labeled cells with a 561 nm laser ( ~5 J/cm2 ) caused a decrease in mitochondrial and an increase in cytoplasmic signal , indicative of MOMP , and accompanied by a globular swelling of mitochondria within min ( Figure 2A , Video 4 ) .",
"Remarkably , laser treatment on Mito-Red-labeled cells resulted in the translocation of GFP-Rab5 to mitochondria , marked by increased colocalization with Mito-Red compared to the untreated ( Figure 2B ) .",
"Furthermore , the frequency of GFP-Rab5 EE in close proximity to the stressed mitochondria increased ( Figure 2A , Post-laser arrowheads ) .",
"This effect was specific to Mito-Red treatment , because cells labeled with MitoTracker Green FM or transfected with RFP-MTS retained their tubular mitochondrial structures under the same laser treatment and did not recruit GFP-Rab5 to the mitochondria ( Figure 2—figure supplement 1A , B , respectively ) .",
"The presence GFP-Rab5 on mitochondria was independently confirmed by staining with antibodies against Rab5 and the outer mitochondrial membrane ( OMM ) protein TOM20 ( Figure 2—figure supplement 1C ) .",
"Next , we examined the kinetics of GFP-Rab5 recruitment to Mito-Red-labeled mitochondria upon laser-induced stress .",
"Time-lapse analysis revealed that the recruitment of Rab5 to rounded mitochondria ( Figure 2C , Pre- vs Post-laser ) was visible within 5 min and reached its peak in signal intensity at ~15 min post-laser treatment ( Figure 2D , E; data correspond to the inset region of Figure 2C ) .",
"The GFP-Rab5 ring-like signal persisted for >60 min ( Figure 2—figure supplement 2 , arrowheads ) .",
"In our time-lapse movies , we did not observe evidence of endosomal fusion with mitochondria ( Figure 2E , Video 5 ) , although we cannot exclude fusion with dim vesicles that may have escaped detection .",
"Our interpretation is that the bulk of Rab5 is recruited on mitochondria from the cytosolic pool .",
"These results suggest that upon acute oxidative stress , Rab5 translocates to mitochondria on the time-scale of minutes .",
"We next asked if GFP-Rab5 translocation to mitochondria is a general response to the overall cell stress or can be elicited locally on individual mitochondria .",
"To test this , we photoirradiated a small area of the cell labeled with Mito-Red ( Figure 2F , Pre , inset ) and monitored the GFP-Rab5 signal after 10 and 20 min .",
"Despite the localized perturbation , the morphological alterations extended to most mitochondria which changed from being tubular to rounded ( Figures 2F , 10 min ) .",
"This is consistent with the fact that mitochondria form a dynamically interconnected network ( Lackner , 2014 , Wang et al . , 2015 ) that appears to react to local damage as an ensemble .",
"However , the recruitment of Rab5 was limited to only the laser-induced area ( Figures 2F , 20 min , inset , arrowheads ) .",
"Similar to Figure 2A , distinct GFP-Rab5-positive endosomes contacting mitochondria became visible ( Figure 2F , inset , double arrowheads ) .",
"These results suggest that Rab5 is recruited to mitochondria in response to signal ( s ) originating from individually stressed mitochondria .",
"The laser-induced stress to either an entire cell or a localized region led to the appearance of Rab5-positive endosomes in close proximity to the swollen mitochondria ( Figure 2A , F , arrowheads ) .",
"By live-cell imaging , these endosomes also appeared to dock stably onto mitochondria ( Video 6 , boxed regions , Video 7 ) .",
"However , due to the diffraction limit of standard light microscopy , we could not resolve distances that are closer than 200 nm and endosomes that are <200 nm in diameter .",
"To confirm that these are indeed organelle contacts , we complemented our study by correlative light and electron microscopy ( CLEM ) in order to obtain ultrastructural details .",
"We designed our experimental setup to ( 1 ) perform live-imaging of an entire cell , ( 2 ) visualize the translocation of GFP-Rab5 and GFP-Rab5-positive endosomes onto mitochondria upon laser-induced stress , and ( 3 ) re-image the same cell by serial section transmission electron microscopy ( TEM ) ( Figure 3A ) .",
"GFP-Rab5 cells labeled with Mito-Red were plated on gridded culture dishes , laser-treated and imaged live on the spinning disk confocal microscope .",
"Upon mitochondrial rounding , cells were immediately fixed and re-imaged to assess organellar morphology following fixation .",
"The fluorescent images showed many distinct GFP-Rab5-positive puncta in close proximity to mitochondria ( Figure 3A ) .",
"Samples were then processed for serial section TEM , and an inset area tracked by live-cell imaging ( Figure 3B , red arrowhead ) was re-located in the thin sections by both nuclear membrane ( Figure 3B , dotted line ) and mitochondria ( Figure 3B , red stars ) acting as fiducial markers .",
"TEM images from three different serial sections of the inset area revealed that the GFP-Rab5-positive structure corresponds to a tubular-cisternal structure that is approximately 400 nm in diameter , with the typical morphology of an early endosome ( Figure 3C , green ) .",
"Serial section analysis showed that the endosomal membrane was in very close contact ( <5 nm ) with the adjacent mitochondrion of a diameter of ~1 . 5 µm ( Figure 3C , red ) , which showed few cristae .",
"The tomogram showed the presence of membrane contact sites between the rounded mitochondrion and a cisternal structure that could correspond to the ER , but did not reveal the presence of double or multiple membranes indicative of mitophagy ( Youle and Narendra , 2011 ) .",
"Our data suggest that upon mitochondrial stress , two events involving early endosomes and mitochondria occur: ( 1 ) Rab5 translocates to mitochondria and ( 2 ) endosomes and mitochondria engage in membrane contacts .",
"Following mitochondrial stress , both the fast kinetics ( min ) of Rab5 recruitment on the rounded mitochondria and the absence of wrapped double-membrane structures argue against mitophagy ( Narendra et al . , 2008; Novak et al . , 2010 ) .",
"We searched for additional evidence to rule out this mechanism .",
"Mitophagy requires the E3 ubiquitin ligase Parkin ( Narendra et al . , 2008 ) , which is normally located in the cytosol but recruited to damaged mitochondria , followed by the formation of LC3-positive autophagosomes and fusion with Lamp1-positive lysosomes in a process that occurs in hours ( hr ) to days ( Dolman et al . , 2013 ) .",
"To test whether the swollen mitochondria observed in our system undergo this process , we examined the localization of these markers .",
"We first labeled BAC GFP-Rab5 cells with Mito-Red in order to image mitochondria and endosomes at steady state ( Figure 4A , C , E , G , Untreated ) prior to triggering laser-induced stress .",
"Upon fixation , cells were immunostained with specific antibodies to detect endogenous LC3 , Lamp1 , and Parkin .",
"The cells were seeded on a gridded dish in order to re-locate the same cells following the immunostaining .",
"This allowed us to assess changes to the localization of the markers as a result of stress when compared to neighboring untreated cells ( Figure 4—figure supplement 1 ) .",
"Cells were then incubated for 60 min at 37°C in order to maximize the time window that these markers might be recruited following GFP-Rab5 translocation to mitochondria .",
"In all laser-induced cells , GFP-Rab5 was specifically enriched around mitochondria when compared to the untreated ( Figure 4B , D , F ) .",
"Although we observed a marginal increase in LC3 and Lamp1 colocalization with Mito-Red in stress-induced cells , the signals were mostly concentrated near the perinuclear region ( Figure 4B , D , I ) .",
"On the other hand , Parkin remained mostly cytoplasmic and did not show enrichment around mitochondria in stress-induced cells ( Figure 4F , I ) .",
"As an additional validation of the immunostaining result , we also tested all three markers in HeLa BAC cell lines expressing GFP-tagged LC3 , Lamp1 , and Parkin by live-cell imaging ( Figure 4—figure supplement 2 ) .",
"Following laser irradiation , cells were monitored live for 60 min .",
"Similar to the endogenous LC3 and Lamp1 staining , a fraction of the puncta was enriched in the perinuclear region , overlapping with small fragmented mitochondria , but not with the rest of the mitochondria ( Figure 4—figure supplement 2A , B ) , suggesting that a low level of autophagy is activated .",
"The GFP-Parkin signals were mostly cytoplasmic in untreated cells but showed a few GFP-Parkin-positive ring-like structures around small fragmented mitochondria , whereas most were devoid of signal in the laser-induced condition ( Figure 4—figure supplement 2C , arrowheads ) .",
"Even after 3 hr post-treatment , we observed no significant increase in the number of GFP-Parkin mitochondria ( data not shown ) .",
"The GFP-Parkin ring-like recruitment to these small fragmented mitochondria could be a result of over-expression , inducing some level of mitophagy ( Rana et al . , 2013 ) .",
"Nevertheless , similar to endogenous staining , Parkin was not strongly recruited to the majority of mitochondria .",
"Altogether , the fast kinetics of Rab5 recruitment to stressed mitochondria ( <10 min ) , the absence of a double membranous structure ( Figure 3C ) , and the lack of significant Parkin , LC3 and Lamp1 recruitment argue for a mechanism distinct from autophagy and mitophagy .",
"The low level of autophagic and mitophagic response raise the question of whether apoptosis is involved .",
"Apoptosis is a programmed cell death pathway which occurs via one of two signaling cascades termed intrinsic and extrinsic pathways ( Tait and Green , 2010 ) .",
"The intrinsic pathway is initiated through the activation of the Bax/Bak-mediated MOMP , which leads to the release of cytochrome c to activate effector caspases ( Tait and Green , 2010 ) .",
"Because photoirradiation with Mito-Red engenders oxidative stress in mitochondria ( Hsieh et al . , 2015 ) , we also examined the localization of endogenous Bax via immunostaining .",
"At steady state , Bax signals were visible as cytoplasmic puncta ( Figure 4G ) .",
"Upon laser-induced stress , we observed increased Bax puncta around mitochondria compared to the untreated ( Figure 4H , I ) .",
"As a positive control for the specificity of the antibody , we treated cells with 10 µM protonophore carbonyl cyanide m-chlorophenyl hydrazone ( CCCP ) , which has been shown to cause Bax translocation to mitochondria ( Mikhailov et al . , 2001; Saikumar et al . , 1998 ) .",
"Indeed , cells treated with CCCP exhibited higher Bax fluorescence intensity and colocalization with Mito-Red compared to control cells ( Figure 4—figure supplement 3 ) .",
"The observed enrichment of Bax on mitochondria upon laser-induced stress led us to ask whether artificially activating apoptosis would also drive Rab5 translocation to mitochondria .",
"One method of triggering mitochondrial-associated apoptosis is the over-expression of the truncated BH3 interacting death domain agonist ( tBid ) , which is a potent inducer of apoptosis by activating Bax and/or self-oligomerization on mitochondria ( Grinberg et al . , 2002 ) .",
"To this end , we infected BAC GFP-Rab5 cells with an adenoviral vector expressing tBid ( Ad-tBid ) for 12 hr at 37°C followed by TOM20 immunostaining .",
"We found a strong enrichment of GFP-Rab5 on mitochondria only in cells infected with Ad-tBid but not in Ad-control cells ( Figure 4—figure supplement 4 ) .",
"Our results suggest a mechanism of Rab5 translocation to mitochondria in response to apoptotic signals .",
"What could be the signal that drives Rab5 recruitment ?",
"Several possible scenarios such as morphological changes to mitochondria and/or release of mitochondrial-derived factor ( s ) may account for this .",
"Morphological changes such as matrix condensation or swelling of mitochondria are often associated with MOMP by Bax activation , cytochrome c release and subsequent activation of caspases ( Gottlieb et al . , 2003 ) .",
"However , this is not a prerequisite .",
"For example , the mitochondrial uncoupler CCCP causes mitochondrial swelling and rounding but without immediate cytochrome c release or cell death ( Gao et al . , 2001; Lim et al . , 2001 ) .",
"On the other hand , hydrogen peroxide ( H2O2 ) induces mitochondrial rounding associated with increased Bax expression ( Gutiérrez-Venegas et al . , 2015 ) , cytochrome c release and caspase activation ( Gutiérrez-Venegas et al . , 2015; Takeyama et al . , 2002 ) .",
"For these reasons , we first investigated the effect of CCCP and H2O2 on Rab5 localization .",
"Whereas mitochondria were mostly tubular in control cells ( Figure 5A , B , top panels ) , the exposure of cells to either CCCP or H2O2 for 2 hr resulted in mitochondrial rounding and fragmentation ( Figure 5A , B , bottom panels ) , as previously reported ( Narendra et al . , 2008; Pletjushkina et al . , 2006 ) .",
"Interestingly , CCCP did not cause Rab5 enrichment on mitochondria , which was only observed in H2O2-treated cells ( Figure 5A , B , arrowheads ) , quantified as colocalization with Mito-Red ( Figure 5C , D ) .",
"Consistent with previous findings , we found that the release of cytochrome c into the cytosol was upregulated in H2O2-treated , but not in CCCP-treated cells ( Figure 5E ) .",
"Since cytochrome c is a known factor for activating caspase-dependent programmed cell death , we also assessed the activity of caspase 3/7 via a 4-amino acid peptide ( DEVD ) conjugated to a DNA-binding dye .",
"Cleavage of the DEVD peptide by caspase 3/7 releases the DNA-binding fragment , yielding a fluorescent signal .",
"Using flow cytometry , we detected ~62% of cells showing strong fluorescent signal in H2O2-treated cells , and merely ~0 . 6% and~6 . 2% in control and CCCP-treated cells , respectively .",
"As additional controls of the treatment , we showed that only CCCP , but not H2O2 , resulted in the recruitment of GFP-Parkin as well as endogenous Parkin on mitochondria ( Figure 5—figure supplement 1A , B ) .",
"Given the primary role of mitochondria in cellular metabolism , we asked whether the observed differences between CCCP and H2O2 could be related to altered mitochondrial respiration .",
"To test this , we measured the oxygen consumption rate ( OCR ) in live HeLa cells grown in the presence of galactose in order to force cells to rely primarily on oxidative phosphorylation as opposed to glycolysis ( Aguer et al . , 2011 ) .",
"Upon the addition of CCCP , a sharp increase in OCR was recorded compared to the media control ( Figure 5G , purple vs orange curve ) .",
"This is because CCCP causes the collapse of the proton gradient and the disruption of the mitochondrial membrane potential .",
"As a result , electrons flow unhindered through the electron transport chain , boosting the OCR .",
"In contrast , injection of H2O2 resulted in an initially sharp decline in OCR , but quickly returned to its earlier steady state within 20 min ( Figure 5G , green curve ) , suggesting that the effect on OCR is reversible .",
"The decrease in respiration rate by H2O2 was similarly reported in intact cardiac mitochondria , in which α-ketoglutarate was used as a respiratory substrate ( Nulton-Persson and Szweda , 2001 ) .",
"Altogether , our data show that , in addition to activating caspase-dependent apoptotic program , H2O2 also induces the translocation of Rab5 to mitochondria without disrupting the membrane potential .",
"Given the key role of Rab5 in the biogenesis of the endosomal system ( Zeigerer et al . , 2012; Zerial and McBride , 2001 ) , the translocation of Rab5 to mitochondria upon oxidative stress by H2O2 led us to investigate the connection between endocytosis and oxidative stress .",
"Our live-cell imaging data ( Figures 2 and",
"3 ) argue that GFP-Rab5 is not delivered to mitochondria by fusion with EE .",
"Rather , it may be released from EE and recruited to mitochondria via a cytosolic intermediate .",
"To test this , we collected the total membrane ( M ) and cytosolic ( C ) fractions by subcellular fractionation from HeLa cells treated with/out H2O2 and immunoblotted for Rab5 , the endosomal tether Rab5 effector EEA1 , GAPDH ( as cytosolic marker ) , and TOM20 ( a mitochondrial marker ) ( Figure 6A ) .",
"It was previously shown that H2O2-induced stress causes increased Rab5-GDI complex in BHK cells ( Cavalli et al . , 2001 ) .",
"Consistent with this , upon 1 hr H2O2 treatment , we detected a ~ 0 . 5-fold increase in the amount of Rab5 in the cytosolic fraction compared to the control ( Figure 6A , lane 2 , 4 ) , supporting the view that the cytosolic pool increases at the expense of the membrane-associated pool .",
"The cytosolic levels of Rab5 then decreased at 2 hr time point ( Figure 6A , lane 4 , 6 , Figure 6B ) .",
"We also detected an increase in the EEA1 pool to the cytosol in the long exposure upon H2O2 treatment ( Figure 6A , right blot ) .",
"Since Rab5 in the M fraction includes both the EE and mitochondrial pool , we specifically measured the EE-associated Rab5 by estimating the colocalization of Rab5 with EEA1 by confocal immunofluorescence microscopy .",
"We found a marked ( ~35% ) decrease in the colocalization in H2O2-treated compared to untreated cells ( Figure 6C ) .",
"Altogether , our results suggest that oxidative stress induces the solubilization of a fraction of Rab5 from the endosomal membrane into the cytosol and its translocation to the mitochondrial membrane .",
"The partial dissociation of Rab5 and EEA1 from the endosomal membrane suggests that endocytic trafficking may be affected .",
"To test this , we stimulated HeLa cells with Alexa-647 Tfn continuously for 5 min at 37°C .",
"In the absence of H2O2 , transferrin was efficiently internalized into endosomes ( Figure 6D , Untreated ) .",
"In contrast , cells pre-treated with H2O2 for 10 min showed a severe block in Tfn uptake with an accumulation of Tfn signal on the cell surface ( Figure 6D , E ) , confirming the inhibition of endocytic uptake .",
"In the laser-induced stress conditions , we observed a high occurrence of EE contacting stressed mitochondria ( Figure 2A , F , Figure 3 ) , and these interactions appeared to be specific to EE ( Figure 1 ) .",
"The effects by H2O2 prompted us to further probe whether differential interactions exist between mitochondria and Tfn- vs . EGF-positive endosomes .",
"To test this , HeLa cells stained with MitoRed were continuously labeled with either Tfn-488 or EGF-488 for 10 min at 37°C and then incubated for 50 min with/out H2O2 .",
"There was a significant increase in the colocalization between Mito-Red and both Tfn-488 and EGF-488 in H2O2-treated compared to non-treated cells ( Figure 1C , D ) .",
"Intriguingly , we also found an increase in the total signal intensity in H2O2 conditions ( Figure 1E ) , in which Tfn signals accumulated more prominently in the perinuclear region ( typical of RE ) , whereas EGF was more evenly distributed compared to untreated ( Figure 1C ) .",
"Increased colocalization with H2O2 is likely attributed to a block in recycling of Tfn ( as uptake is inhibited ) and sorting of EGF to LE/lysosome , thereby resulting in cargo accumulation in EE .",
"Altogether , our data suggest that oxidative stress induced by laser irradiation or H2O2 leads to Rab5 translocation from EE to mitochondria , increased EE-mitochondrial MCS , and a defect in endosomal sorting .",
"By live-cell imaging , we found that mitochondria respond to H2O2 treatment with different kinetics within a cell ( Video 8 ) .",
"Distinct regions of the mitochondrial network were more prone to rounding and membrane permeabilization than others , as revealed by the differential loss of Mito-Red during H2O2 treatment ( Figure 7A , inset image ) .",
"Regions containing stressed rounded mitochondria correlated exclusively with the Rab5 ring-like recruitment ( Figure 7A , inset image , arrowheads ) , suggesting that Rab5 may be involved in either facilitating or preventing the apoptotic process .",
"Therefore , we asked whether Rab5 plays a role in regulating cytochrome c release .",
"For these experiments , we transiently over-expressed GFP-Rab5 ( or GFP as control ) in HeLa cells and measured the amount of cytosolic cytochrome c at different time points after incubation with H2O2 .",
"We found a significant delay in the cytochrome c release from mitochondria in GFP-Rab5 over-expressing cells compared to control cells ( Figure 7B , C ) .",
"Our results suggest that Rab5 plays a protective role in mitochondrial-induced apoptosis by down-regulating the release of pro-apoptotic factor ( s ) to the cytosol .",
"We reasoned that if the translocation of Rab5 to mitochondria induced by oxidative stress is a pro-survival response by lowering the apoptotic potential , then the process should be reversible when stress is removed and mitochondria may recover their normal state .",
"The fast recovery rate of mitochondrial respiration following H2O2 injection ( Figure 5E ) also supports this prediction .",
"We initially tested a range of H2O2 concentrations ( 100 µM to 1 mM ) and incubation times ( 2 to 24 hr ) , in order to find an optimal balance between a measurable level of Rab5 translocation to mitochondria and minimal cell death .",
"We found that concentrations up to 500 µM did not cause a noticeable cell rounding by 2 hr ( data not shown ) .",
"Therefore , we pre-incubated cells with 250 µM H2O2 over a period of 24 hr and quantitatively measured TOM20 levels and Rab5-mitochondria colocalization as a means to assess mitochondrial mass and interaction dynamics .",
"Cells were incubated in the presence or absence of H2O2 for 6 , 12 and 24 hr , and either lysed for western blot analysis or fixed for immunostaining .",
"Interestingly , we found that the levels of TOM20 started to increase after 6 hr , suggesting that the mitochondrial mass increased ( Figure 8A , left panels ) .",
"In immunostained cells , we found that the colocalization between endogenous TOM20 and Rab5 ( Figure 8B ) peaked at 6 hr and started to taper off after 12 hr ( Figure 8C ) .",
"Decreased colocalization correlated with increased TOM20 protein levels ( Figure 8A ) .",
"Remarkably , the removal of H2O2 at 12 hr followed by an additional 12 hr incubation in complete medium not only fully restored the morphology of mitochondria from rounded to tubular ( Figure 8B , bottom panel ) , but also returned TOM20:Rab5 colocalization to steady state levels ( Figure 8C ) .",
"The response was dose-dependent as cells exposed to 500 µM H2O2 showed arrested TOM20 levels and were unable to be rescued despite H2O2 removal at 12 hr ( Figure 8A , right panels ) .",
"Our data show that Rab5 translocation to mitochondria induced by oxidative stress is a reversible and protective process responding to apoptotic signals via the regulation of mitochondria .",
"Because Rab5 translocates from EE to mitochondria with a consequent reduction in endocytic uptake , we next asked whether the endosomal Rab5 effectors are also recruited to mitochondria .",
"We systematically assessed the localization of various endosomal effectors such as Rabenosyn-5 , EEA1 , APPL1 and APPL2 in BAC GFP-Rab5 HeLa cells labeled with Mito-Red via immunostaining by pair-wise combinations .",
"We deliberately chose to detect the endogenous because the tagged proteins often severely perturb the endosomal system , as assessed by quantitative endocytic trafficking ( Kalaidzidis et al . , 2015 ) .",
"Specific antibodies were first tested in untreated control cells , which showed significant levels of colocalization with GFP-Rab5 ( Figure 9—figure supplement 1 ) .",
"Upon laser-induced stress , the appearance of GFP-Rab5 rings around mitochondria provided an immediate visual cue and served as a positive control .",
"Cells were fixed after 30-min incubation post-laser treatment .",
"Of the tested effectors , we detected a strong enrichment of Rabenosyn-5 , but not EEA1 , on mitochondria in the same cell ( Figure 9A , C ) .",
"Neither APPL1 nor APPL2 showed enrichment around mitochondria , despite a robust Rab5 recruitment ( Figure 9B , C ) .",
"Unlike Rabenosyn-5 , EEA1 , APPL1 and APPL2 remained well distributed in endosomal-like vesicles in both treated and untreated cells ( Figure 9A , B , Figure 9—figure supplement 1B , C , D ) .",
"As Rabenosyn-5 and EEA1 are recruited to endosomes via both Rab5 and PI ( 3 ) P-binding FYVE motifs ( Nielsen et al . , 2000 ) , we asked whether phosphatidylinositol 3-phosphate ( PI ( 3 ) P ) was present on mitochondria in our stress conditions .",
"To test this , we over-expressed the PI ( 3 ) P probe GFP-2xFYVEHrs ( Gillooly et al . , 2000 ) in HeLa cells and monitored GFP signals in live cells upon laser-induced stress .",
"Fluorescence signals were present as vesicle-like puncta ( Figure 9—figure supplements 2 , 0 min ) , as previously reported ( Gillooly et al . , 2000 ) .",
"After 60 min , stressed and swollen mitochondria were observed , but these were completely devoid of GFP signals , which remained on vesicle-like puncta ( Figure 9—figure supplements 2 , 60 min ) .",
"To corroborate the immunostaining data with an independent method , we also tested the effect of H2O2 on Rab5 and Rab5 effectors in association with mitochondria by subcellular fractionation .",
"We isolated cytosolic ( C ) and mitochondrial fractions ( Mito ) via differential centrifugation and probed them with different effector antibodies by western blot ( Figure 9D ) .",
"Consistent with our immunostaining results , Rab5 and Rabenosyn-5 , but not EEA1 and APPL1/2 , were found to be specifically enriched in the mitochondrial fraction ( marked by TOM20 ) treated with H2O2 compared to non-treated ( Figure 9D , lane 2 , 4 ) .",
"Altogether , our findings reveal a selective mechanism of Rab5 translocation and activation on mitochondria in the absence of PI ( 3 ) P .",
"Translocation and recruitment of effectors imply that Rab5 must be activated on the mitochondrial membrane .",
"Activation of Rab GTPases on organelle membranes depends on a family of GEFs ( Blümer et al . , 2013; Pfeffer , 2013; Zerial and McBride , 2001; Zhen and Stenmark , 2015 ) .",
"We first examined the localization of Rabex-5 , a known GEF of Rab5 on the endosomal membrane , by immunostaining in BAC GFP-Rab5 cells .",
"For reasons described above , we visualized the endogenous protein because tagged Rabex-5 constructs proved not to be functional as judged by their perturbations on the endosomal system ( Kalaidzidis and Zerial , unpublished data ) .",
"The formation of GFP-Rab5 rings upon laser treatment served as a positive control .",
"Despite a modest enrichment on mitochondria upon laser-induced stress , endogenous Rabex-5 remained mostly cytosolic and on cytoplasmic puncta ( Figure 10A ) , consistent with its endosomal localization ( Figure 10—figure supplement 1A ) .",
"The localization pattern of Rabex-5 led us to hypothesize that another GEF might be principally involved .",
"We turned our attention to Alsin based on several lines of evidence .",
"Alsin is the gene product of ALS2 , which is mutated in multiple neurodegenerative disorders such as juvenile amyotrophic lateral sclerosis ( ALS ) , juvenile primary lateral sclerosis ( JPLS ) , and infantile-onset ascending hereditary spastic paralysis ( IAHSP ) .",
"Alsin comprises three GEF domains: ( 1 ) a RCC1-like GEF domain for Ran GTPase , ( 2 ) a DH-PH domain for Rho GTPase , and ( 3 ) a C-terminal VPS9 domain for Rab5 ( Topp et al . , 2005 ) ( Figure 10—figure supplement 1B ) .",
"Functional studies in ALS mouse models have associated Alsin with neuronal survival ( Kanekura et al . , 2004; Panzeri et al . , 2006 ) and endolysosomal trafficking ( Hadano et al . , 2016; Hadano et al . , 2010 ) .",
"Moreover , corticospinal motor neuron ( CSMN ) in Alsin KO mice display selective defects in mitochondrial morphology ( Gautam et al . , 2016 ) .",
"At steady state , Alsin localized to vesicular structures , showing partial overlap with Rab5 ( Figure 10—figure supplement 1C ) , consistent with the reported localization of Alsin ( Kanekura et al . , 2004; Topp et al . , 2004 ) .",
"However , after laser treatment , Alsin exhibited a strong and uniform staining around mitochondria ( Figure 10B ) , where it showed significant colocalization with GFP-Rab5 and Mito-Red ( Figure 10C ) .",
"The spatial and functional connection between Alsin and Rab5 suggest that Alsin may also be implicated in stress-induced response on mitochondria .",
"We tested this idea by over-expressing either Alsin or WT Rab5 in HeLa cells and found that both prevented caspase 3/7 activation as revealed by the weak fluorescence signals ( due to the lack of cleavage on the DEVD-conjugated DNA-binding dye ) compared to control cells , when challenged with H2O2 ( Figure 10—figure supplement 2A , B ) .",
"Our results point to Alsin as a candidate GEF for activating Rab5 on mitochondria upon stress induction .",
"Several mouse models have been generated for the studies on Alsin .",
"However , these models have failed to recapitulate the phenotypes observed in human patients ( Cai et al . , 2008 ) .",
"It has recently been reported that the absence of Alsin appears to specifically affect the health of corticospinal motor neurons ( Gautam et al . , 2016 ) .",
"Therefore , in order to directly probe the role of Alsin in a more physiological background without compromising our ability for genetic and chemical manipulations , we generated Alsin CRISPR knockout cells in human-induced pluripotent stem cells ( iPSCs ) .",
"We confirmed the deletion of the Alsin gene by sequencing ( not shown ) and RT-PCR , and the encoded protein by western blot ( Figure 11—figure supplement 1A , B ) .",
"We were then able to differentiate both WT and mutant ( Alsin-/- ) iPSCs into spinal motor neurons ( iPSC-sMNs ) using a previously reported protocol ( Reinhardt et al . , 2013 ) .",
"In short , we induced neural progenitor cells ( NPC ) through embryonic bodies formation by growing iPSC in a medium supplemented with transforming growth factor-ß ( TGF- ß ) and bone morphogen protein ( BMP ) small molecule inhibitors ( SB431542 and dorsomorphin , respectively ) , and WNT and Sonic Hedgehog signaling activators ( CHIR99021 and PMA , respectively ) .",
"Differentiation and maturation stages were achieved by culturing cells in retinoic acid ( RA ) , cAMP , and neurotrophic factors ( BDNF and GDNF ) ( Figure 11A ) .",
"As a quality control , high expression of pluripotency markers such as Oct4 and Lin28 were observed in our iPSCs as well as Nestin , Sox2 and Pax6 expression in our neuro-progenitor cells ( NPCs ) ( Figure 11—figure supplement 1C ) .",
"Differentiation into mature spinal motor neurons was validated by the expression of choline acetyltransferase ( ChAT ) , HB9 , and Islet-1 ( ISL1 ) ( Figure 11—figure supplement 1D , E ) .",
"These cells showed extensive axonal network as revealed by the MAP2 staining ( Figure 11—figure supplement 1E ) .",
"Finally , mature spinal motor neurons were re-stained for the expression of Alsin in both WT and Alsin-/- cells ( Figure 11—figure supplement 1F ) .",
"We first examined the steady-state localization of Rab5 and morphology of mitochondria by immunostaining for endogenous Rab5 and TOM20 .",
"Similar to hippocampal neurons ( de Hoop et al . , 1994 ) , Rab5 was ubiquitously present on endosomal-like vesicles in the soma , dendrites and axon in iPSC-sMNs .",
"The mitochondrial network in iPSC-sMNs was less tubular and contained more numerous and smaller rounded mitochondria than those in HeLa cells ( Figure 11B , Ctrl ) .",
"Next , we tested whether iPSC-sMNs would exhibit the same mitochondrial response to oxidative stress as observed in HeLa cells .",
"Noticeably , we found iPSC-sMNs to be more susceptible to detachment and cell rounding than HeLa cells when challenged with 250 µM H2O2 for 2 hr under the same conditions ( data not shown ) .",
"We thus lowered the H2O2 concentration to 100 µM such that no immediate cell detachment nor rounding were observed during the treatment .",
"We then examined the morphology of mitochondria , the association of Rab5 with mitochondria , and the release of cytochrome c into the cytosol .",
"At steady state , we did not observe significant alterations in mitochondria morphology in both WT and Alsin-/- iPSC-sMNs .",
"However , WT iPSC-sMNs challenged with H2O2 showed a robust enrichment of Rab5 on mitochondria , but not in Alsin-/- iPSC-sMNs ( Figure 11B , H2O2 ) .",
"To corroborate these results , we also performed subcellular fractionation of iPSC-sMNs .",
"In control cells , endogenous Rab5 was detected primarily in the cytosolic fraction and minimally in the mitochondrial fraction ( Figure 11C , lane 1 , 2 , 5 , 6 ) .",
"On the other hand , cells challenged with H2O2 showed a strong enrichment of Rab5 co-fractionating with the mitochondrial fraction in WT iPSC-sMNs , but only weakly in Alsin-/- iPSC-sMNs ( Figure 11C , lane 3 , 7 ) .",
"The lack of Rab5 enrichment in Alsin-/- cells also correlated with a greater susceptibility to H2O2-induced apoptotic signaling , as assessed by the rapid release of cytochrome c into the cytosol within 1 hr and subsequent accumulation , when compared to WT cells ( Figure 11D ) .",
"Collectively , our findings demonstrate that Alsin is a key regulator for recruiting Rab5 to mitochondria , which altogether , impart a cytoprotective function for cells against oxidative stress ."
],
[
"We discovered a novel cytoprotective mechanism during oxidative stress entailing the translocation of Rab5 from EE to mitochondria .",
"Interestingly , the activation of Rab5 requires Alsin , which has been implicated in early onset ALS .",
"Our results provide an unexpected mechanistic link between the endosomal system and mitochondria that could be of primary importance for understanding the mechanistic cause of ALS and other neurodegenerative diseases .",
"Different nutrient or environmental perturbations can affect mitochondria morphology and metabolic activities such as oxidative phosphorylation and programmed cell death ( Galloway and Yoon , 2013 ) .",
"Mitochondria can elicit adaptive responses to oxidative stress that may lead to hypoxia adaptation , inflammation , or programmed cell death ( Sena and Chandel , 2012 ) .",
"Our findings suggest that the endocytic system is a primary responder to mitochondria under oxidative stress .",
"Laser- or exogenous ROS ( e . g . H2O2 ) -induced damage causes MOMP , mitochondrial swelling , and release of cytochrome c , leading to caspase activation and apoptosis .",
"Under these conditions , the endosomal system appears to rapidly respond to damaged mitochondria through a rescue pathway , which results in the recruitment of Alsin and Rab5 to mitochondria , inhibition of cytochrome c release , decrease in mitochondrial oxygen consumption and hence , increased overall cell viability ( Figure 12 ) .",
"In the course of this study , a mitochondrial clearance mechanism was reported where Rab5-positive EE sequester mitochondria via the ESCRT machinery when cells are treated with the proton uncoupler FCCP ( Hammerling et al . , 2017 ) , an analog of CCCP .",
"Our mechanism appears to be distinct from this as well as the canonical autophagic/mitophagic mechanisms .",
"First , we did not observe the engulfment of mitochondria into Rab5-positive EE but rather , the recruitment of Alsin , Rab5 , and Rabenosyn-5 on mitochondria , as well as an increase in early endosomal-mitochondrial MCS in response to stress .",
"This is also distinct from the intra-mitochondrial recruitment of Rab5 and endo-lysosomes upon over-expression of the apoptotic factors ( Hamacher-Brady et al . , 2014 ) .",
"Second , we did not observe engulfing membraneous structures around stressed mitochondria nor upon CCCP treatment .",
"One explanation could be the use of different cell types and the lower concentration of CCCP employed in our experiments .",
"Third , the recruitment of Rab5 to damaged mitochondria occurs rapidly , that is within min , well preceding any autophagic components that we analyzed in this study .",
"We found that autophagy is restricted to only a subset of small mitochondrial fragments that are LC3+ , whereas the majority are devoid of known autophagic markers such as Parkin , LC3 and Lamp1 .",
"We could not rule out that mitochondrial clearance mechanism may still be activated at a later time .",
"However , our data with H2O2 show that the mechanism described here is reversible ( Figure 8 ) and argue for a mitochondrial-protective role rather than a degradative process .",
"In fact , the reversal and recovery of cells from late-stage apoptosis ( i . e . following cytochrome c release and caspase activation ) have recently been reported in multiple cells lines including HeLa cells and brain cells in a process called ‘anastasis’ ( Sun et al . , 2017; Tang et al . , 2012 ) , suggesting that this may be a general mechanism to cope with cellular stress .",
"We attempted to track the fate of individually damaged mitochondria in a localized region after laser treatment ( data not shown ) , but the continuous photoirradation required to achieve a high spatio-temporal resolution also led to a quick decrease in MitoTracker Red signal and undesirable additional stress to the cell over time , preventing us from determining its precise outcome .",
"The loss of Mito-Red signal is likely due to MOMP , as evident by the release of cytochrome c , and not a result of mitochondrial clearance because the outer mitochondrial membrane can be stained by TOM20 and visualized by the presence of Rab5 ring-like formation .",
"Which molecular mechanism is responsible for the dissociation of Rab5 from EE and its recruitment to mitochondria ?",
"On EE , the levels of Rab5 depend on the equilibrium between the cytosolic pool of Rab5 complexed to Rab GDI and the membrane-associated pool sustained by the Rabex-5/Rabaptin-5 complex and a plethora of Rab5 effectors ( Del Conte-Zerial et al . , 2008; Lippé et al . , 2001; Zerial and McBride , 2001; Zhang et al . , 2014 ) .",
"Prior studies have shown that such equilibrium , and consequently endocytic trafficking , is adaptive to stress and apoptotic signal .",
"For example , Rabaptin-5 is selectively cleaved by caspase-3 during apoptosis , thus affecting its interaction with Rab5 and reducing the overall endocytic capacity ( Cosulich et al . , 1997; Swanton et al . , 1999 ) .",
"The activation of p38 MAPK by H2O2 also stimulates the formation of the GDI:Rab5 complex , thus favoring the extraction of Rab5 from the early endosomal membrane ( Cavalli et al . , 2001 ) .",
"In addition , p38 MAPK modulates the endosomal function via phosphorylation and membrane association of Rab5 effectors ( Macé et al . , 2005 ) .",
"Such a mechanism may account for the mobilization of Rab5 from the endosomal membrane , which is suggested by increased in cytosolic Rab5 and decreased colocalization of Rab5 with the endosomal EEA1 ( Figure 6A–C ) .",
"These phenotypes correlated to a defect in endosomal sorting ( Figure 1E ) and a block in transferrin uptake ( Figure 6D , E ) , which may be yet another protective mechanism in order to avoid iron overload and toxicity associated with neurodegeneration ( Núñez et al . , 2012 ) .",
"Interestingly , hippocampal HT-22 neurons exposed to excess iron exhibit mitochondrial fragmentation and a decrease in cell viability ( Park et al . , 2015 ) .",
"The metabolic effect of H2O2 on OCR is intriguing .",
"Mitochondria require oxygen to produce ATP to drive energy-consuming reactions ( Bratic and Trifunovic , 2010 ) .",
"The observed decrease in OCR ( Figure 5E ) may serve to lower cellular respiration and prevent further ROS production .",
"If the stress response triggers solubilization of Rab5 from EE , then it must also catalyze the activation of Rab5 on mitochondria .",
"We discovered that this step depends on Alsin .",
"The C-terminal VPS9 domain of Alsin has GEF activity towards Rab5 , and plays a role in Rab5 endosomal localization and dynamics ( Otomo et al . , 2003; Topp et al . , 2004 ) .",
"However , the physiological role of Alsin with respect to Rab5 and endosomal activity has remained somewhat mysterious .",
"At steady state , Alsin is mainly cytosolic with a fraction localizing to vesicular-like structures ( Figure 10—figure supplement 1C ) ( Millecamps et al . , 2005 ) .",
"In stress-induced conditions , however , Alsin re-localizes to mitochondria .",
"The N-terminal RLD of Alsin has been shown to exhibit an autoinhibitory effect on its VPS9 domain ( Otomo et al . , 2003 ) .",
"We posit that mitochondrial-induced stress triggers structural changes in the protein , releasing the autoinhibitory effect of RLD , thereby exposing the VSP9 domain for Rab5 activation and recruitment .",
"Alsin was required for Rab5 translocation to mitochondria as this was severely diminished in Alsin-/- iPS-sMN cells .",
"The presence of ( low levels ) Rabex-5 on mitochondria ( Figure 10A , C ) detected in HeLa cells suggests that other GEFs may contribute to some Rab5 activation , depending on the cell types , but cannot fully compensate for the loss of Alsin function .",
"A homologous gene , ALS2CL , containing only the carboxyl-terminal half of ALS2 , may also play a role by specifically binding to Rab5 and forming a homodimer with the full-length Alsin to membranous compartments ( Hadano et al . , 2004; Suzuki-Utsunomiya et al . , 2007 ) .",
"What is the function of the assembly of Rab5 and Rab5 machinery ( Alsin and Rabenosyn-5 ) on mitochondria besides its protective role ?",
"The stress response triggers remodeling of mitochondria to confer molecular features characteristic of the endocytic system .",
"The Rab5 machinery may be used to bring mitochondria in close proximity to EE and form MCS ( Figure 1 ) .",
"These MCS may mediate the transfer of lipids and metabolites ( Helle et al . , 2013 ) , or involve in ‘patching’ up mitochondrial wounds by recruiting the ESCRT machinery for closure , or other endomembranes for fusion , both of which are observed in the PM-repair response ( Jimenez et al . , 2014; Reddy et al . , 2001 ) .",
"Worth noting from our EM study , we observed ER-like membranous structure in contact with stressed mitochondria ( Figure 3C ) and considering the role of ER-mitochondria contacts in Ca2+-regulated apoptosis ( Pinton et al . , 2008 ) , one may postulate that an orchestrated three-way organelle crosstalk exists .",
"Considering that Rab5 is necessary for the biogenesis of the endolysosomal system ( Zeigerer et al . , 2012 ) , the Rab5 translocation may be a priming step of a stress response pathway that subjects mitochondria to interact with the entire endolysosomal system , in order to modulate the mitochondrial apoptotic potential .",
"One quality control mechanism is the formation of mitochondrial-derived vesicles ( MDVs ) , which are involved in the transport of oxidized or damaged cargo to LE and lysosomes for degradation ( Soubannier et al . , 2012 ) .",
"This process depends on PINK1/Parkin ( McLelland et al . , 2014 ) , but can also occur independently ( Matheoud et al . , 2016 ) .",
"Rab5 may play a role in MDV formation , although we could not detect vesicle budding events in our experimental conditions .",
"Once recruited onto mitochondria , Rab5 activity may not be limited to the recruitment of its effectors , but initiate a more extensive endosomal Rab cascade via the Rab coupling/conversion mechanism .",
"On EE , Rab5 interacts with divalent effectors , coupling its activity to other Rab proteins ( e . g . Rab4 , Rab11 ) that are required for receptor recycling ( de Renzis et al . , 2002; Vitale et al . , 1998 ) .",
"Rab5 also initiates the activation of Rab7 , resulting in the conversion of EE into LE ( Rink et al . , 2005 ) .",
"The Rab coupling/conversion may also be initiated on the mitochondria .",
"Therefore , it is possible that the mitochondria-endosome MCS may evolve over time leading to a Rab7-dependent mitophagic pathway ( Jimenez-Orgaz et al . , 2018 ) , the engulfment of mitochondria by the EE ( Hammerling et al . , 2017 ) , or conventional autophagic processes ( Ao et al . , 2014; Stolz et al . , 2014 ) .",
"Future work exploring the dynamics of other endosomal Rab GTPases in relation to Rab5 will be necessary to elucidate the precise role of the endosomal system on mitochondria .",
"The physiological role of Alsin , although elusive , has been linked to both endosomes and mitochondria .",
"Cultured hippocampal neurons from Alsin knockout mice display an accumulation of enlarged Rab5 endosomes and a reduced endosomal motility ( Lai et al . , 2009 ) .",
"Mutational and linkage analysis of Alsin from human patients show that the VPS9 domain is critical for Alsin function ( Daud et al . , 2016; Verschuuren-Bemelmans et al . , 2008 ) .",
"A recent EM study on the corticospinal motor neurons ( CSMN ) from Alsin KO mice reveals a selective morphological defect in mitochondria with enlarged core and broken cristae ( Gautam et al . , 2016 ) .",
"Interestingly , WT vs Alsin KO CSMN show no change in Parkin expression , suggesting that mitophagy does not play a major role ( Gautam et al . , 2016 ) .",
"We postulate that the pathological condition of mitochondrial defects in Alsin KO cells is related to a deficiency in Rab5 recruitment to mitochondria , thereby leading to a decline in protection from ROS and oxidative stress associated with aging .",
"In ALS patients , motor neurons likely accumulate more damaged mitochondria as age progresses , which eventually become an overburden for cells .",
"The primary cause for ALS is still unclear , but oxidative stress is considered to be a major contributor .",
"Mutations in the antioxidant enzyme , superoxide dismutase 1 ( SOD1 ) , are associated with motor neuron degeneration .",
"In mouse models , an accumulation of the SOD1 mutant proteins results in mitochondrial swelling and increased oxidative damage ( Jaarsma et al . , 2001 ) .",
"Interestingly , loss of Alsin in the mutant SOD1 transgenic mice exacerbates and accelerates disease progression ( Hadano et al . , 2010 ) .",
"These studies , along with our findings , corroborate the protective role of Alsin during oxidative stress .",
"The mechanistic link between Rab5 and Alsin may present a general or related mechanism in other neurodegenerative diseases .",
"In Parkinson disease , the most common mutation in the multidomain Leucine-rich repeat kinase 2 ( LRRK2 ) protein leads to a hyper-activation of the kinase domain , resulting in hyper-phosphorylation of a number of Rab GTPase substrates including Rab5 ( Steger et al . , 2017; Steger et al . , 2016 ) .",
"This may present yet another mechanism of regulating Rab5 localization and function on mitochondria .",
"Future work using different neurodegenerative disease models in differentiated human neurons will provide deeper insights into the disease etiology ."
],
[
"The following cell lines have been validated and tested negative for mycoplasma contamination: HeLa ( Kyoto ) cell line , BAC HeLa GFP expressing cell lines , and human KOLF_C1 iPSC ( kindly provided by Bill Skarnes , Sanger Institute ) .",
"HeLa cells were cultured in high-glucose DMEM ( Gibco ) with 10% fetal bovine serum , 100 U/ml penicillin , 100 µg/ml streptomycin , and 2 mM glutamine ( all reagents from Sigma-Aldrich ) with 5% CO2 at 37°C .",
"All plasmids were transfected using Effectene transfection reagent ( Qiagen , Germany ) according to the manufacturer’s protocol .",
"All bacterial artificial chromosome ( BAC ) transgene HeLa cell lines expressing different markers were obtained from the BAC recombineering facility at MPI-CBG ( Dresden , Germany ) and generated using the method previously described ( Poser et al . , 2008 ) .",
"Construction of the pEGFP-C3-2xFYVE was made using mouse Hrs FYVE domain containing a linker ( QGQGS ) ( Raiborg et al . , 2001 ) .",
"Human Rab5c cDNA was subcloned into the pEGFP-C3 plasmid ( Addgene ) .",
"Human Alsin cDNA subloned into the pEF1/Myc-His ( Invitrogen ) plasmid was a kind gift from Dr . Ikuo Nishimoto ( Kanekura et al . , 2004 ) .",
"Alexa-conjugated transferrin ( Invitrogen; T13342 ) and EGF ( Invitrogen; E13345 ) were used at 25 µg/ml and 2 µg/ml , respectively .",
"Carbonyl cyanide 3-chlorophenylhydrazone ( CCCP ) was purchased from Sigma Aldrich ( C2759 ) .",
"Stock solution was prepared to a final concentration of 10 mM in DMSO .",
"100 mM Hydrogen peroxide ( H2O2 ) ( Merck Millipore; 7722-84-1 ) stock solution was prepared in PBS .",
"Cells were seeded either in a 35-mm glass-bottom dish or ibidi µ-Dish 35 mm , high Grid-500 .",
"Before imaging , medium was replaced with HEPES-buffered DMEM without phenol red ( Gibco ) .",
"Time-lapse imaging was performed using the Nikon TiE inverted stand microscope equipped with spinning disc scan head ( CSU-X1; Yokogawa ) , fast piezo objective z-positioner [Physik Instrumente] , and back-illuminated EMCCD camera ( iXon EM +DU-897 BV; Andor ) .",
"Imaging was done with an Olympus UPlanSApo 100 × 1 . 4 Oil and Nikon Apo 100 × 1 . 49 Oil DIC 0 . 13–0 . 20 objectives ( illumination by lasers: DPSS-488nm , DPSS-561nm , DPSS-640nm ) .",
"Individual planes were recorded at ~11 frames/s with Z-stacks of three planes ( step 0 . 3 µm ) .",
"Cells were incubated with MitoTracker Red CMXRos ( ThermoFisher; M7512 ) at a final concentration of 100 nM at 37°C for 30 min , 5% CO2 incubator , and followed by 2X PBS wash before irradiating with 561 nm laser on the spinning disc Andor-Olympus-IX71 at a low-power dosage of ~5 J/cm2 for 60 s .",
"Cells were grown on a gridded dish ( ibidi µ-Dish 35 mm , high Grid-500 ) .",
"Cells in different locations were laser-treated with 561 nm laser for 30 s .",
"Cells were fixed in 2 . 5% glutaraldehyde/PBS for 30 min at room temperature .",
"Post-fixation and embedding were performed using 1% osmium tetroxide/1 . 5% potassium ferrocyanide and Epon Lx112 , respectively .",
"Sectioning of 150 nm thick UA sections was performed on a Leica Ultracut UCT ( Leica Microsystem , Wetzlar , Germany ) with a diamond knife .",
"Samples were post-stained with 2% uranyl acetate and lead citrate .",
"2D images were acquired on a Tecnai T12 ( FEI , Hillsboro , OR ) .",
"Cells were seeded on a ibidi Grid-500 glass bottom .",
"After laser or H2O2 treatment , cells were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature .",
"Cells were washed twice with PBS and permeabilized in PBS containing 0 . 1% saponin , and 1% BSA for 30 min at room temperature .",
"Cells were immunostained with the corresponding primary antibodies: anti-rabbit Rabenosyn-5/ZFYVE20 ( Sigma Aldrich: HPA044878 ) , anti-mouse EEA1 ( BD Biosciences: 610457 ) , anti-rabbit TOM20 ( Santa Cruz Biotechnology: sc-11415 ) , anti-rabbit APPL1 ( Abcam: ab59592 ) , anti-mouse APPL2 ( home-made ) , anti-mouse Rab5 ( BD Biosciences: 610724 ) , anti-mouse cytochrome c ( Abcam: ab6311 ) , and anti-rabbit Alsin ( Novus Biological: NBP2-14284 ) antibodies .",
"Alexa fluor-conjugated ( ThermoFisher ) were used as secondary antibodies .",
"Samples were mounted with Mowiol ( Sigma-Aldrich ) on glass slides and examined using the Zeiss LSM 880 inverted single photon point scanning confocal system with Quasar detector ( 32 spectral detection channels in the GaAsP detector plus 2PMTs ) and transmitted light detector .",
"Acquired images were processed and saved using the Zeiss ZEN software .",
"For immunofluorescence on iPSCs , smNPCs , and sMNs , cells were fixed with 4% formaldehyde for 15 min , washed three times with wash buffer ( 0 . 3% Triton-X in PBS ) for 5–10 min , and blocked with blocking buffer ( 5% goat serum , 2% BSA , and 0 . 3% Triton-X in PBS ) at room temperature for 1 hr .",
"Cells were incubated with primary antibodies in blocking buffer overnight at 4°C .",
"After three washes with PBS for 10 min , cells were incubated with secondary antibodies in wash buffer for 2–3 hr at room temperature followed by three washes in PBS for 10 min .",
"Primary antibodies used include: goat anti-ChAT ( 1:100 ) ( Millipore , #AB144P ) , mouse anti-HB9 ( 1:50 ) ( DSHB , #81 . 5C10 , conc . ) , rabbit anti-ISL1 ( 1:100 ) ( Abcam , #ab20670 ) , mouse anti-LIN28 ( 1:1000 ) ( Cell signaling , #5930S ) , chicken anti-MAP2 ( 1:1000 ) ( Novus Biologicals , #NB300-213 ) , mouse anti-Nestin ( 1:150 ) ( R and D Systems , #MAB1259 ) , rabbit anti-OCT4 ( 1:500 ) ( Abcam , #ab19857 ) , rabbit anti-PAX6 ( 1:300 ) ( Covance , #PRB-278P ) , and rabbit anti-SOX2 ( 1:500 ) ( Abcam , #ab97959 ) .",
"Ad-Ctrl and Ad-tBid ( a kind gift from Dr . Heidi McBride ) were used at 1:200 PFU/cell .",
"Cells were seeded in a 384-well plate and incubated with either complete medium or in the presence of 250 μM H2O2 at 37°C for 2 hr .",
"Cells were then pulsed with Alexa-647 Tfn ( 10 µg/ml ) for 5 min , followed by 3x PBS wash , fixed with 3 . 7% PFA for 15 min , and then stained with DAPI ( 1:1000 ) and CellMask Blue ( 1:2000 ) ( ThermoFisher ) .",
"Image acquisition was performed via the automated confocal imaging system , CV7000S Yogokawa .",
"Images analysis were performed using MotionTracking software .",
"Cytosolic and mitochondrial fractions were performed using the mitochondria isolation kit , according to the manufacturer’s protocol with minor modification ( ThermoFisher: cat89874 ) .",
"Cells ( ~1×107 ) were resuspended in 400 µl Mitochondrial Isolation Reagent A . Cells were chemically lysed by adding 5 µl of Reagent B . After 5 min incubation on ice , 400 µl of Reagent C was added to each sample and centrifuged at 720 x g for 10 min .",
"The post-nuclear supernatant ( PNS ) was transferred to a new eppendorf tube and centrifuged at 3000 x g for at 4°C for 15 min .",
"For the total membrane and purified cytosolic fractions , the PNS was clarified at 200 , 000 g at 4°C for 1 hr .",
"The resulting supernatant was collected and trichloroacetic acid ( TCA ) /acetone precipitation was performed to obtain the final cytosolic fraction .",
"The remaining pellet was washed by adding 500 µl of Reagent C and centrifuged at 15 , 000 x g for 5 min .",
"Final samples were resuspended in SDS loading buffer .",
"Cells were seeded on a 12-well plate .",
"For hydrogen peroxide treatment , reagent was added directly into the well to achieve the appropriate concentration .",
"Separation of mitochondrial and cytosolic fractions were performed using the mitochondrial isolation kit from ThermoFisher ( cat:89874 ) with an additional step of trichloroactic acid precipitation of the cytosolic fraction .",
"The final pellet was dried in a 95°C heat block for 2–3 min before resuspending it in the SDS loading buffer .",
"Cell lysates were separated by SDS-PAGE , transferred onto the nitrocellulose membrane and blocked in 5% milk in PBS containing 0 . 1% Tween .",
"Primary and secondary antibodies were diluted in the blocking buffer and incubated at room temperature for 2 hr .",
"The bands were detected using the electrochemiluminescence reagent and exposure onto x-ray films .",
"The following antibodies were used in western blot: anti-mouse cytochrome c ( Abcam: ab13575 ) , anti-rabbit gamma tubulin ( Sigma-Aldrich: T6557 ) , anti-rabbit Rabenosyn-5/ZFYVE20 , anti-rabbit Alsin ( Sigma Aldrich: SAB4200137 ) , anti-mouse EEA1 ( BD Biosciences: 610457 ) , anti-rabbit APPL1 ( Abcam: ab59592 ) , anti-mouse APPL2 ( home-made ) , anti-mouse GAPDH ( Sigma Aldrich: G8795 ) , anti-mouse gamma tubulin ( Sigma Aldrich: T6557 ) , and anti-rabbit TOM20 ( Santa Cruz Biotechnology: sc-11415 ) .",
"The caspase-3/7 activation in CCCP- vs H2O2- treated cells were measured using the caspase-3/7 green flow cytometry assay kit ( ThermoFisher: C10427 ) .",
"After 2 hr treatment , cells were scraped off per well from a six-well dish , gently resuspended in 1 ml of PBS containing 1 µl of green detection reagent , and incubated at 37°C for 20 min .",
"Samples were analyzed using the 488 nm excitation with standard fluorescence compensation and emission filter ( 530/30 BP ) in the FACS Calibur ( Beckton Dickinson ) .",
"The gating was set based on the background signal in the DMSO control .",
"The total cell count was set to 5000 .",
"The caspase-3/7 activation in Alsin and Rab5 over-expressed cells was measured using the caspase-3/7 green detection reagent ( ThermoFisher: C10423 ) .",
"Cells were incubated in a complete medium containing 5 µM of green detection reagent at 37°C for 30 min prior to fixation .",
"Cells were imaged using the Zeiss LSM 880 microscope .",
"20 , 000 HeLa cells per well were seeded in a XFe96-well plate ( Seahorse Bioscience ) , and grown to ~80% confluency after overnight incubation .",
"Cells were then equilibrated with a bicarbonate-free DMEM medium containing 4 mM glutamine and 10 mM galactose in a 37°C ambient CO2 incubator for 1 hr , before starting the experiment .",
"CCCP and H2O2 compounds were prepared fresh and diluted in the assay medium , and were injected from the reagent ports at the indicated time .",
"Oxygen consumption rates ( OCR ) were measured using a Seahorse Extracellular Flux Analyzer .",
"Human KOLF_C1 iPSC were cultured in feeder-free conditions on Matrigel with TeSR-E8 media ( StemCell , Germany ) .",
"For ALS2/Alsin knockout using CRISPR/Cas9 genome editing , 350 , 000 cells were detached using Accutase , washed once with PBS and electroporated using the Neon Transfection System ( Invitrogen , Germany , 10 µl kit , 1000V , 20 ms , three pulses ) .",
"The genomic sequence of human Alsin was analyzed for CRISPR/Cas9 target sites by Geneious 8 . 1 . 6 software ( Biomatters ) , and two pairs of guides flanking a critical exon ( exon3 ) were selected ( 5’-GCTAAAGTACTGAATTTTGG-3’ and 5’-AATAAAATCAGCAGGTGTGG-3’; 5’-GAATTTCTACAAAGTGCAGG-3’ and 5’-TAGCCTGGATGATGGCCGTT-3’ ) and were used together to cause a frame shifting exon deletion .",
"The in vitro efficiency of these gRNAs was assessed by generating a genomic PCR cleavage template of 3 . 4 bp ( primers used: for-CCTCCCTTCCCAGGATCTGA and rev-TGCTCAACTCGAGTGCCTTT; for-CAGGGTGAGCATCCCACATT and rev-AGGAGTTCCAGTCAACCAGT ) and incubating with recombinant Cas9 .",
"All gRNAs used in vitro were identical in sequence to the DNA sense strand and not complementary to the mRNA sequence .",
"The RNAs employed in this method were chemically modified and length optimized variants of the native guide RNAs ( Alt-RTM CRISPR crRNAs and tracrRNA , Integrated DNA Technologies , Coralville , IA ) .",
"The recombinant Cas9 ( provided by Protein Expression Facility at MPI-CBG ) protein from Streptococcus pyogenes was used .",
"The crRNAs were mixed with trRNA and NLS-Cas9 ( 1 µg/µl ) .",
"The guide RNA complex was formed by mixing the crRNAs and tracrRNAs in equal amounts in Buffer R ( Invitrogen , Germany ) at 100 µM .",
"Five days after electroporation , cells were pooled and seeded for clonal dilution .",
"Single clones were mechanically picked and amplified .",
"Next , genomic DNA was isolated using QuickExtract DNA Extraction Solution ( EpiCentre , USA ) .",
"Homozygous deletions were verified by PCR and sequencing .",
"All procedures were performed as previously described ( Reinhardt et al . , 2013 ) .",
"Briefly , for smNPC generation , iPSC colonies detached from Matrigel-coated wells ( by 1 mg/ml dispase ) were resuspended in hESC medium ( DMEM/F12 , 20% KnockOUT Serum Replacement , 1% Penicillin/Streptomycin/Glutamine , 0 . 1 mM Non-Essential Amino Acids Solution , 0 . 05 mM beta-mercaptoethanol , without bFGF supplemented with 10 μM SB431542 ( Tocris , #1614 ) , 1 μM dorsomorphin ( Tocris , #3093 ) , 3 μM CHIR99021 ( Axon Medchem , #Axon-1386 ) and 0 . 5 μM purmorphamine ( STEMCELL Technologies , #72202 ) , and cultured in non-coated petri dishes .",
"After 2 days , hESC medium was replaced by N2B27 medium ( 1:1 mixture of DMEM/F12 and Neurobasal medium , 1% Penicillin/Streptomycin/Glutamine , 1:100 B-27 supplement minus vitamin A , 1:200 N-2 supplement ) and supplemented with the same small molecules as listed above .",
"After another 2 days , culture medium was replaced by smNPC expansion medium ( N2B27 medium supplemented with 150 μM ascorbic acid ( Sigma , #A4403 ) , 3 μM CHIR99021 and 0 . 5 μM purmorphamine ) .",
"On day 6 of neural induction , embryonic bodies were broken into smaller clumps by titration and plated in six wells of a Matrigel-coated 12-well plate .",
"On day 9 , cells were passaged for the first time using Accutase at a 1:3 split ratio and seeded in four wells of a Matrigel-coated six-well plate .",
"Afterwards , cells were passaged once a week and seeded at a density of 1 × 106 cells per well .",
"To obtain a highly pure smNPC culture , smNPCs were propagated for at least 10 passages in smNPC expansion medium .",
"For differentiation of smNPC to MNs , smNPCs were seeded at a density of 1 . 5 × 106 cells per one well of a Matrigel-coated six-well plate and cultured in N2B27 medium supplemented with 1 μM purmorphamine for the first 2 days of differentiation .",
"The cells were then cultured in N2B27 medium supplemented with 1 μM purmorphamine and 1 μM retinoic acid ( Sigma , #R2625 ) until day 9 of differentiation .",
"On day 9 , cells were dissociated using Accutase and plated on polyornithine/laminin-coated ibidi μ-slides ( at a density of 150000 cells per well ) or Nunc four-well plates ( at a density of 300 , 000 cells per well ) in maturation medium ( N2B27 medium supplemented with 0 . 5 mM cAMP ( Sigma , #D0627 ) , 10 ng/ml BDNF ( Peprotech , #450-02-10 ) , and 10 ng/ml GDNF ( Peprotech , #450-10-10 ) ) .",
"Cells were maintained in maturation medium until analysis on day 28 .",
"Image resizing , cropping and brightness were uniformly adjusted in Fiji ( http://fiji . sc/ ) .",
"Colocalization analysis was performed using MotionTracking software ( Rink et al . , 2005 ) ( http://motiontracking . mpi-cbg . de/get/ ) and described previously ( Gilleron et al . , 2013 ) .",
"The y-axis is expressed as the ratio of co-localized objects ( e . g . A to B ) to total objects found in A . Final images were assembled using Adobe Photoshop and Illustrator .",
"Densitometry quantification were performed in Fiji following the previously described protocol ( http://www . yorku . ca/yisheng/Internal/Protocols/ImageJ . pdf ) .",
"p Values were calculated by two-tailed t-test using GraphPad Prism7 ."
]
] | [
"Mitochondrial stress response is essential for cell survival , and damaged mitochondria are a hallmark of neurodegenerative diseases .",
"Thus , it is fundamental to understand how mitochondria relay information within the cell .",
"Here , by investigating mitochondrial-endosomal contact sites we made the surprising observation that the small GTPase Rab5 translocates from early endosomes to mitochondria upon oxidative stress .",
"This process is reversible and accompanied by an increase in Rab5-positive endosomes in contact with mitochondria .",
"Interestingly , activation of Rab5 on mitochondria depends on the Rab5-GEF ALS2/Alsin , encoded by a gene mutated in amyotrophic lateral sclerosis ( ALS ) .",
"Alsin-deficient human-induced pluripotent stem cell-derived spinal motor neurons are defective in relocating Rab5 to mitochondria and display increased susceptibility to oxidative stress .",
"These findings define a novel pathway whereby Alsin catalyzes the assembly of the Rab5 endocytic machinery on mitochondria .",
"Defects in stress-sensing by endosomes could be crucial for mitochondrial quality control during the onset of ALS ."
] | [
"The inside of a human cell is divided into compartments called organelles , which are surrounded by membranes .",
"Each organelle plays a specific role in keeping the cell healthy and also has unique mix of molecular markers on its surface .",
"These markers allow other molecules to identify the different organelles , meaning that specific organelles can communicate with each other and coordinate their activities .",
"One way that organelles can do this is via so-called membrane contact sites , which are small areas where the compartments’ outer membranes come close together .",
"Mitochondria are organelles that release energy inside human cells .",
"These compartments also work to keep the levels of toxic chemicals called reactive oxygen species in the cell within a safe range .",
"This is important because cells can die if these levels become too high – a state known as oxidative stress .",
"Mitochondria also communicate with other organelles called endosomes , which receive materials from the cell surface , sort and direct them to different destinations throughout the cell .",
"In many diseases affecting the nervous system , the mitochondria and endosomes in nerve cells do not work properly .",
"These cells also have higher than normal levels of oxidative stress .",
"Hsu et al . therefore wanted to find out if mitochondria and endosomes worked together to help cells to cope with this kind of stress .",
"Hsu et al . triggered oxidative stress in human cancer cells by exposing them first to a dye that stained the mitochondria and then to intense light .",
"In stressed cells , a subset of endosomes called early endosomes formed many more membrane contact sites with mitochondria than in non-stressed cells .",
"At the same time , the protein Rab5 , usually found on early endosomes , relocated to the surface of mitochondria .",
"Human cells previously engineered to produce larger than normal amounts of Rab5 were also more likely to survive oxidative stress .",
"These experiments suggested that early endosomes cooperate with mitochondria , via Rab5 , to protect cells from oxidative stress .",
"So , how is Rab5 relocated to mitochondria ?",
"Hsu et al . searched for activators of Rab5 and found that Alsin also migrated to mitochondria in stressed cells .",
"The gene for Alsin is also mutated in amyotrophic lateral sclerosis ( ALS ) , a degenerative nerve disorder that remains poorly understood .",
"Next , Hsu et al . deleted the gene for Alsin from human stem cells growing in the laboratory and coaxed these cells into becoming nerve cells .",
"Experiments with these cells showed that the absence of Alsin prevented Rab5 from moving to the mitochondria .",
"Nerve cells lacking Alsin were also more susceptible to oxidative stress than normal cells .",
"Together , these findings show that early endosomes work with mitochondria to sense and ward off oxidative stress .",
"They also reveal an unexpected connection between this process and a gene mutated in ALS .",
"Further experiments are now needed to explore if problems with endosomes or mitochondria , and specifically with molecules like Alsin and Rab5 , are responsible for other neurodegenerative disorders , like Parkinson’s disease and Huntington’s disease ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex | elife-05115-v2 | [
[
"Macroautophagy ( hereafter , ‘autophagy’ ) is a conserved eukaryotic pathway for cellular self-preservation through cellular self-consumption ( Mizushima et al . , 2011; Rubinsztein et al . , 2012b; Reggiori and Klionsky , 2013; Green and Levine , 2014 ) .",
"The most ancient role for autophagy is surviving during starvation .",
"Bulk cytosol is captured in a growing double membrane structure termed the phagophore .",
"Upon sealing , the resulting double membrane vesicle is known as an autophagosome .",
"The autophagosome fuses with the lysosome or vacuole , resulting in the degradation of its contents and the recycling of biosynthetic precursors by export across the lysosomal membrane .",
"In higher eukaryotes , autophagy has acquired many additional roles in cellular protection .",
"For example , the core autophagy protein BECN1 ( Beclin 1 ) is a tumor suppressor .",
"BECN1 is deleted in 40–75% of human breast , ovarian , and prostate cancers ( Liang et al . , 1999 ) .",
"Autophagy is also thought , under most conditions , to protect cells from the accumulation of pathogenic inclusions that can lead to Huntington's , Parkinson's , and other neurodegenerative diseases ( Rubinsztein et al . , 2012a; Nixon , 2013 ) .",
"During the initiation of autophagy , an autophagy-specific form of the class III phosphatidylinositol 3-kinase complex ( PI3KC3-C1 ) is activated and recruited to the site of phagophore nucleation .",
"Class III PI3Ks synthesize phosphatidylinositol 3-phosphate ( PI ( 3 ) P ) from phosphatidylinositol ( PI ) , as compared to the class I and II PI3Ks that generate PI ( 3 , 4 , 5 ) P3 and PI ( 3 , 4 ) P2 , respectively ( Backer , 2008 ) .",
"In yeast , where the complex was first characterized , there are two PI 3-kinase complexes , known as complexes I and II , with clear-cut distinctions in their functions ( Kihara et al . , 2001 ) .",
"Both yeast complexes contain the core subunits Vps34 , Vps15 , and Atg6 .",
"Vps34 contains the catalytic domain responsible for lipid kinase activity , as well as a putative lipid-binding C2 domain and a helical domain ( Schu et al . , 1993; Miller et al . , 2010 ) .",
"Vps15 is a large ( 150 kDa ) protein essential for the activity of the complex ( Stack et al . , 1993 ) .",
"Vps15 contains a protein kinase domain whose function and possible substrates are uncertain , as well as HEAT and WD40 repeats .",
"Atg6 is the yeast ortholog of the human tumor suppressor BECN1 and contains an intrinsically disordered region , a coiled coil , and a BARA domain .",
"Yeast complex I further contains the coiled coil subunit Atg14 and is the complex that is involved in autophagy initiation .",
"Yeast complex II contains instead another coiled coil protein , Vps38 , and functions in endosome maturation .",
"The yeast complexes are prototypes of two human PI 3-kinase complexes ( Volinia et al . , 1995 ) , which both contain the common core subunits VPS34 ( also known as PI3KC3 ) , VPS15 , and BECN1 ( Backer , 2008; Funderburk et al . , 2010; He and Levine , 2010; Wirth et al . , 2013 ) ( Figure 1A ) .",
"The human cognate of PI3KC3 complex I contains ATG14 ( also known as ATG14L or BARKOR ) ( Itakura et al . , 2008; Sun et al . , 2008; Matsunaga et al . , 2009; Zhong et al . , 2009 ) .",
"We will refer to this complex as PI3KC3-C1 .",
"ATG14 is specifically recruited to sites of autophagosome initiation ( Matsunaga et al . , 2010; Fogel et al . , 2013; Graef et al . , 2013; Hamasaki et al . , 2013; Wirth et al . , 2013; Ge et al . , 2014 ) .",
"A second human PI3KC3 contains , in place of ATG14 , the UV resistance-associated gene product , UVRAG .",
"We will refer to this complex as PI3KC3-C2 .",
"PI3KC3-C2 has been proposed to function at later stages in autophagy ( Liang et al . , 2006 ) but is not currently thought to participate in the initiation of autophagosome biogenesis .",
"The overall objective of this study was to define the architecture of the two human PI3KC3 complexes .",
"One aspect of the larger goal was to ascertain whether there are gross structural differences between PI3KC3-C1 and -C2 that might affect their relative ability to target the ER and initiate autophagy . 10 . 7554/eLife . 05115 . 003Figure 1 . Reconstitution and 3D reconstruction of the PI3KC3-C1 complex .",
"( A ) Domain structures of the four subunits of PI3KC3-C1 .",
"The small unlabeled yellow box between VPS34 C2 and helical domains is the CHIL motif , described below .",
"( B ) Purification of PI3KC3-C1 .",
"Coomassie-stained SDS-PAGE gel of purified PI3KC3-C1 .",
"( C ) Thin-layer chromatography of radiolabeled PI ( 3 ) P generated by PI3KC3-C1 from PI and [γ-32 P] ATP .",
"( D ) Reference-free class averages of PI3KC3-C1 , each containing ∼200 particles .",
"The first row represents particles that were selected for the 3D reconstruction .",
"The lower two rows represent particles excluded from the 3D reconstruction , the arrowhead indicating the position of a density dislodged from the main part of the complex .",
"All class averages shown are calculated from data acquired on untilted grids , except the top right class average which is from 45° tilt .",
"( E ) 3D reconstruction calculated from ∼39 , 000 particles in four orientations and displayed at a threshold determined from subsequent docking analyses . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 003 The pivotal role of PI3KC3 in the basic mechanism of autophagy induction and the potential importance of PI3KC3 modulators in treating cancer , neurodegenerative , and other diseases are driving interest in the structure of the complex .",
"To date , crystal structures have been obtained for the catalytic core and helical domains of Drosophila Vps34 ( Miller et al . , 2010 ) , the WD40 propeller domain of yeast Vps15 ( Heenan et al . , 2009 ) , the coiled coil domain of human BECN1 ( Li et al . , 2012 ) , and the BARA domains of yeast and human Atg6/BECN1 ( Huang et al . , 2012; Noda et al . , 2012 ) .",
"Structures of homologs of the Vps34 C2 domain and the Vps15 HEAT repeats and protein kinase domains are available .",
"These various regions have to work together in a single complex in autophagy initiation .",
"Currently , there are essentially no data on the structure of the 361 . 8 kDa quaternary assembly of these subunits with each other in PI3KC3-C1 ( Hurley and Schulman , 2014 ) .",
"We view this information as essential , if we are to begin to understand how autophagy is initiated and how it is regulated and to address the potential for therapeutic modulation of PI3KC3-C1 .",
"To this end , we reconstituted active human PI3KC3-C1 and PI3KC3-C2 by co-expression of all four subunits and imaged the complex by electron microscopy ( EM ) .",
"The complexes are elongated , loosely connected , and dynamic; yet by careful selection of class averages and Bayesian data processing , we were able to generate a three-dimensional reconstruction of PI3KC3-C1 at 28-Å resolution .",
"We have mapped the positions of the various domains of the subunits by tagging with maltose binding protein ( MBP ) .",
"Insight into large-scale conformational fluctuations of PI3KC3-C1 was obtained from EM , while local dynamics was mapped by hydrogen–deuterium exchange .",
"Taken together , the data led us to a model for the subunit architecture and dynamics of PI3KC3-C1 .",
"The main architectural principles also hold for the PI3KC3-C2 complex .",
"The structural model suggests that the VPS15 kinase domain acts as a latch to regulate PI 3-kinase activity in both the PI3KC3-C1 and -C2 ."
],
[
"In order to generate sufficient quantities of compositionally homogeneous PI3KC3-C1 , synthetic DNA constructs encoding VPS34 , VPS15 , BECN1 , and ATG14 were co-transfected in HEK293 cells .",
"The resulting complex contained all four subunits at apparently equal stoichiometry ( Figure 1B ) and the subunits co-migrated as a single peak on gel filtration chromatography ( Figure 2A ) .",
"The material was enzymatically active as judged by ATP hydrolysis in the presence of PI ( Figure 2B ) and by the formation of PI ( 3 ) P from PI as assessed by thin layer chromatography ( Figure 1C ) .",
"ATP hydrolysis by PI3KC3-C1 was essentially completely blocked by the PI 3-kinase inhibitor wortmannin ( Figure 2B ) , confirming that the ATPase activity was due to the lipid kinase domain and not either the VPS15 kinase domain or contaminants . 10 . 7554/eLife . 05115 . 004Figure 2 . Characterization of the PI3KC3-C1 complex .",
"( A ) Size-exclusion chromatography of PI3KC3-C1 showing that the complex elutes as a single peak well separated from the void volume ( Vo ) .",
"( B ) ATP hydrolysis by PI3KC3-C1 is inhibited by wortmannin .",
"RLU , relative luminescence units × 106 . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 004 PI3KC3-C1 samples were stained with uranyl formate and imaged by EM ( Figure 3A ) .",
"Two-dimensional class averages for PI3KC3-C1 are shown in Figure 1D .",
"The class averages revealed a V-shaped particle with two arms of approximately 20 nm in length .",
"There was a strongly preferred orientation with the V view visible face-on in projection ( Figure 3B ) .",
"Edge-on views were less common .",
"An ab initio 3D reconstruction was generated by the random conical tilt ( RCT ) method ( Radermacher et al . , 1987 ) and refined using particles acquired at 0° , 30° , and 45° .",
"Class averages from ∼160 , 000 particles were culled of aggregates , contaminants , lower resolution classes , and those with sparsely populated conformations , leading to a final set of ∼40 , 000 high-quality particles .",
"The final reconstruction ( Figure 1E ) was calculated using a single 3D class in RELION ( Scheres , 2012 ) , with the initial RCT structure low-pass filtered to 80 Å as the initial reference .",
"This reconstruction has a resolution of 28 Å according to the gold standard FSC criterion as implemented in RELION ( Figure 3C ) and was used for docking of known crystal structures or homology models of domains .",
"The reconstructed density shows some distortion in the direction normal to the plane of the preferred particle orientation , due to the missing data for orientations at >45° tilt .",
"The chirality of the structure was validated by tilt-pair analysis ( Rosenthal and Henderson , 2003 ) with image pairs collected at 0° and 15° ( Figure 3D ) . 10 . 7554/eLife . 05115 . 005Figure 3 . Electron microscopy of PI3KC3-C1 . ( A ) Representative unprocessed micrograph of PI3KC3-C1 .",
"( B ) Euler angle distribution of particles used for the 3D reconstruction shown in Figure 1E .",
"The views are the same as those shown in Figure 1E .",
"Long/red bars mean many particles contributing to the view along that bar .",
"( C ) Gold-standard Fourier shell correlation ( FSC ) plot of the 3D reconstruction shown in Figure 1E .",
"( D ) Tilt-pair analysis to validate the absolute hand of the 3D reconstruction shown in Figure 1E .",
"Plots calculated from image pairs acquired at 0° and 15° for PI3KC3-C1 ( left panel ) and as control for E . coli 70S ribosomes ( right panel ) .",
"The ribosome plot was calculated using a previously published map deposited in EMDB with accession code EMD-1849 ( Agirrezabala et al . , 2011 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 005 The 28 Å reconstruction described above lacked the detail , on its own , to unambiguously place most of the structures of the various subunit domains .",
"The exceptions were the donut-shaped region corresponding to the WD40 domain of VPS15 and the arch-shaped density at the base of the V corresponding to the HEAT repeat of the same subunit .",
"Therefore , we generated and purified a series of N- and C-terminal MBP fusion constructs to map the domains using EM ( Figure 4A , B ) .",
"Both N- and C-terminal MBP fusions of VPS34 and BECN1 and the N-terminal MBP fusions of ATG14 and VPS15 were successfully purified and imaged . 10 . 7554/eLife . 05115 . 006Figure 4 . MBP-tagging identifies PI3KC3-C1 subunits .",
"( A ) Six different MBP-tagged versions of the complex were used to identify the position of the different PI3KC3-C1 subunits .",
"A cartoon diagram indicating the position of the MBP tags is shown on the right .",
"The two left columns show the reference-free 2D class averages of the MBP-labeled samples and arrowhead highlights the MBP tag position .",
"The middle column shows the corresponding class for the unlabeled sample .",
"Two right columns show the difference map calculated by subtracting the unlabeled reference class from the labeled and the dotted circle represents the MBP density .",
"( B ) 3D reconstruction of the PI3KC3-C1 complex highlighting the position of the six MBP tags used for domain mapping . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 006 With respect to the canonical ‘V’ view of the complex , the VPS34 kinase subunit is at the tip of the right-hand arm of the V . Its N-terminus is near the crotch of the V . In turn , BECN1 is aligned with the left arm of the complex .",
"Its N-terminus is near the junction of the V and its C-terminus at the tip of the left arm .",
"The N-terminus of scaffolding VPS15 subunit is located near the C-terminus of VPS34 in the right arm .",
"Finally , the N-terminus of ATG14 is near that of BECN1 .",
"Strikingly , this suggests that the BECN1 and ATG14 coiled coils must be parallel to each other , in contrast to the antiparallel coiled coil described for the BECN1 homodimer ( Li et al . , 2012 ) .",
"Given the added guidance provided by MBP tags , we docked the known crystal structures or homology models of the structured domains of VPS34 , VPS15 , and BECN1 on to the density and the docked volume accounted for 65% of the total density .",
"( Figure 5A , C ) .",
"The helical and catalytic domains of VPS34 are integrated into a single structural unit referred to as the ‘HELCAT’ region ( Miller et al . , 2010 ) ( Figure 1A ) , which matches in size and shape the density identified near the C-terminal MBP tag in this subunit ( Figures 4B and 5C ) .",
"Thus , the HELCAT region was positioned such that its C-terminal helix was proximal to the position of the C-terminal MBP tag ( Figures 4B and 5C ) .",
"A density feature matching the size and shape of the C2 domain is located between the HELCAT and the N-terminal MBP tag ( Figures 4B and 5C ) .",
"There is no available crystal structure for VPS34-C2 .",
"However , a model based on the closely related structure of the C2 domain of PI3KC2α ( Liu et al . , 2006 ) was docked into this density , and a reasonable fit was obtained ( Figure 5C ) . 10 . 7554/eLife . 05115 . 007Figure 5 . Subunit architecture of PI3KC3-C1 . ( A ) Segmented volume representation of the 3D reconstruction highlighting the different domains of the complex colored as shown in ( B ) .",
"Arrows indicate regions assigned to the BECN1-ATG14 coiled coil .",
"( B ) Domain structures of the four subunits of PI3KC3-C1 .",
"( C ) 3D reconstruction of the complex with the docked structures shown in a ribbon representation .",
"The MBP label positions are represented as dotted circles .",
"( D ) 3D reconstruction of the complex with the docked structures in ribbon representation in different rotations . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 007 The crystal structure of the BARA domain ( also sometimes referred to as the ‘ECD’ ) of human BECN1 ( Huang et al . , 2012 ) was unambiguously identified as a match to the size and shape of the only density feature near the BECN1 C-terminal MBP tag ( Figures 4B and 5C ) .",
"The remaining large density features were assigned to VPS15 , beginning with the N-terminally tagged kinase domain .",
"A homology model for the VPS15 kinase catalytic domain was derived from the structure of protein kinase A ( PKA ) ( Knighton et al . , 1991 ) .",
"The model was docked into a major density feature adjoining the position of the tag and matching its size and shape ( Figures 4B and 5C ) .",
"A model for the 498-residue HEAT repeat domain of VPS15 was generated by threading the corresponding sequence onto the backbone of the HEAT repeat portion of the exportin subunit Cse1 ( Matsuura and Stewart , 2004 ) and provided an excellent match to the arch-shaped density adjoining the C-terminus of the VPS15 kinase domain ( Figures 4B and 5C ) .",
"Indeed , the kinase and HEAT repeat domains of VPS15 seem to be fused into a single contiguous unit .",
"The most C-terminal domain of VPS15 , the WD40 propeller region , is connected to the HEAT by a 180-residue linker .",
"This propeller was assigned to the last unaccounted for density feature , located next to the BECN1 BARA domain , which has a donut-like shape that matches the crystal structure of the yeast Vps15 propeller domain ( Heenan et al . , 2009 ) ( Figures 4B and 5C ) .",
"Collectively , the docked or assigned regions of the VPS34 C2 and HELCAT , VPS15 kinase , HEAT , and WD40 , BECN1 BARA domains account for 2162 residues of the 3187 present in ATG14–PI3KC3 .",
"Two long , narrow tubes of density are present in the area where the ATG14 and BECN1 coiled coil regions would be expected ( Figure 5A , D third panel , see arrows ) .",
"One tube is adjacent to the VPS15 WD40 domain .",
"It points towards the BECN1 BARA domain at one end and towards the second tube at the other .",
"Following a gap in the density between the two tubes , the second one continues until it merges with the density for the VPS15 HEAT domains .",
"The BARA domain marks the C-terminus of the BECN1 coiled-coil , and MBP marks the N-termini of ATG14 and BECN1 coils , so we assigned this region to the coiled coil dimer .",
"Because there is a break between the two tubular regions and the BARA density , we did not build an explicit molecular model of this region .",
"One of the few unassigned density features projects away from the VPS15 HEAT domain ( Figure 5A , C ) , very near the MBP markers for the N-termini of ATG14 and BECN1 ( Figure 4B ) .",
"This suggests that at least parts of the N-termini of one or both of these subunits have some ordered structure .",
"The resulting model consists of a 190-Å long right arm and a 210-Å long left arm , joined at a 90o angle ( Figure 5 and Video 1 ) . 10 . 7554/eLife . 05115 . 008Video 1 . Architecture of PI3KC3-C1 . A 360° rotation of the docked model shown in Figure 5D . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 008 While both PI3KC3-C1 and -C2 have been implicated in autophagy , PI3KC3-C1 is the complex involved in autophagosome initiation ( Matsunaga et al . , 2010 ) .",
"UVRAG is larger than ATG14 and contains a unique N-terminal C2 domain ( Figure 6B ) .",
"In order to gain insight into the structural basis for the differences in localization and function between the two complexes , PI3KC3-C2 was expressed , purified , assayed , and prepared for EM imaging ( Figure 6A ) using the same procedures as for PI3KC3-C1 .",
"2D class averages of this complex show the same characteristic V-morphology and preferential face-on orientations as seen for PI3KC3-C1 .",
"As for PI3KC3-C1 , the majority of particles belonged to classes in which the VPS34 HELCAT domain was dislodged from the rest of the complex .",
"The main difference in the images is the consistent presence of additional density at the base of the V ( Figure 6C , arrows ) .",
"The location of the density corresponds to the expected location of the UVRAG C2 domain .",
"The main conclusion from the 2D characterization of the PI3KC3-C2 is that the overall conformation and architecture are essentially identical to that of the PI3KC3-C1 as seen at the present resolution of our analysis .",
"Therefore , the functional differences between the two complexes cannot be attributed to gross differences in architecture or conformation . 10 . 7554/eLife . 05115 . 009Figure 6 . Reconstitution and EM analysis of PI3KC3-C2 . ( A ) Purification of PI3KC3-C2 .",
"Coomassie-stained SDS-PAGE gel of purified PI3KC3-C2 .",
"( B ) Domain structures of ATG14 and UVRAG .",
"( C ) The top row shows the reference-free 2D class averages for the PI3KC3-C2 complex .",
"The bottom four rows show the corresponding class averages of the PI3KC3-C2 and arrowhead highlights the additional density attributed to the UVRAG C2 domain . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 009 Having assigned 65% of the density to known well-ordered regions of PI3KC3-C1 subunits , approximately 1025 out of the 3187 residues were not accounted for .",
"These comprise the extended N-terminal regions of BECN1 and ATG14 , the long C-terminal region of ATG14 , and the linkers between the C2 and helical domains of VPS34 and the HEAT and WD40 domains of VPS15 .",
"In order to characterize these structurally undefined regions , we carried out hydrogen–deuterium exchange ( HDX ) as detected by mass spectrometry ( MS ) ( Englander , 2006; Engen , 2009; Chalmers et al . , 2011 ) .",
"Excellent peptide coverage was obtained for all of VPS15 ( 167 peptides were analyzed , 89 . 9% protein coverage ) and all of VPS34 ( 119 peptides were analyzed , 74 . 7% protein coverage ) with the exception of the C2 domain ( Figure 7A ) .",
"Throughout the ordered domains of VPS15 and VPS34 , partial exchange was observed , consistent with the folded nature of the domains ( Figure 7A , B ) .",
"Complete exchange was observed throughout the VPS15 HEAT-WD40 linker at incubation times as short as 10 s ( Figure 7B ) , indicating that this region is essentially completely flexible and intrinsically disordered .",
"The linker between the helical and kinase domains of VPS34 also manifested complete exchange , consistent with the absence of density for this region in the VPS34 crystal structure ( Miller et al . , 2010 ) . 10 . 7554/eLife . 05115 . 010Figure 7 . Local dynamics of PI3KC3-C1 assessed by hydrogen–deuterium exchange . PI3KC3-C1 was incubated for 10 s in D2O .",
"Percent deuterium incorporation plotted vs the central residue number of peptides from VPS34 ( A ) and VPS15 ( B ) .",
"( C ) Percent change in deuteration for VPS34 region 205–247 across various time points ( 10 s to 1 hr ) .",
"Green bars above refer to peptide coverage .",
"( D ) Conservation of the C2-Helix Internal Linker ( CHIL ) motif in VPS34 . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 010 One significant and unexpected zone of protection was discovered within the linker regions , a ‘cold spot’ comprising residues 218–237 of VPS34 ( Figure 1A , yellow box between C2 and helical domains; Figure 7A , C ) .",
"This region is part of the C2-helical domain linker .",
"It is highly conserved from yeast to humans ( Figure 7D ) , so we christened it the C2-Helical Internal Linker ( CHIL ) motif .",
"Peptides covering this region show very little increase in deuteration over the interval from 10 s to 1 hr ( Figure 8 ) .",
"Because this 20-residue motif is too small to form a folded entity on its own , we presume that it assembles with a nearby unit , likely the VPS15 HEAT or the VPS34 helical domain . 10 . 7554/eLife . 05115 . 011Figure 8 . Time course of deuteration of CHIL motif peptides . Hydrogen–deuterium incorporation as a function of time ( 10 s to 1 hr ) for selected peptides in the CHIL motif of VPS34 .",
"The deuteration level of an individual peptide was determined via HDexaminer , and it was assumed that the first residue in a peptide is non-deuterated . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 011 The 2D class averages calculated from all raw images of PI3KC3-C1 revealed a high degree of structural plasticity of the complex ( Figures 1D and 9 ) .",
"Whilst a structurally homogeneous subset of images ( Figure 1D , top row ) was selected for the 3D reconstruction described above , the excluded images provide additional insights into the structural dynamics of PI3KC3-C1 .",
"Most strikingly , many class averages show the density identified as VPS34 HELCAT being dislodged from the rest of the complex ( Figure 1D , lower two rows ) .",
"Additionally , some class averages appear to entirely lack the density for VPS34 HELCAT .",
"In principle , this could either be due to dissociation of VPS34 from the complex or due to the averaging out of HELCAT densities in a multitude of positions .",
"The latter possibility seems more likely , given that the dislodged HELCAT positions generally lack contacts with the rest of the complex . 10 . 7554/eLife . 05115 . 012Figure 9 . Global conformational changes of PI3KC3-C1 . ( A ) Representative class averages of the PI3KC3-C1 complex with the VPS15 KINHEAT in the open ( far left ) to closed ( far right ) conformation and the dynamic VPS35 HELCAT .",
"( B ) Class averages with docked VPS15 KINHEAT , VPS15 WD-40 , and BECN1 BARA domains overlaid .",
"( C ) Schematic of the PI3KC3-C1 complex showing the open and closed conformations of the Vps15 KINHEAT and the dynamics of the Vps34 HELCAT .",
"The VPS34 C2 domain is yellow and the CHIL motif is orange .",
"( D ) Percentage of well-resolved particles sorting into class averages with a lodged , dislodged , or undiscernible VPS34 HELCAT . DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 012 The untilted images recorded for the PI3KC3-C1 3D reconstruction were revisited to determine the abundance of states with a dislodged HELCAT .",
"Of the particles that sorted into high-quality class averages , 48 . 5% had a lodged HELCAT , the remaining particles mainly having a dislodged but discernible HELCAT ( Figure 9D ) .",
"The same analysis of PI3KC3-C2 revealed 34 . 0% particles in the lodged state and a larger percentage of particles having no discernible HELCAT density ( Figure 9D ) .",
"The class averages in Figure 1D show the range of motion of the dislodged HELCAT , spanning a radius of up to 18 nm from the proposed position of the VPS34 C2 domain to the center of mass of the HELCAT .",
"A second mode of flexibility of PI3KC3-C1 takes place at the VPS15 HEAT ( Figure 9 , Video 2 ) .",
"This flexibility is seen both in the lodged and dislodged HELCAT state and leads to a movement of the VPS15 kinase domain relative to the WD40 .",
"Comparison of individual class averages indicate that one pivoting point for this motion is between the WD40 domain and the C-terminus of the HEAT repeat , with additional flexibility between the HEAT and kinase domains ( Video 2 ) . 10 . 7554/eLife . 05115 . 013Video 2 . Conformational changes of PI3KC3-C1 . A sequence of 2D class averages arranged according to the pivoting motion of VPS15 , from open to more-closed .",
"The sequence also shows several different lodged and dislodged states of VPS34 HELCAT ( in no particular order ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05115 . 013"
],
[
"The EM reconstruction and docking model for PI3KC3-C1 provides the first overall view of VPS15 , VPS34 , BECN1 , and ATG14 in their functional context .",
"The HDX-MS experiments together with the EM analysis highlight the dynamic nature of the complex .",
"At the available resolution , the overall architecture is essentially the same for PI3KC3-C2 and PI3KC3-C1 .",
"The reconstruction thus provides a unified model for VPS34 complex organization .",
"The overall structure allows us to rationalize a variety of previous data on subunit organization .",
"The BARA domain of BECN1 is essential for the incorporation of BECN1 into the PI3KC3-C1 complex ( Furuya et al . , 2005 ) , consistent with its interactions with the VPS15 WD40 domain in the EM structure .",
"Similarly , the ATG14 coiled coil region is essential for its association with VPS34 ( Itakura et al . , 2008 ) , consistent with the interactions of both the ATG14 coiled coil and VPS34 with the central VPS15 scaffold .",
"The C2 domain of VPS34 was previously shown to be the main locus for the incorporation of VPS34 into the complex ( Liang et al . , 2006 ) .",
"This fits with the structural observation that VPS34-C2 is integral to the core architecture , while the C-terminal HELCAT region is readily dislodged .",
"An interaction was reported between the C-terminal helix of VPS34 and the VPS15 kinase-HEAT region ( Budovskaya et al . , 2002 ) , consistent with the contacts we observed between the kinase domains of VPS15 and VPS34 .",
"The C-terminal helix of VPS34 is critically involved in membrane interaction upon activation ( Miller et al . , 2010 ) , and this raises the interesting speculation that VPS34 could be regulated via inter-subunit sequestration of the same helix .",
"The congruence of the size and shape of density features with subunit domains , the connectivity of the domains within VPS34 and VPS15 , the localization of the tags , and the consistency with published interactions all lend high confidence to our structural assignments and the defined architecture of the complex .",
"The 2D class averages highlight the globally dynamic nature of the VPS34 HELCAT module relative to the rest of the complex .",
"Many of these class averages show that VPS34-HELCAT is dislodged from the rest of the complex , even though the rest of the complex is intact .",
"The class averages also provide some insight into the nature of the motions .",
"It should be emphasized that , while there is evidence for domain movements , the exact extent and direction of the movements are unknown .",
"The range of movements may be constrained in our experiments by the presence of the carbon film on which the complex is imaged .",
"Thus , the movements noted here may be a subset of the range that could occur in solution .",
"VPS34-HELCAT makes contacts principally with the kinase domain of VPS15 .",
"In many of the class averages , the VPS15 kinase domain pivots toward the other arm of the V . The pivoting motion includes the VPS15 HEAT repeat domain .",
"Thus the VPS15 kinase and HEAT regions function as a single module .",
"By analogy to the HELCAT terminology for VPS34 , we refer to it as the KINHEAT module .",
"VPS15 is not obviously a pseudokinase on the basis of its sequence ( Taylor et al . , 2013 ) , yet there is no compelling evidence that VPS15 can phosphorylate substrates ( Backer , 2008 ) .",
"The VPS15-KINHEAT physically contacts the VPS34-HELCAT region , which suggests that regulation could occur through direct and stable interactions without covalent phosphorylation .",
"While the V-shaped PI3KC3-C1 complex flexes on a large scale , the EM and HDX data also show that there are limits to the types of motions that occur .",
"The HDX data show that the VPS15 HEAT-WD40 linker is essentially completely disordered , yet the WD40 domain is firmly lodged in the complex in all class averages .",
"The WD40 is thus clearly not undergoing tethered diffusion or ‘fly-casting’ in the context of the assembled complex .",
"The only part of the structure that we observed to undergo fly-casting is the VPS34 HELCAT module .",
"Even in this case , the motions are constrained .",
"Although the linker between the VPS34 C2 and helical domains spans 130 residues , the presence of the anchored CHIL domain in the center of this linker sharply restricts the range of motion .",
"There are ∼50–65 residues between the CHIL and the boundaries of the nearest ordered modules , which would limit the maximum reach of the fully extended linker to ∼20 nm .",
"This is consistent with the maximum 18 nm displacement of the HELCAT region seen in EM images .",
"It is striking that no direct contacts are seen between the ATG14 and BECN1 module and VPS34 .",
"Given the resolution of the reconstruction , it is certainly possible that direct interactions exist though they were not visualized .",
"Nevertheless , the structure highlights a central and extensive role for VPS15 in scaffolding and bridging between the ATG14:BECN1 regulatory subcomplex and the VPS34 catalytic subunit .",
"VPS15 is capable of pivoting about the KINHEAT-WD40 junction .",
"The base of the V is mainly formed from the HEAT repeats of VPS15 .",
"The base of the V is also the locus for the unstructured or unresolved N-terminal regions of ATG14 and BECN1 .",
"Most of the known signaling inputs into PI3KC3-C1 converge on the N-terminus of BECN1 .",
"This region is subject to activating phosphorylations by the upstream autophagy initiating kinase ULK1 ( Russell et al . , 2013 ) and the AMP-activated kinase AMPK ( Kim et al . , 2013 ) .",
"The BECN1 BH3 domain responsible for binding to Bcl-2 , which inhibits autophagy , is located in this region ( Pattingre et al . , 2005 ) .",
"Along the same lines , a Cys-rich region of ATG14 immediately N-terminal to the coiled coil has been proposed to be the site of ER targeting ( Matsunaga et al . , 2010 ) .",
"Thus , this region of the PI3KC3-C1 structure might act as an allosteric reporter for localization to the ER membrane .",
"It is tempting to speculate that the right arm of the complex transmits signals from the region of the BECN1 N-terminus to the VPS34 lipid kinase domain .",
"It is exciting to finally have a structural context in which to test how the many signals that impinge on BECN1 and PI3KC3-C1 are processed ."
],
[
"The full length DNAs encoding VPS15 , VPS34 , BECN1 , ATG14 , and UVRAG were codon optimized for expression in HEK293 cells .",
"Synthetic genes encoding VPS15 , VPS34 , BECN1 DNAs were amplified by PCR and cloned into the pCAG vector coding for an N-terminal twin-STREP-FLAG tag using KpnI and XhoI restriction sites .",
"The ATG14 and UVRAG DNAs were cloned in the pLEXm vector encoding an N-terminal GST tag followed by TEV restriction site .",
"The pCAG vector encoding an N-terminal GST tag followed by a TEV restriction site was used for expression of ATG14 .",
"HEK293 cells adapted for suspension were grown in Freestyle media ( Invitrogen , Grand Island , NY ) supplemented with 1% FBS ( Invitrogen ) at 37°C , 80% humidity , 5% CO2 , and rocked at 140 rpm .",
"Once the cultures reached 1 . 5–2 million cells ml−1 in the desired volume , they were transfected as followed .",
"For a 1 l transfection , 3 ml PEI ( 1 mg ml−1 , pH 7 . 0 ) was added to 33 ml Hybridoma media and 1 mg of total DNA in another 33 ml hybridoma media .",
"1 mg of transfection DNA contained equal mass ratio of PI3KC3-C1 and -C2 complex expression plasmids ( pLEXm-GST-TEV-ATG14 [or pLEXm-GST-TEV-UVRAG] , pCAG-twinSTREP-FLAG-VPS15 , pCAG-twinSTREP-FLAG-VPS34 , and pCAG-twinSTREP-FLAG-BECN1 ) .",
"PEI was added to the DNA , mixed and incubated for a further 20 min at room temperature .",
"66 ml of the transfection mix was then added to each 1 l culture .",
"Cells were harvested after 3 days .",
"Cells were lysed by gentle shaking in lysis buffer ( 50 mM Tris , pH 8 . 0 , 200 mM NaCl , 2 mM MgCl2 , 10% ( vol/vol ) glycerol , 1% ( vol/vol ) Triton X-100 , 1 mM TCEP , and EDTA free proteinase inhibitors [Roche , Basel , Switzerland] ) at 4°C .",
"Lysates were clarified by centrifugation ( 18 , 000×g for 60 min at 4°C ) and incubated with 10 ml glutathione Sepharose 4B ( GE Healthcare , Uppsala , Sweden ) for 1 hr at 4°C with gentle shaking .",
"The glutathione Sepharose 4B matrix was applied to a gravity column , washed four times with 50 ml wash buffer ( 50 mM Tris , pH 8 . 0 , 200 mM NaCl , 2 mM MgCl2 , and 1 mM TCEP ) , and purified complexes were eluted with 50 ml wash buffer containing 50 mM reduced glutathione .",
"Eluted complexes were treated with TEV protease at 4°C overnight .",
"TEV-treated complexes were loaded on a 2 . 5 ml Strep-Tactin Sepharose gravity flow column ( IBA GmbH , Göttingen , Germany; at 4°C ) .",
"The Strep-Tactin Sepharose column was washed five times with 2 . 5 ml wash buffer , and purified complexes were eluted with 6 ml wash buffer containing 10 mM desthiobiotin ( Sigma-Aldrich , St . Louis , MO ) .",
"Eluted complexes were purified to homogeneity by injection on Superose 6 16/50 ( GE Healthcare ) column that was pre-equilibrated in gel filtration buffer ( 20 mM Tris–HCl , pH 8 . 0 , 200 mM NaCl , 2 mM MgCl2 , and 1 mM TCEP ) .",
"ATP consumption in the presence of lipids was determined using the ADP-Glo Kinase Assay kit ( Promega , Madison , WI ) .",
"The kinase reaction was performed in 96-well NBS white plates ( Corning , Corning , NY ) , in 1 . 25× kinase reaction buffer ( 12 . 5 mM HEPES , pH 7 . 0 , 125 mM NaCl , 2 . 5 mM MnCl2 ) , and 2 μg PI:phosphatidylserine ( PS ) substrate solution .",
"The reaction was initiated by adding 5 μl of 125 μM ATP to 20 μl of 1 . 25× kinase reaction buffer .",
"The reaction was carried out for 1 hr at 37°C , then 25 μl of ADP-Glo reagent containing 10 mM MgCl2 was added to the reaction mixture and incubated at 23°C for 30 min to stop the enzyme reaction and deplete unconsumed ATP .",
"After depletion of ATP , 50 μl of Kinase Detection Reagent was added to convert ADP to ATP and introduce luciferase and luciferin to detect ATP .",
"The reaction mixture was further incubated 23°C for 30 min and the luminescence was measured with a GloMax-Multi detection system ( Promega ) .",
"Production of PI ( 3 ) P from PI was assayed as follows .",
"Recombinant PI3KC3 was pre-incubated in 73 µl reaction buffer ( 20 mM Tris–HCl , pH 8 . 0 , 100 mM NaCl , 10 mM MgCl2 ) containing 20 μg sonicated phosphatidylinositol ( Avanti Polar Lipids , Alabaster , AL ) for 20 min on ice .",
"The reaction was started by adding 6 µl cold ATP ( 0 . 5 mM in reaction buffer ) and 1 µl ATP [γ-32P] ( 10 µCi , PerkinElmer ) .",
"After incubation at room temperature for 20 min , the reaction was terminated by the adding 20 μl 8 M HCl .",
"The organic phase was extracted with 160 µl methanol/chloroform ( 1:1 , vol/vol ) .",
"Extracted phospholipid products were resolved by TLC using a silica-coated gel ( Whatman , Little Chalfont , United Kingdom ) and a solvent composed of chloroform:methanol:4 M ammonium hydroxide ( vol/vol/vol , 9/7/2 ) , followed by visualization with a phosphorimager ( Typhoon Trio , GE Healthcare ) .",
"Negatively stained samples of PI3KC3-C1 and -C2 were prepared on continuous carbon grids that had been plasma cleaned in a 10% O2 atmosphere for 10 s using a Solarus plasma cleaner ( Gatan Inc . , Pleasanton , CA ) .",
"4 μl of PI3KC3 complexes at a concentration of 25 nM in 20 mM Tris , pH 8 . 0 , 200 mM NaCl , 2 mM MgCl2 , 1 mM TCEP , and 3% trehalose were placed on the grids and incubated for 30 s .",
"The grids were floated on four successive 50 μl drops of 1% uranyl formate solution incubating for 10 s on each drop .",
"The stained grids were blotted to near dryness with a filter paper and air-dried .",
"For the MBP tag analysis , native and tagged samples were imaged using an FEI Tecnai 12 electron microscope ( FEI , Hillsboro , OR ) operated at 120 keV at a nominal magnification of 49 , 000 ( 2 . 18 Å calibrated pixel size at the specimen level ) using a defocus range of −0 . 7 to −1 . 5 μm with an electron dose of 35e−/Å2 .",
"Images were acquired on a TVIPS TemCam F-416 4049 × 4096 pixel CMOS detector ( TVIPS GmbH , Gauting , Germany ) using the automated Leginon data collection software ( Suloway et al . , 2005 ) .",
"For the initial 3D random conical tilt reconstruction , tilt-pair images were recorded at 0° and 50° at 30 , 000 nominal magnifications with the same setup ( 3 . 56 Å/pixel at the specimen level ) .",
"Subsequent 3D structure refinement was performed using larger datasets with images acquired using an FEI Tecnai F20 transmission electron microscope operated at 120 kV at a nominal magnification of 80 , 000 using a defocus range of −0 . 7 to −1 . 5 μm with an electron dose of 35e−/Å2 at tilts of 0° , 30° , and 45° .",
"Images were acquired on a Gatan UltraScan4000 4049 × 4096 pixel CCD detector using Leginon with a 1 . 51 Å calibrated pixel size at the specimen level ( Suloway et al . , 2005 ) .",
"The initial steps of image processing and classification were performed using the Appion image processing environment ( Lander et al . , 2009 ) .",
"Particles were first selected manually from micrographs and subjected to 2D iterative reference-free classification and alignment using a topology-representing network classification and IMAGIC ( Image Science Software GmbH , Berlin , Germany ) multi-reference alignment ( MRA ) ( van Heel et al . , 1996; Ogura et al . , 2003 ) .",
"The 2D class averages thus generated served as templates for subsequent automated particle selection .",
"Template-based automated particle picking was performed using the FindEM program ( Roseman , 2004 ) .",
"The contrast transfer functions ( CTFs ) of the micrographs were estimated using the CTFFIND for untilted images and CTFTILT for tilted images ( Mindell and Grigorieff , 2003 ) .",
"CTF correction of the micrographs was performed by Wiener filter using ACE2 ( Mallick et al . , 2005 ) for the micrographs used for subunit localization and by tilt- and position-dependent phase flipping using EMAN ( Ludtke et al . , 1999 ) for micrographs used for the 3D reconstructions .",
"Particles were extracted using a 192 × 192 ( T12 ) or 256 × 256 ( F20 ) pixel box size and binned by a factor of 2 .",
"Each particle was normalized to remove pixels whose values were above or below 4 . 5 σ of the mean pixel value using the XMIPP normalization program ( Scheres et al . , 2008 ) .",
"In order to remove incorrectly selected protein aggregates or other artifacts , images whose mean or standard deviation deviated a lot from the typical values of the dataset were removed .",
"The remaining particles were subjected to five rounds of iterative classification using a topology-representing network and 2D MRA in IMAGIC ( van Heel et al . , 1996; Ogura et al . , 2003 ) .",
"The resulting 2D class averages were manually inspected to remove protein aggregates , contaminants , lower-resolution classes , as well as particles with a dislodged VPS34 HELCAT domain .",
"The remaining particles were subjected to another five rounds of classification and MRA to produce the final 2D class averages , each being calculated from an average of 200–250 raw images .",
"Datasets consisting of 53 , 649 , 36 , 997 , 3799 , 20 , 065 , 40 , 854 , and 12 , 804 particles were collected for the MBP-VPS34 , VPS34-MBP , MBP-BECN1 , BECN1-MBP , MBP-ATG14 , and MBP-VPS15 , respectively .",
"For the subunit and domain localization , reference-free 2D class averages were generated as described above for each tagged dataset and the native dataset .",
"Class averages from each dataset were grouped by projection matching to re-projections of a filtered reference volume of the native complex using SPIDER ( Shaikh et al . , 2008 ) .",
"The corresponding class averages in the different projection groups of the native and tagged datasets were manually examined to locate the position of the tag .",
"For the difference map generation class averages in the same projection group were individually normalized and the native class averages were subtracted from the tagged class averages .",
"Extra density for the MBP tags was clearly visible in 31% of the class averages for MBP-VPS34 , 9 . 1% for VPS34-MBP , 10% for MBP-BECN1 , 23 . 5% for BECN1-MBP , 14% for MBP-ATG14 , and 3 . 6% for MBP-VPS15 .",
"An ab initio structure was determined by random conical tilt ( RCT ) using SPIDER routines integrated into Appion ( Radermacher et al . , 1987; Lander et al . , 2009 ) .",
"Briefly , 23 tilt-pair images yielded an initial dataset of 12 , 027 tilt-pair particles .",
"The untilted particles were aligned and classified in 2D using reference-free classification and MRA as described above , followed by a single round of 2D reference-based alignment using Spider's AP MQ command ( Frank et al . , 1996 ) .",
"The final 14 classes ranged from 725 to 1198 untilted particles each and class-volumes were calculated from the matching tilted particles by backprojection .",
"The class-volumes were then refined against the entire dataset ( both tilted and untilted ) using an iterative projection matching procedure combining SPARX and EMAN2 libraries ( Baldwin and Penczek , 2007; Tang et al . , 2007 ) .",
"Each class-volume that corresponded to the whole complex converged to the same 3D reconstruction ( with a final resolution of 27–30 Å , according to the FSC at 0 . 5 cutoff criterion ) .",
"This map was used as the starting model for all subsequent structure refinements .",
"Structure refinement was performed using images acquired at 0° , 30° , and 45° .",
"The initial particle selection gave 109 , 776 particles at 0° , 24 , 085 at 30° , and 25 , 694 at 45° .",
"Reference-free 2D class averages were calculated as described above , with images recorded at different tilt angles treated separately to allow assessment of tilt-dependent orientations and image quality .",
"Initial attempts to perform 3D classification using RELION ( Scheres , 2012 ) with respect to the position on the VPS34 HELCAT region did not result in robust 3D classes .",
"Thus , particles belonging to class averages of lower resolution , as well as class averages with a dislodged VPS34 HELCAT region , were excluded from further processing .",
"This resulted in a dataset of 24 , 116 particles at 0° , 8643 at 30° , and 5986 at 45° .",
"The final structure ( deposited at EMDB with accession number 2846 ) was calculated using a single 3D class in RELION ( Scheres , 2012 ) , with the main RCT structure low-pass filtered to 80 Å as initial reference .",
"The distribution of Euler angles assigned by RELION was visualized with UCSF Chimera ( Pettersen et al . , 2004 ) .",
"The structure had a resolution of 28 Å according to the gold standard FSC criterion as implemented in RELION and was used for docking of homology models of domains .",
"The density map was further validated by tilt-pair analysis ( Rosenthal and Henderson , 2003 ) with image pairs collected at 0° and 15° using software made available by John Rubinstein ( U Toronto ) .",
"As a control for the tilt-pair analysis , image pairs were collected on negatively stained Escherichia coli 70S ribosomes under identical conditions , co-mapping them to a published 70S ribosome map ( EMDB 1849 ) ( Agirrezabala et al . , 2011 ) .",
"Amide hydrogen exchange mass spectrometry ( HDX-MS ) was initiated by a 20-fold dilution of stock PI3KC3-C1 ( 2 μM ) into D2O buffer containing 20 mM Tris–HCl ( pD 8 . 0 ) , 200 mM NaCl , 2 mM MgCl2 , and 1 mM TCEP at 30°C .",
"Incubations in deuterated buffer were performed at intervals from 10 s to 1 hr .",
"Backbone amide exchange was quenched at 0°C by the addition of ice-cold quench buffer ( 400 mM KH2PO4/H3PO4 , pH 2 . 2 ) .",
"Quenched samples were injected onto a chilled HPLC setup with in-line peptic digestion and desalting steps .",
"Desalted peptides were eluted and directly analyzed by an Orbitrap Discovery mass spectrometer ( Thermo Scientific , Waltham , MA ) .",
"The HPLC system was extensively cleaned between samples .",
"Initial peptide identification was performed via tandem MS/MS experiments .",
"A PEAKS Studio 7 ( www . bioinfor . com ) search was used for peptide identification .",
"Initial mass analysis of the peptide centroids was performed using HDExaminer version 1 . 3 ( Sierra Analytics , Modesto , CA ) , followed by manual verification of each peptide .",
"The deuteron content of the peptic peptides covering PI3KC3-C1 was determined from the centroid of the molecular ion isotope envelope .",
"The deuteron content was adjusted for deuteron gain/loss during pepsin digestion and HPLC .",
"Both non-deuterated and fully deuterated PI3KC3-C1 were analyzed .",
"Fully deuterated PI3KC3-C1 was prepared by four cycles of drying and resolubilization in D2O containing 6 M guanidinium hydrochloride ."
]
] | [
"The class III phosphatidylinositol 3-kinase complex I ( PI3KC3-C1 ) that functions in early autophagy consists of the lipid kinase VPS34 , the scaffolding protein VPS15 , the tumor suppressor BECN1 , and the autophagy-specific subunit ATG14 .",
"The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM , revealing a V-shaped architecture .",
"All of the ordered domains of VPS34 , VPS15 , and BECN1 were mapped by MBP tagging .",
"The dynamics of the complex were defined using hydrogen–deuterium exchange , revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain .",
"VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex .",
"Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V . The N-terminus of BECN1 , the target for signaling inputs , resides near the pivot point .",
"These observations provide a framework for understanding the allosteric regulation of lipid kinase activity ."
] | [
"To survive starvation and other hard times , cells have developed a unique recycling strategy: they can scavenge the resources they need from within the cell itself .",
"To do this , the cell forms a double-layered envelope around particular sections of the cell to seal them off from the rest .",
"Then , the contents of the envelope are taken apart and the resulting raw materials are sent elsewhere in the cell where they can be used as required .",
"This process is called autophagy .",
"In more complex organisms like humans , autophagy can have additional roles .",
"One of the key proteins involved in autophagy—called BECN1—suppresses the growth of tumors , and the gene that makes BECN1 is missing in 40–70% of human breast , ovarian , and prostate cancers .",
"Autophagy may also help to prevent Huntington's disease and other similar conditions by stopping disease-causing proteins or broken cell parts from building up inside brain cells .",
"The BECN1 protein does not work alone .",
"Instead , it becomes part of a group , or ‘complex’ , of several proteins that are required to form the envelope made during autophagy .",
"However , the three-dimensional structure of the protein complex is unclear .",
"Baskaran et al . used electron microscopy and other techniques to investigate this structure and found that the complex forms a V shape with two arms , which is held together by its largest protein , VPS15 .",
"This protein also acts as a bridge between BECN1 and another protein that is a target for new cancer drugs , called VPS34 .",
"Next , Baskaran et al . used a different set of techniques to determine how the complex moves .",
"This revealed that many of the connections between proteins in the complex are flexible .",
"However , one of the arms is inflexible and this limits the ability of the VPS34 protein to move .",
"Understanding this structural constraint may help us to design drugs that are able to target the protein complex more efficiently ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Human intracranial recordings link suppressed transients rather than 'filling-in' to perceptual continuity across blinks | elife-17243-v2 | [
[
"The perceived continuity of the visual input despite its frequent interruptions by spontaneous eye blinks is a ubiquitous and powerful dissociation between sensation and perception .",
"As such , it offers an ecological test of candidate neural correlates of visual awareness .",
"The perceptual omission of one's own eye blinks cannot be explained by the blinks' apparent briefness: blinks occlude the pupil for a considerable amount of time , typically 100–150 ms ( Riggs et al . , 1981 ) .",
"External darkenings of such duration have been shown to cause a robust percept ( Riggs et al . , 1981 ) .",
"Strikingly , since blinks normally occur at least 1000 times an hour ( Cruz et al . , 2011 ) , about three or four percent of our waking hours are unknowingly spent with our eyes closed .",
"While spontaneous blinks are invisible , voluntary blinks are not .",
"However , their perception is also not veridical: they are experienced as shorter and less dim than physically comparable artificial darkenings ( Riggs et al . , 1981 ) .",
"Human psychophysical experiments have found evidence for a decrease in visual sensitivity during blinks even when the optical-retinal impact of blinks was neutralized ( Volkmann et al . , 1980 ) .",
"This effect could be mediated by an extra-retinal suppression of the neural response in early visual cortices during blinks , as indicated by feline V1 single unit recordings ( Buisseret and Maffei , 1983 ) and human fMRI ( Bristow et al . , 2005b ) .",
"Whereas the evidence for blink-related suppression of early visual activity may explain why visual sensation is reduced during blinks , it does not readily explain the perceived continuity of the visual scene across blinks .",
"Consider a reduction of retinal input driven by an external origin , such as when someone briefly turns off the lights .",
"Such a reduction will diminish low-level visual activity as well , yet , unlike blinks , this external reduction is clearly visible .",
"Hence , it seems that a neural basis for the special perceptual status of blinks requires a representation that is both unperturbed by blinks and yet still sensitive to perceived external darkenings .",
"Furthermore , if one assumes that perceived continuity relies on a read-out of an ongoing representation of the visual scene , this entails an active 'filling-in' of this visual representation during blinks but not during external darkenings ( Billock , 1997 ) .",
"Operationally , a filling-in mechanism would be reflected in continuous neural activity across blinks but not across gaps despite the decrease in retinal input common to both .",
"This hypothetical effect is in an opposite direction to the previously reported neuronal suppression .",
"Critically , testing these predictions using human neuroimaging requires sufficient spatiotemporal resolution to distinguish between activity changes that occur prior , during and following the blink event across different visual regions .",
"In particular , the sluggish BOLD-fMRI signal ( used in previous related studies , e . g . , Bristow et al . , 2005a ) can register only a temporal average of the total blink-related changes , potentially summing over antagonistic positive and negative components .",
"Here , we overcame this limitation by examining the effect of spontaneous and voluntary eye blinks and brief external image disappearances ( 'gaps' ) on visual representations in human patients undergoing intra-cranial electrocorticographic evaluation for intractable epilepsy .",
"This approach allowed us to test how these brief events interact with object-related human visual responses on a millisecond/millimeter-scale across multiple visual regions recorded simultaneously .",
"Following previous findings in paradigms unrelated to blinks ( Fisch et al . , 2009; Moutard et al . , 2015 ) , we hypothesized that the high-frequency broadband power envelope ( HFB ) response of the local field potential in human high-level ventral visual cortex would reflect the perceptual distinction between extrinsic disappearance of the stimuli and their disappearance due to spontaneous eye blinks .",
"Furthermore , we sought to directly test the intuitive yet untested conjecture that missing content due to blinks is actively filled-in by sustained neuronal activity , whereas perceived external stimulus disappearances leave 'dips' in neural responses .",
"In brief , we found a posterior-anterior gradient in blink versus gap representations .",
"In early visual cortex , these perceptually-distinct events elicited similar HFB responses , whereas in higher-level visual cortex , the termination of gaps elicited considerable overshoot beyond baseline levels , an effect absent in spontaneous blinks .",
"Intriguingly , both low and high-level cortical sites failed to exhibit a differential filling-in for blinks compared to gaps , suggesting that the perceived continuity of the visual scene might not depend on a continuous neural representation in category-selective visual areas but on the lack of representation of discontinuities , that is , stimulus disappearances and reappearances ."
],
[
"Whereas blinks may be correlated with neural responses also in non-visual regions ( e . g . , motor , somatosensory responses or even default mode network , see Nakano et al . , 2013 ) , in the present study we were concerned exclusively with the modulation of visual representations by blinks .",
"Therefore , we first tested which electrodes reliably responded to the presented stimuli ( e . g . faces ) themselves .",
"Since we were interested in local field potential correlates of average spiking rate , all of our analyses were conducted on the high-frequency broadband power envelope ( sampled between 70 and 150 Hz ) .",
"A number of previous studies have shown the HFB signal to be a good index of the aggregate firing rate ( Mukamel et al . , 2005; Ray et al . , 2008; Rasch et al . , 2008; Manning et al . , 2009; Nir et al . , 2007 ) .",
"Following standard preprocessing and HFB computation , we tested the onset response ( 50–350 ms , uncontaminated by either gaps or blinks ) to each electrode optimal object stimuli , compared with the inter-block gray blank baseline periods ( Figure 2 ) .",
"In agreement with previous reports ( Noy et al . , 2015a ) , responsivity of a considerable effect size was found almost entirely within the anatomically defined visual cortex , showing only minimal responses in more anterior cortical regions . 10 . 7554/eLife . 17243 . 006Figure 2 . High-frequency broadband ( HFB , 70–150 Hz ) visual responses to object images . HFB responses from all participants , sampled from 50 to 350 ms following the transition from one object image to another , compared with the HFB activity sampled during inter-block blanks are presented .",
"Each circle marks the location of one electrode on a common cortical template .",
"The response strength for each electrode's optimal ( maximally responding ) category is measured in standard deviations of the baseline ( Glass' Δ ) and is color-coded as the circles' face color saturation .",
"Electrodes showing significant face-selectivity are presented in a red hue and the others are presented in a cyan hue .",
"Electrodes that passed the inclusion criteria for the subsequent analyses ( corrected significance < 0 . 05 and an effect size of at least two standard deviations ) are encircled in black .",
"The colored labels on the cortical surface were derived from a surface-based atlas of retinotopic areas ( Wang et al . , 2014 ) and from Destrieux Atlas ( Destrieux et al . , 2010 ) as implemented in FreeSurfer 5 . 3 ( Fusiform gyrus , in red ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 00610 . 7554/eLife . 17243 . 007Figure 2—source data 1 . Individual electrode data for Figure 2 . Each row describes an electrode , with its SUMA standardized mesh nearest vertex ( index , hemisphere and MNI coordinates ) , its FWE-corrected p-value for stimuli HFB greater than baseline HFB , best responding category , the effect-size of the best responding category ( in baseline standard deviations ) , whether the electrode was selected for further analyses and face-selectivity ( uncorrected ) p-value . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 007 143 Electrodes out of a total of 1585 were found to show visual responses that were both significant ( Wilcoxon rank-sum test , p<0 . 05 , Bonferroni-corrected within-patient across electrodes ) and of a considerable effect size ( Glass' Δ ≥ 2 , see Materials and methods ) .",
"These electrodes were qualified for subsequent analyses .",
"We chose face-preference as a model for ventral category-selectivity , motivated by its prevalent occurrence in previous recordings ( Privman et al . , 2007 ) .",
"Thus , responsive electrodes were assessed for face-selectivity by comparing the responses to face-stimuli with each non-face category .",
"This analysis identified 19 electrodes ( found in 10 of the 14 patients , see Table 1 ) that responded significantly more strongly to faces than to any other category ( Wilcoxon rank sum test , p<0 . 05 for all contrasts , uncorrected ) , located mostly in high-level ventral visual cortex .",
"In order to isolate the effect of interruptions ( blinks and gaps ) from the stimulus-driven responses , the response to the stimulus itself had to be accounted for first .",
"We have approached this using time-domain deconvolution .",
"This is similar to the way signals are unmixed in the analysis of fast event-related fMRI ( Burock and Dale , 2000 ) .",
"This procedure was implemented as a multiple linear regression of the observed timecourse with a set of finite impulse response bases ( see Figure 3 and Materials and methods ) .",
"Its end result is estimates of the contribution of each experimental event to the observed HFB timecourse over time , after the contributions of other experimental events were accounted for .",
"See Figure 3—figure supplement 1 for a demonstration of the advantage of this approach over standard event-related averaging . 10 . 7554/eLife . 17243 . 008Figure 3 . A schematic illustration of the general linear model ( GLM ) design matrix used in the deconvolution of the neural responses . The observed time series in each electrode is modeled as a linear sum of overlapping responses triggered by the displayed stimuli and by the different interrupting events – gaps , voluntary blinks and spontaneous blinks ( for simplicity , only a single set of blinks predictors appears in the illustration ) .",
"Each response is composed of a sequence of non-overlapping unit pulses ( 4 ms-wide pulses were used for the actual 250 Hz HFB timecourse , here a less detailed , 10 Hz model is presented ) .",
"Note that in this example both the stimuli and the interruptions are modeled separately for face and non-face trials .",
"See Figure 3—figure supplement 1 for a demonstration of the advantage of this approach over standard-event related averaging . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 008Face photographs: Owen Lucas .",
"Available on Flickr under the Public Domain Mark 1 . 0 https://creativecommons . org/publicdomain/mark/1 . 0/ ) .",
"https://www . flickr . com/photos/144006675@N05/27487033282 .",
"https://www . flickr . com/photos/owen_lucas_photography/7454467582 .",
"Accessed on August 2016 . 10 . 7554/eLife . 17243 . 009Figure 3—figure supplement 1 . Bias in the estimation of gap-related responses due to unaccounted overlap of stimulus and gap responses and its correction by the deconvolution approach . All responses here are from a representative V3d electrode in patient P20 , sampled during both face and non-face trials .",
"( a ) Trials with early , middle and late gaps .",
"The red dashed rectangle marks the window used for gap-related event-related averaging .",
"The red solid line marks the moment of image disappearance ( gap-onset ) .",
"( b ) Trials without any gaps or blinks , used as a control .",
"The orange rectangle marks the window used for event-related averaging of 'pseudo-gaps' , null-events situated at similar latencies as the true gaps .",
"( c ) Gap-related response as estimated by standard event-related averaging .",
"Note the artifacts caused by unaccounted response-overlap , appearing at the beginning and end of the trace .",
"( d ) Pseudo-gap related response .",
"Since this trace is based on event-related averaging of trials that did not contain true gaps , it reflects only the overlap-related artifacts .",
"( e ) Deconvolved gap-related response .",
"Middle horizontal green line marks zero HFB contribution .",
"Note the lack of overlap-artifacts . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 009 Figure 4 presents examples of the gap-related , voluntary blink- and spontaneous blink-related responses in two pairs of cortical sites , each sampled within an individual patient .",
"In both patient P46 ( Figure 4a ) and patient P39 ( Figure 4b ) there were concurrent recordings of ventral early ( V1 and V2 , respectively , defined by a surface-based probabilistic atlas , Wang et al . , 2014 ) and ventral high-level ( Ventral Occipital and Fusiform gyrus ) visual sites .",
"In both cases , activity in early visual sites was strongly modulated by blinks , undergoing a dip in activation levels following eye closure and then overshooting beyond expected activation levels as the eyes re-opened and the visual image reappeared .",
"Gaps induced a qualitatively comparable effect – a negative dip in response to the onset of the display of the blank screen followed by a positive overshoot triggered by the reappearance of the stimulus .",
"By contrast , the two higher-level ventral electrodes showed considerable reappearance-related responses following the gaps , but largely , no such responses to blinks .",
"In P39 , the high-level electrode was face-selective; by estimating blink and gap-related responses separately for face and non-face trials , we found that the reappearance-related overshoot induced by gaps ( and to a lesser extent by voluntary blinks ) in this electrode was face-selective as well . 10 . 7554/eLife . 17243 . 010Figure 4 . Deconvolved high-frequency broadband responses to gaps , voluntary and spontaneous blinks , simultaneously sampled at the early visual cortex and at the ventral high-level cortex . Two pairs of electrodes from two patients are presented .",
"All responses here are locked to the stimulus reappearance .",
"Error bounds show the standard error of the regression coefficients .",
"Horizontal bars mark response timepoints significantly different from zero ( p<0 . 05 , FDR-corrected within-participant , see Materials and methods ) .",
"n is the number of event occurrences .",
"Note the activation-dip followed by a reappearance-related overshoot for gaps , voluntary blinks and spontaneous blinks in early visual sites in both participants .",
"By contrast , the two ventral high-level sites showed almost no disappearance-related dip for all three interruptions and a reappearance-related overshoot response only following gaps .",
"( a ) In participant P46 , events from all trials ( face and non-face alike ) are depicted in black .",
"( b ) In patient P39 , the high-level electrode showed greater responses to faces .",
"By estimating the contribution of gaps , voluntary and spontaneous blinks separately during face and non-face trials ( red and black traces , correspondingly ) , it is evident that the high-level reappearance-overshoot effect is dependent on the category of the reappearing stimulus . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 01010 . 7554/eLife . 17243 . 011Figure 4—figure supplement 1 . Deconvolved high-frequency broadband responses to gaps , voluntary and spontaneous blinks , simultaneously sampled at the early visual cortex and at the dorsal high-level cortex . Two pairs of electrodes from two patients are presented .",
"Responses in the upper panels ( a , b ) are locked to stimulus disappearance and responses in the lower panels ( c , d ) are locked to stimulus reappearance .",
"Error bounds show the standard error of the regression coefficients .",
"Horizontal black bars mark timepoints significantly different from zero ( p<0 . 05 , FDR-corrected within patient ) .",
"( a/c )",
"Patient P57 .",
"For visualization proposes , the brain image has been horizontally flipped .",
"Note the similar responses to gaps and to spontaneous blinks in the V1 site .",
"By contrast , the MST site responds exclusively to gap-related disappearance with a burst of HFB activation increase .",
"This patient was not instructed to produce voluntary blinks .",
"( b/d )",
"Patient P20 .",
"In this case , two adjacent electrodes over dorsal V3 and V3a showed a sharp transition between similar responses to gaps and blinks in V3 and a positive response to gap-related image disappearance and gap-related image reappearance in V3a ( seen as two positive bumps , the latter smeared due to jitter ) but not for blink-related disappearance or reappearance . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 011 Whereas most of our high-level visual electrodes in this study covered ventral regions , a similar yet distinct mode of transformation between early and late responses to blinks and gaps was evident in two cases with high-level dorsal coverage ( Figure 4—figure supplement 1 ) .",
"Patient P57 ( Figure 4—figure supplement 1a , c ) had concurrent recordings in V1 and in a site in a high-level dorsal stream region , over the middle temporal gyrus , slightly less than 1 cm anterior to the border of anatomically defined MST/TO2 .",
"The dorsal high-level electrode showed a positive HFB response triggered by the image disappearance ( Figure 4—figure supplement 1a , compare with 1c for image-reappearance-lock ) due to gaps and no response whatsoever for blinks .",
"By contrast , both gaps and spontaneous blinks were associated with a considerable dip in V1 activation ( that particular patient was not instructed to voluntarily blink ) .",
"In patient P20 ( Figure 4—figure supplement 1b , d ) , who had adjacent electrodes in anatomically defined dorsal V3 and V3a , a sharp short-range transformation was observed .",
"The V3 site showed a response pattern compatible with the early sites in the previous three patients , whereas the V3a site showed strong positive responses both to stimulus disappearance ( Figure 4—figure supplement 1b ) and to stimulus reappearance ( Figure 4—figure supplement 1d ) when these were caused by gaps but not when they were caused by voluntary or spontaneous blinks .",
"Moving from these four example electrode pairs to the entire sample of fourteen patients , we parceled the visually responsive electrodes into seven regions of interest ( ROIs ) , ensuring that each ROI contained electrodes from at least six different patients .",
"The resulting ROIs included five retinotopic regions , V1 , V2 , V3 , V4 and VO , defined according to the surface-based probabilistic atlas ( Wang et al . , 2014 ) , face-selective electrodes ( defined functionally , see above ) and high-level non-face selective electrodes defined as being situated outside and away of any of the retinotopic areas specified by the surface-based atlas ( Figure 5—figure supplement 1a , see Materials and methods for electrode localization and parcellation details ) .",
"Figure 5a depicts the grand-averages of the deconvolved stimulus reappearance-related responses for gaps , voluntary blinks and spontaneous blinks within each ROI , separated into face and non-face trials ( see Figure 5—figure supplement 1b for disappearance-locked grand-averages ) .",
"Responses followed a consistent hierarchical progression: in V1 and V2 , gaps , voluntary blinks and spontaneous blinks all produced qualitatively similar ( but not identical ) responses – which consisted of a dip in activation in response to the image disappearance , followed by an overshoot beyond baseline levels triggered by the image reappearance .",
"Progressing along the visual hierarchy , the response to gaps and blinks diverged: the response to the image reappearance was sustained only when triggered by the termination of an external gap , and not by the termination of a blink .",
"The negative activation dip reduced its amplitude along the hierarchy , even more so for gaps than to blinks .",
"This is in stark contrast with the expected filling-in during blinks but not gaps .",
"As an alternative analysis , we derived traditional event-related averages of the HFB signal of uninterrupted stimuli and subtracted them from each trial ( instead of the deconvolution procedure ) before computing gap and blink-related event-related-averages .",
"This procedure yielded very similar results ( Figure 5—figure supplement 1c ) , which rules out the possibility that the observed results were somehow an artifact of the deconvolution analysis we adopted .",
"Figure 5b depicts the response to the object images themselves , after accounting for the effects of gaps and blinks .",
"Note the considerable decline within several hundred milliseconds in response amplitude .",
"In order to examine the possible effect of this decline on the HFB-dips , gap-related HFB responses were grouped by the gaps' latency relative to trial onset ( Figure 5—figure supplement 2 ) .",
"The result of this analysis revealed similar dip magnitudes for gaps happening earlier and later in the trials in high-order regions . 10 . 7554/eLife . 17243 . 012Figure 5 . Grand averages of deconvolved high-frequency broadband responses .",
"( a ) Responses to gaps , voluntary blinks and spontaneous blinks along the visual hierarchy .",
"All of the traces here are locked to the image reappearance , marked as t = 0 .",
"See Figure 5—figure supplement 1b for the analogous stimulus-disappearance locked traces .",
"After within-electrode estimation , the traces were averaged first within individuals and then across individuals , such that each grand-average is derived from independent individual traces .",
"n is the total number of averaged electrodes for each trace .",
"Error bounds show the standard error of the mean across individuals .",
"Note the gradual appearance of differential responses to gaps compared with voluntary and spontaneous blink as the visual signal traveled forward .",
"( b ) Responses to object images ( face and non-faces ) .",
"Note that each stimulus was presented for one whole second . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 012Face photograph: Owen Lucas .",
"Available on Flickr under the Public Domain Mark 1 . 0 https://creativecommons . org/publicdomain/mark/1 . 0/ ) .",
"https://www . flickr . com/photos/144006675@N05/27487033282 .",
"Accessed on August 2016 . 10 . 7554/eLife . 17243 . 013Figure 5—figure supplement 1 . ROI parcellation and additional grand-averages .",
"( a ) Parcellation of visually responsive electrodes into ROIs .",
"( b ) Grand-averages of deconvolved disappearance-locked responses to gaps , voluntary and spontaneous blinks .",
"t = 0 is the moment in which the object image disappeared due to gap/blink onset .",
"Compare with Figure 5a which is reappearance-locked .",
"Note the lack of greater activation-dip for gaps compared with blinks in higher-level visual ROIs , which would have been expected if blinks ( but not gaps ) were filled-in by these regions .",
"( c ) Grand-averages of reappearance-locked responses to gaps , voluntary and spontaneous blinks , calculated by the subtraction of the uninterrupted response template followed by averaging , instead of deconvolution .",
"Note the similarity of the estimated traces to these in Figure 5a . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 01310 . 7554/eLife . 17243 . 014Figure 5—figure supplement 2 . Effect of gap latency . Grand averages of deconvolved high-frequency broadband responses to gaps , shown separately for early ( 350 ms ) , middle ( 550 ms ) and late gaps ( 750 ms ) .",
"All traces are locked to object image disappearance ( i . e . gap onset ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 014 In order to provide a more direct visualization of the data , we generated videos depicting the reappearance-locked responses to gaps ( Video 1 ) and spontaneous blinks ( Video 2 ) timepoint by timepoint , across all of the 143 electrodes .",
"Following the gaps' termination , a wave of activation that began in V1 and spread up to the anterior edge of the visual cortex , can be observed .",
"By contrast , the blinks' termination triggered a much more localized positive activation that remained confined to V1–V3 . 10 . 7554/eLife . 17243 . 015Video 1 . Response to gaps ( stimulus-reappearance lock ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 015Face photograph: Owen Lucas .",
"Available on Flickr under the Public Domain Mark 1 . 0 https://creativecommons . org/publicdomain/mark/1 . 0/ ) .",
"https://www . flickr . com/photos/144006675@N05/27487033282 .",
"Accessed on August 2016 .",
"10 . 7554/eLife . 17243 . 016Video 2 . Response to spontaneous blinks ( stimulus-reappearance lock ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 016Face photograph: Owen Lucas .",
"Available on Flickr under the Public Domain Mark 1 . 0 https://creativecommons . org/publicdomain/mark/1 . 0/ ) .",
"https://www . flickr . com/photos/144006675@N05/27487033282 .",
"Accessed on August 2016 .",
"In order to statistically test the reproducibility and generalizability of the observed response patterns , we quantified the two most recurring components of the gap and blink-related responses , the negative activation dip following image disappearance ( interruption onset ) and the positive overshoot following the image reappearance ( interruption offset ) , on an individual electrode basis .",
"Specifically , for each electrode and interrupting event ( gap/spontaneous blink/voluntary blink ) , we searched the reappearance-related response for clusters of contiguous above-baseline timepoints .",
"The cluster of largest activation integral that followed the reappearance was marked as the electrode's reappearance-related overshoot component .",
"Similarly , the contiguous below-baseline cluster in the disappearance-locked response with the largest ( negative ) activation integral was marked as the electrode's negative-dip component .",
"This procedure directly addressed the response-latency variability across electrodes , reflected in the travelling wave-like nature of the responses across the visual hierarchy ( see Videos 1 and 2 ) .",
"The effects within each estimated component were statistically tested by two complementary approaches: the first was multiple-comparisons-corrected permutation testing in each individual electrode , as commonly done in electrocorticographic ( ECoG ) studies .",
"The second was an ROI based mixed-effects group analysis directly assessing the generalizability across electrodes and patients' ROIs .",
"It should be emphasized that in both tests , the potential effect of selection bias ( commonly referred to as 'circular analysis' ) on these measurements was eliminated .",
"In the permutation tests , both the real ( unshuffled ) and permuted data were subject to the same level of selection bias .",
"The ROI-based mixed-effects group analysis was performed on unbiased response estimates derived by a split-half approach ( see Materials and methods ) .",
"In accordance with the pattern seen in the grand averages , significant electrode-level differences between the reappearance-related overshoot responses for gaps and spontaneous blinks emerged in higher-level visual cortex ( Figure 6 ) .",
"51 sites ( out of 143 ) showed a significant advantage for gaps over spontaneous blinks ( two-tailed permutation test , pFDR < 0 . 05 ) .",
"The effect was not uniformly distributed across the ROIs: V1 showed no significant electrodes out of 17 electrodes , V2 – 0/12 , V3 – 5/15 , V4 – 2/7 , VO – 4/8 , face-selective – 10/19 and high-level face non-selective ROI – 18/30 .",
"Randomization test of independence confirmed the apparent inhomogeneity of effect occurrence ( χ2 ( 6 ) = 17 . 219 , p=0 . 00001 ) .",
"Comparing voluntary blinks with gaps ( Figure 6—figure supplement 1 ) revealed a similar , but weaker pattern: 35 of 108 electrodes recorded in the 10 patients who performed voluntary blinks showed significant advantage of gaps over voluntary blinks ( two-tailed permutation test , pFDR < 0 . 05 ) , and the effect was more prevalent in higher-level ROIs: V1 – 1/13 , V2 – 1/11 , V3 – 2/11 , V4 – 1/6 , VO – 4/6 , face selective – 10/18 , face non-selective – 13/22 ( χ2 ( 6 ) = 13 . 137 , p=0 . 001 ) .",
"No electrodes showed significantly greater reappearance-related overshoot for voluntary blinks than for gaps . 10 . 7554/eLife . 17243 . 017Figure 6 . Electrode-level permutation testing of the reappearance-related response overshoot for gaps compared with the same measure for spontaneous blinks . See Figure 6—figure supplement 1 for a comparison of gaps with the voluntary blinks .",
"Each circle stands for one of 143 visually responsive electrodes polled across 14 participants , colored according to the FDR-adjusted permutation test p-value ( logarithmic color scale ) .",
"The partially filled bars on the right show the percentage of electrodes showing a significant gap overshoot > blink overshoot effect within-each region of interest .",
"None of the visually responsive electrodes exhibited a significant inverse effect ( blink overshoot>gap overshoot ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 01710 . 7554/eLife . 17243 . 018Figure 6—source data 1 . Individual electrode data for Figure 6 . Each row describes an electrode , with its SUMA standardized mesh nearest vertex ( index , hemisphere and MNI coordinates ) , its ROI ( if assigned ) , unbiased ( split-half based ) integral under the gap overshoot response , unbiased integral under the spontaneous blink overshoot response , FDR-corrected p-value for gaps greater than spontaneous blinks and FDR-corrected p-value for spontaneous blinks greater than gaps . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 01810 . 7554/eLife . 17243 . 019Figure 6—figure supplement 1 . Electrode-level permutation testing of the reappearance-related response overshoot for gaps compared with the same measure for voluntary blinks . See Figure 6 for the same measure compared between gaps with spontaneous blinks and for a full legend . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 01910 . 7554/eLife . 17243 . 020Figure 6—figure supplement 1—source data 1 . Individual electrode data for Figure 6—figure supplement 1 . Each row describes an electrode , with its SUMA standardized mesh nearest vertex ( index , hemisphere and MNI coordinates ) , its ROI ( if assigned ) , unbiased ( split-half based ) integral under the gap overshoot response , unbiased integral under the voluntary blink overshoot response , FDR-corrected p-value for gaps greater than voluntary blinks and FDR-corrected p-values for voluntary blinks greater than gaps . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 020 For the activation dip component , there was evidence of dips significantly greater for spontaneous blinks than for gaps in 26 of 143 electrodes ( two-tailed permutation test , pFDR < 0 . 05 ) , found mostly in early visual cortex – V1 – 6/17 , V2 – 7/12 , V3 – 2/15 , V4 – 3/7 , VO – 1/8 , face-selective 0/19 and high-level non-face selective 4/30 ( χ2 ( 6 ) = 16 . 498 , p=0 . 001 ) .",
"No electrode showed significantly greater dips for gaps than for spontaneous blinks , again a result inconsistent with the selective filling-in hypothesis .",
"Voluntary blinks showed higher occurrence of significantly greater dip for blinks than for gaps ( 51 of 108 electrodes ) , with no evidence for non-uniform distribution across ROIs: V1 – 9/13 , V2 – 8/11 , V3 – 7/11 , V4 – 3/6 , VO – 4/6 , face-selective – 7/18 , high-level face non-selective – 11/22 ( Randomization test of independence n . s . , χ2 ( 6 ) = 2 . 299 , p=0 . 53 ) .",
"No electrode showed evidence of greater dips for gaps than voluntary blinks .",
"In order to test the generalization of these results across electrodes and patients , we fitted the reappearance-related overshoot component ( and alternatively , the disappearance-related activation dip component ) with a three-way mixed-effects model with the fixed factors of trial-category ( face/non-face ) , interruption type ( gap/voluntary blink/spontaneous blink ) and ROI and the random factors of electrode and patient .",
"The results of this analysis can be interpreted similarly to the more commonly used repeated-measures ANOVA , however , since the mixed-effects approach naturally allows for missing values , it is well suited for data sampled with varying anatomical coverage .",
"The estimated responses are presented in Figure 7 .",
"We first describe the results for the reappearance-related overshoot ( Figure 7a–b ) .",
"The two ANOVA tests of primary interest are",
"( a ) the interaction of ROI with interruption , which was significant ( F ( 12 , 471 . 29 ) = 4 . 38 , p<0 . 001 ) , indicating that gaps , voluntary blinks and spontaneous blinks indeed affected different ROIs differently and",
"( b ) the three-way interaction between ROI , interruption and category , which was not significant ( F ( 12 , 463 . 31 ) = 0 . 59 , p=0 . 85 ) , potentially because category appeared to modulate interruption only in a single ROI ( face-selective electrodes ) .",
"The significant ROI and interruption interaction was further investigated by testing for simple effects between spontaneous blinks , voluntary blinks and gaps within each ROI .",
"In agreement with our previous analysis , in face-trials , gaps showed significantly larger reappearance-related overshoot responses compared with voluntary blinks both in VO , the face-selective and high-level face non-selective ROIs ( pFDR < 0 . 05 ) .",
"Compared with spontaneous blinks , gaps had significantly larger reappearance-related overshoot response also in V3 ( pFDR < 0 . 05 ) .",
"In non-face trials ( Figure 7b ) , gaps had a significantly larger overshoot response compared with voluntary blinks in the high-level face non-selective ROI .",
"Compared with spontaneous blinks , gaps had significantly larger reappearance-related overshoot ( in non-face trials ) also in V3 and VO . 10 . 7554/eLife . 17243 . 021Figure 7 . Mixed-effects response estimates . The bars depict estimated average magnitudes ( mixed-effects least squares means and their SEs ) of reappearance-related overshoots ( upper panels ) and disappearance-related activation-dips ( lower panels ) for gaps , voluntary blinks and spontaneous blinks , occurring during face trials ( left panels ) and during non-face trials ( right panels ) .",
"Asterisks mark significant within-ROI simple effects ( pFDR < 0 . 05 – * , pFDR < 0 . 01 – ** , pFDR < 0 . 001 – *** ) .",
"( a ) Average reappearance-related overshoot magnitudes ( face trials ) .",
"( b ) The same measurement for events occurring during non-face trials .",
"( c ) Disappearance-related activation-dips magnitudes ( deeper activation-dips are more negative ) during face-trials .",
"( d ) The same measurement for events occurring during non-face trials . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 02110 . 7554/eLife . 17243 . 022Figure 7—source data 1 . Mixed-effect model outputs used to create Figure 7 . This excel workbook includes parameter estimates for each experimental condition and ROI as well as within-ROI simple effects , both calculated by lsmeans R package .",
"Simple effects are accompanied with FDR-corrected p-values .",
"Data for overshoot ( Figure 7a , b ) and dip components ( Figure 7c , d ) are provided in different excel spreadsheets . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 02210 . 7554/eLife . 17243 . 023Figure 7—figure supplement 1 . Mixed-effects group analysis of gap and blink events matched for onset latency . Only events occurring during face-trials were used .",
"Asterisks mark significant FDR-corrected within-ROI simple effects ( p<0 . 05 – * , p<0 . 01 – ** , p<0 . 001 – *** , FDR-corrected ) .",
"( a ) Individual onset latency histograms of matched gaps and voluntary blinks .",
"( b ) The equivalent data for matched gaps and spontaneous blinks .",
"( c ) Average HFB reappearance-related overshoot magnitudes for the matched gaps and voluntary blinks ( mixed-effects least-square means and their standard errors ) .",
"Compare with Figure 7a – the same diminishing of the responses for voluntary blinks but not gaps is evident .",
"( d ) HFB reappearance-related overshoot magnitudes for the matched gaps and spontaneous blinks .",
"Compare with Figure 7b .",
"( e ) Disappearance-related activation-dip magnitudes for matched gaps and voluntary blinks .",
"Compare with Figure 7c .",
"( f ) Disappearance-related activation-dip for matched gaps and voluntary blinks .",
"Compare with Figure 7d . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 02310 . 7554/eLife . 17243 . 024Figure 7—figure supplement 2 . Mixed-effects group analysis of gap and blink events matched for duration . Asterisks mark significant FDR-corrected within-ROI simple effects ( p< . 05 – * , p< . 01 – ** , p< . 001 – *** , FDR-corrected ) .",
"( a ) Individual duration histograms of matched gaps and voluntary blinks .",
"( b ) The equivalent data for matched gaps and spontaneous blinks .",
"( c ) Average HFB reappearance-related overshoot magnitudes for the matched gaps and voluntary blinks ( mixed-effects least-squares means and their standard errors ) .",
"Compare with Figure 7a .",
"( d ) HFB reappearance-related overshoot magnitudes for the duration matched gaps and spontaneous blinks .",
"Compare with Figure 7B .",
"( e ) Disappearance-related activation-dip magnitudes for the duration matched gaps and voluntary blinks .",
"Compare with Figure 7c .",
"( f ) Disappearance-related activation-dip for the duration matched gaps and voluntary blinks .",
"Compare with Figure 7d . DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 02410 . 7554/eLife . 17243 . 025Figure 7—figure supplement 3 . Controls for gap low-level properties . The panels depict mixed-effects group analysis of responses to gap and spontaneous blink events , with control of the gap luminance ( black or gray , panels a-b ) or the gap temporal gradient ( gradual or sharp , panels c-d ) .",
"The values shown are mixed-effects least-square means and their standard errors .",
"Asterisks mark significant FDR-corrected within-ROI simple effects ( p<0 . 05 - * , p<0 . 01 - ** , p<0 . 001 - *** , FDR-corrected ) .",
"( a ) Reappearance-related overshoot magnitudes for gray gaps , black gaps and spontaneous blinks ( face-trials ) .",
"Note how the gray gaps caused less low-level impact than blinks yet they still triggered a significantly greater response than blinks in high-level visual areas .",
"( b ) Reappearance-related overshoot magnitudes for gray gaps , black gaps and spontaneous blinks ( non-face trials ) .",
"( c ) Reappearance-related overshoot magnitudes for gradual gaps ( fading-in and out ) , sharp gaps ( abruptly appearing ) and spontaneous blinks ( face-trials ) .",
"( d ) Reappearance-related overshoot magnitudes for gradual gaps ( fading-in and out ) , sharp gaps ( abruptly appearing ) and spontaneous blinks ( non-face trials ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17243 . 025Face photograph: Owen Lucas .",
"Available on Flickr under the Public Domain Mark 1 . 0 https://creativecommons . org/publicdomain/mark/1 . 0/ ) .",
"https://www . flickr . com/photos/144006675@N05/27487033282 .",
"Accessed on August 2016 .",
"Fitting the disappearance-related activation-dip component with the same mixed-effects model ( Figure 7c–d ) also found a significant interaction of ROI and interruption ( F ( 12 , 466 . 48 ) = 4 . 26 , p<0 . 001 ) .",
"As in the reappearance-related overshoot data , the three-way interaction of interruption , category and ROI was not significant ( F ( 12 , 463 . 10 ) = 0 . 45 , p=0 . 94 ) .",
"Inspecting the effects between the three kinds of interruptions within each ROI found that the activation dip triggered by gaps was smaller ( more shallow ) compared to that triggered by voluntary blinks in most ROIs ( see Figure 7c–d for detailed simple effects results , pFDR < 0 . 05 ) and it was also smaller than the dip triggered by spontaneous blinks in V1 and V2 ( both face and non-face trials , pFDR < 0 . 05 ) .",
"Importantly , no evidence was observed for greater ( deeper ) activation dip for gaps compared with either spontaneous or voluntary blinks in any ROI , and except for the high-level face non-selective ROI during non-face trials , the non-significant trend in all ROIs was inverse ( gaps had smaller dips than both kinds of blinks ) , once again , a result that is inconsistent with selective filling-in of blinks but not of gaps .",
"Repeating the mixed-effects analysis after normalizing each ROI by its total response to the object-images ( e . g . normalizing by the integrals of the traces depicted in Figure 5b ) yielded very similar results .",
"Note that both the electrode-level permutation testing and group mixed-effects analysis demonstrated a compatible pattern of effects .",
"Since the limitations involved with the clinical setting did not allow us to present the gaps in an exact physical replay of the individual patients' blink latencies and durations , differences in these properties could have confounded our results .",
"We tested that possibility by post-hoc selecting subsets of gaps and blinks such that their latency or duration histograms will tightly match ( Figure 7—figure supplements 1 , 2 , correspondingly ) .",
"The matching procedure was done independently for voluntary and spontaneous blinks , using face-trials .",
"Whereas this post-hoc selection led to considerably increased sampling error estimates ( due to the usage of only a fraction of the original trials ) , the observed pattern of results largely reproduced the results reported above and was qualitatively indistinguishable from that produced by the entire dataset , both for matching the events latencies ( Figure 7—figure supplement 1c–f ) and for matching the events durations ( Figure 7—figure supplement 2c–f ) .",
"Specifically , in all matched analyses , we still observed significant advantages of the reappearance-related overshoot for gaps compared with spontaneous and voluntary blinks in the higher-level ROIs ( face-selective and non-face selective high-level visual electrodes , pFDR < 0 . 05 ) .",
"The finding of greater ( deeper ) activation dip for blinks compared with gaps was even more pronounced than in the original ( unmatched ) analysis , with no ROI showing even a non-significant trend in the inverse direction .",
"To further control for potential low-level differences between the spontaneous blinks and gaps we included two additional experimental controls .",
"First , in nine patients , half of the gaps were implemented with a gray instead of a black blank .",
"This manipulation has the effect of decreasing both the amplitude of the image transients caused by the gap and their spatial extent; since the same gray-level was used as the screen background , only the central part of the screen was perturbed instead of its entirety .",
"If transients produced by gaps persisted in higher-level visual cortex due to greater low-level impact , this manipulation should reduce this effect .",
"However , using gray instead of black gaps produced qualitatively similar results ( Figure 7—figure supplement 3a–b ) , keeping the pattern of high-level advantage of gaps over blinks intact .",
"A second control , conducted in seven patients , consisted of displaying half of the gaps gradually , fading through three frames of 25% , 50% and 75% intermediate mixing levels ( manipulated orthogonally of the gray/black gap manipulation ) , each fade-in or fade-out lasting a total of 66 ms . Similarly to the gray-gaps manipulation , this lower-level manipulation of the gaps did not alter the results qualitatively ( Figure 7—figure supplement 3c–d ) .",
"To control for potential blink artifacts , the ECoG analysis pipeline ( HFB computation followed by deconvolution ) was applied also to the EOG channel .",
"This analysis found a positive blink-related artifact that peaked roughly at the same time as our eye-tracker derived blink onsets ( mean±SD 1 . 43 ms ± 23 . 8 ) and about 130 ms ( −134 ms ± 30 . 2 ) before the eye tracker derived blink offsets .",
"There were no negative HFB responses in the EOG channels , excluding an artifactual source for the ECoG blink-related HFB-dips ."
],
[
"By inspecting the high-frequency broadband responses to visual images interrupted by spontaneous blinks , voluntary blinks and blank frames ( gaps ) , we observed three main results:",
"( a ) early visual cortex ( V1 and V2 ) responded in a similar fashion to blinks and gaps: in both cases , HFB activity dipped following the stimulus disappearance and then increased beyond the uninterrupted activation levels , triggered by the stimulus' reappearance .",
"( b ) The activation overshoot triggered by blinks' termination gradually decreased along the cortical hierarchy , whereas the overshoot produced by gaps' termination was sustained , resulting in an increasing difference between the responses to gaps' and blinks' termination in higher-level visual cortex .",
"( c ) We found no supporting evidence for the filling-in hypothesis , which predicts a sustained activation during blinks but not during gaps; rather , our results showed an inverse trend in most of the ROIs .",
"Due to technical limitations imposed by the clinical setting , the gaps employed in the current study were only an approximate simulation of the retinal impact of eye blinks .",
"Some of the potential low-level differences between these two conditions concerned the events' durations , their latency in relation with the trials' onsets , their luminance levels , their visual field extent and the relative abruptness of their onsets and offsets .",
"Our series of controls argue against the possibility that these low-level differences could account for the observed effects – neither the direct matching of gaps and blinks by duration or latency nor the deliberate manipulation of the other stimulus aspects qualitatively altered the observed pattern of results ( see Figure 7—figure supplements 1–3 ) .",
"Nevertheless , even after considering these controls , the gaps and the blinks are still distinct physical events with retinal impacts that cannot be assumed to be precisely equivalent .",
"Can these potential retinal differences account for the differential response to gaps and blinks we observed in high-level visual cortex ?",
"We believe that the observed similarity between gap and blink-related responses in early visual cortex empirically argues against this possibility .",
"Put simply , the responses in early visual cortex can be viewed as an 'internal control' of the low-level effects of these two types of events- and these early responses were quite similar for gaps and blinks ( see Figure 5a , left column ) .",
"This interpretation is supported by a large body of data demonstrating the preferential sensitivity in early visual cortex to low-level aspects of the stimulus compared to higher order representations ( for a review , see Grill-Spector and Malach , 2004 ) .",
"It could be argued that the lack of differential HFB-dip for gaps compared with blinks in high-order regions may be due to the substantial decline in the sustained responses to the object images in these regions ( Figure 5b ) .",
"Such a decline could have resulted in a 'floor-effect' for those gaps and blinks that occurred later in the trial when the signal was already low .",
"However , early and late-onset gaps showed similar depression of activity following image disappearance ( Figure 5—figure supplement 2 ) .",
"Oculo-motor-related effects ( resulting from eye-muscle activity or eye and eyelid movements ) can represent another potential source of artifactual differences in ECoG signals between blinks and gaps .",
"In scalp EEG recordings , such effects are indeed a major concern .",
"However , previous research has demonstrated that in intracranial recordings these artifacts are limited to regions more anterior than the visual responsive sites that were used in our study ( Ball et al . , 2009 ) .",
"Moreover , even without relying on the electrodes' anatomical locations , the timing of our reported effects , following the blinks by tens to hundreds of milliseconds , unequivocally indicates their neural source ( see Results ) .",
"Lastly , the different number of gap and spontaneous blink events might have posed a statistical issue .",
"Importantly , blink and gap-related responses were tested against each other , a comparison that is not biased by SNR differences .",
"Furthermore , the pattern of results was unaltered even when the number of events was equated ( Figure 7—figure supplements1 , 2 ) ."
],
[
"Intracranial recordings were obtained from fourteen individuals ( four females , mean±SD age 34 ± 10 . 7 ) , monitored for pre-surgical evaluation due to pharmacologically intractable epilepsy ( see Table 1 for individual demographic , clinical and experimental details ) .",
"All patients gave fully informed consent , including consent to publish , according to NIH guidelines , as monitored by the institutional review board at the Feinstein Institute for Medical Research , in accordance with the Declaration of Helsinki .",
"Data was obtained as part of protocol number 07–125 .",
"Patients had the opportunity to consent prior to electrode implantation and were informed that they may choose to decline or later withdraw from the study without affecting their clinical care .",
"Consent includes agreement to participate with studies of cognitive and sensorimotor processes and publication of any deidentified data obtained .",
"Risks include tedium and potential breach of medical information and are minimized by giving ample breaks and implementation of protocols to deidentify data close to the time of recording .",
"Benefits to the subject include increased monitoring of the electrocorticogram and involvement of research methods to help localize electrodes with respect to preoperative MRI .",
"Five additional patients performed the experiment but their recordings were excluded after initial data quality inspection .",
"Reasons for exclusion were independent of the main analyses and consisted of: ( 1 ) widespread interictal spikes , ( 2 ) no visual coverage , ( 3 ) severe visual field impairments ( 4 ) contamination of the ECoG signal by synchronization triggers and ( 5 ) a case in which the patient misunderstood the instructions to voluntary blink and executed prolonged eye closures ( mean duration of 642 ms ) instead .",
"The subjects were seated in bed in front of an LCD monitor , approximately 70 cm from the screen .",
"Blocks of grayscale still images of faces , houses , tools , abstract patterns or animals , embedded in a uniform gray background , were presented at a constant pace of 1 Hz , with no blanks between consecutive stimuli .",
"The images subtended a visual angle of 15 . 8° .",
"Stimuli were grouped in blocks of ten consecutive images of mixed categories , separated by between-block intervals of blank gray screen ( three seconds long ) .",
"A total of 32 blocks per subject were considered for this study ( additional 12 blocks included sudden spatial displacements of the stimuli and were not included in the analysis ) .",
"In 10 of the 14 patients ( see Table 1 ) , twelve of the blocks were preceded by an auditory cue to blink at a steady pace of one blink per second until a stop signal that followed the block's end .",
"Blinks occurring during these blocks were categorized as 'voluntary' and blinks occurring outside these blocks were classified as 'spontaneous' .",
"In the rest of the blocks , gaps in the visual stimulation were pseudo-randomly introduced in 60% of the trials at 350 , 550 or 750 ms post stimulus transition .",
"We applied a wide range of gap durations ( 100–200 ms in most patients , see Figure 1—figure supplement 1 ) in order to allow for post-hoc matching of blinks and gaps .",
"The subjects' main task was to click the mouse button when an image of an animal appeared .",
"These trials were later excluded from the analyses .",
"Each patient participated in a single session that was divided by three short breaks in which the patients were required to rest with their eyes closed .",
"Including the breaks , an entire session typically lasted about 15 min .",
"As the experiment proceeded , additional controls for the gap event were introduced: In nine of the subjects ( starting at P36 ) , half of the gaps were gray ( intensity level 183/255 , where the object stimuli mean intensity level was 160/255 ) instead of black ( 0/255 intensity level ) and in seven of these patients ( starting at P46 ) , half of the gaps were faded in and out through four frames ( 16 . 6 ms each ) of 25% , 50% and 75% and 100% mixing levels between the image and the blank screen ( manipulated orthogonally of the gray/black gap manipulation ) .",
"The task was implemented using Presentation ( Neurobehavioral Systems ) .",
"Recordings were conducted at Northshore University Hospital , Manhasset , NY , USA .",
"The patients were implanted with subdural grids , strips , and/or depth electrode shafts ( Ad-Tech Medical Instrument , Racine , Wisconsin ) .",
"In the subdural grids and strips , each recording site was 2 mm in diameter with 1 cm separation , whereas in the depth electrodes each recording site was 1 mm in diameter with 5 mm separation .",
"A subgaleal electrode at the vertex was used as a reference and the acquired signals were filtered between 0 . 07 Hz and 200 Hz ( half-power boundaries ) and sampled at a rate of 500 Hz by an XLTEK EMU128FS system ( Natus Medical , Pleasanton , California ) or filtered between 0 . 1 Hz and 256 Hz and sampled at a rate of 512 Hz by a BRAINBOX EEG-1166 system ( Braintronics , Almere , Netherlands ) .",
"Stimulus-triggered electrical pulses were recorded along with the electrophysiological data to allow precise timing of neural responses in relation with the stimuli and the video eye-tracking data .",
"Prior to electrode implantation , patients underwent a T1 weighted 1 mm isometric anatomical MRI scan on a 3-tesla Signa HDx scanner ( GE Healthcare , Chicago , Illinois ) .",
"Following the implant , a thin-slice computed tomography ( CT ) and a T1-weighted anatomical MRI scan on a 1 . 5-tesla Signa Excite scanner ( GE Healthcare ) were acquired in order to aid electrode localization .",
"The pre-implant and post-implant MRI scans were rigidly co-registered using FSL's Flirt ( Jenkinson and Smith , 2001; Jenkinson et al . , 2002; RRID:SCR_002823 ) .",
"A similar co-registration was performed on the post-implant MRI and post-implant CT scans .",
"Concatenating these two co-registrations allowed visualizing the post-implant CT scan on top of the pre-operative MR scan while minimizing localization error due to potential brain shift caused by surgery and implantation .",
"Individual contacts were then identified by an expert inspection of the thresholded CT along with the post-op MR and were marked in each subject's pre-operative MRI native space , aided by BioImage Suite ( Papademetris et al . , 2006; RRID:SCR_002986 ) .",
"A 3d model of each patient's cortical surface was segmented and reconstructed from the pre-implant MRI using FreeSurfer 5 . 3 ( Dale et al . , 1999; RRID:SCR_001847 ) .",
"Each electrode contact was snapped to the nearest vertex on the cortical surface ( e . g . Dykstra et al . , 2012 ) .",
"Contacts that were farther away than 10 mm from the surface were excluded from further analyses .",
"In order to allow for integration of observations across subjects , the three-dimensional mesh of each individual cortical surface was standardized by resampling its unfolded spherical form using SUMA ( Argall et al . , 2006; RRID:SCR_005927 ) .",
"This resulted with a cortex-based alignment of each patient to a common template , allowing the visualization of electrodes from different individuals on a single cortical surface while adhering to the electrodes' location in relation with individual gyri and sulci .",
"All preprocessing was done using in-house made Matlab ( The MathWorks , Inc . ) scripts , except for filtering for which EEGLAB's two-way least-squares FIR filtering ( Delorme and Makeig , 2004; RRID:SCR_007292 ) was employed .",
"Each channel was downsampled to 500 Hz ( if required ) , re-referenced by the common average across all intracranial channels within the individual patient ( following an established practice , see Liu et al . , 2015 ) and then notch-filtered to remove 60 Hz line noise .",
"Next , channels were manually inspected for interictal spikes , remaining electromagnetic interferences , eye or eyelid movement artifacts and other noticeable signal contaminations .",
"An additional channel exclusion criterion was the physical position of the electrodes over lesions or ictogenic zones identified by a certificated neurologist .",
"The common average reference was then recomputed using only non-excluded channels and only these channels were considered for further analysis .",
"Following this initial preprocessing , high-frequency broadband ( HFB ) response between 70 and 150 Hz was estimated .",
"Eight 10 Hz wide band pass filters were used to split the signal into narrow frequency bands .",
"The momentary amplitude of each band was then estimated by the absolute value of its Hilbert transform .",
"Each of the resulting timecourses was then divided by its mean across time .",
"Next , the eight bands were recombined into a single timecourse by averaging .",
"This procedure is aimed at sampling the relevant frequency range with equal weighting , overcoming the 1/f amplitude spectrum of the LFP signal ( Fisch et al . , 2009; Ossandón et al . , 2011 ) .",
"HFB signals were then automatically scanned for widespread outliers by the following procedure: the discrete-time derivative of each channel's HFB was z-normalized and its absolute value was taken .",
"Any timepoint in which the across-channels median of the absolute values z-scores exceeded the value of two was marked for rejection , as well as timepoints distanced up to 200 ms away from it .",
"In the subsequent analyses , any trials including rejected timepoints were excluded .",
"Finally , the HFB time series of each electrode was downsampled to 250 Hz and normalized again by dividing it by the mean activation during the inter-block gray screen intervals , transforming the time series' units into percent signal change with the inter-block intervals as a 0% change baseline .",
"The mean HFB activation during 50 ms to 350 ms following the beginning of each trial ( marked by the transition from one stimulus exemplar to another ) was compared with the mean HFB activation measured during the inter-block baseline periods , in which a gray screen was presented .",
"Only trials that contained no blinks ( or gaps , by design ) during that interval or in the preceding 100 ms were considered for this analysis ( see subsection on blink detection below ) .",
"Whereas statistical testing against baseline was done using all of the object images , effect-size estimation was based on the optimal ( maximally responding ) category of each electrode .",
"This response was subtracted by the mean baseline HFB and then divided by the standard deviation of baseline periods , yielding a Glass' Δ effect-size estimate .",
"This estimate was used in the visualization in Figure 2 and in the effect size criterion for electrode inclusion .",
"In order to enable averaging across electrodes and patients , the visually responsive electrodes were parceled into seven ROIs ( V1 , V2 , V3 , V4 , VO , face-selective and high-level non-face selective electrodes , see Figure 5—figure supplement 1 and Table 1 ) based on functional and anatomical criteria .",
"First , electrodes functionally qualifying as 'face-selective' ( see Results ) were assigned to the 'face-selective' ROI .",
"For each of the remaining electrodes , we compared its location on the SUMA standardized template with a surface-based probabilistic topographic atlas , based on a sample of fMRI retinotopic mappings ( Wang et al . , 2014 ) .",
"Each electrode was associated with the topographic map of the maximal probability value over the electrode's snapped vertex , but only if that probability value was at least 25% .",
"When this condition was not met , we searched for qualifying vertices in a 5 mm radius from the snapped vertex .",
"Electrode locations still unlabeled by this procedure were then tested against the V1 , V2 ( Fischl et al . , 2008 ) and MT+ ( Malikovic et al . , 2007 ) labels derived from post-mortem histology mappings , as provided by FreeSurfer 5 . 3 ( RRID:SCR_001847 ) .",
"Since these labels are cytoarchitectonically defined , they extend further than the effective visual field of the functional retinotopic mapping .",
"Whereas the atlas by Wang and colleagues ( 2014 ) describes 25 topographic maps , we required that each potential ROI to be sampled by at least six patients , in order to enable reliable generalization .",
"This resulted with five retinotopic ROIs: V1 , V2 , V3 , V4 and VO ( Ventral-Occipital ) .",
"Dorsal and ventral subdivisions of V1 , V2 and V3 were combined .",
"35 visually responsive electrodes that were associated with maps which were not sufficiently sampled across patients were not assigned to an ROI , hence they were not included in the grand averages and the group mixed-effects analysis , but they are included in the within-electrode analysis ( Figure 5 and Figure 5—figure supplement 1 ) .",
"Finally , the remaining electrodes that were more than 5 mm farther from any of the retinotopic maps or histological labels were grouped together as the 'high-level non-face selective' ROI .",
"All of these electrodes were located anterior to the retinotopic atlas ( Figure 5—figure supplement 1 ) .",
"In order to isolate the effect of blinks ( and gaps ) on the neural response , the contribution of the stimuli displayed in the background ( e . g . face images ) has to be accounted for .",
"A simple approach is to form a template of the response to uninterrupted stimuli and subtract it from the timecourse prior to the segmentation and averaging of blink ( or gap ) related responses .",
"We opted for a more statistically efficient method that utilizes the entire timecourse without selection ( but see Figure 5—figure supplement 1c ) .",
"As often done in the analysis of fast-event related fMRI ( Burock and Dale , 2000 ) and recently introduced to the analysis of EEG ( Dandekar et al . , 2012 ) , the timecourse was modeled as a sum of finite impulse response ( FIR ) basis functions , expressing the contributions of events of different experimental conditions over pre-specified ranges of time lags .",
"By estimating a corresponding linear multiple regression model , the contribution of each experimental condition at each time lag was estimated while taking into account the contributions of all other events .",
"The structure of the regression design matrix is illustrated in Figure 3 .",
"The onsets of new face stimuli ( i . e . the beginning of face trials ) and the onsets of new non-face stimuli ( excluding animals ) were modeled by two sets of 375 FIR predictors spanning from 0 to 1500 ms post stimulus onset .",
"The onsets of spontaneous blinks , that is , the image disappearances due to spontaneous blinks , were also modeled separately for face and non-face trials: Image disappearances due to spontaneous blinks occurring during were modeled using a set of 187 FIR predictors , spanning from –250 to 500 ms post blink onset , with separate predictors for blinks occurring at face trials and at non-face trials .",
"Each unit pulse within an FIR predictor spanned exactly one timepoint ( 4 ms ) .",
"Gaps and voluntary blinks were modeled by four additional predictor sets ( again , modeling separately events that happened during face and non-face trials ) of the same parameters .",
"In principle , blink and gap offsets ( i . e . the reappearance of the stimuli ) could have been modeled in parallel with the onsets within the same model .",
"However , we found that such a model suffers from issues related with collinearity .",
"Therefore , the effects of image reappearance were tested by fitting an alternative model in which blink and gap offsets were used instead of onsets .",
"A model incorporating both onsets and offsets simultaneously was used only for the plotting of the stimulus-related responses ( Figure 5b ) .",
"Timepoints during animal trials , timepoints rejected due to signal outliers or eye tracking problems and timepoints during the rest breaks were all excluded from this analysis by the inclusion of a dummy nuisance predictor for each excluded timepoint .",
"For conciseness and ease of visualization , the non-selective single electrode responses ( Figure 4a and Figure 4—figure supplement 1 ) and the group gap-blink response map ( Figure 6 ) were derived from a simpler regression model built without the distinction between face and non-face stimuli .",
"Given an HFB timecourse and a design matrix , the ordinary least square estimator was used to derive regression coefficients: β^= ( XTX ) −1XTy , where β is the p-long vector of the estimated regression coefficients , X is the design matrix ( n × p ) and y is the observed timecourse .",
"p stands for the number of predictors and n stands for the number of timepoints in the observed time course .",
"The standard errors of these regression coefficients were estimated by an HC3 heteroskedasticity-consistent standard error estimator ( Davidson and Mackinnon , 1993 ) : HC3= ( XTX ) −1XTdiag[ ei2 ( 1−hii ) 2 ]X ( XTX ) −1 , where HC3 is the p-long vector of estimated regression coefficients' standard errors , X is the design matrix , diag[ v ] is a diagonal matrix with vector v on its main diagonal ( as in matlab 'diag' function ) , ei is the residual at timepoint i and hii is the 'leverage value' of timepoint i , defined as the i-th entry of the main diagonal of the 'hat' matrix H . H is the matrix that transforms an observed timecourse into a predicted timecourse , defined as H=X ( XTX ) −1XT .",
"The motivation behind using this more involved standard error estimator is that unlike the homoscedastic standard error estimator commonly applied in fMRI GLM analyses , a heteroskedasticity-consistent estimator does not yield error bars of a uniform height across time .",
"Instead , the estimated standard-error reflects the variability of the particular samples that contributed to each timepoint in the deconvolved trace ( see Hayes and Cai , 2007 for introduction and review ) .",
"For the testing of single-electrode individual timepoints ( Figure 4 and Figure 4—figure supplement 1 ) , the estimated regression coefficients were divided by their standard errors and compared to a standard normal distribution ( a large-sample approximation ) .",
"The resulting p-values were then FDR-corrected in time with a q-value equal to 0 . 05 divided by the number of visual electrodes within the patient .",
"In each electrode and for each interruption type ( gap , spontaneous blinks and voluntary blinks ) we quantified the two following components:",
"( a ) reappearance-related overshoot , which was measured from the offset ( reappearance ) -locked deconvolved HFB response and",
"( b ) activation-dip , which was measured from the onset ( disappearance ) -locked deconvolved HFB response .",
"For the reappearance-related overshoot , we measured the integrals ( area under curve ) below each contiguous cluster of above-zero timepoints following the reappearance and picked the largest one .",
"Similarly , for the activation-dip , we picked the maximal integral above clusters of contiguous below-zero timepoints that followed the stimulus disappearance .",
"The units of both integrals were percent signal change times second .",
"For the group mixed-effects analysis , the interruptions were randomly split into two halves , each giving rise to a distinct set of predictors .",
"This resulted with two independent deconvolved traces for each interruption type .",
"One trace was used to determine the clusters' temporal extents while the other was used to measure the activation integrals during these intervals .",
"These measurements were averaged across 30 random splits of the data .",
"This procedure guaranteed the response estimates to be non-circular ( i . e . free from selection-bias ) , while preserving the flexibility of tailoring each cortical site with its optimal time windows .",
"In order to test the significance of the observed differences between gap-related and blink-related responses at each recording site , we estimated the empirical null-distribution of the difference between them using a random permutation test .",
"Differences in activation-dips and reappearance-related overshoots were tested separately .",
"In each simulation iteration , we shuffled the labels of the single gap-events with the single blink-events ( either voluntary or spontaneous blinks , the two blink types were contrasted with gaps in separate tests ) .",
"This shuffling was restricted such that the counts of gaps and blinks occurring within face trials and non-face trials were kept constant .",
"Then , the deconvolved response traces were re-estimated using the shuffled labels and the difference in activation-dip or reappearance-related overshoot components between gaps and blinks was recorded .",
"The observed differences derived from the original , unshuffled events were assigned with a p-value equal to p=b+1m+1 where b is the number of random permutations with a difference at least as big as the observed difference and m is the number of total simulations ( Phipson and Smyth , 2010 ) , which was set to 2000 .",
"The resulting p-values were corrected for 2-tailed testing and then were FDR-corrected across all visual responsive electrodes .",
"In order to test for non-uniformity of the proportions of significant results across ROIs , we used a randomization test of independence ( McDonald , 2009 ) .",
"This was implemented by measuring the following test statistic: χ2=∑i=17 ( Oi−Ei ) 2Ei where Oi is the observed number of significant electrodes in the i-th ROI and Ei is the expected number of significant electrodes that ROI , assuming uniformity across ROIs ( i . e . the number of electrodes in that ROI multiplied by the overall proportion of significant electrodes across all ROIs ) .",
"The observed statistic was compared with an empirical null-distribution generated by randomly assigning significant electrodes across ROIs while keeping the total number of electrodes within each ROI and the total number of significant electrodes across ROIs fixed .",
"Mixed-effects group analysis was implemented using the LME4 package ( Bates et al . , 2014 ) of the R language ( R Core Team , 2013 ) .",
"The magnitude of event-related responses ( activation-dip or reappearance-related overshoot , each component tested independently ) was entered as the dependent variable .",
"The independent variables were interruption type ( gap , voluntary blink or spontaneous blink ) , stimulus category ( face or non-face ) and ROI ( V1 , V2 , V3 , V4 , VO , Face-selective or high-level non face-selective ) .",
"A random intercept model was formulated as response ~ interruption*category*ROI+ ( 1|patient/electrode ) .",
"Whereas including random slopes when applicable is generally recommended ( Barr et al . , 2013 , but see Bates et al . , 2015 ) , these could not be included for our dataset since they led to over-parameterization ( model undefinability ) .",
"The analyses of duration and latency matched events ( Figure 7—figure supplements 1 , 2 ) used only face-trials; hence their model included only two independent variables , interruption ( gap/blink ) and ROI .",
"This model was fitted separately for each matching procedure ( matching of event onset latency and matching of event duration ) .",
"Main effects and interactions were tested using Type III ANOVA with Kenward-Roger approximation for degrees of freedom implemented by the afex R package ( Singmann et al . , 2015 ) .",
"Simple effects were tested within each ROI using lsmeans R package ( Lenth and Hervé , 2015 ) and were FDR-corrected for multiple comparisons .",
"Since initial inspection of the data found greater variability in conditions with greater observed values , we log-transformed all values prior to model fitting in order to better conform to the model's homoscedasticity assumption .",
"This transformation did not qualitatively change the subsequent results .",
"Prior to the log-transform , the data were uniformly shifted in order to avoid negative values and the sign of the activation-dip magnitudes was inverted .",
"For visualization purposes ( in Figure 7 , Figure 7—figure supplements 1–3 ) , the estimated coefficients and the locations of their standard error estimates were transformed back to the original scale by the corresponding inverse transforms .",
"Patients' blinks were monitored by a video eye tracker ( ET ) operated monocular at 500 Hz ( EyeLink 1000 , SR research , Ontario , Canada ) and by a single EOG electrode placed above one of the patient's eyebrows ( referenced to the ECoG common-average ) .",
"In order to register the occurrence of a blink , the concurrent presence of EOG and pupil-size ( measured by the ET ) blink-related artifacts was required .",
"Trials including ambiguous events or missing eye tracking data were excluded from later analysis .",
"In general , both measures picked up reliable blink-related artifacts and were in high agreement .",
"In two of the patients , in which no video-tracking was available ( P20 and P25 ) , blinks were detected exclusively by the EOG channel .",
"As the eyelid gradually covers the pupil , the pupil size estimate of the ET rapidly diminishes , producing a distinctive artifact .",
"Blink onsets ( blink-related image disappearances ) were registered at the peak acceleration of the measured pupil-size decrease ( corresponding with the eyelid passing the pupil's midline ) .",
"In cases in which tracking was lost before the occurrence of such an acceleration peak , blink onset was marked at the last sample prior to signal loss .",
"Respectively , blink offset ( blink-related image reappearance ) was marked by the peak acceleration of the pupil-size increase measured following the reacquisition of the pupil signal .",
"For the two patients where no eye tracking was available ( P20 and P25 ) , electrooculogram ( EOG ) -based blink timing was used .",
"The EOG signal was filtered ( FIR bandpass , 0 . 2–40 Hz ) .",
"Remaining slow noise fluctuations were removed by fitting the EOG trace around each blink ( ±750 ms ) with a sum of a fifth degree polynomial baseline and a trapezoid ( modeling the blink artifact ) with height , onset , sustain and decay times as free parameters .",
"The estimated polynomial baseline was then removed from the trace .",
"Next , the baseline-removed trace was thresholded: the sample that first passed the threshold marked the blink onset and the first sample that crossed back below it marked the blink offset .",
"The individual threshold level for each of the two patients was determined empirically from the other 12 patients that did have concurrent EOG and eye tracking: for each such patient , the median amplitude of the EOG blink-artifact produced by spontaneous blinks was estimated .",
"We scanned the range between 0 and 100% and found the threshold of 22% to best predict the eye-tracker based blink duration from the EOG artifact .",
"Repeating the analyses reported in the results section with EOG-based blink detection and timing for the entire group of 14 patients yielded highly comparable results to those described in the results section above .",
"The onset matching procedure of blinks with gaps was performed separately for spontaneous and voluntary blinks .",
"The procedure was done within-patient and used only gaps and blinks that occurred during face-trials .",
"Matching was done iteratively: In each matching iteration , one gap and one blink were paired .",
"In order to be eligible to pairing , the two events had to have a latency difference of no more than 50 ms . The decision criterion among multiple possible pairings was the minimization of the difference between the average latency of the paired gaps subset and the average latency of the paired blinks subset .",
"This was repeated until no eligible pairings were available .",
"This procedure resulted with a subset of gaps and a subset of blinks of an equal number of events and of highly similar latency histograms and means ( Figure 7—figure supplement 1a–b ) .",
"The data from patients whose events could not be matched ( no eligible pairings ) were excluded from the matched analysis ( P50 for both voluntary and spontaneous blinks , P25 and P32 for spontaneous blinks ) .",
"Alternatively , gaps and blinks were matched for duration in the same manner as they were matched for onset latency .",
"The pairing criterion was no more than 15 ms difference in duration and the choice among multiple pairings was by minimizing the difference between the average duration of the paired gaps subset and the average duration of the paired blinks subset .",
"As in the latency matching , this resulted in equally-sized events subsets with highly similar histograms and means ( Figure 7—figure supplement 2a–b ) .",
"Patient P50's events could not be matched using this procedure for both voluntary and spontaneous blinks .",
"Patient P44's spontaneous blinks ( but not voluntary blinks ) were also excluded .",
"All FDR corrections applied in the study used 'FDR p-value adjustment' described by Yekutieli and Benjamini ( 1999 , eq . 3 ) , implemented by Winkler ( 2011 ) , ensuring that individual p-values control false discover rate as defined by Benjamini and Hochberg ( 1995 ) ."
]
] | [
"We hardly notice our eye blinks , yet an externally generated retinal interruption of a similar duration is perceptually salient .",
"We examined the neural correlates of this perceptual distinction using intracranially measured ECoG signals from the human visual cortex in 14 patients .",
"In early visual areas ( V1 and V2 ) , the disappearance of the stimulus due to either invisible blinks or salient blank video frames ( 'gaps' ) led to a similar drop in activity level , followed by a positive overshoot beyond baseline , triggered by stimulus reappearance .",
"Ascending the visual hierarchy , the reappearance-related overshoot gradually subsided for blinks but not for gaps .",
"By contrast , the disappearance-related drop did not follow the perceptual distinction – it was actually slightly more pronounced for blinks than for gaps .",
"These findings suggest that blinks' limited visibility compared with gaps is correlated with suppression of blink-related visual activity transients , rather than with \"filling-in\" of the occluded content during blinks ."
] | [
"The average person blinks once every few seconds , each time shutting off their view of the world for about a tenth of a second .",
"Nevertheless , we rarely notice a blink .",
"By contrast , we readily notice a single blank frame in a movie , even if the frame lasts far less than a blink .",
"The fact that we do not usually notice our spontaneous blinks is a striking example of the discrepancy between the images we perceive versus the information that enters our eyes .",
"This dissociation between the information that the eyes receive and what we perceive raises a number of questions .",
"First , which brain areas represent the actual information from the eyes , and at what point do brain areas start to represent our subjective perception instead ?",
"Second , how does the brain \"stabilize\" our perception of vision despite the frequent interruptions that occur whenever we blink ?",
"In short , does the brain \"fill in\" the missing images or “edit out” the gaps ?",
"To answer these questions , Golan et al . turned to human patients who were undergoing a surgical procedure related to the treatment of epilepsy .",
"In the course of such procedures , and strictly for diagnosis purposes , electrodes are temporarily placed directly on the surface of the brain – the cortex – making it possible to monitor the activity of individual cortical areas .",
"Towards the back of the brain , where cortical processing of visual signals begins , neurons responded in a way that was consistent with the physical information the eye actually received rather than the perception of vision .",
"Thus , neurons showed the same responses to easily seen blank frames in a movie as to unnoticeable blinks .",
"However , as the signals streamed forward to down-stream brain regions involved in vision , neurons in successive areas were increasingly likely to distinguish between the perceptually visible blank frames versus the invisible blinks .",
"Unexpectedly , Golan et al . found no evidence that the brain fills in the missing picture during blinks .",
"Instead , it seems that the brain generates a continuous perception by actively \"deleting\" the brief neural signals that are turned on when our visual input has been shut off .",
"The brain only does this for blinks but not for artificial interruptions – such as blank movie frames – which explains why we notice the latter but not the former .",
"A future challenge will be to isolate the pathway that leads from the brain regions that generate blinks to the regions that deal with vision , and that enables us to tell blinks from blanks ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"medicine",
"neuroscience"
] | Stimulus-induced gamma rhythms are weaker in human elderly with mild cognitive impairment and Alzheimer’s disease | elife-61666-v2 | [
[
"Alzheimer’s disease ( AD ) is a predominant cause of dementia ( decline in cognitive abilities ) of old age and substantially contributes to the socioeconomic burden in the geriatric population , necessitating early diagnosis .",
"Advances in our understanding of cellular pathology of AD in rodent models and its link to gamma rhythms in brain have spurred interest to investigate diagnostic and therapeutic potential of gamma rhythms in AD and other forms of dementia ( Mably and Colgin , 2018; Palop and Mucke , 2016 ) .",
"Gamma rhythms are narrow-band oscillations in brain’s electrical activity with center frequency occupying ~30–80 Hz frequency range ( Gray et al . , 1989 ) .",
"These are suggested to be generated from excitatory-inhibitory interactions of pyramidal cell-interneuron networks ( Buzsáki and Wang , 2012 ) involving parvalbumin ( PV ) and somatostatin ( SOM ) interneurons ( Cardin et al . , 2009; Sohal et al . , 2009; Veit et al . , 2017 ) .",
"These have been proposed to be involved in certain cognitive functions like feature binding ( Gray et al . , 1989 ) , attention ( Chalk et al . , 2010; Fries et al . , 2001; Gregoriou et al . , 2009 ) , and working memory ( Pesaran et al . , 2002 ) .",
"Some studies have reported abnormalities in gamma linked to interneuron dysfunction in AD .",
"For example , Verret et al . , 2012 reported PV interneuron dysfunction in parietal cortex of AD patients and hAPP mice ( models of AD ) .",
"They found aberrant gamma activity in parietal cortex in such mice .",
"Further , some recent studies have suggested therapeutic benefit of entraining brain oscillations in gamma range in rodent models of AD .",
"For example , Iaccarino et al . , 2016 suggested that visual stimulation using light flickering at 40 Hz entrained neural activity at 40 Hz and correlated with decrease in Aβ amyloid load in visual cortices of 5XFAD , APP/PS1 mice models of AD .",
"Based on such reports in rodents in both visual and auditory modalities , some investigators have suggested a paradigm termed GENUS ( gamma-entrainment of neural activity using sensory stimuli ) and have claimed to show neuro-protective effects in rodent models of AD ( Adaikkan et al . , 2019; Martorell et al . , 2019 ) .",
"Recent studies in human EEG ( Murty et al . , 2020; Murty et al . , 2018 ) and MEG ( Pantazis et al . , 2018 ) have reported existence of two gamma rhythms ( slow: ~20–34 Hz and fast: ~36–66 Hz ) in visual cortex , elicited by Cartesian gratings .",
"Age-related decline in power and/or frequency of these stimulus-induced gamma rhythms has been shown in cognitively healthy subjects ( Gaetz et al . , 2012; Murty et al . , 2020 ) .",
"However , abnormalities in such stimulus-induced visual narrow-band gamma rhythms in human patients of mild cognitive impairment ( MCI , a preclinical stage of dementia [Albert et al . , 2011; Petersen et al . , 1999; Sosa et al . , 2012] ) or AD have not been demonstrated till date .",
"We addressed this question in the present double-blind case-control EEG study involving a large cohort of elderly subjects ( N = 244; 106 females , all aged >49 years ) recruited from urban communities in Bangalore , India .",
"These were classified as healthy ( N = 227; see Murty et al . , 2020 ) , or suffering from MCI ( N = 12 ) or AD ( N = 5 ) based on clinical history and Clinical Dementia Rating ( CDR; Hughes et al . , 1982; Morris , 1993 ) scores .",
"We studied narrow-band gamma rhythms induced by full-screen Cartesian gratings in all these subjects .",
"We also examined steady-state visually evoked potentials ( SSVEPs ) at 32 Hz in a subset of these subjects ( seven MCI and two AD subjects ) .",
"We monitored eyes using an infrared eye tracker to rule out differences in gamma power due to potential differences in eye position or microsaccade rate ( Yuval-Greenberg et al . , 2008 ) ."
],
[
"First , we examined how the two gamma rhythms differed in MCI subjects as compared to their healthy age- and gender-matched controls .",
"We averaged spectral data for all analyzable bipolar electrodes ( as described in Murty et al . , 2020 ) from nine occipital and parieto-occipital pairs ( marked in black enclosures in Figure 2D; see EEG data analysis subsection in 'Materials and methods' ) for each subject .",
"We compared the change in power spectral densities ( PSDs ) for each MCI with the mean change in power of their corresponding age- and gender-matched controls .",
"Figure 2A shows the median stimulus-induced change in PSDs for 12 MCIs ( yellow ) and their controls ( dark orange; light shaded regions show ± SD of median after bootstrapping for 10 , 000 iterations ) .",
"While both slow and fast gamma ‘bumps’ were conspicuously visible for MCI as well as control groups , power in both slow gamma ( 20–34 Hz ) and fast gamma ( 36–66 Hz ) ranges ( but not alpha , 8–12 Hz range ) was significantly lower in the MCI group compared to the control group ( Kruskal-Wallis [K-W] test , significance as shown in Figure 2A ) .",
"This could also be seen in the median time-frequency change in power spectrograms ( baseline: −500–0 ms of stimulus onset ) for cases and controls in Figure 2B .",
"Change in band-limited power was significantly less for both gamma bands in the MCI group compared to the control group ( Figure 2C; slow gamma: χ2 ( 23 ) = 4 . 09 , p=0 . 043; fast gamma: K-W test , χ2 ( 23 ) = 5 . 61 , p=0 . 018 ) .",
"However , alpha power was not significantly different ( χ2 ( 23 ) = 0 . 33 , p=0 . 56 ) .",
"Results were similar when we combined both gamma bands to a single band ( 20–66 Hz; χ2 ( 23 ) = 5 . 08 , p=0 . 024 ) or used the ‘traditional’ gamma band ( 30–80 Hz; χ2 ( 23 ) = 5 . 34 , p=0 . 021 ) .",
"Figure 2—figure supplement 1 shows the time-frequency change in power spectra of individual MCI subjects and the mean of their controls , sorted by increasing gamma power in the MCI subjects .",
"Although there was substantial variability across subjects ( also observed in Figure 2C ) , only 3/12 MCIs showed higher slow gamma power and only 2/12 MCIs showed higher fast gamma than controls .",
"We note that we have used very stringent conditions for computation of gamma power , similar to our previous work ( Murty et al . , 2020; Murty et al . , 2018 ) .",
"For example , for all subjects , we used the same set of electrodes over which gamma was computed , as well as same time and frequency ranges .",
"Further , we computed the total power within a band by simply summing the absolute power values within the band separately in baseline and stimulus periods and then taking a ratio .",
"This estimate has larger contribution from lower frequencies in the band because of the power-law distribution of PSDs in baseline/stimulus periods .",
"Consequently , if the traces are overlapping at lower frequencies within the band and diverge at higher frequencies , which was the case in the slow gamma range , the total change in power in the band may not be significantly different .",
"Therefore , these results could be further improved by customizing the low frequency limit of the gamma band for each subject , as well as choosing only electrodes that show stronger gamma .",
"For example , taking slow and fast gamma ranges as 26–34 Hz and 44–56 Hz improved the p-values ( slow gamma: χ2 ( 23 ) = 7 . 69 , p=0 . 005; fast gamma: χ2 ( 23 ) = 8 . 34 , p=0 . 004 ) .",
"Although we have refrained from such customization here because we wanted to study the efficacy of a simple and subject-independent computational procedure , such data-driven subject specific optimization holds promise for improving the efficacy of a gamma-based biomarker .",
"Figure 2D shows the median scalp maps ( see EEG data analysis subsection in 'Materials and methods' ) of change in band-limited power across 112 bipolar electrode pairs ( shown as discs ) for alpha , slow , and fast gamma bands .",
"Stimulus-induced change in power across all three bands was most prominent in the nine electrode pairs as above .",
"However , this change in power was less in the MCI group compared to the control group in slow and fast gamma bands ( but not alpha band ) as noted in Figure 2C .",
"For two MCI subjects ( M1 and M4 as shown in Figure 2—figure supplement 1 ) , slow gamma power was less than 0 dB .",
"This could be because visual stimuli typically suppress power at low frequencies ( <30 Hz ) , and if the visual stimulus does not induce sufficiently strong slow-gamma rhythm , there is an overall reduction in power between 20 and 35 Hz .",
"However , our results remained consistent even when these two MCI subjects were removed from analysis ( χ2 ( 19 ) = 4 . 81 , p=0 . 028 , for slow gamma between 26 and 34 Hz ) .",
"Similarly , our results did not change when we removed one MCI subject ( subject M5 ) who had negative fast gamma power ( χ2 ( 21 ) = 4 . 56 , p=0 . 032 ) .",
"Our study had many control subjects for each case subject ( range 4–19 ) .",
"To rule out the possibility that our results were influenced by this imbalance , we first ordered the control subjects based on the difference in experiment date from the case subject and then chose only the first N control subjects .",
"We tried different values of N and found that our results remained consistent in all cases , with less slow and fast gamma power in cases vs controls .",
"For example , Figure 2—figure supplement 2 shows the results for N = 1 ( a single control for each case ) .",
"Although the error bars shown in Figure 2C appear smaller for controls than cases , that is only because each data point for the control group is already an average across many subjects .",
"When a single control subject was used per case ( Figure 2—figure supplement 2 ) , the error bars were comparable ( standard error of the medians: 0 . 33 , 0 . 20 , and 0 . 34 for controls vs 0 . 28 , 0 . 08 , and 0 . 36 for cases , for slow gamma , fast gamma , and alpha , respectively ) .",
"Similarly , if we pooled all the control subjects in one group without averaging ( N = 74 controls vs 12 MCI subjects ) , the standard deviations of slow gamma , fast gamma , and alpha power were 0 . 94 , 0 . 72 , and 0 . 67 for controls and 0 . 79 , 0 . 36 , and 1 . 00 for cases .",
"Therefore , the variability in power was comparable in the two groups .",
"Figure 2—figure supplement 3 shows the results for individual spatial frequencies as well as after pooling all three spatial frequencies .",
"MCI subjects had less slow and fast gamma than controls at all spatial frequencies , although the effect was stronger at spatial frequencies of 2 and 4 cpd .",
"We also tested whether the event-related potentials ( ERPs ) varied across MCIs and healthy controls .",
"Consistent with literature , we noticed three prominent peaks in the ERPs for these subjects: P1 , N1 , and P2 ( Figure 2—figure supplement 4 ) .",
"The ERPs were not different between MCIs and controls ( Figure 2—figure supplement 4 , panel A ) .",
"Specifically , we did not find any significant difference between P1/N1/P2 peak amplitude ( Figure 2—figure supplement 4 , panel B ) .",
"These analyses indicate that the differences that we observed for MCIs and controls were limited only to slow and fast gamma power .",
"Figure 3 shows the same results for the five AD subjects in our cohort .",
"We interpret these results with caution , since the number of subjects is small ( although the study would have benefitted from a larger sample size for both MCI and AD categories , it was not possible to increase the sample size due to the COVID-19 pandemic ) .",
"Nonetheless , we observed a strong reduction in the slow and fast gamma bands ( Figure 3A and B ) , which was significant for both slow gamma ( χ2 ( 9 ) = 4 . 81 p=0 . 028 ) and fast gamma ( χ2 ( 9 ) = 3 . 94 , p=0 . 047 ) , but not alpha ( χ2 ( 9 ) = 0 . 01 , p=0 . 92 ) .",
"Data from individual AD subjects and their controls are shown in Figure 3—figure supplement 1 .",
"All five AD subjects had less slow gamma power , and only one AD subject ( A5 ) had more fast gamma power relative to the controls .",
"Further , similar to MCI subjects , AD subjects had less slow and fast gamma than controls at all spatial frequencies , although the effect was stronger at spatial frequencies of 2 and 4 cpd ( Figure 3—figure supplement 2 ) .",
"Because four out of five AD subjects had only mild AD ( CDR = 1 ) , we also tested the results after combining the MCI and AD data sets ( Figure 3—figure supplement 3 ) .",
"While slow gamma ( χ2 ( 33 ) = 9 . 51 , p=0 . 002 ) and fast gamma ( χ2 ( 33 ) = 8 . 88 , p=0 . 003 ) power were strongly reduced in cases vs controls , there was no difference in alpha power ( χ2 ( 33 ) = 0 . 25 , p=0 . 61 ) or frequencies higher than ~65 Hz .",
"We further tested the dependence of power in gamma/alpha bands on CDR score using a linear regression model ( see Regression analysis section in 'Materials and methods' ) while accounting for age and gender .",
"In the matched condition in which data from all the age- and gender-matched control subjects for each case were averaged ( yielding 17 cases and 17 controls ) , the coefficient for CDR was significantly negative for both gamma bands ( βCDR = –0 . 57/–0 . 25 , p=0 . 0017/0 . 0063 for slow/fast gamma ) .",
"Results were similar for the unmatched condition , in which all the controls were considered separately ( 112 healthy , 12 MCI , and 6 AD subjects ) , albeit the CDR slope ( βCDR ) was significantly negative only for slow gamma ( βCDR = –0 . 62/–0 . 29 , p=0 . 0071/0 . 0702 for slow/fast gamma ) .",
"On the other hand , similar to the results in previous analyses , alpha power did not depend on CDR ( βCDR = –0 . 30/–0 . 30 , p=0 . 14/0 . 21 for matched/unmatched conditions ) .",
"We have previously shown that gamma power decreases with age in healthy elderly , and females have more gamma than males ( Murty et al . , 2020 ) .",
"Consistent with these results , we found that coefficient for age was significantly negative ( βAGE = –0 . 018/–0 . 0094 , p=0 . 014/0 . 06 for slow/fast gamma ) , while coefficient for gender was significantly positive ( βGENDER = 0 . 33/0 . 35 , p=0 . 0070/3 . 15 × 10−5 for slow/fast gamma ) when the regression analysis was performed on the full set of healthy subjects ( N = 227 ) .",
"However , when the regression analysis was performed with cases and controls as described above , the coefficients were not significant ( matched: βAGE = 0 . 0042/4 . 13 × 10−5 , p=0 . 73/0 . 99 and βGENDER = –0 . 036/0 . 11 , p=0 . 87/0 . 36 for slow/fast gamma; unmatched: βAGE = –0 . 0071/–0 . 0009 , p=0 . 51/0 . 91 and βGENDER = 0 . 17/0 . 19 , p=0 . 30/0 . 10 for slow/fast gamma ) .",
"This also suggests that CDR is a stronger predictor of gamma power than age or gender .",
"These results suggest that the alternate hypothesis ( gamma power in controls was greater than cases ) was more likely than the null hypothesis ( controls and cases had comparable gamma power ) .",
"We quantified this by estimating the Bayes factor ( BF ) , which is the ratio of the marginal likelihood of the alternate hypothesis and the null hypotheses , given the data that we observed ( see 'Materials and methods for details ) . For the MCI group ( N = 12 ) , BF computed using single-tailed paired t-test was ~1 . 89 for both slow and fast gamma . However , as before , choosing a more ‘sensitive’ range for slow gamma ( 26–34 Hz ) and fast gamma ( 44–56 Hz ) improved the BF to 3 . 34 and 4 . 60 , respectively , suggesting substantial evidence for the alternate hypothesis over the null hypothesis . For the AD group ( N = 5 ) , BF was 2 . 85 for slow gamma and 1 . 83 for fast gamma , suggesting weak evidence , which did not improve substantially when more sensitive ranges were used . However , when both MCI and AD groups were combined , BF increased to 13 . 70 for slow gamma ( 26–34 Hz ) and 8 . 60 for fast gamma ( 44–56 Hz ) , further strengthening the evidence in favor of alternate hypothesis . On the other hand , evidence for alpha band power was in favor of the null hypothesis ( BF was 0 . 088 when only MCIs were considered , 0 . 29 for ADs , and 0 . 077 when both MCI and AD subjects were considered ) . Previous studies have correlated increases in gamma power with occurrence of small involuntary eye movements called microsaccades ( Yuval-Greenberg et al . , 2008 ) . These have been described in previous literature using plots called ‘main sequence , ’ which show peak velocity on ordinate and maximum velocity on abscissa on a log-log scale . These plots reveal the ballistic nature of microsaccades , that is , the initial velocity and maximum displacement of the eye in the visual field are correlated during microsaccadic movements ( Engbert , 2006 ) . We compared eye data between MCI/AD subjects and their respective controls and found comparable eye positions and microsaccade profiles ( Figure 4A ) , similar main sequence ( Figure 4B ) , and similar pupillary reactivity ( Figure 4C ) to stimulus presentation ( measured as coefficient of variation of pupil diameter across time; see Murty et al . , 2020 ) . Further , the trends described in Figures 2 and 3 did not change qualitatively when we reanalyzed the data after removing stimulus repeats containing microsaccades ( see 'Materials and methods' ) from analysis ( Figure 4—figure supplement 1 ) , although these did not reach significance due to lesser number of trials ( ~45% of original analysis ) and fewer subjects compared to the original analysis ( see figure legend for details ) .",
"Similarly , the trends held true when we reanalyzed only those repeats that had at least one microsaccade ( Figure 4—figure supplement 2 ) .",
"These results indicate that the trends described in Figure 2 are independent of the presence or absence of microsaccades .",
"We also tested if the trends described in Figures 2 and 3 were seen for absolute band-limited power in the baseline condition .",
"The cases ( MCIs or ADs ) and their respective controls had comparable PSDs ( Figure 5 ) and slopes of PSDs ( Figure 5—figure supplement 1 ) in the baseline condition .",
"Further , baseline power in alpha , slow gamma , and fast gamma frequency ranges did not differ significantly between cases and controls ( K-W test; for MCIs: χ2 ( 23 ) = 0 . 48/0/0 , p=0 . 49/0 . 95/1 for alpha/slow/fast gamma , respectively; for AD: χ2 ( 9 ) = 0 . 27/0 . 53/0 . 27 , p=0 . 60/0 . 46/0 . 60 for alpha/slow/fast gamma , respectively ) .",
"Thus , we concluded that the trends described in Figures 2 and 3 were specific to stimulus-induced change in slow/fast gamma power and did not depend on baseline absolute power or slopes of PSDs .",
"We next tested whether power of SSVEPs in gamma range also decreased in the MCI group as compared to the control group .",
"We tested for SSVEPs at 32 Hz by presenting full-screen gratings that phase-reversed at 16 Hz ( as described in Figure 1B ) .",
"Ten of the 12 MCIs participated in this study , out of which data of only seven could be analyzed ( data from three MCIs were discarded due to noise , see 'Materials and methods' for details ) .",
"Figure 6A and B show median change in power spectral density plots ( in 250–750 ms window of stimulus onset ) and change in power time-frequency spectrograms ( from a baseline period of −500–0 ms of stimulus onset ) for these seven MCIs and their respective age- and gender-matched controls ( as done for analyses in Figure 2A and B ) .",
"Figure 6C and D show bar plots and scalp maps for 112 bipolar electrodes for change in SSVEP power at 32 Hz ( during 250–750 ms of stimulus onset from a baseline of −500–0 ms , same as in Figure 2C and D ) for control and MCI groups , respectively .",
"We did not observe any reduction in SSVEP power at 32 Hz in the MCI group as compared to the control group ( K-W Test , significance at each frequency of the change in power spectra is shown in Figure 6A; significance at 32 Hz: χ2 ( 13 ) = 0 . 04 , p=0 . 85 ) .",
"For comparison , we reanalyzed data for slow and fast gamma power for the Gamma experiment ( as in Figure 2 ) with the same set of seven MCI subjects and their respective controls .",
"MCIs had less slow and fast gamma power compared to controls as seen in Figure 6—figure supplement 1 ( same format as Figures 2 and 3 ) , although these trends did not reach significance due to small sample size ( K-W test , χ2 ( 13 ) = 0 . 49/2 . 56 , p=0 . 48/0 . 11 for slow/fast gamma , respectively ) .",
"As before , choosing the more sensitive slow-gamma frequency range between 26 and 34 Hz yielded a significant difference between the two groups ( K-W test , χ2 ( 13 ) = 3 . 93 , p=0 . 047 ) .",
"Alpha followed a similar insignificant trend as in Figure 2 ( χ2 ( 13 ) = 0 , p=0 . 949 ) .",
"Further , adding the two AD subjects yielded significant differences in both slow and fast gamma power ( K-W test , slow gamma between 26 and 34 Hz in Gamma experiment: χ2 ( 17 ) = 4 . 31 , p=0 . 038; fast gamma: χ2 ( 17 ) = 4 . 69 , p=0 . 03 ) , but not for SSVEP power ( for change in power at 32 Hz in SSVEP experiment: χ2 ( 17 ) = 0 . 05 , p=0 . 82 ) .",
"Trends for SSVEPs were not different when we performed time-frequency analysis on trial-averaged time-amplitude waveforms for each subject ( Figure 6—figure supplement 2A–C ) , instead for averaging time-frequency spectra of individual repeats for each subject , as done above for Gamma and SSVEP analyses .",
"Further , stimulus onset-related responses ( 0–250 ms ) were comparable between MCIs and their respective controls ( Figure 6—figure supplement 2D ) .",
"The RMS amplitude , peak amplitude , and time of the peak of the stimulus-onset response also did not differ among MCI subjects and their respective controls ( Figure 6—figure supplement 2D–E ) .",
"To conclude , change in SSVEP power at 32 Hz for cases was comparable to that of their controls , like alpha but unlike slow and fast gamma oscillations ."
],
[
"Our sample is a representative of urban population in India as we adopted community-based sampling instead of hospital-based sampling .",
"Importantly , out of the 257 subjects that we collected data from ( 247 used for analysis plus 10 subjects whose data was noisy and thus rejected , see 'Materials and methods' ) , there were 13 MCIs ( ~5% ) and 6 AD subjects ( 2 . 3% ) .",
"These figures match closely to the previously reported prevalence of MCI and AD in India ( Kalaria et al . , 2008; Mathuranath et al . , 2012; Sosa et al . , 2012 ) .",
"Within the recruited sample , most cases ( ~70% ) had MCI , a condition that is conceptualized as intermediate stage between normal aging and AD .",
"Criteria used to diagnose MCI are not strong and hence there is a need for a valid biomarker .",
"Our study highlights the potential use of gamma oscillations in EEG in that direction .",
"Moreover , we had limited our analyses to sensor ( electrode ) level instead of reconstructing the neural sources and performing analyses at that level .",
"This has allowed us to present a screening technique that is easy to replicate in a clinical setting .",
"Furthermore , as our metrics were derived directly from neural activity , these could serve as a more objective assessment of the clinical status of the individual .",
"Future studies should examine if different other stimulus paradigms like annular gratings ( Muthukumaraswamy and Singh , 2013 ) and drifting gratings ( Orekhova et al . , 2015 ) could add to the robust evidence that is presented in this study .",
"Gamma rhythms are generated by excitatory-inhibitory interactions in the brain ( Buzsáki and Wang , 2012 ) .",
"These interactions could be influenced by many structural factors ( Buzsáki et al . , 2013 ) that could get abnormal in AD ( such as cortical thinning and atrophy; see Dickerson et al . , 2009; Serrano-Pozo et al . , 2011 ) .",
"However , how such structural derailments influence gamma recorded over scalp is unknown .",
"A few studies in MEG had reported significant positive correlations between gamma frequency and cortical thickness as well as volume of cuneus ( Gaetz et al . , 2012 ) and thickness of pericalcarine area ( Muthukumaraswamy et al . , 2010 ) .",
"However , such results could not be replicated in later studies ( Cousijn et al . , 2014 ) and have been shown to be confounded by age ( Robson et al . , 2015 ) .",
"Age is as a common factor that influences both macroscopic structure ( Lemaitre et al . , 2012; Salat et al . , 2004; van Pelt et al . , 2018 ) as well as gamma power and frequency ( Murty et al . , 2020 ) .",
"Our main aim in this study was to examine the potential of gamma as a biomarker .",
"However , as structural changes in AD brain are more evident and drastic compared to cognitively healthy aging ( Dickerson et al . , 2009; Serrano-Pozo et al . , 2011 ) , future studies should examine correlations of gamma power/frequency and macroscopic structure such as cortical thickness in healthy/MCI/AD subjects while controlling for age .",
"Moreover , as gamma rhythms have been correlated with many higher level cognitive functions such as attention , working memory , etc . ( see Introduction ) , attempts must be made to extend and validate these findings in clinical populations , such as AD .",
"Some investigators have suggested neuroprotective effects of entraining neural oscillations using flickering light/sound at 40 Hz ( analogous to our SSVEP paradigm ) in rodent models of AD ( Adaikkan et al . , 2019; Martorell et al . , 2019; Thomson , 2018 ) .",
"Although we did not find any significant trend for SSVEP at 32 Hz in cases compared to controls ( unlike our observations with narrow-band gamma ) , we cannot directly compare the results from abovementioned studies with our study for several reasons .",
"First , the frequency of entrainment was different ( 40 Hz vs 32 Hz ) .",
"Second , the model organism was different ( human vs rodent ) .",
"Finally , we did not measure any cognitive or pathological outcome of the visual stimulation .",
"Indeed , while the previous studies have focused on therapeutic aspect of flicking stimulation , we only studied its potential for diagnosis .",
"It is possible that entrainment of neural oscillations to visual stimulation in gamma frequency range gets deranged only in advanced stages of AD ( as in the rodent models of Iaccarino et al . , 2016 ) .",
"It may thus have therapeutic benefit but may not reflect as abnormal on testing early on ( as in our case ) .",
"Further , as discussed in the Methods , SSVEP study was always done at the end of the experiment in our study and the total number of stimulus repeats were much less than the gamma study .",
"Nonetheless , the differences in trends for gratings and SSVEP evoked gamma presented in this study suggest that these two phenomena might be sub-served by different , yet unknown mechanisms .",
"These differences have to be borne in mind while designing screening/therapeutic tools for MCI and AD .",
"Stimulus-induced change in visual narrow-band gamma power has the potential to be a simple , low-cost , easy to replicate , and objective biomarker for screening of MCI and AD .",
"How this gamma-based biomarker compares against other methods used in diagnosis ( MRI , PET , cognitive tests , etc . ) , whether addition of this biomarker to other standard methods improves the overall diagnosis , and the specificity of this biomarker for AD compared to other causes of dementia remain open questions that will require further research ."
],
[
"We recruited 257 elderly subjects ( 109 females ) aged 50–92 years from the Tata Longitudinal Study of Aging ( TLSA ) cohort from urban communities in Bangalore between July 2016 and July 2019 .",
"They were clinically diagnosed by psychiatrists ( authors BN/AML ) and/or a neurologist ( author MJ ) as cognitively healthy ( N = 236 ) or suffering from MCI ( N = 15 ) or AD ( N = 6 ) through clinical history and a semi-structured clinical interview ( Clinical Dementia Rating; see Table 1 ) .",
"Five out of the six AD subjects were directly referred to the study by the neurologist .",
"Diagnosis of all MCI/AD subjects was further reviewed by a panel of four experts for consensus ( see Appendix; criteria used by the panel are given in Tables 1–3 in Supplementary file 1 ) , who reclassified two MCI subjects as healthy .",
"Data from these two subjects was not used for further analysis .",
"Subjects went through the experiments only once .",
"However , there were a few subjects who had undergone the experiments more than once ( annually as part of a different longitudinal study ) .",
"For such subjects , we used only data from the first year .",
"However , in the first year of study , four subjects did not have eye data and data of one participant was noisy .",
"Further , one participant was diagnosed as MCI in the second year .",
"Hence , for these six subjects , data from the first year was discarded and data from the second year was used instead .",
"From the rest , we discarded data of ten subjects due to noise ( nine healthy and one MCI; see Artifact Rejection subsection ) .",
"Finally , we discarded one AD patient ( male , aged 92 years ) as he did not have any healthy age- and gender-matched control .",
"We were thus left with 227/12/5 ( females: 101/3/2 ) healthy/MCI/AD subjects for analysis .",
"For the purpose of this study , we called the MCI/AD subjects as cases and their respective age- and gender-matched healthy subjects as controls .",
"Since the subjects were directly recruited from the community based on advertisements without any prior knowledge of their clinical status ( which was determined during the study itself and revealed to the experimenters after the EEG recordings were over ) , no explicit power calculation was done .",
"All subjects reported normal or corrected-to-normal vision and were instructed to wear spectacles if prescribed earlier .",
"They participated in the study voluntarily and were monetarily compensated for their time and effort .",
"We obtained informed consent from all subjects before the experiment .",
"The Institute Human Ethics Committees of Indian Institute of Science , NIMHANS , and MS Ramaiah Hospital , Bangalore approved all procedures .",
"This article is in compliance with the STROBE statement ( von Elm et al . , 2007 ) .",
"Experimental setup , EEG recordings , and analysis were same as what we had described in our previous study ( Murty et al . , 2020 ) .",
"Briefly , we recorded raw EEG signals from 64 active electrodes using BrainAmp DC ( Brain Products GmbH , Germany ) according to the international 10–10 system , referenced online at FCz .",
"We filtered raw signals online between 0 . 016 Hz and 1000 Hz and sampled at 2500 Hz .",
"We rejected electrodes whose impedance was more than 25 KΩ ( 4 . 0% , 3 . 4% , and 0 . 6% for healthy , MCI , and AD subjects , respectively ) .",
"Impedance of the final set of electrodes was 5 . 5 ± 4 . 2 , 5 . 9 ± 4 . 4 , and 3 . 8 ± 3 . 5 KΩ for healthy , MCI , and AD subjects , respectively .",
"All subjects sat in a dark room in front of an LCD screen with their head supported by a chin rest .",
"The screen ( BenQ XL2411 , resolution 1280 × 720 pixels , refresh rate 100 Hz ) was gamma-corrected and placed at a mean ± SD distance of 58 . 1 ± 0 . 8 cm from the subjects ( 53 . 8–61 . 0 cm ) according to their convenience ( thus subtending a width of at least 52° and height of at least 30° of visual field for full-screen gratings ) .",
"We calibrated the stimuli to the viewing distance in all cases .",
"Subjects performed a visual fixation task , as described in Figure",
"1 . They performed the main ‘Gamma’ experiment in 2–3 blocks ( total 543 blocks across 257 subjects ) according to their comfort .",
"We also tested 32 Hz SSVEPs on these subjects in the SSVEP experiment .",
"We chose 32 Hz SSVEP ( induced by gratings of temporal frequency of 16 cycles per second or cps ) for two reasons .",
"First , in a separate set of experiments , we had recorded spikes and local field potentials ( LFP ) in the primary visual cortex of awake monkeys while presenting counterphasing gratings at varying temporal frequencies and found that the SSVEP gain was highest for gratings with temporal frequencies of 12-16 cps ( Salelkar and Ray , 2020 ) .",
"Second , 32 Hz was between slow and fast gamma bands , hence within the available time for the experiment , we were able to record SSVEP activity closest to both the gamma rhythms .",
"Subjects completed both experiments during a single session .",
"We considered only those subjects for analysis in SSVEP experiment who had analyzable data for the Gamma experiment ( see Artifact Rejection section below ) .",
"This gave us a total of 222/11/5 subjects ( 99/3/2 females ) for healthy/MCI/AD categories for the SSVEP experiment .",
"We recorded eye signals ( pupil position and diameter data ) using EyeLink 1000 ( SR Research Ltd ) for all subjects ( except for one subject each in healthy/MCI/AD categories ) .",
"Eye data for Gamma experiment is shown in Figure 4 .",
"We rejected stimulus repeats with fixation breaks ( eye blinks or shifts in eye position outside a square window of width 5° centered on the fixation spot ) during −0 . 5 s to 0 . 75 s of stimulus onset ( mean ± SD: 16 . 7 ± 14 . 2% , 12 . 8 ± 12% , and 31 . 6 ± 18 . 4% for Gamma experiment; and 16 . 7 ± 15 . 1% , 9 . 1 ± 16% , and 46 . 4 ± 1% for SSVEP experiment , for healthy , MCI , and AD subjects , respectively ) .",
"For the remaining repeats , all the subjects were able to maintain fixation with a standard deviation of less than 0 . 5° , 0 . 3° , and 0 . 6° for Gamma experiment and 0 . 6° , 0 . 4° , 0 . 6° for SSVEP experiment for healthy , MCI , and AD subjects , in either directions .",
"We used a pipeline to reject artifact-containing data as described in Murty et al . , 2020 .",
"Briefly , we applied a repeat-wise thresholding process on both time-domain waveforms and multitapered PSD ( between −500 ms and 750 ms of stimulus onset ) to select bad repeats across electrodes .",
"We discarded those electrodes that had more than 30% of all repeats marked as bad , and subsequently labelled any repeat as bad if it occurred in more than 10% of total number of remaining electrodes .",
"We next discarded those electrodes that had PSD slopes ( calculated in 56-84 Hz range as described briefly in EEG data analysis subsection; see Murty et al . , 2020 for details ) less than",
"0 . Further , we discarded any block that did not have at least a single clean bipolar electrode pair ( see Data Analysis subsection below ) in any of the following three groups of bipolar electrodes: PO3-P1 , PO3-P3 , POz-PO3; PO4-P2 , PO4-P4 , POz-PO4 and Oz-POz , Oz-O1 , Oz-O2 .",
"Despite these strict criteria , we ended up rejecting only 53/497 , 4/31 , and 1/15 blocks for healthy , MCI , and AD subjects; and we rejected only 5 . 5 ± 6 . 4% , 5 . 7 ± 3 . 6% , and 4 . 1 ± 1 . 9% of electrodes for healthy , MCI , and AD subjects , among those blocks that were analyzed .",
"We then pooled data across all good blocks for each subject for final analysis .",
"Those subjects who did not have any analyzable blocks ( 9 of 236 healthy subjects and 1 of 15 MCIs , respectively ) were discarded from further analysis .",
"We used a similar procedure for the SSVEP experiment .",
"Note that we did this experiment always towards the end , and therefore the signal quality could be poorer than the Gamma experiment .",
"Consequently , we rejected 25/222 , 5/12 , and 3/5 blocks for healthy , MCI , and AD subjects; and 6 . 6 ± 7 . 2% , 8 . 5 ± 7 . 4% , and 5 . 7 ± 1% of electrodes among those blocks that were analyzed .",
"Hence , we rejected data from 25/4/3 out of 222/11/5 subjects as they did not have any analyzable blocks , leaving 197/7/2 ( 93/1/1 females ) healthy/MCI/AD subjects for analysis for the SSVEP experiment .",
"For all analyses we re-referenced data at each electrode offline to its neighboring electrodes ( bipolar reference ) .",
"We thus obtained 112 bipolar pairs out of 64 unipolar electrodes ( Murty et al . , 2020 ) .",
"We considered the following nine bipolar electrodes for analysis: PO3-P1 , PO3-P3 , POz-PO3 , PO4-P2 , PO4-P4 , POz-PO4 , Oz-POz , Oz-O1 , Oz-O2 , which are inside the black encapsulation shown in Figure 2D .",
"We discarded a bipolar electrode if either of its constituting unipolar electrodes was marked bad during artifact rejection .",
"Data was pooled for the rest of the bipolar electrodes for further analysis .",
"We analyzed all data using custom codes written in MATLAB ( The MathWorks , Inc , RRID:SCR_001622 ) as described in Murty et al . , 2020 .",
"We computed PSD and the time-frequency power spectrograms using multi-taper method with a single taper using Chronux toolbox ( Mitra and Bokil , 2008; http://chronux . org/ , RRID:SCR_005547 ) .",
"We chose baseline period between −500 ms and 0 ms of stimulus onset , and stimulus period between 250 ms and 750 ms , to avoid stimulus onset-related transients , yielding a frequency resolution of 2 Hz for the PSDs .",
"We calculated time frequency power spectra using a moving window of size 250 ms and step size of 25 ms , giving a frequency resolution of 4 Hz .",
"We calculated change in power in different frequency bands as follows:ΔPower=10 ( log10∑fST ( f ) ∑fBL ( f ) ) where ST and BL are stimulus and baseline power spectra ( across frequencies of interest , f ) averaged across all analyzable repeats and the nine bipolar electrodes as described above .",
"As mentioned in the Results , we computed the spectra for gratings with spatial frequency of 2 and 4 cpd and all four orientations for the Gamma experiment ( unless otherwise mentioned ) .",
"The total number of repeats that were thus analyzed were 184 . 2 ± 58 . 3 , 174 . 2 ± 52 . 1 , and 126 . 4 ± 29 . 3 for healthy , MCI , and AD subjects .",
"To test the dependence of alpha/gamma power on different orientations ( see results ) , we calculated orientation selectivity for each subject using the following ( Murty et al . , 2018 ) :Orientationselectivity=|∑i=1NRie ( j⋅2θi ) |∑i=1NRiwhere θi and Ri are the orientations and absolute power ( µV2 , for 250-750 ms after stimulus onset in the frequency band of interest ) , for each i = [0° 45° 90° 135°] ( thus , N = 4 ) .",
"We averaged absolute power values across spatial frequencies for calculating the orientation selectivity .",
"For SSVEP experiment , we analyzed only the counter-phasing condition .",
"There were 30 . 2 ± 6 . 9 , 32 . 9 ± 7 . 5 , and 17 . 5 ± 2 . 1 repeats for healthy , MCI , and AD subjects , respectively .",
"We took the power at 32 Hz ( twice the counter-phasing frequency , i . e . , 16 cps ) for analysis .",
"Static gratings were presented in SSVEP experiment mainly to prevent adaptation and were discarded from analysis .",
"We generated scalp maps using the topoplot . m function of EEGLAB toolbox ( Delorme and Makeig , 2004 , RRID:SCR_007292 ) , modified to show each electrode as a colored disc .",
"We calculated slopes ( for Figure 5—figure supplement 1 ) by fitting baseline PSD averaged across all analyzable repeats and bipolar electrodes with a power law function using fminsearch in MATLAB:P ( f ) =A . f-β , where P is the PSD across frequencies f , A is scaling factor , and β is the slope .",
"We detected microsaccades using a threshold-based method described earlier ( Murty et al . , 2018 ) , initially proposed by Engbert , 2006 , for the analysis period of −0 . 5 s to 0 . 75 s of stimulus onset for the Gamma experiment .",
"After removing the microsaccade-containing repeats ( 85 . 6 ± 47 . 6 , 76 . 5 ± 36 . 5 , 70 ± 17 . 2 ) , there were 98 . 5 ± 46 ( minimum 7 ) repeats for healthy subjects ( n = 226 ) , 96 . 2 ± 35 . 7 ( min: 54 ) for MCI ( n = 11 ) , and 64 . 3 ± 14 . 4 ( min: 52 ) for AD subjects ( n = 4 ) , respectively , excluding three subjects for whom eye data could not be collected .",
"We used coefficient of variation ( CV , ratio of standard deviation to mean ) of pupil diameter across time for every repeat as a measure of pupillary reactivity to stimulus for that repeat ( Murty et al . , 2020 ) .",
"We calculated CV for each analyzable repeat separately and calculated mean CV across repeats for every subject for comparison .",
"All our statistical interpretations were based on non-parametric tests on medians using K-W test ( results were similar if we used Wilcoxon sign-rank test ) .",
"We used two-way ANOVA for comparing change in alpha/gamma power across spatial frequencies and orientations for 227 healthy subjects ( see Results section ) .",
"The study is controlled at p<0 . 05 regardless of the sample size , since the false alarm rate does not depend on the sample size .",
"BF is the likelihood ratio of the marginal likelihood of alternate hypothesis to the likelihood of null hypothesis , given the data .",
"This allows for comparing the alternate with null hypothesis , rather than just infer from the evidence for null hypothesis ( Jarosz and Wiley , 2014; Keysers et al . , 2020 ) .",
"In the present case , we used the Bayesian paired t-test with the Cauchy prior scaling set to",
"1 . In this scheme , BF values below one suggests the absence of effect and evidence in favor of the null hypothesis , BF values between 1 and 3 provide anecdotal evidence in favor of the alternate hypothesis , while values between 3 and 10 provide substantial evidence and values above 10 provide strong evidence in favor of the alternative ( Jeffreys , 1998 ) .",
"For gamma power , we calculated BF for right-tailed paired t-test ( power ( controls ) >power ( cases ) ) , while for alpha power , we calculated BF for left-tailed paired t-test ( power ( controls ) <power ( cases ) ) .",
"We used the MATLAB toolbox on BF by Bart Krekelberg ( Krekelberg , 2021 ) based on Rouder et al . , 2012 .",
"We considered a linear regression model: ΔPower = β0 + βCDR . CDR + βAGE . AGE + βGENDER . GENDER + ε .",
"Categorical variable GENDER was converted to numerical variable by considering ‘0’ for males and ‘1’ for females .",
"The variable AGE was considered as the subject’s age in years , approximated to the integer .",
"We used the fitlm ( ) function in MATLAB to implement linear regression model .",
"The function outputs significance ( p-values ) for the t-statistic of the null hypothesis test over coefficient of each corresponding regressor variable in the model .",
"All spectral analyses were performed using Chronux toolbox ( version 2 . 10 ) , available at http://chronux . org .",
"Relevant data and codes are available at the following GitHub repository: https://github . com/supratimray/TLSAEEGProjectPrograms ( Murty , 2021 copy archived at swh:1:rev:5860e435fea06a49599ac81907bd63099e46581b ) ."
]
] | [
"Alzheimer’s disease ( AD ) in elderly adds substantially to socioeconomic burden necessitating early diagnosis .",
"While recent studies in rodent models of AD have suggested diagnostic and therapeutic value for gamma rhythms in brain , the same has not been rigorously tested in humans .",
"In this case-control study , we recruited a large population ( N = 244; 106 females ) of elderly ( >49 years ) subjects from the community , who viewed large gratings that induced strong gamma oscillations in their electroencephalogram ( EEG ) .",
"These subjects were classified as healthy ( N = 227 ) , mild cognitively impaired ( MCI; N = 12 ) , or AD ( N = 5 ) based on clinical history and Clinical Dementia Rating scores .",
"Surprisingly , stimulus-induced gamma rhythms , but not alpha or steady-state visually evoked responses , were significantly lower in MCI/AD subjects compared to their age- and gender-matched controls .",
"This reduction was not due to differences in eye movements or baseline power .",
"Our results suggest that gamma could be used as a potential screening tool for MCI/AD in humans ."
] | [
"Alzheimer’s disease is one of the most common forms of dementia , characterised by declining memory and thinking skills , and behavioural changes that worsen over time .",
"It affects millions of people worldwide , mostly in older age , and yet early indicators of the disease are lacking .",
"Most cases are only diagnosed once a person’s brain function becomes noticeably impaired , even though known biological changes underpin the disease .",
"Detecting Alzheimer’s disease early could aid diagnosis and enable early intervention , while also improving the chances of finding treatments to halt or reverse the disease .",
"Currently , brain function is measured by performing cognitive tests , such as remembering a set of words , imaging the brain with MRIs or CT scans , and blood or spinal fluid tests .",
"Many of these tests can be invasive and expensive , so researchers are exploring whether measuring oscillations in the brain’s electrical activity can be a non-invasive and chepaer way of testing brain function .",
"Gamma oscillations are rhythmic signals , thought to be involved in attention and working memory .",
"Animals used to study Alzheimer’s disease have shown some abnormalities in gamma oscillations , and studies of healthy humans have also observed a decline in the strength and frequency of these oscillations with age .",
"These findings have spurred an interest in understanding the link between gamma oscillations and AD in humans .",
"To investigate this link , Murty et al . measured patterns of brain activity in elderly people chosen from the community using electrodes placed on their scalps ( a technique called electroencephalography ) .",
"These participants watched certain images previously shown to elicit gamma oscillations .",
"Participants who were later diagnosed with early Alzheimer’s disease had weaker gamma oscillations than their cognitively healthy peers in the part of the brain that processes visual images .",
"These results build upon previous findings from animal research suggesting that gamma oscillations may be disrupted in early Alzheimer’s disease .",
"The work by Murty et al . could lead the way to new ways of diagnosing Alzheimer’s disease , where early indicators are urgently needed ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | The DEG/ENaC cation channel protein UNC-8 drives activity-dependent synapse removal in remodeling GABAergic neurons | elife-14599-v3 | [
[
"Neural circuits are reorganized during development to produce a mature , functional nervous system .",
"Circuit refinement involves both the elimination of specific synapses and the strengthening of others .",
"These developmental changes in brain architecture are temporally coordinated with critical periods in which neuronal wiring can be shaped by activity .",
"The establishment of binocular vision in the mammalian brain , for example , depends on visual input during a distinct developmental period in which the circuit is uniquely sensitive to neural activity .",
"GABA signaling regulates the onset and duration of this critical period of plasticity in the visual circuit , but the molecular mechanisms that drive synaptic remodeling in this pathway are poorly defined ( Deidda et al . , 2015; Hensch , 1998 ) .",
"Calcium influx by voltage-gated calcium channels ( VGCCs ) triggers neurotransmitter secretion in active neurons ( Catterall et al . , 2008 ) .",
"Elevated intracellular calcium can also regulate synaptic strength by controlling gene expression and protein function ( Flavell and Greenberg , 2008; Baumgärtel and Mansuy , 2012 ) .",
"For example , the calcium-sensitive phosphatase , calcineurin , antagonizes long term potentiation ( LTP ) through dephosphorylation of an AMPA receptor subunit that results in its endocytosis from the postsynaptic membrane ( Baumgärtel and Mansuy , 2012; Winder et al . , 1998 ) .",
"Recent work has shown that functional synaptic components can also be removed by the canonical apoptotic protease , caspase-3 ( Ertürk et al . , 2014; Wang et al . , 2014 ) .",
"In C . elegans , the cell death pathway component CED-3/caspase-3 and its upstream regulator CED-4/Apaf1 promote synaptic disassembly by activating the F-actin severing protein gelsolin; the calcium sensitivity of this pathway points to a potential role for neuronal activity in synaptic remodeling ( Pinan-Lucarre et al . , 2012; Meng et al . , 2015; Liu et al . , 2011 ) .",
"Degenerin/Epithelial Sodium Channels ( DEG/ENaCs ) are voltage-insensitive , trimeric cation channels .",
"The term degenerin arises from the finding that constitutively active forms of DEG/ENaC channels induce neurodegeneration ( Bianchi and Driscoll , 2002 ) .",
"ENaCs are expressed in mammalian epithelial tissues , where they promote sodium reabsorption ( Wemmie et al . , 2002 ) .",
"Emerging evidence indicates that DEG/ENaCs may also influence neuronal plasticity and synaptic function ( Wemmie et al . , 2002 , 2003; Kreple et al . , 2014 ) .",
"For example , the acid-sensing DEG/ENaC protein , ASIC1a , is expressed in distinct brain regions where it promotes learning and memory ( Wemmie et al . , 2003 , 2004; Zha et al . , 2006; Ziemann et al . , 2009 ) .",
"Models to explain this role of DEG/ENaCs in synaptic plasticity have posited that depolarizing DEG/ENaC-dependent cation transport activity could enhance the activation of voltage-sensitive postsynaptic ion channels ( Wemmie et al . , 2006 ) .",
"DEG/ENaCs can also function in the presynaptic compartment to elevate neurotransmitter secretion ( Cho and Askwith , 2008; Voglis and Tavernarakis , 2008; Younger et al . , 2013 ) .",
"In this case , the depolarizing activity of a presynaptic ENaC channel is proposed to enhance neurotransmitter release by elevating calcium import at local VGCCs ( Younger et al . , 2013 ) .",
"Here , we describe a DEG/ENaC protein that likely exerts a similar effect on intracellular calcium , but with the strikingly different downstream outcome of presynaptic destruction .",
"In the nematode C . elegans , a simple , well-defined GABAergic circuit is remodeled during development in a mechanism that is accelerated by neural activity ( White et al . , 1978; Hallam and Jin , 1998; Park et al . , 2011; Thompson-Peer et al . , 2012 ) .",
"Dorsal D ( DD ) motor neurons initially form synapses with ventral muscles ( Figure 1A ) , which are later eliminated and relocated to dorsal muscles ( Figure 1B ) .",
"Ventral D ( VD ) motor neurons generate ventral synapses and express the transcriptional repressor protein UNC-55 , which prevents these cells from remodeling; thus both DD and VD synapses are remodeled in unc-55 mutants ( Figure 1B , C ) ( Walthall and Plunkett , 1995; Zhou and Walthall , 1998; Shan et al . , 2005 ) .",
"Several lines of evidence indicate that UNC-55 blocks the native DD remodeling program: ( 1 ) Ectopic expression of UNC-55 in DD neurons prevents synaptic remodeling ( Shan et al . , 2005 ) ; ( 2 ) Ventral VD synapses are initially established and then removed in unc-55 mutants in a sequence that parallels the DD remodeling program ( Petersen et al . , 2011; Meng et al . , 2015 ) ; ( 3 ) VD neurons form ectopic but functional synapses with dorsal muscles in unc-55 mutants ( Thompson-Peer et al . , 2012 ) ; ( 4 ) The transcription factor genes irx-1 and hbl-1 , which UNC-55 negatively-regulates , normally function in DD neurons to promote remodeling ( Petersen et al . , 2011; Thompson-Peer et al . , 2012 ) .",
"We exploited the anti-remodeling role of UNC-55 in a screen designed to detect key components of the DD remodeling program as RNAi hits that prevent the ectopic remodeling phenotype of unc-55 mutants .",
"This approach revealed a molecularly diverse array of genes that drive removal of GABA synapses ( Petersen et al . , 2011 ) .",
"Here we demonstrate that one of these UNC-55 targets , the DEG/ENaC protein UNC-8 , functions to dismantle the presynaptic apparatus in remodeling GABA neurons ( Figure 1D , E ) .",
"The necessary roles of UNC-2/VGCC and TAX-6/Calcineurin in this synapse elimination mechanism argue that intracellular calcium is involved .",
"Our results indicate that UNC-8 promotes synapse removal downstream of TAX-6 and thus could be potentially activated by TAX-6/Calcineurin .",
"In turn , we suggest that the depolarizing activity of UNC-8 DEG/ENaC could enhance UNC-2/VGCC function .",
"The net positive feedback loop involving UNC-2/VGCC , TAX-6/Calcineurin and UNC-8/DEG/ENaC could elevate presynaptic calcium above a critical threshold to activate a downstream caspase-dependent pathway leading to destabilization of the presynaptic apparatus ( Meng et al . , 2015 ) .",
"Thus , we propose a model in which UNC-8 triggers a synaptic removal mechanism that is regulated by the intersection of a genetic program that controls UNC-8 expression and a calcium-signaling pathway that promotes neurotransmission as well as synapse elimination . 10 . 7554/eLife . 14599 . 003Figure 1 . GABAergic neuron synaptic remodeling is transcriptionally controlled and depends on UNC-8 . ( A ) Dorsal D ( DD ) GABAergic motor neurons ( dark blue ) synapse with ventral muscles during embryonic development .",
"( B ) DD synapses are relocated to dorsal muscles at the end of the first larval stage ( L1 ) .",
"Ventral D ( VD ) GABAergic motor neurons ( light blue ) are generated in the late L1 and innervate ventral muscles .",
"( C ) The COUP/TF transcription factor UNC-55 is expressed in VD neurons and blocks remodeling; VD neurons relocate synapses to the dorsal side in loss-of-function unc-55 mutants .",
"( D/E )",
"UNC-8/DEG/ENaC expression is negatively regulated by UNC-55 and RNAi knockdown of unc-8 suppresses ectopic VD remodeling in unc-55 mutants .",
"These results suggest that UNC-8 may also promote DD remodeling .",
"Schematics modified from Petersen et al . ( 2011 ) .",
"( F ) unc-8 expression in remodeling neurons is visualized with a punc-8::GFP reporter gene .",
"Strong punc-8::GFP ( green ) expression was observed in DD motor neurons in wild type , but was also detected in VD motor neurons in unc-55 animals .",
"GABAergic motor neurons are labeled with pttr-39::mCherry ( magenta ) .",
"Scale bar is 10 μm .",
"( G ) Normalized fluorescence intensity is plotted on the Y-axis in arbitrary units ( A . U . ) .",
"punc-8::GFP expression is enhanced in VDs , but not DDs , in unc-55 mutants ( ***p<0 . 001 , ns is not significant , One-Way ANOVA with Bonferroni correction ) .",
"n ≥ 26 DDs and n ≥ 51 VDs per genotype , data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 00310 . 7554/eLife . 14599 . 004Figure 1—figure supplement 1 . UNC-8 is expressed in remodeling GABA neurons .",
"( A ) A GFP C-terminal fusion with the intact UNC-8 protein was generated by recombineering ( see Materials and methods ) .",
"( B ) UNC-8::GFP shows mosaic expression in the ventral nerve cord ( VNC ) of early L2 animals in GABAergic DD neurons ( asterisks ) and in cholinergic DA and DB neurons ( upper left ) .",
"Schematic denotes VNC neurons that express UNC-8::GFP ( upper right ) .",
"The six DD neurons are labeled with punc-25::mCherry .",
"mCherry-labeled cell bodies between the DD neurons are the newly born VD neurons ( lower left ) .",
"Merged image of UNC-8::GFP fosmid and punc-25::mCherry-labeled GABA neurons ( lower right ) .",
"Asterisks denote an expression of UNC-8::GFP in DD3 and DD4 .",
"Scale bar is 20 μm .",
"( C ) UNC-8::GFP is expressed in DD and VD neurons in unc-55 mutants .",
"GABA neurons are labeled with wdIs90 [punc-25::mCherry::RAB-3] .",
"UNC-8::GFP-expressing GABA neurons are outlined .",
"Scale bar is 10 μm .",
"UNC-8::GFP rescues the unc-55 remodeling phenotype in unc-55; unc-8 animals ( ***p<0 . 001 , n ≥ 20 animals , One-Way ANOVA with Bonferroni correction , data are mean ± SD ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 004"
],
[
"Our previous microarray data indicated that unc-8 expression is elevated in GABA neurons in unc-55 mutants ( Petersen et al . , 2011 ) .",
"As an independent test of this finding and to determine if unc-8 is also expressed in DD neurons , we used a GFP reporter gene ( punc-8::GFP ) that includes a 1 . 6 kb genomic region upstream of the unc-8 coding sequence ( Etchberger et al . , 2007 ) .",
"In wild-type adults , punc-8::GFP is expressed in DD GABAergic neurons , but shows little detectable GFP signal in VD motor neurons ( Figure 1F , G ) .",
"In an unc-55 mutant , however , punc-8::GFP expression was clearly visible in both DD and VD neurons .",
"As an additional test of this model , we fused GFP to the UNC-8 C-terminus in a fosmid reporter gene that spans the unc-8 locus to include large flanking regions .",
"The resultant UNC-8::GFP fusion protein is functional in vivo and is expressed in DD neurons in the wild type and also in VD neurons in an unc-55 mutant .",
"In this case , we also detected strong UNC-8::GFP expression in adjacent ventral cord DA and DB cholinergic neurons as predicted by the selective degeneration of these cells in a mutant that encodes a constitutively active UNC-8 protein ( Wang et al . , 2013 ) .",
"These results suggest that the fosmid reporter likely includes gene regulatory regions that direct expression of the native UNC-8 protein ( Figure 1—figure supplement 1A–C ) .",
"Together , these findings establish that UNC-8 expression is correlated with the execution of a genetically controlled program that promotes remodeling of GABAergic synapses .",
"Our RNAi results suggested that UNC-8 promotes synapse removal in remodeling GABA neurons ( Figure 1D , E ) ( Petersen et al . , 2011 ) .",
"To confirm this finding , we generated a loss-of-function allele unc-8 ( tm5052 ) that deletes 197 nucleotides of the first transmembrane domain and shifts the reading frame to introduce a premature stop codon ( Figure 2A ) .",
"In this configuration , unc-8 ( tm5052 ) should result in an unstable unc-8 mRNA and likely null allele . 10 . 7554/eLife . 14599 . 005Figure 2 . The DEG/ENaC subunit UNC-8 promotes removal of ventral DD synapses .",
"( A ) Schematic of the unc-8 gene and predicted UNC-8 protein .",
"DEG/ENaC channel subunits contain two transmembrane domains ( TM domains ) and a large extracellular loop ( gray bars ) .",
"The unc-8 deletion allele tm5052 is indicated ( blue bar ) .",
"( B ) DD GABA neuron synapses ( green ) with ventral muscles are relocated to the dorsal side during development .",
"DD-specific GFP-tagged RAB-3 ( pflp-13::GFP::RAB-3 ) labels synapses in the ventral nerve cord of early L1 larvae and the dorsal nerve cord of adults .",
"Asterisk denotes DD4 soma .",
"Scale bar is 10 μm .",
"( C ) DD remodeling was quantified by counting GFP::RAB-3 puncta during larval stages .",
"Removal of ventral DD synapses is significantly delayed in unc-8 animals ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , ns is not significant , Student’s t-test , data are mean ± SD ) .",
"( D ) Representative images of wild-type and unc-8 adult ventral nerve cords ( asterisk denotes DD5 soma ) .",
"Ventral DD synapses labeled with GFP-tagged synaptobrevin ( pflp-13::SNB-1::GFP ) are retained in unc-8 mutant adult animals .",
"Scale bar is 10 μm .",
"Inset shows pixel intensity over a 20 μm region ( indicated by dashed boxes ) of the ventral nerve cord in wild-type and unc-8 animals .",
"( E ) Removal of DD::SNB-1::GFP puncta is defective in unc-8 mutant adults; however , dorsal DD synaptic assembly is not affected ( ***p<0 . 001 , n ≥ 20 , ns is not significant , Student’s t-test , data are mean ± SD ) .",
"( F ) Ventral localization of GFP-tagged UNC-8 in GABA neurons .",
"Asterisk denotes DD5 soma .",
"Scale bar is 10 μm .",
"( G ) UNC-8 promotes removal of ventral presynaptic components in DD neurons , but is not required for dorsal synapse formation . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 00510 . 7554/eLife . 14599 . 006Figure 2—figure supplement 1 . UNC-8 removes ventral synapses , but is not required for assembly of dorsal synapses .",
"( A ) DD-specific expression of SNB-1::GFP ( pflp-13::SNB-1::GFP ) labels DD synapses .",
"In the wild type , all SNB-1::GFP puncta are ventrally localized in early L1 larvae , but are strictly dorsal in adults after DD remodeling .",
"Scale bar is 10 μm .",
"Asterisk denotes DD4 cell soma .",
"( B ) Loss of unc-8 function does not delay dorsal DD synapse formation .",
"Dorsal SNB-1::GFP puncta were counted in developing wild-type and unc-8 mutant L1/L2 larvae .",
"( C ) GABA neuron development is not delayed in unc-8 .",
"GABA neurons ( DD + VD ) in the ventral cord , marked with SNB-1::GFP , were counted in developing wild-type and unc-8 mutant L1/L2 larvae .",
"( D ) DD remodeling was quantified by counting GFP::RAB-3 puncta during the L1-L2 larval stages .",
"Assembly of dorsal DD synapses is not significantly different between wild-type and unc-8 animals ( Student’s t-test , data are mean ± SD ) .",
"Results for ( B–D ) were pooled from 3 independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 006 To ask if UNC-8 promotes DD synapse removal , we utilized a DD-specific promoter ( pflp-13 ) to drive expression of the presynaptic vesicle-associated proteins , GFP::RAB-3 and SNB-1::GFP/synaptobrevin .",
"DD remodeling is initiated toward the end of the first larval stage ( L1 ) and involves the relocation of ventral presynaptic components to the dorsal side ( Figure 2B , Figure 2—figure supplement 1A ) ( Hallam and Jin , 1998; Petersen et al . , 2011 ) .",
"Dorsal and ventral synapses were quantified throughout the remodeling period in early stage larval animals by monitoring GFP::RAB-3 puncta .",
"This experiment revealed that the removal of ventral DD synapses was significantly delayed in unc-8 mutants compared to wild type ( Figure 2C ) .",
"This effect is unlikely due to slower overall development as the birth of post-embryonic VD neurons is unaffected in unc-8 mutants ( Figure 2—figure supplement 1C ) .",
"In addition , residual ventral SNB-1::GFP puncta are visible in the DD neurons of adult unc-8 animals , but rarely in wild type indicating that unc-8 is required for the complete removal of ventral GABA synapses in remodeling DD neurons ( Figure 2D , E ) .",
"In contrast , assembly of dorsal DD synapses was not perturbed in unc-8 animals ( Figure 2E , Figure 2—figure supplement 1B , D ) .",
"These results suggest that unc-8 is required for the efficient removal of presynaptic components in the DD remodeling program , but is not necessary for assembly of nascent DD synapses with dorsal muscles ( Figure 2G ) .",
"The idea that unc-8 promotes removal of ventral presynaptic components in the DD remodeling program is consistent with our finding that expression of a GFP-tagged UNC-8 protein in GABA neurons results in UNC-8::GFP puncta that are exclusively localized to the ventral nerve cord ( Figure 2F ) .",
"Having shown that unc-8 is required for the timely and efficient removal of ventral synapses in remodeling DD neurons , we next used unc-8 ( tm5052 ) to confirm this function in VD neurons that remodel in unc-55 mutants .",
"This test is important because previous evidence suggests that VD remodeling in unc-55 mutants is driven by the ectopic activation of the native DD remodeling program ( Shan et al . , 2005; Petersen et al . , 2011 ) .",
"Expression of the GABAergic presynaptic marker SNB-1::GFP in wild-type adults shows a uniform pattern of SNB-1::GFP puncta in the ventral nerve cord that corresponds to VD synapses with ventral muscles ( Figure 3A ) ( Hallam and Jin , 1998 ) .",
"As previously reported , ventral SNB-1::GFP clusters are largely depleted in unc-55 mutant animals due to ectopic VD remodeling ( Petersen et al . , 2011 ) .",
"We constructed an unc-55; unc-8 double mutant to ask if unc-8 is required for the removal of ventral synapses in remodeling GABA neurons .",
"Both fluorescence intensity measurements as well as quantification of SNB-1::GFP puncta number revealed significant retention of the SNB-1::GFP signal in the ventral nerve cord of unc-55;unc-8 mutant animals in comparison to unc-55 alone ( Figure 3A–D , Figure 3—figure supplement 1A ) .",
"We also performed time course experiments to confirm that the retention of ventral GABA synapses during larval development in unc-55; unc-8 animals persists into adulthood ( Figure 3—figure supplement 1B ) .",
"Because this assay should detect ventral SNB-1::GFP puncta in both DD and VD neurons , we co-labeled these synapses with the DD-specific marker , flp-13::mCherry::RAB-3 to identify the fraction of each GABA neuron type .",
"This experiment revealed that approximately 40% of residual GABA synapses in unc-55; unc-8 animals are derived from DD neurons and the balance ( ~60% ) from VD neurons ( Figure 3—figure supplement 1C ) .",
"Our results indicate that the wild-type unc-8 gene is capable of promoting the removal of ventral synapses in both classes of GABA neurons thus supporting the hypothesis that the DD and VD remodeling programs utilize shared sets of active components ( Shan et al . , 2005; Petersen et al . , 2011 ) . 10 . 7554/eLife . 14599 . 007Figure 3 . UNC-8 drives removal of ventral GABAergic synapses .",
"( A ) Ventral GABA synapses labeled with GFP-tagged synaptobrevin ( punc-25::SNB-1::GFP ) for 10 adult animals .",
"Wild-type and unc-8 ( tm5052 ) show similar distributions of SNB-1::GFP puncta .",
"Ventral SNB-1::GFP is depleted from unc-55 due to VD remodeling , but partially restored in unc-55; unc-8 animals .",
"( B ) Data for Figure panels A , C , D were collected from the ventral nerve cord between VD4 and VD5 .",
"( C ) SNB-1::GFP fluorescent intensity measurements from each genotype .",
"Each line represents the pixel intensity over a 20 μm region of the VNC from a single representative animal .",
"( D ) Cumulative frequency curves for SNB-1::GFP fluorescence intensity for each genotype ( n > 10 animals ) .",
"unc-55 animals show a significant loss of ventral SNB-1::GFP fluorescence ( p< 0 . 0001 vs wild type ) .",
"SNB-1::GFP fluorescence is partially restored in unc-55; unc-8 animals , demonstrating the role of UNC-8 for synapse removal ( p<0 . 0001 vs unc-55 ) .",
"p values calculated with Kruskal-Wallis and Dunn’s post test .",
"( E ) Knockdown of unc-8 by GABA-neuron-specific RNAi ( unc-8 csRNAi ) restored SNB-1::GFP puncta to the VNC of unc-55; juIs1 animals vs control animals expressing the empty vector RNAi ( control csRNAi ) .",
"GABAergic neurons are labeled with punc-25::mCherry ( magenta ) .",
"Asterisks denote GABA neuron cell bodies and arrowheads point to SNB-1::GFP-labeled ventral synapses .",
"( F ) Quantification of ventral synapses in the region anterior to each cell body expressing the RNAi construct ( n > 60 animals ) .",
"RNAi knockdown of unc-8 in unc-55 mutant GABA neurons significantly suppresses synapse removal ( ***p<0 . 001 , Student’s t-test . Data are mean ± SD ) .",
"Scale bars are 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 00710 . 7554/eLife . 14599 . 008Figure 3—figure supplement 1 . UNC-8 promotes synapse disassembly in remodeling GABAergic neurons . Fluorescent puncta for the presynaptic protein SNB-1::GFP were counted in the ventral nerve cord from VD3 to VD11 .",
"( A ) SNB-1::GFP puncta are removed from ventral synapses in unc-55 and this effect partially depends on unc-8 ( ***p<0 . 001 , *p<0 . 05 , n ≥ 10 ) .",
"One-Way ANOVA with Bonferroni correction , data are mean ± SD .",
"( B ) Time course experiments with unc-55 and unc-55; unc-8 animals show the removal of ventral GABAergic synapses ( punc-25::SNB-1::GFP ) over time .",
"Ventral VD synapses are initially established at 24-hr post lay in both unc-55 and unc-55; unc-8 and these synapses are largely removed in unc-55 animals .",
"Loss of unc-8 function disrupts ventral synapse removal in unc-55 animals and results in the retention of significantly more ventral synapses .",
"( ***p<0 . 001 , ns is not significant , Student’s t-test ) .",
"n ≥ 78 animals per genotype , data are mean ± SD .",
"( C ) Ventral GABA synapses in unc-55; unc-8 animals were co-labeled with punc-25::SNB-1::GFP , which marks all synapses ( DD and VD ) and pflp-13::mCherry::RAB-3 , which labels DD synapses only .",
"The proportion of ventral DD synapses ( labeled with pflp-13::mcherry::RAB-3 ) in unc-55; unc-8 adults are shown in gray as a percentage of total ventral synapses . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 00810 . 7554/eLife . 14599 . 009Figure 3—figure supplement 2 . UNC-8 functions cell autonomously and is sufficient to promote ventral synapse elimination in GABA neurons .",
"( A ) UNC-8 function is cell-autonomous .",
"Ventral synapses were counted in unc-55; unc-8 animals injected with GABA::unc-8cDNA and the GABA marker punc-25::mCherry ( left ) .",
"Asterisk denotes cell expressing unc-8cDNA and punc-25::mCherry .",
"Scale bar is 10 μm , anterior to left .",
"GABA neurons expressing unc-8cDNA showed fewer ventral puncta than neighboring unc-55; unc-8 neurons that do not express the unc-8cDNA transgene , indicating that UNC-8 functions in GABA neurons to promote synapse removal ( ***p<0 . 001 , n ≥ 15 animals , Student’s t-test , data are mean ± SD ) .",
"( B ) Forced expression of unc-8cDNA in GABA neurons induces disassembly of of ventral synapses .",
"VD neurons expressing unc-8cDNA ( co-labeled with GABA::mCherry , VD8 denoted with arrow ) show significantly fewer ventral SNB-1::GFP ( punc-25::SNB-1::GFP ) puncta than neighboring VD neurons that do not express unc-8cDNA which are unaffected ( e . g . , VD9 denoted with arrowhead ) .",
"Anterior to left .",
"Ectopic expression of unc-8cDNA in VD neurons has no effect on dorsal synapses ( ***p<0 . 001 , ns is not significant , Student’s t-test , n ≥ 51 VDs ( ventral ) and n = 7 VDs ( dorsal ) , data are mean ± SEM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 009 Having established that UNC-8 is necessary for the complete removal of GABAergic presynaptic proteins ( Figures 2 , 3 ) and that punc-8::GFP is expressed in remodeling DD and VD neurons ( Figure 1F ) , we next asked if UNC-8 function is cell-autonomous .",
"To address this question , we used cell-specific RNA interference ( csRNAi ) to knock down unc-8 expression in GABA neurons .",
"unc-55 animals expressing empty vector RNAi display a stereotypical loss of ventral GABA synapses as predicted .",
"In contrast , unc-55 mutant VD neurons that express the unc-8 ( csRNAi ) transgene show significant retention of GABAergic synapses in the ventral nerve cord ( Figure 3E , F ) , thus demonstrating that unc-8 functions in GABA neurons to promote the elimination of ventral synapses .",
"As an additional test of this model , we restored wild-type unc-8 function to unc-55;unc-8 mutant animals by expressing unc-8 cDNA in GABA neurons .",
"In this case , SNB-1::GFP puncta are efficiently removed from the ventral nerve cord , thus confirming that unc-8 exerts a cell-autonomous role in the disassembly of ventral GABAergic synapses ( Figure 3—figure supplement 2A ) .",
"Finally , we determined that forced expression of unc-8 cDNA in wild-type VD neurons induces synapse elimination whereas the ventral synapses of neighboring VD neurons not expressing the UNC-8 transgene are maintained .",
"In addition to confirming the active role of UNC-8 in GABA neuron synapse elimination , this experiment also demonstrates that expression of UNC-8 alone is sufficient to promote synaptic disassembly in a GABA neuron that otherwise maintains a stable , functional presynaptic apparatus ( Figure 3—figure supplement 2B ) .",
"Having confirmed that wild-type unc-8 promotes the removal of the presynaptic proteins SNB-1::GFP and GFP::RAB-3 from the ventral nerve cord in remodeling GABA neurons ( Figure 2C–E , Figure 3 , Figure 3—figure supplement 1 ) , we next used additional fluorescent markers to ask if the unc-8 gene also drives the elimination of other key components of the presynaptic apparatus .",
"For this experiment , we monitored fluorescent markers for the presynaptic density protein α-liprin/SYD-2 ( Dai et al . , 2006 ) and the membrane-associated endocytic protein endophillin/UNC-57 ( Schuske et al . , 2003 ) .",
"As observed for SNB-1::GFP , all the additional presynaptic proteins examined appear as distinct puncta that localize to the presynaptic membrane in wild-type and unc-8 GABA neurons .",
"This ventral localization is substantially reduced in unc-55 animals .",
"The partial recovery of fluorescent puncta in unc-55; unc-8 animals relative to unc-55 shows that UNC-8 promotes the elimination of ventral SNB-1::GFP , UNC-57::GFP , mCherry::RAB-3 , and SYD-2::GFP puncta ( Figure 3—figure supplement 1A , Figure 4A–E ) .",
"Because these components mark synaptic vesicles ( SNB-1 , RAB-3 ) as well as the presynaptic membrane ( SYD-2 , UNC-57 ) , their removal predicts that unc-8 could be required for dismantling the overall presynaptic apparatus .",
"To investigate this possibility , we simultaneously imaged mCherry::RAB-3 and UNC-57::GFP to monitor the organization of the presynaptic domain in unc-55; unc-8 animals .",
"RAB-3 cycles on and off synaptic vesicles in a GTP/GDP-dependent manner and UNC-57/endophilin recycles between vesicles and the presynaptic membrane ( Schuske et al . , 2003; Nonet et al . , 1997 ) .",
"As expected , mCherry::RAB-3 and UNC-57::GFP are strongly co-localized at ventral synapses in wild-type animals ( r2 = 0 . 72 ± 0 . 03 ) ( Figure 4F ) .",
"Although fewer presynaptic clusters are detected at ventral synapses in unc-55; unc-8 mutants , residual mCherry::RAB-3 and UNC-57::GFP puncta are comparably co-localized ( r2 = 0 . 67 ± 0 . 04 ) ( Figure 4G , Figure 4—figure supplement 1A–C ) . 10 . 7554/eLife . 14599 . 010Figure 4 . UNC-8 promotes disassembly of the presynaptic apparatus in GABAergic motor neurons . Fluorescent puncta for presynaptic proteins ( UNC-57::GFP , mCherry::RAB-3 , and SYD-2::GFP ) were counted in the ventral nerve cord from VD3 to VD11 .",
"( A/C )",
"Representative images ( A ) and quantification ( C ) of endophilin/UNC-57 indicate that unc-8 promotes removal of UNC-57::GFP from ventral synapses in remodeling neurons ( *p<0 . 05 , ***p<0 . 001 , n ≥ 25 ) .",
"( B/D )",
"Representative images ( B ) and quantification ( D ) show reduced removal of the presynaptic G protein RAB-3 in unc-55; unc-8 animals ( ***p<0 . 001 , n ≥ 21 ) .",
"Scale bars are 10 μM .",
"( E ) Efficient removal of the presynaptic density protein α-liprin/SYD-2 from ventral synapses in unc-55 requires unc-8 ( ***p<0 . 001 , n ≥ 21 , One-Way ANOVA with Bonferroni correction , data are mean ± SD ) .",
"( F/G )",
"GFP-tagged endophilin ( punc-25::UNC-57::GFP ) and mCherry::RAB-3 ( punc-25::mCherry::RAB-3 ) are co-localized in GABA neurons of wild-type and unc-55;unc-8 animals .",
"Representative images and normalized fluorescence intensity plots for a 20 μm region of the ventral nerve cord are shown .",
"Scale bar is 10 μm .",
"r2 is Pearson’s correlation coefficient ( n > 10 , mean ± SEM ) .",
"Presynaptic components are co-localized in wild type ( r2 = 0 . 72 ± 0 . 03 ) and unc-55;unc-8 ( r2 = 0 . 67 ± 0 . 04 ) .",
"Average r2 value for unc-55;unc-8 is not statistically different from the average r2 value for wild type ( p<0 . 001 , Mann-Whitney test , see Figure 4—figure supplement 1 ) .",
"( H/I )",
"Electron micrographs of GABA synapses with ventral muscles in ( H ) wild type and ( I ) unc-55;unc-8 .",
"No ventral GABA presynaptic densities were detected in unc-55 .",
"Arrows point to presynaptic density , scale bars are 200 nm .",
"( J ) Synaptic vesicles were quantified in ventral GABAergic synapses .",
"Synapses in wild-type , unc-8 and unc-55;unc-8 animals contain comparable numbers of synaptic vesicles ( N > 5 for each genotype , ns is not significant ) .",
"( K ) Representative traces of ventral mini-iPSCs from each genotype .",
"( L ) The high frequency of ventral mini-iPSCs in wild-type and in unc-8 animals were not observed in unc-55 or unc-55;unc-8 ( ***p<0 . 001 , ns is not significant , n ≥ 5 , data are mean ± SEM , One-Way ANOVA with Bonferroni correction ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 01010 . 7554/eLife . 14599 . 011Figure 4—figure supplement 1 . Ventral synapses in unc-55;unc-8 mutants are well-organized .",
"( A–B ) unc-55 and unc-8 animals expressing GFP-tagged endophilin ( punc-25::UNC-57::GFP ) and mCherry::RAB-3 ( punc-25::mCherry::RAB-3 ) were examined for co-localization .",
"Representative images and normalized fluorescence intensity plots for a 20 μm ventral cord region of unc-55 and unc-8 animals are shown .",
"Scale bar is 10 μm .",
"r2 is the coefficient of determination ( n > 10 , mean ± SEM ) .",
"UNC-57::GFP and mCherry::RAB-3 puncta are co-localized in unc-8 ( r2 = 0 . 65 ± 0 . 05 ) ; but not in unc-55 mutants which show few residual ventral VD synapses ( r2 = 0 . 37 ± 0 . 06 ) .",
"( C ) Average coefficient of determination for UNC-57::GFP and mCherry::RAB-3 .",
"Wild-type , unc-8 , and unc-55; unc-8 animals show no significant differences; whereas , unc-55 animals show much lower r2 values ( n ≥ 11 , data are mean ± SEM , Mann-Whitney test , ***p<0 . 001 , ns is not significant ) .",
"( D ) Representative electron micrograph of a ventral GABAergic synapse in an unc-8 animal .",
"Arrow points to presynaptic density , scale bar is 200 nm .",
"( E ) Representative traces of spontaneous activity ( GABAergic and cholinergic ) in ventral muscles .",
"( F ) Frequency of ventral spontaneous events ( GABAergic and cholinergic ) demonstrate that the loss of GABA activity in unc-55 and unc-55; unc-8 animals is not due to defects in neurotransmission to ventral muscles ( n ≥ 5 animals , ns is not significant , mean ± SEM , One-Way ANOVA with Bonferroni correction ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 01110 . 7554/eLife . 14599 . 012Figure 4—figure supplement 2 . GABAergic and cholinergic synapses are detectable in electron micrographs of wild-type and unc-55; unc-8 animals .",
"( A/B )",
"Ventral GABAergic ( A ) and cholinergic ( B ) presynaptic densities are visible in electron micrographs from wild-type animals .",
"( C/D )",
"Ventral GABAergic ( C ) and cholinergic ( C/D ) presynaptic densities were also detected in unc-55; unc-8 animals , suggesting that wild-type unc-8 function is required for the complete removal of ventral GABA synapses in unc-55 mutants .",
"Arrows denote presynaptic densities , PD ( presynaptic density ) , G ( GABAergic neuron ) , A ( cholinergic neuron ) , m ( muscle arm ) , dotted lines are adherens or gap junctions .",
"( E ) Serial slices from the ventral nerve cord of the unc-55; unc-8 animals .",
"10 serial slices span a region with GABAergic neuron terminal labeled 'G' and cholinergic neuron terminal labeled 'A' .",
"Each micrograph slice number is listed in lower right corner .",
"Cholinergic ( arrowhead ) and GABAergic ( arrow ) presynaptic densities are labeled .",
"GABAergic and cholinergic neuron synapses were distinguished by previously established criteria ( see Materials and methods ) .",
"Scale bars are 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 01210 . 7554/eLife . 14599 . 013Figure 4—figure supplement 3 . The postsynaptic UNC-49 GABAA receptor co-localizes with the presynaptic domains of remodeling GABAergic neurons . Presynaptic regions ( GABAergic neurons ) are labeled with mCherry::RAB-3 ( magenta ) and the postsynaptic compartment ( body muscle ) is marked with UNC-49::GFP ( green ) ; arrows denote regions of co-localization .",
"Arrowheads point to GABA neuron cell soma .",
"Asterisks mark intestinal autofluorescent granules .",
"( A ) .",
"Pre-remodeling DD synapses with ventral muscles ( early L1 larva , 2 hr post hatch ) .",
"( B ) Pre-remodeling VD synapses with ventral muscles ( L2 larva , 18 hr post hatch ) .",
"Note co-localization of mcherry::RAB-3 and UNC-49::GFP in both unc-55 and unc-55; unc-8 animals before remodeling .",
"Scale bars are 10 μm .",
"( C ) Ventral GABAergic synapses in young adults .",
"Few ventral mCherry::RAB-3-marked presynaptic domains are detected in the unc-55 mutant background due to ectopic remodeling but are abundant in both unc-8 and unc-55; unc-8 .",
"Scale bars are 25 μm .",
"( D ) Average correlation coefficient for ventral UNC-49::GFP and mCherry::RAB-3 puncta before remodeling .",
"unc-8 is not significantly different from wild type ( p>0 . 05 , n ≥ 3 L1 larvae , ns is not significant ) and unc-55; unc-8 is not significantly different from unc-55 ( p>0 . 05 , n ≥ 5 L2 larvae , ns is not significant , data are mean ± SD , Mann-Whitney test ) .",
"( E ) Average coefficients of determination for ventral UNC-49::GFP and mCherry::RAB-3 puncta after remodeling are not significantly different among wild-type , unc-8 and unc-55; unc-8 animals .",
"unc-55 shows significantly lower values likely due to the relative depletion of mCherry::RAB-3 vs UNC-49::GFP ( **p<0 . 01 , ns is not significant , n ≥ 5 young adults , data are mean ± SD , Kruskal-Wallis with Dunn’s multiple comparisons test ) .",
"Mutant strains were unc-55 ( e1170 ) and unc-8 ( tm5052 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 013 We used electron microscopy ( EM ) to test this idea by examining the structure of the GABAergic synapses in unc-55; unc-8 .",
"At the EM level , both GABAergic and cholinergic synapses show electron-dense presynaptic terminals and abundant synaptic vesicles , but can be distinguished by morphological criteria ( see Materials and methods ) .",
"We identified ventral GABAergic synapses in wild-type , unc-8 and in unc-55; unc-8 animals ( wild type = 6 synapses/20 . 08 μm , n = 3 animals , unc-8 = 5 synapses/12 . 88 μm , n = 2 animals , unc-55; unc-8 = 2 synapses/12 . 16 μm , n = 2 animals , Figure 4H–J , Figure 4—figure supplement 1D , and Figure 4—figure supplement 2 ) .",
"As expected , no ventral GABAergic synapses were detected in unc-55 mutants ( 0 synapses/9 . 84 μm , n = 2 animals ) ; whereas ventral cholinergic motor neuron synapses were preserved ( data not shown ) .",
"The finding that GABAergic synapses were detectable in unc-55;unc-8 double mutants is consistent with the partial restoration of fluorescently-labeled presynaptic markers to ventral GABA neuron processes in unc-55; unc-8 animals ( Figure 3A , Figure 3—figure supplement 1 , Figure 4 A–G ) and therefore argues that the wild-type unc-8 gene promotes disassembly of multiple components of the presynaptic apparatus .",
"Having confirmed that unc-55; unc-8 animals contain GABAergic presynaptic domains we next used electrophysiological recordings to ask if these residual synapses are functional .",
"Spontaneous inhibitory postsynaptic events ( iPSCs ) arising from GABA release were recorded from ventral body muscles ( see Materials and methods ) .",
"Robust , iPSCs were detected for wild-type and unc-8 animals ( Figure 4K , L ) .",
"Ventral iPSCs were not produced in unc-55 mutants as expected since these animals lack ventral GABAergic synapses ( Figure 3A ) ( Petersen et al . , 2011 ) .",
"Interestingly , ventral iPSCs were not restored in unc-55; unc-8 animals despite the presence of organized clusters of fluorescent presynaptic proteins ( Figure 3A , Figure 4A–G ) and electron dense active zones with abundant synaptic vesicles ( Figure 4I–J ) .",
"Total ventral synaptic activity ( cholinergic and GABAergic ) was measured to confirm that spontaneous ventral cholinergic activity was unaffected by the unc-55 and unc-8 mutations ( Figure 4—figure supplement 1E–F ) .",
"We see no significant change in the expression or clustering of postsynaptic GABAA receptors ( UNC-49::GFP ) in unc-55; unc-8 animals in comparison to wild type; UNC-49::GFP is closely co-localized with presynaptic mCherry::RAB-3 at ventral unc-8 and unc-55; unc-8 GABAergic synapses both before remodeling and in adults ( Figure 4—figure supplement",
"3 ) ( Gally and Bessereau , 2003; Petersen et al . , 2011 ) .",
"Together , these data argue against the possibility that the absence of IPSCs in unc-55; unc-8 animals is caused by a defective postsynaptic response to GABA and therefore favor the hypothesis that presynaptic dysfunction limits GABA secretion .",
"To summarize , we have shown that unc-55 animals lack GABAergic neuromuscular junctions and that presynaptic components are partially restored in unc-55; unc-8 mutants .",
"Despite the presence of both presynaptic domains with abundant synaptic vesicles and clusters of postsynaptic GABAA receptors , residual GABAergic synapses in unc-55; unc-8 animals are not functional .",
"Taken together , these results indicate that UNC-8 is required for the efficient removal of the ventral presynaptic apparatus , but that the presynaptic defect of unc-55; unc-8 animals could be due to an additional UNC-8-independent pathway , potentially involving the previously described homeodomain transcription factor irx-1 ( Petersen et al . , 2011 ) , acting in parallel to promote the disassembly of the ventral synaptic architecture of remodeling GABAergic neurons .",
"We have shown that UNC-8 protein preferentially gates sodium when expressed in Xenopus oocytes ( Wang et al . , 2013 ) .",
"We therefore considered the hypothesis that UNC-8 channel function is required for synaptic elimination .",
"This idea is consistent with our finding that the unc-8 mutant disrupts DD and VD synapse removal ( Figures 2C and 3A ) .",
"As a specific test of this hypothesis , we treated unc-55 mutant animals with Benzamil , which has previously been shown to inhibit DEG/ENaC channel activity ( Kleyman and Cragoe , 1988 ) .",
"In this experiment , unc-55 mutant animals were grown on media containing 3 mM Benzamil ( see Materials and methods ) .",
"Ventral SNB-1::GFP puncta were scored in adult animals .",
"As expected , unc-55 animals from control plates show few ventral SNB-1::GFP puncta in GABA neurons ( Figure 5A , B ) .",
"In contrast , treatment of unc-55 animals with Benzamil results in a significantly larger number of residual SNB-1::GFP puncta on the ventral side ( Figure 5A , B ) .",
"However , Benzamil treatment of unc-55;unc-8 animals does not induce any additional enhancement of ventral SNB-1::GFP puncta .",
"This result argues that Benzamil antagonizes synaptic removal by specifically inhibiting UNC-8 ( Figure 5—figure supplement 1A ) .",
"To confirm that Benzamil inhibits UNC-8 channel activity , we expressed the constitutively active UNC-8 ( G387E ) protein in Xenopus oocytes for electrophysiological analysis ( Figure 5—figure supplement 1B ) .",
"We have previously established that the UNC-8 ( G387E ) protein shows robust sodium transport activity in an extracellular solution that is depleted of calcium and other divalent cations ( Figure 5C , Figure 5—figure supplement 1C ) ( Wang et al . , 2013 ) .",
"Under these conditions , Benzamil strongly inhibits UNC-8 ( G387E ) sodium transport with a Ki ~ 119 μM ( Figure 5C , D ) .",
"In the presence of extracellular calcium , Benzamil acts as a more potent inhibitor ( Ki = 47 μM ) suggesting that calcium ions enhance Benzamil binding ( Figure 5—figure supplement 1C ) .",
"A similar effect of extracellular divalent cations on inhibitor potency was previously observed for the DEG/ENaC inhibitor Amiloride ( Wang et al . , 2013 ) .",
"Treatment with Amiloride also antagonizes the removal of ventral SNB-1::GFP puncta in unc-55 mutant neurons ( Figure 5B ) , but does not alter ventral SNB-1::GFP puncta in either unc-8 or unc-55;unc-8 animals ( Figure 5—figure supplement 1A ) .",
"Taken together , these results show that Amiloride and Benzamil disrupt the GABA neuron synaptic removal and are consistent with the idea that this effect is due to the inhibition of sodium transport by an UNC-8-containing DEG/ENaC channel ( Figure 5E ) . 10 . 7554/eLife . 14599 . 014Figure 5 . UNC-8 cation channel activity promotes the removal of ventral synapses in remodeling GABA neurons .",
"( A ) Representative images and fluorescence intensity plots ( generated from 20 μm dashed region ) of SNB-1::GFP-marked ventral GABA neuron synapses in unc-55 animals treated with either 3 mM Benzamil or water ( control ) .",
"Scale bar is 10 μm .",
"( B ) Benzamil and Amiloride antagonize the removal of ventral GABA synapses in unc-55 mutant animals .",
"Ventral GABA neuron synapses were quantified by counting SNB-1::GFP puncta ( ***p<0 . 001 , ns is not significant , n ≥ 25 animals , data are mean ± SD , One-Way ANOVA with Bonferroni correction ) .",
"( C ) Benzamil blocks UNC-8 ( G387E ) current in Xenopus oocytes .",
"Representative currents from oocyte expressing UNC-8 ( G387E ) in a bath of divalent cation-free solution plus EGTA .",
"Currents elicited by 20mV voltage steps from -160mV to +100mV .",
"The holding potential was -30mV .",
"The gray line represents the zero current level ( left ) .",
"The same oocyte exposed to 500µM Benzamil ( right ) .",
"( D ) Benzamil dose-response curve in divalent cation-free bath solution .",
"Currents recorded with Benzamil were normalized against recordings in divalent cation-free bath solution plus EGTA at -100mV .",
"Data were fitted to the Boltzmann's equation to derive Ki = 119 µM ( n = 10 oocytes ) .",
"Data are mean ± SEM .",
"( E ) Pharmacological inhibition of UNC-8 channel activity with either Benzamil or Amiloride blocks removal of ventral synapses in remodeling GABA neurons synaptic remodeling ( EC is extracellular ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 01410 . 7554/eLife . 14599 . 015Figure 5—figure supplement 1 . UNC-8 is required for the inhibitory effect of Benzamil and Amiloride on synaptic removal .",
"( A ) Treatment of unc-8 and unc-55; unc-8 animals with either 3 mM Amiloride or 3 mM Benzamil does not limit removal of ventral synapses .",
"Vehicle control for Amiloride and Benzamil treatment is water ( n ≥ 10 , **p<0 . 01 , ***p<0 . 001 , ns is not significant , mean ± SEM , One-Way ANOVA with Bonferroni correction ) .",
"( B ) The point mutant G387E renders the UNC-8 channel constitutively active and was used for in vitro oocyte experiments .",
"Schematic shows the unc-8 genomic region and denotes the locations of transmembrane domains TM1 and TM2 and the predicted extracellular loop region .",
"( C ) UNC-8 ( G387E ) currents recorded in physiological solution plus the DEG/ENaC inhibitor Benzamil at -100mV were normalized against currents recorded in physiological solution at -100mV .",
"Data were fitted with the Boltzmann's equation for Ki = 46 . 6 µM ( n = 10 oocytes ) .",
"Data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 015 Previous work has shown that neuronal activity promotes DD presynaptic remodeling; the dorsal DD synapse formation is accelerated in mutants that enhance synaptic activity and delayed by genetic defects that block neurotransmission ( Thompson-Peer et al . , 2012 ) .",
"We predicted that neuronal activity is also required for the removal of ventral synapses in remodeling GABA neurons .",
"Voltage-gated calcium channels ( VGCCs ) promote synaptic vesicle fusion and neurotransmitter release by elevating intracellular calcium ( Catterall et al . , 2008 ) .",
"In C . elegans , the unc-2 gene encodes a CaV2 α1 subunit of a P/Q-type calcium channel ( Mathews et al . , 2003 ) .",
"UNC-2 is localized to the presynaptic terminals of ventral cord motor neurons and is required for neurotransmitter release ( Gracheva et al . , 2008; Saheki and Bargmann , 2009 ) .",
"We determined that unc-55; unc-2 double mutants show a significantly greater number of SNB-1::GFP puncta on the ventral side in comparison to unc-55 animals ( Figure 6A , B ) .",
"This result indicates that wild-type unc-2 promotes synapse removal in remodeling GABA neurons .",
"Because comparable levels of ventral SNB-1::GFP signal were observed in unc-55; unc-8 animals , we next conducted a genetic test to ask if unc-2 and unc-8 eliminate ventral synapses by either independent or shared mechanisms .",
"We observed no additional increase in the number of ventral cord SNB-1::GFP puncta in unc-55; unc-8; unc-2 animals compared to either double mutant ( i . e . , unc-55;unc-8 or unc-55;unc-2 ) .",
"This result favors a model in which unc-2 and unc-8 act together in a linear pathway to remove ventral synapses in remodeling GABA neurons ( Figure 6A , B , E ) . 10 . 7554/eLife . 14599 . 016Figure 6 . UNC-8 drives synaptic remodeling in an activity-dependent pathway that requires neurotransmitter release .",
"( A ) Loss-of-function mutations in either unc-2 ( VGCC ) or unc-8 impairs removal of ventral SNB-1::GFP in unc-55 animals .",
"The unc-8 mutation does not enhance the unc-55;unc-2 remodeling defect , demonstrating that UNC-2/VGCC and UNC-8/DEG/ENaC function in a common pathway to promote GABA synapse removal ( ***p<0 . 001 vs unc-55 , One-Way ANOVA with Bonferroni correction , data are mean ± SD , n ≥ 17 ) .",
"( B ) Representative images and insets show fluorescence intensity plots over a 20 μm region of the ventral nerve cord for each genotype .",
"Scale bar is 5 μm .",
"( C ) DD synapses are precociously remodeled in tom-1 mutants .",
"This effect is suppressed in unc-8; tom-1 animals ( *p<0 . 05 , ***p<0 . 001 vs wild type , ††p<0 . 01 vs tom-1 Student’s t-test , results pooled from > 3 independent experiments per genotype ) Plots are normalized for the total number of GABA neurons labeled with SNB-1::GFP in the ventral cord to account for developmental delay in tom-1 mutants .",
"( D ) Optogenetic stimulation of channelrhodopsin ( ChR2 ) -induced activity in GABA neurons for 13 hr ( 0 . 5 Hz ) results in precocious appearance of SNB-1::GFP marked DD synapses in the dorsal nerve cord ( n ≥ 18 animals , ***p<0 . 001 , data are mean ± SEM , One-Way ANOVA with Bonferroni correction ) , ATR is all-trans retinal .",
"Mutant alleles were unc-2 ( e55 ) and tom-1 ( ok2437 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 016 We performed an additional genetic experiment to confirm that UNC-8 functions in an activity-dependent pathway in remodeling DD motor neurons .",
"The tomosyn protein , TOM-1 , normally down-regulates neurotransmitter release by inhibiting synaptic vesicle ( SV ) priming ( Gracheva et al . , 2006; McEwen , 2006 ) .",
"We confirmed that a loss-of-function tom-1/tomosyn mutant , which enhances evoked SV fusion , results in precocious assembly of dorsal DD synapses ( Figure 6C ) ( Thompson-Peer et al . , 2012 ) .",
"This effect was not observed , however , in tom-1;unc-8 double mutants which suggests that wild-type unc-8 function is necessary for the accelerated remodeling of DD neurons in tom-1 mutant animals ( Figure 6C ) .",
"Although we previously determined that UNC-8 is exclusively involved in the removal of ventral DD synapses in wild-type animals , this result suggests that UNC-8 is also necessary for the precocious assembly of dorsal DD synapses in tom-1 mutants .",
"Perhaps UNC-8 exerts an indirect effect on dorsal assembly in this context in which remodeling is triggered prematurely when potential recycling of presynaptic components to nascent dorsal synapses could be limiting ( Park et al . , 2011 ) .",
"Our genetic results indicate that UNC-8 functions in an activity-dependent synaptic remodeling pathway .",
"Because this conclusion is based on the analysis of mutants that alter neural activity throughout the C . elegans nervous system , we performed an experiment to ask if the GABA neuron activity alone is sufficient to drive synaptic remodeling .",
"This requirement predicts that optogenetic activation of GABAergic neurons should accelerate the remodeling process .",
"To test this idea , we used a GABA neuron-specific promoter to drive channelrhodopsin ( ChR2 ) expression in DD neurons ( Liu et al . , 2009 ) .",
"Blue light exposure of these GABA::ChR2 transgenic animals evoked muscle relaxation with an immediate cessation of locomotion as expected for a treatment that enhances GABA release ( data not shown ) ( Schuske et al . , 2004 ) .",
"Synchronized populations of GABA::ChR2 animals were exposed to blue light at 0 . 5 Hz for 13 hr .",
"DD neurons were assayed for precocious remodeling by counting the number of dorsal SNB-1::GFP puncta at the end of this period .",
"The results of this experiment show that optogenetic activation of GABA neurons accelerates DD remodeling and that both blue light and exogenous all-trans retinal ( ATR ) are required for this effect; DD neurons that express ChR2 ( GABA::ChR2 ) remodel earlier with significantly more dorsal puncta after 13 hr of light exposure in comparison to controls ( Figure 6D ) .",
"This finding argues that neuronal activity in DD neurons is sufficient to drive synaptic remodeling and thus suggests that a mechanism linking neural activity and unc-8 function could be cell autonomous to GABA neurons .",
"We have shown that calcium signaling through UNC-2 induces removal of ventral GABA synapses and that UNC-8 functions in this pathway to promote synapse disassembly ( Figure 6A ) .",
"In considering potential mechanisms for this effect , we performed a genetic experiment to test the idea that elevated intracellular calcium arising from UNC-2 function could activate cytoplasmic signaling proteins that drive synapse disassembly .",
"One of these candidate downstream effectors , the calcium/calmodulin-activated phosphatase , calcineurin , is highly expressed throughout the nervous system and has been implicated in activity-dependent mechanisms that regulate synaptic maintenance and function ( Baumäartel and Mansuy , 2012; Winder et al . , 1998 ) .",
"Calcineurin is composed of two protein components , the catalytic subunit calcineurin A and the regulatory subunit calcineurin B . To determine if calcineurin function is required for the GABA neuron remodeling mechanism , we first tested the role of the C . elegans calcineurin A homolog , TAX-6 ( Figure 7A ) .",
"We found that a tax-6 loss-of-function allele impedes the removal of ventral synapses in remodeling GABA neurons ( Figure 7B ) .",
"To ask if tax-6 is expressed in GABA neurons , we determined that a translational reporter gene , ptax-6::TAX-6::GFP is co-localized with the GABA neuron-specific marker , punc-47::mCherry ( Figure 7C ) .",
"We then used a loss-of-function allele of the calcineurin B homolog cnb-1 to confirm the role of calcineurin in synaptic disassembly .",
"Our results indicate that cnb-1 function is required for the efficient removal of ventral synapses in remodeling GABA neurons .",
"In addition , genetic ablation of unc-8 in unc-55; cnb-1 animals do not enhance this phenotype , which suggests that UNC-8 and calcineurin promote synapse elimination in a common pathway ( Figure 7D ) . 10 . 7554/eLife . 14599 . 017Figure 7 . The calcium/calmodulin-dependent phosphatase calcineurin promotes GABA synapse removal in the UNC-8 pathway .",
"( A ) Calcineurin A and B subunits ( TAX-6 and CNB-1 , respectively ) require calcium and calmodulin ( CaM ) to activate phosphatase activity .",
"( B ) Loss of tax-6 partially suppresses the unc-55 remodeling phenotype in GABA neurons ( ***p<0 . 001 , ns is not significant , One-Way ANOVA with Bonferroni correction , data are mean ± SD , n ≥ 25 ) .",
"( C ) GFP-tagged TAX-6 under the control of the tax-6 promoter region ( ptax-6::TAX-6::GFP ) is expressed in GABA neurons ( punc-47::mCherry ) .",
"Scale bar is 20 μm .",
"( D ) CNB-1 is required for remodeling in unc-55 animals .",
"Loss of cnb-1 function does not enhance the unc-55; unc-8 remodeling defect , suggesting that calcineurin and UNC-8 promote synapse removal in a common genetic pathway ( left , ***p<0 . 001 compared to wild type , tttp<0 . 001 compared to unc-55 , ns is not significant , One-Way ANOVA with Bonferroni correction , data are mean ± SD , wild type n = 10 , mutants n = 20 ) .",
"Representative images of ventral nerve cords in unc-55 , unc-55; cnb-1 and unc-55; unc-8; cnb-1 animals .",
"Asterisks denote GABA neuron soma , scale bar is 20 μm ( right ) .",
"( E ) Gain-of-function tax-6 ( tax-6d ) mutants remodel precociously and this effect is suppressed by Benzamil .",
"Percentage of ventral ( blue ) vs dorsal ( gray ) DD synapses ( pflp-13::GFP::RAB-3 , **p<0 . 01 , ***P<0 . 001 , ns is not significant , One-Way ANOVA with Bonferroni correction , n ≥ 8 animals per timepoint , data are mean ± SEM ) .",
"Results for tax-6d and for the tax-6d control for Benzamil treatment ( see Materials and methods ) are combined because they were not significantly different .",
"Benz denotes 3mM DEG/ENaC inhibitor Benzamil .",
"Mutant alleles were tax-6 ( p675 ) , tax-6d ( jh107 ) , cnb-1 ( ok276 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 017 These results indicate that calcineurin promotes the removal of ventral synapses and therefore predict that the constitutively active gain-of-function allele tax-6 ( d ) should induce precocious synapse disassembly .",
"To test this idea , we used the presynaptic marker GFP::RAB-3 to determine that DD ventral synapses are removed prematurely in tax-6 ( d ) mutants; dorsal synaptic assembly is also precocious and occurs significantly earlier than in wild-type controls .",
"Additionally , we determined that treatment of tax-6 ( d ) animals with the DEG/ENaC inhibitor Benzamil significantly delays the precocious remodeling phenotype ( Figure 7E ) .",
"These results argue that intracellular calcium acting through TAX-6/Calcineurin promotes the overall synaptic remodeling mechanism and that this effect requires UNC-8 channel activity , thereby suggesting that unc-8 functions downstream of tax-6 .",
"To summarize , our findings are consistent with the hypothesis that neuronal activity drives synaptic remodeling through a calcium-dependent signaling pathway in which calcineurin and unc-8 function together to promote disassembly of the presynaptic apparatus .",
"Previous work has shown that the canonical apoptotic pathway promotes the removal of ventral GABA synapses in remodeling DD neurons .",
"In this mechanism , the adaptor protein CED-4/Apaf1 and its downstream effector , CED-3/caspase activate the actin-severing protein gelsolin to destabilize a presynaptic F-actin network during synapse elimination ( Meng et al . , 2015 ) .",
"Because synaptic removal is activated during a discrete developmental period , a specific signal is likely required to trigger this pathway .",
"Based on our finding that calcium signaling and UNC-8 function together to promote GABA synapse elimination and previous work that detected roles for calcium in caspase activation and gelsolin function ( Pinan-Lucarre et al . , 2012; Liu et al . , 2011 ) , we considered the possibility that these components function in a common pathway and that calcium activates the overall mechanism .",
"To test this idea , we first asked if the cell death gene ced-4 is required for GABA synapse removal in unc-55 mutants in which both DD and VD neurons remodel .",
"A loss-of-function mutation in ced-4 results in significant retention of ventral GABA synapses in comparison to unc-55 mutant animals and therefore confirms that ced-4 is necessary for the efficient removal of ventral synapses in both classes ( e . g . , DD and VD ) of remodeling GABA neurons ( Figure 8A , [Meng et al . , 2015] ) .",
"The ced-4-dependent effect on remodeling in unc-55 animals is comparable to that of unc-55; unc-8 double mutants .",
"Furthermore , the combination of ced-4 and unc-8 mutants did not further enhance the restoration of ventral synapses over that of either unc-55; unc-8 or unc-55; ced-4 animals ( Figure 8A ) .",
"Together , these results suggest that the DEG/ENaC protein UNC-8 functions in a common pathway with ced-4 to eliminate ventral GABA synapses .",
"Because we have also shown that calcium and unc-8 function in a common pathway to promote removal of GABA synapses , we propose that a rise in intracellular calcium in active GABA neurons is sufficient to trigger ced-4-dependent synapse elimination ( Figure 8B , see Discussion ) . 10 . 7554/eLife . 14599 . 018Figure 8 . Model of UNC-8-driven synapse disassembly in the GABA neuron remodeling program .",
"( A ) A loss-of-function mutation in the pro-apoptotic gene ced-4 partially suppresses GABA neuron remodeling in unc-55 animals .",
"Genetic ablation of unc-8 in the triple mutant unc-55; ced-4; unc-8 mutants does not enhance the remodeling defect and thus suggests that UNC-8 and CED-4 promote synapse elimination in a common pathway ( ***p<0 . 001 compared to wild type , tttp<0 . 001 compared to unc-55 , ns is not significant , One-Way ANOVA with Bonferroni correction , data are mean ± SD , wild type n = 10 , mutants n ≥ 20 ) Mutant allele is ced-4 ( n1162 ) .",
"( B ) Model of a calcium-dependent mechanism for removal of the presynaptic apparatus .",
"A DEG/ENaC channel containing UNC-8 is not active ( gray ) in quiescent GABA motor neurons ( top panel ) .",
"( 1 ) GABA neuron depolarization activates the voltage-gated calcium channel ( VGCC ) , UNC-2 , to allow calcium entry ( black circles bottom panel ) .",
"( 2 ) Intracellular calcium activates the calcium/calmodulin-dependent phosphatase , calcineurin ( CaN/TAX-6 ) .",
"( 3 ) CaN phosphatase may activate UNC-8 , which results in the movement of sodium ions ( white circles ) into the presynaptic GABA neuron , further depolarizing the presynaptic membrane and activating VGCCs .",
"This positive feedback loop is predicted to further elevate intracellular calcium .",
"( 4 ) Our results show that UNC-8 drives the removal of presynaptic components and functions in a common genetic pathway with calcium signaling and with the apoptotic protein CED-4 .",
"Therefore , we hypothesize that selective expression of UNC-8 in remodeling GABA neurons effectively boosts the level of intracellular calcium to activate a CED-4-dependent pathway for removal of the presynaptic apparatus . DOI: http://dx . doi . org/10 . 7554/eLife . 14599 . 018"
],
[
"Previous work has detected roles for DEG/ENaC proteins in synaptic function and neural plasticity .",
"For example , members of the ASIC ( acid-sensing ion channels ) class of the DEG/ENaC family proteins are activated by low pH and have been implicated in learning and memory ( Wemmie et al . , 2002 , 2004; Zha et al . , 2006 ) .",
"In one possible mechanism to explain this role , acidification of the synaptic cleft that accompanies the neurotransmitter release is thought to trigger ASIC-mediated sodium influx .",
"The resultant membrane depolarization is proposed to enhance postsynaptic signaling by activating local voltage gated ion channels and NMDA receptors ( Wemmie et al . , 2013 ) .",
"Presynaptic roles are also possible as suggested by studies in C . elegans that detected an ASIC-like channel that promotes neurotransmitter secretion in a learning and memory paradigm ( Voglis and Tavernarakis , 2008 ) .",
"Our finding that a reconstituted UNC-8 channel is in fact inhibited by low pH , however , suggests that UNC-8 is unlikely to function as an ASIC-like protein in vivo ( Wang et al . , 2013 ) .",
"This observation is important because non-ASIC members of the DEG/ENaC family are also expressed in the brain , but their roles in synaptic function are largely unknown ( Giraldez et al . , 2013; Yamamura et al . , 2004; Waldmann , 1995 ) .",
"A recent study established that the DEG/ENaC protein , pickpocket , exerts a homeostatic function that maintains neurotransmitter release at the Drosophila neuromuscular junction .",
"In this mechanism , sodium influx arising from elevated pickpocket transcription and membrane insertion is proposed to result in local depolarization that activates a nearby voltage-gated calcium channel ( VGCC ) and consequent neurotransmitter release ( Younger et al . , 2013 ) .",
"This model is consistent with the finding that the CaV2 . 1 VGCC channel subunit cacophony is required for synaptic homeostasis ( Müller and Davis , 2012 ) .",
"Our results are suggestive of a similar presynaptic role for UNC-8 of enhancing intracellular calcium influx with the important difference that UNC-8 ultimately eliminates neurotransmitter secretion by triggering destruction of the presynaptic apparatus .",
"In addition , our findings provide a plausible explanation for the radically different outcomes of pickpocket and UNC-8 channel function .",
"In this study , we demonstrate that UNC-8 promotes the removal of presynaptic domains in remodeling GABAergic neurons .",
"This UNC-8 function requires its intrinsic cation channel activity ( Figure 5 ) which likely involves sodium influx since electrophysiological studies of a reconstituted UNC-8 channel in Xenopus oocytes indicate that UNC-8 preferentially transports sodium and is impermeant to calcium ( Wang et al . , 2013 ) .",
"Because our evidence points to punctate UNC-8 localization within the ventral nerve cord ( Figure 2F ) , it is reasonable to predict that UNC-8-dependent sodium import would activate nearby VGCC channels ( Wemmie et al . , 2013 ) .",
"This idea is consistent with the observation that VGCC function is required for ASIC-induced elevation of intracellular calcium in hippocampal neurons ( Zha et al . , 2006 ) .",
"Furthermore , our results show that synaptic removal in remodeling C . elegans GABA neurons is disrupted by genetic ablation of the CaV2 channel subunit , UNC-2 .",
"As a key component of the presynaptic apparatus , UNC-2 mediates calcium influx to drive neurotransmitter release ( Richmond et al . , 2001 ) .",
"We suggest that the UNC-2-dependent rise in cytoplasmic calcium in active GABA neurons is enhanced by UNC-8 cation channel function .",
"Moreover , because our genetic results indicate that unc-2 and unc-8 function in a common pathway ( Figure 6A ) , the elevation of intracellular calcium arising from this interaction could be required for efficient removal of the presynaptic apparatus in remodeling GABA neurons ( see below ) .",
"The idea of a downstream role for calcium in synaptic remodeling is underscored by our finding that the calcineurin homolog , TAX-6 , also functions in the unc-8-dependent mechanism of synapse elimination ( Figure 7 ) .",
"The serine-threonine phosphatase activity of calcineurin/CaN is calcium and calmodulin-dependent and has been shown to regulate neural plasticity .",
"For example , calcineurin/CaN antagonizes LTP in a mechanism that dephosphorylates the GluR1 subunit of the AMPA receptor to promote its removal from the postsynaptic membrane ( Winder et al . , 1998; Gorski et al . , 2012; Lee et al . , 1998 ) .",
"TAX-6/Calcineurin could exert a parallel role in the removal of the presynaptic apparatus in GABA neurons by activating the UNC-8 channel .",
"Our finding that the DEG/ENaC inhibitor , Benzamil , potently blocks the accelerated remodeling phenotype of a constitutively active TAX-6 protein is consistent with this idea ( Figure 7E ) .",
"The resultant positive feedback loop involving UNC-2 , TAX-6/Calcineurin and UNC-8 should further elevate intracellular calcium ( Figure 8 ) .",
"In an analogous mechanism , phosphorylation by CaMKII activates ASIC1a and the resultant VGCC-dependent elevation of intracellular calcium in turn promotes CaMKII activity ( Zha et al . , 2006; Gao et al . , 2005; Zha and Zha , 2013 ) .",
"In this model , UNC-8 would act as a synaptic amplifier to drive a self-reinforcing signaling pathway that elevates intracellular calcium .",
"The key role of calcium signaling in this model is consistent with previous work showing that removal of the GABA presynaptic apparatus requires components of the canonical apoptotic pathway including the caspase-3 homolog CED-3 and its upstream activator CED-4/Apaf1 .",
"In this mechanism , the CED-3 protease cleaves the actin-severing protein gelsolin to release an active gelsolin domain that in turn destabilizes an F-actin network at the presynaptic membrane ( Meng et al . , 2015 ) .",
"Notably , both CED-3 and gelsolin function in calcium-dependent pathways ( Pinan-Lucarre et al . , 2012; Liu et al . , 2011 ) .",
"Because our genetic evidence argues that unc-8 and ced-4 function in a common pathway ( Figure 8A ) , we propose a model in which UNC-8 promotes the elevation of intracellular calcium above a critical threshold that then triggers the apoptotic pathway to dismantle the presynaptic region .",
"The localization of apoptotic components to the presynaptic membrane ( Meng et al . , 2015 ) may ensure that a local rise in calcium near the synapse is sufficient to activate this mechanism , but also protects the neuron from apoptotic death .",
"The conserved role of CED-3/caspase-3 in synapse elimination in other species ( Ertürk et al . , 2014; Wang et al . , 2014;Li et al . , 2010 ) argues that similar DEG/ENaC-dependent mechanisms could be widely employed to trigger synaptic destruction .",
"Our results show that unc-8 promotes the removal of ventral synapses in remodeling DD and VD motor neurons , but is generally not necessary for the nascent assembly of dorsal synapses ( Figure 2E ) .",
"The proposed downstream role of the apoptotic pathway is similarly limited to synaptic removal ( Meng et al . , 2015 ) .",
"In addition , the elimination of GABAergic synapses is only partially dependent on this unc-8 pathway ( Figure 2C , Figure 3 , Figure 3—figure supplement 1A , Figure 4 ) ( Meng et al . , 2015 ) .",
"These findings point to important roles for additional components in GABA neuron synaptic remodeling ( Howell et al . , 2015; He et al . , 2015 ) .",
"For example , the cyclin box-containing protein CYY-1 and cyclin-dependent kinase CDK-5 function together to promote synaptic removal and reassembly , respectively ( Park et al . , 2011 ) .",
"In previous work , we have shown that the Iroquois family homeodomain transcription factor , IRX-1 , promotes synaptic removal .",
"IRX-1 also promotes dorsal DD synapse formation , suggesting that IRX-1 orchestrates expression of multiple genes that drive DD synaptic remodeling ( Petersen et al . , 2011 ) .",
"The exact function of the IRX-1 pathway in synaptic disassembly is unknown , but likely involves the extraction of components that are required for GABA release since functional GABA synapses are restored to the ventral nerve cord of unc-55 animals that are also mutant for irx-1 ( Petersen et al . , 2011 ) .",
"In the future , it will be interesting to define the specific roles of each of these effectors in the mechanism of GABA neuron synaptic remodeling .",
"Despite the necessity for additional pathways functioning in parallel to unc-8 to execute a comprehensive GABA neuron remodeling program , our results also determined that UNC-8 alone is sufficient to trigger synapse elimination ( Figure 3—figure supplement 2B ) .",
"This finding argues that transcriptional regulation of unc-8 expression effectively functions as a genetic switch that determines the developmental fate of the presynaptic signaling apparatus in active GABAergic neurons ."
],
[
"Strains were maintained at 20°C on NGM plates seeded with OP50 ( E . coli ) , unless otherwise stated ( Brenner , 1974 ) .",
"The wild type strain is N2 and only hermaphrodite animals were analyzed .",
"The unc-55 ( e1170 ) and unc-8 ( tm5052 ) alleles were used for these studies .",
"For a complete list of the strains used in this study , see Supplementary file 1A .",
"The unc-8 ( tm5052 ) allele was obtained from a UV/TMP mutagenized library , as described previously ( Gengyo-Ando and Mitani , 2000 ) and was identified by PCR amplification with primers spanning the deleted region .",
"tm5052 likely corresponds to an unc-8 null allele because it deletes a portion of the fifth and entire sixth exon ( 197 base pairs ) with the insertion of CT resulting in a premature stop codon prior to the first transmembrane domain .",
"We used the following primers to detect the tm5052 mutation: Forward 5’-TGGGGCCCTAATAATTTCGA-’3 and Reverse 5’- AGTGACAGTATGAAGCCAGG-’3 .",
"Construction of GABA csRNAi transgenic lines RNAi plasmids used for GABA neuron-specific knock down of unc-8 target the first 7 exons of the unc-8 coding region .",
"To clone the unc-8 sense construct pSA76 , a 2 . 3 kb region of unc-8 cDNA was amplified with the following primers containing 5’AscI/3’SacII adaptors: Forward 5’- GGCGCGCCATGTCACCTTTGCTGACGT-3’ and Reverse 5’-GCCAGGAGGTGATATTCTAGCCGCGG-3’ .",
"This fragment was cloned into pCR2 . 1 via TOPO-TA reaction ( Invitrogen , Waltham , MA ) to yield pSA75 .",
"The 2 . 3 kb unc-8 cDNA fragment was then subcloned into the existing GABAergic cell-specific RNAi ( csRNAi ) plasmid pSA47 via AscI/SacII to yield pSA76 ( Petersen et al . , 2011 ) .",
"pSA76 contains the DD/VD specific promoter , pttr-39 and the unc-119 wild-type mini gene ( Maduro and Pilgrim , 1995 ) .",
"To construct the unc-8 antisense plasmid pSA78 , the 2 . 3 kb unc-8 cDNA fragment was amplified with the following primers containing 5’’SacII/3’AscI adaptors: Forward 5’-CCGCGGATGTCACCTTTGCTGACGTG-3’ and Reverse 5’- CCAGGAGGTGATATTCTAGGGCGCGCC-3’ .",
"The unc-8 antisense fragment was subcloned into pCR2 . 1 via TOPO-TA reaction ( Invitrogen ) to yield pSA73 .",
"The unc-8 fragment from pSA73 was then inserted into the GABA neuron-specific RNAi ( csRNAi ) plasmid pSA47 via AscI/SacII to yield pSA77 .",
"The unc-8-containing region of pSA77 between ScaI and SacII was then inserted into plasmid pSA49 to yield pSA78 .",
"The pttr-39 promoter in pSA78 drives expression of the 2 . 3 kb unc-8 antisense fragment and mCherry .",
"pSA76 ( unc-8 sense ) and pSA78 ( unc-8 antisense ) were linearized and ligated , then transformed into unc-119 worms via microparticle bombardment to yield a spontaneous integrant ( strain NC2601 ) as indicated by 100% transmission of rescued ( unc-119+ ) movement ( indicating unc-8 sense ) and mCherry expression in all GABAergic motor neurons ( indicating unc-8 antisense ) ( Praitis et al . , 2001 ) .",
"A control plasmid was also created containing pttr39-driven mCherry and the unc-119 rescuing gene , which was transformed into unc-119 worms via microparticle bombardment .",
"The UNC-8::GFP fosmid was recombineered as previously described ( Tursun et al . , 2009 ) .",
"Briefly , the 30 kb fosmid WRM0635cA02 containing the unc-8 genetic locus was obtained from GeneService ( Nottingham , UK ) and purified .",
"Fosmid DNA was transformed into electrocompetent SW105 cells and was verified by PCR .",
"A GFP-galK recombineering cassette was amplified with 50 kb homology arms from pBALU1 and gel-purified .",
"The GFP-galK PCR product was transformed into electrocompetent , λRed recombinase-activated , fosmid-containing SW105 cells .",
"The cells containing the fosmid and GFP-galK were grown for more than 60 hr at 32°C and streaked on MacConkey and galactose plates with chloramphenicol to ensure the insertion of recombineering cassette .",
"To excise galK from the GFP intron , colonies were incubated with 0 . 1% arabinose to create an unc-8::GFP expression fosmid .",
"This unc-8::GFP fosmid was then purified and confirmed by sequencing .",
"The fosmid was injected into unc-8 ( tm5052 ) animals at 25 ng/μl with co-injection marker pceh-22::GFP at 15 ng/μl .",
"UNC-8 cDNA was PCR-amplified from pSGEM/pTWM60 ( Wang et al . , 2013 ) with primers that span the UNC-8 cDNA sequence and exclude the 3’ stop codon .",
"The primer sequences are: Forward 5’-ATGAGCGCAAGGAGTAGT-3’ and Reverse 5’- TTTGCTCATTAACTCCTTTGT-3’ .",
"Primers include either 5’-AscI or 3’-SacII adaptors for inserting UNC-8 cDNA into pMLH260 ( punc-25::coq1cDNA::GFP::unc-54 ) in place of the coq-1 fragment .",
"The resultant plasmid , pTWM62 ( punc-25::UNC-8::GFP ) was injected ( 10 ng/μl ) with co-selectable marker pmyo-2::mCherry::unc-54 ( 2 . 5 ng/μl ) into unc-8 ( tm5052 ) animals .",
"UNC-8 cDNA was PCR-amplified from pSGEM/pTWM60 ( Wang et al . , 2013 ) with primers that span the UNC-8 cDNA sequence .",
"The primer sequences are: Forward 5’-ATGAGCGCAAGGAGTAGT-3’ and Reverse 5’- TTTGCTCATTAACTCCTTTGT-3’ .",
"Primers include either 5’-AscI or 3’-EcoRI adaptors for inserting UNC-8 cDNA into pTWM35 ( pttr39::arx-5::GFP::unc-54 ) in place of the ARX-5::GFP fragment .",
"The resultant plasmid , pTWM92 ( pttr-39::UNC-8cDNA ) was injected ( 25 ng/μl ) with co-selectable markers pmyo-2::mCherry::unc-54 ( 2 ng/μl ) and punc-25::mCherry::RAB-3 ( 5 ng/μl ) into unc-55; unc-8 ( tm5052 ) juIs1 or juIs1 animals .",
"For time-course experiments , 100 adult hermaphrodites from each genotype were picked to fresh 60 mm plates and allowed to lay eggs for one hour .",
"The mid-point at which the eggs were laid is considered T0 .",
"All adults were removed from the plates after 1 hr .",
"Plates were maintained at 23°C until assayed .",
"Puncta arising from localization of fluorescent presynaptic markers were counted with a Zeiss Axiovert microscope ( 63X oil objective ) in immobilized animals .",
"For timecourse experiments puncta were counted between DD1 and DD6 ( Figures 2C , 6C , D , 7E , Figure 2—figure supplement 1B–D ) or from VD3 to VD11 in adults ( Figure 3—figure supplement 1B ) .",
"Data were pooled from 3 separate experiments .",
"In young adults , labeled puncta were counted in the ventral nerve cord region between VD3 and VD11 ( Figures 2E , 4A–E , 5B , 6A , 7B , D , 8A , Figure 1—figure supplement 1C , Figure 3—figure supplement 1 , Figure 5—figure supplement 1A ) .",
"For experiments featuring mosaic expression of either unc-8 ( csRNAi ) ( Figure 3F ) or unc-8cDNA ( Figure 3—figure supplement 2 ) , puncta were counted from individual DD and VD neurons .",
"The examiner was blinded to genotype .",
"Animals were immobilized with 15 mM levamisole/0 . 05% tricaine and mounted on a 2% agarose pad in M9 buffer as previously described ( Smith et al . , 2010 ) .",
"Z-stack images were collected on a Leica TCS SP5 confocal microscope using a 63X oil objective ( 0 . 5 μm/step ) , spanning the focal depth of the ventral nerve cord GABA neurons and synapses .",
"Leica Application Suite Advanced Fluorescence ( LAS-AF ) software was used to generate maximum intensity projections .",
"Images in Figure 3A were collected from 10 animals of each genotype .",
"Ventral nerve cord images between VD4 and VD5 were straightened using an ImageJ plug-in and aligned in rows .",
"All fluorescence intensity plots were created by drawing a line through the ventral nerve cords of each animal and calculating the fluorescence intensity value in arbitrary units over the distance in micrometers with the ImageJ plot profile tool .",
"For Figure 1 , DD and VD neurons were identified by the GABA-specific marker pttr-39::mCherry .",
"The cells were traced in ImageJ and the background was subtracted .",
"The unc-8::GFP fluorescence intensity was then normalized to the pttr-39::mCherry fluorescence intensity for each cell .",
"Fluorescence intensity plots in Figure 4 and Figure 4—figure supplement 1 were created with the ImageJ plot profile tool , analyzing the same region of the ventral nerve cord in both GFP and RFP channels .",
"Fluorescence intensity values were normalized for each channel .",
"The coefficient of determination ( r2 ) was calculated in ImageJ using the Manders coefficients macro , from at least 10 animals for each genotype .",
"r2 values were averaged and presented as mean ± SEM .",
"An r2value of 0 represents no co-localization , whereas r2 = 1 represents complete co-localization .",
"Amiloride hydrochloride hydrate ( Sigma , #A7410 ) stock solution was prepared in sterile water ( 50 mg/ml ) and stored at -20°C .",
"A final concentration of 3 mM Amiloride diluted in OP50 bacteria was seeded on NGM plates .",
"Control NGM plates contained the same volume of sterile water added to OP50 .",
"Benzamil hydrochloride hydrate ( Sigma , #B2417 , St . Louis , MO ) stock solution was prepared in sterile water ( 1 mg/ml ) and stored at 4°C .",
"A final concentration of 3 mM Benzamil diluted in OP50 bacteria was seeded on NGM plates .",
"Control NGM plates contained the same volume of sterile water added to OP50 .",
"Plates were stored at 4°C for up to one week .",
"Five adult unc-55; juIs1 animals were placed on either Amiloride , Benzamil , or control plates at room temperature and progeny examined at the young adult stage ( Figure 5A , B and Figure 5—figure supplement 1A ) .",
"Adult tax-6 ( d ) ; wyIs202 animals were grown on Benzamil or control plates and their larval progeny were collected on control or Benzamil plates for timecourse assays ( Figure 7E ) .",
"The number of ventral puncta was counted using a Zeiss Axiovert microscope ( 63X oil objective ) and Z-stack images were captured on a Leica TCS SP5 confocal microscope using a 63X oil objective ( 0 . 5 μm/step ) .",
"The examiner was blinded to genotype and treatment condition .",
"All-trans retinal ( Sigma , #R2500 ) was dissolved in ethanol to prepare a 100 mM stock and stored at -20°C .",
"300 μM of all-trans retinal stock solution ( ATR plates ) or ethanol ( control plates ) was added to OP50 bacteria and seeded onto NGM plates .",
"Plates were protected from light and were stored at 4°C for up to one week .",
"100 adult hermaphrodites were placed on either ATR or control plates , allowed to lay eggs for 1 hr and then removed from the plate .",
"The midpoint of this hour is considered T0 .",
"Plates were exposed to blue light pulses with a 470-nm LED light ( #M470L2 , Thor Labs , Newton , NJ ) for 13 hr ( 0 . 5 Hz , 2 mW/mm2 measured with Solartech Inc . Solar Meter 9 . 4 radiometer ) .",
"Light stimulation was controlled using NI Max software through TTL signals generated by a digital function generator ( National Instruments , Austin , TX ) .",
"13 hr after egg laying , animals were assayed for DD remodeling by counting the number of dorsal SNB-1::GFP puncta .",
"The examiner was blinded to the treatment .",
"Data were collected from three independent time course experiments with at least six animals per treatment .",
"The C . elegans dissection and electrophysiological methods were as previously described ( Richmond and Jorgensen , 1999 ) .",
"Animals were immobilized along the dorsal axis with Histoacryl Blue glue , and a lateral cuticle incision was made with a hand-held glass needle , exposing ventral medial body wall muscles .",
"Muscle recordings were obtained in the whole-cell voltage-clamp mode using an EPC-10 patch-clamp amplifier and digitized at 1 kHz .",
"The extracellular solution consisted of 150 mM NaCl , 5 mM KCl , 5 mM CaCl2 , 4 mM MgCl2 , 10 mM glucose , 5 mM sucrose , and 15 mM HEPES ( pH 7 . 3 , ~340 mOsm ) .",
"The low Cl intracellular patch pipette solution used to isolate outward GABA minis at a 0 mV holding potential was composed of 115 mM KGluconate , 25 mM KCl , 0 . 1 mM CaCl2 , 1 mM BAPTA and 50 mM HEPES .",
"Data were acquired using Pulse software ( HEKA , Southboro , Massachusetts , United States ) run on a Dell computer .",
"Subsequent analysis and graphing was performed using Pulsefit ( HEKA ) , Mini analysis ( Synaptosoft Inc . , Decatur , Georgia , United States ) and Igor Pro ( Wavemetrics , Lake Oswego , Oregon , United States ) .",
"UNC-8 ( G387E ) cRNA was synthesized using T7 mMESSAGE mMACHINE kit ( Ambion , Waltham , MA ) .",
"cRNA was purified and examined on a denaturating agarose gel to confirm correct size and integrity .",
"cRNA quantification was performed spectroscopically .",
"Stage VI defolliculated oocytes from Xenopus Laevis were purchased from Ecocyte Bioscience US LLC ( Austin , Texas ) .",
"Oocytes were injected with 10 ng/oocyte of cRNA and incubated in OR2 solution ( 82 . 5 mM NaCl , 2 . 5 mM KCl , 1 mM CaCl2 , 1 mM MgCl2 , 1 mM Na2HPO4 , 0 . 5 g/liter polyvinyl pyrolidone , and 5 mM HEPES , pH 7 . 2 , supplemented with penicillin and streptomycin ( 0 . 1 mg/ml ) and 2 mM Na-pyruvate ) plus 500 µM amiloride ( to prevent channel hyperactivation-dependent cell death ) at 20°C for 2–3 d before recordings .",
"Currents were measured using a two-electrode voltage-clamp amplifier ( GeneClamp 500B; Axon Instruments , Sunnyvale , CA ) at room temperature .",
"Electrodes ( 0 . 2–0 . 5 MΩ ) were filled with 3 M KCl , and oocytes were perfused with a physiological NaCl solution ( 100 mM NaCl , 2 mM KCl , 1 mM CaCl2 , 2 mM MgCl2 , and 10 mM HEPES , pH 7 . 2 ) and divalent cation free plus EGTA NaCl solution ( 110 mM NaCl , 2 mM KCl , 1 mM EGTA , and 10 mM HEPES , pH 7 . 2 ) .",
"pH was adjusted at the indicated values using NaOH .",
"The oocyte membrane was clamped at −30 mV and stepped from −160 to +100 mV .",
"Benzamil was added to the solutions from a stock of 10 mM .",
"A saturating concentration of benzamil ( 1mM ) was added at the end of each experiment to confirm that endogenous/leak currents were similar in amplitude to non-injected oocytes within each oocyte batch .",
"Oocytes that had larger endogenous/leak currents were not further analyzed .",
"We used the pCLAMP suite of programs ( Axon Instruments ) for data acquisition and analysis .",
"Currents were filtered at 200 Hz and sampled at 1 kHz .",
"We used OriginPro 8 ( OriginLab Corporation , Northampton , MA ) to generate graphs , Ki , and for statistical analysis .",
"Young adult hermaphrodites of each strain were prepared for high-pressure freeze ( HPF ) fixation as described ( Rostaing et al . , 2004 ) .",
"10–15 animals were loaded into a specimen chamber filled with E . coli .",
"The specimens were frozen rapidly in a high-pressure freezer ( Bal-Tec HPM010 ) at −180°C and high pressure .",
"Freeze substitution was performed on frozen samples in a Reichert AFS machine ( Leica , Oberkochen , Germany ) with 0 . 1% tannic acid and 2% OsO4 in anhydrous acetone .",
"The temperature was kept at −90°C for 107 hr , increased at 5°C/hr to −20°C , and kept at -20°C for 14 hr .",
"The temperature was then increased by 10°C/h to 20°C .",
"Fixed specimens were embedded in Epon resin after infiltration in 50% Epon/acetone for 4 hr , 90% Epon/acetone for 18 hr , and 100% Epon for 5 hr .",
"Embedded samples were incubated for 48 hr at 65°C .",
"All specimens were prepared in the same fixation procedure and labeled with anonymous tags so that the examiner was blinded for genotype .",
"Ultra thin ( 40 nm ) serial sections were cut using an Ultracut 6 ( Leica ) and collected on formvar- covered , carbon-coated copper grids ( EMS , FCF2010-Cu ) .",
"Grids were counterstained in 2% aqueous uranyl acetate for 4 min , followed by Reynolds lead citrate for 2 min .",
"Images were obtained on a Jeol JEM-1220 ( Tokyo , Japan ) transmission electron microscope operating at 80 kV .",
"Micrographs were collected using a Gatan digital camera ( Pleasanton , CA ) at a magnification of 100k .",
"Images were quantified using NIH ImageJ software .",
"Dorsal and ventral cords were distinguished by size and morphology .",
"GABAergic synapses were identified by previously established criteria , including position in the cord as well as the morphology of the synapse ( White et al . , 1986; Jin et al . , 1999 ) .",
"GABAergic synapses are larger than their cholinergic motor neuron counterparts , and the active zones in these synapses form a direct , perpendicular angle with muscle arms .",
"On the other hand , the presynaptic density in cholinergic synapses orient at an acute angle to the muscle , generally 30–45° and are often dyadic .",
"Some images were collected at 30 k to aid in identifying synaptic identity based on terminal position in the cord .",
"Two colleagues with expertise in EM reconstruction of the C . elegans ventral nerve cord independently reviewed synapse images from each strain to verify identification .",
"Each profile represents an image taken of a 40 nm section .",
"A synapse was defined as a set of serial sections containing a presynaptic density and flanking sections from both sides without presynaptic densities .",
"Synaptic vesicles were identified as spherical , light gray structures with an average diameter of ~30 nm .",
"At least two animals were analyzed for each genotype .",
"Numbers of profiles analyzed for each genotype were: wild type = 502 , unc-8 = 322 , unc-55 = 246 , unc-55; unc-8 = 304 for ventral GABAergic synapse evaluation; wild type = 502 , unc-8 = 322 , unc-55 = 246 , unc-55; unc-8 = 304 for ventral cholinergic synapse evaluation ."
]
] | [
"Genetic programming and neural activity drive synaptic remodeling in developing neural circuits , but the molecular components that link these pathways are poorly understood .",
"Here we show that the C . elegans Degenerin/Epithelial Sodium Channel ( DEG/ENaC ) protein , UNC-8 , is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons .",
"UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons , but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor , UNC-55 .",
"We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus .",
"Thus , UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons ."
] | [
"The brain contains billions of nerve cells , or neurons , that communicate with one another through connections called synapses .",
"As the brain develops , these circuits are extensively modified as new synapses are created and others are removed .",
"Neurological disorders may emerge if these processes are not regulated correctly .",
"Identifying the biological pathways that control the addition and removal of synapses could therefore provide new insights into how to treat human brain diseases .",
"To communicate across a synapse , the signaling neuron releases chemicals called neurotransmitters that alter the activity of the receiving neuron .",
"Some neurotransmitters , such as GABA , inhibit the activity of the receiving neuron .",
"The activity of a neuron – and hence how often it releases neurotransmitters – depends on different ions moving into and out of the neuron through proteins called ion channels that are embedded in the cell membrane .",
"For example , the movement of calcium ions into the neuron can trigger the release of neurotransmitters .",
"The roundworm Caenorhabditis elegans is often used as a model organism to study how the brain develops .",
"During development , the worm nervous system eliminates synapses that release GABA and reassembles them at new locations .",
"However , the nervous system does not eliminate these synapses at random .",
"Miller-Fleming , Petersen et al . now show that a C . elegans protein called UNC-8 is responsible for this effect .",
"UNC-8 forms part of an ion channel that allows sodium ions to enter the neuron and is selectively produced in GABA neurons that are destined for remodeling .",
"Miller-Fleming , Petersen et al . found that inside GABA-releasing neurons , calcium ions stimulate an enzyme called calcineurin that may in turn activate UNC-8 .",
"Sodium ions then enter the neuron through UNC-8 channels .",
"This boosts the activity of the calcium ion channels , which further increases how many calcium ions enter the cell .",
"Ultimately , the amount of calcium inside the neuron becomes high enough to activate an additional pathway that eliminates the synapse .",
"This downstream pathway involves components of a cell-killing ( or “apoptotic” ) mechanism that is repurposed in this case to remove the GABA release apparatus at the synapse .",
"Other proteins are likely to help UNC-8 sense the activity of neurons and destroy synapses in response .",
"Further work is required to investigate these additional components and to determine how they work with UNC-8 to remove synapses in the nervous system during development ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Visual pursuit behavior in mice maintains the pursued prey on the retinal region with least optic flow | elife-70838-v1 | [
[
"The visual system of mice serves a variety of seemingly opposing functions that range from detection of predators , to finding shelter and selection of food and mates , and is required to do so in a diverse set of environments ( Boursot et al . , 1993 ) .",
"For example , foraging in open areas where food is available involves object selection , and in the case of insect predation ( Badan , 1986; Tann et al . , 1991 ) , involves prey tracking and capture ( Hoy et al . , 2016; Langley , 1983; Langley , 1984; Langley , 1988 ) , but the visual system can also simultaneously be relied on for avoidance of predation , particularly from airborne predators ( Hughes , 1977 ) .",
"Like with many ground-dwelling rodents ( Johnson and Gadow , 1901 ) , predator detection in mice is served by a panoramic visual field which is achieved by the lateral placement of the eyes in the head ( Dräger , 1978; Hughes , 1979; Oommen and Stahl , 2008 ) combined with monocular visual fields of around 200 degrees ( Dräger and Olsen , 1980; Hughes , 1979; Sterratt et al . , 2013 ) .",
"In mice , the panoramic visual field extends to cover regions above the animal’s head , below the animal's snout and laterally to cover ipsilaterally from behind the animal's head to the contralateral side , with the overlapping visual fields from both eyes forming a large binocular region overhead and in front of the animal ( Hughes , 1977; Sabbah et al . , 2017 ) .",
"In addition , eye movements in freely moving mice constantly stabilize the animal’s visual field by counteracting head rotations through the vestibulo-ocular reflex ( VOR ) ( Meyer et al . , 2020; Meyer et al . , 2018; Michaiel et al . , 2020; Payne and Raymond , 2017 ) maintaining the large panoramic overhead view ( Wallace et al . , 2013 ) critical for predator detection ( Yilmaz and Meister , 2013 ) .",
"Given the VOR stabilized panoramic field of view , it is not clear what part of the visual field mice use to detect and track prey ( but see: Johnson et al . , 2021 ) .",
"Mouse retina contains retinal ganglion cells ( RGCs ) , the output cells of the retina , with a broad diversity of functional classes ( Baden et al . , 2016; Bleckert et al . , 2014; Franke et al . , 2017; Zhang et al . , 2012 ) .",
"Given the lateral eye position , the highest overall density faces laterally ( Dräger and Olsen , 1981; Sabbah et al . , 2017; Salinas-Navarro et al . , 2009; Stabio et al . , 2018 ) .",
"Further , as the functionally defined ganglion cells ( Baden et al . , 2016; Bleckert et al . , 2014; Franke et al . , 2017; Zhang et al . , 2012 ) and cone sub-types ( Szél et al . , 1992 ) are segregated into retinal subregions within the large stabilized field of view , recent studies suggest that retinal subregions are tuned for specific behavioral tasks depending on what part of the world they subtend ( Baden et al . , 2016; Bleckert et al . , 2014; Hughes , 1977; Sabbah et al . , 2017; Szatko et al . , 2020; Zhang et al . , 2012 ) .",
"The challenge is to measure what part of the visual field the mouse is attending to during a visually based tracking task ( Hoy et al . , 2016 ) and the location of all objects within the field of view during the behavior .",
"While recent studies have implied the relationship between prey and retina through tracking head position ( Johnson et al . , 2021 ) or measured both the horizontal and vertical eye rotations ( Meyer et al . , 2020; Meyer et al . , 2018 ) during pursuit behavior ( Michaiel et al . , 2020 ) to uncover a large proportion of stabilizing eye-rotations , what is missing is the extent and location of the area used when detecting and pursuing prey , and the relationship to the retina ( Bleckert et al . , 2014 ) .",
"Here , we measured the position of a cricket in the visual fields of freely moving mice performing a prey pursuit behavior , using head and eye tracking in all three rotational axes , namely horizontal , vertical , and torsional .",
"Eye tracking included an anatomical calibration to accurately account for the anatomical positions of both eyes .",
"To quantify object location in the animal’s field of view and generate optic flow fields , head and eye rotations were combined with a high-resolution digital reconstruction of the arena to form a detailed visual map from the animal’s eye perspective .",
"Given that mice use multisensory strategies during prey pursuit ( Gire et al . , 2016; Langley , 1983; Langley , 1988 ) and can track prey using auditory , visual , or olfactory cues ( Langley , 1983; Langley , 1988 ) , we developed a behavioral arena that isolated the visual aspect of the behavior by removing auditory and olfactory directional cues to ensure that the behavior was visually guided .",
"To transfer the retinal topography onto the corneal surface , we developed an eye model capturing the optical properties of the mouse eye .",
"We show that during prey detection mice preferentially position prey objects in stable foci located in the binocular field and undertake direct pursuit .",
"Prey objects remain in the functional foci through the stabilizing action of the VOR , and not through active prey-pursuit eye movements .",
"The stabilized functional foci are spatially distinct from the regions of highest total retinal ganglion cell density , which are directed laterally , but coincides with the regions of the visual field where there is minimal optic flow and therefore minimal motion-induced image disturbance during the behavior , as the mouse runs towards the cricket .",
"Lastly , by building an optical model that allows corneal spatial locations to be projected onto the retina , we suggest that the functional foci correspond to retinal subregions containing a large density of Alpha-ON sustained RGCs that have center-surround receptive fields and project to both superior colliculus and dLGN ( Huberman et al . , 2008 ) and possess properties consistent with the requirements for tracking small and mobile targets ( Krieger et al . , 2017 ) ."
],
[
"To measure what part of the visual field mice use during prey capture while also considering that mice can use multisensory strategies during prey pursuit ( Gire et al . , 2016; Langley , 1983; Langley , 1988 ) , we first developed an arena which isolated the visual component of prey pursuit by masking olfactory and auditory spatial cues ( Figure 1A , see Materials and methods for details ) .",
"By removing both olfactory and auditory cues , the average time to capture a cricket approximately doubled compared to removal of auditory cues alone ( time to capture , median ± SD , control 24 . 92 ± 16 . 77 s , olfactory and auditory cues removed , 43 . 51 ± 27 . 82 s , p = 0 . 0471 , Wilcoxon rank sum test , N=13 control and 12 cue removed trials from N = 5 mice ) .",
"To track mouse head and eye rotations during prey capture , we further developed a lightweight version of our head mounted oculo-videography and camera-based pose and position tracking system ( Wallace et al . , 2013; Figure 1B and Materials and methods ) .",
"This approach allowed quantification of head rotations in all three axes of rotation ( pitch , roll , and yaw ) , as well as eye rotations in all three ocular rotation axes ( torsion , horizontal , and vertical , Figure 1C , Figure 1—figure supplement 1A and B ) .",
"The same camera-based system was used to track and triangulate the position of the cricket ( see Materials and methods and Figure 1—figure supplement 1C ) .",
"To quantify the position and motion of the environment and cricket in the mouse's field of view , we also developed a method that enabled a calibrated environment digitization to be projected onto the corneal surface .",
"This approach utilized a combination of laser scanning and photogrammetry , giving a resolution for the reconstruction of the entire experimental room of 2 mm , as well as a detailed measurement of eye and head rotations ( Figure 1D–E , and see Materials and methods ) .",
"Mice , like rats ( Wallace et al . , 2013 ) , have a large visual field of view which extends to also cover the region over the animal’s head ( Figure 1F ) .",
"To ensure the entire visual fields of the mouse could be captured during behavior , we digitized the entire experimental room and contents ( Figure 1E , Figure 1—figure supplement 1D–F , Video 1 ) .",
"The coordinate systems of the environmental digitization and mouse and cricket tracking systems were registered using 16–20 fiducial markers identified in both the overhead camera images and the digitized environment .",
"The average differences in position of fiducial points between the two coordinate systems were less than 1 mm ( mean ± SD , x position , 0 . 18 ± 3 . 1 mm , y position , 0 . 07 ± 1 . 6 mm , z position , 0 . 66 ± 1 . 8 mm , N=54 fiducial points from three datasets ) .",
"The next step was to re-create the view for each eye .",
"First , and for each mouse , the positions of both eyes and nostrils were measured with respect to both the head-rotation tracking LEDs and head-mounted cameras , then calibrated into a common coordinate system ( Figure 1B ) .",
"Together , this enabled a rendered representation of the digitized field of view for each combination of head and eye rotations .",
"This rendered image , from the animal’s point of view , contained all the arena and lab objects ( Figure 1G–H , Video 2 and 3 , Figure 1—figure supplement 1G ) .",
"In addition to object position and distance ( Figure 1I ) , the motion of the environment and each object in the field of view could be quantified as the mouse performed prey capture behaviors ( Figure 1J , and Figure 1—figure supplement 1H ) .",
"Crickets ( Acheta domesticus ) , shown previously to be readily pursued and preyed upon by laboratory mice ( Hoy et al . , 2016 ) , provided a prey target that could successfully evade capture for extended periods of time ( total time for each cricket before capture: 64 . 4 ± 39 . 3 s , average time ± SD , N = 21 crickets and three mice , Video 4 and 5 ) .",
"To ensure that only data where the mouse was actively engaged in the detection and tracking of the cricket were used , we identified occasions where the mouse either captured the cricket , or contacted the cricket but the cricket escaped ( see Materials and methods for definitions ) , and then quantified the trajectories of both mouse and cricket leading up to the capture or capture-escape ( Figure 2A ) .",
"Within these chase sequences we defined three behavioral epochs ( detect , track , and capture , Figure 2B , see Materials and methods for definition details ) based on the behavior of mouse and cricket , and similar to previous studies ( Hoy et al . , 2016 ) .",
"Upon cricket detection , mice oriented and ran towards the cricket , resulting in a significant adjustment to their trajectory ( Δ target bearing: 40 . 2 ± 35 . 1° , P = 6 . 20 x 10−10 , Δ speed: 10 . 2 ± 7 . 4 cm/s , P = 1 . 91 x 10−10; N=57 detect-track sequences N = 3 mice; Paired Wilcoxon’s signed rank test for both tests ) , and a rapid reduction in the Euclidean distance to the cricket ( Figure 2C ) .",
"During tracking , the cricket was kept in front of the mouse , resulting in a significant reduction in the spread of target bearings compared to during detect epochs ( Figure 2D , Target bearing: detect 6 . 2 ± 62 . 1° , track: 2 . 5 ± 25 . 6° , mean ± SD , Brown-Forsythe test p = 0 , F statistic=7 . 05x103 , N=4406 detect and 13624 track frames , N=3 mice ) , consistent with previous findings ( Hoy et al . , 2016 ) .",
"To avoid the closing phase of the pursuit being associated with whisker strikes ( Shang et al . , 2019; Zhao et al . , 2019 ) , tracking periods were only analyzed when the mouse was more than 3 cm from the cricket , based on whisker length ( Ibrahim and Wright , 1975 ) .",
"Using the detailed digitization of the behavioral arena and surrounding laboratory ( Figure 1E , Video 1 ) , an image of the cricket and objects in the environment was calculated for each head and eye position during the predator-prey interaction ( Video 2 and 3 ) .",
"Using this approach , we addressed the question of what area of the visual field was the cricket located in during the various behavioral epochs .",
"In the example pursuit sequence in Figure 2E , the cricket was initially located in the peripheral visual field and then transitioned to the lower nasal binocular quadrant of the cornea-view during pursuit and capture ( red trace in left eye to blue trace in both eyes ) .",
"Correspondingly , an average probability density map calculated for all animals during the detect epoch showed a very broad distribution of cricket positions across the visual field ( Figure 2F , Figure 2—figure supplement 1A and B ) .",
"Upon detection the mouse oriented toward the cricket , bringing it toward the lower nasal binocular visual field ( Figure 2E , Video 6 ) .",
"When averaged for all pursuit sequences from all animals , projected cricket positions formed a dense cluster on the cornea of both eyes ( Figure 2G and H , Figure 2—figure supplements 1A , C–D , 50% contour center for left and right eye respectively , radial displacement from optical axis 64 . 3 ± 7 . 5° and 63 . 3 ± 9 . 9° , rotational angle 126 . 2 ± 8 . 9° and −115 . 7 ± 6 . 1° , mean ± SD , N = 3 mice ) , which was significantly different from the cluster in the detect epoch ( average histogram of the location of cricket image during tracking phase vs average histogram of the location of cricket during detect phase: Left eye P = 3 . 54 x 10−46 , Right eye P = 1 . 08 x 10−81 , differences calculated by taking the Mean Absolute Difference with bootstrapping , N=57 detect-track sequences , N = 3 mice ) .",
"Thus , during the tracking and pursuit behavior the image of the prey consistently fell on a local and specific retinal area that we refer to from here on as the functional focus .",
"The functional focus fell within the binocular field , while the region of elevated density of RGCs has been found to be located near the optical axis ( Dräger and Olsen , 1981 ) , which suggests that the location of the retinal specialization may not overlap with the functional focus .",
"To establish the relation between the identified functional focus and the density distribution of RGCs , we made a mouse eye-model ( Figure 3A ) , modified from previous models ( Barathi et al . , 2008 ) .",
"Using the eye model , retinal spatial locations could be projected through the optics of the mouse eye to the corneal surface .",
"We first reconstructed the isodensity contours quantifying the distribution of all RGCs ( Dräger and Olsen , 1981 ) to define the retinal location with the highest overall ganglion cell density ( Figure 3—figure supplement 1A–C , note that these contours are also in agreement with other recently published maps of total RGC density [Bleckert et al . , 2014; Zhang et al . , 2012] ) .",
"The lens optical properties were based on a GRIN lens ( present in both rats [Hughes , 1979; Philipson , 1969] and mice [Chakraborty et al . , 2014] ) .",
"To determine the optical characteristics of this lens , we developed a method which combined models of the lens surface and refractive index gradient ( Figure 3A , Figure 3—figure supplement 1D and Tables 1 and 2 , see Materials and methods for details ) .",
"Using this model , the contours representing the retinal specializations were projected through the eye model onto the corneal surface to determine equivalent corneal locations ( Figure 3B , Figure 3—figure supplement 1E ) .",
"Comparing this location to the functional focus location showed that the region with the highest overall RGC counts and the functional focus ( Figure 3B ) occupied distinct retinal locations ( Figure 3C ) .",
"Viewed from above the animal’s head , the functional foci were directed at the region in front of the animal’s nose and within the region of stable binocular overlap ( azimuth: 1 . 4 ± 8 . 8° and −4 . 4 ± 9 . 3° , elevation 5 . 7 ± 2 . 1° and 4 . 9 ± 1 . 4° for left and right eyes respectively , N = 13641 frames , N=3 mice ) , while the retinal specialization was directed laterally ( azimuth: −66 . 2 ± 6 . 7° and 70 . 3 ± 4 . 7° , elevation: 30 . 8 ± 12 . 2° and 41 . 0 ± 13 . 5° for left and right eyes respectively , N = 13641 frames N=3 mice . Figure 3D , Figure 3—figure supplement 1F–G ) .",
"Given that density distributions for different subtypes of RGCs can be spatially heterogeneous with density peaks in distinctly different retinal locations , and that the region of peak density for Alpha-ON sustained RGC’s is spatially located on the dorso-temporal retina ( Bleckert et al . , 2014 ) , consistent with projecting to the front of the animal , we next quantified whether this region overlapped with the functional focus observed here ( Figure 3E ) .",
"The average 50% contour of the functional focus was overlapped by the highest density of Alpha-ON sustained RGC's by 35% and 67% for left and right eye respectively ( Figure 3E , black , mean ± SD for left and right eye , 35 . 1 ± 19 . 8% , 66 . 7 ± 0 . 09% , p = 0 . 095 and 0 . 019 , one-sided Student’s t-test ) , and the overlap with the second highest density was 83% and 95% ( mean ± SD for left and right eye , 82 . 8 ± 20 . 1% , 94 . 8 ± 24 . 7% , p = 0 . 042 and 0 . 003 , one-sided Student’s t-test ) , suggesting a high degree of correspondence between the highest density of Alpha-ON sustained RGC’s and the functional focus during pursuit behavior .",
"Viewed from above the animal’s head the functional foci were directed at the region in front of the animal’s nose azimuth: 1 . 4 ± 8 . 8° and −4 . 4 ± 9 . 3° , elevation: 5 . 7 ± 2 . 1° and 4 . 9 ± 1 . 4° for left and right eyes respectively , N = 13641 frames , N=3 mice .",
"The Alpha-ON sustained RGC’s were also directed in front of the animal’s nose ( mean ± SD , elevation:16 . 0 ± 6 . 9° and 10 . 8 ± 11 . 0° , azimuth: −3 . 6 ± 0 . 7° and 5 . 8 ± 7 . 9° for left and right eyes respectively , N = 168400 frames , N = 3 mice , Figure 3F ) .",
"Together this suggests that objects falling within the functional foci are processed by Alpha-ON sustained RGC’s .",
"Eye movements in freely moving mice , like with rats ( Wallace et al . , 2013 ) , can be large and rapid ( Meyer et al . , 2020; Payne and Raymond , 2017 ) , and counter head rotations through the VOR , enabling the large field of view around the animals head to be stabilized while the animal is moving .",
"The relationships between head rotations and both the horizontal and vertical eye rotations have recently been quantified , and in addition it has been reported that both during exploration and hunting , mice also have abrupt gaze shifts brought about by the combination of head rotation and conjugate saccade-like horizontal eye rotations ( Meyer et al . , 2020; Michaiel et al . , 2020 ) .",
"We also observed both forms of eye movements in the current study ( Figure 4—figure supplement 1 ) .",
"However , how these rotations combine with torsional rotations is not known .",
"If mouse VOR operates similar to that observed in the rat ( Wallace et al . , 2013 ) , torsional rotations in the mouse will play a significant role in stabilizing the visual field particularly during changes in head pitch .",
"As with the vertical and horizontal rotations ( Meyer et al . , 2018 ) , torsional rotations in freely moving mice spanned a wide range of rotation angles ( Figure 4—figure supplement 2A–D ) , and were correlated with head pitch ( Pearson’s correlation coefficient ( r ) : detect −0 . 72 , 0 . 58 , track: −0 . 60 and 0 . 53 for left and right eyes respectively , N=4406 detect and 13624 track frames , N=3 mice , Figure 4—figure supplement 2C–D ) as well as head roll ( Pearson’s correlation coefficient ( r ) : detect: −0 . 46 , –0 . 47 track: −0 . 45 and −0 . 48 for left and right eyes respectively , N=4406 detect and 13624 track frames , N=3 mice , Figure 4—figure supplement 2L–M ) , as found previously for freely moving rats ( Wallace et al . , 2013 ) .",
"As with rats , the rotational relationship between the two eyes was dynamic with different forms of coordination ( Figure 4—figure supplement 2E–I ) , including episodes of in- and excyclovergence ( torsional rotation of both eyes toward or away from the nose , respectively ) as well as dextro- and levocycloversion ( torsional rotation of both eyes to the animal’s right or left , respectively ) .",
"We next analyzed how effectively rotations of the eye around multiple rotational axes combined to compensate the rotation of the head ( Figure 4A , Figure 4—figure supplement 3A–G ) .",
"We compared movement of the head around its rotational axes and eye movements around the same rotational axes ( Figure 4A ) , effectively defining alternative rotational axes for the eyes to match the axes of the head .",
"Rotation of the eye around these re-defined axes would involve simultaneous rotations in multiple of the eye’s anatomical axes .",
"The gain of this compensation was relatively linear and less than unity for both pitch- and roll-axes , indicating on average under-compensation of the head rotation ( slope ( gain ) of relation for pitch axis , −0 . 45 ± 0 . 12 and −0 . 48 ± 0 . 06; roll axis −0 . 51 ± 0 . 12 and −0 . 62 ± 0 . 05 for left and right eye respectively , 168852 frames , N=3 mice ) .",
"The relatively linear relationships between head and eye rotation around the head pitch and roll axes ( Figure 4B ) with a transition through the origin suggests that the horizontal , vertical and torsional eye movements are combined to effectively compensate pitch- and roll-related head movements .",
"We next digitally froze each individual eye rotation axis ( torsion , vertical , and horizontal ) and measured the effect on countering the head rotation ( Figure 4C ) .",
"For rotations around the head x-axis ( head pitch changes ) the gain of compensation was most affected by freezing torsional rotations ( Figure 4C , gain mean ± SD , control: −0 . 45 ± 0 . 12and-0 . 48 ± 0 . 06; torsion frozen −0 . 24 ± 0 . 1 and −0 . 24 ± 0 . 01 , for left and right eyes respectively , N = 168852 frames , N=3 mice ) , while freezing vertical or horizontal rotations had more minor effects ( Figure 4C , Table 3 ) .",
"The gain of compensation for rotations around the head y-axis ( head roll changes ) was dramatically affected by freezing vertical rotations ( Figure 4C , gain mean ± SD , control: −0 . 51 ± 0 . 12 and −0 . 62 ± 0 . 05 , vertical frozen −0 . 16 ± 0 . 14 and −0 . 17 ± 0 . 03 , for left and right eyes respectively , N = 168852 frames , N=3 mice ) , with freezing torsion also reducing compensation gain but to a lesser extent ( Figure 4C , Table 3 ) .",
"We next quantified the stability and alignment of the animal’s binocular visual field during the pursuit sequences and determined the location of the functional foci within the stabilized region .",
"Similar to rats , left and right visual fields overlapped extensively ( Dräger and Olsen , 1980; Hughes , 1979 ) , with eye movements creating variability in the extent of the overlap at the edges of the two visual fields , the transition from monocular to binocular ( Figure 4D ) .",
"The functional foci for both eyes were predominately contained within the region of continuous binocular overlap .",
"A horizontal transect through the optical axis for all animals showed a gradual transition from continuous binocular coverage to zero binocular coverage commencing just nasal of the optical axis ( Figure 4D , Figure 4—figure supplement 3H and I ) , indicating that the region of highest overall RGC density spans this transitional region whereas the functional foci are , on average , contained within the binocular region ( Figure 4—figure supplement 3H ) .",
"We next quantified the variability of alignment of the left and right visual fields within the binocular region , and specifically in the functional focus location ( Figure 4E ) by using the center of mass ( 50% isodensity contour center ) of the left eye functional focus as an initial reference point and projecting this point to the boundary of a hypothetical sphere surrounding the head .",
"This contact point on the sphere was then re-projected into the right eye to identify the matching location of the left eye ( Figure 4E ) .",
"We then followed the trajectory of the re-projected point in the right eye to get a measurement of alignment variability ( Figure 4F , for comparison with the locations in the right eye projected into the left eye see Figure 4—figure supplement 3I–K ) .",
"While pursuing crickets , alignment precision varied through time ( Figure 4G ) with the mean alignment of the reference point over all animals and data segments being ~8–9° , which is around the size of V1 cortical neuron receptive fields ( ~5–15° [Niell and Stryker , 2008] , Figure 4H , mean ± SD , left eye projected into right eye 8 . 8 ± 6 . 9°; right eye projected into left eye 8 . 6 ± 6 . 7° ) .",
"Repeating this analysis for all points within the region where the probability of binocular overlap was greater than 5% showed that there was a relatively uniform alignment over the entire region ( Figure 4I ) , and that the average alignment error in the functional foci was 8–10° .",
"Coordination of eye movements was important for alignment , as freezing the movements of one eye to its mean position resulted in a significant increase in the alignment error when comparing individual cricket tracking sequences ( left all rotations vs . left eye frozen P = 1 . 78 x 10−10 , right eye all rotations vs . right eye frozen P = 7 . 12 x 10−11 , N=52 sequences , unpaired Student’s t-test ) , and a ~54% increase in the mean alignment error over all frames for the reference location ( Figure 4I , left eye projected into right eye ( left eye frozen ) 13 . 4 ± 8 . 3°; Right eye projected into left eye ( right eye frozen ) 13 . 4 ± 8 . 3° , mean ± SD , 159318 frames , N=3 mice ) , which also resulted in a uniform increase in alignment error over the whole overlap region ( Figure 4J and Figure 4—figure supplement 3J–L ) .",
"In summary , during pursuit behavior the functional foci are located in a stable binocular region of the mouse’s visual field .",
"However , in the absence of a mechanism for voluntarily directing its gaze toward a specific target , such as smooth pursuit , tight coupling of VOR-evoked eye movements to head rotations would seem to be restrictive to an animal’s ability to move the target into a specific part of their visual field during pursuit .",
"We therefore next measured what mechanisms mice use to bring the prey into their functional focus .",
"At detection , mice orient toward their target , aligning their head with the prey and running towards it ( Figure 2D ) , keeping the cricket within a narrow window around its forward direction .",
"This provides a direct way for mice to hold their prey within their binocular visual fields ( Figure 4D ) .",
"We next measured whether additional head or eye movements are used to keep the target within the functional foci .",
"If the mice were actively maintaining the prey within a fixed location of their visual fields , the position of the cricket image should not change as the mouse approaches the cricket .",
"The cricket image location could be maintained by either a head or eye rotation .",
"If they were not actively maintaining the prey in a fixed location , the cricket images should shift downwards in the visual fields as the mouse approaches .",
"To distinguish between these two possibilities , we plotted the cricket positions in the eye views color-coded by the distance between the mouse and cricket ( Figure 5A ) .",
"As the mouse approached the cricket during the track behavioral epoch , the projected cricket positions systematically shifted lower in the visual field ( Figure 5A lower ) .",
"This suggests that the mice did not use additional head or eye movements ( Figure 5—figure supplement 1 ) to bring the cricket into the functional foci , but rather manipulated the cricket’s position in the eye view by orienting and moving towards the target .",
"Consistent with this , head pitch remained stable as the mice approached the crickets ( Figure 5B ) .",
"Furthermore , there was no significant difference in head pitch as a function of distance to the cricket between non-tracking and tracking periods ( non-tracking head pitch: −3 . 7 ± 26 . 5° , mean ± SD , median = −11 . 3° , tracking head pitch: −12 . 9 ± 15 . 7° , mean ± SD , median = −14 . 6° , Ks test , P = 0 . 709 , paired Student’s t-test P = 0 . 197 , N = 18 non-tracking and track sequences , N = 3 mice ) .",
"In addition , and consistent with previous findings ( Michaiel et al . , 2020 ) , mice did not significantly converge their eyes as they approached the crickets ( non-tracking head vergence: 7 . 6 ± 13 . 5° , mean ± SD , median = 8 . 6° , tracking head vergence: 2 . 5 ± 16 . 7° , mean ± SD , median = 3 . 2° . Ks test , P = 0 . 425 , paired Student’s t-test P = 0 . 225 , N=18 non-tracking and track sequences , N = 3 mice , Figure 5—figure supplement 1J and Table 4 ) .",
"These observations suggest that the primary role for the eye movements is stabilizing the visual fields .",
"If mice successfully track and capture prey by retaining the target in front of them then this should be reflected in the trajectories taken by the mice during the tracking epoch compared to the non-tracking behavioral epochs .",
"During cricket tracking periods , mice ran directly toward the cricket , and their trajectories were significantly straighter than during equivalent non-tracking phases ( Figure 5C–G ) .",
"Lateral deviation at the half-way point ( Figure 5E , non-tracking 4 . 3 ± 4 . 0 cm , tracking 1 . 4 ± 2 . 0 cm , p = 0 . 009 ) , maximum lateral deviation ( Figure 5F , non-tracking , 7 . 7 ± 4 . 9 cm , tracking 2 . 8 ± 2 . 0 cm , p = 0 . 0006 ) and the area between the trajectory and minimum distance path to the target ( Figure 5G , area under the curve , non-tracking 135 . 6 ± 102 . 7 cm2 , tracking 51 . 3 ± 45 . 8 cm2 , p = 0 . 0029 ) were all significantly smaller in the tracking epochs ( all comparisons mean ± SD , N=13 tracking and non-tracking sequences , N=3 mice , Wilcoxon’s Rank Sum Test ) .",
"Together this suggests that mice do not make compensatory vertical head movements , tracking eye movements or vergence eye movements to keep prey within their functional foci , but instead retain their target within a restricted bearing by running straight towards it .",
"This raised the question of what is unique about the position of the functional focus on the cornea ?",
"Optic flow is the pattern of object motion across the retina that can be self-induced , through eye , head or translational motion , or induced by motion of objects in the environment , or combinations thereof ( for review see: Angelaki and Hess , 2005 ) .",
"In a freely moving animal in a still environment , translational motion results in a pattern of optic flow that consists of a radial flow-fields emanating from a point of zero-optic flow ( Figure 6A ) .",
"While optic flow is used by many species for both navigation and the estimation of the motion properties of moving objects , motion induced blur degrades image formation on the retina and decreases resolution depending on the animal’s direction of travel ( Land , 1999 ) .",
"Optic flow is minimized in the direction of travel directly in front of the animal ( Sabbah et al . , 2017 ) , with flow fields directed away from the travel direction and forming a second minimum directly behind the animal’s head ( Figure 6A , see also Angelaki and Hess , 2005 ) .",
"To measure the characteristics of optic flow in in both eyes of freely moving mice and to relate this flow pattern to the location of the functional foci , we next calculated average optic flow from freely moving data during pursuit behavior using the digitized environment and eye-views ( Figure 6B ) .",
"First , we calculated optic flow in the idealized case of forward translation motion when all surrounding surfaces were equidistant ( Figure 6C ) .",
"As mice have laterally facing eyes ( optical axis = 59 . 9 ± 19 . 8° and −62 . 3 ± 14 . 7° lateral of frontal for the left and right eyes respectively , N=3 mice ) , idealized forward motion resulted in the region of minimal optic flow in each eye being located off optical axis in the ventro-medial corneal region representing the animal’s forward direction ( radial displacement from optical axis 36 . 64 ± 0 . 92° and −41 . 11 ± 2 . 27° , rotational angle 122 . 95 ± 17 . 05° and −107 . 94 ± 9 . 96° , for the left and right eyes respectively , mean ± SD , N=2 mice , Figure 6C ) .",
"During free movement both the distance from the eyes to objects in the environment , as well as head and eye-rotations had a strong influence on the optic flow fields .",
"We visualized the average flow fields during free motion by calculating the optic flow on the cornea during multiple pursuit trials ( N=20 prey chases containing 52 tracking sequences , initial Euclidean distance mouse-cricket >20 cm ) .",
"The resulting optic flow density maps were complex with a wide range of average speeds ( 133 . 44 ± 221 . 42 °/s , mean ± 1SD , median 28 . 64 °/s , interquartile range 4 . 57–137 . 18 °/s , N=2 mice , Figure 6D ) .",
"The area of lowest optic flow extended from the nasal part of the field of view to overhead ( Figure 6D ) but , unlike the simulated case ( Figure 6C ) , optic flow was not symmetric around the regions of minimal optic flow .",
"Optic flow in the 30x30° region surrounding the ventro-medial point of minimal optic flow was significantly lower than that in an equivalent region in the ventro-temporal region during free movement , but not in the simulated case ( free movement: nasal 46 . 3 ± 9 . 8 °/s , temporal: 199 . 4 ± 29 . 0 °/s , p = 0 . 0014 , simulated: nasal 163 . 6 ± 82 . 2 °/s , temporal: 833 . 0 ± 416 . 5 °/s , p = 0 . 0662 , mean ± SD , two-sided t-test , unequal variance , N=2 mice ) .",
"Optic flow was higher in the lower visual field and considerably lower in the upper visual field ( lower left eye visual field: 262 . 44 ± 106 . 50 °/s , upper left eye visual field: 44 . 87 ± 24 . 31 °/s , p = 1 . 78x10−20 , lower right eye visual field: 361 . 91 ± 168 . 80 °/s , upper right eye visual field: 40 . 59 ± 22 . 79 °/s , p = 6 . 68x10−19 , Two-sided t-test , unequal variance , N=2 mice ) , due to the greater distance between ceiling and mouse ( distance to floor 2 ± 1 cm , distance to ceiling 308 ± 107 cm , 9873 frames , N=3 mice ) .",
"Given the advantage of low optic flow to mammalian vision , we next quantified the position of least optic flow during prey tracking .",
"We calculated the location of the translational optic flow minimum in each frame for each eye , and created a probability map of this location over the visual field ( Figure 6E ) .",
"The region of highest likelihood for the presence of the optic flow minimum overlapped considerably with the functional foci in both eyes during the tracking epochs of the pursuit behavior ( overlap of optic flow 95% minima and functional foci 50% regions: 100% and 99 ± 1% , overlap of optic flow 50% minima and functional foci 50% regions: 61 ± 14% and 72 ± 4% in left and right eyes respectively , N=3 mice , Figure 6E ) .",
"Together this shows that the behavioral strategy employed by mice during hunting , consisting of orienting themselves to directly face the prey and following a straight and direct course to it , results in the image of their prey coinciding with the region of reduced optic flow during pursuit , where the retinal image of their prey is least distorted due to motion induced image blur ."
],
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"We developed a technique for reconstructing the visual fields in a freely moving mouse during prey pursuit to quantify the spatial relationship between the environment , cricket and the mouse .",
"Using this approach , we show that during pursuit of crickets , the hunting behavior employed by mice results in the image of the prey consistently falling within a localized region of their visual field , termed here the functional focus .",
"The positional maintenance of the cricket was not achieved by active eye movements that followed the prey , but rather by the animal’s change in behavior , specifically the head-movement and orientation toward the prey during pursuit .",
"While eye rotations stabilized the visual field via the vestibulo-ocular reflex by countering head rotations , the rotations were not specific to either prey detection or prey tracking .",
"This strongly suggested that eye-rotations in mice , like in rats , primarily stabilize their large field of view and that all three rotational axes , including ocular torsion , combine to counter head rotations .",
"In addition , we also show that eye rotations cannot be predicted from head rotations in any one axis as has been suggested by recent studies of mouse eye motion ( Meyer et al . , 2020; Meyer et al . , 2018; Michaiel et al . , 2020 ) but rather by a combination of all head rotations ( Figure 4—figure supplement 2 ) .",
"As the eye rotations were predominately associated with countering head-rotations , this raised the question of whether the mouse can use a large fraction of its stabilized visual field to pursuit crickets , or whether a specific region is utilized .",
"To accurately determine the correspondence between the animal’s visual field and the retinal image , we developed a quantitative model of the mouse eye and optics .",
"Using this we show that the location of the functional focus occurs within a dorso-temporal retinal region in an area with the highest density of Alpha-ON sustained RGCs , whose general properties have been previously proposed to be well suited for this purpose ( Bleckert et al . , 2014 ) .",
"Finally , we used the detailed , digitally rendered reconstruction of the arena and surrounding room to calculate the realistic optic flow in the visual fields ( Gibson et al . , 1955; Sabbah et al . , 2017; Saleem , 2020 ) of the mice as they pursued crickets , which showed that the functional foci coincide with the region of the visual fields with minimal optic flow during the cricket pursuit , and presumably are thereby minimally distorted by motion-induced image blur ( for review see Angelaki and Hess , 2005 ) .",
"Critical to this finding was the ability to isolate the visual sense , generate a detailed reconstruction of both the local environment and the animal’s ocular anatomy and optical pathways , but also record eye motion in all three optical axes especially ocular torsion , something that has only been achieved in rats ( Wallace et al . , 2013 ) .",
"Lastly , by building an optical model and establishing the relationship between the retinal surface and the corneal surface we were able to relate the data generated from published studies on retinal anatomy ( Bleckert et al . , 2014; Dräger and Olsen , 1981; Sterratt et al . , 2013 ) and physiology ( Dhande et al . , 2015; Martersteck et al . , 2017; Murphy and Rieke , 2006; Pang et al . , 2003; Sabbah et al . , 2017; van Wyk et al . , 2009 ) to our behavioral data .",
"Both estimates of the field of view of the mouse eye ( Dräger , 1978 ) and electrophysiological measurements of receptive field locations of visually responsive neurons ( Dräger and Olsen , 1980; Wagor et al . , 1980 ) have established that the binocular region of the visual field in mice is contained within the nasal visual field of each eye , and spans a region of 30-40° in front of the animal ( Wagor et al . , 1980 ) .",
"We present here , that similar to the rat ( Wallace et al . , 2013 ) , the overlapping monocular fields that make up the binocular overlap are not constantly maintained ( Figure 4H ) but fluctuate at the margins as the eyes rotate to counter head rotations ( Figure 4D ) , resulting in a region where there is a transition from one area with near continuous binocular coverage , through to a region that is invariably monocular .",
"The functional focus described here lies within the region of high probability of maintained binocular overlap .",
"This region of the visual field projects onto the temporal retina , which contains both ipsilaterally projecting ( uncrossed ) RGCs ( Dräger and Olsen , 1980; Reese and Cowey , 1986 ) and RGCs which form part of the callosal projection pathway ( Laing et al . , 2015; Olavarria and Van Sluyters , 1983; Ramachandra et al . , 2020 ) , both of which are considered central to binocular visual processing .",
"In addition , the current study adds to the significance of these previous findings and suggests that the functional focus location is well placed to support stereoscopic depth perception , assuming that this form of visual processing is available to and employed by the mouse ( La Chioma et al . , 2019; La Chioma et al . , 2020; Samonds et al . , 2019; Scholl et al . , 2013; Scholl et al . , 2015 ) .",
"Further supportive of the importance and relevance of the region of binocular overlap , another recent study provides strong evidence to suggest that ipsilaterally projecting RGCs in the ventro-temporal retina are important in the final phase of cricket pursuit ( mouse to cricket distance less than 6 cm ) , with selective ablation of these RGCs reducing the probability that coming into close proximity with the cricket resulted in its capture ( Johnson et al . , 2021 ) .",
"This finding is complimentary to the current study , in that it deals with the section of the hunting behavior excluded from analysis in the current study , that being behavioral segments where the distance between mouse and cricket is < 3 cm .",
"This criteria was used in the current study to mitigate the possibility that the mouse was using its mystacial whiskers to detect or assist in detection of the cricket location .",
"In the current study , we find that the location of the image of the cricket systematically shifts nasally and slightly ventrally on the cornea ( temporally and slightly dorsally on the retina ) as the mouse closes in on the cricket , and this may place the cricket’s image within the retinal region containing the ipsilaterally-projecting RGCs .",
"As the mouse closes further , beyond our distance threshold , these RGCs may become increasingly important , particularly when the cricket is within grasping distance , where binocular vision and stereopsis may be most relevant .",
"While the overall highest density of retinal ganglion cells in mice is located in the region around the optical axis ( Dräger and Olsen , 1981 ) , a recent study examining the distributions of various different subclasses of RGCs has shown that the highest density of Alpha-ON sustained RGCs resides in the superior-temporal retina ( Bleckert et al . , 2014 ) in a region which would approximately coincide with the functional focus .",
"These Alpha-ON sustained RGCs have center-surround receptive fields , a rapid response and fast conducting axon , and are thought to be ‘spot detectors’ ( for review see Dhande et al . , 2015 ) .",
"In addition , the Alpha-ON sustained RGCs in this particular retinal region differ from the same RGC-type in other regions of the retina as they have a significantly smaller dendritic tree radius and subtend a smaller area of physical space as well as have overlapping receptive fields ( Bleckert et al . , 2014 ) .",
"Taken together , the cellular properties as well as the region in-front of the animal which provides their input are consistent with the requirements for tracking small and mobile targets ( Bleckert et al . , 2014; Dean et al . , 1989; Lettvin et al . , 1959; Procacci et al . , 2020 ) .",
"A recent study has shown that both wide-field and narrow-field neuronal types in the mouse superior colliculus play central roles in the detection and pursuit phases of this pursuit task , respectively ( Hoy et al . , 2019 ) , and consistent with this , Alpha-ON sustained RGCs having projections to the superior colliculus ( Martersteck et al . , 2017 ) .",
"It is currently unclear how the primary visual cortex ( V1 ) contributes to this behavior , but some role is possible if not probable , which would also be supported by the strong Alpha RGC projection to the dorsal lateral geniculate nucleus and thus V1 ( Martersteck et al . , 2017 ) .",
"Additionally , an increased cortical magnification factor occurs in the region corresponding to the nasal , binocular visual field ( Garrett et al . , 2014; Schuett et al . , 2002 ) .",
"Finally , we show that the region that contains these Alpha-ON sustained RGCs also coincides with the region of minimum optic flow and therefore reduced image blur during translation pursuit , a feature which would support accurate localization of small targets by Alpha-ON sustained RGCs .",
"Patterns of optic flow are thought to be an important component of perception of self-motion ( Lappe et al . , 1999 ) .",
"Mechanistically supporting this , global alignment across the retina of the preferred orientation of direction-selective retinal ganglion cells with the cardinal directions of optic flow during idealized motion has been shown in mice ( Sabbah et al . , 2017 ) .",
"The average optic flow measured here was , perhaps not surprisingly , strikingly different from that observed with idealized motion , resulting in large part from the large differences to objects in the environment in which the behaviors were performed .",
"For fast moving , ground dwelling animals like mice , considerable asymmetry in optic flow across the visual field may be the more normal case , considering that objects above the animal are , in general , likely to be more distant .",
"In freely moving rats , it has been shown that ocular torsion is correlated with head pitch such that nose-up rotation of the head is counteracted by incyclotorsion ( rotation toward the nose ) of both eyes , with nose-down pitch counteracted by excyclotorsion ( Wallace et al . , 2013 ) .",
"These rotations have the effect of stabilizing the horizontal plane of the retina with respect to the horizon .",
"The considerable radial separation between the optical axis of the eye and both the functional foci observed in the current study as well as the highest density region of Alpha-ON sustained RGCs ( Bleckert et al . , 2014 ) renders the direction in which these regions point highly sensitive to torsional rotation .",
"Consequently , torsional rotation also has an important effect on alignment of the left and right visual fields in addition to its role in visual field stabilization .",
"We show here that torsional rotation in freely moving mice is also dynamic , with episodes showing in- and excyclovergence as well as dextro- and levocycloversion .",
"Further , while the correlation between torsional rotation and head pitch observed in rats was measured , there was also an additional relation between ocular torsion and head roll consistent with VOR-evoked dextro- and levocycloversion .",
"Consequently , prediction of torsion using a model based on head pitch alone resulted in an average error of around 7° , while an expanded model including roll as well performed better ( Figure 4—figure supplement 2J–O ) .",
"In summary , we show here that during pursuit in mice the image of the intended prey falls consistently in a localized region of their visual fields , referred to here as the functional focus , and that this occurs through the animal orientating their head and body and running directly toward the prey rather than with specific eye movements .",
"The location of the functional focus is within the binocular visual field , but in addition also coincides with the region of minimal optic flow during the pursuit , and presumably also minimally distorted by motion blur ."
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[
"Experiments were carried out in accordance with protocols approved by the local animal welfare authorities ( Landesamt für Natur Umwelt und Verbraucherschutz , Nordrhein-Westfalen , Germany , protocol number 84–02 . 04 . 2017 . A260 ) .",
"Experiments were carried out using male C57Bl/6J mice acquired from Charles River Laboratories .",
"At the time of the cricket hunting experiments , mice ( n=9 ) were between 2 and 8 months old , and weighed between 21 and 29 g .",
"Mice were maintained on a 12 hr light/dark cycle .",
"Crickets ( Acheta domesticus , Bugs-International , Germany ) were housed in 480x375x210 cm cages with ad lib water and food ( powdered mouse chow ) .",
"During experiments in which head and eye rotations were recorded cricket body sizes ranged from 1 cm to 2 cm ( 1 . 8 ± 0 . 3 cm , mean ± SD , n=25 ) .",
"Animals were anesthetized using fentanyl , medetomidine , and midazolam ( 50 µg/kg , 5 mg/kg , and 0 . 5 mg/kg , delivered i . p . , respectively ) , and analgesia was provided with carprofen ( 7 mg/kg delivered s . c . ) .",
"Body temperature was maintained using a thermostatically regulated heating pad .",
"Respiration rate and depth of anesthesia was monitored throughout the procedure .",
"Following opening of the skin and removal of connective tissue overlying the sagittal suture and parietal bones , the skull was cleaned with H2O2 ( 3% ) .",
"A custom-made implant , consisting of a flat circular attachment surface for attachment to the skull , and implant body with three anti-rotation pins and a magnet ( Figure 7A–B ) , was fixed to the dried skull using a UV-curing dental adhesive ( Optibond FL , Kerr Corporation , Orange , California , USA ) and a UV-curing dental composite ( Charisma , Kulzer GmbH , Hanau , Germany ) .",
"The implant attachment surface and body were made from light-weight , bio-compatible dental resin ( Dental SG , Formlabs , Germany ) .",
"Skin margins were closed with 5/0 Vicryl sutures ( Ethicon Inc , Somerville , NJ , USA ) and a cyanoacrylate adhesive ( Histoacryl , B . Braun , Melsungen , Germany ) .",
"The injectable anesthetic combination was antagonized with naloxone , atipamezole and flumazenil ( respectively 1 . 2 mg/kg , 0 . 5 mg/kg and 0 . 75 mg/kg , delivered i . p . ) , and the animal was allowed to recover .",
"The eye cameras for oculo-videography were mounted on mounting arms which were attached to a baseplate with complementary holes to the anti-rotation pins on the implant and fitted with a magnet of complementary polarity .",
"During positioning of the head-camera , mice were anesthetized with isoflurane ( induction: 3–5% isoflurane , maintenance: 2 . 0% isoflurane in air ) .",
"Anesthetic depth and body temperature were monitored as above .",
"The cameras were positioned to have a sharp image of the entire eye , with the mounting arms adjusted such that the cameras and mounting system caused minimal disruption to the mouse’s lateral and frontal field of view .",
"Mounting arms were secured with cyanoacrylate adhesive glue ( Histoacryl , B . Braun , Melsungen , Germany ) .",
"The eye-camera system was then removed and the animal allowed to recover .",
"Mice were acclimated to cricket capture in their home cage .",
"Individual crickets were placed in the mouse’s home cage overnight , in addition to their standard ad lib mouse food .",
"Mice were handled and habituated to the experimenter , the head cameras , and the head tracking mounts .",
"The ability of each mouse to visually track the crickets was assessed using the protocol of Hoy et al . , 2016 .",
"Briefly , the ability of the mice to track and capture crickets in a white walled , 480 x 375 x 210 cm arena was assessed in lit and dark conditions ( Figure 7C ) .",
"Mice were given 2 min in which to capture the crickets .",
"Prior to the assessment mice were food deprived overnight before the trial .",
"Crenellations along the iridial-pupil border were less distinct in mice than those previous described in rats ( Wallace et al . , 2013 ) .",
"Ocular torsion changes were therefore measured by tracking the rotations of small spots of titanium dioxide ( TiO2 ) paste dots ( ~300 μm ) applied to ventral and/or temporal locations on the cornea as described in van Alphen et al . , 2010 .",
"The TiO2 paste consisted of TiO2 powder ( Kronos Titan GmBH , Leverkusen , Germany ) mixed with a small quantity of sterile artificial cerebrospinal fluid solution with the following composition ( in mM ) : 135 NaCl , 5 . 4 KCl , 1 . 8 CaCl2 , 1 MgCl2 , 5 HEPES , pH balanced to 7 . 2 ( 300 mOsm/l ) .",
"Application of the TiO2 spots was performed with the animal anesthetized with isoflurane ( induction: 5% isoflurane , maintenance: 0 . 5–1 . 0% isoflurane in air , total time anesthetized 5-10mins ) .",
"Anesthetic depth and body temperature was monitored as above .",
"Following application of TiO2 spots , mice were allowed to recover for >45 min prior to a cricket hunt .",
"The presence of the TiO2 marks did not significantly change the animal’s cricket hunting performance as assessed by the average time taken to capture crickets ( Figure 7C ) .",
"Initially , mice were allowed to explore the experimental arena ( 1x1x0 . 26 m ) without head camera mounts .",
"During subsequent training sessions , mice were acclimated to cricket hunting , with the head cameras on , in the experiment arena .",
"Auditory white noise ( 60–65 dB , NCH-Tone generator v 3 . 26 , NCH Software , Inc Greenwood Village , USA ) was provided through four speakers ( Visaton , Germany ) , one on each wall of the arena .",
"Olfactory noise was provided by ventilating the arena ( TD-1000/200 Silent fan , S and P , Barcelona , Spain ) through a perforated floor ( 5 cm perforation spacing ) with air blown through a cage containing live crickets ( cricket cage dimensions 480x375x210cm ) .",
"During experiments the arena was lit by a single lamp ( 4000 K , 9W , Osram , Munich , Germany ) positioned ~1 m above the arena .",
"During each experiment , the mouse was given 5 min to explore the arena without head cameras .",
"After this period , the mouse was removed from the arena and the head cameras were mounted .",
"At the commencement of each hunt , the cricket was released at a variable location into the central region of the arena .",
"Head and eye tracking was performed as described in Wallace et al . , 2013 , with modifications as described below .",
"The eye camera mount and implant were re-designed to be smaller , lighter and stronger ( Figure 7A–B ) .",
"The camera system body , camera holders and mounting arms were produced using a Formlabs Form2 SLA 3D printer ( Formlabs Inc , USA ) , with Dental SG Resin ( Formlabs Inc , USA ) as the primary construction material .",
"The cable used for position tracking LEDs power inputs and for data transfer and camera were custom cables ( Axon Kabel GmbH , Leonberg , Germany ) combined with custom-designed flexible flat cables ( IBR Ringler , Bad Rappenau , Germany ) for the cameras , to reduce stiffness over the last 30 cm .",
"Eye movements were recorded at 60 Hz ( camera resolution 752x480 pixels ) , with illumination provided by a ring of three IR-LEDs ( λ=850 nm , OSRAM SFH4050 or SFH4053 @ 70mA , RS Components , Germany ) surrounding the camera lens .",
"The mouse’s head position and head rotations were tracked using seven IR-LEDs ( λ = 950 nm , OSRAM SFH4043 @ 70mA , RS Components , Germany ) mounted on three struts of carbon fiber that projected from the body of the camera system .",
"The resultant total system weight was ~3 g , including effective cable weight .",
"The positions of the cricket within the arena were recorded using four cameras ( 488 x 648 px , recorded at 200 Hz , piA640-210gm , Basler cameras , Basler Ahrensburg , Germany ) fitted with NIR-blocking filters ( Calflex X , Qioptiq , Germany ) .",
"Cameras were located ~1 . 5 m above the arena and were positioned so that the arena was covered at all points by two or more cameras from differing vantage points .",
"Mouse IR-head tracking LEDs were recorded at 200 Hz using four cameras ( piA640-210gm , Basler cameras , Basler Ahrensburg , Germany ) .",
"Image acquisition , synchronization , and mouse head rotation calculations were performed as described previously ( Wallace et al . , 2013 ) .",
"Head mount features and mouse anatomical features ( medial canthi and nostril positions ) were recorded at 50 Hz using four synchronized cameras ( acA2040-90um , Basler cameras , Basler Ahrensburg , Germany ) fitted with 25 mm focal length objectives ( CCTV lens , Kowa Optical Products Co . Ltd , Japan ) calibrated as described below .",
"Cameras were positioned to provide images of the animal and headset from different angles to allow triangulation of the anatomical features ( Figure 7D ) .",
"During acquisition of the calibration images , the animal was illuminated with 12 IR-LED modules , ( λ = 850 nm , Oslon Black PowerStar IR-LED module , ILH-IO01-85ML-SC201_WIR200 , i-led . co . uk , Berkshire , UK ) run at 1 A . Position tracking LEDs , medial canthi , nares , mirror corner and camera chip corner positions were marked in two or more camera views , in multiple synchronized frames .",
"Based on the triangulated positions of anatomical features , head cameras and position tracking LEDs the mouse’s eye position could be placed a common coordinate system .",
"To establish the animal’s horizontal plane from the head tracking LEDs , a position for the animal’s nose was first defined by averaging to 3D positions of the marked nostrils .",
"A pre-forward vector was calculated using the direction between mean of eyes and nose and a pre-up vector as vector orthogonal to the pre-forward and vector between the eyes .",
"Next , the left vector was defined as orthogonal to pre-forward and pre-up .",
"Finally , the system was rotated by 40° around the left vector such that forward vector was elevated .",
"This established a head-fixed forward-left-up coordinate system that was based on the bregma-lambda sagittal plane by tilting the eyes-nose plane by an angle of 40° .",
"Head tracking frame rates were 200 Hz , while eye tracking cameras recorded at 60 Hz .",
"Eye positions were consequently interpolated as follows: LetRt1 , Rt2∈SO ( 3 ) be two rotations that transform the vector 0 , 0 , -1t into the gaze vectors vt1 , vt2 in head fixed coordinates at times t1 , t2 .",
"Then for a time t′ witht′=t1+s⋅ ( t2−t1 ) , 0<s<1the corresponding rotation Rt' is interpolated such that vt' is placed on the geodesic defined by vt1 , vt2 with an angle of s*∠ ( vt1 , vt2 ) to vt1 , and the rotation of a vector perpendicular to ( 0 , 0 , -1 ) t is continuous and uniform between t1 and t2 .",
"Overhead cameras for animal position and cricket tracking , were calibrated as previously described for the overhead cameras ( Wallace et al . , 2013 ) , with the addition of automated detection of corresponding points in the calibration images using openCV and the eye camera calibration performed as described in Wallace et al . , 2013 .",
"Pupil boundary tracking , compensation for eye image displacement , and gaze vector calculation was performed as described previously in Wallace et al . , 2013 .",
"Where contrast between pupil and iris was insufficient to allow automated pupil position tracking , pupil positions were manually tracked .",
"The TiO2 spots for tracking ocular torsion were tracked manually in each image frame .",
"Torsional rotations were determined based on the tracked TiO2 spot positions as follows .",
"Total rotation of the eye was defined as previously described in Wallace et al . , 2013 , as:Reye=RϕRθRψ=1000cosϕ-sinϕ0sinϕcosϕcosθ0-sinθ010sinθ0cosθcosψ-sinψ0sinψcosψ0001where ϕ = vertical , θ = horizontal and ψ = torsional rotations .",
"The mouse’s gaze vector has the coordinates 00-1T for the reference position of the eye , and in each frame:vgaze=Reye[00−1] With the eye in its reference position , we assume that the marked TiO2 spot is located in the x-y plane of the eye camera ( Wallace et al . , 2013 ) .",
"The anatomical location of this marked spot can then be described by two unknown parameters r ( where r>1 is the 3D distance of the eyeball surface to the eyeball center , and a distance of 1 describes the rotation radius of the pupil ) and α is the fixed angle between the TiO2 mark and the gaze vector .",
"After eye rotation the 3D location of the TiO2 is:vmark=Reye[rsinα0−rcosα]and the predicted pixel coordinates of the spot in the image are:pmark=[aECbEC]+fz0[v1markv2mark]where aEC and bEC are the location in the image of the center of the eye ball and fz0 a scaling factor , both of which are determined in the calibration procedure for pupil boundary tracking , described in full in Wallace et al . , 2013 .",
"When r and α are known the value ψ can be determined .",
"Using the Matlab function fminbnd the squared 2D distance|pmarkpmark|22between the predicted and marked locations of the TiO2 mark is minimized .",
"This method is used to determine the ocular torsion based on the TiO2 spot location , both during and after calibration .",
"Calibration was performed as follows: For a given r and α choice , ψ can be calculated as above .",
"The sum of square errors in pixel locations is then calculated over all frames .",
"We optimized over r and α using the Matlab function fminsearch .",
"To initialize r , we make use of the fact that the pupil model , pmark and r together determine the 3D location of the mark vmark in each image .",
"For each frame , we first calculated:Δa=p1mark−aECf/z0Δb=p2mark−bECf/z0m=min1 , rΔa2+Δb2vinitmark=[mΔamΔb−r2−m2 ( Δa2+Δb2 ) ]αinit=cos−1 ( vgaze . vinitmark/r ) Using this method , αinit is estimated separately for each frame , and if the choice of r is correct then these values should agree .",
"We can use fminbind to minimize the following with respect to r:Var ( αinit ) = ( αinit−αinit¯ ) 2¯ After r is initialized , αinit is calculated , with α initialized using the mean over frames .",
"Torsional rotations were normalized by calculating a mean torsion value for the 0 . 01% of frames that were closest to both median pitch and roll of the head .",
"Torsional values in other tracked frames were then normalized to this mean torsion value .",
"Cricket body positions were automatically tracked using the method and algorithm described for tracking eye corners , as described in the section ‘Compensation for lateral eyeball displacement – tracking of anatomical landmarks around the eye’ in Wallace et al . , 2013 .",
"To increase the contrast between the region around the cricket in the image and the cricket , ~100 background image frames ( in which neither mouse nor cricket was present ) were averaged and subtracted from frames in which the cricket was present .",
"In frames where automated cricket position tracking was not possible , frames were tracked manually .",
"As the cameras used for cricket tracking had been calibrated along with the animal position tracking cameras ( see above ) , the three-dimensional location of the cricket could be triangulated in a common coordinate system with the animal’s position .",
"To decrease the effects of tracking noise and rapid head rotations , mouse velocity , target bearing and inter-animal Euclidean distances were first filtered using a 50 ms sliding window Gaussian filter .",
"The criteria used to classify the different hunt phases were based on those described in Hoy et al . , 2016 .",
"In an initial step , behavioral end points ( tend ) for capture periods were identified by manual inspection of the tracking videos .",
"Further identification of the behavioral start points ( tstart ) and tend points for the different hunt sequence epochs were then identified as described below .",
"The tend points were defined as: The start points of the hunt epochs were defined as follows: Cases in which the eye cameras were dislodged by the animal during the chase ( n=4 hunt sequences ) were included in the dataset up until the point where the cameras were dislodged .",
"Target bearing was defined as the angle between the cricket position and the mouse’s forward head direction in the horizontal plane .",
"For the digital reconstruction , the company 3dScanlab ( Cologne , Germany ) was engaged to create a complete scan , photo series and 3D mesh model of the arena and room , which they performed using an RTC 360 3D laser scanner ( Leica , Germany ) .",
"The 3D point cloud produced by the laser scanner was converted to a 3D mesh model , to which textures of the experiment arena obtained from photographs ( Nikon D810 , 36 Mpx ) were baked .",
"The camera tracking coordinate system , in which the mouse and cricket positions were tracked , and the scanned coordinate system of the 3D mesh model were aligned based on 16 fiducial points which could be clearly identified in both tracking camera images and the scan .",
"Crickets were modeled as 2 cm diameter , 1 cm thick disks centered on their tracked position with the disk's axis oriented parallel to gravity .",
"Each eye was modeled as a hemisphere with a 180° field of view whose equator was perpendicular to the animal’s gaze vector .",
"For the projection of the environment onto the cornea , frame-wise animal’s eye views for both eyes were created with custom written software in C++ ( g++ 7 . 5 . 0 , QMake 3 . 1 , Qt 5 . 9 . 5 , libopenexr 2 . 2 . 0 , libpng 1 . 6 . 34 and OpenGL-core-profile 4 . 6 . 0 ) on a GeForce RTX 2070 ( NVidia driver 450 . 66 ) , using first cube mapping followed by a transformation into a spherical coordinate system .",
"To do this , individual frame-wise coordinate transformations were made using the eye locations and orientations determined as described above to transform the mesh model of the arena and cricket to a static eye coordinate system using custom written vertex shaders to perform the coordinate transformation and the fragment shaders to texture the mesh .",
"A cube-map ( 1024 x 1024 pixels per face ) was created by performing such coordinate transformations for a 90 degree view in the direction of the optical axis of the eye and four mutually orthogonal directions .",
"Custom written code was then used to transform the cube-map into a spherical coordinate system , with a 180 degree opening angle , using fragment shaders , resulting in a 1024 x 1024 pixel frame exported as png and OpenEXR files .",
"In addition to the color map , maps of depth ( pixel-wise object intersection distance ) , object identification , optic flow and 3D position of the object intersection point in the contralateral eye’s coordinate system were also generated .",
"For generation of the prey image probability density maps , animal’s eye views were rendered that contained the cricket only ( i . e . without inclusion of arena and room ) .",
"Density maps from multiple detect-track sequences , and multiple animals , were made by averaging .",
"Ocular alignment was defined as the consistency of the projection of a given point in the eye view of one eye into the other in an infinitely distant environment .",
"This is equivalent to a projection in an idealized finite-distant spherical environment while assuming a distance between the animal’s eyes of 0 .",
"For calculation , the radius of the sphere can then be set to 1 ( without loss of generality ) .",
"A point , located at the center of mass of the functional focus in each eye , was chosen from which to calculate the degree of inter-ocular alignment .",
"This point was projected from one eye to the sphere surface and into the contralateral eye .",
"The degree of alignment between the two eyes was calculated as follows: LetRi , Li:R3→R3be the affine transformations for the left and right eye , and letE⊆R3be the idealized environment .",
"For a given direction u∈S2 we calculate the projection into the right eye pi∈R3 by:pi=Li−1∘Ri∘u The average alignment is then calculated using the formula:∑¯2⋅arcsin ( |pi−⟨∑¯pi⟩|/2 ) where ∑¯pi denotes mean and ⟨⟩ denotes normalization .",
"Visual field overlap was analyzed in the idealized finite-distant spherical environment described above for ocular alignment .",
"Visual overlap was calculated from the frame-wise maps of 3D object intersection points in the contralateral eye ( see above section ‘Generation of animal’s eye view’ ) generated for the ocular alignment analysis: pixels whose 3D object intersection points had an angle of less than 90° to the optical axis were considered part of the overlapping field of view .",
"Probability maps of overlap were calculated by averaging .",
"For analyses of the effect of freezing eye movements , eye rotations ( horizontal , vertical , and torsional ) were set to the mean rotation in one eye , and the effect quantified in the other eye view .",
"To calculate the optic flow in a given pixel for a given eye , we consider the difference vector between the 3D positions in the static eye coordinate system of the object intersection point for this pixel one frame before and after the frame of interest , divided by 2∙dt and mapped to unit distance by dividing by the distance between eye and interception point .",
"This yields a 3D motion vector which is independent of influences of the frame rate and rendering resolution .",
"The spherical projection used in the rendering process described above is a non-conformal , locally non-isometric map , meaning that angles between lines and distances between points are not preserved .",
"This makes it necessary to evaluate the flow in each point in a local , orthonormal 3D coordinate system defined by the direction vector between the eye position and the object intersection point and derivative vectors along the angular coordinates vθ and vφ at that point .",
"Thus , we define the 2D flow at a given point as the orthogonal projection of the 3D flow vector onto the local plane spanned by vθ and vφ .",
"In this study , we only use the first two components of the vector , while the third component contains the motion in radial direction to the eye .",
"In Figure 5C , optic flow was calculated for the animal in the idealized spherical environment described above , meaning the animal’s head was equidistant to the surrounding at all points .",
"This simplified scene was characterized as follows .",
"Leth∈R3be the coordinate of the center of the mouse’s head , then the scene around it was defined as{p∈R3||p−h|=r}with r = 50 cm .",
"For optic flow calculations , the sphere is considered fixed in global coordinates , and the flow is evaluated at the point where the mouse is in the center of the sphere translating forward at a speed of 1 cm/s .",
"In Figure 5E , optic flow was calculated with the animal in the digitally reconstructed environment ( see above ) .",
"The points in the scatter plot of optic flow poles in mouse corneal views were color-coded for the density of neighboring points using a two-dimensional Gaussian smoother with standard deviationσ=2π180 For a given point , the density was calculated as:si=∑j∈F12πσ2exp-xi-xj22∙σ2/|F|where F is the set of all considered frame indices , andxi=∂hpi∂hpiwhere ∂h[p]i is the discrete central difference quotient of the mouse’s eye trajectory p in frame i , in the coordinate system of the respective eye , evaluated over h=4 frames .",
"When constructing the eye model , we took experimentally determined values from Barathi et al . , 2008 ( see Table 1 ) .",
"While we recognize that this study employed a different strain of mice to the one used here , the methodology used provides estimates of physical and optical parameters measured under conditions closest to those relevant for the current study .",
"Further , variation of these parameters was not found to change the model to an extent that would influence the conclusions drawn from analyses involving the eye model ( see below ) .",
"These values distinctly define the spatial shapes and positions of the refractive components of the model eye ( Figure 3A ) , as well as refractive indices for all but the lens , nlens .",
"We further assume a pupil radius of 594 µm , which is the mean of constricted and dilated mouse pupil sizes from Pennesi et al . , 1998 .",
"We define the focal point of a bundle of rays as the point with minimal least squares distance to the rays .",
"To optimize the missing refractive index nlens:Ω→R+ inside the lens body Ω⊂R3 , we first calculated two lens models and optimized them such that the focal point of 10000 rays emitted from an object at 10 cm distance on the optical axis lay on the retina .",
"The first model , for optimization of the lens surface , was derived with optimal constant refractive index nc∈R+ over the volume .",
"The second model , for lens gradient optimization , was derived with a smooth transition of refractive index to the anterior and posterior lens boundary , that is , nb=1 . 333 on ∂Ω .",
"We then used Poisson’s equation δng=c , and optimized the strength of the gradient c∈R+ .",
"We assumed the final lens model as a linear combination of these two models:nlens=α⋅nc+ ( 1−α ) ⋅ngwith α∈0 , 1 , where we optimized α as described for the above models , but from a point 10 cm away and 45° off optical axis .",
"The derived refractive indices ( Table 2 ) were within the range measured in Cheng et al . , 2019 .",
"To test the sensitivity of the model to changes in assumed physical parameters , we systematically changed the radius of curvatures listed in Table 1 , and the thickness listed in Table 2 by 10 , 50 , and 100 µm ( several different values were used , to check the linearity of the dependence ) .",
"We calculated the propagation of uncertainty through the eye model by analyzing the variation of radial elevation on the retina of the 45 rays ( above ) , taking the numerical differentiation of each input variable that was used in the model .",
"Lens optimization was performed for each newly generated eye model ( as described above ) .",
"The maximum deviations were 0 . 4 , 1 . 38 and 2 . 76 degrees for the 10 , 50 , and 100 µm changes , respectively ( Figure 7E ) , and overall , none of the observed effects on the model would influence the conclusions drawn from the analyses performed using the eye model .",
"The refractive elements in the rodent eye do not behave like ideally corrected optical elements , with the result that there is a distribution of incident rays with slightly varying angles of incidence on the cornea which converge on any given point on the retina .",
"Projection from retina to cornea therefore requires an estimate of the distribution of outside world angles of incidence for any point of interest on the retina .",
"To do this , we used a Monte-Carlo simulation to back-trace through the optics a set of randomly chosen rays emerging from the point of interest on the retina .",
"Since the intensity of light on a surface with an incoming angle of θ is proportional to cos ( θ ) , this function was also chosen for the probability density distribution of ray exit angles .",
"The rays were then traced until they either hit any opaque surface , resulting in the affected ray being discarded , or passed through the anterior cornea , in which case the ray was accepted and its angle added to the distribution of passing exit angles for the respective point on the retina .",
"Refraction on boundary layers between different indices of refraction was performed analytically according to Snell’s law .",
"In volumes with a continuous variable refractive index ( i . e . gradient-index ( GRIN ) optics ) , we used a finite-elements model .",
"We first discretized the lens as a 40x40x40 lattice of side length 2 . 4 mm .",
"We then started from initial conditions where s ( 0 ) is the point of incidence and v ( 0 ) is the vector of incidence multiplied by the speed of light c .",
"The subsequent discrete trajectory and direction of propagation is then calculated step-wise according tosti+1:=sti+υ ( ti ) ∙ ( ti+1-ti ) υ~ti+1:=υ~ti+∇lognsti+1∙ ( ti+1-ti ) υti∶=υ~ti|υ~ti|2 The gradient is calculated in the lens lattice as the three-dimensional difference quotient , and then trilinearly interpolated to the exact position sti of the ray .",
"To determine the corneal location corresponding to the histologically identified retinal specialization in the mouse , isodensity lines were redrawn from Dräger and Olsen , 1980 in Illustrator and digitized using Matlab .",
"Isodensity lines enclosing regions containing the highest and second highest density of retinal ganglion cells , as well as the optic disc and outline of the retinal whole mount , were redrawn directly from Figure 3A in Dräger and Olsen , 1981 , with horizontal being taken as horizontal ( nasal-temporal ) in the figure .",
"The isodensity lines were scaled to match the eye diameter used for model eye , then placed into the model eye such that the center of mass of the optic disc reconstructed with the retinal ganglion cell contours was coincident with the intersection of the optic axis and retina in the eye model ( Figure 2—figure supplement 1A-C ) .",
"As the eye model was rotationally symmetrical , no further alignment between the histology and eye model was necessary .",
"The high retinal ganglion cell density regions were then back-projected from retina to cornea as described above ( Figure 2—figure supplement 1D-E ) .",
"To quantify the effect of head rotations on VOR evoked eye movements in a common coordinate system , head rotations were normalized such that the average pitch and roll were 0 .",
"Axes were labeled X and Y respectively and eye rotations were represented using this horizon-aligned X-Y coordinate system .",
"Positive head X values indicate head pitched up , while negative head X values indicate head pitched down .",
"Negative head Y values indicate roll left , while positive Y values indicate roll right .",
"Comparisons of the relationship between head and eye rotations were carried out using differential rotations between frame and average pose , defined in the following way:l′:L→G , r′:R→G , h:H→Gare the affine transformations between Cartesian global coordinate system G , head-fixed coordinate system H and left/right-eye coordiante systems L/R .",
"The transformations from L/R respectively to H are:l=h-1∙l'r=h-1∙r' We calculate the left and right eye differential rotations as:ldelta=l⋅l¯−1rdelta=r⋅r¯−1where l¯ and r¯ denote the average transformations over all frames ( chordal L2 mean , implementation from SciPy 1 . 4 . 1 ) .",
"Within one experimental trial , the experimentally measured variables of interest are highly correlated with each other .",
"This fact prevents us from using standard statistical tests on the whole time-trace to establish if any difference we observed in the data across different experimental conditions are significant or not , as one requirement of these kind on tests is that the samples from the populations being compared are independent of each other .",
"However , we realized that trial-to-trial variability is the dominant source of variability in the data , whereas within-trial variability explains a smaller fraction of the total variance observed ( a more detailed report is found in Table 4 ) .",
"For this reason , we decided to represent each temporal trace by its median value .",
"We used the median and not the mean , because the former is more resistant to the presence of outliers and it is better suited to represent the ‘average’ value of a variable in this context .",
"This operation reduced the size of the dataset to one data points per trial , which we can reasonably assume to be independent of each other ."
]
] | [
"Mice have a large visual field that is constantly stabilized by vestibular ocular reflex ( VOR ) driven eye rotations that counter head-rotations .",
"While maintaining their extensive visual coverage is advantageous for predator detection , mice also track and capture prey using vision .",
"However , in the freely moving animal quantifying object location in the field of view is challenging .",
"Here , we developed a method to digitally reconstruct and quantify the visual scene of freely moving mice performing a visually based prey capture task .",
"By isolating the visual sense and combining a mouse eye optic model with the head and eye rotations , the detailed reconstruction of the digital environment and retinal features were projected onto the corneal surface for comparison , and updated throughout the behavior .",
"By quantifying the spatial location of objects in the visual scene and their motion throughout the behavior , we show that the prey image consistently falls within a small area of the VOR-stabilized visual field .",
"This functional focus coincides with the region of minimal optic flow within the visual field and consequently area of minimal motion-induced image-blur , as during pursuit mice ran directly toward the prey .",
"The functional focus lies in the upper-temporal part of the retina and coincides with the reported high density-region of Alpha-ON sustained retinal ganglion cells ."
] | [
"Mice have a lot to keep an eye on .",
"To survive , they need to dodge predators looming on land and from the skies , while also hunting down the small insects that are part of their diet .",
"To do this , they are helped by their large panoramic field of vision , which stretches from behind and over their heads to below their snouts .",
"To stabilize their gaze when they are on the prowl , mice reflexively move their eyes to counter the movement of their head: in fact , they are unable to move their eyes independently .",
"This raises the question: what part of their large visual field of view do these rodents use when tracking a prey , and to what advantage ?",
"This is difficult to investigate , since it requires simultaneously measuring the eye and head movements of mice as they chase and capture insects .",
"In response , Holmgren , Stahr et al . developed a new technique to record the precise eye positions , head rotations and prey location of mice hunting crickets in surroundings that were fully digitized at high resolution .",
"Combining this information allowed the team to mathematically recreate what mice would see as they chased the insects , and to assess what part of their large visual field they were using .",
"This revealed that , once a cricket had entered any part of the mice’s large field of view , the rodents shifted their head – but not their eyes – to bring the prey into both eye views , and then ran directly at it .",
"If the insect escaped , the mice repeated that behavior .",
"During the pursuit , the cricket’s position was mainly held in a small area of the mouse’s view that corresponds to a specialized region in the eye which is thought to help track objects .",
"This region also allowed the least motion-induced image blur when the animals were running forward .",
"The approach developed by Holmgren , Stahr et al . gives a direct insight into what animals see when they hunt , and how this constantly changing view ties to what happens in the eyes .",
"This method could be applied to other species , ushering in a new wave of tools to explore what freely moving animals see , and the relationship between behaviour and neural circuitry ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"structural biology and molecular biophysics"
] | Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients | elife-62611-v3 | [
[
"Integral membrane proteins serve diverse and critical cellular roles , including signal transduction , lipid biosynthesis , adhesion , and transport of molecules across the bilayer .",
"In eukaryotic cells , the endoplasmic reticulum ( ER ) serves as the primary site of integral membrane protein synthesis , targeting ( co- or post-translationally ) , insertion , folding , and quality control ( Ellgaard et al . , 2016; Costa et al . , 2018 ) .",
"However , the features of membrane-spanning regions ( e . g . low hydrophobicity , charged residues , non-optimal lengths , lipid- and ion-binding sites and hairpins or kinked transmembrane helices ) that mediate important functions pose particular challenges for transmembrane protein biosynthesis and folding .",
"Consequently , membrane protein biogenesis is prone to failure , and this can lead to cellular stress and disease ( Marinko et al . , 2019 ) .",
"Thus , it is important to understand the cellular factors that facilitate proper membrane protein biogenesis for such challenging clients .",
"The ER membrane protein complex ( EMC ) has emerged as a conserved player in the process of membrane protein biogenesis .",
"It was first identified in Saccharomyces cerevisiae as an abundant and stable multi-protein membrane complex whose disruption results in stress mirroring that caused by misfolded membrane proteins ( Jonikas et al . , 2009 ) .",
"Loss of the EMC in mammalian cells is associated with failed biogenesis and degradation of a subset of membrane proteins ( Christianson et al . , 2012 ) .",
"Accordingly , the EMC has been implicated in several mechanistically distinct steps of membrane protein biogenesis , stabilization , and quality control ( Bircham et al . , 2011; Richard et al . , 2013; Satoh et al . , 2015; Savidis et al . , 2016; Shurtleff et al . , 2018; Volkmar et al . , 2019; Tian et al . , 2019 ) .",
"One well-established EMC function is as an insertase for terminal transmembrane helices .",
"EMC’s insertase function has been demonstrated for two classes of clients: low hydrophobicity tail-anchored proteins ( i . e . those that contain C-terminal membrane anchors ) and a subset of polytopic transmembrane proteins in which the first helix is inserted with the N-terminus in the lumen ( Guna et al . , 2018; Chitwood et al . , 2018 ) .",
"However , many studies indicate EMC functions beyond initial insertion of N- or C-terminal helices .",
"The EMC has been implicated in the biogenesis and stability of many membrane protein classes that do not require a terminal transmembrane insertase ( Bircham et al . , 2011; Louie et al . , 2012; Richard et al . , 2013; Shurtleff et al . , 2018; Coelho et al . , 2019; Luo et al . , 2002; Volkmar et al . , 2019; Talbot et al . , 2019; Petkovic et al . , 2020 ) .",
"Recent studies have shown that the EMC is required for stability of internal transmembrane helices of human and viral multi-pass membrane proteins ( Hiramatsu et al . , 2019; Lin et al . , 2019; Ngo et al . , 2019; Coelho et al . , 2019; Xiong et al . , 2020 ) .",
"Additionally , the human EMC ( hEMC ) physically interacts with the NS4A-B region of the Dengue Virus polyprotein following Sec61-dependent translocation and signal peptidase cleavage , suggesting roles in post-translational stabilization of polytopic membrane proteins ( Ngo et al . , 2019; Lin et al . , 2019 ) .",
"Similarly , the S . cerevisiae EMC ( yEMC ) co-immunoprecipitated with full-length polytopic transmembrane clients , including Pma1p ( Luo et al . , 2002 ) , Mrh1p , and Fks1p ( Shurtleff et al . , 2018 ) .",
"In addition to varying types of transmembrane protein clients , the EMC also associates with a range of regulatory factors , including many general and substrate-specific chaperones in the cytoplasm and in the ER lumen ( Bagchi et al . , 2016; Coelho et al . , 2019; Kudze et al . , 2018; Richard et al . , 2013; Shurtleff et al . , 2018 ) .",
"The complex architecture of the EMC provides additional support for multifunctionality in membrane protein biogenesis .",
"The EMC is an eight ( yeast ) or nine ( mammalian ) component , 248–284 kDa complex with considerable mass in the ER lumen , membrane , and cytosol .",
"The cytoplasmic domain contains conserved tetratricopeptide repeats ( TPR ) repeats in EMC2 , and the human complex accommodates an additional subunit , EMC8/9 , whose function is not yet understood .",
"The ER lumenal domain in yeast does not contain an N-terminal EMC1 expansion seen in hEMC .",
"Notably , the ER lumenal domain has been linked to a number of disease-associated phenotypes ( Junes-Gill et al . , 2011; Probert et al . , 2015; Harel et al . , 2016; Abu-Safieh et al . , 2013; Diamantopoulou et al . , 2017; Marquez et al . , 2020 ) , and presents the possibility of additional functions for the human lumenal domain .",
"One EMC subunit ( EMC3 ) shares limited sequence homology with a family of insertases that are evolutionarily related to the bacterial insertase YidC ( Samuelson et al . , 2000; Kumazaki et al . , 2014; Borowska et al . , 2015; Anghel et al . , 2017 ) , perhaps explaining the insertase function of the complex .",
"During the preparation of our manuscript , studies describing the structures of the yeast Get1/Get2/Get3 structures , human WRB/CAML/TRC40 ( McDowell et al . , 2020 ) , translocon bound to Nicalin-TMEM147-NOMO ( McGilvray et al . , 2020 ) , human structures of the EMC ( O'Donnell et al . , 2020; Pleiner et al . , 2020 ) , and the yeast structure of the EMC ( Bai et al . , 2020 ) were published .",
"Those studies focused on the insertase activities of these proteins from the individual species; however , the elaboration of the EMC compared to other known membrane protein biogenesis factors and a diverse client range points to additional functionality that has so far eluded mechanistic explanation .",
"Notably , a systematic structure-based functional analysis across species , conformations , the three distinct EMC domains , and including non-insertase client proteins and mutagenesis of the extensive lumenal domain had not been done .",
"Here , we determined high-resolution cryo-EM structures of yeast EMC bound to a Fab and two conformations of the human EMC structure .",
"Furthermore , we characterized the phenotypes of three distinct classes of EMC clients associated with a series of structure-based EMC mutants .",
"Both yEMC and hEMC structures reveal a path for transmembrane helix insertion from the cytoplasm into the membrane via a conserved cavity .",
"Our structures and mutants also revealed a second lipid-filled cavity with regions of importance for all three client types probed .",
"Analysis of human disease mutations in hEMC1 and our structure-informed mutations enabled us to decouple the EMC insertase function from non-insertase functions and reveal a potential role of the EMC in differentially controlling the biogenesis of distinct classes of client proteins .",
"These structure-function studies collectively establish that the EMC adopts a modular architecture enabling its diverse functions in membrane protein biogenesis ."
],
[
"To comprehensively dissect both conserved and species-specific functions of the EMC , we developed approaches to produce EMC for structure determination and broad mutational analysis ( Figure 1A–D ) .",
"We developed systems to produce robust quantities of pure intact yEMC and hEMC to determine structures for the two organisms in which different facets of EMC function have been described in detail ( Jonikas et al . , 2009; Christianson et al . , 2012; Guna et al . , 2018; Shurtleff et al . , 2018 ) .",
"Parallel efforts converged on an approach involving FLAG affinity-tagging of the EMC5 C-terminus , which was performed for endogenous yEMC and recombinant hEMC in human embryonic kidney ( HEK ) cells ( Figure 1—figure supplements 1–2 , Supplementary file 1 ) .",
"In parallel , to enable testing of hypotheses based on structures , we created a suite of human ( K562 ) knockout cell lines deleted for individual hEMC subunits - hEMC1 ( lumen ) , hEMC2 ( cytoplasm ) , hEMC3 , and hEMC5 ( transmembrane ) - and a series of reporters of EMC-dependent transmembrane protein biogenesis ( Figure 1—figure supplements 3–4 ) .",
"Reintroduction of the wild-type hEMC subunits in the respective knockout cells fully rescued the knockout phenotype ( Figure 1—figure supplements 5–6 ) .",
"This allowed for introduction of structure-based mutations in hEMC subunits into the respective knockout cells to determine features supporting biogenesis of fluorescently tagged versions of three different types of EMC clients: the transmembrane domain of a C-terminal tail-anchored transmembrane protein ( squalene synthase , SQS378-410 ) ( Guna et al . , 2018 ) , a polytopic transmembrane protein that depends on the EMC N-terminal insertase activity ( Beta 1 adrenergic receptor , B1AR ) ( Chitwood et al . , 2018 ) , and a polytopic transmembrane protein ( Sigma intracellular receptor 2 , TMEM97 ) whose biogenesis requires the Sec61 translocon but does not require a terminal helix insertase ( Figure 1—figure supplements 3–6 ) .",
"Three individual EMC clients were fused to mCherry fluorescent protein and GFP separated by a P2A ribosomal skipping sequence .",
"Translation of the described mRNA generates two products due to peptide bond skipping at the P2A sequence .",
"For each molecule of the client-mCherry fusion , there is one GFP molecule .",
"Reduction in mCherry levels relative to GFP reflects post-translational degradation of the client fused to mCherry .",
"Each of the client reporters were introduced into five separate cell lines: wild-type K562 cells , hEMC1 knockout K562 cells , hEMC2 knockout K562 cells , hEMC3 knockout K562 cells , and hEMC5 knockout K562 cells .",
"Monitoring the effect of an hEMC mutation on fluorescent reporter levels provided a quantitative measure of its impact on EMC-dependent biogenesis of each class of client protein .",
"A number of mutations of varying severity , varying conservation between yeast and human ( Figure 1—figure supplements 7–8 ) , were designed and tested spanning the hEMC structure .",
"Subsequently , these 49 mutations were mapped onto the structure grouped by reporter phenotype ( Video 1 , Supplement File 2 ) .",
"To allow for direct comparison of our structure-guided mutant phenotypes with those published recently by others ( Pleiner et al . , 2020; Bai et al . , 2020; O'Donnell et al . , 2020 ) , we summarized all mutant data ( Supplementary file 3 ) .",
"A subset of the mutant cell lines was validated by genotyping ( Figure 1—figure supplement 9 ) .",
"Western blots against the endogenous hEMC subunits allowed us to control for mutational effects on the production and stability of the hEMC complex itself .",
"We concurrently blotted for three clients , SQS , TMEM97 , and BCAP31 , to assay changes in endogenous protein levels for each of the mutations ( Figure 1—figure supplements 5–6 , Supplementary file 4 ) .",
"This strategy thus distinguishes effects resulting from a global disruption of the EMC complex from those caused by specific disruption of EMC function .",
"These functional assays of the hEMC show a broad dependence of all these clients on the EMC , consistent with previous work ( Shurtleff et al . , 2018; Guna et al . , 2018; Chitwood et al . , 2018; Volkmar et al . , 2019; Tian et al . , 2019 ) .",
"In order to understand the mechanism of action , we will now go in more detail through several of the mutants with the strongest functional phenotypes in differing regions of the three-dimensional structure .",
"We determined structures of yEMC and hEMC — all showing overall compositional similarity , with regional conformational differences between the yeast and human complexes ( Figure 2A–D ) .",
"We obtained reconstructions of yEMC bound to an antigen binding fragment ( Fab ) and hEMC reconstituted both in detergent micelles and lipid nanodiscs , with the latter strategy yielding the most isotropic and highest resolution data .",
"For yEMC+FabDH4 and hEMC , the global map resolutions reached 3 . 2 Å and 3 . 4 Å , respectively ( Table 1 , Figure 2—figure supplements 1–4 ) .",
"The cryo-EM maps allowed for de novo model building of both human and yeast complexes ( Figure 2—figure supplements 5 and 6 ) .",
"As described in the following sections , our multiple EMC structures enable a broad survey of its conserved architecture , with variations between the structures pointing to conformational and compositional differences ( Figure 2—figure supplements 7–8 ) .",
"We note that our maps and models are consistent with recent cryo-EM data from yeast EMC ( Bai et al . , 2020 ) , human EMC ( O'Donnell et al . , 2020; Pleiner et al . , 2020 ) , and a crystal structure of human EMC2-EMC9 ( O'Donnell et al . , 2020; Figure 2—figure supplement 9 ) .",
"The EMC is comprised of cytoplasmic , transmembrane , and lumenal domains arranged similarly for yeast and human , despite significant evolutionary separation ( Figure 2A–B ) .",
"For both species , subunits encompassing EMC2 to EMC7 form an interconnected core complex , while there is additional density capping both the cytoplasmic and lumenal domains of hEMC , occupied by an hEMC8/9 and an hEMC1 N-terminal expansion , respectively ( Figure 2C–D ) .",
"hEMC8 and hEMC9 are paralogs of each other , which have not been identified in yeast ( Wideman , 2015 ) .",
"We modeled and depict only hEMC8 for clarity , but due to the 44% sequence identity with hEMC9 and both being present in the recombinant system we refer to this as hEMC8/9 .",
"The large hEMC1 insertion in hEMC constitutes the majority of a membrane distal beta-propeller domain protruding into the lumen , a feature missing from S . cerevisiae .",
"Compared to other ER-resident proteins implicated in membrane protein biogenesis ( Suloway et al . , 2009; Pfeffer et al . , 2017; Ramírez et al . , 2019; McDowell et al . , 2020; McGilvray et al . , 2020 ) , the arrangement of domains of the EMC is unusual with the transmembrane domain connecting prominent cytoplasmic and lumenal domains ( Figure 2E ) .",
"On a global level , the structure suggests complexities beyond those of some other ER machineries fulfilling select functions in transmembrane protein biogenesis .",
"The exterior interface of the cytoplasmic domain is formed by EMC2 , EMC3 , EMC4 , and parts of hEMC8/9 ( in human ) , while parts of EMC5 , EMC2 , and EMC8/9 are shielded from the cytoplasm ( Figure 3A–B ) .",
"The helical fold of EMC2 constitutes the central organizer of this platform , established by five or six TPR motifs in human versus yeast , respectively ( Figure 3C ) .",
"TPR domains are commonly found mediating protein-protein interactions and are present in numerous well-characterized chaperone-protein and other interaction networks ( Blatch and Lässle , 1999; Scheufler et al . , 2000; Schlegel et al . , 2007; Assimon et al . , 2015; Krysztofinska et al . , 2017; Graham et al . , 2019 ) .",
"Yeast EMC2 features a more curved helical arrangement with N- and C-terminal domains in closer proximity to each other than seen in hEMC2 .",
"Notably , the canonical peptide-binding TPR groove is occupied by the partially helical C-terminus of EMC5 , which forms a large interaction surface with EMC2 .",
"To test the functional roles of this interaction , we mutated three residues within the hEMC2 TPR motif ( hEMC2K125E + R126D + K127E ) or a single hEMC5 residue buried in the TPR-binding groove ( hEMC5F90A ) .",
"The mutations on both sides of the interface decreased hEMC integrity by western blot , with a modest decrease of hEMC subunits for hEMC5F90A and a strong reduction in the levels of several hEMC subunits for hEMC2K125E + R126D + K127E ( Figure 3C , Figure 3—figure supplements 1–2 , Figure 1—figure supplements 5–6 ) .",
"This suggests that this interface might be critical for EMC complex assembly rather than EMC function .",
"The multi-protein cytoplasmic cap has distinct elements between hEMC and yEMC .",
"Capping the cytoplasmic domain in hEMC is hEMC8/9; the functional roles of this cap-like structure are not yet clear .",
"An hEMC8-9 heterodimer is not observed and our cryo-EM permits tracing with both the hEMC8 or hEMC9 amino acid sequence ( Figure 3—figure supplement 3 ) .",
"Mass spectrometric analysis of our hEMC preparations reveals slightly higher abundance of hEMC8 to hEMC9 ( Figure 1—figure supplement 2E–F , Supplementary file 1 ) , so we modeled the cytoplasmic cap structure with the hEMC8 sequence .",
"A groove on hEMC8/9 cradles an N-terminal peptide of hEMC4 , which proceeds into the EMC4 segment that traverses over hEMC2 and the three-helix bundle of hEMC3 ( Figure 3D ) .",
"Although yEMC lacks EMC8/9 , yEMC4 follows a similar binding trajectory along cytoplasmic yEMC2 and yEMC3 surfaces .",
"A stretch of 20 hEMC4 amino acids ( residues 23–42 ) after the hEMC8/9 binding site is only poorly resolved in our cryo-EM maps and predicted to be disordered ( 40% glycine content ) .",
"This loop contains primarily polar amino acids , and traverses the top of the hEMC2 TPR domain .",
"To see whether this dynamic hEMC2-hEMC4 interface played a role in client stabilization , we mutated two charged patches on hEMC2 to alanines ( hEMC2E146A+E149A+Q150A , hEMC2E168A+D170A+K173A ) , lying in close vicinity to hEMC423-42 ( Figure 3E–F ) .",
"These mutants lead to a modest accumulation of the tail-anchored client ( SQS378-410 ) but did not affect polytopic client abundance or decrease of hEMC subunits ( Figure 3E–F; Figure 1—figure supplement 5 ) .",
"Several mutants across the cytoplasmic domain showed similar phenotypes , supporting a key role in tail anchor protein biogenesis ( Figure 3—figure supplements 1–2 ) .",
"The transmembrane core of EMC is predicted to include contributions from each subunit except for EMC2 and , in humans , hEMC8/9 ( Figure 2C–D ) .",
"The EMC presents two distinct and structurally conserved cavities on opposite sides of the transmembrane core that differ in size , shape , subunit compositions and apparent function ( Figure 4A–B ) .",
"One cavity , which we refer to as the lipid-filled cavity , appears contiguous with the ER lipid environment ( Figure 4A ) .",
"The second cavity , which we refer to as the gated cavity , appears to open toward the cytoplasm in our structures and is more occluded by a transmembrane helix gate from the lipid environment ( Figure 4B ) .",
"Notable structural hallmarks present in both species include a superimposable core of nine transmembrane helices , a set of flexible gate helices , and an amphipathic EMC1 brace helix ( Figure 4C ) .",
"Evaluating potential client paths from the cytoplasm into the transmembrane domain revealed a cavernous opening at the membrane-cytoplasmic interface of the gated cavity , wide enough to allow passage of a client helix , and tapering toward the lumen ( Figure 4D ) .",
"Consistent with its potential role as a cytoplasmic conduit into the EMC , the EMC3 portion of the cytoplasmic domain , which delineates this opening , sits approximately 45 Å from the lumenal side of the gated cavity .",
"This dimension exceeds the thickness of the ER membrane ( Mitra et al . , 2004; Heberle et al . , 2020; Cornell et al . , 2020; Figure 4D ) .",
"This cavity is lined primarily by EMC3 , EMC4 , and EMC6 ( Figure 5A ) .",
"Simulating the dimension of the first transmembrane helix of a known terminal insertase-client ( B1AR - Chitwood et al . , 2018 ) suggests that there is sufficient space for a client helix even in the client-free state of the EMC ( Figure 5B ) .",
"The gated cavity is hydrophilic on the cytoplasmic side and becomes increasingly hydrophobic toward the lumenal side ( Figure 5C ) .",
"The entrance into the gated cavity interior ( Figure 5A ) is formed primarily by the EMC3 cytoplasmic domain .",
"To test its function , charge swap mutations were introduced along the rim of this opening ( hEMC3E63K + D213K + E223K , hEMC3R59E + R62E + K216E ) ( Figure 5D ) .",
"These mutants resulted in loss of the tail-anchored client ( SQS378-410 ) and partial loss of the N-terminal insertase-dependent polytopic client ( B1AR ) , reflecting a failure to support insertase activity .",
"These mutants had no appreciable effect on the abundance of the polytopic transmembrane client ( TMEM97 ) reporter ( Figure 5E , Figure 5—figure supplements 1–2 ) .",
"A similar phenotype was observed with alanine substitutions for a pair of lysines at the periphery of this cytoplasmic rim ( hEMC3K42A + K43A ) ( Figure 5—figure supplement 2 ) .",
"Having identified a functionally important entry route for terminal helix insertase clients , we next considered potential surfaces inside the cavity that might accommodate a client helix .",
"A polar patch close to the membrane interior of this cavity was conspicuous , even though the specific amino acid residues are not strictly conserved ( Figure 1—figure supplement 8 ) .",
"Mutating a pair of adjacent asparagine residues to equivalently sized but negatively charged aspartates ( hEMC3N114D+N117D ) resulted in a dramatic decrease in SQS378-410 reporter levels and no significant decrease in the other two client reporter levels ( Figure 5F ) .",
"Western blot analysis for this mutant showed wild-type rescue levels of hEMC subunits and a decrease in endogenous SQS levels ( Figure 1—figure supplement 6 ) .",
"Meanwhile , mutating a neighboring positively charged residue to an alanine ( hEMC3R180A ) , a residue that is conserved in some of the YidC-superfamily insertase proteins ( Anghel et al . , 2017 ) , resulted in partial loss of only the tail-anchored insertase client ( SQS378-410 ) ( Figure 5—figure supplements 1 and 3 ) .",
"Lastly , we surveyed residues closer to the hydrophobic lumenal side of the gated cavity .",
"Lipid density was resolved at positions along the cavity in hEMC and yEMC cryo-EM maps ( Figure 4B ) and the properties of this hydrophobic seal to the lumen are conserved ( Figure 5—figure supplement 4A–B ) .",
"The importance of this hydrophobic seal is suggested by the strong effect of a structurally mild mutation of a conserved methionine to a leucine ( hEMC3M151L ) , which caused significant decrease in both SQS378-410 and B1AR abundance ( Figure 5G ) .",
"Mutation of a neighboring aromatic residue ( hEMC3F148L ) , contacting both a lipid and a hEMC4 C-terminal transmembrane helix , caused a decrease in all three client types without altering the levels of hEMC subunits ( Figure 5—figure supplements 1–2 , Figure 1—figure supplement 6 ) .",
"Together these results indicate that proper EMC insertase function depends on the exact composition of the cavity and not simply on its hydrophobic nature .",
"While the core transmembrane helices of the gated cavity are superimposable in all four of our EMC structures , the adjacent gate helices appear in different relative orientations .",
"The structural variability likely reflects dynamics of the gate ( Figure 4C ) .",
"Comparing detergent and nanodisc maps for both species identified two major gate conformations ( Video 2 , Figure 5—figure supplement 5A ) .",
"One of the conformations , referred to as the closed-gate conformation , results in a more occluded membrane cavity .",
"The other conformation , referred to as the open-gate conformation , would provide space for client accommodation .",
"The C-terminal transmembrane helix of EMC4 and ensuing lumenal segment are well resolved in all four structures; however , other regions of EMC4 , including the segment connecting the cytoplasmic domain to the transmembrane gate helices , were poorly resolved , perhaps owing to mobility .",
"The yEMC detergent map , yEMC nanodisc map , and hEMC detergent map all show the unassigned helices in the closed conformation , preventing client residence in the gated cavity .",
"By contrast , the hEMC nanodisc map reveals an open-gate conformation with the unassigned helices shifted away from the transmembrane core to provide space for a client ( Figure 5B ) .",
"Consistent with our observations , the closed transmembrane gate conformation can also be seen in recently published cryo-EM maps of hEMC ( O'Donnell et al . , 2020 ) and yEMC ( Bai et al . , 2020 ) , which studied LMNG and digitonin-solubilized complexes , respectively ( Figure 5—figure supplement 5B ) .",
"We note that the conformational heterogeneity and concomitant lower resolution of the gate likely accounts for the challenges in making unambiguous subunit assignments ( Figure 5—figure supplement 5C–E ) , reflected by the three different interpretations reported in recent structures ( Pleiner et al . , 2020; O'Donnell et al . , 2020; Bai et al . , 2020 ) .",
"Considering the apparent flexibility of the gate , we sought to mutate the hEMC4 interfaces resolved in the cytoplasm versus the membrane .",
"As described above , mutating residues that together form a composite-binding surface for the cytoplasmic domain of hEMC4 ( hEMC2E146A+E149A+Q150A , hEMC2E168A + D170A + K173A , Figure 3E–F ) , we observed a modest accumulation of the tail-anchored insertase client ( SQS378-410 ) .",
"Likewise , mutating residues in the center of the gated cavity , close to one of the unassigned helices in the closed-gate conformation ( hEMC3V118A + I122A ) ( Video 2 , Figure 5—figure supplement 1 ) led to an increase of SQS378-410 .",
"This SQS378-410 accumulation effect stands in contrast to mutating a residue that contacts the lumenal anchor of hEMC4 ( hEMC3F148L ) , which caused a reduction of SQS378-410 levels ( Figure 5—figure supplement 1 ) .",
"In addition to the gated cavity , the EMC harbors another membrane-accessible cavity .",
"The surface of the lipid-filled cavity includes contributions from EMC1 , EMC3 , EMC5 , and EMC6 ( Figure 6A ) .",
"In our structures , the EMC2 N-terminus occludes cytoplasmic accessibility to this cavity ( Figure 4D , Figure 6A–B ) .",
"However , this cavity may be accessible from the membrane or the ER lumen .",
"The respective distance from the cytoplasmic EMC2 N-terminus to the lumenal side of the lipid-filled cavity is approximately 35 Å across , which is close to the average ER membrane thickness ( Mitra et al . , 2004 ) .",
"The lipid-filled cavity features a uniformly hydrophobic surface ( Figure 6C ) and superimposes across our ensemble of EMC structures .",
"As noted , we resolved several lipids in our cryo-EM maps lining the cavity wall and modeled four POPC ( 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphatidylcholine ) molecules in the hEMC nanodisc map ( Figure 6C ) .",
"The residues in close proximity to these lipids are moderately conserved ( Figure 5—figure supplement 4C–D ) .",
"To characterize the functional role of the lipid-filled cavity , we mutated cavity-lining and lipid-proximal residues ( Figure 6D , Figure 6—figure supplements 1–2 ) .",
"Most of these mutations resulted in an increased abundance of the tail-anchored reporter ( SQS378-410 ) and wild-type rescue levels for the other two reporters ( B1AR , TMEM97 ) .",
"However , one lipid-proximal mutant showed decreased levels of all three client reporter types with varying severity ( hEMC3R13E ) without altering overall EMC levels ( Figure 6E ) .",
"Western blotting for the endogenous SQS and TMEM97 revealed a decrease in endogenous SQS and TMEM97 levels for this mutant ( Figure 1—figure supplement 6 ) .",
"An analogous mutation in Drosophila EMC3 was recently was reported to cause reduced levels of Rh1 in this mutant background ( Xiong et al . , 2020 ) .",
"The amphipathic EMC1 brace helix , which packs against the transmembrane helices of EMC5 , is a structural hallmark of the lipid-filled cavity ( Figure 6D ) .",
"Here , mutating interfacial residues from hEMC5 ( hEMC5H19L+S23A+Q26L ) caused a marked decrease in the N-lumenal polytopic reporter ( B1AR ) and no effect on either the tail-anchored client ( SQS378-410 ) or the polytopic client reporter ( TMEM97 ) ( Figure 6F ) .",
"Unexpectedly , mutating interfacial residues from hEMC1 ( hEMC1F473Y+R487K ) showed a diametrically opposed phenotype in which B1AR was unaffected , increased SQS378-410 levels , and TMEM97 levels markedly decreased ( Figure 6G ) .",
"Another mutation in this brace ( hEMC1M483A+R487H+Q491N ) resulted in a decrease in TMEM97 and no significant effect on the other two client reporters .",
"An adjacent hEMC5D44K mutations in the interfacial brace had yet different resulting client flow cytometry profiles , with an increase in SQS378-410 and no effect on either of the polytopic client reporters ( Figure 6—figure supplement 2B–C ) .",
"The pleiotropic client phenotypes across the panel of interfacial brace mutants suggest that this feature is critical for multiple EMC functions .",
"Composed primarily of EMC1 , EMC7 , and EMC10 , the extensive EMC lumenal domain ( Figure 7A ) is important for polytopic client biogenesis and interactions with lumenal chaperones ( Luo et al . , 2002; Shurtleff et al . , 2018; Hiramatsu et al . , 2019; Coelho et al . , 2019 ) .",
"EMC7 and EMC10 are scaffolded on two beta-propellers of EMC1 , one distal and the other proximal to the membrane .",
"The lumenal cap differs between hEMC and yEMC , with a four-bladed distal beta-propeller in yeast and eight-bladed distal propeller the human complex ( Figure 7B ) .",
"All three lumenal EMC subunits have structural folds known to participate in protein-protein interactions ( Reinisch and De Camilli , 2016 ) .",
"Mutations in this lumenal domain have been linked to loss of the EMC complex ( Bircham et al . , 2011 ) , a trafficking delay for membrane protein Pma1 ( Luo et al . , 2002 ) , and male infertility ( Zhou et al . , 2018 ) .",
"Several regions of the lumenal domain form stabilizing interactions with the membrane cavities .",
"The gate helices of the gated cavity are anchored via the embedding of EMC4’s C-terminus within the membrane-proximal EMC1 propeller .",
"The lipid-filled cavity is connected to the ER lumenal domain via the amphipathic EMC1 brace helix , which is tethered to the membrane-proximal EMC1 beta-propeller .",
"The connections between the lumenal domain and the transmembrane cavities could allow for conformational coupling during client handling .",
"Indeed , superimposing the two conformations presented above , the open- and closed-gate states , revealed not only differences in the transmembrane domain but also a rotation of the lumenal domain relative to the membrane cavities ( Video 3 ) .",
"The lumenal positioning is consistent for all three of our closed-gate conformation reconstructions ( hEMC detergent , yEMC nanodisc , yEMC detergent ) .",
"By contrast , the one map with an open gated cavity displayed a lumenal rotation and concomitant shifts in position of the hEMC1 brace helix ( Figure 7—figure supplement 1 ) .",
"Indeed , our set of interfacial hEMC1 brace mutants described above ( Figure 6F–G , Figure 6—figure supplement 2B–C ) , showed differing client phenotypes when mutated from either the hEMC1 or the hEMC5 side .",
"This suggests a complex conformational interplay between lumenal and transmembrane domains during the engagement of diverse client types .",
"We investigated several known disease mutations in both conserved and human-specific regions of hEMC1 ( Figure 7C–D , Figure 7—figure supplements 2–3; Harel et al . , 2016; Abu-Safieh et al . , 2013; Amberger et al . , 2019 ) .",
"One of these disease-associated residues sits near the anchor point for the lumenal hEMC4 transmembrane gate helix ( hEMC1R881C ) , while the majority are found farther from the membrane ( hEMC1G868R , hEMC1A144T , hEMC1T82M ) ( Figure 7C–D , Figure 7—figure supplement 2B ) .",
"Incorporating each of these disease mutations into our EMC functional assay resulted in lower levels of the N-cytoplasmic polytopic client ( TMEM97 ) and an increase in the level of the tail-anchored client ( SQS378-410 ) , discussed in more detail below .",
"Two different hEMC1 mutants associated with cerebellar atrophy , visual impairment , and psychomotor retardation ( hEMC1T82M , hEMC1G868R ) map to the hinge region between the hEMC1 beta propellers where hEMC7 binds ( Figure 7D ) .",
"Both the mutants at this protein-protein interface resulted in depletion of the N-cytoplasmic polytopic client ( TMEM97 ) .",
"EMC7 and EMC10 form beta-sandwich domains on either side of the membrane-proximal beta-propeller of EMC1 and contact each other across the EMC1 surface .",
"Consistent with our structures , coupling of these subunits is supported by the prior finding that in the absence of EMC7 , EMC10 is also lost from the complex while the other EMC components appear unaffected ( Shurtleff et al . , 2018 ) .",
"EMC7 and EMC10 have been proposed to be auxiliary components with weaker phenotypes compared to core EMC subunits ( Jonikas et al . , 2009; Shurtleff et al . , 2018; Dickinson et al . , 2016 ) .",
"Upon deleting yEMC7 , multi-pass transmembrane clients are retained in the ER but tail-anchored clients , including SQS-homolog Erg9 , decrease in abundance ( Shurtleff et al . , 2018 ) .",
"Several features of our data suggest dynamic association of hEMC7 .",
"Density for the hEMC7 beta-sandwich at the hinge between the two hEMC1 beta propellers was relatively weak in the consensus hEMC nanodisc map ( Figure 2—figure supplement 4 ) .",
"Additional rounds of 3D classification revealed two distinct classes , one with clear density for hEMC7 , and one with weak density in this region .",
"Mass spectrometric analysis of purified hEMC , however , revealed that the abundance of hEMC7 was similar to that of the other hEMC components ( Figure 1—figure supplement 2; Supplementary file 1 ) .",
"Both reconstructions , with and without density for the hEMC7 lumenal domain , displayed well-resolved density for hEMC10 .",
"Together , we conclude that hEMC7 is associated with hEMC1 in two different conformational states of hEMC7 with potentially distinct functions .",
"The OMIM database ( Amberger et al . , 2019 ) lists a mutation of unknown significance linked to retinitis pigmentosa ( hEMC1A144T ) residing in the EMC1 distal propeller ( Figure 7—figure supplement 2 ) .",
"Additionally , we also generated mutations in two surface exposed patches of the membrane-distal EMC1 beta-propeller projecting into the lumen ( hEMC1D31K , hEMC1R69D , hEMC1G71S , hEMC1H93D + E138D + N282K , Figure 7E , Figure 7—figure supplements 2–3 ) .",
"Overall , these mutations displayed the same client effect: a decrease in the N-cytoplasmic polytopic client reporter ( TMEM97 ) , no change in the N-lumenal polytopic client reporter ( B1AR ) , and accumulation of the tail-anchored client reporter ( SQS378-410 ) .",
"Upon identifying antibodies against yEMC , we observed that the top two antibodies bind to a similar extended loop in the distal yEMC1 beta-propeller , perhaps suggesting that this site is accessible for co-factor binding in the ER .",
"Intriguingly , this region of the lumenal domain corresponds to the region where hEMC1 has an expanded distal beta-propeller .",
"Taken together , the data provide evidence that the lumenal domain is functionally coupled to the broader EMC role in transmembrane client stabilization .",
"Moreover , these data support that the EMC is acting as a holdase chaperone to shield polytopic clients from degradation while they are folding to their functional form ."
],
[
"EMC3’s fold at the interface between the cytoplasm and membrane forms the core of the gated cavity and is reminiscent of proteins from the YidC family of insertases ( Borowska et al . , 2015; Dalbey and Kuhn , 2015; Anghel et al . , 2017 ) .",
"Indeed , mutations in either the cytoplasmic or transmembrane domains of EMC3 establish that these features are critical for terminal helix insertase activity .",
"In light of our observation of multiple gate conformations , we speculate that these conformations modulate insertion and release into the ER membrane .",
"Notably , mutating the surface of the cytoplasmic cap , which extends beyond the EMC3 cytoplasmic helices toward EMC8/9 , resulted in an unexpected increase in C-tail anchor client ( SQS378-410 ) abundance .",
"Of the three clients analyzed , SQS was the only one to show enhanced levels .",
"It is unclear if this enhancement is SQS-specific or representative more broadly of all post-translationally targeted EMC tail-anchored clients .",
"Future studies will be required to address if this is due to regulated insertion of SQS by the EMC , parallel pathways for inserting SQS into the membrane ( i . e . mediated by TRC40/GET ) , and/or slower cytoplasmic clearance of chaperone-bound SQS .",
"Post-translational insertase clients have previously been shown to be targeted to the ER by cytoplasmic chaperones ( Guna et al . , 2018 ) .",
"Structural analysis and coupled mutagenesis , from our and recent studies ( O'Donnell et al . , 2020; Pleiner et al . , 2020; Bai et al . , 2020 ) , suggest that clients then engage the cytoplasmic domain of the EMC , the transmembrane gate opens , the terminal helix is inserted into the EMC-gated cavity , and then another conformational change would allow for release into the lipid bilayer ( Figure 8A–B ) .",
"Further studies are needed to establish the precise C-terminal client range , as most tail anchor clients have been shown to be inserted by the GET ( in yeast ) or WRB ( in human ) complexes ( Denic et al . , 2013; Mateja and Keenan , 2018 ) .",
"Both the N-terminal ( B1AR ) and C-terminal insertase ( SQS ) clients depend on the EMC-gated cavity .",
"Indeed , both the SQS tail-anchored helix and the first transmembrane helix of B1AR are moderately hydrophobic , with polar residues near the cytoplasmic end of the transmembrane helix , and both showed a strong dependence on the gated cavity .",
"Nevertheless , our panel of mutants revealed some notable differences in the handling of these two client types .",
"B1AR showed more dependence than SQS on the lipid-filled cavity in contrast to mutants elsewhere in the complex .",
"Consistent with this , a number of mutations , primarily in the gated cavity , show residues of importance to both SQS and B1AR .",
"However , there are also a number of mutations that appear to only affect SQS .",
"One possible reason could be due to differences in the mechanism of initial engagement: SQS is targeted to the ER by cytoplasmic chaperones , while B1AR is targeted by SRP .",
"Another key difference is that B1AR is polytopic and needs to overcome the additional challenge of tertiary transmembrane packing to reach its folded state .",
"This work provides support for a model where the EMC inserts both types of terminal transmembrane helices into the gated cavity with differences in initial targeting and perhaps release into the lipid environment ( Figure 8C–D ) .",
"Future work will address the interplay between B1AR synthesis and its co-translational engagement with the translocon to ascertain whether there is a direct handoff between the translocon and the EMC or the EMC acts post-translationally to insert the N-terminal helix of B1AR .",
"Unlike the two terminal insertase clients we investigated , TMEM97 biogenesis was negatively impacted by mutation of the lumenal EMC1 .",
"The depletion of TMEM97 observed in these mutant backgrounds is consistent with the lumenal domain contributing to a holdase chaperone function , passively shielding its client while it is being synthesized and/or folded ( Zhang et al . , 2017 ) .",
"Interestingly , the diametrically opposed phenotype of mutants in the EMC lumenal domain on SQS raises the possibility that occupancy by one type of client can support an EMC conformation that is unfavorable for receiving the other .",
"Alternative conformations could establish competition between client types for EMC occupancy .",
"One explanation for this observation is that there is a conformational change between the insertase-active versus the holdase-active states .",
"Interestingly , we identified at least two EMC conformations in our collection of structures , and EMC may adopt different conformations in various client and cofactor-engaged states .",
"In yeast , the polytopic clients co-purifying with the EMC are also glycosylated .",
"One possible model is that the putative carbohydrate-binding domains in EMC7 or EMC10 directly contribute to engagement with client proteins .",
"We speculate post-translational modifications on clients and the EMC could modulate function including client binding , chaperone binding , or regulating signaling in response to cellular cues .",
"Multi-pass transmembrane proteins require membrane factors to assist after insertion into the membrane to pack transmembrane helices in the correct order and topology .",
"We propose that the EMC may act as a chaperone holdase to facilitate one of the following: helix and lipid packing , shielding from degradation while synthesis is in progress , or assisting in the assembly of multi-protein transmembrane complex formation .",
"This is consistent with observations that in the absence of the EMC numerous integral membrane proteins are degraded ( Shurtleff et al . , 2018; Volkmar et al . , 2019; Tian et al . , 2019 ) .",
"Direct interactions with multi-pass transmembrane proteins have been shown previously ( Shurtleff et al . , 2018; Coelho et al . , 2019 ) .",
"Furthermore , EMC dependence of internal transmembrane domain segments has also been established ( Ngo et al . , 2019; Hiramatsu et al . , 2019 ) .",
"In the absence of yEMC7 , a primarily lumenal subunit , a polytopic membrane protein was retained for longer in the ER , suggesting the possibility that yEMC7 may be involved in client release from the EMC .",
"We propose a model where the EMC engages polytopic clients either during or directly after translation and remains bound until the client is released either to the membrane environment directly or handed off to client-specific and general ER chaperones ( Figure 8E–F ) .",
"It remains to be seen whether these polytopic clients directly engage with the lipid-filled cavity or the gated cavity or the lumen domain , what the determinants for engaging with a client or release into the membrane are , and how the EMC fits into the broader ER lumenal chaperone network .",
"Why does the cell use a multifunctional EMC molecular machinery rather than specialized machinery for each of the functions encompassed by the EMC ?",
"Considering that the cell already has general machinery ( Sec61 translocon ) and tail-anchor insertase machinery ( GET/TRC complex ) , we speculate that the EMC coordinates biogenesis of diverse membrane proteins .",
"Several observations suggest broader roles of the EMC as an integrator of information sensing the protein and lipid environment and coordinating its multiple activities , including the regulating the biogenesis of membrane proteins .",
"For example , the initial identification of the EMC included numerous genetic interactions with both protein and lipid synthesis factors in yeast ( Jonikas et al . , 2009 ) and these disparate interdependencies have been subsequently observed in numerous species including human EMC ( Lahiri et al . , 2014; Tang et al . , 2017; Guna et al . , 2018; Volkmar et al . , 2019; Volkmar and Christianson , 2020 ) .",
"Also , several client proteins are enzymes or cofactors involved in multiple stages of lipid synthesis or trafficking , and this may provide a unifying explanation for the range of genetic interactions and co-essentiality observations reported to date ( Guna et al . , 2018; Shurtleff et al . , 2018; Volkmar et al . , 2019; Tian et al . , 2019; Wainberg et al . , 2019; Corradi et al . , 2019; Volkmar and Christianson , 2020 ) .",
"Perhaps by facilitating the insertion of sterol synthesis protein SQS , the EMC allows for modulation of local membrane thickness and lipid composition to accommodate differences within the broad range of membrane proteins being synthesized .",
"In this regard , one structural feature of particular interest is the EMC1 amphipathic brace , which resides adjacent to the lipid-filled cavity .",
"This conserved feature sits within the interfacial membrane boundary , raising the possibility that it can modulate the lipid or protein composition of this cavity .",
"Notably , several other membrane proteins involved in ER homeostasis , including Opi1 and Ire1 , also contain amphipathic helices that have been proposed to sense the properties of the lipid bilayer ( Volmer et al . , 2013; Jacquemyn et al . , 2017; Halbleib et al . , 2017; Hofbauer et al . , 2018; Cho et al . , 2019 ) .",
"Future work will explore how the EMC overall , and the EMC1 brace helix in particular , govern client release into the membrane , interface with the local structure of the lipid bilayer , and play roles in specific client-lipid interactions .",
"In addition to the three client classes we investigate here , it is clear that EMC has a broader range of clients including multi-protein assemblies ( Richard et al . , 2013; Talbot et al . , 2019 ) , lipid-modulating proteins ( Volkmar et al . , 2019 ) , lipid-binding proteins ( Salas-Estrada et al . , 2018; Sejdiu and Tieleman , 2020 ) , and those with helices that do not span the bilayer ( Lin et al . , 2019; Ngo et al . , 2019 ) .",
"The compartmentalization and interdependence that we observe for effects of mutations on client handling provide a foundation for understanding this multifunctionality .",
"We propose that the complexity of the EMC machine , combining insertase and holdase chaperone functions within one molecular machine , has arisen to mitigate the error prone biogenesis of a diverse range of membrane spanning proteins in the dynamic environment of the ER ."
],
[
"K562 dCas9 KRAB cells were grown in RPMI 1640 ( GIBCO ) with 25 mM HEPES , 2 mM l-glutamine , 2 g/L NaHCO3 and supplemented with 10% ( v/v ) fetal bovine serum ( FBS ) , 100 units/mL penicillin , 100 μg/mL streptomycin , 2 mM l-glutamine .",
"HEK293T cells were grown in Dulbecco’s modified eagle medium ( DMEM , GIBCO ) with 25 mM d-glucose , 3 . 7 g/L NaHCO3 , 4 mM l-glutamine and supplemented with 10% ( v/v ) FBS , 100 units/mL penicillin , 100 μg/mL streptomycin .",
"All cell lines were grown at 37°C .",
"All cell lines were periodically tested for Mycoplasma contamination using the MycoAlert Plus Mycoplasma detection kit ( Lonza ) .",
"Lentivirus was generated by transfecting HEK39T cells with standard fourth-generation packaging vectors using TransIT-LT1 Transfection Reagent ( Mirus Bio ) .",
"Media was changed 10 hr post-transfection .",
"Viral supernatant was harvested 60 hr after transfection , filtered through 0 . 45 μm PVDF filters and frozen prior to transduction .",
"A single and dual knockout guide system was developed in the pX458 backbone ( Addgene plasmid # 48138 ) with guides targeting hEMC1 , hEMC2 , hEMC3 , or hEMC5 ( Key Resources table ) .",
"Targeting guides were selected using the Broad’s guide selection tool ( https://portals . broadinstitute . org/gpp/public/analysis-tools/sgrna-design ) .",
"For the single hEMC5 knockout system , an hEMC5 targeting guide was cloned into pX458 by digesting with BbsI and ligating to annealed oligos for the hEMC5 sgRNA .",
"For the dual knockout system , a four-step cloning process generated the final knockout plasmid: ( 1 ) Each of the two guides targeting the same locus were individually cloned into pX458 .",
"( 2 ) Then pX458_sgRNA1 was digested with XbaI .",
"( 3 ) SgRNA2 cassette from pX458_sgRNA2 was PCR amplified with oligos containing overhangs spanning the XbaI cloning site and purified .",
"( 4 ) Finally , the final dual guide vector was generated by Gibson cloning ( NEBuilder ) .",
"To generate the hEMC knockout cell lines , K562 dCas9 KRAB cells were nucleofected with the respective hEMC knockout plasmids using Lonza SF Cell Line 96-well Nucleofector Kit ( V4SC-2096 ) .",
"Two days post-nucleofection , GFP-positive cells were single cell sorted into 96-well plates using BD FACS AriaII .",
"After colonies from single cells grew out , genomic DNA was isolated using QuickExtract ( Lucigen ) , the sgRNA-targeted sites were PCR amplified and then NGS-sequenced via Genewiz’s EZ-Amplicon service .",
"Sequencing data was analyzed and aligned to the respective reference alleles in the human genome .",
"Clones whose alleles harbored only indel mutations for hEMC1 , hEMC2 , hEMC3 , and hEMC5 ( full knockouts ) respectively were further validated on the protein level .",
"Dual client reporters for TMEM97 , ADRB1 ( protein name: B1AR ) , and FDFT1 ( protein name: SQS ) were introduced lentivirally into each of the EMC1 , EMC2 , EMC3 , and EMC5 knockout cell lines .",
"TMEM97 and ADRB1 full-length sequences were used with a C-terminal tag -mCherry-P2A-GFP .",
"The sequence for FDFT1 transmembrane domain ( SQS378-410 ) was tagged N-terminally with GFP-P2A-mCherry- and an opsin tag on the C-terminus as used in a prior study ( Guna et al . , 2018 ) .",
"Three days post-transduction , GFP/mCherry-positive cells were sorted on BDAriaII .",
"Sequences for these constructs are available in the Supplementary file 5 .",
"The EMC mutant genes were synthesized and cloned by Twist into pKDP119-SFFV-[insert site]-IRES-Puro-P2A-BFP .",
"For hEMC subunit mutation details refer to the Key Resources Table , for sequences refer to Supplementary file 5 .",
"Mutant hEMC cell lines were generated by lentiviral introduction of the respective hEMC mutant subunit into the respective knockout cell lines ( hEMC1 , hEMC2 , hEMC3 , or hEMC5 ) containing the dual fluorescent reporters for each EMC client ( pKDP110_ADRB1_mCherry_P2A_GFP , pKDP111_TMEM97_mCherry_P2A_GFP , or GFP_P2A_mCherry_FDFT1_TMD_opsintag ) .",
"The expression of each fluorescent reporter was read out 6 days after puromycin selection in each of the hEMC mutant cell lines .",
"For each hEMC mutant cell line , 20 , 000 live cells were recorded on Attune NxT flow cytometer .",
"FlowCal flow analysis package was used for analysis in Python .",
"First , live cells were gated based on FSC/SSC .",
"Then GFP ( BL1-A ) and mChery ( YL2-A ) were plotted for each mutant and control cell line .",
"mCherry:GFP intensity ratios were calculated for individual cells in each cell line .",
"Fluorescence ratios for each substrate in an hEMC mutant cell line were normalized to the mCherry:GFP ratio of the same substrate in the hEMC wild-type rescue cell line .",
"Distributions of fluorescence ratios were plotted as histograms in Python using seaborn .",
"We performed bootstrap estimates of the mean of normalized mCherry/GFP ratio from the FACS data .",
"For bootstrapping , we performed 1000 iterations with 50 cells/iteration to fit normal distributions .",
"We performed two separate one-sided T-tests at a p-value cutoff of 0 . 01 between each mutant and the respective subunit WT to test for significant decreases or increases in ratios based on bootstrapped estimates of the mean .",
"These statistics are contained in the files ‘filtered_final_pvalues . 01cutoff . lo . csv’ and ‘filtered_final_pvalues . 01cutoff . hi . csv’ respectively .",
"Statistics were generated for the EMC-independent membrane protein controls ( ‘stats . membrane . controls . lo’ , ‘stats . membrane . controls . hi’ ) and for the mCherry-p2a-GFP control ( ‘ . mcherry . p2a . gfp . control . lo’ , ‘stats . mcherry . p2a . gfp . control . hi’ ) .",
"Values can be found in Supplementary file 2 .",
"Cell pellets were lysed using lysis buffer ( 20 mM Tris pH 7 . 5 , 150 mM NaCl , 5 MgCl2 , 1% Triton x-100 , 1 mM DTT , 24 U/ml ) Turbo DNase ( Ambion ) .",
"Clarified lysate was quantified and samples were boiled with 4x LDS sample ( Thermo Fisher , NP0007 ) buffer for 5 min at 95°C .",
"Samples were separated on 4–12% or 12% Bolt Bis-Tris Plus Gels ( Invitrogen , NP0322PK2 ) .",
"Proteins were transferred onto nitrocellulose membranes using Bio-Rad Trans-Blot Turbo transfer system .",
"Membranes were blocked in Odyssey Blocking Buffer ( LI-COR , 927–50000 ) for an hour at room temperature .",
"Blocked membranes were incubated with primary antibody diluted in TBST and incubated overnight at 4°C on a shaker .",
"Primary antibodies were detected by incubating membranes with 1:10 , 000 dilution of IRDye-conjugated ( LI-COR ) secondary anti-mouse and anti-rabbit antibodies for 1 hr at room temperature .",
"Blots were visualized using LI-COR imaging system .",
"The primary antibodies used in this study are listed in the Key Resources table .",
"Strain BY4741 and BY4742 were used as the wild-type parental strains for the creation of the yEMC overexpression strain .",
"Yeast homologous recombination ( Rothstein , 1991 ) was used to generate yeast strains .",
"For the overexpression strain , the endogenous promotor for each yEMC subunit ( yEMC1 , yEMC2 , yEMC3 , yEMC4 , yEMC5 , yEMC6 , yEMC7 , yEMC10 ) were replaced with a TEF2 promoter .",
"In addition , EMC5 was tagged at the C-terminus with linker-TEV-linker-3xFlag .",
"Auxotrophic markers and drug selection markers in both BY4741 and BY4742 were employed to add this promoter modification to all these eight subunits and the two strains were crossed to create the resulting BY4743 strain used for immunoprecipitation .",
"Endogenous EMC yeast strain was made using W303a wild-type parental background ( leu2-3 , −112; his3-11 , −15; trp1-1; ura3-1; ade2-1; can1-100; MATa ) .",
"Homologous recombination was used to integrate a linker-TEV-linker-3xFlag at the C-terminus of yEMC coding sequence .",
"Genomic PCR was conducted to verify integration .",
"Fabs were identified as described in these studies ( Kim et al . , 2011; Wu et al . , 2012 ) .",
"Overexpressed yEMC solubilized in DDM as described above was biotinylated and streptavidin magnetic beads were used to capture yEMC , which was then subjected to a Fab phage library .",
"Unbound Fabs were washed away and then binding Fabs were eluted and analyzed by ELISA .",
"Two Fabs were identified binding yEMC , Fab DH4 and DE4 .",
"Plasmid with either Fab DH4 or DE4 were transformed into BL21 Gold Star cells and plated onto agarose plates with 2x YT + 2% glucose + Ampicillin .",
"Cultures were inoculated from resulting colonies for overnight growth at 30°C into 2xYT + 2% glucose + Amp .",
"In the morning dilute overnight culture to OD600 of 0 . 05 in 1L , in a 2 . 8 L flask of 2xYT + 0 . 1% glucose + Amp .",
"Grow the culture at 180 rpm at 37°C shaker until OD600 of 0 . 6 , then , switch to shaking at 19°C for 1 hr .",
"Next , induce with 0 . 4 mM IPTG .",
"Shake at 180 rpm at 19°C for 18–20 hr .",
"Spin 1L cultures down at 3500 rpm in large Beckman Centrifuge at 4°C for 20 min in ( 8 . 1 rotor ) .",
"Discard media and gently resuspend cell pellet in ice-cold 20 ml in Buffer 1 ( 0 . 2 M Tris pH 8 . 0 , 0 . 5 mM EDTA , 0 . 5 M Sucrose ) on ice .",
"Transfer the resuspended cells from step 2 into two smaller JLA 25 . 5 centrifuge tubes .",
"Add 20 mL of ice cold ddH2O with 2x protease inhibitor cocktail ( Roche Complete Ultra , Millipore Sigma 5056489001 ) from step 3 to the resuspended pellets .",
"Incubate at on ice for 1 hr occasionally swirling samples gently .",
"Spin periplasmic fractions at 13 , 000 x g for 15 min , 4°C , rotor 25 . 50 .",
"Wash 500 µL Ni resin ( Qiagen , Ni-NTA , 30210 ) per periplasmic fraction four times in Buffer 2 ( 50 mM Tris pH 8 . 0 , 250 mM NaCl ) .",
"Add MgCl2 and imidazole to a final concentration of 10 mM to each periplasmic fraction .",
"Add beads to periplasmic fractions and nutate at 4°C for 2 hr .",
"Spin down beads at 2000 x g , 10 min , 4°C .",
"Transfer beads either to a 50-mL gravity column .",
"Wash the beads with 20 column volumes of Buffer 3 ( 50 mM Tris pH 8 . 0 , 500 mM NaCl , 20 mM Imidazole ) .",
"Elute protein with three column volumes of Buffer 4 ( 50 mM Tris pH 8 . 0 , 500 mM NaCl , 300 mM Imidazole ) .",
"Analyze eluate by SDS-PAGE 4–12% Invitrogen ( Invitrogen , NP0321PK2 ) .",
"Fabs as two bands run around 30 kDa in reducing conditions , or 50 kDa in non-reducing conditions .",
"Dialyze eluate O/N in Dialysis cassette 10 kD molecular weight cutoff at 4°C against 150 mM KOAc , 20 mM HEPES pH 6 . 8 .",
"The OE-Emc5-3xflag yeast strain were grown in YEPD media in a 40 L fermenter , harvested and flash frozen in liquid nitrogen .",
"Cell pellets were thawed and diluted in lysis buffer ( 50 mM HEPES pH 6 . 8 , 150 mM KOAc , 2 mM MgOAc , 1 mM CaCl2 , 0 . 2M Sorbital , 2x Protease Inhibitor ) .",
"Bead beating ( 10 times → 1 min on , 2 min off ) was used to lyse cells .",
"For 25 g of cells , 0 . 1 mm cold beads were added and lysis buffer up to the top of the 50 mL canister .",
"After lysis , beads were filtered and solution centrifuged at 10 , 000 xg for 10 min .",
"Supernatants were ultracentrifuged at 42 , 000 RPM ( Ti 45 rotor ) for 2 hr .",
"Supernatant was discarded .",
"Membrane pellet was combined with the lipid layer , and resuspended in lysis buffer and then a precooled dounce homogenizer was used to dounce 20 times .",
"Membranes were aliquoted and flash frozen in liquid nitrogen .",
"On ice , 150 mL of solubilization buffer ( 50 mM HEPES pH 6 . 8 , 150 mM KOAc , 2 mM MgOAc , 1 mM CaCl2 , 15% glycerol , 1% b-DDM , 2x Protease Inhibitor ) was added incrementally to 7 . 5 g of thawing membranes , nutated at 4°C for 1 hr in JA 25 . 5 rotor tubes , and centrifuged at 20 , 000 rpm for 45 min .",
"Meanwhile 2 . 5 mL of αFLAG agarose beads ( Millipore A2220 ) were rinsed in 50 mL of low-salt buffer ( 50 mM HEPES pH 6 . 8 , 150 mM KOAc ) .",
"Supernatant was added to αFLAG beads and nutated at 4°C for 2 hr .",
"Resulting solution was applied over a glass column .",
"After flowing through unbound solution , αFLAG beads were washed with 100 mL low-salt buffer , 100 mL high-salt buffer ( 50 mM HEPES pH 6 . 8 , 300 mM KOAc , 0 . 05% b-DDM ) , and 100 mL low-salt buffer .",
"αFLAG beads were resuspended in 10 mL of low-salt buffer and 300 µL of TEV ( 1 . 15 mg/mL ) was added and nutated overnight at 4°C .",
"Removed supernatant from beads by low-speed spin and applied over 500 µL of NiNTA beads equilibrated with low-salt buffer to remove excess TEV .",
"Flow through glass column and collect supernatant .",
"Using a 100 kD concentrator ( Millipore , UFC910008 ) solution was concentrated to 2 mg/mL .",
"Concentrated EMC protein was applied to the Akta Explorer Superose 6 Increase column ( Cytiva , 29091596 ) for size exclusion chromatography in the size exclusion buffer ( 20 mM HEPES pH 6 . 8 , 150 mM KOAc , 0 . 05% b-DDM ) .",
"Fractions were evaluated by SDS-PAGE Coomassie stain and negative stain electron microscopy then EMC peak fractions were pooled and incubated with 2x molar excess of Fab , either Fab DH4 or Fab DE4 , for 30 min on ice .",
"Solution was applied to Akta Explorer Superose 6 Increase for size exclusion of Fab bound EMC .",
"Resulting EMC-Fab fractions were evaluated by SDS-PAGE Coomassie stain and EMC-Fab peak fractions were pooled .",
"Yeast was grown in rich media ( YPAD ) in a 65L fermenter until OD 2 . 6 .",
"Cell pellets were harvested and flash frozen in liquid nitrogen .",
"Pellets were ground using three cycles in a French press .",
"As above , the resulting solution was ultracentrifuged to separate membranes , dounced to homogenize , and flash frozen in liquid nitrogen .",
"Thawed membranes were solubilized in 1% b-DDM ( Anatrace , D310 ) nutating at 4°C for 1 hr then centrifuged to separate solubilized membranes from the pellet .",
"Supernatant was applied to equilibrated αFLAG beads , nutated at 4°C for 1 hr , and applied over a disposable plastic column at 4°C .",
"αFLAG beads were washed with low-salt buffer and high salt buffer .",
"Then washed with low-salt buffer with b-DDM+CHS ( Anatrace , CH210 ) ( 10:1 ) in place of b-DDM .",
"αFLAG beads were then transferred to a 15-mL Eppendorf tube for TEC cleavage and nanodisc reconstitution .",
"Bio-Beads SM-2 ( Bio-Rad ) were prepared ~400 µL biobeads , rinsing with EtOH , and then water four times .",
"Yeast Extract Total ( Avanti Polar Lipids , 190000 C-100mg ) was prepared by transferring chloroform resuspended solution to a glass vial , drying the lipids into a film with nitrogen gas , drying in a vacuum desiccator overnight , and then solubilizing the lipids first in water and then in size exclusion buffer with DDM+CHS by bath sonication , aliquots stored at −20°C until use .",
"A total of 200 µL of TEV protease ( 5 mg/mL ) and 150 µL of 1 mg/mL Yeast Total Extract solubilized in b-DDM+CHS , at room temperature for 30 min .",
"Then added MSP1D1 , purified as described previously ( Ritchie et al . , 2009 ) , to a ratio of 200:10:1 ( Yeast total extract:MSP1D1:EMC ) , at 4°C for 10 min .",
"Then activated Bio-Beads SM-2 ( Bio-Rad ) , ~300 µL , were added and nutated overnight .",
"On-bead reconstitution employed adapted from Laverty et al . , 2019 .",
"In the morning , ~100 µL more Bio-Beads SM-2 ( Bio-Rad ) were added and 2x molar excess of FabDH4 , nutated for another hour .",
"Beads and solution applied to an EconoPac column ( Bio-Rad ) .",
"Flow through was collected and solution was applied to a 100 kD ( Amicon ) concentrator .",
"Resulting concentrated EMC was applied to the Akta Explorer Superose 6 Increase column for size exclusion chromatography .",
"Peak fractions were pooled for SDS-PAGE Coomassie stain , negative stain , and cryo-EM evaluation .",
"Key reagents used are provided in the Key Resource Table .",
"Structural biology applications used in this project were compiled and configured by SBGrid ( Morin et al . , 2013 ) .",
"The yeast EMC structure was built de novo using Coot ( version 0 . 8 . 7 and 0 . 9 ) and UCSF ChimeraX ( Goddard et al . , 2018 ) .",
"Visible secondary structure was built by hand for the entire structure using overlays of the yEMC detergent consensus map as well as the yEMC nanodisc unsharpened and sharpened map .",
"Starting with the best resolved transmembrane helices , sequence was placed for each of the predicted transmembrane helices , using TMHMM ( Krogh et al . , 2001 ) , in the yEMC proteins .",
"Visual inspection for landmark residues ( tryptophan , tyrosine , leucine , and proline ) in the sequences that correlated with the position of well densities as well as fit correlation in UCSF Chimera was computed to assign identities for yEMC1 , yEMC3 , yEMC5 , and yEMC6 .",
"Connectivity between the EMC1 assigned helix to the lumenal domain was used to start assigning sequence for the lumenal portion of EMC1 .",
"Secondary structure prediction was computed for all yEMC proteins using Phyre2 ( Kelley et al . , 2015 ) and Quick2D , a tool within the Max-Plank Institute for Developmental Biology Bioinformatics Toolkit that visualizes several different secondary structure predictors ( Jones , 1999; Cuff and Barton , 2000; Ouali and King , 2000; Rost , 2001; Lupas et al . , 1991; Jones et al . , 1994; Ward et al . , 2004; Peng et al . , 2006; Obradovic et al . , 2005 ) .",
"Secondary structure prediction was used to check and guide sequence assignment of beta strands and helices .",
"Next several homology models were computed and overlain for yEMC2 , with a predicted TPR structural domain , using Robetta ( Raman et al . , 2009; Song et al . , 2013 ) , I-TASSER ( Zhang , 2008; Roy et al . , 2010; Yang et al . , 2015 ) , Phyre2 ( Kelley et al . , 2015 ) , and RaptorX ( Källberg et al . , 2012 ) .",
"These were used in addition to secondary structure prediction to guide sequence assignment , loop building , and helical packing .",
"Fab DH4 starting structure was computed using Phyre2 1-to-1 threading against a crystal structure of a monoclonal Fab ( PDB 1M71 , Vyas et al . , 2002 ) .",
"EMC3 , EMC5 , and EMC6 were built off of the transmembrane helices using sphere refinement , real space refinement , regularization , and visual monitoring of the Ramachandran plot in Coot .",
"EMC7 and EMC10 both form beta sandwich folds on the exterior of the EMC1 lumenal domain , beta strand sequence was placed for both in both densities , position of aromatic residues and loop length differed between the two allowing assignment of each .",
"After building EMC1-3 , EMC5-7 , and EMC10 , there remained several transmembrane helices and a beta strand fitted into the lumen but not connected to EMC1 , EMC7 , or EMC10 .",
"The resolution of the lumenal domain is better than 3 Å in most parts allowing for sequence placement of the EMC4 C-terminus and C-terminal transmembrane helix .",
"The connectivity of the transmembrane helix to the cytoplasmic domain was not resolved .",
"However , there was an additional poorly resolved short helix and loop density in the cytoplasmic domain which was assigned to EMC4 .",
"Two poorly resolved transmembrane helices remained , however , due to the fact they did not have clear connectivity to any built strand , poly alanine alpha helices were built in but not assigned to a yEMC protein ( Figure 5—figure supplement 5 ) .",
"EMC4 had density in the cytoplasmic domain as well as the lumenal domain , suggesting that it has either one or three transmembrane passes .",
"EMC7 and EMC10 were predicted to have transmembrane helices , however , the connection between the lumenal densities and those predicted transmembrane helices was not clear .",
"Additional density that was not built into was visualized in UCSF ChimeraX ( Goddard et al . , 2018 ) and allowed for subsequent assignment of several glycosylated residues and one POPC molecule .",
"Each subunit was built in a separate pdb file and subjected to iterative rounds of phenix . real_space_refine ( Adams et al . , 2011; Liebschner et al . , 2019 ) into segmented maps preceded and followed by adjustment in Coot .",
"Manual assignment of secondary structure restraints was used and improved during Phenix refinement .",
"Once all of the well-resolved secondary structure was assigned to yEMC subunits , PDBs were combined and subjected to iterative rounds of phenix . real_space_refine ( Adams et al . , 2011; Afonine et al . , 2018; Liebschner et al . , 2019 ) in the unsharpened and then sharpened maps .",
"Loops were built back where the connectivity was clear and then refined again in Phenix and Coot .",
"PDBs were prepared for refinement steps using phenix . reduce to add hydrogens throughout refinement steps , ReadySet to generate cif restraints , and Phenix PDB preparation tool for creating mmCIF files for deposition .",
"Representative regions of the model as well as the map-to-model FSC can be found in Figure 2—figure supplement 5 .",
"Model for the yEMC nanodisc sample was used to generate reference model restraints for phenix real space refinement of yEMC DDM model .",
"T-coffee PSI-Coffee extension ( Notredame et al . , 2000 ) was used to compute sequence alignments between yEMC , hEMC , and homologous proteins ( Figure 1—figure supplements 6–7 , Figure 3—figure supplement 3 , Figure 5—figure supplement 3 ) .",
"Outputs of these alignments were visualized in Jalview ( Waterhouse et al . , 2009 ) for figure creation and colored by ClustalX convention .",
"All figures were assembled and edited in Adobe Illustrator .",
"Figure 1 and Figure 1—figure supplement 4 were created using BioRender .",
"All the visualization , structure figures , and structure videos were made using UCSF ChimeraX 1 . 0 ( Goddard et al . , 2018 ) and UCSF Chimera 1 . 14 ( Pettersen et al . , 2004 ) .",
"Flow cytometry plots were generated in Python and labeled in Adobe Illustrator ."
]
] | [
"Membrane protein biogenesis in the endoplasmic reticulum ( ER ) is complex and failure-prone .",
"The ER membrane protein complex ( EMC ) , comprising eight conserved subunits , has emerged as a central player in this process .",
"Yet , we have limited understanding of how EMC enables insertion and integrity of diverse clients , from tail-anchored to polytopic transmembrane proteins .",
"Here , yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies .",
"Structure-based functional studies distinguish between two separable EMC activities , as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis .",
"These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation , and a non-insertase EMC function mediated by the EMC lumenal domain .",
"Our studies illuminate the structural and mechanistic basis of EMC’s multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes ."
] | [
"Cells are surrounded and contained by a plasma membrane consisting of a double layer of fats and proteins .",
"These proteins monitor and facilitate the movement of food , oxygen and messages in and out of the cell , and help neighboring cells communicate .",
"Membrane proteins are manufactured in a cell compartment called the endoplasmic reticulum .",
"Cellular machines called ribosomes visit this compartment’s membrane to manufacture proteins that need to be secreted or embedded into the cell’s membranes .",
"As these proteins are made , they are pulled into the endoplasmic reticulum so they can be folded correctly and inserted in the membrane .",
"A cellular machine in this compartment’s membrane that aids this process is the endoplasmic reticulum membrane protein complex ( EMC ) .",
"Many steps can go wrong during protein assembly , so to control protein quality , the EMC has to accommodate the variety of complex physical features that proteins can have .",
"To explore the activity of the EMC , Miller-Vedam , Bräuning , Popova et al . studied the normal structure of the EMC in both yeast and human cells grown in the lab .",
"These snapshots of the complex in different species had a lot in common , including how the complex was arranged within and around the membrane .",
"Next , Miller-Vedam , Bräuning , Popova et al . generated 50 mutant versions of the EMC in human cells to determine how changing different parts of the complex affected the production of three proteins that rely on the EMC to fold correctly .",
"These proteins were an enzyme called squalene synthase , a signaling protein called the beta adrenergic receptor and sigma intracellular receptor 2 , a protein involved in the regulation of cholesterol levels .",
"Mutations in the section of the EMC outside of the endoplasmic reticulum , within the main cellular compartment , negatively impacted the stability of squalene synthase .",
"This section of the EMC provides a platform where proteins can associate before entering the membrane .",
"The part of EMC that spans the membrane contains both a fat-filled cavity and a cavity with a ‘door’ that is either open or closed .",
"Mutations in this section disrupted the insertion of both squalene synthase and the beta adrenergic receptor into the membrane , a role performed by the cavity with the door .",
"The specific role of the fat-filled cavity is still not fully understood , but a mutation affecting this cavity disrupts the correct production of all three proteins studied .",
"The largest section of the complex , which sits inside the endoplasmic reticulum , protected proteins as they folded , ensuring they were not destroyed for being folded incorrectly before they were fully formed .",
"Mutations in this part of the EMC negatively impacted the stability of sigma intracellular receptor 2 without negatively affecting the other proteins .",
"This molecular dissection of the activity of the EMC provides insights into how membrane proteins are manufactured , stabilized , coordinated , and monitored for quality .",
"These findings could contribute towards the development of new treatments for certain congenital diseases .",
"For example , cystic fibrosis , retinitis pigmentosa , and Charcot-Marie-Tooth disease are all thought to be caused by mutations within membrane proteins that require the EMC during their production ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"short report",
"structural biology and molecular biophysics",
"neuroscience"
] | Disease-associated mutations in the human TRPM3 render the channel overactive via two distinct mechanisms | elife-55634-v2 | [
[
"Transient Receptor Potential Melastatin 3 ( TRPM3 ) is a Ca2+ permeable , non-selective cation channel activated by heat ( Vriens et al . , 2011 ) and chemical activators such as the neurosteroid pregnenolone sulfate ( PregS ) ( Wagner et al . , 2008 ) and the synthetic compound CIM0216 ( Held et al . , 2015 ) .",
"TRPM3 is a well-established temperature sensor in peripheral sensory neurons of the dorsal root ganglia ( DRG ) ; its genetic deletion in mice leads to defects in noxious heat sensation as well as reduced inflammatory heat hyperalgesia ( Vriens et al . , 2011; Vandewauw et al . , 2018 ) .",
"Inhibitors of TRPM3 also reduced both acute heat sensation and inflammatory heat hyperalgesia ( Straub et al . , 2013; Krügel et al . , 2017 ) .",
"While TRPM3-/- mice show defects in noxious heat sensation , the channel shows increased activity well below the noxious range , when temperature is increased from 15°C to 26°C , with further increases in activity at 37°C ( Vriens et al . , 2011 ) .",
"TRPM3 is also inhibited by activation of Gi-coupled receptors such as μ-opioid receptors and GABAB receptors in DRG neurons , and agonists of those receptors reduced nocifensive reactions to local injection of TRPM3 agonists ( Badheka et al . , 2017; Dembla et al . , 2017; Quallo et al . , 2017 ) .",
"TRPM3 is also expressed in tissues other than peripheral sensory neurons , where its functional roles are not well understood .",
"In pancreatic β-cells , it was shown that application of the TRPM3 agonist PregS induced an increase in insulin secretion ( Wagner et al . , 2008 ) , but TRPM3-/- mice showed no impairment in glucose homeostasis ( Vriens et al . , 2011 ) .",
"TRPM3 is also expressed in vascular smooth muscle cells , and PregS was reported to induce contractile responses in freshly isolated aorta , but the concentration required for this response was higher than the plasma levels of this compound ( Naylor et al . , 2010 ) .",
"TRPM3 is also expressed in various regions of the brain , where its functional role is essentially unexplored ( Oberwinkler and Philipp , 2014 ) .",
"Very little is known about the physiological and pathophysiological roles of TRPM3 in humans .",
"A recent paper showed that two missense mutations in TRPM3 are associated with a neurodevelopmental disorder with intellectual disability , hypotonia and epilepsy , pointing to important roles of this channel in the human brain ( Dyment et al . , 2019 ) .",
"Seven of the eight patients had a de novo Val to Met substitution in the S4-S5 loop , while one patient had a Pro to Gln substitution in the extracellular segment of S6 .",
"The effects of these mutations on channel function however were not reported .",
"Here , we tested the functional effects of the two disease-associated mutations using electrophysiology and intracellular Ca2+ measurements .",
"We find that both disease-associated mutations render the channel overactive .",
"Both mutants showed constitutive activity that was inhibited by the TRPM3 antagonist primidone .",
"As primidone is a clinically used medication ( Krügel et al . , 2017 ) , our findings offer potential clinical intervention to treat this channelopathy .",
"We also find that the Val to Met substitution in the S4-S5 loop induced a larger left shift in the concentration response relationship to PregS and CIM0216 than the Pro to Gln substitution close to the pore-loop in the extracellular segment of S6 .",
"The increase in heat activation on the other hand was more pronounced in the S6 mutant .",
"We conclude that both reported mutants of TRPM3 are gain of function , but the mechanism of increased channel activity is different for the two mutants ."
],
[
"Here we used , patch clamp electrophysiology and intracellular Ca2+ measurements in HEK293 cells and two-electrode voltage clamp electrophysiology in Xenopus oocytes to study the effects of disease-associated mutations on TRPM3 function .",
"TRPM3 has a large number of splice variants ( Oberwinkler and Philipp , 2014 ) , but there is no information available which splice variants are expressed in the human brain , and relatively little is known about the functional differences between splice variants .",
"To ensure that our results do not only apply to one variant , we used two commonly used and well-characterized splice variants of TRPM3 in our experiments .",
"In HEK293 cells , we expressed the human orthologue of the most studied mouse variant TRPM3α2 that was originally cloned from mouse brain ( Oberwinkler et al . , 2005; Vriens et al . , 2011 ) .",
"In Xenopus oocytes , we expressed the human TRPM3 splice variant we used in several previous studies ( Badheka et al . , 2015; Badheka et al . , 2017 ) , originally described in Grimm et al . ( 2003 ) also called the TRPM31325 variant ( Oberwinkler et al . , 2005 ) .",
"Most alternatively spliced exons are in the cytoplasmic N-terminus; thus , the numbering of mutated residues varies between splice variants .",
"The more common S4-S5 segment mutant described as V837M ( Dyment et al . , 2019 ) corresponds to V990M in the hTRPM3 splice variant we expressed in Xenopus oocytes , and to V992M in the hTRPM3α2 variant we expressed in HEK293 cells .",
"The S6 mutant P937Q in Dyment et al corresponds to P1090Q in the hTRPM3 variant we used in oocytes , and it is P1092Q in the hTRPM3α2 we used in HEK293 cells .",
"Figure 1—figure supplement 1 shows the location of these residues in the TRPM3 sequence and putative structure .",
"First , we co-expressed the Ca2+ indicator GCaMP6f ( Chen et al . , 2013 ) and the hTRPM3α2 isoform and its mutants in HEK293 cells and performed intracellular Ca2+ measurements in a 96-well plate reader .",
"We found that the concentration-response relationship to PregS ( Figure 1A–D ) and CIM0216 ( Figure 1E–H ) were left shifted in the mutant channels; V992M showed a much larger shift than P1092Q for both agonists .",
"The V992M mutant also showed a larger increase in basal Ca2+ levels than P1092Q at room temperature ( 21°C ) , and the application of the TRPM3 antagonist primidone decreased basal Ca2+ levels for both mutants in a concentration-dependent manner ( Figure 1I–L ) .",
"Primidone had no effect on basal cytoplasmic Ca2+ levels in cells transfected with the wild type TRPM3 indicating negligible basal activity of the wild type channel at room temperature ( Figure 1I ) .",
"Primidone robustly inhibited Ca2+ signals evoked by EC50 concentrations of PregS both for wild type and mutant channels ( Figure 1—figure supplement 2 ) .",
"We also measured PregS responses at 37°C .",
"Consistent with earlier results ( Vriens et al . , 2011 ) , sensitivity of wild-type TRPM3 to PregS increased at 37°C; the EC50 of activation decreased to 0 . 99 μM at 37°C compared to 7 μM at room temperature ( Figure 1—figure supplement 3A , B ) .",
"Basal Ca2+ levels at 37°C were substantially elevated in cells expressing wild-type channels , which is consistent with the low temperature threshold of TRPM3 ( Vriens et al . , 2011 ) .",
"Both mutant channels showed very high basal Ca2+ levels , which were not further increased by PregS , indicating substantial Ca2+ overload when kept at 37°C continuously ( Figure 1—figure supplement 3C , D ) .",
"Primidone ( 50 μM ) reduced basal Ca2+ levels at 37°C in wild type channels and to a smaller extent in the mutant channels ( Figure 1M–P ) .",
"Next , we transfected HEK293 cells with the mutant and wild-type hTRPM3α2 and used fura-2 Ca2+ imaging to study the effects of acutely increased temperatures ( Figure 2 ) .",
"We first increased the temperature to 37°C , followed by 10 μM primidone at room temperature to facilitate return of Ca2+ to baseline .",
"Then we applied 25 μM PregS ( in the absence of primidone ) , and compared the Ca2+ responses induced by 37°C to that induced by PregS .",
"In cells transfected with wild-type TRPM3 , the temperature-induced Ca2+ response was , on average , 29 . 4% of that induced by PregS , for the V992M mutant it was 76 . 5% , whereas for the P1092Q mutant it was ~122 . 5% ( Figure 2D , E ) .",
"In cells not expressing TRPM3 increasing temperature to 37°C induced only negligible Ca2+ signals ( not shown ) .",
"Cytoplasmic Ca2+ is an indirect measure of TRPM3 activity , thus next , we performed whole cell patch clamp experiments to compare currents induced by increased temperatures and by PregS .",
"These measurements were performed in the absence of extracellular Ca2+ to avoid indirect effects of increased cytoplasmic Ca2+ such as Ca2+ induced desensitization .",
"We stimulated each cell with a temperature ramp from 23°C to 36°C followed by a saturating concentration of PregS ( 100 μM ) at room temperature ( Figure 3A–C ) .",
"Since the current amplitudes induced by PregS were highly variable , presumably due to different expression levels of the channel ( Figure 3H ) , we normalized the currents induced by increased temperatures to those evoked by PregS and plotted these relative currents as a function of temperature ( Figure 3D–F ) .",
"Figure G shows that the slope of the increase of these currents as a function of temperature were significantly steeper for the P1092Q mutant than for V992M , and both mutants were significantly steeper than the wild type .",
"Next , we expressed the wild-type hTRPM31325 and its V990M and P1090Q mutants in Xenopus oocytes and performed full concentration response measurements with the TRPM3 agonist PregS .",
"Consistent with our Ca2+ measurements , the concentration response relationships for PregS were left-shifted for both mutants compared to wild-type , but the effect of the V990M mutation was much more pronounced than that of the P1090Q ( Figure 4A–D ) .",
"Both disease-associated mutations are de novo , and all known patients are heterozygous .",
"To mimic heterozygous conditions , we co-injected oocytes with wild type cRNA and either mutant in a 1:1 ratio .",
"The PregS dose response was still markedly left shifted for the V990M:TRPM3 combination , but it was only marginally shifted in the P1090Q:TRPM3 combination compared to WT TRPM3 ( Figure 4E–G ) .",
"To assess basal current levels , we applied 50 μM primidone at room temperature ( 20–22°C ) .",
"Consistent with our Ca2+ measurements , primidone evoked a significantly larger inhibition of basal activity in the V990M than in the P1090Q mutant both at 100 and −100 mV ( Figure 4—figure supplement 1B-D ) .",
"Primidone did not induce any inhibition in non-injected oocytes ( not shown , n = 5 ) , but it evoked a small reduction in oocytes expressing TRPM3 ( Figure 4—figure supplement 1A , D ) .",
"Current amplitudes induced by 100 μM PregS were not different between the two mutants and wild type , at +100 mV , but the V990M mutants showed somewhat larger amplitudes at −100 mV than the wild type or the P1090Q mutant ( Figure 4—figure supplement 1E ) .",
"We also compared the basal current amplitudes before applying any stimuli , and found that oocytes expressing the V990M mutant showed higher currents than those expressing P1090Q , and both mutants had higher basal currents than wild-type TRPM3 ( Figure 4—figure supplement 1F ) .",
"We also compared the currents induced by increased temperatures to those evoked by PregS in channels expressed in Xenopus oocytes ( Figure 4—figure supplement 2 ) .",
"We found that the ratio of currents induced by 30°C over those induced by 50 μM PregS were significantly larger in the P1090Q mutant compared to V990M , and both mutants showed significantly larger current ratios than wild type TRPM3 .",
"Increasing temperature to 30°C induced negligible currents in non-injected oocytes , but those currents became larger at higher temperatures ( not shown ) , which prevented us from testing higher temperatures in this experimental setting .",
"TRPM3 has been shown to be inhibited by activation of Gi-coupled receptors via direct binding of Gβγ ( Badheka et al . , 2017; Dembla et al . , 2017; Quallo et al . , 2017 ) .",
"To test if the mutations alter receptor-induced inhibition , we co-expressed TRPM3 or its mutants with Gi-coupled muscarinic M2 receptors in Xenopus oocytes .",
"Figure 4—figure supplement 3A–F show that when we applied acetylcholine ( ACh ) to stimulate M2 receptors , it evoked a ~ 50% inhibition of TRPM3 currents induced by 50 μM PregS .",
"The inhibition of the P1090Q mutant was similar to wild type , but the V990M mutant was essentially not inhibited .",
"To test whether the lack of inhibition was due to allosteric effects of the increased sensitivity to PregS , we stimulated the oocytes expressing the V990M mutant with 5 μM PregS where ACh induced a ~ 35% inhibition ( Figure 4—figure supplement 3C , E ) .",
"We also tested inhibition at the EC50 of PregS for wild type ( 17 μM ) and mutant channels , 0 . 6 μM for V990M and 7 μM for P1090Q .",
"Figure 4—figure supplement 3G-K shows that wild type and V990M mutants were inhibited to a similar extent , the P1090Q mutant was inhibited somewhat more than the wild-type channel .",
"These data show that while the V990M mutation affects receptor-induced inhibition at high PregS concentrations , it is not likely to be the primary mechanism of its gain-of-function phenotype ."
],
[
"Overall , our data show that disease-associated mutations in TRPM3 render the channel overactive .",
"Both mutants showed basal activity even at room temperature , which was reduced by the TRPM3 antagonist primidone .",
"Basal activity of the V992M mutant at room temperature was higher than that of the P1092Q mutant , but at 37°C , the difference in basal activity between the two mutants became negligible .",
"Given the increased constitutive activity of the mutants at body temperature , increased neuronal excitability and/or Ca2+-induced neuronal damage is a possible disease-causing mechanism .",
"Primidone is a clinically approved antiepileptic drug; it is thought to exert its effects by being converted to barbiturate by the liver , but it crosses the blood brain barrier ( Nagaki et al . , 1999 ) , and directly inhibits TRPM3 activity even below its therapeutic concentration ( Krügel et al . , 2017 ) .",
"Our data showing that primidone inhibited the basal activity of the mutant channels , suggests a potential therapy for this newly described channelopathy .",
"The V990/V992M mutation showed a larger increase in basal activity at room temperature , and also induced a larger left shift in the concentration response curves of the agonists PregS and CIM0216 than the P1090/1092Q mutation .",
"This is expected if both mutations increase the stability of the open state of the channel , with the V990/992M mutation having a larger effect .",
"If increased open state stability is the only explanation for the over-activity of the mutants , we would expect V990/992M to be also more sensitive to activation by increased temperatures than P1090/1092Q .",
"This is however not what we observed .",
"We consistently find that the P1090/1092Q mutant showed more pronounced activation by increased temperatures than the V990/992M mutant .",
"This indicates that the mechanism of over-activity is different for the two mutants .",
"These two residues are in different locations; V990/992 is in the S4-S5 linker whereas P1090/1092 is in the outer portion of S6 ( Figure 1—figure supplement 1 ) .",
"The S4-S5 linker plays essential roles in channel gating , and has been shown to be a hotspot of disease-associated gain-of-function mutations in a number of TRP channels , including TRPV3 , TRPV4 , TRPM4 and TRPA1 ( Hofmann et al . , 2017 ) .",
"This channel segment also plays a role in binding of hydrophobic , or amphipathic ligands in TRPM channels ( Huang et al . , 2020 ) .",
"For example , in TRPM8 channels the S4-S5 linker is in direct contact with both the cooling agent icilin , and the menthol analog WS12 ( Yin et al . , 2019 ) .",
"While PregS is thought to activate TRPM3 by directly binding to the channel ( Drews et al . , 2014 ) , its binding site in the channel is not known , and currently there is no structural information available for TRPM3 .",
"Because of the clear increase in basal activity of the V990/992M mutant , it is quite likely that the mutation primarily increased the stability of the open state and the decrease in EC50 of agonist activation is a consequence of the change in activation equilibrium constant ( Colquhoun , 1998 ) .",
"It cannot be excluded , however , that the mutation also affected PregS binding concurrently , given the general role of this segment in ligand binding in TRP channels .",
"The membrane phospholipid phosphatidylinositol 4 , 5-bisphosphate ( PIP2 ) is also required for PregS-induced TRPM3 activity ( Badheka et al . , 2015; Tóth et al . , 2015; Uchida et al . , 2016 ) .",
"In TRPM8 , the equivalent of V990 is two positions upstream from a residue that is in close contact with PIP2 , and it is 4 and 7 positions downstream from two residues in close contact with icilin and the menthol analog WS12 ( Yin et al . , 2019; Figure 1—figure supplement 1A ) .",
"PIP2 is located adjacent to the menthol analogue WS12 and the cooling agent icilin in the TRPM8 structures , and menthol has been shown to allosterically affect PIP2 activation ( Rohács et al . , 2005 ) .",
"Therefore , it is also possible that the V990/992M mutation affects PregS activation indirectly via PIP2 .",
"Increased Ca2+ levels in cells expressing mutated channels may also modify cellular PI ( 4 , 5 ) P2 levels , which may alter channel activity .",
"Differentiating between these possibilities will require future studies .",
"While the mechanism of temperature activation of TRP channels is not fully understood ( Clapham and Miller , 2011; Islas , 2017; Arrigoni and Minor , 2018; Castillo et al . , 2018 ) , large-scale unbiased mutagenesis studies on TRPV1 ( Grandl et al . , 2010 ) and TRPV3 ( Grandl et al . , 2008 ) show that mutations in the pore region and the outer portion of S6 in these channels selectively abolished heat- , but not agonist-induced channel activation .",
"The P1090/1092Q mutation in TRPM3 is located in the outer portion of S6 , and it had a stronger effect on heat activation than the V990/992M mutation , therefore it is possible that the primary effect of the P1090/1092Q mutation is increasing heat sensitivity .",
"Increased temperatures synergize with PregS in activating wild-type TRPM3 ( Vriens et al . , 2011 ) , see also Figure 1—figure supplement 3B , therefore it is possible that the increased PregS sensitivity of the P1090/1090Q mutant is secondary to its increased heat activation .",
"Overall our data show that both disease-associated mutations render TRPM3 overactive , but likely with different mechanisms ."
],
[
"Intracellular Ca2+ measurements were performed using a Flexstation-3 96‐well plate reader with rapid well injection ( Molecular Devices ) as described earlier ( Hughes et al . , 2019 ) with some modifications .",
"Briefly Human Embryonic Kidney 293 ( HEK293 ) cells were purchased from American Type Culture Collection ( ATCC ) , Manassas , VA , ( catalogue number CRL-1573 ) , RRID:CVCL_0045; cell identity was verified by STR analysis by ATCC .",
"HEK293 cells were cultured in MEM supplemented with 10% FBS and 100 IU/ml penicillin plus 100 µg/ml streptomycin in 5% CO2 at 37°C .",
"Additional cell authentication was not performed , but passage number of the cells was monitored , and cells were used up to passage number 25–30 from purchase , when a new batch of cells was thawed with low passage number; cells were tested for the lack of mycoplasma infection .",
"HEK293 cells were transfected with hTRPM3α2-GFP , or its mutants ( 200 ng ) and GCaMP6 ( 1 μg ) using the Effectene reagent ( Qiagen ) .",
"The human orthologue of the mouse splice variant TRPM3α2 in the pCDNA3 . 1 ( + ) vector ( hTRPM3 variant 10; NM_001366141 . 2 ) tagged with GFP on its N terminus , and its V992M and P1092Q mutants were purchased from Genescript , GCaMP6f was a kind gift from Dr . Lawrence Gaspers .",
"After 24 hr , transfected cells were plated on poly-D-lysine coated black-wall clear-bottom 96-well plates and measurements were performed 24–48 hr after plating .",
"Before experiments , the MEM media was replaced with a solution containing ( in mM ) 137 NaCl , 5 KCl , 1 MgCl2 , 2 CaCl2 , 10 HEPES and 10 glucose , pH 7 . 4 and the plate was measured at around 21°C .",
"GCaMP6 signal was measured at excitation wavelengths 485 nm and fluorescence emission was detected at 525 nm .",
"Sampling interval was 0 . 86 s and four parallel reads were performed for each condition .",
"For most experiments , 2 μM ionomycin was applied to determine the maximum response .",
"Primidone was purchased form Sigma , CIM0216 from Calbiochem , ionomycin and PregS from Cayman Chemicals .",
"For measurements at 37°C the MEM medium used to culture the cells was replaced with the measurement solution preheated to 37°C , and the plate was placed in the plate reader warmed to 37°C using its built-in temperature controller .",
"Ca2+ imaging experiments were performed using an Olympus IX-51 inverted microscope equipped with a DeltaRAM excitation light source ( Photon Technology International , PTI ) , as described earlier ( Badheka et al . , 2017 ) .",
"HEK293 cells were transfected with hTRPM3α2-GFP or its mutants using the Effectene reagent ( Qiagen ) .",
"Cells were loaded with 1 μM fura-2 AM ( Invitrogen ) for 40 min before the measurements at 37°C , and dual-excitation images at 340 and 380 nm excitation wavelengths were detected at 510 nm with a Roper Cool-Snap digital CCD camera .",
"Measurements were conducted at room temperature in extracellular solution containing 137 mM NaCl , 5 mM KCl , 1 mM MgCl2 , 2 mM CaCl2 , 10 mM HEPES and 10 mM glucose , pH 7 . 4 .",
"PregS , and primidone were applied with a gravity-driven whole chamber perfusion system .",
"Temperature stimulation was performed using a custom-built system as described earlier ( Badheka et al . , 2017 ) by pushing bath solution through a spiral tubing immersed in hot water using a 60 ml syringe while monitoring the temperature of the perfusion chamber using a CL-100 Warner Instruments temperature controller .",
"The analogue signal from the CL-100 unit was fed into the Digidata digitizer and the temperature curve was collected in Clampex .",
"Data analysis was performed using the Image Master 5 software ( PTI ) .",
"HEK293 cells were transiently transfected with cDNA encoding the hTRPM3α2-GFP , or its mutants with 0 . 2 µg of constructs using the Effectene reagent ( Qiagen ) according manufacturer’s protocol and were used in experiments 48–72 hr later .",
"Measurements were carried out on GFP positive cells , in an extracellular solution containing ( in mM ) 137 NaCl , 5 KCl , 1 MgCl2 , 10 HEPES and 10 glucose , pH 7 . 4 .",
"The intracellular solution contained ( in mM ) 140 potassium gluconate , 5 EGTA , 1 MgCl2 , 10 HEPES , and 2 NaATP , pH 7 . 3 .",
"Patch clamp pipettes were prepared from borosilicate glass capillaries ( Sutter Instruments ) using a P-97 pipette puller ( Sutter Instrument ) and had a resistance of 2–4 MΩ .",
"In all experiments after formation of GΩ-resistance seals , the whole-cell configuration was established and currents were recorded using a ramp protocol from −100 mV to +100 mV over 500 ms preceded by a −100 mV step for 100 ms; the holding potential was 0 mV , and this protocol was applied once every 1 s .",
"The currents were measured with an Axopatch 200B amplifier , filtered at 5 kHz , and digitized through Digidata 1440A interface .",
"In all experiments , cells that had a passive leak current over 100 pA were discarded .",
"Data were collected and analyzed with the PClamp10 . 6 ( Clampex ) acquisition software ( Molecular Devices , Sunnyvale , CA ) , and further analyzed and plotted with Origin 2019b ( OrigiLab , Northampton , MA ) .",
"Heat stimulation was performed as described for the Ca2+ imaging experiments .",
"Xenopus laevis oocytes were prepared as described earlier ( Badheka et al . , 2015 ) .",
"All animal procedures were approved by the Institutional Animal Care and Use Committee at Rutgers New Jersey Medical School .",
"In brief , frogs were anesthetized in 0 . 25% ethyl 3-aminobenzoate methanesulfonate solution ( MS222; Sigma-Aldrich ) ; bags of ovaries were removed surgically from the anesthetized frogs .",
"Individual oocytes were obtained by overnight digestion at 16°C in 0 . 2–0 . 3 mg/ml type 1A collagenase ( Sigma-Aldrich ) , dissolved in a solution containing 82 . 5 mM NaCl , 2 mM KCl , 1 mM MgCl2 , and 5 μM HEPES , pH 7 . 4 ( OR2 solution ) .",
"The next day the collagenase containing solution was discarded and the oocytes were washed multiple times with OR2 solution .",
"The oocytes were maintained in OR2 solution supplemented with 1 . 8 mM CaCl2 , 100 IU/ml penicillin , and 100 μg/ml streptomycin at 16°C .",
"cRNA was transcribed from the linearized human TRPM3 ( hTRPM3 ) cDNA clone ( Grimm et al . , 2003 ) , or its mutants in the pGEMSH vector using the mMessage mMachine kit ( Thermo Fisher Scientific ) .",
"cRNA ( 40 ng ) was microinjected into individual oocytes , using a nanoliter-injector system ( Warner Instruments ) .",
"For combined injection of wild-type and mutant TRPM3 for Figures 4E–G , 40 μg total cRNA was injected in a 1:1 ratio .",
"The V990M and P1090Q mutants were generated using the QuikChange II XL Site-Directed Mutagenesis Kit ( Agilent Technologies ) .",
"For the GPCR regulation of these mutants , we injected cRNA of human M2 muscarinic receptors together with TRPM3 or mutants at 1:1 ratio .",
"Oocytes were used for electrophysiological measurements 48–72 hr after microinjection .",
"The hTRPM31325 clone in a mammalian expression vector was provided by C . Harteneck ( Eberhard Karls University Tübingen , Tübingen , Germany ) , and it was subcloned into the pGEMSH oocyte vector using standard molecular biology techniques .",
"Two electrode voltage clamp experiments were performed as described ( Badheka et al . , 2015 ) .",
"In brief , oocytes were placed in extracellular solution ( 97 mM NaCl , 2 mM KCl , 1 mM MgCl2 , and 5 μM HEPES , pH 7 . 4 ) , and currents were recorded with thin-wall inner filament– containing glass pipettes ( World Precision Instruments ) filled with 3 M KCl in 1% agarose .",
"Currents were measured with a ramp protocol from −100 to 100 mV once every 0 . 5 s with a GeneClamp 500B amplifier and analyzed with the pClamp 9 . 0 software ( Molecular Devices ) .",
"PregS , ACh and primidone were applied with a gravity driven whole chamber perfusion system .",
"Temperature stimulation was performed the same way as for the whole cell patch clamp and Ca2+ imaging experiments in HEK293 cells .",
"Statistical analysis was performed with Origin 2019b and Prism6 .",
"Data are plotted as mean ± SEM and scatter plots .",
"No statistical method was used to predetermine sample sizes , but our sample sizes are similar to those generally employed by the field .",
"Experiments were performed in random order .",
"Data were analyzed with t-test , or one-way analysis of variance with Bonferroni’s post hoc test , p values are reported in the figures ."
]
] | [
"Transient Receptor Potential Melastatin 3 ( TRPM3 ) is a Ca2+ permeable non-selective cation channel activated by heat and chemical agonists such as pregnenolone sulfate and CIM0216 .",
"TRPM3 mutations in humans were recently reported to be associated with intellectual disability and epilepsy; the functional effects of those mutations , however , were not reported .",
"Here , we show that both disease-associated mutations in the human TRPM3 render the channel overactive , but likely via different mechanisms .",
"The Val to Met substitution in the S4-S5 loop induced a larger increase in basal activity and agonist sensitivity at room temperature than the Pro to Gln substitution in the extracellular segment of S6 .",
"In contrast , heat activation was increased more by the S6 mutant than by the S4-S5 segment mutant .",
"Both mutants were inhibited by the TRPM3 antagonist primidone , suggesting a potential therapeutic intervention to treat this disease ."
] | [
"Inherited brain disorders often cause severe problems for those affected by them .",
"One example is a group of diseases , collectively termed “developmental and epileptic encephalopathies” , or DEE for short .",
"People with these diseases usually have both epilepsy and intellectual disabilities , and in some patients these conditions are associated with two mutations that change a gene called TRPM3 .",
"The TRPM3 gene encodes a protein called an ion channel .",
"Ion channels form pores on the surfaces of cells .",
"When channels are active , the pores open , allowing charged particles – which , in the case of TRPM3 , are sodium and calcium ions – to pass through , carrying tiny electrical currents .",
"In the nervous system , ion channels help nerve cells communicate and also allow them to sense changes in the environment .",
"The TRPM3 channel is known to open in response to heat and certain chemical “activators” .",
"In mice , TRPM3 is found in sensory nerve cells , where it acts as a heat sensor .",
"Although altering TRPM3 in mice affects their ability to sense intense or painful heat stimuli , they are otherwise completely normal and have no symptoms resembling human DEE disorders .",
"Although TRPM3 is found in the human brain , little is known about its role there or what effects the DEE-associated mutations have on its activity .",
"Zhao et al . therefore set out to determine , whether each of the mutation was a ‘loss of function’ , meaning that it stopped the channel from opening , or a ‘gain of function’ , meaning it made the channel open more often .",
"Frog egg cells and mammalian cells grown in the laboratory were engineered to produce the TRPM3 ion channel .",
"Measurements of electrical activity on these cells revealed that the two mutations seen in people with DEE were both ‘gain of function’ .",
"Both mutants were more sensitive to heat and chemical activators than the normal protein .",
"They were also more active overall , even without any stimuli .",
"However , one mutation had a greater effect on heat sensitivity , while the other caused a larger increase in chemical-induced activity .",
"Imaging experiments revealed that both mutant channels also increased the amount of calcium inside the cells .",
"This could explain why the mutations cause disease , since abnormally high calcium levels can damage nerve cells .",
"In addition , the epilepsy drug primidone switched off the mutant channels , pointing to potential treatment of this disease using primidone ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology",
"biochemistry and chemical biology"
] | A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop | elife-13664-v1 | [
[
"Artemisinin , a C15 isoprenoid ( sesquiterpene ) naturally produced in the wild plant Artemisia annua ( sweet wormwood , native to temperate Asia ) , is the main ingredient of artemisinin combination therapies ( ACTs ) , currently the only effective cure of malaria ( Okell et al . , 2014 ) .",
"As ACTs are the mainstay of malaria treatment and no alternative to artemisinin derivatives is expected to enter the market in the foreseeable future , there is a steadily increasing demand for ACTs which reached nearly 400 million treatment courses in 2013 ( http://www . who . int/malaria/publications/world_malaria_report_2014 ) .",
"The mechanism of action of artemisinin on the malaria parasites Plasmodium falciparium and P . vivax is not entirely clear , but it is generally believed that the reactive endoperoxide bridge present in the molecule ( Figure 1 ) is responsible for its medicinal properties .",
"In addition to their antimalarial activity , artemisinin and its derivatives are currently also considered as promising anti-cancer , antiviral and anti-inflammatory agents ( e . g . , Willoughby et al . , 2009 ) .",
"In A . annua , artemisinin is produced in the cytosol of the glandular trichomes of leaves and flowers ( Tang et al . , 2014 ) .",
"The biosynthesis initiates with the conversion of the isoprenoid building blocks IPP and DMAPP into farnesyl pyrophosphate ( FPP ) which is then converted into amorpha-4 , 11-diene by amorphadiene synthase ( ADS ) , the enzyme catalyzing the first committed step of the pathway ( Figure 1 ) .",
"Amorpha-4 , 11-diene is a volatile compound that is oxidized to artemisinic alcohol and subsequently to artemisinic aldehyde by the cytochrome P450 monooxygenase CYP71AV1 ( CYP ) and its redox partner , the cytochrome P450 reductase ( CPR ) .",
"Artemisinic aldehyde is then further oxidized to artemisinic acid by the same enzyme pair , or alternatively , is reduced to dihydroartemisinic aldehyde by the double bond reductase 2 ( DBR2; Figure 1; Zhang et al . , 2008 ) .",
"Artemisinic acid can be efficiently and cheaply converted to artemisinin by chemical means ( Paddon et al . , 2013; Kopetzki et al . , 2013 ) and , therefore , represents a high-value precursor for the industrial production of artemisinin-based pharmaceuticals ( Paddon and Keasling , 2014 ) . 10 . 7554/eLife . 13664 . 003Figure 1 . Metabolic pathway of artemisinin biosynthesis . The canonical pathway of artemisinin synthesis starts with the conversion of IPP/DMAPP ( C5 isoprenoids produced by the MVA pathway in the cytosol or the MEP pathway in the chloroplast ) into farnesyl pyrophosphate ( FPP ) , catalyzed by farnesyl pyrophosphate synthase ( FPS ) .",
"Amorpha-4 , 11-diene synthase ( ADS ) converts FPP into amorpha-4 , 11-diene in the first committed step of the pathway .",
"Amorpha-4 , 11-diene is then successively oxidized to artemisinic alcohol , artemisinic aldehyde and artemisinic acid by the cytochrome P450 monooxygenase CYP71AV1 ( CYP ) and its redox partner , the cytochrome P450 reductase ( CPR ) .",
"In A . annua , artemisinic aldehyde is converted to dihydroartemisinic aldehyde by DBR2 , and then to dihydroartemisinic acid by ALDH1 .",
"Artemisinin is generated by the spontaneous oxidation of dihydroartemisinic acid in planta , and can be produced by chemical conversion of artemisinic acid in vitro .",
"Enzymes depicted in red improve the efficiency of different oxidation steps in yeast ( Paddon et al . , 2013; Paddon and Keasling , 2014 ) .",
"See text for details . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 003 In view of the great medicinal value of artemisinic compounds , their low accumulation levels in A . annua and the unstable supply of the plant , enormous efforts have been undertaken to produce artemisinic compounds synthetically or in heterologous biological systems .",
"Currently , the semisynthetic synthesis in yeast ( Paddon et al . , 2013 ) represents the most efficient heterologous production system for artemisinic acid , the immediate precursor of artemisinin ( Figure 1 ) .",
"However , the production costs are still high and ACTs remain unaffordable to many people in the tropical and subtropical regions of Africa and Asia that are most severely afflicted with malaria .",
"Since production in yeast requires large volumes of costly synthetic culture media and large-capacity bioreactors run under sterile conditions , production in plants can potentially provide a much cheaper , renewable and easily scalable source of artemisinic acid .",
"Although the production of artemisinic compounds at low levels has been shown to be feasible in heterologous plant systems ( Wu et al . , 2006; van Herpen et al . , 2010; Zhang et al . , 2011; Farhi et al . , 2011 ) , the development of an efficient production system for the drug precursor artemisinic acid has not been achieved .",
"Here we have pursued a novel synthetic biology approach towards high-level production of artemisinic acid in chloroplasts of tobacco ( Nicotiana tabacum ) , a fast-growing crop that produces high amounts of biomass at very low cost .",
"We show that by implementing the core pathway in the chloroplast and subsequently selecting for optimum combinations and expression levels of additional pathway enzymes from a large population of combinatorially supertransformed transplastomic lines , artemisinic acid can be produced in tobacco leaves to levels of more than 120 mg/kg fresh weight ."
],
[
"The core enzymes to synthesize artemisinic acid are FPS , ADS , CYP and CPR ( Figure 1 ) .",
"Accessory enzymes ( indicated in red in Figure",
"1 ) and additional enzymes facilitating more efficient biosynthesis of artemisinin are CYB5 , ADH1 , ALDH1 and DBR2 .",
"We first implemented the canonical pathway from FPP to artemisinic acid in tobacco chloroplasts using stable plastid genome transformation ( Svab and Maliga , 1993; Bock , 2015 ) .",
"To this end , we designed a number of synthetic operons ( Zhou et al . , 2007; Lu et al . , 2013 ) that combine the genes for the four core enzymes ( FPS , ADS , CYP and CPR; Figure",
"1 ) in different arrangements and under the control of different expression signals ( Figure 2A ) .",
"Four synthetic artemisinic acid operon constructs ( pAO1-4 ) were built and introduced into the chloroplast ( plastid ) genome of tobacco plants by particle gun-mediated transformation .",
"Chloroplast-transformed ( transplastomic ) lines were selected on regeneration medium with spectinomycin and purified to homoplasmy by additional rounds of selection and regeneration ( Svab and Maliga , 1993; Bock , 2015 ) .",
"Restriction fragment length polymorphism ( RFLP ) analysis verified integration of the synthetic operon constructs into the plastid genome by homologous recombination and successful elimination of all wild-type copies of the highly polyploid chloroplast genome ( Figure 2B ) .",
"Homoplasmy of the transplastomic lines was additionally verified by seed assays that confirmed lack of segregation of the spectinomycin resistance and uniparentally maternal inheritance ( Figure 2C ) . 10 . 7554/eLife . 13664 . 004Figure 2 . Implementation of the canonical pathway of artemisinic acid biosynthesis in chloroplasts . Synthetic codon-optimized genes for the four enzymes required to produce artemisinic acid ( Figure",
"1 ) were introduced into the tobacco plastid genome by stable genetic transformation with four different synthetic operon constructs ( pAO1-4 ) .",
"The constructs differ in gene arrangement and in the translation signals that drive synthesis of the key pathway enzyme ( ADS ) catalyzing the first committed step .",
"( A ) Physical map of the plastid genome region ( ptDNA ) used for integration of the synthetic artemisinic acid operons and maps of the transgenic loci in the generated transplastomic tobacco lines ( Nt-AO1-4 ) .",
"The artemisinic acid operon genes are depicted as light blue boxes .",
"Chloroplast promoters and terminators are shown in green , the aadA selectable marker gene for chloroplast transformation is represented as a white box , and genes in flanking plastid sequences used for transgene targeting via homologous recombination are in black .",
"Genes above the line are transcribed from left to right , genes below the line are transcribed in the opposite direction .",
"The four transgenes are arranged in two dicistronic operons .",
"FPS and CYP are driven by the Chlamydomonas reinhardtii plastid ribosomal RNA operon promoter ( Cr Prrn ) and the g10 leader sequence from phage T7 ( T7 Lg10 ) .",
"The second operon containing ADS and CPR is driven by the C . reinhardtii psbA promoter ( Cr PpsbA ) and either the T7 Lg10 or the psbA leader sequence from C . reinhardtii ( Cr LpsbA ) .",
"This operon is arranged either in sense and downstream of the first operon ( AO1 ,",
"3 ) or in antisense , downstream of the aadA cassette ( AO2 , 4 ) .",
"The genes in each operon are separated by an intercistronic expression element ( IEE ) conferring intercistronic RNA processing and , in this way , enhancing expression of downstream cistrons of the operon ( Zhou et al . , 2007; Drechsel and Bock , 2010 ) .",
"The BamHI restriction sites used in RFLP analyses and the expected fragment sizes are indicated .",
"The location of the hybridization probe is shown as a black bar .",
"Cr: C . reinhardtii; Nt: N . tabacum; T7: bacteriophage T7; P: promoter; L: leader sequence; T: terminator; SD: Shine-Dalgarno sequence .",
"( B ) RFLP analysis of transplastomic plants .",
"Two independently isolated transplastomic lines are shown for constructs pAO1-3 and one for pAO4 .",
"( C ) Seed assays confirming the homoplasmic state of the transplastomic plants .",
"Seeds were germinated on medium containing 500 mg/L spectinomycin ( Nt-AO2-1 , Nt-AO3-1 , Nt-wt ) or antibiotic-free medium ( Nt-wt ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 004 All homoplasmic transplastomic lines grew autotrophically under greenhouse conditions and produced viable seeds .",
"However , transplastomic lines obtained with constructs pAO1 and pAO3 ( Figure 2A ) displayed a slightly pale-green phenotype and a subtle growth delay at the juvenile stage ( Figure 3A; Figure 3—figure supplement 1 ) .",
"This phenotype could be due to toxicity of artemisinic metabolites produced in these plants ( Bharati et al . , 2012 ) or , alternatively , depletion of isoprenoid precursors from other metabolic pathways in the cell , such as carotenoid and chlorophyll biosyntheses .",
"Measurement of chlorophylls and carotenoids confirmed that , indeed , both pigment classes are significantly reduced in plants exhibiting the mild phenotype ( Figure 3—figure supplements 1 and 2 ) .",
"Metabolite profiling ( see Materials and Methods ) of the transplastomic lines revealed that all lines accumulated the volatile artemisinic acid precursor amorpha-4 , 11-diene and its first oxidation product artemisinic alcohol ( Figure 1; Figure 3 ) .",
"Interestingly , amorpha-4 , 11-diene accumulated to lower levels in the lines displaying the subtle phenotype , whereas artemisinic alcohol was detected in similar amounts in all transplastomic plants .",
"Accumulation of artemisinic acid correlated with the altered phenotype in Nt-AO1-1 and Nt-AO3-1 , suggesting that a more efficient conversion of amorpha-4 , 11-diene to downstream metabolites could be the cause of the phenotype .",
"This hypothesis gained support from the analysis of a series of developmental stages and leaf ages which revealed that , while in Nt-AO2 plants , artemisinic acid accumulates only in mature leaves of young and flowering plants , it accumulates throughout development in Nt-AO3 plants .",
"These analyses also confirmed the inverse relationship between artemisinic acid and amorpha-4 , 11-diene accumulation ( Figure 4A–C ) . 10 . 7554/eLife . 13664 . 005Figure 3 . Phenotype of transplastomic tobacco plants and accumulation of artemisinic compounds .",
"( A ) Transplastomic lines Nt-AO1-1 and Nt-AO3-1 display a slightly pale and growth-delayed phenotype at the juvenile stage .",
"WAT: weeks after transfer from tissue culture to soil; scale bars: 10 cm .",
"( B ) Amorpha-4 , 11-diene is synthesized in all transplastomic lines , but accumulates to lower levels in the lines displaying an altered phenotype ( purple bars ) .",
"( C ) Artemisinic alcohol is detected in similar amounts in all transplastomic plants .",
"( D ) Accumulation of artemisinic acid correlates with the altered phenotype of Nt-AO1-1 and Nt-AO3-1 .",
"Relative accumulation of amorpha-4 , 11-diene was profiled by GC-MS analysis of volatile organic compounds ( VOCs ) .",
"Relative accumulation of the sum of free and conjugated artemisinic alcohol and artemisinic acid were determined by GC-MS analysis of the soluble metabolite fraction after saponification ( see Materials and methods; Figures 6 and 7 ) .",
"In agreement with previous reports ( van Herpen et al . , 2010 ) , these compounds were found to be present mainly as conjugates .",
"Expanding leaves of 5–6 plants per line were used for each measurement .",
"Error bars represent the SD .",
"Different letters above the bars indicate significant differences as determined by One-way ANOVA ( p<0 . 001 ) and the Holm-Sidak post-hoc test . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 00510 . 7554/eLife . 13664 . 006Figure 3—figure supplement 1 . Phenotypes of Nt-AO2-1 and Nt-AO3-1 plants throughout development . Six plants per line ( Nt-wt , Nt-AO2-1 and Nt-AO3-1 ) were grown under standard greenhouse conditions and photographs were taken of one representative plant per line at different time points: young plants ( before flowering , stage 1 ) , flowering plants ( stage",
"2 ) and old plants ( seed capsules formed , stage 3 ) .",
"Light-green leaves and slightly delayed growth of line Nt-AO3-1 are more evident at the young stage .",
"At later stages , all Nt-AO lines display a wild type-like phenotype and produce viable seeds in indistinguishable amounts .",
"y: young leaf; i: expanding ( intermediate ) leaf; m: fully expanded ( mature ) leaf . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 00610 . 7554/eLife . 13664 . 007Figure 3—figure supplement 2 . Isoprenoids levels throughout development in wild-type Nicotiana tabacum plants ( Nt-wt ) and the transplastomic lines Nt-AO2-1 and Nt-AO3-1 . Plants were grown under standard greenhouse conditions and samples were taken from young ( y ) , expanding ( i ) , and fully expanded ( m ) leaves at three developmental stages ( 1–3; cf . Figure 3—figure supplement 1 ) .",
"Metabolite levels were determined by UPLC analysis .",
"The values represent the peak height for each compound divided by 10 . 000 and normalized to the fresh weight ( FW ) , resulting in the normalized response/FW .",
"Error bars represent the SD ( n = 3 plants per line ) .",
"Different letters above the bars indicate significant differences as determined by One-way ANOVA ( p<0 . 05 ) and the Holm-Sidak post-test . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 007 To identify the molecular basis of the striking metabolic differences between the different operon constructs , a series of northern blot experiments was conducted .",
"In view of the commonalities of the Nt-AO2 and Nt-AO4 plants versus the Nt-AO1 and Nt-AO3 plants , it seemed reasonable to assume that the relative orientation of the two operons ( Figure 2A ) is causally responsible for the different visual and metabolic phenotypes .",
"When the expression of the four transgenes was assayed , a striking difference was observed in the CYP/CPR expression ratio in that high artemisinic acid accumulation correlated with a high CYP/CPR expression ratio in Nt-AO3 plants ( Figure 4D–G; Figure 4—figure supplement 1 ) .",
"In nature , CYPs are often found in excess to their CPR counterparts , with ratios of 10–100:1 or higher ( reviewed , e . g . , in Guengerich , 2002 ) .",
"Even though there is currently no consensus explanation for this observation , it is known that CPRs can activate molecular oxygen , thereby producing superoxide radicals and wasting redox capacity of the cell ( Manoj et al . , 2010 ) .",
"A high CYP/CPR ratio would prevent this CPR-mediated toxicity and result in a more efficient use of the redox power of the cell for artemisinic acid synthesis , as observed in our Nt-AO1 and Nt-AO3 transplastomic lines .",
"This explanation is also in agreement with published data on transcript accumulation and protein abundance for these two enzymes in A . annua .",
"While the levels of the CPR transcript and the CPR protein remain constant during development of the plant and in different organs , the transcript and protein levels of CYP increase in the developmental stages and organs where artemisinin synthesis is induced ( Olofsson et al . , 2011; Zeng et al . , 2008 ) .",
"Especially the final oxidation step from artemisinic aldehyde to artemisinic acid appears to require an effective monooxygenase ( Ting et al . , 2013 ) , suggesting that the higher levels of artemisinic acid in our transplastomic Nt-AO1 and Nt-AO3 lines are most likely related to their higher CYP/CPR expression ratio .",
"However , determination of the CYP and CPR protein accumulation levels ( and enzyme activities ) would be necessary to precisely assess these ratios and ultimately confirm their impact on metabolite conversion in the pathway . 10 . 7554/eLife . 13664 . 008Figure 4 . Production of artemisinic acid is maintained throughout plant development and correlates with a high CYP/CPR expression ratio . Artemisinic compounds and expression levels of the transgenes were measured in young ( stage 1 ) , flowering ( stage",
"2 ) and old plants ( stage 3; see Figure 3—figure supplement 1 ) .",
"( A ) Amorpha-4 , 11-diene accumulates to higher levels in line Nt-AO2-1 than in Nt-AO3-1 .",
"( B ) Artemisinic alcohol is present at similar levels in early ( 1 ) and late stages ( 3 ) of development in lines Nt-AO2-1 and Nt-AO3-1 , but it is slightly higher in the flowering stage ( 2 ) of line Nt-AO3-1 .",
"( C ) Artemisinic acid accumulates to high levels during all developmental stages of line Nt-AO3-1 , whereas in line Nt-AO2-1 , it is detectable only in mature leaves of young and flowering plants .",
"Relative accumulation of amorpha-4 , 11-diene and artemisinic alcohol was profiled , the tissue content of artemisinic acid was quantified using an authenticated reference standard ( n = 5–6 plants per line; Figures 6 and 7 ) .",
"The sum of free and conjugated artemisinic alcohol and artemisinic acid were determined .",
"y: young leaf; i: expanding ( intermediate ) leaf; m: fully expanded ( mature ) leaf .",
"Error bars represent the SD .",
"( D–G )",
"Northern blot analysis of the expression of the four transgenes .",
"Total RNA samples from N . tabacum wild-type ( Nt-wt ) plants and the transplastomic lines Nt-AO2-1 and Nt-AO3-1 ( at the developmental stages 1–3 ) were separated in denaturing 1 . 5% agarose gels , blotted and hybridized to strand-specific RNA probes .",
"Below each blot , the rRNA-containing region of the ethidium bromide-stained gel prior to blotting is shown as a control for RNA integrity and equal loading .",
"The Nt-wt sample corresponds to RNA extracted from a fully expanded leaf of a N . tabacum wild-type plant at developmental stage 2 .",
"The smallest labeled band in each blot corresponds to the monocistronic mRNA .",
"Larger bands represent unprocessed polycistronic precursor transcripts and read-through transcripts ( which are common in plastids; e . g . , Elghabi et al . , 2011; Lu et al . , 2013 ) .",
"CYP transcripts accumulate to higher levels in line Nt-AO3-1 , while CPR transcripts accumulate to higher levels in line Nt-AO2-1 , resulting in a higher CYP/CPR expression ratio in line Nt-AO3-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 00810 . 7554/eLife . 13664 . 009Figure 4—figure supplement 1 . Quantitation of the expression of CYP and CPR by qRT-PCR analysis . Samples from young ( y ) , expanding ( intermediate; i ) and fully expanded ( mature; m ) leaves were measured in three technical replicates for each line , in early , flowering and late developmental stages ( 1–3; cf . Figure 3—figure supplement 1 ) .",
"The wild-type sample ( wt ) corresponds to a sample from a fully expanded leaf of N . tabacum cv .",
"Petit Havana at the flowering stage .",
"The qRT-PCR data confirm the northern blot analyses that had revealed a higher CYP to CPR expression ratio in transplastomic line Nt-AO3-1 than in line Nt-AO2-1 . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 009 Having successfully implemented the canonical pathway of artemisinic acid synthesis into the chloroplast of tobacco plants , we next sought to maximize artemisinic acid production .",
"In our best-performing transplastomic plants ( Nt-AO3 ) , artemisinic acid accumulation reached a maximum of 2–4 mg/kg fresh weight ( FW ) , equivalent to approximately 20–40 mg/kg dry weight ( DW; or 0 . 002–0 . 004% DW ) , a level significantly lower than artemisinin accumulation in A . annua ( varying between 0 . 01 and 1% DW; Liu et al . , 2011; Bryant et al . , 2015 ) .",
"Recently , a number of accessory A . annua enzymes have been identified that enhance the flux through the pathway , including a cytochrome b5 ( CYB5 ) that promotes electron transfer to P450 monooxygenases ( Schenkman and Jansson , 2003 ) , a new alcohol dehydrogenase ( ADH1 ) that improves the oxidation of artemisinic alcohol to artemisinic aldehyde ( Paddon et al . , 2013 ) and an aldehyde dehydrogenase ( ALDH1 ) that catalyzes the conversion of dihydroartemisinic aldehyde into dihydroartemisinic acid and , in yeast , also enhances the conversion of artemisinic aldehyde into artemisinic acid ( Paddon et al . , 2013; Figure 1 ) .",
"We also considered two additional enzymes: The double bond reductase 2 ( DBR2 ) from A . annua introduces a branch point into the pathway by reducing artemisinic aldehyde to dihydroartemisinic aldehyde ( Zhang et al . , 2008 ) and , therefore , potentially can lead to the synthesis of artemisinin ( Figure 1 ) .",
"Finally , the 1-deoxy-D-xylulose-5-phosphate reductoisomerase ( DXR ) from the cyanobacterium Synechocystis , a key regulatory enzyme in the MEP pathway of isoprenoid biosynthesis , was selected because its expression may improve precursor availability ( Figure 1 ) .",
"Since the quantitative contributions of these enzyme activities to artemisinic acid biosynthesis are not well understood and , moreover , the optimum enzyme activities required to mediate maximum flux through the pathway are unknown , we decided to pursue a combinatorial supertransformation approach .",
"Combinatorial transformation involves the mixing of multiple single-gene transformation constructs and their biolistic co-transformation followed by large-scale screening of many transgenic lines by their metabolic ( or other ) phenotypes ( Zhu et al . , 2008; Naqvi et al . , 2009 ) .",
"Individual transgenic lines generated by this approach differ in the transgene combination they harbor as well as in transgene copy numbers and expression levels , thus facilitating selection of optimized genotypes that condition the desired metabolic output ( Naqvi et al . , 2009 ) .",
"We applied combinatorial nuclear transformation to our transplastomic Nt-AO2-1 ( high accumulation of amorpha-4 , 11-diene but low levels of artemisinic acid ) and Nt-AO3-1 ( low accumulation of amorpha-4 , 11-diene and high accumulation of artemisinic acid ) lines , assuming that artemisinic acid production can be substantially increased by identifying the optimum combination and expression levels of the additional pathway enzymes .",
"Combinatorial supertransformation of transplastomic lines encoding a canonical metabolic pathway with a plasmid cocktail containing additional and/or accessory pathway enzymes represents a new approach in synthetic biology that we refer to as COSTREL ( for COmbinatorial Supertransformation of Transplastomic REcipient Lines ) .",
"Genes for the five candidate enzymes ( CYB5 , ADH1 , ALDH1 , DBR2 , DXR ) were cloned into individual expression cassettes , the resulting plasmids were mixed and co-bombarded with a kanamycin resistance gene into the nuclear genomes of transplastomic Nt-AO2-1 and Nt-AO3-1 plants .",
"612 kanamycin-resistant shoots ( Nt-AO-CS lines ) were generated by supertransformation of the transplastomic recipient lines Nt-AO2-1 and Nt-AO3-1 .",
"After rooting in kanamycin-containing medium , 512 plantlets were transferred to soil and grown to maturity under standard greenhouse conditions .",
"At the onset of flowering , a fully expanded leaf was harvested for preliminary profiling of artemisinic acid and its precursors by GC-MS ( see Materials and Methods ) .",
"Based on growth , phenotype and fertility of the plants , 199 COSTREL lines were selected for metabolic screening of artemisinic compounds: 79 Nt-AO2-CS and 120 Nt-AO3-CS lines ( Figure 5—source data 1 ) .",
"The various lines displayed great variation with respect to the accumulation levels of the compounds assayed ( amorpha-4 , 11-diene , artemisinic alcohol , dihydroartemisinic alcohol , dihydroartemisinic acid and artemisinic acid ) .",
"Importantly , COSTREL lines could be identified that contained strongly increased levels of the drug precursor artemisinic acid ( Figure 5—source data 1 ) .",
"In combinatorial transformation , all transgenes that simultaneously enter the nucleus of the recipient cell usually integrate into the same genomic locus ( most likely into a transient DNA double-strand break ) , and therefore co-segregate into the next generation ( Naqvi et al . , 2009 ) .",
"This feature allowed us to raise a T1 generation of supertransformed lines from seeds and repeat the metabolite profiling with T1 leaf material grown under highly standardized conditions .",
"These analyses confirmed the results obtained with the T0 plants and revealed that , in the case of the Nt-AO2-CS lines , the highest increase in artemisinic acid content occurred in line 132 showing a 33-fold increase compared to its transplastomic recipient line Nt-AO2-1 , whereas among the Nt-AO3-CS lines , line 180 reached an even 77-fold increase compared to transplastomic line Nt-AO3-1 ( Figure 5—source data 1; Figure 5A ) .",
"The trait artemisinic acid content was stable across generations , and the highest producer , line Nt-AO3-CS180 , reached levels of 120 . 4 ± 42 mg per kg FW in the T1 generation . 10 . 7554/eLife . 13664 . 010Figure 5 . Isolation of combinatorially supertransformed transplastomic lines with a strong increase in artemisinic acid accumulation . Transplastomic lines Nt-AO2-1 and Nt-AO3-1 were combinatorially supertransformed with genes for additional enzymes of the pathway ( Figure",
"1 ) to facilitate large-scale screening for increased artemisinic acid production .",
"( A ) Relative artemisinic acid levels ( given in response/FW; see Materials and methods ) in the T1 generation of two combinatorially supertransformed lines obtained with transplastomic recipient line Nt-AO2-1 ( Nt-AO2-CS ) and eight lines obtained with transplastomic recipient line Nt-AO3-1 ( Nt-AO3-CS ) .",
"An up to 77-fold increase in artemisinic acid was achieved ( line Nt-AO3-CS180 ) in comparison to recipient line Nt-AO3-1 .",
"For a complete list of screened supertransformed lines , see Figure 5—source data 1 .",
"( B ) Inverse relationship between artemisinic acid accumulation and artemisinic alcohol accumulation in supertransformed lines .",
"Fully expanded leaves of 5–6 plants per line ( at the flowering stage ) were used for metabolite profiling .",
"The sum of free and conjugated artemisinic alcohol and artemisinic acid were determined .",
"( C ) qRT-PCR analysis of transgene expression suggests a predominant role of ALDH1 in boosting artemisinic acid synthesis .",
"2–3 plants per line were measured , and the expression levels were ranked after One-way ANOVA comparison ( p<0 . 05 ) .",
"Brown color indicates absence of gene expression .",
"( D ) Combinatorially supertransformed lines with a high increase in artemisinic acid ( Nt-AO2-CS132 and Nt-AO3-CS180 ) display a similar phenotype as the corresponding transplastomic recipient line .",
"WAT: weeks after transfer from tissue culture to soil; scale bars: 10 cm . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 01010 . 7554/eLife . 13664 . 011Figure 5—source data 1 . Metabolic screening ( phenotyping ) and genotyping of the T0 generation of combinatorially supertransformed Nt-AO-CS lines . The supertransformed lines Nt-AO2-CS and Nt-AO3-CS are arranged according to their artemisinic acid content , from low to high .",
"Fresh weight corrected response values ( R/FW ) for amorpha-4 , 11-diene were multiplied by 1 , 000 and expressed as Rx1000/FW .",
"Dihydroartemisinic acid was only detectable in line Nt-AO3-CS180 at a low level of 0 . 03 R/FW .",
"Asterisks mark the selected candidate lines further analyzed in the T1 generation .",
"Lines were clustered according to artemisinic acid content using hierarchical cluster analysis based on Ward’s method .",
"Cnd: cluster nd ( artemisinic acid not detected ) ; C1: cluster 1; C2: cluster 2; C3: cluster 3; C4: cluster 4; C5: cluster 5 .",
"Genes detected in the genomic PCR assays are numbered as follows: 1: dxr; 2: CYB5; 3: ADH1; 4: ALDH1; 5: DBR2; 0: no gene detected; ?",
": unclear result .",
"nd: not detected; nm: not measured; -: not determined .",
"Amorpha-4 , 11-diene values are from one measurement per line , values for artemisinic alcohol , dihydroartemisinic alcohol and artemisinic acid represent averages of three technical replicates per line .",
"SD: standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 01110 . 7554/eLife . 13664 . 012Figure 5—figure supplement 1 . Amorpha-4 , 11-diene , dihydroartemisinic alcohol and dihydroartemisinic acid accumulation in the T1 generation of combinatorially supertransformed plants . Levels of amorpha-4 , 11-diene in Nt-AO-CS lines do not correlate with the strong increase in artemisinic acid ( Figure 5 ) .",
"Dihydroartemisinic alcohol accumulates to higher levels in lines Nt-AO2-CS95 and Nt-AO3-CS145 .",
"Dihydroartemisinic acid is detectable only in two of the best-performing COSTREL lines .",
"Amorpha-4 , 11-diene levels were determined by GC-MS of VOCs .",
"Dihydroartemisinic alcohol and dihydroartemisinic acid were measured by GC-MS analysis of soluble saponified metabolites .",
"The sum of free and conjugated artemisinic compounds was determined .",
"Error bars represent the SD ( n = 5–6 plants per line ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 01210 . 7554/eLife . 13664 . 013Figure 5—figure supplement 2 . Measurements of plant height and total leaf biomass of COSTREL line Nt-AO3-CS180 ( progeny of four different T1 lines ) , its transplastomic progenitor line Nt-AO3-1 and the wild type ( wt ) .",
"( A ) Comparison of plant height at the same age .",
"Plants were measured when the wild type started to flower .",
"( B ) Comparison of plant height at the same developmental stage .",
"Transplastomic line Nt-AO3-1 and COSTREL line Nt-AO3-CS180 were measured five days later than the wild type to compensate for their slightly delayed onset of flowering .",
"( C ) Comparison of total leaf biomass ( fresh weight , FW ) at the same plant age .",
"Plants were measured when the wild type started to flower .",
"( D ) Comparison of total leaf biomass at the same developmental stage .",
"Transplastomic line Nt-AO3-1 and COSTREL line Nt-AO3-CS180 were measured five days later than the wild type to compensate for their delayed flowering .",
"Note that , at the same plant age , transplastomic line Nt-AO3-1 and COSTREL line Nt-AO3-CS180 are slightly shorter and produce less total leaf biomass than the wild type .",
"Once the transplastomic plants and the COSTREL lines flower ( same developmental stage , five days later ) , the COSTREL plants reach a height and a total leaf biomass that is close to the values measured for the wild type .",
"On average , the COSTREL plants are 7% shorter and produce 13% less leaf biomass than wild-type plants .",
"No significant difference in either height or total leaf FW was observed between COSTREL plants and their transplastomic progenitor line Nt-AO3-1 .",
"Error bars represent the SD ( n = 6 ) .",
"Different letters above the bars indicate significant differences as determined by One-way ANOVA ( p<0 . 05 ) and the Holm-Sidak post-hoc test . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 013 To obtain insights into pathway regulation and identify bottlenecks in artemisinic acid synthesis , we investigated correlations between pathway metabolites and between artemisinic acid accumulation and the set of transgenes expressed in the nucleus of Nt-AO2-CS and Nt-AO3-CS COSTREL lines .",
"Increased amounts of artemisinic acid in the Nt-AO-CS lines were negatively correlated with the accumulation of artemisinic alcohol ( Figure 5A , B ) , indicating that the efficiency of oxidation of the alcohol represents a key bottleneck in the pathway that we alleviated by supertransformation with the additional pathway genes .",
"Importantly , artemisinic alcohol was reduced to nearly undetectable levels in the best-performing line Nt-AO3-CS180 suggesting that maximum conversion efficiency has been achieved ( Figure 5B; Figure 5—source data 1 ) .",
"Another significant correlation at the metabolite level was a strong positive correlation between amorpha-4 , 11-diene and artemisinic alcohol ( Tables 1 and",
"2 ) which may be a consequence of the enzymatic limitation downstream of artemisinic alcohol .",
"By contrast , amorpha-4 , 11-diene was not significantly correlated with artemisinic acid accumulation .",
"Dihydroartemisinic alcohol ( presumably generated by an endogenous enzymatic activity in tobacco; Ting et al . , 2013; Zhang et al . , 2011 ) , while accumulating in some lines , showed only a weak positive correlation with artemisinic acid in the Nt-AO2-CS but not in the Nt-AO3-CS lines ( Figure 5—figure supplement 1; Tables 1 and 2; Figures 6 and 7 ) .",
"Dihydroartemisinic acid , the direct precursor of artemisinin , was detected only in line Nt-AO3-CS180 in the T0 generation and in lines Nt-AO3-CS53 and Nt-AO3-CS180 in the T1 generation ( Figure 5—source data 1 and Figure 5—figure supplement 1; Figure 7C ) , and therefore had to be excluded from the correlation analysis . 10 . 7554/eLife . 13664 . 014Table 1 . Correlation analysis of artemisinic compounds and transgenes introduced into transplastomic line Nt-AO2-1 by combinatorial supertransformation .",
"The levels of the artemisinic compounds amorpha-4 , 11-diene , artemisinic alcohol , dihydroartemisinic alcohol and artemisinic acid , and the presence of the transgenes dxr , CYB5 , ADH1 , ALDH1 and DBR2 were correlated using Spearman’s method in the 39 Nt-AO2-CS lines analyzed by genomic PCR in the T0 generation ( see Figure 5—source data",
"1 ) using the SPSS software .",
"Dihydroartemisinic acid was excluded from this analysis , because it was not detectable in any of the Nt-AO2-CS lines .",
"CC: correlation coefficient .",
"Positive values indicate positive correlations and negative values indicate negative correlations .",
"*: p<0 . 05; **: p<0 . 01; N: number of samples where both variables are present . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 014Nt-AO2-CSAmorpha-4 , 11-dieneArtemisinic alcoholDihydroartemi-sinic alcoholArtemisinic aciddxrCYB5ADH1ALDH1DBR2Amorpha-4 , 11-dieneCC1 . 0000 . 602**0 . 147-0 . 1120 . 067-0 . 020-0 . 009-0 . 0150 . 156N352913243535353535Art .",
"alcoholCC0 . 602**1 . 000-0 . 557*-0 . 141-0 . 159-0 . 165-0 . 1700 . 013-0 . 217N292913242929292929Dihydroart .",
"alcoholCC0 . 147-0 . 557*1 . 0000 . 642*0 . 4620 . 2480 . 3830 . 1800 . 496N131313131313131313Art .",
"acidCC-0 . 112-0 . 1410 . 642*1 . 0000 . 4040 . 1590 . 1790 . 2070 . 317N242413242424242424dxrCC0 . 067-0 . 1590 . 4620 . 4041 . 0000 . 643**0 . 402*0 . 546**0 . 578**N352913243939393939CYB5CC-0 . 020-0 . 1650 . 2480 . 1590 . 643**1 . 0000 . 1920 . 507**0 . 793**N352913243939393939ADH1CC-0 . 009-0 . 1700 . 3830 . 1790 . 402*0 . 1921 . 0000 . 372*0 . 270N352913243939393939ALDH1CC-0 . 0150 . 0130 . 1800 . 2070 . 546**0 . 507**0 . 372*1 . 0000 . 420**N352913243939393939DBR2CC0 . 156-0 . 2170 . 4960 . 3170 . 578**0 . 793**0 . 2700 . 420**1 . 000N35291324393939393910 . 7554/eLife . 13664 . 015Table 2 . Correlation analysis of artemisinic compounds and transgenes introduced into transplastomic line Nt-AO3-1 by combinatorial supertransformation .",
"The levels of the artemisinic compounds amorpha-4 , 11-diene , artemisinic alcohol , dihydroartemisinic alcohol and artemisinic acid , and the presence of the transgenes dxr , CYB5 , ADH1 , ALDH1 and DBR2 were correlated using Spearman’s method in the 61 Nt-AO3-CS lines analyzed by genomic PCR in the T0 generation ( see Figure 5—source data",
"1 ) using the SPSS software .",
"Dihydroartemisinic acid had to be excluded from this analysis , because it was detectable only in one of the Nt-AO3-CS lines in the T0 generation .",
"Note that the negative correlation between artemisinic alcohol and artemisinic acid ( Figure 5A , B ) is restricted to those lines that display increased artemisinic acid contents , and therefore is not statistically significant over all COSTREL lines analyzed ( cf . Figure 5—source data 1 ) .",
"CC: correlation coefficient .",
"Positive values indicate positive correlations and negative values indicate negative correlations .",
"*: p<0 . 05; **: p<0 . 01; N: number of samples where both variables are present . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 015Nt-AO3-CSAmorpha-4 , 11-dieneArtemisinic alcoholDihydroartemi-sinic alcoholArtemisinic aciddxrCYB5ADH1ALDH1DBR2Amorpha-4 , 11-dieneCC1 . 0000 . 363**0 . 0790 . 2370 . 0060 . 0820 . 439**-0 . 0320 . 049N605839596060606060Art .",
"alcoholCC0 . 363**1 . 0000 . 124-0 . 081-0 . 088-0 . 1190 . 0860 . 026-0 . 009N585939595959595959Dihydroart .",
"alcoholCC0 . 0790 . 1241 . 0000 . 029-0 . 1690 . 1530 . 3060 . 1950 . 219N393939393939393939Art .",
"acidCC0 . 237-0 . 0810 . 0291 . 0000 . 2410 . 1830 . 2170 . 386**0 . 141N595939606060606060dxrCC0 . 006-0 . 088-0 . 1690 . 2411 . 0000 . 1530 . 2240 . 415**0 . 170N605939606161616161CYB5CC0 . 082-0 . 1190 . 1530 . 1830 . 1531 . 0000 . 1850 . 322*0 . 503**N605939606161616161ADH1CC0 . 439**0 . 0860 . 3060 . 2170 . 2240 . 1851 . 0000 . 339**0 . 252*N605939606161616161ALDH1CC-0 . 0320 . 0260 . 1950 . 386**0 . 415**0 . 322*0 . 339**1 . 0000 . 444**N605939606161616161DBR2CC0 . 049-0 . 0090 . 2190 . 1410 . 1700 . 503**0 . 252*0 . 444**1 . 000N60593960616161616110 . 7554/eLife . 13664 . 016Figure 6 . Chromatograms and mass spectra of amorpha-4 , 11-diene and artemisinic alcohol . Characteristic peaks for one specific fragment at the expected retention time or index are displayed for each compound .",
"( A ) Amorpha-4 , 11-diene-specific mass feature 119 at a retention time of 1564 s .",
"This metabolite is present in all Nt-AO lines , and at slightly higher levels in lines Nt-AO2-1 and Nt-AO4-1 .",
"( B ) Artemisinic alcohol-specific mass feature 202 at a retention index of 1784 .",
"The compound is present at similar levels in all Nt-AO lines .",
"Both compounds are absent from the wild-type sample .",
"In addition to the chromatograms , the characteristic mass spectrum ( m/z ) of each compound is shown for the standard and for one of the artemisinic acid operon lines .",
"EPY224: yeast strain that produces amorpha-4 , 11-diene ( Ro et al . , 2006 ) .",
"One representative plant per line is depicted .",
"Mass spectra and mass features of trimethylsilylated artemisinic alcohol are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 01610 . 7554/eLife . 13664 . 017Figure 7 . Chromatograms and mass spectra of the artemisinic compounds artemisinic acid , dihydroartemisinic alcohol and dihydroartemisinic acid . Characteristic peaks for one specific fragment at the expected retention index are shown for each compound .",
"( A ) Artemisinic acid-specific mass feature 216 is shown at a retention index of 1850 .",
"This compound accumulates to higher levels in lines Nt-AO1-1 and Nt-AO3-1 .",
"( B ) Dihydroartemisinic alcohol-specific mass feature 162 at a retention index of 1789 .",
"The compound is present at high levels in COSTREL line Nt-AO2-CS95 , but is absent from transplastomic line Nt-AO2-1 .",
"( C ) Dihydroartemisinic acid-specific mass feature 163 at a retention index of 1859 .",
"This compound accumulates in COSTREL line Nt-AO3-CS180 , but is absent from transplastomic line Nt-AO3-1 .",
"All compounds are absent from the wild-type sample .",
"In addition to the chromatograms , the characteristic mass spectrum of each compound is shown for the standard and for one of the artemisinic acid operon lines .",
"Mass spectra and mass features of trimethylsilylated artemisinic compounds are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 017 To correlate the genotype of the COSTREL lines with their metabolic phenotypes , the transgene sets present in the nucleus of Nt-AO2-CS and Nt-AO3-CS lines were determined ( Figure 5—source data 1 ) .",
"Statistical analysis revealed that elevated artemisinic acid levels in Nt-AO3-CS lines were most strongly correlated with the presence of the ALDH1 transgene .",
"Weaker ( and statistically not significant ) correlations were observed between high artemisinic acid levels and the presence of dxr and ADH1 , and , in Nt-AO2-CS lines , also the DBR2 and ALDH1 transgenes ( Tables 1 and 2 ) .",
"These results indicate that ALDH1 and dxr are most likely the genes with the greatest impact on the increase in artemisinic acid content .",
"As transgene presence is not necessarily indicative of transgene expression , we measured mRNA accumulation in the T1 generation of a selected set of COSTREL lines by qRT-PCR analyses .",
"The results support the importance of dxr , ADH1 , ALDH1 and DBR2 in boosting artemisinic acid synthesis and revealed that the best-performing line ( Nt-AO3-CS180 ) expresses dxr , ADH1 , ALDH1 and DBR2 to high levels ( Figure 5C ) .",
"To test whether artemisinic acid accumulation is correlated with a visible phenotype of the plants , the best-performing COSTREL lines were compared with their transplastomic recipient lines .",
"No significant phenotypic differences were observed and even line Nt-AO3-CS180 ( that showed the strongest increase in artemisinic acid accumulation; Figure 5A ) was nearly indistinguishable from its transplastomic recipient Nt-AO3-1 ( Figure 5D ) .",
"This suggests that artemisinic acid is not toxic to plant cells ( and that a further increase in artemisinic acid might be achievable ) .",
"Growth and biomass measurements confirmed that there are no significant differences between transplastomic line Nt-AO3-1 and the best-performing COSTREL line Nt-AO3-CS180 , and revealed only a small reduction in total leaf biomass ( by on average 13% ) of the COSTREL line compared to the wild type ( Figure 5—figure supplement 2 ) .",
"None of our transplastomic lines and none of the analyzed COSTREL lines accumulated detectable levels of artemisinin ( see Materials and Methods ) .",
"This could be because the set of transgenes introduced into tobacco was insufficient to obtain conversion of artemisinic acid into artemisinin .",
"However , some of our best-performing COSTREL lines accumulated detectable amounts of dihydroartemisinic acid ( Figure 5—source data 1 and Figure 5—figure supplement 1; Figure 7 ) , the immediate precursor of artemisinin , indicating that DBR2 and ALDH1 can function in plastids .",
"The chloroplast is likely to produce sufficient amounts of singlet oxygen ( which is a regular by-product of photosynthetic electron transfer ) to facilitate the spontaneous conversion of dihydroartemisinic acid into artemisinin ( Kopetzki et al . , 2013; Figure 1 ) .",
"An alternative explanation for the lack of artemisinin accumulation could be that COSTREL lines that produce artemisinin were not recovered , because artemisinin is highly toxic to photosynthetically active cells ( Bharati et al . , 2012 ) .",
"The fact that no artemisinin could be detected and only low amounts of dihydroartemisinic acid were obtained in a few lines , whereas artemisinic acid accumulated to high amounts , may indicate that future efforts should be focused on maximizing the production of artemisinic acid ."
],
[
"In the course of this work , we have developed a new synthetic biology approach that combines chloroplast transformation with combinatorial nuclear transformation and large-scale metabolic screening of supertransformed plant lines .",
"This strategy enabled the transfer of an entire biochemical pathway of secondary metabolism from a medicinal plant to a high-biomass crop .",
"For the foreseeable future , ACTs will remain the most powerful weapon in the world’s battle against malaria ( http://www . who . int/malaria/areas/treatment/overview/en/ ) .",
"When used as an oral monotherapy , artemisinin can promote the development of resistance in the parasite ( Mok et al . , 2015; Straimer et al . , 2015; Mbengue et al . , 2015 ) and , therefore , ACTs are based on fixed-dose co-formulations that combine two different active ingredients in one tablet .",
"Development of an inexpensive and sustainable production method that is suitable to meet the constantly growing demand for artemisinin and its derivatives has remained a grand challenge .",
"Enormous breeding efforts are currently underway to produce new varieties of A . annua that accumulate higher and more consistent levels of the compound ( Graham et al . , 2010 ) .",
"However , as A . annua produces artemisinin only in a very small fraction of the leaf cells ( the glandular trichomes ) and its cultivation is inefficient , slow and vulnerable to adverse environmental conditions , the development of a production method that is independent of A . annua is highly desirable ( Bryant et al . , 2015 ) .",
"If accomplished in a high-biomass non-food/non-feed crop , this would provide a stable supply of the feedstock that can be scaled up at will and at short notice , and take full advantage of the existing agricultural infrastructure .",
"In the course of this work , we have established tobacco as an efficient production factory for artemisinic acid .",
"Tobacco is a high-biomass crop , grown in large acreages , for which alternative uses ( that are unrelated to smoking ) have long been sought .",
"Since tobacco is well suited for cultivation at high cropping densities and multiple harvests ( 4–5 ) per season are possible , 40 t of biomass can be obtained from a single acre of tobacco field at a cost of only around $100 per ton ( http://tobacco . ces . ncsu . edu/wp-content/uploads/2012/07/tobacco-production-cost-2011-1 . pdf ? fwd=no ) .",
"Thus , with our best-performing COSTREL line , production levels of ~4 . 8 kg artemisinic acid per acre can be obtained , suggesting that the current world demand ( of ~100 t artemisinin ) can be met by cultivating tobacco on an area of ~200 km2 , which is less than the area of the city of Boston ( assuming ~50% loss during extraction and conversion of artemisinic acid to artemisinin; Paddon et al . , 2013; Kopetzki et al . , 2013 ) .",
"Whereas in A . annua the artemisinin biosynthetic pathway is confined to glandular trichomes , our COSTREL tobacco lines produce artemisinic acid in chloroplasts and , thus , in the whole leaf .",
"Together with the absence of toxic effects of artemisinic acid on the chloroplast ( Figure 5D; Figure 5—figure supplement 2 ) , this offers great potential for further enhancement of the pathway by addressing the bottlenecks that limit flux in our current best-performing lines .",
"Furthermore , previous transgenic work has shown that the redox environment in the cytosol of tobacco cells favors reduction of aldehydes to alcohols rather than their oxidation to acids , thus limiting the ability of the cytosolically located pathway to produce high quantities of artemisinic acid ( Zhang et al . , 2011 ) .",
"The high levels of artemisinic acid achieved in this work by implementing the pathway into plastids suggest that the chloroplast offers a more favorable redox milieu that allows the quantitative conversion of artemisinic alcohol into artemisinic acid ( Figure 5A , B ) .",
"Although tobacco leaves also possess glandular trichomes ( where artemisinin is produced in A . annua ) , the trichomes in our COSTREL plants are unlikely to accumulate large amounts of artemisinic acid .",
"This is because transgene expression from the plastid genome is generally very low in non-photosynthetic tissues and cell types .",
"It can be significantly enhanced by designing specific ( chimeric ) expression signals that confer high transgene activity in non-green tissues ( Zhang et al . , 2012; Caroca et al . , 2013 ) , but the expression signals used to drive our synthetic artemisinic acid operons ( Figure 2 ) are not suitable to trigger efficient gene expression in non-photosynthetic plastids .",
"The chloroplast represents an attractive site for engineering new metabolic pathways into plants .",
"Being the biosynthetic center of the plant cell , the chloroplast contains large pools of diverse metabolites that can be tapped .",
"Expression of genes for metabolic enzymes from the plastid genome has a number of attractions , including high expression levels , simple stacking of multiple transgenes in synthetic operons ( Lu et al . , 2013; Gnanasekaran et al . , 2016 ) and high-precision engineering via homologous recombination ( Maliga , 2004; Bock , 2015 ) .",
"Previously , plastid transformation was employed to enhance endogenous metabolic pathways ( Apel et al . , 2009; Lu et al . , 2013 ) or to produce novel metabolites , such as ketocarotenoids and biopolymers ( Hasunuma et al . , 2008; Bohmert-Tatarev et al . , 2011 ) .",
"Recently , two ER-resident cytochrome P450 enzymes of the dhurrin pathway ( a cyanogenic glucoside from sorghum ) were successfully expressed from a synthetic operon in tobacco chloroplasts ( Gnanasekaran et al . , 2016 ) .",
"Together with the third pathway enzyme , a glucosyltransferse , the two P450 enzymes catalyzed the formation of dhurrin from tyrosine .",
"The activity of the P450 enzymes was strictly light-dependent , indicating that the electrons used come from the photosynthetic electron transport chain ( Gnanasekaran et al . , 2016 ) .",
"This suggests that , at least when P450 enzymes are anchored to the thylakoid membrane , reduced ferredoxin can replace the NADPH-dependent native reductase ( Gnanasekaran et al . , 2016 ) , thus making the chloroplast a superb compartment for the implementation of secondary metabolic pathways that involve P450-catalyzed reactions .",
"By transplastomic introduction of the core pathway for artemisinic acid synthesis , our COSTREL approach takes advantage of the stability and high efficiency of transgene expression from the plastid genome ( Maliga , 2004; Bock , 2015 ) .",
"Subsequent combinatorial supertransformation of the nuclear genome with genes for auxiliary and regulatory factors then allows fine-tuning of the pathway and optimization of metabolic flux by screening metabolic phenotypes of hundreds of transgenic lines that differ in the set of transgenes they harbor in the genome and the expression levels of the transgenes ( Zhu et al . , 2008; Naqvi et al . , 2009; 2010 ) .",
"Importantly , this approach requires no prior knowledge about the contributions of the individual factors to metabolic flux and the optimum expression strength of each transgene .",
"Previous metabolic engineering work in microorganisms has demonstrated that the success is often more dependent on achieving the optimum balance of enzyme activities than on the absolute levels of enzyme ( over ) expression ( e . g . , Peralta-Yahya et al . , 2012 ) .",
"The use of combinatorial supertransformation , therefore , provides a significant advantage over the construction of large transformation vectors expressing multiple pathway genes , because the great variation between transgenic events in",
"( i ) the transgene combination present ,",
"( ii ) the copy numbers of the individual transgenes and",
"( iii ) the absolute and relative expression strengths of the transgenes ( depending , e . g . , on the integration site in the genome and the structure of the transgenic locus ) is likely to yield at least some events that harbor the optimum combination of transgenes and provide the right balance of enzyme activities .",
"Moreover , the characterization of these elite events can provide valuable information about pathway regulation , limiting steps and bottlenecks that should be the target of future engineering and optimization efforts .",
"In sum , our COSTREL strategy provides a new synthetic biology tool that facilitates the efficient transfer of complex metabolic pathways into new host organisms while , at the same time , maximizing the metabolic output ."
],
[
"Tobacco plants ( Nicotiana tabacum cv . Petit Havana ) were grown under sterile conditions on agar-solidified MS medium ( Murashige and Skoog , 1962 ) supplemented with 30 g/L sucrose .",
"Genetically modified plants were selected , propagated and rooted in the same medium containing additionally 500 mg/L spectinomycin ( transplastomic plants ) or 50 mg/L kanamycin ( combinatorially supertransformed plants ) .",
"For sampling and seed production , plants were transferred to soil and grown under standard greenhouse conditions .",
"The synthetic operon constructs for chloroplast transformation ( pAO1-4 ) are based on plastid transformation vector pKP9 ( Zhou et al . , 2008 ) .",
"They all contain the four genes required for the canonical artemisinic acid biosynthetic pathway in Artemisia annua: FPS ( AF112881 ) , ADS ( AF138959 ) , CYP71AV1 ( CYP , DQ268763 ) and CPR ( DQ318192; Figure 1 ) .",
"The genes were codon optimized for expression in the chloroplast and chemically synthesized ( GeneArt , Regensburg , Germany ) .",
"The four genes were then assembled into synthetic operons as follows .",
"The CYP71AV1 ( CYP ) gene was synthesized with a Shine-Dalgarno ( SD ) sequence derived from the chloroplast rbcL gene and with the flanking restriction sites NheI ( at the 5’ end ) and XbaI ( at the 3’ end ) .",
"The gene was cloned into pZF1 replacing the P24 gene ( Zhou et al . , 2008 ) and generating construct pZF83 .",
"pZF1 is an intermediate cloning construct that contains the promoter from the rRNA operon from tobacco ( Prrn ) , the leader sequence from the gene 10 of bacteriophage T7 ( T7 Lg10 ) , the P24 capsid protein gene of HIV-1 and the terminator of the chloroplast rbcL gene ( TrbcL; Zhou et al . , 2008 ) .",
"A fragment containing the rRNA operon promoter from Chlamydomonas reinhardtii ( Cr Prrn ) , the T7 Lg10 , the gfp gene , the terminator of the atpA gene from the chloroplast genome of C . reinhardtii ( Cr TatpA ) and the intercistronic expression element ( IEE; Zhou et al . , 2007 ) was excised with SacI and NheI from a modified version of construct pDK139 in which the ClaI , SalI and XhoI restriction sites between Cr TatpA and IEE were removed by XhoI/HindIII digestion and blunting of the overhanging ends by a fill-in reaction with Klenow enzyme .",
"pDK139 is a chloroplast transformation construct based on vector pHK20 ( Kuroda and Maliga , 2001 ) .",
"The excised fragment was cloned into pZF83 , replacing the region spanning Prrn and T7 Lg10 and generating construct pZF84 .",
"Next , the FPS gene was synthesized flanked by NdeI and PacI restriction sites at the 5’ and 3’ ends , respectively .",
"The excised NdeI/PacI restriction fragment was cloned into the identically digested pZF84 , replacing the gfp gene and giving rise to plasmid pZF85 .",
"The complete fragment from Cr Prrn to TrbcL was then cut out from pZF85 with SacI and ClaI and ligated into chloroplast transformation vector pKP9 ( Zhou et al . , 2008 ) , producing clone pZF90 .",
"The ADS gene was synthesized ( flanked by NcoI and EcoRV restriction sites ) and cloned into vector pKCZaphA-6 , replacing the aphA-6 gene and giving rise to plasmid pZF86 .",
"pKCZaphA-6 ( Fleischmann et al . , 2011 ) is an intermediate cloning construct that contains the C . reinhardtii psbA promoter ( Cr PpsbA ) , the C . reinhardtii psbA leader ( Cr LpsbA ) , the aphA-6 gene for kanamycin resistance and the C . reinhardtii rbcL terminator ( Cr TrbcL ) .",
"Next , the terminator of the tobacco rps16 gene ( Trps16 ) was amplified by PCR with primers containing EcoRV and PstI restriction sites at the 5’ and 3’ ends , respectively , and cloned into pZF86 digested with the same enzymes , generating vector pZF87 .",
"The CPR gene was synthesized as a PstI/SphI restriction fragment with the rbcL SD sequence and an IEE element at its 5’ end .",
"The fragment was cloned into pZF87 digested with the same enzymes , giving rise to pZF88 .",
"Artemisinic acid operon constructs pAO1 and pAO2 were generated by digesting pZF88 with ClaI ( releasing the cassette containing the ADS-CPR dicistron between Cr PpsbA and Cr TrbcL ) and cloning this cassette into pZF90 digested with the same enzyme .",
"In vector pAO1 , the ADS-CPR cassette is integrated in sense orientation , downstream of the FPS-CYP cassette , whereas in construct pAO2 , the fragment is integrated in antisense ( Figure 2A ) .",
"For generation of pAO3 and pAO4 , the Cr PpsbA - Cr LpsbA fragment was eliminated from pZF88 by digestion with MluI and NcoI and subsequently replaced by a PCR-amplified Cr PpsbA - T7 Lg10 fragment obtained by digestion with the same enzymes , thus generating plasmid clone pZF89 .",
"pZF89 was then digested with ClaI and cloned into pZF90 in a similar way as for generation of pAO1 and pAO2 .",
"Construct pAO3 originates from integration of the ADS-CPR cassette in sense orientation , whereas pAO4 harbors the cassette in antisense orientation ( Figure 2A ) .",
"Constructs pCS1-5 for combinatorial supertransformation contain the genes dxr ( BA000022 ) from Synechocystis sp .",
"and CYB5 ( JQ582841 . 1 ) , ADH1 ( JF910157 . 1 ) , ALDH1 ( FJ809784 . 1 ) and DBR2 ( EU704257 . 1 ) from A . annua .",
"The genes were codon optimized for expression in the nucleus and synthesized ( Eurofins MWG Operon ) .",
"The five constructs are derivatives of pUC18 and contain the terminator from the nopaline synthase gene ( Tnos ) , the transit peptide from RBCS and either the 35S promoter from the cauliflower mosaic virus ( CaMV ) , the mannopine synthase gene promoter from Agrobacterium tumefaciens ( Pmas ) or the ubiquitin-10 promoter from Arabidopsis thaliana ( PUBIQ10 ) .",
"To generate these constructs , the RBCS transit peptide ( TP ) was amplified by PCR with primers introducing XbaI/XhoI , ApaI/XhoI or SpeI/XhoI restriction sites into the 5’ and 3’ ends of the amplification product , respectively .",
"The TP was then digested with the corresponding restriction enzymes and cloned into a P35S-Tnos cassette ( opened with XbaI/XhoI ) , a Pmas-Tnos cassette ( opened with ApaI/XhoI ) and a PUBIQ10-Tnos cassette ( opened with SpeI/XhoI ) , producing constructs pPF28 , pPF29 and pPF30 , respectively .",
"Constructs pCS1 and pCS2 are derivatives of pPF28 and were generated by cloning the synthetic genes dxr and CYB5 into pPF28 as XhoI/SacI fragments .",
"Constructs pCS4 and pCS5 are derivatives of pPF29 and were obtained by cloning the synthetic genes ALDH1 and DBR2 into pPF29 as XhoI/SacI fragments .",
"Finally , construct pCS3 was obtained in a similar way , by cloning the synthetic gene ADH1 into pPF30 as an XhoI/XmaI restriction fragment .",
"The plasmid cocktail for combinatorial transformation was produced by mixing equal quantities of constructs pCS1-5 ( each at a concentration of 2 µg/µL ) and plasmid pPH200 that contains the nptII gene for kanamycin resistance between the CaMV 35S promoter and terminator .",
"For chloroplast transformation , young leaves harvested from aseptically grown wild-type tobacco plants were bombarded with gold particles covered with plasmid-DNA ( pAO1-4 ) using the DuPont PDS1000He biolistic gun .",
"Spectinomycin-resistant shoots were selected on plant regeneration medium with 500 mg/L spectinomycin ( Svab and Maliga , 1993 ) .",
"Primary transformants were identified by Southern blot analysis and at least one additional regeneration round was performed to obtain homoplasmic plants .",
"Independently generated transplastomic lines are designated by the construct number followed by the number of the individual line ( e . g . , Nt-AO1-2 stands for Nicotiana tabacum plant obtained with construct pAO1 , transplastomic line number 2 ) .",
"Homoplasmy was confirmed by Southern blot analyses and seed assays .",
"Young leaves from transplastomic plants Nt-AO2-1 and Nt-AO3-1 grown under aseptic conditions were harvested and bombarded with gold particles coated with a plasmid DNA mixture containing pCS1-5 and pPH200 using the DuPont PDS1000He biolistic gun .",
"Kanamycin-resistant shoots were selected on plant regeneration medium containing 50 mg/L kanamycin .",
"Resistant shoots were rooted in the same medium , then transferred to soil and grown to maturity under standard greenhouse conditions .",
"Material from T0 plants was used for initial molecular analyses and preliminary metabolite profiling experiments .",
"To generate standardized material for metabolite measurements and molecular analysis of the T1 generation , seeds from candidate supertransformed lines were surface-sterilized and sown on MS medium with 200 mg/L kanamycin .",
"After three weeks , six green ( resistant ) seedlings per line were transferred to soil and raised under standard greenhouse conditions .",
"Plant height and total leaf biomass were determined for six plants each of N . tabacum wild type ( wt ) , the transplastomic line Nt-AO3-1 and the progeny of four Nt-AO3-CS180 T1 lines .",
"Measurements were performed at two different stages .",
"The first measurement was done when the wild-type plants started to flower ( 'same age' ) .",
"The second measurement was done when the Nt-AO3-1 and Nt-AO3-CS180 plants started to flower ( typically five days after the first measurement ) , to compensate for the slightly delayed development of the transplastomic plants and the COSTREL plants .",
"The height was measured from the top of the pot to the top of the inflorescence .",
"The total leaf biomass ( fresh weight , FW ) was determined by weighing all leaves of an individual plant .",
"Total plant DNA was extracted from frozen leaf material by a CTAB-based protocol ( Doyle and Doyle , 1990 ) .",
"For total RNA extraction , samples of 300–400 mg of frozen powdered plant material were extracted with the peqGOLD Trifast reagent ( Peqlab GmbH , Erlangen , Germany ) , following the manufacturer’s instructions .",
"The RNA pellet was resuspended in 100 µL of RNase-free water and mixed with 250 µL buffer RA1 from the NucleoSpin RNA Plant kit ( Macherey-Nagel , Düren , Germany ) .",
"350 µL of 70% EtOH were mixed with the RNA solution , passed through the RNA-binding column and purified following the protocol of the supplier .",
"Finally , the RNA was eluted in 45 µL of RNase-free water and stored at -80°C until use .",
"Prior to reverse transcription , isolated RNAs were tested for the presence of contaminating DNA by a standard PCR using 1 ng of RNA as template .",
"If no DNA amplification was observed , cDNA was synthesized as follows .",
"1 . 5 µg of RNA were incubated with 1 µL of oligo ( dT ) primer ( 10 µM ) and 1 µL of dNTPs ( 10 mM ) for 5 min at 65°C .",
"Then , 7 µL of a master mix were added ( 4 µL of 5x First Strand buffer , 1 µL 0 . 1 M DTT , 40 U RNaseOUT and 200 U SuperScript III Reverse Transcriptase; Invitrogen , Carlsbad , CA ) and incubated for 1 hr at 50°C , followed by an inactivation step of 15 min at 70°C .",
"Alternatively , cDNA was synthesized using the QuantiTect Reverse Transcription kit ( Qiagen , Hilden , Germany ) following the manufacturer's instructions .",
"The quality of the cDNA was tested by standard PCR .",
"Quantitative RT-PCR was performed in a LightCycler 480 ( Roche , Mannheim , Germany ) using cDNA as template in 5 µL reactions containing 1 µL of each gene-specific primer ( 1 . 25 µM; Table 3 ) , 2 . 5 µL of the LightCycler 480 SYBR green I Master mix and 0 . 5 µL of a 1:50 cDNA dilution .",
"Three biological ( independent plants ) and three technical replicates per line were analyzed .",
"The relative transcript levels were determined using the formula ( 1+E ) -ΔΔCt where E is the binding efficiency of the primers ( Pfaffl , 2001 ) .",
"E was calculated from the slope of the expression level of each gene in a dilution series of a given cDNA .",
"Results were normalized to the mRNA levels of ACTIN as a housekeeping gene ( Table 3 ) , and relative mRNA accumulation levels were calculated according to the delta-delta Ct method .",
"To identify the key genes involved in the increased levels of artemisinic acid in supertransformed plants , the expression levels of each transgene ( in all lines were it was present ) were compared by One-way ANOVA analysis ( p<0 . 05 ) .",
"The results were expressed as a heat map , where the darkest green color represents the highest expression level ( brown: no expression ) . 10 . 7554/eLife . 13664 . 018Table 3 . List of oligonucleotides used in this study .",
"The reverse primers ( _R ) for amplification of the genes FPS , ADS , CYP and CPR contain the sequence of the T7 promoter ( bold ) to facilitate in vitro transcription . DOI: http://dx . doi . org/10 . 7554/eLife . 13664 . 018GenePrimerSequence ( 5'→3' ) PurposedxrDXR_FCAACCTATGTACGTTGTTGGAGAAGAGGGqPCRDXR_RCTGGAGCACCAGCAATCAATGTCTCCYB5CYB5_FCCAGGAGGAGATGAAGTTCTTTTGGCTGqPCRCYB5_RGCTGGAGGAACGTAAGCTCTCTTCTTTGADH1qADH1_F2TCCAGGTCATGAAGGTGTTGqPCRqADH1_R2ATTGTCCACACTCACCAAGGALDH1ALDH1_FCCTGTTTCTTTGGAATTGGGTGGTAAGTCqPCRALDH1_RCAGCAACACACATCTCACCTTTGTTAGTGDBR2DBR2_FGAGCAAGTTGAGGGTTGGAAGAAAGTTGqPCRDBR2_RTAGAAGAGATAGGAGCAGCTCCACCTGCYPCYP_qFCCTGAACCTTGGAGATTACCqPCRCYP_qRGCCCATTTAGGAGAAGATACAACCPRCPR_qFCCTGTTGGAATGGGTGATGqPCRCPR_qRCCTACAGCAGCAGTATAAGGAGACTINqTac9actin fCCTGAGGTCCTTTTCCAACCAqPCRqTac9actin rGGATTCCGGCAGCTTCCATTFPSFPS_probe_FCCTGCTTTTGAATTTGATGATGRNA probeFPS_probe_RTAATACGACTCACTATAGGGCGAAACCAACAAGGTTGTCCADSADS_probe_FCTGAAGCTGTTGAAAGATGGTCRNA probeADS_probe_RTAATACGACTCACTATAGGGGGAGCAGATACAGCCCATTCCYPCYP_probe_FTCCTCATCGAGGAGTACGAGRNA probeCYP_probe_RTAATACGACTCACTATAGGGTTACAGGTCGTCCAGATCCAGCPRCPR_probe_FATGATTGGTCCTGGAACTGGRNA probeCPR_probe_RTAATACGACTCACTATAGGGGCCATTCCTTTAGCATCTCC For Southern blot analysis , samples of 2–3 µg DNA were digested with BamHI , separated by electrophoresis in 0 . 8% agarose gels and transferred onto Hybond XL nylon membranes ( GE Healthcare , Little Chalfont , UK ) by capillary blotting .",
"For northern blot analysis , samples of 4–5 µg total RNA were separated in denaturing formaldehyde-containing agarose gels ( 1 . 5% ) and transferred onto nylon membranes .",
"As RFLP probe , a 550-bp fragment of the psaB gene was amplified by PCR using primer pair P7247 / P7244 ( Wurbs et al . , 2007 ) and purified .",
"The probe was labeled with [α32P]dCTP by random priming ( Multiprime DNA labeling kit; GE Healthcare ) .",
"Probes for FPS , ADS , CYP and CPR were generated by in vitro transcription and radioactive labeling with [α32P]UTP .",
"PCR fragments of 200–300 bp were amplified for each gene using specific primers ( Table 3 ) that contain the T7 promoter sequence in the reverse primer .",
"Radiolabeled probes were generated by incubating 5 µL PCR product with 4 µL H2O , 2 µL 10x buffer , 3 µL of an equimolar mixture of ATP , CTP and GTP , 2 µL T7 RNA polymerase ( 15 U/µL ) and 4 µL [α32P]UTP ( 40 µCi ) for 30 min at 37°C .",
"Hybridizations were performed overnight at 65°C .",
"Following standard washing steps , autoradiographic screens were exposed to the membranes for 3–4 hr and then scanned in a Typhoon TRIO+ scanner ( GE Healthcare ) .",
"The following genetically engineered strains of Saccharomyces cerevisiae were used as reference strains for artemisinic metabolites ( Ro et al . , 2008 ) :",
"( i ) EPY300 ( MATαα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 PGAL1-tHMGR PGAL1-upc2-1 erg9::PMET3-ERG9 PGAL1-tHMGR PGAL1-ERG20; Ro et al . , 2008 ) , a control strain that does not produce artemisinic compounds ,",
"( ii ) EPY224 ( EPY300 transformed with plasmid pRS425-Leu::ADS; Ro et al . , 2006 ) , a strain that produces amorpha-4 , 11-diene , and",
"( iii ) EPY302 ( EPY300 transformed with plasmids pRS425-Leu::ADS and pESC-Ura::AMO/CPR; Ro et al . , 2006 ) , a strain that produces artemisinic acid and artemisinic acid pathway intermediates .",
"Plasmid pRS425-Leu::ADS complements the leucine auxotrophy and plasmid pESC-Ura::AMO/CPR complements the uracil auxotrophy of strain EPY300 .",
"The three yeast strains were kindly provided by Dr . Jay D . Keasling ( UC Berkley , USA ) .",
"Yeast strains were maintained on solid synthetically defined ( SD ) medium supplemented with 2% ( w/v ) sucrose and 0 . 002% ( w/v ) uracil for EPY224 , and additionally 0 . 01% ( w/v ) leucine for EPY300 .",
"Induction of the synthesis of artemisinic compounds in strains EPY224 and EPY302 was done by adding 1 . 8% ( w/v ) galactose and 1 mM methionine to liquid SD medium and reducing the sucrose content to 0 . 2% ( w/v ) .",
"Yeast strains were incubated for 120 hr at 30°C and 160 rpm , until they reached an OD600 of 1 . 4 .",
"Control strain EPY300 was incubated under the same conditions in liquid SD medium supplemented with 2% ( w/v ) sucrose , 0 . 002% ( w/v ) uracil and 0 . 01% ( w/v ) leucine .",
"For GC-MS profiling of volatile organic compounds ( VOCs ) , leaves of N . tabacum plants were collected , immediately frozen in liquid nitrogen and processed in a cryogenic grinding robot ( Labman , North Yorkshire , UK ) .",
"Aliquots of 500 ± 10 mg of frozen powdered leaf tissue were weighed in frozen microcentrifuge tubes , and then transferred to frozen 20 mL head-space screw cap vials .",
"The powdered plant material was kept at 15°C in the closed vials for at least 1 hr and then incubated for 10 min at 50°C prior to VOC analysis .",
"VOCs were sampled in a replicated randomized block sequence design by solid phase micro extraction ( SPME ) using a StableFlex™ SPME fiber with 65 μm polydimethylsiloxane/divinylbenzene coating ( Supelco , Bellefonte , USA ) , and profiled as described previously ( Agudelo-Romero et al . , 2015; 2013 ) using a DB-624 capillary column of 60 m length , 0 . 25 mm internal diameter and 1 . 40 µm film thickness ( Agilent Technologies Deutschland GmbH , Waldbronn , Germany ) .",
"VOCs were analyzed by gas chromatography coupled to electron impact ionization/quadrupole mass spectrometry ( GC-EI/QUAD-MS ) using an Agilent 6890N24 gas chromatograph connected to an Agilent 5975B VL mass spectrometer ( Agilent Technologies , Böblingen , Germany ) .",
"Data files were visually controlled , exported in NetCDF file format and baseline-corrected using the Agilent ChemStation software and the MetAlign software ( Lommen , 2009 ) .",
"Data processing into a standardized numerical data matrix and compound identification were performed using the TagFinder software ( Luedemann et al . , 2008 ) .",
"Criteria for manually supervised metabolite identification were the presence of at least three specific and selective mass fragments and a retention time deviation <1 . 0% .",
"The relative accumulation of amorpha-4 , 11-diene in VOC profiles of leaf tissue was analyzed using the mass spectral intensity of specific and selective mass fragments ( Response ) after normalization to fresh weight ( Response/FW ) .",
"Amorpha-4 , 11-diene was identified in VOC profiles with the help of the reference substance obtained from cultures of the genetically engineered yeast strain EPY224 ( Ro et al . , 2006 ) .",
"To this end , the strain was grown in 25 mL of inducing SD medium for 120 hr at 30°C under vigorous shaking ( 160 rpm ) , until an OD600 of ~1 . 4 was reached .",
"Additionally , control strain EPY300 was grown in 25 mL of SD medium supplemented with 2% ( w/v ) sucrose , 0 . 002% ( w/v ) uracil and 0 . 01% ( w/v ) leucine under the same conditions .",
"Amorpha-4 , 11-diene was identified by differential display of 1 mL cell suspensions in 20 mL head-space screw cap vials comparing the VOC profiles of the compounds obtained from strain EPY224 with those from control strain EPY300 , and analysis of the main differential VOC .",
"VOC profiles obtained from leaf material of A . annua were used to further validate the identification of amorpha-4 , 11-diene .",
"Amorpha-4 , 11-diene present in the VOCs of tobacco leaf material from transplastomic and combinatorially supertransformed lines was annotated by mass spectral ( m/z ) and retention time matching to the reference data in the Golm Metabolome Database ( GMD , http://gmd . mpimp-golm . mpg . de/; Kopka et al . , 2005 ) .",
"For compound information and reference data , see the GMD entry for amorpha-4 , 11-diene ( GMD identifier: A149010; http://gmd . mpimp-golm . mpg . de/search . aspx ) .",
"The retention time of amorpha-4 , 11-diene in the VOC analysis ( Agudelo-Romero et al . , 2015 ) of tobacco plant samples was on average 1563 s , with less than 1% deviation between independent experiments .",
"The specific fragments used for verification of the identity of the compound in complex samples were m/z 93 , 105 , 119 , 133 , 189 and 204 ( Figure 6A ) .",
"For preparation and GC-MS profiling of lipophilic saponification products from total leaf tissue containing artemisinic acid and/or intermediates of artemisinic acid biosynthesis , aliquots of 150 ± 5 mg of frozen powdered N . tabacum leaves were mixed with 500 µL of 2 N KOH/methanol , and incubated at 70°C for 1 hr with gentle shaking ( at 800 rpm ) .",
"After acidification of the saponified samples with 100 µL of 12 M HCl , 300 µL of hexane were added and the samples were vortexed for 1 min .",
"After centrifugation for 5 min at 14 , 000 rpm , 200 µL of the hexane extract were transferred into a clean microcentrifuge tube and concentrated under a mild N2 flow to near dryness .",
"Samples were manually trimethylsilylated .",
"Trimethylsilylation was performed by adding 50 μL of a mixture of N , O-bis ( trimethylsilyl ) trifluoroacetamide ( BSTFA ) and an n-alkane standard in hexane ( 7:1 , v/v ) followed by incubation at 37°C for 30 min with gentle shaking ( 800 rpm ) .",
"Metabolite profiling was performed as detailed previously ( Erban et al . , 2007 ) by gas chromatography coupled to electron impact ionization/time-of-flight mass spectrometry ( GC-EI/TOF-MS ) using an Agilent 6890N24 gas chromatograph ( Agilent Technologies ) connected to a Pegasus III time-of-flight mass spectrometer ( LECO Instrumente GmbH , Mönchengladbach , Germany ) .",
"Retention indices were calibrated in the range relevant for the intermediates of artemisinic acid biosynthesis by addition of a C15/C18/C19 alkane reference mixture to each sample ( Strehmel et al . , 2008 ) .",
"Chromatograms were acquired , visually controlled , baseline-corrected and exported in NetCDF file format using the ChromaTOF software ( Version 4 . 22; LECO , St . Joseph , USA ) .",
"Data analysis of GC-EI/TOF-MS profiles of lipophilic saponification products was performed as described for the VOC analysis .",
"Relative quantification of the intermediates of artemisinic acid biosynthesis was performed by calculating normalized responses/FW values using the response of the C18 n-alkane and the fresh weight of the sample .",
"Intermediates of artemisinic acid biosynthesis were initially identified by comparing GC-EI/TOF-MS profiles from yeast strain EPY224 ( synthesizing amorpha-4 , 11-diene ) to those of yeast strain EPY302 ( synthesizing artemisinic acid and also accumulating all pathway intermediates ) and control strain EPY300 ( that does not express any of the pathway enzymes ) .",
"Strain EPY302 was cultured in the same way as strain EPY224 , but without addition of uracil .",
"All extractions were performed in duplicate omitting saponification .",
"One of the two sample sets was trimethylsilylated as described above , while the other sample set remained non-derivatized .",
"Non-derivatized samples from yeast were compared to trimethylsilylated samples to unambiguously link the non-derivatized soluble metabolic intermediates of artemisinic acid ( A188031 ) and artemisinic alcohol ( A177023 ) to their respective trimethylsilylated analytes ( artemisinic acid 1TMS , A185023; artemisinic alcohol 1TMS , A178029 ) .",
"Dihydroartemisinic alcohol 1TMS ( A179026 ) and dihydroartemisinic acid 1TMS ( A186033 ) were identified after trimethylsilylation .",
"To further validate the identity of the artemisinic compounds , the trimethylsilylated and non-derivatized GC-EI/TOF-MS profiles from yeast were compared to equivalently processed leaf material of A . annua .",
"Finally , GC-EI/TOF-MS profiles from authenticated reference compounds ( kindly provided by Andreas Pallidis and Dr . Alexander R . van der Krol , Wageningen University , The Netherlands ) were used to unambiguously confirm identification of artemisinic acid ( AA ) , artemisinic aldehyde ( AAA ) , artemisinic alcohol ( AAOH ) , dihydroartemisinic acid ( DHAA ) , dihydroartemisinic aldehyde ( DHAAA ) and dihydroartemisinic alcohol ( DHAAOH ) .",
"Artemisinic alcohol , dihydroartemisinic alcohol , dihydroartemisinic acid and artemisinic acid were identified as trimethylsilylated chemical derivatives in complex profiles according to their mass spectrum ( m/z ) and retention time index relative to the C15/C18/C19 n-alkanes , using reference data from the Golm Metabolome Database .",
"Guidelines for manually supervised metabolite identification were the presence of at least 3 specific mass fragments per compound and a retention index deviation <1 . 0% ( Strehmel et al . , 2008 ) .",
"The average retention index of artemisinic alcohol ( 1TMS ) was 1785 and the specific fragments used for verification were m/z 91 , 105 , 119 , 132 , 162 , 187 and 202 .",
"The average retention index of dihydroartemisinic alcohol ( 1TMS ) was 1789 and the specific fragments used for verification were m/z 91 , 105 , 162 , 189 and 204 .",
"The average retention index of dihydroartemisinic acid ( 1TMS ) was 1859 and the specific fragments used for verification were m/z 91 , 105 , 119 , 130 , 162 , 163 , 293 and 308 .",
"The average retention index of artemisinic acid ( 1TMS ) was 1851 and the specific fragments used for verification were m/z 91 , 105 , 119 , 188 , 216 , 291 and 306 ( Figures 6 and 7 ) .",
"Retention indices of each compound showed a deviation of less than 1% in all measurements performed .",
"For quantification purposes , the most abundant and specific among the selective mass features of each artemisinic metabolite was chosen , i . e . , m/z 162 or 202 for artemisinic alcohol ( Figure 6B ) , m/z 162 or 204 for dihydroartemisinic alcohol ( Figure 7B ) , m/z 163 for dihydroartemisinic acid ( Figure 7C ) , and m/z 188 or 216 for artemisinic acid ( Figure 7A ) .",
"For absolute quantification of artemisinic acid , we first determined the percentage of recovery of artemisinic acid spiked into wild-type tobacco leaf tissue samples in comparison to the recovery of pure artemisinic acid processed without saponification and in the absence of leaf material .",
"To this end , 150 ± 5 mg of powdered frozen leaf material from N . tabacum was mixed with 10 µL of an artemisinic acid standard of known concentration ( 2 mg/mL in methanol ) and subjected to the saponification protocol .",
"The matrix-free artemisinic acid standard was prepared by dissolving 2 mg of artemisinic acid powder ( Apin Chemicals , Oxon , UK ) in 1 mL of methanol .",
"All spiked samples were prepared and measured in six replicates and compared to the non-saponified matrix-free artemisinic acid reference samples .",
"The average of the artemisinic acid response values obtained from the reference samples was set to 100% , and the percentage of recovery of artemisinic acid from the leaf tissue matrix after saponification was calculated to be 66 ± 16% .",
"This value was used to correct for the final amount of total artemisinic acid in saponified extracts from plant samples .",
"The artemisinic acid concentration in transplastomic and combinatorially supertransformed plants was calibrated using a dilution series of the commercial non-saponified standard .",
"GC-EI/TOF-MS analysis was as described above .",
"The final quantification of artemisinic acid in line Nt-AO3-CS180 was done as described above except that , due to the high amounts , only 1/10 of the standard extract volume was used .",
"For identification of artemisinin or degradation products of artemisinin , aliquots of 1 . 2 ± 0 . 01 g of frozen powdered leaf tissue were placed in 20 mL head-space screw cap vials , mixed with 3 . 6 mL hexane and incubated for 1 hr in a water bath at 69°C .",
"The tubes were shortly vortexed and opened every 10 min to release the vapor pressure .",
"Samples were then centrifuged for 5 min at 14 , 000 rpm .",
"300 µL of the hexane extracts were transferred to 1 . 1 mL Chromacol vials and reduced to 50 µL under a mild flow of N2 .",
"For identification of artemisinin or its degradation products , 500 , 1000 or 2500 ng of an artemisinin standard ( 1 mg/mL; Sigma-Aldrich , Steinheim , Germany ) were subjected to the same procedure .",
"GC-EI/TOF-MS profiling was performed as described for soluble metabolites using the whole tissue saponification protocol .",
"As reported previously , only the degradation products of artemisinin ( peaks A and B; Sipahimalani et al . , 1991 ) , were detected , likely due to thermal instability of artemisinin .",
"Peaks A and B were only detected in samples that contained the artemisinin reference compound , but not in any of the plant samples .",
"For UPLC analysis of pigments , samples of 40 ± 2 mg of frozen powdered leaf tissue were extracted with 500 µL HPLC grade acetone .",
"A stainless steel ball was added to the mixture and the samples incubated for 20 min at 30°C and 1 , 400 rpm in the dark .",
"After centrifugation for 5 min at 12 , 000 rpm and 4°C , the upper phase was collected in a new microcentrifuge tube and stored on ice in darkness .",
"The acetone extraction was repeated two more times , using 250 µL of acetone each time and combining the three upper phases .",
"Following centrifugation for 5 min at 12 , 000 rpm and 4°C to precipitate any remaining insoluble material , 600 µL of the acetone extracts were transferred to 9 mm glass vials .",
"Samples were analyzed using a Waters UPLC Class H ( Milford , USA ) equipped with an autosampler , Quaternary Solvent Manager , and eλ PDA detector .",
"Pigments were separated in a Waters ACQUITY UPLC BEH C18 1 . 7 µm C18 2 . 1 × 50 mm column at 28°C , using UPLC solutions A and B . Elution was carried out at a flow rate of 0 . 5 mL/min with the following gradient: 100–0% of solution A from 0 to 5 min , 100% solution B from 5 to 6 min , 0–100% solution A from 6 to 6 . 5 min , and 100% solution A from 6 . 5 to 7 . 5 min .",
"Carotenoids were detected at 450 nm and chlorophylls at 640 nm .",
"Three biological replicates ( i . e . , independent plants ) per condition were measured and data were analyzed with the Empower 3 software ."
]
] | [
"Artemisinin-based therapies are the only effective treatment for malaria , the most devastating disease in human history .",
"To meet the growing demand for artemisinin and make it accessible to the poorest , an inexpensive and rapidly scalable production platform is urgently needed .",
"Here we have developed a new synthetic biology approach , combinatorial supertransformation of transplastomic recipient lines ( COSTREL ) , and applied it to introduce the complete pathway for artemisinic acid , the precursor of artemisinin , into the high-biomass crop tobacco .",
"We first introduced the core pathway of artemisinic acid biosynthesis into the chloroplast genome .",
"The transplastomic plants were then combinatorially supertransformed with cassettes for all additional enzymes known to affect flux through the artemisinin pathway .",
"By screening large populations of COSTREL lines , we isolated plants that produce more than 120 milligram artemisinic acid per kilogram biomass .",
"Our work provides an efficient strategy for engineering complex biochemical pathways into plants and optimizing the metabolic output ."
] | [
"Malaria is by far the most devastating tropical disease in the world .",
"It affects hundreds of millions of people – mainly in Africa and Asia – with almost half a million deaths every year .",
"The most effective therapies against malaria all include the drug artemisinin , which is naturally found in an Asian plant called Artemisia annua .",
"Unfortunately , the artemisinin content of A . annua plants is relatively low and the demand for this drug outstrips the supply of the plant .",
"The costly production process makes artemisinin-based treatments inaccessible to many of the people in the most badly affected regions , and so researchers have been trying to find new ways to produce this drug .",
"Genetically modifying crop plants , such as tobacco , to produce artemisinin or related compounds could potentially provide a more sustainable and cheaper source of the drug .",
"Inside plant cells , a structure called the nucleus contains DNA that encodes most of a plant’s genes , but compartments called mitochondria and chloroplasts also contain some DNA .",
"Existing methods to genetically modify plants are able to insert a few genes into either the nucleus or the chloroplasts at a time .",
"However , the production of artemisinin in A . annua involves many different genes that act at different stages of the process , and the precise roles played by many of them remain unclear .",
"Fuentes et al . developed a new approach to insert many of the A . annua genes involved in artemisinin production into tobacco plants at the same time , instead of one-by-one .",
"The new method , referred to as COSTREL , takes advantage of the researchers’ ability to insert new genes into both the nucleus and the chloroplast of the tobacco plants .",
"In the first step , Fuentes et al . inserted a core set of genes that are essential to make artemisinin into the chloroplast .",
"This enabled the plants to produce a molecule called artemisinic acid , which the researchers can extract from the plants and convert into artemisinin by simple chemical reactions .",
"After testing different arrangements of the genes in the chloroplast , the plant line that had the highest levels of artemisinic acid was used to introduce a set of “accessory” genes into the nuclear DNA .",
"These accessory genes are not strictly required to make the drug , but they help to regulate the process in a largely unknown manner .",
"The experiments generated hundreds of genetically modified plant lines that each have different combinations of the accessory genes .",
"Fuentes et al . examined these lines and were able to identify plants that could produce large amounts of artemisinic acid .",
"Therefore , these findings lay the foundations for a cheap way to produce this life-saving drug in tobacco .",
"In the future , the COSTREL method developed by Fuentes et al . could also be used to genetically engineer other complex biochemical processes into plants ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"tools and resources",
"neuroscience"
] | Mapping quantal touch using 7 Tesla functional magnetic resonance imaging and single-unit intraneural microstimulation | elife-12812-v2 | [
[
"The primary somatosensory cortex ( S1 ) has been extensively explored in animal studies where it has been shown that this area displays multiple , fine-grained representations of the body ( Paul et al . , 1972; Kaas et al . , 1979; Favorov et al . , 1987 ) .",
"Penfield and Boldrey ( Penfield & Boldrey 1937 ) derived the first maps of the somatotopic human body representation in S1 using electrical stimulation of the cortical surface .",
"Somatosensory research in humans has involved using psychophysical ( Klatzky et al . , 1985; Gescheider et al . , 2002 ) , microneurographic ( Vallbo & Johansson 1984; Johansson & Vallbo 1983 ) , and neuroimaging ( McGlone et al . , 2002; Martuzzi et al . , 2014; Servos et al . , 2001 ) techniques to study different stages and levels of detail in somatosensory function .",
"Functional magnetic resonance imaging ( fMRI ) has been used extensively for the non-invasive study of the somatosensory cortices in humans ( Nelson and Chen , 2008; McGlone et al . , 2002; Sanchez-Panchuelo et al . , 2010 ) .",
"Most fMRI studies have investigated the spatial pattern of cortical activation in response to vibrotactile ( Francis et al . , 2000; Sanchez-Panchuelo et al . , 2010 ) or pneumatic ( Huang and Sereno , 2007; Overduin and Servos , 2008 ) mechanical stimulation of the digits , or to electrical stimulation of the skin ( Blankenburg et al . , 2003 ) or median nerve ( Kampe et al . , 2000; Ferretti et al . , 2007 ) .",
"These approaches excite large populations of different classes of mechanoreceptive afferents resulting in relatively diffuse activations in contralateral S1 and bilateral secondary somatosensory cortex ( S2 ) .",
"Microneurography provides a method to record the spike discharge activity of a single mechanoreceptive afferent in conscious humans ( Vallbo and Hagbarth , 1968 ) to determine its response to skin contact and the properties of its receptive field , i . e . location , size , and shape .",
"In this manner , mechanoreceptive afferents innervating the glabrous skin of the hand can be categorized into one of four types: fast-adapting type 1 ( FA1 ) , fast-adapting type 2 ( FA2 ) , slowly-adapting type 1 ( SA1 ) , and slowly-adapting type 2 ( SA2 ) ( Vallbo and Johansson , 1984 ) .",
"In intraneural microstimulation ( INMS ) , single mechanoreceptive afferents are selectively activated by passing a small ( 1–7 μA ) current through the recording microelectrode , thus evoking a quantal sensation in the projected sensory field , which matches the physiological qualities of the recorded mechanoreceptive afferent ( Torebjörk et al . , 1987 ) .",
"Microstimulation of an FA1 afferent evokes a well-defined , local sensation of ‘flutter’ or ‘buzzing’ , while microstimulation of an SA1 afferent evokes a sensation of continuous pressure or inward pulling ( Vallbo et al . , 1984; Ochoa and Torebjörk , 1983 ) .",
"Microstimulation of an FA2 afferent evokes a diffuse sensation of vibration over a larger area , whereas microstimulation of an SA2 afferent does not produce a consistent , conscious sensory experience ( Vallbo et al . , 1984; Ochoa and Torebjörk , 1983 ) .",
"It has been shown in a small number of previous studies that INMS of single mechanoreceptive afferents can be combined with noninvasive imaging methods to advance our understanding of the effects of mechanoreceptive afferent activity in somatosensory cortices .",
"For example , INMS of FA1 and SA1 afferents in the median nerve produces frequency-following electroencephalography responses within contralateral S1 ( Kelly et al . , 1997 ) .",
"The single previous study combining INMS with fMRI ( Trulsson et al . , 2001 ) , using a 3 T scanner and a surface coil positioned over the parietal lobe contralateral to the site of stimulation , showed that INMS of FA1 and SA1 afferents induced activity in S1 and S2 , which overlapped with regions activated by applying mechanical vibration to the relevant units’ receptive fields .",
"However , a detailed characterization of the specificity of single unit INMS activations within the representation of the digits in S1 has yet to be performed .",
"Several studies have previously assessed the cortical response to vibrotactile stimulation of the glabrous skin of the human hand , and shown that this evokes a hemodynamic response in multiple primary and secondary cortical areas , including contralateral S1 , bilateral S2 , primary motor cortex ( M1 ) , supplementary motor area ( SMA ) , cingulate cortex , posterior parietal cortex ( PPC ) , and insula cortex ( McGlone et al . , 2002; Trulsson et al . , 2001; Gelnar et al . , 1998 ) .",
"Ultra-high field ( 7T ) fMRI has also recently been used in conjunction with vibrotactile stimulation to map individual digit representations and resolve the fine , within-digit organization ( base-to-tip ) , thus revealing functional subdivisions of areas in S1 ( Sanchez-Panchuelo et al . , 2010; Sanchez-Panchuelo et al . , 2012 ) .",
"Compared to lower field measurements , 7T fMRI provides greatly increased sensitivity and blood-oxygenation level dependent ( BOLD ) signal contrast , coupled with improved intrinsic spatial specificity ( Gati et al . , 1997 ) .",
"Here , we used 7T fMRI to resolve whole-brain cortical activation patterns evoked by INMS of single mechanoreceptive afferent units in the glabrous skin of the hand , and to assess the precise spatial localization of INMS-evoked BOLD responses in contralateral S1 , in comparison to activation due to mechanical vibrotactile stimulation ."
],
[
"Clear and reproducible BOLD responses were found in somatosensory regions , when INMS was perceived .",
"Occasionally , participants reported that the sensation evoked by the INMS stopped , likely due to a minor dislodgement of the microelectrode .",
"This occurred for U7 where a projected sensation was perceived prior to scanning , but no sensation was felt during the fMRI run .",
"For some units , the sensation was weak ( U2 , U3; possibly due to difficulty in attending to the stimulus sensation when inside the scanner ) , or lost during the fMRI run ( U5 , U6 , U8 ) .",
"We compared the location of fMRI responses of all perceived INMS units in contralateral S1 with the digit representation obtained from both vibrotactile stimulation of the microstimulated unit’s receptive field and the fMRI somatotopy maps formed from the traveling-wave ( phase-encoding ) vibrotactile paradigm ( Figure 2 ) .",
"We found that fMRI responses to INMS of single units ( all except for U1; Figure 3—figure supplement 1 ) were spatially localized within the relevant S1 digit representation identified from vibrotactile stimulation .",
"Figure 2a shows example maps of digit somatotopy defined from the vibrotactile traveling-wave paradigm for Participant 4 in the right and left hemispheres ( left and right of the figure , respectively ) .",
"Figure 2b shows the BOLD response to INMS of U11 ( right ) and U9 ( left ) for Participant 4 .",
"These responses are well-localized within regions of the somatotopic map for digit 4 of the left hand and digit 1 of the right hand , respectively .",
"Figure 2c shows the activation generated in S1 by applying vibrotactile stimulation to the receptive field of U11 ( right ) and U9 ( left ) .",
"Fits to the hemodynamic responses evoked in S1 by INMS and the application of vibrotactile stimulation to the unit’s receptive field can be seen in Figure 2d . 10 . 7554/eLife . 12812 . 005Figure 2 . Spatial localization of INMS-induced versus vibrotactile-induced responses in contralateral S1 . Activation maps related to stimulation of two different afferents in Participant 4 are rendered onto a flattened cortical patch spanning the central sulcus of the right ( left of figure ) and left ( right of figure ) hemispheres .",
"Dark gray represents the sulci and light gray the gyri .",
"( a ) Digit somatotopy , where phase values ( in radians ) and corresponding preferred stimulus location ( fingertip ) are shown .",
"Orderly representation of the digits is found on the posterior bank of the central sulcus ( white line ) and the post-central gyrus ( dashed black line ) , corresponding to S1 .",
"( b ) Statistical maps ( Z > 3 . 08 , FDR-adjusted ) from INMS of U11 ( left ) and U9 ( right ) .",
"BOLD activation is localized within the expected digit ROI identified from digit somatotopy , as shown by the blue ( digit 4 ) and red ( digit 1 ) lines , which denote phase values encoded by the blue ( 3 . 77–5 . 03 rad ) and orange ( 0–1 . 26 rad ) colors respectively .",
"The solid black line indicates the SI hand mask ( calculated by dilating the somatotopy map by 5 voxels ) within which FDR correction was performed .",
"( c ) Statistical maps ( Z > 3 . 08 , FDR-adjusted ) for vibrotactile stimulation of the corresponding receptive fields of U11 ( top ) and U9 ( bottom ) .",
"( d ) HRF estimated from the GLM analysis for INMS and vibrotactile stimulation averaged across voxels of the ROI ( U10 , top; U9 , bottom ) .",
"Error bars show voxel-wise parameter standard errors averaged across voxels of the ROI . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 00510 . 7554/eLife . 12812 . 006Figure 3 . Spread of activation across the digit ROIs identified from the somatotopy .",
"( a ) Statistical maps ( Z > 3 . 08 , FDR-adjusted ) from INMS of seven single units in participants 2 , 3 and 4 .",
"In each case the activation map is rendered onto a flattened cortical patch spanning the central sulcus of the right hemisphere .",
"Dark gray represents the sulci and light gray the gyri .",
"The solid black line indicates the SI hand mask ( calculated by dilating the somatotopy map by 5 voxels ) within which FDR correction was performed .",
"Activation is localized within the expected digit ROI ( black line ) identified from the digit somatotopy ( see color legend ) .",
"( b ) Statistical maps ( Z > 3 . 08 , FDR-adjusted ) for vibrotactile stimulation of the corresponding receptive field of units .",
"( c ) Z-scores ( FDR-corrected ) of the INMS BOLD response averaged across voxels for each of the digit ROIs identified from the traveling-wave analysis .",
"Error bars indicate standard error across voxels in ROI .",
"( d ) Proportion of voxels activated by the INMS paradigm at Z>3 . 08 ( FDR-corrected ) for each digit ROI .",
"The source data for plots in panels",
"( c ) and",
"( d ) are available in the Figure 3—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 00610 . 7554/eLife . 12812 . 007Figure 3—source data 1 . Source files for plots of Z-score and Proportion of active voxels in each Digit ROI . This matlab file contains variables for each individual unit ( U4 , U5 , U6 , U8 , U9 , U11 ) with fields ‘micro_stats’ and ‘vibro_stats’ containing a structure with the results for single unit INMS and vibrotactile stimulation of the unit’s receptive field , respectively .",
"Each structure has the following fields: ‘zetaMean’ , ‘betaSem’: ( 5 digits x 1 ) -vector containing mean Z-score ( FDR-corrected ) and standard error across voxels for each Digit ROI; ‘PropActVox’: ( 5 digits x 1 ) -vector containing proportion of active voxels ( Z>3 . 08 , FDR-corrected ) in each Digit ROI; and ‘betaMean’ , ‘betaSem’: ( 5 digits x 1 ) -vector containing mean GLM parameter estimate and standard error across voxels for each Digit ROI .",
"GLM parameter estimates are not plot in Figure 3 but are used for subsequent group analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 00710 . 7554/eLife . 12812 . 008Figure 3—figure supplement 1 . Comparison of contralateral S1 responses to different paradigms for Participant 1 . Statistical maps overlaid on a high resolution T2*-weighted structural image .",
"( a ) Digit somatotopic maps obtained with the traveling-wave paradigm for both hands , showing the location of the maps in the posterior bank of the central sulcus .",
"( b ) Map of veins identified using T2*-weighted magnitude and phase images .",
"Phase images are unwrapped and high-pass filtered .",
"A map of veins is approximated by thresholding the unwrapped , filtered phase image and convolving the identified voxels with a 2 mm kernel .",
"( c ) Statistical maps ( Z > 3 . 08 , FDR-adjusted ) for INMS of U1 .",
"Note , there is no activation in the S1 hand area , as shown by the ROIs delineating each of the digits .",
"( d ) Time series of the BOLD response to INMS of U1 for the digit 2 ROI , denoted by the green line in image ( upper panel ) and of a region of activation co-localized with a vein as indicated by the white circle ( lower panel ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 008 Figure 3 shows the spatial localization of the activation produced in S1 by the seven perceived INMS units ( U4-U6 , U8-U11 ) ( Figure 3a ) and corresponding vibrotactile stimulation of each units’ receptive field ( Figure 3b ) .",
"In general , the BOLD responses due to INMS and vibrotactile stimulation were well localized within the expected digit ROI , as defined from the traveling-wave somatotopy paradigm .",
"Figure 3c plots the average INMS z-score ( FDR corrected ) in each digit ROI , and Figure 3d shows the proportion of active voxels to the INMS paradigm that were classified to each digit ROI ( z>3 . 08 , FDR corrected ) .",
"As expected , the average z-score and proportion of active voxels in the digit ROIs corresponding to digits in which the INMS was sensed was higher than in the neighboring digits .",
"Figure 4 plots the group-level response to show the spatial spread of the INMS and vibrotactile response to neighboring digits .",
"Figure 4a shows the mean z-score , Figure 4b the proportion of active voxels and Figure 4c the GLM parameter estimate to INMS ( top ) and vibrotactile stimulation of the unit’s receptive field ( bottom ) .",
"ANOVA results showed a significant difference in mean Z-score ( F4 , 30=14 . 08 , p<10-5; F4 , 30=12 . 97 , p<10-5 ) , proportion of active voxels ( F4 , 30=16 . 12 , p<10-6; F4 , 30=17 . 64 , p<10-6 ) and GLM parameter estimates ( F4 , 30=13 . 52 , p<10-5; F4 , 30=14 . 1 , p<10-5 ) across the stimulated and neighboring digit classification ( INMS; vibrotactile ) .",
"A multiple pairwise comparison , adjusted for multiple comparisons , showed that measures for the stimulated digit were significantly higher than those of the neighboring digits for mean Z-score ( p<0 . 0001 INMS; p<0 . 005 vibrotactile stimulation ) , proportion of active voxels ( p<0 . 00005 for INMS and vibrotactile stimulation ) and GLM parameter estimates ( p0 . 01 for INMS and vibrotactile stimulation ) . 10 . 7554/eLife . 12812 . 009Figure 4 . Group analysis ( N = 7 units ) of the BOLD response to INMS and vibrotactile stimulation of the unit’s receptive field , showing the stimulated digit compared to the neighboring digits .",
"( a ) Z-scores ( FDR-corrected ) of INMS response in digit ROIs ( defined from digit somatotopy ) averaged across ROIs for the stimulated digit ( N = 7 ) compared to neighboring digits ( 1st degree neighbors , N = 11; 2nd degree neighbors , N = 9 , 3rd degree neighbors , N = 5 , 4th degree neighbors , N = 3 ) .",
"The z-score for the stimulated digit was significantly different to that of neighboring digits .",
"***p<0 . 0001 , **p<0 . 005 , statistical significance corrected for multiple comparison using Bonferroni correction .",
"( b ) Proportion of voxels activated by the INMS ( top ) and vibrotactile ( bottom ) paradigm at Z>3 . 08 ( FDR-corrected ) for the stimulated digit compared to the neighboring digits .",
"Mean and standard error across ROIs .",
"The proportion of active voxels in the stimulated digit ROI was significantly different to that of neighboring digits .",
"***p<0 . 00005 , statistical significance corrected for multiple comparison using Bonferroni procedure .",
"( c ) GLM parameter estimates of the INMS ( top ) and vibrotactile ( bottom ) paradigm for the stimulated digit compared to the neighboring digits .",
"The parameter estimate in the stimulated digit ROI was significantly higher than that of neighboring digits .",
"**p<0 . 01 , statistical significance corrected for multiple comparison using Bonferroni procedure .",
"For all plots ( a–c ) the mean and standard error across N measures is shown .",
"The source data used for the ANOVA tests are available in the Figure 4—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 00910 . 7554/eLife . 12812 . 010Figure 4—source data 1 . Source files for ANOVA tests . This matlab file contains the 2D-matrices ( 11 x 5 ) , related to each panel in Figure 4 , that were used for the 1-way analysis of variance ( performed using the ‘anova1’ matlab command ) .",
"Each matrix row contains data for each of the 7 units ( there are up to eleven 1st degree neighboring digit ROIs ) and each matrix columns represents the ‘proximity’ to the stimulated digit ROI ( stimulated digit ROI , 1st degree , 2nd degree , 3rd degree and 4th degree neighboring digit ROIs ) .",
"‘Zeta_micro’ and ‘Zeta_vibro’ are the matrices containing the Z-score ( FDR-corrected ) values , ‘PerVox_micro’ and ‘PerVox_vibro’ contain the proportion of active voxels ( Z>3 . 08 , FDR-corrected ) and’ Beta_micro’ and ‘Beta_vibro’ contain the GLM parameter estimates for INMS and vibrotactile stimulation respectively .",
"ANOVA results show a significant difference in mean Z-score ( F4 , 30=14 . 08 , p<10-5; F4 , 30=12 . 97 , p<10-5 ) , proportion of active voxels ( F4 , 30=16 . 12 , p<10-6; F4 , 30=17 . 64 , p<10-6 ) and GLM parameter estimates ( F4 , 30=13 . 52 , p<10-5; F4 , 30=14 . 1 , p<10-5 ) across the stimulated and neighboring digit classification ( INMS; vibrotactile ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 010 For those units lost during the fMRI run ( U5 , U6 , U8 ) , no areas were found to show a significant correlation with an additional ( parametric ) regressor when modelling linear reductions in induced response over time ( to model gradual losses of unit responses ) , likely due to the sudden rather than gradual loss of the unit .",
"Thus parameter estimates to INMS stimulation were not significantly different between the GLM including a parametric regressor and modelling the INMS stimulation alone .",
"Participants freely described the mechanical , point-vibrotactile stimulus applied to each unit’s receptive field as feeling very similar in extent and quality to the INMS , especially for the sensations generated from FA1 units .",
"Figure 5a compares the mapping of INMS-induced fMRI responses ( yellow ) for all FA1 single units to maps of the responses produced by applying vibrotactile stimulation to the units’ receptive fields ( blue ) .",
"Overlapping cortical responses are shown in green .",
"Activation maps show the conjunction of the individual FA1 unit responses , using the same statistical threshold ( Z > 3 . 08 , false discovery rate ( FDR ) correction ) for both INMS and vibrotactile stimulation .",
"BOLD responses to single unit INMS were detected in a number of sensory-related brain areas , including S1 , S2 ( Brodmann areas ( BA ) 40 and 43 ) , premotor cortex ( PMC; SMA and dorsal PMC ) , M1 , insula ( anterior insula cortex ( AIC ) and posterior insula cortex ( PIC ) ) , prefrontal cortex ( PFC ) and PPC .",
"Table 2 details the location and statistical significance ( mean and standard error across units ) of the BOLD responses produced in these areas by INMS of the five FA1 single units in the left hand .",
"Common areas of activation for INMS and vibrotactile stimulation included S1 , S2 , PMC , M1 , and contralateral PIC; however , INMS gave rise to significant activity in additional brain regions , including the AIC , PPC and contralateral PFC ( Table 2 ) .",
"Figure 5b shows that the HRFs generated in these regions by INMS were similar in both onset and duration to the INMS-elicited responses in S1 and S2 . 10 . 7554/eLife . 12812 . 011Figure 5 . fMRI activation patterns and time courses in cortical areas .",
"( a ) Cortical activation patterns in MNI space .",
"Transverse slices and surface reconstructions showing areas of activation in response to INMS ( red clusters ) and mechanical vibrotactile stimulation applied directly to the respective unit’s receptive field ( blue clusters ) , as well as areas of overlap ( green clusters ) .",
"Clusters represent common regions of significant activation from all single FA1 units on the left hand ( U1 , U4 , U6 , U8 , and U11 ) .",
"Individual statistical maps for each afferent were thresholded at Z < 3 . 08 after correcting for multiple comparisons ( FDR ) and cluster-corrected at p=0 . 01 , prior to forming the conjunction map .",
"( b ) BOLD time courses due to INMS for U4 in different cortical areas .",
"Responses contralateral ( right ) to the hand stimulation site are shown in red and ipsilateral ( left ) responses are shown in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 01110 . 7554/eLife . 12812 . 012Table 2 . Cortical areas showing significant activation to INMS of single mechanoreceptive afferents and the corresponding vibrotactile stimulation .",
"Results show the mean and standard error across the five FA1 mechanoreceptive afferents subject to INMS at 30 Hz and corresponding vibrotactile stimulation of the perceived sensation , showing the number of units showing significant activation , MNI coordinates , beta values , Z-score and number of voxels in ROI .",
"R=contralateral , L=ipsilateral .",
"Source files for Table 2—source data 1 and Table 2—source data 2 contain single unit INMS and vibrotactile stimulation results , respectively , for each of the 5 ( U1 , U4 , U6 , U8 , U11 ) individual units . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 01210 . 7554/eLife . 12812 . 013Table 2—source data 1 . Source files for single unit INMS . This matlab file contains 2D-matrices ( 19x5 ) with the results for single unit INMS for each of the 5 individual units ( U1 , U4 , U6 , U8 , U11 ) in each of the 19 ROIs .",
"'BetaValues’ contains mean across voxels of the beta values , ‘Z-score’ contains the mean Z_score ( FDR- corrected ) across voxels and ‘NumberVoxels’ contains the number of significant active voxels ( Z > 3 . 08 , FDR-corrected ) in the ROI .",
"Table 2 summarizes the results by showing the mean and standard error across the 5 units . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 01310 . 7554/eLife . 12812 . 014Table 2—source data 2 . Source files for vibrotactile stimulation . This matlab file contains 2D-matrices ( 19 ROIs x 5 units ) with the results for vibrotactile stimulation applied to the receptive field for each of the 5 individual units ( U1 , U4 , U6 , U8 , U11 ) in each ROI .",
"‘BetaValues ‘contains mean across voxels of the beta values , ‘Z_score’ contains the mean Z-score ( FDR- corrected ) across voxels and ‘NumberVoxels’ contains the number of significant active voxels ( Z > 3 . 08 , FDR-corrected ) in the ROI .",
"Table 2 summarizes the results by showing the mean and standard error across the 5 units . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 014Single unit INMSVibrotactile stimulationROINo .",
"Unitsx , y , z MNI co-ordinatesBetaZVoxelsBetaZVoxelsSI R SI L4 354 , -12 , 46 -52 , -12 , 441 . 4 ± 0 . 2 1 . 2 ± 0 . 25 . 9 ± 0 . 5 5 . 6 ± 0 . 838 ± 7 20 ± 91 . 3 ± 0 . 3 1 . 6 ± 0 . 35 . 4 ± 0 . 3 5 . 2 ± 0 . 241 ± 12 19 ± 1BA 40 R BA 40 L5 460 , -22 , 16 -60 , -22 , 161 . 4 ± 0 . 2 1 . 5 ± 0 . 44 . 9 ± 0 . 2 5 . 3 ± 0 . 256 ± 5 73 ± 51 . 4 ± 0 . 1 1 . 4 ± 0 . 24 . 8 ± 0 . 2 5 . 0 ± 0 . 154 ± 7 72 ± 12BA 43 R BA 43 L2 360 , -4 , 10 -58 , -12 , 141 . 1 ± 0 . 4 1 . 0 ± 0 . 45 . 4 ± 0 . 1 4 . 8 ± 0 . 345 ± 6 33 ± 81 . 2 ± 0 . 4 1 . 7 ± 0 . 34 . 4 ± 0 . 2 4 . 2 ± 0 . 230 ± 20 26 ± 11SMA R SMA L5 54 , 0 , 60 -2 , 0 , 601 . 2 ± 0 . 2 1 . 2 ± 0 . 24 . 8 ± 0 . 3 4 . 5 ± 0 . 393 ± 27 66 ± 191 . 3 ± 0 . 2 1 . 2 ± 0 . 14 . 8 ± 0 . 2 4 . 5 ± 0 . 343 ± 21 29 ± 6PMC R PMC L4 554 , 0 , 50 -52 , -2 , 500 . 8 ± 0 . 2 1 . 1 ± 0 . 14 . 7 ± 0 . 2 5 . 5 ± 0 . 336 ± 11 37 ± 71 . 1 ± 0 . 2 1 . 2 ± 0 . 15 . 0 ± 0 . 2 4 . 3 ± 0 . 146 ± 9 20 ± 8M1 R M1 L3 254 , -6 , 48 -52 , -6 , 480 . 9 ± 0 . 2 1 . 5 ± 0 . 25 . 2 ± 0 . 5 6 . 3 ± 0 . 151 ± 20 66 ± 360 . 8 ± 0 . 2 1 . 3 ± 0 . 15 . 0 ± 0 . 7 5 . 3 ± 0 . 531 ± 10 21 ± 3PIC R PIC L5 546 , -2 , 10 -42 , -2 , 100 . 8 ± 0 . 2 0 . 8 ± 0 . 14 . 2 ± 0 . 2 4 . 4 ± 0 . 245 ± 12 38 ± 140 . 8 ± 0 . 2 -4 . 7 ± 0 . 2 -27 ± 3 -AIC R AIC L4 434 , 26 , 4 -32 , 26 , 41 . 2 ± 0 . 1 1 . 1 ± 0 . 14 . 7 ± 0 . 2 4 . 4 ± 0 . 2146 ± 20 106 ± 21- -- -- -PPC R PPC L4 538 , -48 , 50 -38 , -48 , 561 . 2 ± 0 . 1 1 . 0 ± 0 . 14 . 4 ± 0 . 3 4 . 4 ± 0 . 3168 ± 44 172 ± 43- -- -- -PFC R442 , 34 , 181 . 2 ± 0 . 24 . 5 ± 0 . 378 ± 22---"
],
[
"The principal finding of our present work is the detailed localization in contralateral S1 of cortical responses to the electrical microstimulation of single , first-order mechanoreceptive afferents , and the demonstration of spatial alignment of these responses with somatotopic maps derived from mechanical skin stimulation .",
"This was achieved through the combined usage of two techniques: intra-neural microstimulation ( INMS ) , to stimulate single mechanoreceptive afferents , and 7T fMRI , to map the cortex with superior spatial resolution .",
"This work also shows that activity generated by stimulation of a single mechanoreceptive afferent can be perceptually characterized and produces a network of cortical responses .",
"Only one previous study has combined single unit INMS with fMRI , at 3T ( Trulsson et al . , 2001 ) , but this was only able to resolve activation in contralateral S1 and S2 as the use of a surface coil limited the spatial extent of activation maps .",
"The greater signal-to-noise ratio and improved BOLD contrast afforded by 7T fMRI allowed us to improve the spatial resolution , with a reduction in the voxel volume by a factor of 6 compared to previous work at 3T ( Trulsson et al . , 2001 ) .",
"We have exploited the improved spatial resolution to provide a detailed characterization of the location and extent of the cortical network involved in encoding inputs from single mechanoreceptive afferents , as well as in comparing these responses to somatotopical maps created from vibrotactile skin stimulation .",
"Measurements of cortical activity elicited by INMS demonstrated that when a singular , quantal touch from the stimulation of a single mechanoreceptive afferent is consciously felt , a precise area in contralateral S1 is active .",
"The response in S1 was well-localized within the expected region , identified from maps of digit somatotopy obtained from vibrotactile stimulation of the fingertips .",
"The extent of the S1 responses to INMS was less than that elicited by vibrotactile stimulation to the unit’s receptive field , although the response produced by single unit INMS was relatively extensive , considering that vibrotactile stimulation simultaneously engages a large number of afferents ( Johansson and Vallbo , 1979; Vallbo and Johansson , 1984 ) .",
"Robust responses were found within the expected digital cortical area for all perceived microstimulated afferents ( Figures 2 and 3 ) , except for U1 , for which no significant responses were found , in either contralateral or ipsilateral S1 , despite the fact that the participant exhibited a complete somatotopic map of the digits in both hemispheres and reported feeling the sensation throughout INMS .",
"To explore this finding further , we used the delineation of digit 2 from the somatotopic map obtained with the vibrotactile traveling-wave paradigm to inspect the time series of S1 responses evoked by INMS for U1 ( located on the palm below digit 2 ) .",
"We also interrogated the BOLD response produced in contralateral S1 when vibrotactile stimulation was applied to the receptive fields of U1 .",
"In S1 , we found negative BOLD responses ( Figure 3—figure supplement 1 ) for both INMS and vibrotactile stimulation applied to the receptive field of the INMS .",
"The negative BOLD response in this subject is possibly due to a steal effect from the nearby vasculature draining from the active cortex ( Bianciardi et al . , 2011 ) since draining venous regions are highly modulated by block paradigms with periods of 'on’ and ‘off’ stimulation , as used to study the response to INMS and vibrotactile stimulation of the receptive field .",
"In contrast , using the traveling-wave paradigm a complete map of the digits in S1 is seen .",
"This is expected , as we have previously shown that a traveling-wave design is insensitive to the non-specific BOLD contributions from large veins that drain blood from across the whole hand representation in S1 ( Uğurbil et al . , 2003; Besle et al . , 2013 ) , thus suppressing the venous signal modulations found in the block INMS/vibrotactile stimulation data .",
"In order to estimate the spatial spread of INMS BOLD responses to neighboring digits , we show that , at the group level , the z-score , proportion of active voxels and GLM parameter estimates are significantly higher ( p<0 . 01 ) in the stimulated ROI than in the neighboring digits ( Figure 4 ) .",
"These results are in-line with our previous findings reported for vibrotactile stimulation ( Besle et al . , 2013 ) .",
"The network of cortical areas activated by both INMS of single mechanoreceptive afferents and mechanical vibrotactile stimulation of the units’ receptive field , included somatosensory areas such as S1 , S2 , and PIC , as well as areas involved in motor control , including M1 , SMA and PMC .",
"Although M1 has previously been shown to be activated by tactile input ( e . g . Francis et al . , 2000; Ackerley et al . , 2012 ) , we cannot exclude the possibility that the M1 activation observed in this study may originate from spatial blurring of somatosensory activation ( given that M1 and S1 are located on opposite banks of the central sulcus ) .",
"When comparing responses to INMS and vibrotactile stimulation applied to the afferents’ receptive fields , INMS activated a number of additional areas , specifically the AIC , PPC and PFC .",
"Exploration of the INMS BOLD time series for these areas ( Figure 5b ) suggests that the activity in these areas is locked to the S1/S2 activity and is not due to anticipation .",
"Both insula and parietal cortices have been shown to contribute to the perception of touch ( Preusser et al . , 2014 ) , and a previous study of tactile attention ( Burton et al . , 2008 ) has shown that a fronto-parietal network , which includes PFC and PPC , is involved in attention .",
"Although identical paradigm timings were used for INMS and vibrotactile stimulation in order to compare the spatial localization of the BOLD response , there were differences in the attentional focus between the INMS and vibrotactile tasks .",
"During the INMS fMRI runs , participants were aware that perception might be lost and hence had to concentrate on the stimulus and report any lack of sensation at the end of the run .",
"In contrast , the vibrotactile stimulus was delivered at a suprathreshold level and participants did not have to monitor that the sensation was still present during the vibrotactile fMRI run .",
"Hence , the increased activity in AIC , PFC and PPC observed in the present study may reflect the increased attentional effects ( i . e . , baseline or gain effects on evoked responses ) during the INMS protocol compared to vibrotactile stimulation .",
"However , this is a preliminary finding and requires further investigation with larger sample sizes and more quantitative analysis to be corroborated .",
"The capability of combining INMS with 7T fMRI has the following theoretical implications for human somatosensory research .",
"Although the notion that peripheral input from the skin is represented directly by four cytoarchitectonic areas ( BA 3a , 3b , 1 and 2 ) in S1 , each containing an orderly somatotopic map of the body surface has been supported by findings from animal studies ( Kaas et al . , 1979; Paul et al . , 1972; Favorov et al . , 1987; Tommerdahl et al . , 2010 ) and 7T fMRI in humans ( Sanchez-Panchuelo et al . , 2010; Sanchez-Panchuelo et al . , 2012; Martuzzi et al . , 2014 ) , a simple point-to-point topographical correspondence between skin surface and cortical representation does not hold .",
"In reality , there is integration and processing through axonal synapsing in the dorsal column nuclei and thalamus prior to mechanoreceptive information entering the cerebral cortex .",
"There appears to be a preserved transmission from single , mechanoreceptive second-order neurons in the dorsal column ( Vickery et al . , 1994 ) .",
"At the level of the thalamus , an axon of a single ventral posterolateral nucleus terminates over a fairly wide , roughly 0 . 5 mm , cortical territory ( Rausell and Jones , 1995 ) , where many individual thalamocortical axons spread out in discrete patches over several millimeters of S1 ( Landry et al . , 1987 ) .",
"This spread corresponds well with our finding that the cortical activation from a single mechanoreceptive afferent extends over an area that is not dissimilar to the area activated by input from many afferents through point-vibrotactile stimulation .",
"Also , neurons in S1 cortical columns have extensive lateral excitatory connections , not only with neighboring neurons , but also with neurons several millimeters away in the same cortical area ( Burton and Fabri , 1995 ) .",
"We have shown that single unit INMS produces bilateral somatosensory activation , as well as influencing motor areas and cognitive networks ( e . g . PPC , PFC ) .",
"Such a wide spreading of stimulus-evoked activity has been clearly documented in microelectrode recording studies ( Reed et al . , 2010 ) .",
"Overall , the spatiotemporal pattern of S1 response to vibrotactile stimulation is far from simple and its functional significance remains to be unraveled .",
"Translational insights from in vivo neurophysiological studies in non-human primates have driven much of the theoretical understanding of cortical mechanisms that govern human tactile perception , but operative procedures , especially those which alter the neurochemistry of cortical synaptic transmission ( Masamoto et al . , 2009 ) , may confound relating such findings to normal functioning of the human brain .",
"This demonstration of the feasibility of combining INMS with 7T fMRI opens up the possibility of a range of further neuroimaging studies that will allow interrogation of the precise anatomical and physiological properties of the fundamental encoding of touch .",
"These include systematic investigation of the sub-cortical ( e . g . thalamic ) responses and laminar-specific cortical responses to INMS of different mechanoreceptive afferent classes using a variety of electrical stimulation patterns ."
],
[
"In Step 1 , the median nerve was accessed at the wrist in order to isolate single axonal responses from mechanoreceptive afferents in the volar hand , on which to perform INMS ( Trulsson et al . , 2001 ) .",
"A high-impedance ( ~300–500 kΩ ) , insulated , tungsten recording/stimulating electrode ( 15 mm length , shaft diameter 0 . 2 mm , tip diameter ~5 µm; FHC , Bowdoin , ME ) was inserted percutaneously into the skin , ~3 cm from the wrist fold between the flexor carpi radialis and the flexor palmaris longus tendons .",
"An uninsulated reference electrode was inserted subcutaneously 3–5 cm away , on the ulnar side of the recording/stimulating electrode , and a ground electrode was attached further up the participant’s arm ( Figure 6 ) .",
"The recording/stimulating electrode was advanced into the median nerve , which was located 0 . 3–1 cm below the skin surface .",
"The preamplifier was taped to the participant’s arm , and the acquisition hardware and stimulator were located at the outer edge of the scanner room ( Figure 6 ) .",
"Differential responses were amplified ( x10 , 000 ) using a preamplifier ( NeuroAmpEX; ADInstruments , Castle Hill , Australia ) , band-pass filtered ( 0 . 3–5 kHz ) and sampled at 10 kHz using PowerLab hardware and LabChart 7 software ( ADInstruments , Castle Hill , Australia ) . 10 . 7554/eLife . 12812 . 015Figure 6 . Figure of the experimental setup . The PowerLab , NeuroAmp EX and ML180 stimulator were placed just inside the magnet room at a field strength not exceeding 5 mT . Placement of the interface equipment within the magnet room was preferred for safety reasons , as isolated cables connected to the participant did not then pass into the control room .",
"The USB interface and trigger cables were passed through the radio frequency shield via a waveguide aperture .",
"An amplifier and loudspeaker was driven from the NeuroAmp EX audio output to give audio feedback to the microneurographer .",
"In addition , a projection of the computer screen could be viewed for visual confirmation of nerve signals .",
"A switch was used to connect the electrodes to either the stimulator or the NeuroAmp head-stage pre-amplifier .",
"In addition , a resistive shunt was placed across the stimulation leads to remove any build-up of charge before connecting or disconnecting the stimulator .",
"Disconnection of the stimulator was necessary because of the high level of noise introduced when it was connected .",
"Star-quad cable was used within the magnet environment to reduce the likelihood of induced currents due to scanner operation affecting the stimulus presentation . DOI: http://dx . doi . org/10 . 7554/eLife . 12812 . 015 The microneurographer delivered light , stroking touch to the palm to evoke activity in low-threshold mechanoreceptive afferents .",
"A loudspeaker in the scanner room allowed the microneurographer to hear the nerve activity and a projector displayed the recording onto the scanner exterior for visual inspection .",
"The microneurographer systematically searched for the nerve until modulations of the signal from the electrode corresponded to mass activity from mechanoreceptive afferents as a result of touch were heard .",
"Using fine adjustments , the electrode was manipulated within the nerve to an intra-fascicular location and single units were searched for by stroking the participant’s hand .",
"Single mechanoreceptive afferents were characterized by their audio and visual signals , and the extent of the receptive field of each afferent was explored using a wooden stick .",
"The location of the receptive field was mapped using von Frey monofilaments and the minimal force required for mechanoreceptor activation noted .",
"Afferents were identified as being myelinated Aβ mechanoreceptors , namely FA1 , SA1 , FA2 or SA2 afferents ( Vallbo and Johansson , 1984 ) .",
"The middle of the receptive field was marked on the skin .",
"Recordings of individual mechanoreceptive afferents in response to mechanical stimulation were made ( e . g . Figure 1a , b ) and analyzed in MATLAB ( The Mathworks; Natick , MA ) .",
"Data were preprocessed to verify the single-unit nature of all recorded mechanoreceptive afferents with an offline pattern-matching algorithm .",
"Once a single mechanoreceptive afferent was identified , INMS was carried out to ascertain the sensation produced by a low-current electrical pulse sequence ( Step 2 ) .",
"Trains of 30 Hz pulses ( 200 µs , positive , square-wave pulses over 0 . 5 s ) were delivered ( via Stimulus Isolator; ADInstruments , Castle Hill , Australia and controlled using the LabChart 7 software ) .",
"The experimenter delivered 2–3 pulse sequences , while the current was increased slowly from 0 µA , in 1 µA steps , until the participant felt a sensation .",
"Once a clear sensation was felt , the precise location of the sensation and its quality were recorded and tested to confirm whether the previously mapped receptive field spatially aligned with that perceived by the participant during INMS .",
"This was done by a process of questioning the participant to determine whether mechanical touch to the receptive field matched the projected sensory field sensation during INMS to within ~1 mm .",
"If so , it was deemed that microstimulation was being applied to the afferent from which recordings had been made .",
"If the participant felt a clear small , point-sensation in the projected sensory field that did not align with the mapped receptive field , the stimulated unit was nevertheless explored .",
"These units were included if the perceived sensation ( e . g . pressure from an SA1 ) was similar in quality to those in matched physiology-INMS trials ( e . g . perceived size , shape , sensation ) ( see Table 1 ) .",
"The stimulating current intensity which generated a sensation was recorded , along with the stimulation currents delivered during each fMRI run .",
"INMS of a stable , single mechanoreceptive afferent could be carried out successfully for up to ~45 min , although Step 3 was completed successfully for only a subset of mechanoreceptive afferents ( see Results ) .",
"Each fMRI run consisted of a block paradigm , comprising 8 cycles of alternating periods of 8 s INMS followed by 23 s rest ( acquisition time ~4 mins ) .",
"The 8 s INMS period consisted of 0 . 5 s burst of stimulation ( 30 Hz pulse frequency; 200 μs pulse width ) each second .",
"For each afferent , 1–3 fMRI repeats of the INMS paradigm were conducted .",
"In some cases , the stimulation current was adjusted between runs , e . g . due to loss of perception ( Vallbo et al . , 1984 ) , to ensure a clear and stable sensation .",
"If the INMS-induced sensation remained stable , other parameters were also tested , including changing the stimulation frequency to 60 Hz , and increasing the stimulation current to investigate the effect of recruiting further mechanoreceptive afferents ( Vallbo et al . , 1984 ) .",
"After Steps 1–3 , fMRI of mechanical vibrotactile stimulation at each microstimulated afferent’s receptive field was carried out with identical timings to the INMS paradigm .",
"Vibrotactile stimuli were delivered at 30 Hz to ~1 mm2 of the skin using a piezo-electric device ( Dancer Design , St-Helens , UK ) .",
"In addition , the digit tips of each participant’s left hand ( and right hand for participant",
"4 ) were stimulated with 5 independently-controlled piezo-electric devices using a traveling-wave or phase-encoding paradigm ( Sanchez-Panchuelo et al . , 2010 ) , analogous to that used in retinotopic mapping , in which each individual digit of the hand is sequentially stimulated to create a travelling wave of activity across cortical regions containing a somatotopic map of the hand .",
"Vibrotactile stimulation at 30 Hz was delivered to each digit tip in periods of 4 s ( intermittent stimulation with 0 . 1 s gap every 0 . 5 s ) , over a 20 s cycle .",
"Data were collected during two runs of 12 cycles each; with stimulation delivered in a forward ( digit 1 to",
"5 ) and reverse order ( digit 5 to 1 ) .",
"MRI data were collected on a 7T scanner ( Achieva; Philips , Amsterdam , Netherlands ) using a head volume transmit coil and 32-channel receive coil ( Nova Medical; Wilmington , MA ) .",
"Functional data were acquired using T2*-weighted , multi-slice , single-shot gradient-echo , echo-planar imaging ( EPI ) with echo time ( TE ) 25 ms , repetition time ( TR ) 2000 ms , flip angle ( FA ) 75° , SENSE reduction factor 3 in the right-left direction .",
"The in-plane spatial resolution was 1 . 5 mm , field of view of 174 × 192 mm2 in right-left and anterior-posterior directions .",
"A slice thickness of 2 . 5 mm was used to achieve full brain coverage ( 80 mm in foot-head direction ) within the TR period .",
"For the traveling-wave paradigm , the slice thickness was reduced to 1 . 5 mm ( 48 mm coverage ) as it was only necessary to span S1 .",
"Following the functional runs , a high-resolution T2*-weighted FLASH dataset was acquired with the same slice prescription and coverage as the functional data ( 0 . 5 × 0 . 5 × 1 . 5 mm3 resolution; TE/TR = 9 . 3/458 ms , FA = 32° , SENSE factor = 2 ) , and a whole-head structural T1-weighted MPRAGE dataset ( 1 mm isotropic resolution , linear phase encoding order , TE/TR 3 . 7/15 ms , FA 8º , inversion time 1184 ms , TR-FOCI pulse [Hurley et al . , 2010] ) to allow projections of functional maps onto flattened reconstructions of the cortical space and MNI space .",
"fMRI raw time series and structural MRI scans for each subject can be found at figshare ( Sanchez Panchuelo , RM; Ackerley , R; Glover , PM; Bowtell , RW; Wessberg , J; Francis , ST; McGlone , F | 2016 | fMRI to intraneural microstimulation of single mechanoreceptive afferents | Available at figshare under a CC0 Public Domain . ) fMRI data sets were realigned to the last volume of the data set using AFNI ( http://afni . nimh . nih . gov/afni ) , and statistical analysis performed using mrTools ( http://www . cns . nyu . edu/heegerlab ) in MATLAB .",
"To account for scanner drift and other low-frequency signals , all time-series were high-pass filtered ( 0 . 01 Hz cut-off ) and data converted to percent signal change .",
"To address the key aims , three analyses were performed:"
]
] | [
"Using ultra-high field 7 Tesla ( 7T ) functional magnetic resonance imaging ( fMRI ) , we map the cortical and perceptual responses elicited by intraneural microstimulation ( INMS ) of single mechanoreceptive afferent units in the median nerve , in humans .",
"Activations are compared to those produced by applying vibrotactile stimulation to the unit’s receptive field , and unit-type perceptual reports are analyzed .",
"We show that INMS and vibrotactile stimulation engage overlapping areas within the topographically appropriate digit representation in the primary somatosensory cortex .",
"Additional brain regions in bilateral secondary somatosensory cortex , premotor cortex , primary motor cortex , insula and posterior parietal cortex , as well as in contralateral prefrontal cortex are also shown to be activated in response to INMS .",
"The combination of INMS and 7T fMRI opens up an unprecedented opportunity to bridge the gap between first-order mechanoreceptive afferent input codes and their spatial , dynamic and perceptual representations in human cortex ."
] | [
"The skin contains multiple types of sensory nerves that inform the brain about events occurring on the surface of the body .",
"One way to study how this process works is to insert a very fine needle through the skin to stimulate a single sensory nerve with a small electrical current .",
"This technique – known as intraneural microstimulation – can activate touch responses in the brain without an object actually contacting the skin .",
"Another technique called functional magnetic resonance imaging ( fMRI ) has been used to measure brain activity .",
"These studies have revealed that when objects come into contact with the skin of the fingers , they stimulate several sensory nerves at the same time , which results in brain activity in a region called the somatosensory cortex .",
"Sanchez Panchuelo , Ackerley et al . combined fMRI and intraneural microstimulation to map brain activity in response to the activation of individual sensory nerves in the fingers of human volunteers .",
"The experiments show that intraneural stimulation activates many areas of the brain that are also activated by mechanical contact .",
"Future work will use this new method to study the brain's response to signals from different types of sensory nerves ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"ecology"
] | Little evidence that Eurasian jays protect their caches by responding to cues about a conspecific’s desire and visual perspective | elife-69647-v2 | [
[
"Theory of mind is thought to provide a causal and flexible cognitive framework that allows humans to navigate complex social interactions ( FeldmanHall and Shenhav , 2019; Tamir and Thornton , 2018 ) .",
"Through this framework , different observable social cues can be used to infer otherwise imperceptible mental states ( e . g . , perspectives , desires , knowledge , beliefs ) , such that the behaviour of other individuals can be interpreted , predicted , and manipulated based on an interplay of different mental states ( Baker et al . , 2017; Bartsch and Wellman , 1989; Bennett and Galpert , 1992 ) .",
"In humans , theory of mind is thought to emerge as a stepwise process , with a meta-representational framework in place by at least 5 years of age ( Wellman , 2018; Wellman and Liu , 2004 ) .",
"However , even before they develop this meta-representational theory of mind , infants can already respond to multiple pieces of social information in an integrated manner ( e . g . , Moses et al . , 2001; Repacholi et al . , 2014 ) .",
"Multiple accounts exist , arguing that they may do so with or without representing others’ mental states as such ( Apperly and Butterfill , 2009; Carruthers , 2020; Scott and Baillargeon , 2017; Southgate , 2019 ) .",
"At a minimum , though , infants seem to implicitly register social cues and exhibit adaptive responses accordingly ( Apperly and Butterfill , 2009; Butterfill and Apperly , 2013 ) .",
"Consequently , different mechanisms may allow individuals to integrate information from multiple social cues .",
"Inspired by Premack and Woodruff , 1978 , the past few decades have seen growing efforts to investigate whether non-human animals also possess something akin to theory of mind ( e . g . , primates: Buttelmann et al . , 2007; Drayton and Santos , 2014; Flombaum and Santos , 2005; Hare et al . , 2000; Hare et al . , 2001; Kano et al . , 2019; Krupenye and Call , 2019; dogs: Horowitz , 2011; Maginnity and Grace , 2014; corvids: Bugnyar et al . , 2016; Dally et al . , 2006; Emery and Clayton , 2001; Ostojić et al . , 2013; Shaw and Clayton , 2012 ) .",
"This issue is significant , given foundational debates in cognitive science about whether minds can represent mental state concepts in the absence of language as well as a long-standing interest in the evolution of the key cognitive traits that make us human ( e . g . , Penn et al . , 2008 ) .",
"However , within this line of research , studies most often focus on testing whether a given species/group has the ability to attribute one specific type of mental state , that is , exclusively focusing on belief , or exclusively focusing on desire , within a single study .",
"As a result , very little is known about whether other species can – like humans ( e . g . , Baker et al . , 2017 ) – integrate multiple social cues that correlate with others’ mental states and exhibit appropriate responses accordingly .",
"This question is far from trivial: in real life scenarios , an individual’s behaviour is likely to result from the interplay of multiple factors ( e . g . , their perspective , desires , and previous knowledge ) that can be indirectly perceived simultaneously by another agent during social interactions .",
"Therefore , by focusing on non-human animals’ ability to respond to a single social cue at a time , comparative psychologists may overlook a crucial aspect of social cognitive complexity .",
"Additionally , investigating whether and how other species may integrate different social cues can help shift the focus of social cognition studies in animals away from binary questions – for example does species X understand false beliefs ?",
"– towards more process-oriented and nuanced research questions , such as: what are the relative contributions of mechanisms A and B to how species X perform behaviour Y ?",
"( Buckner , 2013; Heyes , 2015 ) .",
"In doing so , this work may also identify mechanisms of varying complexity that operate in the absence of language and may feed into uniquely human social cognition .",
"Corvids are a group of large-brained birds that are hypothesised to have evolved sophisticated cognitive abilities independently from primates ( Clayton and Emery , 2015; Emery and Clayton , 2004; Güntürkün and Bugnyar , 2016; Osvath et al . , 2014; Seed et al . , 2009 ) .",
"This group represents a good model for this line of research because they might be capable of responding – independently – to social cues correlating with different types of mental states ( perspective: Bugnyar et al . , 2016; Dally et al . , 2004; Dally et al . , 2005; Legg et al . , 2016; Legg and Clayton , 2014; Shaw and Clayton , 2013; Stulp et al . , 2009; desires: Ostojić et al . , 2013 , Ostojić et al . , 2016 , Ostojić et al . , 2017Apperly and Butterfill , 2009; knowledge: Bugnyar and Heinrich , 2005; Dally et al . , 2006; Emery and Clayton , 2001 ) .",
"In particular , previous research has reported that Eurasian jays ( Garrulus glandarius ) may be able to adjust their behaviour according to cues that correlate with the perspective and current desire of a conspecific .",
"In the presence of a conspecific competitor , these jays have been reported to preferentially cache food in less visible locations ( e . g . , behind barriers , at distance ) , or in non-noisy substrates , which has been interpreted as a potential response to the visual ( Legg et al . , 2016; Legg and Clayton , 2014 ) or acoustic perspective ( Shaw and Clayton , 2013 ) of a potential pilferer .",
"In parallel , research investigating food-sharing behaviour found that male Eurasian jays change the type of food shared with their female partner , depending on which food the female has been sated on , and therefore which she desires ( Ostojić et al . , 2013; Ostojić et al . , 2014; Ostojić et al . , 2016 ) .",
"In a recent study , a similar response to another’s satiety has also been reported in the context of caching , whereby Eurasian jays and Western scrub-jays preferentially cached food that an observer , and thus potential pilferer , was sated on Ostojić et al . , 2017 .",
"Notably , the effect reported in this study was unlikely to be based on theory of mind because the caching jays showed this effect also when they did not know what food the observer was pre-fed on and when the only cue available was the observer’s behaviour during the caching event itself ( Ostojić et al . , 2017 ) .",
"Thus , taken together , these studies seem to indicate that Eurasian jays employ a variety of cache protection strategies to limit the risk of cache loss , by responding to cues correlating with the perspective or current desire of a potential pilferer .",
"Although this evidence is not sufficient to demonstrate mental state attribution , nor to pinpoint the exact underlying cognitive mechanism , it does suggest that corvids are capable of behaving flexibly on the basis of different types of social cues .",
"The aim of the current study was to investigate whether Eurasian jays can integrate multiple cues that correlate with different types of mental states to solve social problems .",
"Previous work has to some extent already , at least implicitly , tested whether animals can integrate perspective cues from different sensory modalities ( corvids: Shaw and Clayton , 2013; Stulp et al . , 2009; primates: Bray et al . , 2014; Santos et al . , 2006 ) .",
"While these studies investigate the ability to integrate different cues regarding the same type of mental state , namely another individual’s perspective in assessing the significance of visual and acoustic perceptual cues , our study concerns the integration of cues correlating with different types of mental states , namely perspectives and desires .",
"Building on previous studies in the caching context , we tested whether jays can integrate information about a conspecific’s perspective and current desire to selectively protect those caches that are at most risk of being pilfered .",
"To this end , we first conducted two experiments in which we manipulated the observer’s visual access to caching locations and its current desire for different foods .",
"We measured the caching pattern of the birds when viewed by this observer .",
"Our manipulation of the observer’s perspective followed the procedure by Legg and Clayton , 2014 .",
"The authors gave Eurasian jay cachers access to two locations , one that could and one that could not be seen from an adjacent compartment and thus by an observer bird ( when present ) .",
"One caching tray was positioned behind an opaque barrier ( out-of-view tray ) , and the other behind a transparent barrier ( in-view tray ) .",
"Legg and Clayton’s experiment encompassed three conditions: jays could cache when an observer bird was housed in the adjacent compartment – the observer was either a higher ranked individual ( Observed by a dominant condition ) or a lower ranked individual ( Observed by a subordinate condition ) – or when no conspecific was present ( Private condition ) .",
"The authors compared the jays’ caching pattern in the Private condition with that in the Observed conditions ( the two observed conditions were merged together ) , and found that jays cached a higher proportion of food items in the out-of-view tray in the Observed than in the Private conditions .",
"Our manipulation of the observer’s desire for different foods followed the procedure by Ostojić et al . , 2017 .",
"The authors investigated whether Eurasian jays and California scrub-jays can protect their caches by preferentially caching the type of food that the observer was not currently motivated to pilfer .",
"A cacher and an observer jay were housed in adjacent testing compartments .",
"In the pre-feeding phase , the observer could feed to satiety on a specific type of food: maintenance diet in the baseline trial , and either food A or B in the two test trials .",
"This procedure subsequently reduces the individual’s motivation for eating and caching that specific food ( but not different kinds of food ) , a phenomenon known as ‘specific satiety’ ( Balleine and Dickinson , 1998; Clayton and Dickinson , 1999; Dickinson and Balleine , 1994 ) .",
"Ostojić et al . , 2017 found that the jays’ preference for caching food A over food B was larger after the observer was sated on food A than after the observer was sated on food B . Interestingly , this pattern was exhibited not only in the Seen Condition – when the cacher had witnessed which particular food had been provided to the observer – but also in the Unseen Condition – when the cacher had not seen the pre-feeding of the observer – thus indicating that the observer’s behaviour at the time of caching may have played a key role in the decision-making process of the cacher .",
"Note that , when data were re-analysed for Eurasian jays only , the effect was still statistically significant in the Seen condition – although it was not statistically significant in the Unseen condition ( Crosby , 2019 ) .",
"In the current study we combined these two protocols .",
"In Experiment 1 , cacher jays were provided with only one type of food – which on one trial was the same food on which the observer sated , and on another trial was a different food from that on which the observer was sated – and two caching trays , one that the observer could see and one that the observer could not see .",
"Thus , here , the jays could choose between two caching locations , one that was in-view and one that was out-of-view of the conspecific , allowing them to selectively cache food out-of-view when it was desired by the observer .",
"In Experiment 2 , cacher jays were provided with a single caching tray – which on one trial could be seen by the observer and on another trial could not be seen by the observer – and two types of food , one of which had previously been pre-fed to the observer .",
"Thus , here , the jays could choose between two food types , one on which the observer was sated and one on which the observer was not sated , allowing them to selectively cache the less desired food when the observer could see them .",
"Consequently , the designs of the two experiments were complementary , such that jays could most effectively protect their caches by deciding where to cache in Experiment 1 , and what to cache in Experiment 2 ."
],
[
"In Experiment 1 , we tested whether jays can integrate multiple cues to decide where to hide food to protect it from being pilfered .",
"To do so , we manipulated the observer’ visual access to two caching trays through a ‘T’-shaped Perspex barrier ( henceforth T-barrier ) .",
"This T-barrier – which was the same barrier originally designed and used in Legg and Clayton , 2014 – consisted of three plastic panels: one transparent panel forming one arm of the ‘T’ and two opaque panels forming the second arm and stem of the ‘T’ ( see Materials and methods ) .",
"The T-barrier could be placed around two caching trays ( Figure",
"1 ) such that an observer could see the tray behind the transparent arm ( in-view tray ) but could not see the tray behind the opaque arm ( out-of-view tray ) .",
"To ensure that the birds ( n = 9 ) were comfortable caching in trays when these were placed in proximity of each of the two arms of the T-barrier , they initially received two familiarisation trials in private , in which only a single tray was present .",
"The tray was placed once behind the opaque and once behind the transparent arm of the barrier .",
"All birds except two reached the criterion in the familiarisation , that is they cached at least one item on each trial , and therefore proceeded to the test ( leading to n = 7 for the test ) .",
"Following the basic design of Ostojić et al . , 2017 , the test trials comprised a pre-feeding phase and a caching phase ( Figure 1 ) .",
"In the pre-feeding phase , the cacher jay could see a conspecific ( the observer ) eat a specific type of food ( macadamia nuts , M , or peanuts , P ) in an adjacent indoor compartment .",
"In the subsequent caching phase , the cacher jay was presented with two caching trays , each placed behind one of the two arms of the T-barrier , and was allowed to cache while the observer jay was still present in the adjacent compartment .",
"Birds were tested in two conditions ( one trial per condition; Figure 1 ) .",
"In the Different Food Condition , the type of food received by the observer in the pre-feeding phase differed from the type of food received by the cacher in the caching phase ( e . g . , M for the observer , and P for the cacher ) .",
"In the Same Food Condition , the observer and the cacher received the same type of food ( e . g . , P for the observer , and P for the cacher ) .",
"If the jays can integrate information from the different cues correlating with the observer’s desire and perspective , their caching pattern should meet two predictions .",
"First , their preference for caching in the out-of-view tray should be greater in the Different Food condition than in the Same Food condition .",
"This is because it is in the Different Food condition that the observer has a stronger desire toward the cacher’s food , such that the caches would be more at risk from being stolen in this condition .",
"Second , in the Different Food condition , the cacher should exhibit a clear preference for caching in the out-of-view tray , therefore in this condition the amount of caches in the out-of-view tray should be higher than expected by chance , namely if the cacher distributed its caches randomly across the out-of-view and the in-view trays .",
"The jays’ preference to cache in a specific location can be indexed either as a proportion of the items cached in the out-of-view tray ( over the items cached in both trays ) or as a difference score of the number of items cached in the out-of-view tray minus the number of items cached in the in-view tray .",
"Because robust findings would necessitate that the two different indices do not lead to drastically different results , we conducted the analyses with both indices ( for details see Analysis in the Materials and methods section ) .",
"The results from the analyses with both indices were consistent .",
"In contrast to the first prediction , a comparison of the proportion of items cached in the out-of-view tray between the Different Food and the Same Food conditions did not detect a statistically significant difference ( Wilcoxon signed-rank test: n = 7 , W = 13 , P = 0 . 21; Figure 1 ) .",
"In contrast to the second prediction , the proportion of items cached in the out-of-view tray was not significantly different from that expected by chance in the Different Food condition ( one-sample Wilcoxon signed-rank test: n = 7 , W = 25 , p=0 . 11 ) .",
"As an additional analysis , we conducted the same comparison in the Same Food condition and also detected no statistically significant difference from chance ( one-sample Wilcoxon signed-rank test: n = 7 , W = 25 , p=1 ) .",
"The same pattern was found when the difference score – number of items cached in the out-of-view tray minus the number of items cached in the in-view tray , [Cachesout-of-view – Cachesin-view] – was used .",
"No statistically significant difference in the difference score was detected between the Different Food and the Same Food conditions ( Wilcoxon signed-rank test: n = 7 , W = 9 , p=0 . 40; Figure",
"1 ) and a comparison of the difference score to chance ( i . e . ,",
"0 ) detected no statistically significant difference in the Different Food condition ( one-sample Wilcoxon signed-rank test: n = 7 , W=-7 , p=0 . 61 ) .",
"The same comparison in Same Food condition also detected no statistically significant difference from chance ( one-sample Wilcoxon signed-rank test: n = 7 , W = 1 , p=1 ) .",
"Notably , the numerical pattern of the jays’ caching was in the opposite direction to the prediction: jays’ caching was more biased towards the out-of-view tray in the Same Food than the Different Food condition ( Proportion of items cached out-of-view: MedianSame Food = 0 . 5 , MedianDifferent Food = 0 . 17; Caches out-of-view minus Caches in-view: MedianSame Food = 0 , MedianDifferent Food = –2 ) .",
"Consequently , the observed data in Experiment 1 cannot be interpreted as supporting the conclusion that the jays were integrating the information from multiple cues to protect the caches that are most at risk of being pilfered by a conspecific .",
"In Experiment 2 , we used a complementary design to test whether jays can integrate multiple cues to decide which type of food to hide to protect their caches from being pilfered .",
"To do so , jays had access to one caching location at a time ( either in-view or out-of-view to the observing conspecific ) and two types of food ( one on which the observer was sated and one on which the observer was not sated ) .",
"Following the general structure of Experiment 1 , in the pre-feeding phase , the cacher bird was first able to see an observer eat one particular type of food ( macadamia nuts or peanuts ) to satiety .",
"In the subsequent caching phase , the cacher bird was presented with a single caching tray and the two types of food ( macadamia nuts and peanuts ) .",
"To manipulate the observer’s visual access to the caching location , the tray was placed behind an ‘U'-shaped Perspex barrier ( henceforth U-barrier ) that consisted of two lateral panels and one central panel forming two angles of approximately 45° ( see Materials and methods; Figure 2 ) . The U-barrier was either transparent , thereby allowing the observer to see the caching location , or opaque , thereby preventing the observer from seeing the caching location . Birds ( n = 8; for details see Materials and methods ) first received two familiarisation trials in private to ascertain that they were comfortable caching both types of food in a tray placed in proximity of each of the barriers . All birds except one reached the familiarisation criteria , namely to cache: ( 1 ) at least one item of both types of food across the two trials , and ( 2 ) at least one item ( of any type of food ) in a tray placed in proximity of both the transparent and the opaque U-barrier . These birds ( n = 7 ) were subsequently tested with the transparent barrier ( In-view condition ) and with the opaque barrier ( Out-of-view condition ) . In each condition , the birds received two trials , one in which the observer was pre-fed one type of food and one in which it was pre-fed the other type ( Figure 2 ) . If the jays can integrate information from the different cues available and which should correlate with the observer’s desire and perspective , their caching pattern might be expected to meet two predictions . First , the jays’ preference to cache P when the observer was sated on P relative to when the observer was sated on M , should be higher in the In-view than in the Out-of-view condition .",
"This is because it is in the In-view condition that the observer can see the caching locations such that here the caching bird could protect its caches by caching preferentially more of the food that the observer is sated on .",
"Second , in the In-view condition , the preference to cache P should be higher when the observer was sated on P than when the observer was sated on M . As in Experiment 1 , both proportion and difference scores were used as indexes to analyse the birds’ preference ( for details see Analysis in the Materials and methods section ) .",
"Again , the results from the analyses using both indices were consistent .",
"A comparison of the proportion of P cached between the In-view and the Out-of-view conditions – [Pcached/ ( Pcached+ Mcached ) ]pre-fed P – [Pcached/ ( Pcached+ Mcached ) ]pre-fed M – did not detect a statistically significant difference ( MedianIn-view = 0 , MedianOut-of-view = 0; Wilcoxon signed-rank test: n = 7 , W = 3 , p=0 . 83; Figure 2 ) .",
"In the In-view condition , no statistically significant difference in the proportion of P cached could be detected between the trials in which observer was sated on peanuts and the trials in which the observer was sated on macadamia nuts ( Medianpre-fed P = 0 . 33 , Medianpre-fed M = 0 . 5; Wilcoxon signed-rank test: n = 7 , W = 3 , p=0 . 83 ) .",
"Thus , neither prediction could be supported .",
"An additional analysis of the same comparison for the Out-of-view condition also did not detect a statistically significant difference in the proportion of P cached between the trials ( Medianpre-fed P = 0 , Medianpre-fed M = 0 . 06; Wilcoxon signed-rank test: n = 7 , W = 1 , p=1 ) .",
"The same pattern of results was found when the jays’ preference was analysed using the other index , namely difference scores .",
"No statistically significant difference in the preference to cache P over M when the observer is sated on P relatively to when the observer is sated on M – that is the difference of difference score: [Pcached – Mcached]pre-fed P – [Pcached – Mcached]pre-fed M – was detected between the In-view and the Out-of-view conditions ( MedianIn-view = –1 , MedianOut-of-view = 0; Wilcoxon signed-rank test: n = 7 , W = −4 , p=0 . 80; Figure 2 ) .",
"Furthermore , in the In-view condition , no statistically significant difference was detected between the trials in which observer was sated on peanuts and the trials in which the observer was sated on macadamia nuts ( Medianpre-fed P = –1 , Medianpre-fed M = 0; Wilcoxon signed-rank test: n = 7 , W=-3 , p=0 . 86 ) .",
"The additional analysis of the same comparison for the Out-of-view condition also did not detect a statistically significant difference between trials ( Medianpre-fed P = –1 , Medianpre-fed M = –1; Wilcoxon signed-rank test: n = 7 , W=-1 , p=1 ) .",
"Consequently , as in Experiment 1 , the observed data cannot be interpreted as support for the hypothesis that jays could integrate the information from multiple cues to protect their caches when these were most at risk of being pilfered by a conspecific .",
"The two experiments thus yielded consistent results .",
"However , a clear interpretation of the results is impeded by the likely low power of our designs to detect the smaller effect sizes that would be consistent with jays integrating both cues .",
"This is likely due to the small sample size and limited number of trials per condition of our experiments , two features that – despite being relatively representative of the research in this area , including the previously published studies on this topic – may have produced imprecise estimates ( Farrar et al . , 2020; Farrar and Ostojic , 2019 ) .",
"Therefore , to strengthen our confidence that Eurasian jays may not be able to integrate multiple cues to protect their caches , it will be essential for future research to conduct additional studies , ideally by employing larger sample sizes and procedures that can increase the precision of the analyses .",
"Although birds could have used multiple cues to guide their caching decisions in Experiments 1 and 2 , they could also have adjusted their caching preference according to just one single type of cue , that is either the cues correlating with the observer’s desire or the cues correlating with the observer’s perspective .",
"Both experiments used an experimental manipulation that , when applied separately , has already been reported – in previous studies – to have elicited a behavioural response that has been interpreted as a cache-protection strategy .",
"Specifically , the caching phase of Experiment 1 involved the same procedure and set-up used by Legg and Clayton , 2014 , except for the specific types and quantities of food provided to the jays .",
"Similarly , the In-view condition of Experiment 2 and the Seen condition of Ostojić et al . , 2017’s experiment employed the same procedure , with the exception that in the former the observers could see the caching location through a transparent barrier , whereas in the latter no barrier was present .",
"However , in contrast to these previous studies , the results obtained in our experiments did not show a directional caching pattern in the predicted direction in these situations .",
"Again , this may be a result of the low statistical power of our design , or possibly from the greater demands associated with tracking and integrating multiple cues to inform decision-making .",
"However , the inconsistencies with previous research could also be due to previously reported effects not being reliable enough to form the basis of follow-up studies .",
"Therefore , we conducted three further experiments to explore the reliability of the effects reported by Legg and Clayton , 2014 and Ostojić et al . , 2017 .",
"In Experiments 3 and 4 , we attempted a replication of Legg and Clayton , 2014’s findings .",
"In Experiment 3 , birds were tested in one trial per condition , mirroring Experiment 1 , while in Experiment 4 , we conducted a complete direct replication of the original study that encompassed two trials in the Private condition and two trials in each of the two Observed conditions .",
"Finally , in Experiment 5 , we tested whether the presence or absence of a transparent barrier – that is the minor difference in the set-up between Experiment 2 and Ostojić et al . , 2017’s experiment – may have affected the Eurasian jays’ response in this caching situation .",
"In Experiment 3 , we investigated whether jays use information about an observer’s visual perspective to protect their caches in a simplified version of Legg and Clayton , 2014’s experiment , that is the jays received only one trial in each of the two testing conditions .",
"This mirrors the procedure in Experiment 1 , where the same set-up was used and only one trial per testing condition was conducted .",
"Following the original study , we presented cacher jays with two caching trays and manipulated the observer’s visual access to cache locations by using the T-barrier .",
"However , while Legg and Clayton , 2014 tested jays in three conditions – Observed by Dominant , Observed by Subordinate and Private – and gave them two trials in each condition , in Experiment 3 , jays ( n = 8 ) received only two trials: one with a conspecific present in the adjacent compartment ( Observed condition ) and one with no conspecific present ( Private condition ) .",
"Seven birds met the inclusion criterion ( see Material and methods for details ) .",
"The two analyses with the different indices yielded consistent results .",
"The proportion of the items cached in the out-of-view tray was not significantly higher in the Observed condition than in the Private condition ( MedianObserved = 0 . 71 , MedianPrivate = 0 . 54; Wilcoxon signed-rank test , n = 7 , W = 2 , pone-tailed = 0 . 59 ) .",
"The same pattern was found when the difference score , that is , the number of items cached in the out-of-view tray minus the number of items cached in the in-view tray [Cachesout-of-view – Cachesin-view] , was analysed .",
"The difference score was not significantly higher in the Observed than in the Private condition ( MedianObserved = 1 , MedianPrivate = 0 . 5; Wilcoxon signed-rank test , n = 7 , W = 11 , pone-tailed = 0 . 84 ) .",
"In a subsequent experiment , Experiment 4 , we conducted a direct replication of Legg and Clayton , 2014’s experiment .",
"Here , the design and procedure were identical to those of the original study ( see Materials and methods; Figure 3 ) .",
"Thus , in contrast to Experiment 3 , in Experiment 4 , jays were tested in three conditions – Observed by Dominant , Observed by Subordinate and Private – and received two trials in each condition .",
"For this experiment only , we also tested the same colony of jays that originally participated in Legg and Clayton , 2014’s experiments .",
"Because these birds had not recently participated in testing using the experimental set-up employed here and the T-barrier , we first conducted a familiarisation that followed the same procedure as that used in Experiment 1 .",
"Nine birds passed the familiarisation and proceeded to the test .",
"In this experiment only , we conducted the same analyses as for all other experiments ( i . e . , Wilcoxon signed-rank tests ) but also an additional one , namely the same analysis ( permutation tests for paired data ) that was also used in Legg and Clayton , 2014 .",
"Again , a strong claim of an effect would require consistent results regardless of the analyses used .",
"In line with the original study , we found that the average number of total items cached across both trays was not significantly higher when the jays were observed by a conspecific than when they were in private ( Permutation test , n = 9 , Z = 0 . 79 , p=0 . 43 ) .",
"Two birds cached no items in any of the Private and Observed trials , thereby they were excluded from further analyses of proportion scores because , given their performance , it was not possible to compare the proportion of items cached in the out-of-view tray between conditions .",
"In the same analysis that was used by Legg and Clayton , 2014 , the average proportion of items cached in the out-of-view tray was not significantly higher in the Observed condition than in the Private condition ( MedianObserved = 0 . 5 , MedianPrivate = 0 . 44; Permutation test , n = 7 , Z = 0 . 15 , pone-tailed=0 . 56 ) .",
"The same results were found in the two analyses that used the same statistical test as in the other experiment reported in this study: average proportion of items cached in the out-of-view tray ( Wilcoxon signed-rank test , n = 7 , W = 2 , pone-tailed=0 . 59 ) ; average difference of the number of items cached in the out-of-view tray minus the number of items cached in the in-view tray ( MedianObserved = 0 , MedianPrivate = –0 . 5; Wilcoxon signed-rank test , n = 9 , W = 13 , pone-tailed=0 . 20 ) .",
"Taken together , Experiments 3 and 4 consistently did not detect the effect originally reported by Legg and Clayton , 2014 , whereby Eurasian jays adjusted their caching pattern to the transparency and opaqueness of the barrier around the caching tray specifically when an observer was present during the caching event ( Figure 3 ) .",
"In addition , the results from Experiments 3 and 4 appear consistent with the negative results from the Different Food condition in Experiment 1 .",
"In Experiment 5 , we investigated whether a minor difference in the set-up , that is the presence of a transparent barrier , may have caused the inconsistency in the results between Experiment 2 and the results reported in Ostojić et al . , 2017’s study .",
"To this end , we employed the same experimental set-up and procedures used in Experiment 2 , except that here , in one condition , jays were presented with the transparent U-barrier ( Barrier condition ) and in another condition , with no barrier ( No-barrier condition ) .",
"All birds ( n = 8 ) passed the familiarisation .",
"In the test , one bird consistently cached no items , such that data of seven birds were analysed ( see Materials and methods for details ) .",
"The two analyses using the two different indices yielded consistent results ( Figure 4 ) .",
"No statistically significant difference could be detected in difference of the proportion of P cached when the observer was sated on P minus the proportion of P cached when observer was sated on M – [Pcached / ( Pcached+ Mcached ) pre-fed P] – [Pcached / ( Pcached+ Mcached ) pre-fed M] – between the Barrier and No-barrier conditions ( MedianBarrier = 0 , MedianNo Barrier = −0 . 04; Wilcoxon signed-rank test: n = 7 , W = 11 , p=0 . 18 ) .",
"In addition , in both conditions , no statistically significant difference could be detected in the proportion of P cached between the two pre-feeding trials ( Barrier condition: Medianpre-fed P = 0 . 17 , Medianpre-fed M = 0 . 1 , Wilcoxon signedrank test , n = 7 , W=-2 , pone-tailed=0 . 43; No-barrier condition: Medianpre-fed P = 0 , 12 , Medianpre-fed M = 0 , 25 , n = 7 , W=-9 , pone-tailed=0 . 91 ) .",
"The same patterns of results were observed when the difference score of the number of P cached minus the number of M cached was analysed .",
"No statistically significant difference could be detected in the differences of difference score – [Pcached – Mcached]pre-fed P – [Pcached – Mcached]pre-fed M – between the Barrier and No-barrier conditions ( MedianBarrier = 0 , MedianNo Barrier = 0; Wilcoxon signed-rank test: n = 7 , W = 15 , p=0 . 14 ) .",
"In addition , we detected no statistically significant in the difference score between the two pre-feeding trials , in either condition ( Barrier condition: Medianpre-fed P = –4 , Medianpre-fed M = –5 , Wilcoxon signed-rank test , n = 7 , W = 1 , pone-tailed = 0 . 50; No-barrier condition: Medianpre-fed P = –4 , Medianpre-fed M = –1 , Wilcoxon signed-rank test n = 7 , W=-9 , pone-tailed = 0 . 91 ) .",
"Thus , the results from Experiment 5 cannot be interpreted as providing support for the idea that the presence of the barrier may be the reason why the results in Experiment 2 did not detect the same pattern as the one reported in Ostojić et al . , 2017 .",
"Crucially , like in the In-view condition in Experiment 2 , both conditions in Experiment 5 also consistently could not detect the effect reported in Ostojić et al . , 2017 ."
],
[
"In Experiments 1 and 2 , we investigated whether Eurasian jays can take into account two types of social cues simultaneously and perform the most advantageous behavioural output accordingly .",
"Specifically , we tested whether caching birds can integrate information from cues correlating with a conspecific observer’s desire and perspective to most effectively protect their caches .",
"Consistently across these two experiments , we did not detect effects that would support such integration of information from different cues .",
"In Experiment 1 , jays did not show a higher preference for caching in the out-of-view tray when they could cache a food that was highly desired by the observer relative to when they could cache a food that was less desired by the observer .",
"In addition , in the former case ( i . e . , Different Food condition ) , jays did not cache more in the out-of-view tray than expected by chance .",
"In Experiment 2 , jays did not show a higher preference for caching the food for which the observer had a decreased desire when the observer could see them relative to when the observer could not see them .",
"In addition , in the former case ( i . e . , In-view condition ) , jays did not cache the food for which the observer had a decreased desire more than the food for which the observer had a higher desire .",
"The negative results we obtained in both experiments appear inconsistent with previous effects in the literature , despite the use of set-ups that were very similar to those used in the original studies .",
"Specifically , the negative results in the Different Food condition in Experiment 1 appear incompatible with the effect reported in Legg and Clayton , 2014 , where jays were found to preferentially cache in an out-of-view tray specifically when they were observed by a conspecific .",
"Similarly , the negative results in the In-view condition in Experiment 2 appear incompatible with the effect reported in Ostojić et al . , 2017 , where jays were found to preferentially cache a specific food when the observer was pre-fed on that food relative to when the observer was pre-fed on a different food .",
"Thus , we conducted three follow-up experiments to explore the reliability of the two previous findings ( Legg and Clayton , 2014; Ostojić et al . , 2017 ) that our first two experiments were built on .",
"In Experiments 3 and 4 , we attempted to replicate the effect reported by Legg and Clayton , 2014 , but – in contrast to the original study – no statistically significant difference between the experimental conditions was detected .",
"Similarly , in Experiment 5 no statistically significant difference was detected between the experimental conditions , a result that contrasts with the effect reported by Ostojić et al . , 2017 .",
"Thus , Experiments 3–5 also yielded negative results .",
"However , evaluating the ‘success’ of a replication study from the statistical significance of a finding alone is overly simplistic , particularly for comparative cognition research , where – like in our experiment – the sample size of replication studies are often as equally small as that of the original studies ( Farrar et al . , 2020 ) .",
"Nevertheless , the finding that we could not detect any significant effect in line with the original experiments of Legg and Clayton , 2014 and Ostojić et al . , 2017 across all five of our experiments was surprising , especially given that they were conducted in the same lab , with many of the same birds and experimenters .",
"Specifically , in four of the seven tests of the hypothesis that the jays could use social cues to protect their caches , the results were not in the direction of the prediction: Experiment 1 , prediction 1; Experiment 2 , predictions 1 and 2; Experiment 5 , prediction 2 , No-barrier condition .",
"In the remaining three tests in which we had a directional prediction – Experiments 3 and 4 , and Experiment 5 prediction 2 , Barrier condition – the effects were in the correct direction but were non-significant and much smaller than the previously reported effects .",
"It is not possible to provide a single , clear answer for why our study was unable to detect effects that are consistent with the previous literature .",
"We propose two explanations that could have played a role , namely low power and our re-use of a unique sample of birds whose behaviour may change across time .",
"First , each of our experiments likely had low power to detect theoretically meaningful , and perhaps the most theoretically plausible , effect sizes .",
"This means that even if the jays were responding to the perspective and/or desires of the observer during our experiments , the effect size might have just been too small for us to detect .",
"However , that we failed to find any significant results across five experiments with similar designs to the original studies shows that no extremely large effect sizes were present in our jays and suggests that the original effect sizes might be overestimated – which would be expected if the original studies came from a series of underpowered research combined with a publication bias ( Farrar et al . , 2020; Fiedler and Prager , 2018; Hedges , 1984 ) .",
"Second , our five studies used the same populations of birds as tested in the previous studies ( Legg and Clayton , 2014; Ostojić et al . , 2017; Table 1 ) , but they were around 5 years older .",
"It is possible that the behaviour of these birds has changed over time , for example as a result of aging and/or learning effects .",
"That is , the original studies might have reported effects without much overestimation , but when we tested the jays for these experiments , they may no longer have had the motivation to protect their caches .",
"The overall number of cached items ( as a proxy for cache motivation ) does not appear too dissimilar in the current experiments and the previous studies ( see Figure 5 for a comparison of the number of items cached per bird per trial across all of the experiments ) .",
"Thus , any differences relating to motivation of the birds would have to be specific to cache protection , rather than just caching .",
"With regard to the potential motivational changes due to ageing , it is possible that ontogenetic variations in sociality could influence the performance in caching tasks .",
"Although data on corvids specifically are limited , research in primates has shown that motivations potentially relevant to cache protection , such as those driving socialisation and social influence , change dramatically throughout life , declining substantially in some taxa ( Machanda and Rosati , 2020 ) .",
"A notable example of this process is provided by rhesus macaques , which , similarly to humans , show a reduced propensity to follow gaze in older age ( Rosati et al . , 2016 ) .",
"When it comes to corvids , the only data at the moment concern age-related shifts in non-social predisposition , such as neophobia ( Greggor et al . , 2020 ) .",
"Thus , age-related changes in the jays’ motivation to attend to others’ social cues are speculative , but in light of the negative results reported here , may present a line of enquiry for future studies .",
"With regard to potential changes due to prior experience and learning , previous studies with Western scrub-jays suggested that the caching behaviour itself is underpinned by a motivational system that acts as a compulsion to cache and that is not sensitive to reinforcement or other external conditions and that a second motivational system influences decision making during caching through sensitivity to conditions at cache retrieval ( Clayton et al . , 2005; de Kort et al . , 2007 ) .",
"In our laboratory , caching experiments are conducted such that an observer is located in an adjacent compartment and cannot access the conspecific’s caches , and in retrieval sessions the jays also receive feedback that their caches are unaffected by conditions at caching .",
"If the jays’ cognitive system tracks conditions at retrieval , then all experiments in which caches are unaffected , i . e . , when jays regularly find them intact in the retrieval phase of a study , may favour a decrease in the birds’ motivation to employ cache-protection strategies in the next similar situation .",
"Thus , it cannot be excluded that the very first time when jays are presented with a novel set-up , their motivation to protect caches is higher than in subsequent presentations , but at the moment this must remain a speculation until studies directly test this possibility .",
"As such , already Experiments 1 and 2 but especially the replication attempts in Experiments 3–5 may have low validity , despite them using natural caching behaviours .",
"This issue is likely exacerbated by the fact that our Eurasian jays regularly cache outside of testing time , such that laboratory conditions do not impair the natural intensity of caching behaviour exhibited by this species ( Goodwin , 1951; Goodwin , 1986 ) .",
"In particular , our jays frequently save several food items ( e . g . , nuts , insect larvae ) to take with them ( e . g . , store items in the crop ) when released from the testing compartments to be cached in the aviary , as well as cache maintenance food ( e . g . , seeds , vegetables , eggs ) during their normal daily activities given that their aviaries offer plenty of opportunities .",
"This behaviour is in line with one of the experiments reported by de Kort et al . , 2007 in which the scrub-jays learned to inhibit caching at one location from which caches were pilfered if another location was available immediately afterwards in which caches were left intact .",
"To investigate the possible influences on the jays’ motivational systems described above , one would ideally use a new sample of birds lacking an extensive experimental history with caching experiments .",
"Unfortunately , it is unlikely that a study like the one above could be conducted in a near future on the same species of corvids because Eurasian jays are not currently housed in any other laboratory in the world , and there is no guarantee that a new colony will be acquired by our laboratory .",
"There seem to be few other ways to test the motivation of our jays to display cache-protection behaviours because to validate their motivation to protect caches would require them to demonstrate the cache-protection behaviours that we set out to test .",
"From negative results alone , it is unlikely that we can dissociate the possibility that the jays cannot display cache-protection behaviours from the possibility that the jays are not motivated to display them .",
"Ultimately , we believe that these issues around the interplay between cognitive and motivational systems are not trivial and that they will need to be considered – alongside issues of low power – by researchers working in the field when addressing issues of reliability and validity of results , including replication attempts , especially given that many studies in the field of comparative cognition also rely on re-using the same samples of animals .",
"Our results conflict not just with the two studies that our research was built on Legg and Clayton , 2014; Ostojić et al . , 2017 , but also , more generally , with a larger body of literature on cache-protection strategies in corvids ( Bugnyar et al . , 2016; Dally et al . , 2004; Dally et al . , 2005; Emery and Clayton , 2001; Heinrich and Pepper , 1998 ) and Eurasian jays in particular ( Legg et al . , 2016; Shaw and Clayton , 2013 ) .",
"We were unable to elicit the cache-protection strategies that this literature implies are consistently observable across corvid species , including in our Eurasian jays .",
"In addition to the two possible explanations raised above , it is important to discuss a third possibility .",
"While it is possible our findings were only local failures to find these effects , it is also possible that the general research practices and methods that have produced the corvid social cognition literature are liable to producing unreliable findings or overestimated effect sizes .",
"If the findings of the present study were shown to be indicative of a broader pattern in the field , it could be necessary to substantially revise our current understanding of corvid social cognition and their exhibition of particular cognitive phenomena .",
"Crucially however , while our data clearly show a local failure to elicit previously reported effects , these data are not sufficient to draw strong conclusions beyond these local failures .",
"This means that , despite posing a challenge to the reliability of the effects described by Legg and Clayton , 2014 and Ostojić et al . , 2017 , in the absence of broader negative results , our findings cannot confute the notion that Eurasian jays are capable of employing cache-protection strategies by responding to cues about the visual perspective and current desire of a conspecific .",
"Furthermore , in the lack of additional negative findings in other corvids or about different cognitive phenomena in the Eurasian jays , it is unclear to what extent our findings allow us to make inferences beyond the species and focus of this study .",
"For instance , the fact that we could not find support that Eurasian jays respond to cues about others’ current mental states in the caching context does not necessarily challenge the reliability of the evidence about similar behaviours in other contexts .",
"For example , in the cooperative context of food sharing , the effect whereby males adjust the pattern of food shared with the female to her specific satiety ( Ostojić et al . , 2013 ) has been reliably shown by the same males in subsequent studies ( Legg , 2015; Ostojić et al . , 2014; Ostojić et al . , 2016 ) .",
"One difference between the cache-protection and the food-sharing context may be the motivation of the jays to reliably exhibit the behaviour in question .",
"Food sharing is an integral part of jays’ courtship behaviour and is important not only in establishing but also maintaining the pair bond ( Goodwin , 1951 ) .",
"Thus , males are likely to be motivated to respond to what the female likes or wants to eat across different time points .",
"In contrast , as discussed earlier , after jays’ experience that their caches are not affected by any perceived risks , the motivation to employ cache-protection strategies in a very similar context may decrease .",
"We currently do not know how many other studies have produced negative cache-protection results but have not been published , and understanding the magnitude of the publication bias ( Fanelli , 2012; Scheel et al . , 2020 ) in this literature is therefore a necessary step to evaluating the evidential strength within the field .",
"Concerning our failed replication of Ostojić et al . , 2017 , a slightly different reasoning applies as , to our knowledge , no similar study has been conducted in another laboratory .",
"As such , we believe we have access to all the data on this topic .",
"These are the current study , the Ostojić et al . , 2017 study , and a further , unpublished , replication attempt that also did not detect the originally reported effects ( Crosby , 2019 ) .",
"Overall , the data on these effects seem too uncertain to draw any firm conclusions about Eurasian jay cognition .",
"Our difficulty with replicating previous research , even in the same laboratory as the original findings and with many of the same birds and experimenters , highlights two ways in which research on corvid social cognition – and likely , on comparative cognition research more broadly – could make progress .",
"First , understanding the extent of publication bias in our literature is key to understanding their evidential value .",
"Retrospectively , this may be achieved through meta-analysis techniques , and prospectively through effective pre-registering of hypothesis-testing research .",
"Second , before building on findings , researchers can , where appropriate , include reliability tests into their research programmes .",
"For example , replication and extension studies , conducted in a two-step process , such as have been traditionally employed in comparative cognition ( Beran , 2018 ) , present a useful procedure .",
"First , the replication stage of the study tests whether an effect previously reported in the literature can be elicited under the circumstances ( e . g . , specific sample of individuals , testing facility or methodological procedure ) in which the novel experimental question will be investigated .",
"Only as a second step , the main study , the extension part , is performed .",
"Such reliability tests may be especially important for previous findings where the effects of publication bias are unknown , as well as when the same animals are tested in follow-up tests ( as is the case in this study ) to probe the reliability of the behavioural patterns over time .",
"To this end , particular efforts should be put into the designing of experiments , for instance by favouring within-subject designs as a way to account for individual-level variation , and by increasing the power of analyses through designs encompassing a larger number of test trials .",
"We believe that these approaches are necessary to strengthen or reshape our understanding of corvid cognition .",
"For instance , if subsequent evidence in Eurasian jays backs up the results of the current study , then belief that these corvids take into account information about the perspective or desire of a conspecific to protect their caches would diminish .",
"However , because of the low likelihood of independent replication of many studies in comparative cognition , it is questionable whether it is possible to produce data that convincingly refute previous claims ( Boyle , 2021 ) .",
"This situation in which some important and influential claims in the literature cannot be directly assessed presents a substantial challenge for comparative cognition research and warrants special and careful consideration and discussion within the field .",
"In conclusion , the current study presents five experiments that are inconsistent with the previous literature on caching in Eurasian jays .",
"Across all experiments , the effects were non-significant , and often in the opposite direction to predictions derived from the published literature .",
"This suggests that previous effect sizes are likely overestimated , or at the very least , that the effects cannot be consistently elicited in the same or similar samples of birds .",
"Therefore , future studies should consider this new evidence when referring to the original studies for subsequent inferences about corvid social cognition .",
"In Experiments 1 and 2 , we investigated a follow-up question that assumed the reliability of previously reported statistical effects , which we later could not replicate in Experiments 3 , 4 , or 5 .",
"The current series of experiments demonstrate the necessity to investigate the uncertainty of such effects and to adjust the claims – including those in previously published literature – accordingly .",
"In regard to the behavioural effects investigated in this study , the caching patterns interpreted as cache-protection strategies in Legg and Clayton , 2014 and Ostojić et al . , 2017 do not seem to be reliable enough to form the basis for follow-up studies such as the ones reported in Experiments 1 and 2 , at least in our sample of jays .",
"It would be informative , but unfortunately not currently possible , to replicate these studies at other laboratories across the world ."
],
[
"Fourteen adult Eurasian jays from two separate colonies were tested in this study ( Table 1 ) .",
"Most of the jays took part in multiple experiments and had previously been tested in experiments that involved caching in a similar set-up as that used in the current study ( details about which jay participated in which experiment ( s ) are given in Table 1 ) .",
"All of the jays were hand-raised , having been taken as chicks from wild nests or from the natural nests of birds in a breeding programme .",
"The birds from each colony were housed as a group in large outdoor aviaries each measuring 20 m long × 10 m wide × 3 m high in Clayton’s Comparative Cognition Lab at the Sub-Department of Animal Behaviour , University of Cambridge , Madingley , UK .",
"At one end , the aviaries were divided such that birds had access to multiple smaller aviaries ( approximately 6 × 2 × 3 m ) and from these smaller aviaries birds could access indoor ( colony",
"1 ) or fully sheltered ( colony",
"2 ) testing compartments ( 2 × 1 × 3 m ) .",
"Birds of colony 2 were housed in pairs in indoor cages until 2009 or 2010 .",
"Outside of testing the birds had ad libitum access to their maintenance diet of vegetables , eggs , seed , and fruits .",
"Water was available at all times .",
"All procedures were approved by the University of Cambridge Animal Ethics Committee ( reference n . ZOO35/17 ) .",
"Birds were tested in the testing compartments measuring 2 m long × 1 m wide × 3 m high , which were accessible from the smaller aviaries through flap windows .",
"In trials requiring the presence of an observer , two birds – a cacher bird and an observer bird – were located in adjacent compartments .",
"These compartments were separated by wire mesh and additional opaque sheeting .",
"A little mesh window ( 30 × 55 cm ) was not covered by the opaque sheeting and through it the birds had visual access to the adjacent compartment .",
"Testing compartments contained a suspended platform ( 1 × 1 m ) approximately 1 m from the ground , onto which food bowls , caching trays , and Perspex barriers could be placed .",
"Each type of food used in the experiments was presented in a bowl of a specific colour , and these colours were kept consistent for all birds to minimise the likelihood of experimenter errors .",
"Rectangular seedling trays ( 5 × 3 pots filled with sand ) were used as caching trays .",
"Trays were painted different colours and were trial specific to minimise the probability that birds’ caching behaviour in one trial would be influenced by its memory from previous trials .",
"In Experiments 1 , 3 , and 4 , a T-barrier was used to manipulate the observer’s visual access to the caching trays .",
"It was the same T-barrier that Legg and Clayton , 2014 used .",
"This barrier consisted of three plastic panels ( 25 × 40 cm ) forming two arms and one stem .",
"One arm of the ‘T’ was constructed out of transparent Perspex , while the other arm and the stem were constructed out of white opaque Perspex .",
"The T-barrier could be placed around two caching trays in the cacher’s compartment , such that the observer could see the tray behind the transparent arm ( in-view tray ) but could not see the tray behind the opaque arm ( out-of-view tray ) .",
"Due to the height of the barrier , the observer could always see the cacher when the latter was standing upright in proximity to the trays .",
"However , the observer could not see the exact location where the cacher hid the food when it was caching in the out-of-view tray .",
"In Experiments 2 and 5 , a U-barrier was used to manipulate the observer’s visual access to the caching trays .",
"The barrier consisted of two lateral Perspex panels ( 26 × 25 cm ) and one central Perspex panel ( 53 × 25 cm ) forming two angles of approximately 45° .",
"In Experiment 2 , we used two U-barriers , one made of transparent Perspex and another made of white opaque Perspex .",
"In Experiment 5 , only the transparent barrier was used .",
"The U-barrier was placed around a single tray in the cacher’s compartment , and if opaque , it impaired the observer’s visual access to the caching tray .",
"In all experiments , the birds’ maintenance diet was removed from the aviary approximately 1 . 5 hr prior to the start of each trial to ensure that the birds were mildly hungry and thus likely to interact with food provided during testing .",
"During the pre-feeding phase , cachers ( n = 7; Table",
"1 ) could see a conspecific eat a specific type of food: either the same type of food they were going to receive in the subsequent caching phase ( Same Food condition ) or a different one ( Different Food condition; Figure 1 ) .",
"The order in which the birds experienced the Different Food and Same Food conditions was counterbalanced across birds .",
"In the subsequent caching phase , cachers were provided with the same food used in the familiarisation and with two caching trays , each one placed behind one of the two arms of the T-barrier ( Figure 1 ) .",
"The food given to the observer during the pre-feeding phase and to the cacher during the caching phase was either 50 macadamia nut halves or 50 whole peanuts with skin .",
"All birds received one trial per condition , that is , two test trials in total .",
"If a bird cached no items on both trials , it was paired with a different observer and the two trials were repeated .",
"If it again did not cache on both trials , these data were not included in the analysis .",
"In contrast , if the bird cached with the second observer , then these data were included in the analysis .",
"This procedure was decided during data collection , after one bird ( Lima ) did not cache any food across both trials , but before the analysis was conducted .",
"For all other birds , test trials were not repeated .",
"The analysis included the data of all seven birds .",
"Experiment 1 was conducted from October to November 2017 by LO , BF , and PA .",
"In all experiments , we recorded the number and type of food items cached on each trial by manually checking the trays .",
"The experimenters were not blind to the conditions while counting the food items .",
"These data were used to test whether the birds had a preference for caching a specific type of food or for caching in a specific tray .",
"In all experiments , we also recorded ( 1 ) the number of items taken from the bowl by observers ( during pre-feeding ) and by cachers and ( 2 ) the number of items recovered by cachers during retrieval sessions .",
"These data were collected such that all data available for each trial are archived and available , but these data were not relevant to the experimental question so that they were not analysed .",
"The birds’ preference for a specific type of food or tray was analysed according to two indices: proportion scores ( e . g . , the proportion of items cached in one location out of total number of items cached in both locations ) and difference scores ( e . g . , number of items cached in one location minus the number of items cached in the other location ) .",
"As stated in the pre-registrations of Experiments 4 and 5 ( https://osf . io/8p4tx/ ) , we originally planned to analyse the data of all five experiments only through proportion scores .",
"However , when a bird caches no item in a trial , then the individual performance in that specific trial cannot be analysed through the proportion scores , yet it can still be analysed through the difference scores .",
"This issue is relevant only to Experiment 4 , where – in line with the procedure of the original study by Legg and Clayton , 2014 , and in contrast with the procedure of Experiments 1 , 2 , 3 , and 5 – the trials in which no item was cached were not repeated .",
"Nevertheless , after the study was conducted , we decided to analyse the data of all experiments – not only the data of Experiment 4 – also by using the difference scores .",
"We reasoned that , if there are large discrepancies between the results obtained with both types of indices , then this may be important information regarding the robustness of any effects because such discrepancies would show that results from small sample sizes are easily susceptible to change based on the type of analysis used .",
"No power analysis was conducted to establish an appropriate sample size for the study .",
"In each experiment , we used all the individuals out of all the Eurasian jays housed in the facility , that were available for testing at the time when the experiment was being conducted and were familiar with the general set-up of caching experiments ( i . e . , the jays that would cache in caching trays placed in indoor testing compartments ) ."
]
] | [
"Eurasian jays have been reported to protect their caches by responding to cues about either the visual perspective or current desire of an observing conspecific , similarly to other corvids .",
"Here , we used established paradigms to test whether these birds can – like humans – integrate multiple cues about different mental states and perform an optimal response accordingly .",
"Across five experiments , which also include replications of previous work , we found little evidence that our jays adjusted their caching behaviour in line with the visual perspective and current desire of another agent , neither by integrating these social cues nor by responding to only one type of cue independently .",
"These results raise questions about the reliability of the previously reported effects and highlight several key issues affecting reliability in comparative cognition research ."
] | [
"Eurasian jays , Garrulus glandarius , are members of the crow family .",
"These large-brained birds hide food when it is abundant , and eat it later , when it is scarce .",
"Previous studies have found that jays avoid theft by other jays by carefully deciding what food to hide , and where .",
"In one study , they preferred to hide their food behind an opaque barrier , rather than a transparent one , when another jay was watching .",
"In a second study , they preferred to hide food that the watching jay had already eaten enough of , and thus did not want .",
"These studies suggest that jays have flexible cognitive skills when it comes to protecting their food .",
"They respond to whether a potential thief can see their hiding place and to how much a thief might want the food they are stashing .",
"The next question is , can Eurasian jays combine these two pieces of information ?",
"For example , if a jay has two types of food they could hide when another jay is present , but only has one place to hide them ( either in view or out-of-view of the other jay ) , does the first jay prefer to stash the food that the second jay has already eaten , and therefore does not want anymore , only when the hiding place is visible to second jay ?",
"To find out , Amodio et al . watched Eurasian jays hiding macadamia nuts or peanuts in the presence of another jay .",
"In the first setup , jays were given one food to hide and two possible hiding places , one opaque and one transparent , while being watched by a jay that had either had its fill of the food , or not tried it .",
"In the second setup , jays were given both foods to hide , but only had one place to hide them ( either transparent or opaque ) ; while being watched by a jay that had eaten enough of one of the foods .",
"Contrary to expectations , the jays did not seem to be able to combine the information about what the other jay could see and what it had been eating .",
"In fact , they seemed unable to respond to either piece of information .",
"When Amodio et al . repeated the original experiments , the jays did not seem to prefer to hide food out of sight , or to hide food that the watcher had already eaten .",
"These results raise questions about the repeatability of experiments on food hiding strategies in birds of the crow family .",
"It suggests that previous findings should be further investigated , potentially to identify important factors that might affect the repeatability of food-hiding tactics .",
"Repeating the experiments may show how best to investigate behavioural patterns in jays in the future ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation | elife-02409-v2 | [
[
"The organization of the cytoplasm into functionally distinct compartments is fundamental to life .",
"Eukaryotes in particular have achieved a high level of organizational complexity by restricting specific biochemical reactions to membrane-bound organelles in the cytoplasm .",
"A different but equally effective strategy for localized biochemistry involves the formation of phase-separated macromolecular assemblies , which are built de novo from proteins and RNAs ( Brangwynne , 2011; Hyman and Simons , 2012; O'Connell et al . , 2012; Wilson and Gitai , 2013 ) .",
"These non membrane-bound assemblies are often large , occupying an intermediate length scale situated between the nanoscale of individual macromolecules and the microscale of cells .",
"Such intermediate-sized ( or mesoscale ) assemblies often have specific functions that are intricately linked to their higher order complexity .",
"Mesoscale assemblies are structurally and functionally diverse , ranging from dynamic RNA granules , such a P bodies and stress granules , to highly ordered microcompartments , such as bacterial carboxysomes .",
"Based on their physicochemical properties they can be categorized into two distinct groups: liquid-like and crystalline .",
"Liquid-like assemblies are disordered and very dynamic ( the components turn over on the order of seconds to minutes ) ( Brangwynne et al . , 2009 , 2011; Li et al . , 2012 ) , whereas crystalline assemblies are more ordered and static ( Yeates et al . , 2010; Wilson and Gitai , 2013 ) .",
"The distinctive properties of these assemblies also entail different geometries: liquid-like assemblies are usually spherical , whereas crystalline assemblies adopt a broader variety of shapes , ranging from linear polymers and two-dimensional polymer sheets to extensively cross-linked three-dimensional crystals .",
"Modern imaging techniques are now enabling us to unravel the various ways in which nature uses such organizational principles to functionally diversify the subcellular landscape .",
"The number of identified mesoscale assemblies in prokaryotic and eukaryotic cells is increasing rapidly ( Yeates et al . , 2010; Hyman and Simons , 2012; Wilson and Gitai , 2013 ) .",
"Metabolic enzymes in particular are able to self-assemble into higher order structures ( O'Connell et al . , 2012 ) , suggesting an important role in regulating the metabolism of cells .",
"These findings establish a link to earlier work from Paul Srere and colleagues , who proposed that metabolic enzymes undergo static and dynamic interactions to spatiotemporally organize and regulate metabolic pathways ( Srere , 1987 ) .",
"Such organizational specificity was believed to be necessary to ensure metabolic efficiency .",
"Additional studies suggest that self-assembling metabolic enzymes can be co-opted for other functional roles .",
"A particularly striking example is that of cytidine triphosphate synthase , a bacterial enzyme that forms cytoskeletal filaments with cell morphological functions ( Ingerson-Mahar et al . , 2010; Barry and Gitai , 2011 ) .",
"Recent large-scale studies in budding yeast identified several metabolic enzymes that form punctate or filamentous structures in the cytoplasm .",
"Examples include cytidine triphosphate synthase ( Noree et al . , 2010 ) , an enzyme involved in the synthesis of cytosine nucleotides , and glutamine synthetase ( Narayanaswamy et al . , 2009 ) , an enzyme that promotes the conversion of glutamate into glutamine .",
"The metabolic reactions catalyzed by these newly identified enzymes were diverse , but the conditions under which they assembled appeared to be similar: filament formation was most frequently observed , when cells entered stationary phase or were depleted of their primary energy source: glucose .",
"This suggests a functional association with the energy-depleted cellular state , an assumption that has so far remained untested .",
"Moreover , it is not yet clear whether these assemblies are catalytically active , serve as enzyme storage compartments , or are protein aggregates .",
"We conducted a comprehensive analysis of the molecular underpinnings and functions of filaments formed by metabolic enzymes during advanced conditions of starvation .",
"Using yeast glutamine synthetase ( Gln1 ) as a model enzyme , we show that filament formation involves the repeated stacking of homo-decameric enzyme complexes by a back-to-back mechanism .",
"We further demonstrate that filament assembly is triggered by a starvation-induced drop in the intracellular pH and results in enzymatic inactivation and the formation of enzyme storage depots .",
"Importantly , these findings also extend to other filament-forming proteins , arguing that filament formation by metabolic enzymes is a specific adaptation that allows yeast to endure and recover from severe starvation conditions ."
],
[
"Gln1 is an essential metabolic enzyme that catalyzes the ATP-dependent synthesis of glutamine from glutamate and ammonium .",
"In agreement with previous studies ( Narayanaswamy et al . , 2009 ) , we found that GFP-tagged Gln1 was evenly distributed in dividing cells but coalesced into fluorescent foci in cells that were maintained in a phosphate buffer ( Figure 1—figure supplement 1 ) .",
"While most of these foci had a punctate appearance , we noticed a few that displayed a rod-like shape .",
"This suggested that Gln1 might be able to assemble into filamentous structures , but at the same time it implied that the efficiency of filament formation was very low .",
"To exclude a potential interference from the GFP tag , we made use of the fact that the homo-oligomeric structure of Gln1 allows mixed oligomers composed of tagged and untagged monomers .",
"Indeed , when we co-expressed untagged and GFP-tagged Gln1 in yeast , the punctate localization pattern disappeared , and Gln1-GFP assembled into a few filaments per cell ( Figure 1—figure supplement 2 ) .",
"To verify this finding , we replaced endogenous Gln1 with a version containing a C-terminal tetracystein tag .",
"This stretch of six amino acids can be labeled with small dyes such as FlAsH ( Adams et al . , 2002 ) .",
"Indeed , FlAsH-labeled Gln1 assembled into filaments in cells that were transferred from media to buffer ( Figure 1—figure supplement 3 ) .",
"In a search for a protein-encoded fluorescent tag that is compatible with the filamentous state , we replaced the GFP tag in the endogenous GLN1 locus with mCherry .",
"Indeed , unlike GFP-tagged Gln1 , mCherry-tagged Gln1 assembled into filaments ( Figure 1A ) .",
"The number of filaments per cell as well as the kinetics of filament formation was comparable to our previous experiment with predominantly untagged Gln1 ( Figure 1—figure supplement 2 ) .",
"These data indicate that mCherry is compatible with the filamentous state and therefore a suitable fluorophore to study the localization of Gln1 in living cells . 10 . 7554/eLife . 02409 . 003Figure 1 . Gln1 assembles into filaments in energy-depleted yeast cells .",
"( A ) Yeast cells expressing mCherry-tagged Gln1 from the endogenous promoter were washed twice with water and resuspended in synthetic media ( left , control ) or citrate buffer of pH 6 ( right , ‘starved’ ) .",
"White lines are the cell boundaries .",
"The scale bar is 5 μm .",
"The numbers in yellow give the percentage of cells with fluorescent foci .",
"At least 200 cells were counted .",
"( B ) Log phase yeast cells expressing mCherry-tagged Gln1 were washed twice with water and resuspended in synthetic media without ( left ) or with ( right ) 2% glucose .",
"Images were taken 4 hr after onset of glucose starvation .",
"( C ) Log phase cells expressing mCherry-tagged Gln1 were washed twice with water and resuspended in a phosphate–citrate buffer of pH 6 without ( left ) or with ( right ) 2% glucose .",
"Images were taken 4 hr after onset of starvation .",
"( D ) Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate–citrate buffer of pH 6 to induce starvation ( time point 0 ) .",
"Filament formation was followed by time-lapse microscopy .",
"Individual time points are indicated in minutes .",
"The white arrow designates an emerging filament .",
"The scale bar is 5 μm .",
"Also see the corresponding Video 1 .",
"( E ) Same as ( D ) except that filament dissolution was investigated by re-adding glucose to cells that had been starved for 4 hr .",
"The white arrow points to a small filament .",
"The red arrow designates the emerging bud .",
"Also see the corresponding Video 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 00310 . 7554/eLife . 02409 . 004Figure 1—figure supplement 1 . GFP-tagged Gln1 predominantly forms punctate structures . Yeast cells expressing GFP-tagged Gln1 from the endogenous promoter were washed twice with water and resuspended in synthetic media ( left , control ) or buffer of pH 6 ( right , ‘starved’ ) .",
"White lines are the cell boundaries .",
"The scale bar is 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 00410 . 7554/eLife . 02409 . 005Figure 1—figure supplement 2 . Co-expression of untagged Gln1 transforms the localization pattern from punctate to filamentous . Yeast cells expressing GFP-tagged Gln1 from the endogenous promoter were washed twice with water and resuspended in synthetic media ( left , control ) or buffer of pH 6 ( right , ‘starved’ ) .",
"The cells co-expressed untagged Gln1 from a plasmid .",
"White lines are the cell boundaries .",
"The scale bar is 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 00510 . 7554/eLife . 02409 . 006Figure 1—figure supplement 3 . Filamentation is not caused by the tag . Yeast cells expressing tetracystein-tagged Gln1 were incubated over night with FIAsH-EDT2 to label Gln1 .",
"The cells were washed twice with water and resuspended in a phosphate–citrate buffer to induce starvation ( pH 6 ) .",
"Images were taken 4 hr after onset of starvation .",
"White lines denote the cell boundaries .",
"The scale bar is 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 006 Using live cell microscopy , we found that mCherry-tagged Gln1 was diffusely localized in dividing cells but formed filaments when the growth medium lacked a carbon source ( 33% of the cells had filaments after 4 hr of glucose starvation ) ( Figure 1B ) .",
"Importantly , when we transferred the cells into a phosphate buffer that contained no metabolizable nutrients , filaments were detectable in all cells ( Figure 1C ) .",
"Thus , under conditions of severe starvation , the filament formation phenotype becomes fully penetrant .",
"This suggests that filament formation by metabolic enzymes is a starvation-induced cellular adaptation .",
"Here , we refer to this specific cellular state as the state of advanced starvation .",
"On average , filament assembly started 50 min ( n = 179; SD = 43 . 9 min ) after onset of advanced starvation conditions ( Figure 1D and Video 1 ) .",
"However , we observed extensive variation from cell to cell , suggesting that yeast vary in their ability to deal with sudden energy depletion ( Video 2 ) .",
"Importantly , addition of glucose to buffer was sufficient to prevent filament formation ( Figure 1C ) .",
"We also found that cells can fully recover from the state of advanced starvation .",
"Upon resupply of nutrients , filament dissolved very rapidly , and shortly after filament dissolution cells re-entered into the cell cycle ( Figure 1E , Videos 3 and 4 ) .",
"On average , filament dissolution was completed ∼18 min after nutrient addition ( n = 65; SD = 11 . 6 min ) .",
"We conclude that Gln1 dynamically and reversibly assembles into filaments when yeast cells are exposed to conditions of advanced starvation . 10 . 7554/eLife . 02409 . 007Video 1 . Gln1 forms filaments in starved yeast .",
"Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation ( time point 0 ) .",
"Filament formation was followed by time-lapse microscopy .",
"Time points are indicated in minutes . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 00710 . 7554/eLife . 02409 . 008Video 2 . Gln1 forms filaments in starved yeast .",
"Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation ( time point 0 ) .",
"Filament formation was followed by time-lapse microscopy .",
"Time points are indicated in minutes . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 00810 . 7554/eLife . 02409 . 009Video 3 . Gln1 filaments dissolve upon glucose addition to starved cells .",
"Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation ( time point 0 ) .",
"The cells were incubated for 4 hr to induce the formation of filaments .",
"At time point 0 , glucose ( 2% ) was added and filament dissolution was followed by time-lapse microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 00910 . 7554/eLife . 02409 . 010Video 4 . Gln1 filaments dissolve upon glucose addition to starved cells .",
"Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation ( time point 0 ) .",
"The cells were incubated for 4 hr to induce the formation of filaments .",
"At time point 0 , glucose ( 2% ) was added and filament dissolution was followed by time-lapse microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 010 A recent study reported a high-resolution crystal structure for yeast Gln1 ( He et al . , 2009 ) .",
"The enzyme is composed of two pentameric rings that interact to form a face-to-face homo-decamer .",
"This structural arrangement is identical to those of mammalian and plant glutamine synthetases .",
"Curiously , however , the crystal structure of yeast Gln1 also revealed a new back-to-back association between two decamers ( Figure 2A ) .",
"The biological significance of this novel interface , however , remained undetermined . 10 . 7554/eLife . 02409 . 011Figure 2 . Gln1 assembles by a back-to-back stacking mechanism .",
"( A ) The crystal structure of Gln1 as reported by He at al . ( 2009 ) .",
"The putative assembly interface and mutations introduced in this study are indicated .",
"( B ) Chromosomally encoded Gln1 was replaced with mCherry-tagged wild-type or variant Gln1 expressed from a plasmid .",
"The strains were washed twice with water and resuspended in synthetic media ( top , control ) or buffer ( bottom , ‘starved’ ) .",
"The numbers in yellow give the percentage of cells with fluorescent foci .",
"At least 200 cells were counted .",
"The scale bar is 5 μm .",
"( C ) Wild type or variant 6xHis-Gln1-V5 was purified from yeast and analyzed by blue native PAGE .",
"Proteins were detected by silver staining ( top ) or immunoblotting with an antibody that recognized a C-terminal V5 tag ( bottom ) .",
"The red arrow denotes a band that corresponds to two stacked decamers .",
"The calculated size of a Gln1 decamer is 420 KDa .",
"( D ) Correlative light electron microscopy ( CLEM ) was performed on yeast cells expressing the R23E variant of Gln1 as mCherry fusion .",
"The black dots are fluorescent beads , which were introduced to facilitate the alignment of the fluorescence and TEM images .",
"The scale bar is 500 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01110 . 7554/eLife . 02409 . 012Figure 2—figure supplement 1 . Detailed structural view of the decamer–decamer interface . All critical residues are shown within a range of 10 Å .",
"Residues mutated in this study are highlighted in red .",
"The subunit identifier is given in brackets .",
"The red arrow denotes the disordered N terminus .",
"The mutations introduced were E186K , R23E , and T49E . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01210 . 7554/eLife . 02409 . 013Figure 2—figure supplement 2 . Detailed structural view of the decamer–decamer interface . All critical residues are shown within a range of 10 Å .",
"Residues mutated in this study are highlighted in red .",
"The subunit identifier is given in brackets .",
"The red arrow denotes the disordered N terminus .",
"The mutations introduced were R23E , T49E , and Y81A . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01310 . 7554/eLife . 02409 . 014Figure 2—figure supplement 3 . Detailed structural view of the decamer–decamer interface . All critical residues are shown within a range of 10 Å .",
"Residues mutated in this study are highlighted in red .",
"The subunit identifier is given in brackets .",
"The red arrow denotes the disordered N terminus .",
"The mutation introduced was P83R . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01410 . 7554/eLife . 02409 . 015Figure 2—figure supplement 4 . Yeast cells expressing mCherry-tagged R23E Gln1 were subjected to staining with Thioflavin T . Only the control filaments ( formed by the yeast prion Rnq1 ) were stainable by ThT , suggesting that the formation of cross-β structure is not required for Gln1 filamentation . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01510 . 7554/eLife . 02409 . 016Figure 2—figure supplement 5 . Lysates from yeast cells expressing wild-type or R23E Gln1 were subjected to semi-denaturing detergent-agarose gel electrophoresis ( SDD-AGE ) .",
"Only the control filaments ( formed by the yeast prion Rnq1 ) were resistant to SDS , indicating that Gln1 filamentation does not involve the formation of cross-β structure . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01610 . 7554/eLife . 02409 . 017Figure 2—figure supplement 6 . 6xHis-Gln1 ( R23E ) -mCherry was affinity purified from yeast , subjected to negative staining and investigated by electron microscopy . Note the presence of filaments with a diameter of ∼120 Å and a repeating unit of ∼100 Å in size .",
"These proportions are consistent with the reported dimensions of Gln1 decamers ( He et al . , 2009 ) .",
"The scale bar is 20 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01710 . 7554/eLife . 02409 . 018Figure 2—figure supplement 7 . Transmission electron microscopy ( TEM ) was performed on yeast cells expressing untagged Gln1 ( R23E ) .",
"The red arrow points to filamentous structures in the cytoplasm .",
"Similar structures were absent from control cells expressing untagged Gln1 .",
"The scale bar is 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 018 The presence of the decamer–decamer interface led us to hypothesize a simple mechanism for filament formation: assembly by repeated back-to-back stacking of decamers .",
"To test this assumption , we mutated specific residues in the interface ( Figure 2A and Figure 2—figure supplements 1 , 2 and 3 ) .",
"The resulting Gln1 variants were fused to mCherry and tested in yeast for their ability to assemble into filaments .",
"We found three mutations ( E186K , P83R , T49E ) that abrogated the ability of Gln1 to form filaments in starved cells ( Figure 2B ) .",
"Intriguingly , we also identified two mutants that formed filaments in growing cells .",
"The Y81A mutant had a slightly increased propensity to form filaments , whereas the R23E mutant formed filaments constitutively , regardless of the growth conditions .",
"To test whether the R23E mutant effect could be neutralized by a second mutation , we introduced the inhibitory T49E mutation into the R23E variant .",
"Indeed , the double mutant ( R23E , T49E ) was no longer able to form filaments ( Figure 2B ) , indicating that the increased propensity to form filaments can be overcome by an inhibitory second site mutation .",
"These findings indicate that the propensity to form filaments can be modulated by specific mutations in Gln1 .",
"They also suggest that the decamer–decamer interface is biologically important .",
"To provide further evidence for the proposed mechanism of assembly , we purified wild-type and mutant Gln1 from yeast cells .",
"From this point on we will use a color code to facilitate discrimination between the different mutant forms of Gln1 .",
"Mutants with inhibitory effects on filament formation will be highlighted in red , whereas constitutively filament-forming mutants will be highlighted in green .",
"First , we investigated the oligomeric state of wild-type and variant Gln1 by blue native PAGE and immunoblotting .",
"As can be seen in Figure 2C , the predominant form of Gln1 was a decamer .",
"Importantly , however , we also identified distinct bands that corresponded to higher molecular weight forms of the enzyme .",
"These assemblies were predominantly detectable for the wild-type and the R23E variant .",
"Moreover , when compared to wild-type Gln1 , the constitutively filament-forming R23E variant showed an increased number of these higher order assemblies .",
"To eliminate the possibility that formation of these assemblies involves cross-β structure—a hallmark of other filament-forming proteins such as Pmel17 ( Fowler et al . , 2006 ) or Curli ( Chapman et al . , 2002 ) —we performed two additional control experiments .",
"First , we determined whether Gln1 filaments could be stained with Thioflavin T ( a dye that specifically binds to cross-β structure ) , and second , we subjected lysates from filament-containing cells to semi-denaturing detergent–agarose gel electrophoresis ( a method that detects cross-β structure based on its resistance to detergent ) ( Alberti et al . , 2010 ) .",
"As can be seen in Figure 2—figure supplements 4 and 5 , we could not find evidence for cross-β structure .",
"As a next step , we isolated His-tagged R23E Gln1 from yeast cells and investigated the purified protein by negative staining and electron microscopy .",
"We identified short filamentous structures , which , upon closer inspection , revealed that they were formed by a repeating unit that precisely matched the dimensions of a Gln1 decamer ( Figure 2—figure supplement 6 ) .",
"Thus , we conclude that Gln1 retains a near-native structure when it assembles into filaments .",
"As a next step , we performed correlative light and electron microscopy ( CLEM ) experiments on cells overexpressing the R23E variant as mCherry fusion .",
"The ultrastructural features of the mCherry-positive structures are shown in Figure 2D .",
"The electron micrographs show a large number of filaments , which are laterally aligned into higher order bundles .",
"This side-by-side bundling is consistent with the growth pattern of the filaments , which was predominately in the longitudinal direction but also included some increase in circumference over time ( see Video 1 ) .",
"To exclude that the bundling was caused by the mCherry tag , we performed a careful ultrastructural analysis of yeast cells that expressed untagged R23E .",
"Indeed , we could also identify fibrillar structures in the cytoplasm of R23E-expressing cells but not in control cells ( Figure 2—figure supplement 7 ) .",
"However , we noticed that the filaments were in closer contact , suggesting that the mCherry tag has some influence on the packing density .",
"Together , these findings indicate that Gln1 assembles into filaments by a back-to-back stacking mechanism .",
"Once formed , these filaments can organize into higher order fibrils .",
"Is Gln1 able to self-assemble or does it need additional factors ?",
"To investigate this question , we purified wild-type and variant Gln1 from bacteria .",
"The purified proteins were investigated by dynamic light scattering and gel exclusion chromatography for their ability to assemble into high molecular weight forms .",
"As can be seen in Figure 3A and Figure 3—figure supplement 1 , R23E Gln1 had a strongly increased propensity to assemble into higher order forms .",
"This propensity was reduced in wild-type Gln1 and absent from a variant that had lost the ability to assemble into filaments in yeast cells ( E186K ) .",
"We next analyzed purified R23E Gln1 by electron microscopy ( Figure 3—figure supplement 2 ) .",
"Our analysis revealed abundant cylindrical particles , consistent with the reported structure of Gln1 ( He et al . , 2009 ) .",
"Interestingly , these particles were organized into chains , providing further support for the proposed back-to-back assembly mechanism .",
"However , we were unable to identify higher order structures that resembled previously observed fibrillar structures in yeast , suggesting that an important factor was missing . 10 . 7554/eLife . 02409 . 019Figure 3 . Self-assembly into filaments is driven by macromolecular crowding .",
"( A ) Equal amounts of 6xHis-tagged wild-type and variant Gln1 purified from bacteria were subjected to dynamic light scattering .",
"Shown is the volume distribution that was derived from the intensity distribution .",
"Note the different scales of the x axes .",
"( B ) Yeast cells expressing Gln1 ( R23E ) -mCherry were spheroplasted and lysed .",
"Images were acquired from harvested , spheroplasted , and lysed cells .",
"Images were taken at the same intensity settings .",
"The inset is the corresponding DIC image .",
"The scale bar is 5 μm .",
"( C ) Cells were treated as in ( A ) except that the lysis buffer contained Ficoll 70 at a concentration of 200 mg/ml .",
"( D ) Gln1-mCherry was purified from yeast and incubated in a phosphate-citrate buffer of pH 7 with or without a crowding agent for 1 hr .",
"Samples were analyzed by fluorescence microscopy and images were taken at the same intensity settings .",
"The scale bar is 3 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 01910 . 7554/eLife . 02409 . 020Figure 3—figure supplement 1 . Gel filtration of wild type and variant 6xHis-tagged Gln1 purified from bacteria . Fractions were applied onto a nitrocellulose filter by using a dot blot apparatus .",
"Molecular weight markers were thyroglobulin ( 660 kDa ) , ferritin ( 440 kDa ) , catalase ( 230kD ) , bovine serum albumin ( 67 kDa ) , and ovalbumin ( 43 kDa ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 02010 . 7554/eLife . 02409 . 021Figure 3—figure supplement 2 . Electron microscopy of 6xHis-tagged R23E Gln1 purified from bacteria . Note the formation of chains , which indicate enzyme stacking by a back-to-back mechanism .",
"The scale bar is 20 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 02110 . 7554/eLife . 02409 . 022Figure 3—figure supplement 3 . 6xHis-Gln1-mCherry was affinity purified from yeast and assembled in the presence of a crowder . The sample was investigated by fluorescence microscopy .",
"Acquired images were deconvolved to increase the signal to noise ratio .",
"The shown image is a maximum intensity projection of 20 individual images that were acquired with 200 nm sectioning .",
"Note the presence of filamentous structures of varying thickness ( numbers 1 , 2 and 3 ) , indicating the formation of higher order fibrils from laterally aligning filaments . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 022 What could be the missing factor ?",
"A first hint came from our attempts to purify filaments from yeast cells .",
"When we lysed filament-containing yeast , the filaments were unstable ( Figure 3B ) .",
"To identify components that are necessary for filament integrity , we performed experiments with modified lysis buffers .",
"One obvious difference to yeast cytoplasm was that the lysis buffer lacked a high background concentration of macromolecules .",
"We therefore tested the influence of macromolecular crowders on filament stability .",
"Strikingly , when we added the crowding agent Ficoll 70 at a concentration close to the physiological concentration of macromolecules ( 200 mg/ml ) , the filaments remained intact ( Figure 3C ) .",
"To further investigate the role of macromolecular crowding , we purified mCherry-tagged R23E from yeast cells and incubated it for 1 hr in the presence or absence of a crowder .",
"The samples were subsequently analyzed by fluorescence microscopy for the formation of higher order structures .",
"Intriguingly , we found that R23E Gln1 formed filamentous structures in the presence of a crowder but not in its absence ( Figure 3D ) .",
"Importantly , similar structures could not be detected when we replaced R23E with the assembly-incompetent variant P83R .",
"Further inspection of in vitro formed Gln1 filaments revealed structures of varying thickness ( Figure 3—figure supplement 3 ) .",
"This suggests that in vitro reconstituted filaments are able to assemble into higher order bundles , as in yeast cells .",
"Thus , we conclude that the assembly of Gln1 is strongly dependent on macromolecular crowding but independent of other cellular components .",
"Wild-type Gln1 only formed filaments in energy-depleted cells , in contrast to the R23E variant , which assembled also in dividing cells .",
"This raised questions about the trigger for assembly in starved yeast .",
"Again experiments with lysates of R23E-expressing cells were revealing .",
"These experiments showed that the stability of R23E filaments could not only be increased by crowders but also by acidifying the lysis buffer .",
"A cell lysate prepared with a lysis buffer of pH 5 contained abundant filaments , while the filaments began to fall apart when the lysis buffer was adjusted to pH 6 , and they were absent from lysates adjusted to pH 7 or 8 ( Figure 4A ) .",
"This raised the possibility that intracellular pH changes are the trigger for filament formation . 10 . 7554/eLife . 02409 . 023Figure 4 . A drop in intracellular pH triggers filament formation .",
"( A ) Yeast cells expressing Gln1 ( R23E ) -mCherry were spheroplasted and lysed in phosphate buffers of different pHs .",
"Images were acquired immediately after lysis .",
"The scale bar is 5 μm .",
"Note that the lysis buffer did not contain a crowder .",
"( B ) Yeast cells expressing mCherry-tagged Gln1 were washed twice with water and resuspended in a glucose-containing buffer of the indicated pHs with or without the proton carrier 2 , 4-dinitrophenol ( DNP ) .",
"Images were taken 1 hr after addition of the buffer .",
"The scale bar is 5 μm .",
"( C ) Yeast cells expressing Gln1-mCherry were washed twice with water and resuspended in phosphate buffers of different pHs to induce starvation .",
"The buffers contained proton carriers and energy inhibitors for rapid equilibration of inside and outside pH . The scale bar is 5 μm .",
"( D ) Gln1-mCherry was purified from yeast and incubated in an acidic or basic buffer containing a crowding agent for 1 hr .",
"Samples were analyzed by fluorescence microscopy .",
"The scale bar is 3 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 02310 . 7554/eLife . 02409 . 024Figure 4—figure supplement 1 . Equal amounts of 6xHis-tagged wild type and E186K Gln1 purified from bacteria were mixed with an acidic buffer and subjected to dynamic light scattering . Shown is the volume distribution that was derived from the intensity distribution . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 02410 . 7554/eLife . 02409 . 025Figure 4—figure supplement 2 . 6xHis-tagged wild-type Gln1 purified from bacteria was subjected to Far-UV CD at pH 7 . 4 and 6 . The degree of helicity is essentially unchanged as evident from the almost unaffected 222 nm signal .",
"This indicates that Gln1 retains a near-native structure in conditions that induce assembly into higher order structures . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 025 The intracellular pH of yeast cells drops significantly when yeast are depleted of their primary energy source glucose ( Orij et al . , 2009; Dechant et al . , 2010; Orij et al . , 2011 ) .",
"This is because yeast cells have to continuously expend energy to maintain the proton gradient across the plasma membrane ( usually yeast media are acidic ) .",
"To test whether pH changes are sufficient to trigger the assembly of Gln1 , we artificially acidified the cytoplasm by adding the protonophore 2 , 4-dinitrophenol ( DNP ) to dividing yeast .",
"Strikingly , acidified yeast abundantly formed filamentous structures , despite the presence of glucose ( Figure 4B ) .",
"Next , we tested whether a neutral or basic outside pH could prevent the formation of filaments in energy-depleted yeast .",
"Indeed , starved yeast maintained in a buffer of pH 7 contained shorter and smaller filaments than yeast in a buffer of pH 5 ( Figure 4C ) .",
"Moreover , filaments were largely absent when the buffer was adjusted to pH 8 .",
"Intracellular pH changes could directly or indirectly affect the assembly of Gln1 .",
"To differentiate between these two possibilities , we purified mCherry-tagged Gln1 from yeast and incubated it for 1 hr in a buffer of pH 5 or 8 .",
"Afterwards , the samples were examined by fluorescence microscopy for the formation of higher order structures .",
"Indeed , Gln1 formed filamentous structures in the acidic buffer but not in the control buffer with a basic pH ( Figure 4D ) .",
"Importantly , this effect was specific , because the assembly-deficient variant P83R did not assemble into higher order structures in an acidic buffer .",
"To follow pH-induced assembly over time , we exposed bacterially purified Gln1 to an acidic buffer and monitored the formation of higher order structures by dynamic light scattering .",
"As can be seen in Figure 4—figure supplement 1 , Gln1 progressively assembled into higher order structures , whereas a mutant version of Gln1 ( E186K ) did not .",
"Moreover , exposure to an acidic buffer did not lead to an extensive alteration of the secondary structure of Gln1 ( Figure 4—figure supplement 2 ) , suggesting that the protein retains its structural integrity under these conditions .",
"Thus , we conclude that Gln1 assembly is directly regulated by protons and that the trigger for filament formation in yeast cells is a starvation-induced drop in the intracellular pH . A recent study reported that several protein complexes assemble into filaments as yeast cells approach stationary phase ( Noree et al . , 2010 ) .",
"One of these filaments consisted of only one protein ( Glt1 ) , while the others were composed of multiple proteins with two ( Ura7/Ura8 ) or more ( Gcd2 , Gcd6 , Gcd7 , Gcn3 , Sui2 ) distinct subunits .",
"To study whether cytosolic acidification is also required for formation of these assemblies , we tagged one protein in each filament with GFP .",
"We then determined the frequency of filaments in yeast cells that were resuspended in different pH-adjusted buffers .",
"Because these buffers lacked an energy source , the starved cells rapidly adopted the outside pH . Intriguingly , we found that all three proteins formed filaments in a strongly pH-dependent manner ( Figure 5A ) .",
"Moreover , filament formation was almost completely abrogated at a neutral or basic pH . We then tested if we could induce filament formation in growing cells by adding the protonophore DNP .",
"Indeed , cells suspended in a DNP-containing buffer of pH 5 or pH 6 showed widespread filament formation , despite the presence of glucose ( Figure 5B ) .",
"However , filaments were absent from cells maintained in a DNP-containing buffer of pH 7 .",
"This indicates that pH changes are a general trigger for the formation of filaments by metabolic enzymes . 10 . 7554/eLife . 02409 . 026Figure 5 . Other metabolic enzymes form filaments in a pH-dependent manner .",
"( A ) Yeast cells expressing sfGFP ( V206R ) -tagged Ura8 , Glt1 , or Gcn3 were washed twice with water and resuspended in buffers of different pHs to induce starvation .",
"Images were taken 2 hr after onset of starvation .",
"The scale bar is 5 μm .",
"( B ) Yeast cells expressing sfGFP ( V206R ) -tagged Ura8 , Glt1 , or Gcn3 were washed twice with water and resuspended in a buffer containing 2% glucose and the proton carrier 2 , 4-dinitrophenol ( DNP ) .",
"Images were taken 1 hr after addition of the buffer .",
"The numbers in yellow give the percentage of cells with fluorescent foci .",
"At least 200 cells were counted .",
"The scale bar is 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 026 To investigate the functional implications of filament formation , we first tested whether filament formation modulates the activity of Gln1 .",
"Indeed , Gln1 was progressively inactivated under conditions of advanced starvation ( Figure 6A ) , and the extent of inactivation correlated with the amount of filament formation ( Figure 6—figure supplement 1 ) .",
"Next , we compared the enzymatic activities of wild-type and mutant Gln1 .",
"As can be seen in Figure 6B , the R23E variant displayed a strongly reduced synthetase activity .",
"Because these activity measurements were performed with cell lysates , we hypothesized that the actual enzymatic inactivation is much stronger in intact cells .",
"To investigate this possibility , we inspected wild-type and variant Gln1 variants for growth phenotypes .",
"Consistent with the essential function of Gln1 , the constitutively assembling R23E variant had a strongly diminished ability to grow ( Figure 6C ) .",
"Importantly , this growth deficiency was abrogated by a second site mutation that prevents filament formation ( T49E ) , indicating that filament formation is the cause of the reduced growth rate .",
"Moreover , cells growing on glutamine as the sole nitrogen source ( Figure 6—figure supplement 2 ) showed no growth defect , suggesting that this deficiency was due to the inability of the cells to produce sufficient amounts of the growth-promoting amino acid glutamine .",
"Based on these findings , we conclude that filament formation during advanced starvation conditions silences the enzymatic activity of Gln1 . 10 . 7554/eLife . 02409 . 027Figure 6 . Assembled Gln1 is catalytically inactive but becomes active again after disassembly .",
"( A ) Lysates were prepared form wild-type cells exposed to advanced starvation conditions for 3 , 6 , or 24 hr , and the glutamine synthetase activity was determined as described previously ( Mitchell and Magasanik , 1984 ) .",
"The obtained values were normalized to the amount of Gln1 in the cell lysate based on immunoblotting experiments .",
"The shown data is the mean of five biological replicates ( ±SEM ) .",
"( B ) Lysates were prepared from yeast cells expressing wiltype or variant Gln1-V5 and the glutamine synthetase activity was determined as in ( A ) .",
"The values were normalized to the amount of Gln1 contained in the lysate based on immunoblotting experiments ( *p value <0 . 05 ) .",
"( C ) Endogenous Gln1 was substituted with the indicated wild type or variant versions expressed from an ADH1 promoter-containing plasmid .",
"Cells were grown over night and equal amounts of late log phase cells were spotted onto synthetic plates in serial 1:5 dilutions .",
"( D ) Cells expressing Gln1-mCherry from a GAL-inducible promoter were washed twice with water and starved in a phosphate–citrate buffer of pH 6 for 4 hr .",
"Filament dissolution was followed after re-addition of glucose by time-lapse imaging .",
"The red arrow denotes the newly formed bud .",
"Note that filament dissolution and bud emergence take longer as the cells had to readjust to a new carbon source ( glucose instead of galactose ) .",
"The scale bar is 5 μm .",
"Also see corresponding Video 5 .",
"( E ) Wild-type and mutant ( Y81A ) yeast were exposed to advanced starvation conditions and regrowth was monitored after addition of nutrients ( we used only 0 . 5% glucose in this assay as this led to slower regrowth and a more pronounced lag phase ) .",
"Note that the Y81A cultures reached a slightly higher optical density , probably because of compensatory changes in the gene expression network .",
"The values show the mean of four technical replicates .",
"The data shown are representative of four independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 02710 . 7554/eLife . 02409 . 028Figure 6—figure supplement 1 . Gln1 filaments form progressively after exposure of yeast to advanced starvation conditions . Cells expressing Gln1-mCherry were washed twice with water and resuspended in a phosphate–citrate buffer of pH 6 to induce starvation .",
"Images were acquired 0 , 3 , 6 , and 24 hr after onset of starvation .",
"The total cellular mCherry signal was segmented into a diffuse and filament fraction using the Squassh segmentation tool from the Mosaic Suite of the Fiji image analysis software .",
"The values shown are the mean ( ±SEM ) of three independent experiments ( p<0 . 05 ) .",
"The y-axis gives the fraction of the signal bound up in filaments .",
"The values were normalized to the 24-hr time point as no further filament formation was observed beyond this time point . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 02810 . 7554/eLife . 02409 . 029Figure 6—figure supplement 2 . The R23E growth defect can be rescued by adding glutamine to the growth medium . Endogenous Gln1 was deleted and substituted with wild-type or variant Gln1 expressed from a plasmid .",
"Cells were grown over night and equal amounts of late log phase cells were spotted onto synthetic plates in serial 1:5 dilutions .",
"The plate media lacked amino acids and contained ammonia or glutamine as the only nitrogen source .",
"The strain background was a prototrophic variant of W303 ( Klosinska et al . , 2011 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 02910 . 7554/eLife . 02409 . 030Figure 6—figure supplement 3 . The Y81A mutation does not affect growth . Endogenous Gln1 was substituted with the indicated wild-type or variant versions expressed from an ADH1 promoter-containing plasmid .",
"Cells were grown over night and equal amounts of late log phase cells were spotted onto synthetic plates in serial 1:5 dilutions . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 03010 . 7554/eLife . 02409 . 031Figure 6—figure supplement 4 . Filament dissolution is impaired in Y81A mutants . Cells expressing WT or Y81A Gln1-mCherry were washed twice with water and resuspended in a phosphate-citrate buffer of pH 6 to induce starvation .",
"After 4 hr of starvation , nutrients were resupplied and images were acquired 0 , 20 , 30 , and 55 min .",
"The y-axis gives the fraction of cells containing filaments .",
"Mean values are shown ( ±SEM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 031 Do these inactive filaments serve as storage depots for Gln1 ?",
"To address this question , we generated a yeast strain that expressed mCherry-tagged Gln1 from the inducible GAL promoter .",
"Because of the essential nature of Gln1 , this strain had to be maintained in the presence of galactose .",
"To test whether assembled Gln1 can become enzymatically active again , we grew the strain in the presence of galactose and then induced starvation by transferring it into a phosphate buffer .",
"After 4 hr of starvation the great majority of the cells had formed filaments .",
"We then added glucose-containing growth medium to induce reentry into the cell cycle .",
"Importantly , under these conditions , the GAL promoter is switched off , and Gln1 is no longer synthesized de novo .",
"Therefore , the cells can only enter the cell cycle when they manage to reactivate enzyme complexes that were previously stored in filaments .",
"Indeed , after the filaments had dissolved , the cells started to divide again ( Figure 6D and Video 5 ) .",
"Growth only stopped after several rounds of cell division , presumably because the amount of Gln1 was diluted below a critical concentration .",
"Thus , we conclude that starvation-induced filaments can serve as storage depots for Gln1 . 10 . 7554/eLife . 02409 . 032Video 5 . Filamentous Gln1 can be reactivated upon entry into the cell cycle .",
"Cells expressing Gln1-mCherry from a GAL-inducible promoter were washed twice with water and starved in a phosphate-citrate buffer of pH 6 for 4 hr .",
"At time point 0 , glucose ( 2% ) was added and filament dissolution was followed by time-lapse microscopy .",
"Note that filament disassembly and bud emergence take longer , because the cells had to readjust to a new carbon source ( glucose instead of galactose ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 032 What is the physiological function of filament formation ?",
"To investigate this question , we monitored the regrowth of yeast after a short episode of starvation .",
"We hypothesized that the ratio of assembled to unassembled Gln1 should specifically affect the initial growth phase of recovering yeast .",
"To investigate this possibility , we observed the growth of Y81A mutants immediately after nutrient re-supply ( the Y81A mutant displayed slower filament dissolution kinetics , but no growth defect in plating assays , see Figure 6—figure supplements 3 and 4 ) .",
"As can be seen in Figure 6E , the Y81A strain showed significantly slower growth after exit from starvation than wild-type cells .",
"Thus , we conclude that exit from starvation is dependent on stored enzymes and that the rate of filament dissolution determines the timing of re-growth .",
"Our findings so far suggest that filament formation may regulate the ability of yeast cells to resume growth after advanced starvation conditions .",
"But does filament formation also promote the survival of and the recovery from starvation ?",
"To investigate this possibility , we interfered with the ability of yeast to form filaments by starving them in buffers of neutral or basic pH for extended times .",
"As can be seen in Figure 7A , the regrowth of yeast was severely impaired in filamentation-inhibiting buffer ( pH 7 or 8 ) , whereas cells maintained in filamentation-promoting buffer ( pH 6 ) showed fast recovery .",
"This suggests that filament formation by metabolic enzymes is required for recovery from severe starvation .",
"Thus , we conclude that filament formation is a specific adaptation that enables yeast to endure severe energy depletion stress . 10 . 7554/eLife . 02409 . 033Figure 7 . Mechanism of filament formation by Gln1 and its potential role in starvation survival .",
"( A ) Acidification of the yeast cytosol promotes survival and recovery from starvation .",
"Yeast cells were exposed to advanced starvation conditions using buffers with a pH of 6 , 7 or 8 for 3 days .",
"Upon re-addition of SD medium containing a limited amount of glucose ( 0 . 5% ) , regrowth was monitored in a plate reader .",
"The values show the mean of four technical replicates ( ±SEM ) .",
"The data shown are representative of four independent experiments .",
"( B ) Mechanism of pH-induced filament formation by metabolic enzymes .",
"Gln1 assembles into filaments by a back-to-back enzyme stacking mechanism .",
"Transitions between unassembled Gln1 decamers ( left ) , filaments ( middle ) , and fibrils ( right ) are driven by pH changes and excluded volume effects .",
"Assembly requires an acidic pH , whereas disassembly requires a basic pH . Gln1 enzyme complexes are shown in green .",
"Blue spheres denote inert macromolecules that are excluded from the space that is occupied by Gln1 .",
"This inaccessible space is indicated by the dotted lines .",
"The bottom diagram illustrates the cumulative excluded volume effect that entropically drives filament assembly and fibril formation .",
"The colors of the bottom diagram correspond to the colors of the excluded volume areas above . DOI: http://dx . doi . org/10 . 7554/eLife . 02409 . 033"
],
[
"How cells survive and recover from severe starvation is a largely unresolved question .",
"Our findings suggest that this may involve extensive changes in the organization of the cytoplasm .",
"We demonstrate that starvation induces the self-assembly of yeast glutamine synthetase into filaments by a simple back-to-back stacking mechanism ( see Figure 7B for a model of assembly ) .",
"We further show that filament formation is triggered by starvation-induced acidification of the cytosol and results in enzyme inactivation .",
"This catalytic inactivation is reversible , arguing that filaments are temporary storage depots for Gln1 .",
"Other filament-forming enzymes show a strikingly similar sensitivity to pH , and their growth-associated functions are probably not needed during advanced starvation .",
"This suggests that the formation of inactive enzyme assemblies may be a general principle , which allows cells to adapt to low energy levels .",
"Consistent with this , we found that filament formation regulates the regrowth of yeast after severe starvation .",
"Recent findings indicate that quiescence-associated subcellular structures , such as proteasome storage granules and actin bodies , also form in a pH-dependent manner ( Peters et al . , 2013 ) .",
"Moreover , pH changes have been implicated in the regulation of stress-induced G protein signaling ( Isom et al . , 2013 ) .",
"This strongly reinforces the notion that intracellular pH changes serve as a global messenger to signal the depletion of energy during starvation and cellular quiescence ( Dechant et al . , 2010; Orij et al . , 2012 ) .",
"In fact , pH-induced formation of filamentous structures may be a more widespread phenomenon in nature , as shown for example by the finding that spider silk formation is induced by low pH ( Kronqvist et al . , 2014 ) .",
"How does the pH regulate the formation of Gln1 filaments ?",
"We noticed that Gln1 has a theoretical isoelectric point of around 6 .",
"Starvation-induced changes in the cytosolic pH will therefore strongly reduce its net charge .",
"Thus , an overall reduction of repulsive interactions or altered charge distributions at the interface—which the mutations introduced in this study may modulate—are likely to be the driving force for assembly .",
"An alternative scenario is that protons act as allosteric effectors that induce structural changes at the decamer–decamer interface .",
"Accordingly , the behavior of the Gln1 mutants could also be explained by an increased or decreased propensity to undergo structural changes .",
"However , currently we have no evidence that protonation induces large conformational changes .",
"Regardless of the specific mechanism , what is evident is that a drop in intracellular pH induces the formation of an attractive interface at the two ends of a decameric Gln1 particle .",
"The steric self-compatibility of this interface and the highly symmetric cylindrical structure of Gln1 then promote the assembly of Gln1 into extended filaments .",
"Once formed , these filaments are able to associate side by side to form higher order bundles ( Figure 7B ) .",
"Where does the energy for assembly come from ?",
"The individual stacking reactions seem to be quite stable at low pH , whereas the final filamentous structure was vulnerable to even mild perturbations .",
"This suggests that the higher order assembly of filaments into microscopically visible fibrils is a cooperative and less favorable process .",
"In agreement with this , we found that the formation of fibrils was strongly dependent on a macromolecular crowding agent .",
"This indicates that the assembly reaction is not only driven enthalpically by chemical interface–interface interactions but also entropically by excluded volume effects ( Figure 7B ) .",
"Consistent with this , protein associations are often enhanced by macromolecular crowding ( Minton , 2001 ) , and crowding effects become particularly relevant in a self-assembling structure with multiple units .",
"Accordingly , a recent theoretical study proposed an important role for crowding in the formation of mesoscale assemblies ( Marenduzzo et al . , 2006 ) .",
"This suggests that macromolecular crowding may have a key role in reorganizing the cytoplasm of starved cells .",
"Gln1 filaments dissolve rapidly when yeast cells re-enter the cell cycle , and the disassembled enzyme complexes are fully functional .",
"This finding and the highly ordered structural arrangement of Gln1 into fibrils argues against the possibility that these filaments are protein aggregates consisting of misfolded proteins .",
"Thus , we conclude that starvation-induced filament formation is not a chaotic protein aggregation event but a desired outcome of a protective cellular program .",
"During its lifetime , a given yeast cell repeatedly transitions into and out of starvation , suggesting that on evolutionary time scales the yeast cytoplasm must have undergone numerous pH fluctuations .",
"This makes it very likely that specific adjustments have been made to optimize the behavior of protein complexes in an energy-depleted and acidified cytoplasm .",
"This consideration is also supported by the fact that other protein complexes assemble into distinct , starvation-induced structures that do not intermix ( Sagot et al . , 2006; Laporte et al . , 2008; Laporte et al . , 2011 , 2013; Liu et al . , 2012 ) .",
"How this remarkable specificity of assembly is achieved in the crowded environment of quiescent cells remains to be determined , but some of the principles presented here may apply more generally .",
"Filament formation inactivates the enzymatic activity and results in the formation of storage depots for Gln1 .",
"How filament formation induces enzymatic inactivation is unclear , but we suspect that assembled Gln1 can no longer undergo the conformational changes that are required for enzymatic activity .",
"It is also conceivable that the substrate cannot gain access to the catalytic site .",
"However , when starved yeast cells are re-exposed to nutrients , intracellular pH levels rise and Gln1 filaments disassemble , thus shifting the enzyme back into its active form .",
"In agreement with this , we found that the ability to dissolve filaments specifically affects the ability of yeast to re-grow after starvation ( Figure 6E ) .",
"Moreover , preventing the starvation-induced decline in intracellular pH severely impaired the ability of yeast to recover from prolonged starvation ( Figure 7A ) , suggesting that filament formation by metabolic enzymes is required for survival of extensive energy depletion stress .",
"What could be the advantage of forming filaments during severe starvation ?",
"We consider four possible scenarios as likely .",
"First , filaments may be resistant to bulk autophagy , allowing yeast cells to spare vital enzymes from degradation during prolonged starvation .",
"Second , filament formation may promote the transition into or recovery from a more solid and probably protective material state of the cytoplasm , as has been proposed for bacteria ( Parry et al . , 2014 ) .",
"Third , filament formation may promote entry into a hypometabolic state that makes energy-depleted cells more resistant to metabolic fluctuations ( enzyme inactivation by filament formation may provide a buffer against metabolic fluctuations , thus preventing accidental re-entry into the cell cycle ) .",
"Fourth , enzyme inactivation may help conserve energy as part of a cell-wide energy conservation program , as has been proposed for mammalian cells containing ADF/cofilin filaments ( Bernstein et al . , 2006; Bernstein and Bamburg , 2010 ) .",
"Discrimination between these possibilities has to await the results from further experimental studies .",
"Our findings suggest that yeast cells can extensively rearrange the cytoplasm to adjust their metabolism in response to environmental changes .",
"We reveal the cytosolic pH as a key regulator of this process and show that assembly into higher order structures can modulate the activity of proteins .",
"Several earlier studies reported intracellular pH changes in response to environmental perturbations .",
"The slime mold Dictyostelium for example experiences a drop in cytosolic pH during starvation ( Gross et al . , 1983 ) and in response to stress ( Pintsch et al . , 2001 ) .",
"Additional findings in bacteria and metazoans point to an important role of the intracellular pH in controlling transitions into and out of hypometabolic states .",
"Among others , pH changes have been shown to control entry into dormancy in bacteria ( Setlow and Setlow , 1980 ) , brine shrimp ( Busa and Crowe , 1983; Hand et al . , 2011 ) , and land snails ( Barnhart and Mcmahon , 1988 ) , as well as hibernation in mammalians , amphibians , and reptiles ( Malan , 2014 ) .",
"The intracellular pH also regulates cellular metabolism more generally in mammalian cells ( Busa and Nuccitelli , 1984; Moolenaar , 1986 ) , and promotes tumorigenesis ( Webb et al . , 2011 ) and the exit from quiescence ( Zetterberg and Engstrom , 1981 ) .",
"These collective findings reveal intracellular pH changes as an evolutionarily ancient signal that directs developmental programs and mediates metabolic restructuring during energy limitation .",
"It will be interesting to revisit these and other cases in the light of our findings ."
],
[
"Cloning procedures were performed as described previously using the Gateway system ( Alberti et al . , 2007 , 2009 ) .",
"For a list of plasmids used in this study please see Supplementary file 1A .",
"The media used were standard synthetic media or rich media containing 2% D-glucose or 2% D-galactose .",
"The yeast strain backgrounds were W303 ADE+ ( leu2-3112; his3-11 , -15; trp1-1; ura3-1; can1-100; [psi-]; [PIN+] ) , BY4741 ( his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; [psi-]; [PIN+] ) or a prototrophic W303 ( Klosinska et al . , 2011 ) .",
"The strain used in Figure 1—figure supplements 1 and 2 is the same as used in a previous publication ( Narayanaswamy et al . , 2009 ) .",
"Yeast gene deletions were performed using a PCR-based approach ( Gueldener et al . , 2002 ) .",
"C terminal tagging of yeast genes was performed as described previously ( Sheff and Thorn , 2004 ) .",
"For a list of used strains please see the Supplementary file 1C .",
"To induce filaments , yeast strains were grown in YPD until early to mid log phase .",
"The cells were then washed once or twice with water and resuspended in a 0 . 1 M phosphate citrate buffer ( pH 5 , 6 or",
"7 ) or a 0 . 1 M phosphate buffer ( pH 8 ) .",
"To ensure rapid equilibration of the intracellular and extracellular pH , some buffers contained proton carriers ( 75 μM monensin , 10 μM nigericin ) and inhibitors to rapidly deplete cells of energy ( 10 mM NaN3 , 10 mM 2-deoxyglucose ) , according to what was described in a previous study ( Brett et al . , 2005 ) .",
"To acidify yeast cells in the presence of glucose , 2 mM 2 , 4-dinitrophenol was added to pH-adjusted phosphate buffers containing 2% glucose in agreement with a previously published procedure ( Dechant et al . , 2010 ) .",
"General fluorescence microscopy and time-lapse videos were acquired using a Deltavision microscope system with softWoRx 4 . 1 . 2 software ( Applied Precision ) .",
"The system was based on an Olympus IX71 microscope , which was used with a 100x 1 . 4 NA ( or 150x 1 . 45 NA ) objective .",
"The images were collected with a Cool SnapHQ camera ( Photometrics ) as 352x352 ( or 384x384 ) pixel files using 1x1 ( or 2x2 ) binning .",
"Images were deconvolved using standard softWoRx deconvolution algorithms ( enhanced ratio , high to medium noise filtering ) .",
"Images were maximum intensity projections of at least 20 individual images .",
"Representative cells are shown and each experiment was performed independently three times .",
"Cell boundaries ( indicated by the white outline in fluorescence microscopy images ) were introduced by changing the contrast of the DIC image and overlaying it with the fluorescent image .",
"Fluorophores used were mCherry or superfolder GFP with a mutation to prevent self-association ( V206R ) .",
"FlAsH-labeling of tetracystein-tagged Gln1 was essentially performed as described previously ( Adams et al . , 2002; Andresen et al . , 2004 ) .",
"More specifically , 200 μl of SD containing 1 μl of a 1 , 2-ethanediol stock ( 5 μM in HEPES , pH 7 . 5 ) and 4 μl of a FlAsH-EDT2 stock ( 4 μM in 1 M Tris–HCl , pH 7 . 5 ) were inoculated with 10 μl of mid log phase yeast cells .",
"The cells were grown overnight at 25°C .",
"The culture was harvested by centrifugation and excess dye was washed away with PBS .",
"The cells were incubated in PBS on a rotating wheel for 30 min and destained in enthanediol-containing PBS .",
"Subsequently the cells were analyzed by fluorescence microscopy .",
"Yeast cells were grown to log phase , vacuum filtered , mixed with 20% BSA , high pressure frozen ( EMPACT2 , Leica Microsystems , Wetzlar , Germany ) and freeze-substituted with 0 . 1% uranyl acetate and 4% water in acetone at −90°C .",
"Samples were transitioned into ethanol at −45°C , before infiltration into a Lowicryl HM-20 resin ( Polysciences , Inc . , Eppelheim , Germany ) , followed by UV polymerization at −25°C .",
"Semi-thin ( 150 nm thick ) sections were mounted on formvar-coated mesh EM grids and stained for 3 min with lead citrate .",
"Imaging was done in a Tecnai-12 biotwin TEM ( FEI Company , Eindhoven , The Netherlands ) at 100 kV with a TVIPS 2k CCD camera ( TVIPS GmbH , Gauting , Germany ) .",
"For CLEM , yeast cells expressing wild type or R23E Gln1-mCherry were processed for TEM .",
"To allow alignment of EM and light microscopy ( LM ) images , unstained sections on EM grids were incubated with quenched 200 nm Blue ( 365/415 ) FluoSpheres as fiducials .",
"Grids were mounted on a glass slide with VectaShield ( Vector Laboratories , Inc . , Burlingame , USA ) and viewed in both red ( for mCherry ) and UV ( for the fiducials ) channels .",
"After staining for TEM , regions of interest in LM were relocated in TEM .",
"Montaged images were acquired at multiple magnifications to facilitate the correlation .",
"LM and TEM images were overlayed in ZIBAmira ( Zuse-Institut , Berlin , Germany ) .",
"Gln1 ( R23E ) -mCherry expressing yeast were grown to an OD600 of ∼0 . 4 .",
"The cells were harvested by centrifugation and washed twice with water .",
"1 ml of XL buffer ( 1 . 2 M Sorbitol , 5 mM EDTA , 0 . 1 M KH2PO4/K2HPO4 pH 7 . 5 ) and 12 μl 10 mg/ml lyticase were added to 10 ODs of harvested cells and the cells were incubated for 40 min at 30°C on a rotator .",
"The spheroplasts were harvested by centrifugation and resuspended in lysis buffer ( 100 mM potassium phosphate , pH 6 . 3 , 150 mM KCl , 20 mM NaCl2 , 5 mM MgCl2 , 1 mM DTT , 1% Triton X-100 ) containing protease inhibitors ( 1 . 25 mM benzamidin , 10 μg/ml pepstatin , 10 μg/ml chymostatin , 10 μg/ml aprotinin , 10 μg/ml leupeptin , 10 μg/ml E−64 and 0 . 4 mM PMSF ) .",
"Cells were lysed on a bead beater for 20 min and immediately transferred onto a glass slide for analysis by fluorescence microscopy .",
"The buffer in experiment Figure 3B contained 200 mg/ml Ficoll 70 and in Figure 4A the pH was adjusted to 5 , 6 , 7 or 8 .",
"For purification of Gln1 from bacteria , wild-type and variant ( E186K , R23E ) Gln1 were expressed as 6xHis fusions in E . coli BL21 DE3 .",
"His-tagged proteins were purified to ∼99% purity from bacterial lysates using a Histrap HP column ( GE Healthcare ) and a gradient elution profile according to the manufacturer's protocol .",
"For purification of Gln1 from yeast , 6xHis-Gln1-V5 and 6xHis-Gln1-mCherry were expressed in W303 ADE + gln1::SpHIS5 cells .",
"Cell lysis was carried out in lysis buffer ( 50 mM KH2PO4/K2HPO4 , pH 8 , 150 mM KCl , 20 mM NaCl ) containing protease inhibitors ( 1 . 25 mM benzamidin , 10 μg/ml pepstatin , 10 μg/ml chymostatin , 10 μg/ml aprotinin , 10 μg/ml leupeptin , 10 μg/ml E−64 and 0 . 4 mM PMSF ) .",
"His-tagged proteins were purified at 4°C using Ni-NTA agarose according to the manufacturer's protocol for native purification in a batch format ( Qiagen ) .",
"Relative concentrations of purified proteins were determined by Western blotting using anti-V5 or anti-mCherry antibodies ( see Supplementary file 1B ) .",
"Yeast-purified mCherry-tagged Gln1 was mixed 1:5 with a 0 . 1 M phosphate-citrate ( pH 5 or",
"7 ) or phosphate buffer ( pH",
"8 ) and incubated at room temperature for one hour .",
"During the incubation period the samples were shaken in a thermomixer at 1000 rpm .",
"Crowding samples contained polyethyleneglycol ( average molecular weight 20 kDa ) at a concentration of 70 mg/ml .",
"After one hour the samples were transferred onto a glass slide and analyzed by fluorescence microscopy .",
"DLS measurements were performed using a Zetasizer instrument ( Malvern ) .",
"100 µg of wild-type or variant Gln1 were diluted in equal volumes of a HEPES buffer ( pH 6 . 5 ) containing 150 mM KCl , 20 mM NaCl , 5 mM MgCl and 1 mM DTT in a low binding microcentrifuge tube .",
"The samples were rotated for 1 hr at room temperature .",
"50 µl of the samples were transferred into a small volume DLS cuvette and placed inside the measurement chamber .",
"The shown values are average values obtained from 20 measurements at room temperature .",
"100 μg of purified wild-type and variant Gln1 was loaded onto a Superose 6HR 10/30 column ( GE Healthcare ) .",
"Eluates were collected as 300 μl fractions and were applied onto a nitrocellulose membrane by using the mini-fold dot blot system ( Whatman ) .",
"Proteins on the membrane were detected by immunoblotting with an anti-His antibody ( see Supplementary file 1B ) .",
"ThT staining of yeast cells containing Gln1 filaments was essentially performed as described previously ( Alberti et al . , 2010 ) .",
"Semi-denaturing detergent agarose gel electrophoresis ( SDD-AGE ) with lysates from filament-containing cells was performed essentially as described previously ( Alberti et al . , 2010 ) .",
"Samples were measured in a 1 mm cuvette on a JASCO J-815 spectropolarimeter .",
"Samples were diluted 1:3 with a phosphate–citrate buffer of pH 7 . 4 or 6 and scaled to the same concentration as determined from UV-Vis absorption at 280 nm .",
"Different dilutions of purified 6xHis-tagged Gln1 ( R23E ) were prepared in buffer A ( 100 mM potassium phosphate , pH 6 . 3 , 150 mM KCl , 20 mM NaCl , 5 mM MgCl2 , 1 mM DTT ) .",
"3 μl of the sample was deposited for 1 min on glow-discharged , carbon-coated EM mesh grids .",
"After blotting , samples were fixed with 1% glutaraldehyde in water .",
"The grids were washed once in water and stained with 2% uranyl acetate .",
"After drying , grids were imaged in TEM ( same setup as used for TEM on yeast cells ) .",
"Synthetase assays were carried out with yeast cell lysate as described previously ( Mitchell and Magasanik , 1984 ) ."
]
] | [
"One of the key questions in biology is how the metabolism of a cell responds to changes in the environment .",
"In budding yeast , starvation causes a drop in intracellular pH , but the functional role of this pH change is not well understood .",
"Here , we show that the enzyme glutamine synthetase ( Gln1 ) forms filaments at low pH and that filament formation leads to enzymatic inactivation .",
"Filament formation by Gln1 is a highly cooperative process , strongly dependent on macromolecular crowding , and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils .",
"Other metabolic enzymes also assemble into filaments at low pH . Hence , we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation .",
"These findings have broad implications for understanding the interplay between nutritional stress , the metabolism and the physical organization of a cell ."
] | [
"Life is based on a series of chemical reactions that control how cells live , grow , and divide .",
"Various metabolic enzymes allow cells to control the rate at which these reactions occur .",
"Recently , researchers have noticed that metabolic enzymes can form filaments in cells , usually when the cells are deprived of energy or nutrients .",
"Petrovska , Nüske et al . now reveal more about how and why an enzyme called glutamine synthetase ( Gln1 ) forms filaments in yeast cells .",
"Gln1 has a cylindrical shape .",
"This shape means that stacking the enzymes end-to-end under the right conditions is enough to make them bond into a long filament .",
"In addition , a zip-like mechanism enables neighboring filaments to fuse to create thicker fibres .",
"These filaments are more likely to form if there are high concentrations of large background molecules around—a condition known as macromolecular crowding .",
"Petrovska , Nüske et al . also found evidence that the filaments are part of a strategy to help cells survive starvation .",
"Enzymes were more likely to construct filaments when the cell division cycle had stopped , which commonly occurs due to a lack of nutrients .",
"In addition , Gln1 filaments only form if the cytoplasm of the cell becomes acidic—which is a response to the cell starving .",
"This has been seen for other metabolic enzymes as well , suggesting that acidification is a signal to reprogram the metabolism of a cell .",
"The Gln1 enzymes in a filament are inactivated , but become active again after the filament breaks up .",
"In addition , preventing Gln1 filament formation makes it harder for cells to recover from a period of starvation .",
"Petrovska , Nüske et al . therefore suggest that the filaments act as a storage depot for the enzymes during starvation .",
"Further experiments are now needed to uncover exactly how these manage to help the starving cell to survive and recover ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"genetics and genomics"
] | Epigenetic profiling of growth plate chondrocytes sheds insight into regulatory genetic variation influencing height | elife-29329-v2 | [
[
"Human height , a main outcome of skeletal growth , is the product of many biological and environmental interactions spanning numerous cell and tissue types acting during pre- and post-natal development .",
"The growth plate , located at the distal ends of long bones such as the femur , is among the most important tissues influencing height .",
"In the growth plate , condensations of mesenchymal cells differentiate into chondrocytes in the process of endochondral ossification .",
"Chondrocytes in the growth plate reside in longitudinally oriented columns divided in zones ( resting , proliferative , pre-hypertrophic , and hypertrophic ) each with important functions in allowing the bone to elongate and mature ( see Liu et al . , 2017 and Samsa et al . , 2017 for reviews ) ( Liu et al . , 2017; Samsa et al . , 2017 ) .",
"This process of growth plate chondrocyte differentiation and maturation promotes elongation of long bones , which ultimately determines much of human height ( Baron et al . , 2015 ) .",
"These processes are under the influence of many extrinsic and intrinsic signals ( Samsa et al . , 2017; Lui et al . , 2012; Kronenberg , 2003 ) .",
"Height has been studied for centuries as a model genetic trait , as it is easily measured and highly heritable ( typically , 70–90% of variation in height within a population is attributable to genetic variation ) ( Silventoinen et al . , 2003; Perola et al . , 2007; Visscher et al . , 2006; Visscher et al . , 2007; Galton , 1886 ) .",
"Height and body proportions are likely selected and thus are interesting from an evolutionary standpoint ( Turchin et al . , 2012 ) .",
"Studies of the genetics of height can shed insight into the mechanisms of childhood growth and into developmental biology of the skeleton ( Baron et al . , 2015; Dauber et al . , 2014 ) .",
"Additionally , abnormalities of growth—short stature or overgrowth—are among the most common childhood disorders and their pathophysiology is poorly understood .",
"Thus , genetic studies of height could further our understanding of the genetic basis of skeletal disease , evolution , biology , and normal human growth and development .",
"Recently , genome-wide association studies ( GWAS ) have been used to study the genetics of height , as well as many other traits and disorders ( Wood et al . , 2014 ) .",
"This approach systematically tests each common variant in the genome for association with a phenotype of interest , such as adult height , with the goal of identifying relevant biology , either from the identities of the genes near the associated variants , or by deducing mechanism from the associated variants themselves .",
"GWAS for height are among the largest conducted thus far ( sample sizes > 250 , 000 ) and have identified more associations than any other phenotype , with nearly 700 regions of the genome ( or loci ) robustly associated with height ( Wood et al . , 2014 ) .",
"The finding of so many loci influencing height , with the number expected to increase with larger sample sizes , supports the assertion that height is highly polygenic .",
"Although each of the variants at these loci have only a small effect on height ( typically much less than 0 . 5 cm per allele ) ( Wood et al . , 2014 ) , the genes within the associated loci as a group highlight important mechanisms influencing growth .",
"For example , height variants are enriched for genes implicated in numerous biological processes relevant to growth , including embryonic stem cell function , long bone development , and spine length , among a number of other processes ( Wood et al . , 2014 ) .",
"Height variants are also preferentially located near genes that are differentially expressed in the growth plate ( Lui et al . , 2012 ) .",
"However , as with all GWAS , moving from genetic associations to biological mechanisms at any individual locus can be challenging .",
"There are two primary reasons for this difficulty .",
"First , there is extensive linkage disequilibrium ( LD ) in the human genome , meaning that the genotypes at nearby genetic variants are often tightly correlated ( Gabriel et al . , 2002 ) .",
"The consequence of LD is that at any particular locus , there will be many variants that have indistinguishable associations , and determining which variant ( s ) are in fact causal ( rather than associated due to LD with a causal variant ) remains difficult .",
"Second , most GWAS variants ( >80% ) reside in non-coding regions ( Gusev et al . , 2014 ) , with no nearby coding variants that can account for the associations .",
"Non-coding variants are challenging to interpret because there is no universal regulatory code to infer function/mechanism , and these variants can act to influence gene expression at long distances and in highly tissue-specific manners ( Bernstein and ENCODE Project Consortium , 2012 ) .",
"Various approaches have been pursued for identifying the causal variants and/or mechanisms , including overlapping associated variants with epigenetic datasets in appropriate tissues/cell-types , statistical genetic methods leveraging subtle variations in LD patterns ( called ‘fine mapping’ ) , and in vitro and in vivo functional assays ( Tak and Farnham , 2015 ) .",
"It is also becoming increasingly clear that GWAS variants tend to fall in regions that have particular epigenetically defined states ( such as enhancers or repressors ) ( Finucane et al . , 2015; Farh et al . , 2015 ) .",
"Thus , the overlap between GWAS and the appropriate epigenetic annotations can help refine the identities of causal variants , helping to decipher mechanism .",
"However , epigenetic data may not exist for the biologically relevant cell type , and there is no universally successful approach to translating non-coding associations from GWAS into biological mechanisms .",
"Although the large number of associated variants offers advantages in making biological inferences for the set of associated variants as a whole ( Lango Allen et al . , 2010 ) , defining functional mechanisms of individual height GWAS variants faces specific challenges .",
"Height variants likely act in many tissues and stages of development , including not only the growth plate but also embryonic cells , endocrine tissues such as the pituitary , and bone , among others ( Wood et al . , 2014 ) .",
"Human growth is also a whole-organism phenotype and is less amenable to study in cell-intrinsic models , making functional approaches particularly challenging .",
"Finally , skeletal tissues can be highly heterogeneous and challenging to isolate because in their mature form they are surrounded by a hard extracellular matrix and strongly adhering soft tissues such as tendons , ligaments , and muscles .",
"These factors make it difficult to isolate the large numbers of biologically relevant cells that are needed for functional experiments or for traditional epigenomic assays such as ChIP-seq ( Furey , 2012 ) .",
"In this study , we profile the epigenetic landscape of mouse growth plate chondrocytes using a new approach called ATAC-seq ( Buenrostro et al . , 2015 , 2013 ) , which allows for accurate profiling of open chromatin regions using relatively few cells ( ~50 , 000 ) .",
"We then use the epigenetic landscape in growth plate chondrocytes to gain new insights into the mechanisms of non-coding elements in the genome influencing expression of growth plate genes .",
"Finally , we use these epigenetic marks , in the context of statistical fine-mapping results and gene expression profiles , to better understand height GWAS variants and the mechanisms by which they may function to influence height in humans .",
"These studies illustrate a path that can help decipher mechanism from GWAS , even when the relevant cell type is relatively inaccessible in humans ."
],
[
"In order to identify open chromatin regions involved in long bone development , we performed ATAC-seq ( Buenrostro et al . , 2015 , 2013 ) on E15 . 5 ( Theiler Stage 23 ) mouse proximal and distal femoral growth zones , regions comprised predominately of the growth plate proper , the overlying perichondrium , and the cartilaginous super-structures that dictate adult bony morphology ( Materials and methods ) .",
"This stage of mouse embryogenesis corresponds to approximately E48-51 ( Carnegie Stage 19 ) of human development and reflects a developmental window when the growth plate proper possesses all chondrocyte zones ( resting , proliferative , pre-hypertrophic , hypertrophic zone ) and is undergoing rapid cell proliferation and differentiation during endochondral ossification .",
"Given that it remains unclear which sub-region ( s ) ( i . e . chondrocyte zones ) of the growth plate are under genetic influence to drive height variation , we performed our experiments on tissues that include all chondrocyte zones ( including perichondrium ) and cartilaginous tissues , excluding regions indicative of the osteogenic invasion in the developing bone collar ( Figure 1a ) .",
"We used ATAC-seq to isolate open chromatin regions from the DNA , which were subsequently subjected to next-generation sequencing ( Materials and methods; Supplementary file 1 ) .",
"This approach yielded 28 , 257 proximal femur peaks and 27 , 958 distal femur peaks , with 23 , 764 shared peaks , 4493 peaks specific to the proximal femur , and 4194 specific to the distal femur ( Figure 1b ) .",
"The proximal and distal peaks span approximately 0 . 79% and 0 . 76% of the genome , respectively .",
"We also assessed the genomic distribution of proximal and distal femur ATAC-seq peaks and found a characteristic enrichment near gene transcriptional start sites and more distal intronic and intergenic non-coding sequences ( Figure 1—figure supplement 1 ) .",
"Since a main goal was to understand the gene regulatory functions of variants influencing human height , we next mapped the locations of each mouse femur open chromatin region to the human genome ( Materials and methods ) .",
"Our liftover resulted in 24 , 805 proximal femur peaks and 24 , 788 distal femur peaks , representing approximately 87 . 7% and 88 . 6% of sites identified in mouse , respectively .",
"These sites that were capable of liftover to human likely reflect relatively conserved genes involved in vertebrate musculoskeletal development ( Shubin et al . , 1997; Petit et al . , 2017 ) .",
"The union of the two sets contained 25 , 829 peaks ( Figure 1—figure supplement 1 ) .",
"To determine whether ATAC-seq peaks are enriched near gene sets/pathways with specific functions , we performed an enrichment analysis using Genomic Regions Enrichment of Annotations Tool ( GREAT ) ( McLean et al . , 2010 ) , which identifies curated gene sets enriched near epigenetic peaks .",
"Analyses of each proximal , distal , and union femur datasets yielded general GO Biological Process and GO Molecular Processes terms related to basic cell biological processes , as well as phenotypes related to a number of biological tissues including skeletal tissues ( Supplementary file 2–4 ) .",
"They also yielded phenotype terms related to general skeletal development , chondrogenesis , and long bone skeletal phenotypes , such as ‘Metaphyseal widening’ , ‘Abnormality of the femoral epiphyses’ , and ‘Abnormality of the femoral head’ ( Figure 1c ) .",
"Thus , ATAC-seq datasets generated on each specific femoral growth plate in mice reflect in part highly relevant anatomy , as well as general aspects of skeletal development in humans ( see Discussion ) .",
"The ATAC-seq peaks we identified also highlight biological processes that are important in the growth plate .",
"For example , GREAT analyses uncovered enrichment for several relevant GO Biological Processes such as ‘Cartilage condensation’ , ‘Positive regulation of chondrocyte differentiation’ , and ‘Chondrocyte proliferation’ ( Supplementary file 2–4 ) .",
"Additionally , we illustrate ATAC-seq peaks at several well-known growth plate genes including Col2a1 , Fgfr1 , and Pth1r ( Figure 1—figure supplement 2 ) ( Long and Ornitz , 2013 ) .",
"We next wanted to validate that our ATAC-seq datasets reflect open chromatin regions important to mouse chondrocyte biology .",
"To do so , we analyzed a previous dataset that used ChIP-seq to profile Sox9 binding in mouse rib chondrocytes ( Ohba et al . , 2015 ) .",
"We found extensive overlap between Sox9-occupied sites and our open chromatin ATAC-seq peaks .",
"For example , 86 . 4% of Class I Sox9 elements ( i . e . elements clustering near transcriptional start sites of highly expressed non-chondrocyte-specific genes ) , 44 . 6% of Class II Sox9 elements ( i . e . elements directing chondrocyte-related gene activity through direct Sox9 dimer binding ) , and 93 . 8% of Sox9 super-enhancer regions ( i . e . enhancer complexes involved in chondrocyte cell identity ) overlap with our femur ATAC-seq peaks ( p<0 . 001 for all three sets , Materials and methods , Figure 2a ) .",
"We also examined overlap between our dataset and various histone modifications in the mouse rib chondrocytes ( Materials and methods ) ( Figure 2b ) ( Ohba et al . , 2015 ) .",
"We found that 85 . 1% of RNA Pol II transcriptionally active promoters , 56 . 9% of H3K4me2 active promoter sites , 86 . 7% of H3K4me3 active promoter sites , 87 . 4% of H3K27ac active enhancer sites , and 59 . 4% of p300 histone acetyltransferase active enhancer sites overlapped with femur ATAC-seq peaks .",
"Conversely , our ATAC-seq datasets displayed relatively lower overlap with markers of gene repression; that is , only 33 . 7% of H3K27me3-repressed enhancer sites overlap with femur ATAC-seq signals ( Figure 2b ) .",
"Based on permutation analyses , the enrichments of all these histone modification sets were significant at p<0 . 001 .",
"Thus , we found that femoral ATAC-seq peaks show marked evidence of active chromatin state and/or gene transcription .",
"We also wanted to determine if our ATAC-seq peaks reflect regulatory elements involved in human limb development and chondrocyte biology .",
"First , we used a ChIP-seq dataset comprised of H3K27ac peaks found in E47 human limb buds ( equivalent to E13 . 5 mouse limb buds ) that are shared with mouse limb buds ( Cotney et al . , 2013 ) .",
"We found that 45 . 2% of these E47 human limb bud H3K27ac peaks overlapped with ATAC-seq peaks , indicating that many of our mouse femur ATAC-seq peaks reflect active human limb enhancers involved in chondrogenesis ( p<0 . 001 , Figure 2c ) .",
"Second , we used an H3K27ac ChIP-seq dataset generated on human adult cultured bone marrow mesenchymal stem-cell-derived chondrocytes ( BMDCs ) ( Herlofsen et al . , 2013; Kundaje et al . , 2015 ) and found that 13 . 3% of human adult BMDC H3K27ac peaks overlapped our mouse distal femur dataset ( p<0 . 001 ) ( Figure 2c ) , which indicates that our femur ATAC-seq dataset also captures cell-intrinsic properties of chondrogenesis .",
"However , the greater overlap seen in the limb bud dataset as compared to the BMDCs suggests that our dataset may be capturing epigenetic properties present in developing in vivo tissues that are poorly modeled by cell culture systems .",
"Our ATAC-seq data were also able to identify 14 of 26 previously-discovered active regulatory elements residing near key cartilage genes detected at different time-points and using different assays ( e . g . see Figure 2d , Supplementary file 5 ) .",
"This suggests that mouse femoral ATAC-seq regions have in vivo enhancer activity as determined in independent assays .",
"Overall , these data indicate that ATAC-seq datasets generated on the mouse femoral growth plates match previously expected patterns of regulatory usage important to chondrocyte biology .",
"We next sought to use our ATAC-seq dataset to gain insight into height-associated variants identified by GWAS .",
"To do so , we intersected our orthologous human ATAC-seq peaks with 688 height GWAS loci from Wood et al . ( 2014 ) ( nine loci were excluded; see Materials and methods ) ( Wood et al . , 2014 ) .",
"Since we do not know which variants are causal and which are associated with height due to LD , for each locus , we identified potentially causal ‘proxy’ SNPs: those with a correlation of r2 >0 . 5 to the lead SNP based on the European subset of 1000 Genomes Phase 3 ( Auton et al . , 2015 ) .",
"The number of proxy SNPs ranged from 1 to 3548 for each of these 688 loci ( Figure 3—figure supplement 1 ) .",
"Across the 688 height loci , we found that 317 loci ( or ~46% ) contained at least one variant that overlapped with an ATAC-seq peak .",
"To test the significance of the observed overlap , we applied GoShifter ( see Materials and methods ) , which performs a shuffling-based approach to evaluate the overlap between an epigenetic data set and GWAS data .",
"We found strong enrichments for the ATAC-seq peak set and height GWAS data ( p=0 . 0059 ) ( Figure 3—figure supplement 1a ) .",
"We repeated this analysis using the Sox9 binding and histone modifications in mouse rib chondrocytes , human BMDCs , and mouse embryonic limb datasets from above , as well as DNaseI hypersensitivity sites from mouse brain , liver , and lung ( Bernstein and ENCODE Project Consortium , 2012; Ohba et al . , 2015; Cotney et al . , 2013; Yue et al . , 2014 ) .",
"Only the mouse rib chondrocyte H3K4me2 peaks reached statistical significance ( p=0 . 0237 ) , and was far less significant than our ATAC-seq data .",
"This suggests that our ATAC-seq data captures functional genetic variation influencing height that is not captured by the other datasets .",
"Also , as no enrichment was observed for other mouse tissues from ENCODE , this suggests that the observed enrichment is unlikely due to artifacts stemming from our use of mouse tissues ( e . g . from conserved regions being lifted over ) .",
"To further demonstrate the specificity of our ATAC-seq peaks for capturing height GWAS signals , we repeated the GoShifter analysis by examining the enrichment of GWAS results for other complex traits/diseases at our ATAC-seq peaks .",
"These other traits/diseases were chosen as they have no obvious connection to height and included age of menarche ( Day et al . , 2017 ) , body mass index ( Locke et al . , 2015 ) , coronary artery disease risk ( Schunkert et al . , 2011 ) , LDL cholesterol ( Teslovich et al . , 2010 ) , mean corpuscular volume ( van der Harst et al . , 2012 ) , schizophrenia risk ( Ripke and Schizophrenia Working Group of the Psychiatric Genomics Consortium , 2014 ) , and type 2 diabetes risk ( Morris et al . , 2012 ) .",
"These additional traits/diseases all demonstrated little to no enrichment at ATAC-seq peaks ( Figure 3—figure supplement 1 ) , suggesting that the growth plate ATAC-seq peaks are specifically capturing height/growth biology .",
"As a second approach to evaluate overlap between our ATAC-seq peak set and height GWAS data , we generated 1000 sets of random loci which were matched to the actual height GWAS loci based on factors including SNP density , minor allele frequency , number of proxy SNPs in the locus , and gene density ( Materials and methods ) ( Pers et al . , 2015 ) .",
"For each of the 1000 matched sets of random loci , we repeated the overlap with the ATAC-seq peaks .",
"As none of the matched sets had as much overlap as our height GWAS loci , the enrichment we observed for the height GWAS data is highly significant ( p<0 . 001; z-score: 11 . 88 ) ( Figure 3c ) .",
"Notably , we observed that the actual number of SNPs that overlap an ATAC-seq peak for each set was higher than the number of represented loci .",
"In total , there were 928 variants that overlapped with an ATAC-seq peak; these 928 variants were distributed across 317 loci ( Supplementary file 6; Figure 3—figure supplement 1 ) .",
"This finding of more than one overlapping variant per locus may indicate multiple causal variants at each locus , as has been shown for other traits ( Roman et al . , 2015 ) .",
"An alternative method to narrow down potentially causal signals within GWAS loci is to use statistical fine-mapping , which leverages the association statistics and patterns of LD at each locus to identify which variants at a given locus are most likely to be causal .",
"We applied PICS ( Farh et al . , 2015 ) to perform statistical fine-mapping of the ( Wood et al . , 2014 ) height GWAS results ( Materials and methods ) .",
"For each of 688 height-associated loci , we calculated credible set probabilities for all SNPs with an r2 >0 . 5 to the lead variant based on the European subset of 1000 Genomes Phase 3 ( Auton et al . , 2015 ) .",
"For each locus , we then determined the 95% credible set , which represents the minimum set of SNPs at that locus such that the truly causal SNP has at least a 95% probability of being in the set .",
"Across 688 loci , this yielded a 95% credible set of 32 , 846 SNPs , which represents approximately a 48 . 2% reduction from the total number of variants in the GWAS loci .",
"Intersecting these credible set SNPs with femur open chromatin regions revealed 468 overlapping variants spanning 192 of the 688 loci ( Figure 4a ) .",
"A previous study profiled expression of mouse growth plate genes and identified a set of 427 genes that were differentially expressed in the growth plate ( Lui et al . , 2012 ) .",
"That study also revealed that height GWAS loci ( using a previous smaller height GWAS ) are enriched near differentially expressed growth plate genes ( Lui et al . , 2012; Lango Allen et al . , 2010 ) .",
"We expanded on those results using the most recent height GWAS from Wood et al . ( 2014 ) and indeed found that height GWAS loci are enriched near 427 differentially expressed growth plate genes ( within 100 kb ) when compared to the 1000 matched random GWAS loci ( p<0 . 001 , z-score: 8 . 54 , Figure 5a ) .",
"Since we detected enrichment of height GWAS variants near femoral open chromatin regions ( see Figure 3 ) and reasoned that many of the variants likely function by influencing expression of growth plate genes , we next identified the number of GWAS variants that fit the criteria of residing in an open chromatin region near a differentially expressed gene ( within 100 kb ) .",
"We again used the 688 GWAS loci from Wood et al . ( 2014 ) and their proxy SNPs ( r2 >0 . 5 ) and found that 46/688 loci ( or ~6 . 7% ) contained at least one GWAS variant that overlapped with an ATAC-seq peak and resided near a differentially expressed gene ( Supplementary file 6 ) .",
"Using the same 1000 sets of matched random loci as described above , we found that these overlaps were highly significant ( p<0 . 001 , z-score: 6 . 36 , Figure 5b ) .",
"These loci likely represent combinations of functional variants that overlap an epigenomic element in growth plates that then modulates expression of nearby genes influencing height .",
"Wood et al . , had previously observed that height GWAS SNPs that represent cis eQTLs in whole blood are enriched for expression in cartilage ( Wood et al . , 2014 ) .",
"Here , we sought to evaluate whether differentially expressed growth plate genes that are also eQTLs genes in whole blood are enriched at ATAC-seq peaks .",
"Among the 427 differentially expressed growth plate genes , 157 had previously been identified as eQTL genes in whole blood ( Westra et al . , 2013 ) .",
"Using the GoShifter permutation analysis as described above , we demonstrated that the corresponding eQTL variants for these 157 genes are significantly enriched at ATAC-seq peaks ( p=0 . 0004 ) Of the 46 loci in the ATAC-seq femur comparison that fall near genes differentially expressed in growth plates , 44 different genes have been identified as possibly being modulated by these putative regulatory variants ( note , some genomic loci have multiple genes and/or GWAS loci ) ( Supplementary file 6 ) .",
"These genes represent major signaling molecules ( e . g . IHH , HHIP ) , transcription factors ( e . g . RUNX2 ) , extracellular matrix factors ( e . g . CHSY1 , EXTL1 ) , among other factors involved in the growth plate in both humans and mice .",
"We next examined some of the most compelling loci from a genetic and mechanistic perspective .",
"We filtered for loci where a height GWAS variant in the PICS 95% credible set overlapped an ATAC-seq peak , and was near a differentially expressed growth plate gene ( ±100 kb ) .",
"In total , 59 variants distributed across 26 loci met these criteria .",
"These 59 variants thus have strong evidence for being potentially causal variants and might be prioritized for downstream study ( Supplementary file 6 ) .",
"For example , at the CHSY1 locus , four variants ( rs8042551 , rs11639408 , rs9920291 , and rs3911964 ) met these criteria .",
"Disruption of Chsy1 in mice and humans leads to severe skeletal phenotypes including long bone length reductions ( Wilson et al . , 2012; Temtamy et al . , 1998; Tian et al . , 2010; Li et al . , 2010 ) .",
"Of the four variants , rs9920291 is predicted to alter HOXD13 binding , as suggested by data generated using universal protein binding microarray technology that demonstrates that the eight base-pair sequence matching the immediate rs9920291 locus disrupts in vitro binding by HOXD13 ( Figure 6a , Supplementary file 7 ) , a homeodomain transcription factor that is expressed in the growth plate ( Kuss et al . , 2014; Reno et al . , 2016 ) and has known activating and repressive roles in skeletal development ( Johnson and Tabin , 1997 ) .",
"We performed a series of functional assays to test the effects of allelic variation at rs9920921 .",
"First , using a luciferase reporter assay in T/C-28a2 human chondrocytes , we found that the open chromatin region surrounding rs9920291 acts as a repressor of expression ( p=7 . 83×10−6 and 3 . 6 × 10−4 for variants carrying the reference and alternate alleles respectively ) ( Figure 6b ) .",
"In addition , the alternate T allele , ( i . e . the height-increasing allele of rs9920291 ) makes the regulatory element act as a weaker repressor ( p=0 . 0014 ) .",
"This is consistent with data from GTEx ( Lonsdale J , GTEx Consortium , 2013 ) , where rs9920291 acts as an eQTL for CHSY1 in transformed fibroblasts ( p=1 . 0×10-7; Figure 6—figure supplement 1 ) , with the T allele correlating with increased CHSY1 expression levels .",
"Additionally , we leveraged the fact that in HEK-293FT cells , rs9920291 is heterozygous , along with several additional SNPs in the coding region of CHSY1 .",
"Assaying a heterozygous exonic coding SNP ( rs28364839 ) and a 3’UTR SNP ( rs11433 ) , we demonstrate that rs9920291 displays allelic skew in expression ( p<0 . 001 for both assayed variants; Figure 6d ) .",
"To further demonstrate that the region surrounding rs9920291 displays regulatory activity for CHSY1 , we performed CRISPR-Cas9 targeting of a 135 bp sequence spanning a conserved portion of the regulatory element containing rs9920291 ( guides sg1 and sg4 ) , or an 17 bp sequence immediately surrounding rs9920291 ( guides sg6 and sg8 ) .",
"Either deletion led to a significant upregulation of CHSY1 expression in vitro in T/C-28a2 cells ( p=7 . 83×10−6 for the 135 bp deletion and p=3 . 64×10−4 for the 17 bp deletion; Figure 6d ) , confirming the regulatory element’s repressor activity .",
"Consistent with our computational predictions that rs9920291 perturbs HOXD13 binding , we found that overexpression of HOXD13 in T/C-28a2 cells significantly upregulated CHSY1 expression ( p=1 . 63×10−5; Figure 6e , Figure 6—figure supplement 1 ) .",
"We also highlight a previously identified height-associated variant ( rs4911178 ) at the GDF5 locus , which resides in an ATAC-seq peak active in femoral growth plates ( Figure 6—figure supplement 2 ) .",
"We have shown in separate work that the underlying enhancer drives GDF5 expression in growth plates and that rs4911178 has allele-specific activity ( Capellini et al . , 2017 ) .",
"Thus , our approach of integrating genetic and growth plate epigenetic data has the ability to identify previously discovered cis-regulatory targets that mediate human height variation .",
"To expand upon our understanding of transcription factor binding at ATAC-seq open chromatin regions , we performed a series of motif enrichment analyses using HOMER ( see Materials and methods ) .",
"First , we performed a de novo motif analysis on the union of distal and proximal femoral ATAC-seq peaks in mouse to identify enriched transcription-factor-binding motifs .",
"We identified a number of highly enriched motifs relevant to chondrogenesis and other skeletal developmental processes , including Atf3 ( James et al . , 2006 ) , Msx2 ( Amano et al . , 2008 ) , Pbx1 ( Selleri et al . , 2001 ) , and Ctcf ( Jerković et al . , 2017 ) ( Figure 7a , Supplementary file 8 , sheet 1 ) .",
"We performed a similar analysis on the liftover human ATAC-seq regions and found enrichments for a similar set of motifs ( Figure 7a , Supplementary file 8 , sheet 2 ) .",
"We next characterized the effect of height GWAS variants that intersect with these motifs within ATAC-seq peaks .",
"Further characterization of the GWAS variants within CTCF motifs indicated a tendency toward disruptive effects ( Figure 7b ) .",
"Comparing the height GWAS variants intersecting ATAC-seq peaks to 500 sets of random GWAS variants revealed an enrichment for variants that are predicted to impact CTCF-binding ( p<0 . 01 , z-score: 6 . 01 , Figure 7c ) ( see Materials and methods ) .",
"An example of a variant within a CTCF motif is shown in Figure 7d .",
"Additionally , we detected a strong enrichment of height GWAS variants overlapping predicted PBX1 motifs within ATAC-seq regions , when compared to height GWAS variants across the genome ( adjusted p value=1 . 91×10−108 , Supplementary file 9; see Materials and methods ) ."
],
[
"Like many anthropometric traits , height is highly heritable and influenced by a large number of distinct genetic inputs that influence the development and/or growth of a number of tissues ( Visscher et al . , 2010 ) .",
"Of these tissues , the growth plate plays a key role in determining overall height .",
"Despite decades of research on chondrocyte growth plate biology , we have been limited in our understanding of how height GWAS variants—most of which are non-coding—act in the growth plate to influence height .",
"This is due in part to the paucity of epigenetic data related to the regulatory landscape of the growth plate , as these datasets have been technically challenging to generate .",
"Here , we leveraged a new method ( ATAC-seq ) ( Buenrostro et al . , 2015 , 2013 ) to overcome previous technical challenges and reveal the open chromatin regulatory landscape of growth plate chondrocytes .",
"We then analyzed height GWAS data in the context of the femur growth plate epigenetic landscape to gain insight into how height GWAS variants act at the growth plate .",
"Our analyses revealed that ATAC-seq datasets generated from mouse growth plate chondrocytes overlap previously identified signals of chondrocyte biology in the mouse , as well as in humans .",
"The ATAC-seq peaks reside near genes enriched for human phenotypes related to skeletal biology , such as ‘metaphyseal widening . ’ These peaks also reside near genes with known roles in chondrocyte biology and include biological processes such as ‘cartilage condensation’ and ‘chondrocyte proliferation . ’ We also show that our open chromatin profiles overlap quite faithfully with locations of active regulatory enhancers and Sox9-binding sites as assessed using ChIP-seq on mouse rib chondrocytes ( Ohba et al . , 2015 ) .",
"Additionally , our datasets overlap with evidence of regulatory control during chondrogenesis and human limb embryogenesis ( Cotney et al . , 2013; Herlofsen et al . , 2013; Kundaje et al . , 2015 ) .",
"These findings indicate that general aspects of chondrocyte regulation , in part shared between humans and mice , are captured by our ATAC-seq growth plate datasets .",
"While we note that appropriate human datasets generated from growth plate chondrocytes are still unavailable and that there is evidence of substantial functional divergence in the genic and non-coding usage in cell types between species ( Bernstein and ENCODE Project Consortium , 2012 ) , the strong overlap between our mouse ATAC-seq data and human embryonic limb and human BMDCs suggests considerable conservation in the regulatory profiles at the growth plate .",
"While our datasets capture aspects of general chondrocyte biology , we also found that ATAC-seq peaks acquired on femoral tissues show enrichment in human phenotypes related to the anatomy of femur .",
"This finding points to a fine-grained , modularized control of skeletal element-specific biology .",
"This is also supported by our independent identification of a number of previously published long bone growth plate chondrocyte enhancers in mice and humans , as well as the growing evidence that a number of important skeletal development genes , such as Bmp5 ( Guenther et al . , 2015 ) , Gdf6 ( Mortlock et al . , 2003; Indjeian et al . , 2016 ) , and Gdf5 ( Capellini et al . , 2017; Chen et al . , 2016 ) have conserved modularized cis-regulatory architectures .",
"As height is encoded by a number of genetic inputs , undoubtedly variants that influence general , as well as element-specific regulatory enhancers ( e . g . tibia versus femur ) , will be important factors shaping height variation in humans .",
"Given that over 80% of GWAS height loci are non-coding , our study demonstrates that height GWAS variants are significantly enriched in growth plate chondrocyte regulatory regions and that the majority reside in intergenic portions of the genome .",
"Importantly , this enrichment of height GWAS loci was not observed nearly as strongly in other chondrocyte or limb bud tissues , which suggests that our dataset is capturing functional genetic variation influencing height that is not captured by other existing datasets .",
"No enrichment was observed for DNaseI hypersensitivity sites from mouse brain , liver , or lung , which suggests that our enrichment with height GWAS variants is tissue-specific and also not due to an artifact from the liftover process from mouse to human .",
"These ATAC-seq peaks overlapping height GWAS variants are enriched for nearby differentially expressed growth plate genes .",
"GWAS results for other traits/diseases that are not clearly related to growth/height did not show enrichment at the ATAC-seq peaks , demonstrating that the ATAC-seq peaks are specifically capturing growth/height biology .",
"Finally , our motif analysis additionally revealed that a number of these human height variants may alter the binding of transcription factors with important roles in chondrogenesis and gene regulatory processes .",
"Combining our statistical fine-mapping and functional fine-mapping has allowed us to narrow down to a much smaller subset of putatively causal variants for downstream functional testing .",
"As an example , one putatively causal variant ( rs9920291 ) resides in the Chondroitin Sulfate Synthase 1 ( CHSY1 ) locus .",
"Human mutations in CHSY1 result in Temtamy preaxial brachydactyly syndrome , characterized by short stature , preaxial brachydactyly , and hyperphalangism of digits 1–3 ( 45 , 46 ) , as well as a number of additional soft and hard tissue disorders .",
"rs9920291 is in the fine-mapped credible set , resides in regulatory elements active in human adult chondrocytes and embryonic limb tissues , and displays evidence of binding by Sox9 in mouse chondrocytes .",
"rs9920291 additionally modifies a critical nucleotide ( C to T ) and disrupts the experimentally determined in vitro binding motif of HOXD13 , a homeodomain transcription factor that is expressed in chondrocytes ( Kuss et al . , 2014; Reno et al . , 2016 ) and has known roles in skeletal development ( Johnson and Tabin , 1997 ) .",
"We performed extensive experimental characterization of rs9920291 at the CHSY1 locus .",
"We demonstrated that rs9920291 displays allelic regulatory activity in human chondrocyte cell lines .",
"We also deleted the regulatory region surrounding rs9920291 using CRISPR-Cas9 and demonstrated that these deletions alter CHSY1 expression .",
"Thus , our results elucidate how common regulatory variation at this locus influences height by modulating CHSY1 expression .",
"Height , given that it is generally not a cell-intrinsic phenotype , has traditionally not been conducive to functional experimentation .",
"Our results highlight how we can pinpoint likely causal variants and mechanisms to make experimentation more tractable for a challenging phenotype such as height .",
"To our knowledge , along with GDF5 , this is the only other height GWAS locus with functional validation ( Capellini et al . , 2017 ) .",
"A number of other variants , listed in the browsable Supplementary file 6 , are available for functional testing .",
"When more extensive height GWAS data ( ideally imputed to a dense reference panel ) are generated and more sophisticated fine-mapping methods are developed , additional evidence of enrichment for fine-mapped variants may be revealed using our approach .",
"When these experiments are combined with high-throughput functional assays to test variant functionality , a more refined picture of height biology will emerge ."
],
[
"All experiments were performed following protocols approved by the Harvard University IACUC committee .",
"Timed matings were established between FVB mice , and pregnant females were euthanized at E15 . 5 in order to acquire embryonic long bone growth plates .",
"Embryos were dissected in PBS1X on ice under a dissection scope and the proximal and distal growth zones of the right and left femur were stripped clear of soft tissues .",
"Each proximal or distal cartilaginous end was then micro-dissected from the bony diaphysis ( Figure",
"1 ) and separately pooled from a single litter , consisting on average of eight animals .",
"Two biological replicates were collected in line with previous ATAC-seq studies ( Gehrke et al . , 2015 ) .",
"The samples were collected in micro-centrifuge tubes containing 200 ul 5% FBS/DMEM .",
"To generate a single-cell chondrocyte suspension , each pooled sample was then subjected to 1% Collagenase II ( VWR 80056–222 , Radnor , Pennsylavania ) digestion for 2 hr at 37°C rocking , mixing every 30 min .",
"After placing on ice , samples were filtered using a micro-centrifuge filter set-up by gently mashing the residual tissues through the filter followed by rinsing with 5% FBS/DMEM .",
"Samples were then spun down at 500 g at 4°C for 5 min .",
"We next performed cell counting methods using trypan blue and a hemocytometer and performed subsequent ATAC-seq steps on those samples that had cell death rates well below 25% .",
"On average we acquired 500 , 000–1 , 000 , 000 living cells per harvest .",
"Next , cells were re-suspended in concentrations of 50 , 000 cells in 1x PBS .",
"Cell samples then subjected to the ATAC-seq protocol as described in Buenrostro et al . , 2015Buenrostro et al . , 2015 , modifying the protocol by using 2 µl of transposase per reaction .",
"The transposase reaction product was then purified using the Omega MicroElute DNA Clean Up Kit following manufacturers protocols , eluted in 10 µl of warmed ddH20 , and stored at −20°C .",
"Next , samples were subjected to PCR amplification and barcoding following Buenrostro et al . , 2015Buenrostro et al . , 2015 .",
"All primers used in this step are listed in Supplementary file 1 .",
"Ten microliters of transposed DNA were then placed in a reaction containing NEBNext High-Fidelity PCR Master Mix , ddH20 , and primers .",
"After amplification , samples were transferred to micro-centrifuge tubes and subjected to the OMEGA Bead Purification Protocol following manufacturers protocol .",
"The samples were eluted in 30 µl of TE , run on a nanodrop , diluted to 5 ng/µl and run on a bioanalyzer .",
"Prior to sequencing sample concentrations were determined using the KAPA Library Quantification Complete Kit ( KK4824 ) .",
"Samples were then sent out to the Harvard University Bauer Core Facility for sequencing on one lane of the Illumina NextSeq 500 ( see Supplementary file 1 ) .",
"Sequencing yielded approximately 400 million reads per lane and an average of 100 million per sample .",
"Quality control statistics are presented in Supplementary file 1 .",
"Sequenced reads were next aligned to the mouse reference mm10 genome assembly using Bowtie2 ( Langmead and Salzberg , 2012 ) .",
"Duplicated reads were removed and significant peaks determined by using MACS2 software ( version 2 . 1 . 0 ) ( Johnson and Tabin , 1997 ) .",
"Peaks were assessed for reproducibility between biological replicates using the IDR statistical test ( Li et al . , 2011 ) at various statistical cut-offs , with an IDR threshold of <0 . 05 selected to define reproducible peaks for all subsequent analyses .",
"Datasets for other IDR score thresholds are available upon request and when used in all analyses show similar statistical enrichments .",
"Intersections between proximal and distal femur peaks were done with IDR-filtered narrow peak files , with the proximal/distal-common set generated by merging overlapping peaks .",
"Annotations of the narrow peak files generated on all datasets were made using HOMER annotatePeaks . pl ( http://homer . salk . edu/homer/motif/ ) .",
"Raw sequencing fastq files and processed peak bed files have been deposited on NCBI GEO ( GSE100585 ) .",
"GREAT ( Genomic Regions Enrichment of Annotations Tool ) ( McLean et al . , 2010 ) identifies curated gene sets that display enrichment near regulatory elements .",
"The software first identifies likely target genes of each regulatory element based on distance .",
"GREAT then tests the enrichment of these genes localized near regulatory elements in curated gene sets that reflect pathways and molecular processes .",
"To increase the specificity of our results , we removed peaks that were also seen in ATAC-seq from the mouse brain .",
"ATAC-seq results were then lifted over to hg19 for analysis .",
"We used the ‘Significant By Region-based Binomial view’; otherwise , all default input parameters and output display settings available at the GREAT website ( http://great . stanford . edu/ ) were used .",
"GREAT v3 . 0 . 0 was used .",
"Mouse chondrocyte data: ChIP-seq profiles Sox9 binding in mouse rib chondrocytes was obtained from Ohba et al . ( 2015 ) .",
"We also obtained ChIP-seq of RNA Pol II , H3K4me2 , H3K4me3 , H3K27ac , p300 and H3K27me3 in mouse chondrocytes from Ohba et al . ( 2015 ) .",
"Human data: H3K27ac ChIP-seq of E47 human limb buds that are shared with mouse was obtained from Cotney et al . , 2013 ( Cotney et al . , 2013 ) .",
"H3K27ac ChIP-seq of human adult cultured BMDCs was obtained from http://egg2 . wustl . edu/roadmap/web_portal/processed_data . html ( Herlofsen et al . , 2013; Kundaje et al . , 2015 ) .",
"The consolidated epigenome BroadPeak file was used .",
"Mouse ENCODE data: DNA-seq data were downloaded from the mouse ENCODE project ( Yue et al . , 2014 ) .",
"All samples were from Mus musculus C57BL/6 and included brain male embryo ( 14 . 5 days ) ( ENCSR000COE ) , liver male adult ( 8 weeks ) ( ENCSR000CNI ) , and lung male adult ( 8 weeks ) ( ENCSR000CNM ) .",
"Processed NarrowPeak bed files were downloaded for each tissue and then lifted over to hg19 as described above .",
"All intersection analyses of ATAC-seq peaks with GWAS loci , genes , or ChIP-seq peaks were performed using BEDTools v2 . 18 ( Quinlan and Hall , 2010 ) and custom R scripts ( R version 3 . 1 . 1 ) ( see Source code 1 ) .",
"For overlaps of genes with GWAS loci , an overlap was called for a given height GWAS locus if the gene’s transcriptional boundaries were within 100 kb of any SNP in the locus ( defined as r2 >0 . 5 to lead SNP ) .",
"For overlap of ChIP-seq datasets , any overlap ( >=1 bp ) was considered to represent the same feature .",
"To evaluate the significance of overlap , a permutation-based procedure was implemented in the R package regioneR ( Gel et al . , 2016 ) .",
"We used the ‘circularRandomizeRegions’ function to generate random permutations .",
"In this function , each chromosome is ‘circularized’ , and the peaks bed file is shifted at random along the circularized chromosome and evaluated for overlap .",
"We used 1000 permutations to calculate the empiric p value .",
"GoShifter v0 . 2 was used to evaluate the statistical enrichment of the overlap between height GWAS loci and ATAC-seq peaks ( or other epigenetic peaks ) ( Trynka et al . , 2015 ) .",
"The European ( EUR ) subset of 1000 Genomes Phase 3 ( Auton et al . , 2015 ) was used to identify proxy SNPs and calculate LD .",
"An LD cutoff of r2 >0 . 5 to the index SNP was used .",
"A window size of 100 kb was used to find LD SNPs ( ‘window’ option ) .",
"All other parameters were set to the software default .",
"100 , 000 permutations were run .",
"GoShifter analyses for additional traits/diseases were performed in the same fashion .",
"SNPSNAP ( Pers et al . , 2015 ) was used to generate matched loci for the height GWAS loci .",
"The European ( EUR ) subset of 1000 Genomes Phase 3 ( Auton et al . , 2015 ) was used to identify proxy SNPs and calculate LD .",
"Loci were defined as variants with r2 > 0 . 5 to the index SNP .",
"Loci were matched using default criteria: MAF ± 5% , gene density ± 50% , distance to nearest gene ±50% and LD buddies ± 50% .",
"SNPSNAP was able to generate matched gene sets for 676 out of the 697 loci from Wood et al . ( 2014 ) .",
"1000 matched sets of random GWAS loci were generated .",
"A list of 427 differentially expressed mouse genes was downloaded from Lui et al . ( 2012 ) .",
"These 427 differentially expressed genes were determined in the Lui et al . ( 2012 ) paper based on meeting two of three criteria: ( 1 ) spatial regulation in the growth plate , ( 2 ) temporal regulation during growth plate formation , and/or ( 3 ) specific expression in the growth plate ( as compared to other tissues such as the lung , kidney , heart ) .",
"Orthologous human genes and their respective positions were identified using Ensembl Biomart ( Kinsella et al . , 2011 ) .",
"The ‘Ensembl Genes 86’ database was used .",
"Using the set of 427 differentially expressed growth plate genes ( see above ) , we identified 157 genes that were also genes for cis-eQTLs in whole blood ( FDR < 0 . 05 ) as identified by Westra et al . ( 2013 ) .",
"For each of these 157 eQTL genes , the corresponding eQTL variant with the strongest p value was used .",
"Using this set of eQTL variants , GoShifter was run as described above to test for enrichment at ATAC-seq peaks .",
"The Genotype-Tissue Expression ( GTEx ) Project ( Lonsdale J , GTEx Consortium , 2013 ) was supported by the Common Fund of the Office of the Director of the National Institutes of Health , and by NCI , NHGRI , NHLBI , NIDA , NIMH , and NINDS .",
"The data used for the analyses described in this manuscript were obtained from the GTEx Portal on 10/27/2017 .",
"ATAC-seq peak centers from proximal/distal tissues were aggregated and padded outwards to a fixed length of 200 bp ( based on the distribution of ATAC-seq peak sizes ) .",
"HOMER ( Jerković et al . , 2017 ) de novo motif analysis was performed for the sequence set utilizing a 10x random shuffling as a background set .",
"De novo motifs were compared to a vertebrate motif library provided by HOMER , which includes the JASPAR database ( Visscher et al . , 2010 ) , with matches scored using Pearson’s correlation coefficient of vectorized motif matrices , with neutral frequencies ( 0 . 25 ) substituted for non-overlapping positions .",
"Lifted over human ATAC-seq peaks were then intersected with height GWAS variants from Wood et al . ( 2014 ) .",
"Variants were characterized for selected motifs which appeared in both the mm10 and hg19 de novo motifs using motifbreakR ( Coetzee et al . , 2015 ) .",
"Motif matches were scored using log-odds scoring ( with 0 . 25 A/C/G/T background frequencies ) for both reference and mutated sequences .",
"Effects on position-weighted matrix ( PWM ) scoring were calculated as differences between the log-odds score of reference and mutant motif hits , with negative differences representing a disrupted motif .",
"To assess enrichment for CTCF-disrupting variants in the ATAC-intersected variant set , 500 randomly sampled subsets ( n = 928 ) of the height GWAS variants ( excluding those intersecting ATAC-seq peaks ) were generated and counted for CTCF-disrupting variants .",
"Counts were standardized and statistical significance was assessed using a continuous distribution function of the standard normal distribution .",
"To assess enrichment of predicted transcription factor binding near variants intersecting ATAC-seq peaks , a set of hg19 DNA sequences was first constructed using 30 bp windows centered on each variant , generating an ATAC-intersected subset and whole GWAS superset .",
"A utility from HOMER ( findmotifs . pl ) was then used to scan this sequence set using its provided vertebrate motif library to search for motif hits .",
"Motifs from JASPAR 2016 were filtered ( 386 motifs ) and counted within each of the two sets .",
"Hypergeometric tests were performed for all motifs using Benjamini-Hochberg FDR correction ( Benjamini and Hochberg , 1995 ) .",
"CRISPR-Cas9 PX458 vector was obtained from Addgene ( Cambridge , Massachusetts ) .",
"pGL4 . 23 luciferase and pGL4 . 74 renilla vectors were acquired from Promega ( Madison , Wisconsin ) .",
"Plasmids encoding HOXD13-FLAG and HOXD13-HA were generously gifted from Dr . Vincenzo Zappavigna ( University of Modena and Reggio Emilia ) ( Caronia et al . , 2003 ) .",
"Human embryonic kidney ( HEK-293FT ) cells were acquired from Dr . Pardis Sabeti ( Harvard University and Broad Institute ) .",
"T/C-28a2 human chondrocyte cells were acquired from Dr . Li Zeng ( Tufts University ) courtesy of Dr . Mary Goldring ( The Hospital for Special Surgery ) .",
"Both cell lines were cultured at 5% CO2 at 37°C in Dulbecco's Modified Eagle's Medium ( DMEM ) , 10% fetal bovine serum ( FBS ) , and 1% penicillin-streptomycin ( P/S ) .",
"Media was replaced every 2–3 days and the cells were sub-cultured every 5 days .",
"All guide RNAs surrounding the human CHSY1 regulatory element or rs9920291 were designed using MIT CRISPR Tools ( http://crispr . mit . edu ) , synthesized by Integrated DNA Technologies , Inc ( Coralville , Iowa ) , and cloned into the PX458 vector following published protocols ( Ran et al . , 2013 ) .",
"The sequence of all sgRNAs are listed in Supplementary file 10 and their locations with respect to the targeted CHSY1 regulatory element and rs9920291 are found in Figure 6—figure supplement 1 .",
"Guide RNAs were initially tested for ability to induce efficient deletions of the human element in cultured HEK-293FT cells .",
"After two days of culture at 37°C , transfected HEK-293FT cells were examined under a GFP-microscope to verify successful transfection and GFP expression .",
"DNA was then extracted using E . Z . N . A Tissue DNA Kit , and the CHSY1 regulatory element region was amplified using PCR and primers surrounding the guide RNA design sites ( Primers: ‘rs9920291 Forward’ and ‘rs9920291 Reverse’ , Supplementary file 10 ) .",
"Amplification products were isolated from 1% agarose gels ( E . Z . N . A Gel Extraction Kit ) and Sanger sequenced to verify deletions of various sizes ( see below ) .",
"After confirmation that sgRNAs worked in HEK-293FT cells , we performed in vitro deletion in the T/C-28a2 chondrocyte cell line ( Kokenyesi et al . , 2000; Finger et al . , 2003 ) .",
"The same sgRNAs were used as above .",
"T/C-28a2 cells were maintained in DMEM ( Gibco , Gaithersburg , Maryland ) supplied with 10% FBS ( Gibco ) and 1% Pen/Strep ( 0 . 025% ) , and seeded in a six-well plate 1 day prior to transfection .",
"After culturing at 37°C , we scanned cells under a GFP-microscope to verify successful transfection efficiency ( i . e . , >70% of cells ) and GFP expression ( N = 3 ) .",
"DNA was then extracted using E . Z . N . A Tissue DNA Kit , and the CHSY1 regulatory element region was amplified using PCR primers surrounding the guide RNA design sites ( MiSeq Primers Forward and MiSeq Primers Reverse , Supplementary file 10 ) .",
"Amplification products were isolated from 1% agarose gels ( E . Z . N . A Gel Extraction Kit ) .",
"Mi-Seq sequencing at the MGH CCIB DNA Core was used to verify successful targeting of the larger CHSY1 regulatory region ( hg19: chr15:101 , 749 , 730-101 , 749 , 865 ) with a modification efficiency of approximately 10% and smaller CHSY1 regulatory region around rs9920291 ( hg19: chr15:101 , 749 , 797–101 , 749 , 813 ) with a modification efficiency of approximately 8 . 5% .",
"RNA was also extracted from control and CRISPR-Cas9 targeted T/C-28a2 cells .",
"RNA was DNase-treated and converted to cDNA for qPCR ( see below ) .",
"Total RNA was extracted from HEK-293FT and T/C-28a2 cells and prepared using the Trizol Reagent ( Thermo Fisher Scientific , Springfield Township , New Jersey ) and Direct-zolTM RNA Miniprep kit ( ZYMO ) .",
"Three micrograms of total RNA were used to synthesize first-strand cDNA using SuperScript III First-Strand Synthesis System ( Thermo Fisher Scientific ) .",
"qRT-PCR analysis was then performed with specific primers and Applied Biosystems Power SYBR master mix ( Thermo Fisher Scientific ) with GAPDH as a reference gene .",
"Primers used for qRT-PCR are listed in Supplementary file 10 .",
"Prior to in vitro transfection of CHSY1 regulatory sequences into HEK-293FT and T/C-28a2 cells ( see below ) , primers containing KpnI/HindIII linker sequences were used to amplify the putative regulatory element surrounding rs9920291 .",
"( see above and Supplementary file 10 ) .",
"The PCR protocol was initiated at 98°C for 30 s , followed by 34 cycles at 98°C for 20 s , 60°C for 20 s , and 72°C for 30 s , and then followed by a 72°C for 5 min final extension .",
"Coriell ( Camden , New Jersey ) sample NA12286 was used to amplify the region containing the reference allele of rs9920291 and NA19119 was used to amplify the region containing the variant allele of rs9920291 .",
"Each amplicon was then ligated into a KpnI/HindIII containing pGL4 . 23 firefly luciferase vector using NEBuffer 2 . 1 ( NEB B7202S ) for digestion and T4 DNA Ligase ( NEB M0202S ) for ligation according to manufacturer's protocols ( New England Biolabs , Beverly , Massachusetts ) .",
"Ligates for constructs containing inserted or non-inserted ( ‘empty’ ) sequence were next transformed into DH10B cells , and streaked on ampicillin plates .",
"Single colonies were then picked , PCR screened , sequenced , and purified using E . Z . N . A . Endo-Free Plasmid DNA Maxi Kits ( Omega D6926-03 ) .",
"The PCR protocol used for screening is listed above .",
"Sanger sequencing was used to confirm the orientation as well as sequence identity of each insert as well as the entire pGL4 . 23 firefly vector .",
"pGL4 . 74 renilla vector was similarly transformed , amplified , purified and sequence verified .",
"Prior to HOXD13 over-expression experiments , HOXD13 constructs were transformed into DH10B cells , streaked on ampicillin plates , individual colonies were grown and purified via Endo-Free Plasmid DNA Maxi Kits ( Omega D6926-03 ) .",
"Purified plasmids underwent diagnostic digest and sequence verification .",
"HEK-293FT cells and T/C-28a2 chondrocytes were passaged using standard conditions ( see above ) prior to reporter gene transfection and HOXD13 over-expression experiments .",
"Prior to transfection , cells were first cultured in DMEM with 10% FBS for 24 hr in 96-well dishes at a seeding density of 3 × 104 cells/well .",
"The volume of media per well was then brought to 100 µl .",
"For luciferase reporter experiments , cells were then transiently transfected with 100 ng firefly luciferase reporter vector or empty pGL4 . 23 luciferase vector , along with 5ng pGL4 . 74 renilla vector .",
"Transfections were performed using Lipofectamine 2000 ( Invitrogen 11668–019 ) at a Lipofectamine:DNA ratio of 4:1 according to manufacturer instructions .",
"Luciferase activity was measured 48 hr after transfection using the 96-welll Dual-Glo Luciferase Assay System ( Promega E2940 ) following manufacturer protocols on a SpectraMax L Microplate Reader ( Molecular Devices; Cat# SpectraMax L Config ) with a 1 min dark adapt , 5 s integration , and max range settings .",
"For cell culture experiments , to compare expression between the reference and alternate allele CHSY1 constructs ( Figure 6b ) , we performed at least four independent transfection experiments at each concentration containing eight technical replicates ( i . e . individual wells of a 96-well plate ) per construct .",
"We first normalized each firefly luciferase value per well by its corresponding renilla luciferase value per well , and then compared the mean expression of the reference allele construct ( normalized by empty vector ) to that of the mean expression of the alternate allele construct ( normalized by empty vector ) using a two-sample directional Student's t-test .",
"For HOXD13 overexpression experiments , HEK-293FT and T/C-28a2 cells were transfected as above but with 0 , 2 and 4 µg of HOXD13 expression or control constructs ( N = 3 biological replicates per condition ) , and after 24 , 48 , and 72 hr RNA was collected for qRT-PCR experiments ( see above ) .",
"Both HEK-293FT cells and T/C-28a2 chondrocytes were screened using Sanger sequencing to identify genotype rs9920291 .",
"Sanger sequencing results confirmed preliminary findings using the HEK-293 browser ( http://hek293genome . org/v2/ ) that HEK-293FT cells were heterozygous at rs9920291 , unlike T/C-28a2 cells which were homozygous for the reference C allele .",
"Thus , only HEK-293FT cells could be used for allele-specific expression .",
"Next , using the HEK-293 browser with follow-up verification using Sanger sequencing , we identified two heterozygous transcript variants ( exon 3 coding variant [rs28364839] and 3’UTR variant [rs11433] ) .",
"For allelic skew analyses on untreated HEK-293FT samples , five independent biological replicates were grown and harvested .",
"RNA was isolated separately from each sample using the Trizol Reagent ( 15596–026 , Ambion by Life Technologies , Norwalk , Connecticut ) and the RNA Clean and ConcentratorTM-5 Kit ( supplied with DNase I , Zymo ) .",
"Samples were then run on a bioanalyzer to achieve RNA Integrity Numbers greater than 8 .",
"These RNA samples were then reverse transcribed using a SuperScript IV First Strand cDNA Synthesis Reaction kit ( 18090010 , Life Technologies ) following manufacturer’s recommendations .",
"cDNA samples were then sent to EpigenDx for allelic skew analysis assay design and execution .",
"SNPs in the coding regions of CHSY1 ( rs11433 and rs28364839 ) , were validated by EpigenDx ( Hopkinton , Massachusetts ) .",
"Pyrosequencing for SNP genotyping ( PSQ H96A , Qiagen Pyrosequencing ) is a real-time sequencing-based DNA analysis that quantitatively determines the genotypes of single or multiple mutations in a single reaction .",
"Briefly , 1 ng of sample cDNA was used for PCR amplification .",
"PCR was performed using 10X PCR buffer ( Qiagen Inc . , Maryland ) at 3 . 0 mM MgCl2 , 200M of each dNTP , 0 . 2 µM each of the forward and reverse primers ( available through EpigenDx ) , and 0 . 75 U of HotStar DNA polymerase ( Qiagen Inc . ) , per 30 µl reaction .",
"PCR cycling conditions were: 94°C 15 min; 45 cycles of 94°C 30 s; 60°C 30 s; 72°C 30 s; 72°C 5 min .",
"One of the PCR primer pairs was biotinylated to convert the PCR product to single-stranded DNA sequencing templates using streptavidin beads and the PyroMark Q96 Vacuum Workstation .",
"Ten microliters of the PCR products were bound to streptavidin beads and the single strand containing the biotinylated primer was isolated and combined with a specific sequencing primer ( available through EpigenDx ) .",
"The primed single stranded DNA was sequenced using the Pyrosequencing PSQ96 HS System ( Qiagen Pyrosequencing ) following the manufacturer’s instructions ( Qiagen Pyrosequencing ) .",
"The genotypes of each sample were analyzed using Q96 software AQ module ( Qiagen Pyrosequencing ) .",
"Pyrosequencing results for each CHSY1 exonic or 3’UTR SNP were used to calculate the allelic ratios in the heterozygous state .",
"For regulatory element transfection experiments , CRISPR-Cas9 deletion experiments , and HOXD13 over-expression experiments , the means ± SEM of multiple independent measurements were calculated .",
"The unpaired two-tailed Student’s t test was used to determine the significance of differences between means .",
"P values smaller than 0 . 05 ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 ) were considered to be statistical significant .",
"To find upstream transcription factors ( TF ) predicted to bind at rs9920291 near CHYS1 we used UniProbe ( Hume et al . , 2015 ) .",
"UniProbe ( http://the_brain . bwh . harvard . edu/uniprobe/about . php ) is built on experimental measurements of binding affinities between large numbers of expressed TFs and all possible 8-mer target oligonucleotides ( Hume et al . , 2015 ) .",
"For two 15 base-pair sequences , each centered at rs9920291 but different only at the C/T variant ( Reference [REF] TGCTAAACAAAAATA and Alternate [ALT] TGCTAAATAAAAATA ) , we imported each sequence separately into the ‘Search for TF Binding Sites’ window on the UniProbe browser ( http://the_brain . bwh . harvard . edu/uniprobe/ ) with the following conditions: Score Threshold: 0 . 4; Species: Homo sapiens .",
"We acquired 18 TF predictions for REF and 61 TF predictions for ALT .",
"We next imported the results into Supplementary file 7 ( sheet 1: REF TGCTAAACAAAAATA and sheet 2: ALT TGCTAAATAAAAATA ) and performed an intersection ( sheet 3: REF ( red ) vs . ALT ( blue ) ) to identify those TF-binding preferences that were reduced/gained between the two sequences .",
"For a TF-binding site to be considered reduced or gained , the site must exhibit an enrichment score change of greater than at least 0 . 10 , which corresponded to significant changes in z-score ( e . g . , Figure 6a ) .",
"To identify the change in HOXD13 binding affinity , we re-ran UniProbe on the REF sequence but at a lower enrichment score of E >= 0 . 2 .",
"This yielded 341 TF predictions ( sheet 4 ) , which were then compared to the 61 TF predictions generated at E >= 0 . 4 ( sheet 5 ) .",
"Finally , to generate the z-score , we downloaded HOXD13 experiment information from UniProbe ( http://the_brain . bwh . harvard . edu/uniprobe/browse . php ) and identified the corresponding z-score for each 8-mer .",
"Lists of potential upstream regulators exhibiting marked reduction/gain in binding affinity were then screened using expression and phenotypic data to identify those TFs also expressed or functionally required in growth plates .",
"To carry out this analysis , we used data in VisiGene ( http://genome . ucsc . edu/cgi-bin/hgVisigene ) , Eurexpress ( http://www . eurexpress . org/ee/ ) , Genepaint ( http://www . genepaint . org/Frameset . html ) , and the Mouse Genome Informatics expression and phenotypic databases ( http://www . informatics . jax . org ) .",
"HOXD13 met the above criteria .",
"HOXD13 had enrichment scores below the recommended UniProbe threshold of 0 . 4 for the REF sequence but much higher ( above 0 . 4 ) for the ALT sequence , and this corresponded to a significant change in z-score ( Figure 6a ) ."
]
] | [
"GWAS have identified hundreds of height-associated loci .",
"However , determining causal mechanisms is challenging , especially since height-relevant tissues ( e . g . growth plates ) are difficult to study .",
"To uncover mechanisms by which height GWAS variants function , we performed epigenetic profiling of murine femoral growth plates .",
"The profiled open chromatin regions recapitulate known chondrocyte and skeletal biology , are enriched at height GWAS loci , particularly near differentially expressed growth plate genes , and enriched for binding motifs of transcription factors with roles in chondrocyte biology .",
"At specific loci , our analyses identified compelling mechanisms for GWAS variants .",
"For example , at CHSY1 , we identified a candidate causal variant ( rs9920291 ) overlapping an open chromatin region .",
"Reporter assays demonstrated that rs9920291 shows allelic regulatory activity , and CRISPR/Cas9 targeting of human chondrocytes demonstrates that the region regulates CHSY1 expression .",
"Thus , integrating biologically relevant epigenetic information ( here , from growth plates ) with genetic association results can identify biological mechanisms important for human growth ."
] | [
"Humans vary considerably in height , a trait that is partly inherited from each individual's parents .",
"Studies have identified hundreds of small changes in DNA that contribute to differences in human height .",
"These small changes often swap out just one of the four letters that make up the DNA code .",
"Some changes occur within genes , yet most occur in stretches of DNA that do not contain genes .",
"These stretches of DNA likely control whether genes important for the growth of the skeleton are switched on or off .",
"Cartilage cells , also known as chondrocytes , are important for height .",
"These cells are found near the end of bones in the growth plates , where the bones grow during childhood and adolescence .",
"The on and off switches for growth genes in chondrocytes are unknown .",
"Identifying these genetic switches could help scientists understand how hundreds of small changes in DNA help determine how tall a person will be .",
"Now , Guo , Liu , Willen et al . identify thousands of switches that turn on and off genes in chondrocytes .",
"In their experiments , chondrocytes were removed from mouse bones and a type of genetic sequencing called ATAC-seq was used to identify the stretches of DNA that act as on/off switches for genes in these cells .",
"Further analysis revealed that many of these same on/off switches occur in human chondrocytes too .",
"Importantly , the experiments showed that many of the small changes in DNA that contribute to differences in human height are also found within these DNA switches .",
"Guo , Liu , Willen et al . next tested how these switches and height-linked genes interact , and found , for example , that one switch acts to shut off the gene for a protein called chondroitin sulfate synthase 1 .",
"People with mutations in this gene can have unusually short stature .",
"However , people with DNA changes in its nearby switch can be taller or shorter than average based on how the DNA influences the switch .",
"More studies might help scientists understand the evolutionary significance of these DNA changes .",
"They will also help determine if the genetic switches in chondrocytes contribute to diseases that affect the bones and joints , like bone cancer or arthritis ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"neuroscience"
] | Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses | elife-24845-v3 | [
[
"At chemical synapses , neurotransmitters such as glutamate are contained in synaptic vesicles ( SVs ) and released by exocytosis .",
"After exocytosis , SVs are retrieved by endocytosis , refilled with neurotransmitter , and transported to release sites to be reused ( Heuser and Reese , 1973 ) .",
"Although cellular and molecular mechanisms of exocytosis and endocytosis have been extensively studied ( Sudhof , 2004; Jahn and Fasshauer , 2012 ) , movements of SVs between endocytosis and exocytosis are least understood .",
"Previous studies have reported a wide range of SV mobility , several orders of magnitude different in their diffusion coefficient ( D ) , between different types or even in the same type of presynaptic terminal ( Holt et al . , 2004; Rea et al . , 2004; Jordan et al . , 2005; Westphal et al . , 2008; Kamin et al . , 2010; Lee et al . , 2012 ) .",
"Within nerve terminals , at both hippocampal and neuromuscular synapses , SVs reportedly display low mobility ( Lee et al . , 2012; Lemke and Klingauf , 2005; Gaffield and Betz , 2007 ) , whereas SVs traveling across distant hippocampal presynaptic boutons show higher mobility ( Lee et al . , 2012; Darcy et al . , 2006; Fernandez-Alfonso and Ryan , 2008; Staras et al . , 2010 ) .",
"It is also controversial whether stimulation can affect SV mobility ( Gaffield et al . , 2006; Peng et al . , 2012 ) or not ( Betz and Bewick , 1992; Westphal et al . , 2008; Kamin et al . , 2010 ) .",
"In spite of the wealth of molecular and cellular knowledge on neurotransmission , much less is known about the mechanisms regulating SV trafficking and supply in nerve terminals , and only few factors , modulating SV movements , have been identified thus far .",
"Synapsin-1 has long been identified as a tether anchoring and releasing SVs in a phosphorylation-dependent manner ( Llinás et al . , 1985 ) and is thought to regulate SV mobility in various nerve terminals ( Jordan et al . , 2005; Gaffield et al . , 2006; Rothman et al . , 2016 ) .",
"Actin network has also been implicated in the regulation of fast trafficking of SVs between synaptic sites in central synapses ( Darcy et al . , 2006 ) .",
"The roles of another cytosketal element , microtubules ( MTs ) and their associated molecular motors are well established for axonal transport ( Hirokawa et al . , 2009 , 2010 ) , but their contribution in the regulation of SV dynamics within nerve terminals remains to be elucidated .",
"Mechanical factors such as membrane tensions are proposed to promote accumulation of SVs in nerve terminals ( Siechen et al . , 2009 ) or to increase their mobility ( Ahmed et al . , 2012 ) .",
"More recently , experimentally constrained models suggested that physical determinants such as hydrodynamic interaction and vesicle collision influence SV movements in presynaptic terminals ( Rothman et al . , 2016 ) .",
"However , biological factors and mechanisms underlying the diversity of SV mobility in nerve terminals remain poorly understood .",
"In mammalian central synapses , imaging studies of SV mobility have been restricted to small hippocampal synapses .",
"Here , by taking advantage of our newly developed giant glutamatergic synapses formed in primary culture between mouse auditory brainstem neurons ( Dimitrov et al . , 2016 ) , we visualized SVs using real-time confocal microscopy , and analyzed their movements by automatically tracking large populations of fluorescently labelled vesicles within presynaptic terminals .",
"We then performed spatio-temporal analyses of more than 35 , 000 vesicle trajectories to quantify their intrinsic dynamic properties ( i . e . maximum speed and track length ) , their modalities of displacement ( i . e . diffusive or active motions ) , and their overall mobility ( i . e . diffusion coefficient ) .",
"We compared these parameters between giant calyceal terminals and small hippocampal presynaptic boutons , between morphologically mature and immature calyceal terminals , and between calyceal terminals over-expressing two distinct vesicular proteins , VGLUT1 or VGLUT2 .",
"We also tested the effects of pharmacological disruption of the microtubule ( MT ) network , as well as of various stimulation protocols on SV movements within calyceal terminals .",
"Altogether , our results revealed several factors influencing SV mobility and trafficking in the CNS: the type of synapse ( giant calyceal or small conventional terminals ) , the spatial localization of vesicles within a terminal ( intra-swelling or inter-swelling ) , and the molecular composition of vesicles ( vesicular glutamate transporter subtypes ) .",
"Our results also suggest that MTs play essential roles in inter-synaptic movements of SVs and that synaptic stimulation does not induce any appreciable increase in SV mobility ."
],
[
"To visualize SVs by fluorescence confocal microscopy , primary cultures over-expressing GFP in presynaptic neurons were incubated in the presence of Q655-labeled synaptotagmin-2 antibody ( Q655-Syt2 ) from 1 hr to overnight to allow spontaneous uptake of the fluorescent marker into SVs .",
"After image acquisition , automatic spot detection of individual SVs , on 2D confocal section , was performed using IMARIS software ( see Materials and methods ) .",
"Q655-Syt2 fluorescent spots distributed throughout the entire calyceal terminal over-expressing GFP ( Figure 1A , Video 1 ) .",
"The average number of labeled vesicles per terminal significantly increased with length of exposure to Q655-Syt2 , reaching ~1500 SVs per terminal after 16 hr ( Figure 1B ) .",
"The use of synaptotagmin-2 antibody significantly increased the efficiency of SV labeling , in comparison to quantum dots only ( Figure 1—figure supplement 1A , B ) .",
"The fluorescence intensity distribution in 150 nm confocal spots for Q655-Syt2-labeled SVs was similar to the one from 40 nm FITC-beads ( Figure 1—figure supplement 1C ) , suggesting single vesicle detection in our tracking experiments .",
"Compared with Q655 , the broader distribution of C5E fluorescence intensity results from the pH sensitivity of the dye emission . 10 . 7554/eLife . 24845 . 003Figure 1 . Autoregressive motion analysis reveals high and broad range of SV mobilites in cultured giant terminals .",
"( A ) Confocal z-stack imaging of a giant presynaptic terminal expressing cytosolic GFP- ( Green ) and Q655-Syt2 ( Red ) -labeled vesicles; corresponding volume rendering of GFP terminal and SV detection ( see Video 1 ) .",
"( B ) The number of labeled SVs detected in whole presynaptic terminal .",
"( C ) Live confocal imaging and SV tracking with the autoregressive motion ( Red ) or Brownian motion ( Blue ) algorithm ( see Video 2 ) .",
"( D ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces , color-coded as in ( C ) .",
"( E ) Comparison of SV displacements and diffusion coefficients for Q655-Syt2- ( n = 12 terminals ) , Q585-Syt2- ( n = 12 ) or C5E-Syt2- ( n = 12 ) labeled vesicles , Q655-Syt2-labeled vesicles after chemical fixation ( n = 12 ) and 40 nm beads ( n = 12 ROI ) .",
"Two-tailed unpaired t-test ( *p<0 . 05; ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 00310 . 7554/eLife . 24845 . 004Figure 1—source data 1 . Data and statistics for Figure 1E . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 00410 . 7554/eLife . 24845 . 005Figure 1—source data 2 . Data and statistics for Figure 1—figure supplement 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 00510 . 7554/eLife . 24845 . 006Figure 1—source data 3 . Data and statistics for Figure 1—figure supplement 2D . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 00610 . 7554/eLife . 24845 . 007Figure 1—source data 4 . Data and statistics for Figure 1—figure supplement 3D . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 00710 . 7554/eLife . 24845 . 008Figure 1—figure supplement 1 . Q655-Syt2 labels SV more efficiently than Q655 alone .",
"( A ) Volume rendering of GFP terminal and SV detection in calyceal terminals loaded with Q655 only or Q655-Syt2 after 16 hr . ( B ) Comparison of the number of SVs detected in calyceal terminals loaded with Q655 alone ( Black , n = 5 terminals ) , Q655-Syt2 ( Red , n = 5 ) or C5E-Syt2 ( Magenta , n = 5 ) after overnight incubation .",
"Two-tailed unpaired t-test ( *p<0 . 05 ) .",
"( C ) Fluorescence intensity distribution in 150 nm confocal spots for 40 nm FITC-beads ( Green ) , SVs loaded with Q655-Syt2 ( Red ) or with C5E-Syt2 ( Magenta ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 00810 . 7554/eLife . 24845 . 009Figure 1—figure supplement 2 . Labeling and tracking of SVs with C5E-Syt2 . ( A ) Confocal z-stack imaging of a giant presynaptic terminal expressing cytosolic GFP ( Green ) and C5E-Syt2 ( Red ) -labeled vesicles .",
"( B ) Visualization and quantification of exocytosis induced by bath application of 65 mM KCl .",
"Upper left panel: confocal image before KCl application , lower left panel: confocal image after KCl application .",
"Right panel: Measurement of C5E fluorescence intensity from ROI ( white box on left panels ) of five different terminals .",
"Black trace: average of 5 traces ( color coded ) , Red trace: Boltzman fitting .",
"( C ) Tracking of SVs in interconnected swellings before ( upper panels ) and after application of 65 mM KCl ( lower panels ) .",
"( D ) Fluorescence recovery after photobleaching in swellings ( upper panels ) or finger-like structures ( lower panels ) .",
"( E ) FRAP analysis showing fluorescence intensity recovery profile ( ROI ~1 . 5 µm , white boxes ) and estimation of the mobile and immobile fraction of SVs in swellings ( Green ) and finger-like structures ( Red ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 00910 . 7554/eLife . 24845 . 010Figure 1—figure supplement 3 . Newly retrieved SVs have lower mobilities and restricted distributions in giant terminals .",
"( A ) Confocal images showing the co-localization of SVs labeled overnight with Q655-Syt2 ( Red ) and for 1 hr or 3 hr with Q585-Syt2 ( Green ) , co-localization ( white ) .",
"( B ) Comparison of the Pearson co-localization coefficient of Q655- and Q585-labeled vesicles after 1 , 2 , or 3 hr post-endocytosis .",
"( C ) Tracking of Q655- ( Red ) and Q585- ( Green ) labeled vesicles after 1 or 3 hr from terminals shown in ( A ) .",
"( D ) Diffusion coefficient of Q655- and Q585-labeled vesicles after 1 ( n = 3 terminals ) , 2 ( n = 3 ) , or 3 hr ( n = 3 ) post-endocytosis .",
"Two-tailed unpaired t-test ( *p<0 . 05; ns , not significant ) .",
"( E ) Dynamic properties and displacement analysis of Q655- ( Red ) and Q585-labeled vesicles after 1 hr ( Blue ) or 3 hr ( Green ) post-endocytosis . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01010 . 7554/eLife . 24845 . 011Video 1 . Volume rendering of GFP-expressing giant calyceal terminal and spot detection of Q655-Syt2-labeled SVs ( color-coded according to their z position ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 011 Two different tracking algorithms were used to analyze their mobility within the terminal: a Brownian motion algorithm to detect non-directional diffusive movements and an autoregressive motion algorithm to detect both diffusive and active directional movements ( Video 2 ) .",
"Analysis of vesicle trajectories , according to their maximum speeds and trajectory lengths , showed that Brownian motion algorithm often failed to trace trajectories with long directional runs , while autoregressive algorithm could trace both directionless random fluctuation and long directional runs ( Figure 1C and D ) with parameters determined empirically .",
"The diffusion coefficient ( D ) , calculated from the mean square displacement ( MSD ) curves of trajectories identified with the autoregressive algorithm ( D = 0 . 065 ± 0 . 004 µm2/s ) , was seven times greater than that calculated from the MSD determined by the Brownian algorithm ( D = 0 . 009 ± 0 . 001 µm2/s ) .",
"This suggests that vesicle mobility could be underestimated by analysis based solely on the Brownian motion algorithm ( Figure 1E ) .",
"Thus , we used autoregressive algorithm for the tracking of SV trajectories in this study . 10 . 7554/eLife . 24845 . 012Video 2 . Tracking of Q655-Syt2-labeled SVs using autoregressive ( Red ) or Brownian ( Blue ) motion algorithm . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 012 We next determined background mobility in our experimental conditions .",
"Aldehyde fixation of labeled giant terminals lowered SV mobility ( D = 0 . 008 ± 0 . 001 µm2/s ) to the level of mobility observed for 40 nm fluorescent polystyrene beads adsorbed onto glass coverslips ( D = 0 . 008 ± 0 . 001 µm2/s ) , indicating that the signal-to-noise ratio in our tracking experiments was ~8 ( Figure 1E ) .",
"To ensure that SVs loaded with Syt-2 antibody can undergo exocytosis , Syt-2 was labeled with the pH-sensitive fluorochrome CypHer5E ( C5E ) instead of quantum dots ( Q655 ) .",
"C5E shows high-fluorescence intensity at low pH = 5 . 5 and low intensity at neutral pH , allowing us to monitor SV exo/endocytic cycles and to track endocytosed SVs .",
"Labeling of SVs with C5E-Syt2 ( Figure 1—figure supplement 2A ) was identical to labeling of SVs with Q655-Syt2 ( Figure 1A ) .",
"Stimulation with 65 mM KCl induced an 80% decrease in the fluorescence intensity of C5E-Syt2-loaded vesicles , showing that a large proportion of labeled SVs underwent exocytosis ( Figure 1—figure supplement 2B ) .",
"Furthermore , diffusion coefficients of SVs labeled with Qdots ( Q655 and Q585 ) or with C5E were similar ( Figure 1E ) , indicating that labeling methods did not affect their dynamic or functional properties .",
"Using C5E-Syt2-labeled vesicles , we performed fluorescence recovery after photo-bleaching ( FRAP ) experiments ( Figure 1—figure supplement 2D , E ) to compare SV mobility with those measured from MSD ( Figure 1E ) .",
"Data pooled from three different terminals showed that in presynaptic swellings , ~40% of SVs were mobile ( T1/2 = 13 . 2 ± 1 . 1 s ) with a calculated diffusion coefficient ( Axelrod et al . , 1976 ) of 0 . 029 ± 0 . 007 µm2/s , whereas in finger-like processes , ~80% of SVs were mobile ( T1/2 = 6 . 3 ± 0 . 7 s ) with a diffusion coefficient of 0 . 061 ± 0 . 007 µm2/s .",
"Although we observed lower D in swellings calculated by FRAP , D in fingers remained comparable to the value estimated by autoregressive MSD curves ( Figure 1E ) .",
"In addition , it has been reported that the mobility of newly retrieved SVs in hippocampal synapses is highest after endocytosis and gradually decreases thereafter ( Kamin et al . , 2010 ) .",
"We examined whether it might also apply to giant synapses .",
"Vesicles were first loaded with Q655-Syt2 by overnight incubation to achieve homogenous labeling of the resting vesicular pool .",
"The next day , the same terminals were exposed to Q585-Syt2 for 1 to 3 hr and Q655- and Q585-Syt2-loaded vesicles were tracked ( Figure 1—figure supplement 3A ) .",
"One hour after the second loading , SVs newly labeled with Q585-Syt2 remained in restricted regions of the terminal , not mixing with previously retrieved Q655-Syt2-labeled vesicles ( Pearson co-localization index , p=0 . 087 ± 0 . 06; Figure 1—figure supplement 3B ) , and showed relatively low mobility ( D = 0 . 045 ± 0 . 001 µm2/s ) .",
"Two to three hours after the second loading , SVs labeled with Q585-Syt2 increased their mobility ( D = 0 . 066 ± 0 . 005 µm2/s after 3 hr; Figure 1—figure supplement 3C , D ) and gradually mixed with the pre-existing pool of SVs labeled with Q655-Syt2 ( p=0 . 266 ± 0 . 035; Figure 1—figure supplement 3A , E ) .",
"We first characterized the basic properties of movements ( maximum speed , trajectory length , modality of displacement , and diffusion coefficient ) of SVs loaded with Q655-Syt2 within giant calyceal terminals .",
"We categorized vesicle trajectories into three groups according to their individual trajectory lengths relative to the average size ( ~2 . 1 µm ) of large presynaptic swellings ( Figure 2—figure supplement 1 ) : short ( S ) trajectories ( length <2 µm , intra-swelling trafficking ) , medium ( M ) trajectories ( 2 µm < length < 4 µm , intermediate trafficking ) and long ( L ) trajectories ( 4 µm < length , inter-swelling trafficking; Figure 2A and Video 3 ) .",
"During SV trafficking , longer trajectories were consistently accompanied by faster maximal speeds ( Figure 2B and Video 4 ) , and movements of SVs were highly heterogeneous between ( Figure 2B ) and within ( Figure 2—figure supplement 2A , B ) individual trajectories .",
"Of all SVs examined in 12 giant terminals , 61% had short trajectories and slow motility , whereas 39% had long ( up to 6 µm ) and fast ( up to 0 . 8 µm/s ) directional movements ( Figure 2C ) .",
"We next analyzed displacement modalities of individual vesicles along their trajectories .",
"Most SVs moving over long trajectories accelerate and decelerate , while SV speed remained more constant for medium and short trajectories ( Figure 2—figure supplement 2A , B ) resulting in a broad variety of displacements ( Figure 2D ) .",
"We categorized displacement modalities of SVs into two groups: one with diffusive motion and the other with active ( facilitated or impeded displacements ) motion ( Figure 2—figure supplement 2C ) , and found that ~20% of labeled SVs move actively in calyceal terminals ( Figure 2D ) . 10 . 7554/eLife . 24845 . 013Figure 2 . Fast and heterogeneous SV movements occur at giant calyceal synapses .",
"( A ) Live confocal imaging of a giant calyceal terminal expressing cytosolic GFP- and Q655-Syt2-labeled vesicles , with SV tracking , color-coded over time , or sorted according to trajectory lengths ( Blue <2 µm , Green 2–4 µm and Red >4 µm , see Video 3 ) .",
"( B ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces ( see Video 4 ) , color-coded as in ( A ) .",
"( C ) Classification and quantification of SV movements in three groups based on their maximum speed and trajectory length ( n = 6175 trajectories ) .",
"( D ) Displacement curves and displacement modalities ( Red: diffusive motion , Blue: active motion ) of identified traces ( n = 6175 trajectories ) .",
"( E ) Diffusion coefficient of SVs at 37o C ( n = 12 terminals ) or 25°C ( n = 4 ) ; or in the presence of 2 . 5 µM OA at 37°C ( n = 9 ) .",
"Two-tailed unpaired t-test ( *p<0 . 05 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01310 . 7554/eLife . 24845 . 014Figure 2—source data 1 . Data and statistics for Figure 2E . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01410 . 7554/eLife . 24845 . 015Figure 2—source data 2 . Data and statistics for Figure 2—figure supplement 2E and H . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01510 . 7554/eLife . 24845 . 016Figure 2—source data 3 . Data and statistics for Figure 2—figure supplement 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01610 . 7554/eLife . 24845 . 017Figure 2—figure supplement 1 . Comparison of calyceal and hippocampal cultures .",
"( A ) Maximum intensity projection image of confocal z-stack showing giant calyceal terminals ( arrowheads ) over-expressing cytosolic GFP after 18 days in culture ( left panel ) and hippocampal neurons over-expressing cytosolic GFP after 15 days in culture ( right panel ) .",
"( B ) Comparison of the size distribution of calyceal swellings ( Red ) and hippocampal boutons ( Blue ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01710 . 7554/eLife . 24845 . 018Figure 2—figure supplement 2 . Mobility and displacement modality of SVs in giant calyceal terminals .",
"( A ) Live confocal imaging of a calyceal terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles , and individual SV tracking sorted according to trajectory lengths ( Blue <2 µm , Green 2–4 µm and Red >4 µm ) .",
"( B ) Speed variation profiles during short ( Blue ) , medium ( Green ) or long ( Red ) trajectories .",
"( C ) Displacement modalities .",
"Representative displacement curves showing different modes of movements .",
"Each representative curve for diffusive motion and active motion ( facilitated and impeded ) was calculated and plotted from an average of 12 different curves extracted from displacement plots similar to Figure 2D .",
"( D ) Confocal imaging of a giant calyceal terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles .",
"White circles and black lines represent swelling and finger areas , respectively .",
"( E ) Comparison of the diffusion coefficient of SVs in swellings or fingers in various conditions ( Control , 30 µM nocodazole , 2 . 5 µM OA , 65 mM KCl , 500 mM sucrose or 1 Hz electrical stimulation , n = 6 terminals for each condition ) .",
"( F ) 3D tracking of SVs labeled with Q655-Syt2 in an individual swelling .",
"Left panel: SV trajectories ( time color-coded ) , right panel: Displacement vectors of SV trajectories .",
"( G ) Schematic diagram showing the proportion of SVs with displacement vectors going toward the synaptic cleft ( Green ) , away from the synaptic cleft ( Red ) , or moving laterally ( Blue ) in an individual calyceal swelling .",
"( H ) Comparison of SV mobilities between 2D and 3D tracking .",
"Trajectory length analysis ( Left panel ) and diffusion coefficient ( Right panel ) in single 2D confocal section ( Blue , n = 3 ) and 3D confocal z-stack ( Red , n = 3 ) .",
"Two-tailed unpaired t-test ( *p<0 . 05; ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01810 . 7554/eLife . 24845 . 019Figure 2—figure supplement 3 . Data acquisition rate does not affect SV tracking .",
"( A ) Syt2-C5E-loaded SV tracking sorted according to trajectory lengths ( Blue <2 µm , Green 2–4 µm and Red >4 µm ) at three different image acquisition speed ( 0 . 5 s , 1 s and 2 s per image ) .",
"Trajectory length analysis in single 2D confocal section 0 . 5 s per image ( Blue , n = 3 ) , 1 s per image ( Black , n = 3 ) and 2 s per image ( Red , n = 3 ) .",
"Two-tailed unpaired t-test for comparison between two groups and two-way ANOVA for multi groups comparison ( ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 01910 . 7554/eLife . 24845 . 020Video 3 . Tracking of Q655-Syt2-labeled SVs color-coded according to SV trajectory lengths ( Blue: short , Green: intermediate and Red: long trajectories ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 02010 . 7554/eLife . 24845 . 021Video 4 . Scatter plot of SV trajectory lengths and maximum speeds during 30 s time-series . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 021 We next examined effects of various factors known to influence SV mobility .",
"At mouse hippocampal synapses ( Kraszewski et al . , 1995; Jordan et al . , 2005 ) and frog neuromuscular junctions ( Betz and Henkel , 1994; Gaffield et al . , 2006 ) , the phosphatase inhibitor , okadaic acid ( OA ) dramatically increases SV mobility .",
"At giant calyceal terminals , OA increased SV mobility by ~43% ( from D = 0 . 065 ± 0 . 004 µm2/s to 0 . 093 ± 0 . 008 µm2/s , Figure 2E ) , a significant , but moderate increase compared with those reported previously .",
"As suggested from FRAP experiments ( Figure 1—figure supplement 3D , E ) , SV mobility was ~1 . 4 times higher in finger-like processes ( D = 0 . 059 ± 0 . 004 µm2/s ) than in swellings ( D = 0 . 044 ± 0 . 003 µm2/s , Figure 2—figure supplement 2D , E ) .",
"Consistently , OA also enhanced SV mobility ~1 . 4 times in both regions ( Figure 2—figure supplement 2E ) .",
"Lowering the temperature from physiological ( 37°C ) to non-physiological ( 25°C ) conditions decreased the diffusion coefficient of SVs by ~30% ( from D = 0 . 065 ± 0 . 004 µm2/s to D = 0 . 046 ± 0 . 003 µm2/s , Figure 2E ) , with a temperature coefficient ( Q10 ) of 1 . 6 , indicating that SV movements were moderately more temperature-dependent than diffusion ( Q10 = 1 . 3 ) .",
"We further analyzed SV movements and distributions in 3D within presynaptic terminals and found that under resting conditions , 25% of labeled vesicles move outward toward the postsynaptic cell , 14% move inward , and 35% move laterally near the synaptic cleft ( Figure 2—figure supplement 2F , G ) .",
"Trajectory proportion and diffusion coefficient were similar between 2D or 3D tracking ( Figure 2—figure supplement 2H ) , suggesting a minimum bias in our tracking method cause by movements of different SV in and out of the focal plan .",
"Four-fold changes in our data acquisition rate also did not significantly affect SV tracking and trajectory proportion ( Figure 2—figure supplement 3 ) , indicating that long SV trajectories are not likely resulting from the detection of different SVs sequentially moving throughout the focal plan .",
"Altogether , these data demonstrate that movements of SVs in calyceal terminals were highly dynamic and heterogeneous under resting conditions .",
"We next applied the same imaging techniques and analytical methods used in giant calyceal terminals to small hippocampal terminals ( Figure 2—figure supplement 1A ) .",
"As observed in giant calyceal terminals , Q655-Syt2-labeled vesicles were distributed throughout hippocampal terminals over-expressing cytosolic GFP ( Figure 3A ) .",
"The distribution of trajectories according to their maximum speed and length from nine hippocampal terminals showed that SVs in small hippocampal boutons had predominately ( 89% ) short trajectories and slow speeds ( Figure 3B and C ) .",
"We next compared modalities of displacements of SVs in small hippocampal boutons ( Figure 3D ) with those in giant calyceal terminals ( Figure 2D ) .",
"In hippocampal boutons , 97% of SVs moved by diffusion while only 3% of SVs moved actively .",
"The diffusion coefficient of SVs in resting hippocampal boutons was 0 . 024 ± 0 . 003 µm2/s ( Figure 3E ) , ~3 times lower than that in giant calyceal terminals ( D = 0 . 065 ± 0 . 004 µm2/s , Figure 2E ) .",
"No differences in D values , calculated using Brownian motion analysis , were observed between hippocampal ( D = 0 . 009 ± 0 . 001 µm2/s , Figure 3E ) and calyceal ( D = 0 . 009 ± 0 . 001 µm2/s , Figure 2E ) terminals . 10 . 7554/eLife . 24845 . 022Figure 3 . Small and homogeneous SV movements occur at small conventional synapses .",
"( A ) Live confocal imaging of a hippocampal bouton expressing cytosolic GFP- and Q655-Syt2-labeled vesicles , with SV tracking color-coded over time , or sorted according to trajectory lengths ( Blue <2 µm , Green 2–4 µm and Red >4 µm ) .",
"( B ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces , color-coded as in ( A ) .",
"( C ) Classification and quantification of SV movements in three groups based on their maximum speed and trajectory length ( n = 2958 trajectories ) .",
"( D ) Displacement curves and displacement modalities ( Red: diffusive motion , Blue: active motion ) of identified traces ( n = 2958 ) .",
"( E ) Diffusion coefficients of SVs in hippocampal terminals calculated from autoregressive ( Red ) or Brownian ( Blue ) analysis ( n = 9 terminals ) .",
"Two-tailed unpaired t-test ( *p<0 . 05 ) .",
"( F ) Comparison of SV mobility in hippocampal boutons ( n = 9 ) or calyceal swellings ( n = 9 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 02210 . 7554/eLife . 24845 . 023Figure 3—source data 1 . Data and statistics for Figure 3E . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 023 To examine whether different SV mobility resulted from size differences of presynaptic terminals , we compared SV diffusion coefficients between calyceal swellings and hippocampal boutons .",
"Although the size of calyceal swellings was about twice that of hippocampal boutons ( Figure 2—figure supplement 1B ) , when SV movements were compared between swellings and boutons having similar areas , mobility of SVs in swellings was consistently ~2-fold higher than that in boutons ( Figure 3F ) .",
"Within calyceal terminals , SV mobility increased with the size of the swellings ( r = 0 . 85 ) , whereas there was no positive correlation ( r = −0 . 05 ) between the size of boutons and SV mobility in hippocampal terminals .",
"These results indicated that between two types of central synapses , with different terminal sizes and morphologies , SV mobility differs substantially; giant synapses show faster and more heterogeneous vesicle movements than small synapses .",
"Thus , the type and the morphology of central synapses appear to significantly impact SV mobility in nerve terminals .",
"As at the developing calyx of Held , giant calyceal terminals in culture undergo significant morphological rearrangements and functional maturation , which were initially classified into four stages ( Dimitrov et al . , 2016 ) .",
"During morphological maturation of synapses , the volume and surface area of presynaptic terminals ( Figure 4—figure supplement 1A , B ) , as well as the number of labeled vesicles ( Figure 4—figure supplement 1C ) increased gradually .",
"Hence , we categorized developing giant terminals into two groups: ‘immature’ ( stages 1 and 2 ) and ‘mature’ ( stages 3 and 4 ) , and compared SV dynamics between them .",
"Immature terminals were characterized by prominent finger-like processes and presynaptic volumes below 1000 µm3 , whereas mature terminals were composed of numerous swellings interconnected with finger-like branches and presynaptic volumes above 1000 µm3 .",
"Q655-syt2-labeled vesicles distributed throughout immature and mature terminals and their movements were analyzed in finger-like processes and interconnected swellings ( Figure 4A and B ) .",
"SV movements displayed wider heterogeneity in immature ( trajectory length , 0–14 µm; maximum speed , 0–1 . 8 µm/s ) than in mature ( trajectory length , 0–9 µm; maximum speed , 0–1 . 4 µm/s ) terminals ( Figure 4C ) .",
"The distribution of trajectories according to their maximum speed and length from nine immature terminals and nine mature terminals showed that morphological maturation of terminals significantly reduced the length ( from 6 . 83 ± 0 . 46 µm to 5 . 57 ± 0 . 07 µm ) and speed ( from 0 . 82 ± 0 . 02 µm/s to 0 . 74 ± 0 . 02 µm/s ) of SVs with long ( L ) trajectories ( Figure 4D ) .",
"The proportion of slow and short vesicles movements increased from immature ( 50% ) to mature ( 70% ) terminals , whereas fast , long directional movements decreased from 50% to 30% during morphological maturation of terminals ( Figure 4D ) .",
"As the calyceal terminal morphology advanced , diffusive motions relative to active directional motions increased by ~1 . 8 fold ( Figure 4E ) .",
"Consistently , SV mobility was significantly reduced after morphological development of calyceal terminals , from immature stage 2 ( D = 0 . 072 ± 0 . 005 µm2/s ) to mature stage 4 ( D = 0 . 052 ± 0 . 003 µm2/s ) terminals ( Figure 4F and Figure 4—figure supplement 1D ) .",
"SV mobility transiently increases from stage 1 ( D = 0 . 062 ± 0 . 002 µm2/s ) to stage 2 ( D = 0 . 075 ± 0 . 004 µm2/s ) and stage 3 ( D = 0 . 071 ± 0 . 002 µm2/s ) , to finally decrease in stage 4 ( D = 0 . 055 ± 0 . 001 µm2/s ) .",
"These results indicate that after a transient increase , SV mobility is globally down-regulated after the morphological maturation of giant calyceal terminals . 10 . 7554/eLife . 24845 . 024Figure 4 . SV mobility decreases after morphological maturation of giant terminals .",
"( A ) Live confocal imaging of a giant immature terminal expressing cytosolic GFP and Q655-Syt2-labeled vesicles , with SV tracking color-coded over time , or sorted according to trajectory length ( Blue <2 µm , Green 2–4 µm and red >4 µm ) .",
"( B ) Confocal imaging of a giant mature terminal as described in ( A ) .",
"( C ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces from immature ( left panel ) or mature ( right panel ) calyceal terminals , color-coded as in ( A ) .",
"( D ) Classification and quantification of SV movements in three groups based on their maximum speeds and trajectory lengths in immature ( Red , n = 9 terminals ) and mature ( Blue , n = 9 ) terminals .",
"( E ) Displacement modalities and diffusion coefficients of SVs in immature ( Red , n = 9 ) and mature ( Blue , n = 9 ) terminals .",
"Two-tailed unpaired t-test ( *p<0 . 05 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 02410 . 7554/eLife . 24845 . 025Figure 4—source data 1 . Data and statistics for Figure 4D and E . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 02510 . 7554/eLife . 24845 . 026Figure 4—source data 2 . Data and statistics for Figure 4—figure supplement 1D . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 02610 . 7554/eLife . 24845 . 027Figure 4—figure supplement 1 . Morphological maturation of giant terminals involves a developmental switch in SV mobility .",
"( A ) Confocal image showing GFP-over-expressing presynaptic neurons overlaid with 3D rendering of giant terminals from different stages of maturation ( stages 1–2: immature terminals; Stages 3–4: mature terminals ) .",
"( B ) Cluster analysis of giant terminals in culture based on their volume and surface area ( Blue: Stages 1–2 , immature terminals; Green: Stages 3–4 , mature terminals ) .",
"( C ) Number of labeled vesicles in immature ( Red: Stages 1–2 ) and mature ( Blue: Stages 3–4 ) terminals .",
"( D ) Variation of the diffusion coefficient of SVs according to terminal volume during morphological maturation ( n = 5 for each developmental stage ) .",
"Two-tailed unpaired t-test for comparison between two groups and two-way ANOVA for multi groups comparison ( *p<0 . 05 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 027 Vesicular glutamate transporters ( VGLUTs ) transport glutamate into synaptic vesicles ( Takamori et al . , 2000; Fremeau et al . , 2004 ) and their subtypes VGLUT1 and VGLUT2 reportedly produce different release probabilities ( Weston et al . , 2011 ) .",
"As giant calyceal terminals in culture express both VGLUT1 and VGLUT2 ( Dimitrov et al . , 2016 ) , we questioned whether expression of different VGLUT subtypes might affect dynamic properties of SVs in nerve terminals .",
"Venus-VGLUT1 and Venus-VGLUT2 were overexpressed in different presynaptic neurons and both localized on SVs throughout giant calyceal terminals ( Figure 5A and B ) , similar to the distribution of endogenous VGLUT1 and VGLUT2 ( Figure 5—figure supplement 1 ) .",
"After 21 days in culture , expression levels of Venus-VGLUT1 and Venus-VGLUT2 , deduced from their fluorescence intensity in the terminals , were similar ( Figure 5C ) .",
"Venus-VGLUT1- or Venus-VGLUT2-expressing SVs were visualized and their movements tracked ( Figure 5D ) and analyzed , as described for vesicles labeled with Q655-Syt2 .",
"The distribution of trajectories , according to their maximum speed and length , showed higher heterogeneity in movements of SVs expressing Venus-VGLUT1 than of SVs expressing Venus-VGLUT2 ( Figure 5E ) .",
"Analysis of trajectories , displacement modalities , and diffusion coefficients of vesicles pooled from six terminals over-expressing Venus-VGLTU1 and 6 terminals over-expressing Venus-VGLUT2 remarkably revealed that VGLUT1-containing SVs had wider ranges and faster movements than VGLUT2-containing SVs ( Figure 5E ) .",
"Surprisingly , the generally observed positive correlation between trajectory length and maximum speed was absent in VGLUT2-containing SVs , but was retained in VGLUT1-containing SVs ( Figure 5F ) .",
"VGLUT1-containing SVs displayed ~1 . 6 times ( 28 . 9% ) more active displacements than VGLUT2-containing SVs ( 18 . 2% , Figure 5G ) .",
"Furthermore , the mobility of vesicles expressing Venus-VGLUT1 ( D = 0 . 072 ± 0 . 005 µm2/s ) was ~1 . 7 times higher than that of those expressing Venus-VGLUT2 ( D = 0 . 043 ± 0 . 008 µm2/s , Figure 5H ) .",
"Assuming a one-to-one expression ratio in calyceal terminals , the average diffusion coefficient of Venus-VGLUT1 and Venus-VGLUT2 expressing SVs ( D = 0 . 058 ± 0 . 006 µm2/s ) was similar to those of Q655-Syt2-labeled SVs ( D = 0 . 065 ± 0 . 004 µm2/s ) or C5E-Syt2-labeled SVs ( D = 0 . 063 ± 0 . 003 µm2/s ) .",
"These results indicated that expression of VGLUT1 conferred higher mobility upon SVs than expression of VGLUT2 , and suggest that the molecular composition of vesicles can influence their dynamic properties . 10 . 7554/eLife . 24845 . 028Figure 5 . Vesicular glutamate transporter isoforms regulate SV dynamic properties .",
"( A ) Confocal z-stack imaging of giant presynaptic terminal expressing Venus-VGLUT1 ( Green ) .",
"( B ) Confocal z-stack imaging of giant presynaptic terminal expressing Venus-VGLUT2 ( Green ) .",
"( C ) Quantification of the fluorescence intensity of Venus-VGLUT1 ( Red , n = 6 terminals ) and Venus-VGLUT2 ( Blue , n = 6 ) in giant calyceal terminals after 21 days in culture .",
"( D ) Upper panels: Live confocal imaging of a calyceal terminal expressing Venus-VGLUT1 , and SV tracking sorted according to trajectory lengths ( Blue <2 µm , Green 2–4 µm and red >4 µm ) .",
"Lower panels: Live confocal imaging of a calyceal terminal expressing Venus-VGLUT2 , and SV tracking sorted according to trajectory lengths ( Blue <2 µm , Green 2–4 µm and red >4 µm ) .",
"( E ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in Venus-VGLUT1 ( Red ) or Venus-VGLUT2 ( Blue ) overexpressing terminals .",
"( F ) Dynamic properties of SVs expressing Venus-VGLUT1 or Venus-VGLUT2 .",
"( G ) Displacement modalities and diffusion coefficients of Venus-VGLUT1 expressing vesicles ( Red , n = 6 ) and Venus-VGLUT2 expressing vesicles ( Blue , n = 6 ) .",
"Two-tailed unpaired t-test ( *p<0 . 05; ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 02810 . 7554/eLife . 24845 . 029Figure 5—source data 1 . Data and statistics for Figure 5C , F and G . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 02910 . 7554/eLife . 24845 . 030Figure 5—figure supplement 1 . Localization of endogenous vesicular glutamate transporter isoforms in cultured giant calyceal terminals . Confocal imaging of calyceal terminals labeled with antibodies against VGLUT1 or VGLUT2 . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 030 Roles of microtubules ( MTs ) are well established in axons ( Conde and Cáceres , 2009 ) , but poorly understood in nerve terminals .",
"Our data indicate that a significant number of vesicles move within giant terminals with speed comparable to that of molecular motors along MTs involved in intracellular organelle transport ( Hirokawa et al . , 2009 , 2010 ) .",
"Synaptic growth involves reorganization of the neuronal cytoskeleton ( Roos et al . , 2000 ) , which in turn could affect SV mobility .",
"Hence , we examined whether cytoskeletal elements such as MTs and kinesins could be involved in transport of SVs in giant calyceal terminals .",
"Immunofluorescence analysis revealed that giant presynaptic terminals were enriched in de-tyrosinated α-tubulin-containing polymers that interconnect neighboring and distant presynaptic swellings filled with SVs ( Figure 6A and Figure 6—figure supplement 1A ) .",
"The molecular motor , KIF1A ( Okada et al . , 1995 ) , was also found in calyceal terminals and partially co-localized with the vesicular protein , synaptophysin , within swellings ( Figure 6—figure supplement 1B , C ) .",
"KIF1A also co-localized with VGLUT1-containing SVs in regions that interconnect swellings ( Figure 6—figure supplement 1D , E ) , suggesting that KIF1A might carry SVs along MTs within and between swellings .",
"To test the possible involvement of MT-based transport in SV mobility , we disrupted presynaptic MT networks by bath application of 30 µM nocodazole .",
"Fluorescence intensity of Silicon-Rhodamine ( SiR ) -tubulin-containing polymers decreased by ~30% after 90 min incubation with nocodazole ( Figure 6B and C ) , indicating that a significant fraction of MTs in calyceal terminals were depolymerized .",
"Depolymerization of one third of MT networks was sufficient to significantly impair fast , long directional SV movements within terminals ( Figure 6D and E ) .",
"The distribution of trajectories according to their maximum speed and length ( from eight untreated and eight nocodazole-treated terminals ) showed that MT disruption reduced the proportion of SVs with long directional trajectories and concurrently increased the proportion of SVs with short displacements ( Figure 6F ) .",
"However , the apparently lower mobility observed after nocodazole treatment ( D = 0 . 057 ± 0 . 004 µm2/s ) was not significant compared to controls before treatment ( D = 0 . 066 ± 0 . 005 µm2/s , Figure 6G ) .",
"On the other hand , disruption of actin network , which essentially localized within discreet regions of presynaptic swellings ( Figure 6—figure supplement 2A , B ) , did not affect the movement of SVs between swellings ( Figure 6—figure supplement 2C ) .",
"These results indicate that MT depolymerization selectively reduces the maximum speed and the proportion of SVs travelling long distances , suggesting a dominant role of MT-based transport for SVs undergoing inter-swelling trafficking in giant terminals . 10 . 7554/eLife . 24845 . 031Figure 6 . Presynaptic MT network regulates long and rapid directional SV movements .",
"( A ) Confocal z-stack imaging of a calyceal terminal labelled with antibodies against de-tyrosinated α-tubulin ( Red ) , VGLUT1 ( Green ) and DAPI ( Blue ) .",
"( B ) Live confocal imaging of a calyceal terminal over-expressing GFP and labeled with SiR-Tubulin before and after treatment with 30 µM Nocodazole .",
"( C ) Quantification of GFP- and SiR-Tubulin fluorescence intensity during nocodazole treatment .",
"( D ) Live confocal imaging of a calyceal terminal expressing cytosolic GFP and Q655-Syt2 labeled vesicles , and SV tracking ( long tracks displayed only ) .",
"( E ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control ( left panel ) or nocodazole-treated terminals ( right panel ) , color-coded ( Blue <2 µm , Green 2–4 µm and red >4 µm ) .",
"( F ) Classification and quantification of SV movements in three groups based on their maximum speeds and trajectory lengths in control ( Red , n = 8 terminals ) and nocodazole-treated ( Blue , n = 8 ) terminals .",
"( G ) Diffusion coefficient of SVs in control ( Red , n = 8 ) and nocodazole-treated ( Blue , n = 8 ) terminals .",
"Two-tailed unpaired t-test ( *p<0 . 05; ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 03110 . 7554/eLife . 24845 . 032Figure 6—source data 1 . Data and statistics for Figure 6F and G . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 03210 . 7554/eLife . 24845 . 033Figure 6—figure supplement 1 . Microtubules and kinesins localize in giant calyceal terminals .",
"( A ) Upper panels: confocal z-stack imaging of giant terminals in culture for 18 days and labeled with antibodies against de-tyrosinated α-tubulin ( Red ) , VGLUT1 ( Green ) and DAPI ( Blue ) .",
"Lower panels: 3D rendering of images shown above .",
"( B ) Confocal images of a giant terminal labeled with antibodies against synaptophysin ( Red ) and KIF1A ( Green ) .",
"( C ) Expanded view of the presynaptic swelling delimited in ( B ) in three consecutive z-stacks; dotted line represents the putative position of the synaptic cleft relative to the swelling .",
"( D ) Confocal z-stack imaging of a giant terminal labeled with antibodies against VGLUT1 ( Red ) and KIF1A ( Green ) .",
"( E ) Orthogonal view of the terminal shown in ( D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 03310 . 7554/eLife . 24845 . 034Figure 6—figure supplement 2 . Actin network localizes in presynaptic swellings .",
"( A ) Live confocal imaging of SiR-Actin in giant cultured terminals over-expressing cytosolic GFP .",
"( B ) Volume rendering of SiR-Actin and SiR-Tubulin in GFP-overexpressing giant terminals .",
"( C ) Proportion SV trajectories and diffusion coefficient in control ( Red , n = 8 ) and 10 µM latrunculin-A-treated ( Blue , n = 4 ) terminals .",
"Two-tailed unpaired t-test ( ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 034 It is controversial whether presynaptic stimulation increases SV mobility ( Peng et al . , 2012 ) or not ( Lemke and Klingauf , 2005; Kamin et al . , 2010; Joensuu et al . , 2016 ) .",
"We examined this issue on C5E-Syt2-labeled SVs , using three different stimulation protocols;",
"( i ) bath-application of 65 mM KCl to induce massive exocytosis ,",
"( ii ) bath-application of 500 mM sucrose to induce SV exocytosis from the readily releasable pool ( RRP ) ( Stevens and Tsujimoto , 1995 ) , and",
"( iii ) sustained electrical field stimulation at 1 Hz to trigger physiological SV exocytosis .",
"In giant terminal swellings , KCl stimulation decreased the number of labeled SVs ( Figure 1—figure supplement 2B ) and redistributed SVs to smaller areas ( Figure 1—figure supplement 2C ) .",
"The overall distribution of SV trajectories did not change significantly after KCl stimulation ( Figure 7A ) .",
"However , long trajectories markedly decreased while the proportion of intermediate trajectories increased and short trajectories remained unchanged ( Figure 7D ) .",
"SV displacement modalities were also unaffected by KCl stimulation ( Figure 7E ) .",
"Surprisingly , SV mobility was reduced ~2 . 7 times ( from D = 0 . 059 ± 0 . 004 µm2/s to 0 . 022 ± 0 . 002 µm2/s ) after KCl stimulation ( Figure 7F ) .",
"However , this reduction was only observed in swelling regions , but not in finger-like regions ( Figure 2—figure supplement 2E ) . 10 . 7554/eLife . 24845 . 035Figure 7 . Synaptic stimulation does not increase SV mobility . Analysis of C5E-Syt2-labeled SVs in giant calyceal terminals .",
"( A ) KCl stimulation: Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control terminals ( Red ) or terminals incubated with 65 mM KCl ( Blue ) .",
"( B ) Sucrose stimulation: Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control terminals ( Red ) or terminals incubated with 500 mM sucrose ( Blue ) .",
"( C ) Electrical simulation: Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces in control terminals ( Red ) or terminals during 1 Hz electrical field stimulation for 30 s ( Blue ) .",
"( D ) Trajectory length analysis in control ( Red ) and KCl-treated terminals , sucrose-treated terminals , or 1 Hz-stimulated ( Blue ) terminals .",
"( E ) Displacement modality analysis in control ( Red ) and KCl-treated terminals , sucrose-treated terminals , or 1 Hz-stimulated ( Blue ) terminals .",
"( F ) Diffusion coefficient analysis in control ( Red ) and KCl-treated terminals , sucrose-treated terminals , or 1 Hz-stimulated ( Blue ) terminals .",
"( KCl treatment: n = 6; sucrose treatment: n = 6; 1 Hz stimulation: n = 6 in D , ( E and F ) .",
"Two-tailed unpaired t-test ( *p<0 . 05; ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 03510 . 7554/eLife . 24845 . 036Figure 7—source data 1 . Data and statistics for Figure 7D , E and F . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 03610 . 7554/eLife . 24845 . 037Figure 7—source data 2 . Data and statistics for Figure 7—figure supplement 1C . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 03710 . 7554/eLife . 24845 . 038Figure 7—figure supplement 1 . SV mobility does not change after spontaneous or stimulated uptake .",
"( A ) Upper panels: confocal images of C5E-Syt2 labeled vesicles loaded during spontaneous activity for 1 hr or during train of 1 Hz electrical stimulation .",
"Lower panel: Number ( N ) and fluorescence intensity ( FI ) of C5E-Syt2-labeled vesicles loaded spontaneously or during 1 Hz stimulation ( Blue bar ) .",
"( B ) Scatter plot of SV trajectory lengths and maximum speeds superimposed with individual trajectory traces during spontaneous ( Red ) or stimulated activity ( Blue ) .",
"( C ) Diffusion coefficients of C5E-Syt2-labeled vesicles loaded during spontaneous ( Red , n = 3 terminals ) or stimulated ( Blue , n = 3 ) activity .",
"Two-tailed unpaired t-test ( ns , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 038 Bath application of hypertonic sucrose solution had no effect on SV trajectories ( Figure 7B ) , nor on the proportion of long , short , and intermediate movements ( Figure 7D ) .",
"Interestingly , hypertonic sucrose induced a ~2 . 2-fold decrease in the proportion of active motions ( from 14 . 2% to 6 . 3% , Figure 7E ) .",
"However , hypertonic sucrose had no effect on SV mobility in the RRP ( Figure 7F and Figure 2—figure supplement 2E ) .",
"We next stimulated calyceal terminals electrically at 1 Hz .",
"Although this stimulation induced significant endocytosis ( Figure 7—figure supplement 1A ) , SV trajectories ( Figure 7B ) , displacement modalities ( Figure 7C ) , and overall mobility ( Figure 7F and Figure 2—figure supplement 2E ) remained unchanged .",
"It has been postulated that SVs undergoing spontaneous and stimulation-evoked release belong to different pools in presynaptic terminals ( Chung et al . , 2010 ) .",
"Thus , we investigated whether SVs , labeled spontaneously or during electrical stimulation , might show different dynamic properties .",
"Electrical stimulation in the presence of C5E-Syt2 , significantly increased the number of C5E-loaded vesicles in terminals , as well as their fluorescence intensity ( Figure 7—figure supplement 1A ) , as reported previously in KCl-stimulated hippocampal neurons ( Kraszewski et al . , 1995 ) .",
"However , the mobility of SVs loaded upon electrical stimulation ( D = 0 . 052 ± 0 . 003 µm2/s ) was not different from that of those loaded spontaneously for 1 hr before stimulation ( D = 0 . 048 ± 0 . 005 µm2/s , Figure 7—figure supplement 1B , C ) .",
"The mobility of SVs loaded with Syt2-C5E spontaneously or during synaptic stimulation was also consistent with the reduced mobility of newly endocytosed Q585-loaded SVs observed after 1 hr ( Figure 1—figure supplement 3D ) , suggesting that the mobility of SVs labeled spontaneously or during stimulation were similar .",
"Thus , while the number of moving vesicles might vary , neither chemical nor electrical stimulation significantly increased their dynamic properties and mobility in giant calyceal terminals .",
"Finally , we tested whether stimulation might affect SV dynamics near active zones ( AZs ) .",
"We labeled surface GluR1/2 on post-synaptic neurons to localize the release sites on the pre-synaptic terminal previously loaded with syt2-C5E ( Figure 8A ) , and compared SV movements within and outside from putative AZs ( Figure 8B ) .",
"We showed that during field electrical stimulation at 1 Hz , the proportion of SVs significantly increased by ~1 . 8 times near release sites ( from 21% to 38% , Figure 8C ) .",
"However , the mobility of SVs inside ( Din = 0 . 027 ± 0 . 008 µm2/s ) and outside ( Dout = 0 . 044 ± 0 . 003 µm2/s ) of AZs remained largely unaffected during stimulation ( Din = 0 . 024 ± 0 . 007 µm2/s and Dout = 0 . 037 ± 0 . 005 µm2/s; Figure 8D ) .",
"The apparent lower mobility of SVs within AZs compared to SVs outside AZs was also not statistically significant before ( p=0 . 08 , n = 6 ) and during stimulation ( p=0 . 18 , n = 6 ) .",
"Thus , our data indicate that electrical stimulation recruits SVs to AZs without altering their overall mobility . 10 . 7554/eLife . 24845 . 039Figure 8 . Electrical stimulation does not affect SV mobility within or outside of active zone .",
"( A ) Live confocal imaging of surface GluR1/2-Cy3 and Syt2-C5E-loaded SVs in GFP over-expressing giant calyceal terminal .",
"( B ) SV tracking color-coded according to trajectory length ( Blue < 2 µm , Green 2–4 µm and red >4 µm ) and within ( Cyan ) AZs ( Magenta ) .",
"( C ) Quantification of the number of SV trajectories inside or outside of AZs in control ( Black , n = 3 terminals ) and during electrical stimulation at 1 Hz for 30 s ( Red , n = 3 ) .",
"( D ) Comparison of diffusion coefficient of SVs inside and outside of AZs between control ( Black , n = 6 terminals ) and electrical stimulation at 1 Hz ( Red , n = 6 ) .",
"Two-tailed unpaired t-test ( *p<0 . 05; ns , not significant ) .",
"Rich media files . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 03910 . 7554/eLife . 24845 . 040Figure 8—source data 1 . Data and statistics for Figure 8C and D . DOI: http://dx . doi . org/10 . 7554/eLife . 24845 . 040"
],
[
"We performed a spatio-temporal analysis of fluorescently labeled SVs in mammalian central synapses to characterize their dynamic properties and movements in presynaptic terminals .",
"Comparative analyses of SV trajectories between",
"( i ) giant calyceal and small hippocampal terminals ,",
"( ii ) morphologically mature and immature calyceal terminals and",
"( iii ) SVs over-expressing VGLUT1 or VGLUT2 , together with analyses of the effects of cytoskeletal perturbation and various types of synaptic stimulation , revealed fast and heterogeneous SV movements in giant terminals , involvement of the MT cytoskeleton in inter-swelling trafficking , unchanged SV mobility in response to stimulation , and an influence of presynaptic morphology and vesicular protein composition on SV dynamics and movements .",
"Imaging and tracking methods , as well as various analytical approaches used in this study , permitted a detailed analysis of SV movements in nerve terminals .",
"Automated tracking of large numbers of SVs , based on an autoregressive motion algorithm , enabled characterization of complex vesicle movements , complexity that could not be assessed by simple Brownian motion analysis .",
"Characterization of SV dynamic properties , such as maximum speed or track length , and SV displacement modalities along individual trajectories , substantially enhanced the analysis of SV movements compared with analyses based solely on diffusion coefficients ( D ) , where subtle changes in active vesicle dynamics may simply escape detection .",
"In calyceal giant terminals , the diffusion coefficients calculated from MSD curves were comparable to those calculated from FRAP experiments .",
"However , we speculate that the discrepancy between the diffusion coefficients in swelling estimated from MSD and FRAP experiments might result from the different number of interconnected fingers/swellings between terminals analyzed in each experiment that would significantly influence the proportion of long and fast SV movements .",
"This indicates minimal bias of our automated tracking method , and allowed us to compare SV mobility in calyceal terminals with those previously reported in other types of synapses ( Holt et al . , 2004; Rea et al . , 2004; Lemke and Klingauf , 2005; Gaffield and Betz , 2007; Shtrahman et al . , 2005; Rothman et al . , 2016 ) .",
"Our present analysis revealed at least three groups of SVs with different dynamic properties and trajectories: intra-swelling trafficking ( slow and short ( S ) trajectories ) , intermediate trafficking ( moderate speed and medium ( M ) trajectories ) , and inter-swelling trafficking ( fast and long ( L ) trajectories ) .",
"We showed that SV movements are highly heterogeneous in giant calyceals , combining both diffusional and active motions .",
"These data are in agreement with recent study showing heterogeneous SV motions in rat hippocampal neuron culture ( Joensuu et al . , 2016 ) .",
"Taken together Joensuu et al . and our current work demonstrated that SV movements in CNS terminals are much more heterogeneous than previously thought .",
"Among all the individual trajectories analyzed in our study , we observed a clear , positive correlation between SV trajectory length and SV traveling speed , implying that the time required for SVs to move over short or long distances could be normalized by this mechanism .",
"This suggests that SVs remote from active zones ( AZs ) could potentially reach their release sites with the same efficiency as SVs closer to AZs .",
"These findings are complementary to a report showing that recycling SVs undergoing release at neuromuscular junctions do not have preferential spatial distributions , but are scattered randomly within nerve terminals ( Rizzoli and Betz , 2004 ) .",
"To date , numerous publications have reported a wide range of SV mobility based on analyses of diffusion coefficients obtained by different methodologies at physiological or non-physiological temperatures , and/or at different types of synapses , such as in mouse hippocampal terminals ( D ~ 1×10−2 µm2/s at RT , D = 4–9×10−4 µm2/s; Lemke and Klingauf , 2005; Shtrahman et al . , 2005 ) , goldfish retinal bipolar cells ( D = 1 . 5×10−2 µm2/s at RT; Holt et al . , 2004 ) , lizard retinal cone cells ( D = 0 . 1 µm2/s at RT; Rea et al . , 2004 ) , or in frog ( D = 2 . 6×10−3 µm2/s ) ( Gaffield et al . , 2006 ) or mouse ( D = 5×10−3 µm2/s ) ( Gaffield and Betz , 2007 ) neuromuscular junctions .",
"Although these data might suggest that SV mobility varies among terminal types , the lack of direct and consistent comparison between synapses raises the question of whether synapse types or morphologies really influence SV mobility .",
"Hence , we analyzed SV movements between giant calyceal and small hippocampal terminals under the same conditions and imaging methods .",
"We found threefold higher SV mobility in calyceal synapses ( D = 6 . 5×10−2 µm2/s ) than in hippocampal synapses ( D = 2×10−2 µm2/s ) .",
"SV mobility tended to be higher at larger calyceal swellings , but this tendency was absent at hippocampal boutons .",
"Thus , terminal size is clearly one of the biological factors influencing SV mobility .",
"Interestingly , Peng et al . ( 2012 ) suggested that discrepancies in SV mobility reported thus far might arise from differences in molecular composition of SVs between and/or within synapses .",
"SV proteins are commonly divided into two classes , based on their functions: transport proteins ( proton pump , VGLUT , etc . ) that mediate neurotransmitter uptake , and trafficking proteins ( rab , synaptotagmin , etc . ) involved in SV intracellular transport and movements ( Sudhof , 1999 ) .",
"VGLUT isoforms have different regional and developmental expression ( Liguz-Lecznar and Skangiel-Kramska , 2007 ) , and reportedly produce different release probabilities ( Weston et al . , 2011 ) .",
"Here , we showed that over-expression of VGLUT1 or VLGUT2 also confers different dynamic properties upon SVs and influences their mobility in calyceal presynaptic terminals .",
"Particularly , VGLUT1-expressing SVs moved faster and traveled farther than VGLUT2-expressing SVs .",
"Remarkably , VGLUT2-expressing vesicles lost the positive correlation between speed and distance , suggesting that their capacity to reach the AZ might be less than of VGLUT1-expressing SVs , when their initial position is distant from the AZ .",
"Speculatively , dynamic properties of VGLUT1- and VGLUT2-expressing vesicles might result from different affinities for tethering/scaffolding proteins or molecular motors .",
"It is likely that the ratio between SVs containing specific molecular markers , as well as their distribution and positioning within terminals , explain differences in SV mobility observed at various synapses .",
"We have identified a large population , ~15% of labeled vesicles , moving between presynaptic swellings at high speeds and with long , directional trajectories .",
"This population of SVs undergoing inter-swelling exchange in giant calyceal terminals appears to be three times larger than that observed in conventional-sized hippocampal synapses ( Alabi and Tsien , 2012 ) and previously designated as the ‘super pool . ’ In hippocampal terminals , actin ( Darcy et al . , 2006 ) and BDNF ( Staras et al . , 2010 ) are reportedly involved in SV movements from the super pool .",
"In contrast , our data in calyceal terminals revealed a significant contribution of MT networks to the transport and movements of SVs between swellings .",
"We speculate that a large ‘super pool’ and presynaptic MTs may be required to coordinate fast and efficient cycling of SVs among the numerous release sites present in single giant terminals , compared to the constitutive sharing and replenishment of SVs observed in multiple individual conventional boutons .",
"Our data show that presynaptic morphology can significantly influence SV dynamics and movements .",
"During development , giant terminals in culture undergo significant reorganization of their presynaptic compartments ( i . e . increased surface , volume , and complexity ) , leading to formation of mature synaptic connections and associated with a 1 . 5-fold reduction in SV dynamics and movements .",
"Although we cannot exclude the possibility that other molecular mechanisms occurring during synaptic growth and maturation regulate SV movements , the high SV mobility observed in immature terminals might arise from two independent , but integrative factors:",
"( i ) co-transport of AZ components and other synaptic proteins with SV precursors ( Okada et al . , 1995; Yonekawa et al . , 1998; Stagi et al . , 2005 ) and",
"( ii ) changes in mechanical tension during synaptic development .",
"In immature terminals , SV mobility is high and transiently increases from stage 1 to stage 2 in parallel with increased presynaptic volume , whereas in mature terminals , SV mobility decreases significantly from stage 3 to stage 4 ( see Figure 4—figure supplement 1D ) .",
"This high and increasing SV mobility observed in immature terminals could result from the necessity to coordinate vesicle trafficking with rapid transport and delivery of synaptic components prior to establishment of stable synaptic contacts ( Bury and Sabo , 2011 ) .",
"On another hand , the rise in mechanical tension associated with expansion of the presynaptic area during development ( Siechen et al . , 2009; Ahmed et al . , 2012 ) could increase the probability of fast active motions , as observed in Aplysia neurons ( Ahmed and Saif , 2014 ) .",
"After formation of stable and mature synaptic contacts during stages 3 and 4 , coordinated trafficking of SVs and AZ components may diminish and mechanical tensions may lessen , simultaneously reducing active transport and SV mobility .",
"These factors , in addition to the structural organization of the MT cytoskeleton , might also account for the decrease ( 1 . 4 times ) in SV mobility observed between finger-like processes and swellings .",
"In giant calyceal terminals , neither chemical nor electrical stimulation increased SV mobility , in agreement with previous reports at the neuromuscular junction ( Betz and Bewick , 1992 ) or at hippocampal synapses ( Lemke and Klingauf , 2005; Kamin et al . , 2010 ) .",
"These results imply that SV trafficking between endocytosis and exocytosis remain largely unchanged upon stimulation .",
"However , our results do not exclude the possibility that SVs , undergoing exocytosis , might transiently change their mobility during stimulation , and image analysis at higher spatial and temporal resolution might resolve putative changes in SV movements involved in neurotransmitter release .",
"Nevertheless , our analysis has revealed some alterations of SV dynamics after KCl stimulation , inducing clustering of SVs in calyeceal swellings , and a marked reduction in long trajectory SV movements .",
"Presumably , after KCl stimulation , SVs were immobilized near release sites .",
"Likewise , hypertonic sucrose stimulation , which depletes SVs from the RRP ( Stevens and Tsujimoto , 1995 ) significantly reduced the number of actively moving SVs , suggesting that SVs depleted from the RRP during exocytosis were replenished from a recycling pool of SVs previously moving with active displacements .",
"Direct support of synaptic transmission might be provided by fast diffusive and subtle local changes in SV mobility near release sites as recently reported ( Rothman et al . , 2016 ) , rather than diverse and heterogenous SV movements prior to release .",
"The latter may contribute to distribute SVs in optimal locations for the functional and structural maintenance of presynaptic terminals .",
"In this regard , during the process of SV labeling , newly endocytosed SVs had low mobility with their distribution confined near endocytic regions for the first hour .",
"Low SV mobility near exo/endocytic regions is likely caused by tethering of SVs around release sites .",
"Classically , synapsin-1 is thought to tether SVs in its dephosphorylated form ( Llinás et al . , 1985 ) .",
"The broad-spectrum phosphatase inhibitor OA increases SV mobility by ~10 times in hippocampal terminals ( Jordan et al . , 2005 ) or at the neuromuscular junction ( Gaffield et al . , 2006 ) .",
"In calyceal terminals , OA only increased SV mobility by ~1 . 4 times , similar to that recently reported at cerebellar mossy fiber terminals ( ~2 times; Rothman et al . , 2016 ) , suggesting higher abundancy of untethered SVs in these terminals and/or phosphorylation independent SV tethering with molecules such as Basson ( Hallermann et al . , 2010 ) or Unc analogs ( Böhme et al . , 2016 ) .",
"We have also demonstrated that SVs accumulate at release sites during electrical stimulation without significantly changing their mobility compared to resting condition .",
"Although we do not exclude the possibility that SV dynamics at release sites might be affected by synaptic activity , higher temporal and spatial resolution imaging methods would be required to assess putative changes in the mobility of SVs associated with neurotransmitter release .",
"Altogether , our data indicate that in central nervous system , SV movements are highly heterogenous and that large synapses possess higher basal SV mobility than smaller synapses .",
"Our results also suggest that SV movements and supply can be influenced by morphological characteristics of presynaptic terminals and by molecular signatures of vesicles .",
"Although the underlying molecular mechanisms remain to be characterized , presynaptic morphology and vesicular composition appeared to be major biological determinants characterizing SV dynamics and trafficking in central synapses ."
],
[
"Giant synapse primary cultures were established as described previously ( Dimitrov et al . , 2016 ) .",
"Briefly , mouse brains from E18 to P1 were extracted in ice cold HBSS ( Life Technologies , USA ) and the cochlear nuclei ( CN ) and the medial nuclei of the trapezoid body ( MNTB ) regions were micro-dissected and stored separately in ice cold HBSS .",
"CN and MNTB regions were dissociated using Nerve Cell Dissociation Medium ( Sumitomo Bakelite , Japan ) according to the manufacturer's instructions .",
"Dissociated neurons were then plated at an equal ratio of CN and MNTB neurons to a final density of 160 , 000–180 , 000 cells per 35 mm culture dish ( Ibidi , Germany ) , previously coated with 100 μg/ml poly-D-lysine ( Millipore , USA ) , in ‘Nerve Cell Culture Medium’ ( Sumitomo Bakelite , Japan ) supplemented with NGF2 . 5S 100 ng/ml ( Life Technologies , USA ) , hBDNF 25 ng/ml ( R and D Systems , USA ) , hFGF2 5 ng/ml ( Peprotech , USA ) , 50 ng/ml hNT-3 ( Peprotech , USA ) and 20 mM KCl ( Nacalai Tesque , Japan ) .",
"At DIV 8 , 5 µM AraC ( Sigma Aldrich , japan ) was added to the medium to inhibit cell proliferation .",
"Medium without AraC was exchanged every 4 days throughout the culture .",
"Hippocampal neurons were prepared as described previously ( Guillaud et al . , 2008 ) and dissociated hippocampal neurons were plated to a final density of 150 , 000 cells per 35 mm culture dish .",
"When needed and before plating , transfection of dissociated VCN or hippocampal neurons with pCAG-AcGFP ( Dimitrov et al . , 2016 ) , Venus-VGLUT1 or Venus-VGLUT2 vectors was performed by electroporation using the Neon transfection system ( Life Technologies , USA ) according to the manufacturer’s instructions .",
"Primary antibodies: Vesicular Glutamate Transporter 1 ( VGLUT1 , Millipore , USA ) , Synaptophysin ( Synaptic System , Germany ) , Synaptotagmin-2 lumenal domain ( Synaptic System ) , detyrosinated α-tubulin ( Glu-α-tubulin , Synaptic System ) , α-tubulin ( Sigma Aldrich , Japan ) , Kinesin motor protein KIF1A ( AbCam , USA ) , GluR1/2-Cy3 extracellular domain ( BIOSS antibodies ) .",
"Secondary antibodies: AlexaFluor 405 , 488 , 568 and 647 , DAPI , Quantum dots Q655 and Q585 ( all from Life Technologies , USA ) .",
"CypHer5E ( C5E , Synaptic System ) .",
"Primary cell cultures grown in 35-mm culture dishes were fixed in PBS 4% paraformaldehyde for 20 min at room temperature or overnight at 4°C .",
"After fixation , cells were permabilized in PBS 0 . 2% saponin for 12 min and blocked in PBS 3% bovine serum albumin ( BSA ) for 45 min at room temperature .",
"Primary antibodies , diluted in PBS 0 . 02% saponin and 0 . 3% BSA , were incubated overnight at 4°C .",
"Cells were then washed three times in PBS 0 . 02% saponin for 10 min and fluorescent secondary antibodies were incubated in PBS 0 . 02% saponin and 0 . 3% BSA for 1 hr at room temperature .",
"After washing three times in PBS 0 . 02% saponin for 10 min , cells were mounted in PBS or Prolong gold antifade reagent ( Life Technologies , USA ) .",
"Confocal imaging was performed on a confocal laser scanning LSM780 microscope equipped with Plan-Apochromat 63x , 1 . 4 NA or Plan-Neofluar 100x , 1 . 45 NA oil immersion lenses ( Carl Zeiss , Germany ) .",
"Both fluorescent-conjugated antibodies directed against the luminal domain of synaptotagmin and fluorescent nanoparticles have been used to label and assess SV cycling in cultured mammalian synapses ( Kraszewski et al . , 1995; Zhang et al . , 2007; 2009; Lee et al . , 2012 ) .",
"Here , SVs from cultured giant terminals were labeled with rabbit polyclonal antibodies directed against the intravesicular ( lumenal ) domain of synaptotagmin-2 tagged with either quantum dots Q655 or Q585 , or with the pH-sensitive fluorophore CypHer5E ( C5E ) .",
"Briefly , 1 µg of synaptotagmin-2 antibody was incubated with 2 . 5 µg of secondary F ( ab ) ’2 antibody ( Q655 or C5E ) in a total volume of 10 µl for 45 min at room temperature before adding it to the culture .",
"Synaptotagmin-2 solution was then applied to cultures between DIV 15 and DIV 21 for 1–16 hr ( typically overnight ) at 37°C and 5% CO2 .",
"This procedure ensured that only vesicles fused to the plasma membrane during spontaneous exocytosis were labeled and we expected that the labeled vesicle pool comprised ≥1–2% of all vesicles found in the terminals .",
"Before imaging , culture medium was replaced with standard Tyrode’s solution ( pH = 7 . 4 ) and live imaging was performed on an LSM 780 confocal laser scanning microscope equipped with a temperature-controlled ( Tokai Heat , Japan ) Plan-Apochromat 63x , 1 . 45 NA oil immersion lens ( Carl Zeiss ) .",
"Tyrode’s solution was continuously perfused with a Dynamax peristaltic pump ( Rainin , Switzerland ) connected to a dual automatic temperature controller TC-334B ( Warner Instruments Corp . , USA ) , to maintain a constant physiological temperature of 36 . 5°C throughout the imaging period .",
"After localizing giant presynaptic terminals over-expressing cytosolic GFP , a region of interest ( ROI ) containing several interconnected presynaptic swellings , located in the upper region of the calyceal terminal ( z = 10–15 µm ) , was identified in order to achieve an effective scanning speed of 1–1 . 25 frame per second for up to 120 s , on single 2D optical section ( initial image resolution 512 × 512 pixels , pixel dwell time 3 . 15 µs ) .",
"For 3D tracking , four optical sections ( ~300 nm ) covering the height of presynaptic swellings ( ~1 . 5 µm ) were acquired with a pixel dwell time of 1 . 27 µs .",
"Raw confocal images were filtered using the median filter algorithm in ZEN 2 . 1 ( Carl Zeiss ) before further analysis .",
"Accurate automatic and simultaneous tracking of a large population of objects , such as vesicles , requires robust and sophisticated algorithms that can identify independent objects , predict their future positions based on their previous speed , directionality , and weighted intensity .",
"Here , IMARIS 8 . 1 with IMARIS Track , Measurement Pro and Vantage plugins ( Bitplane , Switzerland ) , commonly used to analyze organelle movements ( Johnson et al . , 2011; Wong and Munro , 2014; Grassart et al . , 2014; Maucort et al . , 2014; Varela et al . , 2015 ) , was used to track SV movements and to perform spatio-temporal analyses of several thousand vesicle trajectories .",
"Spot detection and tracking was performed on 30 s sequences using either the autoregressive or Brownian motion algorithm with an initial spot size of 150 nm ( Gaussian fitted ) , a maximum distance between spots on two consecutive frames of 0 . 8 µm without frame gap .",
"The distance between spots on two consecutive frames was set to 0 . 8 µm because kinesin-driven transport in mammalian neurons has an average speed of 0 . 7–1 µm/s ( Guillaud et al . , 2003 ) ; thus , we conservatively estimated the maximum distance travelled by a vesicle associated with kinesin within a 1 s interval would be ~0 . 8 µm .",
"For rendering and visualization , synaptic vesicles were color-coded according to their respective speeds , and tracks were color-coded according to time or to their trajectory length .",
"Analysis and comparison of SV trajectories and dynamic properties were performed in mature terminals unless stated otherwise .",
"Statistical data sets regarding pre-synaptic volume and surface , vesicle speed and track length , etc . were exported from IMARIS Measurement Pro to Prism 6 ( Graphpad software Inc . , USA ) and MS Excel ( Microsoft , USA ) .",
"The mean square displacement ( MSD ) analysis and estimation of the diffusion coefficient ( D ) from vesicle trajectories was performed using the MSD Matlab plugin for IMARIS ( Tarantino et al . , 2014 ) .",
"The diffusion coefficient from FRAP experiments was calculated as D = [0 . 224x ( ω2/T1/2 ) ] , where ω represents the length of the bleached region of interest and T1/2 , the time to recover half of the fluorescence intensity ( Axelrod et al . , 1976 ) .",
"The Pearson coefficient ( P ) for co-localization was calculated in IMARIS .",
"Labeling of microtubule and actin networks in live terminals was performed with SiR-tubulin and SiR-actin ( Cytoskeleton Inc . , USA ) according to the manufacturer’s instructions .",
"Briefly , primary cultures were exposed to 1 µM SiR-Tubulin or SiR-Actin in Tyrode’s solution for 30 min at 37°C .",
"Labeling solution was replaced with fresh Tyrode’s solution without SiR-Tubulin or SiR-Actin before imaging .",
"Surface labeling of AMPA receptors in giant calyceal culture was performed by incubation with 10 µg/ml mouse monoclonal antibodies directed against the extracellular domain of GluR1/2 tagged with Cy3 , for 15 min at room temperature before live imaging .",
"Projection of the surface area labeled with GluR1/2-Cy3 on to the pre-synaptic terminal was used to delineate release sites and compare SV mobility inside and outside putative active zones .",
"De-polymerization of microtubules and disruption of actin filaments were performed by bath application of 30 µM nocodazole ( Tocris , USA ) or 10 µM latrunculin-A ( Focus Biomolecules , USA ) , respectively .",
"Okadaic acid ( Tocris , USA ) was used at 2 . 5 µM ( Betz and Henkel , 1994; Kraszewski et al . , 1995 ) .",
"Electrical field stimulation ( Ohhashi et al . , 1980 ) of giant terminals in culture was performed at a frequency of 1 Hz , with voltage ranging from 50 to 100 V and a pulse duration of 0 . 3 ms . No statistical method was used to predetermine sample size .",
"Experiments were not randomized and investigators were not blinded to allocation during experiments .",
"Each experiment was repeated three times independently to ensure reproducibility and adequate statistical power .",
"All data sets were compared using two-tailed , unpaired Student’s t-tests and two-way ANOVA in Prism 6 ( Graphpad Software Inc . ) .",
"Data are presented as mean ± s . e . m . pooled from at least three independent experiments .",
"Statistical significance ( * ) was assumed when p≤0 . 05 ( data sets and exact p values are provided in source data files ) .",
"All experiments have been performed in accordance to the regulations of OIST animal care and use committee ( protocol #2015–128 ) .",
"OIST animal facilities and animal care and use program are accredited by AAALAC International ( reference #1551 ) ."
]
] | [
"Transport of synaptic vesicles ( SVs ) in nerve terminals is thought to play essential roles in maintenance of neurotransmission .",
"To identify factors modulating SV movements , we performed real-time imaging analysis of fluorescently labeled SVs in giant calyceal and conventional hippocampal terminals .",
"Compared with small hippocampal terminals , SV movements in giant calyceal terminals were faster , longer and kinetically more heterogeneous .",
"Morphological maturation of giant calyceal terminals was associated with an overall reduction in SV mobility and displacement heterogeneity .",
"At the molecular level , SVs over-expressing vesicular glutamate transporter 1 ( VGLUT1 ) showed higher mobility than VGLUT2-expressing SVs .",
"Pharmacological disruption of the presynaptic microtubule network preferentially reduced long directional movements of SVs between release sites .",
"Functionally , synaptic stimulation appeared to recruit SVs to active zones without significantly altering their mobility .",
"Hence , the morphological features of nerve terminals and the molecular signature of vesicles are key elements determining vesicular dynamics and movements in central synapses ."
] | [
"In the brains of mammals , communication between cells called neurons is vital for learning and memory .",
"Pairs of neurons communicate at junctions called synapses .",
"At a synapse , the first neuron releases chemical messengers into the gap between the cells , which then bind to and activate receptor proteins on the surface of the second neuron .",
"The chemical messengers are released from bubble-like packages called synaptic vesicles that fuse with the first neuron’s membrane and empty their contents into the synapse .",
"This same neuron then retrieves and reassembles the components of the vesicle , ready to be filled again with the chemical messengers .",
"Neurons must continually retrieve and refill vesicles in order to continue transmitting information at synapses .",
"But while the mechanisms of vesicle fusion and retrieval are well characterized , it remains unclear what triggers the movement and supply of vesicles inside synapses or how these processes are regulated .",
"Deciphering these mechanisms will help us better understand how synapses work in healthy as well as diseased brains .",
"Using high-resolution microscopy , Guillaud et al . have now studied the movements of fluorescently labeled vesicles inside mouse brain synapses grown in the laboratory .",
"This revealed that synaptic vesicles move in much more varied and complex ways than previously thought .",
"The movement of vesicles changed depending on the type and developmental stage of the synapses .",
"It also depended on the identity of particular proteins within the membranes of the vesicles themselves .",
"These proteins , known as transporters , enable vesicles to take up the chemical messengers .",
"Vesicles with different transporters showed different patterns of movement .",
"Disrupting components of the internal skeleton of the neuron – specifically protein filaments called microtubules – also disrupted vesicle movement .",
"By contrast , changes in the activity level of the synapse had no such effect .",
"The next step is to determine exactly how these factors regulate the movement of vesicles at synapses .",
"Studies can then examine whether these processes are disrupted in neurological disorders , in which communication at synapses is often impaired ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Methods"
] | [
"developmental biology"
] | Cellular resolution models for even skipped regulation in the entire Drosophila embryo | elife-00522-v1 | [
[
"A detailed knowledge of transcriptional control will have profound consequences for our understanding of myriad biological processes , including development , homeostasis , and evolution of new phenotypes .",
"To this end , through a combination of genomic , genetic , and molecular experiments , the field continues to accumulate considerable information documenting components of regulatory systems and regulator-target interactions ( Gerstein et al . , 2010; The modENCODE Consortium , 2010; The ENCODE Project Consortium , 2012 ) .",
"At present however , many of these descriptions are qualitative .",
"A major goal going forward is to interpret these data in a quantitative manner ( Wilczynski and Furlong , 2010 ) : how do regulators and regulatory interactions convert input signals to the appropriate output expression pattern ?",
"In general , answering these questions remains a significant challenge .",
"The experiments needed to probe regulatory functions in detail are technically demanding; moreover , many systems involve multiple layers of control that cannot be investigated within a single experimental set-up .",
"Theoretical models can help advance experimental investigations by providing a framework for deriving general principles and developing testable hypotheses ( Reeves et al . , 2006; Tomlin and Axelrod , 2007; Lewis , 2008; Oates et al . , 2009; Davidson , 2010 ) .",
"An effective model should be able to define and predict expression accurately by describing how and by how much regulators influence target gene expression ( Hasty et al . , 2001; Segal and Widom , 2009 ) .",
"Transcription in animals is controlled by interaction among transcription factors ( TFs ) , enhancers , core promoters , silencers , insulators , and chromatin structure ( Lemon and Tjian , 2000; Arnosti , 2003; Levine , 2010; Ohler and Wassarman , 2010; Dean , 2011 ) .",
"It is thought that core promoter elements and chromatin structure provide general competence for transcription at transcription start sites ( Lenhard et al . , 2012 ) , whereas more distant enhancers up-regulate expression of genes under specific conditions ( Bulger and Groudine , 2011; Ong and Corces , 2011 ) .",
"A single gene can be regulated by multiple enhancers , each directing a portion of the overall gene expression pattern in space and time .",
"Enhancers operate by binding TFs , which in turn recruit regulatory co-factors and/or interact directly with the core promoter where RNA polymerase acts ( Spitz and Furlong , 2012 ) .",
"A comprehensive model of transcriptional regulation would therefore include many factors , such as regulatory DNA sequence , DNA conformation , TF concentrations and nucleosome position among others ( Segal and Widom , 2009 ) .",
"However , many of the parameters in such a model are currently impossible to measure .",
"In the absence of such measurements , a partial yet predictive model based on available data is still valuable .",
"Here , we propose models of transcriptional control that are highly predictive of target gene expression given only TF concentrations at cellular resolution .",
"Our goal is to make few assumptions about the underlying molecular mechanism .",
"Instead , by generating models that predict experimental measurements as accurately as possible , we infer probable biological mechanisms and insights suggested by the parameters of the models .",
"To achieve this , we focus on modeling the functional link between TF inputs and the resulting output ( i . e . , the ‘regulatory input function‘ ) .",
"These models are specific to individual enhancers: they capture how genomic loci interpret TF concentrations to control the output expression level of their target genes .",
"Though multiple previous modeling studies have explicitly included protein–DNA interactions ( e . g . , in Drosophila , see He et al . , 2010; Janssens et al . , 2006; Junion et al . , 2012; Kazemian et al . , 2010; Segal et al . , 2008; Zinzen et al . , 2009 ) , here , we choose to model the relationship between inputs and outputs directly as this offers several advantages .",
"First and most importantly , this type of model encapsulates numerous relevant levels of biophysical interactions ( i . e . , TF-DNA , TF-TF , enhancer-promoter etc ) .",
"Second , it enables us evaluate the utility of higher-order interactions between TFs , propose potential regulators and consider alternative hypotheses of experimental results .",
"Third , in the context of developmental biology , it allows us to explore the minimal information required to define positional information in the early embryo .",
"Finally , focusing on input and output measurements means that the approach is applicable to relatively uncharacterized systems , for instance where enhancer regions have not yet been identified , or in assessing the conservation of regulatory input functions between species ( Wunderlich et al . , 2012 ) .",
"We develop and test our models in the context of the well-studied even skipped ( eve ) enhancers in order to demonstrate their accuracy and utility .",
"eve is expressed in a symmetrical pattern of seven stripes that subdivide the embryo along the anteroposterior axis ( Nüsslein-Volhard and Wieschaus , 1980 ) .",
"Each stripe is only a few nuclei wide and any regulatory input function of an enhancer must define at least two borders at a high level of precision .",
"A number of well-characterized enhancers direct expression of the seven eve stripes individually or in pairs ( Goto et al . , 1989; Harding et al . , 1989; Fujioka et al . , 1999 ) .",
"Here , we focus on the enhancers eve 2 and eve 3+7 , which have been shown to control stripe 2 and stripes 3 and 7 respectively ( Goto et al . , 1989; Harding et al . , 1989; Stanojevic et al . , 1991; Small et al . , 1992 , 1996 ) .",
"Many of the input TFs and their roles in regulating eve expression have been defined; however , there remain unexplained properties underlying their regulation .",
"An advantage of modeling eve is that we can use the available information as independent validations of our ability to recover known regulators and predict the outcome of regulatory perturbations , while also producing new insights .",
"It is notable that though there has been some success in simulating the simpler gap gene expression pattern and in predicting eve expression on a portion of the anteroposterior axis , modeling pair-rule expression accurately across the whole embryo has remained a significant challenge ( Jaeger et al . , 2004; Janssens et al . , 2006; Papatsenko and Levine , 2008; Segal et al . , 2008; Kazemian et al . , 2010; Kim et al . , 2013 ) .",
"To fit regulatory input functions , we require accurate measurements of expression levels for both the regulating TFs and eve .",
"The Virtual Embryo from the Berkeley Drosophila Transcription Network Project provides the best available data for this purpose ( Fowlkes et al . , 2008 ) .",
"It is a cellular resolution , spatiotemporal atlas of gene expression and morphology for a whole Drosophila melanogaster blastoderm embryo .",
"The dataset contains the three-dimensional coordinates for 6078 nuclei , along with mRNA expression measurements of 95 different genes at six time points during the 50 min leading to gastrulation: these genes include critical TFs that direct patterning in the early Drosophila embryo .",
"Using our modeling framework , we",
"( i ) predict expression patterns with accuracy and explanatory power at cellular resolution across the whole embryo;",
"( ii ) recover previously described regulatory relationships and test whether they provide sufficient positional information to define the resulting expression pattern;",
"( iii ) propose potential new regulatory relationships by comparing alternative models; and",
"( iv ) predict expression patterns under perturbation of input TFs , capturing the outcome of knockdown and misexpression experiments .",
"Given the high level of accuracy of our models , we conclude with observations regarding mechanism and principles of enhancer function ."
],
[
"Our strategy is to find the logistic regression coefficients that most accurately describe the relationship between measured regulator concentrations and specific stripes of eve expression ( Figure 1 ) .",
"We first train our models using the known regulators described in the literature to evaluate if they are sufficient for determining eve expression .",
"At this stage , we also test the model for consistency across different subsets of the data .",
"Next , we ask generally which regulators are able to specify eve expression ( regulator discovery ) and consider the plausibility of concentration-dependent dual regulation .",
"Finally , we assess whether our models are able to predict beyond the conditions of the training data: specifically , we test whether our models can predict expression under perturbation , such as mutation of TFs and their cognate enhancer binding sites , by comparing our predictions with independently published experimental results . 10 . 7554/eLife . 00522 . 003Figure 1 . Schematic representation of method used to model eve expression .",
"( A ) Logistic regression is used to calculate the probability pi that eve is ON in a given nucleus i , given TF concentrations .",
"A logistic model linearly combines the values of independent variables ( in this case , the concentrations , xki , of regulators 1 to k ) to produce a prediction; the predictor , ηi , is then transformed by the logistic function to give the probability , pi , of eve being ON .",
"The weight parameters βk are optimized to provide the best fit with the training data: positive weights indicate activators and negative weights indicate repressors .",
"( B ) Schematic representation of the data preparation , model training and prediction steps using eve stripe 2 as an example .",
"The plots represent how logistic regression operates; the lateral perspectives of the embryo show the Virtual Embryo and processed expression data for eve and four regulators ( Bcd , Hb , Kr , and Gt ) .",
"In Step 1 , eve’s expression is discretized whereas TF concentrations are retained as continuous values .",
"Each nucleus corresponds to a data point .",
"In Step 2 , the logistic model is trained to classify whether eve is ON or OFF using all nuclei in stripe 2 , and all OFF nuclei in the embryo .",
"In Step 3 , the trained model is used to predict eve expression in every nucleus of the entire embryo using the concentrations of the relevant regulators within them ( shown in green for activators , and purple for repressors ) .",
"In Step 4 , the effects of perturbations are predicted by adjusting the concentration of the regulator under consideration ( in this case , Hb ) , but without changing any model parameters . DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 00310 . 7554/eLife . 00522 . 004Figure 1—figure supplement 1 . Expression of eve across the anteroposterior axis . Each strip shows the expression of eve in a narrow lateral band ( 10 μm either side of the lateral midline ) along the anteroposterior ( A-P ) axis for each time point ( 1–6 ) in the Virtual Embryo .",
"The interval between the time points is approximately 10 min .",
"The horizontal line is the threshold of 0 . 2 used in training the models .",
"The rug plot indicates which nuclei are considered within the stripes according to this threshold . DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 004 First we focused on the expression of the second stripe of eve , as it is directed by a very well-characterized enhancer , eve",
"2 . Through detailed molecular analysis , it is known that eve stripe 2 is controlled by the gap genes ( Frasch and Levine , 1987; Stanojevic et al . , 1991; Small et al . , 1992 ) , a class of TFs that are present in broad regions of the early embryo .",
"In the generally accepted minimal mechanism , two activators Hunchback ( Hb ) and Bicoid ( Bcd ) enable broad permissive eve expression in the region of stripe 2 , while two repressors Giant ( Gt ) and Kruppel ( Kr ) define the anterior and posterior borders respectively by suppressing eve outside the stripe .",
"Having successfully applied the model using known regulators , we next developed a method to identify a parsimonious set of regulators from the dataset informative for the target enhancer’s expression .",
"Such techniques are broadly applicable in discovering potential regulators of uncharacterized enhancers , and therefore useful in producing testable hypotheses .",
"We tested a stepwise selection process , but found that it generally includes more regulators than necessary for a good visual fit ( e . g . , a stepwise selection procedure for eve 2 with the Bayesian information criterion finds 11 regulators ) .",
"The stopping point ( i . e . , the penalty for adding an extra parameter ) is effectively arbitrary in this case , or at least difficult to determine a priori in a justifiable manner .",
"Additionally , stepwise selection does not consider all models exhaustively .",
"Instead , since we are particularly interested in identifying parsimonious models that can explain eve expression , and logistic models are fast to fit , we took the approach of fitting all possible models of four regulators out of the possible 38 in the dataset ( 73 , 815 models ) and used the log likelihood of each fitted model as its score ( or equivalently here , the Akaike information criterion ) .",
"Gratifyingly , the best-scoring model comprises the known regulators Hb , Bcd , Gt , and Kr .",
"To make use of the scores more generally , we developed a method that summarizes the scores for all 73 , 815 predictions and highlights regulators that work well together ( Figure 2D ) .",
"For each possible pair of regulators ( the fixed pair ) , we determined the best-scoring model of four regulators containing the pair .",
"The two regulators of the fixed pair are shown on the axes on the heat map , with the highest score for the pair depicted in the intersecting cell on a color scale from light ( the minimum score on the heat map ) to dark ( the maximum score ) .",
"Dark bands crossing the heat map highlight individual regulators that consistently make informative contributions to stripe 2 expression .",
"Hence , it is clear that Bcd , Hb , and Gt are key regulators .",
"Although Kr is actually in the top-scoring model , the heat map does not show it as consistently informative .",
"The linear model successfully recapitulates stripe 2 expression; however , it identifies Bcd as a repressor , whereas most existing literature defines the TF as an activator .",
"Despite the apparent consensus , Bcd’s function is not straightforward .",
"The need for Bcd-binding sites for successful eve expression suggests an activating function ( Small et al . , 1992 ) ; but this does not explain why the enhancer is inactive in the anteriormost region of the embryo despite Bcd being present at high concentrations and the known repressors Gt and Kr having low concentrations ( Figure 2A ) .",
"Our linear models reflect this apparent paradox: Bcd is highlighted as one of the most important TFs during regulator discovery in spite of consistently having a negative coefficient , but a model trained by excluding the anterior region of the embryo assigns Bcd an activating function ( Figure 2—figure supplement 1 ) .",
"These observations strongly suggest that Bcd—as both a repressor and activator—provides useful positional information to eve .",
"We asked whether these two functions could be reconciled if Bcd’s regulatory effect were dependent on its concentration , either directly , or mediated through other factors or post-translational modifications ( Janody et al . , 2000 , 2001; Andrioli et al . , 2002 ) .",
"This is readily modeled by adding a single parameter: a quadratic term for Bcd ( Figure 2E–G ) .",
"The result is clear: the modified model retains a repressive function for Bcd in the anterior of the embryo where it is present in high concentrations , but enables an activating function in the region of stripe 2 where it has lower concentrations ( Figure 2E ) .",
"The modification doesn’t lead to over-fitting on small training subsets and in fact improves the model’s ability to generalize to the whole embryo from an anteroposteriorly restricted training subset ( Figure 2—figure supplements 2 and 4 ) .",
"In addition , regulator discovery now identifies all four TFs as important , with a more consistently informative role for Kr than in the simple linear model ( Figure 2H ) .",
"We next tested whether our model is predictive of experimental perturbations .",
"We considered experiments that test the role of eve 2 regulators by either knocking down the input TF ( Stanojevic et al . , 1991 ) , or by mutating binding sites for that TF in the eve 2 enhancer ( Arnosti et al . , 1996 ) .",
"To simulate these perturbations , we set the concentrations of Bcd or Hb to zero without further adjustment of the coefficients .",
"Strictly speaking , this models the direct effect of the perturbation and is akin to the removal of the relevant binding sites from the enhancer .",
"The results of these perturbations are shown in Figure",
"3 . Only the quadratic model correctly predicts the expression pattern in a Bcd null mutant ( Figure 3C ) .",
"In the linear model , Bcd is designated a repressor and so its mutant causes broad eve expression in the anterior of the embryo in contrast to the experimental result ( Figure 3C ) .",
"In the quadratic model the lack of either activator ( Bcd or Hb ) abolishes the expression of stripe 2 as expected ( Figure 3C , D ) .",
"In both the linear and quadratic models , the loss of the repressors Gt or Kr causes eve expression to extend towards the anterior and posterior of the embryo respectively , in line with their roles in defining the stripe borders ( Figure 3A , B ) .",
"10 . 7554/eLife . 00522 . 011Figure 3 . The quadratic model accurately predicts eve 2 expression under perturbation of input TFs . The effects of regulatory perturbations on stripe 2 expression are predicted by altering regulator concentrations but keeping all the model coefficients unchanged; for TF deletion or binding site mutants , this involves setting the relevant regulator’s concentrations to 0 .",
"Predictions are made for perturbations using the linear and quadratic models .",
"Comparisons to experiments provide robust , independent validations of model predictions .",
"Loss of ( A ) gt or ( B ) Kr causes eve expression to extend towards the anterior and posterior of the embryo respectively , in excellent agreement with experimental evidence .",
"( C ) For the bcd mutant , the linear model predicts expression at the anterior of the embryo , something that is not observed in experiments .",
"In contrast , the quadratic model does not suffer from this .",
"( D ) Perturbing hb leads to complete loss of eve stripe 2 for both models .",
"The better agreement between predictions and experimental evidence suggests that the quadratic is a more plausible model of eve 2 regulation . In situ images in panels 1 , 2 , and 6 are reproduced from Figure 4B–C and 6C , Small et al . ( 1992 ) , The EMBO Journal; Nature Publishing Group has granted permission to reproduce these images under the terms of the Creative Commons Attribution 3 . 0 Unported License ( CC BY 3 . 0 ) .",
"DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 011© 1991 , Cold Spring Harbor Laboratory Press , All Rights Reserved1991Cold Spring Harbor Laboratory PressThe in situ image in panel 3 is reprinted with permission from Figure 2D , Small et al . ( 1991 ) , Genes & Development .",
"© 1991 , American Association for the Advancement of Science , All Rights Reserved1991American Association for the Advancement of ScienceIn situ images in panels 4 and 5 are reprinted with permission from Figure 3A and 3C , Stanojevic et al . ( 1991 ) , Science .",
"© 1996 , The Company of Biologists , All Rights Reserved1996The Company of BiologistsThe in situ image in panel 7 is reproduced with permission from Figure 6B , Arnosti et al . ( 1996 ) , Development .",
"Both models predict the observed response to binding site mutations: the expansions of stripe 2 in the correct directions and extent along the length of the embryo .",
"The models demonstrate that the extent of posterior extension in the Kr mutant is restricted because of decreasing activator concentrations ( Figure 3B ) .",
"For the anterior extension in the Gt mutant , the restriction requires a repressor since activator concentrations remain high to the end of the embryo ( Figure 3A; Andrioli et al . , 2002 ) .",
"Bcd can provide this repression in both linear and quadratic models: however , only the quadratic can reconcile this with Bcd’s known activating function .",
"For the linear model to work with a Bcd activator , one would need a fifth regulator as an anterior repressor .",
"Indeed , multiple studies have searched for a repressor in this region , and multiple candidates have been identified though none have been conclusive ( Bellaïche et al . , 1996; Janody et al . , 2000; Andrioli et al . , 2002; Zhao et al . , 2002; Singh et al . , 2005 ) .",
"eve stripes 3 and 7 are regulated together by a single enhancer ( Small et al . , 1996; Clyde et al . , 2003; Struffi et al . , 2011 ) .",
"Such an arrangement requires appropriate TF concentrations for eve activation to be present in nuclei separated by some distance .",
"We tested whether our modeling framework can contend with the challenge of specifying two extra stripe borders using the available regulator concentrations .",
"As with eve 2 , we can further compare the models by predicting the outcomes of regulatory perturbations of input TFs ( Figure 5 ) .",
"Here we consider perturbations of kni and hb , the best characterized regulators of eve 3+7 .",
"It is again important to distinguish between expression in a mutant background , which reveals both direct and indirect interactions , and corresponding binding site mutations within the eve 3+7 enhancer , which probe only direct interactions . 10 . 7554/eLife . 00522 . 021Figure 5 . Linear and quadratic logistic models accurately predict eve 3+7 expression under perturbation of Kni and Hb . The effects of regulatory perturbations on eve 3+7 expression are predicted as described in the main text .",
"( A ) Perturbation of kni and its binding sites cause full reporter expression between the stripes .",
"The linear model predicts this observed extension , but the quadratic does not .",
"( B ) Perturbation of hb causes stripe 3 to expand and move anteriorly , and stripe 7 to expand slightly .",
"Binding site mutations show similar effects , though perhaps without the anterior shift of stripe",
"3 . The linear model provides good prediction of both stripes .",
"The quadratic produces a good stripe 3 prediction , including its anterior shift , but fails to predict any expression in stripe 7 .",
"( C–F )",
"Given the initial preference for the quadratic , we considered minor and biological plausible assumptions that allow the model to make accurate predictions .",
"For the kni mutants , these are ( C ) the minor adjustment of the intercept and ( D ) inclusion of indirect effects of kni on hb by increasing Hb by 50% of wild-type kni .",
"For the hb mutants , these are ( E ) the inclusion of residual maternal Hb in the posterior and ( F ) simulating the effects of residual Hb binding sites . In situ images in panels 1 and 3 are reprinted with permission from Figure 4B–C , Small et al . ( 1996 ) , Developmental Biology ( © copyright Elsevier , 1996 , All Rights Reserved ) .",
"In situ images in panels 2 and 4 are reproduced with permission from Figures 4H and 6D , Struffi et al . ( 2011 ) , Development ( © copyright The Company of Biologists , 2011 , All Rights Reserved ) .",
"DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 02110 . 7554/eLife . 00522 . 022Figure 5—figure supplement 1 . Indirect effects and the quadratic model can explain the expansion and retreat of expression observed in the eve 3+7 reporter in a kni mutant . Since Kni represses Hb , the loss of kni may lead to an increase of Hb towards steady state .",
"To approximate this , Hb was added in increasing proportion , from 20% to 150% , of wild-type kni expression .",
"The resulting eve 3+7 quadratic prediction is shown in a kni mutant . DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 02210 . 7554/eLife . 00522 . 023Figure 5—figure supplement 2 . An adjustment to the intercept in the quadratic logistic model for eve 3+7 results in a slight expansion of expression between the stripes . This prediction shows the effect of increasing the intercept by 4 . 5 .",
"This change in the intercept corresponds to potential differences in expression between the endogenous gene and the transgenic reporter . DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 02310 . 7554/eLife . 00522 . 024Figure 5—figure supplement 3 . Hb binding site mutants may dampen or remove Hb repression at higher concentrations . Dampening the effect of Hb at higher concentrations , for example by 12 ( 1− e−2Hb ) as shown in the center plot , changes the regulatory impact of Hb in the embryo ( left and right plots ) .",
"The left plot shows Hb activating at low concentrations and repressing at high concentrations; the right plot shows an attenuation of this effect leading instead to a weakening in activation .",
"The corresponding predictions are shown below . DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 024 We also tested the linear and quadratic models for both eve 2 and eve 3+7 on the two previous time points in the Virtual Embryo , which were not used for training ( Figure 6 ) .",
"The results for eve 2 show a wider stripe forming before it narrows to the boundaries of stripe",
"2 . This mirrors published results for an eve 2 reporter as well as the endogenous expression of eve ( Small et al . , 1992; Andrioli et al . , 2002 ) .",
"The predictions for eve 3+7 are also consistent in terms of the positions of the stripes , although they have stripe 3 appearing earlier than stripe 7 .",
"This timing difference is not obvious in the endogenous eve expression recorded in the Virtual Embryo , although stripe 7 does appear relatively weak in some transgenic reporters ( Small et al . , 1996 ) .",
"At the earlier time points the difference in sharpness of the stripe borders between the quadratic and linear model is more pronounced suggesting that the interpretation of positional information by the quadratic model is more stable and precise . 10 . 7554/eLife . 00522 . 025Figure 6 . Models predict eve 2 and 3+7 expression at earlier time points . Model predictions for earlier time points in the Virtual Embryo are shown for the ( A ) eve 2 and ( B ) eve 3+7 linear and quadratic models .",
"The time points are labeled from the start of the dataset; the third time point is the one used throughout the main text .",
"For eve 2 , the linear and quadratic models show a wider stripe at the second time point and a well-defined stripe at time point",
"3 . This matches the in situ images below from Andrioli et al . ( 2002 ) which show a transgenic reporter at early and mid cycle .",
"The predictions for eve 3+7 are consistent in terms of the positions of the stripes , with stripe 3 appearing earlier than stripe 7 .",
"At the earlier time points the difference in sharpness of the stripe borders between the quadratic and linear model is more pronounced suggesting that the interpretation of positional information by the quadratic model is more stable and precise . The in situ image for eve 3+7 is reprinted with permission from Figure 2C , Small et al . ( 1996 ) , Developmental Biology ( © copyright Elsevier , 1996 , All Rights Reserved ) .",
"In situ images for eve 2 are reproduced with permission from Figure 4A , B , Andrioli et al . ( 2006 ) , Development ( © copyright The Company of Biologists , 2006 , All Rights Reserved ) .",
"DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 025 As a final test of our models , we compare our predictions to the misexpression work of Clyde and colleagues ( Figure 7 , Figure 7—figure supplement 1; Clyde et al . , 2003 ) .",
"In this study , the authors constructed transgenes with a snail promoter that misexpressed hb or kni in the ventral region of the embryo and recorded the effects of expressing one or two copies of these transgenes .",
"The experiments confirmed that Hb and Kni repress eve 3+7 and 4+6 at different concentrations as suggested by Fujioka et al . ( 1999 ) .",
"However , the experiments also revealed some curious observations that are not easily explained .",
"First , stripes 3 and 7 respond differently to the same additional concentrations of Hb despite being regulated by the same enhancer .",
"Secondly , and most intriguingly , the results show substantial bending of stripes: in the presence of one copy of the hb transgene , stripe 3 extends towards the posterior of the embryo , and with two copies , stripe 7 bends towards the anterior .",
"These behaviors cannot be explained readily by simple , qualitative inspection of the embryos and they were not explored in the original study . 10 . 7554/eLife . 00522 . 026Figure 7 . Quadratic models accurately predict fine-scale features of expression patterns due to input misexpression . The study by Clyde et al . ( 2003 ) misexpressed hb and kni along the ventral surface of the embryo using transgenes driven by a snail promoter and recorded the effects of one or two copies of these transgenes on eve expression .",
"We replicated these experiments using quadratic models for eve 2 and eve 3+7 ( trained on stripe 3 ) , by adding Hb and kni in proportion to the distribution of snail in the Virtual Embryo dataset .",
"As described in the main text , we also added an indirect effect from Hb activating Kr .",
"( A ) With kni misexpression , the model accurately predicts the thinning ( x1 transgene ) , then cutting of stripe 3 ( x2 ) .",
"( B ) With Hb misexpression , the model successfully predicts the bulging , then cutting and bending of stripe 3 ( x2 ) , and the bulging of stripe 7 ( x2 ) .",
"Stripe 2 remains unaffected in both perturbations , in agreement with the experimental results .",
"The accuracy of the predictions indicates that the quadratic model for eve 3+7 can explain the experimental results very well .",
"In contrast the linear models are unable to predict these results . In situ images are reproduced from Figures 1F–H and 1K–M , Clyde et al . ( 2003 ) , published in Nature; Nature Publishing Group has granted permission to reproduce these images under the terms of the Creative Commons Attribution 3 . 0 Unported License ( CC BY 3 . 0 ) .",
"DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 02610 . 7554/eLife . 00522 . 027Figure 7—figure supplement 1 . Supplementary misexpression predictions of the eve 2 and eve 3+7 linear ( A , B ) and quadratic ( C–E ) models .",
"( A , C and E ) have no indirect effects , whereas ( B and D ) include a hypothetical indirect effect mediated by Hb activating Kr .",
"This is modeled by adding Kr in proportion ( 50% ) to the increase of Hb .",
"The eve 3+7 model in ( A–D ) was trained on stripes 3 and 7 , whereas in ( E ) it was trained on stripe 3 only .",
"The predictions are shown for one ( x1 ) and two ( x2 ) copies of the hb and kni transgenes as described in the main text . DOI:http://dx . doi . org/10 . 7554/eLife . 00522 . 027 To model this experiment , we added Hb and kni to the Virtual Embryo in proportion to the measured distribution of snail at different concentrations ranging from 0 . 1 to 0 . 4 ( ∼10% to ∼40% of maximal expression ) .",
"We simulated the responses of eve 2 and eve 3+7 using both the linear and quadratic models above and a quadratic model trained on stripe",
"3 . Figure 7 and Figure 7—figure supplement 1 display the predictions of these models at 0 . 2 and 0 . 4 of Hb and kni , simulating the effects of one or two copies of the transgene respectively .",
"Figure 7 includes a putative indirect effect on eve 2 via Kr ( see below ) .",
"The quadratic models predict the fine-scale effects of this misexpression experiment with remarkable accuracy whereas the linear models do not ( compare Figure 7 to Figure 7—figure supplement 1A , B ) .",
"Specifically , the quadratic model trained on stripe 3 successfully reproduces the bending and bulging of both stripes 3 and 7 under hb misexpression ( Figure 7B ) , as well as the repression seen with kni misexpression ( Figure 7A ) .",
"Our models for eve 2 predict that since Hb is an activator , increasing the concentration of Hb should lead to increased activation of eve 2 and a resultant broadening of the stripe ( Figure 7—figure supplement 1A , C , E ) ; but this is not in fact observed in the misexpression study .",
"Indirect effects of Hb on another factor , such as Kr , can resolve this discrepancy: adding Kr in proportion ( 50% ) to the increase of Hb does indeed prevent stripe 2 from expanding , but only in the quadratic model and not in the linear ( Figure 7 and Figure 7—figure supplement 1B , D ) .",
"Given all the evidence provided here , on balance we conclude that the quadratic models are the most likely for both eve 2 and eve 3+7 ."
],
[
"Our goal was to understand the regulatory system underlying spatiotemporal patterning in the early Drosophila embryo by fitting regulatory input functions to the output of individual enhancers .",
"Our models are accurate , predictive and simple to apply and interpret .",
"We showed that simple functional forms relating TF concentrations to eve expression outputs are highly predictive of wild-type and mutant expression patterns .",
"In doing so , we have demonstrated that precise positional information—in other words , information interpreted by individual nuclei to produce an expression pattern—is available in the early embryo .",
"We determined whether TFs are most informative when serving as activators or repressors of each enhancer , and we also explored whether a dual-regulatory role for some TFs improved expression predictions .",
"Here we discuss our work in relation to other models of regulatory function , the insights our models provide into transcriptional regulation and positional information in the embryo , and the experimentally testable hypotheses proposed by our models .",
"The regulation underlying anteroposterior patterning of the Drosophila blastoderm has long been a favorite system for modeling work; for recent reviews , see for example Jaeger et al . ( 2009 ) and Papatsenko ( 2009 ) .",
"Some models have been successful in reproducing the gap gene patterns ( Jaeger et al . , 2004 , 2007; Bieler et al . , 2011; Papatsenko and Levine , 2011 ) , but none have succeeded in accurately predicting precise stripes of even skipped across the whole anteroposterior axis of the embryo ( Levine , 2008 ) .",
"In general , previous models have focused on utilizing information contained in the cis-regulatory sequence; for example predicting expression and evaluating potential TFs of a 1 . 7kb region of regulatory DNA upstream of eve ( Janssens et al . , 2006 ) , fitting models to fusions of eve enhancers and predicting expression from different regulatory DNA ( Kim et al . , 2013 ) , testing models of TF binding and synergy by predicting expression across many enhancers ( Segal et al . , 2008; He et al . , 2010 ) and identifying enhancer sequences within the genome based on the fit between predicted and observed expression patterns ( Kazemian et al . , 2010 ) .",
"Our choices in modeling the regulatory function of enhancers differ from these previous studies in a number of important respects .",
"First , our models are highly accurate in fitting the eve expression pattern in the entire embryo .",
"This is in part because we chose to model the regulatory function of each enhancer separately , rather than fitting a single model that applies across many enhancers simultaneously .",
"By defining parameters that are specific for each enhancer , we are able to assign the regulatory roles for TFs in a context-specific manner .",
"Second , our models also perform well because , unlike previous studies , we do not impose any biological mechanisms on our models ( e . g . , a ‘thermodynamic score’ for protein–DNA interactions ) .",
"Instead we worked the other way round: we tested models that fit data as accurately as possible and then inferred the underlying mechanisms .",
"This simple framework nonetheless allows us to propose experimentally testable hypotheses .",
"Third , our modeling framework is quick to apply .",
"This allowed us to search comprehensively for informative regulators , a property that is particularly valuable for studying poorly characterized enhancers .",
"Since the models are accurate and predictive , they may reflect the underlying molecular mechanism for transcriptional regulation .",
"Further , the models are relatively easy to interpret , so we can infer what they mean in terms of biological mechanism .",
"Here we highlight three features .",
"Our models predict which input TFs are relevant for a given enhancer , and whether they act as activators or repressors .",
"In the case of eve 2 and eve 3+7 , we showed that many of these predictions are confirmed by independent experiments already in the literature .",
"These studies involve either perturbing a candidate regulator by mutation , over-expression or misexpression , or mutagenizing binding sites for a candidate regulator in an enhancer sequence , and then measuring the expression of eve .",
"To confirm our predictions , we made qualitative comparisons between published data ( in the form of a single representative image ) and our model predictions .",
"Having validated our modeling framework on these well-characterized enhancers , we can now broadly apply this framework to discover regulators for less well-characterized enhancers in this system .",
"While many enhancers in this network have been mapped by computational studies and functional genomics ( Berman et al . , 2002; Schroeder et al . , 2004; Kazemian et al . , 2010; Négre et al . , 2011; Schroeder et al . , 2011 ) , our knowledge of most of their regulatory input functions remains incomplete .",
"Our modeling framework complements existing functional genomic and bioinformatics approaches: combined they will allow a comprehensive description of the relevant inputs of each of these enhancers , and how those inputs work together to produce an output expression pattern .",
"Our models also point to a role for concentration-dependent effects of Hb and Bcd on their targets .",
"We hypothesize that this is due to concentration-dependent differences in protein-protein interactions , perhaps mediated by the arrangement of TF binding sites in an enhancer , as has been proposed for Hb ( Papatsenko and Levine , 2008 ) .",
"To test whether binding site arrangements are important , the binding sites for Bcd and Hb can be rearranged within the eve 2 and eve 3+7 enhancers , and the output of these mutated enhancers measured .",
"To test which parts of the TFs are involved in mediating protein-protein interactions , the TFs themselves can be mutated , and protein–protein interactions can be assayed by in vitro binding studies .",
"Finally , to test the concentration-dependent effects directly , the concentration of Hb and Bcd can be manipulated in vivo by over-expression , knock-down and misexpression .",
"Our modeling framework is especially useful in this last case , as predictions with and without concentration-dependent effects can be compared .",
"We propose that misexpression studies are likely to be particularly informative , based on the fine-scale differences such as stripe bending and bulging that we were able to predict .",
"Instead of making qualitative comparisons to experimental data , it would be ideal to test our models quantitatively at cellular resolution .",
"This is possible if we create additional Virtual Embryo data where perturbations , both to input TFs and enhancer sequences , are measured .",
"For knock-down , over-expression or misexpression of TFs , we will need to create a new Virtual Embryo for each perturbation .",
"This will capture all of the direct and indirect consequences of perturbing the TF .",
"We can assess the consequences of mutating enhancer sequences by integrating transgenic reporters into any given Virtual Embryo dataset , as in Wunderlich et al . , 2012 .",
"Creating these new datasets is not a trivial undertaking technically but it would provide the framework for us to directly compare the output of our model predictions to experimental data at cellular resolution to detect fine-scale differences , and without making assumptions about indirect effects .",
"For example , this would allow us to test our proposed role for tll in repressing the posterior border of eve stripe 7 , where classic experiments have been inconclusive and to validate future predictions for other enhancers in the segmentation network .",
"We fully anticipate that analyzing this type of data will lead to further refinements of our models .",
"Clearly , our model depends on the quality of the data in the Virtual Embryo , which was derived from many in situ hybridization images of the Drosophila blastoderm ( Keränen et al . , 2006; Luengo Hendriks et al . , 2006; Fowlkes et al . , 2008 ) .",
"To predict spatiotemporal expression patterns , it’s important that the measurements are quantitative and at the resolution of individual cells .",
"One advantage of the blastoderm is that the relevant nuclei are near the surface of the embryo , making it easier to segment the overall fluorescence signal and assign it to individual nuclei .",
"However , microscopy and other techniques such as single-cell transcriptomics are continually improving ( Kalisky et al . , 2011 ) ; we anticipate that many comparable datasets will become available over time , both for other developmental time points in Drosophila , and in other model systems .",
"Our study demonstrates how theoretical models can be applied to such data in order to make new biological discoveries ."
],
[
"Release 2 . 0 of the Virtual Embryo dataset was downloaded from the Berkeley Drosophila Transcription Network Project website ( http://bdtnp . lbl . gov/Fly-Net/ ) ( Fowlkes et al . , 2008 ) .",
"The release contains composited mRNA expression measurements for 95 genes in 6078 nuclei at six time points ( or ‘cohorts’ ) .",
"Also provided are protein expression data for four gene products ( Bcd , Hb , Kr , and Gt ) for some of the time points .",
"Data for the current study were extracted from a ‘comma-separated values’ ( CSV ) format Virtual Embryo file ( D_mel_wt__alas_r2 . vpc ) : each row corresponds to a nucleus in the embryo , with columns containing measurements including three-dimensional coordinates , average expression level for a given gene , time point for measurement etc .",
"Expression measurements are provided as relative values for each nucleus , ranging from ‘0’ for minimum expression across all six time points to a little over ‘1’ for maximum expression ( e . g . , , the maximum for eve is 1 . 11 and for Hb it is 1 . 05 ) .",
"The variability in the maximum is a result of the method used to determine the relative variation between nuclei across different time points in the Virtual Embryo ( Fowlkes et al . , 2008 ) .",
"The coordinates of the Virtual Embryo are along the anteroposterior",
"( x ) , left-right",
"( y ) and dorsoventral",
"( z ) axes .",
"The difference between the minimum and maximum is 404 μm for the x-coordinate , 154 μm for the y-coordinate and 155 μm for the z-coordinate .",
"Training was performed using expression measurements at the third time point ( Cohort 3 ) .",
"6 , 078 nuclei were classed as ON ( 2444 ) or OFF ( 3634 ) depending on whether eve’s expression is above or below the threshold of 0 . 2 ( approximately 20% of maximum ) .",
"Nuclei were grouped into the seven eve stripes making use of the neighboring nuclei information provided in the Virtual Embryo ( stripe 2 = 348 nuclei , stripe 3 = 342 nuclei , stripe 7 = 383 nuclei ) .",
"mRNA expression measurements for 34 genes were included in the training data ( brk , bun , cad , CG10924 , CG17786 , CG4702 , cnc , croc , Cyp310a1 , D , Dfd , Doc2 , emc , fj , fkh , hkb , kni , knrl , oc , path , rho , sala , slp1 , slp2 , sna , sob , srp , term , tll , Traf1 , trn , tsh , twi , zen ) .",
"For four TFs ( Bcd , Hb , Kr , Gt ) , we used the protein expression measurements instead .",
"Logistic regression was used to model eve expression by linking the regulator concentrations as continuous input variables , to eve’s expression state as the binary output .",
"For a nucleus i , the predictor , ηi , is calculated as a linear combination of concentrations:ηi=β0+β1x1i+ . . . +βkxkiwhere xki is the expression measurement of the kth gene for the ith nucleus with the β to be estimated .",
"For the quadratic models , a single quadratic term was added for the regulator , q , in question: The predictor is linked to the estimated probability pi of eve being ON in the ith nucleus: Models for eve 2 were trained using the 348 nuclei defined as ON in stripe 2 , as well as the nuclei defined as OFF excluding the nuclei of other stripes and their immediate neighbors ( 2588 nuclei ) .",
"Similarly , models for eve 3+7 were trained using 725 ON nuclei in stripes 3 and 7 , and 2756 OFF nuclei .",
"The models were fitted using the R function glm from the stats package , which uses Iteratively Re-weighted Least Squares .",
"For our best fitting models , glm issued a fitting and evaluation warning message .",
"This was because most of the logistic models that classify the eve stripes successfully have some fitted probabilities very near 0 or 1 .",
"( The nuclei on the borders of the stripes have intermediate values ) .",
"Although this can suggest problems in certain situations , here , in agreement with Ripley ( Ripley , 2008 ) it is viewed as a desirable outcome of classification .",
"The trained model was then used to predict eve expression in all 6078 nuclei across the entire embryo , using the concentrations of the relevant regulators .",
"The consistency of the model across different training subsets was tested in several ways .",
"( i ) Each model was trained on a subset of the training dataset and then used to predict eve expression for the whole embryo .",
"Subsets used included: nuclei within 20 μm either side of the lateral midline; nuclei within the relevant stripe ( s ) and only their immediate neighbors; and a cross-validation test , which was the average of 100 predictions each trained on a random subset of 50 nuclei .",
"For eve 3+7 , two extra subsets excluded the ON nuclei from either stripe 3 or 7 .",
"Less consistent models produce poor predictions after training on some subsets .",
"( ii ) Each model was trained using all 38 regulators and then used to predict eve expression for the whole embryo .",
"Models suffering from localized over-fitting show fragmented eve expression .",
"( iii ) Models were trained for each of the stripes in turn , using the regulators of the best-fitting models ( such as Bcd , Hb , Gt , and Kr for eve 2 ) .",
"This showed that the given regulators are not able to fit any arbitrary stripe well .",
"The effects of regulatory perturbations were simulated by adjusting the concentrations of the relevant regulator without changing any model parameters ( i . e . , without retraining ) , and then predicting eve expression across the whole embryo .",
"Binding site mutations and null mutants were simulated by setting the regulator concentration to ‘0’ in all nuclei .",
"Where indicated , indirect effects were simulated by adjusting the expression level of downstream regulators and again , predicting eve expression without any model adjustments .",
"Other types of regulatory perturbations , such as the misexpression studies , were performed similarly by adjusting regulator concentrations as described in the main text .",
"Model predictions of wild-type and mutant eve expression are displayed graphically for each nucleus in the embryo .",
"The Virtual Embryo contains three-dimensional coordinates for each nucleus , making it possible to show the predictions in their spatial context .",
"In most figures , embryos are shown from two perspectives: lateral and three-dimensional .",
"In the lateral perspective , each nucleus is plotted using the ( x , z ) coordinate , ignoring the y coordinate .",
"The x- and z-axes are aligned to the anteroposterior ( left to right ) and dorsoventral ( top to bottom ) axes respectively , so showing a view from the left side of the embryo .",
"Since predictions for the left and right sides are similar , all nuclei ( i . e . , both left and right ) are plotted in one composite view from the left side of the embryo .",
"The three-dimensional perspective is plotted using the cloud function from the lattice package in R , similarly from an anterior perspective .",
"Nuclei are colored according to the model’s prediction , from p=0 ( light ) to p=1 ( dark ) .",
"The color scale for predictions within stripes is grey-scale and predictions outside of stripes are shown on a red scale , with peach for values below 0 . 15 .",
"To accompany the visual display of wild-type predictions , we also calculated percentage accuracies to aid comparison between alternative models .",
"These values provide good indications of model performance in predicting the stripe boundaries .",
"For each model , we calculated the proportion of nuclei predicted as being ON ( p>0 . 5 ) within the stripe ( s ) under consideration ( i . e . , true positives ) , in nuclei immediately adjacent to the stripe nuclei , and two nuclei away ( i . e . , false positives ) .",
"The identities of neighboring nuclei are provided by the Virtual Embryo dataset .",
"For eve 2 , we trained all possible linear models using four out of 38 regulators in the dataset ( total 73 , 815 models ) , using the log likelihood of each fitted model as its score .",
"A similar approach was used for exploring quadratic models , except that any model containing Bcd and/or Hb also included the corresponding quadratic term ( s ) .",
"The results are summarized as heat maps as shown in Figure 3 .",
"Analysis was performed with R version 2 . 15 . 1 ( R Core Team , 2012 ) , using colors from the ColorBrewer palettes in the RColorBrewer package .",
"Plots made use of the lattice , ggplot2 and RBGL packages .",
"The graph package was used to select neighboring nuclei ."
]
] | [
"Transcriptional control ensures genes are expressed in the right amounts at the correct times and locations .",
"Understanding quantitatively how regulatory systems convert input signals to appropriate outputs remains a challenge .",
"For the first time , we successfully model even skipped ( eve ) stripes 2 and 3+7 across the entire fly embryo at cellular resolution .",
"A straightforward statistical relationship explains how transcription factor ( TF ) concentrations define eve’s complex spatial expression , without the need for pairwise interactions or cross-regulatory dynamics .",
"Simulating thousands of TF combinations , we recover known regulators and suggest new candidates .",
"Finally , we accurately predict the intricate effects of perturbations including TF mutations and misexpression .",
"Our approach imposes minimal assumptions about regulatory function; instead we infer underlying mechanisms from models that best fit the data , like the lack of TF-specific thresholds and the positional value of homotypic interactions .",
"Our study provides a general and quantitative method for elucidating the regulation of diverse biological systems ."
] | [
"The transcription of genes into messenger RNA ( mRNA ) molecules is one of the most important processes in biology , but our present understanding of this process is largely qualitative .",
"Molecules such as transcription factors and regions of DNA other than the region that codes for the mRNA are known to interact with each other to influence the onset of transcription , and also the rate at which it occurs .",
"However , given the cellular concentrations of transcription factors in a developing organism , it is not known if it is possible to accurately predict their effects on transcription .",
"Being able to make such predictions would greatly improve our understanding of how transcription and the development of an organism are controlled .",
"Ilsley et al . have tackled this problem by analysing a large volume of data called the Virtual Embryo dataset: produced by the Berkeley Drosophila Transcription Network Project , this dataset includes the results of mRNA expression measurements on 95 different genes at six different times during the early development of Drosophila melanogaster , a species of fruit fly .",
"In particular , Ilsley et al . focussed on the expression at one point in time of the even skipped ( eve ) gene , a widely studied gene that is important for embryo development in these fruit flies .",
"The eve gene is one of the genes responsible for dividing the fly into segments which form part of its body plan .",
"Without making any assumptions about the biological mechanisms that might be involved , Ilsley et al . built a statistical model that was able to predict the pattern of gene expression for a fruit fly , given the concentrations of the relevant transcription factors in the various cells within the embryo as input .",
"The model was also able to predict the patterns of gene expression observed in other experiments involving mutations and the misexpression of fruit fly genes .",
"Moreover , Ilsley et al . have made various predictions involving the genes Bicoid and Hunchback that can be tested experimentally in future studies ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Intrinsic monitoring of learning success facilitates memory encoding via the activation of the SN/VTA-Hippocampal loop | elife-17441-v2 | [
[
"In the last decade , multiple studies have shown that external rewards can enhance human learning and memory through Long Term Potentiation ( LTP ) processes triggered by the co-activation of a dopamine-dependent loop formed by the ventral striatum ( VS ) , the substantia nigra/ventral tegmental area complex ( SN/VTA ) , and the hippocampus ( HP; hereafter referred to as the SN/VTA-HP loop; Lisman and Grace , 2005; Goto and Grace , 2005; Lisman et al . , 2011; Shohamy and Adcock , 2010 ) .",
"According to this model , the activation of this reward-memory loop starts when new information that needs to be stored in memory ‘arrives’ at the HP .",
"A signal is then sent to the SN/VTA through the VS , which is thought to integrate affective , motivational , and goal-directed information into the loop ( Lisman and Grace , 2005; Goto and Grace , 2005 ) .",
"SN/VTA neurons are then disinhibited , facilitating dopaminergic signalling into the HP ( Lodge and Grace , 2006; Shohamy and Adcock , 2010 ) , which in turn enhances memory formation .",
"In accord with this model , recent evidence suggests that both the anticipation of an explicit reward ( e . g . , the possibility to win money; Adcock et al . , 2006; Wittmann et al . , 2005; Wolosin et al . , 2012; Callan et al . , 2008 ) and also intrinsic motivational states ( e . g . , curiosity about future events; Gruber et al . , 2014 ) can modulate memory formation through the activation of the SN/VTA-HP loop .",
"Whereas these previous studies manipulated participants' expectations ( i . e . , money/curiosity ) in order to enhance memory , in everyday life we constantly engage in learning processes in which the possibility to obtain an external reward is mostly absent .",
"If neither extrinsic feedback regarding the learning success nor external reward is given , some internal system must still decide whether the output of the learning experience ( i . e . , new information ) is valid and should thus be stored into long-term memory .",
"One could predict that successful internally-guided learning ( i . e . , learning without explicit feedback ) might trigger intrinsic reward-related processes after positive monitoring ( related to a feeling of efficacy; White , 1959 ) , favouring the encoding of the learning episodes into the memory system .",
"Concordantly , psychological theories on self-regulated learning have proposed that internal monitoring and trial-by-trial evaluation of learning success may be crucial determinants of learning in complex environments ( Bjork et al . , 2013 ) .",
"Considering the importance of the SN/VTA-HP loop in the regulation of extrinsically motivated learning ( Adcock et al . , 2006; Wittmann et al . , 2005; Callan et al . , 2008; Wolosin et al . , 2012; Gruber et al . , 2014 ) , we hypothesized that the recruitment of this reward-memory loop should be instrumental in storing successful learning experiences also in the absence of explicit feedback , especially after positive intrinsic evaluation of the learning outcome .",
"Related to this , we recently developed a learning task ( Ripollés et al . , 2014; see also Mestres-Missé et al . , 2007 ) that mimicked our capacity to learn the meaning of new-words presented in verbal contexts , a process that usually occurs without external guidance and that is considered one of the most important sources of vocabulary learning during childhood years ( Nagy et al . , 1985 ) .",
"Indeed , there is an extensive body of evidence showing that children ( as young as eight years-old ) can learn new words from written contexts ( e . g . , by reading ) on their own , not only when explicitly instructed to do so , but also during incidental learning conditions ( Jenkins et al . , 1984; Konopak et al . , 1987; Kuhn and Stahl , 1998; Nagy et al . , 1985 , 1987; Werner and Kaplan , 1950 ) .",
"Moreover , as adult learners , we also constantly face the problem of learning the meaning of new-words in our own native or non-native languages , and in most cases , the repeated presentation of a new-word in different verbal contexts can allow to discover the meaning of the new-word ( Nation , 2001 ) .",
"Therefore , our word-learning task is ideally suited to test internally-guided learning as:",
"( i ) in our task participants are able to learn the meaning of artificially created new-words by using contextual information , without the need for explicit feedback or reward; and",
"( ii ) our paradigm mimics a learning process that occurs in real-world environments .",
"Importantly , we recently showed that , in our paradigm , successful meaning extraction enhanced fMRI-signals within the VS . Moreover , this activity was not related to novelty , attention or exertion of effort ( Ripollés et al . , 2014 ) .",
"While our previous results suggested that learning the meaning of a new word triggered reward-related signals within the VS , we never assessed the effect that these internally elicited signals had on the formation of longer-lasting memory traces .",
"Thus , an important question arises: can positive monitoring of learning success—in the absence of explicit feedback or external reward—mediate the entrance of new information into long-term memory via the modulation of the SN/VTA-HP loop ?",
"In order to address this question , we first re-analyzed the functional magnetic resonance imaging ( fMRI ) data from our previous work ( Exp .",
"1 ) using a region-of-interest ( ROI ) analysis that focused on all the areas of the SN/VTA-HP loop ( constrained to be reward-related by means of a meta-analysis on reward; NeuroSynth , Yarkoni et al . , 2011 ) .",
"In addition , we used a post-scan retrieval test to assess the possible memory benefits induced by the activation of the SN/VTA-HP loop during successful encoding .",
"We hypothesized that increased brain activity and functional connectivity within the areas of the loop , in the absence of any external reward , should be associated with enhanced memory formation ( i . e . , greater activity and connectivity during encoding for later remembered vs . later forgotten items ) .",
"Furthermore , considering that the effects of dopamine are stronger during the late stage of LTP ( Murayama et al . , 2014; Adcock et al . , 2006; Gruber et al . , 2014 ) , we carried out a second experiment using a modified version of our word-learning task ( Exp .",
"2 ) in which an additional surprise recognition test was carried out 24 hr after encoding .",
"Based on our previous findings suggesting a strong relation between language learning and reward processing ( Ripollés et al . , 2014 ) and also on studies showing that uncovering the solution to a problem is closely tied to an increase in subjective pleasantness ( Kizilirmak et al . , 2015; Bechara and Damasio , 2005 ) , we recorded subjective self-reported ratings of arousal and pleasantness during the encoding phase of our task , along with objective physiological measures ( electrodermal activity ) .",
"Indeed , evidence suggests that emotion-related signals—assisting cognitive processes such as learning and decision making—can be captured by electrodermal activity ( EDA; Bechara and Damasio , 2005 ) .",
"In particular , skin conductance responses ( SCRs ) have been linked to enhanced memory formation , motivational behavior ( Cahill et al . , 1998 ) , explicit reward-related processes ( Lole et al . , 2014; Mas-Herrero et al . , 2014 ) , and also to a modulation of the orbitofrontal cortex , the amygdala , and the striatum ( Critchley et al . , 2000 ) .",
"Finally , a third experiment ( Exp . 3 ) was carried out to replicate the behavioural effects of Exp . 2 over longer retention intervals ( one week ) .",
"We hypothesized that if internal monitoring of learning success would indeed trigger reward-related processes that ultimately enhance memory formation , new-words remembered after longer retention periods should be associated with increased pleasantness ratings and enhanced SCRs during the initial encoding phase ."
],
[
"To answer this question , 36 participants completed an fMRI version of our word-learning task ( see Figure 1A ) , in which the meaning of a new-word could be learned from the context provided by two sentences built with an increasing degree of contextual constraint ( Mestres-Missé et al . , 2010 ) .",
"Only half of the pairs of sentences disambiguated multiple meanings , allowing the encoding of a congruent meaning of the new-word during its second presentation ( M+ condition; see Material and methods ) .",
"For the other pairs , the new-word was not associated with a congruent meaning across the sentences , and could not be learned ( M- condition ) .",
"In addition , non-readable sentences ( NR ) were presented as a control .",
"During encoding , participants recognized the correct meaning of ( mean , std ) 60 ± 15% of M+ new-words and correctly indicated an absence of coherent meaning in 61 ± 22% of M- cases ( see Appendix 1 for more details ) .",
"Given our explicit a-priori hypothesis regarding the VS , HP , and SN/VTA , an ROI analysis was performed .",
"In order to avoid circularity , the ROIs were created using independent data and were restricted to those regions related to reward-related processes ( see Materials and methods ) . 10 . 7554/eLife . 17441 . 003Figure 1 . Schematic overview of trials and conditions in the word-learning paradigm .",
"( A ) Participants completed 10 short encoding blocks .",
"Four pairs of sentences of each condition ( M+ , M- ) and two pairs of non-readable sentences ( NR , only for the fMRI experiment ) were presented per block .",
"Note that first sentences for each condition are always presented prior to and in a different order than second sentences .",
"( B ) Each trial in the fMRI experiment started with a fixation cross lasting 500 ms followed by 6 German words of the sentence for 2 s and 1 s of dark screen .",
"Finally , the new-word was presented for 500 ms . Between trials , there was a variable inter-trial interval of 1 to 6 s ( Poisson distribution , Hinrichs et al . , 2000 ) .",
"( C ) Each trial in Exp . 2 started with a fixation cross lasting 1000 ms , continued with the 7 first Spanish words of the sentence presented for 2 s , and was followed by a 1 s duration dark screen .",
"The new-word was presented for 1000 ms . and was followed by 7 s of a small fixation point presented in the middle of the screen .",
"For first sentences , a new trial was presented after 3 s of dark screen .",
"For second sentences , after this period , a screen with the word Answer appeared and subjects had 3 s to produce a verbal answer .",
"Then , the SAM scales for pleasantness and arousal were sequentially presented ( the experiment did not continue until participants provided a rating ) .",
"Finally , a new second sentence trial started after 3 s of dark screen .",
"M+ ( meaning extraction possible during second presentation ) ; M- ( correct meaning extraction not possible during second presentation ) ; NR sentences ( non readable ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 003 In addition to reward , several structures , including the VS and the SN/VTA , are also activated by the novelty or salience of the stimuli ( Guitart-Masip et al . , 2010; Bunzeck and Düzel , 2006 ) , by attentional processes , by task-difficulty/exertion of effort ( Boehler et al . , 2011 ) , or by the response accuracy itself—regardless of successful meaning extraction ( i . e . , correct M- trials ) .",
"The design of our paradigm allows us to address these alternative explanations by including the incongruent condition ( M- , no learning ) and the order of presentation ( first or second sentence ) in our analyses ( note that participants were equally prompted to complete the task for both M+ and M- conditions ) .",
"Regarding the possible effort-related interpretation of the SN/VTA activation ( Boehler et al . , 2011 ) , previous studies using a similar paradigm have shown that incongruent conditions ( M- ) can be more difficult and effortful to resolve than congruent ones ( M+; e . g . Mestres-Missé et al . , 2014 ) .",
"Finally , the inclusion of the M- condition and the order of presentation also account for possible novelty and accuracy or subjective meaning confounds , as:",
"( i ) new-words are equally novel to participants for M+ and M- conditions;",
"( ii ) novelty effects should be more apparent during the first than during the second presentation of a particular new-word;",
"( iii ) generalized effects of accuracy or subjective meaning attribution should be apparent during correct rejection or false alarms of M- trials .",
"In order to rule out all aforementioned alternative explanations , a full-factorial ROI analysis was performed ( for a similar approach , see e . g . , Adcock et al . , 2006; Gruber et al . , 2014 ) .",
"Individual beta coefficients for each participant were extracted from the fMRI word-learning task and submitted to a 2 × 2 × 3 × 2 × 2 repeated measures analysis of variance ( ANOVA ) with the factors Hemisphere ( Left , Right ) , Order ( first sentence , second sentence ) , ROI ( VS , HP , SN/VTA ) , Condition ( M+ , M- ) , and Response ( Correct , Incorrect ) .",
"We then tested whether the effects revealed during encoding in the HP , VS , and SN/VTA really reflected signal changes due to successful meaning extraction or rather to unspecific effects of accuracy , second word presentation , or experimental condition .",
"Corroborating our hypothesis , we found a significant interaction of Order × Condition × Response [F ( 1 , 35 ) = 4 . 254 , p<0 . 047 , partial η2 = 0 . 108] .",
"Moreover , this triple interaction was further affected by region [F ( 2 , 70 ) = 4 . 258 , p<0 . 018 , partial η2 = 0 . 108; the effect is somewhat smaller for the HP ROIs , but still significant; see paired t-tests below] but not by hemisphere .",
"This interaction reflects greater activation for correct versus incorrect trials only for second sentences and for the M+ condition .",
"Importantly , there was no main effect of condition ( p>0 . 19 ) , further emphasizing that the reported effects were driven by the M+ correct trials .",
"Concordantly , paired t-test comparisons for all correct versus incorrect conditions showed significant differences in all ROIs only for M+ correct versus incorrect trials ( see Figure 2 ) during the second sentence presentation [left VS , t ( 35 ) = 6 . 29 , p<0 . 001 , d = 1 . 31; left HP , t ( 35 ) = 3 . 59 , p<0 . 002 , d = 0 . 59; left SN/VTA , t ( 35 ) = 4 . 50 , p<0 . 001 , d = 0 . 79; right VS , t ( 35 ) = 4 . 73 , p<0 . 001 , d = 0 . 89; right HP , t ( 35 ) = 3 . 28 , p<0 . 003 , d = 0 . 56; right SN/VTA , t ( 35 ) = 4 . 25 , p<0 . 001 , d = 0 . 76; two-tailed , at p<0 . 05 FDR-corrected] .",
"In addition , no significant Order × Condition × Response [F ( 1 , 35 ) = 0 . 354 , p>0 . 55 , partial η2 = 0 . 010] interaction was found when using a control ROI based at the primary visual cortex ( see Materials and methods ) , which further supports the specificity of our results .",
"Nevertheless , one could have expected that this loop should also have been activated during incorrect meaning attribution ( false alarms in the M- condition ) , as participants reported meaning acquisition .",
"However , the results of Exp . 3 ( in which subjects also provided confidence ratings , see Figure 7 , Material and methods and Appendix 1 ) , revealed that confidence ratings were significantly lower for false alarms in the M- incorrect condition than for correct responses in the M+ ( see below for a similar pattern of results for pleasantness ratings and electrodermal activity ) .",
"Together , these results strongly suggest that the observed effects were not driven by novelty , attention , task-difficulty , exertion of effort or accuracy , but rather by successful meaning extraction: the VS , HP , and SN/VTA were only engaged when participants correctly learned the meaning of the new-word . 10 . 7554/eLife . 17441 . 004Figure 2 . ROI analysis controlling for novelty and task difficulty . Blue areas depict independent ROIs used for beta-extraction overlaid on a canonical T1-weighted template ( VS , HP ) or the mean proton density normalized template from all subjects ( SN/VTA ) .",
"Bar graphs show mean beta coefficients within ROI for each condition of interest ( M+ correct first sentence , M+ incorrect first sentence , M- correct first sentence , M- incorrect first sentence , M+ correct second sentence , M+ incorrect second sentence , M- correct second sentence , M- incorrect second sentence; the NR condition is shown as a control ) with standard error of the mean ( dark grey for M+; light grey for M-; white for NR ) .",
"Paired t-test comparisons for all correct versus incorrect conditions revealed significant differences in all ROIs only for M+ correct versus incorrect trials during the second sentence presentation , when participants successfully learned the meaning of a new-word .",
"L , Left Hemisphere; M+ , congruent meaning extraction possible; M- , congruent meaning extraction impossible; NR , non-readable sentences; VS , Ventral Striatum; HP , Hippocampus; SN/VTA , Substantia Nigra/ Ventral Tegmental Area .",
"***p<0 . 001; **p<0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 004 In Exp . 1 , approximately thirty minutes after the word-learning experiment was completed , all M+ and M- new-words were tested again in a surprise memory test outside the scanner ( mean time between encoding and testing: 54 min; range: 37–72 min ) .",
"Participants still recognized the correct meaning of 68 ± 15% of M+ new-words learned during the test inside the scanner .",
"Participants correctly ascribed no meaning to 68 ± 23% of M- new-words that had been correctly rejected during the prior test .",
"For a second analysis testing the strength of memory formation , M+ correct trials were divided into those in which subjects learned the new-word inside the scanner and still remembered it in the test after the encoding session ( remembered condition ) and those in which the new-word was not correctly identified in the post-scan test ( forgotten condition; for a similar approach , see Gruber et al . , 2014 ) .",
"For the analysis of the M+ condition , three subjects with less than 3 forgotten trials had to be excluded .",
"Thus , data from thirty-three subjects were used ( average number of trials with standard deviation for each condition: 17 . 15 ± 6 . 41 remembered trials and 7 . 54 ± 3 . 01 forgotten trials ) .",
"Beta coefficients were again submitted to a 2 × 2 × 3 × 2 repeated measures ANOVA with the factors Hemisphere ( Left , Right ) , Order ( first sentence , second sentence ) , ROI ( VS , HP , SN/VTA ) , and Memory ( Remembered , Forgotten ) .",
"We found a significant interaction of Order × Memory [F ( 1 , 32 ) = 6 . 398 , p<0 . 017 , partial η2 = 0 . 167] which was not affected by region or hemisphere ( all ps>0 . 812 ) .",
"This interaction reflects greater activation in remembered versus forgotten trials only during the second sentence presentation .",
"Concordantly , subsequent paired t-test comparisons showed significant differences ( see Figure 3 ) in remembered versus forgotten trials in the left and right VS , HP , and SN/VTA during the second sentence presentation [left VS , t ( 32 ) = 2 . 78 , p<0 . 009 , d = 0 . 61; right VS , t ( 32 ) = 2 . 40 , p<0 . 022 , d = 0 . 47; left HP , t ( 32 ) = 2 . 60 , p<0 . 014 , d = 0 . 55; right HP , t ( 32 ) = 2 . 58 , p<0 . 015 , d = 0 . 56; left SN/VTA , t ( 32 ) = 2 . 79 , p<0 . 009 , d = 0 . 62; right SN/VTA , t ( 32 ) = 3 . 05 , p<0 . 005 , d = 0 . 65; two-tailed , FDR-corrected at p<0 . 05; see also Appendix 2 for further supporting analyses in a subset of participants with balanced number of trials in remembered and forgotten conditions] .",
"Importantly , the analysis of remembered vs . forgotten M- trials ( eight subjects with fewer than 3 forgotten trials had to be excluded , see Materials and methods; average number of trials with standard deviation for each condition: 17 . 14 ± 7 . 03 remembered trials and 7 . 32 ± 3 . 24 forgotten trials ) revealed no significant interaction of Order × Memory [F ( 1 , 27 ) = 0 . 004 , p>0 . 948 , partial η2 = 0 . 0001; not affected by region or hemisphere , all ps>0 . 301] further underlining the specificity of our results to successful meaning extraction ( M+ condition ) .",
"In addition and , as expected , no significant interaction of Order × Memory was found for the control ROI located at the primary visual cortex for M+ or M- trials [F ( 1 , 32 ) = 0 . 53 , p>0 . 46 , partial η2 = 0 . 017 and F ( 1 , 27 ) = 0 . 023 , p>0 . 88 , partial η2 = 0 . 001 , respectively] which further supports the specificity of our results to reward and memory related regions . 10 . 7554/eLife . 17441 . 005Figure 3 . ROI analysis of memory effects . Blue areas depict independent ROIs used for beta-extraction overlaid on a canonical T1-weighted template ( VS , HP ) or the mean proton density normalized template from all subjects ( SN/VTA ) .",
"Bar graphs show mean beta coefficients within ROI for M+ correct trials in which the learned new-word was still remembered during the test performed after the scanning session ( remembered ) and for M+ correct trials in which the new-word was not properly recognized in the post-scan test ( forgotten ) with standard error of the mean ( dark grey for M+ correct remembered; light grey for M+ correct forgotten; the M+ incorrect condition is shown in white as a control ) .",
"Paired t-tests showed greater fMRI activity within all ROIs for remembered than for forgotten words , only during the second sentence presentation .",
"M+ , congruent meaning extraction possible; VS , Ventral Striatum; HP , Hippocampus; SN/VTA , Substantia Nigra/ Ventral Tegmental Area .",
"***p<0 . 005; **p<0 . 01; *p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 005 Moreover , if the SN/VTA , HP , and VS form a functional network that promotes memory formation , connectivity among them should also predict word-learning .",
"Thus , we performed a functional connectivity analysis ( psychophysiological interaction , PPI; Friston et al . , 1997 ) which focused on enhanced inter-regional coupling during the congruent condition , when meaning is successfully extracted and remembered in the post-scan test ( M+ remembered second sentence ) versus when it is not learned ( M+ incorrect second sentence ) .",
"Subject-specific word-learning success was positively correlated ( p-values for peaks are provided using within-ROI family-wise error correction ) with the change in connectivity between the left VS ( source ) and the left HP [see Figure 4A: 168 voxels; maximum at x = −20 , y = −10 , z = −22; t ( 34 ) = 4 . 44; p<0 . 005 , d = 1 . 48] .",
"The correlation of the left VS with the SN/VTA , however , did not survive a correction for multiple comparisons [left SN/VTA , maximum at x = −2 , y = −16 , z = −14 , t ( 34 ) = 1 . 73 , p=0 . 342 , d = 0 . 58; right SN/VTA , maximum at x = 6 , y = −16 , z = −16 , t ( 34 ) = 2 . 58 , p=0 . 098 , d = 0 . 86] .",
"Word-learning was also positively correlated with the change in functional connectivity ( see Figure 4B ) between the left HP ( source ) and the left VS [100 voxels , maximum at x = V16 , y = 2 , z = −12 , t ( 34 ) = 4 . 34 , p<0 . 004 , d = 1 . 45] .",
"Again , the correlation with the SN/VTA did not survive a correction for multiple comparisons [left SN/VTA , maxima at x = −10 , y = −20 , z = −10 , t ( 34 ) = 2 . 19 , p=0 . 176 , d = 0 . 73; right SN/VTA maxima at x = 10 , y = −14 , z = −12 , t ( 34 ) = 2 . 47 , p=0 . 112 , d = 0 . 82] .",
"Finally , subject-specific word-learning was positively correlated with the change in functional connectivity ( see Figure 4C ) between the left SN/VTA ( source ) and the left HP [64 voxels , maximum at x = −30 , y = −10 , z = −20 , t ( 34 ) = 3 . 56 , p<0 . 037 , d = 1 . 19] and also with the left VS [10 voxels , maximum at x = −24 , y = −8 , z = −10 , t ( 34 ) = 3 . 33 , p<0 . 04 , d = 1 . 11] .",
"Moreover , there was an overlap at the left VS between the analysis in which the seed was the left HP and that in which the left SN/VTA was used as a source .",
"The same happened at the HP when using the left SN/VTA or the left VS as seeds ( see Figure 4D ) .",
"In contrast , analyses using seeds in the right SN/VTA , HP , or VS revealed no significant correlations with individual word-learning in any area of interest .",
"In addition , no significant correlations were found at the control ROI located at the primary visual cortex for any of the seeds .",
"Finally , we computed several additional PPI analyses in order to better characterize the nature of the reported effects .",
"First , we repeated the main PPI calculations but focusing on enhanced inter-regional coupling driven by main effects of condition ( all M+ versus all M- trials during the second sentence presentation ) .",
"As expected , no significant correlations with the number of remembered words were found .",
"In the same manner , no effects were found if the PPI analyses focused on M- remembered vs . M- incorrect trials , which further supports the specificity of the connectivity results to the M+ remembered condition .",
"Together , the PPI results from left-sided seeds demonstrate that subject-specific longer lasting word-learning success was related to increased coupling among the VS , HP , and SN/VTA in addition to the intraregional changes in activity within each of these areas in isolation . 10 . 7554/eLife . 17441 . 006Figure 4 . Changes in subject-specific connectivity due to individual word-learning success . Blue areas in A , B , C depict the seeds for connectivity-analysis overlaid on a canonical T1-weighted template ( VS , HP ) or the mean proton density normalized template from all subjects ( SN/VTA ) .",
"Significant correlations between word-learning ( learned new-words during the encoding session which were still correctly recognized in the post-scan test ) and the change in connectivity in: ( A ) the left VS ( source ) and the left HP; ( B ) the left HP ( source ) and the left VS; ( C ) the left SN/VTA ( source ) and the left HP and the left VS . Results are shown at a p<0 . 005 uncorrected threshold , with all main peaks within each cluster surviving a p<0 . 05 FWE corrected threshold within the ROI .",
"The scatter plots display the correlation between the number of learned-words and the mean change in functional connectivity for each particular cluster of interest .",
"Panel ( D ) shows , on the left , the overlap at the left VS ( blue ) between the correlations with the source at the left SN/VTA ( red ) and at the left HP ( light blue ) .",
"On the right , the overlap ( yellow ) between the correlations with the source at the left SN/VTA and at the left VS ( green ) is shown .",
"Neurological convention is used in both images with MNI coordinates at the bottom left of each slice .",
"L , Left Hemisphere; VS , Ventral Striatum; HP , Hippocampus; SN/VTA , Substantia Nigra/ Ventral Tegmental Area . DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 006 For the fMRI experiment , the average time interval between encoding of a new-word and its meaning and the presentation of that new-word during the post-scan test was approximately 54 min .",
"If the intrinsic reward modulated LTP , then an enhancement in memory formation should also be evident for longer retention intervals .",
"For this reason , 24 participants completed an additional behavioural experiment ( Exp . 2 ) .",
"The experimental design of the fMRI task ( Exp . 1 ) and Exp . 2 were identical except for the three following differences:",
"( i ) on-line verbal answers were recorded;",
"( ii ) in order to further confirm that our results were reward-related , participants provided subjective arousal and pleasantness ratings using the nine-point visual Self-Assessment Manikin scale ( SAM; Bradley and Lang 1994 ) ; and",
"( iii ) subjects completed the retrieval test ( which followed the same structure of the fMRI recognition test , see Material and methods; chance level was 33% as three choices were available: no consistent meaning , consistent meaning A , consistent meaning ( B ) after approximately 24 hr .",
"In addition , SCRs were recorded during the encoding phase as an additional objective measure of emotional processing .",
"In Exp . 2 , participants ascribed correct meaning to 62 ± 16% of new-words from the M+ condition during the encoding phase .",
"In 59 ± 16% of the M- trials , participants correctly indicated an absence of coherent meaning .",
"Thus , the pattern of results of experiments 1 and 2 ( and also 3 , see Appendix 1 ) was very similar , despite the slight differences in its design: a statistical comparison confirmed that encoding success for all experiments was not different ( p>0 . 91; see Appendix 1 ) .",
"We then directly tested ( as with the fMRI experiment ) whether the encoding of a new-word and its meaning modulated SCRs and whether this effect was actually driven by successful meaning extraction or rather by unspecific effects of accuracy , attention , novelty , cognitive load , second word presentation , or experimental condition .",
"Mean normalized SCRs time-courses for all conditions of interest ( see Figure 5A; one subject was excluded due to problems with data collection ) showed an increase in EDA only for M+ correct trials during the second sentence presentation , with a latency of approximately 3 s .",
"Indeed , we found a significant triple interaction of Order × Condition × Response [F ( 1 , 22 ) = 32 . 07 , p<0 . 001 , partial η2 = 0 . 593; calculated using the mean normalized SCR signal between seconds 4 and 8; the effect is still significant if the mean signal is calculated between seconds 1 to 8 , see Appendix 3] .",
"Concordantly , two-tailed paired t-test comparisons for all correct versus incorrect conditions showed significant differences only for M+ correct versus incorrect trials during the second sentence presentation [t ( 22 ) = 4 . 92 , p<0 . 001 , d = 0 . 944 , FDR-corrected; p>0 . 6 for all other comparisons; see Figure 5B] .",
"These results strongly suggest that the reported effects were not driven by novelty , attention , or cognitive load: SCRs were only enhanced when participants learned the meaning of a new-word . 10 . 7554/eLife . 17441 . 007Figure 5 . SCR signals and pleasantness/arousal scales during the encoding phase of the second experiment .",
"( A ) Time-course of normalized skin conductance response associated to M+ and M- correct and incorrect conditions during first ( left ) and second ( right ) sentence presentation .",
"Solid lines indicate the averaged SCR signal with the corresponding standard error of the mean ( red for M+ correct; green for M+ incorrect; light orange for M- correct; light blue for M- incorrect ) .",
"( B ) Mean ( averaged using the signal form seconds 4 to 8 ) normalized SCR for each condition of interest with standard error of the mean ( dark grey for M+; light grey for M- ) .",
"Paired t-test comparisons for all correct versus incorrect conditions showed significant differences only for M+ correct versus incorrect trials during second sentence presentation , when participants successfully learned the meaning of a new-word .",
"( C ) Mean pleasantness/arousal ratings ( scale between −4 and 4 ) for each condition of interest with standard error of the mean ( dark grey for M+; light grey for M- ) .",
"Paired t-test comparisons showed that ratings for pleasantness , but not arousal , were greater for M+ correct trials .",
"See Figure 7A for a replication of these effects .",
"M+ , congruent meaning extraction possible; M- , congruent meaning extraction impossible .",
"***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 007 Turning to the subjective pleasantness and arousal ratings collected during the encoding phase , we found a significant interaction of Condition × Response for pleasantness [F ( 1 , 23 ) = 15 . 38 , p<0 . 001 , partial η2 = 0 . 401] but not for arousal [F ( 1 , 23 ) = 0 . 71 , p>0 . 4 , partial η2 = 0 . 030 , see Figure 5C] .",
"Two-tailed paired t-test comparisons revealed that pleasantness ratings after correct versus incorrect trials were higher for the M+ [t ( 23 ) = 4 . 87 , p<0 . 001 , d = 0 . 867 , FDR-corrected] but not for the M- condition [t ( 23 ) = 0 . 58 , p>0 . 56 , d = 0 . 063] .",
"Thus , high pleasantness ratings were only associated with successful meaning extraction .",
"Importantly , the subjective ratings cannot be explained by participants’ compliance with explicit task demands: when asked about the purpose of the study , most subjects answered that it was to measure the effects of reading load on mood ( the explanation given to them during briefing , see Materials and methods ) .",
"None of them answered that the specific purpose of the study was to assess the effect of reward on learning ( or similar ) .",
"Nevertheless , 23 out of 24 subjects did state that successful meaning extraction was rewarding .",
"These results further show that during our task , successful meaning extraction was associated with increased feelings of reward .",
"Finally , participants carried out an unexpected recognition test 24 . 03 ± 3 . 88 hr after the encoding phase .",
"After this 24-hr retention delay , participants still recognized the correct meaning of 42 ± 15% of learned new-words during the encoding phase , significantly above chance level [t ( 23 ) = 2 . 80 , p<0 . 010 , d = 0 . 56; chance level was set at 33% , see Materials and methods] .",
"Note that , as expected , participants only recognized the correct meaning of 12 ± 10% of M+ new-words which were not learned during encoding [significantly below the recognition rate for learned M+ new-words , t ( 23 ) = 8 . 05 , p<0 . 001 , d = 2 . 23] .",
"Regarding the incongruent condition , participants correctly indicated that 55 ± 27% of M- new-words identified during the encoding phase had no meaning ascribed in the 24-hr test , significantly above chance level [t ( 23 ) = 3 . 88 , p<0 . 001 , d = 0 . 78] .",
"However , the 24-hr recognition rate for M- new-words which were not correctly identified during the encoding phase was 48 ± 27% , which is not significantly different from the 24-hr recognition rate for M- new-words correctly identified during encoding [t ( 23 ) = 1 . 76 , p>0 . 090 , d = 0 . 24] .",
"Importantly , in Exp . 3 ( which followed the same task structure as Exp . 2 , see Materials and methods ) participants still recognized 40 ± 15% of learned new-words after a one week retention period [seven days and 4 . 97 ± 15 hr; t ( 22 ) = 5 . 16 , p<0 . 001 , d = 1 . 03] , while the percentage of correctly rejected M- words was below chance level after this period of time ( 16 . 49 ± 17% ) .",
"We then tested whether enhanced EDA was related to long-term memory effects ( i . e . , enhanced SCRs for remembered vs . forgotten M+ new-words in the 24-hr test , only during the second sentence presentation ) .",
"For this analysis , two subjects with fewer than 3 remembered trials were excluded .",
"Mean normalized SCRs time-courses ( see Figure 6A ) showed enhanced EDA for remembered M+ correct trials during second sentence presentation ( M+ incorrect trials are also shown as a control ) .",
"We found a significant interaction of Order × Memory [F ( 1 , 20 ) = 11 . 57 , p<0 . 003 , partial η2 = 0 . 367 , calculated using the mean normalized SCR signal between seconds 4 and 8; the effect is still significant if the mean signal is calculated between seconds 1 to 8 , see Appendix 3 ) . Statistical comparisons for all correct versus incorrect conditions showed significant differences for M+ remembered versus forgotten trials during second [t ( 20 ) = 3 . 45 , p<0 . 003 , d = 0 . 78 , FDR-corrected] but not during first sentence presentation [t ( 20 ) = −0 . 12 , p>0 . 9 , d = −0 . 031; see Figure 6B] .",
"As expected , when computing the same analysis for M- trials ( five subjects with fewer than threeremembered or forgotten trials were excluded ) , no significant interaction of Order × Memory was found when using the mean normalized SCR signal between seconds 4 and 8 [F ( 1 , 17 ) = 0 . 01 , p>0 . 915 , partial η2 = 0 . 001] or between seconds 1 to 8 [the whole time-course , F ( 1 , 17 ) = 0 . 05 , p>0 . 818 , partial η2 = 0 . 003] .",
"These results indicate that EDA signals were not modulated by memory effects related to the M- condition .",
"Together , the EDA results indicate that remembered new-words elicited enhanced SCRs during the encoding phase as compared to forgotten ones . 10 . 7554/eLife . 17441 . 008Figure 6 . SCR signals and pleasantness/arousal scales in the second experiment for remembered new-words after a 24-hr retention delay .",
"( A ) Time-course of normalized skin conductance response associated to M+ new-words that were remembered or forgotten in the 24 hr test during the first ( left ) and second ( right ) sentence presentation .",
"The M+ incorrect conditions are shown as control .",
"Solid lines indicate the averaged SCR signal with the corresponding standard error of the mean ( red for M+ remembered; green for M+ forgotten; light blue for M+ incorrect ) .",
"( B ) Bar graphs depict mean normalized SCR ( averaged 4 to 8 s post-stimulus ) for each condition of interest with standard error of the mean ( dark grey for M+ remembered; light grey for M+ forgotten ) .",
"The M+ incorrect condition is shown in white as a control .",
"Paired t-test comparisons for remembered versus forgotten conditions showed significant differences only for M+ words during second sentence presentation .",
"( C ) Mean pleasantness/arousal ratings ( scale between −4 and 4 ) for M+ correct trials in which the new-word was still remembered during the test performed 24-hr after the encoding session ( remembered , dark grey ) and for those in which the new-word was forgotten ( forgotten , light grey ) .",
"The M+ incorrect condition is shown in white as a control .",
"Paired t-tests revealed significant differences between remembered and forgotten items for pleasantness , but not for arousal .",
"See Figure 7B for a replication of these effects .",
"M+ , congruent meaning extraction possible; M- , congruent meaning extraction impossible .",
"***p<0 . 005 . DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 008 Concordantly , subjective pleasantness ratings were also higher for remembered than for forgotten M+ new-words in the 24-hr recognition test [t ( 23 ) = 4 . 30 , p<0 . 001 , d = 0 . 454; see Figure 6C] , while no difference in arousal ratings was found [t ( 23 ) = 0 . 68 , p>0 . 25 , d = 0 . 055] .",
"These effects were replicated in Exp . 3 for a 1-week retention period ( see Appendix 1 and Figure 7 ) .",
"Once again , and , as expected , the same analysis for the M- condition ( three subjects were excluded from this analysis as they did not correctly identify in the 24-hr test any of the M- new-words correctly rejected during the encoding phase ) showed no difference in subjective pleasantness [t ( 20 ) = −1 . 44 , p>0 . 16 , d = −0 . 12] or arousal ratings [t ( 20 ) = −0 . 33 , p>0 . 73 , d = −0 . 039] for M- remembered vs . forgotten trials .",
"Thus , neither pleasantness nor arousal scales were modulated by memory effects related to the M- condition .",
"All in all , these results suggest that intrinsic reward , derived from an internal evaluation of learning success for the M+ condition only , had a modulatory effect on long-term memory . 10 . 7554/eLife . 17441 . 009Figure 7 . Exp .",
"3 . ( A ) Mean pleasantness/arousal/confidence ratings ( scale between −4 and 4 ) for each condition of interest with standard error of the mean ( dark grey for M+; light grey for M- ) .",
"M+ incorrect trials have been divided into incorrect-incongruent ( subjects say Incongruent instead of providing a meaning ) , incorrect-other ( subjects provide a wrong meaning ) and misses ( no answer ) .",
"M- incorrect trials have been divided into incorrect-other ( false alarms: subjects provide a meaning for an incongruent new-word ) and misses ( no answer ) .",
"( B ) Exp . 3: Mean pleasantness/arousal/confidence ratings ( scale between −4 and 4 ) for M+ correct trials in which the new-word was still remembered during the test performed 24-hr after the encoding session ( remembered , dark grey ) and for those in which the new-word was forgotten ( forgotten , light grey ) .",
"( C ) Exp . 3: Mean pleasantness/arousal/confidence ratings ( scale between −4 and 4 ) for M+ correct trials in which the new-word was still remembered during the test performed one week after the encoding session ( remembered , dark grey ) and for those in which the new-word was forgotten ( forgotten , light grey ) .",
"M+ , congruent meaning extraction possible; M- , congruent meaning extraction impossible . DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 009"
],
[
"The goal of the present study was to test whether an intrinsic signal , triggered by successful internally-guided learning , could enhance memory formation through the activation of a network formed by the VS , the HP , and the SN/VTA .",
"The fMRI-results demonstrate that successful meaning extraction for a new-word , in the absence of any external feedback , enhanced fMRI signals within the entire SN/VTA-VS-HP loop , with the observed activity not being caused by novelty , attention , task-difficulty , or exertion of effort , but rather by reward-related effects ( Figure 2 ) .",
"Moreover , in a second experiment , objective physiological measures ( EDA ) and subjective pleasantness ratings were only enhanced during successful meaning extraction ( Figure 5 ) , which further demonstrates that learning was associated with increased reward processing .",
"Regarding our main hypothesis—the effect of intrinsic reward on memory—new-words learned and later remembered elicited greater fMRI activity and functional connectivity within the VS , HP , and SN/VTA than those that were forgotten ( Figures 3 and 4 ) .",
"Finally , and most importantly , Exp . 2 also showed that remembered new-words after a 24-hr retention delay ( and after seven days in Exp . 3 ) were closely tied to enhanced SCRs and increased ratings of pleasantness during encoding , suggesting that intrinsic reward—driven by self-monitoring of correct performance—did have an effect on memory formation ( Figures 6 and 7 ) .",
"This study extends our previous results ( Ripollés et al . , 2014 ) by showing that successful learning , in the absence of external feedback or reward , engages a complex subcortical network—that includes not only reward , but also memory and dopamine related regions—which seems to modulate the entrance of new information into long-term memory .",
"All in all , this is the first study , to the best of our knowledge , to provide a neural mechanism—the SN/VTA-HP loop—which might subserve reward-related memory enhancements when the reward is intrinsic and triggered by an internal evaluation of correct performance .",
"Thus , learning , under certain circumstances , could be fuelling itself through intrinsic reward-related processes .",
"Remarkably , this engagement was only observed for the meaningful condition and not during incorrect meaning attribution in the incongruent condition ( M- incorrect ) .",
"Likewise , neither subjective pleasantness ratings nor EDA measures were affected by M- incorrect trials , suggesting that the incorrect behavioural responses are somehow corrected by the internal monitoring system and thus did not trigger learning ( see confidence ratings for Exp . 3 in Appendix 1 and Figure 7 ) .",
"Our results are in line with those of Kizilirmak et al . ( 2015 ) who showed that , behaviourally , successful generation of a solution to a problem in the absence of explicit feedback was related to both positive affect and enhanced memory formation .",
"Importantly , we extend those findings to meaningful learning of new-words and show that objective measures of emotional responses ( EDA ) are affected as well .",
"In this vein , our results are in accord with accumulating evidence suggesting that emotions can influence decision making and learning ( Dunsmoor et al . , 2015; Adcock et al . , 2006; Kizilirmak et al . , 2015; Bechara and Damasio , 2005 ) .",
"In addition , the intrinsic nature of the reward signals triggered by successful learning in the present experiments finds parallels in reinforcement learning models which show that learning systems incorporating intrinsic ( in addition to extrinsic ) reward signals surpass those based only on extrinsic ones ( Schultz , 2015; Barto et al . , 2004 ) .",
"Crucially , in our paradigm , internally generated signals were also fundamental: fMRI activity within the SN/VTA , HP and VS was only modulated by successful learning ( i . e . , when participants realized that they had extracted a correct meaning; also , confidence and pleasantness ratings were only high during correct trials , see Figure 7 ) .",
"Importantly , there is accruing evidence suggesting that the dopaminergic release at the HP is fundamental to promote stable memories ( Bethus et al . , 2010; Frey et al . , 1990; Hansen and Manahan-Vaughan , 2014; Huang and Kandel , 1995; McNamara et al . , 2014; Rossato et al . , 2009 ) .",
"Indeed , several studies have used stimuli that trigger the release of dopamine—especially reward—to induce memory enhancements in human adults ( i . e . , activating the SN/VTA by a rewarding stimulus such as money enhances the memory for events present during , after and even before the reward was delivered; Shohamy and Adcock , 2010; Krebs et al . , 2009; Wittman et al . , 2005 , 2008; Adcock et al . , 2006; Murty and Adcock , 2014; Wolosin et al . , 2012 ) .",
"Importantly , not only extrinsic signals but also intrinsic motivational states can enhance memory formation .",
"For example: in a recent study , both the VS and HP showed enhanced activity during the anticipation of trivia answers that were later remembered , only when participants were engaged in states of high curiosity ( Gruber et al . , 2014 ) .",
"Thus , both anticipation of explicit rewards and intrinsic motivational states can promote memory formation , and both engage the SN/VTA , HP and VS . Likewise , a very recent study demonstrated that after neurofeedback training , human adults displayed the ability to sustain the VTA activation without the need for an external reward ( MacInnes et al . , 2016 ) .",
"MacInnes et al . ( 2016 ) also showed that connectivity between the VTA and the HP was enhanced during and after neurofeedback training ( thus relating their results to the SN/VTA-Hippocampal loop ) , although they did not study the impact of this volitional sustained VTA activation on learning .",
"Another source of evidence linking dopamine and memory enhancements comes from pharmacological studies that tried to rise the levels of dopamine in the human brain .",
"In this regard , research has shown memory benefits after the intake of dexamphetamine , which blocks dopaminergic and adrenergic re-uptake , and levodopa ( a dopamine precursor; Breitenstein et al . , 2004; Whiting et al . , 2007 , 2008; Shellshear et al . , 2015; Knecht et al . , 2004; Chowdhury et al . , 2012; Bunzeck et al . , 2014 ) .",
"To summarize , there is converging evidence that explicit reward ( i . e . , money ) , intrinsic motivational states ( i . e . , curiosity ) and even pharmacological manipulation ( i . e . , levodopa intake ) can increase the levels of dopamine at the HP , potentially inducing memory benefits ( Shohamy and Adcock , 2010; Gruber et al . , 2014 ) .",
"We suggest that the activity within the SN/VTA-HP loop can also be modulated by an intrinsic reward—inherent to the process of learning itself and triggered by internal self-monitoring of correct performance—which ultimately promotes the storage of new information into long-term memory via dopaminergic modulation of the midbrain ( see Figure 8 ) .",
"Our results do support this working hypothesis , as a activity within all three areas of interest was larger for remembered than for forgotten learned new-words in the fMRI experiment .",
"In this vein , dopamine release at the HP can mediate both early ( minutes ) and late ( hours ) LTP , although stronger effects have been reported for the latter ( Otmakhova and Lisman , 1996; Bethus et al . , 2010; Smith et al . , 2005; for a review see Lisman et al . , 2011 ) .",
"The results from Exp . 2—in which pleasantness ratings and SCRs were higher for new-words that were remembered after a 24-hr retention delay—are crucial to support this notion , suggesting that the emotional impact associated to successful learning had an effect on long-term memory formation ( see also Exp . 3 for a replication of the behavioural effects after a 1-week retention period ) .",
"Note that the new-words to be learned were all novel and that the meanings associated with them were equally familiar .",
"Therefore , it is unlikely that subjects may have learned some new-words over others based on their specific characteristics .",
"In addition , the SN/VTA-HP loop was not activated by M- new-words , suggesting that in our paradigm this loop was only triggered by meaningful learning .",
"However and , in spite of all this evidence and although fMRI signals within the SN/VTA have been linked to dopaminergic signalling ( Düzel et al . , 2009; Schott et al . , 2008; Knutson et al . , 2007; Salimpoor et al . , 2011; Ferenczi et al . , 2016 ) , enhanced fMRI activity in isolation only provides indirect evidence for dopamine release .",
"Nevertheless , from a comparative neurobiological perspective , our results converge with previous animal findings revealing that midbrain dopaminergic neurons signal not only primary but also cognitive rewards ( Bromberg-Martin et al . , 2009 ) and with a recent study showing that midbrain-dopaminergic neurons of songbirds contribute to song learning , which is also mediated by an internal evaluation of correct performance ( i . e . , songbirds learn to sing by comparing their song to their memory trace of an adult's song; Mandelblat-Cerf et al . , 2014 ) . 10 . 7554/eLife . 17441 . 010Figure 8 . The SN/VTA-Hippocampal loop . In the downward arm of the loop , activation starts when new information that needs to be stored in memory ‘arrives’ at the HP via cortical inputs .",
"A signal is then sent to the SN/VTA through the subiculum of the hippocampus , the VS , and the ventral pallidum .",
"Neurons at the SN/VTA are disinhibited by the arriving signal from the HP , which facilitates their dopaminergic firing .",
"In the upward arm of the loop , dopamine is released back into the hippocampus , which in turn enhances memory formation and learning through long term potentiation processes .",
"We suggest that—in the same manner as an extrinsic reward modulates the HP , VS , and SN/VTA , and promotes memory benefits—the activity within the SN/VTA-HP loop can be induced by an intrinsic reward inherent to the process of learning itself and triggered by an internal monitoring of correct performance; and that this intrinsic reward ultimately promotes the storage of new information into long-term memory via dopaminergic modulation of the midbrain .",
"We hypothesize that the prefrontal cortex is fundamental for self-monitoring of correct performance ( see Ripollés et al . , 2014 for results showing activity within the inferior , middle and superior frontal gyrus while participants were engaged in the same learning task ) .",
"PFC , prefrontal cortex . DOI: http://dx . doi . org/10 . 7554/eLife . 17441 . 010 Importantly , functional connectivity results further corroborated our interpretation , as they showed that the connection strength among the HP , VS , and SN/VTA predicted subject-specific word-learning success for remembered words in the post-scan test ( see Figure 4 ) .",
"These connectivity results are of utmost importance , as they reveal that the coupling among the HP , VS , and SN/VTA predicted subject-specific memory enhancements ( see Adcock et al . , 2006; Wolosin et al . , 2012 for similar effects of external reward on SN/VTA-HP connectivity ) .",
"Regarding the connectivity results , an important limitation of the current work is that , given the poor temporal resolution of fMRI , we cannot fully address the temporal sequence of events during successful word learning .",
"In principle , effective connectivity analyses could have been computed in order to assess the directionality of the reported effects .",
"However , we acquired fMRI volumes in an interleaved order , which is suboptimal for Dynamic Causal Modeling and other types of effective connectivity analyses ( Stephan et al . , 2010 ) .",
"Future research could take advantage of concurrent EEG and fMRI set-ups and a continuous acquisition of fMRI data to further assess the relationships among the different regions of the SN/VTA-HP loop .",
"In addition , future studies could also try to directly measure the release of dopamine at the HP during learning by means of Positron Emission Tomography ( PET ) or by using pharmacological interventions ( e . g . , levodopa intake ) .",
"Although our results ( coming from fMRI data , EDA signals and subjective pleasantness ratings ) suggest that the SN/VTA-HP loop was engaged by an intrinsic reward-related signal that was associated with the participants' monitoring of their learning success , recent research proposes several alternative interpretations on how this circuit could be engaged in the absence of feedback .",
"On the one hand , the simple act of choosing ( i . e . , participants had perceived control over their learning ) can induce enhancements in memory , with the latter being associated with enhanced connectivity between the striatum and the HP ( Murty et al . , 2015 ) .",
"Importantly , Murty and colleagues emphasized that , in their task , participants were active learners that had a perceived sense of agency over their environment .",
"In a similar manner , in our paradigm participants managed to actively guide their own learning and to successfully monitor their performance , and this also had an effect on memory success ( see Figure 7 ) .",
"However , participants were asked to choose in both M+ and M- conditions , making decisions in both circumstances .",
"Therefore , differences in choice-related activity should have less influence in our paradigm .",
"On the other hand , Duncan et al . ( 2014 ) showed that connectivity between the HP and the SN/VTA during encoding of a classical associative task , in the absence of feedback , was predictive of long-term memory success , providing further support for the SN/VTA-HP loop .",
"It is important to emphasize that according to the SN/VTA-HP model ( Lisman and Grace , 2005 ) this network will be activated when the HP detects that novel information has to be encoded ( i . e . , by a novelty signal ) .",
"Although the temporal resolution of fMRI data prevents us from fully disentangling the origin of the detected activity within the SN/VTA-HP loop , given that the ROIs were selected using a meta-analysis on reward and taking into account that the M- and NR condition serve as a control for novelty effects , we suggest that the reported effects are related to intrinsic reward-related ( rather than to novelty-related ) signals that increased activity within the loop and ultimately enhanced learning .",
"In addition , subjective goals can also modulate activity within the striatum ( Han et al . , 2010 ) and , therefore , in our paradigm , participants' internal motivation towards correctly completing the task could also have modulated activity within the areas of the loop .",
"Note , however , that the incongruent condition ( M- ) partially controls for this possibility , as participants were equally prompted to correctly complete the M+ and M- conditions and in both cases a successful solution could be achieved .",
"Moreover , research in human adults has shown that reward and dopamine related regions can use internally generated signals of self-performance to guide perceptual learning in the absence of external feedback ( see for a review , Daniel and Pollmann , 2014 ) .",
"In particular , in two recent studies , activity within the VS ( Daniel and Pollmann , 2012 ) and within the VS and SN/VTA ( Guggenmos et al . , 2016 ) was related to the generation of a reward/confidence prediction error ( i . e . , activity was induced if participants thought that they performed better than they had expected in a perceptual task ) .",
"In Daniel and Pollmann ( 2012 ) , Guggenmos et al . ( 2016 ) and in the present work , learning occurs without external feedback , is driven by mesolimbic areas and internally generated signals of correct performance play a fundamental role .",
"However the output to be learned and the mechanism subserving this learning could still be different: while the results of the two aforementioned studies are based on the well-known role of dopamine in the codification of a reward prediction error ( Schultz , 1998 ) , we suggest that our learning is driven by the effects that dopamine has in LTP when it is released at the HP ( Shohamy and Adcock , 2010 ) .",
"Finally , our results also have specific implications for language learning .",
"The fact that the SN/VTA-HP loop can mediate the entrance of new-words into memory fits well with current perspectives which suggest that language requires the convergence of multiple neural functions—many shared with other species—such as memory , attention , and , crucial to our hypothesis , reward-related mechanisms ( Fitch , 2010; Ripollés et al . , 2014 ) .",
"In this vein , several studies have linked the intake of levodopa with an enhancement in word-learning ( Knecht et al . , 2004; Shellshear et al . , 2015 ) .",
"By showing that the coupling between the SN/VTA , VS , and HP can modulate successful word-learning from context—a natural learning process that is thought to be related to vocabulary growth during our lifespan ( Nagy et al . , 1985 ) and that occurs without the need of explicit reward , feedback or external guidance—we provide a mechanistic explanation on how dopamine could enhance certain forms of second language acquisition .",
"We suggest that language models should be extended to include the reward-memory SN/VTA-HP loop , as it might partially subserve specific word-learning processes ( see Figure 8; Davis and Gaskell , 2009; Rodríguez-Fornells et al . , 2009 ) .",
"In conclusion , we show that an intrinsic signal , triggered by successful learning , can modulate the entrance of new information into memory through the activation of the entire SN/VTA-VS-HP loop .",
"We propose that this signal is reward-related and that dopamine release plays a crucial role in this process .",
"Finally , the fact that word-learning can fuel itself through reward-related mechanisms that have major implications for second language acquisition and , most importantly , for the recovery from neurological disorders such as aphasia ."
],
[
"Forty German speakers were recruited from the student population at Otto-von-Guericke University ( Magdeburg , Germany ) for fMRI-Exp .",
"1 .",
"Four participants were rejected due to excessive head movements during the MRI session ( abrupt head motion exceeding 4 mm ) .",
"For Exps .",
"2 and 3 , twenty-four different Spanish speakers were recruited from the student population at the Universitat de Barcelona ( Barcelona , Spain ) .",
"All participants were right handed , gave written consent , and received compensation for their participation in accord with local ethics .",
"Thus , the final group consisted of thirty-six participants ( mean age , SD = 24 . 75 ± 4 . 7 , 17 women ) for Exp . 1 ( same group as in Ripollés et al . , 2014 ) , 24 subjects ( 25 . 16± 3 . 73 years , 15 women ) for Exp . 2 and 24 for Exp . 3 ( 21 . 16 ± 3 . 27 years , 18 women ) .",
"For fMRI Exp . 1 , the sample size was chosen based on the recommendation that , in order to achieve 80% of power , at least 30 participants should be included in an experiment in which the expected effect size is medium to large ( Cohen , 1988 ) .",
"We decided to recruit 40 participants anticipating possible problems ( e . g . , participant exclusion due to excessive movement ) .",
"To determine sample sizes for Experiments 2 and 3 we took into account behavioural results from Exp . 1 , in which participants still remembered 68% ( SD = 15 ) of learned new-words in the test carried out approximately 30 min after the encoding session ended ( chance level was 33%; effect size of Cohen's d = 2 . 13 ) .",
"Since testing occurred after a 24-hr retention delay , we assumed a more conservative ( but still large ) effect size of 0 . 8 ( Cohen , 1988 ) .",
"A sample size analysis , calculated using the G*Power program , showed that a sample size of 12 was required to ensure 80% of power to detect a significant effect of encoding at the 5% significance level .",
"We decided to double the calculated a priori sample size , as we expected the 24-hr retention delay to lower the recognition success as compared to Exp . 1 ."
]
] | [
"Humans constantly learn in the absence of explicit rewards .",
"However , the neurobiological mechanisms supporting this type of internally-guided learning ( without explicit feedback ) are still unclear .",
"Here , participants who completed a task in which no external reward/feedback was provided , exhibited enhanced fMRI-signals within the dopaminergic midbrain , hippocampus , and ventral striatum ( the SN/VTA-Hippocampal loop ) when successfully grasping the meaning of new-words .",
"Importantly , new-words that were better remembered showed increased activation and enhanced functional connectivity between the midbrain , hippocampus , and ventral striatum .",
"Moreover , enhanced emotion-related physiological measures and subjective pleasantness ratings during encoding were associated with remembered new-words after 24 hr .",
"Furthermore , increased subjective pleasantness ratings were also related to new-words remembered after seven days .",
"These results suggest that intrinsic—potentially reward-related—signals , triggered by self-monitoring of correct performance , can promote the storage of new information into long-term memory through the activation of the SN/VTA-Hippocampal loop , possibly via dopaminergic modulation of the midbrain ."
] | [
"Research shows that a reward such as money , or even simply the promise of such a reward , can boost the formation of long-term memories .",
"However , in our everyday lives , we continually gain new knowledge and make new memories in the absence of any obvious immediate reward .",
"Rewards activate a network of brain regions that includes the hippocampus , which has a key role in memory , plus several areas that release the chemical messenger dopamine , which boosts memory formation .",
"However , it was not clear whether this network of brain regions also supports learning that is driven internally rather than by external rewards or incentives .",
"Ripollés et al . have now tested this idea by asking thirty-six volunteers to try and learn the meaning of new words by reading pairs of sentences , all while lying down inside a brain scanner .",
"Half of the paired sentences provided a clear and obvious meaning for the new word .",
"As such , the volunteers were reasonably aware when they’d learned the meaning of a new word without any external feedback .",
"This approach confirmed that the activity of the brain’s reward-memory loop did indeed increase whenever a volunteer learned a new word .",
"Next , outside the brain scanner , the volunteers performed the same task but this time they had to rate how engaging and enjoyable they found it after each trial .",
"Emotional responses such as enjoyment trigger sweating , which alters the electrical activity of the skin .",
"Ripollés et al . observed greater changes in this “electrodermal” activity when the volunteers learned words that they would go on to remember one day later , than when they learned words that they would quickly forget .",
"The volunteers also reported greater enjoyment when learning the words that they would subsequently remember better , even after seven days .",
"Overall , these findings suggest that internally driven learning is in itself rewarding , and that under certain circumstances at least it can activate the brain’s reward-memory circuit .",
"A key question for the future is whether tapping into intrinsically rewarding forms of learning might be a more effective educational strategy than relying on external feedback and incentives .",
"This could be crucial to improving the design of educational programs – for example , in teaching literacy and foreign languages – and for improving the recovery of verbal skills lost after stroke ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Vascular permeability in retinopathy is regulated by VEGFR2 Y949 signaling to VE-cadherin | elife-54056-v2 | [
[
"Blood vessel dysfunction in the retina is a critical component of several blinding eye diseases .",
"In particular , the retina is sensitive to the fluid accumulation that can result from excessively leaking blood vessels in the eye and reduction of the ensuing edema is a therapeutic goal in the clinic ( Daruich et al . , 2018 ) .",
"Vascular endothelial growth factor A ( VEGFA ) is one of the key factors controlling endothelial cell biology in the eye ( Daruich et al . , 2018 ) .",
"The importance of VEGFA signaling is underscored by the clinical success of antibodies ( bevacizumab , ranibizumab ) neutralizing VEGFA , or soluble receptor fusion proteins ( aflibercept ) , neutralizing several VEGF family members .",
"These drugs preserve vision in many patients , but over time disease progression may resume , and for others there is no benefit even from the initial anti-VEGF treatment ( Bressler et al . , 2017 ) .",
"The role of VEGFA for survival of VEGFR2-expressing retinal neurons presents an additional challenge ( Nishijima et al . , 2007 ) .",
"Moreover , anti-VEGF treatment has been linked to capillary loss and rarefaction of normal adult vasculature ( Kamba and McDonald , 2007 ) .",
"While a focus in the development of new therapies to treat retinopathies has been on increasing the potency of anti-VEGFA therapy , the detrimental effects of long-lasting VEGFA suppression are now being recognized ( Usui-Ouchi and Friedlander , 2019 ) .",
"Taken together , the development of alternative treatments to block excessive vascular leakage in the eye while sparing other VEGF functions is very important .",
"The retina is protected by a blood retinal barrier ( BRB ) which , under normal conditions , limits the extravasation of blood components into the tissue .",
"The BRB consists of an inner barrier at the level of retinal vessels and an outer barrier at the cell junctions of the retinal pigment epithelium and it serves to stringently protect the tissue from pathogens , edema and inflammation ( Zhao et al . , 2015 ) .",
"The BRB is established in conjunction with vessel entry in the central nervous system during early development ( Chow and Gu , 2017 ) .",
"In eye diseases such as diabetic retinopathy , hypoxia and the ensuing increased production of VEGFA is accompanied by breakdown of the BRB ( Campochiaro , 2015 ) .",
"The molecular mechanisms downstream of VEGFA leading to this disruption have not been defined .",
"VEGFA , originally identified as vascular permeability factor ( VPF ) ( Senger et al . , 1986 ) , binds to VEGFR2 inducing receptor phosphorylation and the propagation of signaling cascades regulating endothelial survival , proliferation and motility ( Simons et al . , 2016 ) .",
"Acute responses to VEGFR2 activity include destabilization of endothelial junctions leading to increased permeability .",
"Notably , phosphorylation of VEGFR2 at Y949 ( 951 in human ) enhances permeability via the disruption of adherens junctions in the dermal vasculature , but also in pathologies such as neuroendocrine cancer , melanoma and glioblastoma ( Li et al . , 2016a ) .",
"Phosphorylation of Y949 creates a binding site for the adaptor molecule T cell specific adaptor ( TSAd ) , which in turn binds c-Src at endothelial junctions ( Li et al . , 2016a; Matsumoto et al . , 2005; Sun et al . , 2012 ) .",
"c-Src is implicated in phosphorylation of vascular endothelial ( VE ) -cadherin ( Li et al . , 2016a; Adam et al . , 2010; Vestweber , 2008; Wallez et al . , 2007 ) .",
"VE-cadherin is the main component of endothelial adherens junctions , forming homophilic interactions between endothelial cells , and is critical in regulation of permeability in response to VEGFA as well as inflammatory cytokines ( Li et al . , 2016a; Akla et al . , 2018; Lampugnani et al . , 2018 ) .",
"Tyrosine phosphorylation of VE-cadherin is associated with different biological functions .",
"Thus , phosphorylation of Y658 and Y685 is associated with the control of vascular permeability ( Orsenigo et al . , 2012 ) , and angiogenic sprouting ( Gordon et al . , 2016 ) , and Y658 with junction stability ( Garrett et al . , 2017; Schulte et al . , 2011 ) , whereas phosphorylation of Y731 in VE-cadherin is linked to leukocyte extravasation ( Wessel et al . , 2014 ) .",
"Serine phosphorylation of VE-cadherin at S665 is also implicated in regulation of endothelial junctions ( Gavard and Gutkind , 2006 ) .",
"Here , the molecular mechanisms underlying vessel leakage in eye diseases were studied using mouse models deficient in the VEGFR2-TSAd-VE-cadherin pathway .",
"Interruption of the VEGFR2 pY949F pathway resulted in suppressed vascular leakage at the choroid or at the superficial retinal vasculature in mouse models of age-related macular degeneration and diabetic retinopathy , respectively .",
"Furthermore , disrupting this pathway correlated with reduced VE-cadherin pY685 levels and reduced pathological neoangiogenesis of the superficial retinal vasculature .",
"These data identify VEGFR2 pY949 signaling as an important contributor to edema in retinopathies which may serve as a basis for development of new therapies selectively suppressing VEGFA-dependent disruption of the vascular barrier ."
],
[
"The Vegfr2Y949F/Y949F mouse ( henceforth denoted KdrY949F/Y949F ) , lacks the pY949 phosphosite of VEGFR2 and therefore fails to induce activation of c-Src .",
"We have previously shown that KdrY949F/Y949F exhibits suppressed vessel permeability in the dermis specifically in response to VEGFA ( Li et al . , 2016a ) .",
"The stringent BRB of the retinal vasculature is not expected to be regulated by VEGFA , however , in ocular disease such as retinopathy , the BRB may be broken down ( Urias et al . , 2017 ) ; in accordance , proliferative retinopathies are characterized by increased transvessel flow and edema ( Campochiaro , 2015; Kim et al . , 2016; Luo et al . , 2011; Stahl et al . , 2010a ) .",
"We therefore set out to determine whether the pY949 signaling pathway regulates pathologic leakage in the setting of proliferative retinopathy .",
"To induce retinopathy , KdrY949F/Y949F mice and their wild-type littermates ( henceforth referred to as Kdr+/+ ) were exposed to a laser-induced chorodial neovascularization ( CNV ) model .",
"The vascular lesions established after laser pulse disruption of the Bruch’s membrane mimic the progression of exudative age-related macular degeneration ( Lambert et al . , 2013 ) .",
"The lesions develop from the choroidal vessels over the course of several days as a consequence of hypoxia and elevated production of VEGFA ( André et al . , 2015 ) .",
"Choroids of CNV-treated Kdr+/+ and KdrY949F/Y949F mice were collected 14 days post-laser injury , a timepoint corresponding to a phase of completed neoangiogenesis and relative maturation of vessels lesions ( André et al . , 2015 ) .",
"Before collection , vessel leakage from the lesions was examined by monitoring extravasation of circulating 100 nm fluorescent microspheres , a particle size selected as being the smallest that would not simply leak through fenestrated pores of the choroid ( Gupta et al . , 2015 ) .",
"After 2 min of circulation , the microspheres remaining in circulation were flushed away by cardiac perfusion and choroid tissue was collected , immunostained , and analyzed by confocal microscopy for lesion size and microsphere accumulation .",
"Lesions were of equal size ( Figure 1A–B; 42038 µm2 ± 2514 , Kdr+/+; 44399 µm2 ± 3454 , KdrY949F/Y949F ) , still , microsphere accumulation was significantly reduced in the vicinity of KdrY949F/Y949F lesions as compared to the Kdr+/+ lesions ( Figure 1C–D ) .",
"We conclude that pathological leakage in the choroid can be suppressed by attenutation of pY949 VEGFR2 signaling and that this decrease is not simply due to an anti-angiogenic effect of interrupted VEGFA signaling .",
"To extend this finding we applied another common retinopathy model , oxygen-induced retinopathy ( OIR ) .",
"In the OIR model , mice are exposed to 75% oxygen during postnatal ( P ) days 7–12 after which they are returned to normal atmosphere ( 21% oxygen ) .",
"During the first stage , VEGFA expression is suppressed which leads to apoptosis of capillaries in the central region of the retina ( Lange et al . , 2009 ) .",
"In the second stage , retinal VEGFA expression is induced as a consequence of moving mice from high oxygen to normal atmosphere ( relative hypoxia ) , leading to the formation of pathological neovascular tufts , characterized by being disorganized and leaky ( Connor et al . , 2009 ) .",
"To determine the role of VEGFR2 pY949 signaling in regulating leakage from neovascular tufts , 25 nm fluorescent microspheres , a particle size chosen as the smallest commercially available , were injected in the tail vein of P17 pups , and allowed to circulate for 15 min before the remaining microspheres were flushed away by cardiac perfusion .",
"As expected , retinas from P17 Kdr+/+ pups submitted to OIR had a considerable central avascular region remaining following the early phase of vessel destruction and also abundant retinal neovascular tuft formation .",
"Of note , at P17 , KdrY949F/Y949F mice had a reduction in tuft formation compared to the Kdr+/+ mice ( Figure 2A–C; see Figure 2—figure supplement 1A for images without overlay ) .",
"This was not due to differences in body weight of P17 pups ( Table 1 ) .",
"The reduced tuft formation was a result of suppressed pathological angiogenesis during the P12-17 period when mice were kept in normal atmosphere following the hyperoxia period .",
"At P12 , the morphology of the retina vasculature was similar for KdrY949F/Y949F and Kdr+/+ littermates ( Figure 2—figure supplement 1B–C ) .",
"Importantly , extravascular accumulation of microspheres in regions of neovascular tuft growth was significantly suppressed in KdrY949F/Y949F retinas compared to Kdr+/+ ( Figure 2D–E ) .",
"Note that vessel-proximal microspheres , sticking to the either side of the wall of the disorganized tufts , were not included in the quantification .",
"We conclude that disrupting pY949 signaling during OIR leads to a suppression of pathological vessel leakage and also to a reduction in pathological neoangiogenesis .",
"The decrease in tuft formation in itself results in reduced overall leakage , however , it should be noted that remaining tufts leaked less in OIR-challenged KdrY949F/Y949F mice ( Figure 2E ) .",
"Moreover , the extent of infiltration of inflammatory CD68+ and CD45+ cells were similar at P17 for the KdrY949F/Y949F and Kdr+/+ strains indicating that the enhanced barrier in the KdrY949F/Y949F pups was specific for macromolecules and did not exclude inflammatory cells ( Figure 2—figure supplement 2A–C ) .",
"The canonical signalling pathway downstream of the pY949 phosphosite consists of the Src Homology 2 ( SH2 ) containing adaptor-protein TSAd ( gene designation Sh2d2a ) , which binds to pY949 with high affinity .",
"TSAd in turn recruits c-Src , leading to the phosphorylation of VE-cadherin ( illustrated in Figure 3A; Li et al . , 2016a; Sun et al . , 2012 ) .",
"To assess the role of these downstream players in retinopathy , we expanded our studies using mice treated with tamoxifen at P12 to specifically delete TSAd expression in endothelial cells , Sh2d2afl/fl; Cdh5-CreERT2 ( denoted Sh2d2a iECKO ) .",
"After OIR challenge of Sh2d2aiECKO mice along with their wild-type littermates ( Sh2d2afl/fl; referred to as Sh2d2aiECWT ) , tuft formation was reduced in Sh2d2a iECKO mice to a similar extent as seen with KdrY949F/Y949F mice ( Figure 3B–C: see Figure 3—figure supplement 1A for images without overlay ) while the avascular area was similar in the Sh2d2aiECKO and Sh2d2aiECWT mice at P17 ( Figure 3D ) .",
"Cre recombination in the Sh2d2aiECKO model was approximately 80% as assessed by vessel area ( Figure 3—figure supplement 1B , C ) , in agreement with immunoblotting analysis performed earlier to show the efficiency of Sh2d2a excision in this model ( Gordon et al . , 2016 ) .",
"The distribution of recombined endothelial cells was similar between neoangiogenic tufts and the normal vasculature .",
"The cytoplasmic tyrosine kinase c-Src phosphorylates VE-cadherin in vitro ( Adam et al . , 2010; Orsenigo et al . , 2012 ) and its activity correlates with detection of phosphorylated VE-cadherin in vivo ( Orsenigo et al . , 2012 ) .",
"Immunostaining for pY418 , a tyrosine residue in c-Src kinase domain required for maximal kinase activity , revealed active c-Src at the periphery of the retina and elevated levels in the neovascular tufts of KdrY949F/Y949F and Kdr+/+ mice ( Figure 3E ) .",
"Of interest , some pY418 c-Src immunostaining was localized at cell-cell junctions as indicated by co-staining with VE-cadherin .",
"Notably , the level of junctional pY418 c-Src was similar in tufts from KdrY949F/Y949F and Kdr+/+ mice ( Figure 3F ) , indicating that pY949 signaling does not account for c-Src activity at cell-cell junctions in this model .",
"Thus , signaling through pY949 in VEGFR2 appeared not to contribute to regulation of c-Src kinase activity at endothelial junctions in the retina vasculature .",
"As VE-cadherin is the major component of adherens junctions and thus vital for vessel integrity ( Giannotta et al . , 2013 ) , we performed immunostaining of P17 KdrY949F/Y949F and Kdr+/+ retinas after OIR , to examine VE-cadherin expression and junction morphology .",
"Neovascular tufts exhibited junctions with an irregular VE-cadherin morphology compared to non-tuft regions ( Figure 3G , H , compare VE-cadherin morphology in tuft and non-tuft panels showing Kdr+/+ ) , suggestive of increased internalization and degradation of VE-cadherin in the tufts ( Bentley et al . , 2014; Dejana et al . , 2008 ) .",
"In agreement , tuft endothelial junctions were detected by antibodies against two known phosphorylation sites in VE-cadherin , pY658 and pY685 ( Figure 3G , H ) , associated with VE-cadherin turnover ( Orsenigo et al . , 2012 ) .",
"The level of pY658 immunostaining was similar between KdrY949F/Y949F and Kdr+/+ tufts ( Figure 3I ) .",
"In contrast , pY685 levels were significantly lower in the KdrY949F/Y949F retinal tufts compared to Kdr+/+ ( Figure 3J ) .",
"The underlying vascular plexus showed positive immunostaining with the VE-cadherin phosphoantibodies as well , though to a lesser degree ( Figure 3G , H ) .",
"Arteries throughout the retina , including in the neovascularized regions , essentially lacked immunostaining for pY685 and pY658 ( Figure 3—figure supplement 1C ) , in keeping with previous findings on the lack of arterial VE-cadherin phosphorylation at these residues ( Orsenigo et al . , 2012 ) .",
"To corroborate the role of pY685 VE-cadherin in VEGFR2-regulated neovascular lesion formation , we performed immunostaining for VE-cadherin and pY685 VE-cadherin on choroid tissue from KdrY949F/Y949F and Kdr+/+ mice after CNV .",
"Lesions at D14 displayed pY685 VE-cadherin immunostaining , though the intensity of the pY685 signal was significantly lower in the KdrY949F/Y949F lesions ( Figure 4A , B ) .",
"This result suggests that junctional VE-cadherin phosphorylation at Y685 is dependent on pY949 VEGFR2 signaling also in the choroid vasculature .",
"The importance of VE-cadherin Y685 phosphorylation in the development of retinopathy was further demonstrated using a VE-cadherin Y685F mutant mouse model and its corresponding wild-type construct ( VEC-Y685F and VEC-WT , respectively ) ( Wessel et al . , 2014 ) , in the OIR model .",
"In these strains , wild-type and mutant CDH5 cDNA is introduced , replacing the endogenous mouse Cdh5 gene .",
"The two strains were maintained separately and each therefore had their separate C57Bl/6 wild-type littermates carrying the intact mouse Cdh5 gene .",
"The extent of oxygen-induced neovascular tuft formation as well as the avascular area in VEC-WT P17 mice was indistinguishable from wild-type C57Bl/6 littermates ( Figure 4—figure supplement 1A–C ) and the avascular area at P12 was likewise indistinguishable for these strains ( Figure 4—figure supplement 1D–E ) .",
"At P17 , however , tuft formation was reduced in the VEC-Y685F pups compared to VEC-WT controls ( Figure 4C–D ) , to a similar extent as seen for the KdrY949F/Y949F and Sh2d2aiECKO strains in relation to their respective controls ( Figures 2 and 3 ) .",
"As for the KdrY949F/Y949F and Sh2d2aiECKO strains strains , the avascular area after high oxygen-exposure was equivalent between VEC-Y685F and VEC-WT mice ( Figure 4E ) .",
"Immunostaining with the anti-VE-cadherin phosphoantibodies revealed complete loss of pY685 immunostaining in the VEC-Y685F retinas , validating the specificity of the antibody ( Figure 4—figure supplement 1F ) .",
"There was no difference in the levels of total VE-cadherin or VE-cadherin pY658 detected by immunostaining , in the VEC-Y685F tufts compared to WT ( Figure 4—figure supplement 1F ) .",
"Neovascular tuft leakage following OIR was examined following the same protocol as above .",
"Microsphere accumulation was significantly reduced in the tufts of VEC-Y685F mice following OIR , compared to VEC-WT ( Figure 4G–H ) .",
"As a control , microsphere leakage was assessed in VEC-WT and littermate C57Bl/6 P17 mice after OIR , which showed no difference in extravascular accumulation ( Figure 4—figure supplement 1G–H ) .",
"Combined , these data show an essential role for VEGFR2 and downstream VE-cadherin Y685 phosphorylation in elevated vascular leakage in retinopathies ."
],
[
"Here we demonstrate that blocking VEGFA-induced VEGFR2 pY949 signaling at any of several steps in the downstream pathway , leads to reduced vascular leakage in two independent retinopathy models , OIR and CNV .",
"While exposure to high oxygen in the OIR procedure causes pathological vascularization in the superficial retinal vessels , the CNV laser injury targets the choroidal vasculature .",
"Thus , we implicate VEGFR2 pY949 signaling in exaggerated vascular leakage in two very different vascular beds in the retina .",
"Importantly , we identify components of a signaling axis that can be targeted in order to control excessive vascular leakage and pathological neoangiogenesis without interfering with physiological functions of VEGFA .",
"Although pathological vascular tuft formation was diminished in the KdrY949F/Y949F strain , careful characterization of the vasculature in different organs of this strain has not revealed deficiencies in vessel density or morphology during development ( Li et al . , 2016a ) .",
"Of note , the improved barrier properties in the KdrY949F/Y949F vasculature do not exclude infiltration of CD68/CD45+ inflammatory cells ( Li et al . , 2016a and Figure 2—figure supplement 2 ) .",
"The effect of bacterial infections and wound healing properties remain to be tested .",
"Nevertheless , based on the currently available information , we favor the notion that suppressed macromolecular leakage in response to VEGFA plays an important role in pathologies while it is dispensable in physiology .",
"We do not exclude that leakage suppression to some extent interferes with neoangiogenesis due to the lack of a fibrinogen mesh for the new vessel to grow on Dvorak et al . ( 1995 ) .",
"However , as leakage was suppressed in the KdrY949F/Y949F retinas in both the CNV and OIR ( normalized to neovascular area ) models , our data support the concept that leakage is regulated separately from angiogenesis per se .",
"We conclude that in a range of tissues , either in the CNS or in peripheral organs , the VEGFR2 – TSAd – VE-cadherin pathway regulates vascular leakage in the context of disease , independent of angiogenesis .",
"A considerable strength of our study is in the use of intravenously injected microspheres to assess leakage , as we are able to address both pathological vessel growth and leakage simultaneously .",
"This is superior to traditional methodologies using Evans blue injection , to determine the area of dye leakage over the retina surface , or measure the relative amount of dye extracted from retinas using formamide ( Xu et al . , 2001 ) .",
"Unlike the methodology described here , neither of these methods allow for the precise visualization of the leakage or how it relates to vessel morphology .",
"Studies in rodent models have suggested that a complete loss of VEGFA-induced signaling in the eye can lead to atrophy of the neural layers , which mechanistically can be explained by the presence of VEGFR2 on retinal neurons ( Okabe et al . , 2014; Saint-Geniez et al . , 2008 ) .",
"In rats , anti-VEGFA treatment does not lead to damage of neural cells or loss of photoreceptor function ( Long et al . , 2018 ) , indicating that VEGFA might indeed be dispensable for the neural retina in this species .",
"However , retrospective and long term follow-up studies in human patients treated with anti-VEGFA therapies do provide examples of rare negative side effects such as hemorrhages , potentially linked to a systemic reduction of VEGFA ( Avery , 2014; Falavarjani and Nguyen , 2013; Keir et al . , 2017 ) .",
"Furthermore , anti-VEGFA treatment may contribute to the progression of geographic atrophy due to insufficient vascularization or to the fact that VEGFA may serve as a vital survival factor for the retinal pigment epithelium ( RPE ) ( Byeon et al . , 2010 ) .",
"Increased treatment frequency in patients receiving ranibizumab or bevacizumab is associated with greater loss of RPE , indicating geographic atrophy ( Enslow et al . , 2016; Grunwald et al . , 2014; Rofagha et al . , 2013 ) .",
"The necessity of repeated intraocular injections ( Patel et al . , 2013 ) for the administration of current anti-VEGFA therapy reduces patient compliance ( Polat et al . , 2017 ) and results in rare but serious instances of eye damage caused by the injection procedure , such as endophthalmitis or retinal detachment ( Falavarjani and Nguyen , 2013; Li et al . , 2016b ) .",
"Ultimately , the interaction between VEGFR2 pY949/pY951 and TSAd would be an ideal target to specifically block leakage but save other aspects of VEGFR2 biology .",
"The literature strongly implicates Src family kinases ( SFK ) in the phosphorylation of tyrosine residues on VE-cadherin , thereby playing an essential role in controlling vessel permeability ( Adam et al . , 2010; Orsenigo et al . , 2012; Wessel et al . , 2014; Trani and Dejana , 2015 ) .",
"In the context of OIR , direct inhibition of c-Src activation by kinase inhibitors ( Seo and Suh , 2017; Werdich and Penn , 2006 ) , indirect inhibition of c-Src kinase ( Toutounchian et al . , 2017 ) , or suppressed c-Src expression using siRNA ( Zhang et al . , 2010 ) , all result in decreased tuft formation .",
"Here , we found that neovascular tufts displayed abundant pY418 c-Src at endothelial junctions .",
"Although we may have expected decreased p418 c-Src levels in the KdrY949F/Y949F tufts , the levels were similar in the mutant and Kdr+/+ littermates .",
"These data indicate that while activation of c-Src alone may be required for induction of permeability and tuft formation , it may not be sufficient to induce these events .",
"In agreement , a triggering event , in addition to c-Src activity , is needed for opening of paracellular junctions ( Adam et al . , 2010; Adam et al . , 2016 ) .",
"Also , Orsenigo and coworkers detected constitutive , flow-dependent pY418 c-Src in leakage-permissive venules , but described an additional triggering event required for leakage to become established ( Orsenigo et al . , 2012 ) .",
"Our data suggests the scenario that VEGFR2 pY949 signaling leads to close proximity and complex formation between VEGFR2 and VE-cadherin potentially involving c-Src ( Li et al . , 2016a ) , which may be activated in a VEGFR2-independent manner .",
"A caveat in the interpretation of these data is that the pY418 antibody used to detect activated c-Src may cross-react with the related Yes and Fyn antibodies .",
"Currently , reagents specifically recognizing only c-Src are missing .",
"The acute stimulation of blood vessels with bradykinin or histamine to induce leakage is linked to a strong and transient drop in VE-cadherin phosphorylation due to internalization and degradation ( Orsenigo et al . , 2012 ) .",
"We find that in the chronic disease models studied here , regions of greater VE-cadherin phosphorylation displayed increased leakage .",
"The decrease in leakage observed in the KdrY949F/Y949F mice challenged with OIR or CNV , correlated with a reduction in VE-cadherin pY685 immunostaining , identifying this phosphosite as a key mediator in leakage regulation .",
"This finding is in line with the observation that sites of leakage in a cremaster model correlated with staining for VE-cadherin pY685 ( Wessel et al . , 2014 ) .",
"The mechanism underlying the important role of VE-cadherin pY685 compared to pY658 in vascular permeability is not understood .",
"The pY685 phosphosite serves as the binding site for C-terminal Src Kinase ( CSK ) , a negative regulator of c-Src activity .",
"CSK phosphorylates c-Src at pY527 , leading to its inactivation ( Latour and Veillette , 2001 ) .",
"Reduced pY685 VE-cadherin levels , as seen in the KdrY949F/Y949F mice , would predict less CSK at the junction and ultimately increased c-Src activity ( Baumeister et al . , 2005; Ha et al . , 2008 ) , which we did not observe .",
"Moreover , CSK deletion in vitro does not alter barrier properties ( Adam et al . , 2010 ) .",
"Another important aspect of the role of VE-cadherin pY685 is its regulation of pathological angiogenesis .",
"Notably , tuft area was decreased per se , in the VEC-Y685F mutant ( Figure 3B , C ) .",
"VE-cadherin has been implicated in regulation of VEGFR2 signaling by limiting receptor internalization .",
"In the presence of VE-cadherin , VEGFR2 is dephosphorylated by density-enhanced phosphatase ( DEP ) 1 , thus decreasing the levels of active VEGFR2 and proliferative signaling ( Lampugnani et al . , 2006 ) .",
"We hypothesize that VE-cadherin pY685 downstream effectors are critical in VEGFR2 signaling at junctions .",
"The VE-cadherin Y658 site has also been identified as an integral regulator of junctional stability .",
"Phosphorylation of the site acts to displace bound p120 catenin leading to destabilized adherens junctions ( Garrett et al . , 2017; Schulte et al . , 2011 ) .",
"VE-cadherin Y658 is phosphorylated by Src family kinases , with low levels of shear stress leading to maximal pY658 immunostaining ( Orsenigo et al . , 2012; Conway et al . , 2017 ) .",
"In the OIR model , tufts are known to have perturbed perfusion , which may drive the high pY658 intensity in tufts , to a similar extent in the WT and mutant models , as observed in this study .",
"At this point , VEGFA-targeting is the only pharmacological strategy exploited clinically and it is therefore of particular interest to understand how edema is established in response to VEGFA .",
"Other molecular regulators of pathological leakage have been described that may operate in concert with or independently of the VEGFR2 Y949 signaling .",
"Semaphorin 3A ( Cerani et al . , 2013 ) , Neuropilin 1 ( Fantin et al . , 2017 ) , activin-like kinase receptor type I ( Akla et al . , 2018 ) , angiopoietin-like 4 ( Babapoor-Farrokhran et al . , 2015 ) , VE-PTP and Tie2 ( Frye et al . , 2015; Shen et al . , 2014 ) have all been implicated in the control of junctional integrity in different vascular beds .",
"Here , we provide information on the specific VEGFR2 signaling pathway leading to edema that can be tested as a readout in other systems .",
"We also present an effective methodology to quantify leakage in relation to pathological angiogenesis , which we foresee will aid in exploration of new targets to treat retinopathies ."
],
[
"Mouse husbandry and oxygen-induced retinopathy ( OIR ) challenge took place at Uppsala University , and the University board of animal experimentation approved all animal work for those studies .",
"Choroidal neovascularization ( CNV ) experiments took place at Karolinska Institutet , St . Erik Eye Hospital , Stockholm; the procedures were approved by the Stockholm’s Committee for Ethical Animal Research .",
"Animal handling was in accordance to the ARVO statement for the Use of Animals in Ophthalmologic and Vision Research .",
"All animal experiments were repeated at least three independent times with wildtype and mutant mice compared within the same litter when possible .",
"Sample size was chosen to ensure reproducibility and allow stringent statistical analysis .",
"Randomization of mice and blinding of the investigators were not performed .",
"No mice were excluded from analyses , though CNV lesions that fused with neighboring lesions were excluded from analysis .",
"A knock-in mutation in the VEGFR2 gene , Kdr , was created by homologous recombination using VelociGene technology ( Regeneron Pharmaceuticals , New York , USA ) , wherein the tyrosine ( Y ) at position 949 was replaced with phenylalanine ( F ) ( Li et al . , 2016a ) .",
"The KdrY949F/Y949F mice , initially on mixed 129S6/C57BL/6 background , were extensively backcrossed to C57BL/6J ( Taconic Biosciences ) .",
"Sh2d2afl/fl; Cdh5-CreERT2 mice , referred to as inducible Endothelial Cell-specific Knock Out Sh2d2aiECKO mice ( previously denoted TsadiECKO mice ) , were generated as described ( Gordon et al . , 2016 ) .",
"Inducible deletion was by intraperitoneal injection of tamoxifen ( Sigma , T5648 ) at P12 , upon removal from hypoxia , and 24 hr later at P13 ( 400 µg/dose ) .",
"To track recombination , the Sh2d2aiECKO strain was crossed with the mT/mG strain ( JAX stock #007676 ) .",
"Cre-driven recombination resulted in mTmG-dependent conversion to green fluorescent protein ( GFP ) ( Muzumdar et al . , 2007 ) .",
"VE-cadherin ( VEC ) -Y685F and VEC-WT mice were generated using either WT or Y685F mutant human CDH5 ( VE-cadherin gene designation ) cDNA to replace the endogenous mouse Cdh5 gene ( Wessel et al . , 2014 ) .",
"A standard OIR model was used as described with minor modification ( Connor et al . , 2009 ) .",
"Briefly , each litter of pups was placed , along with the mother , into a chamber that maintained an oxygen concentration of 75 . 0% ( ProOx 110 sensor and A-Chamber , Biospherix , Parish , NY ) .",
"Mice remained in the chamber for 5 days , beginning at P7 and extending until P12 , when they were returned to normal atmosphere ( ~21% oxygen ) until termination at P17 .",
"While the pups remained at 75% oxygen throughout , lactating adult females were removed from the chamber on P8 , P9 , P10 , and P11 for 2 hr a day , to prevent oxygen toxicity-related death .",
"At P17 , pups were weighed and sacrificed and eyes enucleated and fixed in 4% paraformaldehyde ( PFA ) at room temperature for 30 min .",
"For microsphere extravasation experiments , mice at P17 were briefly warmed under a heat lamp to dilate tail veins before a tail vein-injection of microspheres ( 1% solution of 25 nm green fluorescent polystyrene beads; 50 µl per mouse ( ThermoFisher Cat . no . G25 ) .",
"Microsphere size was chosen based on previous experience in analyzing VEGFA-regulated leakage from non-fenestrated vessels ( Li et al . , 2016a ) .",
"Microspheres were allowed to circulate for 15 min before final sacrifice and tissue collection .",
"To remove blood and microspheres from the retinal vessels , mice were perfused with phosphate-buffered saline ( PBS ) .",
"Mice were fully anesthetized using isofluorane inhalation or alternately by an intraperitoneal injection of a mixture of ketamine/xylazine ( 100 mg/kg ketamine; 20 mg/kg xylazine ) , after which room temperature PBS was flushed through the vasculature .",
"Litters of mice exposed to OIR protocol were excluded from analysis when the average weight of pups at P17 was less than 5 . 5 grams , as low weight may indicate maternal neglect or other reasons for inability to thrive ( Stahl et al . , 2010b ) .",
"See Table 1 for average weight of pups at P17 .",
"A standard protocol of laser-induced CNV was employed ( Lambert et al . , 2013; André et al . , 2015 ) .",
"Briefly , 6–14 week-old KdrY949F/Y949F mice were anesthetized ( ketamine/xylazine; 30 mg/kg , 5 mg/kg respectively ) and pupils dilated using a topical administration of tropicamide ( 0 . 5%; Alcon , Puurs , Belgium ) .",
"Choroidal neovascularization lesions were induced in both eyes by diode laser ( 532 nm; IRIS Medical , Mountain View , CA , USA ) with settings: 75 µm spot , 200 mW intensity , 100 ms duration .",
"All visual hemorrhagic lesions were excluded from the study .",
"After laser-induction , animals were treated twice with 1 mL of saline ( 9 mg/mL NaCl; B . Braun , Melsungen , Germany ) subcutaneously under the back skin to prevent dehydration and the eyes were kept lubricated by topical administration of a paraffin and Vaseline mix ( APL , Gothenburg , Sweden ) .",
"At post-laser day 8 , or 14 , mice were culled and eyes immediately enucleated .",
"The retina tissue was carefully dissected away to expose the choroid and CNV lesions .",
"At day 14 , prior to sacrifice , mice were warmed on heating pads and given a tail vein-injection of microspheres ( 1% solution of 100 nm Blue-fluorescent polystyrene beads; 100 µl per mouse ( ThermoFisher Cat . no . B100 ) followed by 2 min of circulation and perfusion with fixative via cardiac puncture under isoflurane anesthesia .",
"A larger microsphere size ( 100 nm ) was chosen than for the OIR analyses ( 25 nm ) to avoid spontaneous passage of small microspheres through the fenestrated choroidal vasculature .",
"When the CNV lesions created in one eye grew and fused together , all fused lesions were excluded from further analysis .",
"Retinal vasculature and CNV lesions were immunostained with directly conjugated Alexa Fluor 488-Isolectin B4 , Alexa Fluor 594-Isolectin B4 , or Alexa Fluor 647-Isolectin B4 ( Sigma ) .",
"Endothelial cell junctions and phosphorylated VE-cadherin were stained with anti-VE-cadherin antibody ( 1:100; BD Rat 555289 ) and affinity purified rabbit antibodies against VE-cadherin pY658 and pY685 ( Orsenigo et al . , 2012 ) .",
"Phosphorylated c-Src was assessed using anti-phospho-Src ( Tyr418 ) Antibody ( 1:100; Invitrogen Rabbit 44–660G ) .",
"Secondary antibodies used were Alexa488 anti-Rat ( 1:500; Invitrogen Donkey A-21208 ) and Alexa555 anti-Rabbit ( 1:500; Donkey A-31572 ) .",
"Inflammatory cells were stained with anti-CD45 ( 1:300; BD Biosciences Goat 553076 ) and anti-CD68 ( 1:300; BioRad Rat MCA1957 ) .",
"Secondary antibodies used were Alexa488 anti-Rat ( 1:500; Invitrogen Donkey A-21208 ) .",
"Alexa555 anti-Goat ( 1:500; Invitrogen Donkey A-21432 ) .",
"Whole mount immunostaining of retinas and choroids was performed following OIR and CNV experiments .",
"Dissected issues , fixed in PFA were first incubated in blocking buffer ( 1% bovine serum albumin/2% fetal calf serum/0 . 05% Na-deoxycholate/0 . 5% Triton X-100/0 . 02% Na Azide in PBS ) for 2 hr to block unspecific binding .",
"Incubation with primary antibodies and secondary antibodies was carried out sequentially over night at 4°C on a rocking platform .",
"Tissues were mounted on slides with Fluormount G mounting media ( SouthernBiotech ) .",
"Microscopy was done using a Zeiss LSM700 microscope or a Leica SP8 confocal microscope .",
"Images were acquired with the 20x , 40x or 63x objective .",
"Processing and quantification of images was done with ImageJ software ( NIH ) .",
"Neovascular tuft formation and avascular area were determined by immunostaining retinas followed by image analysis .",
"Quantification of total vascularized area , central avascular area , and tuft area was performed by outlining images manually in ImageJ .",
"Using a tilescan of the IB4 channel for each whole mounted retina , the freehand selection tool was used to demarcate the vascular front , creating an ROI ( region of interest ) for the total vascularized area .",
"The freehand selection tool was also used to outline IB4 positive vessels from neovascular tufts – regions with disorganized dilated vessels often with markedly intense IB4 staining .",
"The ROIs for tufts were merged into a single ROI corresponding to the all the neovascular tuft area for a given retina .",
"The tuft area normalized to the total vascularized area was reported as a percentage of the total retina that contained tufts .",
"Similarly , the avascular region was determined using the freehand selection tool to outline the central avascular regions .",
"Regions where the superficial layer of capillaries was absent were determined and merged to form a single ROI corresponding to all of the avascular regions for a given retina .",
"The avascular area normalized to the total vascularized area was reported as a percentage of the total retina that was still avascular .",
"The researcher was blinded to the genotype of the sample when performing quantifications .",
"The quantity of extravasated fluorescent microspheres , marking sites of leakage , was measured in digital fluorescent images of CNV lesions from images taken with a Leica SP8 confocal microscope ( 63X objective ) equipped with single and dual fluorescence filters charge-coupled device ( CCD ) camera .",
"Camera settings were constant for images from all groups in each experiment .",
"Using ImageJ software ( NIH , Bethesda , MD ) , a guassian blur filter ( sigma 1 ) was applied to each image and a threshold was applied to the microsphere channel ( 405 for Blue fluorescence ) using the Triangle algorithm .",
"Microsphere area was calculated using the Analyze Particles function with an upper limit of 500 pixels to avoid the inclusion of large staining artifacts .",
"For the OIR model , the quantity of extravasated fluorescent microspheres , marking sites of leakage , was measured in digital fluorescent images of regions of retina over 10 to 15 images in each eye taken with a Leica SP8 confocal microscope ( 63X objective ) equipped with single and dual fluorescence filters charge-coupled device ( CCD ) camera .",
"Camera settings were constant for images from all groups in each experiment .",
"Using ImageJ software ( NIH , Bethesda , MD ) , the microsphere channel and IB4-vessel channel ( 488 for Green fluorescence and 647 for IB4 ) were adjusted with threshold ( Huang for Ib4 and Triangle for FITC ) for each channel .",
"Extravasated microsphere area was calculated by measuring the signal in the green fluorescence channel after removing any signal contained within the ROI ( region of interest ) corresponding to the IB4-positive area .",
"The Analyze Particles function was employed to quantify the microspheres .",
"A lower limit of 10 pixels was selected to distinguish the microsphere signal from background noise .",
"The mean area density for each group of mice was calculated from the median value of all images of the eyes of each mouse ( Fuxe et al . , 2011 ) .",
"To quantify leakage based on microscopic images , the amount of tracer extravasation was normalized to blood vessel density .",
"The researcher was blinded to the genotype of the sample when performing quantifications .",
"Immunostaining for CD68 , CD45 , and IB4 was performed on retinas from Kdr+/+ and KdrY949F/Y949F mice .",
"Confocal images were analyzed using ImageJ software for the presence of inflammatory cells within neovascular tufts .",
"For each image , the tuft area was manually outlined using the freehand selection tool and then each channel was thresholded ( CD68 with Zen; CD45 with Triangle ) and the positive area within these tuft regions was calculated as the percentage of total tuft area .",
"The average of five regions per retina are presented for each animal .",
"Statistical analysis was performed with GraphPad Prism ( GraphPad ) .",
"An unpaired Student’s T test was used to compare means between experimental groups .",
"A Mann Whitney U test was used to compare the medians between experimental groups with similar outcome .",
"All tests were two-tailed and p<0 . 05 was considered a statistically significant result .",
"Values shown are the mean , with standard deviation used as the dispersion measure .",
"Biological replicates refer to individual mice for OIR experiments and to individual lesions for CNV experiments .",
"Independent experiments refer to experiments done in different days with independently generated material .",
"A statistical method of sample size calculation was not used during study design .",
"For in vivo experiments , we used an average of 6 animals per experiment , with a minimum of 3 ( detailed number of animals used is given in figure legends ) .",
"The investigators were blind to the genotype of the animal for data analysis ."
]
] | [
"Edema stemming from leaky blood vessels is common in eye diseases such as age-related macular degeneration and diabetic retinopathy .",
"Whereas therapies targeting vascular endothelial growth factor A ( VEGFA ) can suppress leakage , side-effects include vascular rarefaction and geographic atrophy .",
"By challenging mouse models representing different steps in VEGFA/VEGF receptor 2 ( VEGFR2 ) -induced vascular permeability , we show that targeting signaling downstream of VEGFR2 pY949 limits vascular permeability in retinopathy induced by high oxygen or by laser-wounding .",
"Although suppressed permeability is accompanied by reduced pathological neoangiogenesis in oxygen-induced retinopathy , similarly sized lesions leak less in mutant mice , separating regulation of permeability from angiogenesis .",
"Strikingly , vascular endothelial ( VE ) -cadherin phosphorylation at the Y685 , but not Y658 , residue is reduced when VEGFR2 pY949 signaling is impaired .",
"These findings support a mechanism whereby VE-cadherin Y685 phosphorylation is selectively associated with excessive vascular leakage .",
"Therapeutically , targeting VEGFR2-regulated VE-cadherin phosphorylation could suppress edema while leaving other VEGFR2-dependent functions intact ."
] | [
"The number of people with impaired vision and blindness is increasing in Western society due to the aging population and the increased prevalence of diabetes .",
"This has led to eye diseases , such as age-related macular degeneration and diabetic retinopathy becoming more common .",
"In both these eye diseases , new blood vessels grow in the retina – the light-sensitive part of the eye – to bring oxygen and nutrients to the tissue .",
"However , these new blood vessels are leaky and allow molecules to leave the bloodstream and enter the retinal tissue .",
"This causes the retina to swell and impair a person’s vision .",
"The leaky blood supply also reduces the amount of oxygen that gets to the tissue , resulting in further damage to the retina .",
"When tissues experience low levels of oxygen , cells start making a protein called vascular endothelial growth factor ( or VEGF for short ) .",
"Whilst this protein is important for helping form new blood vessels , it also makes these vessels leaky .",
"Current treatments for age-related macular degeneration and diabetic retinopathy decrease swelling in the eye by blocking the action of VEGF .",
"However , these treatments also cause existing blood vessels and nerve cells to die , leading to irreversible damage .",
"Now , Smith et al . have set out to find whether the effects of VEGF can be blocked without causing further damage to existing cells .",
"To investigate this possibility , the eyes and retinas of mice were treated with a laser or exposed to changing oxygen levels to create injuries that resembled human age-related macular degeneration and diabetic retinopathy .",
"Each of the tested mice had specific mutations in proteins known to interact with VEGF .",
"Fluorescent particles were injected into the bloodstream of the mice to assess how these different mutations affected blood vessel leakage: if fluorescent particles could no longer be detected outside the blood vessels , this suggested that the mutation had stopped the vessels from leaking .",
"Further experiments showed these specific mutations affected leakage and did not prevent new blood vessels from forming .",
"In the future it will be important to see if drugs , rather than mutations , can also decrease the leakiness of blood vessels in the retina .",
"Such chemical compounds could then be tested in mouse experiments .",
"If successful , these drugs might be used to treat patients with age-related macular degeneration and diabetic retinopathy ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"structural biology and molecular biophysics"
] | A family of fluoride-specific ion channels with dual-topology architecture | elife-01084-v1 | [
[
"Fluoride pervades our biosphere , appearing in groundwater , sea , and soil typically at 10–100 μM levels ( Weinstein and Davison , 2004 ) .",
"This ubiquitous inorganic xenobiotic inhibits two enzymes essential for glycolytic metabolism and nucleic acid synthesis: enolase and pyrophosphatase ( Marquis et al . , 2003; Samygina et al . , 2007 ) .",
"Accordingly , many unicellular organisms , as well as green plants , use F− exporter proteins in their cell membranes to keep cytoplasmic F− concentration low , thereby minimizing the anion’s toxic effects ( Baker et al . , 2012; Stockbridge et al . , 2012 ) .",
"Two separate , phylogenetically unrelated families of F− exporters are now known: CLCF-type F−/H+ antiporters , a subclass of the widespread CLC superfamily of anion-transport proteins , and a group of small membrane proteins known as the ‘crcB’ family or , as we rename them here ( to avoid confusion with the CLCs ) , the ‘Fluc’ family .",
"Fluc proteins are widespread among unicellular organisms .",
"Deletion of the single Fluc gene in Escherichia coli produces hypersensitivity to F− , and this growth-phenotype may be rescued by F− exporter genes from a variety of bacterial species ( Baker et al . , 2012; Stockbridge et al . , 2012 ) .",
"By expressing , purifying , and functionally reconstituting several bacterial Fluc homologs , we demonstrate that these are highly selective F−-conducting ion channels constructed as dimers of identical or homologous membrane-embedded domains arranged in an inverted-topology fashion .",
"This type of molecular architecture is unprecedented among ion channels but is reminiscent of the dual-topology construction of small multidrug transporters ( Rapp et al . , 2006; Schuldiner , 2009; Morrison et al . , 2012 ) and of the inverted structural repeats appearing in many membrane transport proteins ( Forrest et al . , 2010 ) ."
],
[
"The predicted transmembrane topology of Fluc ( Figure 1B ) was experimentally tested with Bpe , a naturally cysteine-free homologue poised for specific labeling at a unique cysteine substituted near the N-terminus ( T3C ) .",
"Lipid vesicles reconstituted with this mutant were treated with LysC protease to exclusively remove the C-terminal His tags exposed to the outside of the liposomes .",
"Figure 1C shows that Fluc proteins are randomly oriented in the liposomes .",
"About half of the protein population has an externally exposed C-terminus and is susceptible to cleavage , as indicated by faster migration on SDS-PAGE .",
"The other half is protected , with the C-termini exposed to the protease-inaccessible liposome interior .",
"After proteolysis , a membrane-impermeant , thiol-reactive fluorophore was added to the liposome suspension to label externally exposed N-termini .",
"This treatment labels only the lower band containing the polypeptides with externally exposed C-termini , thereby demonstrating that the N- and C-termini are exposed on the same side of the membrane , as anticipated by the Fluc hydropathy profile .",
"To further confirm the predicted four-TM topology , similar experiments were performed with unique cysteines ( N31C and E94C ) placed in the first or third loops ( Figure 1C ) .",
"Here , the label reacts exclusively with the proteins that retain the C-terminal His-tag , thus placing these loops and the C-terminus on opposite sides of the membrane .",
"Purified Fluc proteins are functional , as seen in F− and Cl− efflux experiments in reconstituted liposomes ( Stockbridge et al . , 2012 ) , with ion-specific electrodes following the appearance of the anions in the external solution .",
"Liposomes were reconstituted under ‘Poisson-dilution’ conditions , that is , with Ec2 or Bpe at protein densities so low that 30–50% of the liposomes are devoid of protein , and most of the protein-containing liposomes carry only a single functional unit ( Walden et al . , 2007 ) .",
"The liposomes were loaded with 150 mM each of KF and KCl and suspended in iso-osmotic solution containing 1 mM KF and KCl along with 300 mM K-isethionate ( 2-hydroxyethanesulfonate , a membrane-impermeant monovalent anion ) .",
"Valinomycin ( Vln ) , a K+ ionophore , was then added to start the efflux by allowing the anions to move down their gradients .",
"After protein-catalyzed ion efflux had reached completion , detergent was added to disrupt all the liposomes and reveal the protein-free fraction .",
"Two striking results are apparent from the F− and Cl− efflux kinetics ( Figure 2A ) .",
"First , the proteoliposomes are selectively permeable to F−; Cl− efflux is undetectable on this timescale , and similar experiments in the absence of F− recapitulate this lack of Cl− transport .",
"Second , Fluc-mediated F− efflux is so fast that an initial rate measurement is precluded by the 1-s dead-time of the stirred-cuvette system .",
"An estimate based on a 35-nm liposome radius leads to a lower limit of ∼30 , 000 s−1 for the single-Fluc transport rate .",
"Since this is substantially higher than turnover of any conformational-cycle based membrane transporter ( Jayaram et al . , 2008 ) , it intimates that Fluc might be a F− channel . 10 . 7554/eLife . 01084 . 007Figure 2 . F− transport and selectivity in Fluc proteins .",
"( A ) Liposome flux assays for Fluc Ec2 and Bpe ( ∼10 pmol protein/mg lipid ) .",
"Anion efflux from liposomes containing KF+ KCl , 150 mM each , was initiated by addition of 1 µM Vln ( filled triangle ) .",
"Anions trapped in protein-free liposomes were released with 50 mM β-OG ( open triangles ) .",
"Anion appearance in the external solution was monitored ( F− red , Cl− black traces ) , and signals normalized to final levels .",
"( B ) Timecourse of insertion of Fluc-Ec2 reconstituted liposomes ( 5 µg/mg ) into a planar bilayer under salt-gradient conditions ( 300 mM NaF/30 mM NaF ) at −100 mV .",
"Arrow indicates addition of 0 . 5-µl liposomes .",
"Dashed line is zero-current level .",
"( C ) Macroscopic I–V relations under ionic conditions indicated .",
"Inset: Current responses to voltage pulses from −90 mV to +90 mV in 20-mV increments under salt-gradient conditions as in ( B ) .",
"Reversal potentials from 4–5 separate bilayers were 53 ± 1 mV and 119 ± 3 mV for salt-gradient and bi-ionic conditions , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 007 Accordingly , we recorded ionic currents mediated by Fluc-Ec2 inserted into planar phospholipid bilayers .",
"Initial experiments aimed at maximizing the electrical signal used liposomes with high protein density , 10–50 Fluc copies per liposome .",
"When fused into planar bilayers , these liposomes evoke a robust increase in bilayer conductance in discrete steps ( Figure 2B ) , each of which reports a single liposome inserting its packet of ion-conducting proteins .",
"The heterogeneity in step-size reflects the wide distribution of liposome size ( Miller and Racker , 1976; Accardi et al . , 2004 ) .",
"After a few minutes , liposomes are perfused away to stabilize the bilayer conductance , thereby allowing examination of its properties .",
"We applied a series of pulses to voltages from −100 mV to +150 mV and recorded the resulting currents to determine current-voltage ( I–V ) relations under various ionic conditions .",
"As shown in the inset to Figure 2C , recorded in the presence of a salt gradient ( 300 mM NaF//30 mM NaF ) , the current appears as a ‘leak’ without any time- or voltage-dependent gating over a wide voltage range .",
"The Fluc-mediated conductance is ideally selective for F− over Na+ , as seen from the zero-current voltage ( reversal potential ) of 53 mV , indistinguishable from the Nernst potential for F− in the eightfold ionic activity gradient .",
"High selectivity is further illustrated by the I–V relation under bi-ionic conditions ( 300 mM NaF//300 mM NaCl ) , whose high reversal potential ( 119 mV ) sets a lower limit of ∼100 on the F−/Cl− permeability ratio .",
"To detect single-channel currents , we prepared Fluc-reconstituted liposomes under Poisson-dilution conditions and fused them as above into planar bilayers .",
"Figure 3A shows examples of Fluc-insertion events in three separate bilayers , along with a histogram of Fluc insertion-step amplitudes taken from ∼50 bilayers ( Figure 3B , upper panel ) .",
"Most of these insertion-steps are 1 . 8 pA under these conditions , equivalent to 7 pS , while a minority ( <20% ) are 30–50% of this main step-size ( illustrated in the third trace ) .",
"The current shows occasional discrete fluctuations of two types: subsecond-timescale closing events and rare millisecond-timescale excursions to a substate of 50–60% of the main-state amplitude .",
"The ‘full-open’ state persists >95% of the time at all voltages ( −200 mV to +200 mV ) .",
"Its high open probability further shows itself in an extended recording with three channels in the bilayer ( Figure 3A , red trace ) and from the amplitude histogram of a typical single channel ( right panel lower histogram ) .",
"These channels display voltage-independent , anion-selective characteristics in harmony with the macroscopic currents reported above , and their appearance is strictly dependent on reconstituting Fluc protein into the liposomes .",
"Accordingly , we conclude that these unitary current-steps reflect activity of single Fluc proteins , with single-molecule turnover of ∼107 F− ions/sec at −200 mV , a rate typical for ion channels and three to four orders of magnitude higher than the fastest known transporters .",
"Thus , Fluc-Ec2 is a highly F−-selective channel , a protein with a transmembrane pore through which F− ion moves thermodynamically downhill by electrodiffusion . 10 . 7554/eLife . 01084 . 008Figure 3 . Single Fluc channels .",
"Fluc-Ec2 reconstituted liposomes ( 0 . 1 µg/mg ) were inserted into planar bilayers under salt-gradient conditions , and current fluctuations ( downward opening ) were recorded at −200 mV .",
"( A ) Upper traces: three examples of the first Fluc insertion events ( arrows ) after adding liposomes to the bilayer .",
"The two top traces are representative of most of the Fluc insertions , while the third trace illustrates the rarer low-conductance events .",
"Lower trace in red is taken from a bilayer containing three Fluc channels ( open levels marked with dashed lines ) .",
"( B ) Upper panel: first-insertion current histograms for 63 separate insertion events taken from ∼50 bilayers .",
"Lower panel: all-points amplitude histogram for a single Fluc channel ( lower panel ) recorded for ∼20 s .",
"In both histograms , zero current is defined as the mean level of the fully closed channel . DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 008 Fluc-Ec2 runs as a homodimer in detergent micelles on a size-exclusion column , as determined by combined measurements of UV absorbance , refractive index , and static light scattering of the eluting peak ( Figure 4A ) .",
"Since an ion channel pore formed on a dimer axis is unprecedented , we also assessed Fluc’s oligomeric state in its native habitat , the lipid bilayer .",
"For this , we prepared Fluc-Bpe for single-molecule total internal reflection fluorescence ( TIRF ) photobleaching experiments ( Tombola et al . , 2008 ) by labeling a unique cysteine mutant , R29C , with Cy5-maleimide and reconstituting the protein at a very low density to favor single-channel liposomes .",
"Upon illumination of these liposomes immobilized on a glass surface , the covalently attached fluorophores bleach in several minutes ( Figure 4B , C ) , mostly in single and double steps ( Figure 4D ) .",
"Given the measured labeling efficiency ( 72% ) , the preponderance of single- and double-bleach events is fully consistent with a dimer ( Figure 4E ) .",
"Fewer than 5% bleach in three or four steps , consistent with co-localization of the liposomes , but not with trimeric or tetrameric architecture for the channel .",
"The same conclusion was obtained in a second dataset using a different Bpe mutant , T3C ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 01084 . 009Figure 4 . Fluc homodimer in micelles and membranes .",
"( A ) SEC-SLS/UV/RI elution profile for Fluc-Ec2 with the molar mass of the eluting peak calculated using the ASTRA technique .",
"( B ) TIRF field of immobilized liposomes containing Bpe-R29C labeled with Cy5-maleimide .",
"( C ) Representative one- and two-step photobleaching events , integrated intensity in arbitrary units .",
"( D ) Observed populations of 1- , 2- , 3- , and 4-step photobleaching for Bpe R29C-conjugated Cy5 in liposomes .",
"Vertical axis is fraction of observations .",
"( E ) Expected populations of photobleaching events for dimer , trimer , and tetramer channel architectures given a 72% labeling efficiency .",
"A second dataset with BPe T3C gave similar results ( Figure 4—figure supplement 1 ) .",
"( F ) Representative measurements of F− efflux from liposomes reconstituted with increasing amounts of Ec2 .",
"F− efflux from liposomes loaded with 300 mM KF , as in Figure 2 .",
"( G ) Poisson-dump analysis for data in ( F ) .",
"Solid curves ( Equation 1 ) are determined by calibrating the system with a 104 kDa CLC protein ( Figure 4—figure supplement 2 ) , with oligomer number indicated ( n = 1–4 ) .",
"Similar results were obtained with Bpe ( Figure 4—figure supplement 3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 00910 . 7554/eLife . 01084 . 010Figure 4—figure supplement 1 . Single-Fluc photobleaching . Observed photobleaching events for Bpe T3C , along with number of steps expected for a dimer , trimer , and tetramer with 62% labeling efficiency . DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 01010 . 7554/eLife . 01084 . 011Figure 4—figure supplement 2 . Calibration of Poisson-dump experiments . CLC-ec1 E148A Y445A , a known homodimer of 104 kDa , was reconstituted at indicated density as described for Fluc in main text .",
"Red curve , an exponential fit to the data points , determines the black curves , expected for monomer , trimer , and tetramer ( Equation 1 , main text ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 01110 . 7554/eLife . 01084 . 012Figure 4—figure supplement 3 . Poisson-dump data for Fluc-Bpe with expected for the monomer , dimer , trimer , and tetramer curves . DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 012 We further tested the oligomeric state of Fluc in membranes by exploiting the F−-transport function of the channel in ‘Poisson-dump’ experiments .",
"The crux of this approach is the Poisson statistics of channel insertion into liposomes during reconstitution ( Goldberg and Miller , 1991; Maduke et al . , 1999; Walden et al . , 2007 ) ; as an increasing amount of protein is reconstituted into a fixed amount of liposomes , the fraction of liposomes devoid of protein , fo , decreases exponentially .",
"This protein-free fraction may be readily determined from F− efflux experiments , as above .",
"Upon Vln addition , F− exits the liposomes that contain functionally active channels , but remains trapped within the protein-free fraction f0 , which is determined by detergent addition ( Figure 4F ) : ( 1 ) f0= exp ( −ρ/nMk ) , where ρ is the experimentally varied protein density ( moles Fluc protein/mg lipid ) , M is the molecular weight of the Fluc subunit , n is the number of subunits in the active channel , and k is a constant dependent on the poorly known size and shape distribution of the liposomes .",
"This constant is determined by calibrating the system with a channel of known molecular weight; for calibration we use Cl− efflux mediated by a high-turnover mutant of the Cl−/H+ antiporter CLC-ec1-E148A/Y445A ( Jayaram et al . , 2008 ) , a homodimer of 52 kDa subunits ( Figure 4—figure supplement 2 ) .",
"Figure 4G shows fo curves expected for monomer , dimer , trimer , and tetramer architecture , along with experimental points from multiple preps , which coincide well with the dimer curve .",
"The best exponential fit to the Fluc-Ec2 data corresponds to a molecular weight of 31 . 7 kDa for the functional channel , in unreasonably good agreement with the predicted size of the homodimer , 31 . 6 kDa .",
"Similar results were obtained in parallel experiments with Fluc-Bpe ( Figure 4—figure supplement 3 ) .",
"In about half of the bacterial genomes in which it is found , Fluc appears as a single gene , as with the homodimeric channels Ec2 and Bpe ( Figure 5A ) .",
"Other prokaryotes carry a pair of homologous Fluc genes arranged in tandem ( ∼25% sequence identity ) , strongly suggestive of primeval gene duplication .",
"To test the function of paired Fluc proteins , we individually expressed , purified and reconstituted each of the twin Flucs of Lactobacillus acidophilus , La1 and La2 ( Figure 1 ) .",
"Although the low yield of La2 precludes detailed biochemical analysis , functional examination of these fraternal twins produces an unambiguous result ( Figure 5B ) .",
"Neither La1 nor La2 alone catalyzes F− efflux from liposomes , but co-reconstituting both proteins produces rapid F− efflux , indicating that the active channel requires heteromeric assembly . 10 . 7554/eLife . 01084 . 013Figure 5 . Assembly and architecture of Fluc channels .",
"( A ) Arrangement of Fluc genes in bacterial and eukaryotic genomes .",
"( B ) Heterodimeric assembly of homologous Fluc subunits .",
"F− efflux was followed by the light-scattering method from liposomes reconstituted with La1 only ( 40 pmol/mg lipid , black ) , La2 ( 40 pmol protein/mg , blue ) , or both ( 20 pmol each/mg lipid , red ) .",
"( C and D )",
"Sequence-based evidence for dual-topology assembly .",
"Distribution of arginines and lysines ( red circles ) on loops for Ec2 , a singleton-type Fluc or for La1 and La2 , homologous-pair Flucs . DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 013 Comparison of Fluc sequences of singletons vs fraternal twins shows striking differences in charge distributions on the loops connecting the helices .",
"The twin Flucs possess abundantly charged loops; for one of the pair , a positive charge bias resides on the N- and C-termini and loop 2 , while its homologous partner exhibits a converse bias , with excess positive charge on loops 1 and 3 ( Figure 5D ) .",
"Thus , according to the positive-inside rule for charge distribution in bacterial membrane proteins , ( von Heijne , 1989 ) one of the twins is predicted to insert into the membrane with the termini in the cytoplasm , and the second should be oppositely oriented .",
"In striking contrast , the singletons display relatively balanced charge distributions on either face .",
"In a genomic analysis of small membrane proteins , including Fluc , Rapp and colleagues ( Rapp et al . , 2006 ) proposed that a balanced Arg+Lys distribution in loops indicates dual-topology oligomeric construction , in which subunits insert randomly in both transmembrane orientations , and those of opposite orientations associate together ( Figure 5C ) .",
"Do singletons like Ec2 and Bpe find partners in the membrane from a pool of randomly oriented Flucs to form inverted-topology homodimers ?",
"Do the fraternal Flucs find their homologous companions oppositely oriented in the membrane ?",
"A strong indication of antiparallel assembly appears in eukaryotic Fluc genomes .",
"These genes all code for a pair of homologous Fluc sequences within a single open reading frame , and the linker connecting them contains a predicted TM helix ( Figure 5A ) .",
"This ‘inversion linker’ would force the second Fluc domain to adopt a transmembrane topology upside down with respect to the first .",
"These genomic characteristics of Fluc channels constitute compelling evidence for dual-topology construction , but since no previously known ion channels are built in this way , experimental support is needed to test such sequence-based suggestions .",
"We approach the question in two ways .",
"First , amino-group cross-linking of Fluc subunits is examined in detergent micelles .",
"Even though the Ec2 subunit contains four amino groups , the dimer cannot be cross-linked by glutaraldehyde ( Figure 6A ) .",
"Since these amines are all located on the same face of the protein—in loop 2 and both termini—glutaraldehyde would be expected to cross-link a conventional parallel dimer but not an antiparallel dimer .",
"With an additional lysine substituted on the opposite face , in loop 1 ( R25K ) or loop 3 ( N95K ) , glutaraldehyde treatment now leads to the appearance of a dimer band on the PAGE gel ( Figure 6A ) .",
"Similarly , lysine-less Bpe is not cross-linked by glutaraldehyde , despite its solvent-accessible N-terminal amino group .",
"Single lysine mutations on the side opposite to the N-terminus , in loop 1 ( R29K ) or loop 3 ( R95K ) , beget a cross-linked dimer ( Figure 6B ) , whereas solvent-accessible lysines on loops 2 ( R68K ) , on the C-terminus ( R130K ) , or on both do not ( Figure 6B ) .",
"These results are in natural harmony with antiparallel orientation and are difficult to understand in terms of a parallel-topology dimer , but do not by themselves provide rigorous proof . 10 . 7554/eLife . 01084 . 014Figure 6 . Fluc architecture .",
"( A and B )",
"Crosslinking patterns .",
"15% SDS-PAGE of WT Ec2 ( A ) or Bpe ( B ) and indicated lysine mutants treated with 0 . 125% glutaraldehyde for the indicated times .",
"Locations of all primary amino groups predicted for antiparallel dimers are indicated ( stars ) .",
"( C ) Design of engineered constructs that force La1 and La2 into parallel or antiparallel orientation , with icons envisioning pore-symmetry in each case .",
"( D ) F− efflux from liposomes for parallel construct LapA ( 7 . 5 pmol/mg ) .",
"( E ) F− and Cl− efflux from liposomes for antiparallel construct Laf-TM ( 7 . 5 pmol/mg ) .",
"( F ) Poisson-dump analysis for Laf-TM .",
"Predicted curves are as in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 01084 . 014 A second test of the dual-topology idea applies the fused-dimer strategy of Schuldiner and colleagues ( Steiner-Mordoch et al . , 2008; Nasie et al . , 2010 ) to the Fluc La1/La2 heterodimer .",
"We expressed several constructs in which La1 and La2 are fused together with linkers designed to force the two domains into parallel or antiparallel orientations ( Figure 6C ) .",
"For the antiparallel construct , Laf-TM , the domains are linked with a non-dimerizing glycophorin-A TM helix ( Lemmon et al . , 1992; Fleming et al . , 1997 ) .",
"Guided by eukaryotic Fluc sequences , we made loop 4 long and hydrophilic ( 18 residues ) , and loop 5 short ( 8 residues ) .",
"The domains of the parallel constructs , LapA and LapB , are connected by hydrophilic linkers of 14 or 26 residues , respectively .",
"All three fused constructs were expressed , purified , and reconstituted into liposomes , Laf-TM giving the best yield and chromatographic monodispersity ( Figure 1—figure supplement 2 ) .",
"Both parallel constructs have minimal transport activity , as shown for LapA ( Figure 6D ) .",
"In marked contrast , Laf-TM behaves quantitatively in Poisson-dump experiments like a fully active monomeric F− channel ( Figure 6E , F ) with high selectivity for F− over Cl− .",
"This result strongly implies that functional Fluc channels are built with the two 4-TM subunits or domains in antiparallel transmembrane topology , an arrangement that prohibits axial symmetry along the pore ( Figure 6C ) ."
],
[
"In this work , we have taken four initial steps towards understanding the molecular character of the Fluc family of F− exporters .",
"The results demonstrate that the mechanism by which Fluc transports F− is thermodynamically passive electrodiffusion through a transmembrane channel , that the channel is unusually F−-selective , and that it is constructed as a dimer of subunits or homologous domains .",
"In addition , the two subunits in the functional dimer are very likely oriented antiparallel to each other within the membrane .",
"How could a passive channel move F− out of the cell against an environmental F− challenge ?",
"It might seem that an active transport mechanism would be required , as in bacteria that use CLCF–type F−/H+ antiporters .",
"However , Fluc channels can work to effectively expel F− in two ways .",
"First , the negative membrane potential maintained by many classes of metabolizing cells tilts the anion’s equilibrium towards expulsion .",
"Second , the high pKa of HF ( ∼3 . 4 ) , anomalous among haloacids , dictates that in weakly acidic environments , a significant amount of extracellular HF is present ( Baker et al . , 2012 ) .",
"This membrane-permeant acid readily enters the cell and dissociates at the higher cytoplasmic pH . F− would therefore accumulate far above its extracellular concentration if no conductive pathway for the anion were present in the membrane , but a passive F− channel would undermine this weak-acid accumulation effect .",
"A deeper enigma is Fluc’s remarkable discrimination between F− , which is so difficult to desolvate , and Cl− , which is orders of magnitude more abundant in the environment .",
"Liposome flux experiments establish a far more dramatic F−/Cl− selectivity than the 100-fold lower limit set by electrical recording .",
"Anion-efflux assays measure Cl− turnover <30 ions/s , while in the same assays with F− , unitary turnover rates exceed 30 , 000 s−1 , and direct single-channel recording yields a turnover of 106–107 F−/sec .",
"These values conservatively put F−/Cl− selectivity >104-fold , the highest value known between close analogs for any ion channel .",
"It is this formidable selectivity that allows a bacterium or yeast to express a constitutively open F− channel in its energy-coupling membrane while avoiding a massive Cl− conductance that would otherwise catastrophically collapse its membrane potential .",
"High ion selectivity requires that F− be largely desolvated as it passes through the Fluc channel ( Hille , 2001 ) , an energetically demanding step ( >100 kcal/mol ) that has been an obstacle to the development of F−-selective small-molecule ligands ( Cametti and Rissanen , 2009 ) .",
"This chemical problem motivates future work to identify selectivity regions in Fluc sequences and , ultimately , structures .",
"The molecular architecture inferred here is unconventional and unexpected .",
"Most ion channel proteins are built on a barrel-stave plan in which three to seven similar subunits surround an axial pore running perpendicular to the membrane plane , the wider pores requiring a larger number of subunits .",
"But an ion-conduction pore formed by a two-stave barrel—a dimer—is unprecedented until now .",
"We are obliged to point out , however , that Fluc’s high open probability precludes a definitive conclusion that the observed single-channel currents are mediated by a single pore rather than two in parallel , especially in light of the infrequent subconductance ( Figure 3 ) .",
"However , this substate is not precisely half the condunctance of the fully open channel , and the protein’s small size makes a two-pore dimer , along the lines of CLC channels ( Middleton et al . , 1994; Ludewig et al . , 1996; Middleton et al . , 1996 ) , unlikely , but a picture like this is not firmly ruled out at our investigation’s early stage .",
"A final surprise that provides an intriguing glimpse into the evolution of membrane transport proteins is the dual-topology construction inferred for Fluc .",
"Unprecedented among known ion channels , dual topology has been observed in the SMR family of proton-coupled multidrug antiporters such as EmrE ( Schuldiner , 2009 , 2012; Morrison et al . , 2012 ) , and it recalls the inverted structural repeats that provide the mechanistic scaffolds of many transporters ( Forrest et al . , 2010 ) .",
"Oppositely oriented repeats within subunits are rare in ion channels , and so it is unclear whether this architecture provides particular mechanistic advantages for Flucs .",
"The inverted structural repeats of transporters almost certainly evolved from gene duplication and subsequent fusion of sequences coding for dimeric proteins with an odd number of TM helices ( Zuckerkandl and Pauling , 1965; Forrest and Rudnick , 2009 ) .",
"The Fluc family obligingly provides examples in modern genomes of all stages along such an evolutionary pathway , including duplicated gene-pairs in bacteria and fused homologues in eukaryotes .",
"Fusion of the latter genes must have required evolutionary gymnastics to capture a TM helix as an inversion linker for maintaining antiparallel topology of subunits with an even number of membrane crossings .",
"Finally , we note a conspicuous analogy to Fluc construction: the aquaporin channel .",
"This protein’s narrow , water-permeable pore is formed within a single 6-TM subunit on the interface between two structurally similar domains in inverted orientation with respect to each other ( Harries et al . , 2004 ) .",
"It is thus entirely feasible to construct a narrow pore at a dual-topology dimer interface .",
"The Fluc-aquaporin analogy is also of interest since the desolvated F− ion , with a radius of 1 . 3 Å , is almost the same size as H2O ."
],
[
"Chemicals from Sigma-Aldrich ( St . Louis , MO ) were of highest grade obtainable .",
"E . coli mixed phospholipids ( EPL ) , 1-palmitoyl , 2-oleoylphosphatidylethanolamine ( POPE ) , 1-palmitoyl , 2-oleoylphosphatidylcholine ( POPC ) , and 1-palmitoyl , 2-oleoylphosphatidylglycerol ( POPG ) were obtained from Avanti Polar Lipids , detergents from Anatrace , and fluorophores from Invitrogen or GE-Biosciences .",
"K-isethionate solutions were prepared from isethionic acid ( Wako Chemicals , Richmond , VA ) titrated with KOH .",
"Constructs used in this study are summarized in Table 1 .",
"Synthetic gene constructs for Fluc-Ec2 , Fluc-Bpe , Fluc-La1 , and Fluc-La2 were inserted into a pASK vector with a C-terminal LysC recognition site and hexahistidine tag ( TRKAASLVPRGSGGHHHHHH ) ( Maduke et al . , 1999 ) .",
"The fused constructs Laf-TM , LapA , and LapB were formed from the sequence of La1 followed by a linker sequence ( Table 1 ) leading into the sequence of La2 ( without the first methionine ) , to which a C-terminal hexahistidine tag was appended .",
"Laf-TM contained a transmembrane linker composed of a non-dimerizing glycophorin A helix ( Lemmon et al . , 1992 ) .",
"Site-directed mutagenesis was performed using standard PCR techniques .",
"All mutants were confirmed functionally active in a liposome-based F− efflux assay .",
"E . coli ( BL21-DE3 ) transformed with the pASK-IBA2 plasmid bearing Fluc constructs was induced with anhydrotetracycline at an optical density of 0 . 5–1 . 0 , and protein was expressed for 1 hr , 37°C .",
"Cells were lysed by sonication and extracted with 60 mM decylmaltoside ( DM ) for 2 hr at 4°C .",
"After pelleting the cell debris , the supernatant was passed over cobalt affinity beads ( Talon , 1 ml/l culture ) , washed with 100 mM NaCl , 45 mM imidazole , and eluted with 300 mM imidazole .",
"The eluate was diluted 10-fold into ion-exchange ( IE ) buffer , 10 mM NaCl , 5 mM DM , 10 mM 2- ( N-morpholino ) ethanesulfonic acid ( MES ) -NaOH pH 6 . 5 , and applied to a 0 . 5-ml cation-exchange column ( Poros 50 HS ) .",
"After washing with 10 vol of IE buffer , protein was eluted with IE buffer + 400 mM NaCl and was further purified on a Superdex 200 size-exclusion column ( SEC ) in 100 mM NaCl , 10 mM NaF , 10 mM ( 4- ( 2-hydroxyethyl ) -1-piperazineethanesulfonic acid [HEPES] ) , pH 7 , 5 mM DM .",
"Typical protein yields were 50–150 μg/l culture for Bpe , Ec2 , and La1 , ∼30 μg/l for the fused La constructs , and ∼5 μg/l for La2 .",
"Proteoliposomes were formed by dialysis of a micellar mix of protein ( 0 . 02–2 μg protein/mg lipid ) , lipid ( 10–25 mg/ml ) , and 35 mM 3-[ ( 3-Cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( CHAPS ) against the desired intraliposomal solution at room temperature for 36 hr .",
"Liposomes were stored in aliquots at −80°C until use .",
"For cysteine labeling applications , the protein was prepared in the presence of 10 mM Tris ( 2-carboxyethyl ) phosphine ( TCEP; Toronto Research Chemicals , Toronto , Canada ) until it was removed at the size exclusion step .",
"10 mM stocks of AlexaFluor 647-C2-maleimide and fluorescein succinimidyl ester were prepared in anhydrous DMSO and kept in the dark at −80°C until use .",
"The Bpe construct contained unique cysteine mutations T3C , N31C , or E94C for maleimide labeling , with an R29K background to increase labeling by the succinimidyl ester .",
"Proteoliposomes ( POPC/POPG , 20 mg/ml ) were prepared with 2 μg Bpe/mg lipid , with an intraliposomal solution of 300 mM NaF , 25 mM HEPES pH 7 . 5 , and 1 mM cysteine .",
"A 100-μl sample of the liposomes was treated with LysC ( 0 . 05 U , 1 hr , 22°C ) to completely cleave all externally exposed C-terminal His tags and was then centrifuged through a 1 . 5-ml G-50 column equilibrated with 300 mM NaF , 25 mM HEPES pH 7 . 5 to remove cysteine and LysC from the external solution .",
"AlexaFluor maleimide ( 50 μM ) was then added to label external cysteine residues , and after an hour freshly prepared phenylmethylsulfonyl fluoride ( PMSF ) was added to quench residual LysC , and 10 mM cysteine to quench the maleimide .",
"Liposomes were disrupted by 150 mM β-octylglucoside , and fluorescein succinimidyl ester ( 50 μM ) was added for 1 hr to stain protein amino groups before quenching with 10 mM Tris .",
"Samples were run on 10–20% SDS-PAGE gels and fluorescent bands were visualized using a Typhoon 9410 Variable Mode Imager ( GE Healthcare ) .",
"Cysteine-conjugated protein was visualized with the red laser ( 633 nm excitation , 670 nm emission ) , and fluorescein-conjugated total protein with the blue laser ( 488 nm excitation , 526 nm emission ) .",
"Fluorescein was used for staining total protein because of the large amount of lipid in the sample , which interferes with Coomassie or silver stain .",
"Anion efflux assays were performed as described previously ( Stockbridge et al . , 2012 ) .",
"In short , liposomes prepared with 10 mg/ml E . coli polar lipids containing 150 mM KF , 150 mM KCl , and 25 mM HEPES pH 7 were extruded 21 times through a 400-nm membrane filter and passed over a 1 . 5-ml G-50 Sephadex column equilibrated in external buffer composed of 300 K-isethionate , 25 mM HEPES pH 7 , 1 mM KF or KCl , according to the ion measured .",
"Liposomes were diluted 20-fold into external buffer in a stirred cuvette , and efflux was initiated by addition of 1 μM K+ ionophore valinomycin .",
"After ∼30 s , 50 mM β-octylglucoside was added to disrupt the liposomes .",
"Cl− or F− appearance in the external solution was monitored using homemade Ag/AgCl or Cole-Parmer LaF3/EuF3 electrodes , respectively; under the ionic conditions used here , these electrodes are ideally selective for Cl− or F− , and they show no cross-reactivity towards the other anion .",
"In some experiments , F− efflux was monitored by 90° light scattering at 600 nm in a fluorimeter .",
"A liposome sample containing 300 mM KF , 25 mM HEPES-KOH pH 7 was diluted 200-fold into 2 ml of a degassed isotonic solution containing 300 mM K- or Na-isethionate , 25 mM HEPES-KOH pH 7 in a stirred cuvette , and efflux was initiated by 1 μM Vln .",
"Water efflux maintaining osmotic balance leads to time-dependent shrinking and flattening of the initially spherical liposomes , accompanied by a ∼10% increase in 90° light scattering ( Jin et al . , 1999; Stockbridge et al . , 2012 ) .",
"After transport was complete , p-trifluoromethoxyphenyl hydrazine ( FCCP ) was added to the cuvette , effectively making the protein-free liposomes specifically permeable to F− to provide a quantitative measurement of the fraction of liposomes , f0 , free of Fluc channels .",
"For Poisson-dump experiments , data were collected for proteins from four independent preparations of CLC-ec1 and Fluc-Ec2 , and two independent preparations of Fluc-Bpe , Fluc-Laf , Fluc-LapA , and Fluc-LapB .",
"Planar bilayer recording was as previously described ( Accardi et al . , 2004 ) , using a POPE/POPG phospholipid mixture to form the bilayer and 150 mM NaF-1 . 5% agar salt bridges to connect the recording chambers to the Ag/AgCl electrodes through 1 M KCl wells .",
"Liposomes ( 0 . 5 μl added to the cis solution ) were fused into the bilayer , with 300 mM or 30 mM NaF , 15 mM Mops-NaOH pH 7 , in the cis or trans chamber respectively , trans defined as electrical ground .",
"Ionic conditions for recording were established by perfusion of either chamber .",
"Macroscopic I–V relations were determined with families of voltage pulses ( 0 . 5–1 s ) from a holding potential of zero to command-voltages from −100 to +150 mV in 10-mV increments .",
"Current output from a Axopatch 200A amplifier was low-pass filtered at 500 Hz and sampled at 2 kHz in pCLAMP software .",
"Voltages were corrected for junction potential , <3 mV .",
"Signals for single-channel analysis were subsequently filtered digitally at 100 Hz .",
"The molecular mass of Fluc in detergent was determined using the static light scattering/refractive index method ( Folta-Stogniew , 2006; Slotboom et al . , 2008 ) implemented at the W . M . Keck Foundation Biotechnology Resource Laboratory , Yale University .",
"Briefly , purified protein was passed over a Superdex-75 SEC column equilibrated in 400 mM NaF , 10 mM MES pH 6 . 5 , 5 mM DM and coupled in-line with light scattering , refractive index , and UV detectors .",
"Ovalbumin , transferase , bovine serum albumin , and carbonic anhydrase were used as standards .",
"The molecular mass was determined in two ways: by the three-detector method ( Hayashi et al . , 1989 ) , and from light scattering intensity at several angles ( ASTRA software , Wyatt Technology Corp . ) .",
"For the labeling reaction , freshly prepared Bpe with a single cysteine substitution ( T3C or R29C ) was incubated with a 20-fold molar excess of Cy5-maleimide for 1 hr at room temperature in the dark and quenched with a 100-fold molar excess of cysteine .",
"To wash away excess fluorophore , the protein was bound to cobalt affinity beads and washed with 45 column volumes of cobalt wash buffer .",
"The protein was eluted with 400 mM imidazole and the labeling efficiency was calculated from the protein absorbance at 280 nm ( εBpe = 38 , 960 M−1 cm−1 , calculated from protein sequence ) , and the Cy5 absorbance at 655 nm ( εCy5 = 2 . 5 × 105 M−1 cm−1 ) , with spillover correction of 0 . 017 .",
"Parallel experiments with wildtype , cysteine-free Bpe showed that the background labeling was <2% under the same conditions .",
"Protein was reconstituted into liposomes at very low density ( 0 . 15 µg/mg lipid ) .",
"Flow chambers were prepared using two glass coverslips that were cleaned by sonication in 2% micro-90 detergent ( Cole-Parmer , Vernon Hills , IL ) , ethanol and 0 . 1 M KOH .",
"Liposomes were formed immediately prior to analysis .",
"BPe T3C liposomes were sonicated vigorously for 10 min until clarity , yielding ∼30 nm liposomes .",
"BPe R29C liposomes were extruded 21 times through a 100 nm filter , then 21 times through 30 nm filter ( Avestin , Ottawa , Canada ) to form small vesicles .",
"Liposomes were diluted ∼105-fold in reconstitution buffer , and loaded into the flow chamber , where they spontaneously adhere to the glass surface .",
"The fluorescence from each liposome was measured using a custom through-objective TIRF microscope built for single molecule detection ( Friedman et al . , 2006 ) .",
"Cy5 fluorescence was measured by illuminating the sample with a helium-neon 633 nm laser ( 75 µW power ) and collecting the emitted light through a 633 nm long-pass filter ( 1 s per frame ) .",
"Each spot was centered inside a 3 × 3 pixel area using linear interpolation and drift-correction , and the intensity was integrated over the duration of the experiment .",
"For glutaraldehyde cross-linking experiments , ∼0 . 1 mg/ml freshly purified protein in 20 mM 3- ( N-morpholino ) propanesulfonic ( MOPS ) , pH 7 . 5 , 100 mM NaCl , 10 mM NaF , and 5 mM DM was incubated with 0 . 125% glutaraldehyde ( ∼500-fold molar excess ) for 5–120 min .",
"The reaction was quenched with a 10-fold molar excess of Tris-HCl , pH 7 . 5 , and samples were run on a 15% SDS-PAGE gel ."
]
] | [
"Fluoride ion , ubiquitous in soil , water , and marine environments , is a chronic threat to microorganisms .",
"Many prokaryotes , archea , unicellular eukaryotes , and plants use a recently discovered family of F− exporter proteins to lower cytoplasmic F− levels to counteract the anion’s toxicity .",
"We show here that these ‘Fluc’ proteins , purified and reconstituted in liposomes and planar phospholipid bilayers , form constitutively open anion channels with extreme selectivity for F− over Cl− .",
"The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation .",
"This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family , and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins ."
] | [
"Fluorine is the thirteenth-most abundant element in the Earth’s crust , and fluoride ions are found in both soil and water , where they accumulate through the weathering of rocks or from industrial pollution .",
"However , high levels of fluoride ions can inhibit two processes essential to life: the production of energy by glycolysis and the synthesis of DNA and RNA bases .",
"In polluted areas , organisms such as bacteria , algae and plants must remove fluoride ions from their cells in order to survive .",
"Since ions cannot freely cross lipid membranes , organisms use proteins called channels or carriers to move ions into and out of their cells .",
"Channel proteins form a pore , or channel , in the cell membrane , through which ions can quickly move from areas of high concentration to areas of low concentration .",
"In contrast , carrier proteins can transport ions in both directions—that is , to and from areas of high concentration—but they are slower than channel proteins .",
"A family of proteins that export fluoride from microbe and plant cells , thus allowing them to grow in the presence of this toxic ion , was discovered recently , but it was not clear if these proteins function as channels or as carrier proteins .",
"Now , Stockbridge et al . find that these proteins , called Fluc proteins , are fluoride channels with an unusual architecture .",
"Fluc proteins are found in many species of bacteria , and Stockbridge et al . show that a number of these , when purified and inserted into a lipid membrane , are channel proteins .",
"Additionally , they do not transport related ions such as chloride , which means that they are unusually selective for ion channels .",
"Two Fluc polypeptides associate to form a channel in the cell membrane , and Stockbridge et al . show that these two subunits are arranged in an antiparallel formation .",
"Although this architecture is unprecedented among ion channels , it has been observed in carrier proteins in a range of organisms , and may indicate that Fluc proteins offer an evolutionary model for many carrier proteins ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | A glucose-starvation response regulates the diffusion of macromolecules | elife-09376-v2 | [
[
"Eukaryotic cells expend a significant amount of energy to establish and maintain a high degree of intracellular organization in order to regulate and orchestrate their complex metabolism .",
"This includes the active enrichment of macromolecules into organelles , which in turn allows for the separation of biochemical pathways and enhances their efficiency by increasing local concentrations of enzymes and metabolites .",
"Similarly , active transport by motor proteins on the cytoskeleton enables large eukaryotic cells to overcome the limits of Brownian motion in which the diffusion time scales with the square of the distance .",
"Nevertheless , many cellular pathways and biochemical interactions are dependent on Brownian diffusion .",
"Much research has therefore been performed to characterize diffusional processes that occur within eukaryotic cells .",
"These studies have revealed that the very complex and extremely crowded cytosol of eukaryotic cells cannot be viewed as an ideal liquid but , instead , can be modeled as a polymer gel ( Luby-Phelps , 2000; Clegg , 1984; Knull and Minton , 1996 ) or soft colloidal glass ( Fabry et al . , 2001; Mandadapu et al . , 2008; Luby-Phelps , 2000 ) .",
"Still , a comprehensive understanding of the biophysical properties of eukaryotic cells is lacking , and it remains poorly understood how these characteristics influence macromolecular movement .",
"Furthermore , little is known about the biological determinants of intracellular diffusion including whether cells functionally regulate their diffusional properties in response to changes in growth conditions or the environment .",
"A particularly well-studied model of intracellular movement is the motion of chromatin in the nucleus .",
"Various groups have analyzed chromatin mobility , but a coherent mechanistic understanding of this process has yet to emerge ( reviewed in Hübner and Spector , 2010 ) .",
"For example , it was shown that chromosomes wiggle in a manner consistent with constrained diffusion and it was proposed that chromosome movement results from Brownian motion rather than from active motility ( Marshall et al . , 1997 ) .",
"In contrast , other studies have suggested that chromatin movement is ATP dependent ( Heun et al . , 2001 ) , and it was hypothesized that chromatin movement is driven by a multitude of ATP-dependent processes along the length of the chromosome ( Neumann et al . , 2012 ) .",
"Similarly , the role of the cytoskeleton in chromatin movement has remained controversial .",
"For example , both microtubule-dependent and -independent movement was reported ( Heun et al . , 2001 , Marshall et al . , 1997 ) , and several recent studies have also implicated the actin cytoskeleton in chromatin mobility ( Chuang et al . , 2006; Koszul et al . , 2008; Spagnol and Dahl , 2014; Spichal et al . , 2016 ) .",
"To better characterize the diffusional properties of eukaryotic cells , we have analyzed here the movement of chromatin and mRNPs in budding yeast under changing growth conditions .",
"Our results demonstrate that cells dramatically change their biophysical properties in response to glucose starvation , which causes a confinement of macromolecules and affects the mechanical properties of the cell .",
"This effect cannot be explained by changes in ATP levels or pH but can be induced by a loss of cell volume without any change in cell mass .",
"This response seems to be conserved as bacteria similarly restrict macromolecular mobility in response to starvation ( Parry et al . , 2014 ) , and we show here that starvation also induces a volume loss in bacterial cells and severely affects the rigidity of fission yeast cells .",
"Our results suggest a novel mechanism by which cells regulate their biophysical properties in order to adapt to environmental stress ."
],
[
"To examine the effects of changes in growth conditions on nuclear chromatin dynamics in budding yeast , we analyzed the movement of various gene loci .",
"LacO repeats were integrated at the POA1 locus on chromosome II , the URA3 locus on chromosome V , and on a centromeric plasmid ( pLacO ) .",
"Co-expression of LacI-GFP allowed us to visualize these three loci and track their mobility over minute-long sequences .",
"Whereas several changes in growth conditions , including growth in different carbon sources or nitrogen starvation , had no obvious effect on chromatin mobility ( data not shown ) , acute glucose starvation induced a dramatic cessation of chromatin movement ( Figure 1A ) .",
"This suggests that chromatin mobility is regulated by the presence of glucose . 10 . 7554/eLife . 09376 . 003Figure 1 . Acute glucose starvation confines macromolecular mobility in the nucleus and cytoplasm ( Figure 1—figure supplement 1 ) .",
"( A ) Minute-long trajectories of the POA1 locus from both ( + ) glucose ( blue ) and ( – ) glucose ( red ) conditions projected on bright field images .",
"Log-growing cells in ( + ) glucose were acutely starved for glucose , ( – ) glucose , for 30 min minutes prior to imaging .",
"Scale bar: 4 µm .",
"( B ) Mean square displacement ( MSD ) curves for POA1 mobility .",
"Upper panel: individual MSDs were averaged into an aggregate MSD for each condition .",
"Error bars represent standard error of the mean ( SEM ) .",
"Lower panel: log-log MSD plot of the same data .",
"( C ) Log-log MSD plot of the pLacO plasmid during exponential growth and after acute glucose starvation .",
"( D ) Minute-long trajectories of GFA1 mRNPs from both ( + ) glucose ( blue ) and ( – ) glucose ( red ) conditions projected on bright field images .",
"( E ) Mean square displacement ( MSD ) curves for GFA1 mRNP mobility .",
"Upper panel: individual MSDs were averaged into an aggregate MSD for each condition .",
"Error bars represent SEM .",
"Lower panel: log-log MSD plot of the same data .",
"( F ) Log-log MSD plot of the FBA1 mRNP during exponential growth and after acute glucose starvation .",
"Dashed gray lines represent a slope of one to guide the eye . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 00310 . 7554/eLife . 09376 . 004Figure 1—figure supplement 1 . Glucose starvation affects the mobility of nuclear and cytoplasmic objects .",
"( A ) Individual log-log MSD plots of POA1 loci in non-starved ( left ) and starved ( right ) cells .",
"( B ) Individual log-log MSD plots of GFA1 mRNP particles in non-starved ( left ) and starved ( right ) cells .",
"Dashed gray lines represent a slope of one to guide the eye . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 00410 . 7554/eLife . 09376 . 005Figure 1—figure supplement 2 . Starvation confines macromolecular mobility .",
"( A ) Log-log MSD plot of the URA3 locus during exponential growth and after acute starvation .",
"( B ) Log-log MSD plot of the GFA1 mRNP during exponential growth and quiescence ( see 'Materials and methods' ) .",
"( C ) Log-log MSD plot of the FBA1 mRNP mobility during exponential growth and quiescence .",
"Dashed gray lines represent a slope of one to guide the eye . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 005 To quantify the dramatic changes in chromatin mobility , we calculated ensemble-averaged mean square displacements ( MSDs ) for the chromatin loci ( n = 183–1172 trajectories each ) ( Figure 1B and C; Figure 1—figure supplement 1A; Figure 1—figure supplement 2A ) .",
"These plots express the magnitude of diffusion for a given particle , quantifying the average displacement per unit time and are used to compute their effective diffusion coefficients ( Qian et al . , 1991 ) .",
"We find that the confinement of chromatin upon glucose starvation ( Figure 1B and C; Figure 1—figure supplement",
"2 ) leads to an approximately three-fold reduction of the apparent diffusion coefficient ( K ) : for instance , KPOA1 decreased from 5 . 7 x 10–3 µm2/s to 2 . 3 x 10–3 µm2/s upon starvation ( Table 1 ) .",
"The change in mobility at this time scale was not caused by a change in the anomaly of the diffusion process as the anomalous diffusion exponent ( α ) , which is given by the slope of the curves in the MSD log-log plot , is not affected ( see also Table 1 ) . 10 . 7554/eLife . 09376 . 006Table 1 . Effective diffusion coefficients ( K; µm2/s ) and anomalous diffusion exponents ( α ) for macromolecules in each condition . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 006Condition POA1 LocuspLacO Plasmid URA3 Locus GFA1 mRNP FBA1 mRNPKαKαKαKαKα ( + ) Glucose0 . 00570 . 690 . 00670 . 780 . 00760 . 650 . 04200 . 830 . 05010 . 85 ( - ) Glucose0 . 00230 . 640 . 00210 . 800 . 00220 . 730 . 01310 . 770 . 01390 . 77 ( - ) Glucose pH 7 . 40 . 00150 . 65--------0 . 01200 . 75----DMSO0 . 00590 . 560 . 00460 . 700 . 00600 . 610 . 04910 . 830 . 05410 . 83Nocodazole0 . 00400 . 480 . 00250 . 570 . 00460 . 510 . 03640 . 850 . 03970 . 85Latrunculin A0 . 00380 . 500 . 00240 . 630 . 00380 . 550 . 04760 . 820 . 05500 . 81Nocodazole + LatA0 . 00280 . 480 . 00140 . 490 . 00260 . 520 . 03030 . 810 . 03670 . 822 mM K+Sorbate0 . 00560 . 730 . 00510 . 800 . 00590 . 680 . 04020 . 820 . 02960 . 804 mM K+Sorbate0 . 00500 . 750 . 00440 . 720 . 00480 . 710 . 04060 . 780 . 02420 . 796 mM K+Sorbate0 . 00390 . 700 . 00180 . 660 . 00270 . 660 . 03780 . 760 . 02700 . 788 mM K+Sorbate0 . 00230 . 640 . 00120 . 610 . 00140 . 610 . 03400 . 760 . 01640 . 770 . 4 M NaCl0 . 00300 . 690 . 00260 . 75----0 . 01290 . 830 . 01460 . 850 . 6 M NaCl0 . 00120 . 600 . 00110 . 58----0 . 00470 . 830 . 00570 . 840 . 8 M NaCl0 . 00090 . 630 . 00110 . 63----0 . 00130 . 670 . 00160 . 77Quiescence------------0 . 00040 . 290 . 00150 . 680 . 02% Azide ( Wash ) 0 . 00370 . 74--------0 . 02930 . 82----0 . 02% Azide ( Spike ) 0 . 00120 . 67--------0 . 01550 . 81---- To analyze whether glucose starvation uniquely affects chromatin dynamics in the nucleus , or whether it also influences the mobility of other macromolecules , we imaged the movement of cytoplasmic mRNPs , which can be conveniently tracked as single particles ( Shav-Tal et al . , 2004 ) .",
"24-PP7 stem-loops were integrated into the 3’ UTR of GFA1 and FBA1 , essential genes involved in distinct processes ( Lagorce et al . , 2002; Schwelberger et al . , 1989 ) , and the movement of individual mRNPs was examined upon co-expression of the coat-binding protein , CP-PP7-3xYFP .",
"Cumulative track projections revealed substantially higher mobility for mRNPs than chromosomal loci in glucose , which is expected given the significantly smaller size of mRNPs compared to chromosome fibers ( Figure 1D ) ( Thompson et al . 2010; Zarnack and Feldbrügge , 2007 ) .",
"Yet , upon glucose starvation , GFA1 and FBA1 mRNPs also exhibited a dramatic reduction in their mobility ( Figure 1E and F; Figure 1—figure supplement 1B ) .",
"Removal of glucose led to a three- to four-fold decrease in the diffusion coefficient of both GFA1 ( KGFA1reduced from 4 . 2 x 10–2 µm2/s to 1 . 3 x 10–2 µm2/s ) and FBA1 ( KFBA1reduced from 5 . 0 x 10–2 µm2/s to 1 . 4 x 10-2 µm2/s ) mRNPs without affecting the anomalous exponent ( α ) .",
"These values are similar to the relative change that we observed in the diffusion of chromatin ( Table 1 ) .",
"Depletion of glucose by growth into quiescence also confined the mobility of mRNPs; therefore , the effects we observe are not a consequence of our cell washes ( Figure 1—figure supplement 2B and C ) .",
"The decrease in mobility of both chromosomal loci and mRNP particles suggests that glucose starvation causes a confinement of macromolecules in the nucleus as well as the cytoplasm .",
"To begin to understand the nature and mechanism of the starvation-induced reduction in chromatin and mRNP mobility , we first focused on the cytoskeleton .",
"In eukaryotes , the movement of many macromolecules is directly influenced by the dynamics of the cytoskeleton .",
"For instance , mRNPs can be actively transported by motor proteins along the cytoskeleton and both the actin cytoskeleton and microtubules were reported to influence chromatin dynamics ( Thompson et al . , 2010; Heun et al . , 2001; Koszul et al . , 2008; Spagnol and Dahl , 2014; Spichal et al . , 2016 ) .",
"Interestingly , actin filaments were also shown to rapidly depolymerize upon starvation ( Uesono et al . , 2004; Sagot et al . , 2006 ) .",
"We therefore wanted to explore whether the starvation-induced confinement of macromolecular mobility resulted from changes in cytoskeletal dynamics .",
"Indeed , our starvation condition lead to ablation of actin filaments in nearly all cells ( Figure 2A ) and a nearly four-fold reduction of microtubule elongation events in G1 cells as visualized by Tub1-GFP ( Figure 2B ) . 10 . 7554/eLife . 09376 . 007Figure 2 . Starvation confines both cytoskeleton-independent macromolecular mobility and mobility influenced by the cytoskeleton ( Figure 2—figure supplement 1 ) .",
"( A ) Quantification of filamentous actin during logarithmic growth , ( + ) glucose , and after acute starvation , ( – ) glucose .",
"Cells were fixed and stained with phalloidin .",
"Z-stack projections were then processed and cells were classified based on the presence or absence of filamentous actin .",
"Error bars are standard error for three biological replicates ( n = 406–709 cells per replicate ) .",
"( B ) Average number of microtubule projections per G1 cell during logarithmic growth , ( + ) glucose , and after acute starvation , ( – ) glucose .",
"Error bars are standard deviation ( SD ) of the mean from three biological replicates ( n = 10–1510-15 cells per replicate ) .",
"The p-value for a two-tailed t-test for unpaired values assuming equal variance is shown .",
"( C ) Log-log MSD plot of the POA1 locus after treatment with nocodazole and/or latrunculin-A ( LatA ) for 20 min prior to imaging .",
"( D ) Log-log MSD plot of the GFA1 mRNP after treatment as described in ( C ) .",
"Dashed gray lines represent a slope of one to guide the eye . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 00710 . 7554/eLife . 09376 . 008Figure 2—figure supplement 1 . Starvation confines the cytoskeleton-independent mobility of mRNPs and the cytoskeleton-influenced mobility of chromatin .",
"( A ) Log-log MSD plot of the FBA1 mRNP after treatment as described in Figure 2C .",
"( B ) Log-log MSD plot of the URA3 locus after treatment as described in Figure 2C .",
"( C ) Log-log MSD plot of the pLacO plasmid after treatment as described in Figure 2C .",
"( D ) Graphic depicting the quantification of intranuclear displacements between the PES4 locus ( green ) and the POA1 locus ( red ) .",
"( E ) Log-log interchromosomal MSDs quantifying the displacement between POA1 and PES4 during logarithmic growth , ( + ) glucose , and after acute starvation , ( – ) glucose .",
"Images were acquired every 1 . 5 s for a total of 150 s , and the intranuclear displacements between the POA1 locus and the PES4 locus were tracked over time by determining the absolute distance between the two loci .",
"( F ) Log-log interchromosomal MSDs quantifying the displacement between POA1 and PES4 after inhibition of the cytoskeleton ( nocodazole + LatA ) and in a DMSO-treated control .",
"Cells were treated as described in Figure 2C and imaged as in ( E ) .",
"Dashed gray lines represent a slope of one to guide the eye . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 008 If the changes in the mobility of chromatin or mRNPs upon glucose withdrawal were due to the reduction of cytoskeletal dynamics , we would predict that drug-induced inhibition of actin and/or microtubule polymerization mimics the starvation response .",
"In order to test this hypothesis , we treated cells with the actin depolymerizer , latrunculin A ( LatA ) , and/or the microtubule depolymerizing drug , nocodazole ( Ayscough et al . , 1997; Jacobs et al . , 1988 ) .",
"Indeed , treatment with either drug reduced chromatin movement , although the effect was insufficient to mimic the confinement of chromatin during starvation ( Figure 2C; Figure 2—figure supplement 1 ) .",
"Concurrent treatments with LatA and nocodazole reduced chromatin mobility in an additive manner to substantially reduce chromatin mobility ( Figure 2C; Figure 2—figure supplement 1 ) .",
"Moreover , log-log MSD plots indicate that the effect of inhibiting the cytoskeleton is most apparent at longer time intervals and may augment the anomalous behavior of chromatin motion ( αPOA1-DMSO = 0 . 56 versus αPOA1-Noc+LatA = 0 . 48; Figure 2C; Figure 2—figure supplement 1; Table 1 ) .",
"The actin and microtubule cytoskeleton could either act on chromatin itself or affect chromatin mobility indirectly by influencing overall nuclear motion .",
"To differentiate between these two possibilities , we tracked the distance between two labelled chromosomal loci ( POA1 , chromosome II and PES4 , chromosome VI ) over time ( Figure 2—figure supplement 1 ) .",
"This approach allows for the exclusive quantification of intranuclear movements because translational changes in the positioning of the nucleus affect both loci identically and thus , do not influence intranuclear distance measurements ( Marshall et al . , 1997 ) .",
"This two-locus analysis confirmed the LatA and nocodazole-induced reduction in chromatin mobility as the interchromosomal distance between the two loci still decreased considerably with simultaneous drug treatment ( Figure 2—figure supplement 1 ) .",
"Of note , however , this result cannot differentiate between cytoplasmic or nuclear cytoskeletal dynamics in modulating chromatin mobility ( e . g . via deformations of the nucleus ) .",
"In conclusion , actin and microtubule dynamics independently contribute to the mobility of yeast chromatin .",
"In contrast to chromatin mobility , mRNP mobility was only moderately affected by perturbation of cytoskeletal dynamics ( Figure 2D; Figure 2—figure supplement",
"1 ) suggesting that the mobility of the GFA1 and FBA1 mRNPs is largely independent of the cytoskeleton .",
"Overall , our results show that glucose starvation restricts cytoskeleton-independent mobility as well as the mobility of macromolecules influenced by the cytoskeleton .",
"Our results so far could be explained by two alternative models:",
"1 ) starvation impacts macromolecular diffusion through multiple , distinct mechanisms , or",
"2 ) a singular , starvation-induced pathway restricts the mobility of macromolecules , and leads to both the collapse of cytoskeletal dynamics and the restriction of mRNP mobility .",
"The acute withdrawal of glucose in fermenting yeast cells is expected to have dramatic consequences on cellular physiology .",
"For example , the cellular ATP concentration drops ( Ashe et al . , 2000 ) and the intracellular pH decreases in starved cells ( Orij et al . , 2009 ) .",
"We therefore tested whether these global changes in cellular physiology lead to the observed changes in macromolecular mobility .",
"First , we investigated the changes in intracellular ATP concentration during starvation .",
"Upon glucose starvation , the ATP concentration rapidly decreased by ~70% .",
"Remarkably , after this initial drop , ATP levels were relatively stable at ~30% of the initial concentration for the remainder of the experiment ( Figure 3A ) .",
"Of note , the maintenance of this reduced ATP level required oxidative phosphorylation as the cellular ATP concentration quickly dropped to nearly undetectable levels when cells deficient in mitochondrial function were starved ( cbp2∆; Shaw and Lewin , 1997 ) ( Figure 3—figure supplement 1 ) .",
"In contrast , when cells washed in sugar-free medium were back-diluted into media containing glucose , ATP levels recovered to the initial concentration within 30 .",
"Thus , glucose starvation induces a rapid ~70% reduction of intracellular ATP levels in agreement with previously published results ( Özalp et al . , 2010 ) . 10 . 7554/eLife . 09376 . 009Figure 3 . A ~70% reduction of intracellular ATP is insufficient to replicate the macromolecular confinement of glucose starvation ( Figure 3—figure supplement 1 ) .",
"( A ) Intracellular ATP concentrations of acutely glucose-starved yeast were back-diluted into media containing 2% dextrose ( n = 2 experiments ) , 2% dextrose + 0 . 02% azide ( n = 2 experiments ) , or maintained in ( – ) glucose media ( n = 3 experiments ) .",
"Intracellular ATP concentrations were determined using a luciferase-based ATP assay ( Ashe et al . , 2000 ) and normalized to pre-treatment levels .",
"The zero min time point was taken immediately after back-dilution into the described media .",
"Error bars represent SD .",
"( B ) Log-log MSD plot of the POA1 locus .",
"Cells were treated with azide as described in ( A ) .",
"The azide treatment fails to replicate the confinement of glucose-starved cells from Figure 1B .",
"( C ) Log-log MSD plot of the GFA1 mRNP .",
"Cells were treated with azide as described in ( A ) .",
"The azide treatment fails to replicate the confinement of glucose-starved cells from Figure 1E . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 00910 . 7554/eLife . 09376 . 010Figure 3—figure supplement 1 . Respiration maintains intracellular ATP concentrations after acute glucose starvation . Log-growing wild-type ( WT ) and cbp2∆ yeast cells were acutely starved for glucose and back-diluted into media containing 2% dextrose ( WT ) or maintained in ( – ) glucose media .",
"Intracellular ATP concentrations were determined as in Figure 3A .",
"The zero minute time point was taken immediately after back-dilution into the described media .",
"Error bars represent SD ( n = 2 experiments ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 010 To determine whether a ~70% reduction in intracellular ATP levels is sufficient to confine cytoskeleton-independent macromolecular mobility as well as mobility influenced by the cytoskeleton , we reproduced this decrease in ATP concentrations in the presence of glucose .",
"This was achieved by back-diluting washed cells into glucose media containing 0 . 02% sodium azide ( NaN3 ) , an inhibitor of oxidative phosphorylation ( Figure 3A ) .",
"After 0 . 02% azide treatment , ATP levels dropped to the same level as in glucose-starved cells , but the mobility of chromatin and mRNPs did not decrease to the same degree as in starvation ( Figure 3B and C ) .",
"We therefore conclude that a ~70% reduction of intracellular ATP , as observed in glucose-depleted cells , is insufficient to fully explain the confinement of macromolecular mobility during glucose starvation .",
"Next , we investigated the contribution of intracellular pH to macromolecular confinement upon starvation .",
"The intracellular pH ( pHi ) of budding yeast was reported to decrease from pH ~7 . 3 to pH ~6 . 4 in a time frame consistent with the observed confinement of macromolecular mobility upon glucose deprivation ( Orij et al . , 2009; Young et al . , 2010 ) .",
"Using the pH-sensitive GFP analog , pHluorin , as a pHi biosensor ( Miesenböck et al . , 1998 ) , we confirmed in our experimental conditions that glucose starvation causes a reduction in pHi from ~7 . 4 to ~6 . 4 ( Figure 4A ) .",
"We then manipulated intracellular proton concentrations by the addition of the weak acid potassium sorbate ( K+Sorbate ) .",
"K+Sorbate traverses the plasma membrane , releases a proton , and reduces pHi in a concentration-dependent manner ( Bracey et al . , 1998; Piper et al . , 2001 ) .",
"Varying the extracellular concentration of K+Sorbate from 0 mM to 8 mM enabled us to titrate pHi from pH 7 . 4 to pH 5 . 9 , thus covering more than the pHi range observed in cells grown either in ( + ) glucose or ( – ) glucose conditions , with little effect on intracellular ATP levels ( Figure 4A and B ) .",
"Remarkably , exposing cells to increasing concentrations of K+Sorbate induced macromolecular confinement , and both chromatin and mRNP mobility could be titrated with decreasing pHi ( Figure 4C–F; Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 09376 . 011Figure 4 . A drop in intracellular pH ( pHi ) can reduce macromolecular mobility but cannot explain the confinement observed in glucose-starved cells ( Figure 4—figure supplements 1 and 2 ) .",
"( A ) Boxplots of the pHi of cells acutely starved for glucose or treated with varying concentrations of potassium sorbate ( K+Sorbate ) .",
"Log-growing yeast cells expressing phluorin ( Miesenböck et al . , 1998 ) were acutely starved of glucose or treated with K+Sorbate for 30 min before ratiometric imaging .",
"Intracellular pH was then estimated from a calibration curve ( see 'Materials and methods'; Figure 4—figure supplement 1 ) .",
"Values from three biological replicates were pooled and compiled into boxplots .",
"Whiskers represent the minimum and maximum respectively .",
"( B ) Intracellular ATP concentrations after treatment with either 2 mM or 8 mM K+Sorbate .",
"Intracellular ATP concentrations were determined as in Figure 3A .",
"The zero minute time point was taken immediately after each treatment .",
"Error bars represent SD ( n =2 ) .",
"( C ) Log-log MSD plot of the POA1 locus after treatment with K+Sorbate as described in ( A ) .",
"( D ) Log-log MSD plot of the GFA1 mRNP after treatment as described in ( A ) .",
"E ) Log-log MSD plot of the pLacO plasmid after treatment as described in ( A ) .",
"( F ) Log-log MSD plot of the FBA1 mRNP after treatment as described in ( A ) .",
"( G ) Log-log MSD plot of the POA1 locus after acute glucose starvation into starvation media ( SC ) adjusted to pH 7 . 4 .",
"( H ) Log-log MSD plot of the GFA1 mRNP after treatment as described in ( G ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 01110 . 7554/eLife . 09376 . 012Figure 4—figure supplement 1 . A drop in intracellular pH ( pHi ) titrates macromolecular mobility .",
"( A ) Intracellular pH calibration curve ( Orij et al . , 2009 ) ( see 'Materials and methods' ) .",
"Error bars represent SD .",
"( B ) Log-log MSD plot of the URA3 locus .",
"Cells were treated as described in Figure 4A . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 01210 . 7554/eLife . 09376 . 013Figure 4—figure supplement 2 . Sodium azide induces a pleiotropic reduction in intracellular pH , which may explain the subsequent confinement of macromolecular mobility .",
"( A ) Intracellular ATP concentrations after differing treatments with 0 . 02% sodium azide ( ‘wash’ versus ‘spike’ ) ( see 'Materials and methods' ) .",
"Intracellular ATP concentrations were determined as in Figure 3A .",
"The zero minute time point was taken immediately after each treatment .",
"Error bars represent SD ( n = 2 experiments ) .",
"( B ) Boxplots of the intracellular pH of cells treated as in ( A ) .",
"Log-growing cells were treated as described with sodium azide for 30 min before ratiometric imaging .",
"Values from three biological replicates were pooled and compiled into boxplots .",
"Whiskers represent the minimum and maximum respectively .",
"Outliers ( black circles ) are defined as points further from the quartile-box than 1 . 5 times the length of the quartile-box .",
"( C ) Log-log MSD plot of the POA1 locus after each treatment described in ( A ) overlaid from Figure 1B .",
"( D ) Log-log MSD plot of the GFA1 mRNP after each treatment described in ( A ) overlaid from Figure 1E . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 013 Importantly , however , treatment with the highest concentration of K+Sorbate ( 8 mM ) was necessary to fully recapitulate the starvation-induced confinement of both chromatin and mRNPs ( Figure 4C–F ) .",
"Under this condition , pHi dropped to 5 . 9 , which is below the physiologically observed pHi of 6 . 4 in glucose-starved cells ( Figure 4A ) .",
"When pHi was lowered to 6 . 4 ( by the addition of 2 mM K+Sorbate ) , the movement of both chromatin and mRNPs was only moderately reduced ( Figure 4C–F ) .",
"Furthermore , starvation in ( – ) glucose media adjusted to pH 7 . 4 , which prevents a drop in intracellular pH ( Dechant et al . , 2010 ) , failed to inhibit the confinement of chromatin and mRNPs upon glucose starvation ( Figure 4G and H ) .",
"Thus , the starvation-induced reduction of pHi is neither sufficient nor necessary and cannot fully explain the observed confinement of macromolecular mobility .",
"In the course of our experiments , we found that the nuclear volume decreased upon glucose withdrawal ( Figure 5A ) .",
"Since nuclear volume and cellular volume are generally tightly linked ( Neumann and Nurse , 2007; Jorgenesen et al . , 2002 ) , we utilized a Coulter Counter to examine whether cell size changes upon glucose starvation .",
"Indeed , the median cell volume decreased from 101 . 6 fL ( σ = 54 . 6 ) in glucose to 86 . 1 fL ( σ = 45 . 6 ) in starved cells , corresponding to a volume reduction of ~15% ( Figure 5B ) .",
"In addition , we observed that the yeast vacuole , an organelle involved in various processes including protein degradation and metabolite storage , swelled in size under glucose starvation conditions ( Figure 5C ) .",
"In non-starved cells , the vacuole-to-cell volume ratio was 0 . 25 ± 0 . 02 , whereas for starved cells this ratio increased to 0 . 40 ± 0 . 01 ( mean ± standard error for three independent experiments ) ( Figure 5D ) .",
"In combination , this vacuolar volume expansion together with the decrease in total cell and nuclear volume , reduces the cytoplasmic space that is available for diffusing molecules upon glucose starvation by ~30% ( 'Materials and methods' ) . 10 . 7554/eLife . 09376 . 014Figure 5 . Starvation induces a constriction in cell size and an expansion in vacuolar volume ( Figure 5—figure supplements 1 and 2 ) .",
"( A ) Nuclear volume after acute glucose starvation .",
"Histograms of nuclear volumes measured by reconstruction from three-dimensional image stacks using Imaris .",
"The p-value resulting from a two-tailed t-test on the average volume in each condition ( unpaired values assuming equal variance ) is p<0 . 001 .",
"( B ) Histograms of cell volumes of log-growing and acutely starved yeast cells .",
"Log-growing cells , ( + ) glucose , were acutely starved of glucose , ( – ) glucose , and cell volume was measured using a Beckman Coulter Multisizer 3 ( see 'Materials and methods' ) .",
"Approximately 50 , 000 cells were measured for each condition .",
"( C ) Cytoplasmic free GFP ( green ) and vacuolar membrane protein Vph1-mCherry ( magenta ) fluorescence images .",
"Scale bar: 10 μm .",
"( D ) Quantification of vacuole-to-cell volume ratio .",
"Error bars represent standard errors about the mean ( N = 3 independent experiments with > 55 cells per experiment ) .",
"( E ) Log-log MSD plot of the POA1 locus after treatment with increasing concentrations of NaCl .",
"Cells were imaged approximately 10 min after hyperosmotic shock .",
"( F ) Log-log MSD plot of the GFA1 mRNP after treatment as described in ( E ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 01410 . 7554/eLife . 09376 . 015Figure 5—figure supplement 1 . Histograms of cell volumes after various treatments .",
"( A ) Cells were treated with K+Sorbate as in Figure 4 and cell volume measured as in Figure 5B .",
"( B ) Cells were starved of glucose in either low or high pH ( 5 . 0 or 7 . 4 ) and cell volume was measured . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 01510 . 7554/eLife . 09376 . 016Figure 5—figure supplement 2 . Hyperosmotic shock increasingly confines macromolecular mobility without affecting intracellular ATP concentrations or intracellular pH .",
"( A ) Intracellular ATP concentrations after treatment with either 0 . 4 M or 0 . 8 M NaCl .",
"Cells were treated as described in Figure 5E and intracellular ATP concentrations were determined as in Figure 3A .",
"The zero minute time point was taken immediately after each treatment .",
"Error bars represent SD ( n = 2 experiments ) .",
"( B ) Boxplots of intracellular pH after hyposmotic shock as described in Figure 5E .",
"Cells were treated with hyperosmotic shock media for 10 min before ratiometric imaging .",
"Values from two biological replicates were pooled and compiled into boxplots .",
"Whiskers represent the minimum and maximum respectively .",
"Outliers ( black circles ) are defined as points further from the quartile-box than 1 . 5 times the length of the quartile-box .",
"( C ) Log-log MSD plot of the pLacO plasmid .",
"Cells were treated as described in Figure 5E .",
"( D ) Log-log MSD plot of the FBA1 mRNP .",
"Cells were treated as described in Figure 5E . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 016 We hypothesized that such a large reduction of the accessible cellular volume may be sufficient to induce the observed macromolecular confinement , for example , by an increase in macromolecular crowding or changes in cellular viscosity ( Luby-Phelps , 2000 ) .",
"If the hypothesis was correct that a decrease of cell volume was the singular response that confines macromolecular mobility , two predictions could be made: ( 1 ) lowering the pHi by the addition of K+Sorbate might induce a decrease in cellular volume since high concentrations of potassium sorbate can replicate the confinement of both chromatin and mRNPs during starvation , and ( 2 ) manipulation of cellular volumes , for example , by changes in osmolarity should recapitulate the effects of glucose starvation on cytoskeleton-independent mobility ( mRNPs ) and on mobility influenced by the cytoskeleton ( chromatin ) .",
"To test our first prediction , we explored the effect of pHi changes on cell volume .",
"Remarkably , lowering pHi by the addition of K+Sorbate also induced an increasing reduction in cell volume .",
"Strikingly , addition of 8 mM K+Sorbate , which led to the same mobility decrease as glucose starvation , reduced the median cell volume to 87 . 1 fL ( σ = 46 . 3 ) , a level virtually identical to that of glucose-starved cells ( Figure 5—figure supplement 1A and Figure 5B ) .",
"Although intracellular acidification led to a decrease in cell volume , it was not required for this decrease since starvation in ( – ) glucose media adjusted to pH 7 . 4 , preventing an intracellular pH drop ( Dechant et al . , 2010 ) , also reduced the cell volume ( Figure 5—figure supplement 1B ) .",
"Thus , the effects of K+Sorbate are better explained in terms of volume decrease than pH changes .",
"These data suggest a strong relationship between cell size and macromolecular mobility ( Figure 4C–F; Figure 5—figure supplement 1 ) .",
"To further test our model , we manipulated the cellular volume by changing the extracellular osmolarity .",
"We treated cells with increasing concentrations of NaCl and monitored the effects on chromatin and mRNPs .",
"Notably , addition of NaCl had little effect on pHi or cellular ATP levels , allowing us to uncouple the effects of cell volume from these factors ( Figure 5—figure supplement 2 ) .",
"As shown in Figure 5E and F and Figure 5—figure supplement 2C and D , both chromatin and mRNP mobility were progressively confined by increasing the magnitude of osmotic shock , consistent with recent results demonstrating that osmotic shock affects intracellular diffusion ( Babazadeh et al . , 2013; Miermont et al . , 2013 ) .",
"Therefore , a reduction of cell volume is sufficient to explain the starvation-induced confinement of macromolecular mobility .",
"We next explored how decreased cell volume could impede the movement of macromolecules .",
"In the very densely packed environment of the cell , a reduction in the volume accessible to diffusing molecules can alter protein-protein interactions and induce complex phase changes such as liquid-to-gel-like or liquid-to-glass-like transitions ( Doliwa and Heurer , 1998; Weeks et al . , 2000 ) .",
"To test whether glucose starvation causes an increase in molecular crowding , we examined whether the observed volume loss is compensated by a reduction in the total biomass of the cell .",
"We performed lyophilization mass measurement experiments in which starved and non-starved yeast cells were freeze-dried to remove their water content , and their masses were measured and normalized against the total number of cells ( Figure 6A ) .",
"We saw no significant difference between the non-starved and starved samples ( 11 . 5 ± 0 . 5 pg/cell and 11 . 9 ± 0 . 4 pg/cell , respectively; mean ± standard deviation for three independent experiments ) .",
"Importantly , yeast cells grown continuously in raffinose , which are known to be smaller than cells grown on glucose ( Tyson et al . , 1979 ) and thus served as a positive control , showed a 23% reduction in cell mass ( 8 . 9 pg/cell ) compared to cells grown in glucose .",
"Thus , acute glucose starvation does not induce a decrease in cell mass despite a significant reduction in the accessible cellular volume .",
"This increase in mass density suggests that the intracellular environment experiences increased molecular crowding conditions , which may lead to broad changes in the material properties of the cell . 10 . 7554/eLife . 09376 . 017Figure 6 . A conserved glucose starvation response alters the mechanical properties of the cytoplasm ( Figure 6—figure supplements 1 and 2 ) .",
"( A ) Dry mass measurements of non-starved and starved yeast .",
"Yeast cells in normal glucose , glucose deprivation , and raffinose growth conditions were lyophilized , and their cellular dry masses were determined .",
"Error bars represent standard deviations about the mean ( N = 3 independent experiments for non-starved and starved conditions; N = 1 for raffinose growth condition ) .",
"( B ) AFM experiments measure a mean stiffness increase from 18 to 22 nN·μm-1 for non-starved and starved cells , respectively , along with an increase in cell-to-cell variability ( Kolmogorov-Smirnov test , p = 0 . 013 ) .",
"Box plot: red line represents the mean , blue lines represent the 1st and 3rd quartiles , and whiskers represent the minimum and maximum values .",
"Each data point represents the mean value of eight measurement cycles on a single cell ( see 'Materials and methods' ) .",
"( C ) Mating pheromone-treated budding yeast cells ( shmoos ) remain elongated after starvation .",
"Shmooed budding yeast cells prior to spheroplasting ( left ) , non-starved shmooed spheroplasts ( center ) , and starved shmooed spheroplasts ( right ) .",
"Scale bar: 10 μm .",
"( D ) Fission yeast cells remain elongated after starvation .",
"Fission yeast cells prior to spheroplasting ( left ) , non-starved spheroplasts ( center ) , and starved spheroplasts ( right ) .",
"Scale bar: 10 μm .",
"Spheroplasting efficiency in the experiments described in ( B ) , ( C ) , and ( D ) was equally efficient in starved and non-starved cells and assessed via lysis in water .",
"( E ) Histograms of cell volumes of log-growing wildtype E . coli after 30 min of treatment with 2 mM DNP or the solvent control ( EtOH ) .",
"Approximately 100 , 000 cells were measured in each condition using a Beckman Coulter Multisizer 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 01710 . 7554/eLife . 09376 . 018Figure 6—figure supplement 1 . Cell stiffness negatively correlates with cell size . Both glucose-starved and non-starved budding yeast cells show an anti-correlation of cell stiffness with cell diameter .",
"Linear fitting R2 value for non-starved cell data = 0 . 54; R2 value for starved cell data = 0 . 71 .",
"Each data point represents the mean value of eight measurement cycles on a single cell ( see 'Materials and methods' ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 01810 . 7554/eLife . 09376 . 019Figure 6—figure supplement 2 . The actin cytoskeleton is a major consumer of intracellular ATP . Intracellular ATP concentrations of log-growing yeast were determined as in Figure 3A after treatment with latrunculin A or the solvent control DMSO .",
"The zero minute time point was taken immediately after each treatment .",
"Error bars represent SD from four biological replicates .",
"p-Values shown are for a two-tailed t-test for unpaired values assuming equal variance . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 019 To examine whether the starvation-induced changes in macromolecular mobility are accompanied by changes in the viscoelastic properties of cells we measured the effective stiffness of budding yeast upon acute glucose starvation using atomic force microscopy ( AFM ) .",
"Our setup used wedged AFM cantilevers for parallel plate compression to characterize yeast cell morphology .",
"These live-cell micro-compression experiments provide measurements of cellular viscoelasticity and cell stiffness under various conditions ( Stewart et al . , 2013; Ramanathan et al . , 2015 ) .",
"After removal of the cell wall , glucose-starved and non-starved yeast spheroplasts were subjected to AFM measurements .",
"Force-response curves were recorded while compressing single cells and analysis showed that glucose starvation induced a change in the cells’ mechanical properties increasing their mean stiffness from 18 to 22 nN·µm–1 as well as increasing the cell-to-cell variability in stiffness ( Figure 6B ) .",
"Consistent with the cell size measurements described above ( Figure 5B ) , these AFM experiments also suggest that starved yeast cells are smaller than the corresponding control cells and that there appears to be a negative correlation between cell size and stiffness ( Figure 6—figure supplement 1 ) .",
"In a separate approach , we also investigated the rigidity of the cytoplasm by examining cell morphology after removal of the cell wall .",
"Without an intact cell wall , the cytoplasmic turgor pressure causes the yeast spheroplast to ‘ball up’ into a sphere .",
"Since budding yeast cells are naturally round to begin with , we treated cells with the mating pheromone α-factor to generate shmoos , mating projections that cause the cell to become substantially elongated .",
"Spheroplasted shmooed cells which were not starved of glucose behaved as expected and formed spherical spheroplasts ( Figure 6C , Video 1 ) .",
"Intriguingly , starved shmooed spheroplasts remained predominantly in an elongated form ( ~four-fold less spherical spheroplasts in starved versus non-starved conditions ) ( Figure 6C , Video 2 ) .",
"This resistance to the internal turgor pressure suggests that the starved yeast cytoplasm is significantly more rigid than that of non-starved yeast , consistent with our AFM measurements .",
"Furthermore , this stiffening is reversible as re-addition of glucose to the starved spheroplasts returned them to a spherical form ( Video 3 ) , suggesting that sensory feedback pathways may exist to actively regulate this stiffening mechanism .",
"Taken together our results demonstrate that budding yeast cells undergo dramatic biophysical changes upon acute glucose starvation affecting their viscoelastic properties in addition to their intracellular macromolecular diffusion . 10 . 7554/eLife . 09376 . 020Video 1 . Spheroplasted budding yeast shmoos . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 02010 . 7554/eLife . 09376 . 021Video 2 . Spheroplasted starved budding yeast shmoos . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 02110 . 7554/eLife . 09376 . 022Video 3 . Re-addition of glucose to spheroplasted starved budding yeast shmoos . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 022 To examine whether similar biophysical changes can also be observed in other unicellular eukaryotes upon glucose withdrawal , we monitored the starvation-response of the fission yeast , Schizosaccharomyces pombe .",
"Budding and fission yeast separated evolutionarily several hundred million years ago , and they are as different from each other as either is from metazoans ( Sipiczki , 2000 ) .",
"Since S . pombe is naturally rod-shaped , we performed analogous spheroplasting experiments as described in Figure 6C .",
"Upon digestion of the cell wall , S . pombe cells round up and become spherical ( Figure 6D , Video 4 ) .",
"Conversely , glucose-starved cells maintain their rod-shape , reminiscent of the shmooed budding yeast cells , again demonstrating that glucose-withdrawal leads to a remarkable stiffening of the cytoplasm ( Figure 6D , Video 5 ) .",
"As for budding yeast , the stiffening is also reversible as addition of glucose to starved S . pombe spheroplasts returns them to a spherical shape ( Video 6 ) . 10 . 7554/eLife . 09376 . 023Video 4 . Spheroplasted fission yeast . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 02310 . 7554/eLife . 09376 . 024Video 5 . Spheroplasted starved fission yeast . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 02410 . 7554/eLife . 09376 . 025Video 6 . Re-addition of glucose to spheroplasted starved fission yeast . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 025 It was recently reported that the bacterial cytoplasm has glass-like properties and can undergo liquid-like to solid-like transitions upon starvation or treatment with 2 , 4-Dinitrophenol ( DNP ) , an uncoupler of oxidative phosphorylation ( Parry et al . , 2014 ) .",
"This condition results in confinement of macromolecules and disproportionally affects large particles above ~30 nm ( Parry et al . , 2014 ) .",
"The underlying mechanism for this cytoplasmic transition into a solid-like state in bacteria remains unexplored , but Parry et al . ( 2014 ) suggested that metabolic activity is needed to keep the bacterial cytoplasm fluid .",
"Although the cytoplasmic organization of prokaryotic and eukaryotic cells have dramatic differences , the similarity between these observations in bacteria and our findings in yeast prompted us to test whether there is a common underlying mechanism and whether bacteria also use a reduction in cell volume to increase crowding and confine macromolecular mobility .",
"Strikingly , a 30 min treatment of E . coli with 2 mM DNP caused a significant reduction in the volume of E . coli , inducing a ~9 . 5% modal volume contraction ( SD = ± 4 . 1% ) ( Figure 6E ) .",
"It should be noted , however , that the treatment utilized here , for technical reasons , is longer than the DNP-incubation time reported by Parry et al . ( 2014 ) .",
"Nevertheless , this result suggests that the reported liquid-to-solid like phase transition of the bacterial cytoplasm may also be triggered by cellular volume loss and suggests that the starvation-induced reduction of cell size could be a deeply conserved starvation response ."
],
[
"Our results identify a novel cellular response that severely alters the biophysical properties of cells and restricts intracellular diffusion in response to glucose starvation .",
"The dramatic macromolecular confinement observed upon starvation of budding yeast cannot be explained by a reduction of intracellular ATP ( Figure 3 ) , by a drop in pH ( Figure 4 ) or inhibition of global metabolism , as respiration persists upon glucose depletion ( Figure 3—figure supplement 1 ) .",
"Instead , we propose that increased macromolecular crowding induced by an acute volume loss fundamentally alters the mechanically properties of cells leading to macromolecular confinement .",
"Our results further suggest that both eukaryotic and prokaryotic cells regulate the level of crowding in order to control intracellular diffusion and macromolecular interactions .",
"In this context , it is intriguing that metazoan cells also seem to regulate their cellular volume as it was recently reported that tissue culture cells rapidly swell during cell division , which might aid with the mitotic separation of large chromosomes ( Zlotek-Zlotkiewicz et al . , 2015; Son et al . , 2015 ) .",
"At this point , it is unknown how glucose starvation induces a reduction in cellular volume and more work is needed to elucidate the signaling pathways and mechanisms by which glucose starvation causes cell size contraction and vacuolar volume increase .",
"Upon acute glucose starvation , budding yeast cells undergo a notable cytosolic acidification lowering their intracellular pH to ~6 . 4 , presumably by the rapid inhibition of the major plasma membrane proton pump Pma1 ( Figure 4; Orij et al . , 2009; Young et al . , 2010; Dechant et al . , 2010 ) .",
"Whereas this acidification might contribute to macromolecular confinement and enhance changes of biophysical properties within cells ( Fels et al . , 2009 ) , pH drop alone is neither necessary nor sufficient to recapitulate the macromolecular confinement , and the volume loss observed in glucose starved cells can occur in the absence of acidification ( Figure 4 and Figure 5—figure supplement 1 ) .",
"An increase of the intracellular proton concentration below 6 by the addition of increasing concentrations of K+Sorbate is able to induce a complete confinement of macromolecules ( Figure 4C–F ) but also significantly reduces the available cytoplasmic volume .",
"Interestingly , inhibition of cellular metabolism by 2-deoxyglucose and antimycin also provokes an enhanced drop of intracellular pHto 5 . 5 , which in turn triggers macromolecular confinement and leads to profound changes in the mechanical properties of the cell consistent with a sol to gel-like transition of the cytoplasm ( Munder et al . , personal communication ) .",
"Our data also support a mechanism of chromatin mobility in which the movement of yeast chromatin is strongly influenced by the joint dynamics of both actin and microtubules .",
"Precedence for such a mechanism is evidenced by previous work describing similar microtubule-dependent motion and reported interactions between microtubules , spindle pole bodies , and centromeres throughout the cell cycle ( Heun et al . , 2001; Tanaka and Tanaka , 2009 ) .",
"Moreover , the actin cytoskeleton is known to propel the movement of meiotic chromosomes and to influence the diffusion of other large macromolecules , suggesting a central function throughout the life cycle of yeast ( Koszul et al . , 2008; Brangwynne et al . , 2009a; Zhou et al . , 2011 ) .",
"The long-range directional movement of chromosomal loci is also reported to be dependent on the actin cytoskeleton ( Chuang et al . , 2006 ) .",
"This is consistent with our data , as the cytoskeletal inhibitors latrunculin-A and nocodozale appeared to more strongly affect the mobility of chromatin at longer timescales and have minor effects at shorter ones ( Figure 2; Figure 2—figure supplement 1 ) .",
"Finally , recent work detailing the existence of actin filaments in the nucleus substantiates the possibility of a network of nuclear actin ( Baarlink et al . , 2013 ) .",
"However , because latrunculin A depolymerizes all actin , our work cannot currently differentiate between the effects of cytoplasmic and nuclear actin in modulating chromatin mobility .",
"Paradoxically , previous experiments exploring the ATP dependence of chromatin mobility reached opposing conclusions in that treatment with sodium azide had only minor effects on the mobility of chromosomes in one study ( Marshall et al . , 1997 ) but drastically reduced their mobility in another ( Heun et al . , 2001 ) .",
"By tracking both chromatin and mRNPs in conditions that closely replicated the perceived differences in the two published protocols ( Marshall et al . , 1997; Heun et al . , 2001 ) , we were indeed able to duplicate the distinct results ( Figure 4—figure supplement 2 ) .",
"Importantly , however , we also observed that azide treatment lowered pHi and that the distinct treatments reduced intracellular pH to dramatically different extents ( Figure 4—figure supplement 2 ) .",
"Thus , a pleiotropic effect of azide treatment on pHi might be sufficient to reconcile the observed differences in the confinement of macromolecules .",
"These results also show that the azide treatment used here ( Figure 3 ( wash ) ; Figure 4—figure supplement 2 ) replicates both the decrease in intracellular ATP concentrations and the drop in pHi in glucose-starved cells .",
"This indicates that changes in ATP and pHi do not have additive effects and are neither alone nor in combination sufficient to explain the effects on macromolecular mobility that we observe in glucose starvation .",
"As expected ( Luby-Phelps , 2000 ) , our single particle-tracking experiments show that both chromatin and mRNPs display anomalous subdiffusion at the timescales that we investigated .",
"Interestingly , while glucose starvation dramatically decreases the diffusivity of the tracked objects , it does not change the anomaly of the diffusion .",
"Anomalous subdiffusion is usually considered to be a transient phenomenon that occurs in complex , disordered media such as the intracellular environment ( Höfling and Franosch , 2013 ) .",
"At short timescales , objects explore a pure viscous environment thus displaying normal Brownian motion .",
"Yet , at intermediate timescales , the motion of the objects is hindered by static structures , leading to anomalous subdiffusion .",
"Finally , at long timescales , that is , when the traveled distances are much larger than the sizes of the structures that impede movement , diffusion is again pure Brownian motion .",
"The fact that glucose deprivation affects diffusivity but not anomaly suggests that starvation induces a dramatic increase of the 'microscopic' viscosity without affecting the higher order cellular structures that impede macromolecule motion ( Huet et al . , 2014 ) .",
"Based on our results and according to the classical molecular crowding theory ( Zimmerman and Minton , 1993 ) this change of microscopic viscosity is probably the consequence of an increase in crowding upon cell size contraction .",
"As the cell cytosol becomes more viscous and restrictive to diffusional motion under glucose starvation , we posit that it must also become more resistive to external perturbations on the cytoplasm .",
"Indeed , we find that upon starvation the cytoplasm of budding and fission yeast undertake an astonishing transition leading to an increase in the viscoelastic stiffness of the cell ( Figure 6 ) .",
"Our observations are reminiscent of recently published observations in bacteria describing a glassy liquid-to-solid transition upon glucose starvation or DNP treatment ( Parry et al . , 2014 ) .",
"Furthermore , we demonstrate that bacteria also undergo a volume loss similar to starved yeast ( Figure 6E ) .",
"This observation suggests that molecular crowding also triggers the reported phase transition in bacteria , and similar mechanisms might be activated in both pro- and eukaryotes in response to glucose starvation .",
"Notably , this modulation of intracellular diffusion represents a mechanism to globally regulate the biochemistry of cells .",
"As described for bacteria ( Parry et al . , 2014 ) , we envision glucose starvation as a dormant state which might hold cells in stasis until provisions in the environment improve .",
"Consequently , we propose that cells functionally regulate intracellular diffusion in order to establish a cellular organization that is restrictive to some molecular interactions , perhaps thereby conserving energy , while simultaneously permissive to essential biochemistry .",
"The conservation of cell contraction between prokaryotes and eukaryotes strongly suggests a vital function during starvation .",
"On the one hand , the reduction in macromolecular mobility may allow for the rapid inhibition of excessive energy expenditure by diminishing unnecessary molecular interactions .",
"This is consistent with previous observations describing the rapid inhibition of principal ATP consumers upon starvation .",
"For example , Pma1p , required for regulation of intracellular pH and thought to use upwards of 20% of cellular ATP , is immediately inhibited upon glucose starvation ( Bracey et al . , 1998 ) .",
"Ribosomes also disengage from mRNA , resulting in a global collapse of polysome profiles ( Ashe et al . , 2000 ) .",
"Additionally , we found that depolymerization of the actin cytoskeleton may function , in part , to conserve ATP , as treatment with latrunculin A rapidly increases intracellular ATP concentrations ( Figure 6—figure supplement 2 ) .",
"Because osmotic shock promptly inhibits translation initiation and depolymerizes the actin cytoskeleton , it is conceivable that the starvation-induced cytoskeletal breakdown and collapse of polysome profiles , as well as other starvation-associated phenotypes , could be partly driven by molecular crowding ( Chowdhury et al . , 1992; Ueseono et al . , 2004; Ashe et al . , 2000; Uesono and Toh-EA , 2002 ) .",
"Therefore , the confinement of intracellular diffusion through molecular crowding may prove vital for economical biochemistry and represent a very upstream event upon carbon starvation .",
"On the other hand , molecular crowding-enhanced aggregation and confined macromolecular mobility may promote sustained interactions between complexes during starvation .",
"In agreement with this hypothesis , starvation is known to induce the formation of compartmentalized biochemistry and potential pockets of liquid-liquid unmixing ( Narayanaswamy et al . , 2009; Brangwynne et al . , 2009b ) .",
"For example , P-bodies , macromolecular complexes composed of mRNPs and mRNA decay machinery , form in the absence of glucose and potentially aggregate through unstructured regions of the component proteins ( Ramachandran et al . , 2011 ) .",
"Moreover , stationary phase induces the formation of foci and filaments for large numbers of proteins ( Narayanaswamy et al . , 2009 ) .",
"This aggregation was hypothesized to enhance interactions between members of the same pathway and promote more efficient biochemistry ( Narayanaswamy et al . , 2009 ) but was more recently also shown to cause enzymatic inactivation in some instances ( Petrovska et al . , 2014 ) .",
"As a consequence , prolonged starvation and confinement of macromolecular mobility may also pose unique challenges to cells , as complexes with elongated stretches of unstructured regions may prove more prone to extensive aggregation .",
"It will now be interesting to explore whether other cell types , including metazoan cells , respond similarly to starvation or growth factor depletion and if limited diffusion indeed stimulates aberrant protein aggregation , a process which has been implicated in a wide variety of human diseases ( Ross and Poirier , 2004 ) ."
],
[
"Yeast strains used in this study are of the W303 strain-background , except for strains containing labeled mRNPs which are heterozygous diploids of W303 and S288c backgrounds .",
"The E . coli strain utilized for volume measurements is wild type BW25113 .",
"Genotypes for every strain are listed in Table 2; plasmids are described in Table 3 .",
"Unless otherwise indicated , yeast cells were grown at room temperature in synthetic complete ( SC ) media at pH 5 . 0 ( titrated with 1 M HCl ) containing 2% dextrose ( SCD ) .",
"Specific dropout media was used to maintain plasmids when necessary .",
"E . coli were grown at 30ºC in LB . 10 . 7554/eLife . 09376 . 026Table 2 . Yeast strains used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 026StrainGenotypeSourceKWY165W303; MATa ura3-1 leu2-3 his3-11 , 15 trp1-1 ade2-1This StudyKWY1622W303; MATα ybr022w::256LacO::LEU2 his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1Green et al . , 2012KWY3541KWY 1622 , cbp2∆::KANMXThis StudyKWY3538W303; MATα his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1 256LacO::URA3This StudyKWY2848W303; MATα his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1 yel021w::128LacO::URA3This StudyKWY4586W303; MATa ybr022w::112TetO::URA3 yfr023w::256LacO::LEU2 his3::LacI-GFP_TetR-3XmCherry::HIS3This StudyKWY970KWY 165 , ura3::pHIS-GFP-TUB1::URA3This StudyKWY3661W303; MATa pHluorin::URA3This StudyKWY4796W303; MATα trp1::dsRED-HDEL::TRP1 leu2::TetR-GFP::LEU2This StudyKWY4736W303 MATa/S288c MATα NDC1/ndc1::NDC1-tdTomato::KANMX GFA1/gfa1::GFA1-24PP7 3xYFP-PP7-CFP::HIS3This StudyKWY4737W303 MATa/S288c MATα NDC1/ndc1::NDC1-tdTomato::KANMX FBA1/fba1::FBA1-24PP7 3xYFP-PP7-CFP::HIS3This StudyKWY5112W303; MATa ura3-1 leu2-3 his3-11 , 15 trp1-1 ade2-1 VPH1::VPH1-mCherry::KANMX leu2::GFP::CaURA3This StudyKWY6241CAF13 ( S . pombe wildtype ) This StudyyYB5978S288c; MATa his3∆1 leu2∆0 ura3∆0 met15∆0 LYS2 ADE2 TRP1 bar1::kanMXCaudron and Barral , 201310 . 7554/eLife . 09376 . 027Table 3 . Plasmids used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 09376 . 027PlasmidDescriptionSourcepKW2734pHMX-256LacO ( CEN , URA3 ) This StudypKW2957pYES2-PACT1-pHluorin ( CEN , URA3 ) Orij et al . , 2009pKW544MET25pro-PP7-CP-3xYFP ( CEN , HIS3 ) This Study Time-lapse epifluorescence imaging was performed using an inverted Nikon TE2000 microscope equipped with an Andor CCD camera , a motorized stage ( Applied Scientific Instrumentation , Inc . , Eugene , OR ) , 60x/1 . 49NA APO TIRF oil immersion objective , and controlled with Metamorph® software ( Molecular Devices , LLC , Sunnyvale , CA ) .",
"Cells were stabilized by coating the glass-bottom of MatTek® dishes ( PG35G-1 . 5-14-C; MatTek Corporation , Ashland , MA ) with 0 . 2% concanavalin A and aspirating the solution prior to adding 300 µL of OD600 0 . 2–0 . 3 cells .",
"All imaging was performed at room temperature .",
"Images for particle tracking were collected at a frame rate of 0 . 5 s for 60 s , unless otherwise indicated .",
"Particles were tracked using custom MATLAB® ( The MathWorks , Inc . , Natick , MA ) scripts based on the 2D feature finding and particle tracking software made available by Dr . Maria Kilfoil ( Pelletier et al . , 2009; http://people . umass . edu/kilfoil/downloads . html ) .",
"An average mean square displacement ( MSD ) was calculated for each condition using the well-described equation:MSDτ= ( r ( t+τ ) −rt ) 2 Effective diffusion coefficients , K , were computed by finding the slope of the best-fit line between the first and tenth time points of the averaged MSD .",
"The anomalous diffusion exponents of the log-log MSDs , α , were determined by fitting the first 40 time intervals of the averaged MSD to a power law .",
"Five milliliters of cells grown in SCD media ( OD600 = 0 . 4–0 . 7 ) were collected by centrifugation ( 3000 rpm ) for 2 min .",
"The supernatant was then removed and cells resuspended in 1 mL of SC media .",
"Four additional wash steps followed , with two 2 min spins ( 6000 rpm ) succeeded by two 1 min spins .",
"The OD600 of the final cell suspension was determined and cells diluted into fresh SC or SCD media for analysis .",
"Time-lapse epifluorescence imaging was performed 30 min after the wash .",
"Logarithmic cells growing in SCD media ( pH 5 . 0 ) were incubated with an appropriate concentration of a stock 20 mM K+Sorbate in SCD ( pH 5 . 0 ) to give rise to the corresponding final concentration of K+Sorbate .",
"Cells were treated for 30 min before imaging .",
"All experiments involving sodium azide ( NaN3 ) took place in SC or SCD media at pH ~5 . 8 .",
"Cells were treated with a final concentration of 0 . 02% NaN3 ( in water ) by two distinct protocols .",
"In one treatment , sodium azide was directly added , or 'spiked' , into a logarithmic culture .",
"In the other , cells were first washed ( via the acute starvation method ) in SC media before being resuspended into SCD media containing 0 . 02% NaN3 .",
"This treatment is referred to as 'wash' and is the condition described in the main text .",
"Cells were treated with either 15 µg/mL nocodazole ( from a 1 . 5 mg/mL stock in DMSO; Sigma-Aldrich , St . Louis , MO ) , and/or 200 µM latrunculin A ( from a 10 mM stock in DMSO; Sigma-Aldrich ) for 20 min before imaging .",
"This results in 3% DMSO as the control .",
"Cells grown in SCD media ( OD600 = 0 . 4–0 . 7 ) were collected by centrifugation ( 3000 rpm ) for 2 min and resuspended in SCD media containing the appropriate concentration of NaCl ( 0 . 4 , 0 . 6 , or 0 . 8 M ) .",
"Cells were then imaged after 10 min .",
"The protocol employed was only marginally modified from Ashe et al . ( 2000 ) .",
"Briefly , 20 µL of sample ( KWY1622 cells in culture ) was added to 20 µL of 10% TCA and immediately vortexed for 1 min .",
"Of the 40 µL solution , 10 µL was then added to 990 µL of ATP reaction buffer ( 25 mM HEPES , 2 mM EDTA , pH 7 . 75 ) .",
"After the time course , 50 µL of a luciferin/luciferase kit ( ATP Determination Kit , Sensitive Assay; B-Bridge International , Inc . , Cupertino , CA ) was directly added to 50 µL of the buffered-ATP solutions and briefly mixed by pipetting the solutions in a Corning 96-well opaque white plate ( Corning , Inc . , Tewksbury , MA ) .",
"Reactions took place in the dark for 10 min at room temperature .",
"Luminescence was then measured using a Tecan Infinite 200 Pro microplatereader ( Tecan Group Ltd . , Männedorf , Switzerland ) which was set for a 10 s integration time and no attenuation .",
"Sample aliquots were taken at 0 , 10 , 30 and 60 min and standardized to a pre-treatment control .",
"ATP concentrations were also corrected for OD600 at the beginning and end of each time course .",
"ATP standards were used to generate a standard curve to insure linearity and estimate intracellular ATP concentrations .",
"We utilized a CellASIC ONIX microfluidic profusion platform and accompanying microfluidic plates to track particles in quiescent cells ( EMD Millipore , a division of Merck KGaA , Darmstadt , Germany ) .",
"Quiescence was established by allowing cells to grow within the same SCD media for 7 days ( Laporte et al . , 2011 ) .",
"Seven-day-old media , collected from the culture by centrifuging 1mL of cells and harvesting the supernatant , was used as perfusion media in the microfluidic plate at a rate of 2 psi .",
"Cell volume measurements were performed as described in Goranov et al . ( 2009 ) .",
"In short , 1 mL of cells ( KWY4736 ) were briefly sonicated ( 1 s , 2X ) and diluted ( 1:100 ) into Isoton II Dilutent ( Beckman Coulter , Inc . , Brea , CA ) such that saturation levels fluctuated between 1–3% during measurements on a Beckman Coulter Multisizer 3 .",
"If oversaturation triggered warnings at any point , the run was terminated and sample repeated .",
"The aperture was also cleaned and flushed between each run .",
"For yeast , the particle count was set to 50 , 000 and for bacteria , 100 , 000 .",
"To quantify filamentous actin , KWY165 cells were fixed with formaldehyde at a final concentration of 4% for 15 min .",
"Fixed cells were washed twice with potassium phosphate buffer and resuspended into PBS-BSA .",
"Cells were then stained with Alexa Fluor 568-phalloidin ( Invitrogen , Thermo Fisher Scientific , Waltham , MA ) and Hoechst stain .",
"Samples were incubated in the dark at room temperature for 1 hr and washed again with PBS-BSA prior to imaging ( Laporte et al . , 2011 ) .",
"Background was subtracted from z-stacks ( 0 . 15 µm steps over 3 µm ) taken with a 100x/1 . 49NA APO TIRF oil immersion objective using a rolling ball radius of 1 pixel in ImageJ ( NIH , Bathesda , MD ) .",
"Maximal intensity projections were then blinded and cells classified based on the presence or absence of apparent filamentous or bundled actin .",
"The phluorin calibration protocol was slightly adapted from Orij et al . , ( 2009 ) to establish a standard curve .",
"50 µg/mL ( in PBS ) of digitonin was used to permeabilize cells for 10 min .",
"After washing cells in PBS and placing them on ice , cells were then incubated in citric acid/Na2HPO4 buffer with pH values ranging from pH 5 . 0 to pH 8 . 0 .",
"phluorin emission intensity ratios were then calculated from calibration images ( taken with a 100x/1 . 49NA APO TIRF oil immersion objective ) using a custom script in ImageJ ( NIH ) and compared to the pH values of the corresponding buffer .",
"The resulting curve was used to estimate experimental intracellular pH values generated from emission ratios produced from the same ImageJ script .",
"The standard curve and all experiments were performed using KWY3661 .",
"Nuclear volume measurements were conducted on KWY4796 cells expressing TetR-GFP ( and DsRed-HDEL ) .",
"Cells were grown in complete synthetic medium with 2% dextrose overnight at 25ºC to OD 0 . 5–0 . 8 .",
"Cells were then treated as indicated and transferred to Nunc Lab-Tek chambers ( Thermo Fisher Scientific ) coated with concanavalin A at OD 0 . 2 .",
"To wash cells into different medium conditions , cells were pelleted at 6000 rpm in a table top centrifuge and washed four times with synthetic medium lacking dextrose and then resuspended in synthetic medium either lacking dextrose or supplemented with 2% dextrose .",
"Three-dimensional stacks with 0 . 24 µm z stepping were acquired on a Zeiss Axio Observer Z1 spinning disk confocal microscope equipped with a motorized piezo stage ( Applied Scientific Instrumentation , Inc . ) and an EMCCD camera using a 100x 1 . 46 Oil , alpha Plan Apochromat objective .",
"TetR-GFP was excited with a 488 nm laser line and scanned using a Quad Band 'RQFT' 405/488/568/647 dichroic and a 520/35 band pass emission filter .",
"Image stacks were processed in Imaris ( Bitplane AG , Zurich , Switzerland ) to reconstruct nuclear volume .",
"S . cerevisiae cells with GFP-Tub1 integrated at the URA3 locus were grown overnight to mid-log phase in SCD media ( pH 5 . 0 ) and then treated by acute glucose starvation ( as above ) .",
"The cells were then adhered to Concanavalin A treated 384-well glass plates while maintaining the experimental condition .",
"Cells were imaged by epifluorescence time-lapse microscopy for 20 min: a z-stack of five images , each 1 µm apart was acquired once every minute .",
"The resulting images were processed by a maximum intensity projection and the number of microtubule elongation events per minute was manually scored by an experimental blinded to the sample identity .",
"KWY165 yeast cells were grown in 400 mL synthetic complete + 2% dextrose ( SCD ) media and harvested during log-phase growth ( OD600 ~ 0 . 8 ) .",
"Cells were spun down , supernatant removed , and cells re-suspended in 50 mL synthetic complete without dextrose ( SC ) media , and then repeated with two further wash steps .",
"The sample was then finally re-suspended into 4 mL SC media .",
"2 mL were added to 48 mL SCD media , and 2 mL were added to 48 mL SC media .",
"Both samples were incubated at 25°C for 30 min .",
"Then the samples were spun down , supernatant removed , and cells re-suspended into 1 mL water , which was repeated two more times .",
"After the final wash step , the supernatant was removed , and the cell pellet was lyophilized under 0 . 040 mbar pressure at -50°C for 24 hr using the Alpha 2–4 LDplus freeze-dryer ( Martin Christ GmbH , Osterode am Harz , Germany ) .",
"The dry mass of the cells were measured , and the cells were re-suspended in water and counted using a hemocytometer to determine the average mass per cell for both the non-starved and starved cells .",
"For mass measurements with raffinose , the protocol was the same except dextrose was replaced with raffinose .",
"KWY5112 cells expressing GFP and N-terminally tagged Vph1-mCherry were grown in SCD media and harvested during log-phase growth .",
"The standard acute glucose starvation protocol was performed , and the cells were placed on concanavalin A-coated Lab-Tek chambered slides ( Thermo Fisher Scientific Inc , Waltham , MA ) and imaged on a spinning disk confocal microscope ( Carl Zeiss AG , Jena , Germany ) .",
"The entire cell volume was imaged using a z-stack ( 0 . 24 µm z-slice height ) under 488 nm ( GFP ) and 561 nm ( mCherry ) laser excitation with a 100x α Plan-Apochromat objective .",
"The z-stack images were segmented and the vacuolar and cellular volumes were computed using custom Python scripts .",
"To calculate the change in accessible cytoplasmic volume after glucose starvation , the total cell shrinkage ( 15% ) , nuclear volume reduction ( 4 . 60 fL to 4 . 12 fL ) , and vacuolar volume expansion ( 25 to 40% of cellular volume ) were taken into account .",
"The accessible cytoplasmic volumes were computed using:ANS=CNS−VNS−NNSAS=CS−VS−NSCS=0 . 85·CNSVNS=0 . 25·CNSVS=0 . 40·CS where A , C , V and N denote the accessible cytoplasmic volume , total cell volume , vacuolar volume , and nuclear volume , respectively , the subscripts NS and S denote the non-starved and starved conditions , and NNS = 4 . 60 fL and NS = 4 . 12 fL .",
"For stiffness measurements of yeast cells an AFM ( Cellhesion 200 , JPK Instruments ) was mounted on an inverted microscope ( Observer . Z1 , Zeiss ) .",
"DIC images were recorded using a 40x/1 . 2 C-Apochromat water immersion objective ( Zeiss ) .",
"Tip-less cantilevers ( NSC12/noAl , Mikromasch , 350 µm long , nominal spring constant 150 mN·m-1 ) were modified with PDMS ( Sylgard 184 , Dow Corning ) wedges to correct for tilt angle of the cantilever and to be able to confine yeast cells between two parallel plates [Stewart et al . , 2013] .",
"KWY165 cells in log-phase growth were spheroplasted for 1 hr in 200 µg·mL-1 zymolyase 100T ( Amsbio ) in SCD plus 1 M sorbitol at 30°C .",
"Spheroplasts were then either washed into SCD plus 1 M sorbitol ( non-starved treatment ) or SC plus 1 M sorbitol ( starved treatment ) for 1 hr at room temperature before plated onto concanavalin A-coated glass-bottomed Petri dishes ( WPI ) .",
"For each stiffness measurement , an isolated spheroplast was identified and DIC imaged .",
"The image was used to determine the spheroplast diameter .",
"Thereafter , the cantilever wedge was positioned ~20 µm above the spheroplast and lowered at 1 µm·s-1 until an upward force of 10 nN was recorded .",
"The cantilever height was maintained for 10 s before the cantilever was raised 10 µm at 1 µm·s-1 .",
"The cantilever was then lowered onto the same cell , paused and raised successively for seven further cycles .",
"The cantilever height and the force acting on the cantilever were recorded at 40 Hz .",
"The slope ( least-squares fit , nN·µm-1 ) of the data points between 3 and 7 nN of force of the downward phase of each cycle was determined .",
"The mean of these slopes was used as the measure of spheroplast stiffness .",
"For budding yeast cells , yYB5978 was grown overnight in YPD ( 1% yeast extract , 2% peptone , 2% dextrose ) at 30°C and diluted to OD600 = 0 . 1 in 25 mL YPD .",
"Two hours later , mating pheromone ( α-factor , Sigma T-6901-1mg ) was added at a concentration of 8 ng/mL in YPD containing 20 µg/mL casein as a blocking agent .",
"After 4–5 hr , the shmooed cells were washed into either SCD or SC ( for non-starved and starved conditions respectively ) and incubated at room temperature for 1 hr .",
"Cells were then spun down and resuspended into either SCD or SC containing 1 M sorbitol and 200 µg/mL zymolyase 100T ( Amsbio ) .",
"Cells were incubated at 30°C for 1 hr .",
"Spheroplasting efficiency was examined upon cell lysis in water .",
"For fission yeast cells , KWY6241 was grown overnight in Edinburgh minimal medium + 2% dextrose ( EMMD ) to log-phase , then washed into either EMMD or EMM ( EMMD with no dextrose ) and incubated at room temperature for 1 hr .",
"Cells were then spun down and resuspended into either EMMD or EMM containing 1 . 2 M sorbitol and 5 mg/mL lysing enzmes from Trichoderma harzianum ( Sigma L1412 ) and 0 . 5 mg/mL zymolyase 100T ( Ambsio ) .",
"Cells were incubated at 30°C for 1 hr .",
"Spheroplasting efficiency was examined upon cell lysis in water .",
"For experiments in which the cells were returned to dextrose media , the cells were spun down and resuspended into dextrose-containing medium ( SCD + 1 M sorbitol for S . cerevisiae or EMMD + 1 . 2 M sorbitol for S . pombe ) ."
]
] | [
"The organization and biophysical properties of the cytosol implicitly govern molecular interactions within cells .",
"However , little is known about mechanisms by which cells regulate cytosolic properties and intracellular diffusion rates .",
"Here , we demonstrate that the intracellular environment of budding yeast undertakes a startling transition upon glucose starvation in which macromolecular mobility is dramatically restricted , reducing the movement of both chromatin in the nucleus and mRNPs in the cytoplasm .",
"This confinement cannot be explained by an ATP decrease or the physiological drop in intracellular pH . Rather , our results suggest that the regulation of diffusional mobility is induced by a reduction in cell volume and subsequent increase in molecular crowding which severely alters the biophysical properties of the intracellular environment .",
"A similar response can be observed in fission yeast and bacteria .",
"This reveals a novel mechanism by which cells globally alter their properties to establish a unique homeostasis during starvation ."
] | [
"Most organisms live in unpredictable environments , which can often lead to nutrient shortages and other conditions that limit their ability to grow .",
"To survive in these harsh conditions , many organisms adopt a dormant state in which their metabolism slows down to conserve vital energy .",
"When the environmental conditions improve , the organisms can return to their normal state and continue to grow .",
"The interior of cells is known as the cytoplasm .",
"It is very crowded and contains many molecules and compartments that carry out a variety of vital processes .",
"The cytoplasm has long been considered to be fluid-like in nature , but recent evidence suggests that in bacterial cells it can solidify to resemble a glass-like material under certain conditions .",
"When cells experience stress they stop dividing and alter their metabolism .",
"However , it was not clear whether cells also alter their physical properties in response to changes in the environment .",
"Now , Joyner et al . starve yeast cells of sugar and track the movements of two large molecules called mRNPs and chromatin .",
"Chromatin is found in a cell compartment known as the nucleus , while mRNPs are found in the cytoplasm .",
"The experiments show that during starvation , both molecules are less able to move around in their respective areas of the cell .",
"This appears to be due to water loss from the cells , which causes the cells to become smaller and leads to the interior of the cell becoming more crowded .",
"Joyner et al . also observed a similar response in bacteria .",
"Furthermore , Joyner et al . suggest that the changes in physical properties are critical for cells to survive the stress caused by starvation .",
"A separate study by Munder et al . found that when cells become dormant the cytoplasm becomes more acidic , which causes many proteins to bind to each other and form large clumps .",
"Together , the findings of the studies suggest that the interior of cells can undergo a transition from a fluid-like to a more solid-like state to protect the cells from damage when energy is in short supply .",
"The next challenge is to understand the molecular mechanisms that cause the physical properties of the cells to change under different conditions ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine"
] | The CUL4-DDB1 ubiquitin ligase complex controls adult and embryonic stem cell differentiation and homeostasis | elife-07539-v2 | [
[
"Stem cells posses the unique properties of self-renewal and the capacity to differentiate to multiple cell types .",
"In the case of hematopoietic stem cells ( HSC ) they are rare and specialized cells , which are able to give rise to all blood lineages .",
"The balance between HSC self-renewal and differentiation needs to be tightly regulated in order to keep the HSC pool size as well as to constantly replenish mature blood cells ( Orkin and Zon , 2008 ) .",
"HSC function is governed extrinsically by cytokines ( Zsebo et al . , 1990; de Sauvage et al . , 1996 ) and developmental signals ( Stier et al . , 2002; Zhang et al . , 2003 ) and intrinsically by transcription factors ( Wilson et al . , 2004; Tothova et al . , 2007; Reavie et al . , 2010 ) , cell cycle regulators ( Cheng et al . , 2000 ) and metabolic pathways ( Nakada et al . , 2010 ) .",
"However , little is known about how HSCs are regulated at the post-translational level .",
"The ubiquitin-dependent proteasome degradation system ( UPS ) is a primary mechanism that controls protein turnover and activation .",
"UPS acts via three sequential enzymes: an E1 ubiquitin activating enzyme , an E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase ( Crusio et al . , 2010 ) .",
"Among these three enzymes , E3 ubiquitin ligases confer substrate specificity .",
"An E3 ubiquitin ligase is a multi-subunit complex that recognizes and binds specific target proteins via substrate recognizing subunits .",
"In HSCs , it has been shown that Fbw7 , an E3 ubiquitin ligase member , governs quiescence of HSCs ( Matsuoka et al . , 2008; Thompson et al . , 2008 ) .",
"Itch and c-Cbl , other two E3 ubiquitin ligases , have been reported to negatively regulate HSC homeostasis and function ( Rathinam et al . , 2008; Rathinam et al . , 2011 ) .",
"In other stem cell systems , it has been shown that Huwe1 , a HECT domain containing E3 ubiquitin ligase , regulates proliferation and differentiation of neural progenitor cells as well as embryonic stem cells ( ESC ) ( D'Arca et al . , 2010 ) .",
"Protein levels of OCT4 and NANOG , transcription factors required for pluripotency , are modulated in an ubiquitin-dependent manner ( Xu et al . , 2009; Ramakrishna et al . , 2011; Buckley et al . , 2012 ) , suggesting key roles of E3 ligase complexes for ESC differentiation .",
"Recently we mapped the ubiquitinated protein landscape in mouse ESC and identified critical UPS members regulating ESC pluripotency and differentiation ( Buckley et al . , 2012 ) .",
"DNA damage binding protein 1 ( Ddb1 ) , a component of the Cullin4-containing E3 ubiquitin ligase , was originally identified as a protein involved in the nucleotide excision repair pathway .",
"DDB1 heterodimerizes with DDB2 and shows high affinity for UV-induced DNA damage sites ( Batty et al . , 2000 ) .",
"Once bound to a damaged site , the CUL4-DDB1 complex ubiquitinates DDB2 and targets it for degradation , facilitating subsequent repair events ( Sugasawa et al . , 2005 ) .",
"The CUL4-DDB1ligase is a multi-component complex .",
"Through its C-terminus , CUL4A or CUL4B binds to the RING finger protein to interact with the E2 conjugating enzyme .",
"On its N-terminus , CUL4A or CUL4B binds to DDB1 to recruit CUL4-DDB1 associated factors ( DCAF ) , a family of WD40 repeat proteins which confer substrate specificity ( Angers et al . , 2006; He et al . , 2006; Higa et al . , 2006 ) .",
"The CUL4-DDB1 ligase has been shown to target several substrates for ubiquitin dependent degradation .",
"The list of substrates include the DNA replication licensing factor Cdt1 ( Higa et al . , 2003; Hu et al . , 2004 ) , the cell cycle inhibitor p27Kip1 ( Bondar et al . , 2006 ) and Cdkn1aCip1 ( Abbas et al . , 2008; Nishitani et al . , 2008 ) , the histone methyltransferase PR-Set7 ( Oda et al . , 2010; Tardat et al . , 2010 ) , and Epe1 , a JmjC domain-containing histone demethylase in fission yeast ( Braun et al . , 2011 ) .",
"More recently it has been found that the CUL4-DDB1 complex can be modulated by binding of the drug , lenalidomide leading to degradation of lymphoid transcription factors IKZF1 and IKF3 in multiple myeloma cells ( Fischer et al . , 2014; Kronke et al . , 2014 ) .",
"These findings suggest that the CUL4-DDB1 ligase complex has numerous substrates that it affects a variety of cellular functions , and that the complex can be altered with targeted therapeutics .",
"Intriguingly , germline Cul4a deleted mice are viable and display no gross abnormality ( Liu et al . , 2009 ) , possibly due to redundancy with Cul4b , whereas Ddb1 deletion is embryonic lethal and embryos are not seen past E12 . 5 ( Cang et al . , 2006 ) .",
"Conditional inactivation of Cul4a in the skin leads to resistance to UV-induced skin carcinogenesis ( Liu et al . , 2009 ) .",
"Specific deletion of Ddb1 in brain results in elimination of neuronal progenitor cells , hemorrhages in brain , and neonatal lethality ( Cang et al . , 2006 ) .",
"DDB1 also plays a role in ESC self-renewal , and silencing of Ddb1 led ESC to differentiate ( Buckley et al . , 2012 ) .",
"To investigate the role of the DDB1 in hematopoietic stem cells , we inactivated the Ddb1 gene in hematopoietic stem and progenitor cells ( HSPC ) and at different developmental stages .",
"Here we report that Ddb1 loss impairs HSPC function in both the adult bone marrow and the fetal liver .",
"More specifically , Ddb1 deletion leads to induction of DNA damage , rapid induction of apoptosis , and Trp53 response , resulting in bone marrow failure and acute lethality .",
"However , deletion of Ddb1 had no effect on resting mature lymphoid cells and whereas in proliferating embryonic stem cells ( ESC ) silencing of Ddb1 led to loss of pluripotency without effects on cell survival .",
"Our results demonstrate CUL4-DDB1 is a novel regulator of stem cell homeostasis ."
],
[
"To study the role of distinct ubiquitin ligases in the biology of HSCs , we initially performed a meta-analysis of genome-wide expression in lineage-Sca1+cKit+ ( LSK ) cells , a population enriched for HSCs , and found several E3 ligases among the top 20% highly expressed genes , including the already reported HSC regulators Fbw7 ( Thompson et al . , 2008 ) , Cul1 , Itch ( Rathinam et al . , 2011 ) and c-Cbl ( Rathinam et al . , 2008 ) ( Figure 1a ) .",
"Both genes of the Cul4DDB1 complex were also highly expressed in LSKs ( Figure 1a ) , suggesting that this E3 complex could be important in early hematopoiesis .",
"We further examined the expression of Ddb1 in long-term HSCs ( LT-HSC , CD150+CD48-LSK ) and downstream progenitor populations .",
"It was found that Ddb1 was expressed at a low level in quiescent LT-HSCs , and significantly upregulated in multipotent progenitors ( MPP , CD150-CD48+LSK ) , a proliferating progenitor subset .",
"Ddb1 expression remained constant in later progenitor populations ( Figure 1b ) .",
"The expression pattern of Ddb1 suggests its potential role in hematopoiesis . 10 . 7554/eLife . 07539 . 003Figure 1 . Ddb1 is highly expressed in the hematopoietic system .",
"( a ) Expression ranking of Cul4-Ddb1 components in LSKs compared to all probes available in microarray .",
"Microarray was performed on LSK cells .",
"The expression value of all probes were ranked from high to low .",
"( b ) Quantitative PCR of Ddb1 in hematopoietic populations . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 003 To investigate the importance of Ddb1 function in hematopoiesis , we generated Ddb1f/f::Vav1Cre+ mice .",
"The Vav1 promoter drives the expression of Cre recombinase in entire hematopoietic compartment during embryonic development ( ~E13 . 5 ) from HSC and progenitors to mature cells .",
"Efficient deletion of Ddb1 in bone marrow was confirmed by qPCR ( Figure 2a ) .",
"Ddb1f/f::Vav1Cre+ mice were born at normal frequencies and were indistinguishable from littermates .",
"However , Ddb1f/f::Vav1Cre+ animals died rapidly after birth ( Figure 2b ) .",
"Peripheral blood analysis at day 7 showed that Ddb1f/f::Vav1Cre+ mice had significantly decreased counts of white blood cells , red blood cells and platelets compared to littermates ( Figure 2c , d ) .",
"Moreover , the cellularity and size of thymus and spleen were significantly reduced ( Figure 2e , f ) .",
"When analyzed by flow cytometry , lineage-Sca1+cKit+ ( LSK ) cells , a population enriched for HSCs , and cKit+ progenitors were undetectable ( Figure 2g ) .",
"Mature lymphoid ( CD4+CD8+ in thymus , B220+IgM+ in spleen ) and myeloid ( Gr1+Mac1+ in spleen ) cells were severely reduced ( Figure 2h ) .",
"Since Vav1Cre expression starts as early as embryonic day 13 . 5 ( E13 . 5 ) ( Stadtfeld and Graf , 2005 ) , we hypothesized that the pan-cytopenia in Ddb1f/f::Vav1Cre+ neonates was due to defects initiated during fetal hematopoiesis .",
"Analysis of E16 . 5 fetal liver of Ddb1f/f::Vav1Cre+ mice showed that the deletion of Ddb1 in fetal hematopoietic cells led to reduction of the LSK and cKit+ progenitors , as well as mature CD19+ B-lymphoid and Gr1+ myeloid cells ( Figure 2i ) .",
"Interestingly , the distribution of LT-HSC and MPP was skewed with higher frequency of LT-HSC and lower frequency of MPP cells ( Figure 2i ) .",
"Genome-wide gene expression analysis revealed that Ddb1-deficient LSKs up-regulated genes associated with stem cell identity ( Mecom , Thy1 , Angpt1 ) and down-regulated genes associated with differentiation ( Figure 2j , k ) , consistent with the phenotypic enrichment of the HSC population .",
"Overall , our data demonstrate that Ddb1-deletion leads to cytopenia and neonatal lethality , confirming that Ddb1 expression is absolutely required for fetal hematopoiesis . 10 . 7554/eLife . 07539 . 004Figure 2 . Abrogation of fetal hematopoiesis in Ddb1f/fVav1Cre+ mice .",
"( a ) Quantitative PCR of Ddb1 in control and Ddb1f/f::Vav1Cre+ mice .",
"( b ) Survival curves of control and Ddb1f/f::Vav1Cre+ mice ( n = 18 per group ) .",
"( c ) Giemsa staining of peripheral blood smears from 7-day old mice .",
"( d ) Peripheral blood counts in 7-day old mice ( n = 4 per group ) .",
"WBC: white blood cells ( p=0 . 0054 ) .",
"RBC: red blood cells ( p=0 . 0007 ) .",
"PLT: platelets ( p=0 . 10 ) .",
"Black bar indicates average .",
"( e ) Total cell numbers in thymi ( p=0 . 0050 ) and spleens ( p=0 . 0016 ) of 7-day old mice ( n = 4 per group ) .",
"Black bar indicates average .",
"( f ) Representative pictures of spleens from 7-day old mice .",
"( g ) Representative FACS plots of bone marrow of 7-day old mice .",
"( h ) Representative FACS plots of thymi and spleens of 7-day old mice .",
"( N=3 ) .",
"( i ) Representative FACS plots of fetal livers at embryonic day 16 . 5 .",
"( n=3 )",
"( j ) Gene set enrichment analysis ( GSEA ) of gene expression analysis performed on fetal LSKs at embryonic day 16 . 5 .",
"( k ) Heatmap of gene expression analysis performed on fetal LSKs at embryonic day 16 . 5 .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 004 To circumvent the neonatal lethality of Ddb1f/f::Vav1Cre+ mice , we turned to the Mx1Cre strain that can be induced to delete the target gene .",
"In Ddb1f/f::Mx1Cre+mice , Ddb1 was deleted in hematopoietic compartment of adult mice by injecting polyinosine-polycytidine ( polyI:C ) to induce interferon α response .",
"The floxed allele of Ddb1 was recombined efficiently after polyI:C injection in all hematopoietic tissues including the bone marrow ( Figure 3a ) .",
"As a result , the Ddb1 protein was undetectable in polyI:C injected Ddb1f/f::Mx1Cre+ bone marrow ( Figure 3b ) .",
"Strikingly , all Ddb1f/f::Mx1Cre+ animals died within 3 weeks post Ddb1 deletion ( Figure 3c ) .",
"To investigate the cause of the lethality , we analyzed Ddb1 deficient mice 7 days after polyI:C injection .",
"Peripheral blood counts showed that the Ddb1 loss resulted in significant reduction in the total number of white blood cells and platelets ( Figure 3d ) .",
"No reduction was found in red blood cells , however enucleated red blood cells have a half-life of approximately 40 days suggesting DDB1 deficient mice succumb to hematopoietic failure prior to red blood cell turnover .",
"Ddb1f/f::Mx1Cre+ bone marrow hypo-cellularity was evident from histological examination ( Figure 3e ) , which was mostly accounted by cellularity decrease of myeloid lineage ( Figure 3f , g ) .",
"To understand the kinetics of Ddb1 deletion effects , we analyzed HSC and progenitor cells at different time points after Ddb1 deletion .",
"cKit+ progenitors ( both MP and LSK ) were decreased in cell number at day 5 and further more at day 7 ( Figure 3h , i ) .",
"We used the SLAM markers ( CD150 and CD48 ) to further characterize the long-term HSCs ( LT-HSC , CD150+CD48-LSK ) , short-term HSCs ( ST-HSC , CD150+CD48+LSK ) and multipotent progenitors ( MPP , CD150-CD48+LSK ) populations .",
"It was found that the frequencies of LT-HSC , ST-HSC and MPP subsets were distorted upon Ddb1 deletion , as there was a significant relative over-representation of LT-HSCs ( Figure 3h ) , similar to the findings in fetal hematopoiesis .",
"In terms of cell number , MPPs , but not ST- and LT-HSCs , were decreased at day5 , which was followed by the decrease of all HSPCs subsets at day7 ( Figure 3h ) .",
"This data suggest that Ddb1 deletion has a greater impact on proliferative populations , in agreement to the expression analysis presented earlier .",
"Together , these results demonstrate that the deletion of Ddb1 leads to acute loss of proliferating HSPCs and their downstream progeny , resulting in bone marrow failure and lethality . 10 . 7554/eLife . 07539 . 005Figure 3 . Deletion of Ddb1 in Ddb1f/f::Mx1Cre+ mice leads to bone marrow failure and acute lethality .",
"( a ) PCR on genomic DNA from tail or polyI:C injected bone marrow to detect wild type ( wt ) , “floxed” ( flox ) and recombined ( del ) alleles of Ddb1 locus .",
"( b ) Western blot of bone marrow cells from polyI:C injected animals .",
"( c ) Survival curve of mice after polyI:C injection ( n = 8 per group ) .",
"( d ) Peripheral blood counts 7 days after polyI:C injection ( n = 6 per group ) .",
"( WBC and PLT p=<0 . 0001 ) .",
"Black bar indicates average .",
"( e ) H&E staining of tibia sections 7 days after polyI:C injection .",
"( f ) Representative FACS plots of bone marrow cells .",
"( g ) Cellularity of bone marrow 7 days after poly:C injection .",
"N=5 per group .",
"( h ) Representative FACS plots of bone marrow cells after polyI:C injection .",
"( i ) Cell numbers of total bone marrow and stem and progenitor cell populations in mice after polyI:C injection ( n = 5 per group ) .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 .",
"( j ) Non-polyI:C injected Ddb1f/f::Mx1Cre+ mice were lethally irradiated and transplanted with wild type bone marrows .",
"Eight weeks after engraftment , DDB1 deletion was induced by polyI:C injection .",
"Survival of these chimera mice ( N= 4 per group ) was followed and compared to polyI:C injected Ddb1f/f::Mx1Cre+ mice . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 005 The Cre recombinase under control of the Mx1 promoter is also expressed in other IFNα-responsive tissues besides hematopoietic cells , including the liver , lungs , and heart ( Kuhn et al . , 1995 ) , however no gross abnormality was found in Ddb1f/f::Mx1Cre+ mice at the time-point of the analysis ( data not shown ) .",
"To further establish that Ddb1 deficient mice died of bone marrow failure , we transplanted wild type bone marrow cells into lethally irradiated non-polyI:C injected Ddb1f/f::Mx1Cre+ and control mice .",
"Eight weeks after bone marrow transplant , polyI:C was injected into the transplanted recipient mice to induce Ddb1 deletion .",
"In this case , the majority of chimeric Ddb1f/f::Mx1Cre+ mice survived significantly longer than Ddb1 deficient mice ( Figure 3j ) , strongly suggesting that the acute lethality observed in polyI:C injected Ddb1f/f::Mx1Cre+ mice is due to bone marrow ablation .",
"Next , we further addressed the function of the DDB1-deleted progenitors and stem cells both in vitro and in vivo Initially , methylcellulose cultures and CFU-S assays were performed to test the differentiation function of DDB1 deficient HSPCs .",
"Strikingly , DDB1 deficient bone marrow cells from polyI:C injected Ddb1f/f::Mx1Cre+ animals were not able to form colonies in cytokine-supplemented in vitro culture as well as in spleens of host mice ( Figure 4a , b ) .",
"Identical results were obtained when we deleted Ddb1 in progenitor cells using a Cre-expressing retrovirus ( Figure 4c ) .",
"These data reveal that Ddb1 deletion impairs differentiation ability of the HSC and progenitor cells .",
"HSC function is assayed by bone marrow transplantation ( BMT ) .",
"To this end , we transplanted DDB1 deficient bone marrow cells and littermate control cells into lethally irradiated recipient mice .",
"All mice receiving Ddb1 deficient cells died within 3 weeks post BMT while those receiving control cells had a normal life span ( Figure 4d ) , indicating that Ddb1 deficient cells are not able to repopulate the hematopoietic system . 10 . 7554/eLife . 07539 . 006Figure 4 . Ddb1 deletion impairs the differentiation of hematopoietic stem and progenitor cells .",
"( a ) Colony numbers and representative images from methylcellulose assay with bone marrow cells from polyI:C injected mice .",
"( b ) Colony numbers and representative images from CFU-S assay with bone marrow cells from polyI:C injected mice .",
"( c ) Bone marrow progenitor cells of Ddb1f/f::Mx1Cre+ mice were infected with retrovirus expressing either control GFP or Cre recombinase .",
"Colony numbers were scored in methylcellulose assays .",
"( d ) Survival curve of recipient mice after bone marrow transplantation ( n = 6 per group ) .",
"Donor cells were from Ddb1f/f::Mx1Cre+ or control mice injected with polyI:C .",
"( e ) Representative FACS plots of bone marrow cells in recipient mice 7 days post Ddb1 deletion .",
"Donor cells were from non polyI:C injected mice , and Ddb1 deletion was induced in recipient mice 8 weeks after engraftment .",
"( f ) Chimerism of peripheral blood in recipient mice ( n=5 per group ) .",
"Donor cells were a mixture at 50:50 ratios of wild type CD45 . 1+ cells and Ddb1f/f::Mx1Cre+ CD45 . 2+ cells ( or control CD45 . 2+ cells ) .",
"Ddb1 deletion was induced in recipient mice 8 weeks after engraftment .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 006 To rule out non-cell autonomous effects of Ddb1 deletion ( i . e . effects in HSC niches ) , we transplanted cells from non-polyI:C injected Ddb1f/f::Mx1Cre+mice into lethally irradiated wild type recipient mice .",
"Eight weeks after engraftment , polyI:C was injected into the recipients to induce the Ddb1 deletion in hematopoietic cells of the recipients .",
"DDB1-deficient cells ( CD45 . 2+ ) lost representation ( Figure 4e ) .",
"Similar to non-transplant settings , cKit+ progenitors and stem cells derived from DDB1 deficient donor cells were significantly reduced and more apoptotic as shown by AnnexinV/7AAD staining ( Figure 4e ) .",
"These results further prove that the effects of Ddb1 deletion on HSPC are cell-autonomous .",
"Next , we performed competitive BMT .",
"Ddb1f/f::Mx1Cre+ and control bone marrow cells ( CD45 . 2+ ) were mixed with equal number of wild type counterparts ( CD45 . 1+ ) and transplanted into lethally irradiated recipients .",
"Eight weeks after engraftment , polyI:C was injected into the recipients to induce Ddb1 deletion .",
"Peripheral blood of the recipients was analyzed at different time points after the Ddb1 deletion .",
"Control cells maintained CD45 . 2+ chimerism through out the time course of the analysis .",
"However , DDB1-deficinet cells were not able to compete with wild type counterparts ( Figure 4f ) .",
"DDB1-deficient myeloid cells ( Mac1+Gr1+ ) were significantly reduced compared to wild type cells as early as 2 weeks after the Ddb1 deletion .",
"DDB1-deficient lymphoid cells ( CD3+ and B220+ ) were also reduced with slower kinetics ( Figure 4f ) .",
"These data suggest that Ddb1 deletion specifically targets expanding progenitors and highly proliferating cells inhibiting the stem and progenitor populations to replenish the hematopoietic system .",
"To gain further insights into DDB1-mediated mechanisms of action , we examined apoptosis status on whole tibia section by TUNEL analysis .",
"We found that DDB1-deficient bone marrow displayed significant apoptosis ( Figure 5a ) .",
"More specifically , we found that DDB1-deficient progenitors ( both LSKs and MPs ) were more apoptotic as shown by the AnnexinV staining , but not lineage+ cells ( Figure 5b ) .",
"In line with these results , DDB1-deficient progenitor cells had elevated protein levels of phospho-Trp53 , the activated form of Trp53 .",
"In addition , cyclin-dependent kinase inhibitor 1A ( Cdkn1aCip1 ) , a transcription target of Trp53 , was accumulated at the protein and mRNA levels ( Figure 5c , d ) .",
"Signs of DNA damage were also observed in Ddb1-deficient LT- , ST-HSCs and MPPs , but not in lineage+ cells , as revealed by γH2Ax staining , a marker for double strand DNA breaks as well as by 53BP1 foci ( Figure 5e , f ) .",
"Collectively these results demonstrate that Ddb1 deletion leads to DNA damage , rapid apoptosis and Trp53 pathway activation in HSPCs . 10 . 7554/eLife . 07539 . 007Figure 5 . Ddb1 deletion induces DNA damage and apoptosis in progenitor cells .",
"( a ) TUNEL staining on tibia sections after polyI:C injection .",
"( b ) Percentage of AnnexinV+ cells gated on different populations after polyI:C injection .",
"Western blot",
"( c ) and qPCR",
"( d ) in lineage negative bone marrows after polyI:C injection .",
"( e ) Histogram of intracellular γH2Ax staining gated on HSPC sub-populations and lineage positive cells after polyI:C injection .",
"( f ) Immunofluoresence staining of γH2Ax and 53BP1 on flow-sorted LSK cells after polyI:C injection .",
"( g ) Representative FACS plots of bone marrow cells 5 days post polyI:C injection .",
"( h ) Frequency and absolute number of progenitor cells ( n = 4 per group ) .",
"One of 2 independent experiments was shown .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 .",
"n . s . p>0 . 05 .",
"( i ) Survival curves of control , Trp53-/-::Ddb1f/f::MxCre+ mice , and Ddb1f/f::MxCre+ mice ( n = 5 per group , one independent experiment ) .",
"Age matched littermates on a mixed 129xC57BL/6 background were used for g-i . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 007 To access whether Ddb1 hematopoietic phenotypes were dependent on Trp53 activity , we generated Trp53-/-::Ddb1f/f::Mx1Cre+ mice and examined stem and progenitor subsets .",
"In the hematopoietic system , Trp53 deletion partially rescued the Ddb1-/- phenotype ( Figure 5g , h , i ) .",
"Both the percentage and absolute number of ST-HSC ( CD150+CD48+LSK ) , but not LT-HSC and MPP , were increased in Trp53-/-::Ddb1f/f::Mx1Cre+ compared with Ddb1f/f::Mx1Cre+animals ( Figure 5g , h ) .",
"These results suggest that Trp53 activation takes place in response to Ddb1 deletion but this activation does not account for the full spectrum of the phenotype .",
"Next , we examined whether the abrogated hematopoiesis resulted from Ddb1 silencing was specific for stem/progenitor cells .",
"To this end , Ddb1 was conditionally deleted in mature T cells using the Cd4Cre+ strain .",
"In this mouse model , Cre recombinase expression initiates at the CD4+CD8+T-lymphocyte stage of development , a population characterized by minimal cell proliferation .",
"DDB1 was efficiently deleted at protein and mRNA levels in total thymocytes ( Figure 6a , b ) .",
"The residual Ddb1 expression in total thymocytes could be attributed to the existence of DN cells in which the Cre recombinase was not expressed .",
"We then examined T cell profiles in thymi and peripheral lymphoid tissue of Ddb1F/F::Cd4Cre+ mice .",
"We found that these mice had normal thymocyte numbers ( Figure 6c ) , and normal CD4/CD8 cell profiles ( Figure 6d ) , confirming our hypothesis that Ddb1 deletion in mature/resting T cells did not affect T cell development .",
"To test this hypothesis further , we stimulated peripheral CD4+ cells using anti-CD3/CD28 treatment in vitro .",
"When activated , the control T cells entered cell cycle , incorporated BrdU , an analogue of thymidine which is incorporated during DNA synthesis , and underwent several rounds of cell division as shown by the dilution of CFSE labeling .",
"Strikingly , the DDB1 deficient CD4+ cells failed to proliferate ( Figure 6e ) .",
"Instead , more AnnexinV positive cells were found in the culture of DDB1-deficient CD4+ cells ( Figure 6f ) , suggesting induction of cell death .",
"Furthermore , we labeled anti-CD3/CD28 treated cells with EdU , an alternative of BrdU and easier for detection by immunoflourescence , and found that there were once more significantly fewer cells incorporating EdU when Ddb1 was deleted ( Figure 6g , h ) , suggesting Ddb1 deletion interferes with cell cycle progression .",
"Combination of BrdU and DAPI labeling clearly showed that , when stimulated to proliferate , DDB1 deficient resting cells were unable to enter S phase of the cell cycle .",
"Consistent with this result , the Cdkn1acip1 protein , a potent CDK and cell cycle inhibitor , was accumulated in DDB1-deficient cells at the protein level , but not mRNA level ( Figure 6i , j ) .",
"When proteasome-dependent protein degradation was inhibited by MG132 , Cdkn1acip1 was significantly accumulated in control cells , however less protein accumulation following proteasome inhibition was seen in DDB1-deficient cells ( Figure 6i ) , demonstrating that Cdkn1acip1 degradation is dependent on DDB1 function .",
"To prove direct interaction , Cdkn1acip1 was co-immunoprecipitated with Cul4 and DDB1 complex ( Figure 7a ) , suggesting that Cdkn1acip1 is indeed a substrate of Cul4aDDB1 .",
"In contrary , p27kip1 , another cell cycle inhibitor and suggested DDB1 substrate ( Bondar et al . , 2006 ) , was not co-immuno-precipitated with the CUL4DDB1 complex ( Figure 7b ) .",
"The above observation led us to test the hypothesis whether DDB1-deficiect phenotype in HSPCs can be restored by silencing Cdkn1acip .",
"To this end , Cdkn1a-/- mice were crossed to Ddb1f/f::Mx1Cre+ and polyI:C was administered .",
"We did not observe the restoration of stem or progenitor cell numbers ( Figure 7c ) .",
"Overall , these data demonstrate that DDB1 function is dispensable for resting T-lymphocyte homeostasis but becomes pivotal when cells enter S phase of the cell cycle . 10 . 7554/eLife . 07539 . 008Figure 6 . Ddb1 deletion is dispensable for mature T cells .",
"( a ) Western blot of DDB1 expression in total thymocytes .",
"( b ) Relative Ddb1 mRNA expression in thymocytes .",
"( c ) Total cell number of thymus of 6-week old mice ( n = 4 ) .",
"( d ) Representative FACS plots of thymus and spleen .",
"( e ) Spleen CD4+ cells were sorted and stimulated with 1 μg/ml anti-CD3/CD28 in vitro .",
"BrdU incorporation and CFSE dilution was analyzed .",
"( Anti-CD3/CD28 treated CD4+ cells were stained for AnnexinV+ by FACS and percentage of AnnexinV+ cells was shown .",
"( f ) Anti-CD3/CD28 treated CD4+ cells were stained for AnnexinV+ by FACS and percentage of AnnexinV+ cells was shown .",
"( g ) Anti-CD3/CD28 treated CD4+ cells were labeled and stained with EdU .",
"( h ) Percentage of EdU+ cells was shown .",
"More than 300 cells were included in each analysis .",
"( i ) Spleen CD4+ cells were treated with 10 μM MG132 for 2 hr and Western blot was performed .",
"Relative Cdkn1acip1 protein levels normalized to β-actin were indicated below the corresponding lanes .",
"( j ) qPCR was performed on spleen CD4+ cell .",
"Representative results of two independent experiments were shown .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 .",
"n . s . p>0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 00810 . 7554/eLife . 07539 . 009Figure 7 . Interaction of Cdkn1acip1 but not p27kip1 with the Cul4-DDB1 complex",
"( a ) 293T cells were transiently co-transfected with plasmids expressing Myc-tagged Cul4a and Cdkn1acip1 .",
"Forty-eight hours post transfection , cells were harvested and lysed .",
"Protein lysates were immunoprecipitated with anti-Myc antibody , and Western blotted for Cdkn1acip1 and DDB1 .",
"( b ) Similarly as described in",
"( a ) , p27kip1 was tested for co-immunoprecipitation with the Cul4a-DDB1 complex .",
"( c ) Representative FACS analysis of bone marrows after polyI:C injection .",
"Age matched littermates on a mixed 129xC57BL/6 background were used from 2 independent experiments ( n = 3–4 per group ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 009 Due to the striking consequences of loss of DDB1 on the HSPC proliferation and survival , but not on definitive differentiated populations , we sought to determine the effects of Ddb1 on an additional stem cell population , and more specifically in embryonic stem cells ( ESC ) .",
"ESC are highly proliferative and can differentiate into cell lineages of all three germ layers .",
"We recently reported that pluripotency and differentiation of mouse embryonic stem cells ( ESC ) are regulated at the post-translational level by the ubiquitin-proteasome systems ( UPS ) .",
"Ddb1 was identified as one of the regulators essential for ESC self-renewal in a large interference RNA ( siRNA ) screen against USP members .",
"Depletion of Ddb1 resulted in loss of ESC self-renewal and pluripotency ( Buckley et al . , 2012 ) .",
"Here , we further validated the loss-of-function effects of Ddb1 on ESC using two distinct shRNAs .",
"Consistent with our previous findings , silencing Ddb1 by shRNAs led to loss of ESC colony morphology ( Figure 8a ) and down-regulation of pluripotency factors Nanog and Oct4 , and up-regulation of genes associated with mesoderm ( Meox1 ) , ectoderm ( Gfap ) , and endoderm ( Sox17 ) early differentiation ( Figure 8b , c ) .",
"However , we did not observe aberrant cell cycle or significant change in rates of apoptosis in these cells ( Figure 8d , e ) .",
"Similar to ESC differentiated in the presence of retinoic acid , total Trp53 protein increased following silencing of Ddb1 ( Figure 8c ) .",
"These data demonstrate that DDB1 is essential for ESC pluripotency and self-renewal without being accompanied by increased apoptosis or defects in cell cycle kinetics . 10 . 7554/eLife . 07539 . 010Figure 8 . ESC loss of self-renewal after silencing of Ddb1",
"( a ) Bright field picture of ESC colony 4 day post retroviral transfection .",
"( b ) Relative expression of pluripotency genes Oct4 , and Nanog and genes representing endoderm ( Sox17 ) , mesoderm ( Meox1 ) and ectoderm ( GFAP ) by qRT-PCR 4 days post selection .",
"( c ) Western blot of ESC 4 day post infection or differentiated in the presence of retinoic acid for up to 4 days .",
"( d ) Representative FACS blot of Annexin V positive cells .",
"( n=3 ) n . s . : p>0 . 05 .",
"( e ) Representative of cell cycle analysis .",
"( n=3 ) n . s . : p>0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 010 The different roles of DDB1 in distinct cell types suggested that cellular contexts are important for DDB1 function .",
"One possibility is that the CUL4-DDB1 complex utilizes distinct DCAFs in different cells .",
"To this end , we sought to identify DDB1-interacting proteins .",
"We identified interacting proteins in three distinct cell types .",
"The different cell types expressed DDB1 protein at similar levels ( Figure 9a ) .",
"First , DDB1-interacting proteins were identified in ESC by targeting DDB1 in tandem with StrepII/Flag tags in the Col1A locus and generating a doxycycline-inducible ESC cell line ( Figure 9b , d ) .",
"We also transiently expressed DDB1 in tandem with HA/Flag tags in 293T cells , and stably transduced DDB1 in tandem with Strep/Flag tags in a promyelocytic leukemia cell line capable of hematopoietic differentiation ( HL60 ) ( Figure 9c ) .",
"Interacting proteins were identified using liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) .",
"We identified 140 proteins in ESC , 110 proteins in 293T , and 128 in HL60 cells ( Supplementary file 1 ) .",
"Interestingly very little overlap was found between subsets except CUL4A and proteins that make up the 26S proteasome .",
"Another subset of proteins identified in ESC and 293T analyses were known proteins found in CUL4DDB1 complexes ( CUL4B , CUL4B , VprBP , DDA1 ) ( Figure 10a , b ) .",
"Interaction of VprBP and DDB1 was validated in 293T cells ( Figure 10d ) .",
"One subset of proteins that was of importance was the DCAF proteins since they are the substrate recognizing proteins in the CUL4DDB1 complex ( Lee and Zhou , 2007 ) .",
"In ESC we identified DCAF11 , DCAF15 , DCAF4 , DCAF8 , whereas in the hematopoietic cell line HL60 only DCAF7 was identified suggesting that differing complexes between cell types maybe responsible for the loss of DDB1 phenotype in different cell types ( Figure 7a , c ) .",
"To determine the DCAF proteins that responsible for the ESC DDB1-loss phenotype , we performed an RNAi screen targeting 15 known protein-members of the Cul4DDB1 complex in ESC .",
"Utilizing a reporter Nanog-GFP cell line as a marker of pluripotency we identified that of the DDB1-interacting DCAF proteins found in ESC silencing of DBA1 , VprBP , and DCAF11 led to ESC differentiation ( Figure 10e ) .",
"Furthermore , depletion of DDA1 , VprBP , and DCAF11 led to down-regulation of transcripts associated with ESC pluripotency ( Figure 10g ) and lead to morphology changes consistent with differentiation ( Figure 10f ) .",
"To determine if silencing of these DCAFs ( DDA1 , VprBP or DCAF11 ) in HSPC is also able to affect differentiation and/or maintenance , we transduced bone marrow-purified HSPC ( LSK ) cells with retroviruses expressing shRNAs against the selected DCAF genes .",
"Interestingly , DDA1 silencing had no effect on colony formation in methylcellulose cultures , whereas a mild reduction in colonies was seen when VprBP was silenced .",
"On the other hand , silencing of DCAF11 lead to a significant ( greater than 50% ) reduction in colony formation ( Figure 10h ) .",
"shRNA silencing was confirmed to be greater than 60% with all shRNAs ( Figure 10i ) .",
"These findings are consistent with the levels of DCAF11 expression in both LSK and Lineagenegc-Kit+ HSPC ( Figure 10j ) .",
"These findings suggest that different phenotypes in ESC , HSPC , and T-lymphocytes could be attributed to distinct substrate recognition by DDB1-associated DCAFs . 10 . 7554/eLife . 07539 . 011Figure 9 . Tagged expression of DDB1 in ESC .",
"( a ) Western blot of DDB1 in different cell types .",
"( b-c )",
"Western blot of total protein and immunoprecipitated of tagged-DDB1 following Doxycycline induction in ESC",
"( b ) and HL-60",
"( c ) .",
"( d ) Silver staining of eluted tagged protein used for one mass spectrometry experiment in ESC . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 01110 . 7554/eLife . 07539 . 012Figure 10 . DDB1 interacts with VprBP and DCAF11 in ESC and are required for ESC self-renewal .",
"( a-c )",
"Ingenuity generated network of protein interactions of CUL4-DDB1 complex in",
"( a ) ESC ,",
"( b ) 293T , and",
"( c ) HL-60 .",
"pink; DDB1 associated proteins",
"( d ) ( left ) immunprecipitation in 293T of endogenous VprBP protein followed by blotting for DDB1 .",
"( right )",
"Transfection of Flag-DDB1 in 293T followed by immunopercipitation .",
"( e-g )",
"Nanog-GFP ES cells were transfected with pools of siRNAs under conditions of self-renewal and analyzed by FACS 72 hrs post-transfcetion .",
"( e ) Nanog-GFP expression .",
"( n=3 ) ( pink= proteins identified in ESC mass spectrometry analysis; blue=DDB1 )",
"( f ) Bright field images of ESC colonies .",
"( g ) Representative experiment of relative expression of Nanog , Oct4 , and Meox1 .",
"( n=3 )",
"( h ) colony-forming units of LSK transduced with shRNAs .",
"( i ) Representative experiment of relative expression of following shRNA silencing .",
"( j ) Quantitative PCR of DCAFs in ESC and hematopoietic populations .",
"( Differentiated= ESC differentiated for 48 hrs with retinoic acid ) .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 07539 . 012"
],
[
"In this study we identify Ddb1 as a critical regulator of stem cell homeostasis both in embryonic pluripotent and hematopoietic stem cells .",
"Conditional ablation of Ddb1 in adult and fetal HSPCs , using the Mx1Cre and Vav1Cre strains respectively , led to a complete loss of progenitors and stem cells , cytopenia , and acute lethality .",
"Furthermore , increased levels of apoptosis and DNA damages were associated with acute Ddb1 inactivation in HSPCs , suggesting that Ddb1 regulates a wide range of cellular functions essential for the maintenance of hematopoiesis .",
"Strikingly , inactivation of Ddb1 in resting lymphocytes ( using the CD4Cre strain ) had no significant effects .",
"Whereas , silencing DDB1 in embryonic stem cells leads to loss of pluripotency and self-renewal devoid of alterations in cell cycle or cell survival .",
"These observations demonstrate that Ddb1 is essential for stem cell self-renewal and differentiation in both HSPC and ESC .",
"Previous studies demonstrate that the mechanism of CUL4-DDB1 differs depending on the cell type ( Bondar et al . , 2006; Cang et al . , 2006; Tardat et al . , 2010; Kronke et al . , 2014 ) .",
"Our studies suggest that Ddb1 deletion affects hematopoietic progenitors by triggering DNA damage and Trp53 response , leading to the rapid induction of apoptosis .",
"Knockdown of DDB1 in ESC by shRNA is not as efficient as Cre- mediated genetic deletion .",
"However , the DDB1 knockdown clearly is sufficient to affect ES cell pluripotency and lead to differentiation ( with no effects on apoptosis ) .",
"At the same time , when DDB1 was silenced ( by the same shRNAs ) in 293T cells these cells underwent rapid apoptosis , even if the knockdown was not 100% ( data not shown ) .",
"Similarly , silencing of DDB1 in Lineagenegc-Kit+ progenitors also lead to cell death and absence of colony formation in CFU assays ( data not shown ) .",
"Additionally , deleting DDB1 in the early embryo is lethal , however the embryos survive till around E12 . 5 far past the point of the proliferation of the inner cell mass , suggesting proliferation and differentiation during early embryogenesis could be DDB1-independent ( Cang et al . , 2006 ) .",
"Ddb1 shows an intriguing pattern of expression during hematopoiesis .",
"It is one of the highest expressed genes in the multipotent progenitor ( MPP ) fraction , and is induced as the quiescent HSCs differentiate .",
"It is expressed in all progenitor populations with the highest levels in progenitors of T cells .",
"Several substrates of CUL4-DDB1 complex have been reported .",
"Interestingly , it appears that there is tissue specificity for the various CUL4-DDB1 substrates .",
"CDT1 and p27Kip1 appear to be targeted by the complex in both brain and fibroblasts ( Bondar et al . , 2006; Cang et al . , 2006 ) .",
"c-JUN and Cdkn1acip1 are degraded by the CUL4-DDB1 complex in skin cells ( Cang et al . , 2007 ) .",
"More recently , PR-set7 , a methyltransferase regulating replication origins , was found to be also a substrate for the CUL4-DDB1 ligase ( Tardat et al . , 2010 ) .",
"Ddb1 deletion resulted in accumulation of the Cdkn1acip1 , a CDK and cell cycle inhibitor , protein in bone marrow progenitor populations and T lymphocytes .",
"On the other hand , we failed to demonstrate p27kip1 stabilization in Ddb1-deleted hematopoietic cells ( data not shown ) .",
"Moreover , there was no detectable interaction between p27kip1 and the CUL4-DDB1 complex .",
"In addition , we failed to detect in Ddb1-deleted progenitor cells accumulation of CDT1 , a licensing factor of DNA replication ( Hu et al . , 2004 ) , or PR-set7 ( Tardat et al . , 2010 ) .",
"However , co-silencing Cdkn1a with Ddb1 did not rescue HSPC homeostasis , which suggests it is unlikely that DDB1 exerts its function solely by regulating Cdkn1acip1 turnover in HSPC .",
"Indeed DNA damage and acute induction of cell death were observed upon Ddb1 deletion .",
"Trp53 pathway silencing partially restored HSPC cell number in Ddb1-deleted animals .",
"It is likely that DDB1 controls a broader spectrum of cellular functions , through its interaction with additional novel protein substrates and the proteasome itself ( data not shown ) .",
"However , it should be noted that mice with mixed background were used for Trp53 and Cdkn1a rescue experiments .",
"The phenotype could be strain-dependent due to variation in MHC alleles .",
"More conclusive results would require crossing mice into pure background .",
"There are findings suggesting that DDB1 can exert its function independent of CUL4A/B ( Lv et al . , 2010 ) .",
"However , DDB1 mainly functions as an adapter protein for the CUL4-DDB1 complex .",
"The specificity of the complex comes from the DCAF family of proteins that recognize specific substrates .",
"Expression of DCAF proteins is cell-type specific and as we have shown pluripotent and adult stem cell populations vary in expression .",
"Mass spectrometry analysis of DDB1 interacting proteins in ESC , 293T , and HL60 cell lines demonstrate differential binding to specific DCAF proteins suggesting different substrates in these different cellular contents that may correlate with the different phenotypes .",
"Some of the previous identified substrates have been associated with cell cycle control and DNA damage , however Kronke et al . demonstrates that two lymphoid transcription factors ( IKZF1 and IKZF3 ) are also targeted for degradation by CUL4-DDB1 complexes utilizing CRBN , a member of the DCAF family , as the substrate recognizing protein of the complex ( Kronke et al . , 2014 ) .",
"Although in our mass spectrometry analysis we did not identify CRBN as an interacting partner , we found in hematopoietic ( primitive myeloid ) cells that DCAF7 interacted with the CUL4-DDB1 complex whereas Vprbp and DCAF11 were found to interact with DDB1 and silencing in ESC mimicked the loss of DDB1 phenotype .",
"Interestingly , in multiple myeloma cells treatment with lenalidomide increased ubiquitination and subsequent degradation of IKZF1 and IKZF3 , demonstrating that small molecules can modulate the CUL4-DDB1-DCAF complex ."
],
[
"Ddb1f/f mice and their genotyping were previously reported ( Long et al . , 2001; Zhang et al . , 2001 ) .",
"Ddb1f/f::Mx1Cre+ animals were on C57BL/6 background , and were injected with 10 μg polyI:C per gram of body weight every two days for total two or three injections and analyzed 3 days from last injection depending on the experiments .",
"For mice transplanted with Ddb1f/f::Mx1Cre+ bone marrow , host mice got three polyI:C injections every two days .",
"Ddb1f/fCD4Cre+ mice were analyzed at 4–6 weeks of age .",
"Trp53-/- and Cdkn1acip-/- were purchased from Jackson Laboratory and on a mixed 129xC57BL/6 background , and crossed with Ddb1f/f::MxCre+ to obtain Trp53-/-::Ddb1f/f::MxCre+ and Cdkn1acip-/-::Ddb1f/f::MxCre+ mice respectively .",
"Thus generated double knockout mice and littermates were on a mixed 129xC57BL/6background ) .",
"All animal experiments were done in accordance to the guidelines of the NYU School of Medicine .",
"The breeding schemes for Trp53-/-::Ddb1f/f::MxCre+ double knockout mice were as follows: F1xF1: Trp53+/-::Ddb1f/+::MxCre+x Trp53+/-::Ddb1f/+::MxCre- F2xF2: Trp53+/-::Ddb1f/f::MxCre+x Trp53+/-::Ddb1f/f::MxCre- F3: Trp53-/-::Ddb1f/f::MxCre+ , or Trp53+/+::Ddb1f/f::MxCre+ Littermates were selected for experiments .",
"The breeding schemes for obtaining Cdkn1a-/-:Ddb1f/f::MxCre+ double knockout mice were similar as above .",
"Antibody staining and FACS analysis was performed as previously described ( Aifantis et al . , 1999 ) .",
"All antibodies were purchased from BD-Pharmingen or e-Bioscience .",
"We used the following antibodies: c-kit ( 2B8 ) , Sca-1 ( D7 ) , Mac-1 ( M1/70 ) , Gr-1 ( RB6-8C5 ) , NK1 . 1 ( PK136 ) , TER-119 , CD3 ( 145-2C11 ) , CD19 ( 1D3 ) , CD4 ( RM4-5 ) , CD4 ( H129 . 19 ) , CD8 ( 53–6 . 7 ) , CD25 ( PC61 ) , CD44 ( IM7 ) , CD45 . 1 ( A20 ) , CD45 . 2 ( 104 ) , CD150 ( 9D1 ) , CD48 ( HM481 ) , AnnexinV , 7-AAD .",
"Bone marrow lineage antibody cocktail includes: Mac-1 , Gr-1 , NK1 . 1 , TER-119 , CD3 , CD19 .",
"For DAPI staining , briefly , the cells were first treated with Fix and Perm reagents according to manufacturer’s instruction ( Invitrogen ) , then resuspended in PBS with 5 μg/ml RNaseA and 2 μg/ml DAPI .",
"γH2Ax staining , TUNEL analysis ( Millipore ) and BrdU staining ( BD Pharmingen ) were performed according to manufacturer’s instruction respectively .",
"The following antibodies were used for Western blot analysis: Ddb1 ( Invitrogen ) , Cdkn1a ( C-19 ) ( Santa Cruz ) and phospho-Trp53 ( Ser15 ) ( Cell signaling ) and β-actin ( Millipore ) .",
"DCAF1 antibody was previously described ( McCall et al . , 2008 ) .",
"Total RNA was isolated using the RNeasy Plus Mini Kit ( Qiagen ) and cDNA was synthesized using the SuperScript First-Strand Kit ( Invitrogen ) .",
"Quantitative PCR was performed using iQ SYBR Green Supermix and an iCycler ( Bio-Rad ) using the primer sequences ( Tm=60°C used for all primers ) .",
"Total bone marrow from polyI:C injected Ddb1f/f::Mx1Cre+ or control mice were plated in duplicate ( 200 , 000 cells/35mm dish ) into cytokine-supplemented methylcellulose medium ( MethoCult 3434 , Stem Cell Technologies ) , and the number and morphology of colonies were scored 7 days later .",
"Alternatively , lineage negative bone marrow cells were isolated by using EasySep Kit ( StemCell Technology ) , and infected with retrovirus expressing pMig-IRES-GFP or pMig-Cre-GFP ( see below ) .",
"Similarly for shRNA infection , Ckit+ enriched bone marrow cells ( Automacs technology ) where transduced with pLMP-Renilla , pLMP-VprBP , pLMP-DCAF11 or pLMP-DDA1 ) .",
"Forty-eight hours post infection , Lineage-GFP+ cells were sorted and 4 , 000 cells were plated in methylcellulose .",
"For CFU-S assay , 100 , 000 bone marrow cells were injected into lethally irradiated ( 960 cGy ) host mice ( n=5 per group ) .",
"Spleens were taken at day 8 and fixed in Bouin’s solution overnight and colonies were counted .",
"For bone marrow transplantation , 2×105 bone marrow cells were transplanted by retro-orbital i . v . injections into lethally irradiated ( 960 cGy ) BL6SJL recipient mice .",
"Duplicate of each sample was used .",
"Three mice were pooled from each genotype for the DN3 microarray experiment .",
"Microarray analysis was performed as previously described ( Gao et al . , 2009 ) .",
"Briefly , freshly isolated cells were sorted by surface marker expression , and total RNA was extracted using the RNeasy kit ( QIAGEN , CA ) .",
"In order to generate sufficient sample quantities for oligonucleotide gene chip hybridization experiments , we used the GeneChip Two-Cycle cDNA Synthesis Kit ( Affymetrix , San Jose , CA ) for cRNA amplification and labeling .",
"The amplified cRNA was labeled and hybridized to the MOE430 Plus 2 oligonucleotide arrays ( Affymetrix ) .",
"The Affymetrix gene expression profiling data was normalized using the previously published Robust Multi-array Average ( RMA ) algorithm using the GeneSpring 7 software ( Agilent , Palo Alto , CA ) .",
"The gene expression intensity presentation was generated with MeV software ( http://www . tm4 . org ) .",
"Microarray data were deposited under the GEO database with the accession number ( GSE70658 ) .",
"Gene set enrichment analysis was performed using Gene Set Enrichment Analysis software ( Mootha et al . , 2003; Subramanian et al . , 2007 ) ( http://www . broadinstitute . org/gsea ) using gene set as permutation type , 1 , 000 permutations and log2 ratio of classes as metric for ranking genes .",
"The ‘stem’ and ‘diff’ gene set was from publication ( Ng et al . , 2009 ) .",
"Other gene sets used in the analysis were taken from gene sets already present in the MSig database of the Broad Institute .",
"ESC were cultured and transfected with siRNA as previously described ( Buckley et al . , 2012 ) .",
"Human DDB1 cDNA in tandem with StrepII/Flag tags were cloned into pBS31 cloning vector ( Hochedlinger et al . , 2005; Beard et al . , 2006 ) .",
"Plasmid ( pBS31-N-SF-DDB1 ) was then nucleoporatated ( Amaxa ) along with FlpE plasmid into KH2 ESC .",
"ESCs were selected with hygromycin for 10 days .",
"Expression of tagged protein was confirmed by western blot following treatment with doxyclyine for 3 days .",
"293T cells were transfected with pCDNA-HA-Flag-DDB1 or pCDNA-HA-Flag .",
"48 hr post transfection cells were treated with 10 μM MG132 for 4 hrs prior to collection of cells .",
"HL-60 were transduced with pMIG-N-SF-DDB1 and selected with puromycin for 5 days 48 hrs following transduction .",
"pBS31-N-SF-DDB1 or pBS31-N-SF targeted ESCs were induced with 2 μM doxycycline ( Sigma ) for 3 days and cells were then treated with 10μM MG132 ( Peptides International ) for 2 hrs prior to collection of the cells .",
"Cell pellets were resuspended in Lysis Buffer ( 100 mM Tris-HCl pH7 . 5 , 150 mM NaCl , 1% Triton-X100 , 1 mM EDTA , 2 mM MgCl2 , and supplemented with Complete Mini protease inhibitors ( Roche ) , and 10 mM N-elthylmaleimide ( Sigma ) .",
"Tagged proteins were bound to StrepTactin macroprep beads ( IBA ) , and eluted per manufactuers instructions .",
"Peptides were digested with trypsin and analyzed by LC-MS/MS .",
"The MS/MS spectra were searched against NCBI database using a local MASCOT search engine ( V . 2 . 3 ) .",
"The means of each data set were analyzed using the Student’s t test , with a two-tailed distribution and assuming equal sample variance ."
]
] | [
"Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation .",
"Here we characterize the role of Ddb1 , a component of the CUL4-DDB1 ubiquitin ligase complex .",
"Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis , targeting specifically transiently amplifying progenitor subsets .",
"However , Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes .",
"Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis .",
"The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53 .",
"Conversely , depletion of DDB1 in embryonic stem cell ( ESC ) leads to differentiation albeit negative effects on cell cycle and apoptosis .",
"Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs , the substrate recognizing components of the E3 complex , between cell types .",
"Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation ."
] | [
"Stem cells can develop into other types of cells via a process called “differentiation” .",
"When a stem cell divides in two , it typically produces another stem cell and a cell that goes on to differentiate .",
"Hematopoietic stem cells ( or HSCs ) are found in the bone marrow and give rise to all blood cells throughout the life of an organism .",
"It is therefore crucial that they divide correctly to maintain the balance between renewing their numbers and making new types of cells .",
"Many studies have investigated how stem cells are maintained , but there are still major gaps in our knowledge .",
"Recent research suggested that the cell’s “ubiquitin-proteasome system” might be important for regulating stem cell division .",
"This system rapidly degrades proteins , thereby regulating protein abundance in cells .",
"Enzymes known as E3 ligases form part of this system , and recognize proteins to be marked for destruction with a small protein tag .",
"Gao et al . have now observed that a component of an E3 ligase called DDB1 is highly expressed in hematopoietic stem cells .",
"Further experiments revealed that genetically engineered mice that lack DDB1 in their population of blood cells die soon after they are born and have fewer blood cells .",
"Gao et al . next inhibited the production of DDB1 in adult mice .",
"This stopped the adult mice’s hematopoietic stem cells from dividing , and the mice died because their bone marrow couldn’t produce new blood cells .",
"These results show that DDB1 is necessary for stem cells to renew their numbers and differentiate into blood cells in both developing and adult animals .",
"Next , Gao et al . investigated the how DDB1 regulates stem cell division , and discovered that a protein called p53 , which is a key player in controlling cell division , is regulated by DDB1 .",
"Under normal conditions , p53 levels are kept low in cells .",
"However , in the absence of DDB1 , the levels of p53 rise , which triggers the death of the hematopoietic stem cells .",
"Further experiments revealed that not all dividing cells undergo cell death with the loss of DDB1 .",
"Instead , Gao et al . found that rapidly dividing embryonic stem cells differentiate when DDB1 is lost but do not die .",
"These findings suggest that specific components of the ubiquitin ligase complex play a key role in deciding a stem cell’s fate .",
"In the future , identifying these components will further our understanding of the decision of stem cells to differentiate ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease",
"immunology and inflammation"
] | Rapid localized spread and immunologic containment define Herpes simplex virus-2 reactivation in the human genital tract | elife-00288-v1 | [
[
"The dynamics of most chronic viral infections such as HIV , hepatitis B and C , and cytomegalovirus are assessed by serially sampling blood using polymerase chain reaction ( Perelson et al . , 1996 ) , and by ex vivo quantitation of circulating lymphocytes to estimate immune response ( Sylwester et al . , 2005 ) .",
"For disseminated infections , sampling from blood may reflect true host–pathogen dynamics due to homogeneous mixing of viruses and PBMCs , which originate from thousands of infectious foci .",
"However , many infections are confined to small anatomic regions; localized host–pathogen interactions within specific organ tissue constitutes the predominant site of pathogenesis , and measurements of viral replication and immune response are not accurately reflected in the blood compartment .",
"Moreover , sampling of infected tissue often reveals that the density of virus and host immune cells differ enormously across small distances .",
"Spatial features of infection are a critical , and relatively unexplored , component of viral dynamics .",
"Herpes simplex virus-2 ( HSV-2 ) is useful for studying localized aspects of pathogenesis in humans .",
"HSV-2 replicates in highly accessible genital keratinocytes .",
"Serial genital swabbing reveals frequent , highly variable shedding episodes in most infected persons ( Wald et al . , 1995 , 1997 , 2000; Crespi et al . , 2007; Mark et al . , 2008 ) , and biopsies show dense clusters of CD4+ and CD8+ lymphocytes in focal areas of viral replication ( Zhu et al . , 2007 ) .",
"Some episodes are associated with lesions , and prolonged viral production , while others are asymptomatic and brief ( Mark et al . , 2008 ) .",
"Markedly different episodes occur within 5–10 days of each other , and viral levels fluctuate dramatically even within a single episode ( Mark et al . , 2008; Schiffer et al . , 2011 ) .",
"At least 20% of episodes are notable for complex erratic viral trajectories , including re-expansion phases that represent failure of the immune system to contain replication ( Schiffer et al . , 2011 ) .",
"Past modeling suggests that nearly constant reactivation of HSV in a small percentage of infected ganglionic neurons might account for high frequency of shedding episodes ( Schiffer et al . , 2009 ) .",
"Animal and mathematical models predict that varying T-cell responses account for episode heterogeneity ( Gebhardt et al . , 2009; Schiffer et al . , 2010 ) .",
"However , the field lacks hypotheses to address the complex morphology of prolonged viral shedding episodes .",
"We performed detailed studies with genital sampling performed over four different time intervals , and concurrently at multiple sites across the genital tract , to precisely define spatiotemporal kinetics of genital HSV-2 replication in immunocompetent patients .",
"We then designed a mathematical model as a tool to develop hypotheses that explain virological data from these cohorts .",
"Model simulations suggest very rapid rates of HSV-2 replication and epidermal cell-to-cell spread .",
"Yet , host containment of virus within a single focus of infection occurs in <24 hr , necessitating seeding of adjacent regions to promote increased shedding ."
],
[
"HSV-2 shedding in the genital tract is characterized by intermittent frequent episodes of variable duration ( hours to weeks ) and viral load ( Mark et al . , 2008; Schiffer et al . , 2011 ) .",
"To evaluate viral patterns during individual shedding episodes , we analyzed data from three cohorts of immunocompetent patients who underwent genital tract swabbing at different time intervals ( Table 1; Boxes 1–3 ) .",
"The same collection methods and HSV PCR assay were used in each cohort .",
"Swabs were tested for HSV DNA using a validated quantitative PCR assay with a sensitivity of 1 copy/reaction ( Jerome et al . , 2002; Magaret et al . , 2007 ) .",
"These studies demonstrated that quantity of genital HSV DNA is stable over minutes but expands and decays extremely rapidly over hours , and that prolonged episodes are notable for frequent and erratic peaks in HSV-2 viral load ( Figure 1 , Figure 1—figure supplements 1–3 ) . 10 . 7554/eLife . 00288 . 003Table 1 . Five cohorts of HSV-2 genital tract sheddingDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 003CohortSubjectsTotal swabsSwabbing frequencyTotal episodesSwabbing durationAnatomic swabbing regionPurposeA396Every 5 min34 hr when lesion presentTotal genital tractSwab-to-swab sampling/assay variabilityB520010 times/day ( every 2 hr during the days and 4 hr overnight ) 54–5 days when lesion presentTotal genital tractEpisode expansion , clearance and re-expansion kineticsC2547064 times/day10930–60 days without or with a lesionTotal genital tractAccurate estimates for expansion/decay slopes for clinical and subclinical episodesD2216Daily430 days without or with a lesion23 separate regionsSpatial dispersion of HSVE53114 , 685Daily1020>30 days with or without a lesionTotal genital tractModel fit10 . 7554/eLife . 00288 . 004Box 1 . Cohort ADOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 004Cohort A included three subjects who were seen within 12 hr of HSV-2 recurrence , and who collected genital swabs every 5 min for 4 hr .",
"This experiment assessed whether there were alterations in viral load related to the PCR assay or collection technique .",
"Only small changes occurred during 5-min intervals ( Figure 1A , Figure 1—figure supplement 1A , B , Figure 1—source data 1 ) .",
"The mean absolute value of viral DNA copy difference between successive swabs was 0 . 20 , 0 . 28 , and 0 . 34 log10 genomic copies in the three participants , and generally increased with time between swabs ( Figure 1B , Figure 1—figure supplement 1C ) .",
"The mean difference in HSV DNA copies between swabs correlated tightly with time between swabs in two participants ( Figure 1C , Figure 1—figure supplement 1D ) , with virtually no difference in viral quantity between swabs separated by 5-min intervals in all three subjects ( Figure 1C , Figure 1—figure supplement 1D ) .",
"Two episodes had negative mean differences in viral quantity with increasing time between swabs ( Figure 1C , Figure 1—figure supplement 1D ) , while one episode had a slightly positive mean difference ( Figure 1—figure supplement 1D ) , suggesting that we captured two episodes during a decay phase and one episode near a peak of viral production .",
"The standard deviation of the mean absolute value of viral DNA copy difference between successive swabs was 0 . 16 , 0 . 22 , and 0 . 26 in the three participants , respectively , suggesting that differences of >0 . 5 log10 HSV-2 DNA copies that may occur over hours usually reflect true changes in viral load rather than noise in the data attributable to clinical collection or PCR technique . 10 . 7554/eLife . 00288 . 005Box 2 . Cohort BDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 005To explore patterns of viral expansion and decay of an individual episode over time , we expanded the duration of evaluation and time interval between sampling .",
"For Cohort B , we enrolled five HSV-2 positive participants with genital lesions , and sampled them 10 times daily ( every 2 hr during the day and 4 hr overnight ) for 4–5 days from lesion onset .",
"A key feature of all episodes was a ‘saw tooth’ morphology of viral expansion and contraction ( Figure 1D , Figure 1—figure supplement 2 , Figure 1—source data 1 ) , with 0 . 8–1 . 8 daily peaks ( defined as an expansion of 0 . 5 log10 HSV DNA followed by a decline of 0 . 5 log10 HSV DNA ) .",
"The mean absolute value of viral DNA copy change between successive swabs separated by 2 hr was 0 . 73 , 0 . 72 , 0 . 50 , 0 . 51 , and 0 . 34 log10 HSV DNA copies in the five participants , respectively , suggesting significant variability in viral levels .",
"In addition , each episode was notable for high initial viral peak ( 6–8 log10 HSV DNA ) followed by decay within 24 hr of initiation , highlighting extremely early rapid viral expansion , followed by rapid control . 10 . 7554/eLife . 00288 . 006Box 3 . Cohort CDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 006To expand data available for analysis of early viral expansion and late viral clearance , we performed new analyses from a published cohort of 25 subjects who were sampled every 6 hr for 30–60 days ( Cohort C ) ( Mark et al . , 2008 ) .",
"Among 109 episodes , 75% of which were subclinical , median regression slopes from initiation to peak and peak to termination were 20 . 3 and −8 . 7 log10/day , respectively .",
"Median first and last copy numbers were 3 . 5 and 3 . 3 log10 HSV DNA copies , respectively: these measures occurred a median of 3 hr after episode initiation or before termination , suggesting that rapid changes ( ∼10-fold/hr ) in genomic copy numbers occurred both early and late within an episode ( Mark et al . , 2008 ) .",
"While a majority of episodes in Cohort C were <12 hr , even among the 55 episodes of >24 hr , transition from initial expansion to clearance phase occurred at a median of 12 hr , implying early immunologic pressure against the virus .",
"Rapid expansion and contraction phases continued throughout more persistent shedding episodes .",
"Figure 1E and Figure 1—figure supplement 3 ( Figure 1—source data 1 ) illustrate episodes of 17-day and 8-day duration from two participants with multiple sharp peaks from steep expansion and decay phases . 10 . 7554/eLife . 00288 . 007Figure 1 . HSV-2 levels in the genital tract are stable over minutes , expand and decay markedly over hours , and fluctuate rapidly and unpredictably over days .",
"( A ) Shedding quantity in a participant , who performed genital swabs every 5 min over 4 hr during a lesion , reveals low swab-to-swab variation in viral quantity .",
"Using data from panel ( A and B ) , absolute mean difference ( R2 = 0 . 99 ) , and ( C ) mean difference ( R2 = 0 . 87 ) , in HSV DNA copies between swabs , are a function of time between swabs .",
"( D ) Shedding quantity in a participant , who performed 10 genital swabs per day during a lesion over 4 days ( swabs every 2–4 hr ) , shows a characteristic saw-tooth pattern; arrows denote rapid viral re-expansion; the participant had a negative swab performed before episode onset .",
"( E ) Shedding quantity in a participant , who performed four genital swabs per day over 17 days demonstrates that rapid and frequent viral re-expansion allows for shedding prolongation; four missing data points are left blank . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 00710 . 7554/eLife . 00288 . 008Figure 1—source data 1 . Source data for Figure 1 , Figure 1—figure supplement 1 , Figure 1—figure supplement 2 and Figure 1—figure supplement 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 00810 . 7554/eLife . 00288 . 009Figure 1—figure supplement 1 . Dynamics of HSV-2 shedding over 5-min time intervals .",
"( A ) and ( B ) Shedding quantity in two participants , who performed genital swabs every 5 min over 4 hr during a lesion , reveals low swab-to-swab variation in viral quantity .",
"( C ) Using data from panels ( A and B ) , absolute mean difference in HSV DNA copies between swabs , correlated moderately with time between swabs in one participant ( green line R2 = 0 . 60 ) due to steady viral decay , but was more stable as a function of time in the other participant due to peaking overall viral load ( blue line , R2 = 0 . 16 ) .",
"( D ) Using data from panels ( A and B ) , mean difference in HSV DNA copies between swabs , was a function of time between swabs in the participants ( green line R2 = 0 . 89 , and blue line R2 = 0 . 85 ) presumably because viral load was generally decaying during most of the 4-hr window ( green ) or gradually expanding during most of the 4-hr window ( blue ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 00910 . 7554/eLife . 00288 . 010Figure 1—figure supplement 2 . Dynamics of HSV-2 shedding with every 2-4 hr sampling . Shedding quantity in four participants , who performed 10 genital swabs per day over 4–5 days during a lesion reveal a characteristic saw-tooth pattern; arrows denote re-expansion .",
"Participants had swabbing initiated upon visualization of lesions .",
"The participant in panel d initiated swabbing ∼16 hr after lesion detection . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 01010 . 7554/eLife . 00288 . 011Figure 1—figure supplement 3 . Dynamics of HSV-2 shedding with every 6-hr sampling over 8 days . Shedding quantity in episodes detected in a participant who performed four genital swabs per day over 60 days demonstrates that viral re-expansion allows for shedding prolongation . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 011 Based on the observation that HSV-2 reactivation occurs simultaneously in multiple regions across the genital tract ( Tata et al . , 2010 ) , we designed a fourth cohort of patients who had daily genital swabs performed in 23 locations ( Table 1 , Box 4 ) .",
"This study revealed that HSV-2 is present across the entire genital tract during many episodes , although viral loads are highly variable over space and time ( Figure 2A , Figure 2—figure supplement 1 ) .",
"We next demonstrated that viral re-expansion more commonly follows a period of decay during episodes with higher peak viral loads ( Table 1 , Box 5 , and Figure 2B ) : if each peak during a prolonged shedding episodes ( Figure 1E ) represents viral production from a single focus of infection , then this implies that multiple spatially dispersed foci of replication ( Figure 2A ) may occur due to seeding from areas with high levels of HSV-2 replication .",
"Concurrent viral expansion and clearance in spatially distinct microregions could account for saw-tooth episodes ( Figure 1E ) , and viral migration to new regions might prolong episodes . 10 . 7554/eLife . 00288 . 012Box 4 . Cohort DDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 012We investigated the spatial-temporal dynamics of reactivation in two patients in which we divided the genital tract into 23 separate regions and swabbed each individual for 30 consecutive days ( Cohort D ) .",
"During brief asymptomatic episodes , low HSV-2 quantities were confined to a few regions , in which viral load and location fluctuated over time ( Figure 2A , Figure 2—figure supplement 1 , Figure 2—source data 1 ) .",
"During longer more severe episodes , HSV was detected throughout the genital tract ( Figure 2A ) , although viral density often varied by >4 logs between adjacent regions . 10 . 7554/eLife . 00288 . 013Figure 2 . HSV-2 replicates and is contained in widely dispersed microenvironments across the genital tract .",
"( A ) HSV shedding quantity in a participant , who underwent daily swabs in 23 regions across the genital tract for 30 days; days without sampling are marked with an X; stars denote days with a lesion; virus is widely dispersed and several prolonged episodes with heterogeneous viral loads across the genital tract are noted .",
"( B ) Increasing probability of episode re-expansion ( nonmonotonic episodes ) as a function of peak episode copy number among 1020 episodes from 531 study subjects; individual peaks during episodes may represent virus from a single ulcer that can seed other regions .",
"( C ) A genital lesion consists of numerous round ulcers ( black dotted circle ) clustered in space; contemporaneous presence of multiple ulcers may indicate concurrent viral expansion in decay in multiple regions .",
"( D ) and ( E ) Immunofluorescent staining of biopsies performed ( D ) at the edge , and ( E ) 1 cm away from an ulcer 3 days post-healing; CD8+ T cells ( green ) at the dermal–epidermal junction ( arrow ) are highly localized to ulcer edge ( 287/mm2 ) and are fourfold less dense 1 cm away ( 72/mm2 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 01310 . 7554/eLife . 00288 . 014Figure 2—source data 1 . Source data for Figure 2 and Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 01410 . 7554/eLife . 00288 . 015Figure 2—figure supplement 1 . Spatial features of HSV genital tract shedding . HSV shedding quantity in a study participant , who underwent daily swabs in 23 regions across the genital tract for 30 days; days without sampling are marked with an X . The participant had three brief localized episodes with low viral copy number in three separate localized regions . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 01510 . 7554/eLife . 00288 . 016Figure 2—figure supplement 2 . Spatial features of HSV-2 lesions . A genital lesion consists of numerous round ulcers or vesicles ( black dotted circle ) , clustered in space . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 01610 . 7554/eLife . 00288 . 017Figure 2—figure supplement 3 . Spatial features of CD8+ T-cell response in genital skin . Immunofluorescent staining of a biopsy performed ( A ) at the edge , and ( B ) 1 cm away from an ulcer 3 days post-healing .",
"CD8+ T-cells ( red ) and CD4+ T-cells ( green ) at the dermal epidermal junction ( arrow ) are highly localized to ulcer edge ( 132/mm2 and 447/mm2 , respectively ) , and are less dense 1 cm away ( 91/mm2 and 132/mm2 , respectively ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 01710 . 7554/eLife . 00288 . 018Box 5 . Cohort EDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 018To define episode heterogeneity , we used the largest available database of HSV-2 shedding ( Cohort E ) , which contained HSV PCR data taken from 531 HSV-2 seropositive persons who sampled the entire genital tract once daily for >30 days .",
"This dataset included 14 , 685 swabs and 1020 episodes of symptomatic and asymptomatic shedding ( Schiffer et al . , 2011 ) .",
"Each episode was classified according to six features: duration , first , peak and last positive HSV genomic copy number , and initiation to peak and peak to termination slopes .",
"The heterogeneity of each feature was captured using frequency histograms .",
"Shedding episodes had highly variable duration ( median days: 3; IQR: 1–8 ) and peak HSV DNA copy number ( median log10 copy number: 4 . 8; range: 2–9 . 2 ) .",
"Episode rate was 15 . 3/year , and per swab shedding frequency was 18% ( ∼3% of swabs contained 102–103 , 103–104 , 104–105 , 105–106 , 106–107 , and >107 HSV DNA copies , respectively ) .",
"In Cohort E , 19% of episodes had re-expansion , which we previously defined as 0 . 5 log decay followed by 0 . 5 log re-expansion ( Schiffer et al . , 2011a , 2011b ) .",
"Further evidence for this phenomenon is that herpetic lesions consist of multiple vesicles and ulcers ( Corey et al . , 1983 ) , which occur in clusters and develop sequentially ( Figure 2C , Figure 2—figure supplement 2 ) , suggesting that virus from a single ulcer may lead to ancillary ulcer formation .",
"In addition , we have previously demonstrated that host T-cell responses in the area of genital lesions are highly localized to foci of viral replication ( Zhu et al . , 2007 , 2009 ) .",
"To better classify T-cell heterogeneity over short distances , we performed biopsies in two patients from Cohort B immediately after lesion healing , and enumerated T-cell counts at the edge of an ulcer and 1 cm away .",
"CD8+ and CD4+ T-cell densities in the genital skin were maximal at ulcer edge ( Figure 2D , Figure 2—figure supplement 3A ) , and considerably lower in regions 1 cm away ( Figure 2E , Figure 2—figure supplement 3B ) .",
"Even within a 1-mm biopsy , CD8+ T cells formed cluster-like aggregates ( Figure 2D , Figure 2—figure supplement 3A ) , suggesting that dynamical interactions between acquired immune cells and infected cells occur within tightly spaced microenvironments on a micrometer scale .",
"We infer that individual ulcers , both microscopic and visible to the naked eye , may occur across a wide region of the genital tract during large outbreaks .",
"While each ulcer and microulcer represent a site of intense interface between the virus and T cells , genital skin and mucosa between ulcers may be relatively quiescent .",
"To generate hypotheses that might explain these observations , we sought to design a mathematical model that precisely recreated the shedding patterns observed in our human shedding studies .",
"Rather than fit models to complex individual episodes with erratic expansion and decay phases ( Figure 1D , E ) , we tested competing models for their ability to reproduce key kinetic patterns from a large more generalizable patient base with genital herpes ( Table 1 , Box 5 ) .",
"We attempted to develop a model that could precisely capture episode heterogeneity within and between persons by recreating frequency histograms from Cohort E , and predicting key observations from Cohorts A–D , including rapid viral expansion and decay , multiple peaks per episode , and wide spatial distribution of HSV DNA .",
"After several iterations of model development failed to reproduce key features of our empirical dataset ( ‘Methods’ ) , we designed a spatial model that accounted for observed multiple concurrent foci of viral replication and immune response ( Figure 2 ) .",
"We hypothesized that secondary ulcer formation in separate genital tract regions might occur via two mechanisms: release of HSV-2 from neuron endings may simultaneously occur in spatially distinct genital regions , or dense aggregates of cell-free particles within an ulcer may be locally infectious .",
"Evidence for the latter hypothesis is tight correlation between episode peak copy number and probability of re-expansion in ( Figure 2B ) .",
"In addition , ulcers tend to be closely arrayed in space rather than widely dispersed across vulnerable regions in the genital tract .",
"‘Kissing lesions’ , in which two ulcers form at the site of skin-to-skin contact , are a well-documented phenomenon ( Figure 2C ) .",
"On the other hand , ulcers do not isolate within genital tract dermatomes as occurs with zoster reactivation .",
"These observations suggest that local seeding from epithelial lesions may be responsible for many secondary ulcers .",
"Our spatial model included 300 regions of 6 . 5 mm diameter linked in a spatial arrangement of adjacent hexagons ( Figure 3A , Figure 3—figure supplement 1 ) .",
"The diameter was chosen as an absolute maximal value for ulcer diameter .",
"We populated each region with differential equations ( Figure 3B , ‘Methods’ ) describing infected epithelial cells ( I ) , which produced cell-associated HSV-2 ( Vi ) at a rate p .",
"Cell-associated HSV-2 was infectious to susceptible epithelial cells ( S ) according to an infectivity parameter βI .",
"Infected cells died either due to direct HSV-2 killing at a rate a , or due to CD8+ T-cell killing at a rate ( f × E ) .",
"Cytolytic CD8+ T cell ( E ) expanded at a maximal rate θ .",
"CD8+ expansion rate increased according to number of infected cells , and was half-maximal ( θ/2 ) at a threshold value of infected cells , r .",
"Cell-associated HSV-2 converted to cell-free HSV-2 ( Ve ) following cell lysis .",
"Cell-free viruses and CD8+ T cells decayed at fixed rates ( c and δ ) within each region .",
"We assumed that viruses ( Vneu ) were randomly released into 300 regions by neurons at a rate ϕ , predicted by a previous model ( Schiffer et al . , 2009 ) , and that these viruses could initiate an ulcer in each reason by infecting an epithelial cell . 10 . 7554/eLife . 00288 . 019Figure 3 . Mathematical model .",
"( A ) Microregions are linked virally because cell-free HSV-2 can seed surrounding regions , and immunologically based on overlapping CD8+ T-cell densities between regions ( not shown ) .",
"( B ) Schematic for HSV-2 infection within a single genital tract microenvironment .",
"Equations capture seeding of epithelial cells by neuronal HSV-2 , replication of HSV-2 within epithelial cells , viral spread to other epithelial cells , cytolytic CD8+ T-cell response to infected cells , transition of cell-associated HSV-2 to cell-free HSV-2 following lysis of infected cells , and elimination of free virus and infected cells . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 01910 . 7554/eLife . 00288 . 020Figure 3—figure supplement 1 . Spatial mathematical model . Viruses produced from neurons ( green ) , cell-associated viruses from epidermal cells ( yellow ) , and cell-free viruses ( orange ) that form after rupture of epidermal cells , are distinguished in the model .",
"Neuron-derived viruses are released throughout the genital tract and are responsible for ulcer initiation within specific regions ( grey hexagons ) .",
"Cell-associated HSV particles contribute to ulcer expansion ( white circle ) within a region .",
"Cell-free particles initiate secondary ulcers in adjacent regions ( upper right ) leading to concurrent ulcers where HSV production occurs .",
"Cytolytic CD8+ T-cell ( purple circles ) response is localized within each region .",
"Regions have a maximum diameter of 6 . 5 mm .",
"However , distance between regions is considered in terms of immunologic co-dependence rather than a physical distance .",
"Seven of 300 total model regions are illustrated . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 020 Adjacent regions in the model were linked virally .",
"Cell-associated HSV ( Vi ) drove spread within an ulcer in a single region , while cell-free HSV ( Ve ) could initiate new ulcers at infectivity βe , but only in six contiguous regions surrounding a productive ulcer ( Figure 3A , Figure 3—figure supplement 1 ) .",
"Based on our observation in cell culture that in a single cell infected by a single virus , viral replication does not occur until approximately 12–16 hr , a fixed time delay parameter ( ε ) was included for ulcer formation .",
"The physical distance between regions was not explicitly considered because the 300 regions were not intended to capture the complex three-dimensional topography of genital skin .",
"Rather , the distance between regions was captured in immunologic terms .",
"Based on the gradient of CD8+ T-cell density as distance increases from an ulcer edge ( Figure 2D , E ) , we assumed that contiguous regions might be immunologically codependent , by including a new fitting parameter ( ρ ) to estimate the extent that CD8+ T-cell density in contiguous regions affected CD8+ T-cell density within a new ulcer region ( ‘Methods’ ) .",
"Contiguous regions in the model were therefore assumed to be far enough away for new ulcers to initiate but potentially close enough to be effected by neighboring immune responses .",
"We solved our model by fitting to the data and assuming either 5 or 10 above parameter values as unknown ( ‘Methods’ ) .",
"In both cases , model output closely reproduced the data within Cohort E , including quantitative shedding frequency ( Figure 4A ) , as well as episode rate ( Figure 4B ) , median initiation to peak and peak to termination slopes ( Figure 4C ) , durations ( Figure 4D ) , and first ( Figure 4E ) , last ( Figure 4F ) , and peak HSV DNA copy numbers ( Figure 4G , Figure 4—source data 1 ) .",
"We also performed a sensitivity analysis using 500 episode ( ∼30 years ) simulations in which single parameter values were adjusted to arrive at narrow ranges for parameter values that reproduced our data ( Table 2 ) .",
"These parameter values were generally within an order of magnitude of previous parameter estimates ( Schiffer et al . , 2009 ) . 10 . 7554/eLife . 00288 . 021Figure 4 . The spatial model reproduces all shedding episode characteristics . Colored bars represent results from ( A ) 14 , 685 genital swabs and ( B–G ) 1020 shedding episodes from 531 study participants .",
"The model simulation , represented with black bars in each panel , continued until 1020 episodes were generated; model sampling occurred every 24 hr as in the clinical protocol .",
"Model output reproduced ( A ) quantitative shedding frequency as well as ( B ) rate , ( C ) median initiation to peak and peak to termination slopes , ( D ) Duration , ( E ) first HSV DNA copy number , ( F ) last HSV DNA copy number , and ( G ) peak HSV DNA copy number of episodes . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 02110 . 7554/eLife . 00288 . 022Figure 4—source data 1 . Source data for Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 02210 . 7554/eLife . 00288 . 023Figure 4—figure supplement 1 . Continuous sampling of spatial model output reveals more accurate measures of episode characteristics . We subjected a 30-year simulation to daily and continuous sampling .",
"( A ) Median initiation to peak slope , and ( B ) peak to termination slopes increased substantially with continual sampling .",
"( C ) Shedding frequency was similar regardless of sampling frequency .",
"( D ) Continuous sampling detected 842 episodes ( 28 . 1/year ) vs 450 episodes ( 15 . 0/year ) with daily sampling .",
"The 392 additional episodes were all less than a day in duration and mostly <104 peak HSV DNA copies per milliliter , skewing the distributions of ( E ) episode duration and ( F ) peak HSV DNA copy number .",
"( G ) Total number of episodes at low and high-peak copy numbers increased with continual sampling . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 02310 . 7554/eLife . 00288 . 024Table 2 . Parameter ranges that result in accurate reproduction of model outcomesDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 024ParameterUnitsSymbolBest fit valueGood fitAverage fitLower limitUpper limitLower limitUpper limitCell-associated HSV infectivityDNA copy days/cell ( viruses needed per day to infect one adjacent cell ) βi5 . 4e−8 ( 111 ) 4 . 86e−8 ( 123 ) 7 . 83e−8 ( 76 ) 3 . 78e−8 ( 158 ) 1 . 32e−7 ( 45 ) Cell-free HSV infectivityDNA copy days/cell ( viruses needed per day to initiate one new ulcer ) βe2 . 65e−11 ( 2 . 26e5 ) 1 . 73e−11 ( 3 . 46e5 ) 2 . 78e−11 ( 2 . 15e5 ) 3 . 98e−12 ( 1 . 50e6 ) 5 . 04e−11 ( 1 . 19e5 ) Epidermal HSV replication rateHSV DNA copies per cell per dayp1 . 03e57 . 21e41 . 7e55 . 15e41 . 96e5Neuronal release rateHSV DNA copies per day per genital tractϕ82459041123Free-viral decay ratePer day ( half-life , hours ) c8 . 8 ( 1 . 9 ) 7 . 0 ( 2 . 4 ) 9 . 7 ( 1 . 7 ) 6 . 2 ( 2 . 7 ) 12 . 3 ( 1 . 4 ) Maximal CD8+ T-cell expansion ratePer dayθ2 . 841 . 853 . 271 . 855 . 25CD8+ T-cell decay ratePer day ( half-life , days ) δ1 . 47e−3 ( 471 ) 1 . 12e−3 ( 619 ) 1 . 69e−3 ( 409 ) 6 . 64e−4 ( 1040 ) 2 . 21e−3 ( 314 ) CD8+ T-cell local recognitionInfected cells at which θ is half maximalr423047474CD8+ regional codependence0 = no codependence , 1 =full codependenceρ0 . 690 . 590 . 860 . 380 . 86Viral production lagDaysε0 . 960 . 531 . 10 . 341 . 1 The model was next evaluated for its ability to predict other key features of genital shedding identified in Cohorts A–D .",
"To explore the dynamics of simulated episodes with higher granularity , we varied sampling frequency of model output to include continuous sampling .",
"The model predicted the empirically derived finding that daily sampling substantially underestimates expansion ( Figure 4—figure supplement 1A ) and clearance slopes ( Figure 4—figure supplement 1B ) .",
"Among 842 simulated episodes with sampling every 0 . 001 days , median initiation to peak expansion rate was 25 . 5 log10 HSV DNA copies per day ( versus 20 . 3 log10 copies per day with 6-hr sampling in Cohort C ) , implying that during the early expansion phase , HSV DNA levels increased 10-fold every 57 min and doubled every 17 min .",
"There was also rapid late clearance ( −7 . 4 log10 copies per day vs −8 . 7 log10 copies per day in Cohort C ) .",
"We simulated the model until 100 episodes with >10 , 000 total infected cells within one ulcer were generated .",
"Number of infected cells and viral load peaked at a median of 13 . 5 hr , predicting the finding from Cohort C that transition from expansion to clearance occurs ∼12 hr after episode commencement .",
"To explain episodes of >4 days , the model predicted multiple peaks within single episodes .",
"In addition , prolonged simulated episodes had wide concurrent dispersal of virus ( Video 1 ) . 10 . 7554/eLife . 00288 . 025Video 1 . Spatiotemporal demonstration of a 10-day episode . The left panel represents total cell-free HSV DNA copies per milliliter present over time .",
"The right panel represents spatial spread of virus during the episode , each hexagon contains one region of shedding and virus spreads to contiguous regions .",
"Amount of virus within a single region is displayed according to a heat map adjacent to the spatial map . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 025 While daily sampling accurately measured quantitative shedding frequency with continuous sampling ( Figure 4—figure supplement 1C ) , the model predicted that more frequent sampling increased episode detection by ∼50% ( Figure 4—figure supplement 1D ) and proportion of episodes with low duration ( Figure 4—figure supplement 1E ) and low peak copy number ( Figure 4—figure supplement 1F ) .",
"The model also predicted the finding from Cohort C that ∼75% of HSV-2 episodes are asymptomatic , brief ( <48 hr ) , and associated with a low peak copy number ( Figure 4—figure supplement 1F ) ( Mark et al . , 2008 ) .",
"There were higher absolute numbers of episodes with high copy number ( >107 HSV DNA copies ) because daily sampling rarely captured true episode peak ( Figure 4—figure supplement 1G ) .",
"Our estimates of parameter values suggest that individual cell-associated viruses are rapidly produced by epidermal cells and are highly infectious to surrounding cells .",
"Epidermal cells produced 72 , 000–170 , 000 HSV DNA copies per day; one of 76–123 cell-associated DNA particles infected a neighboring cell per day at ulcer onset ( Table 2 ) .",
"When we simulated the model until 100 episodes with >10 , 000 total infected cells within a single ulcer were generated , median times to 100 , 1000 , and 10 , 000 total infected cells were 5 . 6 , 8 . 7 , and 12 . 9 hr , respectively .",
"Median time interval from becoming infected to infecting a new cell during the expansion phase of the 100 simulated episodes was 6 . 8 hr; median time from becoming infected to infecting a first surrounding cell was 59 min .",
"This finding was surprising because in culture , HSV replication occurs within newly infected cells 4–6 hr post-infection ( Roizman , 2007 ) .",
"Our data suggest that in vivo an HSV-infected keratinocyte might become locally infectious by spreading the virus even before replication initiates within this cell , a phenomenon recently described for vaccinia ( Doceul et al . , 2010 ) .",
"The model results suggested that secondary ulcer formation by cell-free particles is crucial for episode prolongation .",
"In 500 simulated episodes , duration was a function of total ulcers ( Figure 5A ) .",
"We analyzed a simulated prolonged episode ( 10 days ) in detail to assess kinetics within single ulcers: free viral production from the initial viral ulcer lasted 3 days ( Figure 5B ) ; infected cells were eliminated within 24 hr ( Figure 5C , D ) ; peak viral load and number of infected cells were 8 . 1 log10 HSV DNA copies and 4 . 1 log10 cells , respectively , although total amount of viral DNA and infected cells produced within the ulcer were considerably higher ( 8 . 5 log10 HSV DNA copies and 4 . 9 log10 cells , respectively ) , reflecting rapid viral and infected cell turnover .",
"Virions generated during the primary ulcer promoted formation of additional ulcers ( Figure 5E and Videos 1 , 2 ) .",
"Before episode termination , 24 total ulcers were produced .",
"The median duration and peak viral production of the 24 ulcers were 2 . 5 days ( range: 0 . 6–4 . 6 days ) and 5 . 2 log10 HSV DNA copies ( range: 2 . 0–8 . 0 log10 ) , respectively .",
"We calculated diameter of each ulcer during simulations based on the number of missing cells due to death from infection , and defined a lesion as any episode with a >1 mm ulcer ( Schiffer et al . , 2009 ) .",
"Consistent with clinical observations in Cohort D , simulated ulcer size did not exceed 5 mm ( Corey et al . , 1983 ) , and many failed to reach the clinical threshold of 1 mm in diameter ( Video 3 ) . 10 . 7554/eLife . 00288 . 026Figure 5 . Containment of infected cells within a single ulcer is extremely rapid , although secondary ulcers explain prolonged episodes .",
"( A ) Episode duration was a function of the number of ulcers before episode termination during 500 simulated episodes .",
"( B ) – ( E ) A 10-day simulated episode consisting of 24 ulcers: ( B ) Total cell-free virus ( red ) over time reflects the saw-tooth pattern of prolonged episodes; virus produced from the initial ulcer ( red dotted line ) was eliminated within 3 days .",
"( C ) Infected cells were eliminated from the initial viral ulcer ( green dotted line ) within 1 day and there were four periods during the episode when no infected cells were present .",
"( D ) Cell-free virus ( red ) , cell-associated virus ( blue ) , and infected cells ( green ) were eliminated from the primary ulcer with different kinetics; infected cells peaked at 13 hr and were extinguished in <24 hr ( E ) Secondary ulcers prolonged episodes; each thin line represents HSV-2 production from a specific region . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 02610 . 7554/eLife . 00288 . 027Video 2 . Spatiotemporal demonstration of a 14-day episode according to viral production within each single region . The left panel represents cell-free HSV DNA measured over time with each region's production demonstrated with a different color .",
"The right panel represents spatial spread of virus during the episode; colors in the right panel correspond to those in the left panel .",
"Amount of virus within a region is displayed according to a heat map adjacent to the spatial map . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 02710 . 7554/eLife . 00288 . 028Video 3 . Spatiotemporal demonstration of infected cell and viral spread during a 6-day episode . The upper left panel represents spatial spread of cell-free virus during the episode; the upper right panel represents spatial spread of cell-associated virus during the episode; the bottom left panel represents spatial spread of infected cells during the episode; the bottom right panel represents ulcer formation during the episode , ulcers turn from black to red when diameter exceeds 1 mm; quantities are displayed according to a heat map adjacent to each spatial map .",
"There is more rapid decay of infected cells and cell-associated particles than of cell-free particles within each region .",
"Visible ulcers persist after viral production terminates within a region . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 028 Parameter estimates suggest that cell-free HSV was ∼2000-fold less infectious than cell-associated HSV ( Table 2 ) , possibly because HSV in the aqueous environment outside of the cell must contend with genital secretion flow kinetics , mucous , low pH , and neutralizing or ADCC-like antibodies , while cell-associated HSV avoids these hazards by congregating within cellular tight junctions ( Collins and Johnson , 2003 ) .",
"In nonmucosal regions , a layer of dead cells that do not support viral replication protects underlying nucleated keratinocytes in the epidermis against infection , further decreasing the probability that cell-free HSV will contact a viral entry receptor .",
"Due to slower elimination of cell-free particles , the model predicted that the number of ulcers containing cell-free particles often exceeded that of ulcers populated by HSV-infected cells , thus prolonging the opportunity for secondary seeding ( Video 3 ) .",
"To address this question , we conducted a 365-day simulation with infectious and noninfectious cell-free HSV DNA ( Videos 4 and 5 ) and found that noninfectious cell-free HSV , as may occur in the presence of comprehensive neutralizing antibody protection , resulted in a decrease in episode duration and shedding frequency from 18 . 2% to 10 . 8% . 10 . 7554/eLife . 00288 . 029Video 4 . Spatiotemporal demonstration of 365 days of simulated shedding . The left panel represents total cell-free HSV DNA copies per milliliter present over time .",
"The right panel represents spatial spread of virus during the episode; each hexagon contains one region of shedding and virus spreads to contiguous regions .",
"Amount of virus within a single region is displayed according to a heat map adjacent to the spatial map .",
"The simulation is notable for episodes of variable duration and peak HSV DNA copy number .",
"Prolonged episodes at days 8 , 38 , 136 , 158 , and 288 , display several re-expansion phases . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 02910 . 7554/eLife . 00288 . 030Video 5 . Spatiotemporal demonstration of 365 days of simulated shedding with noninfectious cell-free particles . The left panel represents total cell-free HSV DNA copies per milliliter present over time .",
"The right panel represents spatial spread of virus during the episode; each hexagon contains one region of shedding .",
"Amount of virus within a single region is displayed according to a heat map adjacent to the spatial map .",
"The simulation is notable for lack of prolonged episodes and lack of episode re-expansion . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 030 In spatial simulations , despite rapid initial accumulation , infected cells were eliminated rapidly , highlighting the intense local immune response ( Figure 5C , D ) .",
"In the 100 simulated episodes described above , the median time to death of 10 , 000 , 1000 , or 100 infected cells was 13 . 7 , 9 . 7 , or 6 . 9 hr , respectively .",
"We previously described an inverse relationship between HSV-2 episode severity and CD8+ T-cell density at the site of reactivation ( Schiffer et al . , 2010 ) .",
"Our models predict that CD8+ T-cell density is an inverse correlate of the reproductive number ( the number of cells infected by the initially infected cell ) in a region .",
"Once T-cell density reaches a certain threshold , R falls below 1 and HSV-containing cells are cleared before infection of 10 cells can occur ( Schiffer et al . , 2010 ) .",
"We performed a global sensitivity analysis of 500 simulated episodes ( ‘Methods’ ) to evaluate drivers of single episode severity .",
"CD8+ T-cell density within the initially infected region was the most predictive parameter of episode peak viral load and duration .",
"Increased viral replication rate in epidermal cells and decreased CD8+ T-cell expansion rate also correlated with higher peak viral production , while increased viral infectivity and replication rate correlated with longer episode duration ( Table 3 ) . 10 . 7554/eLife . 00288 . 031Table 3 . Predictive model parameters for key model outcomesDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 031Single episode featuresLong-term shedding featuresPeak viral loadDurationShedding rateEpisode rateCD8+ T-cell density at reactivation site−0 . 56−0 . 47NANACell-associated HSV infectivity—0 . 12—0 . 13Cell-free HSV infectivity————Epidermal cell replicate rate0 . 130 . 14−0 . 25−0 . 31Neuronal release rate——0 . 430 . 55Free-viral decay rate——−0 . 2—Maximal CD8+ T-cell expansion rate−0 . 09—0 . 370 . 51CD8+ T-cell decay rate—0 . 09—−0 . 16CD8+ T-cell local recognition————CD8+ regional co-dependence——0 . 320 . 34Viral production lag——0 . 240 . 23Partial correlation coefficients are listed only for parameters that are found to improve predictive effect on outcomes using Akaike information criteria models .",
"Episode features are from 500 single episode simulations .",
"Long-term shedding outcomes were measured over 10-years during 500 simulations .",
"These findings suggest that local T-cell density at the site of neuronal HSV-2 release into genital skin determines short-term viral trajectories .",
"When high CD8+ T-cell density regions enclosed the initial infected region , spread was limited ( Video 6 ) , whereas more prolonged episodes followed a serpiginous route along low CD8+ T-cell density pathways before encountering a high-density dead end ( Video 7 ) .",
"Episodes of >10 days occurred when large genital tract areas were at low CD8+ T-cell density ( R > 1 ) ( Video 8 ) .",
"HSV replication sometimes terminated at edge regions , which is biologically realistic because herpetic lesions rarely form in skin surrounding the genital tract . 10 . 7554/eLife . 00288 . 032Video 6 . Spatiotemporal demonstration of immune response to a pair of 2-day episodes . The upper left panel represents total cell-free HSV DNA copies per milliliter present over time .",
"The upper right panel represents spatial spread of cell-free virus during the episode .",
"The lower left panel represents CD8+ T-cell density within each region .",
"The lower right panel indicates reproductive number within each region .",
"Quantities are displayed according to a heat map adjacent to each spatial map .",
"Areas with high CD8+ T-cell levels and low reproductive numbers do not support high-level viral production .",
"The simulated episodes are short because virus does not spread from the initial plaque to adjacent regions with high CD8+ T-cell density and reproductive numbers less than one . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 03210 . 7554/eLife . 00288 . 033Video 7 . Spatiotemporal demonstration of immune response to a 7-day episode . The upper left panel represents total cell-free HSV DNA copies per milliliter present over time .",
"The upper right panel represents spatial spread of cell-free virus during the episode .",
"The lower left panel represents CD8+ T-cell density within each region .",
"The lower right panel indicates reproductive number within each region .",
"Quantities are displayed according to a heat map adjacent to each spatial map .",
"The simulated episode is medium length because virus spreads from the initial region to adjacent regions with low CD8+ T-cell density and reproductive numbers less than one , but is ultimately contained when it reaches an anatomic edge region outside of the genital tract , and regions of high CD8+ T-cell density and low reproductive number within the genital tract . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 03310 . 7554/eLife . 00288 . 034Video 8 . Spatiotemporal demonstration of immune response to a 14-day episode . The upper left panel represents total cell-free HSV DNA copies per milliliter present over time .",
"The upper right panel represents spatial spread of cell-free virus during the episode .",
"The lower left panel represents CD8+ T-cell density within each region .",
"The lower right panel indicates reproductive number within each region .",
"Quantities are displayed according to a heat map adjacent to each spatial map .",
"The simulated episode is prolonged because virus spreads from the initial region to adjacent regions with low CD8+ T-cell density and reproductive numbers less than one , and is not contained until many regions of the genital tract are infected . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 034 After prolonged episodes , portions of the genital tract were effectively immunized against high-copy episodes for a short period , while other regions remained susceptible to substantial spread of virus .",
"Over years , CD8+ T-cell and reproductive number spatial patterns cycled intermittently ( Video 9 ) , suggesting that HSV causes a chronic dynamic inflammatory state in the genital tract .",
"These predictions are consistent with empirical studies of biopsied tissue of HSV-2–infected patients ( Cunningham et al . , 1985; Zhu et al . , 2007 , 2009 ) , and may explain relatively stable genital shedding rates seen within infected persons over decades . 10 . 7554/eLife . 00288 . 035Video 9 . Spatiotemporal demonstration of immune response over 20 years . The left panel represents CD8+ T-cell density within each region .",
"The right panel indicates reproductive number within each region .",
"Quantities are displayed according to a heat map adjacent to each spatial map .",
"CD8+ T-cells expand rapidly at sites of episodes and then decay slowly over time , correlating with decreases and increases in reproductive number respectively .",
"CD8+ T-cell and reproductive number spatial patterns cycle intermittently between a patchwork of heterogeneous density , broad low density , broad high density , and stark division between regions of high and low density .",
"However , CD8+ T-cell density is virtually always high in at least some regions of the genital tract . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 035 To better define viral reactivation and latency patterns that best explain our shedding datasets , we altered the frequency and spatial dispersion of neuronal HSV release in the model; genital shedding trends were unchanged if HSV was released continuously or in daily , every 3-day , or weekly pulses , provided that the average HSV DNA release rate remained ∼82/day , and that release occurred randomly throughout the genital tract ( Figure 6 ) .",
"Simulations in which release occurred continuously at 82 HSV DNA particles per day into 1 , 5 , or 20 randomly selected regions resulted in a poor fit , due to a higher percentage of rapidly contained episodes and lower shedding rate ( Figure 6—figure supplement 1 ) .",
"HSV-2 release into 50 geographically clustered regions within a confined area led to lower episode rate , less shedding , and poorer overall fit ( Figure 6—figure supplement 2 ) .",
"These data suggest that neuronal HSV release occurs at a frequent slow trickle across wide regions of the genital tract . 10 . 7554/eLife . 00288 . 036Figure 6 . Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics if particles were released continuously , daily , or weekly from neurons . White circles represent results from ( A ) 14 , 685 genital swabs and ( B–G ) 1020 shedding episodes from 531 study participants ( Figure 4 ) .",
"The model simulations represented with colored bars in each panel continued until 1020 episodes were generated .",
"Sampling occurred every 24 hr as in the clinical protocols .",
"Model output with release of virus randomly throughout the 300 regions on a continuous ( pink ) , daily ( purple ) , every 3 days ( blue ) , and weekly ( orange ) basis at an average rate of 82 HSV DNA particles per day reproduced ( A ) quantitative shedding frequency and episode , ( B ) rate , ( C ) median initiation to peak slope and peak to termination slopes , ( D ) Duration , ( E ) first HSV DNA copy number , ( F ) last HSV DNA copy number , and ( G ) peak HSV DNA copy number . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 03610 . 7554/eLife . 00288 . 037Figure 6—figure supplement 1 . Random spatial dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into only a minority of the 300 modeled regions . White circles represent results from ( A ) 14 , 685 genital swabs and ( B–G ) 1020 shedding episodes from 531 study participants ( Figure 4 ) .",
"The model simulations represented with colored bars in each panel continued until 1020 episodes were generated .",
"Sampling occurred every 24 hr as in the clinical protocols .",
"Model output with continuous release ( 82 HSV DNA particles per day ) of virus randomly to 300 ( pink ) , 100 ( blue ) , and 50 ( brown ) regions reproduced ( A ) quantitative shedding frequency and episode , ( B ) rate , ( C ) median initiation to peak slope and peak to termination slopes , ( D ) Duration , ( E ) first HSV DNA copy number , ( F ) last HSV DNA copy number , and ( G ) peak HSV DNA copy number .",
"Model output with continuous release ( 82 HSV DNA particles per day ) of virus randomly to 20 ( yellow ) , 5 ( light blue ) , and 1 ( red ) region underestimated ( A ) quantitative shedding frequency and episode , ( B ) rate , ( D ) Duration , ( E ) first HSV DNA copy number , and ( G ) peak HSV DNA copy number . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 03710 . 7554/eLife . 00288 . 038Figure 6—figure supplement 2 . Dispersion of viral particles from neurons reproduced the full diversity of episode characteristics during simulations in which particles were released into a minority of modeled regions , provided that dispersion was random rather than clustered within a single geographic region . White circles represent results from ( A ) 14 , 685 genital swabs and ( B–G ) 1020 shedding episodes from 531 study participants ( Figure 4 ) .",
"The model simulations represented with colored bars in each panel continued until 1020 episodes were generated .",
"Sampling occurred every 24 hr as in the clinical protocols .",
"Model output with continuous release ( 82 HSV DNA particles per day ) of virus to 50 randomly dispersed regions ( brown ) reproduced ( A ) quantitative shedding frequency and episode , ( B ) rate , ( C ) median initiation to peak slope and peak to termination slopes , ( D ) Duration , ( E ) first HSV DNA copy number , ( F ) last HSV DNA copy number , and ( G ) peak HSV DNA copy number .",
"Model output with continuous release ( 82 HSV DNA particles per day ) of virus to 50 clustered regions ( green ) underestimated ( A ) shedding frequency due only to ( B ) too low of an episode rate with daily sampling . DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 038 We next tested the model's ability to predict substantial variability in shedding , lesion , and episode rates in studies consisting of 30- to 60-day sampling periods ( Wald et al . , 1995 , 1997 , 2000; Crespi et al . , 2007; Mark et al . , 2008 ) .",
"Even when parameter values were not varied , the stochastic model produced realistic outcomes ( Table 4 ) .",
"Lesion and shedding frequency were variable with 30- and 60-day simulations , corresponding to previously published results ( Wald et al . , 1997 , 2000; Mark et al . , 2008 ) .",
"Simulations of 1 and 10 years , generated less varied outcomes ( Table 4 ) , suggesting that the random nature of episode initiation , along with short sampling periods may account for some shedding heterogeneity seen in clinical studies . 10 . 7554/eLife . 00288 . 039Table 4 . Spatial model simulations that varied only according to duration of sampling ( 30 days , 60 days , 365 days , and 10 years ) DOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 039Simulation durationPercent of time with HSV DNA > 150 copies per mLPercent of time with lesions* presentEpisodes per yearLesions per year30 dayMean13 . 77 . 6113 . 2Median3 . 40120Range0–82 . 80–58 . 80–36 . 50–24 . 360 dayMean19 . 09 . 813 . 44 . 4Median18 . 63 . 4126Range0–54 . 20–46 . 90–42 . 60–18365 dayMean19 . 610 . 114 . 34 . 6Median19 . 99 . 9144Range7 . 1–36 . 82 . 3–228–241–910 yearMean17 . 59 . 114 . 54 . 3Median17 . 69 . 014 . 54 . 2Range14 . 8–19 . 81 . 9–12 . 811 . 5–17 . 11 . 4–6*Lesions were defined as > 1 mm diameter ulcers . Sixty simulations were performed at each of the sampling durations .",
"Within shorter sampling duration simulations , lesion , and shedding frequency varied significantly , while ranges narrowed with prolonged sampling .",
"To assess other possible hypotheses for contrasting shedding rates , we conducted another global sensitivity analysis in which 500 Monte Carlo simulations were conducted over a 10-year time frame ( ‘Methods’ ) .",
"Predictors of elevated shedding rate included high neuronal release rate of HSV-2 DNA , high CD8+ T-cell replication rate , high regional co-dependence of CD8+ T-cell density , low HSV-2 DNA replication rate in skin cells , and low free-viral decay rate ( Table 3 ) .",
"The finding that parameter levels which would appear to favor immune control in the mucosa , in fact correlate with higher long-term shedding rates , highlight the complex nonlinear dynamics of the spatial model; parameters that favor lower amounts of viral replication in the short term promote a higher proportion of episodes that are contained rapidly by the immune system; CD8+ T-cell expansion is less substantial during these brief episodes .",
"Therefore , episodes are more commonly initiated at lower CD8+ T-cell densities .",
"As a result , conditions of high CD8+ T-cell replication rate and low HSV-2 DNA replication rate in keratinocytes tend to favor both a higher proportion of very brief episodes ( <1 day ) and more severe episodes that spread to multiple regions .",
"The overall impact is to increase the shedding rate ."
],
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"We used a strategy of intense sampling over intervals ranging from every 5 min to every 24 hr , to characterize temporal and spatial dynamics of genital HSV-2 replication .",
"While we previously identified that a majority of HSV-2 reactivations last less than a day ( Mark et al . , 2008 ) , our current findings demonstrate that the cardinal feature of HSV-2 shedding is extraordinary episode heterogeneity .",
"Longer and higher copy number episodes , which often manifest with genital lesions , are notable for multiple erratic peaks and wide viral dispersion .",
"Viral loads are stable over periods of minutes but can change dramatically over hours .",
"HSV-2 expansion and decay phases in mucosa are considerably more rapid and complex than those of HIV and hepatitis B , which have been characterized by sampling plasma ( Ciupe et al . , 2007; Ribeiro et al . , 2010 ) .",
"To explain these findings , we developed a stochastic spatial model that accurately reproduces empirically derived episode rate , duration , early viral expansion , late viral decay rate , and viral re-expansion frequency , as well as serpiginous spatial distribution of viral ulcers during recurrences .",
"Our findings redefine the pace of viral immune interaction within a single herpetic ulcer .",
"HSV replication within epithelial cells , and spread by cell-to-cell transfer , seem to occur extremely rapidly and efficiently .",
"The model predicts that >108 viral DNA genomes can be produced in one ulcer , even though the immune response becomes dominant only ∼13 . 5 hr after HSV-2 detection .",
"Routes of spread between tightly packed epithelial cells might include transit through tight cellular junctions ( Collins and Johnson , 2003; Farnsworth and Johnson , 2006 ) , distant propulsion when cells violently lyse , and homogenous mixing with susceptible cells as a result of high viral titers packed inside herpetic vesicles ( Spruance et al . , 1977 ) .",
"The model is in agreement with mouse studies , which show that tissue-resident CD8+ T-cell density at the site of viral challenge dictates lesion severity ( Gebhardt et al . , 2009; Shin and Iwasaki , 2012 ) , and further suggests that local CD8+ T-cell count supersedes all other parameters of viral replication and immunologic response in determining the extent of shedding during an episode .",
"While common wisdom is that extended viral replication occurs due to delayed arrival of acquired immune cells to the site of viral pathogenesis , our observations suggest that the immune response in microregions of replication is extraordinarily rapid .",
"Most shedding episodes are contained within hours , when <10 cells are infected ( Schiffer et al . , 2009 ) .",
"Yet , even if thousands of epithelial cells get infected in an ulcer , these cells appear to be eliminated in <24 hr .",
"Our model suggests that cell-free virus plays an important role in episode prolongation: cell-free viruses seed adjacent regions where T-cell density may be lower , leading to the formation of secondary foci of viral replication , which allows wide spread of virus across the genital tract .",
"Thus , the spatial dynamic between virus and host defines episode morphology , and explains variability in shedding severity in an individual over periods of hours to weeks .",
"These results suggest that studies of neutralizing and antibody-dependent cell-mediated cytotoxicity responses in localized mucosal areas are needed , and may explain why antiviral therapies which only impact intracellular replication and do not eliminate prolonged episodes .",
"While the model successfully generates hypotheses to explain complex shedding patterns in humans , it also highlights the difficulty of predicting an individual's short-term shedding pattern , and in particular , the timing and morphology of their next episode .",
"A heterogeneous spatial array of immune cell densities at episode onset may determine the complex trajectory of a shedding episode , but these starting conditions cannot be directly measured in humans .",
"It is similarly not possible to know in which specific microregion an episode will initiate .",
"When we simulated the model over 30-day intervals typically used in our clinical studies , there was heterogeneity in the shedding rate based solely on randomness of episode initiation and high variability of episode duration .",
"As such , we did not employ well-validated methods to infer parameter values by fitting to an individual's nonlinear shedding data ( Ionides et al . , 2006 ) .",
"On the other hand , the model does raise intriguing possible explanations for observed heterogeneity in long-term shedding behavior between infected persons ( Wald et al . , 1997 ) .",
"Surprisingly , while enhanced immune function in the form of lower viral replication rate or viral infectivity has the predictable short-term effect of limiting episode duration , the model suggests that sustained higher T-cell CD8+ replication rate and lower viral replication rate might counterintuitively increase long-term shedding rates: these parameter conditions favor a higher proportion of rapidly contained episodes , which in turn lowers the degree of CD8+ expansion .",
"Therefore , the CD8+ density at episode initiation is on average lower , which also allows for more frequent widely dispersed episodes .",
"Because the overall stability of parameter values over time cannot be easily measured , and because it is unknown if the model's parameters behave independently or in a colinear fashion , these sensitivity analysis predictions are relevant for hypothesis generation only .",
"An area of prospective research is to identify which virological and immune factors account for phenotypic variability between infected persons .",
"For the model to precisely recapitulate episode rate , it is necessary to assume a nearly constant slow drip of HSV-2 from neurons ( Schiffer et al . , 2009 ) .",
"The stochastic model and spatial shedding data both suggest that there is diffuse dispersal of virus from neurons into genital epithelia , rather than focal reactivations only within certain microregions .",
"Such a strategy allows HSV-2 to randomly seed regions where immune cell decay from previous reactivations may allow a high-titer episode to initiate .",
"This does not imply that latency is constantly bypassed within all neurons .",
"Rather , we favor the idea that the vast majority of HSV within ganglia is maintained in a low copy number latent state ( Wang et al . , 2005; Verjans et al . , 2007 ) , while a minority reactivates albeit on a frequent basis .",
"More experimental work is needed to precisely define the true mechanisms that form a balance between HSV latency and replication within ganglia .",
"Our results have interesting implications for HSV-2 transmission during coitus .",
"Most transmissions occur when an infected person is asymptomatic ( Mertz et al . , 1985 , 1998 ) , a finding that can be interpreted within the context of our model's output .",
"Secondary seeding starts to predictably occur in modeled episodes when viral loads in a single ulcer exceed 106 HSV DNA copies .",
"At this viral load , there are roughly 1000 dead cells , which is not enough for a microulcer to become visible to the naked eye .",
"Therefore , HSV-2 can efficiently seed new regions across the genital tract leading to prolonged high copy number episodes even when shedding is not associated with lesions .",
"The goal of this study was to design a model that is realistic and adequately complex to recreate our stringently defined dataset , but simple enough to have identifiable parameters .",
"Hence , one limitation is that the model underestimates immunologic complexity .",
"CD8+ T-cell response is assumed to be a surrogate of the entire immune response .",
"Other key cellular subsets such as CD4+ lymphocytes , natural killer cells , and dendritic cells are not included .",
"Lack of inclusion of innate immunity is another shortcoming as interferon type 1 is present early during lesions ( Spruance et al . , 1982 ) , and contributes to episode containment .",
"Our model generates the hypothesis that chronic HSV-2 infection leads to a dynamic and spatially heterogeneous state of immune readiness .",
"However , the interacting features of host response are not addressed and require further investigation using human and animal models of infection .",
"For convenience , we divided the genital tract into 300 regions .",
"Simulations with 200–400 regions provided similar fit to the data ( not shown ) .",
"To model spatial spread most completely would require following each cell in the genital tract with an agent-based model as well as accurate spatial elaboration of the genital anatomy ( Bauer et al . , 2009 ) : this would entail great computational cost and inclusion of many unknown parameters .",
"The high number of fitting points in Cohort E and limited number of unknown parameters in our model decreases but does not eliminate the possibility that the model has multiple solutions .",
"If swabs only remove a fixed proportion of free-viral DNA , then it is possible that we are underestimating the genital tract viral load .",
"Based on the complex nature of the observational data , the use of a stochastic simulation model , and the inability to manipulate experimental conditions in our longitudinal studies of human shedding , our parameter values are estimates rather than precise solutions .",
"We believe that the most critical hypotheses generated from this study are qualitative , and are relevant even if certain parameter values were not precisely identified .",
"In summary , our data suggest intensely localized competition between HSV-2 and the immune system in genital mucosa .",
"Our model raises the possibility that by rapidly spreading between epidermal cells , HSV-2 ensures high viral production within a single ulcer , despite an intense local immune response .",
"Cell-free HSV-2 may prolong episodes via spread to contiguous sites where immune cell density is lower .",
"Each episode is contained by the mucosal immune system , and HSV-2 is often asymptomatic in immunocompetent hosts .",
"Viral strategies that lead to repeated episodes , rapid episode expansion and frequent re-expansion , allow for high shedding frequency , thereby enhancing transmission and disease manifestations .",
"Interventions that target spread of virus between epidermal cells in a single ulcer , and to new regions of the genital tract , will be of value in controlling HSV-2 infection ."
],
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"Study participants signed informed consent for the human experimentation described in this paper .",
"All potential risks of being involved in the protocols , including discomfort , bleeding and infection , were explained to all participants in detail .",
"The consent process including agreeing to have the experimental data published .",
"Study subjects were reimbursed for clinic visits and the considerable time dedicated to each of the protocols .",
"The University of Washington institutional review board approved all study protocols described in this study .",
"HSV-2 seropositive participants performed swabs of the genital tract , which were subsequently processed for quantitative HSV DNA PCR using polymerase chain reaction ( PCR ) ( Wald et al . , 1997; Magaret et al . , 2007; Mark et al . , 2008 ) .",
"Detection of HSV DNA was performed using a well-validated collection and detection method ( Magaret et al . , 2007 ) .",
"Swabs of genital secretions were placed into vials containing 1 ml of PCR transport medium and refrigerated until laboratory processing .",
"HSV DNA was detected using a quantitative PCR assay , and was expressed as copies per milliliter of medium .",
"The PCR assay uses type-common primers to the HSV gene encoding glycoprotein B . An internal control was included to ensure that HSV-negative swabs were not due to inhibition .",
"Laboratory personnel were blinded to clinical data .",
"We designed 5 separate cohorts , which used common sample protocols and virological assay but differed according to how frequently patients swabbed their genital tract and according to location of sampling ( Table 1 ) .",
"Cohort E was used for fitting the model ( Schiffer et al . , 2011 ) : participants swabbed their entire genital tract every 24 hr whether lesions were present or not for a minimum of 30 days .",
"We used eight summary measures ( five frequency histograms and three median measures , Figure 4 ) , which included a total of 42 data bins , to describe fundamental characteristics of shedding episodes .",
"Three histograms described frequency of different HSV DNA copy number values within logarithmically defined strata ( peak , first , and last positive swab within an episode , respectively ) .",
"Two frequency histograms described episode duration , and frequency of total swabs within logarithmically defined strata .",
"Duration of episodes was measured with interval censoring ( whereby episodes of unknown duration due to initiation before day 0 of the shedding protocol or termination following the end of the swabbing period were excluded ) and no interval censoring ( whereby these episodes were included ) ( Figure 4D ) .",
"Median measures included regression slopes from initiation to episode peak and episode peak to termination , as well as episode frequency ( Figure 4B , C ) .",
"Cohort C was similar to Cohort E , except participants swabbed every 6 hr ( Mark et al . , 2008 ) , allowing for more precise measures of episode rate , early episode expansion rate , and late episode decay rate .",
"Cohort C was not used for direct model fitting because the number of subjects was much larger in Cohort E . During Cohort C , episodes of age 0–6 hr were captured with the first positive swab value: the median episode within the 2 datasets was captured 3 hr ( 0 . 125 days ) after shedding episode initiation .",
"We therefore divided median first positive swab copy number by 0 . 125 to estimate the median early expansion slope .",
"We used an equivalent approach using last positive copy number from episodes to estimate a late decay slope .",
"We calculated initiation to peak slopes in Cohorts E and C , with the assumption that episodes initiated 12 and 3 hr before the first positive swab , respectively , and fit a regression model from this point to episode peak .",
"We calculated peak to termination slopes in Cohorts E and C , with the assumption that episodes ended 12 and 3 hr after the last positive swab , respectively , and fit a regression model from this episode peak to this point .",
"The narrow interval of swabs from Cohort C was likely to represent a more accurate estimate of expansion and decay kinetics .",
"However , median values from Cohort E ( Figure 4B ) were useful as fitting measures because we also sampled daily when fitting the model .",
"As detailed in Table 1 , we performed two shedding protocols ( Cohorts A and B ) when genital lesions were present .",
"To demonstrate spatial analysis of shedding ( Cohort D ) , study participants had a daily detailed genital examination by an experienced clinician .",
"If lesions were present , the lesion location was recorded on genital diagrams .",
"Twenty-three distinct sites were swabbed at each daily visit ( Figure 2A ) .",
"Care was taken to avoid contamination from other anatomic areas during sampling ( Tata et al . , 2010 ) .",
"We defined spatiotemporal dynamics of HSV-2–specific lymphocyte response to a genital recurrence with in situ staining of genital skin biopsy with HSV-2 antigen-specific quantum dot multimers ( Zhu et al . , 2007 , 2009 ) .",
"We then examined biopsy specimens with confocal microscopy .",
"CD8+ lymphocytes appeared at the precise site of HSV release from the neuron termini , the dermal/epidermal junction .",
"Previous studies indicated that at least 10% of CD8+ T cells detected in tissue at the dermal–epidermal junction are HSV-2 specific and that this proportion remains constant as CD8+ T-cell levels fluctuate ( Zhu et al . , 2007 ) .",
"Therefore , we used data derived from in situ staining of CD8+ T cells to define relative densities of HSV-2–specific CD8+ T cells in mucosal tissue .",
"In previous studies , absolute number of CD8+ lymphocytes was measured per square millimeter of genital skin .",
"These measures are underestimates for total number of CD8+ T cells present in the entire lesion area because most lesions are >1 mm2 .",
"Values of sequential CD8+ T-cell densities gathered every 2 weeks therefore estimate relative intensity of the immune response at different time points .",
"All preliminary simulations were performed using Berkley Madonna , while C++ was used for all spatial modeling , high throughput simulations , parameter fitting , sensitivity analyses , and video generation .",
"The goal of our models was to reproduce the 42 data bins within the 8 summary measures of HSV-2 shedding from Cohort E . To this end , we iteratively developed four models with competing equations ( Table 5 ) .",
"We solved models stochastically due to the random nature of shedding episode initiation and clearance , and to account for the frequent presence of low numbers of infected cells: at each time step , integer values for equation terms were drawn randomly from binomial distributions .",
"To assess the degree of fit between models , numerical output and empirical data required prolonged simulations to minimize fluctuations in output due to stochastic effect .",
"For each fitting attempt , we ran the model until 500–1000 episodes were generated .",
"While state variables were updated at a narrow time interval ( 0 . 001 days ) , we assembled the modeled data exactly as it was gathered in the clinical protocols by sampling every 24 hr . 10 . 7554/eLife . 00288 . 040Table 5 . Mathematical models of HSV-2 pathogenesisDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 040ModelEquations ( additions to previous model are denoted in bold ) Variables ( model fitting variable ) New features1 ( Schiffer et al . , 2009; Schiffer et al . , 2010 ) • ΔS=[λ− ( βi×S×V ) − ( βi×S×Vneu ) ] Δt• ΔI=[ ( βi×S×Vi ) + ( βi×S×Vneu ) − ( a×I ) − ( f×I×E ) ] Δt• ΔE=[ ( F ( I ) ×θ×E ) − ( δ×E ) ] Δt• ΔVi=[ ( p×I ) − ( c×Vi ) − ( βi×S×Vi ) ] Δt• ΔVneu=[φ− ( c×Vneu ) − ( βi×S×Vneu ) ] Δt• F ( I ) =I/ ( I+r ) • Vtot=Vi+Vneu• λ=d ( S−S0 ) S , I , E , Vi Vneu , —2• ΔS=[λ− ( βi×S×V ) − ( βi×S×Vneu ) ] Δt• ΔI=[ ( βi×S×Vi ) + ( βi×S×Vneu ) − ( a×I ) − ( f×I×E ) ] Δt• ΔE=[ ( F ( I ) ×θ×E ) − ( δ×E ) ] Δt• ΔVneu=[φ− ( c×Vneu ) − ( βi×S×Vneu ) ] Δt• ΔVi=[ ( p×I ) − ( a×Vi ) − ( βi×S×Vi ) ] Δt• ΔVe=[ ( a×Vi ) − ( c×Ve ) ] Δt• F ( I ) =I/ ( I+r ) • Vtot=Vi+Ve+Vneu• λ=d ( S−S0 ) S , I , E , Vi , Vneu , ( Ve ) Cell-free and cell-associated particles3• ΔS ( i…300 ) ==[λ− ( βi×S×Vi ) − ( βi×S×Vneu ) − ( βe×S×Ve ) ] Δt• ΔI ( i…300 ) =[βi×S×Vi ) + ( βi×S×Vneu ) − ( βe×S×Vetot ) − ( a×l ) − ( f×l×E] Δt• ΔS ( i…300 ) =[ ( F ( I ) ×θ×E ) − ( δ×E ) ] Δt• ΔVneu ( i…300 ) =[φ− ( c×Vneu ) − ( βi×S×Vneu ) ] Δt• ΔVi ( i…300 ) =[ ( p×I ) − ( a×Vi ) − ( βi×S×Vi ) Δt• ΔVe ( i…300 ) = [ ( a×Vi ) − ( c×Ve ) ]Δt• F ( I ) =I/ ( I+r ) • Itot=I1+I2+…+Ie300• Vetot=Ve1+Ve2+…+Ve300• Vitot=Vi1+Vi2+…+Vi300• λ=d ( S−S0 ) S , I , E , Vneu , Vitot , ( Vetot ) Concurrent plaques from cell-free particles4*• S0=1 . 67 e5 per region• ΔS ( i…300 ) =[λ− ( βi×S×Vi ) − ( βi×S×Vneu ) − ( βe×S×Ve ) ] Δt• ΔI ( i…300 ) =[ ( βi×S×Vi ) + ( βi×S×Vneu ) + ( βe×S×Veadj ) − ( a×I ) − ( f×I×E ) ] Δt• ΔE ( i…300 ) =[ ( F ( I ) ×θ×E ) − ( δ×E ) ] Δt• ΔVneu ( i…300 ) =[φ− ( c×Vneu ) − ( βi×S×Vneu ) ] Δt• ΔVi ( i…300 ) =[ ( p×I ) − ( a×Vi ) − ( βi×S×Vi ) ] Δt• ΔVe ( i…300 ) =[ ( a×Vi ) − ( c×Ve ) ] Δt• λ=d ( S−S0 ) • F ( I ) =I/ ( I+r ) • Veadj=Vefrom 6 adjacent regions• Itot=I1+I2+…+I300• Vetot=Ve1+Ve2+…+Ve300• Vitot=Vi1+Vi2+…+Vi300S , I , E , Vneu , Vitot , ( Vetot ) Spatial model*Model 4 has a parameter of regional CD8+ co-dependence ( ρ ) within each plaque-forming region .",
"At episode onset within a region , the CD8+ density is adjusted to infer the spatial co-dependence of CD8 density from surrounding regions: Ei ( time + 0 . 001 ) = ( Ei × ( 1 − ρ ) ) + ( Eavg × ρ ) where Eavg is average of E from the 6 surrounding regions . Models are described in the ‘Methods’ .",
"Units and values of each parameter in the optimized model are listed in Table",
"2 . Variables include: S ( susceptible skin cells ) , I ( infected skin cells ) , E ( CD8+ T cells ) , Vi ( cell-associated HSV DNA particles ) , and Ve ( cell-free HSV DNA particles ) .",
"For each of the four competing mathematical models ( Table 5 ) , we performed repeated simulations while adjusting and narrowing parameter values ( see ‘Parameter value search’ below ) until a weighted least squares approach no longer improved fit to the 8 frequency histograms from Cohort E . For each model fitting simulation , we assigned each of the summary measures ( 1 .",
"episode rate ,",
"2 . episode duration ,",
"3 . median initiation to peak slope ,",
"4 . median peak to termination slope ,",
"5 . first positive copy number of episodes ,",
"6 . last positive copy number of episodes ,",
"7 . peak positive copy number of episodes , and",
"8 . quantitative shedding ) a weighting factor to ensure that each summary measure carried an equivalent weight .",
"First , using the empirical data ( colored bars , Figure 4 ) , the mean value of bins within each of the five histograms ( 1 .",
"episode duration ,",
"2 . first positive copy number of episodes ,",
"3 . last positive copy number of episodes ,",
"4 . peak positive copy number of episodes , and",
"5 . quantitative shedding ) was calculated; the inverse square of this value was then used to generate an ‘initial weighting factor’ , which was then divided by the number of bins within the histogram such that each bin was assigned a ‘bin weighting factor’ .",
"The three median measures ( 1 .",
"episode rate ,",
"2 . median initiation to peak slope , and",
"3 . median peak to termination slope ) only contained one bin such that the initial weighting factors were equal to the bin weighting factors .",
"We calculated a ‘fitting score’ for every simulation of the model .",
"For each bin , the difference between the empirical data and model output was squared and multiplied by the bin weighting factor for the bin , to arrive at a bin score .",
"For the episode duration histogram , if the modeled value fell between the interval censored and noninterval censored empirical values , then the difference was assumed to be zero .",
"Each simulation was given a fitting score equal to the sum of these 42 ‘bin scores’ with a lower score representing better model fit .",
"Using an ad hoc approach , we compared fitting scores with repeated automations of the same model but different parameter sets , to arrive at the optimal set of values for each model; once a model's scores no longer improved , we assigned the model a best fitting score .",
"Best fitting scores were compared between our four models ( Table 6 ) in order to select the superior model . 10 . 7554/eLife . 00288 . 041Table 6 . Model fit to Cohort EDOI: http://dx . doi . org/10 . 7554/eLife . 00288 . 041Summary measureSummed criteria scoresBest fitting scoreAICShedding frequencyEpisode durationFirst positive swabLast positive swabPeak positive swabMedian expansionMedian decayEpisode frequencyModel 12 . 135 . 590 . 050 . 080 . 240 . 070 . 430 . 018 . 61−50Model 21 . 268 . 330 . 380 . 340 . 220 . 040 . 090 . 0310 . 69−41Model 30 . 253 . 750 . 310 . 220 . 610 . 050 . 500 . 225 . 92−62Model 4 solved for 10 parameters0 . 250 . 130 . 290 . 150 . 090 . 010 . 010 . 030 . 96−139Model 4 solved for 5 parameters0 . 390 . 320 . 440 . 210 . 090 . 0400 . 181 . 67−125Summed criteria scores measure the degree of fit for each model according the eight individual shedding episode features using a weighted sum of squares .",
"Model 4 is the spatial model .",
"Models 1–3 are described in the ‘Methods’ .",
"Best fitting score is a sum of all summed criteria scores for a particular model with lower scores indicating better fit .",
"AIC: Akaike information criteria with lower scores indicating better fit .",
"We complemented the best fitting score approach by using ‘Akaike information criteria ( AIC ) scores’ as a standardized measure of model agreement with data .",
"The AIC penalizes for extra parameters but rewards better model fit to the data .",
"For our model , the AIC was calculated as AIC = N × ln ( RSS/N ) + 2k , where N = number of fitting points , k = number of parameters in the model , and RSS is the residual sum of squares , which we equated to the best fitting score .",
"To characterize how well each of the four models reproduced individual fitting criteria , we generated a standardized grading system that accounted for the unique nature of our dataset ( Table 6 ) .",
"For every fitting simulation , we summed the bin scores in each of the eight histograms into individual ‘summed criteria scores’ .",
"We observed that summed criteria scores >1 resulted in significant disparity between the empirical and modeled data , while scores between 0 . 5 and 1 signified only moderate differences .",
"Finally , if sums of the scores within a histogram amounted to <0 . 5 , then there was close similarity between the modeled data .",
"For each simulation , if a summed criteria score exceeded 1 , then we labeled the simulation a ‘poor’ fit to the summary measure .",
"If a summed criteria score exceeded 5 , then we labeled the simulation a ‘very poor’ fit .",
"If a summed criteria score exceeded 0 . 5 but was <1 . 0 , we labeled the simulation a ‘fair’ fit , while scores <0 . 5 were considered a ‘good’ fit .",
"Using this approach , we specified which facets of shedding certain models could not replicate .",
"We used summed criteria scores to provide ranges for optimized parameter values ( Table 2 ) ."
]
] | [
"Herpes simplex virus-2 ( HSV-2 ) is shed episodically , leading to occasional genital ulcers and efficient transmission .",
"The biology explaining highly variable shedding patterns , in an infected person over time , is poorly understood .",
"We sampled the genital tract for HSV DNA at several time intervals and concurrently at multiple sites , and derived a spatial mathematical model to characterize dynamics of HSV-2 reactivation .",
"The model reproduced heterogeneity in shedding episode duration and viral production , and predicted rapid early viral expansion , rapid late decay , and wide spatial dispersion of HSV replication during episodes .",
"In simulations , HSV-2 spread locally within single ulcers to thousands of epithelial cells in <12 hr , but host immune responses eliminated infected cells in <24 hr; secondary ulcers formed following spatial propagation of cell-free HSV-2 , allowing for episode prolongation .",
"We conclude that HSV-2 infection is characterized by extremely rapid virological growth and containment at multiple contemporaneous sites within genital epithelium ."
] | [
"Viruses infect organisms as diverse as unicellular bacteria , plants , and animals .",
"Two well-known human viral infections are herpes simplex virus 1 , which is responsible for most cold sores , and herpes simplex virus 2 ( HSV-2 ) , which causes most cases of genital herpes .",
"The first signs of HSV-2 infection are genital lesions , which usually heal relatively quickly .",
"However , the virus can also enter nerve cells , where it hides from the immune system and survives for the lifetime of the infected host .",
"In a process that is not fully understood , the dormant viruses inside nerve cells periodically reactivate and are transported back to the genital tract where they can cause recurrent genital sores .",
"HSV-2 also commonly replicates in genital skin when lesions are not present , and is efficiently transmitted to other individuals at these times .",
"Most studies of viral infection rely on the examination of blood to determine viral load and characterize the immune response .",
"However , the true battle between HSV-2 and its host occurs within genital tissues .",
"HSV-2 lesions are clearly visible and it is therefore possible to precisely follow the number of viruses and the intensity of the host immune response as a function of time and position .",
"Schiffer et al . follow this approach by examining the levels of HSV-2 DNA in the genital tracts of over 600 individuals , who were sampled at different time intervals and at multiple sites .",
"Dramatic differences in the viral load and the density of CD8+ T cells , which are critical for controlling HSV-2 , are observed across several millimeters of the skin .",
"Viral load also varies dramatically over a few hours .",
"These data are used to develop a mathematical model that characterizes the spatial dynamics of HSV-2 reactivation and suggests how the host immune system responds to reactivation .",
"Historically , it was thought that host immune cells , including CD8+ T cells , required many days to contain an HSV-2 reactivation , allowing prolonged symptoms .",
"However , the model developed by Schiffer et al . indicates that although viral infection can spread from a single skin cell to thousands of cells in 12 hr or so , the host immune cells typically clear all infected skin cells in under 24 hr .",
"Episodes of viral shedding lasting >3 days are predicted to occur due to viral seeding of adjacent regions of genital skin .",
"Schiffer et al . predict that the complex episodic nature of HSV-2 reactivation is heavily influenced by the spatial distribution and density of immune cells within the genital tract , which means that new lesions occur in regions where immune cell density is low .",
"The rapid onset and localized clearance pattern suggests that viral elimination by skin-resident CD8+ T cells is actually highly effective within single genital ulcers .",
"This hypothesis represents an area of intense current investigation for clinicians , virologists , mathematical modelers , and immunologists ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"epidemiology and global health",
"medicine"
] | Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission | elife-58728-v2 | [
[
"Despite the World Health Organisation ( WHO ) advocating widespread testing for SARS-CoV-2 , national capacities for implementation have diverged considerably ( WHO , 2020b; Our World in Data , 2020 ) .",
"In the UK , the strategy has been to perform SARS-CoV-2 testing for essential workers who are symptomatic themselves or have symptomatic household contacts .",
"This approach has been exemplified by recent studies of symptomatic HCWs ( Hunter et al . , 2020; Keeley et al . , 2020 ) .",
"The role of nosocomial transmission of SARS-CoV-2 is becoming increasingly recognised , accounting for 12–29% of cases in some reports ( Wang et al . , 2020 ) .",
"Importantly , data suggest that the severity and mortality risk of nosocomial transmission may be greater than for community-acquired COVID-19 ( McMichael et al . , 2020 ) .",
"Protection of HCWs and their families from the acquisition of COVID-19 in hospitals is paramount , and underscored by rising numbers of HCW deaths nationally and internationally ( Cook et al . , 2020; CDC COVID-19 Response Team , 2020 ) .",
"In previous epidemics , HCW screening programmes have boosted morale , decreased absenteeism and potentially reduced long-term psychological sequelae ( McAlonan et al . , 2007 ) .",
"Screening also allows earlier return to work when individuals or their family members test negative ( Hunter et al . , 2020; Keeley et al . , 2020 ) .",
"Another major consideration is the protection of vulnerable patients from a potentially infectious workforce ( McMichael et al . , 2020 ) , particularly as social distancing is not possible whilst caring for patients .",
"Early identification and isolation of infectious HCWs may help prevent onward transmission to patients and colleagues , and targeted infection prevention and control measures may reduce the risk of healthcare-associated outbreaks .",
"The clinical presentation of COVID-19 can include minimal or no symptoms ( WHO , 2020a ) .",
"Asymptomatic or pre-symptomatic transmission is clearly reported and is estimated to account for around half of all cases of COVID-19 ( He et al . , 2020 ) .",
"Screening approaches focussed solely on symptomatic HCWs are therefore unlikely to be adequate for suppression of nosocomial spread .",
"Preliminary data suggests that mass screening and isolation of asymptomatic individuals can be an effective method for halting transmission in community-based settings ( Day , 2020 ) .",
"Recent modelling has suggested that weekly testing of asymptomatic HCWs could reduce onward transmission by 16–23% , on top of isolation based on symptoms , provided results are available within 24 hr ( Imperial College COVID-19 Response Team , 2020 ) .",
"The need for widespread adoption of an expanded screening programme for asymptomatic , as well as symptomatic HCWs , is apparent ( Imperial College COVID-19 Response Team , 2020; Black et al . , 2020; Gandhi et al . , 2020 ) .",
"Challenges to the roll-out of an expanded screening programme include the ability to increase diagnostic testing capacity , logistical issues affecting sampling and turnaround times and concerns about workforce depletion should substantial numbers of staff test positive .",
"Here , we describe how we have dealt with these challenges and present initial findings from a comprehensive staff screening programme at Cambridge University Hospitals NHS Foundation Trust ( CUHNFT ) .",
"This has included systematic screening of >1000 asymptomatic HCWs in their workplace , in addition to >200 symptomatic staff or household contacts .",
"Screening was performed using a validated real-time reverse transcription PCR ( RT-PCR ) assay detecting SARS-CoV-2 from combined oropharyngeal ( OP ) and nasopharyngeal ( NP ) swabs ( Sridhar et al . , 2020 ) .",
"Rapid viral sequencing of positive samples was used to further assess potential epidemiological linkage where nosocomial transmission was suspected .",
"Our experience highlights the value of programmes targeting both symptomatic and asymptomatic staff , and will be informative for the establishment of similar programmes in the UK and globally ."
],
[
"Between 6th and 24th April 2020 , 1 , 270 HCWs in CUHNFT and their symptomatic household contacts were swabbed and tested for SARS-CoV-2 by real-time RT-PCR .",
"The median age of the HCWs was 34; 71% were female and 29% male .",
"The technical RT-PCR failure rate was 2/1 , 270 ( 0∙2% see Materials and methods ) ; these were excluded from the ‘Tested’ population for further analysis .",
"Ultimately , 5% ( n = 61 ) of swabs were SARS-CoV-2 positive .",
"21 individuals underwent repeat testing for a variety of reasons , including evolving symptoms ( n = 3 ) and scoring ‘medium’ probability on clinical COVID-19 criteria ( Tables 1–2 ) ( n = 11 ) .",
"All remained SARS-CoV-2 negative .",
"Turn around time from sample collection to resulting was 12–36 hr; this varied according to the time samples were obtained .",
"Table 3 outlines the total number of SARS-CoV-2 tests performed in each screening group ( HCW asymptomatic , HCW symptomatic , and HCW symptomatic household contact ) categorised according to the ward with the highest anticipated risk of exposure to COVID-19 ( ‘red’ , high; ‘amber’ , medium; ‘green’ , low; Tables 4–5 ) .",
"In total , 31/1 , 032 ( 3% ) of those tested in the HCW asymptomatic screening group tested SARS-CoV-2 positive .",
"In comparison , 30/221 ( 14% ) tested positive when HCW symptomatic and HCW symptomatic household contact screening groups were combined .",
"As expected , symptomatic HCWs and their household contacts were significantly more likely to test positive than HCWs from the asymptomatic screening group ( p<0∙0001 , Fisher’s exact test ) .",
"HCWs working in ‘red’ or ‘amber’ wards were significantly more likely to test positive than those working in ‘green’ wards ( p=0∙0042 , Fisher’s exact test ) .",
"All users of FFP3 masks underwent routine fit-testing prior to usage .",
"Cleaning and re-use of masks , theatre caps , gloves , aprons or gowns was actively discouraged .",
"Cleaning and re-use of eye protection was permitted for certain types of goggles and visors , as specified in the hospital’s PPE protocol .",
"Single-use eye protection was in use in most Scenario 1 and 2 areas , and was not cleaned and re-used .",
"All non-invasive ventilation or use of high-flow nasal oxygen on laboratory-confirmed or clinically suspected COVID-19 patients was performed in negative-pressure ( −5 pascals ) side rooms , with 10 air changes per hour and use of Scenario 2 PPE .",
"All other aerosol generating procedures were undertaken with Scenario 2 PPE precautions , in negative- or neutral- pressure facilities .",
"General clinical areas underwent a minimum of 6 air changes per hour , but all critical care areas underwent a minimum of 10 air changes per hour as a matter of routine .",
"Surgical operating theatres routinely underwent a minimum of 25 air changes per hour .",
"Viral loads varied between individuals , potentially reflecting the nature of the sampling site .",
"However , for individuals testing positive for SARS-CoV-2 , viral loads were significantly lower for those in the HCW asymptomatic screening group than in those tested due to the presence of symptoms ( Figure 1 ) .",
"For the HCW symptomatic and HCW symptomatic contact screening groups , viral loads did not correlate with duration of symptoms or with clinical criteria risk score ( Figure 1—figure supplement 1 and data not shown ) .",
"Each individual in the HCW asymptomatic screening group was contacted by telephone to establish a clinical history , and COVID-19 probability criteria ( Table 1 ) were retrospectively applied to categorise any symptoms in the month prior to testing ( Figure 2 ) .",
"One HCW could not be contacted to obtain further history .",
"Individuals captured by the HCW asymptomatic screening group were generally asymptomatic at the time of screening , however could be divided into three sub-groups:",
"( i ) HCWs with no symptoms at all ,",
"( ii ) HCWs with ( chiefly low-to-medium COVID-19 probability ) symptoms commencing ≤7 days prior to screening and",
"( iii ) HCWs with ( typically high COVID-19 probability ) symptoms commencing >7 days prior to screening ( Figure 2 ) .",
"9/12 ( 75% ) individuals with symptom onset >7 days previously had appropriately self-isolated and then returned to work .",
"One individual with no symptoms at the time of swabbing subsequently developed symptoms prior to being contacted with their positive result .",
"Overall , 5/1032 ( 0 . 5% ) individuals in the asymptomatic screening group were identified as truly asymptomatic carriers of SARS-CoV-2 , and 1/1032 ( 0 . 1% ) was identified as pre-symptomatic .",
"Box 1 shows illustrative clinical vignettes .",
"For the HCW asymptomatic screening group , nineteen wards were identified for systematic priority screening as part of hospital-wide surveillance .",
"Two further areas were specifically targeted for screening due to unusually high staff sickness rates ( ward F ) , or concerns about appropriate PPE usage ( ward Q ) ( Figure 3 ) .",
"Interestingly , in line with findings in the total HCW population , a significantly greater proportion of HCWs working on ‘red’ wards compared to HCWs working on ‘green’ wards tested positive as part of the asymptomatic screening programme ( ‘green’ 6/310 vs ‘red’ 19/372; p=0 . 0389 , Fisher’s exact test ) .",
"The proportion of HCW with a positive test was significantly higher on Ward F than on other wards categorised as ‘green’ clinical areas ( ward F 4/43 vs other ‘green’ wards 2/267; p=0 . 0040 , Fisher’s exact test ) .",
"Likewise , amongst wards in the ‘red’ areas , ward Q showed significantly higher rates of positive HCW test results ( ward Q 7/37 vs other ‘red’ wards 12/335; p=0 . 0011 , Fisher’s exact test ) .",
"Ward F is an elderly care ward , designated as a ‘green’ area with Scenario 0 PPE ( Tables 4–5 ) , with a high proportion of COVID-19 vulnerable patients due to age and comorbidity .",
"4/43 ( 9% ) ward staff tested positive for SARS-CoV-2 .",
"In addition , two staff members on this ward tested positive in the HCW symptomatic/symptomatic contact screening groups .",
"All positive HCWs were requested to self-isolate , the ward was closed to admissions and escalated to Scenario 1 PPE ( Table 5 ) .",
"Reactive screening of a further 18 ward F staff identified an additional three positive asymptomatic HCWs ( Figure 4 ) .",
"Sequence analysis indicated that 6/9 samples from HCW who worked on ward F belonged to SARS-CoV-2 lineage B∙1 ( currently known to be circulating in at least 43 countries [Rambaut et al . , 2020] ) , with a further two that belonged to B1∙7 and one that belonged to B2∙1 .",
"This suggests more than two introductions of SARS-CoV-2 into the HCW population on ward F ( Figure 4—figure supplements 1–2 , Table 6 ) .",
"It was subsequently found that two further staff members from ward F had previously been admitted to hospital with severe COVID-19 infection .",
"Ward Q is a general medical ward designated as a ‘red’ clinical area for the care of COVID-19 positive patients , with a Scenario 1 PPE protocol ( Tables 4–5 ) .",
"Here , 7/37 ( 19% ) ward staff tested positive for SARS-CoV-2 .",
"In addition , one staff member tested positive as part of the HCW symptomatic screening group , within the same period as ward surveillance .",
"Reactive screening of a further five staff working on Ward Q uncovered one additional infection .",
"4/4 sequenced viruses were of the B∙1 lineage ( Figure 4—figure supplements 1–2 , Table 6; other isolates could not be sequenced due to a sample CT value >30 ) .",
"All positive HCWs were requested to self-isolate , and infection control and PPE reviews were undertaken to ensure that environmental cleaning and PPE donning/doffing practices were compliant with hospital protocol .",
"Staff training and education was provided to address observed instances of incorrect infection control or PPE practice .",
"Ward O , a ‘red’ medical ward , had similar numbers of asymptomatic HCWs screened as ward F , and a similar positivity rate ( 4/44; 9% ) .",
"This ward was listed for further cluster investigation after the study ended , however incorrect PPE usage was not noted during the study period .",
"The majority of individuals who tested positive for SARS-CoV-2 after screening due to the presence of symptoms had high COVID-19 probability ( Table 7 ) .",
"This reflects national guidance regarding self-isolation at the time of our study ( UK Government , 2020a ) ."
],
[
"Through the rapid establishment of an expanded HCW SARS-CoV-2 screening programme , we discovered that 31/1 , 032 ( 3% ) of HCWs tested positive for SARS-CoV-2 in the absence of symptoms .",
"Of 30 individuals from this asymptomatic screening group studied in more depth , 6/30 ( 20% ) had not experienced any symptoms at the time of their test .",
"1/6 became symptomatic suggesting that the true asymptomatic carriage rate was 5/1 , 032 ( 0 . 5% ) .",
"11/30 ( 37% ) had experienced mild symptoms prior to testing .",
"Whilst temporally associated , it cannot be assumed that these symptoms necessarily resulted from COVID-19 .",
"These proportions are difficult to contextualise due to paucity of point-prevalence data from asymptomatic individuals in similar healthcare settings or the wider community .",
"For contrast , 60% of asymptomatic residents in a recent study tested positive in the midst of a care home outbreak ( Arons et al . , 2020 ) .",
"Regardless of the proportion , however , many secondary and tertiary hospital-acquired infections were undoubtedly prevented by identifying and isolating these SARS-CoV-2 positive HCWs .",
"12/30 ( 40% ) individuals from the HCW asymptomatic screening group reported symptoms > 7 days prior to testing , and the majority experiencing symptoms consistent with a high probability of COVID-19 had appropriately self-isolated during that period .",
"Patients with COVID-19 can remain SARS-CoV-2 PCR positive for a median of 20 days ( IQR 17–24 ) after symptom onset ( Zhou et al . , 2020 ) , and the limited data available suggest viable virus is not shed beyond eight days ( Wölfel et al . , 2020 ) .",
"A pragmatic approach was taken to allowing individuals to remain at work , where the HCW had experienced high probability symptoms starting >7 days and ≤1 month prior to their test and had been well for the preceding 48 hr .",
"This approach was based on the following: low seasonal incidence of alternative viral causes of high COVID-19 probability symptoms in the UK ( Public Health England , 2018 ) , the high potential for SARS-CoV-2 exposure during the pandemic and the potential for prolonged , non-infectious shedding of viral RNA ( Zhou et al . , 2020; Wölfel et al . , 2020 ) .",
"For other individuals , we applied standard national guidelines requiring isolation for seven days from the point of testing ( UK Government , 2020b ) .",
"However , for HCW developing symptoms after a positive swab , isolation was extended for seven days from symptom onset .",
"Our data clearly demonstrate that focusing solely on the testing of individuals fitting a strict clinical case definition for COVID-19 will inevitably miss asymptomatic and pauci-symptomatic disease .",
"This is of particular importance in the presence of falling numbers of community COVID-19 cases , as hospitals will become potential epicentres of local outbreaks .",
"Therefore , we suggest that in the setting of limited testing capacity , a high priority should be given to a reactive asymptomatic screening programme that responds in real-time to HCW sickness trends , or ( to add precision ) incidence of positive tests by area .",
"The value of this approach is illustrated by our detection of a cluster of cases in ward F , where the potential for uncontrolled staff-to-staff or staff-to-patient transmission could have led to substantial morbidity and mortality in a particularly vulnerable patient group .",
"As SARS-CoV-2 testing capacity increases , rolling programmes of serial screening for asymptomatic staff in all areas of the hospital is recommended , with the frequency of screening being dictated by anticipated probability of infection .",
"The utility of this approach in care-homes and other essential institutions should also be explored , as should serial screening of long-term inpatients .",
"The early success of our programme relied upon substantial collaborative efforts between a diverse range of local stakeholders .",
"Similar collaborations will likely play a key role in the rapid , de novo development of comprehensive screening programmes elsewhere .",
"The full benefits of enhanced HCW screening are critically dependent upon rapid availability of results .",
"A key success of our programme has been bespoke optimisation of sampling and laboratory workflows enabling same-day resulting , whilst minimising disruption to hospital processes by avoiding travel to off-site testing facilities .",
"Rapid turnaround for testing and sequencing is vital in enabling timely response to localised infection clusters , as is the maintenance of reserve capacity to allow urgent , reactive investigations .",
"There appeared to be a significantly higher incidence of HCW infections in ‘red’ compared to ‘green’ wards .",
"Many explanations for this observation exist , and this study cannot differentiate between them .",
"Possible explanations include transmission between patients and HCW , HCW-to-HCW transmission , variability of staff exposure outside the workplace and non-random selection of wards .",
"It is also possible that , even over the three weeks of the study , ‘red’ wards were sampled earlier during the evolution of the epidemic when transmission was greater .",
"Further research into these findings is clearly needed on a larger scale .",
"Furthermore , given the clear potential for pre-symptomatic and asymptomatic transmission amongst HCWs , and data suggesting that infectivity may peak prior to symptom onset ( He et al . , 2020 ) , there is a strong argument for basic PPE provision in all clinical areas .",
"The identification of transmission within the hospital through routine data is problematic .",
"Hospitals are not closed systems and are subject to numerous external sources of infection .",
"Coronaviruses generally have very low mutation rates ( ~10−6 per site per cycle ) ( Sanjuán et al . , 2010 ) , with the first reported sequence of the current pandemic only published on 12th January 2020 ( GenBank , 2020 ) .",
"In addition , given SARS CoV-2 was only introduced into the human population in late 2019 , there is at present a lack of diversity in circulating strains .",
"However , as the pandemic unfolds and detailed epidemiological and genome sequence data from patient and HCW clusters are generated , real-time study of transmission dynamics will become an increasingly important means of informing disease control responses and rapidly confirming ( or refuting ) hospital acquired infection .",
"Importantly , implementation of such a programme would require active screening and rapid sequencing of positive cases in both the HCW and patient populations .",
"Prospective epidemiological data will also inform whether hospital staff are more likely to be infected in the community or at work , and may identify risk factors for the acquisition of infection , such as congregation in communal staff areas or inadequate access to PPE .",
"Our study is limited by the relatively short time-frame , a small number of positive tests and a lack of behavioural data .",
"In particular , the absence of detailed workplace and community epidemiological data makes it difficult to draw firm conclusions with regards to hospital transmission dynamics .",
"The low rate of observed positive tests may be partly explained by low rates of infection in the East of England in comparison with other areas of the UK ( cumulative incidence 0 . 17% , thus far ) ( Public Health England , 2020 ) .",
"The long-term benefits of HCW screening on healthcare systems will be informed by sustained longitudinal sampling of staff in multiple locations .",
"More comprehensive data will parametrise workforce depletion and COVID-19 transmission models .",
"The incorporation of additional information including staffing levels , absenteeism , and changes in proportions of staff self-isolating before and after the introduction of widespread testing will better inform the impact of screening at a national and international level .",
"Such models will be critical for optimising the impact on occupationally-acquired COVID-19 , and reducing the likelihood that hospitals become hubs for sustained COVID-19 transmission .",
"In the absence of an efficacious vaccine , additional waves of COVID-19 are likely as social distancing rules are relaxed .",
"Understanding how to limit hospital transmission will be vital in determining infection control policy , and retain its relevance when reliable serological testing becomes widely available .",
"Our data suggest that the roll-out of screening programmes to include asymptomatic as well as symptomatic patient-facing staff should be a national and international priority .",
"Our approach may also be of benefit in reducing transmission in other institutions , for example care-homes .",
"Taken together , these measures will increase patient confidence and willingness to access healthcare services , benefiting both those with COVID-19 and non-COVID-19 disease ."
],
[
"Two parallel streams of entry into the testing programme were established and managed jointly by the Occupational Health and Infectious Diseases departments .",
"The first ( HCW symptomatic , and HCW symptomatic household contact screening groups ) allowed any patient-facing or non-patient-facing hospital employee ( HCW ) to refer themselves or a household contact , including children , should they develop symptoms suggestive of COVID-19 .",
"The second ( HCW asymptomatic screening group ) was a rolling programme of testing for all patient-facing and non-patient-facing staff working in defined clinical areas thought to be at risk of SARS-CoV-2 transmission .",
"Daily workforce sickness reports and trends in the results of HCW testing were monitored to enable areas of concern to be highlighted and targeted for screening and cluster analysis , in a reactive approach .",
"High throughput clinical areas where staff might be exposed to large numbers of suspected COVID-19 patients were also prioritised for staff screening .",
"These included the Emergency Department , the COVID-19 Assessment Unit , and a number of ‘red’ inpatient wards .",
"Staff caring for the highest priory ‘shielding’ patients ( Haematology/Oncology , Transplant medicine ) were also screened , as were a representative sample of staff from ‘Amber’ and ‘Green’ areas .",
"The personal protective equipment ( PPE ) worn by staff in these areas is summarised in Table 5 .",
"Inclusion into the programme was voluntary , and offered to all individuals working in a given ward during the time of sampling .",
"Regardless of the route of entry into the programme , the process for testing and follow-up was identical .",
"Wards were closed to external visitors .",
"We devised a scoring system to determine the clinical probability of COVID-19 based on symptoms from existing literature ( Wang et al . , 2020; Giacomelli et al . , 2020; Table 1 ) .",
"Self-referring HCW and staff captured by daily workforce sickness reports were triaged by designated Occupational Health nurses using these criteria ( Table 2 ) .",
"Self-isolating staff in the medium and low probability categories were prioritised for testing , since a change in the clinical management was most likely to derive from results .",
"Self-isolation and household quarantine advice was determined by estimating the pre-test probability of COVID-19 ( high , medium or low ) in those with symptoms , based on the presence or absence of typical features ( Tables 1–2 ) .",
"Symptom history was obtained for all symptomatic HCWs at the time of self-referral , and again for all positive cases via telephone interview when results became available .",
"All individuals who had no symptoms at the time of testing were followed up by telephone within 14 days of their result .",
"Pauci-symptomatic individuals were defined as those with low-probability clinical COVID-19 criteria ( Table 2 ) .",
"Testing was primarily undertaken at temporary on-site facilities .",
"Two ‘Pods’ ( self-contained portable cabins with office , kitchen facilities , generator and toilet ) were erected in close proximity both to the laboratory and main hospital .",
"Outside space was designed to enable car and pedestrian access , and ensure ≥2 m social distancing at all times .",
"Individuals attending on foot were given pre-prepared self-swabbing kits containing a swab , electronically labelled specimen tube , gloves and swabbing instructions contained in a zip-locked collection bag .",
"Pods were staffed by a team of re-deployed research nurses , who facilitated self-swabbing by providing instruction as required .",
"Scenario 1 PPE ( Table 5 ) was worn by Pod nurses at all times .",
"Individuals in cars were handed self-swabbing kits through the window , with samples dropped in collection bags into collection bins outside .",
"Any children ( household contacts ) were brought to the pods in cars and swabbed in situ by a parent or guardian .",
"In addition to Pod-based testing , an outreach HCW asymptomatic screening service was developed to enable self-swabbing kits to be delivered to HCWs in their area of work , minimising disruption to the working routine of hospital staff , and maximising Pod availability for symptomatic staff .",
"Lists of all staff working in target areas over a 24 hr period were assembled , and kits pre-prepared accordingly .",
"Self-swabbing kits were delivered to target areas by research nurses , who trained senior nurses in the area to instruct other colleagues on safe self-swabbing technique .",
"Kits were left in target areas for 24 hr to capture a full cycle of shift patterns , and all kits and delivery equipment were thoroughly decontaminated with 70% ethanol prior to collection .",
"Twice daily , specimens were delivered to the laboratory for processing .",
"The swabbing , extraction and amplification methods for this study follow a recently validated procedure ( Sridhar et al . , 2020 ) .",
"Individuals performed a self-swab at the back of the throat followed by the nasal cavity as previously described ( Our World in Data , 2020 ) .",
"The single dry sterile swab was immediately placed into transport medium/lysis buffer containing 4M guanidine thiocyanate to inactivate virus , and carrier RNA .",
"This facilitated BSL2-based manual extraction of viral RNA in the presence of MS2 bacteriophage amplification control .",
"Use of these reagents and components avoided the need for nationally employed testing kits .",
"Real-time RT-PCR amplification was performed as previously described and results validated by confirmation of FAM amplification of the appropriate controls with threshold cycle ( CT ) ≤36 .",
"Lower CT values correspond to earlier detection of the viral RNA in the RT-PCR process , corresponding with a higher copy number of the viral genome .",
"In 2/1 , 270 cases , RT-PCR failed to amplify the internal control and results were discarded , with HCW offered a re-test .",
"Sequencing of positive samples was attempted on samples with a CT ≤30 using a multiplex PCR based approach ( Quick et al . , 2017 ) using the modified ARTIC v2 protocol ( Quick , 2020 ) and v3 primer set ( Artic network , 2020 ) .",
"Genomes were assembled using reference based assembly and the bioinformatic pipeline as described ( Quick et al . , 2017 ) using a 20x minimum coverage as a cut-off for any region of the genome and a 50 . 1% cut-off for calling of single nucleotide polymorphisms ( SNPs ) .",
"Samples were sequenced as part of the COVID-19 Genomics UK Consortium , COG-UK ) , a partnership of NHS organisations , academic institutions , UK public health agencies and the Wellcome Sanger Institute .",
"As soon as they were available , positive results were telephoned to patients by Infectious Diseases physicians , who took further details of symptomatology including timing of onset , and gave clinical advice ( Table 2 ) .",
"Negative results were reported by Occupational Health nurses via telephone , or emailed through a secure internal email system .",
"Advice on returning to work was given as described in Table 2 .",
"Individuals advised to self-isolate were instructed to do so in their usual place of residence .",
"Particularly vulnerable staff or those who had more severe illness but did not require hospitalisation were offered follow-up telephone consultations .",
"Individuals without symptoms at the time of testing were similarly followed up , to monitor for de novo symptoms .",
"Verbal consent was gained for all results to be reported to the hospital’s infection control and health and safety teams , and to Public Health England , who received all positive and negative results as part of a daily reporting stream .",
"Swab result data were extracted directly from the hospital-laboratory interface software , Epic ( Verona , Wisconsin , USA ) .",
"Details of symptoms recorded at the time of telephone consultation were extracted manually from review of Epic clinical records .",
"Data were collated using Microsoft Excel , and figures produced with GraphPad Prism ( GraphPad Software , La Jolla , California , USA ) .",
"Fisher’s exact test was used for comparison of positive rates between groups defined in the main text .",
"Mann-Whitney testing was used to compare CT values between different categories of tested individuals .",
"HCW samples that gave SARS CoV-2 genomes were assigned global lineages defined by Rambaut et al . , 2020 using the PANGOLIN utility ( O'Toole and McCrone , 2020 ) .",
"As a study of healthcare-associated infections , this investigation is exempt from requiring ethical approval under Section 251 of the NHS Act 2006 ( see also the NHS Health Research Authority algorithm , available at http://www . hra-decisiontools . org . uk/research/ , which concludes that no formal ethical approval is required ) .",
"Written consent was obtained from each HCW described in the anonymised case vignettes ."
]
] | [
"Significant differences exist in the availability of healthcare worker ( HCW ) SARS-CoV-2 testing between countries , and existing programmes focus on screening symptomatic rather than asymptomatic staff .",
"Over a 3 week period ( April 2020 ) , 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital .",
"Symptomatic staff and symptomatic household contacts were additionally tested .",
"Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab .",
"3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2 .",
"17/30 ( 57% ) were truly asymptomatic/pauci-symptomatic .",
"12/30 ( 40% ) had experienced symptoms compatible with coronavirus disease 2019 ( COVID-19 ) >7 days prior to testing , most self-isolating , returning well .",
"Clusters of HCW infection were discovered on two independent wards .",
"Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1 .",
"Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms .",
"This approach will be critical for protecting patients and hospital staff ."
] | [
"Patients admitted to NHS hospitals are now routinely screened for SARS-CoV-2 ( the virus that causes COVID-19 ) , and isolated from other patients if necessary .",
"Yet healthcare workers , including frontline patient-facing staff such as doctors , nurses and physiotherapists , are only tested and excluded from work if they develop symptoms of the illness .",
"However , there is emerging evidence that many people infected with SARS-CoV-2 never develop significant symptoms: these people will therefore be missed by ‘symptomatic-only’ testing .",
"There is also important data showing that around half of all transmissions of SARS-CoV-2 happen before the infected individual even develops symptoms .",
"This means that much broader testing programs are required to spot people when they are most infectious .",
"Rivett , Sridhar , Sparkes , Routledge et al . set out to determine what proportion of healthcare workers was infected with SARS-CoV-2 while also feeling generally healthy at the time of testing .",
"Over 1 , 000 staff members at a large UK hospital who felt they were well enough to work , and did not fit the government criteria for COVID-19 infection , were tested .",
"Amongst these , 3% were positive for SARS-CoV-2 .",
"On closer questioning , around one in five reported no symptoms , two in five very mild symptoms that they had dismissed as inconsequential , and a further two in five reported COVID-19 symptoms that had stopped more than a week previously .",
"In parallel , healthcare workers with symptoms of COVID-19 ( and their household contacts ) who were self-isolating were also tested , in order to allow those without the virus to quickly return to work and bolster a stretched workforce .",
"Finally , the rates of infection were examined to probe how the virus could have spread through the hospital and among staff – and in particular , to understand whether rates of infection were greater among staff working in areas devoted to COVID-19 patients .",
"Despite wearing appropriate personal protective equipment , healthcare workers in these areas were almost three times more likely to test positive than those working in areas without COVID-19 patients .",
"However , it is not clear whether this genuinely reflects greater rates of patients passing the infection to staff .",
"Staff may give the virus to each other , or even acquire it at home .",
"Overall , this work implies that hospitals need to be vigilant and introduce broad screening programmes across their workforces .",
"It will be vital to establish such approaches before ‘lockdown’ is fully lifted , so healthcare institutions are prepared for any second peak of infections ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Neural dynamics between anterior insular cortex and right supramarginal gyrus dissociate genuine affect sharing from perceptual saliency of pretended pain | elife-69994-v2 | [
[
"As social beings , our own affective states are influenced by other people’s feelings and affective states .",
"The facial expression of pain by others acts as a distinctive cue to signal their pain to others , and thus results in sizeable affective responses in the observer .",
"Certifying such responses as evidence for empathy , however , requires successful self-other distinction , the ability to distinguish the affective response experienced by ourselves from the affect experienced by the other person .",
"Studies using a wide variety of methods convergently have shown that observing others in pain engages neural responses aligning with those coding for the affective component of self-experienced pain , with the anterior insula ( aIns ) and the anterior mid-cingulate cortex ( aMCC ) being two key areas in which such an alignment has been detected ( Lamm et al . , 2011; Rütgen et al . , 2015; Jauniaux et al . , 2019; Xiong et al . , 2019; Zhou et al . , 2020; Fallon et al . , 2020 , for meta-analyses ) .",
"However , there is consistent debate on whether activity observed in these areas should indeed be related to the sharing of pain affect or whether it may not rather result from automatic responses to salient perceptual cues – with pain vividly expressed on the face being one particularly prominent example ( Zaki et al . , 2016 , for review ) .",
"It was thus one major aim of our study to address this question .",
"In this respect , contextual factors , individuals’ appraisals , and attentional processes would all impact their exact response to the affective states of others ( Gu and Han , 2007; Hein and Singer , 2008 , for review; Lamm et al . , 2010; Forbes and Hamilton , 2020; Zhao et al . , 2021 ) .",
"Recently , Coll et al . , 2017 have thus proposed a framework that attempts to capture these influences on affect sharing and empathic responses .",
"This model posits that individuals who see identical negative facial expressions of others may have different empathic responses due to distinct contextual information and that this may depend on identification of the underlying affective state displayed by the other .",
"In the current functional magnetic resonance imaging ( fMRI ) study , we therefore created a situation where we varied the genuineness of the pain affect felt by participants while keeping the perceptual saliency ( i . e . , the quality and strength of pain expressions ) identical .",
"To this end , participants were shown video clips of other persons who supposedly displayed genuine pain on their face vs . merely pretended to be in pain .",
"Note that for reasons of experimental control , all painful expressions on the videos had been acted .",
"This enabled us to interpret possible differences between conditions to the observers’ appraisal of the situation rather than to putative visual and expressive differences .",
"This way , we sought to identify the extent to which responses in affective nodes ( such as the aIns and the aMCC ) genuinely track the pain of others , rather than resulting predominantly from the salient facial expressions associated with the pain .",
"Another major aim of our study was to assess how self-other distinction allowed individuals to distinguish between the sharing of actual pain vs . regulating an inappropriate and potentially misleading ‘sharing’ of what in reality is only a pretended affective state .",
"We focused on the right supramarginal gyrus ( rSMG ) , which has been suggested to act as a major hub selectively engaged in affective self-other distinction ( Silani et al . , 2013; Steinbeis et al . , 2015; Hoffmann et al . , 2016; Bukowski et al . , 2020 ) .",
"Though previous studies have indicated that rSMG is functionally connected with areas associated with affect processing ( Mars et al . , 2012; Bukowski et al . , 2020 ) , we lack more nuanced insights into how exactly rSMG interacts with these areas , and thus how it supports accurate empathic responses .",
"Hence , we used dynamic causal modeling ( DCM ) to investigate the hypothesized brain patterns of affective responses and self-other distinction for the genuine and pretended pain situations , focusing on the aIns , aMCC , and their interaction with rSMG .",
"Furthermore , we investigated the relationship between neural activity and behavioral responses as well as empathic traits .",
"In line with the literature reviewed above , we expected that , on the behavioral level , genuine pain would result in – alongside the obvious other-oriented higher pain ratings – higher self-oriented unpleasantness ratings .",
"On the neural level , we predicted aIns and aMCC to show a stronger response to the genuine expressions of pain , but that these areas would also respond to the pretended pain , but to a lower extent .",
"Differences in rSMG engagement and distinct patterns of this area’s effective connectivity with aIns and aMCC were expected to relate to self-other distinction , and thus to explain the different empathic responses to genuine pain vs . pretended pain ."
],
[
"Three repeated-measures ANOVAs were performed with the factors genuineness ( genuine vs . pretended and pain [pain vs . no pain] ) , for each of the three behavioral ratings .",
"For ratings of painful expressions in others ( Figure 1C , left ) , there was a main effect of the factor genuineness: participants showed higher ratings for the genuine vs . pretended conditions , Fgenuineness ( 1 , 42 ) = 8 . 816 , p=0 . 005 , η² = 0 . 173 .",
"There was also a main effect of pain: participants showed higher ratings for the pain vs . no pain conditions , Fpain ( 1 , 42 ) = 1718 . 645 , p<0 . 001 , η² = 0 . 976 .",
"The interaction term was significant as well , Finteraction ( 1 , 42 ) = 7 . 443 , p=0 . 009 , η² = 0 . 151 , and this was related to higher ratings of painful expressions in others for the genuine pain compared to the pretended pain condition .",
"For ratings of painful feelings in others ( Figure 1C , middle ) , there was a main effect of genuineness: participants showed higher ratings for the genuine vs . pretended conditions , Fgenuineness ( 1 , 42 ) = 770 . 140 , p<0 . 001 , η² = 0 . 948 .",
"There was also a main effect of pain , as participants showed higher ratings for the pain vs . no pain conditions , Fpain ( 1 , 42 ) = 1544 . 762 , p<0 . 001 , η² = 0 . 974 .",
"The interaction for painful feelings ratings was significant as well , Finteraction ( 1 , 42 ) = 752 . 618 , p<0 . 001 , η² = 0 . 947 , and this was related to higher ratings of painful feelings in others for the genuine pain compared to the pretended pain condition .",
"For ratings of unpleasantness in self ( Figure 1C , right ) , there was a main effect of genuineness: participants showed higher ratings for the genuine vs . pretended conditions , Fgenuineness ( 1 , 42 ) = 74 . 989 , p<0 . 001 , η² = 0 . 641 .",
"There was also a main effect of pain: participants showed higher ratings for the pain vs . no pain conditions , Fpain ( 1 , 42 ) = 254 . 709 , p<0 . 001 , η² = 0 . 858 .",
"The interaction for unpleasantness ratings was significant as well , Finteraction ( 1 , 42 ) = 73 . 620 , p<0 . 001 , η² = 0 . 637 , and this was related to higher ratings of unpleasantness in self for the genuine pain compared to the pretended pain condition .",
"In sum , the behavioral data indicated higher ratings and large effect sizes of painful feelings in others and unpleasantness in self for the genuine pain compared to the pretended pain condition .",
"Ratings of pain expressions also differed in terms of genuineness , at comparably low effect size , though they were expected to not show a difference by way of our experimental design and the pilot study .",
"We also found a significant correlation between behavioral ratings of painful feelings in others and unpleasantness in self in the genuine pain condition , r = 0 . 691 , p<0 . 001 , while in the pretended pain condition , the correlation was not significant , r = 0 . 249 , p=0 . 107 ( Figure 1D ) .",
"A bootstrapping comparison showed a significant difference between the two correlation coefficients , p=0 . 002 , 95 % Confidence Interval ( CI ) = [0 . 230 , 1 . 060] .",
"Three contrasts were computed: ( 1 ) genuine: pain – no pain , ( 2 ) pretended: pain – no pain , and ( 3 ) genuine ( pain – no pain ) – pretended ( pain – no pain ) .",
"Across all three contrasts , we found activations as hypothesized in bilateral aIns , aMCC , and rSMG ( Figure 2A and Table 1 ) .",
"To identify whether or which brain activity was selectively related to the behavioral ratings described above , we performed a multiple regression analysis where we explored the relationship of activation in the contrast genuine pain – pretended pain with the three behavioral ratings .",
"We found significant clusters in bilateral aIns , visual cortex , and cerebellum that could be selectively explained by the ratings of self-unpleasantness rather than ratings of painful expressions in others or painful feelings in others ( Figure 2B ) .",
"We performed DCM analysis to specifically examine the modulatory effect of genuineness on the effective connectivity between the right aIns and rSMG .",
"More specifically , we sought to assess whether the experimental manipulation of genuine pain vs . pretended pain tuned the bidirectional neural dynamics from aIns to rSMG and vice versa , in terms of both directionality ( sign of the DCM parameter ) and intensity ( magnitude of the DCM parameter ) .",
"If the experimental manipulation modulated the effective connectivity , we would observe a strong posterior probability ( pp>0 . 95 ) of the modulatory effect .",
"Our original analysis plan was to include aMCC in the DCM analyses , but based on the fact that aMCC did not show as strong evidence ( in terms of the multiple regression analysis ) as the aIns of being involved in our task , we decided to use a more parsimonious DCM model without the aMCC .",
"We found strong evidence of inhibitory effects on the aIns to rSMG connection both in the genuine pain condition and in the pretended pain condition ( Figure 1A-C ) .",
"Comparing the strength of these modulatory effects on the aIns to rSMG connection revealed a reduced inhibitory effect for genuine pain as opposed to pretended pain , t41 = 2 . 671 , p=0 . 011 ( meangenuine pain = −0 . 821 , 95% CI = [−0 . 878 , −0 . 712]; meanpretended pain = −0 . 934 , 95% CI = [−1 . 076 , −0 . 822]; Figure 3C ) .",
"There was no evidence of a modulatory effect on the rSMG to aIns connection .",
"To examine how the modulatory effects from the DCM were related to the behavioral ratings , we computed two multiple linear regression models for each condition .",
"For the genuine pain condition , we find that the modulatory effect was significantly related to the rating of painful feelings in others ( t = 2 . 317 , p=0 . 026 ) , but not related to the rating of either painful expressions in others ( t = −1 . 492 , p=0 . 144 ) or unpleasantness in self ( t = 0 . 058 , p=0 . 954 ) .",
"For the pretended pain condition , none of the ratings was significantly related to the modulatory effect ( Figure 3D ) .",
"The variance inflation factors ( VIFs ) for three ratings in both models were calculated to diagnose collinearity , showing no severe collinearity problem ( all VIFs < 5; the smallest VIF = 1 . 132 and the largest VIF = 4 . 387 ) .",
"In addition , we tested two multiple stepwise linear regression models to investigate whether subscales of all three questionnaires could explain modulatory effects for genuine pain and pretended pain .",
"In the genuine pain condition , we found that the modulatory effect was significantly explained by scores of two subscales , that is affective ability and affective reactivity of the ECQ: Fmodel ( 1 , 39 ) = 6 . 829 , p=0 . 003 , R2 = 0 . 270; Baffective ability = 0 . 052 , beta = 0 . 497 , p=0 . 002; Baffective reactivity = −0 . 040 , beta = −0 . 421 , p=0 . 008 .",
"No significant predictor was found with the other questionnaires ( i . e . , IRI and TAS ) .",
"In the pretended pain condition , none of the three questionnaires significantly predicted variations of the modulatory effect .",
"No severe collinearity problem was detected for either regression model ( all VIFs < 2; the smallest VIF = 1 . 011 and the largest VIF = 1 . 600 ) ."
],
[
"In this study , we developed and used a novel experimental paradigm in which participants watched video clips of persons who supposedly either genuinely experienced pain or merely pretended to be in strong pain .",
"Combining mass-univariate analysis with effective connectivity ( DCM ) analyses , our study provides evidence on the distinct neural dynamics between regions suggestive of affect processing ( i . e . , aIns and aMCC ) and self-other distinction ( i . e . , rSMG ) for genuinely sharing vs . responding to pretended , non-genuine pain .",
"With this , we aimed to clarify two main questions: First , whether neural responses in areas such as the aIns and aMCC to the pain of others are indeed related to a veridical sharing of affect , as opposed to simply tracking automatic responses to salient affective displays .",
"And second , how processes related to self-other distinction , implemented in the rSMG , enable appropriate empathic responses to genuine vs . merely pretended affective states .",
"The mass-univariate analyses suggest that the increased activity in aIns for genuine pain as opposed to pretended pain properly reflects affect sharing .",
"As aforementioned , the network of affective sharing and certain domain-general processes ( e . g . , salience detection and automatic emotion processing ) overlap in aIns and aMCC ( Zaki et al . , 2016 , for review ) .",
"This indicates that indeed , part of the activation in these areas could be related to perceptual salience , which is why it has been widely debated as a potential confound of empathy and affect sharing models ( Zaki et al . , 2016 , for review; Lamm et al . , 2019 , for review ) .",
"However , when comparing genuine pain versus pretended pain , activity in these areas was not only found to be stronger in response to genuine pain , but the increased activation in aIns was also selectively correlated with ratings of self-oriented unpleasantness and was not correlated with either other-related painful expressions or painful feelings in terms of the regression analysis .",
"That only aIns and not also aMCC shows such correlation may be explained by previous studies , according to which aIns is more specifically associated with affective representations , while the role of aMCC rather seems to evaluate and regulate emotions that arise due to empathy ( Fan et al . , 2011; Lamm et al . , 2011; Jauniaux et al . , 2019 ) .",
"Taken together , the activation and brain-behavior findings provide evidence that responses in aIns ( and to a lesser extent also the aMCC ) are not simply automatic responses triggered by perceptually salient events ( otherwise the increased aIns activation should also be explained by other behavioral ratings in the sense of shared influence by domain-general effects ) .",
"Rather , they seem to track the actual affective states of the other person , and thus the shared neural representation of that response ( see Zhou et al . , 2020 , for similar recent conclusions based on multi-voxel pattern analyses ) .",
"Our findings are also in line with the proposed model of Coll et al . , 2017 , which suggests that affect sharing is the consequence of emotion identification .",
"More specifically , while part of the activation in the aIns and aMCC is indeed related to an ( presumably earlier ) automatic response , the added engagement of these areas once they have identified the pain as genuine shows that only in this condition , they then also engage in proper affect sharing .",
"Ideally , one should be able to discern these processes in time , but neither the temporal resolution of our fMRI measurements nor the paradigm in which we always announced the conditions beforehand would have been sensitive enough to do so .",
"Thus , future studies including complementary methods , such as EEG and MEG , and tailored experimental designs are needed to pinpoint the exact sequence of processes engaged in automatic affective responses vs . proper affect sharing .",
"Beyond higher activation in affective nodes supporting ( pain ) empathy , increased activation was also found in rSMG .",
"The inferior parietal lobule was shown to be generally engaged in selective attention , action observation , and imitating emotions ( Bach et al . , 2010; Pokorny et al . , 2015; Gola , 2017; Hawco et al . , 2017 ) .",
"Importantly , a specific role in affective rather than cognitive self-other distinction has been identified for rSMG ( Silani et al . , 2013; Steinbeis et al . , 2015; Bukowski et al . , 2020 ) .",
"Based on such findings , it has been proposed that the rSMG allows for a rapid switching between or the integration of self- and other-related representations , as two processes that may underpin the functional basis of successful self-other distinction ( Lamm et al . , 2016 , for review ) .",
"Theoretical models of empathy and related socio-affective responses suggest that such regulation is especially important to avoid so-called empathic over-arousal , which would shift the focus away from empathy and the other’s needs , toward taking care of one’s own personal distress ( Batson et al . , 1987 , for review; Decety and Lamm , 2011 , for review ) .",
"Concerning the current findings , we thus propose that the higher rSMG engagement in the genuine pain condition reflects an increasing demand for self-other distinction imposed by the stronger shared negative affect experienced in this condition .",
"Beyond these differences in the magnitude of rSMG activation , the DCM analysis demonstrated less inhibition on the aIns-to-rSMG connection for genuine pain compared to pretended pain .",
"Note that our focus on the right aIns rather than bilateral aIns was because it is located in the same hemisphere as the right SMG .",
"Various theoretical accounts suggest that areas such as the aIns and rSMG may play a key role in comparing self-related information with the sensory evidence ( Decety and Lamm , 2007 , for review; Seth , 2013 , for review ) .",
"According to recent theories on predictive processing ( Clark , 2013 , for review ) and active inference ( Friston , 2010 , for review ) , the brain can be regarded as a “prediction machine” , in which the top-down signals pass over predictions and the bottom-up signals convey prediction errors across different levels of cortical hierarchies ( Chen et al . , 2009; Friston , 2010 , for review; Bastos et al . , 2015 ) .",
"It is suggested that these top-down predictions are mediated by inhibitory neural connections ( Zhang et al . , 2008; Bastos et al . , 2015; Miska et al . , 2018 ) .",
"Our findings align with such views , by suggesting that the inhibitory connection from aIns to rSMG can be explained as the predictive mismatch between the top-down predictions of self-related information ( e . g . , personal affect ) and sensory inputs ( e . g . , pain facial expressions ) .",
"This suppression of neural activity leads to an explaining away of incoming bottom-up prediction error .",
"This is reflected by the absence of any condition-dependent modulatory effects on the rSMG to aIns connection , suggesting that the influence of the task conditions is sufficiently modeled by the predictions from aIns to rSMG .",
"Therefore , the stronger inhibition for pretended pain , compared to genuine pain , could indicate a higher demand to overcome the mismatch between the visual inputs and the agent’s prior beliefs and contextual information about the situation ( i . e . , “this person looks like in pain , but I know he/she does not actually feel it” ) .",
"We speculate that a dynamic interaction between sensory-driven and control processes is underlying the modulatory effect: when individuals realized after an initial sensory-driven response to the facial expression that it was not genuinely expressing pain , control , and appraisal processes led to a reappraisal of the triggered emotional response , and thus a dampening of the unpleasantness .",
"The reduced inhibition in the genuine pain condition could moreover be a mechanism that explains the higher rSMG activation in this condition .",
"Model comparison showed that the best model to explain the inhibitory effect with the behavioral ratings for both the genuine and pretended pain is the model without interactions between ratings .",
"That is , if any behavioral rating contributed to the modulation of aIns to rSMG , the effect would be more likely coming from single ratings rather than their interactions .",
"Specifically , we found the strength of the inhibitory effect in the genuine pain condition to correlate with ratings of painful feelings in others , but not with the ratings of pain expression in others or unpleasantness in self .",
"For the pretended pain condition , none of the ratings showed a correlation .",
"The latter could in principle be due to a lack of variation in the ratings ( which by way of the design were mostly close to zero or one ) .",
"We deem it more plausible , though , that the correlation findings provide further evidence that the modulation of aIns to rSMG is implicated in encoding others’ emotional states , which serves as a functional foundation for self-other processing when participants engaged in genuine affect sharing .",
"This regulation cannot be totally attributed to domain-general processes , otherwise other ratings should have also explained this variation .",
"It is also interesting to note that the found correlation relates to cognitive evaluations of the other’s pain rather than to own affect , as tracked by the unpleasantness in self-ratings .",
"This would to some extent be in line with DCM findings by Kanske et al . , 2016 .",
"These authors found that the inhibition of the temporoparietal junction ( TPJ ) by the aIns was linked to interactions between Theory of Mind ( ToM ) and empathic distress , that is the interaction of ‘cognitive’ vs . ‘affective’ processes engaged in understanding others’ cognitive and affective states .",
"Note that the right TPJ is an overarching area involved in self-other distinction of which rSMG is considered a part or at least closely connected to Decety and Lamm , 2007 , for review .",
"The correlations between the DCM inhibitory effect and empathic traits assessed via questionnaires provide further refinements for the relevance of rSMG in implementing self-other distinction to allow for an appropriate empathic response .",
"When participants shared genuine affect , the inhibitory effect on the aIns to rSMG connection was positively correlated with affective ability and negatively correlated with affective reactivity .",
"Affective ability reflects the capacity to subjectively share emotions with others , while affective reactivity plays a role in the susceptibility to vicarious distress and thus to more automatic responses to another’s emotion ( Batchelder et al . , 2017 ) .",
"Again , as for the correlations with the three rating scales , we did not find correlations of empathic traits for the pretended pain condition .",
"Taken together , the DCM results and their qualification by the correlation findings suggest that in the genuine pain condition , which requires an accurate sharing of pain , rSMG interacts with aIns to achieve ‘affective-to-affective’ self-other distinction – that is disambiguating affective signals originating in the self from those attributable to the other person .",
"The aIns to rSMG connection in the pretended pain condition may reflect a related , yet slightly distinct mechanism .",
"Here , it seems that ‘cognitive-to-affective’ self-other distinction is at play , which helps resolve conflicting information between the top-down contextual information ( i . e . , that the demonstrator is not actually in pain ) from what seems an unavoidable affective response to the highly salient perceptual cue of the facial expression of pain .",
"Given our behavioral and trait data did not allow us to distinguish more precisely between these different types of self-other distinction , this however remains an interpretation and a hypothesis that will require further investigation .",
"This inhibitory effect might be related to socioemotional disturbances of individuals with autism spectrum disorders ( ASD ) , who show impairments in social cognition , including self- and other-related processing ( Hoffmann et al . , 2016; Lamm et al . , 2016 , for review ) .",
"It is thus likely that ASD individuals exhibit distinct inhibition of the aIns-to-rSMG connectivity pattern compared to healthy controls .",
"Further research with ASD individuals is required .",
"One potential limitation of the study could be the slightly higher ratings of other-oriented pain expressions for genuine pain , which were hypothesized to have no difference , as compared to pretended pain .",
"As we found the enhanced aIns activation in the genuine pain condition mainly tracked personal unpleasantness rather than perceptually domain-general processes , and because the effect size of the pain expression difference was much smaller than for the affect ratings , we consider this difference did not fundamentally influence the interpretation of our findings .",
"An additional limitation was that our study design did not aim to explicitly quantify self-other distinction .",
"Rather , in line with previous research and based on our theoretical framework and rationale , we inferred the engagement of this process from the experimental conditions and the associated behavioral and neural responses .",
"We expect our findings to prompt and inform future research designed to quantify and experimentally disentangle self- and other-related processes more explicitly .",
"Note though that our participants and the targets shown in the videos were balanced with respect to their sex/gender .",
"Yet sex/gender effects ( for a review , though , see Christov-Moore , 2014 ) were outside the scope of the current study , and we thus did not perform any sex/gender-related analyses .",
"In conclusion , the current study advances our understanding of two main aspects of empathy .",
"First , we provide evidence that empathy-related responses in the aIns can indeed be linked to affective sharing , rather than attributing them to responses triggered only by perceptual saliency .",
"Second , we show how aIns and rSMG are orchestrated to track what another person really feels , thus enabling us to appropriately respond to their actual needs .",
"Beyond these basic research insights , our study provides novel avenues for clinical application , and the investigation of contextual and interpersonal factors in the accurate diagnosis of pain and its expression ."
],
[
"Forty-eight participants took part in the study .",
"Five of them were excluded because of excessive head motion ( >15% scans with the frame-wise displacement over 0 . 5 mm in one session ) .",
"Data of the remaining 43 participants ( 21 females; age: Mean = 26 . 72 years , S . D . = 4 . 47 ) were entered into analyses .",
"This sample size was determined on a priori power analysis in Gpower 3 . 1 ( Faul et al . , 2007 ) .",
"We assumed a medium effect size of Cohen’s d = 0 . 5 .",
"After calculation , the minimum sample size statistically required for this study was 34 ( α = 0 . 05 , two-tailed , 1−β = 0 . 80 ) .",
"Participants were pre-screened by an MRI safety-check questionnaire , assuring normal or corrected to normal vision and no presence or history of neurologic , psychiatric , or major medical disorders .",
"All participants were being right-handed ( self-reported ) and provided written consent including post-disclosure of any potential deception .",
"The study was approved by the ethics committee of the Medical University of Vienna and was conducted in line with the latest version of the World Medical Association , 2013 .",
"As part of our study , we developed a novel experimental design and corresponding stimuli , which consisted of video clips showing different demonstrators ostensibly in four different situations: ( 1 ) Genuine pain: the demonstrator’s right cheek was penetrated by a hypodermic needle attached to a syringe , and the demonstrator’s facial expression changed from neutral to a strongly painful facial expression .",
"( 2 ) Genuine no pain: the demonstrator maintained a neutral facial expression when a Q-tip fixed on the backend of the same syringe touched their right cheek .",
"( 3 ) Pretended pain: the demonstrator’s right cheek was approached by the same syringe and the hypodermic needle , with the latter covered by a protective cap; upon touch by the cap , the demonstrator’s facial expression changed from neutral to a strongly painful facial expression .",
"( 4 ) Pretended no pain: the demonstrator maintained a neutral facial expression when a Q-tip fixed on the backend of the same syringe touched their right cheek .",
"To create these stimuli , we recruited 20 demonstrators ( 10 females ) , with experience in acting , and filmed them in front of a dark blue background .",
"An experimenter who stood on the right side of the demonstrators , but of whom only the right hand holding the syringe could be seen , administered the injections and touches .",
"Unbeknownst to the participants , all painful expressions were acted , as the needle was a telescopic needle ( i . e . , a needle that seemed to enter the cheek upon contact , but in reality , was invisibly retracting into the syringe ) .",
"The reason for using a protective cap in the pretended pain condition was to match the perceptual situation that an aversive object was approaching a body part in both pain conditions .",
"In all situations , the demonstrator was instructed to look naturally toward the camera 1 . 5 m in front of them .",
"As soon as the needle or the cap touched the demonstrator’s cheek , the demonstrator made a painful facial expression , as naturally and vividly as possible .",
"In the neutral control conditions , demonstrators maintained a neutral facial expression when a Q-tip fixed at the backend of the syringe touched their cheek .",
"Again , a syringe with a needle attached to the other end was used to perceptually control for the presence of an aversive object in all four conditions .",
"Note that in another set of conditions , demonstrators showed disgusted or neutral expressions .",
"Data from these conditions will be reported elsewhere .",
"All demonstrators signed an agreement that their video clips and static images could be used for scientific purposes .",
"To validate the stimuli , we performed an online validation study with N = 110 participants , who were asked to rate a total of 120 video clips of 2 s duration of the two conditions ( 60 of each condition ) showing painful expressions ( i . e . , the genuine and the pretended pain conditions ) .",
"The main aim of the validation study was to identify a set of demonstrators that expressed pain with comparable intensity and quality , and whose pain expressions in the genuine and pretended conditions were comparable .",
"After each video clip , participants rated three questions on a visual analog scale with nine tick-marks and the two end-points marked as ‘almost not at all’ to ‘unbearable’: ( 1 ) How much pain did the person express on his/her face ?",
"( 2 ) How much pain did the person actually feel ?",
"( 3 ) How unpleasant did you feel to watch the person in this situation ?",
"The order of these three questions was pseudo-randomized .",
"Moreover , eight catch trials randomly interspersed across the validation study to test whether participants maintained attention to the stimuli .",
"Here , participants were asked to correctly select the demonstrator they had seen in the last video , between two static images of the correct and a distractor demonstrator displayed side by side , both showing neutral facial expressions .",
"The validation study was implemented within the online survey platform SoSci Survey ( https://www . soscisurvey . de ) , with a study participation invite published on Amazon Mechanical Turk ( https://www . mturk . com/ ) , a globally commercial platform allowing for online testing .",
"Survey data of 62 of 110 participants ( 34 females; age: mean = 28 . 71 years , S . D . = 10 . 11 ) were entered into analysis ( inclusion criteria: false rate for the test questions < 2/8 , survey duration > 20 min and <150 min , and the maximum number of continuous identical ratings < 5 ) .",
"Based on this validation step , we had to exclude videos of six demonstrators ( three females ) for which participants showed a significant difference in painful expressions in others between the genuine pain and the pretended pain conditions .",
"As a result of this validation , videos of 14 demonstrators ( 7 females ) , which showed no difference in the pain expression rating between genuine and pretended conditions and which overall showed comparable mean ratings in all three ratings , were selected for the subsequent pilot study .",
"In the pilot study , 47 participants ( 24 females; age: mean = 26 . 28 years , S . D . = 8 . 80 ) were recruited for a behavioral experiment in the behavioral laboratory .",
"The aim was to verify the experimental effects and the feasibility of the experimental procedures that we intended to use in the main fMRI experiment , as well as to identify video stimuli that may not yield the predicted responses .",
"Thus , all four conditions described above were presented to the participants .",
"Participants were explicitly instructed that they would watch other persons’ genuine painful expressions in some blocks , while in other blocks , they would see other persons acting out painful expressions ( recall that in reality , all demonstrators had been actors , and the information about this type of necessary deception was conveyed to participants at the debriefing stage ) .",
"They would see all demonstrators’ neutral expressions as well .",
"Participants were instructed to rate the three questions mentioned above .",
"Upon screening for video clips that showed aberrant responses , we excluded videos of two demonstrators ( one female ) , for whom the pain expression rating difference between the pretended vs . genuine expressions was large .",
"Forty-eight videos of 12 demonstrators entered the following analyses .",
"Three separate repeated-measures ANOVAs were respectively performed for the three rating questions .",
"For the main effect of genuineness ( genuine vs . pretended ) , it was not significant and low in effect size for painful expressions in others ( Fgenuineness ( 1 , 46 ) = 2 . 939 , p=0 . 093 , η² = 0 . 060 ) , but was significant with high effect size for the painful feelings in others ( Fgenuineness ( 1 , 46 ) = 280 . 112 , p<0 . 001 , η² = 0 . 859 ) as well as the unpleasantness in self ( Fgenuineness ( 1 , 46 ) = 43 . 143 , p<0 . 001 , η² = 0 . 484 ) .",
"The main effects of pain ( pain vs . no pain ) for all three questions were found significant with high effect size ( the smallest effect size was for the rating of unpleasantness in self , Fpain ( 1 , 46 ) = 82 . 199 , p<0 . 001 , η² = 0 . 641 ) .",
"Our pilot study thus ( 1 ) provided assuring evidence that the novel experimental paradigm worked as expected and ( b ) made it possible to select video clips that we could match for the two conditions ( i . e . , genuine pain and pretended pain ) .",
"More specifically , as expected and required for the main study , participants rated the painfulness of the demonstrators to be substantially higher when it was genuine as compared to those that were pretended , and this also resulted in much higher unpleasantness experienced in the self .",
"It is worth noting that , the two conditions did not differ with respect to the ratings of the painful facial expressions , implying that putative differences in ratings as well as the subsequent brain imaging data could only be attributed to the contextual appraisal of the demonstrators’ actual painful states , rather than the differences in facial pain perception .",
"Based on this pilot study , we thus decided on video clips of 12 demonstrators ( 6 females ) in the main fMRI experiment .",
"The experiment was implemented using Cogent 2000 ( version 1 . 33; http://www . vislab . ucl . ac . uk/cogent_2000 . php ) .",
"MRI scanning took place at the University of Vienna MRI Center .",
"Once participants arrived at the scanner site , an experimenter instructed them that they would watch videos from the four conditions outlined above .",
"Participants were explicitly instructed to recreate the feelings of the demonstrators shown in the videos as vividly and intensely as possible .",
"Based on the validation and pilot study , the painful expressions for the genuine and pretended conditions were matched .",
"We also counterbalanced the demonstrators appearing in the genuine and pretended conditions across participants , thus controlling for differences in behavioral and brain response that could be explained by differences between the stimulus sets .",
"Note that , all video clips were validated and piloted multiple times to ensure the experimental effect ( details can be found in the section above ) .",
"The participant performed the fMRI experiment in two runs ( Figure 1 ) .",
"Each run was composed of two blocks showing genuine pain and two blocks showing pretended pain .",
"In each block , the participant watched nine video clips containing both painful and neutral videos .",
"To remind participants’ the condition of the upcoming block , a label of 4 s duration appeared at the beginning of each block , showing either ‘genuine’ or ‘pretended’ ( in German ) .",
"Each trial started with a fixation cross ( + ) presented for 4–7 s ( in steps of 1 . 5 s , mean = 5 . 5 s ) .",
"After that , the video ( duration = 2 s ) was played .",
"A short jitter was inserted after the video for 0 . 5–1 . 0 s ( in steps of 0 . 05 s , mean = 0 . 75 s ) .",
"After the jitter , the following three questions were displayed ( in German ) one after the other in a pseudo-randomized order: ( 1 ) How much pain did the person express on his/her face ?",
"( 2 ) How much pain did the person actually feel ?",
"( 3 ) How unpleasant did you feel to watch the person in this situation ?",
"Beneath each question , a visual analog scale ranging from 0 ( not at all ) to 8 ( unbearable ) with nine tick-marks was positioned .",
"The participant moved the marker along the scale by pressing the left or right keys on the button box , and they pressed the middle key to confirm their answer .",
"The marker initially was always located at the midpoint ( ‘4’ ) of the scale .",
"When the confirmed key was pressed , the marker turned from black to red .",
"All ratings lasted for 4 s even when the participant pressed the confirmed key before the end of this period .",
"Between the two runs , the participant had a short break ( 1–2 min ) .",
"Before entering the scanner , participants conducted practice trials on the computer to get familiarized with the button box and the experimental interface .",
"After that , participants were moved into the scanner and performed the task .",
"Following the functional imaging runs , a 6 . 5 min structural scanning was employed .",
"When participants finished the scanning session , they were scheduled for a date to complete three questionnaires in the lab: the Empathy Components Questionnaire ( ECQ ) ( Batchelder , 2015; Batchelder et al . , 2017 ) , the Interpersonal Reactivity Index ( IRI ) ( Davis , 1980 ) , and the Toronto Alexithymia Scale ( TAS ) ( Bagby et al . , 1994 ) .",
"For the ECQ , there are 27 items in total to be categorized into five subscales: cognitive ability , cognitive drive , affective ability , affective drive , and affective reactivity , using a 4-point Likert scale ranging from 1 ( ‘strongly disagree’ ) to 4 ( ‘strongly agree’ ) ( Batchelder , 2015; Batchelder et al . , 2017 ) .",
"For the IRI , there are 28 items divided into four subscales: perspective taking , fantasy , empathic concern , and personal distress , using a 5-point Likert scale ranging from 0 ( ‘does not describe me well’ ) to 4 ( ‘describes me very well’ ) ( Davis , 1980 ) .",
"For the TAS , there are 20 items and three subscales – difficulty describing feelings , difficulty identifying feelings , and externally oriented thinking , using a 5-point Likert scale ranging from 1 ( ‘strongly disagree’ ) to 5 ( ‘strongly agree’ ) ( Bagby et al . , 1994 ) .",
"The average interval between the scanning session and the lab survey was 1 week .",
"The participant was debriefed after completing the whole study .",
"We applied repeated-measures ANOVAs to investigate the main effects and the interaction of the two factors genuine vs . pretended and pain vs . no pain , using SPSS ( version 26 . 0; IBM ) .",
"Furthermore , we conducted Pearson correlations to examine whether ratings of painful feelings in others were correlated with unpleasantness in self for the genuine pain and the pretended pain .",
"The correlation coefficients were further compared using a bootstrap approach with the R package bootcorci [https://github . com/GRousselet/bootcorci ( Rousselet , 2021 ) ] .",
"fMRI data were collected using a Siemens Magnetom Skyra MRI scanner ( Siemens , Erlangen , Germany ) , with a 32-channel head coil .",
"Functional whole-brain scans were collected using a multiband-accelerated T2*-weighted echoplanar imaging sequence ( multiband acceleration factor = 4 , interleaved ascending acquisition in multi-slice mode , 52 slices co-planar to the connecting line between anterior and posterior commissure , TR = 1200 ms , TE = 34 ms , acquisition matrix = 96 × 96 voxels , FOV = 210 × 210 mm2 , flip angle = 66° , inter-slice gap = 0 . 4 mm , voxel size = 2 . 2 × 2 . 2 × 2 mm3 ) .",
"Two functional imaging runs , each lasting around 16 min ( ~800 images per run ) , were performed .",
"Structural images were acquired with a magnetization-prepared rapid gradient-echo sequence ( TE/TR = 2 . 43/2300 ms , flip angle = 8° , ascending acquisition , single-shot multi-slice mode , FOV = 240 × 240 mm2 , voxel size = 0 . 8 × 0 . 8 × 0 . 8 mm3 , 208 sagittal slices , slice thickness = 0 . 8 mm ) .",
"Imaging data were preprocessed with a combination of Nipype ( Gorgolewski et al . , 2011 ) and MATLAB ( version R2018b 9 . 5 . 0; MathWorks ) , with Statistical Parametric Mapping ( SPM12; https://www . fil . ion . ucl . ac . uk/spm/software/spm12/ ) .",
"Raw data were imported into BIDS format ( http://bids . neuroimaging . io/ ) .",
"Functional data were subsequently preprocessed using slice timing correction to the middle slice ( Sladky , 2011 ) , realignment to the first image of each session , co-registration to the T1 image , segmentation between gray matter , white matter , and cerebrospinal fluid , normalization to MNI template space using Diffeomorphic Anatomical Registration Through Exponentiated Lie Algebra ( DARTEL ) toolbox ( Ashburner , 2007 ) , and smoothing with a 6 mm full width at half-maximum three-dimensional Gaussian kernel .",
"To improve data quality , we performed data scrubbing of the functional scans for those whose frame-wise displacements ( FD ) were over 0 . 5 mm ( Power et al . , 2012; Power et al . , 2014 ) .",
"In other words , we identified individual outlier scans and flagged the volume indices as nuisance regressors in the general linear model ( GLM ) for the first-level analysis .",
"In order to perform mass-univariate functional segregation analyses , a first-level GLM design matrix was created and composed of two identically modeled runs for each participant .",
"Seven regressors of interest were entered in each model: stimulation phase of the four conditions ( i . e . , genuine pain , genuine no pain , pretended pain , pretended no pain; 2000 ms ) and rating phase of the three questions ( i . e . , painful expressions in others , painful feelings in others , and unpleasantness in self; 12 , 000 ms ) .",
"Six head motion parameters and the scrubbing regressors ( FD > 0 . 5 mm , if applicable ) were additionally entered as nuisance regressors .",
"Individual contrasts of the four conditions and the three ratings ( all across the two runs ) against implicit baseline were respectively created .",
"On the second level , a flexible factorial design was employed to perform the group-level analysis .",
"The design included three factors: a between-subject factor ( i . e . , subject ) that was specified independent and with equal variance , a within-subject factor ( i . e . , genuine or pretended ) that was specified dependent and with equal variance , and a second within-subject factor ( i . e . , pain or no pain ) that was specified dependent and with equal variance ( Gläscher and Gitelman , 2008 ) .",
"Three contrasts were computed: ( 1 ) main effect of genuine: pain – no pain , ( 2 ) main effect of pretended: pain – no pain , and ( 3 ) interaction: genuine ( pain – no pain ) – pretended ( pain – no pain ) .",
"We applied an initial threshold of p<0 . 001 ( uncorrected ) at the voxel level and a family-wise error ( FWE ) correction ( p<0 . 05 ) at the cluster level .",
"The cluster extent threshold was determined by the SPM extension ‘cp_cluster_Pthresh . m’ ( https://goo . gl/kjVydz ) .",
"A multiple regression model was built on the group level to investigate the relationship between specific brain activations and behavioral ratings .",
"In this model , the contrast genuine pain – pretended pain was set as the dependent variable , and differences between conditions for three behavioral ratings were specified as independent variables .",
"The reason that we used the comparison between conditions for both brain signals and behavioral ratings was to control for potential effects of perceptual salience .",
"All covariates were mean-centered .",
"An intercept was added in the model .",
"To test whether the order of entering ratings into the regression model influence the results , we performed five additional regression analyses with all possible orders of three ratings .",
"The results were consistent across all six regression models , and we only showed the result for one regression ( i . e . , expression+ feeling + unpleasantness ) in the Results section .",
"Note that , we performed the regression model with the contrast genuine pain – pretended pain instead of the more exhaustive contrast genuine ( pain - no pain ) - pretended ( pain – no pain ) , and this was because the genuine and the pretended pain conditions were the main focus of our work .",
"Moreover , the pain contrast showed more robust ( in terms of statistical effect size ) and widespread activations across the brain , making it more likely to pick up possible brain-behavior relationships .",
"The same threshold as above was applied in this analysis .",
"We aimed to assess these brain-behavior relationships for the following regions of interest ( ROI ) : ( 1 ) aIns and aMCC , that is two regions associated with affective processes and specifically with empathy for pain , ( 2 ) rSMG , an area implicated in affective self-other distinction .",
"The ROI masks were defined as the conjunction of the averaging contrast between genuine and pretended: pain – no pain ( threshold: voxel-wise FWE correction , P < 0 . 05 ) and the anatomical masks created by the Wake Forest University ( WFU ) Pick Atlas SPM toolbox ( http://fmri . wfubmc . edu ) with the automated anatomical atlas ( AAL ) .",
"The ROI masks were created with Marsbar ROI Toolbox implemented in SPM12 ( Brett et al . , 2002 ) .",
"Note that we specifically selected the ROIs this way , such that they were orthogonal ( i . e . , independent ) to the subsequent analyses of interest .",
"As exploratory analyses found significant correlations mainly in aIns , rather than in aMCC , we will focus in the results section on two ROIs: the right aIns and the rSMG .",
"Focusing on the right aIns instead of the left one was because the right aIns is on the ipsilateral hemisphere as rSMG .",
"To investigate the functional network involved in affective processes and self-other distinction and how it was modulated by our experimental manipulations ( i . e . , genuine pain and pretended pain ) , we used DCM to estimate the effective connectivity between the ROIs based on the tasked-related brain responses ( Stephan and Friston , 2010 , for review ) .",
"The DCM analyses were conducted with DCM12 . 5 implemented in SPM12 ( v . 7771 ) .",
"First , we extracted individual time series separately for each ROI .",
"To ensure the selected voxels engaged in a task-relevant activity but not random signal fluctuations , we determined the voxels both on a group-level threshold and an individual-level threshold ( Holmes et al . , 2020 ) .",
"An initial threshold was set as p<0 . 05 , uncorrected .",
"The significant voxels in the main effect of genuine pain and pretended pain were further selected by an individual threshold .",
"For each participant , an individual peak coordinate within the ROI mask was searched , and an individual mask was consequently defined using a sphere of the 6 mm radius around the peak .",
"As a result , the individual time series for each ROI was extracted from the significant voxels of the individual mask and summarized by the first eigenvariate .",
"One participant was excluded as no voxels survived significance testing .",
"Second , we specified three regressors of interest: genuine pain , pretended pain , and the video input condition ( the combination of genuine pain and pretended pain ) .",
"That we did not specify no-pain conditions was because ( 1 ) the pain conditions were our main focus , and ( 2 ) adding no-interest conditions would inevitably increase the model complexity .",
"Then , a fully connected DCM model for each participant was created .",
"Three parameters were specified: ( 1 ) bidirectional connections between regions and self-connections ( matrix A ) , ( 2 ) modulatory effects ( i . e . , genuine pain and pretended pain ) on the between-region connections ( matrix B ) , and ( 3 ) driving inputs ( i . e . , the video input condition ) into the model on both regions ( matrix C ) ( Zeidman et al . , 2019a ) .",
"To remain parsimonious , we did not set modulatory effects on the self-connections in matrix A . Then the full DCM model was individually estimated .",
"Finally , group-level DCM inference was performed using parametric empirical Bayes ( Zeidman et al . , 2019b ) .",
"We conducted an automatic search over the entire model space ( max . n = 256 ) using Bayesian model reduction and random-effects Bayesian model averaging , resulting in a final group model that takes accuracy , complexity , and uncertainty into account ( Zeidman et al . , 2019b ) .",
"The threshold of the Bayesian posterior probability was set to pp>0 . 95 ( i . e . , strong evidence ) but we reported all parameters above pp>0 . 75 ( i . e . , positive evidence ) for full transparency of the DCM results .",
"Finally , a paired sample t-test was performed to compare modulatory effects between the genuine pain and the pretended pain conditions .",
"To probe whether task-related modulatory effects were associated with behavioral measurements , we performed multiple linear regression analyses of modulatory parameters with ( 1 ) the three behavioral ratings and ( 2 ) the empathy-related questionnaires ( i . e . , IRI , ECQ , and TAS ) .",
"We set up regression models for the genuine pain condition and the pretended pain condition , respectively , in which the DCM parameters of modulatory effects were determined as dependent variables and the ratings of painful expressions in others , painful feelings in others , and unpleasantness in self as independent variables .",
"Considering that interactions between behavioral ratings might contribute to the regression model , we tested five regression models ( with and without interaction; See Supplementary file",
"1 ) for both genuine pain and pretended pain .",
"Results showed that for both genuine pain and pretend pain , the model without any interaction outperformed other models .",
"The results of the winning multiple regression model are reported in the Results section .",
"We performed additional two regression models for both conditions in which DCM modulatory effects were set as dependent variables and scores of each subscale of all questionnaires were set as independent variables , respectively .",
"Considering the number of independent variables was relatively large ( >10 ) , we performed the analyses for questionnaires using a stepwise regression approach .",
"As two participants did not complete all three questionnaires , we excluded their data from the regression analyses .",
"The statistical significance of the regression analysis was set to p<0 . 05 .",
"The multicollinearity for independent variables was diagnosed using the VIF that measures the correlation among independent variables , in the R package car ( https://cran . r-project . org/web/packages/car/index . html ) .",
"Here we used a rather conservative threshold of VIF < 5 as a sign of no severe multicollinearity ( Menard , 2002; James et al . , 2013 ) ."
]
] | [
"Empathy for pain engages both shared affective responses and self-other distinction .",
"In this study , we addressed the highly debated question of whether neural responses previously linked to affect sharing could result from the perception of salient affective displays .",
"Moreover , we investigated how the brain network involved in affect sharing and self-other distinction underpinned our response to a pain that is either perceived as genuine or pretended ( while in fact both were acted for reasons of experimental control ) .",
"We found stronger activations in regions associated with affect sharing ( anterior insula [aIns] and anterior mid-cingulate cortex ) as well as with affective self-other distinction ( right supramarginal gyrus [rSMG] ) , in participants watching video clips of genuine vs . pretended facial expressions of pain .",
"Using dynamic causal modeling , we then assessed the neural dynamics between the right aIns and rSMG in these two conditions .",
"This revealed a reduced inhibitory effect on the aIns to rSMG connection for genuine pain compared to pretended pain .",
"For genuine pain only , brain-to-behavior regression analyses highlighted a linkage between this inhibitory effect on the one hand , and pain ratings as well as empathic traits on the other .",
"These findings imply that if the pain of others is genuine and thus calls for an appropriate empathic response , neural responses in the aIns indeed seem related to affect sharing and self-other distinction is engaged to avoid empathic over-arousal .",
"In contrast , if others merely pretend to be in pain , the perceptual salience of their painful expression results in neural responses that are down-regulated to avoid inappropriate affect sharing and social support ."
] | [
"Empathy enables us to share and understand the emotional states of other people , often based on their facial expressions .",
"This empathic response involves being able to distinguish our own emotional state from someone else’s , and it is influenced by how we recognize that person’s emotion .",
"In real life , knowing and identifying whether the facial expression we are witnessing reflects genuine or pretended pain is particularly important so that we can appropriately react to someone’s emotions and avoid unnecessary personal distress .",
"How our brains manage to do this is still heavily debated .",
"Two areas , the anterior insular ( aIns for short ) and the mid-cingulate cortex , appear to be activated when someone ‘feels’ someone else’s pain .",
"However , these regions might just automatically be triggered by vivid emotional facial expressions , regardless of whether we really respond to that pain .",
"To examine this question , Zhao et al . measured brain activity as healthy adults watched video clips of people either feeling or pretending to feel pain .",
"The activation of aIns was particularly related to the emotional component that someone shared with another person’s genuine pain , but not to pretended pain .",
"This suggests that neurons in the aIns track a truly empathic response when seeing someone who is actually experiencing pain .",
"Effective connectivity analyses which reflect how brain areas ‘crosstalk’ also revealed distinct patterns when people viewed expressions of genuine , as opposed to pretended pain .",
"Zhao et al . focused on the interactions between the alns and the right supramarginal gyrus , a brain region which helps to distinguish another person’s emotions from our own .",
"This crosstalk tracked others’ feelings when participants viewed expressions of genuine but not of pretended pain .",
"Put together , these findings provide a more refined model of empathy and its neural underpinnings .",
"This will help further our understanding of conditions such as autism or depression , in which a person’s social skills and emotional processing are impaired ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease",
"immunology and inflammation"
] | Persistent inflammation during anti-tuberculosis treatment with diabetes comorbidity | elife-46477-v2 | [
[
"It is now well established that diabetes mellitus ( DM ) is associated with increased the risk to become infected with Mycobacterium tuberculosis ( Mtb ) , to progress from latent infection to active pulmonary tuberculosis ( TB ) disease , and to suffer adverse TB outcomes including delayed sputum conversion on treatment , treatment failure , death , and recurrent TB ( Critchley et al . , 2017 ) .",
"The biochemical and cellular mechanisms of increased TB susceptibility in DM are incompletely understood .",
"Emerging evidence ties the complication of diabetic immunopathy to the well-studied diabetic complications of microvascular , macrovascular and renal disease driven by pathways primary driven by chronic hyperglycemia and related oxidative stress ( Giacco and Brownlee , 2010; Martinez and Kornfeld , 2014 ) .",
"Pulmonary TB disease with concurrent DM is associated with a higher burden of immune pathology and systemic inflammation compared to TB in euglycemic hosts ( Martinez and Kornfeld , 2014 ) .",
"Restrepo et al . were the first to describe increased expression of a broad range of cytokines from antigen-stimulated whole blood from patients with concurrent TB and DM as compared to TB alone ( Restrepo et al . , 2008 ) .",
"This was subsequently verified by several studies reporting higher plasma cytokine levels and higher frequencies of cytokine-expressing T cells assessed by flow cytometry in samples from individuals with TBDM comorbidity ( Kumar et al . , 2013a; Kumar et al . , 2013b; Prada-Medina et al . , 2017 ) .",
"Published studies have focused on samples obtained at baseline , before or shortly after the initiation of anti-TB treatment ( ATT ) .",
"We hypothesized that the hyperinflammatory phenotype of TBDM comorbidity is driven by a higher lung bacterial burden and/or by impaired counter regulatory mechanisms that normally limit bystander tissue injury from protective immune responses .",
"To test the prediction that the DM would be associated with delayed resolution of inflammation during TB treatment , we conducted a longitudinal assessment of plasma cytokine and growth factor levels in cohorts of adult pulmonary TB patients with and without DM recruited in India and Brazil ."
],
[
"Adults suspected to have pulmonary TB disease were screened for participation in the Effects of Diabetes on Tuberculosis Severity ( EDOTS ) study on presentation to the government clinic system in Chennai , India .",
"Candidates conforming to the inclusion and exclusion criteria where tested for glycemic status based on medical history , oral glucose tolerance test ( only for those without prior DM history ) , and glycohemogloblin ( HbA1c; for all enrolled participants ) .",
"Follow-up visits occurred at monthly intervals during the 6 month course of ATT for drug-sensitive TB and then quarterly through the final study visit one year after ATT completion ( month-18 ) .",
"Enrolled participants were withdrawn from the study and not included in the current analysis if their baseline sputum culture was negative or if it was positive for multi-drug resistant Mtb .",
"Selected demographic , anthropometric and behavioral variables of the India cohort are shown in Table 1 .",
"Compared to the normoglycemic TB group , the TBDM group had higher median age and higher body mass index ( BMI ) .",
"The median and interquartile range of BMI in the normoglycemic TB group fell below the WHO cutoff for undernutrition of 18 . 5 kg/m2 ( WHO Expert Consultation , 2004 ) .",
"Trends for higher likelihood of self-reported current or past smoking in the normoglycemic TB group did not reach statistical significance .",
"There was no difference in the proportion of TB or TBDM participants who reported current or past alcohol consumption .",
"The median HbA1c in TBDM participants was 10 . 0% , indicating poor glycemic control .",
"There were no statistically significant differences in sex , age , smoking or alcohol consumption between TBDM participants with DM newly diagnosed during screening for this ( NDM ) study vs known DM diagnosis prior to incident TB ( KDM ) , but median BMI was higher in the KDM group ( Table 2 ) .",
"All participants in the non-diabetic TB group were classified as euglycemic based on oral glucose tolerance test but sixteen had HbA1c ≥ 5 . 8% , placing them in the prediabetic range by American Diabetes Association criteria .",
"There was no difference in median 25-hydroxyvitamin D levels between TB and TBDM participants , although the median for both groups fell below a common threshold for insufficiency ( 20 ng/mL ) ( Thacher and Clarke , 2011 ) .",
"There were no significant differences between groups in the TB radiographic severity score , the presence of lower lung zone lesions or cavitary lesions .",
"The Brazil cohort differed from the India cohort in several respects ( Table 3 ) .",
"In that cohort , DM status was classified based only on HbA1c , there was no difference in median age between TBDM and TB participants , roughly equal proportions of male and female participants , and no differences between groups in smoking or alcohol consumption .",
"There was no difference in median BMI between TBDM and TB participants in the Brazil cohort and nearly all participants had BMI above the undernutrition cutoff of 18 . 5 kg/m2 .",
"Median HbA1c was lower in the Brazilian TBDM and TB groups compared to their Indian counterparts , but glycemic control was nonetheless poor ( ≥7 . 5% ) in the majority of Brazilian TBDM participants .",
"A comparison of demographic and clinical characteristics between the cohorts from Brazil and India is shown in Table 4 and further stratified into TB and TBDM subgroups within each cohort in Table 5 .",
"Statistically significant differences included a higher proportion of female sex and higher BMI in the Brazil cohort , while the cohorts did not differ with respect to alcohol or consumption or smoking .",
"Sputum acid-fast bacilli smear grades were significantly higher in TBDM than TB participants from India with a similar trend in the Brazil cohort that did not reach statistical significance ( Figure 1a ) .",
"Sputum smear grade differences between TBDM and TB were highly significant when the combined participants from both countries were considered ( Figure 1b ) .",
"Plasma levels of seventeen cytokines and growth factors were measured in samples from participants in the Indian TBDM and TB groups at baseline ( pre-ATT ) , after the intensive phase of ATT ( month-2 ) , at the completion of ATT ( month-6 ) , and one year after TB treatment completion ( month-18 ) .",
"Hierarchical cluster analysis of log2-transformed and z-score normalized values for each analyte identified two main clusters exclusively comprised of TBDM or TB , respectively , at baseline ( Figure 2a ) .",
"The TBDM group exhibited higher levels of nearly all analytes compared to the TB group .",
"The two discrete clusters comprised only of TBDM or TB individuals were maintained at the month-2 and month-6 timepoints but were lost by month-18 .",
"The separation between TBDM and TB groups based on plasma cytokine levels was also evident on principal component analysis ( PCA; Figure 2b ) .",
"By PCA , there was nearly complete separation of groups at baseline , with a slight trend towards overlap at month-2 and month-6 , culminating in near complete merger of values for the two groups at month-18 , albeit with some outliers among TBDM participants .",
"An identical panel of plasma analytes was measured in samples from the Brazil cohort collected at pre-ATT baseline , at treatment month-2 , and at treatment completion ( month-6 ) .",
"Results for the Brazilian participants were remarkably like those obtained in the India cohort , with complete separation of the TBDM and TB groups at baseline and only partial overlap at the later time points during treatment ( Figure 1c and d ) .",
"These results show that the unique pattern of systemic hyperinflammation associated with concurrent TB and DM was present in Indian and Brazilian populations , despite considerable differences in host genetic , demographic , and behavioral characteristics , and in the local prevalent M . tuberculosis strains in the two study populations ( Suzana et al . , 2017; Vasconcellos et al . , 2014 ) .",
"To compare the temporal pattern of plasma cytokine levels within the TBDM or TB groups over the course of ATT , log2 transformed data were analyzed using one-way ANOVA with linear trend posttest .",
"Results that remained statistically significant ( FDR 1% ) were included the heatmaps for the India and Brazil cohorts .",
"The Indian TBDM group exhibited a linear trend of deceasing plasma levels of most pro-inflammatory cytokines during and after ATT , with rising levels of IL-5 , IL-10 , IL-13 , and TGF-b ( Figure 3a ) .",
"Similar trends were identified in the Brazilian TBDM group ( Figure 4a ) .",
"The trends within the normoglycemic TB groups from India ( Figure 3a ) and Brazil ( Figure 4a ) where mutually consistent and differed from the TBDM groups most notably with declining levels of IL-10 .",
"Analysis of plasma cytokines accounting for differences between the TBDM and TB groups of the India cohort is shown in Figure 3b .",
"There was a statistically significant difference in the levels of fourteen out of seventeen cytokines measured in the India cohort at baseline , of which only one ( IL-10 ) was lower in TBDM than TB .",
"Notably , the fold-difference in baseline IL-10 levels exceeded that of all other analytes at all timepoints .",
"The pattern of higher cytokine levels in TBDM than TB individuals was mostly maintained at month-2 except that IL-10 changed from being significantly lower in TBDM than TB at baseline to significantly higher as compared to TB , while IL-13 that changed from being slightly higher in TBDM at baseline to markedly lower than TB at month-2 .",
"The direction and magnitude of differences in levels of most other measured cytokines were similar in month-6 compared to month-2 , but at month-18 only six cytokines were present at significantly higher levels in TBDM and the magnitude of fold-differences was much reduced .",
"There were no significant differences in cytokine levels between TBDM participants with DM newly diagnosed during screening as compared those known to have DM prior to enrollment ( Figure 3—figure supplement 1 ) .",
"Analysis of between-group cytokine levels in the Brazil cohort showed only five of seventeen total analytes present at significantly different levels in TBDM vs TB participants ( Figure 4b ) .",
"Like the India cohort , Brazilian TBDM participants had significantly lower levels of IL-10 at baseline .",
"The levels of IFN-g , IL-17F , GCSF , and TGF-b were significantly higher in Brazilian TBDM than TB participants at baseline .",
"By month-2 , IL-10 reversed from significantly higher to significantly lower in the TBDM group , as was seen in the India cohort .",
"At the ATT completion ( month-6 ) , eight analytes were present at significantly different levels in Brazilian TBDM vs TB participants .",
"Of these , seven were higher in TBDM than TB , with only IL-1b lower in TBDM .",
"Taken together , these analyses indicated that TBDM individuals had higher levels of circulating pro-inflammatory mediators compared to TB individuals at baseline , and this systemic hyper-inflammatory state persisted through the course of TB treatment with a temporal shift in relative IL-10 levels .",
"Decision tree analysis of plasma cytokine data was performed to identify those biomarkers that differentiated TBDM from TB individuals with the highest accuracy and minimal numbers of markers , which might reflect mechanistic differences between groups within the cohorts ( Figure 5 ) .",
"At baseline , IL-10 exhibited the strongest discriminating effect , with lower levels in the TBDM than the TB groups in both the India and Brazil cohorts ( Figure 5a and c , respectively ) .",
"The patterns in the Indian and Brazilian participants diverged at later time points .",
"At month-2 , lower IL-13 along with higher IL-12p70 levels powered the difference between TBDM and TB in the India cohort .",
"The pattern shifted again at month-6 with higher levels of IL-4 and GM-CSF associating with TBDM .",
"In contrast , IL-10 remained the strongest discriminating factor in the Brazil cohort at all time points evaluated , along with lower IL-17A in the TB group at month-2 and lower IL-8 in the TB group month-6 .",
"Receiver operating characteristic ( ROC ) analysis showed the single or combined cytokines that accurately defined the difference between TBDM and TB in both cohorts at the three timepoints , with area under the curves ≥ 0 . 9577 and P values < 0 . 0001 ( Figure 5b and d ) .",
"The underlying biological mechanisms can only be inferred from the available data , but low baseline levels of the anti-inflammatory cytokine IL-10 in TBDM comports with higher baseline levels of several pro-inflammatory cytokines in that group ( Sugimoto et al . , 2016 ) .",
"Overall , the dynamic pattern demonstrated by these results is consistent with prolonged inflammation and delayed relative rise in pro-resolving cytokines ( IL-10 , IL-4 , IL-13 ) ( Ortega-Gómez et al . , 2013 ) in the TBDM group .",
"A validated method for grading TB severity by chest x-ray interpretation was used to score images obtained at baseline , month-6 , and month-18 in the India cohort of TBDM and TB individuals ( Ralph et al . , 2010 ) .",
"There was considerable individual variation at all timepoints but no statistically significant difference in median radiographic scores between groups at any timepoint ( Figure 6a ) .",
"Spearman correlation matrices were built to examine associations between the radiographic score value at each time point , or the difference in score value between the indicated time points ( fold variation ) , and the individual plasma analyte levels in the entire cohort .",
"The association of baseline HbA1c with plasma analytes at each time point was also examined .",
"As shown in Figure 6b , the strongest associations identified were between plasma biomarkers at baseline with HbA1c , particularly TNF-α , IL-2 , IL-17F , and IL-1β ( positive correlations ) and IL-5 and IL-10 ( negative correlations ) .",
"Biomarkers measured at month-6 remained strongly associated with the HbA1c measured at baseline , but with shift from strong positive to weak negative correlation with IL-1 and from strong negative to moderate positive with IL-10 .",
"While some statistically significant correlations of radiographic scores or change in radiographic score were identified , particularly with month-18 biomarker levels , these were mostly moderate to weak correlations and did not demonstrate a consistent pattern or trend .",
"The number of analytes having significant correlation with baseline HbA1c by this analysis changed from sixteen at baseline , to ten at month-6 , and one only one ( positive correlation with IL-7 ) at month-8 .",
"Interestingly , IL-7 is known to maintain strong cellular immune responses for months , in contrast to more short-lived responses to IL-2 ( Lynch and Miller , 1994 ) ."
],
[
"Increased circulating levels of numerous cytokines and growth factors , many linked to protective immunity , typifies TBDM comorbidity at the time of TB diagnosis ( Kumar et al . , 2013a; Prada-Medina et al . , 2017; Restrepo et al . , 2008 ) .",
"Our goal for the current study was to investigate longitudinal trends in plasma levels of these mediators over the course of treatment for drug-sensitive pulmonary TB in participants rigorously classified as diabetic or normoglycemic at baseline .",
"Plasma samples from two ongoing observational cohort studies , one in India and one in Brazil , were available for this investigation .",
"Seventeen plasma mediators were measured by a combination of Luminex and conventional ELISA .",
"Results from baseline samples were consistent with the previously reported hyper-inflammatory state of TB with concurrent DM prior to the initiation of ATT .",
"Despite clinically efficacious antimicrobial chemotherapy , we found that the hyperinflammatory profile of TBDM participants persisted in both Indian and Brazil cohorts through the intensive phase of TB treatment at two months and treatment completion at six months .",
"Samples obtained from the India cohort one year after treatment completion showed substantial overlap mediator levels between the TBDM and TB groups , consistent with eventual resolution of TB-related inflammation in the majority of TBDM participants .",
"While TBDM was associated with higher levels of many plasma mediators compared to normoglycemic TB participants in the India and Brazil cohorts , lower IL-10 at baseline was the most important parameter accounting for differences between the TBDM and TB groups .",
"IL-10 is a key anti-inflammatory mediator , inhibiting the generation of diverse pro-inflammatory cytokines in myeloid and lymphoid cells ( Robb et al . , 1985 ) .",
"Even in the absence of concurrent infection , hyperglycemia is associated with a systemic inflammatory state characterized by elevated ratios of diverse pro-inflammatory cytokines to IL-10 in plasma ( Butkowski and Jelinek , 2017 ) .",
"Hyporesponsiveness to IL-10 signaling is an additional factor contributing to chronic low-grade inflammation in type 2 DM ( Barry et al . , 2016 ) .",
"A causal relationship between low IL-10 at baseline in TBDM and high levels of pro-inflammatory cannot be established with the data available from our cohort study but there was striking switch from relatively lower to higher IL-10 levels in TBDM following the initiation of ATT .",
"While low IL-10 is commonly associated with greater susceptibility to TB ( Redford et al . , 2011 ) , emerging evidence suggests that poorly controlled inflammation provides a host environment that supports rather than limits Mtb replication ( Mishra et al . , 2017; Mishra et al . , 2013 ) .",
"Among the cytokines analyzed for this study , IL-4 , IL-5 , IL-10 , IL-13 , and TGF-b participate in the resolution of infection-related pulmonary inflammation ( Barbosa et al . , 2006; Bosurgi et al . , 2017; Schett and Neurath , 2018 ) .",
"While there was some variation in the levels of specific analytes across the cohorts , an overall trend for declining levels of pro-inflammatory mediators and increasing levels of pro-resolving mediators was observed in the TBDM and TB groups alike .",
"The data suggest that DM comorbidity is associated with hyperinflammation prior to TB treatment , which resolves more slowly than in normoglycemic TB but is not sustained indefinitely .",
"Plasma cytokine elevation in DM is restricted to the setting of active TB disease since Kumar et al . reported that ESAT-6 or CFP10 stimulated CD4+ T cells from individuals with DM and latent TB infection have lower intracellular expression of Th1 , Th2 , and Th17 cytokines compared to T cells from latently infected normoglycemic individuals ( Kumar et al . , 2016 ) .",
"We speculate that the excessive and prolonged inflammation in TBDM comorbidity is driven by a higher lung bacterial load stimulating innate and adaptive responses and/or a defect in counterregulation intrinsic to the diabetic host that maintains inflammatory foci despite sterilizing antimicrobial treatment .",
"These hypotheses are not mutually exclusive .",
"In support of the high bacterial burden hypothesis , animal studies of the TBDM interaction consistently show higher plateau levels of Mtb colony-forming units in the lung , along with quantitatively more pulmonary immune pathology and elevated levels of pro-inflammatory cytokines compared to normoglycemic control animals with TB ( Martens et al . , 2007; Podell et al . , 2014 ) .",
"Clinical studies have variously reported higher sputum smear and culture scores and greater radiographic severity in TBDM comorbidity , which constitute crude surrogates for bacterial burden and immune pathology ( Alavi et al . , 2014; Jiménez-Corona et al . , 2013; Moreno-Martínez et al . , 2015; Yoon et al . , 2017 ) .",
"In the current study , bacterial burden as reflected by sputum smear score was higher in TBDM than TB ( Figure 1 ) .",
"In support of an intrinsic propensity for hyperinflammatory responses in TBDM , naïve T cells from chronically hyperglycemic mice were shown to have increased proliferation and production of a broad range of cytokines following antigen-receptor activation; a phenotype attributed to basal chromatin decondensation ( Martinez et al . , 2014 ) .",
"Furthermore , impaired wound healing is common diabetic complication that is due , in part , to impaired resolution of inflammation ( Baltzis et al . , 2014 ) .",
"Our study had several limitations .",
"While the cohorts providing clinical data and samples were recruited for prospective longitudinal investigation , the projects were not initially designed for cross-cohort comparison and radiographic scores and plasma samples from one year after TB treatment completion were not available for the Brazil cohort .",
"The sample sizes for these two cohorts was sufficient for comparison of plasma analytes but underpowered for logistic regression .",
"Higher median BMI and higher representation of female sex in the Brazil cohort could account at least in part for lower cytokine levels compared to the India cohort , although there was no difference in radiographic severity of disease between the two cohorts overall and more cavitary TB in the Brazil cohort ( Table 4 and Table 5 ) .",
"Finally , the normoglycemic TB arm of the EDOTS study featured high prevalence of undernutrition that is an independent risk factor for adverse TB outcomes ( Sinha et al . , 2019 ) .",
"This might account for the lack of differences in radiographic severity scores between the TBDM and TB groups in the India cohort .",
"In conclusion , we found that individuals with TBDM comorbidity not only have heightened levels of pro-inflammatory cytokines prior to ATT , but they maintain this state through the course of TB treatment .",
"The clinical significance of excessive and prolonged inflammation in TBDM comorbidity stems from that observation that morbidity and mortality in pulmonary TB largely reflects the consequences of immune-mediated lung damage ( Ravimohan et al . , 2018 ) .",
"On that basis , we predict that pulmonary impairment after TB will be greater in patients with concurrent DM .",
"We further speculate that sustained inflammation contributes to increased risk for TB-related mortality that is associated with DM ( Reed et al . , 2013 ) .",
"Matrix metalloproteinases ( MMPs ) are implicated in immune-mediated lung matrix destruction in TB ( Ong et al . , 2014 ) .",
"The anti-diabetic drug metformin was reported to exert a host-protective anti-inflammatory effect , including reduced MMP expression in normoglycemic mice infected with Mtb by aerosol , and metformin is associated with reduced risk of cavitary TB and mortality in people with concurrent DM by mechanisms that appear to be independent of any effect on glycemic control ( Degner et al . , 2018; Singhal et al . , 2014 ) .",
"We previously reported that TBDM comorbidity is associated with higher plasma MMP levels compared to TB without DM , and that TBDM patients using metformin had significantly lower MMP levels than those on anti-DM treatment regimens without metformin ( Kumar et al . , 2018 ) .",
"Together , these observations suggest that ant-inflammatory host-directed therapies , including metformin , may be particularly beneficial in the large and growing population of TB patients with concurrent DM ."
],
[
"All research presented here was conducted according to the principles expressed in the Declaration of Helsinki .",
"The India cohort study was approved by the Ethics Committee of the Prof . M . Viswanathan Diabetes Research Centre ( ECR/51/INST/TN/2013/MVDRC/01 ) .",
"The Brazil cohort study was approved by the Ethics Committee of the Maternidade Climério de Oliveira , Federal University of Bahia ( CAAE: 0115 . 0 . 054 . 000–09 ) .",
"Written informed consent was obtained from all participants at both sites .",
"Adults ( age 25–60 years ) with newly diagnosed pulmonary TB disease were screened and enrolled in the ongoing EDOTS study currently underway in Chennai , India ( Kornfeld et al . , 2016 ) .",
"Participants with concurrent TB and DM ( TBDM group ) who reached the final study visit ( month-18 ) were selected sequentially for the present investigation of plasma samples and clinical data .",
"The comparison group of normoglycemic EDOTS participants ( TB group ) was matched for age and sex as closely as possible with the TBDM group .",
"Exclusion criteria were: treatment for any prior episode of TB disease , more than 7 days treatment for the current TB episode , more than seven doses of a fluoroquinolone within 30 days , multi-drug resistant Mtb isolated at baseline pregnant or nursing , HIV-seropositive , or taking immunosuppressive drugs .",
"The diagnosis TB disease was established by positive sputum culture on solid media and compatible chest x-ray at baseline .",
"Participants were classified as having DM by HbA1c ≥ 6 . 5% for ( for those with no known prior history of DM ) plasma glucose ≥200 mg/dL at 2 hr after 75 gm glucose challenge ( American Diabetes Association , 2018 ) .",
"Participants were classified as normoglycemic by plasma glucose <140 mg/dL at 2 hr after 75 gm glucose challenge according to World Health Organization ( WHO ) criteria .",
"Self-reported cigarette and alcohol use was categorized as current , past , or never .",
"A total of 43 TBDM and 44 TB participants were included in this study .",
"Treatment for drug-sensitive TB disease was provided by government clinics and staff of the Revised National Tuberculosis Control Program in Chennai .",
"Samples and clinical information used in the present study were selected from an ongoing project performed at the Instituto Brasileiro para Investigação da Tuberculose ( IBIT , Brazilian Institute for TB Investigation ) , Salvador , Bahia , Northeast Brazil .",
"Samples were collected between June 2015 and January 2018 .",
"For the current investigation , data from 26 TB patients with type-2 DM ( HbA1c ≥ 6 . 5% ) from whom cryopreserved plasma samples were available for the immunoassays at month 0 ( pre-ATT ) , month 2 and month 6 of therapy were selected .",
"Additional TB patients without DM ( n = 25 ) , matched by age , sex , BMI , smoking history and alcohol use were selected .",
"Diagnosis of TB at IBIT follows the guidelines of the Brazilian Society of Pulmonology and Tisiology ( Conde et al . , 2009 ) , which is similar to WHO recommendations .",
"For the present study , TB diagnosis was performed at IBIT’s microbiology referral laboratory as the following: three sputum smears were examined by fluorescence microscopy , processed by the modified Petroff’s method and cultured on Lowenstein-Jensen medium .",
"All patients were ≥18 years old , BCG-vaccinated , HIV-unexposed ( confirmed with negative serology ) , had no prior diagnosis of TB , were diagnosed with DM at the time of study enrollment , and were not taking DM drugs .",
"Exclusion criteria were age <18 years , previous diagnosis/treatment of TB or DM , self-reported pregnancy , and presence of psychiatric disease that might hamper proper application of the clinical questionnaire .",
"Alcohol use was assessed by CAGE questionnaire , with alcoholism classified by positive responses to two or more questions ( Ewing , 1984 ) .",
"Plasma samples from both cohorts were purified and stored frozen at −80°C prior to batch-wise Luminex assays ( Bio-Rad , Hercules , CA ) .",
"The parameters analyzed were interferon-γ ( IFN-g ) , tumor necrosis factor-α ( TNF-a ) , interleukin ( IL ) 1-β ( IL-1b ) , IL-2 , IL-4 , IL-5 , IL-6 , IL-7 , IL-8 , IL-10 , IL-12p70 , IL-13 , IL-17A ) , granulocyte colony-stimulating factor ( G-CSF ) and granulocyte macrophage colony-stimulating factor ( GM-CSF ) .",
"Plasma levels of transforming growth factor-β1 ( TGF-b; R and D Systems ) and IL-17F ( BioLegend , San Diego , CA ) were measured by ELISA .",
"The median values with interquartile ranges ( IQR ) were used as measures of central tendency .",
"Fisher's exact test was used to compare frequencies between the study groups .",
"Continuous variables were compared between the study groups using the Mann-Whitney U test ( 2-group comparisons ) , or the Kruskall-Wallis test with Dunn's multiple comparisons ad hoc test ( between three or more groups ) .",
"Hierarchical cluster analyzes were performed using the Ward's method with bootstrap ( 100X ) of z-score normalized values of soluble biomarkers .",
"Dendograms represent Euclidean distance .",
"A principal component analysis model using data on all the soluble biomarkers was performed to compare and visualize the grouping between TBDM and TB .",
"Fold Change Analysis of TBDM patients versus TB patients of log2-transformed and t-test comparisons were performed with the Benjamini–Hochberg false discovery rate ( FDR ) adjustment for multiple testing set at 1% .",
"Decision trees were employed to identify a minimal set of targets allowing separation between the TBDM from TB .",
"This method analyzes all the phenotypic attributes and selects the most relevant attributes that allow group classification ( Fukutani et al . , 2017 ) .",
"As input for tree construction , we used data on all the markers .",
"The J48 algorithm implemented in the WEKA program ( Waikato Environment for Knowledge Analysis , version 3 . 6 . 11 , University of Waikato , New Zealand ) .",
"To estimate the classification accuracy of the decision tree models , we employed a 10-fold cross validation methodology .",
"The sensibility and specificity were measured from the confusion matrix , the receiver operating characteristic curve ( ROC ) and viewed in the scatter plot with the decision tree cutoffs .",
"Correlations were examined using the Spearman test .",
"P value below 0·05 was considered statistically significant after adjustments for multiple comparisons ( FDR 1% ) .",
"The statistical analyses were performed using GraphPad Prism 7 . 0 ( GraphPad Software , La Jolla , CA , USA ) , JMP 13 . 0 ( SAS , Cary , NC , USA ) , and R 3 . 5 . 1 ( R Foundation , Vienna , Austria ) programs ."
]
] | [
"Diabetes mellitus ( DM ) increases risk for pulmonary tuberculosis ( TB ) and adverse treatment outcomes .",
"Systemic hyper-inflammation is characteristic in people with TB and concurrent DM ( TBDM ) at baseline , but the impact of TB treatment on this pattern has not been determined .",
"We measured 17 plasma cytokines and growth factors in longitudinal cohorts of Indian and Brazilian pulmonary TB patients with or without DM .",
"Principal component analysis revealed virtually complete separation of TBDM from TB individuals in both cohorts at baseline , with hyper-inflammation in TBDM that continued through treatment completion at six months .",
"By one year after treatment completion , there was substantial convergence of mediator levels between groups within the India cohort .",
"Non-resolving systemic inflammation in TBDM comorbidity could reflect delayed lesion sterilization or non-resolving sterile inflammation .",
"Either mechanism portends unfavorable long-term outcomes including risk for recurrent TB and for damaging immune pathology ."
] | [
"Tuberculosis is the leading cause of death from infection worldwide .",
"The bacteria that causes tuberculosis infect one in every four people on the planet , though most never develop the disease .",
"People with diabetes are more likely to develop tuberculosis and they develop more severe symptoms , which may contribute to further spread of the disease .",
"As diabetes rates are growing worldwide , particularly in countries with a high burden of tuberculosis , it is becoming to increasingly important to understand how these two conditions interact .",
"People with diabetes often have more severe inflammation at the time they are diagnosed with tuberculosis than tuberculosis patients without diabetes .",
"Inflammation can cause permanent lung damage in patients with tuberculosis , which can have serious consequences .",
"Learning more about how treatment for tuberculosis affects inflammation in people with diabetes could help improve the outcomes for these patients .",
"Now , Kumar , Fukutani et al . show that people with diabetes experience higher levels of inflammation than patients without diabetes throughout the course of treatment for tuberculosis .",
"The analysis compared 17 markers of inflammation in tuberculosis patients with and without diabetes at diagnosis , and at two time periods during the 6-month course of treatment .",
"Kumar , Fukutani et al . also looked at two separate groups of patients , one from India and one from Brazil .",
"Inflammation was measured in the patients in India one year after the completion of treatment .",
"One-year after treatment for tuberculosis in India , inflammation levels were the same in patients with and without diabetes .",
"Persistently higher levels of inflammation likely explain why patients with diabetes experience more severe symptoms and suggests they may have more permanent lung damage after tuberculosis .",
"Scientists are currently developing new treatments that can be used with antibiotics to more quickly cure tuberculosis and protect the lungs by reducing inflammation .",
"Patients with diabetes and tuberculosis may benefit from these new treatments or from existing drugs like metformin or statins that may reduce inflammation ."
] | 2019 |
[
"Introduction",
"Materials and methods",
"Results",
"Discussion"
] | [
"epidemiology and global health",
"medicine"
] | An agnostic study of associations between ABO and RhD blood group and phenome-wide disease risk | elife-65658-v2 | [
[
"The blood group antigens of the ABO and RhD systems play a pivotal role in transfusion medicine because of their role in the safe administration of blood transfusions .",
"In addition , these cell surface antigens have been demonstrated to have direct effects on the susceptibility for several diseases ( Franchini et al . , 2012; Stowell and Stowell , 2019a; Stowell and Stowell , 2019b ) .",
"One of the first such studies was published in 1962 , demonstrating a relationship between the ABO system and ischemic heart disease ( Bronte-Stewart et al . , 1962 ) .",
"Multiple subsequent studies have revealed associations with a range of diseases , with a prominent example being a decreased risk of thromboembolic events and increased risks of some hemorrhagic events in individuals with blood group O ( Franchini et al . , 2012; Edgren et al . , 2010; Vasan et al . , 2016a ) .",
"The difference in thrombotic and hemorrhagic phenotypes has been attributed to variability in levels of Factor VIII and von Willebrand factor , where ABO status may explain as much as 30% of this variability ( Franchini et al . , 2012; Orstavik et al . , 1985; Germain et al . , 2015; Lindström et al . , 2019; O'Donnell et al . , 2002 ) Other prominent examples include associations with risks of a number of infectious diseases , to the extent that the allele distribution of the blood group antigens has evolved to reflect some areas endemic to these infectious diseases ( Franchini and Bonfanti , 2015 ) .",
"This is in part true for infectious disease such as Plasmodium falciparum malaria , Helicobacter pylori , and Vibrio cholera , where ABO blood groups are involved in different aspects of pathogenesis , from microbe attachment and entry into cells to subsequent disease development and severity of disease ( Stowell and Stowell , 2019a; Franchini and Bonfanti , 2015; Cserti and Dzik , 2007; Degarege et al . , 2019 ) .",
"Surface antigens of the ABO blood groups are defined by the immunodominant , terminal sugar residues on certain glycolipids and glycoproteins anchored to the membrane of red blood cells and exposed extracellularly .",
"Even if expressed from a single-gene locus , the ABO gene on chromosome 9 , A and B antigens are present not only on erythroid cells but also in many other tissues , and due to the diverse tissue , expression may result in differences in disease occurrence ( Harmening , 2012 ) .",
"The A and B allelic variants of the ABO locus encode the A and B glycosyltransferases , which differ only by a few amino acid residues , add the donor substrates UDP-N-acetylgalactosamine or UDP-galactose , respectively , to a common acceptor substrate , namely a carbohydrate chain terminating with the so-called H antigen , in turn dependent on fucosyltransferase activity expressed from the FUT1 and FUT2 genes on chromosome 19 .",
"In blood group O , the H antigen is left unaltered due to lack of ABO enzyme activity , most commonly by a single-nucleotide deletion in the ABO coding region ( Storry and Olsson , 2009 ) .",
"The RhD antigen , on the other hand , has a less clear link to health outcomes .",
"RhD status has mainly been linked to alloimmunization of the pregnant women with hemolytic disease of the fetus and newborn as a consequence ( Urbaniak and Greiss , 2000 ) .",
"Beyond these direct effects , little is known about its role in disease pathogenesis .",
"The difference between RhD-positive and -negative blood group is the presence or absence of the RhD protein on the red blood cell surface .",
"However , both individuals with and without RhD possess the homologous RhCE protein and Rh-associated glycoprotein ( RhAG ) on their red cells .",
"Thus , functions carried out by RhD are likely performed RhCE and RhAG in RhD-negative individuals , and this redundancy may in part explain the scarcity of findings related to RhD status ( Avent and Reid , 2000 ) .",
"Using the Scandinavian Donation and Transfusion ( SCANDAT ) database , we have previously studied associations between ABO blood groups and cancer subtypes , cardiovascular and thromboembolic disease , the occurrence of dementia and degradation of bioprosthetic aortic valves in relation to ABO blood group ( Vasan et al . , 2016a; Persson et al . , 2019; Vasan et al . , 2016b; Vasan et al . , 2015 ) .",
"However , these and most other prior studies into the association between ABO blood group and disease outcomes have been limited by potentially misdirected a priori hypotheses and phenome-wide disease associations have not been thoroughly explored in a systematic manner .",
"Therefore , in the current study , we aimed to agnostically investigate the association between ABO and RhD blood group and disease occurrence for a large number of disease phenotypes using large-scale population-based Swedish healthcare registries ."
],
[
"Individuals in the study were identified using an updated version of the Scandinavian donations and transfusion database ( SCANDAT3-S ) .",
"This database includes close to 8 million individuals who have donated blood , received a blood transfusion , or have had blood group testing done for other reasons .",
"Other reasons for blood group testing would typically be pre-emptive testing for example , before surgery or in antenatal care .",
"The database contains detailed information on blood donations , transfusions , as well as blood group antigen and antibody testing results and is thoroughly described elsewhere ( Zhao et al . , 2020 ) .",
"It is nationally complete since 1995 , but information dates back to 1968 with various levels of completeness , mainly depending on the geographical region .",
"Using unique national registration numbers assigned to all inhabitants of Sweden , the SCANDAT3-S database has been linked to a range of national health outcomes registers , for hospital care , cancer , cause of death , and drug prescriptions .",
"From SCANDAT3-S , we extracted information on ABO and RhD blood group and created a main cohort and a validation cohort .",
"The main cohort consisted of all individuals who were born in Sweden where at least one parent was born in Sweden and who , for any reason , had undergone ABO and RhD blood group typing with a conclusive result , but who did not donate blood within 90 days of the test .",
"Person-time for blood donors were excluded from the main cohort to maximize the representativeness of the study population .",
"In the validation cohort , we included all individuals in the SCANDAT3-S database who had ever donated blood .",
"As such , an individual could contribute person-time in both cohorts , such as in the case a person started to donate blood more than 90 days later from a blood grouping test that was initially performed for other reasons .",
"The person-time before blood donation would contribute to the main cohort censoring at entry in the validation cohort starting at the time of blood grouping before the blood donation .",
"We defined and studied a large number of disease categories .",
"Non-cancer disease categories were based on discharge diagnoses from the national patient register , which covers all hospital inpatient care in Sweden since 1987 and all specialist outpatient care since 1997 , and from the Cause of Death register , which records underlying causes of death for all persons in Sweden since 1964 ( Brooke et al . , 2017; Ludvigsson et al . , 2011 ) .",
"Because the 10th revision of the International Classification of Disease ( ICD ) was implemented in 1997 , we limited outcomes ascertainment to events from 1997 or later to avoid inconsistencies between ICD revisions .",
"Cancer outcomes were based on the Cancer Register , which records all incident cancer cases in Sweden since 1958 ( Barlow et al . , 2009 ) .",
"All of these registries are held and maintained by the Swedish National Board of Health and Welfare and have a high level of completeness and accuracy .",
"Dates of death and emigration were obtained from population registers kept by Statistics Sweden .",
"Details of non-cancerous disease categories are presented in Supplementary file 3 .",
"Non-cancer diseases were classified into disease categories based on the first three codes of the diagnosis , according to the ICD-10 codebook .",
"We did not consider external causes of disease , traumatic injuries , or symptom-based codes as these were deemed unlikely to be related to blood group antigens .",
"Cancer disease categories were based on anatomical coding using the 7th revision of the ICD for all non-hematological malignancies and the 8th revision of the ICD for hematological malignancies .",
"For details of cancer categories , see Supplementary file 4 ( SAS code for cancer disease grouping is available upon request ) .",
"In total , we considered 1217 distinct disease categories .",
"After database construction , we excluded disease categories with fewer than 50 events before analysis as we would be unlikely to detect sufficient events in the validation cohort in categories with fewer than 50 events in the main cohort .",
"All persons were followed from the date of the first blood grouping test , from their 18th birthday , or from January 1 , 1997 , whichever occurred last .",
"Follow-up was extended until the first incident event in each disease category , emigration , death or December 31 , 2017 , whichever occurred first .",
"A person could thus be included in follow-up for all disease categories investigated .",
"Descriptive statistics were presented for cohort baseline data .",
"For the main analysis , we used a Poisson regression model .",
"In the model , we incorporated the following covariates: ABO blood group ( A , AB , B or O ) , RhD status ( weak or category expression variants were excluded ) , sex , calendar-period , and age .",
"A restricted cubic spline functions with four or five knots placed according to Harrell’s method were applied to the age and calendar-period covariates ( Harrell , 2015 ) .",
"The regression model was fitted separately to each disease category resulting in incidence rate ratios ( IRR ) for each ABO blood group and RhD status using blood group O and RhD negative as reference , respectively .",
"Wald’s method was used to construct 95% confidence intervals ( CI ) .",
"Equi-dispersion was tested using a Lagrange multiplier test .",
"For disease categories where data demonstrated significant over- or under-dispersion after also performing the same analysis but reducing the number of knots from 5 to 4 , analyses were instead run using quasi-Poisson regression .",
"Multiple testing was handled using a two-stage approach .",
"First , in the exploratory analysis using the main cohort , we applied a false discovery rate ( FDR ) adjustment of raw p-values assuming positive dependency of stochastic ordering between outcomes .",
"Second , in the confirmatory analysis using the validation cohort , we used the disease categories with significant effects from explorative analysis , with results presented both without adjustment and using a Bonferroni adjustment .",
"In effect , this allowed us to limit type one errors presenting confirmed associations with high certainty , but still not to compromise type two errors for future confirmatory analysis in other cohorts ."
],
[
"Characteristics of the main and validation cohorts are presented in Table 1 .",
"When combining the main and validation cohort , there were a total of 5 . 1 million unique individuals .",
"The main cohort consisted of 4 . 2 million individuals who at any point had undertaken an ABO and RhD blood antigen test .",
"The distribution of A , AB , B , and O were 47% , 5% , 10% , and 38% , respectively , and 84% of individuals were RhD positive .",
"Women constituted 60% of the cohort .",
"The median age at cohort entry was 52 years ( interquartile range [IQR] , 30–71 ) and the median year of birth was 1949 ( IQR , 1931–1971 ) .",
"Not accounting for censoring due to disease events , the main cohort accrued a total of 49 . 9 million person-years of follow-up , 23 . 7 million in blood group A , 2 . 3 million in blood group AB , 4 . 9 million in blood group B , and 18 . 9 million in blood group O . Of the original 1217 disease categories , 1090 remained available for analyses after excluding disease categories with fewer than 50 events .",
"The median number of events per disease category in the main cohort was 4748 ( IQR , 869–231 , 166 ) .",
"A meta-summary of results of regression analyses is presented in Table 2 , and graphically in the form of a volcano plot in Figure 1 ( also , as an interactive , online variant as Supplementary file 1 ) .",
"Alternatively , results are also presented as an ICD chapter-based , variant Manhattan plot in Figure 2 ( also as an interactive , online variant as Supplementary file 2 ) .",
"Overall , in the main cohort and before FDR adjustment for multiple testing , there were 343 and 98 statistically significant associations for the ABO and RhD blood group systems and unique disease categories , respectively .",
"Of these , a total of 143 ( 41% ) and 13 ( 13% ) associations between blood group and unique outcome remained statistically significant for ABO and RhD blood group systems , respectively , after FDR adjustment .",
"For the ABO system , IRRs for statistically significant associations after FDR adjustment ranged from 0 . 57 to 0 . 99 for negative associations and from 1 . 01 to 1 . 52 for positive associations .",
"For RhD status , IRRs ranged from 0 . 90 to 0 . 97 for negative associations and from 1 . 02 to 1 . 08 for positive associations .",
"Details of all associations identified after FDR are presented in Supplementary file 5 ( for ABO blood groups ) and Supplementary file 6 ( for RhD ) .",
"In our validation cohort , consisting of almost 1 . 2 million blood donors accruing 22 million person-years of follow-up , we validated the findings from the significant disease categories from the first analysis .",
"Among the 143 and 5 significant disease categories for ABO and RhD , respectively , the median number of events was 7129 ( IQR 2464–19 , 973 ) .",
"Before multiple testing adjustment , we identified 160 associations between a blood group in 143 and 5 disease categories , for the ABO and RhD blood group , respectively .",
"After Bonferroni adjustment , there were 49 and 1 associations remaining between ABO and RhD blood group , respectively ( Table 2 and Figure 3 ) .",
"A number of previously well-established associations were seen among the Bonferroni-adjusted results .",
"For thrombosis , blood group A had a higher risk as compared to blood group O ( e . g . , pulmonary embolism , IRR 1 . 57 [95% CI , 1 . 51–1 . 64] and portal vein thrombosis , IRR 1 . 51 [95% CI , 1 . 25–1 . 83] ) .",
"Bleeding disorders were more frequent in blood group O as compared to blood group A ( e . g . , gastric ulcer , IRR 0 . 92 [95% CI , 0 . 88–0 . 95] and duodenal ulcer , IRR 0 . 86 [95% CI , 0 . 82–0 . 9] ) .",
"Thyrotoxicosis was also less common in blood groups A and AB as compared to blood group O ( with IRRs of 0 . 90 [95% CI , 0 . 86–0 . 93] and 0 . 84 [95% CI , 0 . 77–0 . 92] , for A and AB , respectively ) .",
"Pregnancy-induced hypertension was less common in blood groups A and AB , as compared to blood group O ( with IRRs of 0 . 95 [95% CI , 0 . 92–0 . 97] and 0 . 87 [95% CI , 0 . 83–0 . 92] for A and AB , respectively ) .",
"Pancreatic cancer was the only malignancy that remained associated with a blood group , specifically blood group A as compared to blood group O ( IRR , 1 . 29; 95% CI , 1 . 19–1 . 40 ) .",
"A new finding was that of calculus of the kidney and ureter , which were found to be less common in blood group B as compared to blood group O ( IRR 0 . 93 [95% CI , 0 . 89–0 . 96] ) .",
"Cholelithiasis , which has been disputed , was more common in blood groups A and AB as compared to blood group O ( with IRRs of 1 . 07 [95% CI , 1 . 05–1 . 09] and 1 . 09 [95% CI , 1 . 05–1 . 13] for A and AB , respectively ) .",
"In disease categories not significant after Bonferroni adjustment , some findings exhibited particularly strong effects , such as for viral and other specified intestinal infections , where blood group AB had a significantly lower risk , as compared to blood group O ( IRR 0 . 74; 95% CI , 0 . 62–0 . 87 ) .",
"There was a lower risk for ankylosing spondylitis in blood group AB as compared to blood group O ( IRR 0 . 79; 95% CI , 0 . 67–0 . 94 ) , and for acute pancreatitis , again with a lower risk in blood group AB as compared to blood group O ( IRR 1 . 14; 95% CI , 1 . 04–1 . 24 ) .",
"For the RhD positive as compared to RhD negative , only one disease category remained statistically significant after Bonferroni adjustment , namely pregnancy-induced hypertension ( IRR 1 . 12; 95% CI , 1 . 09–1 . 16 ) .",
"Strong effects identified in the main cohort , but not in the validation cohort were hereditary factor VIII deficiency in blood group B ( IRR 0 . 57; 95% CI , 0 . 42–0 . 77 ) , well-differentiated thyroid cancer in blood groups AB ( IRR 0 . 74; 95% CI , 0 . 62–0 . 88 ) and B ( IRR 0 . 79; 95% CI , 0 . 70–0 . 90 ) , measles in blood group A ( IRR 1 . 72; 95% CI , 1 . 23–2 . 39 ) , as well as both erythema nodosum ( IRR 1 . 32; 95% CI , 1 . 15–1 . 53 ) and sarcoidosis in blood group B ( IRR 1 . 15; 95% CI , 1 . 08–1 . 23 ) , as compared to blood group O ."
],
[
"In this large cohort study of 5 . 1 million unique persons followed over 70 million person-years , we performed an agnostic analysis of associations between the ABO and RhD blood groups and the risk of 1217 distinct disease categories .",
"After multiple testing adjustment and comparison with a validation cohort , 49 and 1 associations between disease categories and blood group for ABO and RhD remained significant , respectively .",
"Overall , we were able to confirm a number of previously known associations such as risk of thrombosis and hemorrhagic events .",
"In addition , we also identified novel associations , some with firm evidence and valid even after conservative Bonferroni adjustment , in the validation cohort , including calculus of the kidney and ureter .",
"Furthermore , being the largest study so far , we also found that blood groups A and AB had a lower risk of gestational hypertension , compared to blood group O , which has previously been disputed ( Clark and Wu , 2008; Franchini et al . , 2016; Lee et al . , 2012 ) .",
"Of the identified associations , we speculate that some associations may be driven by increased screening due to other concomitant diseases that are associated with blood groups , which might be the case for hyperlipidemias that are screened for in heart disease .",
"Most of the investigated disease or disease groups , however , do not seem to be strongly influenced by the ABO blood group of the individual .",
"As to possible mechanisms explaining the associations identified , we may only generate hypothesis to be further tested .",
"It is however peculiar to identify a condition , such as renal caliculi , with highly variable distribution , in regard to geographic region , to have an association to blood group .",
"A possible mechanism could be , when considering the full spectrum of disease categories investigated , that lower urine pH in diabetic patients , a disease category associated with an increased incidence in blood group B , may result in altered stone formation ( Sorokin et al . , 2017 ) .",
"In previous studies , intending to dissect the association between blood groups and disease , there is diversity in findings and non-findings , something that may be a consequence of ethnic or geographic group , sample size , complexity of statistical modeling , or disease classifications used .",
"This , however , imposes difficulties when comparing our results to the vast number of studies available concentrating the mentioned discoveries to a select few disease categories .",
"This is hitherto the largest study investigating blood group antigens and disease occurrence in an effort to find novel and confirm previously known associations .",
"There are some particular strengths to our approach and data .",
"Most notably , the study was based on a very large study population , representing one third of the Swedish population , with long-term and unbiased follow-up .",
"This ensures both the reliability and the generalizability of the results .",
"The agnostic approach also has the advantage of not being based on specific pre-set conceptions of specific disease categories and possible associations of blood group .",
"All disease categories are treated equally and investigated using the same principles effectively removing researcher bias .",
"Moreover , the data has been collected prospectively in various high-quality healthcare registries during a long time period with almost complete coverage .",
"In addition , the fact that all blood group data in the SCANDAT3-S database were collected from clinical transfusion registers – the quality of which is essential for the safe administration of blood transfusions – ensures that there should be little or no errors in the blood group coding .",
"Similarly , while the validity of the outcomes registration certainly varies between the different disease categories , the degree of such misclassification is unlikely to vary between blood groups , and so it should not affect the magnitude of point estimates .",
"The current study is limited by several factors .",
"One such factor is the disease classification scheme used , based primarily on ICD revision 10 categories that in some instances may lack precision .",
"Smaller disease entities were not accounted for and thus there may be true associations that were missed .",
"It is thus possible that some of the associations that we reported were driven by multiple unknown associations within a specific disease category that may have unequal , or even detrimental , effects on the outcome .",
"However , we believe that this limitation is an opportunity for further sub-categorized investigations in the future when even more events and follow-up time are available .",
"Another limitation that prevents strong casual inference is the possibility some of the observed associations between ABO blood group and disease categories were driven by other disease associations with ABO blood group .",
"This might , for example , be the case for the associations between blood group A and diabetes as well as hyperlipidemia , which are potentially driven not by a causal association but possibly instead by an association between blood group A and ischemic heart disease , at the occurrence of which diabetes and lipid disorders are screened for and thus frequently diagnosed .",
"We cannot exclude the possibility that some of the other associations were driven by similar non-causal mechanisms .",
"To limit the possibility of false-positive findings , we handled over-dispersed Poisson models using Quasi-Poisson and also in the main cohort applied the FDR approach , described by Benjamini and Hochberg , and then utilized a Bonferroni adjustment on the sub-grouped outcomes in the validation cohort .",
"The aim of this approach was to reduce type one errors without being overly conservative by first conducting an explorative analysis in the main cohort .",
"We also employed a confirmatory analysis with a validation cohort to further limit the possibility of false-positive findings .",
"However , because the validation cohort was both smaller and consisted only of blood donors , who were selected for their good health , the ensuing smaller number of events may result in failure to detect potentially interesting associations .",
"Furthermore , selecting explicitly healthy donors with no known serious health issue at time of start of donation may result in failure to detect less common or specifically non-acquired or early acquired disease .",
"However , in the validation cohort , only approximately 1% of the categories had fewer events than 50 and all findings from the main cohort can be seen in the live Supplementary files 1 and 2 and Supplementary files 5–8 .",
"It may still be informative to consider also some of the associations from the main cohort that were not corroborated in the validation cohort .",
"This is exemplified by pancreatic cancer where we saw an increased risk in blood groups AB and B in the main cohort ( IRR 1 . 37 and 1 . 129 , p<0 . 00001 and p<0 . 00001 for AB and B , respectively ) and a similar , yet non-significant effect in the validation cohort ( IRR 1 . 14 and 1 . 26 , p-value 0 . 8 and 0 . 6 for B and AB , respectively ) .",
"This also expands to the non-findings in terms of cancerous disease were multiple relationships that have previously been demonstrated , but not in the validation cohort after Bonferroni adjustment ( Vasan et al . , 2016b ) .",
"This strengthens our decision to not limit the presentation of findings to only disease categories identified in the conservative Bonferroni-adjusted analysis .",
"Still , after these limitations we believe that our findings support and generate strong further evidence for previously known associations and indicate new and interesting relationships for disease such as calculus of the kidney and ureter , pregnancy-induced hypertension , well-differentiated thyroid cancer , and sarcoidosis .",
"The new set of associations should be validated in other cohorts but also investigated using a mechanistic approach for a possible causal and biological interaction ."
]
] | [
"There are multiple known associations between the ABO and RhD blood groups and disease .",
"No systematic population-based studies elucidating associations between a large number of disease categories and blood group have been conducted .",
"Using SCANDAT3-S , a comprehensive nationwide blood donation-transfusion database , we modeled outcomes for 1217 disease categories including 70 million person-years of follow-up , accruing from 5 . 1 million individuals .",
"We discovered 49 and 1 associations between a disease and ABO and RhD blood groups , respectively , after adjustment for multiple testing .",
"We identified new associations such as a decreased risk of kidney stones and blood group B as compared to blood group O . We also expanded previous knowledge on other associations such as pregnancy-induced hypertension and blood groups A and AB as compared to blood group O and RhD positive as compared to negative .",
"Our findings generate strong further support for previously known associations , but also indicate new interesting relations .",
"Swedish Research Council ."
] | [
"The blood types that many people are familiar with , such as O-negative or AB-positive , are determined by two systems of antigens or proteins on the surface of the red blood cells: the ABO system and the RhD system .",
"The ABO system types people’s blood as A , B or AB if they have A and/or B antigens , or as type O if they have neither; while the RhD system provides the positive or negative label depending on whether or not the RhD antigen is present .",
"Previous studies have found that some ABO blood groups are linked to increased risk and severity of a variety of conditions , including blood clots in veins , bleeding disorders and gastric ulcers .",
"Despite the known influence that blood groups can have on disease , the connection has not been fully studied in many conditions , particularly for RhD status .",
"Knowing the differences in risk and disease severity between different populations could help clinicians identify individuals that they need to monitor more closely and include blood group information in prediction models .",
"To fill this gap in information , Dahlén et al . systematically looked for relationships between diseases and blood groups using records from 5 . 1 million people on a Swedish national blood donation-transfusion database .",
"Examining 1 , 217 disease categories revealed that the vast majority did not appear to have a connection to either the ABO or RhD systems of classifying blood .",
"However , the analysis identified 49 diseases with links to ABO blood types and one linked to RhD status .",
"One notable finding was that people with blood group B have an decreased risk of kidney stones .",
"The distribution of blood groups varies significantly around the world , so this relationship between disease and blood group may in part explain regional differences in disease occurrence .",
"In the future , identifying relationships with blood groups may help to better understand the underlying biological mechanisms of diseases and lead to new avenues of research ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"structural biology and molecular biophysics"
] | Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication | elife-14158-v3 | [
[
"All cells face the challenging task of copying and passing on genetic information to progeny in an error-free manner as possible ( Fuchs and Fujii , 2013 ; Sutera and Lovett , 2006 ) .",
"DNA synthesis is carried out by large , multi-subunit assemblies , termed replisomes , which are assembled at replication origins in accord with cell cycle cues .",
"Dedicated proteins known as initiators play an essential role in selecting replication start sites , remodeling origin DNA , and providing an appropriate platform for recruiting replicative helicases to origins prior to the onset of strand synthesis ( Costa et al . , 2013; Dutta and Bell , 1997; Kaguni , 2011; Leonard and Grimwade , 2011 ) .",
"Although specific helicase loading mechanisms vary across the three domains of life – archaea , bacteria , and eukaryotes – all appear to rely on replication initiation factors belonging to the AAA+ ( ATPases Associated with various cellular Activities ) superfamily of nucleotide hydrolases .",
"The timely and accurate deposition of replicative helicases onto origin DNA is a highly coordinated and regulated process .",
"In bacteria , replication initiation relies on the DnaA initiator , which recognizes and marks the bacterial replication origin ( Bramhill and Kornberg , 1988a; 1988b; Funnell et al . , 1987; Hsu et al . , 1994 ) .",
"During initiation , DnaA actively opens an AT-rich region of the origin ( Bramhill and Kornberg , 1988a , 1988b; Dixon and Kornberg , 1984; Funnell et al . , 1987; Gille and Messer , 1991; Hsu et al . , 1994; Skarstad et al . , 1990 ) , termed a DNA-unwinding element ( DUE ) ( Kowalski and Eddy , 1989 ) , and helps to recruit two copies of the replicative helicase to the newly melted single strands .",
"In certain Gram-negative bacteria , a protein known as DnaC assists with loading of the helicase ( known in these organisms as DnaB ) ; many Gram-positive species retain a homolog of DnaC termed DnaI .",
"Both DnaC and DnaI are composed of an N-terminal helicase binding domain that connects to a C-terminal AAA+ ATPase domain by a variable linker region of unknown function ( Loscha et al . , 2009 ) ( Figure 1A ) .",
"There is evidence that ATPase activity by DnaC/I proteins controls key aspects of the helicase loading cycle and may be auto-regulated ( Davey et al . , 2002; Ioannou et al . , 2006; Learn et al . , 1997 ) ; however , the mechanism by which this control is exerted is not understood . 10 . 7554/eLife . 14158 . 003Figure 1 . The phage 77 ORF104 protein binds the S . aureus DnaI ATPase domain .",
"( A ) Domain organization of SaDnaI .",
"The N-terminal helicase-binding domain is colored gray , the linker region is magenta , and the AAA+ domain is orange .",
"Numbers refer to amino acid positions .",
"AAA+ ATPase motifs are labeled: Walker-A ( WA ) , Walker-B ( WB ) , Sensor-I ( SI ) , Box VII , Sensor-II ( SII ) , and the initiator/loader specific motif ( ISM ) .",
"( B ) SDS-PAGE analysis of amylose pull-downs between His6MBP-77ORF104 and full-length SaDnaI ( with ATP ) .",
"A DnaI alone control is shown .",
"Supernatant – S/N , W1 and W2 – washes .",
"( C ) Limited trypsin proteolysis and SDS-PAGE analysis of full-length SaDnaI in the presence of 77ORF104 .",
"A ~20kDa band of SaDnaI is stabilized in the presence of 77ORF104 . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 003 Given the marked rise in multi-drug resistant bacterial strains , along with associated human fatalities ( Klein et al . , 2007; 2013; Salgado et al . , 2003 ) , there is renewed interest in exploring bacteriophage genomes to discover new antimicrobial agents .",
"Although bacterial DNA replication proteins are strikingly distinct from those found in eukaryotes and archaea ( Leipe et al . , 1999 ) , antibiotics currently on the market do not target replication components directly , suggesting that this system may be a valuable prospective drug target ( McHenry , 2011; Robinson et al . , 2010; 2012 ) .",
"Along these lines , a small , 52 amino acid protein from phage strain 77 , encoded by the ORF104 gene , has been reported to interact directly with the Staphylococcus aureus helicase loader , DnaI , and to inhibit host DNA replication in vivo ( Liu et al . , 2004 ) .",
"The discovery of 77ORF104 marks the first known instance in which the replication initiation machinery of a bacterium can be inhibited by an exogenous factor ( Liu et al . , 2004 ) ; however , where the inhibitor protein associates with the helicase loader and how it represses helicase loader activity has remained unknown .",
"To gain insights into both bacterial helicase loader mechanisms and how they may be disrupted by an exogenous viral system , we used a combination of structural , biochemical , and comparative studies to define how the phage 77 ORF104 protein inhibits S . aureus DnaI ( SaDnaI ) .",
"We show that the phage protein binds directly to DnaI’s AAA+ ATPase domain , where it both remodels a region critical for loader self-assembly and sterically masks a known loader-loader interaction site .",
"Inhibitor binding , which represses the helicase loader’s ATP hydrolysis activity , is found to exploit a surface region normally occupied by a portion of the linker region that connects the N- and C-terminal domains of DnaI .",
"Together , our data not only establish a new means by which viruses can inhibit DNA replication , but also indicate that bacterial helicase loaders possess an unanticipated auto-regulatory element , located within their variable linker region , that serves to help restrict premature loader self-assembly ."
],
[
"To begin to probe the interaction between 77ORF104 and the helicase loader from S . aureus ( SaDnaI ) , we first cloned , expressed , and purified both full-length proteins .",
"By performing amylose pull-down assays using the tagged inhibitor protein as bait and DnaI as prey ( Figure 1B ) , we confirmed that 77ORF104 associates with SaDnaI as previously reported ( Liu et al . , 2004 ) .",
"We then conducted limited proteolysis studies of SaDnaI in both the presence and absence of 77ORF104 to determine where the phage protein might bind .",
"Inspection of the reactions using SDS-PAGE showed relatively rapid degradation of SaDnaI when the phage protein was omitted .",
"By contrast , when 77ORF104 was added to the full-length loader , a distinct 20kDa species remained protected from digestion ( Figure 1C ) .",
"Having established that 77ORF104 appears to bind to and/or mask a defined portion of SaDnaI , we next sought to define the interacting regions more precisely .",
"We therefore expressed and purified individual domains of SaDnaI , including the N-terminal domain ( NTD ) and linker , the AAA+ domain and linker ( CTD ) , and the AAA+ domain alone .",
"Turning again to pull-down assays with tagged 77ORF104 , we found that the phage protein was capable of retaining both the isolated C-terminal AAA+ ATPase domain ( with or without the preceding linker region ) but not the N-terminal domain on its own ( Figure 2A–B ) .",
"Given this finding , we next tested whether nucleotide was required for SaDnaI’s association with 77ORF104; ATP was found to be dispensable for association with the full-length loader ( compare Figures 1B , 2C ) .",
"Together , these findings establish that the phage inhibitor binds directly to the ATPase fold of DnaI but that this interaction is independent of nucleotide . 10 . 7554/eLife . 14158 . 004Figure 2 . Binding of 77ORF104 to the SaDnaI AAA+ domain is nucleotide-independent . SDS-PAGE analysis of amylose pull-downs between His6MBP-77ORF104 and ( A ) SaDnaICTD , ( B ) SaDnaINTD , or ( C ) full-length SaDnaI in the absence of ATP .",
"DnaI and 77ORF104 alone controls are also shown .",
"( D ) Binding the phage 77 ORF104 protein to SaDnaIAAA+ and SaDnaINTD as measured by a change in fluorescence anisotropy ( ΔFA – change in milli-anisotropy units ) .",
"The X-axis denotes protein concentration .",
"Data points and error bars derive from three-independent experiments .",
"No measurable binding was observed for SaDnaINTD . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 004 To better understand the physical basis for the phage 77 ORF104-SaDnaI interaction , we set out to determine a crystal structure of the complex .",
"To define a minimal helicase loader construct for crystallization screening , we fluorescently labeled the N-terminus of wild-type 77ORF104 ( Materials and methods ) and performed fluorescence anisotropy-based binding assays with various purified SaDnaI truncations .",
"In accord with our pull-down studies , the core AAA+ domain of SaDnaIAAA+ proved sufficient for binding to 77ORF104 , while the N-terminal domain of SaDnaI showed no evidence of binding ( Figure 2D ) .",
"The calculated apparent Kd ( Kd , app = 49 nM ± 19 nM ) for the inhibitor-loader AAA+ domain interaction proved comparable to that previously reported for the 77ORF104 protein and full-length SaDnaI ( Kd = ~50 nM as reported by surface plasmon resonance ) ( Liu et al . , 2004 ) .",
"Together , these data show that only the core AAA+ domain of the S . aureus helicase loader is required for high affinity association with the phage inhibitor protein , and suggested that the SaDnaI ATPase fold might serve as a promising candidate for co-crystallization studies .",
"Following screening , the successful acquisition of crystals , and data collection ( see Materials and methods ) , we determined a crystal structure of the AAA+ domain of SaDnaI bound to both 77ORF104 and an ATP-mimetic , ADP•BeF3 ( Figure 3A ) , using single-wavelength anomalous dispersion for phasing .",
"Following several rounds of building and refinement , the model converged at an Rwork/Rfree of 18 . 0%/21 . 8% for the resolution range of 47 . 4–1 . 9 Å ( Table 1 ) .",
"The final model contains residues 136–300 for SaDnaI and all 52 residues of 77ORF104 , and displays good overall stereochemistry . 10 . 7554/eLife . 14158 . 005Figure 3 . Structure of the ADP•BeF3-bound SaDnaIAAA+•77ORF104 complex and biochemical validation of observed interactions .",
"( A ) Overall structure of ADP•BeF3-bound SaDnaIAAA+ complexed with 77ORF104 .",
"SaDnaIAAA+ is colored orange and 77ORF104 sky-blue .",
"ADP ( dark blue ) , BeF3 ( limon-teal ) and a magnesium ion ( black ) , are shown within the ATP binding site of SaDnaIAAA+ .",
"( B ) Close-up view of the SaDnaI AAA+ ATP binding site .",
"Conserved motifs are colored: Walker-A ( Lys170 ) ( blue ) , Walker-B ( Asp225 and Asp226 ) ( yellow ) , and Sensor-I ( Asn260 ) ( grey ) .",
"Lys39 from 77ORF104 is colored green , a magnesium ion in black and liganding waters in red .",
"Refined 2Fo-Fc electron density is shown for a portion of the region , contoured at 1 . 6 σ .",
"( C ) Analysis of the ADP•BeF3-bound SaDnaIAAA+•77ORF104 interface .",
"Several residues from 77ORF104 that participate in the interaction are shown as sticks and labeled .",
"Three elements selected for mutagenesis studies are colored pink ( Tyr17 ) , green ( Lys39 ) , and red ( residues 47–52 ) .",
"( D ) Competition assay showing the ability of different 77ORF104 mutants to compete away wild-type , N-terminally labeled 77ORF104 from interacting with SaDnaIAAA+ .",
"Competition is evident by a decrease in fluorescence anisotropy ( ΔFA – change in milli-anisotropy units ) as labeled protein is displaced by the unlabeled 77ORF104 competitor . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 00510 . 7554/eLife . 14158 . 006Table 1 . Data collection , phasing and refinement statistics for apo SaDnaIAAA+ and ADP•BeF3-SaDnaIAAA+•77ORF104 structures . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 006Construct:'apo' SaDnaIAAA+77ORF104-SaDnaIAAA+Data CollectionBeamlineBNL NSLS X25BNL NSLS X25Wavelength1 . 5000 . 979Space groupP212121P6522Cell edges ( Å ) 113 . 10 , 126 . 26 , 183 . 3473 . 17 , 73 . 17 , 189 . 72Cell angles ( ° ) 90 . 0 , 90 . 0 , 90 . 090 . 0 , 90 . 0 , 120 . 0Resolution range ( Å ) 48 . 1–2 . 6 ( 2 . 74– 2 . 6 ) 44 . 8–1 . 9 ( 1 . 97–1 . 9 ) aUnique reflections81 , 324 ( 11 , 704 ) 24 , 589 ( 2 , 390 ) Completeness ( % ) 99 . 9 ( 99 . 5 ) 100 . 00 ( 100 . 00 ) Rmerge0 . 16 ( 4 . 00 ) 0 . 165 ( 1 . 55 ) Rmeas0 . 17 ( 4 . 33 ) 0 . 17 ( 1 . 65 ) Rpim0 . 066 ( 1 . 66 ) . 054 ( . 57 ) Redundancy13 . 3 ( 13 . 2 ) 18 . 5 ( 15 . 7 ) I/σ ( I ) 3 . 4 ( 0 . 2 ) 14 . 82 ( 1 . 44 ) Wilson B-Factor29 . 626 . 6CC 1/20 . 996 ( 0 . 684 ) 0 . 999 ( 0 . 693 ) Phasing# of sitesNA4FOMbNA0 . 90 ( 0 . 54 ) RefinementResolution limits ( Å ) 48 . 13 ( 2 . 6 ) 47 . 4 ( 1 . 9 ) Rwork ( Rfree ) b22 . 7 ( 26 . 5 ) 18 . 0 ( 21 . 8 ) No .",
"protein residues1975223No .",
"solvent/ligand molecules67/9209/32RMSD Bond , Å0 . 0030 . 012RMSD Angle , °0 . 5401 . 335Protein geometryRamachandran preferred/outliers ( % ) 95 . 87/0 . 6199 . 5/0Rotamer outliers ( % ) 0 . 480aNumbers in parentheses refer to the highest resolution shell . bFive percent of the total number of reflections were used to calculate Rfree .",
"Examination of the ADP•BeF3-bound SaDnaIAAA+•77ORF104 complex revealed that the phage protein binds to the AAA+ domain of SaDnaI in a 1:1 manner ( Figure 3A ) .",
"SaDnaI’s core AAA+ domain forms an αβα fold typical of the superfamily , and possesses all of the canonical motifs involved in nucleotide binding , such as the Walker-A , Walker-B and Sensor-I elements ( Walker et al . , 1982 ) ( Figure 1A ) .",
"Collectively , these motifs adopt a configuration similar to that seen in structures of other nucleotide-bound AAA+ ATPases ( e . g . , see [Erzberger and Berger , 2006] ) , except that the last six C-terminal residues of the structure , which include the Sensor-II amino acid ( Arg304 ) , were unresolved ( Figure 3B ) .",
"Inspection of the electron density in the active site revealed clear density for nucleotide binding , permitting modeling of ADP , BeF3 , and a single Mg2+ ion and its associated waters .",
"Interestingly , 77ORF104 can be seen to directly engage the bound nucleotide .",
"In this regard , Lys39 of the phage inhibitor protein makes one of the more notable contacts , projecting into the SaDnaIAAA+ active site to engage the BeF3 moiety directly ( Figure 3B ) .",
"Upon inspection of the 77ORF104•SaDnaIAAA+ binding interface , it became evident that the interaction of these proteins can be divided into roughly three 'hotspots' ( Figure 3C ) .",
"One such locus involves the five most C-terminal residues of the protein , which form a β-strand that associates laterally with one edge of the β-sheet in SaDnaI’s AAA+ domain core .",
"The other two loci involve residues such as Tyr17 and Lys39 , which make contacts to or around the nucleotide-binding site of SaDnaI and the associated loader-inhibitor interaction surface .",
"To determine whether specific contacts observed in the SaDnaIAAA+•77ORF104 complex are important for the inhibitor’s association with DnaI , we designed , cloned and purified several 77ORF104 mutants based on the structure and tested them for binding to SaDnaIAAA+ ( Figure 3D ) .",
"Lys39 and Tyr17 were each mutated to glutamate in the full-length 77ORF104 protein , and the last six amino acids of the inhibitor’s C-terminus were also truncated ( 77ORF104Δ47–52 ) .",
"We then developed a competition-based fluorescence anisotropy assay to assess the ability of different mutant proteins to displace fluorescently labeled , wild-type 77ORF104 from the SaDnaI AAA+ domain .",
"As expected , native 77ORF104 proved capable of competing away the dye-labeled 77ORF104 protein from binding to SaDnaIAAA+ ( Ki , app=1 . 625 μM ± 0 . 07 μM ) ( Figure 3D ) , establishing the utility of the assay .",
"Testing of the 77ORF104K39E mutant revealed only a modest reduction in potency relative to the wild-type protein , indicating that this amino acid serves a relatively peripheral role in stabilizing the inhibitor-loader interface ( Figure 3D ) .",
"By comparison , removal of the C-terminal tail of 77ORF104 or mutation of Tyr17 to glutamate completely abrogated the ability of 77ORF104 to compete for binding by the labeled inhibitor protein ( Figure 3D ) .",
"Together , these data corroborate the structural interactions seen in the 77ORF104•SaDnaIAAA+ complex , demonstrating that both Tyr17 and the last five residues of the C-terminal tail of 77ORF104 are particularly critical for the activity of the phage inhibitor .",
"The results with the 77ORF104K39E mutant additionally are consistent with our pull-down studies , which show that the 77ORF104-SaDnaI interaction does not require nucleotide for stable association ( Figure 2A–B ) .",
"Having established how the phage inhibitor engages SaDnaI , we sought next to determine how 77ORF104 binding might interfere with specific loader functions .",
"Most AAA+ ATPase systems oligomerize by inter-ATPase domain interactions that juxtapose the active site of one subunit with a catalytically important basic amino acid ( the arginine finger ) of another subunit ( Wendler et al . , 2012 ) .",
"In the case of 77ORF104 , the protein associates with the nucleotide-binding face of SaDnaI; superposition of the SaDnaI AAA+ domain in our complex onto the AAA+ domain of a single subunit from a nucleotide-assembled dimer of Aquifex aeolicus DnaC ( Mott et al . , 2008 ) shows that the 77ORF104 protein occupies the same location as that of the neighboring protomer ( Figure 4A ) .",
"This arrangement indicates that the phage inhibitor blocks SaDnaI activity by sterically blocking loader self-assembly . 10 . 7554/eLife . 14158 . 007Figure 4 . 77ORF104 alters the local conformation of the SaDnaI ISM but does not block interactions with the SaDnaC replicative helicase .",
"( A ) Superposition of a nucleotide-stabilized Aquifex aeolicus DnaCAAA+ ( AqDnaCAAA+ ) dimer ( PDB ID 3ECC , [Mott et al . , 2008] ) onto the SaDnaIAAA+•77ORF104 structure .",
"The docking results in a steric clash between 77ORF104 and the neighboring protomer of the AqDnaCAAA+ dimer .",
"The inset ( rotated 180˚ ) shows a close-up view of the ATP binding center , highlighting how the dimer-partner of the Aquifex aeolicus DnaCAAA+ domain projects a positive amino acid ( Lys210 ) into the AAA+ active site in a manner similar that seen for Lys39 from 77ORF104 .",
"( B ) Induction of a local conformational change to the SaDnaI ISM by 77ORF104 can be seen in a comparative structural analysis with other DnaI homologs .",
"The inset ( rotated 90˚ ) shows a close-up view of ISM helices in the 77ORF104-inhibited complex aligned to apo SaDnaIAAA+ and Streptococcus pyogenes DnaI ( SpyoDnaIAAA+ ) ( PDB ID 2QGZ , Seetharaman et al . , to be published ) .",
"The blue arrow indicates the direction of ISM bending by the phage inhibitor protein .",
"( C ) 77ORF104 associates with SaDnaI when the helicase loader is bound to the host SaDnaC helicase .",
"SDS-PAGE analysis of amylose pull-down experiments using purified His6MBP-tagged 77ORF104 , SaDnaI , and the SaDnaC replicative helicase .",
"The positions of each protein are indicated on the right; inputs are shown on the left half of the gel .",
"His6MBP-tagged 77ORF104 co-precipitates with SaDnaI alone and SaDnaI bound to the SaDnaC replicative helicase , but not with the SaDnaC helicase alone .",
"Pull-down experiments were performed in the absence of nucleotide .",
"Performing the experiment in the presence of nucleotide generated the same result ( not shown ) , indicating nucleotide is not required for SaDnaI to associate with SaDnaC . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 00710 . 7554/eLife . 14158 . 008Figure 4—figure supplement 1 . 77ORF104 directly interferes with the self-association of SaDnaI .",
"( A ) SDS-PAGE analysis of centrifugal SaDnaI assembly assay .",
"Lanes from reaction containing SaDnaI with or without 77ORF104 are shown and labeled .",
"S/N – supernatant .",
"( B ) Dynamic light scattering plots of SaDnaI alone ( upper ) and SaDnaI in the presence of equimolar 77ORF104 ( lower ) .",
"( C ) Table representing some the physical parameters calculated from DLS measurements of DnaI alone and in the presence of the ORF104 protein . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 00810 . 7554/eLife . 14158 . 009Figure 4—figure supplement 2 . Analytical gel filtration results of SaDnaC , SaDnaI , 77ORF013 and 77ORF104 , both alone and in various combinations . The relative positions of marker proteins are indicated .",
"( A ) Analytical gel filtration chromatographs of SaDnaC and SaDnaI alone , SaDnaC and SaDnaI together , and SaDnaC and SaDnaI with 77ORF104 .",
"( B ) Analytical gel filtration chromatographs of SaDnaC and 77ORF013 alone , SaDnaC and 77ORF013 together , and SaDnaC and 77ORF013 with 77ORF104 .",
"( C ) SDS-PAGE analysis of selected peak fractions for SaDnaC , SaDnaI and 77ORF013 alone .",
"( D ) SDS-PAGE analysis of selected peak fractions for SaDnaC , SaDnaI and SaDnaC + SaDnaI .",
"( E ) SDS-PAGE analysis of selected peak fractions for 77ORF104 + SaDnaC + SaDnaI and 77ORF013 + SaDnaC .",
"( F ) SDS-PAGE analysis of selected peak fractions for 77ORF104 + SaDnaC + 77ORF013 . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 009 Within AAA+ ATPases , one specialized feature that phylogenetically distinguishes helicase loaders such as DnaI and bacterial replication initiator proteins from other superfamily members is the existence of an extra α-helix that is inserted into one edge of the core AAA+ fold ( Iyer et al . , 2004 ) .",
"This element , termed the Initiator/loader-Specific Motif ( ISM ) ( Dueber et al . , 2007 ) , has been shown to be important for self-assembly and function in replicative helicase loaders and initiators ( Mott et al . , 2008; Dueber et al . , 2007; Erzberger et al . , 2006; Duderstadt et al . , 2011 ) .",
"In nucleotide-oligomerized DnaA and DnaC/I structures , the ISM introduces a significant out-of-plane displacement of adjacent ATPase folds , and appears responsible for pushing these assemblies into a helical , as opposed to closed-ring , formation .",
"As anticipated , based on structural alignments with DnaC/I homologs , the ISM of 77ORF104-bound SaDnaIAAA+ forms a V-shaped projection from the central ATPase domain .",
"Inspection of this region , however , revealed that one of the α-helices of the SaDnaI ISM is bent compared to the ISM region of other helicase loaders ( Figure 4B ) .",
"Examination of the crystal contacts in our SaDnaIAAA+•77ORF104 structure indicates that the change in conformation of the ISM is not the result of packing interactions , indicating that binding of the 77ORF104 inhibitor is responsible for introducing this conformational change in SaDnaIAAA+ .",
"To determine whether the conformational change seen in the SaDnaI ISM corresponds to a natural state of S . aureus protein , or as a result of binding to phage 77 ORF104 , we crystallized and determined the structure of the SaDnaI AAA+ domain in absence of the inhibitor protein ( Figure 4B ) .",
"Structural alignment of the apo and 77ORF104-bound SaDnaIAAA+ models shows that the ISM is straight in the absence of the inhibitor , as seen in other helicase loader structures , such as Streptococcus pyogenes DnaI ( shown in Figure 4B ) .",
"This result indicates that the bent conformational change visualized for the SaDnaI ISM in the presence of the inhibitor is a consequence of 77ORF104 binding , rather than a species-specific state of the loader alone .",
"Thus , in addition to sterically blocking partner binding , 77ORF104 remodels a critical self-assembly element in DnaI to further interfere with loader self-association .",
"Given that the N-terminal domain of DnaC/I-type helicase loaders contains the portion of the protein known to bind the replicative helicase , we reasoned that the 77ORF104 inhibitor might not disrupt the ability of DnaI to associate with its cognate target , DnaC ( known as SaDnaC , a DnaB-family helicase not to be confused with the E . coli DnaI homolog and helicase loader , EcDnaC ) .",
"To test this idea , we performed pull-down experiments using tagged 77ORF104 as bait to bind either untagged SaDnaI or SaDnaC-bound SaDnaI as prey ( Figure 4C ) .",
"Analysis of the reactions by SDS-PAGE revealed that both free SaDnaI and 77ORF104-bound SaDnaI were able to bind to the SaDnaC helicase equally well .",
"By contrast , 77ORF104 did not bind to the SaDnaC helicase alone .",
"Overall , this result is consistent with a model in which 77ORF104 inhibits SaDnaI function not by blocking loader/helicase associations , but by preventing loader self-assembly .",
"Both light-scattering and analytical size-exclusion chromotography data corrobate this model ( Figure 4—figure supplement 1; Figure 4—figure supplement 2 ) .",
"If 77ORF104 targets the ability of SaDnaI to self-associate , as opposed to an interaction with the host helicase , then the protein should be expected to affect functions of DnaI that rely on loader-loader interactions .",
"The nucleotide binding cycle of bacterial helicase loaders has been proposed to be coupled to an ability of the loader to self-assemble ( Biswas et al . , 2004; Mott et al . , 2008; Tsai et al . , 2009a ) , an activity that can be stimulated in DnaI/DnaC-family loaders by single-stranded ( ss ) DNA ( Davey et al . , 2002; Ioannou et al . , 2006 ) .",
"To test whether 77ORF104 might impact such a function , we carried out radioactive ATP hydrolysis assays using [γ-32P]-ATP and M13-ssDNA .",
"Although SaDnaI was found to exhibit relatively modest ATPase activity on its own , the presence of M13-ssDNA markedly stimulated nucleotide turnover ( Figure 5A ) .",
"By contrast , when incubated with the 77ORF104 inhibitor in the presence of M13-ssDNA , the observed stimulation of ATP hydrolysis by ssDNA was much reduced ( Figures 5B , C ) .",
"To establish that the increase in ATPase activity we observed was derived from SaDnaI and not from a potentially contaminating ATPase , we cloned , expressed and purified several active site mutants of SaDnaI .",
"Both a Walker A mutant ( K170A ) and an arginine finger mutant ( R288A ) showed reduced ATP hydrolysis activity in the presence of ssDNA , indicating that the ssDNA-stimulated activity seen in our assays was indeed specific to DnaI and did not arise from a contaminating ATPase ( Figure 5D ) .",
"Taken together with our structural and biochemical data ( see also Figure 4—figure supplement 1 ) , our findings support a model in which 77ORF104 inhibits SaDnaI activity by blocking loader self-assembly and preventing proper ATPase function . 10 . 7554/eLife . 14158 . 010Figure 5 . 77ORF104 inhibits ssDNA stimulation of ATP hydrolysis by SaDnaI .",
"( A ) Representative TLC image shown for SaDnaI ATP hydrolysis in the presence ( left ) or absence ( right ) of M13ssDNA .",
"( B ) M13ssDNA stimulates ATP hydrolysis by SaDnaI while 77ORF104 inhibits this effect .",
"( C ) Representative TLC image of SaDnaI ATP hydrolysis experiments in the presence and absence of wild-type 77ORF104 and/or M13ssDNA .",
"( D ) Effects of ATPase mutations on observed hydrolysis activity .",
"Walker-A ( K170A ) and arginine finger ( R288A ) mutations in SaDnaI show reduced stimulation of activity , comparable to that seen in the absence of ssDNA . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 010 Because phage 77 utilizes a specialized protein to inhibit the activity of the host’s helicase loader , we became curious as to how the phage itself might replicate .",
"Interestingly , within the phage 77 genome , the gene next to ORF104 ( termed ORF013 ) is annotated as an AAA+ ATPase .",
"The two genes are located in the same operon in the phage chromosome , suggesting that they might be expressed contemporaneously and function synergistically .",
"To determine the protein family to which 77ORF013 belongs , we searched the database for homologous proteins of known function .",
"Surprisingly , this analysis revealed that the 77ORF013 gene encodes a putative DnaC/DnaI-type helicase loader ( Figure 6A ) , thereby raising the possibility that the 77ORF013 protein might bind to the host’s replicative helicase directly .",
"To test this hypothesis , we cloned , expressed and purified a tagged version of 77ORF013 and performed amylose pull-down experiments using the untagged SaDnaC replicative helicase as prey .",
"SDS-PAGE analysis of different pull-down fractions showed that the phage 77 ORF013 protein was indeed capable of binding the SaDnaC replicative helicase ( Figure 6B ) ; analytical gel filtration chromatography studies corroborate this result ( Figure 4—figure supplement 2 ) .",
"Thus , the partner open reading frame shared by the 77ORF104 inhibitor protein appears to encode for a phage variant of a bacterial replicative helicase loader . 10 . 7554/eLife . 14158 . 011Figure 6 . Phage 77 encodes a bacterial helicase loader homolog ( ORF013 ) that binds to the host SaDnaC replicative helicase .",
"( A ) Phylogenetic tree of the phage encoded ORF013 gene with various initiator/loader clade AAA+ ATPases .",
"77ORF013 clusters with DnaC/DnaI family helicase loaders; a distantly related AAA+ ATPase ( E . coli ClpX ) was included for the purpose of rooting the tree .",
"( B ) SDS-PAGE analysis of amylose pull-down experiments using His6MBP-tagged phage loader 77ORF013 and the S . aureus DnaC replicative helicase .",
"The 77ORF013 helicase loader homolog binds to the host SaDnaC helicase .",
"( C ) SDS-PAGE analysis of amylose pull-downs using His6MBP-tagged 77ORF104 and 77ORF013 helicase loader homolog .",
"77ORF013 does not associate with the 77ORF104 inhibitor protein .",
"( D ) Binding to phage 77ORF104 inhibitor protein to SaDnaIAAA+ , 77ORF013 and E . coli DnaC as measured by a change in fluorescence anisotropy ( ΔFA – change in milli-anisotropy units ) .",
"The X-axis represents protein concentration .",
"Data points and error bars derive from three-independent experiments .",
"No measurable binding was observed for the phage 77 ORF013 or E . coli DnaC .",
"SaDnaIAAA+ is shown as a positive control . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 011 The finding that 77ORF013 is a helicase loader homolog raised the question as to whether the cognate 77ORF104 protein would bind to it as well as to the host SaDnaI protein .",
"To address this question , we performed pull-downs of the purified tagged 77ORF104 inhibitor with 77ORF013 .",
"This assay revealed no association between the putative phage-encoded helicase loader and 77ORF104 ( Figure 6C ) .",
"As measured by anisotropy , 77ORF104 also proved unable to interact with either a Gram-negative , DnaC-type helicase loader ( E . coli DnaC ) or 77ORF013 ( Figure 6D ) , indicating that the association of 77ORF104 with DnaI is specific to the S . aureus helicase loader .",
"Together , these findings indicate that the phage inhibitor protein’s association with SaDnaI is specific for binding only to the helicase loader of a host bacterium targeted by the virus ."
],
[
"In the present study , we have set out to better understand not only the function of DnaI-family bacterial helicase loaders , but also how bacteriophage can interfere with helicase loader activity to block host DNA replication .",
"We determined crystal structures of the AAA+ ATPase region of the SaDnaI helicase loader in a nucleotide-free state and bound to both the ATP mimic ADP•BeF3 and a DnaI-binding protein ( 77ORF104 ) from phage 77 ( Figures 3 and 4A–B ) .",
"The structures reveal that binding of the phage inhibitor not only remodels a region known to be critical for DnaI homo-oligomerization , the Initiator Specific Motif ( ISM ) ( Figure 4B ) , but that it also sterically occludes a principal subunit-subunit interaction surface on the helicase loader ( Figure 4A ) .",
"Association studies between 77ORF104 and SaDnaI show that productive interactions are not dependent on nucleotide binding ( Figure 2C ) , but instead require the last five C-terminal residues of the phage inhibitor protein and a particular tyrosine residue ( Tyr17 ) on the inhibitor protein that is buried in the binding interface with SaDnaI ( Figure 3C–D ) .",
"The observed structural changes and regions masked by 77ORF104 binding would be expected to prevent DnaI self-association .",
"This prediction is borne out by ATPase assays showing that this activity – which relies on DnaI-DnaI interactions to form a competent catalytic center – is repressed by the protein ( Figure 5B ) .",
"Interestingly , ORF104 was not found to disrupt the loader’s association with the replicative helicase SaDnaC ( Figure 4C ) .",
"This result indicates that 77ORF104 could in principle act at multiple stages during the DNA replication cycle where helicase-loading events are utilized , including both initiation and replication restart ( Bell and Kaguni , 2013; Heller and Marians , 2006 ) .",
"During the course of carrying out this work , we discovered that phage 77 encodes another protein , ORF013 , which is homologous to other DnaC/I-family members ( Figure 6A–B ) .",
"Binding studies revealed that this protein binds to the host’s replicative helicase , SaDnaC , but not to the inhibitory 77ORF104 protein that is also produced by phage 77 ( Figure 6C–D ) .",
"These observations suggest that the 77ORF013 and 77ORF104 , which appear to both share a common operon , may serve as a two-pronged mechanism by which host replication can be inhibited and a portion of the cell’s replication machinery co-opted for promoting viral DNA duplication .",
"Surveys of other S . aureus phage genomes , such as phages 80 and 80α , reveal the existence of homologs of both the phage 77 ORF013 protein and the ORF104 inhibitor ( Kwan et al . , 2005; Liu et al . , 2004; Tormo-Más et al . , 2010 ) , indicating that the phage 77 ORF013/ORF104 system may be widespread among Staphylococcus aureus phages .",
"The hijacking of a host replicative helicase for viral replication has been seen before with divergent viruses , including the phages λ , P2 and Mu , as well as influenza ( Kawaguchi and Nagata , 2007; Kruklitis and Nakai , 1994; Mallory et al . , 1990; Odegrip et al . , 2000 ) .",
"However , the co-expression of a putative viral helicase loader with an inhibitor of a host helicase loader has only been seen thus far for phage 77 and its relatives .",
"Although replication initiation has yet to be reconstituted for S . aureus using purified factors , the planned development of such a system will allow for future investigations into the effects of ORF013/ORF104 family proteins on helicase loading and phage replication in vitro .",
"A second unexpected finding obtained from this work derives from an analysis of where 77ORF104 binds to SaDnaI in the context of other bacterial helicase loading proteins .",
"Previous studies of Bacillus subtilis DnaI and the related E . coli DnaC protein have shown that bacterial helicase loaders typically require helicase binding and/or ssDNA binding to promote ATPase activity ( Ioannou et al . , 2006; Learn et al . , 1997; Davey et al . , 2002 ) , a function proposed to directly depend on loader self assembly ( Mott et al . , 2008 ) .",
"Superposition of the 77ORF104-SaDnaI complex with structures of other DnaI orthologs , particularly those that contain a portion of the helicase loaders’ linker regions , reveal that the C-terminal tail of the inhibitor actually occupies a pocket that corresponds to the observed binding site for a portion of the linker itself ( Figure 7A ) .",
"Moreover , inspection of these overlays shows that in associating with its own AAA+ ATPase domain , the inter-domain linker additionally blocks a portion of the surface where subunit-subunit interactions form during loader self-association ( Figure 7A , see inset ) .",
"This observation explains why DnaI ATPase domain constructs containing a portion of the linker crystallized as monomers even in the presence of nucleotide , and indicates that the linker likely serves as a switch that reports on relative status of the N- and C-terminal regions , repressing loader-loader interactions until the correct helicase and DNA targets are bound ( Figure 7B ) .",
"In this regard , the 77ORF104 protein exploits this binding locus , taking advantage of the linker-binding pocket to inactivate the host loader , an activity that would allow the cellular replicative helicase to be re-routed by the phage-encoded loader to promote viral replication ( Figure 7B ) .",
"This interaction , which occurs within a small hydrophobic pocket on the loader AAA+ ATPase domain , suggests that the linker-binding region of the DnaI AAA+ domain could serve as an attractive site for the development of chemical inhibitors to target helicase loading and DNA replication in bacteria .",
"Future efforts will help test and establish these concepts further . 10 . 7554/eLife . 14158 . 012Figure 7 . Insights into bacterial helicase loader mechanism and regulation derived from this study .",
"( A ) The N- and C-terminal domain linker of DnaC/I helicase loaders occupies a region bound by the phage 77 inhibitor protein .",
"The monomeric ATPase domain of G . kaustophilus DnaI ( GkaDnaI AAA+ ) ( PDB ID 2W58 , [Tsai et al . , 2009b] ) is shown superposed onto a nucleotide-assembled dimer of both the A . aeolicus DnaC AAA+ domain ( a construct that lacks the linker , PDB ID 3ECC , [Mott et al . , 2008] ) ) and the SaDnaIAAA+ domain ( as seen in complex with 77ORF104 ) .",
"The C-terminal tail of 77ORF104 forms a β-strand that occupies the same location as the GkaDnaIAAA+ linker ( magenta ) , although the strand runs in the opposite direction .",
"( Inset )",
"Close-up view of the linker/tail binding site .",
"Several amino acids in the linker are shown as space filling ( magenta ) to illustrate how this element would sterically clash with a second , incoming helicase loader ATPase domain to prevent self assembly ( pink spheres , from AqDnaC ) .",
"( B ) Schematic summarizing the auto-regulation of DnaC/I helicase loaders and the ability of phage 77-family viruses to inhibit host helicase loading and co-opt host replicative helicases in S . aureus and related Gram-positive bacteria .",
"The bacterial helicase loader linker region forms an intra-molecular interaction with its associated AAA+ domain to auto-repress inopportune loader/loader interactions; upon binding to the replicative helicase , the linker is proposed to undock from this region to allow self-assembly of the loader ATPase folds and subsequently assist with loading of the replicative helicase onto single-stranded origin DNA .",
"The phage 77 ORF104 inhibitor protein binds to cognate loaders such as SaDnaI , repressing loader self-association , ATPase activity , and presumably its ability to properly participate in the helicase loading reaction .",
"Phage 77 is also found to encode a helicase loader homolog ( ORF013 ) that directly binds to the host replicative helicase , likely as a means to re-direct the host’s replication machinery toward replication of the viral genome . DOI: http://dx . doi . org/10 . 7554/eLife . 14158 . 012"
],
[
"Coding sequences for full-length Staphylococcus aureus DnaI , DnaIAAA+ ( residues 136–306 ) , DnaINTD ( residues 1–117 ) , SaDnaC replicative helicase , and phage 77 ORF104 and ORF013 were cloned into a pET28b ( EMD Millipore , Billerica , MA ) derivative with a tobacco etch virus ( TEV ) protease‐cleavable , N‐terminal hexahistidine-MBP tag and verified by sequencing ( Elim Biopharmaceuticals , Hayward , CA ) .",
"All SaDnaI and phage 77 proteins were expressed in E . coli , using BL21 codon-plus ( DE3 ) RIL cells ( Agilent ) , by inducing at A600= 0 . 4–0 . 5 with 1 mM IPTG at 37°C for 3 hr .",
"Cells were harvested by centrifugation , resuspended in lysis buffer and lysed by sonication .",
"All S . aureus DnaI and phage proteins were purified by Ni2+‐affinity chromatography over a 5‐mL HisTrap HP column ( GE ) .",
"Lysis buffer consisted of 500 mM NaCl , 20 mM imidazole , 50 mM HEPES-KOH pH 7 . 5 , 10 mM MgCl2 , 10% glycerol , and protease inhibitors ( 1 mM phenylmethylsulfonyl fluoride ( PMSF ) , 1 mg/mL Pepstatin A , 1 mg/mL Leupeptin ) .",
"After washing in binding buffer containing 1 M NaCl , proteins were eluted with 300 mM imidazole in elution buffer containing 50 mM NaCl , 50 mM HEPES-KOH pH 7 . 5 , 10 mM MgCl2 , 10% glycerol and protease inhibitors ( 1 mM PMSF , 1 mg/mL Pepstatin A , and 1 mg/mL Leupeptin ) over 5 column volumes .",
"His6-MBP tags were removed with His6-tagged TEV protease ( MacroLab , UC Berkeley ) adding the TEV protease at a ratio of 1:40 TEV:protein and incubating at 4°C overnight .",
"Proteins were then exchanged into binding buffer , repassaged over a HisTrap column , and run over a Sepharose S200 gel filtration column ( GE ) in 500 mM NaCl , 50 mM HEPES-KOH pH 7 . 5 , 10 mM MgCl2 , 10% glycerol , and protease inhibitors ( 1 mM PMSF , 1 mg/mL Pepstatin A , and 1 mg/mL Leupeptin ) .",
"Selenomethionine labeled proteins were purified by the same protocol with the exception that 0 . 5 mM TCEP was added to all buffers .",
"Protein purity was assessed by polyacrylamide gel electrophoresis and Coomassie staining .",
"Proteins were concentrated by centrifugal ultrafiltration ( EMD Millipore Amicon Ultra ) .",
"Concentration was determined by absorption at 280 nm using the following calculated extinction coefficients: 23 , 380 M-1 cm-1 for FL SaDnaI , 72 , 310 M-1 cm-1 for H6MBP-77ORF104 , 14 , 440 M-1 cm-1 for SaDnaIAAA+ ( 136–306 ) , 4470 M-1 cm-1 for 77ORF104 and 7450 M-1 cm-1 for SaDnaINTD ( 1–117 ) , 15 , 930 M-1 cm-1 for SaDnaICTD ( 117–306 ) , 28 , 880 M-1 cm-1 for 77ORF013 , and 27 , 850 M-1 cm-1 for SaDnaC replicative helicase .",
"Mutant Staphylococcus bacteriophage 77ORF104 and S . aureus DnaI proteins were generated using QuickChange ( Agilent ) site‐specific mutagenesis and sequenced ( GeneWiz , Frederick , MD ) .",
"S . aureus DnaI and phage 77ORF104 mutants were purified as described above .",
"His6-MBP tagged SaDnaC was expressed in E . coli , using BL21 codon-plus ( DE3 ) RIL cells ( Agilent ) , by induction at A600 = 0 . 6 with 1 mM IPTG at 37°C for 2 . 5–3 hr .",
"Cells were harvested by centrifugation , resuspended in lysis buffer and lysed by sonication .",
"SaDnaC was purified by Ni2+‐affinity chromatography over a 5‐mL HisTrap HP column ( GE ) .",
"Lysis buffer consisted of 500 mM NaCl , 20 mM imidazole , 50 mM Tris-HCl pH 8 . 0 , 2 mM MgCl2 , 10% glycerol , 0 . 5 mM β-mercaptoethanol and protease inhibitors ( 1 mM PMSF , 1 mg/mL Pepstatin A , 1 mg/mL Leupeptin ) .",
"After washing in binding buffer containing 500 mM NaCl , proteins were eluted with 300 mM imidazole in elution buffer containing 50 mM NaCl , 50 mM Tris-HCl pH 8 . 0 2 mM MgCl2 , 10% glycerol , 0 . 5 mM β-mercaptoethanol and protease inhibitors over 5 column volumes .",
"The elution was then loaded onto a HiTrap Mono Q Sepharose HP Column ( GE Healthcare , United Kingdom ) equilibrated in 150 mM NaCl , 50 mM Tris-HCl pH 8 . 0 , 2 mM MgCl2 , 10% glycerol , 0 . 5 mM β-mercaptoethanol and protease inhibitors and eluted in buffer containing 300 mM NaCl .",
"The His6-MBP tag was removed with His6-tagged TEV protease adding the TEV protease at a ratio of 1:40 TEV:protein and incubating at 4°C overnight .",
"Proteins were then exchanged into binding buffer , repassaged over a HisTrap column , and run over a Sepharose S300 gel filtration column ( GE ) in 800 mM NaCl , 20 mM Tris-HCl pH 8 . 0 , 5 mM MgCl2 , 10% glycerol , 1 mM β-mercaptoethanol , 0 . 01 mM ATP and protease inhibitors .",
"Protein purity was assessed by polyacrylamide gel electrophoresis and Coomassie staining .",
"Proteins were concentrated by centrifugation ( Millipore Amicon Ultra MWCO 30 K ) .",
"Concentration was determined by absorption at 280 nm using a calculated extinction coefficient of 27 , 850 M-1 cm-1 for the SaDnaC replicative helicase .",
"To crystallize the ADP•BeF3-bound SaDnaIAAA+•77ORF104 complex , we expressed and purified both proteins individually , using minimal media containing selenomethionine for SaDnaIAAA+ and 2xYT media for 77ORF104 .",
"Following purification , the SaDnaIAAA+•77ORF104 complex was formed using a two-fold excess of 77ORF104 to SaDnaIAAA+ , and the complex purified by passage over a Sepharose S200 gel filtration column equilibrated in S200 buffer plus 0 . 5 mM TCEP .",
"Following concentration to 6 mg/mL , ADP•BeF3was spiked into the sample to 2 mM final concentration as an ATP mimetic .",
"Crystallization was performed by hanging-drop vapor diffusion by mixing 2 μL of protein complex in S200 sizing buffer with 2 μL well solution ( 50 mM Tris-HCl 8 . 5 , 20 mM MgCl2 , 20% ethanol ) .",
"Large , rod-shaped crystals grew within 1–2 days at 21°C using a protein concentration of 6 . 5 mg/mL .",
"Crystals were cryo-protected by serial exchange into a harvesting buffer containing 25% PEG 400 , 50 mM HEPES 7 . 5 , 500mM NaCl , 5 mM MgCl2 , 10% glycerol , 0 . 5 mM TCEP , and 2 mM ADP•BeF3 before flash freezing in liquid nitrogen .",
"Crystals were stored in liquid nitrogen prior to data collection .",
"Apo SaDnaIAAA+ was expressed and purified as described above with only a minor change made to the S200 sizing buffer ( substituting 500 mM KCl for 500 mM NaCl ) .",
"Sparse matrix screening was performed as described above at a protein concentration of 5 mg/mL .",
"The final crystallization conditions for SaDnaIAAA+ contained a well solution of 0 . 1 M citric acid , 10 mM MgCl2 , and 0 . 8 M ammonium sulfate at pH 4 . 0 .",
"Prior to crystallization , ADP•BeF3 was spiked into the sample to 2 mM final concentration and crystals were grown at 21°C .",
"Crystals were cryo-protected by serial exchange into a harvesting buffer containing 25% glycerol , 0 . 1 M citric acid pH 4 . 0 , 0 . 8 M ammonium sulfate , 50 mM HEPES-KOH pH 7 . 5 , 10 mM MgCl2 , 10% glycerol , and 2 mM ADP•BeF3 before flash freezing in liquid nitrogen .",
"Datasets for both structures were collected at beamline X25 at the National Synchrotron Light Source ( NSLS ) , Brookhaven National Laboratory .",
"A single selenomethionine SAD dataset for the SaDnaIAAA+•77ORF104 complex was collected at a wavelength of 0 . 979 Å and processed in XDS ( Kabsch , 1988 , 2010 ) ( Table 1 ) .",
"One ADP•BeF3-SaDnaIAAA+•77ORF104 complex was found to occupy each asymmetric unit .",
"Selenium positions for the SeMet-SaDnaIAAA+were determined using HYSS ( Hybrid Substructure Search ) as part of the PHENIX package ( Adams et al . , 2010; Grosse-Kunstleve and Adams , 2003 ) .",
"Initial experimental electron density maps were generated from AUTOSOL ( Terwilliger et al . , 2009 ) via phasing by single wavelength anomalous dispersion .",
"Several cycles of model building were performed using COOT ( Emsley and Cowtan , 2004 ) and the PHENIX package was used for refinement ( Adams et al . , 2010 ) .",
"The apo SaDnaIAAA+ dataset was collected at a wavelength of 1 . 5 Å and processed in XDS ( Kabsch , 1988 , 2010 ) .",
"Crystals were determined to belong to the space group P212121 with unit cell dimensions a=113 . 09 Å , b=126 . 26 Å , c=183 . 34 Å , and a solvent content of approximately 52 . 4% ( Table 1 ) .",
"Twelve SaDnaIAAA+ molecules were found in the asymmetric unit .",
"The structure was solved by molecular replacement using MR-PHASER in the PHENIX package ( McCoy et al . , 2007 ) followed by several cycles of model building in COOT ( Emsley and Cowtan , 2004 ) and refinement in PHENIX ( Adams et al . , 2010 ) .",
"The minimal AAA+ core domain of SaDnaI from the complex structure , lacking the ISM , was used as a search model with MR-PHASER .",
"The final , 12-chain model converged at an Rwork/Rfree of 22 . 5/26 . 9% for the resolution range of 48 . 1–2 . 6Å ( Adams et al . , 2010 ) .",
"Each chain contains residues 136–306 SaDnaIAAA+ and a single SO42- ion modeled into the P-loop of the AAA+ ATPase domain .",
"Despite the inclusion of Mg2+ ions and ADP•BeF3 with the protein prior to crystallization , no density for either moiety could be observed , likely because the low pH and high SO4 ion concentration present in the well solution precluded stable binding .",
"The coordinates for the ADP•BeF3-SaDnaIAAA+•77ORF104 complex ( 5HE9 ) and apo SaDnaIAAA+ ( 5HE8 ) structures have been uploaded to the PDB .",
"Both SaDnaI and 77ORF104 proteins were dialyzed overnight into a reaction buffer containing 500 mM KCl , 50 mM HEPES-KOH pH 7 . 5 , 10 mM MgCl2 and 10% glycerol .",
"SaDnaI alone or SaDnaI in the presence of the 77ORF104 protein were incubated on ice for 10 min prior to addition of trypsin .",
"Trypsin protease was added to the reaction resulting in the final concentrations: 5 μM , 10 μM , 20 μM and 80 μM; proteins were then incubated with trypsin for 30 min at 25°C .",
"Reactions were quenched with 200 μM PMSF .",
"After addition of an equal volume of 2X SDS-loading dye , reactions were heated at 95°C for 5 min before running 15 μL of each reaction on a 15% SDS-PAGE gel and staining with Coomassie blue .",
"Amylose pull-downs were performed using His6MBP-tagged proteins and untagged proteins in a total volume of 200 μL .",
"All His6MBP-tagged proteins ( 164 µM ) were dialyzed into binding buffer containing 20 mM HEPES-KOH pH 7 . 5 , 100 mM NaCl , 5 mM MgCl2 , 10% glycerol , and 1 mM DTT at 4°C prior to setting up the protein binding reaction .",
"Pulldowns that included ATP had a final ATP concentration of 2 mM .",
"H6MBP-tagged proteins were incubated with their untagged protein for 10 min at 25°C before the addition of 50 μL of amylose bead slurry , pre-equilibrated in binding buffer .",
"The final reactions contained 10 µM His6MBP-tagged protein and 20 µM untagged protein .",
"After the addition of beads , the binding reaction was incubated for 20 min at 25°C , with gentle mixing every 2 min during the incubation .",
"Amylose binding reactions were spun down at 1500 RPM for 1 min .",
"Amylose beads were then washed twice prior to either elution with maltose elution buffer ( 40 mM maltose , 20 mM HEPES-KOH pH 7 . 5 , 100 mM NaCl , 5 mM MgCl2 , 10% glycerol , and 1 mM DTT ) or the addition of 2X SDS loading dye .",
"Amylose pull-down supernatant , washes , elution and beads samples were run on a 12% SDS-PAGE gel ( a slight excess of total protein was used over the binding capacity of the bead volume , so as to allow for visualization of sample input ) .",
"SDS-PAGE gels were stained for 30 min with SYPRO Orange stain diluted to 1X in 10% glacial acetic acid .",
"Gels were washed with dI-H20 three times before imaging the gels on a Kodak Gel Imager .",
"Purified wild-type 77ORF104 protein was exchanged into amine-labeling buffer ( 50 mM HEPES-KOH pH 7 . 5 , 500 mM KCl , 10% glycerol , 10 mM MgCl2 , 1 mM β-mercaptoethanol ) by centrifugation .",
"The neutral pH favors labeling of the amino terminus of proteins rather than surface lysines ( Sélo et al . , 1996 ) .",
"The protein was then concentrated to a final volume of 500 μL by centrifugation using 500μL VIVASPIN ultrafiltration units ( MWCO 3K , Sartorius ) to a final concentration of 342 μM .",
"AlexaFluor 488 5-SDP ( sulfodicholorphenol ) ester ( Life Technologies ) ( 1 mg ) was dissolved into 20 μL DMSO .",
"The dye ( 20 µL ) was added to the concentrated protein ( 342 μM ) and the reaction incubated at 4°C while wrapped in aluminum foil and rocking for 1 hr .",
"Unreacted dye was quenched by adding 20 μL of 1 M L-lysine in 20 mM Tris-HCl pH 7 . 5 .",
"Reactions were incubated with the quench for 30 min at 25°C .",
"Free dye and quench were separated from dye-protein conjugates using a 10 mL PD-10 desalting column ( GE ) equilibrated in the amine-labeling buffer ( see above ) .",
"The labeled protein was then exchanged into a buffer containing 50 mM HEPES-KOH pH 7 . 5 , 500 mM KCl , 30% glycerol , 10 mM MgCl2 , 1 mM β-mercaptoethanol by centrifugation .",
"The concentrated labeled 77ORF104 protein was aliquoted , snap frozen in liquid nitrogen , and stored at -80°C .",
"Purified proteins were prepared in 2-fold dilutions steps in dilution buffer containing 50 mM HEPES-KOH pH 7 . 5 , 500 mM KCl , 10% glycerol and 10 mM MgCl2 .",
"N-terminally labeled , wild-type 77ORF104 was diluted to 40 nM in a reaction buffer ( 50 mM HEPES-KOH pH 7 . 5 , 10% glycerol , 5 mM MgCl2 , 1 mM DTT ) to make a 2X stock .",
"For serial dilutions , purified proteins were sequentially diluted in 2-fold steps into protein dilution buffer .",
"Proteins were mixed with 10 µL of N-terminally labeled 77ORF104 on ice and incubated for 10 min .",
"The final reaction volume was 20 μL containing the final buffer conditions ( 50 mM HEPES-KOH pH 7 . 5 , 125 mM KCl , 10% glycerol , 5 mM MgCl2 , 1 mM DTT ) .",
"The final concentration of labeled 77ORF104 in each reaction was 20 nM .",
"Anisotropy measurements were recorded using CLARIOStar microplate reader ( BMG LAB TECH ) at 535 nm .",
"All data points are the average of three independent measurements .",
"For the 77ORF104 protein binding assays , data were plotted using GraphPad Prism Version 6 ( GraphPad Prism Software , La Jolla California USA , www . graphpad . com ) and fit by nonlinear regression to the single-site binding equation ( Swillens , 1995 ) .",
"For the 77ORF104 competition experiments , reactions were prepared as previously described except that the reaction mixture contained 20 nM labeled 77ORF104 and 2 μM SaDnaIAAA+ .",
"Wild-type 77ORF104 and protein mutants were serially diluted ( as described above ) in the same dilution buffer and final buffer solution .",
"Assays were performed as described above for the 77ORF104 mutants .",
"Anisotropy measurements were recorded using a CLARIOStar microplate reader ( BMG LAB TECH ) .",
"All data points represent the average of three independent measurements .",
"Error bars represent the standard deviation of three independent measurements .",
"Data points were plotted using GraphPad Prism Version 6 and fit with a simple smooth line for aiding visualization .",
"ATPase assays were performed in 30 µL of reaction buffer containing: 10 nM ( 4500 Ci/mmol ) [γ32P]ATP , 100μM cold ATP , 100 mM KCl , 50 mM HEPES-KOH pH 7 . 5 , 10 mM MgCl2 , 10% glycerol , and M13mp18 ssDNA ( New England Biolabs , Inc . ) added to a final concentration of 80 ng/uL .",
"The final reactions contained 10 µM SaDnaI and 60 µM 77ORF104 .",
"The 77ORF104 or SaDnaI mutant proteins were added to the reactions on ice , after which the tubes were shifted to 37°C for 2 hr . 3 µL was next removed from the reaction and quenched by the addition of 3 µL of 250 mM EDTA pH 8 . 0 and 1% SDS .",
"Quenched reactions were spotted ( 1 µL ) onto thin-layer chromatography sheets coated with polyethyleneimine cellulose ( PEI-Cellulose F; EM Science ) and developed in 0 . 4 M potassium phosphate ( pH 3 . 4 ) for 30 min .",
"[γ32P] ATP and free phosphate [γ32P] migrated differently and were quantitated using a Typhoon FLA 9500 PhosphorImager ( GE ) and ImageJ software ( Schneider et al . , 2012 ) ."
]
] | [
"Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases .",
"How loader activity is appropriately controlled remains unclear .",
"Here , we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of the Staphylococcus aureus replicative helicase loader , DnaI .",
"The viral protein binds to the loader’s AAA+ ATPase domain , allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity .",
"Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins .",
"We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader , which we show binds to the host helicase but not to the inhibitor itself .",
"These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association ."
] | [
"Cells must copy their DNA in order to grow and divide .",
"DNA replication begins when a small region of the DNA double helix is unwound to expose single strands of DNA .",
"A protein called a helicase is then shepherded onto the unwound DNA regions by other proteins known as loaders .",
"Once loaded , the helicase can unwind long stretches of the chromosome in which the DNA is packaged , producing the template required by the replication machinery to duplicate the DNA .",
"This process must be accurately executed to avoid generating errors that could damage the DNA and potentially cause cells to die .",
"DnaI is a helicase loader protein that is found in some types of bacteria .",
"In the disease-causing bacterial species Staphylococcus aureus ( S . aureus ) , an inhibitor protein from a virus that infects the bacteria can interact with DnaI and halt S . aureus DNA replication , leading to cell death .",
"However , it has not been understood how this viral protein controls the activity of the loader molecules .",
"DnaI consists of three regions: one that binds to the helicase , a short 'linker' region , and a third element that harnesses chemical energy ( in the form of a small high-energy molecule called ATP ) to drive the loader’s activity .",
"Using biochemical and structural techniques , Hood and Berger now show that the viral inhibitor protein interacts with the DnaI loader from S . aureus by binding to the loader's ATP-binding region .",
"When the two proteins are bound together , the loader can still bind to its target helicase but it cannot interact with other loader molecules .",
"This defect prevents the loaders from self-assembling into a structure that is required for them to load the replicative helicase .",
"Hood and Berger also found that the region of DnaI targeted by the inhibitor is important for normally ensuring that the loader molecules self-assemble at the correct place and time .",
"A second unexpected discovery was that the virus encodes its own helicase loader , which binds to the bacterial helicase but not to the viral inhibitor protein .",
"The next stage of work will be to determine whether the regions on the helicase loader that are targeted by the inhibitor and that are important for regulating self-assembly can be selectively disrupted by small molecules to interfere with DNA replication in bacteria ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"ecology",
"plant biology"
] | Plant defense phenotypes determine the consequences of volatile emission for individuals and neighbors | elife-04490-v2 | [
[
"In The Origin of Species , Darwin wrote that ‘varieties are species in the process of formation’ ( Darwin , 1876 ) .",
"We now know that Darwin's ‘varieties’ are polymorphic characters resulting from natural genetic variation within species .",
"The geneticist Dobzhansky ( 1973 ) echoed Darwin's view: ‘It is evident that the differences among proteins at the levels of species , genus , family , order , class , and phylum are compounded of elements that vary also among individuals within a species’ .",
"It has also long been known that intraspecific genetic variation in plants can alter ecological community structure; for example , in 1960 , De Wit reported that the reproductive output in mixtures of the grass species Anthoxanthum odoratum and Phleum pratens depended on associations of particular genotypes ( De Wit , 1960; Allard and Adams , 1969 ) .",
"However , efforts to measure the broader consequences for communities are more recent ( Antonovics , 1992; reviewed in Haloin and Strauss , 2008 ) and include the recently described field of community genetics .",
"Community genetics aims to quantify how genetic variation in specific traits affects the structure of ecological communities ( reviewed in Whitham et al . , 2006; Haloin and Strauss , 2008; Whitham et al . , 2012 ) .",
"Genes most likely to have community effects are those with a large effect in the foundation species that structure communities ( Whitham et al . , 2006 ) , and plants are foundation species in almost all communities ( Haloin and Strauss , 2008 ) .",
"Community genetics is a basic level of investigation—meaning it is close to the basic unit acted on by evolution , the gene—within the larger effort to meet what Haloin and Strauss ( 2008 ) refer to as ‘one of the supreme challenges of biology’: understanding how the feedback loops between community ecology and evolution shape biodiversity .",
"The maintenance of biodiversity and the ecosystem services it supports is a matter of increasing concern as the human population grows , and agricultural resource use increases ( Green et al . , 2005 ) .",
"In a meta-analysis of 446 biodiversity measures taken between 1954 and 2004 , of which 312 manipulated plant species biodiversity , species-level biodiversity was shown to positively affect several ecosystem properties including stability , regulation of biodiversity , nutrient cycling , erosion control , and productivity at the primary , secondary , and tertiary levels ( Balvanera et al . , 2006 ) .",
"Functional differences among species are often thought to be the source of effects resulting from species-level biodiversity ( Ebeling et al . , 2014 ) .",
"In fact , a growing number of studies has shown that genetic diversity within species can have large effects on ecological community composition ( e . g . , Crutsinger et al . , 2006; Johnson et al . , 2006; Cook-Patton et al . , 2011 ) .",
"Whitham et al . ( 2012 ) refer to the genetically based tendency for particular genotypes to support different ecological communities as ‘community specificity’ .",
"Some , or even most , biodiversity effects may be explained through the community specificity of particular genetic traits .",
"Four postulates of community genetics provide the roadmap for understanding the community effects of genes ( Wymore et al . , 2011; Whitham et al . , 2012 ) : ( 1 ) the focal species must have a significant effect on its community or ecosystem , ( 2 ) the trait under investigation must be genetically based and heritable , ( 3 ) conspecifics differing genetically in this trait must have quantifiably different effects on community and ecosystem processes , and ( 4 ) a predictable effect on those community and ecosystem processes must follow when the gene of interest is manipulated .",
"As Koch's postulates first allowed medical researchers to identify disease-causing agents , these postulates allow community geneticists to identify genes responsible for structuring specific aspects of ecological communities .",
"So far , although postulates one through three have been satisfied in several ecological systems , postulate four—the critical step in attributing community functions to specific genes—has been tested in only a handful of systems ( Whitham et al . , 2006; 2012 ) .",
"Whitham et al . ( 2012 ) suggest that improved genetic tools will soon permit the widespread testing of postulate four , as has been done for nicotine in the wild tobacco Nicotiana attenuata: an ecological model plant with a transformation system and a well-characterized ecological community ( Krügel et al . , 2002; Steppuhn et al . , 2004; Kessler et al . , 2008; reviewed in; Schuman and Baldwin , 2012 ) .",
"In this study , we built on deep molecular ecological knowledge of N . attenuata to demonstrate that all four postulates for genes controlling a suite of ecologically characterized defense traits ( Kessler and Baldwin , 2001; Kessler et al . , 2004; Allmann and Baldwin , 2010; Schuman et al . , 2012 ) .",
"Specifically , we manipulated direct defenses: traits which directly reduce plant fitness loss to herbivores , and indirect defenses: traits which attract enemies of herbivores which remove or impair herbivores , and thereby indirectly reduce plant fitness loss to herbivores .",
"N . attenuata's direct and indirect defenses , their biosynthesis and ecological functions have been characterized in detail over two decades of research ( reviewed in Schuman and Baldwin , 2012 ) .",
"The direct defenses comprise a suite of chemical traits , most of which are induced by herbivory via jasmonate signaling , and genetically silencing these defenses causes significant changes in herbivore communities ( Kessler et al . , 2004; Gaquerel et al . , 2010; Kallenbach et al . , 2012 ) .",
"N . attenuata also produces a complex blend of herbivore-induced plant volatiles ( HIPVs ) which can be parsed into terpenoids , GLVs and other fatty acid derivatives , and aromatics , and are regulated by elicitors in herbivore oral secretions ( OS ) ( Gaquerel et al . , 2009; Schuman et al . , 2009 ) .",
"Of these , the jasmonate-regulated sesquiterpene ( E ) -α-bergamotene ( TAB ) and the GLVs have been shown to reduce herbivore loads by attracting predators , which for the GLVs has also been shown to result in greater plant fitness ( Halitschke et al . , 2000; Kessler and Baldwin , 2001; Halitschke et al . , 2008; Schuman et al . , 2009; 2012; Allmann and Baldwin , 2010 ) .",
"The majority of N . attenuata's direct and indirect chemical defenses can be abolished by silencing the expression of two biosynthetic genes: LIPOXYGENASE 2 ( LOX2 ) , which provides substrate for GLV biosynthesis; and LOX3 , essential for jasmonate biosynthesis , and thus also for the expression of most direct defense traits and the emission of TAB ( Kessler et al . , 2004; Allmann et al . , 2010 ) .",
"We specifically manipulated the expression of genes controlling direct and indirect defense traits in a factorial design by combining the silencing of LOX2 and LOX3 with the overexpression of Zea mays TERPENE SYNTHASE 10 ( TPS10 ) , which produces TAB and ( E ) -β-farnesene ( TBF ) as its main products ( Köllner et al . , 2009; Schuman et al . , 2014 ) .",
"This allowed us to independently manipulate total direct and indirect defenses , and the specific volatiles , TAB and TBF .",
"We grew these transgenic , otherwise isogenic genotypes during two consecutive field seasons , both in replicated populations of different compositions , and as individuals .",
"Studies of genetic diversity and community specificity most commonly measure the diversity and composition of invertebrate communities on plants , but also invertebrate abundance , and occasionally functional measures such as predation rates ( reviewed in Haloin and Strauss , 2008 ) .",
"We focused on correlates of plant fitness: plant size , apparent health , flowering , and mortality before reproductive maturity; and on functional measures of the effectiveness of plant defense: total canopy damage from herbivores , herbivore abundance , and predation services received by plants in populations .",
"The functional measures were chosen to reflect our observation of plants' ecological communities in both years .",
"Nearly , all of these measures of plant fitness and interactions with herbivores and their predators were significantly affected not only by the genotype of individual plants , but by neighboring genotypes in populations ."
],
[
"Transgenic lines manipulated in the emission of TAB , TBF , and GLVs , and the production of total jasmonate-mediated defenses ( Figure 1 and associated source data files ) were screened and characterized as described in Appendix 1 .",
"To investigate the community-level effects of variation in TAB and TBF in well- and poorly defended plants , we used four genotypes: WT plants , which have jasmonate-mediated induced direct defense compounds and herbivore-induced volatile emission comprising primarily sesquiterpenes and GLVs; TPS10 plants , which are like WT but have both enhanced induced , as well as constitutive emission of the herbivore-induced sesquiterpenes TAB and TBF ( Schuman et al . , 2014 ) ; lox2/3 plants , which are deficient in both jasmonate-mediated direct defense compounds and herbivore-induced volatiles , and lox2/3xTPS10 plants , which are like lox2/3 , but with constitutive emission of TAB and TBF . 10 . 7554/eLife . 04490 . 003Figure 1 . The lox2/3 and TPS10 transgenic constructs , alone and in combination ( lox2/3xTPS10 ) , independently alter herbivore-induced plant defenses and sesquiterpene HIPVs .",
"( A ) The endogenous lipoxygenase genes LOX2 and LOX3 and the Z . mays sesquiterpene synthase gene TPS10 ( TPS10 ) were manipulated in transgenic lines of N . attenuata in order to uncouple the production of two HIPVs—the sesquiterpenes ( E ) -α-bergamotene ( TAB ) and ( E ) -β-farnesene ( TBF ) —from jasmonate-mediated direct defenses and other volatiles .",
"LOX2 and LOX3 provide fatty acid hydroperoxides from 18:2 and 18:3 fatty acids ( 18:2/18:3-OOH ) for the synthesis of green leaf volatiles ( GLVs ) or jasmonic acid ( JA ) and the jasmonate-derived hormones , respectively .",
"GLVs are released in response to herbivore damage , and jasmonates regulate the production of most anti-herbivore defenses , including trypsin protease inhibitors ( TPIs ) , and other HIPVs .",
"LOX2 and LOX3 were transgenically silenced using an inverted repeat ( ir ) construct specifically silencing both ( lox2/3 ) .",
"Jasmonate-regulated volatiles in N . attenuata are mostly sesquiterpenes , of which the most prevalent is TAB , and include trace amounts of the biosynthetically related sesquiterpene TBF .",
"The Z . mays sesquiterpene synthase TPS10 ( Köllner et al . , 2009 ) , which produces TAB and TBF as its main products , was ectopically over-expressed in N . attenuata plants under control of a 35S promoter ( TPS10 ) to uncouple the emission of these volatiles from endogenous jasmonate signaling .",
"( B and C )",
"Plants with lox2/3 and TPS10 constructs , and a cross with both constructs having the transgenes in a hemizygous state ( lox2/3xTPS10 ) had the expected phenotypes both in glasshouse and field experiments .",
"For each compound or group of compounds , the mean WT value was set to 1 ( indicated by dashed lines ) , and all values were divided by the WT mean resulting in mean fold changes ±SEM .",
"A complete description of TPS10 plants including the demonstration of a single transgene insertion and WT levels of non-target metabolites is provided in Schuman et al . ( 2014 ) , and additional information for lox2/3 , TPS10 , and lox2/3xTPS10 plants ( single transgene insertion for lox2/3 , accumulation of target gene transcripts ) is given in Appendix 1 .",
"( B ) Glasshouse-grown plants were treated with wounding and M . sexta OS , and JA , GLVs , TAB and TBF were analyzed at the time of peak accumulation; n = 4 .",
"For lox2/3 and lox2/3xTPS10 , GLVs were not quantifiable ( NQ ) by GC–MS due to inconsistently detected or no detected signals .",
"***p<0 . 001 in Tukey HSD tests following a significant one-way ANOVA ( p<0 . 001 ) of WT , lox2/3 , and lox2/3xTPS10; # plants with the TPS10 construct emit significantly more TAB + TBF ( Holm-Bonferroni-corrected p<0 . 01 in a Wilcoxon rank sum test of WT and lox2/3 vs lox2/3xTPS10 and TPS10 ) ; ns , not significant .",
"TBF was not detectable in lox2/3 or WT samples .",
"For absolute amounts of JA and other phytohormones , GLVs , TAB and TBF measured in glasshouse samples , see Figure 1—source data 1 , 2 .",
"( C ) TPI activity , total detectable GLVs , and total TAB and TBF measured in frozen leaf samples harvested from field-grown plants at the end of experimental season two ( June 28th ) , after plants had accumulated damage from naturally occurring herbivores .",
"TPIs ( n = 11–24 ) were slightly elevated in field samples of TPS10 plants , but TPI activity did not differ from WT plants in glasshouse samples from two independent TPS10 lines including the line used for this study ( Schuman et al . , 2014 ) .",
"Total GLVs , TAB , and TBF were quantified by GC–MS in hexane extracts from the same tissue samples used to measure TPI activity , n = 9–22 ( a few samples had insufficient tissue for the analysis ) .",
"Neither TAB nor TBF could be detected in lox2/3 samples ( NQ ) ; TBF was also not detectable in WT samples .",
"*Corrected p<0 . 05 , ***corrected p<0 . 001 in pairwise Wilcoxon rank sum tests following significant ( corrected p<0 . 05 ) Kruskal–Wallis tests across all genotypes for each category; p-values were corrected for multiple testing using the Holm-Bonferroni method; ns , not significant .",
"For absolute amounts of TPIs , GLVs , TAB and TBF , and non-target volatiles measured in field-collected tissue samples , see Figure 1—source data 3; emission of GLVs , TAB , TBF , and non-target volatiles from field-grown plants is given in Appendix 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 00310 . 7554/eLife . 04490 . 004Figure 1—source data 1 . Absolute values for JA and GLVs shown as fold-changes in Figure 1B in lox2/3 and lox2/3xTPS10 plants , as well as JA-Ile , individual GLVs , and leaf areas for headspace collection ( mean ± SEM ) ; n = 4 . Data are from a single glasshouse experiment .",
"Values for TPS10 ( line 10–3 ) are reported in Schuman et al . ( 2014 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 00410 . 7554/eLife . 04490 . 005Figure 1—source data 2 . Absolute values for TAB and TBF shown as fold-changes in Figure 1B , and leaf areas for headspace collection ( mean ± SEM ) ; n = 4 . Data are from a single glasshouse experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 00510 . 7554/eLife . 04490 . 006Figure 1—source data 3 . Absolute values for analytes shown as fold-changes in Figure 1C ( mean ± SEM ) .",
"Data are from tissue samples of several pooled leaves per plant from field-grown plants , taken at the end of experimental season two ( June 28th ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 006 These genotypes were grown as individuals and in five-plant populations in a field plot in N . attenuata's native habitat ( Figure 2A , Figure 2—figure supplements 1 , 2 ) .",
"A population size of five plants is realistic for natural populations of N . attenuata ( Figure 2—figure supplement 3 ) , particularly before 1995 , after which an invasive brome grass fueled a dramatic increase in fire sizes and consequently N . attenuata population sizes .",
"This choice of population size also allowed us to clearly differentiate between edge and center positions ( Figure 2B ) .",
"Populations were either monocultures , or mixed cultures containing a single TPS10-expressing plant at the center and plants of the same defense- and HIPV level at the edges ( WT around TPS10 , and lox2/3 around lox2/3xTPS10 , Figure 2B ) . 10 . 7554/eLife . 04490 . 007Figure 2 . Field experiments designed to measure genotype-by-population effects of TPS10 volatiles in well-defended ( WT , TPS10 ) or poorly defended ( lox2/3 , lox2/3xTPS10 ) plants . Two similar experiments were conducted in two consecutive field seasons .",
"( A ) Each genotype was planted as individuals and in five-plant populations ( see Figure 2—figure supplement 1 ) .",
"Each individual or population was planted ca .",
"1 . 5 m from the nearest neighboring individual/population , measured perimeter-to-perimeter .",
"Populations consisted of five plants arranged as shown in a 0 . 4 × 0 . 4 m square .",
"( B ) Individual and population types: monocultures and , additionally , mixed cultures were planted in which a TPS10 plant ( TPS10 or lox2/3xTPS10 ) was surrounded by four plants of the same level of jasmonate-mediated defense and GLVs ( WT or lox2/3 ) .",
"Replicates ( n = 12 in season one , 15 in season two of each individual or population type ) were arranged in a blocked design ( see Figure 2—figure supplement 2 ) : blocks consisting of one replicate of each type ( all six randomly-ordered populations followed by all four randomly-ordered individuals ) were staggered such that consecutive blocks were not aligned , and thus populations and individuals were also interspersed .",
"Random order was modified only when necessary to ensure that no two replicate individuals/populations were placed next to each other vertically , horizontally , or diagonally .",
"This planting design reflects typical distributions in native populations of Nicotiana attenuata ( see Figure 2—figure supplement 3 ) , particularly before 1995 , after which an invasive brome grass fueled a dramatic increase in fire sizes and consequently N . attenuata population sizes . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 00710 . 7554/eLife . 04490 . 008Figure 2—figure supplement 1 . Photographs of the field experiment in season one .",
"( A ) Photograph of a closed-headspace volatile trapping showing ca .",
"25% of the experiment on May 7th of season one .",
"( B ) Photograph showing a similar portion of the experiment on May 23rd of season one . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 00810 . 7554/eLife . 04490 . 009Figure 2—figure supplement 2 . Layout in experimental season two , and color codes . R , row of earth between two irrigation lines; not to scale . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 00910 . 7554/eLife . 04490 . 010Figure 2—figure supplement 3 . Example of plant distribution in a native N . attenuata population , photographed in 2004 . Reprinted with permission from Danny Kessler , Copyright 2004 .",
"All rights reserved . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 010 There was no effect of population type ( individual , monoculture , mixed culture ) or plant position ( individual , edge , center ) on genetically engineered levels of GLVs , jasmonate-mediated defense ( trypsin protease inhibitor [TPI] activity served as an indicator ) , TAB and TBF ( Figure 1C , Figure 1—source data 3 and details in Appendix 2 ) , with one exception: trace amounts of TAB were detected in open headspace trappings around young lox2/3 plants at the edges of lox2/3 + lox2/3xTPS10 mixed cultures , but not around lox2/3 individuals or lox2/3 plants at the edges of monocultures ( Figure 3 ) . 10 . 7554/eLife . 04490 . 011Figure 3 . The TPS10 product TAB can be detected in the open headspace around lox2/3 plants in mixed cultures containing a lox2/3xTPS10 plant .",
"( A ) An ‘open headspace’ trapping was conducted during season two ( May 16th ) with rosette-stage individuals and edge plants as shown , using activated charcoal filters shielded from ozone and UV by MnO2-coated copper ozone scrubbers ( black strips in Teflon tubes pointed at plant ) and aluminum foil , respectively .",
"The headspace was sampled during the day , when sesquiterpenes are most abundant ( see Appendix 2 ) .",
"Eluents from all four filters were combined and analyzed using highly sensitive GCxGC-ToF analysis; n = 4 plants .",
"( B ) The TPS10 product trans-α-bergamotene ( TAB ) could be detected in the open headspace of two of four lox2/3 plants at the edges of mixed cultures containing a lox2/3xTPS10 plant , but not in lox2/3 individuals or lox2/3 plants in monocultures .",
"Peak areas were normalized to the internal standard ( IS ) peak .",
"Arrows indicate the plant that was sampled . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 011 Prior to the field experiment , we tested whether TPS10 expression affects defense against a specialist herbivore in a WT or LOX2/3-deficient background via a no-choice assay with larvae of the naturally co-occurring solanaceous specialist Manduca sexta in the glasshouse .",
"M . sexta larvae grew five times as large on lox2/3 or lox2/3xTPS10 plants as they did on WT or TPS10 plants ( Figure 4; Wilcoxon rank sum tests , Holm-Bonferroni-corrected p-values <0 . 001 ) .",
"In contrast , larvae grew to similar sizes on plants that differed only in the TPS10 overexpression construct ( corrected p-values >0 . 2 ) .",
"The progression of M . sexta larval instars was also faster on LOX2/3-deficient plants: all larvae feeding on lox2/3 or lox2/3xTPS10 were in the third instar by day 8 , while 11/17 larvae feeding on WT and 12/15 larvae feeding on TPS10 were still in the second instar ( corrected p<0 . 001 in Fisher's tests for lox2/3 v . WT , lox2/3 v . TPS10 , lox2/3xTPS10 v . WT , and lox2/3xTPS10 v . TPS10 , corrected p=0 . 4440 for WT v . TPS10 , and corrected p=1 for lox2/3 v . lox2/3xTPS10 ) . 10 . 7554/eLife . 04490 . 012Figure 4 . Manduca sexta larvae grow larger on LOX2/3-deficient plants , regardless of TPS10 expression , in a glasshouse experiment . Data are shown as mean ± SEM; n = 15–19 larvae on day 8 and 11–16 larvae on day 12 .",
"One M . sexta neonate per plant ( starting n = 25 larvae ) was placed immediately after hatching on the youngest rosette leaf of an elongated plant .",
"Larvae were weighed at the third and fourth instars ( of 5 total ) , corresponding to days 8 and 12 , after which larvae become mobile between plants .",
"a , b/A , B Different letters indicate significant differences ( corrected p<0 . 001 ) in larval mass on different plant genotypes within each day , in Wilcoxon rank sum tests following significant ( corrected p<0 . 001 ) Kruskal–Wallis tests for each day ( WT vs lox2/3 day 8 , W17 , 19 = 310 , corrected p<0 . 001 , day 12 , W15 , 15 = 217 , corrected p<0 . 001; TPS10 vs lox2/3xTPS10 , day 8 , W15 , 20 = 290 , corrected p<0 . 001 , day 12 , W11 , 14 = 143 , corrected p<0 . 001; WT vs TPS10 day 8 , W17 , 15 = 95 , corrected p=0 . 227 , day 12 , W15 , 11 = 56 , corrected p=0 . 361; lox2/3 vs lox2/3xTPS10 day 8 , W19 , 20 = 249 , corrected p=0 . 201 , day 12 , W16 , 14 = 134 , corrected p=0 . 377 ) .",
"p-values were corrected for multiple testing using the Holm-Bonferroni method . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 012 In the field , total canopy damage from foliar herbivores was assessed on June 9th in season one , and June 2nd in season two .",
"Canopy damage in both seasons was due to both generalist and specialist herbivores from chewing and piercing-sucking feeding guilds .",
"The generalists included noctuid larvae , Trimerotropis spp .",
"grasshoppers , Oecanthus spp .",
"tree crickets , and leaf miner spp .",
"; the specialists included the sphingid larvae M . sexta and Manduca quinquemaculata , flea beetles Epitrix hirtipennis and E . subcrintia , and the mirid Tupiocoris notatus .",
"Canopy damage from herbivores was more than twice as great in season two ( 9–24% total canopy ) as in season one ( 2–9% total canopy ) .",
"In both seasons , lox2/3 and lox2/3xTPS10 plants accumulated twice as much canopy damage as WT and TPS10 plants , regardless of plant position or population type ( Figure 5 and associated source data files ) : there were no significant differences during either season between plants or populations differing only in TPS10 expression , or plants of the same genotype in mono- vs mixed cultures with single TPS10-expressing plants; details of statistical tests are given in Appendix 3 . 10 . 7554/eLife . 04490 . 013Figure 5 . Only LOX2/3 and not TPS10 , plant position , or population type determined total foliar damage from herbivores over two consecutive field seasons . Note differences in y-axis scale .",
"Data were collected on June 9th in season one ( left panels ) , and on June 2nd in season two ( right panels ) from plants at all different positions in the experiment: individual plants ( A–B ) , and plants at the edges ( C–D ) or at the centers ( E–F ) of populations .",
"Black bars denote WT , grey bars TPS10 , red bars lox2/3 , and pink bars lox2/3xTPS10 plants .",
"Total canopy damage reflected the trends in canopy damage caused by individual herbivores ( Figure 5—source data 1 , 2 ) .",
"Total canopy damage in season one was low ( left panels ) , ranging from 5–10% on lox2/3 and lox2/3xTPS10 plants , and only 2–5% on WT and TPS10 plants .",
"Damage levels were about twice as high in season two ( right panels ) , but showed the same relative pattern , with LOX2/3-deficient plans having more damage .",
"Damage levels within a season were similar for plants in different positions ( p>0 . 07 , see Appendix 3 ) .",
"a , b Different letters indicate significant differences ( p<0 . 05 ) between LOX2/3-expressing and LOX2/3-deficient plants ( WT , TPS10 v . lox2/3 , lox2/3xTPS10 ) in minimal ANOVA or linear mixed-effects models on arcsin-transformed data .",
"For individuals and center plants n = 7–13 , and for edge plants n = 16–48 ( up to 4 per population; the blocking effect was accounted for by a random factor in statistical analysis ) ; exact replicate numbers are given in Figure 5—source data 1 , 2 .",
"There were no significant differences in damage between plants differing only in TPS10 expression ( p>0 . 4 ) , plants in mono- vs mixed cultures ( p>0 . 5 ) , or plants of the same genotype at different positions ( individual , edge , or center , p>0 . 07 ) .",
"Statistical models are given in Appendix 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 01310 . 7554/eLife . 04490 . 014Figure 5—source data 1 . Total canopy damage from individual herbivores or groups of herbivores in experimental season one , corresponding to data shown in Figure 5A ( mean ± SEM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 01410 . 7554/eLife . 04490 . 015Figure 5—source data 2 . Total canopy damage from individual herbivores or groups of herbivores in experimental season two , corresponding to data shown in Figure 5B ( mean ± SEM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 015 Damage observed could be attributed to specific herbivores according to typical feeding patterns and the observation of feeding individuals .",
"Individual herbivores generally followed the trend of total herbivore damage , causing greater damage on lox2/3 and lox2/3xTPS10 plants ( Figure 5—source data 1 , 2 ) .",
"The largest and most significant differences ( p-values < 0 . 05 ) were between plants with vs without the lox2/3 silencing construct ( lox2/3 and lox2/3xTPS10 v . WT and TPS10 , statistical comparisons in Figure 5—source data 1 , 2 ) .",
"All significant differences were between plants or populations with vs without the lox2/3 silencing construct , and never between plants or populations differing only in the TPS10 overexpression construct , nor between plants of the same genotype in different population types ( Figure 5 and associated source data files ) .",
"It should be noted that in season one , multiple lepidopteran species were present on plants and their damage could not be clearly distinguished; since these included generalist species as well as Manduca spp .",
", lepidopteran damage in season one also could not be categorized as generalist or specialist damage ( Figure 5—source data 1 ) .",
"In season two , lepidopteran damage not resulting from experimental infestation for predation assays ( not counted toward total canopy damage ) was due to noctuid species ( Figure 5—source data 2 ) .",
"The abundance of arthropods on plant shoots was quantified for a subset of plants on June 26th in season two , at which time T . notatus adults and nymphs and Epitrix spp .",
"adults were the only foliar herbivores observed .",
"Interestingly , total herbivore abundance at the end of the season did not entirely reflect foliar herbivore damage assessed on June 9th ( Figure 5B ) , although a visual assessment of the plants indicated that relative herbivore damage levels still reflected the June 9th data: herbivores were less abundant on plants expressing TPS10 , independently of lox2/3 expression ( Figure 6A and associated source data files ) .",
"Herbivore abundance on WT plants differed depending on plants' location: in a comparison of abundance on individuals vs plants at the edges or centers of monocultures , abundance was highest on individuals and lowest on center plants .",
"In contrast , herbivore abundance on TPS10-expressing plants did not depend on plant location ( Figure 6B , Appendix 3 ) .",
"Foliar herbivore abundance on WT and lox2/3 edge plants was not affected by the presence of a TPS10-expressing neighbor: neither population type ( monoculture or mixed culture ) , nor the interaction of genotype with population type was significant .",
"However for WT and lox2/3 edge plants , herbivore abundance did vary as a function of genotype , with abundance indeed being greater on lox2/3 edge plants ( Figure 6C , Appendix 3 ) , consistent with greater damage to these plants ( Figure 5D ) . 10 . 7554/eLife . 04490 . 016Figure 6 . Foliar herbivore abundance is determined by TPS10 expression , lox2/3 expression , and plant position . Data were collected on June 26th in season two , when herbivores and predators were more abundant ( see Figure 5 ) .",
"Black denotes WT , grey TPS10 , red lox2/3 , and pink lox2/3xTPS10 plants .",
"Counts for individual herbivores and predators are given in Figure 6—source data 1 , 2 .",
"( A ) Herbivore counts on focal plants of each genotype revealed fewer herbivores present on plants expressing TPS10 ( n given in Figure 6—source data 1 ) .",
"a , b Different letters indicate significant differences ( corrected p<0 . 05 ) between genotypes in Tukey contrasts following significant differences in a generalized linear mixed-effects model ( see Appendix 3 ) .",
"( B ) For WT and TPS10 individuals or plants in monocultures ( n given in Figure 6—source data 2 ) , there was a significant effect ( p<0 . 05 ) of plant genotype ( z = −3 . 662 , p=0 . 0003 ) and an interaction of genotype with position ( TPS10 by center vs individual position , z = 2 . 186 , p=0 . 0288 , generalized linear mixed-effects model in Appendix 3 ) .",
"( C ) The presence of a TPS10-expressing neighbor in mixed populations did not significantly affect herbivore abundance on WT or lox2/3 edge plants , but there was a significant difference between WT and lox2/3 genotypes at the edges of populations ( n given in Figure 6—source data 2 , z = 2 . 358 , p=0 . 0183 , generalized linear model in Appendix 3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 01610 . 7554/eLife . 04490 . 017Figure 6—source data 2 . Total and median numbers of herbivores counted on focal plants of different genotypes on June 26th in season two , broken down into different population types and plant locations for WT and TPS10 plants . Replicate numbers of lox2/3 and lox2/3xTPS10 plants were not sufficient for this analysis ( n ≤ 3 per population type and position ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 01710 . 7554/eLife . 04490 . 018Figure 6—source data 1 . Total and median numbers of herbivores and predators counted on focal plants of different genotypes on June 26th in season two . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 018 Thus LOX2 and LOX3 expression increased plants' susceptibility to foliar herbivores , measured as herbivore growth or percentage of plant canopy damaged ( Figures 4 , 5 ) , but the relationship between foliar herbivore abundance and LOX2/LOX3 expression was more complex and dependent on plants' position in populations ( Figure 6 ) .",
"In contrast , herbivore abundance , but not damage or performance , was consistently reduced on TPS10-expressing plants ( Figures 4–6 ) .",
"We monitored plant growth and size , to control for these non-defense traits which could confound the analysis of differences among genotypes , plant positions , and population types , and their interactions with insect communities .",
"Rosette diameter and plant stem height were measured at three times during the main period of vegetative growth in season one ( May 1st–2nd , 8th–11th , and 16th–17th ) , and after plants had transitioned from vegetative growth to reproduction in season two ( June 4th–6th ) , at which time stem diameter was also measured .",
"There were no significant differences in rosette diameter or stem diameter ( p-values >0 . 08 in ANOVAs or linear mixed-effects models with LOX2/3 deficiency and TPS10 expression , plant position , and population type as factors: see statistical approach in Appendix 3 ) .",
"There were few differences in final measurements of plant height .",
"LOX2/3-deficient plants were slightly taller at the final size measurement on May 17th of season one: the stem height of WT plants was 28 . 5 ± 1 . 1 cm , TPS10 plants 27 . 2 ± 1 . 5 cm , lox2/3 plants 35 . 4 ± 1 . 2 cm , and lox2/3xTPS10 plants 32 . 2 ± 1 . 5 cm ( mean ± SEM ) .",
"In season two , final measurements of plant height on June 6th revealed only a minor difference for central plants expressing TPS10 , discussed below .",
"Statistical analyses of plant height are given in Figure 8—source data 1 .",
"We also monitored early flower production by plants as a measure of growth and reproduction rates .",
"To assess the rate of flowering , flowers were counted once shortly after plants first began to flower , and again 1 week later; in season two , plant size and flower production was additionally assessed once immediately prior to quantification of herbivore damage .",
"In season one , in which herbivores damaged <10% of total canopy area ( Figure 5 ) , LOX2/3-deficient plants produced more flowers earlier than WT and TPS10 plants .",
"Shortly after the first plants began to flower in season one ( May 11th ) , more lox2/3 and lox2/3xTPS10 than WT and TPS10 plants were flowering ( 30% v . 14% ) , and this was still true 1 week later , after most plants had flowers ( 95% v . 79%; G-tests of independence , corrected p<0 . 001 on May 11th and 17th; Figure 7A ) .",
"In pairwise comparisons among individual genotypes , there were no significant differences in G-tests of independence between WT and TPS10 ( corrected p-values > 0 . 2 ) or lox2/3 and lox2/3xTPS10 ( corrected p-values >0 . 7 ) .",
"However , more lox2/3 than WT plants were flowering at both timepoints ( May 11th corrected p<0 . 001 , May 17th corrected p=0 . 022 ) , and more lox2/3xTPS10 than TPS10 plants were flowering on May 17th ( corrected p<0 . 001 ) .",
"Both lox2/3 and lox2/3xTPS10 plants also produced more flowers per plant: on May 17th lox2/3 had 6 ( median = mean ) flowers per plant while WT had 4 , and lox2/3xTPS10 had 4/5 ( median/mean ) flowers per plant while TPS10 had 2/3 . 10 . 7554/eLife . 04490 . 019Figure 7 . LOX2/3 deficiency accelerated flowering under low herbivory . Flower production was monitored twice per season: once shortly after plants began to flower and again 1 week later , when most or all plants were flowering .",
"Bars indicate either total percentage of plants of each genotype which were flowering ( left panels ) , or the mean number of flowers per plant ( right panels ) .",
"WT and TPS10 ( black and grey bars ) are shown opposite lox2/3 and lox2/3xTPS10 ( red and pink bars ) .",
"Ratios in bars indicate numbers of flowering/total plants monitored; a few plants were not included in counts which had recently lost their main stem to herbivores and had not yet re-grown .",
"( A ) In season one , when plants suffered less damage from herbivores ( Figure 5 ) , plants with the lox2/3 silencing construct produced more flowers earlier: asterisks indicate pairwise differences between genotypes differing only in the lox2/3 silencing construct .",
"***Corrected p<0 . 001 , **corrected p<0 . 01 , *corrected p<0 . 05 , or no significant difference ( ns ) in G-tests ( percentage flowering ) or in Wilcoxon rank sum tests ( flower number , WT vs lox2/3: May 11th , W120 , 120 = 8582 , p=0 . 0002; May 17th , W120 , 119 = 5043 , p<0 . 0001; TPS10 vs lox2/3xTPS10: May 11th , W84 , 84 = 3801 , p=0 . 2063; May 17th , W84 , 84 = 2520 , p=0 . 0027 ) .",
"( B ) In season two , during which plants received more damage from herbivores ( Figure 5 ) , flowering was monitored at later dates: the first timepoint in ( B ) is comparable to the second timepoint in ( A ) in terms of the proportion of plants flowering and the average number of flowers per plant ( note difference in scale ) .",
"In season two , LOX2/3 deficiency rather decreased early flower numbers in the comparison of WT vs lox2/3 on May 29th , and did not increase flower numbers at either measurement ( WT vs lox2/3: May 29th , W103 , 95 = 3860 . 5 , p=0 . 0091; June 6th , W97 , 69 = 2940 . 5 , p=0 . 3018; TPS10 vs lox2/3xTPS10: May 29th , W87 , 74 = 3646 . 5 , p=0 . 1417; June 6th , W83 , 63 = 2695 , p=0 . 7518 ) .",
"For a complete analysis of the effects of LOX2/3 deficiency and TPS10 expression , plant position , and population type on flowering and plant size , see source data file for Figure 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 019 In season two , when herbivore damage ranged from 10–24% of total canopy ( Figure 5 ) , LOX2/3-deficient plants did not flower earlier ( Figure 7B ) .",
"Plants were in an earlier stage of flowering on May 29th in season two , than on May 17th in season one: both the proportion of flowering plants ( 72% May 29th season two , 87% May 17th season one ) and the mean number of flowers per plant ( 4 on May 29th season two , 5 on May 17th season one ) were lower for the May 29th observation .",
"The differences between plants with or without the lox2/3 silencing construct , which was evident on May 17th in season one , were not observed on May 29th in season two: the proportion of flowering plants was similar for all genotypes ( 68% for lox2/3 and lox2/3xTPS10 v . 75% for WT and TPS10 , G-test of independence , corrected p = 1 ) , as was the average number of flowers per plant: 1/3 ( median/mean ) for lox2/3 , 3/5 for WT , 2/4 for lox2/3xTPS10 , and 2/3 for TPS10 .",
"1 week later , 100% of plants were flowering , and the median number of flowers per plant remained similar regardless of LOX2/3 deficiency ( Figure 7B ) .",
"However , in season two , there were subtler effects on flower production which could be attributed to plant position , LOX2/3 deficiency , and TPS10 expression ( Figure 8 ) .",
"The plant height and flowering data collected immediately prior to the assessment of herbivore damage in season two were analyzed as described in Appendix 3 for effects of LOX2/3 deficiency , TPS10 expression , plant position , and population type ( statistical analysis in the source data file for Figure 8 ) .",
"There were almost no differences in plant height ( Figure 8 , left panels ) , indicating that plant size differed little; the only significant difference was a small negative effect of TPS10 expression for plants in the centers of populations , most pronounced in mixed cultures ( Figure 8E ) .",
"In contrast , flower production of individual plants was strongly decreased by TPS10 expression and only slightly decreased by LOX2/3 deficiency ( Figure 8B ) , but differed little for plants in populations ( Figure 8 , right panels ) , with the exception that the shorter central plants also produced slightly fewer flowers ( Figure 8F ) .",
"Interestingly , for individual plants , there was an interactive effect of TPS10 expression and LOX2/3 deficiency resulting in similar flower production for lox2/3xTPS10 plants and WT plants , although both lox2/3 individuals and TPS10 individuals produced significantly fewer flowers than WT ( Figure 8B , statistical analysis in the source data file for Figure 8 ) . 10 . 7554/eLife . 04490 . 020Figure 8 . TPS10 expression reduced flower production under high herbivory . Plant size as measured by stem length ( left panels ) , and reproduction as measured by flower production ( right panels ) ; n given in Figure 8—source data 1 .",
"Data were collected on June 6th in season two from plants at all different positions in the experiment: individual plants ( A–B ) , and plants at the edges ( C–D ) or at the centers ( E–F ) of populations .",
"Black bars denote WT , grey bars TPS10 , red bars lox2/3 , and pink bars lox2/3xTPS10 plants .",
"Plant height differed little , with only a small negative effect of TPS10 expression for center plants ( E ) , which was more pronounced in mixed- than monocultures , indicating a slight competitive disadvantage for these plants .",
"Individual plants differed much more than plants in populations in terms of flower production , with TPS10-expressing plants at a disadvantage ( B ) ; this effect was greatly reduced in populations . a , b Different letters indicate significant differences ( corrected p<0 . 05 ) in Tukey post-hoc tests on plant genotype following significant effects in a generalized linear model , or generalized linear mixed-effects model ( C–D ) with genotype as a factor; these p-values were corrected using the Holm-Bonferroni correction for multiple testing as the same data were also tested for effects of LOX2/3 deficiency and TPS10 expression ( see Figure 8—source data 1 ) .",
"There was no difference in flower production for edge plants , but a small negative effect of TPS10 expression on flower production in center plants ( F ) corresponding to the slight reduction in height of these plants ( E ) .",
"Statistical models are given in Figure 8—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 02010 . 7554/eLife . 04490 . 021Figure 8—source data 1 . The same statistical approach described in Appendix 3 for Figure 5 was used for plant size and reproduction data from May 17th in season 1 , and June 6th in season two ( the most complete measurements , closest to the date of herbivore damage screens , from each season ) .",
"It should be kept in mind that plants in the season one analysis were at an earlier stage of growth and flowering , for example , had about 10% as many flowers as plants in the season 2 analysis; for an analysis of flowering at similar stages , see Figure 7 .",
"Significant p-values and factors are given in bold , while p-values and factors marked in grey and in bold are marginal , but are required in the minimal model because their removal resulted in poorer ( higher ) AIC values .",
"Other non-significant factors remained in models because they were the least insignificant factor ( if only one factor remained ) , or because they are dependent on other factors which are significant or required . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 021 Overall , these data indicate that accelerated reproduction of LOX2/3-deficient plants in season one was due to a reduced net cost for these plants of LOX2/3-mediated defenses under low herbivore damage , which disappeared under higher herbivore damage in season two ( Figures 7 , 8 and associated source data file ) .",
"In contrast , TPS10 expression had little effect on plant growth and flowering in season one ( Figure 7 , source data file for Figure 8 ) and the effects of TPS10 expression on flowering in season two depended strongly on plant position ( individual , edge or center , Figure 8 and associated source data file ) .",
"This indicates that TPS10 expression did not have large , direct costs for plants in the field , but that the reproductive output of TPS10 plants was sensitive to the presence and identity of neighbor plants .",
"Especially under high herbivory in season two , there were few differences in plant size and these did not correspond to differences in herbivore damage ( Figures 5 , 8 ) .",
"We quantified plant mortality as an unambiguous measure of plant health and fitness ( Figure 9 ) .",
"In our experiments , mortality can be interpreted as a measure of total reproductive potential and thus Darwinian fitness , because we observed mortality of plants before , or during , the peak of their flower production , and ended our experiments when plants would normally produce mature seed .",
"( Production of viable seed is not generally allowed for field-released transgenic plants , and we did not allow our plants to produce seed . ) 10 . 7554/eLife . 04490 . 022Figure 9 . One TPS10-expressing plant per population counteracts a mortality increase in LOX2/3-deficient populations . Bars indicate total percentage mortality for all plants in each population type , and ratios in bars indicate numbers of dead/total plants ( n = 75 ) .",
"WT and TPS10 populations ( left , black , and grey bars ) are compared to the equivalent lox2/3 and lox2/3xTPS10 populations ( right , red , and pink bars ) differing only in the lox2/3 silencing construct .",
"LOX2/3-deficient plants had higher mortality in monocultures , regardless of whether they also expressed TPS10 ( **WT mono v . lox2/3 mono , corrected p<0 . 01; *TPS10 mono v . lox2/3xTPS10 mono , corrected p<0 . 05 in G-tests; p-values were corrected for multiple testing using the Holm-Bonferroni method ) .",
"However , the mortality of plants in mixed lox2/3 + lox2/3xTPS10 populations is similar to that of the plants in mixed WT + TPS10 populations ( ns , not significant ) .",
"There was no significant difference in mortality among mono- and mixed cultures of WT and TPS10 plants ( corrected p>0 . 3 ) , or of lox2/3 and lox2/3xTPS10 plants , although there was a marginal difference between lox2/3 monocultures and lox2/3 + lox2/3xTPS10 mixed cultures ( corrected p=0 . 091 ) .",
"In addition , a health index was assigned to each plant ( discussed in text ) ; this index was correlated with several plant growth and reproduction parameters ( see Figure 9—figure supplement 1 ) .",
"Data were collected on June 29th at the end of experimental season two; in season one , all plants experienced much lower herbivory ( Figure 5 ) and negligible mortality ( 0–5% ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 02210 . 7554/eLife . 04490 . 023Figure 9—figure supplement 1 . The health index assigned to plants is strongly and significantly correlated to several measures of plant size and reproduction . All measures of plant size and reproduction made on June 6th in season two are plotted against simultaneously assigned health indices >1 ( a health index of 1 refers to dead plants ) .",
"Spearman's ρ and the corresponding Holm-Bonferroni corrected p-value are shown for each correlation . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 023 In season one , mean plant mortality was only 1% , or a total of 6 plants , by the end of experiments in mid-June .",
"However , in season two , greater damage from herbivores ( Figure 5 ) was associated with much higher mean plant mortality: 46% , or 236 plants by the end of experiments on June 29th .",
"Overall , LOX2/3-deficient plants had higher mortality: 55% for lox2/3 and lox2/3xTPS10 vs 38% for WT and TPS10 plants ( G-test of independence , corrected p = 0 . 002 ) .",
"Thus , lox2/3 plants in monocultures had significantly higher mortality than WT plants in monocultures ( corrected p=0 . 003 ) , and lox2/3xTPS10 plants in monocultures had significantly higher mortality than TPS10 plants in monocultures ( corrected p=0 . 022 ) ( Figure 9 ) .",
"In contrast , mortality rates for plants in mixed cultures were similar regardless of LOX2/3 deficiency ( corrected p=0 . 870 ) .",
"This was associated with marginally decreased mortality for plants in lox2/3 + lox2/3xTPS10 mixed cultures ( Figure 9 , lox2/3 mono v . lox2/3 + lox2/3xTPS10 mix , corrected p=0 . 092 ) .",
"Interestingly , mortality in WT + TPS10 mixed cultures was slightly higher than in monocultures ( 45% v . 37% for WT monocultures and 31% for TPS monocultures ) , although these differences were also not significant ( corrected p-values >0 . 090 ) .",
"In summary , because plants in lox2/3xTPS10 monocultures had higher mortality than those in TPS10 monocultures , we conclude that expression of TPS10 by LOX2/3-deficient plants did not compensate for the absence of LOX2/3-mediated defenses; yet surprisingly , single lox2/3xTPS10 plants in lox2/3 + lox2/3xTPS10 mixed populations reduced the mortality of the entire population to the same level as in WT + TPS10 mixed populations .",
"Plant health , in terms of turgor , expanded leaf area and senescence , varied noticeably under the higher levels of herbivore damage in season two ( Figure 5 ) , and so plants were given an apparent health rating on a scale of one ( dead ) or two ( low ) to five ( high ) .",
"This scale was sufficiently objective that several observers were able to independently assign the same health index number to randomly-chosen plants , after training on fewer than 10 other plants .",
"Ratings >1 on this health scale ( living plants ) were strongly correlated to several growth and reproduction traits measured ( Spearman's ρ values ≥0 . 55 , corrected p-values <0 . 001 ) : number of buds , stem diameter , number of side branches , rosette diameter , stem length , and number of flowers ( Figure 9—figure supplement 1 ) .",
"LOX2/3 deficiency was associated with significantly poorer apparent health on this scale: excluding dead plants , a median health index of 3 for lox2/3 and lox2/3xTPS10 vs 3 . 5 for WT and TPS10 ( Wilcoxon rank sum test , W153 , 186 = 19 , 057 . 5 , corrected p<0 . 001 ) .",
"In pairwise tests between genotypes for surviving plants , WT appeared significantly healthier than lox2/3 , but not lox2/3xTPS10 ( WT v . lox2/3 , W102 , 82 = 2722 , corrected p<0 . 001; WT v . lox2/3xTPS10 , W102 , 71 = 2687 , corrected p=0 . 059 ) ; whereas TPS10 appeared significantly healthier than both lox2/3 and lox2/3xTPS10 ( TPS10 v . lox2/3 , W84 , 82 = 1996 . 5 , corrected p<0 . 001; TPS10 v . lox2/3xTPS10 , W84 , 71 = 1995 , corrected p=0 . 006 ) .",
"There was no difference in the apparent health of WT vs TPS10 or lox2/3 vs lox2/3xTPS10 ( corrected p-values=1 , TPS10 v . WT , W84 , 102 = 4619; lox2/3 v . lox2/3xTPS10 , W82 , 71 = 2604 . 5 ) .",
"Separate examination of surviving individual , center , and edge plants revealed that LOX2/3 deficiency was only associated with poorer apparent health in edge plants ( lox2/3 and lox2/3xTPS10 v . WT and TPS10: individuals , W21 , 22 = 230 . 5 , corrected p=1; edge plants , W102 , 129 = 9544 , corrected p<0 . 001; center plants , W30 , 35 = 598 . 5 , corrected p=1 ) .",
"There were significant differences in the apparent health of edge plants in different population types: WT edge plants appeared significantly healthier than lox2/3 plants at the edges of monocultures , but not healthier than lox2/3 plants at the edges of lox2/3 + lox2/3xTPS10 mixed cultures ( WT mono v . lox2/3 mono , W42 , 29 = 929 , corrected p = 0 . 003; WT + TPS10 mix v . lox2/3 mono , W40 , 29 = 891 . 5 , corrected p=0 . 003; WT mono v . lox2/3+lox2/3xTPS10 mix , W42 , 34 = 953 , corrected p=0 . 164; WT + TPS10 mix v . lox2/3 + lox2/3xTPS10 mix , W40 , 34 = 892 , corrected p=0 . 249 ) .",
"However , TPS10 plants at the edges of monocultures appeared significantly healthier than lox2/3 or lox2/3xTPS10 edge plants in mixed- or monocultures ( TPS10 mono v . lox2/3 mono , W47 , 29 = 1129 . 5 , corrected p<0 . 001; TPS10 mono v . lox2/3 + lox2/3xTPS10 mix , W47 , 34 = 1209 . 5 , corrected p=0 . 001; TPS10 mono v . lox2/3xTPS10 mono , W47 , 39 = 1389 . 5 , corrected p<0 . 001 ) .",
"No other comparisons between plants of individual population types were significant ( corrected p-values >0 . 08 ) and there was no significant association between apparent health and plant position ( Kruskal–Wallis test across individual , edge , and center plants , χ22 = 2 . 38 , corrected p=1 ) or plants of the same genotype in mono- vs mixed cultures ( Wilcoxon rank sum tests , corrected p-values=1 ) .",
"In summary , as for foliar herbivore damage , growth , flowering , and mortality , there was no significant difference in the apparent health of plants differing only in TPS10 expression .",
"However , TPS10 expression in lox2/3xTPS10 plants was associated with an apparent health similar to WT plants , while lox2/3 plants had a significantly lower apparent health than WT plants .",
"Furthermore , the differences in apparent health were stronger between TPS10 plants and LOX2/3-deficient plants than between WT plants and LOX2/3-deficient plants , indicating a weak , positive effect of TPS10 expression on apparent plant health .",
"Interestingly , lox2/3 plants at the edges of monocultures appeared less healthy than WT edge plants , but lox2/3 plants at the edges of lox2/3 + lox2/3xTPS10 mixed cultures did not , indicating a positive neighbor effect of TPS10 expression on the health of plants in LOX2/3-deficient populations .",
"At the end of season two , we found that 35% of all surviving plants were infested with T . mucorea larvae .",
"An adult T . mucorea is pictured in Figure 10A .",
"Interestingly , TPS10 expression was associated with slightly higher infestation rates: 29% of lox2/3 and 34% of WT plants vs 39% of TPS10 and lox2/3xTPS10 plants .",
"Looking at infestation rates by population type , we discovered a small difference between WT ( 34% infested ) and TPS10 plants ( 39% infested ) which masked a much larger effect: WT plants in WT + TPS10 mixed populations ( 55% infested ) had an infestation rate more than twice that of WT plants in monocultures ( 19% infested , corrected p=0 . 015 , Figure 10B ) .",
"We observed the same tendency in lox2/3 and lox2/3xTPS10 populations: 33% of lox2/3 plants in mixed populations with lox2/3xTPS10 plants were infested , vs only 16% of lox2/3 plants in monocultures; interestingly , lox2/3xTPS10 plants tended to have higher infestation rates than lox2/3 plants in populations ( Figure 10C ) .",
"Likely due to higher plant mortality in lox2/3 populations ( Figure 9 ) , replicate numbers were too low for these effects to be significant ( corrected p-values =1 for the comparison of infestation rates of lox2/3 plants in mono- vs mixed culture , and of lox2/3 vs lox2/3xTPS10 monocultures ) .",
"Likely also due to low replicate numbers ( n = 7–10 ) , there were no significant differences in infestation among individuals ( corrected p-values = 1 in pairwise tests ) , but 30% of TPS10 , 38% of WT , 50% of lox2/3 , and 57% of lox2/3xTPS10 individuals were infested .",
"Based on these trends , had there been a significant effect for individuals , it would have been due to LOX2/3 deficiency and not TPS10 expression . 10 . 7554/eLife . 04490 . 024Figure 10 . TPS10 plants increase the infestation rates of their WT neighbors with the stem-boring weevil T . mucorea .",
"( A ) Photograph of a T . mucorea adult emerging from the stem which it infested as a larva reprinted with permission from Anke Steppuhn , Copyright 2005 .",
"All rights reserved .",
"Adults mate and oviposit on young N . attenuata plants in March and April , and upon hatching , larvae burrow into the growing stem of the plant chosen by their mother ( Diezel et al . , 2011 ) .",
"Infestation was scored as the number of plants with hollow , frass-filled stems ( from which larvae were usually also recovered ) .",
"( B ) T . mucorea infestation more than doubled for WT plants , but did not change for TPS10 plants in mixed cultures vs monocultures .",
"Bars indicate total percentage of plants infested; ratios in bars indicate numbers of infested/total plants .",
"Data were collected at the end of experimental season two ( June 29th ) .",
"*Corrected p<0 . 05 in a Fisher's exact test , n = 31–47 ( all surviving replicates ) .",
"p-values were corrected for multiple testing using the Holm-Bonferroni method .",
"( C ) The presence of lox2/3xTPS10 plants also tended to increase T . mucorea infestation in lox2/3 + lox2/3xTPS10 populations but , likely due to lower replicate numbers caused by higher mortality for plants in lox2/3 and lox2/3xTPS10 monocultures ( Figure 9 ) , differences are not significant ( corrected p-values=1 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 024 Thus , although TPS10 expression did not significantly alter plants' rates of infestation by T . mucorea , it did significantly increase the infestation of neighboring plants .",
"Generalist Geocoris spp .",
"predators co-occur with N . attenuata plants , and prey on their herbivores in response to specific HIPVs including GLVs and TAB , in an example of indirect defense ( Kessler and Baldwin , 2001; Allmann and Baldwin , 2010; Schuman et al . , 2012; 2013 ) .",
"Near the end of experiments in season two , Geocoris pallens were abundant and Geocoris punctipes were also present at the field site .",
"We considered that plants in populations with TPS10-expressing neighbors might either benefit from enhanced attraction of Geocoris spp .",
", or suffer in competition for the predation services of Geocoris spp .",
"We also reasoned that the lack of all other HIPVs in LOX2/3-deficient plants could alter the strength and direction of such a relationship .",
"To test the overarching hypothesis that TPS10-expressing plants in populations alter the distribution of predation by Geocoris spp .",
", we conducted predation activity assays from June 21st to June 27th on edge and center plants that were similar in size and apparent health .",
"We treated LOX2/3-expressing ( WT , TPS10 ) and LOX2/3-deficient ( lox2/3 , lox2/3xTPS10 ) populations as two separate experimental groups because it was not possible to match plants across these two groups for apparent health or replicate numbers ( both were lower for lox2/3 and lox2/3xTPS10 populations ) .",
"Selected lox2/3 and lox2/3xTPS10 populations had a mode of 3 remaining plants , range 2–5; and selected WT and TPS10 populations had a mode of 5 remaining plants , range 3–5; within these two groups , there was no significant difference between mono- and mixed cultures in number of plants remaining ( G-tests , corrected p>0 . 1 ) .",
"Plants were baited with M . sexta eggs and larvae ( Figure 11A ) , and we counted the numbers predated by Geocoris spp .",
"over 24 hr ( source data file for Figure 11 , Kessler and Baldwin , 2001 ) . 10 . 7554/eLife . 04490 . 025Figure 11 . TPS10 and LOX2/3 interact to alter the predation of M . sexta by Geocoris spp from edge vs center plants .",
"( A ) Photograph of a M . sexta egg and first-instar larva , and G . pallens adult on an N . attenuata leaf reprinted with permission from Danny Kessler , Copyright 2006 .",
"All rights reserved .",
"As shown here , one first-instar M . sexta larva and one egg were placed on a lower stem leaf in a standardized position on healthy , size-matched plants ( one leaf/plant ) , and their predation was monitored for 24 hr .",
"Data are total numbers from five consecutive trials conducted between June 21st and June 27th at the end of experimental season two , when Geocoris spp .",
"and herbivores were more abundant ( see Figure 5 ) .",
"( B and C )",
"Predation rates of M . sexta are higher on lox2/3xTPS10 than lox2/3 plants at the edges of monocultures ( B ) , but lower on TPS10 than WT plants at the centers of monocultures ( C ) .",
"Bars indicate the total percentage of M . sexta eggs and larvae predated per population; numbers of predated/total eggs and larvae are given inside bars .",
"The same trends were observed in both egg and larva predation; separate numbers of larvae and eggs predated are given in Figure 11—source data 1 .",
"Plants of the WT background and plants with the lox2/3 silencing construct were separately matched for parallel experiments and thus analyzed separately .",
"( B ) Predation of herbivores from edge plants was similar ( not significant , ns ) regardless of the number of TPS10 plants in WT + TPS10 populations , but increased with increasing numbers of lox2/3xTPS10 plants in lox2/3 + lox2/3xTPS10 populations .",
"For plants at the center of populations , the pattern was reversed: ( C ) predation rates decreased with increasing numbers of TPS10 plants in WT + TPS10 populations , and were similar ( ns ) regardless of the number of lox2/3xTPS10 plants in lox2/3 + lox2/3xTPS10 populations .",
"a , b Different letters indicate significant differences ( corrected p<0 . 05 ) in Fisher's exact tests; p-values were corrected for multiple testing using the Holm-Bonferroni method . DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 02510 . 7554/eLife . 04490 . 026Figure 11—source data 1 . Total numbers of M . sexta larvae and eggs predated by Geocoris spp .",
"in experimental season two ( corresponding to data shown in Figure 10 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04490 . 026 TPS10-expressing plants significantly altered predation rates at the edges and centers of populations , and whether the effect was at the edge or the center depended on LOX2/3 expression ( Figure 11B , C ) .",
"Specifically , TPS10 expression increased predation from edge plants in lox2/3 and lox2/3xTPS10 populations ( Fisher's exact tests , corrected p-values<0 . 05 ) but not in WT and TPS10 populations ( 21–34% , corrected p-values >0 . 3 ) ( Figure 11B ) .",
"At the edges of monocultures , lox2/3xTPS10 plants received 43% predation whereas lox2/3 plants received only 16% ( corrected p=0 . 049 ) , and lox2/3 plants at the edges of lox2/3 + lox2/3xTPS10 mixed cultures were intermediate with 23% ( lox2/3 mono v . lox2/3 + lox2/3xTPS10 mixed , corrected p=0 . 723 , lox2/3xTPS10 mono v . lox2/3 + lox2/3xTPS10 mixed , corrected p=0 . 457 ) .",
"On the other hand , TPS10 expression decreased , rather than increased predation from center plants in WT + TPS10 populations ( from 46% to 12% , corrected p<0 . 04 ) and had no effect on center plants in LOX2/3-deficient populations ( ranging from 13% to 23% , corrected p-values=1 , Figure 11C ) .",
"WT plants received 46% predation at the center of monocultures , while TPS10 plants received 38% predation at the center of mixed cultures and only 12% at the center of monocultures ( WT mono v . WT + TPS10 mixed , corrected p=0 . 618; WT mono v . TPS10 mono , corrected p=0 . 012; WT + TPS10 mixed v . TPS10 mono , corrected p=0 . 031 ) .",
"Thus in populations of lox2/3 and lox2/3xTPS10 plants deficient in all HIPVs except for the engineered TPS10 volatiles , TPS10 expression by edge plants increased predator activity on those plants .",
"In contrast , TPS10 expression by center plants failed to attract more predator activity to the centers of populations .",
"In populations of WT and TPS10 plants with intact WT HIPVs , TPS10 expression did not increase predator activity at all; however , TPS10 expression by edge plants seemed to reduce the penetration of predators to center plants .",
"Thus , depending the richness of the plants' HIPV blends , TPS10 expression had either positive direct effects on predator activity for emitting plants , or negative indirect effects for their neighbors ."
],
[
"It is well known that jasmonate-mediated defenses can be costly in the absence of herbivores ( reviewed in e . g . , Steppuhn and Baldwin , 2008 ) , and under low levels of damage from herbivores in season one ( Figure 5A ) , growth and early flower production was greater in lox2/3 and lox2/3xTPS10 than in WT and TPS10 plants ( Figures 7 , 8 and associated source data file ) .",
"These data indicate that our measurements were sufficiently sensitive to detect developmental costs of defense metabolite production .",
"Attempts to engineer the emission of terpene volatiles have sometimes resulted in plants with stunted growth or symptoms of autotoxicity ( Robert et al . , 2013; reviewed in Dudareva and Pichersky , 2008; Dudareva et al . , 2013 ) .",
"However , we did not detect any consistent costs of TPS10 overexpression: TPS10 plants were generally not smaller than WT ( Figure 8 and associated source data file ) .",
"The same line of TPS10 also grew similarly to WT in a greenhouse competition study ( line 10–3 in Schuman et al . , 2014 ) .",
"However , there were effects of TPS10 expression on flowering which differed depending on plants' position in different populations .",
"In particular , under high herbivory in season two , TPS10 expression strongly decreased flower production for well-defended individual plants , but promoted flower production in LOX2/3-deficient individuals ( Figure 8B ) .",
"In contrast , flower production was not affected by TPS10 expression for edge plants in populations , and was slightly reduced for TPS10-expressing plants in the centers of populations ( Figure 8D , F ) .",
"For central plants , in contrast to individuals , the reduction in flower production due to TPS10 expression was similar both for LOX2/3-expressing and LOX2/3-deficient plants .",
"Overall , these data indicate that TPS10 expression may have altered the relative attractiveness or apparency of TPS10 plants , with results depending strongly on plants' neighbors .",
"The potential mechanisms in terms of herbivore abundance and predation rates , and consequences in terms of health and mortality , are discussed further below .",
"Higher levels of damage from foliar herbivores in season two ( Figure 5B ) were likely the main factor which shifted LOX2/3-mediated defenses from costly , in terms of early flower production ( Figure 7 ) , to beneficial , in terms of reduced plant mortality before and during the reproductive phase , and increased apparent plant health ( Figure 9 and Figure 9—figure supplement 1 ) .",
"Overall , there were few differences in growth and size among genotypes ( Figure 8 and associated source data file , and results reported in text ) , making it reasonable to attribute changes in herbivore damage , mortality , and other interactions to the manipulated chemical traits , rather than plant apparency or attractiveness based on size and development .",
"LOX2/3 deficiency significantly increased damage from foliar herbivores and growth of the specialist M . sexta , whereas TPS10 expression had no effect on either interaction ( Figures 6 , 7 ) .",
"There was , furthermore , no effect of plant population type ( mixed or monoculture ) on damage from foliar herbivores in field experiments ( Figure 5 and associated source data files ) .",
"Together , these data indicate that feeding of M . sexta larvae and other naturally occurring foliar herbivores are not directly affected by TPS10 volatiles .",
"However , TPS10 volatiles may have altered the attraction of herbivores to plants: TPS10-expressing plants had consistently smaller populations of foliar herbivores by the end of season two , despite the fact that these plants tended to look healthier , and independently of LOX2/3 expression ( Figure 6 ) .",
"In contrast , ovipositing T . mucorea mothers may be attracted to TPS10 volatiles ( Figure 10 ) .",
"Interestingly , M . sexta moths laid fewer eggs on plants for which the headspace had been supplemented with TAB ( Kessler and Baldwin , 2001 ) .",
"A direct effect on colonizing herbivores and ovipositing females may result in indirect effects in terms of associational resistance or associational susceptibility for neighboring plants ( Kessler and Baldwin , 2001; Barbosa et al . , 2009 ) .",
"Although such indirect effects were not detected in foliar herbivore abundance on neighboring WT and lox2/3 plants ( Figure 6C ) , TPS10 expression did significantly affect neighbors' mortality ( Figure 9 ) , apparent health , infestation by T . mucorea larvae ( Figure 10 ) , and predation services ( Figure 11 ) .",
"The most straightforward indirect effects of TPS10 expression were evident in open headspace trappings ( Figure 3 ) : the presence of a lox2/3xTPS10 plant in the center of a population was associated with TAB in the headspace of neighboring lox2/3 plants , and TAB was not found in the open headspaces of lox2/3 individuals or monocultures .",
"The most parsimonious explanations are diffusion of volatiles into neighbors' headspace , or adherence and re-release of volatiles from the surface of neighbor plants , as has been shown in birch ( Betula spp . ) by Himanen et al . ( 2010 ) .",
"Because no TAB was detectable in extracts from the leaves of lox2/3 plants even when plants were much larger and more likely to have direct contact with TPS10 neighbors , it is unlikely that the TAB detected in open trappings of lox2/3 headspaces came from the stimulation of TAB biosynthesis in lox2/3 ( Figure 1C , Figure 1—source data 3 ) .",
"Indirect effects of TPS10 volatiles on neighboring plants may be attributed to the volatile compounds themselves , because we found no evidence that TPS10 volatiles affected the defenses of emitting plants , or of neighbor plants in populations ( Appendices 1 , 2 , Figure 1 and associated source data files ) ; and TPS10 plants also did not alter the defense response of neighbors in a glasshouse competition experiment ( Schuman et al . , 2014 ) .",
"In contrast , due to the large suite of defenses regulated by LOX2 and LOX3 products , it would have been more difficult to determine the proximate cause of neighbor effects resulting from manipulation of LOX2 and LOX3 expression .",
"Although several components of plant HIPV blends have been shown to prime the direct and indirect defenses of neighbors in different systems ( reviewed in Baldwin et al . , 2006; Arimura et al . , 2010; see Heil and Kost , 2006 ) , GLVs may often be the active cues ( Engelberth et al . , 2004; Kessler et al . , 2006; Frost et al . , 2008 ) .",
"We did not mix GLV-deficient with GLV-emitting plants in our experimental design , but we can conclude that TPS10 volatiles had no detectable direct effects on neighbor plants in our set-up .",
"We observed multiple significant indirect effects of TPS10 expression in populations , as mentioned above; and two of these were strongest in mixed cultures containing only one TPS10-expressing plant .",
"The mortality of lox2/3 and lox2/3xTPS10 plants in monocultures was significantly higher than that of WT and TPS10 plants in monocultures , but the mortality of lox2/3 + lox2/3xTPS10 plants in mixed cultures was not higher than that of WT + TPS10 plants in mixed cultures ( Figure 9 ) .",
"Thus , a single TPS10 emitter in populations of otherwise defenseless plants reduced overall mortality to levels comparable to well-defended populations containing single TPS10 emitters .",
"This apparent protective effect of TPS10 volatiles was weaker for monocultures of lox2/3xTPS10 plants .",
"This may be because TPS10 volatile emission makes plants more apparent to herbivores like T . mucorea as well as beneficial insects like Geocoris spp .",
"( Figures 9 , 10 ) , and if plants are otherwise poorly defended , greater apparency may be dangerous ( Feeny , 1976 ) .",
"Interestingly , although there were no statistically significant differences in mortality among populations of WT and TPS10 plants , mortality was lowest in TPS10 monocultures; and WT + TPS10 mixed cultures had mortality rates 1 . 5-fold that of TPS10 monocultures ( Figure 9 ) .",
"This indicates that well-defended TPS10 plants may have benefited directly from emitting TPS10 volatiles , and may have additionally increased the mortality of non-emitting neighbors .",
"If so , TPS10 volatiles may increase plants' competitive ability by increasing neighbor mortality .",
"Indeed , TPS10 plants had a dramatic effect on the infestation of their WT neighbors by T . mucorea weevils ( Figure 10 ) .",
"TPS10 volatiles seemed only to affect the infestation rates of plant populations; for individual plants , jasmonate-mediated defense is likely more important ( Diezel et al . , 2011 , and see ‘Results’ ) .",
"Infestation patterns in populations ( Figure",
"10 ) strongly indicate that ovipositing T . mucorea adults seek populations of plants based on their volatile emission and then oviposit on all plants in the population .",
"Infestation rates did tend to be higher for lox2/3xTPS10 plants than for lox2/3 plants in populations ( Figure 10C ) , indicating that the absence of other volatiles such as GLVs may reduce plants' apparency to ovipositing T . mucorea .",
"It is interesting that the TPS10-mediated increase in T . mucorea infestation was greater for WT plants in mixed WT + TPS10 populations than for TPS10 plants in monocultures ( Figure 10 ) : though TPS10 monocultures tended to have higher infestation rates than WT monocultures , this difference was not statistically significant , whereas the difference between WT plants in monocultures and WT plants in mixed cultures was both larger and significant .",
"It is tempting to speculate that T . mucorea may be attracted to lower relative abundance of TPS10 volatiles , and deterred by higher relative abundance of these volatiles in the total mixture of the plant headspace .",
"If true , this could provide a mechanism by which TPS10 emitting plants could inflict an indirect cost of emission on non-emitting neighbors , which would be always slightly higher than their own cost of emission .",
"Furthermore , if attraction or deterrence depended on the ratio of TPS10 volatiles to other plant volatiles , the effects might differ for TPS10 and lox2/3xTPS10 plants ( Bruce et al . , 2005 ) .",
"The infliction of such an indirect cost on neighbors is reminiscent of extortionist strategies in the iterated prisoner's dilemma of evolutionary game theory: extortionist strategies lock a player's payoff to its opponent's payoff plus some positive number , thereby ensuring that the extortionist always benefits compared to its opponent ( Press and Dyson , 2012 ) .",
"Interestingly , such strategies have been shown in simulations not to be evolutionarily stable , but to catalyze the evolution of cooperation in populations ( Hilbe et al . , 2013 ) .",
"Of course it is possible that the manipulation of LOX2 and LOX3 expression within populations would also reveal such extortionist-like effects , for example if T . mucorea preferred to oviposit in TPS10 plants over lox2/3xTPS10 plants in the same population , which would support the hypothesis that T . mucorea preference reflects the ratio between TPS10 volatiles and other HIPVs .",
"Predation rates of M . sexta herbivores from edge and center plants in populations ( Figure",
"11 ) indicate that populations' total HIPV emissions can alter the roles of specific volatiles for indirect defense .",
"In lox2/3 + lox2/3xTPS10 populations , deficient in all HIPVs except those provided by TPS10 expression , lox2/3xTPS10 edge plants of monocultures experienced higher predation rates , whereas predation rates were similar for center lox2/3xTPS10 and lox2/3 plants in mono- and mixed cultures .",
"For TPS10 and WT plants , effects on predation rates were exactly the opposite .",
"There was significantly less predation of M . sexta from TPS10 center plants in monocultures vs mixed cultures , but TPS10 edge plants had predation rates similar to WT .",
"Together , these results indicate that the effect of TPS10 volatiles on indirect defense was positive for lox2/3xTPS10 plants , but negative for TPS10 plants , and only significant for plants in monocultures .",
"In mixed cultures , TPS10 volatiles had no effect on the predation of M . sexta herbivores from emitting plants or their neighbors .",
"It should be noted that due to higher mortality in lox2/3 + lox2/3xTPS10 populations , center plants were sometimes more exposed in these populations as a result of edge plants dying .",
"However , the fact that TPS10 volatiles benefitted only edge plants in these populations indicate that Geocoris spp .",
"predators nevertheless distinguished between edge and center plants .",
"We conclude that TPS10 expression had different consequences for plants' indirect defense , depending on whether one or all plants in a population expressed TPS10 , and whether those plants also emitted other HIPVs .",
"If only one plant in the population expressed TPS10 , or if other HIPVs were also emitted in the population , TPS10 expression did not enhance indirect defense and was sometimes detrimental to emitters .",
"The attraction of most , or all , predators and parasitoids to HIPVs depends on learned associations between HIPVs and prey: naïve predators are just as likely to respond as they are not to respond ( Allison and Hare , 2009 ) .",
"Because LOX2/3-deficient plants were heavily attacked by herbivores ( Figure 5 ) , predators could associate TPS10 volatiles emitted by lox2/3xTPS10 plants with herbivore damage ( though damage did not necessarily reflect abundance—see Figure 6 ) .",
"Because lox2/3 and lox2/3xTPS10 plants had reduced emissions of other HIPVs ( Figure 1 and associated source data files , Appendix 2 ) , there were few other volatiles which could provide the basis for learned associations between HIPVs and prey .",
"On the other hand , TPS10 volatiles emitted constitutively by less heavily attacked TPS10 plants ( Figure 5 ) would be less often associated with feeding herbivores than the endogenous HIPVs released only when herbivores attack , and all the more so if TPS10-expressing plants supported fewer foliar herbivores ( Figure 6 ) .",
"Perhaps because ‘honest’ endogenous HIPVs were also emitted by WT plants , TPS10 plants had no advantage over WT in attracting predators .",
"The concept of honest vs deceptive , or ‘cry wolf’ HIPV emission has been investigated in the context of emission proportionate , or disproportionate to numbers of attacking herbivores ( Shiojiri et al . , 2010 ) .",
"But what happens when plants ‘cry wolf’ by constitutively emitting volatiles which , in nature , are only associated with herbivore damage ?",
"Plants engineered to constitutively emit particular HIPVs can attract more parasitoids when parasitoids are trained to associate engineered volatiles with their prey ( Kappers et al . , 2005; Schnee et al . , 2006 ) , or when all emitting plants are also experimentally infested ( Rasmann et al . , 2005; Degenhardt et al . , 2009 ) .",
"Such studies demonstrate that genetic engineering of HIPVs can enhance indirect defense , but they do not demonstrate that constitutive emission is an effective strategy in the complex environments of agriculture , or nature ( Kos et al . , 2009; Schuman et al . , 2012; Xiao et al . , 2012 ) .",
"From an arthropod's perspective , constitutive HIPV emission is oxymoronic .",
"If there is no effort to artificially associate constitutively emitted volatiles with prey , predators , and parasitoids may not learn to respond , or worse , learn to actively ignore the volatile emissions .",
"Whether achieved via genetic methods or synthetic release stations ( Kaplan , 2012 ) , constitutive emission is unlikely to enhance indirect defense if prey densities are low , or if plants emit other ‘honest’ HIPVs .",
"Moreover , assigning an indirect defense function to HIPVs based on their ability to attract predators or parasitoids to single plants may be misleading .",
"In real populations , the effectiveness of HIPVs likely depends on the density of HIPV emission and the location of emitting plants ( Xiao et al . , 2012 ) .",
"There may be competition among plants in populations either to attract , or to arrest predators and parasitoids of herbivores .",
"Specifically , whether plants benefit from emitting more or less than their neighbors likely depends on the basal level of HIPV emission in a population ( low or high ) , and the location of plants in a population ( edge or center ) .",
"Perhaps , wild-type plants are capable of adjusting their own volatile emission in response to the emission of their neighbors in order to adapt ( Choh et al . , 2004; Baldwin et al . , 2006; Yan and Wang , 2006 ) .",
"In this context , it would be interesting to know whether central lox2/3 plants surrounded by WT edge plants would experience a similar predation disadvantage as central TPS10 plants surrounded by WT edge plants , and whether central WT plants surrounded by lox2/3 edge plants would have an advantage over their neighbors in attracting predators .",
"Now that careful and extensive research has established the importance of biodiversity to ecosystem function , the next step is to understand how the two are linked in order to craft informed socioeconomic and environmental policy ( Midgley , 2012 ) .",
"The community genetics perspective is essential in order to understand biodiversity effects at the level of individual heritable traits ( Whitham et al . , 2006 ) .",
"This is especially true in light of recent research showing that intraspecific , genetic biodiversity is at least as important as interspecific biodiversity for the structure of ecological communities ( Crutsinger et al . , 2006; Johnson et al . , 2006; Cook-Patton et al . , 2011; Moreira and Mooney , 2013 ) and the recognition that interspecific biodiversity is an emergent property of genetic biodiversity ( Johnson and Stinchcombe , 2007; Haloin and Strauss , 2008 ) .",
"The work presented here demonstrates that the population-level effects of an indirect defense gene may be greater than its effects on individual plants , demonstrating that the population perspective is vital to our understanding of gene function .",
"Furthermore , our results indicate that populations comprising individuals differing in only a single genetic trait can functionally imitate more diverse communities .",
"Engineering functional diversity may become an important strategy in a world with over 10 billion humans ( United Nations , Department of Economic and Social Affairs , Population Division , 2013 ) , where every patch of arable land may be planted with custom-engineered high-yield crop monocultures ."
],
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"Seed germination , glasshouse growth conditions , and the Agrobacterium tumefaciens ( strain LBA 4404 ) –mediated transformation procedure have been described previously ( Krügel et al . , 2002; Schuman et al . , 2014 ) .",
"Seeds of the 31st generation of the inbred ‘UT’ line of N . attenuata ( Torr . ex S Watson ) described by Krügel et al . ( 2002 ) were used for the wild-type ( WT ) plants in all experiments .",
"We used homozygous transformed lines of the second transformed generation ( T2 ) , each with a single transgene insertion , to either ectopically overexpress Zea mays TERPENE SYNTHASE 10 ( TPS10 ) under the control of a 35S promoter in the pSOL9 plasmid ( TPS10 line A-09-389-6/10-3 , described in Schuman et al . , 2014 ) , or silence N . attenuata LIPOXYGENASE 2 and LIPOXYGENASE 3 ( LOX2/3 ) via an inverted repeat construct specifically targeting both genes in the pSOL8 plasmid ( line A-07-707-2 , sequences targeting NaLOX2 and NaLOX3 described in Allmann et al . ( 2010 ) ( Figure 1 ) .",
"Vector construction and the pSOL8 and pSOL9 plasmids have been described previously ( Gase et al . , 2011 ) .",
"To combine the ectopic overexpression of TPS10 with the silencing of LOX2/3 , a hemizygous cross was created between the lox2/3 and TPS10 T2 homozygous lines ( lox2/3xTPS10 , Figure 1 ) .",
"Further details of transgenic line screening and characterization are given in Appendix 3 .",
"For field experiments , seedlings were transferred to 50 mm peat pellets ( Jiffy ) 15 days after germination and gradually hardened to the environmental conditions of high sunlight and low relative humidity over 10 days .",
"Small , adapted , size-matched rosette-stage plants were transplanted into a field plot in a native habitat in Utah located at latitude 37 . 146 , longitude 114 . 020 .",
"Field plantations were conducted under APHIS permission numbers 06-242-3r-a3 , 10-349-102r , and 11-350-101r .",
"Plants were watered thoroughly once at planting and as needed over the first 2 weeks until roots were established; all plants received the same watering regime in each year .",
"WT , TPS10 , lox2/3 , and lox2/3xTPS10 plants were arranged as individuals and populations according to the descriptions in Figure 2 and Figure 2—figure supplement 2 ( n = 12 in season one , n = 15 in season two of each individual or population type ) .",
"A distance of 0 . 5 m is sufficient for N . attenuata's herbivores and their predators to distinguish odor from individual plants ( Kessler and Baldwin , 2001; Schuman et al . , 2012 ) : so that insects could perceive plants in a population as a single unit , they were planted in 0 . 4 × 0 . 4 m squares , while the distance between single individuals/populations was ca .",
"1 . 5 m .",
"Individual and population types were marked with small ( ca . 5 cm long , ⌀ 0 . 5 cm ) bamboo sticks having different numbers of horizontal markings , which were checked during experiments only when establishing treatment groups , or confirming current location within the experiment .",
"During experiments and data collection , plants were referred to only by their number or position ( e . g . , n1 population 2 upper left , where upper left was defined from a standardized observer position ) .",
"Plant genotypes were never identified during data collection to avoid bias , and experimental treatments and data collection were spatially and temporally randomized corresponding to the randomized arrangement of individuals and populations in the field plot ( Figure 2—figure supplement 2 ) .",
"Prior to measuring gene transcripts and induced defenses or HIPVs , glasshouse-grown plants , which were not damaged by herbivores , were treated with wounding and M . sexta oral secretions ( W + OS ) as a standardized method to mimic herbivore feeding as described previously ( Schuman et al . , 2012 ) .",
"The first fully-expanded leaf ( node +1 ) was used for quantifying gene transcript ( LOX2 , LOX3 , TPS10 ) and jasmonate ( JA , JA-Ile ) accumulation on a set of bolting plants .",
"The adjacent older leaf ( node +2 ) was used to measure headspace volatiles on the same plants ( data in Appendix 2 , and see ‘Quantification of volatiles in the headspace of glasshouse-grown plants’ ) .",
"A separate set of plants was used following the same procedure for WT and TPS10 jasmonates and GLVs , and the results are reported in Schuman et al . ( 2014 ) and ratios to WT are shown in Figure 1B .",
"Control plants were left untreated .",
"Treated leaves , and leaves at the same position on control plants were cut at the petiole and wrapped in a double layer of aluminum foil , then immediately flash-frozen in liquid nitrogen , and kept at −80°C until processing and analysis .",
"For field-grown plants , five similar , mature , non-senescent stem leaves were harvested after the end of predation assays , on June 28th of experimental season two .",
"Two of these leaves had been damaged by the M . sexta first-instar larvae used for predation assays , and three were systemic leaves with similar levels of damage from other naturally-occurring herbivores .",
"Leaves were cut at the petiole , pooled in a single sample per plant and wrapped in a double layer of aluminum foil , then immediately frozen on dry ice insulated with ice packs frozen at −20°C .",
"Samples were stored at −20°C until transport to the MPICE on dry ice , where they were kept at −80°C until processing and analysis .",
"All sample processing was carried out over liquid nitrogen until the addition of the extraction solvents .",
"Prior to analysis , entire leaves were crushed with a mortar and pestle and each transferred to a 2 mL microcentrifuge tube for storage .",
"For specific measurements , aliquots were weighed into microcentrifuge tubes containing two steel balls and finely ground in a GenoGrinder ( SPEX Certi Prep , Metuchen , NJ ) prior to extraction .",
"JA and the active jasmonate hormone JA-Ile were extracted from 100 mg of leaf tissue ( from n = 4 plants ) using ethyl acetate spiked with internal standards ( IS ) and quantified by LC-MS/MS ( Varian , Palo Alto , CA ) as described by Oh et al . ( 2012 ) , with the modification that 100 ng rather than 200 ng of [2H2]JA , and 20 ng rather than 40 ng of JA-[13C6]Ile were used as internal standards ( IS ) .",
"Individual jasmonates were quantified in ng by comparison to the corresponding IS peak area and normalized per g leaf tissue fresh mass ( FM ) .",
"The quantification of TPI in field tissue samples as an indicator of jasmonate signaling is described in Appendix 3 .",
"For GLVs , the +2 leaf ( n = 4 plants ) was enclosed immediately after W + OS elicitation in two 50 mL PET cups ( Huhtamaki , Finland ) lined on the edges with foam to protect leaves and with an activated charcoal filter attached to one side for incoming air , and secured with miniature claw-style hair clips as described previously ( Schuman et al . , 2009 ) .",
"For TPS10 products in the headspace of glasshouse-grown plants ( n = 4 ) , leaves were enclosed 24 hr after W + OS treatment and the headspace was collected from 24–32 h during the peak emission of sesquiterpenes in N . attenuata ( Halitschke et al . , 2000 ) .",
"Headspace VOCs were collected on 20 mg of Poropak Q ( Tholl et al . , 2006; Sigma–Aldrich , St . Louis , MO ) in self-packed filters ( bodies and materials from ARS Inc . , Gainesville , FL ) by drawing ambient air through these clip cages at 300 mL min−1 using a manifold with screw-close valves set to provide equal outflow , via pushing air at 2 to 3 bar through a Venturi aspirator as described previously ( Oh et al . , 2012 ) .",
"Background VOCs present in ambient air were collected using empty trapping containers and background signals were later subtracted if necessary from raw intensities of plant samples prior to further processing .",
"After trapping , Porapak Q filters were stored at −20°C until extraction by addition of 320 ng of tetralin as an internal standard ( IS ) , and elution of volatiles with 250 mL of dichloromethane ( Sigma–Aldrich ) .",
"Filters were eluted into a GC vial containing a 250 μL glass insert .",
"Samples were analyzed on one of two different GC–MS instruments from Varian with columns from Phenomenex ( Torrance , CA; 30 m × 0 . 25 mm i . d . , 0 . 25 μm film thickness ) .",
"1 μL of each sample was injected in splitless mode , and then the injectors were returned to a 1:70 split ratio from 2 min after injection through the end of each run .",
"He carrier gas was used with a column flow of 1 mL min−1 .",
"GLV samples were analyzed by a CP-3800 GC Saturn 2000 ion trap MS with a polar ZB-wax column and a CP-8200 autoinjector; the GC and MS were programmed as previously described for this instrument ( Schuman et al . , 2012 ) , and compounds were separated by a temperature ramp of 5°C min−1 between 40°C and 185°C .",
"TPS10 products and other VOCs were analyzed on a CP-3800 GC coupled to a Saturn 4000 ion-trap mass spectrometer with a nonpolar ZB5 column and a CP-8400 autoinjector; The GC and MS were programmed as previously described for this instrument ( Oh et al . , 2012 ) , and compounds were separated by a temperature ramp of 5°C min−1 between 40°C and 180°C .",
"Individual volatile compound peaks were quantified using the combined peak area of two specific and abundant ion traces per compound using MS Work Station Data Analysis software ( Varian ) and normalized by the 104 + 132 ion trace peak area from tetralin in each sample .",
"The identification of compounds was conducted by comparing GC retention times and mass spectra to those of standards and mass spectra databases: Wiley version 6 and NIST ( National Institute of Standards and Technology ) spectral libraries .",
"The area of trapped leaves was quantified for comparison by scanning and calculating areas in pixels using SigmaScan ( Systat Software Inc . , San Jose , CA ) , and subsequently converting pixels to cm2 using a size standard which was scanned with leaves .",
"Plant volatiles were quantified in tissue from field-grown plants ( n = 9–22 ) .",
"Frozen tissue ( 100 mg ) was spiked with 800 ng tetralin as an internal standard ( Sigma–Aldrich ) and extracted in 300 µL hexane during an overnight rotating incubation at RT .",
"Tissue was allowed to settle and 100 µL of water- and tissue-free hexane was transferred to a GC vial containing a 250 µL microinsert .",
"Samples were analyzed by a Varian CP-3800 GC-Saturn 4000 ion trap MS connected to a ZB5 column ( 30 m × 0 . 25 mm i . d . , 0 . 25 μm film thickness; Phenomenex ) as described in ‘Quantification of volatiles in the headspace of glasshouse-grown plants’ .",
"Analyte quantities were expressed as percent tetralin IS per 100 mg FM .",
"For the data in Figure 3 , on May 16th of season two , four activated charcoal filters were placed around each plant ( n = 4 ) as shown in Figure 3A and the headspace was not enclosed .",
"The open headspace was sampled for 7 h during the day at which time emission of most volatiles was previously found to be greatest ( Appendix 2 ) .",
"After elution ( see Appendix 3 ) , eluents from all four filters were combined and concentrated to a volume of 15–20 µL under a gentle stream of nitrogen gas .",
"Pooled and concentrated samples were analyzed by Agilent 6890N GC equipped with an Agilent 7683 auto-injector coupled with a LECO ( St . Joseph , MI ) Pegasus III time-of-flight MS with a 4D thermal modulator upgrade .",
"Injected samples were separated first on a nonpolar column ( C1 RTX-5MS , 20 m × 250 µm i . d . × 0 . 5 µm; Restek , Bellefonte , PA ) and every 6 s ( modulation time ) transferred to a midpolar column ( DB-17 , 0 . 890 m × 100 µm i . d . × 0 . 1 µm; Agilent Technologies , Santa Clara , CA ) for the second separation .",
"Chromatography and analysis conditions as well as deconvolution , alignment , and integration of VOC analyte peaks are described in Gaquerel et al . ( 2009 ) .",
"During peak table alignment using the comparison feature imbedded in ChromaToF software ( LECO ) , mass spectra alignment was accepted at a similarity threshold of 500/1000 .",
"Individual volatile compound peaks were normalized by the peak area of the tetralin IS in each sample .",
"Analyte quantities were expressed as percent tetralin IS per plant .",
"As for OS collection , we used M . sexta neonates from an in-house colony at the MPICE .",
"One M . sexta neonate per plant was placed on the youngest rosette leaf of elongated plants ( n = 25 larvae ) .",
"Larval movement was unrestricted within a plant , but plants were spaced on the table so that larvae could not move directly between plants .",
"Larval mass was determined on days 8 and 12 , corresponding on average to the third and fourth larval instars .",
"Plant genotypes were randomized spatially on a single glasshouse table and temporally in placement and weighing of larvae .",
"Due to mortality and to larval movement off of plants , 11–16 larvae remained within each treatment group by day 12 .",
"Total canopy damage due to herbivores occurring naturally on the field plot was quantified on June 9th in season one , and on June 2nd in season two .",
"Damage was calculated as described in Schuman et al . ( 2012 ) by identifying damage from specific herbivores according to their characteristic feeding patterns , counting the number of leaves per plant ( small leaves were counted as 1/5 to 1/2 of a leaf based on leaf area and large leaves were counted as 1 leaf ) , estimating the total percentage of leaf area damage due to each herbivore , and dividing the total leaf area damage from each herbivore by the total number of leaves .",
"Leaf area damage was estimated in categories of 1% , 5% , 10% , 15% , and so on , in steps of 5% .",
"All such damage estimates were made by MCS .",
"Plants with a health index <2 . 5 ( see ‘Ranking of apparent plant health’ ) were excluded from herbivore damage calculations as it was difficult to accurately assess herbivore damage to these plants .",
"In the morning of June 26th in season two , a subset of focal plants with a health index ≥2 was chosen ( see ‘Ranking of apparent plant health’ ) and numbers of herbivorous and predacious arthropods present on the shoots of these plants were counted ( total n's in Figure 6—source data 1 , 2 ) .",
"The counting was done by a team of two people in parallel moving down the plot in the same direction , so as to complete counts as quickly as possible and to avoid disturbing plants prior to counting .",
"Plants were randomly assigned to each counter .",
"Counting proceeded by looking at each branch of the plant in a systematic order from the top to the bottom of the plant , observing both the adaxial and abaxial sides of leaves as well as full areas of stems .",
"In this way especially mobile arthropods which tend to feed near the top of plants were either counted before they fled ( Epitrix spp . ) , or could be counted once they had relocated to the base of the plant ( T . notatus ) .",
"Plant size ( rosette diameter , stem length , and branching ) was measured at three times during the main period of vegetative growth and beginning of reproduction in season one ( May 1st–2nd , 8th–11th , and 16th–17th ) , and after plants had switched from vegetative growth to reproduction in season two ( June 4th–6th ) , at which time stem diameter was also measured .",
"Rosette diameter was measured as the maximum diameter found by gently laying a ruler over the rosette; stem length was measured from the base of the stem to the tip of the apical inflorescence by placing a ruler beside the stem resting at the base of the rosette; stem diameter was measured by placing a calipers at the base of the stem immediately above the rosette; and all side branches 5 cm or longer were counted .",
"Additionally , plant reproductive output was monitored by counting the number of flowers ( counted once the corolla protruded visibly from the calyx ) before removal at two time points after plants began to flower: on May 8th–11th and 16th–17th in season one , and on May 29th and June 4th–6th in season two .",
"Additionally , buds >2 mm long were counted in season two .",
"After these counts , floral meristems were removed as necessary to prevent the distribution of ripe seeds , as is required for field-released transgenic plants .",
"Plant mortality was quantified as the number of plants which died between the end of planting , and the end of the experiment when all plants and remains were removed from the field plot .",
"During removal of plants on June 29th in season two , we split stems open lengthwise to check for T . mucorea frass and larvae ( Diezel et al . , 2011 ) .",
"Plants were counted as infested if the main stem was hollow and filled with T . mucorea frass , or if a larva was found in such a hollow , frass-filled stem or axial branch .",
"These counts were done by tallying totals without reference to specific plant ID's , and thus could only be analyzed using unreplicated statistical tests .",
"In season two , plant health was also ranked by MCS on a scale of 1–5 in increments of 0 . 5 ( 1 , 1 . 5 , 2 , 2 . 5 , … , 5 ) using the index in Figure 7C of Schuman et al . ( 2012 ) : 5 , healthy , <5% of shoot senescent , no chlorosis or signs of water stress; 4 , 5–10% of shoot senescent and may have visible chlorosis; 3 , 10–15% of shoot senescent , some water stress , up to 50% of canopy chlorotic; 2 , >15% of shoot senescent , heavy water stress or visibly sick; 1 , dead .",
"This scale was sufficiently objective that several observers not directly involved in the experiment arrived independently at the same ranks , to a precision of ±0 . 5 , to describe the health indices of randomly-chosen plants , after training on fewer than 10 example plants .",
"Five consecutive predation assays were conducted between June 21st and June 27th at the end of experimental season two , when Geocoris spp .",
"and herbivores were abundant .",
"M . sexta eggs and larvae ( total n's given in the source data file for Figure 11 ) were used as bait for Geocoris spp .",
"predators as described by Kessler and Baldwin ( 2001 ) and Steppuhn et al . ( 2008 ) .",
"Native Manduca spp .",
"did not provide sufficient synchronously oviposited eggs for trials , and so we used M . sexta eggs kindly provided by C Miles from her laboratory colony at Binghamton University , Vestal , New York .",
"Size-matched plants with health indices ≥2 in populations were used .",
"We treated LOX2/3-expressing ( WT , TPS10 ) and LOX2/3-deficient ( lox2/3 , lox2/3xTPS10 ) populations as two separate experimental groups because it was not possible to match plants across these two groups for apparent health or replicate numbers ( both were lower for lox2/3 and lox2/3xTPS10 populations ) .",
"During the five predation assays it was occasionally necessary to select new plants , preventing a repeated measures analysis of the predation data; however , newly selected plants were matched as described .",
"At the end of predation assays , tissue samples were taken from a subset of these plants for the extraction of volatiles and determination of TPI activity ( Figure 1 , Figure 1—source data 3 and preceding methods ) .",
"For each assay , one first-instar M . sexta larva and one egg were placed on one lower stem leaf in a standardized position on each plant , and the predation of eggs and larva was monitored after 24 hr .",
"No other Manduca spp .",
"eggs or larvae were present on the plants used during the assays .",
"Eggs were affixed to the underside of the leaf with a drop of α-cellulose glue ( KVS , Germany ) , which does not damage plants or affect volatile emission ( Kessler and Baldwin , 2001 ) .",
"Feeding by the M . sexta larva ensured the locally elicited emission of HIPVs .",
"Larvae were considered to be predated when either the larva was missing , but clearly identifiable , fresh M . sexta feeding damage was present , or when the predated larval carcass was found on the same plant; eggs were considered predated when the eggshell was empty but intact , except for a small hole which characterizes the typical damage caused by Geocoris spp .",
"feeding as described in Kessler and Baldwin ( 2001 ) and depicted in Figure 3 of Schuman et al . ( 2012 ) .",
"Eggs occasionally collapse during Geocoris spp .",
"predation , but collapsed eggs were not counted unless the eggs were at least 50% empty with a visible feeding hole .",
"Summary statistics were calculated in Microsoft Excel , as were G-tests , Fisher's exact tests , and tests of Spearman's correlation coefficients , which were calculated using Microsoft Excel spreadsheets from JH Macdonald's online Handbook of Biological Statistics ( Macdonald , 2009 ) .",
"All other statistical tests were calculated in R version 2 . 15 . 2 , using the RStudio interface version 0 . 96 . 316 or version 0 . 97 . 449 ( R Core Team , 2012 ) .",
"In some cases , data which were clearly not different upon visual inspection and which had been found in similar data sets not to differ significantly were not statistically tested ( e . g . , rosette diameter in some subsets of data from season one ) .",
"When possible , Welch t-tests were used for pairwise comparisons and ANOVAs , linear mixed-effects models , generalized linear models , or generalized linear mixed-effects models were used for multiple comparisons as appropriate; we checked treatment groups graphically ( quantile–quantile plots , residual v . fitted plots ) and statistically ( Shapiro–Wilk test , Bartlett test ) for compliance with model assumptions , including normality and homoscedasticity .",
"Data containing too many zero values or not meeting model assumptions were analyzed using ranking tests ( Wilcoxon rank sum for pairwise comparisons , Kruskal–Wallis for multiple groups ) .",
"R package nlme was used for linear mixed-effects models ( Pinheiro et al . , 2014 ) , and packages lme4 ( Bates et al . , 2014 , ; 2014b ) and multcomp ( Hothorn et al . , 2008 ) were used for generalized linear mixed-effects models .",
"The optimal fixed structure for ANOVAs , linear mixed-effects models , generalized linear models , and generalized linear mixed-effects models was found by stepwise model simplification and factor level reduction followed by the comparison of the models via the Akaike information criterion ( AIC , Akaike , 1973 ) .",
"Specific examples of model simplification for individual analyses are given in Appendix 3 and Figure 8—source data 1 .",
"Holm-Bonferroni p-value corrections were calculated in Microsoft Excel for families of tests on the same data .",
"Like the Bonferroni correction , the Holm-Bonferroni correction controls the familywise error rate and is simple to calculate , but the Holm-Bonferroni method is more powerful ( Holm , 1979 ) .",
"Briefly , all p-values in a family of tests conducted on the same data set and subsets were listed from smallest to largest .",
"The smallest p-value was multiplied by the total number of tests ( n ) conducted on that data .",
"If the resulting corrected p-value was <0 . 05 , then the next-smallest p-value was multiplied by n − 1 .",
"If the resulting corrected p-value was <0 . 05 , then the third smallest p-value was multiplied by n − 2 , and so on .",
"At the first correction resulting in a p-value ≥0 . 05 , that p-value and all larger p-values were considered non-significant .",
"The corrected p-values reported in the text are the products of the correction procedure , for example , ( smallest p-value ) × n or ( second smallest p-value ) × ( n − 1 ) ."
]
] | [
"Plants are at the trophic base of terrestrial ecosystems , and the diversity of plant species in an ecosystem is a principle determinant of community structure .",
"This may arise from diverse functional traits among species .",
"In fact , genetic diversity within species can have similarly large effects .",
"However , studies of intraspecific genetic diversity have used genotypes varying in several complex traits , obscuring the specific phenotypic variation responsible for community-level effects .",
"Using lines of the wild tobacco Nicotiana attenuata genetically altered in specific well-characterized defense traits and planted into experimental populations in their native habitat , we investigated community-level effects of trait diversity in populations of otherwise isogenic plants .",
"We conclude that the frequency of defense traits in a population can determine the outcomes of these traits for individuals .",
"Furthermore , our results suggest that some ecosystem-level services afforded by genetically diverse plant populations could be recaptured in intensive monocultures engineered to be functionally diverse ."
] | [
"Plants are at the base of many food webs .",
"This means that the different traits and characteristics of the plant species in an ecosystem can have a large impact on the animals and other organisms that live there .",
"Individuals within the same plant species often differ in multiple genes .",
"This ‘genetic diversity’ can affect the populations of other organisms in their ecosystem , for example , by altering which species are present , and the number of individuals .",
"However , previous studies that investigated plant genetic diversity in ecosystems used plants that varied in multiple , usually unknown , genetic traits , which made it difficult to identify specific genetic traits in plants that can influence the whole ecosystem .",
"One way that plants affect their ecosystems involves how they defend themselves against the herbivores that try to eat them .",
"For example , plants can use sharp spines or harmful chemicals to deter herbivores , or attract predators that will attack the herbivores .",
"Here , Schuman et al . carried out a 2-year field study using wild tobacco plants that had been genetically altered to employ different defensive strategies .",
"This was achieved by altering the expression of three genes in the plants in specific combinations .",
"Two of the genes , called LOX2 and LOX3 , are required to make most of the chemicals that tobacco plants use to defend themselves against herbivores .",
"The third gene , called TPS10 , which comes from the crop plant maize , gives plants the ability to release a fragrance that attracts natural predators of their herbivores .",
"Except for these specific alterations , the plants were otherwise genetically identical .",
"These plants were then grown either alone , or in groups of five plants , which reflects the normal size of groups of wild tobacco growing in its natural environment .",
"The groups contained mixtures of plants with different gene alterations .",
"Schuman et al . found that the expression levels of these genes in individual plants could determine how well the whole group fared in several different measures of plant health and defense .",
"For example , plants lacking LOX2 and LOX3 usually appeared to be less healthy and they were less likely to survive long enough to reproduce because they were less able to defend themselves against herbivores .",
"However , if these plants were grown in a group with one plant that expressed TPS10 , all of the plants in the group looked healthier and were more likely to survive long enough to reproduce .",
"Many crops are grown in large fields containing individual plants that are largely genetically identical , which makes them more vulnerable to disease .",
"Schuman et al . 's findings suggest that some of the community-level protective effects provided by diversity in wild populations of plants could be introduced into crop fields by altering the expression of a few specific genes in some individuals ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | A cell atlas of the chick retina based on single-cell transcriptomics | elife-63907-v2 | [
[
"The retina is about as complex as other regions of the vertebrate central nervous system .",
"It differs from many regions , however , in being particularly accessible to study .",
"For example , its neurons can be imaged live without surgical intervention , visual stimuli can be precisely controlled in time and space , and the paucity of long-distance inputs facilitates analysis of the entire circuit ex vivo .",
"These and other technical advantages , along with the intrinsic importance of the retina and the fact that most blinding diseases arise from retinal dysfunction , have combined to make the retina a popular model for analysis of neural structure , function , development , and disease ( Wässle , 2004; Dowling , 2012; Hoon et al . , 2014 ) .",
"Accordingly , retinas of many vertebrate species have been studied in detail , including those of rodents ( e . g . mice and rats ) , carnivores ( e . g . cats and ferrets ) , primates ( e . g . macaques , marmosets , and humans ) , birds ( e . g . chickens and pigeons ) , fish ( e . g . zebrafish and goldfish ) , reptiles ( e . g . turtles and lizards ) , and amphibia ( e . g . salamanders and frogs ) ( Dowling , 2012; Thoreson and Dacey , 2019; Baden et al . , 2020 ) .",
"All these studies depend on classification and characterization of the cell types that comprise the retina .",
"Recently , this enterprise has been greatly enhanced by the introduction of methods for high-throughput single-cell transcriptomic profiling ( scRNA-seq ) , which enable comprehensive and minimally biased sampling of cell types .",
"To date , however , with the exception of a single study on zebrafish RGCs ( Kölsch et al . , 2020 ) , these methods have been applied only to mice and primates ( Macosko et al . , 2015; Shekhar et al . , 2016; Rheaume et al . , 2018; Liang et al . , 2019; Menon et al . , 2019; Peng et al . , 2019; Tran et al . , 2019; Cowan et al . , 2020; Lu et al . , 2020; Orozco et al . , 2020; Yan et al . , 2020a; Yan et al . , 2020b; reviewed in Shekhar and Sanes , 2021 ) , restricting use of non-mammalian models and making it difficult to draw evolutionary relationships among species at the molecular level .",
"Here , we address this limitation by using scRNA-seq to generate a cell atlas of the chick retina .",
"The basic plan of the retina is highly conserved among all vertebrates ( Baden , 2020 ) .",
"Five neuronal classes are arranged in three cellular ( nuclear ) layers separated by two synaptic ( plexiform ) layers: photoreceptors ( PRs ) in an outer nuclear layer ( ONL ) , three sets of interneurons ( horizontal , bipolar , and amacrine cells; HCs , BCs , ACs ) in an inner nuclear layer ( INL ) , and output neurons ( retinal ganglion cells , RGCs ) , along with some ACs , in a ganglion cell layer ( GCL; Cajal , 1892; Masland , 2012; Figure 1A–B ) .",
"PRs form synapses with HCs and BCs in an outer plexiform layer ( OPL ) , while RGCs , BCs , and ACs form synapses in an inner plexiform layer ( IPL ) .",
"Axons of RGCs then exit the eye and travel through the optic nerve to a variety of retinorecipient areas in the brain ( Dhande et al . , 2015; Martersteck et al . , 2017 ) .",
"Each of these classes is divided into types with specific patterns of connectivity among them endowing distinct RGC types with sensitivities to different visual stimuli , such as edges , moving or oriented objects , and color contrasts ( Sanes and Masland , 2015 ) .",
"The retina also contains glial cells: Müller glia in the INL , and in many species , astrocytes and oligodendrocytes in and beneath the GCL ( Reichenbach and Bringmann , 2013; Vecino et al . , 2016 ) .",
"Altogether , transcriptomic and morphological studies have identified a total of >130 neural ( neuronal and glial ) cell types in mice and ~70 in primates ( Yan et al . , 2020a; Yan et al . , 2020b; Peng et al . , 2019 ) .",
"Although the basic retinal plan is conserved , cell types and patterns of connectivity vary among species , serving their visual needs ( Baden et al . , 2020 ) .",
"Birds are highly visual animals with sizable eyes , generally high acuity and sophisticated retinas ( Cook , 2000; Seifert et al . , 2020 ) .",
"For example , although most mammals have two cone PR types , most birds are tetrachromatic , with cone PRs selectively sensitive to red , green , blue , and ultraviolet light ( Hart , 2001; Baden and Osorio , 2019 ) .",
"Many have regions specialized for high acuity vision , akin to the fovea found in primates .",
"Indeed , histological and immunohistochemical studies have suggested that there may be more cell types in avian retinas than in those of mammals ( Cajal , 1892; Mariani and Leure-DuPree , 1977; Hayes , 1982; Quesada et al . , 1988; Naito and Chen , 2004; Karten and Brecha , 1983; Brecha et al . , 1984; Karten et al . , 1990 ) .",
"Among birds , the retina of the domestic chicken ( Gallus gallus domesticus ) has been the most intensively studied .",
"In particular , it has been a favored model for developmental analyses , including studies on the generation and migration of retinal neurons , their diversification into classes and types , the growth and guidance of RGC axons to the optic tectum , and the capacity of retinal neurons and their axons to regenerate ( Adler , 2000; Mey and Thanos , 2000; Thanos and Mey , 2001; Wilken and Reh , 2016; Wisely et al . , 2017 ) .",
"To complement and facilitate these studies , we used scRNA-seq to profile cells from the chick retina .",
"From ~40 , 000 single-cell transcriptomes , we identified cells of all six classes named above ( PR , HC , BC , AC , RGC , and glia ) and used unsupervised methods to divide them into ~150 clusters .",
"We show that 136 of the groups represent putative cell types , with others corresponding to developmental intermediates .",
"We then devised a method for CRISPR-based somatic cell integration of fluorescent reporters into genes shown by scRNA-seq to be expressed by specific types .",
"Using this technique along with other histological methods , we matched molecular profiles to morphology for many neuronal types .",
"We also found a positional signature in Müller glia , with distinct expression patterns based on their location along the anterior-posterior , dorsal-ventral , and central-peripheral retinal axes .",
"Finally , we compared the cell classes and types of chick retina with those of three mammalian species – mouse , macaque , and human – demonstrating conserved molecular features of all classes and some types , along with multiple differences between chick and mammals .",
"Together , our results provide new insights into retinal structure and evolution , as well as a foundation for anatomical , physiological , and developmental studies of avian retina ."
],
[
"All known chick retinal cell types are born by E14 , retinal structure is relatively mature by E18 , and birds are visually competent at hatching ( E21; Figure 1C; Hamburger and Hamilton , 1951; Prada et al . , 1991; Cepko et al . , 1996; Mey and Thanos , 2000; Yamagata and Sanes , 1995a; Yamagata and Sanes , 1995b; Drenhaus et al . , 2003 ) .",
"We used a droplet-based method ( Zheng et al . , 2017 ) to obtain 30 , 022 high-quality single-cell transcriptomes from embryonic day 18 ( E18 ) chick retina ( Figure 1—figure supplement 1A ) .",
"We assigned cells to classes based on expression of previously established markers , using methods described in Peng et al . , 2019 and Yan et al . , 2020a .",
"We identified five neuronal classes ( PRs , HCs , BCs , ACs , and RGCs ) as well as two glial types , Müller glia and oligodendrocytes ( Figure 1D , E , F ) .",
"Of the E18 retinal cells profiled , 620 were RGCs ( Figure 1F ) .",
"This fraction ( ~2% ) was similar to that observed in other species ( Macosko et al . , 2015; Peng et al . , 2019 ) but was insufficient for extensive classification of what we anticipated would be a highly heterogeneous class .",
"We therefore used magnetic beads coated with antibodies to the pan-RGC cell surface marker Thy1 ( French and Jeffrey , 1986; Yamagata et al . , 2002 ) to enrich RGCs from E16 retina before scRNA-seq .",
"We obtained 9159 single-cell transcriptomes , of which 8107 ( 89% ) were RGCs ( Figure 1G; Figure 1—figure supplement 1B ) that were used for subsequent analysis .",
"We combined single-cell transcriptomes for all cells except RGCs from the E18 dataset and for all and only RGCs from the E16 dataset to generate a cell atlas .",
"To resolve cell types within these classes , we reclustered each one separately , and obtained a total of 150 clusters ( Figure 1H ) .",
"Each presumably corresponded to a cell type , a small group of closely related types , a positional variant or , since we profiled embryonic cells , a developmental intermediate .",
"Quality metrics for each clusters are shown in Supplementary file 3 .",
"As one approach to separating definitive types from developmental intermediates , we collected cells from E12 retina , obtaining single-cell transcriptomes from PRs , HCs , BCs , RGCs , and MGs as well as several putative precursor populations ( Figure 1I; Figure 1—figure supplement 1C–E ) .",
"These cells were not used to generate the atlas , but rather to test developmental hypotheses .",
"Generation of a retinal cell atlas for mouse benefited from prior knowledge , numerous well-characterized antibodies , and many transgenic lines that express a reporter in one or a few types ( Macosko et al . , 2015; Shekhar et al . , 2016; Tran et al . , 2019; Yan et al . , 2020a ) .",
"Lacking these advantages for chick , we tested methods for inserting fluorescent reporters into genes shown by scRNA-seq to be expressed by specific types , thereby allowing us to visualize those cells’ morphology .",
"In a method called SLENDR , guide RNAs , and plasmids encoding Cas9 and a reporter are delivered to somatic cells to insert the reporter into a chromosomal site determined by the sequence of the guide RNAs and homologous sequences appended to the reporter ( Mikuni et al . , 2016 ) .",
"We were unsuccessful in applying this method to chick retina , and therefore modified it .",
"We used in ovo electroporation to deliver a mixture consisting of guide RNAs , Cas9 protein , and a single-strand DNA containing a reporter sequence flanked by ~70 bases gene-specific homology arms ( Gurumurthy et al . , 2019; Figure 2A ) .",
"We co-electroporated this mixture with piggyBac transposon reporter/transposase constructs encoding a spectrally distinct reporter to monitor the site and efficiency of electroporation , and optimized reagents to enhance homologous recombination ( see Materials and methods ) .",
"We call the method eCHIKIN for electroporation- and CRISPR-mediated Homology-Instructed Knock-IN .",
"To test eCHIKIN , we targeted genes expressed in most cells of three retinal classes: VSX2 ( Chx10 ) in BCs , TFAP2A in ACs and RBPMS2 in RGCs ( Chen and Cepko , 2000; Bassett et al . , 2007; Piri et al . , 2006 ) .",
"We inserted a nine amino acid hemagglutinin ( HA ) tag at the N-terminus of VSX2 and RBPMS2 and inserted GFP into the TFAP2A locus .",
"In each case , the homologously inserted reporter specifically labeled the expected cell class , as verified by soma position and immunostaining: VSX2-HA-tagged cells were restricted in the outer part of the INL , as appropriate for BCs , and were VSX2-positive and TFAP2A-negative ( Figure 2B–D ) .",
"TFAP2A-GFP cells were present in the inner portion of the INL as appropriate for TFAP2A-expressing ACs ( Figure 2E ) .",
"In this case , GFP was not fused to the endogenous proteins , so it filled the cytoplasm , revealing dendrites of ACs in the IPL .",
"In addition , the labeled cells still express the endogenous gene ( Figure 2E ) , suggesting that only one allele was edited in most cells .",
"Likewise , RBPMS2-HA-tagged cells were confined to the ganglion cell layer , as appropriate for RGCs ( Figure 2F ) .",
"In each case , the spectrally distinct co-electroporated reporter , which had its own regulatory elements and integrated non-homologously , was expressed in cells of all layers ( Figure 2B–F ) .",
"To efficiently label cells that expressed target genes at low levels , we inserted Cre recombinase into theTFAP2A and RBPMS2 loci , and co-electroporated with a Cre-dependent fluorescent protein ( loxP-STOP-loxP-GFP under the control of strong and ubiquitously expressed CAGS promoter and enhancer ) .",
"Cell class specificity was similar in these cases to those in which the loci were tagged directly with a reporter ( Figure 2G , H ) .",
"Thus , eCHIKIN provides a means of matching molecular identity to cellular position , morphology and lamination without germline manipulation .",
"Chicks are tetrachromatic , with cone types that express red opsin ( OPN1LW ) , green opsin ( OPN1MSW ) , blue opsin ( OPN2SW ) , or violet opsin ( OPN1SW ) ( Kram et al . , 2010; Enright et al . , 2015 ) .",
"In addition to these conventional ‘single-cones’ ( SCs ) , the retina also contains rods that express rhodopsin ( RHO ) and OPN1LW-expressing ‘double-cones’ ( DCs ) composed of tightly apposed principal and accessory cells , which together comprise about half of all PRs ( Smith et al . , 1985; López-López et al . , 2008; Oishi et al . , 1990 ) .",
"This composition is unlike that of rodent and primate retinas , which are rod-dominated and contain two or three SC types but no DCs .",
"Reclustering of PRs revealed 12 clusters ( numbered in order of descending abundance; Figure 3A; Figure 3—figure supplement 1A ) , including one each expressing RHO , OPN1MSW , OPN2SW , and OPN1SW ( PR clusters 3 , 5 , 8 , and 11 ) , marking them as rods and green , blue , and violet SCs , respectively ( Figure 3B ) .",
"As expected , in situ hybridizations to opsins labeled non-overlapping cells ( Figure 3C and data not shown ) .",
"These types were also distinguished by other differentially expressed genes , including PDE6B , PDE6G ( phosphodiesterases ) , and MAFA ( a transcription factor ) in rods , SLC27A6 ( a fatty acid transporter ) in blue and violet SCs and LOC101750261 ( a non-coding RNA ) in green SCs .",
"Expression of OPN1LW was detected in four clusters ( PR clusters 1 , 2 , 4 , and 7; Figure 3B ) , representing red SCs and DCs .",
"To distinguish among them , we localized cluster seven with STRA6 , a retinol transporter ( Isken et al . , 2008 ) , and clusters 1 , 2 , and 4 with CALB1 .",
"STRA6+ cones were thin , as expected for SCs .",
"In contrast , CALB1+ cells were broader , as expected for DCs ( Figure 3D–F ) .",
"We also generated an eCHIKIN probe for CALB1 , which labeled DCs ( Figure 3G ) .",
"Thus , we identify PR7 as red SCs and PR1 , 2 , and 4 as components of DCs .",
"PR clusters 1 , 2 , and 4 are quite similar at the gene expression level , so we were unable to distinguish principal from accessory cell components of DCs , or to determine why they formed three clusters rather than the expected two .",
"Their close similarity raises the possibility that besides the gap junctions that connect them ( Smith et al . , 1985 ) there may be additional ways that enable exchange of mRNAs .",
"The transcriptomic relationships among these clusters are demonstrated by a dendrogram in Figure 3B .",
"There were three major branches , of which two corresponded to rods and cones .",
"Among the cones , SCs are more closely related to each other than to any components of DCs .",
"Among SCs , red and green SCs are each other’s closest relatives as are blue and violet SCs .",
"The third clade of PRs , comprising four clusters , expressed the PR fate determination gene , PRDM1 ( Katoh et al . , 2010; Brzezinski et al . , 2013 ) , but did not express opsins , suggesting that they were immature PRs .",
"To relate these clusters to the definitive PRs , we used a supervised classification method , XGBoost ( Chen and Guestrin , 2016 ) that predicts the best match among mature types for each putative immature type ( Figure 3H ) .",
"PR6 and 9 mapped mostly to the DC types , PR10 mapped to three out of the four SC types , and PR12 mapped almost exclusively mapped to rods .",
"Consistent with this relationship , each immature cluster expressed markers of the corresponding mature PRs – for example , PR6 expressed the DC marker GRIK3 , and PR10 expressed the rod markers MAFA and PDE6G .",
"To further test the idea that the opsin-negative PR-like cells represented immature PRs , we queried our E12 dataset together with the E18 PRs for clustering .",
"E12 cells aligned with both immature and mature types of E18 were present ( Figure 3—figure supplement 1B , C ) but , as expected , the proportions differed with age: immature types comprise 91% of the E12 data set but only 16% of the E18 data set .",
"Finally , we analyzed retinas from earlier ( E12 and E16 ) and later ( E20 ) stages by in situ hybridization with probes for ARHGAP18 , and SLIT1 , expressed by PR6 ( immature DCs ) ; OPN1LW ( mature red SCs and DCs ) and STRA6 ( mature SCs ) .",
"All PRs have been born by E12 ( Prada et al . , 1991 ) , At E12 , ARHGAP18 was expressed by PRs in the ONL , but OPN1LW was not detectable .",
"Conversely , OPN1LW was expressed at E20 but ARGHAP18 was not ( Figure 3—figure supplement 2A–D ) , suggesting that ARGHAP18 is transiently expressed in the ONL during development .",
"At E16 , consistent with the fact that retinal differentiation proceeds in a central-to-peripheral gradient ( Kahn , 1974; Spence and Robson , 1989; Prada et al . , 1991; Bruhn and Cepko , 1996 ) , ARHGAP18 and SLIT1 were expressed by ONL cells selectively in peripheral retina , whereas OPN1LW and STRA6 were by ONL cells in central retina ( Figure 3—figure supplement 2E–Q ) .",
"Together , these results strongly suggest that immature and mature PR types co-exist at the same age but in different regions .",
"Initial histological studies of avian retina distinguished two HC types: Type I , which bears an axon , and Type II , which does not ( Cajal , 1892; Mariani and Leure-DuPree , 1977 ) .",
"Later studies combined immunohistochemical and morphological criteria to propose further subdivisions into three or four types ( Fischer et al . , 2007; Edqvist et al . , 2008; Boije et al . , 2016 ) .",
"Clustering of E18 HCs yielded five clusters , HC1-5 ( Figure 4A , Figure 4—figure supplement 1A ) , all expressing the canonical HC marker ONECUT3 .",
"LHX1 and ISL1 were detected in exclusive populations and other markers distinguished each type ( Figure 4B ) .",
"Based on prior immunohistochemical analysis ( Fischer et al . , 2007; Edqvist et al . , 2008 ) , we hypothesized that HC1 and HC3 corresponded to Type I and HC2/HC4/HC5 to Type II HCs .",
"The two type I clusters ( HC1 and HC3; LHX1+ ISL1- ) were closely related , with few genes differentially expressed between them showing large differences ( Figure 4B ) .",
"In situ hybridization for two such genes – IPCEF1 enriched in HC1 and OXT enriched in HC3 – confirmed partially overlapping expression ( Figure 4C ) .",
"The differences between IPCEF1+OXT- and IPCEF1-OXT+ cells might reflect graded expression in time and/or space .",
"Spatiotemporal analysis of expression similar to that described for PRs above , indicated that OXT ( enriched in HC3 ) was expressed throughout the retina at E14 , restricted to peripheral at E18 , and disappeared at E20 ( Figure 4—figure supplement 1B–G ) .",
"In contrast , IPCEF1 ( enriched in HC1 ) was expressed selectively in central retina at E14 and E18 , and strongly throughout retina at E20 ( Figure 4—figure supplement 1H–M ) .",
"Consistent with this conclusion , most of the HCs identified in the E12 collection expressed OXT but not IPCEF1 ( Figure 4—figure supplement 1N–P ) .",
"Thus , we conclude that HC1 is a mature type and HC3 its immature counterpart .",
"The ISL1+LHX1- types HC2 , HC4 , and HC5 were distinguished by selective and non-overlapping expression of distinct receptor-type tyrosine kinases , NTRK1 , EGFR , and LTK , respectively ( Figure 4B , D–I ) .",
"In situ hybridization of en face sections demonstrated that these groups form mosaics of HCs , with the density of each mosaic corresponding roughly to the abundance of the type observed by scRNA-seq ( NTRK1>EGFR>LTK; Figure 4J–L ) .",
"NTRK1 ( TrkA ) is expressed by ‘Candelabrum’ axon-less HCs ( Edqvist et al . , 2008 ) which correspond to the abundant HC2 .",
"Using Golgi staining , Quesada et al . , 1988 identified 14 types of BCs in chick based on dendritic morphology , and noted that further subdivisions might be possible if axonal morphology was also considered .",
"Unsupervised clustering of our data identified 22 groups of BCs ( Figure 5A ) ranging in frequency from 1 . 5 to 10 . 4% of all BCs ( Figure 5—figure supplement 1A ) .",
"All expressed the canonical markers VSX2 and OTX2 , and each was distinguished by selective expression of other genes ( Figure 5B ) .",
"Immunostaining and in situ hybridization for several of these genes confirmed their expression by BC subsets ( Figure 5—figure supplement 1B–O ) .",
"In mammals , BCs are conventionally divided into two groups , based on whether they respond to illumination with depolarizing ( ON ) or hyperpolarizing ( OFF ) responses; BCs innervated by cones can be either ON or OFF , whereas those innervated by rods are all ON type ( Euler et al . , 2014 ) .",
"In mammals , ON BCs are characterized by expression of TRPM1 , ISL1 , and GRM6; OFF BCs express GRIK1; and rod BCs express PRKCA in addition to canonical ON markers ( Morgans et al . , 2009; Morgans et al . , 2010; Shekhar et al . , 2016 ) .",
"In our dataset , the expression of GRIK1 was mutually exclusive with that of TRPM1 and ISL1 in all clusters but one , resulting in 11 likely ON types and 10 likely OFF types ( Figure 5C ) .",
"( GRM6 , the canonical marker of mammalian ON BCs is missing in the current version of the chick genome , so we were unable to assess its expression . )",
"In situ hybridization for GRIK1 and TRPM1 confirmed that these genes are also expressed in a largely non-overlapping pattern in chick retina ( Figure 5—figure supplement 1B , C; see below ) .",
"Supporting this assignment , FEZF2 , which marks some OFF BC types in mice ( Shekhar et al . , 2016 ) was enriched in 5 OFF types .",
"PRKCA was expressed at highest levels in BC19 , a putative ON cluster , suggesting that it might represent rod BCs ( Greferath et al . , 1990 ) .",
"BC10 , which expresses both ON and OFF markers might have mixed properties , as has been seen in fish ( Yn et al . , 2012 ) .",
"In general , ON BCs were transcriptomically more closely related to other ON BCs than to OFF BC and vice versa ( Figure 5B , C ) , but the segregation was not as strict as in mice or primates , in which ON and OFF BCs form separate clades ( Shekhar et al . , 2016; Peng et al . , 2019; Yan et al . , 2020b ) .",
"A key feature of BC types is the IPL sublamina or sublaminae in which their axons arborize .",
"We used eCHIKIN and immunohistochemistry to assess lamination , adopting the convention of dividing the IPL into five strata ( S1-5 ) .",
"We generated eCHIKIN probes for 5 BC types: BC1 ( ANGPT2 ) , BC6 ( TPBGL ) , BC8 ( RRAD ) , BC12 ( IRX3 ) , and BC15 ( SLC6A4 ) .",
"As shown in Figure 5D–H , BC1 axons terminated in S3 , BC6 and BC8 axons in S3 , and BC15 in S4; BC12 axons were bistratified with termini in S1 and S2 .",
"Antibodies to TPBGL ( BC6 ) and SLC6A4 ( BC15 ) stained S3 and S4 , respectively , consistent with these assignments ( Figure 5I , J ) ; anti-STRA6 ( BC3 , 9 ) and anti-ERBB4 ( BC12 ) stained S3-4 and S1-2 , respectively ( Figure 5—figure supplement 1Q , R ) .",
"In mammals , there is a relationship between the axonal lamination of BCs and their response properties , with ON cone , OFF cone and rod BC axons arborizing in the order OFF cone > ON cone > rod proceeding from the INL to the GCL .",
"In general , the lamination patterns observed with eCHIKIN and immunohistochemistry followed this rule with three putative OFF cone types ( BC6 , 8 , and 12 ) laminating in S1 and S2 , two putative ON cone types ( BC3 and",
"18 ) laminating in S3 and S4 , and putative rod bipolars ( BC19; PRKCA+ ) laminating in S5 ( Figure 5K ) .",
"The sole exception was BC15 , which was GRIK+ but laminated in S4 .",
"Finally , we asked whether the positions of BC somata in the INL were related to the positions of their axons in the IPL .",
"Somata of putative OFF ( GRIK1+ ) and ON ( TRPM1+ ) BCs were situated in the outer and inner portions of the INL , respectively , corresponding to the positions of their axon terminals ( Figure 5—figure supplement 1B , C ) .",
"To assess correspondence for individual types , we determined somata position by in situ hybridization and axon position as described above .",
"For most types , somata position in INL was correlated with terminal arborization position in IPL ( quantified in Figure 5—figure supplement 1P , and summarized in Figure 5L ) .",
"Interestingly , the somata of BC10 , the putative ON-OFF type ( see above ) , populate the interface between ON and OFF regions in the INL as revealed by SOX5 immunostaining ( Figure 5—figure supplement 1J , P and Figure 5L ) .",
"This correlation has not , to our knowledge , been observed for mammalian BCs .",
"ACs are a diverse class of interneurons , most of which form inhibitory ( GABAergic or glycinergic ) synapses on BCs , RGCs , and other ACs .",
"In mammals , ACs are the most heterogeneous retinal class ( Yan et al . , 2020a; Yan et al . , 2020b; Peng et al . , 2019 ) .",
"Similarly , ACs formed the most heterogeneous class in chicks , with 59 putative types ( Figures 1G and 6A ) , ranging in frequency from 0 . 4 to 7 . 2% of all ACs ( Figure 6—figure supplement 1A ) .",
"All expressed SLC32A1 , a transporter that loads both GABA and glycine into synaptic vesicles , and each type expressed either GABAergic markers ( GABA transporter , SLC6A1 , and the GABA synthetic enzymes , GAD1 and GAD2 , 40 clusters ) or glycinergic markers ( glycine transporter 1 , SCL6A9 , 19 clusters ) ( Figure 6B ) .",
"AC somata were present in both the INL and the GCL .",
"Expression of two broadly expressed member of the AP2 family of transcription factors , TFAP2A and TFAP2B , distinguished these populations: TFAP2A expression was restricted to ACs in the INL , while TFAP2B was expressed by ACs in both locations ( Figure 6B , D , E ) .",
"We identified markers selectively expressed by one or a few types ( Figure 6C ) .",
"Many were neuropeptides , consistent with classical immunohistochemical studies ( Karten and Brecha , 1983; Brecha et al . , 1984; Karten et al . , 1990 ) ; they included TAC1 ( substance P ) , NPY ( neuropeptide Y ) , PENK ( enkephalin ) , NTS ( neurotensin ) , and NMB ( neuromedin-B ) .",
"We used in situ hybridization or immunohistochemistry to validate selective expression of several markers in AC subsets ( e . g . NMB in AC17; CHODL in AC40; NPY in AC52; NTS in AC31 and 58; PENK highest in AC31 , 34 , and 42; and MAFA in AC35 and 58; Figure 6C and Figure 6—figure supplement 2B–E ) .",
"Double label studies distinguished sets of NTS+PENK+ and NTS+MAFA+ double positive ACs , corresponding to AC31 and AC58 , respectively ( Figure 6—figure supplement 1F–H ) .",
"An eCHIKIN probe for NTS also labeled two AC types , a more abundant one with broad processes that ramify in S1 , S3 , and S4 , morphologically reminiscent of pigeon NTS+ ACs ( Brecha et al . , 1984 ) , and a less abundant one with arbors in S5 ( Figure 6—figure supplement 2J , K ) .",
"We further investigated two AC types that have been studied extensively in mammals .",
"One is the starburst amacrine cell ( SAC ) , the only retinal cholinergic cell type .",
"It comprises cohorts in both layers , called ON ( somata in the GCL , with dendrites in S4 ) and OFF ( somata in the INL , with dendrites in S2; Figure 6F; Millar et al . , 1987 ) .",
"Two AC clusters , AC7 and AC25 , expressed the cholinergic markers choline acetyltransferase ( CHAT ) , choline transporter ( SLC5A7 ) , and vesicular acetylcholine transporter ( SLC18A3 ) , indicating their identity as SACs .",
"We assign AC7 and AC25 to ON and OFF SACs , respectively , because AC7 expressed FEZF1 , a transient marker of developing ON starburst marker in mouse , whereas AC25 expressed TENM3 and ZFHX3 , transient OFF markers SAC markers in mouse ( Peng et al . , 2020; Figure 6—figure supplement 2A ) .",
"The other is an unusual excitatory AC , the VG3 amacrine , which expresses the vesicular glutamate transporter 3 ( SLC17A8 , VGlut3 ) .",
"Two closely related chick AC clusters , AC37 and AC39 were SLC17A8-positive , with levels higher in AC37 than AC39 ( Figure 6C and Figure 6—figure supplement 2B ) .",
"AC37 also expressed the recognition molecule Sidekick 2 ( SDK2; Yamagata et al . , 2002 ) , which is also a selective marker of mouse VG3 ACs ( Krishnaswamy et al . , 2015; Yamagata and Sanes , 2018 ) , suggesting that AC37 is the authentic chick VG3 AC ( Figure 6—figure supplement 2D ) .",
"SDK1 , the homolog of SDK2 , is expressed at highest levels in AC38 , but not in chick VG3 AC ( Figure 6—figure supplement 2B , C ) .",
"We generated an eCHICK probe from ERBB4 , which is expressed at highest levels in AC37 and AC38 among ACs , and used it to mark these cells .",
"They stratified in S2 and S4 ( Figure 6—figure supplement 2E ) , consistent with Sdk1 and Sdk2 localization reported previously ( Yamagata et al . , 2002 ) but distinct from mouse VG3 ACs , which stratify in S3 .",
"From 8107 single RGC transcriptomes , we resolved 41 clusters ( Figure 7A ) ranging in abundance from 0 . 6 to 5 . 1% of all RGCs ( Figure 7—figure supplement 1A ) .",
"Like RGCs in mammals , all expressed the canonical RGC markers RBPMS and THY1 , as well as one or more of the Brn3 ( POU4F ) transcription factors and the RBPMS homolog , RBPMS2 ( Figure 7B ) .",
"Each cluster could be specified by selective expression of one or , in some cases , a few genes ( Figure 7B , C; Figure 7—figure supplement 1B–I ) .",
"They include genes that are expressed by subsets of RGCs in mammals such as SATB1 and SATB2 ( Peng et al . , 2019; Figure 7D , E ) .",
"Among the 41 putative RGC types , cluster 11 selectively expressed OPN4 . 1 , a homolog gene to the defining marker of mammalian intrinsically photosensitive RGCs ( ipRGCs ) , as well as the transcription factor EOMES ( Tbr2 ) ( Figure 7B; Figure 7—figure supplement 1I ) , which is expressed in all but not only ipRGCs ( Chaurasia et al . , 2005; Mao et al . , 2014 ) , indicating that GC11 is a chick ipRGC type .",
"We used eCHIKIN to reveal morphologies of three RGC types: GC23 ( TFAP2D ) , with dendrites in S5; GC18 ( MC5R ) , with dendrites in S5; and GC15 and/or 13 ( ETV1 ) with dendrites in S4; ( Figure 7F–H ) .",
"GC15 selectively expresses the recognition molecule SDK1 , which we have shown to be concentrated in S4 ( Yamagata et al . , 2002; Figure 7—figure supplement 2A , B ) .",
"The main central target of chick RGCs is the optic tectum .",
"Within the tectum axons of distinct RGC populations terminate in one of five retinorecipient laminae , called B , C , D upper , D lower and F ( Yamagata and Sanes , 1995a; Yamagata and Sanes , 1995b; Yamagata et al . , 2006 ) .",
"Based on expression of markers identified in previous studies , we conclude that GC25 targets tectal lamina B ( TAC1[substance P]+ , NMB [neuromedin B]+ , SST [somatostatin I]+ , and RELN [reelin]+ ) and GC1 targets tectal lamina F ( CHRNB2 [neuronal acetylcholine receptor beta2 subunit]+ , SS2 [somatostatin II] , and EZR [ezrin]+ ) ( Yamagata et al . , 2006 ) .",
"GC1 is characterized by expression of a transcription factor RUNX2 ( Figure 7B , Figure 7—figure supplement 2C–E ) .",
"Müller glia , the major retinal glial type , has generally been viewed as a homogeneous population ( see Discussion ) .",
"However , we distinguished five clusters in the single-cell dataset of E18 chick retina ( Figure 8A and B ) .",
"In examining genes differentially expressed among these clusters ( Figure 8C ) , we noted three that had been shown to exhibit topographically biased expression at early stages of retinal development , when most cells are still mitotically active: CHRDL1 ( ventropin ) , expressed in ventral retina was enriched in cluster MG1 ( Sakuta et al . , 2001 ) ; and EPHA3 and FOXD1 ( BF2 ) , expressed in temporal retina , were enriched in MG5 ( Cheng et al . , 1995; Yuasa et al . , 1996; Yamagata et al . , 1999 ) .",
"This suggested that positional differences might underlie Müller glia heterogeneity .",
"We used in situ hybridization to test this idea .",
"We documented selective expression of CHRDL1 ( MG1 ) in ventral retina , WIF1 ( MG2 ) in dorsal retina , FOXI2 ( MG5 ) in temporal retina , and FOXG1 ( BF1 ) in nasal retina ( Figure 8D–M ) .",
"FOXG1 was detected in a subset of cells , that failed to form a single cluster but presumably comprise nasal cells ( Figure 8C ) .",
"Finally , in situ hybridization for genes selectively expressed in MG3 ( PSCA ) and MG4 ( TMEM123 ) indicated that cells in these clusters were associated with central and peripheral retina , respectively ( Figure 8N–Q ) .",
"Expression of all markers was graded with position .",
"Co-staining for glutamine synthetase , an MG marker ( Linser and Moscona , 1979 ) , showed that all these genes were selectively expressed in MG ( Figure 8—figure supplement 1 ) .",
"Quantitative analysis confirmed both the distinct expression patterns of genes expressed by each cluster as well as the partial overlap expected for graded expression and the dual identity of , for example , cells in the ventral quadrant of central retina ( Figure 8—figure supplement 2A ) .",
"Together , these results reveal a striking positional map of gene expression in MG ( Figure 8R ) .",
"In light of the central-to-peripheral developmental gradient documented above we wondered whether MG3 ( central ) and MG4 ( peripheral ) represented authentic positional differences or different developmental stages .",
"To distinguish these alternatives , we performed two additional analyses .",
"First , we queried the E12 dataset .",
"Markers of dorsal , ventral , nasal , and temporal retina were selectively expressed , but the peripheral marker at E18 , TMEM123 , was broadly expressed at E12 , suggesting that it is a marker for an early developmental stage ( Figure 8—figure supplement 2A , B; Figure 8—figure supplement 3A ) .",
"PSCA was barely detectable expressed at this stage , but FGF8 , known to mark a small central retinal region ( da Silva and Cepko , 2017 ) was selectively expressed by a restricted group of cells ( Figure 8—figure supplement 3A ) .",
"Second , we used in situ hybridization to assess the distribution of key genes at E12 and E20 .",
"As development proceeds , PSCA and FGF8 are expressed in nested domains , suggesting further distinctions along the central-peripheral axis ( Figure 8—figure supplement 3B ) .",
"At later stages , FGF8 expression declines and TMEM123 is progressively restricted to peripheral regions ( summarized in Figure 8—figure supplement 3C ) .",
"Thus , MG3 represents a positionally restricted cell group , whereas MG4 may largely represente immature MGs .",
"In summary , our analysis revealed a positional basis for the transcriptomic heterogeneity of MG .",
"Reclustering of oligodendrocytes revealed five clusters ( Figure 9A; Figure 9—figure supplement 1A ) .",
"All expressed the oligodendrocyte marker OLIG2 ( Zhou et al . , 2001 ) , but they exhibited differential expression of other known markers of developing and mature oligodendrocytes ( reviewed in Goldman and Kuypers , 2015 ) , suggesting that they represented different developmental stages .",
"Pseudotime analysis arranged the clusters in the order: OL5 , OL3 , OL1 , OL2 , and OL4 , and selectively expressed markers supported this order .",
"For example , HES1 , a marker of oligodendrocyte precursors is expressed in the order OL5>OL3 , OL1>OL2 , OL4; myelin components such as PLP1 and MBP are expressed at highest levels in OL2 and OL4; and CLDN11 , a component of tight junctions formed in compacted myelin , is selectively expressed by OL4 ( Figure 9B and Figure 9—figure supplement 1B ) .",
"Several other genes exhibited similar differential expression , making them candidate markers of successive stages in oligodendrocyte differentiation ( Figure 9—figure supplement 1C ) .",
"We used in situ hybridization to localize oligodendrocytes in retina , using probes for PLP1 and BCAS1 ( OL2 and OL4 ) and PDGFRA ( OL1 and OL3 ) .",
"PLP1 and BCAS1 were co-expressed , whereas PDGFRA+ cells formed a distinct population ( Figure 9B; Figure 9—figure supplement 1B , C ) .",
"Both populations were confined to the GCL .",
"BCAS1+PLP1+ cells were more abundant in central retina , near the optic disc , than in the periphery , whereas PDGFRA+ cells were more abundant peripherally than centrally ( Figure 9C–L ) .",
"These patterns are consistent with the idea that oligodendrocyte precursors enter from the optic nerve and migrate peripherally , with progeny maturing in a central-to-peripheral gradient ( Ono et al . , 1997; Fischer et al . , 2010 ) .",
"As noted in the Introduction , the basic retinal plan , including the structure and placement of its main cell classes , is conserved among vertebrates .",
"We asked whether this morphological conservation is accompanied by transcriptomic conservation .",
"To this end , we combined data from the E18/E16 chick atlas with those from our previously published retinal atlases of mouse ( Macosko et al . , 2015; Shekhar et al . , 2016; Tran et al . , 2019; Yan et al . , 2020a ) , macaque ( Peng et al . , 2019 ) , and human ( Yan et al . , 2020b ) , and submitted the entire group for clustering .",
"This procedure generated nine clusters , all of which contained cells from all species ( Figure 10A , B ) .",
"Each cluster could be identified with high confidence by expression of class-specific markers ( Figure 10C ) and by reference to prior assignments made when each species was analyzed individually ( Figure 10D ) .",
"Six of the clusters corresponded to major retinal cell classes: rods , cones , HCs , BCs , RGCs , and MG .",
"The other three clusters were composed of ACs distinguished by neurotransmitter type: GABAergic , glycinergic , and cholinergic+GABAergic ( SACs ) ; these distinctions are discussed in the next section .",
"Transcriptomic relationship among classes are shown in Figure 10E .",
"The highest level split is between MG and neurons; the second separates PRs from other neurons; the third separates HCs from other interneurons and RGCs; and the last separates GABAergic from glycinergic ACs .",
"These patterns are consistent with structural and functional data , in that glia and neurons play largely non-overlapping roles , PRs are highly specialized neuron-like cells , HCs are unique among interneurons generally , and the GABAergic and glycinergic ACs are inhibitory interneurons with similar functions and patterns of connectivity .",
"The greater similarily of RGCs to ACs than to BCs is also not unexpected in that ACs and RGCs use conventional neurotransmission mechanisms , whereas BC bear ribbon synapses that are shared with PRs but few other neuronal types .",
"On the other hand , the distance placement of SACs is unexpected .",
"All four species obey the same rules of similarity .",
"In contrast , relationships among species vary by class .",
"Humans and macaques are each other’s closest relatives in only four of nine classes , and mice rather than chicks are outliers in seven of nine cases; both of these patterns are unexpected from phylogenetic considerations .",
"The differences are small , however , and may result from technical considerations: the highly variable genes used for clustering may not be sufficient to distinguish cells within any individual class , as the high dimensional space is saturated by genes for other classes .",
"Indeed , the relationship among species differ when each class is clustered separately ( Figure 11 , see below ) .",
"Nonetheless , when all cells are combined and compared by species , retinal cells from chick and mouse are transcriptomically more similar to each other than either is to primates ( Figure 10F ) .",
"We have no explanation for this seeming anomaly .",
"One possibility is that chick and mouse retina are both more complex in terms of numbers of cell types than either human or macaque retina ( Table 1 ) .",
"Finally , we examined the conservation of cell types within classes for the four species , using two methods: building a dendrogram based on similarity matrix of all types from all species ( Figure 11 and Figure 11—figure supplements 1 and",
"2 ) or pooling cells within each class from all species and submitting the combined data for clustering ( shown for ACs and RGCs in Figure 11—figure supplements 1 and 2 ) .",
"Only mature chick types were used for this analysis .",
"Key findings by class are as follows: Cones ( Figure 11A ) : Mammalian M/L and S cones form separate clades .",
"( Primate M and L cones differ transcriptomically only at the opsin locus [Peng et al . , 2019] and are combined here . Most mouse cones are M type , so we had insufficient power to analyze mouse S cones as a separate type ) .",
"Chick cones are outliers , but red ( L ) SCs are most closely with mammalian M/L cones , and blue and green SCs are most closely related to mammalian S cones .",
"Violet SCs and red DCs are distant relatives of both M/L and S clades , which does not correspond to the sequence relationships of their opsins ( Shichida and Yamashita , 2003; Terakita , 2005 ) but does correspond to lineages deduced from evolutionary considerations ( Baden and Osorio , 2019 ) .",
"HCs ( Figure 11B ) : The most notable distinction among HC cells is that some bear axons and other do not .",
"Primate H1 , chick HC1 and all mouse HCs are axon-bearing , while primate H2 , chick HC2 , 4 , and 5 do not .",
"However , whereas mammalian axon-bearing and axon-less HCs form separate groups , all chick HCs are outliers .",
"BCs ( Figure 11C ) : The principal division among BCs is into ON and OFF subclasses , based on whether they depolarize or hyperpolarize to light .",
"They form separate clades in each of the three mammalian species taken individually ( Shekhar et al . , 2016; Peng et al . , 2019; Yan et al . , 2020a ) .",
"Chick BC types have not been characterized physiologically , but based on position and key markers that have been validated , we tentatively assigned all 21 types to ON or OFF subclasses , with the 22nd being potentially ON-OFF .",
"Chick putative ON and OFF BCs also generally divide into transcriptomically separate groups ( Figure 5B , C ) , but the distinction is less absolute than that for mammals .",
"When all four species are considered together , the distinction remains , but one set of chick ON BCs clusters with most mammalian BC types 4 and 5 , leading to a division of ON BCs into two groups .",
"ACs ( Figure 11—figure supplement 1 ) : Most if not all ACs use GABA or glycine as a transmitter or co-transmitter .",
"These subclasses form separate clusters when all retinal cells are analyzed together ( Figure 10 ) and this distinction is largely maintained when ACs are reanalyzed separately .",
"One type , SACs , form a distinct clade , which includes all five types from all four species ( two in chicks ) ( Figure 10E and Figure 11—figure supplement 1C ) .",
"SACs are unique among ACs in many respects: They are the only cholinergic neuron in the retina , they have been found in all vertebrate retinas studied to date , they arise earlier in development than most other ACs , and once generated , their dendrites serve as scaffolds to patterns those of other retinal cell types ( Peng et al . , 2017; Ray et al . , 2018; Duan et al . , 2018; Yan et al . , 2020a ) .",
"They are transcriptomic outliers among ACs in each species separately; what is noteworthy is that SACs of all species are transcriptomically similar to each other .",
"It is difficult to discern similar chick-mammalian conservation for other AC types , at least in part because so few chick AC types have been characterized by structural or physiological criteria .",
"One exception is the VG3 AC , which is identifiable by expression of the glutamate transporter .",
"However , relating clusters derived from a pool of all ACs from all species to types classified in each species separately ( Figure 11—figure supplement 1C ) provides several candidate chick homologs of characterized mammalian AC types .",
"RGCs ( Figure 11—figure supplement 2 ) : We showed previously that conservation of types between mouse and primates was lower for RGCs than other cell classes ( Peng et al . , 2019 ) .",
"This relationship extends to chicks .",
"Comparison of the two clustering methods described above shows a conservation of intrinsically photosensitive RGCs , but we detect no chick close relatives of well-studied mammalian RGC types such as midget or parasol RGCs ( in primates ) , direction-selective or alpha RGCs ( in mice ) ."
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"Germ-line transgenesis is well established in most commonly used model organisms ( mice , zebrafish , Drosophila , C . elegans ) making it possible to generate reporter lines that can be used to reveal the morphology of molecularly defined cell types .",
"This approach has been a powerful one in our studies of mouse and fish retina ( Shekhar et al . , 2016; Tran et al . , 2019; Yan et al . , 2020a; Kölsch et al . , 2020 ) .",
"Lacking this tool for chick , we modified methods for CRISPR-based genome modification in somatic cells ( Mikuni et al . , 2016; Matsuda and Oinuma , 2019; Mikuni , 2020 ) to insert reporters or Cre recombinase into genes identified as ‘cell-type specific’ in our dataset .",
"The use of short ( 70 bp ) homology arms to direct the reporter to appropriate loci made it straightforward to generate targeting fragments .",
"The method can be used to assess the subcellular distribution of the gene product , by fusing the reporter to the coding sequence , or to assess cell morphology , by inserting a soluble fluorescent protein at the translational start site or coupling Cre with a cre-dependent reporter .",
"Of 20 fragements tested , 16 ( 80% ) labeled cells in predicted patterns , including all cases in which we validated the expression pattern by in situ hybridization or antibodies .",
"One potential drawback of the current method is that gene disruption , particularly if both alleles were disrupted , could alter properties of the targeted cells , including their morphology , but we saw no evidence for this in the cells we examined .",
"The 136 ‘mature’ cell types in our atlas are distributed among seven classes: 8 PRs , 4 HCs , 22 BCs , 59 ACs , 41 RGCs , 1 MG ( lumping positional variants , as discussed below ) , and 1 oligodendrocyte .",
"The number is substantially greater than that deduced from morphological surveys ( 11 BCs , 26 RGCs; Quesada et al . , 1988; Naito and Chen , 2004; reviewed in Seifert et al . , 2020 ) .",
"Nonetheless it may not be complete .",
"First , we did not detect microglia , astrocytes or a variant glial type named diacytes ( Rompani and Cepko , 2010 ) , even though all are known to be present .",
"Second , we do not know whether our RGCs include displaced types with somata in the INL , although these are known to be present in birds ( Britto et al . , 1988 ) .",
"Third , although the retina is quite mature at E16-18 , we cannot exclude the possibility that additional types arise later .",
"Fourth , the least abundant AC and RGC types we identified comprised 0 . 4% ( 28/6642 ACs ) and 0 . 6% ( 52/8107 RGCs ) of the cells in each class .",
"Our sample may have been insufficient to detect still less abundant types: they might have been lumped with related types or missed altogether .",
"As one way to address this possibility , we performed a downsampling test on the three most heterogeneous cell classes – BCs , ACs and RGCs .",
"In this test , one asks how many clusters are obtained from a randomly chosen subset of all cells .",
"Cluster number was unchanged with only 60% of BCs or 80% of RGCs , suggesting that we had profiled enough cells to capture nearly all types in these classes ( Figure 12 ) .",
"In contrast , the number of AC clusters declined with removal of even 10% of cells , indicating that our dataset was likely too small to capture all types within this class .",
"Table 1 compares the number of cell types in the chick atlas to those in mouse , monkey and human .",
"The number of types in chick is similar to those in mouse , and nearly two-fold higher than those in monkey or human .",
"Given the sampling limitation discussed above , however , and the larger number of mouse cells profiled ( ~140 , 000 vs ~40 , 000 in chick ) we suspect that the true number of types is higher in chick than in any mammals sampled to date .",
"Thus , our results confirm the suspicion that the chick retina , and perhaps those of other birds , is more complex by this measure than those of mammals .",
"As noted above , an additional 14 clusters represented developmental or positional variants .",
"We do not include them in the atlas , consistent with current views on the distinction between cell type and cell state ( Zeng and Sanes , 2017; Yuste et al . , 2020 ) .",
"Many of the cell types we identified , such as PRs and HCs , could be matched to types previously characterized morphologically or immunohistochemically ( e . g . Yamagata et al . , 2002; Yamagata et al . , 2006; Yamagata and Sanes , 2012; Fischer et al . , 2007; Edqvist et al . , 2008; Enright et al . , 2015 ) .",
"For nearly all of these , we provide new markers that can be used to learn more about their structure , function , and development of each cell type .",
"For example , HCs types are distinguished by selective expression of receptor-type tyrosine kinases that have been implicated in neuronal differentiation ( LTK , EGFR and NTRK1 ) , and could play roles in their type-specific development or function ( Lemmon and Schlessinger , 2010 ) .",
"Other intriguing cell types include a putative ON-OFF bipolar type ( BC10 ) , a putative serotonergic bipolar type ( BC15 , expresses the serotonin transporter [SLC6A2]; see Millar et al . , 1988 ) , a glutamatergic amacrine type ( AC37 , expresses VGlut3 [SLC17A8] ) related to the mammalian VG3 amacrine type ( Krishnaswamy et al . , 2015 ) , and RGC types corresponding to types we showed previously to project to distinct retinorecipient sublaminae in the optic tectum ( GC25 , B-RGCs; GC1 , F-RGCs ) ( Yamagata and Sanes , 1995a; Yamagata and Sanes , 1995b ) .",
"There is a long-standing debate as to whether or not birds may have a ‘midget-like’ pathway akin to that in the primate retina ( Seifert et al . , 2020 ) .",
"This is an important issue , since midget RGCs comprise 80–90% of all RGCs in humans and other primates .",
"We found a close relationship between chick BC18 and primate ‘flat midget bipolars , ’ one of two BC types that innervate midget RGCs .",
"We did not , however find convincing relationships of any chick RGCs to primate midget RGCs .",
"To date , only a single Müller glial type has been identified in mice and primates using scRNAseq ( Macosko et al . , 2015; Shekhar et al . , 2016; Peng et al . , 2019; Yan et al . , 2020b; Wang et al . , 2017 ) .",
"In contrast , unsupervised analysis of chick Müller glia generated five clusters , which were , however , less well separated from each other than types in other classes .",
"In situ hybridization with probes for genes differentially expressed among these clusters revealed a positional basis for the heterogeneity .",
"The five clusters were enriched in cells derived from dorsal , ventral , temporal , central , and peripheral retina; and a sixth group , which did not cluster separately , contained cells from nasal retina .",
"Several genes have been shown to exhibit position-dependent expression in early embryos ( prior to E8; Cheng et al . , 1995; Yuasa et al . , 1996; Yamagata et al . , 1999; Sakuta et al . , 2001 ) ; in each case , their expression at E18 was consistent with prior data .",
"Five of these six groups appear to be authentically position-dependent , based on studies at earlier ages , while the sixth ( peripheral ) may largely reflect the central-to-peripheral gradient of retinal development that has been observed in several species ( Kahn , 1974; Spence and Robson , 1989; Prada et al . , 1991; Bruhn and Cepko , 1996 ) .",
"We sought , but failed to find , positional variants of other cell types .",
"Müller glia are the most abundant single type in our dataset .",
"Thus , weak positional identities may not have been detected in other types .",
"It may be relevant , however , that most of the positional genes we detected were selectively expressed in Müller glia .",
"Recently , Hoang et al . , 2020 reported single-cell transcriptomic data on P10 chicken retina .",
"Reanalysis of their data suggests that similar graded expression of some of these topographic gene , such as CHRDL1 , persist after hatching ( data not shown ) .",
"This result is tantalizing in light of recent reports on region- and lamina-selective differences in gene expression of mammalian astrocytes ( Batiuk et al . , 2020; Bayraktar et al . , 2020 ) .",
"The positional map we describe for chick MG may provide a useful model for investigating the sources and roles of heterogeneity in glial types that were believed until recently to be indivisible .",
"Although our aim was to profile mature retinal types , we found 10 clusters that fit within cell classes by our criteria , but lacked mature features: four PRrs , one HC , one Müller glia , and four oligodendrocytes .",
"Several lines of evidence suggested that these clusters were composed of developmental intermediates .",
"First , histological analysis showed that they were more abundant at earlier stages ( E10-E12 ) than at E18 and , in some cases , less abundant still at E20 .",
"Second , at intermediate stages , their appearance and disappearance followed the known center to periphery gradient of retinal development ( Kahn , 1974; Spence and Robson , 1989; Prada et al . , 1991; Bruhn and Cepko , 1996 ) – that is , mature types were more abundant in central than peripheral retina and the opposite was true for the developmental intermediates .",
"Third , we observed a larger proportion of immature PRs and HCs at E12 .",
"Finally , in some cases , their gene expression patterns were characteristic of immature cells .",
"For example , immature PRs did not express opsin , but did express genes implicated in PR development ( e . g . ARHGAP18 , Maeda et al . , 2011; SLIT1 , Plump et al . , 2002; PRDM1 , Katoh et al . , 2010; Brzezinski et al . , 2013 ) .",
"Similarly , developing oligodendrocytes expressed genes previously associates with successive stages in oligodendrocyte maturation ( Goldman and Kuypers , 2015 ) as well a variety of other genes that now become candidates for stage-specific markers .",
"A recent study reported on ~5000 single-cell transcriptomes from chick retinas explanted at E1 . 5–3 and maintained in vitro for 2 days ( Ghinia Tegla et al . , 2020 ) .",
"As expected from the early stage , most of their cells appear to be progenitors , which we did not find at later stages .",
"They did find two cone , two HC , and one RGC cluster , however , consistent with the early birth of these cells , as shown in Figure 1B .",
"It has been known since the time of Cajal that the fundamental retinal plan is conserved throughout vertebrates: the same cell classes ( PR , HC , BC , AC , RGC , and MG ) are present in every species studied to date ( Cajal , 1892; Lamb et al . , 2007 ) .",
"Cajal also recognized that in the vast majority of cases , each class is divided into multiple types that differ in morphological detail .",
"Our retinal atlas has now allowed us to address two unanswered questions: Does the resemblance of cell classes across orders extend from morphological to molecular similarity and are types within classes conserved across orders ?",
"The answer to first question is clearly yes .",
"Despite the >300 million years since the mammalian and avian lineages diverged ( Kumar and Hedges , 1998 ) , all classes remained transcriptomically similar among species , and share expression of key genes that have been used to mark each class in mammals ( Figure 10 ) .",
"In contrast , types within classes are less well conserved: in only a minority cases , such as PRs , SACs , VG3-ACs and ipRGCs , do chick types have orthologous mammalian types as their closest relatives .",
"This differences between classes and types supports the idea that classes form a common plan within which species evolve distinct types to enable visual behaviors appropriate for their environment , behavioral repertoire , and other sensory capabilities .",
"An open question is whether an underlying similarity might be discovered by examining additional species and/or probing type-specific transcriptional programs .",
"Studies to test these possibilities are underway .",
"The chick has been used for thousands of studies on the development , structure and function of the retina and its projections to central targets ( Nicol , 2015; Cepko et al . , 1996; Adler , 2000; Mey and Thanos , 2000; Wilken and Reh , 2016; Wisely et al . , 2017; pubmed search for chick+retina retrieves >4000 papers ) .",
"Two bottlenecks in moving this work forward have been",
"( a ) lack of a global classification and characterization of chick retinal cell types and",
"( b ) metrics that can be used to relate chick to mammalian retinal cell types .",
"Our goal in the study reported here has been to address these two challenges .",
"Using the powerful method of high-throughput scRNAseq , we provide the first chick retinal atlas since Cajal , 1892 , and a gene-specific Golgi-like method to study neuronal morphology , discovering new features of retinal diversity and development as well as insights into the extent to which retinal cell classes and types are conserved between chick and mammals .",
"We hope that our results will facilitate further use of chick as a model for retinal structure , function , and development ."
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[
"Animals were used in accordance with NIH guidelines and protocols approved by Institutional Animal Use and Care Committee at Harvard University .",
"Fertilized chicken eggs ( specific pathogen free ) were obtained from Charles River Laboratories ( Wilmington , MA ) , and incubated in a 1550 HATCHER ( GQF MFG , Savannah , GA ) at 38°C .",
"Retinas were dissected in Hanks’ balanced salt solution supplemented with 20 mM HEPES , pH 7 . 4 ( HBSS ) .",
"After removing pigment epithelial cells and pecten , whole retinae from both eyes were dissociated at 37°C for 30 min with papain ( LK003160 , Worthington , Lakewood , NJ ) .",
"The same volume of Neurobasal medium ( Thermo Fischer , Waltham , MA ) and 10 µg of deoxyribonuclease I ( DN25 , Sigma , St . Louis , MO ) were added , and cells were triturated .",
"To remove debris , cells were then washed twice through a cushion of the ovomucoid protease inhibitor/BSA/HBSS in the papain dissociation medium .",
"Cells were counted , resuspended in PBS with acetylated BSA , and processed with the Chromium Next GEM Single Cell 3’ Library Construction Kit ( version 2 ) ( 10x Genomics , Pleasanton , CA; Zheng et al . , 2017 ) .",
"Briefly , single cells are partitioned into oil droplets containing single oligonucleotide-derivatized beads followed by cell lysis , barcoded reverse transcription of RNA , amplification , shearing , and attachment of 5’ adaptor and sample index oligos .",
"Libraries were sequenced on the Illumina HiSeq 2500 ( Paired end reads: Read 1 , 26 bp , Read 2 , 98 bp ) .",
"To enrich RGCs , cells dissociated as above were immunopurified with mouse monoclonal antibody to Thy-1 ( BSJ-1: French and Jeffrey , 1986 ) using goat anti-mouse IgG conjugated magnetic beads ( Miltenyi Biotec , Auburn , CA ) ( Yamagata et al . , 2002 ) .",
"Cells were then resuspended in PBS with acetylated BSA and processed as above .",
"We analyzed scRNA-seq data using a pipeline modified from Peng et al . , 2019 .",
"Steps are as follows: Trajectory analysis of oligodendrocytes was performed using the R package ‘Monocle3’ ( Cao et al . , 2019 ) .",
"For cross species analysis , types from each species were down-sampled to a maximal of 200 before pooling .",
"Genes of non-human species were converted to their human homologene using the R package ‘homologene’ and only those with homologenes in all four species were retained for analysis .",
"Clustering was performed using ‘Seurat’ as described above , but with cells from different species aligned using the ‘Reference-based’ integration with all four species as reference .",
"Dendrograms were then built using scaled data after integration , with a total of 100 trees in each case; 80% of cells and 80% of the highly variable genes were used in each build .",
"Final trees were generated using the ‘consensus’ function from the ‘Clann’ package ( Creevey and McInerney , 2005; Figure 10F , Figure 11 and y-axis of Figure 11—figure supplements 1C and 2C ) .",
"Alternatively , trees were built from clusters identified in the integrated space ( x-axis of Figure 11—figure supplements 1C and 2C ) .",
"Initial assessment of the aligned data showed a complete absence of violet opsin ( OPN1SW ) , which we found to result from incomplete sequence in the genome file GRCg6a .",
"We therefore added the cDNA sequence to the annotation file .",
"A down-sampling test was performed by randomly sampling 10–90% of cells within each class in steps of 10% of the total number of cells used to generate the atlas .",
"At each step , downsampling was performed 10 times , using different randomly selected subsets .",
"Subsets were then clustered by the same methods as described above .",
"Antibodies used in this study were: rabbit polyclonal antibodies to GFP ( Millipore , AB3080P ) ; mouse monoclonal anti-Brn3a ( clone , 5A3 . 2 , Millipore , MAB1585 ) ; rabbit anti-calretinin ( Millipore , AB5054 ) ; rabbit anti-calbindin ( Swant , CB38 ) ; rabbit anti-protein kinase C- α ( PKCα ) ( Sigma , P4334 ) ; rabbit anti-VSX2 ( Chx10 , GeneTex , GTX114143 ) ; rat monoclonal anti-HA tag ( Roche , 3F10 ) ; rabbit anti-Satb1 ( Abcam , ab109122 ) ; mouse monoclonal anti-Satb2 ( Abcam , ab51502 ) ; rabbit anti-Met-enkephalin ( ImmunoStar , 20065 ) ; rabbit anti-neuropeptide Y ( Abcam ab10980 ) .",
"Mouse monoclonal antibodies AP2A ( clone , 3B5 ) , OTX1 ( Otx-5F5 ) , and PAX6 were from Developmental Studies Hybridoma Bank ( Iowa City , IA ) .",
"A mouse monoclonal antibody to neuromedin-B ( NMB1 ) was previously described ( Yamagata et al . , 2006 ) and is available from Developmental Studies Hybridoma Bank .",
"Antibodies to cell surface proteins were generated by immunizing L cells that had been stably transfected with cDNAs ( Yamagata and Sanes , 2012 ) .",
"To generate stable cell lines , full-length cDNAs were generated using SuperScript III ( Thermo Fisher ) , amplified from cDNA obtained from chick retina using a high-fidelity Phusion DNA polymerase ( NEB ) , cloned into a piggyBac transposon vector pXL-CAG-Zeocin-3xF2A ( NotI/AscI sites; Martell et al . , 2016 ) by Gibson assembly ( NEBuilder HiFi DNA Assembly , NEB ) , transfected to L cells together with a piggyBac transposase vector pCAG-PBorf using Transporter five transfection reagent ( Polyscience ) , and selected in 1 mg/ml Zeocin ( Invivogen ) for 10–14 days .",
"Cells resistant to Zeocin were pooled , grown , harvested , and rinsed with PBS three times .",
"Female mice were then injected intraperitoneally with 107 cells in 0 . 5 ml four to five times at 2–3 weeks intervals , beginning at 6 weeks of age .",
"Antiserum was then collected , incubated with paraformaldehyde-fixed untransfected L cells to remove irrelevant antibodies , and used for immunostaining .",
"To validate antibodies , 293T human embryonic kidney cells were transfected with each construct , and immunostained .",
"In each case , the antibody stained cells transfected with a cDNA encoding the cognate immunogen but not untransfected cells or cells transfected with cDNA encoding other immunogents ( data not shown ) .",
"For immunostaining , retinas were fixed with 4% ( w/v ) paraformaldehyde/PBS overnight at 4°C , sunk in 15% ( w/v ) and 30% ( w/v ) sucrose/PBS , and mounted in Tissue Freezing Medium ( EM Sciences , Hatfield , PA ) .",
"Sections were cut in a cryostat , either permeabilized with 0 . 1% ( w/v ) TritonX-100/PBS for 5 min at room temperature or with 100% methanol for 15 min at −20°C , blocked with 5% ( w/v ) skim milk/PBS for 30 min at room temperature , incubated with appropriate antibodies overnight , rinsed , and incubated with appropriate secondary antibodies ( Jackson ImmunoResearch , West Grove , PA ) and NeuroTrace 640 ( ThermoFisher/Invitrogen ) .",
"After rinsing with PBS , sections were mounted in VECTASHIELD ( Vector Labs , Burlingame , CA ) and imaged with a Zeiss Meta510 confocal microscope ( Oberkochen , Germany ) .",
"In situ hybridization cDNA for generating RNA probes were amplified from cDNA obtained from chick retina using SuperScript III ( Thermo Fisher ) and a high-fidelity Phusion DNA polymerase ( NEB ) and cloned into either pCMV vectors using Gibson Assembly or into pCR2 . 1 TOPO ( Thermo Fisher ) by TOPO cloning .",
"Probe sequences are listed in Supplementary file 1 .",
"RNA probes were generated from the linearized plasmids using T7 RNA polymerase ( Thermo Fisher ) and digoxygenin- or fluorescein-labeled nucleotides ( Roche ) , and hydrolyzed to around 500 bp if needed .",
"In situ hybridization using nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate and double color in situ hybridization using TSA Plus ( PerkinElmer ) were performed as previously described ( Yamagata et al . , 1999; Yamagata and Sanes , 2012 ) .",
"For double-color in situ hybridization , slides were incubated with 0 . 1M glycine-HCl , pH 2 . 0 , for 30 min at room temperature following the first color reaction to remove peroxidase-conjugated anti-hapten antibodies and thereby avoid cross-reactivity .",
"Sections were mounted in and imaged with a Zeiss Meta510 confocal microscope .",
"To label and visualize cells that express marker genes , we devised eCHIKIN ( electroporation- and CRISPR-mediated Homology-Instructed Knock-IN ) .",
"nBriefly , our method resembles in several respects two others that adapted the initial SLENDR technology ( Mikuni et al . , 2016 ) to mouse embryos ( Ohtsuka et al . , 2018; Miura et al . , 2018; Gurumurthy et al . , 2019 ) .",
"We introduced CRISPR/Cas9 ribonucleoprotein complexes and single-strand DNA to chick embryos by in ovo electroporation , using reagents from the Alt-R CRISPR-Cas9 System ( IDT , Coralville , IA ) .",
"Cas9 requires a CRISPR RNA ( crRNA ) to specify the DNA target sequence .",
"The crRNA sequence was designed to target the sequence near the initiation codon ( ATG ) based on S . pyogenes PAM sequence and the MIT guide specificity score in the UCSC genome browser ( https://genome . ucsc . edu ) ( Supplementary file 2 ) .",
"The crRNA ( 0 . 1nmole ) was first annealed with an equimolar amount of transactivating crRNA ( tracrRNA ) in 5 µl in the annealing buffer ( GenScript ) by heating at 95°C for 5 min followed by rapid chilling .",
"This product was then incubated with S . pyogenes Cas9 protein ( 5 µg ) for 60 min at room temperature to prepare a ribonucleoprotein complex .",
"This complex was mixed with a single-strand DNA ( 0 . 1–0 . 5 µg ) and the other components described below .",
"Each single-strand DNA contains ~70 base gene-specific homology arms at the both ends ( Supplementary file 2 ) .",
"Short single strand DNAs were purchased from IDT ( Coralville , Iowa ) .",
"Longer single strand DNA was prepared by asymmentric PCR using a 1:100 ratio of two primers .",
"Templates included Venus ( Aequorea coerulescens GFP variant ) or Cre ( Addgene plasmid #14797 ) .",
"Following amplification with EconoTaq PLUS GREEN 2X Master Mix ( Lucigen ) , products were agarose gel-purified to select single strand DNA using the QiaQuick Gel Extraction kit ( Qiagen ) ; 20% ( v/v ) isopropanol was added to the solubilization solution before binding to spin columns .",
"The mixture of ribonucleoprotein and single-strand DNA ( 0 . 1–0 . 5 µg ) was electroporated to developing chick embryos ( Hamburger-Hamilton stage ~10 , E1 . 5 ) together with 1 µM Cas9 electroporation enhancer ( carrier DNA from IDT ) and 0 . 1 mM HDR enhancer ( DNA ligase IV inhibitor from IDT ) to enhance homologous recombination .",
"We also added 1 µg piggyBac transposon reporter ( pXL-CAG-mCherry ) and 0 . 1 µg a transposase construct ( pCAG-PBorf ) to monitor successful electroporation .",
"For Cre activation , 1 µg of pXL-CAG-loxP-STOP-loxP-Venus was also included .",
"All the DNA reagents were prepared in advance as ribonuclease-free by extensive phenol-chloroform extractions followed by ethanol precipitation and rinsing with 70% ( v/v ) ethanol .",
"A total of 0 . 01% ( w/v ) Fast Green ( Sigma ) was added to monitor injection .",
"Electroporation was with six square pulses of 7 V for 25 ms using ECM830 ( Harvard Apparatus ) after immersing electrodes with Hanks’ balanced salt solution supplemented with 50 µg/ml kanamycin .",
"After sealing eggshells with plastic tapes , eggs were returned to 37°C incubator .",
"Images were processed with Adobe Photoshop , and Image-J ( Version 1 . 47d , Fiji ) .",
"Position of spots were measured using Image-J .",
"Single-cell RNA-Seq data were analyzed using R 3 . 6 . 2 ( The R foundation , https://www . r-project . org/ ) ."
]
] | [
"Retinal structure and function have been studied in many vertebrate orders , but molecular characterization has been largely confined to mammals .",
"We used single-cell RNA sequencing ( scRNA-seq ) to generate a cell atlas of the chick retina .",
"We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates – photoreceptor , horizontal , bipolar , amacrine , retinal ganglion , and glial cells .",
"To assess morphology of molecularly defined types , we adapted a method for CRISPR-based integration of reporters into selectively expressed genes .",
"For Müller glia , we found that transcriptionally distinct cells were regionally localized along the anterior-posterior , dorsal-ventral , and central-peripheral retinal axes .",
"We also identified immature photoreceptor , horizontal cell , and oligodendrocyte types that persist into late embryonic stages .",
"Finally , we analyzed relationships among chick , mouse , and primate retinal cell classes and types .",
"Our results provide a foundation for anatomical , physiological , evolutionary , and developmental studies of the avian visual system ."
] | [
"The evolutionary relationships of organisms and of genes have long been studied in various ways , including genome sequencing .",
"More recently , the evolutionary relationships among the different types of cells that perform distinct roles in an organism , have become a subject of inquiry .",
"High throughput single-cell RNA sequencing is a technique that allows scientists to determine what genes are switched on in single cells .",
"This technique makes it possible to catalogue the cell types that make up a tissue and generate an atlas of the tissue based on what genes are switched on in each cell .",
"The atlases can then be compared among species .",
"The retina is a light-sensitive tissue that animals with a backbone , called vertebrates , use to see .",
"The basic plan of the retina is very similar in vertebrates: five classes of neurons – the cells that make up the nervous system – are arranged into three layers .",
"The chicken is a highly visual animal and it has frequently been used to study the development of the retina , from understanding how unspecialized embryonic cells become neurons to examining how circuits of neurons form .",
"The structure and role of the retina have been studied in many vertebrates , but detailed descriptions of this tissue at the molecular level have been largely limited to mammals .",
"To bridge this gap , Yamagata , Yan and Sanes generated the first cell atlas of the chicken retina .",
"Additionally , they developed a gene editing-based technique based on CRISPR technology called eCHIKIN to label different cell types based on genes each type switched on selectively , providing a means of matching their shape and location to their molecular identity .",
"Using these methods , it was possible to subdivide each of the five classes of neurons in the retina into multiple distinct types for a total of 136 .",
"The atlas provided a foundation for evolutionary analysis of how retinas evolve to serve the very different visual needs of different species .",
"The chicken cell types could be compared to types previously identified in similar studies of mouse and primate retinas .",
"Comparing the relationships among retinal cells in chickens , mice and primates revealed strong similarities in the overall cell classes represented .",
"However , the results also showed big differences among species in the specific types within each class , and the genes that were switched on within each cell type .",
"These findings may provide a foundation to study the anatomy , physiology , evolution , and development of the avian visual system .",
"Until now , neural development of the chicken retina was being studied without comprehensive knowledge of its cell types or the developmentally important genes they express .",
"The system developed by Yamagata , Yan and Sanes may be used in the future to learn more about vision and to investigate how neural cell types evolve to match the repertoire of each species to its environment ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"immunology and inflammation",
"genetics and genomics"
] | Siglec receptors impact mammalian lifespan by modulating oxidative stress | elife-06184-v1 | [
[
"Aging is controlled partly by genetic factors , such as insulin/IGF-1 , mTOR , AMPK , and Sirtuin signaling pathways ( Lopez-Otin et al . , 2013 ) .",
"Another important element affecting aging is thought to be cumulative damage to macromolecules by reactive oxygen and nitrogen species ( ROS/RNS ) induced by unbalanced cellular inflammatory responses , or generated via mitochondrial dysfunction ( Berlett and Stadtman , 1997; Dizdaroglu et al . , 2002 ) .",
"A large proportion of reactive oxygen species ( ROS ) formed in vivo is derived from the electron transport chain in mitochondria during cellular respiration .",
"Additionally , ROS are generated in blood and tissue phagocytes upon release of superoxide radicals by NADPH oxidase in response to pathogens ( Finkel and Holbrook , 2000 ) .",
"ROS can also be rapidly induced from resident local cells and recruited leukocytes upon tissue injury .",
"Evolution towards an optimal trade-off between protective and damaging ROS levels in organisms includes the introduction of a number of enzymatic and non-enzymatic anti-oxidant mechanisms to maintain homeostasis and mitigate damage .",
"Accordingly , comparative studies have shown association between the longevity of a species and the capacity of cells in its individuals to resist oxidative stress ( Kapahi et al . , 1999; Andziak et al . , 2006; Brown and Stuart , 2007 ) .",
"Understanding the finer details of this regulatory process might also provide access to measures for alleviating and controlling conditions associated with aging , a pressing medical challenge in a society with increasing lifespan .",
"In this study , we sought to determine whether the CD33rSiglecs impact aging and influence lifespan in mammals .",
"Siglecs are mainly expressed by cells of the immune system and bind broadly to sialylated structures of the same cell or of neighboring cells through their extracellular domain ( Crocker et al . , 2007 ) .",
"Two classes of Siglecs are defined based on sequence homology and conservation .",
"The first group ( Sialoadhesin/Siglec-1 , CD22/Siglec-2 , MAG/Siglec-4 and Siglec-15 ) share low sequence identity but are conserved across mammals .",
"In contrast , the genes encoding CD33rSiglecs underwent extensive rearrangements , including duplication , conversion , and pseudogenization , and therefore vary in number and in sequence between different mammal species ( Cao and Crocker , 2011; Padler-Karavani et al . , 2014; Schwarz et al . , 2015 ) .",
"For instance , mice and humans ( the two best studied organisms in this respect ) express five and ten functional CD33rSiglecs , respectively ( Angata et al . , 2004 ) .",
"CD33rSiglecs in humans are numbered ( e . g . , Siglecs-3 , -5 , -6 , -7 , -8 , -9 , -10 , -11 , -XII , -14 and -16 ) , while murine CD33rSiglecs ( other than Siglec-3 ) are identified by a distinct alphabetical nomenclature ( Crocker et al . , 2007; Macauley et al . , 2014 ) .",
"Although information regarding Siglec expression patterns is not comprehensive , it is known that many members are expressed in a cell type-specific manner .",
"For instance , among the murine CD33rSiglecs , CD33 is expressed mainly in granulocytes , Siglec-E is expressed primarily in neutrophils , monocytes , microglia , and dendritic cells , Siglec-F is mainly found in eosinophils and mast cells , Siglec-G is predominantly expressed in B cells and some dendritic cells , and Siglec-H is primarily expressed in plasmacytoid dendritic cells ( Pillai et al . , 2012 ) .",
"Although it is not possible to identify clear CD33rSiglec orthologs between human and murine Siglecs , due to rapid Siglec evolution and deep divergence time between mice and humans , some Siglec receptors ( for instance , Siglec-E and Siglec-9 ) are considered to be functional homologs ( Läubli et al . , 2014 ) .",
"Notably , there is no evidence so far for a significant degree of functional redundancy among Siglecs .",
"Despite the general low affinity of Siglecs towards the sialylated structures , it appears that each Siglec has unique sialoglycan specificity profile with regard to the type of sialic acid , its linkage and the composition of underlying glycan structure .",
"Interestingly , CD33rSiglecs can transmit inhibitory signals into immune cells by phosphorylation of intracellular ITIM or ITIM-like domains , thus quenching pro-inflammatory cascades ( Crocker et al . , 2007 ) .",
"Recently , it has been shown that Siglecs can directly control Toll-like receptor ( TLR ) signaling by sustaining sialic acid-dependent interactions with TLRs and CD14 ( Chen et al . , 2014; Ishida et al . , 2014 ) .",
"As the development of chronic inflammation is one of the hallmarks of aging ( Franceschi et al . , 2005 ) , we investigated whether the number of CD33rSiglecs has co-evolved to modulate the aging process .",
"We asked if the number of CD33rSIGLEC genes correlates with the maximum lifespan in mammalian species and found there was indeed a strong link , which was maintained after correction for phylogenetic or body mass constraints .",
"We then tested if deletion of Siglec-E impacts longevity in mice .",
"Indeed , Siglec-E-deficient mice exhibited accelerated signs of aging compared to littermate controls .",
"We detected an increased rate of oxidative damage to cellular macromolecules at the systemic level , which we found to be related to a disrupted ROS homeostasis and early signs of aging .",
"Finally , we tested the absence of Siglec-E in survival studies and found that these mice had significantly reduced longevity .",
"Our combined data indicate that CD33rSiglecs regulate inflammatory damage and that the expansion of their number in the genome has coevolved with the extension of lifespan in mammals ."
],
[
"The evolutionary theory of germ line and disposable soma predicts that long-lived species assure their longevity through investments in more resilient somatic tissues ( Moore et al . , 1991; Kirkwood , 1992 ) .",
"It follows then that genes involved in management of cellular stress and repair of damage contribute to lifespan .",
"Indeed , it has been experimentally shown that lifespan of eight mammalian species correlates to the ability of their primary fibroblasts to cope with stress ( Kapahi et al . , 1999 ) .",
"As Siglecs are capable of modulating cellular inflammatory responses and the number of genes encoding CD33rSiglecs varies widely between species ( Angata et al . , 2004 ) , we asked if CD33rSIGLEC gene number correlates with maximum lifespan in mammals .",
"A positive correlation was observed between these two parameters in the 14 mammalian species tested ( R2 = 0 . 7630 ) ( Figure 1A , Figure 1—figure supplements 1 , 2 ) .",
"As the genes encoding CD33rSiglecs are mainly found in a single syntenic cluster in each species , we considered the possibility that the observed correlation could be due to factors associated with the chromosomal environment surrounding these genes or due to hitchhiking effects with adjacent genes .",
"We therefore examined the Kallikrein-related peptidase ( KLK ) gene cluster , which is located in the chromosomal region immediately adjacent to CD33rSIGLECs in most of the 14 species .",
"Strikingly , the number of KLK genes showed a poor correlation with mammalian lifespan ( R2 = 0 . 1825 ) ( Figure 1B ) .",
"Next , we tested if the observed correlation of CD33rSIGLEC/maximum lifespan was due to a general expansion of genes encoding for cell surface receptors that interact with pathogens to initiate immune responses .",
"Therefore , we examined Toll-like receptor ( TLR ) genes , which play important roles in innate recognition of PAMPs and DAMPs ( Janeway and Medzhitov , 2002; Beutler , 2009 ) , and genes for IgG Fc gamma receptors , which bind the Fc region of IgG to regulate immune responses ( Nimmerjahn and Ravetch , 2008 ) .",
"Predicted gene numbers for both families showed only marginal association with maximum lifespan ( Figure 1C , D ) . 10 . 7554/eLife . 06184 . 003Figure 1 . Correlation between gene numbers in gene families and maximum lifespan in mammals . Numbers of CD33rSiglecs ( A ) , KLK ( B ) , IgG Fc receptors ( C ) , and TLRs genes ( D ) and maximum lifespan in 14 mammalian species listed in Figure 1—figure supplement 1 .",
"( E and F )",
"Correlation of CD33rSIGLECs and maximum lifespan after correction for average adult body weight and phylogeny .",
"PGLS: λ = 1 , phylogenetic tree I ( E ) or tree II ( F ) were used .",
"The Pearson's correlation coefficient ( R2 ) for each plot is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 00310 . 7554/eLife . 06184 . 004Figure 1—figure supplement 1 . Data of 14 mammalian species used for analysis of correlation . Maximum lifespan and average adult body weight were retrieved from the AnAge database .",
"The number of genes of each family was either found in the literature or searched following the methodology described in the ‘Materials and methods’ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 00410 . 7554/eLife . 06184 . 005Figure 1—figure supplement 2 . Correlation between number of genes of each family and maximum lifespan . Data are shown in a linear scale , whereas are presented in a logarithmic scale in Figure 1 for statistical reasons .",
"The correlation coefficient ( R2 ) is indicated for each plot . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 00510 . 7554/eLife . 06184 . 006Figure 1—figure supplement 3 . Phylogeny and tree branch information . Phylogeny and tree branch information used in this study were obtained from Prasad et al . ( 2008 ) and trimmed by Archaeopteryx 0 . 957 ( Zmasek and Eddy , 2001; Han and Zmasek , 2009 ) down to the same 14 species .",
"The topology is also indicated in the Newick tree format . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 006 Since closely related species may also share similar traits simply due to their common ancestry , data from different species may not be statistically independent .",
"To control for such effects , we used phylogenetic comparative analysis using Phylogeny Generalized Least-Squares ( PGLS ) or Felsenstein's Independent Contrast ( FIC ) approaches .",
"The correlation between CD33rSiglecs and longevity remained very strong after such phylogenetic correction ( Table 1 and Figure 1—figure supplement 3 ) .",
"Moreover , the correlation was maintained after mathematical correction for body mass represented by average adult body weight ( Table 2 ) , another factor known to correlate with metabolic rate and lifespan ( Manini , 2010 ) .",
"Overall , a positive correlation was shown between the residual maximum lifespan and residual CD33rSIGLEC gene numbers ( controlling for both body mass and phylogeny ) ( Figure 1E , F ) .",
"Since the time that these data were originally collected and evaluated , additional genome sequences have become available .",
"Therefore , in order to further test the strength of the correlation , we included the data from three short-lived primate genomes ( Saimiri boliviensis , Tarsius syrichta , and Otolemur garnettii ) .",
"Interestingly , these genomes were found to have fewer CD33rSIGLEC genes ( 5 , 5 , and 4 genes , respectively ) .",
"Addition of these data to the primary correlation did not change the statistical significance of the association between number of CD33rSIGLEC genes and maximum longevity ( R2 = 0 . 661 in logarithmic scale , R2 = 0 . 752 in linear scale ) .",
"Furthermore , based on the different sample size of different species tested , an adjusted value of maximum longevity of 90 years for humans was considered , in line with previous studies ( Lorenzini et al . , 2005 ) .",
"Notably , the overall correlation between number of CD33rSIGLEC genes and maximum longevity remained strong ( R2 = 0 . 649 in the logarithmic scale , R2 = 0 . 843 in the linear scale ) . 10 . 7554/eLife . 06184 . 007Table 1 . Statistical analysis of the correlation between number of genes and maximum lifespan , corrected for phylogenyDOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 007Gene familyPGLSFICTree I CD33rSIGLECs0 . 000160 . 00012 KLKs0 . 490 . 17 TLRs0 . 380 . 10 IgG Fc receptors0 . 350 . 0016Tree II CD33rSIGLECs0 . 000170 . 00011 KLKs0 . 650 . 23 TLRs0 . 320 . 14 IgG Fc receptors0 . 390 . 0019Phylogenetic comparative analysis was conducted in COMPARE 4 . 6b using Phylogeny Generalized Least-Squares ( PGLS ) or Felsenstein's Independent Contrast ( FIC ) approaches .",
"Student's t-values were computed based on the regression slopes and the standard errors .",
"Two-tailed probability ( p ) value of a Student's t-test was estimated using a degree of freedom of 11 .",
"The phylogenetic relationship of 14 mammalian species represented by Tree I and Tree II are indicated in Figure 1—figure supplement 3 .",
"Note that although FIC analysis obtained a significant p value for IgG Fc receptor gene family , the p value increased to 0 . 56 when human data was excluded .",
"Thus , this correlation is driven by one outlier data point . 10 . 7554/eLife . 06184 . 008Table 2 . Statistical analysis of the correlation between number of genes and maximum lifespan , corrected for body weightDOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 008Gene familyt valuep valueTree I CD33rSIGLECs3 . 5214350 . 004786 KLKs0 . 3606010 . 725226 TLRs−0 . 7018220 . 497370 IgG Fc receptors1 . 6565190 . 125834Tree II CD33rSIGLECs3 . 6609170 . 003748 KLKs0 . 1802270 . 860251 TLRs−0 . 7264650 . 482726 IgG Fc receptors1 . 5895860 . 140235Phylogenetic comparative analysis conducted in CAIC package .",
"Average adult body weight and maximum lifespan of 14 mammalian species were log-transformed and phylogenetic regressions were run using pglmEstLambda in the CAIC package in R . This function uses the PGLS method and estimates λ with the average adult body weight controlled for .",
"Student's t-values and two-tailed probability ( p ) values are shown .",
"The phylogenetic relationship of 14 mammalian species represented by Tree I and Tree II are indicated in Figure 1—figure supplement 3 .",
"Taken together , these data indicate that the number of CD33rSIGLEC genes correlates to lifespan in mammals .",
"This correlation appears to be independent from phylogenetic constraints , from effects of genomic location , from a generally observed rapid evolution of receptors involved in immune responses and from body mass .",
"We decided to use a mouse model to seek experimental evidence for the observed correlation , as mice have a simplified CD33rSiglec profile compared to other mammalian model systems , in terms of number of genes and expression patterns .",
"In fact , mice possess five CD33rSiglecs ( namely , CD33 , Siglec-E , -F , -G , -H ) .",
"Among these , Siglec-E is the dominant receptor and it is strongly expressed on neutrophils , tissue macrophages ( Figure 2—figure supplement 1 ) , and microglia ( Zhang et al . , 2004; Claude et al . , 2013 ) .",
"We monitored the survival of mice lacking Siglec-E ( Siglec-E−/− ) over the course of 100 weeks in comparison to their control wild type littermates ( WT ) ( Figure 2A ) .",
"The survival study was carried out in two sequential cohorts totaling 117 WT and 120 Siglec-E−/− mice .",
"Overall survival of the Siglec-E−/− males was markedly reduced compared to WT ( 48% and 70% remaining , respectively , when the experiment was terminated ) .",
"Similarly , relative to the WT , the median survival of Siglec-E−/− females decreased by 17% .",
"In an attempt to mimic natural conditions of early exposure to inflammatory insults , we exposed all groups of mice to a non-specific antigenic challenge early in life ( heterologous cell membranes mixed with Freund's adjuvant ) .",
"This treatment did not affect the viability of Siglec-E−/− mice over 100 weeks ( Figure 2—figure supplement 2 ) .",
"No differences in general appearance or body weight were noted and no signs of specific pathologies were observed during the study ( Figure 2—figure supplement 3 ) .",
"Hematological and biochemical analysis of blood samples at periodic intervals and at termination of the study did not reveal significant differences between the two groups ( Figure 2—figure supplement 4 ) .",
"Additionally , there was no evidence indicative of systemic chronic disease , such as increased leukocyte counts , microcytic anemia , or hypoalbuminemia .",
"Serum creatinine levels did not suggest diminished renal function .",
"Higher values of alanine aminotransferase were noted for Siglec-E−/− animals but were not statistically different from the controls .",
"Similarly , systematic histological analysis of multiple organs showed no evidence of pathological abnormalities , though we observed sporadic instances of periportal liver inflammation , and a slight increase in lung inflammation compared to control mice ( Figure 2—figure supplement 5 ) .",
"Examination of kidneys showed that more of the Siglec-E−/− mice exhibited minor age-related glomerular changes , with thickening of glomerular tufts , visible on Periodic-Acid Schiff stains .",
"These data were in line with previous work on the same mice at a younger age ( McMillan et al . , 2013 ) . 10 . 7554/eLife . 06184 . 009Figure 2 . Absence of immunomodulatory Siglec-E aggravates aging phenotypes and reduces lifespan in mice .",
"( A ) Survival curves of WT and Siglec-E−/− male ( n = 59–62 ) and female ( n = 58–59 ) littermates .",
"Data are from two independent cohorts ( cohort 1 included 31 WT and 38 Siglec-E−/− males , 31 WT and 33 Siglec-E−/− females . Cohort 2 included 28 WT and 23 Siglec-E−/− males , 27 WT and 26 Siglec-E−/− females ) .",
"Log-rank test analysis showed significant differences in the survival curves both in males and females ( males: χ2 = 5 . 833 , d . f . = 1 and p = 0 . 0157; females: χ2 = 8 . 821 , d . f . = 1 and p = 0 . 0030 ) .",
"( B and C )",
"Mice at 80 weeks were assessed for spatial learning and memory via Barnes maze .",
"Latency to escape ( B ) and number of errors before finding the escape box ( C ) are indicated in 3-day interval .",
"Error bars reflect mean ± s . e . m . ( n = 11 ) .",
"p was calculated with a Student's t test .",
"( D ) Hair graying of males was evaluated by three independent observers in a blind test .",
"Average rank scores for each mouse are indicated .",
"Error bars reflect mean ± s . e . m . ( n = 9–11 ) .",
"p was calculated with a Student's t test .",
"( E ) Surviving mice were sacrificed at 100 weeks of age .",
"Representative field of β-galactosidase staining of liver .",
"Arrows indicate cells with increased localized staining .",
"Scale bar is 100 μm .",
"( F ) Skin epidermal thickness was measured for WT and Siglec-E−/− .",
"Mean and s . e . m . are indicated , n = 12 , p was calculated with a Student's t test . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 00910 . 7554/eLife . 06184 . 010Figure 2—figure supplement 1 . Expression of Siglec-E in mouse tissues . Sections of frozen organs from WT and Siglec-E−/− animals were stained with anti-Siglec-E antibodies .",
"Siglec-E is expressed in splenocytes and in tissue macrophages in liver , heart , and kidney . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01010 . 7554/eLife . 06184 . 011Figure 2—figure supplement 2 . Exposure to human red blood cell membranes does not impact survival of Siglec-E−/− mice . Survival curves of mice intraperitoneally ( IP ) injected with human red blood cell membranes and Freund's adjuvant ( ADJ + HRBC ) .",
"Controls mice were injected with adjuvant and PBS ( ADJ + PBS ) or PBS only .",
"There are no significant differences between groups .",
"Log-rank ( Mantel–Cox ) test: Chi square = 1 . 310 , df = 3 , p = 0 . 7268 .",
"Gehan-Breslow-Wilcoxon test: Chi square = 0 . 6019 , df = 3 , p = 0 . 8960 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01110 . 7554/eLife . 06184 . 012Figure 2—figure supplement 3 . Deletion of Siglec-E does not alter body weight increase . Weights of mice were recorded for mice from the two cohorts characterized in this study .",
"Indicated are mean ± sem , * indicates p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01210 . 7554/eLife . 06184 . 013Figure 2—figure supplement 4 . Hematology and serum chemistry values of male mice at the termination of the study . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01310 . 7554/eLife . 06184 . 014Figure 2—figure supplement 5 . Absence of Siglec-E is associated with overall increased inflammation in liver and lung . Paraffin sections of liver or lungs from WT and Siglec-E−/− animals were analyzed for inflammation using anti-CD45 immunohistochemistry and demonstrate many more areas with accumulation of inflammatory cells in Siglec-E−/− age mice . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01410 . 7554/eLife . 06184 . 015Figure 2—figure supplement 6 . Deletion of Siglec-E does not affect locomotor activity . WT and Siglec-E−/− mice show habituation of activity ( a significant decrease ) across the 2 hr test in the activity chamber .",
"There are no differences between the groups .",
"Indicated are mean ± sem , * indicates p < 0 . 05 , n = 7–11 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 015 We submitted the mice to a series of analyses to test if Siglec-E−/− animals exhibited exacerbated age-related defects .",
"First , 80-week-old Siglec-E−/− mice showed a threefold increase in error rate in the Barnes maze test compared to controls ( Figure 2B , C ) .",
"These results are consistent with previous reports of impairments in learning and in spatial memory in aged mice ( Kennard and Woodruff-Pak , 2011 ) .",
"Deficits in the Barnes maze were not due to alterations in locomotor activity ( Figure 2—figure supplement 6 ) .",
"Secondly , a blind test involving three independent observers noted increased hair graying in Siglec-E−/− males compared to WT ( Figure 2D ) .",
"Hair graying is related to incomplete maintenance of melanocyte stem cells through loss of the differentiated progeny that occurs physiologically during aging ( Nishimura et al . , 2005 ) .",
"After termination of the survival study , immunohistochemistry of liver tissues revealed an increased frequency of focal expression of beta-galactosidase , a marker of senescent cells ( Figure 2E ) .",
"Moreover , examination of the epidermis revealed a 50% reduction in thickness in animals lacking Siglec-E ( Figure 2F ) .",
"Epidermis tends to thin with increasing age through mechanisms that possibly involve senescent cells ( Lopez-Otin et al . , 2013 ) .",
"Collectively , these data indicate that deletion of Siglec-E results in a faster progression of aging and , consequently , to increased frailty leading to an earlier death .",
"Siglec-E regulates inflammatory states upon acute stress ( McMillan et al . , 2013; Chang et al . , 2014 ) .",
"We speculated that aging might act as a chronic stimulus and investigated whether Siglec-E−/− mice exhibited low-grade signs of inflammation .",
"As noted above , some organs showed accumulation of inflammatory cells ( Figure 2—figure supplement 5 ) .",
"Inflammation was not due to anti-nuclear antibodies , which are typical of some autoimmune diseases but were undetectable in the sera of Siglec-E−/− and WT mice .",
"To gain mechanistic insights , we analyzed the role of Siglec-E on the management of oxidative stress in innate immune cells .",
"Primary bone marrow neutrophils from Siglec-E−/− mice were more prone to produce oxidative burst upon stimulation , compared to controls ( Figure 3A and Figure 3—figure supplement 1 ) .",
"Additionally , neutrophils lacking Siglec-E secreted higher ROS per cell ( Figure 3B ) .",
"Similarly , thioglycollate-recruited peritoneal neutrophils showed a 10% increase in ROS ( Figure 3—figure supplement 2 ) , corroborating the notion that Siglec-E controls oxidative stress and that the elimination of CD33rSiglec receptors leads to disordered ROS .",
"These observations were also in line with what was shown with a microglial cell line ( Claude et al . , 2013 ) . 10 . 7554/eLife . 06184 . 016Figure 3 . Altered ROS homeostasis in mice lacking Siglec-E .",
"( A ) Neutrophils purified from bone marrow were incubated with immunocomplexes .",
"Cells producing vacuolar ROS were measured by flow cytometry after 60 min .",
"Representative of three experiments , for each n = 3 .",
"( B ) Neutrophils secrete ROS upon stimulation with PMA for 60 min .",
"Extracellular ROS were detected with a probe that does not cross the plasma membrane ( n = 11–12 ) .",
"( C ) Representative Gstp1 immunohistochemistry in liver from WT or Siglec-E−/− male mice at 100 weeks .",
"Expression pattern is altered in the knockout mice .",
"( D ) Immunoblot analysis and quantification of Gstp1 expression in liver of 100-week-old mice .",
"The level of Gstp1 protein is reduced of about 40% .",
"p was calculated with a Student's t test , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01610 . 7554/eLife . 06184 . 017Figure 3—figure supplement 1 . Neutrophils lacking Siglec-E are more prone to oxidative burst . Neutrophils isolated from bone marrow were incubated with immunocomplexes conjugated with a probe sensitive to ROS .",
"Flow cytometry profiles at 60 min indicate that cells lacking Siglec-E are more prone to produce ROS , whereas the levels of ROS are comparably low before stimulation .",
"Gates indicate cells producing ROS . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01710 . 7554/eLife . 06184 . 018Figure 3—figure supplement 2 . Thioglycollate-elicited neutrophils from Siglec-E−/− produce higher ROS than WT controls . Neutrophils isolated from peritoneum were incubated with immunocomplexes conjugated with a probe sensitive to ROS .",
"The percentage of cells producing ROS after 60 min of incubation is higher when Siglec-E is absent , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 01810 . 7554/eLife . 06184 . 019Figure 3—figure supplement 3 . Gstp1 is found in lower levels in the liver of Siglec-E−/− mice .",
"( A ) Representative images generated during analysis of Gstp1 staining of liver tissues .",
"Original images ( left ) were converted to grayscale to discard color information and only indicate brightness .",
"Normalized images are shown in the second column ( with jet color map ) .",
"Images with pixel labels are in the third column ( red: bright pixels , blue: dark pixels , green: discarded from analysis ) .",
"In the last column , the labeled images are overlayed on the original image to compare pixel labels to the original ( 30% transparency ) .",
"Three images per liver sample were acquired and processed in this way .",
"( B ) Quantification of the Gstp1 staining , as measured by the proportion of dark/bright areas .",
"Mean ± sem , n = 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 019 Since we found evidence of inflammation in the liver , we used a microarray to examine differential gene expression in this organ in aged Siglec-E−/− animals .",
"The liver is a central organ for the regulation of glucose homeostasis , xenobiotic metabolism and detoxification , and steroid hormone biosynthesis and degradation .",
"Gene expression analysis in aged C57BL/6 mice has indicated that 40% of the genes with changes in expression during aging are associated with inflammation ( Lee et al . , 1999; Cao et al . , 2001 ) .",
"Another set of genes undergoing changes is related to stress response and chaperones , followed by genes involved in xenobiotic metabolism .",
"Principal component analysis uncovered a subset of genes whose expression differed significantly between genotypes .",
"Pathway analysis of differentially regulated genes suggested changes in leukocyte-mediated inflammation , including increased activation of leukocytes and granulocyte movement , as well as an increase in ROS metabolism in the Siglec-E−/− mice ( Supplementary file 1 ) .",
"Interestingly , the glutathione S-transferase protein 1 ( gstp1 ) gene was found to be down-regulated .",
"Gstp1 catalyzes nucleophilic attack by reduced glutathione on a variety of electrophilic compounds ( Hayes et al . , 2005 ) .",
"The resulting complexes are usually less toxic and are eventually metabolized and exported via a glutathione-dependent transport system .",
"In humans , early loss of Gstp1 expression due to promoter hypermethylation results in increased cancer susceptibility ( Lin et al . , 2001 ) .",
"In male mice , Gstp1 is quantitatively the principal glutathione transferase in the liver and is found at lower levels in other organs ( Knight et al . , 2007 ) .",
"In liver from WT male mice , anti-Gstp1 antibodies stained hepatocytes of the zone 1 ( Figure 3C ) .",
"A less defined pattern was observed in liver sections of Siglec-E−/− mice .",
"Overall , we observed a substantial difference in staining ( Figure 3—figure supplement 3 ) .",
"Immunoblot analysis confirmed a 40% reduction in Gstp1 expression ( Figure 3D ) .",
"It is interesting to note that Gsto1 gene , encoding for another glutathione S-transferase , was found to be negatively regulated by age in a previous study ( Cao et al . , 2001 ) .",
"Thus , changes in the xenobiotic-metabolizing capacity of the liver appear to be intimately connected to the aging process .",
"Taken together , these data indicate that absence of Siglec-E leads to a dysregulation of ROS metabolism , resulting in increased levels of reactive species .",
"This phenomenon is due to both an increased production of vacuolar ROS and a deficiency of removal of ROS .",
"Many types of ROS that are formed to serve a signaling or protective function can also cause damage spontaneously to lipids , nucleic acids , and proteins .",
"Polyunsaturated fatty acids are a sensitive oxidation targets for ROS because of a damaging chain reaction that takes place once lipid peroxidation is initiated ( Niki , 2009 ) .",
"DNA bases are also very susceptible to ROS attack , and oxidation of DNA is believed to cause mutations and deletions ( Fraga et al . , 1990 ) .",
"Most amino acids in a protein can be oxidized by ROS , with these modifications leading to a loss of function ( Brennan and Hazen , 2003 ) .",
"Such damage occurs constantly , and cells must repair it or replace the impaired molecules .",
"Defects that allow oxidative damage to accumulate can contribute to the origin and progression of cancers and neurodegenerative diseases , and in general contribute to the symptoms of aging ( Berlett and Stadtman , 1997; Halliwell , 2013 ) .",
"Similarly , impairment of the processes that control ROS levels can lead to molecular damage .",
"We looked for signs of molecular damage in the organs of the Siglec-E−/− mice , and found a 1 . 4-fold increase of DNA damage in liver compared to WT ( Figure 4A ) .",
"This was in line with the evidence that glutathione S-transferases protect cells against as much as 90% of the damage induced by electrophiles and other free radicals ( Vasieva , 2011 ) .",
"Brain , spleen , and heart tissues also showed a slight trend towards increase in DNA damage ( Figure 4—figure supplement 1 ) .",
"Notably , these differences were not detected in the organs of 10-week-old mice ( Figure 4—figure supplement 2 ) .",
"We then searched for oxidative adducts in proteins elsewhere in the body and found elevated plasma protein-bound 3-nitrotyrosine levels , a marker of protein modification by nitric oxide ( NO ) -derived oxidants ( Figure 4B ) .",
"Similarly , liver of Siglec-E−/− mice showed a trend towards accumulation of oxidized amino acids in proteins compared to WT ( Figure 4—figure supplement 3 ) .",
"Furthermore , we detected a twofold increase of F2-isoprostanes levels , including 8-iso Prostaglandin F2α and its metabolite 2 , 3-dinor-8-iso PGF2α in the urine ( Figure 4C , D ) .",
"F2-isoprostanes are generated by non-enzymatic peroxidation of arachidonic acid due to free radical species ( Montuschi et al . , 2004 ) .",
"Taken together , these data indicate that elimination of Siglec-E leads to accelerated oxidative modification of DNA , proteins and lipids at the systemic level , via elevated ROS and reactive nitrogen species ( RNS ) production . 10 . 7554/eLife . 06184 . 020Figure 4 . Increased oxidative damage in mice lacking Siglec-E .",
"( A ) Oxidative damage of DNA ( AP sites , apurinic/apyrimidinic sites ) was measured in the liver from WT and Siglec-E−/− mice at 100 weeks , n = 10 .",
"( B ) Nitrotyrosine ( NO2Tyr ) accumulation in plasma proteins of Siglec-E−/− mice , n = 8 .",
"( C and D )",
"Concentrations of F2-isoprostanes in urine derived from free radical-induced oxidation of arachidonic acid .",
"Values were normalized by creatinine levels to account for dilution in urine .",
"F2-Isoprostane levels are significantly higher in Siglec-E−/− mice .",
"Data are mean ± s . e . m . , Student's t test . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 02010 . 7554/eLife . 06184 . 021Figure 4—figure supplement 1 . Spleen and brain of aged WT and mutant mice have equivalent levels of DNA damage . Quantification of apurinic/apyrimidinic ( AP ) sites in DNA from spleen or brain .",
"Mean ± sem is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 02110 . 7554/eLife . 06184 . 022Figure 4—figure supplement 2 . Young Siglec-E−/− mice exhibit levels of DNA damage comparable to WT . Quantification of AP sites in DNA from liver , heart , or spleen of 10-week-old mice .",
"DNA damage is expressed as ratio between DNA damage in Siglec-E−/− and WT animals , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 02210 . 7554/eLife . 06184 . 023Figure 4—figure supplement 3 . Oxidation of hepatic proteins . Protein-bound oxidized amino acid content ( normalized to the precursor amino acid ) in proteins from liver homogenates was determined by stable isotope dilution LC/MS/MS analysis .",
"Methyl-tyrosine ( m-Tyr ) is a stable adduct of phenylalanine ( Phe ) .",
"Brominated tyrosine ( BrTyr ) is formed by brominating oxidants .",
"Di-tyrosine ( Tyr ) is an oxidative crosslink .",
"Indicated is mean ± sem , n = 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 06184 . 023"
],
[
"CD33rSiglecs differ by a great degree in number , sequence and expression pattern among mammalian species .",
"Together with the evidence that many genes involved in the biosynthesis of sialylated glycoconjugates are rapidly evolving and that some bacterial pathogens can also produce sialylated structures , this led to the hypothesis that CD33rSiglecs function to recognize self in the form of the host sialic acids ( ‘self-sialome’ ) , thereby dampening unwanted responses in the steady state by immune cells , wherein CD33rSiglecs are prominently expressed ( Crocker and Varki , 2001 ) .",
"Indeed , it has been shown for several CD33rSiglecs that ligation of sialic acid results in the phosphorylation of tyrosine residues of the intracellular immunoreceptor tyrosine-based inhibitory motifs ( ITIMs ) , followed by the recruitment of phosphatases SHP-1 and SHP-2 that turn off the pro-inflammatory cascade ( Angata et al . , 2002; Ikehara et al . , 2004 ) .",
"Therefore , CD33rSiglecs engage in a dense network of sialic acid-dependent interactions in cis on the cell membrane to provide homeostasis .",
"However , higher affinity ligands on other cells can displace cis interactions to take advantage of the inhibitory properties of CD33rSiglecs in these host cells .",
"For instance , many cancer cells produce a heavily sialylated cell surfaces to contact Siglecs of natural killer cells and neutrophils , allowing successful escape from immune recognition ( Hudak et al . , 2014; Jandus et al . , 2014; Läubli et al . , 2014 ) .",
"Similarly , bacterial pathogens expressing sialic acids can engage CD33rSiglecs ( Carlin et al . , 2009; Chang et al . , 2014 ) and might therefore represent strong driving forces for evolution of this class of receptors ( Varki , 2011 ) .",
"Of course , other yet unexplored functions of Siglecs might also be associated to their variation in number .",
"In this study , we analyzed the expansion of CD33rSIGLEC genes in the context of evolution of aging .",
"To test our hypothesis that inhibitory CD33rSiglecs might affect aging by regulating ROS homeostasis , we used mice as a simplified model system as deletion of Siglec-E results essentially in the removal of most of the CD33rSiglec receptors from macrophages and neutrophils , which are the main producers of ROS upon inflammatory stimuli .",
"Mice lacking Siglec-E are viable , exhibit no apparent developmental defect and reproduce normally ( McMillan et al . , 2013 ) .",
"Upon acute challenge by lipopolysaccharide-induced airway inflammation or by intravenous administration of bacteria , Siglec-E deficient mice develop exaggerated neutrophil recruitment to the lung and produce higher levels of pro-inflammatory cytokines ( McMillan et al . , 2013; Chang et al . , 2014 ) .",
"However , as for other Siglec-deficient mice such as CD33 or Siglec-F null animals , no clear phenotype was reported in absence of acute challenge ( Brinkman-Van der Linden et al . , 2003; Zhang et al . , 2007 ) .",
"Here , we showed that Siglec-E affects ROS homeostasis , and deletion of Siglec-E results in overproduction of ROS .",
"This , together with a secondary impairment in the radical-scavenging enzyme Gstp1 expression leads to higher levels of oxidative adducts of proteins , lipids and DNA , which may lead to acceleration of aging .",
"We thus concluded that Siglec-E impacts aging in mice through regulation of ROS homeostasis .",
"Formally , we cannot state that the upregulation of ROS due to the absence of Siglec-E is the direct cause of the observed molecular damage .",
"In fact , the observed levels of oxidation may be primarily due to the impairment of the detoxification system , which might be controlled upstream by Siglec-E through ROS signaling .",
"Interestingly , the latter hypothesis is in line with a recent revision of the Harman's free radical theory of aging that proposes that ROS generation represents a stress signal to age-dependent damage , rather then being the primary cause of it ( Harman , 1956; Hekimi et al . , 2011 ) .",
"However , our findings do support the concept that alteration of the ROS homeostasis accelerates aging .",
"It is also interesting to note that both genetic and pharmacological intervention to reduce ROS levels has produced contrasting results in reverting aging phenotypes , suggesting that too low or too high ROS levels can be equally deleterious .",
"In light of this , it is likely that overexpression of Siglec-E in mice might not result in lifespan expansion .",
"Additionally , this work complements a recent report showing Siglec-E in ROS management in the fibrinogen/β2-integrin signaling pathway ( McMillan et al . , 2014 ) .",
"However , in our assays , bone marrow and peritoneal neutrophils from Siglec-E−/− mice consistently showed a higher ROS production .",
"Lastly , whilst we suggest here that Siglec-E impacts inflammaging by a ROS-mediated mechanism , Siglec-E might also modulate the ability to recognize and remove senescent cells in aging ( van Deursen , 2014 ) .",
"Even if the correlation between number of CD33rSiglec family members and lifespan is particularly strong , a higher number of SIGLEC genes may not directly translate in a comparable increase of CD33rSiglec pathway activity .",
"Instead , it is quite possible that the observed gene expansion relates to expression patterns in specific cell types that might ultimately influence cellular CD33rSiglec repertoires .",
"We suggest that specific CD33rSiglec members contribute to the regulation of inflammation by interaction through distinct sialylated structures in vivo , leading to improved regulation of inflammatory responses .",
"Our finding that long-living mammals tend to have more CD33rSIGLEC genes is in line with the ‘inflammaging’ theory ( Franceschi et al . , 2000 ) .",
"Inflammaging postulates that lifetime exposure to antigenic load caused by both clinical and subclinical infections , as well as exposure to noninfective agents , generates low-grade inflammation .",
"This yields additional cytokines and results in a vicious cycle that drives immune system remodeling to a chronic proinflammatory state , ultimately leading to aging and common age-related disorders ( Finch and Crimmins , 2004; Finch et al . , 2010 ) .",
"Therefore , age-related diseases may represent the cost of an efficient defense against pathogens conferred by strong inflammation in early life .",
"It also derives that elements that protect against inflammatory damage or mediate repair may have an impact on aging , and that species differences in those elements may translate in distinct patterns of age-dependent disease .",
"The evidence presented in this work is consistent with a role of CD33rSiglecs in modulating aging derived from chronic inflammation .",
"In fact , CD33rSiglecs are receptors of innate immune cells .",
"Their primary function is to recognize self-associated molecular patterns and modulate host immune responses by regulating cellular reactions , survival , and production of cytokine mediators ( Crocker et al . , 2007; Chen et al . , 2009; Cao and Crocker , 2011; Varki , 2011 ) .",
"CD33rSiglecs counteract random molecular damage , which is the main driver of aging .",
"Lastly , CD33rSIGLEC gene number correlates with longevity .",
"In summary , our data provide molecular mechanisms underlying the CD33rSiglec-dependent control of oxidative stress and identify this gene family as modulators of aging pattern and lifespan in mammals ."
],
[
"Siglec-E−/− mice were described in McMillan et al . ( 2013 ) and backcrossed with C57BL/6 animals .",
"Heterozygous mice were used to produce WT and Siglec-E−/− littermates control .",
"Mice were housed in cages of groups of 3–5 that did not change from weaning , at 20 ± 2°C under a 12 hr light/12 hr dark photoperiod .",
"Mice were provided with unlimited access to water and to a soy-based chow ( Dyets , Inc . , AIN-93M , Bethlehem , PA ) supplemented with either 0 . 25 mg/g chow Neu5Gc ( by adding purified porcine submaxillary mucin ) or 0 . 25 mg/g chow Neu5Ac ( by adding edible bird's nest , Golden Nest Inc . , Arcadia , CA ) .",
"Addition of Neu5Gc or Neu5Ac did not significantly increase the caloric content of the chow .",
"Sterile inflammation was induced via intra-peritoneal injection with 200 μg of human erythrocyte membrane ghosts in 200 μl PBS , along with Freund's complete adjuvant , at an age of 10 weeks .",
"Human erythrocyte membrane ghosts were prepared as described previously ( Hedlund et al . , 2008 ) .",
"A booster injection using Freund's incomplete adjuvant with the same amount of immunogen was given 2 and 4 weeks later .",
"Mice were accessed periodically for evaluation of health status , body weight , and blood tests .",
"Deaths were recorded by animal technicians throughout the study .",
"Decisions for euthanasia of aged mice with severely compromised health were taken by animal technicians , following the guidelines of Institutional Animal Care and Use Committee of the University of California , San Diego , and without involving the scientists .",
"At approximately 75 weeks of age WT or Siglec-E−/− male mice were ranked blind in order of visible graying to coat fur .",
"In total 26 mice were ranked , including 6 female mice ( 3 Siglec-E−/− and 3 WT ) which had no obvious graying in the coat and therefore acted as a negative baseline .",
"The highest graying was scored 25 , and the lowest was scored = 0 .",
"The option was available to say that there was no difference between some or all mice , in which case they would be given the same score , but this option was not used by any of the analysts .",
"Organs were extracted from euthanized animals and either fixed in 4% paraformaldehyde ( skin , liver , lung , brain ) or snap-frozen in OCT and stored at −80°C .",
"Fixed tissues were processed and embedded in paraffin .",
"Paraffin sections were de-paraffinized , blocked , and stained with antibodies following protocols of the UC San Diego Mouse Phenotypic Core http://mousepheno . ucsd . edu/ .",
"Antibodies were as following: goat anti-Siglec-E ( R&D Systems , Minneapolis , MN ) , rabbit anti-Gstp1 ( Sigma–Aldrich , St . Louis , MO ) , mouse anti-β-actin ( Sigma–Aldrich ) , rabbit anti-β-actin ( Cell Signaling , Danvers , MA ) , anti-CD45 ( BD Pharmingen , San Jose , CA ) , rat anti-Ly6G ( clone 1A8 , BD Pharmingen ) , mouse anti-Gstp1 ( BD Pharmingen ) , and rabbit anti-β-galactosidase ( Bioss Antibodies , Woburn , MA ) .",
"Secondary antibodies were from LI-COR ( Lincoln , NE ) or Jackson ImmunoResearch Laboratories ( West Grove , PA ) .",
"Dorsal skin was dissected from mice , fixed in 10% paraformaldehyde , and embedded in paraffin .",
"Paraffin sections were prepared at 5-μm thickness and stained with hematoxylin and eosin .",
"Digital photomicrography using the Keyence B6000 ( Keyence , Itasca , IL ) was performed to collect 400× images , and the epidermal thickness was measured with Keyence BZII Analyzer .",
"Bone marrow neutrophils were flushed from femur and tibia and purified by Percoll gradient .",
"Peritoneal neutrophils were obtained from peritoneal exudate 16 hr after intraperitoneal injection of 3% thioglycollate .",
"Purity was evaluated by flow cytometry with an anti-Ly6G antibody .",
"For phagosomal ROS , 1 million neutrophils were incubated for 60 min in 700 μl PBS containing 0 . 5% ( wt/vol ) glucose and 140 μg/ml Fc OxyBURST ( Life Technologies , Grand Island , NY ) .",
"ROS production was measured with a BD FACScalibur ( BD Biosciences ) .",
"For extracellular ROS , half a million neutrophils were incubated with 10 μg/ml OxyBURST Green H2HFF BSA ( Life Technologies ) and phorbol myristate acetate ( PMA ) .",
"ROS production was monitored with a SpectraMax M3 ( Molecular Devices , Sunnyvale , CA ) .",
"Resected liver samples were placed immediately into RNALater ( Qiagen , Valencia , CA ) on ice .",
"Tissues were homogenized using a Kinematica homogenizer .",
"RNA was isolated from tissues using RNeasy kit ( Qiagen ) .",
"Concentration and quality of RNA were measured by a NanoDrop ND-1000 spectrophotometer ( NanoDrop Technologies ) and by a 2100 Bioanalyzer ( Agilent ) .",
"Gene microarray analysis was run by the UC San Diego Biomedical Genomics Microarray Core Facility using a MouseRef-8 v2 Expression BeadChip ( Illumina , San Diego , CA ) .",
"A principal component analysis ( PCA ) was conducted on the signals obtained from the data matrix ( 25 , 697 probes × 6 samples ) with Matlab ( Mathworks , Inc . , Torrance , CA ) .",
"Data generated from gene array were analyzed for differential geneexpression .",
"Genes were considered differentially expressed with a p value <0 . 05 ( Mann–Whitney U test ) , and a Log2 fold change of >1 or < −1 .",
"The generated list was analyzed using Ingenuity Pathway Analysis .",
"For immunoblot analysis , liver tissues were washed with PBS and homogenized in RIPA buffer .",
"Cell lysates were spun at 10 , 000×g .",
"Protein concentration of the supernatant was measured with a BCA kit ( Pierce , Rockford , lL ) .",
"Proteins were run in a SDS-PAGE and transferred to a nitrocellulose membrane .",
"Membranes were incubated with antibodies .",
"Signals were acquired with an Odyssey instrument ( LI-COR ) and analyzed by Image Studio software ( LI-COR ) .",
"For immunohistochemistry analysis , liver were fixed in 10% paraformaldehyde and embedded in paraffin .",
"Paraffin sections were prepared at 5-μm thickness and incubated with antibodies .",
"Images were collected using a B6000 microscope ( Keyence ) .",
"Simple image analysis on the brightness ( grayscale value of the image ) was conducted with the Image Processing Toolbox of Matlab ( Mathworks , Inc . ) .",
"Images were loaded into Matlab , normalized via z score , and then pixel value histograms were inspected .",
"Images were manually inspected for artifact and those pixels were discarded from the analysis ( as shown on the images as white lines on far right inset and marked as green in the image to the left ) .",
"Histograms often showed a bimodal distribution of pixel values indicating a clear demarcation of positive staining .",
"The middle value between this bimodal distribution was used as a criterion to classify between negative and positive staining .",
"Then , the pixels were marked either as bright ( red ) or dark ( blue ) and counted .",
"Visual inspection of the classified image was compared to the original image , and if pixels were misclassified , the midpoint was manually changed until the resulting image most clearly separated the tissue differences .",
"DNA was extracted from tissues with a DNeasy Blood & Tissue Kit ( Qiagen ) .",
"DNA concentrations of each sample were adjusted to 0 . 1 μg/ml .",
"The number of apurinic/apyrimidinic ( AP ) sites was determined using the DNA damage Quantification Kit ( Dojindo , Rockville , MD ) , following the manufacturer's instructions .",
"At the time of harvest , all tissues were immediately rinsed in ice-cold PBS and frozen at −80°C in PBS containing 100 μM diethylenetriamine pentaacetic acid ( DTPA ) and 100 μM butylated hydroxytoluene ( BHT ) in gas-tight containers overlaid with nitrogen .",
"Analysis of oxidative modification of amino acids was done by stable isotope dilution liquid chromatography with on-line tandem mass spectrometry ( LC/MS/MS ) using a HPLC interfaced to an AB SCIEX 5000 triple quadrupole mass spectrometer , as described in Zheng et al . ( 2004 ) .",
"Urine samples were spun to remove potential cellular debris and then frozen at −80°C until the time of analysis .",
"Urinary creatinine ( Cr ) levels were quantified on an Abbott Architect machine ( Abbott Diagnostics , Abbott Park , IL ) , according to the manufacturer's instructions .",
"Immediately after thawing , an internal standard ( 9α , 11α , 15S-trihydroxy-5Z , 13E-dien-1-oic-3 , 3 , 4 , 4-d4 acid; PGF2α-d4; Cayman Chemical Company ) was added to the sample .",
"Urinary levels of F2-IsoProstanes ( PGF2α and 2 , 3-dinor-PGF2α ) were analyzed by stable isotope dilution LC/MS/MS using a HPLC interfaced to an AB SCIEX 5000 triple quadrupole mass spectrometer .",
"To adjust for variations in urinary dilution , the results of F2-IsoProstanes are reported as ratios with urine Cr concentrations .",
"The Barnes maze test is a spatial learning and memory test originally developed in rats ( Barnes , 1979 ) , but also adapted for mice ( Bach et al . , 1995 ) .",
"The Barnes maze task has the benefit of minimizing pain and distress to the animal .",
"The Barnes maze apparatus consists of an opaque Plexiglas platform 75 cm in diameter elevated 58 cm above the floor .",
"20 holes , 5 cm in diameter , are located 5 cm from the perimeter , and a black Plexiglas escape box ( 19 × 8 × 7 cm ) is placed under one of the holes .",
"Distinct spatial cues are located all around the maze and are kept constant throughout the study .",
"On the first day of testing , a training session was performed , which consists of placing the mouse in the escape box and leaving it there for 5 min . 1 min later , the first trial was started .",
"At the beginning of each trial , the mouse was placed in the middle of the maze in a 10-cm high cylindrical black start chamber .",
"After 10 s the start chamber is removed a bright light is turned on , and the mouse is allowed to explore the maze .",
"The trial ended when the mouse entered the escape tunnel or after 3 min elapsed .",
"When the mouse entered the escape tunnel , it remained there for one minute .",
"When the mouse did not enter the tunnel , it is gently placed in the escape box for one minute .",
"The tunnel was always located underneath the same hole ( stable within the spatial environment ) , which is randomly determined for each mouse .",
"Mice were tested once a day for 9 days .",
"On day 10 , a probe test was conducted during which time the escape tunnel was removed and the mouse allowed to freely explore the maze for 3 min .",
"The time spent in each quadrant was determined and the percent time spent in the target quadrant ( the one originally containing the escape box ) was compared with the average percent time in the other three quadrants .",
"Each session was videotaped and scored by an experimenter blind to the genotype of the mouse .",
"Measures recorded include the number of errors made per session and the strategy employed by the mouse to locate the escape tunnel .",
"Errors were defined as nose pokes and head deflections over any hole that did not have the tunnel beneath it .",
"Search strategies were determined by examining each mouse's daily session and classifying it into one of three operationally defined categories: ( 1 ) Random search strategy—localized hole searches separated by crossings through the center of the maze , ( 2 ) Serial search strategy—systematic hole searches ( every hole or every other hole ) in a clockwise or counterclockwise direction , or ( 3 ) Spatial search strategy—reaching the escape tunnel with both error and distance ( number of holes between the first hole visited and the escape tunnel ) scores of less than or equal to 3 .",
"Locomotor activity was measured using an automated monitoring system ( Kinder Associates , San Diego , CA ) .",
"Polycarbonate cage ( 42 × 22 × 20 cm ) containing a thin layer of bedding material was placed into frames ( 25 . 5 × 47 cm ) mounted with photocell beams .",
"Each mouse was tested for 120 min .",
"Sequences of previously reported human and mouse CD33rSiglecs were retrieved from HGNC ( http://www . genenames . org/ ) and MGI ( http://www . informatics . jax . org/ ) , respectively .",
"NCBI annotated CD33rSIGLEC genes from additional mammalian species were used as references for orthologous gene searching .",
"Additional putative CD33rSIGLEC genes were obtained by searching available mammalian genome sequences at UCSC Genome Bioinformatics ( http://genome . ucsc . edu/ ) , Ensembl ( http://www . ensembl . org/index . html ) , and NCBI ( http://www . ncbi . nlm . nih . gov/gene ) .",
"As SIGLEC genes contain introns , we adopted and modified a previously established search strategy ( Shi and Zhang , 2006 ) .",
"First , we used BLAT/TBlastN to identify the genomic location of a putative CD33rSiglec gene in a genome with a previously reported CD33rSiglec as a query .",
"Secondly , Genscan was used to predict the gene structure found in this genome location .",
"Simultaneously , the genomic DNA sequences of the putative CD33rSIGLEC gene and the known CD33rSiglec protein sequence were used to conduct a protein-to-genomic sequence alignment by Wise2 .",
"Furthermore , to ensure the accurate prediction of a CD33rSIGLEC , the obtained putative protein sequence was examined by TMHMM V . 2 . 0 or SPLIT 4 . 0 SERVER for the presence of a transmembrane domain and examined by SignalP 3 . 0 Server for the presence of a signal peptide .",
"Additional domain evaluation was also conducted in Pfam 25 . 0 ( http://pfam . sanger . ac . uk/ ) to find the existence of V-set and C2-set domains in the putative CD33rSiglec-encoding gene .",
"All candidates then underwent BLAST analysis against the entire GenBank to ensure that their best hits are annotated as CD33rSiglecs .",
"This step is important because CD33rSIGLECs are known to be related to other SIGLEC genes ( e . g . , CD22 , MAG , and SIGLEC15 ) , as well as other cell surface Ig-like receptors .",
"The above gene search strategy was also applied for predicting all of the KLKs , TLRs , and IgG Fc receptors in mammals under consideration , though the criteria used in gene structure evaluation were gene family dependent .",
"Based on previous studies on CD33rSiglecs some particular characteristics are considered in order to define a gene as encoding a functional Siglec ( Crocker et al . , 1998 ) .",
"One criterion is that a Siglec protein is capable of binding sialylated glycans .",
"This binding activity requires a conserved arginine residue in the Ig-like V-set domain .",
"The other criterion is that a functional Siglec protein should contain either a cytosolic tail with at least one ITIM motif or a transmembrane domain carrying a positively charged amino acid .",
"The eventually acquired candidate CD33rSiglecs in each species were considered as true orthologs and used in our correlation analysis .",
"Defining a functional gene using our gene prediction approach is not black and white , due to the nature of incomplete genome sequences or genome sequencing errors .",
"Thus , a few rules were considered during our prediction process .",
"First , when entire exons of a gene ( usually one or two ) are missing due to a gap in the genome but ORFs remain undisrupted in the available sequences , we treat the case as a functional gene .",
"Second , different species have variable quality of genome coverage .",
"For example , human and mouse genomes have the highest coverage ( >12× ) out of all mammals , whereas cat and pig genomes have the lowest ones ( <5× ) .",
"Notably , we did not see a trend of higher genome coverage leading to more CD33rSiglec-encoding genes .",
"Moreover , even when we focused only on the species with comparable genome coverage ( opossum , dog , marmoset , cow , rhesus macaque , orangutan , chimpanzee , elephant , horse , and rat ) , the correlation of the number of CD33rSiglecs and maximum life span was still maintained .",
"Therefore , the quality of genome sequencing in mammalian species likely had no impact on our overall findings and conclusion .",
"Finally , we also observed genome sequencing errors in the form of 1 bp mutations or indels in two KLK genes , one IgG Fc receptor gene , and five TLR genes .",
"In this study , such sequences were also considered as functional genes in all species .",
"Notably , the number of TLR genes predicted in several mammalian species using our approach is equal to those reported earlier ( Leulier and Lemaitre , 2008 ) .",
"Data regarding maximum lifespan and average adult body weight for mammalian species are from AnAge: the animal aging and longevity ( http://genomics . senescence . info/species/ ) ( de Magalhães and Costa , 2009 ) .",
"Unpaired Student's t-test was used for comparisons involving two groups .",
"Lifespan analysis was performed using log-rank ( Mantel–Cox ) test .",
"Median survival refers the time at which half the subjects have died .",
"The Pearson's coefficient was used to calculate correlation .",
"All variables except the gene number counts were log-transformed for statistical analyses .",
"Prism 6 Program ( GraphPad , La Jolla , CA ) was used for most of the statistical analyses .",
"PGLS and FIC analysis were conducted in COMPARE 4 . 6b ( http://www . indiana . edu/∼martinsl/compare/ ) using a degree of freedom of 11 , with three ( one for calculating contrast and two for estimating the slope and the intercept ) subtracted from 14 ( the total number of taxa ) .",
"Phylogenetic regressions controlled for the body mass were run using pglmEstLambda in the CAIC package ( Comparative Analysis of Independent Contrasts ) in R . The function of pglmEstLambda uses the PGLS method , estimating λ as an index of the strength of the phylogenetic pattern in the data .",
"The model included CD33rSiglec gene numbers as response , maximum lifespan and body mass as covariates .",
"For λ values , we followed the rationale described in Navarrete et al . ( 2011 ) .",
"All animal studies were approved by the IACUC of the University of California San Diego .",
"Gene expression data are available at the GEO Archive ( GSE64760 ) ."
]
] | [
"Aging is a multifactorial process that includes the lifelong accumulation of molecular damage , leading to age-related frailty , disability and disease , and eventually death .",
"In this study , we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors ( CD33rSiglecs ) and maximum lifespan in mammals .",
"In keeping with this , we show that mice lacking Siglec-E , the main member of the CD33rSiglec family , exhibit reduced survival .",
"Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular , structural , and cognitive level .",
"We found that accelerated aging was related both to an unbalanced ROS metabolism , and to a secondary impairment in detoxification of reactive molecules , ultimately leading to increased damage to cellular DNA , proteins , and lipids .",
"Taken together , our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation , which in turn reduces molecular damage and extends lifespan ."
] | [
"As we get older , we are more likely to become frail , be less mobile and develop heart disease , diabetes , and other age-related diseases .",
"This is partly due to damage to tissues and organs that accumulates over the course of our lifetime .",
"How quickly we age is controlled both by our genetics and by the environment we live in .",
"It is thought that damage to DNA , proteins , and other molecules in the body caused by chemically active molecules called reactive oxygen species ( ROS ) can influence aging .",
"ROS are produced during respiration , immune responses , and other important processes in cells , but in excessive amounts they can be extremely harmful .",
"To avoid damage to DNA and other important molecules , cells have several ways to control the levels of ROS .",
"One of the other hallmarks of aging is the development of chronic inflammation in tissues around the body , which is partly triggered by the immune system in response to cell damage .",
"A group of genes called the CD33rSIGLEC genes are involved in controlling inflammation .",
"The genomes of different mammal species carry different numbers of these genes , but it is not clear whether this alters the aging process in these animals .",
"In this study , Schwarz et al . investigated whether the CD33rSIGLEC genes influence the lifespans of mammals .",
"Species with a higher number of CD33rSIGLEC genes generally have a longer lifespan than those with fewer of these genes .",
"Mice that were missing one of these genes and were subjected to inflammation early in life showed signs of accelerated aging and had shortened lifespans compared with normal mice .",
"As predicted , these mice also had higher levels of ROS , which led to a greater amount of damage to the DNA and other molecules in their bodies .",
"Schwarz et al . 's findings suggest that the CD33rSIGLECs co-evolved in mammals to help control the levels of ROS during inflammation , thereby reducing the damage to cells and extending the lifespan of the animals .",
"Given that individual humans have different numbers of working CD33rSIGLEC genes , it would be interesting to see if this influences human lifespan ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology"
] | Autophagy functions as an antiviral mechanism against geminiviruses in plants | elife-23897-v3 | [
[
"Plants have evolved various defense mechanisms to combat plant pathogens , including viruses .",
"The two major mechanisms for plant antiviral immunity are RNA silencing and resistance ( R ) gene-mediated resistance ( Mandadi and Scholthof , 2013 ) .",
"RNA silencing is a sequence-specific mechanism used to directly defend host cells against foreign invaders such as viruses and transposable elements ( Ding , 2010 ) .",
"By contrast , the activation of R gene-mediated resistance triggers a rapid defense response that often includes localized programmed cell death , known as the hypersensitive response ( HR ) .",
"The HR can prevent local viral infection and elicit systemic acquired resistance to viral infection .",
"Autophagy is an evolutionarily conserved mechanism that recycles damaged or unwanted cellular materials under stress conditions or during specific developmental processes ( Liu and Bassham , 2012 ) , and plays a critical role in multiple physiological processes , including plant biotic stress responses ( Han et al . , 2011 ) .",
"During the plant’s response to incompatible pathogens , autophagy contributes to HR cell death but restricts the spread of programmed cell death beyond the initial infection site ( Liu et al . , 2005; Patel and Dinesh-Kumar , 2008; Hofius et al . , 2009; Yoshimoto et al . , 2009 ) .",
"During compatible plant–pathogen interactions , autophagy positively regulates plant defense responses against necrotrophic pathogens ( Lai et al . , 2011; Lenz et al . , 2011; Kabbage et al . , 2013 ) .",
"However , disrupting autophagy in Arabidopsis thaliana leads to enhanced resistance to the biotrophic pathogen powdery mildew and dramatic pathogen-induced cell death ( Wang et al . , 2011 ) .",
"The role of autophagy in plant defense responses against the bacterial pathogen Pseudomonas syringae DC3000 is controversial ( Patel and Dinesh-Kumar , 2008; Hofius et al . , 2009; Lenz et al . , 2011 ) .",
"However , it is unclear how autophagy links plant immunity in these studies .",
"Autophagy may link plant immunity in different ways , with autophagy playing a role in degrading pathogen effectors or defense-related plant proteins , or pathogen effectors interfering with autophagy .",
"Indeed , viral proteins are reported to promote autophagic degradation of plant host RNAi-related components ( Derrien et al . , 2012; Cheng and Wang , 2016 ) .",
"In addition , 2b protein from Cucumber mosaic virus is thought to be targeted for degradation by autophagy through the calmodulin-like protein rgsCaM ( Nakahara et al . , 2012 ) .",
"Recently , an oomycete effector is reported to interfere with autophagy by depleting the putative selective autophagy cargo receptor Joka2 out of ATG8 complexes ( Dagdas et al . , 2016 ) .",
"However , the role of autophagy in degrading pathogen effectors or plant defense-related proteins and the effect of viral effectors on autophagy remain uncertain in plants .",
"All current findings are based on the data from chemical autophagy inhibitor treatments ( with potential off-target effects ) and/or silencing of autophagy-nonspecific autophagy-related ( ATG ) gene Beclin 1 ( Derrien et al . , 2012; Nakahara et al . , 2012; Cheng and Wang , 2016 ) .",
"Further , in these above studies , no data showed that disruption of classic autophagy really affects pathogen invasion .",
"Moreover , to date , there is no evidence to show that autophagy has a role during any compatible plant-virus interactions .",
"Geminiviruses are a large , diverse group of plant viruses with circular single-stranded DNA genomes , and many geminiviruses cause devastating diseases in different crops .",
"These viruses often occur in disease complexes .",
"For example , Cotton leaf curl Multan virus ( CLCuMuV ) , in association with the disease-specific satellite DNA Cotton leaf curl Multan betasatellite ( CLCuMuB ) , causes cotton leaf curl disease , a major viral disease in cotton ( Sattar et al . , 2013 ) .",
"In addition to cotton , CLCuMuV infects many other plants , including Nicotiana benthamiana .",
"CLCuMuV encodes six proteins , namely C1 , C2 , C3 , C4 , V1 and V2 , whereas CLCuMuB is approximately half the size of the CLCuMuV DNA genome and encodes a single protein , βC1 ( Briddon et al . , 2003 ) .",
"Like most βC1 factors encoded by geminivirus betasatellites , CLCuMuB βC1 is required by CLCuMuV for the induction of disease symptoms in plants .",
"In addition , CLCuMuB βC1 enhances the accumulation of its helper virus , CLCuMuV ( Saeed et al . , 2015; Jia et al . , 2016 ) , and is involved in RNA silencing ( Amin et al . , 2011 ) .",
"Recently , we and other groups reported that geminivirus βC1s can subvert ubiquitination to assist their helper viruses to infect plants ( Jia et al . , 2016; Shen et al . , 2016 ) .",
"In this study , we demonstrated that autophagy targets the virulence factor βC1 of CLCuMuV for degradation .",
"Furthermore , we uncovered that autophagy functions as a novel antiviral mechanism against three geminiviruses in plants ."
],
[
"To investigate the role of CLCuMuB βC1 ( hereafter βC1 ) in plant–virus interactions , we performed yeast two-hybrid screening of a Solanum lycopersicum cDNA library using βC1 as the bait .",
"From this screen , we identified the autophagy-related protein SlATG8f as a βC1-interacting protein .",
"We also found that βC1 interacted with NbATG8f , the closest N . benthamiana homolog of SlATG8f in yeast ( Figure 1A ) . 10 . 7554/eLife . 23897 . 003Figure 1 . CLCuMuB βC1 interacts with NbATG8f in vivo and in vitro .",
"( A ) βC1 interacts with NbATG8f in yeast .",
"SKY48 yeast strains containing AD-NbATG8f transformed with BD-βC1 or BD ( control ) were grown on Leu- selection plates at 28°C for 4 d .",
"The positive interaction was indicated by the blue colony formation on X-gal-containing galactose ( Gala ) and raffinose ( Raf ) but not on plates containing glucose ( Glu ) .",
"( B ) GST pull-down assay to show the in vitro interaction of NbATG8f with βC1 , but not βC1V32A .",
"The total soluble proteins of E . coli expressing NbATG8f-6×His were incubated with GST-βC1 or GST-βC1V32A immobilized on glutathione-sepharose beads and monitored by anti-His antibody .",
"( C ) βC1 was co-immunoprecipitated with NbATG8f .",
"GFP-NbATG8f was transiently co-expressed with and HA-βC1 or its mutant HA-βC1V32A in N . benthamiana leaves .",
"At 60 hr post agroinfiltration ( hpi ) , leaf lysates were immunoprecipitated with anti-GFP beads and then the precipitants were assessed by immunoblotting ( IB ) using anti-HA ( upper panel ) or anti-GFP antibodies ( middle panel ) .",
"( D ) BiFC analyses in N . benthamiana .",
"Representative images of nYFP-βC1 or nYFP-βC1V32A BiFC co-expressed with cYFP-NbATG8f .",
"( E ) Western blot analyses of BiFC construct combinations from the same experiments as in ( D ) .",
"All combinations were detected with anti-GFP polyclonal antibody . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 00310 . 7554/eLife . 23897 . 004Figure 1—figure supplement 1 . N terminus of βC1 is responsible for binding to NbATG8f . Schematic representation of the truncated mutants of βC1 , their interactions with NbATG8f in yeast .",
"Yeast cells transformed with four truncated mutants of βC1 and NbATG8f were selected on X-Gal-containing medium . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 00410 . 7554/eLife . 23897 . 005Figure 1—figure supplement 2 . βC1 co-immunoprecipitated with multiple ATG8 homologs .",
"( A ) Homology tree of NbATG8s .",
"Homology tree of eight homologs of NbATG8 in N . benthamiana .",
"The results were produced by DNAMAN package and the observed divergency method was applied to calculate distance .",
"( B ) GFP-NbATG8s were transiently co-expressed with HA-βC1 in N . benthamiana leaves .",
"At 60 hpi , leaf lysates were immunoprecipitated with anti-GFP beads and then the precipitants were assessed by immunoblotting ( IB ) using anti-HA ( upper panel ) or anti-GFP antibodies ( middle panel ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 00510 . 7554/eLife . 23897 . 006Figure 1—figure supplement 3 . βC1 is co-localized with NbATG8f . CFP-NbATG8f was transiently co-expressed with YFP-βC1 or YFP-βC1V32A in N . benthamiana leaves via agroinfiltration .",
"The confocal microscope images of mesophyll cells were taken at 60 hpi . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 006 We validated the in vivo and in vitro interactions between βC1 and NbATG8f using Glutathione S-Transferase ( GST ) pull-down ( Figure 1B ) and co-immunoprecipitation assays ( Figure 1C ) .",
"Moreover , we identified an 11-amino acid motif from residue 30 to 40 of βC1 that is necessary for its interaction with NbATG8f ( Figure 1—figure supplement 1 ) .",
"Strikingly , GST pull-down and co-immunoprecipitation assays indicated that the mutant protein βC1V32A was unable to interact with NbATG8f ( Figure 1B , C ) .",
"Further , co-immunoprecipitation assays indicated that βC1 also interacted with other three ATG8 isoforms ( Figure 1—figure supplement 2 ) .",
"We then performed a bimolecular fluorescence complementation ( BiFC ) assay to identify the subcellular localization of the βC1–NbATG8f interaction in plant cells .",
"A positive interaction between nYFP-βC1 and cYFP-NbATG8f was observed in both the cytoplasm and vacuoles of plant cells , as indicated by the presence of yellow fluorescence ( Figure 1D ) .",
"However , no such interaction was detected between nYFP-βC1V32A and cYFP-NbATG8f ( Figure 1D ) , although all constructs were successfully expressed ( Figure 1E ) .",
"We further confirmed the vacuolar localization of the βC1–NbATG8f interaction by performing time-lapse observations of mesophyll cells by confocal microscopy , which revealed Brownian motion of fluorescent punctate structures within the central vacuole ( Video 1 ) . 10 . 7554/eLife . 23897 . 007Video 1 . βC1- NbATG8f Interaction Is Localized in Vacuoles . nYFP-βC1 transiently expressed with cYFP-NbATG8f in N . benthamiana leaves and examined by confocal laser scanning microscopy at 60 hpi .",
"Yellow color represents YFP fusion fluorescence and red color for chlorophyll . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 007 In addition , we found that YFP-βC1 , but not YFP-βC1V32A , co-localized with GFP-NbATG8f-positive bodies in both the cytoplasm and central vacuoles of mesophyll cells of N . benthamiana leaf tissue , as revealed by confocal microscopy ( Figure 1—figure supplement 3 ) .",
"The vacuolar deposition of YFP-βC1 was also confirmed by time-lapse observations of mesophyll cells by confocal microscopy , which revealed Brownian motion of fluorescent punctate structures within the central vacuole ( Video 2 ) , which is consistent with the vacuolar localization of the βC1–NbATG8f interaction ( Video 1 ) . 10 . 7554/eLife . 23897 . 008Video 2 . βC1 Is Co-localized with NbATG8f . YFP-βC1 transiently expressed with CFP-NbATG8f in N . benthamiana leaves and examined by confocal laser scanning microscopy at 60 hpi .",
"Cyan color represents CFP-ATG8f , yellow color YFP-βC1 and red color for chlorophyll . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 008 Taken together , these results demonstrate that βC1 specifically interacts with NbATG8s and the V32 residue is essential for the βC1–NbATG8f interaction .",
"Since βC1 specifically interacted with NbATG8s , we hypothesized that autophagy affects the infection of plants by geminiviruses .",
"To test this hypothesis , we investigated whether geminivirus infection could induce autophagy in N . benthamiana .",
"First , we performed quantitative RT-PCR ( qRT-PCR ) analysis , finding that mRNA levels of NbATG2 , NbATG3 , NbATG5 , and NbATG7 were upregulated during the infection of plants with CLCuMuV ( CA ) plus CLCuMuB ( β ) ( hereafter referred to as CLCuMuV infection or CA+β ) ( Figure 2—figure supplement 1 ) .",
"We then used Cyan Fluorescent Protein ( CFP ) -tagged NbATG8f ( CFP-NbATG8f ) as an autophagosome marker ( Han et al . , 2015 ) to visualize possible autophagic activity .",
"In N . benthamiana plants infected with CA+β , we observed increased numbers of autophagosomes ( represented by CFP-NbATG8f puncta ) ( Figure 2A , B ) .",
"Transmission electron microscopy confirmed that viral infection increased the number of autophagic structures in infected cells ( Figure 2C , D ) compared to the control .",
"Further , we tested the effect of CLCuMuV infection on autophagy flux using Joka2/NBR1 , a selective autophagy cargo receptor , as a protein marker .",
"Joka2 has been used as a useful tool to measure autophagy flux in plants because it is degraded in the vacuole once autophagy activity increases ( Zhou et al . , 2013; Xu et al . , 2017 ) .",
"Indeed , we found that CLCuMuV infection ( CA+β ) reduced the protein level of NbJoka2 , although it did not change mRNA level of NbJoka2 ( Figure 2—figure supplement 2 ) , indicating that viral infection enhances autophagic flux .",
"These data demonstrate that CLCuMuV infection induces autophagy . 10 . 7554/eLife . 23897 . 009Figure 2 . CLCuMuV infection activates autophagy .",
"( A ) Representative confocal microscopy images of dynamic autophagic activity revealed by specific autophagy marker CFP-NbATG8f in plants infected with CLCuMuV plus CLCuMuB ( CA+β ) .",
"( B ) Quantification of the CFP-NbATG8f-labeled autophagic puncta per cell from ( A ) .",
"More than 500 mesophyll cells for each treatment were used for the quantification .",
"Relative autophagic activity in virus infected plants was normalized to that of control plants , which was set to 1 . 0 .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 .",
"( C ) Representative TEM images of autophagic structures .",
"Ultrastructure of autophagic bodies ( arrows ) was observed in the vacuoles of mesophyll cells of uninfected control and plants infected with CA+β .",
"V for vacuole .",
"( D ) Autophagosome-like structures from ( C ) were quantified .",
"At least 30 cells for each treatment were used for the quantification .",
"Relative autophagic activity in virus infected plants was normalized to that of control plants , which was set to 1 . 0 .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 00910 . 7554/eLife . 23897 . 010Figure 2—figure supplement 1 . Transcription pattern of autophagy-related genes were altered during CLCuMuV infection . Quantitative RT-PCR was performed using total RNA isolated from the leaves of virus infected plants and non-infected plants .",
"Expression data relative to non-infected plants are normalized to that of NbeIF4α .",
"Values are means ± SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 01010 . 7554/eLife . 23897 . 011Figure 2—figure supplement 2 . Viral infection decreased NbJoka2/NBR1 protein level .",
"( A ) Western blot assays showed that NbJoka2 protein level was reduced due to the increased autophagy flux in CLCuMuV-infected plants .",
"NbJoka2 was detected with anti-NBR1 polyclonal antibody .",
"Stars indicate expected band size .",
"( B ) mRNA level of NbJoka2 was unchanged in CLCuMuV-infected plants .",
"Real-time RT-PCR of NbJoka2 was used to determine mRNA level .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 011 Since autophagy is induced by CLCuMuV infection and βC1 specifically interacts with NbATG8f , we investigated the role of autophagy in CLCuMuV infection .",
"For this purpose , we silenced autophagy-related genes in N . benthamiana using Tobacco rattle virus ( TRV ) -based virus-induced gene silencing ( VIGS ) ( Liu et al . , 2002 ) .",
"Because there is functional redundancy of ATG8 genes due to the presence of multiple homologs in plants , we silenced NbATG5 and NbATG7 in N . benthamiana .",
"Compared to non-silenced control plants , the mRNA levels of NbATG5 and NbATG7 were significantly reduced by gene-specific VIGS ( Figure 3—figure supplement 1A ) , whereas we observed no obvious differences in TRV RNA levels between ATG5-silenced plants , ATG7-silenced plants , and control plants ( Figure 3—figure supplement 1B ) .",
"In addition , autophagy was blocked in ATG5- and ATG7-silenced plants ( Figure 3—figure supplement 2A , B ) .",
"It is worth noting that CLCuMuV infection had no effect on TRV-mediated VIGS ( Figure 3—figure supplement 3 ) .",
"Furthermore , ATG5- and ATG7-silenced plants did not show any abnormal developmental phenotypes .",
"We then infected ATG5- and ATG7-silenced plants with CA+β .",
"We observed that the leaf curl symptoms caused by viral infection were much more severe and appeared 3 days earlier ( Figure 3A , B ) , and CLCuMuV DNA levels were significantly higher in ATG5- and ATG7- silenced plants compared to control plants ( Figure 3C ) .",
"By contrast , silencing of GFP in a N . benthamiana GFP transgenic line 16C had no effect on CA+β infection ( Figure 3—figure supplement 4 ) . 10 . 7554/eLife . 23897 . 012Figure 3 . CLCuMuV DNA accumulation is affected by host cell autophagy .",
"( A ) Viral symptoms in ATG5- and ATG7–silenced plants infected with CLCuMuV plus CLCuMuB ( CA+β ) at 12 dpi .",
"Bar represents 7 cm .",
"( B ) The incidence of viral symptom appearance at different time points of post infection in ATG5- and ATG7-silenced plants .",
"Symptom was indicated as the appearance of curled leaf caused by CA+β .",
"Values represent means ± SE from three independent experiments .",
"( C ) Relative viral DNA accumulation in ATG5- and ATG7–silenced plants infected with CA+β .",
"Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 .",
"( D ) Viral symptoms in GAPCs–silenced plants infected with CA+β at 15 dpi .",
"Bar represents 7 cm .",
"( E ) The incidence of symptom appearance at different time points of post infection in GAPCs-silenced plants .",
"Symptom was indicated as the appearance of curled leaf caused by CLCuMuV infection .",
"Values represent means ± SE from three independent experiments .",
"( F ) Relative viral DNA accumulation in GAPCs-silenced plants .",
"Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 01210 . 7554/eLife . 23897 . 013Figure 3—figure supplement 1 . TRV viral titers were not changed during VIGS .",
"( A ) Reduced mRNA levels of ATG5 and ATG7 in the silenced plants .",
"Real-time RT-PCR was performed using gene-specific primers .",
"NbeIF4α was used as an internal control .",
"Values are means ±S E from three independent experiments .",
"( B ) Relative viral load of TRV in ATG5- and ATG7-silenced plants .",
"Real-time PCR analysis of CP gene from TRV was used to determine viral load .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 01310 . 7554/eLife . 23897 . 014Figure 3—figure supplement 2 . Involvement of ATG5 and ATG7 in autophagy is confirmed by VIGS .",
"( A ) Representative confocal images of dynamic autophagic activity revealed by specific autophagy marker CFP-NbATG8f in ATG5- and ATG7-silenced plants and control plants .",
"( B ) Reduced autophagic activity in ATG5- and ATG7-silenced plants .",
"Quantification of the CFP-NbATG8f-labeled autophagic puncta per cell was performed .",
"More than 500 mesophyll cells for each treatment were used for the quantification .",
"Relative autophagic activity was normalized to that in TRV control plants , which was set to 1 . 0 .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 compared with control . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 01410 . 7554/eLife . 23897 . 015Figure 3—figure supplement 3 . CLCuMuV infection has no effect on TRV-based VIGS of NbPDS .",
"( A ) TRV-based VIGS of PDS still caused a bleached phenotype in plants infected with CLCuMuV ( CA ) plus CLCuMuB ( β ) at 15 dpi .",
"( B ) Relative mRNA level of NbPDS in plants infected with CA+β and non-infection .",
"Leaf tissue was taken from NbPDS-silenced plants or control ( TRV alone ) at 15 dpi and total RNA was isolated .",
"Real-time RT-PCR of NbPDS was used to determine mRNA level in virus infected or control plants .",
"Values represent means ± SE from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 01510 . 7554/eLife . 23897 . 016Figure 3—figure supplement 4 . Silencing of a non-autophagy related gene GFP has no effect on CLCuMuV infection .",
"( A ) Viral symptom in GFP-silenced N . benthamiana GFP-transgenic16c line .",
"The pictures were taken at 12 dpi .",
"( B ) mRNA level of GFP in GFP-silenced N . benthamiana GFP-transgenic16c line .",
"Real-time RT-PCR was performed using GFP-specific primers .",
"eIF4α was used as an internal control .",
"Values are means ± SE from three independent experiments .",
"( * ) p<0 . 05 .",
"( C ) Relative CLCuMuV DNA accumulation in GFP-silenced N . benthamiana GFP-transgenic16c line .",
"Real-time PCR analysis of CLCuMuV V1 gene was used to determine viral DNA level .",
"Values represent means ± SE from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 016 Next , we examined the effect of enhanced autophagy on CLCuMuV infection .",
"Consistent with the observation that down-regulating cytosolic glyceraldehyde-3-phosphate dehydrogenase ( GAPCs ) gene expression significantly activates autophagy ( Han et al . , 2015 ) , we found that VIGS of GAPCs delayed symptom development in plants infected by CA+β ( Figure 3D , E ) and reduced viral DNA accumulation ( Figure 3F ) .",
"These results suggest that autophagy functions as an antiviral mechanism against CLCuMuV .",
"To further explore the biological significance of the βC1-NbATG8f interaction on autophagy-mediated defense against CLCuMuV infection , we generated a CLCuMuB mutant ( βV32A ) by replacing βC1 with its mutant counterpart βC1V32A and inoculating this mutant virus ( CA+βV32A ) onto N . benthamiana leaves .",
"Interrupting the interaction between βC1 and ATG8 accelerated the occurrence of viral symptoms and resulted in much more severe leaf curling symptoms than that caused by CA+β ( Figure 4A , B ) .",
"Leaf curling symptoms caused by CA+βV32A appeared 3 days earlier than the symptoms caused by CA+β ( Figure 4A–B ) .",
"Moreover , we observed increased viral DNA accumulation in plants infected by CA+βV32A versus CA+β ( Figure 4C ) .",
"Since the V32A point mutation eliminates the interaction of βC1 with NbATG8f , these results suggest that the interaction of βC1 with NbATG8 is essential for the antiviral defense mechanism of autophagy against CLCuMuV infection . 10 . 7554/eLife . 23897 . 017Figure 4 . A V32A point mutation in βC1 enhanced CLCuMuV infection .",
"( A ) CLCuMuB mutant ( βV32A ) , which encodes a mutant βC1V32A , caused the enhanced viral symptom compared to wild type CLCuMuB ( β ) when co-infected with CLCuMuV ( CA ) .",
"The pictures were taken at 12 dpi .",
"A V32A point mutation in βC1 ( βC1V32A ) eliminates its interaction with NbATG8f .",
"( B ) The incidence of symptom appearance at different time points of post infection .",
"Symptom was indicated as the appearance of curled leaf caused by the infection with CA+β or CA+βV32A .",
"( C ) Relative viral accumulation of CLCuMuV DNA .",
"Real-time PCR analysis of V1 gene from CLCuMuV was used to determine viral DNA level .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 017 Since we observed the vacuolar localization of the βC1-NbATG8f interaction and co-localization of βC1 with NbATG8f , we guess that βC1 is delivered to the vacuoles by autophagy for its degradation .",
"To test this hypothesis , we investigated the effect of autophagy on the subcellular localization of βC1 by expressing YFP-βC1 or its mutant YFP-βC1V32A in the non-silenced control , ATG5- and ATG7- silenced plants .",
"As expected , we observed YFP-βC1 in the vacuoles in the non-silenced control plants .",
"However , YFP-βC1 accumulated mostly in cytoplasm in ATG5- and ATG7- silenced plants ( Figure 5A ) .",
"Similarly , YFP-βC1V32A also accumulated mostly in cytoplasm in all plants , regardless of whether ATG5 / ATG7 was or not silenced ( Figure 5—figure supplement 1 ) .",
"In addition , silencing of either ATG5 or ATG7 resulted in more accumulation of YFP-βC1 but not YFP-βC1V32A ( Figure 5B and Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 23897 . 018Figure 5 . βC1 proteins is targeted for autophagic degradation .",
"( A ) Confocal microscopy images of YFP-βC1 in mesophyll cells of N . benthamiana leaves .",
"YFP-βC1 was transiently expressed in non-silenced control ( TRV alone ) , ATG5 or ATG7 silenced plants .",
"The confocal microscope images of mesophyll cells were taken at 60 hpi .",
"( B ) Western blot analyses of YFP-βC1 construct from the same experiments as in ( A ) .",
"Level of the fusion protein , YFP-βC1 , was detected with anti-GFP polyclonal antibody .",
"( C ) Silencing of either ATG5 or ATG7 enhanced the accumulation of HA-βC1 , but not HA-βC1V32A .",
"Each expression constructs were agroinfiltrated into N . benthamiana leaf .",
"At 60 hpi leaf lysates were separated by SDS-PAGE and fusion proteins were detected by anti-GFP or anti-HA antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 01810 . 7554/eLife . 23897 . 019Figure 5—figure supplement 1 . Silencing of either ATG5 or ATG7 has no effect on localization of YFP-βC1V32A .",
"( A ) Confocal microscopy images of YFP-βC1V32A in mesophyll cells of N . benthamiana leaves .",
"YFP-βC1 was transiently expressed in non-silenced control ( TRV alone ) , ATG5 or ATG7 silenced plants .",
"The confocal microscope images of mesophyll cells were taken at 60 hpi .",
"( B ) Western blot analyses of YFP-βC1V32A construct from the same experiments as in ( A ) .",
"Level of the fusion protein , YFP-βC1V32A , was detected with anti-GFP polyclonal antibody . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 01910 . 7554/eLife . 23897 . 020Figure 5—figure supplement 2 . Silencing of either ATG5 or ATG7 has no effect on transcript levels of target genes . Real-time RT-PCR was performed to detect mRNA levels of target genes using gene-specific primers and total RNAs extracted from the infiltrated leaves of different plants .",
"eIF4α was used as an internal control .",
"Expression data relative to TRV alone groups are normalized to that of eIF4α .",
"Values are means ± SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 020 We also tested the impact of autophagy on HA-βC1 .",
"Silencing of either ATG5 or ATG7 increased the accumulation of βC1 but did not affect the level of βC1V32A mutant protein or GFP alone ( in the control; Figure 5C ) .",
"However , no difference in the RNA level of the βC1 construct was detected in NbATG5- or NbATG7-silenced plants ( Figure 5—figure supplement 2 ) .",
"These results strongly suggest that βC1 is targeted by autophagy for the degradation , and the interaction of βC1 with NbATG8 is required for the autophagic degradation of βC1 .",
"We then investigated the effects of autophagy on Tomato yellow leaf curl virus ( TYLCV ) and Tomato yellow leaf curl China virus ( TYLCCNV ) .",
"Silencing of ATG5 and ATG7 caused more severe disease symptoms and enhanced viral DNA accumulation compared to the control , and silencing of GAPCs reduced the severity of viral disease symptoms and viral DNA levels in plants infected by TYLCV or TYLCCNV ( Figure 6 ) .",
"These results suggest that autophagy may have evolved as a general antiviral mechanism against various geminiviruses . 10 . 7554/eLife . 23897 . 021Figure 6 . Autophagy regulates viral infection of TYLCV and TYLCCNV .",
"( A ) Viral symptoms in ATG5- and ATG7-silenced plants at 12 dpi .",
"Bar represents 7 cm .",
"( B ) Viral symptoms in GAPCs–silenced ( GAPCs VIGS ) plants at 15 dpi .",
"Bar represents 7 cm .",
"( C ) and ( D ) Relative viral DNA accumulation in ATG5- and ATG7-silenced plants .",
"Real-time PCR analysis of V1 gene from TYLCCNV ( C ) or TYLCV ( D ) was used to determine viral DNA level in infected or control ( TRV alone ) plants .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 .",
"( E ) and ( F ) Relative viral DNA accumulation in GAPCs–silenced plants .",
"Real-time PCR analysis of V1 gene from TYLCCNV ( E ) or TYLCV ( F ) was used to determine viral DNA level in infected or control ( TRV alone ) plants .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 .",
"( G ) , ( H ) , ( I ) and ( J ) .",
"The incidence of symptom appearance at different time points of post infection in VIGS plants .",
"Symptom was indicated as the appearance of curled leaf caused by TYLCCNV in ATG5- and ATG7-silenced ( G ) and GAPCs–silenced ( I ) plants or TYLCV infection in ATG5- and ATG7-silenced ( H ) and GAPCs–silenced ( J ) plants .",
"Values represent means ± SE from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 021 Joka2/NBR1 is the sole known selective autophagy cargo receptor in plants .",
"To investigate the potential role of selective autophagy in antiviral defense against CLCuMuV infection , we silenced Joka2 in N . benthamiana using TRV-based VIGS ( Figure 7A ) .",
"Joka2 mRNA levels were significantly reduced in Joka2-silenced plants compared to non-silenced control plants ( Figure 7B ) .",
"However , silencing of Joka2 had no effect on CLCuMuV infection of plants with CA+β ( Figure 7C ) .",
"These results suggest that Joka2-mediated selective autophagy is not involved in antiviral defense against CLCuMuV infection . 10 . 7554/eLife . 23897 . 022Figure 7 . Silencing of Joka2 , a plant selective autophagy cargo receptor , has no effect on CLCuMuV infection .",
"( A ) Viral symptoms in Joka2-silenced N . benthamiana at 12 dpi .",
"( B ) mRNA level of Joka2 was reduced in Joka2-silenced plants .",
"Real-time RT-PCR of Joka2 was used to determine mRNA level .",
"Values represent means ± SE from three independent experiments .",
"( * ) p<0 . 05 .",
"( C ) Relative viral DNA accumulation in Joka2–silenced plants .",
"Real-time PCR analysis of CLCuMuV V1 gene was used to determine viral DNA level .",
"Values represent means ±SE from three independent experiments .",
"( * ) p<0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 23897 . 022"
],
[
"Autophagy is known to play an important role in disease resistance or susceptibility to various pathogens in plants ( Han et al . , 2011 ) .",
"However , how autophagy is linked to plant immunity remains unknown .",
"In this study , we show that geminivirus CLCuMuV infection activates autophagy and that autophagy targets the virulence protein βC1 for degradation .",
"Further , we demonstrated for the first time that autophagy plays an active role as an antiviral mechanism in compatible plant-virus interactions .",
"Autophagy acts as a defense mechanism against some invading intracellular pathogens in mammalian systems ( Boyle and Randow , 2013; Randow and Youle , 2014; Paul and Münz , 2016 ) .",
"Plant cells also employ autophagy to defend themselves against several pathogens ( Han et al . , 2011; Li et al . , 2016 ) .",
"Autophagy positively regulates plant resistance against necrotrophic pathogens ( Lai et al . , 2011; Lenz et al . , 2011; Kabbage et al . , 2013 ) but negatively affects plant resistance against the biotrophic pathogen powdery mildew ( Wang et al . , 2011 ) .",
"Furthermore , silencing of Joka2 , encoding a selective autophagy cargo receptor for polyubiquitinated cargoes , enhances susceptibility to Phytophthora infestans although it is not reported whether disrupting autophagy has an effect on plant response to this pathogen ( Dagdas et al . , 2016 ) .",
"However , we found that Joka2 silencing had no effect on CLCuMuV infection ( Figure 7 ) , implying that Joka2-mediated selective autophagy is not involved in antiviral defense against CLCuMuV in plants .",
"CLCuMuB βC1 may not interfere with Joka2-mediated selective autophagy by depleting Joka2 out of ATG8 complexes because it has a non-classic ATG8-interacting motif ( see below ) .",
"In addition , silencing of autophagy-related genes caused cell death induced by Tobacco mosaic virus ( TMV ) to spread in inoculated leaves of N . benthamiana plants containing the TMV resistance gene N but had no effect on plant systemic resistance against TMV ( Liu et al . , 2005 ) .",
"However , the role of autophagy in compatible plant-virus interactions has been unclear .",
"Here , we showed that disrupting autophagy enhanced plant susceptibility to three different DNA viruses , while activating autophagy enhanced plant resistance to viral infection ( Figure 3 and Figure 6 ) , suggesting that autophagy plays an active antiviral role in compatible plant-virus interactions .",
"Autophagy can either facilitate or suppress viral infection in mammalian cells ( Orvedahl et al . , 2010; Chiramel et al . , 2013; Paul and Münz , 2016; Wang et al . , 2016b ) .",
"However , little is known about whether and/or how autophagy affects viral infection in plants .",
"Plant viruses encode antiviral RNA silencing suppressors known as VSRs .",
"Based on the data from autophagy inhibitor 3-MA treatment , the VSR protein P0 from Polerovirus is thought to trigger the autophagic degradation of AGO1 , a component of the cellular RNAi-based antiviral defense machinery ( Derrien et al . , 2012 ) .",
"Similarly , the VSR protein VPg from Turnip mosaic virus is also reported to mediate the degradation of the cellular RNAi-based antiviral defense component SGS3 , partially via autophagy ( Cheng and Wang , 2016 ) .",
"Based on the data from an autophagy inhibitor treatment and silencing of an autophagy-nonspecific ATG gene Beclin 1 , 2b protein from Cucumber mosaic virus is thought to be targeted for degradation by autophagy through the calmodulin-like protein rgsCaM ( Nakahara et al . , 2012 ) .",
"In addition , the in vitro application of an autophagy inhibitor wortmannin partially inhibited proteolysis of TYLCV proteins ( Gorovits et al . , 2016 ) .",
"In our study , by silencing of two ATG genes ( ATG5 and ATG7 ) , we clearly show that CLCuMuB βC1 is degraded by autophagy .",
"Further , autophagy genes are transcriptionally up-regulated by infection by some geminiviruses in plants ( Ascencio-Ibáñez et al . , 2008; Miozzi et al . , 2014 ) .",
"These observations indirectly suggest that autophagy may contribute to plant immunity during compatible plant–virus interactions .",
"In this study , we showed that disrupting autophagy reduces plant resistance against three different plant viruses , whereas activating autophagy enhances this resistance .",
"To the best of our knowledge , in this study , we provide the first direct evidence that autophagy functions as an antiviral mechanism in compatible plant-virus interactions .",
"CLCuMuB βC1 is predicted to contain two ATG8-interacting motifs ( AIMs ) ( Kalvari et al . , 2014 ) .",
"Interestingly , we found that an approximately 11-amino-acid motif ( LVSTKSPSLIK ) comprising residues 30 to 40 of βC1 , but not the predicted AIMs , is responsible for the interaction of βC1 with NbATG8f .",
"Furthermore , a point mutation at position 32 from valine to alanine in this motif eliminated the βC1-NbATG8 interaction .",
"In addition , we found that βC1 interacted with at least four NbATG8 homologs .",
"ATG8 proteins interact with some cargo receptors , leading to autophagic degradation of the cargoes ( Johansen and Lamark , 2011 ) .",
"We found that disrupting autophagy increased the accumulation of βC1 protein but had no effect on the accumulation of βC1V32A ( Figure 5 ) , suggesting that βC1 is targeted for autophagic degradation .",
"Furthermore , YFP–βC1 co-localized with CFP-NbAt8f-positive autophagic bodies in vacuoles of leaf mesophyll cells ( Figure 1—figure supplement 3 and Video 1 ) , suggesting that βC1 is targeted for autophagic degradation by binding to ATG8s .",
"In this study , disrupting autophagy reduced plant resistance against TYLCV , while activating autophagy enhanced plant resistance against this virus .",
"Interestingly , autophagy also participates in resistance to TYLCV in whiteflies ( Wang et al . , 2016a ) .",
"TYLCV induces protein aggregation in plants and whiteflies , and viral proteins ( mostly viral coat protein ) and ATG8 co-exist in these TYLCV-induced aggregates ( Gorovits et al . , 2016 ) .",
"Further , some AIMs are evolutionarily conserved among plant and animal proteins ( Kalvari et al . , 2014; Dagdas et al . , 2016 ) , it is possible that autophagy uses similar mechanism to degrade TYLCV protein ( s ) in plants and whiteflies .",
"The βC1 protein from TYLCCNV β-satellite interacts with and is phosphorylated by SnRK1 ( sucrose-nonfermenting1-related kinase 1 ) , a plant ortholog of budding yeast SNF1 and mammalian AMPK ( AMP-activated protein kinase ) ( Shen et al . , 2011 ) .",
"AMPK promotes autophagy by directly phosphorylating different protein substrates involved in the initiation phase of autophagy ( Cardaci et al . , 2012 ) .",
"Interestingly , TYLCCNB βC1 also contains the polypeptide sequence LASTKSPALAK at residues 30–40 , which is similar to the ATG8-binding motif of CLCuMuB βC1 ( LVSTKSPSLIK ) .",
"Moreover , a mutation in this motif affects TYLCCNV infection ( Shen et al . , 2011 ) .",
"It is possible that TYLCCNB βC1 is also targeted for autophagic degradation .",
"Consistent with this hypothesis , disrupting autophagy reduced plant resistance against TYLCCNV , while activating autophagy enhanced plant resistance against this virus ( Figure 6 ) .",
"Disrupting autophagy increased viral DNA accumulation , while enhancing autophagy inhibited this process ( Figure 3 ) .",
"Importantly , the elimination of the interaction of βC1 with NbATG8f resulted in enhanced viral infection , suggesting that the βC1-NbATG8 interaction is essential for the autophagy-mediated antiviral defense response against CLCuMuV .",
"Thus , we provide compelling evidence that autophagy represents a novel antiviral strategy that involves targeting viral proteins for degradation and inhibiting viral infection in plants .",
"This unexpected discovery may facilitate the development of new strategies to protect plants from viral invasion .",
"In summary , we provide direct evidence that autophagy functions as a novel antiviral mechanism in plants .",
"Interestingly , CLCuMuB βC1 is an ATG8-binding protein , as well as a strong silencing suppressor .",
"This finding suggests that a delicate balance between viral pathogenesis and different host antiviral immunity mechanisms , such as autophagy and RNA silencing , has developed during plant–virus co-evolution .",
"Plants may have evolved to suppress viral infection by targeting viral protein ( s ) for autophagic degradation .",
"Indeed , we showed that disrupting autophagy enhanced plant susceptibility to three different viruses , whereas increasing autophagy enhanced plant resistance against these viruses .",
"On the other hand , possessing very strong virulence may not be the best strategy for the long-term survival of viruses in the battle between plants and viruses .",
"In this scenario , plant viruses may have evolved the ability to use the host’s cellular autophagy pathway to reduce their virulence through partial autophagic degradation of some viral virulence factors such as βC1 .",
"Thus , the virus would not completely destroy the plant cell or totally evade other host defense mechanisms such as RNA silencing or DNA methylation .",
"Consistent with this idea , plant viruses can establish latent , mild , or severe infection in plants , but they rarely kill their hosts ."
],
[
"GFP-transgenic16c line or wild type N . benthamiana plants were grown in pots placed in growthrooms at 25°C under a 16-h-light/8-h-dark cycle .",
"Vectors pTRV1 ( Liu et al . , 2002 ) and pTRV2-LIC ( Dong et al . , 2007 ) were described previously .",
"pTRV2-NbATG5 and pTRV2-NbATG7 were described ( Wang et al . , 2013 ) .",
"VIGS construct of NbGAPCs was described ( Han et al . , 2015 ) .",
"pTRV2-NbJoka2 was generated by cloning NbJoka2 cDNA fragment into pTRV2-LIC .",
"Approximately 200 bp of mGFP5-ER were cloned into pTRV2 by specific primers .",
"The full-length infectious clones of CLCuMuV ( GQ924756 . 1 ) and CLCuMuB ( GQ906588 . 1 ) were described ( Jia et al . , 2016 ) .",
"The full-length infectious clones of TYLCCNV and TYLCV were described ( Tao and Zhou , 2004; Zhang et al . , 2009 ) .",
"βC1 and its mutant βC1V32A were cloned into pGEX4T-1 vector to express GST-tagged fusion proteins in Escherichia coli .",
"Full-length cDNA of NbATG8f was cloned into pET28a to express NbATG8f-6×His in E . coli .",
"CFP-NbATG8f , GFP-NbATG8f , GFP-NbATG8c , GFP-NbATG8d , GFP-NbATG8i , cYFP-NbATG8f , nYFP-βC1 , GFP-βC1 , YFP-βC1 , HA-βC1 , HA-nLUC , nYFP-βC1V32A , GFP-βC1V32A , YFP-βC1V32A , HA-βC1V32A , and HA-nLUC were obtained respectively by overlapping PCR , and then cloned between the duplicated CaMV 35S promoter and the NOS terminator of pJG045 , a pCAMBIA1300-based T-DNA vector ( Du et al . , 2013 ) .",
"For co-IP assays , total proteins from N . benthamiana leaves ( 1 g leaf tissues for each sample ) were extracted in ice-cold immunoprecipitation buffer ( 10% [v/v] glycerol , 25 mM Tris , pH 7 . 5 , 150 mM NaCl , 1× protease inhibitor cocktail [Roche] , and 0 . 15% [v/v] Nonidet P-40 ) as described ( Wang et al . , 2015 ) .",
"Protein extracts were incubated with GFP-Trap beads ( ChromoTek ) for 3 hr at 4°C .",
"The precipitations were washed four times with ice-cold immunoprecipitation buffer at 4°C and were analyzed by immunoblot using anti-HA ( Cell Signaling Technology ) , or anti-GFP ( ChromoTek ) antibodies .",
"TRV-mediated VIGS assays were performed as described ( Liu et al . , 2005 ) .",
"Confocal imaging was performed as described ( Han et al . , 2015 ) .",
"The leaves were agroinfiltrated with autophagy marker CFP-NbATG8f for 60 hr expression , followed by an additional infiltration with 20 uM E-64d for 8 hr before being monitored by a Zeiss LSM 710 three-channel microscope with an excitation light of 405 nm , and the emission was captured at 454 to 581 nm .",
"TEM observation was performed as described ( Wang et al . , 2013 ) .",
"GST pull-down assays were performed as described previously ( Zhao et al . , 2013 ) .",
"GST-βC1 and NbATG8f-6×His fusion proteins were produced in BL21 ( DE3 ) cells ( Stratagene ) .",
"For yeast two-hybrid interaction assay , CLCuMuB βC1 was PCR amplified and cloned into yeast vector pYL302 to generate the LexA DNA binding domain ( BD ) containing bait vectors .",
"NbATG8f was PCR amplified and cloned into the B42 activation domain ( AD ) -containing vector pSAH20b .",
"The interaction assay and yeast two-hybrid screen was performed as described ( Du et al . , 2013 ) .",
"For expression analysis of NbATG genes , real-time RT-PCR was performed as described ( Wang et al . , 2013 ) .",
"eIF4a was used as the internal control .",
"For quantification of viral DNA loads , two DNA standard curves were generated .",
"Single copy of target viral genome DNA was cloned into pMD19-T ( TaKaRa , Japan ) and was used as standard viral DNA ( SVD ) while the genome DNA of healthy N . benthamiana was served as standard genome DNA ( SGD ) .",
"Standard curves were generated from ten-fold dilutions of both SVD and SGD .",
"Approximately 200 bp fragment of V1 gene from CLCuMuV/TYLCY/TYLCCNV and 61 bp region of eIF4α gene were amplified by employing SYBR green based real-time PCR to produce standard curves .",
"Viral DNA load in infectious plants was determined according to standard curves of SVD and SGD .",
"Results were expressed as fold change of virus DNA from virus infected plant tissue .",
"For quantification of TRV viral loads , real-time RT-PCR was performed with TRV CP - specific primers ( Zhao et al . , 2013 ) .",
"eIF4a was used as the internal control .",
"For protein analysis , total proteins were extracted from N . benthamiana leaves using 2×Laemmli buffer .",
"After boiling for 10 min , protein extracts were separated by SDS-PAGE for immunoblot analysis with indicated antibodies as described ( Wang et al . , 2013 ) .",
"Citrine Yellow Fluorescent protein ( YFP ) -based Bimolecular Fluorescence Complementation ( BiFC ) assay was performed as described ( Burch-Smith et al . , 2007; Jia et al . , 2016 ) ."
]
] | [
"Autophagy is an evolutionarily conserved process that recycles damaged or unwanted cellular components , and has been linked to plant immunity .",
"However , how autophagy contributes to plant immunity is unknown .",
"Here we reported that the plant autophagic machinery targets the virulence factor βC1 of Cotton leaf curl Multan virus ( CLCuMuV ) for degradation through its interaction with the key autophagy protein ATG8 .",
"A V32A mutation in βC1 abolished its interaction with NbATG8f , and virus carrying βC1V32A showed increased symptoms and viral DNA accumulation in plants .",
"Furthermore , silencing of autophagy-related genes ATG5 and ATG7 reduced plant resistance to the DNA viruses CLCuMuV , Tomato yellow leaf curl virus , and Tomato yellow leaf curl China virus , whereas activating autophagy by silencing GAPC genes enhanced plant resistance to viral infection .",
"Thus , autophagy represents a novel anti-pathogenic mechanism that plays an important role in antiviral immunity in plants ."
] | [
"Plants use a variety of processes to protect themselves against viruses and other disease-causing microbes .",
"Autophagy , for example , is a process that breaks down damaged or unwanted molecules found inside cells , which has also been linked to plant disease resistance .",
"However , it is not precisely clear how autophagy helps plants to resist diseases , because this process seems to make plants more resistant to some disease-causing microbes but more susceptible to others .",
"Now , Haxim , Ismayil et al . show that autophagy helps to protect plants against three viruses belonging to the Geminiviridae family of plant viruses .",
"One of these viruses causes an important disease in cotton plants , called cotton leaf curl disease .",
"This virus can infect many other plant species , including a close relative of tobacco plants , called Nicotiana benthamiana , which is commonly used in plant biology experiments .",
"Haxim , Ismayil et al . show that one of proteins produced by this virus , one called βC1 , interacts with a plant protein called ATG8 and is then sent to be broken down by autophagy .",
"Further experiments then identified a mutation in this protein that stopped it interacting with ATG8 .",
"Viruses carrying this mutated form of βC1 caused more severe symptoms and replicated more in N . benthamiana plants .",
"Interfering with autophagy made the N . benthamiana plants less resistant to the cotton leaf curl disease virus , and to two other geminiviruses that often infect tomatoes .",
"Activating autophagy had the opposite effect , and made the plants more resistant to all three viruses .",
"Together these findings provide direct evidence that autophagy helps to defend plants against a number of viruses , by degrading one or more viral proteins in the plants .",
"In the future , researchers may be able to build on these findings to engineer crop plants to be more resistant to viruses ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine",
"neuroscience"
] | Id4 promotes the elimination of the pro-activation factor Ascl1 to maintain quiescence of adult hippocampal stem cells | elife-48561-v2 | [
[
"Tissue stem cells must maintain their long-term activity while minimising the accumulation of genetic and metabolic damages .",
"In several adult tissues , stem cells can remain inactive for long periods of time in a state of quiescence .",
"Specific stimuli promote the exit from quiescence of different types of adult stem cells , such as hypoxia for stem cells of the carotid body or muscle injury for satellite stem cells ( Dumont et al . , 2015; Sobrino et al . , 2018 ) .",
"Regulation of the transit between quiescent and active compartments is essential to maintain a pool of stem cells able to sustain tissue homeostasis and provide an adequate response to insults over the lifespan of the organism .",
"An excessive retention of stem cells in the quiescent compartment would not produce enough differentiated progeny to maintain functionality , as happens for instance during aging ( García-Prat et al . , 2016; Leeman et al . , 2018 ) .",
"On the other hand , excessive stem cell activity would eventually result in stem cell exhaustion , also leading to loss of functionality ( Castilho et al . , 2009; Ho et al . , 2017 ) .",
"Quiescence is an essential property of cancer stem cells that allows them to evade immune surveillance and results in resistance to treatment ( Agudo et al . , 2018 ) .",
"Despite their relevance for the fields of tissue repair , aging and cancer biology , the mechanisms regulating quiescence in adult stem cells are still largely unknown .",
"In the adult brain , NSC populations in the ventricular-subventricular zone ( V-SVZ ) of the lateral ventricles and in the dentate gyrus ( DG ) of the hippocampus , generate new neurons and glia that integrate into pre-existing neuronal networks ( Bond et al . , 2015; Lim and Alvarez-Buylla , 2016 ) .",
"In both regions , a large fraction of stem cells is quiescent .",
"Extensive work has led to the identification of extracellular signals present in the V-SVZ and DG niches that regulate quiescent and active states ( Choe et al . , 2016; Silva-Vargas et al . , 2013 ) .",
"Notch , BMP4 and the neurotransmitter GABA have been shown to maintain stem cell quiescence while Wnt , Shh and the neurotransmitter glutamate are thought to promote stem cell activity ( Bao et al . , 2017; Choe et al . , 2016; Engler et al . , 2018; Imayoshi et al . , 2010; Lie et al . , 2005; Mira et al . , 2010; Petrova et al . , 2013; Qu et al . , 2010; Song et al . , 2012; Yeh et al . , 2018 ) .",
"In contrast , little is known of the cell intrinsic machinery that NSCs employ to adjust their activity to the different signals received from the niche .",
"Ascl1 is one of the few intrinsic regulators of NSC quiescence described so far .",
"Ascl1 is a basic-helix-loop-helix ( bHLH ) transcription factor that is present in a fraction of dividing stem cells and intermediate progenitors in the adult hippocampus .",
"Loss of Ascl1 completely blocks the activation of adult hippocampal stem cells , inhibits the generation of new neurons and prevents the depletion of the stem cell pool over time ( Andersen et al . , 2014 ) .",
"Ascl1 may therefore determine the balance between quiescence and activity of hippocampal NSCs .",
"Indeed , stabilization of Ascl1 protein by inactivation of the E3 ubiquitin ligase Huwe1 results in over-proliferation of hippocampal stem cells and prevents their return to quiescence ( Urbán et al . , 2016 ) .",
"However , Huwe1 inactivation is not sufficient to trigger the large-scale activation of quiescent stem cells , indicating that additional mechanisms maintain the quiescent state of hippocampal stem cells .",
"The Id ( Inhibitor of differentiation/DNA binding ) proteins are known inhibitors of bHLH transcription factors such as Ascl1 ( Imayoshi and Kageyama , 2014; Ling et al . , 2014 ) .",
"Id proteins contain a conserved HLH domain with which they dimerize with some bHLH proteins .",
"However , they lack a DNA binding domain and therefore prevent bHLHs with which they interact from binding DNA and other bHLH factors ( Benezra et al . , 1990 ) .",
"For instance , Id proteins have previously been shown to sequester E proteins , the dimerization partners of Ascl1 .",
"The resulting monomeric form of Ascl1 can no longer bind DNA and is furthermore rapidly degraded by the proteasome ( Shou et al . , 1999; Viñals et al . , 2004 ) .",
"In mammals , the Id family comprises four genes , Id1-4 .",
"The Id genes are expressed in multiple tissues during development and in adult stem cell niches , and have been shown to promote stemness and proliferation in different systems , including in hematopoietic stem cells and in stem cells of the adult SVZ ( Niola et al . , 2012; Singh et al . , 2018 ) .",
"However single cell transcriptome analysis has also shown that expression of Id3 and Id4 in particular , is highly enriched in quiescent hippocampal NSCs in vivo , thus linking Id genes with NSC quiescence ( Hochgerner et al . , 2018; Shin et al . , 2015 ) .",
"Here we show that Ascl1 mRNA is expressed by hippocampal stem cells independently of their proliferative or quiescent states , but that only active stem cells reach significant levels of Ascl1 protein .",
"This non-transcriptional regulation of Ascl1 is recapitulated in hippocampal stem cell cultures in vitro , where the quiescence-inducing factor BMP4 has no effect on Ascl1 mRNA expression but is sufficient to reduce Ascl1 protein levels .",
"We performed a gene expression screen in these cells and found that Id4 is strongly induced in quiescent NSCs .",
"Accordingly , analysis of the expression of Id1-4 proteins in the hippocampus also showed that Id4 is expressed by the highest percentage of NSCs .",
"We demonstrated that Id4 sequesters the Ascl1 heterodimerization partner E47 and that the resulting Ascl1 monomers are rapidly degraded by the proteasome .",
"Therefore , Id4 blocks the pro-activation transcriptional program driven by Ascl1 and keeps stem cells quiescent .",
"Indeed , elimination of Id4 from the adult brain results in increased Ascl1 protein levels in stem cells of the hippocampus and in their rapid entry into the cell cycle , and also leads to an increase in Id3 expression that might partially compensate for the loss of Id4 and suppresses Ascl1 protein in the absence of that factor ."
],
[
"To investigate how Ascl1 expression is regulated in hippocampal NSCs , we first assessed the transcriptional activity of the Ascl1 locus using the Ascl1KIGFP mouse reporter line , in which the GFP reporter replaces the Ascl1 coding sequence and marks cells that transcribe the Ascl1 gene ( Leung et al . , 2007 ) .",
"For clarity , hippocampal stem cells in vivo will be called hereafter radial glia-like cells ( RGLs ) while hippocampal stem cells in culture will be called NSCs .",
"We identified RGLs by their expression of glial fibrillary acidic protein ( GFAP ) , localization of their nucleus in the subgranular zone of the DG and presence of a radial process extending towards the molecular layer .",
"We found that 82 . 3 ± 3 . 8% of all RGLs were positive for GFP in the hippocampus of P70 Ascl1KIGFP mice and therefore transcribed Ascl1 .",
"In contrast , only 1 . 9 ± 0 . 3% of these cells expressed Ascl1 protein at a level detected with anti-Ascl1 antibodies ( Figure 1A and B ) .",
"Notably , 83 . 8 ± 4 . 1% of the RGLs that did not express Ki67 and were therefore quiescent expressed GFP , indicating a transcriptionally active Ascl1 locus ( Figure 1C and D ) .",
"Moreover , GFP immunolabeling intensity was comparable in active Ki67+ RGLs and quiescent Ki67- RGLs ( Figure 1E ) .",
"We confirmed the presence of Ascl1 transcripts at similar levels in quiescent and active RGLs using single molecule in situ hybridization ( Figure 1F , G ) .",
"These results show that , unexpectedly , Ascl1 is already expressed in quiescent RGLs and that NSC activation is not accompanied by the induction or marked upregulation of Ascl1 transcription .",
"The finding that quiescent and proliferating RGLs transcribe the Ascl1 gene at comparable levels but only proliferating RGLs express detectable levels of Ascl1 protein , indicates that Ascl1 protein expression in quiescent hippocampal RGLs is regulated by a non-transcriptional mechanism .",
"To investigate the mechanism regulating Ascl1 protein levels in quiescent hippocampal stem cells , we used an established cell culture model of NSC quiescence ( Martynoga et al . , 2013; Mira et al . , 2010; Sun et al . , 2011 ) .",
"The signalling molecule BMP4 has been shown to contribute to the maintenance of NSC quiescence in the hippocampus ( Bonaguidi et al . , 2008; Mira et al . , 2010 ) .",
"BMP4 is also able to induce a reversible state of cell cycle arrest in embryonic stem cell-derived NSC cultures ( Martynoga et al . , 2013; Mira et al . , 2010 ) .",
"Similarly , we found that NSCs originating from the adult hippocampus and maintained in culture in the presence of FGF2 stopped proliferating and entered a reversible quiescent state when exposed to BMP4 ( Figure 2—figure supplement 1A–D ) .",
"RNA sequencing analysis revealed that 1839 genes were differentially expressed between NSCs in proliferating and quiescent states ( Figure 2—figure supplement 1E , F ) .",
"Ascl1 RNA levels were not significantly different between these two conditions as verified by QPCR ( Figure 2A ) .",
"The intensity of GFP in cultured hippocampal NSCs derived from Ascl1KIGFP mice was also comparable in proliferating and quiescent conditions ( Figure 2B , C ) , as for GFP expression in the hippocampus of Ascl1KIGFP mice ( Figure 1E ) .",
"In contrast , Ascl1 protein levels were strongly reduced in BMP-treated quiescent NSCs ( Figure 2D–F ) , resembling the absence of Ascl1 protein in quiescent hippocampal RGLs in vivo ( Figure 1B ) .",
"Treatment of quiescent NSCs with proteasome inhibitors significantly increased the levels of Ascl1 protein , suggesting that Ascl1 mRNA is translated in quiescent NSCs but Ascl1 protein is rapidly degraded in a proteasome-dependent manner ( Figure 2G and Figure 2—figure supplement 1G ) .",
"We previously showed that Ascl1 protein is targeted for proteasomal degradation by the E3 ubiquitin ligase Huwe1 in proliferating hippocampal NSCs ( Urbán et al . , 2016 ) .",
"We therefore asked whether Huwe1 is also responsible for the degradation of Ascl1 in quiescent NSCs .",
"We found that Ascl1 protein was similarly reduced in Huwe1 mutant and control NSC cultures upon addition of BMP4 ( Figure 2—figure supplement 1H , I ) , demonstrating that a Huwe1-independent mechanism prompts the down-regulation of Ascl1 protein in quiescent cultured NSCs .",
"Together , these results establish BMP-treated NSC cultures as an appropriate model to characterize the mechanisms controlling Ascl1 protein levels in quiescent hippocampal stem cells .",
"We screened our RNA-Seq data for potential Ascl1 inhibitory factors induced in quiescent conditions ( Figure 3—figure supplement 1 ) .",
"The four Id genes Id1-4 were strongly induced in quiescent NSC cultures ( Figure 3—figure supplement 1A ) .",
"Since Id proteins sequester E proteins , resulting in the degradation of Ascl1 monomers , they are strong candidates to regulate Ascl1 post-translationally in hippocampal stem cells ( Shou et al . , 1999; Viñals et al . , 2004 ) .",
"Although the transcripts for the four Id genes were induced by BMP4 in NSC cultures ( Figure 3A ) , Id2 and Id3 were already expressed at high levels in proliferating conditions and only Id1 and Id4 were clearly upregulated at the protein level upon addition of BMP ( Figure 3B–E and Figure 3—figure supplement 1B , C ) .",
"Of those , Id4 expression was highly variable and was absent from cells presenting high levels of Ascl1 , suggesting a possible negative regulatory relationship between the two proteins , while Id1 was also expressed at various levels but did not anti-correlate with Ascl1 expression levels ( Figure 3C–F and Figure 3—figure supplement 1D–I ) .",
"Id4 therefore represented the most promising Id protein for dynamically regulating Ascl1 protein levels in BMP4-induced quiescent NSCs .",
"Co-immunoprecipitation ( co-IP ) experiments in NSCs showed that the E protein E47 ( a product of the Tcf3/E2A gene ) interacts with Ascl1 in proliferating conditions but with Id4 in quiescence conditions ( Figure 3—figure supplement 1J ) .",
"However , Ascl1 protein levels were lower in quiescent cultures , making the co-IP results difficult to interpret .",
"We therefore also carried an in vitro competition-binding assay with separately transfected E47 , Ascl1 and Id4 gene products ( Figure 3G ) , which confirmed that the interaction between Ascl1 and E47 is disrupted by the presence of Id4 , whilst no interaction was detected between Id4 and Ascl1 ( Figure 3H ) .",
"Therefore , Id4 sequesters E proteins away from their binding partner Ascl1 .",
"In agreement with monomeric Ascl1 being more unstable than the heterodimer with E47 , we found that the half-life of Ascl1 was reduced from 194 min in proliferating cells to 31 min in quiescent cells ( Figure 3—figure supplement 1K , L ) .",
"Next , we examined the expression of Id proteins in the adult DG by immunohistochemistry .",
"Id1 , Id3 and Id4 proteins are clearly expressed in the SGZ of the DG where hippocampal stem cells are located , while Id2 is enriched in granule neurons but not detected in the SGZ ( Figure 3I and Figure 3—figure supplement 2A–F ) .",
"Id1 is expressed by a substantial fraction of RGLs ( 47 . 5 ± 7 . 3% ) and is enriched in proliferating cells ( Figure 3—figure supplement 2A–C ) .",
"Id3 is expressed by a small fraction of mostly quiescent RGLs ( 16 . 9 ± 1 . 9% ) , at similar levels in proliferating and quiescent RGLs ( Figure 3—figure supplement 2E–G ) .",
"In contrast , Id4 is expressed by the vast majority of RGLs ( 89 . 6 ± 2 . 3% ) , at high levels in quiescent hippocampal RGLs and at much lower levels in proliferating RGLs ( Figure 3I–K ) , and co-localizes with Ascl1 mRNA-expressing cells ( Figure 3—figure supplement 2H , I ) .",
"Expression of the three genes encoding Eeins ( Tcf3/E2A , Tcf4/Itf2/E2 . 2 and Tcf12/Heb ) has been reported in RGLs in single cell RNA sequencing studies and we confirmed the presence of Tcf4/Itf2/E2 . 2 protein in RGLs in the DG ( Figure 3—figure supplement 2J , K ) ( Hochgerner et al . , 2018 ) .",
"Altogether , Id4 is a good candidate to suppress Ascl1 protein and promote quiescence in hippocampal stem cells via Eein sequestration , both in culture and in vivo .",
"To address the role of Id4 in Ascl1 regulation and in hippocampal stem cell quiescence , we first asked whether forcing the expression of Id4 in proliferating NSCs would be sufficient to reduce Ascl1 protein level and induce a quiescent state ( Figure 4A ) .",
"Id4 expression was low in control proliferating NSCs ( Figure 3B–D ) and was strongly increased after transfection with an Id4 expression construct ( Figure 4C ) .",
"Id4-transfected NSCs maintained Ascl1 mRNA at levels similar to those of control NSCs but showed markedly reduced Ascl1 protein levels ( Figure 4C–E ) .",
"Moreover , transfection of Id4 resulted in a significant decrease in NSC proliferation ( Figure 4F–I and Figure 4—figure supplement 1A , B ) .",
"This decrease was not due to differentiation , since Id4-expressing cells retained expression of the stem cell markers Sox2 and Nestin ( Figure 4—figure supplement 1C , D ) .",
"The effects of Id4 protein on Ascl1 expression and NSC proliferation suggest that induction of Id4 in BMP-treated NSCs contributes to the degradation of Ascl1 protein and the induction of quiescence ( Figure 4A ) .",
"We then asked whether over-expression of Id1-3 beyond their endogenous levels in proliferating NSCs could also reduce Ascl1 protein levels , by transfecting NSCs in parallel with expression construct for each of the Ids .",
"Over-expressing Id1 , Id2 or Id3 also suppressed Ascl1 protein levels , although Id4 overexpression was most effective ( Figure 4—figure supplement 1E ) .",
"Next , we asked whether inactivating Id proteins could stabilize Ascl1 protein and revert some aspects of the quiescent state in BMP-treated NSCs .",
"Knockdown of Id1-4 in quiescent NSCs by transfection of siRNAs targeting each Id gene separately or by co-transfection of Id4-siRNA with either Id1- , Id2- or Id3-siRNA , did not significantly affect Ascl1 protein levels ( Figure 4—figure supplement 1F ) despite a significant knockdown of each gene at both mRNA and protein levels ( Figure 4—figure supplement 1G–H ) .",
"It is worth noting that Id4 knockdown resulted in an increase in the protein levels of Id1 and Id3 ( Figure 4—figure supplement 1G ) without affecting their mRNA expression ( Figure 4—figure supplement 1H ) , suggesting that Id4 may suppress Id1 and Id3 protein expression .",
"Since Id1-3 can suppress Ascl1 protein when overexpressed ( Figure 4—figure supplement 1E ) , the upregulation of Id1 and Id3 in Id4-silenced cells might constitute a compensatory mechanism that maintains Ascl1 protein at low levels .",
"Because NSCs express the four Id proteins , which have redundant functions , we next chose to neutralise all of them by overexpressing the E protein E47 .",
"Since Id proteins have been shown to strongly bind E proteins , we reasoned that an excess amount of E47 should sequester Id proteins into E47-Id complexes , allowing the formation of Ascl1-E47 complexes and the stabilization of Ascl1 ( Figure 4B ) .",
"Indeed , overexpression of E47 in BMP-treated NSCs resulted in an increase in the levels of Ascl1 protein without significantly affecting Ascl1 mRNA levels ( Figure 4J–L ) .",
"Overexpression of E47 was also sufficient to partially revert the cell cycle arrest of BMP-treated NSCs , and we observed a strong correlation between Ascl1 protein levels and the proliferative state of the cells ( Figure 4M , N and Figure 4—figure supplement 1I , J ) .",
"Together , these results support a model whereby induction of high levels of Id proteins by BMP4 in quiescent NSCs promotes the degradation of Ascl1 by sequestering its dimerization partners ( Figure 4B ) .",
"They also raise the possibility that suppression of the transcriptional activity of Ascl1 is a key feature of the induction of quiescence by Id proteins .",
"All four Id proteins can reduce Ascl1 protein expression when overexpressed in NSCs , but the mutually exclusive expression of Id4 and Ascl1 proteins suggested that Id4 may have the most important role among endogenous Id proteins for the regulation of Ascl1 in quiescent NSCs ( Figure 3F and Figure 3—figure supplement 1D–I ) .",
"To investigate the mechanism by which Id4 induces quiescence in NSCs , we compared the transcriptome of Id4-overexpressing and control proliferating NSCs using RNA-Seq .",
"Expression of Id4 resulted in the up-regulation of 806 genes and down-regulation of 823 genes ( Figure 5A ) .",
"Expression of Ascl1 , Tcf3 , Tcf4 , Tcf12 , Hes1 , Hes5 and Hey1 were not significantly changed by Id4 overexpression in our data set ( Figure 5—figure supplement 1A ) .",
"Id4-regulated genes represented 44 . 2% of the genes regulated by BMP4 in NSCs , including 31 . 1% of the upregulated and 56 . 2% of the downregulated genes , indicating that Id4 has an important role in the induction of the gene expression program of quiescence downstream of BMP4 ( Figure 5B and Figure 5—figure supplement 1B–D ) .",
"The genes commonly regulated by Id4 and BMP4 are involved in cell cycle ( downregulated ) and cell adhesion ( upregulated ) ( Figure 5C–D ) , which are hallmarks of the NSC quiescent state ( Llorens-Bobadilla et al . , 2015; Martynoga et al . , 2013; Shin et al . , 2015 ) .",
"Direct transcriptional targets of Ascl1 were strongly downregulated in Id4-overexpressing NSCs , including genes with important roles in cell cycle progression such as Skp2 , Cdk1 , Cdk2 and Foxm1 , as well as other canonical Ascl1 targets such as Dll1 and Dll3 ( Castro et al . , 2011; Martynoga et al . , 2013 ) ( Figure 5E ) .",
"Overall , our analysis indicates that induction of Id4 by BMP4 and the subsequent degradation of Ascl1 results in the downregulation of its targets , leading to the cell cycle arrest of NSCs .",
"In light of the role of Id4 in inducing a quiescent-like state in NSCs , and since Id4 is highly expressed in stem cells in the adult hippocampus and is particularly enriched in quiescent RGLs , we next assessed the role of Id4 in the maintenance of the quiescent state of RGLs in vivo by analysing the hippocampus of mice carrying a conditional mutant allele of Id4 ( Id4fl ) ( Best et al . , 2014 ) ( Figure 6A and Figure 6—figure supplement 1A ) .",
"To eliminate Id4 from RGLs , we crossed Id4fl/fl mice with the Glast-CreERT2 deleter line ( Mori et al . , 2006 ) and the tdTomato reporter line ( Madisen et al . , 2010 ) ( Figure 6A ) .",
"We administered tamoxifen to the triple transgenic mice for 5 days , which resulted in complete elimination of Id4 protein ( Figure 6—figure supplement 1B ) and we analyzed the brains immediately after ( Id4cKO mice; Figure 6A ) .",
"The fraction of RGLs expressing Ascl1 increased from 4 . 4 ± 0 . 5 in control mice to 15 . 3 ± 2 . 7 in Id4cKO mice , with heterozygote mice showing only a small and non-significant increase in the fraction of Ascl1+ RGLs ( Figure 6B , C ) .",
"Ascl1 protein levels were also upregulated in Ascl1-expressing RGLs from Id4cKO mice while mRNA levels , measured by single molecule in situ hybridization , were lower in RGLs from Id4cKO mice than in control mice ( Figure 6D and Figure 6—figure supplement 1C ) .",
"The fraction of proliferating RGLs increased from 4 . 1 ± 0 . 5 in control mice to 13 . 7 ± 2 . 0 in Id4cKO mice , while heterozygote mice were indistinguishable from control mice ( Figure 6F , G ) .",
"Ascl1 expression was strongly correlated with Ki67 expression in RGLs in control and , particularly , in Id4cKO mice , supporting the direct link between Ascl1 upregulation and RGL activation ( Figure 6—figure supplement 1D–F and Andersen et al . , 2014; Urbán et al . , 2016 ) .",
"When Id4cKO mice were analyzed 30 days after tamoxifen administration and Id4 deletion , the rate of proliferation of RGLs remained significantly higher than in control mice , although the difference was smaller ( 3-fold at 5 days and two-fold at 30 days; Figure 6H ) .",
"Similarly , the fraction of Ascl1+ RGLs at 30 days post-Id4 deletion , was increased to a lesser extent , and non-significantly , than at 5 days ( Figure 6E ) .",
"Since RGL activation is linked to the depletion of the RGL pool ( Encinas et al . , 2011; Pilz et al . , 2018 ) , we quantified the total number of RGLs in Id4cKO and control mice 30 days after Id4 deletion and found no difference between genotypes ( Figure 6—figure supplement 1G ) .",
"To determine whether deletion of Id4 might trigger compensatory mechanisms , we examined the expression of the other Id proteins in Id4cKO mice and found that Id1 and particularly Id3 were strongly upregulated in RGLs in these mice at both 5 days and 30 days after Id4 deletion ( Figure 6I–L and Figure 6—figure supplement 1H , I ) .",
"We also analyzed the co-expression of Id3 protein , Id4 mRNA and Ascl1 mRNA by in situ and immunostaining in wild-type mice to determine whether Id3 could suppress Ascl1 protein independently of Id4 in a subset of RGLs that co-express Id3 and Ascl1 and are negative for Id4 .",
"We found that the majority of Id3+GFAP+ RGLs co-express Ascl1 mRNA ( Figure 6—figure supplement 1J , L ) and of the Id3+Ascl1+ cells , the vast majority also express Id4 ( Figure 6—figure supplement 1K , M ) .",
"This suggests that Id3 might only regulate Ascl1 protein independently of Id4 in a very small number of RGLs , but may become functionally relevant and compensate for loss of Id4 in Id4cKO mice .",
"Together , these findings demonstrate that Id4 expression in hippocampal RGLs contributes to the suppression of Ascl1 protein expression and the maintenance of quiescence , and suggest that compensatory mechanisms involving the upregulation of other Id proteins maintain partially RGL quiescence in the absence of Id4 ."
],
[
"In this study , we show that the repressor protein Id4 promotes the maintenance of adult hippocampal stem cells ( RGLs ) in a quiescent state .",
"The function of Id4 in maintenance of RGL quiescence is in remarkable contrast with its role in promoting the proliferation of progenitor cells in the embryonic cerebral cortex ( Bedford et al . , 2005; Yun et al . , 2004 ) .",
"This difference may reflect the different role of the bHLH proteins that are Id4 targets in embryonic versus adult neural lineages .",
"In the embryonic forebrain , NSCs are in a proliferative state and proneural bHLH proteins mostly act to promote neuronal differentiation .",
"Their inactivation by Id4 during development therefore results in a block of differentiation and extended proliferation .",
"In contrast , RGLs in the adult hippocampus are mostly quiescent , Ascl1 is required to promote their activity , and inactivation of Ascl1 by Id4 results in their failure to proliferate .",
"We also show that Id4 promotes the degradation of the pro-activation factor Ascl1 in RGLs .",
"As Ascl1 protein is only detectable in proliferating RGLs ( Andersen et al . , 2014 ) , we were not expecting the Ascl1 gene to be transcribed by most RGLs including many quiescent cells .",
"We found that despite Ascl1 mRNA being expressed and translated , Ascl1 protein does not accumulate in quiescent RGLs due to its rapid degradation .",
"This surprising finding could be the reason why single cell transcriptomic analysis of hippocampal cells did not identify Ascl1 among the genes differentially expressed between quiescent and active stem cells ( Artegiani et al . , 2017; Hochgerner et al . , 2018; Shin et al . , 2015 ) .",
"This non-transcriptional control of a key activation factor is also found , for instance , in satellite stem cells where the bHLH factor MyoD is transcribed in quiescent cells but its translation is inhibited by an RNA-binding protein to prevent stem cell activation ( de Morrée et al . , 2017 ) .",
"It is well established that Id proteins , including Id4 , form non-functional heterodimers with E proteins , which are dimerization partners of tissue-specific bHLH transcription factors such as Ascl1 ( Imayoshi and Kageyama , 2014; Ling et al . , 2014; Patel et al . , 2015; Sharma et al . , 2015 ) .",
"The three genes encoding for E proteins in mice are all expressed by RGLs , and since they are thought to have redundant functions , it is difficult to investigate their specific contributions to RGL behaviour .",
"Nevertheless , high levels of Ids are expected to result in the sequestration of all E proteins away from functional dimers with Ascl1 .",
"Non-dimerized Ascl1 is not able to bind DNA , and this alone could explain why Ascl1 target genes are downregulated in NSCs upon Id4 overexpression or BMP treatment , which increases the expression of all Id proteins .",
"But how does Id4 prevent Ascl1 protein accumulation ?",
"Exposure of different cell types to BMPs has been shown to trigger the proteolytic degradation of Ascl1 ( Shou et al . , 1999; Viñals et al . , 2004 ) .",
"In lung carcinoma cells , the formation of heterodimers with E47 stabilizes Ascl1 , and induction of Id1 by BMP2 sequesters E47 , resulting in degradation of the unstable monomeric form of Ascl1 ( Viñals et al . , 2004 ) .",
"We show that Ascl1 is more unstable in quiescent than proliferating hippocampal NSCs and that Ascl1-E47 dimers are disrupted by Id4 .",
"Therefore , we propose that a similar mechanism to that in lung carcinoma cells promotes the degradation of Ascl1 when quiescent hippocampal stem cells express high levels of Id4 .",
"To interfere with the function of Id4 in NSCs and circumvent the compensation by other Id proteins , we have overexpressed E47 , which is expected to interact with and titrate all Id proteins .",
"We found that this is indeed sufficient to stabilize Ascl1 protein and promote cell cycle re-entry of BMP-treated NSCs .",
"However , we realize that E47 overexpression might interfere with other factors than Id proteins and Ascl1 .",
"Silencing Id4 in quiescent NSCs in culture was confounded by the functional compensation of the other Id proteins , which are able to suppress Ascl1 protein levels when overexpressed .",
"Loss of Id4 in hippocampal RGLs also resulted in increased Id1 and Id3 protein levels , suggesting that Id4 may suppress these proteins in vivo , and that its loss may be partially compensated by the increase in their expression .",
"Since deletion of Id4 has the limitation of functional compensation by other Ids , more refined tools will be required in the future to dissect the specific mechanisms by which Id4 upregulation leads to Ascl1 degradation .",
"Our transcriptomic analysis suggests that Id4 alone contributes to a large extent to the gene expression program induced by BMP to promote NSC quiescence .",
"Overexpression of Id4 in the absence of BMP induces many of the genes that BMP4 induces , and suppresses many of the genes suppressed by BMP4 .",
"Among the genes suppressed by both BMP4 treatment and Id4 overexpression , an important fraction corresponds to cell cycle regulators , including many Ascl1 targets .",
"Besides regulating the activity of tissue-specific bHLH transcriptional activators such as Ascl1 , Id proteins also interact with bHLH transcriptional repressors of the Hes family ( Bai et al . , 2007 ) .",
"Direct interaction of Id2 with Hes1 blocks the autorepressive activity of Hes1 protein resulting in its stable expression at a high level .",
"Therefore , Id proteins promote a switch of the expression pattern of Hes proteins from oscillating , resulting in oscillatory expression of target genes such as Ascl1 , to stably high , resulting in constant repression of these targets ( Bai et al . , 2007; Boareto et al . , 2017; Sueda et al . , 2019 ) .",
"However , we find that Ascl1 is transcribed in most quiescent RGLs , indicating that Hes proteins do not repress Ascl1 transcription in these cells .",
"Id4 has been shown to inhibit the action of Id1-3 proteins by interacting with stronger affinity with them than with other binding partners ( Patel et al . , 2015; Sharma et al . , 2015 ) .",
"This non-canonical role of Id4 has been proposed to explain that Id4 promotes the proliferation of embryonic neural progenitors by blocking the interaction of Id1-3 with Hes proteins and thus promoting oscillations of Hes proteins – and consequently of Ascl1 – and progenitor proliferation ( Bedford et al . , 2005; Boareto et al . , 2017; Yun et al . , 2004 ) .",
"Id4 might therefore maintain Ascl1 transcription – separately from its role in eliminating Ascl1 protein – by interfering with the role of other Id proteins in stabilising Hes protein expression .",
"In support of this model , Id4 deletion in RGLs in vivo results not only in upregulation of other Id proteins but also in a reduction of Ascl1 transcript levels .",
"The function that we have identified for Id4 in the hippocampus is also distinct from the role reported for other Id factors in the adult V-SVZ , where Id1 and Id3 promote stem cell self-renewal ( Nam and Benezra , 2009 ) and Id1-3 maintain stem cell function by keeping stem cells adherent to their niche environment ( Niola et al . , 2012 ) .",
"Id4 is not the only Id protein expressed in hippocampal RGLs , as Id1 is also expressed in nearly half of them .",
"Nevertheless , Id4’s role in the regulation of stem cell quiescence is clearly different from that of Id1 .",
"While Id4 expression is restricted to RGLs that are quiescent and express low levels or no Ascl1 protein , Id1 protein is found in proliferating RGLs , many of which also express Ascl1 protein , suggesting that contrary to Id4 , Id1 at the level it is expressed in RGLs in homeostasis does not promote stem cell quiescence or the degradation of Ascl1 , although the same factor may have the potential to promote Ascl1 degradation when expressed at higher levels , that is when overexpressed in cultured NSCs .",
"In agreement with this , Id1 has recently been shown to have a role in the activation of hematopoietic stem cells upon stress signals ( Singh et al . , 2018 ) .",
"We have addressed the effect of loss of Id1 from RGLs by examining Smad4cKO mice , where Id1 expression in RGLs is greatly reduced while Id4 expression is unaffected ( Blomfield et al . , 2018 ) .",
"Loss of Smad4 did not affect RGLs , indicating that Id1 is not required to suppress Ascl1 expression or RGL proliferation .",
"It is unclear why Id1 , which has been shown to dimerize with E proteins and promote Ascl1 degradation in another cell type ( Viñals et al . , 2004 ) has no such effect in hippocampal stem cells , but this may be the result of its relative expression levels in RGLs .",
"Id3 expression , on the other hand , is mostly restricted to quiescent RGLs and is therefore a better candidate to compensate for the loss of Id4 in quiescent cells .",
"While Id4 is expressed in the vast majority of quiescent RGLs , Id4 deletion results in loss of quiescence of only a fraction of them at both 5d and 30d post-deletion , suggesting that compensatory mechanisms operate to blunt the Id4cKO phenotype in RGLs .",
"Thus , other Ids - in particular Id3 , because of its expression pattern in quiescent RGLs - or unrelated factors might compensate partially for the loss of Id4 .",
"An increase in the repression of Ascl1 transcription , due to increased Hes-Id protein interactions and Hes protein stabilization , might also contribute to the blunting of the Id4cKO phenotype .",
"In support of this , Ascl1 transcription was lower in Id4cKO mice as early as 5 days after Id4 deletion .",
"We have previously shown that the ubiquitin ligase Huwe1 degrades Ascl1 in proliferating RGLs and allows a fraction of these cells to return to quiescence ( Urbán et al . , 2016 ) .",
"Huwe1 is expressed in quiescent RGLs ( Urbán et al . , 2016 ) and although Id4 might mask its role in degrading Ascl1 in these cells , it might be able , in the absence of Id4 , to eliminate excess Ascl1 and maintain RGL quiescence .",
"Given the important role of Id4 in maintaining RGL quiescence , it seems likely that its expression is regulated by niche signals to control RGL activity .",
"Id genes , including Id4 , are well known effectors of BMP signalling in neural cells and other cell types ( Ling et al . , 2014; Patel et al . , 2015; Samanta and Kessler , 2004 ) .",
"Smad4 deletion strongly reduces Id1 but not Id4 levels in RGLs , indicating that Id4 diverges from other Id proteins not only in its activity but also in the regulation of its expression ( Blomfield et al . , 2018 ) .",
"Id4 has been shown to be directly regulated by Notch signalling in embryonic neural progenitors ( Li et al . , 2012 ) and in adult hippocampal stem cells ( Zhang et al . , 2019 ) but we found that Id4 expression persists in RGLs lacking the essential Notch signalling effector RBPJk ( Blomfield et al . , 2018 ) .",
"Id4 expression is only mildly affected in RGLs lacking both Smad4 and RBPJk , indicating that additional pathways beside BMP-Smad4 and Notch-Rbpjk promote RGL quiescence by maintaining Id4 expression .",
"Id4 is expressed in most quiescent RGLs but it is sharply downregulated in active RGLs .",
"Indeed , it is one of the most differentially expressed genes in quiescent versus active stem cells , both in vivo and in NSC cultures ( Shin et al . , 2015 and this paper ) .",
"Down-regulation of Id4 is crucial for RGLs to produce sufficient levels of Ascl1 protein to leave the quiescent state and become active , emphasizing the importance of this gene in the maintenance of RGL quiescence .",
"An important aim of future research will be to identify the niche signals that induce Id4 expression in quiescent RGLs and reduce its expression in active cells ."
],
[
"To measure immunofluorescence intensity , the nucleus of each identified RGL was manually outlined based on DAPI staining , and the average pixel value of the channel of interest was measured using FIJI software .",
"Every value was normalized to the background level measured in a negative nucleus in the same z-plane as each RGL .",
"At least 200 RGLs in each of at least three mice were quantified for each protein .",
"For in vitro ICC quantification , average pixel intensity for each channel was measured for the area of each nuclei , using FIJI software .",
"For each experiment , at least 100 cells were quantified across at least three biological replicates .",
"To generate the arbitrary units ( A . U . ) for both in vivo and in vitro IHC , all the values within a sample were made relative to the average of the control , and expressed as a % , with the average of control being 100% .",
"For quantification of RNAscope staining , the number of ‘dots’ in each identified RGL nucleus were counted for each probe .",
"In addition , the average pixel intensity in and around each RGL nucleus was measured for each probe , using FIJI .",
"100 RGLs were quantified across five mice .",
"For analysis of Id4 and E47 nucleofected cells , Id4+ or GFP+ ( E47 ) cells were identified by immunostaining for Id4 or GFP respectively , and positive cells compared to negative , non-transfected cells within the same coverslip .",
"Cell counts were done from at least 3 coverslips from three biological replicates .",
"For quantifying RGL density , the DG volume was calculated by multiplying the length , height and depth of the SGZ imaged in mm , and the number of stem cells counted expressed per mm3 .",
"All data were analyzed with masking of genotype/group to avoid bias .",
"For quantification of WB and IP assays , films were scanned and , if appropriate , subjected to band densitometry and quantification using Image J software ( RRID:SCR_002285 ) .",
"Each band value was normalized according to the background of the filter and its loading control .",
"The appropriate sample size ( ‘n’ ) was determined based on previous experiments of identical characteristics from our previous publications ( Andersen et al . ; Urban et al . ) and similar published data from other groups , using a minimum of 3 mice per condition for in vivo experiments , and a minimum of triplicate for in vitro experiments .",
"Throughout this paper , ‘technical replicate’ refers to the same sample being tested multiple times; ‘biological replicate’ refers to independent biological samples .",
"All data collected were included , as variation was considered within expected ranges and variances were non-significant .",
"Statistical analyses were conducted using the GraphPad Prism seven software ( RRID:SCR_002798 ) using a two-sample unpaired t test assuming Gaussian distribution for the comparison of two conditions; paired t test was used for Figure 4E–I , L–N , where the control and treatment conditions for each biological replicate were performed on cultures in parallel; or ordinary one-way ANOVA followed by Tukey’s multiple comparisons test , for the pairwise comparison of three conditions .",
"All error bars represent the mean ± SEM .",
"Significance is stated as follows: p>0 . 05 ( ns ) , p<0 . 05 ( * ) , p<0 . 01 ( ** ) , p<0 . 001 ( *** ) , p<0 . 0001 ( **** ) , confidence intervals of 95% .",
"Statistical details of each experiment can be found in the figure legend .",
"n represents number of independent biological repeats ."
]
] | [
"Quiescence is essential for the long-term maintenance of adult stem cells but how stem cells maintain quiescence is poorly understood .",
"Here , we show that neural stem cells ( NSCs ) in the adult mouse hippocampus actively transcribe the pro-activation factor Ascl1 regardless of their activated or quiescent states .",
"We found that the inhibitor of DNA binding protein Id4 is enriched in quiescent NSCs and that elimination of Id4 results in abnormal accumulation of Ascl1 protein and premature stem cell activation .",
"Accordingly , Id4 and other Id proteins promote elimination of Ascl1 protein in NSC cultures .",
"Id4 sequesters Ascl1 heterodimerization partner E47 , promoting Ascl1 protein degradation and stem cell quiescence .",
"Our results highlight the importance of non-transcriptional mechanisms for the maintenance of NSC quiescence and reveal a role for Id4 as a quiescence-inducing factor , in contrast with its role of promoting the proliferation of embryonic neural progenitors ."
] | [
"Stem cells in embryos give rise to all the tissues in the body .",
"Adults also have stem cells , but they are fewer in number and they are usually dedicated to repairing and regenerating specific tissues .",
"A region of the brain called the hippocampus , which is involved in learning , memory and mood , has a pool of neural stem cells .",
"These cells can produce new brain cells long into adulthood , but maintaining their regenerative potential is a balancing act .",
"Enough new brain cells need to be made to keep up with the brain’s demands , but if every stem cell matured into a brain cell , the brain’s capacity for repair would be lost .",
"So , some neural stem cells hit a metaphorical snooze button to enter a resting state known as quiescence .",
"Stem cells in the hippocampus make a protein called Ascl1 that interacts with DNA to switch on quiescent cells so they will divide and mature .",
"Left unchecked , Ascl1 could deplete the stem cell supply , so resting stem cells must have a way to turn Ascl1 off , but it was previously unknown how .",
"Clues point to the E proteins , which interact with Ascl1 to allow it to bind to DNA .",
"If the E proteins are not present , Ascl1 cannot work as a genetic switch .",
"E proteins can also interact with inhibitor of DNA binding/differentiation proteins , known as Id proteins for short .",
"To find out whether Id proteins affect Ascl1 activity , Blomfield et al . looked at stem cells in the hippocampus of adult mice , and at quiescent stem cells grown in the laboratory .",
"Blomfield et al . showed that all stem cells in the hippocampus make Ascl1 , but its levels are much lower when stem cells are resting .",
"This difference was down to an Id protein called Id4 .",
"In resting stem cells , Id4 interacted with E proteins , preventing them from binding to Ascl1 , and stopping Ascl1 from ‘waking up’ the cells .",
"This not only left Ascl1 unable to activate its target genes , it also made it vulnerable to destruction by the cell's protein recycling system .",
"Mice with no Id4 in their hippocampus stem cells had higher levels of Ascl1 , and their stem cells were more active .",
"The number of stem cells in a resting state increases as we age , and in illnesses like depression , limiting brain cell replacement .",
"Uncovering the signals that switch Id4 on or off could reveal why stem cells rest more with age and illness .",
"This could help us find ways to kick-start the production of new brain cells in adulthood ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"tools and resources",
"neuroscience"
] | NetPyNE, a tool for data-driven multiscale modeling of brain circuits | elife-44494-v3 | [
[
"The worldwide upsurge of neuroscience research through the BRAIN Initiative , Human Brain Project , and other efforts is yielding unprecedented levels of experimental findings from many different species , brain regions , scales and techniques .",
"As highlighted in the BRAIN Initiative 2025 report ( Bargmann et al . , 2014 ) , these initiatives require computational tools to consolidate and interpret the data , and translate isolated findings into an understanding of brain function ( Shou et al . , 2015; Fisher et al . , 2013 ) .",
"Biophysically detailed multiscale modeling ( MSM ) provides a promising approach for integrating , organizing and bridging many types of data .",
"Individual experiments often are limited to a single scale or level: for example , spiking activity in vivo , subcellular connectivity in brain slices , and molecular processes in dissociated or cultured tissue .",
"These data domains cannot be compared directly , but can be potentially integrated through multiscale simulations that permit one to switch readily back-and-forth between slice-simulation and in vivo simulation .",
"Furthermore , these multiscale models permit one to develop hypotheses about how biological mechanisms underlie brain function .",
"The MSM approach is essential to understand how subcellular , cellular and circuit-level components of complex neural systems interact to yield neural function or dysfunction and behavior ( Markram et al . , 2015; Skinner , 2012; MindScope et al . , 2016 ) .",
"It also provides the bridge to more compact theoretical domains , such as low-dimensional dynamics , analytic modeling and information theory ( Churchland and Sejnowski , 2016; Churchland and Abbott , 2016; Cunningham and Yu , 2014 ) .",
"NEURON is the leading simulator in the domain of multiscale neuronal modeling ( Tikidji-Hamburyan et al . , 2017 ) .",
"It has 648 models available via ModelDB ( McDougal et al . , 2017 ) , and over 2000 NEURON-based publications ( https://neuron . yale . edu/neuron/publications/neuron-bibliography ) .",
"However , building data-driven large-scale networks and running parallel simulations in NEURON is technically challenging ( Lytton et al . , 2016 ) , requiring integration of custom frameworks to build and organize complex model components across multiple scales .",
"Other key elements of the modeling workflow such as ensuring replicability , optimizing parameters and analyzing results also need to be implemented separately by each user ( Mulugeta et al . , 2018; McDougal et al . , 2016 ) .",
"Lack of model standardization makes it difficult to understand , reproduce and reuse many existing models and simulation results .",
"We introduce a new software tool , NetPyNE ( Networks using Python and NEURON ) .",
"NetPyNE addresses these issues and relieves the user from much of the time-consuming programming previously needed for these ancillary modeling tasks , automating many network modeling requirements for the setup , run , explore and analysis stages .",
"NetPyNE enables users to consolidate complex experimental data with prior models and other external data sources at different scales into a unified computational model .",
"Users can then simulate and analyze the model in the NetPyNE framework in order to better understand brain structure , brain dynamics and ultimately brain structure-function relationships .",
"The NetPyNE framework provides: ( 1 ) flexible , rule-based , high-level standardized specifications covering scales from molecule to cell to network; ( 2 ) efficient parallel simulation both on stand-alone computers and in high-performance computing ( HPC ) clusters; ( 3 ) automated data analysis and visualization ( e . g . connectivity , neural activity , information theoretic analysis ) ; ( 4 ) standardized input/output formats , importing of existing NEURON cell models , and conversion to/from NeuroML ( Gleeson et al . , 2010; Cannon et al . , 2014 ) ; ( 5 ) automated parameter tuning across multiples scales ( molecular to network ) using grid search and evolutionary algorithms .",
"All tool features are available programmatically or via an integrated graphical user interface ( GUI ) .",
"This centralized organization gives the user the ability to interact readily with the various components ( for building , simulating , optimizing and analyzing networks ) , without requiring additional installation , setup , training and format conversion across multiple tools .",
"NetPyNE’s high-level specifications are implemented as a declarative language designed to facilitate the definition of data-driven multiscale network models by accommodating many of the intricacies of experimental data , such as complex subcellular mechanisms , the distribution of synapses across fully detailed dendrites , and time-varying stimulation .",
"Contrasting with the obscurity of raw-code descriptions used in many existing models ( McDougal et al . , 2016 ) , NetPyNE’s standardized language provides transparent and manageable descriptions .",
"These features in particular promise to increase the reproducibility of simulation results and the reuse of models across research groups .",
"Model specifications are then translated into the necessary NEURON components via built-in algorithms .",
"This approach cleanly separates model specifications from the underlying technical implementation .",
"Users avoid complex low-level coding , preventing implementation errors , inefficiencies and flawed results that are common during the development of complex multiscale models .",
"Crucially , users retain control of the model design choices , including the conceptual model , level of biological detail , scales to include , and biological parameter values .",
"The NetPyNE tool allows users to shift their time , effort and focus from low-level coding to designing a model that matches the biological details at the chosen scales .",
"NetPyNE is one of several tools that facilitate network modeling with NEURON: neuroConstruct ( Gleeson et al . , 2007 ) , PyNN ( Davison , 2008 ) , Topographica ( Bednar , 2009 ) , ARACHNE ( Aleksin et al . , 2017 ) and BioNet ( Gratiy et al . , 2018 ) .",
"NetPyNE differs from these in terms of the range of scales , from molecular up to large networks and extracellular space simulation – it is the only tool that supports NEURON’s Reaction-Diffusion ( RxD ) module ( McDougal et al . , 2013; Newton et al . , 2018 ) .",
"It also provides an easy declarative format for the definition of complex , experimentally derived rules to distribute synapses across dendrites .",
"NetPyNE is also unique in integrating a standardized declarative language , automated parameter optimization and a GUI designed to work across all these scales .",
"NetPyNE therefore streamlines the modeling workflow , consequently accelerating the iteration between modeling and experiment .",
"By reducing programming challenges , our tool also makes multiscale modeling highly accessible to a wide range of users in the neuroscience community .",
"NetPyNE is publicly available from http://netpyne . org , which includes installation instructions , documentation , tutorials , example models and Q&A forums .",
"The tool has already been used by over 50 researchers in 24 labs to train students and to model a variety of brain regions and phenomena ( see http://netpyne . org/models ) ( Dura-Bernal et al . , 2018; Romaro et al . , 2018; Lytton et al . , 2017; Neymotin et al . , 2016b ) .",
"Additionally , it has been integrated with other tools in the neuroscience community: the Human Neocortical Neurosolver ( https://hnn . brown . edu/ ) ( Jones et al . , 2009; Neymotin et al . , 2018 ) , Open Source Brain ( http://opensourcebrain . org ) ( Gleeson et al . , 2018; Cannon et al . , 2014 ) , and the Neuroscience Gateway portal ( http://nsgportal . org ) ( Sivagnanam et al . , 2013 ) ."
],
[
"NetPyNE’s workflow consists of four main stages: ( 1 ) high-level specification , ( 2 ) network instantiation , ( 3 ) simulation and ( 4 ) analysis and saving ( Figure 1 ) .",
"The first stage involves defining all the parameters required to build the network , from population sizes to cell properties to connectivity rules , and the simulation options , including duration , integration step , variables to record , etc .",
"This is the main step requiring input from the user , who can provide these inputs either programmatically with NetPyNE’s declarative language , or by using the GUI .",
"NetPyNE also enables importing of existing cell models for use in a network .",
"The next stages can be accomplished with a single function call – or mouse click if using the GUI .",
"The network instantiation step consists of creating all the cells , connections and stimuli based on the high-level parameters and rules provided by the user .",
"The instantiated network is represented as a Python hierarchical structure that includes all the NEURON objects required to run a parallel simulation .",
"This is followed by the simulation stage , where NetPyNE takes care of distributing the cells and connections across the available nodes , running the parallelized simulation , and gathering the data back in the master node .",
"Here , NetPyNE is using NEURON as its back-end simulator , but all the technical complexities of parallel NEURON are hidden from the user .",
"In the final stage , the user can plot a wide variety of figures to analyze the network and simulation output .",
"The model and simulation output can be saved to common file formats and exported to NeuroML , a standard description for neural models ( Cannon et al . , 2014 ) .",
"This enables exploring the data using other tools ( e . g . MATLAB ) or importing and running the model using other simulators ( e . g . NEST ) .",
"An additional overarching component enables users to automate these steps to run batches of simulations to explore model parameters .",
"The user can define the range of values to explore for each parameter and customize one of the pre-defined configuration templates to automatically submit all the simulation jobs on multi-processor machines or supercomputers .",
"Each of these stages is implemented in modular fashion to make it possible to follow different workflows such as saving an instantiated network and then loading and running simulations at a later time .",
"The following sections provide additional details about each simulation stage .",
"A major challenge in building models is combining the data from many scales .",
"In this respect , NetPyNE offers a substantial advantage by employing a human-readable , clean , rule-based shareable declarative language to specify networks and simulation configuration .",
"These standardized high-level specifications employ a compact JSON-compatible format consisting of Python lists and dictionaries ( Figure 2 ) .",
"The objective of the high-level declarative language is to allow users to accurately describe the particulars and patterns observed at each biological scale , while hiding all the complex technical aspects required to implement them in NEURON .",
"For example , one can define a probabilistic connectivity rule between two populations , instead of creating potentially millions of cell-to-cell connections with Python or hoc for loops .",
"The high-level language enables structured specification of all the model parameters: populations , cell properties , connectivity , input stimulation and simulation configuration .",
"NetPyNE generates a simulatable NEURON model containing all the elements and properties described by the user in the rule-based high-level specifications .",
"As described above , declarations may include molecular processes , cells , connections , stimulators and simulation options .",
"After instantiation , the data structures of both the original high-level specifications and the resultant network instance can be accessed programmatically or via GUI .",
"Traditionally , it has been up to the user to provide an easy way to access the components of a NEURON network model , for example the connections or stimulators targeting a cell , the sections in a cell , or the properties and mechanisms in each section .",
"This feature is absent in many existing models .",
"Hence , inspecting s models requires calling multiple NEURON functions ( e . g . SectionList . allroots ( ) , SectionList . wholetree ( ) and section . psection ( ) ) .",
"Other models include some form of indexing for the elements at some scales , but since this is not enforced , their structure and naming can vary significantly across models .",
"In contrast , all networks generated by NetPyNE are consistently represented as a nested Python structure .",
"The root of the instantiated network is the net object ( Figure 4 ) .",
"net contains a list of cells; each cell contains lists or dictionaries with its properties , sections , and stimulators .",
"Each section sec contains dictionaries with its morphology and mechanisms .",
"For example , once the network is instantiated , the sodium conductance parameter for cell #5 can be accessed as net . cells[5] .",
"secs . soma . mechs . hh . gbar .",
"This data structure also includes all the NEURON objects – Sections , NetCons , NetStims , IClamps , etc . embedded hierarchically , and accessible via the hObj dictionary key of each element .",
"Computational needs for running much larger and more complex neural simulations are constantly increasing as researchers attempt to reproduce fast-growing experimental datasets ( Bezaire et al . , 2016; Markram et al . , 2015; MindScope et al . , 2016; Dura-Bernal et al . , 2018; Hereld et al . , 2005; Lytton et al . , 2016 ) .",
"Fortunately , parallelization methods and high-performance computing ( HPC , supercomputing ) resources are becoming increasingly available to the average user ( Hines , 2011; Hines et al . , 2008; Migliore et al . , 2006; Towns et al . , 2014; Amunts et al . , 2016; Sivagnanam et al . , 2013; Krause and Thörnig , 2018 ) .",
"The NEURON simulator provides a ParallelContext module , which enables parallelizing the simulation computations across different nodes .",
"However , this remains a complex process that involves distributing computations across nodes in a balanced manner , gathering and reassembling simulation results for post-processing , and ensuring simulation results are replicable and independent of the number of processors used .",
"Therefore , appropriate and efficient parallelization of network simulations requires design , implementation and deployment of a variety of techniques , some complex , many obscure , mostly inaccessible to the average user ( Lytton et al . , 2016 ) .",
"NetPyNE manages these burdensome tasks so that the user can run parallelized simulations with a single function call or mouse click .",
"Cells are distributed across processors using a round-robin algorithm , which generally results in balanced computation load on each processor ( Migliore et al . , 2006; Lytton et al . , 2016 ) .",
"After the simulation has run , NetPyNE gathers in the master node all the network metadata ( cells , connections , etc . ) and simulation results ( spike times , voltage traces , LFP signal , etc . ) for analysis .",
"As models scale up , it becomes impractical to store the simulation results on a single centralized master node .",
"NetPyNE offers distributed data saving methods that reduce both the runtime memory required and the gathering time .",
"Distributed data saving allows multiple compute nodes to write information in parallel , either at intervals during simulation runtime , or once the simulation is completed .",
"The output files are later merged for analysis .",
"Random number generators ( RNGs ) are often problematic in hand-written parallelized code; careful management of seeds is required since even use of the same seed or seed-sets across nodes will result in different random streams when the number of nodes is changed .",
"Since random values are used to generate cell locations , connectivity properties , spike times of driving inputs , etc . , inconsistent streams will cause a simulation to produce different results when switching from serial to parallel or when changing the number of nodes .",
"In NetPyNE , RNGs are initialized based on seed values created from associated pre- and post-synaptic cell global identifiers ( gids ) which ensures consistent results across different numbers of cores .",
"Specific RNG streams are associated to purposive seeds ( e . g . connectivity or locations ) and to a global seed , allowing different random , but replicable , networks to be run by modifying the single global seed .",
"Similarly , manipulation of purposive seeds can be used to run , for example , a network with identical wiring but different random driving inputs .",
"We previously performed parallelization performance analyses , demonstrating that run time scales appropriately as a function of number of cells ( tested up to 100 , 000 ) and compute nodes ( tested up to 512 ) ( Lytton et al . , 2016 ) .",
"Simulations were developed and executed using NetPyNE and NEURON on the XSEDE Comet supercomputer via the Neuroscience Gateway ( Sivagnanam et al . , 2013 ) .",
"The Neuroscience Gateway , which provides neuroscientists with free and easy access to supercomputers , includes NetPyNE as one of the tools available via their web portal .",
"Larger-scale models – including the M1 model with 10 thousand multicompartment neurons and 30 million synapses ( Dura-Bernal et al . , 2018 ) and the thalamocortical model with over 80 thousand point neurons and 300 million synapses ( Potjans and Diesmann , 2014; Romaro et al . , 2018 ) – have been simulated on both the XSEDE Comet supercomputer and Google Cloud supercomputers .",
"Run time to simulate 1 second of the multicompartment-neuron network required 47 minutes on 48 cores , and 4 minutes on 128 cores for the point-neuron network .",
"To extract conclusions from neural simulations it is necessary to use further tools to process and present the large amounts of raw data generated .",
"NetPyNE includes built-in implementations of a wide range of visualization and analysis functions commonly used in neuroscience ( Figure 5 ) .",
"All analysis functions include options to customize the desired output .",
"Functions to visualize and analyze network structure are available without a simulation run: ( 1 ) intracellular and extracellular RxD species concentration in a 2D region; ( 2 ) matrix or stacked bar plot of connectivity; ( 3 ) 2D graph representation of cell locations and connections; and ( 4 ) 3D cell morphology with color-coded variable ( e . g . number of synapses per segment ) .",
"After a simulation run , one can visualize and analyze simulation output: ( 1 ) time-resolved traces of any recorded cell variable ( e . g . voltage , synaptic current or ion concentration ) ; ( 2 ) relative and absolute amplitudes of post-synaptic potentials; statistics ( boxplot ) of spiking rate , the interspike interval coefficient of variation ( ISI CV ) and synchrony ( Kreuz et al . , 2015 ) ; power spectral density of firing rates; and information theoretic measures , including normalized transfer entropy and Granger causality .",
"A major feature of our tool is the ability to place extracellular electrodes to record LFPs at any arbitrary 3D locations within the network , similar to the approach offered by the LFPy ( Lindén et al . , 2013 ) and LFPsim ( Parasuram et al . , 2016 ) add-ons to NEURON .",
"The LFP signal at each electrode is obtained by summing the extracellular potential contributed by each neuronal segment , calculated using the ‘line source approximation’ and assuming an Ohmic medium with conductivity ( Parasuram et al . , 2016; Buzsáki et al . , 2012 ) .",
"The user can then plot the location of each electrode , together with the recorded LFP signal and its power spectral density and spectrogram ( Figure 6 ) .",
"The ability to record and analyze LFPs facilitates reproducing experimental datasets that include this commonly used measure ( Buzsáki et al . , 2012 ) .",
"NetPyNE permits saving and loading of all model components and results separately or in combination: high-level specifications , network instance , simulation configuration , simulation data , and simulation analysis results .",
"Saving network instances enables subsequent loading of a specific saved network with all explicit cells and connections , without the need to re-generate these from the high-level connectivity rules .",
"NetPyNE supports several standard file formats: pickle , JSON , MAT , and HDF5 .",
"The use of common file formats allows network structure and simulation results to be easily analyzed using other tools such as MATLAB or Python Pandas .",
"Network instances can also be exported to or imported from NeuroML ( Cannon et al . , 2014 ) , a standard declarative format for neural models , and SONATA ( https://github . com/AllenInstitute/sonata ) , a format standard for neural models proposed by the Blue Brain Project and Allen Institute for Brain Science .",
"These formats are also supported by other simulation tools , so that models developed using NetPyNE can be exported , explored and simulated in other tools including Brian ( Goodman , 2008 ) , MOOSE ( Bower and Beeman , 2012; Ray and Bhalla , 2008 ) , PyNN ( Davison , 2008 ) , BioNet ( Gratiy et al . , 2018 ) or Open Source Brain ( Gleeson et al . , 2018 ) .",
"Similarly , simulations from these other tools can be imported into NetPyNE .",
"This feature also enables any NetPyNE model to be visualized via the Open Source Brain portal , and permits a NeuroML model hosted on the portal to be parallelized across multiple cores ( e . g . on HPC ) using NetPyNE .",
"Support for saving output simulation data to the standardized HDF5-based Neuroscience Simulation Data Format ( NSDF ) ( Ray et al . , 2016 ) is under active development .",
"Long simulations of large networks take a long time to run .",
"Due to memory and disk constraints , it is not practical to save all state variables from all cells during a run , particularly when including signaling concentrations at many locations via the using the reaction-diffusion module .",
"Therefore , NetPyNE includes the option of recreating single-cell activity in the context of spike inputs previously recorded from a network run .",
"These follow-up simulations do not typically require an HPC since they are only running the single neuron .",
"The user selects a time period , a cell number , and a set of state variables to record or graph .",
"Parameter optimization involves finding sets of parameters that lead to a desired output in a model .",
"This process is often required since both single neuron and network models include many under-constrained parameters that may fall within a known biological range of values .",
"Network dynamics can be highly sensitive , with small parameter variations leading to large changes in network output .",
"This then requires searching within complex multidimensional spaces to match experimental data , with degeneracy such that multiple parameter sets may produce matching activity patterns ( Edelman and Gally , 2001; Prinz et al . , 2004; Neymotin et al . , 2016b ) .",
"A related concept is that of parameter exploration .",
"Once a model is tuned to reproduce biological features , it is common to explore individual parameters to understand their relation to particular model features , for example how synaptic weights affect network oscillations ( Neymotin et al . , 2011 ) , or the effect of different pharmacological treatments on pathological symptoms ( Neymotin et al . , 2016b; Knox et al . , 2018 ) .",
"Many different approaches exist to perform parameter optimization and exploration .",
"Manual tuning requires expertise and a great deal of patience ( Van Geit et al . , 2008; Moles , 2003 ) .",
"Therefore , NetPyNE provides built-in support for several automated methods that have been successfully applied to both single cell and network optimization: grid-search ( Achard and De Schutter , 2006 ) and various types of evolutionary algorithms ( EAs ) ( Dura-Bernal et al . , 2017; Neymotin et al . , 2017; Carlson et al . , 2014; Rumbell et al . , 2016; Markram et al . , 2015; Gouwens et al . , 2018 ) .",
"Grid search refers to evaluating combinations on a fixed set of values for a chosen set of parameters , resulting in gridded sampling of the multidimensional parameter space .",
"EAs search parameter space more widely and are computationally efficient when handling complex , non-smooth , high-dimensional parameter spaces ( Moles , 2003 ) .",
"They effectively follow the principles of biological evolution: here a population of models evolves by changing parameters in a way that emulates crossover events and mutation over generations until individuals reach a desired fitness level .",
"NetPyNE provides an automated parameter optimization and exploration framework specifically tailored to multiscale biophysically-detailed models .",
"Our tool facilitates the multiple steps required: ( 1 ) parameterizing the model and selecting appropriate ranges of parameter values; ( 2 ) providing fitness functions; ( 3 ) customizing the optimization/exploration algorithm options; ( 4 ) running the batch simulations; and ( 5 ) managing and analyzing batch simulation parameters and outputs .",
"To facilitate parameter selection and fitness function definitions , all the network specifications and simulation outputs are available to the user via the NetPyNE declarative data structure – from molecular concentrations and ionic channel conductances to long-range input firing rates .",
"This frees the user from having to identify and access parameters or state variables at the NEURON simulator level .",
"Both parameter optimization and exploration involve running many instances of the network with different parameter values , and thus typically require parallelization .",
"For these purposes , NetPyNE parallelization is implemented at two levels: ( 1 ) simulation level – cell computations distributed across nodes as described above; and ( 2 ) batch level – many simulations with different parameter values executed in parallel ( Dura-Bernal et al . , 2017 ) .",
"NetPyNE includes predefined execution setups to automatically run parallelized batch simulations on different environments: ( 1 ) multiprocessor local machines or servers via standard message passing interface ( MPI ) support; ( 2 ) the Neuroscience Gateway ( NSG ) online portal , which includes compressing the files and uploading a zip file via RESTful services; ( 3 ) HPC systems ( supercomputers ) that employ job queuing systems such as PBS Torque or SLURM ( e . g . Google Cloud Computing HPCs ) .",
"Users are able to select the most suitable environment setup and customize options if necessary , including any optimization algorithm metaparameters such as population size or mutation rate for EAs .",
"A single high-level command will then take care of launching the batch simulations to optimize or to explore the model .",
"The GUI enables users to intuitively access NetPyNE functionality .",
"It divides the workflow into two tabs: ( 1 ) network definition and ( 2 ) network exploration , simulation and analysis .",
"From the first tab it is possible to define – or import from various formats – the high-level network parameters/rules and simulation configuration ( Figure 2B ) .",
"Parameter specification is greatly facilitated by having clearly structured and labeled sets of parameters , graphics to represent different components , drop-down lists , autocomplete forms and automated suggestions .",
"The GUI also includes an interactive Python console and full bidirectional synchronization with the underlying Python-based model – parameters changed via the Python console will be reflected in the GUI , and vice versa .",
"In the second tab the user can interactively visualize the instantiated network in 3D , run parallel simulations and display all the available plots to analyze the network and simulation results .",
"An example of a multiscale model visualized , simulated and analyzed using the GUI is shown in Figure",
"7 . A description of this model was provided in the Reaction-diffusion parameters subsection .",
"The GUI is particularly useful for beginners , students or non-computational researchers , who can leverage it to rapidly build networks without advanced programming skills and without learning NetPyNE’s declarative syntax .",
"From there , they can simulate and explore multiscale subcellular , cellular and network models with varying degrees of complexity , from integrate-and-fire up to large-scale simulations that require HPCs .",
"The GUI is also useful for modelers , who can easily prototype new models graphically and later extend the model programmatically using automatically generated Python scripts .",
"Finally , the GUI is useful – independently of expertise level – to explore and visualize existing models developed by oneself , developed by other users programmatically , or imported from other simulators .",
"Understanding unfamiliar models becomes easier when users can navigate through all the high-level parameters in a structured manner and visualize the instantiated network structure , instead of just looking at the model definition source code ( McDougal et al . , 2015 ) .",
"Our recent model of primary motor cortex ( M1 ) microcircuits ( Dura-Bernal et al . , 2018; Neymotin et al . , 2016b; Neymotin et al . , 2017 ) constitutes an illustrative example where NetPyNE enabled the integration of complex experimental data at multiple scales: it simulates over 10 , 000 biophysically detailed neurons and 30 million synaptic connections .",
"Neuron densities , classes , morphology and biophysics , and connectivity at the long-range , local and dendritic scale were derived from published experimental data ( Suter et al . , 2013; Yamawaki et al . , 2015; Yamawaki and Shepherd , 2015; Harris and Shepherd , 2015; Sheets et al . , 2011; Weiler et al . , 2008; Anderson et al . , 2010; Yamawaki et al . , 2015; Kiritani et al . , 2012; Apicella et al . , 2012; Hooks et al . , 2013; Suter and Shepherd , 2015 ) .",
"Results yielded insights into circuit information pathways , oscillatory coding mechanisms and the role of HCN in modulating corticospinal output ( Dura-Bernal et al . , 2018 ) .",
"A scaled down version ( 180 neurons ) of the M1 model is illustrated in Figure",
"8 . Several models published in other languages have been converted to NetPyNE to increase their usability and flexibility .",
"These include models of cortical circuits exploring EEG/MEG signals ( https://hnn . brown . edu/ ) ( Jones et al . , 2009; Neymotin et al . , 2018 ) , interlaminar flow of activity ( Potjans and Diesmann , 2014; Romaro et al . , 2018 ) ( Figure 9A ) and epileptic activity ( Knox et al . , 2018 ) ( Figure 9B ) ; a dentate gyrus network ( Tejada et al . , 2014; Rodriguez , 2018 ) ( Figure 9C ) ; and CA1 microcircuits ( Cutsuridis et al . , 2010; Tepper et al . , 2018 ) ( Figure 9D ) .",
"As a measure of how compact the NetPyNE model definition is , we compared the number of source code lines ( excluding comments , blank lines , cell template files and mod files ) of the original and NetPyNE implementations ( see Table 1 ) ."
],
[
"By providing support for NEURON’s intracellular and extracellular reaction-diffusion module ( RxD ) ( McDougal et al . , 2013; Newton et al . , 2018 ) , NetPyNE helps to couple molecular-level chemophysiology – historically neglected in computational neuroscience ( Bhalla , 2014 ) – to classical electrophysiology at subcellular , cellular and network scales .",
"RxD allows the user to specify and simulate the diffusion of molecules ( e . g . , calcium , potassium or IP3 ) intracellularly , subcellularly ( by including organelles such as endoplasmic reticulum and mitochondria ) , and extracellularly in the context of signaling and enzymatic processing – for example metabolism , phosphorylation , buffering , and second messenger cascades .",
"This relates the scale of molecular interactions with that of cells and networks ( Bhalla and Iyengar , 1999 ) .",
"NetPyNE rules allow users to not only define connections at the cell-to-cell level , but also to compactly express highly specific patterns of the subcellular distribution of synapses , for example depending on the neurite cortical depth or path distance from soma .",
"Such distinct innervation patterns have been shown to depend on brain region , cell type and location; they are likely to subserve important information processing functions and have effects at multiple scales ( Komendantov and Ascoli , 2009; Kubota et al . , 2015; Petreanu et al . , 2009; Suter and Shepherd , 2015 ) .",
"Some simulation tools ( GENESIS [Bower and Beeman , 2012] , MOOSE [Ray and Bhalla , 2008] , PyNN [Davison , 2008] and neuroConstruct [Gleeson et al . , 2007] ) include basic dendritic level connectivity features , and others ( BioNet [Gratiy et al . , 2018] ) allow for Python functions that describe arbitrarily complex synapse distribution and connectivity rules .",
"However , NetPyNE is unique in facilitating the description of these synaptic distribution patterns via flexible high-level declarations that require no algorithmic programming .",
"NetPyNE’s high-level language has advantages over procedural descriptions in that it provides a human-readable , declarative format , accompanied by a parallel graphical representation , making models easier to read , modify , share and reuse .",
"Other simulation tools such as PyNN , NEST , Brian or BioNet include high-level specifications in the context of the underlying procedural language used for all aspects of model instantiation , running and initial analysis .",
"Procedural languages require ordering by the logic of execution rather than the logic of the conceptual model .",
"Since the NetPyNE declarative format is order free , it can be cleanly organized by scale , by cell type , or by region at the discretion of the user .",
"This declarative description is stored in standardized formats that can be readily translated into shareable data formats for use with other simulators .",
"High-level specifications are translated into a network instance using previously tested and debugged implementations .",
"Compared to creating these elements directly via procedural coding ( in Python/NEURON ) , our approach reduces the chances of coding bugs , replicability issues and inefficiencies .",
"The trade-off is that users of a declarative language are constrained to express inputs according to the standardized formats provided , offering less initial flexibility compared to a procedural language .",
"However , NetPyNE has been designed so that many fields are agglutinative , allowing multiple descriptors to be provided together to home in on particular subsets of cells , subcells or subnetworks , for example cells of a certain type within a given spatial region .",
"Additionally , users can add procedural NEURON/Python code between the instantiation and simulation stages of NetPyNE in order to customize or add non-supported features to the model .",
"Developers of several applications and languages , including NeuroML , PyNN , SONATA and NetPyNE , are working together to ensure interoperability between their different formats .",
"NeuroML ( Cannon et al . , 2014 ) is a widely used model specification language for computational neuroscience , which can store instantiated networks through an explicit list of populations of cells and their connections , without higher level specification rules .",
"We are collaborating with the NeuroML developers to incorporate high-level specifications similar to those used in NetPyNE , for example compact connectivity rules ( see https://github . com/NeuroML/NeuroMLlite ) .",
"The hope is that these compact network descriptions become a standard in the field so that they can be used to produce identical network instances across different simulators .",
"To further promote standardization and interoperability , we and other groups working on large-scale networks together founded the INCF Special Interest Group on ‘Standardized Representations of Network Structures’ ( https://www . incf . org/activities/standards-and-best-practices/incf-special-interest-groups/incf-sig-on-standardised ) .",
"To facilitate the exchange of simulation output data , we are currently adding support for the Neuroscience Simulation Data Format ( NSDF ) ( Ray et al . , 2016 ) , which was designed to store simulator-independent multiscale data using HDF5 .",
"Work is also in progress to extend NEURON’s RxD partial support for reading and writing Systems Biology Markup Language ( SBML ) , a standardized declarative format for computer models of biological processes ( Bulanova et al . , 2014 ) .",
"In the future , we aim to provide direct translation of SBML to NetPyNE’s RxD declarative specifications .",
"A major challenge when building complex models is optimizing their many parameters within biological constraints to reproduce experimental results ( Van Geit et al . , 2008; Moles , 2003 ) .",
"Although there can be multiple solutions to observed dynamics , Marder and colleagues demonstrated that these are sparse in the space of possible solutions and that they correspond to physiologically reasonable ranges of the cell and synapse parameters , constrained but not precisely specified by experiment ( Golowasch et al . , 2002; Prinz et al . , 2003 ) .",
"Multiple tools are available to fit detailed single-cell models to electrophysiological data: BluePyOpt ( Van Geit et al . , 2016 ) , Optimizer ( Friedrich et al . , 2014 ) , pypet ( Meyer and Obermayer , 2016 ) or NeuroTune ( https://github . com/NeuralEnsemble/neurotune ) .",
"However , these tools are limited to optimizing parameters and matching experimental data at the single-cell scale .",
"NetPyNE provides a parameter optimization framework that covers the molecular , cellular and circuit scales , thus enabling and encouraging the exploration of interactions across scales .",
"It also closely integrates with the simulator , rather than being a standalone optimizer , avoiding the need for an additional interface to map the data structures in both tools .",
"This integration allows the user to select optimization parameters and specify fitness functions that reference the same data structures employed during model definition and analysis of simulation results .",
"NetPyNE offers multiple optimization methods , including evolutionary algorithms , which are computationally efficient for handling the non-smooth , high-dimensional parameter spaces encountered in this domain ( Moles , 2003; Van Geit et al . , 2008; Svensson et al . , 2012 ) .",
"In addition to the tool itself , we have developed detailed online documentation , step-by-step tutorials ( http://netpyne . org ) , and example models .",
"The code has been released as open source ( https://github . com/Neurosim-lab/netpyne ) .",
"Ongoing support is provided via a mailing list ( with 50 subscribed users ) and active Q and A forums ( 150 posts and over 5000 views in the first year ) : http://netpyne . org/mailing , http://netpyne . org/forum and http://netpyne . org/neuron-forum .",
"Users have rapidly learned to build , simulate and explore models that illustrate fundamental neuroscience concepts , making NetPyNE a useful tool to train students .",
"To disseminate the tool , we have also provided NetPyNE training at conference workshops and tutorials , summer schools and university courses .",
"Several labs are beginning to use NetPyNE to train students and postdocs .",
"Models being developed in NetPyNE cover a wide range of regions including thalamus , sensory and motor cortices ( Dura-Bernal et al . , 2018; Neymotin et al . , 2016b ) , claustrum ( Lytton et al . , 2017 ) , striatum , cerebellum and hippocampus .",
"Application areas being explored include schizophrenia , epilepsy , transcranial magnetic stimulation ( TMS ) , and electro- and magneto-encephalography ( EEG/MEG ) signals ( Sherman et al . , 2016 ) .",
"A full list of areas and applications is available at http://netpyne . org/models .",
"Tools such as NetPyNE that provide insights into multiscale interactions are particularly important for the understanding of brain disorders , which can involve interactions across spatial and temporal scale domains ( Lytton , 2008; Lytton et al . , 2017 ) .",
"Development of novel biomarkers , increased segregation of disease subtypes , new treatments , and personalized treatments , may benefit from integrating details of molecular , anatomical , functional and dynamic organization that have been previously demonstrated in isolation .",
"Simulations and analyses developed in NetPyNE provide a way to link these scales , from the molecular processes of pharmacology , to cell biophysics , electrophysiology , neural dynamics , population oscillations , EEG/MEG signals and behavioral measures ."
],
[
"NetPyNE is implemented as a Python package that acts as a high-level interface to the NEURON simulator .",
"The package is divided into several subpackages , which roughly match the components depicted in the workflow diagram in Figure 1 .",
"The specs subpackage contains modules related to definition of high-level specifications .",
"The sim subpackage contains modules related to running the simulation .",
"It also serves as a shared container that encapsulates and provides easy access to the remaining subpackages , including methods to build the network or analyze the output , and the actual instantiated network and cell objects .",
"From the user perspective , the basic modeling workflow is divided into three steps: defining the network parameters ( populations , cell rules , connectivity rules , etc ) inside an object of the class specs . NetParams; setting the simulation configuration options ( run time , integration interval , recording option , etc ) inside an object of the class specs . SimConfig; and passing these two objects to a wrapper function ( sim . createSimulateAnalyze ( ) ) that takes care of creating the network , running the simulation and analyzing the output .",
"The following standard sequence of events are executed internally to instantiate a network from the high-level specifications in the netParams object: ( 1 ) create a Network object and add to it a set of Population and Cell objects based on netParams . popParams parameters; ( 2 ) set cell properties ( morphology and biophysics ) based on cellParams parameters ( checking which cells match the conditions of each rule ) ; ( 3 ) create molecular-level RxD objects based on rxdParams parameters; ( 4 ) add stimulation ( IClamps , NetStims , etc ) to the cells based on stimSourceParams and stimTargetParams parameters; and ( 5 ) create a set of connections based on connParams and subConnParams parameters ( checking which pre- and post-synaptic cells match the connectivity rule conditions ) , with the synaptic parameters specified in synMechParams .",
"After this process is completed all the resulting NEURON objects will be contained and easily accessible within a hierarchical Python structure ( object sim . net of the class Network ) as depicted in Figure 4 .",
"The network building task is further complicated by the need to implement parallel NEURON simulations in an efficient and replicable manner , independent of the number of processors employed .",
"Random number generators ( RNGs ) are used in several steps of the building process , including cell locations , connectivity properties and the spike times of input stimuli ( e . g . NetStims ) .",
"To ensure random independent streams that can be replicated deterministically when running on different numbers of cores we employed NEURON’s Random123 RNG from the h . Random class .",
"This versatile cryptographic-quality RNG ( Salmon et al . , 2011 ) is initialized using three seed values , which , in our case , will include a global seed value and two other values related to unique properties of the cells involved , for example for probabilistic connections , the gids of the pre- and post-synaptic cells .",
"To run NEURON parallel simulations NetPyNE employs a pc object of the class h . ParallelContext ( ) , which is created when the sim object is first initialized .",
"During the creation of the network , the cells are registered via the pc methods to enable exchange and recording of spikes across compute nodes .",
"Prior to running the simulation , global variables , such as temperature or initial voltages , are initialized , and the recording of any traces ( e . g . cell voltages ) and LFP is set up by creating h . Vector ( ) containers and calling the recording methods .",
"After running the parallel simulation via pc . solve ( ) , data ( cells , connections , spike times , recorded traces , LFPs , etc ) are gathered into the master node from all compute nodes using the pc . py_alltoall ( ) method .",
"Alternatively , distributed saving allows writing the output of each node to disk file and combines these files after the simulation has ended .",
"After gathering , the built-in analysis functions have direct access to all the network and simulation output data via sim . net . allCells and sim . allSimData .",
"NetPyNE enables import of existing cells in hoc or Python , including both templates/classes and instantiated cells .",
"To achieve this , NetPyNE internally runs the hoc or Python cell model , extracts all the relevant cell parameters ( morphology , mechanisms , point processes , synapses , etc ) and stores them in the NetPyNE JSON-like format used for high-level specifications .",
"The hoc or Python cell model is then completely removed from memory so later simulations are not affected .",
"Importing and exporting to other formats such as NeuroML or SONATA requires mapping the different model components across formats .",
"To ensure validity of the conversion , we have compared simulation outputs from each tool , or converted back to the original format and compared to the original model .",
"Tests on mappings between NetPyNE and NeuroML can be found at https://github . com/OpenSourceBrain/NetPyNEShowcase .",
"Exploring or fitting model parameters typically involves running many simulations with small variations in some parameters .",
"NetPyNE facilitates this process by automatically modifying these parameters and running all the simulations based on a set of high-level instructions provided by the user .",
"The two fitting approaches – grid search and evolutionary algorithms – both require similar set up .",
"The user creates a Batch object that specifies the range of parameter values to be explored and the run configuration ( e . g . use 48 cores on a cluster with SLURM workload manager ) .",
"For evolutionary algorithms and optionally for grid search , the user provides a Python function that acts as the algorithm fitness function , which can include variables from the network and simulation output data ( e . g . average firing rate of a population ) .",
"The NetPyNE website includes documentation and examples on how to run the different types of batch simulations .",
"Once the batch configuration is completed , the user can call the Batch . run ( ) method to trigger the execution of the batch simulations .",
"Internally , NetPyNE iterates over the different parameter combinations .",
"For each one , NetPyNE will ( 1 ) set the varying parameters in the simulation configuration ( SimConfig object ) and save it to file , ( 2 ) launch a job to run the NEURON simulation based on the run options provided by the user ( e . g . , submit a SLURM job ) , ( 3 ) store the simulation output with a unique filename , and repeat for the next parameter set , or if using evolutionary algorithms , calculate the fitness values and the next generation of individuals ( parameter sets ) .",
"To implement the evolutionary algorithm optimization we made use of the inspyred Python package ( https://pythonhosted . org/inspyred/ ) .",
"Inspyred subroutines are customized to the neural environment using parameters and fitness values obtained from NetPyNE data structures , and running parallel simulations under the NEURON environment either on multiprocessor machines via MPI or supercomputers via workload managers .",
"The NetPyNE GUI is implemented on top of Geppetto ( Cantarelli et al . , 2018 ) , an open-source platform that provides the infrastructure for building tools for visualizing neuroscience models and data and for managing simulations in a highly accessible way .",
"The GUI is defined using JavaScript , React and HTML5 .",
"This offers a flexible and intuitive way to create advanced layouts while still enabling each of the elements of the interface to be synchronized with the Python model .",
"The interactive Python backend is implemented as a Jupyter Notebook extension which provides direct communication with the Python kernel .",
"This makes it possible to synchronize the data model underlying the GUI with a custom Python-based NetPyNE model .",
"This functionality is at the heart of the GUI and means any change made to the NetPyNE model in the Python kernel is immediately reflected in the GUI and vice versa .",
"The tool’s GUI is available at https://github . com/Neurosim-lab/NetPyNE-UI and is under active development ."
]
] | [
"Biophysical modeling of neuronal networks helps to integrate and interpret rapidly growing and disparate experimental datasets at multiple scales .",
"The NetPyNE tool ( www . netpyne . org ) provides both programmatic and graphical interfaces to develop data-driven multiscale network models in NEURON .",
"NetPyNE clearly separates model parameters from implementation code .",
"Users provide specifications at a high level via a standardized declarative language , for example connectivity rules , to create millions of cell-to-cell connections .",
"NetPyNE then enables users to generate the NEURON network , run efficiently parallelized simulations , optimize and explore network parameters through automated batch runs , and use built-in functions for visualization and analysis – connectivity matrices , voltage traces , spike raster plots , local field potentials , and information theoretic measures .",
"NetPyNE also facilitates model sharing by exporting and importing standardized formats ( NeuroML and SONATA ) .",
"NetPyNE is already being used to teach computational neuroscience students and by modelers to investigate brain regions and phenomena ."
] | [
"The approximately 100 billion neurons in our brain are responsible for everything we do and experience .",
"Experiments aimed at discovering how these cells encode and process information generate vast amounts of data .",
"These data span multiple scales , from interactions between individual molecules to coordinated waves of electrical activity that spread across the entire brain surface .",
"To understand how the brain works , we must combine and make sense of these diverse types of information .",
"Computational modeling provides one way of doing this .",
"Using equations , we can calculate the chemical and electrical changes that take place in neurons .",
"We can then build models of neurons and neural circuits that reproduce the patterns of activity seen in experiments .",
"Exploring these models can provide insights into how the brain itself works .",
"Several software tools are available to simulate neural circuits , but none provide an easy way of incorporating data that span different scales , from molecules to cells to networks .",
"Moreover , most of the models require familiarity with computer programming .",
"Dura-Bernal et al . have now developed a new software tool called NetPyNE , which allows users without programming expertise to build sophisticated models of brain circuits .",
"It features a user-friendly interface for defining the properties of the model at molecular , cellular and circuit scales .",
"It also provides an easy and automated method to identify the properties of the model that enable it to reproduce experimental data .",
"Finally , NetPyNE makes it possible to run the model on supercomputers and offers a variety of ways to visualize and analyze the resulting output .",
"Users can save the model and output in standardized formats , making them accessible to as many people as possible .",
"Researchers in labs across the world have used NetPyNE to study different brain regions , phenomena and diseases .",
"The software also features in courses that introduce students to neurobiology and computational modeling .",
"NetPyNE can help to interpret isolated experimental findings , and also makes it easier to explore interactions between brain activity at different scales .",
"This will enable researchers to decipher how the brain encodes and processes information , and ultimately could make it easier to understand and treat brain disorders ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology"
] | A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana | elife-25474-v2 | [
[
"The pyridine nucleotide NAD+ ( nicotinamide adenine dinucleotide ) not only serves as a ubiquitous coenzymatic redox carrier in metabolic reactions , but also participates in intracellular signal transduction ( Berger et al . , 2004; Noctor et al . , 2006 ) .",
"NAD+ is the precursor of the second messenger cyclic ADP-ribose ( cADPR ) , which triggers calcium ( Ca2+ ) release from intracellular stores in various organisms ( Galione and Churchill , 2000; Lee , 2001; Hunt et al . , 2004; Ziegler , 2005 ) .",
"NAD+ also functions as the ADP-ribose donor and the acetyl group acceptor in protein ADP-ribosylation and deacetylation reactions , respectively ( Jacobson and Jacobson , 1999; Bürkle , 2001; Denu , 2003; Jackson et al . , 2003; Hunt et al . , 2004 ) .",
"In response to environmental stimuli , cellular NAD+ can also be released into the extracellular space by active exocytosis or diffusion through transmembrane transporters in living cells or passive leakage across membrane in collapsed tissues ( Bruzzone et al . , 2001; Contreras et al . , 2003; Seman et al . , 2003; Adriouch et al . , 2007 ) .",
"It has recently been shown that extracellular NAD+ ( eNAD+ ) plays an important signaling role in numerous physiological and pathological processes ( Billington et al . , 2006; Haag et al . , 2007; Adriouch et al . , 2012 ) .",
"In animal cells , eNAD+ can be processed by ectoenzymes such as CD38 , CD157 , and mono ( ADP-ribosyl ) transferases ( ARTs ) ( Billington et al . , 2006 ) .",
"CD38 is a multifunctional enzyme attached to the extracellular surface of the plasma membrane , which utilizes NAD+ as the substrate to produce cADPR ( Ceni et al . , 2003; De Flora et al . , 2004; Krebs et al . , 2005; Malavasi et al . , 2006; Morabito et al . , 2006; Partidá-Sánchez et al . , 2007 ) .",
"ARTs are glycosylphoshpatidylinositol-anchored or secreted ectoenzymes that use NAD+ to ADP-ribosylate lipid raft-associated signaling proteins ( Nemoto et al . , 1996; Han et al . , 2000; Seman et al . , 2003; Bannas et al . , 2005; Zolkiewska , 2005 ) .",
"eNAD+ may also be perceived by cell-surface receptors .",
"Moreschi et al . ( 2006 ) reported that NAD+ promotes intracellular [Ca2+] elevation in the human purinoceptor P2Y11-tranfected astrocytoma cells , but not in untransfected cells .",
"They also showed that , in human granulocytes , treatment with the selective and potent P2Y11 inhibitor NF157 and down-regulation of P2Y11 expression by short interference RNA , both prevented NAD+-induced intracellular [Ca2+] increases and chemotaxis ( Moreschi et al . , 2006 ) .",
"These results demonstrate that P2Y11 is involved in eNAD+-triggered transmembrane signaling .",
"Several other studies using similar approaches have also indicated that purinoceptors , including P2Y1 , P2X1 , P2X4 , and P2X7 , are engaged in eNAD+-mediated signaling ( Mutafova-Yambolieva et al . , 2007; Grahnert et al . , 2009; Klein et al . , 2009 ) .",
"However , NAD+per se has never been demonstrated to bind to purinoceptors , and thus the identity of eNAD+ receptors still remains a mystery in animals .",
"In plants , NAD+ and its derivatives have also been shown to function in stress tolerance and/or defense signaling ( Dutilleul et al . , 2005; Adams-Phillips et al . , 2010; PetriacqPétriacq et al . , 2013 ) .",
"Overexpression of the bacterial NAD+ biosynthesis gene nadC , which increases intracellular NAD+ levels , enhances defense gene expression and bacterial pathogen resistance ( Pétriacq et al . , 2012 ) .",
"In contrast , mutations in FLAGELLIN-INSENSITIVE4 , a de novo NAD+ biosynthesis gene , suppress stomatal immunity ( Macho et al . , 2012 ) .",
"Furthermore , overexpression of Arabidopsis thaliana Nudix hydrolase homolog 6 ( AtNUDT6 ) , which encodes an ADP-ribose/NADH pyrophosphohydrolase , and knockout of AtNUDT6 , AtNUDT7 , or AtNUDT8 lead to alterations of intracellular NADH levels and salicylic acid ( SA ) -mediated defense signaling ( Bartsch et al . , 2006; Ge et al . , 2007; Ishikawa et al . , 2010; Fonseca and Dong , 2014 ) .",
"We have recently shown that exogenous NAD+ induces SA-dependent and -independent PATHOGENESIS-RELATED ( PR ) gene expression and disease resistance ( Zhang and Mou , 2009 ) .",
"Importantly , we found that pathogen-induced hypersensitive response causes leakage of NAD+ into the extracellular fluid at concentrations sufficient to induce PR gene expression and disease resistance ( Zhang and Mou , 2009 ) .",
"These results provided the first line of evidence that NAD+ may also play a signaling role in plant extracellular space .",
"However , since proteins with significant homology to animal CD38/CD157 , ARTs , and purinoceptors are absent in plants , it remains unclear if eNAD+ is an endogenous signaling molecule in plants and if plants use similar mechanisms to process or perceive eNAD+ ( Hunt et al . , 2004; Sánchez et al . , 2004; Zolkiewska , 2005; Zhang and Mou , 2008 ) .",
"We have shown that expression of the human NAD+-metabolizing ectoenzyme CD38 partially compromises systemic acquired resistance ( SAR ) in Arabidopsis ( Zhang and Mou , 2012 ) , which strongly suggests that plants may use different mechanisms to sense eNAD+ .",
"In order to understand eNAD+ and its signaling role in plants , we performed a forward genetic screen in Arabidopsis to identify mutants insensitive to exogenous NAD+ ( ien ) treatment ( Zhang et al . , 2012 ) .",
"Characterization of several ien mutants revealed that the Mediator complex subunits MED14/STRUWWELPETER and MED16/SENSITIVE TO FREEZING6 /IEN1 as well as the Elongator complex function downstream of eNAD+ ( Zhang et al . , 2012 , 2013; An et al . , 2016 ) .",
"However , no receptor genes were identified in the forward genetic screen .",
"In this study , we employed a reverse genetic approach to identify eNAD+ receptors in Arabidopsis .",
"We demonstrate that the lectin receptor kinase ( LecRK ) , LecRK-I . 8 , is a potential eNAD+ receptor and plays a positive role in plant basal immunity .",
"Our findings indicate that cell-surface lectin receptors can potentially act as eNAD+-sensing receptors and present direct evidence for eNAD+ being a novel endogenous signaling molecule in plants ."
],
[
"Thus far , we have only shown that exogenous application of NAD+ induces PR gene expression in the model plant Arabidopsis ( Zhang and Mou , 2009 ) .",
"To identify genes induced by NAD+ at the genome level , we performed a microarray experiment to monitor NAD+-induced transcriptome changes in wild-type Col-0 plants ( National Center for Biotechnology Information Gene Expression Omnibus series number GSE76568 ) .",
"Triplicate experiments were performed independently , and the data were analyzed to identify genes that showed a twofold or higher induction or suppression with a low q value ( ≤0 . 05 ) .",
"Compared to the mock ( water ) treatment , NAD+ addition caused profound transcriptional changes , including upregulation of 2155 genes and downregulation of 2014 genes .",
"In the upregulated genes , those involved in plant immune responses were significantly enriched , whereas in the downregulated genes , those associated with responses to hormone stimuli , such as auxin stimulus , were overrepresented ( Figure 1A ) .",
"The NAD+-upregulated genes include a large number of pathogen-associated molecular pattern ( PAMP ) -triggered immunity ( PTI ) and SA pathway genes ( Supplementary file 1A ) .",
"In contrast , expression of several jasmonic acid ( JA ) /ethylene ( ET ) -mediated defense pathway genes , including the widely used defense marker gene PLANT DEFENSIN1 . 2 , was suppressed by NAD+ treatment ( Supplementary file 1A ) .",
"Interestingly , more than 88% of the NAD+-induced genes were also activated by the bacterial pathogen Pseudomonas syringae pv .",
"tomato ( Pst ) DC3000 carrying the effector avrRpt2 ( Figure 1B ) ( Wang et al . , 2013 ) .",
"These results are in agreement with the strong resistance induced by NAD+ against the hemibiotrophic bacterial pathogen P . syringae ( Zhang and Mou , 2009 ) . 10 . 7554/eLife . 25474 . 003Figure 1 . Exogenous NAD+-induced transcriptome changes .",
"( A ) Gene Ontology ( GO ) term enrichment test of the genes that were upregulated and downregulated by NAD+ treatment at 4 hr showed that genes involved in plant defense such as innate immune response , immune response , and response to chitin were significantly enriched in the upregulated genes , whereas those associated with responses to hormone stimuli , such as auxin stimulus , were overrepresented in the downregulated genes .",
"( B ) Overlap between the genes that were upregulated by NAD+ treatment at 4 hr and that by Pst DC3000/avrRpt2 at least at one time point of 4 , 8 , and 12 hr post-inoculation ( Wang et al . , 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 003 Further analysis of the microarray data revealed that a group of receptor kinase ( RK ) including several cell wall-associated kinase ( WAK ) genes were induced by the NAD+ application ( Supplementary file 1B ) .",
"We isolated transferred DNA ( T-DNA ) insertion lines for fourteen of the RK genes ( Supplementary file 1B ) , tested their responsiveness to NAD+ , and found that NAD+-induced resistance to the bacterial pathogen P . syringae pv .",
"maculicola ( Psm ) ES4326 was reduced in one T-DNA insertion line ( Arabidopsis Biological Resource Center accession code Salk_066416 ) ( Figure 2—figure supplement 1A ) .",
"Salk_066416 carries a T-DNA insertion in the gene At5g60280 , which was predicted to encode the legume-like ( L-type ) lectin receptor kinase-I . 8 ( LecRK-I . 8 ) ( Bouwmeester and Govers , 2009 ) .",
"We then isolated two more T-DNA insertion lines ( Salk_005125 and Salk_206382 ) for further investigation .",
"The three T-DNA insertion lines had reduced transcript levels ( Figure 2—figure supplement 1B and C ) .",
"Salk_066416 has previously been named lecrk-I . 8–2 ( Wang et al . , 2014 ) .",
"Salk_005125 and Salk_206382 were thus named lecrk-I . 8–3 , and lecrk-I . 8–4 , respectively ( Figure 2—figure supplement 1B ) .",
"The lecrk-I . 8–4 mutant accumulated higher transcript levels than lecrk-I . 8–2 and lecrk-I . 8–3 , and was considered a weak allele ( Figure 2—figure supplement 1C ) .",
"NAD+-induced expression of PR1 and resistance to Psm ES4326 were significantly inhibited in all three lecrk-I . 8 alleles but not in the extracellular ATP ( eATP ) receptor mutant dorn1-3 ( Choi et al . , 2014 ) , and induction of PR2 and PR5 was also repressed in lecrk-I . 8–2 and lecrk-I . 8–3 ( Figure 2A–D ) .",
"These results indicate that LecRK-I . 8 is a component of the eNAD+-induced defense signaling pathway .",
"Interestingly , induction of PR2 and PR5 was significantly enhanced in the dorn1-3 mutant ( Figure 2B and C ) , which is consistent with eATP being a negative regulator of SA signaling ( Chivasa et al . , 2009 ) . 10 . 7554/eLife . 25474 . 004Figure 2 . LecRK-I . 8 functions in extracellular NAD+-triggered defense signaling pathway .",
"( A ) to ( C ) NAD+-induced expression of PR1 ( A ) , PR2 ( B ) , and PR5 ( C ) was reduced in the lecrk-I . 8 mutants .",
"Plants were treated with 0 . 2 mM NAD+ solution or water .",
"Leaf tissues were collected 20 hr later for qPCR analysis .",
"Expression levels were normalized against UBQ5 .",
"Data represent the mean of three independent samples with standard deviation ( SD ) .",
"Asterisks indicate significant differences between the wild type ( WT ) and the mutants ( *p<0 . 05 , **p<0 . 01 , two-way ANOVA ) .",
"( D ) NAD+-induced resistance to the bacterial pathogen Psm ES4326 was decreased in the lecrk-I . 8 mutants .",
"Plants were treated as in ( A ) .",
"Five h later , the plants were inoculated with a Psm ES4326 suspension ( OD600 = 0 . 001 ) .",
"The bacterial titers were determined 3 d post-inoculation .",
"Data represent the mean of eight independent samples with SD .",
"Asterisks indicate significant differences between the wild type and the mutants ( *p<0 . 05 , **p<0 . 01 , two-way ANOVA ) .",
"Cfu: colony-forming units .",
"( E ) Expression levels of LecRK-I . 8 were elevated in two 35S:LecRK-I . 8 transgenic lines .",
"Data represent the mean of three independent samples with SD .",
"Different letters above the bars indicate significant differences ( p<0 . 05 , one-way ANOVA ) .",
"( F ) and ( G ) Induction of PR1 ( F ) and PR5 ( G ) by NAD+ was enhanced in the 35S:LecRK-I . 8 lines .",
"The experiments were performed as in ( A ) except that the plants were treated with 0 . 1 mM NAD+ .",
"All experiments were repeated three times with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 00410 . 7554/eLife . 25474 . 005Figure 2—source data 1 . LecRK-I . 8 functions in extracellular NAD+-triggered defense signaling pathway . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 00510 . 7554/eLife . 25474 . 006Figure 2—figure supplement 1 . Exogenous NAD+-induced Psm ES4326 resistance in T-DNA insertion lines of 14 candidate genes and transcript levels of LecRK-I . 8 in three T-DNA insertion lines .",
"( A ) Leaves of 4-week-old soil-grown plants were infiltrated with 0 . 2 mM NAD+ or water .",
"Five h later , the infiltrated leaves were inoculated with a Psm ES4326 suspension ( OD600 = 0 . 001 ) .",
"Eight leaves per genotype per treatment were collected 3 d post-inoculation and pooled to examine the growth of the pathogen .",
"WT: wild type .",
"Cfu: colony-forming units .",
"( B ) The T-DNA insertion sites in Salk_066416 ( lecrk-I . 8–2 ) , Salk_206382 ( lecrk-I . 8–3 ) , and Salk_005125 ( lecrk-I . 8–4 ) and the positions of the three pairs of primers used for real-time qPCR analysis of LecRK-I . 8 transcript levels .",
"( C ) Transcript levels of LecRK-I . 8 in lecrk-I . 8–2 , lecrk-I . 8–3 , and lecrk-I . 8–4 were significantly lower than those in the wild type ( WT ) .",
"Leaves of 4-week-old soil-grown plants were infiltrated with ( + ) or without ( - ) 0 . 2 mM NAD+ solution .",
"Total RNA was extracted 4 hr later and subjected to real-time qPCR analysis .",
"Expression levels were normalized against UBQ5 .",
"Data represent the mean of three independent samples with SD .",
"Different letters above the bars indicate significant differences ( p<0 . 05 , one-way ANOVA of square-root transformed data ) .",
"The comparison was made separately for each treatment .",
"The experiment was repeated with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 00610 . 7554/eLife . 25474 . 007Figure 2—figure supplement 2 . Characterization of 35S:LecRK-I . 8 transgenic lines .",
"( A ) Expression levels of LecRK-I . 8 in 35S:LecRK-I . 8-GFP transgenic lines .",
"Total RNA was extracted from the wild type ( WT ) and 11 single T-DNA insertion homozygous 35S:LecRK-I . 8 transgenic lines and subjected to qPCR analysis .",
"Expression levels were normalized against UBQ5 .",
"Data represent the mean of three independent samples with SD .",
"( B ) Basal resistance of seven 35S:LecRK-I . 8 transgenic lines expressing different levels of LecRK-I . 8 .",
"Plants were inoculated with a Psm ES4326 suspension ( OD600 = 0 . 001 ) .",
"The bacterial titers were determined 3 d post-inoculation .",
"Data represent the mean of eight independent samples with SD .",
"The experiments were repeated with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 007 To test if overexpression of LecRK-I . 8 could enhance Arabidopsis responsiveness to NAD+ , we transformed a 35S:LecRK-I . 8 construct into wild-type Col-0 plants .",
"Intriguingly , we were unable to identify transgenic lines expressing very high levels of LecRK-I . 8 .",
"The expression levels of LecRK-I . 8 in all of the obtained 11 transgenic lines increased less than fourfold ( Figure 2—figure supplement 2A ) , suggesting that overexpression of LecRK-I . 8 may be detrimental to plant growth and development .",
"Nevertheless , although the transgenic lines , which expressed higher levels of LecRK-I . 8 than the wild type , did not show enhanced disease resistance ( Figure 2—figure supplement 2B ) , NAD+-induced expression of PR1 and PR5 in these lines was significantly enhanced ( Figure 2E–2G ) .",
"This result supports that LecRK-I . 8 functions in eNAD+-mediated defense pathway .",
"Both DORN1 and LecRK-I . 8 contain a putative transmembrane domain and two putative arginine-glycine-aspartic acid ( RGD ) -binding motifs that likely mediate plant cell wall-plasma membrane interactions ( Gouget et al . , 2006 ) ( Figure 3A and Figure 3—figure supplement 1 ) , suggesting their possible plasma membrane localization .",
"Indeed , DORN1 has been shown to be localized in the plasma membrane ( Bouwmeester et al . , 2011 ) .",
"To determine the subcellular localization of LecRK-I . 8 , we transformed a 35S:LecRK-I . 8-GFP ( Green Fluorescence Protein ) construct into the lecrk-I . 8–2 mutant .",
"Very low levels of LecRK-I . 8-GFP protein were detected in three out of eleven single insertion homozygous transgenic lines ( Figure 3—figure supplement 2A ) , which is consistent with the hypothesis that overexpression of LecRK-I . 8 may be harmful to plants .",
"Nevertheless , the low level of LecRK-I . 8-GFP complemented the enhanced disease susceptibility phenotype of lecrk-I . 8–2 ( see below ) ( Figure 3—figure supplement 2B ) , indicating that LecRK-I . 8-GFP is biologically active .",
"Unfortunately , we were unable to detect any GFP fluorescence in the transgenic Arabidopsis plants accumulating LecRK-I . 8-GFP .",
"To circumvent this problem , we transiently expressed LecRK-I . 8-GFP in Nicotiana benthamiana .",
"The LecRK-I . 8-GFP fusion protein in N . benthamiana appeared to be localized in the plasma membrane ( Figure 3B ) .",
"To confirm this subcellular localization , we co-expressed LecRK-I . 8-GFP and AHA2-mCherry in N . benthamiana .",
"As shown in Figure 3C , the GFP and mCherry signals in the co-transformed N . benthamiana epidermal cell precisely overlapped with each other .",
"Since AHA2 encodes a well-established plasma membrane localized P-type H+- ATPase ( DeWitt et al . , 1996 ) , this result indicates that LecRK-I . 8 is localized at the plasma membrane . 10 . 7554/eLife . 25474 . 008Figure 3 . Subcellular localization of the LecRK-I . 8-GFP fusion protein .",
"( A ) Putative RGD-binding motifs in DORN1 and LecRK-I . 8 .",
"( B ) Confocal images of N . benthamiana epidermal cells transiently expressing GFP ( Left ) and LecRK-I . 8-GFP ( right ) .",
"( C ) Confocal images of N . benthamiana epidermal cells transiently co-expressing LecRK-I . 8-GFP and AHA2-mCherry .",
"Left: LecRK-I . 8-GFP , middle: AHA2-mCherry , and right: merged image . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 00810 . 7554/eLife . 25474 . 009Figure 3—figure supplement 1 . Alignment between the NAD receptor LecRK-I . 8 and the ATP receptor DORN1 . The amino acid sequences of LecRK-I . 8 and DORN1 were aligned using the CLUSTALW tool at the PBIL ( Pôle Bioinformatique Lyonnais ) .",
"LecRK-I . 8 and DORN1 ( LecRK-I . 9 ) have approximately 81 . 5% amino acid sequence similarity .",
"The transmembrane domain and the kinase domain ( KD ) were underlined in blue and red , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 00910 . 7554/eLife . 25474 . 010Figure 3—figure supplement 2 . Characterization of 35S:LecRK-I . 8-GFP lecrk-I . 8–2 transgenic lines .",
"( A ) LecRK-I . 8-GFP protein levels in 35S:LecRK-I . 8-GFP lecrk-I . 8–2 transgenic lines .",
"Total protein was extracted from the wild type ( WT ) and 11 single T-DNA insertion homozygous 35S:LecRK-I . 8-GFP lecrkI . 8–2 transgenic lines and subjected to SDS-PAGE and analyzed by immunoblot analysis using a monoclonal anti-GFP antibody .",
"The Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate and prolonged exposure ( 1 hr ) of the x-ray film were employed to detect the LecRK-I . 8-GFP band .",
"The asterisk indicates an unspecific band , indicating equal loading .",
".",
"( B ) Basal resistance of the three 35S:LecRK-I . 8-GFP lecrk-I . 8–2 transgenic lines showing LecRK-I . 8-GFP protein .",
"Plants were inoculated with a Psm ES4326 suspension ( OD600 = 0 . 0001 ) .",
"The bacterial titers were determined 3 d post-inoculation .",
"Data represent the mean of eight independent samples with SD .",
"Different letters above the bars indicate significant differences ( p<0 . 05 , one-way ANOVA ) .",
"The experiment was repeated with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 010 LecRK-I . 8 contains a cytoplasmic kinase domain ( KD ) ( Figure 3—figure supplement 1 ) .",
"To test if LecRK-I . 8 is an active kinase , we expressed the LecRK-I . 8KD fragment as a Maltose-Binding Protein ( MBP ) -LecRK-I . 8KD fusion protein in Escherichia coli .",
"The MBP-LecRK-I . 8KD recombinant protein was purified using amylose resin and subjected to kinase activity assay ( Figure 4 ) .",
"Simultaneously purified MBP protein was included in the experiment as a negative control ( Figure 4 ) .",
"As shown in the autoradiograph , the LecRK-I . 8KD protein exhibited a strong autophosphorylation activity and also phosphorylated myelin basic protein , a substrate commonly used for in vitro kinase assays .",
"This result indicates that LecRK-I . 8 possesses kinase activity . 10 . 7554/eLife . 25474 . 011Figure 4 . Kinase activity of the LecRK-I . 8 kinase domain . An autoradiograph ( right panel ) showing that the kinase domain ( KD ) of LecRK-I . 8 is active based on autophosphorylation and phosphorylation of the myelin basic protein .",
"The purified MBP and MBP-LecRK-I . 8KD proteins used for kinase activity assays were separated in a different SDS-PAGE gel ( left panel ) , and the white arrow in the left panel indicates the expected size of the MBP-LecRK-I . 8KD protein band .",
"The experiment was repeated with similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 011 Plasma membrane localization of LecRK-I . 8 plus its active cytoplasmic kinase domain suggest a putative role for LecRK-I . 8 as a receptor for an extracellular ligand .",
"To test whether NAD+ is a ligand binding to LecRK-I . 8 , we generated transgenic Arabidopsis plants expressing the extracellular lectin domain ( amino acids ( AAs ) 23 to 283 ) of LecRK-I . 8 fused to GFP ( eLecRK-I . 8-GFP ) .",
"The eLecRK-I . 8-GFP protein was immunoprecipitated using an anti-GFP antibody ( Figure 5—figure supplement 1A ) and subjected to binding assays with 32P-labeled NAD+ .",
"A significant NAD+ binding activity was detected for the immunoprecipitated eLecRK-I . 8-GFP protein , but not for the GFP protein immunoprecipitated from transgenic plants expressing GFP using the same antibody ( Figure 5A ) , indicating that the NAD+ binding activity is likely specific to eLecRK-I . 8 .",
"A specific NAD+ binding activity was also detected for an eLecRK-I . 8-HA-His fusion protein transiently expressed in N . benthamiana ( Figure 5B and Figure 5—figure supplement 1B ) .",
"Furthermore , NAD+ binding activity was detected for the recombinant fusion protein MBP-eLecRK-I . 8 but not for MBP , MBP-eDORN1 ( AAs 22 to 288 ) , and MBP-eLecRK-I . 3 ( AAs 22 to 286 ) ( Figure 5C and Figure 5—figure supplement 1C ) .",
"The extracellular domain of LecRK-I . 6 ( AAs 22 to 286 ) , which is the closest homolog of LecRK-I . 8 ( Bouwmeester and Govers , 2009 ) , did not bind NAD+ , either ( Figure 5—figure supplement 2 ) , suggesting that LecRK-I . 6 may not be an eNAD+ receptor .",
"Since LecRK-I . 8 , DORN1 , LecRK-I . 3 , and LecRK-I . 6 , all are L-type LecRKs and belong to the LecRK-I clade ( Bouwmeester and Govers , 2009 ) , this result indicates that not all of the extracellular lectin domains of L-type LecRKs can bind NAD+ .",
"In addition , we detected a dramatic increase of NAD+ binding activity in the membrane fractions of the 35S:LecRK-I . 8 transgenic plants and a clear decrease of NAD+ binding activity in the membrane fractions of the lecrk-I . 8–2 mutant plants ( Figure 5D ) .",
"These results are consistent with the increased and decreased NAD+ responsiveness in the 35S:LecRK-I . 8 and lecrk-I . 8–2 plants , respectively ( Figure 2 ) .",
"Interestingly , there were differences in ligand affinities between the membrane fractions of the 35S:LecRK-I . 8 plants and the wild type , which might suggest possible conformation changes when LecRK-I . 8 is overexpressed in plants .",
"Finally , the immunoprecipitated eLecRK-I . 8-GFP protein showed a typical saturation curve for NAD+ binding with a dissociation constant ( Kd ) of 436 . 5 ± 104 . 8 nM ( Figure 5E ) , which falls well below the extracellular NAD ( H ) concentration ( ~0 . 4 mM ) in pathogen-infected leaf tissues ( Zhang and Mou , 2009 ) , and thus indicates a relatively high affinity .",
"On the other hand , we found that 5 μM of NAD+ was able to significantly induce the early PAMP responsive genes GLUTATHIONE S-TRANSFERASE1 ( GST1 ) and FLG22-INDUCED RECEPTOR-LIKE KINASE1 ( FRK1 ) ( Figure 5—figure supplement 3 ) , indicating that the NAD+ concentrations required to trigger defense responses are significantly higher than the Kd value of LecRK-I . 8 , which is rather unusual and difficult to reconcile with a primary ligand sensor function of LecRK-I . 8 . 10 . 7554/eLife . 25474 . 012Figure 5 . LecRK-I . 8 binds NAD+ .",
"( A ) to ( C ) , Binding of 32P-labeled NAD+ to immunoprecipitated GFP and eLecRK-I . 8-GFP proteins ( A ) , purified eLecRK-I . 8-HA-His protein ( B ) , and recombinant MBP , MBP-eLecRK-I . 8 , MBP-eDORN1 , and MBP-eLecRK-I . 3 proteins ( C ) .",
"( - ) in ( B ) is an empty vector control .",
"Approximately 0 . 5 g Arabidopsis leaf tissues , 1 g N . benthamiana leaf tissues , and ~5 μg recombinant proteins were used for each binding assay in ( A ) , ( B ) , and ( C ) , respectively .",
"( D ) Binding of 32P-labeled NAD+ to the microsomal fractions of 35S:LecRK-I . 8 , wild-type ( WT ) , and lecrk-I . 8–2 plants .",
"Specific binding was determined by subtracting the binding in the presence of 1000-fold unlabeled NAD+ from the total binding in the absence of cold competitor .",
"( E ) Saturation binding assay for LecRK-I . 8 .",
"Immunoprecipitated eLecRK-I . 8-GFP proteins were incubated with the indicated concentrations of 32P-labeled NAD+ for 30 min .",
"Free NAD+ was removed by washing .",
"Data were plotted as a specific binding with SD of three experiments .",
"The dissociation constant ( Kd ) was calculated by one site specific binding saturation model using GraphPad Prism 5 ( www . graphpad . com ) .",
"( F ) Competitive binding assay for LecRK-I . 8 .",
"Samples containing 250 nM of 32P-labeled NAD+ in the presence of 100 nM to 1 mM of unlabeled nucleotides were assayed for specific binding of 32P-labeled NAD+ .",
"Inhibition constant ( Ki ) values were calculated in GraphPad Prism 5 using the one site Fit Ki competition model .",
"In ( A ) , ( B ) , ( C ) , and ( E ) , results from three independent experiments were combined ( error bars represent SD ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 01210 . 7554/eLife . 25474 . 013Figure 5—source data 1 . LecRK-I . 8 binds NAD+ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 01310 . 7554/eLife . 25474 . 014Figure 5—figure supplement 1 . Purified eLecRK-I . 8 proteins .",
"( A ) Proteins immunoprecipitated from the wild-type ( WT ) , 35S:GFP and 35S:eLecRK-I . 8-GFP plants using the anti-GFP antibody .",
"( B ) Proteins purified from N . benthamiana leaves infiltrated with Agrobacteria carrying the pCAMBIA1300S-eLecRK-I . 8-HA-His plasmid or the empty pCAMBIA1300S vector ( - ) using the HisPur Cobalt resin .",
"The asterisk indicates an unspecific band .",
".",
"( C ) Proteins purified from E . coli cells expressing the recombinant MBP , MBP-eLecRKI . 8 , MBP-eDORN1 , and MBP-eLecRK-I . 3 fusion proteins using the amylose resin . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 01410 . 7554/eLife . 25474 . 015Figure 5—figure supplement 2 . NAD+ binding assay of the recombinant MBP-eLecRK-I . 6 protein .",
"( A ) Proteins purified from E . coli cells expressing the recombinant MBP , MBP-eLecRKI . 8 , and MBP-eLecRK-I . 6 fusion proteins using the amylose resin .",
"( B ) Binding of 32P-labeled NAD+ to recombinant MBP , MBP-eLecRK-I . 8 , and MBP-eLecRK-I . 6 proteins .",
"Approximately 5 μg recombinant proteins were used for the binding assay .",
"Results from three independent experiments were combined ( error bars represent SD ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 01510 . 7554/eLife . 25474 . 016Figure 5—figure supplement 3 . Induction of several early PAMP-responsive genes by low concentrations of NAD+ .",
"Four-week-old soil-grown wild-type Col-0 plants were treated with the indicated concentration of NAD+ solution .",
"Leaf tissues were collected 30 min later for qPCR analysis .",
"Expression levels were normalized against UBQ5 .",
"Data represent the mean of three independent samples with SD .",
"The experiment was repeated with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 016 However , binding of NAD+ to LecRK-I . 8 suggests that LecRK-I . 8 may be a receptor for eNAD+ .",
"To test the specificity of the binding between NAD+ and LecRK-I . 8 , we analyzed the ability of various unlabeled nucleotides to compete for binding of 32P-labeled NAD+ .",
"While unlabeled NAD+ exhibited strong competition for binding with 32P-labeled NAD+ , other nucleotides including NADP+ , ATP , ADP , and AMP showed little or no competition ( Figure 5F ) .",
"Given the structural similarity between NAD+ and NADP+ and the fact that NADP+ can similarly induce immune responses in plants ( Zhang and Mou , 2009 ) , it is surprising that NADP+ did not efficiently compete for binding with 32P-labeled NAD+ ( Figure 5F ) .",
"These results suggest that LecRK-I . 8 may be a receptor specific for NAD+ .",
"If LecRK-I . 8 is a receptor specific for NAD+ , mutations in LecRK-I . 8 should only affect NAD+-induced defense signaling .",
"To test this hypothesis , we treated the lecrk-I . 8 mutants and wild type with NAD+ and three other defense inducers , NADP+ , flg22 ( a peptide corresponding to the 22 AAs of the conserved N-terminal part of flagellin ) , and SA .",
"As shown in Figure 6A and B , while NAD+ triggered significantly lower levels of PR1 gene expression and Psm ES4326 resistance in the lecrk-I . 8 mutants than those in the wild type , NADP+ , flg22 , and SA induced similar levels of PR1 gene expression and Psm ES4326 resistance in the lecrk-I . 8 mutants and the wild type .",
"These results indicate a specific role for LecRK-I . 8 in eNAD+-triggered signaling .",
"Moreover , LecRK-I . 8 has previously been shown to mediate Pieris brassicae egg extract-triggered PR1 gene expression ( Gouhier-Darimont et al . , 2013 ) , but the identity of the elicitor ( s ) in the egg extracts is unclear .",
"To test if one of the elicitors in insect egg extracts is NAD+ , we treated the previously generated 35S:CD38 transgenic plants ( Zhang and Mou , 2012 ) , lecrk-I . 8–2 , and wild type with Trichoplusia ni ( cabbage looper ) egg extracts following the published protocol ( Gouhier-Darimont et al . , 2013 ) , and analyzed the egg extract-induced PR1 expression .",
"CD38 is a human NAD ( P ) -metabolizing ectoenzyme and has been shown to partially block exogenous NAD+-induced PR1 expression and Psm ES4326 resistance ( Zhang and Mou , 2012 ) .",
"Similarly to P . brassicae egg extracts ( Gouhier-Darimont et al . , 2013 ) , T . ni egg extracts induced PR1 gene expression in the wild-type plants , and the induction was dramatically reduced in the lecrk-I . 8–2 mutant ( Figure 6C ) .",
"Importantly , T . ni egg extract-induced PR1 expression was also significantly inhibited in the 35S:CD38 plants ( Figure 6C ) .",
"Therefore , insect egg extracts may either contain NAD+ as part of their defense inducing activity or induce release of cellular NAD+ into the extracellular space .",
"Taken together , our results strongly suggest that LecRK-I . 8 is a potential receptor for NAD+ . 10 . 7554/eLife . 25474 . 017Figure 6 . Extracellular NADP+- , flg22- , and SA-induced immune responses are not affected in the lecrk-I . 8 mutants .",
"( A ) NAD+- , NADP+- , flg22- , and SA-induced PR1 expression in the wild type ( WT ) and the lecrk-I . 8 mutants .",
"Plants were infiltrated with 0 . 2 mM NAD+ , 0 . 2 mM NADP+ , 1 μM flg22 , or water .",
"For SA treatment , plants were treated with soil drenches plus foliar sprays of 0 . 5 mM SA solution or water .",
"Leaf tissues were collected 20 hr later for qPCR analysis .",
"Expression levels were normalized against UBQ5 .",
"Data represent the mean of three independent samples with SD .",
"Asterisks indicate significant differences between the wild type ( WT ) and the mutants ( **p<0 . 01 , two-way ANOVA ) .",
"( B ) NAD+- , NADP+- , flg22- , and SA-induced Psm ES4326 resistance in the wild type and the lecrk-I . 8 mutants .",
"Plants were treated as in ( A ) .",
"Five h after NAD+ or NADP+ treatment and 24 hr after flg22 or SA treatment , the plants were inoculated with a Psm ES4326 suspension ( OD600 = 0 . 001 ) .",
"The bacterial titers were determined 3 d post-inoculation .",
"Data represent the mean of eight independent samples with SD .",
"Asterisks indicate significant differences between the wild type and the mutants ( *p<0 . 05 , **p<0 . 01 , two-way ANOVA ) .",
"( C ) Insect egg extract-induced PR1 gene expression in 35S:CD38 transgenic plants .",
"Two μL of T . ni egg extracts were dropped onto leaves of the WT , lecrk-I . 8–2 , and 35S:CD38 transgenic plants .",
"The treated leaves without petiole were collected 3 d later for qPCR analysis .",
"Leaf tissues from untreated plants were used as the control .",
"Data represent the mean of three independent samples with SD .",
"Different letters above the bars indicate significant differences ( p<0 . 05 , one-way ANOVA ) .",
"The comparison was made separately for each treatment .",
"All experiments were repeated three times with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 01710 . 7554/eLife . 25474 . 018Figure 6—source data 1 . Extracellular NADP+- , flg22- , and SA-induced immune responses are not affected in the lecrk-I . 8 mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 018 Our results so far have shown that mutations in LecRK-I . 8 partially compromised a low concentration of NAD+-induced PR gene expression and Psm ES4326 resistance .",
"Since PR genes are late defense genes , we compared NAD+-induced expression of several early defense genes , including AZELAIC ACID INDUCED1 ( AZI1 ) , PHYTOALEXIN DEFICIENT4 ( PAD4 ) , NON-INDUCIBLE IMMUNITY1 ( NIM1 ) -INTERACTING1 ( NIMIN1 ) , NIMIN2 , WRKY18 , and WRKY54 , in wild type and the lecrk-I . 8–2 mutant .",
"As shown in Figure 7—figure supplement 1 , induction of these early defense genes by NAD+ was not significantly affected by the lecrk-I . 8–2 mutation .",
"This result indicates that NAD+ perception mechanisms other than LecRK-I . 8 still exist in the lecrk-I . 8–2 mutant .",
"To substantiate this conclusion , we treated wild type and the lecrk-I . 8–2 mutant with different concentrations of NAD+ and tested NAD+-induced defense responses .",
"The expression of PR genes and resistance to Psm ES4326 activated by 0 . 2 and 0 . 4 mM NAD+ were significantly reduced in the lecrk-I . 8–2 plants compared with those in the wild type , whereas 0 . 6 mM NAD+ induced similar levels of PR gene expression and Psm ES4326 resistance in the mutant and the wild type ( Figure 7 ) .",
"These results are consistent with the remaining NAD+ binding activity in the membrane fractions of the lecrk-I . 8–2 mutant , supporting the existence of other NAD+ receptors and/or perception mechanisms in Arabidopsis . 10 . 7554/eLife . 25474 . 019Figure 7 . Immune responses induced by different concentrations of NAD+ in lecrk-I . 8–2 . ( A ) Comparison of different concentrations of NAD+-induced expression of PR1 , PR2 , and PR5 in lecrk-I . 8–2 and the wild type ( WT ) .",
"Leaves of 4-week-old soil-grown plants were infiltrated with the indicated concentrations of NAD+ .",
"Total RNA was extracted from the infiltrated leaves 20 hr later and subjected to real-time qPCR analysis .",
"Expression was normalized against constitutively expressed UBQ5 .",
"Data represent the mean of three independent samples with SD .",
"Asterisks indicate significant differences between lecrk-I . 8–2 and the wild type ( *p<0 . 05 , **p<0 . 01 , two-way ANOVA ) .",
"( B ) Comparison of different concentrations of NAD+-induced resistance to Psm ES4326 in lecrk-I . 8–2 and the wild type .",
"Leaves of 4-week-old soil-grown plants were infiltrated with the indicated concentrations of NAD+ .",
"Five h later , the infiltrated leaves were inoculated with a Psm ES4326 suspension ( OD600 = 0 . 001 ) .",
"The in planta bacterial titers were determined 3 d post-inoculation .",
"Data represent the mean of eight independent samples with SD .",
"Asterisks indicate significant differences between lecrk-I . 8–2 and the wild type ( **p<0 . 01 , two-way ANOVA ) .",
"Experiments were repeated three times with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 01910 . 7554/eLife . 25474 . 020Figure 7—source data 1 . Immune responses induced by different concentrations of NAD+ in lecrk-I . 8-2 . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 02010 . 7554/eLife . 25474 . 021Figure 7—figure supplement 1 . NAD+-induced expression of several early defense-responsive genes in the lecrk-I . 8–2 mutant . Four-week-old soil-grown wild-type ( WT ) and lecrk-I . 8–2 plants were treated with 0 . 2 mM NAD+ solution or water .",
"Leaf tissues were collected 4 hr later for qPCR analysis .",
"Expression levels were normalized against UBQ5 .",
"Data represent the mean of three independent samples with SD .",
"The experiment was repeated with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 021 If eNAD+ is an endogenous signaling molecule and LecRK-I . 8 is an essential cell-surface receptor for eNAD+ , mutations in LecRK-I . 8 should compromise immune responses .",
"However , in Figures 2D , 6B and 7B , where the plants were infected with Psm ES4326 at a high dose ( an inoculum of OD600 = 0 . 001 ) ( Clarke et al . , 2000 ) , the bacterial pathogen grew to similar levels in mock ( water ) -treated wild type and lecrk-I . 8 mutants .",
"The high dose is generally used for disease resistance test and may not be able to resolve differences in disease susceptibility ( Glazebrook et al . , 1996; Clarke et al . , 2000 ) .",
"Therefore , we inoculated wild-type and lecrk-I . 8 plants with a low dose ( an inoculum of OD600 = 0 . 0001 ) .",
"Under this condition , both lecrk-I . 8–2 and lecrk-I . 8–3 exhibited significantly reduced PR1 gene induction and significantly enhanced susceptibility to Psm ES4326 , compared with the wild type and the dorn1-3 mutant ( Figure 8 ) .",
"We also tested biological induction of SAR in the lecrk-I . 8 mutants and found that SAR induction was not affected in all three lecrk-I . 8 alleles ( Figure 8—figure supplement 1 ) .",
"Nevertheless , our results demonstrate that the potential eNAD+ receptor LecRK-I . 8 plays a positive role in plant immunity . 10 . 7554/eLife . 25474 . 022Figure 8 . Basal immunity is compromised in the lecrk-I . 8 mutants .",
"( A ) Psm ES4326-induced PR1 expression was inhibited in the lecrk-I . 8 mutants .",
"Plants were inoculated with ( + ) or without ( − ) a Psm ES4326 suspension ( OD600 = 0 . 0001 ) .",
"Leaf tissues were collected 24 hr post-inoculation for qPCR analysis .",
"Expression was normalized against constitutively expressed UBQ5 .",
"Data represent the mean of three independent samples with SD .",
"Different letters above the bars indicate significant differences ( p<0 . 05 , one-way ANOVA ) .",
"The comparison was made separately for each treatment .",
"WT: wild type .",
"( B ) and ( C ) , The lecrk-I . 8 mutants were more susceptible to Psm ES4326 than the wild type .",
"Plants were inoculated with a Psm ES4326 suspension ( OD600 = 0 . 0001 ) .",
"The bacterial titers in ( B ) were determined immediately and 3 d post-inoculation .",
"Data represent the mean of eight independent samples with SD .",
"Different letters above the bars indicate significant differences ( p<0 . 05 , one-way ANOVA ) .",
"The comparison was made separately for each time point .",
"Photos showing the disease symptoms in ( C ) were taken 3 d post-inoculation .",
"Experiments in ( A ) and ( B ) were repeated three times with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 02210 . 7554/eLife . 25474 . 023Figure 8—source data 1 . Basal immunity is compromised in the lecrk-I . 8 mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 02310 . 7554/eLife . 25474 . 024Figure 8—figure supplement 1 . Biological induction of SAR in lecrk-I . 8 mutants . Three lower leaves on each plant were inoculated with Psm ES4326 ( OD600 = 0 . 002 ) ( +SAR ) or mock-treated with 10 mM MgCl2 ( -SAR ) .",
"Two days later , two upper uninfected/untreated leaves were challenge-inoculated with Psm ES4326 ( OD600 = 0 . 001 ) .",
"The in plants bacterial titers were determined 3 d after challenge inoculation .",
"Data represent the mean of eight independent samples with SD .",
"The experiment was repeated with similar trends . DOI: http://dx . doi . org/10 . 7554/eLife . 25474 . 024"
],
[
"Here , we present several lines of evidence to demonstrate that LecRK-I . 8 is a potential eNAD+ receptor in Arabidopsis .",
"First , the LecRK-I . 8 gene is induced by exogenous NAD+ ( Supplementary file 1B ) , which is consistent with the observation that receptor-encoding genes are often ligand inducible ( Zipfel et al . , 2006 ) .",
"Second , LecRK-I . 8 is localized in the plasma membrane and possesses an active cytoplasmic kinase domain ( Figures 3 and 4 ) .",
"Third , LecRK-I . 8 binds NAD+ , but not NADP+ , ATP , ADP , or AMP ( Figure 5F ) , and three other LecRKs , DORN1 , LecRK-I . 3 , and LecRK-I . 6 , do not bind NAD+ ( Figure 5C and Figure 5—figure supplement 2 ) .",
"Fourth , mutations in LecRK-I . 8 inhibit NAD+-induced , but not NADP+- , flg22- , and SA-induced , defense responses ( Figure 6A and B ) .",
"Finally , mutations in LecRK-I . 8 compromise basal resistance to the bacterial pathogen Psm ES4326 ( Figure 8 ) .",
"NAD+ has long been shown to bind to rat brain synaptic membranes ( Khalmuradov et al . , 1983 ) , and recent studies have also indicated that several purinergic P2X and P2Y receptors are involved in eNAD+-induced biological responses ( Moreschi et al . , 2006; Mutafova-Yambolieva et al . , 2007; Grahnert et al . , 2009; Klein et al . , 2009 ) .",
"However , there has been no direct evidence demonstrating the binding of NAD+ to a known cell-surface receptor .",
"Therefore , the identity of eNAD+-binding receptors has remained unclear .",
"Our identification of a LecRK as a potential eNAD+ receptor suggests that lectin receptors may directly bind NAD+ , leading to transmembrane signaling .",
"As lectin receptors are widely distributed in both the animal and plant kingdoms ( Drickamer and Taylor , 1993; Vaid et al . , 2012 ) , and certain animal and plant lectin domains have convergently evolved similar ligand binding architecture ( Loris , 2002 ) , animal cells might also use lectin receptors to sense eNAD+ .",
"This speculation could be tested using receptor-NAD+ binding assays .",
"Identification of a potential eNAD+ receptor in Arabidopsis provides direct evidence for eNAD+ being a bona fide endogenous signaling molecule in plants ( Zhang and Mou , 2012 ) .",
"This is consistent with the fact that genes involved in plant immune responses are significantly enriched in the genes upregulated by NAD+ and that the majority of the NAD+-induced genes are also activated by the bacterial pathogen Pst DC3000/avrRpt2 ( Figure 1 ) ( Wang et al . , 2013 ) .",
"These results together should eradicate the skepticism concerning the physiological relevance of eNAD+ perception in plants ( Fu and Dong , 2013 ) .",
"Additionally , given the diverse roles played by eNAD+ in animal cells ( Billington et al . , 2006; Iqbal and Zaidi , 2006; Haag et al . , 2007; Adriouch et al . , 2012; Mutafova-Yambolieva and Durnin , 2014 ) , further studies with the Arabidopsis lecrk-I . 8 mutants will likely reveal new biological functions for this signaling molecule in plants .",
"eNAD+ may play a role in plant-insect interactions .",
"It has recently been reported that deposition of P . brassicae egg batches on Arabidopsis leaves induces the SA signaling pathway , which in turn suppresses the JA pathway , thus benefiting the insect progeny ( Little et al . , 2007; Bruessow et al . , 2010 ) .",
"By treatment with P . brassicae egg extracts , which mimics the effect of oviposition , Gouhier-Darimont et al . ( 2013 ) identified LecRK-I . 8 as a potential cell surface receptor for the insect egg-derived elicitors .",
"Although it has been shown that a fraction from purified P . brassicae egg lipids is able to induce PR1 gene expression ( Gouhier-Darimont et al . , 2013 ) , the identity of the elicitors still remains unknown .",
"Three pieces of evidence generated in this study indicate that the defense-inducing activity of the insect egg extract could be , at least , partially attributed to NAD+ , and/or that the perception of egg extract may lead to release of cellular NAD+ into the extracellular space .",
"First , LecRK-I . 8 is potentially a cell surface receptor specific for NAD+ ( Figure 5 ) .",
"Second , the human NAD ( P ) -metabolizing ectoenzyme CD38 inhibits T . ni egg extract-induced PR1 gene expression ( Figure 6C ) .",
"Finally , the majority of P . brassicae oviposition-induced receptor-like kinase genes ( Little et al . , 2007 ) are also upregulated by exogenous NAD+ addition ( Supplementary file 1C ) .",
"Thus , insect eggs appear to use NAD+ to alter the SA-JA signaling balance in plants for the benefit of the insect progeny .",
"It has been shown that eNAD+ and eATP play multiple , partially overlapping roles in animal immune cells ( Haag et al . , 2007 ) .",
"One of the best-studied examples is the mechanism activating the P2X7 purinoceptor by eNAD+ and eATP in T cells and macrophages ( Bartlett et al . , 2014; Rissiek et al . , 2015 ) .",
"While ATP activates P2X7 through direct binding ( Surprenant et al . , 1996; Rassendren et al . , 1997; Chessell et al . , 1998 ) , NAD+ regulates P2X7 in these two different cell types via distinct mechanisms .",
"In T cells , NAD+ promotes ART-mediated ADP-ribosylation of P2X7 , which is sufficient for activation of the purinoceptor ( Seman et al . , 2003; Adriouch et al . , 2008 ) , whereas in macrophages , ADP-ribosylation does not activate P2X7 but rather reduces the threshold concentration of ATP needed to turn on the receptor ( Hong et al . , 2009 ) .",
"Thus , eNAD+ and eATP function additively and synergistically in T cells and macrophages , respectively , to regulate P2X7 signaling .",
"In contrast , available evidence obtained by studies in Arabidopsis indicates that eNAD+ and eATP act antagonistically to modulate the SA signaling pathway .",
"eNAD+ induces SA accumulation and SA-dependent defense gene expression and disease resistance ( Zhang and Mou , 2009 ) , whereas eATP suppresses these defense responses ( Chivasa et al . , 2009 ) .",
"In agreement with these results , induction of PR genes by exogenous NAD+ in the eATP receptor mutant dorn1-3 is significantly enhanced compared with that in the wild type ( Figure 2A–C ) .",
"On the other hand , the dorn1-3 mutation does not significantly affect basal and NAD+-induced resistance to the bacterial pathogen Psm ES4326 ( Figures 2D and 8B ) , which may be attributed to the presence of other eATP receptors .",
"Whether knockout of LecRK-I . 8 influences eATP-mediated suppression of SA signaling needs further investigation .",
"As in animal cells ( Ziegler and Niere , 2004 ) , eNAD+ is likely perceived by multiple receptors and/or mechanisms in plants .",
"Mutations in LecRK-I . 8 only block 0 . 2 and 0 . 4 mM NAD+-induced , but not 0 . 6 mM NAD+-induced , PR gene expression and disease resistance ( Figure 7 ) .",
"Furthermore , induction of several early defense responsive genes by 0 . 2 mM NAD+ is not inhibited by the lecrk-I . 8–2 mutation ( Figure 7—figure supplement 1 ) .",
"These results indicate that LecRK-I . 8 is not the sole eNAD+ perception mechanism in Arabidopsis .",
"Indeed , the Arabidopsis genome contains genes encoding 75 LecRKs ( 32 G-type , 42 L-type , and 1 C-type ) as well as a large number of other RKs and channels ( Arabidopsis Genome Initiative , 2000; Vaid et al . , 2012 ) , many of which are also NAD+ inducible and could potentially encode eNAD+ receptors .",
"Thus , a genome-wide survey via NAD+ binding assays is warranted for identification of other eNAD+ receptors in Arabidopsis .",
"It is currently unclear whether eNAD+ signaling is specific for Arabidopsis or the mustard family ( Brassicaceae ) .",
"Recent genome-wide analyses of LecRKs in multiple plant species revealed that this large family of RKs also exists in other dicots and monocots .",
"For instance , there are 231 LecRKs ( 180 G-type , 50 L-type , and 1 C-type ) in Populus ( Yang et al . , 2016 ) , 173 LecRKs ( 100 G-type , 72 L-type , and 1 C-type ) in rice ( Vaid et al . , 2012 ) , 263 LecRKs ( 177 G-type , 84 L-type , and 2 C-type ) in bread wheat ( Shumayla et al . , 2016 ) , and 113 LecRKs ( 59 G-type , 53 L-type , and 1 C-type ) in foxtail millet ( Zhao et al . , 2016 ) .",
"These results strongly suggest that eNAD+ receptors may be broadly distributed in the plant kingdom .",
"Further investigations are needed to test if eNAD+ signaling is in play in diverse plant species and to identify eNAD+ receptors in these plant species ."
],
[
"The wild type used in this study was the Arabidopsis thaliana ( L . ) Heynh .",
"ecotype Columbia ( Col-0 ) .",
"The T-DNA insertion lines used in this study are listed in Supplementary file 1B , and the dorn1-3 mutant ( SALK_042209 ) was previously described ( Choi et al . , 2014 ) .",
"The T-DNA insertion lines were obtained from either Arabidopsis Biological Resource Center at The Ohio State University ( Columbus , OH ) or Nottingham Arabidopsis Stock Center at The University of Nottingham ( Nottingham , UK ) .",
"Homozygous mutant plants of the T-DNA insertion lines were confirmed with primers flanking the T-DNA insertions and the left border primers LBa1 , LB3 , and o8409 ( Supplementary file 1D ) .",
"The Arabidopsis seeds were sown on autoclaved soil ( Sunshine MVP; Sun Gro Horticulture , Agawam , MA ) and cold-treated at 4°C for 3 days .",
"Plants were germinated and grown at 22°C to 24°C under a 16-hr-light/8-hr-dark regime .",
"Four-week-old soil-grown plants were used for chemical treatment and pathogen infection .",
"NAD+-Na and NADP+-Na were dissolved in water , and the pH of the resulting solutions was adjusted to ~6 . 0 using 0 . 1 M NaOH .",
"Flg22 was dissolved in water to make a 100 μM stock solution , which was freshly diluted before each experiment .",
"For NAD+ , NADP+ , and flg22 treatment , the solution was infiltrated into Arabidopsis leaves using a 1 mL needleless syringe .",
"For SA treatment , plants were soil-drenched with 0 . 5 mM SA , sprayed with 0 . 5 mM SA plus 0 . 1% Tween 20 , and partially covered with a transparent plastic dome .",
"Cabbage looper eggs were ordered from Benzon Research Inc . ( Carlisle , PA ) .",
"Generation of egg extracts and treatment with egg extracts were conducted as previously described ( Gouhier-Darimont et al . , 2013 ) .",
"Inoculation of Arabidopsis plants with the bacterial pathogen Psm ES4326 was performed by pressure-infiltration using a 1 mL needleless syringe .",
"Eight leaves per genotype/treatment from eight plants were collected immediately after the inoculation and/or 3 d post-inoculation to examine the growth of the pathogen .",
"Total RNA extraction , reverse transcription , and real-time qPCR were performed as previously described ( Wang et al . , 2015 ) using gene-specific primers ( Supplementary file 1D ) .",
"Leaf tissues for each independent RNA sample were collected from 12 plants .",
"Protein gel blot analysis was conducted as described previously ( Mou et al . , 2003 ) .",
"For subcellular localization study , the DNA fragments encoding mGFP in pRTL2-mGFP and mCherry in pNDH-OCT were amplified using the primers XbaI-SalI-GFPF/XhoI-GFPR and XbaI-SalI-mCherryF/XhoI-mCherryR , respectively .",
"The primers used for plasmid construction are listed in Supplementary file 1D .",
"The PCR products were digested with XbaI and XhoI and cloned into XbaI/SalI-digested pCAMBIA1300S to create pCAMBIA1300S-GFP and pCAMBIA1300S-mCherry .",
"Then the coding regions of LecRK-I . 8 and AHA2 were amplified using the primers BamHI-flLecRK-I . 8F/SalI-flLecRK-I . 8R and SacI-AHA2F/SalI-AHA2R , respectively .",
"The PCR products were digested with BamHI or SacI and SalI and cloned into the corresponding sites of pCAMBIA1300S-GFP and pCAMBIA1300S-mCherry , resulting in pCAMBIA1300S-LecRK-I . 8-GFP and pCAMBIA1300S-AHA2-mCherry , respectively .",
"For generation of transgenic Arabidopsis expressing eLecRK-I . 8-GFP , the LecRK-I . 8 fragment encoding the extracellular domain was amplified using the primers EcoRI-ATGLecRK-I . 8F and BspHI-eLecRK-I . 8R .",
"The PCR products were digested with EcoRI and BspHI and cloned into EcoRI/NcoI-digested pRTL2-mGFP to generate pRTL2-eLecRK-I . 8-GFP .",
"Then the 35S:eLecRK-I . 8-GFP cassette was recovered using HindIII and subcloned into HindIII-digested and calf intestinal phosphatase-treated pCB302 to produce pCB302-35S:eLecRK-I . 8-GFP .",
"For transient expression in N . benthamiana , the LecRK-I . 8 fragment encoding the extracellular domain was amplified using the primers SacI-eLecRK-I . 8F and SalI-HisHAeLecRK-I . 8R .",
"The PCR products were digested with SacI and SalI and cloned into the corresponding sites of the vector pCAMBIA1300S , resulting in the plasmid pCAMBIA1300S-eLecRK-I . 8-HA-His .",
"For expression of the MBP-eLecRK-I . 8 , MBP-eDORN1 , MBP-eLecRK-I . 3 , MBP-eLecRK-I . 6 , and MBP-LecRK-I . 8KD fusion proteins in Escherichia coli , DNA fragments encoding the corresponding domains of these proteins were amplified using the primers listed in Supplementary file 1D .",
"The PCR products were digested with appropriate restriction enzymes and cloned into the corresponding sites of pMAL-p2X , generating pMAL-p2X-fragment plasmids .",
"The plasmids pCB302-35S:eLecRK-I . 8-GFP , pCAMBIA1300S-eLecRK-I . 8-HA-His , pCAMBIA1300S-LecRK-I . 8-GFP and pCAMBIA1300S-AHA2-mCherry were introduced into the Agrobacterium strain GV3101 ( pMP90 ) and the pMAL-p2X-fragment plasmids were introduced into the E . coli strain BL21 ( DE3 ) by electroporation .",
"Arabidopsis wild-type Col-0 plants were transformed with Agrobacteria carrying the pCB302-35S:eLecRK-I . 8-GFP plasmid following the floral dip protocol ( Clough and Bent , 1998 ) .",
"Transient expression in N . benthamiana was performed as described previously with slight modifications ( Krasileva et al . , 2010 ) .",
"Briefly , Agrobacteria carrying pCAMBIA1300S-LecRK-I . 8-GFP , pCAMBIA1300S-AHA2-mCherry , or pCAMBIA1300S-eLecRK-I . 8-HA-His were suspended in an induction buffer ( 10 mM MES-KOH , pH 5 . 6 , 10 mM MgCl2 and 100 μM acetosyringone ) to an OD600 of 0 . 4 , preinduced for 2 to 3 hr at 28°C , and then infiltrated into N . benthamiana leaves using a 1 mL needleless syringe .",
"Two to 3 d later , the leaves infiltrated with the Agrobacteria carrying pCAMBIA1300S-LecRK-I . 8-GFP/pCAMBIA1300S-AHA2-mCherry and pCAMBIA1300S-eLecRK-I . 8-HA-His were used for microscopy analysis and protein purification , respectively .",
"For immunoprecipitation , leaf tissues from 3-week-old soil-grown 35S:eLecRK-I . 8-GFP and 35S:GFP plants were homogenized on ice in extraction buffer ( 50 mM HEPES , pH 7 . 5 , 50 mM NaCl , 10 mM EDTA , 5 mM DTT , 1% Triton X-100 , and protease inhibitors: 50 μg/mL TPCK , 50 μg/mL TLCK , and 0 . 6 mM PMSF ) .",
"The homogenates were centrifuged at 20 , 000 g at 4°C for 20 min and the supernatants were transferred to new Eppendorf tubes .",
"Monoclonal anti-GFP antibodies were added to the extracts ( 1:200 ) .",
"After incubation at 4°C for 1 hr , the antibody bound proteins were precipitated by adding protein G plus-agarose beads to the extracts ( 20 μL/mL ) , followed by incubation at 4°C overnight .",
"The beads were then precipitated by centrifugation at 2000 rpm for 5 min , washed 3 times with the extraction buffer without detergent , and then used for immunoblotting and NAD+ binding assays .",
"For purification of eLecRK-I . 8-HA-His , agroinfiltrated N . benthamiana leaf tissues were homogenized on ice in extraction buffer ( 50 mM Tris-HCl , pH7 . 5 , 150 mM NaCl , 0 . 1% Triton X-100 , 0 . 2% Nonidet P-40 , 6 mM β-mercaptoethanol , and protease inhibitors: 50 μg/mL TPCK , 50 μg/mL TLCK , and 0 . 6 mM PMSF ) .",
"The homogenates were centrifuged at 20 , 000 g at 4°C for 20 min and the supernatants were transferred to new Eppendorf tubes .",
"The eLecRK-I . 8-HA-His protein was purified using HisPur Cobalt Resin following the manufacturer’s protocol ( Thermo Scientific , Waltham , MA ) .",
"After washing , the resins with bound proteins were used for immunoblotting and NAD+ binding assays .",
"For purification of MBP-eLecRK-I . 8 , MBP-eDORN1 , MBP-eLecRK-I . 3 , MBP-eLecRK-I . 6 , and MBP-LecRK-I . 8KD , a single colony of BL21 ( DE3 ) carrying the corresponding plasmid was cultured overnight at 37°C in 5 mL Lysogeny broth ( LB ) with 50 µg/mL ampicillin .",
"One mL of the seed culture was added to 500 mL fresh LB medium with 50 µg/mL ampicillin and cultured at 37°C with shaking to an OD600 of 0 . 4 .",
"Isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 0 . 3 mM and the culture was allowed to grow for another 16–18 hr at 18°C before the cells were harvested for protein extraction .",
"MBP-fusion proteins were purified with amylose resin according to the protocol supplied by the manufacturer ( New England Biolabs , Ipswich , MA ) .",
"NAD+ binding experiments were performed following a previously described protocol for brassinolide binding assays ( Wang et al . , 2001 ) .",
"Briefly , beads with the bound proteins were re-suspended in binding buffer ( 10 mM HEPES , pH 7 . 5 , 5 mM MgCl2 ) and aliquoted in 86 µL portions for individual binding reactions .",
"For total binding assay , 10 µL binding buffer and 4 µL 32P-labeled NAD+ ( 6 . 25 µM ) were added , resulting in 250 nM 32P-labeled NAD+ in the final 100 µL reaction mixture .",
"32P-labeled NAD+ ( specific activity 800 Ci/mmol ) was purchased from PerkinElmer ( Waltham , MA ) .",
"For nonspecific binding , 10 µL of 2 . 5 mM unlabeled NAD+ and 4 µL 32P-labeled NAD+ were added , resulting in 250 µM unlabeled NAD+ and 250 nM 32P-labeled NAD+ in the final 100 µL reaction mixture .",
"The mixtures were incubated for 30 min at room temperature with gentle mixing every 5 min .",
"The beads containing the binding reactions were then precipitated by centrifugation at 2000 rpm for 5 min , washed 3 times with the binding buffer , re-suspended in 10 mL scintillation counter liquid per sample , and carefully transferred into scintillation vials .",
"The vials were placed in a Beckman Coulter LS6500 Multi-Purpose Scintillation Counter ( Beckman Coulter , Brea , CA ) and bound 32P-labeled NAD+ was quantified by scintillation counting .",
"Specific NAD+ binding was calculated by subtracting the nonspecific binding from the total binding .",
"For saturation binding assay , protein G plus-agarose beads with the bound proteins were incubated with different concentrations ( 50 , 200 , 500 , 1000 , and 1500 nM ) of 32P-labeled NAD+ in the absence ( for total binding ) or presence ( for nonspecific binding ) of additional 1000-fold unlabeled NAD+ in the binding buffer .",
"For competitive binding assay , protein G plus-agarose beads with the bound proteins were incubated with 250 nM of 32P-labeled NAD+ in the presence of different concentrations ( 100 nM , 1 µM , 10 µM , 100 µM and 1 mM ) of unlabeled nucleotides ( NAD+ , NADP+ , ATP , ADP and AMP ) in the binding buffer .",
"The dissociation constant ( Kd ) was calculated by one site specific binding saturation model using GraphPad Prism 5 ( www . graphpad . com ) .",
"Inhibition constant ( Ki ) values were calculated by importing the data points into GraphPad Prism 5 ( www . graphpad . com ) using the one site Fit Ki competition model .",
"For microsome-based binding assays , microsomes were prepared as previously described with minor modifications ( Wang et al . , 2001; Caño-Delgado and Wang , 2009 ) .",
"All steps were conducted at 4°C .",
"Arabidopsis plants were grown under a 15-hr-light/9-hr-dark regime for about 6 weeks .",
"Rosette leaf tissues were homogenized with a mortar and pestle in 1 mL/1 gram chilled membrane extraction buffer ( 20 mM Tris-HCl , pH 7 . 5 , 250 mM mannitol , 5 mM MgCl2 , 0 . 1 mM CaCl2 , and protease inhibitors ) .",
"Homogenates were filtered through two layers of Miracloth and centrifuged at 10 , 000 g for 10 min at 4°C .",
"The supernatant was centrifuged at 100 , 000 g for 1 hr at 4°C to pellet microsomes .",
"The microsomal pellet was resuspended at a protein concentration of 2 mg/mL in binding buffer ( 10 mM MES-KOH , pH 5 . 7 , 5 mM MgCl2 , 0 . 25 mM CaCl2 , 0 . 25 M mannitol , and protease inhibitors ) .",
"Each binding assay contains 50 μL microsomes , indicated amount ( 50 , 200 , 500 , 1000 nM ) of 32P-labeled NAD+ , 1 mg/mL BSA , with 1000-fold excess unlabeled NAD+ for background binding assays .",
"The final reaction volume was brought to 100 μL by adding binding buffer .",
"The binding reactions were incubated for 30 min at room temperature with gentle mixing every 5 min .",
"The bound and free 32P-labeled NAD+ were separated by filtering the mixture through a glass-fibre filter ( Whatman ) and washing with 10 mL ice-cold binding buffer , and were quantified by scintillation counting .",
"Binding data were analyzed and plotted using GraphPad Prism 5 .",
"The kinase assay was performed as described by Choi et al . ( 2014 ) with minor modifications .",
"Briefly , 5 µg of MBP-LecRK-I . 8KD and 2 µg of myelin basic protein in a buffer ( 50 mM Tris-HCl pH7 . 5 , 50 mM KCl , 10 mM MnCl2 , 10 mM MgCl2 ) plus 1 µL of ATP ( 0 . 4 µL 32P-labeled ATP , 0 . 4 µL cold ATP , 0 . 2 µL H2O ) in a total volume of 30 µL were incubated at 30°C for 30 min . 32P-labeled ATP ( specific activity 3000 Ci/mmol ) was purchased from PerkinElmer .",
"After SDS-PAGE electrophoresis , the gel was dried and exposed to X-ray film for 3 hr .",
"Four-week-old soil-grown plants were treated with 1 mM NAD+ or water .",
"Total RNA samples extracted from the treated leaves were subjected to microarray analysis .",
"Briefly , RNA concentration was determined on a NanoDrop Spectrophotometer ( Thermofisher Scientific , Waltham , MA ) and sample quality was assessed using the 2100 Bioanalyzer ( Agilent Technologies , Santa Clara , CA ) .",
"cDNA was synthesized from 200 ng of total RNA and used as a template for in vitro transcription in the presence of T7 RNA Polymerase and cyanine labeled CTP’s using the Quick Amp Labeling kit ( Agilent Technologies ) according the manufacturer’s protocol .",
"The amplified , labeled complementary RNA ( cRNA ) was purified using the RNeasy Mini kit ( Qiagen , Valencia , CA ) .",
"For each array , 1650 ng of Cy three labeled cRNA was fragmented and hybridized with rotation at 65°C for 17 hr .",
"Samples were hybridized to Arabidopsis 4 × 44 k arrays ( Agilent Technologies ) .",
"The arrays were washed according to the manufacturer’s protocol and then scanned on a G2505B scanner ( Agilent Technologies ) .",
"Data were extracted using Feature Extraction 10 . 1 . 1 . 1 software ( Agilent Technologies ) .",
"Data ( individual signal intensity values ) obtained from the microarray probes were background corrected using a normexp+offset method , in which a small positive offset ( k = 50 ) was added to move the corrected intensities away from zero ( Ritchie et al . , 2007 ) .",
"The resulting data were log transformed ( using two as the base ) and normalized between individual samples using quantile normalization ( Smyth , 2005 ) .",
"After normalization , a linear model was fitted on each gene for comparison using the limma package in R ( Ritchie et al . , 2015 ) .",
"To control false discovery rate ( FDR ) and correct for multiple hypothesis testing , a q value was calculated and used to assess the significance of each test ( Benjamini and Hochberg , 1995 ) .",
"A probe-by-probe comparison was performed between different treatments using water treatment as the reference sample .",
"In each comparison , a q value and fold change ( FC ) were computed for each gene locus .",
"The gene expression fold changes were computed based on the normalized log-transformed signal intensity data .",
"The comparison results were further explored to obtain numbers of overlapped genes between NAD+ treatment and Pst DC3000/avrRpt2 infection .",
"Pathway and gene ontology analysis were performed on deferentially expressed genes using DAVID Bioinformatics Resources ( Huang et al . , 2009 ) .",
"N . N . benthamiana leaf tissues were mounted in water and viewed with a Zeiss confocal LSM 5 Pascal microscope ( Jena , Germany ) .",
"GFP was visualized using an excitation wavelength of 488 nm and a bandpass 505 to 530 nm emission filter and mCherry was visualized using an excitation of 543 nm and a bandpass 600 to 680 nm emission filter .",
"Statistical analyses were performed using the one-way ANOVA and the two-way ANOVA in Prism 5 . 0b ( GraphPad Software , La Jolla , CA ) .",
"The accession numbers of the microarrays discussed in this manuscript are GSE76568 and GSE38986 in Gene Expression Omnibus ."
]
] | [
"Nicotinamide adenine dinucleotide ( NAD+ ) participates in intracellular and extracellular signaling events unrelated to metabolism .",
"In animals , purinergic receptors are required for extracellular NAD+ ( eNAD+ ) to evoke biological responses , indicating that eNAD+ may be sensed by cell-surface receptors .",
"However , the identity of eNAD+-binding receptors still remains elusive .",
"Here , we identify a lectin receptor kinase ( LecRK ) , LecRK-I . 8 , as a potential eNAD+ receptor in Arabidopsis .",
"The extracellular lectin domain of LecRK-I . 8 binds NAD+ with a dissociation constant of 436 . 5 ± 104 . 8 nM , although much higher concentrations are needed to trigger in vivo responses .",
"Mutations in LecRK-I . 8 inhibit NAD+-induced immune responses , whereas overexpression of LecRK-I . 8 enhances the Arabidopsis response to NAD+ .",
"Furthermore , LecRK-I . 8 is required for basal resistance against bacterial pathogens , substantiating a role for eNAD+ in plant immunity .",
"Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants ."
] | [
"Plants and animals are generally healthy , despite being surrounded by many different microbes that have the potential to infect them and cause disease .",
"This is because plants and animals are able to sense infections and promptly activate immune responses against them .",
"A molecule known as NAD is involved in many processes inside healthy cells , but it can also act as a warning signal of infection .",
"When an invading microbe damages a host cell , NAD leaks out of the damaged cell .",
"Neighboring healthy cells sense this NAD and activate immune responses .",
"It is thought that specific receptor proteins on the surface of animal and plant cells are responsible for detecting NAD that has leaked out of damaged cells .",
"However , the identities of these receptors were not known .",
"Wang , Zhou et al . used genetics and biochemical techniques to investigate how cells in a plant known as Arabidopsis detect NAD .",
"The experiments reveal that a receptor protein called LecRK-I . 8 can bind NAD via a section of the receptor known as the lectin domain .",
"Arabidopsis plants with mutant forms of LecRK-I . 8 are less able to activate immune responses when exposed to NAD compared to normal plants .",
"Furthermore , the mutant plants are less able to defend themselves against Pseudomonas syringae , a bacterium that can infect many different plants .",
"On the other hand , plants with higher levels of LecRK-I . 8 than normal produce stronger immune responses to NAD .",
"The findings of Wang , Zhou et al . identify the first receptor on the surface of plant cells that can detect NAD .",
"The next challenge is to find out if humans and other animals also use similar proteins to detect NAD during infections .",
"In agriculture , bacterial infections can lead to major losses of crops .",
"Therefore , these findings may help researchers to develop crop varieties that are more resistant to these infections ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"evolutionary biology",
"microbiology and infectious disease"
] | Integrating genotypes and phenotypes improves long-term forecasts of seasonal influenza A/H3N2 evolution | elife-60067-v2 | [
[
"Seasonal influenza virus infects 5–15% of the global population every year causing an estimated 250 , 000 to 500 , 000 deaths annually with the majority of infections caused by influenza A/H3N2 ( World Health Organization , 2014 ) .",
"Vaccination remains the most effective public health response available .",
"However , frequent viral mutation results in viruses that escape previously acquired human immunity .",
"The World Health Organization ( WHO ) Global Influenza Surveillance and Response System ( GISRS ) monitors influenza evolution by sampling currently circulating viruses , or strains , and analyzing these strains with genome sequencing and serological assays .",
"The WHO GISRS uses these data to select vaccine viruses that should best represent circulating viruses in the next influenza season .",
"However , because the process of vaccine development and distribution requires several months to complete , optimal vaccine design requires an accurate prediction of which viruses will predominate approximately one year after vaccine viruses are selected .",
"Historically , the effectiveness of the H3N2 vaccine component has been much lower than the other seasonal influenza subtypes .",
"For example , H3N2’s mean vaccine effectiveness from 2004 to 2015 was 33% compared to 61% for H1N1pdm and 54% for influenza B viruses ( Belongia et al . , 2016 ) .",
"Multiple factors can reduce vaccine effectiveness including selection of a vaccine strain that is not antigenically representative of future populations ( Belongia et al . , 2016; Gouma et al . , 2020 ) and adaptations of the selected strain to egg-passaging during vaccine production that alter the antigenicity of the resulting vaccine component ( Zost et al . , 2017 ) .",
"Even when vaccine strains are well-matched antigenically , they may fail to induce a strong immune response due to previous infection history of vaccine recipients ( Cobey et al . , 2018 ) .",
"While all of these factors must be addressed to increase vaccine effectiveness , substantial effort has focused on the selection of the most representative strain for the next season’s vaccine .",
"Current vaccine predictions focus on the hemagglutinin ( HA ) protein , which acts as the primary target of human immunity .",
"Until recently , the hemagglutination inhibition ( HI ) assay has been the primary experimental measure of antigenic cross-reactivity between pairs of circulating viruses ( Hirst , 1943 ) .",
"Most modern H3N2 strains carry a glycosylation motif that reduces their binding efficiency in HI assays ( Chambers et al . , 2015; Zost et al . , 2017 ) , prompting the increased use of virus neutralization assays including the neutralization-based focus reduction assay ( FRA ) ( Okuno et al . , 1990 ) .",
"Together , these two assays are the gold standard in virus antigenic characterizations for vaccine strain selection , but they are laborious and low-throughput compared to genome sequencing ( Wood et al . , 2012 ) .",
"As a result , researchers have developed computational methods to predict influenza evolution from sequence data alone ( Luksza and Lässig , 2014; Steinbrück et al . , 2014; Neher et al . , 2014 ) .",
"Despite the promise of these sequence-only models , they explicitly omit experimental measurements of antigenic or functional phenotypes .",
"Recent developments in computational methods and influenza virology have made it feasible to integrate these important metrics of influenza fitness into a single predictive model .",
"For example , phenotypic measurements of antigenic drift are now accessible through phylogenetic models ( Neher et al . , 2016 ) and functional phenotypes for HA are available from deep mutational scanning ( DMS ) experiments ( Lee et al . , 2018 ) .",
"We describe an approach to integrate previously disparate sequence-only models of influenza evolution with high-quality experimental measurements of antigenic drift and functional constraint .",
"The influenza community has long recognized the importance of incorporating HI phenotypes and other experimental measurements of viral phenotypes with existing forecasting methods to inform the vaccine design process ( Gandon et al . , 2016; Morris et al . , 2018; Lässig et al . , 2017 ) .",
"Although several distinct efforts have made progress in using HI phenotypes to evaluate the evolution of seasonal influenza ( Steinbrück et al . , 2014; Neher et al . , 2016 ) , published methods stop short of developing a complete forecasting framework wherein the evolutionary contribution of HI phenotypes can be compared and contrasted with new and existing fitness metrics .",
"However , unpublished work by Luksza and Lässig , 2014 to the WHO GISRS network incorporates antigenic phenotypes into fitness-based predictions ( Morris et al . , 2018; M Łuksza , personal communication , June 2020 ) .",
"Here , we provide an open source framework for forecasting the genetic composition of future seasonal influenza populations using genotypic and phenotypic fitness estimates .",
"We apply this framework to HA sequence data shared via the GISAID EpiFlu database ( Shu and McCauley , 2017 ) and to HI and FRA titer data shared by WHO GISRS Collaborating Centers in London , Melbourne , Atlanta and Tokyo .",
"We systematically compare potential predictors and show that HI phenotypes enable more accurate long-term forecasts of H3N2 populations compared to previous metrics based on epitope mutations alone .",
"We also find that composite models based on phenotypic measures of antigenic drift and genotypic measures of functional constraint consistently outperform any fitness models based on individual genotypic or phenotypic metrics ."
],
[
"We developed a framework to forecast seasonal influenza evolution inspired by the Malthusian growth fitness model of Luksza and Lässig , 2014 .",
"As with this original model , we forecasted the frequencies of viral populations one year in advance by applying to each virus strain an exponential growth factor scaled by an estimate of the strain’s fitness ( Figure 1 and Equation 1 ) .",
"Luksza and Lässig , 2014 measured model performance by identifying clades – groups of strains that all share a recent common ancestor – and comparing observed and estimated future clade frequencies .",
"However , as clade definitions are inherently unstable between seasons , we evaluated our models by comparing the genetic composition of observed and estimated future populations with the earth mover’s distance metric .",
"The earth mover’s distance calculates the minimum distance between two populations , given the frequency of each individual within a population and a pairwise ‘ground distance’ between individuals ( Rubner et al . , 1998 ) .",
"We defined distinct amino acid haplotypes as individuals in our observed and estimated future populations .",
"For frequencies of individuals , we used the observed frequencies of haplotypes in the future and our model’s estimated frequencies .",
"We calculated the ground distance between individuals as the Hamming distance between haplotypes .",
"With this implementation , more accurate projections of the future population’s composition produce smaller earth mover’s distances between the observed and estimated future ( Figure 1 ) .",
"We estimated viral fitness with biologically-informed metrics including those originally defined by Luksza and Lässig , 2014 of epitope antigenic novelty and mutational load ( non-epitope mutations ) as well as four more recent metrics including hemagglutination inhibition ( HI ) antigenic novelty ( Neher et al . , 2016 ) , deep mutational scanning ( DMS ) mutational effects ( Lee et al . , 2018 ) , local branching index ( LBI ) ( Neher et al . , 2014 ) , and change in clade frequency over time ( delta frequency ) ( Table 1 ) .",
"All of these metrics except for HI antigenic novelty and DMS mutational effects rely only on HA sequences .",
"The antigenic novelty metrics estimate how antigenically distinct each strain at time t is from previously circulating strains based on either genetic distance at epitope sites or log2 titer distance from HI measurements .",
"Increased antigenic drift relative to previously circulating strains is expected to correspond to increased viral fitness .",
"Mutational load estimates functional constraint by measuring the number of putatively deleterious mutations that have accumulated in each strain since their ancestor in the previous season .",
"DMS mutational effects provide a more comprehensive biophysical model of functional constraint by measuring the beneficial or deleterious effect of each possible single amino acid mutation in HA from the background of a previous vaccine strain , A/Perth/16/2009 .",
"The growth metrics estimate how successful populations of strains have been in the last six months based on either rapid branching in the phylogeny ( LBI ) or the change in clade frequencies over time ( delta frequency ) .",
"We fit models for individual fitness metrics and combinations of metrics that we anticipated would be mutually beneficial .",
"For each model , we learned coefficient",
"( s ) that minimized the earth mover’s distance between HA amino acid sequences from the observed population one year in the future and the estimated population produced by the fitness model ( Equation 2 ) .",
"We evaluated model performance with time-series cross-validation such that better models reduced the earth mover’s distance to the future on validation or test data .",
"The earth mover’s distance to the future can never be zero , because each model makes predictions based on sequences available at the time of prediction and cannot account for new mutations that occur during the prediction interval .",
"We calculated the lower bound for each model’s performance as the optimal distance to the future possible given the current sequences at each timepoint .",
"As an additional reference , we evaluated the performance of a ‘naive’ model that predicted the future population would be identical to the current population .",
"We expected that the best models would consistently outperform the naive model and perform as close as possible to the lower bound .",
"The long-term evolution of influenza H3N2 hemagglutinin has been previously described as a balance between positive selection for substitutions that enable escape from adaptive immunity by modifying existing epitopes and purifying selection on domains that are required to maintain the protein’s primary functions of binding and membrane fusion ( Bush et al . , 1999; Neher , 2013; Luksza and Lässig , 2014; Koelle and Rasmussen , 2015 ) .",
"To test the ability of our models to accurately detect these evolutionary patterns under controlled conditions , we simulated the long-term evolution of H3N2-like viruses under positive and purifying selection for 40 years ( Materials and methods , Figure 2 ) .",
"These selective constraints produced phylogenetic structures and accumulation of epitope and non-epitope mutations that were consistent with phylogenies of natural H3N2 HA ( Figure 3 , Tables 2 and 3 ) .",
"We fit models to these simulated populations using all sequence-only fitness metrics .",
"As a positive control for our model framework , we also fit a model based on the true fitness of each strain as measured by the simulator .",
"We hypothesized that fitness metrics associated with viral success such as true fitness , epitope antigenic novelty , LBI , and delta frequency would be assigned positive coefficients , while metrics associated with fitness penalties , like mutational load , would receive negative coefficients .",
"We reasoned that both LBI and delta frequency would individually outperform the mechanistic metrics as both of these growth metrics estimate recent clade success regardless of the mechanistic basis for that success .",
"Correspondingly , we expected that a composite model of epitope antigenic novelty and mutational load would perform as well as or better than the growth metrics , as this model would include both primary fitness constraints acting on our simulated populations .",
"As expected , the true fitness model outperformed all other models , estimating a future population within 6 . 82 ± 1 . 52 amino acids ( AAs ) of the observed future and surpassing the naive model in 32 ( 97% ) of 33 timepoints ( Figure 4 , Table 4 ) .",
"Although the true fitness model performed better than the naive model’s average distance of 8 . 97 ± 1 . 35 AAs , it did not reach the closest possible distance between populations of 4 . 57 ± 0 . 61 AAs .",
"With the exception of epitope antigenic novelty , all biologically-informed models consistently outperformed the naive model ( Figure 5 , Table 4 ) .",
"LBI was the best of these models , with a distance to the future of 7 . 57 ± 1 . 85 AAs .",
"This result is consistent with the fact that the LBI is a correlate of fitness in models of rapidly adapting populations ( Neher et al . , 2014 ) .",
"Indeed , both growth-based models received positive coefficients and outperformed the mechanistic models .",
"The mutational load metric received a consistently negative coefficient with an average distance of 8 . 27 ± 1 . 35 AAs .",
"Surprisingly , the composite model of epitope antigenic novelty and mutational load did not perform better than the individual mutational load model ( Figure 5—figure supplement 1 ) .",
"The antigenic novelty fitness metric assumes that antigenic drift is driven by nonlinear effects of previous host exposure ( Luksza and Lässig , 2014 ) that are not explicitly present in our simulations .",
"To understand whether positive selection at epitope sites might be better represented by a linear model , we fit an additional model based on an ‘epitope ancestor’ metric that counted the number of epitope mutations since each strain’s ancestor in the previous season .",
"This linear fitness metric slightly outperformed the antigenic novelty metric ( Table 4 ) .",
"Importantly , a composite model of the epitope ancestor and mutational load metrics outperformed all other epitope-based models and the individual mutational load model ( Figure 5—figure supplement 1 ) .",
"From these results , we concluded that our method can accurately estimate the evolution of simulated populations , but that the fitness of simulated strains was dominated by purifying selection and only weakly affected by a linear effect of positive selection at epitope sites .",
"We hypothesized that a composite model of mutually beneficial metrics could better approximate the true fitness of simulated viruses than models based on individual metrics .",
"To this end , we fit an additional model including the best metrics from the mechanistic and clade growth categories: mutational load and LBI .",
"This composite model outperformed both of its corresponding individual metric models with an average distance to the future of 7 . 24 ± 1 . 66 AAs and outperformed the naive model as often as the true fitness metric ( Figure 5 , Table 4 , Table 5 ) .",
"The coefficients for mutational load and LBI remained relatively consistent across all validation timepoints , indicating that these fitness metrics were stable approximations of the simulator’s underlying evolutionary processes .",
"This small gain supports our hypothesis that multiple complementary metrics can produce more accurate models .",
"We validated the best performing model ( true fitness ) using two metrics that are relevant for practical influenza forecasting and vaccine design efforts .",
"First , we measured the ability of the true fitness model to accurately estimate dynamics of large clades ( initial frequency >15% ) by comparing observed fold change in clade frequencies , log10x ( t+Δt ) x",
"( t ) and estimated fold change , log10x^ ( t+Δt ) x",
"( t ) .",
"The model’s estimated fold changes correlated well with observed fold changes ( Pearson’s R2=0 . 52 , Figure 6—figure supplement 1A ) .",
"The model also accurately predicted the growth of 87% of growing clades and the decline of 58% of declining clades .",
"Model forecasts were increasingly more accurate with increasing initial clade frequencies ( Figure 6—figure supplement 1C ) .",
"Next , we counted how often the estimated closest strain to the future population at any given timepoint ranked among the observed top closest strains to the future .",
"We calculated the distance of each present strain to the future as the Hamming distance between the given strain’s amino acid sequence and each future strain weighted by the future strain’s observed or estimated frequency ( Equations 3 and 4 ) .",
"The estimated closest strain was in the top first percentile of observed closest strains for half of the validation timepoints and in the top 20th percentile for 100% of timepoints ( Figure 6—figure supplement 1B ) .",
"Percentile ranks per strain based on their observed and estimated distances to the future correlated strongly across all strains and timepoints ( Spearman’s ρ2=0 . 87 , Figure 6—figure supplement 1D ) .",
"In contrast , the naive model’s forecasts of clade frequencies were considerably less accurate ( Figure 6—figure supplement 2C ) .",
"However , the naive model’s estimated closest strains to the future were consistently in the top fifth percentile of observed distances to the future and the correlation of its estimated percentile ranks and the observed ranks was strong ( Spearman’s ρ2=0 . 78 , Figure 6—figure supplement 2B D ) .",
"These results suggested that estimating a single closest strain to the future is a more tractable problem than estimating the future frequencies of clades .",
"Finally , we tested all of our models on out-of-sample data .",
"Specifically , we fixed the coefficients of each model to the average values across the validation period and applied the resulting models to the next 9 years of previously unobserved simulated data .",
"A standard expectation from machine learning is that models will perform worse on test data due to overfitting to training data .",
"Despite this expectation , we found that all models except for the individual epitope mutation models consistently outperformed the naive model across the out-of-sample data ( Figure 4 , Figure 5 , Figure 5—figure supplement 1 , Table 4 ) .",
"The composite model of mutational load and LBI appeared to outperform the true fitness metric with average distance to the future of 7 . 10 ± 1 . 19 compared to 7 . 38 ± 1 . 89 , respectively .",
"However , we did not find a significant difference between these models by bootstrap testing ( Table 5 ) and could not rule out fluctuations in model performance across a relatively small number of data points .",
"As with our validation dataset , we tested the true fitness model’s ability to recapitulate clade dynamics and select optimal individual strains from the test data .",
"While observed and estimated clade frequency fold changes correlated more weakly for test data ( Pearson’s R2=0 . 14 ) , the accuracies of clade growth and decline predictions remained similar at 82% and 53% , respectively ( Figure 6A ) .",
"We observed higher absolute forecast errors in the test data with higher errors for clades between 40% and 60% initial frequencies ( Figure 6C ) .",
"The estimated closest strain was higher than the top first percentile of observed closest strains for half of the test timepoints and in the top 20th percentile for 16 ( 89% ) of 18 of timepoints ( Figure 6B ) .",
"Observed and estimated strain ranks remained strongly correlated across all strains and timepoints ( Spearman’s ρ2=0 . 80 , Figure 6D ) .",
"The naive model performed comparatively well on these test data with all its estimated closest strains to the future in the top 20th percentile and a slightly higher correlation between observed and estimated percentile ranks than the true fitness model ( Spearman’s ρ2=0 . 82 , Figure 6—figure supplement 3 ) .",
"These results confirmed that our approach of minimizing the distance between yearly populations could simultaneously capture clade-level dynamics of simulated influenza populations and identify individual strains that are most representative of future populations .",
"However , they also supported the earlier finding that clade frequency forecasts may be inherently more challenging than identification of the closest strain to the future .",
"Next , we trained and validated models for individual fitness predictors using 25 years of natural H3N2 populations spanning from October 1 , 1990 to October 1 , 2015 .",
"We held out strains collected after October 1 , 2015 up through October 1 , 2019 for model testing ( Figure 7 ) .",
"In addition to the sequence-only models we tested on simulated populations , we also fit models for our new fitness metrics based on experimental phenotypes including HI antigenic novelty and DMS mutational effects .",
"We hypothesized that both HI and DMS metrics would be assigned positive coefficients , as they estimate increased antigenic drift and beneficial mutations , respectively .",
"As antigenic drift is generally considered to be the primary evolutionary pressure on natural H3N2 populations ( Smith et al . , 2004; Bedford et al . , 2014; Luksza and Lässig , 2014 ) , we expected that epitope and HI antigenic novelty would be individually more predictive than mutational load or DMS mutational effects .",
"Previous research ( Neher et al . , 2014 ) and our simulation results also led us to expect that LBI and delta frequency would outperform other individual mechanistic metrics .",
"As the earliest measurements from focus reduction assays ( FRAs ) date back to 2012 , we could not train , validate , and test FRA antigenic novelty models in parallel with the HI antigenic novelty models .",
"Biologically-informed metrics generally performed better than the naive model with the exceptions of the epitope antigenic novelty and DMS mutational effects ( Figure 8 and Table 6 ) .",
"The naive model estimated an average distance between natural H3N2 populations of 6 . 40 ± 1 . 36 AAs .",
"The lower bound for how well any model could perform , 2 . 60 ± 0 . 89 AAs , was considerably lower than the corresponding bounds for simulated populations .",
"The average improvement of the sequence-only models over the naive model was consistently lower than the same models in simulated populations .",
"This reduced performance may have been caused by both the relatively reduced diversity between years in natural populations and the fact that our simple models do not capture all drivers of evolution in natural H3N2 populations .",
"Of the two metrics for antigenic drift , HI antigenic novelty consistently outperformed epitope antigenic novelty ( Table 6 ) .",
"HI antigenic novelty estimated an average distance to the future of 6 . 01 ± 1 . 50 AAs and outperformed the naive model at 16 of 23 timepoints ( 70% ) .",
"The coefficient for HI antigenic novelty remained stable across all timepoints ( Figure 8 ) .",
"In contrast , epitope antigenic novelty estimated a distance of 7 . 13 ± 1 . 47 AAs and only outperformed the naive model at seven timepoints ( 30% ) .",
"Epitope antigenic novelty was also the only metric whose coefficient started at a positive value ( 1 . 17 ± 0 . 03 on average prior to October 2009 ) and transitioned to a negative value through the validation period ( −0 . 19 ± 0 . 34 on average for October 2009 and after ) .",
"This strong coefficient for the first half of training windows indicated that , unlike the results for simulated populations , the nonlinear antigenic novelty metric was historically an effective measure of antigenic drift .",
"The historical importance of the epitope sites used for this metric was further supported by the relative enrichment of mutations at these sites for the most successful ‘trunk’ lineages of natural populations compared to side branch lineages ( Table 3 ) .",
"These results led us to hypothesize that the contribution of these specific epitope sites to antigenic drift has weakened over time .",
"Importantly , these 49 epitope sites were originally selected by Luksza and Lässig , 2014 from a previous historical survey of sites with beneficial mutations between 1968–2005 ( Shih et al . , 2007 ) .",
"If the beneficial effects of mutations at these sites were due to historical contingency rather than a constant contribution to antigenic drift , we would expect models based on these sites to perform well until 2005 and then overfit relative to future data .",
"Indeed , the epitope antigenic novelty model outperforms the naive model for the first three validation timepoints until it has to predict to April 2006 .",
"To test this hypothesis , we identified a new set of beneficial sites across our entire validation period of October 1990 through October 2015 .",
"Inspired by the original approach of Shih et al . , 2007 , we identified 25 sites in HA1 where mutations rapidly swept through the global population , including 12 that were also present in the original set of 49 sites .",
"We fit an antigenic novelty model to these 25 sites across the complete validation period and dubbed this the ‘oracle antigenic novelty’ model , as it benefited from knowledge of the future in its forecasts .",
"The oracle model produced a consistently positive coefficient across all training windows ( 0 . 80 ± 0 . 21 ) and consistently outperformed the original epitope model with an average distance to the future of 5 . 71 ± 1 . 27 AAs ( Figure 8—figure supplement 1 ) .",
"These results support our hypothesis that the fitness benefit of mutations at the original 49 sites was due to historical contingency and that the success of previous epitope models based on these sites was partly due to ‘borrowing from the future’ .",
"We suspect that our HI antigenic novelty model benefits from its ability to constantly update its antigenic model at each timepoint with recent experimental phenotypes , while the epitope antigenic novelty metric is forced to give a constant weight to the same 49 sites throughout time .",
"Of the two metrics for functional constraint , mutational load outperformed DMS mutational effects , with an average distance to the future of 6 . 14 ± 1 . 37 AAs compared to 6 . 75 ± 1 . 95 AAs , respectively .",
"In contrast to the original Luksza and Lässig , 2014 model , where the coefficient of the mutational load metric was fixed at −0 . 5 , our model learned a consistently stronger coefficient of −0 . 99 ± 0 . 30 .",
"Notably , the best performance of the DMS mutational effects model was forecasting from April 2007 to April 2008 when the major clade containing A/Perth/16/2009 was first emerging .",
"This result is consistent with the DMS model overfitting to the evolutionary history of the background strain used to perform the DMS experiments .",
"Alternate implementations of less background-dependent DMS metrics never performed better than the mutational load metric ( Table 6 , Materials and methods ) .",
"Thus , we find that a simple model where any mutation at non-epitope sites is deleterious is more predictive of global viral success than a more comprehensive biophysical model based on measured mutational effects of a single strain .",
"LBI was the best individual metric by average distance to the future ( Figure 8 ) and tied mutational load by outperforming the naive model at 17 ( 74% ) timepoints ( Table 6 ) .",
"Delta frequency performed worse than LBI and HI antigenic novelty and was comparable to mutational load .",
"While delta frequency should , in principle , measure the same aspect of viral fitness as LBI , these results show that the current implementations of these metrics represent qualitatively different fitness components .",
"The LBI and mutational load might also be predictive for reasons other than correlation with fitness , see Discussion .",
"To test whether composite models could outperform individual fitness metrics for natural populations , we fit models based on combinations of best individual metrics representing antigenic drift , functional constraint , and clade growth .",
"Specifically , we fit models based on HI antigenic novelty and mutational load , mutational load and LBI , and all three of these metrics together .",
"We anticipated that if these metrics all represented distinct , mutually beneficial components of viral fitness , these composite models should perform better than individual models with consistent coefficients for each metric .",
"Both two-metric composite models modestly outperformed their corresponding individual models ( Table 6 , Figure 9 , and Table 5 ) .",
"The composite of mutational load and LBI performed the best overall with an average distance to the future of 5 . 44 ± 1 . 80 AAs .",
"The relative stability of the coefficients for the metrics in the two-metric models suggested that these metrics represented complementary components of viral fitness .",
"In contrast , the three-metric model strongly preferred the HI antigenic novelty and mutational load metrics over LBI for the entire validation period , producing an average LBI coefficient of −0 . 04 ± 0 . 09 .",
"Overall , the gain by combining multiple predictors was limited and the sensitivity of coefficients to the set of metrics included in the model suggests that there is substantial overlap in predictive value of different metrics .",
"As with the simulated populations , we validated the performance of the best model for natural populations using estimated and observed clade frequency fold changes and the ranking of estimated closest strains compared to the observed closest strains to future populations .",
"The composite model of mutational load and LBI effectively captured clade dynamics with a fold change correlation of R2=0 . 35 and growth and decline accuracies of 87% and 89% , respectively ( Figure 10—figure supplement 1A ) .",
"Absolute forecasting error declined noticeably for clades with initial frequencies above 60% , but generally this error remained below 20% on average ( Figure 10—figure supplement 1C ) .",
"The estimated closest strain from this model was in the top first percentile of observed closest strains for half of the validation timepoints and in the top 20th percentile for 20 ( 87% ) of 23 timepoints ( Figure 10—figure supplement 1B ) .",
"This pattern held across all strains and timepoints with a strong correlation between observed and estimated strain ranks ( Spearman’s ρ2=0 . 66 , Figure 10—figure supplement 1D ) .",
"The naive model’s performance repeated the pattern we observed with simulated populations: it made poor forecasts of absolute clade frequencies , but its estimated closest strains to the future were consistently highly ranked among the observed closest strains ( Figure 10—figure supplement 2B C ) .",
"Finally , we tested the performance of all models on out-of-sample data collected from October 1 , 2015 through October 1 , 2019 .",
"We anticipated that most models would perform worse on truly out-of-sample data than on validation data .",
"Correspondingly , only the three models with the HI antigenic novelty metric significantly outperformed the naive model on the test data ( Table 6 ) .",
"The composite of HI antigenic novelty and mutational load performed modestly , although not significantly , better than the individual HI antigenic novelty model ( Table 5 ) .",
"Surprisingly , the best model for the validation data – mutational load and LBI – was one of the worst models for the test data with an average distance to the future of 7 . 70 ± 3 . 53 AAs .",
"The individual LBI model was the worst model , while mutational load continued to perform well with test data .",
"LBI performed especially poorly in the last two test timepoints of April and October 2018 ( Figure 8 ) .",
"These timepoints correspond to the dominance and sudden decline of a reassortant clade named A2/re ( Potter et al . , 2019 ) .",
"By April 2018 , the A2/re clade had risen to a global frequency over 50% from less than 15% the previous year , despite an absence of antigenic drift .",
"By October 2018 , this clade had declined in frequency to approximately 30% and , by October 2019 , it had gone extinct .",
"That LBI incorrectly predicted the success of this reassortant clade highlights a major limitation of growth-based fitness metrics and a corresponding benefit of more mechanistic metrics that explicitly measure antigenic drift and functional constraint .",
"However , we cannot rule out the alternate possibility that the LBI model was overfit to the training data .",
"After identifying the composite HI antigenic novelty and mutational load model as the best model on out-of-sample data , we tested this model’s ability to detect clade dynamics and select individual closest strains to the future for vaccine composition .",
"The composite model partially captured clade dynamics with a Pearson’s correlation of R2=0 . 46 between observed and estimated growth ratios and growth and decline accuracies of 52% and 58% , respectively ( Figure 10A ) .",
"The mean absolute forecasting error with this model was consistently less than 20% , regardless of the initial clade frequency ( Figure 10C ) .",
"The estimated closest strain from this model was in the top first percentile of observed closest strains for half of the validation timepoints and in the top 20th percentile for 100% of timepoints ( Figure 10B ) .",
"Similarly , the observed and estimated strain ranks strongly correlated ( Spearman’s ρ2=0 . 72 ) across all strains and test timepoints ( Figure 10D ) .",
"The estimated strain ranks of the naive model were not as well correlated ( Spearman’s ρ2=0 . 56 ) , but seven of its eight estimates for the closest strain to the future ( 88% ) were in the top fifth percentile of observed closest strains ( Figure 10—figure supplement 3B D ) .",
"We further evaluated our models’ ability to estimate the closest strain to the next season’s H3N2 population by comparing our best models’ selections to the WHO’s vaccine strain selection .",
"For each season when the WHO selected a new vaccine strain and one year of future data existed in our validation or test periods , we measured the observed distance of that strain’s sequence to the future and the corresponding distances to the future for the observed closest strains ( Equation 3 ) .",
"We compared these distances to those of the closest strains to the future as estimated by our best models for the validation period ( mutational load and LBI ) and the test period ( HI antigenic novelty and mutational load ) using Equation 4 .",
"The observed closest strain to the future represents the centroid of the observed future population , while the estimated closest strains are the models’ predictions of that future population’s centroid .",
"The mutational load and LBI model selected strains that were as close or closer to the future than the corresponding vaccine strain for 10 ( 83% ) of the 12 seasons with vaccine updates ( Figure 11 ) .",
"On average , the strains selected by this model were closer to future than the vaccine strain by 1 . 93 AAs ( Figure 11—figure supplement 1 ) .",
"For the two seasons that the model selected more distant strains than the vaccine strain , the mean distance relative to the vaccine strain was 1 . 58 AAs .",
"The HI antigenic novelty and mutational load model performed similarly by identifying strains as close or closer to the future for 11 ( 92% ) seasons with an average improvement over the vaccine strains of 2 . 33 AAs .",
"For the one season that the model selected a more distant strain , that selected strain was 0 . 75 AAs farther from the future than the vaccine strain .",
"Interestingly , the strains selected by the naive model were always better than the selected vaccine strain .",
"Since the naive model predicts that the future will be identical to the present , these strains represent the centroid of each current population .",
"With an average improvement over the vaccine strains of 2 . 19 AAs , the naive model performed consistently better than the LBI-based model and nearly as well as the HI-based model .",
"These results were consistent with our earlier observations that the naive model often performs as well as biologically-informed models when estimating a single closest strain to the future .",
"To enable real-time forecasts , we integrated our forecasting framework into our existing open source pathogen surveillance application , Nextstrain ( Hadfield et al . , 2018 ) .",
"Prior to finalizing our model coefficients for use in Nextstrain , we tested whether our three best composite models could be improved by learning new coefficients per timepoint from the test data .",
"Additionally , we evaluated a composite of FRA antigenic novelty and mutational load .",
"Since the earliest FRA data were from 2012 , we anticipated that there were enough measurements to fit a model across the test data time interval .",
"If modern H3N2 strains continue to perform poorly in HI assays , the FRA-based assay will be critical for future forecasting efforts .",
"Two of three models performed worse after refitting coefficients to the test data than their original fixed coefficient implementations ( Figure 9—figure supplement 1 ) .",
"While , the mutational load and LBI model improved considerably over its original performance , it still performed worse than the naive model on average .",
"These results confirmed that the coefficients for our selected best model would be most accurate for live forecasts .",
"Interestingly , the FRA antigenic novelty metric received a consistently positive coefficient of 1 . 40 ± 0 . 24 in its composite with mutational load .",
"Unfortunately , this model performed considerably worse than the corresponding HI-based model .",
"These results suggest that we may need more FRA data across a longer historical timespan to train a model that could replace the HI-based model .",
"After confirming the coefficients for our best model of HI antigenic novelty and mutational load , we inspected forecasts of H3N2 clades using all data available up through June 6 , 2020 .",
"Consistent with an average two-month lag between data collection and submission , the most recent data were collected up to April 1 , 2020 and made our forecasts from this timepoint to April 1 , 2021 .",
"Of the five major currently circulating clades , our model predicted growth of the clades 3c3 . A and A1b/94N and decline of clades A1b/135K , A1b/137F , and A1b/197R ( Figure 12 ) .",
"To aid with identification of potential vaccine candidates for the next season , we annotated strains in the phylogeny by their estimated distance to the future based on our best model ( Figure 13 ) ."
],
[
"Our evaluation of models by time-series cross-validation and true out-of-sample forecasts revealed substantial potential for model overfitting .",
"We observed overfitting to both specific genetic backgrounds and general historical contexts .",
"A clear example of the former was the poor performance of our DMS-based fitness metric compared to a simpler mutational load metric .",
"Although the DMS experiments provided detailed estimates of which amino acids were preferred at which positions in HA , these measurements were specific to a single strain , A/Perth/16/2009 ( Lee et al . , 2018 ) .",
"When we applied these measurements to predict the success of global populations , they were less informative on average than the naive model .",
"To benefit from the more comprehensive fitness costs measured by DMS data , future models will need to synthesize DMS measurements across multiple H3N2 strains from distinct genetic contexts .",
"We anticipate that these measurements could be used to define and continually update a modern set of sites contributing to mutational load in natural populations .",
"This set of sites could replace the statically defined set of ‘non-epitope’ sites we use to estimate mutational load here .",
"We observed overfitting to historical context in sequence-based models of antigenic drift .",
"The fitness benefit of mutations that led to antigenic drift in H3N2 in the past is well-documented ( Wiley et al . , 1981; Smith et al . , 2004; Wolf et al . , 2006; Koel et al . , 2013 ) .",
"Although the antigenic importance of seven specific sites in HA were experimentally validated by Koel et al . , 2013 , these sites do not explain all antigenic drift observed in natural populations ( Neher et al . , 2016 ) .",
"Other attempts to define these so-called ‘epitope sites’ have relied on either aggregation of results from antigenic escape assays ( Wolf et al . , 2006 ) or retrospective computational analyses of sites with beneficial mutations ( Shih et al . , 2007; Luksza and Lässig , 2014 ) .",
"We found that models based on all of these definitions except for the seven Koel epitope sites overfit to the historical context from which they were identified ( Table 6 ) .",
"These results suggest that the set of sites that contribute to antigenic drift at any given time may depend on both the fitness landscape of currently circulating strains and the immune landscape of the hosts these strains need to infect .",
"Recent experimental mapping of antigenic escape mutations in H3N2 HA with human sera show that the specific sites that confer antigenic escape can vary dramatically between individuals based on their exposure history ( Lee et al . , 2019 ) .",
"In contrast to models based on predefined ‘epitope sites’ , our model based on experimental measurements of antigenic drift did not suffer from overfitting in the validation or test periods .",
"We suspect that this model was able to minimize overfitting by continuously updating its antigenic model with recent experimental data and assigning antigenic weight to branches of a phylogeny rather than specific positions in HA .",
"Even the most accurate models with few parameters will sometimes fail due to the probabilistic nature of evolution .",
"For example , the model with the best performance across our validation data – mutational load and LBI – was also one of the worst models across our test data .",
"Although we cannot rule out the role of overfitting , this model’s poor performance coincided with unusual evolutionary circumstances .",
"The diversity of H3N2 lineages during our test period was higher than the historical average ( Koelle et al . , 2006 ) , with the most recent common ancestor of all circulating strains dating eight years back .",
"This persistence of diversity may have reduced the effectiveness of the LBI metric that assumes relatively rapid population turnover .",
"Additionally , this model’s poorest performance occurred in 2019 when it failed to predict the sudden decline of a dominant reassortant clade , A2/re .",
"Only our models based on HI antigenic novelty and mutational load continued to perform as well or better than the naive model during the same time period .",
"These results highlight the challenge of identifying models that remain robust to stochastic evolutionary events by avoiding overfitting to the past .",
"Correspondingly , we observed that composite models of multiple orthogonal fitness metrics often outperformed models based on their individual components .",
"These results are consistent with previous work that found improved performance by integrating components of antigenic drift , functional constraint , and clade growth ( Luksza and Lässig , 2014 ) .",
"However , the effective elimination of LBI from our three-metric model during the validation period ( Figure 9 ) reveals the limitations of our current additive approach to composite models .",
"The recent success of weighted ensembles for short-term influenza forecasting through the CDC’s FluSight network ( Reich et al . , 2019 ) suggests that long-term forecasting may benefit from a similar approach .",
"By forecasting the composition of future H3N2 populations with biologically-informed fitness metrics , our best models consistently outperformed a naive model ( Table 6 ) .",
"While this performance confirms previously demonstrated potential for long-term influenza forecasting ( Luksza and Lässig , 2014 ) , the average gain from these models over the naive model appears low at 0 . 96 AAs per year for validation data and 0 . 85 AAs per year for test data .",
"However , these results are consistent with the observed dynamics of H3N2 .",
"First , the one-year forecast horizon is a fraction of the average coalescence time for H3N2 populations of about 3–8 years ( Rambaut et al . , 2008 ) .",
"Hence , we expect the diversity of circulating strains to persist between seasons .",
"Second , H3N2 hemagglutinin accumulates 3 . 6 amino acid changes per year ( Smith et al . , 2004 ) .",
"This accumulation of amino acid substitutions contributes to the distance between annual populations observed by the naive model .",
"In this context , our model gains of 0 . 96 and 0 . 85 AAs per year correspond to an explanation of 27% and 24% of the expected additional distance between annual populations , respectively .",
"Several clear opportunities to improve forecasts still remain .",
"Integration of more recent experimental data may improve estimates of antigenic drift .",
"Despite the weak performance of our FRA antigenic novelty model on recent data , continued accumulation of FRA measurements over time should eventually enable models as accurate as the current HI-based models .",
"In addition to these FRA data based on ferret antisera , recent high-throughput antigenic escape assays with human sera promise to improve existing definitions of epitope sites ( Lee et al . , 2019 ) .",
"These assays reveal the specific sites and residues that confer antigenic escape from polyclonal sera obtained from individual humans .",
"A sufficiently broad geographic and temporal sample of human sera with these assays could reveal consistent patterns of the immune landscape H3N2 strains must navigate to be globally successful .",
"Models should also integrate information from multiple segments of the influenza genome and will need to balance the fitness benefits of evolution in genes such as neuraminidase ( Chen et al . , 2018 ) with the costs of reassortment ( Villa and Lässig , 2017 ) .",
"Our forecasting framework makes the inclusion of fitness metrics based on additional gene segments technically straightforward .",
"However , the definition of appropriate fitness metrics for neuraminidase and other genes remains an important scientific challenge .",
"An additional challenge to model training is a relative lack of historical strains for which all genes have been sequenced .",
"Of the 34 , 312 H3N2 strains in GISAID with all eight primary gene segments and collection dates between October 1 , 1990 and 2019 , the majority ( 24 , 466 or 71% ) were collected after October 1 , 2015 .",
"Data availability will therefore inform which gene segments are prioritized for inclusion in future models .",
"Finally , forecasting models need to account for the geographic distribution of viruses and the vastly different sampling intensities across the globe .",
"Most influenza sequence data come from highly developed countries that account for a small fraction of the global population , while globally successful clades of influenza H3N2 often emerge in less well-sampled regions ( Russell et al . , 2008; Rambaut et al . , 2008; Bedford et al . , 2015 ) .",
"Explicitly accounting for these sampling biases and the associated migration dynamics would allow models to weight forecasts based on both viral fitness and transmission .",
"Prediction of future influenza virus populations is intrinsically limited by the small number of data points available to train and test models .",
"Increasingly more complex models are therefore prone to overfitting .",
"Across the validation and test periods , we found that antigenic drift and mutational load were the most robust predictors of future success for seasonal influenza H3N2 populations .",
"Several metrics like the rate of frequency change or epitope mutations are naively expected to have predictive power but do not .",
"Others metrics like the mutational load are not expected to measure adaptation but are predictive .",
"These results point to one aspect that often overlooked when comparing the genetic make-up of an asexual population at two time points: the future population is unlikely to descend from any of the sampled tips but ancestral lineages of the future population merge with those of the present population in the past .",
"Optimal representatives of the future therefore tend to be tips in the present that tend to be basal and less evolved .",
"The LBI and the mutational load metric have the tendency to assign low fitness to evolved tips .",
"The LBI in particular assigns high fitness to the base of large clades .",
"Much of the predictive power , in the sense of a reduced distance between the predicted and observed populations , might be due to putting more weight on less evolved strains rather than bona fide prediction of fitness .",
"In a companion manuscript , Barrat-Charlaix et al . , 2020 show that LBI has little predictive power for fixation probabilities of mutations in H3N2 .",
"Our framework enables real-time practical forecasts of these populations by leveraging historical and modern experimental assays and gene sequences .",
"By releasing our framework as an open source tool based on modern data science standards like tidy data frames , we hope to encourage continued development of this tool by the influenza research community .",
"We additionally anticipate that the ability to forecast the sequence composition of populations with earth mover’s distance will enable future forecasting research with pathogens whose genomes cannot be analyzed by traditional phylogenetic methods including recombinant viruses , bacteria , and fungi .",
"The entire workflow for our analyses was implemented with Snakemake ( Köster and Rahmann , 2012 ) .",
"We have provided all source code , configuration files , and datasets at https://github . com/blab/flu-forecasting ( Huddleston , 2020; copy archived at https://github . com/elifesciences-publications/flu-forecasting ) ."
],
[
"We simulated the long-term evolution of H3N2-like viruses with SANTA-SIM ( Jariani et al . , 2019 ) for 10 , 000 generations or 50 years where 200 generations was equivalent to 1 year .",
"We discarded the first 10 years as a burn-in period , selected the next 30 years for model fitting and validation , and held out the last 9 years as out-of-sample data for model testing ( Figure 2 ) .",
"Each simulated population was seeded with the full length HA from A/Beijing/32/1992 ( NCBI accession: U26830 . 1 ) such that all simulated sequences contained signal peptide , HA1 , and HA2 domains .",
"We defined purifying selection across all three domains , allowing the preferred amino acid at each site to change at a fixed rate over time .",
"We additionally defined exposure-dependent selection for 49 putative epitope sites in HA1 ( Luksza and Lässig , 2014 ) to impose an effect of antigenic novelty that would allow mutations at those sites to increase viral fitness despite underlying purifying selection .",
"We modified the SANTA-SIM source code to enable the inclusion of true fitness values for each strain in the FASTA header of the sampled sequences from each generation .",
"This modified implementation has been integrated into the official SANTA-SIM code repository at https://github . com/santa-dev/santa-sim as of commit e2b3ea3 .",
"For our full analysis of model performance , we sampled 90 viruses per month to match the sampling density of natural populations .",
"For tuning of hyperparameters , we sampled 10 viruses per month to enable rapid exploration of hyperparameter space .",
"To avoid overfitting our models to the relatively limited data from natural populations , we used simulated H3N2-like populations to tune hyperparameters including the KDE bandwidth for frequency estimates and the L1 penalty for model coefficients .",
"We simulated populations , as described above , and fit models for each parameter value using the true fitness of strains from the simulator .",
"We identified the optimal KDE bandwidth for frequencies as the value that minimized the difference between the mean distances to the future from the true fitness model and the naive model .",
"We set the L1 lambda penalty to zero , to reduce variables in the analysis and avoid interactions between the coefficients and the KDE bandwidths .",
"Higher bandwidths completely wash out dynamics of populations by making all strains appear to exist for long time periods .",
"This flattening of frequency trajectories means that as bandwidths increase , the naive model gets more accurate and less informative .",
"Given this behavior , we found the bandwidth that produced the minimum difference between distances to the future for the true fitness and naive models instead of the bandwidth that produced the minimum mean model distance .",
"Based on this analysis , we identified an optimal bandwidth of 212 or the equivalent of 2 months for floating point dates .",
"Next , we identified an L1 penalty of 0 . 1 for model coefficients that minimized the mean distance to the future for the true fitness model .",
"Hemagglutination inhibition ( HI ) and focus reduction assay ( FRA ) measurements were provided by WHO Global Influenza Surveillance and Response System ( GISRS ) Collaborating Centers in London , Melbourne , Atlanta and Tokyo .",
"We converted these raw two-fold dilution measurements to log2 titer drops normalized by the corresponding log2 autologous measurements as previously described ( Neher et al . , 2016 ) .",
"Prior to our analyses , we downloaded all HA sequences and metadata from GISAID ( Shu and McCauley , 2017 ) .",
"For model training and validation , we selected 15 , 583 HA sequences ≥ 900 nucleotides that were sampled between October 1 , 1990 and October 1 , 2015 .",
"To account for known variation in sequence availability by region , we subsampled the selected sequences to a representative set of 90 viruses per month with even sampling across 10 global regions including Africa , Europe , North America , China , South Asia , Japan and Korea , Oceania , South America , Southeast Asia , and West Asia .",
"We excluded all egg-passaged strains and all strains with ambiguous year , month , and day annotations .",
"We prioritized strains with more available HI titer measurements provided by the WHO GISRS Collaborating Centers .",
"For model testing , we selected an additional 7 , 171 HA sequences corresponding to 90 viruses per month sampled between October 1 , 2015 and October 1 , 2019 .",
"We used these test sequences to evaluate the out-of-sample error of fixed model parameters learned during training and validation .",
"Supplementary file 1 describes contributing laboratories for all 22 , 754 validation and test strains .",
"For each timepoint in model training , validation , and testing , we selected the subsampled HA sequences with collection dates up to that timepoint .",
"We aligned sequences with the augur align command ( Hadfield et al . , 2018 ) and MAFFT v7 . 407 ( Katoh et al . , 2002 ) .",
"We inferred initial phylogenies for HA sequences at each timepoint with IQ-TREE v1 . 6 . 10 ( Nguyen et al . , 2015 ) .",
"To reconstruct time-resolved phylogenies , we applied TreeTime v0 . 5 . 6 ( Sagulenko et al . , 2018 ) with the augur refine command .",
"To account for uncertainty in collection date and sampling error , we applied a kernel density estimation ( KDE ) approach to calculate global strain frequencies .",
"Specifically , we constructed a Gaussian kernel for each strain with the mean at the reported collection date and a variance ( or KDE bandwidth ) of two months .",
"The bandwidth was identified by cross-validation , as described above .",
"This bandwidth also roughly corresponds to the median lag time between strain collection and submission to the GISAID database .",
"We estimated the frequency of each strain at each timepoint by calculating the probability density function of each KDE at that timepoint and normalizing the resulting values to sum to one .",
"We implemented this frequency estimation logic in the augur frequencies command .",
"We defined the following fitness metrics per strain and timepoint .",
"Sequence data are available from GISAID using accession ids provided in Supplementary file 1 .",
"Source code , derived data from serological measurements , fitness metric annotations , and resulting fitness model performance data are available in the project’s GitHub repository ( Huddleston , 2020; copy archived at https://github . com/elifesciences-publications/flu-forecasting ) .",
"Raw serological measurements are restricted from public distribution by previous data sharing agreements ."
]
] | [
"Seasonal influenza virus A/H3N2 is a major cause of death globally .",
"Vaccination remains the most effective preventative .",
"Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity .",
"This antigenic drift necessitates regular vaccine updates .",
"Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection .",
"Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences .",
"We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates .",
"Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence-only estimates , while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates .",
"Importantly , the best models integrated estimates of both functional constraint and either antigenic drift phenotypes or recent population growth ."
] | [
"Vaccination is the best protection against seasonal flu .",
"It teaches the immune system what the flu virus looks like , preparing it to fight off an infection .",
"But the flu virus changes its molecular appearance every year , escaping the immune defences learnt the year before .",
"So , every year , the vaccine needs updating .",
"Since it takes almost a year to design and make a new flu vaccine , researchers need to be able to predict what flu viruses will look like in the future .",
"Currently , this prediction relies on experiments that assess the molecular appearance of flu viruses , a complex and slow approach .",
"One alternative is to examine the virus's genetic code .",
"Mathematical models try to predict which genetic changes might alter the appearance of a flu virus , saving the cost of performing specialised experiments .",
"Recent research has shown that these models can make good predictions , but including experimental measures of the virus’ appearance could improve them even further .",
"This could help the model to work out which genetic changes are likely to be beneficial to the virus , and which are not .",
"To find out whether experimental data improves model predictions , Huddleston et al . designed a new forecasting tool which used 25 years of historical data from past flu seasons .",
"Each forecast predicted what the virus population might look like the next year using the previous year's genetic code , experimental data , or both .",
"Huddleston et al . then compared the predictions with the historical data to find the most useful data types .",
"This showed that the best predictions combined changes from the virus's genetic code with experimental measures of its appearance .",
"This new forecasting tool is open source , allowing teams across the world to start using it to improve their predictions straight away .",
"Seasonal flu infects between 5 and 15% of the world's population every year , causing between quarter of a million and half a million deaths .",
"Better predictions could lead to better flu vaccines and fewer illnesses and deaths ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"computational and systems biology"
] | Shadow enhancers can suppress input transcription factor noise through distinct regulatory logic | elife-59351-v2 | [
[
"The first evidence that transcription occurred in bursts , as opposed to as a smooth , continuous process , was observed in Drosophila embryos .",
"Electron micrographs showed that even highly transcribed genes had regions of chromatin lacking associated transcripts in between regions densely associated with nascent transcripts ( McKnight and Miller , 1979 ) .",
"As visualization techniques have improved , it is increasingly clear that transcriptional bursting is the predominant mode of expression across organisms from bacteria to mammals ( Chubb et al . , 2006; Dar et al . , 2012; Sanchez and Golding , 2013; Zenklusen et al . , 2008; Bothma et al . , 2014 ) .",
"These bursts of transcriptional activity , separated by periods of relative silence , have important implications for cellular function , as mRNA numbers and fluctuations largely dictate these quantities at the protein level ( Csárdi et al . , 2015; Hansen et al . , 2018 ) .",
"Such fluctuations in regulatory proteins , like TFs and signaling molecules , can propagate down a gene regulatory network , significantly altering the expression levels or noise of downstream target genes ( Blake et al . , 2003 ) .",
"Given the inevitable fluctuations in regulatory proteins like TFs , it is unclear how organisms establish and maintain the precise levels of gene expression seen during development , where expression patterns can be reproducible down to half-nuclear distances in Drosophila embryos ( Dubuis et al . , 2013; Gregor et al . , 2007 ) .",
"Many mechanisms that buffer against expression noise , either inherent or stemming from genetic or environmental variation , have been observed ( Lagha et al . , 2012; Stapel et al . , 2017; Raj et al . , 2010 ) .",
"For example , organisms use temporal and spatial averaging mechanisms and redundancy in genetic circuits to achieve the precision required for proper development ( Stapel et al . , 2017; Raj et al . , 2010; Erdmann et al . , 2009; Lagha et al . , 2012 ) .",
"Here , we propose that shadow enhancers may be another mechanism by which developmental systems manage noise ( Barolo , 2012 ) .",
"Shadow enhancers are groups of two or more enhancers that control the same target gene and drive overlapping spatiotemporal expression patterns ( Hong et al . , 2008; Barolo , 2012 ) .",
"Shadow enhancers are found in a wide range of organisms , from insects to plants to mammals , particularly in association with developmental genes ( Cannavò et al . , 2016; Osterwalder et al . , 2018; Garnett et al . , 2012; Bomblies et al . , 1999 ) .",
"While seemingly redundant , the individual enhancers of a shadow enhancer group have been shown to be critical for proper gene expression in the face of both environmental and genetic perturbations ( Frankel et al . , 2010; Osterwalder et al . , 2018; Perry et al . , 2010 ) .",
"Such perturbations may exacerbate fluctuations in upstream regulators ( Cheung and Ma , 2015; Chen et al . , 2015 ) .",
"Although shadow enhancers are shown to be pervasive in developmental systems and necessary for robust gene expression , their precise mechanism of action is still unknown .",
"One proposed mechanism is that having multiple enhancers controlling the same promoter reduces the effective ‘failure rate’ of the promoter and ensures a critical threshold of gene expression is reached ( Lam et al . , 2015; Perry et al . , 2011 ) .",
"An alternative , but not mutually exclusive , possibility is that shadow enhancers ensure robust expression by buffering noise in upstream regulators .",
"Several studies suggest that individual enhancers of a shadow enhancer group tend to be controlled by different sets of TFs , which we call a ‘separation of inputs’ ( Wunderlich et al . , 2015; Cannavò et al . , 2016; Ghiasvand et al . , 2011 ) .",
"We hypothesize that this separation of inputs allows shadow enhancers to buffer against fluctuations in upstream TF levels to drive more consistent expression levels .",
"The Drosophila gap gene Kruppel ( Kr ) provides a useful system in which to probe the mechanisms of action of shadow enhancers .",
"During early embryogenesis , Kr expression is critical for thorax formation , and like the other gap genes in the Drosophila embryo , has quite low noise ( Preiss et al . , 1985; Dubuis et al . , 2013 ) .",
"During this time , Kr is controlled by the activity of two enhancers , proximal and distal ( Perry et al . , 2011 ) , that drive overlapping expression in the center of the embryo ( Figure 1—figure supplement 1 ) .",
"We call the two individual enhancers together the shadow enhancer pair .",
"Previous experiments have shown that each enhancer is activated by different TFs ( Figure 1A; Wunderlich et al . , 2015 ) .",
"Here , we focus on differences in activation , as the key repressors of Kr , Knirps and Giant , are likely to regulate both enhancers .",
"By measuring live mRNA dynamics , we can use the Kr system in Drosophila embryos to assess whether and how shadow enhancers act to buffer noise and to identify the sources of noise in the developing embryo .",
"To test our hypothesis , we measured live mRNA dynamics driven by either single Kr enhancer , duplicated enhancers , or the shadow enhancer pair and compared the dynamics and noise associated with each .",
"We showed that the individual Kr enhancers can act largely independently in the same nucleus , while identical enhancers display correlated activity .",
"We constructed a simple mathematical model to describe this system and found that TF fluctuations are necessary to reproduce the correlated activity of identical enhancers in the same nucleus .",
"The shadow enhancer pair drives lower noise than either duplicated enhancer , and using the model , we found that this is a natural consequence of the separation of TF inputs .",
"Additional experiments , including simultaneous measurements of TF levels and expression and a decomposition of noise sources , further demonstrate that the shadow enhancer pair is less sensitive to fluctuations in TF levels than is a single enhancer .",
"Additionally , the shadow enhancer pair is uniquely able to maintain low levels of expression noise across a wide range of temperatures .",
"We suggest that this noise suppression ability is one of the key features that explains the prevalence of shadow enhancers in developmental systems ."
],
[
"To test our hypothesis that the separation of inputs between Kruppel’s ( Kr ) shadow enhancers provides them with noise-buffering capabilities , we needed to first test the ability of each enhancer to act independently .",
"In previous work , we found that the individual enhancers in the shadow enhancer pair are controlled by different activating TFs ( Wunderlich et al . , 2015 ) .",
"These experiments established that the enhancers responded differently to perturbations in key TFs , indicating that each enhancer uses a distinct regulatory logic .",
"The proximal enhancer is activated by Hunchback ( Hb ) and Stat92E , and the distal enhancer is activated by Bicoid ( Bcd ) and Zelda ( Zld ) ( Figure 1A ) .",
"Given this separation of inputs , the shadow enhancer pair could provide a form of noise buffering if variability in gene expression is driven primarily by fluctuations in upstream factors .",
"Conversely , variability in upstream regulators may be low enough in the developing embryo that these fluctuations are not the primary driver of downstream expression noise .",
"If this were the case , the separation of inputs is unlikely to be a key requirement of shadow enhancer function .",
"To investigate these possibilities , we measured and compared the correlation of allele activity in homozygous or heterozygous embryos that carry two reporter genes .",
"Proximal homozygotes contained the proximal enhancer driving a reporter , inserted in the same location on both homologous chromosomes , and distal homozygotes similarly had the distal enhancer driving reporter expression on both homologous chromosomes ( Figure 1B ) .",
"We also made heterozygous embryos , called shadow heterozygotes , which had one proximal and one distal reporter , again in the same location on both homologous chromosomes .",
"To measure live mRNA dynamics and correlations in allele activity , we used the MS2-MCP reporter system ( Figure 1C , D ) .",
"This system allows the visualization of mRNAs that contain the MS2 RNA sequence , which is bound by an MCP-GFP fusion protein ( Bertrand et al . , 1998 ) .",
"In the developing embryo , only the site of nascent transcription is visible , as single transcripts are too dim , allowing us to measure the rate of transcription ( Garcia et al . , 2013; Lucas et al . , 2013 ) .",
"In blastoderm-stage embryos with two MS2 reporter genes , we can observe two distinct foci of fluorescence corresponding to the two alleles ( Figure 1D; Videos 1 , 2 , 3 , 4 , 5 , 6 ) , in line with previous results that suggest there are low levels of transvection at this stage ( Lim et al . , 2018; Fukaya and Levine , 2017 ) .",
"To confirm our ability to distinguish the two alleles , we imaged transcription in embryos hemizygous for our reporter constructs , which only show one spot of fluorescence per nucleus .",
"Our counts of active transcription sites in homozygous embryos correspond well to the expected value calculated from hemizygous embryos ( Figure 1—figure supplement 1 ) .",
"Therefore , we are able to measure the correlation of allele activity , although we cannot identify which spot corresponds to which reporter .",
"We predicted that if variability in gene expression is driven by fluctuations in input TFs , we would observe lower correlations of allele activity in shadow heterozygotes than in either the proximal or distal homozygotes .",
"However , if global factors affecting both enhancers dominate , there would be no difference in allele activity correlations .",
"During the ~1 hr of nuclear cycle 14 ( nc14 ) we found that allele activity is more than twice as correlated in both proximal and distal homozygotes than in shadow heterozygote embryos at 47–57% egg length , which encompasses the central region of Kr expression during this time period ( Figure 1 ) .",
"The difference in our ability to measure allele correlation in the more anterior and posterior regions of the embryo stems from the slightly different expression patterns driven by the proximal and distal enhancers ( Figure 1—figure supplements 1 , 2 ) .",
"The lower allele correlation in shadow heterozygote embryos indicates not only that the individual member enhancers of the shadow enhancer pair can act largely independently in the same nucleus , but that differential TF inputs are likely the primary determinants of transcriptional bursts in this system .",
"Notably , heterozygotes still show marginal allele correlation , indicating that some correlation is induced by either shared input TFs or factors that affect transcription globally .",
"The independence of individual Kr enhancers allows for the possibility that shadow enhancers can act to buffer noise by providing distinct inputs to the same gene expression output .",
"To explore the conditions needed for the two Kr enhancers to act nearly independently within the same nucleus , we generated a simple model of enhancer-driven dynamics .",
"We considered an enhancer Ei that interacts with a transcription factor Ti , which together bind to the promoter to form the active promoter-enhancer complex Ci ( Figure 2A ) .",
"When the promoter is bound by the enhancer , it drives the production of mRNA .",
"Since the MS2 system only allows us to observe mRNA at the site of transcription , we modeled the diffusion of mRNA away from the transcription site as decay .",
"The transcription factor Ti is produced in bursts of ni molecules at a time , and it degrades linearly .",
"For simplicity , the transcription factor Ti is an abstraction of the multiple activating TFs that interact with the enhancer , and Ti corresponds to a different set of TFs for the proximal and distal enhancer .",
"This nonlinear model generalizes the linear model by Bothma et al . , 2015 by explicitly taking into account the presence of TFs .",
"We estimated some model parameters directly from experimental data and others by fitting using simulated annealing .",
"The mRNA degradation parameter α and production parameter r were measured directly from fluorescence data without any input from the model ( see Materials and methods for details ) .",
"The remaining parameters were first estimated using mathematical analysis , then fine-tuned using simulated annealing .",
"We found separate parameter sets for the proximal and distal enhancers that , when used to simulate transcription , fit the experimentally measured characteristics of the transcriptional traces , including transcription burst size , frequency , and duration , as well as the total mRNA produced ( Figure 2—figure supplement 1 ) .",
"We hypothesized that a model that lacks fluctuations in the input TFs could not recapitulate the high correlation of transcriptional activity in homozygotes versus the low correlation in heterozygotes .",
"To test this hypothesis , we generated another model of TF production .",
"We call our original model described above bursting TFs .",
"The other model is one in which TF numbers are constant over time , which we call constant TFs and is equivalent to the model in Bothma et al . , 2015 .",
"If the difference in transcription correlation between homozygotes and heterozygotes is due to fluctuating numbers of TFs , we expected that the bursting TFs model will recapitulate this behavior , while the constant TFs model will not .",
"However , if the constant TFs model is also able to recapitulate the observed difference in correlations , then the correlations are likely a consequence of the identical enhancers simply being regulated by the same set of TFs .",
"For each model , we used the 10 best parameter sets to simulate transcriptional activity in homozygote and heterozygote embryos and analyzed the resulting allele correlations .",
"We found that the bursting TFs model always produced results in which both homozygote allele correlations are significantly higher than the heterozygote , which qualitatively mirrors the experimental observations ( Figure 2B ) .",
"None of the best fitting parameter sets for the constant TF model were able to produce the experimentally-observed behavior and always resulted in near zero correlations for both the homozygote and heterozygote embryos ( Figure 2C ) .",
"Notably , using the bursting TFs model , all the simulated allele correlations were lower than the experimentally observed values , for example the simulated heterozygote allele correlation was near zero , while the experimental value was 0 . 14 at the embryo’s midpoint .",
"We hypothesized that this discrepancy was because the model assumes complete independence of the proximal and distal enhancer input TFs , while in reality , there may be some degree of shared inputs , either of known TFs or a general component of the transcriptional machinery .",
"To test this hypothesis , we generated a model that added a common TF to the bursting TFs model and attempted to fit the model parameters .",
"Some of the best parameter sets recapitulated the nonzero correlation of the heterozygote embryos , indicating that a shared factor may play a role in this system; however , this behavior was inconsistent from one parameter set to the next ( Figure 2—figure supplement 2 ) .",
"Therefore , we concluded that the simpler bursting TFs model , which consistently recapitulated the key features of the allele correlation data , was more suitable for subsequent analysis .",
"In conclusion , in our minimalist model of enhancer-driven transcription , the presence of TF fluctuations is required for the observed differences in allele correlation .",
"These results also demonstrate the advantage of using a single generic TF for each enhancer .",
"By abstracting away TF interactions , we reduced the complexity and number of parameters in the model , which allowed us to explore the relationship between TF production and allele correlation .",
"Both the experimental measurements of allele correlation and the computational model suggest that input TF fluctuations are an appreciable source of noise for enhancer activity .",
"Further , previous experimental work ( Wunderlich et al . , 2015 ) and the low correlation of transcriptional activity in heterozygotes ( Figure 1E ) indicates that each individual Kr enhancer receives different TF input signals .",
"This suggests that the shadow enhancer pair will be less sensitive to an input TF fluctuation than a single enhancer , because the shadow enhancer pair’s activity is dependent on a broader range of TF inputs .",
"To directly observe the relationship between input TF levels and enhancer output , we simultaneously tracked Bcd levels and enhancer activity in individual nuclei ( Figure 3; Supp . Videos 7–8 ) .",
"We measured this relationship for both the distal enhancer , which is activated by Bcd , and the shadow enhancer pair , and predicted that the distal enhancer’s transcription dynamics are more strongly influenced by fluctuations in Bcd levels .",
"To allow for tracking of both Bcd levels and enhancer activity , we crossed female flies that express eGFP-tagged Bcd in the place of endogenous Bcd ( called Bcd-GFP from here on; Gregor et al . , 2007 ) and MCP-mCherry with male flies homozygous for either the shadow pair or distal enhancer reporter .",
"As the Bcd-GFP transgene was inserted in a Bcd null background , the resulting embryos should receive roughly WT levels of Bcd .",
"The females flies were heterozygous for the maternally deposited Bcd-GFP , and therefore , we estimate that roughly half of the Bcd proteins were labeled .",
"Given the previous work demonstrating the normal function and expression levels of tagged Bcd , we expect the Bcd-GFP levels to be a representative sample of total Bcd ( Gregor et al . , 2007 ) .",
"Higher activator TF levels increase enhancer activity .",
"We therefore measured the correlation of nuclear Bcd-GFP levels to the slope of MS2 signal .",
"When the enhancer is active , MS2 signal has a positive slope , when the enhancer is inactive , slope is negative .",
"If the shadow enhancer pair is less sensitive than the distal enhancer to fluctuations in Bcd levels , we would predict higher correlation between Bcd-GFP levels and the activity of the distal enhancer than that of the shadow enhancer pair .",
"We find that the transcription dynamics driven by the distal enhancer are indeed significantly more correlated to nuclear Bcd-GFP levels ( median r = 0 . 18 ) than the dynamics driven by the shadow pair ( median r = 0 . 14; Figure 3F; p-value=6 . 1×10−3 ) , although both correlations are modest ( see Discussion ) .",
"The lower correlation indicates that transcription driven by the shadow pair is less sensitive to Bcd level fluctuations than is the distal enhancer .",
"Our modeling recapitulates this finding , showing that the separated TF inputs of the shadow pair are sufficient to explain the observed decreased sensitivity to TF fluctuations ( median r = 0 . 14 for the distal enhancer; 0 . 11 for the shadow pair; p-value=2 . 2×10−2; Figure 3G ) .",
"These findings indicate that the shadow enhancer pair is better able to buffer fluctuations in a single activating TF than a single enhancer , likely due to the shadow enhancer pair’s separation of TF inputs .",
"We wanted to further test whether the shadow enhancer pair drives less noisy gene expression output than a simple enhancer duplication .",
"We compared the noise in expression driven by the shadow enhancer pair to that driven by two copies of either the distal or proximal enhancer ( Figure 4 ) .",
"If the shadow enhancer pair drives lower noise , this suggests that having two independently acting enhancers is a critical feature of shadow enhancers’ ability to reduce variability and mediate robustness .",
"Alternatively , if duplicated enhancers drive similar levels of expression noise , this suggests that enhancer independence is not critical for shadow enhancer function and that shadow enhancers mediate robustness through a different mechanism , such as ensuring a critical threshold of expression is met ( Lam et al . , 2015; Perry et al . , 2011 ) .",
"We tracked transcriptional activity in embryos expressing MS2 under the control of the shadow enhancer pair , a duplicated proximal enhancer , or a duplicated distal enhancer ( Figure 4 ) .",
"To measure noise associated with each enhancer , we used these traces to calculate the coefficient of variation ( CV ) of transcriptional activity across nc14 .",
"CV is the standard deviation divided by the mean and provides a unitless measure of noise to allow comparisons among our enhancer constructs .",
"We then grouped these CV values by the embryo position of the transcriptional spots and found the average CV at each position for each enhancer construct .",
"All the enhancer constructs display the lowest expression noise at the embryo position of their peak expression ( Figure 4A ) , in agreement with previous findings of an inverse relationship between mean expression and noise levels ( Dar et al . , 2016; Figure 4—figure supplement 1 ) .",
"The shadow enhancer pair’s expression noise is ~30% or 15% lower , respectively , than that of the duplicated proximal or distal enhancers in their positions of maximum expression ( Figure 4C ) .",
"If the primary function of shadow enhancers is only to ensure a critical threshold of expression is reached , we would not expect to also see the lower expression noise associated with the shadow enhancer pair compared to either duplicated enhancer .",
"Furthermore , this decreased expression noise is not simply a consequence of higher expression levels , as the shadow enhancer pair produces less mRNA than the duplicated distal enhancer during nc14 ( Figure 4B ) .",
"The lower expression noise associated with the shadow enhancer pair suggests that it is less susceptible to fluctuations in upstream TFs than multiple identical enhancers .",
"To explore which factors drive the difference in CVs between the duplicated and shadow enhancer constructs , we extended our model to have a single promoter controlled by two enhancers ( Figure 5A ) .",
"To do so , we assumed that either or both enhancers can be looped to the promoter and drive mRNA production .",
"The rate of mRNA production when both enhancers are looped is the sum of the rates driven by the individual enhancers .",
"We assumed that some parameters , for example the TF production rates and mRNA decay rate , are the same as the single enhancer case .",
"We allowed the parameters describing the promoter-enhancer looping dynamics ( the kon and koff values ) to differ , depending on the enhancer’s position in the construct relative to the promoter and whether another enhancer is present .",
"To fit the kon and koff values , we used the medians of the 10 best single enhancer parameter sets as a starting point and performed simulated annealing to refine them .",
"This approach allowed us to examine how the model parameters that describe promoter-enhancer looping dynamics change when two enhancers are controlling the same promoter .",
"We compared the koff and kon values for each enhancer in the two enhancer constructs to their values from the single enhancer model .",
"We generally found that koff values increased and kon values decreased ( Figure 5B ) .",
"The effect is most pronounced in the duplicated distal enhancer , with large changes to the koff and kon values for the enhancer in the position far from the promoter ( position 2 ) .",
"Given that our model assumes that enhancers act additively and only allows for changes in the koff and kon values , these observed effects may indicate that either the presence of a second enhancer interferes with promoter-enhancer looping or that the promoter can be saturated .",
"Our model cannot distinguish between these two possibilities , but these observations are consistent with our ( Figure 5—figure supplement 1 ) and previous results indicating that the Kr enhancers can act sub-additively ( Scholes et al . , 2019 ) .",
"Additionally , the dramatic changes in koff and kon values in the duplicated distal enhancer are consistent with a previous assertion that enhancer sub-additivity is most pronounced in cases of strong enhancers ( Bothma et al . , 2015 ) .",
"We used these models to simulate transcription and predict the resulting CVs from the duplicated enhancer and shadow pair constructs .",
"In line with experimental data , we found the model predicts that the shadow pair construct drives lower noise than the duplicated distal or duplicated proximal enhancer constructs in the middle of the embryo ( Figure 5C ) .",
"This is particularly notable , as we did not explicitly fit our model to reproduce the experimentally observed CVs .",
"There is only one fundamental difference between the shadow pair and duplicated enhancer models , namely the use of separate TF inputs for the shadow pair .",
"Therefore , in our simplified model , we can conclude that the separation of input TFs is sufficient to explain the lower noise driven by the shadow enhancer pair construct .",
"To further understand the sources of noise the shadow enhancer pair is able to buffer , we compared the extrinsic and intrinsic noise associated with the shadow enhancer pair to that associated with either single or duplicated enhancers .",
"To do so , we measured the transcriptional dynamics of embryos with two identical reporters in each nucleus and calculated noise sources using the approach of Elowitz et al . , 2002 .",
"Intrinsic noise corresponds to sources of noise , such as TF binding and unbinding , that affect each allele separately .",
"It is quantified by the degree to which the activities of the two reporters in a single nucleus differ .",
"Extrinsic noise corresponds to global sources of noise , such as TF levels , that affect both alleles simultaneously .",
"It is measured by the degree to which the activities of the two reporters change together .",
"Intrinsic and extrinsic noise are defined such that , when squared , their sum is equal to total noise2 , which corresponds to the CV2 of the two identical alleles in each nucleus in our system ( see Materials and methods ) .",
"Because our data do not meet one key assumption needed to measure extrinsic and intrinsic noise with the two-reporter approach ( see Discussion; Figure 6—figure supplement 1 ) , we use the terms inter-allele noise and covariance in place of intrinsic and extrinsic noise .",
"Based on our separation of inputs hypothesis and CV data , we expected the total noise associated with the shadow enhancer pair to be lower than that associated with the duplicated enhancers .",
"We predicted that the shadow enhancer pair will mediate lower total expression noise through lower covariance , as the two member enhancers are regulated by different TFs .",
"Given the complexity of predicting inter-allele noise from first principles ( Materials and methods; Figure 6—figure supplement 2 ) , we predicted that constructs with two enhancers will have lower inter-allele noise than single enhancer constructs but did not have a strong prediction regarding the relative inter-allele noise among the different two-enhancer constructs .",
"Comparisons of noise between the single and duplicated enhancer constructs would further allow us to discern whether reductions in noise are generally associated with two-enhancer constructs or whether this is a particular feature of the shadow enhancer pair .",
"Neither the duplicated proximal nor distal enhancers drive significantly lower total noise than the corresponding single enhancers , indicating that the addition of an identical enhancer is not sufficient to reduce expression noise in this system ( Figure 6A ) .",
"The shadow enhancer pair drives lower total expression noise than either single or duplicated enhancer , consistent with the temporal CV data in Figure 4 .",
"The median total expression noise associated with the duplicated distal and duplicated proximal enhancers is 1 . 4 or 2 . 4 times higher , respectively , than that associated with the shadow enhancer pair ( Figure 6A ) .",
"Note that for measurements of noise , our distal construct places the enhancer at the endogenous spacing from the promoter , as we wanted to control for positional effects on expression and noise ( Scholes et al . , 2019; Figure 6—figure supplement 3 ) .",
"In line with our expectations , the shadow enhancer pair has significantly lower covariance levels than either single or duplicated enhancer ( Figure 6B ) .",
"The shadow enhancer pair also has lower inter-allele noise than all of the other constructs , though these differences are only marginally significant ( p=0 . 13 ) when compared to the duplicated distal enhancer .",
"Covariance makes a larger contribution to the total noise for the duplicated distal enhancer and the shadow enhancer pair , while inter-allele noise is the larger source of noise for the single distal enhancer and the single or duplicated proximal enhancers ( Figure 6B ) .",
"The lower total noise and covariance of the shadow enhancer pair support our hypothesis that , by separating regulation of the member enhancers , the shadow enhancer pair can buffer against upstream fluctuations .",
"The lower inter-allele noise associated with the shadow enhancer pair warrants further investigation .",
"A simple theoretical approach predicts that two enhancer constructs will have lower inter-allele noise ( Figure 6—figure supplement 2 ) .",
"Given that this is not universally observed in our data , this suggests that there is still much to discover about how inter-allele noise changes as additional enhancers control a gene’s transcription .",
"We showed the Kr shadow enhancer pair drives expression with lower total noise than either single or duplicated enhancer , yet previous studies have generally found individual member enhancers of a shadow enhancer set are dispensable under ideal conditions ( Frankel et al . , 2010; Perry et al . , 2011; Osterwalder et al . , 2018 ) .",
"However , in the face of environmental or genetic stress , the full shadow enhancer group is necessary for proper development ( Frankel et al . , 2010; Osterwalder et al . , 2018; Perry et al . , 2011 ) .",
"We therefore decided to investigate whether temperature stress causes significant increases in expression noise and whether the shadow enhancer pair or duplicated enhancers can buffer these potential increases in noise .",
"Similar to our findings at ambient temperature ( 26 . 5°C ) , the shadow enhancer pair drives lower total noise than all other tested enhancer constructs at 32°C ( Figure 7B ) .",
"At 32°C , the duplicated distal and duplicated proximal enhancers display 35% or 52% , respectively , higher total noise than the shadow enhancer pair .",
"At 17°C , the shadow enhancer pair has approximately 46% lower total noise than either the single or duplicated proximal enhancer , 21% lower total noise than the single distal enhancer , and is not significantly different than the duplicated distal enhancer ( Figure 7A ) .",
"As seen by the variety of shapes in the temperature response curves ( Figure 7C ) , temperature perturbations have enhancer-specific effects , suggesting input TFs may differ in their response to temperature change .",
"The low noise driven by the shadow enhancer pair across conditions is consistent with previous studies showing shadow enhancers are required for robust gene expression at elevated and lowered temperatures ( Frankel et al . , 2010; Perry et al . , 2010 ) ."
],
[
"When measured in fixed embryos , the TFs used in Drosophila embryonic development show remarkably precise expression patterns , displaying errors smaller than the width of a single nucleus ( Dubuis et al . , 2013; Gregor et al . , 2007; Little et al . , 2013; He et al . , 2008 ) .",
"It therefore was unclear whether fluctuations in these regulators play a significant role in transcriptional noise in the developing embryo .",
"By measuring the temporal dynamics of the individual Kr enhancers , each of which is controlled by different transcriptional activators , we show that TF fluctuations do significantly contribute to the noise in transcriptional output of a single enhancer .",
"Within a nucleus , expression controlled by the two different Kr enhancers is far less correlated than expression driven by two copies of the same enhancer , indicating that TF inputs , as opposed to more global factors , are the primary regulators of transcriptional bursting in this system .",
"Our current findings leave open the possibility that additional mechanisms , such as differences in 3D nuclear organization between different reporters , may also contribute to the differences in noise that we see .",
"We also showed that activity driven by the Kr shadow enhancer pair is less sensitive to levels of a single TF than is activity driven by an individual Kr enhancer .",
"While prior work has shown that changes in TF levels precede changes in target transcription ( Bothma et al . , 2018 ) , the sensitivity of individual enhancers to changes in TF levels had not been previously quantified .",
"The correlation between Bcd levels and activity of the distal enhancer is modest , and we expect that this reflects both the influence of additional TF inputs and nuclear heterogeneity that causes the local Bcd levels available to the enhancer to differ from total nuclear levels ( Mir et al . , 2018 ) .",
"We suspect that the correlation between the activity of the distal enhancer and Bcd levels in the microenvironment surrounding the enhancer is higher than what we were able to measure here .",
"New and emerging technologies will likely allow for live measurements of multiple TF inputs at higher spatial resolution , enabling further insights into the dynamics of expression regulation .",
"The finding that the Kr shadow enhancer pair is less sensitive to TF levels helps reconcile our finding that the individual Kr enhancers are influenced by fluctuations in input TFs with previous studies showing that endogenous Kr expression patterns are rather reproducible ( Little et al . , 2013 ) .",
"Previous work has cited the role of spatial and temporal averaging , which buffers noisy nascent transcriptional dynamics to generate more precise expression levels .",
"Shadow enhancers operate upstream of this averaging , driving less noisy nascent transcription than either single enhancers or enhancer duplications .",
"We developed a stochastic mathematical model of Kr enhancer dynamics and mRNA production that recapitulates our main experimental results .",
"This model is based on that by Bothma et al . , 2015 , but it is expanded to include the dynamics of a TF that regulates each enhancer .",
"We placed a strong emphasis on the simplicity of this model , for example by using a single abstract TF for each enhancer .",
"This choice both avoids a combinatorial explosion of parameters and makes the model results and parameters easier to interpret .",
"One of the most notable features of the model is that it recreates the differences in noise between shadow and duplicated enhancer constructs without any additional fitting , indicating that these differences in the model system are a direct result of the separation of input TFs to the proximal and distal enhancers .",
"Future versions of this model can include refinements .",
"For example , in the current model , we do not include the influence of repressive TFs or consider the multiple modes of action used by activating TFs .",
"Future experiments and models can also be designed to identify the mechanism of enhancer non-additivity: changes in promoter-enhancer looping , saturation of the promoter , or other mechanisms .",
"In our investigation of sources of noise , we decomposed total noise into extrinsic and intrinsic components as in Elowitz et al . , 2002 .",
"In that study , the authors showed that the activity of one reporter did not inhibit expression of the other reporter , and therefore their calculations assumed no negative covariance between the reporters’ expression output .",
"In our system , we found a small amount of negative covariance between the activity of two alleles in the same nucleus ( Figure 6—figure supplement 1 ) .",
"For this reason , we called our measurements covariance and inter-allele noise .",
"The negative covariance we observed indicates that activity at one allele can sometimes interfere with activity at the other allele , suggesting competition for limited amounts of a factor necessary for reporter visualization .",
"The two possible limiting factors are MCP-GFP or an endogenous factor required for transcription .",
"If MCP-GFP were limiting , we would expect to see the highest levels of negative covariance at the center of the embryo , where the highest number of transcripts are produced and bound by MCP-GFP .",
"Since the fraction of nuclei with negative covariance is highest at the edges of the expression domain ( Figure 6—figure supplement 1 ) , the limiting resource is likely not MCP-GFP , but instead a spatially-patterned endogenous factor , like a TF .",
"Currently , the field largely assumes that adding reporters does not appreciably affect expression of other genes .",
"However , sequestering TFs within repetitive regions of DNA can impact gene expression ( Liu et al . , 2007; Janssen et al . , 2000 ) , and a few case studies show that reporters can affect endogenous gene expression ( Laboulaye et al . , 2018; Thompson and Gasson , 2001 ) .",
"If TF competition is responsible for the observed negative covariance between reporters , a closer examination of the effects of transgenic reporters on the endogenous system is warranted .",
"In addition , TF competition may be a feature , not a bug , of developmental gene expression control , as modeling has indicated that molecular competition can decrease expression noise and correlate expression of multiple targets ( Wei et al . , 2019 ) .",
"There are likely several features of shadow enhancers selected by evolution outside of their noise-suppression capabilities .",
"Preger-Ben Noon , et al . showed that all shadow enhancers of shavenbaby , a developmental TF gene in Drosophila , drive expression patterns in tissues and times outside of their previously characterized domains in the larval cuticle ( Preger-Ben Noon et al . , 2018 ) .",
"This suggests that shadow enhancers , while seemingly redundant at one developmental stage , may play separate , non-redundant roles in other stages or tissues .",
"Additionally , a recent study investigating shadow enhancer pairs associated with genes involved in Drosophila embryonic development found that CRISPR deletions of the individual enhancers result in different phenotypes , suggesting each plays a slightly different role in regulating gene expression ( Dunipace et al . , 2019 ) .",
"In several other cases , both members of a shadow enhancer pair are required for the precise expression pattern generated by the endogenous locus ( El-Sherif and Levine , 2016; Perry et al . , 2012; Dunipace et al . , 2011; Perry et al . , 2011; Yan et al . , 2017 ) .",
"These sharpened expression patterns achieved by a shadow enhancer pair may reflect enhancer dominance or other forms of enhancer-enhancer interaction and are likely another important function of shadow enhancers ( El-Sherif and Levine , 2016 ) .",
"In the case of Kr , the endogenous expression pattern is best recapitulated by the shadow enhancer pair , with the individual enhancers driving slightly more anterior or posterior patterns of expression ( Figure 1—figure supplement 1; El-Sherif and Levine , 2016 ) .",
"Additionally , the early embryonic Kr enhancers drive observable levels of expression in additional tissues and time points , but these expression patterns overlap those driven by additional , generally stronger , enhancers , suggesting that the primary role of the proximal and distal enhancers is in early embryonic patterning ( Hoch et al . , 1990 ) .",
"Therefore , while we cannot rule out the possibility that the proximal and distal enhancers perform separate functions at later stages , it seems that their primary function , and evolutionary substrate , is controlling Kr expression pattern and noise levels during early embryonic development .",
"Here , we have investigated the details of shadow enhancer function for a particular system , and we expect that some key observations may generalize to many sets of shadow enhancers .",
"Shadow enhancers seem to be a general feature of developmental systems ( Cannavò et al . , 2016; Osterwalder et al . , 2018 ) , but the diversity among them has yet to be specifically addressed .",
"While we worked with a pair of shadow enhancers with clearly separated TF activators , shadow enhancers can come in much larger groups and with varying degrees of TF input separation between the individual enhancers ( Cannavò et al . , 2016; Osterwalder et al . , 2018 ) .",
"To discern how expression dynamics and noise driven by shadow enhancers depend on their degree of TF input separation , we are investigating these characteristics in additional sets of shadow enhancers with varying degrees of differential TF regulation .",
"Our current results combined with data gathered from additional shadow enhancers will inform fuller models of how developmental systems ensure precision and robustness ."
],
[
"The single , duplicated , or shadow enhancers were each cloned into the pBphi vector , upstream of the Kruppel promoter , 24 MS2 repeats , and a yellow reporter gene as in Fukaya et al . , 2016 .",
"We defined the proximal enhancer as chromosome 2R:25224832–25226417 , the distal enhancer as chromosome 2R:25222618–25223777 , and the promoter as chromosome 2R:25226611–25226951 , using the Drosophila melanogaster dm6 release coordinates .",
"The precise sequences for each reporter construct are given in Supplementary file 4 .",
"For the allele correlation experiments , each enhancer was cloned 192 bp upstream of the Kr promoter , separated by the endogenous sequence found between the proximal enhancer and the promoter .",
"For transcriptional noise experiments , the distal enhancer was placed at its endogenous spacing , 2835 bp upstream of the promoter , and the proximal enhancer sequence was replaced by a region of the lambda genome that is predicted to have few relevant TF-binding sites .",
"In the shadow enhancer pair or duplicated enhancer constructs , the two enhancers were separated by the sequence separating the proximal and distal enhancers in the endogenous locus .",
"Using phiC31-mediated integration , each reporter construct was integrated into the same site on the second chromosomes by injection into yw; PBac{y[+]-attP-3B}VK00002 ( BDRC stock #9723 ) embryos by BestGene Inc ( Chino Hills , CA ) .",
"To produce embryos with biallelic expression of the MS2 reporter , female flies expressing RFP-tagged histones and GFP-tagged MCP ( yw; His-RFP/Cyo; MCP-GFP/TM3 . Sb ) were crossed with males containing one of the enhancer-MS2 reporter constructs .",
"Virgin female F1 offspring were then mated with males of the same parental genotype , except in the case of shadow heterozygous flies , which were mated with males containing the other single enhancer-MS2 reporter .",
"Live embryos were collected prior to nc14 , dechorionated , mounted on a permeable membrane , immersed in Halocarbon 27 oil , and put under a glass coverslip as in Garcia et al . , 2013 .",
"Individual embryos were then imaged on a Nikon A1R point scanning confocal microscope using a 60X/1 . 4 N . A . oil immersion objective and laser settings of 40uW for 488 nm and 35uW for 561 nm .",
"To track transcription , 21 slice Z-stacks , at 0 . 5 um steps , were taken throughout the length of nc14 at roughly 30 s intervals .",
"To identify the Z-stack’s position in the embryo , the whole embryo was imaged after the end of nc14 at 20x using the same laser power settings .",
"Later in the analysis , each transcriptional spot’s location is described as falling into one of 42 anterior-posterior ( AP ) bins , with the first bin at the anterior of the embryo .",
"Unless otherwise indicated , embryos were imaged at ambient temperature , which was on average 26 . 5°C .",
"To image at other temperatures , embryos were either heated or cooled using the Bioscience Tools ( Highland , CA ) heating-cooling stage and accompanying water-cooling unit .",
"Tracking of nuclei and transcriptional spots was done using the image analysis Matlab pipeline described in Garcia et al . , 2013 .",
"For every spot of transcription imaged , background fluorescence at each time point is estimated as the offset of fitting the 2D maximum projection of the Z-stack image centered around the transcriptional spot to a gaussian curve , using Matlab lsqnonlin .",
"This background estimate is subtracted from the raw spot fluorescence intensity .",
"The resulting fluorescence traces across the time of nc14 are then subject to smoothing by the LOWESS method with a span of 10% .",
"The smoothed traces were used to measure transcriptional parameters and noise .",
"Traces consisting of fewer than three time frames were removed from calculations .",
"To calculate transcription parameters , we used the smoothed traces to determine if the promoter was active or inactive at each time point .",
"A promoter was called active if the slope of its trace ( change in fluorescence ) between one point and the next was greater than or equal to the instantaneous fluorescence value calculated for one mRNA molecule ( FRNAP , described below ) .",
"Once called active , the promoter is considered active until the slope of the fluorescence trace becomes less than or equal to the negative instantaneous fluorescence value of one mRNA molecule , at which point it is called inactive until another active point is reached .",
"The instantaneous fluorescence of a single mRNA was chosen as the threshold because we reasoned that an increase in fluorescence greater than or equal to that of a single transcript is indicative of an actively producing promoter , while a decrease in fluorescence greater than that associated with a single transcript indicates transcripts are primarily dissociating from , not being produced from , this locus .",
"Visual inspection of fluorescence traces agreed well with the burst calling produced by this method ( Figure 2—figure supplement 3 ) .",
"Using these traces and promoter states , we measured burst size , frequency and duration .",
"Burst size is defined as the integrated area under the curve of each transcriptional burst , from one ‘ON’ frame to the next ‘ON’ frame , with the value of 0 set to the floor of the background-subtracted florescence trace ( Figure 2—figure supplement 3 panel C ) .",
"Duration is defined as the amount of time occurring between the frame a promoter is determined active and the frame it is next determined inactive ( Figure 2—figure supplement 3 panel F ) .",
"Frequency is defined as the number of bursts occurring in the period of time from the first time the promoter is called active until 50 min into nc14 or the movie ends , whichever is first ( Figure 2—figure supplement 3 panel E ) .",
"The time of first activity was used for frequency calculations because the different enhancer constructs showed different characteristic times to first transcriptional burst during nc14 .",
"For these , and all other measurements , we control for the embryo position of the transcription trace by first individually analyzing the trace and then using all the traces in each AP bin ( anterior-posterior; the embryo is divided into 41 bins each containing 2 . 5% of the embryo’s length ) to calculate summary statistics of the transcriptional dynamics and noise values at that AP position .",
"All Matlab codes used for burst calling , noise measurements , and other image processing are available at the Wunderlich Lab GitHub ( Waymack , 2020; copy archived at https://github . com/elifesciences-publications/KrShadowEnhancerCode ) .",
"To compare the sensitivity of the activity of the shadow pair and distal enhancer to Bcd levels , we tracked the fluorescence of Bcd-GFP and MCP-mCherry in individual nuclei across the time of nc14 .",
"To obtain embryos for simultaneous tracking , we crossed female flies heterozygous for Bcd-GFP and MCP-mcherry with male flies homozygous for either the shadow pair or distal enhancer reporter .",
"Bcd-GFP and MCP-mCherry are maternally deposited and thereby allow us to measure levels of Bcd and enhancer activity in individual nuclei of the resulting embryos .",
"Embryo collection and preparation was performed as described above .",
"The same microscope , objective , and Z-step profile were used as described above , but laser settings were switched to 40uW for 561 nm and 35uW for 488 nm .",
"Analysis of transcriptional activity was performed as described above .",
"Time traces of Bcd-GFP levels in individual nuclei were subjected to background correction by subtracting the average fluorescence of the regions of the image not containing a nucleus at each time point from the raw Bcd-GFP fluorescence .",
"The resulting Bcd-GFP time traces were then subjected to smoothing by the MATLAB smooth function , using the LOWESS method with a span of 10% .",
"To measure the sensitivity of enhancer activity to Bcd levels , we correlated the slope of MS2 traces to the corresponding Bcd-GFP levels in the same nucleus .",
"Slope was calculated between the MS2 values at consecutive time points and compared to the Bcd-GFP value at the earlier of the two time points .",
"This process was done for all time points through 50 min into nc14 .",
"To put our results in physiologically relevant units , we calibrated our fluorescence measurements in terms of mRNA molecules .",
"As in Lammers et al . , 2018 , for our microscope , we determined a calibration factor , α , between our MS2 signal integrated over nc13 , FMS2 , and the number of mRNAs generated by a single allele from the same reporter construct in the same time interval , NFISH , using the hunchback P2 enhancer reporter construct ( Garcia et al . , 2013 ) .",
"Using this conversion factor , we can calculate the integrated fluorescence of a single mRNA ( F1 ) as well as the instantaneous fluorescence of an mRNA molecule ( FRNAP ) .",
"With our microscope , FRNAP is 379 AU/RNAP and F1 is 1338 AU/RNAP∙min .",
"With these values , we are able to convert both integrated and instantaneous fluorescence into total mRNAs produced and number of nascent mRNAs present at a single time point , by dividing by F1 and FRNAP , respectively .",
"To calculate the temporal CV each transcriptional spot i , we used the formula:CV ( i ) =standard deviation ( mi ( t ) ) mean ( mi ( t ) ) where mi ( t ) is the fluorescence of spot i at time t .",
"We also decomposed the total noise experienced in each nucleus to inter-allele noise and co-variance , analogous to the approach of Elowitz et al . , 2002 .",
"Inter-allele noise is calculated one nucleus at a time .",
"It is the mean square difference between the fluorescence of the two alleles in a single nucleus:ηIA2=⟨m1t-m2t2⟩2⟨m1t⟩⟨m2t⟩where m1 ( t ) is the fluorescence of one allele in the nucleus at time t , and m2 ( t ) is the fluorescence of the other allele in the same nucleus and the angled brackets indicate the mean across the time of nc14 .",
"Covariance is the covariance of the activity of the two alleles in the same nucleus across the time of nc14:ηCV2=⟨m1tm2t⟩-⟨m1t⟩⟨m2t⟩⟨m1t⟩⟨m2t⟩ The inter-allele and covariance values are defined such that they sum to give the total transcriptional noise displayed by the two alleles in a single nucleus .",
"ηtot2=⟨m1t2+m2t2⟩-2⟨m1t⟩⟨m2t⟩2⟨m1t⟩⟨m2t⟩ This total noise value is equal to the coefficient of variation of the expression of the two alleles in a single nucleus across the time of nc14 .",
"To determine any significant differences in total noise , covariance , or inter-allele noise values between the different enhancer constructs , we performed Kruskal-Wallis tests with the Bonferroni multiple comparison correction .",
"We constructed a model of enhancer-driven transcription based on the following chemical reaction network:T+E⇌koffkonC→rC+R∅→β1nTT→β−1∅R→α∅where E is an enhancer that interacts with a transcription factor T , which together bind to the promoter at a rate kon to form the active promoter-enhancer complex C . When the promoter is in this active form , it leads to the production of mRNA denoted by R , which degrades by diffusion from the gene locus at a rate α .",
"Transcription is interrupted whenever the complex C disassociates spontaneously at a rate koff .",
"In the bursting TFs model , the transcription factor T appears at a rate β1 and degrades at a rate β−1 .",
"To recapitulate Kruppel expression patterns , the value of β1 was assumed to be given by ( 1 ) f",
"( x ) =c12πσ2e− ( x−μ ) 22σ2 , where x is the percentage along the length of the egg and c is a scaling constant .",
"Since Kruppel activity peaks near the center of the egg , we chose µ = 50 , while c and σ were fitted along with the other parameters .",
"Lastly , n was assumed to be fixed across the length of the egg .",
"We also generated a constant TF model , which is an adaptation of the model in Bothma et al . , 2015 .",
"This model implicitly assumes that TF numbers are constant and , therefore , are incorporated into the value of kon as described by the reactionsE⇌koffTkonC→rC+RR→α∅ In this case , the value for T was fitted for each bin in a similar way to β1 , that is the constant number of TFs was assumed to be described by Equation ( 1 ) ( values were rounded to the nearest integer ) .",
"To simulate the transcriptional traces , we implemented a stochastic approach .",
"Individual chemical events such as enhancer-promoter looping take place at random times and are influenced by transcription factor numbers .",
"Individual trajectories of chemical species over time were calculated using the Gillespie algorithm ( Gillespie , 1976 ) , and these trajectories are comparable to the experimentally measured transcriptional traces .",
"Since the enhancer is either bound or not bound to the promoter , we imposed the constraint that C + E = 1 when simulating model dynamics .",
"To yield a starting estimate for the kon and koff parameters , we defined the start and end of a burst as the time when the reactions E→C and C→E occur , respectively .",
"The length of the ith burst was defined as the range of [bi , pi] where bi corresponds to the time of the ith instance of the reaction E→C and pi to the time of the ith instance of the reaction C→E .",
"The time between the ith burst and the i + 1th burst is [pi , bi+1] .",
"The Gillespie algorithm dictates that the time spent in any given state is determined by an exponentially distributed random variable with a rate parameter equal to the product of two parts: the sum of rate constants of the outgoing reactions , and the number of possible reactions .",
"If the enhancer is either bound or unbound , we have that C = 1 or E = 1 , respectively .",
"Therefore , by letting tb be the average time between bursts and td be the average duration of a burst , we can writetb=limM→∞1M∑j=1M ( 1N−1∑i=1N−1 ( bi+1j−pij ) ) =1konET≈1konandtd=limM→∞1M∑j=1M ( 1N∑i=1N ( pij−bij ) ) =1koffC=1koffwhere N is the number of bursts for spot j , bij and pij denote the beginning and end of burst i in spot j respectively , and M denotes the total number of spots in the egg .",
"The right-hand sides are given by the expected value of the exponential distribution and the assumption that , on average , T is close to 1 .",
"While this may not be the case for T , the assumption provides a convenient upper bound for the average time between bursts , which is likely not to have a much smaller value for a lower bound .",
"( A low enough value of tb would imply nearly constant fluorescence intensity instead of bursts . )",
"Finally , the average duration of a burst td can be calculated directly from the data and used to obtain koff by calculating 1/td .",
"Similarly , the average time between bursts tb is readily available from the data giving us kon ≈ 1/tb .",
"We were able to directly estimate mRNA production and degradation rates from the experimental data .",
"To estimate α , we focused on periods of mRNA decay; that is periods where no active transcription is taking place and are thus described byR′=−αR , which in turn can be solved to be ( 2 ) R=ce−tα , where c is a constant of integration .",
"Taking the derivative of Equation 2 yields ( 3 ) R′ ( t ) =−αce−tα , which corresponds to the slope of the decaying burst .",
"We define the interval of decay of the ith burst as [pi , bi+1] .",
"For some point t0 ∈ ( pi , bi+1 ) , let R0=R ( t0 ) =ce−t0α .",
"Solving this expression for c gives that c=R0et0α .",
"Substituting for c in Equation 3 evaluated at t0 results in R′ ( t0 ) =−αR0et0αe−t0α=−αR0 .",
"Then , it follows that ( 4 ) α=−R′ ( t0 ) R0 .",
"In other words , the rate of decay of mRNA fluorescence can be calculated from any trace by taking the ratio of the slope during burst decay and its intensity at a given time t0 ∈ ( pi , bi+1 ) .",
"Adjacent measurements of fluorescence intensity from the single enhancer systems were used to approximate the slope at each point in the traces .",
"Then , Equation 4 was applied to each point .",
"A histogram of all calculated values was generated ( Figure 2—figure supplement 4 ) .",
"In this figure , there was a clear peak , which provided us with an estimate of α ≈ 1 . 95 .",
"The estimation of r was done for periods of active transcription , which are also accompanied by simultaneous mRNA decay .",
"By noting that C = 1 during mRNA transcription , we can approximate these periods as the zeroth order process∅⇌αrR The differential equation associated with this system is given by ( 5 ) R′=r−αR , and has steady state R∗=r/α .",
"Equation 5 can be solved explicitly for R by choosingR ( t ) =rα+ce−tα , where c is a constant of integration .",
"For two adjacent measurements at times t1 and t2 we can write their respective measured amounts of mRNA as ( 6 ) R1=rα+c1e−t1α , and ( 7 ) R2=rα+c2e−t2α , Solving for c1 and c2 givesc1= ( R1−rα ) et1αc2= ( R2−rα ) et2α .",
"The short-term fluctuations of mRNA from R1 to R2 between two adjacent discrete time points in the stochastic system can be approximated by Equations 6 and 7 .",
"This implies that ( R1−rα ) et1α= ( R2−rα ) et2α , which in turn givesr=αR1−R2eαΔt1−eαΔt .",
"Therefore , the estimation of r can be computed given two adjacent measurements of fluorescence and the time between them .",
"Finally , we used a similar approach as done with α to calculate values of r from fluorescence data .",
"However , unlike α , r was calculated for each bin to account for differences in transcriptional efficiency across the length of the embryo .",
"Simulations and parameter fitting were done with MATLAB .",
"Optimization in fitting was done by minimizing the sum of squared errors ( SSE ) between the normalized vectors of burst properties and allele correlations of the experimental and simulated data .",
"In particular , a vector y of experimental data was created by concatenating the following vectors: burst size , integrated fluorescence , frequency , duration , and allele correlation across the length of the embryo .",
"The vector y was subsequently normalized by dividing each burst property by the largest element in their respective vectors ( except correlation which by definition is unitless between −1 and 1 ) .",
"A vector x was created in an analogous fashion to y but using simulated instead of experimental data .",
"However , x was normalized using the same elements that were used to normalize y .",
"Then , the discrepancy between the experimental and simulated data was measured by:SSE=∑i=1n ( yi−",
"xi ) 2 We used a high-performance computing cluster to compute 200 independent runs of parameter fitting with simulated annealing for each model variant .",
"The algorithm requires an initial guess of the parameter set P0 , an initial temperature Γ0 , a final temperature Γ’ , the number of iterations per temperature N , and a cooling factor µ .",
"Then , each iteration is as follows: To generate our results , we chose Γ0 = 1 , Γ’ = Γ0/10 , N = 30 , and µ = 0 . 8 .",
"We observed an improvement in the quality of the fittings by using analysis-derived parameter values as initial guesses instead of values given through random sampling .",
"The sampled space ranged from 10−3 to 103 for all parameters , except n , which was sampled from 100 to 102 , and σ , which was randomly chosen to be an integer between 1 and 20 .",
"Equal numbers of parameter values were sampled at each order of magnitude .",
"The analysis in the section above was used to estimate the parameters in P0 .",
"Parameters that were not estimated in the previous section were given the following initial guesses: n = 10 , β−1 = 1 , σ = 6 , and c = 40 .",
"Initial guesses for c and σ were based on the experimental observation that there is little transcription outside of 20–80% egg length .",
"Based on this observation , simulations were limited to this egg length range , as well .",
"For the constant TFs model , both analysis-derived and random initial parameter values were used to maximize the likelihood of finding any parameter set capable of recapitulating the observed allele correlation .",
"Parameter sets resulting from fitting were sorted in ascending order based on their sum of squared errors , and the 10 lowest error parameter sets are what we called the 10 best parameter sets .",
"For all figures , we simulated 80 spots per bin and simulated each bin five times to generate error bars .",
"Data for the distal enhancer at the proximal location was used to reproduce simulated allele correlations in all cases .",
"Gillespie simulations update the counts of each chemical species at random time intervals .",
"However , for ease of parameter fitting and to better recapitulate the experiments , we generated data in two distinct timescales: one consisting of 30 second intervals after which mRNA counts were recorded , and another consisting of random time intervals generated by the algorithm after which chemical counts were updated .",
"The former one was used for all parameter fitting rounds and generation of figures .",
"To explore two enhancer systems , we expanded our previous model to include an additional enhancer .",
"First , we considered duplicated enhancer systems , which consist of either two proximal or two distal enhancers .",
"Enhancers were denoted by E1 and E2 , which correspond to two identical enhancers that exist in different locations relative to the promoter .",
"They are activated by the same transcription factors as described by the reactionsT+E1⇌koff1kon1C1→r1C1+RT+E2⇌koff2kon2C2→r2C2+R∅→β1nTT→β−1∅R→α∅ Without loss of generality , we used E1 to denote the enhancer at the proximal location and E2 to denote the enhancer at the distal location .",
"This model describes independent enhancer dynamics; that is the behavior of one enhancer does not affect the behavior of the other , and , as such , both enhancers can be simultaneously looped to the promoter .",
"Consequently , to account for potential enhancer interference or competition for the promoter , we assumed distinct kon and koff values for each enhancer in the duplicated enhancer constructs .",
"We also used distinct values of r for each distal enhancer in the duplicated distal construct since fluorescence data was available for this enhancer at the proximal and endogenous location .",
"For proximal enhancers , we assume r1 = r2 .",
"To describe the dynamics of the shadow enhancer pair , we denoted the activators for E1 ( the proximal enhancer ) and E2 ( the distal enhancer ) by T1 and T2 , respectively:T1+E1⇌koff1kon1C1→r1C1+RT2+E2⇌koff2kon2C2→r2C2+R∅→β1n1T1∅→γ1n2T2T1→β−1∅T2→γ−1∅R→α∅ The production rate of T2 , γ1 , was calculated in the same way as production rate of T1 , β1 , but differed in the values of c and σ .",
"The two enhancer models were also used to calculate allele correlation between homozygotes and heterozygotes because a distinction between the mRNA produced by C1 and C2 was made .",
"This approach works because , for example when considering the homozygote embryos , each single enhancer resides in the same nucleus and is therefore affected by the same fluctuating TF numbers .",
"In the duplicated enhancer model , each enhancer E1 or E2 is affected by the same fluctuations in the number of transcription factor T . An analogous logic applies to the heterozygotes .",
"To fit the two enhancer models to experimental data , we retained several parameters from the single enhancer models .",
"Parameters r and α were directly calculated from the data , and , as such , did not vary across models .",
"We assume that parameters concerning transcription factors ( β1 , β−1 , γ1 , γ−1 , n1 , and n2 ) are not affected by the presence of an additional enhancer .",
"Therefore , in our model , only kon and koff are free to change .",
"To fit the values of kon1 , kon2 , koff1 , and koff2 , we set the other model parameters to the median values of the 10 best parameter sets in the respective single enhancer model .",
"We then used a similar simulating annealing approach to fit the kon and koff values .",
"We used the resulting values to simulate transcriptional traces and to calculate the predicted CV values shown in Figure 5 .",
"To make a prediction about the expected change in inter-allele noise between single and two enhancer reporter constructs , we used the theory put forth in Sánchez and Kondev , 2008; Sanchez et al . , 2011 .",
"This formalism can be used to calculate the expected mean and variance of the transcriptional output of a promoter , given the possible states of the promoter , transition rates between states , and the rate of transcription resulting from each state .",
"In these papers , the authors apply their formalism to different promoter architectures .",
"Here , we generate a simpler model , in which we abstract away the individual transcription factor ( TF ) binding configurations , which would be numerous and poorly parametrized , and simply define states by whether an enhancer is looped to the promoter and activating transcription .",
"Since these models do not account for fluctuations that would contribute to extrinsic noise , for example fluctuations in TF or RNA polymerase levels , they can predict the dependence of intrinsic noise on enhancer arrangement .",
"To apply this model to our system , we used these parameters: γ , degradation rate of mRNA; p , production rate of mRNA; k , on rate for enhancer-promoter looping; l , off rate for enhancer-promoter looping .",
"We generated five models that represent different configurations of either one or two enhancers controlling a single promoter .",
"Key assumptions are that the parameters describing this system are independent of both the position of the enhancer relative to the promoter and the presence of a second enhancer controlling the same promoter .",
"In Model 1 , there is a single enhancer controlling one promoter .",
"There are two states , when the enhancer and promoter are not looped ( mRNA production rate of 0 ) , and when the enhancer and promoter are looped ( mRNA production rate of p ) .",
"In Model 2 ( OR model ) , there are two enhancers controlling one promoter , transcription is activated if either enhancer is looped , and both enhancers can’t be bound at the same time .",
"In Model 3 ( additive model ) , there are two enhancers controlling one promoter , transcription is activated if either enhancer is looped , and , if both enhancers are bound , transcription occurs at twice the rate of single enhancer looping states .",
"In Model 4 ( synergistic model ) , there are two enhancers controlling one promoter , transcription is activated if either enhancer is looped , and , if both enhancers are bound , transcription occurs at three times the rate of single enhancer looping states .",
"In Model 5 ( XOR model ) , there are two enhancers controlling one promoter , transcription is activated if either enhancer is looped , and , if both enhancers are bound , no transcription occurs .",
"Results of these models are shown in Figure 6—figure supplement 2 ."
]
] | [
"Shadow enhancers , groups of seemingly redundant enhancers , are found in a wide range of organisms and are critical for robust developmental patterning .",
"However , their mechanism of action is unknown .",
"We hypothesized that shadow enhancers drive consistent expression levels by buffering upstream noise through a separation of transcription factor ( TF ) inputs at the individual enhancers .",
"By measuring the transcriptional dynamics of several Kruppel shadow enhancer configurations in live Drosophila embryos , we showed that individual member enhancers act largely independently .",
"We found that TF fluctuations are an appreciable source of noise that the shadow enhancer pair can better buffer than duplicated enhancers .",
"The shadow enhancer pair is also uniquely able to maintain low levels of expression noise across a wide range of temperatures .",
"A stochastic model demonstrated the separation of TF inputs is sufficient to explain these findings .",
"Our results suggest the widespread use of shadow enhancers is partially due to their noise suppressing ability ."
] | [
"In all higher organisms , life begins with a single cell .",
"During the early stages of development , this single cell grows and divides multiple times to develop into the many different kinds of cells that make up an organism .",
"This is a highly regulated process during which cells receive instructions telling them what kind of cell to become .",
"These instructions are relayed via genes , and a particular combination of activated genes determines the cell’s fate .",
"Specific pieces of DNA , known as enhancers , act as switches that control when and where genes are active , while so-called shadow enhancers are found in groups and work together to turn on the same gene in a similar way .",
"Shadow enhancers are often active during the early stages of life to direct the formation of specialized cells in different parts of the body .",
"But so far , it has been unclear why it is beneficial to the divide the role of activating genes across several shadow enhancers rather than a single one .",
"Here , Waymack et al . examined shadow enhancers around a gene called Kruppel in embryos of the fruit fly Drosophila melanogaster .",
"Manipulating the shadow enhancers showed that they help to make gene activity more resistant to changes .",
"Factors such as fluctuations in temperature have different effects on each shadow enhancer .",
"Having several shadow enhancers working together ensures that , whatever happens , the right genes still get activated .",
"For genes like Kruppel , which are key for healthy development , the ability to withstand unexpected changes is a valuable evolutionary benefit .",
"The study of Waymack et al . reveals why shadow enhancers are involved in the regulation of many genes , which may help to better understand developmental defects .",
"Many conditions caused by such defects are influenced by both genetics and the environment .",
"Genetic illnesses can vary in severity , which may be related to the roles of shadow enhancers .",
"As such , studying shadow enhancers could lead to new approaches for treating genetic diseases ."
] | 2020 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | Designed α-sheet peptides inhibit amyloid formation by targeting toxic oligomers | elife-01681-v1 | [
[
"There are now over 40 different human amyloid diseases , each linked to the buildup of a specific precursor protein or peptide ( Chiti and Dobson , 2009 ) .",
"These diseases involve the conversion of a protein from its soluble native state into insoluble amyloid fibrils , or , in the case of peptides , the conversion from a soluble , loosely structured form to fibrils .",
"Given that many different sequences can form amyloid fibrils of similar architecture , there may be some common structural features of the prefibrillar amyloidogenic intermediates .",
"X-ray fiber diffraction indicates that the insoluble , mature amyloid fibrils are composed of cross β-sheet structure ( Jahn et al . , 2010; Eisenberg and Jucker , 2012 ) .",
"Therefore , it is widely held that the formation of amyloid fibrils involves a transition to β-sheet structure in the amyloidogenic intermediate .",
"However , the mechanism of self-assembly at the atomic level remains elusive .",
"Another feature of these diseases is that soluble oligomeric intermediates , not the insoluble well-ordered fibrils , are preferentially responsible for cellular toxicity ( Bucciantini et al . , 2002; Hardy and Selkoe , 2002; Tomic et al . , 2009; Xue et al . , 2009 ) .",
"Similarly , the soluble oligomeric forms of the prion protein are the most infectious per unit protein ( Silveira et al . , 2005 ) .",
"As such , fibrils may be protective , at least up to a point , as their breakdown to smaller aggregates yields greater toxicity and infectivity .",
"The discovery of a compound that promotes inclusion formation while reducing toxicity and cellular pathology supports this hypothesis ( Bodner et al . , 2006 ) .",
"In a similar vein , Jiang et al . ( 2013 ) demonstrated that binding the fibrillar state of Aβ could reduce toxicity presumably by shifting the equilibrium from oligomer to fibril .",
"Research on the soluble oligomers has become critically important since there is a consensus that the soluble oligomer species is more toxic than mature fibrils ( Chiti and Dobson , 2006; Tomic et al . , 2009; Xue et al . , 2009 ) and , while nontoxic , the fibrils are a reservoir for toxic oligomeric species ( Shahnawaz and Soto , 2012 ) .",
"In fact , structural similarities between soluble oligomers from a range of unrelated proteins/peptides has been demonstrated by generation of an antibody that recognizes a common backbone structure ( Kayed et al . , 2003; Tomic et al . , 2009 ) .",
"Glabe and co-workers developed an antibody ( denoted A11 ) that is specific for soluble oligomeric intermediates derived from a variety of peptides and proteins , including Aβ ( 1-42 ) , α-synuclein , islet amyloid polypeptide , polyglutamine , lysozyme , human insulin , and a prion peptide ( Glabe and Kayed , 2006 ) .",
"The antibody does not , however , bind the corresponding insoluble fibrils ( cross-β structure ) or the natively folded precursors ( various structures ) .",
"Based on the specificity of the antibody for soluble oligomers with various sequences , it was proposed that the antibody might recognize a unique conformation of the backbone .",
"This antibody inhibits toxicity associated with the intermediates , implying a common mechanism of toxicity and offering hope for a broad-based therapeutic agent .",
"Some years ago we ‘discovered’ a novel secondary structure , which we call ‘α-sheet’ , that is populated during molecular dynamics ( MD ) simulations of a range of amyloid proteins ( and peptides ) with different structures and sequences under amyloidogenic conditions ( Figure 1A ) ( Armen et al . , 2004a , 2004b , 2005; Steward et al . , 2008 ) .",
"The position where the α-sheet forms along the sequence coincides with the most amyloidogenic regions of the proteins , as determined experimentally ( Armen et al . , 2004a ) .",
"Consequently , we proposed that α-sheet is a common structure involved in the early stages of protein aggregation ( Armen et al . , 2004a; Daggett , 2006 ) .",
"In the course of characterizing the structure observed by MD , we learned that α-sheet was first predicted by Pauling and Corey and called ‘polar pleated sheet’ .",
"However , they ruled the structure energetically unfavorable and concluded , correctly , that the β-sheet structure would be favored in normal proteins ( Pauling and Corey , 1951 ) .",
"An α-sheet is similar to a β-sheet , but instead of alternating main chain NH and C = O groups along the strands , an α-sheet has the NH groups aligned on one side and the carbonyls on the other .",
"As such , the α-sheet has a molecular dipole and a very different hydrogen-bonding pattern across the sheet compared to a β-sheet .",
"Interestingly , the main chain ( Φ , Ψ ) dihedral angles of the α-sheet alternate between the αL and αR conformations ( Daggett , 2006 ) ( Figure 1A ) .",
"Although locally helical , the alternating dihedral angles form an extended chain resulting in the carbonyl groups and amide groups aligning in a plane .",
"Such an arrangement creates uniform electrostatic faces to aid in the addition of further strands ( Armen et al . , 2004b ) .",
"Once a sheet is formed , a simple peptide plane flip could convert the α-sheet into a β-sheet and ultimately a mature fibril ( Daggett , 2006 ) .",
"α-sheet structure has been observed in a peptide crystal structure ( Di Blasio et al . , 1994 ) , and short stretches of α-strand are present in various proteins in the Protein Data Bank ( Daggett , 2006 ) , although extensive α-sheet formation has not been observed in native proteins . 10 . 7554/eLife . 01681 . 003Figure 1 . α-sheet conversion , conformational properties and peptide designs .",
"( A ) β- to α-sheet conversion of transthyretin ( as reported by Armen et al . , 2004a , 2004b ) .",
"The protein backbone is shown in cartoon representation with the region of interest ( residues 105–121 ) shown as sticks .",
"At 0 ns ( top left ) the residues of interest form a β-hairpin .",
"The dihedral angles for 1 ns of dynamics of these residues are found mainly in the β-region of the Ramachandran plot ( top left quadrant , lower left panel; increasing frequency of occupancy is shown from blue through red ) with several turn residues in the αR conformation ( bottom left quadrant ) .",
"After 30 ns ( top right ) the β-sheet has converted to an α-sheet .",
"The dihedral angles for 1 ns of dynamics of the same residues reveal that the majority of residues have moved from the β-region to the αL ( top right quadrant ) or αR region of the Ramachandran plot ( lower right panel ) .",
"( B ) Intrinsic residue propensities for L- and D-alanine were calculated from 100 ns of MD simulations of a GGXGG peptide system ( Beck et al . , 2008 ) ( D-alanine was simulated using the same protocol ) .",
"The backbone structure ( upper panels ) as well as the Ramachandran plot of the conformation of the alanine residue during the entire simulation , demonstrate the conformational preference for L-alanine to adopt the αR conformation and for D-alanine to favor the αL conformation ( lower panels ) .",
"( C ) Peptide designs reported in this study .",
"All designs are single turn hairpins , with the exception of α3 , which contains a cyclic peptide backbone resulting in two turns .",
"Hairpin peptides are N- and C-terminally acetylated and amidated , respectively , except for β , which had a free N-terminus .",
"D-amino acids are denoted by lower case and are underlined , and turn residues are colored red in the linear peptides and red and blue in the cyclic design . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 003 To investigate the role of α-sheet in amyloid formation , we computationally designed numerous small , stable α-hairpin peptides .",
"We reasoned that if α-sheet is the novel backbone structure in toxic oligomers , then α−sheet peptides designed to be complementary to the structure in the oligomer should bind to the toxic oligomer and inhibit amyloidosis .",
"Our designs began with a backbone template in an ideal α-sheet conformation .",
"We then investigated combinations of residues with high propensities to populate desired regions of conformational space using our Structural Library of Intrinsic Residue Propensities ( SLIRP ) , which is part of our Dynameomics project ( Beck et al . , 2008; van der Kamp et al . , 2010 ) .",
"Owing to the expected transient nature of the αL conformation , we stabilized the structure using D-amino acids , which essentially have inverted conformational propensities compared with their normal L-counterparts ( Figure 1B ) .",
"Peptides containing alternating D- and L-amino acids have previously been shown to form extended structures ( De Santis et al . , 1974; Heitz et al . , 1981 ) , and they populate similar conformational space as our MD-identified α-sheet sequences consisting solely of L-amino acids .",
"MD simulations ( Beck et al . , 2000–2014 ) were performed to assess the stability of the de novo designed amino acid sequences .",
"Sequences designed to adopt β-sheet and random coil conformations were included in our experiments as controls .",
"Several of the most promising peptide designs were selected for experimental characterization in two different amyloid systems: transthyretin ( TTR ) and beta amyloid 1-42 [Aβ ( 1−42 ) or Aβ for short] .",
"Both are associated with amyloid diseases , systemic in the case of TTR and Alzheimer's Disease in the case of Aβ , but they have completely different sequences and structures .",
"Aβ is a largely unstructured peptide fragment in aqueous solution , while TTR is a tetramer composed of immunoglobulin-like β-sandwich domains .",
"Both systems have well characterized aggregation profiles and aggregate under mild , non-denaturing conditions ( Quintas et al . , 1997; Foss et al . , 2005; Hopping et al . , 2013 ) .",
"Here we focus on five peptide designs ( β , rc , α1 , α2 , α3 ) ( Figure 1C ) .",
"β is the designed Trpzip 3 β−hairpin , but with Trp to Leu substitutions to improve spectroscopic properties ( Cochran et al . , 2001a , b ) .",
"The β-design was included as a negative control for TTR and a positive control for Aβ .",
"β-hairpins are known inhibitors of Aβ aggregation ( Yamin et al . , 2009; Cheng et al . , 2012 ) .",
"The rc peptide was designed to be an unstructured random coil to provide a negative control; it is a randomly scrambled version of the β-sheet sequence , β .",
"The remaining three peptides were designed to adopt α-sheet structure .",
"α1 and α2 are linear hairpins containing a sheet of alternating D- and L-amino acids .",
"α1 was designed to have high α-sheet propensity .",
"α2 is a derivative of α1 with modifications aimed to improve stability and the introduction of a Cys for coupling experiments .",
"α3 consists of a sheet of alternating D- and L-amino acids and two turns , creating a cyclic amide backbone .",
"Despite our best efforts we were unable to design a soluble , random coil control based on the α-sheet peptides , i . e . the same composition and length .",
"Shuffling of the amino acid sequences resulted in insoluble peptides that were unusable in the solution-phase assays , so we opted to use smaller but well-defined controls ."
],
[
"First we tested our peptides for anti-amyloidogenic activity in a fibrillization assay using transthyretin ( TTR ) .",
"Four of the five designed peptides ( α2 was sparingly soluble and therefore not tested in any solution-phase experiments ) were co-incubated with TTR ( in excess 20:1 , expressed relative to TTR monomer ) at pH 4 . 5 to trigger dissociation of the native tetramer followed by aggregation ( Quintas et al . , 1997; Foss et al . , 2005 ) .",
"Note that a 10:1 ratio ( and higher ) is common in these aggregation inhibition assays ( Frydman-Marom et al . , 2011; Hochdorffer et al . , 2011 ) .",
"The aggregation was monitored via binding of Congo red ( Figure 2A ) .",
"The percentage inhibition of aggregation was determined at 48 hr , after the aggregation stabilized .",
"At these concentrations , α1 and α3 resulted in 72 and 56% inhibition , respectively , relative to TTR alone at low pH . In contrast , the rc and β controls resulted in little to no inhibition .",
"The neutral pH tetrameric TTR control changed little over time .",
"Moreover , the designs in the absence of TTR did not bind Congo red and were indistinguishable from the buffer-only controls ( Figure 2—figure supplement 1 ) .",
"To ensure that the observed increase in Congo red binding reflected the formation of amyloid fibrils , we performed atomic force microscopy ( AFM ) ( Figure 2—figure supplement 2 ) . 10 . 7554/eLife . 01681 . 004Figure 2 . α-Sheet designs inhibit amyloid formation and selectively bind toxic species .",
"( A ) Peptide designs ( 800 μM ) were co-incubated at pH 4 . 5 with 40 μΜ TTR ( monomer ) at 37°C and aggregation was monitored by Congo red binding .",
"Error bars indicate the standard deviation .",
"( B ) Toxicity of the TTR solution after 24 hr pre-incubation at pH 4 . 5 against the human neuroblastoma cell line SH-SY5Y in a MTT metabolic viability assay .",
"( C ) ThT monitoring of 10 μM Aβ aggregation and inhibitory effects of 100 μM peptides present from the beginning of the aggregation at 37°C .",
"Inhibition values were taken at 12 hr due the decay in ThT fluorescence , particularly for uninhibited samples , which has been described elsewhere ( Yamin et al . , 2009 ) .",
"( D ) Aβ toxicity after 6 hr of aggregation , as probed using the MTT assay and the SH-SY5Y cell line .",
"All data represent average ± SD ( * indicates p<0 . 05 , determined using Student's t test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 00410 . 7554/eLife . 01681 . 005Figure 2—figure supplement 1 . Increase in Congo red binding is not due to peptide aggregation . Congo red binding of peptides incubated under identical aggregation conditions reveals that contributions to dye binding do not come from the designs . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 00510 . 7554/eLife . 01681 . 006Figure 2—figure supplement 2 . AFM spectroscopy reveals aggregation conditions ultimately result in fibrils . Congo red binds multiple soluble species in addition to fibrils , so presence of fibrils was confirmed visually .",
"Application of a TTR solution aggregated for 72 hr revealed the presence of small aggregates and fibrils .",
"Fibrils are indicated with an arrow .",
"Scale = 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 00610 . 7554/eLife . 01681 . 007Figure 2—figure supplement 3 . Increase in ThT fluorescence is not due to peptide aggregation . ThT fluorescence of peptides incubated under identical aggregation conditions indicate that the peptides do not contribute to the observed increase in fluorescence . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 00710 . 7554/eLife . 01681 . 008Figure 2—figure supplement 4 . AFM spectroscopy confirms an increase in fibrillar products . Application of an aggregated Aβ solution reveals the presences of aggregates and fibrils .",
"Fibrils are indicated with arrows .",
"Scale = 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 008 Since our aim is to target the toxic oligomer , we determined when the toxic oligomeric species was present during the course of aggregation .",
"The toxicity of TTR was assessed by monitoring cell viability using the MTT assay after treating SH-SY5Y neuroblastoma cells with TTR that had been allowed to aggregate for different periods of time at pH 4 . 5 .",
"Toxicity was apparent around 24 hr , and the results from this time point are shown in Figure 2B .",
"Under these conditions , the viability of the treated cells was reduced by approximately 20% , indicating that TTR was aggregating via the toxic pathway .",
"Addition of the controls , rc or β , to a 24-hr pre-incubated TTR sample had little to no effect on further aggregation , as detected with the Congo red assay .",
"However , when α1 and α3 were mixed with 24-hr pre-incubated , toxic TTR , they inhibited 81% and 77% , respectively , of the remaining TTR aggregation observed in the absence of inhibitor .",
"Similarly , we co-incubated our designs ( in 10:1 excess ) with Aβ at pH 7 . 4 and followed fibril formation with thioflavin T ( ThT ) fluorescence changes upon binding ( Figure 2C ) .",
"The presence of fibrils was confirmed by AFM spectroscopy ( Figure 2—figure supplement 3 ) .",
"As observed for TTR , both α1 and α3 inhibited the levels of fibril formed .",
"Again α1 was more efficient , inhibiting approximately 87% of fibril formation compared with 66% for α3 , when measured after the reaction stabilized at 22 hr .",
"The β-sheet design , β , also resulted in 62% inhibition of Aβ fibrillization , which began exerting its effect primarily between 0 and 6 hr .",
"In contrast , α1 became inhibitory after aggregation proceeded for approximately 6 hr .",
"The peptide designs alone did not alter the fluorescence of ThT , and , as with Congo red , they were indistinguishable from buffer–only controls over the time course of the assay ( Figure 2—figure supplement 4 ) .",
"Also note that the α-sheet designs are effective at lower concentrations and the efficacy increases at lower temperature; for example , a fourfold excess of α1 essentially completely abolishes Aβ aggregation at 25°C ( unpublished ) .",
"We report the 37°C results here , though , for comparison with TTR , which aggregates more slowly at 25°C .",
"Aβ toxicity was also assessed using the MTT assay and found to reduce the viability of treated SH-SY5Y cells to less than 50% after 6 hr incubation ( Figure 2D ) .",
"Addition of the α-sheet designs to a pre-aggregated ( 6 hr ) , toxic sample of Aβ showed essentially complete inhibition of 97% for α1 and 96% for α3 , compared with the extent of remaining aggregation observed in the absence of inhibitor .",
"Despite much effort over the last few years it has not proved possible to isolate and characterize toxic soluble oligomers .",
"So , to further probe which species our peptide designs are binding to , we immobilized the designs on agarose beads and applied solutions of either fresh or pre-incubated , toxic samples of Aβ and TTR .",
"Immobilization in this manner also allowed us to test the sparingly soluble α2 design by limiting self-aggregation .",
"The peptides were immobilized via their lysine residues on aldehyde-functionalized agarose beads .",
"Their ability to bind TTR or Aβ from solutions at various stages of aggregation was investigated using dot blot analysis of the eluents .",
"All three immobilized α-sheet designs ( α1 , α2 and α3 ) bound significantly more TTR from pre-incubated , toxic oligomer-containing TTR solutions ( pre-incubated at low pH for 24 hr ) than did the rc and β controls ( Figure 3A ) .",
"Note that while α2 does not perform as well as the other two designs ( Figure 3A ) , the binding of TTR by α2 relative to the β and rc controls is significant , as shown by the statistical analysis in Figure 3—figure supplement 1 .",
"The extent of TTR binding by the β and rc controls is the same as that of the column matrix alone , which does bind some TTR in the absence of immobilized peptide ( Figure 3—figure supplement 2 ) .",
"α1 , α2 and α3 also bound pre-aggregated , toxic Aβ preferentially , whereas β and rc did not ( Figure 3B , and see statistical analysis in Figure 3—figure supplement 3 ) .",
"In the case of the β design , it preferentially bound the fresh , monomeric Aβ solutions ( 0 hr ) over the toxic solutions ( 6 hr ) ( Figure 3C ) , indicating that inhibition was due to interactions with the ‘native’ form , not the toxic oligomer .",
"β-hairpins are known inhibitors of Aβ aggregation ( Yamin et al . , 2009 ) .",
"In contrast , α-sheet , as demonstrated with α1 , did not bind native , tetrameric TTR nor fresh , monomeric Aβ but instead preferentially bound species from the toxic , aggregated 6 hr samples ( Figure 3D ) .",
"Moreover , the α-sheet designs did not bind the fibrillar forms of Aβ or TTR acquired by allowing the aggregation reactions to continue for over 3 weeks , as illustrated with immobilized α1 ( Figure 3—figure supplement 4 ) . 10 . 7554/eLife . 01681 . 009Figure 3 . Immobilized designs bind toxic soluble oligomer from solution . Peptide designs were immobilized onto agarose beads and their ability to bind TTR or Aβ from solution at various stages of aggregation was probed using dot blot analysis .",
"The presence of TTR or Aβ in the initial flow through ( FT ) , sequential buffer washes ( W ) , and sequential guanidine hydrochloride elution steps ( E1–E2 and E3–E4 ) ( x-axes ) was detected by the integrated peak density of the dot blot analysis ( y-axes ) .",
"E1–W9 are within the linear range of the immunochemistry .",
"( A ) All three α-sheet designs , α1 , α2 and α3 more strongly bound species from the 24 hr pre-aggregated , toxic TTR solutions than did either control .",
"( B ) Similar results were observed with the α-sheet designs binding to toxic Aβ solutions pre-aggregated for 6 hr .",
"Despite the inhibitory effects seen with the β design in the Aβ fibrillization assay , little Aβ from a pre-aggregated solution bound to the immobilized β design .",
"( C ) Comparison of binding from a fresh ( 0 h ) , or pre-aggregated ( 6 hr ) , toxic Aβ solution .",
"β is the only design that preferentially bound fresh Aβ over the aggregated toxic form , indicating that the inhibition observed was due to interactions with monomeric Aβ .",
"( D ) In contrast to the β control , α1 preferentially bound the pre-aggregated , toxic form of Aβ compared with fresh monomeric Aβ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 00910 . 7554/eLife . 01681 . 010Figure 3—figure supplement 1 . Statistical analysis of data presented in Figure 3A . Relative peak density values from dot blot analyses are given as average values from three independent experiments , with standard deviations .",
"Statistical analysis for E1 , E2 and W8 were performed against the corresponding values for rc and β .",
"t tests that returned a value p≤0 . 05 are indicated with an asterisk ( red when tested against the corresponding value from rc , green when tested against the same value for β ) and were considered significant .",
"Values for E1 , E2 and W8 were also summed and standard deviations propagated ( bottom line ) , and the t test repeated against the corresponding values for rc and β .",
"These results indicate that α1 , α2 and α3 all bind species present in a toxic TTR solution to a greater extent than the random coil or β-sheet control peptides . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 01010 . 7554/eLife . 01681 . 011Figure 3—figure supplement 2 . Nonspecific binding of nonnative TTR to column matrix . While the column-binding assay is not quantitative , nor was it expected to be , we can estimate the confidence in the peak densities by seeing to what extent TTR binds to the column matrix in the absence of coupled peptides .",
"TTR aged for 24 hr was applied to agarose beads blocked only with Tris to establish the effect of the column matrix .",
"As can be seen , the extent of binding to the column matrix ( E1 and E2 ) is comparable to that observed with the rc and β controls .",
"Note also that aggregated TTR elutes earlier from columns blocked with Tris , presumably due to the increased hydrophobicity of TTR as it aggregates and the less favorable interactions with the more hydrophilic Tris matrix . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 01110 . 7554/eLife . 01681 . 012Figure 3—figure supplement 3 . Statistical analysis of data presented in Figure 3B . Relative peak density values from dot blot analyses are given as average values from three independent experiments , with standard deviations .",
"Statistical analysis for E1 , E2 and W8 were performed against the corresponding values for rc and β .",
"t-tests that returned a value p≤0 . 05 are indicated with an asterisk ( red when tested against the corresponding value from rc , green when tested against the same value for β ) and were considered significant .",
"Values for E1 , E2 and W8 were also summed and standard deviations propagated ( bottom line ) , and the t test repeated against the corresponding values for rc and β .",
"These results indicate that α1 , α2 and α3 all bind species present in a toxic Aβ solution to a greater extent than the random coil or β-sheet control peptides . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 01210 . 7554/eLife . 01681 . 013Figure 3—figure supplement 4 . Immobilized α-sheet designs do not bind fibrils . TTR ( red ) or Aβ ( black ) fibrils were applied to agarose beads coated with the α1 design .",
"The elution profile does not show any increase in elution when guanidine is applied to the column , indicating that there are no specific interactions between the fibrils and the immobilized designs . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 013 The instability of α-sheet structure in proteins and peptides containing solely L-amino acids leaves us with no established spectroscopic signatures with which to assess our structures .",
"We can , however , make and test predictions based on the unique conformational and electronic environments resulting from this structure .",
"Circular dichroism ( CD ) signals arise from the differential absorption of left- and right-hand polarized light by chiral molecules .",
"For proteins , the orientation of individual amide bonds is responsible for the resulting CD spectra in the far-UV region .",
"Mirror image structures , formed by replacing whole L-amino acid sequences with D-amino acids , such as gramicidin A , produce mirror-image CD-spectra ( Koeppe et al . , 1992 ) .",
"We anticipate that the α-sheet structure would be effectively invisible due to near equal absorbance of both left and right polarized light , with any residual signal emanating from the turns and terminal residues .",
"The electrostatic interactions between aligned amide groups in an α-sheet ( Armen et al . , 2004a; Daggett , 2006 ) are expected to give rise to strong Fourier-transform infrared ( FTIR ) signals .",
"Torii recently performed density functional theory calculations of three slightly different orientations of α-sheet structures ( Torii , 2008 ) .",
"All models featured a strong high-frequency absorbance in the 1675–1680 cm−1 region , with a weaker band around 1640 cm−1 , which appears to be distinct from α-helix ( ∼1650–1658 cm−1 ) , β-sheet ( ∼1620–1640 cm−1 ) and turn structures ( ∼1670 cm−1 ) ( Barth and Zscherp , 2002 ) .",
"Nuclear magnetic resonance ( NMR ) spectroscopy can provide site-specific conformational information .",
"Owing to the unique alignment of the amide groups in an α-sheet , we expect to see strong sequential dNN Nuclear Overhauser Effect ( NOE ) crosspeaks along the backbone since the NH groups are aligned on one side of the chain instead of alternating between opposite faces as in β-sheet structure .",
"Furthermore , we would not expect to observe the long-range dNN or the strong sequential dαN NOEs characteristic of β-sheets .",
"Thus , CD , FTIR and NMR studies were performed to assess the structures of our peptide designs .",
"The CD spectrum of rc shows a random coil signal with a strong negative absorption around 200 nm ( Figure 4A ) .",
"In its FTIR spectrum we observe multiple small absorbance peaks not corresponding to a predominance of any specific structure ( Figure 4B ) .",
"Our β-sheet control is a modified version of the Trpzip peptide , which forms a stable β-hairpin in solution .",
"In agreement with previous structural work ( Cochran et al . , 2001a ) , β exhibits a CD spectrum reflective of β-structure and random coil , with a minimum near 220 nm and another near 200 nm ( Figure 4A ) .",
"The substitution of Leu for Trp removed the strong exciton-coupling between the Trp residues observed in the parent peptide thereby ‘exposing’ the β-structure CD signal .",
"The β-sheet structure was confirmed by FTIR through its strong absorbance at 1632 cm−1 ( Figure 4B ) .",
"The CD spectra for α1 , α2 and α3 are essentially featureless , as expected for the cancellation of αL and αR signals , except for a slight dip around 200 nm consistent with turn formation ( Figure 4A ) .",
"α1 and α2 exhibited the predicted FTIR α-sheet bands at 1640 and 1675–1680 cm−1 ( Figure 4B ) .",
"These bands were less pronounced in α3 .",
"Cyclization of the amide backbone of α3 through a non-optimal turn may have caused distortion of the structure , as has been reported in other peptide systems ( Clark et al . , 2005 ) , and suggests an area for improvement in future designs .",
"Altogether , these results prove that the designed α-sheet peptides do not form α-helix or β-sheet structure , and that the random coil content is not large ( compare against the scale of the rc control CD spectrum ) .",
"Thus , these results are consistent with and supportive of the designed α-sheet structure . 10 . 7554/eLife . 01681 . 014Figure 4 . Designed peptides display unique spectroscopic signatures expected for α-sheet .",
"( A ) CD spectra for peptide designs reveal a random coil structure for rc and β-structure spectrum with a bit of random coil for β .",
"In contrast , α1 , α2 and α3 have largely featureless CD spectra with some random coil content expected to arise from the turns and tail residues .",
"Note the different scales for the y-axes .",
"All spectra are presented as molar ellipticity , highlighting the difference in intensity of the random coil component for each design compared with the rc spectrum .",
"( B ) FTIR spectra of the peptide designs , displayed as both absorbance ( black line ) and the second derivative ( colored line ) , correlate well with the CD spectra .",
"The β design shows a strong signal at 1632 cm−1 , as expected for β-structure .",
"The α-sheet designs have signals near 1640 and 1675–1680 cm−1 and the absorption is more intense for α1 and α2 .",
"( C ) Fingerprint ( top ) and NH region ( bottom ) of the 1H NOESY spectra for α1 .",
"Sequential assignments are shown in red and multiple sequential NOEs are observed and labeled .",
"( D ) Fingerprint ( top ) and NH region ( bottom ) of the 1H NOESY spectra for α3 .",
"The NH region reflects the predominance of NH–NH interactions and lack of other main-chain interactions characteristic of the common secondary structures .",
"Mapping backbone NOEs on computational models as green bars ( E , α1 and F , α3 ) reveal in-plane alignment of the peptide groups along the majority of the sheet .",
"NOEs in the turn regions determined whether the carbonyl or amide hydrogens pointed up in the structures as oriented in the figure ( N-terminus top left ) .",
"Cα , C , N , H and O atoms are shown in gray , gray , blue , white and red , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 01410 . 7554/eLife . 01681 . 015Figure 4—figure supplement 1 . Distances corresponding to dNN NOEs calculated from MD simulations . r−6 weighted distances calculated from MD simulations of α1 in α-sheet and in β-sheet conformation .",
"α1 is not stable as a β-sheet and did not retain the structure well even in these short simulations .",
"Consequently a β-hairpin of the same size within a protein was also used as a control for expected H–H distances within a dynamic but stable hairpin .",
"Specifically , residues 105–124 of the antiparallel β-hairpin neuronal nitric oxide synthase ( PDB:1QAU ) was used from a 50 ns simulation at 298 K . Triplicate simulations of the isolated hairpins were performed at 298 K for 20 ns .",
"r−6 intensities , which should be proportional to the NOE intensity , were calculated step by step from the distance then averaged across the pooled simulations and time points and converted back to distance ( <r−6 >−1/6 ) .",
"Residue 117 of 1QAU is proline , preventing measurement for itself and the preceding residue .",
"The lower values observed for α1 in a β-hairpin conformation , similar to those of the anti-parallel β-hairpin in 1QAU suggest to us that our α1 does not adopt β-structure .",
"Sheet regions are highlighted . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 01510 . 7554/eLife . 01681 . 016Figure 4—figure supplement 2 . Distances corresponding to intraresidue dαN NOEs calculated from MD simulations . r−6 weighted distances calculated from MD simulations of α1 in α-sheet and in β-sheet conformation .",
"r−6 intensities , which should be proportional to the NOE intensity , were calculated for each structure then averaged across the pooled simulations and time points and converted back to distance ( <r−6 >−1/6 ) .",
"Residue 117 of 1QAU is proline , preventing measurement for itself .",
"Residues 113 and 115 of 1QAU , and residue 14 of α1 are glycines , resulting in two measurements for Hαi–HNi .",
"Sheet regions are highlighted . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 01610 . 7554/eLife . 01681 . 017Figure 4—figure supplement 3 . Distances corresponding to sequential dαN ( i–i+1 ) NOEs calculated from MD simulations . r−6 intensities , which should be proportional to the NOE intensity , were calculated for each structure and then averaged across the pooled simulations and time points and converted back to distance ( <r−6 >−1/6 ) .",
"Residue 117 of 1QAU is proline , preventing measurement for the preceding residue .",
"Residues 113 and 115 of 1QAU , and residue 14 of α1 are glycines , resulting in two measurements for Hαi–HNi+1 .",
"Sheet regions are highlighted . DOI: http://dx . doi . org/10 . 7554/eLife . 01681 . 017 We performed further structural studies utilizing homonuclear NMR spectroscopy .",
"Multiple sequential dNN NOEs were observed along the backbones of both α1 and α3 ( α2 was not soluble enough for NMR ) ( Figure 4C , D ) .",
"No long-range dNN or dαN NOEs indicative of α-helical or β-sheet structure ( Wüthrich , 1986 ) were observed .",
"The NH⋯NH NOEs are mapped onto structural models of α1 and α3 ( Figure 4E , F ) , highlighting the stretches of α-strand structure in both designs .",
"As mentioned above , strong sequential dNN NOEs are expected for α-sheet , but not for β-sheet , and strong sequential dαN NOEs are expected for β-sheet , but not for α-sheet .",
"To test this idea we calculated the ensemble-weighted NH–NH , intraresidue Hα-NH and sequential Hα-NH distances in MD simulations for α1 in an α-sheet conformation and a β-sheet conformation , as well as a natural β-hairpin within a protein ( Figure 4—figure supplements 1–3 ) .",
"The calculated NH–NH distances in the strands are much shorter in the α-sheet , consistent with the strong dNN NOEs observed for both α1 and α3 , and the observed breaks in the patterns ( Figure 4E , F ) reveal vulnerabilities in the structures and provide direction for improved designs .",
"The bulk of the observed intraresidue and sequential dαN NOE intensities in Figure 4C , D are of similar magnitude ( 12/21 for α1 ) or the intraresidue NOE is stronger ( 6/21 for α1 ) , consistent with α-sheet structure ( Figure 4—figure supplements 2 , 3 ) .",
"There are , however , three residues with strong sequential dαN NOEs in α1 but all three of these are involved in dNN NOEs , which is inconsistent with β-structure .",
"We observed no long-range side chain-side chain NOEs despite increasing the mixing time of the NOESY experiments up to 400 ms , perhaps due to residual dynamics in the peptide .",
"The lack of these distance restraints prevented the generation of a well-converged solution structure; however , in support of the CD and FTIR spectroscopic data , the NMR data are consistent with α-sheet secondary structure and inconsistent with α-helix , random coil , and β-sheet structures .",
"Ten years ago a common conformation was demonstrated among soluble oligomeric species from amyloid proteins/peptides of diverse sequence and structures that cross react with the A11 antibody ( Kayed et al . , 2003 ) .",
"Also at that time we identified a novel target structure , α-sheet , through MD simulations and proposed that it is the defining feature of the toxic oligomeric species ( Armen et al . , 2004a; Daggett , 2006 ) .",
"Unfortunately , the precise structure of this toxic intermediate remains elusive , and it has become clear that the oligomers are conformationally heterogeneous ( Carulla et al . , 2009; Bitan et al . , 2005 , and references therein ) .",
"Here we have taken a different approach to probe these soluble oligomeric species through experimental test of our α-sheet hypothesis through peptides designed to be complementary to the proposed α-sheet structure in the oligomeric intermediates .",
"Three of these computationally derived designs were synthesized and characterized experimentally , and they do indeed appear to adopt α-sheet structure ( as shown by FTIR , CD and NMR ) .",
"The two soluble α-sheet designs ( α1 and α3 ) inhibited both TTR and Aβ aggregation in solution .",
"In addition , when immobilized to agarose beads , all three α-sheet designs bound species from the toxic TTR and Aβ preparations preferentially over the nontoxic fresh samples .",
"In contrast , the β control formed a β-hairpin , as supported by CD and FTIR , and it preferentially bound the monomeric form of Aβ and it did not react with TTR .",
"When mature fibrils were applied to the immobilized α−sheet designs , the fibrils did not bind and appeared to have no affinity for the α-sheet structure .",
"While these α-sheet peptides were not designed against a specific protein target , we observed inhibition of aggregation in two very different amyloid systems .",
"These results support our hypothesis that α-sheet structure is involved in the toxic oligomeric stage of aggregation , and they provide a reference to determine spectroscopic signatures that can now be used to investigate the structural changes amyloid proteins undergo during amyloidosis .",
"In addition , the α-sheet designs may aid in capture of the elusive toxic oligomeric species for in-depth characterization .",
"Having demonstrated that the α-sheet structure may constitute a broad-based inhibitor of amyloidosis , our α-sheet designs introduce a novel class of amyloid inhibitors that target the toxic soluble oligomeric state of different amyloidogenic peptides and proteins ."
],
[
"The α-sheet peptides were designed in silico using our database of amyloid protein MD simulations to determine preferred backbone geometries .",
"Our SLIRP database ( Beck et al . , 2008; van der Kamp et al . , 2010 ) was used to select residues with high propensity to adopt the desired structure .",
"MD simulations were performed to assess both turn and α-sheet stability .",
"Ideal α-sheet and β-sheet templates were created and sequences were chosen based on their intrinsic conformational preferences and to consist of a mix of polar and nonpolar amino acids to maintain good inter-strand interactions and solubility .",
"Intrinsic conformational propensities of all 20 L-amino acids in a GGXGG peptide were determined by extensive molecular dynamics ( MD ) simulations ( Beck et al . , 2008 ) .",
"D-amino acid propensities were determined in a similar manner ( manuscript in preparation ) .",
"MD simulations were then performed to assess the stability of our designs .",
"Multiple short simulations were performed , at least 3 × 20 ns , for each peptide at 25°C using our in-house MD package in lucem molecular mechanics ( ilmm ) ( Beck et al . , 2000–2014 ) , with the Levitt et al . ( 1995 ) all atom force field and the F3C water model ( Levitt et al . , 1997 ) .",
"Standard simulation protocols were followed ( Beck and Daggett , 2004 ) .",
"α-Sheet stability was assessed by monitoring both the secondary structure and the turn structure , based on hydrogen bonding over the duration of the simulations .",
"Results were expressed as the fraction of simulation time the atoms were within hydrogen bonding distance .",
"Cα RMSD was used qualitatively to monitor backbone deviation from the ideal hairpin structure in conjunction with the hydrogen bond scoring function to determine promising designs .",
"We took an iterative approach , with the results of the analyses used to modify and refine sequences for further simulation and evaluation .",
"We chose several sequences from a pool of well-behaved simulations for experimental evaluation .",
"High scoring designs were synthesized and their inhibitory effects were determined .",
"Aliqouts of transthyretin ( TTR ) ( 496-11; Lee Biosolutions , St . Louis MO ) were made from a 1 mg/ml solution 20 mM ammonium carbonate , pH 8 .",
"Aliquots were lyophilized and stored at −18°C .",
"Prior to use , TTR was dissolved to 80 μM ( monomer ) in acetate buffer ( 50 mM potassium acetate , 100 mM potassium chloride pH 4 . 5 ) and sonicated for 10 min .",
"The stock solution was centrifuged before use .",
"Peptide designs were added to stock TTR to a final TTR concentration of 40 μM ( monomer ) in acetate buffer ( pH 4 . 5 ) in 500 μl microcentrifuge tubes .",
", which were incubated at 37°C .",
"Periodically , samples were collected from the TTR:peptide mixture by briefly centrifuging , and then carefully pipetting the solution up and down prior to withdrawing a 10 μl sample and diluting it in 190 μl of 10 μM Congo red in an individual well of a 96-well assay plate .",
"Each measurement was performed in triplicate .",
"Absorbance measurements were taken at 477 and 540 nm .",
"Relative Congo red binding was determined using the method of Klunk et al . ( 1989 ) via the following relationship: rCb = ( Abs540/25 , 295 ) - ( Abs477/46 , 306 ) .",
"All datapoints were normalized to the value recorded for TTR alone pH 4 . 5 at 48 hr .",
"Aβ ( 1-42 ) ( AMYD-002; CPC Scientific , Sunnyvale CA ) was stored as 2 mg/ml stock in hexafluoroisopropanol ( HFIP ) at −18 °C .",
"Prior to use , the stock solution was thawed , an aliquot taken and the HFIP was removed under a gentle stream of air .",
"A 1 mg/ml stock solution of Aβ was made in 5 mM NaOH and sonicated for 5–10 min .",
"The stock was filtered through a 0 . 22 μm cellulose filter ( Costar Spin-X; Corning Inc , NY ) .",
"The concentration of stock Aβ was determined by first diluting the stock 1:50 in 5 mM NaOH then taking the absorbance at 220 nm ( ε220 = 50 , 000 cm−1 M−1 ) .",
"Aliquots of the NaOH stock were placed in separate wells of a 96-well black-walled fluorescence plate ( Nunc ) and immediately diluted in PBS ( 11 mM phosphate ) containing 20 μM Thioflavin T ( ThT ) to give 150 μl of 10 μM Aβ at pH 7 . 5 .",
"Peptide inhibitors were added directly to 10 μM Aβ samples from concentrated aqueous stocks .",
"Covered plates were incubated at 37°C and were periodically removed for fluorescence measurements .",
"ThT fluorescence was measured at λex = 450 nm and λem = 480 nm using a Tecan Safire2 plate reader .",
"Peptide designs were immobilized to the Pierce Amino Link resin following the manufacturer's instructions .",
"Peptides were immobilized in a volume of 200 μl of coupling buffer ( 100 mM sodium phosphate , 150 mM sodium chloride , pH 7 . 2 ) and 2 μl cyanoborohydride solution ( 5 M sodium cyanoborohydride in 1 M NaOH ) at a concentration of 358 μM overnight at 4°C .",
"Any residual active sites were blocked with 400 μl quenching buffer ( 1 M tris hydrochloride , pH 7 . 4 ) and 4 μl cyanoborohydride solution for 4 hr at 25°C .",
"Binding experiments were performed from 200 μl amyloid solution ( 5 μM Aβ or 20 μM TTR ( monomer ) diluted to the desired concentration in coupling buffer ) , which was allowed to bind to the peptide-bound agarose beads for 2 hr at 25°C .",
"The solution was then collected by centrifugation ( flow through , FT ) .",
"The beads were resuspended in 300 μl coupling buffer , vortexed to obtain a uniform slurry , and the solution was again collected by centrifugation ( wash 1 , W1 ) .",
"The wash step was performed an additional five times ( W2–W6 ) .",
"One final wash step was performed but after resuspending the resin , the solution was allowed to sit for 5 min before centrifugation ( W7 ) .",
"The resin was next resuspended in 100 μl 2 M guanidine hydrochloride , incubated for 5 min at room temperature , then collected as before .",
"This was performed twice ( E1–E2 ) .",
"The resin was washed again with 300 μl coupling buffer ( W8 ) followed by two elution steps with 6 M guanidine hydrochloride ( E3–E4 ) .",
"One final wash step was performed with 300 μl coupling buffer ( W9 ) .",
"All collected eluents were analyzed by applying triplicate 1 μl spots to nitrocellulose , and then performing standard dot blot analysis as described by Kayed et al . ( 9 ) with an anti-TTR ( sc-13098 , Santa Cruz Biotechnology , Santa Cruz , CA ) or anti-Aβ ( ab39377; Abcam Inc , Cambridge , MA ) primary antibody diluted 1:1000 in 5% or 10% nonfat milk , respectively .",
"The toxicity of aggregates was tested against the human neuroblastoma cell line SH-SY5Y in an MTT cell viability assay .",
"The human neuroblastoma cell line SH-SY5Y ( CRL-226; American Type Culture Collection ) was grown in 75 cm2 flasks in 1:1 DMEM/F12 ( CellGro , Manassas , VA ) supplemented with 10% FBS and 50 units/ml penicillin/50 μg/ml streptomycin ( complete media ) , and incubated at 37°C in humidified 5% CO2 environment .",
"Cells were routinely passaged when they reached 90% confluence by addition of trypsin ( Gibco ) and replated at a ratio of 1:10 in complete media .",
"Cells were plated to a density of 25 , 000 cells/well in a 96-well plate ( 100 μl/well ) and allowed to attach overnight .",
"The cell assay was performed as described by Reixach et al . ( 2004 ) .",
"Far UV CD spectra were recorded on an Aviv model 420 spectrometer ( Aviv Biomedical ) over 200–260 nm in a 1 mm quartz cuvette .",
"All samples were prepared at 100 μM , with the exception of the sparingly soluble design , α2 which was prepared at 35 μM .",
"All samples were prepared in 50 mM phosphate , 100 mM NaCl buffer , pH 5 . 8 , and were recorded at 25°C with a resolution of 0 . 5 nm , a scan speed of 20 nm/min , and a 2 nm bandwidth .",
"Average values from three scans were plotted using the Origin 8 software ( Originlab , Northhampton , MA ) .",
"All spectra were smoothed using the Savitzky-Golay method with 5–12 points/window , and polynomial order 2 .",
"IR spectra were obtained using a Perkin–Elmer Spectrum 100 instrument equipped with a diamond attenuated total reflectance sample unit and an MCT detector .",
"Peptide samples were pelleted and re-suspended as a 1–2 µl slurry .",
"The slurry was applied to the diamond and dried to a film over a few minutes while following the disappearance of the broad liquid water band at ∼1636 cm−1 and the appearance of the protein amide I and amide II bands .",
"The spectra were background-subtracted and comprised of 64 accumulations ( 4 cm−1 resolution; 1 cm/s OPD velocity; strong apodization ) .",
"Spectra shown here were recorded as soon as successively collected spectra ( each recorded over 80 s ) stabilized , indicating little further evaporation of liquid water .",
"This approach was taken to eliminate spectral contributions from free liquid water without desiccating the peptide film any more than necessary .",
"Second derivative spectra were calculated using the instrument software and 13 data points .",
"Peptides were prepared in 50 mM potassium phosphate buffer containing 100 mM KCl pH 5 . 8 .",
"All NMR experiments were performed on Bruker Avance 600 and/or 500 MHz spectrometers equipped with cryogenic triple resonance probes .",
"The sample temperature was kept at 25°C .",
"4 , 4-dimethyl-4-silapentane-1-sulfonic acid ( DSS ) was used for proton chemical shift referencing whereas indirect referencing was used for carbon and nitrogen .",
"The resonance assignments for peptides were carried out using 1H-1H TOCSY and [1H-1H] NOESY spectra recorded in 90% H2O and 10% 2H2O .",
"The assignments thus obtained are translated onto the natural abundance 1H-15N HSQC and 1H-13C HSQC spectra .",
"All spectra were processed with Topspin3 . 0 ( Bruker ) and analyzed using CARA ( http://cara . nmr . ch/doku . php ) or Sparky ( http://www . cgl . ucsf . edu/home/sparky/ ) ; figures were made using CARA .",
"AFM was performed with a Dimension 3100 atomic force microscope using tapping mode and silicon tips ( FESP; Bruker; Camarillo , CA ) .",
"10 μl of 10 μM Aβ ( 1-42 ) or 40 μM TTR were applied directly to freshly cleaved mica and incubated for 10 min . 50 μl of water was added then removed by capillary action with a lint-free lab wipe .",
"A further 50 μl of water was added and incubated for 5 min before removal .",
"The mica surface was then allowed to dry under ambient conditions prior to imaging ."
]
] | [
"Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation , but the amorphous nature of this oligomer prevents its structural characterization by experiment .",
"Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common , nonstandard secondary structure , called α-sheet .",
"Here we report the experimental characterization of peptides designed to be complementary to the α-sheet conformation observed in the simulations .",
"We demonstrate inhibition of aggregation in two different amyloid systems , β-amyloid peptide ( Aβ ) and transthyretin , by these designed α-sheet peptides .",
"When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated , nontoxic species or mature fibrils .",
"The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures , supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics ."
] | [
"The build up of very thin fibres called amyloid fibrils is known to lead to more than 40 different human diseases , including Parkinson’s disease and rheumatoid arthritis .",
"These diseases involve soluble proteins or peptides joining other proteins or peptides to form the fibrils , which are not soluble .",
"However , the damage is done by the time the fibrils form because soluble intermediate structures formed by the proteins and peptides are toxic .",
"The development of methods that can detect these toxic intermediate structures could lead to earlier interventions before significant damage .",
"Amyloid fibrils are known to have a beta-sheet structure that is found in many protein systems .",
"In 2004 , based on computer simulations , researchers predicted that proteins and peptides that go on to form amyloid fibrils would pass through a related but less stable structure called an alpha-sheet , and that this structure would be toxic .",
"Now Hopping et al . , including some of the researchers involved in the 2004 work , have confirmed that the alpha-sheet structure is indeed involved in the formation of amyloid fibrils .",
"To do this Hopping et al . designed peptides with alpha-sheet structures that could bind to the alpha-sheet structures predicted by their simulations .",
"When these complementary designed peptides were added to a solution of peptide that causes Alzheimer’s Disease , or a protein that causes systemic amyloid disease , the designed peptides bound the toxic peptides or proteins and prevented the formation of fibrils .",
"The results of Hopping et al . suggest that designed alpha-sheet compounds might be able to capture peptides and proteins that are implicated in a wide variety of amyloid diseases , independent of their composition and native structure , by targeting the intermediate alpha-sheet structure .",
"Future challenges include showing that most proteins and peptides pass through this intermediate structure as they form fibrils , and improving the sensitivity of the binding in the hope of developing diagnostics for amyloid diseases ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"structural biology and molecular biophysics"
] | Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations | elife-51700-v2 | [
[
"The 13 phospholipase C ( PLC ) isozymes expressed in humans preferentially hydrolyze the membrane phospholipid phosphatidylinositol 4 , 5-bisphosphate ( PIP2 ) to generate the second messengers diacylglycerol and inositol 1 , 4 , 5-trisphosphate ( IP3 ) ( Harden and Sondek , 2006; Kadamur and Ross , 2013 ) .",
"Diacylglycerol is retained within membranes where it recruits and activates numerous proteins including conventional isoforms of protein kinase C . In contrast , IP3 diffuses throughout the cytosol where it binds to IP3 receptors embedded in endoplasmic reticulum leading to mobilization of sequestered calcium .",
"PLC-mediated depletion of PIP2 also modulates the activities of several ion channels and proteins with phosphoinositide-binding domains .",
"Thus , the PLCs coordinate fluctuations in PIP2 levels and the bifurcating signaling pathways emanating from PIP2 hydrolysis to regulate numerous cellular processes , including fertilization and embryogenesis , cell proliferation and differentiation , as well as various types of cell migration ( Asokan et al . , 2014; de Gorter et al . , 2007; Jones et al . , 2005; Mouneimne et al . , 2004 ) .",
"The two PLC-γ isozymes , PLC-γ1 and PLC-γ2 , are unique among the PLCs in that they are directly activated by tyrosine phosphorylation .",
"PLC-γ1 and PLC-γ2 are phosphorylated on equivalent sites , Tyr783 and Tyr759 , respectively , and this phosphorylation is typically required to stimulate phospholipase activity .",
"Several classes of tyrosine kinases phosphorylate and activate the PLC-γ isozymes .",
"These include a large number of receptor tyrosine kinases ( RTKs ) including Trk receptors ( Minichiello et al . , 2002; Vetter et al . , 1991 ) and many growth factor receptors such as epidermal growth factor receptor ( EGFR ) ( Kim et al . , 1991; Nishibe et al . , 1990; Peters et al . , 1992; Takahashi et al . , 2001; Wahl et al . , 1989 ) .",
"A second large group is soluble tyrosine kinases coupled to immune receptors and includes members of the Src , Syk , and Tec families ( Law et al . , 1996; Nakanishi et al . , 1993; Schaeffer et al . , 1999 ) .",
"In this way , the PLC-γ isozymes are poised to transduce signals initiated by a wide variety of extracellular stimuli .",
"The regulated , phosphorylation-dependent activation of PLC-γ1 and -γ2 controls numerous aspects of biology including proper development of the vascular , neuronal , and immune systems during embryonic development , adaptive immune responses , neuronal transmission , bone homeostasis , chemotaxis , and platelet aggregation ( Yang et al . , 2012 ) .",
"The PLC-γ isozymes have also recently emerged as drivers of several human diseases ( Koss et al . , 2014 ) .",
"Notably , genome-wide sequencing studies have demonstrated that PLC-γ1 and PLC-γ2 are frequently and recurrently mutated in several leukemias and lymphomas .",
"In fact , PLC-γ1 is the most frequently ( ~40% ) mutated gene in adult T cell leukemia/lymphoma , where mutant forms of the isozyme are presumed to drive oncogenesis through enhanced phospholipase activity coupled to elevated NFAT- and NF-κB-dependent transcription ( Kataoka et al . , 2015; Vaqué et al . , 2014 ) .",
"Moreover , activating mutations in PLC-γ2 arise with high frequency ( ~30% ) in patients with B cell leukemias treated with ibrutinib ( Woyach et al . , 2014 ) , a covalent inhibitor of Bruton’s tyrosine kinase ( BTK ) .",
"PLC-γ2 is a major BTK substrate and mutations in PLC-γ2 likely function as escape mutations , reactivating pathways controlling cell survival and proliferation that are usually rendered quiescent by ibrutinib treatment .",
"Mutated , and presumably active , forms of the PLC-γ isozymes are also found in patients with angiosarcomas ( Behjati et al . , 2014 ) , and several disorders associated with dysregulated immune responses ( Ombrello et al . , 2012; Zhou et al . , 2012 ) , inflammatory bowel disease ( de Lange et al . , 2017 ) , and familial steroid-sensitive nephrotic syndrome ( Gbadegesin et al . , 2015; Parker et al . , 2019 ) .",
"A naturally occurring variant of PLC-γ2 that is moderately active is strongly associated with protection from late-onset Alzheimer’s disease ( Magno et al . , 2019; Sims et al . , 2017 ) and highlights the notion that aberrant PLC activity can be either deleterious or beneficial depending on the context .",
"However , for the vast majority of mutant forms of PLC-γ1 and PLC-γ2 , an increase in lipase activity has not been demonstrated directly .",
"While the PLC-γ isozymes control essential aspects of both normal and disease-associated cellular processes , the molecular mechanisms controlling these enzymes remain elusive .",
"Broadly , phosphorylation-dependent activation of PLC-γ1 and PLC-γ2 is controlled by an array of regulatory domains unique to these isozymes .",
"In particular , the regulatory array harbors the obligatory sites of tyrosine phosphorylation ( Gresset et al . , 2010; Kim et al . , 1991; Nishibe et al . , 1990 ) , and also includes an SH2 domain ( nSH2 ) required for tyrosine kinase binding ( Bae et al . , 2009 ) .",
"The array also mediates basal autoinhibition of phospholipase activity , since removal or mutation of a second SH2 domain ( cSH2 ) within the array results in robust and constitutive activation of PLC-γ isozymes in vitro and in cells ( Gresset et al . , 2010; Hajicek et al . , 2013 ) .",
"The regulatory array is completed by a split PH ( sPH ) domain and an SH3 domain , which modulate PLC activity by scaffolding numerous signaling and adaptor proteins ( Sherman et al . , 2016; Walliser et al . , 2008 ) .",
"Although these general aspects of the regulation of PLC-γ1 and PLC-γ2 are well-documented , several fundamental questions remain unresolved .",
"For example , the other half of the autoinhibitory interface formed by the cSH2 domain has not been identified , and how this domain enforces autoinhibition of phospholipase activity is unknown .",
"Possibilities include physical occlusion of the lipase active site , steric hindrance that prevents the enzyme from engaging membranes , or a combination of both as is observed for the PLC-β isozymes ( Hicks et al . , 2008 ) .",
"Although the cSH2 domain is presumed to be the primary arbiter of autoinhibition , several reports have also implicated the sPH domain in maintaining an autoinhibited state ( Everett et al . , 2011; Gresset et al . , 2010 ) .",
"How this domain might contribute to autoinhibition is unknown .",
"Similarly unclear is how autoinhibition is relieved by tyrosine phosphorylation .",
"Phosphorylated Tyr783 in PLC-γ1 binds with nanomolar affinity to the cSH2 domain , and this engagement presumably couples a large , but ill-defined conformational rearrangement within the array to activation ( Gresset et al . , 2010; Hajicek et al . , 2013; Poulin et al . , 2005 ) .",
"However , even these few mechanistic details are under debate since an alternative model posits engagement of RTKs by the cSH2 domain as an initial step required for activation ( Huang et al . , 2016 ) .",
"The paucity of mechanistic understanding of the activation of the PLC-γ isozymes is largely attributable to a lack of structural information .",
"While structures of isolated portions of the regulatory array of PLC-γ1 are available , these structures provide an incomplete and sometimes erroneous context for defining how the array integrates the functions of basal autoinhibition and phosphorylation-dependent activation .",
"There are no structures of full-length PLC-γ isozymes .",
"Here , we describe the 2 . 5 Å-resolution crystal structure of essentially full-length PLC-γ1 .",
"The structure highlights a regulatory array exquisitely positioned to prevent membrane engagement of the catalytic core while simultaneously arranged to scaffold tyrosine kinases and additional regulatory proteins into a signaling nexus .",
"Kinases have unfettered access to the nSH2 domain of the regulatory array and will dock here initially .",
"In contrast , the phosphotyrosine binding pocket within the cSH2 domain is buried through interactions with the catalytic core .",
"Docked kinases are well positioned to phosphorylate Tyr783 located within a nearby solvent-exposed loop and phosphorylated Tyr783 ( pTyr783 ) is expected to bind the cSH2 domain to disrupt its interaction with the catalytic core .",
"This disruption is predicted to favor a substantial rearrangement of PLC-γ1 required for productive membrane engagement and hydrolysis of PIP2 .",
"The structure also explains how cancer-associated substitutions disrupt autoinhibition to elevate basal PLC-γ1 activity and contribute to supra-activation of the isozyme in the context of receptor overexpression .",
"The combined effects of substitution and tyrosine phosphorylation suggest that cellular context , for example overexpression of EGFR found in many cancers , will be especially important for understanding diseases modulated by the PLC-γ isozymes ."
],
[
"In addition to the aforementioned array of regulatory domains , the PLC-γ isozymes also possess a set of core domains common to most other isoforms of PLC: an N-terminal PH domain , two pairs of EF hands , a catalytic TIM barrel , and a C2 domain .",
"The regulatory array bisects the TIM barrel , subdividing this domain into the X- and Y-boxes ( Figure 1a , Figure 1—figure supplement 1 ) .",
"To facilitate crystallization , several regions predicted to be disordered were removed from the construct used for structure determination .",
"In particular , 20 and 75 residues were deleted from the N- and C-terminus , respectively .",
"In addition , an internal loop of 25 residues connecting the cSH2 and SH3 domains was removed and replaced with a flexible linker; we refer to this internal deletion as Δ25 ( Figure 1—figure supplement 1 , also see Materials and methods ) .",
"The crystallized construct therefore contains residues 21–765 and 791–1215 of PLC-γ1 .",
"The cSH2/SH3 domain loop contains Tyr783 , which is required for phosphorylation-dependent activation of PLC-γ1 .",
"However , we have demonstrated previously that this loop is not directly required for autoinhibition ( Gresset et al . , 2010 ) , and consistent with this notion , the crystallized form of PLC-γ1 was autoinhibited in cells ( Figure 1—figure supplement 2 ) .",
"While nuanced differences in regulation between the wild-type and crystallized version of PLC-γ1 cannot be excluded , the latter faithfully recapitulated the mutational activation of the wild-type enzyme ( Figure 1—figure supplement 2 ) .",
"In addition , deletion of the cSH2/SH3 domain loop had no measurable effect on the hydrodynamic radius or specific activity of the purified protein ( Figure 1—figure supplement 2 ) , further demonstrating that removal of the loop did not significantly alter the biochemical properties of the enzyme .",
"The 2 . 5 Å crystal structure ( Supplementary file 1 ) of essentially full-length rat PLC-γ1 readily explains the autoinhibition of the PLC-γ isozymes .",
"In particular , the regulatory array sits ‘atop’ the conserved catalytic core and blocks the core from productively engaging membranes ( Figure 1b ) .",
"In addition , the overall structure is highly electronegative ( Figure 1—figure supplement 3 ) , and this property will also inhibit lipase activity by disfavoring interactions with negatively charged membranes .",
"In particular , the overall negative charge of the PH domain indicates that it is unlikely to bind phosphatidylinositol 3 , 4 , 5-trisphosphate as previously reported ( Falasca et al . , 1998 ) .",
"For PLCs to hydrolyze membrane-embedded PIP2 , the hydrophobic ridge of the catalytic TIM barrel must insert into lipid bilayers ( Ellis et al . , 1998 ) .",
"However , in the case of PLC-γ1 , the hydrophobic ridge interacts with portions of the sPH domain in the regulatory array; this arrangement is expected to effectively block membrane engagement .",
"The active site sits beneath the hydrophobic ridge and is readily located by the bound Ca2+ cofactor ( Figure 1—figure supplement 2 ) .",
"As implied by the visibility of the Ca2+ cofactor , the active site is fully solvent-exposed and could presumably hydrolyze soluble substrates not embedded in lipid bilayers .",
"Two major interfaces lock the regulatory array on top of the catalytic core .",
"The first is the aforementioned sPH domain interacting with the hydrophobic ridge of the TIM barrel ( Figure 1c ) .",
"Here , residues from the sPH domain interdigitate with residues of the hydrophobic ridge in a ‘zipper-like’ arrangement .",
"A second interface is formed between loops of the cSH2 domain and the C2 domain of the catalytic core ( Figure 1d ) .",
"In this case , the BG and EF loops of the cSH2 domain clasp prominent turns of the C2 domain—almost as if the loops are pinching the C2 domain .",
"The pinched region of the C2 domain is an additional membrane anchor point in the PLC-δ isozymes , where Ca2+ mediates between the C2 domain and negatively charged membranes ( Ananthanarayanan et al . , 2002; Lomasney et al . , 2012 ) .",
"Based on sequence conservation and overall charge distribution , this region of the C2 domain of PLC-γ1 also seems likely to interact with Ca2+ and membranes , although this idea has not been tested .",
"The analogous portion of the C2 domain of PLC-γ2 is anticipated to engage Ca2+ in a similar manner .",
"Of note , this loop was implicated in the Ca2+-dependent translocation of PLC-γ2 to the plasma membrane necessary for amplification of the Ca2+ signaling cascade in B cells ( Nishida et al . , 2003 ) .",
"The two interfaces between the regulatory array and catalytic core do not overlap , but the sPH and cSH2 domains within the regulatory array brace each other through the C-terminal portion of the SH3 domain that lies between them ( Figure 1e ) : picture an arch with the tail end of the SH3 domain acting as the keystone .",
"The structure of full-length and autoinhibited PLC-γ1 immediately evokes a straightforward mechanism for its activation upon tyrosine phosphorylation .",
"The same BG and EF loops of the cSH2 domain that pinch the C2 domain are also used to engage pTyr783 and surrounding regions ( Hajicek et al . , 2013 ) ( Figure 1f , Figure 1—figure supplement 4 ) .",
"Therefore , when Tyr783 is phosphorylated , we propose that it will compete with the C2 domain for binding to the cSH2 domain .",
"This competition would presumably disengage the cSH2 domain from the C2 domain to initiate a rearrangement of the regulatory array relative to the catalytic core .",
"Moreover , perturbations at the interface of the cSH2 and C2 domains may propagate to the interface between the sPH domain and TIM barrel through keystone residues of the SH3 domain tail to amplify the original structural rearrangements .",
"Indeed , the structure suggests that a wholesale rearrangement of the regulatory array relative to the catalytic core is required for productive membrane engagement by PLC-γ1 ( Figure 1g ) .",
"This idea is consistent with previous studies using small-angle X-ray scattering ( SAXS ) that showed phosphorylation of PLC-γ1 is coupled to a conformational change that has yet to be defined ( Bunney et al . , 2012 ) .",
"The SAXS studies were also used by Bunney and colleagues to analyze the arrangement of domains within PLC-γ1 .",
"They posited a fundamentally distinct arrangement of domains relative to the crystal structure presented here .",
"In particular , the sPH and cSH2 domains were modeled as occupying the central volume of the SAXS envelope with the nSH2 and SH3 domains assuming flanking positions .",
"In this model , the sPH domain does not contact the PLC core .",
"This arrangement of domains would necessarily require a different mode of autoinhibition with the main autoinhibitory interface formed between the cSH2 domain and the TIM barrel .",
"The overall structure of PLC-γ1 also supports the multivalent scaffolding properties of the PLC-γ isozymes required for proper signaling ( Lemmon and Schlessinger , 2010; Rouquette-Jazdanian et al . , 2012; Timsah et al . , 2014 ) .",
"In particular , both the nSH2 and SH3 domains are organized within the overall structure for unfettered access to cognate ligands ( Figure 2 , Figure 1—figure supplement 4 ) .",
"For example , the phosphotyrosine-binding pocket of the nSH2 domain is fully solvent exposed and the major site for engagement of phosphorylated RTKs ( Bae et al . , 2009; DeBell et al . , 1999; Poulin et al . , 2000 ) .",
"Likewise , the canonical polyproline-binding site of the SH3 domain is positioned to readily engage various proteins .",
"Relevant examples include the scaffolding protein SLP-76 ( Deng et al . , 2005 ) , the E3 ubiquitin ligase Cbl ( Tvorogov and Carpenter , 2002 ) , and the guanine nucleotide exchange factor Vav1 ( Braiman et al . , 2006; Knyazhitsky et al . , 2012; Sherman et al . , 2016 ) —all of which must be engaged by PLC-γ1 for the proper clustering of T cell receptors and subsequent downstream signaling .",
"PLC-γ2 mediates similar clustering in response to active B cell receptors ( Wang et al . , 2014 ) and presumably will be structurally similar to PLC-γ1 .",
"In addition , the monomeric GTPase Rac2 binds the sPH domain of PLC-γ2 to elevate lipase activity ( Piechulek et al . , 2005; Walliser et al . , 2008 ) and the equivalent surface within the sPH domain of PLC-γ1 is fully exposed to solvent ( Figure 1—figure supplement 4 ) .",
"This last observation suggests that binding of Rac2 would not disrupt the overall structure of autoinhibited PLC-γ2 .",
"Rather , Rac2 may stabilize an active conformation of PLC-γ2 once PLC-γ2 is engaged with membranes as previously suggested ( Walliser et al . , 2016 ) .",
"In counterpoint to the above examples , the canonical phosphotyrosine-binding site of the cSH2 domain is buried in the structure of PLC-γ1 .",
"This site is presumably reserved as the ‘trigger’ for phospholipase activation upon engagement of pTyr783 .",
"Therefore , pTyr783 is suggested to function as an intramolecular ligand that can effectively compete for the buried surface of the cSH2 domain .",
"This situation is in contrast to intermolecular competitors such as kinases that would need to overcome substantial entropic penalties in order to bind the cSH2 domain .",
"Tyr783 in PLC-γ1 is presumed to be the primary site of phosphorylation coupled to enzyme activation ( Gresset et al . , 2010; Kim et al . , 1991 ) .",
"Eight additional tyrosines are phosphorylated ( positions 186 , 472 , 481 , 771 , 775 , 959 , 977 , and 1254 ) , but these sites appear dispensable for RTK-dependent activation in cells ( Bunney et al . , 2012 ) .",
"In contrast , activation of PLC-γ1 by soluble tyrosine kinases requires phosphorylation of both Tyr775 and Tyr783 ( Serrano et al . , 2005 ) and this situation is similar for PLC-γ2 where the analogous tyrosines ( positions 753 , 759 ) are also phosphorylated during phospholipase activation ( Humphries et al . , 2004; Ozdener et al . , 2002; Rodriguez et al . , 2001; Watanabe et al . , 2001 ) .",
"How dual sites of phosphorylation cooperate to drive phospholipase activity is an open question but presumably shares aspects of regulation described above .",
"Additional tyrosines ( positions 1197 , 1217 ) in PLC-γ2 are also phosphorylated and implicated in regulation ( Watanabe et al . , 2001 ) , but these sites are not conserved in PLC-γ1 .",
"While the structure of PLC-γ1 strongly suggests that it must undergo a substantial rearrangement in order to gain access to its membrane-resident substrate , PIP2 , this idea is speculative without substantiation .",
"We formally tested this idea using two bespoke fluorescent substrates of mammalian PLCs ( Figure 3 ) .",
"The first case , WH-15 , is a soluble analogue of PIP2 ( Huang et al . , 2011 ) .",
"It is predicted to have unimpeded access to the active site of PLC-γ1 and mutations assumed to relieve autoinhibition by wholesale rearrangement should not affect basal specific activity for the hydrolysis of WH-15 .",
"This is in fact the case since wild-type PLC-γ1 and a set of mutated forms that are constitutively active in cells ( Hajicek et al . , 2013 ) ( also see below ) have essentially identical capacity to hydrolyze WH-15 in vitro ( Figure 3a ) .",
"In contrast , XY-69 is a fluorescent substrate of PLCs that was specifically designed to embed into lipid bilayers ( Huang et al . , 2018 ) .",
"When XY-69 in lipid vesicles was presented to the same set of PLCs , there was now a dramatic difference in hydrolytic rates ( Figure 3b ) .",
"Wild-type PLC-γ1 had very low specific activity , while the mutated forms were up to 30-fold more active .",
"This discrimination presumably reflects the capacity of mutations to disrupt the interface between the regulatory domains and the catalytic core to favor a form of PLC-γ1 better able to engage PIP2 in membranes .",
"Discrimination was greatly diminished—albeit not completely eliminated—when XY-69 was solubilized in detergent micelles ( Figure 3c ) .",
"These results are consistent with the postulation that autoinhibition arises from the overall spatial arrangement of PLC-γ1 that prevents it from productively engaging membranes .",
"Mutations that destabilize this arrangement are proposed to concomitantly relieve autoinhibition and allow PLC-γ1 better access to membranes and PIP2 .",
"Accelerated molecular dynamics ( aMD ) simulations support the proposed mechanism of activation .",
"In particular , all-atom simulations of PLC-γ1 reproducibly highlighted a flexible set of regulatory domains relative to a virtually static catalytic core ( Figure 4a , Figure 4—figure supplement 1 ) .",
"Moreover , this flexibility increased for simulations of a constitutively active mutant form of PLC-γ1 harboring a single substitution ( D1165H ) within the C2 domain at the interface with the phosphotyrosine-binding site of the cSH2 domain ( Figure 4—figure supplement 2 ) .",
"Of note , D1165H corresponds to the D1140G substitution in PLC-γ2; PLC-γ2 ( D1140G ) has been identified in patients with relapsed chronic lymphocytic leukemia treated with ibrutinib ( Burger et al . , 2016; Landau et al . , 2017 ) .",
"For both wild-type and mutant PLC-γ1 , the correlated motions indicate that the regulatory domains tended to move as a relatively rigid block ( Figure 4b ) .",
"Comparisons of average structures derived from the aMD simulations highlight increased disorganization within the interface between the cSH2 and C2 domains upon mutation ( Figure 4c ) .",
"For example , Asp1165 resides within the β5/β6 turn of the C2 domain where it participates in two hydrogen bonds that stabilize the turn that forms a major part of the interface with the cSH2 domain .",
"Substitution of Asp1165 to His ( D1165H ) disrupts the proximal hydrogen-bonding network and results in the partial unfolding of the β5 and β6 strands of the C2 domain during simulations .",
"The collapse of this region is linked to an approximately 30° rotation of the cSH2 domain as it moves toward the C2 domain by approximately 10 Å .",
"The relative movements of the C2 and cSH2 domains are propagated to the rest of the regulatory array due to its propensity to move as a block .",
"Movements are essentially identical for a constitutively active mutant form of PLC-γ1 harboring two substitutions ( Y747E+R748E ) within the phosphotyrosine-binding site of the cSH2 domain and on the opposite side of the interface from Asp1165 ( Figure 4—figure supplements 1 , 2 and 3 ) .",
"This result suggests that diverse mutations within the cSH2/C2 domain interface will favor similar movements .",
"The PLC-γ isozymes are frequently mutated in several leukemias ( Burger et al . , 2016; Kataoka et al . , 2015 ) and lymphomas ( Choi et al . , 2015; da Silva Almeida et al . , 2015; Kiel et al . , 2015; Vaqué et al . , 2014 ) .",
"In particular , PLC-γ1 is the most frequently mutated protein in adult T cell leukemia/lymphoma ( Kataoka et al . , 2015 ) .",
"In this disease , sites of substitution in PLC-γ1 are found throughout the entire primary sequence with clusters at several hotspots ( Figure 5a ) .",
"This rather uninformative arrangement is dramatically clarified when the entire set of substitutions is mapped onto the structure of autoinhibited PLC-γ1 ( Figure 5b ) .",
"Now , the majority of sites localize to the interfaces formed between the PLC core and the regulatory array .",
"This three-dimensional clustering strongly suggests that most cancer-associated substitutions in PLC-γ1 disrupt the placement of the regulatory domains atop the core to disfavor autoinhibition .",
"Indeed , in a panel of PLC-γ1 isozymes expressed in HEK293 cells , cancer-associated substitutions at these interfaces produced a spectrum of constitutively active phospholipases—sometimes exceeding 1500-fold greater activity than wild-type PLC-γ1 ( Figure 5c ) .",
"Cancer-associated mutations within the equivalent regions of PLC-γ2 produced similar enhancements , indicating conserved regulation between the two isozymes ( Figure 5—figure supplement 1 ) .",
"Cancer-derived mutations outside the autoinhibitory interfaces generally produced the smallest increases in basal lipase activities—but these increases were nonetheless significant in comparison to the wild-type isozyme ( Figure 5c , inset ) .",
"How might these additional mutations lead to constitutive phospholipase activity ?",
"Based on the sites of mutation within the structure of autoinhibited PLC-γ1 , three mechanisms are likely .",
"First , substitutions may increase the affinity of the active form of PLC-γ1 for membranes .",
"This option is likely the case for R48W located in the PH domain near the presumed interface with membranes .",
"A similar mode leading to elevated phospholipase activation was proposed for a substituted form of PLC-γ2 that causes arthritis in mice and has increased affinity for membranes relative to wild-type PLC-γ2 ( Everett et al . , 2009 ) .",
"Second , substitutions might disrupt interactions provided by the keystone residues of the SH3 domain that buttress the organization of the sPH and cSH2 domains needed to maintain autoinhibition .",
"Representative substitutions include R687W and R753H and additional examples are found in both PLC-γ1 ( Figure 5—figure supplement 2 ) and -γ2 ( Figure 5—figure supplement 1 ) .",
"Of note , R687W is analogous to R665W in PLC-γ2 and arises in patients with relapsed chronic lymphocytic leukemia treated with ibrutinib ( Woyach et al . , 2014 ) .",
"Finally , mutations within the nSH2 domain , for example Q606R and D625Y , are near the binding site for phosphotyrosine ( Bae et al . , 2009 ) and may increase affinity for phosphorylated kinases .",
"The PLC-γ isozymes are normally activated upon phosphorylation , especially by diverse growth factor receptors .",
"Therefore , the cancer-associated mutations in PLC-γ1 were further tested for effects on lipase activity after co-expression of PLC-γ1 and EGFR ( Figure 6a ) .",
"In all cases , a high concentration of EGF used to activate the receptor produced elevated lipase activity relative to wild-type PLC-γ1 .",
"This result indicates an untapped reserve of lipase activity that is , at least partially , released by these cancer-associated mutations in response to EGF .",
"This point is further emphasized for lipase responses measured at varying concentrations of EGF for a representative subset of mutant PLC-γ1 isozymes with varying levels of constitutive activation ( Figure 6b , upper graph ) .",
"Both P867R and D1165H occur at the autoinhibitory interfaces and produced substantially elevated lipase activity relative to wild-type PLC-γ1 at all concentrations of EGF .",
"In contrast , R48W occurs at the predicted interface of the active isozyme with membranes and abnormally elevated lipase activity of PLC-γ1 ( R48W ) manifests only at high concentrations of EGF .",
"This functional difference possibly reflects a mechanistic difference: P867R and D1165H likely destabilize the inactive ensemble of PLC-γ1 while simultaneously favoring active forms of the isozyme; in contrast , R48W has no substantive effect on the inactive population under these conditions and presumably only stabilizes the active isozyme once bound to membranes ( Figure 6b , lower graph ) .",
"Regardless of the mechanistic details , these functional results suggest important biological ramifications .",
"Namely , the lipase activity of mutant PLC-γ isozymes should be dependent on cellular context .",
"For example , PLC-γ1 or -γ2 harboring mutations such as R48W that preferentially stabilize active ensembles may be essentially quiescent until the isozymes are activated by phosphorylation .",
"These situations are relatively nuanced in comparison to more robustly activating mutations , for example P867R and D1165H , that disrupt core aspects of autoinhibition .",
"However , the more subtly activating substitutions may nonetheless contribute to cancer in cells with high levels of active kinases such as upon the overexpression of EGFR or other growth factor receptors—situations with widespread clinical relevance ( Wilson et al . , 2012 ) ."
],
[
"The structure of full-length , autoinhibited PLC-γ1 provides a first clear view of the regulated activation of the PLC-γ isozymes .",
"The overall picture is of a catalytic core that is conserved among all PLCs and that is prevented from spuriously hydrolyzing PIP2 by a set of interdependent regulatory domains stationed to preclude access of the active site to membranes .",
"Additionally , the regulatory domains are organized to integrate numerous molecular inputs that ultimately control phospholipase activity and mediate necessary scaffolding functions ( Figure 7 ) .",
"Importantly , the nSH2 domain is optimally positioned to readily bind phosphorylated kinases and align them to promote the phosphorylation of Tyr783 needed for activation of PLC-γ1 .",
"Although capable of engaging phosphorylated portions of kinases ( Groesch et al . , 2006 ) , the equivalent surface of the cSH2 domain is buried through interactions with the C2 domain and is unlikely to initiate engagement of kinases as previously suggested ( Huang et al . , 2016 ) .",
"However , the two SH2 domains might work in concert upon receptor engagement to facilitate the binding of phosphorylated Tyr783 to the cSH2 domain .",
"This idea is supported by the comparison of the full-length structure of PLC-γ1 with a structure of the two SH2 domains of PLC-γ1 bound to the phosphorylated kinase domain of fibroblast growth factor receptor 1 ( FGFR1 ) ( Bae et al . , 2009 ) .",
"Based on this comparison , the βA/αA loop of the nSH2 domain is rearranged to accommodate pTyr766 of FGFR1 and this rearrangement leads to additional movements of the cSH2 domain ( Figure 7—figure supplement 1 ) .",
"In the full-length structure , equivalent movements upon binding FGFR1 would open the surface of the cSH2 domain that binds pTyr783 , effectively priming it to engage pTyr783 .",
"Engagement of pTyr783 by the cSH2 domain is presumed to unlatch the cSH2 domain from the catalytic core and initiate what is likely to be a relatively massive rearrangement of the regulatory domains with respect to the core before the core can engage membranes and hydrolyze PIP2 .",
"The model of activation described above provides the mechanistic underpinnings for understanding the mutational landscape of the PLC-γ isozymes associated with disease .",
"In particular , most of the substitutions and small deletions in these isozymes that are linked to cancers ( Burger et al . , 2016; Kataoka et al . , 2015; Woyach et al . , 2014 ) or autoimmune disease ( Ombrello et al . , 2012 ) occur at the interfaces between the core and regulatory domains based on the structure of autoinhibited PLC-γ1 .",
"These mutations disrupt these interfaces , release autoinhibition , and favor conformations that engage membranes to promote constitutive phospholipase activity .",
"Shifted conformational equilibria may also explain the supra-activation of mutant forms of PLC-γ1 by EGFR .",
"That is , mutant forms of PLC-γ1 that are predisposed to be ‘open’ may also have a greater propensity to bind EGFR and a lower probability of turning off .",
"Constitutive activation varies greatly , ranging from approximately 10-fold to over 1500-fold , with important cellular implications: highly active forms are expected to drive downstream signaling under all circumstances while more subtly active forms are likely to promote disease only within specific cellular contexts .",
"An excellent example of this latter class includes mutated forms of PLC-γ2 ( R665W or L845F ) that arise in patients treated with ibrutinib .",
"B cells harboring either mutant of PLC-γ2 possess normal calcium homeostasis until B cell receptors are activated , at which point intracellular calcium levels rise and remain elevated , while in the equivalent wild-type case , calcium homeostasis is rapidly reestablished ( Woyach et al . , 2014 ) .",
"Both mutant forms have essentially wild-type phospholipase activity at low levels of expression but are hypersensitive to activation by Rac2 ( Walliser et al . , 2016 ) .",
"These results suggest that once active , the mutant forms of PLC-γ2 are stabilized at membranes by binding to Rac2 .",
"That is , the mutations are cryptic until the isozymes are activated , at which point the mutated PLC-γ2 isozymes are slow to return to their autoinhibited state .",
"Similar context-dependent activation of mutant forms of the PLC-γ isozymes should also occur upon activation by kinases .",
"PLC-γ1 ( R48W ) provides an example: it has essentially wild-type phospholipase activity until co-expressed with high levels of active EGFR .",
"Analogous scenarios may be wide-spread in cancers where tyrosine kinases are constitutively active upon substitution , truncation , fusion , or overexpression—all conditions shown to activate PLC-γ1 ( Arteaga et al . , 1991; Peles et al . , 1991 ) .",
"Alternatively , the activation of RTKs by stromal components contributes to treatment-resistant cancers ( Straussman et al . , 2012 ) and roles for wild-type and mutant PLC-γ isozymes under these conditions need to be explored .",
"On a final note , the interfacial regulation of the PLC-γ isozymes suggests promising avenues for their isozyme-specific , allosteric modulation by small molecules to advance related chemical biology and on-going drug discovery .",
"Namely , compounds that inhibit the movement of the regulatory domains relative to the catalytic core should also prevent membrane engagement and consequent PIP2 hydrolysis .",
"Such compounds might treat cancers and immune diseases driven by constitutively active forms of the PLC-γ isozymes .",
"Conversely , allosteric modulators that stabilize active forms of the PLC-γ isozymes might bolster immunotherapies ( Guittard et al . , 2018 ) or provide promising leads for the treatment of Alzheimer’s disease where a naturally occurring hypermorphic variant of PLC-γ2 is linked to protection from this disease ( Magno et al . , 2019 ) ."
],
[
"Multi-angle light scattering measurements were performed using Wyatt DAWN HELEOS II light scattering instrumentation ( with Wyatt Optilab T-rEX refractometer and Wyatt dynamic light scattering module ) coupled to a Superdex 200 10 mm x 300 mm GL size exclusion column .",
"Following equilibration with buffer containing 20 mM HEPES ( pH 7 . 4 ) , 150 mM NaCl , and 0 . 02% w/v NaN3 , 50 μL of PLC-γ1 ( 21–1215 ) proteins at 2 mg/mL were loaded onto the column .",
"Data analysis was performed with ASTRA software version 6 ( Wyatt Technologies ) .",
"X-ray diffraction data were collected on crystals of native PLC-γ1 ( 21–1215 ) Δ25 at the Southeast Regional Collaborative Access Team ( SER-CAT ) beamline 22-BM at the Advanced Photon Source at Argonne National Laboratory .",
"One scan totaling 200° of data was collected at 100 K on a MAR 200 CCD detector .",
"Each frame was exposed for 10 s and consisted of a 1° oscillation .",
"Gadolinium-derivatized crystals of PLC-γ1 ( 21–1215 ) Δ25 were used to collect a single-wavelength anomalous diffraction ( SAD ) dataset at the gadolinium absorption peak ( λ = 7 , 244 . 3 eV ) .",
"Data were acquired at SER-CAT beamline 22-ID on a Rayonix MX300-HS CCD detector .",
"Each frame consisted of a 1° oscillation and was exposed for 1 s .",
"A total of 240° of data were collected at 100 K in 30° wedges using an inverse beam strategy .",
"Both sets of diffraction data were indexed , integrated , and scaled using HKL2000 ( Otwinowski and Minor , 1997 ) .",
"Phases for the gadolinium-bound form of PLC-γ1 ( 21–1215 ) Δ25 were solved by SAD using the AutoSol routine in the Phenix software suite ( Adams et al . , 2010 ) .",
"A partial model of this structure was built using AutoBuild ( Terwilliger et al . , 2008 ) and used as a molecular replacement search model to solve phases for the structure of native PLC-γ1 ( 21–1215 ) Δ25 .",
"The remainder of the model was then built in an iterative process that consisted of manual model building in Coot ( Emsley et al . , 2010 ) followed by restrained refinement in Phenix .",
"The structure was validated using MolProbity ( Chen et al . , 2010 ) and molecular representations produced with PyMOL ( Schrodinger LCC , 2019 ) .",
"Complete data collection and refinement statistics are shown in Supplementary file 1 .",
"To quantify basal phospholipase activity , HEK293 cells were plated at a density of ~75 , 000 cells/well in 12-well cluster plates and transiently transfected with 100 ng of vector encoding wild-type or mutant forms of PLC-γ1 .",
"Twenty-four hours post-transfection , cells were metabolically labeled overnight in serum-free , inositol-free medium containing 1 μCi of [3H]myo-inositol and 10 mM LiCl .",
"Accumulation of [3H]inositol phosphates was quantified as described previously ( Waldo et al . , 2010 ) .",
"In all experiments , counts that accumulated in cells transfected with empty vector ( ~500–1000 cpm ) were subtracted as background .",
"EGFR-dependent activation of PLC-γ1 and PLC-γ2 was quantified in HEK293 cells transiently co-transfected with 200 ng of vector expressing various forms of the PLC-γ isozymes and 100 ng of vector expressing wild-type EGFR .",
"Cells were metabolically labeled as described above , except LiCl was omitted from the radiolabeling medium .",
"Cells received a 30 min challenge with the indicated concentrations of recombinant human EGF ( Invitrogen ) diluted in Hank’s Balanced Salt Solution containing 20 mM HEPES ( pH 7 . 5 ) , 10 mM LiCl , and 200 μg/mL fatty acid-free bovine serum albumin ( BSA ) .",
"Expression of each form of PLC-γ1 and PLC-γ2 was confirmed by immunoblotting of cell lysates using a monoclonal antibody against the HA epitope ( BioLegend , clone 16B12 ) .",
"Lysates were also probed with a monoclonal antibody against β-actin ( SigmaAldrich , clone AC-15 ) as a loading control .",
"All immunoblots represent a single exposure from one experiment , and the HA epitope and β-actin were detected on the same blot .",
"Immunoblots were loaded with all mutant versions of PLC-γ1 or PLC-γ2 in numerical order; bands were subsequently cropped and then reordered in Photoshop to reflect the order in which data are presented in bar graphs and dose-response curves .",
"The identity of the HEK293 cell line was not authenticated , and testing for mycoplasma contamination was not performed ."
]
] | [
"Direct activation of the human phospholipase C-γ isozymes ( PLC-γ1 , -γ2 ) by tyrosine phosphorylation is fundamental to the control of diverse biological processes , including chemotaxis , platelet aggregation , and adaptive immunity .",
"In turn , aberrant activation of PLC-γ1 and PLC-γ2 is implicated in inflammation , autoimmunity , and cancer .",
"Although structures of isolated domains from PLC-γ isozymes are available , these structures are insufficient to define how release of basal autoinhibition is coupled to phosphorylation-dependent enzyme activation .",
"Here , we describe the first high-resolution structure of a full-length PLC-γ isozyme and use it to underpin a detailed model of their membrane-dependent regulation .",
"Notably , an interlinked set of regulatory domains integrates basal autoinhibition , tyrosine kinase engagement , and additional scaffolding functions with the phosphorylation-dependent , allosteric control of phospholipase activation .",
"The model also explains why mutant forms of the PLC-γ isozymes found in several cancers have a wide spectrum of activities , and highlights how these activities are tuned during disease ."
] | [
"Many enzymes are poised to receive signals from the surrounding environment and translate them into responses inside the cell .",
"One such enzyme is phospholipase C-γ1 ( PLC-γ1 ) , which controls how cells grow , divide and migrate .",
"When activating signals are absent , PLC-γ1 usually inhibits its own activity , a mechanism called autoinhibition .",
"This prevents the enzyme from binding to its targets , which are fat molecules known as lipids .",
"When activating signals are present , a phosphate group serves as a ‘chemical tag’ and is added onto PLC-γ1 , allowing the enzyme to bind to lipids .",
"Failure in the regulation of PLC-γ1 or other closely related enzymes may lead to conditions such as cancer , arthritis and Alzheimer’s disease .",
"However , it remains unclear how autoinhibition suppresses the activity of the enzyme , and how it is stopped by the addition of the phosphate group .",
"Here , Hajicek et al . determine in great detail the three-dimensional structure of the autoinhibited form of the enzyme using a method known as X-ray crystallography .",
"This reveals that PLC-γ1 has two major lobes: one contains the active site that modifies lipids , and the other sits on top of the active site to prevent lipids from reaching it .",
"The findings suggest that when the phosphate group attaches to PLC-γ1 , it triggers a large shape change that shifts the second lobe away from the active site to allow lipids to bind .",
"The three-dimensional structure also helps to understand how mutations identified in certain cancers may activate PLC-γ1 .",
"In particular , these mutations disrupt the interactions between elements that usually hold the two lobes together , causing the enzyme to activate more easily .",
"The work by Hajicek et al . provides a framework to understand how cells control PLC-γ1 .",
"It is a first step toward designing new drugs that alter the activity of this enzyme , which may ultimately be useful to treat cancer and other diseases ."
] | 2019 |
[
"Introduction",
"Results",
"Materials and methods"
] | [
"immunology and inflammation"
] | Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes | elife-19891-v2 | [
[
"Cells interact with their environment through a complex set of biochemical networks that transmit information across the plasma membrane .",
"Often , signal transduction relies on the spatial organization of receptors as well as effector proteins that regulate down-stream signaling activity .",
"In principle , spatial organization in biological membranes can be enforced through varied mechanisms including direct protein-protein interactions ( Douglass and Vale , 2005; Su et al . , 2016b ) , dynamic or passive coupling to cytoskeletal elements ( Wülfing and Davis , 1998; Kaizuka et al . , 2007; DeMond et al . , 2008 ) , adhesion ( Davis and van der Merwe , 2006 ) , curvature mediated forces ( Zhu et al . , 2012; Aimon et al . , 2014 ) , or steady state biochemical networks with spatial heterogeneity ( Chau et al . , 2012 ) .",
"It is also proposed that plasma membrane lipids contribute to the spatial organization of membrane proteins via the same thermodynamic forces that drive the separation of liquid-ordered and liquid-disordered phases in model membranes ( Schroeder et al . , 1994; Lingwood and Simons , 2010 ) .",
"Liquid-ordered like domains are often referred to as lipid rafts or lipid shells ( Simons and Ikonen , 1997; Anderson and Jacobson , 2002 ) , and are hypothesized to impact a broad array of signaling cascades that originate at the plasma membrane ( Simons and Toomre , 2000 ) including B cell receptor signaling ( Cheng et al . , 1999 ) .",
"However , the existence of phase-like membrane domains and their putative roles in signaling pathways remain controversial , largely because the majority of experimental support for this concept is indirect or relies on methodology with well characterized limitations ( Heerklotz , 2002; Munro , 2003; Kwik et al . , 2003; Kenworthy , 2008 ) .",
"Some of the strongest experimental evidence supporting a heterogeneous plasma membrane comes from Förster resonance energy transfer ( FRET ) measurements between membrane components .",
"This method is sensitive to heterogeneity on length-scales smaller than the Förster distance ( ~5 nm ) ( Kenworthy and Edidin , 1998; Varma and Mayor , 1998; Pyenta et al . , 2001; Zacharias et al . , 2002; Sharma et al . , 2004; Rao and Mayor , 2005; Sengupta et al . , 2007; Goswami et al . , 2008 ) .",
"While powerful for detecting interactions between proteins and/or lipids that occur on small length-scales , the FRET signal is highly nonlinear with respect to probe separation distance and depends on both donor/acceptor ratio and probe absolute density , which typically vary in experiments .",
"These complications can lead to weak sensitivity of FRET measurements to changes in local concentration and often modeling is required to interpret experimental findings quantitatively ( Kenworthy and Edidin , 1998; Rao and Mayor , 2005 ) .",
"Here , we apply super-resolution fluorescence localization imaging to complement these past approaches to directly visualize ordered and disordered-like domains in intact cell plasma membranes .",
"This approach allows us to characterize and quantify , in a model-independent manner , the spatial organization of membrane components on length scales between those accessible by FRET-based techniques and conventional optical microscopy .",
"B cells undergo a signaling response when their B cell receptors ( BCRs ) are engaged by antigen , either in the form of solution-phase multivalent antigen ( Minguet et al . , 2010 ) or surface-presented monovalent antigen ( Batista et al . , 2001 ) , however the molecular mechanisms driving BCR signal initiation are still controversial ( Packard and Cambier , 2013 ) .",
"Notably , simply clustering the BCR with antibodies directed against the receptor initiates phosphorylation by Src family kinases , resulting in the binding of downstream kinases and effectors that amplify and propagate the signaling response ( Cambier and Ransom , 1987; Campbell and Sefton , 1990 ) .",
"This supports the idea that , at least in some contexts , the spatial organization of the BCR can function to communicate antigen binding , similar to other transmembrane receptors ( Metzger , 1992 ) , as opposed to a mechanism where receptor binding is conveyed solely through ligand-induced conformational changes ( Campbell and Humphries , 2011; Sounier et al . , 2015 ) .",
"Several mechanistic models have been put forth to explain how BCR clustering could give rise to receptor activation .",
"In one model , BCR clustering initiates signaling via protein-protein interactions between neighboring receptors , such as the transphosphorylation of nearby receptors by receptor-bound kinases ( Sotirellis et al . , 1995; Kurosaki and Kurosaki , 1997 ) .",
"A second model proposes that antigen binding acts to separate pre-clustered BCR , exposing binding sites to kinases that propagate activation ( Yang and Reth , 2010a , 2010b ) .",
"A third model , which is the focus of investigation here , postulates that clustering BCR acts to stabilize an ordered membrane domain that impacts the receptor-proximal distribution of regulatory proteins involved in initiating or modulating the resulting cellular response .",
"Specifically , ordered membrane domains are predicted to support interactions with activating kinases and suppress interactions with deactivating phosphatases .",
"This hypothesis has been strengthened by experimental support ( Pierce , 2002; Sohn et al . , 2006; Gupta and DeFranco , 2007; Sohn et al . , 2008a ) , and among this evidence are observations of changes in FRET upon receptor clustering and activation between BCR and a marker of ordered membrane domains , but not between BCR and a marker of disordered domains ( Sohn et al . , 2006 , 2008b ) .",
"These studies demonstrated that receptor clustering leads to a transient increase in near-neighbor interactions ( within a few lipid diameters ) between BCR and order-preferring lipid probes and suggested that these interactions are important for the initiation of BCR activation by the Src family kinase Lyn .",
"Currently , the role of membrane domains in BCR signaling remains a topic of active investigation ( Pierce and Liu , 2010; Horejsi and Hrdinka , 2014 ) as the field works to put together a more holistic picture of how interactions between proteins and lipids could support signaling function .",
"Here , we use super-resolution fluorescence localization microscopy to characterize the lipid environment around BCR clusters .",
"Using this approach , we directly visualize ordered domains co-localized with clustered BCR .",
"We find that these domains sort key regulatory proteins involved in BCR signaling and provide a local environment that favors tyrosine phosphorylation .",
"We also present a predictive model whereby BCR clustering leads to its phosphorylation through the stabilization of an ordered membrane domain .",
"Our findings suggest that the collective protein-lipid and lipid-lipid interactions responsible for the stabilization of an ordered domain around BCR clusters give rise to emergent signaling function by influencing the local biochemical environment of BCRs within clusters .",
"These measurements detail the molecular redistribution of membrane components around embedded membrane protein clusters , utilizing super-resolution microscopy to gain access to length scales that are smaller than those accessible by conventional microscopy and larger than length scales accessible by FRET .",
"Overall , our imaging studies provide additional direct evidence that clustered BCR associates with a local environment enriched in ordered lipids .",
"Together with our simulation and functional data , this evidence supports a mechanism for clustering-induced activation of BCR that involves lipid-mediated sorting of regulatory proteins .",
"Similar mechanisms may be relevant to other pathways where changes in receptor organization lead to signaling functions ."
],
[
"Membrane heterogeneity was probed using established markers of ordered and disordered membrane domains ( Figure 1a and Figure 1—figure supplement 1 ) .",
"Disordered domains were marked with a short transmembrane peptide ( TM ) and a peptide anchored to the inner leaflet through a polybasic sequence and geranylgeranyl modification ( GG ) ( Pyenta et al . , 2001; Baumgart et al . , 2007; Levental et al . , 2010 ) .",
"Ordered membrane domains were marked with a minimal lipidated peptide anchored to the inner leaflet through saturated palmitoyl and myristol modifications ( PM ) ( Pyenta et al . , 2001; Baumgart et al . , 2007 ) , or with cholera toxin subunit B ( CTxB ) , which binds to the ganglioside GM1 on the outer leaflet of the plasma membrane .",
"Probe partitioning was verified in isolated giant plasma membrane vesicles ( GPMVs ) imaged at low temperature as demonstrated in Figure 1—figure supplement 2 . PM , TM , and GG all lack specific protein interaction domains; therefore their spatial distributions are determined by their interactions with the plasma membrane . 10 . 7554/eLife . 19891 . 003Figure 1 . Clusters of ordered or disordered phase markers create distinct membrane domains .",
"( a ) Schematic representation of minimal anchor peptides and their phase preference as determined from model membranes .",
"Amino acid sequences and chemical structures are shown in Figure 1—figure supplement 1 .",
"( b ) Reconstructed super-resolution images of PM with either clustered TM ( left ) or clustered CTxB ( right ) .",
"Scale-bars are 5 µm and 500 nm in the inset .",
"( c ) Cross-correlation functions , C",
"( r ) , of PM and GG constructs in cells containing clustered TM ( left ) or of PM and TM constructs in cells containing clustered CTxB ( right ) .",
"A value of C",
"( r ) = 1 indicates a random co-distribution , C",
"( r ) > 1 indicates co-clustering , and C",
"( r ) < 1 indicates exclusion .",
"These curves represent an average over multiple individual cells from multiple experiments .",
"Curves are averaged over the following number of cells: TM with PM ( 12 ) or GG ( 8 ) , and CTxB with PM ( 58 ) or TM ( 27 ) .",
"Error-bars indicate the SEM between cells .",
"Curves for individual cells are shown in Figure 1—figure supplement 4 .",
"Additional representative images for all conditions are shown in Figure 1—figure supplement 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 00310 . 7554/eLife . 19891 . 004Figure 1—figure supplement 1 . Amino acid sequences of membrane anchors used in this study . The amino acid sequence and post-translational modification of the four transiently expressed membrane anchors are shown .",
"PM contains the 10 N-terminal amino acids from Lyn , which code for a myristoylation and palmitoylation of the N-terminal glycine and cysteine , respectively .",
"GG contains a polybasic sequence and C-terminal geranylgeranylation , designed from the C-terminal sequence of K-Ras with modification of the CAAX box to code for geranylgeranylation instead of prenylation .",
"CD45TM is the transmembrane domain of CD45 with a FLAG tag , shown in blue , fused to the short extracellular region .",
"Export of CD45TM to the plasma membrane is improved by addition of the signal sequence from HA on the N-terminus .",
"The signal sequence is cleaved in the ER and is not shown here .",
"The full CD45TM sequence is described in the Materials and Methods .",
"TM is the transmembrane domain of Linker for Activation of T Cells ( LAT ) where palmitoylation sites have been mutated .",
"The fluorophore mEos3 . 2 is depicted as a green circle , and linker amino acids between the membrane anchors and mEos3 . 2 are depicted as straight lines . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 00410 . 7554/eLife . 19891 . 005Figure 1—figure supplement 2 . Membrane anchors partition into different phases in GPMVs .",
"( a ) Alexa-555 CTxB and DiD-C16 partition into different phases in GPMVs .",
"CTxB is a well-established marker of the liquid-ordered phase , indicating that DiD-C16 partitions into the liquid-disordered phase .",
"( b ) PM-eGFP and DiD C16 partition into alternate phases , indicating that PM-eGFP partitions with the liquid-ordered phase .",
"These GPMVs were prepared with glutathione instead of DTT to preserve the palmitoylation state of this peptide .",
"( c ) eGFP-GG and Alexa647 CTxB partition into alternate phases , indicating that GG partitions into the liquid-disordered phase .",
"( d ) YFP-TM and Alexa647 CTxB partition into alternate phases , indicating that TM partitions into the liquid-disordered phase .",
"Grayscale images were acquired sequentially for the labels indicated leading to some movement of the vesicle and domains occur between acquisitions .",
"False-colored images represent a simple superposition of the two color channels .",
"The traces at the right denote the fluorescence intensity along the white line shown in the false colored image .",
"In all instances , the vesicle surface was imaged and not a cross-section , leading to fluorescence intensity present in the vesicle interior .",
"The scale bar in all images is 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 00510 . 7554/eLife . 19891 . 006Figure 1—figure supplement 3 . Finite lateral resolution and incomplete spatial sampling impacts measured cross-correlations . An Ising model simulated at T = 1 . 05 times the critical temperature is used to demonstrate how finite lateral resolution and incomplete spatial sampling impacts measurements of cross-correlations between proteins and membrane phase-like domains .",
"( a ,",
"c ) Magenta points represent ordered domain preferring proteins that are either allowed to explore all space",
"( a ) or are forced to reside within a circular domain",
"( c ) .",
"Green pixels in the fully sampled images ( top panels of a and",
"c ) represent disordered components and black pixels represent ordered components .",
"In the under-sampled images , green points are a subset of the green pixels shown in the fully sampled images , at a density of roughly 400/µm2 ( 100 points per 512 by 512 nm box ) .",
"The left-most images in a and c represent simulation snapshots without additional processing .",
"The remaining images are generated from the same information present in the left-most image , but both colors are filtered ( blurred ) by a 2D Gaussian function with standard deviation ( σ ) of 30 nm ( second column ) or 220 nm ( third column ) to simulate the finite localization precision of super-resolution microscopy or traditional resolution of diffraction-limited microscopy , respectively .",
"Scale-bars are 100 nm .",
"( b ,",
"d ) Cross-correlations C",
"( r ) between magenta proteins and green disordered components were tabulated from 100 images similar to those shown in panels of a and c .",
"Solid lines represent C",
"( r ) tabulated from fully sampled images ( top panels of a ,",
"c ) and points with error bounds represent C",
"( r ) tabulated from under-sampled images ( bottom panels of a ,",
"c ) .",
"Without blurring , it is clearly evident that green components are depleted from the local area around proteins ( C",
"( r ) < 1 for short separation distances",
"( r ) in simulations of both clustered and unclustered proteins .",
"Blurring by the super-resolution localization precision ( σ = 30 nm ) dramatically reduces the magnitude of depletion in the absence of protein clustering ( blue points in",
"b ) , but depletion is still apparent when proteins are clustered ( blue points in",
"d ) .",
"This is because the size of the protein cluster is on the same order as the length scale of the Gaussian blurring .",
"In contrast , no significant depletion is observed when blurring is applied to mimic conventional fluorescence microscopy ( σ = 220 nm ) because the length scale of blurring is much longer than the size of protein and membrane phase-like structures .",
"These results visually demonstrate that finite resolution limits reduce the magnitude of observed correlations .",
"Also , in all cases , cross-correlation functions tabulated from under-sampled images reproduce those of the fully sampled images within error bounds even though the images are visually very different .",
"We also note that experimental super-resolution images are not able to accurately capture the fraction of the membrane occupied by one type of phase-like domain .",
"For the example of the unclustered receptor with σ = 30 nm , the fully-sampled image gives the false impression that the majority of the frame is occupied by green ( disordered ) components , even though it is actually only 50% of the total area .",
"In contrast , the under-sampled image gives the visual impression that less than half of the frame is occupied by green disordered components .",
"Both finite lateral resolution and under-sampling in space make it difficult to accurately determine the surface fraction occupied by phase-like domains in experimentally acquired super-resolution images . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 00610 . 7554/eLife . 19891 . 007Figure 1—figure supplement 4 . Correlation functions from individual cells and average curves . Colored lines are cross-correlation curves from individual cells that contribute to the average curves presented in Figure 1c .",
"The large filled symbols represent the average curve and error bars indicate the standard error of the mean between individual curves at each interparticle distance . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 00710 . 7554/eLife . 19891 . 008Figure 1—figure supplement 5 . Distribution of correlation function values closely matches the width expected from single measurement errors . The histograms show the distributions of C ( r < 25 nm ) values obtained from single cell measurements under each of the conditions indicated .",
"The curved lines indicate the best fit Gaussian function to each histogram , demonstrating that values are normally distributed .",
"The horizontal errorbar is centered around the average value and indicates the magnitude of the average error ( dC2 ) determined by estimating error on single cell measurements of C",
"( r ) as described in the Materials and Methods .",
"Since dC2",
"( r ) is not calculated for the first spatial bin ( r < 25 nm ) , we instead use the next bin ( 25 nm < r < 50 nm ) for this comparison which likely underestimates the single cell error .",
"The center of this errorbar is placed at the mean value of C ( r < 25 nm ) which is not always the center of the Gaussian fit .",
"The observation that the average error bar from a single cell measurement is close to the width of the distribution of single cell measurements indicates that errors are dominated by sampling statistics and not by other types of cell-to-cell variation . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 00810 . 7554/eLife . 19891 . 009Figure 1—figure supplement 6 . The cross-correlation amplitude is weakly dependent on lipid probe expression level . The first spatial bin of the cross-correlation C ( r < 25 nm ) is plotted against the density of the mEos3 . 2 probe for individual cells .",
"Surface density of mEos3 . 2 is determined by fitting the autocorrelation as described in Methods .",
"The solid line shows a moving average of the points , the lighter shaded region shows the standard error and the darker shaded region shows the standard error of the mean of C ( r < 25 nm ) .",
"Higher density of lipid probe has negligible effect on TM cross-correlations with clustered CTxB , but has a small effect on PM correlations with clustered CTxB , acting to reduce the cross-correlation at very high densities of mEos3 . 2 expression . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 00910 . 7554/eLife . 19891 . 010Figure 1—figure supplement 7 . Representative images from Figure 1 . Representative images from conditions included in average curves but not shown in Figure 1 .",
"Scale bars are 5 µm .",
"( left )",
"Cells expressing mEos3 . 2-TM were labeled with CTxB-biotin that was then clustered with streptavidin-Atto655 .",
"( right )",
"Cells expressing both mEos3 . 2-GG and YFP-TM .",
"TM was clustered using a biotinylated anti-GFP antibody followed by streptavidin-Atto655 . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 010 We directly observed the sorting of peptides to and away from protein clusters in cell membranes using multi-color super-resolution fluorescence localization imaging ( Betzig et al . , 2006; Hess et al . , 2006; Heilemann et al . , 2009 ) and a quantitative cross-correlation analysis ( Sengupta et al . , 2011; Veatch et al . , 2012; Stone and Veatch , 2015 ) following methods detailed in Materials and methods .",
"First , we stabilized a disordered domain in the plasma membrane by clustering transiently expressed TM through the binding of primary and secondary antibodies , then imaged these clusters in combination with transiently expressed PM or GG peptides ( Figure 1b , left panel ) .",
"Pair cross-correlation functions indicate that TM clusters are enriched in GG and depleted of PM on average compared to the membrane as a whole ( Figure 1c , left panel ) , consistent with the partitioning of these probes into disordered domains in GPMVs .",
"In separate experiments we stabilized an ordered domain by clustering biotinylated CTxB bound to endogenous GM1 with streptavidin , and imaged these clusters in combination with PM or TM peptides ( Figure 1b , right panel ) .",
"Pair cross-correlation functions indicate that PM is enriched and TM is depleted on average within CTxB clusters ( Figure 1c , right panel ) , in good agreement with the partitioning of these probes into ordered domains in GPMVs .",
"The spatial heterogeneity observed within TM or CTxB clusters is subtle; we observed at most 20% enrichment or depletion of the peptide probes within the clustered protein domains , although this is an underestimate of the actual enrichment or depletion due to the finite lateral resolution of the measurement ( Figure 1—figure supplement 3 ) .",
"Relevant to this point , we found that TM clusters ( 68 nm ) were on average larger than CTxB clusters ( 40 nm ) , which were on the order of our cumulative resolution ( 40 nm ) in the measurements summarized in Figure 1c .",
"It is possible that the weaker enrichment and depletion of lipid probes within CTxB clusters as compared to TM clusters is simply due to the limited resolution of these measurements .",
"Cross-correlation curves are aggregated from multiple ( 8-58 ) single-cell measurements , and error bars on Figure 1c indicate the standard error of the mean ( SEM ) between curves generated from single cells ( Figure 1—figure supplement 4 ) .",
"Error in these measurements was dominated by probe sampling statistics ( Figure 1—figure supplement 5 ) because the surface density of probes ( 2–20 μm−2 ) is such that only a few peptides co-localize with nanosized clustered protein domains within an image of a single chemically fixed cell .",
"We note that the apparent surface area occupied by fluorescent peptides is likely a vast under-estimate of the surface area occupied by ordered or disordered phase-like domains since these probes are only one of many membrane components that likely occupy these domains , leading to inherent under-sampling of space ( Figure 1—figure supplement 3 ) ( Stone et al . , 2017 ) .",
"In addition , incomplete spatial sampling by fluorescent peptides can give rise to the appearance of peptide self-clustering , since a single mEos3 . 2 fluorophore can reversibly photo-switch ( Endesfelder et al . , 2011 ) and therefore is likely detected multiple times over the course of a measurement ( Veatch et al . , 2012; Stone et al . , 2017 ) .",
"The cross-correlation analysis used here has advantages over other co-clustering algorithms for images with low spatial sampling , and can detect enrichment or depletion of probes even when this effect is not evident by visual inspection of images , as illustrated in Materials and methods .",
"While in principle the cross-correlation analysis is insensitive to probe surface densities beyond an impact on signal to noise ( Figure 1—figure supplement 3 ) ( Veatch et al . , 2012 ) , we observe a weak expression level dependence of PM recruitment to CTxB clusters , possibly suggesting that probe expression impacts the mixing properties of the plasma membrane as a whole ( Figure 1—figure supplement 6 ) .",
"Both the expression-level dependence of probe partitioning and the small size of CTxB and TM clusters impede us from drawing quantitative conclusions regarding the magnitude of enrichment or depletion of peptides into domains .",
"Instead , we draw conclusions regarding whether probes are enriched or depleted within our sensitivity limits , which is not impacted by either of these factors .",
"Taken together , these findings indicate that clustered plasma membrane proteins can stabilize domains spanning both plasma membrane leaflets that sort established markers of ordered and disordered domains in intact cell membranes .",
"Importantly , we observe depletion of markers from domains of the alternate phase , as well as equivalency between order-and disorder-driven sorting .",
"These are properties of liquid-ordered and liquid-disordered domains in phase separated membranes ( Veatch and Keller , 2005 ) ; therefore we refer to them as phase-like domains .",
"We used similar methods to probe membrane heterogeneity in the vicinity of BCR and BCR receptor clusters .",
"Endogenously expressed BCR was labeled with a biotinylated f ( Ab ) 1 against IgM , BCR was clustered with streptavidin acting as a generic antigen , and BCR clusters were imaged in combination with transiently expressed PM or TM peptides ( Figure 2 ) .",
"The sorting of phase sensitive peptides with respect to BCR clusters was observed in chemically fixed CH27 B cells ( Figure 2a ) , live CH27 B cells ( Figure 2b ) , and chemically fixed primary mouse B cells ( Figure 2c ) .",
"In all cases we found that the PM peptide was enriched and the TM peptide was excluded from BCR clusters .",
"Cross-correlation curves were aggregated from multiple single-cell measurements , and error bars indicate the SEM between curves generated from single cells ( Figure 2—figure supplement 1 ) .",
"Variance in these measurements is dominated by probe sampling statistics ( Figure 2—figure supplement 2 ) and lipid probe expression density only weakly impacts the cross-correlation between BCR and lipid probes ( Figure 2—figure supplement 3 ) .",
"Again , we found that the expression level of phase sensitive peptides impacts the magnitude but not the sign of peptide partitioning with respect to clustered BCR , allowing us to determine the type of domain stabilized by BCR clusters if not its quantitative composition .",
"The direct measurements of peptide sorting shown here are generally consistent with past FRET and biochemical isolation measurements that argued that clustered BCR resides within ordered membrane domains ( Cheng et al . , 1999; Pierce , 2002; Sohn et al . , 2006 , 2008b ) .",
"The association between clustered BCR and the ordered domain marker PM appears more sustained in our imaging measurements than was observed in past reports using FRET ( Sohn et al . , 2006 , 2008b ) , possibly due to the different length-scales probed by these methods . 10 . 7554/eLife . 19891 . 011Figure 2 . BCR clusters localize within ordered membrane domains .",
"( Upper panels )",
"Representative reconstructed super-resolution images of the BCR and PM in chemically fixed",
"( a ) and live",
"( b ) CH27 B cells , and chemically fixed primary B cells",
"( c ) .",
"Scale-bars are 5 µm and 500 nm in the inset .",
"( Lower panels )",
"Average cross-correlation curves , C",
"( r ) , between BCR and phase markers .",
"Error-bars indicate the SEM between cells .",
"In",
"( a ) cells were chemically fixed either 1 min following BCR clustering ( 1 min Ag , left ) or 5 min after BCR clustering ( 5 min Ag , right ) .",
"In",
"( b ) , data was acquired from live cells between 0 and 6 min following BCR clustering .",
"In",
"( c ) , BCR was clustered for 5 min prior to chemical fixation .",
"In all cases , the order-favoring peptide ( PM ) was enriched and the disorder-favoring peptide ( TM ) was depleted from BCR clusters .",
"Curves from individual cells are shown in Figure 2—figure supplement 1 .",
"Curves are averaged over the following number of cells:",
"( a ) 1 min BCR and PM ( 18 ) or TM ( 11 ) ; 5 min BCR and PM ( 21 ) or TM ( 10 ) .",
"( b ) BCR and PM ( 4 ) or TM ( 4 ) .",
"( c ) BCR and PM ( 4 ) or TM ( 5 ) .",
"Correlation curves from right column in",
"( a ) were used to make a schematic figure showing enrichment and depletion of probes around BCR clusters in Figure 2—figure supplement 4 .",
"Representative images for conditions not shown here can be found in Figure 2—figure supplement 10 . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01110 . 7554/eLife . 19891 . 012Figure 2—figure supplement 1 . Correlation functions from individual cells and average curves . Cross-correlation curves from individual cells ( colored lines ) are averaged to obtain the curves shown in Figure 2 ( black lines with errorbars ) .",
"Error bounds indicate the standard error of the mean between curves at each interparticle distance .",
"Average cross-correlations are computed from the number of cells ( N ) indicated on each plot . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01210 . 7554/eLife . 19891 . 013Figure 2—figure supplement 2 . Distribution of correlation function values closely matches the width expected from single measurement errors . The histograms show the distributions of C ( r < 25 nm ) values obtained from single cell measurements in fixed cells where clustered BCR was imaged with the anchor probes indicated .",
"The curved lines indicate a Gaussian fit to each histogram , demonstrating that values are normally distributed .",
"The horizontal errorbar is centered around the average value and indicates the magnitude of the average error ( dC2 ) determined by estimating error on single cell measurements of C",
"( r ) as described in Methods .",
"Since dC2",
"( r ) is not calculated for the first spatial bin ( r < 25 nm ) , we instead use the next bin ( 25 nm < r < 50 nm ) for this comparison which likely underestimates the single cell error .",
"The center of this errorbar is placed at the mean value of C ( r < 25 nm ) which is not always the center of the Gaussian fit .",
"The observation that the average error bar from a single cell measurement is close to the width of the distribution of single cell measurements indicates that errors are dominated by sampling statistics and not by other types of cell-to-cell variation . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01310 . 7554/eLife . 19891 . 014Figure 2—figure supplement 3 . Dependence of cross-correlation amplitudes on lipid probe expression levels . The first spatial bin of the cross-correlation C ( r < 25 nm ) is plotted against the density of the mEos3 . 2 probe for individual cells .",
"Surface density of mEos3 . 2 is determined by fitting the autocorrelation as described in Methods .",
"The solid line shows a moving average of the points , the lighter shaded region shows the standard error and the darker shaded region shows the standard error of the mean of C ( r < 25 nm ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01410 . 7554/eLife . 19891 . 015Figure 2—figure supplement 4 . PM and TM cross-correlation functions have a larger correlation length than BCR autocorrelation functions . Conceptual diagram showing correlation functions from CH27 cells fixed 5 min following BCR clustering that were smoothed and made symmetric about the y axis .",
"This figure highlights the length scale differences between the BCR autocorrelation ( blue ) and cross-correlations between BCR and PM ( black ) and TM ( magenta ) .",
"The range of the BCR autocorrelation is a measure of the size of BCR clusters , and BCR correlations with PM and TM extend to larger distances .",
"The magnitude of the BCR autocorrelation is much greater than that of the cross-correlations shown .",
"To facilitate visual comparisons , the vertical scale for the BCR autocorrelation , G",
"( r ) , is different than the one used for the cross-correlations , C",
"( r ) , as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01510 . 7554/eLife . 19891 . 016Figure 2—figure supplement 5 . Cross-correlations between clustered BCR and PM are reduced in the presence of a signaling inhibitor . C",
"( r ) between PM and BCR in untreated cells and cells treated with 40 µM of the Src kinase inhibitor PP2 and chemically fixed either one minute",
"( a ) or five minutes",
"( b ) following antigen addition .",
"The number of cells imaged is included in the legend .",
"PM enrichment in BCR clusters is reduced in PP2 treated cells , suggesting that downstream signaling acts to amplify formation of an ordered domain around BCR clusters . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01610 . 7554/eLife . 19891 . 017Figure 2—figure supplement 6 . Cross-correlations in live cells are calculated by averaging correlations between non-simultaneous frames . Cross-correlations between probes in live cells suffer from poor statistics when only simultaneous frames are used to determine the cross-correlation .",
"However cross-correlations between non-simultaneous frames can also be averaged when the time evolution of the cross-correlation is sufficiently slow or predictable .",
"Here , we average correlations for probes observed in the same frame ( τ = 0 ) with correlations from frames shifted in time up to 50 frames ( τ = 50 ) or approximately 1 s .",
"Shown are selected spatial bins from the steady state cross-correlation for clustered BCR with PM ( left ) and TM ( right ) from data acquired on a single cell .",
"Since the cross-correlation does not evolve dramatically between τ = 0 and τ = 50 , cross-correlation curves are averaged together over this time window . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01710 . 7554/eLife . 19891 . 018Figure 2—figure supplement 7 . The mobility of lipid probes is not altered when in close proximity to BCR clusters . The mobility of anchors in close proximity to BCR clusters was probed in the same two-color live cell super-resolution experiments presented in Figure 2 . The cumulative step-size distributions shown were either assembled from all steps in probe trajectories ( red and blue curves for BCR and anchor probes , respectively ) or from the subset of steps in probe trajectories detected within 100 nm of a BCR localization in the same image frame ( green bars ) .",
"Step-sizes for correlated probes were determined from the probe positions in the immediately preceding and following frames .",
"Correlated Lyn diffuses somewhat more slowly than the total population of Lyn , most likely due to binding to BCR .",
"When close to BCR , the lipid probes PM and TM exhibit the same mobility as their respective total populations , indicating that membrane domains do not alter protein mobility at the temporal and spatial scales probed .",
"NLyn = 8 , NPM = 14 , NTM = 8 . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01810 . 7554/eLife . 19891 . 019Figure 2—figure supplement 8 . Cross-correlations between clustered BCR and PM are reduced but still observable at physiological temperatures . Average cross-correlation ( C",
"( r ) ) between PM and BCR at room temperature or at growth temperature ( 37°C ) one minute following antigen addition ( NRT = 18 , N37 = 11 ) .",
"PM enrichment in BCR clusters is reduced but still present at 37°C , indicating that domains around BCR persist at physiological temperatures . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 01910 . 7554/eLife . 19891 . 020Figure 2—figure supplement 9 . Cross-correlations between PM and unclustered BCR or CTxB are near detection limits . Representative images ( top ) showing cells expressing PM-mEos3 . 2 and labeled with Atto655 anti-IgM f ( Ab ) 1 ( left ) or Atto655-CTxB ( right ) , where BCR or CTxB labels are not clustered with streptavidin .",
"Scale bars are 5 µm .",
"Average cross-correlations , C",
"( r ) ( bottom ) , between unclustered BCR and lipid probes ( left ) and unclustered CTxB and lipid probes ( right ) .",
"Curves indicate an average over multiple cells ( N ) : BCR and PM ( 14 ) , CTxB and PM ( 18 ) .",
"Subtle enrichment of PM is evident in both cases . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 02010 . 7554/eLife . 19891 . 021Figure 2—figure supplement 10 . Representative images from Figure 2 . Representative images from conditions included in average curves but for which images are not shown in Figure 2 . Scale bars are 5 µm .",
"In all images , IgM BCR is labeled with f ( Ab ) 1 conjugated to Atto655 and clustered with streptavidin .",
"TM is expressed as a mEos3 . 2 fusion protein .",
"From left to right: CH27 cell fixed 1 min following antigen addition; CH27 cell fixed 5 min following antigen addition; live CH27 cell reconstructed from frames following antigen addition; murine primary B cell fixed 5 min following antigen addition . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 021 We find that both PM enrichment and TM depletion extend beyond BCR clusters themselves ( Figure 2—figure supplement 4 ) .",
"It is likely that this extended domain arises from additional signaling structures assembled proximal to BCR , as is observed in other immune-receptor signaling systems ( Balagopalan et al . , 2015 ) .",
"For example , palmitoylated adapter proteins involved in signal transduction such as LAB/LAT2/NTAL may incorporate into activated BCR microclusters and act to extend the domain ( Mutch et al . , 2007; Malhotra et al . , 2009 ) .",
"Further , PM enrichment was reduced in cells treated with the Src kinase inhibitor PP2 prior to receptor clustering and fixation ( Figure 2—figure supplement 5 ) , suggesting that ordered domain stabilization is amplified by receptor activation and the recruitment of down-stream signaling partners .",
"The same magnitude of PM and TM co-localization with BCR clusters was observed in live cells ( Figure 2b ) as in chemically fixed cells , indicating that co-localization is not an artifact of chemical fixation .",
"Here co-localization was quantified using a steady-state cross-correlation function ( Stone and Veatch , 2015 ) .",
"Additional sensitivity was obtained in these measurements by including probe pairs imaged within a time separation of up to 50 frames or approximately 1 s since we did not observe significant changes in steady state correlations over this window ( Figure 2—figure supplement 6 ) .",
"We note that PM proximal to BCR did not exhibit altered mobility in live cells , indicating that PM enrichment arises from weak and/or transient interactions ( Figure 2—figure supplement 7 ) .",
"As a counter-example , Lyn proximal to BCR does exhibit slowed diffusion , likely due to specific Lyn-BCR binding interactions .",
"Videos 1–3 show single molecule localizations compiled over time for BCR-PM , BCR-TM , and BCR-Lyn , respectively .",
"These videos demonstrate that the distributions of PM and TM do not change dramatically upon BCR clustering . 10 . 7554/eLife . 19891 . 022Video 1 . Reconstructed image time lapse of single molecule localizations from live cell measurements of BCR ( magenta ) and PM ( green ) .",
"Individual images are reconstructed using 100 frames ( 2",
"s ) of single molecule images , receptors are crosslinked at time = 0 , and the scale-bar is 5 µm .",
"Single BCR or PM proteins are imaged over multiple frames and each produce clouds of localizations whose extent depend on the protein mobility as well as the typical time that a probe remains activated .",
"This combined with under-sampling of the molecules that are present gives rise to the self-clustered appearance of probes ( especially BCR ) prior to receptor clustering .",
"Overall , there is not an obvious reorganization of PM after BCR is clustered . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 02210 . 7554/eLife . 19891 . 023Video 2 . Reconstructed image time lapse of single molecule localizations from live cell measurements of BCR ( magenta ) and TM ( green ) .",
"Individual images are reconstructed using 100 frames ( 2",
"s ) of single molecule images , receptors are crosslinked at time = 0 , and the scale-bar is 5 µm .",
"Single BCR or TM proteins are imaged over multiple frames and produce clouds of localizations whose extent depend on the protein mobility as well as the typical time that a probe remains activated .",
"This combined with under-sampling of the molecules that are present gives rise to the self-clustered appearance of probes ( especially BCR ) prior to receptor clustering .",
"Overall , there is not an obvious reorganization of PM after BCR is clustered . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 02310 . 7554/eLife . 19891 . 024Video 3 . Reconstructed image time lapse of single molecule localizations from live cell measurements of BCR ( magenta ) and Lyn kinase ( green ) .",
"Individual images are reconstructed using 100 frames ( 2",
"s ) of single molecule images , receptors are crosslinked at time = 0 , and the scale-bar is 5 µm .",
"Single BCR or Lyn proteins are imaged over multiple frames and produce clouds of localizations whose extent depend on the protein mobility as well as the typical time that a probe remains activated .",
"This combined with under-sampling of the molecules that are present gives rise to the self-clustered appearance of probes ( especially BCR ) prior to receptor clustering .",
"A population of Lyn is immobile or diffuses more slowly .",
"These proteins appear as brighter spots since numerous localizations occur in the same location . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 024 We also observed sorting of PM and TM peptides with respect to BCR clusters imaged in primary mouse B cells fixed 5 min following antigen stimulation ( Figure 2c ) that is qualitatively consistent with observations in the CH27 cell line .",
"Interestingly , the magnitude of sorting is increased in primary cells compared to CH27 cells .",
"This may be a biological consequence of the LPS treatment required to maintain cell viability during transient transfection , or due to other differences in membrane composition between these two cell types .",
"PM was also enriched within BCR clusters when CH27 cells were chemically fixed at 37°C ( Figure 2—figure supplement 8 ) , indicating that ordered domains are formed at growth temperatures , although the magnitude of enrichment is reduced compared to the room temperature examples shown in Figure 2a .",
"We note that BCR clustering elicits a cellular response at both temperatures .",
"We additionally observed weak PM enrichment around unclustered BCR in chemically fixed CH27 cells , although the magnitude of this sorting is on the edge of the sensitivity limits of our imaging system ( Figure 2—figure supplement 9 ) .",
"This weak signal could indicate reduced partitioning of monomeric or pre-clustered BCR with ordered domains prior to streptavidin-induced clustering , or could simply reflect that domain sizes are at or below the finite lateral resolution of these measurements ( ~40 nm ) ( illustrated in Figure 1—figure supplement 3 ) .",
"At this time , we cannot comment on the oligomerization state of BCR prior to enforced clustering with streptavidin , because single color images of BCR contain over-counting artifacts that render monomers indistinguishable from small oligomers ( Veatch et al . , 2012 ) .",
"Interestingly , the magnitude of PM sorting with respect to unclustered BCR is comparable to the sorting of this peptide observed with respect to unclustered CTxB ( Figure 2—figure supplement 9 ) .",
"Improved lateral resolution is needed to systematically investigate the lateral organization of receptors and peptides in intact cells without receptor clustering , and may be enabled by recent improvements in fluorophores and imaging modalities ( Huang et al . , 2013; Grimm et al . , 2015 , 2016 ) .",
"Together , the results presented in Figures 1 and 2 support the view that BCR clustering locally stabilizes an ordered lipid domain .",
"Sorting of order- and disorder-preferring probes around BCR clusters follows the same trends observed for clusters of an established marker of ordered domains , CTxB , and is consistent in fixed , live , and primary cells .",
"Domains formed around clustered proteins share many of the features of domains observed in model membranes .",
"Individual lipids are free to diffuse into and out of domains , suggesting that these domains are fluid .",
"The amount of enrichment observed in these experiments is also consistent with expectations from ordered-disordered phase fluctuations in model systems above their transition temperatures ( Veatch et al . , 2008; Machta et al . , 2012; Zhao et al . , 2013 ) , in contrast to the strong recruitment described in early work on membrane rafts ( Simons and Ikonen , 1997 ) .",
"Finally , the symmetry in the sign of correlations observed for order- and disorder-preferring probes and order- or disorder-preferring protein clusters is also consistent with the two liquid phase regime in model systems ( Machta et al . , 2012 ) .",
"At the same time , the effects of lipid phase-mediated sorting are likely superimposed on other interactions present within the complex milieu of the membrane , such as electrostatic interactions between charged peptides and lipids .",
"The observed sorting of minimal peptides suggests that the localization of full-length proteins is also impacted by their membrane anchoring motifs .",
"We investigated the role of membrane domains in the localization of two critical regulators of BCR activation , Lyn kinase and CD45 phosphatase ( Dal Porto et al . , 2004 ) , by comparing their spatial distribution around BCR to that of their membrane anchor peptides ( Figure 3a ) .",
"Both CD45 and its minimal anchor peptide CD45TM were excluded from BCR clusters to the same extent , indicating that membrane domain interactions are sufficient to drive this partitioning .",
"Lyn kinase was more enriched in BCR clusters than its membrane anchor PM , consistent with the direct binding of Lyn to phosphorylated ITAM sequences on BCR mediated by SH2 interaction domains in full-length Lyn ( Johnson et al . , 1995 ) .",
"The effective interaction energy between membrane anchors and BCR is given by the potential of mean force ( PMF ) , which is simply obtained from measured cross-correlation functions ( Veatch et al . , 2012; Stone and Veatch , 2015 ) .",
"PMFs between the BCR and PM or Lyn indicate that the anchor sequence provides over one third of the energy associated with Lyn-BCR interactions ( Figure 3b ) . 10 . 7554/eLife . 19891 . 025Figure 3 . Ordered domains promote tyrosine phosphorylation .",
"( a ) Average cross-correlation functions ( C",
"( r ) , left ) and representative super-resolution images ( right ) demonstrating that full-length proteins and their minimal membrane anchors sort with respect to clusters of both BCR ( top ) and CTxB ( bottom ) .",
"The correlations presented represent an average over multiple ( N ) individual cells: Between BCR and Lyn ( 4 ) , PM ( 21 ) , CD45 ( 10 ) , CD45TM ( 10 ) ; between CTxB and Lyn ( 19 ) , PM ( 58 ) , CD45 ( 11 ) , and CD45TM ( 5 ) .",
"( b ) Lyn and PM distributions with respect to B cell receptor clusters expressed as the potential of mean force ( PMF ) .",
"( c ) Both BCR and CTxB domains are sites of tyrosine phosphorylation ( pY ) as detected through a generic anti-pY antibody ( 4G10 ) , while disordered TM domains were not enriched in pY proteins .",
"Curves are averaged over the following number of cells: BCR and pY ( 14 ) , CTxB and pY ( 13 ) , and TM and pY ( 7 ) .",
"( d ) Activated Lyn ( pY-397 ) was more enriched in CTxB clusters than Lyn as a whole , indicating that ordered domains favor activation of this protein .",
"Curves are averaged over the following number of cells: CTxB and pY Lyn ( 40 ) , CTxB and Lyn ( 49 ) .",
"( e ) Schematic of membrane domains stabilized by BCR and CTxB clusters .",
"Curves and color-scale at bottom are quantitative representations of the relative enrichment or depletion of the components indicated as represented in parts a and c .",
"Scale-bars are 5 µm and 500 nm in the inset .",
"Additional representative images are shown in Figure 3—figure supplement 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 02510 . 7554/eLife . 19891 . 026Figure 3—figure supplement 1 . Cell surface clustering of cholera toxin subunit B elicits calcium mobilization in B cells . Cytosolic calcium levels were monitored in CH27 B cells both before and after biotinylated CTxB was clustered with streptavidin using the calcium indicator Fluo-4 as described in Methods .",
"Colored curves represent raw fluorescence intensity traces for single cells and the average response of 40 cells is shown in black .",
"The blue shaded region denotes +/- one standard deviation between the averaged cells .",
"One reason for the broad width of this distribution is that individual cells oscillate between high and low fluorescent states , as apparent in the single cell traces . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 02610 . 7554/eLife . 19891 . 027Figure 3—figure supplement 2 . CTxB clusters are not highly correlated with BCR . Average cross-correlation between clustered CTxB and BCR indicates that these two proteins are not colocalized .",
"Biotinylated CTxB was clustered by streptavidin conjugated to Atto 655 and cells were fixed prior to labeling BCR with a f ( Ab ) 1 fragment conjugated to Alexa 532 .",
"The lack of pronounced cross-correlation between BCR and clustered CTxB indicates that CTxB is not forcing BCR to be clustered nor was it strongly recruiting BCR .",
"Interestingly , the weak enrichment of BCR within CTxB clusters is similar to the enrichment of membrane anchored probes around clustered CTxB and BCR , suggesting that the enrichment stems from domain partitioning .",
"The curve is an average of 6 cells with errorbars showing the standard error of the mean . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 02710 . 7554/eLife . 19891 . 028Figure 3—figure supplement 3 . Subtle increases in protein phosphotyrosine levels in response to CTxB clustering are suggested by western blots of whole cell lysates . CH27 cells were treated as indicated above lanes , and anti-phosphotyrosine western blots were performed using cell lysates .",
"The top shows a representative western blot , where identity of Syk , Lyn , and BCR bands are estimated from the molecular weight marker shown at right .",
"The actin band was determined by stripping and reblotting for actin .",
"The bottom shows the quantification of four western blots from two biological replicates , where band intensity was background subtracted and normalized by the actin intensity for that lane .",
"Results are suggestive of increased phosphorylation of BCR , Lyn , and Syk following CTxB binding and clustering although none of the conditions reach p=0 . 05 significance levels when probed using a two sample t-test .",
"Errorbars show standard error of the mean between the four blots . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 02810 . 7554/eLife . 19891 . 029Figure 3—figure supplement 4 . Representative images from Figure 3 . Representative images from conditions included in average curves in Figure 3 are shown here .",
"Scale bars are 5 µm .",
"( a ) Cells stained for CD45 ( top ) or expressing CD45tm ( bottom ) where either BCR ( top ) or CTxB ( bottom ) was clustered .",
"Average curves are shown in Figure 3a .",
"( b ) Phosphotyrosine ( pTyr ) was immunolabeled in cells where either BCR ( left ) or TM ( right ) was clustered .",
"Average curves are shown in Figure 3c .",
"( c ) Lyn was immunolabeled in CH27 cells where CTxB was clustered .",
"Average curves are shown in Figure 3d . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 029 We demonstrated that ordered domains are sufficient to sort full length signaling proteins by also monitoring their distribution with respect to CTxB clusters ( Figure 3a ) .",
"Again we found that CD45 was depleted and Lyn was enriched in these ordered domains .",
"In this case , full-length proteins partitioned similarly to their minimal anchor peptides , as expected since CTxB does not bind directly to either of these proteins .",
"The sorting of proteins into ordered membrane domains is also sufficient to locally alter their phosphorylation state .",
"This was seen by visualizing the distribution of phosphorylated protein tyrosine residues ( pY ) with respect to protein clusters using a generic anti-pY antibody ( Figure 3c ) .",
"BCR and CTxB clustering both induce local tyrosine phosphorylation co-localized with clusters , while TM clustering does not .",
"This direct observation agrees with previous indirect measures of localized phosphorylation ( Harder and Simons , 1999; Cheng et al . , 1999 ) .",
"As expected , total pY enrichment was reduced in CTxB clusters compared to BCR clusters , since CTxB itself lacks sites for tyrosine phosphorylation and does not directly bind to additional proteins containing pY .",
"However , enrichment in ordered domains stabilized by CTxB clustering is sufficient for phosphorylation of resident proteins , as is evident from the stronger co-localization of trans-activated ( pY397 ) Lyn with CTxB clusters compared to overall Lyn ( Figure 3d ) .",
"The membrane remodeling that occurs upon CTxB clustering is also sufficient to trigger a cellular response .",
"In agreement with past reports ( Francis et al . , 1992 ) , we found that CTxB clustering leads to Ca2+ mobilization ( Figure 3—figure supplement 1 ) without significantly altering the distribution of BCR ( Figure 3—figure supplement 2 ) .",
"CTxB binding and clustering also resulted in subtle increases in tyrosine phosphorylation of multiple protein species detected within cellular extracts probed via Western blot ( Figure 3—figure supplement 3 ) .",
"The imaging results shown in Figure 3 draw a connection between the lipid-mediated protein sorting observed in Figures 1 and 2 and signaling function .",
"We showed that ordered domains contribute a substantial fraction of the free energy required to concentrate the kinase Lyn and all of the free energy required to deplete the phosphatase CD45 from BCR clusters .",
"Through this sorting , ordered domains provide a local environment that favors tyrosine phosphorylation , as supported by correlations between CTxB clusters and anti-pY antibodies even in the absence of specific recruitment of signaling machinery through protein-protein interactions .",
"It is reasonable to expect that the ~50% increase in the ratio of Lyn to CD45 due to sorting by ordered domains could cause a significant change in BCR phosphorylation levels if we compare to results obtained for the related T cell receptor ( TCR ) system .",
"In a reconstituted system , TCR phosphorylation was shown to have switch-like dependence on the relative concentrations of the Src kinase Lck , which is the analog of Lyn in the TCR system , and CD45 at physiological levels ( Hui and Vale , 2014 ) .",
"As a result , small increases in Lck concentration and decreases in CD45 concentration were shown to produce large shifts in TCR phosphorylation , and this behavior likely also applies to the BCR system investigated here .",
"Additionally , the actual enrichment and depletion of probes around BCR and CTxB is larger than the measured values presented here since the real spatial distributions are convolved with the finite resolution of the measurement to give the observed cross-correlations .",
"Lastly , local activation of Lyn within ordered domains also provides a potential mode of positive regulation .",
"Concentration of Lyn in ordered domains and exclusion of phosphatases would be expected to favor Lyn trans-activation at Y397 and prevent inactivation .",
"This would create an environment within ordered domains where Lyn is not only more concentrated but also more active , as has been suggested previously ( Young et al . , 2003 ) and supported by the results shown in Figure 3d .",
"Thus , distinct membrane environments could influence both the local concentration and activity of proteins , and these effects may amplify or negate one another to determine an overall signaling outcome .",
"Our observations of protein sorting by ordered domains suggest a mechanism for receptors to become phosphorylated upon clustering via differential partitioning of proteins regulating BCR phosphorylation .",
"Figure 4 describes a predictive model that reproduced the sorting behavior of kinase and phosphatase anchor peptides observed experimentally ( Figure 4a and Figure 4—figure supplement 1 ) .",
"The model consists of receptors that can be phosphorylated by kinases and dephosphorylated by phosphatases .",
"In addition , activated receptors can phosphorylate other receptors , mimicking the actions of receptor-bound kinases ( RBKs ) such as Lyn and Syk ( Johnson et al . , 1995 ) .",
"These protein components are embedded in a heterogeneous membrane represented by a 2D Ising model where extended ordered and disordered domains form at equilibrium through interactions between adjacent components ( Machta et al . , 2011 , 2012 ) .",
"This model represents phase-like heterogeneity as extended composition fluctuations that collectively emerge from weak intermolecular interactions when membranes are positioned near a miscibility critical point , and is supported by experiments in both purified and isolated biological membranes ( Veatch et al . , 2008; Honerkamp-Smith et al . , 2008; Zhao et al . , 2013 ) .",
"Receptors and kinases act as typical ordered components and phosphatases act as typical disordered components .",
"Through this set of minimal assumptions , receptors became collectively activated upon clustering ( Figure 4b and Video 4 ) .",
"Clustered receptors were activated to a lesser extent in the absence of RBK positive feedback , but were not activated in a uniform membrane even with RBK feedback ( Figure 4b and Videos 5 and 6 ) .",
"This localized receptor phosphorylation may favor recruitment and assembly of adapter proteins that mediate the cellular immune response ( Su et al . , 2016b ) . 10 . 7554/eLife . 19891 . 030Figure 4 . A model linking receptor clustering to receptor phosphorylation .",
"( a ) Schematic representation of the model described in the main text .",
"Possible biological analogs of model components are indicated , with “RBK” representing receptor-bound kinases .",
"( b ) Simulation snap-shots ( top ) and sample receptor phosphorylation time-traces ( bottom ) indicate receptors are robustly phosphorylated ( pY ) upon clustering when membranes are heterogeneous .",
"Phosphorylation was diminished in simulations without RBKs and absent in simulations with a uniform membrane .",
"All curves have the same vertical scale and time-lapses of the simulations are in Videos 4–6 .",
"( c ) Simulation snap-shots ( top ) and sample receptor phosphorylation time-traces ( bottom ) for simulations where an ordered domain is stabilized without confining receptors to the domain .",
"In this case , more receptors are included to represent the large number of membrane proteins containing tyrosine residues that can be phosphorylated .",
"The efficiency of receptor phosphorylation depends on the size of the stabilized ordered domain for reasons discussed in the main text .",
"All curves have the same vertical scale .",
"Time-lapses of the simulations are shown in Videos 7–9 .",
"All scale bars are 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03010 . 7554/eLife . 19891 . 031Figure 4—figure supplement 1 . Simulations naturally reproduce experimental kinase and phosphatase distributions with respect to the BCR .",
"( a ) Time-averaged positions of the receptor , kinase , and phosphatase in simulations .",
"Kinases are recruited and phosphatases are excluded from clustered BCR .",
"The location of the receptor cluster is indicated by the dashed white line .",
"( b ) Average cross-correlation functions between unclustered receptors ( top ) or clustered receptors ( bottom ) and kinases and phosphatases from simulation snap-shots .",
"On the right , simulation images are convolved with a Gaussian function with a 30 nm standard deviation prior to conducting the cross-correlation to better mimic experimental conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03110 . 7554/eLife . 19891 . 032Video 4 . Simulated time course of receptor activation upon clustering in a heterogeneous membrane . Simulations are conducted as described in Methods .",
"The positions of receptors ( circles ) , kinases ( green squares ) , and phosphatases ( magenta triangles ) are shown at 1 ms intervals ( representing 1000 simulation updates ) .",
"Inactive receptors are shown in blue and activated ( phosphorylated ) receptors are shown in red .",
"Symbols are drawn larger than the pixels that they represent for clarity .",
"The receptor field is turned on at time = 0 to induce receptor clustering .",
"The time trace shown at the bottom is redrawn from Figure 4b . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03210 . 7554/eLife . 19891 . 033Video 5 . Simulated time course of receptor activation upon clustering in a heterogeneous membrane without the positive feedback loop accomplished through receptor bound kinases . Simulations are conducted as described in Methods .",
"The positions of receptors ( circles ) , kinases ( green squares ) , and phosphatases ( magenta triangles ) are shown at 1 ms intervals ( representing 1000 simulation updates ) .",
"Inactive receptors are shown in blue and activated ( phosphorylated ) receptors are shown in red .",
"Symbols are drawn larger than the pixels that they represent for clarity .",
"The receptor field is turned on at time = 0 to induce receptor clustering .",
"The time trace shown at the bottom is redrawn from Figure 4b . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03310 . 7554/eLife . 19891 . 034Video 6 . Simulated time course of receptor activation upon clustering in a uniform membrane . Simulations are conducted as described in Methods .",
"The positions of receptors ( circles ) , kinases ( green squares ) , and phosphatases ( magenta triangles ) are shown at 1 ms intervals ( representing 1000 simulation updates ) .",
"Inactive receptors are shown in blue and activated ( phosphorylated ) receptors are shown in red .",
"Symbols are drawn larger than the pixels that they represent for clarity .",
"The receptor field is turned on at time = 0 to induce receptor clustering .",
"The time trace shown at the bottom is redrawn from Figure 4b . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03410 . 7554/eLife . 19891 . 035Video 7 . Simulated time course of receptor activation upon stabilization of a large ordered domain . Simulations are conducted as described in Methods .",
"The positions of receptors ( circles ) , kinases ( green squares ) , and phosphatases ( magenta triangles ) are shown at 1 ms intervals ( representing 1000 simulation updates ) .",
"Inactive receptors are shown in blue and activated ( phosphorylated ) receptors are shown in red .",
"Symbols are drawn larger than the pixels that they represent for clarity .",
"The field is turned on at time = 0 to induce a circular ordered domain with a radius of 100 nm .",
"The time trace shown at the bottom is redrawn from Figure 4c . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03510 . 7554/eLife . 19891 . 036Video 8 . Simulated time course of receptor activation upon stabilization of a small , ordered domain . Simulations are conducted as described in Methods .",
"The positions of receptors ( circles ) , kinases ( green squares ) , and phosphatases ( magenta triangles ) are shown at 1 ms intervals ( representing 1000 simulation updates ) .",
"Inactive receptors are shown in blue and activated ( phosphorylated ) receptors are shown in red .",
"Symbols are drawn larger than the pixels that they represent for clarity .",
"The field is turned on at time = 0 to induce a circular ordered domain with a radius of 50 nm .",
"The time trace shown at the bottom is redrawn from Figure 4c . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03610 . 7554/eLife . 19891 . 037Video 9 . Simulated time course receptor activation state within a uniform membrane with an applied field . Simulations are exactly as described for Video 7 but unspecified membrane components are all of the same type ( ordered in this case ) .",
"The positions of receptors ( circles ) , kinases ( green squares ) , and phosphatases ( magenta triangles ) are shown at 1 ms intervals ( representing 1000 simulation updates ) .",
"Inactive receptors are shown in blue and activated ( phosphorylated ) receptors are shown in red .",
"Symbols are drawn larger than the pixels that they represent for clarity .",
"The field is turned on at time = 0 but does not impact the organization of components .",
"The time trace shown at the bottom is redrawn from Figure 4c . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 037 Receptor activation was also observed when an ordered domain was stabilized using an external potential without confining receptors to the domain ( Figure 4c ) , mimicking the CTxB clustering result of Figure 3 . Receptor activation was more subtle in these simulations .",
"This is because only a fraction of receptors reside within the ordered domain at a given time , receptors freely leave the protective environment of the ordered domain , and the local concentration of receptors within the domain is reduced compared to simulations with clustered receptors .",
"In these simulations , we also find that the size of the ordered domain impacts the efficiency of receptor activation .",
"This occurs because the residency time of a receptor within the domain increases with increasing domain size , as does the average number of receptors within the domain .",
"Both of these factors increase the likelihood that the RBK positive feedback will amplify receptor activation .",
"This minimal model provides a framework for interpreting our imaging data and for developing hypotheses for the specific role of lipid-mediated sorting in BCR clustering-induced activation .",
"Here , existing small , dynamic phase fluctuations were stabilized through clustering of ordered components to form a larger , stable domain structure of approximately the size of the cluster .",
"This model quantitatively reproduces our imaging measurements of protein sorting by ordered domains , recapitulates clustering-induced BCR activation , and demonstrates that ordered domain formation alone can be sufficient to activate receptors .",
"Further , phase-like heterogeneity is required for robust activation of BCR under these simulation conditions .",
"The dependence of receptor activation on domain size in simulations where ordered domains are formed without BCR clustering ( Figure 4c ) highlights the collective nature of interactions that determine the local environment of receptors .",
"In order to demonstrate this model’s predictive power , we ran further simulations to probe receptor phosphorylation as the surface fraction of ordered components was varied ( Figure 5a ) .",
"Clustered receptors were more phosphorylated in simulations with a larger fraction of disordered components and were less phosphorylated in simulations with more ordered components .",
"This occurs because varying the surface fraction of ordered vs . disordered components acts to enhance ( or suppress ) the local enrichment and depletion of signaling modulators at receptor clusters ( Figure 5—figure supplement 1 ) .",
"This is also reflected in cross-correlation functions calculated from simulations ( Figure 5b ) that report the co-localization of receptors and the order-preferring kinase for the surface fractions indicated . 10 . 7554/eLife . 19891 . 038Figure 5 . Phosphorylation model predicts the response to changing the fraction of ordered and disordered components .",
"( a ) Representative snap-shots ( top ) and histograms showing receptor phosphorylation ( bottom ) in simulations run with different fractions of ordered and disordered components ( grey and white pixels respectively ) .",
"Receptors ( not shown ) were confined within the red dashed circle .",
"Snap-shots corresponding to conditions in the plot are indicated as colored symbols .",
"( b ) Cross-correlations between receptors and ordered components in simulations run with a variable fraction of ordered and disordered components .",
"Simulations shown are a subset of those displayed in",
"( a ) as indicated by symbols .",
"( c ) Acute cholesterol modulation with MβCD altered the surface fraction of ordered ( dark ) and disordered ( bright ) phases in isolated GPMVs ( top ) and modulated calcium mobilization in response to antigen , measured using the cytoplasmic Ca2+ indicator Fluo-4 ( bottom ) .",
"Values were normalized to the maximum response of untreated cells .",
"Colored symbols on vesicle micrographs and plot indicate equivalent treatments in both measurements .",
"Calcium measurements are an average of at least two biological replicates .",
"( d ) Cross correlations between BCR and PM-mEos3 . 2 in CH27 cells treated with indicated amounts of MβCD ( 18 ) , cholesterol-loaded MβCD ( 18 ) , or left untreated ( 21 ) and then fixed 5 min following antigen addition .",
"Scale bars are 100 nm in simulations and 5 µm in micrographs . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03810 . 7554/eLife . 19891 . 039Figure 5—figure supplement 1 . Kinase and phosphatase partitioning into receptor clusters change dramatically as the surface fraction of ordered components is varied . Time-averaged positions of the receptor , kinase , and phosphatase in simulations with ordered components making up 20%",
"( a ) or 80%",
"( b ) of the simulated membrane .",
"The location of the receptor cluster is indicated by the dashed white line . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 03910 . 7554/eLife . 19891 . 040Figure 5—figure supplement 2 . Averaged and baseline-corrected Fluo-4 intensity curves . Relative Fluo-4 intensity of CH27 cells stimulated with anti-IgM f ( Ab ) 2 with either MβCD treatment , cholesterol treatment , or no treatment .",
"The curves were integrated within the gray box to give the values shown in Figure 5c in the main text . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 04010 . 7554/eLife . 19891 . 041Figure 5—figure supplement 3 . Cholesterol treatments do not alter annexin V staining . CH27 cells were treated with either 10 mM MβCD , 10 mM MβCD loaded with cholesterol , or control buffer in an identical manner as cells from Figure 3c where calcium release was tested .",
"A positive control was also performed by heat treating cells at 60°C for 15 min .",
"Following treatment , cells were washed and stained with annexin Vconjugated to Alexa 488 , which binds to PS on the external leaflet of the plasma membrane when cells are beginning to undergo apoptosis .",
"Cells were washed again after binding to remove unbound annexin Vand total fluorescence was measured using a fluorescence plate reader .",
"The minimal difference in annexin staining between cholesterol treated and untreated cells indicates that the calcium release results shown in Figure 5c are not attributable to changes in cell viability . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 041 These simulation results are qualitatively consistent with functional and imaging data obtained in B cells with modulated cholesterol levels ( Figure 5c , d and Figure 5—figure supplement 2 ) .",
"Acute modulation of cholesterol levels in intact cells with methyl β cyclodextrin ( MβCD ) alters the surface fraction of coexisting ordered and disordered phases in GPMVs imaged at low temperature that were isolated from these cells ( Figure 5c ) .",
"This occurs without an effect on cell viability as measured via annexin V binding to the extracellular surface ( Figure 5—figure supplement 3 ) .",
"Past work indicates that this perturbation can also alter domain composition in vesicles at elevated temperature in a way that is quantitatively predicted by our model of membrane heterogeneity ( Zhao et al . , 2013 ) .",
"In intact B cells , we observed increased calcium mobilization upon BCR clustering when cholesterol levels were acutely lowered with MβCD , in agreement with past work ( Awasthi-Kalia et al . , 2001 ) .",
"We also observed decreased calcium mobilization upon BCR clustering in cells pretreated with MβCD-cholesterol complexes , which act to increase cellular cholesterol levels ( Christian et al . , 1997 ) .",
"The observed changes in calcium mobilization are accompanied by changes in PM cross-correlation with BCR clusters that are also in line with model predictions ( Figure 5d ) .",
"PM is more strongly enriched in BCR clusters in cells pretreated with MβCD than in untreated cells , and less strongly enriched in cells pretreated with MβCD-cholesterol complexes .",
"While the model clearly over-simplifies the behavior of the plasma membrane and the complexity of the BCR signaling pathway , it is able to predict both the functional response associated with membrane perturbation and changes in domain-mediated sorting reported by super-resolution imaging experiments .",
"In spite of the acknowledged plieotropic effects of cholesterol modulation with MβCD ( Munro , 2003; Kwik et al . , 2003 ) , its effects on the surface fraction of ordered vs . disordered phases in GPMVs and the cross-correlation of PM with BCR clusters mirror the changes in the fraction of ordered and disordered components enforced in simulations .",
"Therefore , we expect that modulating cholesterol levels using MβCD impacts the composition of the plasma membrane of B cells in a way that parallels the changes in our minimal model: by varying the surface fraction of ordered vs . disordered components .",
"This in turn alters peptide sorting around BCR clusters .",
"The striking correspondence of the structural and functional outputs of the simulations to experimental measures of PM partitioning and BCR activation provides validation of the model and its assumptions .",
"In conclusion , we directly observed domains in cellular plasma membranes that resemble liquid-ordered and liquid-disordered phases in model membranes .",
"In these experiments we visualized both enrichment and , for the first time , depletion of membrane species from phase-like domains in intact cells .",
"This sorting behavior was induced both by clustering generic membrane proteins with known phase partitioning in GPMVs and by clustering BCR .",
"BCR clustering drove spatial co-localization or segregation of minimal membrane anchors from BCR clusters , consistent with the formation of an ordered phase-like domain .",
"These domains also sorted BCR signaling partners into and out of receptor clusters .",
"Experiments that examined functional outcomes of BCR signaling indicated that formation of an ordered domain through protein clustering creates a local membrane environment that favors protein tyrosine phosphorylation .",
"Functional and imaging results are consonant with a simple but predictive model of membrane phase behavior .",
"Based on these observations and supported by the predictive model , we propose a mechanism where receptor clustering alone can initiate collective BCR activation by stabilizing an ordered membrane domain .",
"This mechanism is distinct from some historical ideas of how “lipid rafts” have been thought to play a role in BCR signaling , which pose that BCR actively partitions into pre-existing ordered domains upon clustering .",
"Instead , our findings support the idea that receptor clusters template a lipid domain in the membrane by stabilizing existing but very subtle and transient membrane composition fluctuations .",
"In the absence of clustering , the size and dynamics of fluctuations allows positive and negative regulators to mix with BCRs .",
"When BCR is clustered , the effect on downstream signaling depends on the size and stability of the resulting domain , which sets the level of access signaling partners have to resident BCR .",
"As a consequence , the structure of the BCR cluster and surrounding membrane can influence the activation state of individual BCRs .",
"Thus , the entire cluster and associated domain , as opposed to single BCRs , comprise the basic signaling unit for the activation of B cells in this context .",
"Super-resolution microscopy is particularly well-suited to capture this action-at-a-distance because it can measure interactions that span the length scale of the BCR cluster .",
"While BCR activation in vivo by natural antigens likely involves signal integration from multiple sources , our work suggests that formation of phase-like ordered domains through a collective ensemble of relatively weak protein-lipid and lipid-lipid interactions impacts overall signaling outcomes .",
"Thus , we both conclude that lipid-mediated interactions can play a significant role in signal transduction from BCRs activated through protein clustering and can describe a plausible mechanism for their action .",
"We suggest that the signaling function conferred by formation of phase-like domains around BCR clusters is an emergent property of a system where signaling molecules are compartmentalized based on their interactions with plasma membrane lipids .",
"By extension , these findings suggest that membrane domains are capable of contributing to a broad array of signal transduction pathways by altering the local concentration of regulatory proteins , shifting the balance of biochemical networks .",
"This compliments existing theories that invoke spatial compartmentalization of receptors and regulatory molecules as a means to control receptor signaling , including size-based exclusion ( Choudhuri et al . , 2005; Cordoba et al . , 2013 ) and 3D protein phase separation ( Su et al . , 2016a ) and places lipid-mediated interactions ( Machta et al . , 2012 ) among other canonical forces that dictate protein interactions in membranes , such as electrostatics , curvature , and adhesion ."
],
[
"f ( Ab ) 1 fragment goat antibody to mouse IgM , µ chain specific ( Jackson ImmunoResearch , West Grove , PA; RRID: AB_2338477 ) was simultaneously chemically modified with Atto 655 NHS ester ( Sigma , St . Louis , MO ) and biotin-X , SSE , 6- ( ( Biotinoyl ) Amino ) Hexanoic Acid , Sulfosuccinimidyl Ester , Sodium Salt ( Sulfo-NHS-LC-Biotin ) ( Invitrogen , Grand Island , NY ) .",
"Modifications were carried out in aqueous solution buffered by 0 . 01 M NaH2PO4 with 0 . 01 M NaH2CO3 , pH 8 . 2 for thirty minutes at room temperature .",
"Reaction products were separated by gel filtration on Illustra NAP-5 columns ( GE Healthcare , Piscataway , New Jersey ) to remove unbound dye from labeled protein .",
"CTxB ( Invitrogen ) was biotylated and conjugated to Atto 655 in-house via similar methods , and both CTxB and f ( Ab ) 1 conjugation was also described previously ( Stone and Veatch , 2015 ) .",
"f ( Ab ) 1 was also conjugated to silicon rhodamine ( SiR ) dye ( Spirochrome , Switzerland ) in conjunction with biotin by similar methods .",
"Streptavidin ( Invitrogen ) and anti-mouse IgG2b ( Jackson ImmunoResearch; RRID: AB_2338463 ) were also conjugated to either Alexa 532 ( Invitrogen ) or Atto 655 by similar methods .",
"CTxB that was only biotinylated ( without Atto 655 conjugation ) was purchased directly from Invitrogen .",
"The commonly used STORM dye Alexa 647 was not used in most cases due to issues associated with the presence of near-red fluorophores present in reactive dye stocks ( Stone and Veatch , 2014 ) .",
"Lyn-eGFP , PM-eGFP , and eGFP-GG plasmids ( Pyenta et al . , 2001 ) were a generous gift from Barbra Baird and David Holowka ( Cornell University , Ithaca , NY ) and were cloned using standard techniques to replace eGFP with mEos3 . 2 .",
"Plasmid DNA encoding mEos3 . 2 protein and YFP-TM anchor sequences were gifts from Akira Ono ( University of Michigan , Ann Arbor , MI ) .",
"The YFP and mEos3 . 2 tagged constructs used here are in Clontech N1 plasmid vector background ( Clontech , Mountain View , CA ) .",
"The clathrinHC-GFP and clathrinHC-mEos3 . 1 constructs ( Sochacki et al . , 2014 ) were gifts from Justin Taraska ( National Heart , Lung , and Blood Institute , NIH , Bethesda , MD ) .",
"We also cloned the CD45 transmembrane domain ( termed CD45tm ) with a small number of flanking amino acids de novo from the amino acid sequence for mouse CD45 isoform 1 , UniParc identifier P06800–1 .",
"We included the HA membrane-targeting signal sequence and a FLAG tag ( Guan et al . , 1992 ) upstream of the construct on the N terminus to allow for efficient plasma membrane delivery and detection , respectively .",
"The signal sequence is cleaved from the construct in the ER prior to trafficking to the plasma membrane .",
"The CD45 transmembrane domain was cloned into the HA-FLAG tag plasmid using standard techniques .",
"Amino Acid sequence of CD45tm insert with upstream HA-FLAG tag , where the signal sequence is shown in italics and the FLAG tag is shown in bold: N terminus-MKTIIALSYIFCLVFADYKDDDDANESTNFNAKALIIFLVFLIIVTSIALLVVLYKIYDLRKKR-C terminus CH27 cells ( RRID: CVCL_7178 ) , a mouse B cell lymphoma-derived cell line , was used as a model system for B lymphocyte signaling through the BCR ( Haughton et al . , 1986 ) .",
"Cells were acquired from Neetu Gupta ( Cleveland Clinic ) , and cell line identity was authenticated using several criteria .",
"Surface expression of mouse IgM , which is a specific marker of mouse B lymphocytes , was confirmed through specific labeling with goat anti-mouse IgM f ( ab ) 1 fluorescent conjugates .",
"Cell morphology was typical for the B lymphoma cell type .",
"Growth rates were monitored for consistency over time and cells were not kept in passage for longer than 60 days .",
"Cultures tested negative for mycoplasma contamination .",
"CH27s do not appear on the list of commonly mis-identified cell lines maintained by the International Cell Line Authentication Committee .",
"Cells were maintained in culture as described previously ( Stone and Veatch , 2015 ) .",
"CH27 cells were transiently transfected by Lonza Nucleofector electroporation ( Lonza , Basel , Switzerland ) with electroporation program CA-137 .",
"Generally , 700 , 000 CH27 cells were transfected with 1 µg plasmid DNA , except for Lyn where 500 , 000 cells were transfected with 0 . 7 µg plasmid DNA to avoid cell death .",
"Cells were grown overnight on glass bottom wells ( MatTek Corporation , Ashland , MA ) at 200 , 000 per well .",
"A subset of CH27 cells adhere spontaneously to glass bottom wells via an unknown mechanism but adhesion is not , in our experience , potentiated by first coating wells with fibronectin .",
"For GG expression , cells were grown overnight in flasks , harvested and spun down , washed by pelleting and re-suspending three times in media , and then plated on the same day as labeling and fixation to minimize coverslip-bound mEos3 . 2-GG because this construct is secreted from cells .",
"For cholesterol depletion and addition , cells were incubated for 15 min at 37°C with indicated concentrations of MβCD or cholesterol loaded MβCD ( Sigma ) freshly dissolved in Balanced Salt Solution ( BSS: 135 mM NaCl , 1 mM MgCl2 , 1 . 8 mM CaCl2 , 5 . 6 mM glucose , 20 mM HEPES , pH 7 . 4 ) .",
"Cells were chemically fixed with 4% paraformaldehyde and 0 . 1% glutaraldehyde in PBS buffer or 2% paraformaldehyde and 0 . 15% glutaraldehyde in half-strength PBS for 10 min at room temperature unless otherwise indicated , and fix was quenched by washing with 5 mg/mL BSA .",
"Primary B lymphocytes were purified from a C57BL/6 mouse ( Jackson Laboratories; RRID:IMSR_JAX:000664 ) using a standard negative selection procedure .",
"All experiments were performed in compliance with federal laws and institutional guidelines as approved by the University of Michigan Committee on Use and Care of Animals .",
"Briefly , one mouse was sacrificed using CO2 asphyxiation .",
"Spleen and lymph nodes were harvested in the presence of DNase I and filtered through a 70 µm strainer .",
"Cells were pelleted and resuspended in DMEM with 2% FBS , 10 mM HEPES , 50 IU/mL penicillin , 50 µg/mL streptomycin , and 0 . 2 mg/mL DNAse I . 5 µg/mL CD11c ( clone N418 , Biolegend; RRID: AB_313772 ) and 5 µg/mL CD43 ( clone S7 , BD Biosciences; RRID: AB_2255226 ) biotinylated antibodies were added to cells for 30 min on ice prior to red blood cell lysis with RBC lysis buffer ( 0 . 14 M NH4Cl and 0 . 017 M Tris , pH 7 . 2 ) and washing by pelleting .",
"Remaining cells were incubated with streptavidin MACS beads ( Miltenyi Biotec ) for 20 min on ice and non-B cells were removed using an Automacs ( Miltenyi Biotec ) on the DEPLETES protocol .",
"Primary B cells were then put into a buffer recommended by Lonza: RPMI 1640 supplemented with 10% FCS , 2 mM glutamine , 50 µM 2-mercaptoethanol , and 50 µg/mL LPS for 24 hr .",
"Electroporation was accomplished with the P4 Primary Nucleofector solution with electroporation program DI-100 ( Lonza ) using 600 , 000 cells with 0 . 6 µg plasmid DNA in each well .",
"Cells were grown overnight in flasks , spun down and washed extensively in cell media , and then plated onto fibronectin plates for 2 hr prior to labeling with f ( Ab ) 1biotin Atto 655 , clustering with streptavidin , and fixation as described above .",
"RBL-2H3 cells ( ATCC CRL-2256; RRID: CVCL_0591 ) , a rat basophilic leukemia-derived cell line , were obtained from Barbara Baird and David Holowka ( Cornell University ) .",
"Cell identity was authenticated by expression of the high-affinity receptor for IgE , FcεRI , which was confirmed by specific binding of fluorescent IgE conjugates to the surface of cells .",
"Cells were checked for characteristic morphology ( Siraganian et al . , 1982 ) , growth rates were monitored for consistency over time , and cells were not kept in passage for longer than 90 days .",
"Cultures tested negative for mycoplasma contamination .",
"RBL-2H3 cells do not appear on the list of commonly mis-identified cell lines maintained by the International Cell Line Authentication Committee .",
"RBL-2H3 cells were maintained in minimum essential medium with L-glutamine and phenol red with 20% fetal bovine serum and 0 . 1% gentamycin at 37°C in 5% CO2 , as described previously ( Gosse et al . , 2005 ) .",
"RBL-2H3 cells were transiently transfected with membrane anchor probes using the protocol described above for CH27 cells , with electroporation program DS-138 .",
"HeLa cells were obtained from Akira Ono ( University of Michigan ) and maintained in high-glucose ( 4 mg/ml ) Dulbecco's Modified Eagle Medium with 5% fetal bovine serum and 1% pen strep at 37°C in 5% CO2 .",
"HeLa cells were only used for experiments to demonstrate properties of the analytical methods used ( Figure 6 ) , where cell identity was not pivotal to the interpretation of results .",
"Cells had morphology and adhesive qualities common to this cell line but were not subjected to additional authentication .",
"HeLa cells were transiently transfected using the protocol described above for CH27 cells , with electroporation program CN-114 . 10 . 7554/eLife . 19891 . 042Figure 6 . Our cross-correlation methodology applied to doubly labeled clathrin .",
"( a ) Two-color super-resolution image of a HeLa cell expressing two distinct labeled clathrin heavy chain constructs is shown .",
"Clathrin heavy chains associate strongly in clathrin coated pits , and thus serve as an example of highly correlated co-clustered objects .",
"Individual clathrin coated pits are shown below in the smaller images .",
"Scalebar in large image is 5 µm , scale-bars in small images are 200 nm .",
"( b ) Zoom in of cyan box shown in large image , where position of points are plotted around an arbitrarily chosen central magenta localization .",
"Dotted lines show the spatial bins used for calculating the cross-correlation function , where the number of green localizations within each bin are counted .",
"In the complete cross-correlation these counts would also be summed over all magenta localizations .",
"( c ) Raw histograms of interparticle distances containing all pairs of particles localized within this cell .",
"The red line shows the expected number of pairs in each spatial bin given a random distribution of both magenta and green localizations .",
"The raw histogram is normalized by this curve to yield c",
"( r ) .",
"( d ) Cross-correlation derived from localizations within this cell .",
"Magnitude of the correlation indicates fold increase of pairs detected at the specified inter-particle distance compared to a random distribution .",
"The expected value of the cross-correlation given a random co-distribution is equal to one due to the normalization .",
"Error bounds shown are dC2",
"( r ) and are estimated from the statistics and resolution of the image as defined in Equation 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 04210 . 7554/eLife . 19891 . 043Figure 6—figure supplement 1 . The cross-correlation function detects deviations in the co-distribution of localizations from random . Four simulations of two-color localization distributions are shown , where the spatial distribution of the localization is given by the labels in the top left of the images .",
"For clustered distributions , particles are randomly placed within non-overlapping circular areas .",
"For co-clustered , both green and magenta localizations are placed within the same circular areas .",
"Cross-correlation functions calculated from simulated distributions are shown at right .",
"Only co-clustered objects yield a cross-correlations that is significantly different from a random distribution .",
"Clustering one object and leaving the other randomly distributed or clustering both objects but maintaining a random co-distribution yields distributions of interparticle distances that are not significantly different than a random co-distribution of both localizations .",
"Scale bars are 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 04310 . 7554/eLife . 19891 . 044Figure 6—figure supplement 2 . The amplitude of the cross-correlation reflects differences in enrichment magnitude and interaction strength . Example cells from three different experiments are shown to illustrate the range of values the cross-correlation function can take for various types of interactions .",
"( a ) An example of a strong enrichment is shown for phosphotyrosine ( pY ) and BCR .",
"Nearly every BCR cluster is colocalized with pY localizations , and little pY is observed outside BCR clusters .",
"( b ) Lyn transiently binds to phosphorylated ITAMs within BCR clusters but is also present outside of clusters , leading to a reduced magnitude of the correlation function .",
"( c ) PM is weakly enriched in BCR clusters and a large fraction of PM is found outside of BCR clusters , however PM is more colocalized than expected from a random distribution when the whole cell is analyzed .",
"In whole-cell images ( top ) , scale bars are 5 µm .",
"Boxed regions shown in whole-cell images are enlarged below ( middle ) and the number of localizations in each pixel is given by the colorbars .",
"Scale bars in enlarged regions are 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 04410 . 7554/eLife . 19891 . 045Figure 6—figure supplement 3 . Cross-correlations detect co-clustering even when there is a low surface density of labeled molecules . An Ising model containing kinase ( green ) and clustered receptors ( magenta ) is used to demonstrate how the surface density of labeled proteins impacts correlation functions .",
"( top )",
"Representative histograms indicating simulated positions of kinase and receptors , with each pixel corresponding to 16 nm × 16 nm area .",
"The average surface density per µm2 is shown .",
"( middle )",
"The same image as above but blurred by a Gaussian function mimicking the localization precision .",
"All scale bars are 100 nm .",
"( bottom )",
"Averaged cross-correlation functions for the conditions represented above .",
"The black curve shows an average over 1000 samplings of the same distribution with the average surface density indicated for both kinase and receptor .",
"The colored lines show averages over 100 samplings .",
"If there are roughly 100 receptor clusters per cell , then these curves represent the expected cell-to-cell variation .",
"Most experiments have mEos3 . 2 surface expression between 1–20 per µm2 so are expected to most closely mimic the situation on the far right .",
"Note that even when surface densities are low , the average correlation function remains unchanged beyond differences in signal to noise . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 04510 . 7554/eLife . 19891 . 046Figure 6—figure supplement 4 . Membrane topology gives rise to long-range structure in cross-correlations , but can be removed by careful selection of regions of interest ( ROI ) .",
"( a ) Reconstructed super-resolution image of a CH27 B cell in which part of the cell has detached from the coverslip surface leading to a reduction in localizations in the center of the cell , which is imaged in total internal reflection .",
"Two different regions of interests are shown , with the excluded regions lightened for contrast .",
"Scale bars are 5 µm .",
"( b ) Cross-correlation functions tabulated using the two ROIs shown in a .",
"The loose ROI produces a C",
"( r ) that is correlated and decays slowly with radius .",
"This is largely a reflection of the topology of the ventral membrane , which contains large regions where both probes are excluded due to membrane detachment .",
"This correlation is removed by selecting a ROI which only includes flat regions .",
"( c ) ROI generation is performed by users and has the potential to introduce bias into the analysis .",
"19 cells comprising different labeling and treatment conditions were randomly viewed by users that lacked knowledge of the specific identity of each cell .",
"The three users tended to define masks that yielded correlation functions with similar amplitudes from single cells .",
"( d ) Average cross-correlation curves corresponding to the 19 cells shown in d , with errorbars indicating the SEM between cells for each user .",
"These results overlap within error bounds suggesting that arbitrary user decisions regarding ROI placement do not significantly impact this result . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 04610 . 7554/eLife . 19891 . 047Figure 6—figure supplement 5 . Estimation of the variance associated with a cross-correlation measured on a single super-resolution fluorescence localization image . Simulations containing randomly distributed green and magenta points are used to identify the sources of error typically found in cross-correlations tabulated from images acquired using super-resolution localization microscopy .",
"Here , labeled molecules are distributed randomly as shown in",
"( a ) .",
"When the localization precision ( 30 nm ) is on the order of the pixel size ( 25 nm ) , then it acts to blur the image of molecular centers",
"( b ) .",
"The smooth blurred image represents a fully sampled PSF .",
"In real localization microscopy images , single labeled molecules are typically localized multiple times , but not often enough to fully sample the super-resolved PSF .",
"Instead the distribution represented by the blurred image is under-sampled .",
"The smooth shape of the PSF is still evident when each PSF is sampled many times",
"( c ) , but images appear more pixelated as this sampling is reduced",
"( d ) .",
"All of these factors impact the statistics of measured correlation functions .",
"For all conditions indicated , the top panel shows a representative simulation snapshot for the condition indicated ( scale-bar = 500 nm ) and the two dimensional cross-correlation , C ( r , θ ) , tabulated from this representative image .",
"The next lower panel shows C ( r , <θ> ) obtained by averaging C ( r , θ ) over angles ( red squares ) , as well as <C",
"( r ) > , the correlation function obtained by averaging over 100 simulation replicates ( black circles ) .",
"Error bars on these curves are either determined from the angular average as described in the main text ( red squares , dC ( r , <θ> ) ) , or by taking the SEM over 100 simulation replicates to obtain d<C",
"( r ) > ( black circles ) .",
"The bottom panels show how the square root of the variance ( dC",
"( r ) ) depends on radius for the conditions indicated .",
"Black circular points show ( d<C",
"( r ) > ) .",
"Red squares show the dC ( r , <θ> ) error averaged over the 100 replicates <dC ( r , <θ> ) > .",
"The dC1",
"( r ) and dC2",
"( r ) points are corrections to dC ( r , <θ> ) and are calculated as described in Methods .",
"( a ) dC ( r , <θ> ) is a good estimate of d<C",
"( r ) > in simulations where the localization precision is much less than the pixel size and when there is good sampling of the super-resolved PSF .",
"( b ) dC ( r , <θ> ) under-estimates d<C",
"( r ) > when labeled objects are detected with 30 nm localization precision but when these super-resolved PSFs are fully sampled .",
"This under-estimate can be corrected using the dC1",
"( r ) described in Equation 2 of Methods .",
"( c ) dC1",
"( r ) is sufficient when labeled objects have 30 nm resolution and their super-resolved PSF is well sampled .",
"( d ) An additional correction is needed when the super-resolved PSF is not well sampled , as described by dC2",
"( r ) in Equation 3 of Methods . DOI: http://dx . doi . org/10 . 7554/eLife . 19891 . 047 For BCR experiments , goat anti-mouse IgM ( Jackson ImmunoResearch , West Grove , PA; RRID: AB_2338477 ) f ( Ab ) 1 fragments conjugated to both fluorophores and biotin were used to label endogenous BCR in the plasma membrane .",
"For fixed cell experiments , cells were stained with 5 µg/ml f ( Ab ) 1 conjugated to Atto 655 for 10 min in BSS followed by extensive washing prior to clustering with 1 µg/mL streptavidin in BSS prior to chemical fixation .",
"Cross-correlations and images of fixed CH27 cells are shown for cells stimulated with antigen for 5 min prior to fixation unless otherwise noted .",
"Primary cells were stimulated for 1 min prior to chemical fixation .",
"For live cell experiments , cells were stained with 5 µg/ml f ( Ab ) 1 conjugated to SiR and biotin in BSS for 10 min .",
"Images and data from live cells were acquired between 0 and 6 min after streptavidin was added at 1 µg/mL .",
"For clustered CTxB experiments , labeling of CTxB clusters was accomplished in one of two ways .",
"Plasma membrane GM1 was bound with biotinylated CTxB at a concentration of 1 µg/mL for 10 min at room temperature in BSS .",
"Cells were then washed extensively before adding 50 µg/mL streptavidin conjugated to Atto 655 for 10 min prior to chemical fixation .",
"In some cases , plasma membrane GM1 was bound with 0 . 5 µg/mL biotinylated CTxB conjugated to Atto 655 for 10 min at 37°C .",
"B cells were then washed with 37°C BSS buffer before clustering CTxB with 0 . 1 mg/mL streptavidin for 5 min at room temperature prior to chemical fixation .",
"These two labeling methods produced equivalent results within error .",
"For clustered TM experiments , TM bearing an extracellular YFP tag was transfected into CH27 cells and subsequently clustered with 13 µg/mL anti-GFP rabbit IgG conjugated to biotin ( ThermoFisher; RRID: AB_1090214 ) for 30 min at room temperature in BSS .",
"Cells were then washed with BSS buffer and stained for 10 min with 100 µg/mL streptavidin conjugated to either Atto 655 when TM clusers were imaged in conjunction with mEos3 . 2 or Alexa 532 when TM clusters were imaged in conjunction with Atto 655 .",
"For phosphotyrosine detection , fixed cells were permeablized with 0 . 1% Triton-X 100 in block buffer ( PBS with 3% fish gelatin with 2 mg/mL BSA ) and labeled with a 1:1000 dilution of anti-phosphotyrosine clone 4G10 primary antibody ( Millipore , RRID:AB_916370 ) in block buffer for 1 hr .",
"Cells were washed extensively before adding the secondary antibody , goat anti-mouse IgG 2b subtype specific ( Jackson ImmunoResearch; RRID: AB_2338463 ) .",
"The secondary antibody was conjugated to either Atto 655 ( when observing TM clusters ) or Alexa 532 ( when observing BCR and CTxB clusters ) prior to use in labeling .",
"For CD45 detection , anti-mouse CD45R ( B220 ) primary antibody clone RA3-6B2 conjugated directly to Alexa 532 was used ( eBiosciences , San Diego , CA; RRID: AB_467253 ) .",
"After cells were fixed , 1 µg/mL antibody was allowed to bind to endogenous CD45 for 2 hr at room temperature in block buffer before washing to remove unbound antibody .",
"For CD45tm detection , B cells transfected with FLAG-CD45tm were chemically fixed and then stained with 20 µg/mL mouse monoclonal anti-FLAG M1 ( Sigma; RRID:AB_439712 ) in block buffer for 1 hr at 37°C .",
"Cells were then washed extensively before labeling with 10 µg/mL goat anti-mouse IgG 2b conjugated to Alexa 532 for one hour at 37°C in block buffer .",
"For endogenous Lyn and phosphorylated Lyn detection , chemically fixed B cells were permeablized with 0 . 1% Triton-X 100 in block solution following clustering of CTxB-biotin with streptavidin-Atto 655 .",
"Samples were then incubated with either a 1:50 dilution of anti-Lyn primary antibody ( rabbit polyclonal anti-Lyn IgG clone 44; Santa Cruz Biotech; RRID:AB_2281450 ) or a 1:100 dilution of anti-phospho-Lyn primary antibody ( rabbit monoclonal IgG anti-pY397 Lyn clone EP503Y; Abcam; RRID:AB_776106 ) for 1 hr at room temperature .",
"Samples were washed extensively in block buffer and then incubated with a 1:1000 dilution of Alexa Fluor 532 conjugated goat anti-rabbit secondary antibody ( goat polyclonal anti-rabbit IgG ( H+L ) ; ThermoFisher; RRID: AB_10374433 ) .",
"Samples were washed in block to remove unbound antibody .",
"For BCR and CTxB cross correlation , biotinylated CTxB was clustered by streptavidin conjugated to Atto 655 and cells were fixed prior to labeling BCR with anti-IgM f ( Ab ) 1 fragments conjugated to Alexa 532 .",
"Samples were washed in block to remove unbound antibody .",
"Two-color super-resolution images of clathrin coated pits were obtained by co-expressing two alternatively labeled clathrin heavy chain ( HC ) constructs , clathrinHC-GFP and clathrinHC-mEos3 . 1 , in HeLa cells .",
"One million HeLa cells were co-transfected with 0 . 75 µg clathrinHC-mEos3 . 1 as well as 0 . 75 µg clathrinHC-GFP .",
"Cells were fixed and membranes were permeablized as above , blocked in 2% BSA , and GFP was labeled with a biotinylated anti-GFP primary antibody ( ThermoFisher; RRID: AB_1090214 ) .",
"Subsequently , cells were washed extensively and streptavidin bound to Alexa 647 ( Invitrogen ) was added to label clathrinHC-GFP for imaging .",
"Imaging was performed on an Olympus IX81-XDC inverted microscope with a cellTIRF module , a 100X UAPO TIRF objective ( NA = 1 . 49 ) , and active Z-drift correction ( ZDC ) ( Olympus America , Center Valley , PA ) as described in previous work ( Stone and Veatch , 2014 , 2015 ) .",
"Images were acquired on an iXon-897 EMCCD camera ( Andor , South Windsor , CT ) .",
"Excitation of Atto 655 was accomplished using a 647 nm solid state laser ( OBIS , 100 mW , Coherent , Santa Clara , CA ) when imaged in conjunction with mEos3 . 2 , or a 640 nm diode laser ( CUBE 640-75FP , Coherent ) when imaged in conjuction with Alexa 532 .",
"Excitation of mEos3 . 2 constructs was accomplished using a 561 nm solid state laser ( Sapphire 561 LP , Coherent ) .",
"Photoactivation of mEos3 . 2 was accomplished with a 405 nm diode laser ( CUBE 405-50FP , Coherent ) .",
"Excitation of Alexa 532 was accomplished with a 532 nm diode-pumped solid-state laser ( Samba 532–150 CW , Cobolt , San Jose , CA ) .",
"Laser intensities were adjusted such that single fluorophores could be distinguished in individual images , and were generally between 5 kW/cm2 and 20 kW/cm2 .",
"Excitation and emission was filtered using a LF405/488/561/647 quadband cube ( TRF89902 , Chroma , Bellows Falls , VT ) or a 532/640 dualband cube ( TRF59907 , Chroma ) .",
"Emission was split into two channels using a DV2 emission splitting system ( Photometrics , Tuscon , AZ ) using a T640lpxr dichroic mirror to separate emission , ET605/52m to filter near-red emission , and ET700/75m to filter far-red emission ( Chroma ) .",
"Chemically fixed samples with Atto 655 and mEos3 . 2 were imaged in a buffer suitable for STORM and PALM microscopy: 30 mM Tris , 9 mg/ml glucose , 100 mM NaCl , 5 mM KCl , 1 mM KCl , 1 mM MgCl2 , 1 . 8 mM CaCl2 , 10 mM glutathione , 8 µg/ml catalase , 100 µg/ml glucose oxidase , pH 8 . 5 .",
"Live samples were imaged with the same buffer except with 200 µg/ml catalase at pH 8 , which is more suitable for live cells since it has enhanced reactive oxygen species scavenging and the pH is closer to physiological pH . Fixed samples with Atto 655 and Alexa 532 or with Alexa 647 and mEos3 . 1 were imaged in a buffer more suitable for oxazine and rhodamine dyes ( Heilemann et al . , 2009 ) : 50 mM Tris , 100 mg/mL glucose , 10 mM NaCl , 100 mM 2-mercaptoethanol , 50 µg/ml glucose oxidase , 200 µg/ml catalase , pH 8 .",
"In some cases , glucose oxidase concentration was lowered or it was omitted from the buffer entirely in order to optimize the photoswitching rates of Atto 655 and Alexa 532 .",
"Live cells were imaged at approximately 45 frames per second with an exposure time of 20 milliseconds , and the exposure time for fixed cells varied between 20 and 50 milliseconds .",
"Single molecule fluorescent events were localized by fitting local maxima in background subtracted images to Gaussian functions using standard methods .",
"The ensemble of peaks was then culled to remove outliers in brightness , size , and localization error using in-house MATLAB software ( Veatch et al . , 2012 ) .",
"For live cells , single molecules were localized in raw live cell movies with the ImageJ plugin ThunderSTORM ( Ovesný et al . , 2014 ) , using weighted least-squares fitting of an integrated Gaussian PSF with multi-emitter fitting analysis enabled to detect up to two single molecules within a diffraction-limited area .",
"Localization data were then exported to our in-house MATLAB software for culling and successive post-processing steps ( Veatch et al . , 2012 ) .",
"Localizations in the near-red emission channel were registered with the far-red emission channel using a registration technique published previously ( Churchman et al . , 2005 ) and previously used by our group ( Stone and Veatch , 2014 , 2015 ) .",
"Stage drift correction was performed every 500 frames by finding the maximum in the 2D cross correlation produced by all localizations between successive groups of frames .",
"Super-resolution localizations were used to reconstruct super-resolved images after correcting for stage drift and channel registration by incrementing the intensity of pixels at positions corresponding to localized single molecules .",
"The super-resolved images have an arbitrary pixel size of 25 nm , and the original images have a pixel size of 160 nm , corresponding to the pixel size of the EMCCD camera .",
"For the purposes of display , localizations were grouped such that probes observed within a small ( typically 80 nm ) radius in sequential frames were merged and counted as a single localization .",
"Note that this grouping correction does not account for multiple observations of the probe imaged at different times , for example as a result of reversible activation .",
"Histograms of localized positions were blurred as described in figure captions and image contrast was adjusted for display purposes .",
"The resolution of particle localization was close to 30 nm for all probes , determined by correlation-based methods as detailed previously ( Veatch et al . , 2012 ) .",
"This resolution is larger than the localization precision of the Gaussian fits because it includes contributions from other sources of error ( e . g . from stage drift ) .",
"Regions containing cells were masked by a user-defined region of interest ( ROI ) , and cross correlations were computed from these regions using methodology described previously ( Sengupta et al . , 2011; Veatch et al . , 2012; Stone and Veatch , 2015 ) and summarized here .",
"Cross-correlation functions report on the enrichment or depletion of distinguishable probes with respect to one another , normalized by a random co-distribution of probes .",
"Thus , the magnitude of the cross-correlation yields information about interactions of labeled objects with one another that may cause their co-distributions to deviate from a random co-distribution .",
"This methodology is demonstrated in Figure 6 using imaging of dually-labeled clathrin coated pits as an example .",
"Clathrin coated pits were imaged in HeLa cells transiently expressing two distinct labeled clathrin heavy chain proteins , one conjugated to mEos3 . 1 ( green ) and a second conjugated to GFP that is antibody labeled with Alexa 647 ( magenta ) .",
"Clathrin was chosen for this demonstration because a large number of individual clathrin proteins assemble within clathrin coated pits , which are sparsely distributed within the cell , therefore their co-localization is easily identified when viewing the reconstructed image .",
"A reconstructed image of a HeLa cell showing the distributions of super-resolved localizations arising from both clathrin constructs is shown in Figure 6a .",
"The correlation function can be assembled by tabulating the pair-wise distances between distinguishable probes localized within a masked image , then binning these separation distances to produce the average number of pairs separated by distances between r and r+Δr , where Δr is usually 25 nm in our measurements .",
"The point distribution of distinguishable probes surrounding an example magenta probe is shown in Figure 6b .",
"In this case , there are many more green points located at small separation distances from the example magenta point than at large separation distances because the magenta point chosen was located at the center of a clathrin coated structure .",
"These pairwise distances are collected within the radial bins given by the dotted lines .",
"The complete correlation function tabulates these separation distances around all magenta probes in the image .",
"Figure 6c shows the histogram describing the distribution of separation distances for all pairs from the cell shown in Figure 6a .",
"In general , the number of pairs in each bin increases linearly with increasing radius for large separation distances because the area corresponding to each bin also increases linearly , as A ≈ 2πrΔr .",
"These histograms are then normalized by the total number of observations divided by the area of the cell and multiplied by the area of the bins .",
"This normalization is equivalent to the number of observations expected in each bin given a random distribution of pairs across all bins , and is shown as a red line in Figure 6c .",
"Importantly , this normalization simply accounts for variation in expression level between cells ( Figure 2—figure supplement 3 ) and corrects for boundary effects that arise due to the finite extent and shape of the ROI .",
"The cross-correlation function can be equivalently calculated from reconstructed images of all localizations using fast Fourier transforms , as has been described previously ( Veatch et al . , 2012; Stone and Veatch , 2015 ) .",
"In this case , a two dimensional cross-correlation is tabulated , C ( r , θ ) , and then C",
"( r ) is obtained by averaging over angles .",
"Generally , C",
"( r ) is tabulated from ungrouped images , meaning that localizations detected within a small radius in sequential frames are counted independently .",
"Ungrouped images are used because cross-correlation functions are not impacted by probe over-counting ( Veatch et al . , 2012 ) and this reduces possible errors introduced by the grouping technique .",
"The properly normalized cross-correlation function for this cell is shown in Figure 6d .",
"Cross-correlation functions only indicate significant correlations when the spatial distribution of one probe influences the spatial distribution of the second probe , even when one or both of the probes are clustered themselves .",
"This effect is demonstrated in Figure 6—figure supplement 1 . Error bars on this curve are estimated using the variance within the radial average of the two dimensional C ( r , θ ) , the average lateral resolution of the measurement , and the numbers of probes imaged in each channel , as described in detail below .",
"As expected , the cross-correlation function tabulated from the image shown in Figure 6a indicates that probes are highly co-localized , where the co-localized density within the first spatial bin ( r < 25 nm ) is five times higher than randomly co-distributed probes .",
"In this case , C",
"( r ) ≈ 1 for separation distances much larger than the size of individual clathrin structures , meaning that the pits themselves are roughly randomly distributed on the cell surface .",
"C",
"( r ) is slightly larger than one even at separation distances approaching 1 µm because clathrin structures are more densely localized on the edge of this cell than towards the center .",
"In some instances , we subtract this long distance offset in order to remove long range contributions to C",
"( r ) which are not currently under investigation .",
"The vast majority of cross-correlation functions reported in the main text have much smaller amplitudes than the one shown in this example .",
"This is because co-localization is much weaker and/or domains are more numerous .",
"Examples of single cell correlation functions for various probes that co-localize with BCR clusters along with reconstructed images are shown in Figure 6—figure supplement 2 . A distinct advantage of this cross-correlation function approach is that it involves averaging over multiple domains within an image , and can be further averaged over images .",
"This makes it possible to quantify co-localization that is far too weak or under-sampled to be apparent from visual inspection of images .",
"This is demonstrated in Figure 6—figure supplement 3 , which shows a simulated case where the same weak co-distribution of probes is sampled to varying degrees .",
"When the spatial distributions are well-sampled , then co-localization is easily apparent both visually in the image and quantitatively in the tabulated correlation function .",
"When spatial sampling is low , co-localization is no longer apparent in images , and in fact probes can appear anti-correlated because sampling is so sparse that localizations are unlikely to be overlapping .",
"However , cross-correlation functions can still detect co-localization in many cases , although reduced sampling decreases the signal-to-noise .",
"ROIs are chosen so that only flat regions of the cell surface are analyzed , which in some cases meant that regions of the cell interior were not included in the ROI when the membrane lifts from the TIR field and membrane components are no longer visualized ( Figure 6—figure supplement 4 ) .",
"When included in the ROI , regions of membrane topology produced correlations that extend to large radii ( >200 nm ) in tabulated cross-correlation functions , as shown in Figure 6—figure supplement 4a–b .",
"This is because both probes are necessarily absent in regions where the membrane has lifted from the glass surface , which makes probes correlated .",
"The normalization of the cross-correlation function properly accounts for complex regions of interest .",
"Significant efforts were made to minimize the impact of membrane topology , but in some cases this was complicated by low spatial sampling of labeled proteins and peptides .",
"Especially in cases where spatial sampling is low , user-defined ROI have the potential to introduce systematic bias that could impact cross-correlation results .",
"In some cases , cells were analyzed without user knowledge of the sample condition , and results were indistinguishable within noise .",
"We also found little user-to-user variation in cross-correlations determined from single cells or averaged over a population ( Figure 6—figure supplement 4c–d ) .",
"One major limitation of the super-resolution methods and probes used here is that it is not possible to simply distinguish multiple observations of the same labeled molecule from a small aggregate of labeled molecules .",
"However , it is possible to estimate the average surface density of labeled molecules for cases where probe blinking follows Poisson statistics and where probes are nearly randomly distributed ( Veatch et al . , 2012 ) .",
"This is accomplished by fitting a Gaussian function with standard deviation σ and amplitude A to the autocorrelation function tabulated from a single color image .",
"This single color image is reconstructed from grouped localization data , meaning that localizations detected within a small radius ( 80nm ) in sequential frames are counted as a single localization .",
"Grouping sequential localizations produces images with sampling that better approximates Poison statistics , since localizations are less correlated in time .",
"When labeled proteins are randomly or nearly randomly distributed in space , the area under of the autocorrelation function is inversely proportional to the surface density of labeled proteins according to: ( 1 ) ρ=12πσ21A We expect this to be an accurate estimate of surface density for the majority of mEos3 . 2 conjugated peptides used in this study , since they are expected to be only subtly self-clustered within the membrane .",
"This estimate will be less accurate for the case of proteins with higher-order structure including extended clusters , such as clustered BCR and CTxB where this density is likely better interpreted as the density of clusters , not individual proteins .",
"We can estimate the average number of times each independent protein or peptide structure is sampled by comparing the average density of localizations to the average surface density of labeled proteins or peptides determined using Equation 1 . For the localization data presented in this study , we generally find that independent proteins and peptides are observed between 10 and 50 times over the 5000–10 , 000 raw acquisition frames imaged .",
"While the cross-correlation obtained between reconstructed images of two different probes is not adversely affected by over-counting , over-counting does impact the observed variance , as described below .",
"Estimating error bounds on individual fixed cell measurements is complicated by the presence of over-counting of single labeled proteins in combination with finite localization precision .",
"In the absence of these two effects , the variance in C",
"( r ) can be simply calculated using Poisson statistics to describe the probability of detecting a certain number of average pairs within some specified area given the cross-correlations observed .",
"This strategy has been applied to estimate error on single live cell cross-correlations ( Stone and Veatch , 2015 ) , but it depends strongly on the average densities of the labeled proteins present .",
"In fixed cells , these numbers can be only estimated due to over-counting as described above .",
"Instead , the variance is estimated by calculating dC ( r , <θ> ) , the standard deviation of the mean obtained when averaging the 2D cross-correlation function C ( r , θ ) over angles to extract C",
"( r ) as outlined in Figure 6—figure supplement 5 and described below .",
"In the limit of resolution much smaller than the pixel size , dC ( r , <θ> ) accurately reproduces the variance obtained by observing many replicates of a simulation where two distinguishable probes are distributed randomly , as shown in Figure 6—figure supplement 5a .",
"When the image resolution is on the order of or larger than the pixel size , then there is smoothing of the image and the resulting C ( r , θ ) .",
"In this case , it is not appropriate to simply tabulate the standard error of the mean of pixel values falling within a separation distance range between r and r+Δr because neighboring pixels in the two-dimensional C ( r , θ ) are correlated .",
"When the localization precision is known , this effect can be simply corrected using a multiplicative factor that only depends on the localization precision , σPSF , which is the standard deviation of the super-resolved point spread function ( PSF ) : ( 2 ) dC1",
"( r ) = ( 1+ ( 2σPSF/Δr ) ) × ( 1+e−r2/4σPSF2 ) ×dC ( r , ⟨θ⟩ ) In all instances presented here , σPSF is taken to be 30 nm for the sake of this calculation .",
"At radii much larger than σPSF , this factor simply corrects for the fact that correlated pixels in C ( r , θ ) are contributing to the average over angles , so the number of independent measurements is less than the number of pixels contributing to the average .",
"At short radii , blurring over the super-resolved point spread function also decreases the amplitude of correlations directly , so there is additional under-estimation of variance by the simple angular average method .",
"This correction factor is applied to simulations of blurred randomly distributed points in Figure 6—figure supplement 5b .",
"This multiplicative correction factor of Equation 2 over-estimates the error when the super-resolved point spread function is not well sampled .",
"This under-sampling introduces variance that should not be amplified by the correction factor shown above .",
"This can be corrected further by subtracting a term that depends on the number of observations of each single color label ( N1 and N2 ) , the number of labeled proteins in the image ( n1 and n2 ) , and the size of the super-resolved point spread function ( σPSF ) : ( 3 ) dC2",
"( r ) =dC1",
"( r ) −4πσPSF2Δr2 ( N12n1+N22n2 ) −1× ( 1+4e−r2/4σPSF2 ) The pre-factor on this correction term represents how well the area occupied by all probes ( 4πσPSF2n ) is sampled by pairs of localizations of that color ( N2 ) .",
"This term becomes negligible when there are many localizations per probe in either channel , as is typical in the fixed cell measurements presented here .",
"For this reason it does not contribute significantly to the results presented .",
"The number of labeled proteins in each channel ( n1 and n2 ) is estimated by fitting the autocorrelation to extract the density of independent objects using Equation 1 above and then multiplying this number by the area of the region of interest .",
"Figure 6—figure supplement 5c shows that dC1",
"( r ) is sufficient to describe the simulation-to-simulation variation when objects are sampled 20 times , which is typical of the images investigated in this work .",
"When sampling is lower , this correction is needed to more accurately estimate the the simulation-to-simulation variation as presented in Figure 6—figure supplement 5d .",
"The Gaussian shape of this correction is estimated from simulations and may not apply in all contexts .",
"In the majority of cases where correlation functions are presented within figures , the values plotted are averaged together across cells of the same treatment and condition to obtain the average correlation function , and the error bars represent the standard error of the mean between cells .",
"The number of cells going into each average is shown in figure captions and figure supplements and , with the exception of the primary cell experiments , includes at least two biological replicates where samples were prepared for imaging on separate days .",
"In all cases we have examined closely , the average error estimated from a single measurement of the cross-correlation function is close to the width of the distribution of single cell cross-correlation values , indicating that the observed variation is dominated by counting statistics and not more systematic differences between cells within the population .",
"Examples demonstrating this point are shown in Figure 1—figure supplement 5 and Figure 2—figure supplement 2 . Cross-correlations from live cells were calculated as described previously ( Stone and Veatch , 2015 ) , where the time evolution of the cross correlation was used to better specify the instantaneous cross-correlation .",
"In brief , cross-correlation functions were computed on a frame-by frame basis from localizations in each channel that occurred in the same frame or in frames separated by a time delay τ .",
"Cross-correlations between frames with time separation of up to 50 frames ( 0 s < τ < 1",
"s ) did not decay significantly ( Figure 2—figure supplement 6 ) and were therefore averaged to obtain a steady-state cross-correlation for data collected in a time window between 0 and 6 min after clustering with streptavidin .",
"Long-range gradients in labeling density arise in live-cell data because labeled molecules continually diffuse onto the ventral membrane from the dorsal membrane during the imaging experiment .",
"The dorsal membrane is outside the reach of TIRF illumination and away from the high laser power that both converts probes to a fluorescent 'off' state and slowly bleaches them .",
"Therefore , probes near the edges of the cell footprint are more likely to reside in a fluorescent 'on' state , and as a result these areas are more densely sampled .",
"To compensate for the effects of this long-range structure on our measurement , we normalize steady-state cross-correlations by the cross-correlation function of the masked average images from each channel which are first convoluted with a two-dimensional Gaussian function with σ = 1 µm .",
"This treatment filters structure larger than 1 µm in size from the steady-state cross-correlation function .",
"For step-size analysis , single molecule trajectories were constructed from super-resolution localizations using a tracking algorithm that searches for localizations within 500 nm in subsequent frames and terminates ambiguous trajectories ( Shelby et al . , 2013 ) .",
"The step size distribution for BCR-correlated probes is calculated by finding all instances of probe localization within 100 nm of a simultaneous BCR localization , and comparing that position to the location of the probe in immediately preceding and subsequent frames .",
"These step sizes were compiled over tens of thousands of frames from multiple single-cell experiments .",
"For measurements of calcium mobilization following BCR clustering and activation , 5 million CH27 cells were loaded with 2 µg/mL Fluo-4 AM ( Invitrogen ) for 5 min at room temperature in 1 mL BSS buffer with 0 . 25 mM sulfinpyrazone .",
"The cell suspension was subsequently diluted to a final volume of 15 mL with BSS buffer and incubated for 30 min at 37°C to allow for dye loading .",
"700 , 000 cells in 1 . 8 mL BSS buffer were then treated with either methyl-β-cyclodextrin ( MβCD ) , MβCD loaded with cholesterol ( Sigma ) , or left untreated at 37°C for 15 min .",
"The concentrations of both MβCD+cholesterol and MBCD were determined by the molecular weight of MBCD alone , 1310 Da .",
"For each treatment condition , cells were then spun down and resuspended in 1 mL of calcium-free PBS with 0 . 25 mM sulfinpyrazone .",
"Cells were spun down again and resuspended in 400 µL PBS .",
"Approximately 300 , 000 cells were loaded into individual wells of a black 96 well plate .",
"Fluo-4 was visualized on a fluorescence plate reader ( Omega; BMG Labtech , Ortenberg , Germany ) using excitation centered at 485 nm and emission centered at 520 nm .",
"Cells were stimulated by addition of f ( Ab ) 2 goat anti-mouse IgM ( Jackson Immunoresearch; RRID:AB_2338469 ) to a final concentration of 3 µg/mL .",
"Average calcium mobilization curves were generated from 2–4 wells per treatment condition .",
"Baseline drift was corrected by fitting a line to the Fluo-4 fluorescence trace prior to antigen addition and dividing the entire fluorescence trace by this baseline .",
"Baseline-corrected fluorescence traces therefore reflect the fold increase in signal compared to spontaneous calcium release and fluorescence background .",
"Baseline-corrected curves were then integrated over a two-minute window after antigen addition that captured the peak calcium response , as shown in Figure 5—figure supplement 2 . For calcium measurements with CTxB clustering , adherent CH27 cells were labeled with biotinylated CTxB in the same manner as super-resolution imaging measurements , described above , and then loaded with 0 . 4 µg/ml Fluo-4 AM in BSS buffer at 37°C for 30 min .",
"Cells were washed and imaged in BSS buffer at room temperature at 10x magnification using a FITC filter set with epifluorescence excitation .",
"Cells were imaged every 0 . 2 s for 1 min before and 8 min after addition of 50 µg/mL streptavidin .",
"Data were recorded using a Neo sCMOS camera ( Andor , South Windsor , CT ) .",
"After data acquisition , Fluo-4 intensity traces for individual cells were tabulated through an automated image processing algorithm that localized cells and tracked pixel intensities corresponding to individual cells before and after stimulation .",
"Raw intensity traces are shown in Figure 3—figure supplement 1 and include the non-zero offset of the camera .",
"Western blots were performed on CH27 cell lysates that probed protein tyrosine phosphorylation following binding and clustering of CTxB or clustering of BCR .",
"One million cells at a concentration of 2 million cells/mL were used for each sample .",
"For samples where BCR was clustered , 10 µg/mL f ( Ab ) 2 goat anti-mouse IgM ( Jackson Immunoresearch; RRID:AB_2338469 ) was added to cells for 2 min prior to cell lysis .",
"For samples were CTxB was bound , cells were incubated with 10 µg/mL CTxB biotin for 10 min , followed either by cell lysis or CTxB clustering by incubation with 100 µg/mL streptavidin for an additional 2 min prior to lysis .",
"Cells were lysed on ice for 20 min with shaking in 1X RIPA buffer containing 1X Halt Phosphatase Inhibitor Cocktail , 4 mM EDTA , and 1X solution of cOmplete Mini protease inhibitor tablet .",
"Cell lysates were spun down for 15 min at 16 , 000g and 4°C , and the supernatant was collected .",
"Lysates were flash frozen in liquid nitrogen and stored at −20°C .",
"Samples were run on SDS PAGE gels with 10% acrylamide , and gels were transferred using the iBlot Dry Blotting System ( ThermoFisher ) as per manufacturers recommendations .",
"Phosphotyrosine was detected by incubating blots with a 1:2500 dilution of anti-phosphotyrosine 4G10 Platinum primary mouse antibody ( Millipore , RRID:AB_916370 ) overnight at 4°C .",
"Blots were then incubated in a 1:1000 solution of horseradish peroxidase-conjugated secondary antibody ( goat anti-mouse IgG , Fcγ subclass 2b specific; Jackson ImmunoResearch; RRID:AB_2338515 ) for 2 hr at room temperature .",
"Actin labeling was used as a loading control , and blots were stripped and re-probed with a 1:1000 solution of rabbit polyclonal anti-actin primary antibody ( Cytoskeleton; RRID:AB_10708070 ) followed by a 1:1000 solution of horseradish peroxidase-conjugated secondary antibody ( goat anti-rabbit IgG; Jackson ImmunoResearch; RRID:AB_2307391 ) .",
"Chemiluminescence was captured using a GelDoc system .",
"Blot band intensity was analyzed in MATLAB .",
"Total band intensities were summed within user-defined regions after subtracting the average background intensity estimated from unused lanes .",
"Total band intensities were normalized by corresponding actin band intensities for each lane .",
"For probe partitioning measurements ( Figure 1—figure supplement 2 ) , giant plasma membrane vesicles ( GPMVs ) were made from adherent rat basophilic leukemia cells ( RBL-2H3 , ATCC CRL-2256; RRID: CVCL_0591 ) using established protocols ( Baumgart et al . , 2007; Veatch et al . , 2008; Zhao et al . , 2013; Gray et al . , 2013 ) with minor modifications .",
"Prior to GPMV isolation , adherent cells were labeled with either 2 µg/mL CTxB conjugated to Alexa 647 ( Invitrogen ) for 10 min at room temperature or 3 µg/mL DiD C16 ( Invitrogen ) in 0 . 03% methanol for 10 min at room temperature .",
"When both DiD and CTxB were imaged , CTxB conjugated to Alexa 555 ( Invitrogen ) was used .",
"Cells were rinsed and incubated in a buffer containing dithiothreitol ( DTT; 2 mM ) and formaldehyde ( 25 mM ) in the presence of calcium ( 2 mM ) at 37°C for 2 hr with gentle rocking .",
"GPMVs were harvested and imaged at low temperature between two coverslips on a home built temperature-controlled stage as described previously ( Veatch et al . , 2008; Zhao et al . , 2013; Gray et al . , 2013 ) .",
"Vesicles were imaged on a separate IX81 inverted microscope ( Olympus ) using epifluorescence illumination with a Cy3 filter set ( Chroma ) for CTxB Alexa 555 and a Cy5 filter-set ( Chroma ) for DiD C16 .",
"Images were captured on a Neo SCMOS camera ( Andor ) .",
"The partitioning of eGFP-GG and YFP-TM were examined by imaging GPMVs harvested from cells transiently expressing these constructs using a GFP filter cube ( Chroma ) .",
"DiD C16 was used as a phase marker ( Figure 1—figure supplement 2 ) .",
"To examine PM anchor phase partitioning , GPMVs were prepared from cells expressing PM-eGFP as described above except with 4 mM glutathione substituted for DTT as the reducing agent .",
"Glutathione was used as a reducing agent in these measurements because it is not cell permeable and therefore is not expected to directly impact the palmitoylation state of the PM peptide , whereas some reducing agents have been found to perturb protein palmitoylation in GPMVs ( Levental et al . , 2010 ) .",
"We note that GPMVs prepared using glutathione have lower transition temperatures and a larger surface fraction of ordered phase than GPMVs prepared using DTT .",
"Due to the low phase separation temperature of vesicles prepared in this manner , 6 µM hexadecanol was added to raise the phase separation temperature to about 1°C ( Machta et al . , 2016 ) so that phase separated vesicles could be observed .",
"GPMVs were imaged as described above .",
"To examine how the surface fraction of ordered and disordered phases varies with acute cholesterol variation , adherent CH27 cells were first pre-treated with either 10 mM MβCD or 10 mM MβCD pre-complexed with cholesterol for 10 min .",
"Cells were then labeled with 2 µg/ml DiI-C12 ( Invitrogen ) in 0 . 02% methanol for 10 min at room temperature and GPMVs were prepared and imaged as described above using DTT as the reducing agent .",
"Fewer vesicles were obtained in MβCD or MβCD-chol pretreated cells than in untreated cells , likely because treated cells were less adherent .",
"A conserved order parameter 2D Ising model was simulated on a 256 by 256 square lattice as described previously ( Machta et al . , 2011 ) with minor modifications .",
"Briefly , components that prefer ordered or disordered regions are represented as pixels that have value of S = +1 and S = -1 respectively .",
"The vast majority of +1 and −1 pixels represent unspecified membrane components ( proteins and lipids ) .",
"In addition , 50 pixels with values of +1 are classified as receptors , 100 pixels with values +1 are classified as kinases , and 100 pixels with values −1 are classified as phosphatases .",
"Receptors are clustered by applying a strong attractive circular field ( φR ) at the center of the simulation frame that only acts on receptors .",
"The final Hamiltonian is given by:H=−∑i , jSiSj−∑iRiΦiR The first term sums over the four nearest neighbors ( j ) surrounding the pixel i and applies to all components .",
"The second term only contributes when receptors occupy position i , where Ri=1 , otherwise Ri=0 .",
"The receptor field ΦiR has a circular shape with a radius of 16 pixels ( 32 nm ) and is centered in a simulation box with periodic boundary conditions .",
"When an ordered domain is stabilized in the absence of receptor clustering , a similar Hamiltonian is used with an applied field that is felt by all membrane components .",
"In this case:H=−∑i , jSiSj−∑iSiΦiD The domain field ΦiD has a circular shape with a radius of either 24 pixels ( ~50 nm ) or 48 pixels ( ~100 nm ) and is centered in a simulation box with periodic boundary conditions .",
"The magnitude of this field was chosen to be equal to a single interaction between components , which is one in these units .",
"This magnitude is sufficient to stabilize a robust domain containing ordered components but does not restrict the motions of individual components within the domain .",
"At each update , two random pixels are chosen , the energy cost or gain for exchanging the two pixels is calculated , and the move is either accepted or rejected using a Monte Carlo algorithm that maintains detailed balance .",
"If the resulting configuration is lower or equal in energy , the exchange is always accepted .",
"If the energy is raised , the exchange is accepted stochastically with probability exp ( −β∆H ) where β is the inverse temperature and ΔH is the change in energy between initial and final states .",
"In this scheme , the critical point occurs at TC = 2/ln ( 1+sqrt ( 2 ) ) .",
"All simulations were run at T = 1 . 05 × TC .",
"One pixel is chosen to represent a 2 nm by 2 nm patch of membrane , so that the correlation length varies with temperature in simulations with equal fractions of ordered and disordered components as observed in experimental observations in isolated plasma membrane vesicles ( Veatch et al . , 2008 ) .",
"Most simulations were run such that there were an equal fraction of ordered and disordered unspecified membrane components .",
"In some cases , the fraction of unspecified membrane compositions assigned to be ordered was varied , as indicated in Figure captions .",
"Uniform simulations were run by setting all unspecified membrane components to be disordered .",
"One sweep corresponds to the option to exchange each of the pixels on average twice ( 2562 pixel swaps are proposed ) .",
"All simulations are initially run using non-local exchanges to decrease equilibration times .",
"For simulations recording receptor phosphorylation state , exchanges were then restricted to nearest neighbors in order to better mimic diffusive dynamics .",
"Simulation sweeps are converted to time assuming a diffusion coefficient of roughly 4 μm2/s , with one sweep corresponding to roughly 1 μs .",
"Most simulations were recorded for 1000 sweeps which corresponds to roughly 1 s .",
"If a move is accepted that places a receptor neighboring a kinase , then the receptor is phosphorylated at a low probability ( 0 . 1% ) .",
"If a move is accepted that places a receptor neighboring a phosphatase , then the receptor is dephosphorylated at a high probability ( 100% ) .",
"These probabilities are chosen to produce a low level of phosphorylation in simulations that contain an equal number of kinases and phosphatases with unclustered receptors .",
"Higher probability of dephosphorylation is physiologically relevant because phosphatases such as CD45 are expressed in the plasma membranes of lymphocytes at several-fold higher densities than Src kinases ( e . g . T cells express between 100 , 000 and 500 , 000 CD45 molecules and between 40 , 000 and 120 , 000 Lck molecules per cell ( Olszowy et al . , 1995; Hui and Vale , 2014 ) .",
"In some simulations , receptors have kinase behavior when they are phosphorylated .",
"In this case , a move that places a phosphorylated receptor next to a second receptor results in the second receptor becoming phosphorylated at a low probability ( 0 . 1% ) .",
"To mimic the experimental limitation of finite lateral resolution , cross-correlation functions between receptors and membrane components were also tabulated from simulation snapshots that were first filtered with a Gaussian shaped point spread function with the indicated width .",
"This is equivalent to convolving the raw two dimensional C ( r , θ ) with the autocorrelation of the point spread function gPSF",
"( r ) ( Veatch et al . , 2012 ) .",
"All analyses were carried out in MATLAB ( The MathWorks , Natick , MA; RRID: SCR_001622 ) .",
"Plasmids and reagents can be obtained via request of the corresponding author ."
]
] | [
"Diverse cellular signaling events , including B cell receptor ( BCR ) activation , are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes .",
"This concept remains controversial and lacks direct experimental support in intact cells .",
"Here , we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy , demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation , and present a minimal , predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain .",
"These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways .",
"Overall , these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction ."
] | [
"Membranes made of molecules called lipids surround every living cell to protect the cell's contents .",
"Cells also communicate with the outside environment via their membranes .",
"Proteins in the membrane receive information from the environment and trigger signaling pathways inside the cell to relay this information to the center of cell .",
"The way in which proteins are organized on the membrane has a major influence on their signaling activity .",
"Some areas of the membrane are more crowded with certain lipids and signaling proteins than others .",
"Lipid and protein molecules of particular types can come together and form distinct areas called “ordered” and “disordered” domains .",
"The lipids in ordered domains are more tightly packed than disordered domains and it is thought that this difference allows domains to selectively exclude or include certain proteins .",
"Ordered domains are also known as \"lipid rafts\" .",
"Lipid rafts and disordered domains may help cells to control the activities of signaling pathways , however , technical limitations have made it difficult to study the roles of these domains .",
"The membranes surrounding immune cells called B cells contain a protein called the B cell receptor , which engages with proteins from microbes and other foreign invaders .",
"When the B cell receptor binds to a foreign protein it forms clusters with other B cell receptors and becomes active , triggering a signaling pathway that leads to immune responses .",
"Stone , Shelby et al . examined lipid rafts and disordered domains in B cells from mice using a technique called super-resolution fluorescence microscopy .",
"The results show that clusters of B cell receptors are present within lipid rafts .",
"These clusters made the lipid rafts larger and more stable .",
"A protein that is needed during the early stages of B cell receptor signaling was also found in the same lipid rafts .",
"Another protein that terminates signaling was excluded because it prefers disordered domains .",
"Together , this provides a local environment in certain areas of the membrane that favors receptor activity and supports the subsequent immune response .",
"Future work is needed to understand how cells control the make-up of lipids and proteins within their membranes , and how defects in this regulation can alter signaling activity and lead to disease ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"neuroscience"
] | Ca2+-dependent release of synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes | elife-57154-v3 | [
[
"The release of neurotransmitters by Ca2+-evoked synaptic vesicle exocytosis is an exquisitely regulated process that is critical for communication between neurons .",
"Release involves tethering of synaptic vesicles to presynaptic active zones , priming of the vesicles to a release-ready state , and Ca2+-triggered fusion of the vesicle and plasma membranes ( Südhof , 2013 ) .",
"Extensive research has allowed reconstitution of basic features of synaptic vesicle fusion with the central components of the neurotransmitter release machinery ( Liu et al . , 2016; Ma et al . , 2013 ) and led to defined models for their functions ( Brunger et al . , 2018; Rizo , 2018 ) .",
"The SNAP receptors ( SNAREs ) syntaxin-1 , SNAP-25 and synaptobrevin form a tight four-helix bundle called the SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion ( Hanson et al . , 1997; Poirier et al . , 1998; Söllner et al . , 1993; Sutton et al . , 1998 ) .",
"This complex is disassembled by N-ethylmaleimide sensitive factor ( NSF ) and soluble NSF adaptors proteins ( SNAPs ) ( Söllner et al . , 1993 ) , whereas SNARE complex assembly is organized in an NSF-SNAP-resistant manner by Munc18-1 and Munc13s ( Ma et al . , 2013; Prinslow et al . , 2019 ) .",
"Release is triggered very fast ( <1 ms ) after Ca2+ influx by the Ca2+ sensor Synaptotagmin-1 ( Syt1 ) ( Fernández-Chacón et al . , 2001 ) .",
"Syt1 is a synaptic vesicle protein with two C2 domains ( named C2A and C2B ) that form most of its cytoplasmic region and adopt β-sandwich structures that bind multiple Ca2+ ions through loops at the top of the β-sandwich ( Fernandez et al . , 2001; Sutton et al . , 1995; Ubach et al . , 1998 ) .",
"These loops also mediate Ca2+-dependent binding to phospholipids ( Chapman and Davis , 1998; Frazier et al . , 2003; Zhang et al . , 1998 ) , which is crucial for neurotransmitter release ( Fernández-Chacón et al . , 2001; Rhee et al . , 2005 ) .",
"A polybasic region on the side of the C2B domain β-sandwich also contributes to membrane binding , in part via interactions with phosphatidylinositol 4 , 5-bisphosphate ( PIP2 ) ( Bai et al . , 2004; Li et al . , 2006 ) .",
"In addition , the Syt1 C2B domain can bind simultaneously to two membranes in a Ca2+ dependent manner through its Ca2+-binding loops and two arginines at the bottom of the β-sandwich ( R398 , R399 ) that are crucial for Syt1 function ( Xue et al . , 2008 ) , suggesting that Syt1 cooperates with the SNAREs in bringing the vesicle and plasma membranes together to mediate membrane fusion ( Araç et al . , 2006; Seven et al . , 2013 ) .",
"Induction of membrane curvature by insertion of the C2 domain Ca2+-binding loops into the bilayer was also proposed to stimulate membrane fusion ( Martens et al . , 2007 ) .",
"While these ideas are attractive , the mechanism of action of Syt1 remains enigmatic , in part because dozens of papers have described Syt1-SNARE interactions but it is unclear which of these interactions is physiologically relevant and which ones arise merely from the high promiscuity of these proteins ( Jahn and Scheller , 2006; Rizo et al . , 2006 ) .",
"Moreover , Syt1 is believed to function in a tight interplay with complexins whereby complexins play both inhibitory and stimulatory functions and Ca2+ binding to Syt1 releases the inhibition ( Giraudo et al . , 2006; Schaub et al . , 2006; Tang et al . , 2006 ) , but the underlying mechanism is unknown .",
"Potentially critical insights were provided by three structures of Syt1-SNARE complexes ( Brewer et al . , 2015; Zhou et al . , 2015; Zhou et al . , 2017 ) , but the observed binding modes were drastically different ( Figure 1A–G ) .",
"Analysis in solution by NMR spectroscopy revealed a dynamic interaction involving the polybasic region of the Syt1 C2B domain , while R398 , R399 at the bottom of C2B remain available for membrane binding ( Brewer et al . , 2015; Figure 1A ) .",
"The functional relevance of this binding mode was supported by the finding that release was disrupted by mutations of basic residues that contact the SNAREs ( including R322E , K325E ) , but not by nearby basic residues that do not point toward the SNAREs ( K324E , K326E ) ( Figure 1D ) , while both mutations disrupted PIP2 binding similarly ( Brewer et al . , 2015 ) .",
"The second structure , determined by X-ray crystallography , revealed a so-called primary interface involving two regions of the C2B domain , one formed largely by E295 and Y338 ( region I ) and another including R398 , R399 ( region II ) ( Figure 1B , E , F; Zhou et al . , 2015 ) .",
"In this structure , the polybasic region is available for PIP2 binding , an arrangement that was supported by a low-resolution cryo-electron microscopy ( cryo-EM ) structure of Syt1-SNARE complexes coating lipid tubes ( Grushin et al . , 2019 ) .",
"The physiological relevance of the primary interface was supported by the strong impairments of release caused by mutations in both region I and II ( E295A/Y338W and R398Q/R399Q ) .",
"However , Syt1-SNARE co-immunoprecipitation ( co-IP ) was not significantly altered by the R398Q/R399Q mutation and only moderately disrupted by the E295A/Y338W mutation ( Zhou et al . , 2015 ) .",
"X-ray crystallography of a tripartite Syt1-complexin-1-SNARE complex yielded a third structure ( Zhou et al . , 2017 ) where complexin-1 forms an α-helix that binds to a groove between synaptobrevin and syntaxin-1 , as observed for a binary complexin-1-SNARE complex ( Chen et al . , 2002 ) , and an α-helix of the C2B domain binds to the same groove , continuing the complexin-1 helix ( Figure 1C , G ) .",
"Isothermal titration calorimetry ( ITC ) supported the existence of this interaction in solution and suggested that Syt1 cooperates with complexin-1 for binding to the SNARE complex , providing an explanation for the finding that the dominant negative effect on release caused by mutation of the Syt1 C2B domain Ca2+ binding sites ( Mackler et al . , 2002; Wu et al . , 2017 ) requires complexin-1 ( Zhou et al . , 2017 ) .",
"The functional importance of the tripartite interface was supported by the observation that a L387Q/L394Q mutation disrupted binding and impaired neurotransmitter release .",
"However , as pointed out in Zhou et al . , 2017 , the primary interface was also observed in these crystals , which had a 1:1:1 Syt1-SNARE complex-complexin-1 stoichiometry .",
"Hence , one of the two interfaces might constitute a crystal contact .",
"Moreover , a screen for mutations that abrogate the dominant negative effect of mutations in the Syt1 C2B domain Ca2+-binding sites in Drosophila yielded a large number of mutations in the primary interface but none in the tripartite interface ( Guan et al . , 2017 ) , indicating that the dominant negative effect may require Syt1-SNARE complex binding through the primary rather than the tripartite interface .",
"The role of Ca2+ in Syt1-SNARE interactions has also been enigmatic .",
"Many studies reported that Ca2+ strongly stimulates binding of Syt1 to SNAREs and SNARE complexes both in solution and on membranes [e . g . ( Chapman et al . , 1995; Dai et al . , 2007; Davis et al . , 1999; Gerona et al . , 2000; Huang and Cafiso , 2008; Li et al . , 1995; Lynch et al . , 2007; Zhou et al . , 2013 ) ] .",
"Paradoxically , the Syt1 Ca2+-binding loops are not involved in SNARE complex binding in the structures described above , although Ca2+ is believed to stimulate binding by increasing the positive electrostatic potential of the C2B domain , as the SNARE complex is negatively charged .",
"Note also that Ca2+ disrupted Syt1-SNARE complexes assembled on lipid tubes ( Grushin et al . , 2019 ) , but another study reported binding of Syt1 to membrane-anchored SNARE complex in the absence and presence of Ca2+ ( Wang et al . , 2016 ) .",
"Conversely , ATP was reported to abolish Syt1-SNARE complex at physiological ionic strength , with or without Ca2+ ( Park et al . , 2015 ) .",
"To shed light into this strikingly confusing picture and decipher how the functions of Syt1 and the SNAREs are coupled , we performed a systematic analysis of Syt1-SNARE complex interactions in solution and on membranes .",
"NMR data show that a soluble complexin-1-SNARE complex binds to the polybasic region and the primary interface of the Syt1 C2B domain with similar affinities , but binding to the tripartite interface could not be detected .",
"Analyses of binding of a Syt1 fragment spanning the two C2 domains ( C2AB ) to nanodiscs or nanodiscs containing anchored SNARE complexes using FRET show that C2AB binds to membrane-anchored SNARE complexes under a variety of conditions , and that complexin-1 does not affect the affinity .",
"Binding of the SNARE complex to the primary interface is impaired by the R398Q/R399Q mutation but is enhanced by the E295A/Y338W mutation , both in solution and on nanodiscs , showing that the primary interface is involved in binding to the nanodisc-anchored SNARE complex .",
"Importantly , Ca2+-dependent binding of C2AB to SNARE complexes anchored on PIP2-nanodiscs is almost abolished in the presence of ATP , but Ca2+-independent binding remains .",
"We also observed that the R322E/K325E mutation disrupts Ca2+-dependent binding of C2AB to PIP2-containing nanodiscs much more strongly than the K324E/K326E mutation , in correlation with the effects of these mutations on release ( Brewer et al . , 2015 ) .",
"Together with previous data , these results suggest a model whereby Syt1 binds to the SNARE complex through the primary interface and to PIP2 through the polybasic region before Ca2+ influx; Ca2+ binding to the C2B domain induces a specific , PIP2-dependent interaction with the plasma membrane , disrupting the interaction with the SNARE complex and allowing cooperation between the SNAREs and Syt1 in inducing membrane fusion ."
],
[
"The tendency of the Syt1 C2 domains to precipitate with the SNARE complex formed by the SNARE motifs of synaptobrevin , syntaxin-1 and SNAP-25 , particularly in the presence of Ca2+ , hindered analysis of their interactions in solution by NMR spectroscopy ( Dai et al . , 2007; Zhou et al . , 2013 ) .",
"Inclusion of 125 mM KSCN allowed NMR analyses in the presence of Ca2+ ( Brewer et al . , 2015 ) , but KSCN might have favored binding of the SNARE complex to the C2B domain polybasic region over binding to the primary interface .",
"In attempts to solve the solubility problem without addition of KSCN , we found that including the complexin-1 fragment that we used for crystallization with the SNARE complex [residues 26–83; Cpx1 ( 26-83 ) ] ( Chen et al . , 2002 ) dramatically improved the solubility of mixtures containing the Ca2+-bound C2B domain and the SNARE complex ( Figure 1—figure supplement 1 ) .",
"Taking advantage of this observation , we analyzed the binding sites of the SNARE complex on the Syt1 C2B domain in the presence of Cpx1 ( 26-83 ) using NMR spectroscopy .",
"Note that the Syt1 C2 domains do not bind to complexin-1 under the conditions that we used for the NMR experiments and that Cpx1 ( 26-83 ) binds to the SNARE complex with high affinity ( KD ca . 25 nM ) ( Xu et al . , 2013 ) .",
"Hence , the assembly formed by Cpx1 ( 26-83 ) and the SNARE motifs of synaptobrevin , syntaxin-1 and SNAP-25 can be considered as a single complex that we refer to as CpxSC .",
"For improved sensitivity , we prepared samples of the C2B domain that were 2H , 15N-labeled and specifically 13CH3-labeled at the Ile δ1 and Met methyl groups ( 2H , 15N-IM-13CH3-C2B ) .",
"First we analyzed the perturbations caused by addition of increasing amounts of CpxSC on transverse relaxation optimized spectroscopy ( TROSY ) -enhanced 1H-15N heteronuclear single quantum coherence ( HSQC ) spectra of 2H , 15N-IM-13CH3-C2B .",
"Titrations of 2H , 15N-IM-13CH3-C2B with CpxSC were performed in the presence of Ca2+ at physiological ionic strength and in the absence of Ca2+ at lower salt concentration ( 100 mM KCl ) to enhance binding , as Syt1-SNARE complex binding is weaker in the absence than in the presence of Ca2+ ( Zhou et al . , 2013 ) .",
"Similar cross-peak shifts were observed in both sets of experiments ( Figure 1H , Figure 1—figure supplement 2 ) , indicating that Ca2+ does not affect the binding mode ( s ) .",
"To analyze the data , we obtained assignments of the 1H-15N TROSY-HSQC spectrum of Ca2+-free 2H , 15N-IM-13CH3-C2B ( Figure 1—figure supplement 3 ) based on the assignments obtained previously in a different buffer ( Fernandez et al . , 2001 ) and a titration where the buffer composition was gradually changed from one buffer to the other .",
"Mapping of the residues corresponding to the cross-peaks that exhibited the largest shifts induced by CpxSC ( Figure 1I ) onto the structure of the C2B domain clearly showed that the residues are clustered on two sides of the β-sandwich , one containing the polybasic region and another corresponding to the primary interface ( Figure 1J ) .",
"There were no significant shifts in cross-peaks from residues in the tripartite interface , which is largely formed by the α-helix spanning residues 384–395 , except for residues at the very C-terminus of this helix that are near the primary interface .",
"We could not obtain accurate KDs for the interactions of CpxSC with the polybasic region and the primary interface from these data because we did not achieved saturation at the highest concentration of CpxSC that we reached ( 85 μM ) and , at these concentrations , some cross-peaks broadened beyond detection or exhibited odd behavior that did not correlate with the previous titration points ( illustrated in Figure 1K by the cross-peaks from two residues corresponding to the polybasic region , K325 and K369 , and two residues from the primary interface , V283 and Y339 ) .",
"However , it was clear that the affinities of both binding sites are comparable , and the KDs are larger than 20 μM .",
"We next examined the effects of mutations in the two binding sites of the C2B domain for the SNARE complex , using 2H , 15N-IM-13CH3-labeled samples of C2B domain mutants .",
"Since the C2B domain Ca2+-binding region is not involved in binding and Ca2+ did not affect the binding modes , these experiments were performed in the absence of Ca2+ .",
"We first analyzed the effects of the R322E/K325E mutation that we designed previously to disrupt SNARE complex binding to the polybasic region and strongly impaired neurotransmitter release ( Brewer et al . , 2015 ) .",
"A titration with CpxSC revealed substantial shifts for selected cross-peaks of the C2B mutant HSQC spectrum , but the patterns were somewhat distinct from those observed for the WT C2B domain ( compare Figure 2A with 1H ) .",
"The cross-peaks from the β-strand containing the mutated residues shifted and their positions could not be ascertained .",
"However , we could identify the diagnostic cross-peak from K369 , which is in the same face of the C2B domain as the polybasic region .",
"The position of this cross-peak was unaffected even at 85 μM CpxSC ( Figure 2C ) , confirming that binding to the polybasic region was abolished by the R322E/K325E mutation .",
"We also observed that the diagnostic cross-peaks from the primary interface corresponding to V283 and Y339 shifted faster in the titration of the R322E/K325E mutant than observed for the WT C2B domain ( Figure 2C ) , which is a natural consequence of the lack of competition with the polybasic region for binding to CpxSC .",
"Interestingly , multiple cross-peaks from the primary interface exhibited much more substantial CpxSC-induced shifts for the mutant than for the WT C2B domain , including cross-peaks from R398 and I401 ( Figure 2A , D ) , residues that are near V283 ( Figure 1E ) .",
"These results suggest that binding of CpxSC to the primary interface is more extensive for the R322E/K325E mutant C2B and that the polybasic region not only competes with the primary interface for binding to CpxSC but also hinders full C2B-SNARE engagement at the primary interface .",
"R398 and R399 at the bottom of the C2B domain are in the primary interface ( Figure 1E ) , but the R398Q/R399Q mutation that almost abolishes neurotransmitter release ( Xue et al . , 2008 ) did not significantly impair Syt1-SNARE co-IP ( Zhou et al . , 2015 ) .",
"To investigate the contribution of these tandem arginines to Syt1-SNARE complex binding at the primary interface , we analyzed binding of CpxSC to the R398Q/R399Q mutant C2B domain .",
"We observed that the CpxSC-induced shifts of the cross-peaks from the primary interface were almost abolished ( Figure 2B and expansions for the V283 and Y339 cross-peaks in Figure 2C ) , showing that the mutation strongly impairs binding of CpxSC to this interface .",
"As expected , CpxSC still bound to the polybasic region of R398Q/R399Q C2B ( e . g . K369 cross-peak , Figure 2C ) .",
"Since individual R398Q and R399Q mutations also impair release considerably ( Xue et al . , 2008 ) , we also tested binding of CpxSC to R398Q C2B and R399Q C2B mutants .",
"Both single mutations impaired CpxSC binding substantially ( Figure 2—figure supplement 1 ) , although to a smaller extent than the double R398Q/R399Q mutation .",
"These results show that both R398 and R399 play important roles in binding of Syt1 to CpxSC through the primary interface .",
"We also prepared a mutant C2B domain bearing both the R322E/K325E mutation in the polybasic region and the R398Q/R399Q mutation in the primary interface .",
"Titration with CpxSC did not induce significant shifts in the 1H-15N TROSY-HSQC spectrum of the R322E/K325E/R398Q/R399Q C2B mutant even when CpxSC was added at 85 μM concentration ( Figure 2E ) .",
"Binding of an unlabeled protein or complex to a 15N-labeled protein is expected to cause not only cross-peak shifts but also decreased cross-peak intensities due to the larger size of the resulting complex compared to the isolated 15N-labeled protein ( Rizo et al . , 2012 ) , as illustrated in Figure 2—figure supplement 2A–C by the gradual decreases in intensities of selected cross-peaks of WT C2B caused by increasing CpxSC concentrations .",
"In contrast , the cross-peak intensities of R322E/K325E/R398Q/R399Q C2B did not decrease appreciably as increasing concentrations of CpxSC were added .",
"Thus , the ratios of the intensities in the presence of 85 μM CpxSC versus the intensities in the absence of CpxSC for the same selected cross-peaks of R322E/K325E/R398Q/R399Q C2B were close to 1 ( Figure 2—figure supplement 2A–C ) .",
"Analysis of these ratios for all cross-peaks showed a relatively homogeneous distribution , with an average ratio of 0 . 947 and some natural variability due to the noise in the data , particularly for the weakest cross-peaks ( Figure 2—figure supplement 2D ) .",
"The average ratio for five cross-peaks that are in well-resolved regions of the spectrum and correspond to residues in the α-helix involved in the tripartite interface ( T383 , G384 , L387 , R388 and S391 ) is 0 . 945 .",
"These data show that the quadruple R322E/K325E/R398Q/R399Q mutation abolishes binding of the Syt1 C2B domain to CpxSC and the mutant does not bind through the tripartite interface under these conditions .",
"To corroborate this conclusion , we also analyzed samples where Cpx1 ( 26-83 ) was 2H , 15N-labeled and formed 2H , 15N-CpxSC to analyze perturbations on Cpx1 ( 26-83 ) upon C2B domain binding .",
"Addition of WT Syt1 C2B domain caused substantial broadening in the 1H-15N TROSY-HSQC spectrum of CpxSC , as manifested by decreased intensities in the cross-peaks corresponding to structured parts of Cpx1 ( 26-83 ) in the complex upon binding to C2B ( Figure 3A ) .",
"Importantly , C2B did not induce significant shifts in any of the cross-peaks from residues at or near the tripartite interface ( residues 66–75; see Figure 3A ) .",
"We note that K73 , K74 and K75 are flexible in the complex but their cross-peaks are in unique positions that are very sensitive to changes in their environment ( Chen et al . , 2002; Pabst et al . , 2000; Trimbuch et al . , 2014 ) .",
"Hence , the absence of shifts in these cross-peaks and those of residues 66–75 in general show that the C2B domain does not bind to CpxSC through the tripartite interface under these conditions .",
"Moreover , R322E/K325E/R398Q/R399Q C2B did not cause shifts or intensity decreases in the 1H-15N TROSY-HSQC spectrum of 2H , 15N-CpxSC ( Figure 3B ) , showing again that the quadruple mutation abrogates binding of the Syt1 C2B domain to CpxSC .",
"Hence , we do not find any evidence for binding of the C2B domain to CpxSC through the tripartite interface even after abolishing the interactions involving the polybasic region and the primary interface .",
"These results contrast with ITC data suggesting the existence of a Syt1-complexin-1-SNARE complex interaction involving the tripartite interface in experiments performed with Syt1 C2B bearing seven mutations designed to disrupt binding via the polybasic region and the primary interface ( KA-Q mutant ) , and a complexin-1-SNARE complex bearing five mutations to further disrupt such binding ( Zhou et al . , 2017 ) .",
"We are collaborating with the laboratory of Axel Brunger to determine the reasons underlying these conflicting results , and the results of these efforts will be published elsewhere .",
"Since the L387Q/L394Q mutation in the α-helix of the C2B domain that forms the tripartite interface was reported to disrupt binding through this interfaces based on ITC data ( Zhou et al . , 2017 ) , we tried to analyze whether this mutation perturbs binding of C2B to CpxSC .",
"The 1H-15N TROSY-HSQC spectrum of 2H , 15N-IM-13CH3-labeled L387Q/L394Q C2B exhibited well-dispersed cross-peaks characteristic of a folded protein domain , but also contained broad cross-peaks in the center of the spectrum that are commonly observed in unstable proteins that unfold and/or aggregate ( Rizo et al . , 2012; Figure 3—figure supplement 1 ) .",
"Addition of 20 μM CpxSC led to strong decreases in cross-peak intensities that arose at least in part from sample precipitation .",
"Hence it was not possible to continue the titration .",
"These results show that the L387Q/L394Q causes a considerable destabilization of the Syt1 C2B domain , which is consistent with the finding that this mutation decreases the thermal denaturation temperature of the C2B domain by about 10°C ( Zhou et al . , 2017 ) .",
"Such destabilization may arise because this mutation involves the replacement of a hydrophobic side chain that packs inside the C2B domain and barely contacts the SNAREs ( L394; Figure 1G ) with a polar residue .",
"Hence , it is plausible that the disruption of neurotransmitter release caused by the L387Q/L394Q mutation ( Zhou et al . , 2017 ) arose from a general loss of function caused by protein instability .",
"In summary , our data show that binding of the Syt1 C2B domain to the Cpx1 ( 26-83 ) -SNARE complex in solution involves the polybasic region and the primary interface , while binding via the tripartite interface is undetectable under the conditions of our NMR experiments even when CpxSC is added at 85 μM concentration and binding through the polybasic region and the tripartite interface is abolished .",
"These results indicate that the tripartite interface observed in the complexin-1-Syt1-SNARE complex crystals might have arisen from crystal packing , but further research will be required to clarify this issue .",
"The primary interface involves two regions of the C2B domain , one containing R398 and R399 ( region II ) , and the other E295 and Y338 ( region I ) .",
"The functional importance of region I was demonstrated by the strong disruption of neurotransmitter release caused by the E295A/Y338W mutation in this region ( Zhou et al . , 2015 ) .",
"To examine the effects of this mutation on Syt1-SNARE binding , we performed titrations of 2H , 15N-IM-13CH3-E295A/Y338W C2B with CpxSC .",
"Interestingly , we found that CpxSC caused more extensive changes in the 1H-15N TROSY-HSQC spectrum of this mutant ( Figure 4A ) than those observed for WT C2B domain ( Figure 1H ) .",
"Analysis of these changes showed that cross-peaks from residues in or near the polybasic region shifted less extensively and more slowly than observed for WT C2B , particularly in the initial points of the titration ( Figure 4B , C ) .",
"Conversely , cross-peaks from the primary interface moved faster and more extensively for E295A/Y338W C2B than for WT C2B; indeed , some cross-peaks that did not shift or barely shifted in WT C2B exhibited considerable shifts for E295A/Y338W C2B domain , including the R398 cross-peak ( Figure 4B , D ) .",
"These effects in cross-peaks from the primary interface are reminiscent of those observed for the R322E/K325E mutant C2B domain ( Figure 2A , C , D ) , but the shifts observed for the primary interface of the E295A/Y338W C2B mutant are even larger .",
"These results show that the E295A/Y338W mutation actually increases the affinity of the Syt1 C2B domain for CpxSC rather than impairs binding , and also appears to make the interaction at the primary interface more extensive , as observed for the R322E/K325E mutation .",
"The basis for this behavior is unclear , but the increased affinity caused by the E295A/Y338W mutation may arise from replacing a tyrosine with a tryptophan , which increases the hydrophobic surface area of this residue .",
"This change may be readily accommodated because the packing in this region of the interface with the SNARE complex is not optimal .",
"We note that the movement of some cross-peaks with increasing CpxSC concentration was curved in some cases ( e . g . those of K325 and R398 , Figure 4C , D ) .",
"This finding suggests that there is an interplay between binding of CpxSC to the primary interface and to the polybasic region .",
"CpxSC appears to bind exclusively to one or the other site at low concentrations , likely because of steric hindrance disfavors simultaneous binding of two CpxSC complexes to one C2B domain .",
"However , simultaneous binding to both sites might be allowed at higher CpxSC concentrations by slight alterations in both binding modes , leading to the curved cross-peak movement .",
"Because the WT and mutant C2B domains used for all the titrations with CpxSC were specifically 13CH3-labeled at the Met and Ile δ1 methyl groups , we also acquired 1H-13C heteronuclear multiple quantum coherence ( HMQC ) spectra of each sample , as these spectra offers very high sensitivity ( Ruschak and Kay , 2010 ) .",
"The HMQC spectra contained only a small number of probes ( Figure 4—figure supplement 1A ) and hence provided more limited information than the 1H-15N TROSY-HSQC spectra , but corroborated the conclusions obtained from the latter regarding binding to the primary interface .",
"Thus , CpxSC caused shifts in the cross-peak from the I401 δ1 methyl group at the primary interface that were more marked for the R322E/K325E mutant , were even larger for the E295A/Y338W mutant , and were abolished for the R398Q/R399Q and R322E/K325E/R398Q/R399Q mutants ( Figure 4—figure supplement 1B ) .",
"The cross-peak from the δ1 methyl of I293 , another residue at the primary interface , was shifted by CpxSC only for the E295A/Y338W mutant .",
"Our NMR data show that there are two major binding modes between the Syt1 C2B domain and the SNARE complex in solution , one involving the polybasic region of C2B and the other involving the primary interface , which includes the tandem arginines R398 , R399 .",
"Since the polybasic region and the tandem arginines have been implicated also in membrane binding [e . g . ( Araç et al . , 2006; Bai et al . , 2004; Li et al . , 2006; Xue et al . , 2008 ) ] , it is critical to analyze interactions between Syt1 and membrane-anchored SNARE complexes to assess whether either of these two binding modes still remain in the presence of membranes , or a different type of interaction might occur .",
"For this purpose , we designed a strategy based on anchoring SNARE complexes on nanodiscs .",
"For these studies we used the Syt1 C2AB fragment that spans both C2 domains because the C2A domain contributes to binding of Syt1 to membranes and could contribute to binding to the SNARE complex directly or indirectly , for example through cooperativity between SNARE and lipid interactions of Syt1 .",
"As a scaffold for the nanodiscs , we chose MSP1E3D1 because it yields stable nanodiscs with a diameter of ca .",
"13 nm ( Nath et al . , 2007 ) that can accommodate the SNARE complex and allow potential simultaneous interactions of Syt1 C2AB with the SNARE complex and the lipids .",
"We prepared SNARE complexes with full-length syntaxin-1 and the SNARE motifs of SNAP-25 and synaptobrevin , anchoring the complexes on the nanodiscs through the syntaxin-1 transmembrane ( TM ) region to mimic the configuration expected to occur on the plasma membrane .",
"The stoichiometry of MSP1D3 to syntaxin-1 was adjusted to form nanodiscs that on average contained one SNARE complex .",
"We refer to these macromolecular assemblies as cisSC-NDs , while control nanodiscs prepared without the SNAREs are abbreviated as NDs ( Figure 5A ) .",
"Compared to experiments using liposomes , this modular design facilitates analysis of individual SNARE complex-Syt1-membrane assemblies without complications that might arise from liposome clustering induced by C2AB ( Araç et al . , 2006 ) .",
"To monitor binding of C2AB to NDs or cisSC-NDs by FRET , we generated a single-cysteine C2AB mutant with the cysteine replacing E346 , and labeled it with an Alex488 donor fluorescent probe ( C2AB* ) .",
"This position was chosen because residue 346 is not located on any of the interfaces that have been implicated in SNARE complex binding ( Figure 5—figure supplement 1A ) and placing the fluorescent probe on this residue is not expected to disrupt these interfaces .",
"Unless otherwise indicated , NDs contained 5% rhodamine-labeled phosphatidylethanolamine ( Rho-PE ) , which constitutes a suitable acceptor probe for highly efficient FRET with Alexa488 .",
"Indeed , titration of C2AB* with cisSC-NDs formed with a 80:15:5 mixture of phosphatildylcholine ( PC ) , phosphatidylserine ( PS ) and Rho-PE in the presence of 125 mM KCl and 1 mM Ca2+ led to progressively more efficient FRET that maximized at a FRET efficiency of ca .",
"0 . 88 ( Figure 5B and C , blue curve ) .",
"Control experiments with analogous NDs lacking SNARE complex showed that saturation required higher ND concentrations ( Figure 5C , red curve ) , yielding a substantially higher apparent KD ( 129 . 5 nM compared to 26 . 6 nM for the cisSC-NDs ) .",
"A summary of the KDs obtained under these and other conditions described below is presented in Supplementary file 1 .",
"Supplementary file 2 lists cooperativity factors calculated from KD NDs/KD cisSC-NDs , which yield an idea of the synergy between interactions of C2AB* with the SNAREs and the lipids , for each condition .",
"Supplementary file 1 also lists repeat experiments performed under selected conditions with different nanodisc preparations that show the reproducibility of the data ( see statistics in Materials and methods ) .",
"We observed a natural variability in the apparent KDs that may arise in part from different incorporation of SNARE complexes into the nanodiscs .",
"Note also that some of the KDs are in the low nM range and may not be accurate because the concentration of C2AB* used for all experiments was 50 nM and the corresponding titration curves thus approach saturating binding conditions .",
"Hence , these KDs and corresponding cooperativity factors must be interpreted with caution .",
"For all these reasons , the conclusions described below were obtained by comparing experiments performed on the same day or over period of two days with the same preparations , and were confirmed by additional comparisons made with other preparations on different days .",
"Importantly , the key conclusions are supported by the overall consistency of the data obtained under different conditions .",
"The higher affinity of C2AB* for cisSC-NDs than for NDs ( Figure 5C , blue and red curves , respectively ) shows that C2AB* binds simultaneously to the SNARE complex and the lipids under these conditions .",
"Since Syt1 C2AB is able to bind simultaneously to two membranes in the presence of Ca2+ ( Araç et al . , 2006 ) , which could cooperate with binding to the SNARE complex ( Brewer et al . , 2015 ) , we also investigated binding of C2AB to trans-SNARE complexes formed between two nanodiscs ( transSC-NDs ) .",
"Because we wanted to compare binding to cis and trans SNARE complexes , in these experiments we placed a tetramethylrhodamine ( TMR ) acceptor fluorescent probe on the SNARE complex rather than on the nanodiscs to allow direct quantification of the SNARE complex concentrations from the UV-vis absorption of the probe .",
"We chose residue 34 of SNAP-25 to place the acceptor probe because it is predicted to be sufficiently close to the donor probe at residue 346 of C2AB in any of the three structures of Syt1-SNARE complexes that have been elucidated ( Figure 5—figure supplement 1B ) , such that binding would be detected regardless of which binding mode occurs .",
"Titrations of C2AB* with transSC-NDs and cisSC-NDs yielded similar results ( Figure 5D ) and comparable apparent KDs ( 118 nM and 96 nM , respectively ) .",
"The lower KD obtained with cisSC-NDs labeled with 5% Rho-PE can be attributed to the negative charge added by the labeled lipids , which should increase their membrane affinity for C2AB ( Zhang et al . , 1998 ) .",
"These results suggest that the affinity of C2AB* for cisSC-NDs and transSC-NDs is similar and ensuing experiments were performed with cisSC-NDs for simplicity .",
"We also analyzed binding of C2AB* to cisSC-NDs that were labeled at the N- or C-terminus of the SNARE complex ( residue 16 or 76 of SNAP-25; Figure 5—figure supplement 1B ) .",
"We obtained similar binding curves and comparable KDs to those obtained with the label at residue 34 ( Figure 5E; Supplementary file 1 ) .",
"The FRET efficiencies observed at saturating concentrations were similar for the labels at residues 34 and 76 of SNAP-25 , and somewhat lower for the label at residue 16 .",
"The FRET efficiencies are consistent with the binding modes involving the polybasic region and the primary interface , which predict that the probe on C2AB* is located at comparable , short distances from residues 34 and 76 , and farther from residues 16 , but are not consistent with the tripartite complex , where residue 346 of C2AB* is expected to be much closer to residue 16 than to residue 76 ( Figure 5—figure supplement 1B ) .",
"However , these results are not conclusive , as other binding modes could also be consistent with the observed FRET efficiencies .",
"We also analyzed the effect of including complexin-1 on binding of C2AB* to cisSC-NDs labeled at residue 34 of SNAP-25 and observed similar binding curves and apparent KDs ( Figure 5F; Supplementary file 1 ) , suggesting that complexin-1 does not substantially alter the interaction of C2AB* with the nanodisc-anchored SNARE complex .",
"This observation is also consistent with binding of C2AB to the SNARE complex through the primary and polybasic interfaces , which is not expected to be affected by complexin-1 .",
"In subsequent experiments we focused on comparing binding of C2AB* to NDs and cisSC-NDs labeled with Rho-PE to analyze the increases in affinity caused by the presence of the SNARE complex , and analyzed how the conditions of the experiments affect the underlying affinities .",
"In experiments performed in 125 mM KCl and 1 mM EGTA to analyze Ca2+-independent interactions , binding to NDs was very weak .",
"We lowered the KCl concentration to 50 mM to facilitate binding and were able to observe efficient binding at relatively high ND concentrations ( Figure 5C , black curve ) , with an apparent KD of 8 . 7 μM .",
"Importantly , we observed a much higher affinity for cisSC-NDs ( Figure 5C , green curve ) , with an apparent KD of 141 nM .",
"These results suggest that there is a strong synergy between binding of Ca2+-free C2AB* to the SNARE complex and the nanodisc phospholipids .",
"Since PIP2 enhances binding of the C2B domain to membranes due to interactions with the polybasic region ( Bai et al . , 2004; Li et al . , 2006 ) , we performed titrations with NDs and cisSC NDs containing 1% PIP2 and indeed observed much higher affinities at 50 mM KCl ( Figure 5G , black and red curves; apparent KDs 286 nM and 43 nM , respectively ) .",
"Binding was weaker in 125 mM KCl ( Figure 5G , blue and green curves ) but there was still a large difference in the KDs observed for NDs and cisSC-NDs ( apparent KDs 4 . 9 μM and 366 nM , respectively ) .",
"We also analyzed binding of C2AB* to NDs and cisSC-NDs containing 1% PIP2 in the presence of 1 mM Ca2+ and again observed increased affinity for the latter ( Figure 5H , black and red curves; apparent KDs 22 nM and 12 nM , respectively ) .",
"Since the high affinity for cisSC-NDs containing PIP2 implied that we were close to saturation binding conditions , which hindered analysis of the SNARE-induced enhancement on binding , we also performed experiments with NDs and cisSC-NDs containing 10% instead of 15% PS ( i . e . composed of PC:PS:Rho-PE:PIP2 84:10:5:1 ) and indeed observed somewhat weaker binding ( Figure 5H , green and orange curves; apparent KDs 29 nM and 20 nM , respectively ) .",
"Overall , the increases in affinity caused by simultaneous binding of C2AB* to the nanodiscs and the SNARE complex were considerably lower in the presence than in the absence of Ca2+ , particularly when the nanodiscs contained PIP2 ( Supplementary file 1 , 2 ) .",
"To dissect the contributions of the polybasic region and R398 , R399 at the primary interface to binding of C2AB* to NDs and cisSC-NDs , we performed titrations under various conditions using WT and mutant versions of C2AB* that were labeled with a donor probe at residue 346 and bore the R322E/K325E , R398Q/R399Q or R322E/K325E/R398Q/R399Q mutations ( Figure 6; Figure 6—figure supplements 1–6 ) .",
"Since the much stronger effects of the R322E/K325E mutation than the K324E/K326E mutation on SNARE complex binding and on neurotransmitter release supported the physiological relevance of Syt1-SNARE complex binding through the polybasic region ( Brewer et al . , 2015 ) , we also included C2AB* with the K324E/K326E mutation in these analyses .",
"All NDs and cisSC-NDs included 5% Rho-PE .",
"Ca2+-independent binding of C2AB* to NDs was markedly impaired by all mutations , but mutations in the polybasic region impaired binding to NDs containing PIP2 much more strongly than the R398Q/R399Q mutation ( Figure 6A , C ) .",
"Hence , binding to these nanodiscs is mediated largely by the polybasic region , as expected , but the tandem arginines also participate in binding to some extent .",
"Binding to cisSC-NDs in EGTA was also impaired strongly by mutations in the polybasic region , but in this case the impairment caused by the R398Q/R399Q mutation was almost as strong ( Figure 6B , D ) .",
"All mutations strongly decreased the cooperativity factors calculated from KD NDs/KD cisSC-NDs , which provide an idea of the contribution of C2AB*-SNARE complex interactions to cisSC-ND binding and ranged from 6 . 64 to 62 . 1 for WT C2AB* in the three conditions including EGTA ( Supplementary file 2 ) .",
"Thus , both the tandem arginines ( R398 , R399 ) and the polybasic region are important for Ca2+-independent , simultaneous binding of C2AB* to the SNARE complex and the lipids .",
"These findings suggest that the underlying interactions are dynamic and involve at least two binding modes whereby either the primary interface containing R398 , R399 or the polybasic region interacts with the SNAREs and the other basic sequence ( the polybasic region or R398 , R399 ) binds to the lipids ( Figure 5—figure supplement 2A , B ) .",
"An interestingly different picture emerged in experiments performed in the presence of Ca2+ .",
"Importantly , the R322E/K325E mutation impaired Ca2+-dependent binding to PIP2-containing NDs much more dramatically than the K324E/K326E mutation ( Figure 6G , Figure 6—figure supplement 6A; apparent KDs 49–62 nM for the K324E/K326E mutant and 1 . 5–2 . 9 μM for R322E/K325E ) .",
"This striking difference correlates with the effects of these mutations on neurotransmitter release ( Brewer et al . , 2015 ) and was not observed for Ca2+-dependent binding of C2AB* to NDs lacking PIP2 ( Figure 6E ) , or Ca2+-independent binding to PIP2-containing NDs ( Figure 6A , C ) or liposomes ( Brewer et al . , 2015 ) , all of which were similarly disrupted by the R322E/K325E and K324E/K326E mutations .",
"These findings show that there is a specific Ca2+-dependent binding mode of Syt1 to PIP2-containing membranes that involves R322 , K325 but not K324 , K326 , and suggest that the specific , strong disruption of neurotransmitter release induced by the R322E/K325E mutation but not the K324E/K326E mutation arises from impairment of Ca2+-dependent binding of Syt1 to the ( PIP2-containing ) plasma membrane rather than to the SNARE complex .",
"The cooperativity factors calculated from KD NDs/KD cisSC-NDs in the presence of Ca2+ under various conditions ( Figure 6E–H , Figure 6—figure supplements 4–6 ) ranged from 1 . 39 to 3 . 11 and hence were considerably smaller than those observed in EGTA ( Supplementary file 2 ) , indicating that interactions of WT C2AB* with the SNARE complex contributed much less to binding to cisSC-NDs in the presence of Ca2+ than in its absence .",
"Interestingly , the cooperativity factors observed for WT C2AB* in the presence of PIP2 were particularly small , suggesting that Ca2+-dependent interactions of C2AB* with PIP2 on the nanodiscs preclude interactions with the SNARE complex .",
"The diminished cooperativity factors in the presence of PIP2 were decreased further by the R398Q/R399Q , K324E/K326E and R322E/K325E/R398Q/R399Q mutations , but where substantially increased by the R322E/K325E mutation , presumably because the specific interactions of C2AB* with PIP2 mediated by R322E , K325E were disrupted , allowing binding to the SNARE complex .",
"These findings can be readily rationalized by modeling how the C2B domain binds to a PIP2-containing membrane in a Ca2+-dependent manner .",
"Because Ca2+ induces binding of the C2B domain in an approximately perpendicular orientation to the membrane that favors insertion of both Ca2+-binding loops into the bilayer ( Araç et al . , 2006; Bai et al . , 2004; Rufener et al . , 2005 ) , this orientation allows binding of R322 and K325 to PIP2 , but K324 and K326 point away from the membrane because they are on the opposite side of the same β-strand ( Figure 5—figure supplement 2C; see also Figure 10B ) .",
"In this configuration , interaction of the SNARE complex with the primary interface of the C2B domain is impossible because it would place the syntaxin-1 C-terminus far from the membrane where it is anchored ( Figure 5—figure supplement 2C ) .",
"Simultaneous Ca2+-dependent binding of the C2B domain to the membrane through its Ca2+-binding loops and to the SNARE complex through its polybasic region is in principle compatible with anchoring of syntaxin-1 to the membrane ( Figure 5—figure supplement 2D ) , but this binding mode is expected to be prevented by PIP2 because PIP2 binds to K322 , R325 , the same residues of the polybasic region that are key for SNARE complex binding ( Brewer et al . , 2015 ) .",
"In summary , Ca2+-dependent binding of C2AB* to PIP2-containing membranes is incompatible with the two major Syt1-SNARE complex binding modes , which involve the primary and polybasic interfaces .",
"However , upon Ca2+- and PIP2-dependent membrane binding , R398 and R399 can still be involved in non-specific interactions with negative residues of the SNARE complex , and such interactions are also possible if the C2B domain binds in a more slanted orientation to membranes lacking PIP2 ( Figure 5—figure supplement 2E , F ) .",
"Such non-specific interactions might underlie the modest decreases in Ca2+-dependent binding of C2AB* to cisSC-NDs caused by the R398Q/R399Q mutation ( Figure 6F , H ) and , similarly , the impairments in such binding caused by the K324E/K326E mutation might arise from other non-specific binding modes .",
"We also analyzed the effects of the E295A/Y338W mutation in region I of the primary interface on binding to NDs and cisSC-NDs .",
"Interestingly , this mutation did not alter binding to NDs under various conditions ( Figure",
"7 ) but caused a considerable increase in affinity for cisSC-NDs containing 15% PS and 1% PIP2 in the absence of Ca2+ , compared to WT C2AB* ( Figure 7A; apparent KDs 366 nM for WT C2AB* and 80 nM for the E295A/Y338W mutant ) .",
"In contrast , we observed only very slight increases in Ca2+-dependent binding of E295A/Y338W C2AB* to cisSC-NDs containing 15% PS with or without 1% PIP2 , or 10% PS and 1% PIP2 , compared to WT C2AB* ( Figure 7B–D ) .",
"The results obtained in the absence of Ca2+ correlate with the increased affinity of the Syt1 C2B domain for CpxSC observed in our NMR experiments ( Figure 4 ) and show that this mutations enhances Ca2+-independent binding of C2AB* to the SNARE complex within cisSC-NDs via the primary interface .",
"However , the lack of an overt effect of the E295A/Y338W mutation on binding to cisSC-NDs in the presence of Ca2+ strongly supports the notion that the primary interface is not involved in Ca2+-dependent binding .",
"To gain insights in how the phospholipids in the nanodiscs influence Syt1-SNARE complex interactions , we performed experiments where we monitored direct binding of C2AB* to the SNARE complex in cisSC-NDs by placing the FRET acceptor on residue 76 of SNAP-25 ( Figure 8; Supplementary file 1 ) .",
"In these experiments we focused on WT C2AB* and C2AB* bearing the R398Q/R399Q , R322E/K325E or E295A/Y338W mutations that strongly impair neurotransmitter release .",
"Comparisons of the results obtained with cisSC-NDS and those of parallel experiments performed with soluble SNARE complexes formed with the cytoplasmic region of syntaxin-1 ( residues 2–253 ) ( referred to as solubleSC ) provided information on how interactions of C2AB* with the lipids enhance SNARE complex binding .",
"To examine how negatively charged phospholipid head groups contribute to such enhancement , we performed experiments with cisSC-NDs that contained only PC ( cisSC-PC-NDs ) or 15% PS , 1% PIP2 and 84% PC ( cisSC-PC/PS/PIP2-NDs ) .",
"We also performed experiments with a soluble SNARE complex formed with the syntaxin-1 SNARE motif ( residues 191–253 ) instead of its cytoplasmic region ( Figure 8—figure supplement 1 ) to mimic the SNARE complex used in the NMR experiments ( referred to as mcc ) .",
"The results were similar to those obtained with solubleSC but revealed somewhat weaker affinities for C2AB* that are consistent with the NMR data obtained with the C2B domain and CpxSC ( Figure 1 ) and suggest that the N-terminal region of syntaxin-1 contributes to Syt1-SNARE interactions , as proposed previously ( Fernandez et al . , 1998 ) .",
"In the absence of Ca2+ , C2AB* bound more strongly to cisSC-PC-NDs than to solubleSC ( KD 0 . 93 and 5 . 5 μM , respectively ) , and binding to cisSC-PC/PS/PIP2-NDs was even tighter ( KD0 . 49 μM ) ( Figure 8A ) .",
"Binding of C2AB* to these complexes was strongly impaired by the R322E/K325E mutation and to a lesser extent by the R398Q/R399Q mutation , and was strengthened by the E295A/Y338W mutation ( Figure 8C , E , G ) .",
"The cooperativity factors calculated from KD solubleSC/KD cisSC-PC-NDs and KD solubleSC/KD cisSC-PC/PS/PIP2-NDs were 5 . 91 and 11 . 09 for WT C2AB* , were dramatically decreased by the R322E/K325E and R398Q/R399Q mutations , and slightly increased by the E295A/Y338W mutation ( Supplementary file 2 ) .",
"These findings are fully consistent with the conclusion that interactions of both the polybasic region and R398 , R399 with the lipids contribute to Ca2+-independent binding of C2AB* to cisSC-NDs , and hence that such binding involves at least two types of interactions where either the primary interface binds to the SNAREs and the polybasic region to the lipids , or the polybasic region binds to the SNAREs and R398 , R339 to the lipids ( Figure 5—figure supplement 2A , B ) .",
"As observed in the previous experiments , the E295A/Y338W mutation increased the former of these two binding modes .",
"As expected , the PC/PS/PIP2-NDs enhanced binding to the SNARE complex more than the PC-NDs , but it is noteworthy that even interactions of C2AB* basic residues with neutral phospholipids such as PC can enhance binding to the SNARE complex .",
"As expected , Ca2+ increased the affinity of C2AB* for solubleSC , cisSC-PC-NDs and cisSC-PC/PS/PIP2-NDs , and binding was tightest for the latter ( Figure 8A , B; Supplementary file 1 ) .",
"The effects of the R398Q/R399Q , R322E/K325E and E295A/Y338W mutations on Ca2+-dependent binding of C2AB* to solubleSC and cis-SC-PC-NDs ( Figure 8D , F ) were similar to those observed in the absence of Ca2+ , and highly efficient FRET was also observed at saturating concentrations , as expected for binding of C2AB* to the SNARE complex through the primary or polybasic interfaces with the acceptor probe on residue 76 of SNAP-25 ( Figure 5—figure supplement 1B ) .",
"However , the highest FRET efficiency observed upon binding of WT C2AB* to cisSC-PC/PS/PIP2-NDs was 0 . 4 ( Figure 5B ) .",
"The highest FRET efficiency was increased to some extent by the E295A/Y338W mutation and even more by the R322E/K325E mutation , but was decreased by the R398Q/R399Q mutation , which consistently led to a bimodal binding curve with a maximum at about 20 nM cisSC-PC/PS/PIP2-NDs in four independent experiments ( Figure 8D , F , H ) .",
"These results are consistent with the notion that Ca2+-dependent binding of C2AB* to PIP2-containing membranes is incompatible with SNARE complex binding through the primary and polybasic interfaces .",
"In the absence of interactions with the SNARE complex , C2AB* may bind with similar probabilities to both sides of the nanodiscs , perhaps with some preference for the side lacking SNAREs where there are no steric clashes with the SNARE complex .",
"Non-specific interactions of R398 , R399 of WT C2AB* with acidic residues from the SNAREs may facilitate binding to the SNARE-containing side ( Figure 5—figure supplement 2E ) , leading to some FRET , but mutation of these arginines may lead to preferential binding of the R398Q/R399Q mutant to the other side , particularly at high nanodisc concentrations when membrane availability is not limiting .",
"The highest FRET efficiency was likely increased by the E295A/Y338W mutation because it enhances SNARE binding .",
"High FRET efficiencies were reached for binding of the R322E/K325E mutant to cisSC-PC/PS/PIP2-NDs or for binding of WT C2AB* and the three mutants to solubleSC or cisSC-PC-NDs ( Figure 8D , F , H ) because the specific Ca2+-dependent interaction of C2AB* with PIP2 that competes with SNARE binding is precluded .",
"Physiological conditions including ATP and PIP2-containing membranes were reported to disrupt Syt1-SNARE complex binding , leading to the conclusion that such binding is not biologically relevant ( Park et al . , 2015 ) .",
"However , a fluorescent probe attached to residue 342 of the C2B domain to monitor binding to the SNARE complex in this study may have disrupted binding through the primary interface because residue 342 is very close to this interface ( Figure 5—figure supplement 1A ) , and another study concluded that Syt1-SNARE complex binding persists in the presence of ATP ( Wang et al . , 2016 ) .",
"To clarify this controversy and examine how ATP affects Syt1-SNARE complex interactions , we first analyzed the effects of adding 2 . 5 mM Mg2+ with or without 2 mM ATP on the FRET observed between C2AB* and NDs or cisSC-NDs containing 5% Rho-PE , 15% PS and 1% PIP2 .",
"Mg2+ and Mg-ATP decreased the FRET with both NDs and cisSC-NDs to some extent in the absence of Ca2+ , but the FRET with cisSC-NDs was still much stronger that the FRET with NDs ( Figure 9—figure supplement 1 ) .",
"However , the difference in FRET with cisSC-NDs and NDs was much smaller in the presence of Ca2+ when Mg2+ and ATP were added ( Figure 9—figure supplement 2 ) .",
"These results suggested that Ca2+-independent binding of C2AB* to the SNARE complex persists in the presence of Mg-ATP , but Ca2+-dependent binding is strongly impaired .",
"These conclusions were supported by titrations of C2AB* with NDs and cisSC-NDs , which revealed a considerable difference in affinity in the absence of Ca2+ but a much smaller difference in Ca2+-dependent binding ( Figure 9A , B ) .",
"To investigate the nature of the Ca2+-independent interaction of C2AB* with cisSC-NDs in the presence of Mg-ATP , we analyzed the effects of mutations on Ca2+-independent binding to NDs and cisSC-NDs .",
"Binding to cisSC-NDs was enhanced by the E295A/Y338W mutation and markedly impaired by the R398Q/R399Q , R322E/K325E , K324E/K326E and R322E/K325E , R398Q/R399Q mutations , whereas all the mutations had small effects on ND binding ( Figure 9C–D ) .",
"The E295A/Y338W mutant bound much tighter to cisSC-NDs than to NDs but all other mutants exhibited similar affinity for NDs and cisSC-NDs ( Figure 9—figure supplement 3 ) .",
"These results suggest that , in the presence of Mg-ATP , Ca2+-independent binding of WT C2AB* to the SNARE complex on nanodiscs also involves at least two binding modes mediated by either the primary or the polybasic interface , and the former is stabilized by the E295A/Y338W mutation .",
"We also analyzed whether complexin-1 altered the affinity of WT C2AB* for cisSC-NDs , but did not observe any significant effects ( Figure 9E ) .",
"We also verified that the R322E/K325E mutation disrupts Ca2+-dependent binding to PIP2-containing NDs much more strongly than the K324E/K326E mutation in the presence of Mg-ATP ( Figure 9F ) , confirming the specificity of such impairment under these conditions ."
],
[
"Because of the well-established functions of the SNAREs as the engines of membrane fusion and of Syt1 as the Ca2+ sensor that triggers synchronous neurotransmitter release , elucidating the role ( s ) of Syt1-SNARE interactions is crucial to understand how Ca2+ sensing is coupled to membrane fusion during release .",
"The recent determination of three structures of Syt1-SNARE complexes led to intriguing models of Ca2+-triggered release ( Brewer et al . , 2015; Zhou et al . , 2015; Zhou et al . , 2017 ) , but yielded a confusing picture because of the striking differences among the structures .",
"Paradoxically , Ca2+ was generally believed to enhance Syt1-SNARE binding , but the C2 domain Ca2+-binding loops were not involved in SNARE binding in any of the structures .",
"Together with available data , the study presented here suggests that Syt1 binds to the SNARE complex before Ca2+ influx , most likely through the primary interface , and that Ca2+ actually releases this interaction , inducing tight membrane binding that involves specific interactions of the C2B polybasic region with PIP2 .",
"We propose that the Syt1-SNARE complex keeps the release machinery in a state that hinders membrane fusion but at the same time is ready for fast release when Ca2+ induces binding of the Syt1 Ca2+-binding loops to the membrane , releasing the Syt1-SNARE interaction and enabling cooperation between Syt1 and the SNAREs in membrane fusion ( Figure 10 ) .",
"To rationalize the immense amount of data available on Syt1-SNARE interactions , it is crucial to decipher which data reflect physiologically relevant interactions and which arise from the promiscuity of these proteins , the lack of key components in a reduced system and/or the choice of experimental conditions .",
"This task is hindered by the fact that relevant interactions may be necessarily weak because of the very nature of this dynamic system , which is expected to undergo quick , drastic rearrangements during the events that lead to fast membrane fusion upon Ca2+ influx .",
"Weak interactions can be dramatically enhanced by co-localization within the confines of a primed synaptic vesicle , but they must be distinguished from other , perhaps stronger but irrelevant interactions that are detectable in vitro ( Magdziarek et al . , 2020 ) .",
"Another complicating aspect is that basic sequences such as the polybasic region and R398 , R399 can bind to SNAREs and to membranes , both of which are acidic , and binding to a wrong molecule can occur in the absence of the bona fide target .",
"Perhaps the most confusing factor was the observation by multiple labs that Ca2+ strongly enhanced binding of Syt1 to SNAREs or SNARE complexes in solution ( see introduction ) , leading to the widespread belief that elucidating how Ca2+-bound Syt1 binds to the SNARE complex was a ‘Holy Grail’ to understand neurotransmitter release .",
"However , it now seems clear that Ca2+ releases Syt1 from SNARE complexes anchored on PIP2-containing membranes such as the plasma membrane and that the Ca2+-induced increase in Syt1-SNARE binding in solution arose merely because Ca2+ increases the positive electrostatic potential of the Syt1 C2 domains ( Fernandez et al . , 2001; Shao et al . , 1997 ) .",
"This enhanced Syt1-SNARE affinity is offset by the specific , Ca2+ -dependent interaction of Syt1 with PIP2-containing membranes , which is incompatible with SNARE complex binding and is much stronger .",
"This key conclusion arises from several lines of evidence .",
"First , our titrations with NDs and cisSC-NDs show that physiological conditions including Mg-ATP almost abolish Ca2+-dependent binding of C2AB* to the SNARE complex anchored on PIP2-containing membranes ( Figure 9B ) , consistent with previous results ( Park et al . , 2015 ) .",
"A previous study reported that Syt1-SNARE complex interactions persist in the presence of ATP ( Wang et al . , 2016 ) , but this conclusion was tested only in the absence of Ca2+ and is thus consistent with our results ( Figure 9A ) .",
"Second , and particularly important is the observation of the high specificity of Ca2+-dependent binding of C2AB* to PIP2-containing NDs , which is disrupted much more strongly by the R322E/K325E mutation than by the K324E/K326E mutation ( Figures 6G and 9F ) , in correlation with the effects of these mutations on neurotransmitter release ( Brewer et al . , 2015 ) .",
"Note that , remarkably , the striking difference in the effects of the two mutations on Ca2+-dependent binding of C2AB* to PIP2-containing NDs was not observed in Ca2+-dependent binding to NDs lacking PIP2 ( Figure 6E ) or Ca2+-independent binding to NDs with or without PIP2 , and with or without SNARE complex ( Figure 6A–D ) .",
"As explained above , modeling readily explains these observations , as R322 and K325 ( but not K324 and K326 ) are expected to be well positioned to interact with PIP2 upon Ca2+-induced binding of the C2B domain to the membrane ( Figure 10B ) .",
"In the absence of Ca2+ , insertion of the Ca2+-binding loops into the membrane is hindered by the negative charge of the loops ( Fernandez et al . , 2001 ) and , consequently , more parallel orientations of the C2B domain with respect to the membrane that allow simultaneous binding of the primary interface to the SNARE complex ( Figure 10A ) are favored .",
"These parallel orientations bring K324 and K326 near the membrane and hence PIP2 can readily interact with these residues as well as with R322 and K325 .",
"Third , binding of the C2B domain to the membrane in the perpendicular orientation induced by Ca2+ and PIP2 is incompatible with the binding modes observed in the three structures of Syt1-SNARE complexes that have been determined .",
"The elongated SNARE complex could not remain membrane-anchored or would have strong steric clashes if it remained bound to the C2B domain through the primary or tripartite interfaces , and binding of the SNARE complex to the polybasic region is hindered because this region interacts with PIP2 .",
"These conclusions are supported by the observation that highly efficient FRET between C2AB* and the SNARE complex labeled at residue 76 of SNAP-25 was observed under a variety of conditions except for those that allowed Ca2+-dependent binding of C2AB* to PIP2-containing SNARE complex nanodiscs ( Figure 8 ) .",
"Note also that the physiological relevance of our NMR structure was supported by the differential disruption of C2B-SNARE complex binding and neurotransmitter release caused by the R322E/K325E and K324E/K326E mutations ( Brewer et al . , 2015 ) .",
"However , the physiological data can now be explained by the differential effects of these mutations on Ca2+-dependent binding of C2AB* to PIP2-containing membranes ( Figures 6G and 9F ) , which occurs with high affinity ( in the low nM range ) and is much tighter than SNARE complex binding ( Figure 8D ) .",
"Syt1 does bind to membrane-anchored SNARE complexes in the absence of Ca2+ , even in the presence of ATP ( Figure 9A; Wang et al . , 2016 ) .",
"Our NMR studies show that there are two main Ca2+-independent binding modes between the C2B domain and CpxSC in solution ( Figure 1 ) that are mediated by the primary and polybasic interfaces .",
"The strong disruption of CpxSC binding to the primary interface caused by the R398Q/R399Q mutation ( Figure 2B , C ) shows that R398 , R399 contribute substantially to the energy of binding at this interface .",
"E295 and Y338 likely contribute also to the binding energy , but additional mutations will be necessary to assess this contribution , as the E295A/Y338W mutation actually enhances binding ( Figure 4 ) .",
"These observations contrast with the finding that co-IP of Syt1 with the SNAREs was not substantially affected by the R398Q/R399Q mutation and was moderately disrupted by the E295A/Y338W mutation ( Zhou et al . , 2015 ) .",
"This discrepancy might arise because co-IP likely detects SNARE binding to the polybasic region upon membrane solubilization , and depends not only on affinities but also on off rates .",
"The effects of the R398Q/R399Q and E295A/Y338W mutations observed by NMR correlate with those observed on Ca2+-independent binding of C2AB* to cisSC-NDs ( Figures 6B , D , 7A , 8 and 9D ) , showing that binding to the SNARE complex on the nanodiscs is mediated in part by the primary interface .",
"However , the R322E/K325E and K324E/K326E mutations also decreased the differences in the affinities of C2AB* for NDs and cisSC-NDs ( Figure 6—figure supplements 1–3; Figure 9—figure supplement 3 ) , indicating that there are additional binding modes where the C2B polybasic region interacts with the SNAREs .",
"These other binding modes are unlikely to be biologically relevant , as the K324E/K326E mutation does not impair neurotransmitter release substantially ( Brewer et al . , 2015 ) .",
"The functional importance of binding of Syt1 to the SNARE complex through the primary interface is overwhelmingly supported by physiological data ( Guan et al . , 2017; Zhou et al . , 2015 ) .",
"Hence , it is most likely that binding of C2AB* to the SNARE complex in cisSC-NDs through the polybasic region arises from limitations of our in vitro experiments and that Syt1 is bound to the SNARE complex through the primary interface before Ca2+ influx .",
"Although this interaction is rather weak , it is likely to be dramatically stabilized by co-localization at the site of fusion and may be favored over other binding modes by other factors .",
"For instance , our experiments used 1% PIP2 , which corresponds to the average PIP2 content of the plasma membrane , but clusters containing 6% PIP2 have been detected ( James et al . , 2008; van den Bogaart et al . , 2011 ) .",
"High PIP2 concentration should favor simultaneous binding of the C2B domain polybasic region to this lipid and of the primary interface to the SNARE complex ( Figure 10A , C ) .",
"Such a state has been observed by cryo-EM on lipid nanotubes , albeit at limited resolution ( Grushin et al . , 2019 ) .",
"The structure revealed a slanted orientation of the SNARE complex that should hinder full zippering at the C-terminus and was disrupted by Ca2+ .",
"It is unclear whether the orientation was dictated by helical packing on the nanotubes and whether Ca2+ disrupted such packing , but these results are consistent with the notion that Ca2+ releases the interaction between Syt1 and membrane-anchored SNARE complexes .",
"Even if the SNARE complex is less slanted in the primed state , a more parallel orientation of the SNARE complex with respect to the membrane ( e . g . Figure 10A ) would still hinder C-terminal zippering , which could explain the increase in spontaneous release observed in Syt1 KO mice ( Xu et al . , 2009 ) .",
"The proposed state is also consistent with the observation that complexin-1 does not alter the affinity of C2AB* for cisSC-NDs ( Figure 9E ) , as complexin-1 and Syt1 bind to opposite sides of the SNARE complex ( Figure 10A ) .",
"In this state , the helix formed by complexin-1 would point toward the vesicle membrane ( Figure 10A , C ) , also hindering SNARE complex zippering ( Trimbuch et al . , 2014 ) .",
"This model provides a basis for the findings that complexin-1 is required for the dominant negative effect of Syt1 bearing mutations in the C2B domain Ca2+-binding sites ( Zhou et al . , 2017 ) and that Syt1 is necessary for the inhibition of spontaneous release by complexin in Drosophila ( Jorquera et al . , 2012 ) .",
"Our finding that the R398Q/R399Q mutation strongly disrupts binding of the C2B domain to the SNARE complex through the primary interface ( Figure 2B , C ) suggests that such disruption underlies the dramatic impairment of Ca2+-evoked neurotransmitter release caused by this mutation , as well as the abrogation of clamping of spontaneous release by Syt1 ( Xue et al . , 2008; Zhou et al . , 2015 ) .",
"The E295A/Y338W mutation in the primary interface also disrupted neurotransmitter release but still allowed clamping of spontaneous release , which resembles the phenotype observed for the R322E/K325E mutation in the polybasic region ( Zhou et al . , 2015 ) .",
"These findings are readily explained by our model and our biochemical data , as the E295A/Y338W enhances ( rather than weakens ) Ca2+-independent binding of Syt1 to the SNARE complex ( Figures 4 , 7A , 8 and 9D ) and hence hinders release of the inhibitory Syt1-SNARE interaction .",
"Similarly , the R322E/K325E mutation impairs Ca2+- and PIP2-dependent membrane binding , which is critical to release this inhibitory interaction .",
"Altogether , the available data support the notion that , before Ca2+ influx , the trans-SNARE complex , complexin-1 and Syt1 form a macromolecular assembly between the vesicle and plasma membranes that inhibits release but is ready to trigger fast membrane fusion upon Ca2+ influx because it brings Syt1 close to the SNAREs ( Figure 10C left panel ) .",
"In this model , Ca2+ triggers tight , specific binding of the C2B domain to PIP2 and other lipids in the plasma membrane while binding of C2B to the SNARE complex is released .",
"The mechanisms underlying the last events leading to membrane fusion are still unclear , but we speculate that the Syt1 C2 domains bridge the vesicle and plasma membranes ( Araç et al . , 2006; Figure 10C , middle panel ) .",
"This action could cooperate with the SNAREs in bringing the membranes together , and insertion of the C2 domain Ca2+-binding loops into the membrane can induce membrane curvature ( Martens et al . , 2007 ) to facilitate membrane fusion ( Figure 10C , right panel ) .",
"Note that R398 , R399 are critical for the membrane-membrane bridging activity of Syt1 and therefore the strong disruption of neurotransmitter release caused by mutating these arginines might arise from impairment of this activity ( Araç et al . , 2006; Xue et al . , 2008 ) in addition to abrogating SNARE binding .",
"Clearly , the proposed model needs further testing and multiple aspects need to be unraveled to understand the mechanism of neurotransmitter release .",
"Thus , syntaxin-1 is known to form clusters on the plasma membrane ( Khuong et al . , 2013 ) , and formation of Syt1 oligomers has been proposed to underlie the primed state and to exert an inhibitory activity that is released by Ca2+ ( Wang et al . , 2017 ) .",
"Such clusters and oligomers could not be present in our nanodisc experiments .",
"However , the Syt1 oligomers are compatible with the state proposed in Figure 10A ( Tagliatti et al . , 2020 ) and hence can be readily incorporated into the model of Figure 10C .",
"Note also that , while our NMR studies could not detect binding of the Syt1 C2B domain to CpxSC through the tripartite interface ( Figures 2E and 3 ) , it is premature to completely rule out the relevance of the tripartite structure given the potential functional importance of very weak interactions ( Magdziarek et al . , 2020 ) .",
"Nevertheless , it is also premature to accept this structure as physiologically relevant , and further evidence for such relevance needs to be obtained , ideally using mutations that exclusively replace residues in the interface rather than interior residues that are important for protein stability .",
"The model of Figure 10 provides a strong foundation to address these issues and incorporate additional proteins that may play key roles in the last steps of neurotransmitter release ."
],
[
"Constructs to express the following proteins or protein fragments were described previously: rat synaptobrevin-2 SNARE motif ( residues 29–93 ) , rat synaptobrevin-2 ( residues 49–93 ) , human SNAP-25A fragments encoding its SNARE motifs ( residues 11–82 and 141–203 ) , full-length rat synaptobrevin , full-length rat syntaxin-1A , rat syntaxin-1A ( residues 191–253 ) , rat syntaxin-1A ( residues 2–253 ) , human SNAP-25A ( residues 11–82 and 141–203 ) , rat synaptotagmin-1 C2B domain with a C277A mutation ( residues 271–421; referred to as WT C2B domain ) , the same rat synaptotagmin-1 fragment with a R398Q/R399Q mutation , rat synaptotagmin-1 C2AB fragment with a C277A mutation ( residues 140–421 ) , full-length rat complexin-1 , rat complexin-1 ( residues 26–83 ) and MSP1E3D1 ( pMSP1E3D1 was a kind gift from Stephen Sligar; Addgene plasmid # 20066; http://n2t . net/addgene:20066; RRID:Addgene_20066 ) ( Araç et al . , 2006; Brewer et al . , 2015; Chen et al . , 2006; Chen et al . , 2008; Chen et al . , 2002; Denisov et al . , 2007; Ma et al . , 2013; Xu et al . , 2013; Zhou et al . , 2013 ) .",
"All these proteins were expressed in E . coli BL21 ( DE3 ) cells and purified as previously described in these references , with the following exceptions .",
"The synaptobrevin-2 ( 29–93 ) , syntaxin-1A ( 191–253 ) , SNAP-25A ( 11–82 ) , SNAP-25A ( 141–203 ) and complexin-1 ( 26–83 ) fragments were expressed E . coli BL21 ( DE3 ) cells with the pET-duet vector .",
"Both SNAP-25 fragments included tryptophan at the N-terminus to facilitate detection by UV spectroscopy .",
"Cells were harvested and re-suspended in PBS pH 7 . 4 with 10 mM imidazole and supplemented with Sigma protease inhibitors ( P2714-1BTL ) .",
"Cleared lysates were applied to Ni-NTA resin ( Thermo Fisher ) , washed with PBS pH 7 . 4 with 10 mM imidazole , PBS pH 7 . 4 with 10 mM imidazole and 10% Triton , PBS pH 7 . 4 with 10 mM imidazole and 1 M NaCl , and eluted in PBS pH 7 . 4 , 500 mM imidazole .",
"The affinity tag was cleaved with TEV protease overnight at 4°C in PBS pH 7 . 4 .",
"After affinity tag cleavage , all proteins were further purified using size exclusion chromatography on a Superdex 75 column ( GE 16/60 ) equilibrated with 20 mM Tris pH 7 . 4 125 mM NaCl .",
"MSP1E3D1 was expressed in E . coli BL21 ( DE3 ) cells grown in Terrific broth media to OD600 = 2 . 0 , then induced with 1 mM Isopropylβ-D-1-thiogalactopyranoside ( IPTG ) for 4 hr at 37°C .",
"Cells were re-suspended in 40 mM Tris pH 8 . 0 300 mM NaCl 1% TritonX-100 5 mM Imidazole containing Sigma protease inhibitors ( P2714-1BTL ) and lysed using an Avestin EmulsiFlex-C5 homogenizer .",
"The soluble fraction of the cell lysate was collected after centrifugation at 48 , 000 x g for 45 min and incubated with Ni-NTA resin ( Thermo Fisher ) for 2 hr at 4°C .",
"The resin was washed with the following buffers sequentially: 40 mM Tris pH 8 . 0 300 mM NaCl 1% Triton , 40 mM Tris pH 8 . 0 300 mM NaCl 50 mM Sodium Cholate 20 mM Imidazole and 40 mM Tris pH 8 . 0 300 mM NaCl 50 mM Imidazole followed by elution using 40 mM Tris pH 8 . 0 300 mM NaCl 500 mM imidazole .",
"This was followed by size exclusion chromatography on a Superdex 200 column ( GE 16/60 ) in 20 mM Tris pH 7 . 4 100 mM NaCl 0 . 5 mM EDTA .",
"Expression vectors for mutant proteins were generated using a combination of the QuickChange site-directed mutagenesis kit ( Strategene ) and standard PCR-based techniques with custom designed primers .",
"These mutants included: Syt1 C2B ( residues 271–421 ) E295A/Y338W , L387Q/L394Q , R398Q , R399Q , R322E/K325E , and R322E/K325E/R399Q/R398Q; Syt1 C2AB ( residues 140–421 ) C277A/E346C ( referred to as WT C2AB* ) , C277A/E346C/R398Q/R399Q , C277A/E346C/R322E/K325E , C277A , E346C , R322E/K325E/R398Q/R399Q , C277A/E346C/E295A/Y338W and C277A/E346C/K324E/K326E; and SNAP-25 ( residues 11–82 ) R16C , Q34C and K76C .",
"All mutant proteins were purified as the WT proteins , including 0 . 5 mM TCEP in the final purification step for cysteine containing proteins .",
"Isotopically labeled and perdeuterated Syt1 C2B domain and Cpx-1 ( 26–83 ) fragments were expressed using M9 expression media in 99 . 9% D2O with D-glucose ( 1 , 2 , 3 , 4 , 5 , 6 , 6-D7 , 97–98% ) as the sole carbon source ( 3 g/L ) and 15NH4Cl as the sole nitrogen source ( 1 g/L ) .",
"Specific 13CH3-labeling at the Met and Ile δ1 methyl groups of Syt1 C2B domain fragments was achieved by adding [3 , 3–2H] 13C-methyl α-ketobutyric acid ( 80 mg/L ) and 13C-methyl methionine ( 250 mg/L ) ( Cambridge Isotope Laboratories ) to the cell cultures 30 min prior to IPTG induction .",
"The SNARE motifs were mixed in the equimolar ratio in following order: synaptobrevin-2 ( 29–93 ) , SNAP-25A ( 141–203 ) , SNAP-25A ( 11–82 ) and syntaxin-1A ( 191–253 ) , in the presence of 1 M NaCl .",
"The mixture contained the following protease inhibitors ( protease inhibitor cocktail A ) : Antipain Dihydrochloride 0 . 016 mg/ml ( Thermo Fischer Scientific: 50488492 ) ; Leupeptin 0 . 33 mg/ml ( Gold Bio: L01025 ) ; Aprotinin 0 . 08 mg/ml ( Gold Bio: A655100 ) .",
"The assembly reaction was incubated in room temperature overnight while rotating .",
"The SNARE motifs that did not incorporate into complex were removed by concentration-dilution at room temperature using 30 kDa molecular weight cutoff ( MWCO ) Amicon centrifugation filters .",
"Samples containing 80 µM WT Syt1 C2B domain were mixed with 30 µM SNARE complex +/- Complexin-1 ( 26–83 ) in 20 mM HEPES , 125 mM KCl , 1 mM CaCl2 , pH 7 . 4 .",
"The total reaction volume was 30 µl .",
"After mixing , the UV spectra of both samples were acquired using Nanodrop ( Thermofisher ) .",
"Next , the samples were centrifuged at 13 , 000 rpm for 5 min spin in a benchtop centrifuge ( Eppendorf ) .",
"The UV spectra of both samples were acquired again after centrifugation .",
"All NMR spectra were acquired at 25°C on Agilent DD2 spectrometers operating at 600 or 800 MHz and equipped with cold probes .",
"1H-15N TROSY HSQC and 1H-13 HMQC spectra were acquired on samples with 10% D2O as the solvent .",
"For titrations of WT and mutant Syt1 C2B domains with SNARE-Complexin-1 ( 26–83 ) complex , assembled soluble SNARE complex was concentrated at room temperature to a concentration above 250 μM using 30 kDa ( Amicon ) centrifugation filters and buffer exchanged into 20 mM HEPES containing 1 mM TCEP and 100 or 125 mM KCl 10% D2O buffer using Zeba Spin Desalting Columns , 7K MWCO , 10 mL ( Thermofisher ) .",
"Complexin-1 ( 26–83 ) was also concentrated above 250 μM using 3 kDa ( Amicon ) centrifugation filters and buffer exchanged to matching buffer .",
"SNARE-Complexin-1 ( 26–83 ) complex was preassembled before mixing with C2B Syt-1 fragment with 20% excess Complexin-1 ( 26–83 ) , and 1 mM EDTA or 1 mM CaCl2 were included in the mix depending on the final sample buffer .",
"After cation exchange , the corresponding 2H , 15N-IM-13CH3-C2B domain fragment was buffer exchanged using a PD-10 Desalting Column ( GE Healthcare ) to matching buffer .",
"Final samples containing 32 μM C2B domain were prepared by adding 1 mM EDTA or 1 mM CaCl2 and protease inhibitor cocktail A . 1H-15N TROSY-HSQC and 1H-13C HMQC spectra were acquired first for the isolated C2B domain and then with the same sample after adding increasing concentrations of complexin-1 ( 26–83 ) /SNARE complex .",
"The concentrations for each titration step for each mutant are indicated in the figure legends .",
"For titrations of 2H , 15N-CpxSC with WT and R322E/K325E/R398Q/R399Q mutant Syt1 C2B domains , the proteins were prepared by the same procedures described above .",
"2H , 15N-CpxSC was formed with 2H , 15N-Cpx1 ( 26-83 ) and 12 . 5% excess assembled soluble SNARE complex .",
"1H , 15N TROSY-HSQC spectra were acquired for 2H , 15N-CpxSC alone and for 40 μM 2H , 15N-CpxSC in the presence of 40 μM WT or R322E/K325E/R398Q/R399Q mutant Syt1 C2B domain in 20 mM HEPES 100 mM KCl 1 mM EDTA 1 mM TCEP .",
"All NMR samples included protease inhibitor cocktail A . Total acquisition times ranged from 3 . 5 to 87 . 5 hr , depending on the sensitivity of the spectra .",
"NMR data were processed with NMRPipe ( Delaglio et al . , 1995 ) and analyzed with NMRView ( Johnson and Blevins , 1994 ) .",
"Single cysteine mutants of Synaptotagmin-1 C2AB ( 140-421 ) were labeled with Alexa488 using maleimide reactions with Alexa Fluor488 C5 Maleimide reagent from Thermo Fisher Scientific ( A10254 ) .",
"Synaptotagmin-1 C2AB single cysteine mutants were first buffer exchanged into 20 mM HEPES pH 7 . 4 125 mM KCl 0 . 5 mM TCEP using concentration and dilution .",
"Buffer exchanged proteins at a concentration of 100–150 μM were incubated with 10-fold excess dye for 4 hr at room temperature or overnight at 4°C .",
"The reaction was quenched by addition of 10 mM DTT .",
"Unreacted dye was separated from labeled protein through cation exchange chromatography on a HiTrap SP column ( GE ) in 50 mM Sodium Acetate pH 6 . 2 ( Buffer A ) using a linear gradient from 0 to 1000 mM NaCl .",
"Prior to elution , the column with the bound protein was washed with 100 mL of buffer A . Single cysteine mutants of SNAP-25A ( 11-82 ) were labeled with TMR using maleimide reactions with TMR-5-Maleimide reagent from Thermo Fisher Scientific ( T6027 ) .",
"Proteins were first buffer exchanged into 20 mM HEPES pH 7 . 4 125 mM KCl 0 . 5 mM TCEP using concentration dilution .",
"Buffer exchanged proteins at a concentration of 100–150 μM were incubated with 10-fold excess dye for 4 hr at room temperature or overnight at 4°C .",
"The reaction was quenched by addition of 10 mM DTT .",
"Unreacted dye was separated from the labeled protein by using two runs on PD Miditrap G25 column followed by size exclusion chromatography on Superdex 75 column ( GE 10/300 or 16/60 ) .",
"The concentration and the labeling efficiency of the tagged proteins were determined by using UV-vis absorbance and a Bradford assay .",
"Appropriate lipid mixtures ( specific to each experiment as indicated in the figures and Supplementary file 1 ) were prepared by mixing chloroform stocks in glass test tubes .",
"These mixtures were dried under a stream of nitrogen and stored overnight in a vacuum desiccator .",
"The lipids were solubilized in a 20 mM HEPES pH 7 . 4 125 mM KCl 1% β-OG ( octyl-beta-glucoside ) buffer by vortexing for 5 min .",
"To form nanodiscs , MSP1E3D1 was incubated with solubilized lipids at a ratio of 1:110 in the presence of 1% β-OG ( final concentration ) at 4°C for 30 min .",
"The mixture was passed over a 4 cm-high Thermo Scientific Pierce Detergent Removal Resin ( 87780 ) column ( approximately 3 mL of the slurry; the final volume of the mixture was always between 3–4 mL ) .",
"The nanodiscs were further purified by size exclusion chromatography using a Superdex 200 column ( GE 16/60 ) .",
"Appropriate fractions were pooled and concentrated to a desired concentration using a 30 kDa MWCO Amicon centrigufation filter .",
"To prepare cisSC-NDs , detergent solubilized cisSNARE-complex was formed by incubating 10 μM full-length rat syntaxin-1A with 15 μM of synaptobrevin-2 ( 29–93 ) , SNAP-25A ( 11-82 ) and SNAP-25A ( 141-203 ) in the presence of 1% β−OG , 0 . 5 M NaCl and protease inhibitor cocktail A overnight at 4°C .",
"To obtain transSC-NDs , we first prepared detergent solubilized activated t-SNARE complex [see Prinslow et al . , 2019 by incubating 10 μM full-length syntaxin-1A with 15 μM synaptobrevin-2 ( 49–93 ) , SNAP-25A ( 11-82 ) and SNAP-25A ( 141-203 ) in the presence of 1% β-OG , 0 . 5 M NaCl and protease inhibitor cocktail A overnight at 4°C .",
"For incorporation into nanodiscs , cisSNARE-complex , activated t-SNARE-complex or full-length synaptobrevin-2 were mixed with MSP1E3D1 and solubilized lipids at a ratio of 1:3:300 and incubated at 4°C for 30 min .",
"The detergent was removed using Thermo Scientific Pierce Detergent Removal Resin , as described for the isolated nanodiscs , and size exclusion chromatography using a Superdex 200 column ( GE 16/60 ) .",
"Fractions from size exclusion chromatography were assessed by SDS-PAGE and fractions that contained approximately one cisSNARE-complex , activated t-SNARE-complex or full-length synaptobrevin-2 per 2 MSP1E3D1 molecules were pooled together , mixed with protease inhibitor cocktail A and concentrated .",
"TransSNARE-complex was formed by incubating nanodiscs containing activated t-SNARE complex with 2-fold excess of synaptobrevin nanodiscs overnight at 4°C .",
"This method leads to quantitative formation of trans-SNARE complexes between nanodiscs based on the complete release of the synaptobrevin-2 ( 49–93 ) peptide , which was assessed by labeling the peptide with 15N and acquiring 1H-15N HSQC spectra , or labeling the peptide with Cy5 at residue 79 and monitoring the fluorescence anisotropy of the dye .",
"For experiments comparing binding of C2AB* to cisSC-NDs and transSC-NDs , the cisSC-NDs were formed by incubating nanodiscs containing activated t-SNARE complex with 2-fold excess of synaptobrevin-2 ( 29–93 ) overnight at 4°C .",
"To estimate the concentration of the resulting ND , cisSC-ND or transSC-ND samples , a stock solution of solubilized lipids containing 5% RhoPE was prepared as previously described in the Materials and methods section .",
"Dilutions of this stock solution were made and the absorbance at 570 nm was measured to make a standard curve .",
"Samples were solubilized in 1% b-OG and their absorbance at 570 nm was measured .",
"This measurement and the standard curve were used to estimate the total lipids in nanodisc samples .",
"The total lipid concentration was divided by 200 to get the approximate concentration of the nanodiscs .",
"To estimate the concentration of SNARE complex samples formed using SNAP-25 ( 11–82 ) R16C , Q34C or K76C labeled with tetramethylrhodamine , the dye absorbance at 555 nm was used .",
"For this purpose , the extinction coefficient of tetramethylrhodamine at 555 nm in 20 mM HEPES pH 7 . 4 125 mM KCl buffer was first determined from a standard curve obtained from the absorbance at 555 nm of several samples made after serial dilution of a stock solution of Tetramethylrhodamine-5-Maleimide .",
"The SNARE motifs were mixed in an equimolar ratio in the following order: synaptobrevin-2 ( 29–93 ) , SNAP-25A ( 141–203 ) , SNAP-25A ( 11–82 ) K76C labeled with tetramethylrhodamine and syntaxin-1A ( 191–253 ) ( for mcc ) or syntaxin-1A ( 2–253 ) ( for solubleSC ) , in the presence of 1 M NaCl .",
"The assembly reaction was incubated in the presence of protease inhibitor cocktail A at room temperature overnight while rotating .",
"The SNARE motifs that did not incorporate into the complex were removed by concentration-dilution at room temperature using 30 kDa Amicon centrifugation filters ( Chen et al . , 2002 ) .",
"The concentration of the SNARE complex was estimated by using UV-vis absorbance .",
"A stock solution of 0 . 5 µM Alexa 488-tagged C2AB ( C2AB* ) , containing 5 µM BSA ( Sigma Aldrich: A3059 ) to prevent binding of C2AB* to the cuvette , was prepared in the appropriate buffer ( 20 mM HEPES pH 7 . 4 and varying amounts of KCl and other components ) on the day of the experiment .",
"The ND , cisSC-ND , transSC-ND , solubleSC or mcc stock solutions were buffer exchanged into the proper buffer by using concentration-dilution or during the size exclusion chromatography step .",
"Other components such as 1 mM CaCl2 , 1 mM EGTA , 2 . 5 mM MgCl2 , 2 mM ATP or a combination of these reagents were added after the concentration step .",
"All the stock solutions were kept on ice and the buffers were kept at room temperature .",
"The ND samples can be kept on ice and used for experiments during a period of three days in the presence of protease inhibitor cocktail A . C2AB* fragments at 50 nM concentration were mixed with varying amount of NDs , cisSC-NDs or transSC-NDs in buffer containing 20 mM HEPES pH 7 . 4 and the concentrations of EGTA , Ca2+ , Mg2+ , ATP and KCl indicated in the figure legends .",
"Each sample of a titration was prepared freshly by adding 20 µL of the stock C2AB* solution to an appropriate amount of ND , cisSC-ND or transSC-ND in a 1 . 5 mL Eppendorf tube .",
"Dilutions of the ND stock solutions were used if less than 5 µL of the original stock had to be pipetted to minimize errors caused by pipetting small volumes .",
"The volume was made up to 200 µL by adding buffer .",
"The tube was vortexed for 2-3 s and 180 µL of the solution was pipetted into an SOG cuvette ( Fischer Scientific: NC9040289 ) .",
"Emission scan of the sample was immediately collected on a PTI Quantamaster 400 spectrofluorometer ( T-format ) at room temperature .",
"All slits were set to 1 . 2 mm .",
"The samples were excited at 480 nm and emission spectra from 500 to 620 nm were acquired .",
"After the scan was collected , the sample was discarded , and the cuvette was washed with water followed by ethanol and dried using a stream of air .",
"Multiple cuvettes were used during the experiment .",
"This did not change the emission scans .",
"Collecting the spectra after 5 min incubation at room temperature also did not change the emission scan .",
"FRET efficiency was calculated using the formula: FRET = ( I0 –I ) / I0 , where I0 is the fluorescence intensity of 50 nM Syt1 C2AB* at 518 nm and I is the fluorescence intensity of the sample at 518 nm .",
"The binding data were fitted to a Hill function ( FRET=Bmax∗ ( C∧h ) / ( ( Kd∧h ) + ( C∧h ) ) , where C is the concentration of NDs , cisSC-NDs , transSC-NDs or solubleSCs , using GraphPad PRISM7 .",
"The titrations of WT and mutant C2AB* with NDs and cisSC-NDs under a variety of conditions and with different lipid compositions were performed over a long period of time as the project developed and the results suggested new experiments to be performed .",
"The data shown in Figures 5–9 and associated figure supplements , and summarized in Supplementary file 1 , correspond to the last sets of experiments performed for presentation in this paper , all of which were consistent with the previous data .",
"Each titration involving a given set of reagents and conditions was repeated at least twice and in most cases three or more times over the course of this work , but a comprehensive statistical analysis for groups of data corresponding to each figure panel is hindered by the fact that the groups of experiments performed previously as the project developed varied ( e . g . with regard to which mutants were included ) .",
"The reproducibility of the data that we obtained in this type of experiments is illustrated in Figure 9—figure supplement 4 by the statistical analysis of the results of independent repeat experiments performed under the conditions of Figure 6G , H with the same set of mutants and three different preparations of NDs and cisSC-NDs .",
"This analysis and , more importantly , the overall consistency of the data obtained in the large number of experiments that we performed strongly support the key conclusions drawn from the titrations ."
]
] | [
"The Ca2+ sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release .",
"Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving ‘polybasic’ , ‘primary’ and ‘tripartite’ interfaces of synaptotagmin-1 .",
"We investigated these interactions using NMR and fluorescence spectroscopy .",
"Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution .",
"Ca2+-free synaptotagmin-1 binds to SNARE complexes anchored on PIP2-containing nanodiscs .",
"R398Q/R399Q and E295A/Y338W mutations at the primary interface , which strongly impair neurotransmitter release , disrupt and enhance synaptotagmin-1-SNARE complex binding , respectively .",
"Ca2+ induces tight binding of synaptotagmin-1 to PIP2-containing nanodiscs , disrupting synaptotagmin-1-SNARE interactions .",
"Specific effects of mutations in the polybasic region on Ca2+-dependent synaptotagmin-1-PIP2-membrane interactions correlate with their effects on release .",
"Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca2+ releases this interaction , inducing PIP2/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release ."
] | [
"Inside the brain , cells called neurons relay messages from one place to another in the form of electrical signals .",
"When an electrical signal reaches a junction between two neurons ( known as a synapse ) it triggers small particles called calcium ions to enter one of the cells .",
"This influx of calcium causes vesicles to fuse with the membrane surrounding the neuron and release molecules called neurotransmitters into the small gap between the two neurons .",
"These molecules travel across the gap to activate an electrical signal in the second neuron which then carries the message onwards .",
"A protein known as synaptotagmin-1 senses calcium ions at synapses and works together with a group of proteins known as the SNARE complex to help vesicles fuse with the cell membrane .",
"Previous studies have reported three different structures of synaptotagmin-1 bound to the SNARE complex in a different way .",
"But it was unclear which of these binding states actually result in the release of neurotransmitters .",
"To address this question , Voleti , Jaczynska and Rizo studied how and when synptotagmin-1 and the SNARE complex bind together using two approaches known as NMR spectroscopy and fluorescence spectroscopy .",
"The experiments suggest that before calcium enters the synapse , synaptotagmin-1 is already bound to a surface on the SNARE complex .",
"This binding inhibits the release of neurotransmitters and has been reported in previous studies .",
"Adding calcium ions causes synaptotagmin-1 to be released from the SNARE complex .",
"This allows synaptotagmin-1 to interact with the membrane and cooperate with the SNARE complex to trigger vesicle fusion .",
"Finding out how neurons release neurotransmitters at synapses may help us to understand how the brain works .",
"This could provide new insights into how defects in the synapse lead to neurological disorders , such as schizophrenia , and potentially aid the development of new treatments for such conditions ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals | elife-00013-v1 | [
[
"Eukaryotes evolved in a world filled with bacteria and throughout their shared history these two branches of life have developed a complex set of ways to compete and cooperate with each other .",
"While research on these interactions has historically emphasized bacterial pathogens , bacteria also regulate the biology of eukaryotes in many other ways ( McFall-Ngai 1999; Koropatnick et al . 2004; Mazmanian et al . 2005; Falkow 2006; Hughes and Sperandio 2008; Desbrosses and Stougaard 2011 ) and may have exerted critical influences on animal evolution .",
"Choanoflagellates , microscopic bacteria-eating eukaryotes that are the closest living relatives of animals ( James-Clark 1868; Saville Kent 1880; Hibberd 1975; Carr et al . 2008; King et al . 2008; Ruiz-Trillo et al . 2008 ) , could provide particularly important insights into the mechanisms underlying bacterial influences on animal biology and evolution .",
"Moreover , some choanoflagellates have both solitary and multicellular stages in their life histories ( Leadbeater 1983; Karpov and Coupe 1998; Dayel et al . 2011 ) and understanding the environmental cues that regulate choanoflagellate colony formation could provide a molecular model for animal multicellularity ."
],
[
"In the choanoflagellate Salpingoeca rosetta , rosette-shaped multicellular colonies develop when a single founder cell undergoes multiple rounds of incomplete cytokinesis , leaving neighboring cells physically attached by fine intercellular bridges ( Fairclough et al . 2010; Dayel et al . 2011 ) .",
"Although the original stock of S . rosetta ( ATCC50818 ) was established from a rosette colony ( Dayel et al . 2011 ) , laboratory cultures consistently produced single cells , with small numbers of rosette colonies forming only sporadically ( Figure 1A , Figure 1—figure supplement 1 ) .",
"Serendipitously , we discovered that the bacterial community influences rosette colony development .",
"Treatment of the ATCC50818 culture with an antibiotic cocktail resulted in a culture of S . rosetta cells that proliferated robustly by feeding on the remaining antibiotic-resistant bacteria but never formed rosette colonies , even upon removal of antibiotics ( Figure 1B ) .",
"This culture line is hereafter referred to as RCA ( for ‘Rosette Colonies Absent’ ) .",
"Supplementation of RCA cultures with bacteria from ATCC50818 restored rosette colony development , revealing that S . rosetta cells in the RCA culture remained competent to form colonies and would do so when stimulated by the original community of environmental bacteria . 10 . 7554/eLife . 00013 . 003Figure 1 . Rosette colony development in S . rosetta is regulated by A . machipongonensis .",
"( A ) The original culture of S . rosetta , ATCC 50818 , contains diverse co-isolated environmental bacteria and forms rosette colonies ( arrowheads ) rarely .",
"( B ) Treatment of ATCC50818 with a cocktail of antibiotics reduced the bacterial diversity and yielded an S . rosetta culture line , RCA , in which rosette colonies never formed .",
"( Representative single cells indicated by arrows . ) ( C ) Addition of A . machipongonensis to RCA cultures was sufficient to induce rosette development .",
"Scale bar , 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 00310 . 7554/eLife . 00013 . 004Figure 1—figure supplement 1 . Frequency of rosette colonies in S . rosetta environmental isolate ATCC 50818 , RCA with and without A . machipongonensis and a monoxenic line with A . machipongonensis feeder bacteria ( Px1 ) .",
"Altering bacterial diversity in S . rosetta cultures alters the frequency of rosette colonies .",
"Data are the whisker-box plots of the frequency of colonial cells in ATCC 50818 and a monoxenic culture of S . rosetta fed only A . machipongonensis bacteria ( Px1 ) for three experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 00410 . 7554/eLife . 00013 . 005Table 1 . Species tested for colony inductionDOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 005Species16S rDNA accession numberReferenceRosette coloniesAlgoriphagus machipongonensis PR1NZ_AAXU00000000Alegado et al . ( 2012 ) +Algoriphagus alkaliphilus AC-74AJ717393Tiago et al . ( 2006 ) +Algoriphagus boritolerans T-22AB197852Ahmed et al . ( 2007 ) +Algoriphagus mannitolivorans JC2050AY264838Yi and Chun ( 2004 ) +Algoriphagus marincola SW-2AY533663Yoon et al . ( 2004 ) +Algoriphagus ornithinivorans JC2052AY264840Yi and Chun ( 2004 ) +Algoriphagus vanfongensis KMM 6241EF392675Van Trappen et al . ( 2004 ) +Algoriphagus antarcticus LMG 21980AJ577141Nedashkovskaya et al . ( 2004 ) +Algoriphagus aquimarinus LMG 21971AJ575264Nedashkovskaya et al . ( 2004 ) +Algoriphagus chordae LMG 21970AJ575265Nedashkovskaya et al . ( 2004 ) +Algoriphagus halophilus JC2051AY264839Yi and Chun ( 2004 ) +Algoriphagus locisalis MSS-170AY835922Yoon et al . ( 2005a ) +Algoriphagus ratkowskyi LMG 21435AJ608641Bowman et al . ( 2003 ) +Algoriphagus terrigena DS-44DQ178979Yoon et al . ( 2006 ) +Algoriphagus winogradskyi LMG 21969AJ575263Nedashkovskaya et al . ( 2004 ) +Algoriphagus yeomjeoni MSS-160AY699794Yoon et al . ( 2005b ) +Agrobacterium tumefaciens C58AE007870Wood et al . ( 2001 ) +Aquiflexum balticum BA160AJ744861Brettar et al . ( 2004a ) −Bacillus subtilis 168AL009126Kunst et al . ( 1997 ) ; Burkholder and Giles ( 1947 ) ; Spizizen ( 1958 ) −Bacteroides fragilis NCTC9343CR626927Cerdeno-Tarraga et al . ( 2005 ) −Belliella baltica BA134AJ564643Brettar et al . ( 2004b ) −Caulobacter crescentus CB15AE005673Nierman et al . ( 2001 ) −Croceibacter atlanticus HTCC2559NR_029064Cho and Giovannoni ( 2003 ) −Cyclobacterium marinum LMG 13164AJ575266Raj and Maloy 1990 ) +Cytophaga hutchinsonii ATCC 33406M58768Lewin ( 1969 ) +Dyadobacter fermentans DSM 18053NR_027533Chelius and Triplett ( 2000 ) +Echinicola pacifica KMM 6172NR_043619Nedashkovskaya et al . ( 2006 ) −Escherichia coli MG1655U00096Blattner et al . ( 1997 ) −Flavobacteria johnsoniae UW101CP000685Bernardet et al . ( 1996 ) −Flectobacillus major DSM 103M62787Raj and Maloy ( 1990 ) +Listeria monocytogenes 10403SCP002002Edman et al . ( 1968 ) −Magnetospirillum magneticum AMB-1AP007255Matsunaga et al . ( 2005 ) −Microscilla marina ATCC 23134M123134Garrity ( 2010 ) −Oceanostipes pacificus HTCC2170CP002157Oh et al . ( 2011 ) −Robiginitalea biformata HTCC2501CP001712Cho and Giovannoni ( 2004 ) −Salinibacter ruber DSM13855CP000159Anton et al . ( 2002 ) −Sphingomonas wittichii RW1CP000699Miller et al . ( 2010 ) −Vibrio fischeri ES114CP000021Ruby et al . ( 2005 ) −Zobellia galactonovorans DsijNR_025053Barbeyron et al . ( 2001 ) +Zobellia uliginosa ATCC 14397M62799Matsuo et al . ( 2003 ) +−: no rosette colonies observed; +: rosette colonies observed .",
"To determine which co-isolated bacterial species stimulate rosette colony development in S . rosetta , the RCA cell line was supplemented with 64 independent bacterial isolates from ATCC50818 and monitored for the appearance of rosette colonies .",
"Only one bacterial species from ATCC50818 , the previously undescribed Algoriphagus machipongonensis ( phylum Bacteroidetes; Alegado et al . 2012 ) , induced rosette colony development in the RCA cell line ( Figure 1C ) .",
"S . rosetta cultures fed solely with A . machipongonensis yielded high percentages of rosette colonies ( Figure 1—figure supplement 1 ) , demonstrating that no other co-isolated bacterial species is required to stimulate rosette colony development .",
"What was not clear was whether other bacteria might also be competent to induce rosette colony development .",
"Therefore , representative Bacteroidetes and non-Bacteroidetes bacteria were grown and fed to RCA cultures ( Figure 2 , Table 1 ) .",
"None of the non-Bacteroidetes species tested , including members of the γ-proteobacteria , α-proteobacteria , and Gram-positive bacteria , were competent to induce rosette colony development .",
"In contrast , all 15 Algoriphagus species tested induced rosette colony development , as did six of 16 other closely related species tested in the Bacteroidetes phylum ( Table 1 ) .",
"Therefore , the ability to induce rosette colony development is enriched in Algoriphagus bacteria and their relatives . 10 . 7554/eLife . 00013 . 006Figure 2 . Diverse members of the Bacteroidetes phylum induce rosette colony development . A maximum likelihood phylogeny inferred from 16S rDNA gene sequences reveals the evolutionary relationships among A . machipongonensis , other members of the Bacteroidetes phylum , and representative γ-proteobacteria ( γ ) , α-proteobacteria ( α ) , and Gram-positive ( + ) bacteria .",
"All 15 members of the Algoriphagus genus ( Table 1 ) , as well as six other species in the Bacteroidetes phylum , were competent to induce colony development ( filled squares ) .",
"In contrast , no species outside of Bacteroidetes and most of the non-Algoriphagus bacteria tested failed to induce rosette colony development ( open squares ) .",
"Scale bar , 0 . 1 substitutions per nucleotide position . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 006 Although Bacteroidetes bacteria regulate morphogenetic processes in such diverse lineages as animals , red algae , and green algae ( Provasoli and Pintner 1980; Matsuo et al . 2005; Mazmanian et al . 2005 ) , the bacterially produced chemical cues that regulate most of these partnerships remain obscure .",
"The limited phylogenetic distribution of bacteria capable of inducing rosette colony development suggested that A . machipongonensis and its close relatives may produce a characteristic molecule that could be identified biochemically .",
"The complete absence of rosette colonies in RCA cultures provided the basis for a robust bioassay that we developed to identify the rosette-inducing molecule ( s ) , which we named RIFs ( Rosette-Inducing Factors ) , from A . machipongonensis cultures .",
"Preliminary studies demonstrated that RIF activity was present in conditioned medium from A . machipongonensis , even when grown in the absence of choanoflagellates .",
"Furthermore , the activity was also found in the A . machipongonensis cell envelope and was heat , protease , and nuclease resistant , revealing that the compound is not a protein , RNA , or DNA ( Table 2 ) . 10 . 7554/eLife . 00013 . 007Table 2 . Responses of RCA culture to various supplementsDOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 007TreatmentRosette coloniesInterpretationSea water−CM from ATCC50818+RIF-1 present in environmental isolate ATCC 50818CM from RCA−RIF-1 is absent in RCA linesLive A . machipongonensis ( cell pellet ) ++RIF-1 is produced by A . machipongonensisHeat killed A . machipongonensis ( cell pellet ) ++RIF-1 is resistant to heatA .",
"machipongonensis CM+RIF-1 is released by live AlgoriphagusA .",
"machipongonensis CM , boiled 10 min+RIF-1 is not labileA .",
"machipongonensis CM + Proteinase K+RIF-1 is not a proteinA .",
"machipongonensis CM + DNAse+RIF-1 is not DNAA .",
"machipongonensis CM + RNAse+RIF-1 is not RNAA .",
"machipongonensis CM , MeOH extract+RIF-1 is an organic compoundA .",
"machipongonensis cell pellet , MeOH extract++RIF-1 is present in the Algoriphagus cell envelopeA .",
"machipongonensis cell pellet , Bligh-Dyer extract++RIF-1 is a lipidSphingomyelin ( 20 mg mL−1 ) −Sphingomyelin does not induce rosette colony developmentMonosialoganglioside ( 20 mg mL−1 ) −Monosialoganglioside does not induce rosette colony developmentGalactocerebroside ( 20 mg mL−1 ) −Galactocerebroside does not induce rosette colony developmentN-palmitoyl-DL-dihydrolacto cerebroside ( 20 mg mL−1 ) −N-palmitoyl-DL-dihydrolacto cerebroside does not induce rosette colony development−: no induction; +: low induction; ++: high inductionCM: conditioned medium; RCA: rosette colonies absent; RIF-1: rosette inducing factor 1 .",
"The bacterial cell envelope components lipopolysaccharide ( LPS ) and peptidoglycan ( PGN ) from Gram-negative bacteria have long been known to affect host biology ( Cohn and Morse 1960; Hoffmann et al . 1999; Kopp and Medzhitov 1999; Takeuchi et al . 1999; Medzhitov and Janeway 2000; Kimbrell and Beutler 2001; Koropatnick et al . 2004 ) , but neither LPS nor PGN from A . machipongonensis triggered rosette development , alone or in combination ( Figure 3A ) .",
"Instead , we found that A . machipongonensis crude lipid extracts enriched in sphingolipids robustly induced rosette development ( Figure 3A ) .",
"In animals , sphingolipid signaling pathways regulate developmental processes such as cell death , survival , differentiation , and migration ( Prieschl and Baumruker 2000; Pyne and Pyne 2000; Spiegel and Milstien 2000; Hannun et al . 2001; Merrill et al . 2001; Herr et al . 2003 ) .",
"Moreover , sphingolipids serve essential functions both as structural components of cell membranes and as signaling molecules in diverse eukaryotes ( Hannich et al . 2011 ) .",
"In contrast , the phylogenetic distribution of sphingolipids in bacteria is largely limited to Bacteroidetes and Sphingomonas , where their endogenous functions are poorly understood ( Olsen and Jantzen 2001; An et al . 2011 ) . 10 . 7554/eLife . 00013 . 008Figure 3 . RIF-1 , a sulfonolipid that induces rosette colony development .",
"( A ) Rosette colony development is induced by live A . machipongonensis and the sphingolipid-enriched lipid fraction ( 20 mg mL−1 ) , but not by fresh medium , A . machipongonensis LPS ( 10 mg mL−1 ) , PGN ( 50 mg mL−1 ) , or LPS+PGN .",
"Shown are the whisker-box plots of the % colonial cells/total cells under each condition in three independent experiments .",
"( B ) The molecular structure of RIF-1 deduced from MS and 1D- and 2D-NMR data .",
"The RIF-1 structure , 3 , 5-dihydroxy-2- ( 2-hydroxy-13-methyltetradecanamido ) -15-methylhexadecane-1-sulfonic acid , has two parts: a base ( shown in red ) that defines the capnine , and a fatty acid ( shown in black ) .",
"Features that distinguish RIF-1 from other known capnoids are shown with colored arrows: the 2-hydroxy on the fatty acid ( black ) and the 5-hydroxy on the capnine base ( red ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 00810 . 7554/eLife . 00013 . 009Figure 3—figure supplement 1 . Separation of A . machipongonensis sphingolipids by thin layer chromatography ( TLC ) .",
"Lipids enriched in sphingolipids were separated by TLC after visualization with ammonium molybdate in 10% H2SO4 .",
"Bands ( 1-12 ) as well as regions between bands ( A-F ) were tested for morphogenic activity .",
"Region F possessed activity and was further purified . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 00910 . 7554/eLife . 00013 . 010Figure 3—figure supplement 2 . MS/MS analysis of RIF-1 . A major fragment derived from m/z = 606 ( M-H ) in the MS/MS spectrum of RIF-1 corresponds to amino-sulfonic acid S1 .",
"HRMS m/z calcd for C17H36NO5S ( M-H ) : 366 . 23142 .",
"Found: 366 . 2310 ( M-H ) - .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01010 . 7554/eLife . 00013 . 011Figure 3—figure supplement 3 . Key two-dimensional ( 2D ) correlations of RIF-1: Observed COSY correlations . Red double-head arrows show key 3J or 4J H-H correlations in the head regions ( 1 to 6 and 2′ to 3′ ) of the fatty acid and the capnine base and in the tail regions with geminal dimethyl groups ( 14 to 17 and 12′ to 15′ ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01110 . 7554/eLife . 00013 . 012Figure 3—figure supplement 4 . Key two-dimensional ( 2D ) correlations of RIF-1: Observed HMBC spin system . Blue single-head arrows show key 2J or 3J H-C correlations in the head and tail regions of the fatty acid and capnine base .",
"The correlations between C-1′ and H-2/N-H demonstrated that the fatty acid and capnine base are joined through an amide bond . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01210 . 7554/eLife . 00013 . 013Figure 3—figure supplement 5 . Key two-dimensional ( 2D ) correlations of RIF-1: Observed TOCSY spin system . Green bonds show two key spin systems in RIF-1 - HO-CH- in the fatty acid fragment and -CH2-CH ( NH ) -CH ( OH ) -CH2-CH ( OH ) -CH2- in the capnine base fragment . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01310 . 7554/eLife . 00013 . 014Figure 3—figure supplement 6 . 1H NMR spectrum of RIF-1 . The spectrum exhibits one NH ( δH 8 . 21 ) , three hydroxyl groups ( δH 5 . 52 , 5 . 20 , and 4 . 31 ) , five signals from 2 . 50-4 . 00 ppm ( four methines connected to either nitrogen at δH-2 3 . 88 or oxygens at δH-2′ 3 . 80/δH-3 3 . 71-3 . 78/δH-5 3 . 53-3 . 61 , and one methylene connected to sulfur at δH-1 2 . 56 & 3 . 01 ) , twenty methylenes and four methyls ( δH d , J = 6 . 6 Hz , 12H ) in the high field region ( δH 0 . 75-1 . 75 ppm ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01410 . 7554/eLife . 00013 . 015Figure 3—figure supplement 7 . gHMQC spectrum of RIF-1 . The 1J H-C correlations demonstrate that 2 ( δH 3 . 88 , δC 50 . 89 ) is connected to a nitrogen; 2′ , 3 , and 5 ( δH 3 . 80/δC 71 . 29 , δH 3 . 71-3 . 78/δC 71 . 51 , and δH 3 . 53-3 . 61/δC 70 . 20 , respectively ) are oxygenated; 1 ( δH 3 . 01 and 2 . 56 , δC 51 . 87 ) is adjacent to a sulfonic acid group; and all the other twenty methylenes and four methyls at high filed ( δH 0 . 75-1 . 75/δC 22 . 00-42 . 00 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01510 . 7554/eLife . 00013 . 016Figure 3—figure supplement 8 . gCOSY spectrum of RIF-1 . Indicated are important H-H correlations between NH and H-2 , 2′-OH and H-2′ , 3-OH and H-3 , and 5-OH and H-5 . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01610 . 7554/eLife . 00013 . 017Figure 3—figure supplement 9 . Expanded dqfCOSY spectrum of RIF-1 . In panel A ( δH 1 . 15-1 . 65 ppm/δH 3 . 52-3 . 82 ppm ) , H-3 ( δH 3 . 73 ) shows correlation to H-4a ( δH 1 . 30 ) , H-5 ( δH 3 . 57 ) to H-4a/H-4b ( δH 1 . 30/1 . 47 ) , and H-5 to H2-6 ( δH 1 . 20/1 . 29 ) ; H-2′ ( ∼δH 3 . 8 ) correlates to H2-3′ ( δH 1 . 45 and 1 . 54 ) .",
"Panel B ( δH 2 . 4-4 . 0 ppm/δH 2 . 4-4 . 0 ppm ) demonstrates the correlations between H2-1 ( δH 2 . 56/∼3 . 0 ) and H-2 ( δH 3 . 88 ) , and between H-2 and H-3 .",
"Panel C ( δH 0 . 75-1 . 60 ppm/δH 0 . 75-1 . 60 ppm ) exhibits correlations in the other methylenes and methyl groups . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01710 . 7554/eLife . 00013 . 018Figure 3—figure supplement 10 . Expanded dqfCOSY spectra of RIF-1 . A dqfCOSY spectrum was collected in order to get a clear connectivity in the oxygenated region ( 1-position to 6-position ) in the capnoid base fragment . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01810 . 7554/eLife . 00013 . 019Figure 3—figure supplement 11 . gHMBC spectrum of RIF-1 . Indicated are important 2J or 3J H-C correlations between NH/H-2/H-2′ and C-1′ ( δC 173 . 23 ) , and between NH and C-2/C-3 ( δC 50 . 89/71 . 51 ) .",
"Based on the MS/MS analysis , the fatty acid fragment must be 2-hydroxy-13-methyltetradecanoyl , and the capnine base fragment must be 2-NH-3 , 5-dihydroxy-15-methylhexadecane-1-sulfonate .",
"Hence , the planar structure of RIF-1 is determined as shown in Fig . 3 and Fig . 3 – Figure Supplements 3-5 . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 01910 . 7554/eLife . 00013 . 020Figure 3—figure supplement 12 . Expanded gHMBC spectrum of RIF-1 ( δH 0-4 . 00 ppm/δC 15 . 0-85 . 0 ppm ) .",
"Indicated are correlations between H-2 to C-4/C-1 ( δC 41 . 36/51 . 87 ) , between H-2′ and C-4′/C-3′ ( δC 24 . 99/34 . 85 ) , between H-1 and C-2/C-3 ( δC 50 . 89/71 . 51 ) , and between H-4 and C-5/C-3 ( δC 70 . 20/71 . 51 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 02010 . 7554/eLife . 00013 . 021Figure 3—figure supplement 13 . Expanded gHMBC spectrum of RIF-1 ( δH 0 . 80-1 . 80 ppm/δC 20 . 0-40 . 0 ppm ) .",
"Key correlations are between H-16 and C-14/C-15/C-17 ( δC 38 . 91/27 . 79/22 . 09 ) , between H-17 and C-14/C-15/C-16 ( δC 38 . 91/27 . 79/22 . 09 ) , between H-14′ and C-12′/C-13′/C-15′ ( δC 38 . 91/27 . 79/22 . 09 ) , and between H-15′ and C-12′/C-13′/C-14′ ( δC 38 . 91/27 . 79/22 . 09 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 02110 . 7554/eLife . 00013 . 022Figure 3—figure supplement 14 . TOCSY spectrum of RIF-1 . The spectrum shows correlations from NH to H2-1 and H-5 through H-2 , H-3 and H2-4 , and from 3-OH to H-5 via H2-4 and H2-1 through H-3 and H-2 . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 02210 . 7554/eLife . 00013 . 023Figure 3—figure supplement 15 . Expanded TOCSY spectrum of RIF-1 ( δH 0 . 50-4 . 25 ppm/δC 0 . 50-4 . 25 ppm ) .",
"Another important spin system is clearly demonstrated by the TOCSY correlations between H-2′ and H2-3′/H-4′/H-5′ on the top left of the expanded spectrum . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 023 To isolate and characterize the molecule ( s ) underlying RIF activity , we focused on the fraction enriched in sphingolipids .",
"Lipids isolated from 160 L of A . machipongonensis culture were separated using preparative liquid chromatography–mass spectrometry ( LC-MS ) and the activity of each fraction was measured using the rosette colony induction bioassay .",
"RIF activity tracked with a single fraction , which was further purified by several rounds of preparative thin-layer chromatography ( Figure 3—figure supplement",
"1 ) to yield approximately 700 µg of active compound ( RIF-1 ) with sufficient purity for structural analysis .",
"RIF-1 represents only 0 . 015% of the A . machipogonensis sphingolipid pool .",
"Based on high-resolution mass spectrometry , RIF-1 has a molecular formula of C32H64NO7S ( M-H: exptl . 606 . 44027 , calcd . 606 . 44035 , Figure 3—figure supplement 2 ) .",
"Detailed analysis of one- and two-dimensional ( COSY , HMBC , TOCSY , Figure 3—figure supplements 3–15 ) nuclear magnetic resonance ( NMR , 600 MHz , Table 3 ) spectra revealed the planar structure of RIF-1 , an unusual sulfonolipid shown in Figure 3B . 10 . 7554/eLife . 00013 . 024Table 3 . Table of NMR chemical shiftsDOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 024Positionδ 1H ( multiplicity , J , #H ) 13C ( δ , ppm ) NH8 . 21 ( d , J=9 . 1 Hz , 1H ) 13 . 01 ( dd , J=14 . 2 , 5 . 1 Hz , 1H ) 51 . 872 . 56 ( dd , J=14 . 3 , 3 . 6 Hz , 1H ) 23 . 88 ( ddd , J=13 . 0 , 8 . 6 , 4 . 5 Hz , 1H ) 50 . 8933 . 78 – 3 . 71 ( m , 1H ) 71 . 51OH35 . 20 ( d , J=4 . 2 Hz , 1H ) 41 . 51 – 1 . 47 ( m , 1H ) 41 . 361 . 33 – 1 . 29 ( m , 1H ) 53 . 61 – 3 . 53 ( m , 1H ) 70 . 20OH54 . 31 ( d , J=3 . 5 Hz , 1H ) 61 . 34 – 1 . 29 ( m , 1H ) 37 . 271 . 24 – 1 . 20 ( m , 1H ) 7–131 . 21 – 1 . 27 ( br s , 14H ) 22 . 5–29 . 6141 . 16 – 1 . 11 ( m , 2H ) 38 . 91151 . 52 – 1 . 47 ( m , 1H ) 27 . 7916 , 170 . 84 ( d , J=6 . 6 Hz , 6H ) 22 . 091′173 . 232′3 . 80 ( dd , J=6 . 6 , 4 . 2 Hz , 1H ) 71 . 29OH2′5 . 52 ( d , J=5 . 0 Hz , 1H ) 3′1 . 59 – 1 . 54 ( m , 1H ) 34 . 851 . 50 – 1 . 45 ( m , 1H ) 4′ ?",
"1 . 35 – 1 . 30 ( m , 2H ) 24 . 995′–11′1 . 21 – 1 . 27 ( br s , 14H ) 22 . 5–29 . 612′1 . 16 – 1 . 11 ( m , 2H ) 38 . 9113′1 . 52 – 1 . 47 ( m , 1H ) 27 . 7914′ , 15′0 . 84 ( d , J=6 . 6 Hz , 6H ) 22 . 09 Sulfonolipids like RIF-1 resemble sphingolipids , but there are important differences between the two .",
"In sphingolipids , a sphingoid base ( 1 , 3-dihydroxy-2-aminoalkane ) is linked through an amide bond to a fatty acid .",
"The long alkyl chains of both the sphingoid base and fatty acid vary in length , branching , number of double bonds , and placement of hydroxyl substituents .",
"In RIF-1 , a capnoid base ( 2-amino-3-hydroxy-15-methyl-1-sulfonic acid ) replaces the sphingoid base , and the sphingolipid hydroxyl , which is the attachment point for the major diversifying elements of the sphingolipid family , is replaced by a sulfonic acid .",
"While members of the sphingolipid family , such as the ceramides , glycosphingolipids , sphingomyelins , and gangliosides , differ by the groups attached to the hydroxyl , the sulfonic acid function in sulfonolipids like RIF-1 has no reported diversifying modifications .",
"In this sense sulfonolipid diversity appears more limited than sphingolipid diversity .",
"Significantly , commercial sphingolipids ( sphingomyelin , monosiloganglioside , galactocerebroside , and N-palmitoyl-DL-dihydrolacto cerebroside; Table 2 ) failed to show any activity in our assay system .",
"To our knowledge , RIF-1 is the first sulfonolipid demonstrated to influence developmental processes in eukaryotes .",
"Finally , we investigated the potency of RIF-1 and its ability to induce colony development under plausible environmental conditions .",
"Purified RIF-1 induces rosette formation with a bell-shaped dose-response curve over a broad range of concentrations , from 10−2 to 107 fM or some nine orders of magnitude ( Figure 4 ) .",
"No observable effects were seen below 10−5 fM , and RIF-1 appears to be inactive above 108 fM .",
"A . machipongonensis conditioned medium contains 104 fM RIF-1 ( Figure 4—figure supplements 1–3 ) , and even if this conditioned medium measurement exaggerates natural concentrations by a factor of 106 , S . rosetta could still respond to its presence .",
"The shape of the dose-response curve and the potency of RIF-1 suggest that S . rosetta perceives RIF-1 in a manner consistent with a receptor-ligand interaction , albeit a receptor of exquisite sensitivity and remarkable dynamic range .",
"While RIF-1 is the only molecule detected with rosette-inducing activity , its maximal activity ( 5 . 6 ± 0 . 5% colonial cells/total cells ) differs from that of the sphingolipid-enriched lipid fraction ( 19 . 2 ± 4 . 6% colonial cells/total cells; Figure 4 ) .",
"This difference may be due to delivery issues of the purified and highly hydrophobic molecule , which in nature resides in membranes and potentially in membrane vesicles .",
"Alternatively , the full potency of RIF-1 as an inducer of colony development may require additional A . machipongonensis molecules not identified in this study . 10 . 7554/eLife . 00013 . 025Figure 4 . Purified RIF-1 is active at plausible environmental concentrations . RIF-1 concentrations ranging from 10−2 to 107 fM induce rosette colony development in RCA cultures .",
"Frequency of rosette colony development was quantified in RCA cultures 2 days after treatment with a dilution series of purified RIF-1 .",
"Data are mean ± s . e . from three independent experiments .",
"Line indicates non-linear regression of the RIF-1 activity profile . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 02510 . 7554/eLife . 00013 . 026Figure 4—figure supplement 1 . Detection of purified RIF-1 . RIF-1 did not show absorbance at 210 nm ( top panel ) , but the molecule ( 606 Da , M-H ) was detected between 25 and 26 minutes ( bottom panel ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 02610 . 7554/eLife . 00013 . 027Figure 4—figure supplement 2 . Detection of RIF-1 in the conditioned medium of A . machipongonensis . RIF-1 ( bottom panel ) was detected in the conditioned medium after the broth was concentrated 250 times . DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 02710 . 7554/eLife . 00013 . 028Figure 4—figure supplement 3 . Co-injection of concentrated conditioned medium with purified RIF-1 . The peak of the RIF-1 in the conditioned medium was enhanced after the sample was spiked with purified RIF-1 ( bottom panel ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00013 . 028"
],
[
"These data reveal that RIF-1 , a sulfonolipid produced by the prey bacterium A . machipongonensis , regulates morphogenesis in its predator , S . rosetta .",
"The ecological relevance of this signaling interaction is indicated both by the coexistence of S . rosetta and A . machipongonensis in nature and by the fact that the activity of RIF-1 at femtomolar concentrations makes it markedly more potent than other marine signaling molecules [e . g . , Vibrio autoinducer ( Schaefer et al . 1996 ) and the tripeptide pheromones of the Caribbean spiny lobster ( Ziegler and Forward 2007 ) ] .",
"The potency of RIF-1 signaling compares favorably with that of silkworm moth sex pheromone signaling , in which vapors from an ∼4 fM solution of bombykol , the sex pheromone of the silkworm moth , induce a pronounced wing fluttering response in males ( Butenandt et al . 1961; Agosta 1992; Roelofs 1995 ) .",
"While it is formally possible that RIF-1-dependent rosette colony development is a promiscuous response to sphingolipid-type molecules , only a handful of sulfonolipids like RIF-1 have been reported ( Godchaux and Leadbetter 1980 , 1983 , 1984 , 1988; Drijber and McGill 1994; Kamiyama et al . 1995a , 1995b; Kobayashi et al . 1995 ) and no other A . machipongonensis lipid tested in this study induced rosette colony development .",
"Therefore we favor a model in which A . machipongonensis cell density , as revealed by RIF-1 concentration , provides S . rosetta with an indication of conditions under which rosette colony development would be advantageous , for instance by promoting more efficient capture of planktonic bacteria ( Kreft 2010 ) .",
"In analogy to the chemotaxis system of bacteria ( Falke et al . 1997 ) , the ability of S . rosetta to respond to increasing bacterial cell density likely requires the hypothesized RIF-1 receptor to become less sensitive at higher concentrations .",
"The high concentration cutoff in the dose-response curve reflects a complete loss of sensitivity at high , but non-physiological , RIF-1 concentrations .",
"Although the presence of RIF-1 in the A . machipongonensis cell envelope suggests that it may be encountered by S . rosetta during phagocytosis , it can also function at a distance .",
"We hypothesize that RIF-1 may be released into the environment in membrane vesicles , which have been described in Gram-negative bacteria such as Bacteroidetes ( Zhou et al . 1998; Møller et al . 2005 ) , and that additional membrane constituents might be required for the full potency of RIF-1 .",
"In the future , elucidating RIF-1 delivery , along with determining the three-dimensional structure of RIF-1 and characterizing sulfonolipids from other Bacteroidetes will begin to provide the needed foundation for a molecular understanding of how S . rosetta perceives RIFs .",
"The morphogenetic interaction described here between S . rosetta and A . machipongonensis raises the possibility that bacterially-produced sphingolipids in general , and sulfonolipids in particular , may be essential for the chemical signaling that allows Bacteroidetes to influence cell differentiation and morphogenesis in diverse animals ( Falkow 2006; Mazmanian et al . 2008; Lee and Mazmanian 2010; An et al . 2011 ) .",
"Sulfonolipids like RIF-1 have been reported to have therapeutic activities , but their endogenous functions are not known .",
"Sulfobacins A and B , which were isolated from the culture broth of a Chryseobacterium sp .",
"were reported as von Willebrand factor receptor antagonists , and flavocristamide A , from a related bacterial species , was reported as a DNA polymerase α inhibitor ( Kamiyama et al . 1995a; Kobayashi et al . 1995 ) .",
"The pervasiveness of interactions between Bacteroidetes and animals ( Webster et al . 2004; Wexler 2007 ) , coupled with the close evolutionary relationship between choanoflagellates and animals ( King and Carroll 2001; King 2004; King et al . 2008; Ruiz-Trillo et al . 2008 ) , raise the possibility that the connection between Bacteroidetes and animal development has deep evolutionary roots ( McFall-Ngai 1999 ) .",
"The discovery of RIF-1 and its biological activity toward S . rosetta provides both the molecular basis and model organism for further understanding a new and potentially important class of small molecule information transfer ."
],
[
"The environmental isolate of Salpingoeca rosetta is deposited at the American Tissue Culture Collection ( ATCC ) under the designation ATCC50818 ( King et al . 2003 ) .",
"The Rosette Colonies Absent ( RCA ) culture line was produced from ATCC50818 by serial treatment with chloramphenicol ( 68 μg mL−1 ) , ampicillin ( 50 μg mL−1 ) , streptomycin ( 50 μg mL−1 ) , and erythromycin ( 50 μg mL−1 ) ( Fairclough et al . 2010 ) .",
"A monoxenic line of S . rosetta ( Px1 ) was generated by treating ATCC 50818 with a combination of ofloxacin ( 10 μg mL−1 ) , kanamycin ( 50 μg mL−1 ) , and streptomycin ( 50 μg mL−1 ) antibiotics to kill the undefined environmental bacteria .",
"Following several rounds of serial dilution , a single cell was isolated by FACS and supplemented with A . machipongonensis ( Dayel et al . 2011 ) .",
"All three S . rosetta cell lines ( ATCC 50818 , RCA , and Px1 ) were grown in cereal grass infused seawater at 25°C and maintained by splitting cultures 1:10 into fresh medium every 3 days ( King et al . 2009 ) .",
"Live cells were imaged with a Leica DMI6000B microscope equipped with a DFC350 FX camera .",
"Under laboratory conditions , S . rosetta differentiates into a variety of cell types including attached thecate cells , solitary swimmers , rosette colonies , chain colonies and loose , disorganized associations of cells attached to one another at the collar or to bacterial biofilms ( Dayel et al . 2011 ) .",
"S . rosetta rosette colonies can be distinguished from other cell types in that they contain clusters of at least four closely associated cells with organized polarity; each cell oriented with its flagellum pointing outward from a central focus .",
"In the qualitative bioassay , RCA cultures were diluted in fresh medium to a density of approximately 104–105 cells mL−1 , aliquoted into 24-well flat bottom culture dishes ( Costar , Corning , NY , USA ) , supplemented with various treatments , and scored for the presence or absence of rosette colonies after 48 hr .",
"For quantitative measurements , RCA cultures were diluted as before into six-well flat bottom culture dishes .",
"To measure the percentage of cells within rosette colonies , each well was scraped to detach thecate cells and the total number of cells and the total number of cells in each rosette colony were counted with a Bright-Line hemacytometer ( Hausser Scientific , Horsham , PA , USA ) .",
"A partial representation of the bacterial flora from ATCC50818 was isolated by standard dilution-plating technique on modified Zobell medium agar ( Carlucci and Pramer 1957 ) at 25°C .",
"Individual isolates were tested for their morphogenic activity by supplementing RCA cultures with a single colony of each isolate .",
"Of 64 isolates tested , the only one that restored rosette colony development to the RCA cell line was a species that formed pink-pigmented colonies ( designated strain PR1 ) .",
"Strain PR1 was used to inoculate liquid modified Zobell medium at 25°C and grown with aeration overnight .",
"PR1 cells were harvested by centrifugation , and genomic DNA was isolated using a Bacterial Genomic DNA Mini-prep Kit ( Bay Gene , Burlingame , CA , USA ) according to the manufacturer’s specifications .",
"The 16S rRNA gene was amplified using universal primers 8F ( 5′-AGAGTTTGATCCTGGCTCAG-3′ ) and 1492R ( 5′-ACCTTGTTACGRCTT-3′ ) ( Weisburg et al . 1991 ) ; comparison of the PR1 16S rRNA sequence to the Greengenes 16S rRNA database ( DeSantis et al . 2006 ) revealed strain PR1 to be most closely related to members of the Algoriphagus genus within the Bacteroidetes phylum .",
"PR1 was subsequently named Algoriphagus machipongonensis ( Bradley et al . 2009 ) .",
"To investigate whether the ability to trigger rosette colony development was specific to A . machipongonensis , we tested three classes of bacterial species for their morphogenic capacity: 15 species in the Algoriphagus genus , 16 non-Algoriphagus members of the Bacteroidetes phylum , and eight species representing three additional major clades within Bacteria .",
"Each species was screened for morphogenic activity using the bioassay for rosette colony development .",
"Live cells from individual colonies grown from solid agar plates were added directly to RCA cultures and scored for the presence or absence of rosette colonies 48 hr after inoculation .",
"Each bacterial species was tested three times .",
"To determine the phylogenetic distribution of morphogenic activity in the bacterial species tested ( Table 1 ) , a sequence alignment of 16S rDNA genes from each species was generated by iterative pairwise comparisons using FSA ( Bradley et al . 2009 ) .",
"Poorly aligned regions were removed by Gblocks version 0 . 91b ( Castresana 2000; Talavera and Castresana 2007 ) using default block parameters .",
"A distance matrix ( distance options according to the Kimura two-parameter model ) , including clustering with the maximum likelihood algorithm , was calculated using Phylip version 3 . 67 ( Falenstein 1989 ) .",
"Support for the resulting tree topology was estimated using bootstrap analysis ( 1000 replicates ) .",
"To determine the biochemical nature of RIF-1 , A . machipongonensis cell fractions and conditioned medium were subjected to a battery of treatments .",
"The results of these tests are summarized in Table",
"2 . Conditioned medium ( CM ) was generated by pelleting either choanoflagellates grown in cereal grass infused with seawater or A . machipongonensis cultures grown in seawater complete medium ( Atlas 2004 ) and filtering the culture supernatant through a 0 . 22 μm pore filter ( Millipore ) to remove live bacteria .",
"To test whether RIF-1 activity required live bacteria , A . machipongonensis was grown overnight at 25°C , centrifuged at 16 , 000×g for 1 min to pellet cells , and heated for 30 min at 80°C to kill viable bacteria .",
"To test whether RIF-1 activity might be heat labile ( e . g . , a polypeptide ) , A . machipongonensis CM was boiled for 10 min .",
"To test whether RIF-1 was a protein , A . machipongonensis CM was incubated with 200 μg mL−1 proteinase K ( New England Biolabs , Ipswich , MA , USA ) for 2 hr at 37°C .",
"To test whether RIF-1 was a nucleic acid , 25 mL of A . machipongonensis CM was lyophilized , resuspended in 2 . 5 mL of water , and extracted with 100% ethanol to a final concentration of 80% ( vol/vol ) for 2 hr at −20 °C and the precipitate was collected by centrifugation for 30 min at 4000×g at 4°C .",
"The precipitate was dissolved in 0 . 01 M PBS ( containing 10 mM MgCl2 and 1 mM CaCl2 ) and incubated with either RNase A ( 100 μg mL−1; Sigma ) or DNase I ( 100 μg mL−1; Sigma ) for 2 . 5 hr at 37°C .",
"To test whether RIF-1 activity was in the methanolic extract , A . machipongonensis cell pellet and CM were lyophilized and vortexed with methanol .",
"Each suspension was centrifuged at 8000 rpm for 5 min and the methanol layer recovered and dried .",
"To test whether RIF-1 was a lipid , A . machipongonensis cell pellet was extracted according to the Bligh–Dyer method ( Bligh and Dyer 1959 ) .",
"Briefly , the cell pellet was resuspended in 3 vol of 1:2 ( vol/vol ) CHCl3:MeOH and vortexed .",
"One volume of CHCl3 was added , and the mixture vortexed .",
"One volume of distilled water was then added , and the mixture vortexed .",
"The same was then centrifuged at 1000 rpm for 5 min and the bottom layer recovered and dried .",
"To test whether RIF-1 was a component of lipopolysaccharide ( LPS ) , A . machipongonensis LPS was isolated using a method from Apicella ( 2008 ) .",
"Lyophilized A . machipongonensis cells were ground with a mortar and pestle and suspended in 10 mM Tris–Cl buffer ( pH 8 . 0 ) , containing 2% sodium dodecyl sulfate ( SDS ) , 4% 2-mercaptoethanol , and 2 mM MgCl2 .",
"The mixture was vortexed and incubated at 65°C until solubilized .",
"Proteinase K ( 20 mg mL−1 ) was added to the mixture , and incubated at 65°C for an additional hour , followed by 37°C incubation overnight .",
"3 M sodium acetate was then added and the sample mixed .",
"Following addition of cold absolute ethanol to the cell suspension , the sample was incubated overnight at –20°C to allow precipitate to form .",
"The mixture was centrifuged at 4000×g for 15 min , and the supernatant discarded .",
"The precipitate was suspended in distilled water .",
"3 M sodium acetate was added , and the mixture vortexed .",
"Following addition of cold absolute ethanol , the mixture was vortexed again , and the suspension again allowed to precipitate overnight at –20°C .",
"After the centrifugation , the precipitate was suspended in 10 mM Tris–Cl ( pH 7 . 4 ) , and DNase I ( 100 μg mL−1; NEB ) and RNase ( 25 μg mL−1; NEB ) added .",
"The mixture was incubated at 37°C for 4 hr , then placed in a 65°C water bath for 30 min .",
"Ninety percent phenol preheated to 65°C was added , and allow to set at 65°C for 15 min .",
"The mixture was placed in an ice bath to cool , and then centrifuged at 6000×g for 15 min .",
"The top aqueous layer was removed , and the phenolic layer re-extracted with an equal volume of distilled water .",
"This sample was again incubated at 65°C for 15 min and then placed in ice water .",
"After centrifugation at 6000×g for 15 min , the two aqueous layers were combined and dialyzed against multiple changes of distilled water over 2 days .",
"To test whether RIF-1 was a peptidoglycan , A . machipongonensis peptidoglycan was isolated using a method adapted from de Jonge et al . ( 1992 ) , A . machipongonensis cell pellet was washed with 0 . 8% NaCl .",
"The cells were resuspended in hot 4% SDS , boiled for 30 min , and then incubated at room temperate overnight .",
"The sample was boiled for an additional 10 min and then centrifuged at 15 , 000×g for 15 min at room temperature .",
"The pellet was washed four times with water and resuspended in water .",
"The sample was digested for mutanolysin ( 10 μg mL−1; Sigma ) overnight at 37°C .",
"The enzyme was inactivated by incubation at 80°C for 20 min .",
"A . machipongonensis was cultured in seawater complete medium ( 16×1 L ) at 30°C for 2 days .",
"The cells were harvested by centrifugation and extracted with CHCl3:MeOH ( 2:1 , 4 L ) .",
"The organic extract was filtered , dried over sodium sulfate ( Na2SO4 ) , and concentrated to give approximately 4 g crude lipid extract .",
"The crude extract was dissolved in a minimum amount of CHCl3:MeOH ( 2:1 ) , and purified by preparative high performance liquid chromatography ( HPLC ) .",
"All solvents were purchased from Fisher Scientific unless otherwise noted .",
"Preparative reversed phase HPLC ( RP-HPLC ) was performed on an Agilent Technologies 1200 Series HPLC using a Phenomenex Luna 5 µm C8 ( 2 ) 100 Å 250×21 . 2 mm column .",
"Isolation of RIF-1 continued with a crude fractionation in which compounds were eluted at 10 mL min−1 in a gradient of solvents A ( 0 . 1% NH4OH in water ) and B ( 0 . 1% NH4OH in methanol ) : 65% B increasing to 100% B over 30 min , isocratic at 100% B for 1 min . before returning to 65% B and re-equilibrating over 10 min .",
"Fractions were analyzed by low-resolution mass spectrometry ( LC-MS ) on an Agilent 6130 LC/MS using a Phenomenex Gemini-NX 5 µm C18 110 Å 100×2 mm column .",
"The next stage involved a higher resolution separation in which compounds were eluted at 0 . 5 mL min−1 in a gradient of solvents A ( 0 . 1% NH4OH in water ) and B ( 0 . 1% NH4OH in methanol ) : 65% B increasing to 100% B over 30 min , isocratic at 100% B for 1 min before returning to 65% B and re-equilibrating over 3 min and those which contained a mass peak corresponding to RIF-1 ( [M-H]=606 . 4 ) were combined and concentrated .",
"This material was then purified by preparative TLC ( 1 mm , silica gel 60 ) , eluted with CHCl3:MeOH:AcOH:H2O ( 100:20:12:5 , Rf=0 . 5 ) .",
"RIF-1 was visualized by staining with ammonium molybdate in 10% H2SO4 .",
"The portion of the plate ( Fraction F; Figure 3—figure supplement 1 ) that induced colony formation and contained RIF-1 ( LC/MS: [M-H] 606 ) was scraped off after RIF-1 was visualized by staining with ammonium molybdate in 10% H2SO4 , and the silica was extracted with CHCl3:MeOH ( 5:1 ) .",
"This material was further purified by preparative TLC on a 250 μm TLC plate ( silica gel 60 ) , eluted with CHCl3:MeOH:AcOH:H2O ( 100:20:12:5 ) .",
"From 16 L of A . machipongonensis culture , approximately 50 μg RIF-1 was obtained in sufficient purity .",
"The entire process , from growth of the cells to isolation of pure RIF-1 , was repeated nine times in order to obtain approximately 0 . 7 mg RIF-1 from a total of 160 L of A . machipongonensis culture .",
"High Resolution Mass Spectrometry ( HRMS ) was carried out by Ted Voss at the WM Keck Foundation Biotechnology Resource Laboratory at Yale University on a Bruker 9 . 4T FT-ICR MS . RIF-1 was dissolved in 200 μL DMSO-d6 and transferred into a 3 mm NMR tube .",
"1H , TOCSY , gCOSY and dqfCOSY were recorded on a Varian Inova 600 spectrometer .",
"HMQC and gHMBC experiments were performed on a Bruker Advance ( sgu ) 900 MHz and Varian Unity Inova 600 MHz equipped with a cryoprobe , respectively .",
"Chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance resulting from incomplete deuteration as the internal standard ( DMSO: δ 2 . 50 ) .",
"Data are reported in Table 3 as follows: chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , br = broad , m = multiplet ) , coupling constants , and integration .",
"Optical rotation was measured on a Jasco P-2000 digital polarimeter with a sodium lamp at 21 . 4°C .",
"Unless otherwise noted , all solvents and reagents were purchased from VWR or Fisher and used without further purification .",
"1H NMR ( 600 MHz , DMSO ) and 13C NMR ( 600 MHz , DMSO ) : see Table",
"3 . Optical rotation: [α]D21 . 4 +6 . 4 ( c=0 . 07 , MeOH ) .",
"HRMS m/z calcd for C32H64NO7S ( M-H ) : 606 . 44035 .",
"Found: 606 . 44027 ( M-H ) − .",
"MS/MS analysis: A major fragment derived from m/z=606 ( M-H ) in the MS/MS spectrum of RIF-1 corresponds to amino-sulfonic acid , Figure 3—figure supplement",
"2 . HRMS/MS m/z calculated for C17H36NO5S ( M-H ) : 366 . 23142 .",
"Found: 366 . 2310 ( M-H ) − .",
"Conditioned medium was prepared from A . machipongonensis grown in seawater complete medium ( 750 mL ) at 30°C for 2 days .",
"The conditioned medium was lyophilized and extracted with CHCl3:MeOH ( 2:1; 78 mL ) .",
"The organic extract was filtered , further extracted with CHCl3 ( 60 mL×2 ) , and filtrates were combined and concentrated to dryness under vacuum .",
"The crude extract was suspended in 5 mL 50% MeOH:H2O and was passed through a C-18 SPE ( 1 g ) column .",
"The open column was then washed with 10 mL 90% MeOH:CHCl3 .",
"The organic eluate was concentrated and dissolved in 3 mL CHCl3:MeOH ( 2:1 ) for LC/MS analysis .",
"The chromatography was carried out using an Agilent 6130 Quadrupole LC/MS system with a C18 reverse-phase column ( 4 . 6×100 mm; Phenomenex Luna; 5 μ ) for 30 min in a linear gradient from solvent A ( 60% methanol/water with 0 . 1% ammonium hydroxide ) to solvent B ( 100% methanol with 0 . 1% ammonium hydroxide ) .",
"The RIF-1 was detected in the conditioned medium at a concentration of 80 ng L−1 .",
"The purified RIF-1 was used as the standard ( Figure 4—figure supplements 1–3 ) .",
"The potency of pure RIF-1 was determined using the quantitative bioassay for rosette colony development .",
"Briefly , 100 ug of pure RIF-1 was solubilized in 100 μL DMSO and this 1 g L−1 stock was stored at -80°C .",
"For each experiment , serial dilutions ranging from 10−1 g L−1 down to 10−17 g L−1 were made in DMSO .",
"2 μL of each dilution was premixed with 1 mL of fresh cereal grass infused seawater ( King et al . 2003 ) to avoid precipitation of RIF-1 and the premixed RIF-1 dilution was then added to 1 mL RCA cultures to yield final concentrations ranging from 10−3 to 10−20 g L−1 , equivalent to 1 . 6×109 fM down to 1 . 6×10−8 fM .",
"The percentage of rosette colonial cells was determined as described above in three independent cell lines in triplicate .",
"From the percent rosette colony development , a bell-shaped dose-response model was determined to be the nonlinear regression curve of best fit determined using GraphPad Prism 5 statistical software ."
]
] | [
"Bacterially-produced small molecules exert profound influences on animal health , morphogenesis , and evolution through poorly understood mechanisms .",
"In one of the closest living relatives of animals , the choanoflagellate Salpingoeca rosetta , we find that rosette colony development is induced by the prey bacterium Algoriphagus machipongonensis and its close relatives in the Bacteroidetes phylum .",
"Here we show that a rosette inducing factor ( RIF-1 ) produced by A . machipongonensis belongs to the small class of sulfonolipids , obscure relatives of the better known sphingolipids that play important roles in signal transmission in plants , animals , and fungi .",
"RIF-1 has extraordinary potency ( femtomolar , or 10−15 M ) and S . rosetta can respond to it over a broad dynamic range—nine orders of magnitude .",
"This study provides a prototypical example of bacterial sulfonolipids triggering eukaryotic morphogenesis and suggests molecular mechanisms through which bacteria may have contributed to the evolution of animals ."
] | [
"All animals , including humans , evolved in a world filled with bacteria .",
"Although bacteria are most familiar as pathogens , some bacteria produce small molecules that are essential for the biology of animals and other eukaryotes , although the details of the ways in which these bacterial molecules are beneficial are not well understood .",
"The choanoflagellates are water-dwelling organisms that use their whip-like flagella to move around , feeding on bacteria .",
"They can exist as one cell or a colony of multiple cells and , perhaps surprisingly , are the closest known living relatives of animals .",
"This means that experiments on these organisms have the potential to improve our understanding of animal development and the transition from egg to embryo to adult .",
"Alegado et al . have explored how the morphology of Salpingoeca rosetta , a colony-forming choanoflagellate , is influenced by its interactions with various species of bacteria .",
"In particular , they find that the development of multicellularity in S . rosetta is triggered by the presence of the bacterium Algoriphagus machipongonensis as well as its close relatives .",
"They also identify the signaling molecule produced by the bacteria to be C32H64NO7S; this lipid molecule is an obscure relative of the sphingolipid molecules that have important roles in signal transmission in animals , plants , and fungi .",
"Moreover , Alegado et al . show that S . rosetta can respond to this molecule – which they call rosette-inducing factor ( RIF-1 ) – over a wide range of concentrations , including concentrations as low as 10−17 M . The work of Alegado et al . suggests that interactions between S . rosetta and Algoriphagus bacteria could be a productive model system for studying the influences of bacteria on animal cell biology , and for investigating the mechanisms of signal delivery and reception .",
"Moreover , the molecular mechanisms revealed by this work leave open the possibility that bacteria might have contributed to the evolution of multicellularity in animals ."
] | 2012 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"evolutionary biology"
] | Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians | elife-03728-v2 | [
[
"Transcriptional regulatory networks are indispensable for the generation of the diverse germ layers , anatomical structures , and cell types of multicellular organisms ( Levine and Davidson , 2005 ) .",
"The impact of cis-regulatory DNA sequence changes on the evolution of development is undeniable but not yet fully understood ( Wray , 2007; Wittkopp and Kalay 2011 ) .",
"Phenotypic differences between species have often been traced to differences in expression of orthologous genes .",
"In turn , this differential expression can be attributed to changes in cis-regulatory modules ( or ‘enhancers’ ) , which may alter their binding by sequence-specific transcription factors ( TFs ) ( Sucena and Stern , 2000; Gompel et al . , 2005; Prud'homme et al . , 2006; Miller et al . , 2007; Shirangi et al . , 2009 ) .",
"In contrast , gene expression patterns may be identical in spite of differences in the cis-regulatory DNA sequences controlling their transcription .",
"For instance , orthologous enhancers might be interchangeable between different species without conspicuous DNA sequence similarity ( Maduro and Pilgrim , 1996; Ludwig et al . , 1998; Piano et al . , 1999; Romano and Wray , 2003; Oda-Ishii et al . , 2005 ) .",
"This is often attributed to the conservation of gene regulatory networks ( GRNs ) and the flexibility of TF binding site distribution in a given enhancer , which contribute to conservation of enhancer function ( Ludwig et al . , 2000; Oda-Ishii et al . , 2005; Hare et al . , 2008; Weirauch and Hughes , 2010 ) .",
"This cryptic regulatory turnover is termed ‘developmental system/systems drift’ ( DSD ) ( True and Haag , 2001 ) .",
"This term broadly applies to the divergence in the molecular or morphogenetic basis for the development of identical homologous characters .",
"However , gene expression patterns that appear conserved between species may also be the output of divergent regulatory networks ( Ludwig et al . , 2005 ) .",
"In such cases , one species' trans environment may be unable to interpret the other species' enhancer , even if the expression patterns controlled by the orthologous enhancers are identical between the two species .",
"This particularly acute manifestation of DSD is thought to contribute to developmental defects in interspecies hybrids ( Takano , 1998 ) , contributing to reproductive isolation and speciation as cis/trans combinations become so incompatible as to be lethal ( Porter and Johnson , 2002 ) .",
"There is ample evidence that DSD is pervasive in metazoan evolution ( Kiontke et al . , 2007; Verster et al . , 2014 ) , but it has been difficult to study due to its cryptic nature .",
"Sea squirts , or ascidians , are well suited for investigating the evolution of gene regulation in development ( Satoh , 2013 ) .",
"Species on the opposite branches of the ascidian tree , estimated to have diverged ∼520 million years apart , have extremely conserved embryos that share identical stereotyped cell divisions up until the late gastrula stage and beyond ( Swalla , 2006; Lemaire , 2009 ) .",
"This allows one to unequivocally infer homology between specific embryonic cells , gene expression patterns , and GRNs .",
"This is in contrast to the striking lack of non-coding sequence conservation , revealed by bioinformatic alignment of genomic sequences from disparate ascidian species .",
"This means that any two randomly selected ascidian species are likely to be more genetically divergent from one another than humans are from fish , which share several highly conserved non-coding sequences ( Woolfe et al . , 2004 ) .",
"This dichotomy between near-identical embryos and un-alignable cis-regulatory sequences represents a singular paradox in the study of evolution and developmental biology today ( Lemaire , 2011 ) .",
"Modern molecular tools for embryo manipulation and visualization are available for several ascidian species , including a protocol for transfection en masse by electroporation of plasmid DNA into fertilized eggs of the solitary ascidian Ciona intestinalis ( Corbo et al . , 1997; Christiaen et al . , 2009b ) .",
"This experimental tractability has allowed researchers to begin addressing the ascidian embryological paradox .",
"Regulation of the Otx gene is a prime example , as enhancers upstream of the Otx genes from C . intestinalis and the distantly related Halocynthia roretzi do not show any DNA sequence similarity but are broadly functional in cross-species assays .",
"This was shown to be due to conservation of cis-regulatory logic in spite of a reshuffling of functional TF binding sites ( Oda-Ishii et al . , 2005 ) .",
"On the other hand , comparisons of C . intestinalis and H . roretzi development have also revealed clear examples of acute DSD in regulation of the Brachyury gene ( Takahashi et al . , 1999 ) , cell signaling upstream of pigment cell differentiation ( Darras and Nishida , 2001; Abitua et al . , 2012 ) , secondary tail muscle specification ( Kim and Nishida , 2001; Tokuoka et al . , 2007; Hudson et al . , 2007; Hudson and Yasuo , 2008 ) , and embryonic marginal zone patterning ( Takatori et al . , 2010; Hudson et al . , 2013 ) .",
"These few examples suggest that considerable DSD may have occurred during the evolution of ascidians , even with their presupposed highly constrained mode of embryogenesis .",
"To get a better idea of how DSD may have shaped ascidian evolution , it was necessary to identify species comparable to Ciona spp .",
"in their experimental tractability but phylogenetically distant enough as to maximize the genetic differences between them .",
"Molgula spp .",
"belong to the order Stolidobranchia like H . roretzi , but produce embryos that are more comparable in size and developmental rate to those of C . intestinalis , a member of the order Phlebobranchia ( Figure 1A ) .",
"The genus is also remarkable for containing several species that have independently evolved anural development ( Berrill , 1931; Swalla and Jeffery , 1990; Hadfield et al . , 1995; Jeffery et al . , 1999; Huber et al . , 2000 ) .",
"These anural , or ‘tail-less’ , Molgula species produce immotile larvae lacking the differentiated structures that are required for swimming ( Swalla and Jeffery , 1990; Kusakabe et al . , 1996 ) , and some species even appear to bypass the larval stage altogether ( Tagawa et al . , 1997; Maliska and Swalla , 2010 ) .",
"Thus , Molgula species comprise a unique group in which to study body plan and life cycle evolution . 10 . 7554/eLife . 03728 . 003Figure 1 . The B7 . 5 lineage in M . occidentalis .",
"( A ) Diagram comparing M . occidentalis ( top ) and C . intestinalis ( bottom ) embryogenesis at 24°C .",
"Embryos were stained with Alexa Fluor dye-conjugated phalloidin to visualize cell outlines and DAPI to visualize cell nuclei .",
"( B ) Diagram of mVISTA ( Frazer et al . , 2004; genome . lbl . gov/vista/ ) alignment of M . oculata Mesp ( Moocul . Mesp ) locus to orthologs in M . occulta , M . occidentalis , and C . intestinalis .",
"Shaded peaks indicate sequence conservation above 70% over 100-bp windows ( blue = protein-coding , pink = non-coding ) .",
"Arrows indicate direction of transcription of protein-coding genes .",
"Non-coding sequences upstream of Mesp are only conserved between M . oculata and M . occulta .",
"M . occidentalis and C . intestinalis show considerable divergence even in protein-coding sequences .",
"Note that microsynteny with SLC5A-related gene supports the orthology of these sequences among the Molgulids .",
"( C ) In situ hybridization ( ISH ) for Moocci . Mesp in 110-cell stage embryo ( vegetal view ) , showing mRNA detection ( green ) in B7 . 5 blastomeres .",
"Nuclei were counterstained with DAPI ( blue ) .",
"Staging is given by hours post-fertilization ( hpf ) .",
"( D ) Vegetal view of a 110-cell stage embryo electoporated with Moocci . Mesp>GFP reporter construct .",
"Reporter gene expression was detected by ISH for GFP transcripts ( green ) .",
"Nuclei were stained with DAPI ( blue ) .",
"( E ) Lateral view of a mid-tailbud stage embryo electroporated with Moocci . Mesp>Histone2B::GFP reporter construct .",
"GFP fluorescence reveals B7 . 5 descendants on left side of embryo: two trunk ventral cells ( TVCs ) and two anterior tail muscles ( ATMs ) .",
"( F ) Diagram of B7 . 5 lineage divisions from 110-cell stage to mid-tailbud stage , inferred from previous C . intestinalis studies .",
"Cells are named according to Conklin's method ( Conklin , 1905 ) .",
"The lineage is bilaterally symmetric , but only cells on the left side are indicated and named .",
"Relative staging given for M . occidentalis ( Mo . occi ) and C . intestinalis ( Ci . inte ) .",
"110-cell and late gastrula: vegetal view .",
"Initial tailbud: dorsal view .",
"Mid-tailbud: lateral view .",
"Anterior pole is on the left in all images and illustrations . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 00310 . 7554/eLife . 03728 . 004Figure 1—figure supplement 1 . Alignment of 5′ flanking sequences from Mesp orthologs . Top: diagram of mVISTA ( Frazer et al . , 2004; genome . lbl . gov/vista/ ) alignment of M . occidentalis Mesp ( Moocci . Mesp ) locus to orthologs in ( from top to bottom ) M . oculata , M . occulta , and C . intestinalis .",
"Bottom: diagram of mVISTA alignment of C . intestinalis Mesp ( Moocci . Mesp ) locus to orthologs in ( from top to bottom ) M . oculata , M . occulta , and M . occidentalis .",
"Blue-shaded peaks indicate protein-coding sequence conservation above 70% in 100-bp windows .",
"Arrows indicate direction of transcription of protein-coding genes Slc5a-related and Mesp . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 00410 . 7554/eLife . 03728 . 005Figure 1—figure supplement 2 . The B7 . 5 lineage of the tail-less species M . occulta .",
"( A ) In situ hybridization for M . occulta Mesp ( Mooccu . Mesp ) in 110-cell st/early gastrula embryo , showing expression ( green ) in the B7 . 5 blastomeres .",
"Image is taken from a vegetal view , anterior to the top .",
"( B ) Lateral view of a transiently-transfected M . occulta mid-tailbud-equivalent stage embryo electroporated with a Moocul . Hand-r reporter construct ( Moocul . Hand-r>H2B::GFP ) .",
"The reporter proteins ( Histone2B::GFP , green ) label the cell nuclei descendant of the trunk ventral cells ( TVCs ) , namely the secondary TVCs ( STVCs ) and the first heart precursors ( FHPs ) .",
"Due to leader/trailer mosaic retention of the electroporated plasmid ( Stolfi and Christiaen , 2012 ) , the daughters of the leader cell ( ‘leader STVC’ and ‘leader HFP’ ) are more strongly labeled than the daughters of the trailer cell .",
"Some staining is also seen in what may be anterior endoderm or A7 . 6 mesoderm , other known territories of Hand-r expression in M . occulta and other ascidian species .",
"Anterior is to the bottom left . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 00510 . 7554/eLife . 03728 . 006Figure 1—figure supplement 3 . Moocci . Mesp in situ hybridization at tailbud stage . In situ hybridization for Moocci . Mesp in initial/early tailbud embryo , showing no expression at this stage .",
"Image is a coronal view with anterior to the left . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 006 In order to realize the full potential of Molgulid ascidians as model organisms for molecular and comparative developmental genetics on par with Ciona spp .",
", we sequenced and assembled the genomes of three Molgula species: the tailed species M . occidentalis and M . oculata , and the tail-less M . occulta .",
"Moreover , we adapted the electroporation protocol to M . occidentalis embryos .",
"To illustrate the power of comparative approaches between Molgula and Ciona , we characterized the B7 . 5 lineage in M . occidentalis .",
"In both C . intestinalis and H . roretzi , this lineage is descended from the B7 . 5 pair of blastomeres of the early gastrula embryo and gives rise to the anterior tail muscles of the larva , and to the heart and atrial siphon/pharyngeal muscles of the adult ( Hirano and Nishida , 1997; Davidson and Levine , 2003; Stolfi et al . , 2010 ) .",
"A growing body of research has focused on the molecular basis of B7 . 5 lineage development in C . intestinalis ( reviewed in Tolkin and Christiaen , 2012; Cota et al . , 2013 ) , some of which have guided the discovery of vertebrate heart developmental mechanisms ( Islas et al . , 2012 ) .",
"Given its detailed characterization in C . intestinalis , we chose the B7 . 5 lineage for an in-depth comparative analysis .",
"Our analysis revealed a remarkable degree of conservation of the clonal topology and gene expression patterns ( together referred to as the ‘ontogenetic motif’ ) underlying ascidian cardiopharyngeal mesoderm development ( Wang et al . , 2013 ) , but also uncovered divergent regulatory mechanisms underlying conserved gene expression profiles between Ciona and Molgula ."
],
[
"Genomes of three Molgula species ( M . occidentalis , M . oculata , and M . occulta ) were sequenced using next-generation sequencing technology and assembled .",
"A common metric for judging the quality of a genome assembly is the contig N50 length , which is determined such that 50% of the assembly is contained in contigs of this length or greater .",
"We used the contig N50 length to select the best assembly for each species given the varying ‘k’ parameter ( length of k-mer overlap ) .",
"A ‘k’ of 39 yields the best assembly for both M . occidentalis and M . occulta .",
"The best ‘k’ for M . oculata was 61 .",
"M . occidentalis , M . occulta , and M . oculata N50 lengths were approximately 26 . 3 kb , 13 kb , and 34 kb , respectively ( Table 1 ) . 10 . 7554/eLife . 03728 . 007Table 1 . Genome assembly statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 007SpeciesN50Mean contig lengthTotalTotal number of base pairsCEGMA C1CEGMA P2M .",
"occidentalis26 , 298507251 , 761262 , 547 , 66081 . 4596 . 77M .",
"occulta13 , 011323358 , 489189 , 110 , 56277 . 4298 . 79M .",
"oculata34 , 042627025 , 497159 , 886 , 71689 . 9299 . 19The contig N50 length , mean contig length , total number of contigs , total number of base pairs and CEGMA scores were collected for each draft assembly .",
"The CEGMA scores is a metric of completeness measured against highly Conserved eukaryotic genes .",
"Alignments of 70% or greater of the protein length are called complete ( C1 ) and all other statistically significant alignments are called partial ( P2 ) .",
"In addition to N50 lengths , we also used CEGMA ( Core Eukaryotic Genes Mapping Approach ) scores , in order to evaluate the assemblies' representative completeness ( Parra et al . , 2007 ) .",
"CEMGA reports scores for complete and partial alignments to a subset of core eukaryotic genes .",
"An alignment is considered ‘complete’ if at least 70% of a given protein model aligns to a contig in the assembly , while a partial alignment indicates that a statistically significant portion of the protein model aligns .",
"The partial alignment scores are ∼97% or higher for all assemblies .",
"M . oculata has the best complete alignment score at ∼90% .",
"M . occidentalis and M . occulta have complete alignment scores of 81% and 77% respectively ( Table 1 ) .",
"These scores indicate that our assemblies contain at least partial sequences for the vast majority of protein-coding genes in the genomes of these species .",
"Various factors make it unreliable to predict genome size and gene density based on assembly metrics alone ( Bradnam et al . , 2013 ) .",
"Of the handful of sequences we isolated and analyzed , we found that the sizes of introns and upstream regulatory regions were roughly comparable to those from their Ciona orthologs .",
"This suggests that the Molgula genomes may be as compact as the C . intestinalis genome ( i . e . , ∼150–170 Mb , ∼16 , 000 genes , Laird , 1971; Simmen et al . , 1998; Satou et al . , 2008 ) .",
"Our sequencing efforts revealed extreme genetic divergence not only between Ciona and Molgula , as expected , but even within the Molgulids .",
"For example , we used BLAST to identify the Molgula orthologs of C . intestinalis Mesp ( Ciinte . Mesp , as per the proposed tunicate gene nomenclature rules , see Stolfi et al . , 2014 ) .",
"Ciinte . Mesp is the sole ortholog of vertebrate genes coding for MesP and Mesogenin bHLH transcription factor family members ( Satou et al . , 2004 ) .",
"VISTA alignment shows high sequence similarity between sequences 5′ upstream of the Mesp genes from the closely related M . oculata and M . occulta ( Figure 1B ) .",
"However , there is no conservation of Mesp DNA sequences , coding or non-coding , between M . oculata/occulta and M . occidentalis , nor between C . intestinalis and any of the three Molgula species ( Figure 1—figure supplement 1 ) .",
"In previous phylogenetic surveys , M . occidentalis has been placed as an early-branching Molgula species , often grouped together in a subfamily with species ascribed to the genera Eugyra and Bostrichobranchus instead ( Hadfield et al . , 1995; Huber et al . , 2000; Tsagkogeorga et al . , 2009 ) .",
"Our sequencing results support the view that M . occidentalis is highly diverged from other Molgula spp .",
"Ciinte . Mesp specifies the B7 . 5 cells as the sole progenitors of the cardiopharyngeal lineage ( Satou et al . , 2004; Davidson et al . , 2005; Hirano and Nishida , 1997; Stolfi et al . , 2010 ) .",
"We performed RNA in situ hybridization ( ISH ) for M . occidentalis Mesp ( Moocci . Mesp ) and found that this gene is also expressed only in the B7 . 5 cells of M . occidentalis embryos ( Figure 1C ) .",
"These cells are unequivocally identified due to the perfect conservation of early embryonic cell cleavage patterns in all ascidians .",
"ISH for the M . occulta Mesp gene ( Mooccu . Mesp ) also revealed conserved expression in this tail-less species ( Figure 1—figure supplement 2A ) .",
"We successfully adapted the Ciona electroporation protocol for simultaneous transfection of reporter gene plasmids into hundreds of synchronized M . occidentalis embryos ( Figure 1D , E ) .",
"We were also able to electroporate M . occulta embryos ( Figure 1—figure supplement 2B ) .",
"However , only M . occidentalis was routinely available to us for in vivo studies , so we focused our experiments on this species .",
"Development of M . occidentalis embryos was optimal at 24°C and faster than that of C . intestinalis ( Figure 1A ) .",
"Using electroporation-based transfection , we determined that an ∼1 . 1 kb genomic DNA fragment upstream of Moocci . Mesp is able to drive expression of fused reporter genes specifically in the B7 . 5 cells with no ‘leaky’ expression in other cells as is commonly observed in C . intestinalis ( Figure 1D; Stolfi and Christiaen , 2012 ) .",
"This faithful recapitulation of Moocci . Mesp expression and the persistence of GFP allows for visualization of the descendants of B7 . 5 long after endogenous Moocci . Mesp transcription has ceased ( Figure 1—figure supplement 3; Davidson et al . , 2005 ) .",
"At the tailbud stage , we find that each B7 . 5 blastomere gives rise to four grand-daughter cells ( Figure 1E ) .",
"The two anterior B7 . 5 grand-daughter cells on either side of the bilaterally symmetric embryo migrate anteriorly and are termed the trunk ventral cells ( TVCs ) due to their final position in the C . intestinalis and H . roretzi embryos ( Nishida , 1987 ) .",
"Their posterior sister cells remain in the tail and become anterior tail muscles ( ATMs ) .",
"As far as we can tell , B7 . 5 lineage ontogeny is perfectly conserved between M . occidentalis and C . intestinalis ( Figure 1F ) .",
"Our focus shifted to the TVCs , which in H . roretzi and C . intestinalis have been shown to be multipotent cardiopharyngeal progenitor cells that give rise to the heart and pharyngeal muscles of the atrial siphon ( Hirano and Nishida , 1997; Stolfi et al . , 2010 ) .",
"We performed a small ISH screen for orthologs of transcription factors that are expressed in C . intestinalis TVCs ( Satou et al . , 2004; Davidson et al . , 2005; Davidson and Levine , 2003; Christiaen et al . , 2008; Beh et al . , 2007 ) .",
"These include the orthologs of conserved cardiac regulators Ets1/2 , GATA4/5/6 , and NK4/Nkx2 . 5/tinman ( Cripps and Olson , 2002 ) ( Figure 2 ) . 10 . 7554/eLife . 03728 . 008Figure 2 . Expression of conserved TVC/heart markers in M . occidentalis embryos . In situ hybridization ( ISH ) in M . occidentalis embryos for ( A and A′ ) Moocci . Ets . b , ( B and B′ ) Moocci . Foxf , ( C and C′ ) Moocci . Hand-related ( Moocci . Hand-r ) , ( D and D′ ) Moocci . Gata4/5/6 , ( E and E′ ) Moocci . Nk4 , ( F and F′ ) Moocci . Rhod/f .",
"ISH was performed on initial tailbud ( A–F ) and mid tailbud ( A′–F′ ) stage embryos .",
"Solid arrowheads indicate definitive expression in TVCs .",
"Dotted arrowheads indicate potential expression of Moocci . Foxf in initial tailbud , obscured by strong epidermal expression .",
"Dotted outline indicates probable position of TVCs , not visible due to lack of mRNA hybridization signal .",
"Initial tailbud embryos were imaged ventrally or dorsally , while tailbud embryos were imaged laterally . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 00810 . 7554/eLife . 03728 . 009Figure 2—figure supplement 1 . Ets . b expression in B7 . 5 of M . occidentalis . In situ hybridization ( ISH ) for Moocci . Ets . b in 110-cell st/early gastrula embryo , showing expression ( green ) in the B7 . 5 blastomeres .",
"Nuclei are counterstained with DAPI ( blue ) .",
"Embryo was imaged from a vegetal view , with anterior to the top . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 00910 . 7554/eLife . 03728 . 010Figure 2—figure supplement 2 . In situ hybridizations reveal TVC gene expression in M . occidentalis . Mid-tailbud M . occidentalis embryos electroporated with Moocci . Mesp>nls::lacZ , assayed with β-galactosidase immunodetection ( IHC , green ) couple to in situ hybridization ( ISH , red ) for ( A ) Moocci . Foxf , ( B ) Moocci . Hand-r , ( C ) Moocci . Gata4/5/6 , and ( D ) Moocci . Nk4 .",
"Expression of these genes was detected in the TVCs , with the possible exception of Nk4 .",
"All embryos are oriented with anterior to the left . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01010 . 7554/eLife . 03728 . 011Figure 2—figure supplement 3 . Hand-r and FoxF co-expression reveals TVCs of M . occulta . Coronal view of M . occulta embryos at ( A–C ) 6 . 5 hpf ( equivalent to the initial tailbud stage of tailed M . oculata ) and ( D–I ) 7 . 5 hpf ( equivalent to the mid-tailbud stage ) .",
"Embryos were assayed for expression of Mooccu . Hand-r ( green—A , D , G ) and Mooccu . Foxf ( red—B , E ) or Mooccu . Aldh1a ( red—H ) by double in situ hybridization .",
"Merged green and red channels show co-expression in TVCs ( C , F , I ) .",
"Mooccu . Foxf expression in epidermis seen in F suggests position of TVCs in ventro-lateral regions of the trunk similar to what is observed in M . occidentalis and C . intestinalis .",
"Mooccu . Aldh1a-expressing cells immediately posterior to TVCs are likely the anterior tail muscles .",
"Anterior is to the left in all panels . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01110 . 7554/eLife . 03728 . 012Figure 2—figure supplement 4 . Aldh1a expression in M . occidentalis embryo . In situ hybridization for Moocci . Aldh1a showing expression in TVCs and ATMs in a mid-tailbud embryo . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 012 In M . occidentalis , Ets . b expression is initiated in B7 . 5 blastomeres ( Figure 2—figure supplement 1 ) , is maintained in their daughter cells the cardiopharyngeal founders and in the TVCs during their migration ( Figure 2A ) .",
"This profile is similar to the expression of C . intestinalis Ets . b ( Ciinte . Ets . b ) , previously named Ets/pointed2 or Ets1/2 ( see Supplementary file 2 for list of old and new gene name correspondences ) .",
"In C . intestinalis , Ets . b mediates the FGF/MAPK-dependent induction of TVCs in part by the activation of key regulators such as Foxf , Hand-related ( Hand-r , also known as Hand-like or NoTrlc ) , and Gata4/5/6 ( also known as GATA-a ) prior to the onset of TVC migration ( Davidson et al . , 2006; Beh et al . , 2007 ) .",
"In M . occidentalis , orthologs of Foxf and Hand-r are also activated in the TVCs shortly before and throughout their migration away from the ATMs ( Figure 2B , C ) .",
"Moocci . Gata4/5/6 expression was detected in migrating TVCs but not before migration ( Figure 2D ) .",
"This is slightly different from Ciinte . Gata4/5/6 , which is expressed in C . intestinalis TVCs prior to migration ( Christiaen et al . , 2010; Ragkousi et al . , 2011 ) .",
"Expression of Moocci . Foxf and Moocci . Gata4/5/6 in surrounding epidermis and endoderm , respectively , is identical to the expression domains of their orthologs in C . intestinalis and sometimes obscured TVC expression .",
"However , double ISH/immunohistochemical detection ( ISH/IHC ) of Moocci . Mesp promoter-driven reporter gene clearly shows transcripts in the migrating B7 . 5-derived TVCs ( Figure 2—figure supplement 2A–C ) .",
"Foxf and Hand-r are also expressed in the TVCs of tail-less M . occulta embryos .",
"Because these embryos do not extend a tail and lack functional larval tail muscles , it is unclear whether their TVCs move away from the posterior pole of the embryo ( Figure 2—figure supplement 3A–F ) .",
"However , their TVCs occupy the ventro-lateral regions of the trunk ( Figure 1—figure supplement 2B ) , similar to what is observed in M . occidentalis , suggesting they might be migrating .",
"We cannot draw any further conclusions about the TVCs of M . occulta , but have not found any evidence for fundamental molecular or morphological differences relative to M . occidentalis .",
"Notably , the ATMs appear to be specified in M . occulta , as revealed by expression of Aldh1a ( also known as Raldh2 , Figure 2—figure supplement 3G–I ) , which encodes the rate-limiting enzyme for retinoic acid ( RA ) synthesis .",
"Therefore , although the larval muscles of M . occulta do not differentiate ( Swalla and Jeffery , 1990; Kusakabe et al . , 1996 ) , their ATMs may still be required for RA-mediated embryonic patterning like in C . intestinalis ( Nagatomo and Fujiwara , 2003 ) and probably also in M . occidentalis ( Figure 2—figure supplement 4 ) .",
"Unexpectedly , we could not detect unequivocal expression of the sole M . occidentalis ortholog of NK4/Nkx2 . 5/tinman ( Moocci . Nk4 ) in the TVCs ( Figure 2E ) .",
"Expression in the ventral ectoderm mirrored that seen in C . intestinalis , indicating that the probe used was functional .",
"It is possible that expression of Nk4 in the TVCs of M . occidentalis is below the threshold of reliable detection by ISH .",
"Our ISH data are not entirely incompatible with Moocci . Nk4 being expressed in the TVCs at very low levels ( Figure 2—figure supplement 2D ) .",
"Conserved TVC gene expression patterns were not limited to TF-coding genes .",
"The small GTPase-coding Rhod/f gene was found to be transcriptionally upregulated in M . occidentalis TVCs just prior to migration ( Figure 2F ) .",
"In C . intestinalis , Rhod/f was identified as an effector of cytoskeleton dynamics in TVC migration and a direct transcriptional target of Foxf and activated Ets . b ( Christiaen et al . , 2008 ) .",
"This suggests that the conservation of B7 . 5 development in ascidians extends to the interface between transcriptional regulators and cellular effectors of cell migration .",
"In C . intestinalis , heart and atrial siphon ( pharyngeal ) muscle precursors are derived from TVCs in a two-step process involving asymmetric cell divisions ( Stolfi et al . , 2010; Wang et al . , 2013 ) .",
"First , each TVC undergoes an asymmetric cell division along the medial/lateral ( M/L ) axis to produce a medial First Heart Precursor ( FHP ) and a lateral Secondary TVC ( STVC ) .",
"Each STVC then also undergoes a M/L asymmetric division to give rise to a medial Second Heart Precursor ( SHP ) and a lateral atrial siphon muscle founder cell ( ASMF ) .",
"In C . intestinalis , this step-wise segregation of heart vs pharyngeal muscle fate is coordinated by ( 1 ) activation of Tbx1/10 in the STVCs ( Wang et al . , 2013 ) and ( 2 ) Tbx1/10-dependent activation of Collier/Olf/EBF ( Ciinte . Ebf , previously named COE ) in the ASMFs ( Stolfi et al . , 2010; Wang et al . , 2013; Razy-Krajka et al . , 2014 ) .",
"The shared clonality of heart and pharyngeal muscles and similarity of associated TF expression patterns point to a single evolutionary origin for this cardiopharyngeal ‘ontogenetic motif’ in the last common ancestor of tunicates and vertebrates ( Wang et al . , 2013 ) .",
"In M . occidentalis , we found that the segregation of heart and pharyngeal muscle fates occurs in identical fashion to that of C . intestinalis , albeit on a faster timescale .",
"At 6 hr post-fertilization ( hpf ) the first asymmetric division of the TVCs begins , with each TVC giving rise to a smaller , more ventral/medial cell and a larger , more dorsal/lateral cell .",
"At 7 . 5 hpf , the resulting STVCs also undergo an asymmetric division along the same axis ( Figure 3A ) .",
"The end result is a cluster consisting of two larger cells ( presumptive ASMFs ) lateral to four smaller cells ( presumptive heart precursors ) , on either side of the embryo ( Figure 3B ) . 10 . 7554/eLife . 03728 . 013Figure 3 . Specification of heart vs atrial siphon muscle precursors in M . occidentalis .",
"( A ) First and second asymmetric division of TVCs in M . occidentalis embryos .",
"B7 . 5 lineage was visualized by electroporation of Moocci . Mesp>H2B::GFP ( green ) .",
"The first division occurs at ∼6 hpf at 24°C , and the second division occurs at ∼7 . 5 hpf at 24°C .",
"( B ) Result of two asymmetric divisions of TVCs on left side of embryo electroporated with Moocci . Mesp>H2B::GFP ( green ) .",
"At 8 hpf at 24°C , a cluster of 6 cells derived from the TVCs is located in the ventro-lateral region of the trunk .",
"From top to bottom: 2 atrial siphon muscle founder cells ( ASMFs ) , 2 second heart precursors ( SHPs ) , and 2 first heart precursors ( FHPs ) .",
"( C ) In situ hybridization ( ISH , red ) for Moocci . Tbx1/10 + β-galactosidase immunodetection ( IHC , green ) in embryos electoporated with Moocci . Mesp>nls::lacZ .",
"Moocci . Tbx1/10 nascent transcripts are detected as two dots in the nuclei of Secondary TVCs ( STVCs ) , between the first and second asymmetric divisions .",
"( D ) ISH + IHC for Moocci . Ebf ( red ) in embryos electoporated with Moocci . Mesp>nls::lacZ ( green ) , revealing Moocci . EBF expression in ASMFs after the second asymmetric division .",
"( E ) ISH for Moocci . Ebf in a swimming larva , viewed dorsally , revealing Moocci . Ebf+ migrating atrial siphon muscle precursors ( ASMPs , arrowheads ) .",
"( F ) Lateral view of a M . occidentalis juvenile ( >100 hpf ) electroporated with Moocci . Mesp>H2B::GFP .",
"GFP + nuclei reveal contributions of B7 . 5 lineage to atrial siphon muscle-derived pharyngeal muscles ( PhMs ) and heart .",
"( G ) Diagram of the TVC divisions giving rise to the pharyngeal muscles and heart of the adult .",
"( H ) Ventral view of a M . occidentalis embryo electroporated with Moocci . Mesp>H2B::GFP , just after the first asymmetric division of the TVCs .",
"( H′ )",
"Inset from ( H ) is focused on the distance between FHPs on either side of the embryo ( mean = 57 . 6 μm , n = 9 ) .",
"( I ) Ventrolateral view of a C . intestinalis embryo electroporated with Ciinte . Mesp>H2B::GFP , right after the first asymmetric division .",
"( I′ )",
"The distance between FHPs from either side is very small ( mean = 7 . 5 μm , n = 14 ) since they contact each other at the midline to form a single cluster of cells .",
"( J ) Double ISH for Moocci . Dlx . a ( green ) and Moocci . Ebf ( red ) in a M . occidentalis larva , viewed dorsally .",
"Moocci . Dlx . a marks the siphon primordia , while Moocci . Ebf marks siphon muscle precursors .",
"Arrowheads point to atrial siphon muscle precursors ( ASMPs ) , which have migrated dorsally but do not encounter an atrial siphon primordium around which to encircle .",
"In contrast , oral siphon muscle precursors form a ring around the oral siphon primordium ( OSP ) .",
"Other Moocci . Ebf+ cells seen are neurons or neuronal precursors in the central nervous system ( cns ) .",
"( K ) Double ISH for Ciinte . Dlx . a ( green ) and Ciinte . Ebf ( red ) in a C . intestinalis larva viewed dorsolaterally .",
"Arrowhead indicates Ciinte . Ebf+ ASMPs on one side of the embryo encircling one of two bilaterally paired atrial siphon primordia ( ASP ) .",
"The same configuration of Ciinte . Ebf+ muscle precursors is seen encircling the OSP . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01310 . 7554/eLife . 03728 . 014Figure 3—figure supplement 1 . Mosaic transgene labeling of M . occidentalis suggests bilateral origin of juvenile heart .",
"( A ) Dorso-lateral view of a metamorphosing juvenile ( >100 hpf ) developing from embryo electroporated with Moocci . Mesp>H2B::GFP .",
"H2B::GFP labels nuclei of right side of and subset of atrial siphon/body-wall muscles .",
"( B ) Magnified view of boxed area in A . Mosaicism of H2B::GFP-labeling of juvenile tissues is interpreted as reflecting left/right mosaic uptake and/or retention of Moocci . Mesp>H2B::GFP plasmid during embryonic stages . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01410 . 7554/eLife . 03728 . 015Figure 3—figure supplement 2 . Delayed atrial siphon primordium specification in M . occidentalis .",
"( A ) Dorsal view of hatched larva ( 13 hpf ) stained with Alexa Fluor 546 phalloidin , showing oral siphon primordium ( OSP ) as a distinct structure , while the atrial siphon primordium ( ASP ) has not formed yet .",
"( B ) Dorsal view of metamorphosing juvenile ( >72 hpf ) stained with Alexa Fluor 546 phalloidin showing fully formed ASP as a rosette of apically constricted cells .",
"OSP is out of the plane of view .",
"( C ) Dorsal view of M . oculata hatched larva stained with Alexa Fluor 633 phalloidin , showing formation of both OSP and ASP at this stage . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01510 . 7554/eLife . 03728 . 016Figure 3—figure supplement 3 . Cardiopharyngeal development in C . intestinalis vs . M . occidentalis . Schematic diagram comparing cardiopharyngeal development in C . intestinalis ( top ) and M . occidentalis ( bottom ) embryos .",
"The B7 . 5 cell lineage , which is identical in the two species , is shown in the middle , and each progenitor type is color-coded to match the embryo diagrams .",
"Dashed line in left-most drawings indicates embryonic midline .",
"Although both FHPs and SHPs contribute to the juvenile heart , the relative contributions of their descendants have yet to be elucidated , in either Molgula or Ciona .",
"Refer to text for other details . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 016 Gene expression during heart vs pharyngeal muscle segregation is also conserved .",
"Namely , Moocci . Tbx1/10 is specifically activated in the STVCs immediately following the first asymmetric division ( Figure 3C ) , while Moocci . Ebf is specifically activated in the ASMFs immediately following the second asymmetric division ( Figure 3D ) .",
"Moocci . Ebf expression is maintained in ASMFs and their daughter cells , the atrial siphon muscle precursors ( ASMPs ) during their migration to the dorsal regions of the now swimming larva ( Figure 3E ) .",
"This step-wise progression of gene expression , cell division , and migration are virtually identical to those observed in C . intestinalis , demonstrating deep evolutionary conservation of the cardiopharyngeal ontogenetic motif in ascidians .",
"We allowed embryos electroporated with Moocci . Mesp>H2B::GFP to settle and undergo metamorphosis from the larval to the juvenile stage .",
"Visualization of H2B::GFP revealed contributions of the B7 . 5 lineage to the heart and pharyngeal muscles of juveniles ( Figure 3F ) .",
"Thus , the post-metamorphic tissues derived from B7 . 5 are identical to those previously reported for C . intestinalis and H . roretzi ( Hirano and Nishida , 1997; Stolfi et al . , 2010 ) .",
"A summary of the TVC divisions and their post-metamorphic fates is shown in Figure 3G .",
"Based on the perfectly conserved parallels to C . intestinalis , we hypothesize that specification of ASM vs heart fate in Molgula occurs through a conserved Tbx1/10-Ebf-dependent regulatory cascade .",
"In summary , the cardiopharyngeal mesoderm of Molgula and Ciona display unequivocally homologous ontogenies , as revealed by nearly identical cell divisions , cell fate choices , and gene expression patterns .",
"These data indicate that the key developmental features of the B7 . 5 lineage evolved at the base of the tunicate radiation .",
"Despite perfect conservation of B7 . 5 lineage cell divisions and cell fates between M . occidentalis and C . intestinalis , we noticed a striking species-specific difference in TVC behavior ( summarized in Figure 3 , Figure 3—figure supplement 3 ) .",
"Namely , in M . occidentalis , the TVCs appear to migrate along a more lateral path than their counterparts in C . intestinalis .",
"As a result , the TVCs on one side of the embryo do not contact those on the other side , resulting in two discrete heart precursor clusters during the tailbud stage ( Figure 3H ) .",
"In C . intestinalis , the TVCs converge towards the ventral midline prior to dividing , such that the resulting FHPs and SHPs from the two sides contact each other as soon as they are born , forming a single cluster of heart precursors ( Figure 3I; Davidson and Levine , 2003 ) .",
"However , observation of juveniles electroporated with Mocci . Mesp>H2B::GFP showed some individuals with hearts that were only half-labeled with H2B::GFP , likely due to left/right transgene mosaicism ( Figure 3—figure supplement 1 ) .",
"This indicates that heart precursors from both sides of the embryo ultimately contribute to a single heart , as they do in C . intestinalis .",
"Therefore , we assume this naturally occurring cardia bifida is only a temporary configuration of a single embryonic heart primordium .",
"This odd configuration has not been previously described in the embryos of other ascidians and may represent a Molgula-specific feature .",
"We also noticed a difference in ASM precursor behavior .",
"In C . intestinalis , migrating ASMPs from either side of the embryo migrate dorsally , forming rings around a bilateral pair of atrial siphon primordia ( ASPs ) on the corresponding side ( Stolfi et al . , 2010 ) .",
"The ASPs are placode-like rosettes of molecularly and morphologically distinct ectodermal cells that arise through a retinoic acid/Hox1- and FGF/MAPK-dependent program and are proposed to be homologous to vertebrate otic placodes ( Wada et al . , 1998; Mazet et al . , 2005; Kourakis and Smith , 2007; Sasakura et al . , 2012 ) .",
"In juveniles of C . intestinalis and other phlebobranch ascidians , there are initially two atrial siphons , one on either side of the animal , that eventually fuse into a single one at the end of the ‘1st Ascidian’ stage ( Chiba et al . , 2004; Kourakis et al . , 2010 ) .",
"In contrast , all stolidobranch ascidians initially specify a single ASP , bypassing the two-siphon fusion process ( Manni et al . , 2004; Grave , 1926; Figure 3—figure supplement 2A–C ) .",
"Indeed , we observed the formation of a single ASP in M . occidentalis late in metamorphosis and in the larvae of M . oculata ( Figure 3—figure supplement 2A , B ) .",
"This flexibility in atrial siphon development in ascidians is an example of DSD at the morphogenetic level .",
"It is believed that the dual primordium condition is ancestral and that the specification of a single primordium is a derived , Stolidobranchia-specific trait ( Kourakis et al . , 2010 ) .",
"We found that M . occidentalis ASMPs , marked by Ebf expression , migrate dorsally but remain as a cluster , unlike the characteristic rings of cells stretched around the invaginations of the ASPs , as observed in C . intestinalis ( Figure 3J , K ) .",
"On the other hand , oral siphon muscle precursors form a ring of Moocci . Ebf+ cells around the presumptive oral siphon primordium ( Figure 3J ) .",
"ISH for the siphon primordium marker Dlx . a reveals that only the oral siphon primordium is specified at this stage in M . occidentalis larvae .",
"In C . intestinalis , both oral and atrial siphon primordia expressing Dlx . a are specified by the larval stage and are encircled by Ebf+ siphon muscle precursors ( Figure 3K ) .",
"Thus , we conclude that this difference in ASMP ring formation is related to the heterochrony of atrial siphon formation between M . occidentalis and C . intestinalis .",
"Given the obvious parallels between C . intestinalis and M . occidentalis cardiopharyngeal development , we expected transcriptional regulatory mechanisms to also be highly conserved between the two species .",
"We tested this assumption by electroporating C . intestinalis reporter constructs into M . occidentalis embryos , and vice-versa .",
"We observed that a Ciinte . Mesp reporter construct ( Davidson et al . , 2005 ) , when electroporated into M . occidentalis embryos , drives relatively weak reporter gene expression in B7 . 5 with substantial leaky expression in other tissues ( Figure 4A , Figure 4—figure supplement 1 ) .",
"Conversely , the Moocci . Mesp enhancer fails to drive any reporter gene expression when electroporated into C . intestinalis embryos ( Figure 4B ) , despite recapitulating robust B7 . 5-specific expression in M . occidentalis embryos ( Figure 1D , E ) . 10 . 7554/eLife . 03728 . 017Figure 4 . Developmental system drift of Mesp regulation between C . intestinalis and M . occidentalis .",
"( A ) M . occidentalis tailbud embryo electroporated with a Ciinte . Mesp>H2B::mCherry reporter construct .",
"Weak reporter gene expression was observed in the B7 . 5 lineage and occasionally in other territories including B-line mesenchyme and tail muscle cells , and A-line neural plate derivatives .",
"( B ) C . intestinalis tailbud embryo electroporated with Moocci . Mesp>H2B::GFP reporter .",
"No fluorescence was seen in any cells , indicating complete lack of activity of Moocci . Mesp enhancer in wild-type C . intestinalis embryos .",
"( C ) In situ hybridization ( ISH ) for Moocci . Tbx6-r . a , ( D ) Moocci . Tbx6-r . b , ( E ) Moocci . Lhx3/4 . a , and ( F ) Moocci . Lhx3/4 . b in 110-cell stage embryos .",
"( G ) Double ISH in 110-cell stage embryo reveals co-expression of Moocci . Lhx3/4b ( green ) and Moocci . Tbx6-r . b ( red ) exactly in the B7 . 5 cells of M . occidentalis .",
"( G′ )",
"Magnified view of inset in ( G ) .",
"( H ) Double ISH for Moocci . Lhx3/4 . a ( green ) and ( I ) Moocci . Lhx3/4 . b ( red ) in a mid-tailbud embryo .",
"Moocci . Lhx3/4 . a but not Moocci . Lhx3/4 . b is expressed in motor ganglion neurons ( arrowhead ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01710 . 7554/eLife . 03728 . 018Figure 4—figure supplement 1 . Weak and leaky expression of Ciinte . Mesp reporter in M . occidentalis embryos . In situ hybridization for mCherry mRNA ( green ) in a M . occidentalis early gastrula stage embryo electroporated with Ciinte . Mesp>mCherry .",
"Nuclei counterstained with DAPI ( blue ) .",
"Embryo is viewed vegetally , anterior to the top .",
"Expression in B7 . 5 blastomeres ( solid arrowheads ) was observed in 42% of embryos .",
"Ectopic expression in other B-line cells ( hollow arrowhead ) was seen in 24% of embryos .",
"Ectopic expression in A-line neural precursors ( hollow double arrowhead ) was seen in 8% of embryos .",
"In contrast , in situ hybridization revealed that Moocci . Mesp reporter construct is expressed in the TVCs in 60% of electroporated M . occidentalis embryos , with 0% embryos showing any ectopic reporter gene expression ( data not shown , see Figure 1D for example ) .",
"n = 50 embryos for each construct . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01810 . 7554/eLife . 03728 . 019Figure 4—figure supplement 2 . Configuration of Lhx3/4 protein domains and Tbx6-r locus in M . occidentalis .",
"( A ) Schematic of Moocci . Lhx3/4 . b and Moocci . Lhx3/4 . a proteins .",
"LIM domains ( LD1 and LD2 ) are in yellow , and the homeodomain ( HD ) is in green .",
"Moocci . Lhx3/4 . a retains a more extensive C-terminus including a highly conserved motif ( highlighted in red ) of unknown function .",
"Alignment to Lhx3/4 orthologs from C . intestinalis ( Ciinte ) and humans ( H . sapi . ) is shown in inset .",
"( B ) Schematic representing the Tbx6-related locus in M . occidentalis , showing head-to-head configuration of Tbx6-r . a and Tbx6-r . b . Exons are represented by thick blocks .",
"Tbx6-r . a is encoded by 6 exons , while Tbx6-r . b is encoded by only 2 exons .",
"The region corresponding to the Moocci . Tbx6-r . b driver used in this study is shown in periwinkle . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 01910 . 7554/eLife . 03728 . 020Figure 4—figure supplement 3 . Divergent Molgula Lhx3/4 . b homeodomains are not predicted to have altered DNA binding specificities .",
"( A ) Alignment of homeodomains ( HDs ) from a set of Molgula Lhx3/4 family proteins , with HD recognition positions highlighted in yellow .",
"HD recognition positions are invariant while intervening sequence is highly diverged between Lhx3/4 . a and Lhx3/4 . b .",
"( B ) Logos and matrices for predicted homeodmain specificities for Moocci . Lhx3/4 . a ( top ) and Moocci . Lhx3/4 . b ( bottom ) , generated by the Homeodomain Specificity Prediction web page ( http://stormo . wustl . edu/cgi-bin/flyhd/hd_pred . cgi; Noyes et al . , 2008 ) .",
"The two predicted binding specificities are identical to one another , due to perfect conservation of the HD recognition positions . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 020 These data suggest acute DSD of transcriptional regulatory mechanisms underlying otherwise identical Mesp expression patterns .",
"More specifically , the trans-regulatory environment of the B7 . 5 blastomeres has diverged between Molgula and Ciona , and compensatory changes in the respective Mesp cis-regulatory sequences must have rendered these unable to function adequately outside of that milieu .",
"The drift of Mesp regulation between M . occidentalis and C . intestinalis prompted us to investigate potential species-specific differences in upstream trans-acting regulators .",
"In C . intestinalis , Mesp is transcriptionally activated downstream of two zygotically expressed TFs: Tbx6-related . b ( Ciinte . Tbx6-r . b ) and Lhx3/4 ( Ciinte . Lhx3/4 ) ( Satou et al . , 2004; Davidson et al . , 2005; Christiaen et al . 2009a ) .",
"Ciinte . Tbx6-r . b is directly activated in the posterior mesodermal lineages by the maternal determinant Macho-1/Zic-related . a ( Ciinte . Zic-r . a ) ( Yagi et al . , 2004 ) , while Ciinte . Lhx3/4 is activated in the vegetal pole in response to stabilized maternal Beta-catenin ( Satou et al . , 2001 ) .",
"Since the expression domains of Ciinte . Tbx6-r . b and Ciinte . Lhx3/4 overlap only in B7 . 5 , activation of Ciinte . Mesp is achieved through the synergistic interaction between Ciinte . Tbx6-r . b and Ciinte . Lhx3/4 proteins at the Ciinte . Mesp promoter in these cells ( Christiaen et al . , 2009a ) .",
"In each of the three Molgula genome assemblies , we identified two Tbx6-r genes of uncertain orthology relationships to the four Tbx6-r genes in the C . intestinalis genome ( Stolfi et al . , 2014 ) .",
"In all three Molgula genomes , the two Tbx6-r genes are arranged in head-to-head configuration with ∼2 kb separating the transcript start sites ( Figure 4—figure supplement 2B ) .",
"Despite the uncertain phylogeny of tunicate Tbx6-r genes , we named these genes Tbx6-r . a and Tbx6-r . b in Molgula .",
"In M . occidentalis , both Tbx6-r . a and Tbx6-r . b are expressed in a broad posterior swath of the pre-gastrula embryo in a manner similar to the expression pattern of Tbx6-r genes in C . intestinalis ( Figure 4C , D; Yagi et al . , 2004 ) .",
"We also identified two Lhx3/4 genes in each Molgula genome .",
"This was unexpected because no Lhx3/4 duplications have been identified in any ascidian species ( Christiaen et al . , 2009a; Kobayashi et al . , 2010 ) .",
"We named these genes Lhx3/4 . a and Lhx3/4 . b .",
"In all three Molgula species , Lhx3/4 . a is the more conserved paralog , while Lhx3/4 . b is more divergent , lacking a well-conserved C-terminal motif ( Figure 4—figure supplement 2A ) .",
"However , the amino acid sequence changes are not predicted to change the DNA-binding preference of the homeodomain ( Figure 4—figure supplement 3; Noyes et al . , 2008 ) .",
"In each of the three Molgula species’ genome assemblies , the two Lhx3/4 genes were found to be located on separate contigs .",
"In M . occidentalis , Lhx3/4 . b is expressed in vegetal cells of the early embryo , overlapping with Tbx6-r genes only in the B7 . 5 cells ( Figure 4F , G ) .",
"In contrast , Moocci . Lhx3/4 . a is not zygotically expressed in the early embryo , but is expressed in larval motor neurons ( Figure 4E , H ) , where Moocci . Lhx3/4 . b is not transcriptionally active ( Figure 4I ) .",
"In both C . intestinalis and H . roretzi , transcription of a single Lhx3/4 gene from separate proximal and distal promoters results in two distinct transcript variants .",
"Early vegetal cells express one transcript variant while motor neurons express the other transcript variant ( Wada et al . , 1995; Satou et al . , 2001; Christiaen et al . , 2009a; Kobayashi et al . , 2010 ) .",
"Since this pleiotropic expression profile appears to be strictly partitioned between the two Lhx3/4 paralogs in M . occidentalis , this constitutes a clear example of regulatory sub-functionalization following gene duplication ( Ohno , 1970; Hahn , 2009 ) .",
"In summary , M . occidentalis embryos express Tbx6-r and Lhx3/4 genes in early B7 . 5 blastomeres , where they could potentially synergize to activate Moocci . Mesp following a trans-acting logic shared with C . intestinalis .",
"The next set of experiments sought to test this potentially conserved logic .",
"We asked whether cis/trans co-evolution between Mesp and Tbx6-r and Lhx3/4 could account for incompatibility of Mesp regulatory sequences between M . occidentalis and C . intestinalis .",
"We previously demonstrated that in C . intestinalis , overexpression of Tbx6-r . b in the vegetal pole cells of the embryo , using the cis-regulatory sequences from the Foxd . b gene ( Shi and Levine , 2008 ) , results in ectopic Mesp activation in these cells due to expanded overlap of Tbx6-r . b and Lhx3/4 ( Christiaen et al . , 2009a ) .",
"Similarly , overexpression of Lhx3/4 in the posterior pole of the embryo , using the cis-regulatory sequences from Tbx6-r . b , is sufficient to cause ectopic Mesp expression throughout this territory .",
"We used these gain-of-function assays in C . intestinalis with the orthologous M . occidentalis proteins to test potential conservation of function in synergistic activation of Mesp expression .",
"Overexpression of Moocci . Tbx6-r . b but not Moocci . Tbx6-r . a was sufficient to activate ectopic Ciinte . Mesp reporter construct expression in vegetal pole cells ( Figure 5A–C ) .",
"In this regard , Moocci . Tbx6-r . b function seemed comparable to that of Ciinte . Tbx6-r . b ( Figure 5D ) .",
"Conversely , overexpression of either Lhx3/4 . b or Lhx3/4 . a from M . occidentalis in the posterior C . intestinalis embryo using the Ciinte . Tbx6-r . b promoter caused ectopic expression of both endogenous Ciinte . Mesp ( Figure 5—figure supplement",
"1 ) and Ciinte . Mesp reporter construct ( Figure 5E–G ) .",
"These experiments revealed that both M . occidentalis Lhx3/4 proteins are conserved enough to activate low levels of Ciinte . Mesp expression ( presumably by synergizing with endogenous Ciinte . Tbx6-r . b ) .",
"However , neither was as effective as Ciinte . Lhx3/4 in activating Ciinte . Mesp ( Figure 5H ) .",
"These data suggested that cis/trans or trans/trans co-evolution involving the highly divergent Moocci . Lhx3/4 . b paralog may only partially account for the incompatibility of Mesp cis-regulatory logic between the two species . 10 . 7554/eLife . 03728 . 021Figure 5 . Divergence of Mesp regulation due to changes in cis and trans .",
"( A ) Wild-type C . intestinalis gastrula embryo showing expression of Foxd . b reporter ( red , Shi and Levine , 2008 ) in vegetal pole cells and Ciinte . Mesp reporter ( green ) in the B7 . 5 cells .",
"( B ) Electroporation of Ciinte . Foxd . b>Moocci . Tbx6-r . a has no effect on Ciinte . Mesp reporter expression .",
"( C ) Electroporation of Ciinte . Foxd . b>Moocci . Tbx6-r . b results in ectopic Ciinte . Mesp reporter expression in the vegetal pole in 58% of embryos ( n = 100 ) .",
"( D ) This is indistinguishable from the effect of Ciinte . Foxd . b>Ciinte . Tbx6-r . b , which results in ectopic Ciinte . Mesp reporter activation in 62% of embryos ( n = 100 ) .",
"( E ) Wild-type C . intestinalis gastrula embryo showing expression of Tbx6-r . b reporter ( Christiaen et al . , 2009a , red ) in all B-line cells and Ciinte . Mesp reporter ( green ) in the B7 . 5 cells .",
"( F ) Electroporation of Ciinte . Tbx6-r . b>Moocci . Lhx3/4 . b results in ectopic Ciinte . Mesp reporter expression in other B-line cells in 17% of embryos ( n = 100 ) ( G ) Ciinte . Tbx6-r . b>Moocci . Lhx3/4 . a results in ectopic Ciinte . Mesp reporter expression in 29% of embryos ( n = 100 ) .",
"( H ) Ciinte . Tbx6-r . b>Ciinte . Lhx3/4 induces ectopic Ciinte . Mesp reporter expression in 88% of embryos ( n = 100 ) .",
"( I ) C . intestinalis tailbud embryo showing no expression of Moocci . Mesp reporter , as expected .",
"( J ) Moocci . Mesp reporter expression is not rescued by electroporation of Ciinte . Foxd . b>Moocci . Tbx6-r . a but ( K ) electroporation of Ciinte . Foxd . b>Moocci . Tbx6-r . b is sufficient to activate Moocci . Mesp reporter in 39% of embryos ( n = 100 ) .",
"( K ) Moocci . Mesp reporter is not transactivated upon electroporation of Foxd . b>Ciinte . Tbx6-r . b . ( L ) Wild-type C . intestinalis tailbud embryo , showing no expression of electroporated Moocci . Mesp reporter .",
"( M ) Electroporation of Ciinte . Tbx6-r . b>Moocci . Lhx3/4 . b or ( N ) Ciinte . Tbx6-r . b>Ciinte . Lhx3/4 is not sufficient to activate co-electroporated Moocci . Mesp reporter expression .",
"( O–R )",
"ISH showing Moocci . Mesp expression ( green , arrowheads ) in 110-cell stage M . occidentalis embryos .",
"Ectopic Moocci . Mesp was not observed upon overexpression of any Lhx3/4 orthologs in the posterior embryo using the Moocci . Tbx6-r . b driver ( P–R ) .",
"All gastrula stage embryos are viewed vegetally , with anterior to the top of the image .",
"All tailbud embryos are viewed laterally , anterior to the left .",
"M . occidentalis embryo nuclei were visualized by staining with DAPI ( blue ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 02110 . 7554/eLife . 03728 . 022Figure 5—figure supplement 1 . Lhx3/4 proteins from M . occidentalis are sufficient to activate ectopic expression of endogenous Mesp in C . intestinalis . In situ hybridization for Ciinte . Mesp in 110-cell stage C . intestinalis embryos .",
"( A ) Expression of Ciinte . Mesp is restricted to the B7 . 5 blastomeres ( solid arrowheads ) in control embryos .",
"( B ) Ectopic Ciinte . Mesp expression is seen in a few cells ( hollow arrowheads ) in 39% ( n = 100 ) of embryos electroporated with Ciinte . Tbx6-r . b>Moocci . Lhx3/4 . b .",
"( C ) Ectopic Ciinte . Mesp expression is seen in a few cells ( hollow arrowheads ) in 74% ( n = 100 ) of embryos electroporated with Ciinte . Tbx6-r . b>Moocci . Lhx3/4 . a .",
"( D ) Ectopic Ciinte . Mesp expression is seen in a broad posterior swath of cells in 86% ( n = 50 ) of embryos electroporated with Ciinte . Tbx6-r . b>Ciinte . Lhx3/4 .",
"All embryos viewed vegetally with the anterior to the top . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 022 Having established that M . occidentalis proteins can activate ectopic Ciinte . Mesp expression , we next asked whether these could be sufficient to activate Moocci . Mesp reporter expression in C . intestinalis embryos .",
"Indeed , Moocci . Mesp reporter activation in the vegetal pole was observed upon overexpression of Moocci . Tbx6-r . b ( Figure 5J ) , but not Moocci . Tbx6-r . a ( Figure 5I ) , consistent with their effects on Ciinte . Mesp activation .",
"Surprisingly , overexpression of Ciinte . Tbx6-r . b was not sufficient to activate Moocci . Mesp reporter ( Figure 5K ) .",
"These results hint at cis/trans co-evolution between Moocci . Mesp cis-regulatory sequences and Moocci . Tbx6-r . b , even though the latter retains enough functional information to activate Ciinte . Mesp .",
"In stark contrast to the rescue by Moocci . Tbx6-r . b , Moocci . Mesp reporter expression was never observed in C . intestinalis embryos upon overexpression of Moocci . Lhx3/4 . b or Ciinte . Lhx3/4 in the posterior pole ( Figure 5L–N ) .",
"This indicates that a functional modification extending beyond cis/trans co-evolution accounts for the lack of Moocci . Mesp reporter activity in C . intestinalis embryos .",
"The heterologous experiments in C . intestinalis suggested that Moocci . Mesp regulation could be independent of Moocci . Lhx3/4 . b .",
"To test this hypothesis , we recapitulated the experiments in M . occidentalis embryos .",
"We isolated cis-regulatory sequences from the Moocci . Tbx6-r . b gene ( Figure 4—figure supplement",
"2 ) and used this driver to overexpress Lhx3/4 coding sequences in the posterior pole of M . occidentalis embryos .",
"All Lhx3/4 proteins tested failed to induce ectopic Moocci . Mesp activation ( Figure 5O–R ) .",
"These data suggest that Moocci . Mesp is not responsive to Moocci . Lhx3/4 . b , even in permissive Moocci . Tbx6-r . b-expressing cells .",
"This differs markedly from the results of equivalent experiments in C . intestinalis , indicating major changes to the cis-regulatory logic governing identical Mesp spatiotemporal expression patterns in Ciona and Molgula .",
"Given the cis-regulatory re-wiring of Mesp regulation , we extended our analysis to search for other instances of acute DSD between Molgula and Ciona .",
"We shifted our focus to the next major cell fate decision in the B7 . 5 lineage , namely the fate choice between TVCs and ATMs .",
"In C . intestinalis , the FGF/MAPK pathway , mediated in part by MEK-dependent phosphorylation and activation of Ets . b , is both necessary and sufficient for TVC specification .",
"Inhibition of the FGF/MAPK/Ets pathway converts TVCs to ATMs , inhibiting cell migration and expression of TVC markers ( Davidson et al . , 2006; Christiaen et al . , 2008; Woznica et al . , 2012 ) .",
"Conversely , constitutive activation of the pathway induces ectopic TVCs at the expense of ATMs .",
"We ought to test the requirement of the MAPK pathway for TVC induction in M . occidentalis .",
"We treated embryos with the MEK small molecule inhibitor U0126 prior to the estimated time window of TVC specification ( ∼5 hpf at 22°C ) .",
"U0126-treated embryos displayed inhibited TVC migration relative to DMSO-treated control embryos ( Figure 6A–D ) .",
"Moreover , expression of Moocci . Foxf and Moocci . Hand-r in the TVCs was severely downregulated by U0126 treatment relative to controls ( Figure 6A–D ) .",
"These data suggest a conserved requirement for MAPK pathway activity in TVC induction and expression of important TVC regulators in M . occidentalis . 10 . 7554/eLife . 03728 . 025Figure 6 . Mutual unintelligibility of orthologous Foxf enhancers in spite of shared requirement for the MAPK pathway in TVC fate specification . In situ hybridization ( ISH , red ) + β-galactosidase immunodetection ( IHC , green ) in embryos electoporated with Moocci . Mesp>nls::lacZ and treated with DMSO ( control treatment ) , showing normal TVC induction in 85% of embryos ( n = 49 ) and TVC-localized expression of ( A ) Moocci . Foxf and ( B ) Moocci . Hand-r in 96% ( n = 25 ) and 100% ( n = 23 ) of embryos , respectively .",
"Treatment with MEK inhibitor U0126 inhibited TVC induction and migration in 66% of embryos ( n = 128 ) .",
"Treatment with U0126 also abolished TVC-specific ( C ) Moocci . Foxf expression in 91% of embryos ( n = 64 ) and ( D ) Moocci . Hand-r expression in 91% of embryos ( n = 64 ) .",
"( E ) Wild-type M . occidentalis tailbud embryo with B7 . 5 lineage labeled by electroporation of Moocci . Mesp>H2B::GFP .",
"Note two TVCs and two ATMs .",
"( F ) TVC induction and migration was enhanced upon electroporation with Moocci . Mesp>Ciinte . Ets . b::VP16 , which converted ATMs into TVC-like cells in 30% of electroporated embryos ( n = 200 ) .",
"( G ) A fragment from −1517 to −425 upstream of the Moocci . Foxf start codon ( Moocci . Foxf[TVC] ) , fused to the minimal promoter ( −230/+21 ) from Moocci . Foxf and GFP ( bpMoFF>GFP ) , recapitulates expression in the TVCs of M . occidentalis .",
"( H ) This same exact construct is silent in C . intestinalis embryos .",
"( I ) Conversely , a published TVC enhancer from the Ciinte . Foxf gene ( Beh et al . , 2007 ) fused to bpMoFF>GFP is silent in M . occidentalis embryos .",
"( J ) This same construct is strongly expressed in the TVCs of C . intestinalis .",
"All panels represent lateral views . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 02510 . 7554/eLife . 03728 . 026Figure 6—figure supplement 1 . Alignment of Foxf coding and non-coding sequences . Diagram of mVISTA alignment of M . oculata Foxf ( Moocul . Foxf ) locus aligned to orthologs in ( from top to bottom ) M . occulta , M . occidentalis , and C . intestinalis .",
"Blue-shaded peaks indicate protein-coding sequence conservation above 70% in 100-bp windows .",
"Red-shaded peaks indicate non-coding sequence conservation above 70% in 100-bp windows .",
"Sequence conservation between the Roscovite Molgulids ( M . oculata and M . occulta ) and M . occidentalis is limited to protein-coding portions , and conservation between Molgula and Ciona is limited to a narrow portion of the sequence coding for the Foxf DNA-binding domain .",
"Only the first exon of Foxf is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 02610 . 7554/eLife . 03728 . 027Figure 6—figure supplement 2 . Moocci . Ets . b and Ciinte . Ets . b are functionally very similar . C .",
"intestinalis embryos co-electroporated with Ciinte . Mesp>H2B::GFP ( green ) , Ciinte . Foxf>mCherry ( red ) , and various Ciinte . Mesp-driven perturbation constructs .",
"Percentages represent the frequency of the phenotype seen in the representative image displayed for each condition .",
"Overexpression of full-length Ets . b proteins from both C . intestinalis and M . occidentalis results ectopic Foxf+ TVCs at the expense of ATMs .",
"The same conversion of ATMs to TVCs is seen for overexpression of Ets . b::VP16 fusions .",
"Conversely , Ets . b::WRPW fusions repress TVC induction and Ciinte . Foxf reporter expression .",
"As expected from the lack of sequence divergence predicted to affect DNA-binding specificities , Ets . b proteins from both species appear to be equally suited to trans-activating Ciinte . Foxf in C . intestinalis embryos .",
"n = 100 for all conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 02710 . 7554/eLife . 03728 . 028Figure 6—figure supplement 3 . Ets . b proteins are not sufficient to transactivate Moocci . Foxf reporter construct activation in C . intestinalis embryos . C .",
"intestinalis embryos co-electroporated with Ciinte . Mesp>H2B::GFP ( green ) , Moocci . Foxf>mCherry ( red ) , and various Ciinte . Mesp-driven perturbation constructs .",
"Activation of Moocci . Foxf reporter construct was impervious in C . intestinalis TVCs even upon overexpression of Ets . b full-length or VP16 fusions from either C . intestinalis or M . occidentalis .",
"n = 100 for all conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 028 We also tested the potential involvement of Ets factors in mediating the MAPK-dependent activation of the TVC program .",
"To do so , we expressed a constitutively active fusion of the Ciinte . Ets . b DNA-binding domain to a VP16 transactivation domain ( Ciinte . Ets . b::VP16 , Davidson et al . , 2006 ) , in M . occidentalis B7 . 5 cells using the Moocci . Mesp driver .",
"Electroporation of Moocci . Mesp>Ciinte . Ets . b::VP16 induced ectopic TVCs at the expense of ATMs , in 31% of transfected embryos ( Figure 6E , F ) .",
"In sum , our results are consistent with a conserved function for MAPK-activated Ets . b upstream of the cardiopharyngeal gene regulatory network in M . occidentalis .",
"Given the conservation of the MAPK/Ets pathway in ascidian TVC specification , we reasoned that Foxf cis-regulatory sequences should be compatible in cross-species assays .",
"We identified a sequence upstream of the Moocci . Foxf gene that is sufficient to drive TVC-specific expression in M . occidentalis ( Figure 6G , Figure 6—figure supplement 1 ) .",
"However , this sequence is completely non-functional when tested in C . intestinalis ( Figure 6H ) .",
"Likewise , a previously identified Ciinte . Foxf TVC enhancer ( Figure 6J; Beh et al . , 2007 ) is incapable of driving TVC-specific expression in M . occidentalis embryos ( Figure 6I ) .",
"Thus , to borrow a term from the field of linguistics , these enhancers are said to be ‘mutually unintelligible’ , since the enhancer from M . occidentalis cannot be interpreted by embryos of C . intestinalis , and the enhancer from C . intestinalis cannot be interpreted by M . occidentalis .",
"This mutual unintelligibility is due to differences between the enhancers and not the promoters , since we used the same Moocci . Foxf basal promoter in both constructs .",
"Although the Ciinte . Foxf enhancer is able to interact with and potentiate transcription from Moocci . Foxf promoter , nucleotide sequence is poorly conserved around Foxf proximal promoter regions , even between M . occidentalis and M . occulta/oculata ( Figure 6—figure supplement 1 ) .",
"We asked whether the observed unintelligibility was indicative of changes in Ets . b protein properties between C . intestinalis and M . occidentalis embryo .",
"At first glance , differences in DNA binding domain amino acid sequences between the Ets . b orthologs do not predict a change in binding site preference ( Donaldson et al . , 1996 ) .",
"Indeed , full-length Moocci . Ets . b and Moocci . Ets . b::VP16 were just as effective as their C . intestinalis counterparts to induce ectopic TVCs when expressed in the C . intestinalis B7 . 5 lineage ( Figure 6—figure supplement 2 ) .",
"Moreover , C . intestinalis TVC induction was abolished by expression of the constitutive repressor form ( Moocci . Ets . b::WRPW ) .",
"However , neither full-length Moocci . Ets . b nor Moocci . Ets . b::VP16 was sufficient to induce activation of Moocci . Foxf reporter in C . intestinalis embryos ( Figure 6—figure supplement 3 ) .",
"These surprising results suggest that , even though TVC induction and TVC-specific Foxf expression depends upon Ets . b-mediated MAPK activity in M . occidentalis embryos , there are profound species-specific differences in the regulatory mechanisms acting in parallel to MAPK/Ets . b upstream of Foxf , reflected in the mutual unintelligibility of the orthologous enhancers .",
"Since investigation into both Mesp and Foxf regulation revealed divergent cis-regulatory logic underlying identical gene expression patterns , we asked whether these examples constituted a general trend between C . intestinalis and M . occidentalis .",
"Testing several other candidate enhancers , we identified other examples of cis-regulatory unintelligibility .",
"For instance , Moocci . Tbx6-r . b and Mooci . Hand-r upstream sequences recapitulated their endogenous activity in M . occidentalis embryos ( Figure 7B , E ) , but these sequences were conspicuously silent in the B7 . 5 lineage in C . intestinalis embryos ( Figure 7C , F ) .",
"Notably , activity in other tissues seemed normal: Moocci . Tbx6-r . b reporter expression was robust and precise in C . intestinalis primary tail muscle cells ( Figure 7A , C ) and Moocci . Hand-r reporter recapitulated expression in the C . intestinalis A7 . 6 and anterior endoderm lineages ( Figure 7D , F ) .",
"In the case of Hand-r , this partial intelligibility is due to the modular organization of its cis-regulatory sequences , which in C . intestinalis is comprised of discrete enhancers controlling expression in each of the distinct domains ( Davidson and Levine , 2003; Woznica et al . , 2012 ) . 10 . 7554/eLife . 03728 . 023Figure 7 . Cross-species reporter construct assays reveal multiple cases of cis-regulatory unintelligibility .",
"( A ) C . intestinalis embryo electroporated with Ciinte . Tbx6-r . b>GFP reporter construct ( Christiaen et al . , 2009a ) , which drives GFP expression in tail muscles and the B7 . 5 lineage cells ( arrowheads ) , recapitulating endogenous Ciinte . Tbx6-r . b expression .",
"( B ) M . occidentalis embryo electroporated with Moocci . Tbx6-r . b>GFP reporter construct , which recapitulates expression in tail muscle cells including B7 . 5 lineage cells ( arrowheads ) .",
"( C ) C . intestinalis embryo electroporated with Moocci . Tbx6-r . b>GFP , which drives expression in B-line tail muscle and mesenchyme cells but is excluded from the B7 . 5 lineage .",
"( D ) C . intestinalis embryo electroporated with Ciinte . Hand-r>H2B::GFP reporter ( Davidson and Levine , 2003 ) , which drives H2B::GFP expression in anterior endoderm , A7 . 6 lineage , and TVCs ( arrowheads ) , recapitulating endogenous Ciinte . Hand-r expression .",
"( E ) M . occidentalis embryo electroporated with Moccci . Hand-r>H2B::GFP construct , which recapitulates the same expression pattern .",
"( F ) C . intestinalis embryo electroporated with Moocci . Hand-r>H2B::GFP , which drives expression in endoderm and A7 . 6 lineage , but not in B7 . 5 .",
"( G ) M . occidentalis embryo electroporated with Ciinte . Ebf neuron-specific YFP ( green ) and H2B::mCherry ( red ) reporter constructs electroporated ( Stolfi and Levine , 2011 ) , which drive very weak expression in a limited subset of motor ganglion neurons ( green and red ) .",
"( H ) C . intestinalis embryo electroporated with a Moocci . Ebf>YFP reporter , which drives robust reporter gene expression in several brain , motor ganglion , and nerve cord neurons .",
"( I ) C . intestinalis embryos electroporated with Ciinte . Sox1/2/3 ( left ) and Moocci . Sox1/2/3 ( right ) H2B::mCherry reporter constructs , both of which recapitulate Sox1/2/3 expression in ectoderm .",
"Panels A–F are lateral views of tailbud embryos , panels G is a dorsal view of a tailbud embryo , panel H is a dorso-lateral view of a hatched larva , and panel I shows vegetal views of mid-gastrula stage embryos .",
"Anterior is to the right , except in Panel I , in which anterior is to the top . DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 02310 . 7554/eLife . 03728 . 024Figure 7—figure supplement 1 . Asymmetric intelligibility between Molgula and Ciona Hand-r TVC enhancers .",
"( A ) β-galactosidase immunodetection on M . occidentalis late tailbud embryo electroporated with Ciinte . Hand-r>nls::lacZ .",
", showing reporter gene expression in TVC descendents ( heart precursors and atrial siphon muscle founder cells ) on one side of the embryo .",
"( B ) M . occidentalis embryo electroporated with Moocul . Hand-r>H2B::GFP , showing recapitulation of endogenous Moocci . Hand-r expression in endoderm , A7 . 6 lineage , and TVCs .",
"( C ) C . intestinalis embryo electroporated with Moocul . Hand-r>H2B::GFP showing slight expression in A7 . 6 lineage and B-line mesenchyme cells , but no expression in TVCs ( hollow arrowheads ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03728 . 024 Curiously , we found that the Ciinte . Hand-r reporter can drive reporter gene expression in M . occidentalis TVCs ( Figure 7—figure supplement 1 ) .",
"Thus , unlike the case of Foxf , there is an asymmetric intelligibility of Hand-r TVC enhancers between M . occidentalis and C . intestinalis .",
"Moreover , a M . oculata Hand-r TVC enhancer is functional in M . occidentalis but not in C . intestinalis ( Figure 7—figure supplement 1 ) .",
"Taken together , these data suggest that differences in enhancer logic may have accumulated over the course of the deep evolutionary history between Molgula and Ciona but not between M . occidentalis and M . oculata , and that some enhancers may have evolved asymmetrically in the two branches , retaining greater pan-ascidian ‘intelligibility’ in one or the other .",
"Finally , we found that acute DSD was not restricted to cis-regulatory sequences controlling gene expression in the B7 . 5 lineage .",
"For instance , a Ciinte . Ebf driver that has been shown to recapitulate neuronal Ebf expression ( Stolfi and Levine , 2011 ) was only very weakly active in a subset of M . occidentalis motor ganglion neurons ( Figure 7G ) .",
"However , the orthologous sequence from M . occidentalis recapitulated robust reporter gene expression in several C . intestinalis Ebf+ neurons throughout the central nervous system ( Figure 7H ) .",
"In contrast , expression of a Moocci . Sox1/2/3 reporter construct in the ectoderm of C . intestinalis embryos was found to be indistinguishable from that of the C . intestinalis ortholog ( Figure 7I ) , suggesting that some cis-regulatory sequences may retain mutual intelligibility between Molgula and Ciona without sequence conservation , as is the case for various Otx enhancers between C . intestinalis and H . roretzi ( Oda-Ishii et al . , 2005 ) .",
"In sum , our preliminary cross-species enhancer activity screen suggests that DSD was rampant during ascidian evolution , resulting in mutual unintelligibility of cis-regulatory mechanisms underlying otherwise identical expression patterns of orthologous genes ."
],
[
"By sequencing the genomes of three Molgula species and adapting the electroporation protocol for routine transfection of M . occidentalis embryos , we have further expanded the reach of ascidian molecular genetics .",
"We have demonstrated the usefulness of M . occidentalis as a species amenable to medium-throughput molecular perturbations and assays .",
"While Ciona/Halocynthia comparisons have yielded fundamental insights into chordate development , the lack of a reliable electroporation protocol for transfection of Halocynthia eggs has hampered comparative studies on a larger scale .",
"On the other hand , eggs from species such as Phallusia mammilata can be electroporated , but this species is much more closely related to Ciona .",
"Thus , Molgula species may significantly contribute to understanding the extreme conservation of ascidian embryogenesis in spite of vast genetic differences and deep evolutionary histories .",
"We have shown that de novo sequencing and assembly of Molgulid genomes using next-generation technologies is relatively efficient and cost-effective , largely due to their compact nature .",
"The sequence information obtained can be used to quickly generate a comprehensive set of molecular tools .",
"We predict that a large number of ascidian genomes will be as compact and efficiently sequenced and assembled as these , unlocking the experimental potential of many ascidian species representing several different families ( Tsagkogeorga et al . , 2009 ) .",
"In our survey of B7 . 5 development , we observed that the cell division events and cell fate choices in the lineage are perfectly conserved between Ciona and Molgula , demonstrating the deep evolutionary origins of the cardiopharyngeal ontogenetic motif in tunicates .",
"Regulatory gene expression patterns in the lineage are also nearly identical , the only potential difference being Nk4 , which was not confidently detected in M . occidentalis TVCs .",
"NK4 proteins are intimately linked to cardiac development throughout bilateria ( Akazawa and Komuro , 2005 ) .",
"Moreover , Nk4 is expressed in C . intestinalis TVCs where it functions as a regulator of heart precursor fate ( Davidson and Levine , 2003; Wang et al . , 2013 ) .",
"However , there are distinct early and late phases of Nk4 expression in C . intestinalis heart development .",
"Nk4 is expressed in the TVCs during the mid-tailbud stage , downregulated during the late tailbud and larval stages , and only re-activated in the differentiating heart of metamorphosing juveniles ( Davidson and Levine , 2003 ) .",
"This points to distinct early ( pre-metamorphic ) and late ( post-metamorphic ) roles for Nk4 in C . intestinalis .",
"In C . intestinalis , early Nk4 expression does not appear to be sufficient for heart fate , as it is expressed in the TVCs prior to the segregation of heart and pharyngeal muscle fates ( Davidson and Levine , 2003; Beh et al . , 2007; Christiaen et al . , 2008 , 2010; Ragkousi et al . , 2011; Wang et al . , 2013 ) .",
"Second , this early expression may not be strictly required for heart fate either , as the first heart precursors ( FHPs ) do not adopt a pharyngeal muscle fate upon dominant-negative Nk4 overexpression like the second heart precursor ( SHPs ) do ( Wang et al . , 2013 ) .",
"Therefore , Nk4 acts as partially redundant mechanism to specify heart precursors in C . intestinalis , complementing parallel mechanisms of heart/pharyngeal muscle cell fate choice ( e . g . , activation of Tbx1/10 in the STVCs and Ebf in the ASMFs ) .",
"It is possible that Moocci . Nk4 is only activated during metamorphosis , an early role for it being dispensable in M . occidentalis TVCs .",
"This might be the case if the complementary mechanism for pharyngeal muscle fate choice is more robust than in Ciona .",
"In this way , the divergence of parallel regulatory mechanisms may have contributed to the loss of a key regulator like Nk4 from the early cardiopharyngeal mesoderm GRN of Molgula .",
"A much more detailed investigation will be required to determine a possible role for Nk4 in the Molgula heart development .",
"M . occidentalis and C . intestinalis also display differences in certain morphogenetic behaviors of B7 . 5 derivatives ( summarized in Figure 3 , Figure 3—figure supplement 3 ) .",
"First , M . occidentalis TVCs migrate more laterally and do not meet at the ventral midline , resulting in a temporary condition of cardia bifida .",
"A similar condition can be induced in C . intestinalis embryos by perturbing endoderm development ( Ragkousi et al . , 2011 ) .",
"Since M . occidentalis and C . intestinalis tailbud embryos look quite different in their shape , it will be interesting to assess endoderm formation and its relationship with TVC migration trajectory in Molgula .",
"Secondly , the ASM precursors in M . occidentalis migrate away from the heart progenitors but do not encounter atrial siphon primordia to encircle in the dorsal part of the embryo .",
"This process is uncoupled in C . intestinalis Hox1 mutants , which fail to specify the ASPs .",
"In these mutant larvae , the ASMPs still appear to migrate dorsally but do not form rings of muscle ( Sasakura et al . , 2012 ) .",
"It remains to be determined how the M . occidentalis ASMPs migrate to the general vicinity of the future atrial siphon opening and remain localized there until the later specification and differentiation of the siphon .",
"These two major morphogenetic differences prompted us to rethink our assumptions regarding the interdependence of certain processes in ascidian cardiopharyngeal development , as identified in C . intestinalis .",
"For instance , one might assume that the convergence of Ciona TVCs at the ventral midline to be important for the asymmetric cell divisions and fate choices segregating heart from ASM precursors .",
"Similarly , one might assume the atrial siphon primordia to be the source of a cue required for the dorsal migration of ASMPs .",
"In M . occidentalis , these processes have been naturally uncoupled .",
"These observations serve to emphasize that sampling diverse taxa provides insights into the modularity of specific developmental processes .",
"From our initial survey of a handful of enhancers from C . intestinalis , M . occidentalis , and M . oculata , we encountered several instances of either mutual unintelligibility , or asymmetric intelligibility of enhancers .",
"Qualitative cross-species assays of M . occidentalis and C . intestinalis cis-regulatory sequences indicated that specific orthologous pairs of enhancers may be mutually intelligible ( e . g . , Sox1/2/3 ectodermal enhancer ) while others are mutually unintelligible ( e . g . , Foxf TVC enhancer ) .",
"Some orthologous gene pairs appear to be regulated by a mix of intelligible and unintelligible tissue-specific enhancers ( e . g . , Hand-r ) .",
"The observation that cross-species intelligibility due to DSD occurs in a tissue-specific manner is consistent with the widely documented modularity of cis-regulatory elements controlling complex gene expression ( Arnone and Davidson , 1997; Levine , 2010 ) .",
"These results add to the mounting evidence suggesting that acute and pervasive DSD may have occurred over the course of ascidian evolution , obfuscated by the identical cell lineages and highly conserved gene expression patterns of ascidian embryos ( Swalla , 2004 ) .",
"The multiple examples of cis-regulatory unintelligibility we identified were rather unexpected given",
"( a ) the extremely conserved pattern of expression of orthologous genes from Molgula and Ciona ( Takada et al . , 2002 ) and",
"( b ) previous observations of mutual intelligibility of enhancers between C . intestinalis and H . roretzi ( e . g . , Otx ) , and between C . intestinalis and the more closely related C . savignyi ( Johnson et al . , 2004; Brown et al . , 2007 ) .",
"Large-scale , quantitative cross-species assays and detailed GRN studies will illuminate factors that may contribute to conservation or divergence in regulatory mechanisms .",
"Cis-regulatory unintelligibility can arise due to different underlying factors .",
"We have presented evidence that , in the case of Mesp , this results in part from a change in an upstream activating input .",
"In C . intestinalis , Lhx3/4 is an activator of Mesp , while in M . occidentalis , Mesp activation appears to be independent of Lhx3/4 . b , despite the conserved expression pattern of this gene in vegetal pole cells .",
"Regulation of Mesp in other ascidian species will need to be investigated in order to establish whether Lhx3/4-dependent activation of Mesp in ascidians is ancestral or derived .",
"On the other hand , both C . intestinalis and M . occidentalis have retained a conserved role for Tbx6-r in regulating Mesp , a regulatory connection that may have arisen in the last common ancestor of tunicates and vertebrates ( Yasuhiko et al . , 2006; Davidson et al . , 2005 ) .",
"In hindsight , it appears the weak expression of Ciinte . Mesp reporter constructs observed in the B7 . 5 cells of M . occidentalis embryos is not indicative of GRN conservation .",
"This expression instead appears to be an artifact attributed in part to the conserved , overlapping expression profiles of Moocci . Tbx6-r . b and Moocci . Lhx3/4 . b , even if this overlap is not instructive for endogenous Moocci . Mesp activation .",
"We can only speculate what is the connection , if any , between the re-wiring of Mesp regulation and the Molgula-specific duplication and sub-functionalization of Lhx3/4 .",
"Interestingly , in C . intestinalis and H . roretzi , the early and late functions of Lhx3/4 are partitioned between two transcript variants of the same locus , not between different loci .",
"This substitution of alternate isoforms with gene duplicates has been documented for a few genes in teleost fish ( Altschmied et al . , 2002; Yu et al . , 2003 ) .",
"Although allowing for some degree of sub-functionalization of protein function , alternative transcription/splicing does not completely relax the constraints imposed by pleiotropy .",
"The Lhx3/4 variants in C . intestinalis and H . roretzi still share the bulk of their amino acid sequence , differing only at the portion of the N-terminus encoded by the alternate first exons ( Christiaen et al . , 2009a; Kobayashi et al . , 2010 ) .",
"In Molgula spp .",
"on the other hand , the two paralogs are considerably diverged from one another throughout their coding sequences , even though the divergence does not appear to alter DNA sequence-binding specificity .",
"Pleiotropy has been hypothesized to both promote and suppress evolution , more specifically DSD ( Hansen , 2003 ) .",
"Investigating the full breadth of Lhx3/4 functions in multiple ascidian species will be required in order to determine the relationship between Lhx3/4 duplication and re-wiring of Mesp regulation .",
"The mutual unintelligibility of orthologous Foxf enhancers was unexpected , given that both M . occidentalis and C . intestinalis depend upon a shared MAPK/Ets-based switch for TVC fate induction .",
"This discrepancy could be due to species-specific requirements for additional activating inputs , and/or silencing by species-specific repressors .",
"Our observation that cis-trans complementation with Moocci . Ets . b::VP16 does not rescue the activity of the Moocci . Foxf enhancer in C . intestinalis embryos suggests that the latter possibility may be the case .",
"It is thought that co-evolution between an enhancer and its trans environment is critical in order for it to faithfully direct a conserved pattern of gene expression , even if the overarching GRN is broadly conserved ( Landry et al . , 2005 ) .",
"Therefore , the incompatibility of B7 . 5 lineage-specific enhancers between M . occidentalis and C . intestinalis may reflect key differences in the trans-regulatory milieus of this lineage in the two species .",
"Future comparative expression profiling of M . occidentalis and C . intestinalis B7 . 5 lineage cells will shed light on the nature of these differences .",
"Even though the word ‘drift’ implies a greater role for chance over natural selection , adaptive processes are very likely to play a role in DSD .",
"True and Haag hypothesized as much in their seminal introduction to the concept of DSD , predicting that cis/trans co-evolution and other compensatory changes should result in enhancers resembling species-specific ‘Rube Goldberg machines’ that give superficially similar outputs but are functionally non-interchangeable ( True and Haag , 2001 ) .",
"Models incorporating selection , pleiotropy , and compensation ( SPC ) have refined this idea by ascribing ‘drift’ in one biological process to adaptation in another ( Landry et al . , 2005; Johnson and Porter , 2007; Pavlicev and Wagner , 2012; Martinez et al . , 2014 ) .",
"For instance , if the same pleiotropic gene is involved in both processes , any mutations in that gene that are adaptive for the process under selection will require some compensatory changes simply to maintain the same output of the other process .",
"This may occur with or without deleterious intermediate states , with the latter occurring through a process termed ‘pseudocompensation’ ( Haag , 2007 ) .",
"We speculate that a high frequency of compensatory changes , required for the rapidly evolving ascidians to accommodate the constraints imposed by their invariant embryonic cell lineages and highly compact genomes , has given rise to a preponderance of cross-species cis-regulatory unintelligibility , following the DSD/SPC model .",
"This perfect storm of intrinsic factors may be the key to explaining the dichotomy observed between highly conserved embryos and divergent cis-regulatory structure/function in ascidians ."
],
[
"Genomic DNA was phenol/chloroform extracted from dissected gonads of Molgula occulta ( Kupffer ) and Molgula oculata ( Forbes ) adults from Roscoff , France , and a Molgula occidentalis ( Traustedt ) adult from Panacea , Florida , USA ( Gulf Specimen Marine Lab ) .",
"Genomic DNA was sheared using an M220 Focused-ultrasonicator ( Covaris , Woburn , MA ) .",
"Sequencing libraries were prepared using KAPA HiFi Library Preparation Kit ( KAPA Biosystems , Wilmington , MA ) indexed with DNA barcoded adapters ( BioO , Austin , TX ) .",
"Size selection was performed using Agencourt ( Beckman–Coulter , Brea , CA ) AMPure XP purification beads ( 300–400 bp fragments ) , or Sage Science ( Beverly , MA ) Pippin Prep ( 650–750 bp and 875–975 bp fragments ) .",
"For M . occulta and M . occidentalis libraries , 6 PCR cycles were used .",
"For M . oculata libraries , 8 cycles were used for the 300–400 bp library , and 10 cycles were used for the 650–750 and 875–975 bp libraries .",
"Libraries of different species but same insert size ranges were multiplexed for sequencing in three 100 × 100 PE lanes on a HiSeq 2000 sequencing system ( Illumina , San Diego , CA ) at the Genomics Sequencing Core Facility , Center for Genomics and Systems Biology at New York University ( New York , NY ) .",
"Thus , each lane was dedicated to a mix of species , specifically barcoded libraries of a given insert size range .",
"Raw sequencing reads were deposited as a BioProject at NCBI under the ID# PRJNA253689 .",
"All genomes were assembled on Michigan State University High Performance Computing Cluster ( http://contact . icer . msu . edu ) .",
"Prior to assembly , read quality was examined using FastQC v0 . 10 . 1 ( Babraham Bioinformatics , 2014 ) .",
"Reads were then quality trimmed on both the 5′ and 3′ end using seqtk trimfq ( https://github . com/lh3/seqtk ) which uses Phred algorithm to determine the quality of a given base pair .",
"Seqtk trimfq only trims bases , so no reads were discarded .",
"Each library per species was then abundance filtered using 3-pass digital normalization to remove repetitive and erroneous reads ( Brown et al . , 2012; Schwarz et al . , 2013; Howe et al . , 2014 ) .",
"Genome assembly was done using Velvet v1 . 2 . 08 ( Zerbino and Birney , 2008 ) with k-mer overlap length ( ‘k’ ) ranging from 19 to 69 and scaffolding was done by Velvet , by default .",
"Velvet does not produce separate files for contigs and scaffolds; because Velvet scaffolded conservatively , contigs dominated the assemblies so we refer to both contigs and scaffolds as contigs .",
"CEGMA scores were then computed to evaluate genome completeness ( Parra et al . , 2007 ) .",
"The latest versions of three species' genome assemblies have been deposited on the ANISEED ( Ascidian Network for In Situ Expression and Embryological Data ) database for browsing and BLAST searching at http://www . aniseed . cnrs . fr/ ( Tassy et al . , 2010 ) .",
"Scripts for genome assembly and CEGMA analysis can be found in the following github repository: https://github . com/elijahlowe/molgula_genome_assemblies . git .",
"Putative orthologs of Ciona intestinalis ( Linnaeus ) and Halocynthia roretzi ( Drasche ) protein-coding genes were initially identified by TBLASTN .",
"Identified sequences were aligned to each other to support orthology relationships within Molgula , and then queried by BLASTP against the NCBI non-redundant protein sequence database to further support orthology relationships to known tunicate and vertebrate proteins .",
"cDNAs were cloned by RT-PCR , or by 5′ and/or 3′ RACE ( SMARTer RACE kit , Clontech , Mountain View , CA ) .",
"The template cDNA libraries were prepared from total RNA extracted from embryos of various stages .",
"Upstream regulatory sequences were cloned by PCR from genomic DNA .",
"For non-RACE PCRs , we used Phusion high-fidelity polymerase ( New England Biolabs , Ipswich , MA ) .",
"Refer to Supplementary file 1 for detailed sequence information .",
"Genes are named according to the proposed unified nomenclature system for tunicate genetic elements ( Stolfi et al . , 2014 ) .",
"Refer to Supplementary file 2 for table with previously used gene names and the corresponding new proposed gene symbols and names .",
"M . occidentalis gravid adults were obtained from Gulf Specimen Marine Lab between March and December .",
"Gravid adults were rare between the winter months from December to March .",
"Animals were kept in a small aquarium with circulating artificial sea water at 24°C until needed .",
"All subsequent manipulations using eggs and embryos were performed between 20°C and 24°C , though 24°C appeared to be the optimal temperature for both dechorionation and embryonic development .",
"Individual gonads were dissected in filtered TAPS-buffered artificial sea water ( T-ASW ) to release eggs and sperm .",
"Eggs were allowed to undergo germinal vesicle breakdown for 10 min and then pooled along with sperm to fertilize for 5 min .",
"Eggs were rinsed thoroughly and dechorionated in solution ( 168 μl 10N NaOH , 1 . 2 ml 2 . 5% pronase E , 0 . 3 g sodium thioglycolate in 40 ml T-ASW ) with constant pipetting for 8–10 min ( modified after Christiaen et al . , 2009c ) .",
"Viewing under a high-powered dissecting microscope was often necessary to verify removal of the thin , translucent chorion that becomes wrapped tightly around the egg upon immersion in dechorionation solution .",
"After confirming chorion removal , eggs were quickly rinsed by the passage through multiple dishes of T-ASW .",
"Electroporation conditions and procedures were identical to those previously described for C . intestinalis ( Christiaen et al . , 2009b ) .",
"Unless otherwise specifically noted , all fluorescent reporter genes were fused at the N-terminus to unc-76 tags ( Dynes and Ngai , 1998 ) , to ensure distribution throughout the whole cytosol , electroporated typically at ∼50 μg to 90 μg per 700 μl electroporation solution , while nuclei were visualized with histone2B::fluorescent protein constructs electroporated at ∼5 μg to 25 μg per electroporation unless otherwise specifically noted .",
"Perturbation constructs were electroporated at ∼35 μg to 100 μg per electroporation .",
"To image juveniles , larvae were allowed to settle and metamorphose in plastic Petri dishes or on glass coverslips in Petri dishes for several days with frequent changes of T-ASW .",
"Fertilization of M . occulta eggs was performed as previously described ( Swalla and Jeffery , 1990 ) .",
"Embryos were dechorionated and electroporated as described for M . occidentalis embryos , using a custom-built electroporation machine ( Zeller et al . , 2006 ) .",
"C . intestinalis ( Type A ‘robusta’ ) adults were collected in San Diego , CA ( M-Rep ) .",
"Embryos were treated in 10 μM U0126 ( Cell Signaling Technology , Danvers , MA ) in T-ASW at least 30 min prior to the targeted MAPK signaling event .",
"Fluorescent in situ hybridization assays were performed as previously described , with modifications ( Beh et al . , 2007; Ikuta and Saiga , 2007; Christiaen et al . , 2009d ) .",
"Embryos were fixed in MEM-3 . 2% to 4% paraformaldehyde buffers for at least 2 hr .",
"Pre-hybridization proteinase K concentrations ranged from 0 . 25 μg/ml ( 110-cell stage ) to 1 μg/ml ( tailbud ) and 5 μg/ml ( larvae ) for Molgula spp .",
"embryos .",
"The Ciinte . Ebf probe was synthesized from template plasmid from the C . intestinalis Gene Collection Release 1 ( Satou et al . , 2002 ) .",
"See Supplementary file 1 for more detailed probe template sequence information .",
"Embryos were counterstained with DAPI and/or Phalloidin conjugates ( LifeTechnologies/Thermo Fisher Scientific , Waltham , MA ) .",
"Images were taken using a combination of different Leica Microsystems ( Wetzlar , Germany ) microscopes: TCS SP8 X confocal microscope , TCS SP5 confocal microscope , and DM2500 epifluorescence microscope ."
]
] | [
"Ascidians present a striking dichotomy between conserved phenotypes and divergent genomes: embryonic cell lineages and gene expression patterns are conserved between distantly related species .",
"Much research has focused on Ciona or Halocynthia spp .",
"but development in other ascidians remains poorly characterized .",
"In this study , we surveyed the multipotent myogenic B7 . 5 lineage in Molgula spp .",
"Comparisons to the homologous lineage in Ciona revealed identical cell division and fate specification events that result in segregation of larval , cardiac , and pharyngeal muscle progenitors .",
"Moreover , the expression patterns of key regulators are conserved , but cross-species transgenic assays uncovered incompatibility , or ‘unintelligibility’ , of orthologous cis-regulatory sequences between Molgula and Ciona .",
"These sequences drive identical expression patterns that are not recapitulated in cross-species assays .",
"We show that this unintelligibility is likely due to changes in both cis- and trans-acting elements , hinting at widespread and frequent turnover of regulatory mechanisms underlying otherwise conserved aspects of ascidian embryogenesis ."
] | [
"When two species have features that look similar , this may be because the features arise by the same processes during development .",
"Other features may look similar yet develop by different mechanisms .",
"‘Developmental system drift’ refers to the process where a physical feature remains unaltered during evolution , but the underlying pathway that controls its development is changed .",
"However , to date , there have been only a few experimental studies that support this idea .",
"Ascidians—also commonly known as sea squirts—are vase-like marine creatures , which start off as tadpole-like larvae that swim around until they find a place to settle down and attach themselves .",
"Once attached , the sea squirts lose the ability to swim and start feeding , typically by filtering material out of the seawater .",
"Sea squirts and their close relatives are the invertebrates ( animals without backbones ) that are most closely related to all vertebrates ( animals with backbones ) , including humans .",
"Furthermore , although different species of sea squirt have almost identical embryos , their genomes are very different .",
"Stolfi et al . have now studied whether developmental system drift may have occurred during the evolution of ascidians , by analyzing different species of sea squirt named Molgula and Ciona .",
"Stolfi et al . compared the genomes of Molgula and Ciona and studied the expression of genes in the cells that give rise to the heart and the muscles of the head .",
"As an embryo develops , specific genes are switched on or off , and these patterns of gene activation were broadly identical in the two species of sea squirt examined .",
"Enhancers are sequences of DNA that control when and how a gene is switched on .",
"Given the similarities between the development of heart and head muscle cells in the different sea squirts , Stolfi et al . looked to see if the mechanisms of gene expression , and therefore the enhancers , were also conserved .",
"Unexpectedly , this was not the case .",
"When enhancers from Molgula were introduced into Ciona ( and vice versa ) , these sequences were unable to switch on gene expression—thus enhancers from one sea squirt species could not function in the other .",
"Stolfi et al . conclude that the developmental systems may have drifted considerably during evolution of the sea squirts , in spite of their nearly identical embryos .",
"This reinforces the view that different paths can lead to the formation of similar physical features ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"genetics and genomics"
] | Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates | elife-00348-v1 | [
[
"Short contiguous regions of non-methylated DNA are found associated with most human and mouse gene promoters , where they create a transcriptionally permissive chromatin environment ( Blackledge et al . , 2010; Thomson et al . , 2010; Blackledge and Klose , 2011; Deaton and Bird , 2011; Jones , 2012 ) that opposes the repressive effects of DNA methylation ( Klose and Bird , 2006; Weber and Schubeler , 2007 ) .",
"In non-methylated regions , CpG dinucleotide frequency is elevated compared to surrounding sequence ( Bird et al . , 1985; Bird , 1987 ) .",
"This is due to accelerated methyl-cytosine mutability , which over evolutionary time leads to a reduction in CpG dinucleotide frequency in densely methylated regions of the genome , while CpG frequency is preserved in non-methylated regions ( Coulondre et al . , 1978; Bird , 1980 ) .",
"Taking advantage of the methylation-dependent variations in nucleotide frequency observed in mammals , algorithms were developed to predict non-methylated regions of DNA based primarily on elevated local G+C content and CpG dinucleotide frequency ( Gardiner-Garden and Frommer , 1987; Takai and Jones , 2002 ) .",
"For more than two decades , CpG island ( CGI ) predictions ( and other nucleotide-based analyses; Saxonov et al . , 2006 ) have been used as a proxy for non-methylated DNA in vertebrate comparative genomics , promoter mapping , and epigenetic studies , often despite little or no experimental evidence that CGIs correspond to bona fide regions of non-methylated DNA outside of mammals ( Ioshikhes and Zhang , 2000; Hannenhalli and Levy , 2001; Bock et al . , 2007; Han and Zhao , 2008 ) .",
"In mouse and human roughly 50–65% of transcription starts sites ( TSSs ) overlap with CGI predictions .",
"Interestingly , CGI predictions in cold-blooded vertebrates often reside away from gene promoters , with only 16% of zebrafish and 17% of frog TSSs overlapping predicted CGIs .",
"This has led to the suggestion that non-methylated DNA is a unique feature of gene promoters in endotherms , potentially representing a major divergence in the usage of this epigenetic system between warm-blooded and cold-blooded vertebrates ( Aïssani and Bernardi , 1991; Sharif et al . , 2010 ) .",
"Here we experimentally identify non-methylated islands ( NMIs ) of DNA in the genomes of seven diverse vertebrates , encompassing major evolutionary branch points and including both warm and cold-blooded vertebrates .",
"Interestingly we reveal that CGI prediction does not accurately identify islands of non-methylated DNA , particularly in lower vertebrates .",
"Using our new NMI maps we are able to examine for the first time the relationship between these epigenetically specified features and gene regulatory elements .",
"Interestingly , in contrast to expectation based on CGI predictions in some cold-blooded vertebrates , we now reveal that NMIs are a central and conserved feature of vertebrate gene promoters .",
"Together this work uncovers a unifying set of features that are common to NMI systems across vertebrates and details an unexpected level of epigenetic conservation at vertebrate gene promoters ."
],
[
"In order to understand whether the prevailing views about non-methylated DNA function and proposed divergence amongst vertebrate species based on CGI prediction are correct , we isolated genomic DNA from the testes of seven representative vertebrates and carried out non-methylated DNA profiling using biotinylated CxxC affinity purification ( Bio-CAP ) ( Illingworth et al . , 2010; Blackledge et al . , 2012 ) followed by massively parallel sequencing .",
"We specifically focused our analysis on species covering major evolutionary branch points for both warm and cold-blooded vertebrates , including: two eutherian mammals ( human—Homo sapiens and house mouse—Mus musculus ) , a monotreme ( platypus—Ornithorhynchus anatinus ) , a bird ( chicken—Gallus gallus ) , a lizard ( green anole lizard—Anolis carolinensis ) , a frog ( African clawed frog—Xenopus tropicalis ) , and a teleost fish ( zebrafish—Danio rerio ) .",
"Using these new experimentally identified non-methylated island ( NMI ) maps we initially examined the location of non-methylated DNA across vertebrate genomes by visualising syntenic regions shared among all seven species ( Figure 1A and Figure 1—figure supplement 1A and B ) .",
"In these regions , the location of computationally derived CGIs in relation to orthologous genes differs greatly across the seven species .",
"In contrast , the distribution of our experimentally identified NMIs was strikingly similar across all seven species .",
"This indicates the suggestion based on CGI prediction that NMIs are a unique feature of warm-blooded vertebrate gene promoters is incorrect ( Aïssani and Bernardi , 1991; Sharif et al . , 2010 ) .",
"Based on the disparity between CGI prediction and experimentally-profiled non-methylated DNA , we compared more closely the prediction-based CGI maps with NMIs genome-wide ( Figure 1B ) .",
"In human , mouse and chicken , NMIs encompassed most CGI predictions , although in human and mouse roughly 50–60% of experimental NMIs lay outside of predicted CGIs in agreement with recent genome-wide bisulfite sequencing studies ( Molaro et al . , 2011; Stadler et al . , 2011 ) .",
"Interestingly , the overlap between NMIs and predicted CGIs in lower vertebrates was surprisingly poor revealing that CGI maps in most species fail to accurately identify regions of non-methylated DNA ( Figure 1B ) . 10 . 7554/eLife . 00348 . 003Figure 1 . CpG island predictions do not accurately identify non-methylated islands of DNA in vertebrate genomes .",
"( A ) Non-methylated DNA profiles in testes at a representative syntenic region for seven vertebrate species .",
"Genes are shown in black ( improved annotation of gene TSSs using RNA-seq data is shown in red ) , CpG island predictions in green ( CGI ) , and non-methylated DNA profiles are shown in blue .",
"A phylogenetic tree ( left ) highlights the evolutionary relationship among the seven species .",
"Dashed grey lines highlight the relationship between the gene TSSs across the species .",
"A gap in the zebrafish profile indicates that aptx is found at a separate locus from dnaja1 and smu1 .",
"( B ) The genome-wide overlap between CpG islands ( green ) and non-methylated islands ( blue ) is depicted as a Venn diagram for each of the species .",
"( C ) Nucleotide properties of non-methylated islands and control regions are depicted as density plots .",
"CpG observed/expected ( left ) and GC content ( right ) are shown for NMI and control regions of the genome .",
"Median values are shown as dark vertical lines .",
"Thresholds for CpG island prediction are indicated ( black dashed line ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 00310 . 7554/eLife . 00348 . 004Figure 1—figure supplement 1 . NMIs are a conserved feature of vertebrate promoters as illustrated by two syntenic loci .",
"( A ) and ( B ) Profiles of non-methylated DNA are shown in testes at two representative syntenic regions for seven vertebrate species .",
"Genes are shown in black ( improved annotation of gene TSSs using RNA-seq data is shown in red ) , CpG island predications in green , and non-methylated DNA profiles are shown in blue .",
"A phylogenetic tree ( left ) highlights the evolutionary relationship among the seven species and dashed grey lines highlight the relationship between the gene TSSs across the species . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 004 To understand why CGI prediction algorithms often fail , we analysed in detail the nucleotide features of NMIs for all species with a focus on the ratio of observed CpG over expected CpG dinucleotides ( CpG O/E ) and total G+C nucleotide content ( GC content ) .",
"These are the two features commonly used to identify CGIs genome-wide ( Gardiner-Garden and Frommer , 1987 ) .",
"We hypothesised that the algorithms may struggle when faced with greatly contrasting genome-wide nucleotide compositions characteristic of diverse phyla .",
"As expected , human and mouse NMIs show elevated CpG O/E and GC content compared to control regions of the genome ( Figure 1C ) .",
"However , many human and mouse NMIs have lower CpG O/E and GC content than the CGI predictions , explaining why the CGI predictions do not accurately identify all NMIs in these species .",
"CGI predictions in chicken are surprisingly accurate .",
"This appears to be due to the fact that NMIs in this species have the highest CpG O/E and GC content compared to the surrounding genome amongst all the vertebrates examined .",
"Although platypus NMIs have a similarly high CpG O/E and GC content , its genome is on average more CpG and GC rich than chicken .",
"This causes the algorithm to massively over-predict CGIs .",
"Lizard and frog encode NMIs with CpG O/E content similar to those in mammals , but with lower GC content .",
"In zebrafish , NMI CpG O/E is high yet the GC content is almost indistinguishable from surrounding DNA sequence ( Cross et al . , 1991 ) .",
"Again , in these species this leads to a general failure of CGI prediction to accurately identify NMIs .",
"The failure of CpG island prediction algorithms to accurately identify NMIs in different species is almost certainly dependent on the variation in CpG density and G+C content amongst vertebrate genomes , but also will rely on genome assembly and annotation quality , particularly of repetitive elements .",
"Indeed , based on genome variations in CpG and G+C content , it has been suggested previously that species-specific CpG island annotation may be required ( Glass et al . , 2007 ) .",
"These sequence variations between species are likely driven by the relative strengths of two processes: reductions of G+C content due to imperfect repair of spontaneous 5-methylcytosine deamination events ( Coulondre et al . , 1978; Bird , 1980 ) and increases in G+C content in species and genomic regions that are especially prone to GC-biased gene conversion , an outcome of recombination ( Duret and Galtier , 2009 ) .",
"Species differences in these antagonistic processes are likely to have caused the varying levels of G+C content both among vertebrates and across different regions of most amniotic genomes .",
"Unlike CGI predictions , Bio-CAP identifies NMIs through an affinity based isolation of non-methylated CpGs and therefore does not solely rely on nucleotide content in the same way prediction algorithms do .",
"Nevertheless , we considered whether the efficiency of NMI identification by Bio-CAP among species differs due to non-methylated CpG content and density .",
"In contrast , non-methylated DNA fragments , even with low CpG density , are effectively detected by Bio-CAP ( Blackledge et al . , 2012 ) and a broad distribution of CpG density within NMIs is identified in all species .",
"Therefore , although CGI prediction does function with some degree of accuracy in mammals and bird , CGI prediction maps are in general a poor indicator of where NMIs exist in vivo , presumably due to varying nucleotide content amongst diverse phyla .",
"In light of the fact that CGI prediction maps largely fail to accurately detect experimentally-identified non-methylated regions of DNA , work over the past 25 years that has extensively used these maps as a proxy for non-methylated DNA and evolutionary comparison clearly requires re-evaluation .",
"Using our new genome-wide maps of non-methylated DNA , we first set out to directly examine whether NMIs are a specific feature of warm-blooded vertebrate gene promoters as has previously been suggested ( Aïssani and Bernardi , 1991; Sharif et al . , 2010 ) .",
"In human , mouse , and chicken we observed that most TSSs of protein coding genes overlap with an NMI ( 72% , 66% , 58% , respectively ) ( Figure 2A ) .",
"In zebrafish , a cold-blooded vertebrate , CGI predictions overlap with only 17% of TSSs suggesting that this is not a major feature of gene promoters in this species .",
"In stark contrast , our new NMI maps reveal strong overlap of NMIs and TSSs ( 55% ) in zebrafish .",
"The occurrence of promoter-associated NMIs in lizard and frog appeared initially to be low .",
"However , by using RNA-seq information to refine TSS annotation ( Akkers et al . , 2010; Barbosa-Morais et al . , 2012 ) , the fraction of TSSs overlapping NMIs in these species increased to 45–58% , similar to that observed in other vertebrates ( Figure 2A ) .",
"In platypus the association of NMIs with gene promoters appears lower and does not improve substantially using RNA-seq information ( Brawand et al . , 2011; Julien et al . , 2012 ) ( 19% increases to 30% ) ; however this is likely an inaccurate representation due to a fragmented and incomplete genome assembly which limits our capacity to build accurate gene models .",
"Nevertheless , non-methylated DNA signal centres at TSSs in all seven species ( Figure 2B ) .",
"Therefore , in direct contradiction to the contention from previous work ( Aïssani and Bernardi , 1991; Sharif et al . , 2010 ) and CGI prediction maps that non-methylated islands of DNA are a unique feature of warm-blooded vertebrate gene promoters , we now unequivocally demonstrate that NMIs are instead a central and ancient feature of vertebrate gene promoters . 10 . 7554/eLife . 00348 . 005Figure 2 . Non-methylated islands are associated with gene promoters in vertebrate genomes .",
"( A ) A histogram depicting the proportion of protein-coding transcription start sites ( TSSs ) which are overlapped by an NMI for all seven species .",
"Blue bars indicate overlap with annotated TSSs and red bars indicate overlap with additional TSSs identified using RNA-seq data ( platypus , chicken and lizard ) or Xtev gene sets ( frog ) .",
"( B ) Profiles of non-methylated DNA were plotted over a 6-kb window centred on all TSSs with an NMI ( dark blue ) , without an NMI ( blue ) , and for all transcription termination sites ( TTS , black ) .",
"The non-methylated DNA signal peaks at the TSS of gene promoters in all vertebrates . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 005 Although DNA sequence within gene regulatory elements is often conserved across vertebrate species , it remains almost completely unknown whether epigenetic features are subject to a similar selective pressure .",
"Interestingly , some evidence has emerged recently indicating that certain epigenetic features may be conserved between mammalian species ( Shibata et al . , 2012; Xiao et al . , 2012 ) .",
"Taking advantage of our new NMI maps , we investigated whether NMIs are conserved at the TSSs of orthologous genes in the seven vertebrate species analysed .",
"Pairwise comparison of homologous vertebrate gene promoters revealed a surprisingly high propensity for orthologous genes to have an NMI at their respective promoters ( Figure 3A ) .",
"This is exemplified by the fact that over 90% of NMIs are shared at orthologous gene TSSs between human and each of the other six species examined .",
"Furthermore , a more extensive three-way comparison between human , mouse and zebrafish revealed conservation of NMIs at nearly all gene promoters of one-to-one orthologues , despite 450 million years of divergent evolution ( Figure 3B ) .",
"Therefore , this remarkable degree of conservation reveals a concerted evolutionary drive not only to select for DNA sequence at gene regulatory elements but also to retain NMI identity as an epigenetic feature of gene regulatory elements . 10 . 7554/eLife . 00348 . 006Figure 3 . Non-methylated islands are a highly conserved epigenetic feature of vertebrate gene promoters .",
"( A ) The presence of NMIs at orthologous gene TSSs is preserved as illustrated by a pairwise analysis of NMIs at vertebrate gene orthologues .",
"The percentage of NMIs conserved at orthologous gene TSSs was calculated in a pairwise manner and found to be highly statistically significant for all comparisons across the seven vertebrate species ( p<10−10 , hypergeometric test ) .",
"( B ) A proportional Venn diagram illustrating the three-way comparison of NMI presence at conserved human-mouse-zebrafish gene orthologue TSSs . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 006 In addition to gene associated NMIs , an unexpectedly large proportion of vertebrate NMIs lie within intergenic regions ( Figure 4A ) , suggesting that some NMIs may be functioning in regulatory roles away from known gene promoters .",
"To try and understand how these intergenic NMIs contribute to genome function , we explored whether intergenic NMIs correspond to regulatory elements such as non-protein coding transcripts or enhancers .",
"Recently , histone H3 lysine 4 mono-methylation ( H3K4me1 ) has been found to be strongly associated with enhancer elements ( Barski et al . , 2007; Heintzman et al . , 2007; Aday et al . , 2011 ) .",
"In order to allow comparison with both non protein-coding transcript models and available genome-wide H3K4me1 data ( Aday et al . , 2011; Stadler et al . , 2011 ) we generated NMI maps for mouse embryonic stem ( ES ) cells and zebrafish 24 hr post-fertilisation ( hpf ) embryos .",
"Initially we analysed whether intergenic NMIs were found associated with the TSSs of annotated non-coding RNAs ( ncRNAs ) and found that less than 3% overlapped with the primary transcripts of miRNAs , rRNAs , snRNAs , or snoRNAs ( data not shown ) .",
"However , in mouse and zebrafish 45–64% of known lncRNA TSSs ( Guttman et al . , 2010; Belgard et al . , 2011; Ulitsky et al . , 2011; Pauli et al . , 2012 ) were associated with NMIs suggesting that this class of candidate regulatory RNAs are subject to similar epigenetic regulatory processes as protein coding genes ( Figure 4B ) .",
"In total , association with the TSSs of all known non-coding transcripts accounted for 15% and 9% of intergenic NMIs in mouse and zebrafish respectively ( Figure 4C ) .",
"Intergenic NMIs not associated with annotated non-coding RNAs were then compared with H3K4me1 peaks and a further 17% and 40% of the NMIs , in mouse and zebrafish respectively , corresponded to putative enhancers .",
"Finally , the remaining intergenic NMIs were compared to tissue-specific RNA-seq datasets ( Stadler et al . , 2011 ) to determine whether they overlapped with un-annotated transcriptional initiation .",
"This revealed that up to 14% and 8% of NMIs in mouse and zebrafish overlap with uncharacterised transcribed regions of the genome .",
"Therefore a substantial proportion ( 46–57% ) of NMIs away from the 5′ end of protein coding genes encompass known ncRNA TSSs , putative enhancers , and other transcribed regions , a property which is highly conserved across two diverse vertebrate species .",
"Importantly , this indicates that NMI profiles provide not just a useful tool for resolving TSSs for protein coding genes in organisms where genome annotation is sparse , but also a valuable resource for identifying putative enhancers or un-annotated coding and non-coding genes .",
"Together these observations reveal that vertebrate NMIs may have an unexpectedly important role away from gene promoters in contributing to the regulatory potential of the genome . 10 . 7554/eLife . 00348 . 007Figure 4 . Intergenic NMIs are associated with distal regulatory elements , non-coding RNAs , and unannotated transcripts .",
"( A ) Most NMIs are associated with known protein-coding genes ( left ) but a substantial proportion are located within intergenic regions of the genome ( right ) .",
"( B ) NMIs ( green ) are found at 45% and 64% of all known long non-coding RNA ( lncRNA ) TSSs ( black ) in mouse and zebrafish respectively .",
"( C ) A pie chart depicting the proportion of intergenic NMIs ( >5 kb from a protein-coding gene ) associated with different genomic features in mouse embryonic stem ( ES ) cells and zebrafish 24 hpf embryos .",
"The association was performed hierarchically in the following order: lncRNA TSSs , other non-coding RNA TSSs ( miRNAs , rRNAs , snRNAs , or snoRNAs ) , other TSSs ( pseudogenes and processed transcripts ) , putative enhancer mark H3K4me1 and novel RNA-seq TSSs .",
"This analysis indicates that intergenic NMIs mark novel transcriptional units or regulatory elements . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 007 In general NMIs in mammals are thought to be maintained in the non-methylated state in most tissues , even if their associated gene is not appreciably transcribed .",
"Recently this belief has been challenged and it appears that some NMIs in human and mouse are more susceptible to differential methylation during tissue specification ( Weber et al . , 2007; Farthing et al . , 2008; Mohn et al . , 2008; Straussman et al . , 2009; Illingworth et al . , 2010; Maunakea et al . , 2010 ) .",
"However , it remains unclear whether differential methylation of NMIs is a common strategy for epigenetic regulation in vertebrates .",
"Therefore to address this question we profiled NMIs in liver for all seven species and compared these maps to those already generated for testes .",
"We first separated a core set of NMIs that are shared between the two tissues from a second set of NMIs that are unique to either testes or liver .",
"Unique NMIs from liver or testes were validated by bisulfite sequencing for mouse and zebrafish ( Figure 5—figure supplement 1 ) .",
"The existence of unique NMIs in each of the interrogated tissues suggests that all vertebrates utilise differential methylation to epigenetically regulate a subset of NMIs .",
"To examine in more detail the properties of shared and unique NMIs , heat maps illustrating non-methylated DNA signal were generated for NMI intervals , ranked by NMI length and clustered according to their classification as shared or unique ( Figure 5A and Figure 5—figure supplement 2A ) .",
"Interestingly , it was immediately apparent that shared and unique NMIs stratified into two largely distinct classes .",
"Shared NMIs tended to be longer , have higher CpG density ( Figure 5C , D and Figure 5—figure supplement 2C , D ) and are generally found associated with the TSSs of protein coding genes ( Figure 5B and Figure 5—figure supplement 2B ) .",
"This is consistent with the classical view that mammalian NMI-associated TSSs are generally refractory to DNA methylation in most tissues ( Illingworth et al . , 2010 ) .",
"We refer to these as ‘canonical’ NMIs .",
"In contrast , tissue-specific NMIs that are differentially methylated tended to be shorter , have lower CpG density and are found away from protein-coding gene TSSs ( Figure 5B–D and Figure 5—figure supplement 2B–D ) .",
"For the subset of differentially methylated TSS-associated NMIs , expression differences of the associated gene were compared between testes and liver for human , mouse , platypus and chicken ( Brawand et al . , 2011 ) .",
"This revealed that genes differentially expressed between liver and testes were significantly enriched for genes with tissue-specific NMIs ( Figure 5 and Figure 5—figure supplement 3 ) .",
"Therefore , the presence of DNA methylation closely correlates with gene repression , suggesting that although an infrequent event , methylation-mediated silencing at NMI promoters is a general feature in epigenetic regulation of certain vertebrate protein-coding genes . 10 . 7554/eLife . 00348 . 008Figure 5 . Differential methylation of a subset of NMIs .",
"( A ) All vertebrate genomes have a subset of NMIs that are subject to differential methylation as illustrated by a heat map of non-methylated DNA signal from testes and liver in human , mouse and zebrafish .",
"In each case NMIs are ranked according to length and clustered as shared ( upper ) or unique ( lower ) between the two tissues .",
"A 5-kb window centred at the NMI is shown and read density is indicated by colour intensity .",
"( B ) The overlap of NMIs identified in liver and testes is depicted by Venn diagrams for NMIs associated with protein-coding TSSs ( upper ) and for NMIs away from TSSs ( lower ) .",
"NMIs at TSSs are generally non-methylated in both tissues whereas differentially methylated NMIs tend to be found away from TSSs .",
"( C ) NMI length distribution plots for shared ( Shared NMIs , solid line ) or unique ( Unique NMIs , dashed line ) NMIs from testes ( blue ) or liver ( red ) .",
"Shared NMIs tend to be longer than tissue-specific unique NMIs .",
"( D ) CpG density distribution plots for shared ( solid line ) or unique ( dashed line ) NMIs from testes ( blue ) or liver ( red ) .",
"Shared NMIs tend to have higher CpG density than unique NMIs . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 00810 . 7554/eLife . 00348 . 009Figure 5—figure supplement 1 . Validation of differentially methylated NMIs between liver and testes in mouse and zebrafish by bisulfite sequencing .",
"( A , i–iv )",
"Mouse NMIs unique to liver or testes were analysed by bisulfite sequencing to verify that the regions were indeed differentially methylated .",
"Traces of non-methylated DNA are depicted for differentially methylated regions in mouse liver ( red ) and testes ( blue ) with NMIs depicted as bars under the traces .",
"The y-axis depicts read density .",
"Methylation status of the unique NMIs was confirmed using the indicated bisulfite PCR amplicon ( BA , black rectangle ) .",
"Empty and filled circles represent non-methylated and methylated CpG dinucleotides , respectively .",
"( B , ( i–iii )",
"Zebrafish NMIs unique to liver or testes were validated by bisulfite sequencing as in ( A ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 00910 . 7554/eLife . 00348 . 010Figure 5—figure supplement 2 . Differential methylation of NMIs in platypus , chicken , lizard and frog and length distributions of NMIs from all seven vertebrates .",
"( A ) A heat map of non-methylated DNA signal from testes and liver in platypus , chicken , lizard and frog .",
"In each case NMIs are ranked according to length and clustered as shared ( upper ) or unique ( lower ) between the two tissues .",
"A 5-kb window centred at the NMI is shown and read density is indicated by colour intensity .",
"( B ) Venn diagrams demonstrate that shared NMIs are found predominantly at protein-coding gene TSSs ( upper ) and unique NMIs tend to be found away from TSSs ( lower ) .",
"( C ) NMI length distribution plots for shared ( Shared NMIs , solid line ) or unique ( Unique NMIs , dashed line ) NMIs from testes ( blue ) or liver ( red ) .",
"Shared NMIs tend to be longer than tissue-specific unique NMIs .",
"( D ) CpG density distribution plots for shared ( solid line ) or unique ( dashed line ) NMIs from testes ( blue ) or liver ( red ) .",
"Shared NMIs tend to have higher CpG density than unique NMIs . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 01010 . 7554/eLife . 00348 . 011Figure 5—figure supplement 3 . Genes with TSS-associated testes or liver specific NMIs are over-represented for increased differential expression in the same tissue . MA plots depicting expression differences for genes with TSS-associated NMIs from liver and testes for human , mouse , platypus and chicken .",
"Genes are coloured according to whether they share an NMI in both liver and testes ( grey ) or have an NMI only in liver ( red ) or testes ( blue ) .",
"Genes are further distinguished as being differentially expressed or overexpressed in a tissue-specific manner ( dark , filled circle ) or not ( light , open circles ) .",
"The log mean expression of the gene from both liver and testes is displayed on the x axis ( A ) and the log ratio of gene expression is displayed on the y axis ( M ) .",
"The dotted lines indicate a fold change threshold of two .",
"Genes with tissue-specific NMIs were significantly over-represented in the set of genes which had increased differential expression in seven out of eight cases ( Fisher's exact test , human testes p<10−21 , liver p<10−27; mouse testes p<10−18 , liver p<10−8; platypus testes p<10−2 , liver p<10−17; chicken liver p<10−6 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 011 Most tissue-specific NMIs are intergenic , indicating that differential methylation of these elements in vertebrate genomes is generally found away from known gene promoters ( Figure 5B and Figure 5—figure supplement 2B ) .",
"We have already shown that intergenic NMIs are enriched for regulatory elements ( Figure 4B , C ) , therefore one exciting possibility is that differential methylation at NMIs away from gene promoters could function as part of an epigenetically driven regulatory process .",
"Recently , we and others have demonstrated that NMIs are recognized by a class of ZF-CxxC DNA binding proteins that recruit chromatin modifying enzymes to create a unique chromatin environment including placement of H3K4me3 ( Blackledge et al . , 2010; Thomson et al . , 2010 ) .",
"To examine whether differential methylation of NMIs corresponds to alterations in H3K4me3 , the levels of this modification were examined in testes and liver in both human and mouse .",
"This revealed a very clear segregation of H3K4me3 with the tissue that has the unique NMI ( Figure 6A ) .",
"Similarly , when we extended this analysis to the lower vertebrates frog and zebrafish there was a clear enrichment of H3K4me3 at tissue-specific NMIs ( Figure 6B ) .",
"Together these observations demonstrate that differential DNA methylation at gene promoters is associated with gene repression and that the chromatin modification state of differentially methylated NMIs may be regulated in a switch-like mechanism dependent on the underlying DNA methylation status .",
"This epigenetically ‘plastic’ subset of NMIs suggests that the landscape of non-methylated DNA in vertebrates may contribute an unexpected level of functional diversity across different tissues in an otherwise static DNA sequence . 10 . 7554/eLife . 00348 . 012Figure 6 . Chromatin modification at NMIs depends on their underlying DNA methylation state .",
"( A ) H3K4me3 read density from testes ( blue ) and liver ( red ) is profiled over testes unique ( left ) and liver unique ( right ) NMIs for human ( upper ) and mouse ( lower ) and displayed as an average profile .",
"At differentially methylated loci , the histone H3K4me3 modification is found preferentially in the tissue with the non-methylated NMI .",
"( B ) The H3K4me3 signal ( profiled in frog stage 11–12 embryos and zebrafish 24 hpf ) is present specifically at unique NMIs from frog stage 11–12 and zebrafish 24 hpf ( green ) and not at unique NMIs from the liver ( red ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 012 The majority of vertebrate genes have a punctate peak of non-methylated DNA at their TSS in fitting with the canonical view of an NMI ( Figure 2B ) .",
"Interestingly however , we observed a subset of genes that deviated from this generality and tended to be encompassed by broad regions of non-methylated DNA ( ‘broad NMIs’ ) that often covered the majority of the gene .",
"This striking observation is exemplified by the sp9 and otp genes ( Figure 7A and Figure 1—figure supplement 1A ) and more complex gene clusters including the hox loci ( Figure 7—figure supplement 1 ) .",
"Like canonical NMIs , broad NMIs appear to be H3K4me3 modified over the entire non-methylated region indicating they are also targeted by ZF-CxxC dependent chromatin modifying activities ( Figure 7B , C ) .",
"To begin trying to identify common features shared amongst genes associated with broad NMIs , Gene Ontology ( GO ) analysis was performed ( Figure 7D ) .",
"Interestingly , genes encompassed by broad NMIs are highly enriched for transcription factors and genes involved in development , suggesting that this epigenetic feature may be related to the mechanisms that underpin their transcriptional regulation .",
"Transcription factors and developmental regulators are often subject to regulation by the polycomb repressive complex in early development ( Sawarkar and Paro , 2010 ) .",
"Therefore the polycomb-mediated H3K27me3 chromatin modification was analysed at broad NMIs in mouse ES cells ( Mikkelsen et al . , 2007 ) and frog stage 11–12 embryos ( Akkers et al . , 2009 ) ( Figure 7E ) .",
"Strikingly 45% and 89% of broad NMIs were associated with H3K27me3 in mouse ES cells and frog stage 11–12 tissues respectively , a significantly higher proportion than observed for canonical NMIs ( Fisher’s exact test , odds ratio > 5 . 3 , p<10−35 ) .",
"As with H3K4me3 , H3K27me3 extends across the broad region of non-methylated DNA suggesting that not only are broad NMIs preferentially subject to polycomb silencing but that polycomb complexes may also read the underlying non-methylated DNA state when placing H3K27me3 marks ( Figure 7F ) .",
"This idea is generally in agreement with a recent observation that clustered CGI predictions were often a good predictor of polycomb nucleation ( Orlando et al . , 2012 ) .",
"Although it still remains largely unknown how polycomb repressive complexes mechanistically recognise gene targets in vivo , our cross-species analysis reveals that polycomb repression is preferentially and spatially targeted to the broad class of NMIs .",
"This reveals that there is a clear functional segregation between the canonical and broad class of NMI and provides an exciting new possibility that the capacity to function as a vertebrate polycomb responsive element may rely on properties specific to the broad class of NMI .",
"Together this demonstrates that broad NMIs specify a unique subset of transcription factors and developmental regulators that are preferentially targeted for polycomb repression during early development , a process that appears to be highly conserved over vertebrate evolution . 10 . 7554/eLife . 00348 . 013Figure 7 . A unique class of broad non-methylated islands encompass polycomb-regulated developmental genes .",
"( A ) An example of a broad region of non-methylated DNA associated with the sp9 gene for four representative species ( human , mouse , frog and fish ) .",
"Dashed grey lines highlight the location of the gene TSSs across the four species .",
"( B ) Non-methylated DNA profiles are depicted for genes associated with broad NMIs ( dark blue ) and canonical NMIs ( light blue ) in mouse embryonic stem ( ES ) cells and frog stage 11–12 .",
"The profile is scaled to show an averaged gene with one gene length depicted upstream and downstream .",
"( C ) H3K4me3 ChIP-seq signal from mouse and frog was plotted as in ( B ) .",
"H3K4me3 profiles reflect the underlying non-methylated DNA profiles .",
"( D ) Genes associated with broad NMIs were analysed by gene ontology ( GO ) analysis for mouse ES cell and frog stage 11–12 .",
"Broad NMIs are found to be significantly enriched for GO term categories associated with sequence-specific DNA binding , transcriptional regulation and development .",
"MF: molecular function; BP: biological process .",
"p<10−5 for all GO terms .",
"( E ) H3K27me3 ChIP-seq signal from mouse and frog was plotted for the same gene sets as in ( B ) .",
"The profile is scaled to show an averaged gene with three gene lengths depicted upstream and downstream .",
"As for H3K4me3 , H3K27me3 ChIP-seq profiles correspond to the underlying non-methylated DNA profile .",
"( F ) A representative example of two broadly non-methylated genes gsx1 and nkx2 . 2 for mouse and frog .",
"In both species , the broad non-methylated regions ( green ) are associated with the polycomb repressive mark H3K27me3 ( red ) .",
"In addition , in mouse , polycomb repressive complex 2 ( ezh2 , yellow and suz12 , orange ) and polycomb repressive complex 1 ( ring1b , purple ) components are associated with the broad non-methylated regions .",
"The y-axis depicts read density .",
"Genes are depicted above the profiles in black . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 01310 . 7554/eLife . 00348 . 014Figure 7—figure supplement 1 . Hox gene clusters are characterized by broad NMIs . The hoxa gene cluster from all seven vertebrate species is associated with broad regions of non-methylated DNA .",
"Genes are shown in black and non-methylated DNA profiles are shown in blue and dashed grey lines highlight the relationship between conserved gene TSSs across the species . DOI: http://dx . doi . org/10 . 7554/eLife . 00348 . 014"
],
[
"Although the DNA methylation system is conserved across vertebrate evolution , CGI maps had previously indicated that this epigenetic system may have significantly diverged between vertebrate species and even acquired unique properties at TSSs during the evolution of warm-blooded vertebrates ( Aïssani and Bernardi , 1991; Sharif et al . , 2010 ) .",
"Despite some recent indications that DNA methylation profiles may be more conserved than previously realised ( Feng et al . , 2010; Zemach et al . , 2010; Wu et al . , 2011; Andersen et al . , 2012 ) , a lack of experimentally identified regions of non-methylated DNA outside of eutherian mammals has hindered the capacity to specifically address whether this system has significantly diverged among vertebrates .",
"To address this fundamental question and to better understand the level of evolutionary conservation in epigenetic systems , we identified NMIs genome-wide in seven diverse vertebrate species demonstrating for the first time that NMIs are in fact a highly conserved feature of vertebrate gene promoters .",
"Importantly , this paradigm shift also revealed that three distinct yet highly conserved classes of NMIs have emerged during vertebrate evolution .",
"The first class is a canonical NMI that best fits the classical definition of a CGI .",
"These NMIs are narrow , associated with gene promoters , and generally remain free of DNA methylation regardless of the tissue or associated gene expression state .",
"The second class of plastic NMIs are shorter , have lower CpG density than canonical NMIs , are usually found away from gene promoters at alternative regulatory elements , and are subject to differential methylation between tissues .",
"Importantly , this class of NMI demonstrates that epigenetic plasticity in the form of differential methylation is a highly conserved mechanism utilised by all vertebrates .",
"Finally , a third and unique class of broad NMIs were identified that often cover an entire gene , are specifically associated with transcription factors or developmental genes , and are associated with polycomb mediated silencing during early development .",
"These three classes of NMIs appear to form a highly conserved logic for the utilisation of non-methylated DNA in vertebrate genomes .",
"Therefore , in contrast to the suggestion that NMIs may have diverged during vertebrate evolution ( Aïssani and Bernardi , 1991; Sharif et al . , 2010 ) , we demonstrate that the central properties that underpin the NMI system are instead highly conserved across vertebrates .",
"Perhaps most importantly , TSS-associated NMIs appears to be under strong selective pressure as part of what appears to be a highly conserved epigenetic system used to specify and control gene regulatory elements in large and complex vertebrate genomes ."
],
[
"Samples were obtained either as purified genomic DNA , as fresh-frozen samples or were dissected in-house and fresh-frozen .",
"Samples were subjected to manual homogenisation followed by DNA purification by phenol chloroform extraction or using the QIAGEN 100/G genomic tip kit ( Manchester , UK ) .",
"Bio-CAP was performed as previously described ( Blackledge et al . , 2012 ) .",
"All Bio-CAP experiments were performed in duplicate with matched input controls .",
"Next generation sequencing was performed using two Illumina sequencing platforms: Genome Analyser IIx and HiSeq Systems yielding 51-bp single-end reads .",
"Computationally predicted CpG islands for all seven species were obtained from the UCSC genome browser ( Kent et al . , 2002 ) .",
"LncRNA datasets from recent publications for mouse ( Guttman et al . , 2010; Belgard et al . , 2011; GSE20851 , GSE27243 ) and zebrafish ( Ulitsky et al . , 2011; Pauli et al . , 2012; GSE32900 and GSE32880 ) were obtained from GEO ( Edgar et al . , 2002 ) or from supplementary material .",
"H3K4me1 datasets for zebrafish 24 hpf ( Aday et al . , 2011; GSE20600 ) and mouse ES cell ( Stadler et al . , 2011; GSE30206 , GSE11172 ) were obtained from GEO .",
"Mouse ( Smagulova et al . , 2011; GSE24438 ) and human ( Hammoud et al . , 2009; Bernstein et al . , 2010; GSE15594 and GSE19465 , testes and liver ) , frog ( Akkers et al . , 2009; GSE14025 , stage 11–12 ) and zebrafish ( Aday et al . , 2011; GSE20600; 24 hpf ) H3K4me3 datasets were obtained from GEO .",
"H3K4me3 and H3K27Me3 datasets for mouse ( Mikkelsen et al . , 2007; GSE12241 , ES cells ) and frog ( Akkers et al . , 2009; GSE14025 , stage 11–12 ) were obtained from GEO .",
"RNAseq datasets for human , mouse , platypus and chicken ( Brawand et al . , 2011; Stadler et al . , 2011; Julien et al . , 2012; GSE30352 , GSE30280 and GSE36120 ) were obtained from GEO .",
"RNAseq datasets for lizard were obtained from Kutter and Odom pre-publication ( Barbosa-Morais et al . , 2012; now available at GSE41338 ) and zebrafish RNAseq data were obtained from the EBI ( ERP000016 , Sample ERS000081 ) .",
"The XTev dataset ( Akkers et al . , 2010 ) was obtained from the Veenstra lab website ( http://131 . 174 . 221 . 43/gertjanveenstra/genomedata . asp ) .",
"ChIP-seq data for a number of polycomb factors profiles for mouse ES cells ( Ku et al . , 2008; GSE13084 ) were obtained from GEO .",
"Sequencing reads were aligned to the appropriate reference genome ( anoCar2 , danRer7 , galGal3 , hg19 , mm9 , ornAna1 , xenTro3 ) using the Bowtie short-read aligner ( v0 . 12 . 7 ) ( Langmead et al . , 2009 ) .",
"Only uniquely mapping reads , with a maximum of two mismatches across the entire read length were used .",
"Non-methylated islands ( NMIs ) were identified using MACS ( v1 . 4 . 0 ) ( Zhang et al . 2008 ) using a bandwidth of 300 and an mfold range of 10–30 .",
"Binding intervals were filtered by a q value of 0 . 01 .",
"Data analysis was performed in R and python using bespoke scripts available online ( http://www . cgat . org/hg/cgat/ ) .",
"CpG observed/expected ( CpG O/E ) and GC content were calculated as in Gardiner-Garden and Frommer ( 1987 ) .",
"Both measures were calculated for each NMI and for a control region of the same size 10 kb upstream of each NMI interval .",
"Ensembl ( release 66 ) genes were annotated as having an NMI at their TSS using a single base pair overlap of an NMI with a window extending 1 kb upstream and downstream from each transcript TSS .",
"Multi-tissue RNAseq datasets for platypus , chicken and lizard were used to improve the annotation of gene TSSs .",
"Where transcript models were not provided by the authors TopHat ( Trapnell et al . , 2009 ) and Cufflinks ( Trapnell et al . , 2010 ) were used to construct transcript models from short-read data .",
"Similarly , the XTev gene dataset was used to improve the annotation of TSSs in the frog genome .",
"NMIs were annotated with respect to both Ensembl ( release 66 ) and RNAseq-based genome annotation as being associated with the following features in a hierarchical manner: protein-coding gene TSS ( ±1 kb ) , gene body , upstream or downstream of a gene ( within 5 kb of the annotated gene model ) .",
"Remaining NMIs were annotated as intergenic .",
"For mouse ES cell and zebrafish 24 hpf embyros , published lncRNA models , Ensembl non-coding RNA annotations , tissue-specific H3K4me1 and RNAseq data were used to account for intergenic NMIs in a hierarchical manner .",
"Where short read genomic alignments were not provided by the authors , chromatin mark datasets were aligned to the appropriate reference genome using Bowtie and peaks were called using MACS as above .",
"Throughout , overlap of genomic intervals ( e . g . , NMIs compared to CGIs , Figure 1B ) was assessed using BEDTools ( Quinlan and Hall , 2010 ) and statistical significance calculated using the Genomic Association Tester ( GAT ) ( Ponjavic et al . , 2007 ) .",
"Evolutionary conservation of protein-coding genes was calculated using OPTIC ( Heger and Ponting , 2008 ) .",
"Conserved genes ( pairwise 1:1 orthologues ) were defined as having an NMI or not as above .",
"The conservation score was calculated as: n/min ( x , y ) where n is the number of conserved genes with an NMI at the TSS of both orthologues and x and y are the numbers of conserved genes with a TSS-associated NMI in each species .",
"For three-way conservation , 1:1:1 orthologues from human , mouse and zebrafish were defined as having an NMI in one , two or all species .",
"NMIs were called from non-methylated DNA profiles in both testes and liver using MACS ( as above ) .",
"An NMI was defined as tissue-specific if it did not overlap with an NMI in the other tissue .",
"Broad NMI-associated genes were defined as having greater than 90% of their gene length covered by NMIs .",
"Short NMI-associated genes had less than 10% ( but greater than 0% ) gene coverage by NMIs .",
"DESeq ( Anders and Huber , 2010 ) was used to identify genes differentially expressed between liver and testes in human , mouse , platypus and chicken RNAseq data ( p<0 . 05 , fold change > 2 ) .",
"H3K4me3 signal was profiled across tissue-specific NMIs using sitepro from the CEAS package ( Shin et al . , 2009 ) .",
"Two-way Venn diagrams were generated in R using the ‘VennDiagram’ package ( Chen and Boutros , 2011 ) .",
"The three-way Venn diagram was generated using the EulerAPE drawing tool ( http://www . eulerdiagrams . org/eulerAPE/ ) .",
"Genomic peaks and intervals were visualised using the Integrated Genome Browser ( IGB ) ( Nicol et al . , 2009 ) .",
"Gene Ontology ( GO ) analysis was performed using a hypergeometic test .",
"Terms were clustered using a modified ReVigo ( Supek et al . , 2011 ) script and a representative term from each cluster was plotted using the GO term enrichment score ."
]
] | [
"Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA , called CpG islands ( CGIs ) , which counteract the repressive effects of DNA methylation on chromatin .",
"In cold-blooded vertebrates , computational CGI predictions often reside away from gene promoters , suggesting a major divergence in gene promoter architecture across vertebrates .",
"By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates , we instead reveal that non-methylated islands ( NMIs ) of DNA are a central feature of vertebrate gene promoters .",
"Furthermore , NMIs are present at orthologous genes across vast evolutionary distances , revealing a surprising level of conservation in this epigenetic feature .",
"By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates .",
"Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution ."
] | [
"DNA methylation—the addition of a methyl group to cytosine , one of the four bases found in DNA—is a central process in genetics .",
"By preventing genes from being expressed as proteins , DNA methylation is one of a number of epigenetic mechanisms that can determine which proteins are made in different cell types without changing the underlying DNA sequence .",
"In warm-blooded vertebrates such as mammals most of the genome is methylated , however short regions of non-methylated DNA are known to be associated with gene promoters ( regions of DNA that act as binding sites for the enzymes and transcription factors that transcribe the DNA in the gene into RNA ) .",
"Much of our current understanding of the role of these islands of non-methylated DNA is based on computational predictions rather than experimental data .",
"In cold-blooded vertebrates , for example , computer models often predict that non-methylated islands are not associated with gene promoters , which potentially suggests an evolutionary divergence in the role of methylation amongst vertebrates .",
"However , this idea has not been confirmed by experimental data .",
"Long et al . have performed experiments to compare the location of non-methylated islands in seven different vertebrate species .",
"In general they find that computational models are not a reliable method for identifying non-methylated islands .",
"Moreover they find that non-methylated islands are a central epigenetic feature of gene promoters in all vertebrates analysed–including three mammals , a bird , a lizard , a frog and a fish—and not just in warm-blooded vertebrates as suggested by computational models .",
"This shows that the epigenetic function of these non-methylated islands has been conserved over more than 450 million years of evolution .",
"In addition to the non-methylated islands associated with gene promoters , Long et al . identify two other types: intergenic non-methylated islands that are found away from gene promoters and are said to be ‘plastic’ because the DNA in these islands can acquire methyl groups , and ‘broad’ non-methylated islands that span many of the genes that are involved in embryonic development .",
"By showing that the epigenetic role of non-methylated islands has been conserved over time , and identifying three specific types of island , the work of Long et al . marks an important change in our understanding of epigenetics in vertebrates ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1 | elife-27612-v2 | [
[
"NGLY1 ( N-glycanase",
"1 ) encodes an evolutionarily conserved enzyme that catalyzes the cleavage of N-glycans from glycoproteins ( Suzuki et al . , 2000 ) .",
"Whole-genome and -exome sequencing has recently resulted in the identification of NGLY1 mutations in patients with an autosomal recessive developmental disorder called NGLY1 deficiency ( Caglayan et al . , 2015; Enns et al . , 2014; Heeley and Shinawi , 2015; Need et al . , 2012 ) .",
"NGLY1-deficient patients show a host of phenotypes including global developmental delay , movement disorder , hypotonia , absent tears , peripheral neuropathy , constipation , and small feet and hands ( Enns et al . , 2014; Lam et al . , 2017 ) .",
"The mechanism by which NGLY1 deficiency causes the above-mentioned clinical phenotypes is not known , and neither has NGLY1 been linked to any major developmental signaling pathway .",
"In yeast , N-glycanase 1 ( Peptide: N-glycanase or PNGase , encoded by PNG1 ) has been associated with endoplasmic reticulum-associated degradation ( ERAD ) ( Suzuki , 2007; Suzuki et al . , 2016 ) , a process that plays a crucial role in the proteasome-mediated degradation of misfolded proteins ( Brodsky , 2012; Smith et al . , 2011 ) .",
"However , null mutants for yeast PNG1 do not show apparent phenotypic abnormalities and exhibit normal growth rate and viability under a variety of experimental conditions ( Suzuki et al . , 2000 ) .",
"A recent study has provided strong evidence that in C . elegans , a transcription factor called SKN-1 ( homolog to mammalian NFE2L1/2 ) needs to be retrotranslocated from ER to cytoplasm by ERAD machinery and deglycosylated by NGLY1 ( PNG-1 in worms ) to function properly ( Lehrbach and Ruvkun , 2016 ) .",
"However , deglycosylation does not result in SKN-1 degradation , but instead promotes the activation of SKN-1 so that it can mediate the necessary transcriptional response to proteasome disruption ( Lehrbach and Ruvkun , 2016 ) .",
"Moreover , mouse embryonic fibroblasts lacking NGLY1 did not show impairment and/or delay in the degradation of misfolded proteins , but displayed an unconventional deglycosylation reaction that may generate aggregation-prone proteins harboring N-GlcNAc and result in cell toxicity in the absence of NGLY1 ( Huang et al . , 2015 ) .",
"Therefore , although NGLY1 seems to be functionally associated with the ERAD machinery , it does not seem to directly promote the degradation of misfolded proteins in animals .",
"Human NGLY1 has a single Drosophila homolog called PNGase-like ( Pngl ) , whose loss-of-function mutants result in semi-lethality and sterility ( Funakoshi et al . , 2010 ) .",
"We have used Drosophila to determine the molecular mechanisms by which Pngl regulates animal development .",
"Our data indicate that Pngl is required in the visceral mesoderm ( VM ) during midgut development to regulate bone morphogenetic protein ( BMP ) signaling from VM to endoderm in embryonic midgut and also to ensure midgut clearance before puparium formation .",
"BMP ligands can signal either as homodimers or as heterodimers through homodimeric or heterodimeric BMP type I receptors ( Bangi and Wharton , 2006a; Ray and Wharton , 2001 ) .",
"Our data indicate that loss of Pngl abolishes a BMP autoregulatory loop in the VM mediated by homodimers of one ligand ( Decapentaplegic; Dpp ) acting through homodimers of one receptor ( Thickveins; Tkv ) , and hence link a deglycosylation enzyme to tissue-specific regulation of BMP signaling in flies ."
],
[
"Based on the analysis of three Pngl alleles ( Pnglex14 , Pnglex18 and Pnglex20; Figure 1A ) , it has previously been reported that loss of Drosophila Pngl results in developmental delay and a semi-lethality phenotype , with about 1% adult escapers ( Funakoshi et al . , 2010 ) .",
"Moreover , these phenotypes were rescued by ubiquitous expression of mouse NGLY1 ( Funakoshi et al . , 2010 ) , suggesting functional conservation between Pngl and its mouse homolog .",
"To further examine the degree of conservation between fly Pngl and its mammalian homologs , we used ΦC31-mediated transgenesis ( Bischof et al . , 2007; Venken et al . , 2006 ) to generate transgenic flies capable of overexpressing wild-type ( WT ) human NGLY1 or the NGLY1-ΔR402 mutant , a single amino acid in-frame deletion identified in an NGLY1 deficiency patient ( Enns et al . , 2014 ) , and asked whether they can rescue the homozygous lethality of Pnglex14 and Pnglex18 .",
"Ubiquitous expression of WT human NGLY1 , but not NGLY1-ΔR402 , was able to rescue the lethality in both Pngl alleles ( Figure 1B ) .",
"Both male and female Pngl–/– escaper flies are sterile and short-lived ( Figure 1C and D ) ( Funakoshi et al . , 2010 ) .",
"However , adult Pngl–/–; Act > NGLY1 WT animals do not show these phenotypes ( Figure 1C and D ) .",
"Together , these results underscore the in vivo functional homology between Pngl and NGLY1 .",
"A previous study did not detect PNGase activity in wild-type Drosophila larval extracts in an in vitro assay using 14C-labeled asialofetuin glycopeptide as a substrate and concluded that Drosophila Pngl might not possess N-glycanase activity ( Funakoshi et al . , 2010 ) .",
"To better assess whether Pngl can function as an N-glycanase , we used RTL ( RTAΔ-transmembrane-Leu2 ) spotting assays , which provide a reproducible in vivo model to assess the level of PNGase activity in yeast ( Hosomi et al . , 2010 ) .",
"RTL undergoes ERAD in a PNGase-mediated , deglycosylation-dependent manner and therefore leucine-auxotrophic yeast cells which express functional Saccharomyces cerevisiae PNGase ( Sc-Png1 ) are unable to grow in media lacking leucine .",
"However , yeast cells that lack Sc-Png1 or express a catalytically-inactive version of Sc-Png1 fail to degrade RTL and can therefore grow on leucine-deficient medium ( Hosomi et al . , 2010 ) .",
"As shown in Figure 1E , png1 mutant yeast cells ( png1Δ ) grow well in media with or without leucine when transfected with the RTL plasmid .",
"Expression of Sc-Png1-HA severely decreases the ability of these cells to grow in the absence of leucine , confirming that this phenotype is PNGase-dependent ( Figure 1E ) ( Hosomi et al . , 2010 ) .",
"An HA-tagged version of the fly Pngl suppressed the growth of the yeast cell in media without leucine to the same extent as the Sc-Png1 ( Figure 1E ) .",
"Meanwhile , Pngl harboring a C303A mutation in its putative catalytic domain ( Pngl-HA-C303A ) failed to rescue PNGase function ( Figure 1E ) , even though WT and C303A versions are expressed at comparable levels ( Figure 1F ) .",
"These observations strongly suggest that Drosophila Pngl is able to deglycosylate RTL and facilitate the efficient degradation of the RTL protein .",
"Next , we expressed a FLAG-tagged version of RTAΔ in yeast cells and asked whether Pngl can increase the level of the deglycosylated version of this protein upon inhibition of the protein biosynthesis .",
"In png1Δ mutant yeast cells , almost all RTAΔ was found to be in the glycosylated form ( g1 ) at three time points ( Figure 1G ) .",
"Expression of Sc-Png1 or fly Pngl increased the relative level of the deglycosylated form ( g0 ) in a time-dependent manner ( Figure 1G and H ) .",
"However , Pngl-C303A failed to remove glycans from RTAΔ .",
"Similar experiments indicate that human NGLY1 , but not the C309A catalytic mutant NGLY1 , can also compensate for the lack of Sc-Png1 in the RTL assay ( Figure 1I ) .",
"Of note , NGLY1-ΔR402 showed a weak rescue of the PNGase activity in this assay , suggesting that it is a hypomorphic allele .",
"Altogether , these observations indicate that Drosophila Pngl is indeed an N-glycanase enzyme and that there is high level of functional conservation between Pngl and its yeast and human homologs .",
"Pngl–/– larvae did not show any gross morphological defects .",
"However , inspection of the larval internal organs suggested defects in the mutant larval midguts .",
"In the anterior part of the midgut , control larvae harbor four finger-like structures called gastric caeca ( Figure 2A and B and Figure 2—figure supplement 1 ) , which play key roles in the insect digestive system , including water and ion transport and secretion of enzymes ( Pullikuth et al . , 2006; Volkmann and Peters , 1989 ) .",
"Pngl–/– larvae showed a severe shortening of the gastric caeca ( Figure 2C , red asterisks ) compared to y w and heterozygote controls ( Figure 2B and Figure 2—figure supplement 1 ) .",
"Control larvae harbor a specific region in the middle midgut called the ‘acid zone’ , which has a luminal pH of less than 2 . 3 and is considered the fly stomach ( Figure 2A ) ( Dubreuil , 2004 ) .",
"The acid zone can be visualized by feeding larvae with bromophenol blue ( BPB ) , which is blue in neutral and basic pH but turns yellow as the pH is decreased from 7 . 0 to 1 . 0 ( Figure 2D and Figure 2—figure supplement 1 , red dotted box ) .",
"Pngl–/– larvae are almost completely devoid of the yellow color in the midgut , indicating a loss of acid zone ( Figure 2E ) .",
"Similar to Pnglex14/ex14 larvae , Pnglex18/ex18 and Pnglex20/ex20 animals showed shortened gastric caeca and a loss of acid zone ( Figure 2—figure supplement 2 and not shown ) .",
"Moreover , Pnglex14 and Pnglex18 hemizygous animals ( over a deficiency allele ) showed semi-lethality and midgut phenotypes ( Figure 2—figure supplement 2 and not shown ) , suggesting that ex14 and ex18 deletions are genetic null alleles .",
"Altogether , these data indicate that loss of Pngl leads to specific midgut defects in Drosophila larvae .",
"In Drosophila embryos , a member of the bone morphogenetic protein ( BMP ) family called Decapentaplegic ( Dpp ) signals from VM to endoderm and is required for midgut specification ( Dubreuil , 2004; Panganiban et al . , 1990 ) .",
"Specifically , BMP signaling from VM in parasegment 3 ( PS3 ) and PS7 is transduced by phosphorylated Mothers against dpp ( pMad ) ( Newfeld et al . , 1996 ) in midgut endoderm , resulting in the formation of the gastric caeca and the acid zone , respectively ( Figure 2F ) .",
"Given the striking similarity between Pngl midgut phenotypes and those caused by impaired BMP signaling from VM ( Panganiban et al . , 1990 ) , we examined the effects of loss of Pngl on BMP signaling by staining Drosophila embryos with an antibody against human pSMAD3 , which recognizes Drosophila pMad ( Li et al . , 2016 ) .",
"Control embryos ( y w and Pngl+/– ) showed pMad staining in areas corresponding to PS3 and PS7 ( Figure 2G and G’ and Figure 2—figure supplement 1 ) .",
"However , Pngl–/– embryos showed a dramatic decrease in the level of pMad in PS3 and PS7 ( Figure 2H and H’ ) .",
"Notably , pMad staining in other regions of the embryos , including the ectodermal and head regions , was not affected by the loss of Pngl ( Figure 2H’ , compare to Figure 2G’ and Figure 2—figure supplement 1 ) .",
"Loss of BMP signaling in PS7 results in lack of second midgut constriction and impairs the expression of the acid-secreting ( copper ) cell-specific homeodomain gene labial in PS7 endodermal cells ( Immerglück et al . , 1990; Nellen et al . , 1994; Panganiban et al . , 1990 ) .",
"We used anti-Labial antibody to mark the precursors of the midgut copper cells in the embryonic endoderm and anti-Fas3 ( Fasciclin",
"3 ) antibody to mark VM ( Weiss et al . , 2001 ) and to visualize midgut constrictions and chambers .",
"As reported previously ( Nellen et al . , 1994 ) , at stage 15 , control embryos show a prominent second midgut constriction and Labial expression anterior to it ( Figure 2I and I’ and Figure 2—figure supplement 1 ) .",
"However , in stage 15 Pngl–/– embryos , no Labial signal was detectable in the PS7 region ( Figure 2J and J’ ) .",
"By stage 16 , all three constrictions are clearly visible in control larvae and Labial is expressed in the midgut compartment between first and second constrictions ( Figure 2K and K’ and Figure 2—figure supplement 1 ) .",
"However , stage 16 Pngl–/– embryos lack Labial staining and second constriction ( Figure 2L and L’ ) , in agreement with impaired BMP signaling in PS7 .",
"Together , these data demonstrate that Pngl is required for mesoderm-to-endoderm BMP signaling in Drosophila embryos .",
"To determine which tissues or cell types require the function of Pngl , we performed RNAi-mediated knock-down ( KD ) and rescue experiments .",
"When crossed to the mesodermal driver Mef2-GAL4 , the PnglRNAi KK101641 strain ( http://stockcenter . vdrc . at/control/main ) resulted in 100% lethality at room temperature and recapitulated the gastric caeca shortening and acid zone loss phenotypes observed in Pngl mutants ( Figure 3A–C ) .",
"In addition , Pngl KD by another mesodermal driver called how24B-GAL4 resulted in partial lethality and acid zone defects in larvae ( Figure 3A and E ) .",
"Pngl KD with how24B-GAL4 did not affect gastric caeca formation ( Figure 3D ) , likely because this driver induces transgene expression later than Mef2-GAL4 ( Figure 3—figure supplement 1 ) , after gastric caeca anlagen have already been formed .",
"However , Pngl KD by two endodermal drivers did not result in lethality and gut phenotypes ( Figure 3A and F–I ) .",
"Moreover , overexpression of human NGLY1 with Mef2-GAL4 and how24B-GAL4 rescued the lethality of the Pngl alleles ( Figure 3J ) .",
"In agreement with the severity of their corresponding KD phenotypes and expression patterns , Mef2-GAL4 rescued the lethality more efficiently than how24B-GAL4 ( Figure 3J ) .",
"Together , these data indicate that during Drosophila development Pngl is primarily required in the mesoderm to ensure animal survival and to promote BMP signaling from visceral mesoderm to endoderm .",
"It was previously shown that a Pngl transgene harboring the C303A mutation in the catalytic domain ( Figure 1E and G ) is not able to rescue the lethality of Pngl mutants ( Funakoshi et al . , 2010 ) .",
"To test whether the enzymatic activity of Pngl is required for the regulation of BMP signaling in the midgut , we overexpressed Pngl-C303A by Mef2-GAL4 in Pngl mutants .",
"As shown in Figure 3K and L , overexpression of wild-type Pngl rescues gastric caeca and acid zone defects of Pngl mutants .",
"However , the mutant version fails to rescue Pngl midgut phenotypes ( Figure 3M and N ) , even though Western blot shows that this mutation does not affect the expression level or stability of Pngl ( Figure 3O ) .",
"Together , these data indicate that the BMP signaling impairment observed in Pngl larval midguts is due to the lack of Pngl’s enzymatic activity .",
"Previous reports indicate that in the absence of midgut acidification , larvae are able to reach adulthood ( Dubreuil et al . , 2001 ) .",
"Moreover , regulatory mutations that result in specific loss of dpp expression in PS3 and a complete loss of gastric caeca only show a partial lethality ( Masucci and Hoffmann , 1993 ) .",
"Accordingly , impaired Dpp signaling in the midgut is not sufficient to fully explain the lethality of Pngl mutants , especially given that gastric caeca are not completely lost .",
"Indeed , animals undergoing dpp KD by the same mesodermal drivers showed a higher survival compared to those undergoing Pngl KD ( Figure 3P , compare to 3A ) .",
"Specifically , Mef2 > PnglRNAi and how24B > PnglRNAi animals showed 0% and ~28% survival rate into adulthood , but Mef2 > dppRNAi and how24B > dppRNAi animals showed ~8% and ~80% survival rate into adulthood , respectively .",
"The weaker effects of dppRNAi are not likely to result from inefficient dpp KD , as Mef2 > dppRNAi larvae exhibited a complete loss of gastric caeca and acid zone , and how24B > dppRNAi larvae lost the acid zone but not the gastric caeca , suggesting a loss or severe impairment of mesoderm-to-endoderm BMP signaling corresponding to the spatiotemporal expression pattern of each driver ( Figure 3Q–T ) .",
"These observations indicate that impaired Dpp signaling in the midgut can only partially account for the lethality observed in Pngl mutants .",
"So far our data suggest that Pngl also plays a Dpp-independent role in the mesoderm during Drosophila development .",
"We noticed that during the wandering phase , Pngl–/– larvae failed to empty their guts ( Figure 4A and A’ ) .",
"To better characterize this phenotype , we carried out midgut clearance assays .",
"When larvae are raised on regular food supplemented with bromophenol blue ( BPB ) , they show a dark blue gut approximately 24–48 hours before puparium formation ( early L3 stage ) .",
"At late L3 stage , they stop eating and enter the wandering stage , during which they empty their gut and reach the stationary stage ( Denton et al . , 2008 ) .",
"Pngl+/– ( control ) and Pngl–/– larvae both exhibit a blue gut during early L3 stage , indicative of eating food containing BPB ( Figure 4B ) .",
"Once taken off the BPB food during the wandering stage , control larvae gradually lose the blue color and pupariate , but the Pngl–/– larvae fail to empty their guts and exhibit a delay in pupariation ( Figure 4B ) .",
"Control y w and Pngl+/– larvae typically emptied their gut in 6 hours and reached the pre-pupal stage ( Figure 4C ) .",
"In contrast , most Pngl–/– larvae showed a blue gut throughout the wandering stage ( Figure 4C ) .",
"Indeed , even 24–48 hours later , more than 50% of the Pngl+/– animals were still in the larval stage and retained the blue food in their abdomens .",
"These data indicate that loss of Pngl causes an impairment of gut clearance at late L3 stage .",
"To examine whether impaired BMP signaling can also explain the food accumulation phenotype observed upon loss of Pngl , we compared the dpp and Pngl KD phenotypes by the same mesodermal drivers .",
"BPB feeding assays showed that Mef2 > dppRNAi larvae exhibit a food accumulation phenotype milder than that observed in Pngl mutant and Mef2 > PnglRNAi larvae , and that how24B > dppRNAi larvae do not exhibit any food accumulation phenotype , in contrast to how24B > PnglRNAi animals ( Figure 4D , compare to Figure 4E ) .",
"These data indicate that the food accumulation phenotype observed in Pngl mutants is at least in part independent of impaired BMP signaling .",
"Loss of Pngl affects BMP signaling in the midgut but not in the ectodermal regions of the Drosophila embryos .",
"To shed light on the mechanism by which Pngl regulates BMP signaling in a tissue-specific manner , we stained Pngl mutant and control embryos with a polyclonal anti-Dpp antibody raised against its prodomain ( Akiyama and Gibson , 2015 ) .",
"In WT and Pngl+/– embryos at stage 13 , the Dpp protein is expressed in ectodermal bands , several regions in the anterior part of the embryo and two rather narrow groups of cells corresponding to PS3 and PS7 , where it shows a punctate pattern superimposed on a diffuse signal ( Figure 5A , B , G and H ) .",
"Overall , the staining is somewhat weaker in Pngl–/– embryos , but staining in PS3 and PS7 is observed , with a notable decrease in puncta ( Figure 5C and I ) .",
"By stage 14 , a broad and strong Dpp expression domain can be observed in PS3 and PS7 in control embryos ( Figure 5D , E , J and K ) .",
"In contrast , Dpp expression in the VM remains weak and narrow in PS3 and PS7 of mutant embryos ( Figure 5F and L ) .",
"Notably , the pattern of Dpp expression in the ectoderm is similar in control and Pngl–/– embryos at both stages , even though the staining seems to be slightly weaker in mutant embryos ( Figure 5A–F ) .",
"These results suggest that a failure to expand the Dpp expression domain in the VM leads to impaired mesoderm-to-endoderm BMP signaling in Pngl mutants .",
"Given the weaker Dpp staining in stage 13 Pngl mutant embryos , we asked whether the failure to expand the Dpp expression domain and loss of BMP signaling in the embryonic midgut of Pngl mutants is simply due to reduction of dpp expression in the mesoderm .",
"To address this point , we used the dppS2 allele , which harbors a chromosomal breakpoint in the regulatory elements that control dpp expression in digestive tract ( Masucci and Hoffmann , 1993 ) .",
"Embryos heterozygous for this allele exhibit a decrease in Dpp expression at stage 13 in PS7 , even compared to Pngl–/– embryos ( Figure 5M , compare to Figure 5G , H and I ) .",
"Nevertheless , these embryos show Dpp propagation at stage 14 and proper Dpp signaling from mesoderm to endoderm , as evidenced by analyzing the expression of Dpp targets in the embryonic midgut and gastric caeca and acid zone in larvae ( Figure 5N and Figure 5—figure supplement 1 ) .",
"These data suggest that decreased Dpp expression by itself cannot explain the impaired Dpp propagation and loss of mesoderm-to-endoderm BMP signaling in Pngl mutants .",
"To understand how Dpp spreads in PS7 during embryonic midgut development , we expressed UAS-GFP in embryonic mesoderm by Mef2-GAL4 driver and stained embryos for pMad at stage 13 and 14 .",
"At stage 13 the majority of pMad staining is localized to the VM and only a few cells are stained in the endoderm ( Figure 5O and O’ ) .",
"Later at stage 14 , BMP signaling spreads through the endoderm and expands in PS7 , as evidenced by the presence of many pMad-positive endodermal cells ( Figure 5P and P’; GFP– , round nuclei ) .",
"These observations are in agreement with previous reports suggesting the existence of an autoregulatory Dpp para-autocrine loop in PS7 , which acts through Ultrabithorax ( Ubx ) expression in VM and results in maintenance and accumulation of Dpp expression in VM cells ( Figure 5Q ) ( Bienz , 1997; Hursh et al . , 1993; Staehling-Hampton and Hoffmann , 1994 ) .",
"To test whether Pngl affects Dpp autoactivation in VM , we used a dpp enhancer trap line and stained embryos for pMad and βGAL .",
"In Pngl+/– embryos at stage 13 , we found a row of cells co-expressing pMad and βGAL ( Figure 5R–R’ ) , suggesting para-autocrine activation .",
"In PS7 of Pngl–/– embryos at stage 13 , dpp-lacZ expression can be detected , but pMad staining is absent ( Figure 5S–S’ ) .",
"These results indicate that in Pngl–/– PS7 , the initial , Ubx-dependent dpp expression ( Sun et al . , 1995 ) occurs , but the autoactivation loop is impaired .",
"At stage 14 , the Dpp expression domain expands in Pngl+/– embryos and dpp-lacZ expressing cells can be seen in multiple layers .",
"Most if not all dpp-lacZ expressing cells still co-express pMad , likely due to autoactivation , while endodermal cells , as receiving cells , only express pMad ( Figure 5T–T’ ) .",
"In stage 14 Pngl–/– embryos , dpp-lacZ-expressing cells remain confined to their initial narrow domain in PS7 , and pMad staining is still not detectable ( Figure 5U–U’ ) .",
"This is in contrast to the dorsal ectodermal region , which showed a comparable expression pattern and intensity in the dorsal ectodermal bands of Pngl+/– and Pngl–/– embryos for both dpp-lacZ and pMad ( Figure 5—figure supplement 2 ) .",
"Altogether , these data indicate that Pngl is not required for the initial expression of dpp through Ubx in Drosophila embryos but is required for the autoregulatory role of Dpp in the VM .",
"We next asked whether bypassing the BMP autoactivation loop by mesodermal overexpression of a GFP-tagged version of Dpp ( Teleman and Cohen , 2000 ) can rescue Pngl phenotypes in embryos .",
"In control animals , dpp-GFP overexpression by Mef2-GAL4 resulted in broad pMad staining in embryonic midgut and ectopic pMad expression throughout the ectoderm ( Figure 6A and D ) .",
"Dpp-GFP protein can be seen by GFP staining and shows a continuous pattern going through PS3 to PS7 ( Figure 6D’ ) .",
"In Pngl–/– embryos , mesodermal expression of Dpp-GFP restored pMad expression in PS3 and PS7 , indicating a rescue of BMP signaling from VM to endoderm ( Figure 6B and E , compare to Figure 6C and F ) .",
"Notably , in Pngl–/–; Mef2 > dpp GFP embryos , pMad staining was limited to PS3 and PS7 , and ectopic pMad expression in the ectoderm was almost completely suppressed ( Figure 6B and E ) , indicating that loss of Pngl dramatically decreases the ability of mesodermally-expressed Dpp-GFP to induce ectopic BMP signaling .",
"If BMP autoactivation is impaired in Pngl–/– VM , bypassing the normal autoactivation process by expressing a constitutively active form of the BMP receptor Tkv ( tkvCA ) in the mesoderm should restore BMP signaling in the PS3 and PS7 endoderm .",
"Mef2 > tkvCA embryos exhibited proper pMad staining in PS3 and PS7 in a Pngl+/+ background , although some pMad staining outside of PS7 could be seen in the VM marked by Fas3 ( Figure 6G and G’ , arrows ) .",
"When tkvCA is overexpressed in Pngl–/– embryonic mesoderm , pMad was restored in PS3 and PS7 and still some extra pMad positive cells were detected ( Figure 6H and H’ , arrows ) .",
"Moreover , ~30% of Pngl–/–; Mef2 > tkvCA animals reach adulthood ( Figure 6I ) .",
"These observations further indicate that impaired BMP signaling is partially responsible for the lethality of Pngl–/– animals .",
"This is likely due to gastric caeca shortening ( Masucci and Hoffmann , 1993 ) , although we cannot exclude that tkvCA overexpression affects unknown BMP-related defects in other parts of mesoderm in these mutants .",
"We note that the rescued Pngl–/–; Mef2 > tkvCA adults did not show the acid zone loss and midgut shortening phenotypes observed in Pngl–/– adult escapers ( Figure 6—figure supplement 1 ) .",
"This indicates that the BMP-related midgut phenotypes of Pngl mutants persist to adulthood .",
"Altogether , these results support the notion that BMP signaling in VM requires an autoregulatory step to reach the required threshold for signaling to endoderm and that this is the step during midgut development that is impaired in Pngl mutants .",
"The data also indicate that this developmental defect contributes to the semi-lethality of Pngl mutants .",
"BMPs can function as homodimers or heterodimers to signal through homo- and/or heterodimeric type I receptors ( Hogan , 1996; O'Connor et al . , 2006 ) .",
"To date , three BMP ligands have been identified in Drosophila: dpp , glass bottom boat ( gbb ) and screw ( scw ) ( Arora et al . , 1994; Doctor et al . , 1992; Padgett et al . , 1987; Wharton et al . , 1999 ) .",
"scw is required in early embryogenesis , where it acts in combination with dpp to specify dorsal cells fates ( Arora et al . , 1994 ) .",
"gbb and dpp mutants have both distinct and overlapping phenotypes during and after embryonic development , suggesting that gbb and dpp are required together for proper signaling in some developmental contexts ( Goold and Davis , 2007; Haerry et al . , 1998; Hong et al . , 2016; Khalsa et al . , 1998; Wharton et al . , 1999 ) .",
"To assess whether gbb is involved in BMP signaling and Dpp expression in embryonic midgut and the generation of Pngl–/– phenotypes , we performed Dpp and pMad staining in gbb mutant embryos at stage 14 , focusing on PS3 and PS7 .",
"Unlike control embryos ( Figure 7A and Figure 7—figure supplement 1 ) , gbbD4/D20 and gbbD4/D4 embryos showed reduced pMad staining in PS3 ( Figure 7C and Figure 7—figure supplement 1 ) , in agreement with previous work indicating that gastric caeca are somewhat short in gbb mutant larvae ( Wharton et al . , 1999 ) .",
"However , in gbb–/– embryos pMad staining in PS7 is increased and shifts anteriorly towards PS3 ( Figure 7C and Figure 7—figure supplement 1 ) .",
"In gbbD4/D20 embryos , the Dpp expression domain in PS3 is similar in size to that in control embryos ( Figure 7—figure supplement 2 ) .",
"However , the Dpp expression domain in PS7 is broader and stronger than controls in these embryos and extends anteriorly to PS6 , and additional Dpp-positive puncta can be detected outside of the acid zone region towards PS3 ( Figure 7—figure supplement 2 ) .",
"At stage 16 , gbb–/–embryos show an abnormal expansion of the second midgut compartment and a diffuse Labial staining both anteriorly and posteriorly to the second constriction ( Figure 7D and D’ and Figure 7—figure supplement",
"1 ) compared to controls ( Figure 7B and B’ and Figure 7—figure supplement 1 ) .",
"Therefore , gbb phenotypes in the midgut are quite different from dpp and Pngl loss-of-function phenotypes .",
"The strong and broader than normal pMad expression domain in gbb–/– PS7 indicates that BMP signaling in this region is mediated by Dpp homodimers alone and Gbb likely limits the area in the VM wherein BMP autoactivation and signaling can occur .",
"In Drosophila , two type I receptors Tkv and Saxophone ( Sax ) and one type II receptor ( Punt ) are involved in BMP signaling ( Letsou et al . , 1995; Nellen et al . , 1994; Nellen et al . , 1996; Ruberte et al . , 1995 ) .",
"Given the differential roles of the Dpp versus Gbb ligands in BMP autoactivation in VM , we asked whether the contribution of Tkv and Sax to this process is also different from each other .",
"As shown in Figure 6E , mesodermal knock-down of tkv results in a severe reduction of pMad staining in PS7 and a partial loss of pMad in PS3 .",
"Similar to Pngl–/– embryos , the Dpp expression domain in PS7 fails to expand in Mef2 > tkvRNAi embryos , although tkv KD does not seem to affect the intensity of Dpp staining at stage 13 or the presence of Dpp-positive puncta ( Figure 7—figure supplement 2 ) .",
"In Mef2 > saxRNAi embryos , pMad staining in PS3 seems to be slightly decreased compared to controls , but it looks broader in PS7 and is extended anteriorly ( Figure 7G ) .",
"Furthermore , Mef2 > tkvRNAi embryos show a lack of second constriction and loss of Labial in the PS7 region ( Figure 7F and F’ ) .",
"In contrast , mesodermal knock-down of sax did not show evident midgut constriction defects , but aberrant Labial staining was detectable both anteriorly and posteriorly to the second constriction , similar to gbb–/– embryos ( Figure 7H and H’ ) .",
"These results strongly suggest that BMP autoactivation in PS7 mesoderm is mediated by Dpp homodimers , as ligand , and Tkv homodimers , as receptor .",
"Similar to its vertebrate homologs , Dpp is synthetized as an inactive proprotein ( Figure 7I ) .",
"Following dimerization in the endoplasmic reticulum ( ER ) , Dpp proprotein dimers traffic through the secretory pathway , where they undergo cleavages to release the active dimer ( Christian , 2012; Künnapuu et al . , 2009; Sopory et al . , 2010 ) .",
"Since our results indicate a role for Pngl in Dpp autoactivation mediated by Dpp homodimers ( as opposed to Dpp-Gbb heterodimers ) in PS7 VM , we examined the effects of loss of Pngl on Dpp dimerization and processing by Western blotting on larval extracts using the above-mentioned anti-Dpp antibody ( Akiyama and Gibson , 2015 ) .",
"When ran on a reducing gel , control larvae ( y w and UAS-PnglRNAi without a GAL4 driver ) exhibited two prominent bands: one corresponding in size to the full-length proprotein monomer and the other to the proprotein dimer , suggesting that Dpp dimers are partially resistant to the amount of reducing agents used in our assays ( Figure 7J ) .",
"A third band slightly bigger and much fainter than the proprotein monomer was also observed in control larvae ( Figure 6J , red asterisk ) .",
"Larvae homozygous for Pngl alleles and those ubiquitously expressing Pngl dsRNA showed an accumulation of a band corresponding in size to the prodomain ( cleavage product ) and a significant decrease in the intensity of the band corresponding to Dpp dimers , without an apparent decrease in the intensity of the proprotein monomer band ( Figure 7J ) .",
"Of note , although larvae heterozygous for the Pnglex14 allele did not show any midgut defects ( Figure 2—figure supplement 1 ) , they displayed an intermediate pattern in the Western blot , suggesting that Pngl plays a dosage-sensitive role in Dpp dimerization and/or processing ( Figure 7J ) .",
"To better assess the level of Dpp dimers in a Pngl-deficient background , we performed Western blot analysis on larval extracts ran on a non-reducing SDS gel electrophoresis ( Figure 7K ) .",
"Control larvae showed two rather strong bands corresponding in size to Dpp dimers and a weaker band corresponding to a proprotein monomer .",
"This indicates that in a non-denaturing state , Dpp molecules are primarily found in association with other molecules and form Dpp-Dpp homodimers .",
"We found a significant decrease in Dpp dimers in Pngl mutant and knock-down larvae and a corresponding increase in the level of full-length monomer and the same cleavage product observed in regular Western blots .",
"Again , Pnglex14/+ larvae showed an intermediate band pattern , with a significant decrease in the intensity of one of the two ‘dimer’ bands and an apparent increase in the intensity of the full-length monomer ( Figure 7K ) .",
"Dpp dimers are not exclusively reduced in midguts of Pngl mutants but are also decreased in other parts of the larval body ( Figure 7—figure supplement 3 ) .",
"In summary , these data provide strong evidence that Pngl is required for the formation and/or the stability of Dpp dimers ."
],
[
"The broad phenotypes of children affected with NGLY1 deficiency ( Enns et al . , 2014; Need et al . , 2012 ) and the semi-lethality of Pngl–/– flies ( Funakoshi et al . , 2010 ) indicate that NGLY1 plays important roles during animal development .",
"However , the N-glycanase function has not been linked to any developmental signaling pathway .",
"Here we report that fly Pngl regulates BMP signaling during embryonic midgut development without affecting BMP signaling in ectodermal and head regions of the embryo .",
"Our data indicate that Pngl is not required in the midgut endoderm to receive the BMP signal , but rather is required in the VM to send the BMP signal .",
"It has previously been shown that BMP signaling uses a paracrine/autocrine loop in the VM to sustain and increase the expression of Dpp in PS3 and PS7 of embryonic VM .",
"This loop is proposed to ensure that the level of BMP ligands in the VM is high enough to induce signaling in the endoderm and to specify gastric caeca , the second midgut constriction and the acid zone ( Bienz , 1997; Hursh et al . , 1993; Staehling-Hampton and Hoffmann , 1994 ) .",
"Several lines of evidence indicate that the BMP autoregulation mediated by the para-autocrine loop in the VM is the step which is impaired in Pngl-deficient embryos .",
"First , Pngl is not required for the initial , Ubx-dependent expression of dpp .",
"In fact , even a 50% decrease in the expression of dpp in the visceral mesoderm of dpps2/+ animals does not impair BMP autoactivation and midgut development .",
"Second , despite expressing Dpp at early stages , BMP signaling is not activated in Pngl–/– VM , as evidenced by the lack of pMad staining .",
"Third , overexpression of Dpp-GFP in the mesoderm is able to induce BMP signaling in the endoderm in Pngl–/– embryos .",
"Lastly , bypassing the para-autocrine loop by transgenic expression of a constitutively active BMP receptor in the mesoderm results in restoration of BMP signaling in PS3 and PS7 regions of the endoderm and in partial rescue of lethality in Pngl–/– embryos .",
"In the BMP para-autocrine loop , VM cells both secrete the BMP ligand and respond to it .",
"Therefore , theoretically , Pngl might play a critical role in sending the BMP signal , receiving the BMP signal , or both .",
"Although our data do not allow us to exclude any of these possibilities , based on the following observations , we favor a scenario in which Pngl is required in VM cells to send the Dpp signal not to receive it: ( 1 ) Pngl is not required to receive the BMP signal in the endoderm; ( 2 ) Loss of Pngl and Pngl KD result in a dramatic decrease in the level of Dpp homodimers and the Dpp-positive puncta; ( 3 ) Expression of a constitutively active form of Tkv in the mesoderm is able to restore midgut pMad staining in embryos and the copper cell region in the adult midgut , and partially rescue the lethality of Pngl–/– animals; ( 4 ) Loss of Pngl almost fully suppresses the aberrant BMP signaling caused by mesodermal overexpression of Dpp-GFP .",
"Whole larval protein extracts from Pngl-deficient animals show an increase in the level of the monomeric forms of Dpp ( full-length and a cleavage product ) and a simultaneous decrease in the bands corresponding in size to Dpp dimers .",
"Moreover , Pngl–/– embryos show a decrease in Dpp-positive puncta both in the mesoderm , where signaling is impaired , and in the ectoderm , where signaling is not impaired .",
"Together , these observations indicate that the effect of loss of Pngl on the Dpp protein itself is not limited to the mesoderm .",
"Indeed , protein extracts from Pngl-deficient midgut and carcass ( without midgut ) both show a decrease in Dpp dimer levels .",
"This suggests that either Pngl regulates BMP signaling by affecting Dpp dimer levels in other larval tissues not identified yet , or that Dpp dimers are only important in the midgut and although they are decreased elsewhere , Dpp-Gbb heterodimers compensate for the lack of Dpp dimers in most other tissues .",
"Regardless , we propose that loss of BMP signaling in Pngl mutant midguts results from a requirement for Dpp homodimers in the para-autocrine autoregulatory loop present in the visceral mesoderm .",
"BMP ligands can signal both as homodimers and as heterodimers ( Bragdon et al . , 2011; O'Connor et al . , 2006 ) .",
"In vitro and in vivo studies have shown that in general , BMP heterodimers have stronger bioactivity than their homodimers counterparts ( Aono et al . , 1995; Butler and Dodd , 2003; Israel et al . , 1996; Little and Mullins , 2009; Morimoto et al . , 2015; Valera et al . , 2010 ) .",
"In some cases , the homodimers induce weak to moderate signaling , and in other cases they either do not elicit signaling or even play an antagonistic role ( Bangi and Wharton , 2006b; O'Connor et al . , 2006 ) .",
"Stronger activity of BMP heterodimers can at least in part be explained by differential affinities of individual BMP ligands for different BMP receptors , combined with stronger signal transduction by heterodimeric type I receptors compared to homodimers of each type I receptor .",
"For example , in Drosophila , Dpp has a higher affinity for Tkv , whereas the other two ligands–Gbb and Scw–have a higher affinity for Sax ( Bangi and Wharton , 2006b; O'Connor et al . , 2006; Shimmi et al . , 2005 ) .",
"A similar receptor-ligand binding preference has been observed among the vertebrate orthologs ( Aoki et al . , 2001; Little and Mullins , 2009 ) .",
"In the embryonic dorsal midline and the wing imaginal disc , Dpp/Scw and Dpp/Gbb heterodimers induce high levels of signaling , respectively , through Tkv/Sax heterodimers ( Bangi and Wharton , 2006b; O'Connor et al . , 2006 ) .",
"Comparison of the gbb mutant phenotypes in the midgut with those caused by Pngl loss and by dpp KD indicates that Dpp homodimers are the only productive form of ligand in PS7 .",
"Moreover , mesodermal KD of tkv severely decreases BMP signaling in PS7 , but mesodermal KD of sax not only does not decrease pMad staining in PS7 , but also results in an expansion of pMad expression domain in the PS7 region , similar to gbb mutant embryos .",
"Together , these observations strongly support the notion that the BMP autoregulatory loop in the VM , which is essential for the activation of BMP signaling in the endoderm , relies solely on Dpp and Tkv homodimers , and therefore is impaired in Pngl mutants due to the severe decrease in the level of Dpp homodimers in these animals .",
"Vertebrate and invertebrate BMP proteins and other members of the TGFβ superfamily each harbor several N-linked glycosylation sites , which have been shown to be glycosylated in many cases ( Groppe et al . , 1998; Miyazono and Heldin , 1989; Tauscher et al . , 2016 ) .",
"Various functional roles have been ascribed to N-glycans on these ligands , including enhancing receptor binding of BMP6 ( Saremba et al . , 2008 ) , keeping the TGFβ1 ligand in a latent state ( Miyazono and Heldin , 1989 ) , and promoting inhibin ( α/β ) heterodimer formation at the expense of activin ( β/β ) homodimer formation ( Antenos et al . , 2007 ) .",
"Accordingly , given the significant increase in Dpp monomeric forms and the simultaneous decrease in Dpp dimers upon loss of Pngl , it is possible that Pngl removes one or more N-glycans from Dpp and thereby promotes the formation or the stability of Dpp homodimers .",
"Whether the regulation of Dpp by Pngl is direct or mediated via other proteins will remain to be explored .",
"In agreement with a previous report ( Masucci and Hoffmann , 1993 ) , our data suggest that the lethality of Pngl mutants cannot be fully explained by shortening of the gastric caeca and impairment of BMP signaling in midgut development .",
"Pngl KD with mesodermal drivers leads to a higher degree of lethality compared to dpp KD with the same drivers .",
"Moreover , how24B > PnglRNAi animals show ~70% lethality , even though they do not have gastric caeca defects .",
"Finally , restoring BMP signaling in the midgut by expressing tkvCA only recues the lethality in ~30% of Pngl–/– animals .",
"Phenotypic analysis of Pngl mutants combined with rescue and KD experiments suggest that a failure to properly empty the gut before puparium formation contributes to lethality in these animals .",
"The molecular mechanisms for the food accumulation phenotype and other potential Pngl–/– phenotypes contributing to lethality are still under investigation .",
"In summary , our work indicates that the fly Pngl is an evolutionarily conserved N-glycanase enzyme necessary to sustain BMP autoactivation in the VM mediated by para-autocrine activity of Dpp homodimers through Tkv homodimers .",
"Although we cannot exclude that Pngl plays important roles in other cell types as well , our data indicate that Pngl is primarily required in the mesoderm during midgut development and its loss results in Dpp-dependent and Dpp-independent midgut defects .",
"Given the reports on potential para-autocrine functions of mammalian Dpp homologs ( Grimsrud et al . , 1999; Rege et al . , 2015; Shukunami et al . , 2000; Tokola et al . , 2015 ) and prominent human pathologies associated with dysregulated BMP signaling in ophthalmic , gastrointestinal and musculoskeletal systems ( Wang et al . , 2014 ) , tissue-specific alterations in BMP signaling might contribute to some of the NGLY1 deficiency phenotypes including retinal abnormalities , delayed bone age and osteopenia , small feet and hands , and chronic constipation ( Enns et al . , 2014; Lam et al . , 2017 ) .",
"Finally , understanding the mechanisms underlying the food accumulation phenotype in Pngl–/– larvae might shed light on the pathophysiology of chronic constipation in NGLY1 deficiency patients ."
],
[
"Animals were grown on standard food containing cornmeal , molasses and yeast at room temperature , except for RNAi crosses , which were cultured at 30°C .",
"The following strains were used in this study: y w , w; L/CyO , kr-GAL4 UAS-GFP ( CyO-GFP ) , TM3 , Sb1/TM6 , Tb1 , Act-GAL4 , Mef2-GAL4 , how24B-GAL4 ( Brand and Perrimon , 1993; Staehling-Hampton et al . , 1994 ) , dpp10638 ( dpp-lacZ ) ( Zecca et al . , 1995 ) , dpps2 ( Masucci and Hoffmann , 1993 ) , UAS-dpp-GFP ( Teleman and Cohen , 2000 ) , UAS-dppRNAi ( Liu et al . , 2010 ) , UAS-tkvRNAi , gbbD4 and gbbD20 ( Chen et al . , 1998 ) , UAS-tkvCA ( Adachi-Yamada et al . , 1999 ) , Df ( 2R ) ED1484 , UAS-CD8::GFP ( Bloomington Drosophila Stock Center ) , Pnglex14 , Pnglex18 , Pnglex20 , UAS-Pngl and UAS-Pngl-C303A ( Funakoshi et al . , 2010 ) , NP3207-GAL4 and NP3270-GAL4 ( Tanaka et al . , 2007 ) ( Kyoto Drosophila Stock Center ) , UAS-PnglRNAi KK101641 , UAS-saxRNAi ( Vienna Drosophila Resource Center ) , UAS-attB-NGLY1-WT-VK31 and UAS-attB-NGLY1-ΔR402-VK31 ( this study ) .",
"To identify homozygous animals in sibling crosses , Pngl and gbb mutants were balanced over a CyO , kr-GAL4 UAS-GFP chromosome .",
"To examine dpp transcription in a Pngl–/– background , a dpp-lacZ Pnglex14/CyO-GFP recombinant strain was generated and crossed to Pnglex14/CyO GFP animals .",
"To overexpress NGLY1 in Pngl–/– animals , Pngl– ( ex14 or ex18 ) /CyO; GAL4 ( Actin , Mef2 or how24B ) /TM6 , Tb1 animals were crossed to Pngl–/CyO; UAS-attB-NGLY1-VK31 ( WT or ΔR402 ) /TM6 , Tb1 animals .",
"To overexpress TkvCA or Dpp-GFP in a Pngl–/– background , Pnglex14/CyO-GFP; Mef2-GAL4 animals were crossed to Pnglex14/CyO-GFP; UAS-tkvCA ( or UAS-dpp-GFP ) and absence of the CyO-GFP was used to select the intended genotype .",
"To overexpress Pngl and Pngl-C303A in Pngl–/– animals , Pnglex14/CyO-GFP; Mef2-GAL4 animals were crossed to Pnglex14/CyO-GFP; UAS-Pngl and Pnglex14/CyO-GFP; UAS-Pngl-C303A animals and selected similar to the above crosses .",
"For survival ( eclosion ) tests , the expected ratio of offspring was calculated based on Mendelian inheritance for each genotypic class and the observed/expected ratio is reported as a percentage .",
"Fertility was assessed by placing 2-day-old single male of each genotype with three 4-day-old virgin y w females or three 4-day-old virgin females of each genotype with three 4-day-old virgin y w males .",
"Flies were transferred to fresh vials every 5 days for three times .",
"The total number of progeny produced over 20 days by each animal was counted .",
"Data are represented as mean ±SD of 3 independent sets of experiments .",
"For longevity analyses , newly eclosed males of each genotype were collected and housed at a density of 5 flies per vial .",
"Flies were transferred to fresh food every 3–4 days , and dead flies were counted every day until all died .",
"Data are represented as mean of 3 independent sets of experiments .",
"Human NGLY1 cDNA in pCMV6-AC vector ( clone SC320763 , OriGene ) was used as template for site-directed mutagenesis to introduce the c . 1205_1207del clinical mutation ( Enns et al . , 2014 ) , which results in the generation of NGLY1-ΔR402 .",
"Wild-type and ΔR402 cDNAs were transferred from pCMV6-AC to pUAST-attB vector by EcoRI-XhoI double digestion and ligation , verified by sequencing , and integrated into the VK31 docking site by ΦC31-mediated transgenesis ( Bischof et al . , 2007; Venken et al . , 2006 ) .",
"Larvae were raised on standard food supplemented with 0 . 05% bromophenol blue ( BPB ) .",
"Wandering larvae were collected from the side of the vial with a wet paint brush , transferred to a petri dish lined with wet Whatman paper and monitored for gut clearance until puparium formation .",
"About 30 larvae were scored for each genotype at each time point .",
"Each data point is from three independent experiments .",
"For larval gut acidification studies , 72 hours after egg deposition , larvae were transferred to standard food containing 0 . 05% BPB and dissected after 12 hours .",
"For examination of adult acid zones , one-day old animals were fed on food supplemented with 0 . 05% BPB for two days and then dissected .",
"The images were taken by ToupCam Camera and analyzed by ToupView software .",
"RTL spotting assay was carried out using png1Δ cells ( png1::KanMX4 Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ) and pRS313-GAL4RTL essentially as described previously ( Masahara-Negishi et al . , 2012 ) .",
"In brief , strains harboring the RTL expression plasmid were spotted on to SC-histidine-uracil or SC-histidine-uracil-leucine medium containing 2% galactose ( w/v ) , and plates were incubated at 30°C for 3 days .",
"Photographs of the plates were taken using FUJIFILM LAS-3000 mini ( Fujifilm Co . , Tokyo , Japan ) .",
"png1Δ cells harboring the pRS315-GPDFLAG-RTAΔ ( Hosomi et al . , 2010 ) were grown at 30°C in SC-leucine liquid medium .",
"Cycloheximide was added at t = 0 min ( final concentration , 4 μg/ml ) , and the samples were collected at the indicated times and subjected to SDS-PAGE , followed by immunoblotting with anti-DYKDDDDK antibody 1:10 , 000 ( Wako Cat# 018–22381 , RRID:AB_10659453 ) .",
"Phosphoglycerate kinase ( Pgk1 ) was used as a loading control and was probed with anti-Pgk1 antibody 1:10 , 000 ( Molecular Probes Cat# A-6457 , RRID:AB_221541 ) .",
"Proteins were extracted from whole larvae in lysis buffer containing protease inhibitor cocktail ( Promega ) .",
"The following antibodies were used: rabbit anti-NGLY1 1:500 ( Sigma-Aldrich Cat# HPA036825 , RRID:AB_10672231 ) , mouse anti-tubulin 1:1000 ( Santa Cruz Biotechnology Cat# sc-8035 , RRID:AB_628408 ) , mouse anti-actin 1:1000 ( DSHB Cat# 224-236-1 , RRID:AB_10571933 ) , rabbit anti-Pngl 1:250 ( Funakoshi et al . , 2010 ) , rabbit anti-Dpp 1:1000 ( Akiyama and Gibson , 2015 ) , mouse anti-HA 1:20 , 000 ( Sigma-Aldrich Cat# B9183 , RRID:AB_439706 ) , goat anti-rabbit-HRP and goat anti-mouse-HRP 1:2000 ( Jackson ImmunoResearch Laboratories ) .",
"Western blots were developed using Pierce ECL Western Blotting Substrates ( Thermo Scientific ) .",
"The bands were detected using an ImageQuant LAS 4000 system from GE Healthcare .",
"At least three independent immunoblots were performed for each experiment .",
"The following antibodies were used: rabbit anti-pSMAD3 1:250 ( Abcam Cat# ab52903 , RRID:AB_882596 ) , guinea pig anti-Labial 1: 1000 ( Guo et al . , 2013 ) , rabbit-anti Dpp 1:100 ( Akiyama and Gibson , 2015 ) , mouse anti-βGAL 1:50 ( DSHB Cat# 40-1a , RRID:AB_528100 ) , mouse anti-Fas3 1:50 ( DSHB Cat# 7G10 anti-Fasciclin III , RRID:AB_528238 ) , mouse anti-GFP 1:500 ( Thermo Fisher Scientific Cat# 33–2600 , RRID:AB_2533111 ) , goat anti-rabbit-Cy3 1:500 , goat anti-mouse-Cy5 1:500 ( Jackson ImmunoResearch Laboratories ) .",
"Confocal images were taken with Leica TCS-SP8 microscope .",
"All images were acquired using Leica LAS-SP software .",
"Amira 5 . 2 . 2 and Adobe Photoshop CS6 were used for processing and Figures were assembled in Adobe Illustrator CS6 ."
]
] | [
"Mutations in the human N-glycanase 1 ( NGLY1 ) cause a rare , multisystem congenital disorder with global developmental delay .",
"However , the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known .",
"Here we show that Drosophila Pngl encodes an N-glycanase and exhibits a high degree of functional conservation with human NGLY1 .",
"Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling .",
"Pngl mutant larvae also exhibit a severe midgut clearance defect , which cannot be fully explained by impaired BMP signaling .",
"Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development .",
"Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers .",
"Thus , our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N-glycanase enzyme ."
] | [
"DNA carries the information needed to build and maintain an organism , and units of DNA known as genes contain coded instructions to build other molecules , including enzymes .",
"Sometimes , genes can become faulty and develop mutations that can affect how an embryo develops and lead to diseases .",
"For example , people with mutations in the gene that encodes an enzyme called N-glycanase 1 experience many problems with their nervous system , gut and other organs .",
"Normally , N-glycanase 1 helps the body remove specific sugar molecules from some proteins in the cells , and is also thought to be important during embryonic development .",
"As an embryo develops , its cells undergo a series of transformations , which is regulated by different molecules and signaling pathways .",
"For example , a pathway known as BMP signaling plays an important role in many tissues .",
"Problems with this pathway can lead to many diseases throughout the body , including the gut , where it helps cells to develop .",
"Previous research has shown that fruit flies lacking the gene that codes for an equivalent N-glycanase enzyme ( which is called Pngl in flies ) cannot develop properly into adults .",
"However , until now it was not known what type of cells need the N-glycanase enzyme in any organism , or if NGLY1 is essential for important signaling pathways like BMP signaling .",
"Now , Galeone et al . have used genetically modified flies to test how losing Pngl affected their development .",
"The results first showed that engineering Pngl-deficient fruit flies to produce the human enzyme eliminated their problems; these flies developed and survived like normal flies .",
"This confirmed that that the human and fly enzymes can perform equivalent roles .",
"Galeone et al . then discovered that Pngl plays two distinct roles in a group of cells that surround the fruit fly’s gut tissue and give rise to the cells that eventually form the muscle layer in the gut .",
"In the larvae , Pngl was required to empty the gut , which is a necessary step before the larvae can develop into an adult .",
"Moreover , Pngl is needed for BMP signaling in the gut , and when flies had the enzyme removed , some parts of their gut could not from properly .",
"This study will provide a framework to improve our understanding of how BMP signaling is regulated in humans .",
"A next step will be to test if some of the symptoms experienced by patients without a working copy of the gene for N-glycanase 1 are caused by a faulty BMP-signaling system in specific tissues .",
"If this is the case , it could provide new opportunities to treat some of these symptoms ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | Etv transcription factors functionally diverge from their upstream FGF signaling in lens development | elife-51915-v2 | [
[
"The cell signaling networks are commonly depicted in a hierarchical manner , starting with the binding of extracellular ligands to cell surface receptors , followed by the relay of cytoplasmic mediators , and culminating in the activation of nuclear transcription factors .",
"In this unidirectional view , each signal-regulated transcription factor ( SRTF ) is expected to control a subset of the transcriptional output of upstream signaling ( Cvekl and Zhang , 2017 ) .",
"This model has been confirmed in many systems .",
"For example , the transcriptomic changes induced by BMP , Hedgehog and Wnt signaling can be readily accounted for by their transcription effectors Smad , Gli and β-catenin , respectively .",
"However , whether this principle applies to the SRTFs for FGF signaling remains to be determined .",
"The 28 mammalian E26 transformation-specific ( ETS ) proteins share a highly conserved winged helix-turn-helix DNA binding domain , which recognizes a core GGA sequence motif ( Charlot et al . , 2010 ) .",
"Post-translational modifications of these factors , especially serine/threonine phosphorylation by ERK , directly affect their subcellular localization , DNA binding and transactivation .",
"In particular , FGF-ERK signaling induces expression of the Etv ( Pea3 ) subfamily of ETS transcription factors , Etv1 ( Er81 ) , Etv4 ( Pea3 ) and Etv5 ( Erm ) , in addition to enhancing their transcriptional activities during embryonic development ( Münchberg and Steinbeisser , 1999; Raible and Brand , 2001; Roehl and Nüsslein-Volhard , 2001 ) .",
"Conditional knockouts of these Etv genes disrupted the anterior-posterior patterning of the limb bud and branching morphogenesis of the lacrimal gland controlled by FGF signaling ( Garg et al . , 2018; Zhang et al . , 2009 ) .",
"The significance of Etv factors have also been demonstrated in studies of human cancer , where aberrant activation of ETV genes has been proposed to emulate oncogenic RAS in cellular transformation ( Hollenhorst et al . , 2011 ) .",
"Thus , the Etv transcription factors Etv1 , Etv4 and Etv5 are considered to be SRTFs that are directly downstream of FGF-Ras-ERK signaling .",
"FGF signaling is required during several steps of vertebrate lens development , including induction of the lens vesicle , proliferation of lens epithelial cells , and differentiation of lens fiber cells ( Cvekl and Zhang , 2017; Faber et al . , 2001; Robinson , 2006 ) .",
"The mature lens consists of a single layer of epithelial cells in the anterior and differentiated lens fiber cells in the posterior; the latter accounts for the bulk of the lens tissue .",
"FGF signaling has been proposed to act in a gradient fashion , promoting proliferation in the lens epithelium at low signaling strength and stimulating differentiation in the lens fiber cells at high strength ( Lovicu and McAvoy , 2001; McAvoy and Chamberlain , 1989; McAvoy et al . , 1991 ) .",
"In support of this model , genetic knockouts of FGF receptors disrupted the expression of lens-specific genes Maf , Prox1 , Pax6 , Cdh1 and Crystallins , affecting survival and proliferation of lens epithelial cells and elongation of fiber cells ( Chow et al . , 1995; Collins et al . , 2018; Garcia et al . , 2005; Robinson et al . , 1995a; Zhao et al . , 2008 ) .",
"Apart from FGF receptors , their co-receptors heparan sulphates and downstream mediators Frs2 , Shp2 , and Crk have also been demonstrated to regulate lens cell differentiation and elongation ( Collins et al . , 2018; Li et al . , 2019; Li et al . , 2014; Madakashira et al . , 2012; Pan et al . , 2006; Qu et al . , 2011 ) .",
"The knockout phenotypes of these genes were reproduced by overexpression of negative regulators of FGF-ERK pathway , Sef and Sprouty ( Newitt et al . , 2010; Shin et al . , 2015 ) .",
"On the other hand , transgenic overexpression of Fgf1 or Fgf3 resulted in premature differentiation of lens epithelial cells ( Collins et al . , 2018; Robinson et al . , 1998; Robinson et al . , 1995b ) , whereas over-activation of FGF signaling as a result of Nf1 and Spry1/2 deletion disrupted lens induction and lens fiber cell differentiation , respectively ( Carbe and Zhang , 2011; Kuracha et al . , 2011 ) .",
"These results demonstrated that the FGF signaling cascade is critical for lens development , but the direct downstream transcriptional effectors of FGF signaling were not well understood .",
"In this study , we investigated the role of Etv family transcription factors in the lens by genetically ablating Etv1 , Etv4 , and Etv5 .",
"Instead of the delayed cell differentiation phenotype expected from FGF signaling deficiency , we observed that the lens epithelial cells differentiated prematurely as a result of reduced Notch signaling .",
"On the other hand , the expression of FGF targets Maf and Crystallins were largely preserved in Etv1/4/5 mutant lenses .",
"We also showed that mTOR signaling was aberrantly upregulated , resulting in the failure of nuclei removal in the mature lenses .",
"These results revealed the critical differences between the function of Etv family transcription factors and FGF signaling during lens development , demonstrating that these SRTFs can operate outside the confine of upstream signaling ."
],
[
"Previous studies have shown that Etv transcription factors are controlled by FGF signaling during embryonic development .",
"To confirm this finding in the lens , we generated conditional knockouts of Fgfr1 and Fgfr2 using a lens-specific Cre driver Pax6Le-Cre , also known as Le-Cre , which is active in the lens ectoderm as early as E9 . 5 ( Ashery-Padan et al . , 2000 ) .",
"As we and others have previously reported , genetic ablation of FGF receptors prevented the lens ectoderm from forming the lens vesicle in the Pax6Le-Cre; Fgfr1fl/fl; Fgfr2fl/fl ( Fgfr CKO ) embryo ( Figure 1A–H , arrows ) ( Collins et al . , 2018; Garcia et al . , 2005 ) .",
"Importantly , both ERK phosphorylation and expression of Etv1 , Etv4 and Etv5 were also lost in the Fgfr CKO lens ectoderm ( Figure 1A–H , outlined ) , demonstrating that FGF signaling indeed controls ERK and Etv activities during lens induction .",
"We next ablated Mek and Erk to investigate whether Etv genes were also regulated by MAPK signaling in the lens .",
"Although the lenses were formed in Pax6Le-Cre; Mek1 ( Map2k1 ) fl/fl; Mek2 ( Map2k2 ) -/- ( Mek CKO ) and Pax6Le-Cre; Erk1 ( Mapk3 ) -/-; Erk2 ( Mapk1 ) fl/fl ( Erk CKO ) embryos , they failed to express any of the Etv genes ( Figure 1I–Q , circled ) .",
"These results demonstrated that the Etv family transcription factors are controlled by FGF-ERK signaling during lens development .",
"To investigate the function of Etv family transcription factors in the lens , we used the Pax6Le-Cre transgenic mouse line to conditionally ablate Etv1 and Etv5 in an Etv4-null background .",
"In Pax6Le-Cre; Etv1fl/fl; Etv4-/-; Etv5fl/fl embryos ( Etv TKO ) , the lens induction and lens vesicle formation were unaffected , but the lens size was reduced at E14 . 5 as shown by Hematoxylin and Eosin staining ( Figure 2A and E ) .",
"Consistent with this , apoptosis increased in the epithelium and the transitional zone as indicated by TUNEL staining ( Figure 2B , F and I , arrows ) .",
"On the other hand , proliferation of the lens epithelial cells was reduced as measured by EdU incorporation assay ( Figure 2C , D and I , arrowheads ) .",
"In the control lens , the nascent fiber cells exited the cell cycle after the transitional zone , as shown by the diminution of Cyclin D1 expression and Edu labeling ( Figure 2C , arrows ) .",
"In contrast , the Etv TKO lens displayed ectopic expression of Cyclin D1 in the posterior lens ( Figure 2G , arrow ) and persistent EdU- and pHH3-positive cells in the fiber cell compartment ( Figure 2C , D , G and H , inserts ) .",
"These phenotypes showed that lens development was disrupted by loss of Etv transcription factors .",
"To identify the downstream targets of Etv genes , we performed RNA sequencing analysis to compare the transcriptional landscape of E14 . 5 control to that of Etv TKO mutant .",
"Since Etv genes are predominantly expressed in the transitional zone at the lens equator , we isolated these cells by laser capture microdissection and extracted RNA for cDNA synthesis ( Figure 3A ) .",
"After amplification , the cDNA library was subjected to high-throughput sequencing to examine the global gene expression changes .",
"Cluster and PCA analysis of the top 200 differentially expressed genes showed that the controls and mutants were well segregated across three biological replicates , demonstrating the consistency of the RNA sequencing data ( Figure 3B and Figure 3—figure supplement 1A ) .",
"Among the 834 genes showing statistically significant changes in expression ( p<0 . 05 ) , 288 and 283 genes were up- and down-regulated by at least two folds , respectively .",
"Interestingly , we noticed that the remaining transcripts of three Etv genes were reduced in mutant samples , suggesting that Etv genes regulated their own expressions ( Figure 3C ) .",
"These results indicate that loss of Etv genes causes complex changes in the lens transcriptome .",
"We next focused on the impact of Etv deletion on FGF signaling .",
"The RNA sequencing data indicated that the receptors for FGF signaling , Fgfr1 and Fgfr3 , were down-regulated in the Etv TKO mutant ( Figure 3C ) , which were confirmed by RNA in situ hybridization ( Figure 3—figure supplement 1B ) .",
"Although this suggests a positive feedback regulation of FGF receptors by Etv genes , it is unlikely to affect lens development , because previous studies have shown that Fgfr1 and Fgfr3 double mutant lenses did not exhibit any overt phenotype ( Zhao et al . , 2008 ) .",
"On the other hand , the transcript level of Spry2 , an inhibitor of receptor tyrosine kinases , was also reduced , which we confirmed by RNA in situ hybridization in Etv TKO mutant lenses ( Figure 3D and F , arrows ) .",
"This is consistent with previous ChIP-seq analysis which revealed that SPRY2 is a direct target of PEA3/ETV family genes ( Yan et al . , 2013 ) .",
"Since we have shown above that expression of Etv1 , Etv4 and Etv5 in the lens is dependent on ERK signaling ( Figure 1I–Q ) , it is not surprising that Spry2 expression is also lost in Erk CKO mutants ( Figure 3—figure supplement 1C , arrows ) .",
"Considering that Sprouty proteins are known to inhibit Ras-MAPK signaling ( Hanafusa et al . , 2002 ) , this suggests that Etv regulation of Spry constitutes a negative feedback loop to regulate FGF-ERK activity .",
"Indeed , we notice that ERK phosphorylation was ectopically activated in Etv TKO mutant lenses ( Figure 3E and G , arrows ) .",
"In support of the upregulation of ERK activity , we also observed increasing expression of Egr1 and Fos ( Figure 3C ) , two early response genes for Ras-MAPK signaling .",
"Moreover , gene set enrichment analysis ( GSEA ) showed that Etv deletion enhanced the activity of the MAP kinase pathway ( Figure 3H ) , which is known to be decreased in FGF receptor mutants .",
"Therefore , inactivation of FGF receptors or Etv transcription factors in the lens resulted in diametrically different outcomes in MAPK signaling .",
"We took three approaches to investigate the role of Etv transcription factors in lens cell differentiation: ( 1 ) examining the Etv loss-of-function phenotype , ( 2 ) corroborating our results with ERK signaling knockouts and ( 3 ) performing genetic epistasis using a FGF gain-of-function model .",
"It was previously reported that FGF signaling controls the expression of αA-crystallin ( Cryaa ) in the lens cells via Etv5 and Maf; the latter also contains Etv-binding sites in its regulatory region ( Xie et al . , 2016 ) .",
"Unexpectedly , immunostaining showed that both Maf and α-crystallins proteins were still present in Etv TKO mutant lenses ( Figure 4A , B , F and G ) .",
"Similarly , we did not detect a significant reduction in γA-crystallin expression ( Figure 4C and H ) , which is normally induced after αA-crystallin in more mature lens fiber cells .",
"To avoid the possibility that protein perdurance may obscure the dynamic changes in gene expression , we also performed RNA in situ hybridization , which showed abundant expression of Cryaa and Cryga mRNAs in Etv TKO lenses ( Figure 4D , E , I and J ) .",
"Consistent with this , there were no statistically significant changes in the transcript levels of Cryaa , Cryga and Maf in our RNA sequencing data .",
"Based on these results , lens differentiation can apparently proceed in the absence of Etv transcription factors .",
"To corroborate the lack of lens differentiation phenotype in Etv loss-of-function mutants , we next examined ERK signaling knockouts .",
"We reasoned that , since the entire Ets superfamily transcription factors including Etv are under the control of ERK , inactivation of MAPK may produce a stronger phenotype than Etv knockouts .",
"Indeed , combined deletion of Erk1 ( Mapk3 ) and Erk2 ( Mapk1 ) resulted in a drastic reduction in lens size as previously reported ( Xie et al . , 2016 ) .",
"However , Maf , α- and γ-crystallins mRNA and protein were still expressed in Erk CKO mutant lenses ( Figure 4K–O ) .",
"To further confirm this finding , we also examined the lens-specific knockout of Mek1 ( Map2k1 ) and Mek2 ( Map2k2 ) , two kinases upstream of Erk .",
"In the severely diminished Mek CKO mutant lenses , we again failed to detect significant reduction in the intensity of Maf , α- and γ-crystallins staining ( Figure 4P–T ) .",
"Therefore , genetic ablation of Erk and Etv transcription factors did not abolish lens differentiation as in FGF receptor mutants .",
"The phenotypic dichotomy between Fgfr and Etv mutants raised the question whether Etv transcription factors play any role in the FGF-induced lens fiber differentiation .",
"To address this issue , we took a gain-of-function approach to test the genetic epistasis between Etv and FGF signaling .",
"As we and others have previously reported ( Collins et al . , 2018; Li et al . , 2019; Robinson et al . , 1998 ) , the lens-specific overexpression of Fgf3 in Fgf3OVE391 transgenic animals greatly elevated ERK phosphorylation in the lens ( Figure 5A and B ) .",
"Consistent with regulation of Etv and Spry2 by ERK signaling we showed above , this led to increased expression of Etv1 , Etv4 , Etv5 and Spry2 in the Fgf3OVE391 lens ( Figure 5—figure supplement 1 ) , which was accompanied by ectopic induction of Maf and γ-crystallin but loss of E-cadherin in Pax6-positive anterior lens epithelium ( Figure 5F , J and N , arrows ) .",
"We hypothesized that if Etv is required for FGF signaling to promote lens differentiation , then blocking Etv activity should prevent the Fgf3-overexpression phenotype .",
"Since it was cumbersome to use the triple knockout of Etv1/4/5 to generate compound mutants for the genetic epistasis experiments , we employed the R26EtvEnR allele , which can be induced to express an Etv4 fusion protein linked to the Engrailed repressor domain ( EtvEnR ) after Cre-mediated excision of a transcriptional STOP cassette .",
"This was previously shown to act as a dominant negative protein to suppress the activities of endogenous Etv transcription factors ( Mao et al . , 2009 ) .",
"Consistent with this , Pax6Le-Cre;R R26EtvEnR lenses were reduced in size like those of Etv TKO mutants , while the expression of E-cadherin , Maf and γ-crystallin was maintained ( Figure 5G , K , O and Q ) .",
"After crossing with Fgf3OVE391 , the lens remained smaller in the Pax6Le-Cre; Fgf3OVE391; R26EtvEnR mutant than that of the control ( Figure 5Q ) .",
"Of note , the expression of Etv1 , Etv4 , Etv5 and Spry2 in the Pax6Le-Cre; Fgf3OVE391; R26EtvEnR mutant was diminished but not abrogated compared to that in Fgf3OVE391 ( Figure 5—figure supplement 1 ) , suggesting that Etv activities was partially inhibited by EtvEnR .",
"Nevertheless , in sharp contrast to Fgf3OVE391 , Maf and γ-crystallins were restricted to the lens fiber compartment in Pax6Le-Cre; Fgf3OVE391; R26EtvEnR lenses , while E-cadherin was preserved in the anterior lens epithelium ( Figure 5H , L and P ) .",
"Therefore , inhibition of Etv transcriptional activities prevented FGF signaling from inducing ectopic expression of the fiber-specific genes in the lens epithelium .",
"Taken together , these results showed that Etv transcription factors were not essential for normal lens differentiation at the transitional zone , but they were required for FGF signaling to directly convert lens epithelial cells into lens fibers .",
"Although the fiber cell differentiation genes were still expressed in Etv TKO mutant lenses , we noticed that the transitional zone marked by the boundary between the lens epithelial marker Foxe3 and the fiber cell marker Prox1 was moved anteriorly ( Figure 6A and E , arrows ) .",
"As a result , the length of the lens epithelium marked by Pax6 and E-cadherin staining was shortened compared to the circumference of the posterior lens ( Figure 6B , F and I , arrows ) .",
"This differentiation pattern is reminiscent of defective Notch signaling , which is known to cause the lens progenitor cells to undergo premature differentiation before reaching the lens equator , resulting in an anteriorly shift of the transitional zone ( Jia et al . , 2007; Le et al . , 2009; Li et al . , 2019; Rowan et al . , 2008; Saravanamuthu et al . , 2012 ) .",
"Intriguingly , one of the down-regulated genes in Etv TKO mutants revealed by our transcriptomic analysis was Jag1 ( Figure 3C ) , which encodes the ligand for Notch signaling .",
"By immunostaining , we confirmed that the expression of Jag1 protein was drastically reduced in Etv TKO mutant lenses ( Figure 6C and G , arrowheads ) .",
"Jag1 expressed in the lens fiber cells signals to the Notch receptors in the lens epithelium to maintain the progenitor pool ( Jia et al . , 2007; Le et al . , 2009 ) .",
"In line with the reduced Notch signaling due to Jag1 deficiency , the anterior lens epithelium in Etv TKO mutants exhibited severely diminished staining of the Notch1 intracellular domains ( Notch1-ICD ) , a proteolytic product of Notch1 receptor triggered by Jag1 activation ( Figure 6D and H , arrows ) .",
"These results support that Etv regulates Jag1-Notch signaling to control the timing of lens cell differentiation .",
"To investigate the molecular mechanism of Jag1 regulation by Etv , we turned to cultured lens cells in vitro .",
"By western blot , we showed that Jag1 expression in the lens epithelial cell culture was induced by FGF2 after 5 hr treatment and suppressed by Mek inhibitors U0126 and PD0325901 but not PI3K inhibitor LY294002 ( Figure 6J and Figure 6—figure supplement 1 ) .",
"This is consistent with previous reports that Jag1 expression in the lens is controlled by ERK-mediated FGF signaling ( Li et al . , 2019; Saravanamuthu et al . , 2009 ) .",
"To determine whether Jag1 is a direct transcriptional target of Etv , we used the recently published ATAC-seq analysis ( Zhao et al . , 2019 ) to search the open chromatin regions in the developing lens and identified two Etv-binding sites located within introns 2 and 5 of Jag1 gene ( Figure 6K ) .",
"By chromatin immunoprecipitation ( ChIP ) , we showed that both introns 2 and 5 sites could be pulled down by Etv5 antibodies but not by IgG control ( Figure 6L ) , demonstrating that these two sites in Jag1 were occupied by Etv in the lens cells .",
"We next investigated whether Etv was required for FGF signaling to induce Jag1 expression in vivo .",
"Jag1 was normally restricted to the nascent lens fibers in wild type control lenses ( Figure 5M , arrowheads ) , but it was abolished in Mek and Erk CKO mutants ( Figure 6N and O ) .",
"In Fgf3OVE391 lenses , however , Jag1 expression was expanded to the entire lens epithelium at the expense of E-cadherin expression ( Figure 6P , arrow ) , demonstrating that FGF signaling can induce de novo Jag1 expression in the lens epithelium .",
"In Pax6Le-Cre; R26EtvEnR lenses , Jag1 expression was significantly reduced compared to those of wild-type control ( Figure 6Q , arrowheads ) , consistent with the role of Etv in Jag1 regulation .",
"Suppression of Etv activity in Pax6Le-Cre; Fgf3OVE391; R26EtvEnR lenses reversed the Fgf3 overexpression phenotype , limiting the Jag1 expression domain to the posterior lenses ( Figure 6R , arrowheads ) .",
"Based on these results , we conclude that Etv mediates the FGF-ERK-Notch crosstalk to control the induction of lens differentiation .",
"The final step of lens cell differentiation is the degradation of their cellular organelles , which is crucial for the transparency of the lens ( Bassnett et al . , 2011 ) .",
"To determine whether Etv transcription factors are required for this process , we examined the clearance of lens cell nuclei by histology .",
"In wild-type control , the maturing fiber cells lost their nuclei as they migrated toward the interior of the lens , eventually forming an organelle free zone ( OFZ ) ( Figure 7A and Figure 7—figure supplement 1 , arrowheads ) .",
"In contrast , Etv TKO mutants retained nuclei within the lens core , indicating defective organelle clearance ( Figure 7E and Figure 7—figure supplement 1 , arrowheads ) .",
"Previous studies have shown that inhibition of mTOR signaling accelerated organelle elimination in chick lens explants ( Basu et al . , 2014 ) .",
"Interestingly , our RNA sequencing analysis of Etv TKO mutant lenses showed significant down-regulation of Tsc2 , which forms a heterodimer with Tsc1 to inhibit mTOR activity ( Figure 3C ) .",
"This suggests that the defective nuclei clearance in the Etv TKO mutant may be caused by elevated mTOR signaling .",
"In support of this hypothesis , we observed that phospho-mTOR ( pmTOR ) was localized in the transitional zone of the wild-type control lens , but it expanded into the fiber cell compartment in Etv TKO mutants ( Figure 7B and F , arrowheads ) .",
"Similarly , Etv TKO mutant lenses displayed significant increase in pS6 and p4EBP1 , two known substrates of mTOR kinase ( Figure 7C , D , G and H ) .",
"Therefore , inactivation of Etv genes led to increased mTOR signaling in the lens .",
"To determine whether mTOR activation indeed interfered with nuclei clearance in the lens , we generated Pax6Le-Cre; Tsc1flox/flox ( Tsc1 CKO ) to ablate the Tsc2 binding partner Tsc1 .",
"As expected from the role of Tsc1/2 complex in suppressing mTOR activity , pmTOR was significantly increased in Tsc1 CKO lenses , which also displayed elevated level of pS6 and p4EBP1 ( Figure 7J–L ) .",
"Importantly , whereas the wild-type control lens contains a well-defined OFZ in the center , the nuclei were scattered throughout the Tsc1 CKO lenses ( Figure 7I and Figure 7—figure supplement 1 ) .",
"The lack of an OFZ in Tsc1 deficient lenses supports our model that Etv control of mTOR signaling is required for the terminal differentiation of the lens fiber cells ."
],
[
"FGF signaling plays important roles during vertebrate lens development .",
"As demonstrated by the profound lens defects in FGF receptor mutants , the primary function of FGF signaling is to promote differentiation of the lens epithelial cells into the fiber cells .",
"In this study , we have studied the functions of Etv family genes , which are the well-established downstream transcription factors induced by FGF during embryonic development .",
"Contrary to the defective lens differentiation found in FGF signaling mutants , deletion of Etv genes did not prevent the expression of lens fiber genes in normal development .",
"Instead , it caused premature differentiation of the lens progenitors as a result of reduced Notch signaling .",
"Moreover , Etv mutations led to an increase in the activity of ERK and mTOR signaling , with the latter responsible for the abnormal retention of the nuclei in the adult lens .",
"Therefore , unlike BMP , Hedgehog and Wnt signaling that command their downstream signal-regulated transcription factors ( SRTFs ) as faithful effectors , ETV transcription factors activated by FGF signaling can oppose the intended function of the pathway .",
"Our study of Etv mutants in lens development has revealed several important features of Etv function that deviate from the expected role of a faithful executor for FGF signaling .",
"First , deletion of Etv caused significant derangement of ERK signaling , which is the main intracellular pathway activated by FGF .",
"Contrary to the loss of ERK activity in mutants lacking either FGF receptors or downstream mediators Frs2 and Shp2 ( Collins et al . , 2018; Li et al . , 2014; Pan et al . , 2010; Zhao et al . , 2008 ) , Etv-deficient lenses displayed elevated ERK signaling , which was evident in the increased level of ERK phosphorylation and expression of ERK early response genes Egr1 and Fos .",
"This was likely due to reduced expression of ERK inhibitor Spry2 , which has been shown to be a direct transcriptional target of Etv ( Yan et al . , 2013 ) .",
"In support of this , deletions of Spry genes have been found to result in upregulation of ERK signaling in a wide variety of tissues , including the lens ( Kuracha et al . , 2011 ) .",
"The aberrant ERK activation in Etv mutants may stimulate downstream targets normally silenced by loss of FGF signaling , inducing further compensatory changes that rewire the signaling network .",
"It underscores the intricate role of Etv in stabilizing the intracellular signaling activated by FGF .",
"The second distinction of Etv mutants compared to FGF receptor knockouts is the relatively normal lens differentiation .",
"This was unexpected because a previous study shown that both lens differentiation genes Maf and Cryaa were under direct control of Etv transcription factors in vitro ( Xie et al . , 2016 ) .",
"We presented three lines of evidence in support of our conclusion .",
"First , we performed RNA sequencing , RNA in situ hybridization , and immunohistochemistry in Etv mutant lenses , none of which showed significant reduction in the expression of Maf and Cryaa .",
"Second , we generated lens-specific knockouts of Erk1/2 , which we showed to be required for Etv expression .",
"Even in these ERK signaling mutants , we still observed expression of Maf and Cryaa .",
"Of note , the previous study observed that immunostaining for α-crystallin and Maf were severely reduced or even absent in the Erk1/2 knockout lens ( Xie et al . , 2016 ) .",
"It is not clear what caused the phenotypic discrepancy between ours and the previous study , which could be due to a difference in the genetic background of the animal models or the source of antibodies .",
"It should be noted that γ-crystallin expression appeared to move posteriorly in both Etv and Erk knockouts ( Figure 4H , M and R ) , which may reflect a slight delay of lens fiber terminal differentiation .",
"Nevertheless , even knockout of ERK kinase Mek still failed to abolish Maf and Cryaa expression .",
"In the third approach , we explored the role of Etv in a gain-of-function setting , showing that inhibition of Etv indeed blocked ectopic FGF from stimulating Maf and γ-crystallin expression in the lens epithelium .",
"This result suggests an intrinsic difference between normal differentiation at the lens equator and ectopic differentiation in the lens epithelium , which may be shaped by their unique microenvironment and different dosage of FGF signaling .",
"Taken together , our results demonstrate that Etv genes are dispensable for FGF-induced cell differentiation under the physiological condition during lens development .",
"Genetic ablation of Etv also differed from inactivation of FGF signaling in its impact on the transitional zone of cell differentiation , which was shifted anteriorly after Etv deletion and posteriorly in FGF signaling mutants .",
"We recently showed that FGF plays a dual role in specifying the location of the transitional zone by regulating the timing of lens differentiation; FGF directly promotes differentiation of lens fiber cells , but it also cooperates with PDGF to restrain differentiation of the lens progenitor cells ( Li et al . , 2019 ) .",
"The latter is mediated by Jag1 , which induces Notch signaling in the anterior lens epithelium to suppress progenitor cell differentiation .",
"In the current study , we determined that Jag1 expression is under the direct control of Etv transcription factors and ERK , delineating a complete molecular cascade that connects FGF to Notch signaling .",
"In the absence of Etv , loss of Notch signaling leads to premature differentiation of lens progenitor cells indicated by Foxe3 and Prox1 expression , but the pattern of Crystallin expressions are unchanged .",
"This suggests the terminal differentiation of lens fibers may be slightly delayed relative to onset of progenitor cells differentiation .",
"Nevertheless , Etv mutants proceed to express Crystallin genes at the comparable level as wild-type controls , which is in sharp contrast to the loss of γ-crystallin expression in FGF receptor mutants ( Zhao et al . , 2008 ) .",
"Therefore , Etv mediates the anti-differentiation function of FGF to activate Notch signaling but it is largely dispensable for the pro-differentiation aspect of FGF signaling .",
"Lastly , our study showed that Etv deficiency augmented mTOR phosphorylation .",
"This is likely caused by reduced expression of Tsc2 , a negative regulator of mTOR .",
"Indeed , we also observed significant increase in phosphorylation of S6 and 4EBP1 , two downstream targets of mTOR .",
"Importantly , we observed aberrant retention of the lens fiber cell nuclei in postnatal Etv mutants , which was mimicked by deletion of Tsc2 partner Tsc1 in the lens .",
"Previous studies have shown that inhibition of mTOR by rapamycin promotes degradation of the organelle in chick lens explants although the exact mechanism remained controversial ( Basu et al . , 2014; Morishita and Mizushima , 2016 ) .",
"Our study provides the in vivo evidence that mTOR is indeed an important regulator of organelle removal in lens maturation .",
"Taken together , our genetic study firmly establishes Etv family transcription factors as SRTFs for FGF signaling in lens development .",
"SRTFs constitute a special class of transcription factors that relay intracellular signals to shape the cellular transcriptome , which ultimately determines the identity and status of the cell .",
"Although many SRTFs have proven to be faithful to their upstream signaling , we showed that Etv transcription factors deviate or even oppose the function of FGF-ERK signaling in the lens .",
"This is likely because , unlike other signaling pathways such as BMP , Hedgehog and Wnt that rely on a single downstream effector , FGF signaling can induce multiple transcription factors .",
"This generates diversity among the FGF SRTFs that may evolve to acquire their unique or even opposing functions , which fine tunes the overall transcriptional response .",
"The three PEA3/ETV genes and ERG are the only ETS genes implicated in tumorigenesis , which has been attributed to their ability to mimic the oncogenic effect of Ras signaling ( Hollenhorst et al . , 2011 ) .",
"Our previous work in lacrimal gland development has indeed found that deletion of Etv transcription factors largely reproduced the phenotype of FGF-ERK mutants ( Garg et al . , 2018 ) .",
"In the lens , however , our current study paints a more complex picture of Etv activity , suggesting that their functions deviate considerably from their upstream FGF signaling .",
"As transcription factors attract increasing interests as viable therapeutic targets , it will be important to elucidate the context-dependent function of these SRTFs in development and diseases ."
],
[
"Mice carrying Erk1 ( Mapk3 ) -/- , Mapk1flox , Mek1 ( Map2k1 ) flox and Mek2 ( Map2k2 ) -/- alleles were bred and genotyped as described ( Newbern et al . , 2008; Newbern et al . , 2011 ) .",
"Pax6Le-Cre mice were from Dr . Ruth Ashery-Padan ( Tel Aviv University , Tel Aviv , Israel ) , Etv1flox mice from Dr . Silvia Arber ( University of Basel , Basel , Switzerland ) , Etv4+/- and Etv5flox mice from Dr . Xin Sun ( University of California at San Diego , San Diego , CA ) , R26EtvEnR from Drs .",
"Andrew McMahon ( University of Southern California , Los Angeles , CA ) and James Li ( University of Connecticut Health Center , Farmington , CT ) , Fgf3OVE391 mice were obtained from Dr . Michael Robinson ( Miami University , Oxford , OH ) and Fgfr2flox from Dr . David Ornitz ( Washington University Medical School , St Louis , MO ) ( Ashery-Padan et al . , 2000; Mao et al . , 2009; Patel et al . , 2003; Robinson et al . , 1998; Yu et al . , 2003; Zhang et al . , 2009 ) .",
"Fgfr1flox mice were from Jackson Laboratory ( Stock No: 007671 ) .",
"Tsc1flox mice were originally obtained from Jackson Laboratory ( Stock No:005680 ) and provided by Dr . Stephen Tsang ( Columbia University , New York , NY ) .",
"To generate embryos , females in breeding were checked for vaginal plugs ( considered as 0 . 5 days pc ) .",
"All the animals were maintained in the mixed genetic background .",
"Mouse maintenance and experimentation was performed according to protocols approved by Columbia University Institutional Animal Care and Use Committee .",
"Pax6Le-Cre or Etvflox mice did not display any lens phenotypes and were used as controls .",
"All the experiments were repeated at least three times .",
"Hematoxylin and Eosin staining ( H and E ) was performed as previously described ( Carbe et al . , 2012 ) .",
"Briefly , paraffin blocks were sections at 10 μm and deparaffinized by heating and histosol washes , followed by rehydration through decreasing percentage of ethanol solutions .",
"The slides were dipped into hematoxylin for 3 min followed by 10-15 min wash with tap water .",
"The samples were decolorized with 1% acid alcohol for 15 s , before treatment with Eosin for 1 min .",
"Samples were then dehydrated by passing through increasing concentration of ethanol , and transferred to histosol .",
"The samples were mounted using permount mounting medium .",
"For immunohistochemistry , paraffin samples were deparaffinized as described above and cryosections were briefly washed with PBS to remove OCT .",
"Antigen retrieval was performed with microwave boiling for 1-2 min followed by heating for 10 min at low-power settings in citrate buffer ( 10 mM sodium citrate , pH 6 . 0 ) .",
"Sections were then washed with PBS and blocked with 5% NGS/0 . 1% Triton in PBS .",
"Primary antibody incubation was performed overnight at 4°C in humid chamber followed by incubation with florescent-conjugated secondary antibodies for 1 hr at room temperature in dark .",
"The following primary antibodies were used: Pax6 ( PRB-278P ) , Prox1 ( PRB-238C ) ( both from Covance , Berkeley , CA ) , E-cadherin ( U3254 , Sigma , St Louis , Missouri ) , Maf ( sc-7866 ) , Foxe3 ( sc-377465 ) , Jag1 ( sc-8303 ) ( all from Santa Cruz Biotechnology ) , Lamin A/C ( ab133256 , Abcam ) , pHH3 ( 06-570 , Millpore ) , phospho-4EBP1 ( #2855 ) .",
"phospho-S6 ( #5364 ) , phospho-Akt ( D9E ) , phospho-Erk ( #4370 ) , phospho-mTOR ( #5536 ) , Cyclin D1 ( #55506 ) , Notch1-ICD ( # 4147 ) ( all from Cell signaling Technology ) .",
"Antibodies against α- and γ-crystallins were kindly provided by Dr . Sam Zigler ( National Eye Institute ) .",
"For Notch1-ICD antibody staining , samples were paraffin embedded , sectioned and followed by antigen retrieval for 20 min in a pressure cooker .",
"To detect phospho-Erk , phospho-mTOR , phospho-S6 , phospho-4EBP1 and NICD , HRP-conjugated secondary antibody and Tyramide signal amplification kit ( Perkin Elemer ) were used .",
"The perimeters of the anterior epithelial layer and posterior fiber cells were measured using ImageJ and the ratio was calculated for control and mutant lenses .",
"To quantify the nuclei degradation , the lenses were stained with nuclear markers DAPI and Lamin A/C .",
"The number of nuclei within a concentric circle of the lens at the half of the lens radius were counted .",
"For statistical analysis , at least three embryos of each genotype were taken and two lens sections at the equatorial plane per embryo were analyzed .",
"The statistical significance was calculated by one-way ANOVA .",
"TUNEL assays were performed on 10-μm paraffin sections following the manufacturer's instructions in the Fluoroscein In Situ Cell Death Detection kit ( Roche Applied Science , Indianapolis , IN ) .",
"Apoptosis rates were calculated as the ratio of TUNEL-positive cells to DAPI-positive cells in control and mutant samples , and results were analyzed by t-test .",
"Pregnant females were injected with EdU ( ab146186 , Abcam ) dissolved in DMSO at the dosage of 50 mg/kg body weight .",
"After 2 hr , the mice were sacrificed and embryos collected for cryosection .",
"For EdU detection , the Click-IT EdU Imaging Kit ( C10337 , Invitrogen ) was used according to the manufacturer's instructions .",
"The proliferation rates in the lens epithelium were calculated as the ratios of EdU-positive cells to DAPI-positive cells within the epithelium and the statistical significances between controls and mutants were evaluated by t-test .",
"Section in situ hybridization was performed as described ( Carbe et al . , 2013 ) .",
"Briefly , the cryoblocks were sectioned at 10 μm and hybridized with diluted probes ( 1:500 ) at 65°C overnight .",
"The sections were washed 3X with wash buffer at 65°C for 30 min , followed by 2X wash with MABT for 30 min .",
"The sections were then blocked with blocking buffer for 1 hr at room temperature , followed by incubation with anti-DIG antibody ( 1:1500 ) overnight at 4°C .",
"Next , the slides were washed 4X with MABT for 20 min and 2X alkaline phosphatase buffer for 10 min , before incubating with BM purple for colorimetric reaction for 24 hrs at room temperature .",
"The following probes were used: Pea3 , Erm5 ( from Dr . Bridget Hogan , Duke University Medical Center , Durham , NC , USA ) , Er81 ( from Dr . Gord Fishell , New York University Medical Center , New York , NY , USA ) , Fgfr1 and Fgfr3 ( from Suzanne Mansour , University of Utah , Salt Lake city , UT ) and Sprouty2 ( from Gail Martin , University of California at San Francisco , San Francisco , CA ) .",
"Freshly harvested embryos were frozen in OCT medium ( Sakura Finetek ) , sectioned at 10 μm thickness and transferred to PEN slides ( Ziess ) .",
"To fix and stain the slides , they were dipped in 95% ethanol for 2 min to fix the samples , stained with crystal violet stain ( 3% in ethanol ) on ice .",
"The slides were then rigorously washed in 2 X 70% ethanol for 30-40 s to remove the OCT and dehydrated in 100% ethanol for 2 min .",
"For control and Etv mutant embryos , the lens tissue was micro-dissected from the transitional zone using Laser capture microscope ( Zeiss AxioObserver . Z1 inverted microscope ) .",
"The RNA was extracted using Qiagen Micro Plus kit .",
"Conversion to cDNA and amplification were performed using Clontech SMART-seq v4 Ultra low input RNA kit and the cDNA library construction was performed using Nextera XT DNA library preparation kit by the core facility at Columbia university prior to RNA sequencing .",
"The RNAseq data are available from the GEO repository ( GSE137215 ) .",
"Normalization and differential gene expression analysis for RNA-seq data were performed using De-Seq package in R studio .",
"The GSEA analysis was performed using JavaGSEA desktop software from Broad Institute .",
"Immortalized lens cells were authenticated by immunostaining with lens markers and confirmed to be free of mycoplasma contamination as previously described ( Li et al . , 2019 ) .",
"They were cultured in Dulbecco’s modified Eagle’s medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) and L-glutamine , 1% penicillin-streptomycin .",
"After serum starved for 24 hr , cells were subject to FGF2 ( 50 ng/ml ) stimulation , with or without Mek inhibitor U0126 ( 50 μM ) , PD0325901 ( 50 μM ) or PI3K inhibitor LY ( 50 μM ) for the indicated time periods , and harvested in CelLytic-M lysis buffer ( Sigma ) with proteinase inhibitor cocktail ( Thermo fisher ) .",
"Protein lysates were collected following centrifugation at 12 , 000 g for 10 min and resuspended in SDS buffer .",
"Equal amounts of total protein were loaded for western blot analysis and visualized using an Odyssey SA scanner ( LICOR Biosciences , Lincoln , NE ) .",
"Antibodies used were Jagged1 ( H-114 , Santa Cruz Biotechnology ) , Notch1 ( #4380 , Cell signaling Technology ) , phospho-ERK1/2 ( sc-7383 , Santa Cruz Biotechnology ) , ERK ( #4695 , Cell signaling Technology ) .",
"The Chromatin Immunoprecipitation ( ChIP ) assays were performed in immortalized lens cells as previously described ( Garg et al . , 2017; Li et al . , 2019 ) .",
"The antibodies used were IgG as isotype control ( #2729 , Cell Signaling Technology ) and anti-Etv5 ( #66657 , Proteintech ) .",
"The primers used for the intron 2 site are GGTTTCTGCTCCACCTCTGA and GGGAGTGCAAACTTGATGCT , and for the intron 5 site are AAGAGCCAGCTCAGCTTCAC and AGATCTGTGCCCCAGAGGAT ."
]
] | [
"The signal regulated transcription factors ( SRTFs ) control the ultimate transcriptional output of signaling pathways .",
"Here , we examined a family of FGF-induced SRTFs – Etv1 , Etv 4 , and Etv 5 – in murine lens development .",
"Contrary to FGF receptor mutants that displayed loss of ERK signaling and defective cell differentiation , Etv deficiency augmented ERK phosphorylation without disrupting the normal lens fiber gene expression .",
"Instead , the transitional zone for lens differentiation was shifted anteriorly as a result of reduced Jag1-Notch signaling .",
"We also showed that Etv proteins suppresses mTOR activity by promoting Tsc2 expression , which is necessary for the nuclei clearance in mature lens .",
"These results revealed the functional divergence between Etv and FGF in lens development , demonstrating that these SRTFs can operate outside the confine of their upstream signaling ."
] | [
"Many cells contain proteins known as signal-induced transcription factors , which are poised to receive messages from the environment and then react by activating genes required for the cell to respond appropriately .",
"It is commonly thought that these transcription factors faithfully follow the instructions they receive from the external signal: for instance , if the message was to encourage the cell to grow , the transcription factors would switch on growth-related genes .",
"As the eyes of mice and other mammals develop , a signal known as FGF is required for certain cells to specialize into lens fiber cells: these long , thin , transparent cells form the bulk of the lens , the structure that allows focused vision .",
"Previous studies suggest that FGF activates three transcription factors known as Etv1 , Etv4 and Etv5 , but their precise roles in the development of the lens has remained unclear .",
"Here , Garg , Hannan , Wang et al . confirm that FGF signaling does indeed activate all three proteins .",
"However , mutant mice that lacked Etv1 , Etv4 and Etv5 still created lens fiber cells , suggesting that the transcription factors are largely unnecessary for lens fiber cells formation .",
"Instead , the Etv proteins participated in a cascade of molecular events involving a protein called Notch; as a result , if the transcription factors were absent , the lens fiber cells formed prematurely .",
"In addition , deactivating Etv1 , Etv4 and Etv5 also promoted the activity of a protein which interfered with the removal of internal cell compartments , a process required for lens fiber cells to mature properly .",
"These findings reveal that the roles of Etv1 , Etv4 and Etv5 deviate from and even oppose FGF signaling in the lenses of mice .",
"Transcription factors control the ultimate fate of a cell , and there is therefore increased interest in targeting them for therapy .",
"The work by Garg , Hannan , Wang et al . reveals an unexpected complexity in how these proteins respond to upstream signals , highlighting the importance of further dissecting these relationships ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"cell biology"
] | The synaptonemal complex has liquid crystalline properties and spatially regulates meiotic recombination factors | elife-21455-v2 | [
[
"In most eukaryotes , chromosome pairing during meiosis culminates with synapsis , defined as the assembly of synaptonemal complexes ( SCs ) between homologous chromosomes ( homologs ) .",
"Upon meiotic entry , prior to pairing and synapsis , replicated chromosomes reorganize around a central ‘axis , ’ a linear structure comprised of cohesin complexes and associated meiosis-specific proteins .",
"Once chromosomes establish local interactions with their homologs through recombination or other mechanisms , the central region of the SC nucleates and assembles progressively between paired axes , resulting in close side-by-side alignment of homologous chromosomes along their entire lengths ( MacQueen et al . , 2002; Page and Hawley , 2004; Rog and Dernburg , 2015 ) .",
"SC assembly is required for stable interhomolog pairing , normal levels of crossover ( CO ) recombination , cell cycle progression , and faithful chromosome segregation ( Page and Hawley , 2004 ) .",
"While disruption of the SC dramatically alters the CO distribution in a variety of organisms , the functional contribution of the SC to CO regulation remains hotly debated .",
"Evidence from budding yeast has suggested that the SC may be a passive ‘glue’ that merely stabilizes the physical pairing of homologs to permit efficient CO formation ( Börner et al . , 2004; Fung et al . , 2004; Zickler and Kleckner , 2015 ) .",
"However , evidence from C . elegans has indicated that the SC plays a direct role in the conserved phenomenon of crossover interference , which distributes COs in a non-random , widely spaced pattern along each chromosome ( Hayashi et al . , 2010; Libuda et al . , 2013 ) .",
"The SC was initially observed sixty years ago by thin-section transmission electron microscopy ( TEM ) , which revealed electron-dense linear structures at the interface between meiotic chromosomes ( Moses , 1956 ) .",
"Subsequent observations of meiocytes from diverse eukaryotes led to a consensus view that the SC is a symmetrical , tripartite structure , with two parallel , darkly-staining lateral bands flanking an electron-lucent , transversely striated , central region ( Westergaard and von Wettstein , 1972and Figure 1B ) .",
"Electron microscopy , superresolution fluorescence imaging , and protein-protein interaction analysis have clearly indicated that the proteins that make up this complex form a highly ordered , periodic structure with bilateral symmetry ( Schild-Prüfert et al . , 2011; Schücker et al . , 2015 ) .",
"Serial-section TEM analysis also led to the discovery of ‘recombination nodules’ associated with SCs at sites of genetic exchange ( Carpenter , 1975 ) , and specific recombination factors have been localized to these sites by a variety of cytological methods .",
"However , how the SC assembles between chromosomes , and how this polymer might govern or respond to meiotic recombination are largely mysterious . 10 . 7554/eLife . 21455 . 003Figure 1 . Polycomplexes are 3D lattices of SC proteins that exhibit liquid-like behaviors .",
"( A ) Fluorescence micrograph showing mid-prophase oocyte nuclei from wild type and htp-3 ( tm3655 ) hermaphrodites , immunostained for SYP-2 .",
"Whereas in wild type animals SCs load between homologous chromosomes and appear as long filaments , in the absence of HTP-3 SC proteins form one or more large bodies that contain all of the known SC central region proteins .",
"See Figure 1—figure supplement 1A for images of the entire gonads .",
"( B ) Representative transmission electron micrographs .",
"The top image shows a single nucleus from a wild type hermaphrodites , with a synapsed chromosome pair ( darkly-staining material flanking the SC is chromatin ) .",
"The middle image shows a single nucleus from a htp-3 ( tm3655 ) hermaphrodite , with a polycomplex indicated by the green arrow .",
"The darkly staining region in the center of the nucleus is the nucleolus .",
"The bottom image shows a higher-magnification view of a polycomplex from a different nucleus .",
"The distance between parallel darkly-staining bands is 97 nm , identical to the width of SCs that normally form between homologous chromosomes .",
"Notably , these polycomplexes do not contain any of the known chromosome axis components , including cohesins and the HORMA domain proteins HTP-3 , HIM-3 , HTP-1 and HTP-2 ( Severson et al . , 2009 and our observations ) .",
"This implies that the electron-dark lateral bands of the SC do not correspond to the chromosome axis , as has been long presumed , but are instead part of the structure formed by the central region proteins .",
"See Figure 1—figure supplement 2A for serial sections of the polycomplex in the lower panel .",
"Scale bars = 0 . 2 μm .",
"( C ) Projection images showing a single nucleus from a live recording of a htp-3 ( tm3655 ) ; GFP-SYP-3 hermaphrodite , at selected time points .",
"Elapsed times are indicated as hours:minutes:seconds .",
"Polycomplexes continually undergo deformations and fusions .",
"Here , fusion of two initially separate polycomplexes is observed between 0:09:00 and 0:10:00 , and again between 0:20:00 and 0:21:00 .",
"The full recording is shown in Video 1 .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 00310 . 7554/eLife . 21455 . 004Figure 1—figure supplement 1 . Further characterization of polycomplexes and heat-induced SC aggregates .",
"( A ) Fluorescence micrograph showing gonads from wild type and htp-3 ( tm3655 ) hermaphrodites , immunostained for SYP-2 .",
"Whereas in wild type animals SCs load between homologous chromosomes and appear as long filaments , in the absence of HTP-3 one or more large bodies that contain all of the known SC central region proteins are observed in each nucleus .",
"See Figure 2B for an explanatory diagram of the C . elegans gonad and Figure 1A for high magnification images of pachytene nuclei .",
"( B–D )",
"Heat-induced SC aggregates are distinct from polycomplexes .",
"( B ) Projection images from a real-time recording from a live htp-3; GFP-SYP-3 hermaphrodite that was incubated for 24 hr at 26 . 5°C prior to imaging .",
"Several nuclei are seen in this field .",
"Following extended exposure to heat , the SC protein aggregates undergo limited motion within the nucleus , and maintain their overall shapes throughout the time course ( 50 min ) .",
"They were not observed to undergo fusions when in close proximity .",
"The full recording is shown in Video 5 .",
"Scale bar = 5 μm .",
"( C ) Transmission electron micrograph showing a single mid-prophase meiotic nucleus from an htp-3 ( tm3655 ) mutant hermaphrodite incubated overnight at 26 . 5°C prior to high-pressure freezing .",
"No striated polycomplexes were detected in these samples .",
"The nucleoli appeared to be very electron-dark and fragmented , as also observed by fluorescence microscopy ( Figure 4—figure supplement 4B ) .",
"Scale bar = 0 . 5 μm .",
"( D ) Polycomplexes are more compact than heat-induced aggregates .",
"Compactness was calculated by dividing the surface area of a sphere with the same volume as the given particle to the surface area of the particle , based on images of polycomplexes and aggregates from live htp-3; GFP-SYP-3 hermaphrodites grown at 20°C or 26 . 5°C for 24 hr .",
"Compactness is overestimated in both cases due to the limited resolution of the microscope system; values are thus primarily useful for comparison .",
"The boxes indicate the median and the interquartile range , and the whiskers 1 . 5 times the interquartile range .",
"n = 234 and 289 nuclei for 20°C and 26 . 5°C , respectively .",
"Student’s t-test: p<10−37 . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 00410 . 7554/eLife . 21455 . 005Figure 1—figure supplement 2 . Further characterization of polycomplexes .",
"( A ) Images from serial 70 nm sections through the polycomplex shown in Figure 1B ( bottom panel ) .",
"Scale bars = 0 . 2 μm .",
"( B ) Still images from a representative recording from an intact , live htp-3 ( tm3655 ) mutant hermaphrodite expressing GFP-SYP-3 ( SC/polycomplexes; green ) and mCherry-H2B ( chromatin; red ) .",
"When polycomplexes come into contact within the same nucleus , they frequently merge with one another ( white arrows at t = 36:04–45:26 ) .",
"An example of an oocyte undergoing apoptosis is also observed in this recording .",
"The mCherry-H2B reporter ( or proteolytic fragments ) localizes to the membranes of the engulfing somatic cell .",
"A single large polycomplex persists for a few minutes following engulfment of the apoptotic corpse ( yellow arrows at t = 1:25:25–1:26:45 ) , and then disappears .",
"The full recording is shown in Video 2 .",
"Scale bar = 5 μm .",
"( C ) Polycomplexes are detected in triple kleisin mutants and in htp-3 mutants expressing only fluorescently tagged SYP-3 .",
"Top and middle: transmission electron micrographs showing mid-prophase meiotic nuclei from rec-8 ( ok978 ) ; coh-4 ( tm1857 ) coh-3 ( gk112 ) hermaphrodites , which lack meiotic kleisins .",
"Middle , two polycomplexes within the same nucleus , both adjacent to the darkly-staining nucleolus ( bottom ) .",
"The darkly staining material around the polycomplexes is chromatin .",
"Bottom , an oocyte nucleus from an htp-3 ( tm3655 ) syp-3 ( ok758 ) ; GFP-SYP-3 hermaphrodite .",
"The darkly staining nucleolus is seen at the bottom right corner of the frame .",
"Scale bars = 0 . 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 005 Synapsis of homologous chromosomes typically requires both nucleation factors and structural proteins that localize throughout the SC , which spreads along the interface between aligned chromosomes ( Page and Hawley , 2004; Zickler and Kleckner , 2015 ) .",
"Structural components , defined here as proteins required for SC assembly , have been identified through genetic screens and/or immunocytochemistry in a variety of organisms .",
"Three to five such proteins have been characterized in budding yeast , Drosophila , C . elegans , and mammals , and at least one SC protein has been identified in plants .",
"However , it is not yet clear whether the full cohort of SC structural elements has been defined in any organism .",
"While the dimensions and appearance of the SC are conserved among diverse phyla , these structural proteins show remarkable divergence in their lengths and primary sequences .",
"All known SC structural proteins contain extensive regions that score highly on coiled-coiled prediction scales .",
"Together with the transverse striations observed by TEM , this has led to a prevalent view of the SC as a stable , ‘crosslinking’ polymer assembled through coiled-coil interactions , perhaps resembling intermediate filaments .",
"However , other observations have hinted at a more dynamic structure .",
"In particular , evidence from budding yeast has revealed that SCs can incorporate newly translated proteins after assembly ( Voelkel-Meiman et al . , 2012 ) , consistent with dynamic exchange of subunits .",
"Additionally , in many organisms SCs are known to undergo ‘synaptic adjustment , ’ post-assembly progressive reorganization that tends to simplify their topology ( e . g . , the extent of loop structures and unsynapsed axes is minimized ) ( Page and Hawley , 2004; MacQueen et al . , 2005; Henzel et al . , 2011 ) – a behavior that implies extensive rearrangement of subunits after initial assembly of the complex .",
"Here we probe the biophysical properties of the SC in vivo , and show that it exhibits liquid-like behaviors and likely assembles through a regulated coacervation process .",
"We further report that SCs in C . elegans , budding yeast , and Drosophila are rapidly and reversibly dissolved by aliphatic alcohols , indicating that their integrity relies on weak hydrophobic interactions .",
"Finally , we demonstrate that the dynamic localization and interdependence of two essential CO factors is recapitulated by chromosome-free assemblies of SC proteins , demonstrating how an SC compartment may dynamically partition enzymatic activities to regulate meiotic recombination ."
],
[
"The SC normally assembles between the paired axes of meiotic chromosomes ( Couteau et al . , 2004; Severson et al . , 2009; Kim et al . , 2014 ) .",
"Note that throughout this work we use terminology that differentiates between the SC ( sometimes called the 'central region' ) and the chromosome axis .",
"In the nematode C . elegans , the SC comprised of at least four structural proteins ( SYP-1–4 ) , which are mutually dependent for their chromosome association ( MacQueen et al . , 2002; Colaiácovo et al . , 2003; Smolikov et al . , 2007 , 2009; Schild-Prüfert et al . , 2011 ) .",
"When the chromosome axis is disrupted — for example , in C . elegans mutants lacking either the essential meiotic axis protein HTP-3 , or all three meiotic kleisins ( REC-8 , COH-3 , and COH-4 ) — SC proteins fail to assemble between chromosomes , and instead form large bodies in the nucleoplasm of meiotic cells ( Figure 1A and Figure 1—figure supplement 1A ) ( Goodyer et al . , 2008; Severson et al . , 2009 ) .",
"Using transmission electron microscopy ( TEM ) , we found that these nuclear bodies have a multilaminar internal structure , in which each layer recapitulates the appearance and dimensions of SCs ( Figure 1B and Figure 1—figure supplement 2A and C ) ( Dernburg et al . , 1998; Schild-Prüfert et al . , 2011 ) .",
"Similar periodic , multilaminar structures formed by SC proteins have been observed in wild-type nuclei or cytoplasm of meiocytes or nurse cells from a wide variety of organisms , as well as under perturbed conditions where SCs cannot assemble between chromosomes , and are known as ‘polycomplexes’ ( Roth , 1966; Westergaard and von Wettstein , 1972; Page and Hawley , 2004 ) .",
"In C . elegans these structured bodies have also been observed in wild-type meiocytes prior to SC assembly ( Goldstein , 2013; Rog and Dernburg , 2015 ) .",
"Importantly , polycomplexes in htp-3 mutants do not contain any of the known chromosome axis components ( cohesins , HIM-3 , or HTP-1–3 ) ( Severson and Meyer , 2014 , and our observations ) , indicating that these proteins are not essential for self-assembly of SC proteins into an ordered structure , but only for their assembly as a unilamellar structure between chromosomes .",
"Additionally , this reveals that the electron-dark bands with 100 nm periodicity within polycomplexes , which correspond to the cytologically-defined ‘lateral elements’ of the SC , are not equivalent to chromosome axes .",
"Polycomplexes contain all known SC structural proteins and depend on the full cohort of these proteins for their formation ( Humphryes et al . , 2013 , and our observations ) .",
"Coupled with their structural similarity to SCs , we reasoned that it might be possible to learn more about the SC , independent of its interactions with chromosome axes , by analyzing polycomplex dynamics in living worms .",
"Time-lapse imaging of htp-3 mutant worms expressing GFP-SYP-3 revealed that prophase nuclei frequently contained more than one fluorescent polycomplex .",
"Unexpectedly , we observed that when these came into contact , they readily fused to form a single larger body ( Videos 1 and 2 , Figure 1C and Figure 1—figure supplement 2B ) .",
"We also observed polycomplexes to undergo large-scale structural deformations , particularly towards the end of prophase as the nuclear volume increased ( Video 1 and Figure 1C ) .",
"The ability of such bodies to merge together and to readily change in shape are defining characteristics of liquids , since they imply that molecules can rapidly rearrange within a material .",
"These observations were thus initially very surprising , given the highly ordered appearance of polycomplexes .",
"However , materials with liquid-like properties and ordered arrangements of molecules , known as structured fluids or liquid crystals , are a well-known class of soft condensed matter , and a variety of structures within cells have been postulated to have such properties ( Brown and Wolken , 1979; Rey , 2010 ) . 10 . 7554/eLife . 21455 . 006Video 1 . Polycomplexes fuse upon contacting each other . Oocytes from the diplotene region of an htp-3 ( tm3655 ) hermaphrodite expressing GFP-SYP-3 , corresponding to the stills shown in Figure 1C .",
"Polycomplexes exhibit constant deformations and merge with one another ( between t = 0:09:00 and t = 0:10:00 and between t = 0:20:00 and t = 0:21:00 ) .",
"Meiotic progression in this recording is right to left .",
"Images were acquired every 1 min .",
"Playback is 300x real-time .",
"Scale bar = 5 μm .",
". DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 00610 . 7554/eLife . 21455 . 007Video 2 . Polycomplexes merge upon contact . Pachytene nuclei from an intact htp-3 ( tm3655 ) hermaphrodite expressing GFP-SYP-3 and histone-mCherry , corresponding to the stills in Figure 1—figure supplement 2B .",
"Polycomplexes ( green ) merged with one another ( between t = 0:40:04 and t = 0:42:44 ) .",
"An apoptotic nucleus was being engulfed between t = 1:24:05 and t = 1:25:25 .",
"A polycomplex remains visible until t = 1:26:45 .",
"Meiotic progression in this recording is from left to right .",
"Images were acquired about every 1 min .",
"Playback is 400x real-time .",
"Scale bar = 4 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 007 In vivo imaging also revealed that polycomplexes likely assemble by regulated phase separation .",
"Our early efforts to carry out long-term recordings in living worms consistently led to meiotic arrest , which we deduced to be a physiological response to food withdrawal ( Wynne et al . , 2012; Rog and Dernburg , 2015 ) .",
"Arrest was evident as abatement of the dramatic chromosome movements that normally occur throughout early prophase ( Wynne et al . , 2012 ) , which typically ceased within 15–20 min of immobilization ( Figure 2A and Figure 2—figure supplement 1 and Videos 3 and 4; see Figure 2B for a schematic of the C . elegans gonad ) .",
"In nuclei that had initiated synapsis before arresting , we observed that diffuse GFP-SYP-3 gradually coalesced to join existing segments of SC between chromosomes .",
"These partial SCs became brighter but not longer , likely reflecting addition of subunits to form a multilayered structure , much as they must stack within 3D polycomplexes .",
"Consistent with this idea , EM analysis of lateral and cross-sectional views of SCs has indicated that they contain varying numbers of layers ( Schmekel et al . , 1993a , 1993b ) .",
"The observation that these pre-existing SC segments did not spread along partially synapsed chromosomes following arrest suggests that longitudinal extension is an active process .",
"In earlier , presynaptic nuclei , GFP-SYP-3 coalesced within each nucleus over a period of tens of minutes to form several compact aggregates .",
"Similar coalescence of SC proteins was observed in animals treated with sodium azide , which reversibly arrests meiosis and other physiological processes by depleting ATP ( Figure 2C ) , suggesting that phosphorylation and/or other ATP-dependent mechanisms normally maintain the solubility of SC subunits . 10 . 7554/eLife . 21455 . 008Figure 2 . Condensation of SC proteins following meiotic arrest or ATP depletion .",
"( A ) Still images from a time-lapse recording of an otherwise wild-type hermaphrodite expressing GFP-SYP-3 , sampled at 1 min intervals .",
"A portion of the germline containing early meiotic nuclei , corresponding approximately to the boxed region in panel ( B ) , is shown .",
"During this recording , chromosome movements abated at approximately 0:05:00 .",
"Following this arrest , gradual condensation of fluorescent bodies is observed in nuclei lacking preexisting stretches of SC ( left side of frame ) , while existing SC segments become brighter , but do not elongate ( near the right side of frame ) .",
"Scale bar = 5 μm .",
"The full recording is shown in Video",
"3 . ( B ) The proximal region of a gonad from a C . elegans hermaphrodite , fixed and stained with DAPI , is shown as a reference for other figures and recordings .",
"Each gonad contains a complete progression of meiotic stages; here , nuclei enter and advance through meiotic prophase from left to right .",
"A signal from the distal tip inhibits meiotic entry; once cells move far enough from this signal they enter meiosis and progress through prophase .",
"At the loop region of the gonad , a spatially regulated signal triggers exit from pachytene .",
"( C ) Projection images from deconvolved 3D image stacks , showing premeiotic and early meiotic nuclei from wild-type adult hermaphrodites that were incubated for 1 hr on plates containing 0 . 5% w/v sodium azide before fixation , and control ( non-azide-treated ) animals .",
"Gonads were dissected and fixed immediately following azide treatment , and stained with antibodies against SYP-2 and HTP-3 to localize SCs and chromosome axes , respectively .",
"Small polycomplexes that contain SYP-2 but not HTP-3 are observed in some early meiotic nuclei in the absence of any treatment , as previously described ( Goldstein , 2013 ) , but become much larger and more abundant in response to azide exposure .",
"Note that transition zone nuclei maintain their polarized chromatin morphology during arrest .",
"Scale bars = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 00810 . 7554/eLife . 21455 . 009Figure 2—figure supplement 1 . Condensation of polycomplexes from the nucleoplasm following meiotic arrest .",
"( A–E )",
"Time-lapse recording showing the coalescence of SC proteins to existing SCs and polycomplexes following cessation of meiotic chromosome movements .",
"Images were acquired from hermaphrodites expressing GFP-SYP-3 at 1 min intervals .",
"In these images the direction of meiotic progression is from top right to bottom left .",
"Meiotic chromosome movements cease at approximately 0:20:00 .",
"The full recording is shown in Video",
"4 . Scale bars = 5 μm .",
"( A ) Additive Z projections at the indicated times .",
"( B–E )",
"Coalescence of SC proteins results in loss of diffuse fluorescence from the nucleoplasm .",
"Additive Z projections from the indicated times are shown .",
"Autofluorescence of lipid droplets in the intestine is seen at the upper left corner .",
"( C–E ) show plots of integrated fluorescence intensity , measured within the regions marked by concentric circles in ( B ) as a function of time .",
"The larger circles encompass whole nuclei , while the smaller circles mark nucleoplasmic regions that lack discrete aggregates or filaments .",
"All intensity values were background-subtracted based on an area of the same size outside the gonad .",
"The difference between the final and initial value , based on linear regression , is indicated .",
"( C ) Nucleoplasmic fluorescence remains stable over time in nuclei where no aggregation is seen ( green circle ) .",
"( D ) In nuclei with new aggregates there is a small increase in overall intensity , but a marked decrease in the intensity of nucleoplasmic regions without aggregates , indicating depletion of nucleoplasmic SC proteins ( lilac circle ) .",
"( E ) In nuclei that contain SC filaments at the time when chromosome motions cease , filaments become progressively brighter ( cyan circle ) .",
"As in ( D ) , the overall fluorescence increases marginally , while regions without filaments exhibit marked decrease in fluorescence , indicating depletion of SC proteins from the nucleoplasm .",
"( F ) Aggregates of SUN-1 at the nuclear periphery disperse following meiotic prophase arrest , by contrast to the behavior of SC proteins , which aggregate in response to arrest .",
"Adult hermaphrodites expressing SUN-1-mRuby were immobilized in 1% w/v sodium azide to deplete ATP .",
"Meiosis in these images progresses from left to right .",
"Additive Z projections of premeiotic transition zone nuclei at the indicated times are shown .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 00910 . 7554/eLife . 21455 . 010Video 3 . Aggregation of SC components following cessation of meiotic chromosome movements . Meiotic prophase in a hermaphrodite expressing GFP-SYP-3 .",
"Premeiotic nuclei are at the left , and nuclei enter meiosis and progress from left to right .",
"Chromosome motion ceases , indicative of meiotic arrest , at about t = 5 min .",
"Images were acquired every 1 min .",
"Playback speed is 300x real-time .",
"Scale bar = 5 μm .",
"Stills are shown in Figure 2A . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 01010 . 7554/eLife . 21455 . 011Video 4 . Aggregation of SC components following cessation of meiotic chromosome movements . Recording from a hermaphrodite expressing GFP-SYP-3 .",
"Premeiotic nuclei are at the lower right portion of the images , and meiotic entry/progression is observed from lower right to upper central portion of the images .",
"Fluorescent lipid droplets within the intestine are seen to the left of the gonad .",
"Images were acquired every 1 min .",
"Playback is 300x real-time .",
"Scale bar = 5 μm .",
"Stills and analysis of this recording are shown in Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 011 The liquid-like behaviors we observe for polycomplexes , as well as the spontaneous coalescence of SC components from the nucleoplasm upon arrest , indicate that these nuclear bodies arise through coacervation , or phase separation , as been inferred for other self-assembling subcellular compartments ( Brangwynne et al . , 2009; Hyman et al . , 2014 ) .",
"SC proteins have also been shown to form nuclear aggregates in C . elegans following extended incubation at elevated temperatures , which also renders the animals sterile ( Bilgir et al . , 2013 ) .",
"We found that these heat-induced bodies lack periodic striations in electron micrographs and do not undergo fusions or shape deformations ( Figure 1—figure supplement 1B–D and Video 5 ) , indicating that they lack both the liquid-like properties and internal order of polycomplexes .",
"This irreversible aggregation likely represents conversion to a denatured state , similar to the way that other coacervates can transition to aberrant solid or amyloid-like states ( Kroschwald et al . , 2015 ) . 10 . 7554/eLife . 21455 . 012Video 5 . Heat-induced SC aggregates do not show liquid-like properties . A htp-3 ( tm3655 ) mutant hermaphrodite expressing GFP-SYP-3 was incubated overnight at 26 . 5°C ( corresponding to images in Figure 1—figure supplement 1B ) .",
"SC aggregates show some mobility within the nucleus , but do not merge with each other , and maintain their irregular shapes over the time course .",
"Meiotic progression in this recording is from left to right .",
"Images were acquired every 1 min .",
"Playback is 300x real-time .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 012 We next investigated whether SCs assembled between chromosomes also exhibit liquid-like behaviors by assessing the mobility of SC proteins within these structures .",
"We engineered worm strains expressing SYP-3 fused to the photoconvertible fluorescent protein mMaple3 ( Wang et al . , 2014b ) .",
"We photoconverted subnuclear regions in live worms and imaged these nuclei over time .",
"The photoconverted proteins rapidly redistributed throughout the SCs within the same nucleus , becoming homogeneously dispersed within 22 min ( Figure 3A , C and Figure 3—figure supplement 1A ) .",
"By contrast , when the axis protein HIM-3 was tagged with mMaple3 , we observed no significant redistribution of converted fluorophores over more than 30 min of observation ( Figure 3B–C and Figure 3—figure supplement 1B ) .",
"This implies that other axis components , particularly HTP-3 , which scaffolds the recruitment of HIM-3 ( Kim et al . , 2014 ) , are also stably associated with the axis .",
"We conclude that SC central region proteins are far more mobile than axis components .",
"The relatively slow rate of subunit turnover within the SC compared to that observed for other liquid-like compartments might reflect a high viscosity of the liquid-like phase , and/or other constraints such as confinement within the very thin laminar space , or a slow rate of exchange of subunits between the different SC compartments in the nucleus .",
"Future studies using high-speed imaging and other tools should provide a more complete understanding of the movement of proteins within the SC .",
"Taken together , our observation of coacervation following meiotic arrest , of liquid-like behaviors of polycomplexes , and of the dynamic exchange of subunits all indicate that the SC is a phase-separated compartment with liquid crystalline properties . 10 . 7554/eLife . 21455 . 013Figure 3 . SC proteins , but not axis components , are highly dynamic .",
"( A–B )",
"Images of representative nuclei from time-lapse recordings of hermaphrodites expressing either the SC protein SYP-3 or the axis component HIM-3 fused to the photoconvertible fluorescent protein mMaple3 .",
"A subnuclear volume was photoconverted using 405 nm laser illumination at t = 00:00 .",
"The mMaple3-SYP-3 signal spread throughout the nucleus and to all chromosomes by 22 min ( A ) , whereas the mMaple3-HIM-3 signal remained confined to a small region throughout the time course ( 32 min; B ) .",
"Scale bars = 2 μm .",
"Elapsed times are indicated as min:sec .",
"( C ) The mobility of HIM-3 ( axis ) and SYP-3 ( SC ) were quantified by estimating the volume of individual nuclei containing photoconverted ( red ) signal as a function of time ( see Materials and methods for details ) ; SYP-3 within assembled SCs is far more mobile than HIM-3 ( Student’s t-test: p<10−13 ) .",
"The horizontal dashed line represents the expansion rate for a point source that becomes homogeneously distributed throughout the nuclear volume in one hour . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 01310 . 7554/eLife . 21455 . 014Figure 3—figure supplement 1 . SC proteins , but not axis components , are highly dynamic .",
"( A ) Time-lapse images of representative nuclei from adult hermaphrodites expressing mMaple3-SYP-3 ( A ) or mMaple3-HIM-3 ( B ) .",
"The fluorescent protein was photoconverted within a small region of individual nuclei at t = 00:00 using focused 405 nm light .",
"Scale bars = 3 μm .",
"( A ) Photoconverted SYP-3 spreads throughout the nucleus and to all chromosome during the time course ( 32 min ) .",
"( B ) Photoconverted HIM-3 remains confined to a region of similar volume throughout the time course ( 32 min ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 014 Recent work has illuminated several mechanisms that can promote intracellular phase transitions , including multivalent interactions ( Li et al . , 2012 ) , low-complexity protein domains with β-strand propensity ( Kato et al . , 2012 ) , and hydrophobic interactions ( Schmidt and Görlich , 2015 ) .",
"The latter are thought to be essential for the integrity of the nuclear pore , within which a network of unstructured proteins forms a diffusion barrier , and for the formation of P-granules in the C . elegans germline ( Updike et al . , 2011 ) , among other liquid-like cellular compartments ( Kroschwald et al . , 2015 ) .",
"In support of this idea , these compartments are reversibly disrupted by moderate concentrations ( ~5% ) of 1 , 6-hexanediol or similar aliphatic alcohols , which lower aqueous surface tension ( Romero et al . , 2007 ) and reduce the hydrophobic effect ( Ribbeck and Görlich , 2002 ) .",
"To test the idea that hydrophobic interactions promote coacervation of SC proteins , we extruded worm gonads in physiological buffer and added varying concentrations of 1 , 6-hexanediol .",
"When gonads expressing GFP-SYP-3 were exposed to 5–10% 1 , 6-hexanediol , we observed immediate disruption of SCs and dispersion of the fluorescence throughout the nucleoplasm .",
"By contrast , the distributions of many other chromosome-associated proteins were unaffected by this treatment , including axis components ( HTP-3 , HIM-3 , HTP-1/2 , LAB-1 , and cohesins ) ; the pairing center protein HIM-8; and proteins associated with CO sites ( COSA-1 and ZHP-3 ) ( Figure 4A–B , Figure 4—figure supplement 3A , Videos 6 and 7 , and data not shown; see Figure 4—figure supplement 1C for an explanatory diagram; in all figures , panels showing samples treated with 1 , 6-hexanediol have pink borders ) .",
"In some nuclei a short stretch of SC associated with the X chromosomes persisted after treatment with 1 , 6-hexanediol ( Figure 4—figure supplement 3 ) , perhaps reflecting enhanced recruitment of SC proteins to the paired sex chromosomes ( Couteau et al . , 2004; Hayashi et al . , 2010 ) .",
"SC dissolution following 1 , 6-hexanediol exposure was also observed in wild-type gonads , indicating that it was not a consequence of the fluorescent tag ( Figure 4—figure supplement 3A ) .",
"Polycomplexes showed a very similar response to 1 , 6-hexanediol: they rapidly dissolved upon exposure to the solvent , while heat-induced SC aggregates were resistant to dissolution ( Figure 5B , Figure 4—figure supplement 4 and Video 8 and Video 9 ) . 10 . 7554/eLife . 21455 . 015Figure 4 . SCs in C . elegans , S . cerevisiae and Drosophila melanogaster dissolve in the presence of 1 , 6-hexanediol .",
"( A ) Projection images showing selected time points from time-lapse recordings of extruded gonads from C . elegans hermaphrodites expressing GFP-SYP-3 and HTP-3-mRuby to mark SCs and axes , respectively .",
"Upon exposure to 5% 1 , 6-hexanediol , SYP-3 immediately dispersed throughout the nucleoplasm , while HTP-3 remained associated with chromosomes .",
"Most of the GFP-SYP-3 fluorescence remained in the nucleus for the duration of our experiments .",
"The full recording is shown in Video 6 .",
"Scale bar = 5 μm .",
"( B ) Gonads were treated with 1 , 6-hexanediol as in ( A ) , but then fixed and counterstained with DAPI .",
"Scale bar = 5 μm .",
"( C–D )",
"The SC in Drosophila is also sensitive to 1 , 6-hexanediol .",
"( C ) Projection images showing GFP fluorescence in two adjacent oocytes from a Drosophila female expressing GFP-C ( 3 ) G .",
"C ( 3 ) G ) is an essential component of the SC central region .",
"Ovarioles were dissected in buffer , and imaged before and after exposure to 7 . 5% 1 , 6-hexanediol .",
"Scale bar = 5 μm .",
"( D ) Projection images showing a single oocyte within a germarium from a fly expressing HA-tagged C ( 2 ) M ( a component of the chromosome axis ) .",
"Ovarioles were dissected , exposed to 7 . 5% hexanediol ( or not ) , and fixed with formaldehyde , then stained with antibodies against C ( 3 ) G ( green ) and HA ( red ) .",
"C ( 3 ) G was solubilized by hexanediol , while C ( 2 ) M remained associated with chromosome axes .",
"Similar results were obtained with flies expressing GFP-C ( 3 ) G and stained with antibodies against GFP and C ( 2 ) M ( data not shown ) .",
"Scale bar = 5 μm .",
"( E ) Diploid ndt80 S . cerevisiae strains expressing the indicated GFP fusion proteins were sporulated and arrested in mid-pachytene .",
"Following exposure to 1 , 6-hexanediol , cells were imaged immediately without fixation .",
"The central region component Zip1p dispersed throughout the nuclei upon 1 , 6-hexanediol treatment , while the cohesin subunit Rec8p remained along chromosomes .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 01510 . 7554/eLife . 21455 . 016Figure 4—figure supplement 1 . SC proteins , but not axis components , are highly dynamic .",
"( A ) Electrostatic and hydrophobic interactions contribute to SC stability .",
"As in Figure 4E , diploid ndt80 S . cerevisiae cells expressing Zip1p-GFP were sporulated , arrested in mid-pachytene , and treated with various concentrations of 1 , 6-hexanediol and KCl in buffer .",
"SCs were scored as ‘dissolved’ if no distinct structures remained .",
"( B ) Effect of various di-alcohols on SC stability .",
"As in Figure 4E , diploid ndt80 S . cerevisiae cells expressing Zip1p-GFP were sporulated , arrested in mid-pachytene , and treated with various concentrations of di-alcohols in buffer .",
"For each alcohol , the lowest concentration necessary to completely dissolve the SC is shown .",
"( C ) Explanatory diagram of the effects of SC dissolution by 1 , 6-hexanediol on meiocytes from S . cerevisae , D . melanogaster and C . elegans .",
"Prior to treatment the SC ( green ) and axes ( red ) colocalize on chromosomes in the nucleus ( nucler envelope , blue; plasma membrane , lilac ) .",
"Upon treatment with 1 , 6-hexanediol ( right ) , SC material becomes diffuse in the nucleus and the cytoplasms , while the chromosome axes remain intact . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 01610 . 7554/eLife . 21455 . 017Figure 4—figure supplement 2 . The syp-3 ( me42 ) mutation results in hypersensitivity of SCs to 1 , 6-hexanediol and prevents aggregation in the absence of axes or at high temperatures .",
"( A ) The sensitivity of SCs to hexanediol was compared for various mutants that affect SC assembly and/or structure .",
"Extruded gonads were exposed to 4% 1 , 6-hexanediol , which disrupts only a minor fraction ( 15% ) of gonads in wild-type animals .",
"In syp-3 ( me42 ) mutants , in which the C-terminus of one SC component is truncated by 11 amino acids , resulting in aberrant synapsis ( Smolikov et al . , 2007 ) , 4% hexanediol completely disrupted the SCs ( see images in ( B ) ) .",
"Other mutations that affect CO formation or SC structure did not markedly affect hexanediol sensitivity , including hal-2 mutants , which show defects in SC assembly similar to syp-3 ( me42 ) mutants ( Zhang et al . , 2012 ) .",
"( B ) Nuclei from wild-type and syp-3 ( me42 ) animals were dissected , treated with 4% hexanediol where indicated , fixed with formaldehyde , and stained with antibodies against SYP-2 ( SC; green ) and HTP-3 ( axes; red ) .",
"Scale bar = 3 μm .",
"( C–D )",
"The syp-3 ( me42 ) mutation prevents polycomplex formation and heat-induced aggregation .",
"( C ) Gonads from htp-3 and htp-3 syp-3 ( me42 ) hermaphrodites were fixed and stained with DAPI ( chromatin; blue ) and with antibodies against the SC component SYP-2 ( green ) .",
"SC proteins in htp-3 syp-3 ( me42 ) animals remain diffuse in the nucleoplasm , or concentrate in the nucleoli of some nuclei .",
"Scale bar = 3 μm .",
"( D ) Wild-type and syp-3 ( me42 ) animals were incubated for 24 hr at 26 . 5°C before fixation and stained with antibodies against SYP-2 ( green ) HTP-3 ( red ) .",
"Under these conditions , SC proteins form nuclear aggregates in wild-type animals , as reported ( Bilgir et al . , 2013 ) , but remain diffuse in the nucleoplasm of syp-3 ( me42 ) animals .",
"Scale bar = 3 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 01710 . 7554/eLife . 21455 . 018Figure 4—figure supplement 3 . The X chromosome pairing center protein HIM-8 and a some of the SC on the X chromosome are refractory to 1 , 6-hexanediol .",
"( A ) A representative gonad from a wild-type adult hermaphrodite treated with 7 . 5% 1 , 6-hexanediol prior to fixation , and stained with antibodies against the SC component SYP-2 ( green ) , the axis component HTP-3 ( red ) , and the X chromosome pairing center protein HIM-8 ( blue ) .",
"While most of the SC is dissolved , the chromosomal axes and the X chromosome pairing center remain intact .",
"The small hexanediol-resistant stretch of SC is adjacent the chromosome X pairing center .",
"Scale bar = 5 μm .",
"( B ) Images of meiotic nuclei from adult hermaphrodites expressing GFP-SYP-3 and treated with 7 . 5% 1 , 6-hexanediol prior to fixation .",
"The small hexanediol-resistant SC stretches and puncta are not present in him-8 mutant worms ( bottom ) , in which the X chromosomes do not pair or synapse .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 01810 . 7554/eLife . 21455 . 019Figure 4—figure supplement 4 . Polycomplexes , but not heat-induced aggregates , are dissolved by 1 , 6-hexanediol .",
"( A ) Time-lapse images of gonads from GFP-SYP-3 hermaphrodites grown for 24 hr at 26 . 5°C and then treated with 7 . 5% 1 , 6-hexanediol .",
"The large SC aggregates were resistant to hexanediol , while the few assembled SCs present in some nuclei were readily dissolved .",
"The outlined regions are shown at larger magnification on the right; rescaled to highlight SC filaments , which are dimmer than the aggregates .",
"The full recording is shown in Video",
"8 . Scale bars = 5 μm .",
"( B–C )",
"Projections of 3D fluorescence optical sections .",
"( B ) Extruded gonads from htp-3 ( tm3655 ) hermaphrodites were incubated with 7 . 5% 1 , 6-hexanediol where indicated , then fixed and stained with antibodies against SYP-2 ( green ) and with DAPI ( blue ) .",
"Polycomplexes in samples grown at 20°C are sensitive to 1 , 6-hexanediol .",
"SC aggregates following prolonged incubation at 26 . 5°C are resistant to 1 , 6-hexanediol .",
"Scale bars = 3 μm .",
"( C ) Extruded gonads from rec-8 ( ok978 ) ; coh-4 ( tm1857 ) coh-3 ( gk112 ) hermaphrodites were incubated with 7 . 5% 1 , 6-hexanediol where indicated , then fixed and stained with antibodies against the SC component SYP-2 ( green ) and the axis component HTP-3 ( red ) , and counterstained DAPI ( blue ) .",
"Polycomplexes in this triple kleisin mutant contain the axis protein HTP-3 , which disperses ( along with the SC components ) upon 1 , 6-hexanediol exposure .",
"Scale bars = 3 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 01910 . 7554/eLife . 21455 . 020Figure 5 . Dissolution of SCs and polycomplexes by 1 , 6-hexanediol is reversible .",
"( A ) Projection images from a time-lapse recording showing GFP-SYP-3 before and after dispersion by 5% 1 , 6-hexanediol , followed by dilution with buffer .",
"Pink borders around images indicate times after hexanediol addition , and yellow borders indicate times after dilution of the hexanediol with buffer .",
"Upon dilution , fluorescence reappears along chromosomes .",
"Some small puncta are also observed in the cytoplasm .",
"Chromosome association of SC proteins after longer exposures to 1 , 6-hexanediol ( >2 min ) was not reversible ( data not shown ) , perhaps due to irreversible perturbations of chromosome structure or SC subunits .",
"The full recording is shown in Video",
"9 . Elapsed time is indicated as mm:ss .",
"Larger-magnification images of a representative nucleus are shown on the right .",
"Scale bars = 5 μm .",
"( B ) Projection images from a time-lapse recording of a htp-3 ( tm3655 ) mutant hermaphrodite expressing GFP-SYP-3 .",
"Exposure to 7 . 5% 1 , 6-hexanediol ( pink image borders ) induces rapid dispersion of the SC proteins from polycomplexes .",
"Upon dilution of 1 , 6-hexanediol with buffer ( yellow image borders ) , SYP-3 coalesces into smaller bodies that fuse with each other upon contact ( lilac arrows ) .",
"The full recording is shown in Video",
"10 . Scale bar = 5 μm .",
"( C ) A diploid ndt80 S . cerevisiae strain expressing Zip1p-GFP was sporulated , arrested in mid-pachytene , and treated with 5% 1 , 6-hexanediol .",
"10 volumes of buffer were added after the indicated incubation times with hexanediol , and cells were then mounted and imaged without fixation .",
"Reassociation of Zip1 with chromosomes was observed when 1 , 6-hexanediol was diluted after 2 min or 30 min , but appeared to be less robust after the longer incubation .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 02010 . 7554/eLife . 21455 . 021Video 6 . The central region of the SC , but not the chromosome axis , is dissolved by 1 , 6-hexanediol . This gonad was extruded from a hermaphrodite expressing GFP-SYP-3 and HTP-3-mRuby .",
"Partially synapsed zygotene nuclei are seen at the left , and fully synapsed pachytene nuclei at the right .",
"1 , 6-hexanediol was added to a concentration of 5% at t = 0:00:20 .",
"Images were acquired every 5 s .",
"Playback is 50x real-time .",
"Scale bar = 5 μm .",
"Stills are shown in Figure 4A .",
". DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 02110 . 7554/eLife . 21455 . 022Video 7 . 1 , 6-hexanediol dissolves the SC central region . Recording from a gonad extruded from a hermaphrodite expressing GFP-SYP-3 and HTP-3-mRuby .",
"5% 1 , 6-hexanediol was added at t = 0:00:35 ( yellow flash ) .",
"Meiotic progression in this recording is from right to left .",
"Images were acquired every 5 s .",
"Playback is 25x real-time .",
"Scale bar = 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 02210 . 7554/eLife . 21455 . 023Video 8 . Heat-induced SC aggregates are resistant to 1 , 6-hexanediol . Time-lapse movie of a gonad from GFP-SYP-3 hermaphrodite incubated overnight at 26 . 5°C shown in Figure 4—figure supplement 4A .",
"7 . 5% 1 , 6-hexanediol was added at t = 0:00:10 .",
"SC stretches in the region of the germline at the left side of the image are quickly dissolved , while the large , irregular SC aggregates remain intact .",
"Meiotic progression in this recording is from left to right .",
"Images were acquired every 5 s .",
"Playback speed is 25x real-time .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 02310 . 7554/eLife . 21455 . 024Video 9 . 1 , 6-hexanediol reversibly dissolves the SC . Time-lapse movie of a gonad from GFP-SYP-3 hermaphrodite shown in Figure 5A .",
"7 . 5% 1 , 6-hexanediol was added at t = 0:00:20 , and diluted at t = 0:00:50 and 0:01:35 .",
"Meiotic progression in this recording is from right to left .",
"Images were acquired every 5 s .",
"Playback is 25x real-time .",
"Scale bar = 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 02410 . 7554/eLife . 21455 . 025Video 10 . 1 , 6-hexanediol dissolves polycomplexes . Extruded gonad from an htp-3 ( tm3655 ) mutant expressing GFP-SYP-3 , corresponding to the stills shown in Figure 5B .",
"7 . 5% 1 , 6-hexanediol was added at t = 0:00:10 , and diluted at t = 0:00:45 and t = 0:01:17 .",
"Two small polycomplexes , reformed after 1 , 6-hexanediol addition and dilution , merge to form a single body at t = 0:01:52 .",
"Images were acquired about every 5 s .",
"Playback is 25x real-time .",
"Scale bar = 4 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 025 Despite the widespread conservation of SC structure among eukaryotes , central region proteins are notorious for their divergence in primary sequence among different lineages ( Westergaard and von Wettstein , 1972; Page and Hawley , 2004; Fraune et al . , 2012 ) .",
"We therefore wondered whether the biophysical properties we observed in C . elegans are conserved .",
"We found that SCs in both Drosophila and budding yeast were rapidly dispersed by 1 , 6-hexanediol , while axis markers ( Rec8p in budding yeast; C ( 2 ) M in Drosophila ) remained associated with chromosomes , as in C . elegans ( Figure 4C–E; see Figure 4—figure supplement 1C for an explanatory diagram ) .",
"This indicates that weak hydrophobic interactions are important for SC integrity in diverse lineages .",
"While SCs in budding yeast , Drosophila , and C . elegans meiocytes were readily disrupted by 1 , 6-hexanediol , we found that similar concentrations of the more polar solvents ethanol or dimethyl sulfoxide failed to dissolve these structures ( data not shown ) .",
"Because the effects of 1 , 6-hexanediol on cellular structures are not well understood , we also probed the sensitivity of SCs to disruption by a number of other water-miscible alcohols .",
"These di-alcohols vary in the length of the hydrocarbon chain and the positions of their hydroxyl groups .",
"Most experiments were performed with yeast cells arrested in mid-pachytene , since it is simple to manipulate the media in which these cells are immersed and no dissection or cell permeabilization was required to see the effects of 1 , 6-hexanediol .",
"For each di-alcohol we determined the minimal concentration required for complete SC disruption , based on loss of any discernible structure by fluorescence microscopy of unfixed yeast cells ( Figure 4—figure supplement 1B ) .",
"We observed a clear correlation between the extent of the hydrophobic domains of different solvents and their effects on the SC .",
"For example , 1 , 2-heptanediol disrupted the structure at lower concentrations than 1 , 2-hexanediol , which was more potent than 1 , 2-pentanediol .",
"Similar results were obtained for the series 1 , 7-heptanediol , 1 , 6-hexanediol , and 1 , 5-pentanediol .",
"Together , these results reinforce the idea that solvation by aliphatic alcohols is due to suppression of the hydrophobic effect , and support a major contribution of hydrophobic interactions to SC stability .",
"We also probed the effect of electrostatic interactions on SC stability .",
"We noted that SC central region proteins in various organisms are predominantly polyampholytic , i . e . , they contain an unusually large fraction of charged residues , both acidic and basic , which are well-mixed within the primary sequences .",
"This suggests that electrostatic interactions likely contribute to SC integrity , and that higher salt concentrations , which shield electrostatic interactions ( Veis , 2011 ) , might disfavor assembly .",
"Consistent with this , we found that increasing the concentration of KCl potentiated the ability of 1 , 6-hexanediol to dissolve the SC in budding yeast ( Figure 4—figure supplement 1A; other salts showed similar effects [data not shown] ) , highlighting a role for electrostatic interactions , in addition to hydrophobic interactions , in SC assembly and stability .",
"The effects of increasing salt concentration were fairly modest in light of the highly charged nature of SC proteins; however , only a limited range of ionic strength could be investigated in living cells , and the effects of these buffer manipulations on intracellular ionic strength could not be directly monitored .",
"Both of these trends observed in budding yeast — disruption of SCs by lower concentrations of more hydrophobic diols , and synergy between electrostatic shielding and amphipathic solvents — also held true for C . elegans SCs ( data not shown ) .",
"Experiments conducted using this system were less comprehensive since they required extensive hand dissection and , probably as a consequence of this manipulation , were somewhat less consistent and thus harder to score .",
"Nevertheless , we tested whether various mutations in C . elegans that perturb meiotic recombination or chromosome structure might affect 1 , 6-hexanediol sensitivity , and observed no major differences , except that the aberrant SCs in syp-3 ( me42 ) mutants , which carry a small C-terminal truncation in an SC protein ( Smolikov et al . , 2007 ) , were hypersensitive to 1 , 6-hexanediol ( Figure 4—figure supplement 2A–B ) .",
"We further probed the effects of this mutation , and found that the syp-3 ( me42 ) truncation also prevents polycomplex formation in an htp-3 background ( Figure 4—figure supplement 2C ) , as well as heat-induced aggregation ( Figure 4—figure supplement 2D ) .",
"The correlation between 1 , 6-hexanediol hyper-sensitivity , defects in SC assembly and lack of SC aggregation further underscore the contribution of hydrophobic interactions to the functional integrity of the SC .",
"When SCs in C . elegans were disrupted by 1 , 6-hexanediol , followed by rapid dilution of the solvent with buffer , GFP-SYP-3 immediately relocalized along the chromosomes ( Video 10 and Figure 5A; throughout the figures , panels showing samples subsequent to the dilution are shown with a yellow border ) .",
"Some fluorescent material that had exited the nucleus , perhaps due to transient disruption of the nuclear pores , formed small puncta in the cytoplasm .",
"Notably , reassembly of SCs following 1 , 6-hexanediol dilution occurred much more quickly than physiological SC assembly between axes , which requires 20–30 min per chromosome pair ( Rog and Dernburg , 2015 ) .",
"Polycomplex dissolution was also reversible: upon dilution of 1 , 6-hexanediol , multiple small puncta formed in each nucleus , and rapidly fused with each other to form larger bodies ( Video 9 and Figure 5B ) , qualitatively similar to the behavior of polycomplexes in live animals ( Figure 1C , Figure 1—figure supplement 2B and Videos 1 and 2 ) .",
"This reassembly following hexanediol dilution occurred much more quickly than under metabolic arrest ( Figure 2A and Figure 2—figure supplement 1 ) .",
"Dissolution of the SC by 1 , 6-hexanediol in budding yeast cells was also reversible: we observed some re-association of SC components with chromosomes after both short ( 2 min ) and longer ( >30 min ) incubations of yeast cells arrested at mid-pachytene ( Figure 5C ) .",
"These results provide additional evidence that neither the chromosome axes , which are essential for SC assembly between chromosomes , nor the SC proteins themselves , are irreversibly perturbed by exposure to 1 , 6-hexanediol .",
"Subcellular liquid-like compartments are selectively permeable to macromolecules , and can regulate biochemical reactions by concentrating enzymes and substrates and restricting their diffusion ( reviewed by Hyman et al . , 2014 ) .",
"We therefore wondered whether the SC might regulate and surveil recombination by partitioning CO factors into a distinct compartment between homologous chromosomes .",
"At least one essential CO factor that is not required for SC assembly , the conserved RING-finger protein ZHP-3 , is observed throughout the SC during early prophase , before becoming restricted to designated CO sites ( Figure 6—figure supplement 1A ) ( Jantsch et al . , 2004; Bhalla et al . , 2008 ) , whereupon it colocalizes with COSA-1 , another conserved protein required for COs ( Yokoo et al . , 2012 ) .",
"We found that CO formation altered not only the localization of ZHP-3 but also its sensitivity to 1 , 6-hexanediol: Prior to CO designation , the ZHP-3 distributed throughout SCs was dispersed by the solvent , while CO-associated ZHP-3 foci remained intact even after disruption of the SC by 1 , 6-hexanediol ( Figure 6—figure supplement 1A–B ) .",
"If ZHP-3 concentrates between chromosomes by partitioning into the SC , we expected that it might also localize within polycomplexes .",
"Indeed , we found that in htp-3 mutants , ZHP-3 was concentrated throughout polycomplexes during most of meiotic prophase .",
"However , near the ‘loop’ region of the gonad , where exit from pachytene normally occurs ( see Figure 2B for reference ) , we observed an abrupt change in ZHP-3 localization: The protein largely disappeared from the interior of the polycomplexes and became restricted to a single focus conspicuously abutting the surface of each polycomplex .",
"Intriguingly , at the same stage , COSA-1 , which was not detected earlier , became localized to these same small , peripheral puncta ( Figure 6A–C ) .",
"This dynamic relocalization of ZHP-3 and COSA-1 is highly analogous to what is observed along bona fide SCs upon CO designation ( Yokoo et al . , 2012 ) .",
"We further determined that ZHP-3 is required for the appearance of COSA-1 foci at polycomplexes , while COSA-1 is required for the relocalization of ZHP-3 to the edge of these bodies and its disappearance from the interior ( Figure 6D–E and Figure 6—figure supplement 1C–D ) , mirroring the interdependence of these factors for their localization to CO sites ( Yokoo et al . , 2012 ) .",
"We also found that the earlier ZHP-3 localization throughout polycomplexes is readily disrupted by 1 , 6-hexanediol , whereas the foci of COSA-1 and ZHP-3 foci at the edges of polycomplexes are 1 , 6-hexanediol-resistant ( Figure 6—figure supplement 1E ) , again mirroring the properties of ZHP-3 and COSA-1 associated with SCs and designated CO sites , respectively .",
"Polycomplexes do not appear to be associated with chromosomes and moreover , meiotic recombination does not initiate in htp-3 mutants ( Goodyer et al . , 2008 ) .",
"Thus , localization of COSA-1 and ZHP-3 to foci on polycomplexes does not require their interaction with CO intermediates such as Holliday junctions , or indeed with chromosomes .",
"These results imply that in response to exogenous cell cycle signals , polycomplexes can recapitulate a signaling network that normally designates COs , and hence suggest how a liquid-like SC compartment may promote and regulate CO recombination along meiotic chromosomes . 10 . 7554/eLife . 21455 . 026Figure 6 . The localization of CO factors to polycomplexes recapitulates their dynamic behavior and interdependencies upon CO designation .",
"( A–B )",
"Dynamic relocalization of ZHP-3 and COSA-1 during meiotic progression .",
"Fluorescence micrograph of a representative gonad from htp-3 ( tm3655 ) hermaphrodite expressing GFP-COSA-1 stained with antibodies against SYP-1 ( SC/polycomplexes; magenta ) , ZHP-3 ( orange ) , and GFP ( COSA-1; green ) , and counterstained with DAPI ( blue ) .",
"ZHP-3 is detected throughout the volume of polycomplexes in the mid-pachytene region of the gonad , while GFP-COSA-1 is not detected .",
"In nuclei at the ‘loop’ region of the gonad , where nuclei normally exit pachytene ( see Figure 2B for reference ) , ZHP-3 staining becomes confined to small foci abutting polycomplexes , where it colocalizes with GFP-COSA-1 .",
"An intermediate stage in which COSA-1 and ZHP-3 colocalize at the surface but ZHP-3 also remains throughout the polycomplex is also seen in some nuclei .",
"Scale bar = 10 μm .",
"( B ) A higher magnification image of a similarly stained gonad , except SYP-1 is shown in blue and ZHP-3 is shown in magenta ( DAPI is not shown ) .",
"Early , late and intermediate stage polycomplexes are marked with green , red and yellow arrowheads , respectively .",
"( C ) A model of ZHP-3 and COSA-1 dynamic localization to SCs and polycomplexes .",
"See main text for details .",
"( D ) Early ( left ) and late ( right ) stage pachytene nuclei from adult htp-3 or htp-3 zhp-3 hermaphrodites expressing GFP-COSA-1 .",
"Gonads were dissected and stained with antibodies against SYP-2 ( SC/polycomplexes; red ) and GFP ( COSA-1; green ) and counterstained with DAPI ( blue ) .",
"GFP-COSA-1 does not form foci associated with polycomplexes in the absence of ZHP-3 .",
"Scale bars = 2 μm .",
"( E ) Early ( left ) and late ( right ) stage pachytene nuclei from adult htp-3 or htp-3; cosa-1 hermaphrodites .",
"Gonads were dissected and stained with antibodies against SYP-1 ( SC/polycomplexes; red ) and ZHP-3 ( green ) , and counterstained with DAPI ( blue ) .",
"In the absence of COSA-1 , ZHP-3 remains localized throughout polycomplexes even at late stage of meiotic prophase .",
"Scale bars = 2 μm .",
"The entire gonads for ( D ) and ( E ) are shown in Figure 6—figure supplement 1C–D . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 02610 . 7554/eLife . 21455 . 027Figure 6—figure supplement 1 . Further characterization of the localization and 1 , 6-hexanediol sensitivity of ZHP-3 and COSA-1 . ( A ) Projection images showing meiotic prophase progression in whole gonads from adult hermaphrodites stained with antibodies against SYP-1 ( SC; red ) and the RING finger protein ZHP-3 ( green ) .",
"The images in the lower panels ( pink borders ) are from a gonad that was exposed to 7 . 5% hexanediol just prior to fixation .",
"As previously described ( Bhalla et al . , 2008 ) , ZHP-3 localizes throughout SCs in early pachytene , and then concentrates at designated CO sites .",
"When the SC is dissolved by hexanediol , the diffusely localized ZHP-3 is also solubilized , but after localization to COs the protein becomes resistant to solubilization by hexanediol .",
"The higher-magnification views on the right show individual nuclei from before and after the relocalization of ZHP-3 upon CO designation ( top and bottom , respectively ) .",
"Scale bars correspond to 10 μm ( left ) and 2 µm ( right ) .",
"( B ) COSA-1 localizes to CO sites and is not dissolved by hexanediol .",
"These projection images are from an adult hermaphrodite expressing GFP-COSA-1 and mRuby-SYP-3 .",
"Extruded gonads were exposed to 7 . 5% 1 , 6-hexanediol prior to fixation , and counterstained with DAPI ( blue ) .",
"GFP-COSA-1 ( green ) and mRuby-SYP-3 ( red ) were visualized by their intrinsic fluorescence .",
"Although SC proteins were dispersed by hexanediol , the 6 CO sites are still marked by GFP-COSA-1 .",
"Bottom , magnified view of a single nucleus .",
"Scale bars are 8 μm and 2 μm , for the top and bottom panels , respectively .",
"( C–D )",
"COSA-1 and ZHP-3 are interdependent for their dynamic relocalization during meiotic progression .",
"The outlined regions are shown at higher magnification in Figure 6D–E .",
"( C ) Adult htp-3 or htp-3 zhp-3 hermaphrodites expressing GFP-COSA-1 were dissected and stained with antibodies against SYP-2 ( SC/polycomplexes; red ) and GFP ( COSA-1; green ) and counterstained with DAPI ( blue ) .",
"GFP-COSA-1 does not form foci associated with polycomplexes in the absence of ZHP-3 .",
"Scale bars = 10 μm .",
"( D ) Adult htp-3 or htp-3; cosa-1 hermaphrodites were dissected and stained with antibodies against SYP-1 ( SC/polycomplexes; red ) and ZHP-3 ( green ) , and counterstained with DAPI ( blue ) .",
"In the absence of COSA-1 , ZHP-3 remains localized throughout polycomplexes throughout meiotic prophase .",
"Scale bars = 10 μm .",
"( E ) Dynamic relocalization of ZHP-3 and COSA-1 is accompanied by different hexanediol sensitivity .",
"Gonads from htp-3 hermaphrodites expressing GFP-COSA-1 were dissected and stained with antibodies against SYP-1 ( SC/polycomplexes; magenta ) , ZHP-3 ( orange ) , and GFP ( COSA-1; green ) , and counterstained with DAPI ( blue ) .",
"Top , higher-magnification view of polycomplexes showing examples of early and late localization patterns .",
"Bottom , upon dissolution of polycomplexes with 1 , 6-hexanediol , small foci positive for ZHP-3 and GFP-COSA-1 remain visible .",
"The solubilized SC proteins are largely excluded from the volume occupied by chromosomes , resulting in a somewhat filamentous appearance .",
"Scale bars = 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 027"
],
[
"Sixty years of observation by EM has revealed that SC structure , as well as the tendency of its components to assemble into ordered polycomplexes , are widely conserved ( Roth , 1966; Westergaard and von Wettstein , 1972; Page and Hawley , 2004 ) .",
"Here we report another conserved property of SCs and polycomplexes: they assemble through coacervation of polypeptides into compartments with liquid crystalline properties .",
"While the symmetric , transversely striated appearance of the SC , combined with known properties of coiled-coil proteins , has suggested a filamentous and stable internal structure , our observations indicate that SCs instead resemble smectic ( planar ) liquid crystal bilayers , structurally analogous to lipid bilayers .",
"Moreover , the SC can also assemble into its canonical smectic structure when its normal association with chromosome axes is prevented .",
"The preferential assembly of SC proteins into a thin lamellar structure between two axes is further evidenced by our observation that upon meiotic arrest , nuclear aggregates appear only if no pre-assembled SC bilayers are present ( Figure 2 ) .",
"While biological membranes are well-known examples of smectic liquid crystals , to our knowledge , the SC is the first known example of such a highly ordered , liquid-like structure arising from protein-protein interactions .",
"However , a variety of biological assemblies of less regular structure have been described as liquid crystals ( Brown and Wolken , 1979; Rey , 2010 ) , and many others have been created in vitro by mixing synthetic peptides in aqueous solution .",
"Liquid crystals are considered to be a distinct phase of matter from liquids , which are disordered and anisotropic .",
"Thus , it is perhaps unsurprising that the proteins that assemble to form SCs are markedly different from those that have been implicated in the assembly of other liquid-like compartments within cells .",
"Many such proteins have low-complexity , intrinsically disordered domains that are essential for their phase transition behavior ( Hyman et al . , 2014; Zhu and Brangwynne , 2015 ) , and some of these have been shown to adopt cross-beta strand structures in vitro ( Kato et al . , 2012 ) .",
"By contrast , SC proteins are predicted to be largely alpha-helical in structure , and have extensive domains predicted to form coiled-coils .",
"Smectic liquid crystals assemble from elongated , amphipathic rod-like molecules ( Gennes and Prost , 1993 ) .",
"It is known that some of the longer SC proteins , particularly SYCP1 in mammals and Zip1p in budding yeast , have a head-to-head orientation within the SC , with their N-termini near the center and their C-termini near the chromosome axes ( Liu et al . , 1996; Schmekel et al . , 1996; Dong and Roeder , 2000; Anderson et al . , 2005 ) .",
"Interaction of their C-termini with axis-associated proteins seems to promote their coacervation with other SC proteins , possibly by promoting high local concentrations .",
"Axis association also favors the formation of single bilayers ( lamellae ) , rather than the stacks of bilayers that make up polycomplexes .",
"By specifying the orientation of these ‘transverse filament’ proteins , the chromosome axes may also act as a ‘director’ for the liquid crystalline array .",
"Many of the best-studied coiled-coil proteins form highly stable interactions through regions of superhelical coiling .",
"Some , such as muscle myosin , keratins , and lamins , form long-lived filamentous networks , which has suggested that the SC might have similar properties .",
"However , it has long been appreciated that interruptions in the phasing of the canonical heptad repeat sequence disrupt supercoiling and reduce the stability of interactions ( Brown et al . , 1996 ) .",
"We note that even the ‘transverse filament’ proteins SYCP1 ( vertebrates and other metazoans ) , Zip1p ( budding yeast ) , C ( 3 ) G ( Drosophila ) , and SYP-1 ( Caenorhabditis ) ( Page and Hawley , 2004 ) contain very few perfect heptads arranged in tandem , which may underlie the lability of the SC .",
"Meiotic synapsis can be disrupted by small increases in temperature , as seen in C . elegans ( Bilgir et al . , 2013 ) and many other organisms , and we propose that this stems from the liquid crystalline properties of the SC .",
"While the C . elegans Bristol N2 strain remains fertile at temperatures up to 25°C , at 26 . 5°C hermaphrodites are sterile , and in such animals we observed SC protein assemblies that were neither crystalline nor liquid-like .",
"Because liquid crystals depend on ordered but weak interactions , they are considered to be a ‘mesophase’ between liquids and crystalline solids .",
"They are often exquisitely sensitive to variations in temperature and/or other perturbations such as electric fields , making synthetic materials useful as thermometers ( on fish tanks and mood rings ) and in video displays ( Gennes and Prost , 1993; Carlton et al . , 2013 ) .",
"Thus strong selective forces to maintain the responsive , liquid-like properties of the SC , which plays a central role in fertility and fitness , may underlie the sequence diversity observed among its constituent proteins , especially among ectothermic organisms .",
"In support of this idea , direct evidence has recently indicated that SC proteins , among other chromosome structural components , are subject to rapid positive selection in plant populations growing at different temperatures ( Wright et al . , 2015 ) .",
"Our observation that meiotic arrest or azide treatment promotes the coalescence of SC components is consistent with the idea that phosphorylation and/or other modifications regulate the coacervation of these proteins .",
"These modifications presumably tune the strength of interactions among SC proteins to control the timing and location of assembly and disassembly , as has been described for other cellular coacervation processes , such as the formation of P-granules ( Wang et al . , 2014a ) .",
"Consistent with this , PLK-2 , a kinase that promotes SC disassembly at the end of meiotic prophase , localizes to SCs ( Harper et al . , 2011 ) , and we have found that several of the SYP proteins can be phosphorylated by PLK-2 in vitro ( Y . Kim and AFD , unpublished ) .",
"Because liquid crystals are ultrasensitive to perturbations of their intermolecular interactions ( Gennes and Prost , 1993; Carlton et al . , 2013 ) , we envision that posttranslational modifications may also modulate the dynamic properties of assembled SCs during meiotic progression .",
"Phase-separated liquid-like compartments in vitro and in vivo are selectively and tunably permeable to macromolecules .",
"This property can concentrate specific cellular activities while excluding others ( Hyman et al . , 2014 ) .",
"While the liquid crystalline properties of the SC set it apart from other compartments that resemble amorphous liquids , we provide evidence that it may have some analogous properties .",
"Specifically , this assembly dynamically regulates the localization of at least two conserved recombination factors , ZHP-3 and COSA-1 ( Figure 6 and Figure 6—figure supplement 1 ) , which are not required for SC assembly , in response to meiotic progression .",
"Previous work has shown that Zip3p , the budding yeast homolog of ZHP-3 , also localizes throughout polycomplexes , while the CO factors Zip2p , Zip4p , Msh5p and the 9-1-1 clamp proteins localize to one or two foci abutting polycomplexes , sometimes referred to as a ‘capping complex’ ( Tsubouchi et al . , 2006; Shinohara et al . , 2015 ) , very similar to the physical distribution we observe for ZHP-3 and COSA-1 during late meiotic prophase .",
"Homologs of ZHP-3 in plants , mammals and fungi also localize to SCs before concentrating at sites of COs ( Chelysheva et al . , 2012; Reynolds et al . , 2013; De Muyt et al . , 2014; Jahns et al . , 2014; Qiao et al . , 2014 ) , suggesting that spatial regulation of COs by the SC is widely conserved .",
"We envision that the SC acts as an active ‘scaffold’ to localize and concentrate ‘client’ proteins ( Banani et al . , 2016 ) , defined here as SC-resident proteins that are not required for SC assembly .",
"These factors , including ZHP-3 , are recruited by the SC to control the processing of double-strand breaks that are induced during meiosis .",
"We also speculate that the SC may share an evolutionary relationship with other liquid-like nuclear compartments , such as repair foci , which concentrate repair factors at sites of DNA damage ( Zhu and Brangwynne , 2015 ) .",
"Our observations support a central role for the SC in CO interference , which acts over many microns and even over entire chromosomes ( Page and Hawley , 2004; Libuda et al . , 2013 ) .",
"Evidence that SCs share physical properties with liquid crystals , a particularly responsive class of active materials , illuminates how they might contribute to the transduction of a long-range , cis-acting interference signal ( Figure 6C ) .",
"In particular , the knowledge that macromolecules can diffuse laterally within the plane of smectic liquid crystals suggests that the dynamics of diffusion within the SC likely determine the rate at which biochemical signals are propagated along the chromosomes .",
"( By analogy , 2D diffusion within lipid membranes profoundly impacts the spatial organization and kinetics of signaling by membrane-associated proteins . )",
"Thus , our discovery that the SC acts as a non-membrane-bound compartment suggests how COs could be regulated in cis through a reaction-diffusion mechanism , which is consistent with quantitative analysis of CO interference in several organisms ( Fujitani et al . , 2002 ) .",
"This idea is also consistent with evidence that reduction in the expression levels of SC proteins , which likely results in discontinuities of the SC along individual chromosomes , can permit additional COs ( Hayashi et al . , 2010; Libuda et al . , 2013 ) .",
"We further report that both SCs and polycomplexes undergo an abrupt biochemical change in response to cell cycle signals , which results in the appearance of a single CO-like focus and removal of ZHP-3 from the interior , indicating that this material can mediate switch-like regulation that spans an entire compartment .",
"We have found that depletion of the ERK kinase MPK-1 , which has been implicated in regulating late meiotic prophase events in C . elegans ( Nadarajan et al . , 2016 ) , does not affect the relocalization of COSA-1 or ZHP-3 on SCs or polycomplexes ( Liangyu Zhang , OR and AFD , unpublished ) , so the nature of the ‘exit pachytene’ signal that triggers this switch is unknown .",
"Finally , we note that long-range order within liquid crystals allows propagation of structural transitions , known as ‘disclinations , ’ over long distances ( Rey , 2010; Bukusoglu et al . , 2016 ) , similar to a ‘domino effect . ’ Energy can also be stored within liquid crystals as elastic strain , and released through abrupt topological changes ( Bukusoglu et al . , 2016 ) .",
"This property could provide a rapid response to crossover formation .",
"Specifically , we imagine that structural changes could be induced within the SC at sites of CO formation and transmitted laterally , in which case interference might be mediated by a mechanochemical rather than a purely biochemical signal .",
"This idea is analogous to a ‘beam-film’ model that has been proposed to explain interference ( Zhang et al . , 2014 ) , but posits that the medium that stores strain is the SC , or more precisely , the SC as confined by its interaction with chromosome axes .",
"Ultrastructural analysis of SC organization before and after CO formation may eventually allow this idea to be tested .",
"Additionally , future studies of the role of SC organization and dynamics in CO interference may enable development of new synthetic materials with long-range signaling capabilities ."
],
[
"All strains were cultured using standard methods ( Brenner , 1974 ) .",
"Worms were maintained at 20°C , except that animals expressing mMaple-HIM-3 and mMaple-SYP-3 were cultured at 25°C to enhance transgene expression .",
"Where indicated , worm plates were incubated overnight at 26 . 5°C to induce SC aggregation ( Bilgir et al . , 2013 ) .",
"See Table 1 for a full list of the strains used . 10 . 7554/eLife . 21455 . 028Table 1 . Strains used in this study . DOI: http://dx . doi . org/10 . 7554/eLife . 21455 . 028Strain NameGenotypeCA257him-8 ( tm611 ) IVCA277unc-24 ( e138 ) him-8 ( e1489 ) spo-11 ( ok79 ) / mIs11 IVCA795htp-1 ( gk174 ) / nT1 [unc- ? ( n754 ) let- ? ] ( IV;V ) CA821htp-3 ( tm3655 ) I / hT2 [bli-4 ( e937 ) let- ? ( q782 ) qIs48] ( I;III ) CA826cra-1 ( tm2144 ) III / hT2 [bli-4 ( e937 ) let- ? ( q782 ) qIs48] ( I;III ) CA861syp-3 ( ok758 ) I / hT2 [bli-4 ( e937 ) let- ? ( q782 ) qIs48] ( I;III ) CA899syp-3 ( me42 ) I / hT2 [bli-4 ( e937 ) let- ? ( q782 ) qIs48] ( I;III ) CA904rec-8 ( ok978 ) / nT1 IV; coh-4 ( tm1857 ) coh-3 ( gk112 ) / nT1 [qIs51] VCA1010meIs8 [pie-1p::GFP::cosa-1 + unc-119 ( + ) ] IICA1095rec-8 ( ok978 ) IV / nT1[qIs51] ( IV;V ) CA1122cosa-1 ( me13 ) / qC1 [dpy-19 ( e1259 ) glp-1 ( q339 ) qIs26] IIICA1234syp-3 ( ok758 ) I; ieSi63 [cbunc-119+ , psyp-3::mMaple3::syp-3] II; unc-119 ( ed3 ) III . CA1237htp-3 ( tm3655 ) I; cosa-1 ( ie98 ) IIICA1238htp-3 ( tm3655 ) I/hT2 [bli-4 ( e937 ) let- ? ( q782 ) qIs48] ( I , III ) ; meIs8 [pie-1p::GFP::cosa-1 + unc-119 ( + ) ] IICA1239htp-3 ( tm3655 ) zhp-3 ( ie97 ) I; meIs8 [pie-1p::GFP::cosa-1 + unc-119 ( + ) ] IICA1253syp-3 ( ok758 ) I; ieSi11 [cbunc-119+ , Psyp-3::EmeraldGFP::syp-3] II; unc-119 ( ed3 ) IIICA1255htp-3 ( tm3655 ) syp-3 ( ok758 ) I; ieSi11 [cbunc-119+ , Psyp-3::EmeraldGFP::syp-3] II; ieSi17 [cbunc-119 , Phtp-3::htp-3::mRuby] IVCA1255htp-3 ( tm3655 ) syp-3 ( ok758 ) I; ieSi11 [cbunc-119+ , Psyp-3::EmeraldGFP::syp-3] II; ieSi17 [cbunc-119 , Phtp-3::htp-3::mRuby] IVCA1297syp-3 ( ok857 ) I; ieSi11 [cbunc-119+ , Psyp-3::EmeraldGFP::syp-3] II; him-8 ( tm611 ) IVCA1298meIs9 [gfp::syp-3]; unc-119CA1299hal-2 ( me79 ) / qC1 [dpy-19 ( e1259 ) glp-1 ( q339 ) qIs26] III; meIs9 ( gfp::syp-3 ) ; unc-119CA1300syp-3 ( ok857 ) htp-3 ( tm2655 ) I; ieSi11 [cbunc-119+ , Psyp-3::EmeraldGFP::syp-3] IICA1303syp-3 ( ok857 ) I; ieSi11 [cbunc-119+ , Psyp-3::EmeraldGFP::syp-3] II; htp-1 ( gk174 ) /nT1 [unc- ? ( n754 ) let- ? ] ( IV;V ) CA1309unc-119 ( ed3 ) III; ieSi21[cbunc-119+ , Psun-1::sun-1::mRuby] IVCA1350him-3 ( ie34[mMaple3::him-3] ) IV HTP-3-mRuby ( ieSi17 ) and mRuby-SYP-3 ( ieSi19 ) were constructed and inserted using MosSCI , essentially as previously described for GFP-SYP-3 and HTP-3-GFP ( Kim et al . , 2014; Rog and Dernburg , 2015 ) .",
"A transgene including a 3x ( Gly-Gly-Ser-Gly ) linker and mRuby ( Kredel et al . , 2009 ) immediately downstream of the coding sequnce of htp-3 was constructed using the MosSCI repair template pCFJ178 .",
"This construct was integrated at the cxTi10882 IV site by MosSCI ( Frøkjaer-Jensen et al . , 2008 , 2012 ) , and later homozygosed .",
"mMaple3-SYP-3 was constructed using a codon-optimized synthetic DNA sequence , with modifications to improve germline expression ( Wang et al . , 2014b; Frøkjær-Jensen et al . , 2016 ) , and inserted by MosSCI .",
"All SYP-3 and HTP-3 transgenes were crossed into mutants lacking the corresponding wild-type proteins ( syp-3 ( ok758 ) or htp-3 ( tm3655 ) , respectively ) .",
"mMaple3-HIM-3 was constructed by inserting a codon-optimized mMaple3 sequence at the N-terminus of the endogenous him-3 gene using CRISPR-Cas9 ( Dickinson et al . , 2013 ) .",
"Analysis of htp-3 mutant animals was performed on homozygous progeny of balanced heterozygotes , and their heterozygous siblings served as wild-type controls .",
"A cosa-1 ( ie98 ) htp-3 ( tm3655 ) strain was constructed by inserting a premature stop codon to disrupt the cosa-1 gene using CRISPR-Cas9 in htp-3 ( tm3655 ) /hT2 animals .",
"A zhp-3 ( ie97 ) htp-3 ( tm3655 ) ; COSA-1-GFP strain was constructed by inserting a premature stop codon disrupting the zhp-3 gene using CRISPR-Cas9 in COSA-1-GFP; htp-3 ( tm3655 ) /hT2 animals .",
"Adult hermaphrodites were immobilized for imaging as previously described ( Rog and Dernburg , 2015 ) .",
"Briefly , worms were placed on freshly prepared agarose pads ( 7 . 5% in water ) overlaid with 100 nm polystyrene beads ( Polysciences , cat#00876 ) ( Kim et al . , 2013 ) .",
"The pad and beads included freshly-dissolved serotonin creatinine sulfate ( Sigma-Aldrich , St . Louis , MO ) at a final concentration of 25 mM .",
"Worms were overlaid with high-performance coverslips ( 0 . 17 ± 0 . 005 mm; Schott ) that were sealed to the slide with VALAP ( 1:1:1 vaseline:lanolin:paraffin ) , and imaged immediately afterwards .",
"Time-lapse images were recorded from the posterior gonad arm , the motion of which was less perturbed by pharyngeal pumping than the anterior arm .",
"Only gonads for which chromosome motion was detected during the entire recording were analyzed , except where otherwise indicated .",
"Hexanediol treatment during time-lapse imaging was performed by dissecting adult hermaphrodites in 30 μL 1X egg buffer ( EB ) on poly-L-lysine covered coverslips ( Neuvitro , Vancouver , WA ) .",
"Extruded gonads were gently pressed against the coverslip to promote adhesion .",
"At the indicated times , an equal volume of hexanediol in EB was added to the drop to attain the indicated final concentrations of hexanediol .",
"Where indicated , the buffer was subsequently diluted with additional EB lacking hexanediol .",
"Images were acquired using a Marianas spinning-disc confocal microscope ( Intelligent Imaging Innovations , Inc . [3i] , Denver , CO ) at ambient temperature ( 19°C–24°C ) , using a 100 × 1 . 46 NA oil immersion objective , yielding a pixel spacing of 133 × 133 nm .",
"3D image stacks of 11–17 sections at 0 . 5 µm z-spacing were acquired over 1–3 s .",
"Raw image stacks were analyzed using Imaris 7 . 3 or 8 . 0 ( Bitplane AG , Zurich , Switzerland ) .",
"Time-lapse series were segmented , tracked and aligned based on overall fluorescence using the Spots tool .",
"Compactness was measured using the Surface tool , analyzing at least three different gonads for each condition .",
"Photoconversion of mMaple3 fusion proteins was performed using the Marianas spinning-disc confocal system described above , equipped with a Vector FRAP module .",
"Two-color 3D image stacks of 11 sections at 0 . 8 µm z-spacing were acquired over 1–3 s .",
"The first time point included activation of 10 × 10 pixel regions ( 1 . 33 × 1 . 33 µm ) using a 405 nm laser at each focal position .",
"Following photoconversion , 3D stacks were acquired every 1–5 min , as indicated .",
"Our recordings indicate that photoconverted molecules can spread both along SCs and between SCs on different chromosomes .",
"We thus assume that this movement has multiple rate components .",
"At the temporal and spatial resolution we could achieve in living animals with spinning disk confocal microscopy , it was not possible to directly measure diffusion rates or the surface tension of individual SC compartments .",
"Instead , we compared the spreading rates of SYP-3 ( SC ) and HIM-3 ( axis ) proteins by calculating the rate of change of the volume of the nucleus containing photoconverted ( red ) signal , based on 3D images from time-lapse recordings .",
"The volume of the nucleus occupied by photoconverted molecules at each time point was placed in one of four bins ( 25% , 50% , 75% or 100% ) , which were then plotted as a function of time elapsed since photoconversion .",
"At least 15 nuclei were scored for each fluorescent marker .",
"The expansion rate was defined as the slope of a line fitted to the data .",
"These values were compared to simple simulations in which a point source of fluorescence became homogeneously distributed throughout the nucleus in 1 hr .",
"Immunofluorescence was performed as previously described ( Phillips et al . , 2009 ) , except that where indicated , dissected hermaphrodites were briefly incubated with 1 , 6-hexanediol in egg buffer immediately before adding fixative .",
"The following antibodies , all of which have been previously described , were used: anti-SYP-2 ( rabbit , affinity purified , 1:1 , 000 ) , anti-SYP-1 ( goat , affinity purified , 1:1 , 000 ) , anti-GFP ( mouse monoclonal , Roche , Indianapolis , IN , 1:400 ) , anti-SUN-1 ( rabbit , affinity-purified , Novus Biologicals , Littleton , CO , 1:10 , 000 ) , anti-HTP-3 ( guinea pig , 1:500 ) , anti-HIM-8 ( rat , 1:500 ) , anti-ZHP-3 ( rabbit , affinity-purified , Novus Biologicals , 1:10 , 000 ) and secondary antibodies conjugated to Alexa 488 , Cy3 , or Cy5 ( Jackson ImmunoResearch , West Grove , PA or Molecular Probes , Thermo-Fisher , Waltham , MA; 1:300 ) .",
"Imaging was performed with a Marianas spinning-disc confocal microscope system ( Intelligent Imaging Innovations , Inc . [3i] ) using a 100 × 1 . 46 NA oil objective , or with a DeltaVision Elite system ( GE Healthcare , Pittsburgh , PA ) equipped with an Olympus 100 × 1 . 4 NA oil-immersion objective .",
"Wide-field images were deconvolved using the SoftWoRx Suite ( GE Healthcare ) .",
"3D stacks were visualized in Imaris 7 . 3 or 8 . 0 ( Bitplane ) .",
"To evaluate the sensitivity of the SC to hexanediol in various mutants ( Figure 4—figure supplement 2A ) , at least 3 slides with >10 gonads per slide were scored .",
"The data are presented as the average value for a set of slides .",
"Gonads were scored as ‘dissolved’ if no filaments were visible , other than short SC stretches on the X chromosome and/or within apoptotic nuclei .",
"The following yeast strains were used: Brün292: MATa/alpha; ndt80::LEU2:ndt80::LEU2 flo8::KanMX6/flo8::KanMX6 ZIP1::GFP ( 700 ) / ZIP1::GFP ( 700 ) ( SK1 strain background ) Brün793: MATa/alpha; GAL-NDT80::TRP1/GAL-NDT80::TRP1 ura3::pGPD1-GAL4 ( 848 ) .",
"ER::URA3/ura3::pGPD1-GAL4 ( 848 ) .",
"ER::URA3 leu2::URA3p-tetR-tdTomato::LEU2 CENV::tetOx224::HIS3 REC8-GFP-URA3/REC8-GFP-URA3 ( SK1 strain background ) Sporulation and pachytene arrest of ndt80 strains was was carried out as described ( Carlile and Amon , 2008 ) .",
"Where indicated , 1 , 6-hexanediol diluted in sporulation medium was added to cells immediately prior to imaging .",
"Where indicated , cells were treated with various concentration of 1 , 6-hexanediol and KCl by mixing a 2x concentrated solution with cells washed 3 times with 20 mM phosphate buffer ( pH 7 ) .",
"‘Washing’ of hexanediol was performed by mixing the treated cells with 10x excess of 20 mM phosphate buffer ( pH 7 ) .",
"Unfixed cells were imaged using a Marianas spinning-disc confocal microscope , as described above .",
"To analyze the effects of hexanediol on Drosophila melanogaster SCs , ovaries from young , mated c ( 3 ) G-GFP; c ( 3 ) G/+ females fed on yeast paste were dissected in PBS ( Page and Hawley , 2001 ) , separated into ovarioles , and transferred to a drop of PBS in a glass-bottomed petri dish .",
"3D wide-field images were acquired using a DeltaVision microscope equipped with a 100 × 1 . 4 NA objective .",
"An equal volume of 2x 1 , 6 hexanediol in PBS was added during imaging .",
"For immunofluorescence , ovarioles from c ( 3 ) G-GFP; c ( 3 ) G/+ or from GAL4-nos/+; UASp-c ( 2 ) M-3xHA/+ females were dissected in Modified Robb’s Saline ( MRS ) , incubated briefly with hexanediol in MRS ( or just MRS ) and fixed with 4% formaldehyde in MRS , following by immunostaining as described ( McKim et al . , 2009 ) .",
"The following antibodies were used: anti-GFP ( 1:400 , mouse , Roche ) , anti-CID ( 1:500 , chicken ) , anti-C ( 2 ) M ( 1:500 , rabbit ) , anti-HA ( mouse , 1:500 ) , anti-C ( 3 ) G ( Rabbit , 1:3000 ) , and appropriate secondary antibodies conjugated to dyes ( 1:300 ) .",
"Samples were imaged on a Marianas spinning-disc confocal microscope , as described above .",
"C . elegans adult hermaphrodites were high-pressure frozen and freeze-substituted according to previously described methods ( McDonald and Webb , 2011; McDonald , 2014 ) .",
"Briefly , worms were frozen in 50 µm deep specimen carriers in a HPM-010 high pressure freezer ( Bal-Tec , Balzers , Liechstenstein ) , freeze-substituted in 1% osmium tetroxide plus 0 . 1% uranyl acetate in acetone over 2 . 5 hr , and embedded in Epon-Araldite resin over a period of 2 . 5 hr .",
"Serial sections ( 70 nm thick ) were picked up on slot grids , post-stained with uranyl acetate and lead citrate , and imaged in Tecnai 12 transmission electron microscope operating at 120kV ( FEI , Hillsboro , OR ) , using an Ultrascan 1000 charge coupled device camera ( Gatan , Pleasanton , CA ) .",
"All movies show maximum-intensity projection images from 3D time-lapse recordings of C . elegans gonads acquired with a spinning disk confocal microscope .",
"In most cases the worms were intact and alive during recording; in experiments involving 1 , 6-hexanediol treatment , gonads were extruded by nicking the cuticle with a scalpel blade shortly before imaging began .",
"Elapsed times are indicated at the bottom right corner of each frame as hours:minutes:seconds: ( milliseconds ) ."
]
] | [
"The synaptonemal complex ( SC ) is a polymer that spans ~100 nm between paired homologous chromosomes during meiosis .",
"Its striated , periodic appearance in electron micrographs led to the idea that transverse filaments within this structure ‘crosslink’ the axes of homologous chromosomes , stabilizing their pairing .",
"SC proteins can also form polycomplexes , three-dimensional lattices that recapitulate the periodic structure of SCs but do not associate with chromosomes .",
"Here we provide evidence that SCs and polycomplexes contain mobile subunits and that their assembly is promoted by weak hydrophobic interactions , indicative of a liquid crystalline phase .",
"We further show that in the absence of recombination intermediates , polycomplexes recapitulate the dynamic localization of pro-crossover factors during meiotic progression , revealing how the SC might act as a conduit to regulate chromosome-wide crossover distribution .",
"Properties unique to liquid crystals likely enable long-range signal transduction along meiotic chromosomes and underlie the rapid evolution of SC proteins ."
] | [
"The genetic information in cells is encoded within long molecules of DNA called chromosomes .",
"In most human cells , the two copies of each chromosome – the one inherited from our mother and the one from our father – are physically separated and behave independently .",
"However , in the reproductive cells that give rise to eggs or sperm , each chromosome must pair with its partner .",
"Pairing first occurs at one or more positions along each chromosome .",
"This triggers a protein-based polymer called the “synaptonemal complex” to assemble between the paired chromosomes , and then spread along the interface between the partners until they are fully lined up side-by-side .",
"Chromosomes in reproductive cells must pair in this particular way to exchange genetic information and generate new combinations of traits .",
"The synaptonemal complex was first observed over 60 years ago , but it remains enigmatic .",
"Though its structure is highly ordered and looks very similar in different organisms from yeast to humans , little is known about how this polymer forms or what it does between chromosomes .",
"Some evidence has suggested that the synaptonemal complex helps to regulate how much information can be transferred between each pair of chromosomes , but not all studies have supported this conclusion .",
"Several lines of evidence suggest that the synaptonemal complex might be fundamentally different from other protein-based polymers , such as those that form filamentous skeletal structures within cells , namely actin filaments and microtubules .",
"Now , Rog et al . have tested the idea that the synaptonemal complex might actually have liquid-like properties , despite its highly ordered appearance .",
"The experiments showed that the proteins that make up the synaptonemal complex in yeast , worms and fruit flies are weakly bound to each other and can move around within the assembled structure .",
"These are considered to be defining properties that distinguish liquids from solid materials .",
"Together with its regular , repetitive organization , these findings indicate that the synaptonemal complex behaves like a liquid crystal .",
"This intriguing class of materials has properties between those of conventional liquids and those of solid crystals , and is particularly sensitive to environmental conditions .",
"Rog et al . believe that this discovery helps to explain how signals are transmitted along the length of chromosomes to regulate the transfer of genetic information .",
"In support of this idea , further experiments showed that proteins that are required for this recombination process were also found within the synaptonemal complex .",
"As reproductive cells transition from one stage of their development to the next , these proteins abruptly move to a new location , indicating that a switch-like signal rapidly spreads throughout the synaptonemal complex .",
"Together the findings suggest that the liquid crystal-like properties of the synaptonemal complex allow signals to be transmitted along the interface between pairs of chromosomes .",
"The next challenges are to understand what triggers these signals and to explore whether they are based upon physical or chemical changes within the synaptonemal complex .",
"Further research is also needed to uncover how this information is propagated along the length of a chromosome ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | Neuronal sources of hedgehog modulate neurogenesis in the adult planarian brain | elife-19735-v2 | [
[
"Once thought to be a non-existent phenomenon , homeostatic adult neurogenesis is a common trait shared by many disparate organisms including rodents , birds , flies , and humans ( Altman , 1962 , 1969; Doetsch et al . , 1999; Goldman and Nottebohm , 1983; Eriksson et al . , 1998; Fernández-Hernández et al . , 2013 ) .",
"However , levels of neuronal turnover are tightly limited in these animals ( Obernier et al . , 2014 ) .",
"In fact , the highest known site of adult homeostatic neurogenesis in the human central nervous system ( CNS ) is the hippocampus , where annual neuronal turnover rates are estimated to be only 1 . 75% ( Spalding et al . , 2013; Sanai et al . , 2011; Bergmann et al . , 2012 ) .",
"Adult neurogenesis in most animals depends on the action of ectodermally derived neural stem cells , which have radial-glial character and are integrated into a stable niche microenvironment .",
"Extrinsic signals such as wingless ( Wnt ) , sonic hedgehog ( Shh ) , and bone morphogenetic proteins ( BMPs ) act to finely control neural stem cell proliferation and differentiation ( Silva-Vargas et al . , 2013; Lehtinen et al . , 2011 ) .",
"The asexual strain of the freshwater planarian , Schmidtea mediterranea ( S . mediterranea ) , is a constitutive adult flatworm ( Lophotrochozoan ) that challenges the dogma of low rates of adult neurogenesis .",
"Not only does S . mediterranea constantly turnover its brain , but also it is capable of complete brain regeneration within only two weeks following decapitation ( Cebria , 2007; Reddien and Sánchez Alvarado , 2004; Newmark and Sánchez Alvarado , 2002 ) .",
"In addition , the uninjured planarian CNS is known to be a highly dynamic organ , which can adjust its size through the addition or subtraction of mature neurons to maintain consistent proportions with the rest of the body as it grows and shrinks , respectively ( Baguñá and Romero , 1981 ; Hill and Petersen , 2015 ) .",
"Amazingly , these regenerative feats and high levels of homeostatic neurogenesis are accomplished in the absence of a recognizable neuroepithelium , and without any definitive neural stem cells ( van Wolfswinkel et al . , 2014; Zhu et al . , 2015 ) .",
"Recently , brain-derived Wnt signals have been shown to influence the neurogenic output of planarian stem cells ( neoblasts ) during regeneration ( Hill and Petersen , 2015 ) .",
"However , little is known about the specific extracellular signals and transcription factors that modulate neoblast activity within this body region to balance cell proliferation and neuronal differentiation , which undoubtedly involves many overlapping regulatory systems .",
"Here , we have identified two planarian homeodomain transcription factors , Smed-nkx2 . 1 and Smed-arx ( henceforth referred to as nkx2 . 1 and arx ) , which act to specify cholinergic , GABAergic , and octopaminergic neurons within the ventral-medial ( VM ) region of the planarian CNS .",
"Interestingly , these very same VM neural cell types are a major source of the planarian hedgehog ligand ( Smed-hh; henceforth referred to as hh ) ( Rink et al . , 2009; Yazawa et al . , 2009 ) .",
"We also describe a novel neoblast population , which are nkx2 . 1+ or arx+ , express the core machinery for the reception of hh and Wnt signals , and are located adjacent to hh+ VM neurons .",
"Finally , we present evidence that consistent hh signaling within this neoblast microenvironment is required to promote normal homeostatic neurogenesis of the VM neuronal population .",
"In total , we identify a hh signaling axis that positively modulates VM neurogenesis through distinct progenitor cells ."
],
[
"nkx2 . 1 and arx were originally cloned and isolated during an RNAi screen aimed at identifying planarian transcription factors with potential roles in neuronal specification .",
"A unique behavioral defect was observed in all nkx2 . 1 ( RNAi ) animals , characterized by tonic muscular contractions that bend the head dorsally , so that it is perpendicular with the rest of the body .",
"Interestingly , some of these nkx2 . 1 ( RNAi ) worms end up on their lateral body edge , causing these animals to move in tight circles , compared to control ( RNAi ) animals which move in straight lines on their ventral surface ( Videos 1 and 2 ) .",
"In contrast , arx ( RNAi ) animals do not display overt behavioral defects , except when flipped onto their dorsal side where these worms have difficulty to corkscrew in order to reorient onto their ventral surface compared to control ( RNAi ) and wild-type animals ( Videos 1 and 3 ) . 10 . 7554/eLife . 19735 . 003Video 1 . Control ( RNAi ) worms exhibiting normal locomotion and ability to flip over from dorsal surface back onto ventral surface . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 00310 . 7554/eLife . 19735 . 004Video 2 . nkx2 . 1 ( RNAi ) worms exhibiting abnormal behaviors , including tonic muscular contractions . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 00410 . 7554/eLife . 19735 . 005Video 3 . arx ( RNAi ) worms exhibiting abnormal muscular contractions and inability to flip back onto dorsal surface . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 005 In order to investigate the root cause of these behavioral defects , and whether nkx2 . 1 and arx might function in neuronal specification and maintenance , whole-mount in situ hybridizations ( WISH ) and cross-sections were first performed on wild-type animals .",
"nkx2 . 1 exhibited broad expression throughout the body but was clearly present within the CNS ( black arrow ) , whereas arx expression was spatially restricted to the medial CNS , including the anterior brain and the full length of the ventral nerve cords ( Figure 1A ) .",
"Transverse sections through the anterior-posterior ( A-P ) midpoint of the brain enabled imaging in greater spatial detail and highlighted the concentrated expression of arx within the ventral-medial region of each brain lobe ( Figure 1B ) .",
"In addition , expression of nkx2 . 1 and arx was observed within a few isolated cells in between the brain lobes , a region largely populated by adult stem cells ( Figure 1B , yellow arrows ) . 10 . 7554/eLife . 19735 . 006Figure 1 . nkx2 . 1 and arx are expressed by VM neural cell types .",
"( A ) WISH images of nkx2 . 1 and arx expression .",
"Black arrow highlights CNS expression .",
"( B ) Single confocal plane from transverse sections of FISH images depicting expression of nkx2 . 1 and arx within the CNS .",
"Yellow arrows highlight nkx2 . 1 and arx expression within stem cell-rich area .",
"( C–E )",
"For each set of images , the upper left panels are projections of confocal images from FISH experiments showing all cholinergic , GABAergic , or octopaminergic neurons in wild-type animals .",
"Solid red boxes represent region of interest for the right panels , which are single confocal planes from dFISH experiments , showing co-expression of nkx2 . 1 or arx within a given neuron subtype .",
"Solid yellow lines in ( C ) demarcate the division line between the VM and DL brain regions .",
"White arrows in dFISH panels indicate double-positive cells .",
"Solid white lines represent the border of the animal .",
"Dashed white lines represent the approximate border of the CNS .",
"Red dashed lines indicate approximate plane for transverse sections .",
"Scale bar = 100 µm .",
"CNS , Central nervous system; DL , Dorsal-lateral; FISH , Fluorescent in situ hybridization; VM , Ventral-medial . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 00610 . 7554/eLife . 19735 . 007Figure 1—figure supplement 1 . nkx2 . 1 and arx are not expressed by DL GABAergic , dopaminergic , or serotonergic neurons .",
"( A ) Projected confocal images showing the full populations of GABAergic , dopaminergic , or serotonergic neurons .",
"Dashed lines represent outer border of the CNS .",
"Solid red box indicates region of interest for panels to the right .",
"Scale bars = 100 µm .",
"( B ) Single confocal planes from dFISH images showing limited expression of nkx2 . 1 and arx , within DL GABAergic , dopaminergic , and serotonergic neurons ( white arrows ) .",
"Scale bars = 20 µm .",
"( C ) Table summarizing the percentage of neurons from each sub-population that express nkx2 . 1 and arx .",
"Values are percentage of neurons ± standard deviation .",
"CNS , Central nervous system; DL , Dorsal-lateral . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 007 In order to determine which neuronal subtypes express nkx2 . 1 and arx , double fluorescent in situ hybridization ( dFISH ) was performed , comparing either of these two transcription factors with established markers of either cholinergic , GABAergic , octopaminergic , dopaminergic , or serotonergic neurons ( Nishimura et al . , 2007a , 2007b , 2008a , 2008b , 2010 ) .",
"Both nkx2 . 1 and arx were found to be expressed by neural cell types that occupy the VM region of the planarian brain ( cholinergic , GABAergic , and octopaminergic neurons ) but were largely excluded from dopaminergic and serotonergic neurons ( Figure 1C–E; Figure 1—figure supplement 1 ) .",
"Cholinergic neurons are by far the most populous cell type in the planarian CNS and are densely distributed throughout the brain ( Figure 1C ) , making it difficult to assess the level of expression of nkx2 . 1 and arx within this entire neuronal population .",
"Therefore , a more focused region of interest was imaged to quantify the degree of expression of nkx2 . 1 and arx within cholinergic neurons ( chat+ ) ( Nishimura et al . , 2010 ) .",
"Transverse sections were first taken at the A-P midpoint of the brain , and individual brain lobes were imaged .",
"Each lobe was then divided into a VM and dorsal-lateral ( DL ) half , with the line of division occurring between the widest point of each brain lobe ( Figure 1C; yellow line ) .",
"A considerable proportion of cholinergic neurons expressed nkx2 . 1 in both the VM ( 36 . 89 ± 5 . 46% ) and DL ( 23 . 33 ± 5 . 13% ) brain regions ( Figure 1C ) .",
"In contrast , arx expression was more heavily biased toward the VM brain region , where 63 . 21 ± 4 . 35% of cholinergic neurons expressed this transcription factor ( Figure 1C ) .",
"Planarian GABAergic neurons ( gad+ ) are present in two spatially distinct VM and DL subgroups ( Figure 1D ) ( Nishimura et al . , 2008b ) .",
"Within the VM GABAergic neuron population , virtually all cells that were imaged expressed nkx2 . 1 ( 98 . 82 ± 2 . 63% ) , whereas expression of arx was not detected ( Figure 1D ) .",
"In contrast , the DL subgroup of GABAergic neurons did not exhibit significant expression of either transcription factor ( Figure 1—figure supplement 1 ) .",
"Due to the relatively modest size of the octopaminergic neuronal subpopulation ( tbh+ ) , and the focal grouping of these cells within the medial region of the CNS , this neural subtype was assessed as a whole .",
"Interestingly , expression of both transcription factors was observed within this neural cell group , with 76 . 9 ± 6 . 66% of octopaminergic neurons exhibiting nkx2 . 1 expression , while a lower proportion were arx+ ( 33 . 44 ± 6 . 54% ) ( Figure 1E ) .",
"Taken together , these expression data suggested a potential role for nkx2 . 1 and arx in the specification and/or maintenance of VM cholinergic , GABAergic , and octopaminergic neurons .",
"In order to test the functional roles in neuronal specification , nkx2 . 1 and arx were knocked down by RNAi , and the cholinergic , GABAergic , and octopaminergic neuronal subtypes were assayed for any changes to cell number during homeostasis only ( see Materials and methods ) .",
"Compared to control ( RNAi ) animals , a loss of arx resulted in a visible reduction of cholinergic neurons in the VM region of the CNS ( Figure 2A; white arrows ) , while nkx2 . 1 ( RNAi ) worms appeared relatively unaffected ( Figure 2—figure supplement 1 ) .",
"In order to quantify the loss of cholinergic neurons in arx ( RNAi ) animals , transverse sections of single brain lobes were imaged as described above , and 20 µm of confocal depth was recorded .",
"Subsequently , the number of chat+ cholinergic neurons were counted within each of the VM and DL brain regions .",
"Strikingly , arx ( RNAi ) worms exhibited a near 40% reduction in the number of VM cholinergic neurons , whereas DL neurons were largely unchanged ( Figure 2A , D , Figure 2—figure supplement 1 ) .",
"In contrast , nxk2 . 1 ( RNAi ) worms displayed no detectable changes in the average number of both VM and DL cholinergic neurons ( Figure 2—figure supplement 1 ) .",
"This very specific loss of VM cholinergic neurons in arx ( RNAi ) worms , combined with the evidence that arx was expressed by >60% of these neurons , demonstrated that arx was required for the maintenance of VM cholinergic neurons . 10 . 7554/eLife . 19735 . 008Figure 2 . nkx2 . 1 and arx are required for the maintenance of VM neural cell types .",
"( A–C )",
"Projected confocal image stacks showing cholinergic , GABAergic , or octopaminergic neuronal populations in RNAi-treated animals .",
"VM neural subpopulations are highlighted by white arrows .",
"Regions of interest are represented by cartoons at the right side of the figure .",
"Solid white lines represent the outer border of the animal , and dashed white lines mark the outer border of the CNS .",
"Solid yellow line demarcates the border between the VM and DL brain regions .",
"Scale bar = 50 µm .",
"( D–F )",
"Quantification of VM cholinergic , VM GABAergic , and octopaminergic neurons in RNAi-treated animals .",
"Significance levels in dot plots: **p<0 . 01 , ***p<0 . 001 , error bars are standard deviation .",
"CNS , Central nervous system; DL , Dorsal-lateral; VM , Ventral-medial . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 00810 . 7554/eLife . 19735 . 009Figure 2—figure supplement 1 . nkx2 . 1 and arx are not required for maintenance of DL neural cell types .",
"( A–B )",
"Projected confocal images showing cholinergic or GABAergic neurons in RNAi-treated animals .",
"Cartoons depict region of interest for all confocal projections .",
"Solid white lines represent outer border of the animal , dashed white lines represent the borders of the brain and solid yellow lines demarcate the plane of division between the VM and DL brain regions .",
"Scale bars = 50 µm .",
"( C–D )",
"Quantification of DL cholinergic and GABAergic neurons .",
"Graphs are dot plots , n ≥ 5 .",
"( E ) Table showing number of neurons for each subtype found in RNAi-treated animals .",
"Values are mean number of neurons ± standard deviation .",
"DL , Dorsal-lateral; VM , Ventral-medial . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 00910 . 7554/eLife . 19735 . 010Figure 2—figure supplement 2 . nkx2 . 1 and arx are not required for the maintenance of dopaminergic or serotonergic neurons Projected confocal images showing dopaminergic neurons or serotonergic neurons . Dashed lines represent the outer border of the CNS .",
"Scale bars = 50 µm .",
"CNS , Central nervous system . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 010 nkx2 . 1 represented an excellent candidate to act in the specification of VM GABAergic neurons based on its expression within nearly all of these cells .",
"As there are many fewer GABAergic than cholinergic neurons in the planarian brain , imaging and quantification of this entire neuronal population was performed .",
"As expected , arx ( RNAi ) animals exhibited no loss of GABAergic neurons in either the VM or the DL subgroups ( Figure 2B , E , Figure 2—figure supplement 1 ) .",
"In contrast , nkx2 . 1 ( RNAi ) worms exhibited a complete loss of all VM GABAergic neurons , while leaving DL neurons intact ( Figure 2B , E , Figure 2—figure supplement 1 ) .",
"Combined with the above dFISH data , the ablation of all VM GABAergic neurons demonstrated that nkx2 . 1 acts in the maintenance of this neural subtype .",
"Interestingly , both nkx2 . 1 ( RNAi ) and arx ( RNAi ) animals exhibited a significant reduction in the number of octopaminergic neurons , which correlated with the degree of expression for each transcription factor within this neural cell type ( Figure 2C , F ) .",
"While nkx2 . 1 ( RNAi ) animals exhibited a > 60% reduction in octopaminergic neurons , arx ( RNAi ) worms displayed a more modest but significant ~ 25% reduction ( Figure 2C , F ) .",
"It should also be noted that the neurons lost in nkx2 . 1 ( RNAi ) or arx ( RNAi ) animals were located in the VM region of the planarian brain ( Figure 2C ) .",
"As expected , RNAi of nkx2 . 1 or arx had no appreciable effect on the dopaminergic and serotonergic neuron populations ( Figure 2—figure supplement 2 ) .",
"Therefore , the maintenance roles of nkx2 . 1 and arx are limited to the VM cholinergic , GABAergic , and octopaminergic neuronal cell populations .",
"Previous studies have shown that the planarian CNS is a major site of expression for the single hedgehog ligand in planarians ( Smed-hh; henceforth referred to as hh ) ( Figure 3A ) ( Rink et al . , 2009; Yazawa et al . , 2009 ) .",
"However , it is unknown what specific cells express this signal and what cells respond to it .",
"Transverse sections through the brain highlighted hh expression within the VM regions of each brain lobe , similar to that of arx ( Figure 3B ) .",
"More specifically , hh expression was observed in the same VM neural cell types as described above for nkx2 . 1 and arx .",
"As with arx , hh expression within cholinergic neurons was heavily biased toward the VM region of the brain lobe , where 23 . 17 ± 2 . 88% of these neurons expressed the signaling molecule ( Figure 3C ) .",
"In addition , hh was expressed by nearly all VM GABAergic neurons ( 98 . 08 ± 3 . 85 ) , as well as a considerable proportion of octopaminergic neurons ( 33 . 7 ± 2 . 23% ) ( Figure 3D–E ) .",
"Although a limited number of DL GABAergic , dopaminergic , and serotonergic neurons exhibited hh expression ( Figure 3—figure supplement 1 ) , VM neurons appear to be the main source of the planarian hh signaling molecule . 10 . 7554/eLife . 19735 . 011Figure 3 . hh is expressed in VM neural cell types .",
"( A ) FISH image of whole-mount hh expression .",
"( B ) FISH image of hh expression in a brain cross-section ( C–E ) Single confocal plane of dFISH images depicting hh expression in cholinergic , GABAergic , and octopaminergic neurons ( white arrows ) .",
"( F ) Projected confocal image stacks displaying hh expression within the brain in RNAi-treated animals .",
"White arrow highlights reduced hh expression in the VM region of the brain .",
"Dashed lines represent the outer border of the brain lobe and yellow lines represent the plane of division between the VM and DL brain regions .",
"( G ) Quantification of VM hh+ neurons .",
"Graph is a dot plot , ***p<0 . 001 , error bars are standard deviation .",
"( H ) Violin plot showing relative expression of hh within sequenced single cells across various tissue types .",
"( I ) Heatmap depicting normalized expression levels of mature neuron markers and genes associated with neuronal specification within nine individually sequenced neurons that also express arx or nkx2 . 1 .",
"Scale bars = 100 µm .",
"DL , Dorsal-lateral; FISH , Fluorescent in situ hybridization; VM , Ventral-medial . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 01110 . 7554/eLife . 19735 . 012Figure 3—figure supplement 1 . hh is not significantly expressed in DL GABAergic , dopaminergic , or serotonergic neurons .",
"( A ) Projectedconfocal images showing GABAergic , dopaminergic , and serotonergic neurons .",
"Dashed white lines represent the outer border of the CNS , and solid red boxes represent regions of interest for panels to the right .",
"Scale bars = 100 µm .",
"( B ) Single confocal planes from dFISH images showing a lack of expression of hh within DL GABAergic , dopaminergic and serotonergic neural populations .",
"Scale bars = 20 µm .",
"( C ) Table showing the percentage of mature neural subpopulations that express the hh signaling molecule .",
"Values are mean percentage of neurons ± standard deviation .",
"CNS , Central nervous system; DL , Dorsal-lateral; dFISH , Double fluorescent in situ hybridization . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 01210 . 7554/eLife . 19735 . 013Figure 3—figure supplement 2 . nkx2 . 1 is not required for neuronal hh expression .",
"( A ) Projected confocal images showing hh+ neurons in the CNS .",
"Top panels show the entire brain , while bottom panels show single brain lobes from transverse sections through the anterior-posterior midpoint of the brain .",
"Solid white lines represent the outer border of the animal , dashed white lines represent the outer border of the CNS , and solid yellow line demarcates the plane of division between the VM and DL brain regions .",
"( B ) Quantification of hh+ neurons in the DL brain region .",
"Graph is a dot plot n ≥ 5 , error bars are standard deviation .",
"( C ) Table showing the number of hh+ neurons in RNAi-treated animals .",
"Values are mean number of hh+ neurons ± standard deviation .",
"CNS , Central nervous system; DL , Dorsal-lateral; VM , Ventral-medial . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 01310 . 7554/eLife . 19735 . 014Figure 3—figure supplement 3 . nkx2 . 1 and arx are expressed in hh+ cells in the brain ( A , B ) Single confocal planes showing co-expression of nkx2 . 1 and arx within hh+ cells in the CNS .",
"Highlighted cells in the solid white boxes are enlarged and individual channels are separated in the panels to the right .",
"Scale bars = 100 µm .",
"Co-expression quantification is at the bottom . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 014 Although only partially overlapping , the finding that hh was co-expressed within the same VM neural cell types as nkx2 . 1 and arx ( Figure 3C–E ) suggested that these two transcription factors may have upstream regulatory function on neuronal hh expression .",
"WISH experiments demonstrated a clear reduction in hh expression within the medial CNS of arx ( RNAi ) worms ( Figure 3F; white arrows ) , while silencing nkx2 . 1 had little effect on hh expression ( Figure 3—figure supplement 2 ) .",
"This reduction in hh expression was quantified in the same manner described above for cholinergic neurons , by imaging transverse sections through single brain lobes , and counting the number of hh+ cells in the VM and DL brain regions , within 20 µm of confocal depth .",
"This analysis revealed a near 60% reduction in the number of VM hh+ neurons in arx ( RNAi ) animals ( Figure 3F–G ) , but no significant loss of hh+ cells in the DL brain ( Figure 3—figure supplement 2 ) .",
"In contrast , silencing of nkx2 . 1 did not significantly alter hh expression levels in either brain region ( Figure 3G , Figure 3—figure supplement 2 ) .",
"While it is unclear whether arx directly regulates hh expression in VM neurons , or whether reduced hh expression is related to a loss of VM cholinergic neurons , it is clear that hh expression in the VM domain of the planarian brain requires arx gene function .",
"Recent studies have begun using single-cell RNA sequencing ( scRNAseq ) technologies , to investigate the complete transcriptional profiles of individual planarian stem cells and mature cell types ( Wurtzel et al . , 2015; Molinaro and Pearson , 2016 ) .",
"From the scRNAseq dataset generated by Wurtzel et al . , all cells from uninjured animals ( including stem cells , neurons , gut , epithelial , muscle , and parapharyngeal cells ) as well as neurons from injured worms were analyzed for expression of hh .",
"From this group of sequenced cells , hh expression was detected in 4/39 neurons , and a single stem cell , but no other cell types ( Figure 3H ) , supporting previous data that hh expression is largely restricted to the nervous system .",
"In addition , these 39 sequenced neurons were examined for expression of nkx2 . 1 and arx , which were detected in two and six neurons , respectively , with two of these cells displaying co-expression with hh ( Figure 3I ) .",
"Expression of nkx2 . 1 or arx within hh+ neurons in the brain was also directly observed by dFISH ( Figure 3—figure supplement 3 ) .",
"High expression levels of common mature neuron markers ( Smed-synaptotagmin , Smed-synapsin , Smed-pc2 ) were found in all nine sequenced cells , and more specifically , five of these cells were chat+ cholinergic neurons ( Figure 3I ) ( Agata et al . , 1998; Cebrià , 2008 ) .",
"In addition , each of these nine neurons expressed a unique combination of neuropeptides ( Collins et al . , 2010 ) , transcription factors ( Cowles et al . , 2013 , 2014; Currie and Pearson , 2013; Pineda et al . , 2002 ) , RNA-binding proteins ( Higuchi et al . , 2008; Koushika et al . , 1996 ) , and even 2 Wnts with known or putative roles in neuronal specification and patterning ( Figure 3I ) .",
"This in silico data acts to further confirm that the planarian hh ligand is expressed by mature neurons .",
"Considering that VM planarian neurons are a major source of the hh signaling molecule ( Figure",
"3 ) ( Rink et al . , 2009; Yazawa et al . , 2009 ) , it was of great interest to discover what cells and tissues might be the target of this brain-derived signaling molecule .",
"Previous observations have shown that hh signaling activity can influence global proliferation levels ( Rink et al . , 2009 ) .",
"Therefore , the adult stem cells ( ASCs ) that occupy the space in between the two brain lobes were examined as a potential target of planarian hh .",
"Reception and effective transduction of the hh signaling pathway requires the receptors Patched ( ptc ) and Smoothened ( smo ) as well as the Gli transcription factors ( Ho and Scott , 2002 ) .",
"Confirming previous reports , WISH experiments for the planarian homologs of these key signal transduction effectors ( Smed-ptc , Smed-smo , and Smed-gli-1 ) ( Rink et al . , 2009 ) demonstrated relatively ubiquitous expression throughout the body of the adult animal , with Smed-gli-1 showing a particularly strong signal within the gastrovascular system ( Figure 4A ) .",
"However , when piwi-1+ ASCs ( Reddien et al . , 2005b ) were visualized in between the two brain lobes , many were observed to co-express the ptc receptor ( Figure 4B ) .",
"Similarly , smo and gli-1 expression was detected in brain-adjacent ASCs ( Figure 4B ) , providing support for the notion that planarian stem cells might be a target of brain-derived hh signals . 10 . 7554/eLife . 19735 . 015Figure 4 . Planarian neoblasts express hh and Wnt signal transduction effectors .",
"( A ) WISH images for ptc , smo , and gli-1 .",
"( B ) Single confocal planes from dFISH images showing expression of ptc , smo , and gli-1 within piwi-1+ stem cells ( white arrows ) in between the two brain lobes in cross-section .",
"Dashed lines mark the outer border of the brain lobes .",
"Cell in solid white box is enlarged and channels are split in two right panels .",
"Scale bars for dFISH images = 10 µm .",
"( C ) Heatmap depicting normalized expression levels of key hh and Wnt signal transduction genes and stem cell markers , within 168 individually sequenced head stem cells ( X1 ) or stem cells + progeny ( X2 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 01510 . 7554/eLife . 19735 . 016Figure 4—figure supplement 1 . Planarian neoblasts express Wnt signal transduction effectors .",
"( A ) WISH images for all planarian fz receptors .",
"( B ) Single confocal planes from dFISH images showing expression of all fz receptors within piwi-1+ stem cells in between the two brain lobes .",
"Cartoon depicts region of interest for all dFISH images .",
"Scales bar for WISH images = 100 µm .",
"Scale bars for dFISH images = 10 µm .",
"( C ) Heatmap depicting normalized expression levels of key hh and Wnt signal transduction genes and stem cell markers , within 84 individually sequenced stem cells ( Wurtzel et al . , 2015 ) .",
"Stem cells are binned based on their known subtype ( sigma , zeta , or gamma ) .",
"( D ) Heatmap depicting normalized expression of stem cell markers , genes associated with neurons , hh , and Wnt transduction genes , within 10 individually sequenced stem cells that also express arx or nkx2 . 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 016 In order to support these dFISH findings , we examined gene expression from a recent dataset ( Molinaro and Pearson , 2016 ) containing 96 single stem cells in the head ( isolated from a FACS gate termed X1 ) as well as 72 cells from a FACS gate termed X2 that contains a mixture of stem cells and their immediate division progeny ( Zhu et al . , 2015; Hayashi et al . , 2006 ) .",
"These cells were examined for expression of ptc , smo , or gli-1 .",
"Individual neoblasts were first examined for expression of established stem cell markers and cell cycle regulators ( piwi-1 , piwi-2 , piwi-3 , Smed-bruli , Smed-tdrd1L2 , Smed-chd4 , Smed-mcm2 , Smed-pcna , Smed-cyclinB-2 ) ( Zhu et al . , 2015; van Wolfswinkel et al . , 2014; Guo et al . , 2006; Reddien and Sánchez Alvarado , 2004 ) .",
"These stem cells were then binned as either X1 or X2 and arranged in descending order based on ptc then smo then gli-1 expression ( Figure 4C ) .",
"Approximately 30% ( 56/168 cells ) expressed at least one of ptc , smo , or gli-1 ( Figure 4C ) .",
"Interestingly , approximately half of these sequenced cells ( 81/168 ) also expressed at least one of the nine planarian Frizzled ( fz ) receptors ( which bind Wnt ligands ) ( Gurley et al . , 2008 , 2010 ) .",
"These in silico findings were also confirmed by performing dFISH experiments , which showed expression of all nine fz receptors within piwi-1+ ASCs in between the two brain lobes ( Figure 4—figure supplement 1 ) .",
"Parallel expression analyses were performed on another scRNAseq dataset of 84 ASCs from the whole body of the worm with similar conclusions ( Figure 4—figure supplement",
"1 ) ( Wurtzel et al . , 2015 ) .",
"Taken together , these dFISH and in silico data suggested that planarian stem cells adjacent to the CNS are one of the cellular targets of brain-derived hh and Wnt signals .",
"Recent studies have highlighted an interesting property of planarian transcription factors: they are expressed not only by the cells and tissues that they act to specify , but also within ASCs nearby the mature organ in question ( Currie and Pearson , 2013; Cowles et al . , 2013; Scimone et al . , 2011 , 2014; Lapan and Reddien , 2011 , 2012 ) .",
"Therefore , ASCs nearby the VM brain region were investigated for expression of the nkx2 . 1 and arx transcription factors .",
"By examining transverse sections through the brain , stem cells , which express piwi-1 mRNA and PIWI-1 protein , can be observed in great numbers , and are largely excluded from the two brain lobes ( Figure 5A–F ) .",
"In addition , their post-mitotic progenitors , which shut down piwi-1 expression , but briefly maintain detectable amounts of PIWI-1 protein , can be visualized in this region ( Guo et al . , 2006; Wenemoser and Reddien , 2010 ) .",
"Some of these progenitors can be found several cell diameters within the mature brain lobes ( Figure 5A–F; green arrowheads ) .",
"In this specific spatial region , expression of nkx2 . 1 or arx was observed within a significant number of stem cells or post-mitotic progenitors ( Figure 5A–F ) .",
"Quantification of piwi-1+ stem cells revealed that a significant percentage displayed expression of nkx2 . 1 ( 7 . 99 ± 0 . 95% ) , whereas arx expression was less frequent ( 1 . 74 ± 0 . 99% ) .",
"Quantification of all PIWI-1+ cells ( encompassing both stem cells and their post-mitotic progeny ) demonstrated an expansion of nkx2 . 1 and arx expression , to include 13 . 29 ± 1 . 96% and 8 . 11 ± 1 . 83% , respectively , of all cells counted . 10 . 7554/eLife . 19735 . 017Figure 5 . nkx2 . 1 and arx are expressed in stem cells and post-mitotic progenitors .",
"( A–F )",
"Single confocal planes of dFISH images combined with immunolabelling , displaying expression of nkx2 . 1 and arx within stem cells ( yellow arrows ) and post-mitotic progenitors ( white arrows ) .",
"Green arrowheads highlight post-mitotic progenitors within the brain lobes .",
"Boxed cells are enlarged below and individual channels are split .",
"Dashed lines mark the outer border of the brain lobes .",
"Cartoon depicts region of interest for all confocal images .",
"Scale bars = 20 µm .",
"( G ) Heatmap depicting normalized expression of stem cell markers , genes associated with neurons , hh , and Wnt transduction genes , within 31 individually sequenced head stem cells ( X1 ) or stem cells + progeny ( X2 ) that also express arx or nkx2 . 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 01710 . 7554/eLife . 19735 . 018Figure 5—figure supplement 1 . nkx2 . 1 and arx are expressed in ptc+/PIWI-1+ cells .",
"( A–B )",
"Single confocal planes from dFISH images combined with immunolabelling , displaying expression of nkx2 . 1 and arx within ptc+/PIWI-1+ stem/progenitor cells .",
"Merged image is to the left , with separated color channels to the right .",
"DAPI is in grey .",
"Scale bars = 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 018 In order to support the expression of nkx2 . 1 and arx within planarian neoblasts , we again examined the pool of 168×1 and X2 cells from the head of the animal ( Molinaro and Pearson , 2016 ) .",
"Both nkx2 . 1 and arx were found to be expressed within these cells and a total of 31/168 cells expressed at least one factor ( Figure 5G ) .",
"Importantly , nkx2 . 1+ or arx+ stem cells demonstrated high expression levels of established stem cell markers/regulators ( piwi-1 , piwi-2 , piwi-3 , Smed-bruli , Smed-tdrd1L2 , Smed-chd4 ) , and cell cycle genes ( Smed-mcm2 , Smed-pcna , Smed-cyclinB-2 ) ( Figure 5G ) ( Zhu et al . , 2015; van Wolfswinkel et al . , 2014; Guo et al . , 2006; Reddien and Sánchez Alvarado , 2004 ) .",
"In addition , many cells displayed expression of established and putative neuronal transcription factors ( Smed-coe , Smed-cas , Smed-hb-2 , Smed-mitfl-2 , Smed-da , Smed-isl-1 ) and mature neural markers ( Smed-synapsin , Smed-pc-2 ) ( Figure 5G ) ( Cowles et al . , 2013 , 2014; Hayashi et al . , 2011; Scimone et al . , 2014 ) .",
"Several of these nkx2 . 1+ or arx+ stem cells also exhibited expression of ptc , smo , or gli-1 ( 7/31 cells ) and at least one of the nine fz receptors ( 14/31 ) ( Figure 5G ) .",
"Parallel expression analyses were performed on the dataset of 84 single ASCs from the whole body of the worm with similar conclusions ( Figure 4—figure supplement",
"1 ) ( Wurtzel et al . , 2015 ) .",
"Finally , ptc expression within nkx2 . 1+ and arx+ neural progenitors was also observed in vivo based on dFISH combined with immunostaining against the PIWI-1 protein ( Figure 5—figure supplement 1 ) .",
"Together , these data demonstrated that putative neural progenitor cells , located adjacent to the mature brain , express the machinery to transduce hh signals and may be committed to producing arx+ or nkx2 . 1+ neurons .",
"Based on the expression of hh within VM neurons and the hh-transduction machinery within adult stem cells , the hh signaling pathway was next assessed for a potential role in the regulation of neural progenitor cells and the ultimate production of new neurons .",
"This was performed by RNAi knockdown of the hh ligand , resulting in decreased signaling activity , or by knockdown of the ptc receptor ( a negative regulator of pathway activity ) , resulting in increased hh signaling levels ( Rink et al . , 2009 ) .",
"Neural progenitor cells were then visualized by taking transverse sections through the brain , and imaging PIWI-1+ stem and progenitor cells in between the brain lobes for expression of several transcription factors of putative neural stem/progenitors ( nkx2 . 1 , arx , lhx1/5–1 , coe , and pax6a ) ( Figure 6A ) ( Currie and Pearson , 2013; Cowles et al . , 2014; Pineda et al . , 2002; Scimone et al . , 2014 ) .",
"hh ( RNAi ) and ptc ( RNAi ) animals were investigated for changes to the percentage of PIWI-1+ stem/progenitor cells expressing these neural transcription factors .",
"Silencing of the planarian hh ligand resulted in a significant reduction of expression for all five neural transcription factors within PIWI-1+ cells ( Figure 6B ) .",
"In contrast , levels of neural progenitor cells were largely unchanged in ptc ( RNAi ) animals , with the exception of coe and pax6a which exhibited a small but significant increase in PIWI-1+ cells ( Figure 6B ) . 10 . 7554/eLife . 19735 . 019Figure 6 . hh signaling regulates homeostatic neurogenesis .",
"( A ) Single confocal plane images displaying examples of neural progenitor cells ( white arrow ) , PIWI-1+ stem/progenitor cells that express neural transcription factors .",
"Scale bar = 10 µM .",
"( B ) Quantification of neural progenitor levels in animals with altered levels of hh signaling .",
"Graphs are dot plots measuring the percentage of PIWI-1+ cells that express each neural transcription factor .",
"n = 10 , *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , error bars are standard deviation .",
"( C–E )",
"Single confocal planes displaying newly generated cholinergic neurons in the planarian brain 6 days after a single BrdU pulse ( white arrows ) .",
"Scale bar = 100 µm .",
"( F ) Dot plot quantifying the number of new cholinergic neurons .",
"n ≥ 10 , ***p<0 . 001 , error bars are standard deviation .",
"( G ) Model displaying nkx2 . 1+ and arx+ neural stem and progenitor cells and the mature neurons that they produce .",
"These same neural cell types also express the hh signaling molecule , which signals back onto adult stem cells , maintaining normal proliferation levels , and homeostatic neurogenesis . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 01910 . 7554/eLife . 19735 . 020Figure 6—figure supplement 1 . hh signaling levels do not affect existing neuronal subpopulations in the intact brain .",
"( A ) Projected confocal images showing cholinergic neurons from 20 µm – thick recordings , of single brain lobes from transverse sections through the anterior-posterior midpoint of the brain .",
"The plane of division ( yellow line ) separating the VM and DL brain halves occurs at the naturally occurring dorsal and ventral vertices of the brain lobe .",
"( B ) Quantification of cholinergic neurons observed in the VM and DL regions of a single brain lobe .",
"( C ) Projected confocal image showing GABAergic neurons .",
"( D ) Quantification of VM and DL GABAergic neurons .",
"( E ) Projected confocal images showing octopaminergic neurons .",
"( F ) Quantification of octopaminergic neurons .",
"All images are from animals subjected to 10 RNAi feedings .",
"Dashed lines represent the outer border of the CNS .",
"Solid white lines represent the outer border of the animal .",
"Scale bars = 100 µm .",
"All graphs are dot plots showing value mean ± standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 02010 . 7554/eLife . 19735 . 021Figure 6—figure supplement 2 . hh signaling levels do not affect existing neuronal subpopulations in the intact brain Projected confocal images showing dopaminergic and serotonergic neurons after six RNAi feedings to knockdown hh or ptc . Dashed lines represent the outer border of the CNS .",
"Scale bars = 50 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 02110 . 7554/eLife . 19735 . 022Figure 6—figure supplement 3 . hh signaling levels do not affect other planarian progenitor populations .",
"( A , D , G , J )",
"WISH images showing expression domains of planarian tissue markers for the eye spots ( ovo ) , pharynx ( foxA ) , epidermis ( prog-2 ) , and gut ( hnf4 ) .",
"( B , E , H , K )",
"Single confocal planes showing tissue specific progenitors after RNAi knockdown of hh or ptc .",
"Region of interest is represented by the white boxes in ( A , D , G , and J ) .",
"Scale bars = 100 µm .",
"( C , F , I , L )",
"Quantification of tissue progenitors after hh or ptc RNAi knockdown .",
"All graphs are dot plots , showing the value mean ± standard deviation .",
"*p<0 . 05 , **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 02210 . 7554/eLife . 19735 . 023Figure 6—figure supplement 4 . hh signaling levels do not affect BrdU incorporation into other planarian tissues .",
"( A , D , G , J )",
"WISH images showing expression domains of planarian tissue markers for the eye spots ( ovo ) , pharynx ( laminin ) , epidermis ( vim-1 ) , and gut ( mat ) .",
"( B , E , H , K )",
"Single confocal planes showing BrdU incorporation into mature tissues after RNAi knockdown of hh or ptc .",
"Region of interest is represented by the white boxes in ( A , D , G , and J ) .",
"Scale bars = 100 µm .",
"( C , F , I , L )",
"Quantification of BrdU incorporation after hh or ptc RNAi knockdown .",
"All graphs are dot plots , showing the value mean ± standard deviation .",
"**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 19735 . 023 The thymidine analog bromodeoxyuridine ( BrdU ) , which is incorporated into dividing stem cells during DNA synthesis and subsequently chases into their post-mitotic progeny , has proven key for planarian lineage-tracing experiments ( Newmark and Sánchez Alvarado , 2000 ) , some of which have revealed that the intact planarian CNS maintains high levels of continuous neurogenesis ( van Wolfswinkel et al . , 2014; Zhu et al . , 2015 ) .",
"Therefore , in order to investigate whether hh signaling levels regulate neuronal homeostasis and the production of new cholinergic neurons , a BrdU pulse-time chase experiment was performed .",
"Following gene silencing of hh or ptc , RNAi-treated animals were exposed to a single pulse of BrdU .",
"After 6- and 9-day chase periods , newly generated cholinergic neurons were detected by FISH for chat expression as well as nuclear BrdU staining ( Figure 6C–E ) .",
"The level of neurogenesis was then quantified by counting the number of chat+BrdU+ cells observed within 30-µm-thick confocal stacks from within the planarian brain .",
"hh ( RNAi ) animals consistently displayed significantly lower cholinergic neural homeostasis both after 6-day ( ~35% reduction ) and 9-day chase periods ( >40% reduction ) , while silencing of ptc had little effect ( Figure 6C–F ) .",
"To investigate whether this decrease in neuronal homeostasis levels affected the intact nervous system , long-term RNAi experiments were performed ( 10 feedings ) , and all five neuronal populations were examined .",
"Of particular interest were the VM cholinergic , GABAergic , and octopaminergic neural populations , which express the hh ligand ( Figure 3C–E ) .",
"However , decreased hh signaling did not affect the intact population of any neuronal cell type ( Figure 6—figure supplements 1 and 2; see Discussion ) .",
"To determine whether brain-derived hh signals were required specifically to maintain normal levels of neurogenesis , or whether this ligand acts globally to stimulate the production of all planarian cell types , progenitor cells and BrdU incorporation were examined for four additional tissues; the eyes , pharynx , epidermis , and gut .",
"Importantly , these other planarian tissues were largely unaffected by perturbations to hh signaling , except for the eyes , which exhibited reduced progenitor cell numbers in hh ( RNAi ) animals , and increased BrdU incorporation after ptc knockdown ( Figure 6—figure supplements 3 and 4 ) .",
"These combined experiments , demonstrated that neuronal sources of the planarian hh ligand specifically regulate their own homeostasis through distinct progenitors in the uninjured brain ."
],
[
"Planarians not only constantly turnover their CNS at relatively high rates , but must also be able to respond to massive injury to regenerate an entire brain de novo .",
"This work has demonstrated that two planarian homeodomain transcription factor homologs , nkx2 . 1 and arx , function in the long-term maintenance of cholinergic , GABAergic , and octopaminergic neurons in the VM region of the planarian CNS ( Figure 2 ) .",
"Surprisingly , these same three VM neural cell types were also found to express the hh signaling molecule ( Figure 3C–E ) .",
"Neuronal hh expression was also confirmed by using a scRNAseq dataset that included mature neurons ( Wurtzel et al . , 2015 ) , and additionally revealed that all four of the sequenced hh+ neurons co-expressed a planarian Wnt ligand , Smed-wnt11-6 ( Figure 3I ) .",
"Specialized nkx2 . 1+ or arx+ neoblasts were also identified , which were typically localized adjacent to the VM region of the planarian CNS , and express key members of the hh and Wnt signal transduction machinery ( Figure 5 ) .",
"Finally , the planarian hh pathway was found to be required to maintain steady levels of neurogenesis in the intact brain , as a reduction in hh signaling activity , led to decreased production of neural stem/progenitor cells and newly generated cholinergic neurons ( Figure 6G ) .",
"Similar to planarian muscle cells , many of which express important signaling molecules that maintain positional control along the body axis and during regeneration events ( Witchley et al . , 2013; Scimone et al . , 2016 ) , mature planarian neurons express hh and Wnt ligands which regulate neurogenesis levels and maintain patterning ( Hill and Petersen , 2015 ) .",
"Previous studies have shown that the Wnt ligand Smed-wnt11-6 and Wnt antagonist Smed-notum are expressed by cholinergic neurons , and that this Wnt signal acts on planarian stem cells to repress neuron production and prevent posterior expansion of brain tissue during regeneration ( Hill and Petersen , 2015 ) .",
"Interestingly , we show that several planarian neurons simultaneously express this repressive Wnt ligand as well as the hh signaling molecule , which promotes the production of neurons ( Figures 3I and 6G ) .",
"Conceptually , it makes sense that signals originating from the mature CNS , would act to repress neurogenesis programs .",
"This would help to establish a steady-state system , which modulates neurogenesis levels depending on the size of the existing CNS .",
"However , our observations suggest that contrary to this model , neuronal sources of hh actively promote neurogenesis levels in both the brain and of eye photoreceptors neurons ( Figure 6—figure supplements 3 and 4 ) .",
"It is unclear , exactly how the planarian hh signaling pathway achieves rates of neuronal homeostasis , whether it acts as a permissive cue , influencing neoblasts adjacent to the brain to produce neural cell types , or as a mitogenic signal , to control proliferation levels of neural-biased stem cells .",
"Similar to mechanisms of Hh action in other systems , the regulatory loops are likely to be complex in planarians due to the fact that long-term hh ( RNAi ) , surprisingly , did not produce robust neural deficits over a span of five weeks ( Figure 6—figure supplements 1 and 2 ) .",
"Perhaps with body-wide changes in proliferation in hh ( RNAi ) , rates of neural cell death are also decreased , leading to little change in neuronal populations ( Rink et al . , 2009 ) .",
"Similarly , in the mammalian CNS , expression of Shh from differentiating and mature neurons is required to maintain normal proliferation levels of embryonic neural precursors and adult neural stem cells , respectively ( Álvarez-Buylla and Ihrie , 2014; Ihrie et al . , 2011; Fuccillo et al . , 2006 ) .",
"Unlike most organisms where neural stem cells are packed within an organized neuroepithelium , planarian neural-biased stem cells are situated in the mesenchymal space in between the two brain lobes ( Figure 5 ) .",
"While this loose grouping of stem cells offers few similarities to a true neuroepithelium in terms of structure or cellular origin , the local signaling microenvironment may fulfill a similar function .",
"Computationally , neural-committed stem cells , termed νNeoblasts , were recently detected and may be the targets of brain-derived signals ( Molinaro and Pearson , 2016 ) .",
"However , while nkx2 . 1+ and arx+ stem cells exhibit a spatial distribution and transcriptional profile suggestive of neuronal lineage commitment ( Figure 5G ) , without definitive lineage-tracing studies , these neoblasts cannot yet be classified as true neural stem cells .",
"In chordate nerve cord development , Hh is expressed ventrally , along the long body axis and has strong ventralizing roles in cell fate determination ( Briscoe and Ericson , 2001; Jessell , 2000 ) .",
"This is not typical of arthropods ( Matise , 2007; Arendt and Nübler-Jung , 1999 ) .",
"It is interesting to speculate that VM neural fates in planarians are specified using mechanisms more associated with chordates .",
"In support of this idea , the role of nkx2 . 1 and arx in the maintenance of planarian cholinergic and GABAergic neurons was particularly noteworthy ( Figure 2 ) , as their mammalian counterparts ( Nkx2 . 1 and Arx ) are known to fulfill similar roles and also be under the control of sonic hedgehog ( Jessell , 2000; Colasante et al . , 2008 ) .",
"In the embryonic rodent ventral telencephalon , Nkx2 . 1 acts in upstream neural progenitor cells to produce both cortical GABAergic interneurons and striatal cholinergic interneurons ( Butt et al . , 2008; Lopes et al . , 2012; Sussel et al . , 1999 ) , whereas Arx functions downstream in the terminal differentiation and migration of cortical GABAergic interneurons ( Vogt et al . , 2014 ) .",
"Notwithstanding this slight deviation in cell fate determinism , it appears that these two transcription factors have retained a remarkable degree of functional conservation across this significant evolutionary gap ."
],
[
"Smed-nkx2 . 1 and Smed-arx were found by homology to mammalian orthologs in the sequenced and assembled planarian genome and transcriptomes as previously described ( Currie and Pearson , 2013; Robb et al . , 2008 ) .",
"Primers were designed and full length genes were cloned by 3’ RACE .",
"Smed-nkx2 . 1 is transcript number dd_Smed_v6_13898_0_1 from ( http://planmine . mpi-cbg . de/ , Brandl et al . , 2016 ) and Smed-arx was deposited to Genbank under the accession number KX961610 .",
"Asexual S . mediterranea CIW4 strain were reared as previously described ( Sánchez Alvarado et al . , 2002 ) .",
"RNAi experiments were performed using previously described expression constructs and HT115 bacteria ( Newmark et al . , 2003 ) .",
"Briefly , bacteria were grown to an O . D . 600 of 0 . 8 and induced with 1 mM IPTG for 2 hr .",
"Bacteria were pelleted and mixed with liver paste at a ratio of 500 µl of liver per 100 ml of original culture volume .",
"Bacterial pellets were thoroughly mixed into the liver paste and frozen as aliquots .",
"The negative control , ‘control ( RNAi ) ’ , was the unc22 sequence from C . elegans as previously described ( Reddien et al . , 2005a ) .",
"All RNAi food was fed to 7-day starved experimental worms every third day for six total feedings , and fixed 10–14 after the final RNAi feeding .",
"All animals used for immunostaining were 2–3 mm in length and size-matched between experimental and control worms .",
"WISH , dFISH , and immunostaining were performed as previously described ( Currie et al . , 2016 ) .",
"Colorimetric WISH stains were imaged on a Leica M165 fluorescent dissecting microscope .",
"dFISH and fluorescent mouse-anti-PIWI-1 ( gift from Dr . Jochen Rink ) stains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head .",
"Cell counts and co-localizations were quantified using freely available ImageJ software ( http://rsb . info . nih . gov/ij/ ) .",
"Significance was determined by a two-tailed Student’s t-test .",
"All experiments were , at minimum , triplicated and at least five worms were used per stain and per time point .",
"All labeling images were post-processed using Adobe Photoshop .",
"Raw scRNAseq data from head-specific X1 and X2 cells from ( Molinaro and Pearson , 2016 ) can be downloaded under NCBI Gene Expression Omnibus ( GEO accession GSE79866 ) .",
"Raw scRNAseq data from uninjured cells ( including stem cells , neurons , gut , epithelial , muscle and parapharyngeal cells ) as well as neurons following injury generated by Wurtzel et al . , were obtained from the NCBI Sequence Read Archive ( SRA:PRJNA276084 ) .",
"Reads were aligned to the SmedASXL transcriptome assembly under NCBI BioProject PRJNA215411 using bowtie2 ( Langmead and Salzberg , 2012 ) with 15 bp 3’ trimming .",
"The read count data were log2-transformed ( log2 ( count + 1 ) ) , violin plot was produced using modified source code from ( Macosko et al . , 2015 ) and the heatmaps were produced using the modified heatmap . 3 source code from ( Molinaro and Pearson , 2016 ) ."
]
] | [
"The asexual freshwater planarian is a constitutive adult , whose central nervous system ( CNS ) is in a state of constant homeostatic neurogenesis .",
"However , very little is known about the extrinsic signals that act on planarian stem cells to modulate rates of neurogenesis .",
"We have identified two planarian homeobox transcription factors , Smed-nkx2 . 1 and Smed-arx , which are required for the maintenance of cholinergic , GABAergic , and octopaminergic neurons in the planarian CNS .",
"These very same neurons also produce the planarian hedgehog ligand ( Smed-hh ) , which appears to communicate with brain-adjacent stem cells to promote normal levels of neurogenesis .",
"Planarian stem cells nearby the brain express core hh signal transduction genes , and consistent hh signaling levels are required to maintain normal production of neural progenitor cells and new mature cholinergic neurons , revealing an important mitogenic role for the planarian hh signaling molecule in the adult CNS ."
] | [
"Most animals can continue to generate and add new neurons in their nervous system into adulthood , though the process is often tightly regulated .",
"In adult humans , only a small number of neurons are made or lost , such that the fewer than 2% of the neurons in the nervous will change over , or “turnover” , the course of a year .",
"The turnover of neurons in some other animals is much higher than it is in humans .",
"A freshwater flatworm , called Schmidtea mediterranea , is one example of such an animal that can even regenerate an entirely new brain if its head is decapitated .",
"These flatworms have a large population of adult stem cells , which makes these high rates of neuron production and regeneration possible .",
"However , it is largely unknown if this population contains stem cells that can only become new neurons , in other words “dedicated neuronal stem cells” .",
"Moreover , it is also not clear what kinds of signals communicate with these stem cells to promote the production of new neurons .",
"In animals from flies to humans , a signaling molecule encoded by a gene called hedgehog forms part of a signaling pathway that can promote neuron production during development .",
"Therefore , Currie et al . asked if the hedgehog signaling molecule might communicate with the stem cells in adult flatworms to control how many new neurons they produce .",
"The experiments revealed that the hedgehog signaling molecule is almost exclusively produced by the flatworm’s brain and the pair of nerve cords that run the length of the flatworm .",
"Currie et al . then found a smaller group of cells close to the flatworm’s brain that looked like dedicated neural stem cells .",
"These cells can receive the hedgehog signals , and further experiments showed that flatworm’s brain requires hedgehog signaling to be able to produce new neurons at its normal level .",
"The hedgehog signaling molecule is likely only one of many signaling molecules that regulate the production of new neurons in flatworms .",
"It will be important to uncover these additional signals and understand how they work in concert .",
"In the future , a better understanding of this process will help efforts to devise ways to induce humans to replace neurons that are lost following injury or neurodegenerative diseases ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"immunology and inflammation"
] | Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema | elife-09623-v2 | [
[
"Tobacco smoking is linked to a long and growing ( Barnes , 2014; Carter et al . , 2015 ) list of fatal illnesses ( e . g . , emphysema , cancer , and stroke ) and is the major preventable cause of human death .",
"Despite public awareness of the harmful effects of smoking , in many large developing countries the prevalence of smoking is growing ( Eriksen et al . , 2014 ) .",
"Compounding this risk is particulate air pollution due to the combustion of organic materials including biomass fuels , slash and burn agriculture , and coal ( Furlaneto et al . , 1969; Arif et al . , 1993; Dadvand et al . , 2014 ) .",
"While our understanding of the immune basis of smoke-induced sterile inflammation has increased , the molecular mechanism underlying emphysema and its persistence despite smoking cessation remains unclear ( Cosio et al . , 2009; Kheradmand et al . , 2012 ) .",
"Even less is known regarding the health-related inhalation effects of atmospheric and workplace airborne carbon particulates .",
"Innate immune cells such as alveolar macrophages and neutrophils are recruited to the lungs in response to cigarette smoke ( Salvi , 2014 ) .",
"Several human studies and pre-clinical models of smoke-induced emphysema have also confirmed that lymphocytes ( T and B cells ) and lung antigen presenting cells ( APCs ) play pathogenic roles in chronic lung inflammation in smokers ( Shan et al . , 2009 , 2014; Churg et al . , 2012b ) .",
"Prior work has shown that increased concentrations of pro-inflammatory cytokines such as IL-6 , IL-1β , and IL-17A are among the most important hallmarks of smoke-mediated lung inflammation ( Shan et al . , 2009 , 2014; Chang et al . , 2014 ) .",
"We and others have previously shown that IL-17A plays a critical role in smoke-induced emphysema in humans and in mouse models of disease ( Shan et al . , 2012; Kurimoto et al . , 2013; Zhang et al . , 2013; Chang et al . , 2014 ) .",
"Further , adoptive transfer of lineage-negative CD11c+ myeloid dendritic cells ( mDCs ) isolated from the lungs of smoke-exposed mice to naive mice recapitulates smoke-induced lung disease , indicating a direct causal relationship between mDC and emphysema ( Shan et al . , 2012 ) .",
"Despite these advances , the mechanism by which smoke induces the inflammatory mDC phenotype remains completely unknown .",
"Tobacco smoke contains many noxious chemicals ( e . g . , carbon monoxide , sulfur , nitrogen dioxide , nitric oxide , and methane ) , aromatics ( e . g . , benzene , toluene , and xylene ) and chlorinated ( e . g . , methyl chloride , chloroethene , and chloroform ) volatile organic compounds , as well as particulate matter ( Wang et al . , 2012; Perfetti and Rodgman , 2013; Salvi , 2014 ) .",
"One or more of these agents is thought to underlie the carcinogenic potential of smoke , involving at least eight different cancers; accordingly , the role of volatile carcinogens found in smoke has been studied extensively ( Pope et al . , 2011; Carter et al . , 2015 ) .",
"Far less is known about the pathogenic effects of particulate matter that is suspended in smoke and which includes nanoparticulate carbon , metal oxides , and inorganic salts .",
"Histopathological analysis of the lungs of heavy smokers invariably reveals dark-staining anthracotic pigment often attributed to poorly soluble material found in tobacco smoke ( Mitchev et al . , 2002 ) .",
"Anthracotic pigment is also found in the lymph nodes of smokers ( Churg et al . , 2005 ) , but its chemical composition and potential contribution to smoking-related diseases have not been explored .",
"In this study , we show that the anthracotic material found in the lung of human smokers is nanoparticulate carbon black ( nCB ) , a hydrophobic material that accumulates specifically in highly activated CD1a+ lung APCs .",
"Raman spectroscopy , hyperspectral imaging , and high-resolution transmission electron microscopy ( HR-TEM ) were used to confirm this observation and show that nCB further accumulates in the lung and APC of mice exposed to smoke .",
"Moreover , we show that nCB given by inhalation to mice in amounts that are comparable to human exposures is alone sufficient to cause sterile inflammation and induce robust T helper 17 cell ( Th17 ) responses and emphysema , implicating the potential health risks of airborne nCB that contaminates a wide range of workplace and domestic environments ( IARC Working Group on the Evaluation of Carcinogenic Risks to Humans , 2010 ) ."
],
[
"In contrast to the white or pink appearance of normal lungs , the lungs of heavy smokers are typically dark brown or black ( Churg et al . , 2005 ) .",
"During the routine preparation of lung phagocytic cells including CD1a+ mDCs and APCs , we observed the same anthracotic pigmentation in lung cells from smokers ( Figure 1A , B ) , whereas the same cells isolated from non-smokers lacked the pigment .",
"We previously showed that activated mDCs from smokers and smoke-exposed mice promote Th1 and Th17 responses ( Shan et al . , 2009 , 2012 ) .",
"To determine the nature of the anthracotic pigment from mDCs , we first performed HR-TEM of the residual black material after complete proteolytic digestion of human emphysematous lung and observed the aggregates that were composed of 20–50 nm spheroids ( Figure 1C ) .",
"To further extend these observations , we examined CD1a+ mDCs from human emphysematous lung tissue using Raman spectroscopy and hyperspectral mapping which showed the signature for pure nCB and not inorganic salts or metal oxides ( Figure 1D–H ) .",
"Thus , nCB is extensively deposited as solid material in the lungs of smokers and specifically accumulates in lung APC . 10 . 7554/eLife . 09623 . 003Figure 1 . Carbon black ( CB ) deposition in the lungs of patients with emphysema .",
"( A ) Representative images of lung CD1a+ cells from a smoker with emphysema and a control subject .",
"Scale bar: 10 μm .",
"( B ) Lung CD1a+ cells from a patient with emphysema , detected by transmission electron microscopy ( TEM ) .",
"Arrow indicates black substance in the vesicles .",
"Scale bar: 1 μm .",
"( C ) Structure of the residual black material from digested human emphysema lung tissue , detected by high-resolution transmission electronic microscopy ( HRTEM ) .",
"Scale bar: 10 nm .",
"( D ) Raman spectrum yielded by the black material in the cells .",
"The bifid spectral peaks between 1000 and 2000 cm−1 are the typical Raman signature for CB .",
"Representative hyperspectral image of lung CD1a+ cells from a patient with emphysema ( E–H ) : a reference sample of nanoparticulate carbon black ( nCB ) was used to generate a signature spectral library ( E ) using CytoViva Hyperspectral Imaging System .",
"Each colored spectra represents the spectral profile of a distinct area of the nCB sample , which were used in combination to map nCB present in cells .",
"( F ) Bright field ( BF ) , ( G ) dark field ( DF ) , and ( H ) overlay CB signature spectrum of lung CD1a+ cells .",
"Positive signals were pseudo-colored red to aid visualization .",
"Scale bar: 20 μm .",
"( I ) Raman spectrum yielded in lung CD11c+ and macrophages isolated from lungs of mice exposed to smoke for 4 months; CB reference ( CB Ref ) signal indicates solid CB sample .",
"SMK: 4 months of cigarette smoke .",
"Inset images for cell type correspond to Raman spectra indicating the subcellular localization of CB .",
"The brightness of each 2 µm × 2 µm pixel , representing one spectrum , indicates the height of the graphitic band of CB at 1600 cm−1 compared to the background , such that brighter pixels indicate more CB . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 003 Next , using our established mouse model of cigarette smoke-induced emphysema ( Shan et al . , 2012 , 2014 ) , we exposed mice to 4 cigarettes daily or air for 4 months to examine whether nCB accumulates in lung APCs .",
"When compared to air-exposed mice , we confirmed that isolated CD11c+ APCs and cells present in bronchoalveolar ( BAL ) fluid ( macrophages >90% ) ( Shan et al . , 2012 ) contain particulate matter with the Raman spectral signature of nCB within each cell ( Figure 1I ) .",
"These findings indicate that both humans and mice chronically exposed to cigarette smoke accumulate nCB within phagocytic lung APCs .",
"We have previously recapitulated smoke-induced lung sterile inflammation and emphysema by adoptively transferring lineage-negative CD11c+ mDCs isolated from the lungs of smoke-exposed mice to naive mice , which revealed the direct , causal role of mDCs in emphysema ( Shan et al . , 2012 ) .",
"As we found that these mDCs contained nCB , we sought to determine if nCB was alone sufficient to induce emphysema .",
"We first determined that the commercial nCB does not desorb polycyclic aromatic hydrocarbons ( PAHs ) , as determined by Soxhlet extraction followed by gas chromatography mass spectroscopy ( GCMS ) ( Harwood and Moody , 1989 ) .",
"Mice were then exposed twice weekly for 6 weeks to hydrocarbon free , hydrophobic , neutral surface charged nCB ( average particle size 15 nm ) , to achieve a total lung dose of ∼1% of wet lung weight ( mg/g ) , which approximates human lung nCB burdens ( Figure 2—figure supplement 1 ) .",
"4 weeks after the last intranasal instillation of nCB , harvested lungs were extensively anthracotic , similar in appearance to lungs of long-term smokers ( Figure 2A ) .",
"Physiologically , the nCB challenge-induced enlargement of the alveolar spaces ( Figure 2B ) concomitant with significant increases in total lung volume quantified by micro-CT imaging and unbiased lung morphometry measurement ( mean linear intercept; MLI ) ( Figure 2C , D ) , both hallmarks of emphysema .",
"Mice exposed to nCB showed significantly increased numbers of macrophages , neutrophils , and lymphocytes in BAL fluid as compared to vehicle ( PBS ) -challenged control animals ( Figure 2E ) .",
"Consistently , increased lung inflammation was accompanied by higher concentrations of inflammatory cytokines and chemokines ( Figure 2—figure supplement 2 ) as well as elastolytic matrix metalloproteinases ( MMPs ) 9 and 12 ( Figure 2F ) , all of which are characteristic features of cigarette smoke-induced emphysema in human patients and animal models of this disease ( Shan et al . , 2009; Churg et al . , 2012a ) .",
"Similarly , both lung parenchymal CD11c+ mDCs and BAL fluid macrophages showed an accumulation of nCB as detected by hyperspectral imaging ( Figure 2G–I ) . 10 . 7554/eLife . 09623 . 004Figure 2 . Carbon black-induced emphysema mouse model .",
"( A ) Representative image of fresh lungs harvested from mice exposed to vehicle ( PBS ) or nanoparticulate carbon black ( nCB ) as described in Figure 2—figure supplement 1 .",
"( B ) Representative Hematoxylin and eosin ( H&E ) staining of formalin-fixed lung sections .",
"Scale bar: 100 μm .",
"( C ) Micro-CT quantification of lung volume .",
"( D ) MLI measurement was done on the same groups of mice .",
"( E ) Total and differential cell count in bronchoalveolar ( BAL ) fluid: macrophages ( Mac ) , neutrophils ( Neu ) , and lymphocytes ( Lym ) .",
"Quantitative PCR of Mmp9 and Mmp12 ( F ) gene expression in BAL cells isolated from PBS- or CB-challenged mice .",
"Representative lung CD11c+ cells isolated from mice challenged with nCB under bright field ( BF ) ( G ) , dark field ( H ) , and overlap images ( pseudo-red area ) ( I ) signifying nCB signature spectrum .",
"Scale bar: 20 μm .",
"Data are mean ± SEM and representative of three independent experiments; ***p < 0 . 001 , **p < 0 . 01 as determined by the Student's t-test; n = 5 per group . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 00410 . 7554/eLife . 09623 . 005Figure 2—figure supplement 1 . Schematic representation of nCB-induced lung inflammation and emphysema protocol . Mice were lightly anesthetized with isoflurane and challenged with 50 µl of 107 ng/ml of CB or vehicle ( PBS with 1% sucrose ) twice weekly for 6 weeks; 4 weeks following the last challenge , mice underwent CT scan of chest and were euthanized . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 00510 . 7554/eLife . 09623 . 006Figure 2—figure supplement 2 . nCB induces pro-inflammatory cytokines and chemokines in the lung . Concentration of pro-inflammatory cytokines and chemokines detected via MILLIPLEX Assay ( Millipore , Billerica , MA ) in the lung homogenate collected from mice in each group .",
"n = 5 per group .",
"***p < 0 . 001 , **p < 0 . 01 , *p < 0 . 05 as determined by Student's t-test and data are mean ± SEM and representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 00610 . 7554/eLife . 09623 . 007Figure 2—figure supplement 3 . nCB persists in the lungs 18 months after the last challenge . Representative image of left and right lobes of the lungs harvested from mice 18 months after the last nCB challenge . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 00710 . 7554/eLife . 09623 . 008Figure 2—figure supplement 4 . nCB-induced emphysema persists in the lungs . Micro-CT quantification of lung volume in mice rested for 7 month after the last nCB or PBS challenge .",
"***p < 0 . 001 as determined by the Student's t-test , and data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 00810 . 7554/eLife . 09623 . 009Figure 2—figure supplement 5 . nCB-induced immune cell infiltration persists in the lungs . BAL fluid analysis of the mice rested for 7 month after the last nCB or PBS challenge showing the cell number of macrophages ( Mac ) and neutrophils ( Neu ) .",
"***p < 0 . 001 , **p < 0 . 01 as determined by the Student's t-test , and data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 009 Gross and microscopic examination of the lungs at 7 and 18 months following the last nCB exposure showed persistence of nCB , lung inflammation , and hyperinflation ( Figure 2—figure supplement 3–5 ) .",
"Thus , nCB—as an insoluble byproduct of tobacco combustion as shown above and also delivered through a non-smoking model—accumulates in lung and airway APCs , is poorly cleared from the lungs , and is alone sufficient to cause lung inflammation and emphysema in mice .",
"We previously determined that cigarette smoke induces lung APC activation in human patients and mice , which then induces Th17 cell differentiation in naive T cells ( Shan et al . , 2009 ) .",
"To determine whether nCB specifically induces Th17 responses in vivo , we first examined lung mDCs from nCB intranasal-challenged mice .",
"Lung CD11c+CD11bhi mDCs were significantly increased in the lungs of nCB-challenged mice when compared with controls ( Figure 3A , B ) .",
"nCB also selectively induced lung Th17 but not Th1 responses relative to control animals ( Figure 3C , D and Figure 3—figure supplement 1 ) .",
"Lung CD11c+ APCs isolated from nCB-challenged mice secreted significantly more of the Th17 cell growth factors IL-6 and IL-1β , along with other pro-inflammatory cytokines and chemokines , but not IL-12 or IL-4 ( IL-4 was undetectable in both PBS and nCB groups ) , which promote Th1 and Th2 cell differentiation , respectively ( Figure 3—figure supplement 2 ) .",
"To determine if lung APCs from nCB-challenged mice induce specific T cell differentiation programs in vitro , we co-cultured naive splenic CD4+ T cells with CD11c+ cells isolated from lungs of nCB- or PBS-challenged mice .",
"Lung APCs from nCB-challenged mice induced significantly more IL-17A , but neither IFN-γ nor IL-4 production , when compared to controls ( Figure 3—figure supplement 3 ) .",
"Lung Th17 responses persisted for at least 7 months following the last nCB challenge ( Figure 3—figure supplement 4 ) .",
"Further , Il-17a−/− mice were resistant to nCB challenge as assessed by their attenuated increases in lung volume , lung immune cell infiltration , and the reduced destruction of alveoli ( Figure 3E–H ) when compared to identically treated WT mice .",
"Thus , in vivo nCB selectively induces chronic lung Th17 responses , which are crucial for CB-induced emphysema in mice . 10 . 7554/eLife . 09623 . 010Figure 3 . nCB promotes Th17 responses . Representative staining ( A ) and cumulative analysis ( B ) of the percentage of CD11c+CD11bhigh cells in lung B220− cell subset .",
"Representative intracellular staining ( C ) and cumulative analysis ( D ) of IL-17A+ cells expressing lung CD4+ T cell ( Th17 ) subset .",
"( E ) Micro-CT quantification of lung volume in WT and Il-17a−/− mice .",
"( F ) Lung MLI was determined in the same group of mice .",
"( G ) BAL fluid analysis of the indicated groups of mice showing the total cells including macrophages ( Mac ) , neutrophils ( Neu ) , and lymphocytes ( Lym ) .",
"***p < 0 . 001 , **p < 0 . 01 , *p < 0 . 05 as determined by the one-way ANOVA and Bonferroni's multiple comparison test .",
"N = 4 to 6 per group .",
"Data are mean ± SEM .",
"( H ) Representative H&E staining of formalin-fixed , 5-μm lung sections in indicated groups of mice .",
"Scale bar: 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01010 . 7554/eLife . 09623 . 011Figure 3—figure supplement 1 . nCB did not induce Th1 responses . Cumulative analysis of intracellular cytokine staining of IFNγ in lung CD4+ T cell subsets in PBS or nCB-challenged mice .",
"Data are mean ± SEM and representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01110 . 7554/eLife . 09623 . 012Figure 3—figure supplement 2 . Lung APCs of nCB-challenged mice secrete Th17 cell-specific pro-inflammatory cytokines and chemokines . Concentration of pro-inflammatory cytokines and chemokines detected by Multiplex system in the supernatant of overnight cultured of lung CD11c+ cells isolated from indicated groups .",
"*p < 0 . 05 as determined by the Student's t-test .",
"n = 3 per group .",
"Data are mean ± SEM and representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01210 . 7554/eLife . 09623 . 013Figure 3—figure supplement 3 . Lung APCs of nCB-challenged mice-induced Th17 responses . Concentration of IL-17A , IFN-γ , and IL-4 expressed in the supernatant of lung CD11c+ cells isolated from CB- or PBS-challenged mice and co-cultured with splenic CD4+ T cells in the presence of anti-CD3 ( 1 μg/ml ) .",
"*p < 0 . 05 as determined by the Student's t-test .",
"n = 5 per group .",
"Data are mean ± SEM and representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01310 . 7554/eLife . 09623 . 014Figure 3—figure supplement 4 . nCB-induced Th17 responses persist in the lungs . Cumulative analysis of intracellular cytokine staining of IL-17A in lung CD4+ T cell subsets in the mice rested for 7 month after the last nCB or PBS .",
"***p < 0 . 001 as determined by the Student's t-test , and data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01410 . 7554/eLife . 09623 . 015Figure 3—figure supplement 5 . Direct effect of nCB on T helper cell differentiation in vitro .",
"( A ) Flow cytometric analysis of intracellular cytokine staining of IFN-γ ( Th1 ) , IL-17A ( Th17 ) , and Foxp3/CD25 surface expression ( Tregs ) .",
"Diff .",
"is the differentiation conditions for Th1 , Th17 , and Tregs ( as described in the methods ) .",
"Y-axis of both Th1 and Th17 panel is empty channel .",
"( B ) Cumulative summary of four independent experiments for Treg differentiation .",
"**p < 0 . 01 as determined by the one-way ANOVA and Bonferroni's multiple comparison test .",
"Data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 015 We next explored whether nCB plays a direct role ( i . e . , independent of APCs ) on T helper cell differentiation .",
"To address this question , we polarized T cells toward Th1 , Th17 , and regulatory ( Treg ) phenotypes in the presence or absence of nCB in vitro .",
"We found that nCB did not affect Th1 or Th17 cell differentiation directly ( Figure 3—figure supplement 5A ) .",
"However , nCB treatment significantly inhibited Treg differentiation ( Figure 3—figure supplement 5A , B ) .",
"These findings indicate that nCB promotes sterile inflammation by inducing Th17 differentiation indirectly through APCs and directly by inhibiting Treg differentiation .",
"The previous findings demonstrated that when deposited in the lungs , nCB activates mDCs and induces durable Th17-dependent inflammation and emphysema in mice .",
"To determine the mechanism of nCB-mediated lung pathology , we next investigated whether its physicochemical properties could account for its immunostimulatory function .",
"Whether manufactured or found in the lungs of smokers with emphysema , nCB is very hydrophobic and completely insoluble in aqueous media .",
"Conjugating polyethylene glycol to nCB ( PEG-nCB ) renders the material hydrophilic and miscible with aqueous solutions ( Hwang et al . , 2014 ) .",
"Mice challenged with intranasal PEG-nCB using the same protocol ( Figure 2—figure supplement 1 ) failed to develop emphysema as assessed by quantitative CT-based lung volume measurements , MLI and microscopic evaluation of the lungs ( Figure 4A , B , C ) .",
"Further , we detected less anthracotic pigment in the lung parenchyma , suggesting that in contrast to hydrophobic nCB , PEG-nCB could be cleared from the lungs ( Figure 4C ) .",
"Microscopic inspection of isolated BAL fluid cells from PEG-nCB-challenged mice showed intact phagocytic cells compared to that of hydrophobic nCB , suggesting that the latter may induce less cytotoxic effects on phagocytic cells ( Figure 4—figure supplement 1 ) .",
"In support of this , we found that the release of lactate dehydrogenase ( LDH ) , an indicator of cytotoxicity , was enhanced in macrophage-like RAW 264 . 7 cells exposed to nCB as compared to PEG-nCB ( Figure 4—figure supplement 2 ) . 10 . 7554/eLife . 09623 . 016Figure 4 . Hydrophobicity of nCB is important for its pathogenesis . Micro-CT quantification of lung volume ( A ) and MLI measurement of lung morphometry ( B ) in vehicle ( PBS ) , nCB , and PEG-nCB treated mice .",
"( C ) Representative H&E staining of lung sections Scale bar: 100 μm .",
"( D ) Total and differential cell count in bronchoalveolar ( BAL ) fluid; macrophages ( Mac ) , neutrophils ( Neu ) , and lymphocytes ( Lym ) .",
"Quantitative PCR of Mmp9 ( E ) and Mmp12 ( F ) gene expression in BAL cells isolated from the above group of mice .",
"Lung homogenate collected from indicated groups of mice were measured for IL-6 ( G ) and IL-1β ( H ) by ELISA .",
"Representative intracellular staining ( I ) or cumulative analysis ( J ) of Th17 cells in the lungs .",
"***p < 0 . 001 , **p < 0 . 01 , *p < 0 . 05 as determined by the one-way ANOVA and Bonferroni's multiple comparison test .",
"n = 4 to 6 per group , and data are mean ± SEM and representative of two independent studies . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01610 . 7554/eLife . 09623 . 017Figure 4—figure supplement 1 . nCB-induced cell damage compared with PEG-nCB . Representative image of H&E stained cytospin preparation of BAL cells isolated from indicated groups of mice .",
"Scale bar: 50 μm .",
"Data are representative of two independent studies . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01710 . 7554/eLife . 09623 . 018Figure 4—figure supplement 2 . nCB-induced cell death compared with PEG-nCB . Lactate dehydrogenase ( LDH ) release from RAW 264 . 7 cells after 24 hr of the indicated treatment .",
"Maximum LDH release was the amount of LDH released from lysed cells .",
"***p < 0 . 001 as determined by the one-way ANOVA and Bonferroni's multiple comparison test .",
"n = 5 per group .",
"Data are mean ± SEM and representative of two independent studies . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 01810 . 7554/eLife . 09623 . 019Figure 4—figure supplement 3 . nCB-induced strong lung inflammation compared with PEG-nCB . Multiplex analysis of pro-inflammatory cytokines and chemokines in lung homogenate of indicated groups .",
"***p < 0 . 001 , **p < 0 . 01 , *p < 0 . 1 as determined by the one-way ANOVA and Bonferroni's multiple comparison test .",
"n = 5 per group .",
"Data are mean ± SEM and representative of two independent studies . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 019 Consistent with the failure to induce emphysema and cell death , exposure to PEG-nCB also resulted in attenuated recruitment of macrophages , neutrophils , and lymphocytes to the lung when compared with hydrophobic nCB ( Figure 4D ) .",
"This reduction in inflammation was accompanied by reduced expression of Mmp9 and Mmp12 transcripts in BAL fluid cells as compared with nCB-challenged mice ( Figure 4E , F ) .",
"The markedly reduced inflammatory nature of PEG-nCB was further underscored by the reduced concentrations of pro-inflammatory cytokines and chemokines detected from freshly collected lung homogenates of PEG-nCB-challenged mice ( Figure 4—figure supplement 3 ) , including decreased IL-6 and IL-1β levels ( Figure 4G , H ) .",
"Critically , PEG-nCB failed to induce lung Th17 cells when compared to nCB-exposed animals ( Figure 4I , J ) .",
"Thus , the pro-inflammatory potential of nCB is intimately tied to its hydrophobic surface and ability to induce cytotoxicity of phagocytic cells .",
"We conducted additional studies to determine how nCB activates APCs to secrete pro-inflammatory cytokines ( e . g . , IL-6 and IL-1β ) and chemokines .",
"In response to nCB , but not PEG-nCB , reverse phase protein array ( RPPA ) identified the activation of several DNA damage ( e . g . , PARP , p-Chk2 , p-ATM ) and MAPK/Erk ( p-ERK , p-MEK1/2 ) -response proteins ( Figure 5A and Figure 5—figure supplement 1 ) .",
"Consistent with these data , we found that nCB , but not PEG-nCB , induced DNA double strand breaks ( DSB ) as determined by phosphorylation of Histone 2AX ( H2AX ) on serine 129 ( γH2AX ) ( Figure 5B , C ) .",
"Further , the induction of DSB was inversely dependent on the size of nCB as we observed progressively fewer DSB with increasing nCB size ( Figure 5—figure supplement 2 ) .",
"We next examined whether CB-induced DSB could account for the pro-inflammatory responses seen in APC .",
"Human monocyte-derived dendritic cells ( MDDCs ) treated with Nu7026 , an inhibitor of the DNA-dependent protein kinase catalytic subunit ( Wilmore et al . , 2004; Zhou et al . , 2014 ) , exhibited reduced IL-6 production in a dose-dependent manner in response to nCB but not LPS ( Figure 5D ) .",
"Moreover , in nCB-exposed RAW 264 . 7 cells , transfection of a specific siRNA against ataxia telangiectasia mutated ( ATM ) —a serine–threonine kinase that coordinates repair of double-stranded DNA breaks ( Guo et al . , 2010 ) —significantly reduced expression of IL-6 and TNFα , two inflammatory cytokines that are induced through ATM ( Figure 5—figure supplement 3 ) . 10 . 7554/eLife . 09623 . 020Figure 5 . nCB activates APCs by the induction of DNA damage and Erk signaling .",
"( A ) Heat map ( reverse phase protein array ) of protein expression and phosphorylation level in RAW 264 . 7 cells stimulated with vehicle ( PBS ) , nCB ( 105 ng/ml ) , and PEG-CB ( 105 ng/ml ) .",
"p: phosphorylated .",
"Blue is relatively low ( −0 . 5 ) and yellow high ( 0 . 5 ) based on log2 ratio of the value for expression level .",
"( B ) RAW 264 . 7 cells under indicated conditions immunostained for nuclear DNA ( DAPI , blue ) and anti-γH2AX ( green ) to detect double strand break ( DSB ) .",
"Scale bar: 50 μm .",
"( C ) Quantitative summary of panel B indicating the percentage γH2AX positive RAW cells in indicated groups .",
"( D ) IL-6 concentration detected by ELISA after 48 hr in the supernatant of MDDC treated with CB or LPS in the presence of increasing dose of Nu7026 or vehicle ( DMSO ) .",
"( E ) IL-17A concentration detected by ELISA after 72 hr co-culture of splenic CD4 T cells and lung CD11c+ cell isolated from the mice after challenged with PBS or nCB and anti-CD3 ( 1 μg/ml ) in the presence of Nu7026 ( 100 nM ) , Ku55933 ( 100 nM ) , or vehicle control ( DMSO ) .",
"( F ) Western blot of protein extracted from BMDC treated with different concentration of nCB targeting phosphorylated-Erk .",
"Data are representative of two independent experiments .",
"( G ) IL-6 concentration detected by ELISA in the supernatant of MDDC treated with nCB in the presence of increasing dose of U0126 ( MEK1/2 inhibitor ) for 48 hr . n = 4 to 7 per group and data are mean ± SEM and representative of two independent experiments ( C , D , E , G ) .",
"***p < 0 . 001 , **p < 0 . 01 as determined by the one-way ANOVA and Bonferroni's multiple comparison test . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 02010 . 7554/eLife . 09623 . 021Figure 5—figure supplement 1 . Heat map depicting molecules whose expression and phosphorylation level differed when RAW 264 . 7 cells were treated with nCB compared with PBS or PEG-nCB treated groups detected by reverse phase protein array . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 02110 . 7554/eLife . 09623 . 022Figure 5—figure supplement 2 . Larger nCB size correlates with weak induction of DNA double strand breaks ( DSB ) .",
"( A ) RAW 264 . 7 cells were treated with vehicle ( PBS ) , nCB ( 105 ng/ml ) with different size in diameter for overnight and immunostained for nuclear ( DAPI ) ( blue ) and anti-γH2AX ( green ) to detect double strand break ( DSB ) .",
"Scale bar: 100 μm .",
"( B ) Quantitative summary of panel A indicating the percentage γH2AX-positive RAW cells in indicated groups .",
"***p < 0 . 001 as determined by the one-way ANOVA and Bonferroni's multiple comparison test .",
"n = 3 fields per group .",
"Data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 02210 . 7554/eLife . 09623 . 023Figure 5—figure supplement 3 . ATM is required for nCB-induced inflammatory factor upregulation in RAW cells .",
"( A ) Atm mRNA expression in RAW 264 . 7 cells transfected with scramble or Atm siRNA .",
"( B ) Il6 and Tnf-α mRNA expression in RAW cells transfected with scramble or Atm siRNA followed by overnight treatment with nCB or vehicle ( PBS ) .",
"***p < 0 . 001 , **p < 0 . 01 , *p < 0 . 1 as determined by One-way ANOVA and Bonferroni's multiple comparison test .",
"n = 4 per group .",
"Data are mean ± SEM and representative of two independent studies . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 02310 . 7554/eLife . 09623 . 024Figure 5—figure supplement 4 . Inhibition of DNA damage does not affect Th1 or Th2 responses . IFN-γ ( A ) and IL-4 ( B ) concentrations were measured using ELISA in the supernatant of anti-CD3 ( 1 μg/ml ) treated CD4+ T cells co-cultured with lung CD11c+ cell isolated from the mice challenged with PBS or nCB in the presence of vehicle ( DMSO ) or inhibitors of DNA damage ( Nu7026 or Ku55933 at 100 nM ) .",
"n = 5 per group , and data are mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 024 To further examine whether induction of Th17 responses is dependent on nCB-mediated DNA damage , CD11c+ lung mDCs isolated from nCB-challenged mice were co-cultured with splenic CD4 T cells in the presence of either Nu7026 or Ku55933 , an inhibitor of ATM ( Li and Yang , 2010 ) , for 3 days .",
"As expected , mDCs isolated from nCB-challenged mice promoted Th17 cell differentiation , which was significantly reduced in response to Nu7026 or Ku55933 ( Figure 5E ) while Th1 and Th2 cell differentiation remained unchanged ( Figure 5—figure supplement 4 ) .",
"Together , these findings suggest that nCB-mediated DNA damage is required for the induction of pro-inflammatory cytokines in mDCs and Th17 cell differentiation .",
"Moreover , nCB exposure in a dose- and time-dependent way increased phosphorylation of Erk ( Figure 5F ) , and similar inhibition of MEK1/2 with U0126 , an inhibitor of MAP kinases ( Newton et al . , 2000 ) , reduced IL-6 production in response to nCB exposure ( Figure 5G ) .",
"Together , these findings indicate that hydrophobic nCB activates DNA damage responses and induces MAPK/Erk signaling coincident with the induction of Th17 responses .",
"The inflammasome detects danger signals released in response to cell injury and sterile inflammation and the adaptor protein ASC ( apoptosis-associated speck-like protein containing CARD ) was shown to be required for inflammasome-dependent caspase-1–mediated conversion of pro-IL-1β to mature IL-1β ( Kono et al . , 2012 ) .",
"In response to nCB exposure , lung CD11c+ mDCs increased IL-6 and IL-1β expression and RAW 264 . 7 cells released more LDH , consistent with the concept that nCB induces both sterile inflammation and necrotic cell death .",
"To determine if ASC is also required for nCB-induced Th17 responses and emphysema , Pycard−/− mice were challenged intranasally with nCB .",
"When compared to WT mice treated identically , Pycard−/− mice showed attenuated emphysema ( Figure 6A–C ) and reduced macrophage , neutrophil , lymphocyte , and mDC infiltration into the lungs ( Figure 6D , E ) .",
"Consistently , lung mDCs of Pycard−/− mice produced less IL-6 and IL-1β and poorly activated splenic T cells to differentiate into Th17 cells when compared with WT mDC ( Figure 6F–H ) .",
"Freshly collected lung homogenates from Pycard−/− mice challenged with nCB also showed reduced inflammatory chemokine production compared with WT mice ( Figure 6—figure supplement 1 ) .",
"Thus , the earliest immunological events induced by nCB include ASC activation and inflammasome assembly , which are in turn required for nCB-mediated Th17 responses and emphysema . 10 . 7554/eLife . 09623 . 025Figure 6 . ASC-mediated inflammasome pathway is required for nCB-induced Th17 responses and emphysema .",
"( A ) Representative H&E staining of lung sections from WT and Pycard−/− mice exposed to nCB or vehicle ( PBS ) as described in Figure 2—figure supplement 1 .",
"Scale bar: 100 μm .",
"( B ) Micro-CT quantification of lung volume in indicated groups of mice .",
"( C ) Lung MLI measurement in the same group of mice .",
"( D ) Total and differential cell count in bronchoalveolar ( BAL ) fluid: macrophages ( Mac ) , neutrophils ( Neu ) , and lymphocytes ( Lym ) .",
"( E ) Relative abundance of lung mDCs ( CD11c+CD11bhigh ) isolated from whole lung tissue in the same group of mice .",
"IL-6 ( F ) and IL-1β ( G ) concentrations detected by ELISA in the supernatant of lung CD11c+ cells isolated from indicated group of mice after overnight culture .",
"( H ) IL-17A concentration detected by ELISA in the supernatant of splenic CD4+ T cells co-cultured with lung CD11c+ cells isolated from indicated group of mice for 3 days in the presence of anti-CD3 ( 1 μg/ml ) .",
"***p < 0 . 001 , **p < 0 . 01 , *p < 0 . 05 as determined by the one-way ANOVA and Bonferroni's multiple comparison test; n = 3 to 7 per group , and data are mean ± SEM and representative of two independent studies . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 02510 . 7554/eLife . 09623 . 026Figure 6—figure supplement 1 . Pycard−/− mice produce less pro-inflammatory chemokines in the lungs in response to nCB challenge .",
"( A ) Multiplex detection of indicated chemokines in freshly harvested lung homogenate from different groups of mice .",
"***p < 0 . 001 , *p < 0 . 05 as determined by the one-way ANOVA and Bonferroni's multiple comparison test .",
"n = 5 per group .",
"Data are mean ± SEM and representative of two independent studies . DOI: http://dx . doi . org/10 . 7554/eLife . 09623 . 026"
],
[
"Evidence from experimental systems and human translational studies strongly support a role for chronic inflammation—and Th17 cells in particular—in the initiation and progression of emphysema in smokers ( Shan et al . , 2012; Eppert et al . , 2013; Kurimoto et al . , 2013 ) .",
"A characteristic feature of the anthracotic pigment of smokers' lungs is that such discoloration persists even long after smoking has ceased ( Churg et al . , 2005 ) .",
"In this study , we addressed the role of insoluble anthracotic pigment that is universally found in the lungs of smokers with emphysema in driving this pathological response .",
"Although several chemical identities have been proposed , our study is the first to clearly identify the anthracotic pigment as nCB and show that it accumulates specifically in human lung phagocytic cells .",
"Our functional studies are also the first to show that nCB administered to the lungs in pathophysiologically relevant amounts can induce sterile inflammation and emphysema that is indistinguishable from disease induced by exposure to cigarette smoke in mice .",
"Thus , nCB is likely the major component of smoke that causes long-term lung toxicity .",
"Furthermore , our findings have major implications regarding the safety of activities involving the chronic inhalation of smoke and the need to control the particulate composition of air .",
"Since nCB is used extensively in the rubber , plastics , and composites industries , the exposure levels should also be controlled in the workplace .",
"Our findings also elucidate both the nature of and the mechanism by which inflammation in the lungs of heavy cigarette smokers is perpetuated even long after cessation of cigarette smoking .",
"Both chronic exposure to cigarette smoke and inhalation of nCB mediate similar inflammatory responses that are characterized by the activation of lung mDCs , differentiation and accumulation of Th17 cells , and lung parenchymal destruction ( emphysema ) ( Shan et al . , 2014 ) .",
"In part , this sterile inflammatory response to nCB is due to activation of the inflammasome pathway .",
"Specifically , we found that inhaled nCB induces the production of IL-1β and IL-6 , two pro-inflammatory cytokines that are required for mDC-mediated differentiation of Th17 cells and emphysema development .",
"This inflammation persists and lung damage continues to accumulate even after smoking cessation due to the insoluble nature of nCB .",
"Although readily taken up by phagocytic lung cells that could theoretically be expectorated or migrate out of the lungs via the lymphatics ( Corry et al . , 1984 ) , these cells most likely undergo cell death too rapidly in response to nCB ingestion for any of these potential clearance mechanisms to operate efficiently .",
"The nCB is then released in the lung by the cells it kills , only to be taken up again and kill subsequent phagocytes .",
"nCB thus establishes an unending cycle of cell death that , if sufficiently pronounced , will trigger activation of the inflammasome pathway in response to the release of danger-associated molecular patterns ( DAMPs ) from dying cells ( Piccinini and Midwood , 2010 ) .",
"Immune responses both rapidly kill invading pathogens and solubilize antigenic and adjuvant-like pathogen-derived substances to facilitate their removal and thus terminate the potentially deleterious inflammation .",
"Both of these fundamental immune functions are thwarted in the context of nCB accumulation , leading to a perpetual cycle of lung inflammation and damage .",
"Our findings , therefore , raise concerns that other insoluble environmental nanoparticles may , if inhaled , accumulate in lung phagocytic cells and induce similar pathology .",
"In support of this , inflammasome-activated IL-1β has been shown to play a major role in lung sterile inflammation induced by other nanoparticles associated with lung diseases ( Merget et al . , 2002 ) .",
"ASC is required for the assembly of pro-caspase 1 in order to yield caspase 1 for the activation of pro-IL-1β to IL-1β ( Franchi and Nunez , 2012 ) .",
"We show that inhaled nCB can activate the inflammasome pathway that results in production of mature IL-1β .",
"In addition , inflammasome sensors activated by nCB-damaged cells require ASC activation because lung mDCs isolated from Pycard−/− mice failed to increase IL-1β and showed attenuated Th17 responses .",
"A critically important physical feature of nCB , accounting in large part for its pro-inflammatory potential , is its hydrophobic character .",
"Exposure to large quantities of hydrophobic nCB has been shown to induce cell injury , pyroptosis and generate reactive oxygen species ( ROS ) in cultured cells ( Reisetter et al . , 2011 ) .",
"One particular characteristic of nCB that correlates with its toxicity is its large surface area; larger forms of elemental carbon have much less potential to induce cell injury ( Oberdörster et al . , 2005 ) and , as we have shown here , damage DNA .",
"A single burning cigarette can generate approximately 1012 particles that vary in size from 1 micron to a few nanometers in diameter ( Sahu et al . , 2013 ) .",
"The deposition site of particulate matter in the lungs of smokers is governed largely by size , with larger particles depositing in the mouth and upper airway while smaller particles are deposited in progressively smaller and more distal airways ( Adam et al . , 2006; Baker and Dixon , 2006 ) .",
"For our studies , we used nCB spheroids with a nominal size of 15 nm that aggregate in clusters of 3–4 , forming 50–75 nm per particle .",
"However , in aqueous solution , this material forms macro-aggregates that fail to distribute evenly in the lung after intranasal challenge as does nCB delivered by smoke inhalation .",
"We were partially successful in alleviating this confounding factor by adding sucrose to the nCB in aqueous solution .",
"Nonetheless , although we endeavored to deliver nCB to mice in amounts that matched actual burdens found in human lung , it is likely that we did not fully recapitulate the in vivo particle size and distribution of nCB acquired through smoke inhalation .",
"Further studies are required to define how nCB size affects in vivo toxicity as defined in these studies .",
"Thermal and chemical analyses have shown that a combustion heat of 350–550°C yields black carbon ( BC ) that contains PAHs that are linked to inflammation ( Bleck et al . , 2006 ) , but combustion at much higher temperatures ( 650–1100°C ) produces PAH-free CB ( Watson et al . , 2005 ) ; we used this material heated to higher temperatures for intranasal administration in mice .",
"A critical question related to our studies is , therefore , which form of carbon , BC or CB , is deposited in the lung during smoking , and how much do PAHs contribute to smoking-related lung inflammation .",
"The nCB used in our studies lacked detectable PAHs by gas chromatography mass spectrometry analysis , which has a sensitivity limit of 1 part in 1010 by mass .",
"While PAHs are present in soot and diesel exhaust particles ( Garza et al . , 2008 ) , neither Raman spectroscopy nor hyperspectral imaging can distinguish CB from BC as could be found in human lungs .",
"However , X-ray analysis of the melting of small metal particles has revealed the temperature distribution inside a burning cigarette as 850–920°C during active inhalation , decreasing to 700°C during the smoldering phase; this temperature regime is consistent with the creation of predominant CB during smoking ( Baker , 1974 ) .",
"Moreover , as PAH-free CB recapitulates almost entirely the pathology induced by smoke exposure , we conclude that sterile inflammation and pulmonary emphysema are primarily the result of CB accumulation in the lung and not that of BC or PAHs therein during smoking .",
"Among signaling pathways that are important for APC activation and inflammation , we found that phosphorylation of Erk was significantly upregulated in cells exposed to CB treatment in a dose- and time-dependent manner while phosphorylation of p38 or JNK were not changed .",
"The most notable , and unexpected , result of our RPPA analysis was the upregulation of several DNA damage enzymes by nCB exposure .",
"This led us to confirm that nano-sized CB induces DSB in DNA as detected by the expression of γH2AX , an ATM-regulated pathway .",
"We show that larger forms of CB result in attenuation of DSB , indicating that the size of CB is an important factor in its genotoxicity .",
"Activation of this DNA repair pathway is in turn linked to the production of pro-inflammatory cytokines such as IL-6 and IL-1β that are required for Th17 differentiation .",
"In addition , microRNA-22 ( miR-22 ) has been shown to be upregulated in lung APC of mice exposed to smoke or nCB and is critical in Th17 responses through activation of AP-1 complexes and histone deacetylase ( HDAC ) 4 ( Lu et al . , 2015 ) .",
"Thus , together with DAMPs released by cells killed by nCB , nCB-induced DNA damage accounts for much of the inflammatory nature of nCB .",
"The mechanism by which nCB cleaves DNA and the additional biological consequences of this adverse property remain active areas of investigation in our laboratory .",
"In summary , our findings show that inhalation of cigarette smoke leads to the accumulation of nCB in airway APCs ( macrophages and mDCs ) .",
"This insoluble material promotes perpetual Th17 cell-mediated lung inflammation in part through the double-stranded cleavage of nuclear DNA .",
"These findings largely explain the persistent and incurable nature of smoking-related lung disease .",
"Because no medical means of removing accumulated lung nCB exists , our findings underscore the need for all individuals and societies to minimize the production of and exposure to smoke-related particulate air pollution and industrial nCB ."
],
[
"C57BL/6J mice were purchased from the Jackson Laboratory ( Bar Harbor , ME ) .",
"Pycard−/− mice ( C57BL/6 background; Pycard encodes for Asc protein [Mariathasan et al . , 2004] ) were obtained from Dr Vishva Dixit ( Genentech , South San Francisco , CA ) .",
"Il-17a−/− mice ( C57BL/6 background ) were obtained from Dr Chen Dong ( The University of Texas MD Anderson Cancer Center , Houston , TX ) .",
"All mice were bred in the transgenic animal facility at Baylor College of Medicine .",
"All experimental protocols ( AN-4589 ) used in this study were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and followed the National Research Council Guide for the Care and Use of Laboratory Animals .",
"MEK1/2 inhibitor U0126 was purchased from Cell Signaling ( Danvers , MA ) .",
"DNA-PKc inhibitor Nu7026 was purchased from Tocris ( Bristol , UK ) .",
"ATM inhibitor Ku55933 was purchased from Millipore ( Billerica , MA ) .",
"Pierce LDH Cytotoxicity Assay Kit was purchased from Life Technologies ( Grand Island , NY ) , and LDH release was measured according to the manufacturer's instructions .",
"Various sizes of carbon black nanoparticles ( nCB ) were obtained from Cabot Corporation ( 15 nm , Monarch 1100 , Lot 1278105; 35 nm , Vulcan 9A32 , Lot: CS-5822; 70 nm diameter , Sterling NS1 , Lot 761510 , CAS# for CB: 1333-86-4; Alpharetta , GA ) .",
"The nCB , although listed to be 15 nm , is more precisely described by the manufacturer to have 15-nm CB particles that are arranged in clusters of 3–5 particles , much as grape clusters , so the actual size is ∼50–75 nm diameter clusters .",
"Conjugation of polyethylene glycol to nCB ( PEG-nCB ) was performed as described ( Zhou et al . , 2014 ) .",
"Briefly , 15-nm nCB ( 250 mmol ) was dispersed in tetrahydrofuran ( THF ) using bath sonication for 3 hr .",
"Then , 4 , 4′-azobis ( 4-cyanopentanoic acid ) ( ACPA ) was added to the nCB dispersion in a three-step process .",
"The first portion of ACPA ( 7 mmol ) was added and stirred continuously for 24 hr at 70°C .",
"This addition was repeated at 24 hr and 48 hr .",
"The mixture was cooled to room temperature , filtered through a 45-µm pore Teflon membrane , washed with THF , ethanol and acetone for three times , and vacuum dried ( 60°C , 100 Torr ) , producing carboxyl-functionalized nCB .",
"Carboxyl-functionalized nCB ( 2 mmol ) was dispersed in dimethylformamide for 30 min , then mixed with N , N′-dicyclohexylcarbodiimide ( 0 . 8 mmol ) , mPEG-NH2 ( 0 . 04 mmol ) , and dimethylaminopyridine ( 2 flakes ) .",
"The reaction was stirred for 24 hr , transferred to a dialysis bag ( molecular weight cut-off , 5 . 0 kDa ) , dialyzed in running deionized water for 1 week , and filtered through a 0 . 22-µm pore Teflon membrane .",
"X-ray photoelectron spectroscopy performed on an a PHI Quantera SXM scanning X-ray microprobe with 26 eV passing energy , 45° takeoff angle , and a 100-μm beam size .",
"Thermogravimetric analysis performed on a TA Instruments Q-600 Simultaneous TGA/DSC .",
"FTIR spectra were recorded using a Nicolet FTIR with an ATR attachment .",
"By TGA , PEG-nCB contains 42–50% PEG by mass .",
"By X-ray photoelectron spectroscopy , the PEG-nCB surface is 15% carbon , 56% oxidized carbon , 1 . 2% nitrogen , and 28% oxygen .",
"FTIR: 840 ( m ) , 960 ( m ) , 1060 ( w ) , 1090 ( vs ) , 1150 ( m ) , 1240 ( m ) , 1280 ( m ) , 1340 ( m ) , 1360 ( m ) , 1470 ( m ) , 1570 ( w ) , 1620 ( w ) , 2880 ( s ) cm−1 .",
"We suspended 20 mg of endotoxin-free nCB or 47 mg PEG-nCB ( containing 20 mg of nCB by weight ) in pre-warmed 1 ml tert-butyl alcohol with 1% sucrose .",
"The mixture was then frozen at −80°C for 24 hr and was placed on a vacuum pump and lyophilized until dry for 24 hr . nCB or PEG-nCB mixed with 1% sucrose were rehydrated in sterile PBS to achieve the concentration of 0 . 5 mg/50 µl , vortexed and sonicated in a water bath for 10 min before administration .",
"Mice were deeply anesthetized with isoflurane , and 50 µl droplets containing 1% sucrose in PBS ( vehicle ) or nCB or PEG-nCB ( mixed with 1% sucrose in PBS ) were applied to the nares .",
"Mice were challenged twice per week for 6 weeks and were sacrificed 1 month after the last challenge .",
"The severity of lung parenchymal destruction ( emphysema ) was determined by computed tomography ( CT ) methods ( Shan et al . , 2012 ) .",
"Briefly , mice were anesthetized with etomidate ( 30 mg/kg ) and placed in an animal CT scanner ( Gamma Medica , Salem , NH ) , and completed images of the chest were obtained by the Animal Phenotyping Core in Baylor College of Medicine .",
"Amira 3 . 1 . 1 software ( FEI , Hillsboro , OR ) was used to process the images and quantification of emphysema in three dimensions .",
"The MLI used for measurement of mouse lung destruction ( e . g . , lung morphometry ) was calculated as previously described ( Shan et al . , 2014 ) .",
"Briefly , an unbiased observer randomly selected ten fields from the left lobe ( large airways and vessels were excluded ) .",
"Paralleled lines were placed on serial lung sections and MLI was calculated by multiplying the length and the number of lines per field , divided by the number of intercepts .",
"BALF and lung tissue were collected as previously described ( Goswami et al . , 2009 ) .",
"Briefly , mice were anesthetized with etomidate and BALF was collected by instilling and withdrawing 0 . 8 ml of sterile PBS twice through the trachea .",
"Total and differential cell counts in the BALF were determined with the standard hemocytometer and HEMA3 staining ( Biochemical Sciences Inc , Swedesboro , NJ ) using 200 µl of BALF for cytospin slide preparation .",
"Mouse lungs were dissected to prepare single-cell suspensions; alternatively , lungs were fixed with instillation of 4% paraformaldehyde solution via a tracheal cannula at 25-cm H2O pressure followed by paraffin embedding and were sectioned for histopathological studies .",
"Hematoxylin and eosin ( H&E ) staining was performed as described ( Goswami et al . , 2009 ) .",
"Mouse lung RBC-free single-cell suspension were stimulated with phorbol 12-myristate 13-acetate ( PMA , 10 ng/ml; Sigma–Aldrich , St . Louis , MO ) and ionomycin ( 1 µg/ml; Sigma–Aldrich ) for overnight supplemented with brefeldin A ( 10 µg/ml; Sigma–Aldrich ) for the last 6 hr .",
"Cells were stained for surface markers with anti-CD3 , anti-CD4 , anti-CD8 , and anti-γδTCR antibodies and then fixed with FACS lysing solution ( BD BioSciences , San Jose , CA ) , permeabilized with 0 . 5% saponin ( Sigma–Aldrich ) , and stained with anti-IFNγ and anti-IL-17A antibodies for analysis of intracellular cytokine production by flow cytometry .",
"Mouse lung or spleen single-cell suspensions were prepared by mincing whole organs through a 40-μm cell strainer ( BD Falcon , San Jose , CA ) followed by red blood cell ( RBC ) lysis ( ACK lysis buffer , Sigma–Aldrich ) for 3 min .",
"For isolation of lung APCs , RBC-free whole lung cells were labeled with anti-CD11c-conjugated magnetic beads ( Miltenyi Biotec , San Diego , CA ) and then isolated by autoMACS ( Miltenyi Biotec ) .",
"For isolation of spleen CD4 T cells , RBC-free whole splenocytes were labeled with anti-CD4 conjugated magnetic beads ( Miltenyi Biotec ) and then isolated by autoMACS .",
"Mouse BMDCs were prepared as previously described with some modification ( Lutz et al . , 1999 ) Femurs and tibias of 4- to 8-week-old female were isolated and freed from the surrounding tissue .",
"Intact bones were kept in 70% ethanol for 3 min followed by a PBS wash .",
"Both ends of the bones were cut with scissors , and the marrow was flushed out with RPMI-1640 medium through a syringe with 26 . 5 needle .",
"RBCs were then removed by ACK lysis buffer and cell debris or tissue clusters were filtered out .",
"Cells from bone marrow were cultured in a 6-well plate with 20 ng/ml mouse GM-CSF and 10 ng/ml mouse IL-4 ( R&D Systems , Minneapolis , MN ) for 5 to 6 days .",
"Human lung single cell suspensions were prepared as previously described ( Shan et al . , 2009 ) .",
"Briefly , fresh lung tissue was cut into 0 . 1-cm pieces in Petri dishes and treated with 2 mg/ml of collagenase D ( Roche Pharmaceuticals , Basel , Switzerland ) in HBSS and incubated for 30 to 40 min at 37°C .",
"Single cells were collected by mincing the digested lung tissue through a 40-μm cell strainer ( BD Falcon ) followed by RBC lysis .",
"Lung CD1a+ DCs were isolated by labeling RBC-free lung cells with anti-CD1a-conjugated magnetic beads ( Miltenyi Biotec ) and then isolated by autoMACS .",
"PBMCs were isolated by Ficoll–Paque ( GE Healthcare Life Sciences , Pittsburgh , PA ) density gradient centrifugation .",
"Human MDDCs were prepared as previously described ( Shan et al . , 2009 ) .",
"Briefly , RBC-free PBMCs were seeded in 6-well plates for 2 hr at 37°C and then nonadherent cells were removed by washing with PBS .",
"Adherent cells were cultured with 50 ng/ml human GM-CSF and 10 ng/ml human IL-4 for 5 to 6 days .",
"CD11c+ cells isolated from mouse lung , BMDCs , monocyte-derived ( MD ) DCs or RAW 264 . 7 cells ( mouse leukemic monocyte/macrophage cell line ) ( ATCC , Manassas , VA ) were treated with indicated amount of nCB for 1 or 2 days , were washed and placed in co-culture assays with or without T cells ( at 1:10 ratio ) .",
"Mouse APCs were co-cultured with congenic splenic CD4+ T cells ( 1:10 ratio ) in the presence of anti-mouse CD3 ( 1 μg/ml; BD Biosciences ) for 3 days .",
"ELISA ( BD BioSciences ) or Multiplex kit ( Millipore ) were used for the measurement of concentration of IL-17A , IFNγ , IL-4 , IL-6 , IL-1β , IL-1α , IL-12p70 , TNFα , MIP-1α , MIP-1β , KC , RANTES , MCP-1 , IP-10 in either lung homogenate or supernatant collected from cultured cells .",
"Mouse ATM siRNA and scramble siRNA were purchased from Sigma–Aldrich .",
"Mouse RAW 264 . 7 cells were transfected with Nucleofection kit ( Lonza , Basel , Switzerland ) according to the manufacturer's instructions .",
"RAW cells treated with siRNA were incubated for 6 hr before CB treatment for overnight .",
"Flow cytometry was performed with a BD LSR II ( BD BioSciences ) , and data were analyzed with FlowJo software ( Tree Star Inc . , Ashland , OR ) .",
"The following anti-mouse antibodies were purchased from BD Pharmingen and used: Pacific Blue-CD3 ( 500A2 ) , PE-Cy5-CD4 ( RM4-5 ) , APC-Cy7-CD8 ( 53-6 . 7 ) , PE-IL-17A ( TC11-18H10 ) and APC-IFNγ ( XMG1 . 2 ) .",
"FITC-γδTCR ( eBioGL3 ) , eFluro450-B220 ( RA3-6B2 ) , PE-CD11b ( M1/70 ) , and APC-CD11c ( N418 ) were purchased from eBioscience ( San Diego , CA ) and used .",
"RAW 264 . 7 cells or BMDCs were harvested , pelleted , washed with PBS and lysed in RIPA ( Radioimmunoprecipitaiton Assay ) buffer ( Sigma–Aldrich ) with a cocktail of proteinase and phosphatase inhibitor ( Thermo Scientific , Waltham , MA ) .",
"The protein concentration of whole cell lysate was detected by BCA kit ( Thermo Scientific ) .",
"Equivalent amounts of protein in each sample were resolved by SDS-PAGE and transferred into nitrocellulose membranes .",
"Membranes were blocked in 5% nonfat-dried milk in PBS with 0 . 05% Tween 20 .",
"Rabbit anti-mouse phospho-Erk ( Cell Signaling , Danvers , MA ) was used for protein detection .",
"Cytospins of single cell suspensions were fixed with 4% formaldehyde , permeabilized with 0 . 5% saponin , and blocked with 3% BSA and Fc receptor Blocker ( BD BioSciences ) .",
"Then cells were stained with anti-γH2AX ( Millipore ) for overnight and detected by antibodies labeled with DAPI ( 4′ , 6-diamidino-2-phenylindole ) and Alexa Fluor 488 .",
"Images were detected with Nikon ECLIPSE TE2000 and NIS-Elements software version 2 . 30 and Leica DFC300 FX .",
"RPPA analysis was performed at the University of Texas MD Anderson Proteomic Core facility .",
"Control RAW cells ( untreated ) and nanoparticle treated ( 100 μg/ml nCB and 100 μg/ml PEG-nCB ) for 24 hr in triplicates were washed , pelleted , and subjected to RPPA analysis .",
"A detailed description of sample processing and data analysis is available on the website of the core facility .",
"Heatmaps were generated by the softwares Cluster and Treeview .",
"Naive CD4+ T cells were isolated using anti-CD4-conjugated magnetic beads ( Miltenyi Biotec ) and were isolated with an autoMACS cell separator .",
"Cells were differentiated under Th1 , Th17 , or Treg polarizing conditions .",
"In brief , 2 to 2 . 5 × 106/ml cells were activated with 1 . 5 μg/ml plate-bound anti-CD3 and 1 . 5 μg/ml soluble anti-CD28 antibodies in addition to: 10 μg/ml anti-IL-4 antibodies , 50 U/ml IL-2 and 20 ng/ml IL-12 ( Th1 polarizing condition ) , or 10 μg/ml anti-IL-4 antibodies , 10 μg/ml anti-IFNγ antibodies , 50 U/ml IL-2 , 40 ng/ml IL-6 and 6 ng/ml TGFβ ( Th17 polarizing conditions ) or 10 μg/ml anti-IL-4 antibodies , 10 μg/ml anti-IFNγ antibodies , 50 U/ml IL-2 and 6 ng/ml TGFβ ( Treg polarizing conditions ) .",
"In some experimental groups 100 ng/ml nCB or vehicle control were added .",
"Cells were cultured for 3 to 5 days , were harvested and washed for intracellular staining of IL-17 , IFNγ or Foxp3 and the surface staining of CD25 to determine Th17 , Th1 , or Treg differentiation .",
"Cell pellets were treated with TRIzol ( Life Technologies ) , and mRNA was extracted as previously described ( Shan et al . , 2014 ) .",
"All probes , mouse Mmp9 ( Mm00600164_g1 ) , mouse Mmp12 ( Mm00500554_m1 ) , mouse Il6 ( Mm00446190_m1 ) , mouse Atm ( Mm01177457_m1 ) , and mouse Tnf ( Mm00443258 ) were purchased from Applied Biosystems ( Foster City , CA ) .",
"All data were normalized to 18S ribosomal RNA ( Hs99999901_s1 ) expression .",
"TEM: human lung CD1a+ cells were fixed with modified Karnovsky's fixative in 0 . 1 M Millonig's phosphate buffer , osmicated , minced into 1-mm cubes , dehydrated , and stained with saturated uranyl acetate ( Watson , 1958 ) .",
"Cells cubes were embedded in resin and polymerized at 68°C for 2 days ( Spurr , 1969 ) .",
"Sections of 55–65 nm were cut and collected on 150 hex-mesh copper grids and counterstained with Reynold's lead citrate ( Reynolds , 1963 ) .",
"The stained sections were viewed with a Hitachi H7500 transmission electron microscope , and images were captured by Gatan US1000 digital camera and Digital Micrograph , v1 . 82 . 366 software .",
"HR-TEM: the human emphysematous lung tissue was digested with proteinase K overnight followed by wash with ethanol and air-dried .",
"The residual black substance was drop-cast directly on a lacey carbon TEM grid ( Ted Pella , Inc . , Redding , CA ) and vacuum dried for 6 hr before usage .",
"The HR-TEM image was taken with the JEM-2100F field emission gun transmission electron microscope ( JEOL USA , Inc . , Peabody , MA ) operated at 200 kV .",
"Samples were imaged and analyzed by CytoViva .",
"Darkfield hyperspectral imaging was performed using a CytoViva dark field microscope system equipped with CytoViva Hyperspectral Imaging System 1 . 2 ( Auburn , AL ) .",
"The Raman spectrum of 15 nm carbon black from Cabot ( Lot: 1278105 ) was acquired using a Renishaw inVia Raman Microscope ( Hoffman Estates , Il ) with a 514 . 5-nm laser and a 50× objective lens .",
"Similarly , Raman spectra were acquired from individually dispersed cells drop cast onto a glass slide and was fixed with 4% paraformaldehyde .",
"The spectra from each cell type were combined , and background cellular fluorescence was subtracted .",
"Raman maps were taken with a pixel size of 2 µm × 2 µm .",
"Soxhlet extraction with dichloromethane and direct extraction with ODCB were used to determine PAH contamination of CB ( Harwood and Moody , 1989 ) .",
"GCMS was used to probe for PAHs , which failed to detect below the limit of detection ( 1 part in 1010 by mass ) as standardized by Pyrene and Anthracene ( Pilla et al . , 2009 ) .",
"For the comparison of cytokine production and gene expression of mice challenged with nanoparticles , cells treated with nanoparticles and reagents , and CT quantification of mouse lung volume , we used the Student's t-test or one-way analysis of variance ( ANOVA ) test and Bonferroni's multiple comparison test .",
"All data shown are the mean ± standard error of the mean ( SEM ) , and all analyses were performed with the Prism software ( GraphPad Software ) ."
]
] | [
"Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema .",
"The effect of carbon black ( CB ) , a universal constituent of smoke derived from the incomplete combustion of organic material , in smokers and non-smokers is less known .",
"In this study , we show that insoluble nanoparticulate carbon black ( nCB ) accumulates in human myeloid dendritic cells ( mDCs ) from emphysematous lung and in CD11c+ lung antigen presenting cells ( APC ) of mice exposed to smoke .",
"Likewise , nCB intranasal administration induced emphysema in mouse lungs .",
"Delivered by smoking or intranasally , nCB persisted indefinitely in mouse lung , activated lung APCs , and promoted T helper 17 cell differentiation through double-stranded DNA break ( DSB ) and ASC-mediated inflammasome assembly in phagocytes .",
"Increasing the polarity or size of CB mitigated many adverse effects .",
"Thus , nCB causes sterile inflammation , DSB , and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers ."
] | [
"Smoking for many years damages the lungs and leads to a disease called emphysema that makes it difficult to breathe and is often deadly .",
"There are thousands of chemicals in cigarette smoke and many of them have been linked to the development of lung cancer , although it has been difficult to pinpoint those that are responsible for smoking-related emphysema .",
"Moreover , cigarette smoke also contains large numbers of small particles and relatively little is known about the role played by these particles in smoking-related disease .",
"One of the hallmarks of long-term smoking is a blackening of the lung tissue that persists even if someone stops smoking .",
"Previously , little was known about the composition of the substance that causes this blackening , or its significance in the development of emphysema .",
"Now , by studying lung tissue taken from smokers with emphysema , You et al . have shown that this black substance is made of nano-sized particles of a material called carbon black ( which is also known as elemental carbon ) .",
"These nanoparticles are produced by the incomplete combustion of the cigarettes .",
"You et al . also confirmed that nanoparticles of carbon black can cause emphysema in mice .",
"Closer examination of the lung damage caused by the nanoparticles revealed that they trigger breakages in DNA , which leads to inflammation of the lung .",
"And because the nanoparticles cannot be cleared , they are released into the lung when cells die , which perpetuates lung inflammation and damage .",
"You et al . then went on to show that nanoparticles of carbon black can be modified in a way that allows them to be cleared from the lungs .",
"Such modifications could potentially protect people who are exposed to carbon black nanoparticles in the environment or in workplaces where carbon black is used , such as factories that produce automobile tires and other rubber products ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"neuroscience"
] | Dynamic range adaptation in primary motor cortical populations | elife-21409-v1 | [
[
"The range of natural stimuli a person or animal encounters in a single day is constantly changing .",
"A common example is walking out of a dark movie theater and into a brightly lit lobby .",
"Although the illuminance of light may change over three orders of magnitude between these two rooms , within seconds we are able to see clearly .",
"This ability can be explained by the behavior of neurons that encode information about light intensity .",
"Neurons in primary visual cortex and the retina encode the intensity of light with their firing rates ( Kuffler , 1953; Hubel and Wiesel , 1962 ) .",
"These tuning relationships typically follow monotonic curves between firing rates of zero and the maximum firing rates of the neurons ( i . e . the dynamic range of the neurons ) .",
"The resolution in the relationship is determined by how many steps in the firing rate can be distinguished .",
"The resolution is an information metric and is a function of both the slope of the curve and the amount of noise in the firing rate .",
"When the level of light intensity changes from the dark theater to the bright lobby , the neural tuning curves shift and adjust their slopes to provide resolution over the new range of light intensity ( Shapley and Enroth-Cugell , 1984; Ohzawa et al . , 1985; Smirnakis et al . , 1997; Ohzawa et al . , 1982; Harris et al . , 2000 ) .",
"This phenomenon is commonly referred to as dynamic range adaptation .",
"In addition to this example in the visual system , dynamic range adaptation has also been documented in other sensory areas , including the auditory ( Kvale and Schreiner , 2004; Ulanovsky et al . , 2004; Dean et al . , 2005; Nagel and Doupe , 2006; Rabinowitz et al . , 2011 ) and somatosensory ( Maravall et al . , 2007; Katz et al . , 2006 ) systems .",
"This phenomenon has been described as optimizing the transmission of information about input stimuli within the limited tuning ranges of the neurons ( Brenner et al . , 2000; Fairhall et al . , 2001; Wark et al . , 2007; Atick , 2011 ) .",
"In sensory systems , meaningful input variables can be described concretely .",
"In motor systems , controlled parameters are still being debated .",
"Force was one of the earliest output variables described , and many studies have demonstrated that firing rates of neurons in primary motor cortex ( M1 ) modulate with output force levels in monotonic relationships ( Evarts , 1966 , 1968; Humphrey et al . , 1970; Hepp-Reymond et al . , 1978; Georgopoulos et al . , 1992 ) .",
"Humphrey et al . briefly noted in their work that the value of regression coefficients relating motor neuronal activity to force scaled with the range of forces in their task ( Humphrey et al . , 1970 ) .",
"This finding could be explained by dynamic range adaptation .",
"As the range of stimuli ( output forces ) changes , the slope of the tuning curve changes to encode the range of forces within a limited range of firing rates .",
"Hepp-Reymond et al . later explored this phenomenon in greater detail ( Hepp-Reymond et al . , 1999 ) .",
"They demonstrated that motor cortical neurons encoding force during an isometric pinching task undergo dynamic range adaptation with changes in the task context and cued expectation of the task context .",
"In later work , the direction of arm movement received attention as a controlled variable ( Georgopoulos et al . , 1982 , 1988; Schwartz et al . , 1988 ) .",
"These studies demonstrated a cosine-tuning relationship between movement direction and neuronal firing rates .",
"Each unit was described as having a preferred direction where the firing rate was maximal for movements in that direction and minimal for movements in the opposite direction .",
"In this classical model , the firing rates linearly modulate between the minimum and maximum rates with the cosine of the angle between the movement direction and preferred direction .",
"This tuning model has been demonstrated for movements in both two- and three-dimensional arm reaches .",
"Notably , the model predicts a lower range in firing rates for 2D movements than 3D movements when the preferred direction is not coplanar with the 2D movement space .",
"This raises the unique question of whether changing the output movement statistics from 3D movements to purely 2D movements would lead to dynamic range adaptation .",
"Studying these differences with actual arm movements is difficult .",
"Regardless of a 2D or 3D context , arm movements are still within a dynamic environment subject to many external factors including gravity , inertia , and arm kinematics to which neurons may modulate .",
"In an effort to enforce a direct relationship between firing rates and movement direction , we investigated this question using a brain-computer interface ( BCI ) .",
"Essentially , the BCI task allowed us to null out those external factors and purely investigate the differences in tuning between 3D and 2D movements .",
"We trained monkeys to move a cursor on a 3D computer screen under brain control , using either a population vector algorithm ( PVA ) or an optimal linear estimator ( OLE ) decoder ( Chase et al . , 2009 ) .",
"The monkeys controlled the cursor by modulating the firing rates of neurons recorded in the motor cortex as they performed a classic center-out task in both 2D and 3D environments .",
"We found that neurons within M1 undergo a change in tuning between the 2D and 3D contexts .",
"We then leveraged the simultaneity of the recordings to test several hypotheses about population-based mechanisms of these changes .",
"We found that the results are consistent with dynamic range adaptation ."
],
[
"Our first finding is that firing rates during the virtual reaches depend on whether the task context is 2D or 3D .",
"Firing rates for each successful trial were calculated in time windows from target presentation until target acquisition .",
"The rates were averaged across repeated trials to each target , to compute the trial-averaged firing rates .",
"For the set of common targets between contexts , we calculated the average firing rates separately for each context .",
"In Figure 2AB , we plot the average firing rates to the targets that lay in the xy-plane for both contexts .",
"The blue lines show the trial-averaged firing rates of the neurons to all targets from the 2D context , plotted as a function of target direction ( azimuth ) .",
"The pink lines show the trial-averaged firing rates of the neurons for the eight targets in the xy-plane during the 3D context , again as a function of azimuth .",
"The horizontal dotted lines show the minimum and maximum trial-averaged firing rates amongst the entire set of 26 targets during the 3D context .",
"As expected , the azimuthal preferred directions are nearly the same between the two contexts ( Figure 2C ) .",
"The changes in azimuthal preferred directions are insignificant under the Moore test of paired samples of angles for both monkeys ( Moore , 1980; Zar , 2010 ) .",
"However , the ranges in the tuning curves are not identical between the two contexts .",
"Instead , the range of firing rates for all 16 targets during the 2D context approaches , and occasionally exceeds , the range observed for all 26 targets during the 3D context .",
"This finding is even more striking when considering the firing rates for the set of eight targets that were identical between contexts .",
"The units show greater maximal firing rates and lower minimal firing rates in the 2D context compared to the 3D context for the BCI task . 10 . 7554/eLife . 21409 . 005Figure 2 . Tuning differences between the 2D and 3D contexts .",
"( A , B )",
"Selected tuning curves for the targets in the xy-plane during the 3D ( pink ) and 2D ( dark blue ) contexts from Monkey A ( panel A ) and Monkey C ( panel B ) .",
"Firing rates show the mean ± SE for each target .",
"There were 10 trials per target in the 3D task for all units shown with the exception of the last unit in ( B ) where there were 12 trials per target .",
"There were 15 trials per target in the 2D task for all units shown with the exception of the first two units in ( panel A ) where there were 27 trials per target .",
"The dashed horizontal lines show the minimum and maximum target-averaged firing rates for all 26 targets in the 3D context .",
"( C ) Changes in azimuthal preferred direction for Monkey A ( top ) and Monkey C ( bottom ) .",
"Firing rates were used to fit log-linear tuning models independently for the 2D and 3D contexts to calculate the preferred directions of the units .",
"The preferred direction from the 2D context was compared to the x and y-components of the preferred direction from the 3D context to calculate the change in the azimuthal preferred direction .",
"The changes are insignificant under the Moore test of paired samples of angles for both Monkey A ( R′=0 . 817 , n=471 , P>0 . 1 ) and Monkey C ( R′=0 . 887 , n=409 , P>0 . 05 ) .",
"( D ) Histograms of the differences in tuning ranges for the set of eight identical targets between the 2D and 3D contexts for Monkey A ( top ) and Monkey C ( bottom ) .",
"For both panels: solid vertical lines , means of distributions; solid horizontal lines , mean ± SE; dashed vertical lines , point of equality between the two tuning ranges .",
"In both subjects , the 2D planar dynamic range was larger than the 3D planar dynamic range ( Monkey A: ***P≪10-20 , Wilcoxon signed-rank test , W=88059 , ◊◊◊P<10−18 , sign test , S=334 , n=471 units; Monkey C: **P=0 . 0032 , W=48975 , ◊P=0 . 0134 , S=230 , n=409 units ) .",
"( E ) Histograms of the differences in tuning ranges for the full set of targets between the 2D and 3D contexts for Monkey A ( top ) and Monkey C ( bottom ) .",
"The full tuning range was greater in the 2D context for Monkey A ( **P=0 . 0081 , W=63402 , ◊P=0 . 0426 , S=258 , n=471 ) and in the 3D context for Monkey C ( ***P<10-5 , W=31208 , ◊◊P=0 . 0011 , S=171 , n=409 ) .",
"Data to recreate the tuning curves and histograms are available in Figure 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 00510 . 7554/eLife . 21409 . 006Figure 2—source data 1 . Example tuning curves , preferred directions , and tuning ranges dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 00610 . 7554/eLife . 21409 . 007Figure 2—figure supplement 1 . Units removed from analysis due to signal loss .",
"( A–E )",
"Five units from Monkey A . ( F ) One unit from Monkey C . Tuning curves from the first , second , and third block of trials are shown as the curves from dark to light orange .",
"Firing rates are drawn as the mean ± SE across the trials in the block .",
"Blocks from the 3D context are drawn as dashed lines and those from the 2D context are drawn as solid lines .",
"Data to recreate these plots are available in Figure 2—figure supplement 1—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 00710 . 7554/eLife . 21409 . 008Figure 2—figure supplement 1—source data 1 . Tuning curves of neurons removed from analysis dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 00810 . 7554/eLife . 21409 . 009Figure 2—figure supplement 2 . Invariance of tuning range changes .",
"( A ) Histograms comparing the tuning ranges from both monkeys for the set of identical targets in the 2D and 3D contexts when blocks of 2D trials preceded blocks of 3D trials ( left ) and when blocks of 3D trials preceded blocks of 2D trials ( right ) .",
"Wl = 178635 , ***P<≪10-38 , Sl = 477 , ◊◊◊P<10−26 , nl = 671 , Wr = 273191 , ***P<10-25 , Sr = 564 , ◊◊◊P<10−16 , nr = 880 .",
"W , Wilcoxon signed-rank tests , S , sign tests , n , number of analyzed units .",
"( B ) Histograms comparing the tuning ranges for the set of identical targets in the 2D and 3D contexts for all recording sessions from both monkeys that used the PVA decoder ( left ) and those that used the OLE decoder ( right ) .",
"WPVA = 144603 , ***P<10-15 , SPVA = 404 , ◊◊◊P<10−9 , nPVA = 649 , WOLE = 20273 , ***P<10-10 , SOLE = 160 , ◊◊◊P<10−8 , nOLE = 231 .",
"( C ) Histograms comparing the tuning ranges from both monkeys for the set of identical targets in the 2D and 3D contexts for the subset of recording sessions where the population used for BCI control was identical between the two contexts ( left ) and those where the population changed ( right ) .",
"Wl = 34298 , ***P<10-14 , Sl = 212 , ◊◊◊P<10−11 , nl = 300 , Wr = 114164 , ***P<10-12 , Sr = 352 , ◊◊◊P<10−6 , nr = 580 .",
"( D ) Histograms comparing the tuning ranges for both monkeys for the set of identical targets in the 2D and 3D contexts for the subset of recording sessions where the decoder was re-adapted between the 3D and 2D contexts ( left ) and those were the decoder was identical with the z-dimension zeroed out in the 2D context ( right ) .",
"Wl = 58688 , ***P<10-6 , Sl = 251 , ◊◊◊P<10−3 , nl = 425 , Wr = 78290 , ***P<10-20 , Sr=313 , ◊◊◊P<10−14 , nr = 455 .",
"For all panels: solid vertical lines , means of distributions; solid horizontal lines , mean ± SE; dashed vertical lines , point of equality between the two tuning ranges .",
"Data to recreate these plots are available in Figure 2—figure supplement 2—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 00910 . 7554/eLife . 21409 . 010Figure 2—figure supplement 2—source data 1 . Context order , decoder type , changed population , and re-adapted decoder tuning range dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 01010 . 7554/eLife . 21409 . 011Figure 2—figure supplement 3 . Tuning range changes for Monkey A for one recording session . The plots show the tuning curves for the targets lying on the xy-plane in the 2D ( dark blue ) and 3D ( pink ) contexts .",
"The firing rates are shown as the mean ± SE ( n2D=24; n3D=10 ) .",
"The minimum and maximum average firing rates amongst the set of all 26 targets from the 3D context are drawn as the dotted horizontal lines .",
"The titles above each plot show the elevation angle , ϕ , that the unit’s preferred direction vector estimated from the 3D data makes with the xy-plane .",
"Units that showed a greater tuning range amongst the set of identical targets in the 2D context than the 3D context are marked with a red asterisk at the top-left corner of the plot .",
"Data to recreate these plots are available in Figure 2—figure supplement 3—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 01110 . 7554/eLife . 21409 . 012Figure 2—figure supplement 3—source data 1 . Single session tuning curves dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 01210 . 7554/eLife . 21409 . 013Figure 2—figure supplement 4 . Difference in tuning ranges in single sessions .",
"( A , B )",
"The plots show the histograms for the difference tuning ranges for the set of eight identical targets between the 2D and 3D contexts for Monkey A ( panel A ) and Monkey C ( panel B ) for the single recording session corresponding to the data shown in Figure 2—figure supplement 3 and a similar single recording session from Monkey C . For both panels: solid vertical lines , means of distributions; solid horizontal lines , mean ± SE; dashed vertical lines , point of equality between the two tuning ranges .",
"WA = 333 , **P=0 . 0031 , SA = 22 , ◊◊P=0 . 0037 , nA = 28 , WC = 731 , **P=0 . 0018 , SC = 31 , ◊◊P = 0 . 0054 , nC = 43 .",
"W , Wilcoxon signed-rank tests , S , sign tests , n , number of analyzed units .",
"Data to recreate these plots are available in Figure 2—figure supplement 4—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 01310 . 7554/eLife . 21409 . 014Figure 2—figure supplement 4—source data 1 . Single session tuning range dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 01410 . 7554/eLife . 21409 . 015Figure 2—figure supplement 5 . Minimal changes with repeated contexts .",
"( A ) The plots show the histograms of the tuning range difference between repeats of the 2D context for Monkey A ( left ) and Monkey C ( right ) .",
"( B ) The plots show the histograms of the tuning range difference between repeats of the 3D context for Monkey A ( left ) and Monkey C ( right ) .",
"In all plots , the difference is calculated where positive values represent increases in tuning range for the repeated context relative to the initial set of trials .",
"For Monkey A , there is some significant difference in tuning ranges between repeats of the 2D context ( 2D context: *P=0 . 0047 , W=2233 , n . s . P=0 . 0904 , S=47 , n=113 units; 3D context: n . s . P=0 . 1189 , W=3829 , n . s . P=0 . 6396 , S=60 , n=114 units ) .",
"For Monkey C , there is a significant difference in tuning ranges between repeats of the 3D context ( 2D context: n . s . P=0 . 2525 , W=3946 , n . s . P=0 . 0829 , S=56 , n=176 units; ***P<10-12 , W=10952 , ◊◊◊P<10−10 , S=88 , n=291 units ) .",
"Notably , for all cases where there is a significant difference between repeat of the same context , the means lie to the left of zero .",
"For all panels: solid vertical lines , means of distributions; solid horizontal lines , mean ± SE; dashed vertical lines , point of equality between the two tuning ranges .",
"Data to recreate these plots are available in Figure 2—figure supplement 5—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 01510 . 7554/eLife . 21409 . 016Figure 2—figure supplement 5—source data 1 . Repeat context tuning range dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 016 To quantify this change , we computed the planar tuning ranges as the difference between the minimum and maximum average firing rate amongst the set of eight identical targets for the two contexts .",
"We found that 334 of 471 units in Monkey A and 230 of 409 units in Monkey C exhibited an increase in planar tuning range in the 2D context relative to the 3D context .",
"The mean ±standard error ( SE ) increase in tuning range was 4 . 02 ± 0 . 33 Hz for Monkey A and 0 . 81 ± 0 . 25 Hz for Monkey C ( Figure 2D ) .",
"The range in firing rates for a set of identical stimuli changes with the context of the task .",
"For the purposes of comparison , the differences in the tuning ranges for the set of all targets between each context are shown in Figure 2E .",
"In our investigation , we varied the experimental conditions to analyze the stability of the tuning changes .",
"While Figure 2D shows the tuning difference when the 2D context follows the 3D context , we found that similar tuning changes occur when the order is reversed ( Figure 2—figure supplement 2A ) .",
"We also explored the effect that a different choice in decoder might have on the result .",
"This is important , because the PVA explicitly normalizes every neuron by its modulation depth , while the OLE does not .",
"The findings reported above are results inclusive of all recording sessions regardless of decoders .",
"When we separately looked at sessions using either the PVA decoder or the OLE decoder , we found that the results were individually significant for both decoder types ( Figure 2—figure supplement 2B ) .",
"Additionally , in a subset of the recording sessions , the entire population used to control cursor movement was identical between the two contexts .",
"When we looked at the population response from these sessions , we again found similar changes in tuning range ( Figure 2—figure supplement 2C ) .",
"Additionally , to test the effects of zeroing out the z-dimension , we conducted a series of recording sessions in which we simply re-adapted the decoder for the 2D context rather than use the decoder from the 3D context with the z-dimension zeroed out .",
"When we separately looked at sessions using either re-adapted decoders or identical decoders with the z-dimension zeroed out in the 2D context , we found that the results were individually significant for both types of sessions ( Figure 2—figure supplement 2D ) .",
"The changes in tuning ranges are also apparent when looking at firing rates from a single recording session from Monkey A ( Figure 2—figure supplement 3 ) .",
"The 2D and 3D planar tuning ranges for this recording session and a similar single recording session from Monkey C are compared in Figure 2—figure supplement",
"4 . As shown in Figure 1B , there was a subset of recording sessions where a task context was repeated after the alternative context .",
"These sessions allowed us to investigate the stability of the tuning ranges for identical contexts across the duration of a recording session ( Figure 2—figure supplement 5 ) .",
"We found a slight decrease in the tuning ranges for repeats of the 2D context for Monkey A and repeats of the 3D context for Monkey C: the second presentation tended to have decreased tuning relative to the first .",
"This may be due to fatigue or satiation .",
"Notably , this decrease in tuning on repeated presentations is in the opposite direction as the increase in tuning we observe when moving from the 3D to the 2D context .",
"This suggests that our results from Figure 2D may be a slight underestimate of the actual tuning increase .",
"We next investigated several mechanisms that might be driving the context-dependent firing rate changes we observed during the BCI task .",
"We considered three possible mechanisms: ( 1 ) a re-aiming mechanism , in which subjects aim at points other than the targets ( Jarosiewicz et al . , 2008; Chase et al . , 2010 ) , ( 2 ) a speed gain mechanism , in which subjects change intended speed between the two contexts ( Moran and Schwartz , 1999 ) , and ( 3 ) a dynamic range adaptation mechanism , in which units adapt to utilize their available dynamic range in both contexts ( Brenner et al . , 2000 ) .",
"These strategies each make different predictions about how tuning changes will correlate across populations of simultaneously recorded cells .",
"Below we discuss each of these mechanisms in turn .",
"The adaptation process appeared to occur rapidly with the switch from the 3D to 2D context .",
"In order to investigate the timecourse of this process , we devised a method to estimate changes in dynamic range on a trial-by-trial basis ( see Materials and methods ) .",
"Briefly , we fit cosine tuning models from trials in both the 2D and 3D contexts .",
"These models represent a prediction of the expected population response for each context that can be assessed on a trial-by-trial basis .",
"By investigating the error in the fit of each model when the subject experiences a transition between 3D and 2D , we can assess how quickly the population switches from its expected 3D response to its expected 2D response .",
"To mitigate noise , we normalized the expected error for each neuron by the average residual error for the 2D and 3D contexts , and we averaged across all neurons and across the multiple recording sessions for each monkey .",
"Note that all errors were cross-validated: we fit separate models for even and odd trials , and used them to predict the results on odd and even trials , respectively .",
"We estimated the error for the first 120 trials in the 2D context as well as the last 60 planar target trials from the preceding 3D context .",
"These normalized , averaged errors are shown in Figure",
"5 . The plots demonstrate a very rapid shift in the tuning of the population as the context changes from 3D to 2D .",
"Immediately on transition from 3D to 2D ( on trial 1 ) , the 3D model error grows considerably .",
"Presumably , the lack of movement in the z-dimension cues the subject to the context change .",
"The 2D model error then reduces .",
"We fit exponential models to the reduction in 2D model error for each monkey , and found exponential decay coefficients ( with 95% confidence bounds ) of 5 . 3 ( 3 . 2 , 16 . 7 ) trials for Monkey A and 4 . 6 ( 3 . 2 , 10 . 5 ) trials for Monkey C . Because each trial takes approximately 1 s to perform , the retuning effect nears completion within seconds .",
"The 2D model error is immediately lower than the 3D model error on the first 2D trial and rapidly reduces to a baseline state within the first few trials . 10 . 7554/eLife . 21409 . 022Figure 5 . Time course of adaptation . The two panels show the time courses of adaptation for Monkey A ( A ) and Monkey B ( B ) .",
"The ordinate axes in the plots show the standardized error between the firing rates in the population and the expected response from tuning fits .",
"Each plot shows two different errors: the error based on tuning models from the 2D context ( dark blue ) and from the 3D context ( pink ) .",
"These standardized errors are averaged across multiple recording sessions for Monkey A ( 18 ) and Monkey C ( 11 ) .",
"The abscissa in the plots show the trial number before and after the change from the 3D context to the 2D context .",
"The data points to the right of zero represent the standardized error for the first 120 trials in the 2D context .",
"The data points to the left of zero represent the last 60 planar target trials of the the 3D context .",
"The dashed black vertical line at zero represents the system down-time while the context was switched .",
"We also plotted exponential fits ( black ) to the 2D tuning model error for the first 120 trials of the 2D context .",
"The data were fit to the equation: y=a+bexpcx .",
"For Monkey A , the fit parameters ( with 95% confidence bounds ) were: a = 0 . 815 ( 0 . 807 , 0 . 8233 ) ; b = 0 . 176 ( 0 . 087 , 0 . 264 ) ; c = −0 . 189 ( −0 . 317 , −0 . 060 ) ; r2 = 0 . 229 .",
"For Monkey C , the fit parameters were: a = 0 . 809 ( 0 . 800 , 0 . 818 ) ; b = 0 . 250 ( 0 . 150 , 0 . 348 ) ; c = −0 . 202 ( −0 . 309 , −0 . 095 ) ; r2 = 0 . 328 .",
"Data to recreate these plots is available in Figure 5—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 02210 . 7554/eLife . 21409 . 023Figure 5—source data 1 . Trial-by-trial tuning model error dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 023 We also assessed the completeness of the dynamic range adaptation .",
"To be complete , the dynamic range in the 2D context should equal the full dynamic range in the 3D context , when all targets are taken into account .",
"We developed the following metric to quantify the completeness of the adaptation: ( 2 ) Adaptation=ρ2D−ρ3Dρ3Dfull−ρ3D Here , ρ represents the tuning range under a specified context and target set .",
"The subscript denotes the context .",
"The superscript ‘full’ denotes the entire set of targets for a given context , whereas the lack of a superscript represents the set of targets identical between contexts .",
"The terms used in computing this metric are described in greater detail in the Tuning Range Analysis subsection of the Methods .",
"This metric normalizes the change in tuning range such that no adaptation would lie at zero and complete adaptation would lie at one .",
"This metric is problematic for neurons where the elevation angle is near the xy-plane and the difference between the full and planar tuning ranges approaches zero .",
"In order to avoid this possible complication , we limited our analysis to those neurons where both the elevation angle was greater than 30∘ and the denominator was greater than 2 Hz .",
"The resulting histograms from the metrics for Monkeys A and C are shown in Figure",
"6 . For both monkeys , the histograms are distributed significantly away from zero under both a sign test and a Wilcoxon signed-rank test .",
"Furthermore , the means ( ±SE ) of the metrics are 0 . 5157 ( ±0 . 1121 ) for Monkey A and 0 . 6793 ( ±0 . 1233 ) for Monkey C . These results suggest that the dynamic range adaptation is more than halfway complete for both monkeys .",
"It is worthwhile to note that dynamic range adaptation is not always complete , even in sensory systems .",
"In the auditory system , Dean and colleagues showed a range of retuning abilities in their example neurons ( cf .",
"Figure 1 of Dean et al . ( 2005 ) ) .",
"Ohzawa developed an adaptability index to characterize the contrast gain adaptation of cells in visual cortex , and reported values in line with our results ( cf .",
"Figure 5 of Ohzawa et al . ( 1985 ) ) . 10 . 7554/eLife . 21409 . 024Figure 6 . Completeness of adaptation . The two panels show histograms of the completeness metric for Monkey A ( A ) and Monkey C ( B ) .",
"For both panels: solid vertical lines , means of distributions; solid horizontal lines , mean ± SE; dashed vertical lines , zero point representing no dynamic range adaptation .",
"For both monkeys , histograms were distributed significantly away from the zero point ( Monkey A: ***P<10-5 , Wilcoxon signed-rank test , W=3580 , ◊◊◊P<10−3 , sign test , S=68 , n=95 units; Monkey C: ***P<10-4 , W=570 , ◊◊◊P<10−3 , S=29 , n=35 units ) .",
"The means ( ± SE ) of the metrics were 0 . 5157 ( ± 0 . 1121 ) for Monkey A and 0 . 6793 ( ± 0 . 1233 ) for Monkey C . Data to recreate these plots is available in Figure 6—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 02410 . 7554/eLife . 21409 . 025Figure 6—source data 1 . Adaptation metric dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 025 BCI provides a powerful framework for addressing basic scientific questions about motor control , including the dynamic range adaptation effects observed here , because the effect each neuron has on cursor movement is fully specified by the experimenter , and because we can obviate complicating factors such as gravity and inertia ( Golub et al . , 2016 ) .",
"However , it is interesting to compare the BCI results to a similar task performed in hand-control .",
"We had a third monkey ( Monkey N ) perform a center-out reaching task with the cursor controlled by the position of a 3D tracking marker placed on the monkey’s hand .",
"For the 2D movements , the z-position of the marker was zeroed out so that the depth of the marker had no effect on the cursor .",
"We computed the tuning ranges for the directionally tuned neurons amongst the set of identical targets for each context ( Figure 7 ) .",
"Unlike with the BCI task , we see no evidence of dynamic range adaptation in the hand-control version of this task .",
"We speculate on the reasons for this in the Discussion section . 10 . 7554/eLife . 21409 . 026Figure 7 . Tuning range in hand-control task . The plot shows the histogram of the tuning range difference between the 2D and 3D contexts for the planar targets in the hand-control task .",
"For the plot: solid vertical line , mean of distribution; solid horizontal line , mean ± SE; dashed vertical line , point of equality between the two tuning ranges .",
"For Monkey N , the tuning ranges were not significantly different between the 2D and 3D contexts ( n . s . P=0 . 6349 , Wilcoxon signed-rank test , W=969 , n . s . P=0 . 9007 , S=31 , n=64 units ) .",
"Data to recreate these plots are available in Figure 7—source data 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 02610 . 7554/eLife . 21409 . 027Figure 7—source data 1 . Hand-control task tuning range dataset . DOI: http://dx . doi . org/10 . 7554/eLife . 21409 . 027"
],
[
"Neurons in sensory regions encode and transmit information about the external environment ( Borst and Theunissen , 1999 ) .",
"Sensory neurons also undergo dynamic range adaptation with contextual changes in the environment ( Ohzawa et al . , 1985; Dean et al . , 2005 ) .",
"In our study , M1 neurons demonstrate significant changes in their firing rates to identical targets , when the task context switches from 3D to 2D .",
"These changes are widespread across the recorded population .",
"The observed changes are not consistent with a re-aiming strategy during the 2D context .",
"Furthermore , the observed changes are not consistent with changes in intended speed between the two contexts .",
"Instead , the observed changes appear to be consistent with dynamic range adaptation .",
"During the 2D context , the z-dimension gets added to the task-irrelevant null space .",
"Without the observed adaptation , the portion of the dynamic range encoding the z-dimension during the 3D context would not be utilized during the 2D context .",
"Our results showed that units begin to utilize that portion of the dynamic range to encode targets in the xy-plane as the task context switches from 3D to 2D .",
"These results are markedly similar to the dynamic range adaptations that occur in sensory regions .",
"Just as neurons in the retina and visual cortex adjust their tuning to encode the current range of light intensities , the recorded units adjusted their tuning to encode the current range of targets in the given context of the BCI task .",
"Researchers have described dynamic range adaptation in sensory systems as functioning to optimize information throughput of the neural populations ( Brenner et al . , 2000; Fairhall et al . , 2001; Wark et al . , 2007 ) .",
"The same process appears to be functioning here .",
"Just as the slopes of tuning curves in the visual system increase to transmit more information about a narrower range of light intensities , the slopes of the tuning curves of the M1 units increase to transmit more information about a narrower range of target directions .",
"Other studies have noted tuning changes of individual neurons during BCI learning tasks ( Jarosiewicz et al . , 2008; Ganguly and Carmena , 2009; Ganguly et al . , 2011; Chase et al . , 2012 ) .",
"However , the dynamic range adaptation we see appears to be fundamentally different from the effects shown in those studies .",
"First , the time scale of dynamic range adaptation is on the order of seconds ( Figure 5 ) .",
"The time scale of BCI-learning-related changes in neural tuning is on the order of tens of minutes ( Jarosiewicz et al . , 2008; Chase et al . , 2012 ) to days ( Ganguly and Carmena , 2009; Ganguly et al . , 2011 ) .",
"Second , the dynamic range adaptation is restricted to changes in tuning depth; changes in preferred direction are minimal .",
"In BCI learning studies , preferred directions typically change as well .",
"Third , dynamic range adaptation occurs without any overt learning pressure .",
"In studies of BCI learning , the mapping between neural activity and cursor movements is changed in such a way to impair task performance .",
"That was not the case here: if the subject continued using the same firing rates in the 3D and 2D contexts , performance would have been unaffected .",
"For these reasons , we speculate that dynamic range adaptation represents an additional degree of flexibility in the responses of neurons in the motor system .",
"While we observed consistent dynamic range adaptation between the 2D and 3D contexts in the BCI task , we did not observe it in the hand-control version of this task .",
"There are a number of possible reasons for this .",
"In hand control , hundreds of thousands of neurons likely contribute to arm movements , and the overall change in dynamic range might be distributed over all of them , making them difficult to observe .",
"In BCI , only the recorded neurons contribute to cursor movement , which could potentially make the effect easier to see .",
"Another possibility is that the number of movement parameters that an individual neuron tunes for in hand control is quite large , and when moving from 3D to 2D , the percentage change in number of control parameters is small .",
"In BCI , each neuron contributes to only 3D or 2D movements of the cursor , which could also make the effect easier to observe .",
"A third intriguing possibility is that hand control and BCI movements invoke fundamentally different control schemes .",
"The Hepp-Reymond study noted a rescaling of tuning when the output range of required forces was changed ( Hepp-Reymond et al . , 1999 ) .",
"That study was performed under isometric conditions , that is , movements were not physically required .",
"Humphrey and colleagues had subjects perform flexion/extension movements of the wrist ( Humphrey et al . , 1970 ) under various load conditions , and noted that regression coefficients for decoding scaled with the load .",
"Here , we used a BCI task in which subjects were not physically required to move , and our subjects tend to exhibit very little physical movement during these tasks .",
"In contrast , the hand-control version of this task required physical arm movements , but not variations in output force .",
"It is possible that dynamic range adaptation in the motor system only occurs under conditions of changing force .",
"If this is true , it suggests that isometric force production is a closer analog to BCI than the overt reaching movements to which it is typically compared .",
"Direction as an important component of the control signal is undeniable .",
"Although directional tuning has been shown to change in a context-dependent manner , it should be emphasized that directional tuning is stable across repetitions within the same condition .",
"Tuning function changes of preferred direction and modulation depth have been observed between brain control and hand control ( Taylor et al . , 2002; Carmena et al . , 2003; Lebedev et al . , 2005 ) , between movements in different speed ranges ( Moran and Schwartz , 1999; Churchland and Shenoy , 2007 ) , and between movements in different postures ( Scott and Kalaska , 1995; Sergio and Kalaska , 2003 ) .",
"Tuning can change during learning , when adapting to visuomotor perturbations ( Wise et al . , 1998; Paz et al . , 2003; Mandelblat-Cerf et al . , 2009 ) , force field perturbations ( Gandolfo et al . , 2000; Arce et al . , 2010 ) , BCI decoder changes ( Jarosiewicz et al . , 2008; Ganguly and Carmena , 2009; Hwang et al . , 2013 ) , or even the consistent coupling of sensory cues with rewarded actions ( Zach et al . , 2008 ) .",
"Here , we find another example of tuning change: dynamic range adaptation .",
"One feature that distinguishes dynamic range adaptation from these other sorts of tuning changes is that , using a principle of information processing ( namely , that the total range of firing is rescaled according to the output statistics ) , the tuning curves from the 2D context may be predicted from the 3D context .",
"Certain types of dynamic range adaptation , such as contrast gain normalization in the visual system , have been linked to a divisive normalization mechanism ( Carandini et al . , 1997 ) .",
"By better defining which aspects of behavior lead to tuning changes and from more complete descriptions of the factors driving these changes , we will gain a deeper understanding of the neural operations taking place during the control of movement .",
"These results support the hypothesis that neurons in M1 serve to efficiently transmit contextually encoded information .",
"Motor learning in dynamic environments can be viewed as learning efficient ways to encode information .",
"Changes in the environmental context , such as tool use , can lead to changes in the association between M1 activity and motor output ( Quallo et al . , 2012 ) .",
"Even during typical reaches in a static environment , M1 neurons efficiently encode information about the task .",
"The common linear tuning function ( Georgopoulos et al . , 1982 ) maximizes the output entropy for a uniform distribution of target directions in a 3D environment .",
"This ‘histogram equalization’ maximizes the information transmission in the low-noise limit ( Nadal and Parga , 1994; Laughlin , 1981 ) .",
"In our studies , the units in M1 have limited dynamic ranges with which to encode cursor movement under the different contexts .",
"Rather than using a subset of the available tuning ranges , the population adaptively displays new coding schemes that fully utilize the ranges of the units comprising the population .",
"This adaptive learning of the M1 population allows for the efficient encoding of task relevant information in changing environments ."
],
[
"The behavioral training and decoding methods closely followed those described by Jarosiewicz et al . ( 2008 ) .",
"Briefly , two male Rhesus monkeys were trained to perform a center-out reaching task in a 3D virtual environment .",
"The monkeys sat in a primate chair facing a mirror that reflects a 3D image from a stereoscopic computer monitor .",
"Before electrode implantation , the monkey was trained to move its hand , fitted with an optical marker , to move a cursor from the center of an imaginary cube to targets at lying at one of the eight corners for a liquid reward ( Reina et al . , 2001 ) .",
"Once the monkeys became proficient at the 3D center-out reaching task , they were implanted with chronic recording arrays .",
"Both monkeys were implanted with a 96-channel array ( Cyberkinetics Neurotechnology Systems , Inc . ) .",
"The implantations were visually placed in the proximal arm area of primary motor cortex .",
"Recordings were amplified , filtered , and sorted on-line with a 96-channel Plexon MAP system ( Plexon Inc . ) .",
"Each monkey was trained to perform the center-out task with both arms restrained by modulating the spiking activity of the recorded units to control cursor velocity .",
"Both monkeys became proficient at the brain-control center-out task in both the 2D and 3D contexts from training sessions prior to starting this study .",
"Each trial began with the monkey holding the cursor at the task space origin for a randomized period ( 50–160 ms ) .",
"The target was presented after this period .",
"The task required the monkey to reach the target within the 2 s reach period and hold the cursor at the target position for a randomized period ( 0–100 ms ) .",
"The cursor was then automatically returned to the home position for the start of the next trial .",
"A water reward was delivered for successful trials .",
"The targets were placed at a distance of 85 mm from the center .",
"The cursor and targets’ radii were 8 mm each .",
"All procedures were performed in accordance with the guidelines of the University of Pittsburgh’s Institutional Animal Care and Use Committee .",
"Each recording session began with a calibration brain control session .",
"The calibration session followed the methods described by Chase et al . ( 2009 ) .",
"The decoding parameters were initially randomized .",
"Eight targets from the 3D context ( those lying on the corners of a cube ) were then presented , individually in random order , and remained until the end of the movement period .",
"Although the cursor did not move much during the movement period due to the randomized parameters , the firing rates were still modulated to target presentation .",
"Once a cycle set comprising one presentation of each of the eight targets was completed , the firing rates were regressed using the regress function in Matlab ( Mathworks , Inc . , Natick , MA ) against the target direction according to the linear tuning model: ( 3 ) ri=b0 , i+mi𝐝⋅𝐩i In a subset of recording sessions , separate calibration sessions were conducted for the 2D and 3D trials .",
"These calibration sessions were conducted immediately prior to the start of the respective context block .",
"The calibration session for the 2D trials differed from the previously described calibration methods only in that the target set comprised 8 targets spaced equally around a circle centered in the xy-plane .",
"For all other recording sessions , the decoding parameters derived from the initial calibration session were used for the entirety of the recording session .",
"Linear decoders ( PVA and OLE ) were used to decode neural signals into cursor movement .",
"The recorded population was assumed to follow the linear tuning function described in Equation 3 For each unit , i , spike counts were measured at 30 Hz and converted into firing rates , ri , by dividing by the sampling interval .",
"Rates were averaged across the last five time bins and normalized by subtracting the fit baseline firing rate and dividing by the fit modulation depth: ( 4 ) ui=ri-b0 , imi The normalized rates of the N units comprising the population were grouped in the vector u=[u1 , u2 , … , uN]⊺ .",
"Similarly , the preferred direction vectors of the N units were grouped in the matrix P=[𝐩1 , 𝐩2 , … , 𝐩N] .",
"For the PVA decoder , normalized rates , 𝐮 , were converted to cursor velocity , 𝐯 , as: ( 5 ) v=ksnDNP⊺u where nD is the number of movement dimensions ( either 2 or 3 ) and ks is the speed factor to convert the normalized speed to a physical speed in mm/s ( values used in our study ranged from 65 to 120 ) .",
"For the OLE decoders , the normalized rates were converted to cursor velocity as: ( 6 ) v=ks ( B⊺B ) −1B⊺ ( ri−b0 , i ) where B=[m1p1 , m2p2 , … , mNpN] .",
"Cursor position , 𝐜 , was updated every sampling interval as: ( 7 ) 𝐜 ( t ) =𝐜 ( t-Δt ) +Δt𝐯 ( t ) For trials in the 2D context , the z-component of the decoded velocity was set to zero to constrain movement to the xy-plane .",
"The analyses in this work focuses on the firing rates of the recorded units , ri .",
"These rates are functions of many different variables: the task context ( i . e . number of movement dimensions , nD ) , the target direction ( 𝐝 ) , the trial number for the target ( j ) , and the time points within the trial ( t ) .",
"For a given monkey and recording session , the rates of the N units comprising the population were grouped in the vector r=[r1 , r2 , … , rN]⊺ .",
"In this work , we analyzed the average firing rate over the trial duration , ⟨𝐫⟩t .",
"These time-averaged firing rates were also averaged over the trials for a given context and target , ⟨𝐫⟩t , j .",
"These trial- and time-averaged firing rates remain a function of the task context and target , 𝐟 ( nD , 𝐝 ) =⟨𝐫⟩t , j .",
"We defined the target set , A , as the target set in the 3D context , A={d:nD=3} .",
"This set comprised 26 targets , |A|=26 .",
"We defined the target set , B , as the target set in the 2D context , B={d:nD=2} .",
"This set comprised 16 targets , |B|=16 .",
"There were eight targets which were common to the two sets , |A∩B|=8 .",
"We defined that set intersection as the set of common targets , C=A∩B .",
"The 3D planar range for the units in the population was defined as: ( 8 ) ρ3D=max{f ( nD , d ) :nD=3 , d∈C}−min{f ( nD , d ) :nD=3 , d∈C} Although all targets in the 2D context lie in the xy-plane , we calculated the 2D range over the set of common targets to directly compare the ranges: ( 9 ) ρ2D=max{f ( nD , d ) :nD=2 , d∈C}−min{f ( nD , d ) :nD=2 , d∈C} Our analysis compared the difference between the 3D planar range ( ρ3D ) and the 2D range ( ρ2D ) for the units comprising the population .",
"For comparisons of the full tuning ranges , the 2D full range was calculated over the entire set of 16 2D targets , d∈B: ( 10 ) ρ2Dfull=max{f ( nD , d ) :nD=2 , d∈B}−min{f ( nD , d ) :nD=2 , d∈B} Similarly , the 3D full range was calculated over the entire set of 26 3D targets , d∈ ( A ) : ( 11 ) ρ3Dfull=max{f ( nD , d ) :nD=3 , d∈A}−min{f ( nD , d ) :nD=3 , d∈A} In order to test a re-aiming hypothesis that would explain the change in observed tuning ranges , we devised a method to estimate the optimal re-aiming points for that hypothesis .",
"For each recording session that used the identical set of units to control the BCI task between contexts , the firing rate data from brain control in the 3D context between target presentation and acquisition was used to fit a log-linear tuning model ( Koyama et al . , 2010 ) to each unit in the population .",
"The log-linear tuning model is similar to the linear tuning model in Equation 3 with the exception of the exponential relationship in the log-linear model: ( 12 ) rll , i ( 𝐝 ) =exp ( bll , 0 , i+mll , i𝐝⋅𝐩ll , i ) The subscripts , ll , are used to distinguish the equation and its tuning parameters from those used in the linear tuning model .",
"The tuning parameters were fit for each neuron using the glmfit function in Matlab with the distribution and link parameters set as Poisson and log , respectively .",
"For each of the eight shared targets , an optimal re-aiming point for the 2D context was estimated using the log-linear tuning models and the decoders from each recording session .",
"The optimization was run in Matlab as a constrained minimization to move the cursor closest to the target after one time-step .",
"The optimization was constrained to ensure that the single re-aiming point for each target and recording session lies in S2 , the space of unit vectors in R3 .",
"The constrained minimization could be written as: ( 13 ) min{‖85d−ΔtksnDN∑i=1Npirll , i ( d∗ ) −b0 , imi‖:d∗∈S2} The scalar , 85 , is used to scale the target direction to the target distance of 85 mm used in the studies .",
"Also , note that the predicted firing rates , rll , i , are derived from the log-linear tuning model in Equation 12 and decoded using the tuning parameters used in the original decoder .",
"Thus , for all target directions , 𝐝 , in the set of common targets , C , we derived optimal re-aiming points , 𝐝* , which we grouped into the set , D . The optimal re-aiming ranges were calculated as: ( 14 ) ρre−aim=max{rll ( d∗ ) :d∗∈D}−min{rll ( d∗ ) :d∗∈D} Similarly , the tuning range predicted by the log-linear model was calculated as: ( 15 ) ρll=max{rll ( d ) :d∈C}−min{rll ( d ) :d∈C} For each unit and recording session , we calculated the sign of the difference in tuning ranges predicted by re-aiming , sgn ( ρre−aim , i−ρll , i ) , and the sign of the observed difference in tuning ranges , sgn ( ρ2D , i−ρ3D , i ) , summing the results in a contingency table .",
"In order to evaluate the speed at which the neural population adapted with changes in context , we created a normalized error metric based upon tuning models from each context .",
"In building the tuning models , we considered only the firing rates for trials where the target was in the set of common targets .",
"For each session and context , the trials were split into two separate groups: even and odd numbered trials .",
"We fit separate linear tuning models for each group .",
"The error for each trial was calculated as the difference between the population response for that trial and the expected response based upon the tuning model fit from the opposite group as that trial .",
"The errors were calculated using both the tuning models created from the 2D context and that from the 3D context .",
"These errors were normalized by dividing by the standardized noise of the neurons from the tuning fits .",
"The noises were estimated as the dispersion parameter from the Matlab glmfit function used to fit the tuning models .",
"In order to standardize the normalization between the 2D and 3D contexts , we used the square root of the product of the dispersion parameters estimated from each context .",
"The absolute value of the normalized errors were averaged over the neurons in the population .",
"For each monkey , the trial-ordered errors were averaged across multiple recording sessions to create the final normalized error metrics displayed in Figure 5 .",
"For the 3D context , the errors could only be estimated for the planar targets .",
"Subsequently , all trials for non-planar targets were removed from the analysis .",
"The remaining planar target trials were ordered as the last planar target trial , the second to last , and so on for the purposes of averaging across recording sessions .",
"While plotting these normalized errors , we noticed a trend for a rapid decrease in the 2D tuning model error in the first few trials of the 2D context .",
"In order to model that drop in the error , we fit an exponential function to the session-averaged data .",
"The exponential function followed the equation: y=a+bexp ( cx ) , where y was the error metric and x was the trial number .",
"In our work , we use the fit parameter c to describe the rapid decrease in the error metric .",
"These fits are displayed in Figure 5 .",
"In order to study whether dynamic range adaptation occurs under a hand-control paradigm , we conducted a similar experiment where the cursor movement was controlled directly via the hand of a monkey .",
"A third monkey ( Monkey N ) was trained to use its hand to control the movement of a computer cursor in a center-out reaching task ( Velliste et al . , 2014 ) .",
"An infrared marker was placed on the back of the monkey’s hand and tracked using an Optotrak 3020 optical tracking system ( Northern Digital , Waterloo , Canada ) .",
"The marker position was used in real time to update the position of the cursor on the stereoscopic computer monitor .",
"The cursor and targets in the hand control paradigm were identical to those used in the brain-control study .",
"We investigated the neural response for trials from both the 3D and 2D contexts , where the 3D context preceded the 2D context .",
"During the trials in the 2D context , the cursor position was fixed in the xy-plane .",
"In other words , the cursor would remain stationary if the monkey moved its hand closer or farther from its body while maintaining the same lateral and vertical position .",
"Neural firing rates were calculated in the time windows between target presentation and acquisition .",
"These rates were averaged across trials of the same target and used to estimate the tuning ranges in each context .",
"This analysis was limited to neurons that were directionally tuned .",
"For each context , we fit a cosine tuning model to the firing rate responses to the set of all targets .",
"Neurons that had a fit modulation depth less than 4 Hz in both the 2D and 3D contexts were excluded from the analysis .",
"In Figure 7 , we compare the difference in tuning ranges for the set of common targets between the 2D and 3D contexts in a similar manner as Figure 2D .",
"This additional study and all associated procedures were performed in accordance with the guidelines of the University of Pittsburgh’s Institutional Animal Care and Use Committee ."
]
] | [
"Neural populations from various sensory regions demonstrate dynamic range adaptation in response to changes in the statistical distribution of their input stimuli .",
"These adaptations help optimize the transmission of information about sensory inputs .",
"Here , we show a similar effect in the firing rates of primary motor cortical cells .",
"We trained monkeys to operate a brain-computer interface in both two- and three-dimensional virtual environments .",
"We found that neurons in primary motor cortex exhibited a change in the amplitude of their directional tuning curves between the two tasks .",
"We then leveraged the simultaneous nature of the recordings to test several hypotheses about the population-based mechanisms driving these changes and found that the results are most consistent with dynamic range adaptation .",
"Our results demonstrate that dynamic range adaptation is neither limited to sensory regions nor to rescaling of monotonic stimulus intensity tuning curves , but may rather represent a canonical feature of neural encoding ."
] | [
"Most cameras are equipped with an auto-contrast feature that enables them to take high quality pictures in a wide range of lighting conditions .",
"Auto-contrast works by increasing the sensitivity of the camera to light in dimly lit surroundings , but reducing it in bright conditions to ensure that images do not become saturated .",
"Our visual system is equipped with a similar feature .",
"Neurons in the visual system increase or decrease their sensitivity to light as appropriate to enable us to see in both dimly lit rooms and dazzling sunshine .",
"This process , which is known as dynamic range adaptation , also occurs in neurons that are sensitive to sound or touch .",
"Rasmussen et al . therefore wondered whether the same might hold true for neurons that encode non-sensory stimuli such as the direction of movement .",
"Would these neurons change their sensitivity to direction if presented with a wide range of possible directions instead of a narrow range ?",
"If so , this would suggest that dynamic range adaptation occurs throughout the nervous system .",
"To find out , Rasmussen et al . trained two rhesus macaque monkeys to use their brain activity to move a cursor on a virtual reality screen in either 2D or 3D .",
"Studying this brain activity showed that neurons became less sensitive to the cursor’s direction of movement when the task switched from 2D to 3D .",
"This makes sense because in a 3D task , which also features depth , the neurons have a greater range of possible movement directions to encode .",
"Conversely , the neurons became more sensitive to the direction of movement when the task switched from 3D to 2D .",
"Under these circumstances the neurons can use activity that was previously dedicated to encoding depth to instead represent the 2D space in finer detail .",
"These results presented by Rasmussen et al . raise several additional questions .",
"Are the mechanisms that support dynamic range adaptation the same in sensory and motor neurons ?",
"If these neurons also encode other aspects of movement , such as speed , would these also be included in the same range as direction or is the adaptation process segregated by specific parameter categories ?",
"And how do these changes in sensitivity affect the movements that animals produce ?"
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Local GABA concentration is related to network-level resting functional connectivity | elife-01465-v1 | [
[
"There has been a surge of recent interest in so-called ‘resting state networks’ ( RSNs ) in the human brain .",
"These robust , distributed networks , most commonly detected using functional MRI , show correlated fluctuations in their resting signal and have revealed networks formed from spatially widespread but anatomically and functionally closely linked regions ( Fox and Raichle , 2007; Snyder and Raichle , 2012 ) .",
"Understanding the basis of these resting state networks is of increasing interest , as variation in the strength of functional coupling within these networks has been shown to be highly sensitive to a wide variety of clinical , genetic , or cognitive states ( Filippini et al . , 2009; Pievani et al . , 2011 ) .",
"However , the neurochemical basis for such variations remains poorly understood .",
"Resting correlations have long been thought to be a functional marker of excitatory connections ( Vincent et al . , 2007; Shmuel and Leopold , 2008 ) .",
"However , recent simulation studies have predicted that patterns of local oscillatory activity can emerge spontaneously in a coherent fashion across large networks with specific connectional architectures ( Cabral et al . , 2011 ) .",
"Such local neuronal dynamics depend on neurochemical inhibitory tone , as shown , for example , by changes in local activity within the primary motor cortices ( M1s ) caused by pharmacological manipulations ( Hall et al . , 2011 ) .",
"If correlated activity spontaneously emerges through attractor dynamics in distant cortical regions , then extent of these functional correlations should be best predicted by local GABA concentrations .",
"Here , we carried out a series of experiments to address this hypothesis .",
"In the first three studies , we directly quantified GABA within the left M1 using Magnetic Resonance Spectroscopy ( MRS ) to test the prediction that the strength of functional connectivity within the motor RSN is related to GABA concentration within M1 , a major node of the network , and that this relationship is specific in both neurochemical and anatomical terms .",
"In a fourth study , we performed an intervention study—to directly assess this relationship by testing whether application of anodal ( excitatory ) transcranial direct current stimulation ( tDCS ) to M1 , an intervention known to decrease local GABA concentration ( Stagg et al . , 2009; 2011a ) , resulted in strengthening of functional connectivity within the motor RSN ."
],
[
"Experiment 1 considered a cohort of 12 young , healthy subjects , of whom 11 had spectra of sufficient quality for inclusion .",
"Using ICA , we demonstrated a significant inverse correlation between functional connectivity within the motor RSN and GABA concentration within M1 ( M1-GABA ) in healthy young volunteers ( r = −0 . 71 , p=0 . 01; Figure 2C ) .",
"To test the anatomical specificity of this result , we assessed the default mode network ( DMN ) , as this is a well-characterized RSN that does not include M1 ( Figure 2B ) .",
"There was no significant correlation between M1-GABA and functional connectivity within the DMN ( r = 0 . 25 , p=0 . 44 ) ( Figure 2D ) , and M1-GABA and functional connectivity within the DMN was significantly less correlated than M1-GABA and functional connectivity within the motor RSN ( Fisher’s R-to-Z transformation , Z = −2 . 29 , p=0 . 02 ) .",
"To test the neurochemical specificity of this result , we measured M1 glutamate ( assessed via Glx , a composite measure of glutamate and glutamine ) .",
"There was no significant correlation between motor RSN functional connectivity and M1-Glx ( r = −0 . 35 , p=0 . 36; Figure 2E ) , and the correlation between M1-GABA and motor RSN functional connectivity persisted when M1-Glx was co-varied out ( r = −0 . 67 , p=0 . 03 ) .",
"Additionally , using a seed-based correlation analysis , we demonstrated a significant inverse relationship between the degree of correlation between the two M1s and GABA concentration ( r = −0 . 60 , p=0 . 047; Figure 3A ) .",
"There was a trend towards an inverse relationship between the degree of correlation between left M1 and left PMD and GABA concentration ( r = −0 . 49 , p=0 . 12; Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 01465 . 007Figure 3 . The degree of correlation between the left and right primary motor cortices ( M1s ) was significantly related to M1 GABA levels . Values shown are raw Pearson’s correlation coefficients for ease of display .",
"As these are not normally distributed all statistical analyses were performed on log-transformed data ( see ‘Materials and methods’ ) .",
"( A ) Experiment 1 ( r = −0 . 60 , p=0 . 047 ) .",
"( B ) Experiment 4: the correlation between left and right M1s was significantly increased after anodal tDCS ( t ( 9 ) = 1 . 94 , p=0 . 04 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01465 . 00710 . 7554/eLife . 01465 . 008Figure 3—figure supplement 1 . There was a trend towards a relationship between the degree of correlation between the left M1 and the left dorsal premotor cortex ( PMd ) and M1 GABA levels .",
"( A ) Experiment 1 ( r = −0 . 49 , p=0 . 12 ) .",
"( B ) Experiment 2 ( r = −0 . 40 , p=0 . 11 ) .",
"( C ) Experiment 3 ( r = −0 . 67 , p=0 . 02 ) .",
"( D ) Experiment 4: the correlation between left and right M1s showed a trend towards an increase after anodal tDCS ( t ( 9 ) = 1 . 87 , p=0 . 09 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01465 . 008 We broadly replicated these findings in two further separate cohorts: once in a group of 16 young healthy subjects ( experiment 2; see Figure 2—figure supplement 1 ) and one in a group of older adults ( experiment 3; see Figure 2—figure supplement 2 ) .",
"Experiment 4 aimed to investigate whether decreasing GABA within M1 using anodal tDCS ( Stagg et al . , 2009; 2011a ) , increased functional connectivity within the motor RSN .",
"We acquired resting fMRI data before and immediately after anodal tDCS applied to the left M1 in a separate group of 10 subjects .",
"A significant increase in network functional connectivity after tDCS was observed in the motor RSN ( pre: 20 . 7 ± 3 . 34 , post: 26 . 7 ± 2 . 97; t ( 9 ) = 2 . 59 , p=0 . 02; Figure 4 ) .",
"There was also a significant increase in the degree of M1-M1 correlation ( t ( 9 ) = 1 . 94 , p=0 . 04; Figure 3B ) . 10 . 7554/eLife . 01465 . 009Figure 4 . Anodal tDCS applied to M1 , which is known to decrease GABA levels , significantly increased functional connectivity within the motor RSN ( t ( 9 ) = 2 . 59 , p=0 . 02 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01465 . 009"
],
[
"In this study , we investigated the basis of the long-range fluctuations seen in resting fMRI .",
"In three separate cohorts of subjects we have demonstrated a significant negative correlation between the levels of GABA in M1 and the strength of functional connectivity within the motor RSN .",
"This relationship was specific both anatomically and neurochemically: no relationship between glutamate levels and resting connectivity was demonstrated .",
"We believe that the fundamental relationship between resting connectivity within the motor network and M1 GABA demonstrated here is robust , as it is replicated across both ICA and seed-based approaches; whether the MRS and fMRI data were collected on the same day or different days; across different scanners; and in young and old healthy cohorts .",
"Further , we have shown that a technique that is known to decrease GABA within M1 significantly increases functional connectivity of the motor RSN .",
"What do these findings tell us about how activity at a cellular level relates to long-range network connectivity ?",
"The activity within the motor RSN has been shown to be related to fluctuations in the power of beta oscillations ( Brookes et al . , 2011 ) , which have in turn been related to GABA activity ( Hall et al . , 2011 ) .",
"A recent simulation-based paper suggested a positive relationship between local oscillatory power in the gamma band and resting state functional connectivity ( Cabral et al . , 2011 ) , something supported in the visual network by a relationship between RSN connectivity and local gamma frequency oscillatory activity ( Shmuel and Leopold , 2008 ) .",
"Evoked gamma power is also known to be inversely related to extra-synaptic GABA tone ( Towers et al . , 2004 ) .",
"GABA is present in the human brain in three major pools—in pre-synaptic vesicles , as a metabolite in the cytoplasm , and in the extracellular fluid , where it underlies extra-synaptic GABA tone .",
"It is likely that MRS-assessed GABA , which is sensitive to the total amount of GABA within the voxel , more closely reflects extra-synaptic rather than synaptic GABA activity ( Stagg et al . , 2011b ) .",
"While it is not yet clear what the relationship is between local gamma oscillations and beta oscillations , the negative correlation found between motor RSN functional connectivity and MRS-assessed GABA in M1 is consistent with the idea that fluctuations in the power of oscillations underlie long-range resting functional connectivity .",
"In combining observational studies ( Experiments 1–3 ) and an intervention study ( Experiment 4 ) , the data presented here significantly extends and brings coherence to previous findings .",
"One previous study has observed a correlation between GABA in the posteromedial cortex and the strength of the default mode network ( Kapogiannis et al . , 2013 ) .",
"Taken together with our findings , this suggests that the relationships we report are not specific to the motor system , but rather a general feature of resting state functional connectivity networks .",
"It is interesting to note , however , that in addition to a relationship with GABA , Kapogiannis et al . demonstrate a significant positive correlation between glutamate in the posteromedial cortex and the strength of the default mode network .",
"There are a number of reasons why we did not find a similar relationship between glutamate and resting state connectivity .",
"Our MRS approach allowed quantification of Glx , a composite measure of glutamate and glutamine , and therefore may be less sensitive to relationships with glutamate specifically .",
"Alternatively , it may be that different networks have different neurochemical profiles , although this seems less likely .",
"Other previous studies have explored the relationship between task-evoked BOLD responses and GABA: a relationship between GABA levels and BOLD signal in response to a task has been demonstrated in anatomically plausible regions for both motor and visual tasks ( Muthukumaraswamy et al . , 2009; Stagg et al . , 2011a ) , such that higher local GABA concentrations are associated with smaller task-related BOLD signals .",
"Motor-task related BOLD signals have also been shown to correlate with the strength of the motor RSN across individuals ( Kannurpatti et al . , 2012 ) .",
"The relationships demonstrated here , build on this prior work and provide a mechanistic explanation for the previous finding that motor learning , which decreases GABA ( Floyer-Lea et al . , 2006 ) , increases RSN functional connectivity ( Albert et al . , 2009 ) .",
"Taken together , these findings relate RSN functional connectivity to local inhibitory tone within the motor cortex , and thereby may reveal an important neural mechanistic explanation for the RSN changes observed in a wide range of states ."
],
[
"12 volunteers ( 6 male; mean age 23 years [range 21–31 years] ) participated .",
"MR data were acquired on a 3T Siemens/Varian MRI System .",
"MRS data was acquired using a 1 channel transmit/receive head-coil from a 2 × 2 × 2 cm voxel centred on the hand-knob area within the motor cortex in the left hemisphere .",
"First , a standard PRESS sequence ( TR = 3 s , TE = 68 ms ) was used to acquire an unedited spectrum with 32 averages .",
"Then , a MEGA-PRESS sequence ( TR = 3 s , TE = 68 ms ) was used , with 20 ms double-banded Gaussian inversion pulses for simultaneous spectral editing and water suppression; the water suppression band was set to a frequency of 4 . 7 ppm , the editing band alternated between 1 . 9 ppm and 7 . 5 ppm , to collect an edited spectrum with 256 averages .",
"FMRI data were acquired on a separate day using the same scanner as above with a 4 channel receive head coil .",
"200 axial echo planar volumes were acquired ( 43 × 3 mm axial slices , TE = 28 ms , TR = 3000 ms , FOV = 192 × 192 ) .",
"Subjects lay at rest with their eyes open for the duration of the FMRI scan , and had performed no tasks prior to this data being acquired .",
"MRS and fMRI data were acquired from 16 volunteers ( 2 male , mean age 24 years , [range 20–39 years] ) on a 3T Siemens Verio in the same session using a 32 channel receive coil .",
"MRS data were acquired as described in experiment 1 , with 288 averages .",
"Data were pre-processed using in-house scripts to combine data from each channel , remove motion corrupted signal averages and correct for frequency drifts during acquisition .",
"128 axial echo planar volumes of fMRI data were acquired ( 44 × 3 mm axial slices , TE = 30 ms TR = 2410 ms , FOV = 192 × 100 ) .",
"12 volunteers ( 6 male , mean age 61 years [range 45–72 years] ) participated .",
"MRS data were acquired on a 3T Siemens/Varian MRI System exactly as described in experiment 1 .",
"FMRI data were acquired on a 3T Siemens Trio system with a 12 channel receive head coil ( 60 vol , 35 × 3 . 8 mm axial slices , TE = 40 ms , TR = 2000 ms , FOV = 192 × 192 ) .",
"10 volunteers ( 3 male , mean age 22 . 7 years [range 21–24 years] ) participated .",
"FMRI data were acquired exactly as in Experiment 1 , before and immediately after tDCS application , which was delivered while subjects lay supine in the MR scanner .",
"tDCS electrodes ( fitted with 5 kΩ resistors; Easycap , GmbH; Germany ) were sited prior to entry into the scanner in a standard montage .",
"The active electrode was placed over the left motor cortex , centered 5 cm lateral and 2 cm anterior to Cz .",
"The reference electrode was placed on the right supraorbital ridge .",
"As described previously ( Stagg et al . , 2009 ) , high-chloride EEG paste was used as a conducting medium .",
"Anodal , facilitatory , tDCS was delivered via a DC stimulator ( Eldith GmbH; Germany ) , the current had a ramp-up time of 10 s , was held at 1 mA for 10 min and then ramped down over 10s .",
"FMRI data processing was carried out using Multivariate Exploratory Linear Optimized Decomposition into Independent Components ( MELODIC; version 3 . 10 ) part of FSL ( FMRIB’s Software Library , www . fmrib . ox . ac . uk/fsl ) ( Smith et al . , 2004; Beckmann et al . , 2005; Jenkinson et al . , 2012 ) .",
"Individual pre-statistics processing consisted of motion correction brain extraction; fieldmap-based EPI unwarping spatial smoothing using a Gaussian kernel of FWHM 6 . 0 mm and high-pass temporal filtering equivalent to 150 . 0 s ( 0 . 007 Hz ) .",
"Functional data was aligned to structural images ( within-subject ) initially using linear registration ( FMRIB’s Linear Image Registration Tool , FLIRT ) , then optimized using Boundary-Based Registration approach ( Greve and Fischl , 2009 ) .",
"Structural images were transformed to standard space using a non-linear registration tool ( FNIRT ) , and the resulting warp fields applied to the functional statistical summary images .",
"Pre-processed functional data for each subject were temporally concatenated across subjects to create a single 4D dataset .",
"Between-subject analysis was performed using a dual regression technique , as described previously ( Filippini et al . , 2009 ) .",
"Briefly , this approach consisted of three stages .",
"First , the concatenated fMRI data set was decomposed using ICA into 25 components and RSNs of interest were identified using spatial correlations against previously defined maps ( Beckmann et al . , 2005 ) .",
"Next , the dual-regression approach was used to identify the subject-specific RSN maps .",
"This process involved using all 25 components to perform a spatial regression against each separate fMRI data set and then using the resulting normalized time-course matrices to perform a temporal regression to estimate subject-specific maps that reflect the subject specific strength of functional connectivity .",
"The resulting subject-specific RSN map was then masked by the group mean RSN map and the mean value within this region extracted for each subject , used as a measure of the strength of functional connectivity within the RSN .",
"Motion is known to have complex effects on resting state connectivity on a subject-by-subject basis ( Power et al . , 2012 ) , but effects of head motion are greatly ameliorated by the dual regression approach used here , compared against simpler single-regression-based analyses ( Zuo et al . , 2010 ) .",
"Data were preprocessed and registered to standard space as described above .",
"For Experiments 1–3 , the MRS voxel of interest each subject was registered to standard space to give a region of interest ( ROI ) for the left M1 , and then flipped about the midline to give a ROI for the right M1 .",
"An ROI of the PMd was derived from a connectivity-based parcellation of the lateral premotor cortex ( Tomassini et al . , 2007 ) , and was masked by the group mean RSN mask .",
"The timecourse of the fMRI signal from these ROIs was then extracted from the fMRI data , pre-processed as described above .",
"For each subject left and right M1s and left M1 and left PMd were correlated using Pearson’s correlation coefficient .",
"As Pearson’s correlation coefficients are non-normally distributed , the r value for each subject was then log-transformed to give a parametric measure of the degree of functional correlation between the ROIs .",
"This was then correlated for each subject with GABA levels acquired from the left M1 .",
"For Experiment 4 , where no MRS was acquired , the mean MRS voxel location from all subjects in Experiments 1–3 was calculated and this was used as the ROI ."
]
] | [
"Anatomically plausible networks of functionally inter-connected regions have been reliably demonstrated at rest , although the neurochemical basis of these ‘resting state networks’ is not well understood .",
"In this study , we combined magnetic resonance spectroscopy ( MRS ) and resting state fMRI and demonstrated an inverse relationship between levels of the inhibitory neurotransmitter GABA within the primary motor cortex ( M1 ) and the strength of functional connectivity across the resting motor network .",
"This relationship was both neurochemically and anatomically specific .",
"We then went on to show that anodal transcranial direct current stimulation ( tDCS ) , an intervention previously shown to decrease GABA levels within M1 , increased resting motor network connectivity .",
"We therefore suggest that network-level functional connectivity within the motor system is related to the degree of inhibition in M1 , a major node within the motor network , a finding in line with converging evidence from both simulation and empirical studies ."
] | [
"Even when your body is at rest , your brain remains active .",
"Subjects lying in brain scanners without any specific task to perform show coordinated and reproducible patterns of brain activity .",
"Areas of the brain with similar functions , such as those involved in vision or in movement , tend to increase or decrease their activity in sync , and these coordinated patterns are referred to as resting state networks .",
"The functions of these networks are unclear—they may support introspection , memory recall or planning for the future , or they may help to strengthen newly acquired skills by enabling the brain to replay previous learning episodes .",
"There is evidence that resting state networks are altered in disorders such as Alzheimer’s disease , autism and schizophrenia , but little is known about how these changes arise or what they might mean .",
"Now , Stagg et al . have used a type of brain scan called magnetic resonance spectroscopy to gain insights into the mechanisms by which one particular network—the resting motor network—is generated .",
"This network consists of areas involved in planning , monitoring and executing movements , and includes the primary motor cortex , which initiates movements by sending instructions to the spinal cord .",
"The levels of a chemical called GABA—a neurotransmitter molecule that tends to inhibit the activity of nerve cells—were measured in the primary motor cortex of young healthy volunteers as they lay idle in a scanner .",
"GABA levels were negatively correlated with the amount of coordinated activity within the resting motor network .",
"By contrast , no relation was seen between coordinated activity and the levels of the neurotransmitter glutamate , which tends to increase the activity of nerve cells .",
"Furthermore , when a weak electric current was applied through the subjects’ scalp to their primary motor cortex—a technique previously shown to lower levels of GABA in the region—the resting motor network became stronger .",
"In addition to providing new information on how the rhythmic patterns of activity seen in the resting brain arise , the work of Stagg et al . contributes to the more general effort to understand the complex patterns of connections within the human brain ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials",
"Methods"
] | [
"cell biology",
"cancer biology"
] | A kinase-independent function of AKT promotes cancer cell survival | elife-03751-v2 | [
[
"The serine–threonine kinase AKT is a non-receptor kinase that plays critical roles in growth and metabolism ( Pearce et al . , 2010 ) .",
"AKT is aberrantly activated in many human cancers , usually due to mutations in upstream components of the Phosphatidyl-Inositol-3-Kinase ( PI3K ) pathway .",
"AKT signaling is required for tumor maintenance in certain genetic contexts , as has been shown for some cancer cell lines harboring PI3K mutations or amplification of the HER2 receptor tyrosine kinase ( She et al . , 2008 ) .",
"Encouraged by these results , several ATP-competitive and allosteric AKT inhibitors have been identified ( Engelman , 2009 ) , but optimal strategies for the clinical development of these agents are currently unknown ."
],
[
"To address this question , we compared the activity of the allosteric AKT inhibitor MK2206 ( Hirai et al . , 2010 ) with the ATP-competitive AKT inhibitors GSK690693 ( Heerding et al . , 2008; Rhodes et al . , 2008 ) and GDC0068 ( Lin et al . , 2013 ) .",
"Each of these AKT inhibitors dose dependently inhibited AKT kinase activity in the non-small-cell lung carcinoma ( NSCLC ) cell line EBC1 as determined by phosphorylation of three AKT substrates ( PRAS40 , GSK3β , and BAD ) in whole-cell lysates ( Figure 1A and Figure 1—figure supplement 1 ) .",
"Consistent with previous reports ( Okuzumi et al . , 2009 ) , the ATP-competitive AKT inhibitors induced hyper-phosphorylation of AKT at the two regulatory phosphorylation sites threonine 308 and serine 473 .",
"When we compared the ability of the AKT inhibitors to induce cell death using a trypan blue exclusion assay , we observed significantly greater cell death induction by the allosteric inhibitor MK2206 compared to the ATP-competitive AKT inhibitors GSK690693 ( Figure 1B—source data",
"1 ) and GDC0068 ( Figure 1C—source data",
"1 ) despite less complete inhibition of AKT substrate phosphorylation at these drug concentrations ( Figure 1A ) . 10 . 7554/eLife . 03751 . 003Figure 1 . Differential sensitivity of human cancer cells to different classes of AKT inhibitors .",
"( A ) Effect of AKT inhibitors on phosphorylation of AKT and AKT protein substrates .",
"EBC1 cells were treated with the indicated doses of MK2206 and GSK690693 ( top ) , or GDC0068 ( bottom ) for 24 hr .",
"Treated cells were lysed and analyzed by immunoblot with the indicated antibodies .",
"Phosphorylation of PRAS40 , BAD , and GSK3β was quantified by image densitometry ( middle ) .",
"( B ) Allosteric AKT inhibitor MK2206 induces more cell death than ATP-competitive inhibitor GSK690693 in EBC1 human lung cancer cells .",
"Cells were treated with drug or vehicle for 96 hr .",
"Cell death was determined by the trypan blue method .",
"Error bars denote standard error of the mean ( t-test *p ≤ 0 . 05 , **p ≤ 0 . 01 , treatment vs vehicle ) .",
"( C ) Allosteric AKT inhibitor MK2206 induces more cell death than ATP-competitive inhibitor GDC0068 .",
"Experimental conditions were as in Figure 1B .",
"( D ) Allosteric AKT inhibitor MK2206 induces more cell death than the ATP-competitive inhibitor GSK690693 in MDA-MB-361 human breast cancer cells .",
"Cell death was assessed on day 1 , day 3 , day 5 , and day 7 following treatment with vehicle or 2 µM of MK2206 or 2 µM of GSK690693 ( top ) ( t-test *p ≤ 0 . 05 , **p ≤ 0 . 01 , treatment vs vehicle ) .",
"An additional plate from each treatment group was lysed 24 hr after drug treatment and analyzed by Western blot with the indicated antibodies ( bottom ) .",
"( E ) Suppression of MK2206-induced cell death by a drug-resistant allele of AKT1 .",
"MDA-MB-361 cells were stably transduced with HA-tagged wild-type AKT1 or AKT1-W80A and treated with 2 µM MK2206 for 96 hr .",
"Cell death was assessed as above .",
"Expression of the transgenes was confirmed by immunoblot using an HA antibody , and loading was controlled with a vinculin antibody ( inset ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 00310 . 7554/eLife . 03751 . 004Figure 1—source data 1 . Contains source data for Figure 1 and all accompanying Figure 1—figure supplements . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 00410 . 7554/eLife . 03751 . 005Figure 1—figure supplement 1 . MK2206 and GSK690693 cause sustained suppression of AKT-dependent BAD phosphorylation . EBC1 cells were treated with either 2 µM MK2206 or 2 µM GSK690693 and lysates were collected at the indicated times .",
"Ser136 BAD phosphorylation was analyzed by immunoblot as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 00510 . 7554/eLife . 03751 . 006Figure 1—figure supplement 2 . MET-amplified cancer cell lines die in response to an allosteric but not an ATP-competitive AKT inhibitor . EBC1 , H1993 , H1648 , and GTL6 cells were treated with vehicle , 2 µM GSK690693 , or 2 µM MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue method as detailed in the ‘Methods’ section . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 00610 . 7554/eLife . 03751 . 007Figure 1—figure supplement 3 . Breast cancer cell lines with HER2 amplification and/or activating PIK3CA-mutations die in response to an allosteric but not an ATP competitive AKT inhibitor . MDA-MB-361 , MCF7 , and BT-474 breast cancer cells were treated with the indicated doses of GSK690693 or MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue method . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 00710 . 7554/eLife . 03751 . 008Figure 1—figure supplement 4 . Non-small-cell lung cancer ( NSCLC ) cell lines with EGFR gene amplification and/or activating EGFR mutations are resistant to cell death induction by AKT inhibitors . HCC827 ( EGFR-Δ74-750/AMP ) ( top right ) , HCC4006 ( EGFR-Δ747-749/AMP ) ( bottom left ) , and H1975 ( EGFR-T790M-L858R ) ( bottom right ) lung cancer cells were treated with the indicated doses of GSK690693 or MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue method . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 00810 . 7554/eLife . 03751 . 009Figure 1—figure supplement 5 . A drug-resistant allele of AKT2 suppresses MK2206-induced cell death . MDA-MB-361 cells were stably transduced with HA-tagged wild-type AKT2 or AKT2-W80A and treated with the indicated concentrations of MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue method ( left ) .",
"Expression of the transgenes was confirmed by immunoblot using an anti-HA antibody , and anti-vinculin was used to control for loading ( right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 00910 . 7554/eLife . 03751 . 010Figure 1—figure supplement 6 . W80A mutation renders AKT1 and AKT2 resistant to MK2206 . MDA-MB-361 cells were stably transduced with HA-tagged AKT1-W80A or AKT2-W80A and treated with 2 µM MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue method ( left ) .",
"Parental MDA-MB-361 cells or MDA-MB-361 cells stably expressing AKT1-W80A or AKT2-W80A were treated with the indicated doses of MK2206 for 24 hr and lysed .",
"To assess drug effects , lysates were subjected to immunoblot analysis with the indicated antibodies ( right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01010 . 7554/eLife . 03751 . 011Figure 1—figure supplement 7 . Near-complete inhibition of tumor cell proliferation by both allosteric and ATP-competitive inhibitors of AKT . PIK3CA-mutant MDA-MB-361 cells were treated with vehicle , 2 µM MK2206 , or 2 µM GSK690693 ( left panel ) , or vehicle , 2 µM MK2206 , or 4 µM GDC0068 ( right panel ) .",
"The number of viable cells was counted on day 1 , day 3 , day 5 , and day 7 . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01110 . 7554/eLife . 03751 . 012Figure 1—figure supplement 8 . Comparison of AKT kinase inhibition by GSK690693 and MK2206 . BT474 cells ( left ) and MDA-MB-361 cells ( right ) were treated with the indicated doses of either MK2206 or GSK690693 for 24 hr and lysed .",
"Lysates were analyzed by immunoblot with the indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01210 . 7554/eLife . 03751 . 013Figure 1—figure supplement 9 . De-inhibition of ErbB3 and p-IGF-IRβ by GSK690693 and MK2206 . EBC1 and BT474 cells were treated with the indicated doses of MK2206 and GSK690693 for 24 hr and lysates were analyzed by Western blot with the indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 013 We had selected EBC1 lung cancer cells for our initial studies because they harbor amplification of MET , a receptor tyrosine kinase that mediates critical oncogenic signals through the PI3K/AKT axis ( Gherardi et al . , 2012 ) .",
"To test whether other MET-amplified human cancer cells show a similar drug response pattern , we extended our experiments to MET-amplified H1993 and H1648 NSCLC cell lines and GTL-16 gastric cancer cells .",
"MK2206 again induced significantly more cell death than GSK690693 ( Figure 1—figure supplement 2 , Figure 1—source data 1 ) .",
"In cell lines with other PI3K pathway alterations previously associated with ‘AKT-dependence’ ( She et al . , 2008 ) , we also found that MK2006 induced significantly more cell death than ATP-competitive AKT inhibitors .",
"These cell lines included breast cancer cells with HER2 amplification and/or PIK3CA-mutation ( Figure 1D and Figure 1—figure supplement 3 , Figure 1—source data 1 ) .",
"In contrast , MK2206 did not induce cell death in non-transformed bronchial epithelial cells ( data not shown ) .",
"MK2206 also did not induce cell death in lung cancer cell lines with activating EGFR mutations ( Figure 1—figure supplement 4 , Figure 1—source data",
"1 ) which have been linked with aberrant PI3K pathway activation ( Engelman et al . , 2005 ) , suggesting that the mode of PI3K activation is critical in establishing AKT addiction in cancer .",
"To exclude the possibility that the greater cell death induction by the allosteric AKT inhibitor was due to additional effects on proteins other than AKT , we stably expressed a synthetic AKT mutant in which a residue critical for allosteric inhibitor binding ( tryptophan 80 ) ( Green et al . , 2008; Calleja et al . , 2009; Wu et al . , 2010 ) had been replaced by an alanine .",
"Expression of AKT1 W80A or AKT2 W80A but not wild-type AKT1 or wild-type AKT2 completely rescued MK2206-induced cell death ( Figure 1E and Figure 1—figure supplement 5 , Figure 1—source data 1 ) .",
"Western blot analysis confirmed that these AKT alleles are biochemically insensitive to MK2206 and prevented the induction of PARP cleavage , a marker of apoptosis ( Figure 1—figure supplement 6 , Figure 1—source data 1 ) .",
"Of note , all three inhibitors were able to induce near-complete proliferation arrest ( Figure 1—figure supplement 7 , Figure 1—source data 1 ) .",
"These results demonstrate that allosteric and ATP-competitive AKT inhibitors differ in their ability to block AKT-mediated survival , and that this survival signal can be mediated by AKT1 or AKT2 .",
"AKT regulates survival through phosphorylation of several protein substrates .",
"We were therefore surprised that ATP-competitive inhibitors induced less cell death than MK2206 despite greater or equal inhibition of AKT substrate phosphorylation ( Figure 1A and Figure 1—figure supplement 8 ) .",
"We could not explain these results by differential effects on induction of ErbB3 expression or IGF-IRβ phosphorylation ( Figure 1—figure supplement 9 ) which can accompany AKT inhibition and mediate resistance to AKT inhibitors ( Chandarlapaty et al . , 2011 ) .",
"One potential explanation for our observations is that ATP-competitive AKT inhibitors might induce an AKT conformation which can serve as a platform for a kinase-independent survival signal ( Figure 2A ) .",
"If true , ATP-competitive AKT inhibitors might antagonize cell death induction by a variety of cell death stimuli , for example , inhibition of the MET kinase in MET-amplified EBC1 cells .",
"We found this to be the case .",
"Despite inducing a modest amount of cell death as single agent , GSK690693 antagonized the ability of crizotinib to kill EBC1 cells .",
"The allosteric AKT inhibitor MK2206 , in contrast , did not compromise the ability of crizotinib to induce cell death ( Figure 2B , Figure 2—source data 1 ) . 10 . 7554/eLife . 03751 . 014Figure 2 . Catalytically inactive AKT supports the survival of AKT-dependent human cancer cells .",
"( A ) Cartoon illustrating how ATP-competitive AKT inhibitors may promote a kinase-independent pro-survival function of AKT .",
"In this model , ATP-competitive inhibitors of AKT block kinase activity , induce AKT hyper-phosphorylation , and induce the formation of a pro-survival complex .",
"( B ) EBC1 cells were treated with either 25 nM crizotinib , 1 µM GSK690693 , 1 µM MK2206 , or the indicated combinations for 96 hr .",
"Cell death was assessed following treatment by the trypan blue assay .",
"( C ) MCF7 cells or a sub-line generated by stable transduction with kinase-deficient AKT1-K179M were treated with either vehicle or 1 µM MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue method ( left ) .",
"A duplicate set of plates was lysed and analyzed by immunoblot with the indicated antibodies ( right ) to confirm kinase inhibition and transgene expression .",
"( D ) MDA-MB-361 cells or a sub-line stably expressing AKT1-K179M were treated with the indicated doses of MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue assay .",
"( E ) Parental EBC1 cells of EBC1 cells stably expressing AKT1-K179M or AKT2-K181M were treated with MK2206 as indicated .",
"After 96 hr , cell death was assessed as elsewhere .",
"( F ) EBC1 cells expressing doxycycline-inducible hairpins targeting both hAKT1 and hAKT2 and a sub-line expressing a hairpin-resistant kinase-deficient mAKT1-K179M allele were grown in the presence or absence of 2 . 5 µg/ml doxycycline for 7 days .",
"Cell death was assessed on day 7 by the trypan blue assay ( bottom ) .",
"Replicate plates were cultured with or without doxycycline for the indicated times and lysed .",
"Lysates were analyzed by immunoblot ( top ) using the indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01410 . 7554/eLife . 03751 . 015Figure 2—source data 1 . Contains source data for Figure 2 and Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01510 . 7554/eLife . 03751 . 016Figure 2—figure supplement 1 . Kinase-dead AKT2-K181M but not wild-type AKT2 protects from MK2206-induced cell death . EBC1 cells were stably transduced with either HA-tagged wild-type AKT2 or AKT2-K181M .",
"Cells were treated with the indicated doses of MK2206 for 96 hr and cell death was analyzed by the trypan blue method ( left ) .",
"Expression of the transgenes was confirmed by immunoblot with the indicated antibodies ( right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 016 To provide genetic evidence for the kinase-independent AKT function , we took advantage of a synthetic AKT mutant which is catalytically inactive due to substitution of lysine by methionine at position 179 at the ATP-binding site ( AKT K179M ) ( Franke et al . , 1995 ) .",
"When we expressed this mutant in MCF7 breast cancer cells , MDA-MB-361 breast cancer cells and EBC1 lung cancer cells , catalytically inactive AKT ( Figure 2C/D/E , Figure 2—source data",
"1 ) but not wild-type AKT ( Figure 2—figure supplement 1 , Figure 2—source data 1 ) , protected from MK2206-induced cell death .",
"Compared to the complete cell death protection provided by the drug-resistant W80A-AKT allele ( Figure 1E , Figure 1—source data 1 ) , the protection by the K179M-AKT allele was only partial , consistent with the coexistence of kinase-independent and kinase-dependent AKT signals .",
"We also used an AKT knockdown strategy to minimize the effect of AKT overexpression on the stoichiometry of the AKT signaling complex .",
"For this purpose , we first generated EBC1 cells with doxycycline-inducible human-specific shRNAs for both AKT 1 and AKT 2 and then expressed a kinase-deficient ( AKT1-K179M ) AKT1 cDNA of murine origin that is partially resistant to knockdown due to mismatches in the mouse sequence .",
"Protein levels of the hairpin-resistant AKT allele after doxycycline induction were similar to the levels of endogenous AKT in parental EBC1 cells ( Figure 2F , top , lanes 6 and 8 vs lanes 1 and 3 ) .",
"Similar to our results with pharmacological AKT blockade , catalytically inactive AKT protected from cell death following doxycycline treatment and AKT depletion of these cells ( Figure 2F , bottom , Figure 2—source data 1 ) .",
"We next examined the effects of AKT inhibitors on phosphoinositide ( PI ) binding .",
"In the absence of growth factors , AKT is inactive because intermolecular interactions between the pleckstrin homology ( PH ) and kinase domain ( KD ) keep the kinase in a closed conformation ( Calleja et al . , 2009 ) .",
"Following growth factor stimulation , AKT shifts to an open conformation and becomes accessible to phosphorylation on threonine 308 and serine 473 , a process that requires localization of AKT to the plasma membrane through the interaction with the phospholipid products of PI3K , phosphatidyl-inositol-3 , 4 , 5-trisphosphate [PtdIns ( 3 , 4 , 5 ) P3] , and PtdIns ( 3 , 4 ) P2 .",
"Using a PI pull-down assay , we observed that MK2206 markedly impaired phosphoinositide binding ( Figure 3A ) .",
"This result is consistent with findings from structural studies that allosteric AKT inhibitors lock AKT in a closed conformation with its phospholipid binding site blocked by the kinase domain ( Wu et al . , 2010 ) .",
"In contrast to our findings with MK2206 , incubation with the ATP-competitive inhibitor GSK690693 increased binding of AKT to PtdIns ( 3 , 4 ) P2 and PtdIns ( 3 , 4 , 5 ) P3 and this was associated with increased localization of AKT to the plasma membrane ( Figure 3B ) . 10 . 7554/eLife . 03751 . 017Figure 3 . Kinase-dead AKT protects from drug-induced cell death in a PH-domain-dependent manner .",
"( A ) HCT116 AKT1−/− AKT2 −/− cells stably transduced with human influenza hemagglutinin ( HA ) epitope-tagged AKT2 were treated with vehicle , MK2206 , or GSK690693 for 4 hr and lysed .",
"Lysates were subjected to a PIP-binding assay to assess phosphoinositide binding preference .",
"AKT binding was assessed by immunoblot using an AKT-specific antibody .",
"( B ) The localization of AKT2 was assessed in the same cells as in A by HA immunofluorescence following 24 hr treatment with the indicated drugs .",
"Arrows indicate HA staining .",
"( C ) Parental MCF10A cells and MCF10A cells stably transduced with HA-tagged wild-type ( WT ) AKT1 ( lane",
"2 ) or AKT1-K179M ( lane",
"3 ) were analyzed by Western blot with the indicated antibodies .",
"( D ) The localization of WT and K179M-AKT1 in MCF10A cells was assessed by HA immunostaining .",
"Shown are representative immunofluorescence images .",
"Arrows indicate HA staining .",
"( E ) Parental MDA-MB-361 cells or sub-lines stably expressing AKT1-K179M or ΔPH-Myr-AKT1-K179M were treated with the indicated doses of MK2206 for 96 hr .",
"Cell death was assessed by the trypan blue assay .",
"( F ) Parental MDA-MB-361 cells , or sub-lines stably expressing kinase-dead AKT1-K179M , or the PIP-binding-deficient variant AKT1-R86A-K179M were treated with the indicated doses of MK2206 for 96 hr .",
"Cell death was measured by the trypan blue assay . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01710 . 7554/eLife . 03751 . 018Figure 3—source data 1 . Contains source data for Figure 3 and Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01810 . 7554/eLife . 03751 . 019Figure 3—figure supplement 1 . Kinase-deficient AKT mutants display enhanced activation loop phosphorylation at threonine 308 . HCT116 AKT1−/− AKT2 −/− cells were stably transduced with wild-type and kinase-deficient alleles ( K179M for AKT1 and K181M for AKT2 ) of either AKT1 or AKT2 .",
"Stably transduced cells were lysed and analyzed by immunoblot with the indicated antibodies .",
"The ratio of phosphorylated AKT ( Thr308 ) /total AKT was determined for each AKT isoform by image quantification densitometry and is indicated below each lane . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 01910 . 7554/eLife . 03751 . 020Figure 3—figure supplement 2 . Kinase-deficient mutant AKT2 exhibits enhanced membrane localization . EBC1 cells stably transduced with HA-tagged wild-type AKT2 ( top ) or kinase-dead AKT2-K181M ( bottom ) were stained with antibodies against HA .",
"Cells were also stained with DAPI and phalloidin , which binds F-actin and marks membrane ruffles .",
"Note , increased membrane localization of mutant AKT2 even in the absence of membrane ruffles .",
"Shown are representative confocal images of each sample . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 02010 . 7554/eLife . 03751 . 021Figure 3—figure supplement 3 . Biochemical effects of PH-domain removal in kinase-dead AKT . Parental MDA-MB-361 cells or MDA-MB-361 cells stably expressing AKT1-K179M or ΔPH-Myr-AKT1-K179M were treated with the indicated doses of MK2206 for 24 hr and lysed .",
"Lysates were subjected to immunoblot analysis with the indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 02110 . 7554/eLife . 03751 . 022Figure 3—figure supplement 4 . Phosphoinositide binding deficient kinase-dead AKT can protect from drug-induced cell death .",
"( A ) To assess AKT and AKT substrate phosphorylation of mutant AKT1 alleles , parental HCT116 AKT1−/− AKT2 −/− cells , or HCT116 AKT1−/− AKT2 −/− cells stably expressing WT AKT1 , AKT1-K179M , or AKT1-R86A-K179M were lysed and analyzed by Western blot with the indicated antibodies .",
"( B ) Parental MDA-MB-361 cells or MDA-MB-361 cells stably expressing AKT1-K179M or AKT1-R86A-K179M were lysed and analyzed by Western blot with the indicated antibodies .",
"Relative levels of AKT1 and vinculin were quantified by image densitometry and are indicated under each lane .",
"( C ) Parental MDA-MB-361 cells or sub-lines stably expressing kinase-dead AKT1-K179M or the PIP-binding-deficient variant AKT1-R25C-K179M were treated with the indicated doses of MK2206 for 96 hr .",
"Cell death was measured by the trypan blue assay .",
"Lysates from MDA-MB-361 cells expressing AKT1-R25C-K179M were collected after treatment with vehicle or 2 µM MK2206 for 24 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 022 We next explored the structural requirements for the pro-survival signal of catalytically inactive AKT .",
"Similar to GSK690693-bound AKT , the AKT1-K179M ( or AKT2-K181M ) mutant was hyper-phosphorylated at T308 ( Figure 3C and Figure 3—figure supplement 1 ) .",
"This phosphorylation event occurs at the plasma membrane ( Stephens et al . , 1998 ) and immunofluorescence in MCF10A and EBC1 cells confirmed that kinase-dead AKT was constitutively localized to the plasma membrane ( Figure 3D and Figure 3—figure supplement 2 ) .",
"To examine whether membrane localization was sufficient for the pro-survival function of AKT1-K179M , we replaced the PH-domain of AKT1-K179M by a membrane-targeting myristoylation signal and expressed this mutant in EBC1 cells .",
"This mutant was unable to inhibit MK2206-induced cell death ( Figure 3E , Figure 3—source data",
"1 ) despite being constitutively phosphorylated in the presence of MK2206 ( Figure 3—figure supplement 3 ) .",
"Lastly , we abolished the ability of the AKT1-K179M mutant to bind phospholipids by introducing a second substitution at an amino acid residue ( R86 ) critical for binding of AKT to PtdIns ( 3 , 4 , 5 ) P3 and PtdIns ( 3 , 4 ) P2 ( Thomas et al . , 2002 ) .",
"Consistent with a loss of membrane localization , the R86A/K179M double mutant showed loss of AKT phosphorylation at threonine 308 when expressed in HCT116 cells that express no endogenous AKT ( Ericson et al . , 2010 ) ( Figure 3—figure supplement 4A ) and it did not protect from MK2206-induced dephosphorylation in cells which do express endogenous AKT ( Figure 3—figure supplement 4B ) .",
"Abrogation of phosphoinositide binding by the R86A substitution did not impair the pro-survival function of kinase-defective AKT ( Figure 3F , Figure 3—source data 1 ) .",
"We observed a similar result with a second mutation ( R25C ) known to abolish phosphoinositide binding ( Thomas et al . , 2002 ) ( Figure 3—figure supplement 4C , Figure 3—source data 1 ) .",
"Mutations in the coding sequence of AKT are rare in human cancers but provide important insights into AKT regulation ( Parikh et al . , 2012 ) .",
"We wondered whether there was precedence in human cancer for kinase-dead AKT mutations .",
"Our survey of published cancer sequencing data identified five AKT2 kinase domain mutations that occurred in at least two independent samples ( G161V/E , R170W , I289M , H355Y , R368C/H ) ( Figure 4A ) .",
"We engineered each of these mutations into an AKT2 expression vector and stably expressed the mutant protein in HCT116 cells with targeted deletions of AKT1 and AKT2 ( HCT116-DKO ) .",
"AKT2-G161V , but none of the other four kinase domain mutants , showed complete loss of in vitro AKT substrate phosphorylation , comparable to the synthetic kinase-dead K181M-AKT2 mutant included as control in our experiments ( Figure 4B ) .",
"Whole-cell lysates from G161V-AKT2 expressing cells showed loss of phosphorylation for the endogenous AKT kinase substrates PRAS40 and BAD ( Figure 4C ) .",
"In the phosphoinositide pull-down assay , AKT2-G161V showed altered phosphoinositide binding with acquired preference for PtdIns ( 4 , 5 ) P2 , again similar to the synthetic kinase-dead AKT2 mutant ( Figure 4D ) . 10 . 7554/eLife . 03751 . 023Figure 4 . AKT mutants found in human cancer can promote cell survival independently of kinase activity .",
"( A ) Distribution of AKT2 mutations that occur in human cancers in 2 or more independent samples .",
"( B ) HA-tagged wild-type and the indicated AKT2 mutant proteins were immunoprecipitated with an HA antibody from stably transduced HCT116 AKT1−/− AKT2 −/− cells and subjected to non-radioactive in vitro kinase assay .",
"‘No kinase’ control consists of an HA-immunoprecipitate from parental HCT116 AKT1−/− AKT2 −/− cells .",
"Substrate phosphorylation , substrate loading , and AKT2 loading were all measured by immunoblot ( for further details see ‘Methods’ ) .",
"( C ) To evaluate the in vivo kinase activity of various AKT2 alleles , cells described in B were also lysed and analyzed by immunoblot with the indicated antibodies .",
"( D ) To determine the PIP-binding preference of WT and mutant AKT2 , HCT116 AKT1−/− AKT2 −/− cells expressing WT , K181M , or G161V alleles of AKT2 were subjected to PIP-binding assay .",
"AKT binding was assessed by immunoblot using an HA antibody .",
"( E ) Parental or AKT2-G161-expressing immortalized human epidermal melanocytes were plated on melanocyte media and allowed to attach overnight .",
"Cells were then given fresh melanocyte media or switched to RMPI media containing 10% fetal bovine serum .",
"96 hr following media switch , cell death was assessed as elsewhere .",
"Expression of the transgene was confirmed by immunoblot ( inset ) .",
"Vinculin was used as a loading control .",
"( F ) Parental EBC1 cells or EBC1 cells stably expressing exogenous WT AKT1 , AKT1-E17K or AKT1-E17K-K179M were treated with the indicated doses of MK2206 for 24 hr and lysed .",
"To asses target inactivation , lysates were analyzed by Western blot with the indicated antibodies .",
"( G ) Cells described in F were treated with the indicated doses of MK2206 for 96 hr .",
"Cell death was assessed as before .",
"( H ) Model of AKT-dependent protection from apoptosis .",
"AKT becomes fully activated following PI3K activation and subsequent phosphorylation at the T308 and S473 regulatory sites .",
"Fully active AKT negatively regulates pro-apoptotic signals such as BAD and FKHR and positively regulates anti-apoptotic signals such as NFκB through phosphorylation ( kinase-dependent functions ) .",
"Fully AKT also regulates survival signals through kinase-independent activities . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 02310 . 7554/eLife . 03751 . 024Figure 4—source data 1 . Contains source data for Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 024 Since AKT2-G161V was found in a human melanoma sample , we explored the pro-survival potential of this mutant in immortalized human melanocytes .",
"These cells required 12-O-tetradecanoylphorbol-13-acetate ( TPA ) for survival in culture , as has previously been reported ( Arita et al . , 1992 ) , but acquired the ability to survive in TPA-deficient media ( RPMI ) after stable expression of AKT2-G161V ( Figure 4E , Figure 4—source data 1 ) .",
"The increased PI ( 4 , 5 ) P2 binding of the kinase-dead G161V-AKT2 mutant ( Figure 4D ) was reminiscent of the most common somatic AKT mutation in human cancer ( E17K-AKT1 ) which localizes to the plasma membrane due to increased affinity for the constitutive plasma membrane lipid PI ( 4 , 5 ) P2 ( Carpten et al . , 2007; Landgraf et al . , 2008 ) .",
"This raised the question whether kinase activity might be dispensable for the pro-survival functions of the oncogenic E17K-AKT1 mutant .",
"To explore this question , we compared the effects of E17K-AKT1 and a kinase-defective allele of this mutant ( E17K/K179M-AKT1 ) in EBC1 cells .",
"Compared to cells transduced with wild-type AKT , cells transduced with the E17K-AKT1 mutant showed residual AKT phosphorylation at equimolar concentrations of MK2206 ( Figure 4F ) , consistent with the reported reduced sensitivity of E17K-AKT1 to allosteric ATP kinase inhibitors ( Calleja et al . , 2009; Wu et al . , 2010; Parikh et al . , 2012 ) .",
"Expression of E17K-AKT1 provided partial protection from MK2206-induced cell death ( Figure 4G , Figure 4—source data 1 ) .",
"Interestingly , the E17K/K179M-AKT1 double mutant provided a similar degree of protection as the E17K-AKT1 single mutant , suggesting that this cancer-associated PH-domain mutant can mediate survival signals independent of its catalytic activity in certain cellular contexts .",
"Lastly , we identified a human endometrial cancer cell line with a kinase domain mutation in AKT1 ( G311D ) ( Barretina et al . , 2012 ) that failed to induce phosphorylation of endogenous AKT substrates in AKT1/2 DKO HCT116 cells ( Figure 5A ) .",
"While knockdown of endogenous AKT1 or AKT2 resulted in growth inhibition , knockdown of AKT1 resulted in substantially greater cell death induction than knockdown of AKT2 ( Figure 5B , Figure 5—source data 1 ) , suggesting a state of addiction to signals provided by the mutant AKT1 . 10 . 7554/eLife . 03751 . 025Figure 5 . Endometrial cancer cells expressing endogenous kinase-deficient AKT1 are sensitive to AKT1 but not AKT2 knockdown .",
"( A ) AKT-deficient HCT116 cells were stably transduced with wild-type or the indicated mutant AKT1 alleles .",
"In cell AKT kinase activity was determined by assessing the phosphorylation of the AKT substrates PRAS40 and BAD by immunoblot as shown .",
"Expression of vinculin was used as a loading control .",
"Note that AKT1-G311D does not promote AKT substrate phosphorylation .",
"( B ) MFE-319 endometrial cancer cells which carry and endogenous AKT1-G311D mutation ( http://www . broadinstitute . org/ccle/home ) were engineered to express tetracycline-inducible hairpins targeting either AKT1 or AKT2 .",
"Cells were grown in the presence or absence of 2 . 5 µg/ml doxycycline for 6 days .",
"Cell death was assessed on day 7 as before ( left ) .",
"To confirm knockdown of AKT and selectivity of the hairpins , a separate set of plates was grown with or without doxycycline for the indicated times and lysed .",
"Vinculin expression was used as a loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 02510 . 7554/eLife . 03751 . 026Figure 5—source data 1 . Contains source data for Figure 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 03751 . 026"
],
[
"The work presented here represents the first example of a kinase-independent function of AKT ( Figure 4H ) .",
"Our findings that catalytically inactivating kinase domain mutations ( AKT1-K179M , AKT2-K181M , AKT2-G161V ) or ATP-competitive inhibitor binding to wild-type AKT can promote phosphoinositide binding document the critical importance of the kinase domain conformation on AKT PH-domain functions .",
"It also suggests that hyper-phosphorylation of AKT in response to ATP-competitive inhibitors ( Okuzumi et al . , 2009 ) may not only be due to impaired phosphatase access ( Chan et al . , 2011 ) but also due to increased phosphorylation at the plasma membrane .",
"Furthermore , given that the loss of phosphoinositide binding does not completely abolish the pro-survival function of kinase-deficient AKT , we suspect that the PH-domain promotes cell survival through regulation of interacting protein partners , and that perturbations in the kinase domain can influence PH-domain function by altering the ability of AKT to reach the appropriate sub-cellular compartment ( likely those richest in PI ( 4 , 5 ) P2 ) and/or interact with specific effector proteins .",
"Further studies are required to identify the effectors of catalytically inactive AKT and define its contribution to other AKT-regulated cellular processes .",
"Our findings have implications for the development of drugs to block AKT in cancer .",
"Previous studies have shown that cancer-associated AKT mutations that favor an open conformation show reduced sensitivity to allosteric AKT inhibitors but retain sensitivity to ATP-competitive inhibitors ( Calleja et al . , 2009; Wu et al . , 2010; Parikh et al . , 2012 ) .",
"Here , we show that cancer cells with aberrant AKT activation by common PI3K pathway lesions ( HER2 , PIK3CA , MET ) show the opposite drug sensitivity profile with reduced sensitivity to ATP-competitive AKT inhibitors .",
"The inability of ATP-competitive inhibitors to suppress non-catalytic AKT functions in certain genetic contexts may represent a liability compared to allosteric AKT inhibitors .",
"This concept remains to be tested in ongoing clinical trials ."
],
[
"MK2206 , GSK690693 , and crizotinib were purchased from Selleck Chemicals ( Houston , TX ) .",
"GDC0068 was purchased from Active Biochem .",
"Antibodies for immunoblots against pPRAS40 ( T246 ) , PRAS40 , AKT , AKT1 , AKT2 , pAKT1 ( S473 ) , pAKT2 ( S474 ) , pAKT ( T308 ) , pAKT ( S473 ) , pAKT substrate ( RXRXXS*/T* ) , BAD , pBAD , cleaved PARP , pMET ( Y1349 ) , Met , pErk1/2 ( T202/Y204 ) , Erk , pGSK3β ( S9 ) , GSK3β , pFoxo3a ( S253 ) , and Foxo3a were purchased from Cell Signaling Technologies ( Danvers , MA , USA ) .",
"HA , vinculin , and β-actin antibodies for immunoblots were purchased from Sigma-Aldrich ( St . Louis , MO ) .",
"HA . 11",
"Clone 16B12 monoclonal antibody for immunofluorescence was purchased from Covance ( Princeton , NJ ) .",
"DAPI , Alexa-488 anti-mouse secondary antibody , Phalloidin 546 , and Prolong Gold Antifade reagent were purchased from Life Technologies ( Carlsbad , CA ) .",
"HA-Tag antibody Sepharose bead conjugate and non-radioactive AKT kinase assay kit were purchased from Cell Signaling Technologies .",
"PIP Beads Sample pack was purchased from Echelon Biosciences Incorporated ( Salt Lake City , UT ) .",
"EBC1 cells were obtained from the Japanese Collection of Research Bioresources ( JCRB ) Cell Bank .",
"GTL-16 cells were the generous gift of Dr Silvia Giordano ( Instituto di Candiolo , Italy ) .",
"HCT116 AKT1−/− AKT2 −/− ( HCT116-DKO ) cells were kindly provided by Dr Bert Vogelstein ( Johns Hopkins University School of Medicine ) .",
"BT474 , H1648 , H1975 , HCC827 , HCC4006 , MCF7 , MCF10A , and MDA-MB-361 cells were purchased from the American Type Culture Collection ( ATCC ) .",
"Human epidermal melanocytes were purchased from ScienCell Research Laboratories ( Carlsbad , CA , Catalog #2200 ) .",
"HCT116-DKO cells were maintained in McCoy’s 5a Medium supplemented with 10% fetal bovine serum ( FBS ) .",
"MCF10A cells were maintained in Mammary Epithelial Cell Growth Medium ( Lonza , Allendale , NJ ) .",
"Human melanocytes were maintained in melanocyte media ( ScienCell , Catalog #2201 ) .",
"All other cell lines were maintained in DMEM media supplemented with 10% FBS .",
"TRIPZ tetracycline-inducible lentiviral shRNAs targeting human AKT1 and AKT2 were purchased from Thermo Scientific ( Lafayette , CO ) ( AKT1 clones: V3THS_358718 and V3THS_358718; AKT2 clones: V2THS_237948 , V3THS_325553 , and V3THS_325558 ) .",
"pLNCX1-HA-mAKT1 ( murine ) was generated by Dr Morris Birnbaum and obtained from Addgene ( Plasmid #15990 ) .",
"pLNCX-HA-mAKT1-K179M was generated by site-directed mutagenesis .",
"All site-directed mutagenesis was carried out using the QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent Technologies ( Santa Clara , CA ) and verified by Sanger sequencing .",
"pLNCX1-ΔPH-Myr-AKT1-K179M was generated by site-directed mutagenesis using pLNCX1-ΔPH myr-AKT1 ( generated by Dr Morris Birnbaum and obtained from Addgene , plasmid #15989 ) as template .",
"pLNCX1-HA-AKT2 ( human #27295 ) was generated by Dr Morris Birnbaum and obtained from Addgene ( Plasmid #15990 ) .",
"From pLNCX1-HA-AKT2 a HindIII/ClaI restriction fragment was used to subclone AKT2 into pLPCX ( Clontech ) to create pLPCX-HA-AKT2 .",
"All AKT2 mutants used in this study were generated by site-directed mutagenesis using pLPCX-HA-AKT2 as template .",
"pLNCX-HA-AKT1 ( human ) was generated by Dr William Sellers and obtained from Addgene ( Plasmid #9004 ) .",
"pLNCX-HA-AKT1-K179M and pLNCX-HA-AKT1-W80A were generated by site-directed mutagenesis using pLNCX-HA-AKT1 as template .",
"pbabe-hTERT+p53DD was generated by Dr Christopher Counter and obtained from Addgene ( Plasmid #11128 ) .",
"Site directed mutagenesis was done using the following primers:AKT1-E17K ( human ) : Sense: 5′-gctgcacaaacgagggaagtacatcaagacctg-3′ , Antisense: 5′-caggtcttgatgtacttccctcgtttgtgcagc-3′ .",
"mAKT1-K179M ( murine ) : Sense: 5′-ccgctactatgccatgatgatcctcaagaaggagg-3′ , Antisense: 5′-cctccttcttgaggatcatcatggcatagtagcgg-3′ .",
"AKT1-K179M ( human ) : Sense: 5′-ccgctactacgccatgatgatcctcaagaaggaag-3′ , Antisense: 5′-cttccttcttgaggatcatcatggcgtagtagcgg-3′ .",
"AKT1-R25C ( human ) : Sense: 5′-aagacctggcggccatgctacttcctcc-3′ , Antisense: 5′-ggaggaagtagcatggccgccaggtctt-3′ .",
"AKT1-R86A ( human ) : Sense: 5′-cagtggaccactgtcatcgaagccaccttccatgtg-3′ , Antisense: 5′-cacatggaaggtggcttcgatgacagtggtccactg-3′ .",
"AKT1-W80A ( human ) : Sense: 5′-tccgctgcctgcaggcgaccactgtcatcg-3′ , Antisense: 5′-cgatgacagtggtcgcctgcaggcagcgga-3′ .",
"AKT2-K181M ( human ) : Sense: 5′- gctactacgccatgatgatcctgcggaagga-3′ , Antisense: 5′- tccttccgcaggatcatcatggcgtagtagc-3′ .",
"AKT2-G161V ( human ) : Sense: 5′-caaactccttggcaaggtaacctttggcaaagtca-3′ , Antisense: 5′-tgactttgccaaaggttaccttgccaaggagtttg-3′ .",
"AKT2-R170W ( human ) : Sense: 5′-aaagtcatcctggtgtgggagaaggccactg-3′ , Antisense: 5′-cagtggccttctcccacaccaggatgacttt-3′ .",
"AKT2-R368C ( human ) : Sense: 5′-tcatggaagagatctgcttcccgcgcacg-3′ , Antisense: 5′-cgtgcgcgggaagcagatctcttccatga-3′ .",
"AKT2-H355Y ( human ) : Sense: 5′-cttctacaaccaggactatgagcgcctcttcgagc-3′ , Antisense: 5′-gctcgaagaggcgctcatagtcctggttgtagaag-3′ .",
"AKT2-I289M ( human ) : Sense: 5′-tggacaaagatggccacatgaagatcactgactttg-3′ , Antisense: 5′-caaagtcagtgatcttcatgtggccatctttgtcca-3′ ."
],
[
"Cells were harvested directly on tissue culture plates with 1% triton cell lysis buffer ( Cell Signaling Technologies ) supplemented with protease and phosphatase inhibitors .",
"Lysates were sonicated , cleared by centrifugation , and normalized to equal amounts of total protein using the DC protein assay ( Biorad ) .",
"For transduction of wild-type and mutant AKT alleles into EBC1 , HCT116 AKT1−/− AKT2 −/− , MCF7 , and MDA-MB-361 cells , amphotropic retroviruses were generated by co-transfection of retroviral constructs carrying the appropriate cDNA and the packaging plasmid pCL-Ampho ( IMGENEX , San Diego , CA ) into 293T cells .",
"Viral particles were collected after 36 and 60 hr following transfection and target cells were infected for 18 hr with the respective virus .",
"Stable expressors were derived through antibiotic selection .",
"To immortalize human epidermal melanocytes ( iHEM ) , primary melanocytes ( ScienCell ) were retrovirally infected with amphotropic pbabe-hTERT + p53DD virus using the above transduction protocol .",
"For lentiviral transduction of Tet-inducible shRNAs targeting AKT1 and AKT2 , viral particles were produced by co-transfection of shRNA constructs ( listed above ) with two packaging plasmids ( pMD2G and pPAX2 ) into 293T cells .",
"Five different viruses were generated ( 2 AKT1 hairpins and 3 AKT2 hairpins ) .",
"Viral particles were collected at 36 and 60 hr after transfection .",
"All five viruses were mixed , and infection of EBC1 cells was carried out for 8 hr after each virus collection .",
"36 hours after the last infection , cells were selected with 10 µM puromycin to derive stable expressors .",
"Following selection , cells were also transduced with pLNCX-HA-mAKT1-K179M retrovirus and selected with 1 mg/ml G148 to generate cells with a hairpin-resistant AKT1-K179M allele .",
"Cells were seeded on 6 cm dishes and allowed to attach overnight .",
"Cells were then treated with the indicated drugs at the indicated doses .",
"Each treatment group was seeded in triplicate .",
"Following treatment , both attached and unattached cells were harvested and counted on a ViCell Cell Viability analyzer .",
"The instrument uses trypan blue to assess cell death .",
"Cell death was expressed as the fraction of trypan blue-positive cells over the total number of cells .",
"Statistical significance was assessed by one-tailed unequal variance t-test ( ns = p > 0 . 05; * = p ≤ 0 . 05; ** = p ≤ 0 . 01 , *** = p ≤ 0 . 001 ) .",
"AKT kinase assays were carried out using the non-radioactive AKT Kinase Assay Kit from Cell Signaling Technologies ( Catalog #9840 ) according to the manufacturer's instructions with the following modification: HA-Tag antibody Sepharose bead conjugate was used to isolate AKT kinase .",
"Briefly , HCT116 AKT1/2 DKO cells expressing exogenous HA-tagged AKT alleles were lysed in 1× lysis buffer and sonicated .",
"Sonicated lysates were normalized to equal amounts of total protein .",
"AKT was then purified from normalized lysates by HA immunoprecipitation for 3 hr at 4°C .",
"HA beads were then washed two times with 500 µl lysis buffer and once with 1× kinase buffer .",
"Beads were then resuspended in 50 µl kinase buffer , and kinase reaction was carried out for 30 min after addition of 1 µg of substrate and ATP to a final concentration of 200 µM .",
"Reaction was terminated by adding 3× Laemmli buffer .",
"10 µl of kinase reaction were analyzed by immunoblot .",
"Substrate phosphorylation and AKT content in each reaction was measured by immunoblot .",
"HCT116 AKT1−/− AKT2 −/− ( with or without exogenous AKT ) cells were grown on a monolayer , washed once with cold PBS , and harvested on the dish using lysis buffer ( 10 mM HEPES pH 7 . 4 , 150 mM NaCl , 1% Triton , and EDTA-free protease and phosphatase inhibitors ) .",
"Lysates were sonicated and cleared by centrifugation .",
"Cleared lysates were then normalized to equal amounts of total protein .",
"The amount of AKT in each lysate was then quantified by Western blot using an HA antibody and the lysates normalized for AKT content using image analysis of AKT immunoreactive bands .",
"Lysate from parental HCT116 AKT1−/− AKT2 −/− cells was used to normalize a second time for total protein amount .",
"Each lysate was then divided into 10 aliquots to verify the final normalization by Western blot and for PIP binding analysis .",
"PIP beads were obtained from Echelon Biosciences Inc . ( Echelon Inc , Salt Lake City , UT ) .",
"Each lysate was incubated with one PIP variant ( 30 μl of 50% bead slurry equivalent to approximately 200 pmoles ) for 3 hr at 4°C .",
"Beads were washed three times with 10 vol of lysis buffer .",
"Bound proteins were eluted by boiling PIP beads in 3× Laemmli Sample Buffer at 95°C .",
"Cells were washed with 1× PBS at room temperature , fixed in 4% PFA for 10 min , and permeabilized for 2 min in 1× PBS containing 0 . 1% Triton X-100 .",
"Coverslips were blocked with 3% bovine serum albumin ( wt/vol ) and 0 . 1% Triton X-100 in PBS for 30 min .",
"Primary antibody incubation was done for 1 hr in blocking solution , followed by five 1× PBS , 0 . 1% Triton X-100 washes .",
"Secondary antibody incubation was carried out for 1 hr followed by five washes and incubated with DAPI for 2 min to visualize DNA .",
"Fluorescent images were acquired on an upright microscope ( Axio imager; Carl Zeiss ) equipped with 100× oil objectives , NA of 1 . 45 , a camera ( ORCA ER; Hamamatsu Photonics ) , and a computer loaded with image-processing software ( Axiovision ) .",
"For confocal analysis , EBC1 cells were plated on fibronectin coated coverslips , washed with 1× PBS , fixed in 4% PFA for 10 min at room temperature , and permeabilized with 0 . 1% Triton X-100 for 10 min .",
"Coverslips were then incubated in blocking solution ( 10% donkey serum , 0 . 1% BSA in PBS ) for 1 hr , followed by incubation with a primary antibody ( anti-HA ) in blocking solution for 1 hr .",
"Alexa-488 anti-mouse antibodies were used for detection .",
"Coverslips were then incubated with Phalloidin 546 for 30 min .",
"Nuclei were stained with DAPI for 3 min .",
"Coverslips were then mounted using Prolong Gold Antifade reagent and images were acquired with a 40× magnification lens in a Leica TCS-SP5 ( WLL ) multiphoton confocal microscope ."
]
] | [
"The serine–threonine kinase AKT regulates proliferation and survival by phosphorylating a network of protein substrates .",
"In this study , we describe a kinase-independent function of AKT .",
"In cancer cells harboring gain-of-function alterations in MET , HER2 , or Phosphatidyl-Inositol-3-Kinase ( PI3K ) , catalytically inactive AKT ( K179M ) protected from drug induced cell death in a PH-domain dependent manner .",
"An AKT kinase domain mutant found in human melanoma ( G161V ) lacked enzymatic activity in vitro and in AKT1/AKT2 double knockout cells , but promoted growth factor independent survival of primary human melanocytes .",
"ATP-competitive AKT inhibitors failed to block the kinase-independent function of AKT , a liability that limits their effectiveness compared to allosteric AKT inhibitors .",
"Our results broaden the current view of AKT function and have important implications for the development of AKT inhibitors for cancer ."
] | [
"To maintain a healthy body , the ability of our cells to survive and divide is normally strictly controlled .",
"If any cells manage to escape these restrictions , they may rapidly divide and form tumors , which can lead to cancer .",
"A protein called AKT can encourage cells to survive and divide , and in healthy cells it is only allowed to be active at specific times .",
"However , in many cancer cells , the genes that make and control AKT activity can be altered by mutations , which can result in AKT being active at the wrong times .",
"Part of the AKT protein acts as an enzyme called a kinase and adds chemical groups called phosphates to other proteins .",
"The phosphate groups can activate or deactivate these proteins to control cell survival and cell division .",
"However , there are other sections to the AKT protein and it is not clear how they are involved in this protein's activity .",
"In this study , Vivanco et al . show that AKT has another role in cell survival that does not depend on its kinase .",
"The experiments show that even when the kinase part of the protein is missing , AKT can help cancer cells to survive drug treatments and external conditions that would normally kill them .",
"This role requires another section of the protein called the PH-domain .",
"There are several chemicals—called inhibitors—that can stop AKT from working properly , and they have the potential to be used to treat some types of cancer .",
"These inhibitors work in different ways: some were able to block the activity of the kinase , but others inhibited AKT by binding to other parts of the protein .",
"Therefore , to develop AKT inhibitors into effective drugs , it will be important to know precisely what role the protein plays in different types of cancers ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"neuroscience"
] | Engineering vanilloid-sensitivity into the rat TRPV2 channel | elife-16409-v2 | [
[
"Transient receptor potential ( TRP ) channels are a large family of cation selective channels , some of which function in sensory terminals to detect a wide array of different stimuli , including temperature , lipid messengers and natural products such as capsaicin , menthol , wasabi and mustard oil ( Ramsey et al . , 2006 ) .",
"The TRPV1 channel is one of the best studied TRP channels , and is activated by vanilloids like capsaicin from hot chili peppers , extracellular acid , the double-knot toxin ( DkTx ) from tarantula venom and noxious heat ( Bohlen and Julius , 2012; Julius , 2013 ) .",
"Structures of TRPV1 have been solved by cryo-electron microscopy ( EM ) ( Cao et al . , 2013; Liao et al . , 2013 ) ( Figure 1A ) , revealing that the transmembrane domain of the TRP channel adopts a structure that is remarkably similar to voltage-activated potassium channels ( Long et al . , 2007 ) , as well inositol trisphosphate and ryanodine receptors ( Yan et al . , 2015; Zalk et al . , 2015 ) .",
"Protons and DkTx are known to activate TRPV1 from the external side of the pore; the residues involved in proton activation have been identified by mutagenesis ( Jordt et al . , 2000; Ryu et al . , 2003; Ryu et al . , 2007 ) , and the DkTx binding site on the pore domain was initially localized by comparing apo and DkTx-bound cryo-EM structures of TRPV1 ( Cao et al . , 2013; Liao et al . , 2013 ) , and subsequently refined by docking the NMR structure of the toxin into the cryo-EM maps of TRPV1 ( Jara-Oseguera et al . , 2016 ) .",
"A Na+ ion binding site within the external pore nearby to where DkTx binds was recently shown to stabilize the closed state of TRPV1 , and to be allosterically coupled with temperature-sensor activation ( Jara-Oseguera et al . , 2016 ) .",
"Thus the external pore appears to be a critical nexus for allosteric gating in TRPV1 .",
"The cryo-EM structure of TRPV1 in the presence of resiniferatoxin ( RTx ) revealed the presence of an additional density at the interface between the S1-S4 domains and the S5-S6 pore domains near the internal side of the membrane ( Figure 1B ) ( Cao et al . , 2013 ) .",
"Although this EM density is insufficient to identify where vanilloids bind , it is consistent with mutagenesis and computational studies ( Elokely et al . , 2016; Gavva et al . , 2004; Jordt and Julius , 2002 ) . 10 . 7554/eLife . 16409 . 003Figure 1 . Structure of TRPV1 and characterization of TRPV1 constructs expressed in yeast .",
"( A ) Side view of the RTx/DkTx bound cryo-EM structures of TRPV1 refined using the NMR structure of DkTx ( Bae et al . , 2016 ) .",
"The EM density tentatively assigned to RTx is colored orange and the backbone of DkTx is colored bright purple .",
"( B ) Close-up view of the RTx EM density ( orange ) with residues closer than 12 . 5 Å from the center of mass of that density shown in stick representation .",
"The four residues studied here ( S512 , T550 , M547 , E570 ) are colored red and the others yellow .",
"( C ) In-gel fluorescence of SDS-solubilized membranes from S . cerevisiae cells overexpressing GFP-tagged TRPV1 domain constructs using the following abbreviations: FL ( Full-lenth ) , p . m . ( pore mutant; deletion of 629–647 ) ( Garcia-Sanz et al . , 2004 ) , N-S4 ( N terminus through 575 ) , N-S6 ( N-terminus through 704 ) , S1-S6 ( 423 to 704 ) , S1-C ( 423 to C terminus ) and S1-S4 ( 431 to 575 ) .",
"( D ) Fluorescence-coupled size exclusion chromatography ( FSEC ) traces of GFP-tagged TRPV1 S1-S4 domain solubilized in DDM .",
"The first peak is the void volume and the second corresponds to monomeric domain .",
"( E ) 3H-RTx binding to intact S . cerevisiae cells expressing full-length TRPV1 and its separate domains , all of which contain GFP tags on their C-termini .",
"Data were normalized to number of cells because the trucated constructs express to higher levels compared to TRPV1 , and data were corrected for non-specific binding as described in Materials and methods .",
"Data points are the mean + S . E . M . for triplicate determinations .",
"Smooth function for full-length GFP-tagged TRPV1 is a fit of the Hill equation to the data with Kd and Hill slope ( nH ) values of 7 . 0 nM and 0 . 78 for TRPV1 .",
"( F ) Binding of 3H-RTx to S . cerevisiae cells containg GFP-tagged TRPV1 , N-terminus His-tagged TRPV1 ( without GFP ) , or dual GFP- and His-tagged TRPV1 construct harboring the Y511A mutation .",
"Smooth functions are fits of the Hill equation to the data with Kd and nH values of 7 . 2 nM and 0 . 82 for TRPV1 , 30 nM and 0 . 6 for TRPV1-His , and >238 nM and 1 . 1 for TRPV1 Y511A .",
"Data points are the mean + S . E . M . for triplicate determinations . DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 003 In contrast to TRPV1 , relatively little is known about the physiological functions and operational mechanism of its closest homolog , the TRPV2 channel .",
"TRPV2 from rats and mice can be activated by extreme temperature above 52°C ( Caterina et al . , 1999; Neeper et al . , 2007; Qin , 2011; Yao et al . , 2011 ) , however , TRPV2 knock-out mice have normal sensitivity to noxious heat ( Park et al . , 2011 ) and the TRPV2 channel from humans is insensitive to heat ( Neeper et al . , 2007; Qin , 2011; Yao et al . , 2011 ) .",
"Thus , temperature sensing does not appear to be a conserved function of the TRPV2 channel ( Peralvarez-Marin et al . , 2013 ) .",
"Indeed , the absence of high-affinity selective activators and inhibitors of TRPV2 has made exploration of this channel and its functions challenging ( Peralvarez-Marin et al . , 2013 ) , although the channel can be activated with low affinity by non-selective agonists such as 2-aminoethoxydiphenyl borate ( 2-APB ) ( Hu et al . , 2004; Juvin et al . , 2007; Neeper et al . , 2007 ) , probenecid ( Bang et al . , 2007 ) and cannabinoids ( Qin et al . , 2008 ) , and inhibited by ruthenium red .",
"The TRPV2 channel is more widely expressed compared to TRPV1 , and has been identified in sensory neurons , spinal cord , brain , pancreas , muscle and the immune system ( Peralvarez-Marin et al . , 2013 ) .",
"Although relatively few functional deficits have been observed in TRPV2 knock-out mice other than increased perinatal lethality ( Park et al . , 2011; Peralvarez-Marin et al . , 2013 ) , the channel has been proposed to play important roles in osmosensation , mechanosensation , innate immunity and cancer ( Katanosaka et al . , 2014; Link et al . , 2010; Monet et al . , 2010; Peralvarez-Marin et al . , 2013; Shibasaki et al . , 2010; Zanou et al . , 2015 ) .",
"The goal of the present study was to confirm the location of the binding site for vanilloids on TRPV1 , and to explore whether TRPV2 channels share common gating mechanisms with TRPV1 .",
"The recent cryo-EM structure of TRPV2 reveals that the protein adopts a structure that is very similar to TRPV1 ( Zubcevic et al . , 2016 ) , suggesting that the gating mechanisms of the two TRP channels may be more similar than their distinct pharmacology and functional properties might suggest .",
"We began by investigating whether biochemically well-behaved domains of TRPV1 could be identified that are competent to bind vanilloids such as resiniferatoxin ( RTx ) .",
"Although we succeeded in producing truncated constructs of TRPV1 , only the full-length channel binds RTx with high affinity .",
"We also discovered that DkTx binding to the external pore of the full-length channel can enhance the affinity of RTx , providing critical support for an allosteric model for activation of TRPV1 ( Jara-Oseguera et al . , 2016 ) .",
"Using the cryo-EM structure of TRPV1 as a guide , we interrogated the putative vanilloid binding pocket with mutations in TRPV1 and our results confirm the location of the vanilloid binding site seen in the cryo-EM structure .",
"We also attempted to engineer vanilloid sensitivity into the TRPV2 channel and made the remarkable discovery that only two mutations in TRPV2 are sufficient to enable vanilloids to bind with high affinity and to activate the channel .",
"Single channel recordings of the vanilloid-sensitive TRPV2 channel show that high open probability ( Po ) can be achieved with RTx , and that the permeation properties of TRPV2 also resemble those of TRPV1 ."
],
[
"Our initial objective was to see if we could define minimal domains of TRPV1 that were competent to bind the vanilloid RTx .",
"Many of the previously identified mutants reported to alter RTx binding or capsaicin activation are localized within the S1-S4 domain of TRPV1 ( Elokely et al . , 2016; Gavva et al . , 2004; Jordt and Julius , 2002; Yang et al . , 2015 ) , and in structurally related Kv channels , the S1-S4 region has been shown to form an autonomous domain ( Jiang et al . , 2003a; 2003b ) that is capable of imparting strong voltage-sensitivity when grafted onto relatively voltage-insensitive K+ channels ( Ben-Abu et al . , 2009; Lu et al . , 2001; 2002 ) .",
"We began by expressing rTRPV1 and a series of truncated constructs in Saccharomyces cerevisiae using a previously described expression vector with a C-terminal GFP tag optimized for fluorescence-based screening of eukaryotic membrane protein expression and stability ( Drew et al . , 2008; Parker and Newstead , 2014 ) .",
"In addition to the full-length rTRPV1 , we expressed the truncated constructs containing the N-terminus through S4 ( N-S4 ) , the N-terminus through S6 ( N-S6 ) , S1 through S6 ( S1-S6 ) and S1 through the C-terminus ( S1-C ) , as well as controls such as the Y511A mutant and a deletion within the outer pore ( pore mutant ) thought to stabilize the full-length rTRPV1 channel ( Garcia-Sanz et al . , 2004 ) .",
"Crude membrane preparations of small-scale S . cerevisiae trials of full-length TRPV1 and truncations yielded clean , prominent bands in Tris-Glycine gels imaged with in-gel fluorescence ( Figure 1C ) , indicating robust expression of the proteins without significant degradation .",
"Constructs containing the S1-S4 or S1-S6 domains of TRPV1 displayed good monodispersity when evaluated with fluorescence-coupled size exclusion chromatography ( FSEC ) using a range of detergents previously used to successfully solubilize eukaryotic membrane proteins ( e . g . Figure 1D ) .",
"Although purification attempts were initially promising for the S1-S4 domain , with clear protein bands visible in elution during the first step of purification , cleavage of GFP with TEV protease led to aggregation and precipitation ( not shown ) .",
"To evaluate whether these constructs were competent to bind vanilloids , we initially measured the binding of [3H]-RTx ( Szallasi and Blumberg , 1999; Szallasi et al . , 1999 ) to intact yeast cells expressing each of the constructs .",
"Although protein levels were comparable for these constructs based on GFP fluorescence , only the full-length TRPV1 channel displayed saturable 3H-RTx binding to intact cells , with a Kd of 7 . 0 nM ( Figure 1E ) .",
"To demonstrate that only the full-length GFP-tagged channel presents specific high-affinity binding to RTx , we studied the Y511A mutant that disrupts vanilloid binding ( Jordt and Julius , 2002; Yang et al . , 2015 ) , and found that the mutant greatly diminished RTx binding to GFP-tagged TRPV1 ( Figure 1F ) .",
"We also measured the binding of RTx to a His-tagged TRPV1 construct and observed that RTx bound with an affinity that was comparable to that observed with GFP-tagged TRPV1 ( Figure 1F ) .",
"The polymodal activation of TRPV1 has been widely conceptualized using allosteric models wherein binding of activators ( such as vanilloids , protons , DkTx or voltage ) are allosterically coupled to opening of the pore ( Brauchi et al . , 2004; Jara-Oseguera et al . , 2016; Matta and Ahern , 2007 ) .",
"The allosteric nature of these interactions has begun to be explored using electrophysiological measurements of channel function ( Jara-Oseguera et al . , 2016; Yao et al . , 2010 ) , but has thus far not been investigated biochemically .",
"We therefore tested whether binding of DkTx can enhance the affinity of TRPV1 for RTx , which would be predicted by an allosteric model wherein the two activators promote opening by binding more tightly to the open state .",
"For these experiments we used a 1D4-tagged TRPV1 construct where expression in S . cerevisiae was driven by a constitutive promoter and we performed binding assays on yeast membranes where the vanilloid would have free access to the channel protein ( see Methods ) .",
"Under these conditions , we measured a Kd for RTx binding to 1D4-tagged TRPV1 of 0 . 6 nM under control conditions , about ten fold higher affinity than what we measured in intact cells using GFP-tagged TRPV1 .",
"When we measured the concentration-dependence of RTx binding to 1D4-tagged TRPV1 in the presence of DkTx , we observed that the tarantula toxin enhanced the affinity for RTx by about 3-fold ( Figure 2A ) .",
"The effects of DkTx were concentration-dependent ( Figure 2B ) and were observed independent of whether the binding assay was performed at 37°C ( our standard temperature ) or at 30°C ( Figure 2A ) .",
"These results demonstrate that DkTx binds to our preparation of TRPV1 , and that binding of DkTx and RTx are allosterically coupled . 10 . 7554/eLife . 16409 . 004Figure 2 . DkTx allosterically modulates the affinity of RTx for the TRPV1 channel .",
"( A ) 3H-RTx binding to S . cerevisiae membranes containg full-length 1D4-tagged TRPV1 in the presence or absence of DkTx ( 0 . 7 µM ) , normalized to total protein per sample .",
"Smooth functions are fits of the Hill equation to the data with Kd and Hill slope ( nH ) values 0 . 53 nM ( nH=1 . 24 ) in control and 0 . 19 nM ( nH=0 . 92 ) in the presence of DkTx at 37°C .",
"At 30°C Kd and nH values were 0 . 8 nM ( nH=1 . 1 ) in control and 0 . 33 nM ( nH=1 ) in the presence of DkTx .",
"Data points are mean + S . E . M . for triplicate determinations .",
"( B ) RTx Kd values in the absence and presence of different concentrations of DkTx measured at 37°C .",
"Values for individual cells are shown as circles and the mean + S . E . M . as bars for between 3 to 5 separate experiments , each of which were determined in triplicate . DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 004 To further investigate the putative vanilloid binding pocket in TRPV1 , we inspected the cryo-EM structure of TRPV1 , looking for residues that are located nearby to the putative RTx density ( Figure 1B; regions/residues highlighted in yellow ) and that differ between TRPV1 and TRPV2 channels ( Figure 3—figure supplement 1; regions highlighted in magenta ) .",
"This analysis identified S512 ( F472 in TRPV2 ) , M547 ( L507 in TRPV2 ) , T550 ( L510 in TRPV2 ) and E570 ( Q530 in TRPV2 ) as potentially important determinants of RTx binding to TRPV1 , consistent with previous experimental demonstrations that mutations of these residues weaken activation of TRPV1 by vanilloids ( Elokely et al . , 2016; Gavva et al . , 2004; Jordt and Julius , 2002; Yang et al . , 2015 ) .",
"To quantify the contribution of each of these residues to TRPV1 channel activation by RTx under our experimental conditions , we mutated each to the corresponding residue in TRPV2 .",
"We then expressed each construct in oocytes and used the two electrode voltage-clamp technique to study activation by RTx and by the non-specific TRPV channel activator 2-APB .",
"We recorded time courses for channel activation by each agonist while stepping the membrane potential from −60 to +60 mV every 3 s from a holding potential of 0 mV using an external recording solution containing 100 mM KCl ( internal K+ concentration in a healthy oocyte is 100 mM ) ( Figure 3A , left ) .",
"We also recorded current-voltage ( I-V ) relations by stepping over a wider range of voltages in the absence and presence of activators , or in the presence of ruthenium red ( RR ) , a non-specific inhibitor of TRP channels ( Figure 3A , right ) .",
"Because RTx has high affinity for TRPV1 and its interaction with perfusion tubing makes it difficult to reliably control its concentration in the low nM range , we applied the vanilloid at 100 nM , a concentration that is saturating based on our RTx binding assay ( Figure 2A ) .",
"In the case of the wild-type TRPV1 channel , 2-APB produced reversible activation of outward and inward currents , RTx produced effectively irreversible activation on the time-scale of our recordings , and RR blocked the RTx-activated currents ( Figure 3A , left ) .",
"The I-V relations we recorded gave results that were consistent with the time courses , and we used these to present results from a population of cells ( Figure 3A , right ) .",
"Similar results were obtained with each of the four TRPV1 mutants ( Figure 3B–E ) , with the exception being the S512F mutant , where we could readily see dissociation of RTx after removing the vanilloid from the recording chamber ( Figure 3B ) , suggesting that this mutant weakens the affinity of RTx .",
"Although previous studies suggest that some of these residues are determinants of vanilloid-sensitivity in TRPV1 ( Gavva et al . , 2004; Jordt and Julius , 2002; Yang et al . , 2015 ) , their individual substitution by the equivalent residues in TRPV2 is not sufficient to produce large disruptions in the activation by a saturating concentration of RTx . 10 . 7554/eLife . 16409 . 005Figure 3 . RTx sensitivity of individual mutations in TRPV1 . ( A ) Voltage protocols used to measure time courses for activation of TRPV1 ( left ) or I-V relations ( right ) .",
"( B , left panel )",
"Representative time course for WT TRPV1 activation in response to 2-APB and RTx measured from outward currents at +60 mV and from inward currents at -60 mV .",
"Pulses were given every 3 s .",
"The colored horizontal lines indicate application of agonists ( 2 mM 2-APB , 100 nM RTx , 50 µM RR ) .",
"The dotted horizontal line indicates the zero-current level .",
"( B , right panel )",
"Mean normalized I-V relations obtained in control ( open symbols , not visible ) , 2-APB ( blue ) , RTx ( red ) and RR immediately after RTx ( black filled symbols ) .",
"Currents were normalized to the value in the presence of saturating 2 mM 2-APB .",
"Data are expressed as mean ± S . E . M . ( n = 4 ) .",
"( C-F )",
"Time courses and I-V relations ( n=3–5 ) for individual mutations as indicated , studied using the same protocols as in ( A , B ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 00510 . 7554/eLife . 16409 . 006Figure 3—figure supplement 1 . Sequences of the transmembrane regions of rat TRPV1-4 channels . Light yellow boxes indicate residues within 12 . 5 Å of the center of mass of the sharpened RTx EM density ( see Figure 1B ) .",
"The four residues near the vanilloid binding pocket ( 512 , 547 , 550 , and 570 in TRPV1 ) that differ between TRPV1 and TRPV2 are shown in magenta . DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 006 We next combined all four mutants to make a quadruple mutant ( TRPV1 QM ) , which completely lost sensitivity to RTx while retaining sensitivity to 2-APB ( Figure 4A ) , suggesting that differences between TRPV1 and TRPV2 at some or all of these positions can explain the differential sensitivity of the two TRP channels to RTx .",
"Although we did not measure the binding affinity of RTx for the TRPV1 QM , leaving open the possibility that the vanilloid binds without activating the channel , this is unlikely because individually the T550I and M547L mutants dramatically weaken RTx binding affinity ( Gavva et al . , 2004 ) .",
"To further define which residues are most critical , we made double mutants where we combined S512F , the mutation that speeds RTx dissociation , with each of the other three mutations .",
"Both S512F/M547L and S512F/T550L channels lost sensitivity to RTx , whereas the S512F/E570Q mutant retained RTx sensitivity ( Figure 4B–D ) , indicating that S512 , M547 and T550 are more important determinants of TRPV1 activation by RTx than E570 .",
"We also examined all four possible triple mutants , and only the mutant that left S512 intact retained sensitivity to RTx ( Figure 4—figure supplement 1 ) .",
"Collectively , these results suggest that",
"1 ) S512 is the most critical determinant of RTx sensitivity ,",
"2 ) M547 and T550 are also important , and",
"3 ) E570 may or may not contribute to the differential sensitivity of TRPV1 and TRPV2 to RTx . 10 . 7554/eLife . 16409 . 007Figure 4 . TRPV1 quadruple mutant and double mutant responses to RTx .",
"( A , left panel )",
"Representative time courses for TRPV1 QM ( S512F , M547L , T550L , E570Q ) activated by 2-APB ( 2 mM ) and RTx ( 100 nM ) measured at +60 and -60 mV .",
"RR ( 50 µM ) was applied after RTx .",
"Same pulse protocol as Figure 3 .",
"The dotted horizontal line indicates the zero-current level .",
"( A , right panel )",
"Mean normalized I-V relations obtained in control ( open circles; not visible ) , 2-APB ( blue ) and RTx ( red ) .",
"Currents were normalized to the value in the presence of 2 mM 2-APB .",
"Data are expressed as mean ± S . E . M . ( n = 3 ) .",
"( B-D Left and right panel )",
"Time course and I-V relations of double mutants of TRPV1 activated by 2-APB ( 2 mM ) or RTx ( 100 nM ) obtained the same way as in ( A ) .",
"For the I-V relations in ( D ) , currents measured when RR was applied immediately after RTx are shown as filled black symbols .",
"Currents were normalized to the value in the presence of 2 mM 2-APB ( n=3–5 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 00710 . 7554/eLife . 16409 . 008Figure 4—figure supplement 1 . TRPV1 triple mutant responses to RTx .",
"( A , left panel )",
"Representative time courses for a TRPV1 triple mutant activated by 2-APB ( 2 mM ) and RTx ( 100 nM ) measured at +60 and -60 mV .",
"RR ( 50 µM ) was applied after RTx .",
"Same pulse protocol as Figure 3 .",
"The dotted horizontal line indicates the zero-current level .",
"( A , right panel )",
"Mean normalized I-V relations under different conditions using the color codes in Figure 3 .",
"Currents were normalized to the value in the presence of 2 mM 2-APB .",
"Data are expressed as mean ± S . E . M . ( n = 3 ) .",
"( B–D Left and right panel ) , Time course and I-V relations of the other TRPV1 triple mutants in either 2-APB ( 2 mM ) or RTx ( 100 nM ) obtained the same way as in ( A ) .",
"Currents were normalized to the value in the presence of 2 mM 2-APB ( n=3–5 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 008 We began investigating the properties of TRPV2 by testing whether the channel is sensitive to RTx .",
"Although TRPV2 could be activated by 2-APB , the channel was completely insensitive to RTx ( Figure 5A ) , consistent with previous reports that this TRP channel is insensitive to capsaicin ( Caterina et al . , 1999 ) .",
"We then introduced all four TRPV1 residues that contribute to RTx sensitivity into TRPV2 to make the TRPV2 QM , and observed that the channel remained insensitive to capsaicin , but was robustly activated by the higher affinity vanilloid RTx ( Figure 5B ) .",
"RTx produced much stronger activation when compared to 2-APB , and we observed little evidence for dissociation of the vanilloid after its removal from the recording chamber ( Figure 5B ) , suggestive of strong and stable binding to TRPV2 QM .",
"To examine the strength of RTx binding to the TRPV2 channel , we undertook binding assays after expressing 1D4-tagged TRPV2 and TRPV2 QM in S . cerevisiae and isolating yeast membranes , similar to the experiments described above with TRPV1 ( Figure 2 ) .",
"Although we only observed weak binding of RTx to TRPV2 , the TRPV2 QM displayed robust and high-affinity RTx binding with a Kd of 18 nM ( Figure 5C ) .",
"The affinity of RTx for the TRPV2 QM is about 30-fold lower compared to TRPV1 , suggesting that other differences between TRPV1 and TRPV2 may influence RTx affinity .",
"Indeed , there are eight other residues located near the RTx binding pocket that differ between TRPV1 and TRPV2 ( see Figure 3—figure supplement 1 ) , which may also explain why the TRPV2 QM is not activated by capsaicin .",
"To examine the relative importance of the four residues for mediating RTx sensitivity in the TRPV2 QM , we constructed triple mutants and evaluated their sensitivity to RTx electrophysiologically .",
"All three triple mutants containing the F472S mutation ( equivalent to S512 in TRPV1 ) were sensitive to RTx , whereas the one triple mutant that retained F472 was insensitive to RTx ( Figure 5D ) , consistent with a dominant role of S512 that we observed in TRPV1 ( Figure 4; Figure 4—figure supplement 1 ) .",
"We also investigated the RTx sensitivity of three double mutants containing the F472S mutation , and observed that either L507M or L510T rendered the TRPV2 channel sensitive to RTx ( Figure 5—figure supplement 1 ) , again matching our results for loss of function experiments in TRPV1 and demonstrating that only two point mutations are required to make the TRPV2 channel sensitive to RTx .",
"Notably , the F472S single mutation is likely not sufficient to confer RTx sensitivity to TRPV2 , as the F472S/Q530E was unresponsive to the vanilloid ( Figure 5—figure supplement 1C ) . 10 . 7554/eLife . 16409 . 009Figure 5 . RTx binds to and activates the TRPV2 QM .",
"( A , left panel )",
"Representative time courses for WT TRPV2 in the presence of 2-APB ( 4 mM ) or RTx ( 100 nM ) measured at +60 and -60 mV .",
"Same pulse protocol as Figure 3 .",
"The dotted horizontal line indicates the zero-current level .",
"( A , right panel )",
"Mean normalized I-V relations under different conditions using the color codes from Figure 3 .",
"Currents were normalized to the value in the presence of 2-APB ( 4 mM ) .",
"Data are expressed as mean ± S . E . M . ( n = 3 ) .",
"( B , left panel )",
"Response of the quadruple mutant of TRPV2 ( F472S , L507M , S510T , Q530E ) to 2-APB ( 4 mM ) ，capsaicin ( 50 µM ) and RTx ( 100 nM ) obtained as in ( A ) .",
"( B , right panel )",
"Mean normalized I-V relations under different conditions using the color codes from Figure 3 .",
"Data are expressed as mean ± S . E . M . ( n=4–8 ) .",
"( C ) Binding of 3H-RTx to membranes containing 1D4-tagged TRPV2 QM ( red ) and 1D4-tagged WT TRPV2 ( black ) .",
"Membranes were prepared from yeast expressing the two constructs to comparable levels as judged by westerns using a 1D4 mAb and equal amounts of membranes were used for assessing binding of RTx to the two TRPV2 constructs , and data were corrected for non-specific binding as described in Materials and methods .",
"Smooth functions are fits of the Hill equation to the data with Kd and nH values of 18 nM and 1 . 7 for TRPV2 QM and of > 200 nM and 0 . 9 for wt TRPV2 .",
"( D ) Summary of the response of TRPV2 , TRPV2 QM and thee triple mutants to RTx .",
"Currents measured in response to RTx ( 100 nM ) at +60 mV are normalized to that measured in response to 2-APB ( 4 mM ) .",
"Values for individual cells are shown as circles and the mean + S . E . M . as bars . DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 00910 . 7554/eLife . 16409 . 010Figure 5—figure supplement 1 . Responses of TRPV2 double mutants to RTx .",
"( A-C )",
"Representative time courses for TRPV2 double mutants activated by 2-APB ( 4 mM ) or RTx ( 100 nM ) measured at +60 and −60 mV .",
"RR ( 50 µM ) was applied as indicated .",
"See Figure 3 for the voltage protocol .",
"The dotted horizontal line indicates the zero-current level .",
"( D ) Summary of the response of TRPV2 double mutants to RTx ( 100 nM ) normalized to that of 2-APB ( 4 mM . ) Values for individual cells are shown as circles and the mean + S . E . M . as bars ( n=3–5 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 010 Previous studies on TRPV1 have shown that vanilloids such as capsaicin promote the open state with high efficacy , with maximal open probabilities ( Po ) of around 0 . 8 to 0 . 9 at saturating concentrations of the agonist ( Hui et al . , 2003; Oseguera et al . , 2007; Premkumar et al . , 2002 ) .",
"To explore the extent to which RTx promotes the open state of the TRPV2 QM , we investigated the unitary properties of the channel using inside-out patch recordings obtained from transiently transfected HEK293 cells .",
"In a patch containing a single TRPV2-QM channel , we observed no spontaneous channel activity prior to application of any agonist ( data not shown ) , but a few seconds of exposure to a solution containing 100 nM RTx resulted in robust channel activation ( Figure 6A ) .",
"The channel entered a mode where the Po was very close to 1 for the entirety of the recording ( approximately 8 min ) , presenting only brief closures ( Figure 6A and E ) .",
"The conductance of the main conducting state was 101 pS in symmetrical 140 mM NaCl at +90 mV ( Figure 6A , D and G ) , but occasional subconducting states could also be detected ( Figure 6C ) .",
"We observed pronounced rectification to the single channel conductance , with a value of 28 pS at -90 mV ( Figure 6B , D and G ) .",
"These results reveal that the single channel conductance of TRPV2 QM is quite high and that it rectifies , two properties that are similar to what has been observed for TRPV1 ( Hui et al . , 2003; Oseguera et al . , 2007; Premkumar et al . , 2002 ) .",
"Two other patches containing two TRPV2 QM channels each presented a slightly lower Po at 100 nM RTx ( Figure 6F ) , as the channels in these patches appeared to enter long-lived resting states between bursts of maximal Po ( Figure 6—figure supplement 1A–C ) .",
"When a higher concentration of RTx was used ( 400 nM ) , long-lived closed states could be detected in two patches with a single channel and two patches with two channels each ( Figure 6F and Figure 6—figure supplement 1D–F ) .",
"From these results we conclude that the permeation properties of TRPV2 channels are similar to TRPV1 channels and that RTx promotes the open state with high efficacy . 10 . 7554/eLife . 16409 . 011Figure 6 . RTx activates the TRPV2 QM channel with high efficacy and affinity .",
"( A ) Single-channel recordings of TRPV2 QM obtained from an inside-out patch at +90 mV in the presence of 100 nM RTx .",
"Currents were elicited by 500 ms pulses to +90 mV from a holding potential of −90 mV applied every 200 ms . A trace without openings obtained in control solution prior to RTx application was used for leak-subtraction .",
"The red continuous horizontal lines indicate the zero-current level ( closed channel - C ) and the dotted lines the current level for an open channel - O . The blue dotted rectangle denotes a portion of the recording that is shown in higher magnification in ( C ) .",
"( B ) Single-channel recordings from the patch in ( A ) obtained at the holding potential of -90 mV .",
"The continuous red lines are the zero current level ( closed channel – C ) and the dotted lines denote an open channel ( O ) .",
"( C ) Magnification of the single-channel recordings in the dotted black box in ( A ) showing the occurrence of subconductance levels , denoted by ( S ) and the dashed black line .",
"( D ) Normalized all-points histograms from all recordings obtained at +90 ( black ) and -90 mV ( blue ) from the patch in ( A-C ) .",
"Histograms were normalized to the total area under the curve .",
"( E ) Po per sweep as a function of recording time calculated for all recordings at +90 ( black ) and -90 mV ( blue ) from the same patch in ( A-D ) .",
"The dotted red line denotes a Po of 0 .",
"( F ) Mean Po values for TRPV2 QM at 100 nM RTx ( open symbols ) and +90 mV calculated from the single-channel patch in ( A-E ) and from two other patches with two channels each ( n = 3 independent patches ) , together with Po values at 400 nM RTx ( closed symbols ) calculated from two single-channel patches and two patches containing two channels each ( n = 4 independent patches ) .",
"( G ) Single-channel current amplitudes for the full-conductance level of an open TRPV2 QM channel in the presence of 100 nM ( open symbols ) or 400 nM ( closed symbols ) RTx at +90 ( black ) or -90 mV ( blue ) obtained from the same patches as the Po values in ( F ) .",
"The dotted red line denotes the zero-current level . DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 01110 . 7554/eLife . 16409 . 012Figure 6—figure supplement 1 . Single-channel activity of TRPV2 QM in the presence of 100 nM and 400 nM RTx .",
"( A ) Recordings from an inside-out patch containing two TRPV2 QM channels in the presence of 100 nM RTx at +90 mV .",
"The voltage protocol used was the same as in Figure 6A .",
"The red continuous horizontal lines indicate the zero-current level ( closed channel - C ) and the dotted lines the current level for an open channel ( O1 ) or two simultaneously open channels ( O2 ) .",
"( B ) Normalized all-points histogram from all recordings obtained at +90 mV from the patch in ( A ) .",
"The histogram was normalized to the total area under the curve .",
"( C ) NPo per sweep ( where N is likely to be",
"2 ) calculated for all recordings at +90 as in ( A ) from the same patch shown as a function of time .",
"The dotted red line denotes an NPo of 0 .",
"( D ) Single-channel TRPV2 QM recordings from an inside-out patch at +90 mV in the presence of 400 nM RTx .",
"The red continuous horizontal lines indicate the zero-current level ( closed channel - C ) and the dotted lines the current level for an open channel ( O ) .",
"( E ) Normalized all points-histogram from recordings at +90 mV and 400 nM RTx from the patch in ( D ) .",
"( F ) Po per sweep as a function of time calculated for the patch in ( D ) at +90 mV and 400 nM RTx .",
"The dotted red line denotes a Po of 0 . DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 012"
],
[
"The goal of the present study was to further interrogate the putative vanilloid binding pocket identified in the recent cryo-EM structures of the TRPV1 channel ( Cao et al . , 2013 ) and to investigate the extent to which the enigmatic TRPV2 channel shares functional mechanisms in common with TRPV1 ( Peralvarez-Marin et al . , 2013 ) .",
"Our biochemical results with expressed TRPV1 constructs and the RTx binding assay demonstrate that the full-length protein expressed in yeast is competent to bind RTx with high affinity , but that the S1-S4 and S1-S6 domains are not sufficient to observe convincing binding of the vanilloid ( Figure 1E , F ) .",
"The RTx binding site identified in the cryo-EM structure of TRPV1 is located at the interface between the S1-S4 domain of one subunit and the S5-S6 domain of the adjacent subunit ( Cao et al . , 2013 ) , and therefore one explanation would be that our truncated constructs do not bind RTx because they are possibly monomeric .",
"We took advantage of the ability of the full-length construct to bind RTx to investigate whether we could observe allosteric coupling between the binding of the vanilloid and that of DkTx , a tarantula toxin that binds to the outer pore of TRPV1 .",
"We observed readily detectable shifts in the Kd for RTx binding in the presence of DkTx ( Figure 2 ) , indicating that both toxins are able to bind to our preparation of TRPV1 and that they are allosterically coupled .",
"Although this type of allosteric coupling is embodied in the allosteric models that have been widely used to understand the complex polymodal gating of TRPV1 seen in electrophysiological studies ( Jara-Oseguera et al . , 2016; Latorre et al . , 2007 ) , this is the first direct biochemical demonstration of allosteric coupling between two ligands that activate TRPV1 .",
"In addition to providing further validation of the RTx binding site inferred from the cryo-EM structure of TRPV1 ( Figure 4; Figure 4—figure supplement 1 ) , our mutagenesis results in TRPV2 provide a new perspective with which to view this poorly understood TRP channel .",
"Our results demonstrate that as few as two TRPV1 residues need to be introduced into TRPV2 in order for RTx to bind to the channel with high affinity and to fully activate the channel ( Figure 5; Figure 5—figure supplement 1; Figure 6 ) .",
"This finding suggests that a quiescent RTx binding pocket already exists in TRPV2 , requiring only subtle engineering to make it competent to bind RTx , and demonstrating that the same basic vanilloid-activation mechanism exists in TRPV2 channels .",
"Our single channel recordings of TRPV2 show that RTx binding can achieve high Po , and that the conductance of the open state is high and exhibits rectification ( Figure 6 ) , properties that are very similar to the TRPV1 channel ( Hui et al . , 2003; Liu et al . , 2009; Oseguera et al . , 2007; Premkumar et al . , 2002 ) .",
"Collectively , these findings fit very well with the recent TRPV2 cryo-EM structure ( Zubcevic et al . , 2016 ) , which shows that the channel adopts a structure that is remarkably similar to TRPV1 ( Liao et al . , 2013 ) ( Figure 7 ) .",
"Our results are also compatible with a computational model of RTx docked into the structure of TRPV1 , where the methoxyl and hydroxyl bearing aromatic ring of RTx hydrogen bond with S512 in TRPV1 ( Elokely et al . , 2016 ) .",
"If we superimpose the cryo-EM structures of TRPV1 and TRPV2 , the native F472 in TRPV2 ( corresponding to S512 ) would likely sterically clash with the methoxyl and hydroxyl groups of RTx , and could not support the stabilizing hydrogen bonds with the hydroxyl side chain of S512 ( Figure 7 ) , explaining why this position is so critical for vanilloid binding to both TRPV1 and TRPV2 channels . 10 . 7554/eLife . 16409 . 013Figure 7 . Comparison of the structures of TRPV1 and TRPV2 and the RTx binding pocket .",
"( A ) Side view of the superposition of the cryo-EM structure of the RTx/DkTx-bound rTRPV1 channel ( gray ) ( Cao et al . , 2013 ) with that of rabbit TRPV2 ( colored by chain ) ( Zubcevic et al . , 2016 ) .",
"RTx is shown in orange as docked by ( Elokely et al . , 2016 ) .",
"( B , C )",
"Enlarged views of the RTx binding pocket showing the side chains in stick representation for the four mutated residues in TRPV2 QM numbered according to the rat sequence of the two proteins .",
"TRPV2 subunits are colored by chain and TRPV1 is shown in gray . DOI: http://dx . doi . org/10 . 7554/eLife . 16409 . 013 The mutants we describe should be useful for obtaining structures of the open state of the TRPV2 channel in the presence of RTx , and for further exploring the gating mechanisms that differ between TRPV1 and TRPV2 channels .",
"Proton activation , as well as regulation by external Na+ ( Jara-Oseguera et al . , 2016 ) , for example , appear to be unique features of TRPV1 channels .",
"In demonstrating that TRPV1 and TRPV2 share vanilloid-activation mechanisms , our results suggest that the TRPV2 channel should be an excellent tool for defining the sites and mechanisms of activation for stimuli that are specific to TRPV1 .",
"In addition , the residues mutated in the TRPV2 quadruple mutant are for the most part conserved among TRPV2 , TRPV3 , and TRPV4 channels ( Figure 3—figure supplement 1 ) , suggesting that despite differing functional properties and physiological roles , it is possible that the vanilloid gating pathway of TRPV1 and TRPV2 is also conserved in TRPV3 and TRPV4 channels ."
],
[
"Following a previously described S . cerevisiae membrane protein expression protocol ( Drew et al . , 2008; Parker and Newstead , 2014 ) , small-scale expression trials were performed in 10 mL of -Leu selective medium with 2% glucose incubated at 30°C in an orbital shaker for ~22 hrs .",
"The overnight culture was then diluted into 10 mL of Leu-containing media with 2% lactate to an OD600 of 0 . 3 , cells were grown to an OD600 of 1 . 0 ( 12–20 hr ) , at which point protein expression was induced by adding 2% ( wt/vol ) galactose .",
"Cells were harvested after 24 hr by centrifugation at 3000 g for 5 min .",
"In-gel fluorescence was also performed on crude membrane preps of small-scale trials using glass beads and vortexing to break cells .",
"To pellet crude membranes , broken cells were centrifuged at 20 , 000g at 4°C for 1 hr and the membrane fraction combined with ( 2x ) Loading Buffer and loaded onto a Tris-Glycine ( 10% , 12% , 15% acrylamide depending on desired protein resolution ) gel along with 2 µL of Invitrogen’s Fluorescent BenchMark Ladder .",
"Gels were imaged using a FLA-3000 fluorescent gel imager from FujiFilm .",
"Constructs that showed high levels of overexpression as assayed by whole cell fluorescence and low levels of degradation measured using in-gel fluorescence were selected for medium scale preparations ( 2L ) to assess detergent solubility and quality of the protein upon solubilization .",
"A 10 ml overnight culture of the –Leu selective medium containing 2% glucose was diluted into 2 L of Leu-containing media with 2% lactate to an OD600 of 0 . 3 , and the cells were then incubated to an OD600 of 1 . 0 ( for 12–20 hr ) , at which point protein expression was induced by adding 2% galactose .",
"S . cerevisiae cells were broken in four passes at 25kpsi , 30kpsi , 35kpsi , and 40kpsi using an Avestin homogenizer .",
"Debris was then removed by centrifugation at 15 , 000g for 10 min .",
"Fluorescence was used to check cell lysis efficiency by measuring the fluorescence before and after spinning down debris .",
"Membranes were then centrifuged from the supernatant at 41 , 000 RPM and 4°C for 2 hr in an OptimaTML-100K ultracentrifuge using a Ti45 rotor .",
"900 µL of membranes were transferred to a clean 1 . 5 mL Eppendorf tube , and 100 µL of 10% detergent ( DDM ) was added to obtain a 1% final detergent concentration .",
"This membrane detergent mixture was then solubilized for 1 hr at 4°C with gentle agitation .",
"Solubilized membranes were then centrifuged out at 100 , 000g for 45 min at 4°C in a desktop ultracentrifuge .",
"The solubilized membranes were then transferred to a clean 1 . 5 mL tube before 500 µL were loaded onto the GE Superose 6 10/300 column coupled to a fluorometer .",
"Once it was determined that a construct showed both promising overexpression levels and indications of monodispersity upon detergent solubilization , large-scale cell cultures were prepared for purification .",
"After membranes were prepared they were detergent solubilized in a beaker with stirring for 1 hr at 4°C .",
"Unsolubilized membranes were removed by centrifugation for 1 hr at 4°C at 41 , 000 RPM in a Beckman Ultracentrifuge with the Ti45 rotor .",
"Solubilization efficiency was calculated by measuring fluorescence before and after centrifugation .",
"Solubilized membranes were then incubated with Ni-NTA resin for 2 hr with stirring at 4°C , the mixture poured through a column , and binding efficiency calculated by measuring the fluorescence of this solution with resin and flow through .",
"The protein was then eluted in 250 mM imidazole , and cleaved during overnight dialysis at 4°C with tobacco etch virus ( TEV ) protease ( 2 mg TEV/1 mg protein as estimated by fluorescence ) in 3 , 500 MWCO dialysis tubing with stirring in 1 . 5 L dialysis buffer ( 150 mM NaCl , 20 mM Tris , 3x CMC detergent ) .",
"In a second step of purification , after dialysis , the presumably cleaved protein solution was filtered with a 22 µM filter , and passed through a Ni Sepharose High Performance HisTrap column .",
"The flow-through , now devoid of His- and GFP tags , as well as His-tagged TEV , was then concentrated to 500 µL using a 50 , 000 MWCO Vivaspin 20 concentrator .",
"Generally , for the S1-S4 and S1-S6 of rTRPV1 , aggregates formed during the TEV cleavage step , and if the second step of purification was performed no cleaved protein was visible in coomassie-stained gels .",
"The full-length TRPV1 and TRPV2 channels with 1D4 affinity tags ( TETSQVAPA ) on their C-termini were cloned into the YEpHIS vector and expressed in the BJ5457 strain of S . cerevisiae using the strong constitutively active PMA1 promoter , as previously described ( Moiseenkova-Bell et al . , 2008 ) .",
"After 48 hr of expression at 30°C in YPD media , cells were harvested by centrifugation and re-suspended in PBS buffer , supplemented with complete protease inhibitor cocktail ( Roche , Indianapolis , IN ) and 4-amidinophenylmethanesulfonyl fluoride hydrochloride ( APMSF ) ( Sigma-Aldrich , St . Louis , MO ) .",
"Cells were broken by passing through an Avestin homogenizer at the pressure of 35 kpsi , cell lysate was cleared by centrifugation at 8 , 000 g for 15 min and membranes were isolated by ultracentrifugation at 190 , 000 g for 1 hr .",
"Membrane pellets were re-suspended in PBS buffer supplemented with protease inhibitors and 200 mM sucrose , flash-frozen in liquid nitrogen and stored at -70°C until RTx binding measurements .",
"S . cerevisiae cells overexpressing TRPV1 constructs to be used in ligand binding assays on intact cells ( Figure 1E , F ) were grown as for small scale expression trials .",
"24 hr after protein induction cells were spun down at 4000g , the supernatant was removed and the pellet resuspended in 1x PBS , normalizing OD600 to 75 for each sample , and aliquoted for storage at -80°C .",
"3H-RTx binding assays on cells were performed at 37°C as previously described ( Feng et al . , 2015 ) , but where non-specific binding of RTx was measured using cells expressing GFP alone .",
"The binding of RTx to TRPV1 and TRPV2 in membranes ( e . g . Figure 2A , B and 5C ) was performed at 37°C unless otherwise stated using membranes prepared from large scale protein preparations of S . cerevisiae containing rat TRPV1 and TRPV2 with a 1D4 tag expressed using a constitutive promoter as in Moiseenkova-Bell et al . , 2008 .",
"Data for binding of RTx to membranes was corrected for non-specific binding , determined using either excess non-radioactive ligand or heat inactivation of the protein , as previously described ( Feng et al . , 2015 ) .",
"The TRPV1-activating double-knot toxin ( DkTx ) was produced recombinantly , folded in vitro and purified as previously described ( Bae et al . , 2012 ) .",
"Yeast membranes were incubated with DkTx solution for 1 hr at 4°C with gentle agitation prior to undertaking RTx binding assays .",
"The WT rat TRPV1 and TRPV2 channels in the pcDNA3 . 1 vector were kindly provided by Dr . David Julius ( UCSF ) ( Caterina et al . , 1997; Caterina et al . , 1999 ) and subcloned into the pGEM-HE vector ( Liman et al . , 1992 ) .",
"Mutations were introduced into rTRPV1 using a two-step PCR mutagenesis technique and the resulting constructs were verified by sequencing .",
"All channel constructs were expressed in Xenopus oocytes and studied following 1–4 days incubation after cRNA injection ( incubated at 17°C in 96 mM NaCl , 2 mM KCl , 5 mM HEPES , 1 mM MgCl2 and 1 . 8 mM CaCl2 , 50 μg/ml gentamycin , pH 7 . 6 with NaOH ) using the two-electrode voltage-clamp recording technique ( OC-725C , Warner Instruments , Hamden , CT ) with a 150 μl recording chamber .",
"Data were filtered at 1–3 kHz and digitized at 20 kHz using pClamp software ( Molecular Devices , Sunnyvale , CA ) .",
"Microelectrode resistances were 0 . 1–1 MΩ when filled with 3 M KCl .",
"For recording macroscopic TRP channel currents , the external recording solution contained ( in mM ) : 100 KCl , 10 HEPES , pH 7 . 6 with KOH .",
"All experiments were performed at room temperature ( ~22°C ) .",
"HEK293 cells were cultured following standard protocols ( Li et al . , 2015 ) and transiently transfected with plasmids for the expression of TRPV2-QM ( pcDNA1 ) and GFP ( pGreen-Lantern , Invitrogen , Carlsbad , CA ) using FuGENE6 ( Promega , Madison , WI ) .",
"Standard whole-cell patch clamp recordings from transiently transfected HEK293 cells at room temperature ( 22–24°C ) were performed .",
"Data was acquired with an Axopatch 200B amplifier ( Molecular Devices , Sunnyvale , CA ) , filtered with an 8-pole low-pass Bessel filter ( model 900 , Frequency Devices , Ottawa , IL ) and digitized with a Digidata 1550A interface and pClamp10 software ( Molecular Devices ) .",
"All data was analyzed using Igor Pro 6 . 34A ( Wavemetrics Inc . , Tigard , OR ) .",
"Pipettes were pulled from borosilicate glass , covered in dental wax and heat-polished to final resistances between 10 – 15 MΩ .",
"The intracellular recording solution consisted of ( in mM ) : 130 NaCl , 10 HEPES , 10 EGTA , pH 7 . 4 ( NaOH/HCl ) , and the extracellular solution ( i . e . pipette solution ) was supplemented with 10 mM MgCl2 .",
"A gravity-fed rapid solution exchange system ( RSC-200 , BioLogic , Claix , France ) was used .",
"Excised patches were placed in front of glass capillaries perfused with different solutions .",
"Data were acquired at 20 kHz and low-pass filtered at 5 kHz , with an additional 2 kHz low-pass filter applied off-line .",
"Single channel currents were recorded using a protocol consisting of 100 ms at a holding potential of −90 mV , 500 ms pulse at +90 mV , followed by another 100 ms at −90 mV , which was applied every 700 ms for the duration of the recording .",
"Data collected at +90 mV and the 100 ms before the step to +90 mV were used for analyzing channel behavior at the two potentials .",
"A series of null-traces were obtained in control solution ( in the absence of agonists ) before application of RTx , and were used for subtraction of the leak currents and the capacitive transients .",
"No spontaneous channel activity was recorded in control solution ( data not shown ) .",
"Recordings in RTx were started as soon as the patch was perfused with the agonist , which was followed by a 10–30 s delay period in which no channel activity was observed , followed by robust channel activation .",
"To reflect the Po of RTx-bound channels , mean Po values shown in Figure 6F were calculated after the initial period where no channel activity was observed .",
"The Po for every sweep was calculated using the 50% threshold crossing technique , which consists in generating idealized traces from the recordings , where data points above a specified threshold are assigned a value of 1 and data points below the threshold are assigned a value of 0 , and the Po is calculated as the mean value of the idealized trace .",
"For recordings at +90 mV , a threshold value of 4 . 5 pA was used ( approximately half of the maximal conductance level of 10 pA ) , whereas a threshold of 1 . 2 pA was used for the recordings at -90 mV .",
"A 5-pass binomial smoothing function was applied to the data at -90 mV prior to calculation of the Po .",
"For multi-channel patches , multiple thresholds were used and idealized traces were assigned a value of 2 when two channels were open simultaneously .",
"For data obtained at +90 mV a threshold slightly lower than 50% of the maximal single-channel current amplitude was used so that subconductance levels were counted as channel openings .",
"The mean Po data in Figure 6F is the average of the Po-values for all sweeps in each patch , with an N = 2 for two-channel patches .",
"For each patch the mean Powas also calculated from the normalized all-points histograms .",
"In this case , histograms compiled from all recorded data-points after leak subtraction at a specified voltage were normalized to the area under the histogram curve .",
"A Gaussian curve was fit to the data centered at 0 pA , and the area under its curve ( AC ) was calculated .",
"The Po was then calculated as Po = 1 – AC .",
"Both methods for calculating Po gave identical results ( data not shown ) .",
"The single-channel current amplitudes were calculated from Gaussian fits to the all-points histogram portion corresponding to the current level of a single open channel ."
]
] | [
"The TRPV1 channel is a detector of noxious stimuli , including heat , acidosis , vanilloid compounds and lipids .",
"The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available , and the threshold for heat activation is extremely high ( >50°C ) .",
"Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures , and identify a putative vanilloid binding pocket near the internal side of TRPV1 .",
"Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin ( RTx ) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2 .",
"Collectively , our results support the interaction of vanilloids with the proposed RTx binding pocket , and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding .",
"Moreover , we show that sensitivity to RTx can be engineered into TRPV2 , demonstrating that the gating and permeation properties of this channel are similar to TRPV1 ."
] | [
"Ion channels form pores in cell membranes and can open and close to allow specific ions to flow from one side of the membrane to the other .",
"Humans and other mammals rely on an ion channel protein called TRPV1 to sense heat , and this protein is activated by the active ingredient in hot chili peppers , a chemical called capsaicin that belongs to a group of compounds called vanilloids .",
"An ion channel called TRPV2 is related to TRPV1 and has a similar shape , but is less well understood .",
"This is partly because there are no chemicals that are known to selectively activate this channel , and it is not activated by the vanilloids that activate TRPV1 .",
"Zhang , Hanson et al . have now investigated where vanilloid compounds bind to activate the rat TRPV1 channel and if the binding site can be created in the rat TRPV2 as well .",
"Like all proteins , these channels are built from smaller units called amino acids .",
"The results from an array of approaches identified four specific amino acids that can be exchanged between TRPV1 and TRPV2 to swap their ability to bind and be activated by resiniferatoxin ( a vanilloid isolated from a cactus-like plant called resin spurge ) .",
"When rat TRPV2 channels were engineered to contain these four amino acids , resiniferatoxin could easily open the channel .",
"In addition , the engineered channel allowed ions to pass through in a way that is similar to TRPV1 , which suggests that the channels are more similar than previously believed .",
"Together the findings show how the TRPV2 channel can be made sensitive to vanilloids .",
"This will help researchers to solve the structure of this ion channel when it is active and open .",
"In addition , the findings may also allow the role of TRPV2 to be explored further in animals , such as mice ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression"
] | Multi-step coordination of telomerase recruitment in fission yeast through two coupled telomere-telomerase interfaces | elife-15470-v2 | [
[
"Telomeres , the physical ends of linear chromosomes , are essential for stable maintenance of chromosomes by facilitating chromosome end replication and preventing them from being degraded or fusing with each other ( Artandi and Cooper , 2009; Palm and de Lange , 2008 ) .",
"In most eukaryotes , telomeres are comprised of short tandem DNA repeats .",
"Rather than a blunt end , the telomere consists of a 3’ single-stranded overhang called the G-tail that provides the substrate for telomerase to counteract iterative telomere shortening after each round of DNA replication .",
"Telomerase is a ribonucleoprotein enzyme that extends telomeres utilizing an RNA component as template for its reverse transcriptase protein subunit ( named TERT in vertebrates and Trt1 in fission yeast ) ( Autexier and Lue , 2006; Collins , 2006 ) .",
"This telomerase-dependent nucleotide addition is rigorously limited to late S/G2 phase .",
"Dysregulation of telomerase and the subsequent perturbation of telomere length homeostasis lead to severe defects in cell proliferation .",
"As a result , a constellation of genetic diseases caused by mutations in the telomere maintenance machinery ( Sarek et al . , 2015 ) , referred to as 'telomeropathies' ( Holohan et al . , 2014 ) or 'telomere syndromes' ( Armanios and Blackburn , 2012 ) , have been continuously identified , and include dyskeratosis congenita ( DC ) , aplastic anemia , and multiple types of cancers .",
"Studies in telomerase regulation and telomere maintenance unveiled a precisely orchestrated process of telomerase recruitment ( Hockemeyer and Collins , 2015; Nandakumar and Cech , 2013; Schmidt and Cech , 2015 ) .",
"In budding yeast , the interaction between the telomere-binding protein Cdc13 and the telomerase holoenzyme component Est1 regulates telomerase activation and its recruitment to the telomere ( Chandra et al . , 2001; Evans and Lundblad , 1999; Pennock et al . , 2001; Qi and Zakian , 2000 ) .",
"Likewise , telomere-associated proteins in humans and fission yeast , which form a highly conserved protein complex called shelterin ( de Lange , 2005; Miyoshi et al . , 2008 ) , are essential to regulate telomere states , recruit telomerase , and govern its activity on telomeres .",
"In fission yeast , the shelterin complex contains the double-stranded DNA ( dsDNA ) binding protein Taz1 ( the homolog of TRF1/TRF2 in humans ) ( Cooper et al . , 1997 ) and the single-stranded DNA ( ssDNA ) binding protein Pot1 ( human POT1 ortholog ) ( Baumann and Cech , 2001 ) , which are bridged via direct protein-protein interactions between Rap1 ( Chikashige and Hiraoka , 2001; Kanoh and Ishikawa , 2001 ) , Poz1 , and Tpz1 ( human RAP1 , TIN2 and TPP1 orthologs , respectively ) ( Miyoshi et al . , 2008 ) .",
"The human system shares a conserved shelterin arrangement by forming a similar 'shelterin bridge' architecture ( Bianchi and Shore , 2008 ) .",
"Recent studies discovered a cluster of residues on human TPP1 , collectively termed TEL-patch , which mediates TPP1-TERT interaction to recruit telomerase to telomeres ( Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) .",
"TEL-patch mediated TPP1-TERT interaction also confers increased telomerase processivity in vitro ( Nandakumar et al . , 2012; Schmidt et al . , 2014 ) .",
"Moreover , a detailed genetic study of TEL-patch mutants revealed multiple functions of TPP1 in telomerase recruitment , activation , and telomere length feedback regulation ( Sexton et al . , 2014 ) .",
"Altered TEL-patch function is clinically manifested as Hoyeraal-Hreidarsson ( HH ) syndrome .",
"HH patients bear extremely short telomeres , even in comparison to other DC patients .",
"A germline mutation causing a single-amino-acid deletion ( K170Δ ) in the human TPP1 TEL-patch that affects its telomerase recruitment function has been identified as the causal mutation of HH ( Kocak et al . , 2014 ) .",
"This signifies the critical telomerase recruitment function of the TEL-patch in normal stem cell development .",
"Interestingly , fission yeast Schizosaccharomyces pombe , which has a similar shelterin architecture to mammals , contains an additional shelterin component called Ccq1 ( Flory et al . , 2004 ) , the functional homolog of which has not yet been identified in mammals .",
"Ccq1 was demonstrated to be a cell cycle-dependent telomerase recruitment factor ( Miyoshi et al . , 2008; Tomita and Cooper , 2008 ) .",
"Further investigation showed that Ccq1 is required to bring telomerase to telomeres through its Tel1ATM/Rad3ATR-mediated Thr93 phosphorylation during late S phase , which creates a binding site for the 14-3-3 domain of Est1 ( Moser et al . , 2011; Webb and Zakian , 2012; Yamazaki et al . , 2012 ) .",
"Est1 is an accessory protein of telomerase holoenzyme and is linked to the telomerase protein subunit ( Trt1 ) via their co-association with telomerase RNA ( TER1 ) ( Leonardi et al . , 2008 ) .",
"Ccq1 also interacts with Tpz1 ( Miyoshi et al . , 2008 ) , and the Ccq1-Tpz1 interaction is therefore thought to bring the telomerase complex ( Est1-TER1-Trt1 ) to the telomere ( Moser et al . , 2009 ) via the Ccq1 Thr93-phosphorylation dependent Ccq1-Est1 interaction .",
"However , in-depth analyses further revealed that Ccq1-Est1 and Ccq1-Tpz1 interactions seem to be mutually exclusive ( Armstrong et al . , 2014 ) .",
"Moreover , Est1-Ccq1 interaction could be disrupted by TER1 in a yeast three-hybrid analysis and Est1 mutations that affect Est1-TER1 interaction also impair Est1-Ccq1 interaction ( Armstrong et al . , 2014; Webb and Zakian , 2012 ) .",
"In addition , based on a Ccq1-centric model , it is hard to explain why the telomeric association of Est1 requires not only Ccq1 , but also Trt1 and TER1 , which are downstream from Ccq1-Est1 interaction ( Webb and Zakian , 2012 ) .",
"Therefore , the hypothetical Tpz1-Ccq1-Est1-TER1-Trt1 interaction chain seems unlikely to form to mediate telomerase recruitment .",
"These results imply that an alternative mechanism exists to directly associate telomerase to other shelterin components , such as Tpz1 , to initiate telomere elongation in fission yeast in response to the cell cycle-dependent Ccq1-Thr93 phosphorylation ( Chang et al . , 2013 ) .",
"In this study , we identified a Tpz1 mutation in the TEL-patch region that results in an ever shorter telomere ( EST ) phenotype , similar to the telomere phenotype of human TPP1 TEL-patch mutants .",
"We observed decreased telomeric association of Trt1 and weakened Tpz1-Trt1 interaction in this Tpz1 TEL-patch mutant , indicating the conserved role of the TEL-patch in telomerase recruitment .",
"Our epistasis analyses demonstrated that the Tpz1 TEL-patch functions by positively regulating Trt1 .",
"Furthermore , we found that telomerase action at telomeres requires formation and resolution of an intermediate state , formed via two cooperative telomere-telomerase interfaces involving cell cycle-regulated Ccq1-Est1 interaction and Tpz1 ( TEL-patch ) -Trt1 interaction .",
"As a result , the temporal information for telomerase recruitment is endowed to the TEL-patch through the phosphorylation status of Ccq1 Thr93 , thus achieving cell cycle-specific telomere elongation ."
],
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"Telomerase exists in low abundance in the cell .",
"Therefore , the interaction between shelterin and telomerase has been proposed to enrich telomerase at chromosome ends .",
"Indeed , a group of surface-exposed amino acids in the human TPP1 OB-domain , termed TEL-patch , are found to be necessary for the telomerase recruitment to telomeres ( Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) .",
"Although the fission yeast shelterin component Ccq1 has been connected to telomerase recruitment in this model organism , it is unlikely to be the sole factor to link Trt1 to telomeres ( Armstrong et al . , 2014 ) .",
"Additional interactions between shelterin components and Trt1 must exist to bring Trt1 directly to the telomere .",
"Given the high conservation of the OB-fold domain arrangement in the N-terminal regions of S . pombe Tpz1 and human TPP1 , we decided to test whether a Tpz1 TEL-patch functions in S . pombe as an interface between telomerase and shelterin .",
"To identify candidate residues for such a TEL-patch in S . pombe Tpz1 , we performed a sequence alignment between fission yeast Tpz1 and human TPP1 in combination with a secondary structure prediction ( Figure 1A ) .",
"We then identified 12 conserved Tpz1 residues in the region corresponding to the human TPP1 TEL-patch as candidate residues for fission yeast Tpz1 TEL-patch .",
"S . pombe cells bearing individual mutations in these 12 residues of Tpz1 were tested for their telomere maintenance .",
"tpz1-I105R and tpz1-V107R mutant strains appear to have destabilized Tpz1 protein and display a similar telomere deprotection phenotype ( Figure 1—figure supplement 1 ) to tpz1Δ strain .",
"The remaining 10 tpz1 mutant strains have comparable levels of Tpz1 expression as the wild-type strain ( Figure 1—figure supplement 2 ) .",
"Strains bearing tpz1-T73A , tpz1-K75E , tpz1-R76E , tpz1-I77R , and tpz1-R81E displayed shortening telomeres ( Figure 1B and Figure 1—figure supplement 1 ) , implicating positive regulatory roles of these residues in telomere length regulation .",
"Although tpz1-T73A , tpz1-K75E , and tpz1-R76E cells all have shortened telomeres , in contrast to the tpz1-I77R and tpz1-R81E cells , their telomeres remain stably short ( Figure 1B and Figure 1—figure supplement 1 ) .",
"This telomere phenotype is similar to that of previously identified fission yeast tpz1-K75A ( Armstrong et al . , 2014 ) or human TPP1-L104A ( Sexton et al . , 2014 ) , proposed to affect telomerase activation and telomere length homeostasis set point , respectively .",
"Strikingly , tpz1-R81E cells showed the classic Ever Shorter Telomere ( EST ) phenotype ( Lundblad and Szostak , 1989 ) , similar to trt1Δ cells ( Figure 1B ) ( Nakamura et al . , 1997 ) .",
"The deterioration in telomere maintenance of tpz1-R81E strain started as early as ~50 generations .",
"Its telomeres shorten to the critical length at 75 generations , and are almost completely lost afterwards .",
"Gradual telomere loss was also observed in tpz1-I77R cells , albeit to a much milder degree than in tpz1-R81E cells .",
"Furthermore , Tpz1-Arg81 and Trt1 appear to have an epistatic relationship because telomere shortening in the tpz1-R81E/trt1Δ double mutant is not additive ( Figure 1C ) .",
"The similar phenotypes observed for tpz1-R81E , trt1Δ , and tpz1-R81E/trt1Δ support a role of Tpz1-Arg81 in directly regulating telomerase activity on telomeres .",
"Similar to the previously identified tpz1-K75A mutant that is defective in telomerase activation ( Armstrong et al . , 2014 ) , tpz1-K75E and tpz1-R76E mutants have a milder telomere loss phenotype and their shortened telomeres are maintained for many generations .",
"These observations clearly distinguish Tpz1-Arg 81 from telomerase activation residues in Tpz1 , i . e . Lys75 or Arg76 . 10 . 7554/eLife . 15470 . 003Figure 1 . Fission yeast Tpz1 TEL-patch mutant leads to an EST phenotype .",
"( A ) A sequence alignment of OB fold-domains between fission yeast Tpz1 , human TPP1 and Oxytricha nova TEBP-β , in combination with a secondary structure prediction .",
"Twelve conserved fission yeast Tpz1 residues subjected to mutagenesis are highlighted in blue with their identities and sequence numbers indicated above the alignment .",
"Residues colored in red are previously identified TEL-patch residues in human TPP1 and mutations of them lead to compromised TPP1-TERT interaction .",
"Black and grey shading indicates sequence identity and similarity , respectively .",
"If the sequence is identical among at least 50% of species , the residues will be shaded in black .",
"The same rule applies to sequence similarity , which is shaded in grey .",
"( B ) Southern blot analysis to measure telomere lengths using EcoRI-digested genomic DNA visualized by the telomere DNA probe for the indicated tpz1 mutant strains from successive re-streaks on agar plates .",
"tpz1-K75E , tpz1-R76E , tpz1-I77R , and tpz1-R81E strains caused telomere shortening and tpz1-R81E cells showed the classic Ever Shorter Telomere ( EST ) phenotype .",
"For the telomere length analysis southern blots presented in all the figures , the 1 kb plus marker from Invitrogen is used .",
"Wild type cells are denoted as 'WT' in the blot .",
"pol1+ indicates the EcoRI-digested pol1+ DNA fragment used as a loading control .",
"( C ) Double-mutant strain tpz1-R81E/trt1Δ shows EST telomere phenotype , similar to the trt1Δ single-mutant strain .",
"( D ) Double-mutant strain tpz1-R81E/poz1Δ shows progressive telomere shortening phenotype . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 00310 . 7554/eLife . 15470 . 004Figure 1—figure supplement 1 . Telomere length measurement of tpz1 mutant strains . Southern blot analysis to measure telomere lengths using EcoRI-digested genomic DNA visualized by the telomere DNA probe for the indicated tpz1 mutant strains from successive re-streaks on agar plates . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 00410 . 7554/eLife . 15470 . 005Figure 1—figure supplement 2 . Evaluation of Tpz1 expression levels in tpz1 mutant strains . Western blot showing expression levels of Tpz1 protein in indicated tpz1 mutant strains . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 00510 . 7554/eLife . 15470 . 006Figure 1—figure supplement 3 . The later generation tpz1-R81E/poz1Δ mutant strain forms circularized chromosomes .",
"( A ) Schematic diagram of chromosome showing sub-telomeric regions .",
"Greek numbers indicate the locations where the PCR primers are designed .",
"( B ) tpz1-R81E/poz1Δ mutant lost sub-telomeric I and II regions at generation 200 .",
"The PCR products ( from I to IV ) are amplified from corresponding regions on chromosomes ( A ) .",
"For S . pombe cells carrying circularized chromosomes due to telomere loss , both sub-telomeric regions I and II are eroded from chromosome ends . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 006 We next asked whether the requirement for TEL-patch residue Tpz1-Arg81 can be bypassed by eliminating the negative shelterin linkage , which keeps telomeres constitutively in the telomerase-extendible state ( Jun et al . , 2013 ) .",
"To this end , we constructed a double-mutant strain , tpz1-R81E/poz1Δ , in which poz1Δ leads to defective shelterin linkage .",
"As shown in Figure 1D , tpz1-R81E/poz1Δ cells presented EST phenotype and the cells senesced at a later generation , similar to the trt1Δ/poz1Δ mutant ( Miyoshi et al . , 2008 ) .",
"A subpopulation survived at ~200 generations by circularizing their chromosomes to bypass the need for telomerase ( Figure 1D and Figure 1—figure supplement 3 ) .",
"This result clearly indicates that Tpz1-Arg81 functions downstream of telomere switching from the non-extendible to the extendible state and upstream of telomerase action , most likely to mediate telomere-telomerase interaction .",
"Therefore , we genetically demonstrated the existence of the TEL-patch in fission yeast Tpz1 that is functionally analogous to the TEL-patch of human TPP1 .",
"Mutations of the TEL-patch residues in human TPP1 disrupt the direct interaction between TPP1 and TERT .",
"Moreover , the TEN domain of human TERT was demonstrated to mediate its interaction with TPP1 ( Schmidt et al . , 2014 ) , providing an interface required for the recruitment of telomerase to telomeres .",
"In fission yeast , both Tpz1 and Ccq1 have been found to interact with Trt1 independent of telomerase RNA ( Armstrong et al . , 2014; Tomita and Cooper , 2008 ) .",
"To directly assess the function of the putative TEL-patch residue Tpz1-Arg81 , we first examined the binding efficiency between Tpz1-R81E and Trt1 utilizing co-immunoprecipitation assay .",
"As shown in Figure 2A , B and Figure 2—figure supplement 1 , whereas Tpz1-E74R , Tpz1-K75A , Tpz1-K75E , and Tpz1-R76E pulled down similar amounts of Trt1 as the wild-type Tpz1 , Tpz1-R81E only immunoprecipitated 30% of Trt1 compared to the wild-type Tpz1 ( Figure 2B and C ) .",
"Considerably reduced Tpz1-Trt1 interaction in the tpz1-R81E mutant correlates well with its progressive telomere shortening phenotype ( Figure 1B ) , further supporting an essential role that Tpz1-Arg81 plays in mediating the interaction between telomerase and shelterin at the telomere .",
"Interestingly , tpz1-I77R cells lost about 50% of the Tpz1-Trt1 interaction ( Figure 2B and C ) .",
"This observation suggests that Tpz1-Ile77 is likely to be part of the TEL-patch as well , consistent with the compromised telomere maintenance in tpz1-I77R cells , albeit milder than that of the tpz1-R81E cells ( Figure 1B ) .",
"The tpz1-R76E mutant retains the wild-type Tpz1-Trt1 interaction ( Figure 2A ) but displays stably shortened telomeres .",
"This implies that Tpz1-Arg76 is involved in telomerase activation , similar to what was previously described for Lys75 ( Figure 2—figure supplement 1 ) ( Armstrong et al . , 2014 ) . 10 . 7554/eLife . 15470 . 007Figure 2 . The Tpz1 TEL-patch mutant is defective in Tpz1-Trt1 interaction .",
"( A ) Co-immunoprecepitation ( Co-IP ) assays evaluating bindings between Trt1 and Tpz1-E74R and Tpz1-R76E .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE ( whole cell extract ) .",
"( B ) Co-IP assays showing that Tpz1-I77R and Tpz1-R81E significantly decrease interaction between Tpz1 and Trt1 .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE .",
"( C ) Quantification of the binding efficiency between Trt1 and Tpz1 mutants from ( B ) .",
"The interaction between Trt1 and Tpz1-WT is set to be 100% .",
"Trt1 levels in the IP were normalized to Tpz1 bound to the beads .",
"( D ) Quantification of the binding efficiency between Ccq1 and Tpz1 mutants from ( B ) .",
"The interaction between Ccq1 and Tpz1-WT is set to be 100% .",
"Ccq1 levels in the IP were normalized to Tpz1 bound to the beads .",
"( E ) Co-IP assays evaluating the binding efficiency between Trt1 and Ccq1 in various Tpz1 mutant cells .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE .",
"( F ) Co-IP assays evaluating the binding efficiency between Trt1 and Tpz1 in ccq1-T93A and ccq1-F157A/K174E .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 00710 . 7554/eLife . 15470 . 008Figure 2—figure supplement 1 . Trt1-Tpz1 interaction is intact in tpz1-K75E and tpz1-K75A strains . Co-immunoprecepitation ( Co-IP ) assays evaluating the binding between full-length Trt1 and Tpz1 mutants K75E and K75A .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE ( whole cell extract ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 008 If the Tpz1-Trt1 interaction were mediated solely via Ccq1 , the reduced Tpz1-Trt1 interaction observed in tpz1-R81E cells would most likely result from reduced Tpz1-Ccq1 interaction , because other components in the proposed Tpz1-Ccq1-Est1-Trt1 interaction-chain remain intact .",
"However , as shown in Figure 2B , D and E , all tested Tpz1 mutants , including Tpz1-R81E , interact with Ccq1 similarly to wild-type Tpz1 .",
"Moreover , the Ccq1-Trt1 interaction remains unchanged for all Tpz1 mutants as well ( Figure 2E ) .",
"Next , we tested whether two Ccq1 mutants , Ccq1-F157A/K174E and Ccq1-T93A , previously shown to be defective in Tpz1-Ccq1 ( Liu et al . , 2015 ) and Ccq1-Est1 ( Moser et al . , 2011 ) interactions , respectively , can completely abolish Tpz1-Trt1 interaction .",
"As shown in Figure 2F , Tpz1-Trt1 interaction is clearly retained in both Ccq1-F157A/K174E and Ccq1-T93A mutants .",
"This was particularly striking for the Ccq1-F157A/K174E mutant , in which Tpz1-Ccq1 interaction is completely disrupted .",
"These data , together with previous studies ( Armstrong et al . , 2014; Webb and Zakian , 2012 ) , argue against Ccq1 as the sole platform that recruits telomerase to telomeres .",
"These results further point to the existence of the TEL-patch in fission yeast Tpz1 and indicate a conserved function in the recruitment of telomerase .",
"To further test whether the failure of the tpz1-R81E mutant to replenish telomeres emanates from its inability to recruit telomerase to telomeres , we directly tested the group of Tpz1 OB-fold domain mutants for in vivo localization of telomerase to telomeres using ChIP assays .",
"As expected , tpz1-R81E , which has significantly reduced Tpz1-Trt1 interaction , exhibited a dramatic decrease in the association of Trt1 with telomeres ( Figure 3A ) .",
"In contrast to the tpz1-R81E mutant , a telomerase activation-defective mutant tpz1-R76E displayed wild-type telomerase localization to telomeres ( Figure 3A ) .",
"The latter result is consistent with the phenotype of another previously described telomerase activation mutant strain tpz1-K75A ( Armstrong et al . , 2014 ) .",
"Interestingly , the tpz1-I77R mutant strain also showed significant decreased localization of Trt1 to telomeres ( Figure 3A ) , consistent with the decreased Tpz1-Trt1 interaction observed in the co-immunoprecipitation assay ( Figure 2B ) .",
"In contrast , in all the tested Tpz1 OB-fold domain mutants , little effect was observed on the telomeric association of both Tpz1 and Ccq1 ( Figure 3B and C ) .",
"These results directly reveal a specific role for Tpz1 N-terminal OB-domain residues Ile77 and Arg81 in mediating telomere-Trt1 interaction and Trt1 recruitment .",
"Moreover , since tpz1-I77R cells have a much more subtle telomere shortening phenotype than tpz1-R81E cells , we speculate that a threshold amount of residual telomerase interaction may be required for telomere maintenance upon disruption of the Tpz1 TEL-patch .",
"Taken together , we redefined the TEL-patch in fission yeast Tpz1 , which functions analogously to the previously characterized TEL-patch in human TPP1 ( Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) . 10 . 7554/eLife . 15470 . 009Figure 3 . The Tpz1 TEL-patch mutant fails to localize telomerase to telomeres .",
"( A–C )",
"Enrichment of Trt1 ( A ) , Ccq1 ( B ) or Tpz1 ( C ) at telomeres is monitored by chromatin immunoprecipitation ( ChIP ) assay .",
"Slot-blot was used to visualize telomere association of Trt1 , Ccq1 or Tpz1 in each indicated tpz1 mutant strains .",
"Error bars in the quantitation of the slot-blot analysis represent standard deviations of two individual repeats . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 009 Direct fusion of telomerase to a shelterin component has been shown to rescue the telomerase recruitment defect , but not other defects , such as telomere activation or telomere length homeostasis regulation in both fission yeast ( Armstrong et al . , 2014 ) and human embryonic stem cells ( hESC ) ( Sexton et al . , 2014 ) .",
"We next tested whether the inability of the Tpz1 TEL-patch mutant to maintain telomere length could be rescued by forcing telomerase to physically associate with telomeres .",
"To this end , we measured telomere lengths of strains with Trt1 fused to Tpz1 mutants as previously described ( Armstrong et al . , 2014 ) .",
"Apparently , the strain bearing fused Trt1—Tpz1 TEL-patch mutant ( trt1—tpz1-R81E ) maintained the same telomere length as the trt1—tpz1 wild-type strain ( Figure 4A ) , indicating rescue of telomere shortening .",
"However , fusion of Trt1 with Tpz1-L449A , which disrupts the Tpz1-Ccq1 interaction , failed to restore telomere maintenance , as previously reported ( Armstrong et al . , 2014 ) .",
"The expression levels of all Trt1—Tpz1 fusion proteins appear to be similar ( Figure 4B ) .",
"These results suggest that the Tpz1-Ccq1 interaction is required for aspects of telomere elongation besides bridging telomerase to telomeres .",
"This interpretation is consistent with our previous genetic study implicating Ccq1 in switching telomeres from non-extendible to extendible state ( Jun et al . , 2013 ) . 10 . 7554/eLife . 15470 . 010Figure 4 . Fusing Trt1 to Tpz1 bypasses the requirement for functional Tpz1 TEL-patch .",
"( A ) Southern blot analysis to measure telomere lengths using EcoRI-digested genomic DNA visualized by the telomere DNA probe for the trt1-tpz1 fusion strains with indicated WT or mutant versions of tpz1 .",
"9PK tags were inserted between trt1 and tpz1 .",
"( B ) Western blot showing expression levels of PK-tagged Trt1 protein and the chimeric Trt1-Tpz1 fusion proteins .",
"Cdc2 was used as a loading control for total proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 010 In fission yeast , Rad3ATR/Tel1ATM-dependent phosphorylation of Ccq1 at Thr93 promotes direct interaction between Ccq1 and Est1—a subunit of the telomerase holoenzyme ( Moser et al . , 2011; Yamazaki et al . , 2012 ) .",
"The 14-3-3–like domain of Est1 was shown to recognize Ccq1 phosphorylated at Thr93 and was proposed to enable telomerase-telomeres association via the Ccq1-Tpz1 complex ( Moser et al . , 2011 ) .",
"Different from the proposed Tpz1-Ccq1-Est1 chain-interaction that connects telomerase to telomere shelterin , Ccq1-Est1 and Ccq1-Tpz1 interaction were demonstrated to be mutually exclusive ( Armstrong et al . , 2014 ) and the binding surfaces for Est1 and Tpz1 in Ccq1 are most likely to overlap .",
"Our recent work utilizing a new strategy , called MICro-MS ( Mapping Interfaces via Crosslinking-Mass Spectrometry ) , mapped the Tpz1-interacting interface on Ccq1 and isolated a group of separation-of-function mutants that specifically disrupt Ccq1-Tpz1 interaction ( Liu et al . , 2015 ) .",
"If Ccq1 interacts with Est1 via an overlapping surface that also mediates Ccq1-Tpz1 interaction , Ccq1 mutants that are defective in Ccq1-Tpz1 interaction is most likely to compromise Ccq1-Est1 interaction too .",
"Indeed , as shown in Figure 5A , Ccq1-F157A/K174E and Ccq1-I175R , which were identified previously to be defective in Tpz1-Ccq1 interaction , both significantly diminished Ccq1-Est1 interaction in co-immunoprecipitation assays , whereas Ccq1-V152R and Ccq1-L177R still retained wild-type binding to Est1 .",
"Unexpectedly , we found that Ccq1-T93A , the Ccq1 mutant in the Rad3ATR/Tel1ATM phosphorylation site Thr93 , did not completely abolish Ccq1-Est1 interaction; rather , it diminished Ccq1-Est1 interaction to a similar degree that was observed for Ccq1-F157A/K174E or Ccq1-I175R mutant . 10 . 7554/eLife . 15470 . 011Figure 5 . Est1 binds to Ccq1 through two different binding sites .",
"( A ) Ccq1-Est1 interaction is diminished but not completely disrupted in the ccq1-T93A , ccq1-F157A/K174E , and ccq1-I175R strains as evaluated by Co-IP assays .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE .",
"( B ) Schematic model of Est1 and Ccq1 interaction showing two binding sites for their interaction as further tested in ( C ) .",
"( C ) Co-IP assays evaluating the contribution of two binding sites to the binding between Est1 and Ccq1 .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE .",
"( D ) Schematic model of Tpz1 C-terminal domain , Est1 and Ccq1 forming an intermediate Est1-Ccq1-Tpz1 complex .",
"( E ) Competitive binding assay showing that Tpz1 can partially compete with Est1 for its interaction with Ccq1; however , the Tpz1-Ccq1 interaction defective mutant Tpz1-L449A cannot . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 011 We therefore speculated that Ccq1 and Est1 probably interact with each other via two binding sites: one provided by phosphorylated Thr93 and the other by the HDAC2/3-like domain at the N-terminus of Ccq1 ( as depicted in Figure 5B ) .",
"As a result , mutating either site only partially disrupts Ccq1-Est1 interaction .",
"Indeed , as expected from the two-binding-site model , a Ccq1 mutant with both binding sites mutated—Ccq1-T93A/F157A/K174E completely lost its ability to bind to Est1 ( Figure 5C ) .",
"Previous mutational analysis of Est1 using the yeast two-hybrid assay identified residues in the predicted Est1 phospho-binding site ( R79 and R180 ) that mediate the interaction between Est1 and Thr93-phosphorylated Ccq1 ( Moser et al . , 2011 ) .",
"Another study found that an additional residue—K252 in the 14-3-3–like domain of Est1 is also important for Est1-Ccq1 interaction , besides for Est1-TER1 interaction ( Webb and Zakian , 2012 ) .",
"In the Est1 structure model , K252 is on a different surface compared to phospho-Thr93 binding residue R79 and R180 .",
"Est1-K252 is therefore very likely to be part of the binding site for the HDAC2/3-like domain at the N-terminus of Ccq1 .",
"In fact , in ccq1-T93A/est1-K252E cells , which bear mutations on Ccq1 and Est1 that in combination disrupt both binding sites , Ccq1-Est1 interaction could not be detected ( Figure 5C ) .",
"However , for strains ccq1-T93A/est1-R79A/R180A and ccq1-F157A/K174E/est1-K252E , in which mutations were introduced to only one of the Ccq1-Est1 binding sites with the other site intact , Ccq1 and Est1 interaction could still be observed at similar levels to single mutants ccq1-T93A or est1-K252E ( Figure 5C ) .",
"Altogether , our results indicate that phospho-Thr93 in Ccq1 contributes to one of the two binding sites that mediate Ccq1-Est1 interaction .",
"The second Est1-interacting surface resides in the HDAC2/3-like domain at the N-terminus of Ccq1 , and overlaps with the Tpz1-interacting surface .",
"To further understand the relationship of Tpz1 and Est1 binding to the HDAC2/3-like domain in Ccq1 , we performed a competitive binding assay .",
"This evaluated the interaction between Est1 and Thr93-phosphorylated Ccq1 in the presence of Tpz1 .",
"In this assay , Flag-tagged Ccq1 was immunoprecipitated from the whole cell extract by α-Flag agarose beads .",
"We then titrated increasing amounts of MBP-Tpz1-CTD to the Ccq1-Est1 complex .",
"As shown in Figure 5E the addition of MBP-Tpz1-CTD partially displaced the Ccq1-bound Est1 at a high concentration of Tpz1 .",
"This did not occur when the Ccq1-binding defective mutant MBP-Tpz1-CTD L449A was added .",
"Because of the entropic advantage provided by the phospho-Thr93-centered Est1-Ccq1 binding site , a high concentration of Tpz1 is needed to compete Est1 off the HDAC2/3-like domain of Ccq1 .",
"Taken together , the mutational analyses and the competitive binding assays indicate that Est1-Ccq1 interaction and Tpz1-Ccq1 interaction are mutually exclusive on the HDAC2/3-like domain of Ccq1 since both association events utilize the same binding surface on Ccq1 containing Phe157 and Lys174 , et al . In addition , because phosphorylated Ccq1-Thr93 provides a secondary binding site for Ccq1-Est1 association , Tpz1-Ccq1-Est1 ternary complex can also form , likely in a transient manner , not as stable as Tpz1-Ccq1 or Ccq1-Est1 binary complexes .",
"Thus , combined with our discovery of the TEL-patch in the N-terminal OB-domain of Tpz1 , we suspect that the cell cycle-regulated Ccq1 ( phospho-Thr93 ) -Est1 interaction can be coupled to the Tpz1 ( TEL-patch ) -Trt1 interaction via the Tpz1-Ccq1-Est1 intermediate complex to coordinate the recruitment of telomerase to telomeres .",
"As shown before , Rad3ATR/Tel1ATM-dependent phosphorylation of Ccq1 at Thr93 is cell cycle-regulated and peaks during late S phase ( Chang et al . , 2013 ) , correlating well with the temporal pattern of telomerase recruitment to the telomere ( Moser et al . , 2009; Webb and Zakian , 2012 ) .",
"Therefore , we asked whether the cell cycle-dependent phosphorylation of Ccq1 actually dictates the TEL-patch-mediated shelterin-telomerase interaction and thus restricts telomerase recruitment to late S phase .",
"To test this , we incubated cdc25-22 cells at non-permissive temperature ( 36°C ) for 3 hr to arrest them in late G2 phase .",
"Cells were then shifted to permissive temperature ( 25°C ) and cell samples were collected every 20 min for co-IP analysis to monitor Tpz1-Trt1 interaction in a 4-hr cell cycle window .",
"As shown in Figure 6A and C , the interaction between Tpz1 and Ccq1 remained almost unchanged throughout the cell cycle .",
"Strikingly , Tpz1 and Trt1 association gradually increased after release from the G2 arrest , peaked during late S phase ( 100–140 min ) , and then decreased to the level at G2 ( Figure 6A and D ) .",
"This temporal pattern of the Tpz1-Trt1 interaction along cell cycle progression correlates very well with that of Ccq1 phosphorylation ( Chang et al . , 2013 ) , and consequently , with that of the Ccq1-Est1 interaction promoted by Ccq1 phosphorylation , which also peaked in late S phase ( Figure 6E and G ) .",
"Consistent with our hypothesis , when the critical Rad3ATR/Tel1ATM phosphorylation site in Ccq1—Thr93 was mutated , neither Tpz1-Trt1 interaction ( Figure 6B and D ) nor Ccq1-Est1 interaction ( Figure 6F and G ) peaked in late S phase .",
"Therefore , we propose that through an intermediate telomerase recruitment complex formed by both Ccq1-Est1 and Tpz1-Trt1 interactions ( Figure 7 ) , the cell cycle information is delivered through Ccq1 Thr93-phosphorylation to the TEL-patch on Tpz1 .",
"This in turn enables Tpz1 to directly position telomerase onto the telomere and elongate it during late S phase , after most of the genome has been replicated . 10 . 7554/eLife . 15470 . 012Figure 6 . Tpz1-Trt1 interaction is cell cycle-regulated .",
"( A ) and ( B ) Co-IP assays evaluating the binding efficiency of Tpz1-Trt1 and Tpz1-Ccq1 interactions during cell cycle progression in ccq1+ ( A ) and ccq1-T93A ( B ) cells .",
"In addition to the indication of time after release from G2 arrest , the levels of S phase cyclin Cig2 , which peak at the G1/S boundary and decline to low levels in G2 and M phase , are also shown .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE .",
"( C ) and ( D ) Quantification of the binding efficiency of Tpz1-Trt1 ( C ) and Tpz1-Ccq1 ( D ) interactions during cell cycle progression as assayed in ( A ) and ( B ) .",
"The lowest level of Tpz1-Trt1 ( C ) or Tpz1-Ccq1 ( D ) interactions is set to be 1 .",
"Plots show mean values ± s . d . for two independent experiments .",
"( E ) and ( F ) Co-IP assays evaluating the binding efficiency of Ccq1-Est1 interaction during cell cycle progression .",
"As in ( A ) and ( B ) , in addition to the indication of time after release from G2 arrest , the levels of S phase cyclin Cig2 , are also shown .",
"Cdc2 was shown as the loading control .",
"Input: 1/30 of input WCE .",
"( G ) Quantification of the binding efficiency of Ccq1-Est1 interaction during cell cycle progression as assayed in ( E ) and ( F ) .",
"The lowest level of Ccq1-Est1 interaction is set to be 1 .",
"Plots show mean values ± s . d . for two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 01210 . 7554/eLife . 15470 . 013Figure 7 . Model of cell cycle-regulated telomerase recruitment via an intermediate state with two coupled telomere-telomerase interactions . A model showing telomerase recruitment in fission yeast through an intermediate state in which Trt1 and Est1 in the telomerase holoenzyme are collaboratively anchored to the telomere via Trt1-Tpz1 TEL patch and Est1-Ccq1 interactions , respectively ( illustrated by two green arrows drawn in the middle panel ) , thereby achieving cell cycle-regulated recruitment of telomerase to telomeres . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 01310 . 7554/eLife . 15470 . 014Figure 7—figure supplement 1 . A speculative two-interface intermediate-state model for human telomerase recruitment . A speculative two-interface intermediate-state model for human telomerase recruitment showing that the C-terminal domain of TIN2 is likely to be the Ccq1-functional equivalent in humans and interacts either with hEST1 or the telomerase RNP directly , forming the second connection between telomeres and telomerase in addition to the TPP1 ( TEL-patch ) -TERT ( TEN domain ) interaction . DOI: http://dx . doi . org/10 . 7554/eLife . 15470 . 014"
],
[
"Our study demonstrates that fission yeast Tpz1 also contains a TEL-patch in its OB-fold domain , analogous to its human ortholog—TPP1 ( Nandakumar et al . , 2012; Sexton et al . , 2012; Zhong et al . , 2012 ) .",
"A TEL-patch mutation causes drastically reduced association of telomerase with telomeres , and consequently an EST phenotype .",
"Similar phenotypic consequences were previously reported for the Ccq1 Thr93 phosphorylation-deficient allele ( Moser et al . , 2011; Yamazaki et al . , 2012 ) .",
"Moreover , our biochemical analyses reveal an unexpected complexity in the Ccq1-Est1 interaction involving two binding sites .",
"As schematically illustrated in Figure 7 , we envision that in late S phase , when the DNA replication machinery completes most of the genome , Rad3ATR/Tel1ATM are activated and phosphorylate the critical Thr93 residue in Ccq1 at telomeres , priming the telomere for telomerase recruitment .",
"Then , telomerase holoenzyme , minimally composed of Trt1-TER1-Est1 , is attracted to the telomere by the cell cycle-regulated , phospho-Thr93-mediated Ccq1-Est1 interaction .",
"At the same time , in a collaborative manner , Trt1 interacts with Tpz1 via the respective TEN domain and the TEL-patch residues , thus forming an intermediate telomerase recruitment complex that further engages the telomerase core enzyme ( Trt1-TER1 ) at the very 3’ end of the telomere for nucleotide additions .",
"Aided by phospho-Ccq1 Thr93 , Est1-Ccq1 interaction at their second binding site takes place , resulting in dissociation of Ccq1 from Tpz1 .",
"Because Est1 may use the same surface to interact with Ccq1 as it interacts with TER1 ( Webb and Zakian , 2012 ) , Est1 is likely to depart from TER1 upon its binding to Ccq1 .",
"With phosphorylation of the critical Thr93 in Ccq1 disappearing after late S phase , Ccq1-Est1 interaction diminishes accordingly .",
"Alignment of the very 3’ end of telomeric ssDNA to the template region in TER1 might also participate in the telomerase recruitment process , and therefore , only Tpz1 on the extreme 3’ end of the telomere , but not the majority of Tpz1 on the internal telomeric regions , is involved in forming the intermediate telomerase recruitment complex .",
"Ever since the very beginning of the telomere research at the molecular genetics level , the noncatalytic , accessory components of the telomerase holoenzyme , such as Est1p and Est3p in budding yeast , have been demonstrated to play equally important roles in telomere elongation as the catalytic core ( telomerase reverse transcriptase and telomerase RNA ) ( Lundblad and Szostak , 1989 ) .",
"Indistinguishable progressive telomere shortening phenotypes are displayed by strains either with deletion of the telomerase RNA , the reverse transcriptase subunit , or accessory proteins ( Leonardi et al . , 2008; Lingner et al . , 1997; Lundblad and Szostak , 1989; Webb and Zakian , 2008 ) , suggesting that all these components work collaboratively to form a functional telomerase .",
"Est1 , whose homologs have been identified from yeasts to humans , has been proposed to recruit and activate telomerase RNP to telomeres in a cell-cycle dependent manner .",
"Unexpectedly , a previous study discovered that the telomerase core , RNP , Trt1 and TER1 RNA , is also required for the association of Est1 with the telomere ( Webb and Zakian , 2012 ) .",
"Our finding that telomerase holoenzyme is recruited to telomeres via an intermediate state involving two-pronged cooperative Est1-Ccq1 and Trt1-Tpz1 interactions , uncovers the active role of Trt1 itself in the recruitment process .",
"In addition , our model explains the intricate interdependence between Trt1 , TER1 , and Est1 for telomeric association of the holoenzyme .",
"What are the advantages of employing two cooperative telomere-telomerase interactions to recruit telomerase to telomeres ?",
"We think that this mode enables temporal and spatial regulation of telomerase recruitment .",
"It has been shown in budding yeast that Tel1ATM prefers shorter telomeres than longer ones .",
"We demonstrate here that the cell cycle-dependent interaction between Ccq1 and Est1 is coupled to the Tpz1 TEL patch-Trt1 interaction .",
"Moreover , in fission yeast , strains with shorter telomeres show a higher level of Ccq1 phosphorylation ( Moser et al . , 2011 ) .",
"Therefore , shorter telomeres could have more access to telomerase due to the higher level of Thr93 phosphorylation in Ccq1 .",
"After locating which telomere to elongate , interaction between the TEL-patch of Tpz1 and the catalytic subunit Trt1 further orients the very 3’ end of telomeric ssDNA via the sequence-specific Pot1/Tpz1-ssDNA interaction to the active site of Trt1 .",
"Two-interface recruitment mode has been observed in other pathways for similar purposes .",
"For example , 53BP1 , an important effector of DNA double-strand-break ( DSB ) response , simultaneously recognizes mononucleosomes containing dimethylated H4K20 ( H4K20me2 ) and H2A ubiquitinated on Lys15 ( H2AK15ub ) ( Fradet-Turcotte et al . , 2013 ) .",
"There , it was proposed that the engagement of H4K20me2 by the Tudor domain of 53BP1 positions its ubiquitination-dependent recruitment ( UDR ) motif in the correct orientation to contact the epitope formed by H2AK15ub , and thus to ensure that 53BP1 responds only to bona fide DSB signaling .",
"Within the fission yeast telomere shelterin complex , Tpz1 physically lies in the middle of the telomeric ssDNA and dsDNA binding proteins .",
"Functionally , Tpz1 is positioned between the positive and negative regulators of the telomerase elongation .",
"Tpz1 directly interacts with three other shelterin components: Poz1 , Ccq1 , and Pot1 .",
"This unique position of Tpz1 in the shelterin complex enables its architectural role in shelterin complex assembly and underscores its potential coordination roles in communicating the dsDNA length and/or structural information to the 3’ end of ssDNA—telomerase’s ultimate destination .",
"In our previous studies , utilizing biochemically identified Tpz1 separation-of-function mutants that can individually but specifically disrupt Tpz1’s interactions with Poz1 , Ccq1 , or Pot1 , we found that Tpz1-mediated complete linkage between telomere dsDNA and ssDNA binding proteins in the shelterin complex is required for defining the telomerase-nonextendible state of telomeres ( Jun et al . , 2013 ) .",
"Disruption of the linkage on either the dsDNA binder or the ssDNA binder side of Tpz1 causes unregulated elongation of telomeres .",
"In addition to maintaining the telomerase-nonextendible state , through its interaction with Ccq1 , Tpz1 may also activate the telomerase-nonextendible state of telomeres by participating in breaking down the 'shelterin bridge' .",
"Moreover , Lys75 , which is close to the TEL-patch in the OB-fold domain of Tpz1 , was demonstrated to have telomerase activation function , the step that follows successful telomerase-telomere association and alters telomerase conformation to become competent for telomere synthesis ( Armstrong et al . , 2014 ) .",
"Recently , SUMOylation of Tpz1-Lys242 in late S phase was shown to enhance the Tpz1-Stn1 interaction , promote Stn1-Ten1 association with telomeres , and thus to coordinate synthesis of the telomeric lagging strand by Polα stimulated via Stn1-Ten1-Polα interaction ( Hannan et al . , 2015; Miyagawa et al . , 2014 ) .",
"The TEL-patch in Tpz1 and its conserved role in telomerase recruitment characterized in this work further extend the versatility of Tpz1 in telomere length homeostasis .",
"Evidently , the ability to regulate the telomeric state together with the role of recruiting/activating telomerase and coordinating Polα are integrated into one single telomeric protein—Tpz1 .",
"This integration ensures timely , accurate , and efficient coupling of conformational transitions of the telomere to the engagement and activation of the telomerase RNP .",
"Although the TEL-patch of the shelterin component TPP1 acting as the telomere-telomerase interface was initially discovered and extensively studied in human cells ( Hockemeyer and Collins , 2015; Schmidt and Cech , 2015 ) , upstream regulatory events of TEL-patch-mediated telomerase recruitment remain to be elucidated .",
"In contrast , regulatory pathways and factors that control telomerase recruitment have been fairly well studied in both budding and fission yeasts due to their convenient and precise genetic manipulability .",
"For instance , in budding yeast , cell cycle-specific assembly and disassembly of active telomerase RNP at telomeres have been shown to restrict telomerase action to late S phase ( Tucey and Lundblad , 2014 ) .",
"Moreover , telomerase tends to preferentially elongate short telomeres and Tel1ATM is enriched in short telomeres to achieve telomere length homeostasis ( Bianchi and Shore , 2007; Sabourin et al . , 2007 ) .",
"Cdk1 was also shown to control the temporal recruitment of telomerase by directing the timing of Cdc13 and Stn1 phosphorylation along cell cycle progression ( Li et al . , 2009; Liu et al . , 2014 ) .",
"Two recent studies unveil the conserved regulatory role of ATM/ATR in human recruitment and telomere elongation ( Lee et al . , 2015; Tong et al . , 2015 ) ; however , the downstream substrate is mostly unknown .",
"In this work , we uncovered the conservation of the TEL-patch in fission yeast Tpz1 , with similar biochemical and functional roles to human TPP1 .",
"This similarity between human and fission yeast makes us speculate that the conservation could probably be extended to other aspects of the telomerase recruitment pathway .",
"Is it possible that human telomerase recruitment also involves two coupled telomere-telomerase interactions ?",
"What is the Ccq1-Est1 interaction equivalent in human cells ?",
"In fact , human TIN2 ( Poz1 homolog ) has been demonstrated to be essential for telomerase recruitment ( Abreu et al . , 2010 ) .",
"DC mutations in the C-terminal region of TIN2 ( not contained in Poz1 ) lead to defective association of TIN2 with telomerase ( Frank et al . , 2015; Yang et al . , 2011 ) .",
"As illustrated in Figure 7—figure supplement 1 , we speculate that the C-terminal domain of TIN2 is likely to be the Ccq1-functional equivalent in humans and interacts either with hEST1 or the telomerase RNP directly , forming the second interface between telomeres and telomerase in addition to the TPP1 ( TEL-patch ) -TERT ( TEN domain ) interaction .",
"Moreover , TIN2 may also be subject to cell cycle-controlled post-translational modifications , and thus mediate its telomerase recruitment function in a highly regulated way ."
],
[
"Fission yeast strains used in this study are listed in Supplementary file 1 .",
"Single-mutant strains were constructed by one-step gene replacement of the entire open reading frame ( ORF ) with the selectable marker .",
"Double- and triple-mutant strains were produced by mating , sporulation , dissection , and selection followed by PCR verification of genotypes .",
"Genes were fused to specific epitope-tags at the C-terminus by homologous recombination; the pFA6a plasmid modules were used as templates for PCR ( Bähler et al . , 1998; Sato et al . , 2005 ) .",
"Point mutations were made by site-directed mutagenic PCR using the high fidelity polymerase Pfu ( Agilent ) .",
"All mutations were confirmed by DNA sequencing ( Eton , San Diego , CA ) .",
"The Trt1-Tpz1 fusion strains were constructed based on previously published strains ( Armstrong et al . , 2014 ) .",
"S . pombe cells grown in 5 ml YEAU overnight were harvest for genomic DNA extraction .",
"EcoRI-digested genomic DNA was separated on 1% agarose gel at 70 V for 18 . 5 hr , and then the gels were incubated in 0 . 25 M hydrochloric acid for 15 min followed by 0 . 5 M sodium hydroxide and 1 . 5 M sodium chloride buffer for 30 min and 0 . 5 M Tris-HCl ( pH 7 . 0 ) and 1 . 5 M sodium chloride for 30 min .",
"DNA was transferred to Amersham Hybond-N+ membrane ( GE Healthcare Life Sciences ) via capillary blotting .",
"DNA was cross-linked to the membrane .",
"The telomeric probe was prepared as previously described ( Jun et al . , 2013 ) .",
"The template of pol1+ was amplified with 5’ primer ( GGTGCAGAAGACGGTCTG CAAG ) and 3’ primer ( CTTAGCATGCAGAAGCATGCGC ) , and both probes were labeled by random hexamer labeling using [α-32P]-dCTP and High Prime ( Roche ) .",
"Hybridizations were carried out with 6 million cpm of probe in Church-Gilbert buffer at 55°C .",
"Blots with both telomeric and pol1+ probe were expose to PhosphorImager screens that were visualized using a Typhoon scanner ( GE Healthcare ) .",
"Frozen cell pellets were cryogenically disrupted with FastPrep MP with three pulses ( 60 s ) of bead-beating in ice-cold lysis buffer ( 50 mM HEPES at pH 7 . 5 , 140 mM NaCl , 15 mM EGTA , 15 mM MgCl2 , 0 . 1% NP40 , 0 . 5 mM Na3VO4 , 1 mM NaF , 2 mM PMSF , 2 mM benzamidine , Complete proteinase inhibitor [Roche] ) .",
"Centrifuge clarified whole cell extracts were adjusted to 13 mg/ml .",
"300 μl cell extracts were incubated with either anti-Flag M2 affinity gel ( Sigma ) , anti-Ccq1 rabbit serum plus Protein G beads ( Roche ) , or c-myc antibody ( Santa Cruz ) plus Protein G beads for 2–4 hr at 4°C .",
"The beads were resuspended in SDS loading buffer , boiled , and subjected to western blotting .",
"Western blot analysis was performed using monoclonal anti-Flag ( M2-F1804 , from Sigma ) , monoclonal anti-PK ( from Abcam ) , anti-Ccq1 rabbit serum , monoclonal anti-Myc ( 9E10 , from Covance ) , and anti-Cdc2 ( y100 . 4 , from Abcam ) .",
"20 μg whole cell extract were used for input control .",
"Fresh S . pombe cells in liquid culture were fixed with 1/10 ( vol/vol ) ratio of an 11% formaldehyde solution ( 11% formaldehyde , 100 mM NaCl , 1 mM EDTA at pH 8 . 0 , 0 . 5 mM EGTA , 50 mM Tris-HCl at pH 8 . 0 ) for 20 min , followed by the termination with 125 mM glycine for 5 min .",
"Cell pellets were disrupted in 400 μL of lysis buffer ( 50 mM Hepes at pH 7 . 5 , 140 mM NaCl , 1 mM EDTA , 1% Trition X-100 , 0 . 1% sodium deoxycholate , Complete proteinase inhibitor [Roche] , 1 mM PMSF , 1 mM benzamidine , 1 mM Na3VO4 , 1 mM NaF ) with FastPrep MP .",
"After three pulse ( 1 min ) of beads-beating , at least 90% cells were broken .",
"Cell extracts were sonicated one time for 30 s in 45 cycles using a Bioruptor ( Diagenode ) .",
"Clarified cell extracts were incubated with anti-Myc resin ( 9E10 , Santa Cruz ) , anti-Flag M2 affinity gel ( Sigma ) or anti-Ccq1 rabbit serum followed by protein G-agarose ( Roche , Indianapolis , IN ) for 3 hr at 4°C .",
"Then , the beads were washed sequentially , each twice , with lysis buffer , lysis buffer with 500 mM NaCl , wash buffer , and 1x TE buffer .",
"Each sample was added with 100 μl of 10% Chelex100 resin and boiled for 15 min , followed by 20 μg proteinase K treatment for 30 min at 55°C .",
"The recovered DNA were denatured with 0 . 4 M NaOH and transferred to a Hybond-XL membrane by using a slot module .",
"The blots were hybridized with telomeric probe; the same blot was then re-probed with rDNA probe after stripping off the telomere probe .",
"Myc-tagged Est1 under the nmt1 promoter was expressed in a wild-type strain or a strain expressing Flag-tagged Ccq1 , these cells were harvested and cryogenically disrupted to obtain clarified extract followed by co-immunoprecipitation using anti-Flag M2 affinity gel ( Sigma ) as described above .",
"After incubation , the beads were washed three times with 800 μl lysis buffer .",
"The competition assay was carried out by adding 100 μl lysis buffer with increasing concentrations ( 5 , 100 , 2000 nM ) of purified recombinant MBP-Tpz1 ( 406–508 ) or MBP-Tpz1 ( 406–508 ) -L449A to the beads .",
"The mixture was incubated at 4°C for 2 hr with the tube rotating .",
"After extensive washing , the beads were resuspended in SDS-PAGE loading buffer , boiled , and subjected to Western blotting with anti-Flag ( M2-F1804 , from Sigma ) , anti-Myc ( 9E10 , from Covance ) , or anti-MBP ( N-17 , from Santa Cruz ) antibodies .",
"cdc25-22 cells bearing ccq1+ or ccq1-T93A grown in 600 ml YEAU overnight at 25°C to OD ~0 . 3 were shifted to restrictive temperature ( 36°C ) for 3 hr to arrest cells in late G2 phase .",
"Synchronous cultures were then generated by releasing these cdc25-22 cells to the permissive temperature ( 25°C ) .",
"50 ml cultures were collected every 20 min for 4 hr and were subsequently subjected to co-immunoprecipitation analyses to evaluate Est1-Ccq1 and Tpz1-Trt1 interactions .",
"Anti-Cdc2 ( y100 . 4 , from Abcam ) was used as a control for input ."
]
] | [
"Tightly controlled recruitment of telomerase , a low-abundance enzyme , to telomeres is essential for regulated telomere synthesis .",
"Recent studies in human cells revealed that a patch of amino acids in the shelterin component TPP1 , called the TEL-patch , is essential for recruiting telomerase to telomeres .",
"However , how TEL-patch—telomerase interaction integrates into the overall orchestration of telomerase regulation at telomeres is unclear .",
"In fission yeast , Tel1ATM/Rad3ATR-mediated phosphorylation of shelterin component Ccq1 during late S phase is involved in telomerase recruitment through promoting the binding of Ccq1 to a telomerase accessory protein Est1 .",
"Here , we identify the TEL-patch in Tpz1TPP1 , mutations of which lead to decreased telomeric association of telomerase , similar to the phosphorylation-defective Ccq1 .",
"Furthermore , we find that telomerase action at telomeres requires formation and resolution of an intermediate state , in which the cell cycle-dependent Ccq1-Est1 interaction is coupled to the TEL-patch—Trt1 interaction , to achieve temporally regulated telomerase elongation of telomeres ."
] | [
"The genetic blueprints for animals , plants and fungi are mostly contained within long strands of DNA and packaged into more compact thread-like structures called chromosomes .",
"As such , most cells need to duplicate their chromosomes before they divide so that the two new cells each get a complete set of genetic instructions .",
"The machinery that copies DNA is unable to make it to the very ends of the chromosomes .",
"Instead , an enzyme called telomerase adds new DNA to the chromosome ends to prevent them becoming too short .",
"Problems with this process can cause serious issues , such as cell death or cancer , and so the activity of telomerase is carefully controlled .",
"Other proteins guide telomerase to the ends of the chromosome only after the rest of the DNA has been copied .",
"However , scientists do not know exactly how cells correctly time the arrival of telomerase .",
"A group of proteins called shelterin protects the chromosome ends , and studies with human cells have shown that telomerase attaches to a specific patch on one of shelterin proteins , called the TEL-patch , to begin its work .",
"Now , Hu , Liu et al . have identified a similar TEL-patch in a shelterin protein from a type of yeast called fission yeast; this patch is also needed to attach telomerase to the chromosome ends .",
"Further experiments with this yeast then showed that telomerase only arrives at the ends of the chromosomes after two parallel interaction interfaces have formed .",
"Importantly , one of these interactions only takes place after most of the chromosomes are have been copied .",
"As such , this “two-pronged interaction” mechanism ensures that the telomerase enzyme arrives at the end of the chromosomes at the right time .",
"Other similarities between human and fission yeast chromosome ends make it plausible that a comparable process controls the timing of telomerase attachment in human cells .",
"However , more studies will be needed to confirm if this is the case ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression"
] | A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype | elife-02313-v1 | [
[
"Epigenetic dysregulation of gene expression plays a major role in the initiation and progression of cancer ( reviewed in Baylin and Jones , 2011; Esteller , 2008; Hassler and Egger , 2012 ) .",
"Among the various epigenetic alterations of cancer genomes , abnormal gains of DNA methylation in normally unmethylated gene promoter CpG islands have been the most extensively investigated .",
"DNA hypermethylation can alter genetic stability and genomic structure , and is associated with transcriptional silencing of gene expression ( commonly referred to as epigenetic silencing; reviewed in Baylin and Jones , 2011; Esteller , 2008; Hassler and Egger , 2012 ) .",
"Numerous studies have identified specific genes affecting cellular growth control that become hypermethylated and transcriptionally silenced in many cancers .",
"Colorectal cancers ( CRCs ) provide a striking example of alterations in DNA methylation that occur during tumor development .",
"A subset of CRCs have a so-called CpG island methylator phenotype ( CIMP ) characterized by aberrant DNA hypermethylation of many genes .",
"In fact , CRCs can be categorized into three distinct subclasses based on their epigenetic and genetic profiles: CIMP-1 ( also called CIMP-high ) , characterized by intense methylation of multiple genes , microsatellite instability and BRAF mutations; CIMP-2 ( also called CIMP-low ) , typified by methylation of a more limited group of genes and mutation in KRAS; and CIMP-negative , distinguished by infrequent methylation and p53 mutation ( Yagi et al . , 2010; Kaneda and Yagi , 2011 ) .",
"Specific panels of CIMP marker genes have been developed to classify CRCs into these three subclasses .",
"These CIMP marker genes have potential clinical value , and are currently being evaluated as biomarkers for risk , diagnosis , prognosis , and prediction of therapeutic responsiveness ( reviewed in Egger et al . , 2012; Gyparaki et al . , 2013 ) .",
"Among the CIMP genes are three well-known tumor suppressors: p14ARF , p16INK4A ( also known as CDKN2A ) , and p15INK4B ( also known as CDKN2B ) ( reviewed in Gil and Peters , 2006 ) .",
"These three genes are located in close proximity to one another ( within a 35 kb region ) at the INK4-ARF locus , yet each is transcribed from a distinct promoter .",
"Interestingly , p14ARF and p16INK4A share exons two and three , but each is translated in a different reading frame , yielding unrelated polypeptides .",
"Inactivation of the INK4-ARF locus is one of the most frequent events in cancers ( reviewed in Kim and Sharpless , 2006 ) .",
"For example , INK4-ARF is transcriptionally silenced in 30–45% of all CRCs and in 70% of CRCs that harbor an activating KRAS mutation ( Burri et al . , 2001; Dominguez et al . , 2003; Lind et al . , 2004 ) .",
"The INK4-ARF locus is also silenced in some non-malignant cells .",
"For example , INK4-ARF is silenced in embryonic , fetal , and adult stem cells , but in more differentiated cells , it becomes poised for expression and increasingly responsive to aberrant mitogenic signals such as those elicited by activated oncogenes ( reviewed in Sherr , 2012 ) .",
"This process is reversed when somatic cells are induced to regain pluripotency through reprogramming .",
"Expression of INK4-ARF limits stem cell self-renewal , suggesting that coordinated INK4-ARF expression may normally act to restrict stem cell numbers .",
"Accordingly , the INK4-ARF locus has been shown to be a barrier for reprogramming ( Li et al . , 2009 ) .",
"In actively growing human diploid fibroblasts , the INK4A-ARF locus is silenced by histone H3 lysine 27 trimethylation ( H3K27me3 ) directed by Polycomb group proteins .",
"When such cells are exposed to cellular stress , such as oncogenic signals , the H3K27me3 mark on the locus is decreased , resulting in expression of INK4A-ARF genes ( Jacobs et al . , 1999; Bracken et al . , 2007; Kotake et al . , 2007 ) .",
"Transcriptional activation is due , at least in part , to upregulation of the H3K27 demethylase JMJD3 , which removes H3K27me3 from INK4A-ARF ( Agger et al . , 2009 ) .",
"Whether the mechanism of INK4-ARF silencing in stem cells and primary differentiated cells is related to , or distinct from , that in cancer cells is unknown .",
"The factors , regulatory pathways , and mechanisms underlying the aberrant promoter hypermethylation and transcriptional silencing characteristic of CIMP-positive CRCs remain to be determined .",
"In addition , the relationship between the initiating genetic events responsible for tumorigenesis ( e . g . , acquisition of activating mutations in oncogenes ) and the epigenetic alterations in CIMP-positive CRCs is not understood .",
"To begin to address these questions , in this study , using p14ARF as a representative CIMP gene , we perform an RNA interference ( RNAi ) screen to identify factors required for p14ARF silencing .",
"Our results reveal a KRAS-directed pathway that mediates silencing of the entire INK4-ARF locus , is responsible for CIMP in CRCs , and is related to the pathway that silences INK4-ARF in human embryonic stem cells ( hESCs ) ."
],
[
"To screen for factors involved in transcriptional silencing of INK4-ARF , we generated a reporter construct in which the p14ARF promoter was used to direct expression of the blasticidin-resistance ( BlastR ) gene ( Figure 1A ) .",
"This p14ARF-BlastR reporter construct was stably transduced into DLD-1 cells , a human CRC cell line in which endogenous p14ARF is transcriptionally silenced ( Zheng et al . , 2000; Figure 1B ) .",
"We selected cells in which the reporter gene had been silenced , as evidenced by acquisition of blasticidin resistance ( Figure 1C ) , transcriptional derepression ( Figure 1B ) , and decreased DNA hypermethylation ( Figure 1D ) following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine . 10 . 7554/eLife . 02313 . 003Figure 1 . Derivation and validation of the DLD-1/p14ARF-BlastR reporter cell line .",
"( A ) Schematic of the shRNA screen .",
"( B ) qRT-PCR analysis monitoring p14ARF expression in parental DLD-1 cells , or p14ARF and BlastR expression in DLD-1/p14ARF-BlastR cells , following treatment with either DMSO or 5-aza-2′-deoxycytidine ( AZA ) .",
"The results were normalized to that observed upon DMSO treatment , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 .",
"( C ) Viability of DLD-1 or DLD-1/p14ARF-BlastR cells treated with DMSO or AZA for 3 days and then 0 , 5 , or 10 µM blasticidin for 6 days .",
"Cells were stained with crystal violet .",
"( D ) Bisulfite sequencing analysis of the endogenous p14ARF promoter in parental DLD-1 cells or the p14ARF-BlastR reporter in DLD-1/p14ARF-BlastR cells treated in the absence or presence of AZA .",
"( Top )",
"Schematic of the p14ARF promoter; positions of CpGs are shown to scale by vertical lines .",
"( Bottom )",
"Each circle represents a methylated ( black ) or unmethylated ( white ) CpG dinucleotide .",
"Each row represents a single clone . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 003 A genome-wide human small hairpin ( shRNA ) library ( Silva et al . , 2005 ) comprising ∼62 , 400 shRNAs was divided into 10 pools , which were packaged into retrovirus particles and used to stably transduce the DLD-1/p14ARF-BlastR reporter cell line .",
"Blasticidin-resistant colonies , indicative of derepression of the reporter gene , were selected and the shRNAs identified by sequence analysis ( Figure 1A ) .",
"Positive candidates identified in the primary screen were validated by stably transducing DLD-1 cells with an shRNA directed against each candidate gene , followed by the analysis of endogenous p14ARF expression by quantitative RT-PCR ( qRT-PCR ) .",
"Using this approach , we identified eight genes that , following shRNA-mediated knockdown , resulted in derepression of endogenous p14ARF ( Figure 2A , Figure 2—figure supplement 1 ) .",
"qRT-PCR analysis confirmed that each shRNA reduced target gene expression ( Figure 2—figure supplement 2 ) .",
"For all genes , a second shRNA whose sequence was unrelated to that isolated from the primary screen also resulted in target gene knockdown ( Figure 2—figure supplement 3A ) and derepression of endogenous p14ARF ( Figure 2—figure supplement 3B ) . 10 . 7554/eLife . 02313 . 004Figure 2 . Identification of a ZNF304-corepressor complex required for transcriptional silencing of INK4-ARF in CRCs .",
"( A ) qRT-PCR analysis monitoring INK4-ARF expression in DLD-1 cells expressing a non-silencing ( NS ) or ZNF304 shRNA .",
"The results were normalized to that obtained with the NS control , which was set to 1 .",
"( B ) Immunoblot analysis monitoring INK4-ARF levels in DLD-1 cells expressing a NS or ZNF304 shRNA .",
"α-tubulin ( TUBA ) was monitored as a loading control .",
"( C ) qRT-PCR analysis monitoring INK4-ARF expression in DLD-1 cells expressing a NS , KAP1 , SETDB1 , DNMT1 , DNMT3A , or DNMT3B shRNA .",
"( D ) ChIP assay monitoring binding of ZNF304 , KAP1 , SETDB1 and DNMT1 to INK4-ARF promoters in DLD-1 cells expressing a NS or ZNF304 shRNA .",
"The results were normalized to that obtained with IgG , which was set to 1 .",
"( E ) Bisulfite sequencing analysis of the p14ARF promoter in DLD-1 cells expressing a NS , KAP1 , SETDB1 , or DNMT1 shRNA .",
"( F ) Tumor formation assay .",
"DLD-1 cells expressing a NS and ZNF304 ( left ) or DNMT1 ( right ) shRNA were subcutaneously injected into the flanks of nude mice ( n = 3 ) , and tumor formation was measured .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 .",
"Results from experiments showing validation of candidates from the RNAi screen , and ZNF304 corepressors , for a role in INK4-ARF transcriptional silencing in DLD-1 cells are presented in Figure 2—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 00410 . 7554/eLife . 02313 . 005Figure 2—figure supplement 1 . Validation of candidates from the RNAi screen for a role in INK4-ARF transcriptional silencing in DLD-1 cells .",
"( A ) List of candidate genes obtained from the primary RNAi screen .",
"( B ) qRT-PCR analysis monitoring expression of p14ARF , p15INK4B , and p16INK4A in DLD-1 cells stably expressing an shRNA targeting a gene isolated from the RNAi screen or , as a control , a non-silencing ( NS ) shRNA .",
"The results were normalized to that obtained with the NS shRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 00510 . 7554/eLife . 02313 . 006Figure 2—figure supplement 2 . Knockdown efficiencies of candidate shRNAs isolated from the RNAi screen . qRT-PCR analysis monitoring knockdown efficiency for each candidate using an shRNA isolated from the primary screen .",
"Values are given relative to expression of each gene following treatment with a NS shRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 00610 . 7554/eLife . 02313 . 007Figure 2—figure supplement 3 . Validation of candidates from the RNAi screen for a role in p14ARF transcriptional silencing in DLD-1 cells using a second shRNA .",
"( A ) qRT-PCR analysis monitoring knockdown efficiency for each candidate using a second shRNA unrelated to that isolated in the primary screen .",
"Values are given relative to expression of each gene following treatment with a NS shRNA , which was set to 1 .",
"( B ) qRT-PCR analysis monitoring p14ARF expression in DLD-1 cells stably expressing an shRNA targeting a candidate gene , using a second , unrelated shRNA against the same target gene .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 00710 . 7554/eLife . 02313 . 008Figure 2—figure supplement 4 . Validation of ZNF304 corepressors for a role in INK4-ARF transcriptional silencing in DLD-1 cells .",
"( A ) qRT-PCR analysis monitoring knockdown efficiency of KAP1 , SETDB1 , DNMT1 , DNMT3A , and DNMT3B .",
"Values are given relative to expression of each gene following treatment with a NS shRNA , which was set to 1 .",
"( B ) qRT-PCR analysis monitoring knockdown efficiency using a second , unrelated shRNA against the same target gene .",
"( C ) qRT-PCR analysis monitoring expression of p14ARF , p15INK4B , and p16INK4A in DLD-1 cells stably expressing a second , unrelated shRNA against KAP1 , SETDB1 , DNMT1 DNMT3A , or DNMT3B .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 008 Our previous studies have shown that transcriptional silencing of tumor suppressor genes ( TSGs ) involves a sequence-specific DNA-binding protein ( Gazin et al . , 2007; Palakurthy et al . , 2009 ) .",
"Therefore , we elected to focus on ZNF304 , a zinc finger DNA-binding protein that contains a KRAB repressor domain ( Sabater et al . , 2002 ) .",
"The qRT-PCR and immunoblot results of Figure 2A , B show that in addition to p14ARF , knockdown of ZNF304 derepressed p15INK4B and p16INK4A , which are also transcriptionally silenced in DLD-1 cells ( Zheng et al . , 2000; Ishiguro et al . , 2006 ) .",
"KRAB domain proteins function by recruiting a corepressor complex that includes the scaffolding protein KAP1 and the histone methyltransferase SETDB1 ( Iyengar and Farnham , 2011 ) .",
"Knockdown of KAP1 or SETDB1 ( Figure 2—figure supplement 4A ) resulted in derepression of p14ARF , p15INK4B , and p16INK4A ( Figure 2C ) .",
"Similar results were obtained using a second , unrelated KAP1 or SETDB1 shRNA ( Figure 2—figure supplement 4B , C ) .",
"p14ARF , p15INK4B , and p16INK4A were also substantially derepressed following knockdown of DNMT1 but not the other DNA methyltransferases , DNMT3A , or DNMT3B ( Figure 2C , Figure 2—figure supplement 4 ) .",
"The identification of KAP1 , SETDB1 , and DNMT1 as ZNF304 corepressors indicates that our shRNA screen , like other large-scale shRNA screens ( reviewed in Mullenders and Bernards , 2009 ) , was not saturating .",
"The chromatin immunoprecipitation ( ChIP ) assay of Figure 2D shows that ZNF304 , as well as KAP1 , SETDB1 and DNMT1 , were bound to the p14ARF , p15INK4B , and p16INK4A promoters in DLD-1 cells .",
"Notably , knockdown of ZNF304 substantially decreased binding of KAP1 , SETDB1 , and DNMT1 .",
"Knockdown of ZNF304 , KAP1 , SETDB1 or DNMT1 also decreased p14ARF promoter hypermethylation ( Figure 2E ) .",
"We predicted that the loss of ZNF304 , which results in derepression of the INK4-ARF locus , would reduce tumorigenicity .",
"Consistent with this prediction , Figure 2F shows that shRNA-mediated knockdown of ZNF304 in DLD-1 cells significantly suppressed tumor growth in mouse xenografts .",
"shRNA-mediated knockdown of DNMT1 similarly suppressed tumor growth , consistent with previous results ( Morita et al . , 2013 ) .",
"DLD-1 cells contain an activated KRAS ( G13D ) mutation and we therefore investigated the relationship between KRAS and silencing of INK4-ARF .",
"shRNA-mediated knockdown of KRAS in DLD-1 cells ( Figure 3—figure supplement 1A , B ) resulted in derepression of p14ARF , p15INK4A , and p16INK4B ( Figure 3A , Figure 3—figure supplement 1C ) and substantially reduced binding of ZNF304 and its corepressors to all three promoters ( Figure 3B ) .",
"Similarly , treatment with manumycin A , a RAS farnesyltransferase inhibitor ( Hara et al . , 1993 ) , also resulted in derepression of p14ARF , p15INK4B , and p16INK4A ( Figure 3C ) and reduced binding of ZNF304 and its corepressors to the three promoters ( Figure 3D ) .",
"Notably , shRNA-mediated knockdown or pharmacological inhibition of KRAS markedly reduced ZNF304 protein levels ( Figure 3E ) .",
"Likewise , addition of the phosphoinositide 3-kinase ( PI3K ) inhibitor LY294002 ( Vlahos et al . , 1994 ) or PI-103 , which blocks the PI3K-AKT signaling pathway downstream of activated KRAS ( Hayakawa et al . , 2006 ) , also derepressed p14ARF , p15INK4B , and p16INK4A and reduced ZNF304 levels ( Figure 3F ) .",
"By contrast , following shRNA-mediated knockdown or pharmacological inhibition of KRAS , ZNF304 mRNA levels were not significantly affected ( Figure 3G ) .",
"Thus , upregulation of ZNF304 by activated KRAS is predominantly post-transcriptional .",
"Consistent with this idea , the reduction of ZNF304 protein levels following KRAS inhibition could be counteracted by proteasome inhibition ( Figure 3H ) . 10 . 7554/eLife . 02313 . 009Figure 3 . Activated KRAS-mediated upregulation of ZNF304 is required for transcriptional silencing of INK4-ARF .",
"( A ) qRT-PCR analysis monitoring INK4A-ARF expression in DLD-1 cells expressing a NS or KRAS shRNA .",
"( B ) ChIP analysis monitoring binding of ZNF304 , KAP1 , SETDB1 , and DNMT1 to INK4-ARF promoters in DLD-1 cells expressing a NS or KRAS shRNA .",
"( C ) qRT-PCR analysis monitoring INK4A-ARF expression in DLD-1 cells treated with DMSO or manumycin A ( Man . A ) .",
"The results were normalized to DMSO , which was set to 1 .",
"( D ) ChIP analysis monitoring binding of ZNF304 , KAP1 , SETDB1 , and DNMT1 to INK4-ARF promoters in DLD-1 cells treated with DMSO or Man .",
"A . ( E ) Immunoblot analysis showing INK4-ARF levels in DLD-1 cells treated with a NS or KRAS shRNA , or DMSO or Man .",
"A . ( F ) Immunoblot analysis showing INK4-ARF levels in DLD-1 cells treated with DMSO , LY294002 , or PI-103 .",
"( G ) qRT-PCR analysis monitoring ZNF304 expression in DLD-1 cells treated with a NS or KRAS shRNA , or DMSO or Man .",
"A . ( H ) Immunoblot analysis showing ZNF304 levels in DLD-1 cells treated with Man .",
"A for 24 hr and 0–10 µM MG-132 for 4 hr . ( I ) PAT-ChIP analysis monitoring binding of ZNF304 to INK4-ARF promoters in matched adjacent normal ( N ) and KRAS-positive CRC human tumor ( T ) samples .",
"Results were normalized to normal samples , which were set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 .",
"Results from experiments validating KRAS knockdown efficiency and the role of KRAS in repressing p14ARF expression , as well as experiments validating the role of ZNF304 and its corepressors in INK4-ARF silencing in other KRAS-positive CRC cell lines , are presented in Figure 3—figure supplements 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 00910 . 7554/eLife . 02313 . 010Figure 3—figure supplement 1 . Validation of a role for KRAS in p14ARF transcriptional silencing in DLD-1 cells .",
"( A and B )",
"Validation of KRAS shRNA knockdown efficiency .",
"qRT-PCR analysis monitoring KRAS expression in DLD-1 cells stably expressing a NS or KRAS shRNA ( A ) or a second , unrelated KRAS shRNA ( B ) .",
"Values are given relative to expression of KRAS following treatment with a NS shRNA , which was set to 1 .",
"( C ) qRT-PCR analysis monitoring p14ARF expression in DLD-1 cells stably expressing a second , unrelated KRAS shRNA .",
"Values are given relative to expression of p14ARF following treatment with a NS shRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01010 . 7554/eLife . 02313 . 011Figure 3—figure supplement 2 . ZNF304 and its corepressors bind to the INK4-ARF promoters in other KRAS-positive human CRC cell lines . ChIP analysis monitoring binding of ZNF304 , KAP1 , SETDB1 , and DNMT1 at the promoters of p14ARF , p15INK4B , and p16INK4A in HCT116 ( left ) and HCT15 ( right ) cells .",
"As a negative control , binding of the factors was also monitored at an irrelevant DNA region ( negative control [NC] DNA ) .",
"The results were normalized to that obtained with an IgG control antibody , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01110 . 7554/eLife . 02313 . 012Figure 3—figure supplement 3 . Validation of a role for ZNF304 and KRAS in INK4-ARF transcriptional silencing in other KRAS-positive human CRC cell lines .",
"( A ) qRT-PCR analysis monitoring expression of p14ARF , p15INK4B , and p16INK4A in HCT116 ( left ) and HCT15 ( right ) cells stably expressing an NS , ZNF304 , or KRAS shRNA .",
"The results were normalized to that obtained with the NS shRNA , which was set to 1 .",
"( B ) qRT-PCR analysis monitoring knockdown efficiency of the ZNF304 and KRAS shRNAs in HCT116 ( left ) and HCT15 ( right ) cells .",
"( C ) qRT-PCR analysis monitoring expression of p14ARF , p15INK4B , and p16INK4A in HCT116 ( left ) and HCT15 ( right ) cells treated with DMSO ( as a control ) or manumycin A ( Man . A ) .",
"The results were normalized to that obtained with DMSO , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01210 . 7554/eLife . 02313 . 013Figure 3—figure supplement 4 . The p14ARF promoter is hypermethylated in KRAS-positive human CRC samples . Bisulfite sequencing analysis of the p14ARF promoter in matched adjacent normal colon ( N ) and KRAS-positive CRC human tumor samples ( T ) samples .",
"( Top )",
"Schematic of the promoter; positions of CpGs are shown to scale by vertical lines .",
"( Bottom )",
"Each circle represents a methylated ( black ) or unmethylated ( white ) CpG dinucleotide .",
"Each row represents a single clone . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 013 To determine the generality and clinical relevance of these results , we analyzed other KRAS-positive human CRC cell lines and tumor samples .",
"In HCT116 and HCT15 CRC cell lines , in which INK4-ARF is transcriptionally silenced ( Yagi et al . , 2010 ) , ZNF304 and its corepressors were associated with the p14ARF , p15INK4B , and p16INK4A promoters ( Figure 3—figure supplement 2 ) .",
"Moreover , shRNA-mediated knockdown of ZNF304 or KRAS ( Figure 3—figure supplement 3A , B ) or treatment with manumycin A ( Figure 3—figure supplement 3C ) derepressed p14ARF , p15INK4B , and p16INK4A expression .",
"We next used a pathology tissue ChIP ( PAT-ChIP ) assay ( Fanelli et al . , 2011 ) to measure association of ZNF304 with p14ARF , p15INK4B , and p16INK4A promoters in KRAS-positive human CRC tumor samples and , as a control , adjacent matched normal colon .",
"Figure 3I shows that ZNF304 was substantially enriched at the three promoters in CRC tumor samples relative to adjacent normal colon .",
"Bisulfite sequencing analysis confirmed p14ARF promoter hypermethylation in the KRAS-positive CRC tumors but not in the matched normal colon ( Figure 3—figure supplement 4 ) .",
"Another factor isolated in our primary RNAi screen was USP28 ( Figure 2—figure supplement 1 ) , a nuclear-localized deubiquitinase ( Popov et al . , 2007 ) .",
"We therefore asked whether USP28 was responsible for stabilization of ZNF304 .",
"Similar to the results with KRAS , knockdown of USP28 in DLD-1 cells substantially reduced ZNF304 protein ( Figure 4A ) but not mRNA ( Figure 4B ) levels .",
"The co-immunoprecipitation experiment of Figure 4C shows that USP28 and ZNF304 were physically associated .",
"Moreover , co-transfection of DLD-1 cells with wild-type USP28 , but not a catalytically inactive USP28 ( C171A ) mutant ( Popov et al . , 2007 ) , reduced ubiquitination of ZNF304 ( Figure 4D ) .",
"Notably , shRNA-mediated knockdown or pharmacological inhibition of KRAS led to reduced levels of USP28 protein ( Figure 4E ) and mRNA ( Figure 4F ) . 10 . 7554/eLife . 02313 . 014Figure 4 . Activated KRAS upregulates ZNF304 through the deubiquitinase USP28 . ( A ) Immunoblot analysis showing ZNF304 levels in DLD-1 cells expressing a NS or USP28 shRNA .",
"( B ) qRT-PCR analysis monitoring ZNF304 expression in DLD-1 cells expressing a NS or USP28 shRNA .",
"( C ) Co-immunoprecipitation analysis .",
"DLD-1 cell extracts were immunoprecipitated with a ZNF304 , USP28 or control ( IgG ) antibody , and the immunoprecipitate was analyzed for ZNF304 or USP28 by immunoblotting .",
"( D ) HA-ubiquitination pull-down assay .",
"Extracts from 293T cells expressing HA-tagged ubiquitin , FLAG-tagged ZNF304 , and FLAG-tagged wild-type ( WT ) or mutant ( C171A ) USP28 were immunoprecipitated using an HA antibody , and the immunoprecipitate was analyzed using a ZNF304 antibody .",
"The arrowhead indicates the position of full-length ( FL ) ZNF304 .",
"( E ) Immunoblot analysis showing USP28 levels in DLD-1 cells treated with a KRAS shRNA or inhibitor .",
"( F ) qRT-PCR analysis monitoring USP28 expression in DLD-1 cells treated with a KRAS shRNA or inhibitor .",
"( G ) ChIP analysis monitoring binding of cJUN to the USP28 promoter or an irrelevant negative control ( NC ) DNA region .",
"( H and I ) qRT-PCR analysis monitoring USP28 ( H ) or INK4-ARF ( I ) expression in DLD-1 cells expressing a control scrambled ( SC ) or cJUN siRNA .",
"Experiments validating the role of cJUN in regulating USP28 expression are presented in Figure 4—figure supplements 1 , 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01410 . 7554/eLife . 02313 . 015Figure 4—figure supplement 1 . The USP28 promoter contains consensus cJUN-binding sites . Schematic diagram showing the positions and sequences of two putative cJUN-binding sites in the USP28 promoter .",
"The extended consensus AP-1 ( cJUN ) -binding sequence is also shown . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01510 . 7554/eLife . 02313 . 016Figure 4—figure supplement 2 . cJUN transcriptionally stimulates USP28 expression in KRAS-positive DLD-1 cells .",
"( A ) qRT-PCR analysis monitoring knockdown efficiency of cJUN using two unrelated siRNAs .",
"The results were normalized to that obtained with a control scrambled ( SC ) siRNA , which was set to 1 .",
"( B and C ) qRT-PCR analysis monitoring expression of USP28 ( B ) or INK4-ARF ( C ) in DLD-1 cells expressing a SC or cJUN siRNA that was unrelated in sequence to that used in Figure 4H , G .",
"The results were normalized to that obtained with the SC siRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 016 The results of Figure 4F showed that KRAS promoted the transcriptional upregulation of USP28 .",
"To gain insight into the basis of KRAS-mediated regulation of USP28 transcription , we performed bioinformatic analysis on the USP28 promoter and identified two putative binding sites for the transcription factor cJUN ( Figure 4—figure supplement 1 ) .",
"Notably , previous studies have shown that KRAS increases cJUN activity ( Mechta et al . , 1997 ) .",
"The ChIP results of Figure 4G show , consistent with the bioinformatic prediction , that in DLD-1 cells cJUN was bound to the USP28 promoter .",
"Moreover , knockdown of cJUN ( Figure 4—figure supplement 2A ) resulted in decreased expression of USP28 ( Figure 4H , Figure 4—figure supplement 2B ) and derepression of p14ARF , p15INK4B , and p16INK4A ( Figure 4I , Figure 4—figure supplement 2C ) .",
"Thus , cJUN is responsible , at least in part , for RAS-mediated transcriptional upregulation of USP28 .",
"Previous studies have shown that deubiquitinase–substrate interactions are often regulated by phosphorylation ( Kessler and Edelmann , 2011 ) .",
"Notably , USP28 contains two predicted phosphorylation sites for PRKD1 ( consensus sequence LxRxxS , [Nishikawa et al . , 1997]; Figure 5A ) , a serine/threonine protein kinase ( Johannes et al . , 1994 ) isolated in our RNAi screen ( Figure 2—figure supplement 1 ) that is dysregulated in a variety of cancers ( Sundram et al . , 2011 ) .",
"We therefore analyzed the role of PRKD1 in KRAS-mediated stabilization of ZNF304 .",
"shRNA-mediated knockdown of PRKD1 in DLD-1 cells resulted in decreased ZNF304 protein levels ( Figure 5B ) , whereas ZNF304 mRNA levels were not significantly affected ( Figure 5C ) .",
"Furthermore , treatment of DLD-1 cells with a PRKD1 chemical inhibitor , CRT0066101 ( Harikumar et al . , 2010 ) , also resulted in decreased ZNF304 protein levels ( Figure 5D ) , derepression of p14ARF , p15INK4B , and p16INK4A ( Figure 5E ) , and loss of ZNF304 , KAP1 , SETDB1 , and DNMT1 binding to the three promoters ( Figure 5F ) .",
"Similar to the results with USP28 , PRKD1 protein and mRNA levels decreased following shRNA-mediated knockdown or pharmacological inhibition of KRAS ( Figure 5G , H ) . 10 . 7554/eLife . 02313 . 017Figure 5 . Role of the protein kinase PRKD1 in KRAS-mediated stabilization of ZNF304 . ( A ) Multiple sequence alignment of the two putative PRKD1 phosphorylation sites in USP28 .",
"Blue indicates conserved leucine and arginine residues in the PRKD1 phosphorylation consensus sequence , and yellow indicates the putative phosphorylated serine residue .",
"The alignment was performed using NCBI’s HomoloGene; amino acid numbers refer to the human protein .",
"( B and C )",
"Immunoblot ( B ) and qRT-PCR ( C ) analysis monitoring ZNF304 in DLD-1 cells expressing a NS or PRKD1 shRNA .",
"( D ) Immunoblot analysis showing p14ARF levels in DLD-1 cells treated with DMSO or CRT0066101 ( CRT ) .",
"( E ) qRT-PCR analysis monitoring INK4-ARF expression in DLD-1 cells treated with DMSO or CRT .",
"( F ) ChIP monitoring ZNF304 and corepressor binding to INK4-ARF in CRT0066101-treated DLD-1 cells .",
"( G and H )",
"PRKD1 immunoblot ( G ) and qRT-PCR ( H ) in DLD-1 cells treated with a KRAS shRNA or inhibitor .",
"( I ) Co-immunoprecipitation analysis .",
"DLD-1 cell extracts were immunoprecipitated with a PRKD1 , USP28 or control ( IgG ) antibody , and the immunoprecipitate was analyzed for PRKD1 or USP28 by immunoblotting .",
"( J ) ( Left ) In vitro kinase assay .",
"Purified wild-type ( WT ) or kinase dead ( KD ) PRKD1 was incubated with USP28 peptides ( shown on the right ) and γ-ATP and analyzed for incorporation of the radiolabel by autoradiography .",
"( K ) HA-ubiquitination pull-down assay as described in Figure 4D except 293T cells expressed WT or mutant ( S899A ) USP28 .",
"( L ) ChIP analysis monitoring binding of CDX1 to the PRKD1 promoter or an irrelevant DNA region ( NC ) DNA .",
"( M and N ) qRT-PCR analysis monitoring PRKD1 ( M ) or INK4-ARF ( N ) expression in DLD-1 cells expressing a control scrambled ( SC ) or CDX1 siRNA .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 .",
"Control experiments related to Figure 5 are presented in Figure 5—figure supplements 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01710 . 7554/eLife . 02313 . 018Figure 5—figure supplement 1 . Confirmation of in vitro autophosphorylation activity of wild-type PRKD1 but not a kinase-dead PRKD1 mutant . Purified wild-type ( WT ) and kinase-dead ( KD ) PRKD1 proteins were incubated in a standard in vitro kinase reaction with radioactive γ-ATP .",
"The reaction mixture was run on a gel , and incorporation of the radiolabel was detected by autoradiography .",
"The silver-stained ( SS ) gel shows the abundance of each protein .",
"The results show that only wild-type PRKD1 was capable of autophosphorylation , indicative of PRKD1 kinase activity . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01810 . 7554/eLife . 02313 . 019Figure 5—figure supplement 2 . USP28-mediated deubiquitination of ZNF304 does not occur in the presence of the PRKD1 chemical inhibitor CRT0066101 . HA-ubiquitination pull-down assay .",
"Extracts from 293T cells expressing HA-tagged ubiquitin , FLAG-tagged ZNF304 , and FLAG-tagged wild-type ( WT ) USP28 treated in the presence or absence of 10 µM CRT0066101 were immunoprecipitated using an HA antibody , and the immunoprecipitate was analyzed by immunoblotting using a ZNF304 antibody .",
"The arrowhead indicates the position of full-length ( FL ) ZNF304 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 01910 . 7554/eLife . 02313 . 020Figure 5—figure supplement 3 . The PRKD1 promoter contains consensus CDX1-binding sites . Schematic diagram showing the positions and sequences of the putative CDX1-binding sites in the PRKD1 promoter .",
"The extended consensus CDX1-binding sequence , according to the TRANSFAC database , is also shown . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02010 . 7554/eLife . 02313 . 021Figure 5—figure supplement 4 . CDX1 transcriptionally stimulates PRKD1 expression in KRAS-positive DLD-1 cells .",
"( A ) qRT-PCR analysis monitoring knockdown efficiency of CDX1 using two unrelated siRNAs .",
"The results were normalized to that obtained with a control scrambled ( SC ) siRNA , which was set to 1 .",
"( B and C ) qRT-PCR analysis monitoring expression of USP28 ( B ) or INK4-ARF ( C ) in DLD-1 cells expressing a scrambled or CDX1 siRNA that was unrelated in sequence to that used in Figure 5M , N .",
"The results were normalized to that obtained with the SC siRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 021 We next asked whether USP28 is a substrate of PRKD1 .",
"Consistent with this possibility , we found that USP28 and PRKD1 were stably associated in a co-immunoprecipitation assay ( Figure 5I ) .",
"We then performed an in vitro kinase assay with purified PRKD1 whose activity was verified in an autophosphorylation assay ( Figure 5—figure supplement 1 ) .",
"Figure 5J shows that PRKD1 could phosphorylate a USP28 peptide containing the second , more conserved predicted phosphorylation site ( Figure 5A ) .",
"Notably , unlike wild-type USP28 , a USP28 derivative containing a mutation in the PRKD1 phosphorylation site , USP28 ( S899A ) , was unable to reduce ZNF304 ubiquitination ( Figure 5K ) .",
"Likewise , treatment with the PRKD1 inhibitor CRT0066101 prevented USP28 from reducing ubiquitination of ZNF304 ( Figure 5—figure supplement 2 ) .",
"The results of Figure 5H demonstrated that KRAS promoted the transcriptional upregulation of PRKD1 .",
"To gain mechanistic insight into this process , we performed bioinformatic analysis on the PRKD1 promoter and identified consensus binding sites for the intestine-specific transcription factor CDX1 ( Figure 5—figure supplement 3 ) .",
"Notably , previous studies have shown that KRAS positively regulates CDX1 activity through the mitogen-activated protein kinase pathway ( Lorentz et al . , 1999 ) .",
"The ChIP results of Figure 5L show , consistent with the bioinformatic prediction , that CDX1 was bound to the PRKD1 promoter in DLD-1 cells .",
"Moreover , knockdown of CDX1 ( Figure 5–figure supplement 4A ) resulted in decreased expression of PRKD1 ( Figure 5M , Figure 5—figure supplement 4B ) and derepression of p14ARF , p15INK4B , and p16INK4A ( Figure 5N , Figure 5—figure supplement 4C ) Thus , CDX1 is responsible , at least in part , for RAS-mediated transcriptional upregulation of PRKD1 .",
"As stated above , approximately 70% of CRCs containing activated KRAS are CIMP-positive as defined by aberrant hypermethylation of a representative panel of ∼50 CIMP marker genes ( Yagi et al . , 2010; Kaneda and Yagi , 2011 ) .",
"Notably , these CIMP marker genes include p14ARF and p16INK4A , as well as other TSGs , which prompted us to ask whether ZNF304 and its corepressors have a general role in the aberrant hypermethylation and transcriptional silencing characteristic of CIMP-positive CRCs containing activated KRAS .",
"To address this possibility , we knocked down KRAS or ZNF304 in DLD-1 cells and analyzed CIMP marker gene expression by qRT-PCR .",
"Remarkably , knockdown of either KRAS or ZNF304 derepressed expression of all 50 CIMP marker genes analyzed ( Figure 6A ) .",
"Interestingly , knockdown of either KRAS or ZNF304 also derepressed VIM and SEPT9 ( Figure 6—figure supplement 1 ) , whose DNA hypermethylation is used to diagnose CRC ( Gyparaki et al . , 2013 ) .",
"Bisulfite sequencing analysis of a representative subset of CIMP marker genes , which included p14ARF , p16INK4A and seven additional genes , revealed that shRNA-mediated knockdown of KRAS or ZNF304 also decreased promoter hypermethylation ( Figure 2E , Figure 6—figure supplement 2 ) .",
"ChIP analysis showed significant enrichment of ZNF304 , KAP1 , SETDB1 , and DNMT1 on the promoters of the nine CIMP marker genes ( Figures 2D and 6B ) , whose expression was also derepressed by the knockdown of the ZNF304 corepressors ( Figures 2C and 6C ) . 10 . 7554/eLife . 02313 . 022Figure 6 . The ZNF304 corepressor complex mediates CIMP in KRAS-positive CRCs .",
"( A ) qRT-PCR analysis monitoring expression of CIMP marker genes in DLD-1 cells expressing a ZNF304 or KRAS shRNA .",
"The results were normalized to that obtained with the NS control , which was set to 1 .",
"( B ) ChIP analysis monitoring binding of ZNF304 , KAP1 , SETDB1 , and DNMT1 to CIMP promoters or an irrelevant DNA region ( NC ) .",
"( C ) qRT-PCR analysis monitoring expression of CIMP marker genes in DLD-1 cells expressing a NS , KAP1 , SETDB1 , or DNMT1 shRNA .",
"( D ) PAT-ChIP analysis monitoring binding of ZNF304 to CIMP promoters in matched adjacent normal ( N ) and KRAS-positive CRC human tumor ( T ) samples .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 .",
"( E ) Model for ZNF304-corepressor-mediated transcriptional silencing of INK4-ARF and CIMP marker genes in CRCs .",
"Experiments validating the role of ZNF304 and corepressors in silencing of CIMP genes in KRAS-positive CRC cell lines , and experiments showing that the promoters of CIMP genes are hypermethylated in KRAS-positive CRC tumor samples , are presented in Figure 6—figure supplements 1–6 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02210 . 7554/eLife . 02313 . 023Figure 6—figure supplement 1 . Knockdown of KRAS or ZNF304 derepresses expression of VIM and SEPT9 in DLD-1 cells . qRT-PCR analysis monitoring expression of VIM and SEPT9 in DLD-1 cells stably expressing a ZNF304 or KRAS shRNA .",
"The results were normalized to the expression level obtained using a NS shRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02310 . 7554/eLife . 02313 . 024Figure 6—figure supplement 2 . Knockdown of KRAS or ZNF304 decreases promoter hypermethylation of representative CIMP genes in DLD-1 cells . Bisulfite sequencing analysis of the promoters of CIMP marker genes in DLD-1 cells stably expressing a NS , KRAS or ZNF304 shRNA .",
"( Top )",
"Schematic of the promoter; positions of CpGs are shown to scale by vertical lines .",
"( Bottom )",
"Each circle represents a methylated ( black ) or unmethylated ( white ) CpG dinucleotide .",
"Each row represents a single clone . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02410 . 7554/eLife . 02313 . 025Figure 6—figure supplement 3 . Confirmation of CIMP in other KRAS-positive human CRC cell lines . Bisulfite sequencing analysis of the promoters of representative CIMP marker genes in HCT116 ( top ) and HCT15 ( bottom ) cells .",
"( Top )",
"Schematic of the promoter; positions of CpGs are shown to scale by vertical lines .",
"( Bottom )",
"Each circle represents a methylated ( black ) or unmethylated ( white ) CpG dinucleotide .",
"Each row represents a single clone . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02510 . 7554/eLife . 02313 . 026Figure 6—figure supplement 4 . ZNF304 and its corepressors are associated with the promoters of representative CIMP genes in other KRAS-positive human CRC cell lines . ChIP analysis monitoring binding of ZNF304 , KAP1 , SETDB1 , and DNMT1 at the promoters of CIMP marker genes in HCT116 ( top ) and HCT15 ( bottom ) cells .",
"As a negative control , binding of the factors was also monitored at an irrelevant DNA region ( negative control [NC] DNA ) .",
"The results were normalized to that obtained with an IgG control antibody , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02610 . 7554/eLife . 02313 . 027Figure 6—figure supplement 5 . Knockdown of KRAS or ZNF304 derepresses expression of representative CIMP genes in other KRAS-positive human CRC cell lines . qRT-PCR analysis monitoring expression of CIMP marker genes in HCT116 ( top ) and HCT15 ( bottom ) cells stably expressing an NS , ZNF304 or KRAS shRNA .",
"The results were normalized to that obtained with the NS shRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02710 . 7554/eLife . 02313 . 028Figure 6—figure supplement 6 . Confirmation of CIMP in KRAS-positive human CRC tumor samples . Bisulfite sequencing analysis of the AOX1 , CACNA1G and IRF8 promoters in matched adjacent normal colon ( N ) and KRAS-positive CRC human tumor samples ( T ) samples .",
"( Top )",
"Schematic of the promoter; positions of CpGs are shown to scale by vertical lines .",
"( Bottom )",
"Each circle represents a methylated ( black ) or unmethylated ( white ) CpG dinucleotide .",
"Each row represents a single clone . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 028 We next asked whether ZNF304 and its corepressors silenced CIMP marker genes in other KRAS-positive human CRC cell lines and tumor samples .",
"In CIMP-positive HCT116 and HCT15 CRC cells ( Zheng et al . , 2000; Figure 6—figure supplement 3 ) , ZNF304 , KAP1 , SETDB1 , and DNMT1 were associated with the promoters of the nine CIMP marker genes ( Figure 3—figure supplement 2 , Figure 6—figure supplement 4 ) , whose expression was derepressed by shRNA-mediated knockdown of ZNF304 or KRAS ( Figures 2A and 3A , Figure 6—figure supplement 5 ) .",
"The PAT-ChIP results of Figures 3I and 6D show that ZNF304 was substantially enriched at the promoters of the nine CIMP marker genes in KRAS-positive human CRC tumors relative to matched normal colon .",
"Bisulfite sequencing analysis of representative CIMP marker genes confirmed promoter hypermethylation in all KRAS-positive CRC tumor samples ( Figure 6—figure supplement 6 ) .",
"These results and those described above reveal a specific pathway that mediates CIMP in KRAS-positive CRCs that is summarized in Figure 6E and discussed below .",
"As described above , the INK4-ARF locus is also transcriptionally silenced in undifferentiated hESCs and becomes poised for expression following differentiation ( Sherr , 2012 ) .",
"We therefore analyzed a possible role for ZNF304 and its corepressors in H9 cells , a well-characterized hESC line .",
"The immunoblot results of Figure 7A show that in undifferentiated H9 hESCs , ZNF304 was present at high levels , which markedly decreased following differentiation by retinoic acid treatment .",
"Moreover , in undifferentiated H9 hESCs ZNF304 , KAP1 , SETDB1 , and DNMT1 were substantially enriched at the p14ARF , p15INK4B , and p16INK4A promoters ( Figure 7B ) , which was largely lost following differentiation .",
"Finally , knockdown of ZNF304 in undifferentiated H9 hESCs ( Figure 7—figure supplement 1 ) resulted in decreased association of KAP1 , SETDB1 , and DNMT1 with the p14ARF , p15INK4B , and p16INK4A promoters ( Figure 7C ) and increased expression of p14ARF , p15INK4B , and p16INK4A ( Figure 7D ) .",
"Collectively , these results indicate that ZNF304 and its corepressors are also responsible for transcriptional silencing of INK4-ARF in undifferentiated hESCs . 10 . 7554/eLife . 02313 . 029Figure 7 . ZNF304 also directs transcriptional silencing of INK4-ARF in hESCs .",
"( A ) Immunoblot analysis showing ZNF304 levels in undifferentiated ( DMSO ) or retinoic acid ( RA ) -treated hESCs .",
"( B and C )",
"ChIP analysis monitoring binding of ZNF304 , KAP1 , SETDB1 , and DNMT1 to INK4-ARF in undifferentiated or RA-treated hESCs ( B ) or in hESCs expressing a NS or ZNF304 shRNA ( C ) .",
"( D ) qRT-PCR analysis monitoring INK4-ARF expression in hESCs expressing a NS , ZNF304 , KAP1 , SETDB1 , or DNMT1 shRNA .",
"( E ) Bisulfite sequencing analysis of the p14ARF and p16INK4A promoters in H9 hESCs and DLD-1 cells .",
"( F and G )",
"ChIP analysis monitoring enrichment of H3K27me3 , H3K9me3 , and H3K4me3 ( F ) and EZH2 and BMI1 ( G ) at INK4-ARF or an irrelevant DNA region ( NC ) in H9 hESCs and DLD-1 cells .",
"( H and I )",
"ChIP analysis monitoring binding of EZH2 and BMI1 ( H ) and H3K27me3 , H3K9me3 and H3K4me3 ( I ) at INK4-ARF in H9 hESCs and DLD-1 cells expressing a NS or ZNF304 shRNA .",
"( J ) qRT-PCR analysis monitoring INK4-ARF expression in H9 hESCs and DLD-1 cells expressing an NS , EZH2 , or BMI1 shRNA .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 .",
"Control experiments related to Figure 7 are shown in Figure 7—figure supplements 1 , 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 02910 . 7554/eLife . 02313 . 030Figure 7—figure supplement 1 . Knockdown efficiencies in H9 hESCs . qRT-PCR analysis monitoring knockdown efficiency of ZNF304 , KAP1 , SETDB1 , and DNMT1 in H9 hESCs .",
"The results were normalized to that obtained with the NS shRNA , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 03010 . 7554/eLife . 02313 . 031Figure 7—figure supplement 2 . Assessment of the role of EZH2 and BMI1 in INK4-ARF transcriptional silencing in H9 hESCs and DLD-1 cells .",
"( A and B ) qRT-PCR analysis monitoring knockdown efficiency of two unrelated EZH2 and BMI1 shRNAs in H9 hESCs and DLD-1 cells .",
"The results were normalized to that obtained with the NS shRNA , which was set to 1 .",
"( C ) qRT-PCR analysis monitoring INK4-ARF expression in H9 hESCs and DLD-1 cells expressing an NS shRNA , or an shRNA against EZH2 or BMI1 that is unrelated to that used in Figure 7J .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 031 As described above , in DLD-1 and other KRAS-positive CRC cell lines and tumors , the INK4-ARF locus is extensively hypermethylated .",
"However , the bisulfite sequencing results of Figure 7E show that unlike DLD-1 cells , in H9 hESCs the p14ARF and p16INK4A promoters were not hypermethylated .",
"We therefore sought to investigate how ZNF304 promoted INK4-ARF silencing in H9 hESCs in the absence of DNA hypermethylation .",
"As mentioned above , previous studies have described a role for Polycomb repressive complexes ( PRCs ) in silencing of INK4-ARF in several non-malignant cell types including fibroblasts , mouse ESCs , and adult stem cells ( Jacobs et al . , 1999; Molofsky et al . , 2005; Bracken et al . , 2007; Li et al . , 2009 ) .",
"We therefore investigated the possible role of PRCs in INK4-ARF silencing in H9 hESCs and , for comparison , in KRAS-positive DLD-1 CRC cells .",
"We first analyzed the INK4-ARF locus for the presence of the inhibitory histone mark , H3K27me3 , which is catalyzed by the PRC2 subunit EZH2 .",
"The ChIP results of Figure 7F show substantial enrichment of H3K27me3 at the p14ARF , p15INK4B , and p16INK4A promoters in H9 hESCs , whereas in DLD-1 cells the level of H3K27me3 was substantially lower .",
"By contrast , the level of histone H3 lysine 9 trimethylation ( H3K9me3 ) at the p14ARF , p15INK4B , and p16INK4A promoters was much higher in DLD-1 cells than in H9 hESCs .",
"As expected , there was no significant enrichment of histone H3 lysine 4 trimethylation ( H3K4me3 ) , a mark associated with transcription activity , in either DLD-1 cells or H9 hESCs .",
"The results described above indicated that although INK4-ARF is silenced in both KRAS-positive CRC cells and H9 hESCs , the inhibitory chromatin marks differ .",
"To determine whether the differential inhibitory marks resulted from selective recruitment of repressive cofactors , we performed ChIP experiments .",
"The results of Figure 7G show that in both H9 hESCs and DLD-1 cells , the PRC2 subunit EZH2 and the PRC1 subunit BMI1 were associated with the p14ARF , p15INK4B , and p16INK4A promoters .",
"Thus , although the inhibitory marks on INK4-ARF differ in H9 hESCs and DLD-1 cells , the same set of repressive cofactors is present .",
"To determine whether ZNF304 was responsible for recruitment of PRC1 and PRC2 , we performed ChIP experiments following depletion of ZNF304 .",
"Figure 7H shows that in both H9 hESCs and DLD-1 cells , knockdown of ZNF304 decreased binding of EZH2 and BMI1 to the p14ARF , p15INK4B , and p16INK4A promoters , which , as expected , was accompanied by a loss of H3K27me3 ( Figure 7I ) .",
"Also , as expected , in both H9 hESCs and DLD-1 cells , the knockdown of ZNF304 resulted in decreased H3K9me3 and increased H3K4me3 at the p14ARF , p15INK4B , and p16INK4A promoters ( Figure 7I ) .",
"The differential levels of H3K27me3 on INK4-ARF described above raised the possibility that PRCs might have a more important role in silencing of p14ARF , p15INK4B , and p16INK4A in H9 hESCs compared to DLD-1 cells .",
"Consistent with this idea , the knockdown of EZH2 or BMI1 ( Figure 7—figure supplement 2A , B ) increased expression of p14ARF , p15INK4B , and p16INK4A in H9 hESCs but not in DLD-1 cells ( Figure 7J , Figure 7—figure supplement 2C ) ."
],
[
"In this report , we have identified a specific pathway that mediates CIMP in KRAS-positive CRCs ( Figure 6E ) .",
"The pathway is initiated on DNA by binding of the transcriptional repressor , ZNF304 , which recruits a corepressor complex that includes SETDB1 , KAP1 and DNMT1 , leading to promoter hypermethylation and transcriptional silencing .",
"Activated KRAS regulates the pathway by maintaining high levels of ZNF304 , which drives DNA binding .",
"The basis by which activated KRAS increases ZNF304 levels is transcriptional upregulation of PRKD1 , a serine/threonine kinase , and USP28 , a deubiquitinase , two of the factors that were isolated in our primary RNAi screen .",
"We further showed that PRKD1 phosphorylates USP28 , which interacts with and stabilizes ZNF304 from proteolytic degradation .",
"ZNF304 is a member of large family of transcription factors .",
"About two-thirds of the approximately 1500 transcription factors encoded by mammalian genomes contain C2H2 zinc-fingers and more than half of these harbor an N-terminal KRAB transcriptional repression domain ( KRAB-ZFP proteins; reviewed in Lupo et al . , 2013 ) .",
"In a previous study , we have found that silencing of the Fas tumor suppressor in RAS-transformed NIH 3T3 cells requires ZFP354B ( Gazin et al . , 2007 ) , which like ZNF304 is a KRAB-ZFP protein .",
"Moreover , like ZNF304 , ZFP354B levels are increased by activated RAS .",
"These findings suggest that KRAB-ZFP transcription factors may have a widespread role in transcriptional silencing of TSGs in cancer cells .",
"Approximately 70% of CRCs containing activated KRAS are CIMP-positive .",
"However , whether activated KRAS is merely associated with or is directly responsible for CIMP remained to be determined .",
"In this study , we have shown that activated KRAS directs a pathway that silences INK4A-ARF and a large number of other genes characteristic of CIMP-positive CRCs .",
"Our results show how a single oncoprotein-directed pathway can silence multiple , unrelated genes .",
"Although we have shown that in CRC cells KRAS directs and is required to maintain transcriptional silencing of INK4A-ARF , in primary cells oncogenic signals ( such as activated KRAS ) induce transcription of INK4A-ARF , which leads to p53 and Rb pathway activation and ultimately growth arrest ( Sherr , 2012 ) .",
"Thus , transformation of a primary to a cancer cell involves a switch converting KRAS from an activator to a repressor of INK4-ARF .",
"Consistent with this idea , we found , as expected , that expression of activated KRAS ( G12V ) in non-transformed WI-38 fibroblasts transcriptionally activated p14ARF , p15INK4B , and p16INK4 ( Figure 8A ) .",
"However , in contrast to the results in KRAS-positive CRC cell lines , in WI-38 cells KRAS failed to increase ZNF304 protein levels ( Figure 8B ) or significantly stimulate USP28 and PRKD1 transcription ( Figure 8C ) , explaining at least in part why KRAS expression does not result in INK4-ARF silencing .",
"These results in WI-38 cells reinforce the pivotal role of ZNF304 and the KRAS-directed pathway we describe in mediating INK4-ARF silencing in KRAS-positive CRCs .",
"We speculate that linking INK4-ARF silencing directly to KRAS may ensure that the locus is not reactivated in CRC tumor cells . 10 . 7554/eLife . 02313 . 032Figure 8 . Expression of activated KRAS in non-transformed WI-38 fibroblasts increases expression of INK4-ARF and does not activate the ZNF304 pathway .",
"( A ) qRT-PCR analysis monitoring INK4-ARF expression in WI-38 cells expressing vector or KRAS ( G12V ) .",
"The results were normalized to the expression level obtained in vector-expressing cells , which was set to 1 .",
"( B ) Immunoblot analysis showing levels of ZNF304 , phosphorylated ERK ( p-ERK ) and total ERK ( t-ERK ) in WI-38 cells expressing vector or KRAS ( G12V ) .",
"( C ) qRT-PCR analysis monitoring expression of USP28 and PRKD1 in WI-38 cells expressing vector or KRAS ( G12V ) .",
"The results were normalized to the expression level obtained in vector-expressing cells , which was set to 1 .",
"Data are represented as mean ± SD .",
"*p≤0 . 05 , **p≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 02313 . 032 Collectively , our results indicate that the ZNF304 pathway mediates transcriptional silencing of p14ARF , p15INK4B , and p16INK4A and other TSGs thereby facilitating RAS-driven tumorigenicity .",
"Thus , in addition to its well-established role in promoting cellular proliferation and preventing apoptosis through downstream signaling pathways ( reviewed in Karnoub and Weinberg , 2008 ) , RAS induces ‘secondary’ oncogenic events by inactivating TSGs through transcriptional silencing .",
"This additional activity may explain , at least in part , why RAS is such a potent oncoprotein and why activating RAS mutations are found at high frequency in human tumors .",
"Finally , the continual requirement of the components of the RAS-directed ZNF304 pathway to maintain silencing has therapeutic implications .",
"The approval of DNA demethylating agents and histone deacetylase inhibitors for treatment of lymphoma patients clearly established the potential to reverse tumor-specific epigenetic alterations ( Mann et al . , 2007; Kelly et al . , 2010 ) .",
"However , DNA methyltransferase and histone deacetylase inhibitors broadly and non-selectively interfere with silencing .",
"More efficacious therapeutics may be obtained by selectively inhibiting the silencing pathway initiated by the relevant oncoprotein .",
"Thus , factors such as USP28 and PRKD1 may provide attractive anti-cancer targets .",
"Unexpectedly , we have found that silencing of INK4A-ARF in undifferentiated hESCs is also mediated by ZNF304-directed recruitment of repressive cofactors .",
"We note that the fold-increase of p14ARF , p15INK4B , and p16INK4A expression following knockdown of ZNF304 or repressive cofactors in H9 hESCs was less than that observed in KRAS-positive CRC cell lines , but is entirely consistent with results of other studies analyzing the role of INK4-ARF repressors in non-malignant cells ( e . g . , Bracken et al . , 2007 ) .",
"This lack of robust re-expression of INK4-ARF in H9 hESCs and other non-malignant cells is presumably due to the absence of oncogenic signals that would strongly stimulate transcription of INK4-ARF genes .",
"ZNF304 is present at high levels in hESCs despite the absence of activated RAS .",
"Interestingly , in ESCs KRAB-ZFPs , and their corepressor KAP1 , have been shown to play an important role in silencing of endogenous retroviruses ( Rowe et al . , 2013 ) .",
"Thus , there may be some general mechanism for activation of KRAB-ZFPs in ESCs .",
"Our finding that ZNF304 levels are elevated in both KRAS-positive CRC cells and hESCs is consistent with studies reporting a variety of similarities between cancer cells and stem cells ( reviewed in Kim and Orkin , 2011 ) .",
"In non-malignant differentiated cells INK4-ARF is reversibly silenced through association with PRCs .",
"Although ZNF304 mediates recruitment of PRCs to INK4-ARF in undifferentiated H9 hESCS , following differentiation ZNF304 levels dramatically decline and there is no longer significant binding of ZNF304 at the p14ARF , p15INK4B , and p16INK4A promoters .",
"Several other transcription factors such as TWIST1 , ZFP277 , HLX1 , HOXA9 and the long non-coding RNA ANRIL have been reported to recruit PRCs and repress INK4-ARF in various differentiated non-malignant cells that presumably lack high levels of ZNF304 ( Negishi et al . , 2010; Yang et al . , 2010; Yap et al . , 2010; Martin et al . , 2013; Ribeiro et al . , 2013 ) .",
"In ESCs , many developmentally important genes are reversibly silenced by PRCs and contain the repressive H3K27me3 mark .",
"In fact , in ESCs H3K27me3 is a more predominant silencing mechanism than DNA hypermethylation , which is believed to be because H3K27me3 is more readily reversible .",
"Several studies have identified subgroups of genes in which the H3K27me3 present in ESCs is replaced by abnormal DNA hypermethylation in cancer cells .",
"This phenomenon , which has been referred to as ‘epigenetic switching’ , results in the permanent silencing of key regulatory genes that may contribute to cell proliferation and tumorigenesis ( reviewed in Sharma et al . , 2010 ) .",
"This differential repression pattern is exactly what we found for INK4-ARF in hESCs and KRAS-positive CRC cells .",
"One model to explain the two repression patterns is differential recruitment of corepressors , such as PRCs or DNMTs , in non-malignant and cancer cells .",
"However , we found that in hESCs and KRAS-positive CRC cells the same set of corepressors are recruited to INK4-ARF .",
"Moreover , corepressor recruitment is dependent upon the same sequence-specific DNA-binding protein , ZNF304 .",
"Our results suggest a model involving differential enzymatic activity of a common , promoter-bound corepressor complex resulting in predominantly DNA hypermethylation , in KRAS-positive CRC cells , or predominantly H3K27me3 , in hESCs ."
],
[
"DLD-1 , HCT15 , and HCT116 cells were obtained from ATCC ( Manassas , VA ) and grown as recommended by the supplier .",
"H9 hESCs were grown in mTeSR1 media ( STEMCELL Technologies , Vancouver , Canada ) under feeder-free conditions on plates coated with Matrigel ( BD Biosciences , San Jose , CA ) .",
"DLD-1 cells were treated with 10 µM manumycin A ( Calbiochem , Darmstadt , Germany ) for 24 hr , 20 µM LY294002 ( Calbiochem ) for 24 hr , 10 µM PI-103 ( Cayman Chemical ) for 24 hr , 0–10 µM MG-132 ( Cayman Chemical , Ann Arbor , MI ) for 4 hr , or 10 µM CRT0066101 ( Cancer Research Technology , London , UK ) for 24 hr .",
"To induce differentiation , H9 hESCs were treated with 10 µM retinoic acid ( Sigma–Aldrich , St . Louis , MO ) for 3 ( Figure 7A ) or 4 ( Figure 7B ) days .",
"WI-38 cells were obtained from ATCC and cultured as recommended by the supplier .",
"To produce retroviruses , HEK293T cells were tranfected with pBabe K-Ras12V ( 12544; Addgeneplasmid; Khosravi-Far et al . , 1996 ) or empty vector ( pBABE-puro; 1764; Addgene plasmid ) .",
"WI-38 cells were seeded with the KRAS or empty vector retrovirus and 3 days later selected with puromycin .",
"The cells were harvested for total RNA for qRT-PCR analysis on day 4 , or for total protein for immunoblot analysis on day 7 .",
"To construct the p14ARF-BlastR reporter , 3 . 98 kb of the p14ARF promoter was PCR amplified from a BAC using primers engineered with BglII and SalI restriction sites , and cloned into a derivative of pDsRed2-N1 ( Clontech , Mountainview , CA ) in which the CMV promoter had been excised , the BlastR gene ( PCR amplified from pEF6/V5-HisB; Invitrogen , Grand Island , NY ) had been inserted in-frame with DsRed2 , and the TK gene ( PCR amplified from the HSV-1-TK gene; Addgene ) had been inserted in-frame with DsRed2-BlastR .",
"The plasmid was linearized and stably transfected into DLD-1 cells using an Amaxa nucleofactor .",
"Immediately after nucleofection , the cells were placed in complete growth medium for 72 hr .",
"Viable cells were allowed to grow into colonies and then selected with 500 µg/ml G418 ( Calbiochem ) .",
"Surviving colonies were individually isolated , expanded , and tested for blasticidin sensitivity; to minimize non-specific survival in the shRNA screen , clones with the lowest resistance to blasticiden , indicating complete reporter silencing , were chosen for further characterization .",
"Clones were treated with 10 µM 5-aza-2′-deoxycytodine ( Calbiochem ) every 24 hr for 72 hr .",
"After 24 hr treatment , 0 , 5 , or 10 µM blasticidin ( Sigma-Aldrich ) was added for 6 days , and cells were fixed and stained with 0 . 1% crystal violet to assess viability .",
"Treatment with 5-aza-2′-deoxycytodine and subsequent challenge with blasticidin was used to identify a clone with robust survival when treated with both drugs .",
"The human shRNAmir library ( release 1 . 20; Open Biosystems/Thermo Scientific , Pittsburgh , PA ) was obtained through the UMass Medical School RNAi Core facility ( Worcester , MA ) .",
"Retroviral pools were generated and used to transduce DLD-1/p14ARF-BlastR cells as previously described ( Gazin et al . , 2007 ) .",
"The cells were selected with puromycin ( 4 μg/ml ) for 3 days , and the puromycin-resistant population was challenged with blasticidin ( 10 µg/ml ) for 14 days .",
"The cells that bypassed the basticidin challenge formed colonies that were isolated and individually expanded , and shRNAs were identified by sequence analysis as previously described ( Gazin et al . , 2007 ) .",
"Individual knockdown cell lines were generated by stable transduction of 1 × 105 cells with a single shRNA ( Supplementary file 1 ) followed by puromycin selection .",
"Total RNA was isolated and reverse transcription was performed as described ( Gazin et al . , 2007 ) , followed by qRT-PCR using Power SYBR Green PCR Master Mix ( Applied Biosystems , Grand Island , NY ) .",
"GAPDH was used as an internal reference gene for normalization .",
"See Supplementary file 2 for primer sequences .",
"Cell extracts were prepared by lysis in Laemmli buffer in the presence of protease inhibitor cocktail ( Roche , Indianapolis , IN ) .",
"The ZNF304 antibody was generated ( by 21st Century Biochemicals , Marlboro , MA ) against a peptide corresponding to amino acids GFWCEAEHEAPSEQSV .",
"The following commercial antibodies were used: p14ARF ( Cell Signaling Technology , Danvers , MA ) , p15INK4B ( Abcam , Cambridge , MA ) , p16INK4A ( Cell Signaling Technology ) , USP28 ( Bethyl Laboratories , Montgomery , TX ) , PRKD1 ( Cell Signaling Technology ) , phospho-ERK1/2 and total ERK1/2 ( both from Cell Signaling Technology ) .",
"The α-tubulin ( TUBA ) antibody was generated in-house .",
"ChIP assays were performed as previously described ( Gazin et al . , 2007 ) using the following antibodies: ZNF304 ( described above ) , KAP1 ( Bethyl Laboratories ) , SETDB1 ( Millipore , Billerica , MA ) , DNMT1 , DNMT3A , and DNMT3B ( all from Imgenex , San Deigo , CA ) , cJUN ( Millipore ) , H3K27me3 ( Cell Signaling Technology ) , H3K9me3 ( Millipore ) , H3K4me3 ( Abcam ) , EZH2 ( Millipore ) and BMI1 ( Abcam ) .",
"The CDX1 antibody ( Chan et al . , 2009 ) was kindly provided by Walter Bodmer ( University of Oxford , UK ) .",
"ChIP products were analyzed by qRT-PCR ( see Supplementary file 1 for primers ) .",
"Samples were quantified as percentage of input , and then normalized to an irrelevant region in the genome ( ∼3 . 2 kb upstream from the transcription start site of GCLC ) .",
"Fold enrichment was calculated by setting the IgG control IP sample to a value of 1 .",
"Bisulfite modification was carried out using an EpiTect Bisulfite Kit ( QIAGEN , Germantown , MD ) followed by assay kits from EpigenDX ( Hopkinton , MA ) or nested PCR primers .",
"Multiple independent clones were sequenced from each PCR product within each cell line ( see Supplementary file 2 for primer sequences ) , of which six representative clones are displayed .",
"DLD-1 cells ( 2 × 106 ) expressing either a NS , ZNF304 or DNMT1 shRNA were suspended in 100 µl of serum-free RPMI and injected subcutaneously into the right flank of athymic BALB/c ( nu/nu ) mice ( Taconic ) ( n = 3 mice per shRNA ) .",
"Tumor dimensions were measured every 7 days for 4 weeks and tumor volume was calculated using the formula π/6 × ( length ) × ( width ) 2 .",
"All experiments were performed in accordance with the Institutional Animal Care and Use Committee ( IACUC ) guidelines .",
"This study was approved by the institutional review board at the University of Massachusetts Medical School ( UMMS ) .",
"Archived specimens ( 2010–2012 ) with sufficient tissue for analysis were obtained from the Department of Pathology at UMMS , and the CRC diagnosis was made by a UMMS pathologist .",
"KRAS mutational analysis was performed by the UMass Memorial Laboratory of Diagnostic Molecular Oncology ( Worcester , MA ) .",
"Formalin-fixed paraffin embedded tissue sections of matched adjacent normal colon and tumor samples isolated from individuals with invasive or metastatic KRAS-positive CRC were de-paraffinized in Histolemon-Erba RS solution ( Carlo Erba Reagents , France ) four times for 10 min at room temperature .",
"The tissue was then resuspended in 100% ethanol , incubated for 10 min at room temperature , spun down and resuspended in 95% ethanol .",
"The washing/resuspension steps were repeated , gradually increasing the percentage of water ( to achieve 70% , 50% , 20% , 0% ethanol ) to rehydrate the tissue .",
"The resulting material was then processed as previously described ( Fanelli et al . , 2011 ) .",
"DLD-1 cell lysate was immunopreciptated with a ZNF304 , USP28 , PRKD1 or control ( IgG ) antibody , and the immunoprecipitate was analyzed for ZNF304 , USP28 or PRKD1 by immunoblotting .",
"Input lanes represent 10% of immunoprecipitated lanes .",
"ZNF304 was PCR amplified from a BAC using primers engineered with HindIII and BamH1 sites and cloned into p3XFLAG-myc-CMV-26 ( Sigma-Aldrich ) to generate p3XFLAG-ZNF304 .",
"p3XFLAG-USP28 and p3XFLAG-USP28 ( C171A ) ( Popov et al . , 2007 ) were kindly provided by Stephen Elledge ( Harvard Medical School , Boston , MA ) ; p3XFLAG-USP28 ( S899A ) was generated by patch PCR using p3XFLAG-USP28 as a template .",
"293T cells ( 2 × 106 ) were plated on 10-cm dishes and transfected with 1 µg p3XFLAG-ZNF304 , 1 µg p3XFLAG-USP28 ( wild-type or mutant ) , 1 µg pcDNA3 . 1-HA-Ubiquitin ( Addgene ) , and 0 . 5 µg pmaxGFP ( Lonza Biologics Inc . , Hopkinton , MA ) using Effectene reagent ( QIAGEN ) .",
"To ensure equivalent transfection efficiency , eGFP expression was monitored 48 hr later .",
"Cells were harvested in NETN-150 buffer ( 20 mM Tris–HCl , pH 8 . 0 , 150 mM NaCl , 1 mM EDTA and 0 . 05% NP-40 ) plus 1X protease inhibitor cocktail ( Roche ) .",
"Pull-downs were performed using an HA antibody ( Cell Signaling Technology ) and anti-rabbit Trublot beads ( eBioscience , San Diego , CA ) .",
"Beads were incubated with lysate for 18 hr , washed three times using NETN-150 buffer , and eluted in 2X sample buffer .",
"Input samples were probed with a FLAG-M2 ( Sigma-Aldrich ) or TUBA antibody , and immunoprecipitated samples were probed with a ZNF304 antibody .",
"Plasmids expressing His-tagged wild-type and kinase-dead PRKD1 proteins were constructed by digesting plamids HA . PKD ( Storz and Toker , 2003 ) and HA . PKD . K/W ( Storz et al . , 2003 ) ( 10808 and 10809; Addgene plasmids ) , respectively , with BamHI and XhoI and ligating into pRSET A ( Life Technologies , Grand Island , NY ) .",
"To ensure activity of the purified wild-type protein , a 20-µl reaction was set up as follows: 1 µl 32P-γ-ATP ( 10 mCi ) , 1 µl 10 µM ATP , 0 . 2 mM microcystin , 4 µl 5X kinase buffer ( 23 mM MOPS , 11 . 5 mM β-glycerphosphate , 23 mM MgCl2 , 4 . 6 mM EGTA , 1 . 8 mM EDTA , 0 . 25 mM DTT [pH 7 . 0] ) , and 60 nM purifiedHis-PRKD1 diluted in 1X kinase buffer .",
"Reactions were incubated for 30 min at 30°C and stopped using 2X Laemmli Sample Buffer .",
"Autophosphorylation of the wild-type protein was confirmed by immunoblotting with a PRKD1-Ser916 antibody ( Cell Signaling Technology ) .",
"To monitor phosphorylation of USP28 , the above reaction was carried out using 10 µM substrate ( peptides corresponding to amino acids 543–557 [TCLQRWRSEIEQDIQ] or 892–906 [YSLFRKVSVYLLTGL] in USP28 ) diluted in 1X kinase buffer .",
"Incorporation of the radiolabel into the peptide was monitored by autoradiography .",
"The USP28 and PRKD1 promoters were analyzed using the TRANSFAC database ( www . gene-regulation . com/pub/databases . html ) to identify putative transcription factor binding sites .",
"All quantitative data were collected from experiments performed in at least triplicate , and expressed as mean ± standard deviation .",
"Differences between groups were assayed using two-tailed student t test using Microsoft Excel .",
"Significant differences were considered when p<0 . 05; *p≤0 . 05 , and **p≤0 . 01 ."
]
] | [
"Approximately 70% of KRAS-positive colorectal cancers ( CRCs ) have a CpG island methylator phenotype ( CIMP ) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes .",
"The factors involved in , and the mechanistic basis of , CIMP is not understood .",
"Among the CIMP genes are the tumor suppressors p14ARF , p15INK4B , and p16INK4A , encoded by the INK4-ARF locus .",
"In this study , we perform an RNA interference screen and identify ZNF304 , a zinc-finger DNA-binding protein , as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS .",
"In KRAS-positive human CRC cell lines and tumors , ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes .",
"Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1 , resulting in DNA hypermethylation and transcriptional silencing .",
"KRAS promotes silencing through upregulation of ZNF304 , which drives DNA binding .",
"Finally , we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells ."
] | [
"Colorectal cancer , which affects the large intestine , is a leading cause of cancer deaths worldwide , ranking fourth after cancers of the lung , stomach , and liver .",
"Like these other cancers , this disease is caused by mutations to genes that allow cells to multiply in an out of control manner .",
"Mutations that change the gene encoding a protein called KRAS are found in many different types of cancer .",
"Moreover , about 70% of colorectal cancers with a KRAS mutation also have an excess of small chemical marks on other genes , some of which are known to suppress the growth of tumors .",
"These marks ‘switch off’ these genes , and although the identities of the enzymes that typically leave these marks on DNA are known , the link between these enzymes and the KRAS protein is unknown .",
"Now Serra , Fang et al . have identified a protein , called ZNF304 , that is required by KRAS to switch off a large number of genes , including multiple tumor suppressors .",
"In the absence of ZNF304 , these tumor suppressor genes remained switched on in cancer cells with the KRAS mutation , so the growth of the tumor was slowed down .",
"ZNF304 is a protein that binds to stretches of DNA , including regions of DNA at the start of several tumor suppressor genes , and it recruits the enzymes that add the chemical marks that switch off these genes .",
"Serra , Fang et al . found that the levels of ZNF304 protein were elevated in colorectal cancer cells with the mutated KRAS , and showed that this was due to the combined activities of two other proteins that prevented ZNF304 from being broken down in the cell .",
"Mutant KRAS caused an increase in the levels of these two proteins , which in turn caused the elevated ZNF304 levels and the excessive marking of the DNA in the tumor suppressor genes .",
"Furthermore , some of these same tumor suppressor genes are switched off in the earliest cells in a human embryo—which have the potential to become any of 200 or so cell types in the human body .",
"In these embryonic stem cells , Serra , Fang et al . showed that ZNF304 , but not KRAS , was also involved in keeping these genes switched off until the stem cells started changing into specific types of cells .",
"Since they are a crucial part of the pathway linking a cancer-causing mutation to increased tumor growth , the proteins identified by Serra , Fang et al . could represent promising targets for the development of new anti-cancer drugs ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease",
"immunology and inflammation"
] | An extrafollicular pathway for the generation of effector CD8+ T cells driven by the proinflammatory cytokine, IL-12 | elife-09017-v2 | [
[
"The activation of naïve CD8+ T cells occurs in the T cell areas of secondary lymphoid organs ( Mempel et al . , 2004 ) .",
"Once activated , CD8+ T cells are thought to undergo rapid clonal expansion and differentiate into IFN-γ-producing effector cytotoxic lymphoid cells ( CTLs ) through an ‘autopilot’ mechanism ( Mercado et al . , 2000; Bevan and Fink , 2001; Kaech and Ahmed , 2001; van Stipdonk et al . , 2001 ) .",
"Consistent with this paradigm , CD8α+ dendritic cells ( DCs ) , which are resident in the splenic white pulp , are critical for both antigen cross-presentation ( den Haan et al . , 2000 ) and production of a key pro-inflammatory cytokine , IL-12 ( Reis e Sousa et al . , 1997; Mashayekhi et al . , 2011 ) .",
"IL-12 is an important signal 3 cytokine that drives clonal proliferation of activated CD8+ T cells in vitro ( Curtsinger et al . , 1999 ) as well as their effector cytokine production in vivo ( Schmidt and Mescher , 1999 , 2002; Valenzuela et al . , 2002; Liu et al . , 2006; Wilson et al . , 2008 ) .",
"However , following their activation and migration into the inner PALS ( Reis e Sousa et al . , 1997 ) , CD8α+ DCs become hyporesponsive and cease IL-12 production ( Reis e Sousa et al . , 1999 ) , raising the question of whether this initial early burst of IL-12 is sufficient to drive end-stage effector CD8+ T cell differentiation to completion .",
"Recently , there has been mounting evidence that additional events occurring outside the T cell area are critical for primary TH1 and CTL effector differentiation .",
"CXCR3 deficiency results in an inefficient generation of cytokine-producing effector cells and instead favors a response skewed towards memory cells ( Kohlmeier et al . , 2011; Kurachi et al . , 2011; Groom et al . , 2012 ) .",
"CXCR3-mediated outmigration of T cells into the splenic marginal zone ( MZ ) ( Kurachi et al . , 2011 ) or the peripheral medullary areas of lymph nodes ( Groom et al . , 2012 ) enables interactions with IL-12 producing myeloid cells .",
"The role of CXCR3 in lymphocyte peripheralization also extends to the memory response and is essential for secondary effector T cell generation from central memory T cells ( Sung et al . , 2012; Kastenmuller et al . , 2013 ) .",
"These recent findings argue that an initial exposure to IL-12 and other signal 3 cytokines during the early stages of T cell activation may not be sufficient for effector T cell differentiation .",
"Instead , a subsequent exposure to IL-12 at extrafollicular sites appears to drive effector CD8+ T cell differentiation to completion .",
"It is also possible that this requirement may stem from the initial lack of a robust CD8α+ DC- derived IL-12 during viral and bacterial immune responses ( Dalod et al . , 2003; Edelson et al . , 2011 ) .",
"During CD8+ T cell priming , the inflammatory milieu governs effector CD8+ T cell generation .",
"In particular , the cytokine IL-12 potently promotes CD8+ T cell effector differentiation and function , through T-bet-dependent induction of KLRG1 expression and IFN-γ production ( Joshi et al . , 2007; Wilson et al . , 2008 , 2010 ) .",
"In addition to these effects , IL-12 appears to also control the migratory potential of primary and effector T cells , by negatively regulating CXCR3 expression ( Slutter et al . , 2013 ) .",
"Given the possibility of IL-12 being produced both within the T cell area and at extrafollicular sites , it remains unclear how IL-12 temporarily and spatially controls these key facets of the primary effector CTL response .",
"Furthermore , although it is assumed that these IL-12 effects are CD8+ T cell-autonomous , the possibility exists that regulation may be indirectly mediated through the actions of this cytokine on other IL-12 responsive immune cells .",
"In order to address these questions , we used a Toxoplasma gondii vaccination model to conduct a detailed analysis of the role of IL-12 in the generation , function and migratory potential of parasite-specific effector CD8+ T cells .",
"We had previously identified this tgd057-reactive H-2Kb-restricted CD8+ T cell response in mice vaccinated with an attenuated , uracil auxotrophic strain of T . gondii ( CPS ) , known to elicit CD8+ T cell-dependent protective immunity ( Fox and Bzik , 2002 ) and have demonstrated a strict in vivo requirement for IL-12 to generate KLRG1+ effector CTLs ( Wilson et al . , 2008 , 2010 ) .",
"Our results reveal that the sequence of differentiative events that culminate in the production of primary end-stage effector CD8+ T cells occurs over a protracted period and that IL-12 exerts regulatory functions at both early and late phases of effector cell generation .",
"The effects of IL-12 in upregulating KLRG1 expression and priming for IFN-γ production require CD8+ T cell intrinsic cytokine signaling .",
"In contrast , we found that the belated downregulation of CXCR3 on effector CD8+ T cells is indirectly regulated by IL-12 and is instead controlled by a pathway in which IFN-γ and IFN-γ-inducible chemokines mediate this downmodulation .",
"Using an in vivo intravascular staining method ( Olson et al . , 2013; Anderson et al . , 2014 ) , we were able to reveal that these later stages of effector CD8+ T cell differentiation occur extrafollicularly , involving DCs as cellular sources of both non-CD8α+ DC-derived IL-12 and CXCR3-ligands .",
"Surprisingly , we also found extensive proliferation of both KLRG1− and KLRG1+ CD8+ T cells in the MZ and red pulp ( RP ) .",
"Taken together with earlier studies ( Lauvau et al . , 2001; Cockburn et al . , 2010 ) , our findings argue against the notion that effector CTL generation occurs through an ‘autopilot’ sequence and , instead , involves a multi-leveled progression of effector T cell precursors through distinct splenic microenvironments , where their differentiation is controlled by a complex interplay with locally positioned activated immune cells ."
],
[
"To determine the early effects of IL-12 on CD8+ T cell proliferation and differentiation during T . gondii infection , we used a tetramer-based enrichment method ( Klenerman et al . , 2002; Moon et al . , 2007 ) to enumerate H-2Kb-restricted CD8+ T cells specific for the T . gondii antigen tgd057 ( Wilson et al . , 2010 ) in wild-type ( WT ) and IL-12p35 deficient hosts following CPS vaccination .",
"The tetramer-based enrichment method allows for a ∼2-log increase in detection of tgd057-specific CD8+ T cells ( Figure1—figure supplement 1 ) .",
"To accurately enumerate the exact numbers of tgd057-specific CD8+ T cells when their frequencies are very low we mixed a known number of naïve Thy1 . 1+-marked tgd057-specific CD8+ T cells from a somatic cell nuclear transfer ( SCNT ) mouse ( Kirak et al . , 2010a ) with spleen cells from individual naïve mice ( Figure 1—figure supplement 2 ) .",
"We are able to detect 2093 ± 273 and 1200 ± 138 endogenous tgd057-specific CD8+ T cells in spleens of naïve WT and IL-12p35 deficient mice , respectively ( Figure 1—figure supplement 2 ) .",
"Figure 1A shows that there is little change in absolute cell numbers of tgd057-specific CD8+ T cells between day 0 and day 4 post-vaccination in the spleen .",
"However , we can detect a 10-fold clonal expansion occurring between days 4 and 5-post infection in spleens of both WT and IL-12 deficient mice .",
"The absolute numbers of tgd057-specific CD8+ T cells continue to increase at similar rates through day 7 in WT and IL-12 deficient mice .",
"These results indicate that the lack of IL-12 signals does not affect the timing or the magnitude of the proliferative response of tgd057-specific CD8+ T cells during T . gondii vaccination . 10 . 7554/eLife . 09017 . 003Figure 1 . CD8+ T cell proliferative response is IL-12 independent while effector cell differentiation is IL-12 dependent .",
"( A ) Absolute numbers of tgd057-specific CD8+ T cells in spleen after CPS vaccination in wild-type ( WT ) and IL-12p35−/− mice .",
"( B ) tgd057-specific CD8+ T cells were analyzed for CD44 , CD62L and KLRG1 cell surface expression .",
"Pie charts represent frequency averages of F1 ( CD62L+ KLRG1− ) , F2 ( CD62L− , KLRG1− ) , F3 ( CD62L− , KLRG1+ ) , and F4 ( CD62L+ , KLRG1+ ) subsets of tgd057-specific CD8+ T cells from WT and IL-12p35−/− spleens D0–D7 post CPS vaccination .",
"Data shown include only CD44hi cells on D4–D7 and include all tgd057-specific CD8+ T cells on D0–D3 .",
"( C ) Absolute numbers of tgd057-specific CD8+ T cells in peritoneal exudate cells ( PECs ) of WT and IL-12p35−/− mice after CPS vaccination .",
"Cells were characterized based on tgd057-specific CD8+ T cells .",
"( D ) Average of frequencies of F1 ( CD62L+ KLRG1− ) , F2 ( CD62L− , KLRG1− ) , F3 ( CD62L− , KLRG1+ ) , and F4 ( CD62L+ , KLRG1+ ) subsets of tgd057-specific CD8+ T cells in PECs D0–D7 .",
"Data represent 5 independent experiments with 3–5 mice per group per experiment .",
"Data were analyzed using two-way ANOVA and ( A ) Holms-Sidak or ( C ) Bonferroni post-hoc tests; *p < 0 . 05 .",
"See Figure 1—figure supplements 1 , 2 for the methodology employed to enumerate absolute numbers of tgd057-specific CD8+ T cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 00310 . 7554/eLife . 09017 . 004Figure 1—figure supplement 1 . Tetramer-based enrichment increases detection of epitope-specific naïve CD8+ T cells in spleen . Samples from pre- and post-tetramer-based enrichment of D4 and D5 post CPS vaccinated spleens , showing a 2-log increase in detection of tgd057:H-2Kb+ specific CD8+ T cells .",
"Data represent 5 independent experiments with 3–5 mice per group per experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 00410 . 7554/eLife . 09017 . 005Figure 1—figure supplement 2 . Accurate determination of endogenous naïve tgd057:H-2Kb-specific CD8+ T cells . To quantify absolute numbers of endogenous tgd057-specific CD8+ T cells , naïve B6 spleens were pulsed with 50 naïve monoclonal Thy1 . 1+ SCNT CD8+ T cells .",
"Representative flow cytometry plots of tetramer binding cells ( doubly stained with APC and PE-labeled tetramers ) partitioned according to Thy1 . 1 staining are shown .",
"Absolute numbers of naïve endogenous tgd057-specific CD8+ T cells in a naïve spleen was calculated based on the ratio of endogenous CD8+ T cells to the pulsed Thy1 . 1+ cells .",
"Total numbers of tgd057-specific CD8+ T cells in a naïve spleen were found to be 2093 ± 273 in WT hosts and 1200 ± 138 cells in IL-12p35−/− hosts .",
"Data represent 5 independent experiments with 3–5 mice per group per experiment .",
"Scatter plots are mean ± SEM , data were analyzed using paired t test and Holms-Sidak post-hoc test; **p ≤ 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 005 Despite the apparent lack of a role for IL-12 in the proliferative recruitment of tgd057- specific CD8+ T cells , IL-12 could play a crucial role in the early diversification of CD8+ T cells into end-stage effector CD8+ T cells vs memory precursor type cells .",
"Indeed , previous studies in our laboratory and others have shown that IL-12 is crucial for the differentiation of KLRG1+ effector CTLs through T-bet dependent induction of effector genes ( Joshi et al . , 2007; Wilson et al . , 2008 , 2010 ) .",
"To determine how IL-12 signals affect the early differentiation of tgd057-specific CD8+ T cells , we monitored the expression of CD62L and KLRG1 following T . gondii vaccination .",
"We have previously used these cell surface markers to define four specific CD8+ T cell stages: F1 ( TCM: CD62L+ , KLRG1− ) , F2 ( TEM: CD62L− , KLRG1− ) , F3 ( TEFF: CD62L− , KLRG1+ ) and F4 ( CD62L+ , KLRG1+ ) ( Wilson et al . , 2008 , 2010 ) .",
"While F1 and F2 are determined to be central and effector memory CD8+ T cells , respectively; F3 are the late stage highly IFN-γ-producing effector CD8+ T cells , little is still known about the phenotype and function of the F4 stage CD8+ T cells .",
"We do not observe consistent changes in CD8+ T cell stage distribution until day 3 in either WT or IL-12 deficient hosts ( Figure 1B ) .",
"However , by day 3 , ∼7–8% of the tgd057-specific CD8+ T cells start to commit to putative TEM and TEFF cells by downregulating CD62L and increasing KLRG1 expression in the WT hosts ( Figure 1B ) .",
"In IL-12p35 deficient hosts , L-selectin downregulation is also observed starting on day 3 , however KLRG1 upregulation is absent ( Figure 1B ) .",
"On day 4 , there is a dramatic increase in the frequency of tgd057-specific CD8+ T cells that have downregulated CD62L , a third of which also acquire KLRG1 in WT mice .",
"However , in the absence of IL-12 , there is an attenuation of KLRG1 expression resulting in an even larger frequency of F2 tgd057-specific CD8+ T cells ( Figure 1B ) .",
"The frequency of KLRG1+ tgd057-specific CD8+ T cells continues to increase through day 7 post-vaccination in WT hosts; yet , in IL-12 deficient mice , KLRG1 upregulation remains attenuated and the frequencies of F3 stage tgd057-specific CD8+ T cells do not reach levels seen WT hosts ( Figure 1B ) .",
"These results indicate that IL-12 signaling has an important role on the differentiation of KLRG1+ effector tgd057-specific CD8+ T cells , and that this occurs earlier than the proliferative burst seen after day 4 .",
"In contrast , downregulation of CD62L and subsequent clonal expansion are IL-12 independent .",
"In spite of the fact that IL-12 signals did not play a crucial role in proliferation of tgd057-specific CD8+ T cells in the spleen , we observed that case to be different at the site of infection .",
"We enumerated tgd057-specific CD8+ T cells in the peritoneal exudate cells ( PECs ) using the SVLAFRRL:H-2Kb tetramer ( Figure 1C ) .",
"Surprisingly , we found that the absolute numbers of tgd057-specific CD8+ T cells in the PECs in an IL-12 deficient environment by day 6 were attenuated by one log in absolute numbers as the height of the adaptive immune response was reached ( Figure 1C ) .",
"The observation that tgd057-specific CD8+ T cells traffic to the peritoneum is attenuated when IL-12 signals are lacking prompted us to investigate the role of IL-12 for effector tgd057-specific CD8+ T cells migration .",
"The T-box protein , T-bet , is a master regulator of the differentiation and functional activity of effector CD8+ T cells .",
"It has been shown to associate with the promoters of genes for KLRG1 ( Joshi et al . , 2007 ) , IFN-γ ( Joshi et al . , 2007 ) , as well as the chemokine receptor , CXCR3 ( Beima et al . , 2006; Harms Pritchard et al . , 2015 ) .",
"We hypothesized that since KLRG1 expression and the production of IFN-γ by F3 late stage effector CD8+ T cells is IL-12 dependent ( Wilson et al . , 2008 , 2010 ) , CXCR3 expression may also be IL-12 dependent , possibly explaining the attenuation of effector cells to the site of infection in our model .",
"Analysis of CXCR3 expression as CD8+ T cells differentiate from F1 ( TCM ) to F2 ( TEM ) cells in the spleen show that CXCR3 expression is highly expressed when CD8+ T cells are CD62L+ or CD62L− ( Figure 2A , B ) .",
"Interestingly , our data demonstrate that the early expression of CXCR3 is IL-12 independent , but T-bet dependent ( Figure 2A , B ) and as these cells differentiate into end-stage effector IFN-γ producing KLRG1+ CD8+ T cells , they downregulate CXCR3 ( Figure 2A , B ) , indicating that IL-12 has late downregulatory effects on CXCR3 expression . 10 . 7554/eLife . 09017 . 006Figure 2 . During effector CD8+ T cell differentiation , expression of CXCR3 is downregulated in an IL-12 dependent manner . CD62L and KLRG1 surface expression ( A ) and CXCR3 surface expression ( B ) on total CD8+ T cells were assessed by flow cytometry 7 days post-vaccination .",
"Data represent 3–4 independent experiments with 4–5 mice per group per experiment .",
"Mean ± SEM , data were analyzed using unpaired t test , and Holms-Sidak post-hoc test , *p ≤ 0 . 05 , **p ≤ 0 . 01 , ***p ≤ 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 006 We next wished to determine where in the spleen KLRG1 upregulation and CXCR3 downregulation occurs as tgd057-specific CD8+ T cells differentiate into late-stage effectors .",
"In order to study the white pulp and RP distribution of the CD8+ T cells within the spleen , we intravenously injected mice with a fluorescently conjugated anti-CD8α antibody on days 4 and 7 post CPS vaccination ( Figure 3A ) .",
"Tissue was isolated after a brief delay to quickly label only cells exposed to the blood and RP within the spleen , but excluded cells in white pulp ( Olson et al . , 2013; Anderson et al . , 2014 ) .",
"Interestingly , our results suggest that on day 4 , tgd057-specific CD8+ T cells upregulate KLRG1 expression after trafficking to the RP ( Figure 3B ) .",
"By day 7 post-vaccination , the tgd057-specific CD8+ T cells are found to be equally localized to the white pulp and the RP prior to increasing their KLRG1 expression , where , once KLRG1 expression is upregulated , the majority ( ∼80% ) of the cells is located in the RP .",
"Thus , the early upregulation of KLRG1 expression on tgd057-specific CD8+ T cells occurs mainly in the RP , where they may be encountering a secondary signal of the pro-inflammatory cytokine , IL-12 , for later differentiation into end-stage effector CD8+ T cells .",
"Because the preferential localization of CD62L− KLRG1− and CD62L− KLRG1+ tgd057-specific CD8+ T cells is in the RP on day 4 , we next determined whether CXCR3 is concomitantly downregulated as KLRG1 expression is increased .",
"As Figure 3C indicates , at day 4 CXCR3 expression in both KLRG1− and KLRG1+ CD8+ T cells is not substantially decreased , but by day 7 , the KLRG1+ CD8+ T cells which are predominantly RP-localized have completely downregulated CXCR3 .",
"Taken together , these results demonstrate that prior to the upregulation of KLRG1 , effector precursor CD8+ T cells expressing high levels of CXCR3 can migrate to the RP , where they upregulate KLRG1 and then downregulate CXCR3 as they differentiate into late-stage effector CD8+ T cells . 10 . 7554/eLife . 09017 . 007Figure 3 . Downregulation of CXCR3 expression on CD8+ T cells occurs following KLRG1 induction in the splenic red pulp ( RP ) .",
"( A ) Representative FACS profile identifying splenic RP and white pulp by differential staining with i . v injected APC conjugated anti-CD8α antibody .",
"( B ) Compiled data of RP distribution of F1 , F2 and F3 tgd057-specific CD8+ T cells in spleen on D4 and D7 post CPS vaccination .",
"( C ) CXCR3 expression on F2-KLRG1− and F3-KLRG1+ tgd057-specific CD8+ T cells on D4 and D7 post CPS vaccination .",
"Data are representative of 3 independent experiments with 6–8 mice per experiment .",
"Mean ± SEM , data were analyzed using unpaired t test , and Holms-Sidak post-hoc test , ***p ≤ 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 007 Our data thus far suggest that IL-12 exerts early effects on the differentiation of effector tgd057-specific CD8+ T cells during T . gondii infection by upregulating KLRG1 even prior to clonal expansion ( Figure 1 ) , but also plays a later role in the downregulation of CXCR3 expression ( Figure 2 ) .",
"IL-12 may be produced only early during vaccine priming and programs the entire effector differentiation pathway over time .",
"Alternatively , IL-12 may be produced at both early and late time points , potentially by distinct APCs .",
"To address the latter scenario , we neutralized IL-12 late ( D3 ) and compared its effects on CD8+ T cell differentiation to early ( D0 ) and continuous neutralization ( D0 and D3 ) following CPS vaccination .",
"Consistent with our earlier results , the blockade of IL-12 at early , late or both time points did not affect absolute tgd057-specific CTL numbers ( Figure 4A ) .",
"When IL-12 is neutralized only on day 3 , KLRG1 expression and IFN-γ-production were still attenuated , albeit less markedly compared to the effects of early and continuous blockade of the cytokine ( Figure 4B , C ) .",
"Importantly , we find that late neutralization of IL-12 interrupts the downregulation of CXCR3 expression on late-stage effector tgd057-specific CD8+ T cells ( Figure 4D ) .",
"These results confirm that IL-12 is required for early programming of effector differentiation , and indicate that IL-12 production occurs days after initial CPS vaccine exposure and continues to induce further differentiation of effector-fated CD8+ T cells . 10 . 7554/eLife . 09017 . 008Figure 4 . IL-12 exerts both early and late effects on CD8+ T cell differentiation , function and chemokine receptor expression . Anti-IL-12p40 mAb was administered i . p . on D0 , D3 or both days post CPS vaccination .",
"Spleens were harvested on D5 post CPS vaccination .",
"( A ) Absolute numbers of tgd057-specific CD8+ T cells , ( B ) average frequency of F1–F4 stages defined by cell surface expression of CD62L and KLGR1 , ( C ) IFN-γ production and ( D ) CXCR3 downregulation on tgd057-specific CD8+ T cells .",
"Data are from 3 independent experiments with 3–4 mice per group per experiment .",
"Mean ± SEM , data were analyzed using multiple unpaired t test , and Holm-Sidak post-hoc test , *p ≤ 0 . 05 , **p ≤ 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 008 The observation that IL-12-mediated downregulation of CXCR3 can be temporally dissociated from its effects on KLRG1 upregulation ( Figure 2B , C ) , which was previously shown to be a CD8+ T cell-autonomous effect of the cytokine ( Slutter et al . , 2013 ) , prompted us to ask if the IL-12 effects are CD8+ T cell-intrinsic or extrinsic .",
"In addition , CXCR3 downregulation on KLRG1+ CD8+ T cells occurs in the RP ( Figure 3B , C ) , where IL-12 producing cell types other than CD8α+ DCs may be involved .",
"Specifically , the late-acting IL-12 may be secreted by inflammatory-monocyte derived DCs , and has been shown to require NK ( Goldszmid et al . , 2012 ) or TH1-CXCR3+ CD4+ T cell -derived IFN-γ ( Cohen et al . , 2013 ) for DC activation .",
"This scenario raises the possibility that other cells besides the developing effector CD8+ T cells may be the targets of IL-12 signaling .",
"In order to determine if IL-12 mediates CXCR3 downregulation directly , we adoptively transferred a 1:1 mixture of naïve WT and IL-12rβ2 deficient monoclonal tgd057-specific CD8+ T cells ( derived via SCNT cloning ) into a CD45 . 1+ WT B6 host ( Figure 5A ) .",
"7 days after CPS-vaccination , we observe that the lack of IL-12 signaling does not alter the frequency of IL-12rβ2 deficient donor CD8+ T cells ( Figure 5B ) , but CXCR3 downregulation is inhibited ( Figure 5C ) .",
"These results confirm earlier conclusions that IL-12 is not essential for T cell expansion in vivo and are consistent with the notion that IL-12 directly signals the downregulation of CXCR3 expression .",
"However , when we further compared CXCR3 expression between KLRG1− vs KLRG1+ stages , we found that the F3 stage KLRG1+ CD8+ T cells had downregulated CXCR3 regardless of their ability to signal IL-12 ( Figure 5D ) .",
"We conclude that CXCR3 is downregulated in a non-CD8+ T cell autonomous manner , which is not consistent with previous results ( Slutter et al . , 2013 ) .",
"The apparent lack of CXCR3 downregulation is due to the predominant deficiency in KLRG1 expression on the IL-12Rβ2 deficient CD8+ T cells ( Figure 5E ) .",
"Unlike CXCR3 , KLRG1 expression and IFN-γ production , that are known to be directly regulated by IL-12 signaling , behave in a cell autonomous manner ( Figure 5E , F ) . 10 . 7554/eLife . 09017 . 009Figure 5 . IL-12 mediates CXCR3 downregulation in a CD8+ T cell-extrinsic manner .",
"( A ) A schematic representation of adoptive co-transfer of naive 500 WT somatic cell nuclear transfer ( SCNT , CD45 . 2+ , Thy1 . 1+ ) and naïve 500 il12rβ2−/− SCNT ( CD45 . 2+ , Thy1 . 2+ ) CD8+ T cells into naïve CD45 . 1+ WT recipients 1 hr prior to CPS vaccination .",
"( B ) Representative FACS profile shows frequency of antigen specific polyclonal endogenous and monoclonal donor CD8+ T cells D7 post CPS vaccination .",
"( C ) CXCR3 expression on total tgd057-specific donor CD8+ T cells on D7 post vaccination ( left ) and expressed as MFI ( right ) .",
"( D ) tgd057-specific donor CD8+ T cells were analyzed for CXCR3 expression by first gating for CD62L and KLRG1 cell surface expression , then studying expression of CXCR3 in F2 and F3 stages .",
"( E ) Frequency of F1–F4 stages of WT CD8+ T cells and IL-12rβ2−/− SCNT CD8+ T cells D7 post-CPS vaccination .",
"( F ) IFN-γ production by WT SCNT and IL-12rβ2−/− SCNT CD8+ T cells was analyzed after CPS restimulation .",
"Data are from 3 independent experiments consisting of 4–8 mice per group per experiment .",
"Mean ± SEM .",
"Data were analyzed using unpaired t test , and ( D ) Bonferroni post-hoc test .",
"( E ) Mann–Whitney post-doc test; *p < 0 . 01 , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 009 To explore the mechanism of CD8+ T cell-extrinsic downregulation of CXCR3 expression , we considered the role of IFN-γ because it promotes the expression of CXCR3 chemokine ligands and because IL-12 may be acting indirectly through IFN-γ production by non-CD8+ T cells .",
"To first test if blocking IFN-γ alone can prevent the downregulation of CXCR3 on CD8+ T cells in vivo , we neutralized IFN-γ late ( D3 ) during CPS vaccination .",
"We found that in vivo blockade of IFN-γ as late as D3 disrupts the downregulation of CXCR3 expression in KLRG1-expressing tgd057-specific CD8+ T cells ( Figure 6A ) without attenuating KLRG1 upregulation or IFN-γ production ( Figure 6B , C ) . 10 . 7554/eLife . 09017 . 010Figure 6 . Neutralization of IFN-γ is sufficient to prevent CXCR3 downregulation on effector CD8+ T cell precursors . Anti-IFN-γ mAb was administered i . p . on D3 post-CPS vaccination .",
"Mice were sacrificed on D5 post-CPS vaccination and tgd057-specific CD8+ T cells were analyzed for ( A ) CXCR3 expression , ( B ) CD62L and KLRG1 surface expression on tgd057-specific CD8+ T cells and ( C ) IFN-γ production post in vitro CPS restimulation in tgd057-specific CD8+ T cells .",
"Data are from 2 experiments consisting of 3–5 mice per group per experiment .",
"Mean ± SEM , paired t test and Holm-Sidak post-hoc test , ***p < 0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 010 The fact that blockade of IFN-γ can also prevent the downregulation of CXCR3 on KLRG1+ CD8+ T cells to the same extent as neutralization of IL-12 does , prompted us to ask if IFN-γ can suppress CXCR3 on CD8+ T cells similarly to IL-12 .",
"We exploited the fact that IL-12 deficient F3 stage KLRG1+ CD8+ T cells fail to decrease CXCR3 and used these cells as an in vitro model system to directly interrogate what factors can cause CXCR3 downregulation .",
"Upon exposure and binding to its chemokine ligands , surface expressed CXCR3 is rapidly downregulated , endocytosed and degraded , but is quickly replenished by newly synthesized protein ( Meiser et al . , 2008 ) .",
"We therefore used CXCL10 exposure to ‘strip’ pre-existing CXCR3 from D7 CPS vaccinated IL-12p35 deficient splenocytes and determined whether IFN-γ or IL-12 can maintain the expression of CXCR3 on CD8+ T cells at a low level .",
"As expected , the addition of IP-10 rapidly decreases the expression of CXCR3 on CD8+ T cells lacking IL-12 signals ( Figure 7A , left ) , but importantly , the addition of IFN-γ and not the addition of CXCL10 alone , maintains the low levels of CXCR3 on these CD8+ T cells , to a similar extent as the addition of IL-12 ( Figure 7A , right ) .",
"This result suggests that IFN-γ may be acting directly on the developing CD8+ T cell to suppress CXCR3 expression downstream of IL-12 .",
"Consistent with this notion , addition of IFN-γ alone to highly purified CD8α+ TCRβ+ T cells from D7 CPS vaccinated IL-12p35 deficient spleens still maintains the low levels of CXCR3 expression ( Figure 7B ) without the need for IL-12-producing accessory cells , indicating that IFN-γ can act directly on the CD8+ T cell . 10 . 7554/eLife . 09017 . 011Figure 7 . An alternative pathway involving IFN-γ and IFN-γ-inducible chemokines mediates suppression of CXCR3 on late-stage effector CD8+ T cell precursors . CXCL10 , IL-12 , or IFN-γ was added in vitro to ( A ) total splenocytes or ( B ) purified CD8+ T cells from D7 IL-12p35−/− mice post CPS vaccination .",
"Cells were first ‘stripped’ of CXCR3 surface expression by addition of CXCL10 for 30 min ( left panel ) , washed , and then incubated overnight ( ON ) with chemokine or cytokines .",
"KLRG1+ CD8+ T cells were analyzed for CXCR3 expression .",
"( C ) Representative FACS profiles of ex vivo ICS of IL-12 and chemokines from 4 hr or D3 after CPS vaccination .",
"Splenocytes were analyzed for IL-12p40 , CXCL9 ( MIG ) and CXCL10 ( IP-10 ) production in CD11c+ CD8α+ dendritic cells ( DCs ) , and CD11c+ CD11b+ myeloid DCs ( mDCs ) .",
"( D ) Representative FACS profiles of IFN-γ production from NK , CD4+ and CD8+ T cells in spleens of D3 CPS vaccinated WT mice .",
"( E ) Three cell model showing that CD8+ T cells are not the direct targets of IL-12 .",
"Rather IL-12 acts through bystander T cell production of IFN-γ , which targets both the developing CD8+ T cells and mDCs to secrete IFN-γ-inducible chemokines .",
"CXCR3 chemokine ligands together with IFN-γ directly act on the developing CD8+ T cells to downregulate CXCR3 expression .",
"( A and B )",
"Data are from 4 experiments of 3–5 pooled IL-12p35−/− CPS vaccinated mice each , Mean ± SEM .",
"( C and D )",
"Data are from 2 experiments with 3–4 WT mice each . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 01110 . 7554/eLife . 09017 . 012Figure 7—figure supplement 1 . mDCs producing IL-12 and CXCL9 are CCR2+ and their depletion attenuates CD8+ T cell differentiation .",
"( A ) mDCs from D3 CPS vaccinated mice producing both IL-12 and CXCL9 are over 95% CCR2-positive .",
"CPS vaccinated CCR2-DTR+ mice and CCR2-DTR− control littermates were injected i . p with 250 ng of diphtheria toxin on D2 . 5 and sacrificed on D5 .",
"Tgd057-specific CD8+ T cells from D5 spleens were analyzed for ( B ) fractional distribution based on CD62L and KLRG1 expression and for ( C ) CXCR3 expression in KLRG1− ( F2 subpopulation ) and KLRG1+ ( F3 subpopulation ) .",
"Data represent 5 mice per group . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 012 Overall , our data suggests there is late production of IL-12 , which indirectly ( putatively through an IFN-γ/CXCL9/10 mediated pathway ) acts on the precursor effector CD8+ T cells to downregulate CXCR3 in the RP .",
"To directly examine if there is late production of IL-12 , we analyzed splenocytes from D3 CPS vaccinated mice after brefeldin A injection .",
"CD11c+ CD11b+ DCs ( myeloid DCs [mDCs] ) , but not CD8α+ DCs produced IL-12 ( Figure 7C , D3 ) .",
"This result confirms that there is late production of IL-12 by a subset of mDCs distinct from the CD8α+ DCs known to be the primary producers of IL-12 during early T . gondii infection ( Figure 7C , 4 hr ) .",
"These mDCs may also be located in the RP where the precursor effector CD8+ T cells are trafficking to and subsequently downregulating CXCR3 ( Figure 3 ) .",
"We also found that CD4+ and CD8+ T cells ( Figure 7D ) , and to a much lesser extent NK cells , are producing IFN-γ in the spleen on day 3 , suggesting that these cell types are putatively responding to the IL-12 derived from these myeloid APCs .",
"Finally , we find that the same myeloid APC subsets producing IL-12 are also the same cells producing CXCL9 and CXCL10 ( Figure 7C ) presumably in response to the IFN-γ derived from TH1 CD4+ and CD8+ T cells .",
"To determine whether the IL-12-producing mDCs are derived from inflammatory monocytes ( Nakano et al . , 2009 ) , we vaccinated CCR2-GFP reporter mice ( Serbina et al . , 2009; Espinosa et al . , 2014 ) .",
"Indeed , nearly all of the IL-12/chemokine -positive DCs on D3 are CCR2+ ( Figure 7—figure supplement 1A ) .",
"To evaluate the role of this late IL-12 producing APC population , we transiently depleted CCR2+ monocytes/derivatives on D2 . 5 by administering diptheria toxin to CCR2-DTR mice ( Hohl et al . , 2009 ) .",
"Transient ablation resulted in markedly diminished KLRG1+ ( F3 and F4 ) subpopulations and attenuation in CXCR3 downregulation in the KLRG1+ CD8+ T cells .",
"Although our data shows that CD8+ T cells are major IFN-γ producers at day 3 , this response is probably not parasite antigen specific because , as indicated by the data above ( Figure 1A ) , clonal bursting does not occur until after day 4 .",
"Taken together , our data suggests a three-cell model for how IL-12 indirectly leads to CXCR3 downregulation ( Figure 7E ) .",
"In the RP , bystander TH1 CD4+ and CD8+ T cells are the cellular targets of the IL-12 produced by inflammatory monocyte-derived mDCs , which in response produce IFN-γ .",
"IFN-γ then can directly target both mDCs to produce chemokines and the developing CD8+ T cells .",
"The downregulation of CXCR3 on CD8+ T cells is driven by both chemokine-induced receptor downmodulation and by IFN-γ-mediated suppression of further CXCR3 expression .",
"Taken together , our data suggest that most of the differentiative steps effector CD8+ T cells undergo occur extrafollicularly , raising the question of where their effector precursor proliferation takes place .",
"A predominant view is that activation and proliferation of CD8+ T cells occurs at the outer zone of the T cell area bordering B cell follicles and subsequently exits via bridging channels to the splenic RP ( Khanna et al . , 2007 ) .",
"Therefore , we sought to determine whether CD8+ T cell proliferation occurs mainly in the white pulp and completes their differentiation in the RP as post-mitotic cells .",
"We combined EdU labeling with intravascular staining during day 5 CPS-vaccination to localize where CD8+ T cell proliferation occurs in the context of the sequence of their differentiation .",
"We found that a significantly greater proportion of proliferating CD8+ T cells ( labeled within a narrow 2 hr period ) were localized to the RP ( Figure 8A , left ) and comprised predominantly of CD62L− CD8+ T cells ( F2 and F3 ) ( Figure 8A , right ) .",
"In fact , on a per cell basis , the proliferative rate increases as cells progress from F1 to F3 stages ( Figure 8B , left ) and exhibit the same skewed extrafollicular localization of cycling CD62L− CD8+ T cells ( Figure 8B , right ) .",
"A similar pattern of increased EdU labeling of CD62L− fractions ( F2 and F3 ) and a skewing towards RP localization of proliferative cells were evident in tgd057-specific CD8+ T cells ( Figure 8C , D ) .",
"These results indicate that , unlike the predominant view , CD8+ T cell proliferation is not restricted to the T cell area .",
"To more precisely localize the extrafollicular sites of CD8+ T cell proliferation , we performed immunohistochemistry of D5 . 5 spleens immediately after IV injection of FITC-labeled anti-CD8α .",
"As shown in Figure 8E , EdU-positive cells double-labeled with IV injected anti-CD8α and ex vivo stained anti-CD8α antibodies are readily observed in the MZ ( marked with yellow arrows in Figure 8E ) and in bridging channels ( Figure 8—figure supplement 1 ) .",
"Extrafollicular proliferative CD8+ T cells are also observed in the RP , many of which are also KLRG1+ ( marked with pink arrows in Figure 8F ) .",
"Interestingly , these data also indicate that clonal proliferation does not cease when CD8+ T cells acquire the effector cell marker , KLRG1 .",
"This raises the question of whether CXCR3 downregulation occurs concomitantly or subsequent to CD8+ T cell proliferation in the KLRG1+ ( F3 ) compartment .",
"As Figure 8G indicates , CXCR3 downregulation is observed in non-proliferating KLRG1+ CD8+ T cells . 10 . 7554/eLife . 09017 . 013Figure 8 . Generation of terminally differentiated CD8+ T cells occurs after cessation of proliferation in the splenic RP . D5 . 5 post CPS vaccinated WT mice were first injected i . p . with EdU 2 hr prior to sacrifice , then injected i . v . with 4 μg of anti-CD8-FITC antibody and sacrificed within 2–3 min .",
"The differential localization and fractional distribution of F1–F4 stages in EdU− and EdU+ ( A ) for total CD8+ T cells and ( C ) tgd057-specific CD8+ T cells are shown .",
"The rate of EdU-positivity as a function of differentiation stage ( B , D left panels ) or differentiation stage and i . v . anti-CD8 accessibility ( B , D right panels ) are shown for total CD8+ T cells ( B ) and tgd057-specific CD8+ T cells ( D ) .",
"Immunofluorescence images showing D5 . 5 splenic localization of EdU+ and double-positive for IV α-CD8 and ex vivo α-CD8 antibodies ( E ) , and KLRG1 ( F ) staining .",
"Splenic compartments are represented as follows: dashed line , MZ border; RP–red pulp; BC–bridging channel; MZ–marginal zone .",
"Sections were stained for IV α-CD8 ( green , CD8 ) , ex vivo α-CD8 ( red , EX CD8 ) , EdU ( blue ) , α-KLRG1 ( white ) .",
"Key to symbols: Yellow arrows: EdU+ IV CD8+ EV CD8+ , Pink Arrows: EdU+ KLRG1+ IV CD8+ EV CD8+ .",
"Expression of CXCR3 in proliferating and non-proliferating CD8+ T cells ( G ) .",
"Data are from 2 experiments of 3–5 CPS vaccinated mice each .",
"Immunofluorescence represents more than six sections per mouse taken from three mice .",
"Mean ± SEM .",
"Student's t-test or ANOVA analysis was done Holms-Sidak post hoc test , *p < 0 . 01 , **p < 0 . 001 and ***p < 0 . 0001 .",
"Refer to Figure 8—figure supplement 1 for IHC image of EdU+ CD8+ T cells in splenic bridging channels . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 01310 . 7554/eLife . 09017 . 014Figure 8—figure supplement 1 . CD8+ T cells in bridging channels are proliferating . D4 . 5 post CPS vaccinated WT mice were injected i . p . with EdU 2 hr prior to sacrifice and then injected i . v . with 4 μg anti-CD8-FITC antibody and sacrificed within 2–3 min . 6 μm splenic sections were stained for α-CD8α antibody and developed for EdU .",
"Immunofluorescence showing localization of double stained CD8α+ T cells that are proliferating ( EdU+ , yellow arrows ) or non-proliferating ( Edu− , blue arrows ) within the bridging channel and MZ .",
"Enlarge inset depicts proliferating and non-proliferating double stained CD8+ T cells in the bridging channel .",
"Splenic compartments are represented as follows: dashed lines–MZ border; orange lines–bridging channel; RP–red pulp; BC–bridging channel; MZ–marginal zone .",
"Sections were stained for IV CD8 ( green , CD8 ) , CD8 ex vivo ( red , EX CD8 ) , EdU ( blue ) .",
"Key to symbols: Yellow arrows: EdU+ CD8+ T cells , Blue arrows: EdU− CD8+ T cells .",
"Data are representative of more than 4 sections per mouse taken from three mice . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 014"
],
[
"In this report , we identify the sequence of events ( schematically depicted in Figure 9 ) that naïve CD8+ T cells undergo as they differentiate into effector CD8+ T cells in response to infection with the intracellular protozoan pathogen , T . gondii .",
"We further delineate the steps where the pro-inflammatory cytokine , IL-12 , is required for effector CD8+ T cell differentiation .",
"Upon activation , CD44 , a surface receptor necessary for T cell adhesion to extracellular matrix proteoglycans , is rapidly upregulated and then L-selectin ( CD62L ) is downregulated in an IL-12-independent manner ( Figure 1 ) .",
"CXCR3 is expressed early in the CD44Hi , CD62L+ ( F1 ) CD8+ T cell stage , and remains highly expressed through the CD44Hi , CD62L− ( F2 ) CD8+ T cell stage ( Figure 2 ) .",
"This early expression of CXCR3 is T-bet dependent but IL-12 independent ( Figure 2 ) .",
"While still in the white pulp and even prior to CD8+ T cell expansion ( Figures 1 , 3 ) , a few CD44Hi , CD62L− CD8+ T cells begin to acquire KLRG1 surface expression in an IL-12 dependent manner .",
"As CD8+ T cell numbers increase and outmigrate to the RP , KLRG1 expression escalates ( Figure 3 ) .",
"As shown in Figure 8 we are able to reveal , for the first time , that CD8+ T cell proliferation does not only occur exclusively in the white pulp ( Figure 8A–D ) .",
"Unexpectedly , we found that most of the proliferating CD8+ T cells are CD62L− ( F2 and F3 stage CD8+ T cells ) and many of these cycling effector precursors are observed to be outmigrating into the MZ ( Figure 8 ) via bridging channels ( Figure 8—figure supplement",
"1 ) ( Khanna et al . , 2007 ) .",
"Finally , these extrafollicular KLRG1+ precursor effector CD8+ T cells downregulate CXCR3 expression in an IL-12 dependent fashion .",
"CXCR3 downregulation by IL-12 is indirectly mediated by IFN-γ and IFN-γ-dependent chemokines , which are produced by other T lymphocytes and mDCs , respectively ( Figure 7 ) , in the MZ where the extrafollicular CD8+ T cells have been shown to cluster and interact with inflammatory myeloid cells producing IL-12 in a CXCR3 dependent manner ( Kurachi et al . , 2011 ) .",
"Our report outlines a detailed order of events during the differentiation of naïve CD8+ T cells to KLRG1+ CXCR3− effector CD8+ T cells and , together with several recent reports ( Khanna et al . , 2007; Kohlmeier et al . , 2011; Kurachi et al . , 2011; Groom et al . , 2012; Sung et al . , 2012 ) , highlights the importance of the MZ and RP as extrafollicular sites for their proliferation and differentiation . 10 . 7554/eLife . 09017 . 015Figure 9 . Schematic depicting the sequence of events leading to the generation of late-stage KLRG1+ effector CD8+ T cells . 1: While in the white pulp , CD8α+ DCs activate CD8+ T cells causing the upregulation of CXCR3 expression .",
"2: CXCR3Hi CD62LLo CD8+ T cells outmigrate towards RP and begin to proliferate .",
"3: Upon outmigration through bridging channels , CXCR3Hi CD62LLo CD8+ T cells continue to proliferate in clusters found in the MZ .",
"4: Exposure to IL-12 produced by mDCs in the MZ/RP areas induces KLRG1 upregulation .",
"5: Subsequent exposure to IFN-γ and IFN-γ-inducible chemokines downregulate CXCR3 expression on RP-localized KLRG1+ effector CD8+ T cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09017 . 015 Our in-depth analysis of IL-12 regulated checkpoints during the early effector CD8+ T cell programming in response to T . gondii vaccination has provided several new insights .",
"While IL-12 has been thought to be the major signal 3 cytokine needed for in vitro proliferation ( Curtsinger et al . , 1999; Valenzuela et al . , 2002 ) and augmentation of the immune synapse ( Markiewicz et al . , 2009 ) , we found that IL-12 does not provide an obligatory signal for T . gondii-specific CD8+ T cell clonal expansion in vivo , but rather is critical for the early KLRG1 expression on precursor effector CD8+ T cells .",
"Although KLRG1 , IFN-γ and CXCR3 are under transcriptional control of T-bet , the expression pattern of CXCR3 is opposite to the expression patterns of KLRG1 and IFN-γ , such that CXCR3 expression is increased early during CD62L+ F1 and CD62L− F2 stages of CD8+ T cell differentiation , while KLRG1 is expressed during the later stages of effector differentiation ( Figure 2 ) .",
"While CXCR3 is expressed early and its upregulation is IL-12 independent , we show that that once precursor effector CD8+ T cells upregulate KLRG1 , CXCR3 expression is downregulated in an IL-12 dependent manner ( Figures 2 , 4 ) .",
"Even though IL-12 is required for both the upregulation of KLRG1 and the downregulation of CXCR3 , these two effects can be temporally and spatially dissociated ( Figures 3 , 4 ) .",
"Furthermore , while the IL-12 effects on KLRG1 and IFN-γ are acting on the CD8+ T cells themselves , we found , surprisingly , that downregulation of CXCR3 is regulated by IL-12 in a non-CD8+ T cell autonomous fashion ( Figure 5 ) .",
"This latter finding seems to be at odds with a previous report that IL-12 directly regulates the suppression of CXCR3 expression of effector CD8+ T cells ( Slutter et al . , 2013 ) .",
"In this previous study , Slutter et al . ( 2013 ) also used a chimeric approach with IL-12rβ1 deficient and WT OT-1 cells to demonstrate that IL-12 receptor signaling has negative regulatory effects on CXCR3 expression during influenza priming and that this control is CD8+ T cell-intrinsic .",
"Our results indicate a similar conclusion when we analyzed CD8+ T cells without regard for KLRG1 expression ( Figure 5 ) .",
"However , when we analyze CXCR3 expression on KLRG1− vs KLRG1+ CD8+ T cells expression , we arrive at a different conclusion ( Figure 5 ) .",
"In the absence of IL-12 receptor signaling , we show that KLRG1+ CD8+ T cells maintain low CXCR3 expression ( Figure 5 ) .",
"The appearance of CD8+ T cell autonomous control can be explained by population skewing at the expense of KLRG1+ CD8+ T cells , which is the only stage where CXCR3 is decreased .",
"Thus , even in the influenza system , IL-12 probably indirectly downregulates CXCR3 .",
"The data from both our chimeric transfer and late in vivo IFN-γ neutralization experiments indicate that IL-12 does not directly regulate CXCR3 downmodulation during precursor effector CD8+ T cell development ( Figures 6 , 7 ) , but instead acts through an indirect pathway by which IFN-γ suppresses CXCR3 expression .",
"In support of this model , we find that there is production of IFN-γ by bystander TH1 T cells and that IFN-γ maintains CXCR3 suppression in vitro .",
"Thus , we propose a three-cell model ( Figure 7 ) for CXCR3 downregulation , in which the developing effector CD8+ T cell is the direct target of IFN-γ derived from bystander TH1 T cells and IFN-γ-induced chemokines produced by mDCs .",
"In this model , IL-12 acts by inducing IFN-γ production from bystander TH1 T cells rather than the developing effector CD8+ T cells themselves .",
"The mechanism of CXCR3 suppression by IFN-γ is currently not clear , but it will be interesting to find out whether it uses the same ID2-mediated pathway described for IL-12 ( Yang et al . , 2011; Knell et al . , 2013 ) .",
"It is also unclear what signals drive the production of IL-12 by mDCs at later time points after the initial IL-12 production by CD8α+ DCs has waned .",
"It is possible that residual non-replicating T . gondii parasites persist ( Dupont et al . , 2014 ) and , in addition , IFN-γ itself acting together with accessory signals from bystander lymphocytes provides inductive signals for mDC production of IL-12 ( Ariotti et al . , 2014; Slutter and Harty , 2014 ) .",
"Regardless of these uncertainties , our identification of IFN-γ as the critical mediator for CXCR3 downregulation provides a common mechanism for how multiple proinflammatory cytokines ( Yang et al . , 2011 ) can redundantly downmodulate CXCR3 on developing effector CD8+ T cells .",
"Our report highlights that the expression pattern of CXCR3 is highly complex as CD8+ T cells progress through distinct stages of differentiation .",
"Although CXCR3 expression has been classically associated with IL-12 driven type-1 effector adaptive immune responses , we show that CXCR3 is expressed very early in the CD62L+ KLRG1− F1 stage and thus allowing CD62L− KLRG1− F2 stage effector precursor CD8+ T cells to outmigrate to the MZ and the RP , where they are further exposed to proinflammatory signals and differentiate into effector CD8+ T cells .",
"The same CXCR3 mediated outmigration has been reported for primary CD4+ , CD8+ T cells and central memory T cells ( Kurachi et al . , 2011; Groom et al . , 2012; Sung et al . , 2012; Kastenmuller et al . , 2013 ) , arguing for the existence of a common extrafollicular pathway for type-1 effector cell generation .",
"Following proinflammatory cytokine and chemokine exposure KLRG1+ CXCR3Hi precursor effector CD8+ T cells gain the ability to produce high levels of IFN-γ while downregulating CXCR3 expression .",
"The observation that outmigrating differentiated effector CD8+ T cells found in the splenic RP are CXCR3Lo while published studies have clearly shown that tissue associated effector CD8+ T cells are mostly CXCR3Hi ( Kohlmeier et al . , 2011; Harris et al . , 2012; Slutter et al . , 2013; Ochiai et al . , 2015 ) raises the question of whether they reacquire CXCR3 once they reach infected tissue sites .",
"A recent study of Mtb-infected lung tissues has demonstrated the existence of two distinct subpopulations of CD4+ TH1 cells that differ in their accessibility to an intravenous fluorescent antibody tracer ( Sakai et al . , 2014 ) .",
"The tracer-accessible subpopulation resembled our F3 stage effector CD8+ T cells being KLRG1+ CXCR3Lo CX3CR1Hi and was IFN-γHi .",
"In contrast , the other subpopulation more deeply localized in the tissue parenchyma were CXCR3Hi and exhibited a highly activated phenotype with expression of CD69 and PD-1 , but lower in KLRG1 expression and IFN-γ production .",
"Therefore , one interesting scenario is by virtue of their CX3CR1 expression , the F3 stage effector cell circulates in the blood and serves a patrolling function similar to CX3CR1Hi monocytes ( Auffray et al . , 2007 ) .",
"They could then regain CXCR3 expression and function as effector CD8+ T cells in parasite-infected tissues .",
"Future studies should investigate the reciprocal patterns of expression of CXCR3 and CX3CR1 and their counter regulation .",
"Using immunohistochemistry methods Khanna et al . ( 2007 ) demonstrated the outmigration of antigen-specific CD8+ T cells through ‘bridging channels’ into the MZ and splenic RP .",
"Kurachi et al . ( 2011 ) further reported that CXCR3 enabled CD8+ T cells to cluster with IL-12 producing CD11c+ DCs in the MZ and RP .",
"Now , we show that precursor effector CD8+ T cells in the bridging channels and MZ are , in fact , actively proliferating and that they receive IL-12 signals to complete their differentiation in the RP .",
"Collectively , these findings highlight a previously unrecognized extrafollicular pathway for the generation and maturation of primary effector CD8+ T cells .",
"Interestingly , in the B cell response , there is an analogous and well-recognized extrafollicular differentiation pathway of IgG class switched plasma cells producing the primary antibody response to T cell-dependent antigens ( MacLennan et al . , 2003; Kurosaki et al . , 2015 ) .",
"By analogy to the way that the non-germinal center plasma cell response is favored by strong B cell receptor signaling and antigen presentation by MZ localized DCs ( Paus et al . , 2006; Phan et al . , 2006; Chappell et al . , 2012 ) , the extrafollicular generation of effector CD8+ T cells is also likely regulated by differential T cell receptor signaling ( Bernhard et al . , 2015 ) and local cytokine and chemokine milieu created by specialized pathogen-engaged APCs interacting with bystander lymphocytes in the MZ and RP .",
"The exposure of effector precursors to this extrafollicular tissue niche could provide major extrinsic factors that determine the observed interclonal and , perhaps , intraclonal variability in effector lymphocyte fates that is independent of their intrinsic TCR-peptide:MHC affinity and dwell time ( Plumlee et al . , 2013; Tubo et al . , 2013 ) ."
],
[
"WT C57BL/6J , IL-12rβ2−/− ( B6 . 129S1-Il12rb2tm1Jm/J ) male , CD45 . 1+ recipients ( B6 . SJL-PtprcaPepc/BoyJ ) and T-bet−/− ( B6 . 129S6-Tbx21tm1Glm/J ) mice were purchased from The Jackson Laboratory ( Bar Harbor , ME ) .",
"IL12p35−/− mice ( B6 . 129S1-Il12atm1Jm/J ) were bred in house .",
"SCNT mice cloned from a single CD8+ T cell specifically expressing TCRαβ for tgd057/SVLAFRRL antigen were a generous gift from Hidde L Ploegh at MIT ( Kirak et al . , 2010b ) .",
"To generate Thy1 . 1+ tgd057-specific SCNT mice , SCNT females were crossed with a Thy1 . 1+ male .",
"The F1 progeny were backcrossed to SCNT females and F2 progeny expressing Thy1 . 1 and TCRαβ specific for tgd057/SVLAFRRL antigen were selected .",
"To generate IL-12rβ2−/− tgd057-specific SCNT mice , SCNT females were crossed with IL-12rβ2−/− males .",
"The F1 progeny with tetramer-binding CD8+ T cells were intercrossed .",
"The F2 progeny were then genotyped to select for IL-12rβ2−/− using tail or ear tissue and phenotyped for inheritance of TCRαβ specific for tgd057/SVLAFRRL antigen .",
"CCR2-DTR depleter ( CCR2-DTR ) and CCR2-reporter ( CCR2-GFP ) strains previously generated on a C57BL/6 background as previously described were used to investigate the role of mDCs during CD8+ T cell differentiation ( Hohl et al . , 2009; Serbina et al . , 2009; Espinosa et al . , 2014 ) .",
"Breeding , handling and housing of all mice was under specific pathogen-free conditions at Rutgers University ( formerly University of Medicine and Dentistry of New Jersey ) in Newark , NJ and according to the Rutgers Institutional Animal Care and Use Committee guidelines .",
"All mice used for experiments were age- and sex-matched .",
"Monolayers of human foreskin fibroblasts ( HFF ) were infected with T . gondii tachyzoites that have a disruption in the carbomyl phosphate synthetase ( cps 1-1 or CPS ) II gene , cultured in DMEM ( Invitrogen , Carlsbad , CA ) supplemented with uracil ( Fox and Bzik , 2002 ) .",
"Fresh tachzyoites were isolated from approximately 80% lysed HFF cultures and irradiated with 150gy 137Cs before injection .",
"Mice were sacrificed on specified days post CPS vaccination .",
"Spleens of infected and uninfected mice were isolated by physical disruption and passage through 70 μm nylon mesh ( BD Falcon ) , washed and erythrocytes lysed using a hypotonic Tris-buffered NH4Cl solution .",
"PECs were isolated by peritoneal lavage with RPMI 1640 ( Invitrogen ) supplemented with 2% FBS , 1% P/S , 50 μM β-mercatopethanol .",
"Live cells from spleens and PECs were counted using trypan blue dye exclusion .",
"To restimulate cells in vitro , 4–6 × 106 cells from spleens and PECs of infected and uninfected animals were plated and restimulated with live CPS tachyzoites ( MOI of 0 . 1 or MOI of 0 . 3 ) and incubated for 10 hr at 37°C .",
"GolgiStop ( BD Bioscience , San Jose , CA ) was added 4 hr prior to end of in vitro incubation .",
"Splenocytes were stained with PE-streptavidin-biotin SVLAFRRL:H2-Kb tetramers for 1 hr on ice in the dark , washed with FACS buffer , then incubated with anti-PE magnetic microbeads ( Miltenyi Biotec , Auburn , CA ) according to manufacturer's instructions .",
"Mix was then applied to LS magnetic column ( Miltenyi Biotec ) , and positively selected cells were collected and analyzed by flow cytometry .",
"Cell surface antibody and tetramer staining was performed concurrently in FACS buffer for 1 hr on ice in the dark , washed and fixed .",
"To stain for intracellular cytokines , samples from in vitro restimulated cultures were permeabilize and stained with cytokine antibodies .",
"Samples were washed and resuspended and FACS analyzed .",
"Flow cytometry data were acquired on BD LSRII and analyzed with FlowJo software ( Tree Star , Ashland , OR ) .",
"Mouse specific antibodies were purchased from eBioscience ( San Diego , CA ) : CD44-PE Cy7 , IFN-γ-eFluor450 , CD8α-Alexa Fluor 700 , TCRβ-PerCP-Cy5 . 5 , KLRG1-APC , Thy1 . 1-eFluor 450 or from BD Bioscience: CD62L-APC-eFluor780 and CXCR3-FITC .",
"PE-streptavidin-biotin SVLAFRRL: H-2Kb tetramers were prepared as previously described ( Wilson et al . , 2010 ) .",
"CD8+ T cells were negatively selected from naïve Thy1 . 1+ tgd057-specific SCNT mice , and IL12rβ2−/− tgd057-specific SCNT mice .",
"Negative selection was performed using magnetic micro beads ( Miltenyi Biotec ) .",
"A small aliquot of cells were surface stained for CD8α , TCRβ , and tetramer , fixed and analyzed for purity .",
"The frequency of CD8α+ tgd057-specific cells was used to normalize the numbers of tgd057-specific CD8+ T cells from the negatively selected samples .",
"For adoptive transfer studies , Thy1 . 1+ tgd057-specific SCNT CD8+ T cells were resuspended in PBS and intravenously ( i . v . ) injected into either naïve WT or IL-12p35−/− recipients .",
"For adoptive co-transfer experiments , a 1:1 mix of 500 donor Thy1 . 1+ tgd057-specific SCNT CD8+ T cells and 500 donor IL12rβ2−/− tgd057-specific SCNT CD8+ T cells were mixed and i . v . injected into naïve WT CD45 . 1+ congenic recipient mice .",
"D4 , D5 , D5 . 5 or D7 CPS vaccinated mice were i . v . injected with 2 . 5–4 μg of fluorescently labeled anti-CD8α antibody .",
"The mice were sacrificed 2–3 min post injection , and splenocyte single-cells suspensions were prepared and stained for CD8α in a different fluorochrome along with other surface antibodies for flow cytometry analysis .",
"Cells in contact with blood circulation are labeled by intravenously injected anti-CD8+ antibody , while cells in the splenic white pulp are not .",
"D4 or D5 CPS vaccinated mice were i . p . injected with EdU labeling reagent ( 10 ml/kg body weight , Invitrogen , C-10418 ) dissolved in sterile PBS 2 hr prior to sacrifice .",
"EdU detection was performed according to manufacturer's manual using Click-iT EdU- Pacific Blue labeling kit after splenocytes were surface stained and fixed .",
"D4 . 5 and D5 . 5 CPS vaccinated spleen were snap frozen in OCT compound ( Tissue-Tek , Sakura Finetek , Torrance , CA ) .",
"6–8 μm sections were sectioned and briefly rehydrated in sterile PBS , surface stained for anti-CD8α and anti-KLRG1 , fixed with 4%PFA then EdU detection was performed according to manufacturer's manual ( Invitrogen , C-10418 ) using Click-iT-EdU Pacific Blue labeling kit .",
"Sections were mounted with Prolong Gold Antifade Mountant ( Invitrogen , P10144 ) .",
"Immunofluorescent images were captured on a Nikon A1R Si confocal microscope equipped with a 20× Plan Apo VC dry objective and analyzed using NIS Elements C3 . 13 . 01 software ( Nikon , Melville , NY , United States ) .",
"On day 0 , day 3 or both days , each mouse was treated via intraperitoneal ( i . p . ) injection with 200 μg of anti-IL-12p40 monoclonal antibody ( C17 . 8 ) , anti-IFN-γ ( XMG . 1 ) or the isotype control , 2A3 clone .",
"4 hr post antibody injection , on day 0 , mice were vaccinated with 2 . 5 × 106 irradiated CPS parasites .",
"Spleen and PECs were harvested for analysis 5 days post CPS vaccination .",
"CCR2+ monocytes were depleted in CCR2-DTR mice and control CCR2-DTR negative littermates by i . p . administration of 250 ng of diphtheria toxin 2 . 5 days post CPS vaccination ( Espinosa et al . , 2014 ) .",
"Diphtheria Toxin was purchased from List Biological Laboratories ( Campbell , CA ) , and reconstituted at 1 mg/ml in PBS .",
"Aliquots were stored in 80°C .",
"Spleens were harvested on D5 post CPS vaccination , and tgd057-specific CD8+ T cells were analyzed .",
"Data are represented as Mean ± SEM .",
"All error bars represent SEM .",
"p values were calculated with Student's t-tests or ANOVA ( GraphPad Prism ) as indicated .",
"p values of less than 0 . 05 were considered significant ."
]
] | [
"The proinflammatory cytokine IL-12 drives the generation of terminally differentiated KLRG1+ effector CD8+ T cells .",
"Using a Toxoplasma vaccination model , we delineate the sequence of events that naïve CD8+ T cells undergo to become terminal effectors and the differentiation steps controlled by IL-12 .",
"We demonstrate that direct IL-12 signaling on CD8+ T cells is essential for the induction of KLRG1 and IFN-γ , but the subsequent downregulation of CXCR3 is controlled by IL-12 indirectly through the actions of IFN-γ and IFN-γ-inducible chemokines .",
"Differentiation of nascent effectors occurs in an extrafollicular splenic compartment and is driven by late IL-12 production by DCs distinct from the classical CD8α+ DC .",
"Unexpectedly , we also found extensive proliferation of both KLRG1− and KLRG1+ CD8+ T cells in the marginal zone and red pulp , which ceases prior to the final KLRG1Hi CXCR3Lo stage .",
"Our findings highlight the notion of an extrafollicular pathway for effector T cell generation ."
] | [
"The immune system helps to protect us from cancer , infection by microbes and other diseases .",
"There are several different types of immune cells that each have particular roles .",
"For example , cytotoxic T cells can kill other cells in the body that are damaged or infected .",
"These cells are found in various locations around the body—including a region of the spleen known as the white pulp—where they wait in an inactive state until they detect signals from a damaged or infected cell .",
"These T cells divide and mature to produce populations of active T cells known as effector cytotoxic lymphoid cells ( or CTLs for short ) , a process which is thought to occur within the white pulp .",
"A small protein called cytokine IL-12 is involved in the production of CTLs .",
"The cytokine is released from other immune cells and causes the activated T cells to divide and mature .",
"It has long been believed that IL-12 produced in the white pulp early on in the process is sufficient to drive this process , but more recent work suggests that sustained production of IL-12 in other areas of the spleen that are accessible to the bloodstream may be needed .",
"Here , Shah et al . studied the generation of cytotoxic T cells in mice that had been exposed to a vaccine against a disease called Toxoplasmosis .",
"Their experiments show that IL-12 drives both the early and late stages of CTL production .",
"In the early stages , the T cells respond to IL-12 that is secreted by a group of ‘lymphoid dendritic’ cells in the white pulp .",
"However , in the later stages , the T cells move away from the white pulp to other parts of the spleen known as the marginal zone and red pulp , where a distinct group of ‘myeloid dendritic’ cells also produce IL-12 and direct the final maturation of the CTLs .",
"Shah et al . 's findings also show that the process in which cytotoxic T cells divide and later mature to produce CTLs involves a series of tightly controlled events that mostly occur outside of the white pulp .",
"These observations provide a new perspective on how to develop vaccines and other treatments that more efficiently generate the CTLs needed to protect against infections and cancer ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Node of Ranvier length as a potential regulator of myelinated axon conduction speed | elife-23329-v2 | [
[
"During development or learning , an adjustment of myelin thickness or internode length may be used to tune the conduction speed of myelinated axons ( Fields , 2008; Tomassy et al . , 2014; Ullén , 2009; Kimura and Itami , 2009 ) .",
"This can promote synchronous neuronal firing ( Lang and Rosenbluth , 2003; Sugihara et al . , 1993 ) , make impulse propagation time less dependent on the spatial trajectory of the axon transmitting information between areas ( Salami et al . , 2003 ) , or adjust propagation delays to mediate sound localization ( Carr and Konishi , 1990; McAlpine and Grothe , 2003; Seidl et al . , 2010; Ford et al . , 2015 ) .",
"Magnetic resonance imaging of humans and cellular studies of rodents suggest that myelination can increase while learning motor tasks such as piano playing ( Bengtsson et al . , 2005 ) , juggling ( Scholz et al . , 2009 ) , reaching ( Sampaio-Baptista et al . , 2013 ) and running ( McKenzie et al . , 2014 ) .",
"Although most interest has focussed on the effect of changes of myelin thickness or internode length , the node of Ranvier is another potential determinant of action potential conduction speed .",
"Increasing the length of the node will increase the node capacitance and the axial resistance for current flow into the internode , which will both decrease conduction speed .",
"On the other hand , a greater length could increase the number of voltage-gated Na+ channels at the node ( if the channel density is constant ) , which may increase conduction speed .",
"Given the potential influence of node length on conduction speed , we quantified heterogeneity of the length of the node of Ranvier in the white matter of the rat optic nerve and corpus callosum , and in the grey matter of rat cerebral cortex .",
"Computer modelling was then used to explore the effects on conduction speed of the range of node lengths observed ."
],
[
"We first measured the length of nodes of Ranvier and axon diameter in adult rat optic nerve using both confocal and serial electron microscopy ( Figure 1 , Figure 1—figure supplement 1 ) .",
"Using electron microscopy ( EM ) we found the mean node length was 1 . 08 ± 0 . 02 µm ( mean ± s . e . m . , n = 46 nodes ) .",
"Node lengths varied 2-fold , from 0 . 7 to 1 . 4 µm with a standard deviation of 0 . 15 µm ( Figure 1—figure supplement 1 ) .",
"Node lengths were not significantly correlated with axon diameter at the node , which had a mean value of 0 . 80 ± 0 . 03 µm ( standard deviation 0 . 19 µm; Figure 1—figure supplement 1 , the slope of the regression line is not significantly different from zero , p=0 . 46 ) .",
"Thus , the different node lengths observed did not simply reflect axons being of different sizes . 10 . 7554/eLife . 23329 . 003Figure 1 . Heterogeneity of Ranvier node lengths in the optic nerve and cerebral cortex .",
"( A ) Confocal image of a single optic nerve node of Ranvier showing the node labelled with antibody to NaV1 . 6 ( green ) and paranodes labelled for Caspr ( red ) .",
"( B ) Intensity profile of Caspr staining for the node in A . Node length was measured as the distance between the half maximum intensity for each paranode .",
"( C , D )",
"Confocal images of nodes in the optic nerve ( C ) and layer V of the cortex ( D ) highlighting the different range of node of Ranvier sizes in these areas .",
"( E ) Mean ± s . e . m . of the node lengths measured in optic nerve ( ON , red ) and layer V of the cortex ( blue ) .",
"( F , G )",
"Distribution of node lengths from data in E for optic nerve ( F ) and cortex ( G ) .",
"( H ) Node diameter as a function of node length in the optic nerve ( red ) and in grey matter of the cerebral cortex ( blue ) .",
"Slope of regression lines in H are not significantly different from zero ( p=0 . 14 ( optic nerve ) and p=0 . 42 ( cortex ) ) .",
"( I ) NaV1 . 6 immunolabel intensity ( summed over each node ) as a function of node length in cerebral cortex ( each point is one node ) .",
"NaV1 . 6 labelling is correlated with node length ( slope of regression line is significantly greater than zero , p=1 . 2×10−15 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23329 . 00310 . 7554/eLife . 23329 . 004Figure 1—figure supplement 1 . Heterogeneity of Ranvier node lengths in the optic nerve . Top: EM picture of node of Ranvier in the adult optic nerve , showing surrounding oligodendrocytes ( paranodal loops false-coloured green ) , an astrocyte ( pink ) and the node length .",
"In practice node length was measured from the total thickness of the transverse sections across the nerve that were between the surrounding oligodendrocytes .",
"Bottom: Node length as a function of node diameter in the optic nerve for 46 nodes measured by EM .",
"Slope of regression line is not significantly different from zero ( p=0 . 46 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23329 . 004 In contrast to EM , confocal microscopy allowed us to measure a greater number of nodes in different parts of the CNS .",
"In addition , given that oligodendrogenesis and myelination continues well into adulthood ( Dimou et al . , 2008; Young et al . , 2013 ) , confocal microscopy of antibody-labelled nodes allowed us to distinguish developing nodes from mature nodes , ensuring that any differences in size observed were not due to different developmental stages .",
"Mature nodes of Ranvier were identified from their NaV1 . 6 staining ( a marker of mature nodes: Boiko et al . , 2001; Kaplan et al . , 2001 ) flanked by Caspr-labelled paranodes ( Figure 1A ) , and node length was measured from the Caspr intensity profile across the node ( Figure 1B , see Materials and methods ) .",
"We first assessed whether , using confocal microscopy ( Figure 1C ) , we could observe variability of node lengths in the rat optic nerve similar to that found when using EM .",
"We again found node length variability ( Figure 1C , E , F , H ) , the mean node length was not significantly different ( p=0 . 06 ) from that obtained with EM ( 1 . 02 ± 0 . 02 μm , standard deviation 0 . 29 µm , n = 164 , Figure 1E ) , and there was again no significant dependence on either node diameter ( Figure 1H , p=0 . 14 ) or axon diameter at the paranode ( measured as the diameter of the Caspr labelling: p=0 . 89 ) .",
"However , the node length range was slightly broader , covering a 4 . 4-fold range ( Figure 1F , H ) , perhaps due to the ~4 fold greater number of nodes quantified .",
"In the grey matter of the adult cerebral cortex ( layer V of motor cortex ) an even larger , 8 . 7-fold , range of node lengths was observed ( Figure 1D , E , G , H ) , from 0 . 43 to 3 . 72 µm ( mean value ± s . e . m 1 . 50 ± 0 . 05 , standard deviation 0 . 58 µm , n = 158 ) .",
"Again there was no significant dependence on node diameter ( Figure 1H , p=0 . 42 ) , the mean value of which was 0 . 64 ± 0 . 01 µm ( n = 158 , standard deviation 0 . 14 µm ) , or on axon diameter at the paranode ( p=0 . 98 ) .",
"Thus , variability of node length is a general feature of myelinated axons .",
"The greater variability of node length observed in the cortex than in the optic nerve has parallels with the far greater variability of myelination seen in the adult cortex ( Tomassy et al . , 2014 ) and could reflect tuning of individual axon conduction speeds to meet information processing needs .",
"To investigate whether this node length variability was accompanied by a similar variability in sodium channel number , we summed the intensity of NaV1 . 6 staining over individual nodes in cortex and compared this with node length ( Figure 1I ) .",
"We found that there is a linear correlation between summed NaV1 . 6 staining and node length ( p=1 . 2×10−15 ) indicating that the number of sodium channels at nodes is approximately proportional to node length ( cf . Rios et al . , 2003 ) , consistent with larger nodes being generated by the insertion of more NaV-containing membrane .",
"However , at any given sodium channel labelling intensity there was still a large variation in node lengths , suggesting that sodium channel density is not absolutely constant , and that it may be possible to vary node length in a manner independent of sodium channel number .",
"If adjustment of node length is used to tune conduction speed , one might expect all the nodes along one axon to have similar lengths ( e . g . all long or all short ) .",
"To assess whether the variability in node lengths mainly occurs along axons or between axons we iontophoretically injected a fluorescent dye into the cortex of adult rats ( see Materials and methods ) .",
"This was taken up into axons and diffused along them , allowing us to measure the lengths of up to 13 successive nodes ( mean 6 . 7 ± 0 . 8 nodes in 18 axons ) along single fluorescently labelled axons in the corpus callosum ( Figure 2A ) .",
"Remarkably , along individual axons ( Figure 2B , C ) , the distribution of node lengths was much narrower than that observed over all callosal axons examined ( Figure 2C ) , with a 48 . 8% ± 3 . 5% lower coefficient of variation ( s . d . /mean , Figure 2D , p=1 . 1×10−10 , one sample t-test , n = 18 axons ) .",
"Importantly , even when excluding axons with mean node lengths >2 . 25 µm ( which were found less frequently , as predicted by the distribution in Figure 1G–H ) , the coefficient of variation for single axons was 38 . 8% ± 4 . 7% lower than that observed over all axons ( p=6 . 6×10−7 ) .",
"Node length was not correlated with internode length ( p=0 . 1 , Figure 2E ) . 10 . 7554/eLife . 23329 . 005Figure 2 . Node of Ranvier lengths are correlated along an axon .",
"( A ) Composite confocal image of a single axon in the corpus callosum iontophoretically labelled with tetramethylrhodamine dextran ( red ) .",
"Three consecutive nodes of Ranvier are highlighted and shown in high resolution images .",
"Nodes of Ranvier are identified as NaV1 . 6 positive clusters ( green ) flanked by Caspr positive paranodes ( blue ) .",
"( B ) Successive node lengths along three example axons with different mean node lengths .",
"The mean node length for each axon is plotted as a dashed line .",
"( C ) Distributions of node lengths of 18 individual axons ( in 0 . 5 µm bins , centered on the median node length for each axon ) show that the variability along each axon is much less than the variability between axons .",
"( D ) Mean coefficient of variation for node lengths along 18 individual axons and the overall coefficient of variation for all axons examined .",
"( E ) Mean internode length for each axon plotted against the mean node length for that axon .",
"Each axon is represented by a different colour , and that colour is maintained for panels B , C and E . Regression line slope is not significantly different from zero ( p=0 . 1 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23329 . 005 Thus , node lengths are similar along axons but differ significantly between axons .",
"This raises the possibility that individual axons consistently adjust their node length to tune conduction speed .",
"To examine the consequences of nodes of Ranvier having different lengths , we simulated action potential propagation in optic nerve and cortical grey matter myelinated axons , as described in the Materials and methods .",
"The differential equations of the model were derived and solved as in Halter and Clark ( 1991 ) .",
"Details of the parameters used are summarised in Table 1 .",
"The conduction speeds predicted for the mean node lengths observed ( 2 . 95 m/s for the optic nerve and 2 . 61 m/s for the cortex ) were within the range of values observed experimentally in the adult rat optic nerve ( 2 . 5–15 m/s: Foster et al . ( 1982 ) ; Sefton and Swinburn ( 1964 ) ; Sjöström et al . ( 1985 ) ) and for different classes of rat cortical grey matter output axons ( 1 . 8–5 . 9 m/s: Kelly et al . , 2001 ) . 10 . 7554/eLife . 23329 . 006Table 1 . Electrical and geometrical parameters of the models . DOI: http://dx . doi . org/10 . 7554/eLife . 23329 . 006Parameter Symbol Value Units Nodal Na+ conductance*gNa3000 mS/cm2Nodal K+ conductance*gKs80mS/cm2Nodal persistent Na+ conductance*gNap5mS/cm2Leakage conductanceNode*InternodegL 800 . 1 mS/cm2mS/cm2Myelin membrane conductancegmy1 . 0mS/cm2Axon membrane capacitance†NodeInternode cax 0 . 90 . 9 μF/cm2μF/cm2Myelin membrane capacitance† , ‡cmy0 . 9μF/cm2Axoplasmic resistivityρax70Ω .",
"cmPeriaxonal resistivityρp70Ω .",
"cmResting potentialEr−82 mVLeakage potentialELk−83 . 38 mVNa+ reversal potentialENa50mVK+ reversal potentialEK−84 mVNode diameterOptic nerveCortex 0 . 730 . 64 μmμmNode lengthOptic nerveCortex 1 . 021 . 50 μmμmParanode lengthOptic nerveCortex 2 . 111 . 90 μmμmParanodal effective periaxonal spaceOptic nerveCortex 0 . 00770 . 0123 nmnmInternodal axon diameterOptic nerveCortex 0 . 820 . 73 μmμmInternodal periaxonal space 15nmG ratioOptic nerveCortex 0 . 780 . 81 Number of myelin wrapsOptic nerveCortex 75 Internode lengthOptic nerveCortex 139 . 2681 . 7 μmμm*Values for standard node length: 1 . 02 µm in optic nerve , 1 . 50 µm in cortex; these are constant for simulations with fixed nodal conductance density , but scaled inversely with node length for simulations where number of nodal channels is kept constant .",
"†Membrane capacitance values are from Gentet et al . ( 2000 ) .",
"‡Figures are per myelin membrane .",
"There are two membranes per myelin lamella .",
"Our data suggest a positive correlation between the number of NaV1 . 6 channels and node length ( indicating a fixed channel density ) , but also raise the possibility of node length varying in a manner independent of channel number ( Figure 1I ) .",
"We therefore modelled two extreme situations , for both the optic nerve and the cortical axons studied: either the density of nodal ion channels was assumed to be constant ( so the number of ion channels increases in proportion to node length ) , or the number of ion channels at the node was held constant at the values assumed for the mean node length observed ( so the density of channels varies inversely with node length ) .",
"Figure 3A and B show that , when the number of channels was held constant at each node , the predicted conduction speed falls with increasing node length ( dashed curves ) .",
"This occurs for two reasons: the increase in node length increases the nodal capacitance ( so each node takes longer to charge ) , and the intracellular axial resistance to current flow from the node into the internode is increased .",
"The changes in conduction speed for the optic nerve are shown in Figure 3A ( the range of measured node lengths is shown for comparison ) .",
"Increasing the node length from its mean value of 1 . 02 µm to the largest value observed ( 2 . 2 µm ) is predicted to decrease the conduction speed by 6 . 5% , while decreasing the node length to the smallest value measured ( 0 . 5 µm ) increases the speed by 3 . 2% ( giving a speed that is 10 . 3% larger than at a length of 2 . 2 µm ) .",
"For cortical axons ( Figure 3B ) the predicted changes are larger , partly because , with a 1 . 5-fold longer node and a 1 . 7-fold shorter internode length , the nodal membrane contributes a larger fraction of the total membrane capacitance ( 14% in cortical axons versus 8% in optic nerve axons ) .",
"The node length variation observed in rat cortex ( Figure 1G–H and 0 . 43 µm to 3 . 7 µm ) results in conduction speeds that are 11 . 6% slower ( for the longest node ) and 7% faster ( for the shortest node ) than the speed for the mean node length of 1 . 5 µm .",
"Thus , altering node length from 3 . 7 to 0 . 43 µm would increase the speed by approximately 21% . 10 . 7554/eLife . 23329 . 007Figure 3 . Predicted effect on conduction speed of different node lengths .",
"( A–F )",
"Calculated conduction speed as a function of node length for axons in ( A ) the optic nerve and ( B–F ) the cortical grey matter .",
"For panels A–F , simulations were carried out assuming either that the density of ion channels at the node is constant as the node length is changed ( solid lines ) , or that the number of ion channels is kept constant ( dashed lines ) at the value assumed for the mean node length .",
"( C–D )",
"Simulations for cortex as in B but examining the effect of altering internode length ( INL , given by each curve ) .",
"( E–F )",
"Calculated dependence of conduction speed of cortical axons on internode length for different assumed node lengths ( NL ) .",
"The observed range of each abscissa parameter is indicated on the graphs .",
"( G ) .",
"Change in membrane area needed , in the myelin sheath ( myelin wrap ) or node of Ranvier ( node length , NL ) , to change the conduction speed by 8 . 6% in optic nerve or 10 . 5% in cortical axons , when nodal ion channel density is held constant ( note logarithmic scale ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23329 . 007 When the nodal ion channel density is kept constant another factor affects the predicted conduction speed , in addition to the change of capacitance and axial resistance at the node: as the node length is decreased the reduction in the number of ion channels present leads to a decrease of conduction speed .",
"Consequently , the plot of speed against node length shows a maximum ( solid curves in Figure 3A–B: note that , above this maximum , increasing node length decreases speed for the reasons stated above , despite the increase in number of sodium channels at the node ) .",
"Decreasing the optic nerve node length from its mean value of 1 . 02 µm to the lowest length observed ( 0 . 5 µm ) is predicted to decrease conduction speed by 14 . 1% , while increasing node length to the value generating the maximum conduction speed ( 1 . 7 µm ) increases the speed by 3 . 3% ( Figure 3A ) , to a value that is 20 . 2% higher than at a length of 0 . 5 µm .",
"Similarly , for the cortex , decreasing the node length from the mean value of 1 . 5 µm to the smallest observed value of 0 . 43 µm decreases the conduction velocity by 19 . 6% while increasing the length slightly to 1 . 7 µm increases the speed by 0 . 2% to a value 24 . 6% larger than at a node length of 0 . 43 µm ( Figure 3B ) .",
"Although the node length is similar at successive nodes along individual axons ( Figure 2B ) , some variation does exist ( with a mean standard deviation of 0 . 25 ± 0 . 06 µm in 18 axons ) , so we assessed the effect of this by simulating a situation where alternate nodes had a length 0 . 25 µm shorter and 0 . 25 µm longer than the mean node length .",
"For example , for a mean node length of 1 . 0 µm , alternate nodes had lengths of 0 . 75 µm and 1 . 25 µm .",
"When the number of channels was held constant at each node , the approximately linear relationship between conduction velocity and node length ( Figure 3A , B ) resulted in a slightly faster propagation through shorter nodes and a slightly slower propagation through longer nodes , the effects of which cancel out with no overall effect on conduction velocity .",
"When the nodal ion channel density was kept constant , the concave-downwards dependence of speed on node length in Figure 3A–B resulted in marginally slower conduction speeds ( <2% reduced ) with variable length nodes , compared to when all the nodes along a particular axon had exactly the same length .",
"It has recently been reported that internode lengths may vary significantly in the cortex ( Tomassy et al . , 2014; Chong et al . , 2012 ) .",
"We measured internode lengths in dye-filled axons in rat layer V cortex by measuring the distance between NaV1 . 6 positive nodes .",
"Similar to previous studies we found a large variability in the length of 30 internodes , the shortest being 27 µm and the largest 154 µm , with a mean value of 82 . 7 ± 6 . 3 µm .",
"Given this large variation , we examined how internode length ( assumed , for simplicity , to be the same for all internodes along an axon ) affects the tuning of conduction speed by changes of node length ( Figure 3C–F ) .",
"For cortical axons ( with a constant nodal sodium channel density ) , the peak of the dependence of conduction speed on node length is displaced to longer node lengths when the internode length is increased ( Figure 3C ) , and in an axon with internode lengths of 154 µm the range of node lengths measured could generate changes in conduction speed of up to 42% .",
"When the number of channels was held constant at each node , the dependence of conduction speed on node length was greater in axons with short internodes ( 27 µm ) , resulting in changes in conduction speed of up to 38% over the range of measured node lengths ( Figure 3D ) .",
"Changes of node length also affect the predicted dependence of conduction speed on internode length ( Figure 3E , F ) .",
"This relationship rises with internode length at low values of internode length , because more of the axon is myelinated , but decreases at large internode lengths as the spread of depolarization between nodes becomes less efficient .",
"This relationship shows a sharper peak for shorter node lengths ( Figure 3E , F ) .",
"We assessed how powerful node length changes can be for tuning conduction speed , compared to altering the amount of myelin around the axon .",
"With the node length and internode length set at the mean values observed and the nodal conductances constant , if the number of wraps of myelin is decreased by 1 ( from the normal 7 to 6 for the optic nerve and from the normal 5 to 4 for the cortex ) , the conduction speed is predicted to decrease by 8 . 6% for the optic nerve and 10 . 5% for the cortex ( for simplicity no change of paranode length was assumed for these simulations , although the paranode may be shorter with less myelin wraps ) .",
"For comparison , a similar speed decrease is predicted ( with no change of wrap number ) if the nodal NaV density is reduced by 26% in the optic nerve or 34% in the cortex , or if the internode length is increased by 36% in the optic nerve or 74% in the cortex .",
"These same speed decreases can be achieved with no change of myelin wrapping or ion channel density by decreasing the node length from 1 . 02 µm to 0 . 625 µm in the optic nerve , or decreasing it from 1 . 5 µm to 0 . 635 µm in cortical axons .",
"( Whether the internode needs to be lengthened by the same amount , to maintain axon length , and the consequences of this , are considered in the Simulations section of the Materials and methods ) .",
"Strikingly , to produce these speed changes , the change of membrane area needed ( shown in Figure 3G ) when altering node length is 1006-fold less in the optic nerve , and 273-fold less in the cortex , than that needed when altering the myelin sheath .",
"Thus , tuning conduction speed by altering node length is far more efficient than altering myelination when considering the amount of lipid and protein synthesis or breakdown needed , and would probably also be faster ."
],
[
"By combining anatomical measurements with computational modelling we have established a proof of principle for the idea that node of Ranvier length may be adjusted to tune axon conduction speed , and hence alter action potential arrival time .",
"This differs from previous ideas on white matter plasticity ( which have focussed on the effect of changes of myelin thickness and internode length ( Fields , 2008; Ullén , 2009 ) ) , and demonstrates that the geometry of the node of Ranvier is also a crucial determinant of action potential conduction speed .",
"Even at constant axon diameter , we found that node length displayed a surprising variability , both in the optic nerve and in the grey matter of the cortex ( Figure 1 ) .",
"Remarkably , this node length variation was largely between different axons , while the nodes on any given axon tended to have similar lengths ( Figure 2 ) .",
"The range of node lengths observed is sufficient to produce large variations in action potential conduction speed ( Figure 3 ) , comparable to those produced by adding or removing a wrap of myelin .",
"These data suggest that axons may be able to adjust their node lengths in order to tune their conduction speed , and thus the arrival time of the information that they transmit .",
"There is now evidence of oligodendrogenesis and myelination in the adult CNS ( Dimou et al . , 2008; Young et al . , 2013 ) which presumably results in the formation of new nodes of Ranvier .",
"To avoid these , we studied only nodes that expressed the mature node marker NaV1 . 6 ( Boiko et al . , 2001; Kaplan et al . , 2001 ) , and the fact that the variability in node length was observed across a large fraction of the nodes measured makes it improbable that this variation is solely due to nodes being at different developmental stages .",
"The node length was approximately proportional to the amount of sodium channel labelling at the node ( Figure 1I ) , suggesting that node length may mainly be adjusted by the insertion or removal of membrane containing sodium channels ( although conceivably it is possible to vary node length in a manner independent of sodium channel trafficking by endo- or exocytosis of vesicles lacking sodium channels in their membrane ) .",
"The fact that node lengths are similar over long distances along an axon ( Figure 2B ) raises three mechanistic questions .",
"First , when the node length is set to a different mean value in different axons , by what mechanism is the nodal ion channel density controlled ( Figure 1I ) ?",
"Second , what signal regulates node length , in order to adjust the arrival time of action potentials at the end of the axon ?",
"Conceivably a signal could be passed back along the axon from a postsynaptic cell by dynein-based motors , as occurs for BMP signalling from postsynaptic cells to the nuclei of presynaptic neurons ( Smith et al . , 2012 ) .",
"Third , what local molecular mechanism regulates the length of each node , how accurately can this be controlled , and is the internode length shortened when the node is elongated ( to preserve overall axon length ) ?",
"Node length regulation may involve modifying the paranodal cell adhesion between the myelin and the axon , mediated by the molecules Caspr , neurofascin 155 and contactin , as well as altering ankyrin G mediated scaffolding within the axon that locates voltage-gated Na+ channels at the node ( Arancibia-Carcamo and Attwell , 2014 ) .",
"Interestingly , nodal amyloid precursor protein has been proposed as a regulator of node length ( Xu et al . , 2014 ) , prompting the speculation that changes in the processing of this molecule could alter node length in Alzheimer’s disease .",
"Computer simulations of the propagation of action potentials along myelinated axons ( Figure 3 ) show that rather small changes in node length can produce quite significant changes of conduction speed .",
"The range of node lengths seen in the optic nerve ( 0 . 5–2 . 2 µm ) can alter the conduction speed by up to 20% ( for a constant nodal channel density in Figure 3A ) , while the range seen in the cortex ( 0 . 43–3 . 7 µm ) can produce a larger change of up to 25% ( for a constant density of nodal channels in Figure 3B ) in axons with 82 µm long internodes , or 38% for axons with 27 µm long internodes ( Figure 3D ) .",
"The effect of altering node length is larger in cortical axons than in the optic nerve , partly because the 1 . 5-fold longer nodes contribute a larger fraction of the total axon capacitance in the cortex where the internodes are also 1 . 7-fold shorter .",
"Our data and simulations suggest that modulation of node length could be a viable strategy for adjusting the propagation time of action potentials to meet information processing needs .",
"For an intercortical callosal axon of length 1 cm ( in rat ) or 6 cm ( in human ) , with the properties that we assume for our simulations , a 20% decrease of axon conduction speed ( from the value occurring for the mean observed node length ) would increase the time needed to propagate information between the cortices from 3 . 8 to 4 . 8 ms in rat and from 23 to 29 ms in humans .",
"Such modulation has been suggested to occur during chronic stress and major depression ( Miyata et al . , 2016 ) which shorten the node , as well as high frequency action potential activity ( Huff et al . , 2011; Trigo and Smith , 2015 ) , acoustic over-exposure ( Tagoe et al . , 2014 ) , pathological release of glutamate ( Fu et al . , 2009 ) and hypoxic conditions ( Reimer et al . , 2011 ) , all of which lengthen the node .",
"Altering node length offers the advantage that very small changes of membrane area , which could easily be produced rapidly by exocytosis or endocytosis at the node , produce large changes of conduction speed .",
"In comparison , to produce the same speed changes by altering the number of myelin wraps requires the energetically expensive ( Harris and Attwell , 2012 ) , and probably more time consuming , synthesis or disassembly of a membrane area that is 273–1006 fold larger .",
"In practice , both mechanisms might be used on different time scales ."
],
[
"For the optic nerve , 3 male ( 8–10 weeks old ) Sprague-Dawley rats were anaesthetised and perfused through the heart with fixative containing 2 . 5% glutaraldehyde and 2% paraformaldehyde in 0 . 1 M cacodylate buffer .",
"The optic nerves were dissected out , post-fixed with 1% OsO4 in 0 . 1 M cacodylate buffer , embedded in EPON and polymerised .",
"Serial ultrathin ( 70 nm ) sections , perpendicular to the nerve’s long axis , were cut on an ultramicrotome and picked up on an osmium-coated glass slide .",
"Back-scattered images were obtained on a scanning electron microscope ( Hitachi SU8010 ) with a working distance of 2 mm , 1–1 . 5 kV accelerating voltage , scan speed of 40 or 80 s , and a typical pixel size of 1 . 65 nm at x30 , 000 magnification , and were analysed with ImageJ ( FIJI ) .",
"Node length ( assessed from the number of sections containing the node ) and mean axon diameter at the node ( assessed as axon perimeter/π ) were measured ( uncorrected for tissue shrinkage during fixation ) .",
"Four male 8–10 week old rats were anaesthetized with isoflurane and killed by cervical dislocation in accordance with United Kingdom animal experimentation regulations .",
"After decapitation the brain was carefully dissected from the skull and 1 mm thick coronal slices containing the corpus callosum were obtained from the forebrain ( from 4 to 8 mm rostral of the olfactory bulb ) using a tissue cutter block .",
"A 10% solution of tetramethylrhodamine dextran ( MW 3000 , Invitrogen , Paisley , UK ) was iontophoretically injected into the cortical grey matter .",
"Thereafter slices were incubated in oxygenated aCSF containing ( in mM ) 124 NaCl , 26 NaHCO3 , 1 NaH2PO4 , 2 . 5 KCl , 2 MgCl2 , 2 CaCl2 , 10 glucose , bubbled with 95% O2/5% CO2 for 2 hr at room temperature to allow for diffusion of the tracer .",
"After incubation slices were immersion fixed in PFA and resliced at 80–100 µm for subsequent immunohistochemical labelling of nodal ( NaV1 . 6 ) and paranodal ( Caspr ) marker proteins .",
"Optic nerves and 4 mm thick coronal sections of fronto-parietal ( motor ) cortex ( from 4 to 8 mm rostral of the olfactory bulb ) from brains of 4 male ( 8–10 week old ) Sprague-Dawley rats were either perfusion or immersion-fixed in 4% paraformaldehyde in PBS .",
"Fixed tissue was then cut into 50 µm slices using a Leica vibratome VT1200S or , for NaV1 . 6 density experiments , cut into 10 µm sections using a cryostat .",
"Slices were blocked and permeabilised in 10% horse serum and 0 . 5% Triton X-100 in PBS .",
"Immunofluorescence labelling was performed over 3 days with the following primary antibodies: rabbit anti-NaV1 . 6 ( Alomone , 1:500 ) ; mouse anti-Caspr clone K65/35 ( Neuromab , UC Davies , 1:100 ) .",
"Slices were then washed extensively ( 3 × 20 min ) and incubated overnight with secondary antibodies: anti-rabbit AlexaFluor488 ( Invitrogen , 1:500 ) , anti-mouse Dy-Light 647 ( Jackson Immunoresearch , 1:500 ) .",
"Slices were then washed 3 × 10 min in PBS and mounted with Dako Fluorescent Mounting Medium .",
"Slices were viewed using an LSM700 or LSM780 confocal microscope using a 63x ( NA 1 . 4 ) oil immersion lens , and images were acquired with LSM software with the pinhole set to 1 Airy unit for the Caspr signal , resulting in an optical slice of 0 . 8 µm .",
"Pixel size was 39 . 7 nm for Figure 1A–H and 99 . 2 nm for Figure 1I and 52 . 7 nm for node measurements in Figure 2 and 263 . 6 nm for internode measurements in Figure",
"2 . For node length analysis , confocal images were analysed using ImageJ software .",
"Images were background subtracted and only nodes that lay approximately parallel to the plane of section ( i . e . displayed nodal NaV1 . 6 labelling with flanking Caspr-labelled paranodes all within a single 0 . 8 µm optical slice ) were selected .",
"Measuring the angle of the axon to the plane of the slice for a subset of 10 randomly chosen axons showed that the apparent node length measured in this way underestimated the actual node length by only 1 . 7% ± 0 . 6% .",
"A maximum intensity projection was generated of the sections in which Caspr labelling was present for a particular node ( up to five interleaved confocal slices at 0 . 38 µm intervals , with a maximum stack thickness of 2 . 32 µm ) , and a line intensity profile ( the thickness of which was slightly less than the Caspr labelling thickness ) was drawn spanning both Caspr-labelled paranodes .",
"The size of the node was then calculated using a MATLAB ( The MathWorks , Inc . ) script which measures the distance between the half maximum intensity for each paranode .",
"Node diameter , paranode length and axon diameter were measured using the line tool in ImageJ over NaV1 . 6 staining ( for node diameter ) and over the Caspr staining ( for axon diameter and paranode length ) .",
"NaV1 . 6 staining was summed over the nodal area to obtain a parameter assumed to be proportional to sodium channel number .",
"Internode length was measured in three dimensions in FIJI using the simple neurite tracer plugin ( Longair et al . , 2011 ) .",
"Data were not corrected for tissue shrinkage during fixation .",
"To simulate action potential propagation along myelinated axons , we implemented , in MATLAB , model C of Richardson et al . ( 2000 ) ( at 37°C , with the unphysiologically low membrane capacitance of Richardson et al . ( 2000 ) corrected to a normal value ) .",
"The differential equations of the model were derived and solved as in Halter and Clark ( 1991 ) .",
"In brief , the axon is divided into compartments representing the node , paranode and internode .",
"For each time step , current flow across the axonal or total myelin membrane is calculated from the values of voltage ( and its rate of change ) , and the membrane capacitance and membrane conductances present per unit length ( simultaneously solving the differential equations that define activation and inactivation of the voltage-gated currents present at the node ) , and intracellular and periaxonal axial current flow are calculated from the intracellular or periaxonal resistance per unit length and the gradient of intracellular or periaxonal voltage .",
"Details of the parameters used are summarised in Table",
"1 . The MATLAB code used can be obtained immediately on request from the authors; it will be written up and documented as a resource for free access from GitHub by August 1st 2017 .",
"Simulations were carried out as in Bakiri et al . ( 2011 ) except that the periaxonal space under the myelin was included ( 51 nodes were simulated and conduction speed was measured between nodes 20 and 30 ) .",
"This model includes fast and persistent Na+ , and slow K+ , voltage-gated channels at the node , but omits voltage-gated K+ channels at the juxtaparanode ( which are little activated because the 100 mV voltage change of the action potential is distributed across the 11–15 membranes of the 5–7 myelin wraps and the axon , implying only a 7–9 mV voltage change across the axonal membrane ) .",
"For simplicity , the node length was usually assumed to be the same at all nodes on the axon , i . e . we ignored the variability in node length along the same axon described in Figure",
"2 . The node diameter was set to the mean value measured experimentally , i . e . 0 . 73 µm ( in 164 nodes ) for the optic nerve , and 0 . 64 µm ( in 158 nodes ) for cortex .",
"The region between two nodes , 139 . 3 µm long for the optic nerve ( Butt et al . , 1994 ) and 81 . 7 µm long for the cortical axons ( the mean value measured in layer V from 30 internodes , see main text ) , was kept constant when node length was varied ( both for simplicity , and because there was no correlation of node length and internode length: Figure 2E ) .",
"This internodal region was divided ( along its length ) into 66 and 86 compartments for the optic nerve and the cortex , respectively , the end 2 . 11 and 1 . 90 µm parts of which represent the paranode where the myelin attaches to the axon ( values measured in 164 and 158 nodes , respectively , from the length of the Caspr labelling; the number of compartments used has to be large for the simulation to be accurate , and needs to be chosen so that an integral number of compartments can represent the paranodal junction; the number was adjusted appropriately when simulating different internode lengths ) .",
"The internodal axon diameter is larger than the diameter at the node ( Halter and Clark , 1991; Berthold and Rydmark , 1983 ) , although this difference is a much smaller percentage for small than for large axons ( Rydmark and Berthold , 1983 ) .",
"The internodal and paranodal axon diameters were set to 0 . 82 µm and 0 . 73 µm for the optic nerve and cortex , respectively ( mean values obtained from 164 axons in optic nerve and 158 cortical axons from the diameter of the paranodal Caspr labelling ) , so that the node diameters were 88% and 86% of the internodal axon diameters respectively .",
"Apart from at the paranodes , the internodal axon was assumed to be surrounded by a periaxonal space of thickness 15 nm ( Robertson , 1959; Mierzwa et al . , 2010; Möbius et al . , 2016 ) , and to have a g ratio ( axon diameter/myelin diameter ) of 0 . 79 in the optic nerve and 0 . 8 in the cortex ( Sugimoto et al . , 1984; Oorschot et al . , 2013; to obtain an integral number of myelin wraps these values were adjusted slightly , to 0 . 78 and 0 . 81 respectively ) .",
"This led to the optic nerve and cortical axons having 7 and 5 myelin wraps , respectively assuming a myelin wrap periodicity of 15 . 6 nm ( Agrawal et al . , 2009; Harris and Attwell , 2012 ) .",
"The periaxonal space at the paranode , because of the structure of the attachment of the myelin to the axon at the paranode , is thought to comprise ( Mierzwa et al . , 2010 ) a pathway of cross sectional area A = 170 nm2 , which spirals around the axon ( from the node to the periaxonal space of the internode ) for a total distance of approximately D = π .",
"d . Nwraps where Nwraps is the number of myelin wraps and d is the axon diameter .",
"A periaxonal space of width w , along a paranode of length L , would have the same resistance as this pathway ifL/ ( π . d . w ) =D/Aorw=A . L/ ( π . D . d ) =A . L . /[ ( π . d ) 2 . Nwraps] The effective value of w used to model this spiral pathway for the optic nerve and cortical axons was thus 0 . 0077 nm and 0 . 0123 nm respectively .",
"In the main text we present calculations showing that a given change of conduction speed can be produced far more efficiently ( in terms of the change of membrane area needed ) by shortening of the node than by adding another wrap of myelin .",
"Those calculations ignore the possibility that , when the node is shortened , the internode needs to be lengthened by the same amount in order to maintain the axon length .",
"It is unclear whether the sub-micron node length changes postulated in our calculation would actually require remodelling of the adjacent myelin sheath – conceivably slackness in the somewhat non-straight internode would allow the change of node length to be accommodated without a change of internode length , and furthermore node length might change by an eversion of the paranodal loops closest to the node without any other significant change to the myelin sheath ( reviewed by Arancibia-Carcamo and Attwell , 2014 ) .",
"Thus , small changes in nodal length might well occur without major remodelling of the myelin sheath .",
"Nevertheless , if one assumes that node shortening by X µm absolutely does require an X µm elongation of the myelin sheath , then more membrane changes are needed than are accounted for in our simple calculation .",
"One can show mathematically that the sheath membrane area increase is larger than the area decrease at the node by a factor of 2x ( number of myelin wraps ) x ( mean radius of wraps ) / ( node radius ) , which is roughly 18 . 4 for the optic nerve and 13 for the cortex .",
"Accounting for these area changes ( and noting that , in this situation , there is no change of total axon length ) would reduce the ratio of the membrane area changes needed to produce a given speed change ( when adding a layer of myelin to the sheath versus changing the node length ) from 1006-fold to 55-fold for the optic nerve and from 273-fold to 21-fold for the cortex , but these ratios remain impressively large , and so the energetic argument favouring speed tuning by alteration of the node length still holds .",
"Data are shown as mean±s . e . m . Comparisons are via 2-tailed Student’s t-tests unless otherwise stated .",
"Assessment of whether the slope of linear regressions differed significantly from zero was obtained using the t-statistic for the slope ."
]
] | [
"Myelination speeds conduction of the nerve impulse , enhancing cognitive power .",
"Changes of white matter structure contribute to learning , and are often assumed to reflect an altered number of myelin wraps .",
"We now show that , in rat optic nerve and cerebral cortical axons , the node of Ranvier length varies over a 4 . 4-fold and 8 . 7-fold range respectively and that variation of the node length is much less along axons than between axons .",
"Modelling predicts that these node length differences will alter conduction speed by ~20% , similar to the changes produced by altering the number of myelin wraps or the internode length .",
"For a given change of conduction speed , the membrane area change needed at the node is >270-fold less than that needed in the myelin sheath .",
"Thus , axon-specific adjustment of node of Ranvier length is potentially an energy-efficient and rapid mechanism for tuning the arrival time of information in the CNS ."
] | [
"Information is transmitted around the nervous system as electrical signals passing along nerve cells .",
"A fatty substance called myelin , which is wrapped around the nerve cells , increases the speed with which the signals travel along the nerve cells .",
"This allows us to think and move faster than we would otherwise be able to do .",
"The electrical signals start at small “nodes” between areas of myelin wrapping .",
"Originally it was thought that we learn things mainly as a result of changes in the strength of connections between nerve cells , but recently it has been proposed that changes in myelin wrapping could also contribute to learning .",
"Arancibia-Cárcamo , Ford , Cossell et al . investigated how much node structure varies in rat nerve cells , and whether differences in the length of nodes can fine-tune the activity of the nervous system .",
"The experiments show that rat nerve cells do indeed have nodes with a range of different lengths .",
"Calculations show that this could result in electrical signals moving at different speeds through different nerve cells .",
"These findings raise the possibility that nerve cells actively alter the length of their nodes in order to alter their signal speed .",
"The next step is to try to show experimentally that this happens during learning in animals ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease"
] | Heterogeneity in surface sensing suggests a division of labor in Pseudomonas aeruginosa populations | elife-45084-v3 | [
[
"Pseudomonas aeruginosa is an opportunistic pathogen that engages in a range of surface-associated behaviors and is a model bacterium for studies of surface-associated communities called biofilms .",
"Biofilms are dense aggregates of cells producing extracellular matrix components that hold the community together .",
"The biofilm mode of growth is beneficial for bacteria in that it allows cells to maintain close proximity to nutrients , promotes exchange of genetic material , and confers cells protection from a variety of chemical and environmental stresses ( e . g . nutrient limitation , desiccation , and shear forces ) , as well as engulfment by protozoa in the environment or by phagocytes in a host ( Davey and O'toole , 2000 ) .",
"Collectively , these advantages make biofilm formation integral to prokaryotic life .",
"The secondary messenger signaling molecule cylic diguanylate monophosphate ( c-di-GMP ) controls the transition between the planktonic to the biofilm mode of growth .",
"In many bacterial species , including P . aeruginosa , elevated c-di-GMP results in repression of flagellar motility genes , while promoting expression of genes involved in producing a biofilm matrix ( Römling et al . , 2013 ) .",
"The P . aeruginosa biofilm matrix is composed of a combination of polysaccharides ( including Pel and Psl ) , proteins ( including the adhesin CdrA ) , and extracellular DNA ( Ma et al . , 2009 ) .",
"Biofilm matrix production is an energetically costly process that is regulated at multiple levels ( Wei and Ma , 2013 ) .",
"The cdrA , pel and psl genes are all transcriptionally induced under conditions of high c-di-GMP ( Starkey et al . , 2009 ) .",
"For many species , the initial step in biofilm formation involves adherence of free swimming planktonic cells to a surface and the initiation of surface sensing .",
"P . aeruginosa has at least two distinct surface sensing systems , the Wsp and the Pil-Chp systems , that when activated , lead to biofilm formation .",
"The Wsp system senses an unknown surface-related signal ( recently proposed to be membrane perturbation [Chen et al . , 2014] ) through WspA , a membrane-bound protein homologous to methyl-accepting chemotaxis proteins ( MCPs ) .",
"Activation of this system stimulates phosphorylation of the diguanylate cyclase WspR , which leads to the formation of aggregates of phosphorylated WspR ( WspR-P ) in the form of visible subcellular clusters .",
"This aggregation of WspR-P potentiates its activity , increasing c-di-GMP synthesis ( Huangyutitham et al . , 2013 ) .",
"In comparison , the Pil-Chp chemosensory-like system initiates a hierarchical cascade of second messenger signaling in response to a surface ( Luo et al . , 2015 ) .",
"First , an increase in cellular cAMP levels occurs through activation of the adenylate cyclase CyaB by the Pil-Chp complex .",
"This increases expression of genes involved in type IV pilus biogenesis , including PilY1 .",
"PilY1 is associated with the type IV pilus and harbors a Von Willebrand motif , which is involved in mechanosensing in eukaryotic systems ( Kuchma et al . , 2010 ) .",
"Thus , it has been proposed that this protein may be involved in the mechanosensing of surfaces ( Persat et al . , 2015 ) .",
"The output of this second signal is through the diguanylate cyclase , SadC , resulting in an increase in cellular c-di-GMP levels .",
"Unlike the Wsp system , which localizes laterally along the cell ( O'Connor et al . , 2012 ) , PilY1 is required to be associated with polarly-localized type IV pili in order to stimulate c-di-GMP production ( Luo et al . , 2015; Kuchma et al . , 2010 ) , suggesting that P . aeruginosa deploys both polar and laterally localized systems to promote c-di-GMP synthesis in response to a surface .",
"Bacteria in biofilms have long been appreciated to exhibit phenotypic heterogeneity due to chemical variation within the biofilm itself , including gradients of oxygen ( Wessel et al . , 2014 ) , nutrients ( Schreiber et al . , 2016 ) , and pH ( Vroom et al . , 1999 ) .",
"These environmental conditions are sensed by individual bacterial cells , leading to differential gene expression and metabolic activities even within a genetically homogeneous population ( Stewart and Franklin , 2008 ) .",
"Specifically , the term ‘division of labor’ refers to cases where genetic or phenotypic heterogeneity results in subpopulations of cells cooperating to perform distinct tasks that provide an overall fitness benefit to the population ( West and Cooper , 2016 ) .",
"Through task allocation , subpopulations of cells can engage in behaviors that are impossible to perform simulataneously ( e . g . bet-hedging strategies between biofilm and planktonic cells [Lowery et al . , 2017] ) , energetically costly to switch between ( e . g . task-switching [Goldsby et al . , 2012] ) , or are metabolically incompatible ( e . g . photosynthesis and nitrogen fixation in cyanobacteria ( [Rossetti et al . , 2010] ) .",
"In particular , there is a rich body of literature demonstrating that genetic ( Kim et al . , 2016; Dragoš et al . , 2018; Klausen et al . , 2003 ) and phenotypic variation ( Klauck et al . , 2018; Serra et al . , 2015; Haagensen et al . , 2007 ) in surface motility and polysaccharide production among individual bacterial cells within a biofilm can represent a division of labor that is required to achieve the architecture and structural integrity of the biofilm matrix ( van Gestel et al . , 2015 ) .",
"Beyond heterogeneity as a result of variation in environmental signals , recent single-cell analyses have revealed that c-di-GMP signaling can drive phenotypic heterogeneity among populations of single cells exposed to the same environmental inputs .",
"Planktonic P . aeruginosa have been shown to achieve heterogeneity among very low levels of c-di-GMP through assymetrical partitioning of a diguanylate cyclase during cell division , leading to diverse swimming molitity behaviors ( Kulasekara et al . , 2013 ) .",
"More recently , this same assymetrical cell division mechanism was shown to generate two populations of P . aeruginosa , one piliated and one flagellated , that are each required for efficient tissue colonization ( Laventie et al . , 2019 ) .",
"Together , these studies support a role for c-di-GMP heterogeneity in generating diverse bacterial behaviors during both biofilm and planktonic growth .",
"Here , we examined the dynamics of c-di-GMP production and bacterial surface motility at the single-cell level during early stages of biofilm formation .",
"We used a plasmid-based , transcriptional reporter of intracellular c-di-GMP to follow the downstream fate of cells producing varying levels of c-di-GMP in response to surface attachment .",
"Within a clonal population of P . aeruginosa , we found that levels of c-di-GMP vary among individual cells as they sense a surface , leading to a division of labor between two energetically costly behaviors associated with early biofilm formation: surface exploration and polysaccharide production ."
],
[
"We initially compared levels of c-di-GMP between P . aeruginosa PAO1 cells growing attached to a silicone surface and subjected to constant flow for 4 hr to those grown planktonically for 4 hr .",
"As expected , we observed that PAO1 cellular c-di-GMP levels are 4 . 4-fold higher ( ±0 . 78 SD , N = 3 , p≤0 . 05 ) after 4 hr of growth attached to a surface compared to planktonic growth ( Figure 1A ) .",
"Because direct measurement of c-di-GMP by LC-MS/MS is limited by our ability to generate enough biomass at earlier time points , we used qRT-PCR to monitor pel transcript levels as a readout of c-di-GMP .",
"We found that after just 30 min of surface attachment , pelA transcript levels had increased almost 10-fold compared to planktonically grown cells ( Figure 1—figure supplement 1 ) .",
"This is consistent with previously published literature showing that transcription of the pel operon is directly and positively controlled by high cellular levels of c-di-GMP ( Hickman and Harwood , 2008; Baraquet et al . , 2012 ) .",
"Next , we sought to visualize early c-di-GMP signaling events at the single cell level .",
"To this end we used a plasmid-based , c-di-GMP responsive transcriptional reporter , pPcdrA::gfpASV ( Rybtke et al . , 2012 ) in two commonly-studied P . aeruginosa strains , PAO1 and PA14 .",
"Planktonic cells ( a condition where the reporter is inactive due to low c-di-GMP levels ) were used to inoculate flow cell chambers .",
"We imaged individual cells of each reporter strain hourly for up to 6 hr after surface attachment ( Figure 1B and Figure 1—figure supplement 2 ) .",
"As expected , we saw minimal GFP fluorescence at the 0 hr time point ( right after surface attachment ) .",
"However , by 1 hr , the reporter was activated in a subset of surface attached cells , as defined by background subtracted GFP fluorescence ≥321 fluorescence units ( referred to as reporter ‘on’ subpopulations ) .",
"Interestingly , between 4 and 6 hr post inoculation , we consistently observed that the c-di-GMP reporter was only active in a subset of cells in both strains ( Figure 1C ) .",
"In PA14 , the reporter was activated in 10% of the population over 6 hr , whereas PAO1 displayed greater reporter activity , with 40–60% of the cells displaying reporter activity through 12 hr ( Figure 1B ) .",
"An analysis of single cell fluorescence supported these observations .",
"We plotted each cell’s individual fluorescence values over time and observed populations of cells with low and high c-di-GMP reporter activity at each timepoint after the 0 hr ( Figure 1—figure supplement 3 ) .",
"We also observed long tails of high GFP fluorescence , particularly at 2 and 4 hr .",
"This suggests that the reporter ‘on’ subpopulation represents cells with a range of high c-di-GMP levels .",
"This wide range of high reporter activity cells between 2 and 4 hr could be indicative of an early ‘spike’ in c-di-GMP production that levels off over time ( Figure 1—figure supplement 3 ) .",
"We next examined the change in distribution of fluorescence intensity over time for single cells by bootstrap sampling of the single cell fluorescence intensity values ( Figure 1—figure supplement 4 ) .",
"The purpose of this bootstrapping analysis is to examine whether the distributions of fluorescence intensity differ at each time point .",
"We found that the median fluorescence intensity was significantly different at every time point except between 4 and 6 hr , and between 8 , 10 , and 12 hr ( Figure 1—source data 2 ) .",
"Together , these single cell analyses support a model in which c-di-GMP signaling is initiated rapidly upon surface attachment in the first 4–6 hr for a subpopulation of attached cells , while the rate of c-di-GMP increase tends to level off at later timepoints .",
"We confirmed the microscopy results from comparing PAO1 and PA14 reporter fluorescence using flow cytometry to assess the proportion of attached cells that were fluorescent ( Figure 1—figure supplement 5D , E ) .",
"To be sure that the promoter of cdrA is representative of c-di-GMP-regulated gene expression , we replaced PcdrA with the promoter of siaA , a gene that is also highly expressed under conditions of elevated c-di-GMP ( Starkey et al . , 2009; Baraquet and Harwood , 2016 ) .",
"We found that pPsiaA::gfpASV reporter activity resembled that of pPcdrA::gfpASV in response to a surface ( Figure 1—figure supplement 6 ) .",
"Thus , reporter activity is indeed linked to cellular levels of c-di-GMP .",
"We then wanted to confirm that subpopulations of surface-attached P . aeruginosa cells with high and low c-di-GMP reporter activity are truly physiologically distinct from one another .",
"We used TRITC-labeled lectins to stain for two c-di-GMP-induced exopolysaccharides , Psl and Pel ( Zhao et al . , 2013; Jennings et al . , 2015 ) , the presence of which is indicative of biofilm formation by PAO1 and PA14 , respectively .",
"After 4 hr of attachment to glass , we observed an enrichment of TRITC-conjugated lectin staining in the population of cells with high c-di-GMP reporter activity ( Figure 1D and Figure 1—figure supplement 7 ) , demonstrating that the subpopulation of cells with high c-di-GMP is producing more exopolysaccharide than their low c-di-GMP counterparts .",
"This correlation was weaker for Psl than Pel , probably due to the fact planktonic populations can make low levels of Psl ( though not the case for Pel ) ( Wei and Ma , 2013 ) .",
"As a complementary approach , we separated 4 hr surface-grown cells of the reporter strain into reporter ‘on’ and ‘off’ subpopulations using flow-assisted cell sorting ( FACS; Figure 1—figure supplements 8 and 9 ) .",
"We then applied qRT-PCR to compare Pel and Psl transcript levels in these two populations .",
"Both the pel and psl operon transcripts were elevated in the reporter ‘on’ subpopulation , relative to the reporter ‘off’ subpopulation ( Figure 1E ) .",
"These data support that , with respect to c-di-GMP signaling , there are at least two distinct subpopulations that arise shortly after surface attachment .",
"We next evaluated the relative contributions of the Wsp and Pil-Chp surface sensing systems to surface-induced c-di-GMP production .",
"Strains with mutations in the Pil-Chp chemosensory system were not significantly defective in surface sensing activity .",
"Deletion of the diguanylate cyclase activated through the Pil-Chp system ( PAO1 ΔsadC ) and the gene encoding the putative sensor PilY1 ( PAO1 ΔpilY1 ) did not significantly influence reporter activity in response to a surface ( Figure 2—figure supplement 1A , B ) .",
"Whereas both the SadC and PilY1 mutants displayed wild type levels of reporter activity , a mutant lacking the main Type IV pilus filament protein ( PAO1 ΔpilA ) did show a statistically significant defect in reporter activity by 6 hr ( Figure 2—figure supplement 1B; p<0 . 05 by T-test ) .",
"We then mutated the c-di-GMP cyclase gene , wspR , to inactivate the Wsp system .",
"In addition , we deleted the gene encoding the methylesterase wspF , which locks the system into the active state , regardless of whether cells are surface-associated .",
"We found that the PAO1 ΔwspR strain exhibited extremely low levels of reporter activity during the first 6 hr after surface attachment ( Figure 2A and Figure 2—figure supplement 2 ) .",
"Complementation of PAO1 ΔwspR restored wild type levels of activity at all time points ( Figure 2—figure supplement 3 ) .",
"As expected , PAO1 ΔwspF had a high proportion of reporter active cells ( Figure 2A ) .",
"We repeated these experiments in the lab strain PA14 and saw a similar trend for Wsp mutants ( Figure 2—figure supplement 4 ) .",
"Kulesekara et al . ( Kulasekara et al . , 2013 ) used a FRET-based c-di-GMP reporter to show that planktonic P . aeruginosa has heterogeneous , albeit very low , concentrations of c-di-GMP which it achieves through assymetrical partitioning of a phosphodiesterase ( PA5017 , also called Pch or DipA ) to the flagellated cell pole during cell division .",
"However , we found no evidence of heterogeneity in c-di-GMP within the planktonic cell inoculum contributing to our observations .",
"We ruled out the phosphodiesterase ( PA5017 ) as responsible for the heterogeneity we see during surface sensing by examining a deletion mutant of PA5017 and showing that this strain still exhibited heterogeneity during surface sensing ( Figure 2—figure supplement 5 ) .",
"Since the Pil-Chp surface sensing apparatus is polarly localized and the Wsp system is localized laterally along the length of the cell body , we examined whether reporter activity correlated with polar versus lateral attachment to the surface .",
"We found that reporter activity was very low in polarly attached cells , while cells attached along the entire length of the cell body displayed a higher proportion of reporter-activated cells at that same time point ( Figure 2B ) .",
"( Our analysis does not account for the time period each cell spends in either the polarly or non-polarly attached states . )",
"This finding is consistent with the localization of the Wsp system and its role for early c-di-GMP signaling during surface sensing .",
"The specific activity of purified WspR increases as a function of WspR concentration when the protein is treated with beryllium fluoride to mimic phosphorylation , supporting the idea that formation of subcellular clusters of WspR-P potentiates its diguanylate cyclase activity and leads to elevated c-di-GMP ( Huangyutitham et al . , 2013 ) .",
"Fewer than 1% of wild-type cells grown in broth have a visible WspR-YFP cluster .",
"However , after a short period of growth on an agar surface , WspR-YFP clusters were visible in 30–40% of wild type PAO1 cells , and this is dependent on sensing by the membrane-bound protein WspA , which is laterally distributed in cells ( Kuchma et al . , 2010 ) .",
"To directly link WspR cluster formation with diguanylate cyclase activity at the cellular level and with surface sensing , we constructed a version of the c-di-GMP reporter that expresses mTFP1 instead of GFP ( pPcdrA::mTFP1 ) to avoid the issue of spectral overlap with WspR-YFP .",
"We monitored reporter activity in two point mutants of WspR ( L170D and E253A ) that are driven by an inducible promoter , translationally fused to eYFP , and have been previously shown to form large subcellular WspR clusters in a higher percentage of cells than wild-type WspR .",
"The WspR[L170D] protein is highly active for c-di-GMP production , and it forms subcellular clusters in about 75% of agar surface-grown cells .",
"A WspR[E253A] point mutation abolishes diguanylate cyclase activity , but this protein still forms clusters in about 70% of surface-grown cells ( Huangyutitham et al . , 2013 ) .",
"As expected , in the presence of inducer , we observed a large increase in c-di-GMP reporter activity in WspR[L170D] , but not WspR[E253A] ( Figure 3A , B ) .",
"We then asked whether the heterogeneity in reporter activity in response to surface attachment correlates with WspR clustering in the WspR[L170D] strain .",
"We found that pPcdrA::mTFP1 activity was significantly higher in cells with at least one subcellular WspR-eYFP focus in the WspR[L170D] strain compared to cells without a WspR-eYFP focus ( Figure 3C and Figure 3—figure supplement 1 ) .",
"These data indicate that the heterogeneity observed in c-di-GMP signaling after surface attachment is due to the heterogeneity in the activity of the Wsp system , as reflected by subcellular clustering of active WspR-P .",
"For phenotypic heterogeneity to represent a division of labor , it must result in a fitness benefit to the population .",
"Therefore , we next asked whether the observed heterogeneity in c-di-GMP signaling in response to a surface has a meaningful influence on biofilm formation .",
"This was particularly important since previously published results indicated that a wspR mutation had only a small impact on biofilm production ( Kulasakara et al . , 2006 ) .",
"However , these studies assessed biofilm formation at later stages of biofilm growth that were well beyond initial surface attachment .",
"Therefore , we chose to compare a wspR mutant to wild type at earlier biofilm stages .",
"We performed in vitro biofilm assays and observed that a PAO1 ΔwspR mutant was defective for biofilm formation relative to wild type PAO1 at 2 , 4 , and 6 hr post-attachment ( Figure 4A ) .",
"However , at later stages of development ( 24 hr ) , the wspR mutant caught up and produced similar amounts of biofilm biomass relative to wild type .",
"Complementation of the ΔwspR strain in trans restored wild type levels of biofilm formation at all time points ( Figure 4A ) .",
"These data suggest that the Wsp system rapidly responds to surface contact to generate elevated levels of c-di-GMP in a subpopulation of cells , which accelerates biofilm production .",
"Given the importance of c-di-GMP signaling in biofilm production , the fact that the ΔwspR strain can ultimately attain wild-type levels of biofilm biomass suggests that one of the many other known c-di-GMP cyclases present in P . aeruginosa may ultimately compensate for c-di-GMP production in the absence of WspR .",
"We hypothesized that heterogeneity in c-di-GMP signaling dictated by the Wsp complex could impact the surface behavior of the two observed subpopulations .",
"We predicted that the subpopulation of cells with high c-di-GMP after surface attachment would produce biofilm matrix exopolysaccharides and contribute to initial microcolony formation , while the cells with low c-di-GMP would exhibit increased surface motility and detachment , which is known to be inhibited by exopolysaccharide production .",
"To test this hypothesis , we tracked both reporter activity and surface behavior for cells within a single field of view for 40 hr .",
"From our single-cell tracking data , we generated family trees across at least four generations of cells , using a previously described technique ( Lee et al . , 2018 ) .",
"We tracked the time-averaged PcdrA::gfpASV reporter activity ( Ic-di-GMP ) , surface motility behavior ( Fmotile , defined as the fraction of time that cells are motile ) , and detachment behavior ( tree asymmetry λ , where λ = 0 represents both daughter cells remaining attached to the surface and λ = 1 represents when one daughter cell detaches or travels outside the field of view ) .",
"In P . aeruginosa , surface exploration is mainly accomplished by type IV pili-mediated twitching motility , and does not appear to be influenced by levels of intracellular c-di-GMP when analyzing single cells ( Ribbe et al . , 2017 ) .",
"Interestingly , when analyzing correlations between c-di-GMP and motility for entire lineages in family trees , we found clear inverse correlations between Ic-di-GMP and Fmotile ( Figure 4B , ρ = −0 . 53 , p=0 . 0012 ) and between Ic-di-GMP and λ ( Figure 4C , ρ = −0 . 45 , p=0 . 0068 ) , suggesting that c-di-GMP levels are strongly inversely correlated with surface motility behavior and detachment behavior over multiple generation of cells .",
"Analyzing these correlations across multiple cell divisions is important because it tells us when and how bacteria respond to c-di-GMP signaling events in terms of their surface motility and detachment .",
"When looking at correlations between c-di-GMP and surface motility , the time between seeing a signaling event ( i . e . , rise or drop in reporter fluorescence intensity ) and seeing a response ( i . e . , change in motility ) can span a broad range ( from a few minutes to well over a cell division time ) .",
"Using a cell’s entire lineage history ( i . e . , tracking daughter cells across cell divisions ) will capture all of these events , whereas using single cell history ( i . e . , within one generation ) will only capture a portion of them .",
"For example , this lineage-level tracking of the influence of signaling events on bacterial behavior was recently shown for correlations between cyclic AMP signaling and P . aeruginosa surface motility ( Lee et al . , 2018 ) .",
"In this study , Lee et al . found that correlations between cell motility and signaling activity were stronger when they took into account lineage history , rather than using single cell history .",
"However , if there are enough instances where the time between a signaling event and a cell’s corresponding behavioral response are within a single generation , then correlations can still be found when using single cell history .",
"For wild type PAO1 ( WT ) , we observe weaker correlations when looking at individual cells in these lineages .",
"Ic-di-GMP vs Fmotile for single cells had a Spearman correlation value ρ = −0 . 40 ( p<0 . 0001 ) , which is smaller than the lineage-level correlation value , suggesting that lineage-level correlations are stronger .",
"To illustrate these correlations , we chose three representative families , with either high , intermediate , or low Ic-di-GMP and plotted their family trees ( Figure 4D ) and spatial trajectories ( Figure 4E ) .",
"Families with the highest Ic-di-GMP had the lowest Fmotile and λ ( Family 1 , Figure 4B–E ) .",
"In these families , daughter cells remained attached following cell division , exhibited continuously elevated c-di-GMP , did not move appreciable distances on the surface , and ultimately produced small microcolonies .",
"In contrast , families of cells with low Ic-di-GMP had the highest Fmotile and λ .",
"For these families , daughter cells frequently detached or traveled outside the field of view , had lower c-di-GMP levels , traveled larger distances on the surface , and ultimately did not form microcolonies ( Family 3 , Figure 4B–E ) .",
"Tracking and lineage analyses were also performed on a PAO1 mutant with the Wsp system inactivated ( PAO1 ΔwspR ) .",
"We observed that the range of tree asymmetry values is like that of WT and that the ΔwspR mutant eventually reaches WT c-di-GMP levels despite initially being lower , which is consistent with the observation that the mutant eventually forms WT-like biofilms .",
"What our analysis revealed about the ΔwspR strain was quite unexpected: The WspR mutant has overall lower surface motility and , importantly , lacks correlations between c-di-GMP , surface motility , and detachment behavior ( both at the level of lineages and at the level of single cells ) .",
"For single cell surface motility , 23% of ΔwspR mutant cells ( 48 of 210 cells ) have non-zero Fmotile ( the metric for surface motility ) compared to 44% of WT cells ( 251 of 565 cells; Figure 4—figure supplement 1 ) .",
"The overall lowered surface motility and the lack of correlations between c-di-GMP and surface motility in the ΔwspR mutant suggest that the Wsp system is involved in translating the heterogeneous c-di-GMP signaling events into the corresponding motility-related responses for cells and their progeny .",
"Therefore , the Wsp system’s multi-generational temporal propagation of surface sensing signaling and behavior is important for the generation of heterogeneous populations of surface motile and immotile cells during early biofilm formation .",
"One important question is what happens to early biofilm development if we were to effectively remove heterogeneity in c-di-GMP output rooted in the WspR surface sensing system .",
"To address this question , we used a strain in which c-di-GMP production could be easily controlled using an optogenetic system .",
"The precise control of c-di-GMP expression in individual cells was made possible by the use of a chimeric protein that fused a diguanylate cyclase domain to a bacteriophytochrome domain .",
"Flow chambers were seeded with the optogenetic strain encoding a heme oxygenase ( bphO ) and light-responsive diguanylate cyclase ( bphS ) ( Ryu and Gomelsky , 2014 ) .",
"We initially characterized the optogentic strain and verified the reporter activity increased with exposure of the optogentic strain to red light ( Figure 5—figure supplement 1 ) and that the laser light did not impact growth or motility ( data not shown ) .",
"Following validation of the strain , cells inoculated on a glass surface were tracked and continuously stimulated with red-light over ~8 hr using adaptive tracking illumination microscopy ( ATIM ) , which allows for precise stimulation of the initial attached cells and their offspring and ensures sustained intracellular c-di-GMP production for a fixed number of surface cell generations ( Figure 5—figure supplement 2 ) .",
"Cellular lineages ( a cell and all of its offspring ) and c-di-GMP reporter activity were continually monitored for at least 12 hr .",
"Families that were not stimulated with light demonstrated a heterogeneous surface response ( Video 1 and Figure 5B , D ) similar to that of Families 1–3 in Figure 4B–E .",
"Some lineages were dominated by surface explorers , whereas others were seen to commit to microcolony formation .",
"In contrast , in families stimulated with light for more than one generation , the resulting c-di-GMP production artificially forced lineages to have low surface motility and commit to microcolony production ( Video 1 and Figure 5A , C ) similar to that of Family one in Figure 4B–E .",
"Families stimulated with light in this manner had higher Ic-di-GMP and lower λ values than those that were not stimulated ( Figure 5—figure supplement 3 ) .",
"We also found that optogenetic control of c-di-GMP results in phenotypes that are consistent with the wild-type behavior presented in Figure 4 , with illuminated cells ( high c-di-GMP ) displaying the least motility and control ( non-illuminated ) cells displaying comparatively greater surface motility ( Figure 5—figure supplement 3 ) .",
"Interestingly , families stimulated with light for one generation or less are not significantly different from un-illuminated controls ( data not shown ) .",
"Our data show that the generation of c-di-GMP can deterministically lead to the creation of an entire lineage of sessile cells with post-division surface persistence , low motility , and initiation of microcolony formation .",
"Altogether , these results show that c-di-GMP levels , surface motility , and detachment are inversely correlated at the lineage level , and that the time scale for this occurs over multiple generations ."
],
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"Collectively , our data show that heterogeneity in cellular levels of c-di-GMP generated by the Wsp system in response to surface sensing , leads to two physiologically distinct subpopulations that each contribute to surface colonization .",
"Phenotypic heterogeneity of single cells is a common phenomenon in bacteria that can be beneficial at the population level by allowing a single genotype to survive sudden environmental changes .",
"Sources of phenotypic heterogeneity among genetically homogeneous populations include bistability ( Dubnau and Losick , 2006 ) and stochasticity ( Elowitz et al . , 2002 ) of gene expression , unequal partitioning of proteins during cell division due to low abundance ( Elowitz et al . , 2002 ) , epigenetic modifications resulting in phase variation ( Casadesús and Low , 2006 ) , or through asymmetrical cell division ( Kulasekara et al . , 2013; Laventie et al . , 2019 ) .",
"In this study , we show that the Wsp system generates heterogeneity in c-di-GMP signaling , and it is never fully activated in 100% of wild-type , surface-attached cells .",
"One possible outcome of phenotypic heterogeneity is a division of labor between costly behaviors that support the growth and survival of the population ( Ackermann , 2015 ) .",
"We found that abolishing c-di-GMP heterogeneity through inactivation of WspR leads to defects in early biofilm formation .",
"This suggests that the subpopulations of high c-di-GMP , polysaccharide producers and low c-di-GMP , surface explorers are both required for efficient biofilm formation and that they represent a division of labor during early biofilm formation .",
"The data show that Wsp-generated c-di-GMP heterogeneity results in phenotypic changes for entire family lineages of cells .",
"It is interesting that correlations between c-di-GMP , surface motility , and surface detachment probability are stronger when considered for an entire lineage in a bacterial family tree , but weaker when considered at the individual cell level .",
"We think that this difference in the strength of correlations between c-di-GMP and bacterial behavior at the lineage versus single cell level is biologically meaningful and likely reflects the complex relationship between c-di-GMP signaling and type IV pili-mediated motility ( Ribbe et al . , 2017; Jain et al . , 2012; Jain et al . , 2017 ) .",
"The time between initiation of a signaling event ( i . e . , increased intracellular c-di-GMP ) and the associated response ( i . e . , attenuation of motility or initiation of polysaccharide production ) can span a large range , depending on the behavior .",
"When examining polysaccharide production , signal propagation appears to be quick , with high c-di-GMP cells initiating exopolysaccharide production within minutes of P . aeruginosa encountering a surface .",
"However , when examining a different bacterial behavior—surface motility—we found the strongest correlation between c-di-GMP signaling and cellular behavior at the lineage level .",
"For many cells , there was a lag between when c-di-GMP first increased and surface motility decreased .",
"While a mother cell may still be surface motile upon initiating c-di-GMP signaling , we found that following cell division , daughter cells with high c-di-GMP eventually became immotile .",
"Thus , whereas an increase in c-di-GMP relatively quickly results in increased biofilm matrix production , this same increase in c-di-GMP likely indirectly influences surface motility .",
"Supporting this , P . aeruginosa is known to produce Psl polysaccharide while engaging in type IV pili mediated motility across a surface , leaving behind a trail of Psl ( Zhao et al . , 2013 ) .",
"Finally , this apparent multigenerational influence of c-di-GMP signaling on bacterial behavior resembles the recently observed multigenerational memory of cAMP signaling in P . aeruginosa ( Lee et al . , 2018 ) .",
"Additional work is needed to determine whether surface-naïve daughter cells have any ‘memory’ of surface attachment by a mother cell , through the maintenance of elevated c-di-GMP across one or more cell divisions .",
"Another future direction of this work is to examine whether other bacterial signaling modalities , including other nucleotide or non-nucleotide signaling systems ( e . g . ppGpp or quorum sensing ) , may exhibit similar multigenerational features .",
"We observed that the Pil-Chp surface sensing system did not play a role in early c-di-GMP signaling in our experimental system .",
"There are numerous potential explanations for this .",
"For example , it is possible that the Pil-Chp system is the dominant surface sensing mechanism under different environmental conditions than the ones we used for this study .",
"Alternatively , the Pil-Chp system might not contribute to general cytoplasmic pools of c-di-GMP ( for which the reporter is sensitive ) and instead participates in specific localized c-di-GMP signaling events .",
"In support of this notion , inactivation of individual diguanylate cyclases is well known to lead to distinct changes in c-di-GMP-regulated behaviors ( Kulasakara et al . , 2006; Merritt et al . , 2010 ) .",
"It is interesting to note that whereas the other Pil-Chp inactivation mutants tested did not have a phenotype , the pilA deletion mutant strain displayed a slight defect in surface sensing in our study , although why is currently unknown .",
"If we overwhelm WspR-generated c-di-GMP heterogeneity by using optogentically-induced sustained c-di-GMP production , we find that phenotypic heterogeneity is lost , and that illuminated cells deterministically become sessile and form microcolonies .",
"Interestingly , our optogenetic experiments show that sustained c-di-GMP production for more than one generation is required before commitment to the sessile lifestyle .",
"This observation is consistent with the fact that we see stronger correlations between c-di-GMP levels and motility behavior at the lineage level compared to the individual cell level .",
"Moreover , since the Wsp surface sensing system generates heterogeneous c-di-GMP levels , this requirement of sustained c-di-GMP production for more than one generation is inherently difficult for wild-type cells to meet , and virtually guarantees the simultaneous existence of motile and sessile subpopulations .",
"The heterogeneity we observed in Wsp signaling shares many similarities with phenotypic heterogeneity generated from other c-di-GMP signaling ( Petersen et al . , 2019 ) and quorum sensing systems ( Grote et al . , 2015; Ramalho et al . , 2016 ) .",
"Nucleotide second messenger and quorum sensing ( QS ) signaling systems are traditionally thought to coordinate cellular behavior in response to information regarding the cell’s environment .",
"However , rather than functioning to initiate a completely homogeneous response at the population level to environmental conditions , a growing body of literature suggests that a common theme of these signaling systems is that they introduce some level of behavioral heterogeneity ( Grote et al . , 2015 ) .",
"For example , QS-induced phenotypic heterogeneity in Vibrio harveyi is attributable to variability in the phosphorylation state of LuxO and influences bioluminencence and biofilm formation ( Anetzberger et al . , 2009 ) .",
"In P . aeruginosa and Caulobacter crescentus , heterogeneity in the very low levels of c-di-GMP present during planktonic growth is achieved through asymmetrical cell division and influences swimming motility ( Kulasekara et al . , 2013 ) .",
"Thus , P . aeruginosa appears to have at least two distinct mechanisms of generating c-di-GMP heterogeneity , which it employs during different modes of growth .",
"In the case of the Wsp system , this phenotypic heterogeneity , which has been ‘hardwired’ into the structure of the Wsp surface sensing network , allows for a division of the labor during early biofilm formation , with one subpopulation committing to initiating the protective biofilm lifestyle , while the other subpopulation is free to explore the surface and potentially colonize distant , perhaps more favorable , locations ."
],
[
"The strains , plasmids , and primers used in this study are listed in Table 1 .",
"Escherichia coli and P . aeruginosa strains were routinely grown in Luria–Bertani ( LB ) medium and on LB agar at 37°C .",
"For the flow cell experiments , P . aeruginosa was grown in either LB or FAB minimal medium supplemented with 10 mM or 0 . 6 mM glutamate at room temperature ( Zhao et al . , 2013 ) .",
"For flow cytometry experiments , P . aeruginosa was grown in either LB medium or in Jensen’s defined medium with glucose as the carbon source ( a growth medium in which Pel is more abundantly produced than in LB ) ( Jennings et al . , 2015 ) .",
"For the tube biofilm and c-di-GMP measurements , P . aeruginosa strains were grown in Vogel-Bonner Minimal Medium ( VBMM; Vogel and Bonner , 1956 ) .",
"Antibiotics were supplied where necessary at the following concentrations: for E . coli , 100 μg/mL ampicillin , 10 μg/mL gentamicin , and 10 or 60 μg/mL tetracycline; for P . aeruginosa , 300 μg/mL carbenicillin , 100 μg/mL gentamicin , and 100 μg/mL tetracycline .",
"PcdrA::gfpASV reporter and vector control plasmids were selected with 100 µg/mL gentamicin for P . aeruginosa strains and 10 µg/mL gentamicin for E . coli .",
"PAO1 ΔpilY1 was constructed using two-step allelic exchange following conjugation of wild type PAO1 with E . coli S17 . 1 harboring pENTRPEX18Gm::ΔpilY1 ( a gift from Joe Harrison ) as previously described ( Hmelo et al . , 2015 ) .",
"PAO1 ΔpilY1 was identified by colony PCR using primers PAO1pilY1-SEQ-F and PAO1pilY1-SEQ-R .",
"PAO1 ΔdipA was constructed similarly by conjugation of wild type PAO1 with E . coli S17 . 1 harboring pENTRPEX18Gm::ΔdipA ( a gift from Joe Harrison ) .",
"PAO1 ΔdipA was identified by colony PCR using primers PAO1dipA-SEQ-F and PAO1dipA-SEQ-R .",
"PA14 ΔwspR and ΔwspF deletion mutants were confirmed by PCR using primers PA14wspR-SEQ-F and PA14wspR-SEQ-R or PA14wspF-SEQ-F and PA14wspF-SEQ-R , respectively .",
"To create MPAO1 attTn7::P ( A1/04/03 ) ::GFPmut , the miniTn7 from pBT270 was integrated into the chromosome of P . aeruginosa PAO1 with the helper plasmid pTNS2 , as previously described ( Choi and Schweizer , 2006 ) .",
"pBT270 was created by introducing the constitutive A1/04/03 promoter ( Lanzer and Bujard , 1988 ) and removing the trc promoter from pBT223 using the QuikChange Lightning Kit ( Agilent Technologies ) and the oligonucleotides OBT314 and OBT315 .",
"pBT223 was constructed via recombineering of pBT200 , pUC18-miniTn7T2-Gm-GW , and pBT212 using Multisite Gateway technology ( Invitrogen ) .",
"pBT212 was constructed by cloning the gfpmut3 from AKN66 using OBT268 and OBT269 , and recombining the PCR product with pDONR221 P1-P5r .",
"Chromosomal insertion of bphS was achieved using the mini-CTX system and these strains were marked with different fluorescent proteins by mini-Tn7 site-specific transposition essentially as previously described ( Choi and Schweizer , 2006; Hoang et al . , 2000 ) .",
"First , a bphS fragment obtained from the plasmid pIND4 was cloned into the vector mini-CTX2 with the PA1/O4/O3 promoter upstream of the MCS via a two-piece ligation .",
"The constructed plasmid was electroporated into PAO1 and the corresponding recombinant strain was identified by screening on LB agar plates containing 1 mM IPTG and 100 μg/mL tetracycline .",
"Then , the strains were electroporated with a pFLP2 plasmid and distinguished on LB agar plates containing 5% ( w/v ) sucrose for the excision of the resistance marker .",
"The c-di-GMP reporter plasmid and mCherry/EGFP marked bphS mutants were constructed as described above .",
"The c-di-GMP reporter plasmid ( PcdrA::gfpASV ) was electroporated into the mCherry-marked strain harboring bphS to monitor the intracellular c-di-GMP level .",
"To validate the optogenetic reporter strain in Figure 5—figure supplement 1 , strains were grown on LB agar plates at 37°C for 24 hr from frozen stocks .",
"Monoclonal colonies were inoculated and cultured with a minimal medium ( FAB ) at 37°C overnight , adding 1 uM FeCl3 and 30 mM glutamate as the carbon source , until the culture reached an OD600 of approximately 2 . 1 .",
"Then , the bacterial culture was further diluted ( 1:100 ) in fresh FAB medium to OD600 0 . 5 .",
"When required , gentamicin was added to medium at 30 μg/mL .",
"Plates and tubes were wrapped with aluminum foil to achieve a dark condition .",
"Finally , the culture was diluted ( 1:50 ) in fresh FAB medium and 6 μL diluted culture was spotted onto an FAB agarose ( 2% ) pad , with 30 mM glutamate and 1 μM FeCl3 .",
"The agarose pad was pressed on a coverglass before cells were illuminated .",
"Measurement of c-di-GMP in tube biofilm cells was performed as previously described ( Colvin et al . , 2011 ) .",
"Transcriptional analysis of PelA expression in tube biofilms was performed as described in the ‘FACS and qRT-PCR of c-di-GMP reporter cells’ section .",
"Crystal violet assays were performed essentially as previously described to measure biofilm biomass , except using gentle washing after 2–6 hr of static incubation ( Armbruster et al . , 2016 ) .",
"To measure biofilm biomass at 24 hr , the crystal violet assay was performed as previously described without gentle washing ( Colvin et al . , 2012 ) .",
"P . aeruginosa cells harboring the pPcdrA::gfpASV reporter plasmid or a promotorless vector control ( pMH489 ) were grown to mid-log in LB with 100 µg/mL gentamicin ( Gm100 ) from LB Gm100 plates or from overnight broth cultures in FAB +10 mM glutamate .",
"Mid-log cells were back diluted into 1% LB or FAB +0 . 6 mM glutamate and flow chambers were inoculated at a final OD600 0 . 1 and inverted for 10 min to allow cells to attach before induction of flow .",
"Clean media was used to wash non-attached cells by flow at 40 mL per hour for 20 min .",
"Flow was then reduced to a final constant flow rate of 3 mL per hour and bacteria were imaged immediately on a Zeiss LSM 510 scanning confocal laser microscope ( t = 0 hr ) .",
"Flow cells were incubated at a constant flow rate at room temperature and imaged hourly for up to 24 hr .",
"For every strain and time point , 5 fields of view and a minimum of 300 cells were captured using identical microscope settings to image GFP fluorescence across all experiments .",
"Images were analyzed using using Volocity software ( Improvision , Coventry , UK ) .",
"We binned cells by their mean GFP fluorescence intensity per pixel , in incremints of 20 fluorescence units , and determined the cut-off bin that corresponded to cells clearling produced GFP when images were examined by eye .",
"Therefore , cells were counted as pPcdrA::gfpASV reporter ‘on’ if their mean GFP fluorescence intensity per pixel was ≥321 fluorescence units .",
"For all summary figures depicting the percentage of cells with the reporter ‘on’ , data are presented in terms of the percentage of cells with an average GFP fluorescence per pixel of ≥321 fluorescence units ( pPcdrA::gfpASV reporter ‘on’ ) .",
"Microscopy images were artificially colored to display GFP fluorescence as green .",
"A region 259 bp upstream through 21 bp into the coding sequence of siaA was amplified from PAO1 genomic DNA using primers BamH1-Psia-F and SiaA-BamH1-R , then gel purified using a QIAquick gel extraction kit ( Qiagen , Hilden , Germany ) digested with BamH1 , then column purified with a QIAquick PCR purification kit ( Qiagen , Hilden , Germany ) to remove BamH1 .",
"The GFP expression vector pMH487 , which contains the gfpmut3 gene with an RNase III splice site and lacking a promoter ( Borlee et al . , 2010 ) , was digested with BamH1 , treated with Antarctic phosphatase ( New England Biolabs , Ipswich , MA ) , then column purified with a QIAquick PCR purification kit ( Qiagen , Hilden , Germany ) to remove BamH1 .",
"The PsiaA allele was ligated into digested pMH487 , then transformed into E . coli DH5α , purified , and sequenced using primer M13F ( −21 ) ( Genewiz ) .",
"The reporter pPsiaA::gfp was electroporated into P . aeruginosa as previously described and maintained under gentamycin selection at 100 μg/mL .",
"Wild type PAO1 harboring the pPcdrA::gfpASV reporter was grown shaking for 20 hr in FAB media with 6 mM glutamate .",
"The flow cell inoculum was prepared by diluting the culture to a final OD600 of 0 . 01 in FAB with 0 . 6 mM glutamate .",
"The flow cell inoculum was injected into the flow cell ( Department of Systems Biology , Technical University of Denmark ) and allowed to incubate for 10 min at 30°C prior to flushing with media at 30 mL/h for 10 min .",
"Experiments were performed under a flow rate of 3 mL/hour for a total of 40 hr .",
"Images were acquired with an Olympus IX81 microscope equipped with a Zero Drift Correction autofocus system , a 100 × oil objective with a 2 × multipler lens , and an Andor iXon EMCCD camera using Andor IQ software .",
"Bright-field images were recorded every 3 s and GFP fluorescence every 15 min .",
"Acquisition continued for a total recording time of 40 hr , which resulted in approximately 48000 bright-field images , and 160 fluorescence images .",
"Images were analyzed in MATLAB to track bacterial family trees , GFP fluorescence , and surface motility essentially as previously described ( Lee et al . , 2018 ) with the following modifications .",
"Image analysis , family tracking and manual validation , family tree plotting , and tree asymmetry λ calculations were performed as previously described ( Lee et al . , 2018 ) without modification .",
"GFP fluorescence intensities were normalized by calculating the distribution of intensities per cell per frame ( extracted by using the binary image as a mask ) and then setting the minimum and maximum intensities to the 1st and 99th percentiles of this distribution for each dataset .",
"Ic-di-GMP ( relative normalized c-di-GMP reporter intensity ) was calculated by averaging the normalized fluorescence intensities across all members of a family .",
"Fmotile ( fraction of time that cells in a family are motile ) was calculated as follows .",
"For each family , every cell trajectory in the family was divided into time intervals .",
"For each time interval , presence or absence of motility was determined using a combination of metrics , including Mean Squared Displacement ( MSD ) slope , radius of gyration , and visit map .",
"MSD slope quantifies the directionality of movement relative to diffusion .",
"Radius of gyration and visit map are different metrics for quantifying the average distance traveled on the surface .",
"Fmotile was then calculated by the fraction of these time intervals that have motility .",
"This calculation was modified from the ‘TFP activity metric’ previously described ( Lee et al . , 2018 ) .",
"Figure 5—figure supplement 2 shows a schematic of the Adaptive Tracking Illumination Microscopy ( ATIM ) setup .",
"An inverted fluorescent microscope ( Olympus , IX71 ) was modified to build the ATIM .",
"The modification includes: 1 ) a commercial DMD-based LED projector ( Gimi Z3 ) was used to replace the original bright-field light source , in which the original lenses in the projector were removed and three-colored ( RGB ) LEDs were rewired to connect to an external LED driver ( ThorLabs ) controlled by a single chip microcomputer ( Arduino UNO r3 ) ;",
"2 ) the original bright-field condenser was replaced with an air objective ( 40× NA = 0 . 6 , Leica ) ; and",
"3 ) an additional 850 nm LED light ( ThorLabs ) was coupled to the illumination optical path using a dichroic mirror ( Semrock ) for the bright-field illumination .",
"The 850 nm LED bright field light source does not affect optogenetic manipulation .",
"An inverted fluorescent microscope ( Olympus , IX71 ) equipped with a 100× oil objective and a sCMOS camera ( Zyla 4 . 2 Andor ) was used to collect bright-field images with 0 . 2 frame rate .",
"The bright-field images were further analyzed to track multiple single cells in real time using a high-throughput bacterial tracking algorithm coded by Matlab .",
"The projected contours of selected single cells were sent to the DMD ( 1280 × 760 pixels ) that was directly controlled by a commercial desktop through a VGA port .",
"The manipulation lights were generated by the red-color LED ( 640 nm ) , and were projected on the single selected cells in real time through the DMD , a multi-band pass filter ( 446/532/646 , Semrock ) and the air objective .",
"Our results indicated that feedback illuminations could generate projected patterns to exactly follow the cell movement ( Figure 5 – Supplemental 2B ) or single cells divisions ( Figure 5 – Supplemental 2C ) in real time .",
"The bacterial strain PAO1-bphS-PcdrA-GFP-mCherry was inoculated into a flow cell ( Denmark Technical University ) and continuously cultured at 30 . 0 ± 0 . 1 °C by flowing FAB medium ( 3 . 0 mL/h ) .",
"The flow cell was modified by punching a hole with a 5 mm diameter into the channel , and the hole was sealed by a coverslip that allows the manipulation light to pass through .",
"An inverted fluorescent microscope ( Olympus , IX71 ) equipped with a 100× oil objective and a sCMOS camera ( Zyla 4 . 2 Andor ) was used to collect bright field or fluorescent images with 0 . 2 or 1/1800 frame rate respectively .",
"The power density of the manipulation lights was determined by measuring the power at the outlet of the air objective using a power meter ( Newport 842-PE ) .",
"GFP or mCherry was excited using a 480 nm or 565 nm LED lights ( ThorLabs ) and imaged using single-band emission filters ( Semrock ) : GFP ( 520/28 nm ) or mCherry ( 631/36 nm ) .",
"Initial-attached cells were selected to be manipulated using ATIM with the illumination at 0 . 05 mW/cm2 , which allowed us to compare the results arising from illuminated or un-illuminated mobile cells in one experiment .",
"The c-di-GMP levels in single cells were gauged using the ratio of GFP and mCherry intensities .",
"Glass culture tubes were inoculated with 1 mL of P . aeruginosa in LB or Jensen’s minimal media at an OD600 0 . 8 and incubated statically at 37°C for 4 hr .",
"Non-adhered cells were removed by washing three times with 2 mL sterile phosphate buffered saline ( PBS ) .",
"Biofilm cells were harvested by vortexing in 1 mL PBS with tetramethylrhodamine ( TRITC ) conjugated lectins ( TRITC-labeled WFL lectin ( 100 μg/mL; Vector Laboratories ) for Pel , TRITC-labeled HHA ( 100 μg/mL; EY Laboratories ) for Psl ) and incubated on ice for 5 min .",
"Cells were washed 3 times to remove non-adhered lectin , resuspended in PBS , and immediately analyzed for GFP and TRITC fluorescence on a BD LSRII flow cytometer ( BD Biosciences ) .",
"Events were gated based on forward and side scatter to remove particles smaller than a single P . aeruginosa cell and large aggregates .",
"We used PAO1 cells that did not express GFP ( wild type PAO1; Figure 1—source data",
"2 ) or constitutively expressed GFP ( PAO1 Tn7::P ( A1/04/03 ) ::GFPmut; Figure 1—source data",
"2 ) to define a gate for high GFP fluorescence .",
"We validated this gate using a strain in which we expect very high levels of reporter activity ( surface grown PAO1 ΔwspFΔpelAΔpslBCD harboring pPcdrA::gfpASV ) and saw that 91 . 6% of cells had high GFP levels ( Figure 1—source data 2 ) , in agreement with our flow cell characterization of this strain ( Figure 2A ) .",
"We determined gating for TRITC using cells that had not been stained with TRITC-conjugated lectin ( Figure 1—figure supplement 6A ) , as well as two strains that overproduced either Psl ( Figure 1—figure supplement 6B ) or Pel ( Figure 1—figure supplement 6C ) that were stained with the appropriate TRITC-conjugated lectin .",
"Our flow cytometry gating procedure accurately gated 99 . 7% of wild type PAO1 cells ( without the PcdrA reporter or lectin-staining ) as low GFP and low TRITC ( Figure 1—figure supplement 6D ) .",
"Static biofilm reporter cells were grown as described above and harvested without lectin staining .",
"Cells were fixed with 6% paraformaldehyde for 20 min on ice , then rinsed once with sterile PBS prior to analysis with a FACSAriaII ( BD Biosciences , San Jose , CA ) .",
"Events were gated first to remove debris and large cellular aggregates , and then gated into cells with low and high GFP fluorescence intensity .",
"The low GFP gate was drawn using wild type PAO1 cells without the gfp gene ( Figure 1—figure supplement 7A ) and the high GFP gate was drawn using both PAO1 Tn7::P ( A1/04/03 ) ::GFPmut ( Figure 1—figure supplement 7B ) and PAO1 ΔwspF ΔpelA ΔpslBCD PcdrA::gfpASV reporter ( Figure 1—figure supplement 7C ) .",
"As expected , wild type PAO1 pPcdrA::gfpASV reporter cells that had been harvested after 4 hr of surface attachment to glass in static LB liquid culture displayed subpopulations of high GFP , reporter ‘on’ cells ( 30 . 8% of the population ) and ‘off’ ( 57 . 2% ) cells ( Figure 1—figure supplement 7D ) , whereas this same strain grown to mid-log planktonically in LB displayed mostly reporter ‘off’ cells ( Figure 1—figure supplement 7E ) .",
"Cells were sorted at 4°C by flow assisted cell sorting ( FACS ) to collect 100 , 000 events into TRIzol LS ( Thermo Fisher Scientific , Waltham , MA ) .",
"RNA was extracted from sorted cells by boiling immediately for 10 min and following the manufacturer’s instructions for RNA isolation .",
"DNA was digested by treating with RQ1 Dnase I ( Promega , Madison , WI ) and samples were checked for genomic DNA contamination by PCR to detect rplU .",
"Expression of pelA , pslA , and ampR was measured by quantitative Reverse Transcriptase PCR ( qRT-PCR ) using the iTaq Universal SYBR Green One-Step kit ( Biorad , Hercules , CA ) and a CFX96 Touch Real-Time PCR detection system ( Bio-Rad , Hercules , CA ) .",
"The ΔΔCq was calculated for three independent samples of sorted wild type PAO1 PcdrA::gfpASV reporter biofilm cell populations by normalizing PelA and PslA to relative levels of AmpR expression .",
"Data were presented as the average fold change in PelA or PslA expression in the PcdrA::gfpASV sorted ‘on’ population ( high GFP ) relative to the ‘off’ population ( low GFP ) for the three biological replicates .",
"A version of the pPcdrA reporter was constructed in the pBBR1MCS5 plasmid to express mTFP1 instead of GFP , for use with YFP-tagged WspR proteins .",
"The PcdrA promoter and an enhanced ribosomal binding site from the gene 10 leader sequence of the T7 phage ( g10L ) was amplified from pUC18-miniTn7T2-PcdrA-RBSg10L-gfpAGA using primers SacI-PcdrA-F and SOE-PcdrA-RBSg10L-R .",
"The primers mTFP1-F and KpnI-mTFP1-R were used to amplify the mTFP1 gene from plasmid pNCS-mTFP1 ( Allele Biotech , San Diego , CA ) .",
"The PcdrA::RBSg10L::mTFP1 allele was constructed by SOE-PCR using primers SacI-PcdrA-F and Kpn1-mTFP1-R , then pBBR1MCS5 and the SOE PCR product were doubly digested with SacI/KpnI .",
"Digested pBBR1MCS5 was treated with Antarctic phosphatase , then both digests were gel purified and ligated .",
"The ligation was transformed into E . coli DH5α , and plasmid from clones growing on LB with 10 μg/mL gentamycin were sequenced with primers M13F and M13F ( −21 ) ( GeneWiz ) .",
"Fluorescence of the pPcdrA::mTFP1 reporter was measured in Wsp mutants in a fluorimeter ( BioTek Synergy H1 Hybrid Reader , BioTek Instruments , Inc , Winooski , VT , USA ) and in flow cells to confirm its activity resembled that of pPcdrA::gfpASV .",
"The pPcdrA::mTFP1 reporter was electroporated into P . aeruginosa strains with the native WspR deleted and harboring an arabinose-inducible copy of WspR-YFP on its chromosome ( Huangyutitham et al . , 2013 ) .",
"Cells were grown on LB agar plates with 100 μg/mL gentamycin and 1% arabinose for 10 hr , then transferred to an agar pad for imaging of Differential Interference Contrast ( DIC ) , YFP , and TFP .",
"WspR-YFP foci and mTFP1 fluorescence was imaged using a Nikon Ti-E inverted wide-field fluorescence microscope with a large-format scientific complementary metal-oxide semiconductor camera ( sCMOS; NEO , Andor Technology , Belfast , United Kingdom ) and controlled by NIS-Elements .",
"WspR-YFP foci were detected and pPcdrA::mTFP1 reporter activity were analyzed using NIS-Elements AR software ( Nikon Instruments , Melville , NY , USA ) .",
"Regions of interest ( ROI ) corresponding to individual cells were determined using DIC images and the average mTFP1 fluorescence was measured within these ROIs .",
"Next , we used essentially the same protocol as previously described for detecting WspR-eYFP foci ( Huangyutitham et al . , 2013 ) , by examining the ratio of the maximum eYFP signal to the mean eYFP signal for each ROI .",
"We verified by eye that the previously determined cut-off ratio of 1 . 7 accurately represented cells with at least one visible WspR-eYFP focus ( Huangyutitham et al . , 2013 ) ."
]
] | [
"The second messenger signaling molecule cyclic diguanylate monophosphate ( c-di-GMP ) drives the transition between planktonic and biofilm growth in many bacterial species .",
"Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence .",
"Current thinking in the field is that once cells attach to a surface , they uniformly respond by producing c-di-GMP .",
"Here , we describe how the Wsp system generates heterogeneity in surface sensing , resulting in two physiologically distinct subpopulations of cells .",
"One subpopulation has elevated c-di-GMP and produces biofilm matrix , serving as the founders of initial microcolonies .",
"The other subpopulation has low c-di-GMP and engages in surface motility , allowing for exploration of the surface .",
"We also show that this heterogeneity strongly correlates to surface behavior for descendent cells .",
"Together , our results suggest that after surface attachment , P . aeruginosa engages in a division of labor that persists across generations , accelerating early biofilm formation and surface exploration ."
] | [
"Bacteria can adopt different lifestyles , depending on the environment in which they grow .",
"They can exist as single cells that are free to explore their environment or group together to form ‘biofilms’ .",
"The bacteria in biofilms stick to a surface , and produce a slimy ‘matrix’ that covers and thereby protects them .",
"Biofilms have been found in lung infections that affect people with the genetic disorder cystic fibrosis , and can also form on the surface of medical implants .",
"Because the biofilm lifestyle protects bacteria from the immune system and antimicrobial drugs , learning about how biofilms form could help researchers to discover ways to prevent and treat such infections .",
"Many bacteria switch between the free-living and biofilm lifestyles by altering their levels of a signaling molecule called cyclic diguanylate monophosphate ( called c-di-GMP for short ) .",
"Bacteria living in biofilms have much higher levels of c-di-GMP than their free-living counterparts , and bacteria that have high levels of c-di-GMP produce more biofilm matrix .",
"Bacteria called Pseudomonas aeruginosa use a protein signaling complex called the Wsp system to sense that they are on a surface and increase c-di-GMP production .",
"Questions remained about how quickly this change in production occurs , and whether bacteria pass on their c-di-GMP levels to the new descendant cells when they divide .",
"Armbruster et al . monitored individual cells of P . aeruginosa producing c-di-GMP as they began to form biofilms .",
"Unexpectedly , not all cells increased their c-di-GMP levels when they first attached to a surface .",
"Instead , Armbruster et al . found that there are two populations – high and low c-di-GMP cells – that each perform complementary and important tasks in the early stages of biofilm formation .",
"The high c-di-GMP cells represent ‘biofilm founders’ that start to produce the biofilm matrix , whereas the low c-di-GMP cells represent ‘surface explorers’ that spend more time traveling along the surface .",
"Armbruster et al . found that the Wsp surface sensing system generates these two populations of cells .",
"Moreover , the c-di-GMP levels in a bacterial cell even affect the behavior of the descendant cells that form when it divides .",
"This effect can persist for several cell generations .",
"More work is needed to examine exactly how the biofilm founders and surface explorers interact and influence how biofilms form , and to discover if blocking c-di-GMP signaling prevents biofilm formation .",
"This could ultimately lead to new strategies to prevent and treat infections in humans ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cancer biology"
] | FXR1 regulates transcription and is required for growth of human cancer cells with TP53/FXR2 homozygous deletion | elife-26129-v3 | [
[
"p53 , a critical tumor suppressor , primarily serves as a transcription factor to control various cellular stress-response signaling pathways , including cell cycle arrest , DNA repair , senescence and apoptosis ( Vousden and Lane , 2007 ) .",
"Loss of p53 function through mutation or deletion of its encoding TP53 is a common feature in a majority of human cancers , resulting in the escape from tumor-suppressor activities .",
"Numerous strategies have been explored to reverse dysregulated p53 suppressor function , including stabilizing p53 expression by antagonizing the p53–MDM2 interaction in cancers harboring normal TP53 copy number , and restoring p53's tumor suppressor activity in TP53-mutated cancer ( Khoo et al . , 2014; Soragni et al . , 2016 ) .",
"In cancer , tumor suppressor genes are often inactivated by genomic deletions that encompass the deletion of neighboring genes .",
"Such bystander genes often belong to multigene families; therefore , their deletion is tolerated due to genetic redundancy .",
"The newly proposed ‘collateral lethality’ concept suggests that such passenger deletion predisposes cancer cells to vulnerabilities that are induced by further inhibition of the remaining genes in the family , whose functions are essential and redundant ( Muller et al . , 2012 , 2015; Nijhawan et al . , 2012 ) .",
"This concept therefore opens the avenue to anti-cancer drug development targeting cancers containing co-deletion of tumor suppressor genes and neighboring genes without affecting wild-type cells .",
"Drugging TP53-deleted cancers had been a challenging task until a recent report demonstrated inhibition of a neighboring gene POLR2A , which is located about 200 kb downstream of TP53 on chromosome 17 and undergoes heterozygous deletion in colorectal cancers containing TP53 heterozygous deletion ( Liu et al . , 2015 ) .",
"Homozygous deletion , resulting in inactivation of both alleles , occurs less frequently and is more focal than heterozygous deletion .",
"There is no documented therapeutic strategy targeting homozygous TP53-deleted cancers .",
"POLR2A is co-deleted in a majority of tumors with TP53 homozygous deletion , and thus its inhibition would not be relevant .",
"FXR2 ( Fragile X-related Protein 2 , also known as FXR2P ) , located 100 kb downstream of TP53 , is also a neighboring gene of TP53 at chromosome 17p13 . 1 .",
"It belongs to the fragile X gene family that has essential functions in binding and regulating mRNA stability , transportation and translation ( Ascano et al . , 2012; Chen et al . , 2014; Darnell et al . , 2001; Siomi et al . , 1996 ) .",
"In this study , we investigated whether FXR2 passenger deletion at the TP53 homozygous deletion locus would result in subsequent cancer-specific vulnerability to inhibition of its family member , FXR1 ( Fragile X-related Protein 1 , also known as FXR1P ) .",
"The fragile X gene family contains three mammalian members , including fragile X mental retardation protein FMR1 ( also called as FMRP ) and its structural homologs FXR1 and FXR2 ( Siomi et al . , 1993 , 1995 ) .",
"These proteins are highly conserved in many species and share a high degree of sequence similarity in major functional domains , including tandem Tudor , KH and RGG box domains ( Kirkpatrick et al . , 2001 ) .",
"They all participate in RNA-binding , regulation of mRNA metabolism , ribosome-binding , and translation ( Ascano et al . , 2012; Chen et al . , 2014; Darnell et al . , 2001; Siomi et al . , 1996 ) .",
"These proteins contain nuclear localization and nuclear export signals which allow them to be shuttled between cytoplasm and nucleus ( Eberhart et al . , 1996 ) .",
"FMR1 , which is highly expressed in neurons , plays a critical role in synaptic plasticity and its silencing results in Fragile X Syndrome , an inherited intellectual disability and the major cause of autism ( Consortium , 1994; Darnell et al . , 2011; Santoro et al . , 2012; Verkerk et al . , 1991 ) .",
"FXR1 is ubiquitously expressed and has potential roles in cardiac and skeletal muscle development ( Huot et al . , 2005; Mientjes et al . , 2004; Van't Padje et al . , 2009; Whitman et al . , 2011 ) .",
"Increasing evidence suggests that FXR1 and FXR2 possess both common and distinct functions in post-transcriptional regulation ( Ascano et al . , 2012; Cavallaro et al . , 2008; Darnell et al . , 2009; Say et al . , 2010; Xu et al . , 2011 ) .",
"FXR1 copy number was amplified and demonstrated oncogenic activity in lung squamous cell carcinoma ( Comtesse et al . , 2007; Qian et al . , 2015 ) .",
"A recent study suggested that FXR1 downregulates p21 by binding and reducing its mRNA stability and/or by modulating p53 expression to avoid senescence in cancer ( Majumder et al . , 2016 ) .",
"At the N-terminus , all three members of the FMR1 family contain a tandem Tudor domain belonging to the Royal family of chromatin-binding proteins ( Adams-Cioaba et al . , 2010; Hu et al . , 2015; Myrick et al . , 2015 ) .",
"The tandem Tudor domain in FMR1 was reported to recognize methylated lysine on histones at the chromatin interface , thus resulting in the protein's subsequent localization to the DNA damage loci , thereby facilitating the repair process ( Adams-Cioaba et al . , 2010; Alpatov et al . , 2014 ) .",
"Nevertheless , the function of FXR1 in cancer and the underlying molecular mechanisms remain elusive .",
"From an analysis of The Cancer Genome Atlas ( TCGA ) and Cancer Cell Line Encyclopedia ( CCLE ) databases , we observed that FXR2 undergoes concomitant homozygous deletion in combination with TP53 homozygous deletion in a significant proportion of human cancers .",
"Our data show that FXR1 inhibition blocks cell growth in TP53 and FXR2 co-deletion cell lines , but not in copy number normal or single deletion cell lines .",
"The results were further confirmed in CRISPR-Cas9-generated TP53 and FXR2 knockout cell clones .",
"Through a comprehensive analysis of chromatin immunoprecipitation followed by mass spectrometry ( ChIP-MS ) and ChIP coupling with high-throughput sequencing ( ChIP-seq ) , we uncovered the molecular mechanism through which FXR1 regulates cell proliferation .",
"FXR1 is located in the gene promoter region together with histone H3 lysine 4 trimethylation ( H3K4me3 ) , and recruits transcription factor STAT1 or STAT3 at gene promoters to facilitate transcription regulation .",
"In an agreement with a role in the FXR1-assocaited mechanism , inhibition of STAT1 or STAT3 target genes can also reduce cell proliferation .",
"Taken together , our findings not only demonstrate the possibility of inhibiting FXR1 in TP53 and FXR2 homozygous co-deletion cancers as a potential treatment option , but also reveal the novel role of FXR1 in gene transcription ."
],
[
"Copy number data in various human tumors published in TCGA database ( access through cBioPortal ( http://www . cbioportal . org; accessed 18 Aug 2016 ) ( Gao et al . , 2013 ) show that homozygous TP53 genomic deletion occurs in various human cancers , at frequencies ranging from 1% to 15% ( Figure 1A , Figure 1—figure supplement 1A ) .",
"One of the neighboring genes , FXR2 , located around 100 kb downstream of TP53 at chromosome 17p13 . 1 , undergoes concomitant deletion in most tumors carrying TP53 homozygous deletion with only a few exceptions ( Figure 1A and B ) .",
"Consistently , in the CCLE database ( Barretina et al . , 2012 ) , the majority of cancer cell lines carrying TP53 homozygous deletion also contain FXR2 deletion ( Figure 1—figure supplement 1B ) .",
"However , FXR2 single deletion is rarely observed in human tumors .",
"In TP53/FXR2 co-deletion tumors , the copy number of FXR1 and FMR1 ( Figure 1A , Figure 1—figure supplement 1B ) is largely unaltered .",
"Of note , FXR1 or FMR1 copy number gain was also observed in tumors , although this wasmutually exclusive with TP53/FXR2 deletion ( Figure 1A , Figure 1—figure supplement 1B ) .",
"The recent discovery of the collateral lethality concept prompted us to hypothesize that concomitant deletion of passenger FXR2 in TP53-deleted cancer cells might make cell growth dependent on FXR1 .",
"We therefore tested whether TP53/FXR2 co-deletion renders cancer cells sensitive to FXR1 inhibition .",
"We selected four cancer cell lines harboring co-deletion of TP53 and FXR2: KATOIII , HL-60 , H358 , and KMS-11 , as well as four cell lines harboring the normal copy number of TP53 and FXR2: MKN45 , AGS , HepG2 , and A549 .",
"The mRNA and protein level of p53 , FXR2 , FXR1 and FMR1 were assessed using q-RT-PCR and Western Blot ( WB ) , respectively .",
"Our data showed that cancer cells with homozygous co-deletion of TP53 and FXR2 exhibit either absent or significantly lower mRNA and protein levels of p53 and FXR2 .",
"By contrast , the copy-number-normal and co-deleted cell lines have comparable FXR1 and FMR1 levels ( Figure 1—figure supplement 2 ) .",
"It is worth noting that p53 protein levels were assessed under a stress condition induced by doxorubicin .",
"Next , we monitored cell proliferation rate upon FXR1 inhibition using inducible short hairpin RNAs ( shRNAs ) .",
"Five doxycycline ( Dox ) -inducible FXR1 shRNAs were tested and most of them resulted in robust downregulation of FXR1 protein level and exhibited anti-proliferative activity in the TP53/FXR2 co-deleted cancer cell line KATOIII , but not in the copy-number-normal cell line MKN45 ( Figure 1—figure supplement 3A ) .",
"We selected shRNA 2 and 3 ( FXR1-sh2 , FXR1-sh3 ) , which showed profound knockdown efficiency for the following studies .",
"Consistently , FXR1 inhibition only suppressed cell proliferation in the cell lines with TP53/FXR2 co-deletion ( H358 , HL-60 , KATOIII and KMS-11 ) but not in the copy-number-normal cells ( AGS , A549 , MKN45 and HepG2 ) , as indicated in Figure 1C and also in Table 1 and Figure 1—figure supplement 3 .",
"Furthermore , we used cell imaging and Matrigel assays to confirm FXR1-knockdown-induced anti-proliferative effects ( Figure 1—figure supplement 4 ) .",
"To determine whether the observed cell growth reduction is attributed to FXR1 inhibition , we rescued FXR1 expression by ectopic expression of shRNA-resistant FXR1 ( harboring mutations on the shRNA-targeting sequence , FXR1m ) ( Figure 1—figure supplement 5 ) .",
"In multiple cancer cell lines , the growth rate was recovered to a level comparable to that of the control , indicating that FXR1 inhibition was responsible for the growth phenotype ( Figure 1—figure supplement 5 , Figure 1D , Figure 3B ) .",
"Consistent with the FXR1m data , expression of FXR2 also rescued the anti-proliferative phenotype induced by FXR1 inhibition , suggesting that FXR2 and FXR1 have redundant functions ( Figure 1D ) .",
"However , ectopic expression of FXR2 only showed partial rescue compared with FXR1m , suggesting that the proteins may also have some distinct functions .",
"Interestingly , FMR1 did not rescue FXR1 knockdown-induced anti-proliferation ( Figure 1E ) .",
"Furthermore , FMR1 knockdown had no impact on proliferation in both TP53/FXR2 copy-number-normal and co-deleted cancer cells ( Figure 1—figure supplement 6 ) .",
"The observation that cancer cells harboring deletion of both TP53 and FXR2 exhibited sensitivity to FXR1 inhibition suggested a collateral lethality correlation between FXR1 and TP53 deletion .",
"To investigate whether TP53/FXR2 co-deletion is essential in determining cells’ sensitivity to FXR1 inhibition , we monitored cell growth rate upon FXR1 downregulation in cell lines carrying homozygous deletion of either TP53 ( H1299 , L540 , MG-63 , SKOV3 ) or FXR2 ( Hep3B ) alone .",
"The levels of p53 , FXR2 and FXR1 were confirmed by q-RT-PCR and WB ( Figure 1—figure supplement 2 ) .",
"In the tested cell lines , the proliferation rate was unaltered ( Figure 1F ) , suggesting that concomitant deletion of FXR2 and TP53 is necessary for triggering FXR1 inhibition-induced cell lethality .",
"The role of FXR1 in controlling cell proliferation in TP53/FXR2 co-deleted cancer cells was further confirmed in an in vivo xenograft .",
"Dox-induced FXR1 downregulation significantly reduced tumor growth in the TP53/FXR2 co-deleted cell line HL-60-derived xenograft model ( Figure 1G ) , but had no impact on growth in the copy-number-normal cell A549-derived xenograft ( Figure 1—figure supplement 3C ) .",
"To confirm the above observations , we engineered the copy-number-normal cell line AGS that is suitable for use with CRISPR ( clustered regularly interspaced short palindromic repeat ) /Cas9 to generate the isogenic homozygous TP53 and FXR2 double knockout ( DKO ) cell clone , a TP53 single KO ( TP53 KO ) cell clone , and a FXR2 single KO ( FXR2 KO ) cell clone .",
"The knockout strategy is illustrated in Figure 2—figure supplement",
"1 . Both the mRNA and the protein levels of p53 and FXR2 in the individual knockout clones were determined by WB ( Figure 2A ) and q-RT-PCR ( Figure 2—figure supplement 2A ) .",
"Consistent with the phenotypes observed in cancer cell lines , shRNA-induced FXR1 downregulation significantly inhibited cell proliferation only in the TP53 and FXR2 double knockout clones ( Figure 2B ) and had no effect on the TP53 single KO ( Figure 2C ) or FXR2 single KO ( Figure 2D ) clones .",
"The FXR1-inhibition-induced anti-proliferative activity observed in TP53/FXR2 double knockout cells was confirmed in the Matrigel assay ( Figure 2—figure supplement 2B ) .",
"Resistance to FXR1 inhibition in both FXR2 single knockout clones ( Figure 2D ) and the FXR2 homozygous deletion cell line Hep3B ( Figure 1E ) suggested that p53 deficiency is required for FXR1 to function in regulating cell proliferation .",
"We downregulated p53 using RNAi in FXR2 KO cells that stably expressed FXR1 shRNA to further investigate the necessity for p53 deficiency in FXR1’s function in proliferation control .",
"In line with its tumor suppressor function , TP53 knockdown by siRNA enhanced cell growth in the tested cells ( Figure 2—figure supplement 2C ) .",
"Interestingly , TP53 knockdown granted FXR1 the capability to regulate cell proliferation: diminished FXR1 suppressed the growth of TP53 siRNA-treated FXR2 KO cells but not of control siRNA-treated FXR2 KO cells ( Figure 2—figure supplement 2C ) .",
"Moreover , knockout of TP53 and FXR2 didn’t change the expression of FXR1 or FMR1 ( Figure 2—figure supplement 2D ) .",
"To confirm the redundant function shared by FXR1 and FXR2 , we also used the same system to engineer a TP53/FXR1 DKO clone and stably expressed FXR2 shRNA-2 or FXR2 shRNA-3 .",
"We observed that knockdown of FXR2 upon Dox induction suppressed proliferation ( Figure 2—figure supplement 3 ) , further confirming the redundancy between FXR1 and FXR2 .",
"Table 1 summarizes the studies in the cancer cell lines and CRISPR-Cas9 knockout clones .",
"Using an arbitrary 30% proliferation inhibition as a cut off , there was a clear pattern of FXR1 regulation of cell proliferation in TP53 and FXR2 co-deleted cancer cells .",
"Therefore , our data validate the role of FXR1 inhibition in blocking cell proliferation in TP53-deleted cancers in a collateral lethality manner upon passenger deletion of FXR2 .",
"FXR1 contains multiple functional domains including the newly discovered N-terminal tandem Tudor domain , KH domains and the RGG box , as illustrated in Figure 3A .",
"In order to pinpoint the domain responsible for regulating cell proliferation , we examined the ability of FXR1 isoforms to rescue FXR1-inhibition-induced cell growth reduction .",
"There are three major isoforms , including full-length FXR1_a , C-tail truncation FXR1_b , and N-terminal tandem Tudor truncation FXR1_c ( isoform gene codes are listed in Materials and methods ) .",
"Ectopic expression of the shRNA-resistant plasmids encoding the three isoforms restored the expression of individual proteins in cells depleted of endogenousFXR1 ( Figure 3B ) .",
"Interestingly , only the full-length ( FXR1_a ) could rescue FXR1 shRNA-induced anti-proliferation .",
"The Tudor-domain-truncated version FXR1_c completely lost its ability to rescue cell proliferation ( Figure 3B ) .",
"The C-tail truncation FXR1-b only partially rescued cell proliferation ( data not shown ) .",
"These data indicate that the tandem Tudor domain is necessary for FXR1 to regulate cell proliferation .",
"It has recently been reported that the tandem Tudor domain of FMR1 can recognize methylated lysine at histone H3 and recruit DNA repair protein , thus playing a role in DNA damage repair ( Alpatov et al . , 2014 ) .",
"We used the histone methyl lysine analog ( MLA ) protein pulldown assay to examine whether histones are bound to the FXR1’s tandem Tudor domain .",
"The Tudor domain showed binding to histone H3 containing methylated lysine at various positions , especially H3K4me1 , H3K4me3 , H3K9me3 , H3K27me2 , H3K36me1 , and H3K79me2 ( Figure 3C ) .",
"The histone binding pattern of the tandem Tudor domain of FXR1 is similar to that of FMR1 .",
"These data indicate that the tandem Tudor domain can potentially bring FXR1 to the chromatin interface , thus playing a role in proliferation control .",
"We then investigated how FXR1 regulates proliferation in TP53/FXR2 co-deleted cancer cells .",
"In order to obtain a comprehensive assessment of FXR1’s function , we conducted chromatin immunoprecipitation using a validated FXR1-specific antibody , followed by mass spectrometry ( ChIP-MS ) , aiming to capture both the chromatin-associated and chromatin-free protein complexes in the cells .",
"Proteins identified in the FXR1 pull-down complex from both H358 and KATOIII cells were analyzed .",
"They are listed in Supplementary file 1 and exemplified in Figure 4A .",
"The enrichment analysis using the Database for Annotation , Visualization and Integrated Discovery ( DAVID ) clustered the potential FXR1-interacting proteins according to their function ( Supplementary file 2 ) .",
"Proteins involved in mRNA binding , stability , splicing/metabolism , transportation , and translation were most enriched ( Figure 4b , Supplementary file 2 ) , consistent with the known function of FXR1 .",
"Interestingly , proteins participating in chromatin/chromosome organization and transcription signaling were also enriched ( Figure 4B and Supplementary file 2 ) .",
"Next , we confirmed the protein interactions using ChIP followed by WB ( ChIP-WB ) .",
"In agreement with our DAVID enrichment analysis , FXR1 interacted with multiple proteins that are involved in chromatin and transcriptional signaling , including the transcription factors STAT1 and STAT3 , the chromatin regulator CHD4 , SNF2H , histone H2B and H3K4me3 , and DNA topoisomerase TOP2A in H358 cells ( Figure 4C ) .",
"We discovered that phosphorylated STAT1 or STAT3 can also interact with FXR1 from the ChIP-WB analysis in the same cells ( Figure 4C , lower panel ) .",
"Importantly , the in vitro pull-down assay using the purified tagged proteins showed that FXR1 may directly interact with both STAT1 and STAT3 ( Figure 4D ) .",
"Interestingly , FXR1 had no impact on the phosphorylation of STAT1 or STAT3 or on their shuttling from the cytoplasm to the nucleus ( Figure 4—figure supplement 1 ) .",
"When combined together evidence of FXR1's capacity to bind methylated histone H3 , these results suggest that FXR1 may be involved in chromatin signaling and gene transcription .",
"To confirm the role of FXR1 , we conducted chromatin immunoprecipitation coupled with high-throughput sequencing ( ChIP-seq ) in the TP53/FXR2 co-deleted H358 cells using FXR1- , STAT1- , STAT3- , H3K4me3- , H3K9me3- , or H3K27me3-specific antibodies .",
"We identified unfiltered 882 peaks of FXR1 ( Supplementary file 3 ) , primarily located at gene promoters close to transcriptional start sites ( TSS ) ( Figure 5A–B and Supplementary file 3 ) .",
"In agreement with our hypothesis , STAT1 , STAT3 and H3K4me3 were also primarily located at TSS ( Figure 5B and Figure 5—figure supplement 1A and Supplementary file 3 ) .",
"Interestingly , the location of FXR1 significantly overlapped with those of STAT1 , STAT3 , and H3K4me3 at their target genes ( Figure 5C and Supplementary file 3 ) .",
"The percentage of overlapping increased when we focused on TSS ( Figure 5C and Supplementary file 3 ) .",
"The FXR1-H3K4me3- or FXR1-STAT1/3-overlapped peaks primarily located in TSS regions , whereas the non-overlapped peaks primarily located in intergenic regions ( Figure 5—figure supplement 1B and Supplementary file 3 ) .",
"At TSS , a total of 210 peak-associated genes were identified for FXR1 ( Figure 5C and Supplementary file 3 ) .",
"Among them , 145 , 147 and 200 peak-associated genes overlapped with STAT1 , STAT3 and H3K4me3 , respectively ( Figure 5C and Supplementary file 3 ) .",
"FXR1 peaks rarely overlap with H3K9me3 or H3K27me3 ( for examples , see Figure 5D , Figure 5—figure supplement 2 , and Supplementary file 3 ) , indicating FXR1's potential role in facilitating active transcription .",
"Peaks aligning FXR1 , STAT1 , STAT3 and selective histone markers of representative genes — CASC4 , ARID1A , GLI1 , SSBP2 , CITED1 , NPAS3 and TRAPPC9 — are depicted in Figure 5D and Figure 5—figure supplement",
"2 . We selected FXR1-peak-associated putative target genes ( 239 genes listed in Supplementary file 5 ) and examined FXR1’s role in their expression using q-RT-PCR .",
"Upon inducible FXR1 knockdown , genes ARID1A , CASC4 , TRAPPC9 , GLI1 , CITED1 , NPAS3 , and SSBP2 were consistently downregulated in both H358 and KATOIII cells .",
"Data from H358 cells are shown in Figure 5E .",
"Using ChIP-PCR , we further investigated whether FXR1 directly occupies the promoter of these target genes .",
"On the basis of the ChIP-seq peak position , we designed and selected two PCR primer pairs against each target gene’s promoter region ( Figure 5—figure supplement 2B ) to capture FXR1’s localization in the ChIP-PCR assay .",
"The promoter regions of the target gene were detected in the FXR1 ChIP pull-down complex , indicating that FXR1 is located at the target gene promoters ( Figure 5F , shCtrl/Dox- and FXR1-sh3/Dox- ) .",
"Dox treatment did not reduce FXR1’s occupancy in the shRNA control cells ( shCtrl/Dox+ , Figure 5F ) , but it evicted FXR1 from the gene promoter in FXR1-shRNA-expressing H358 cells ( FXR1-sh3/Dox+ , Figure 5F ) , confirming the specificity of the FXR1 ChIP assay .",
"Using the same approach , STAT1 or STAT3 were located in the vicinity of FXR1 at the target gene promoter ( Figure 5F ) .",
"More interestingly , FXR1 knockdown resulted in STAT1 and STAT3 eviction from the gene promoters ( Figure 5F ) , suggesting that FXR1 is required to recruit or stabilize STAT1 and STAT3 at gene promoters .",
"On the other hand , STAT3 did not affect FXR1’s occupancy of sites at gene promoters because its knockdown had no effect on the localization of FXR1 at promoters ( Figure 5G , Figure 5—figure supplement 3 ) .",
"To further confirm the redundant function shared between FXR1 and FXR2 as well as to investigate the interaction between FXR1/2 and STAT1/3 , we conducted FXR1 , FXR2 , STAT1 , STAT3 , and H3K4me3 ChIP-seq in AGS cells .",
"The data indicated that the four proteins were enriched at TSS regions in the genome ( Figure 5H , Figure 5—figure supplement 4A and Supplementary file 3 ) .",
"FXR1 peak-associated genes had 89 . 95% ( 2935/3263 FXR1-peak-associated genes ) overlap with FXR2 peak-associated genes in AGS cells ( Figure 5I ) , and this percentage increased when we focused on TSS ( Figure 5—figure supplement 4B and Supplementary file 3 ) , suggesting a shared function of FXR1 and FXR2 at gene promoters .",
"Only a small percentage of FXR2 binding-site-associated or peak-associated genes overlapped with those similarly associated with FXR1 in AGS cells .",
"The difference is probably due to the weaker capability of the FXR1 ChIP antibody in pull down assay when compared to the FXR2 ChIP antibody , as reflected by the difference in the peak-associated gene numbers for FXR1 and FXR2 ( 3263 and 15 , 346 ) , respectively ( Figure 5I ) .",
"There was also significant overlapbetween FXR1-associated genes and STAT1/3-associated genes , suggesting at least partially , that FXR1 mediates STAT1/3 transcriptional activity in AGS cells ( Figure 5I , Figure 5—figure supplement 4B ) .",
"The ChIP-seq peaks at the promoter region of the representative gene ARID1A in AGS cells are shown in Figure 5—figure supplement 4C .",
"Our ChIP-PCR results revealed a similar localization pattern for FXR2 as for FXR1 , with both proteins occupying the promoters of target genes ( Figure 5J upper panel and Figure 5—figure supplement 4D ) .",
"Furthermore , the expression levels of target genes remained constant after FXR2 knockdown except for a slight inhibition of CASC4 and TRAPPC9 expression , further confirming the redundant function shared by FXR1 and FXR2 ( Figure 5J , lower panel ) .",
"Taken together , our data confirm that FXR1 is required to recruit or stabilize STATs on gene promoters , thus facilitating FXR1’s transcriptional activity , and also show that FXR1 and FXR2 share similar function .",
"Our study showed that only a small portion of STAT1/3 peaks in the genome ( 0 . 7–1 . 57% ) overlap with FXR1 peaks , indicating that FXR1 cooperates with STAT1/3 in a small number of STAT target genes’ transcription in H358 cells ( Supplementary file 3 ) .",
"By contrast , 24 . 38–25 . 74% of FXR1 peaks overlap with STAT1 and STAT3 peaks , suggesting a significant role for STATs in FXR1’s transcriptional activity ( Supplementary file 3 ) .",
"As STAT1 and STAT3 participate in FXR1 transcriptional activity , we hypothesized that a JAK inhibitor could repress FXR1 target gene expression .",
"Indeed , we found that the JAK inhibitor S-Ruxolitinib suppressed STAT1 phosphorylation in a dose-dependent manner and inhibited the expression of some but not all FXR1 target genes in H358 cells ( Figure 5K , Figure 5—figure supplement 5 ) .",
"Note that JAK inhibitor didn’t suppress FXR1 expression ( Figure 5K ) .",
"These data confirm the involvement of STAT in FXR1’s transcriptional activity .",
"We performed RNA-seq to analyze gene expression in H358 cells with biological triplicates .",
"We selected FXR1-knockdown-induced gene expression changes using fold change log2 >0 . 6 as the cutoff ( Supplementary file 6 ) .",
"Among the seven FXR1 target genes identified by ChIP-seq , five showed significant downregulation upon FXR1 knockdown .",
"The remaining two genes showed moderate downregulation ( Supplementary file 7 ) .",
"Among the genes whose promoters are occupied by FXR1 , 45 out of 210 genes showed regulation upon FXR1 knockdown in RNA-seq assay ( Figure 5—figure supplement 1D and Supplementary file 7 ) .",
"RNA-seq revealed significantly more genes regulated by FXR1 than ChIP-seq .",
"The discrepancy may be attribute primarily to the fact that FXR1 is a well-known mRNA-binding protein , regulating RNA metabolism and stability in addition to the newly discovered function in transcription regulation revealed in this study .",
"Therefore , FXR1 knockdown could impact on both populations of genes , those regulated by FXR1’s transcriptional activity and those regulated by FXR1’s RNA-binding function .",
"In addition , the scope and limitation of RNA-seq and ChIP-seq could also contribute to the discrepancy .",
"In order to assess whether target genes mediate FXR1’s function in proliferation regulation , we utilized siRNAs to knockdown target genes and monitored the change in cell proliferation .",
"The knockdown efficiency of the target genes by siRNAs was assessed using q-RT-PCR ( Figure 6—figure supplement 1A ) .",
"Among the seven target genes , knockdown of GLI1 showed the most consistent anti-proliferative effect whereas ARID1A , CITED1 , or SSBP2 knockdown showed less inhibitory effect in a 7-day proliferation assay ( Figure 6—figure supplement 1B ) .",
"We further evaluated the combinatorial effect on cell proliferation of downregulation of both FXR1 and its target genes .",
"We performed combinatorial partial knockdowns of both FXR1 and its target genes by applying lower concentration of siRNA ( 5 nM ) and shortening Dox treatment duration ( to 4 days ) .",
"As shown in the cell image and MTS detection ( Figure 6A ) , the combined knockdown of both the target genes and FXR1 showed an additive effect on cell growth inhibition .",
"Amongst the target genes , GLI1 downregulation by siRNA showed the most dramatic combinatorial effect with FXR1 knockdown in suppressing cell proliferation .",
"Upon FXR1 inhibition , cells underwent blebbing , suggesting a role of FXR1 in cell apoptosis .",
"Following this lead , we found that FXR1 knockdown induced the cleavage of PARP in H358 cells ( Figure 6B ) , a signal event of cell apoptosis .",
"Furthermore , FXR1 inhibition increased caspase 3/7 activity ( Figure 6B ) .",
"Consistently , knockdown of FXR1 target gene GLI1 also induced apoptotic signals including the cleavage of PARP and activation of caspase 3/7 in the same cells ( Figure 6C ) .",
"We observed the similar effect of FXR1 shRNA in inducing PARP cleavage and caspase 3/7 activity in another cell line , HL-60 ( Figure 6—figure supplement 2 ) .",
"More interestingly , the effect on PARP cleavage could be rescued by ectopic expression of shRNA-resistant full-length FXR1m_a , indicating a direct role of FXR1 in cell apoptosis signaling ( Figure 6—figure supplement 2 ) .",
"These data suggest that downregulation of FXR1 inhibits cell growth through the activation of cell apoptotic signaling , at least partially through regulating target gene transcription .",
"Taken together , the findings of our study indicate that FXR1 inhibition suppresses cell proliferation in cancer cells containing TP53 and FXR2 homozygous deletions in a collateral lethality manner .",
"FXR1 regulates gene transcription , at least in part , to enable control of cell proliferation ."
],
[
"The importance of p53 in cancer has led to tremendous efforts to target its activity by therapeutic approaches .",
"For cancers containing normal TP53 copy number , there has been good progress in interrupting MDM2–p53 interactions to stabilize and promote p53 tumor suppressor activity .",
"For TP53 mutated cancers , the current strategy is to restore p53 anti-tumor activity ( Khoo et al . , 2014; Soragni et al . , 2016 ) .",
"No therapeutic strategies have been proposed to target TP53 deletion cancers until a recent report demonstrated that TP53 heterozygous deletion predisposes cancer cells to further suppression of POLR2A , a neighboring gene undergoing concomitant deletion ( Bradner , 2015; Errico , 2015; Liu et al . , 2015 ) .",
"However , this strategy will not be applicable to cancers with homozygous TP53 deletions in which POLR2A is co-deleted in most cases ( Figure 1—figure supplement 1C ) .",
"Here , for the first time , we report an approach to target cancers with concomitant homozygous TP53 and FXR2 deletion by inhibiting FXR1 .",
"The passenger deletion of FXR2 in the TP53 deletion locus renders cancer vulnerable to FXR1 inhibition , which echoes with the recently raised concept of collateral lethality in tumor suppressor gene deletion ( Muller et al . , 2015; Muller et al . , 2012; Nijhawan et al . , 2012 ) .",
"In this study , several lines of evidence confirmed the redundant function between FXR1 and FXR2:",
"1 ) both FXR1 and FXR2 can rescue FXR1 shRNA-inhibited cell proliferation ( Figure 1D ) ,",
"2 ) FXR1 and FXR2 share a significant portion of overlapping binding sites in the genome ( Figure 5I , Figure 5—figure supplement 4B–C ) , and",
"3 ) knockout of either protein sensitizes TP53 homozygous deletion-containing cancer cells for the inhibition of the remaining member ( Figure 2D , Figure 2—figure supplement 2 ) .",
"Although another family member , FMR1 , shares structural and functional similarity with FXR1 and FXR2 , our data suggest that FMR1 does not participate in the regulation of proliferation or in rescuing the FXR1 inhibition in the tested cell models .",
"TP53 homozygous deletion is observed in 1–15% of human tumors , the majority of which have FXR2 homozygous deletions according to the TCGA database .",
"This study therefore opens the avenue to the development of FXR1-based targeted therapies to treat TP53 homozygous deletion cancers , in which there is a significant unmet medical need .",
"Interestingly , we have found that TP53 deletion is required for cancer cells to respond to FXR1 inhibition .",
"This was confirmed in our engineered FXR2 single knockout cell clones which were only sensitive to FXR1 knockdown when p53 was downregulated .",
"However , the mechanism of action remains elusive .",
"There have been reports suggesting that p53 deficiency promotes the activation of JAK/STAT signaling , which may lead to cellular dependency on FXR1 because it regulates the localization of STAT1/3 at gene promoters ( Guo et al . , 2014; Spehlmann et al . , 2013 ) .",
"Considering that FXR1 can both suppress p53 to escape senescence ( Majumder et al . , 2016 ) and activate gene transcription ( evidence from this study ) , p53 deletion may augment FXR1’s role in transcription regulation in controlling cancer cell proliferation .",
"Given the various critical functions of p53 in governing cellular behavior , its deficiency may create a cell dependency on FXR1 and FXR2 activities .",
"The underlying molecular mechanism requires further investigation .",
"Some studies have explored the oncogenic property of FXR1 in lung cancer with FXR1 overexpression or amplification ( Comtesse et al . , 2007; Qian et al . , 2015 ) .",
"FXR1 copy number gain occurs in some types of cancers , particularly in lung squamous carcinoma ( Comtesse et al . , 2007; Qian et al . , 2015 ) and its occurrence is mutually exclusive with TP53 and FXR2 homozygous deletion according to TCGA database ( Figure 1A ) .",
"Together with our study , this evidence suggests that FXR1 controls tumor growth in cancers with either TP53/FXR2 homozygous deletion or FXR1 amplification .",
"Furthermore , we reveal a novel function of FXR1 in transcriptional regulation and elucidate its molecular mechanism in regulating cell proliferation , in addition to its well-established role in mRNA binding .",
"Our ChIP-MS study revealed that FXR1 interacts with chromatin regulators and transcription factors .",
"In addition , about a half of FXR1’s peaks in the ChIP-seq assay co-localize with H3K4me3 peaks at gene promoters , suggesting a new role for FXR1 in facilitating active transcription .",
"FXR1 interacts with and recruits or stabilizes STAT1 or STAT3 at the target gene promoters , suggesting that cooperation with STATs is required for its transcriptional activity .",
"Indeed , the inhibitor of the STAT upstream kinase JAK suppressed some of FXR1’s target genes expression .",
"Reciprocally , FXR1 is required for the localization of STATs on its target gene promoters .",
"Generally , it is believed that JAK-catalyzed STAT1/3 phosphorylation drives their shuttling to the nucleus and localization at gene promoters in order to regulate transcription ( Levy and Darnell , 2002 ) .",
"Our findings advance the current views about the regulation of transcription by STAT1/3 by revealing the necessity for FXR1 in regulating STAT occupancy at promoters .",
"STAT is well established in regulating cytokine gene transcription .",
"We have also observed that FXR1 knockdown inhibited the expression of multiple cytokines ( data not shown ) , consistent with a recent report of FXR1 function in positively regulating cytokine expression in monocytes ( Le Tonqueze et al . , 2016 ) .",
"Notably , only about 0 . 7–1 . 57% of the STAT1/3 peaks overlap with FXR1 peaks in our ChIP-seq study , suggesting that FXR1 only participates in the transcription of a very small fraction of JAK-STAT target genes .",
"Although JAK inhibition suppressed the expression of some FXR1 target genes , it had no effect on inhibiting cancer cell proliferation in FXR1 shRNA-sensitive cells ( Figure 5—figure supplement 6 ) .",
"This is in line with the fact that JAK-STAT signaling pathway governs a much broader spectrum of gene expression and cellular behaviors .",
"It w worth investigating whether other epigenetic regulators are involved in STAT transcriptional activity .",
"Among the FXR1 peaks , approximately 25% overlapped with STAT peaks according to our study in H358 cells .",
"FXR1 may also cooperate with other transcription factors in regulating transcription; examples include BTF ( Ma et al . , 2014 ) which was also identified in an FXR1 protein complex in our ChIP-MS study .",
"In addition , we identified several other FXR1-interacting proteins that have essential function in transcription regulation , including TOP2A/2B , GTF2A , and FACT80 .",
"Further investigations are needed to dissect the underlying mechanism of FXR1 in transcription regulation .",
"FXR1 is an RNA-binding protein that regulates mRNA stability , transportation of RNA from the nucleus to the cytoplasm , and translation through its functional KH domains and RGG box .",
"Our ChIP-MS data confirmed the established role of FXR1 , which binds to and regulates mRNA processing and translation ( Figure 4B ) .",
"Furthermore , we confirmed ChIP-MS findings and discovered that FXR1 binds to the mRNA of target genes such as CASC4 , TRAPPC9 , GLI1 , and SSBP2 in the mRNA binding assays ( Figure 6—figure supplement 3 ) .",
"Our study confirmed that , in addition to its direct function in transcription , FXR1 binds to some but not all of its target gene mRNAs , which may contribute to its role in proliferation regulation .",
"This raises the possibility that FXR1 has dynamic functions in transcription and subsequent mRNA processing .",
"The newly discovered N-terminal tandem Tudor domain has a high degree of similarity among the three Tudor family members ( Adams-Cioaba et al . , 2010; Hu et al . , 2015; Myrick et al . , 2015 ) .",
"The Tudor domain in FMR1 was reported to bind to multiple methylated lysine at histone H3 and to participate in DNA damage repair ( Alpatov et al . , 2014 ) .",
"Our study shows that the tandem Tudor in FXR1 binds to methylated lysine in histone H3 in a similar manner as that in FMR1 and it is required for FXR1’s function in regulating cancer cell proliferation .",
"Further investigation is needed to study the amino acid residues and the structure of the aromatic cage within the FXR1 tandem Tudor domain that are responsible for histone binding , which might provide the foundation for development of Tudor domain inhibitors .",
"The observation that FXR1 knockout mice die shortly after birth and knockdown of FXR1 causes abnormalities in striated muscle and heart in zebrafish ( Mientjes et al . , 2004; Zarnescu and Gregorio , 2013 ) raise potential safety concerns with regard to inhibiting FXR1 in cancer patients .",
"However , the inducible FXR1 knockout in adult mice didn’t display the lethal phenotype ( Mientjes et al . , 2004 ) , suggesting the potential of FXR1 as a drugging target .",
"Overall , this study provides an important approach to targeting human cancers with TP53 homozygous deletions .",
"The novel function of FXR1 in transcription advances our knowledge regarding FXR1’s function in gene expression on top of its known roles in mRNA processing and translation ."
],
[
"Copy-number analysis in human tumor tissues was performed using data from The Cancer Genome Atlas ( TCGA ) ( http://www . cbioportal . org/cross_cancer . do ) as previously described ( Gao et al . , 2013 ) .",
"Gene copy numbers in human cancer cell lines were downloaded from the Cancer Cell Line Encyclopedia database ( CCLE ) ( http://www . broadinstitute . org/ccle ) and analyzed using an R package ComplexHeatmap ( https://bioconductor . org/packages/release/bioc/html/ComplexHeatmap . html ) .",
"KATOIII , HL-60 , H358 , MKN45 , AGS , HepG2 , A549 , H1299 , MG-63 , SKOV3 , Hep3B , and HEK 293 T cell lines were purchased from American Type Culture Collection ( ATCC , Manassas , Virginia , USA , http://www . atcc . org ) .",
"The KMS-11 cell line was obtained from Horizon Discovery Ltd . ( Cambridge , UK ) .",
"The L540 cell line was purchased from Biovector Science Lab , Inc ( Beijing , China ) .",
"Although L540 is listed as a misidentified cell line by ICLAC ( International Cell Line Authentication Committee ) , we used it as a TP53 single deletion cell model after comfirming the abscence of p53 mRNA and protein expression .",
"The cell lines were cultured in RMPI 1640 medium ( GIBCO ) or DMEM medium ( GIBCO ) supplemented with 10% fetal bovine serum ( FBS , GIBCO ) and 100 U/mL penicillin-streptomycin ( GIBCO ) at 37°C with 5% CO2 .",
"All cell lines were authenticated by Short Tandem Repeat ( STR ) profiling and routinely monitored for mycoplasma contamination .",
"The source and mycoplasma status as well as the Research Resource Identifiers ( RRIDs ) of each cell line are listed in Supplementary file 8 .",
"Cell proliferation rate was measured by MTS assay .",
"Cell apoptosis was determined by analyzing Caspase3/7 activity .",
"The luminescence signal in the Caspase3/7 assay was recorded by an Envision 2104 Multilabel reader ( Perkin Elmer , Waltham , MA ) .",
"The detailed procedure and information are in the Supplementary methods .",
"ChIP sample preparation follows the manual of SimpleChIP Enzymatic Chromatin IP Kit ( Magnetic Beads , CST , #9003 ) .",
"The experimental procedure and reagent information for ChIP-WB , ChIP-MS , ChIP-seq , or ChIP-PCR are in the Supplementary methods .",
"In cell assays , three independent replicates were included in each experiment .",
"Student T Test was performed to compare the differences between two groups with normal distribution .",
"All data were analyzed using GraphPad Prism 6 .",
"Gene-specific shRNA sequences were obtained from the Sigma website ( http://www . sigmaaldrich . com/ ) , and the hairpin sequences were cloned into the doxycycline ( Dox ) -inducible lentiviral vector pLKO-Tet-On with Puromycinas as a selection marker .",
"Five shRNAs targeting each gene ( FXR1 , FXR2 and STAT3 ) were screened .",
"Lentiviral particles were packaged in HEK 293 T cells by transient transfection .",
"Briefly , 6 µg of shRNA encoding lentiviral plasmid , 0 . 6 µg of helper PCG10 plasmid and 5 . 4 µg of helper PCG41 plasmid were mixed with 30 µl X-tremeGENE HP DNA Transfection Reagent ( Roche , 06366236001 ) and co-transfected into HEK 293 T cells at 70–80% confluency in a 10 cm Petri dish .",
"At 72 hr post transfection , the supernatant containing virus particles was collected and filtered through a 0 . 45 µm PVDF Millex syringe filter .",
"Each cell line was subjected to a puromycin tolerance test to determine the optimal concentration for establishing stable cell lines .",
"To generate a shRNA-expressing stable cell line , cells were infected with lentiviral particles with 8 µg/ml polybrene .",
"After 48 hr , the cells were selected by 0 . 5–2 µg/ml puromycin ( GIBCO , A11138-03 ) for 7 days .",
"shRNA expression was induced by 0 . 1–1 µg/ml Dox ( Sigma , D9891 ) , and the knockdown efficiency was tested by WB assay or q-RT-PCR .",
"Dox concentration was optimized for each stable cell line to achieve the most optimal knockdown efficiency at the minimum dose .",
"For ectopic expression of proteins of interest , cells were infected with the lentiviral pLenti6 . 3-MCS construct encoding the FXR1 , FXR2 or FMR1 gene open reading frame and selected using 2–6 µg/ml blasticidin ( GIBCO , A11139-03 ) for stable cell line generation .",
"The inserted open reading frames are: FXR1_a , NM_005087 . 3; FXR1_b , NM_001013438 . 2; FXR1_c , NM_001013439 . 2; FXR2 , – NM_004860 . 3; and FMR1 , NM_002024 . 5 .",
"The sequence of the FXR1 shRNA resistant mutation was CGCCAGGTTCCATTTAATGAA mutated to CGTCAAGTACCGTTCAACGAG and TTGCGAAGTATTCGTACGAAGTTG mutated to TTACGGAGCATCCGAACAAAATTA .",
"FXR1 shRNAs were inserted in adenoviral vector pAd/CMV/IRES/GFP .",
"The adenoviruses were produced in HEK 293 T cells by transient transfection of the plasmids .",
"Virus titer was determined by counting GFP-positive cells after adenoviral infection .",
"In the proliferation assay , adenoviruses were added to the cell culture medium and replenished 4 days afterwards to achieve efficient FXR1 knockdown .",
"The shRNA target sequence is listed in Supplementary file 8 .",
"Cells were lysed using RIPA lysis buffer ( CST , 9806S ) with complete protease inhibitor cocktail ( Sigma , P8340 ) and phosphatase inhibitor cocktail ( Sigma , P5726 ) .",
"The protein concentration was detected by BCA kit ( Thermo Scientific , Cat#23225 ) .",
"The protein lysate was denatured by adding NuPAGE sample reducing agent ( Novex , Invitrogen , NP0009 ) and NuPAGE LDS Sample buffer ( Novex , Invitrogen , NP0007 ) followed by heating at 95°C for 5 min .",
"Equal amounts of protein lysate was loaded to the NuPAGE Electrophoresis System ( Life Technologies ) according to the manufacturer's protocol .",
"Proteins in the gel were transferred to the nitrocellulose membrane in iBlot 2 Transfer Stacks ( IB23001 and IB23002 ) using the iBlot 2 Dry Blotting System ( Life Technology , IB21001 ) .",
"The membrane was blocked in SuperBlock Blocking Buffer in TBS ( Pierce , 37535 ) , incubated with the specific primary antibodies for the protein of followed by reaction with the secondary antibodies .",
"The protein signals were detected using SuperSignal West Femto Maximum Sensitivity Substrate ( Thermo , 34095 ) , and were scanned and quantified using ChemiDoc MP System ( BIO-RAD ) .",
"The antibodies and their Research Resource Identifiers ( RRIDs ) used in this study are listed in Supplementary file 8 .",
"Cell proliferation rate was detected by MTS assay .",
"Briefly , the cells were seeded in 96-well plates at optimal density in 100 µl medium per well followed by treatment .",
"Cells were added with CellTiter 96 AQueous One Solution Reagent ( Promega , G3581 ) at the indicated time by following the manual instructions and subjected to absorbance recording at 490 nm using a PowerWave HT Microplate Spectrophotometer ( BioTek ) .",
"The cell proliferation rate in MTS was determined by measuring absorbance at 490 nm .",
"In the duplicated experiment , cell images were scanned after fixing the cells with cold methanol and staining with 0 . 1% crystal violet .",
"In some cases , the cells were seeded on the solidified Matrigel ( BD Bioscience ) layer in 96-well plate ( 50 µl per well ) to measure the organoid growth .",
"Cell apoptosis was determined by analyzing Caspase3/7 activity using the Caspase-Glo3/7 assay kit ( Promega , G8092 ) according to the kit instructions or by detecting the protein level of cleaved PARP ( antibody from Cell Signaling Technology , 9541 ) in Western blot .",
"The luminescence signal in the Caspase3/7 assay was recorded by an Envision 2104 Multilabel reader ( Perkin Elmer ) .",
"The HL-60-derived xenograft model in female BALB/c nude mice was used to determine FXR1’s function in tumor growth .",
"Briefly , the female BALB/c nude mice ( age 6–8 weeks ) housed under pathogen-free conditions were inoculated subcutaneously in the right flank with HL-60 cells stably expressing control shRNA ( shCtrl ) or Doxycycline-inducible FXR1-sh3 .",
"Each mouse received 5 × 106 cells in 0 . 2 ml PBS:Matrigel ( 1:1 mixture ) for tumor development .",
"When tumor size reached approximately 180 mm3 on day 14 , mice were randomly grouped ( n = 6 ) and Doxycycline ( 2 mg/ml ) was added to drinking water .",
"Doxycycline was replenished twice per week .",
"Tumor volume and body weight were measured twice a week .",
"Tumor size was measured using a calliper , and tumor volume was calculated according to the following equation: tumor volume ( mm3 ) = length ( mm ) × width2 ( mm2 ) × 0 . 5 .",
"The experiment was terminated when the tumor volume reached 2 , 000 mm3 according to animal welfare guidelines .",
"All animal studies were carried out under IACUC number R20150728-Mouse and Rat approved by the Institutional Animal Care and Use Committee ( IACUC ) of WuXi AppTec .",
"Summary statistics , including mean and the standard error of the mean ( s . e . m ) , were provided for the tumor volume of each group at each time point .",
"The A549-derived xenograft model was studied using a similar method .",
"Guide RNA ( gRNA ) was designed according to the formula of GN20GG or CCN20C in the forward or reverse strand of genomic DNA , respectively ( Cong et al . , 2013; Ran et al . , 2013; Shalem et al . , 2014; Wang et al . , 2013 ) .",
"Two sets of gRNAs targeting the 5’ and 3’ sequences of the genomic region of interest ( TP53-to-FXR2 , TP53 alone , FXR1 alone , or FXR2 alone ) were designed .",
"The synthesized double-strand DNA of the gRNA was inserted into the pU6-gRNA_SPycas9-2Acherry vector expressing Cas9 protein ( Mavrakis et al . , 2016 ) .",
"To test gRNA efficiency , gRNA-expressing plasmids were transfected into cells using XtremeGene reagent ( Roche ) .",
"Genomic DNA was extracted using the Purelink Genome DNA mini kit ( Invitrogen , K1820-02 ) .",
"PCR was performed using genomic DNA as the template and the Accuprime Pfx SuperMix PCR system ( Invitrogen , 12344040 ) to amplify the sequence containing the gRNA PAM position , and PCR product was sequenced to determine the successful cut by gRNA-guided Cas9 by monitoring the miscellaneous peaks .",
"The efficient gRNA target sequences were: TP53-5’-gRNA , GGGCAGCTACGGTTTCCGTCTGG; TP53-3’-gRNA , GGTGTGCGTCAGAAGCACCCAGG; FXR2-5’-gRNA , GGAGGCGGTGGCGGCGCCATGGG; FXR2-3’-gRNA , GGGGGGTGGCACAGCAGCTTGGG; FXR1-5’-gRNA , GTGGAGGTTCGCGGCTCTAA; FXR1-3’-gRNA , GAAAAAAAAGTTGCTGGCTAT .",
"The donor plasmid contains the recombination sequences compatible with the 5’ and 3’ arms of the genomic region of interest and the GFP-coding sequence in vector pcDNA3 . 1 .",
"The mixture of gRNA-Cas9 plasmids targeting the 5’ or 3’ sequence of the genomic region and donor plasmid at ratio 1:1:1 was transfected into the candidate cells in a 10 cm Petri dish using XtremeGene .",
"One week post-transfection , the GFP-expressing cells were sorted and seeded into a 96-well plate at a density of one cell per well using FACSArisII ( BD ) .",
"The single cell clones were cultured for two weeks to identify and filter out the cells with transient GFP signal .",
"The clones with strong GFP signal , which would have potential insertion and replacement of the genomic region of interest by the donor sequence , were subjected to knockout validation by genomic PCR-sequencing , q-RT-PCR , and Western blot .",
"The sequence of genomic PCR primers is listed in Supplementary file 8 .",
"ChIP sample preparation: ChIP samples were prepared using SimpleChIP Enzymatic Chromatin IP Kit ( Magnetic Beads , CST , #9003 ) according to the manufacturer’s protocol .",
"Briefly , cells cultured in 15 cm Petri dishes were crosslinked with 1% formaldehyde when reaching 70–80% confluence .",
"Crosslinking was terminated by adding 10 × glycine followed by rinsing the cells twice with cold PBS , and 4 × 107 cells were collected into one 15 ml conical tube .",
"The cell pellet was resuspended and incubated in Buffer A on ice for 10 mins for cell lysis .",
"The nuclei were collected by centrifugation at 3 , 000 rpm for 5 mins at 4°C and resuspended in Buffer B followed by centrifugation and supernatant removal .",
"The nuclei pellets were resuspended in 1 . 0 ml Buffer B , and transferred to a 1 . 5 ml microcentrifuge tube .",
"7 . 5 µl of Micrococcal Nuclease was added and incubated for 20 mins at 37°C to digest the DNA to lengths of approximately 150–300 bp .",
"After the termination of digestion by adding 100 µl of 0 . 5 M EDTA , the nuclei pellets were collected by centrifuging at 13 , 000 rpm for 1 min at 4°C .",
"The nuclear pellets were resuspended in 1 ml ChIP buffer and separated into two tubes , and incubated on ice for 10 mins followed by subsequent sonication of lysates at 9W , 10 s ON/30 s OFF , nine cycles .",
"Lysates were collected by centrifugation at 10 , 000 rpm for 10 mins at 4°C .",
"The supernatant ( single nucleosome lysate ) was transferred to a new tube , and stored at −80°C for the following experiments .",
"50 µl of lysate was used for the analysis of chromatin digestion .",
"Pulldown: The single nucleosome lysate was diluted with ChIP buffer with protease inhibitor cocktail ( for each pulldown , 200 μl lysate was added into 300 μl 1 × ChIP Buffer ) .",
"10 µl of sample was saved as input .",
"To reduce nonspecific binding , the lysate was pre-cleared with 10% BSA and 30 μl ChIP Grade Protein G Magnetic Beads ( CST , #9006 ) at 4°C with rotation for 1 hr .",
"The supernatant was collected by placing the tube on a magnetic separation rack .",
"Antibodies of interest or IgG control were added and incubated on rotation at 4°C overnight followed by incubation with 30 µl of magnetic beads for 2 hr at 4°C .",
"Beads were washed with low salt wash buffer ( 100 µl 10 × ChIP Buffer in 900 ml water ) three times and with high salt wash buffer ( 100 µl 10 × ChIP Buffer and 70 µl 5 M NaCl in 830 µl water ) once on rotation .",
"The antibodies used in ChIP pulldown were: anti-FXR1 ( Sigma , HPA018246 , RRID: AB_1849204 ) ; anti-FXR2 ( Invitrogen , PA5-28979 , RRID: AB_2546455 ) ; anti-STAT1 ( Santa Cruz , sc-345 , RRID: AB_675903; CST , 14994S ) ; anti-STAT3 ( Santa Cruz , sc-482 , RRID: AB_632440; CST , 12640S , RRID: AB_2629499 ) ; rabbit IgG ( CST , 2729 , RRID: AB_1031062 ) ; anti-H3K4me3 ( CST , 9727 , RRID: AB_561095 ) ; anti-H3K9me3 ( Active Motif , 39161 , RRID: AB_2532132 ) ; and anti-H3K27me3 ( Millipore , 07–449 , RRID: AB_310624 ) .",
"For ChIP-WB , protein G beads were added with 30 μl of DTT-containing 2 × SDS sample buffer and denatured at 95°C in thermomixer with gentle vortexing ( 1 , 200 rpm ) for 5 min .",
"Eluted proteins were separated in SDS-PAGE gel and analyzed by WB .",
"For ChIP-MS , eluted proteins were separated in SDS-PAGE gel followed by gel fixing in buffer containing methanol , acetic acid , and water at 5:1:4 ratio .",
"The gel was stained in EZBlue Gel Staining Reagent ( Sigma , G1041 ) and subjected to mass spectrometry ( MS ) analysis .",
"For ChIP-seq or ChIP-PCR , beads were eluted with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing ( 1 , 200 rpm ) in thermomixer .",
"Eluted supernatant was incubated with 6 µl 5 M NaCl and 2 µl Proteinase K at 65°C for 2 hr in thermomixer to digest the protein .",
"DNA was purified using spin columns , and sent for ChIP-seq or ChIP-PCR analysis .",
"DNA from ChIP pull down complexes was subjected to library preparation according to Illumina's instructions ( Part # 11257047 ) .",
"Sequencing was performed on Illumina HiSeq 2000 .",
"ChIP-seq data quality was analyzed using FastQC ( Andrews , 2010 ) .",
"Reads in the fastq files were trimmed by 5 bp in the 5´-end , and then mapped to the reference human genome ( HG19 ) using BOWTIE2 .",
"Multiple mapped reads and duplicate reads were removed .",
"FXR1 binding genomic regions ( called ChIP-seq peaks ) were identified by the Model-based Analysis of ChIP-Seq ( MACS ) algorithm using the default p-value cutoff of 1 × e−5 .",
"The distribution of FXR1 ChIP-seq peaks were investigated by the annotations on the human genome .",
"The peaks were binned into eight groups according to the positions relative to annotated genes: 5’ Distal ( up to 100 kb upstream TSS ) , 5’ Proximal ( up to 10 kb upstream TSS ) , 5’ TSS ( 5 kb upstream TSS to 1 kb downstream TSS ) , Intragenic ( 1 kb downstream TSS to 1 kb upstream TTS ) , 3’ TTS ( 1 kb upstream TTS to 5 kb downstream TTS ) , 3’ Proximal ( up to 10 kb downstream TTS ) , 3’ Distal ( up to 100 kb downstream TTS ) , and the remaining sequence defined as Intergenic region .",
"The aggregation signals of FXR1 , H3K4me3 , STAT1 , and STAT3 at genebody ( TSS to TTS ) plus upstream and downstream 5 kb region were extracted and plotted using in-house perl and MATLAB code .",
"A hub for the UCSC genome browser was constructed in order to investigate the detailed information of the peaks .",
"The summit positions of the FXR1 ChIP-seq peaks were extended to 100 bp both upstream and downstream as input region for HOMER ( Heinz et al . , 2010 ) to call known and de novo motifs .",
"The overlap between ChIP-seq peaks was determined by 1 bp overlap of two peaks that were extended by 500 bp both upstream and downstream .",
"The TSS associated genes in each ChIP-seq were determined by overlapping peaks to refseq genes coordinates .",
"Venn diagrams were generated using online web tool VENNY 2 . 1 ( http://bioinfogp . cnb . csic . es/tools/venny/index . html ) .",
"Protein function enrichment was analyzed using DAVID ( Huang et al . , 2008 , 2009 ) .",
"The ChIP-seq peaks were visualized using IGV ( http://www . broadinstitute . org/igv/ ) .",
"The sequence of ChIP-PCR primers is listed in Supplementary file 8 .",
"FXR1 or IgG control ChIP pulldown complexes were separated in SDS-PAGE gel .",
"The gel was stained with EZblue ( Sigma , G1041 ) .",
"The stained gel was cut into multiple parts based on protein molecular weight .",
"Gel pieces were destained in solution containing 50 mM NH4HCO3 ( Sigma Aldrich ) and Acetonitrile ( ACN ) ( Fisher Scientific ) at 1:1 ratio , dried with SpeedVac , rehydrated in 300 μl of 10 mM DTT ( Sigma Aldrich ) solution at 56°C for 1 hr , and reacted in 25 mM IAA ( Sigma Aldrich ) at 37°C for 30 mins .",
"After washing with 25 mM NH4HCO3 ( Sigma Aldrich ) for 10 mins , the gel pieces were digested with trypsin solution ( 0 . 01 g/L trypsin in 25 mM NH4HCO3 ) ( Promega ) at 37°C for 12 hr .",
"The digested peptides were extracted using 300 μl of 50% ACN-0 . 1% FA .",
"The extracted solution was collected and concentrated with SpeedVac .",
"The samples were resolved with 0 . 1% FA and analyzed by nanoLC-MS/MS ( Thermofisher ) .",
"MS/MS was performed in a data-dependent mode in which the top 10 most abundant ions ( S/N > 30 ) for each MS scan were selected for MS/MS analysis by HCD .",
"Parameters used during searching included enzyme , trypsin; missed cleavages , One; dynamic modifications , oxidation ( M ) ; static modifications , Carbamidomethyl ( C ) ; precursor mass tolerance , 10 ppm; fragment mass tolerance , 0 . 1 Da .",
"Parameters in data filtration with Prohits included kinds of exclusion proteins: Heat Shock , Ribosomal , Cytoskeleton , Keratin , Artifact Protein , Rib , Nucleoprotein and Albumin; Max SAINT < 0 . 7 ( Liu et al . , 2012; Liu et al . , 2010 ) .",
"All MS and MS/MS data were searched in the combined mode against Uniprot Human database ( August , 2013 ) using the Proteome Discoverer 1 . 3 software ( Thermofisher ) .",
"A score greater than 31 ( confidence interval values > 95% ) obtained with the Mascot search engine ( Matrix Science , U . K . ) was considered as significant .",
"All the searching results were filtered with Prohits software to remove non-specific binding proteins ( Max SAINT < 0 . 7 ) ( Liu et al . , 2010 , 2012 ) .",
"FXR1 interacting proteins were classified according to the GO ( gene ontology ) database .",
"Protein function clustering was analyzed with DAVID .",
"Visualization and network map of FXR1-interacting proteins were delineated using BioGRID in Cytoscape .",
"Protein–protein interaction was analyzed using the KEGG ( Kyoto Encyclopedia of Genes and Genomes ) pathway database .",
"Total RNA was extracted using RNeasy Mini Kit ( Qiagen , 74106 ) and Qiagen QIAcube 230 V w . starter Pack-2 machine , and reverse-transcribed using Oligo ( dT ) primer and the SuperScript III First-Strand synthesis system ( Invitrogen , 18080–051 ) .",
"q-RT-PCR using cDNA product as the template was performed using Power SYBR Green PCR Master Mix ( Applied Biosystems ( AB ) , 4367659 ) and an ABI 7900HT Fast Real-Time PCR-3 Machine with specific primers .",
"Gene expression was quantified using the comparative Ct ( cycle threshold ) method and the results were normalized to GAPDH .",
"Sequences of q-RT-PCR primers are listed in Supplementary file 8 .",
"Each RNA-seq sample had three biological replicates .",
"RNA isolation was performed using Trizol and RNeasy MinElute Cleanup Kit from cells .",
"Following extraction , RNA quantity was assessed using Nanodrop and the integrity of total RNA using the RNA 6000 Nano Kit on an Aligent 2100 Bioanalyzer .",
"An RNA library was constructed using the TruSeq RNA Sample Preparation Kit .",
"The first step in the workflow involved purifying the poly-A containing mRNA molecules using oligo-dT-attached magnetic beads .",
"Following purification , mRNA was fragmented into small pieces using divalent cations under elevated temperature .",
"The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers .",
"Second-strand cDNA synthesis followed , using DNA PolymeraseI and RNase H . The cDNA fragments then went through an end repair process , involving the addition of a single ‘A’ base , and then ligation of the adapters .",
"The products were then purified and enriched with PCR to create the final cDNA library .",
"The clusters of the cDNA library were generated on HiSeq X HD PE flow cell .",
"Then the clusters were sequenced on a HiSeq X system .",
"RNA-seq data quality was analyzed using FastQC ( Andrews , 2010 ) .",
"Reads in the fastq files were trimmed by five base pairs at the 5´-end , and then mapped to the reference human genome ( HG19 ) with the algorithm STAR using default parameters .",
"Multiple mapped reads and duplicate reads were removed to extract raw reads of refseq genes using HOMER .",
"Differential expressed genes were identified using R package DESeq2 and determined using log2FC cutoff 0 . 6 and adjusted p value cutoff 0 . 01 .",
"KEGG pathway enrichment was investigated using GSEA ( Subramanian et al . , 2005; Mootha et al . , 2003 ) .",
"Histone H3 MLA proteins with methylation at various residues were purchased from Active Motif ( 31208–31222 ) .",
"His-tagged-FXR1-Tudor ( 2-132 ) protein were expressed and purified from an Escherichia coli system , and pulldown assay was performed as previously described ( Alpatov et al . , 2014 ) .",
"In brief , in each reaction , 2 µg of MLA protein and 2 µg of FXR1-Tudor protein were added in 500 µl of Histone MLA Binding Buffer ( 50 mM Tris pH 7 . 5 , 1 M NaCl , 2 mM MgCl2 , 0 . 5% Triton X-100 , and 10 mM Imidazole ) ( save 10 µl as input ) , followed by rotation at 4°C for 30 mins .",
"The reaction solution was spun down ( 10 , 000 g , 2 min ) to collect the supernatant in a new tube .",
"15 μl of Ni-NTA beads ( Qiagen ) was added to the collected supernatant followed by rotation at 4°C for 1 hr .",
"Beads were collected by spinning at 1 , 000 g for 2 min , washed with 1 ml of Histone MLA Binding Buffer five times , suspended in 30 μl of 2 × SDS sample buffer and boiled to denature the proteins .",
"The FXR1-bound histone MLA proteins were detected in a WB assay .",
"JAK inhibitor S-Ruxolitinib ( dual JAK1/2 inhibitor tool compound ) was purchased from selleck ( http://www . selleck . cn/ ) .",
"Owing to the instability of the compound , the medium with inhibitor was replaced every 5 days .",
"Gene-specific siRNA sequence was designed using the software in Hannon Lab ( http://cancan . cshl . edu/RNAi_central/RNAi . cgi ? type=siRNA ) .",
"The siRNA oligonucleotides were produced by Invitrogen , and the MISSION siRNA Universal Negative Control #2 ( Sigma ) was used as a negative control .",
"Five siRNAs for each gene were tested .",
"siRNAs were introduced into cells at a final concentration of 5–20 nM using Lipofectamine RNAiMAX Transfection Reagent ( Invitrogen , 13778–150 ) and Opti-MEM ( Gibco , 31985–062 ) according to the manufacturer’s protocol .",
"Knockdown efficiency was assessed 48 hr post-transfection by q-RT-PCR or WB .",
"Sequences of siRNAs are listed in Supplementary file 8 .",
"Flag-tagged FXR1 or HA-tagged-STAT1 or -STAT3 proteins were expressed in HEK 293 T cells .",
"Cell lysates were prepared using lysis buffer ( 50 mM Tris-Cl , pH 7 . 5 , 500 mM NaCl , 1% Triton X-100 ) with complete protease inhibitor cocktail ( Sigma , P8340 ) , phosphatase inhibitor cocktail ( Sigma , P5726 ) , and PMSF ( Sigma , 93482–50 ml-F ) .",
"Lysate was incubated , at 4°C overnight , with 100 μl Flag-M2 magnetic beads ( Sigma , M8823 ) to purify Flag-tagged-FXR1 protein or with 100 μl Influenza Hemagglutinin ( HA ) magnetic beads ( Thermo Fisher , 88837 ) to purify HA-tagged-STAT protein .",
"Beads were washed with 1 ml lysis buffer three times and with 600 μl elution buffer ( 50 mM Tris-Cl , pH7 . 5 , 500 mM NaCl , 10% Glycerol ) once .",
"Target protein was eluted with 30 μl of Flag peptide solution ( 5 mg/ml , in elution buffer , Sigma , F4799-4MG ) to obtain Flag-tagged-FXR1 protein or with HA peptide ( 2 mg/ml , in elution buffer , Sigma , I2149-1MG ) to harvest HA-tagged-STAT protein .",
"For in vitro pulldown , 25 µl purified Flag-tagged-FXR1 protein was incubated with 75 µl purified HA-tagged-STAT1 or -STAT3 protein in 500 µl binding buffer ( 50 mM Tris pH 7 . 5 , 150 mM NaCl , 0 . 1% NP-40 , 1 mM DTT , phosphatase inhibitor and protease inhibitor cocktail ) at 4°C overnight .",
"30 µl Flag or HA magnetic beads were added and incubated at 4°C for another 3 hr .",
"Beads were washed with wash buffer ( 50 mM Tris-pH 7 . 5 , 300 mM NaCl , 0 . 1% NP-40 ) three times .",
"The eluted products in 30 µl elution buffer were subjected to WB analysis .",
"H358 cells were lysed in lysis buffer ( 100 mM NaCl , 10 mM MgCl2 , 30 mM Tris-HCl , pH 7 . 5 , 1 mM dithiothreitol [DTT] , protease inhibitor cocktail , 100 U/ml RNasin , 0 . 1% Nonidet P40 [NP40] ) and divided into two parts .",
"2% of lysate was saved as input .",
"The lysate was pre-cleared by 10% BSA and 30 µl ChIP Grade Protein G Magnetic Beads ( CST , #9006 ) on rotation for 1 hr at 4°C .",
"The supernatant was collected by a magnetic separation rack , incubated with FXR1 antibody ( Sigma , HPA018246 ) or IgG control ( CST , 2729 ) for 1 hr at 4°C , and incubated with 30 µl of magnetic beads for 1 hr at 4°C on rotation .",
"Beads were washed with NT2 buffer ( 50 mM Tris-Cl , 150 mM NaCl , 1 mM MgCl2 , and 0 . 05% NP-40 ) five times .",
"Endogenous RNA protein complexes ( RNP ) were eluted from antibody/Protein G beads by incubation with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing ( 1200 rpm ) in thermomixer .",
"Proteins in the elution were digested by Proteinase K . RNA was purified using spin columns from RNeasy Mini Kit ( Qiagen , 74106 ) and reverse-transcribed using Oligo ( dT ) primer and the SuperScript III First-Strand synthesis system ( Invitrogen , 18080–051 ) .",
"The cDNA was subjected to RT-PCR assay using AccuPrime Pfx SuperMix ( Invitrogen , 12344040 ) ( Qian et al . , 2015 ) .",
"The PCR products are detected by electrophoresis in agarose gels and stained with ethidium bromide , visualized by ChemiDoc .",
"The non-target genes GAPDH and Actin serve as the negative controls .",
"ChIP-seq data reported in this paper have been deposited in the Gene Expression Omnibus ( GEO ) database under accession number GSE79707 .",
"RNA-seq data reported in this paper have been deposited in the Gene Expression Omnibus ( GEO ) database under accession number GSE101754 ."
]
] | [
"Tumor suppressor p53 prevents cell transformation by inducing apoptosis and other responses .",
"Homozygous TP53 deletion occurs in various types of human cancers for which no therapeutic strategies have yet been reported .",
"TCGA database analysis shows that the TP53 homozygous deletion locus mostly exhibits co-deletion of the neighboring gene FXR2 , which belongs to the Fragile X gene family .",
"Here , we demonstrate that inhibition of the remaining family member FXR1 selectively blocks cell proliferation in human cancer cells containing homozygous deletion of both TP53 and FXR2 in a collateral lethality manner .",
"Mechanistically , in addition to its RNA-binding function , FXR1 recruits transcription factor STAT1 or STAT3 to gene promoters at the chromatin interface and regulates transcription thus , at least partially , mediating cell proliferation .",
"Our study anticipates that inhibition of FXR1 is a potential therapeutic approach to targeting human cancers harboring TP53 homozygous deletion ."
] | [
"Healthy human cells employ many tricks to avoid becoming cancerous .",
"For example , they produce proteins known as tumor suppressors , which sense if a cell shows early signs of cancer and instruct the cell to die .",
"A gene known as TP53 produces one of the most important tumor suppressor proteins , and this gene is inactive or missing in many types of human cancer .",
"Treating cancers that have completely lost the TP53 gene is particularly difficult .",
"One way to develop new treatments for these conditions would be to target other proteins that these cancers need to survive; but these proteins first need to be identified .",
"Fan et al . have now identified one such protein in human cancer cells lacking TP53 .",
"Searching databases of DNA sequences from human cancer cells revealed that those without the TP53 gene often also lose a neighboring gene called FXR2 .",
"Cancer cells survive without FXR2 because a similar gene , called FXR1 , can compensate .",
"Fan et al . therefore decided to experimentally lower the activity of the FXR1 gene and , as expected , cancer cells without TP53 and FXR2 stopped growing .",
"Normal cells , on the other hand , were unaffected by the deletion of the FXR1 gene since FXR2 is still there .",
"This phenomenon , in which cancer cells become vulnerable after the loss of certain genes but only because they have already lost important tumor suppressors , is called “collateral lethality” .",
"Further experiments showed that the protein encoded by FXR1 coordinates with other proteins to activate genes that contribute to cell growth .",
"These findings suggest new ways to treat human cancers that have lost TP53 .",
"For example , if scientists can find small molecules that inhibit the protein encoded by FXR1 and show that these molecules can block the growth of tumors lacking TP53 and FXR2 , this could eventually lead to a new anticancer drug .",
"However , like any new drug , these small molecule inhibitors would also need to be extensively tested before they could be taken into human clinical trials ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"ecology"
] | Group size and composition influence collective movement in a highly social terrestrial bird | elife-59902-v1 | [
[
"Movement behaviour fundamentally influences how individuals encounter resources that are critical for their fitness .",
"In highly social animals , movements are determined by how efficiently a group can coordinate their behaviour ( Chapman and Chapman , 2000; Couzin et al . , 2002; Strandburg-Peshkin et al . , 2017; Strandburg-Peshkin et al . , 2015 ) .",
"Coordination requires effectively deciding where and when to move ( Conradt and Roper , 2005 ) as well as maintaining cohesion during movement ( Gall and Manser , 2017 ) .",
"Both these factors could be shaped by intragroup characteristics .",
"For example , it has become increasingly evident that group composition ( Farine et al . , 2015 ) , such as the presence of uniformed individuals ( Couzin et al . , 2011 ) or the mixture of individual personality types ( Aplin et al . , 2014; Jolles et al . , 2017 ) , can shape the behavioural properties of groups .",
"In societies where individuals form stable groups , group size is another potentially important group characteristic that may also significantly impact fine-scale collective movement patterns .",
"However , while the balance of benefits ( such as decreased predation risk according to the ‘many eyes hypothesis’; Pulliam , 1973 ) and costs ( increased intra-group competition over resources ) associated with group size have been extensively explored ( Krause and Ruxton , 2002 ) , we still have very little information on whether group size translates to differences in movement behaviour and ranging patterns of naturally occurring groups .",
"Theory predicts that larger group size can benefit animal groups that move collectively through complex and heterogeneous landscapes .",
"If group members pool their information , then larger groups should be able to reach more accurate estimates of their environment ( Cantor et al . , 2020; Couzin et al . , 2011 ) , as highlighted by the ‘wisdom of the crowd’ theory ( Galton , 1907 ) .",
"This process can translate to navigational accuracy , via the ‘many wrongs hypothesis’ , as pooling the imperfect individual estimations of directions can generate accurate group-level estimates that allow groups to move more directly towards resources ( Simons , 2004 ) .",
"Larger groups are also more likely to contain individuals with knowledge relevant to the current position or situation of a group ( the ‘pool of competence hypothesis’; Giraldeau , 1984 ) .",
"For example , studies on killer whales ( Brent et al . , 2015 ) and elephants ( McComb et al . , 2001 ) have shown that older individuals become leaders under conditions of extreme resource shortages .",
"Such mechanisms should therefore allow larger groups to more effectively navigate their environment , and therefore exhibit a larger home-range size .",
"Effective navigation , and subsequently larger home ranges , should be beneficial for group members .",
"Larger areas encompass a greater diversity and abundance of resources needed to meet group members’ nutritional needs ( Corriale et al . , 2013; Hubbs and Boonstra , 1998; Maher and Burger , 2011 ) , and moving to more new areas increases the probability of discovering new food resources .",
"Using larger areas can also allow groups to be more variable in their movements , which could make them more unpredictable to predators ( Roth and Vetter , 2008 ) and thus decreases predation risk ( Richardson et al . , 2018 ) .",
"Thus , all else being equal , such as environmental characteristics , the members of a group with a relatively larger home range should have higher survival in periods of resource limitations and/or greater reproductive output than an identical group with a smaller home range .",
"In reality , home-range size is often limited by many factors , including competition for space with other groups ( Christensen and Radford , 2018 ) .",
"However , many group-living species live in non-territorial societies , where groups can benefit from exploiting a home range without paying the costs of intergroup fights and competition .",
"In such species , groups can interact affiliatively and preferentially with specific other groups , resulting in a multilevel society .",
"In such societies , having a larger home-range size might generate more chances to associate with conspecifics from other groups ( Grueter et al . , 2020 ) , which , in turn , can increase mating opportunities ( by increasing contact with members of the opposite sex; Grueter et al . , 2015 ) , dispersal opportunities ( as sub-adults often disperse to neighbouring groups; Städele et al . , 2015 ) and information sharing about food resources or predation risk ( Whitehead et al . , 2012 ) .",
"Thus , there are many ways that groups can benefit from maximizing their space use , up to the point where the costs of travel and navigation do not override the benefits gained .",
"However , living in large groups can also introduce challenges , particularly in species that maintain stable group membership .",
"Collective movement requires maintaining cohesion , a challenge that increases with group size and in more complex environments ( Codling et al . , 2007 ) .",
"From a movement perspective , coordination among many individuals could result in slower decision-making , due to both coordination challenges as well as larger groups being likely to harbour more conflicts of interest .",
"For example , one study of collective movement in baboons found that the probability that an individual baboon followed one initiator versus the other was much lower when both disagreement among initiators on where to go and the number of initiators increased ( Strandburg-Peshkin et al . , 2015 ) .",
"Larger groups are also likely to have slower movement speeds while ‘on-the-go’ .",
"We all have personal experiences of how much slower it can be to move or make decisions ( such as where to have lunch ) when we are in a large group , as the group generally may have to conform to the slowest members ( Herbert-Read et al . , 2013 ) or satisfy a diversity of preferences ( Strandburg-peshkin et al . , 2017 ) .",
"Finally , it may be more challenging for individuals to influence larger groups to move to new areas , and thus to extend the group’s home-range .",
"These factors suggest that the consensus costs borne by larger groups will ultimately limit the size of the home-range that such groups can exploit .",
"Given that both positive and negative relationships between group size and home-range size may exist , we a priori predict a non-linear relationship between the size of stable groups and collective movement characteristics .",
"This non-linear relationship is likely to represent an optimal group size for movement that reflects the point where the difference between navigational accuracy and coordination problems is maximized .",
"To our knowledge , this relationship remains largely unexplored in wild , free-living groups .",
"A first challenge in quantifying the relationship between home-range size and group size requires long-term tracking across multiple replicated groups and , ideally , replication in time .",
"A second challenge is that many species that live in stable groups ( at least most of the well-studied species ) either experience increased intra-group competition as group sizes increase ( e . g . baboons; Markham et al . , 2015 ) or they are territorial ( e . g . meerkats; Bateman et al . , 2015 ) .",
"Territoriality introduces several confounding effects .",
"For example , the greater resource-holding potential of larger groups ( Bateman et al . , 2015; Markham et al . , 2015 ) can translate to larger home-ranges .",
"Further , different territorial groups can vary in the density and distribution of resources within their territory , which could make it appear that smaller groups have larger home-ranges if they live in marginal areas with low resource densities .",
"As a result , the relationship between group movement characteristics and group size has remained relatively unclear .",
"In this study , we examine the effect of group size on the movement and space use patterns of wild groups of vulturine guineafowl ( Acryllium vulturinum ) , and whether resulting patterns correspond to differences in reproductive output among groups .",
"Our study species lives in a multilevel society consisting of large , stable groups with multiple breeding units and groups form stable associations with specific other groups ( Papageorgiou et al . , 2019 ) .",
"The stable social structure and the lack of territoriality—the entire population inhabits a small area of approximately 500 ha and all groups can easily access all parts of our study area—makes vulturine guineafowl an ideal species to investigate how group size influences movement characteristics because groups can range without major constraints on ranging imposed by other groups .",
"Further , their large body size ( ~1 . 5 kg ) allow guineafowl to be fitted with high-resolution solar-powered GPS tags to collect both fine-scale data on their movement as well as long-term data on their ranging behaviour .",
"We use data from GPS tags fitted to one individual from a total of 21 distinct groups , collected over five 2-month-long non-breeding seasons ( Figure 1 ) .",
"We fit a continuous-time movement model ( ctmm ) to each individual GPS dataset ( one bird per group per season ) using GPS locations collected every 5 min .",
"From each model , we calculated ( 1 ) home-range size , ( 2 ) the tendency for groups to use the same areas on consecutive days , and ( 3 ) daily travel distance .",
"We then used high-resolution ( 1 Hz ) GPS location data from each individual to quantify ( 4 ) the movement speed of its group while ‘on the go’ .",
"Because groups move through heterogeneous environments , larger groups should be able to navigate more effectively .",
"Their large size should also result in larger daily travel distances as they search for the resources needed to survive .",
"By contrast , moving as a large group also introduces consensus costs , and these could limit the home-range size of the largest groups by affecting their speed or their tendency to move to new areas .",
"As a result of the interaction between these two effects , we expect that there will be a non-linear relationship between group size and movement , which , if it results in more effective use of available resources , could correspond to an optimal group size for group movement characteristics .",
"We predict that intermediate groups will have the largest home-range sizes .",
"This will arise because while larger groups should travel further , intermediate groups will have lower day-to-day home-range fidelity .",
"We further predict that intermediate size groups will present the fastest speed while ‘on the go’ .",
"However , these factors may be tempered by group composition .",
"Groups in our population reproduced several times over our study period , and the presence of young group members could restrict the movements of groups .",
"Thus , we run our analysis while controlling for the number of chicks present in the groups , and predict that groups with more chicks should travel slower and shorter distances , thereby restricting their home-range size .",
"Finally , we predict that a non-linear relationship between group size and movement should translate to corresponding peak in fitness , measured as the number of chicks in a group .",
"Such a pattern would suggest that maximising the difference between navigation and coordination represents an optimal group size at which groups are most efficient in exploiting their environment ."
],
[
"Our movement analysis , based on 1 , 274 , 434 five-min GPS positions , spanned 45 seasonal observations of individuals ( groups ) over five 2-month-long seasons ( Figure 1 , see Supplementary file 1A for a summary of the data ) .",
"Group sizes , recorded during daily census observations , were widely distributed , with intermediate groups being observed less often ( Figure 2 ) .",
"Fitting generalised estimating equations ( GEEs ) with the number of adults ( group size ) and the number of chicks as predictors ( while accounting for repeated measures of the same groups across seasons ) revealed that home-range size was significantly predicted by group size and its quadratic term , but that this relationship was modulated by the presence of chicks .",
"Intermediate groups had the largest home-range , but home-range size decreased with increasing number of chicks ( Table 1A , see Supplementary file 1B for all model fits ) .",
"Average temporal overlap in space use was also significantly predicted by group size and its quadratic term , but was not significantly modulated by the presence of chicks ( Table 1B , see Supplementary file 1C for all model fits ) .",
"Average daily distance travelled increased linearly with group size ( Table 1C , see Supplementary file 1D for all model fits ) .",
"Speed ‘on-the-go’ was not explained by group size or by number of chicks in the group ( Table 1D , see Supplementary file 1E for all model fits ) .",
"Overall , smaller and larger groups expressed smaller home-range sizes and increased average temporal overlap in space use , while larger groups expressed longer daily travel distances than groups of smaller size ( see Figure 3 and Figure 4 ) .",
"The number of chicks in the group was also significantly predicted by group size and its quadratic term ( Table 1E , Figure 3D , see also Supplementary file 1F for all model fits ) .",
"We found that intermediate-sized groups had more chicks , with the peak in fitness ( group size = 33 ) closely matching the peak in home-range size ( group size = 36 ) and average daily overlap ( group size = 37 ) .",
"Number of adults ( group size ) and its quadratic term significantly predicted ( A ) home-range size and ( B ) average temporal overlap in space use .",
"The presence of chicks modulated the relationship between group size and home-range size , reducing the overall contribution of the number of adults to home-range size ( the negative interaction with number of adults ) and reducing the quadratic effect ( the positive interaction with number of adults squared , which offsets the negative quadratic coefficient ) .",
"( C ) The average daily distance travelled increased with number of adults ( see Figure 3C ) .",
"( D ) Speed while travelling was not significantly impacted by group size or its quadratic term .",
"( E ) The number of chicks per group was significantly predicted by group size and its quadratic term ( see Figure 3D ) .",
"Parameter estimates , significance , and R2 were generated using the most parsimonious fitted GEEs based on QIC ( see Materials and methods ) .",
"All models tested are summarized in the Supplementary file 1B to 1F ."
],
[
"Our study shows that group size and composition can impact collective movement characteristics .",
"Groups of vulturine guineafowl that have an intermediate size exhibit larger home-ranges and explore more new areas day-by-day .",
"However , this relationship exists despite intermediate groups travelling shorter distances each day compared to larger groups .",
"Even though the presence of chicks does not impact how far or how fast groups travel , having many chicks restricts the home range of their group .",
"Further , our study also shows that groups of intermediate size have higher fitness as they produce more offspring than larger or smaller groups .",
"Our results are therefore consistent with our a priori prediction that a non-linear relationship should exist between group size and collective movement , reflecting the increasing navigation ability with increasing group size and the increasing challenges of coordinating movement in larger groups .",
"The relationship between group size and reproductive output further suggests that intermediate-sized groups are more effective at exploiting space , providing evidence for an optimal group size for collective movement .",
"The relationships between group size and group movement outcomes provide new insights into how differently sized animal groups interact with their environment .",
"Larger groups cover larger distances during the day , which is likely to be important for encountering sufficient resources to meet their energetic demands , as proposed by the ecological constraints model ( Chapman and Chapman , 2000; Ganas and Robbins , 2005; Gillespie and Chapman , 2001; Teichroeb and Sicotte , 2009 ) .",
"However , by exploiting larger areas over a whole season , groups of intermediate size are likely to benefit from having access to a greater abundance and/or diversity of food resources ( Maher and Burger , 2011 ) , they potentially also benefit both from being less predictable to predators ( Richardson et al . , 2018; Roth and Vetter , 2008 ) , and from having access to a more diverse social landscape .",
"In addition , intermediate-sized groups achieve larger home ranges while travelling shorter distances than larger groups , implying that they have lower energy expenditure .",
"It is worth noting that we focused our analysis on 2-month periods each capturing similar ecological conditions .",
"Future studies could investigate whether these non-linear relationships between group-size and movement characteristics change across different environmental conditions and especially in seasons where energy might be under tighter constraints , such as during droughts .",
"Evidence for this could bring important insights into the drivers of group size variation .",
"While we predicted that group size would translate to differences in movement while ‘on-the-go’ , our measure of moment-by-moment movement ( speed while travelling ) did not reveal any relationship between group size and movement speed .",
"One reason for this could be that larger groups could have better knowledge of the environment , meaning that they can offset consensus costs ( i . e . periods of slow movement due to conflict within the group regarding movement directions ) with more effective movement once departed ( Couzin et al . , 2005 ) .",
"For example , the speed and polarization , and subsequently the directionality of group motion , increases with the size of fish schools ( Tunstrøm et al . , 2013 ) .",
"As group size increases in locusts , they transition from disordered to ordered motion ( Buhl et al . , 2006 ) .",
"Thus , it is possible that while the realised movement speed does not differ with group size , the characteristics of how the movement is achieved could vary between smaller and larger groups .",
"Alternatively , the challenge of reaching a consensus could mean that larger groups end up repeating the same routes more often , which they can achieve by moving faster as individuals all know where they are going .",
"This explanation is consistent with our findings that larger groups were less variable in where they travelled each day .",
"Such mechanisms linking collective movement with ranging behaviour warrant further investigation .",
"Several studies in the past have investigated the relationship between group size and ranging behaviour .",
"Most notably , Markham et al . , 2015 found that intermediate-sized groups of wild baboons ( Papio cynocephalus ) utilized smaller areas , covered shorter distances per day , and used space less evenly than smaller and larger groups—patterns which are largely opposite to our findings .",
"Stevenson and Castellanos , 2000 found that woolly monkeys ( Lagothrix lagothricha ) in groups of intermediate size travelled less during the day than larger and smaller groups .",
"Finally , research on meerkats ( Suricata suricatta ) Bateman et al . , 2015 found no relationship between group size and group movement characteristics .",
"However , in most previously studied systems , group movement characteristics are likely to be impacted not only by the ability for groups to move , but also by intra-group contest competition and inter-group competition ( Markham et al . , 2015 ) .",
"For example , smaller groups have the weakest resource-holding potential , meaning that they should have the smallest territories .",
"At the same time , they might be excluded from the best , most resource-rich , habitats by larger groups ( who need more resources due to intra-group competition ) , meaning that they could also end up having to use equally sized or even larger areas ( Markham et al . , 2015; Stevenson and Castellanos , 2000 ) .",
"In contrast to previous studies , our evidence for a non-linear relationship between group size and movement characteristics comes from a species that not only presents minimum intergroup competition but also groups use largely overlapping areas .",
"These features reduce the confounds on the link between group size and group movement behaviours .",
"Our study is therefore more likely to reveal effects arising from movement-based mechanisms without confounding effects that are exogenous to the group .",
"Vulturine guineafowl live in a multilevel society , where groups preferentially associate with other groups ( Papageorgiou et al . , 2019 ) .",
"In such societies , intergroup associations can bring many important benefits , such as information sharing on predators and resources , as well as mating and dispersal opportunities ( Grueter et al . , 2020 ) .",
"A large home range size in a multilevel society could provide groups with access to more communal roosts ( we have observed up to five groups in a single roost , Papageorgiou et al . , 2019 ) and more glades , and thus more opportunities to associate with more groups .",
"However , the benefits of associating with other groups might not increase linearly with home range size , as associating with a large number of conspecifics can also imply costs , such as disease transmission or increased competitions for mates ( Krause and Ruxton , 2002 ) .",
"More work is needed on the interactions between ranging behaviour of groups and the broader social environment in multilevel societies .",
"We built on existing insights from studies of collective behaviour to develop an a priori prediction that the relationship between group size and movement characteristic should be non-linear .",
"Our prediction proposed that maximising the use of space ( i . e . a larger home range ) through effective movement ( faster movement and less repetitive ranging ) should result in more efficient exploitation of the resources available to a group .",
"A key question is whether groups actually benefit from this maximisation ?",
"By analysing data on the reproductive success across the groups , we found striking evidence for a peak in fitness , with intermediate-sized groups having more chicks .",
"This peak , which closely matches the group size peaks in movement characteristics ( home range size and average daily overlap ) , suggests that intermediate group sizes might be linked to higher reproductive success .",
"Our findings therefore provide evidence for an optimal group size in the context of collective movement .",
"An interesting additional finding from our study is that optimally sized groups ( 33–37 individuals ) were relatively rare .",
"Instead , the distribution of group size appears to be bimodal with most groups being either smaller or larger than the intermediate group size .",
"This observation aligns with the hypothesis that while an optimal group size results in maximum individual fitness ( Pride , 2005 ) groups at the optimal size should be unstable and therefore rarely observed ( Sibly , 1983 ) .",
"Groups in our study grow either through reproduction or through immigration .",
"We found that intermediate-sized groups have high reproductive success , which naturally pushes them beyond the optimal .",
"Further , because dispersing individuals should prefer optimal size groups , they should also turn intermediate-sized groups into groups that are larger than the optimal size .",
"Future studies should examine whether groups near the optimal size for collective movement also highly attract more dispersers .",
"The observed variation in results between our study and previous studies investigating the effect of group size on ranging behaviour in mammals could also potentially arise due to methodological differences .",
"For example , we used high-resolution tracks and continuous-time movement modelling to get much more precise estimates of movement characteristics .",
"By contrast , other studies have used much lower sampling resolutions , often relying on hand-held GPS devices ( Bateman et al . , 2015; Markham et al . , 2015 ) or used simplistic minimum convex polygons to model home-ranges ( Markham et al . , 2015 ) .",
"Minimum convex polygons are unlikely to be very good estimates of home-ranges as they are sensitive to outliers ( e . g . from transcription errors or poor GPS fixes ) and capture only one element of space use ( the extreme boundaries , which could be influenced by a range of factors such as habitat geometry; He et al . , 2019 ) .",
"However , despite these potential methodological differences , we believe that conflicting findings are more likely to suggest that the effect of group size on ranging behaviour and collective movement varies according to the properties of the different animal social systems .",
"For example , we predict that the outcome of the many factors that contribute to the ranging characteristics of social groups will be modulated by the distribution of food resources each species exploits .",
"Vulturine guineafowl have very little need to defend areas from other groups or compete for resources within groups , as their food source ( insects and small seeds ) are widespread and they roost on the top of dense stands of trees , a habitat feature which is common in the study area .",
"When competition is decreased , we predict that group size could become a more important driver of collective movement outcomes .",
"We hope that by collecting more data on this question across species there will soon be sufficient studies to fully test this prediction .",
"In addition to the effects of the number of adults in the group , we also revealed the impact of having young present in the group .",
"Vulturine guineafowl are precocial , meaning that they are very small when they join the group , and mature over long periods of time—at 1 year of age they are only approximately 65% of the full adult size .",
"For a number of months after hatching , chicks are unlikely to contribute to making decisions , but are highly vulnerable to predators—causing them to remain more in cover .",
"At the same time , chicks are likely to move more slowly , or cover less ground , due to their smaller body size .",
"We found that having chicks in the group has a profound effect on home-range size .",
"Our results indicate that groups with more chicks use smaller areas , thereby limiting groups of intermediate sizes from exhibiting larger home-ranges .",
"The limitations that chicks have on group movements raise interesting questions about the costs of group living .",
"The fact that groups of vulturine guineafowl remain cohesive across seasons and years suggests that any costs borne from collective movement will be shared by all group members , including by individuals which have failed to reproduce ( vulturine guineafowl are plural breeders; Papageorgiou et al . , 2019 ) or by subadults who have yet to disperse .",
"Thus , our data reveals a potentially important source of conflict among group members .",
"Such conflicts may be common in plural-breeding groups , which are commonly found in multilevel societies .",
"We have shown that using synchronous and high-resolution GPS tracking of multiple social groups that reside in the same area can reveal new insights into the movement and social ecology of species .",
"Specifically , we demonstrated that group size and composition can predict collective movement and space use characteristics .",
"Our results are consistent with the prediction that increased navigation accuracy but decreased coordination ability could produce a non-linear relationship between group size and collective movement .",
"However , the mechanisms that underlie the interaction between coordination maintenance and navigation accuracy in free-living groups of various sizes remain to be discovered .",
"Further , we have identified dimensions of movement—specifically that intermediate-sized groups have larger home-ranges and use a larger diversity of areas—that translate into intermediate-sized groups having greater efficacy at exploiting their environment , and , therefore , higher fitness .",
"These results suggest the presence of an optimal group size for collective movement .",
"A further interesting observation is that the benefits of having an optimal group size are not present when groups have offspring , and that few groups are of an optimal size , highlighting a potentially important feedback between current and future reproductive success .",
"The ability to develop and test our novel hypotheses relating to collective movement of animals in the wild was only possible by conducting a large-scale multi-group tracking study in a species that does exhibit little intra-group and inter-group competition .",
"We hope that cheaper , more reliable , and smaller devices , will continue to reveal similar insights into the lives of wild animals across the diversity of social systems ."
],
[
"We studied a population of vulturine guineafowl that resides in an area of approximately 500 ha in the southern part of the Mpala Research Conservancy ( MRC ) in Laikipia , Kenya .",
"Vulturine guineafowl are large , predominantly terrestrial , and highly gregarious birds .",
"They primarily forage on the ground , feeding on small grass root buds , seeds , fresh grass and small invertebrates , in other words widely dispersed food sources that have been predicted to limit within- and between-group contest competition ( Dammhahn and Kappeler , 2009 ) .",
"Based on 3 years of observations of colour-marked individuals , we found that they live in social groups , which range in size from 13 to 65 adults , have stable membership across years and remain completely cohesive ( with individuals rarely out of sight of other group members ) during non-breeding seasons ( typically January to March and July to October ) ( Papageorgiou et al . , 2019 ) .",
"Within a local area , groups form a multilevel society , which includes groups roosting communally ( with roosts containing several hundred birds ) and groups regularly encountering other groups during the day ( at which times between-groups agonistic interactions are rarely observed ) ( Papageorgiou et al . , 2019 ) .",
"Intergroup encounters and foraging are most common on glades ( Papageorgiou et al . , 2019 ) , which are small deforested areas that were previously used as overnight shelters for cattle and are therefore are rich in nutrients ( Young et al . , 1995 ) .",
"All vulturine guineafowl groups have access to glades as they are numerous in our study area .",
"On glades , vulturine guineafowl are prone to predation by raptors , such as martial eagles ( Naude et al . , 2019 ) , while moving through dense vegetation makes them prone to ambush by carnivorous mammals , such as jackals or leopards .",
"We trapped nearly all the adult members from each of the groups in our study area using large walk-in traps .",
"All trapped individuals were kept inside covered cages ( 1 m*50 cm*50 cm cage with 2 cm plastic mesh covered by a canvas ) to minimize stress and allow birds to remain standing while awaiting processing .",
"Birds were removed one by one , measured , and ringed with an individually numbered National Museums of Kenya stainless steel ring ( fitted on the tarsus of the right leg ) and a unique combination of four plastic colour bands fitted on each leg , on the tibiotarsus , for field identification .",
"For more details on trapping and marking processes , see Papageorgiou et al . , 2019 .",
"In each group , we fitted high-resolution solar-powered GPS tags ( 15 g Bird Solar , e-obs Digital Telemetry , Grünwald , Germany ) to between one and five individuals .",
"The devices were elevated using neoprene pads to prevent feathers from covering the tag’s solar panel , and were attached to the body using a backpack harness design ( Teflon ribbon ) , which included a cotton thread safety release mechanism .",
"GPS tags stayed on the birds from 6 months to 2 years , before the cotton breaks .",
"Groups were trapped once per year to mark new members and check the condition of the birds which carry GPS tags .",
"Since the start of the study in 2016 , no bird has ever been found with problems due to the GPS tags .",
"Each tag ( tag , pads , ribbons , crimps ) weighed approximately 20 . 5 g , far less than the recommended 3% of the birds’ body weight ( Kenward , 2001 ) .",
"Each device was programmed to record one data point ( date , time , coordinates ) every second when the battery had a high charge ( approximately every 2nd to 3rd day , for up to 8 hr continuously ) .",
"When the battery was at the next lower threshold , we set tags to record one point per second for the first 10 s of every 5th minute .",
"If battery charge was very low ( this setting was rarely used during our study period ) , tags were set to record one point every 15 min .",
"Data were remotely downloaded every 2 or 3 days using a BaseStation II ( e-obs Digital Telemetry , Grünwald , Germany ) .",
"Field-testing suggested that that the relative spacing of tags was accurate to within 1 m , 95% of the time tracked .",
"We uploaded all of our data to the Movebank repository ( Wikelski and Kays , 2018 ) , see https://www . movebank . org/cms/webapp ?",
"gwt_fragment=page=studies , path=study475851705 for further details .",
"We selected seasons with intermediate rainfall and greenness , as described in Papageorgiou et al . , 2019 .",
"Vulturine guineafowl groups temporarily split during the breeding ( wet ) season as breeding pairs form , subadults disperse from their natal groups and females lay and incubate their eggs ( Papageorgiou et al . , 2019 ) .",
"It is therefore challenging to quantify membership accurately during the breeding season .",
"Breeding seasons can occur twice a year ( during April or May and during October or November ) .",
"We also excluded periods of drought , as these cause groups to regularly travel to large bodies of water , such as the river that crosses our study area .",
"This unusual situation ( guineafowl are adapted to rarely require access to standing water under normal conditions ) would make the estimation of home-range size confounded by how far from the river each group roosts .",
"We thus made our selection of seasons based on the seasonal conditions where groups remain cohesive but do not make extraordinary movements , while remaining blind to the specific movement patterns of the GPS-tagged birds .",
"We defined seasons as being 2 months , which corresponds to period changes in conditions at our study site .",
"Given that our entire study population inhabits a small area , approximately 500 ha , and that all groups can easily access all parts of our study area , we assume that groups’ relative ranging behaviour was not restricted by the environment during these intermediate seasons .",
"Data for this study were collected from May 2017 to February 2020 , and the specific time periods that fulfilled the criteria listed above were categorised as Season A ( 30 . 5 . 2017 to 1 . 8 . 2017 ) , Season B ( 15 . 12 . 2017 to 15 . 2 . 2018 ) , Season C ( 1 . 8 . 2018 to 29 . 9 . 2018 ) , Season D ( 10 . 6 . 2019 to 9 . 8 . 2019 ) , and Season E ( 14 . 1 . 2020 to 16 . 3 . 2020 ) .",
"Census data was collected daily from a vehicle driving along the roads in our study area .",
"Every time a group was encountered , the identities of the marked birds were recorded , and a total number of birds present was counted ( including unmarked birds ) .",
"Each observation of a ‘group’ is defined as being a single or a multiple group , depending on whether the group moves away cohesively or whether individuals move away in different directions .",
"Counts are split into the number of adults and the number of chicks , with chicks being easily distinguishable until ~ 9 months of age .",
"Within each season , we selected groups containing one or more GPS tagged individuals for which we had precise group-size counts where observers could clearly see and count all the group members being present ( i . e . groups were counted more than once by more than one observer , the group size was found consistent across counts ) .",
"We included data on 21 different observational datasets of groups , with some groups replicated in more than one of the seasons ( Supplementary file 1A , Figure 1 ) .",
"For seasons in which a group had chicks , we calculated the median number of chicks per group , across all observations , in each study season .",
"We used the GPS data from each group in each season to quantify movement behaviour and resulting home-range characteristics .",
"In groups containing more than one GPS-tagged individual , we randomly selected one to represent that group’s movement , given that groups move very cohesively ( typically group members are within 29 m of each other 95% of the time tracked; Papageorgiou et al . , 2019 ) .",
"We calculated ( 1 ) the core home-range size ( 50% auto-correlated kernel density estimate ) , ( 2 ) average temporal overlap in space use , ( 3 ) average daily distance travelled .",
"We also explored ( 4 ) speed while moving .",
"We used the built-in features from the Movebank data repository to remove the very rare outliers in our dataset that were falling outside Kenya .",
"For ( 1 , 2 ) and ( 3 ) we used data points collected on the tenth second of every fifth minute , sub-setting this from high-resolution ( 1 Hz ) data and from data when the tag was set to collect 10 points every 5th minute .",
"This approach allowed us to ensure that the data points from all GPS-tagged birds were precisely synchronised in time .",
"For core home-range size ( 1 ) , we calculated home-range sizes by fitting a continuous-time movement model ( ctmm ) to each individual GPS dataset ( one bird per group per season ) .",
"These models follow a continuous-time stochastic process from which the maximum likelihood Gaussian home-range area can be extracted after the best fitted model is selected based on AIC .",
"Because ctmms model the actual movement , they better predict home-ranges than classical approaches , such as minimum convex polygons , that are typically prone to being highly influenced by just a few data points ( Fleming et al . , 2015 ) .",
"We used the maximum likelihood home-ranges to determine the area in which each bird was located 50% of the time .",
"This procedure was done using the ‘ctmm’ R package ( Calabrese et al . , 2016; Fleming and Calabrese , 2018 ) .",
"For average temporal overlap in space use ( 2 ) , we measured the inverse of the exploration of new areas , for each bird in each season .",
"We first fitted a ctmm to the 5-min data from each bird for the first 21 days it was tracked in each season ( longer time spans were not computationally feasible for this particular analysis ) .",
"We then calculated the overlap of the daily auto-correlated kernel density estimation for home-range of each bird , using the ‘overlap’ function of the ‘ctmm’ R package ( Winner et al . , 2018 ) .",
"Specifically , we calculated the overlap between daily ranges that were up to 3 days apart .",
"Finally we calculated the mean of the spatial overlap of one group with itself across each consecutive sets of 3 days , for the entire 21-day period , for each group .",
"For average daily distance travelled ( 3 ) , we used the 5-min data from above ( one bird per group per season ) and the function ‘speed’ from the ctmm R package , which provides the mean travel distance per day but not the speed while ‘on the go’ .",
"To estimate the mean speed ‘on the go’ ( 4 ) for each individual in each season we used only a subset of the data representing the times when the GPS tags were collecting one point per second ( 1 Hz ) .",
"From these data , we created a histogram of the log of the movement speeds ( distance travelled between consecutive points ) , revealing that speeds were multimodally distributed for each individual .",
"We found that the minima between the second and the third mode corresponded to −3 . 5 ( or 0 . 03 ms−1 ) and classified speeds exceeding this value as ‘moving’ and speeds slower than this value as ‘stationary’ ( Bender et al . , 2011; Wilson et al . , 2015 ) .",
"We then used the data classified as ‘moving’ to calculate the mean of the distance travelled in consecutive seconds of the 1 Hz data .",
"We fitted GEEs using the R package ‘geepack’ to test whether group size and number of chicks predicted home-range size , average temporal overlap in space use , average daily distance travelled and speed ‘on the go’ .",
"Our primary predictor variable was the number of adults in the group .",
"Because chicks could modulate the relationship between the number of adults ( our measure of group size ) and movement response variables , we also allowed models to include a term for the number of chicks .",
"Because we expected a non-linear relationship between group size and movement parameters , we also allowed models to include the number of adults squared ( the quadratic term ) .",
"Further , because we did not know how chicks could modulate group movement , we allowed the number of chicks and the number of chicks squared to be added in interactions with the number of adult and the number of adults squared .",
"We created all combinations of response variables and interactions ( only between terms containing adults and chicks ) , but ensuring that models contained at least the number of adults as a predictor , and always included the number of chicks when the number of chicks squared was included .",
"We used QIC to select the most parsimonious models ( lowest QIC ) ( Hocking , 2014 ) and we also calculated the R2 of each GEE to estimate their goodness of fit ( Zheng , 2000 ) .",
"For all models , we fitted group ID as a repeated measure to account for multiple measures of the same group across seasons .",
"All calculations and statistical tests were conducted in R version 3 . 5 . 1 ( R Development Core Team , 2018 ) with the significance threshold set at p≤0 . 05 .",
"We investigated the relationship between group size and reproductive success by fitting a GEE containing group size and it’s quadratic term as predictors of the median number of chicks in the group in a given season .",
"We limited this analysis to seasons during which there were chicks in our study population .",
"In Seasons A and D , the study population had no chicks since they had not bred for more than 10 months .",
"During the rest of the three study seasons ( Seasons B , C and E ) , the chicks where between 3 and 5 months old , and each season represented a different cohort of chicks .",
"The code to run the ctmms and conduct the statistics is available on https://github . com/DanPapageorgiou/Group_size; Papageorgiou , 2020; copy archived at swh:1:dir:fcd6639f112b1d96bfe8d122f96a2c0ed2a1673b . All data used in this study are stored on Movebank under the study name Avulturinum_Farine: https://www . movebank . org/cms/webapp ?",
"gwt_fragment=page=studies , path=study475851705 ."
]
] | [
"A challenge of group-living is to maintain cohesion while navigating through heterogeneous landscapes .",
"Larger groups benefit from information pooling , translating to greater ‘collective intelligence’ , but face increased coordination challenges .",
"If these facets interact , we should observe a non-linear relationship between group size and collective movement .",
"We deployed high-resolution GPS tags to vulturine guineafowl from 21 distinct social groups and used continuous-time movement models to characterize group movements across five seasons .",
"Our data revealed a quadratic relationship between group size and movement characteristics , with intermediate-sized groups exhibiting the largest home-range size and greater variation in space use .",
"Intermediate-sized groups also had higher reproductive success , but having more young in the group reduced home-range size .",
"Our study suggests the presence of an optimal group size , and composition , for collective movement ."
] | [
"Many social animals live in stable groups that stay together for years , or even lifetimes .",
"Being in a group offers a range of benefits , such as safety from predators , information on where to find food or water , and more accurate navigation .",
"But these benefits come at a cost .",
"The larger the group , the harder it is to make decisions that balance the needs of each individual .",
"So , while members of a large group should be better at locating resources and finding their way , they may take longer to decide where to go next .",
"In nature , groups of the same species can vary greatly in size and can have large or small numbers of offspring .",
"This raises the question of whether there is an optimal group size where the benefits of living together are maximized relative to the costs ?",
"To help answer this question , Papageorgiou and Farine studied the group behaviour of vulturine guineafowl , a social , ground-dwelling bird found in the savannahs of East Africa .",
"A lightweight GPS tracker was fitted to the members of 21 different groups of vulturine guineafowl to see how group size affects the movement of these birds .",
"The tags collected data every five minutes from dawn until dusk each day , and remained active over five two-month spans of similar weather conditions .",
"This revealed that groups of intermediate size , which contain 33 to 37 birds , ranged over larger areas allowing them to access more diverse resources , and used less energy by travelling shorter distances .",
"Birds in these groups also explored more new areas , decreasing their chances of encountering a predator , and produced more chicks , suggesting that their collective behaviour gave them a reproductive advantage .",
"These findings suggest that intermediate sized groups display an optimal level of movement compared to larger or smaller groups .",
"Understanding how social groups of different sizes interact with their environment can aid conservation planning .",
"Future work should focus on how this relationship changes with the seasons .",
"This could reveal more about the effects of group size during challenging conditions , like drought ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience",
"genetics and genomics"
] | Taar1 gene variants have a causal role in methamphetamine intake and response and interact with Oprm1 | elife-46472-v3 | [
[
"Amphetamine-like stimulants , including methamphetamine ( MA ) , remain the second most common class of illicit drugs used worldwide , behind cannabis-containing drugs ( United Nations Office on Drugs and Crime , 2016 ) .",
"MA is the most widely abused psychostimulant ( Chomchai and Chomchai , 2015 ) , and the consequences of MA addiction pose major societal and health concerns ( National Institute on Drug Abuse , 2019 ) .",
"MA-associated deaths in the United States were relatively stable between 2005 and 2013 , but have since been on an increasing trajectory , rising from about 3600 individuals in 2013 to almost 11 , 000 in 2017 ( National Institute on Drug Abuse , 2018 ) .",
"Discovering and understanding genetic factors that contribute to MA addiction risk , and uncovering the linked molecular and cellular processes , may help to curb this trend and improve interventions and long-term treatment success .",
"Whereas use of large human cohorts is effective at mapping loci and nominating candidate genes , precise experimental control over genetic composition , development , and drug exposure history is practical only in animal models .",
"Evidence that mouse models continue to contribute critical information to our understanding of pathological conditions was recently reviewed ( Nadeau and Auwerx , 2019 ) .",
"Selected lines of rodents have been derived to model genetic risk factors for drug use .",
"These include , particularly , those bred for various drug intake phenotypes , such as our mice bred for different amounts of voluntary MA intake ( Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .",
"In previous studies , we demonstrated a strong genetic contribution to level of voluntary MA consumption using multiple replicate sets of mice that we bidirectionally , selectively bred for MA high drinking ( MAHDR ) and MA low drinking ( MALDR ) , which for simplicity are also referred to here as the ‘high’ and ‘low’ lines .",
"Multiple lines of evidence support the involvement of the trace amine-associated receptor 1 gene , Taar1 , in differences in MA intake , including: quantitative trait locus ( QTL ) mapping in the high and low lines ( Belknap et al . , 2013 ) ; the direct agonist activity of MA at the trace amine-associated receptor 1 ( TAAR1 ) ( Bunzow et al . , 2001; Wolinsky et al . , 2007 ) ; the presence of a single nucleotide polymorphism ( SNP ) in Taar1 ( Taar1m1J ) that renders the receptor it expresses ( TAAR1 ) non-functional in response to MA and to other direct agonists ( Harkness et al . , 2015; Shi et al . , 2016 ) ; fixation of the Taar1m1J allele in high line mice of all five replicates , and retention of the wildtype , Taar1+ allele in all low line replicates ( Harkness et al . , 2015; Reed et al . , 2018 ) ; greater level of MA intake in Taar1 knockout mice compared to wildtype mice ( Harkness et al . , 2015 ) ; strong association of Taar1 genotype with MA intake in multiple genetic models ( Reed et al . , 2018 ) ; and strong association of Taar1 genotype with additional MA-related traits that correspond with level of MA intake , including level of sensitivity to MA-induced hypothermia and conditioned taste aversion ( Harkness et al . , 2015; Reed et al . , 2018 ) .",
"In the current studies , to demonstrate a causal role for Taar1 , we used CRISPR-Cas9 technology to replace the Taar1m1J allele found in high line mice with the Taar1+ allele , creating a knock-in with functional TAAR1 .",
"These mice were then tested for MA consumption in comparison to an unaltered high line control , with the prediction that knock-in of Taar1+ would reduce MA intake .",
"In addition , we tested knock-in and control mice for MA-induced change in body temperature to determine if this gene has a pleiotropic effect on this MA trait .",
"We predicted that insertion of the Taar1+ allele would increase sensitivity to MA-induced hypothermia in the knock-in mice .",
"Our existing data indicate that Taar1 genotype has a major impact on MA consumption , and gene mapping results demonstrate that a chromosome 10 QTL , at the location of Taar1 , accounts for as much as 60% of the genetic variance in risk for MA intake ( Belknap et al . , 2013 ) .",
"Other undefined variants must account for the remaining genetic variance .",
"We previously examined the µ-opioid receptor 1 gene , Oprm1 , as a quantitative trait gene ( QTG ) residing at the proximal end of mouse chromosome 10 , but it was eliminated as a risk factor for MA intake ( Eastwood et al . , 2018 ) .",
"Although Oprm1 variants do not predict risk , Oprm1 is involved in MA consumption via regulation by a top-ranked transcription factor network of genes that is differentially expressed between the MA drinking lines .",
"We found that Oprm1 and its product , the µ-opioid receptor ( MOP-r ) , are more highly expressed in the prefrontal cortex of low line mice , relative to high line mice ( Belknap et al . , 2013; Eastwood et al . , 2018 ) .",
"In addition , when MOP-r agonists were given to high line mice to test the hypothesis that increased MOP-r-regulated activity reduces MA intake , the acquisition of MA intake was attenuated , simulating the voluntary intake phenotype of the low line ( Eastwood et al . , 2018; Eastwood and Phillips , 2014a ) .",
"High line mice possess the same mutant Taar1m1J/m1J genotype as their progenitor DBA/2J ( D2 ) strain and are more likely to also possess the Oprm1 variant from the D2 strain , due to the relatively close proximity of Taar1 and Oprm1 on chromosome 10 ( 17 Mb apart ) .",
"Due to this linkage , we have been unable to explore independent and interactive effects of the Taar1 and Oprm1 genotypes on MA intake or other MA-related traits in the MA drinking lines .",
"Instead , we explored this in the present studies , using a family of related recombinant inbred mouse strains derived from a C57BL/6J ( B6 ) x D2 inbred strain intercross ( collectively the BXD strains ) in which the linkage is disrupted .",
"We examined the independent and interactive effects of these genes on MA intake and sensitivity to MA-induced change in body temperature , and performed QTL mapping for both traits using the BXD strain data ."
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"We predicted that MA intake would be reduced in high line mice in which the Taar1m1J allele was removed and replaced with the Taar1+ allele .",
"In the initial repeated measures analyses of variance ( ANOVAs ) , there were no interactions involving sex for either mg/kg MA consumption or total volume consumed , so we performed additional analyses with data for the sexes combined .",
"For MA consumption ( Figure 1a; Figure 1—source data 1 ) , there was a significant MA concentration x genotype interaction ( F[1 , 36]=66 . 8 , p<0 . 0001 ) .",
"Replacement of the Taar1m1J allele with the Taar1+ allele converted high MA intake to low MA intake for both MA concentrations .",
"Control mice , which are homozygous Taar1m1J , consumed significantly more MA at the 40 mg/l concentration , compared to the 20 mg/l concentration , whereas low and comparable levels of MA consumption were found for both MA concentrations in knock-in mice .",
"For total volume consumed from the water and MA tubes , during the time that MA was available ( Figure 1b; Figure 1—source data 1 ) , there was only a significant main effect of MA concentration ( F[1 , 36]=21 . 4 , p<0 . 0001 ) .",
"Total volume consumed was greater when mice were offered 40 mg/l MA , compared to 20 mg/l MA .",
"Thermal response to MA is a genetically-correlated response to selection for level of MA intake; thus , low MA drinking line mice display MA-induced hypothermia , whereas high line mice do not ( Harkness et al . , 2015 ) .",
"We tested the hypothesis that Taar1 genotype has a causal role .",
"Knock-in of Taar1+ in high line mice restored a hypothermic response to MA that did not occur in control mice ( Figure 2; Figure 2—source data 1 ) .",
"In the initial repeated measures ANOVA there were significant independent interactions of sex with genotype ( F[1 , 90]=7 . 1 , p=0 . 009 ) , MA dose ( F[1 , 90]=4 . 9 , p=0 . 03 ) and time ( F[4 , 360]=2 . 9 , p=0 . 02 ) .",
"However , sex did not determine the pattern of response to saline vs . MA in each genotype across time .",
"There was a significant 3-way genotype x MA dose x time interaction ( F[4 , 360]=10 . 2 , p<0 . 0001 ) that did not interact with sex .",
"We next examined the effects of genotype and time within each dose group .",
"In the saline-treated mice ( Figure 2a ) , there were significant main effects of time ( F[4 , 188]=35 . 9 , p<0 . 0001 ) and genotype ( F[1 , 47]=4 . 7 , p=0 . 04 ) .",
"Temperature declined by 0 . 3°C over time , regardless of genotype , and knock-in mice had 0 . 25°C higher body temperature than control mice , regardless of time .",
"In the MA-treated mice ( Figure 2b ) , the genotype x time interaction was significant ( F[4 , 188]=13 . 0 , p<0 . 0001 ) .",
"Knock-in mice had a significant hypothermic response to MA at T30 compared to T0 , whereas control mice were resistant to the hypothermic effects of MA , instead exhibiting significant MA-induced increases in body temperature at all post-MA treatment time points , compared to T0 .",
"Body temperatures were significantly higher in knock-in than control mice at baseline , by about 0 . 6°C , but significantly lower by about 1 . 2°C , at T30 .",
"Initial analyses characterized 18 BXD strains for MA consumption ( Figure 3; Figure 3—source data 1 ) .",
"There was a significant MA concentration x strain interaction ( F[17 , 215]=17 . 2 , p<0 . 0001 ) .",
"MA intake differed among the strains for both the 20 ( Figure 3a; p<0 . 0001 ) and 40 ( Figure 3b; p<0 . 0001 ) mg/l MA concentrations , and all Taar1m1J/m1J strains , but not Taar1+/+ strains , exhibited higher levels of MA intake from the 40 mg/l MA concentration than from the 20 mg/l concentration ( p=0 . 0006 to<0 . 0001 ) .",
"For total volume consumed when mice were offered water vs . MA , there was a main effect of strain ( F[17 , 215]=27 . 7 , p<0 . 0001 ) and a main effect of MA concentration ( F[1 , 215]=58 . 6 , p<0 . 0001 ) .",
"Total volume consumed differed among the strains by as much as 4 ml ( Figure 3c ) , but the strain distribution pattern for total volume did not correspond with the distributions for MA intake at either MA concentration .",
"Total volume consumed was significantly less when water and 20 mg/l MA were available , compared to when water and 40 mg/l MA were available ( mean ± SEM = 5 . 3±0 . 1 and 5 . 7 ± 0 . 1 for 20 and 40 mg/l , respectively ) .",
"We next examined the data from the BXD mice with regard to Taar1 and Oprm1 genotype to detect potential independent and epistatic effects on MA consumption .",
"First , to identify potential independent effects , we calculated Pearson’s correlations of each genotype with individual 20 and 40 mg/l MA intake amounts .",
"MA intake was significantly associated with Taar1 genotype ( r = 0 . 74 and 0 . 81 , ps <0 . 0001 , for 20 and 40 mg/l MA , respectively ) , but not Oprm1 genotype ( r = 0 . 06 and 0 . 10 , p=0 . 36 and 0 . 13 , for 20 and 40 mg/l MA , respectively ) .",
"Next , we considered main and interaction effects by repeated measures ANOVA with MA concentration , Oprm1 genotype , Taar1 genotype , and sex as factors .",
"There were no significant interactions involving sex , so further analyses were performed with data for the sexes combined .",
"Data are summarized in Figure 4 ( Figure 4—source data 1 ) , with mice grouped according to their four possible Taar1/Oprm1 genotypes ( Table 1 ) .",
"For MA consumption ( Figure 4a ) , there was a significant Oprm1 genotype x Taar1 genotype x MA concentration interaction ( F[1 , 225]=17 . 6 , p<0 . 0001 ) .",
"For consumption of MA from the 20 mg/l MA concentration , main effects of the Oprm1 ( F[1 , 229]=8 . 6 , p=0 . 004 ) and Taar1 ( F[1 , 229]=301 . 3 , p<0 . 0001 ) genotypes indicated that both genes influenced MA intake , with greater MA intake in Taar1m1J/m1J and Oprm1D2/D2 mice , compared to Taar1+/+ and Oprm1B6/B6 mice , respectively .",
"However , there was no significant Oprm1 genotype x Taar1 genotype interaction .",
"In contrast , for MA consumption from the 40 mg/l MA concentration , there was a significant Oprm1 genotype x Taar1 genotype interaction ( F[1 , 229]=13 . 9 , p=0 . 0002 ) .",
"MA intake was significantly higher in Taar1m1J/m1J mice , compared to Taar1+/+ mice , regardless of Oprm1 genotype ( ps <0 . 0001 ) .",
"However , Taar1m1J/m1J/Oprm1D2/D2 mice consumed significantly more MA than Taar1m1J/m1J/Oprm1B6/B6 mice .",
"Oprm1 genotype did not significantly impact MA intake in Taar1+/+ mice .",
"For total volume consumed ( Figure 4b ) , there were significant main effects of Oprm1 genotype ( F[1 , 225]=24 . 8 , p<0 . 0001 ) , Taar1 genotype ( F[1 , 225]=4 . 7 , p=0 . 03 ) , and MA concentration ( F[1 , 225]=75 . 7 , p<0 . 0001 ) , but no significant interactions .",
"Oprm1D2/D2 mice consumed more total volume than Oprm1B6/B6 mice , and Taar1m1J/m1J mice consumed more than Taar1+/+ mice .",
"In addition , total volume consumed was greater when mice were offered 40 mg/l MA , compared to 20 mg/l MA .",
"We used a similar approach to analyze body temperature data that were collected after saline or MA treatment in 15 BXD strains ( Figure 5; Figure 5—source data 1 ) .",
"For ease of viewing , the data in Figure 5 are separated by saline/MA treatment and Taar1 genotype for strain responses across time .",
"A repeated measures ANOVA detected a significant strain x MA dose x time interaction ( F[56 , 1172]=10 . 0 , p<0 . 0001 ) .",
"In saline-treated mice ( Figure 5a , b ) , the BXD strains differed at each time point ( ps = 0 . 04 to<0 . 0001 ) , except T0 , and body temperature differed across time in strains 161 ( Taar1+/+/Oprm1D2/D2 ) , 171 , 194 ( both Taar1m1J/m1J/Oprm1B6/B6 ) , 186 , 210 ( both Taar1m1J/m1J/Oprm1D2/D2 ) , and 218 ( Taar1+/+/Oprm1B6/B6 ) ( ps = 0 . 004 to<0 . 0001 ) .",
"In MA-treated mice ( Figure 5c , d ) , the BXD strains differed at each time point ( ps <0 . 0001 ) , except T0 , and body temperature differed across time in all strains ( ps = 0 . 007 to<0 . 0001 ) , except 160 , 171 , 194 ( all Taar1m1J/m1J/Oprm1B6/B6 ) , 178 , 186 , and 210 ( all Taar1m1J/m1J/Oprm1D2/D2 ) .",
"The maximum mean drop in body temperature in saline-treated mice was 0 . 9°C ( Figure 5a , b ) , whereas there was a maximum mean drop of 4°C in MA-treated Taar1+/+ mice ( Figure 5c ) , and a maximum increase of 1°C in MA-treated Taar1m1J/m1J mice ( Figure 5d ) .",
"We next examined the BXD data with regard to Taar1 and Oprm1 genotype to detect potential independent and epistatic effects on the hypothermic effect of MA .",
"Again , to identify potential independent effects , we calculated Pearson’s correlations of each genotype with MA-induced change in body temperature from T0 to T30 .",
"There was a significant correlation of body temperature change with Taar1 genotype ( r = 0 . 71 , p<0 . 0001 ) , but not with Oprm1 genotype ( r = 0 . 11 , p=0 . 16 ) .",
"Next , we considered main and interaction effects by repeated measures ANOVA with time , Oprm1 genotype , Taar1 genotype , sex and MA dose as factors .",
"There were significant independent interactions of sex with MA dose ( F[1 , 307]=8 . 0 , p=0 . 006 ) and time ( F[4 , 1228]=5 . 0 , p=0 . 0007 ) .",
"However , sex did not play a significant role in the pattern of response of each genotype to each dose across time .",
"There was a significant Oprm1 genotype x Taar1 genotype x MA dose x time interaction ( F[4 , 1228]=8 . 0 , p<0 . 0001 ) that did not interact with sex .",
"We next examined the effects of genotype and time within each dose group ( Figure 6; Figure 6—source data 1 ) .",
"In the saline-treated mice ( Figure 6a ) , the Oprm1 genotype x time interaction was significant ( F[4 , 596]=2 . 6 , p=0 . 04 ) .",
"Both Oprm1B6/B6 and Oprm1D2/D2 mice exhibited reductions in body temperatures at T60-120 ( ps <0 . 001 ) , compared to T0 .",
"However , these differences in body temperature were of no more than 0 . 3°C on average .",
"In the MA-treated mice ( Figure 6b ) , there was a significant 3-way interaction of Oprm1 genotype x Taar1 genotype x time ( F[4 , 664]=11 . 4 , p<0 . 0001 ) .",
"Taar1+/+/Oprm1D2/D2 mice had significantly lower body temperatures at all post-MA treatment time points , compared to Taar1+/+/Oprm1B6/B6 mice .",
"Taar1+/+/Oprm1B6/B6 mice exhibited a significant hypothermic response to MA at T30-90 , compared to T0 , and recovered to baseline ( T0 ) temperatures by T120 , whereas Taar1+/+/Oprm1D2/D2 mice exhibited a significant hypothermic response to MA at T30-120 , and remained below baseline for the duration of testing .",
"However , the rate of increase after the initial decrease at T30 was similar for the two genotypes .",
"Regardless of Oprm1 genotype , Taar1m1J/m1J mice were resistant to the hypothermic effects of MA , exhibiting increases in body temperature at all post-MA treatment time points , compared to T0 .",
"The QTL results for both traits are displayed in Figure 7 ( Figure 7—source data 1 ) .",
"A significant QTL in the same region of chromosome 10 emerged for both MA consumption ( Figure 7a ) and body temperature response to MA ( Figure 7b ) .",
"This QTL is at the location of the Taar1 SNP ( 23 . 9 Mb ) .",
"The correlations between the strain means and the Taar1 SNP were r = 0 . 94 , p<0 . 0001 for MA consumption and r = 0 . 82 , p=0 . 02 for temperature response to MA .",
"In addition , suggestive QTLs were detected on chromosomes 17 and 18 for MA effect on body temperature .",
"There was a strong statistical trend for an increase in the chromosome 10 logarithm of the odds ( LOD ) score for MA intake ( from 8 . 2 to 8 . 8; p=0 . 07 ) , and a significant increase for the chromosome 10 LOD score for temperature response ( from 3 . 7 to 4 . 2; p=0 . 01 ) , when results were compared for interval mapping and composite interval mapping .",
"Composite mapping identified a suggestive QTL on chromosome four for MA intake that had not reached the suggestive significance threshold in initial mapping; however , the LOD change from 2 . 5 to 2 . 7 was not statistically significant ( Figure 7c ) .",
"The LOD scores for the suggestive QTLs on chromosomes 17 and 18 for temperature response were comparable for initial and composite mapping ( Figure 7d ) ."
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"We leveraged CRISPR-Cas9 technology to replace the mutant Taar1m1J allele with the common Taar1+ allele in selectively bred high line mice .",
"The patterns of MA consumption in mice with the different Taar1 genotypes mimicked the patterns previously observed in the MA drinking lines ( Harkness et al . , 2015; Shabani et al . , 2011; Wheeler et al . , 2009 ) .",
"Thus , control mice escalated their MA consumption with increasing MA concentration , similar to previous findings in high line mice , whereas MA consumption was low and not concentration-dependent in the knock-in mice , similar to previous findings in the selectively bred low line mice .",
"Although oral is not the most common route of administration , some MA users do take MA orally , and when taken via this route , MA has a half-life similar to that for other routes of administration ( Cruickshank and Dyer , 2009 ) .",
"Furthermore , oral consumption , like other routes of administration , can lead to dependence ( Galloway et al . , 2010 ) .",
"On average , mice with absent TAAR1 function consume about 6 mg/kg/18h from a 40 mg/l MA solution ( Harkness et al . , 2015; Hitzemann et al . , 2019; Reed et al . , 2018; Shabani et al . , 2011; Wheeler et al . , 2009 ) .",
"High line mice will consume an average of as much as 14 mg/kg/18h , when MA is offered as a 140 mg/l concentration ( Shabani et al . , 2016 ) .",
"Daily MA use in humans ranges from 300 to 800 mg/day on average ( Cho et al . , 2001; Simon et al . , 2002 ) , which translates to 3 . 9 to 10 . 4 mg/kg/day in a 77 kg individual .",
"Whereas the half-life of MA is shorter in rodents compared to humans ( Cho et al . , 2001 ) , relevant levels of MA are attained in our mouse model , with locomotor stimulant effects observed in the high line mice at consumed doses as low as 0 . 4 mg/kg in 1h ( Shabani et al . , 2012a ) .",
"In rats with functional TAAR1 , TAAR1 agonists delivered systemically or to specific subregions of the mesocorticolimbic system , decreased MA seeking and cocaine seeking ( Liu et al . , 2017; Pei et al . , 2017 ) , suggesting that treatments that increase TAAR1 activity could be beneficial therapeutics .",
"However , when TAAR1 is non-functional , receptor agonists are not an option , leaving us to consider downstream mechanisms of TAAR1 activation .",
"Details of the mechanisms enlisted by TAAR1 activation remain elusive .",
"Expressing TAAR1 using a heterologous expression system to identify its messenger system ( s ) has proved difficult , potentially due to the predominant intracellular localization of the receptor .",
"Studying the function of TAAR1 is also complicated by the lack of good quality antibodies and selective antagonists ( Liu and Li , 2018; Rutigliano et al . , 2017 ) .",
"There is one available TAAR1 antagonist , EPPTB ( Bradaia et al . , 2009 ) , but it has a high rate of clearance ( Rutigliano et al . , 2017; Stalder et al . , 2011 ) and poor solubility , which limit in vivo use .",
"Our attempts to develop better antagonists with chemist collaborators have not yet met with success , and other investigators in the field have had a similar experience ( Lam et al . , 2018 ) .",
"One strategy that could be considered for reducing MA consumption is to target mechanisms that are impacted by the absence of TAAR1 function .",
"For example , TAAR1 modulates monoaminergic neurotransmission ( Espinoza et al . , 2015a; Leo et al . , 2014; Lindemann et al . , 2008; Revel et al . , 2011; Xie and Miller , 2008 ) , and recently has been implicated in glutamatergic neurotransmission ( Espinoza et al . , 2018; Espinoza et al . , 2015b ) .",
"High line mice are hyperglutamatergic at baseline , compared to low line mice , in both the nucleus accumbens ( NAc ) and medial prefrontal cortex ( mPFC ) .",
"They also exhibit a larger glutamate response to MA in the NAc , but not mPFC ( Lominac et al . , 2016; Szumlinski et al . , 2017 ) .",
"In addition , compared to low line mice , high line mice have blunted dopamine levels in both the NAc and mPFC , and exhibit a heightened dopaminergic response to MA in the mPFC , but not NAc ( Lominac et al . , 2014 ) .",
"Whether these differences are related to Taar1 genotype is unknown , but they suggest certain manipulations to be examined for their role in MA intake .",
"Our previous findings in the MA drinking lines demonstrated that the thermal response to MA is genetically-correlated with level of MA consumption .",
"Thus , high line mice are resistant to the hypothermic effect of MA , whereas low line mice are highly sensitive to this effect of MA ( Harkness et al . , 2015 ) .",
"Here , we performed QTL analysis using data from the BXD strains and identified a significant QTL on chromosome 10 for the effect of MA on body temperature .",
"This QTL is in the SNP-poor region of chromosome 10 ( Shi et al . , 2016 ) , where we mapped the QTL for MA consumption in the MA drinking lines and BXD strains .",
"QTL mapping was previously performed in BXD strains for the effects of 4 , 8 and 16 mg/kg MA on body temperature recorded 48 min after administration .",
"Although a QTL was mapped on chromosome 10 for the 4 mg/kg dose , it was considerably distal to the current QTL ( Grisel et al . , 1997 ) , and not likely to be the same one for several reasons .",
"First , the MA doses and time after administration were considerably different from those in our studies .",
"But , more importantly , the Grisel et al . ( 1997 ) paper was published well before the Taar1m1J mutation arose in D2 mice ( Reed et al . , 2018 ) ; therefore , all of the BXD strains in that study shared the common Taar1+/+ genotype .",
"We have confirmed the Taar1+/+ genotype of many of the BXD strains that were derived prior to when the mutation appeared ( Reed et al . , 2018; Shi et al . , 2016 ) .",
"Sensitivity to MA-induced hypothermia corresponds with low levels of MA consumption in MAHDR-Taar1+/+ knock-in mice , MALDR line mice , the wildtype littermates of Taar1 knockout mice , and BXD strains with the Taar1+/+ genotype .",
"These data could indicate that Taar1 has independent pleiotropic effects on the two traits .",
"Another possibility is that the subjective experience of MA-induced hypothermia in mice with functional TAAR1 limits MA consumption , thereby playing a protective role .",
"Not known is whether MA-induced hypothermia is subjectively unpleasant .",
"However , hypothermia prolonged the associative period during which aversion could be conditioned in a conditioned taste aversion ( CTA ) procedure ( Misanin et al . , 1998; Misanin et al . , 2002 ) , and mice with functional TAAR1 form MA-induced CTA , whereas those without functional TAAR1 do not ( Harkness et al . , 2015; Shabani et al . , 2012b; Wheeler et al . , 2009 ) .",
"Low line and high line mice voluntarily consume similar amounts of MA on the first day that MA is offered , but low line mice then decrease consumption on the subsequent day , and remain at low intake levels from then on ( Eastwood et al . , 2014; Shabani et al . , 2012a ) .",
"It is possible that initial MA consumption induces hypothermia in low line mice , so that initial MA consumption is associated with this potentially unpleasant physiological effect , and reduces the desire to consume more MA .",
"Additional research is needed to determine if MA drinking produces changes in body temperature , as occurs in response to an IP injection of MA .",
"Regardless of the chicken-egg question , the Taar1+ allele replacement in high line mice produced MA intake levels like those found in low line mice , indicating that this gene has a major role in determining level of MA intake .",
"Similarly , both the pattern and magnitude of hypothermic response to MA in the knock-in mice resembled those of low line mice .",
"Low line mice had a maximal response of 1–2°C ( depending upon replicate line ) after treatment with 2 mg/kg MA ( Harkness et al . , 2015 ) , and the response in the knock-in mice was also maximal at 30 min and of about 1 . 2°C .",
"Therefore , Taar1 appears also to have a primary role in the hypothermic response to MA .",
"However , Taar1 genotype does not impact basal body temperature , nor the hypothermic response to ethanol ( Harkness et al . , 2015 ) .",
"Further , the Taar1 mutation does not appear to affect locomotor activity at baseline or in response to the 2 mg/kg dose of MA used here ( Shabani et al . , 2011; Wheeler et al . , 2009 ) , making it unlikely that differences in thermal response to MA in mice with different Taar1 genotypes are due to differences in movement after treatment .",
"In addition to the significant QTL on chromosome 10 for MA intake and MA-induced thermal response , we identified other genomic regions at the suggestive level of significance .",
"None of these overlapped for the two traits , and the chromosome 4 MA intake QTL that reached the suggestive significance threshold with composite mapping was not identified in our previous studies of the MA drinking lines ( Belknap et al . , 2013 ) .",
"There may be additional alleles that account for smaller amounts of genetic variance that our analysis did not have the power to detect .",
"The need for large sample sizes to detect rare variants and alleles with smaller independent effects for complex traits is recognized ( Belknap , 1998; Belknap et al . , 1996; Buchner and Nadeau , 2015; Flint , 2011; Solberg Woods , 2014 ) .",
"Previous findings indicate that Oprm1 regulation and opioid system activity contribute to MA consumption in mice ( Belknap et al . , 2013; Eastwood et al . , 2018; Eastwood and Phillips , 2014a; Eastwood and Phillips , 2014b ) , and MA dependence/psychosis in humans ( Ide et al . , 2004; Ide et al . , 2006 ) .",
"Our results in the BXD strains with different combinations of Taar1/Oprm1 genotypes support a potential epistatic interaction in the effects of these genes on MA consumption and on the hypothermic response to MA .",
"The combined effects of Oprm1 and Taar1 genotype on each trait was non-additive .",
"Oprm1 genotype impacted MA consumption in Taar1m1J/m1J mice , but not Taar1+/+ mice .",
"The opposite pattern of effect emerged for MA-induced hypothermia; Oprm1 genotype impacted this trait in Taar1+/+ mice , but not Taar1m1J/m1J mice .",
"For both traits , the Oprm1D2/D2 mice exhibited the more robust effects , compared to Oprm1B6/B6 mice , and Oprm1 genotype had an impact only in mice with the stronger MA trait .",
"Thus , Taar1m1J/m1J/Oprm1D2/D2 mice consumed more MA than Taar1m1J/m1J/Oprm1B6/B6 mice , and Taar1+/+/Oprm1D2/D2 mice showed greater MA-induced hypothermia compared to Taar1+/+/Oprm1B6/B6 mice .",
"Low levels or absence of the MA-related phenotype could preclude the Oprm1 genotype from having an effect .",
"This interaction between Taar1 and Oprm1 was supported by composite interval QTL mapping for MA-induced body temperature change , in which controlling for Oprm1 genotype resulted in a significantly increased LOD score , specifically for the chromosome 10 QTL .",
"For MA intake , the LOD score increase did not meet the significance threshold , but the strong statistical trend deserves further consideration , which could be accomplished by testing more BXD strains of appropriate genotypes , as they become available , to increase power .",
"When we assessed the independent associations of each genotype with these MA-related traits , significant correlations were found only with Taar1 genotype .",
"That Oprm1 genotype alone did not correspond with amount of MA consumed is consistent with our previous findings , indicating that Oprm1 genotype is not a risk factor for MA intake , and its interactive effect with Taar1 may be associated with the regulation of Oprm1 by a significant MA intake risk gene network ( Belknap et al . , 2013 ) .",
"The interactive effect of Oprm1 with Taar1 , in the absence of an independent effect of Oprm1 , is consistent with other studies demonstrating epistatic interactions involving polymorphisms that do not have significant independent associations with phenotypes ( Lehner , 2011 ) .",
"Epistasis has been proposed as a reason for variability in disease occurrence among individuals with disease risk mutations .",
"However , like the many intermolecular epistatic interactions with unknown mechanisms ( Lehner , 2011 ) , the mechanism ( s ) underlying the impact of Oprm1 genotype on these Taar1-associated traits is not yet known .",
"One possible mechanism to explore relates to differential Oprm1 expression .",
"A polymorphism in the promoter region of Oprm1 was related to promoter activity , in which D2 mice had greater Oprm1 promoter activity compared to B6 mice ( Doyle et al . , 2006 ) .",
"It was hypothesized that this might alter MOP-r expression .",
"Doyle et al . ( 2014 ) found greater Oprm1 expression in the frontal cortex of D2 , compared to B6 mice .",
"It is possible that greater expression of Oprm1 could result in greater MA intake in Taar1m1J/m1J/Oprm1D2/D2 mice , and greater MA-induced hypothermia in Taar1+/+/Oprm1D2/D2 mice .",
"However , Eastwood et al . ( 2018 ) examined MOP-r protein density in D2 vs . B6 mice in the prefrontal cortex and did not find a difference .",
"On the other hand , our low line mice , which consumes little MA , is sensitive to MA-induced hypothermia and is largely of the Taar1+/+ genotype , had both greater Oprm1 expression and MOP-r density exclusively in the prefrontal cortex , compared to our high line mice , which has the opposite MA-related phenotypes and Taar1m1J/m1J genotype ( Belknap et al . , 2013; Eastwood et al . , 2018 ) .",
"Thus , the hypothesis that greater Oprm1 expression results in greater MA-induced hypothermia in Taar1+/+ genotype mice is consistent with these findings , but the hypothesis that greater Oprm1 expression results in greater MA intake in Taar1m1J/m1J genotype mice is not consistent with the existing data .",
"In fact , greater MA intake is associated with reduced prefrontal cortex Oprm1 and MOP-r expression in the high line , and peripheral application of drugs that increase MOP-r activity reduced MA consumption in high line mice ( Eastwood et al . , 2018; Eastwood and Phillips , 2014a ) .",
"We are currently creating Taar1+/+ knock-in mice on a D2 strain background and Taar1m1J/m1J knock-in mice on a B6 background .",
"Epistatic behavioral and molecular effects may be more approachable for study on these homogeneous backgrounds .",
"We observed genotype-dependent effects on total volume consumed from the MA and water tubes , but conclude that genotypic differences in MA consumption were not an artifact of overall fluid intake differences .",
"The total volume consumption difference between Taar1m1J/m1J and Taar1+/+ mice was small , compared to their robust difference for MA intake .",
"Furthermore , the interactive effect of Taar1 and Oprm1 was specific to MA intake .",
"We previously reported greater total fluid intake in high line mice , compared to low line mice , of about the same magnitude ( ~0 . 5 ml ) in some , but not all , replicate sets of lines ( Hitzemann et al . , 2019; Shabani et al . , 2011; Shabani et al . , 2019; Wheeler et al . , 2009 ) .",
"Locomotor behavior has been found to be positively associated with MA intake in the low and high lines ( Shabani et al . , 2012a ) , and larger intake volumes in mice consuming more MA could be related to stimulant effects that impact fluid needs .",
"But , because a difference in total fluid intake has not always been found , another interpretation is that , due to greater reinforcing effects of MA in mice of the Taar1m1J/m1J genotype , such as the high line mice ( Shabani et al . , 2012a ) , avidity for MA is greater , leading to larger intake volumes .",
"We observed some differences in body temperature between the knock-in and control mice that were unrelated to MA treatment .",
"However , these baseline differences did not appear to account for the genotype-dependent MA response .",
"Although the knock-in mice had higher baseline temperatures and therefore , a greater opportunity to experience a reduction in body temperature when treated with MA , the difference at baseline was of about 0 . 5°C , whereas the difference at 30 min post MA treatment was over 1°C .",
"This was , in part , because the MA responses were qualitatively opposite , with the knock-in mice exhibiting decreases in body temperature , similar to MALDR mice , and the controls exhibiting a time-dependent increase , similar to MAHDR mice ( Harkness et al . , 2015 ) .",
"In our previous studies , sex differences for MA consumption and the thermal response to MA were inconsistently found , and some are summarized in Reed et al . ( 2018 ) .",
"Generally , when there have been differences , females have consumed about 15% or 0 . 5 mg/kg more MA than males , but in no case were there differences between the MAHDR and MALDR lines or between mice with the different Taar1 genotypes in only one sex ( Eastwood and Phillips , 2014a; Harkness et al . , 2015; Reed et al . , 2018; Shabani et al . , 2011; Shabani et al . , 2016; Wheeler et al . , 2009 ) .",
"Likewise , sex did not have a significant impact on genotype-dependent differences in MA consumption in the present studies .",
"Similarly , there were some sex effects in the body temperature studies , but sex did not determine the pattern of response to saline vs . MA in each genotype across time .",
"Thus , these sex effects did not impact overall interpretations of data for either trait .",
"One consideration regarding Taar1 involvement in MA consumption and MA thermal response is the potential for differential potency of agonists interacting with the two TAAR1 isoforms .",
"However , our analyses indicate that the receptor expressed by Taar1m1J is non-functional , rather than sub-functional .",
"Thus , the D2-like isoform of TAAR1 , expressed by Taar1m1J , exhibits no cAMP response to a wide range of MA concentrations , or to TAAR1-specific agonists ( Harkness et al . , 2015; Shi et al . , 2016 ) .",
"If the Taar1 mutation reduces potency of agonists , we would expect a shift in the agonist dose-response curve for cAMP accumulation .",
"Further , at doses of MA up to 16 mg/kg , high line mice fail to exhibit MA-induced hypothermia , whereas low line mice exhibit hypothermia at doses as low as 1 mg/kg that reverses toward hyperthermia at higher doses ( Harkness et al . , 2015 ) .",
"Thus , the hypothermic effect of MA via TAAR1 may compete with the expression of MA-induced hyperthermia occurring via a non-TAAR1 mechanism in low mice .",
"The gRNA used to produce the MAHDR-Taar1+/+ knock-in mice was a perfect match for the sequence on either side of the chromosome 10 Taar1 SNP , with imperfect mapping to some additional chromosomes .",
"In our original QTL analysis for MA drinking and in the current analysis ( Figure 7 ) , there were no significant or suggestive QTLs mapped to those additional chromosomes ( Belknap et al . , 2013; and data herein ) .",
"This suggests that if off-target deletions or insertions occurred at these locations , they would be unlikely to have the profound impact on the MA-related traits that we have observed .",
"The chromosome 10 QTL accounts for 60% of the genetic variance in MA intake , and Taar1 genotype-phenotype correlations range from r = 0 . 81 to 0 . 96 for 40 mg/L MA intake , and r = 0 . 71 to 0 . 82 for 2 mg/kg MA-induced hypothermia ( Reed et al . , 2018; and data herein ) .",
"Therefore , the large effects we observed would be expected for replacement of Taar1m1J with Taar1+ .",
"The high line mice possess a genetically heterogeneous background; thus , sequencing the CRISPR knock-in and control mice would not provide specific information about off-target effects , because the existing genetic variation would not be separable from such effects .",
"Further , there appears to be some consensus that off-target effects of CRISPR-Cas9 are rare and indistinguishable from the background rate of de novo mutation ( e . g . , Anderson et al . , 2018; Ayabe et al . , 2019; Iyer et al . , 2018; Mianné et al . , 2016; Nakajima et al . , 2016; Willi et al . , 2018 ) .",
"The knock-in and control mice used in our studies were produced by independent breeding pairs .",
"As noted in Materials and methods , this interbreeding of like-Taar1-genotype individuals is a consistent feature for all other existing populations in which the Taar1 SNP exists .",
"The only exception has been for the B6 x D2 F2 mice that were produced to create the high and low selected lines , which existed as multi-Taar1-genotype littermates .",
"An equally strong association of Taar1 genotype with MA intake was found in these mice ( Reed et al . , 2018 ) .",
"In summary , these data demonstrate that Taar1 is a major contributor to MA intake and MA-induced hypothermia , and provide evidence for an interaction between Taar1 and Oprm1 in their effects on these MA traits .",
"Genetic variation in the human Oprm1 gene has been associated with MA dependence/psychosis ( Ide et al . , 2004; Ide et al . , 2006 ) , but additional research results in this area have not appeared in the literature .",
"The potential impact of human TAAR1 genetic variation on risk for MA use or on the magnitude of MA-related phenotypes is not yet known .",
"There are over 200 non-synonymous SNPs with the potential for missense variants in human TAAR1 ( Rutigliano et al . , 2017 ) Initial examination of some of these human TAAR1 variants indicates that TAAR1 proteins with variable levels of function are expressed ( Shi et al . , 2016 ) .",
"The success of TAAR1 agonists in treatment is dependent upon at least partial receptor function that can be enhanced .",
"Whether human TAAR1 variants are relevant to MA use and addiction in humans , and whether TAAR1 agonists are a viable treatment option , are important research questions to pursue .",
"Further , research into potential interactive effects between human TAAR1 and OPRM1 variants for their impact on MA use risk and response would be valuable ."
],
[
"All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals ( National Research Council , 2011 ) and were approved by the Institutional Animal Care and Use Committee of the VA Portland Health Care System ( VAPORHCS ) .",
"Male and female mice were group housed ( 2–5 per cage ) in filtered polycarbonate shoebox cages ( 28 . 5 × 17 . 5×12 cm ) that were lined with Bed-o’Cobs bedding ( The Andersons , Inc , Maumee , OH ) and fitted with wire tops , except during MA drinking studies .",
"For these studies , mice were individually housed in the same type of caging and provided with a cotton fiber nestlet for enrichment .",
"All mice were maintained on a 12:12h light:dark schedule ( lights on at 0600h ) , and had ad libitum access to laboratory rodent block food ( PicoLab Laboratory Rodent Diet 5LOD , 4 . 5% fat content; Animal Specialties , Woodburn , OR ) and tap water .",
"The Taar1m1J mouse SNP , rs33645709 , encodes a non-synonymous proline to threonine mutation at amino acid position 77 that originally occurred in D2 mice in 2001–2003 as a spontaneous mutation ( Reed et al . , 2018 ) .",
"Proline is often highly conserved , because it is essential for conservation of a particular protein fold .",
"In fact , this mutation renders TAAR1 non-functional , and the SNP is fixed ( homozygous ) in high line mice ( Harkness et al . , 2015; Reed et al . , 2018; Shi et al . , 2016 ) .",
"CRISPR-Cas9 technology ( Jinek et al . , 2012; Zhang , 2012 ) was used to replace the mutant Taar1m1J allele with the reference Taar1+ allele in high line mice to generate a homozygous Taar1+/+ knock-in on the high MA drinking line background at the Oregon Health & Science University Transgenic Mouse Models Shared Resource .",
"CRISPR in vitro transcribed guide RNAs ( gRNAs ) , targeting the specified region of Taar1 , and donor ( 100b ) oligo single strand DNA ( ossDNA ) for incorporation of the Taar1+ sequence were designed and synthesized by ThermoFisher Scientific ( Carlsbad , CA , USA ) ( Figure 8a ) .",
"During the development of the gRNA , in silico analysis with the basic local alignment search tool or BLAST was used to assure specificity of the sequence .",
"The gRNA chosen for use was a perfect match for Taar1 , with no mismatches , except at the SNP location .",
"The gRNA sequence had similarity to nine additional regions on chromosomes 1 , 3 , 5 , 9 , 11 and 19 , but with 2–3 mismatches in each case .",
"This gRNA had the sequence tctgataatgAcctgcagcatgg .",
"The location of the SNP is indicated by the capital A , encoding the mutant genotype present in the high line .",
"The location of the SNP within the Taar1 ossDNA sequence , which is the reference genotype , is indicated below by the capital C , with the gRNA sequence underlined .",
"The gene editing replaces A with C: cFFctggctccttcactccatggccattgtcgactttctgctgggctgtctgataatgCcctgcagcatggtgagaactgttgagcgctgttggtatZZt .",
"The ossDNA was designed without a silent mutation and to be asymmetric to provide increased efficiency of the gRNA .",
"Cas9 mRNA ( catalog number L-7606 ) was purchased from TriLink ( San Diego , CA , USA ) .",
"High line mice were embryo donors .",
"Three-four week old , female mice were super-ovulated , mated with high line males , and 0 . 5 day old eggs were isolated as previously described ( Hogan et al . , 1994 ) .",
"A mixture of Cas9 mRNA , Taar1 gRNA , and donor ossDNA for Taar1+ ( at concentrations of 100 ng/µl , 50 ng/µl , and 100 ng/µl , respectively ) was injected into the pronuclei of one-cell fertilized eggs .",
"The injected eggs were transferred into the oviducts of pseudo pregnant recipient CD1/NCrl foster dams ( Charles River , San Diego , CA , USA ) , and the surviving offspring were sequenced ( see Genotyping and sequencing ) to determine if they had retained the Taar1m1J allele or if an alteration resulting in insertion of Taar1+ had occurred .",
"Control mice ( MAHDR-Taar1m1J/m1J ) were derived at the same time , from those mice in which the Taar1m1J allele was not successfully altered .",
"These offspring were transported to the VAPORHCS vivarium and bred to produce the MAHDR-Taar1+/+ knock-in and MAHDR-Taar1m1J/m1J control mice that participated in our experiments .",
"Knock-in and control mice were maintained as within-line breeding pairs in a shared colony room .",
"This breeding of individuals with the same Taar1 genotype is consistent with the maintenance of all other populations in which the Taar1 SNP exists ( e . g . , MAHDR vs . MALDR; BXD strains; D2 mice from The Jackson Laboratory vs . D2 mice from other suppliers; Reed et al . , 2018 ) .",
"Representative chromatographs for the Taar1m1J ( control ) and Taar1+ ( knock-in ) sequences are displayed in Figure 8b and c , respectively .",
"Separate cohorts of knock-in and control mice were tested for two-bottle choice MA intake or MA-induced change in body temperature , as detailed below and in our previous publications ( Harkness et al . , 2015; Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .",
"For MA intake , 20 knock-in and 20 control mice were tested in a single cohort ( n = 10/genotype/sex ) , and the mice were 87–90 days old at the start of testing .",
"For body temperature , 48 knock-in and 50 control mice were tested in 3 cohorts of 25–46 mice ( final n = 12–13/genotype/dose/sex ) , and the mice were 79–94 days old on the day of testing .",
"Breeding pairs that produced the BXD mice tested in these studies were obtained from RWW ( University of Tennessee Health Science Center , Memphis , TN ) , and established within the VAPORHCS vivarium .",
"Specific strains were chosen according to Taar1 and Oprm1 genotypes to allow identification of potential independent and interactive effects on MA-related phenotypes .",
"Separate cohorts of offspring were tested for two-bottle choice MA intake or MA-induced body temperature change .",
"Mice were homozygous for either the reference Taar1+ or mutant Taar1m1J allele , as well as for the B6 or D2 Oprm1 allele , in all possible combinations ( Table 1 ) .",
"A total of 233 BXD mice ( 134 female and 99 male ) , ranging from 53 to 114 days of age , were tested for MA intake in 4 cohorts of 27–97 mice .",
"A total of 325 BXD mice ( 171 female and 154 male ) , ranging from 49 to 124 days of age , were tested for MA-induced body temperature change in 16 cohorts of 8–36 mice .",
"Numbers of BXD mice with the four potential combined Taar1/Oprm1 genotypes ( Table",
"1 ) were n = 22–45/genotype/sex for MA intake , and n = 14–25/genotype/sex/MA dose for the body temperature study .",
"We previously established and described genotyping methods for the Taar1 SNP ( Harkness et al . , 2015; Reed et al . , 2018 ) .",
"Genomic DNA was extracted from ear punch or tail snip samples , using QuickExtract DNA Extraction Solution ( Lucigen , Middleton , WI , USA ) .",
"For samples from the BXD mice and the original mice created by the CRISPR-Cas9 procedure , the Taar1 region was PCR amplified using a Hotstart DNA polymerase kit ( Qiagen , Valencia , CA , USA ) , with sequence-specific primers surrounding the region of interest ( see Harkness et al . , 2015 for details ) .",
"PCR products were sequenced at the Oregon Health & Science University Sequencing Core and aligned with the mouse Taar1 sequence ( NM_053205 . 1 ) to identify which allele ( s ) was present based on the rs33645709 SNP .",
"All knock-in and control offspring of the original mice were genotyped with an updated protocol that uses a standard Taqman ( ThermoFisher Scientific ) with fluorescent probes to detect and differentiate the Taar1 alleles ( Reed et al . , 2018 ) .",
"We performed Oprm1 genotyping for BXD mice as previously described ( Ferraro et al . , 2005 ) .",
"Sequence-specific primers for exon 10 of Oprm1 were used as the forward primer ( 5'-ggttatgcctctctggattag-3' ) and reverse primer ( 5'-tccatcgcttacatcttacca-3' ) .",
"A SNP in exon 10 of the B6 Oprm1 gene ( Oprm1B6 ) creates a DdeI restriction site ( Ferraro et al . , 2005 ) , so that two bands ( 235 and 168 bp ) are created when amplified DNA from mice with Oprm1B6 is digested with the DdeI restriction enzyme; DdeI does not excise the D2 Oprm1 gene ( Oprm1D2 ) , resulting in one band .",
"After amplification , PCR products were digested with DdeI , run on a 4% agarose gel , and visualized using ethidium bromide staining .",
"A representative gel is displayed in Figure 8d to indicate differentiation of homozygous ( B6/B6 , D2/D2 ) and heterozygous ( B6/D2 ) Oprm1 genotypes .",
"However , all BXD mice used in these studies were homozygous .",
"( + ) MA hydrochloride was purchased from Sigma ( St . Louis , MO , USA ) and dissolved in tap water for drinking or in sterile 0 . 9% saline ( Baxter Healthcare Corp . , Deerfield , IL , USA ) for injection .",
"For the body temperature studies , saline and MA were administered by intraperitoneal injection at a volume of 10 ml/kg body weight .",
"Methods were consistent with those we used to measure two-bottle choice MA intake during the production of the MA drinking lines ( Hitzemann et al . , 2019; Shabani et al . , 2011; Wheeler et al . , 2009 ) .",
"During the initial two days of the study , mice had access to two tubes filled with tap water to familiarize them with the novel drinking apparatus .",
"Starting on the third day , one water tube was replaced with a tube containing MA dissolved in tap water to which mice had access for 18h/day , beginning 3h before the lights turned off .",
"The MA tube was removed for the remaining 6h of each day .",
"Unpublished data ( Phillips ) indicate that the 6h forced abstinence periods between periods of 18h MA access are important for higher MA intake levels , compared to 24h MA access periods .",
"The MA concentration was 20 mg/l for 4 days , and was increased to 40 mg/l for an additional 4 days .",
"The MA and water tube positions were alternated every other day to account for potential side bias .",
"MA consumption was indexed as the average intake on the second and fourth days of access to each MA concentration , as these days represent the day after the tube positions were switched , when mice should be familiar with the relative locations of the water and MA tubes .",
"MA consumption was measured in ml ( accuracy = 0 . 2 ml ) , and then converted to mg/kg , based on body weight measured every two days .",
"Total volume consumed ( ml ) from the water and MA tubes during the 18h MA access periods was also measured .",
"Methods for determining MA-induced change in body temperature were consistent with those we established previously ( Harkness et al . , 2015 ) .",
"Mice were tested after injection of saline or 2 mg/kg MA at an ambient temperature of 21 ± 2°C , for 120 min beginning at 0800h , two hours after lights on .",
"The MA dose was chosen from previous results demonstrating that mice with functional TAAR1 exhibit a robust hypothermic response to 2 mg/kg MA 30 min after administration that is absent in mice with non-functional TAAR1 ( Harkness et al . , 2015; Reed et al . , 2018 ) .",
"Treatment groups ( saline or MA ) were assigned so that males and females were equally represented , and mice from each family were distributed equally between the groups .",
"Mice were weighed and then placed into individual perforated acrylic plastic cubicles ( 8 × 19×8 cm in W x H x L ) that served to prevent huddling-associated effects on body temperature ( Crabbe et al . , 1987; Crabbe et al . , 1989 ) .",
"Mice were undisturbed for 1h before a baseline rectal temperature was obtained at time 0 ( T0 ) by inserting a glycerin-coated , 5 mm probe attached to a Thermalert model TH-8 digital thermometer ( Sensortek , Clifton , NJ , USA ) 2 . 5 cm into the rectum for 5 s .",
"Saline or MA was then administered , and mice were returned to their cubicles .",
"Rectal temperature was again measured at 30 , 60 , 90 , and 120 min post-injection ( T30-T120 ) .",
"QTL analyses using BXD strain means were conducted using the WebQTL mapping module of GeneNetwork ( www . genenetwork . org; RRID:SCR_002388 ) .",
"The traits mapped were:",
"1 ) MA intake when the 40 mg/l MA concentration was offered , which is the phenotype used for selective breeding ( Shabani et al . , 2011; Wheeler et al . , 2009 ) , and",
"2 ) MA-induced body temperature change at 30 min post-treatment .",
"The 30 min time point is when the hypothermic effect of the 2 mg/kg dose of MA is largest ( Harkness et al . , 2015; Reed et al . , 2018 ) .",
"The change score was calculated by subtracting temperatures at 30 min from baseline temperatures taken immediately before treatment .",
"QTL mapping was initially performed using the interval mapping function and then composite interval mapping was applied , with Oprm1 genotype at rs29351111 as a co-factor , to assess the potential interaction of Oprm1 and Taar1 .",
"QTL significance was assessed using the LOD score obtained after 1000 or 2000 permutations , for interval or composite interval mapping , respectively , and was considered significant if the genome-wide p-value was <0 . 05 , and considered suggestive if the genome-wide p-value was <0 . 63 , which yields one false positive per genome scan on average .",
"These are the standard settings used for GeneNetwork QTL mapping .",
"R version 3 . 6 . 0 ( The R Foundation for Statistical Computing , https://www . r-project . org/foundation/; RRID:SCR_001905 ) was used to analyze changes in LOD scores computed from interval vs . composite interval QTL mapping with a Chi-squared test comparing additive vs . interactive models for the effects of Taar1 and Oprm1 genotype .",
"MA consumption and MA-induced body temperature change means for the BXD strains are available in GeneNetwork .",
"For additional information about using GeneNetwork for QTL mapping , see Mulligan et al . ( 2017 ) .",
"Statistica 13 . 3 ( TIBCO Software , Inc , Palo Alto , CA , USA ) was used for statistical analyses other than QTL mapping .",
"For MA drinking studies , MA consumption ( mg/kg ) and total volume consumed ( ml ) data were analyzed by repeated measures ANOVA , with MA concentration as the repeated measure , and genotype ( or strain ) and sex as possible independent variables .",
"Body temperature data were analyzed by repeated measures ANOVA , with time as the repeated measure , and genotype ( or strain ) , sex , and MA dose as possible independent variables .",
"For BXD MA drinking and body temperature data , initial analyses characterizing the strains did not include sex as a factor , due to group sizes that were too small .",
"However , analyses examining associations between Taar1/Oprm1 genotypes and MA-related phenotypes did examine the potential impact of sex .",
"For these analyses , to examine potential interaction effects on the measured phenotypes , data for the entire BXD population were analyzed with sex , Taar1 genotype , and Oprm1 genotype coded as separate independent variables ( Reed et al . , 2018 ) .",
"Effects were considered statistically significant at p<0 . 05 .",
"Complex interactions were simplified by ANOVAs at levels of a particular factor .",
"Two-way interactions were further interpreted using simple main effects analysis , with Bonferroni correction .",
"To compare body temperature data at post-injection time points ( T30-120 ) to T0 within a particular genotype , Dunnett’s post hoc test was used .",
"Pearson’s r was used to calculate correlations between phenotypes and Taar1 or Oprm1 genotype .",
"Sample sizes for these studies were based on our previous MA drinking and body temperature studies ( e . g . , Harkness et al . , 2015; Reed et al . , 2018 ) ."
]
] | [
"We identified a locus on mouse chromosome 10 that accounts for 60% of the genetic variance in methamphetamine intake in mice selectively bred for high versus low methamphetamine consumption .",
"We nominated the trace amine-associated receptor 1 gene , Taar1 , as the strongest candidate and identified regulation of the mu-opioid receptor 1 gene , Oprm1 , as another contributor .",
"This study exploited CRISPR-Cas9 to test the causal role of Taar1 in methamphetamine intake and a genetically-associated thermal response to methamphetamine .",
"The methamphetamine-related traits were rescued , converting them to levels found in methamphetamine-avoiding animals .",
"We used a family of recombinant inbred mouse strains for interval mapping and to examine independent and epistatic effects of Taar1 and Oprm1 .",
"Both methamphetamine intake and the thermal response mapped to Taar1 and the independent effect of Taar1 was dependent on genotype at Oprm1 .",
"Our findings encourage investigation of the contribution of Taar1 and Oprm1 variants to human methamphetamine addiction ."
] | [
"People who misuse drugs often do so partly in response to the environment they find themselves in , and partly because of their genetics .",
"The genetic component of someone’s risk is influenced by many different genes , and most research has found that each gene has a small individual effect .",
"A method called quantitative trait locus ( QTL ) analysis can help find parts of the genome that influence someone’s risk of misusing drugs .",
"In 2013 , researchers found one region on chromosome 10 in mice has a particularly large influence on how much methamphetamine an individual mouse will ingest if the drug was available in one of its two water bottles .",
"A gene called Taar1 was particularly important in this region and another gene , called Oprm1 , may also play a significant role .",
"When the Taar1 gene is switched off , mice consume larger amounts of methamphetamine , have a heightened reward response from the drug , and are insensitive to the adverse effects – such as hypothermia .",
"But whether Taar1 directly caused these effects , and whether Taar1 and Oprm1 interact , had not yet been determined .",
"If these genes played a causal role , they could be useful targets for treatment of methamphetamine-use disorder .",
"Stafford , Reed et al . – who include several of the researchers involved in the 2013 work – now report that when a particular variant of Taar1 was present in mice they consumed large amounts of methamphetamine .",
"The variant codes for a faulty version of a receptor protein .",
"When this variant was replaced with a working version using gene editing , the mice consumed less methamphetamine and also became sensitive to hypothermia induced by the drug .",
"This confirms that this gene does play a causal role in methamphetamine consumption and hypothermia .",
"Next , Stafford , Reed et al . tested mice with different combinations of variants of Oprm1 and Taar1 to see how the genes interacted .",
"The results showed that the effects of Taar1 on both consumption of the drug and hypothermia depended on the Oprm1 variant present .",
"The findings suggest that variants of these two genes in humans could influence an individual’s risk of addiction to methamphetamine .",
"It is possible that in future the disorder could be treated by drugs that modify the brain activity impacted by these receptors .",
"But first , it will be important to find out if these genes play a similar role in humans as they do in mice ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"epidemiology and global health"
] | A user-friendly, open-source tool to project impact and cost of diagnostic tests for tuberculosis | elife-02565-v2 | [
[
"Infectious disease transmission models are important tools for translating the best current knowledge of the natural history and epidemiology of infectious diseases into projections of epidemiological impact ( e . g . , incidence , mortality ) and costs under alternative strategies for disease control ( Garnett et al . , 2011 ) .",
"Currently , most published transmission models are either loosely calibrated to reflect global/regional outcomes or more tightly fit to specific epidemiological settings; in either case , model results may be difficult for local decision-makers in the majority of public health settings to utilize .",
"Simplified models designed for in-country use by decision-makers , most notably the Spectrum suite of models supported by the Futures Institute ( Stover et al . , 2010 ) , have been used to inform decision-making in the fields of reproductive health and human immunodeficiency virus ( HIV ) for over a decade ( Stover , 2004 ) .",
"Estimates from the Spectrum models are now routinely incorporated into official global and country-level estimates of HIV disease burden ( Brown et al . , 2010 ) and intervention impact ( Farnham et al . , 2013 ) .",
"Other simplified models are readily available for impact projections related to non-infectious diseases , where transmission assumptions are less important ( Betz Brown et al . , 2000; Walker et al . , 2013 ) .",
"However , to date , simple , user-friendly transmission models have not been widely used for decision-making related to many infectious diseases other than HIV .",
"Diagnosis of active tuberculosis ( TB ) is an example of a public health intervention for which transmission models may provide guidance on both global ( Dowdy et al . , 2006; Abu-Raddad et al . , 2009 ) and country-specific levels ( Menzies et al . , 2012 ) .",
"Specifically , an unprecedented number of new diagnostic strategies for active TB are now recommended by the World Health Organization ( WHO ) , including same-day microscopy ( World Health Organization , 2011 ) , microcolony-based culture techniques ( Leung et al . , 2012 ) such as the microscopic-observation drug-susceptibility ( MODS ) assay ( Moore et al . , 2006 ) , line-probe assays for drug susceptibility testing ( Bwanga et al . , 2009 ) , and Xpert MTB/RIF ( ‘Xpert’ ) , a molecular assay capable of providing results ( including rifampin resistance ) in 90 min with minimal human resource requirements ( Boehme et al . , 2010 , 2011 ) .",
"TB program decision-makers must repeatedly determine when to invest in scaling up a novel diagnostic test , which test ( s ) to promote , and whether the implementation strategy should differ by epidemiological situation ( Cobelens et al . , 2012 ) .",
"Without transmission models to provide locally relevant estimates of cost and impact under alternative implementation strategies , such decisions will be made without systematically considering the implications of available scientific evidence .",
"To aid in this decision-making process , we created a flexible , simple modeling tool that allows non-expert users to define their local situation according to three key epidemiological parameters ( TB incidence , proportion of new TB cases that are multidrug-resistant [MDR] , and adult human immunodeficiency virus [HIV] prevalence ) and local unit costs of TB diagnosis and treatment .",
"This tool then incorporates those estimates into a combined decision analysis-transmission framework to generate 5-year projections of TB incidence , mortality , and control costs for nine diagnostic strategies ( Figures 1 and 2 ) .",
"These strategies are:‘Baseline’: Sputum smear microscopy for each diagnostic attempt , with liquid-media TB culture only to evaluate smear-positive cases with a history of previous TB treatment for drug resistance .",
"( Cultures in all scenarios trigger drug-susceptibility testing if positive . ) ‘TB culture if previously treated’: Sputum smear microscopy used for patients without a history of TB treatment; smear plus liquid-media culture used to diagnose TB in any previously treated individual with symptoms ( regardless of smear status ) .",
"‘Xpert if HIV-positive’: Xpert MTB/RIF for HIV-infected patients only , with a positive test for rifampin resistance triggering treatment for MDR-TB .",
"Xpert is assumed to be deployed at the district level , such that results cannot generally be provided during the same clinical encounter ( Lawn et al . , 2012 ) .",
"This strategy is conceived as a ‘best-case’ scenario for HIV-targeted TB testing: if individuals unaware of their HIV status are not tested with Xpert , this strategy will overestimate effectiveness , and if those unaware of their status are tested , it will underestimate costs .",
"‘Xpert if Smear-Positive’: Xpert MTB/RIF for smear-positive patients only ( i . e . , for rapid DST ) , with a positive test for rifampin resistance triggering treatment for MDR-TB .",
"‘Xpert for All’: Xpert MTB/RIF for all patients .",
"‘Xpert with Culture DST Confirmation’: Xpert MTB/RIF for all patients , but treatment for MDR-TB only initiated if rifampin resistance is confirmed by culture .",
"‘MODS/TLA’: Sputum smear , plus microcolony-based TB culture ( e . g . , MODS or thin-layer agar , TLA ) for all patients .",
"‘Same-Day Microscopy’: Double the per-test cost of sputum smear microscopy , in exchange for the ability to provide results to patients in the same clinical encounter ( e . g . , with peripheral , unbatched reading of sputum smears ) .",
"‘Same-Day Xpert’: Double the per-test cost of Xpert MTB/RIF , in exchange for the ability to provide results to patients in the same clinical encounter ( e . g . , peripheral deployment , with greater costs reflecting lower volume per machine [Vassall et al . , 2011] ) . 10 . 7554/eLife . 02565 . 003Figure 1 . Overview of user-friendly model . Users are asked , via open-source computer script or Web interface , to select one of the nine diagnostic strategies and to provide unit costs and three basic epidemiological parameters ( TB incidence , MDR-TB prevalence among new cases , and adult HIV prevalence ) .",
"The selected diagnostic strategy is used to populate a decision tree that calculates",
"( a ) the probability of missed diagnosis , unsuccessful treatment , and successful treatment ,",
"( b ) costs , and",
"( c ) diagnostic delays .",
"These outputs depend on patients' TB ( yes/no , and drug susceptibility status ) , HIV , and TB treatment history status .",
"The selected epidemiological parameters are then used to populate a dynamic transmission model , creating a steady-state population that reflects local TB epidemiology .",
"The decision tree—which inputs user-defined unit costs—is then incorporated into the transmission model to project outcomes under the selected diagnostic scenario .",
"Users can sequentially select multiple diagnostic scenarios for comparison , and the computer script ( though not the Web interface ) allows users to manipulate input parameters at their discretion . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 00310 . 7554/eLife . 02565 . 004Figure 2 . Transmission model of TB diagnosis . Boxes represent sub-populations in the model , and arrows represent rates of movement between those sub-populations .",
"Parallel structures exist for:",
"( a ) HIV-infected vs HIV-uninfected;",
"( b ) never-treated vs previously treated ( for TB ) ; and",
"( c ) among TB-infected individuals , drug-susceptible vs isoniazid-monoresistant vs rifampin-resistant ( including MDR ) .",
"‘Pre-diagnostic’ TB refers to individuals who are infectious but have not yet begun to seek care .",
"Mortality occurs from all sub-populations ( not shown ) , and at a higher rate among those with HIV and active TB . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 004 For purposes of illustration , we evaluated each of these diagnostic strategies in four emblematic epidemiological settings , defined by TB incidence , MDR-TB prevalence among new cases , and adult HIV prevalence:‘Reference/High-Incidence Setting’ ( e . g . , Southeast Asia ) : TB incidence 250 per 100 , 000/year ( twice the global incidence [World Health Organization , 2012] ) , MDR-TB prevalence of 3 . 7% in new TB cases ( the estimated global prevalence [World Health Organization , 2012] ) , adult HIV prevalence of 0·83% ( the estimated global prevalence [UNAIDS , 2012] ) ;‘Low-Incidence Setting’ ( e . g . , United States , Western Europe ) : TB incidence at entry of 8 . 9 per 100 , 000/year and declining , with similar MDR-TB ( as a proportion of new cases ) and HIV prevalence as above;‘High MDR Setting’ ( e . g . , former Soviet Union ) : TB incidence 100 per 100 , 000/year , MDR-TB prevalence of 10% in new TB cases , adult HIV prevalence of 0 . 83%; and‘High HIV Setting’ ( e . g . , sub-Saharan Africa ) : Adult HIV prevalence of 20% , TB incidence 500 per 100 , 000 , and MDR-TB prevalence among new cases of 3 . 7% .",
"In each setting , we used a uniform set of costs for purposes of comparison ( Table 1 ) .",
"Although these four settings form the basis of the results presented here , decision-makers can use the open-source model program ( included as Supplementary file 1 , written in the open-source programming language Python , Version 2 . 7 , www . python . org ) to re-define any parameter in Table 1 according to their best local knowledge; a manual for doing so is also included as Supplementary file 2 .",
"Capacity to create non-equilibrium settings ( e . g . , declining TB incidence , increasing MDR-TB ) is included .",
"We also provide a web-based interface ( flexdx . modeltb . org ) that allows users to input local TB incidence , MDR-TB prevalence , HIV prevalence , and unit costs; this interface provides customized results without the requirement to manipulate programming code .",
"The program corresponding to the web version is also available on a public repository ( https://github . com/JJPennington/FlexDx-TB-Web-Django ) . 10 . 7554/eLife . 02565 . 005Table 1 . Model input parameters*DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 005ParameterValueReference ( s ) /RationaleTB and HIV TransmissionTransmission rate , per smear-positive/highly infectious person-yearCalibrated to user-defined TB incidence†Proportional reduction in per-case transmission rate , MDR-TBCalibrated to user-defined MDR-TB prevalence†Proportional reduction in fitness , isoniazid-monoresistant TB25% of MDR-TB reductionAssumptionHIV incidence rate , per yearCalibrated to user-defined HIV prevalence†Relative transmission rate from smear-negative/less infectious TB0 . 22 ( Behr et al . , 1999 ) Proportion of pulmonary TB that is smear-positive/highly infectious HIV-negative0 . 63 ( Steingart et al . , 2006a; Steingart et al . , 2006b ) HIV-infected0 . 50 ( Getahun et al . , 2007 ) TB ProgressionEndogenous reactivation rate HIV-negative0 . 0005/year ( Horsburgh et al . , 2010 ) HIV-infected0 . 05/year ( Antonucci et al . , 1995 ) Proportion of recent infections resulting in rapid progression HIV-negative0 . 14 ( Vynnycky and Fine , 1997; Dye et al . , 1998 ) HIV-infected0 . 470 . 75 without ART , ( Daley et al . , 1992 ) 75% reduction if on ART , ( Williams et al . , 2010 ) 50% ART coverageReduction in TB rapid progression probability due to latent TB infection ( HIV-negative only ) 0 . 79 ( Andrews et al . , 2012 ) TB Mortality and ResolutionLife expectancy at age 1555 years ( World Bank , 2012 ) Annual mortality from HIV0 . 05/year ( UNAIDS , 2012 ) Annual mortality from TB HIV-negative , smear-positive/highly infectious0 . 23/year ( Tiemersma et al . , 2011 ) HIV-negative , smear-negative/less infectious0 . 07/year ( Tiemersma et al . , 2011 ) HIV-infected1 . 0/year ( Corbett et al . , 2003; Corbett et al . , 2007; Wood et al . , 2007 ) Rate of spontaneous TB resolution ( HIV-negative only ) Smear-positive/highly infectious0 . 1/year ( Tiemersma et al . , 2011 ) Smear-negative/less infectious0 . 27/year ( Tiemersma et al . , 2011 ) TB Treatment Outcomes and Emergence of Drug ResistanceProbability of failure or relapse ( within 1 year ) Drug-susceptible0 . 04 ( World Health Organization , 2012 ) INH-monoresistant , first-line therapy0 . 21 ( Menzies et al . , 2009b ) INH-monoresistant , retreatment or 2nd-line0 . 16 ( Menzies et al . , 2009b ) MDR-TB , first-line or retreatment0 . 50 ( Espinal et al . , 2000 ) MDR-TB , second-line therapy0 . 30 ( World Health Organization , 2010 ) Proportion of one-year recurrence due to failure Drug-susceptible0 . 14 ( Lew et al . , 2008 ) INH-monoresistant0 . 33 MDR-TB0 . 56Probability of acquired drug resistance ( per treatment course ) Susceptible becoming INH-monoresistant0 . 001 ( Menzies et al . , 2009a; Menzies et al . , 2009b ) Susceptible becoming MDR-TB0 . 002 INH-monoresistant becoming MDR-TB0 . 045 If treated with 2 effective drugs for >6 mos0 . 017Behavioral ParametersInfectious months before starting to seek care HIV-negative9 months ( Dowdy et al . , 2013 ) HIV-infected1 month ( Corbett et al . , 2004 ) Diagnostic frequency while seeking care5 . 0/year ( Storla et al . , 2008; Sreeramareddy et al . , 2009 ) Probability of treatment in a TB patient whose microbiological test is negative0 . 25 ( Wilkinson et al . , 2000; Dowdy et al . , 2008 ) Loss to follow-up between diagnostic presentation and treatment initiation Sputum smear or GXP ( not same-day ) 0 . 15 ( MacPherson et al . , 2014 ) Culture ( microcolony or commercial liquid ) 0 . 25 ( Dowdy et al . , 2008 ) Same-day diagnosis0AssumptionDiagnostic AccuracySensitivity for smear-negative/less-infectious TB Sputum smear microscopy0 Xpert MTB/RIF0 . 72 ( Brownell et al . , 2012 ) Culture ( microcolony or commercial liquid ) 0 . 85 ( Cruciani et al . , 2004; Leung et al . , 2012 ) Specificity for TB ( Steingart et al . , 2006; Boehme et al . , 2011; Leung et al . , 2012 ) Sputum smear microscopy0 . 98 Xpert MTB/RIF0 . 98 Microcolony culture0 . 98Sensitivity for drug resistance ( if TB detected ) Microcolony culture ( rifampin and isoniazid ) 0 . 98 ( Minion et al . , 2010 ) Xpert MTB/RIF ( rifampin only ) 0 . 94 ( Boehme et al . , 2011 ) Specificity for drug resistance ( if TB detected ) Microcolony culture ( isoniazid ) 0 . 96 ( Minion et al . , 2010 ) Microcolony culture ( rifampin ) 0 . 99 ( Minion et al . , 2010 ) Xpert MTB/RIF ( rifampin ) 0 . 98 ( Boehme et al . , 2011 ) Diagnostic Delay and non-TB Care-SeekingDays from presentation to treatment initiation Sputum smear or Xpert MTB/RIF7 daysAssume 1 week Microcolony or commercial liquid culture30 days ( Boehme et al . , 2011 ) Months of therapy before a failing regimen will be changed , or before default and recurrence6 monthsAssumptionAnnual rate of diagnostic evaluation for TB , among people who do not have active TB0 . 01/year10% of suspects have TB , high-incidence settingCost Parameters ( user-defined; values below for comparison purposes only ) Per-patient cost of TB therapy First-lineUS$500User-defined RetreatmentUS$1000 ( Vassall et al . , 2011 ) Second-line/MDRUS$5000 ( Vassall et al . , 2011 ) Outpatient visit ( diagnosis or follow-up ) US$10 ( Vassall et al . , 2011 ) Per-test cost: Sputum smearUS$2 ( Vassall et al . , 2011 ) Same-day sputum smearUS$10Assumption Xpert MTB/RIFUS$15 ( Vassall et al . , 2011 ) Same-day Xpert MTB/RIFUS$30Assumption Microcolony culture ( with DST ) US$5 ( Solari et al . , 2011 ) Commercial liquid-media cultureUS$20 ( Vassall et al . , 2011 ) Commercial liquid-media culture + DSTUS$40 ( Vassall et al . , 2011 ) *In the actual model program ( Supplementary file 1 ) , users can change any parameter based on local values .",
"†For reference , the transmission rate ( in infections per person-year during diagnosis-seeking active TB ) is 36 . 9 in the reference scenario , 14 . 0 in the low-incidence scenario , 25 . 4 in the high MDR scenario , and 12 . 9 in the high HIV scenario .",
"Corresponding proportional reductions in MDR-TB transmission rate are 0 . 23 , 0 . 23 , 0 . 21 , and 0 . 19; and HIV incidence estimates ( per 1000 adult person-years ) are 0 . 7 , 0 . 6 , 0 . 6 , and 18 . 9 ."
],
[
"To validate the model , we compared selected model outcomes to published global estimates .",
"In the high-incidence setting , our model estimated TB mortality at 14% of incidence ( 95% uncertainty range: 7–20% , WHO global estimate 14% [World Health Organization , 2012] ) , HIV-associated TB at 13% of all incident TB ( 95% uncertainty range: 4–14% , global estimate 13% [World Health Organization , 2012] ) , previously treated cases at 13% of all incident cases ( 95% uncertainty range: 9–34% , global estimate 14% [World Health Organization , 2012] ) , and duration of TB disease at 1 . 2 years ( 95% uncertainty range 0 . 8–2 . 1 , global estimate 1 . 4 years [World Health Organization , 2012] ) .",
"Our model estimated that MDR-TB prevalence in previously treated cases was 15 . 4% ( WHO estimate 20% [World Health Organization , 2012] ) , but unlike our model , WHO notifications often count failure and recurrence after default ( in the same person ) as two separate cases .",
"At steady-state in the model , 80% of incident TB was due to recent infection rather than reactivation .",
"Figure 3 shows the projected incremental 5-year cost and impact of each of nine selected diagnostic strategies ( described in greater detail in the ‘Materials and methods’ section ) , in the high-incidence scenario .",
"In general , both the cost and impact of targeted strategies ( culture for retreatment , Xpert for HIV-positive , Xpert for smear-positive ) on incidence were small relative to broader diagnostic strategies ( Xpert for all , MODS/TLA , same-day Xpert ) .",
"The incremental cost-effectiveness , in terms of cost per case averted ( i . e . , slope of the line from origin to each point in Figure 3 ) , was similar across all strategies with the exception of same-day smear , which was cost-saving and had greater effectiveness than the baseline .",
"Same-day microscopy remained cost-saving by year 5 as long as same-day results could be provided at less than five times the per-smear cost of routine results ( i . e . , <$10/test ) .",
"Among the targeted strategies , Xpert for smear-positives was the most expensive but had the greatest impact on MDR-TB cases averted , whereas Xpert for HIV-infected individuals and culture for retreatment cases offered smaller gains at lower cost .",
"Among the broad strategies , culture confirmation of rifampin-resistant tests on Xpert saved costs with little reduction in effectiveness .",
"There was little difference between culture-confirmed Xpert and MODS/TLA , and same-day Xpert was the most expensive and most effective strategy . 10 . 7554/eLife . 02565 . 006Figure 3 . Incremental 5-year cost and impact of TB diagnostic strategies , high-incidence setting . Shown are cumulative projected 5-year costs and impact ( averted TB cases [panel A] or MDR-TB cases [panel B] ) of each diagnostic strategy described in the Introduction , incremental to the baseline strategy , per 100 , 000 population .",
"Strategies with greater impact appear to the right on the x-axis; more costly strategies appear higher on the y-axis .",
"The same-day smear strategy is cost-saving but shown at an incremental cost of $0 for simplicity . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 006 The projected impacts and costs of the nine diagnostic strategies relative to the baseline strategy , in each of four selected epidemiological settings , are shown in Figure",
"4 . In all four settings , the ranking of diagnostic strategies remained similar for all outcomes , although the cost of broader diagnostic strategies relative to targeted strategies fell substantially over 5 years in higher-incidence settings as the broader strategies generated declines in TB incidence .",
"In the low-incidence setting , where a higher proportion of TB treatments are false-positive and more incident TB is also due to reactivation ( 60% of all new cases ) , the relative cost of improved TB diagnosis was the highest , while the relative impact was the least .",
"In the high HIV setting , the impact of diagnostic interventions on TB incidence was diminished relative to the high-incidence setting , though the impact on TB mortality was similar .",
"Additionally in this setting , the Xpert for HIV-positive strategy was substantially more costly , but also more effective , than in other settings . 10 . 7554/eLife . 02565 . 007Figure 4 . Relative impact of diagnostic strategies in emblematic settings . Shown are projected changes in TB incidence , MDR-TB incidence , TB mortality , and costs ( in Year 1 and Year 5 after immediate implementation ) , relative to baseline ( Strategy 1 ) after implementing each of the diagnostic strategies described in the text .",
"Epidemiological outcomes are measured at the end of Year",
"5 . Panel A ( high incidence ) shows a setting with TB incidence of 250 per 100 , 000/year , stable MDR-TB prevalence of 3 . 7% among new cases , adult HIV prevalence of 0 . 83% , and cost of $500 to treat one case of TB with first-line therapy .",
"In panel B ( low incidence ) , the TB incidence is reduced to 8 . 3 per 100 , 000/year ( implemented by gradual decline in incidence over 50 years ) .",
"In panel C ( high MDR ) , MDR-TB prevalence among new cases is set at 3 . 7% in the beginning of year 1 , increasing to 10 . 7% by the end of year",
"5 . In panel D ( high HIV ) , adult HIV prevalence is set to 20% and TB incidence is set to 500 per 100 , 000/year . DOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 007 The impact of the ‘Xpert for all’ strategy on TB incidence ( selected a priori as the primary outcome for sensitivity analysis ) was most sensitive to three parameters , both in terms of absolute effects on impact estimates and partial rank correlation coefficients .",
"These three parameters were: ( 1 ) the proportion of TB patients who would be empirically treated even if microbiologic testing yielded a negative result ( ‘empiric treatment proportion’ ) , ( 2 ) duration of infectiousness before seeking care ( ‘pre-diagnostic delay’ ) , and ( 3 ) rate of reactivation from latent infection to active disease ( ‘reactivation rate’ ) .",
"For this latter parameter , in-depth investigation revealed that the key determinant was not the reactivation rate per se , but rather the proportion of active TB representing recent vs remote infection .",
"If the empiric treatment proportion was increased from 25% to 37 . 5% , the projected reduction in TB incidence fell from 20% to 13% .",
"By contrast , when only 12 . 5% of false-negative patients were started on therapy , ‘Xpert for all’ achieved a 31% reduction in incidence .",
"Corresponding reductions in incidence with ‘Xpert for all’ ( 20% at baseline ) included: 27% if pre-diagnostic delay was shortened from 9 months to 4 . 5 months , 14% if pre-diagnostic delay was lengthened to 13 . 5 months , 15% if the reactivation rate was doubled from 0 . 05% to 0 . 1% per year , and 23% if reactivation was halved to 0 . 025% per year .",
"The projected 5-year reduction in incidence for this strategy did not fall outside the range of 12–25% under variation of any other model parameter , up to 50% of that parameter's baseline value ( Table 1 ) .",
"Both costs and incremental costs were more sensitive to the cost of TB treatment than the cost of diagnostics .",
"For example , doubling the cost of first-line therapy ( from $500 to $1000 per person ) augmented the incremental year 1 costs for ‘Xpert for all’ from a 57% increase over baseline to a 77% increase , and doubling the cost of MDR therapy ( from $5000 to $10 , 000 per person ) generated an 86% increase in incremental year 1 costs , whereas doubling the unit cost of Xpert ( from $15 to $30 ) only resulted in a 72% increase .",
"Unit costs other than those for first-line treatment , MDR treatment , and the diagnostic modality under study in each scenario were not important determinants of incremental costs ."
],
[
"To date , most transmission models of infectious disease control interventions present results that are not directly usable by decision makers because they are not customizable to local conditions .",
"We present a flexible , user-friendly model of TB diagnosis and transmission that allows users without modeling expertise to define an epidemiologic setting ( according to TB incidence , MDR-TB prevalence , and HIV prevalence ) and unit costs , evaluating various diagnostic strategies in that setting in terms of population-level costs and impact .",
"While this model cannot precisely replicate the epidemiological situation in any given location , it applies a standardized methodology across a wide range of settings , thereby illustrating important interactions between epidemiological parameters and projected impact .",
"We provide both a web interface for rapid calculations and full model code whereby users can change any model parameter for ‘personalized’ sensitivity analysis .",
"Our model results suggest that the rank-ordering of diagnostic strategies may be relatively stable across epidemiological settings , but that the actual population-level costs and impact differ dramatically .",
"This model also serves as an example of how epidemiologists can provide decision-makers across a wide variety of local settings with rapid access to customizable ‘first-pass’ projections of cost and impact from transmission models without the need to construct tightly fitted models to represent all epidemiological settings .",
"Our results provide important guidance to TB decision-makers .",
"Specifically , in settings where little additional up-front investment is possible ( <25% increase in TB control budget ) , same-day microscopy has the greatest impact on TB incidence with no increase in overall cost at 5 years , provided that same-day microscopy can be feasibly delivered for less than $10 per patient .",
"For settings in which containing MDR-TB is the most important consideration , Xpert for smear-positives has the greatest effectiveness for this outcome , but at substantial price and very little impact on overall TB incidence and mortality .",
"Xpert for HIV-positives and culture for retreatment cases both offer meaningful , albeit small , gains; the cost and impact of these strategies are the highest in HIV-endemic settings .",
"In settings where more initial investment is possible ( about 50% increase in TB control budget ) , broader scale-up of either Xpert or MODS/TLA for all TB diagnosis can offer substantially greater benefits , both in terms of reduced incidence ( often 10 times more TB cases averted than with the more narrowly-targeted strategies above ) and long-term costs ( in that the incremental cost of these strategies declines greatly by Year 5 ) .",
"In general , culture confirmation of positive Xpert results before committing to a course of second-line therapy is preferred .",
"Finally , where the greatest impact is sought , combination of Xpert for all plus infrastructure for same-day diagnosis achieves this aim in all settings , but at the highest cost .",
"Although this model represents a highly simplified framework , it compares well to other global estimates to which it was not fit ( e . g . , WHO estimates of TB mortality , previously treated TB , HIV-associated TB , and MDR-TB prevalence among previously treated cases ) .",
"Its results are also similar to those of other mathematical models of TB diagnosis that are fit to specific locations .",
"For example , Menzies et al . ( 2012 ) modeled scale-up of Xpert for people living with HIV in southern Africa , estimating a 6% reduction in incidence , 21% reduction in TB mortality , 25% reduction in MDR-TB incidence that declines to <10% over a longer time frame , and 40% increase in costs ( not including costs of antiretroviral therapy ) .",
"Our corresponding estimates for the Xpert for HIV-positive strategy in the high-HIV setting are 5% reduction in incidence , 25% reduction in TB mortality , 7% reduction in MDR-TB incidence , and increase in costs from 41% ( year 1 ) to 28% ( year 5 ) .",
"For purposes of deciding between alternative strategies of scaling up TB diagnostics , the estimates from these two models are likely to provide similar guidance .",
"For any such simplified transmission model to have an impact on decision-making , it is important to consider who the eventual users of such a model might be , and to develop the model in consultation with those groups .",
"In this case , we identified a number of potential end-user organizations including technical assistance agencies ( e . g . , KNCV Tuberculosis Foundation ) , funding bodies ( e . g . , Global Fund for AIDS , Tuberculosis , and Malaria ) , non-governmental organizations ( e . g . , Medecins Sans Frontiers ) , National Tuberculosis Programs , and regional offices of the World Health Organization .",
"We then invited representatives of these organizations to attend a 1-day workshop ( April 2014 , The Hague , The Netherlands ) describing methods and challenges related to modeling of TB diagnostics in general , including this transmission model in particular .",
"We developed ‘hands-on’ exercises for participants to better learn the model and solicited specific feedback , which is being incorporated into the model structure and web interface in ongoing fashion .",
"We next intend to develop a series of informal ‘case studies’ whereby the use of this model for in-country decision-making can be demonstrated and disseminated .",
"As with any modeling analysis , this research has important limitations .",
"In order to provide sufficient flexibility and generalizability , we make a number of strong assumptions that include a constant population , homogeneous mixing , no change in parameter values over time , and simplistic incorporation of HIV and drug resistance .",
"Without making such simplifying assumptions , it is impossible to deliver a flexible modeling framework that can generate transparent , customizable , rapid results ( i . e . , without complex statistical fitting to each individual epidemiological scenario ) .",
"This model can replicate user-defined TB incidence , MDR-TB prevalence , and HIV incidence but does not describe the breakdown of these values in key subpopulations ( e . g . , congregate settings or geographical ‘hotspots’ ) , nor does it incorporate operational aspects of the TB diagnostic system in any one setting .",
"Thus , this model does not ameliorate the need for more detailed models in settings where precise estimates are needed; rather it provides access to ‘first-pass’ estimates in settings ( i . e . , the vast majority of local decision-makers ) where such tightly calibrated model projections are not available .",
"This model focuses on transmission dynamics and thus does not include pediatric TB and extrapulmonary TB; these largely non-infectious disease manifestations remain very important components of the TB epidemic that are not captured here .",
"We validate our model against global estimates and other models; ideally , further validation would be performed over time using field data across a wide variety of settings .",
"Our model allows users to define three key epidemiological parameters ( as well as other model assumptions within the program ) , but data to inform even these three parameters—as well as unit costs—are unlikely to be available on sub-national levels .",
"As a result , users wishing to adapt the model to a smaller geographic scale will need to perform additional data-gathering exercises to inform these estimates if they wish to maximize the utility of the model .",
"Nevertheless , even if high-quality data are not available at the local level , this model allows decision-makers to estimate epidemiological and economic values according to reasonable assumptions ( e . g . , comparison of TB notification rates or budget line-items to those in the published literature or at the national level ) , vary those assumptions in real-time , and obtain corresponding projections of comparative impact that incorporate the best available current data on epidemiological or natural history parameters ( e . g . , TB progression and reactivation ) .",
"Future efforts might also provide flexibility to specify operational characteristics ( e . g . , health system capacity ) as well .",
"A final concern is that , by providing users the ability to specify TB incidence , MDR-TB prevalence , and adult HIV prevalence , a number of scenarios can be created ( e . g . , low TB incidence and very high adult HIV prevalence ) that are not epidemiologically realistic .",
"Although there is danger in allowing uninformed users to make projections for such scenarios ( and the model will reject or alert users to highly implausible values ) , we believe that this risk is outweighed by the benefit of providing full flexibility to model epidemiological scenarios ( e . g . , sub-district level data ) that will never be captured by a limited number of closely-calibrated TB transmission models .",
"In summary , we have created a flexible modeling framework that allows users without modeling expertise to generate simulated populations with locally relevant values for TB incidence , MDR-TB prevalence , adult HIV prevalence , and TB treatment costs .",
"By comparing an array of diagnostic options across emblematic epidemiological scenarios , we provide guidance to decision-makers who seek to ascertain the optimal diagnostic strategy to achieve their selected disease control targets , and to do so using a standardized methodology .",
"Success in the fight against infectious disease generally , and TB specifically , depends on our ability to place global knowledge in the hands of local decision-makers , enabling them to choose those interventions that are likely to have the greatest impact , given existing resources and local epidemiological realities .",
"This flexible modeling framework of diagnostic interventions takes an important step in that direction ."
],
[
"Using previously published models of TB diagnostics as a guide ( Dye et al . , 1998; Abu-Raddad et al . , 2009; Menzies et al . , 2012; Dowdy et al . , 2013 ) , we constructed a transmission model of TB using ordinary differential equations .",
"This model categorizes patients according to HIV status ( positive or negative ) , TB treatment status ( never treated or previously treated ) , TB disease status ( as shown in Figure 2 ) , and among those who are infected with TB , drug resistance status ( susceptible , isoniazid-monoresistant , and rifampin-resistant including MDR ) , and level of infectiousness ( smear-negative/less-infectious and smear-positive/highly infectious ) .",
"Individuals enter the model at age 15 , and TB with no pulmonary component ( i . e . , not infectious ) is not included .",
"For purposes of transparent communication , we chose a population size of 100 , 000 ( to match standard reporting of TB outcomes ) and assumed a constant population with no net population growth or immigration/emigration .",
"After constructing the transmission framework , we used decision analysis to estimate",
"( a ) the probability of each diagnostic outcome;",
"( b ) the diagnostic delay; and",
"( c ) the cost of TB diagnosis and treatment under each of the nine diagnostic strategies , assuming immediate implementation at the beginning of a given year ( ‘Year 1’ ) .",
"Two separate authors ( DWD and PJD ) independently coded the model; these models gave comparable results .",
"After setting probabilities and costs under each diagnostic strategy , we then created a flexible modeling structure capable of generating epidemiological scenarios as a function of three variables , which the user specifies: HIV prevalence ( assuming a global mean level of antiretroviral therapy coverage ) , TB incidence , and MDR-TB prevalence among new TB cases .",
"We accomplished this by allowing three key parameters to vary across model scenarios: annual HIV incidence , rate of TB transmission per smear-positive/highly infectious person-year , and relative per-case transmission rate of rifampin-resistant TB .",
"We also allow users to specify all relevant unit costs for TB diagnosis and treatment; other model parameters were estimated from existing literature ( Table 1 ) .",
"We then created a program that numerically generates a unique steady-state population meeting the user-defined values; this population serves as the baseline strategy ( Strategy 1 above ) at the beginning of the time frame under evaluation .",
"The program has flexibility to create its steady-state population 50 years in the past , allowing it to replicate the protracted , slow declines in TB incidence as seen in many lower-incidence settings; this is done automatically for any scenario with a target TB incidence less than 50 per 100 , 000/year .",
"The mathematical model consists of the following TB compartments:Uhp , UninfectedLhdp , Latently infectedEhdp , Early active ( infectious status i = 0 ) Ahdip , Late activePhdip , Active ‘pre-treatment’: diagnosis in progress , will lead to appropriate therapyIhdip , Active ‘inappropriate treatment’: receiving therapy that ends in default or failure In these compartments , the subscript h refers to HIV status ( h = 0 if HIV-uninfected , 1 if HIV-infected ) , d refers to drug resistance status ( d = 0 if drug-susceptible , 1 if isoniazid [INH]-monoresistant , and 2 if multidrug-resistant [MDR] ) , i refers to infectious status ( i = 0 if smear-negative/less infectious and 1 if smear-positive/highly infectious ) , and p refers to previous treatment status ( p=0 if never treated , 1 if previously treated ) .",
"Infectious status can be conceptualized as an individual's sputum smear status , if two smears were to be performed in a quality-assured laboratory at any given point in time .",
"The model assumes an adult population of stable size with no immigration or emigration: the number of individuals entering the uninfected compartment U is set as equal to the number who die ( whether from TB or other causes ) from all other compartments .",
"Pediatric and purely extrapulmonary TB are not explicitly considered because the diagnostic considerations for these manifestations are different .",
"In the short-term , however , to the extent that these forms of TB are non-infectious and equally fatal as adult pulmonary TB , their effects on TB incidence and mortality may be approximated by dividing the model's projected incidence and mortality by ( 1 − proportion of TB that is not adult pulmonary ) , to obtain a new incidence/mortality estimate .",
"Thus , if 20% of all TB in a given location is extrapulmonary or paediatric , the rough projected total TB incidence would be ( projected TB incidence ) / ( 0 . 8 ) .",
"In this model , we consider latent TB infection to be asymptomatic and non-infectious , with a constant rate of reactivation and ongoing risk of exogenous reinfection leading to active TB; individuals successfully treated for TB are assumed to return to this compartment upon initiation of effective therapy ( i . e . , therapy that will result in completion , with no relapse for 2 years ) .",
"Upon developing active TB , individuals enter a ‘pre-diagnostic’ phase that is characterized by a low level of infectiousness and mortality ( equivalent to smear-negative TB ) and during which individuals do not actively seek diagnosis .",
"The duration of this phase ( 9 months ) was selected a priori based on an existing model ( Dowdy et al . , 2013 ) in which the total duration of disease after incorporating this phase reflected the global ratio of prevalence to incidence , as estimated by the World Health Organization .",
"We compared the total duration of disease to this ratio as part of our model validation and assumed that this ‘pre-diagnostic’ phase is much shorter for HIV-infected vs HIV-uninfected individuals .",
"Upon completing this ‘pre-diagnostic’ phase , individuals progress to a diagnosis-seeking phase of active disease , which is characterized by separation into highly infectious ( ‘smear-positive’ ) and less infectious ( ‘smear-negative’ ) compartments .",
"Among HIV-uninfected individuals , the highly infectious compartment also carries higher mortality risk .",
"Diagnosis-seeking active TB implies active seeking of diagnosis at a defined rate; the probability that any single diagnostic attempt will result in effective therapy is calculated as a function of diagnostic sensitivity , probability of empiric therapy ( i . e . , without bacteriological confirmation ) , prior treatment status , and losses to follow-up , as described below .",
"Each diagnostic attempt , if successful , leads either to effective therapy ( which is initiated after a defined diagnostic delay ) or to ineffective therapy ( defined as leading to failure or default ) .",
"In order to focus on differences between the nine selected strategies above in a tractable framework , we subsumed all other diagnostic tests and procedures ( e . g . , chest X-ray , antibiotic trials ) as a probability of non-microbiologic diagnosis , without attempting to specify the associated cost or diagnostic delay .",
"Once effective therapy is initiated , it is assumed to immediately render the individual non-infectious , with no residual risk of mortality .",
"Upon initiation of ineffective therapy , individuals are assumed to remain infectious ( at the ‘smear-negative’/less infectious level ) for a defined period before either failing ( followed by another round of therapy , which can be either appropriate/curative or ineffective ) or default .",
"Reasoning that default will occur , on average , at the midpoint between receipt of fully-ineffective and fully-effective therapy , half of defaulters are presumed to develop recurrent TB ( which is assumed to occur immediately ) , while the other half return to the latent TB compartment ( from which reactivation or reinfection remains possible ) .",
"All individuals who relapse within 1 year are included as failures; thus , no specific parameter for relapse is incorporated .",
"Individuals who are effectively treated , or whose disease is contained without therapy , return to the latent compartment following the convention of other TB models ( Dye et al . , 1998; Abu-Raddad et al . , 2009 ) .",
"As the goal of this model is to focus on TB-related interventions , HIV is modeled as occurring at a defined annual incidence , calibrated to achieve a given user-defined prevalence at baseline .",
"We do not explicitly model HIV infection in dynamic fashion ( i . e . , the HIV incidence rate does not depend on the number of HIV-infected individuals in the model ) .",
"HIV infection is assumed to affect all parameters related to TB disease , including the level of immune protection afforded by latent infection ( assumed zero if HIV-infected ) , mortality rate ( increased ) , rate of reactivation from latent TB ( increased ) , duration of ‘pre-diagnostic’ TB ( decreased ) , and risk of ‘primary’ progression to active disease upon infection ( increased ) .",
"For purposes of maintaining a simple model structure , we do not explicitly model CD4 counts or antiretroviral therapy ( ART ) , but instead assume the global average of ART coverage , as estimated by UNAIDS ( 2012 ) .",
"We weight all HIV-related parameters according to this estimated probability of ART receipt; this probability can be modified by users .",
"We assume infection with TB strains of three different drug resistance levels: fully susceptible , INH-monoresistant , and MDR .",
"Dual infection with multiple strains is not considered in this model , but superinfection ( i . e . , reinfection of a latently infected individual with a different strain , resulting in primary progression to disease with the reinfecting strain ) is allowed , as is acquisition of resistance ( i . e . , change of strain from more susceptible to less susceptible ) as a result of therapy .",
"A key consideration with any TB diagnostic strategy is the role of the diagnostic test as applied to individuals who do not have TB ( i . e . , specificity ) .",
"We included this element by considering that a small proportion of the population without TB would present with TB-like symptoms each year .",
"This proportion ( selected such that 10% of individuals being evaluated in the high-incidence setting actually have underlying TB ) remains constant across all scenarios , such that the pre-test probability of TB is higher in settings of high TB incidence , and declines over time as successful TB control strategies are employed .",
"Individuals without TB who are ( inappropriately ) treated for TB incur costs of TB therapy and are also marked as ‘previously treated’ for purposes of diagnostic evaluation in the future .",
"Economic parameters are estimated using a unit-costing approach , whereby the unit cost of a TB diagnostic attempt and a TB treatment course ( separately for first-line , category two , and second-line therapy ) is enumerated under each scenario , and this cost is multiplied by the number of diagnostic attempts and treatment courses performed .",
"For simplicity , we adopt the perspective of a TB control program for our costing; additional costs of HIV care and general health services ( e . g . , hospitalization ) are not included .",
"The decision tree below is used to estimate the cost per diagnostic attempt or treatment course , conditional on an individual's HIV , drug resistance , and previous treatment status .",
"Individuals who default or die on therapy are assumed to incur half the cost of a treatment course .",
"Given the short time horizon ( 5 years ) , the focus on costs and outcomes ( rather than cost-effectiveness per se ) , and the desire to compare costs in year 1 and at the end of year 5 in equivalent terms , we did not discount future costs or outcomes for this analysis .",
"All costs are reported in US dollars , assuming the year of costs that is specified by the user .",
"Under each scenario , we use decision analysis to ascertain the following quantities related to each diagnostic attempt:Probability of receiving successful treatment . Probability of receiving ineffective treatment ( resulting in failure or default , including the probability of acquired resistance ) .",
"Cost of treatment ( conditional on whether treatment is successful or ineffective ) .",
"Cost of diagnosis . Diagnostic delay incurred before treatment initiated .",
"These quantities are calculated conditional on each patient's infectious ( smear ) status , drug resistance status , HIV status , and prior treatment status .",
"These probabilities are calculated for each diagnostic attempt , with the result fed back into the transmission model for purposes of appropriately allocating flows between compartments .",
"Inputs into the decision model include the probabilities of failure/recurrence , probability of empiric therapy , loss to follow-up before treatment , diagnostic accuracy , diagnostic delay , and economic parameters as shown in Table 1 .",
"Outputs from the decision tree appear as parameters in the model , as described in Table 2 and the following equations . 10 . 7554/eLife . 02565 . 008Table 2 . Model parameters and symbolic representationsDOI: http://dx . doi . org/10 . 7554/eLife . 02565 . 008ParameterRepresentationBaseline value ( see Table 1 ) Transmission rate ( transmission events per highly infectious person-year ) βCalibrated to TB incidenceProportional reduction in per-case transmission rate Drug-susceptible TBφ01 . 0 Isoniazid-monoresistant TBφ125%* of φ2 MDR-TBφ2CalibratedHIV incidence rate , per yearθCalibrated to HIV prevalenceRelative transmission rate from smear-negative/less infectious TBζ0 . 22Proportion of pulmonary TB that is smear-positive/highly infectious HIV-negativeψ00 . 63 HIV-infectedψ10 . 50Endogenous reactivation rate , per year HIV-negativeε00 . 005 HIV-infectedε10 . 05Proportion of recent infections resulting in rapid progression HIV-negativeπ00 . 14 HIV-infectedπ10 . 47Reduction in TB rapid progression probability due to latent TB infection HIV-negativeι0 . 79 HIV-infectedNot included0Baseline mortality rate , per yearμbl1/55 = 0 . 018Additional HIV-related mortality rate , per yearμh0 . 05Additional untreated TB-related mortality rate , per year HIV-negative , smear-positive/highly infectiousμt10 . 23 HIV-negative , smear-negative/less infectiousμt00 . 07 HIV-infectedμth1 . 0Rate of spontaneous TB resolution , per year Smear-positive/highly infectiousν10 . 1 Smear-negative/less infectiousν00 . 27 HIV-infectedNot included0Rate of starting diagnosis-seeking in active TB , per year HIV-negativeδe01 . 33 ( 9 months ) HIV-infectedδe112 ( 1/month ) Rate of progression: ineffective therapy to repeat therapy ( failure ) or active TB ( relapse ) , per yearδf6/12 = 0 . 5Rate of diagnostic evaluation for TB , per year Late active TBInput into decision tree5 . 0 No active TBτ00 . 01Decision tree outputs ( in addition to unit costs ) :Vary by intervention Successful diagnosis rate of late active TB , per yearσhdip Rate of movement from successful diagnosis to treatment ( 1/diagnostic delay ) , per yearρhdip Ineffective diagnosis rate of late active TB , per yearκhdip Rate of diagnosis and treatment leading to new resistance , per year susceptible to INH-monoresistantαsihip susceptible to MDRαsmhip INH-monoresistant to MDRαimhip*Calculated such that ( 1−φ1 ) = 0 . 25* ( 1−φ2 ) .",
"Our primary outcomes under each scenario were TB incidence , TB mortality , MDR-TB incidence , and incremental TB diagnostic and treatment costs ( during year 1 and at the end of the 5-year period ) relative to the baseline strategy .",
"By giving flexibility to change model inputs , we provide users the ability to conduct any sensitivity analysis desired .",
"However , for illustrative purposes , we also conducted a series of one-way sensitivity analyses in which each model parameter in Table 1 was varied by ±50% of its listed value ( for proportions , 50% of the difference between the value and either zero or one ) .",
"Our primary outcome for sensitivity analysis was the change in TB incidence , comparing the ‘Xpert for all’ strategy to baseline in the high incidence setting .",
"We also conducted multivariable uncertainty analyses by calculating partial rank correlation coefficients ( Kendall , 1942 ) between each natural history parameter and the outcomes of TB incidence , TB mortality , and 5-year costs .",
"In addition , we constructed 95% uncertainty intervals around our estimates of outcomes in each individual country by simultaneously varying each model parameter by ±10% over a uniform distribution and each target value ( i . e . , TB incidence , HIV prevalence , and MDR-TB prevalence ) over its reported uncertainty range .",
"In this fashion , we constructed 10 , 000 simulations using Latin Hypercube Sampling ( McKay et al . , 1979 ) and took 95% uncertainty ranges as the 2 . 5th and 97 . 5th percentiles of outcomes in these simulations; these ranges are provided in the web-based version of the model for each country .",
"Rates of flow between compartments are governed by the system of ordinary differential equations listed in Equations 1–6 .",
"We first define the forces of infection ( according to resistance status ) and total mortality for simplicity .",
"( 1 ) dUhp ( t ) dt=Ih=0 , p=0*μtot ( t ) −[λ0 ( t ) +λ1 ( t ) +λ2 ( t ) +μbl ( t ) ]*Uhp ( t ) −Ih=0*[θ*U0p ( t ) ]+Ih=1*[θ*U0p ( t ) −μh*U1p ( t ) ]−Ip=0*[τ0* ( 1−sh ) *Uh0 ( t ) ]+Ip=1*[τ0* ( 1−sh ) *Uh0 ( t ) ]where μtot is the sum of all mortality ( to maintain a constant population ) , λd is the force of infection for a given drug resistance strain , μbl is the baseline mortality rate , μh is the HIV-specific mortality rate , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , θ is the HIV incidence rate , τ0 is the rate of seeking diagnosis among people without TB , and sh is the specificity of the diagnostic test .",
"Thus , uninfected individuals exit through infection and death , acquire HIV according to the HIV incidence rate , and become previously treated for TB ( inappropriately ) according to the specificity of the test .",
"The HIV-uninfected , not previously treated compartment is replenished at a rate that matches total mortality and thereby maintains a constant population .",
"( 2 ) dLhdp ( t ) dt=λd ( t ) * ( 1−πh ) *{Uhp ( t ) +∑d[Lhdp ( t ) * ( 1−Ih=0*ι ) ]}+Ip=1*∑i , p[ρhdip*Phdip ( t ) ]+Ih=0*{ν0*E0dp+∑i[νi* ( A0dip+P0dip+I0dip ) ]}−{[ ( λ0 ( t ) +λ1 ( t ) +λ2 ( t ) ) * ( 1−Ih=0*ι ) +εh+μbl+Ih=1*μh]*Lhdp ( t ) }−Ih=0*[θ*L0dp ( t ) ]+Ih=1*[θ*L0dp ( t ) ]where λd is the strain-specific force of infection , πh is the proportion of recent infections that progress rapidly to active TB , ι is the relative reduction in rapid progression after infection among people with latent TB , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , ρhdip is the rate of treatment after successful diagnosis is initiated , νi is the spontaneous recovery rate , εh is the endogenous reactivation rate , μbl is the baseline mortality rate , μh is the HIV-specific mortality rate , and θ is the HIV incidence rate .",
"Thus , individuals enter the latently infected compartment through initial TB infection ( without rapid progression ) , reinfection ( without rapid progression , and accounting for immune protection ) , initiation of successful treatment , or spontaneous resolution .",
"Individuals completing treatment only enter the previously treated compartment ( p=1 ) .",
"Individuals exit this compartment through TB reinfection , endogenous reactivation , and death , and they acquire HIV infection at a constant rate .",
"( 3 ) dEhdp ( t ) dt=λd ( t ) *πh*{Uhp ( t ) +∑d[Lhdp ( t ) * ( 1−Ih=0*ι ) ]}+Lhdp ( t ) *εh−{[μbl+Ih=0* ( μt0+ν0+δe0 ) +Ih=1* ( μh+μth+δe1 ) ]*Ehdp ( t ) }−Ih=0*[θ*E0dp ( t ) ]+Ih=1*[θ*E0dp ( t ) ]where λd is the force of infection , πh is the proportion of recent infections that progress rapidly to active TB , ι is the relative reduction in rapid progression after infection among people with latent TB , Ih=0 is an indicator function of HIV status ( = 1 if h = 0 , 0 otherwise ) , εh is the endogenous reactivation rate , μbl is the baseline mortality rate , μt0 is the TB-specific mortality rate for less-infectious TB , ν0 is the spontaneous recovery rate for less-infectious TB , δeh is the HIV-specific rate of progression to late active TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , and θ is the HIV incidence rate .",
"Thus , individuals enter this compartment through rapid progression of recent infection or endogenous reactivation of latent infection .",
"They exit through progression to late active disease , spontaneous cure ( if HIV-uninfected ) , or death , and they acquire HIV infection at a constant rate .",
"( 4 ) dAhdip ( t ) dt=δeh*[Ii=1*ψh+Ii=0* ( 1−ψh ) ]*Ehdp ( t ) +δf*Ihdip ( t ) −Ahdip ( t ) *[μbl+Ih=0* ( μti+νi+σ0dip+κ0dip+Id=0*αsi0ip+Id=0*αsm0ip+Id=1*αim0ip ) +Ih=1* ( μh+μth+σ1dip+κ1dip+Id=0*αsi1ip+Id=0*αsm1ip+Id=1*αim1ip ) ]−Ih=0*[θ*A0dip ( t ) ]+Ih=1*[θ*A0dip ( t ) ]where δeh is the rate of progression from early active TB , Ieq is an indicator function ( = 1 if the condition eq is met , 0 otherwise ) , ψh is the proportion of active TB that is highly infectious ( smear-positive ) , δf is the rate ( 1/duration ) of ineffective therapy , μbl is the baseline mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , σhdip is the rate of diagnosis ultimately leading to successful treatment , κhdip is the rate of placing individuals on ineffective treatment that does not generate acquired resistance , αsihip is the rate of placing individuals on ineffective treatment that generates INH monoresistance , αsmhip and αimhip are the rates of placing individuals on ineffective treatment that generates MDR-TB , and θ is the HIV incidence rate .",
"Thus , individuals enter this compartment through progression from early active disease or relapse/failure after ineffective treatment .",
"They exit through spontaneous recovery , diagnosis leading to successful treatment , initiation of ineffective treatment ( which can , in turn , generate acquired drug resistance ) , or death , and they acquire HIV infection at a constant rate .",
"This compartment consists of individuals who have initiated a diagnostic attempt that will lead to successful treatment , yet remain infectious while awaiting initiation of treatment .",
"Inclusion of this compartment is designed to capture the effects of diagnostic delays .",
"( 5 ) dPhdip ( t ) dt=σhdip*Ahdip ( t ) −Phdip ( t ) *[μbl+Ih=0* ( μti+νi+ρ0dip ) +Ih=1* ( μh+μth+ρ1dip ) ]−Ih=0*[θ*P0dip ( t ) ]+Ih=1*[θ*P0dip ( t ) ]where σhdip is the rate of initiating successful diagnostic attempts , Ih=0 is an indicator function of HIV status ( = 1 if h = 0 , 0 otherwise ) , μbl is the non-TB mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , ρhdip is the rate of starting therapy after initiating a successful diagnostic attempt ( 1/diagnostic delay ) , and θ is the HIV incidence rate .",
"Thus , individuals enter this compartment by initiation of successful diagnostic attempts and exit through initiation of treatment , spontaneous cure , or death .",
"They acquire HIV infection at a constant rate .",
"This compartment contains all individuals being treated whose treatment course ends in default or failure .",
"Unlike the previous ( successful treatment ) compartment , diagnostic delay is not explicitly incorporated into this compartment , as to do so would prolong the duration of time until individuals who default re-enter the ‘late active’ compartment .",
"Inclusion of a diagnostic delay before this compartment does not materially affect results .",
"Individuals exit this compartment either in default after partial therapy—which has a defined probability of achieving cure despite not being completed—or failure .",
"Failure leads immediately to another course of treatment , which can either be successful ( i . e . , transition to the latent compartment ) or unsuccessful ( i . e . , remain in the inappropriate treatment compartment—which results in transition to the ‘previously treated’ compartment , p=1 , not shown below ) .",
"( 6 ) dIhdip ( t ) dt=κhdip*Ahdip ( t ) +Id=1*αsihdip*Ah0ip ( t ) +Id=2*[αsmhip*Ah0ip ( t ) +αimhip*Ah1ip ( t ) ]−Ihdip ( t ) *[μbl+Ih=0* ( μt0+νi+δf ) +Ih=1* ( μh+μth+δf ) ]−Ih=0*[θ*I0dip ( t ) ]+Ih=1*[θ*I0dip ( t ) ]where κhdip is the rate of placing individuals on ineffective treatment that does not generate acquired resistance , αsihip is the rate of placing individuals on ineffective treatment that generates INH monoresistance from drug-susceptible TB , αsmhip is the rate of placing individuals on ineffective treatment that generates MDR-TB from drug-susceptible TB , αimhip is the rate of placing individuals on ineffective treatment that generates MDR-TB from INH-monoresistant TB , μbl is the non-TB mortality rate , μti is the TB-specific mortality rate for non-HIV-associated TB , μh is the HIV-specific mortality rate , μth is the TB-specific mortality rate for people living with HIV , νi is the spontaneous recovery rate , δf is the rate ( 1/duration ) of ineffective therapy , and θ is the HIV incidence rate .",
"Thus , individuals enter this compartment through ineffective treatment from the late active compartment ( conditional on whether that treatment also generates new drug resistance ) and exit through completion of a course of ineffective therapy , spontaneous resolution , or death .",
"They acquire HIV infection at a constant rate .",
"The equations above comprise a series of 100 ordinary differential equations .",
"In order to generate an equilibrium population according to user specifications of TB incidence , MDR-TB prevalence , and HIV prevalence , it is necessary to solve for the roots of a system of these 100 equations , plus three additional equations to account for the user inputs .",
"We accomplish this using the ‘fsolve’ routine in SciPy ( www . scipy . org ) .",
"To solve for these three additional equations , we first match each user input to a single parameter: TB incidence to the transmission rate β , MDR-TB prevalence to the relative reduction in transmission for MDR-TB φ2 , and HIV prevalence to the HIV incidence rate θ .",
"We then constrain the system of equations such that the total population remains constant at 100 , 000 , and we add the following three equations to the system:dβ/dt= ( calculated TB incidence ) − ( user-defined TB incidence ) dφ2/dt= ( calculated MDR-TB prevalence ) − ( user-defined MDR-TB prevalence ) dθ/dt= ( calculated HIV prevalence ) − ( user-defined HIV prevalence ) By solving for the roots at which this system of equations equals zero , we generate an equilibrium ( steady-state ) population that also defines β , φ2 , and θ such that the user-defined targets are also met .",
"Additional complexity is added to account for non-equilibrium in TB incidence and MDR-TB prevalence .",
"We accomplish this by fitting an equilibrium defined by the user-specified target of HIV prevalence , and the user-specified ‘initial’ values of TB incidence and MDR-TB prevalence .",
"This equilibrium is set to be 50 years in the past; at this time , the system of equations is solved as above .",
"We then allow for the parameters β and φ2 to be altered from their original values ( corresponding to the equilibrium condition ) such that a second set of targets are attained after 50 years ( start of the analysis ) .",
"If these calculated values at the end of 50 years do not match the user-defined targets , the parameter values are changed accordingly , and the model is re-run from equilibrium until the appropriate parameter value is identified that generates the user-defined TB incidence and MDR-TB prevalence , within a relative tolerance of 0 . 05 in each variable .",
"These parameters may then be further modified such that they change over the analysis frame of five years ( e . g . , to describe an epidemic with increasing MDR-TB prevalence over time ) .",
"In the primary version of the model , this is only automated for low-incidence scenarios in which the user inputs a TB incidence of less than 50 per 100 , 000/year .",
"In such cases , the model generates an equilibrium population 50 years in the past with a TB incidence of 50 per 100 , 000/year and reduces the transmission parameter β until the user-defined TB incidence is achieved 50 years later ( i . e . , the start of the analytic period ) .",
"This accounts for the fact that most low-incidence settings have substantially more latent TB infection than would be estimated by an equilibrium population with a very low TB incidence—thereby more appropriately reflecting a higher proportion of TB due to reactivation rather than recent infection .",
"However , we include code ( described in the user manual below ) that allows users to define high incidence scenarios that are likewise not at equilibrium , as well as ‘emerging MDR’ scenarios in which the prevalence of MDR-TB is increasing rather than stable ."
]
] | [
"Most models of infectious diseases , including tuberculosis ( TB ) , do not provide results customized to local conditions .",
"We created a dynamic transmission model to project TB incidence , TB mortality , multidrug-resistant ( MDR ) TB prevalence , and incremental costs over 5 years after scale-up of nine alternative diagnostic strategies .",
"A corresponding web-based interface allows users to specify local costs and epidemiology .",
"In settings with little capacity for up-front investment , same-day microscopy had the greatest impact on TB incidence and became cost-saving within 5 years if delivered at $10/test .",
"With greater initial investment , population-level scale-up of Xpert MTB/RIF or microcolony-based culture often averted 10 times more TB cases than narrowly-targeted strategies , at minimal incremental long-term cost .",
"Xpert for smear-positive TB had reasonable impact on MDR-TB incidence , but at substantial price and little impact on overall TB incidence and mortality .",
"This user-friendly modeling framework improves decision-makers' ability to evaluate the local impact of TB diagnostic strategies ."
] | [
"Tuberculosis is an infectious bacterial disease caused predominantly by the microorganism Mycobacterium tuberculosis .",
"Although the number of deaths from tuberculosis has been falling in recent years , the disease still kills more than 1 million people every year , mainly in developing countries .",
"Tuberculosis can be treated with antibiotics , but the emergence of bacteria that are resistant to existing drugs is threatening efforts to eradicate the disease .",
"Preventing the spread of tuberculosis is heavily dependent on accurate diagnosis of individuals with the disease .",
"This is challenging because the initial symptoms are often mild , usually just a cough , which means that someone can spread the disease to many others over a period of several months before the symptoms become worse—fever , night sweats , and weight loss—and they realize that they are sick .",
"Multiple diagnostic strategies are available , from the relatively low-tech—examining sputum samples under a microscope to detect tuberculosis bacteria—to more sophisticated tests that can detect bacterial DNA and determine whether the bacteria are drug-resistant in less than 2 hr .",
"Choosing which diagnostic strategy to adopt can be challenging because the optimal solution in a region will depend on the specific local conditions .",
"To overcome this problem , Dowdy et al . have developed a computer program that enables decision-makers to input four key parameters that describe the tuberculosis situation in their region , and to obtain 5-year projections of the rate of new infections , mortality , and total costs likely to result from adopting any of nine different diagnostic strategies .",
"The four parameters are the number of new cases of tuberculosis each year ( incidence ) , the proportion of new cases that are multi-drug resistant , the proportion of the adult population that has HIV , and the local costs of various diagnostic techniques and treatments .",
"Since the entire computer program is written in a freely available open-source programming language ( Python ) , any user can tweak these parameters to provide a more precise fit to their own region .",
"Alternatively , the standard version of the program can be run directly from a website without any need to interact with computer code .",
"This model is the first to enable local decision-makers to evaluate the impact of different diagnostic strategies for tuberculosis under the conditions specific to their region .",
"The model predicts , for example , that in areas where there is little money available for up-front investment , same-day microscopy analysis of sputum samples and starting patients on treatment is the most cost-effective strategy for reducing the rate of new infections .",
"Given the wide variation in conditions within even small geographical areas , this more flexible approach should lead to the more efficient use of resources and may , ultimately , help to reduce the spread of tuberculosis ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"tools and resources"
] | DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics | elife-64940-v3 | [
[
"The 23 intervertebral discs ( IVDs ) in the human spine provide stability , mobility , and flexibility .",
"IVD degeneration ( IDD ) , most common in the lumbar region ( Saleem et al . , 2013; Teraguchi et al . , 2014 ) , is associated with a decline in function and a major cause of back pain , affecting up to 80% of the world’s population at some point in life ( Rubin , 2007 ) , presenting significant socioeconomic burdens .",
"Multiple interacting factors such as genetics , influenced by ageing , mechanical and other stress factors , contribute to the pathobiology , onset , severity , and progression of IDD ( Munir et al . , 2018 ) .",
"IVDs are large , avascular , extracellular matrix ( ECM ) -rich structures comprising three compartments: a hydrated nucleus pulposus ( NP ) at the centre , surrounded by a tough annulus fibrosus ( AF ) at the periphery , and cartilaginous endplates of the adjoining vertebral bodies ( Humzah and Soames , 1988 ) .",
"The early adolescent NP is populated with vacuolated notochordal-like cells , which are gradually replaced by small chondrocyte-like cells ( Risbud et al . , 2015 ) .",
"Blood vessels terminate at the endplates , nourishing and oxygenating the NP via diffusion , whose limited capacity mean that NP cells are constantly subject to hypoxic , metabolic , and mechanical stresses ( Urban et al . , 2004 ) .",
"With ageing and degeneration , there is an overall decline in cell ‘health’ and numbers ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) , disrupting homeostasis of the disc proteome .",
"The ECM has key roles in biomechanical function and disc hydration .",
"Indeed , a hallmark of IDD is reduced hydration in the NP , diminishing the disc’s capacity to dissipate mechanical loads .",
"Clinically , T-2 weighted MRI is the gold standard for assessing IDD , that uses disc hydration and structural features such as bulging or annular tears to measure severity ( Pfirrmann et al . , 2001; Schneiderman et al . , 1987 ) .",
"The hydration and mechanical properties of the IVD are dictated by the ECM composition , which is produced and maintained by the IVD cells .",
"To meet specific biomechanical needs , cells in the IVD compartments synthesise different compositions of ECM proteins .",
"Defined as the ‘matrisome’ ( Naba et al . , 2012 ) , the ECM houses the cells and facilitates their inter-communication by regulation of the availability and presentation of signalling molecules ( Taha and Naba , 2019 ) .",
"With ageing or degeneration , the NP becomes more fibrotic and less compliant ( Yee et al . , 2016 ) , ultimately affecting disc biomechanics ( Newell et al . , 2017 ) .",
"Changes in matrix stiffness can have a profound impact on cell-matrix interactions and downstream transcriptional regulation , signalling activity , and cell fate ( Park et al . , 2011 ) .",
"The resulting alterations in the matrisome lead to vicious feedback cycles that reinforce cellular degeneration and ECM changes .",
"Notably , many of the associated IDD genetic risk factors , such as COL9A1 ( Jim et al . , 2005 ) , ASPN ( Song et al . , 2008 ) , and CHST3 ( Song et al . , 2013 ) , are variants in genes encoding matrisome proteins , highlighting their importance for disc function .",
"Therefore , knowledge of the cellular and extracellular proteome and their spatial distribution in the IVD is crucial to understanding the mechanisms underlying the onset and progression of IDD ( Feng et al . , 2006 ) .",
"Current knowledge of IVD biology is inferred from a limited number of transcriptomic studies on human ( Minogue et al . , 2010; Riester et al . , 2018; Rutges et al . , 2010 ) and animal ( Veras et al . , 2020 ) discs .",
"Studies showed that cells in young healthy NP express markers including CD24 , KRT8 , KRT19 , and T ( Fujita et al . , 2005; Minogue et al . , 2010; Rutges et al . , 2010 ) , whereas NP cells in aged or degenerated discs have different and variable molecular signatures ( Chen et al . , 2006; Rodrigues-Pinto et al . , 2016 ) , such as genes involved in TGFβ signalling ( TGFA , INHA , INHBA , BMP2/6 ) .",
"The healthy AF expresses genes including collagens ( COL1A1 and COL12A1 ) ( van den Akker et al . , 2017 ) , growth factors ( PDGFB , FGF9 , VEGFC ) , and signalling molecules ( NOTCH and WNT ) ( Riester et al . , 2018 ) .",
"Although transcriptomic data provides valuable cellular information , it does not faithfully reflect the molecular composition .",
"Cells represent only a small fraction of the disc volume , transcriptome-proteome discordance does not enable accurate predictions of protein levels from mRNA ( Fortelny et al . , 2017 ) , and the disc matrisome accumulates and remodels over time .",
"Proteomic studies on animal models of IDD , including murine ( McCann et al . , 2015 ) , canine ( Erwin et al . , 2015 ) , and bovine ( Caldeira et al . , 2017 ) , have been reported .",
"Nevertheless , human-animal differences in cellular phenotypes and mechanical loading physiologies mean that these findings might not translate to the human scenario .",
"So far , human proteomic studies have compared IVDs with other cartilaginous tissues ( Önnerfjord et al . , 2012 ) and have shown increases in fibrotic changes in aging and degeneration ( Yee et al . , 2016 ) , a role for inflammation in degenerated discs ( Rajasekaran et al . , 2020 ) , the presence of haemoglobins and immunoglobulins in discs with spondylolisthesis and herniation ( Maseda et al . , 2016 ) , and changes in proteins related to cell adhesion and migration in IDD ( Sarath Babu et al . , 2016 ) .",
"The reported human disc proteomes were limited in the numbers of proteins identified and finer compartmentalisation within the IVD , and disc levels along the lumbar spine have yet to be studied .",
"Nor have the proteome dynamics in term of ECM remodelling ( synthesis and degradation ) in young human IVDs and changes in ageing and degeneration been described .",
"In this study , we presented DIPPER ( the Big Dipper are point-reference stars for guiding nautical voyages ) , a comprehensive disc proteomic resource , comprising static spatial proteome , dynamic proteome and transcriptome , and a methodological flow , for studying the human IVD in youth , ageing , and degeneration .",
"First , we established a high-resolution point-reference map of static spatial proteomes along the lateral and anteroposterior directions of IVDs at three lumbar levels , contributed by a young ( 16M ) and an aged ( 59M ) cadavers with no reported scoliosis or degeneration .",
"We evaluated variations among the disc compartments and levels by principal component analysis ( PCA ) , analysis of variance ( ANOVA ) , and identification of differentially expressed proteins ( DEPs ) .",
"We discovered modules containing specific sets of proteins that describe the directional trends of a young IVD , and the deconstruction of these modules with ageing and degeneration .",
"Using a LASSO regression model , we identified proteins ( the hydration matrisome ) predictive of tissue hydration as indicated by high-resolution MRI of the aged discs .",
"Finally , we showed how the point-reference proteomes can be utilised , to integrate with other independent transcriptome and dynamic proteome ( SILAC and degradome ) datasets from 15 additional clinical disc specimens , elevating the robustness of the proteomic findings .",
"An explorable web interface hosting the data and findings is presented , serving as a useful resource for the scientific community ."
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"DIPPER comprises 94 genome-wide measurements from lumbar disc components of 17 individuals ( Figure 1A; Table 1 ) , with data types ranging from label-free proteomics , transcriptomics , SILAC to degradome ( López-Otín and Overall , 2002 ) .",
"High-resolution static spatial proteomes were generated from multiple intact disc segments of young trauma-induced ( 16 M ) and aged ( 57 M ) cadaveric spines .",
"T1- and T2-weighted MRI ( 3T ) showed the young discs ( L3/4 , L4/5 , L5/S1 ) were non-degenerated , with a Schneiderman score of 1 from T2 images ( Figure 1B ) .",
"The NP of young IVD were well hydrated ( white ) with no disc bulging , endplate changes , or observable inter-level variations ( Figure 1B; Figure 1—figure supplement 1A ) , consistent with healthy discs and were deemed fit to serve as a benchmarking point-reference .",
"To investigate structural changes associated with ageing , high-resolution ( 7T ) MRI was taken for the aged discs ( Figure 1C ) .",
"All discs had irregular endplates , and annular tears were present ( green arrowheads ) adjacent to the lower endplate and extending towards the posterior region at L3/4 and L4/5 ( Figure 1C ) .",
"The NP exhibited regional variations in hydration in both sagittal and transverse images ( Figure 1C ) .",
"Morphologically , the aged discs were less hydrated and the NP and AF structures less distinct , consistent with gross observations ( Figure 1—figure supplement 1A ) .",
"Scoliosis was not detected in these two individuals .",
"Information of the disc samples used in the generation of other profiling data are described in Materials and methods and Table",
"1 . They are clinical samples taken from patients undergoing surgery .",
"The disc levels and intactness varied , and thus are more suitable for cross-validation purposes and some are directly relevant to IDD .",
"The intact discs from the two cadaveric spines enabled us to derive spatial proteomes for young and aged human IVDs .",
"We subdivided each lumbar disc into 11 key regions ( Figure 1D ) , spanning the outer-most ( outer AF; OAF ) to the central-most ( NP ) region of the disc , traversing both anteroposterior and lateral axes , adding valuable spatial information to our proteomic dataset .",
"Since the disc is an oval shape , an inner AF ( IAF ) region was assigned in the lateral directions .",
"A ‘mixed’ compartment between the NP and IAF with undefined boundary was designated as the NP/IAF in all four ( anteroposterior and lateral ) directions .",
"In all , this amounted to 66 specimens with different compartments , ages , directions and levels , which then underwent LC-MS/MS profiling ( Supplementary file 1 ) .",
"Systematic analyses of the 66 profiles are depicted in a flowchart ( Figure 1A ) .",
"A median of 654 proteins per profile were identified for the young samples and 829 proteins for the aged samples , with a median of 742 proteins per profile for young and aged combined .",
"The proteome-wide distributions were on similar scales across the profiles ( Figure 1—figure supplement 1B ) .",
"Of the 3100 proteins detected in total , 418 were matrisome proteins ( 40 . 7% of all known matrisome proteins ) and 2682 non-matrisome proteins ( ~14% of genome-wide non-matrisome genes ) ( Figure 1E; Figure 1—figure supplement 1C ) , and 983 were common to all four major compartments , namely the OAF , IAF , NP/IAF , and NP ( Figure 1E , upper panel ) .",
"A total of 1883 proteins were identified in young discs , of which 690 ( 36% ) were common to all regions .",
"Additionally , 45 proteins ( 2 . 4% ) were unique to NP , whilst NP/IAF , IAF , and OAF had 86 ( 4 . 6% ) , 54 ( 2 . 9% ) , and 536 ( 28% ) unique proteins , respectively ( Figure 1E , middle panel ) .",
"For the aged discs , 2791 proteins were identified , of which 803 ( 28 . 8% ) were common to all regions .",
"NP , NP/IAF , and IAF had 34 ( 12% ) , 80 ( 28 . 7% ) , and 44 ( 15% ) unique proteins , respectively , with the OAF accounting for the highest proportion of 1314 unique proteins ( 47% ) ( Figure 1E , lower panel ) .",
"The aged OAF had the highest number of detected proteins with an average of 1156 , followed by the young OAF with an average of 818 ( Figure 1F ) .",
"The quantity and spectrum of protein categories identified suggest sufficient proteins had been extracted and the data are of high quality .",
"We divided the detected matrisome proteins into core matrisome ( ECM proteins , encompassing collagens , proteoglycans , and glycoproteins ) and non-core matrisome ( ECM regulators , ECM affiliated , and secreted factors ) , according to a matrisome classification database ( Naba et al . , 2012 ) ( matrisomeproject . mit . edu ) ( Figure 1F ) .",
"Despite the large range of total numbers of proteins detected ( 419 to 1920 ) across the 66 profiles ( Figure 1F ) , all six subcategories of the matrisome contained similar numbers of ECM proteins ( Figure 1F and G; Figure 1—figure supplement 2A ) .",
"The non-core matrisome proteins were significantly more abundant in aged than in young discs ( Figure 1—figure supplement 2A ) .",
"On average , 19 collagens , 18 proteoglycans , 68 glycoproteins , 52 ECM regulators , 22 ECM affiliated proteins , and 29 secreted factors of ECM were detected per profile .",
"The majority of the proteins in these matrisome categories were detected in all disc compartments , and in both age groups .",
"A summary of all the comparisons are presented in Figure 1—figure supplement 1C–E , and the commonly expressed matrisome proteins are listed in Table",
"2 . Even though there are approximately three times more non-matrisome than matrisome proteins per profile on average ( Figure 1F ) , their expression levels in terms of label-free quantification ( LFQ ) values are markedly lower ( Figure 1—figure supplement 2B ) .",
"Specifically , the expression levels of core-matrisome were the highest , with an average log2 ( LFQ ) of 30 . 65 , followed by non-core matrisome at 28 . 56 , and then non-matrisome at 27 . 28 ( Figure 1—figure supplement 2B ) .",
"Within the core-matrisome , the expression was higher ( p=6 . 4 × 10−21 ) in young ( median 30 . 74 ) than aged ( median 29 . 72 ) discs ( Figure 1—figure supplement 2C & D ) .",
"This difference between young and aged discs is consistent within the subcategories of core and non-core matrisome , with the exception of the ECM regulator category ( Figure 1H ) .",
"The non-core matrisome and non-matrisome , however , exhibited smaller cross-compartment and cross-age differences in terms of expression levels ( Figure 1—figure supplement 2E–H ) .",
"That is , the levels of ECM proteins in each compartment of the disc declines with ageing and possibly changes in the relative composition , while the numbers of proteins detected per matrisome subcategory remain similar .",
"This agrees with the concept that with ageing , ECM synthesis is not sufficient to counterbalance degradation , as exemplified in a proteoglycan study ( Silagi et al . , 2018 ) .",
"Although 86 . 5% ( 2682 ) of the detected proteins were non-matrisome , their expression levels were considerably lower than matrisome proteins across all sample profiles ( Figure 1F ) .",
"A functional categorisation according to the Human Genome Nomenclature Committee gene family annotations ( Yates et al . , 2017 ) showed many categories containing information for cellular components and activities , with the top 30 listed in Figure 1I .",
"These included transcriptional and translational machineries , post-translational modifications , mitochondrial function , protein turnover and importantly , transcriptional factors , cell surface markers , and inflammatory proteins that can inform gene regulation , cell identity , and response in the context of IVD homeostasis , ageing , and degeneration .",
"These functional overviews highlighted 77 DNA-binding proteins and/or transcription factors , 83 cell surface markers , and 175 inflammatory-related proteins , with their clustering data presented as heatmaps ( Figure 1—figure supplement 3 ) .",
"Transcription factors and cell surface markers are detected in some profiles ( Figure 1—figure supplement 3A and B ) .",
"The heatmap of the inflammatory-related proteins showed that more than half of the proteins are detected in the majority of samples , with four major clusters distinguished by age and expression levels ( Figure 1—figure supplement 3C ) .",
"For example , one of the clusters in the aged samples showed enrichment for complement and coagulation cascades ( False Discovery Rate , FDR q = 1 . 62 × 10−21 ) and clotting factors ( FDR q = 6 . 05 × 10−9 ) , indicating potential infiltration of blood vessels .",
"Lastly , there are 371 proteins involved in signalling pathways , and their detection frequency in the different compartments and heat map expression levels are illustrated in Figure 1—figure supplement 3D .",
"Cellularity within the IVD , especially the NP , decreases with age and degeneration ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) .",
"We assessed whether cellularity of the different compartments could be inferred from the proteomic data .",
"Quantitation of histones can reflect the relative cellular content of tissues ( Wiśniewski et al . , 2014 ) .",
"We detected 10 histones , including subunits of histone 1 ( HIST1H1B/C/D/E , HIST1H2BL , HIST1H3A , HIST1H4A ) and histone 2 ( HIST2H2AC , HIST2H2BE , HIST2H3A ) , with four subunits identified in over 60 sample profiles that are mutually co-expressed ( Figure 1—figure supplement 4A ) .",
"Interestingly , histone concentrations , and thus cellularity , increased from the inner to the outer compartments of the disc , and showed a highly significant decrease in aged discs compared to young discs across all compartments ( Figure 1J , Wilcoxon p=5 . 6 × 10−4 ; Figure 1—figure supplement 4B ) .",
"GAPDH and ACTA2 are two commonly used reference proteins , involved in the metabolic process and the cytoskeletal organisation of cells , respectively .",
"They are expected to be relatively constant between cells and are used to quantify the relative cellular content of tissues ( Barber et al . , 2005 ) .",
"They were detected in all 66 profiles .",
"GAPDH and ACTA2 amounts were significantly correlated with a Pearson correlation coefficient ( PCC ) of 0 . 794 ( p=9 . 3 × 10−9 ) ( Figure 1—figure supplement 4C ) , and they were both significantly co-expressed with the detected histone subunits , with PCCs of 0 . 785 and 0 . 636 , respectively ( Figure 1K; Figure 1—figure supplement 4D ) .",
"As expected , expression of the histones , GAPDH , and ACTA2 was not correlated with two core-matrisome proteins , ACAN and COL2A1 ( Figure 1—figure supplement 4E ) , whereas ACAN and COL2A1 were significantly co-expressed ( Figure 1—figure supplement 4F ) , as expected due to their related regulation of expression and tissue function .",
"Thus , cellularity information can be obtained from proteomic information , and the histone quantification showing reduced cellularity in the aged IVD is consistent with the reported changes ( Rodriguez et al . , 2011; Sakai et al . , 2012 ) .",
"To gain a global overview of the data , we performed PCA on a set of 507 proteins selected computationally , allowing maximal capture of valid values , while incurring minimal missing values , followed by imputations ( Figure 2—figure supplement 1; Materials and methods ) .",
"The first two principal components ( PCs ) explained a combined 65 . 5% of the variance , with 39 . 9% and 25 . 6% for the first and second PCs , respectively ( Figure 2A and B ) .",
"A support vector machine with polynomial kernel was trained to predict the boundaries: it showed PC1 to be most informative to predict age , with a clear demarcation between the two age groups ( Figure 2A , vertical boundary ) , whereas PC2 distinguished disc sample localities , separating the inner compartments ( NP , NP/IAF , and IAF ) from the OAF ( Figure 2A , horizontal boundary ) .",
"PC3 captured only 5 . 0% of the variance ( Figure 2C & D ) , but it distinguished disc level , separating the lowest level ( L5/S1 ) from the rest of the lumbar discs ( L3/4 and L4/5 ) ( Figure 2D , horizontal boundary ) .",
"Samples in the upper level ( L3/4 and L4/5 ) appeared to be more divergent , with the aged disc samples deviating from the young ones ( Figure 2D ) .",
"To extract the most informative features of the PCA , we performed proteome-wide associations with each of the top three PCs , which accounted for over 70% of total variance , and presented the top 100 most positively and top 100 most negatively correlated proteins for each of the PCs ( Figure 2E–G ) .",
"As expected , the correlation coefficients in absolute values were in the order of PC1 > PC2 > PC3 ( Figure 2E–G ) .",
"The protein content is presented as non-matrisome proteins ( grey colour ) and matrisome proteins ( coloured ) that are subcategorised as previously .",
"For the negatively correlated proteins , the matrisome proteins contributed to PC1 in distinguishing young disc samples , as well as to PC2 for sample location within the disc , but less so for disc level in PC3 .",
"Further , the relative composition of the core and non-core matrisome proteins varied between the three PCs , depicting the dynamic ECM requirement and its relevance in aging ( PC1 ) , tissue composition within the disc ( PC2 ) and mechanical loading ( PC3 ) .",
"PC1 of young discs identified known chondrocyte markers , CLEC3A ( Lau et al . , 2018 ) and LECT1/2 ( Zhu et al . , 2019 ) , hedgehog signalling proteins , HHIPL2 , HHIP , and SCUBE1 ( Johnson et al . , 2012 ) , and xylosyltransferase-1 ( XYLT1 ) , a key enzyme for initiating the attachment of glycosaminoglycan side chains to proteoglycan core proteins ( Silagi et al . , 2018; Figure 2E ) .",
"Most of the proteins that were positively correlated in PC1 were coagulation factors or coagulation related , suggesting enhanced blood infiltration in aged discs .",
"PC2 implicated key changes in molecular signalling proteins ( hedgehog , WNT and Nodal ) in the differences between the inner and outer disc regions ( Figure 2F ) .",
"Notably , PC2 contains heat shock proteins ( HSPA1B , HSPA8 , HSP90AA1 , HSPB1 ) which are more strongly expressed in the OAF than in inner disc , indicating the OAF is under stress ( Takao and Iwaki , 2002 ) .",
"Although the correlations in PC3 were much weaker , proteins such as CILP/CILP2 , DCN , and LUM were associated with lower disc level .",
"To investigate how age , disc compartment , level , and direction affect the protein profiles , we carried out ANOVA for each of these phenotypic factors for the categories of matrisome and non-matrisome proteins ( Figure 2—figure supplement 1H–J ) .",
"In the young discs , the dominant phenotype explaining the variances for all protein categories was disc compartment .",
"It is crucial that each disc compartment ( NP , IAF , and OAF ) has the appropriate protein composition to function correctly ( Figure 2—figure supplement 1I ) .",
"This also fits the understanding that young healthy discs are axially symmetric and do not vary across disc levels .",
"In aged discs , compartment is still relevant for non-matrisome proteins and collagen , but disc level and directions become influential for other protein categories , which is consistent with variations in mechanical loading occurring in the discs with ageing and degeneration ( Figure 2—figure supplement 1J ) .",
"In the combined ( young and aged ) disc samples , age was the dominant phenotype across major matrisome categories , while compartment best explained the variance in non-matrisome , reflecting the expected changes in cellular ( non-matrisome ) and structural ( matrisome ) functions of the discs ( Figure 2—figure supplement 1H ) .",
"This guided us to analyse the young and aged profiles separately , before performing cross-age comparisons .",
"PCA of the 33 young profiles showed a distinctive separation of the OAF from the inner disc regions on PC1 ( upper panel of Figure 3A ) .",
"In PC2 , the lower level L5/S1 could generally be distinguished from the upper lumbar levels ( lower panel of Figure 3A ) .",
"The detected proteome of the young discs ( Figure 3B ) accounts for 9 . 2% of the human proteome ( or 1883 out of the 20 , 368 on UniProt ) .",
"We performed multiple levels of pairwise comparisons ( summarised in Figure 3—figure supplement 1A ) to detect proteins associated with individual phenotypes , using three approaches ( see Materials and methods ) : statistical tests; proteins detected in one group only; or proteins using a fold-change threshold .",
"We detected a set of 671 DEPs ( Supplementary file 2 ) ( termed the ‘variable set’ ) , containing both matrisome and non-matrisome proteins ( Figure 3D ) , and visualised in a heatmap ( Figure 3—figure supplement 2 ) , with identification of four modules ( Y1-Y4 ) .",
"To investigate how the modules are associated with disc components , we compared their protein expression profiles along the lateral and anteroposterior axes .",
"The original log2 ( LFQ ) values were transformed to z-scores to be on the same scale .",
"Proteins of the respective modules were superimposed on the same charts , disc levels combined or separated ( Figure 3E–H; Figure 3—figure supplement 3 ) .",
"Module Y1 is functionally relevant to NP , containing previously reported NP and novel markers KRT19 , CD109 , KRT8 and CHRD ( Anderson et al . , 2002 ) , CHRDL2 , SCUBE1 ( Johnson et al . , 2012 ) , and CLEC3B .",
"Proteins levels in Y1 are lower on one side of the OAF , increases towards the central NP , before declining towards the opposing side , forming a concave pattern in both lateral and anteroposterior directions , with a shoulder drop occurring between IAF and OAF ( Figure 3E ) .",
"Module Y2 was enriched in proteins for ECM organisation ( FDR q = 8 . 8 × 10−24 ) ; Y3 was enriched for proteins involved in smooth muscle contraction processes ( FDR q = 8 . 96 × 10−19 ) ; and Y4 was enriched for proteins of the innate immune system ( FDR q = 2 . 93 × 10−20 ) .",
"Interestingly , these modules all showed a tendency for convex patterns with upward expression toward the OAF regions , in both lateral and anteroposterior directions , in all levels , combined or separated ( Figure 3F–H; Figure 3—figure supplement 3B–D ) .",
"The higher proportions of ECM , muscle contraction , and immune system proteins in the OAF are consistent with the contractile function of the AF ( Nakai et al . , 2016 ) , and with the NP being avascular and ‘immune-privileged’ in a young homeostatic environment ( Sun et al . , 2020 ) .",
"The most distinctive pattern on the PCA is the separation of the OAF from the inner disc , with 99 proteins expressed higher in the OAF and 55 expressed higher in the inner disc ( Figure 3I , J ) .",
"Notably , OAF and inner disc contained different types of ECM proteins .",
"The inner disc regions were enriched in collagens ( COL3A1 , COL5A1 , COL5A2 , and COL11A2 ) , matrillin ( MATN3 ) , and proteins associated with ECM synthesis ( PCOLCE ) and matrix remodelling ( MXRA5 ) .",
"We also identified in the inner disc previously reported NP markers ( KRT19 , KRT8 ) ( Risbud et al . , 2015 ) , in addition to inhibitors of WNT ( FRZB , DKK3 ) and BMP ( CHRD , CHRDL2 ) signalling ( Figure 3I , J ) .",
"Of note , FRZB and CHRDL2 were recently shown to have potential protective characteristics in osteoarthritis ( Ji et al . , 2019 ) .",
"The TGFβ pathway appears to be suppressed in the inner disc where antagonist CD109 ( Bizet et al . , 2011; Li et al . , 2016 ) is highly expressed , and the TGFβ activity indicator TGFBI is expressed higher in the OAF than in the inner disc .",
"The OAF is enriched with various collagens ( COL1A1 , COL6A1/2/3 , COL12A1 and COL14A1 ) , basement membrane ( BM ) proteins ( LAMA4 , LAMB2 and LAMC1 ) , small leucine-rich proteoglycans ( SLRP ) ( BGN , DCN , FMOD , OGN , PRELP ) , and BM-anchoring protein ( PRELP ) ( Figure 3I , J ) .",
"Tendon-related markers such as thrombospondins ( THBS1/2/4 ) ( Subramanian and Schilling , 2014 ) and cartilage intermediate layer proteins ( CILP/CILP2 ) are also expressed higher in the OAF .",
"Tenomodulin ( TNMD ) was exclusively expressed in 9 of the 12 young OAF profiles , and not in any other compartments ( Supplementary file 2 ) .",
"This fits a current understanding of the AF as a tendon/ligament-like structure ( Nakamichi et al . , 2018 ) .",
"In addition , the OAF was enriched in actin-myosin ( Figure 3I ) , suggesting a role of contractile function in the OAF , and in heat-shock proteins ( HSPA1B , HSPA8 , HSPB1 , HSP90B1 , HSP90AA1 ) , suggesting a stress response to fluctuating mechanical loads .",
"We sought to identify transitions in proteomic signatures between adjacent compartments .",
"The NP and NP/IAF protein profiles were highly similar ( Figure 3—figure supplement 1B ) .",
"Likewise , NP/IAF and IAF showed few DEPs , except COMP which was expressed higher in IAF ( Figure 3—figure supplement 1C ) .",
"OAF and NP ( Figure 3—figure supplement 1R ) and OAF and NP/IAF ( Figure 3—figure supplement 1O ) showed overlapping DEPs , consistent with NP and NP/IAF having highly similar protein profiles , despite some differences in the anteroposterior direction ( Figure 3—figure supplement 1P & Q ) .",
"The clearest boundary within the IVD , between IAF and OAF , was marked by a set of DEPs , of which COL5A1 , SERPINA5 , MXRA5 were enriched in the IAF , whereas LAMB2 , THBS1 , CTSD typified the OAF ( Figure 3—figure supplement 1D ) .",
"These findings agreed with the modular patterns ( Figure 3E–H ) .",
"Of the 1 , 880 proteins detected , 1 , 204 proteins were not found to vary with respect to the phenotypic factors .",
"The majority of these proteins were detected in few profiles ( Figure 3B ) and were not used in the comparisons .",
"We set a cutoff for a detection in >1/2 of the profiles to prioritise a set of 245 proteins , hereby referred to as the ‘constant set’ ( Figure 3C ) .",
"Both the variable and the constant sets contained high proportions of ECM proteins ( Figure 3D ) .",
"Amongst the proteins in the constant set that were detected in all 33 young profiles were known protein markers defining a young NP or disc , including COL2A1 , ACAN , and A2M ( Risbud et al . , 2015 ) .",
"Other key proteins in the constant set included CHAD , HAPLN1 , VCAN , HTRA1 , CRTAC1 , and CLU .",
"Collectively , these proteins showed the common characteristics shared by compartments of young discs , and they , alongside the variable set , form the architectural landscape of the young disc .",
"PCA was used to identify compartmental , directional , and level patterns for the aged discs ( Figure 4A ) .",
"Albeit less clear than for the young discs ( Figure 3A ) , the OAF could be distinguished from the inner disc regions on PC1 , explaining 46 . 7% of the total variance ( Figure 4A ) .",
"PC2 showed a more distinct separation of signatures from lumbar disc levels L5/S1 to the upper disc levels ( L4/L5 and L3/4 ) , accounting for 21 . 8% of the total variance ( Figure 4A ) .",
"As with the young discs , we performed a series of comparative analyses ( Figure 4C; Figure 4—figure supplement 1A–H; Supplementary file 3 ) .",
"Detection of DEPs between the OAF and the inner disc ( Figure 4B ) , showed that 100 proteins were expressed higher in the OAF , similar to young discs ( Figure 3C ) .",
"However , in the inner regions , only nine proteins were significantly expressed higher , in marked contrast to the situation in young discs .",
"Fifty-five of the 100 DEPs in the OAF region overlapped in the same region in the young discs , but only 3 of the 9 DEPs in the inner region were identified in the young disc; indicating changes in both regions but more dramatic in the inner region .",
"This suggests that ageing and associated changes may have initiated at the centre of the disc .",
"The typical NP markers ( KRT8/19 , CD109 , CHRD , CHRDL2 ) were not detectable as DEPs in the aged disc; but CHI3L2 , A2M and SERPING1 ( Figure 4B ) , which have known roles in tissue fibrosis and wound healing ( Lee et al . , 2011; Naveau et al . , 1994; Wang et al . , 2019a ) , were detected uniquely in the aged discs .",
"A comparative analysis of the protein profiles indicated that the aged OAF retained 55% of the proteins of a young OAF .",
"These changes are primarily reflected in the class of SLRPs ( BGN , DCN , FMOD , OGN , and PRELP ) , and glycoproteins such as CILP , CILP2 , COMP , FGA/B , and FGG .",
"From a cellular perspective , 45 proteins enriched in the aged OAF could be classified under ‘responses to stress’ ( FDR q = 1 . 86 × 10−7; contributed by CAT , PRDX6 , HSP90AB1 , EEF1A1 , TUBB4B , P4HB , PRDX1 , HSPA5 , CRYAB , HIST1H1C ) , suggesting OAF ECM and cells are responding to a changing environment such as mechanical loading and other stress factors .",
"To map the relative changes between inner and outer regions of the aged discs , we performed a systematic comparison between compartments ( Figure 4—figure supplement 1A–D ) .",
"The most significant observation was a weakening of the distinction between IAF and OAF that was seen in young discs , with only 17 DEPs expressed higher in the OAF of the aged disc ( Figure 4—figure supplement 1C ) .",
"More differences were seen when we included the NP in the comparison with OAF ( Figure 4—figure supplement 1D ) , indicating some differences remain between inner and outer regions of the aged discs .",
"While the protein profiles of the NP and IAF were similar , with no detectable DEPs ( Figure 4—figure supplement 1A & B ) , their compositions shared more resemblance with the OAF .",
"These progressive changes of the protein profiles and DEPs between inner and outer compartments suggest the protein composition of the inner disc compartments becomes more similar to the OAF with ageing , with the greatest changes in the inner regions .",
"This further supports a change initiating from the inner region of the discs with ageing .",
"We investigated protein composition in the young disc modules ( Y1-Y4 ) across the lateral and anteroposterior axes in the aged discs ( Figure 4C–F ) .",
"For module Y1 that consists of proteins defining the NP region , the distinctive concave pattern has flattened along both axes , but more so for the anteroposterior direction where the clear interface between IAF and OAF was lost ( Figure 4C; Figure 4—figure supplement 1I ) .",
"Similarly for modules Y2 and Y3 , which consist of proteins defining the AF region , the trends between inner and outer regions of the disc have changed such that the patterns become more convex , with a change that is a continuum from inner to outer regions ( Figure 4D , E; Figure 4—figure supplement 1J , K ) .",
"These changes in modules Y1-3 in the aged disc further illustrate the convergence of the inner and outer regions , with the NP/IAF becoming more OAF-like .",
"For Y4 , the patterns along the lateral and anteroposterior axes were completely disrupted ( Figure 4F; Figure 4—figure supplement 1L ) .",
"As Y4 contains proteins involved in vascularity and inflammatory processes ( Supplementary file 5 ) , these changes indicate disruption of cellular homeostasis in the NP .",
"The protein profiles of the aged disc levels , consistent with the PCA findings , showed similarity between L3/4 and L4/5 ( Figure 4—figure supplement 1E ) , but , in contrast to young discs , differences between L5/S1 and L4/5 ( Figure 4—figure supplement 1F ) , and more marked differences between L5/S1 and L3/4 ( Figure 4—figure supplement 1G , H ) .",
"Overall , the findings from PCA , protein profiles ( Figure 2B–D ) , and MRI ( Figure 1C ) agree .",
"As compared to young discs , the more divergent differences across the aged disc levels potentially reflect progressive transmission from the initiating disc to the adjacent discs with ageing .",
"To further investigate the aetiologies underlying IDD , cross-age comparisons are needed .",
"Next , we performed extensive pair-wise comparisons between the young and aged samples under a defined scheme ( Figure 5—figure supplement 1A; Supplementary file 4 ) .",
"First , we compared all 33 young samples with all 33 aged samples , which identified 169 DEPs with 104 expressed higher in the young and 65 expressed higher in the aged discs ( Figure 5A ) .",
"A simple gene ontology ( GO ) term analysis showed that the most important biological property for a young disc is structural integrity , which is lost in aged discs ( Figure 5B; Supplementary file 5 ) .",
"The protein classes most enriched in the young discs were related to cartilage synthesis , chondrocyte development , and ECM organisation ( Figure 5B ) .",
"The major changes in the aged discs relative to young ones , were proteins involved in cellular responses to an ageing environment , including inflammatory and cellular stress signals , progressive remodelling of disc compartments , and diminishing metabolic activities ( Figure 5C; Supplementary file 5 ) .",
"For young versus old discs , we compared DEPs of the whole disc ( Figure 5A ) with those from the inner regions ( Figure 5D ) and OAF ( Figure 5E ) only .",
"Seventy-five percent ( 78/104 ) of the downregulated DEPs ( Figure 5F ) were attributed to the inner regions , and only 17% ( 18/104 ) were attributed to the OAF ( Figure 5F ) .",
"Similarly , 65% ( 42/65 ) of the upregulated DEPs were solely contributed by inner disc regions , and only 18 . 4% ( 12/65 ) by the OAF .",
"Only 5 DEPs were higher in the OAF , while 49 were uniquely upregulated in the inner discs ( Figure 5G ) .",
"The key biological processes in each of the compartments are highlighted in Figure 5F and G . Expression of known NP markers was reduced in aged discs , especially proteins involved in the ECM and its remodelling , where many of the core matrisome proteins essential for the structural function of the NP were less abundant or absent .",
"While this is consistent with previous observations ( Feng et al . , 2006 ) , of interest was the presence of a set of protein changes that were also seen in the OAF , which was also rich in ECM and matrix remodelling proteins ( HTRA1 , SERPINA1 , SERPINA3 , SERPINC1 , SERPINF1 , and TIMP3 ) and proteins involved in fibrotic events ( FN1 , POSTN , APOA1 , APOB ) , suggesting these changes are occurring in the ageing IVDs ( Figure 5F , G ) .",
"Proteins associated with cellular stress are decreased in the aged inner disc , with functions ranging from molecular chaperones needed for protein folding ( HSPB1 , HSPA1B and HSPA9 ) to modulation of oxidative stress ( SOD1 ) ( Figure 5F ) .",
"SOD1 has been shown to become less abundant in the aged IVD ( Hou et al . , 2014 ) and in osteoarthritis ( Scott et al . , 2010 ) .",
"HSPB1 is cytoprotective and a deficiency is associated with inflammation reported in degenerative discs ( Wuertz et al . , 2012 ) .",
"We found an increased concentration of clusterin ( CLU ) ( Figure 5G ) , an extracellular chaperone that aids the solubilisation of misfolded protein complexes by binding directly and preventing protein aggregation ( Trougakos , 2013; Wyatt et al . , 2009 ) , and also has a role in suppressing fibrosis ( Peix et al . , 2018 ) .",
"Inhibitors of WNT ( DKK3 and FRZB ) , and antagonists of BMP/TGFβ ( CD109 , CHRDL2 , DCN FMOD , INHBA and THBS1 ) signalling were decreased or absent in the aged inner region ( Figure 5F , G ) , consistent with the reported upregulation of these pathways in IDD ( Hiyama et al . , 2010 ) and its closely related condition osteoarthritis ( Leijten et al . , 2013 ) .",
"Targets of hedgehog signalling ( HHIPL2 and SCUBE1 ) were also reduced ( Figure 5F ) , consistent with SHH’s key roles in IVD development and maintenance ( Rajesh and Dahia , 2018 ) .",
"TGFβ signalling is a well-known pathway associated with fibrotic outcomes .",
"WNT is known to induce chondrocyte hypertrophy ( Dong et al . , 2006 ) , that can be enhanced by a reduction in S100A1 ( Figure 5F ) , a known inhibitor of chondrocyte hypertrophy ( Saito et al . , 2007 ) .",
"To gain an overview of the disc compartment variations between young and aged discs , we followed the same strategy as in Figure 3—figure supplement 2 to aggregate three categories of DEPs in all 23 comparisons ( Figure 5—figure supplement 1A ) and created a heatmap from the resulting 719 DEPs .",
"This allowed us to identify six major protein modules ( Figure 5H ) .",
"A striking feature is module 6 ( M6 ) , which is enriched for proteins involved in the complement pathway ( GSEA Hallmark FDR q = 4 . 9 × 10−14 ) and angiogenesis ( q = 2 . 3 × 10−3 ) .",
"This module contains proteins that are all highly expressed in the inner regions of the aged disc , suggesting the presence of blood .",
"M6 also contains the macrophage marker CD14 , which supports this notion .",
"We visualised the relationship between the young ( Y1-4 ) and young/aged ( M1-6 ) modules using an alluvial chart ( Figure 5I ) .",
"Y1 corresponds primarily to M1b that is enriched with fibrosis , angiogenesis , apoptosis , and EMT ( epithelial to mesenchymal transition ) proteins .",
"Y2 seems to have been deconstructed into three M modules ( M2b > M3 > M4 ) .",
"M2b and M3 contain proteins linked to heterogeneous functions , while proteins in M4 are associated with myogenesis and cellular metabolism , but also linked to fibrosis and angiogenesis .",
"Y3 primarily links to M2a with a strong link to myogenesis , and mildly connects with M3 and M4 .",
"Y4 has the strongest connection with M6a , which is linked to coagulation .",
"Both the variable and the constant sets of the young disc were also changed in ageing .",
"In the constant set , there is a higher tendency for a decrease in ECM-related proteins , and an increase in blood and immune related proteins with ageing , that may reflect an erosion of the foundational proteome , and infiltration of immune cells ( Figure 5—figure supplement 1L ) .",
"In all , the IVD proteome showed that with ageing , activities of the SHH pathways were decreased , while those of the WNT and BMP/TGFβ pathways , EMT , angiogenesis , fibrosis , cellular stresses , and chondrocyte hypertrophy-like events were increased .",
"The proteome reflects both current and past transcriptional activities .",
"To investigate upstream cellular and regulatory activities , we obtained transcriptome profiles from two IVD compartments ( NP and AF ) and two sample states ( young , scoliotic but non-degenerated , YND; aged individuals with clinically diagnosed IDD , AGD ) ( Table 1 ) .",
"The transcriptome profiles of YND and AGD are similar to the young and aged disc proteome samples , respectively .",
"After normalisation ( Figure 6—figure supplement 1A ) and hierarchical clustering , we found patterns reflecting relationships among IVD compartments and ages/states ( Figure 6—figure supplement 1B–D ) .",
"PCA of the transcriptome profiles showed that PC1 captured age/state variations ( Figure 6A ) and PC2 captured the compartment ( AF or NP ) differences , with a high degree of similarity to the proteomic PCA that explained 65 . 0% of all data variance ( Figure 2A ) .",
"We compared the transcriptome profiles of different compartments and age/state-groups ( Figure 6—figure supplement 1E–H ) .",
"We detected 88 DEGs ( Materials and methods ) between young AF and young NP samples ( Figure 6—figure supplement 1E ) , in which 39 were more abundant in young NP ( including known NP markers CD24 , KRT19 ) and 49 were more abundant in young AF , including the AF markers , COL1A1 , and THBS1 .",
"In the AGD samples , 11 genes differed between AF and NP ( Figure 6—figure supplement 1F ) , comparable to the proteome profiles ( Figure 4C ) .",
"Between the YND and AGD AF , there were 45 DEGs , with COL1A1 and MMP1 more abundant in YND and COL10A1 , WNT signalling ( WIF1 , WNT16 ) , inflammatory ( TNFAIP6 , CXCL14 , IL11 ) , and fibrosis-associated ( FN1 , CXCL14 ) genes more abundant in AGD ( Figure 6—figure supplement 1G ) .",
"The greatest difference was between YND and AGD NP , with 216 DEGs ( Figure 6—figure supplement 1H ) , with a marked loss of NP markers ( KRT19 , CD24 ) , and gain of AF ( THBS1 , DCN ) , proteolytic ( ADAMTS5 ) , and EMT ( COL1A1 , COL3A1 , PDPN , NT5E , LTBP1 ) markers with age .",
"Again , consistent with the proteomic findings , the most marked changes are in the NP , with the transcriptome profiles becoming AF-like .",
"We partitioned the DEGs between the YND and AGD into DEGs for individual compartments ( Figure 6B ) .",
"The transcriptomic ( Figure 6B ) Venn diagram was very similar to the proteomic one ( Figure 5F–G ) .",
"For example , WNT/TGFβ antagonists and ECM genes were all downregulated with ageing/degeneration , while genes associated with stress and ECM remodelling were more common .",
"When we directly compared the transcriptomic DEGs and proteomic DEPs across age/states and compartments ( Figure 6C–F ) , we observed strong concordance between the two types of datasets for a series of markers .",
"In the young discs , concordant markers included KRT19 and KRT8 , CHRDL2 , FRZB , and DKK3 in the NP , and COL1A1 , SERPINF1 , COL14A1 , and THBS4 in the AF ( Figure 6C ) .",
"In the AGD discs , concordant markers included CHI3L2 , A2M and ANGPTL4 in the NP and MYH9 , HSP90AB1 , HBA1 , and ACTA2 in the AF ( Figure 6D ) .",
"A high degree of concordance was also observed when we compared across age/states for the AF ( Figure 6E ) and NP ( Figure 6F ) .",
"Despite the transcriptomic samples having diagnoses ( scoliosis for YND and IDD for AGD ) , whereas the proteome samples were cadaver samples with no reported diagnosis of IDD , the changes detected in the transcriptome profiles substantially support the proteomic findings .",
"A surprising indication from the transcriptome was the increased levels of COL10A1 ( Lu et al . , 2014 ) , BMP2 ( Grimsrud et al . , 2001 ) , IBSP , defensin beta-1 ( DEFB1 ) , ADAMTS5 , pro-inflammatory ( TNFAIP6 , CXCL ) and proliferation ( CCND1 , IGFBP ) genes in the AGD NP ( Figure 6—figure supplement 1E–H ) , reaffirming the involvement of hypertrophic-like events ( Melas et al . , 2014 ) in the aged and degenerated NP .",
"The genome-wide transcriptomic data included over 20 times more genes per profile than the proteomic data , providing additional biological information about the disc , particularly low abundance proteins , such as transcription factors and surface markers .",
"For example , additional WNT antagonists were WIF1 ( Wnt inhibitory factor ) and GREM1 ( Figure 6B Leijten et al . , 2013 ) .",
"Comparing the YND NP against YND AF or AGD NP ( Figure 6—figure supplement 1E , H ) , we identified higher expression of three transcription factors , T ( brachyury ) , HOPX ( homeodomain-only protein homeobox ) , and ZNF385B in the YND NP .",
"Brachyury is a well-known marker for the NP ( Risbud et al . , 2015 ) , and HOPX is differentially expressed in mouse NP as compared to AF ( Veras et al . , 2020 ) , and expressed in mouse notochordal NP cells ( Lam , 2013 ) .",
"Overall , transcriptomic data confirmed the proteomic findings and revealed additional markers .",
"The proteomic data up to this point is a static form of measurement ( static proteome ) and represents the accumulation and turnover of all proteins up to the time of harvest .",
"The transcriptome indicates genes that are actively transcribed , but does not necessarily correlate to translation or protein turnover .",
"Thus , we studied changes in the IVD proteome ( dynamic proteome ) that would reflect newly synthesised proteins and proteins cleaved by proteases ( degradome ) , and how they relate to the static proteomic and transcriptomic findings reported above .",
"We performed ex vivo labelling of newly synthesised proteins using the SILAC protocol ( Ong et al . , 2002; Figure 7A; Materials and methods ) on AF and NP samples from four YND individuals and one AGD individual ( Table 1 ) .",
"In the SILAC profiles , light isotope-containing signals correspond to the pre-existing unlabelled proteome , and heavy isotope-containing signals to newly synthesised proteins ( Figure 7B ) .",
"The ECM compositions in the light isotope-containing profiles ( Figure 7B , middle panel ) are similar to the static proteome samples of the corresponding age groups described above ( Figure 1F ) .",
"Although for NP_YND152 , the numbers of identified proteins in the heavy profiles are considerably less than NP_YND151 due to a technical issue during sample preparation , it is overall still more similar to NP_YND152 than to other samples ( Figure 7—figure supplement 1B ) , indicating that its biological information is still representative of a young NP and the respective AF samples are similar .",
"In contrast , the heavy isotope-containing profiles contained fewer proteins in the AGD than in the YND samples ( Figure 7B , left panel ) and showed variable heavy to light ratio profiles ( Figure 7B , right panel ) .",
"To facilitate comparisons , we averaged the abundance of the proteins detected in the NP or AF for which we had more than one sample , then ranked the abundance of the heavy isotope-containing ( Figure 7C ) , light isotope-containing ( Figure 7D ) proteins .",
"The number of proteins newly synthesised in the AGD samples was about half that in the YND samples ( Figure 7C ) .",
"This is unlikely to be a technical artefact as the total number of light isotope-containing proteins detected in the AGD samples is comparable to the YND , in both AF and NP ( Figure 7D ) , and the difference is again well illustrated in the heavy to light ratios ( Figure 7—figure supplement 1A ) .",
"Reduced synthesis of non-matrisome proteins was found for the AGD samples ( GAPDH ) as a reference point ( dotted red lines ) ( Figure 7C and D; Figure 7C and E , grey portions ) .",
"Of the 68 high abundant non-matrisome proteins in the YND NP compartment that were not present in the AGD NP , 28 are ribosomal proteins ( Figure 7—figure supplement 1C ) , suggesting reduced translational activities .",
"This agrees with our earlier findings of cellularity , as represented by histones , in the static proteome ( Figure 1J , K ) .",
"Changes in protein synthesis in response to the cell microenvironment affects the architecture of the disc proteome .",
"To understand how the cells may contribute and respond to the accumulated matrisome in the young and aged disc , we compared the newly synthesised matrisome proteins of AGD and YND samples rearranged in order of abundance ( Figure 7E ) .",
"More matrisome proteins were synthesised in YND samples across all classes .",
"In YND AF , collagens were synthesised in higher proportions than in AGD AF with the exception of fibril-associated COL12A1 ( Figure 7E , top panel ) .",
"Similarly , higher proportions in YND AF were observed for proteoglycans ( except FMOD ) , glycoproteins ( except TNC , FBN1 , FGG and FGA ) , ECM affiliated proteins ( except C1QB ) , ECM regulators ( except SERPINF2 , SERPIND1 , A2M , ITIH2 , PLG ) , and secreted factors ( except ANGPTL2 ) .",
"Notably , regulators that were exclusively synthesised in young AF are involved in collagen synthesis ( P4HA1/2 , LOXL2 , LOX , PLOD1/2 ) and matrix turnover ( MMP3 ) , with enrichment of protease HTRA1 and protease inhibitors TIMP3 and ITIH in YND AF compared to AGD AF .",
"In AGD NP , overall collagen synthesis was less than in YND NP ( Figure 7E , lower panel ) ; however , there was more synthesis of COL6A1/2/3 and COL12A1 .",
"Furthermore , AGD NP synthesised more LUM , FMOD , DCN , PRG4 , and PRELP proteoglycans than YND NP .",
"Notably , there was less synthesis of ECM-affiliated proteins ( except C1QC and SEMA3A ) and regulators – particularly those involved in collagen synthesis ( P4HA1/2 , LOXL2 , LOX ) – but an increase in protease inhibitors .",
"A number of newly synthesised proteins in AGD NP were similarly represented in the transcriptome data , including POSTN , ITIH2 , SERPINC1 , IGFBP3 , and PLG .",
"Some genes were simultaneously underrepresented in the AGD NP transcriptome and newly synthesised proteins , including hypertrophy inhibitor GREM1 ( Leijten et al . , 2013 ) .",
"The degradome reflects protein turnover by identifying cleaved proteins in a sample ( López-Otín and Overall , 2002 ) .",
"When combined with relative quantification of proteins through the use of isotopic and isobaric tagging and enrichment for cleaved neo amine ( N ) -termini of proteins before labelled samples are quantified by mass-spectrometry , degradomics is a powerful approach to identify the actual status of protein cleavage in vivo .",
"We employed the well-validated and sensitive terminal amine isotopic labelling of substrates ( TAILS ) method ( Kleifeld et al . , 2010; Rauniyar and Yates , 2014 ) to analyse and compare six discs from six individuals ( two young and non-degenerated , YND; and four aged and/or degenerated , AGD ) ( Table 1; Figure 7F; Kleifeld et al . , 2010; Rauniyar and Yates , 2014 ) using the 6-plex tandem mass tag ( TMT ) -TAILS ( labelling six independent samples and analysed together on the mass spectrometer ) ( Figure 7F ) .",
"Although shotgun proteomics is intended to identify the proteome components , N-terminome data is designed to identify the exact cleavage site in proteins that also evidence stable cleavage products in vivo .",
"Here , TAILS identified 123 and 84 cleaved proteins in the AF and NP disc samples , respectively .",
"Performing hierarchical clustering on the data we found that the two YND samples ( 136 and 141; Table 1 ) tend to cluster together in both AF and NP ( Figure 7G , H; Figure 7—figure supplement 2A , B ) .",
"Interestingly , the trauma sample AGD143 ( 53 year male ) , who has no known IDD diagnosis , tend to cluster with other clinically diagnosed AGD samples , in both AF and NP .",
"This might be because AGD143 has unreported degeneration or ageing is a dominant factor in degradome signals .",
"We identified two protein/peptide modules in the AF ( Figure 7G ) , corresponding to more degradation/cleaving in YND AF ( magenta ) and AGD AF ( blue ) , respectively .",
"There are only 13 unique proteins for proteins/peptides more degraded in the YND AF , the most common of which is COL1A1/2 , followed by COL2A1 .",
"In comparison , the module corresponding to more degradation in AGD AF recorded 24 unique proteins , 7 ( CILP , CILP2 , COL1A1 , COMP , HBA1 , HBB , PRELP ) of which are in strong overlap ( χ2 p=2 . 0 × 10−71 ) with the 99 proteins higher in outer AF in the spatial proteome ( Figure 3I ) .",
"This indicates that key proteins defining a young outer AF is experiencing faster degradation in aged and degenerated samples .",
"Similarly , we identified two modules in the NP ( Figure 7H ) , whereby one ( magenta ) corresponds to more degradation in the YND , and the other ( blue ) corresponds to more degradation in the AGD .",
"Only 10 unique proteins were recorded in the magenta module ( for YND ) , with COL2A1 being the most dominant ( 928 peptides ) ; whereas 32 were recorded for the blue module ( for AGD ) .",
"Overall , there are more unique proteins involved in faster degradation in AGD AF and NP .",
"We tested for a correlation between MRI signal intensity and proteome composition .",
"In conventional 3T MRI of young discs , the NP is brightest reflecting its high hydration state while the AF is darker , thus less hydrated ( Figure 1B; Figure 8—figure supplement 1B ) .",
"Since aged discs present with more MRI phenotypes , we used higher resolution MRI ( 7T ) on them ( Figure 1C; Figure 8A ) , which showed less contrast between NP and AF than in the young discs .",
"To enhance robustness , we obtained three transverse stacks per disc level for the aged discs ( Figure 8B; Figure 8—figure supplement 1D ) , and averaged the pixel intensities for the different compartments showing that overall , the inner regions were still brighter than the outer ( Figure 8C ) .",
"Next , we performed a level-compartment bi-clustering on the pixel intensities of the aged disc MRIs , which was bound by disc level and compartment ( Figure 8D ) .",
"The findings resembled the proteomic PCA ( Figure 2 ) and clustering ( Figure 3—figure supplement 1V–W ) patterns .",
"We performed a pixel intensity averaging of the disc compartments from the 3T images ( Figure 8—figure supplement 1D ) , and a level-compartment bi-clustering on the pixel intensities ( Figure 8—figure supplement 1C ) .",
"While the clustering can clearly partition the inner from the outer disc compartments , the information value from each of the compartments is less due to the lower resolution of the MRI .",
"In all , these results indicate a link between regional MRI landscapes and proteome profiles , prompting us to investigate their potential connections .",
"The MRI and the static proteome were done on the same specimens in both individuals , so we could perform proteome-wide associations with the MRI intensities .",
"We detected 85 significantly correlated ECM proteins , hereby referred to as the hydration matrisome ( Figure 8E ) .",
"We found no collagen to be positively correlated with brighter MRI , which fits current understanding as collagens contribute to fibrosis and dehydration .",
"Other classes of matrisome proteins were either positively or negatively correlated , with differential components for each class ( Figure 8E ) .",
"Positively correlated proteoglycans included EPYC , PRG4 and VCAN , consistent with their normal expression in a young disc and hydration properties .",
"Negatively correlated proteins included OAF ( TNC , SLRPs ) and fibrotic ( POSTN ) markers ( Figure 8E ) .",
"Given this MRI-proteome link and the greater dynamic ranges of MRI in the aged discs enabled by the higher resolution 7T MRIs ( Figure 8D ) , we hypothesised that the hydration matrisome might be used to provide information about MRI intensities and thus disc hydration .",
"To test this , we trained a LASSO regression model ( Tibshirani , 1996 ) of the aged MRIs using the hydration matrisome ( 85 proteins ) , and applied the model to predict the intensity of the MRIs of the young discs , based on the young proteome of the same 85 proteins .",
"Remarkably , we obtained a PCC of 0 . 689 ( p=8 . 9 × 10−6; Spearman = 0 . 776 ) between the actual and predicted MRI ( Figure 8—figure supplement 1E ) .",
"The predicted MRI intensities of the young disc exhibited a smooth monotonic decrease from the NP towards IAF , then dropped suddenly towards the OAF ( Figure 8F , right panel ) , with an ROC AUC ( receiver operating characteristics , area under the curve ) of 0 . 996 between IAF and OAF ( Figure 8—figure supplement 1F ) .",
"In comparison , actual MRIs exhibited a linear decrease from NP to OAF ( Figure 8F , left panel ) .",
"On reviewing these two patterns , we argue that the predicted intensities may be a more faithful representation of the young discs’ water contents than the actual MRI , as it reflects the gross images ( Figure 1—figure supplement 1A ) , PCA ( Figure 3A ) and Y1 modular trend in the young discs ( Figure 3E ) .",
"This exercise not only revealed the inherent connections between regional MRI and regional proteome , but also identified a set of ECM components that is predictive of MRI relating to disc hydration , which may be valuable for future clinical applications ."
],
[
"Here , we present DIPPER – a human IVD proteomic resource comprising point-reference genome-wide profiles .",
"The discovery dataset was established from intact lumbar discs of a young cadaver with no history of skeletal abnormalities ( e . g . scoliosis ) , and an aged cadaver with reduced IVD MRI intensity and annular tears .",
"Although these two individuals may not be representative of their respective age-groups , this is the first known attempt to achieve high spatial resolution profiles in the discs , adding a critical and much needed dimension to the current available IVD proteomic datasets .",
"We showed that our spatiotemporal proteomes integrate well with the dynamic proteome and transcriptome of clinical samples , demonstrating their application values with other datasets .",
"In creating the point-references , we use a well-established protein extraction protocol ( Önnerfjord et al . , 2012 ) , and chromatographic fractionation of the peptides prior to mass spectrometry , we produced a dataset of the human IVD comprising 3 , 100 proteins , encompassing ~400 matrisome and 2 , 700 non-matrisome proteins , with 1 , 769 proteins detected in three or more profiles , considerably higher than recent studies ( Maseda et al . , 2016; Rajasekaran et al . , 2020; Ranjani et al . , 2016; Sarath Babu et al . , 2016 ) .",
"The high quality of our data enabled the application of unbiased approaches including PCA and ANOVA to reveal the relative importance of the phenotypic factors .",
"Particularly , age was found to be the dominant factor influencing proteome profiles .",
"Comparisons between different compartments of the young disc produced a reference landscape containing known ( KRT8/19 for the NP; COL1A1 , CILP and COMP for the AF ) and novel ( FRZB , CHRD , CHRDL2 for the NP; TNMD , SLRPs , and SOD1 for the AF ) markers .",
"The young healthy discs were enriched for matrisome components consistent with a healthy functional young IVD .",
"Despite morphological differences between NP and IAF , the inner disc compartments ( NP , NP/IAF , and IAF ) display high similarities , in contrast to the large differences between IAF and OAF , which was consistent for discs from all lumbar disc levels .",
"This morphological-molecular discrepancy might be accounted for by subtle differences in the ECM organisation , such as differences in GAG moieties on proteoglycans , or levels of glycosylation or other modifications of ECM that diversify function ( Silagi et al . , 2018 ) .",
"Nonetheless , we partitioned the detected proteins into a variable set that captures the diversity , and a constant set that lays the common foundation of all young compartments , which work in synergy to achieve disc function .",
"Clustering analysis of the 671 DEPs of the variable set identified four key modules ( Y1-Y4 ) .",
"Visually , Y1 and Y2 mapped across the lateral and anteroposterior axes with opposing trends .",
"Molecularly , module Y1 ( NP ) contained proteins promoting regulation of matrix remodelling , such as matrix degradation inhibitor MATN3 ( Jayasuriya et al . , 2012 ) and MMP inhibitor TIMP1 .",
"Inhibitors of WNT and BMP signalling were also present .",
"Module Y2 ( OAF ) included COL1A1 , THBS1/2/3 , CILP1/CILP2 , and TNMD , consistent with the OAF’s tendon-like features ( Nakamichi et al . , 2018 ) .",
"It also included a set of SLRPs that might play roles in regulation of collagen assembly ( Robinson et al . , 2017; Taye et al . , 2020 ) , fibril alignment ( Robinson et al . , 2017 ) , maturation and crosslinking ( Kalamajski et al . , 2016 ) , while others are known to inhibit or promote TGFβ signalling ( Markmann et al . , 2000 ) .",
"Notably , the composition of the IAF appeared to be a transition zone between NP and OAF rather than an independent compartment , as few proteins can distinguish it from adjacent compartments .",
"Classes of proteins in both Y3 ( smooth muscle feature ) and Y4 ( immune and blood ) resemble Y2 , which reflects the contractile property of the AF ( Nakai et al . , 2016 ) and the capillaries infiltrating or present at the superficial outer surface of the IVD .",
"In the aged disc , the DEPs between the inner and outer regions of the discs suggests extensive changes in the inner compartment ( s ) .",
"Mapping the aged data onto modules Y1-Y4 allowed a visualisation of the changes .",
"The flattening of the Y1 and Y2 modules along both the lateral and anteroposterior axes indicated a convergence of the inner and outer disc .",
"This is supported by the observed rapid decline of NP proteins and increase of AF proteins in the inner region .",
"Fewer changes were seen in the aged OAF , which concurs with the notion that degenerative changes originate from the NP and radiate outwards; however , infringement of IAF into the NP cannot be excluded .",
"The most marked change was seen in module Y4 ( blood ) , where the pattern was inverted , characterised by high expression in the NP but low in OAF .",
"While contamination cannot be excluded and there are reports that capillaries do not infiltrate the NP even in degeneration ( Nerlich et al . , 2007 ) , our finding is consistent with other proteomic studies showing enrichment of blood proteins in pathological NP ( Maseda et al . , 2016 ) .",
"The route of infiltration can be from the fissured AF or cartilage endplates .",
"Calcified endplates are more susceptible to microfractures , which can lead to blood infiltration into the NP ( Sun et al . , 2020 ) .",
"Of interest is the involvement of an immune response within the inner disc .",
"This corroborates reports of inflammatory processes in ageing and degenerative discs , with the upregulation of pro-inflammatory cytokines and presence of inflammatory cells ( Molinos et al . , 2015; Wuertz et al . , 2012 ) .",
"The SILAC and degradome studies provided important insights into age-related differences in the biosynthetic and turnover activity in the IVD .",
"The SILAC data indicated that protein synthesis is significantly impaired in aged degenerated discs .",
"These findings correlate with reports of reduced cellularity in ageing ( Rodriguez et al . , 2011 ) , which we have also ascertained by leveraging the relationship of histones and housekeeping genes with cell numbers .",
"From the TAILS degradome analysis , we observed more cleaved protein fragments in aged compartments , particularly for structural proteins important for tissue integrity such as COMP and those involved in cell-matrix interactions such as FN1 , which was coupled with the enrichment of the proteolytic process GO terms in the aged static proteome .",
"Collectively , this reveals a systematic modification and replacement of the primary proteomic architecture of the young IVD with age that is associated with diminished or failure in functional properties in ageing or degeneration .",
"Despite known transcriptome-proteome discordance ( Fortelny et al . , 2017 ) , our identification of their concordance allows insights into active changes in the young and aged discs .",
"For example , inhibitors of the WNT pathway and antagonists of BMP/TGFβ signalling ( Leijten et al . , 2013 ) were down-regulated in the aged discs in both the proteome and transcriptome .",
"Interestingly , the activation of these pathways is known to promote chondrocyte hypertrophy ( Dong et al . , 2006 ) , and hypertrophy has been noted in IDD ( Rutges et al . , 2010 ) .",
"This suggests a model whereby the regulatory environment suppressing cellular hypertrophy changes with ageing or degeneration , resulting in conditions such as cellular senescence and tissue mineralisation that are part of the pathological process .",
"In support , S100A1 , a known inhibitor of chondrocyte hypertrophy ( Saito et al . , 2007 ) is downregulated , while chondrocyte hypertrophy markers COL10A1 and IBSP ( Komori , 2010 ) are upregulated in the aged disc .",
"Similar changes have been observed in ageing mouse NP ( Veras et al . , 2020 ) as well as in osteoarthritis ( Zhu et al . , 2009 ) where chondrocyte hypertrophy is thought to be involved in its aetiology ( Ji et al . , 2019; van der Kraan and van den Berg , 2012 ) .",
"Given that WNT inhibitors are already in clinical trials for osteoarthritis ( Wang et al . , 2019b ) , this may point to a prospective therapeutic strategy for IDD .",
"A key finding of our study is the direct demonstration , within a single individual , of association between the hydration status of the disc as revealed by MRI , and the matrisome composition of the disc proteome .",
"The remarkable correlation between predicted hydration states inferred from the spatial proteomic data and the high-definition phenotyping of the aged disc afforded by 7T MRI has enormous potential for understanding the molecular processes underlying IDD .",
"In conclusion , we have generated point-reference datasets of the young and aged disc proteome , at a significantly higher spatial resolution than previous works .",
"By means of a methodological framework , we revealed compartmentalised information on the ECM composition and cellular activities , and their changes with ageing .",
"Integration of this point-reference with additional age- and protein-specific information of synthesis/degradation helps gain insights into the underlying molecular pathology of degeneration ( Figure 8G and H ) .",
"The richness of information in DIPPER makes it a valuable resource for cross referencing with human , animal and in vitro studies to evaluate clinical relevance and guide the development of therapeutics for human IDD ."
],
[
"Two human lumbar spines were obtained through approved regulations and governing bodies , with one young ( 16M ) provided by L . H . ( McGill University ) and one aged ( 59M ) from Articular Engineering , LLC ( IL , USA ) .",
"The young lumbar spine was received frozen as an intact whole lumbar spine .",
"The aged lumbar spine was received frozen , dissected into bone-disc-bone segments .",
"The cadaveric samples were stored at −80⁰C until use .",
"Clinical specimens were obtained with approval by the Institutional Review Board ( references UW 13–576 and EC 1516–00 11/01/2001 ) and with informed consent in accordance with the Helsinki Declaration of 1975 ( revision 1983 ) from another 15 patients undergoing surgery for IDD , trauma or adolescent idiopathic scoliosis at Queen Mary Hospital ( Hong Kong ) , and Duchess of Kent Children’s Hospital ( Hong Kong ) .",
"Information of both the cadaveric and clinical samples are summarised in Table 1 .",
"The discs were thawed overnight at 4°C , and then pre-equalised for scanning at room temperature .",
"For the young IVD , these were imaged together as the lumbar spine was kept intact .",
"T2-weighted and T1-weighted sagittal and axial MRI , T1-rho MRI and Ultrashort-time-to-echo MRI images were obtained using a 3T Philips Achieva 3 . 0 system at the Department of Diagnostic Radiology , The University of Hong Kong .",
"For the aged discs , the IVD were imaged separately as bone-disc-bone segments , at the Department of Electrical and Electronic Engineering , The University of Hong Kong .",
"The MRS and CEST imaging were performed .",
"The FOV for the CEST imaging was adjusted to 76 . 8 × 76 . 8 mm2 to accommodate the size of human lumbar discs ( matrix size = 64 × 64 , slice thickness = 2 mm ) .",
"All MRI experiments were performed at room temperature using a 7 T pre-clinical scanner ( 70/16 Pharmascan , Bruker BioSpin GmbH , Germany ) equipped with a 370 mT/m gradient system along each axis .",
"Single-channel volume RF coils with different diameters were used for the samples based on size ( 60 mm for GAG phantoms and human cadaveric discs ) .",
"The MRI images in the transverse view were then assessed for intensity of the image ( brighter signifying more water content ) .",
"Three transverse MRI images per IVD were overlaid with a grid representing the areas that were cut for mass-spectrometry measurements as outlined previously .",
"For each region , the ‘intensity’ was represented by the average of the pixel intensities , which were graphically visualised and used for correlative studies .",
"The endplates were carefully cut off with a scalpel , exposing the surface of the IVD , which were then cut into small segments spanning seven segments in the central left-right lateral axis , and five segments in the central anteroposterior axis ( Figure 1C ) .",
"In all , this corresponds to a total of 11 locations per IVD .",
"Among them , four are from the OAF , two from the IAF ( but only in the lateral axis ) , one from the central NP , and four from a transition zone between IAF and the NP ( designated the ‘NP/IAF’ ) .",
"Samples were stored frozen at −80⁰C until use .",
"NP and AF disc tissues from spine surgeries ( Table 1 ) were cultured in custom-made Arg- and Lys-free α-MEM ( AthenaES ) as per formulation of Gibco α-MEM ( Cat #11900–024 ) , supplemented with 10% dialysed FBS ( 10 , 000 MWCO , Biowest , Cat# S181D ) , penicillin/streptomycin , 2 . 2 g/L sodium bicarbonate ( Sigma ) , 30 mg/L L-methionine ( Sigma ) , 21 mg/L ‘heavy’ isotope-labelled 13C6L-arginine ( Arg6 , Cambridge Isotopes , Cat # CLM-2265-H ) , 146 mg/L ‘heavy’ isotope-labelled 4 , 4 , 5 , 5-D4 L-Lysine ( Lys4 , Cambridge Isotopes , Cat # DLM-2640 ) .",
"Tissue explants were cultured for 7 days in hypoxia ( 1% O2 and 5% CO2 in air ) at 37⁰C before being washed with PBS and frozen until use .",
"The frozen samples were pulverised using a freezer mill ( Spex ) under liquid nitrogen .",
"Samples were extracted using 15 volumes ( w/v ) of extraction buffer ( 4M guanidine hydrochloride ( GuHCl ) , 50 mM sodium acetate , 100 mM 6-aminocaproic acid , and HALT protease inhibitor cocktail ( Thermo Fischer Scientific ) , pH 5 . 8 ) .",
"Samples were mechanically dissociated with 10 freeze-thaw cycles and sonicated in a cold water bath , before extraction with gentle agitation at 4°C for 48 hr .",
"Samples were centrifuged at 15 , 000 g for 30 min at 4°C and the supernatant was ethanol precipitated at a ratio of 1:9 for 16 hr at −20°C .",
"The ethanol step was repeated and samples were centrifuged at 5000 g for 45 min at 4°C , and the protein pellets were air dried for 30 min .",
"Protein pellets were re-suspended in fresh 4M urea in 50 mM ammonium bicarbonate , pH 8 , using water bath sonication to aid in the re-solubilisation of the samples .",
"Samples underwent reduction with TCEP ( 5 mM final concentration ) at 60°C for 1 hr , and alkylation with iodoacetamide ( 500 mM final concentration ) for 20 min at RT .",
"Protein concentration was measured using the BCA assay ( Biorad ) according to manufacturer’s instructions .",
"A total of 200 μg of protein was then buffer exchanged with 50 mM ammonium bicarbonate with centricon filters ( Millipore , 30 kDa cutoff ) according to manufacturer’s instructions .",
"Samples were digested with mass spec grade Trypsin/LysC ( Promega ) as per manufacturer’s instructions .",
"For SILAC-labelled samples , formic acid was added to a final concentration of 1% , and centrifuged and the supernatant then desalted prior to LC-MS/MS measurements .",
"For the cadaveric samples , the digested peptides were then acidified with TFA ( 0 . 1% final concentration ) and quantified using the peptide quantitative colorimetric peptide assay kit ( Pierce , catalogue 23275 ) before undergoing fractionation using the High pH reversed phase peptide fractionation kit ( Pierce , catalogue number 84868 ) into four fractions .",
"Desalted peptides were dried , re-suspended in 0 . 1% formic acid prior to LC-MS/MS measurements .",
"Samples were loaded onto the Dionex UltiMate 3000 RSLC nano Liquid Chromatography coupled to the Orbitrap Fusion Lumos Tribid Mass Spectrometer .",
"Peptides were separated on a commercial Acclaim C18 column ( 75 μm internal diameter ×50 cm length , 1 . 9 μm particle size; Thermo ) .",
"Separation was attained using a linear gradient of increasing buffer B ( 80% ACN and 0 . 1% formic acid ) and declining buffer A ( 0 . 1% formic acid ) at 300 nL/min .",
"Buffer B was increased to 30% B in 210 min and ramped to 40% B in 10 min followed by a quick ramp to 95% B , where it was held for 5 min before a quick ramp back to 5% B , where it was held and the column was re-equilibrated .",
"Mass spectrometer was operated in positive polarity mode with capillary temperature of 300°C .",
"Full survey scan resolution was set to 120 , 000 with an automatic gain control ( AGC ) target value of 2 × 106 , maximum ion injection time of 30 ms , and for a scan range of 400–1500 m/z .",
"Data acquisition was in DDA mode to automatically isolate and fragment topN multiply charged precursors according to their intensities .",
"Spectra were obtained at 30 , 000 MS2 resolution with AGC target of 1 × 105 and maximum ion injection time of 100 ms , 1 . 6 m/z isolation width , and normalised collisional energy of 31 .",
"Preceding precursor ions targeted for HCD were dynamically excluded of 50 s .",
"Raw data were analysed using MaxQuant ( v . 1 . 6 . 3 . 3 , Germany ) .",
"Briefly , raw files were searched using Andromeda search engine against human UniProt protein database ( 20 , 395 entries , Oct 2018 ) , supplemented with sequences of contaminant proteins .",
"Andromeda search parameters for protein identification were set to a tolerance of 6 ppm for the parental peptide , and 20 ppm for fragmentation spectra and trypsin specificity allowing up to two miscleaved sites .",
"Oxidation of methionine , carboxyamidomethylation of cysteines was specified as a fixed modification .",
"Minimal required peptide length was specified at seven amino acids .",
"Peptides and proteins detected by at least two LFQ ion counts for each peptide in one of the samples were accepted , with a false discovery rate ( FDR ) of 1% .",
"Proteins were quantified by normalised summed peptide intensities computed in MaxQuant with the LFQ option enabled .",
"A total of 66 profiles were obtained: 11 locations × 3 disc levels × 2 individuals; with a median of 665 proteins ( minimum 419 , maximum 1920 ) per profile .",
"The high-resolution , high mass accuracy mass spectrometry ( MS ) data obtained were processed using Proteome Discoverer ( Ver 2 . 1 ) , wherein data were searched using Sequest algorithm against Human UniProt database ( 29 , 900 entries , May 2016 ) , supplemented with sequences of contaminant proteins , using the following search parameters settings: oxidised methionine ( M ) , acetylation ( Protein N-term ) , heavy Arginine ( R6 ) and Lysine ( K4 ) were selected as dynamic modifications , carboxyamidomethylation of cysteines was specified as a fixed modification , minimum peptide length of 7 amino acids was enabled , tolerance of 10 ppm for the parental peptide , and 20 ppm for fragmentation spectra , and trypsin specificity allowing up to two miscleaved sites .",
"Confident proteins were identified using a target-decoy approach with a reversed database , strict FDR 1% at peptide and PSM level .",
"Newly synthesised proteins were heavy labelled with Arg6- and Lys4 , and the data was expressed as the normalised protein abundance obtained from heavy ( labelled ) /light ( un-labelled ) ratio .",
"Degradome analyses was performed on NP and AF from three non-degenerated and three degenerated individuals ( Table 1 ) .",
"Frozen tissues were pulverised as described above and prepared for TAILS as previously reported ( Kleifeld et al . , 2010 ) .",
"After extraction with SDS buffer ( 1% SDS , 100 mM dithiothreitol , 1X protease inhibitor in deionised water ) and sonication ( three cycles , 15 s/cycle ) , the supernatant ( soluble fraction ) underwent reduction at 37°C and alkylation with a final concentration of 15 mM iodoacetamide for 30 min at RT .",
"Samples were precipitated using chloroform/methanol , and the protein pellet air dried .",
"Samples were re-suspended in 1M NaOH , quantified by nanodrop , diluted to 100 mM HEPES and 4M GnHCl and pH adjusted ( pH6 . 5–7 . 5 ) prior to 6-plex TMT labelling as per manufacturer’s instructions ( Sixplex TMT , Cat# 90061 , ThermoFisher Scientific ) .",
"Equal ratios of TMT-labelled samples were pooled and methanol/chloroform precipitated .",
"Protein pellets were air-dried and re-suspended in 200 mM HEPES ( pH8 ) , and digested with trypsin ( 1:100 ratio ) for 16 hr at 37°C , pH 6 . 5 and a sample was taken for pre-TAILS .",
"High-molecular-weight dendritic polyglycerol aldehyde polymer ( ratio of 5:1 w/w polymer to sample ) and NaBH3CN ( to a final concentration of 80 mM ) was added , incubated at 37°C for 16 hr , followed by quenching with 100 mM ethanolamine ( 30 min at 37°C ) and underwent ultrafiltration ( MWCO of 10 , 000 ) .",
"Collected samples were desalted , acidified to 0 . 1% formic acid and dried , prior to MS analysis .",
"Samples were analysed on a Thermo Scientific Easy nLC-1000 coupled online to a Bruker Daltonics Impact II UHR QTOF .",
"Briefly , peptides were loaded onto a 20 cm x 75 µm I . D . analytical column packed with 1 . 8 µm C18 material ( Dr . Maisch GmbH , Germany ) in 100% buffer A ( 99 . 9% H2O , 0 . 1% formic acid ) at 800 bar followed by a linear gradient elution in buffer B ( 99 . 9% acetonitrile , 0 . 1% formic acid ) to a final 30% buffer B for a total 180 min including washing with 95% buffer B . Eluted peptides were ionised by ESI and peptide ions were subjected to tandem MS analysis using a data-dependent acquisition method .",
"A top17 method was employed , where the top 17 most intense multiply charged precursor ions were isolated for MS/MS using collision-induced-dissociation , and actively excluded for 30 s .",
"MGF files were extracted and searched using Mascot against the UniProt Homo sapiens database , with semi-ArgC specificity , TMT6plex quantification , variable oxidation of methionine , variable acetylation of N termini , 20 ppm MS1 error tolerance , 0 . 05 Da MS2 error tolerance and two missed cleavages .",
"Mascot .",
"dat files were imported into Scaffold Q+S v4 . 4 . 3 for peptide identification processing to a final FDR of 1% .",
"Quantitative values were calculated through Scaffold and used for subsequent analyses .",
"AF and NP tissues from four individuals were cut into approximately 0 . 5 cm3 pieces , and put into the Dulbecco's modified Eagle's medium ( DMEM ) ( Gibco ) supplemented with 20 mM HEPES ( USB ) , 1% penicillin-streptomycin ( Gibco ) and 0 . 4% fungizone ( Gibco ) .",
"The tissues were digested with 0 . 2% pronase ( Roche ) for 1 hr , and centrifuged at 200 g for 5 min to remove supernatant .",
"AF and NP were then digested by 0 . 1% type II collagenase ( Worthington Biochemical ) for 14 hr and 0 . 05% type II collagenase for 8 hr , respectively .",
"Cell suspension was filtered through a 70 µm cell strainer ( BD Falcon ) and centrifuged at 200 g for 5 min .",
"The cell pellet was washed with phosphate buffered saline ( PBS ) and centrifuged again to remove the supernatant .",
"RNA was then extracted from the isolated disc cells using Absolutely RNA Nanoprep Kit ( Stratagene ) , following manufacturer’s protocol , and stored at −80°C until further processing .",
"The quality and quantity of total RNA were assessed on the Bioanalyzer ( Agilent ) using the RNA 6000 Nano total RNA assay .",
"cDNA was generated using Affymetrix GeneChip Two-Cycle cDNA Synthesis Kit , followed by in vitro transcription to produce biotin-labelled cRNA .",
"The sample was then hybridised onto the Affymetrix GeneChip Human Genome U133 Plus 2 . 0 Array .",
"The array image , CEL file and other related files were generated using Affymetrix GeneChip Command Console .",
"The experiment was conducted as a service at the Centre for PanorOmic Sciences of the University of Hong Kong .",
"CEL and other files were loaded into GeneSpring GX 10 ( Agilent ) software .",
"The RMA algorithm was used for probe summation .",
"Data were normalised with baseline transformed to median of all samples .",
"A loose filtering based on the raw intensity values was then applied to remove background noise .",
"Consequently , transcriptomic data with a total of 54 , 675 probes ( corresponding to 20 , 887 genes ) and eight profiles were obtained .",
"The detected proteins were compared against the transcription factor ( TF ) database ( Vaquerizas et al . , 2009 ) and the human genome nomenclature consortium database for cell surface markers ( CDs ) ( Braschi et al . , 2019 ) , where 77 TFs and 83 CDs were detected ( Figure 1—figure supplement 3A ) .",
"Excluding missing values , the LFQ levels among the data-points range from 15 . 6 to 41 . 1 , with a Gaussian-like empirical distribution ( Figure 2—figure supplement 1A ) .",
"The numbers of valid values per protein were found to decline rapidly when they were sorted in descending order ( Figure 2—figure supplement 1B , upper panel ) .",
"To perform PCAs , only a subset of genes with sufficiently large numbers of valid values ( i . e . non-missing values ) were used .",
"The cutoff for this was chosen based on a point corresponding to the steepest slope of descending order of valid protein numbers ( Figure 2—figure supplement 1B , second panel ) , such that the increase of valid values is slower than the increase of missing values beyond that point .",
"Subsequently , the top 507 genes were picked representing 59 . 8% of all valid values .",
"This new subset includes 12 . 4% of all missing values .",
"Since the subset of data still contains some missing values , an imputation strategy was adopted employing the Multiple Imputation by Chained Equations ( MICE ) method and package ( Buuren and Groothuis-Oudshoorn , 2011 ) , with a max iteration set at 50 and the default PMM method ( predictive mean matching ) .",
"To further ensure normality , Winsorisation was applied such that genes whose average is below 5% or above 95% of all genes were also excluded from PCA .",
"The data was then profile-wise standardised ( zero-mean and one standard deviation ) before PCA was applied on the R platform ( R Development Core Team , 2013 ) .",
"To assess the impact of the spatiotemporal factors on the proteomic profiles , we performed Analysis of Variance ( ANOVA ) , correlating each protein to the age , compartments , level , and directionality .",
"To draw the soft boundaries on the PCA plot between groups of samples , support vector machines with polynomial ( degree of 2 ) kernel were applied using the LIBSVM package ( Chang and Lin , 2011 ) and the PCA coordinates as inputs for training .",
"A meshed grid covering the whole PCA field was created to make prediction and draw probability contours for −0 . 5 , 0 , and 0 . 5 from the fitted model .",
"Hierarchical clustering was performed with ( 1- correlation coefficient ) as the distance metrics unless otherwise specified .",
"To address the problem of ‘dropout’ effects while avoiding extra inter-dependency introduced due to imputations , we adopted three strategies in calculating the DEPs , namely , by statistical testing , exclusively detected , and fold-change cutoff approaches .",
"First , for the proteins that have over half valid values in both groups under comparison , we performed t-testing with p-values adjusted for multiple testing by the false discovery rate ( FDR ) .",
"Those with FDR below 0 . 05 were considered statistical DEPs .",
"Second , for the proteins where one group has some valid values while the other group is completely not detected , we considered the ones with over half valid values in one group to be exclusive DEPs .",
"For those proteins that were expressed in <50% in both groups , the ones with fold-change greater than two were also considered to be DEPs .",
"To fit the lateral and anteroposterior trends for the modules of genes identified in the young samples , a Gaussian Process Estimation ( GPE ) model was trained using the GauPro package in R Development Core Team , 2013 .",
"Pathway analyses was conducted on the GSEA ( Subramanian et al . , 2005 ) .",
"Signalling proteins was compiled based on 25 Signal transduction pathways listed on KEGG ( Kanehisa et al . , 2019 ) .",
"For transcriptomic data , we used a thresholding approach to detect DEGs ( differentially expressed genes ) , whereby a gene was considered a DEG if the log2 ( fold-change ) is greater than three and the average expression ( logarithmic scale ) is greater than 10 ( Figure 6—figure supplement 1E–H ) .",
"The LASSO model between MRI and proteome was trained using the R package ‘glmnet’ , wherein the 85 ECMs were first imputed for missing values in them using MICE .",
"Nine ECMs were not imputed for too many missing values , leaving 76 for training and testing .",
"The best value for λ was determined by cross-validations .",
"A model was then trained on the aged MRIs ( dependent variable ) and aged proteome of the 76 genes ( independent variable ) .",
"The fitted model was then applied to the young proteome to predict MRIs of the young discs .",
"The custom scripts for processing and analysing the data were housed at github . com/hkudclab/DIPPER; Tam , 2021; copy archived at swh:1:rev:49e4da79786de1dfa496d7c7472343f3b865696c .",
"An interactive web interface for the data is available at http://www . sbms . hku . hk/dclab/DIPPER/ ."
]
] | [
"The spatiotemporal proteome of the intervertebral disc ( IVD ) underpins its integrity and function .",
"We present DIPPER , a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals .",
"To begin with , protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs .",
"They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs .",
"Machine learning methods predicted a ‘hydration matrisome’ that connects extracellular matrix with MRI intensity .",
"Importantly , the static proteome used as point-references can be integrated with dynamic proteome ( SILAC/degradome ) and transcriptome data from multiple clinical samples , enhancing robustness and clinical relevance .",
"The data , findings , and methodology , available on a web interface ( http://www . sbms . hku . hk/dclab/DIPPER/ ) , will be valuable references in the field of IVD biology and proteomic analytics ."
] | [
"The backbone of vertebrate animals consists of a series of bones called vertebrae that are joined together by disc-like structures that allow the back to move and distribute forces to protect it during daily activities .",
"It is common for these intervertebral discs to degenerate with age , resulting in back pain and severely reducing quality of life .",
"The mechanical features of intervertebral discs are the result of their proteins .",
"These include extracellular matrix proteins , which form the external scaffolding that binds cells together in a tissue , and signaling proteins , which allow cells to communicate .",
"However , how the levels of different proteins in each region of the disc vary with time has not been fully examined .",
"To establish how protein composition changes with age , Tam , Chen et al . quantified the protein levels and gene activity ( which leads to protein production ) of intervertebral discs from young and old deceased individuals .",
"They found that the position of different mixtures of proteins in the intervertebral disc changes with age , and that young people have high levels of extracellular matrix proteins and signaling proteins .",
"Levels of these proteins decreased as people got older , as did the amount of proteins produced .",
"To determine which region of the intervertebral disc different proteins were in , Tam , Chen et al . also performed magnetic resonance imaging ( MRI ) of the samples to correlate image intensity ( which represents water content ) with the corresponding protein signature .",
"The data obtained provides a high-quality map of how the location of different proteins changes with age , and is available online under the name DIPPER .",
"This database is an informative resource for research into skeletal biology , and it will likely advance the understanding of intervertebral disc degeneration in humans and animals , potentially leading to the development of new treatment strategies for this condition ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | SIR-2.1 integrates metabolic homeostasis with the reproductive neuromuscular excitability in early aging male Caenorhabditis elegans | elife-01730-v1 | [
[
"Although lifespan is well studied in the model organism Caenorhabditis elegans ( Kenyon , 2010 ) , the aging process per se is still under intense research ( Jin , 2010 ) .",
"For example , behavioral and/or mental ability starts to decline during early aging , prior to any dramatic structural or morphological dysfunction ( Salthouse , 2004; Guo et al . , 2012 ) .",
"The decline could be due to physiological alterations toward a non-optimal state , which leads to the failure of proper behavioral execution .",
"Therefore , it is urgent to uncover the molecular mechanism ( s ) underlying the physiological changes , so that certain actions could prevent or postpone the non-optimal modifications that occur during aging .",
"Our previous studies used C . elegans males to establish a behavioral model for studying physiological alterations that occur during early aging ( Guo et al . , 2012 ) .",
"The spicule intromission motor step , which occurs during C . elegans male copulation , is sensitive to changes that occur during early aging ( Garcia et al . , 2001; Garcia and Sternberg , 2003 ) .",
"Through pharmacological , optogenetics , and genetic analyses , we found a correlation between the increased excitability of the spicule intromission circuit and the decline of mating at early adulthood ( Guo et al . , 2012 ) .",
"However , the molecular mechanisms underlying this correlation were unknown .",
"Caloric restriction is an effective way to extend lifespan ( Hursting et al . , 2003 ) .",
"In C . elegans , caloric deprivation such as transient starvation for 3 to 18 hr during early adulthood can reduce excitability in the spicule intromission circuitry and prolong the mating potency of wild type ( LeBoeuf et al . , 2011 ) .",
"We showed that the effect of transient starvation is partially mediated by UNC-43/CaMKII and up-regulation of the unc-103 and egl-2-encoded ERG-like K+ channels ( LeBoeuf et al . , 2011; Guo et al . , 2012 ) .",
"Although males with mutations in both K+ channels abrogate most of the beneficial effects of transient starvation , we still observed a small increase in the mating ability of aged double-mutant males , suggesting that other mechanisms are applied by transient starvation to improve mating ( Guo et al . , 2012 ) .",
"One possibility is that metabolic alteration , induced by transient starvation , compensates for the physiological changes that occur during aging .",
"The connection between metabolic change and physiological status might be the generation of reactive oxygen species ( ROS ) .",
"First , metabolic status determines the oxidative stress burden: ROS is the byproduct of oxidative phosphorylation ( Murphy , 2009 ) .",
"Second , emerging evidence shows that ROS modulates the excitability of neuromuscular system through modification of ion channels ( Taglialatela et al . , 1997; Cai and Sesti , 2009; Aggarwal and Makielski , 2013 ) .",
"However , there are few comprehensive in vivo studies , which link ROS-mediated physiological changes to complex behavior alteration .",
"To address this , we investigated how a mutation in the C . elegans metabolism regulator , SIR-2 . 1 , alters behavior .",
"SIR-2 . 1 is an ortholog of yeast SIR2 ( Tissenbaum and Guarente , 2001 ) .",
"Yeast SIR2 , a NAD+-dependent histone deacetylase , with a role in chromatin regulation , can silence ribosome DNA expression and recombination ( Gottlieb and Esposito , 1989 ) .",
"When overexpressed , sir2 extends yeast lifespan , whereas deletion of the gene shortens the lifespan by 50% ( Kaeberlein et al . , 1999 ) .",
"Although there is controversy on whether overexpression of invertebrate sir2 ortholog extends lifespan ( Burnett et al . , 2011; Viswanathan and Guarente , 2011 ) , sirtuin family proteins have been shown to regulate glucose and fat metabolism ( Houtkooper et al . , 2012 ) .",
"For example , the C . elegans SIR-2 . 1 inhibits lipid synthesis during fasting ( Walker et al . , 2010 ) .",
"In addition , sirtuin proteins mediate an oxidative stress response by regulating antioxidant gene expression through transcriptional factors such as FOXO in a 14-3-3-dependent manner ( Berdichevsky et al . , 2006; Webster et al . , 2012; Merksamer et al . , 2013 ) .",
"Overall , sirtuin proteins could be involved in age-related diseases , such as type II diabetes and neurodegenerative diseases ( Satoh et al . , 2011; Houtkooper et al . , 2012 ) .",
"Taken together , it is possible that sirtuin proteins regulate the cell’s metabolic status and physiology , which further alters the coordination of specific behaviors .",
"In this study , we used C . elegans male mating behavior to study the molecular and physiological alterations underlying the behavioral decline that occur during early aging .",
"We found that wild-type males require SIR-2 . 1 to maintain mating potency , and sir-2 . 1 mutant males show a premature decline in copulation behavior , consistent with oxidative stress-induced hyperexcitability of their mating circuit .",
"We propose that in sir-2 . 1 ( 0 ) males , enhanced glycolysis/fatty acid oxidation , coupled with a compromised anti-stress system , contribute to premature mating decline .",
"However , in mutant and aged wild-type males , pyruvate carboxylase and phosphenolpyruvate carboxykinase are up-regulated , likely as a compensatory mechanism to shunt excess pyruvate from glycolysis and the TCA cycle to lipid biosynthesis , gluconeogenesis , and glyceroneogenesis ."
],
[
"Previously , we reported that C . elegans male mating behavior deteriorates during early aging .",
"N2 and him-5 ( e1490 ) C . elegans ( henceforth , will be referred to as wild type ) males’ mating capability begins to decline at day 3 of their adulthood , although their median lifespan is 11–12 days ( Guo et al . , 2012 ) .",
"We demonstrated that transient starvation of young males can extend their mating span , partially through up-regulation of ether-a-go-go K+ channels ( LeBoeuf et al . , 2011 ) ; however , our data also suggested that transient starvation can improve mating through additional mechanisms ( Guo et al . , 2012 ) .",
"Considering that metabolism is altered in food-deprived males ( Tan et al . , 2011 ) , we tested whether perturbing the histone deacetylase metabolic regulator , sir-2 . 1 , affects the functional span of copulation behavior in fed and transiently starved/re-fed males .",
"In adult hermaphrodites , animals that lack sir-2 . 1 have increased intestinal lipids ( Walker et al . , 2010 ) , a phenotype opposite of starved animals .",
"Likewise , we found that 1-day-old sir-2 . 1 ( ok434 ) null ( 0 ) males also contain more lipids than wild type ( Figure 1A ) .",
"In addition , we observed that 2-day-old wild-type males have more fat ( Figure 1A ) .",
"Thus , we asked if sir-2 . 1 ( 0 ) males might have altered mating due to metabolic dysregulation .",
"Allowing the males to mate for 5 hr , we found that well-fed aging sir-2 . 1 ( 0 ) males’ mating ability drops prematurely , compared to wild-type males ( Figure 1B",
"( i ) ) .",
"Even under unlimited mating conditions , the mating potency of 2-day-old sir-2 . 1 ( 0 ) drops to 42% ( p<0 . 0001 , n = 47 ) ( Figure 1B",
"( ii ) ) . 10 . 7554/eLife . 01730 . 003Figure 1 . sir-2 . 1 ( 0 ) males have altered lipid content and their mating ability deteriorates prematurely .",
"( A ) 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild-type males have more lipid content than 1-day-old wild type .",
"Left: quantification of fat staining , mean ± SEM , unpaired t-test .",
"Right: representative images of fat staining .",
"( B ) Mating potency of wild-type and sir-2 . 1 ( 0 ) males .",
"Copulations were allowed to occur for 5 hr",
"( i ) or for an unlimited time",
"( ii ) .",
"The number of males in each assay is listed at the bottom of each bar .",
"The numerical percentage of wild-type males that mated on day 1 was normalized to 100% .",
"The normalization factor was then applied to the other experimental conditions .",
"The normalized percentages for each day are listed on the top .",
"Fisher’s exact test was used to compare the mating potency prior to normalization .",
"( C ) Transient starvation reduces sir-2 . 1 ( 0 ) mating deficiency .",
"( D ) Mating potency of sir-2 . 1 ( 0 ) and rescued strain sir-2 . 1 ( 0 ) ; rgEX399 [Psir-2 . 1:sir-2 . 1::yfp] .",
"ns , not significant .",
"Asterisks * , ** and *** indicate the p<0 . 05 , 0 . 01 , and 0 . 0001 in this paper , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00310 . 7554/eLife . 01730 . 004Figure 1—figure supplement 1 . ( A ) Tissue specific expression of sir-2 . 1 does not rescue the reduced mating potency of sir-2 . 1 ( 0 ) males at day 2 . The lev-11 promoter expresses sir-2 . 1 in all body wall and sex muscles .",
"The ges-1 promoter expresses sir-2 . 1 in the intestine .",
"The aex-3 promoter expresses sir-2 . 1 in all neurons .",
"( B ) Adult lifespan of wild-type ( circles ) and sir-2 . 1 ( 0 ) ( squares ) males ( n = 50 for wild-type males; n = 76 for sir-2 . 1 ( 0 ) males ) ( Log-rank [Mantel-Cox] Test ) .",
"( C ) Muscle fiber organization in the genital muscles of 1-day-old and 2-day-old sir-2 . 1 ( 0 ) males .",
"Asterisks indicate the diagonal muscles , arrow head indicates the oblique muscles .",
"( D ) In vitro sperm activation assays of 2-day-old wild-type and sir-2 . 1 ( 0 ) males .",
"Representative images are shown on the top of percentage bars .",
"Arrow indicates the activated sperm with pseudopod , and solid arrow head indicate the inactivated sperm .",
"No significant differences were observed between wild type and sir-2 . 1 ( 0 ) males ( unpaired t-test , N = 5 trials ) .",
"In each trial , 50–60 sperm cells were analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 004 We then asked if transient starvation can suppress the mating defect in sir-2 . 1 ( 0 ) .",
"To do so , we starved sir-2 . 1 ( 0 ) males for ∼20 hr from L4 , and conducted a 5 hr mating potency assay .",
"Transient starvation improved mating of 2-day-old sir-2 . 1 ( 0 ) males from 13% to 75% ( p<0 . 0001 , Figure 1C ) , but at day 3 , the mating potency of transiently starved sir-2 . 1 ( 0 ) males was still lower than wild type .",
"Thus , similar to wild type , the metabolic alteration and/or up-regulated EAG K+ channel functions caused by starvation alleviate some of the dysfunction caused by the sir-2 . 1 deletion .",
"However , the mutant’s reduced mating potencies between day 1 and 3 under both conditions indicate that SIR-2 . 1 contributes to the functionality of the mating circuits during this period .",
"To confirm that premature mating deterioration in sir-2 . 1 ( 0 ) males is caused by the ok434 allele , we introduced into sir-2 . 1 ( 0 ) animals a rescuing transgene containing the sir-2 . 1 endogenous promoter driving the sir-2 . 1 genomic sequence fused to yfp .",
"The extrachromosomal expression of sir-2 . 1 significantly improved the mating potency of 2-day-old sir-2 . 1 ( 0 ) males from 26% to 75% ( p<0 . 0001 , Figure 1D ) .",
"sir-2 . 1 is expressed broadly in C . elegans ( Bamps et al . , 2009 ) , thus we further conducted tissue-specific rescue assays , but found that none of the tissue-specific promoters driving the expression of sir-2 . 1 , including neuronal , muscle , and intestinal promoters can rescue the premature mating decline ( Figure1—figure supplement 1A ) , suggesting that sir-2 . 1 is required in multiple tissues to maintain male mating .",
"To exclude the possibility that sir-2 . 1 ( 0 ) mating deficiency at day 2 is due to a shorter lifespan , we conducted a lifespan assay and found that sir-2 . 1 ( 0 ) males lived as long as wild type ( Figure 1—figure supplement 1B ) .",
"Another possibility for the mating deterioration is morphological deformities of the sex musculature .",
"However , in 2-day-old sir-2 . 1 ( 0 ) males expressing a functional yfp:act-1 transgene ( Figure 1—figure supplement 1C ) , we did not observe any obvious muscle fiber disorganization , which normally occurs in 8-day-old wild-type males ( Guo et al . , 2012 ) .",
"Although we did not inspect neural morphology , published studies showed that neural morphology does not change in C . elegans during aging ( Herndon et al . , 2002 ) .",
"Another potential explanation for the lower mating efficiency is that sperm activity in 2-day-old sir-2 . 1 ( 0 ) males is defective .",
"C . elegans male sperm are stored in the seminal vesicle as a non-activated form and become activated after transfer into a hermaphrodite .",
"Regulated by proteases , individual sperm goes through a morphological change to form a pseudopod .",
"This pseudopod provides mobility for the sperm to fertilize the hermaphrodite oocyte ( Smith and Stanfield , 2011 ) .",
"To test whether the low mating potency of 2-day-old sir-2 . 1 ( 0 ) males is due to failure in sperm activation , we did an in vitro sperm activation assay and found that similar to wild type , 92 . 0 ± 4 . 3% of sperms from 2-day-old sir-2 . 1 ( 0 ) can be artificially activated by pronase ( Figure 1—figure supplement 1D ) .",
"Taken together , we speculate that the premature mating decline in sir-2 . 1 ( 0 ) is due to physiological changes , rather than the structural degeneration of either neuromuscular circuits or sperm .",
"We showed that wild-type mating deterioration at day 3 is correlated with an increased excitability in the mating circuitry ( Guo et al . , 2012 ) .",
"Hence , we hypothesized that sir-2 . 1 ( 0 ) mating decline might also be due to a premature increase in the cellular excitability .",
"To test this , we used two acetylcholine ( ACh ) agonists , levemisole ( LEV ) and arecoline ( ARE ) to determine the responses of wild-type and sir-2 . 1 ( 0 ) males at multiple ages .",
"In the male spicule intromission circuit , LEV binds to ionotropic ACh receptors , whereas ARE is a nonselective ACh agonist ( Liu et al . , 2007; Correa et al . , 2012 ) .",
"Activation of ACh receptors depolarizes the male’s neurons and muscles , and ultimately causes sex muscle contractions; as a result , males protrude their copulatory spicules .",
"We found that at day 1 , sir-2 . 1 ( 0 ) and wild-type males had similar response to a sub-threshold effective concentration of ARE ( 50 μM ) ( Figure 2A",
"( i ) ) .",
"However , 2-day-old sir-2 . 1 ( 0 ) males were more sensitive to agonist stimulation and required significantly less time to respond ( Figure 2A",
"( ii ) ) .",
"Additionally , 2-day-old sir-2 . 1 ( 0 ) males were more sensitive to sub-threshold LEV stimulation .",
"58% sir-2 . 1 ( 0 ) compared to 35% of wild type protracted their spicules in 500 nM LEV ( p<0 . 05 , n > 30 ) ( Figure 2B",
"( ii ) ) .",
"These results indicate that the loss of sir-2 . 1 in males alters the spicule intromission circuit’s excitability during early aging . 10 . 7554/eLife . 01730 . 005Figure 2 . sir-2 . 1 ( 0 ) males’ sex circuitry becomes more excitable during aging , and those males display ejaculation defects .",
"( A ) 1-day-old wild-type and sir-2 . 1 ( 0 ) males ( n = 30 ) have similar response to the ACh agonist arecoline ( ARE ) .",
"The time required for those males to protrude their spicules out in 50 μM ARE solution are not significantly different",
"( i ) ( unpaired t-test ) , whereas 2-day-old sir-2 . 1 ( 0 ) males require significantly less time to respond to ARE",
"( ii ) ( unpaired t-test ) .",
"Mean and SEM are indicated .",
"( B ) 1-day-old wild-type and sir-2 . 1 ( 0 ) males ( n = 30 ) have similar response to the ACh agonist levamisole ( LEV )",
"( i ) .",
"However , 2-day-old sir-2 . 1 ( 0 ) males are more sensitive to LEV",
"( ii ) .",
"( Fisher’s exact test ) .",
"( C , D ) 2-day-old sir-2 . 1 ( 0 ) males have an ejaculation defect .",
"( C ) The percentages of 2-day-old wild-type and sir-2 . 1 ( 0 ) males that ejaculated during copulation .",
"( Fisher’s exact test ) .",
"( D ) The numbers of cross progeny produced by individual 2-day-old wild-type and sir-2 . 1 ( 0 ) with unc-64 ( e240 ) hermaphrodites .",
"Mean and SEM are indicated ( unpaired t-test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00510 . 7554/eLife . 01730 . 006Figure 2—figure supplement 1 . ( A ) A cartoon illustration of C . elegans male mating behavior .",
"( B ) 2-day-old sir-2 . 1 ( 0 ) males have no defect in turning behavior , ( C ) sensing the vulva of the hermaphrodite and ( D ) staying at the vulva . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 006 To address how hyperexcitability disrupts copulation , we observed the mating behavior of 2-day-old sir-2 . 1 ( 0 ) and wild-type males .",
"We found that 2-day-old sir-2 . 1 ( 0 ) males performed most of the mating steps similarly to the wild-type control ( Figure 2— figures supplement 1A , B , C , D ) .",
"Although 2-day-old sir-2 . 1 ( 0 ) males can effectively insert their spicules , a significant number of them failed to transfer sperm into the hermaphrodite ( Figure 2C ) .",
"Upon spicule insertion , sperm moved out from the seminal vesicle and traveled through the vas deferens; however , they remained stuck in the vas deferens and did not drain through the cloacal opening ( Videos 1 , 2 and 3 ) .",
"Even the exceptional sir-2 . 1 ( 0 ) males that successfully ejaculated , transferred less sperm and produced fewer progeny ( Figure 2D ) . 10 . 7554/eLife . 01730 . 007Video 1 . Wild-type male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00710 . 7554/eLife . 01730 . 008Video 2 . 2-day-old sir-2 . 1 ( 0 ) male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 00810 . 7554/eLife . 01730 . 009Video 3 . 2-day-old sir-2 . 1 ( 0 ) male’s ejaculation . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 009 The male copulatory spicules are attached to three sets of sex muscles: the retractor , protractor , and anal depressor muscles .",
"Contraction of the protractor muscles leads to spicules insertion into the vulva ( Garcia et al . , 2001 ) .",
"During the normal ejaculation step of mating behavior , after spicule penetration , the posterior gubernaculum erector and retractor muscles contract , presumably to pull the proctodeum posteriorly , so that sperm can drain from the vas deferens ( Figure 3—figure supplement 1 ) ( Liu et al . , 2007 ) .",
"In sir-2 . 1 ( 0 ) males , we speculated that after spicule insertion , the abnormal increased cell excitability causes the spicule protractor and anal depressor muscles to hypercontract , which during sperm transfer , would pinch close the vas deferens opening .",
"To test this , we imaged the Ca2+ in the spicule-associated dorsal protractor and anal depressor muscles ( region-of-interest , ROI , indicated in Figure 3A , B ) , by expressing G-CaMP3 in these sex muscles of both sir-2 . 1 ( 0 ) and wild-type males ( Tian et al . , 2009; Guo et al . , 2012 ) .",
"During the mating behavior of 2-day-old wild-type males , the G-CaMP3 ΔF/F0 increased to 129 . 0 ± 32 . 5% ( n = 5 ) at the time of spicule insertion , and the Ca2+ signal started to decline to 86 . 7 ± 30 . 8% during the 10-s period after spicule insertion ( Figure 3A and Video 4 ) .",
"This indicates that the spicule protractor muscles partially relax after spicule insertion .",
"However , in 2-day-old sir-2 . 1 ( 0 ) males , ΔF/F0 increased up to 204 . 3 ± 97 . 5% ( n = 5 ) upon spicule insertion .",
"Unlike wild type , Ca2+ transients did not decrease as much , and the ΔF/F0 fluctuated at about 129 . 8 ± 33 . 0% ( Figure 3B and Video 5 ) .",
"The sustained higher Ca2+ levels in 2-day-old sir-2 . 1 ( 0 ) males suggest that spicule protractor and anal depressor muscles are hypercontracted and pinch the vas deferens opening , thus blocking sperm release . 10 . 7554/eLife . 01730 . 010Figure 3 . Ca2+ imaging of spicule-associated muscles in mating males . Pseudo-colored images of Ca2+ in the spicule muscles of 2-day-old wild-type and sir-2 . 1 ( 0 ) males during mating ( A ) and ( B ) are representative frames to show the Ca2+ levels of the spicule-associated muscles during spicule insertion attempts , penetration and the start of sperm transfer ( ∼10 s after insertion for wild type ) or 19 s after insertion ( for sir-2 . 1 ( 0 ) males ) .",
"The asterisks indicate the hermaphrodite vulva .",
"Below the images , the Ca2+ transients in the protractor and anal depressor muscles ( indicated by the black rectangle in A and B ) are plotted for 5 individual wild-type ( A ) and sir-2 . 1 ( 0 ) males ( B ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01010 . 7554/eLife . 01730 . 011Figure 3—figure supplement 1 . A cartoon illustrating the contractile changes of the spicule-associated muscles during intromission and ejaculation behaviors of a 2-day-old wild-type and sir-2 . 1 ( 0 ) male , respectively . Muscles displaying warmer colors signify the increasing intensity of the contractile events .",
"The square box refers to the region of interest used for calcium imaging measurements shown in Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01110 . 7554/eLife . 01730 . 012Video 4 . Ca2+ transient in a 2-day-old wild-type male . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01210 . 7554/eLife . 01730 . 013Video 5 . Ca2+ transient in a 2-day-old sir-2 . 1 ( 0 ) male . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 013 C . elegans hermaphrodite studies showed that SIR-2 . 1 promotes the expression of antioxidant genes through its association with the FOXO/DAF-16 transcription factor ( Berdichevsky et al . , 2006 ) .",
"sir-2 . 1 ( 0 ) hermaphrodites are more sensitive to stresses such as reactive oxygen species ( ROS ) ( Rizki et al . , 2011 ) .",
"Therefore , we asked if ROS-induced damage contributes to the premature mating deterioration .",
"We confirmed that similar to hermaphrodites , sir-2 . 1 ( 0 ) males are also more sensitive to paraquat , a ROS-generator .",
"Mutant males are less viable in 10 mM paraquat after 24 hr; 89% of sir-2 . 1 ( 0 ) males survived , compared to 99% of wild type ( p<0 . 01 , n > 100 ) ( Figure 4A ) .",
"When exposure time reached 48 hr , the difference between two strains became more obvious , 39% of wild-type males survived , compared to 4% of sir-2 . 1 ( 0 ) ( p<0 . 001 ) ( Figure 4A ) . 10 . 7554/eLife . 01730 . 014Figure 4 . ROS contributes to the mating deterioration .",
"( A ) Survival rates of wild-type and sir-2 . 1 ( 0 ) males on NGM containing 10 mM paraquat at 24 hr and 48 hr post paraquat exposure .",
"( B ) Mating potency of 1-day-old wild-type males exposed to 0 . 01 , 0 . 1 , and 1 mM paraquat .",
"( C ) The percentages of males with their spicules protruding out ( SpOUT ) in response to 1 μM levamisole ( LEV ) after treatment with 1 mM paraquat .",
"( D–G )",
"Exposing males to N-acetyl-cystine ( NAC ) improves mating .",
"The percentages of 3-day-old wild-type ( D ) and 2-day-old sir-2 . 1 ( 0 ) ( E ) males that protrude their spicules out in response to 100 nM LEV after NAC exposure .",
"Mating potency of 3-day-old wild-type ( F ) and 2-day-old sir-2 . 1 ( 0 ) ( G ) males after NAC exposure ( Fisher’s exact test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 014 Since sir-2 . 1 ( 0 ) males are more sensitive to oxidative stress , we hypothesized that during aging , accumulated ROS from metabolism might contribute to the decreased mating efficiency and to the increased excitability of the spicule intromission circuit .",
"To test this , we grew wild-type males on plates containing 1 mM paraquat from late L4 to adult and assayed their mating ability and genital muscle excitability .",
"After exposure to paraquat for 24 hr , males showed significant decline in mating potency ( Figure 4B ) .",
"Additionally , these males also displayed increased genital muscle sensitivity to the day 1 EC50 concentration ( 1 μM ) of LEV .",
"56% of wild type protracted their spicules; however , 83% males exposed to paraquat responded to the ACh agonist ( p<0 . 05 , n = 54 ) ( Figure 4C ) .",
"To further test if ROS contributes to the copulation decline , we supplemented the males’ media with the antioxidant N-acetyl-cysteine ( NAC ) ( Schulz et al . , 2007 ) , and asked if NAC can delay genital muscle excitability changes and improve fertility .",
"Indeed , when we exposed wild-type and sir-2 . 1 ( 0 ) males to NAC from L4 to adulthood day 3 and adulthood day 2 , respectively , the antioxidant decreased the males’ sensitivity to 100 nM LEV ( the EC50 concentration for older males [Guo et al . , 2012] ) ( Figure 4D , E ) and increased their mating potency ( Figure 4F , G ) .",
"These results are consistent with the idea that ROS contributes to the behavioral decline .",
"Next , we asked why 2-day-old sir-2 . 1 ( 0 ) males are more sensitive to ROS .",
"Metabolism as a major source of endogenous ROS stress might contribute to behavioral decline .",
"SIR-2 . 1’s role in regulating metabolic processes has not been well described in C . elegans hermaphrodites , and scarcely in males .",
"Therefore , we compared the expression levels of 55 genes involved in multiple metabolic processes including: glycolysis , gluconeogenesis/glyceroneogenesis , citrate acid cycle , glyoxylate cycle , fatty acid metabolism and electron transport chain ( ETC ) /oxidative phosphorylation ( OXPHOS ) ( Castelein et al . , 2008 ) between age-matched sir-2 . 1 ( 0 ) and wild-type males .",
"Out of the 55 genes we surveyed , 17 showed statistically significant changes ( Figure 5 ) ; the information for all the genes is tabulated in the Supplementary file 1 . 10 . 7554/eLife . 01730 . 015Figure 5 . sir-2 . 1 ( 0 ) males have altered expression of metabolic genes . Relative mRNA expression level of genes involved in metabolic processes such as glycolysis ( A ) , TCA cycle ( B ) , fatty acid oxidation ( C ) , Gluconeogenesis/glyceroneogenesis/lipid synthesis ( D ) , Glyoxylate cycle ( E ) , and ETC ( F ) in 2-day-old wild type , 1-day-old , and 2-day-old sir-2 . 1 ( 0 ) males relative to 1-day-old wild type .",
"d1 WT refers to day1 wild type; d2 WT refers to day 2 wild type; d1 s2 refers to day1 sir-2 . 1 ( 0 ) ; d2 s2 refers to day 2 sir-2 . 1 ( 0 ) ( unpaired t-test compared to 1-day-old wild type ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 015 Through real-time PCR analyses , we found that mRNAs encoding key enzymes involved in the initiation of glycolysis ( hexokinase [F14B4 . 2] and glucose-6-phosphate isomerase [Y87G2A . 8] ) and fatty acid oxidation ( fatty acid acyl-CoA synthetase [C46F4 . 2] ) were up-regulated in 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild-type males , relative to 1-day-old wild type ( Figure 5A , B ) .",
"This is consistent with the published observation that hexokinase is also up-regulated in the whole body of conditional sirt1 knock-out mice ( Gomes et al . , 2013 ) .",
"Most enzymes involved in the TCA cycle did not change in their levels ( Supplementary file 1 ) .",
"In contrast , expression of ETC/OXPHOS components ( cco-1 , W09C5 . 8 ) was reduced in sir-2 . 1 ( 0 ) ( Figure 5F ) .",
"Other genes that were significantly up-regulated include key anabolic enzymes like fatty acid desaturase ( fat-5 , 6 , 7 ) , pyruvate carboxylase ( PC ) ( pyc-1 ) and phosphoenolpyruvate carboxykinase ( PEPCK ) ( pck-1 and pck-2 ) , isocytrate lysase ( icl-1 ) and aconitase-cytosol ( aco-1 ) , which are important for fatty acid biosynthesis , gluconeogenesis , glyceroneogensis , and glyoxylate cycle ( Figure 5D , E ) ( Yang et al . , 2009 ) .",
"Consistent with this , hepatic cells without sirt1 also have an up-regualtion of PEPCK gene expression ( Wang et al . , 2011 ) .",
"Fatty acid desaturase plays a critical role in lipid/triglyceride biosynthesis ( Van Gilst et al . , 2005; Flowers and Ntambi , 2008 ) .",
"PC catalyzes the carboxylation of pyruvate to oxaloacetate ( OAA ) , the first step that shunts pyruvate from glycolysis to gluconeogenesis/glyceroneogenesis .",
"Alternatively , OAA , an intermediate of TCA , can be converted to phosphoenolpyruvate ( PEP ) by PEPCK directly inside the mitochondrion or transported and converted to PEP by PEPCK in cytosol ( Figure 6A ) .",
"The up-regulation of fatty acid desaturase is consistent with the increased lipid staining in sir-2 . 1 ( 0 ) males ( Figure 1A ) . 10 . 7554/eLife . 01730 . 016Figure 6 . sir-2 . 1 ( 0 ) males might have enhanced metabolism .",
"( A ) Schematic illustration of main metabolic enzymes which have altered expression in sir-2 . 1 ( 0 ) males .",
"Red arrows indicate catabolic pathways .",
"Blue arrows indicate anabolic pathways .",
"( B ) ATP content measured in 1 , 2 and 3-day-old wild-type and sir-2 . 1 ( 0 ) males .",
"( C ) Glycogen staining in 1-day-old wild type and sir-2 . 1 ( 0 ) .",
"The glycogen staining level is quantified by measuring the mean gray level of the ROI indicated on the top right corner .",
"The mean gray level is inversely correlated with the iodine stain .",
"( D ) Oil Red O staining of wild type and mutant C . elegans males .",
"( E ) sir-2 . 1 ( + ) and sir-2 . 1 ( 0 ) need pck-2 to maintain their mating at day 2 and day 1 respectively .",
"All percentages of mating potency are normalized to that of 1-day-old wild-type male .",
"( F ) sir-2 . 1 ( 0 ) requires pck-1 to maintain their mating at day 1 and day 2 , while sir-2 . 1 ( + ) males do not need pck-2 to maintain their mating at either day 1 or day 2 .",
"( G ) 2% glucose reduces 1-day-old sir-2 . 1 ( 0 ) mating potency , but not 2-day-old wild-type males ( Fisher’s exact test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 01610 . 7554/eLife . 01730 . 017Figure 6—figure supplement 1 . The level of glucose content is similar between 1-day-old sir-2 . 1 ( 0 ) and wild-type males . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 017 To confirm if the changes in mRNA levels of those metabolic enzymes reflect functional alterations in the metabolic processes , we also measured ATP , glucose and glycogen accumulation in wild-type and sir-2 . 1 ( 0 ) males .",
"Consistent with increased expression levels of glycolysis and fatty acid oxidation genes , sir-2 . 1 ( 0 ) males produced significantly more ATP at day 1 .",
"At day 2 , wild-type ATP levels increased to match the level of sir-2 . 1 ( 0 ) males .",
"But at day 3 , sir-2 . 1 ( 0 ) males again accumulated more ATP ( Figure 6B ) .",
"These data suggest that sir-2 . 1 ( 0 ) and older wild-type males might have an enhanced catabolism , consistent with the potential to generate more ROS .",
"Based on metabolic roles of PEPCK ( Yang et al . , 2009 ) , up-regulation of pck genes could lead to excess glucose/glycogen in sir-2 . 1 ( 0 ) males .",
"Although similar amounts of glucose were detected in sir-2 . 1 ( 0 ) and wild-type males ( Figure 6—figure supplement 1 ) , more glycogen was synthesized in the mutant ( Figure 6C ) .",
"In addition to gluconeogenesis , PEPCK catalysis of OAA to PEP is also a key step for the synthesis of glycerol-3-phosphate , which is used in triglyceride biosynthesis ( Figure 6A ) ( Nye et al . , 2008 ) .",
"Indeed , males lacking functional pck-2 , but not pck-1 have less lipid content ( Figure 6D ) .",
"Additionally , sir-2 . 1 ( 0 ) males that lack pck-2 , but not pck-1 , showed reduced lipid staining ( Figure 6D ) , indicating that pck-2 is necessary for the up-regulation of fat synthesis in sir-2 . 1 ( 0 ) .",
"Taking together the real-time PCR results , accumulation of metabolic products and hypersensitivity to paraquat , we propose that in sir-2 . 1 ( 0 ) males , glycolysis and fatty acid oxidation are up-regulated to provide excessive NADH to the electron transport chain .",
"Since we also measured reduced expression of ETC components cytochrome c oxidase , more ROS might be generated via electron leak ( Lee et al . , 2010 ) .",
"However , we hypothesized that the enhanced expression of enzymes involved in anabolic processes might be a suboptimal self-compensatory mechanism to shunt excess pyruvate from being oxidized in the TCA cycle .",
"To test if the up-regulation of pck genes in sir-2 . 1 ( 0 ) is a compensatory response , we assayed the mating potency of pck-1 ( 0 ) and pck-2 ( 0 ) single mutants and sir-2 . 1 ( 0 ) ; pck-1 ( 0 ) and pck-2 ( 0 ) ; sir-2 . 1 ( 0 ) double mutants .",
"At day 1 , sir-2 . 1 ( 0 ) and pck-2 ( 0 ) males mated comparable to wild type ( Figure 6E ) .",
"However at day 2 , the potency of pck-2 ( 0 ) males started to decline similarly to sir-2 . 1 ( 0 ) .",
"In contrast , for males containing both sir-2 . 1 ( 0 ) and pck-2 ( 0 ) , their mating potency dropped at day 1 ( Figure 6E ) .",
"This indicates that without pck-2 , males that contain or lack sir-2 . 1 display accelerated behavioral decline .",
"Similar to the requirement for functional pck-2 , males that lack sir-2 . 1 also needed pck-1 to maintain their mating potency at day 1; however , pck-1 was not required for sir-2 . 1 ( + ) males to mate efficiently at day 2 ( Figure 6F ) .",
"Next , we reasoned that if excessive glycolysis contributes to the behavioral deterioration , artificially adding extra glucose to the males’ media could accelerate their mating decline .",
"To test this , we grew males on UV-killed-OP50 NGM plates supplemented with 2% glucose , from hatched larvae up to the adult age prior to behavioral decline , which is day 1 or day 2 , for sir 2 . 1 ( 0 ) and wild-type males , respectively .",
"We found that the glucose reduced mating potency of 1-day-old sir-2 . 1 ( 0 ) , but not 2-day-old wild type ( Figure 6G ) , indicating that wild type can cope with the extra glucose better than sir-2 . 1 ( 0 ) .",
"We hypothesized that unlike wild type , sir-2 . 1 ( 0 ) males cannot efficiently respond to the oxidative stress generated by the enhanced catabolism .",
"To test this , we used qRT-PCR to measure the mRNA levels of antioxidant genes: superoxide dismutase ( sod-1 , 2 , 3 , 4 and 5 ) , catalase ( ctl-1 , 2 ) , and glutathione transferase ( gst-10 and gsto-1 ) relative to 1-day-old wild-type males ( Figure 7 ) .",
"As expected , the expression of sod-1 , sod-5 , gst-10 and gsto-1 was reduced in 1-day-old sir-2 . 1 ( 0 ) and 2-day-old wild type ( Figure 7 ) .",
"For sod-2 , day 1 expression was also reduced in sir-2 . 1 ( 0 ) , but this gene’s expression increased in both wild type and sir-2 . 1 ( 0 ) at day 2 , possibly a stress response .",
"For sod-3 and ctl-2 , their day 1 expression was similar in both strains; however at day 2 , sod-3 expression became higher and ctl-2 expression became lower in mutants .",
"Finally , sir-2 . 1 ( 0 ) males displayed an increased ctl-1 expression at day 1 , which is also reported in antioxidant-compromised daf-16 ( 0 ) mutant .",
"The enhanced expression of ctl-1 is considered as an adaptive response ( Yanase et al . , 2002 ) .",
"These results indicate that in addition to a potentially altered metabolism , which could generate excessive ROS , sir-2 . 1 ( 0 ) males might also have a comprised antioxidant response , which is consistent with their hypersensitivity to excessive glucose intake ( Figure 6G ) and to the ROS generator ( Figure 4A ) . 10 . 7554/eLife . 01730 . 018Figure 7 . sir-2 . 1 ( 0 ) males have compromised expression of anti-oxidant genes . Relative mRNA expression level of anti-oxidant genes superoxide dismutase ( A ) , catalase ( B ) , and glutathione transferase ( C ) in 1 , 2-day-old wild type and sir-2 . 1 ( 0 ) males ( unpaired t-test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 018 Based on the above results , one could hypothesize that increasing SIR-2 . 1 expression or activity might delay mating deterioration during aging .",
"However , we found that transgenic overexpression of sir-2 . 1 does not improve the mating potency of 3-day-old wild type ( Figure 8A ) .",
"It is unlikely that the fusion with YFP disrupts SIR-2 . 1 function , because the same transgene can rescue the sir-2 . 1 ( 0 ) phenotype .",
"Thus , we speculate that up to a point , the expression level of sir-2 . 1 is not rate limiting for SIR-2 . 1 activity during early aging .",
"However , one could also speculate that the normal endogenous levels of NAD+ limit the function of SIR-2 . 1 .",
"To test this , we grew males in the presence of the NAD+ precursor nicotinamide ( NAM ) at 200 μM concentration ( Houtkooper et al . , 2010 ) , and then conducted the mating potency .",
"Indeed , NAM exposure significantly improves 3-day-old wild-type mating potency , but not 2-day-old sir-2 . 1 ( 0 ) males ( Figure 8B , C ) .",
"This result is consistent with the idea that excess NAD+ might stimulate SIR-2 . 1 activity .",
"But additionally , excess NAD+ might also reduce ROS production by relaxing the demand of oxidizing NADH back to NAD+; as a corollary to this possibility , the lack of excess NAM to positively affect the sir-2 . 1 ( 0 ) male’s behavior might be aggravated by the abnormally high expression of catabolic enzymes in the mutant males .",
"To further test if overexpressing SIR-2 . 1 activity can promote mating behavior in older males , we exposed 3-day-old transgenic SIR-2 . 1 over-expressed males with exogenous NAM , but found that excess SIR-2 . 1 does not amplify the positive effect of NAM ( Figure 8D ) .",
"Thus , we cannot exclude the possibility that NAM or possibly NAD+ additionally promotes behavioral extension through mechanisms parallel to SIR-2 . 1 activity . 10 . 7554/eLife . 01730 . 019Figure 8 . Exogenous nicotinamide improves mating during aging . sir-2 . 1 overexpression cannot increase mating potency of 3-day-old wild type ( A ) .",
"However , feeding with a NAD+ precursor nicotinamide ( NAM ) significantly improve the mating potency of 3-day-old wild type ( B ) but not 2-day-old sir-2 . 1 ( 0 ) males ( C ) .",
"Overexpression of sir-2 . 1 cannot further promote the effect of exogenous NAM ( D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 019"
],
[
"Behavioral decline at advanced age can be attributed to morphological degeneration , such as neuronal death and muscle sarcopenia ( Herndon et al . , 2002; Glenn et al . , 2004 ) .",
"However , during early aging , prior to any significant cellular decay , physiological changes that affect neuronal and muscular functionality can contribute to the reduction of behavioral coordination ( Salthouse , 2004 ) .",
"Male mating in C . elegans is a complex behavior that requires coordination of multiple motor systems to impregnate the hermaphrodite ( Liu et al . , 2011; Correa et al . , 2012 ) .",
"Previously , we showed that male mating behavior declines significantly during early aging .",
"Wild type cannot mate well at adulthood day 3 , and this decline is correlated with an increased excitability in the sex circuitry ( Guo et al . , 2012 ) .",
"Similar to aging males , a recent study using hermaphrodites showed that synaptic transmission in the locomotion circuit is enhanced after 5 days of adulthood ( Mulcahy et al . , 2013 ) .",
"Thus , it is possible that during early aging , the neuromuscular systems of both sexes become hyperexcitable .",
"However , the faster decline in mating suggests that the male reproductive circuitry is more sensitive to age-related physiological changes .",
"We found that SIR-2 . 1 is a modulator of behavior and is required to maintain mating in aging males .",
"1-day-old sir-2 . 1 ( 0 ) males can mate similarly to wild type , suggesting that sir-2 . 1 is not essential for mating .",
"However , unlike wild-type males , the mating ability of sir-2 . 1 ( 0 ) prematurely drops at day",
"2 . This is due to hyperexcitability of the reproductive circuitry that coordinates spicule intromission and ejaculation .",
"The hyperexcitability of the spicule muscles causes the male proctodeum to block the connection between the vas deferens and the cloacal opening , which indirectly obstructs the transfer of sperm .",
"The mutant phenotype resembles the behavioral , physiological , and pharmacological changes that occur in older wild-type males .",
"This indicates that in wild type , SIR-2 . 1 maintains the functional excitability of the intromission and ejaculation circuit , possibly by slowing down the deteriorative events that accumulate during aging .",
"We suggest that as the male ages , SIR-2 . 1 regulates the amounts of catabolic , anabolic , and free radical scavenging enzymes to balance the energy demands needed for rapid reproductive motor responses , with the generation of damaging metabolic by-products , such as ROS .",
"Our data indicate that the male’s cellular physiology is correlated with his ability to mate .",
"We showed previously and in this study that perturbation of the male’s physiology through genetic mutation or through dietary alterations during early adulthood will affect his ability to mate later in life .",
"The physiology of wild-type males is likely changing from day 1 to day 2 , as determined by the level of mRNAs encoding metabolic enzymes and the amount of terminal metabolic products .",
"These physiological changes can ultimately lead to excessive carbon flow into the TCA cycle , and consequently , more NADH to be oxidized by ETC complexes ( Figures 6A and 9 ) .",
"This promotes ROS generation via electron leak ( Federico et al . , 2012 ) , which can be reflected by behavioral decay at day",
"3 . Analysis of sir-2 . 1 ( 0 ) males allowed us to extrapolate how this protein deacetylase regulates male physiology .",
"Lack of SIR-2 . 1 will induce these deleterious changes to occur sooner , and degenerative behavioral responses in the mutant males are measured at day",
"2 . Under standard laboratory conditions , wild-type males are raised constitutively on abundant E . coli until senescence .",
"We speculate that since SIR-2 . 1 uses NAD+ as a cofactor to deacetylate proteins , the physiological changes that occur after 2 days of constitutive feeding in wild type adults might be due to reduction in SIR-2 . 1 function , via the lower ratio of NAD+ to NADH in wild type .",
"The phenomenon of altering NAD+ to NADH levels in vertebrate cells is shown to reduce the activity of SIRT1 ( Braidy et al . , 2011 ) .",
"Decreased activity of SIR-2 . 1 will not only aggravate a bias towards catabolism , but will also decrease the levels of ROS scavengers .",
"This is consistent with a hermaphrodite study , which showed that sir-2 . 1 overexpression can protect the organism from ROS , possibly via HCF-1 and FOXO/DAF-16 , to regulate the expression of stress response genes ( Rizki et al . , 2011 ) . 10 . 7554/eLife . 01730 . 020Figure 9 . A cartoon of the metabolism and behavior that occurs in wild-type and sir-2 . 1 ( 0 ) males during early aging . For successful reproductive behavior , SIR-2 . 1 is required to maintain proper carbon flow to meet the male’s energy demands and balance the generation of ROS .",
"In 1-day-old old sir-2 . 1 ( 0 ) males , catabolism such as glycolysis and fatty acid oxidation is enhanced , and consequently , oxidative phosphorylation and generation of ROS are also increased .",
"Without SIR-2 . 1 , ROS accumulation by day 2 of adulthood can lead to hyperexcitability of the male’s genital neuromuscular circuitry .",
"This results in blocked ejaculation and impotency .",
"It is possible that in 2- to 3-day-old wild-type males , the NAD+-dependent SIR-2 . 1 activity declines due to a lower ratio of NAD to NADH; thus older wild-type males might have a similar physiology as 1-day-old sir-2 . 1 ( 0 ) males . DOI: http://dx . doi . org/10 . 7554/eLife . 01730 . 020 Although our qPCR analyses suggest that enhanced catabolism might be occurring in 1-day-old of sir-2 . 1 ( 0 ) and 2-day-old wild-type males , their mating ability could be facilitated via anabolic compensatory mechanisms .",
"In addition to enhanced expression of catabolic genes , mRNAs encoding enzymes such as pyruvate carboxylase and phosphoenolpyruvate carboxykinase ( PEPCK ) appeared to be also up-regulated .",
"This could be a likely reason for why the males contain more measurable lipids and glycogen .",
"We propose that the up-regulation of anabolic process is a self-compensatory mechanism to divert carbon from the TCA cycle ( Figure 9 ) .",
"sir-2 . 1 ( 0 ) males that are mutant for PEPCK genes , lose their ability to generate fat and fail to mate efficiently on day 1 .",
"Likewise sir-2 . 1 ( + ) males with a mutation in PEPCK genes also display premature mating decline .",
"We hypothesize that anabolic pathways could act as a homeostatic mechanism to reduce ROS production .",
"This idea raises the possibility that obesity as a phenotype might be a compensatory mechanism to alleviate the effects of other underlying metabolic dysfunctions .",
"Indeed , by switching of glycolysis to gluconeogenesis has been shown to be a potential strategy to cure hepatocarcinoma ( Ma et al . , 2013 ) .",
"A recent study showed that diabetic patients , who are not obese , have higher mortality than overweight ones ( Carnethon et al . , 2012 ) .",
"Another study showed that a lifestyle intervention focusing on weight loss did not reduce the rate of cardiovascular events in obese adults with type II diabetes ( Look AHEAD Research Group , 2013 ) , challenging the traditional viewpoint that obesity is a major contributor to metabolic disorders .",
"The free radical aging theory states that aging is the result of free radical-induced molecular damages ( Harman , 1956 ) .",
"Although this notion is challenged by the hormesis theory , which posits that moderate amounts of physiological or environmental insults can reinforce cellular processes that reduce stress-induced damage ( Afanas’ev , 2010; Schulz et al . , 2007 ) , artificially applied ROS is reported to change the excitability of cultured neurons and muscles through chemically damaging ion channels ( Danson and Paterson , 2006 ) .",
"Therefore , aspects of the free radical theory of aging might still apply to physiological changes in neural muscular systems and behavioral decay .",
"Consistent with this , we showed here that genetic and environmental conditions , which can lead to oxidative stress , caused increased excitability of the male mating circuitry and behavioral decay during early aging .",
"There is not a strict correlation between oxidative stress and changes in the electrical properties of neurons and muscles .",
"Oxidation of different types of ion channels changes their conductance and alters the cell’s excitability ( Annunziato et al . , 2002 ) .",
"One C . elegans study indicates that oxidative stress reduces cell excitability by increasing the conductance of K+ channels ( Sesti et al . , 2010 ) .",
"Another mammalian study showed that oxidative stress hyperpolarizes the resting potential , but extends the duration of the action potential in cardiac ganglion ( Whyte et al . , 2009 ) .",
"Mitochondria ROS has been shown to trigger Ca2+ increases in the pulmonary arterial myocytes ( Waypa et al . , 2002 , 2006 ) .",
"In a study using glia , L-type voltage-gated Ca2+ channels ( L-VGCC ) were found to be a target of ROS .",
"After modification by ROS , their conductance of Ca2+ was increased ( Bond and Greenfield , 2007 ) .",
"Different from vertebrate skeletal muscles , L-VGCC in C . elegans propagates action potentials , and the entry of external Ca2+ directly promotes excitation–contraction coupling ( Lee et al . , 1997; Maryon et al . , 1998 ) .",
"Previous work showed that L-VGCC EGL-19 in C . elegans is required for sustained tonic contraction of the copulatory spicule muscles ( Garcia et al . , 2001 ) .",
"Therefore , oxidation of L-VGCC in C . elegans might contribute to the increased excitability of mating circuits .",
"Other major targets of ROS are voltage-gated K+ channels .",
"In C . elegans , oxidation of KVS-1 slows down its inactivation , leading to hyperpolarization and sensory function loss ( Cai and Sesti , 2009 ) .",
"The ERG-like K+ channel UNC-103 is a major excitability regulator of the sex circuit ( Reiner et al . , 2006 ) .",
"Although there is no report of oxidative modification on UNC-103 , human encoded H-ERG channels can be activated by oxidative stress , so that cells become hyperpolarized and less excitable ( Cui and Zhang , 2013 ) .",
"Considering that ROS increases the excitability of the mating circuit , it is possible that L-VGCC is more prone to be oxidized than K+ channels in male reproductive cells ."
],
[
"Worms were grown at 20°C on nematode growth media ( NGM ) plates seeded with E . coli strain OP50 , except for strains containing the pha-1 ( e2123 ) , which were maintained at 15°C .",
"The alleles used in this work included: lite-1 ( ce314 ) on LGX; pck-2 ( ok2586 ) on LGI; pck-1 ( ok2098 ) , pha-1 ( e2123 ) ( Schnabel and Schnabel , 1990 ) , unc-64 ( e240 ) ( Brenner , 1974 ) on LGIII; sir-2 . 1 ( ok434 ) on LGIV; and him-5 ( e1490 ) ( Hodgkin et al . , 1979 ) on LGV .",
"The males were generated by the him-5 ( e1490 ) mutation .",
"Males containing only this mutation are referred to as wild-type; him-5 ( e1490 ) males have been shown to mate efficiently as wild type ( Hodgkin , 1983 ) .",
"pck-2 ( ok2586 ) , pck-1 ( ok2098 ) , and sir-2 . 1 ( ok434 ) were generated by the C . elegans Gene Knockout Consortium .",
"sir-2 . 1 ( ok434 ) animals were out-crossed 4 times with the him-5 ( e1490 ) strain .",
"The deletion in sir-2 . 1 ( 0 ) was detected through PCR using primers listed in the Supplementary file 2 .",
"Altered mediums used here included NGM medium containing glucose ( 2% ) , paraquat , N-acetyl-cystine ( NAC ) ( Sigma , St . Louis , MO ) , and nicotinamide ( NAM ) ( Sigma ) respectively .",
"The latter three were added at the indicated concentration just before pouring the plates .",
"OP50 used for the special medium containing glucose and NAC was UV-killed and concentrated to make sure the worms were not food deprived .",
"We assume that the animals ingest these compounds as they feed on E . coli or absorb them through their cuticle .",
"DNA primers are listed in the Supplementary file 2 .",
"The sir-2 . 1 genomic sequence , plus 2 kb upstream of its ATG , was PCR-amplified from N2 DNA .",
"The PCR product was digested and ligated between the SphI and SalI sites of pSX322YFP to obtain the plasmid pXG5 .",
"To obtain promoters for driving sir-2 . 1 expression , the sir-2 . 1 endogenous promoter was removed via PCR-mutagenesis from pXG5 to construct pXG6 .",
"The Gateway ATTR cassette frame A was inserted in front of the sir-2 . 1 genomic sequence to make the destination clone pXG7 .",
"Plasmids ( pXG8 , pXG9 , and pXG11 ) that promote neuronal , muscular , and intestinal expression of sir-2 . 1 were obtained through Gateway LR reactions between pXG7 and pLR35 ( Paex-3 ) ( LeBoeuf et al . , 2007 ) , pLR22 ( Plev-11 ) ( Gruninger et al . , 2008 ) and pBL50 ( Pges-1 ) ( Urano et al . , 2002 ) , respectively .",
"pXG5 ( 25 ng/μl ) , pXG8 ( 10 ng/μl ) , pXG9 ( 1 ng/μl ) or pXG11 ( 50 ng/μl ) were injected to sir-2 . 1 ( 0 ) hermaphrodites and transgenic animals were selected via YFP fluorescence .",
"pXG5 ( 50 ng/μl ) was injected into wild-type hermaphrodites to obtain strains with overexpression of sir-2 . 1 ( referred as sir-2 . 1 ( OE ) ) .",
"The mating potency assay was performed as described in Guo et al . ( 2012 ) .",
"Briefly , L4 males were isolated from hermaphrodites .",
"One male and one pha-1 ( e2123 ) hermaphrodite were co-transferred to a 5-mm-diameter OP50 mating lawn at 20°C , which is a restrictive temperature for pha-1 ( e2123 ) to sire viable self-progeny .",
"This allowed us to score the mating potency of males , as only cross-progenies would develop to adulthood .",
"To quantify different parameters of mating , we observed , up to 5 min , copulations between males and paralyzed unc-64 ( e240 ) hermaphrodites .",
"The ability of males to sense the vulva was calculated by counting the number of times he stopped at the vulva divided by the total number of times he stopped and/or passed by the vulva .",
"The turning quality was calculated as: the number of smooth turns ( defined as the male tail keeping contact with hermaphrodite and turning without hesitation ) divided by the total number of turns .",
"Ejaculation assays were conducted in two ways .",
"We directly observed sperm transfer after spicule insertion , and determined if sperm drained into the unc-64 ( e240 ) hermaphrodite’s uterus .",
"Additionally , cross-progeny were counted 1–2 days after spicules insertion .",
"Lifespan assay was conducted as described in Guo et al . ( 2012 ) .",
"L4 males were isolated and raised 20 per plate .",
"The males that can respond to gentle touch with a platinum wire were counted and transferred to new plates every day .",
"Males that dried on the wall of the Petri plate were censored from the assay on the day they died .",
"To assess the sensitivity to paraquat , L4 males were transferred to plates containing 10 mM paraquat and scored at 24 and 48 hr .",
"Sperm activation assays were done according to L’Hernault and Roberts ( 1995 ) ; Smith and Stanfield ( 2011 ) .",
"Briefly , three 2-day-old males , isolated at L4 stage , were cut at the posterior portion with a needle in 20 μl sperm media ( 50 mM Hepes , pH7 . 0 , 45 mM NaCl , 25 mM KCl , 1 mM MgSO4 , and 5 mM CaCl2 ) freshly supplemented with polyvinylprolidone ( PVP ) 40 , 000 molecular weight ( Sigma ) and the activator pronase ( Roche , Indianapolis , IN ) at the final concentration of 10 mg/ml and 500 μg/ml on a slide .",
"A coverslip with a thin layer of Vaseline applied around the edge was put on the top of the sperm media to form a chamber over the sperm .",
"After 5-min incubation , activated sperm with a pseudopod and inactive ones were counted using a compound scope fitted with a 100X objective .",
"Approximately , 50–60 sperm cells were counted in each sample section .",
"Arecoline ( ARE ) and levamisole ( LEV ) were dissolved in water at the concentration of 100 mM , and then diluted accordingly , as described in Liu et al . ( 2007 ) .",
"Briefly , 50 μM of ARE , 1 μM and 500 nM of LEV for 1 , 2-day-old males , and 100 nM LEV for 3-day-old males were used .",
"Males were introduced to 1 ml drug baths and scored if they protruded their spicules for more than 5 s within 5 min of drug exposure .",
"Ca2+ imaging was conducted and analyzed as described in Guo et al . ( 2012 ) .",
"50 ng/μl pLR289[Punc-103E:G-CaMP3::sl2:::DsRed] ( Correa et al . , 2012 ) and 50 ng/μl pBX1 ( Granato et al . , 1994 ) were co-injected into pha-1 ( e2123 ) ; him-5 ( e1490 ) ; lite-1 ( ce314 ) hermaphrodites to generate the extrachromosomal array rgEx566[Punc-103E:G-CaMP3::sl2:::DsRed] .",
"rgEx566 was crossed into pha-1 ( e2123 ) ; him-5 ( e1490 ) ; sir-2 . 1 ( 0 ) ; lite-1 ( ce314 ) hermaphrodites .",
"Fluorescence signals from G-CaMP3 and DsRed were recorded simultaneously during the copulations of 2-day-old wild-type and sir-2 . 1 ( 0 ) males with paralyzed hermaphrodites containing the transgene rgEx431[Phsp-16:egl-2 ( gf ) ; Punc-103E:DsRed] .",
"300 day 1 and day 2 adult males were accumulated over a period of time .",
"RNA was extracted by Trizol , and cDNA was synthesized by SuperScript II ( Life technology , Grand Island , NY ) using around 2 μg total RNA , as described in LeBoeuf and Garcia ( 2012 ) .",
"The RT-qPCR reactions were performed using BIO-RAD CFX96 real-time system and SsoFast EvaGreen supermix .",
"11 candidate reference genes were tested to see whether their expression changed from day 1 and day 2 in both wild-type and sir-2 . 1 males ( Hoogewijs et al . , 2008 ) ; from our analyses , act-1 and gpd-2 were selected as the reference to normalize the expression of the metabolic genes .",
"Many of the primers used to detect the expression of metabolic enzymes are described in Castelein et al . ( 2008 ) .",
"Other primers for additional metabolic and antioxidant stress genes are listed in the Supplementary file 2 .",
"Three replicates were conducted on the same RNA samples .",
"We used the t-test to determine which mRNA transcripts in sir-2 . 1 ( 0 ) males were significantly different from their cognate wild-type transcripts .",
"To measure ATP and glucose , 100 males were collected at different ages , frozen and thawed three times .",
"The worms were homogenized , and the supernatant was collected and measured using an ATP determination Kit ( Life technology ) and the Glucose Oxidase Assay Kit ( Life technology ) .",
"The ATP and glucose were normalized to the amount of dsDNA quantified by picoGreen ( Life technology ) .",
"To stain glycogen , 1-day-old sir-2 . 1 ( 0 ) and wild-type virgin males were transferred to 2% agar pads .",
"The pads containing both genotypes were then placed over a bottle of iodine crystals for 30 s ( Frazier and Roth , 2009 ) .",
"The pictures were taken by a Leica compound scope mounted with OLYMPUS DP70 camera .",
"The RGB images were then converted to 16-bit gray scale , and the mean gray levels of the isthmus regions were measured using the SimplePCI image quantification software ( Hamamatsu , Janpan ) .",
"The mean gray level was reversely correlated with the red signal .",
"Oil Red O staining was done according to O’Rourke et al . ( 2009 ) .",
"Briefly , males were collected and washed with PBS , and then fixed with Modified Ruvkuns witches brew ( MRWB ) buffer containing 1% paraformaldehyde ( PFA ) for 1 hr .",
"Worms were then washed with PBS and suspended in 60% isopropanol for 15 min at room temperature .",
"The 60% isopropanol was removed and worms were bathed in 60% Red Oil O staining solution .",
"The RGB images were taken by a Leica compound scope mounted with OLYMPUS DP70 camera .",
"The images were quantified by ImageJ according to Mehlem et al . ( 2013 ) ."
]
] | [
"The decline of aging C . elegans male’s mating behavior is correlated with the increased excitability of the cholinergic circuitry that executes copulation .",
"In this study , we show that the mating circuits’ functional durability depends on the metabolic regulator SIR-2 . 1 , a NAD+-dependent histone deacetylase .",
"Aging sir-2 . 1 ( 0 ) males display accelerated mating behavior decline due to premature hyperexcitability of cholinergic circuits used for intromission and ejaculation .",
"In sir-2 . 1 ( 0 ) males , the hypercontraction of the spicule-associated muscles pinch the vas deferens opening , thus blocking sperm release .",
"The hyperexcitability is aggravated by reactive oxygen species ( ROS ) .",
"Our genetic , pharmacological , and behavioral analyses suggest that in sir-2 . 1 ( 0 ) and older wild-type males , enhanced catabolic enzymes expression , coupled with the reduced expression of ROS-scavengers contribute to the behavioral decline .",
"However , as a compensatory response to reduce altered catabolism/ROS production , anabolic enzymes expression levels are also increased , resulting in higher gluconeogenesis and lipid synthesis ."
] | [
"Although the signs of aging are clear to us all , precisely why we age is less well understood .",
"One possibility is that as cells use oxygen to fuel the breakdown of large molecules into smaller ones to release energy , they also generate by-products called reactive oxygen species that can damage DNA .",
"As we get older , this damage gets worse .",
"Consistent with this idea , it has been shown that a reduced calorie intake can reduce oxidative damage in certain species , in addition to extending lifespan .",
"Many experiments on aging have been performed on worms belonging to the species C . elegans .",
"Male worms of this species live for an average of 11–12 days , but begin to show signs of aging—for example , a reduced ability to mate—as early as day 3 of their adult lives .",
"Now , Guo and García have revealed that a protein called SIR-2 . 1 , which regulates metabolism in worms , also helps to protect the animals from the effects of aging .",
"Male worms in which the gene for this protein has been ‘knocked out’ have a normal lifespan , but show signs of aging earlier than normal males .",
"They are also more susceptible to the damaging effects of reactive oxygen species , suggesting that SIR-2 . 1 may offer protection against oxidative damage .",
"Indeed , levels of ATP—the molecule used to move energy around inside cells—are increased in knockout worms .",
"This suggests that certain metabolic processes and the production of reactive oxygen species , are increased in the knockout worms , which speeds up the aging process .",
"While the link between metabolism and aging is well known , the work of Guo and García offers insights into some of the molecular mechanisms that may form the basis of this relationship ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Ki-67 is a PP1-interacting protein that organises the mitotic chromosome periphery | elife-01641-v1 | [
[
"When mitotic chromosomes are examined by whole mount microscopy the surface chromatin is obscured by a layer of proteins and RNA derived from the dense fibrillar component ( DFC ) and granular component ( GC ) of the nucleolus ( Moyne and Garrido , 1976; Harrison et al . , 1982; Adolph and Kreisman , 1983; Sumner , 1991; Gautier et al . , 1992b; Wanner et al . , 2005 ) .",
"This perichromosomal layer includes pre-rRNA , U3 snoRNAs , and over 20 ribosomal proteins , including nucleolin and Nopp140 , NPM/B23 , Bop1 , Nop52 , PM-Scl 100 , and Ki-67 ( Gautier et al . , 1992a , 1994; Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005; Fomproix et al . , 1998; Angelier et al . , 2005 ) .",
"The perichromosomal layer represents 1 . 4% of the chromosome proteome ( Ohta et al . , 2010 ) .",
"At present , its functional significance remains unstudied ( Van Hooser et al . , 2005 ) .",
"Ki-67 is one of the earliest proteins to bind the perichromosomal layer in mitosis .",
"Ki-67 is an antigen recognised by a monoclonal antibody generated by immunizing mice with nuclei of Hodgkin lymphoma cells ( Gerdes et al . , 1983 ) .",
"Because Ki-67 is nuclear only in proliferating cells , it is widely used as a marker to assess cell proliferation .",
"For example , immuno-histochemical assessment of the proportion of cells staining for nuclear Ki-67 is used to predict the responsiveness or resistance of tumours to therapy ( Dowsett et al . , 2011 ) .",
"Ki-67 protein is predominantly localised in the cortex and dense fibrillar components of the nucleolus during interphase ( Kill , 1996; MacCallum and Hall , 2000b ) .",
"During mitosis it relocates to the periphery of the condensed chromosomes ( Verheijen et al . , 1989b; Isola et al . , 1990 ) .",
"It has been reported that Ki-67 is associated with the nuclear matrix ( Verheijen et al . , 1989b ) , preribosomes ( Isola et al . , 1990 ) , satellite DNA in G1 ( Bridger et al . , 1998 ) , and with the chromosome scaffold of mitotic cells ( Verheijen et al . , 1989a ) .",
"Approximately 40% of the cellular pool of protein is present on isolated mitotic chromosomes ( Ohta et al . , 2010 ) .",
"Previous studies suggested that Ki-67 is regulated by phosphorylation .",
"Hyperphosphorylated Ki-67 does not bind DNA during mitosis ( Endl and Gerdes , 2000; MacCallum and Hall , 1999 ) and is characterized by increased mobility , on mitotic chromosomes measured by FRAP .",
"In interphase , non-phosphorylated Ki-67 binds DNA and does not recover after FRAP ( Saiwaki et al . , 2005 ) .",
"Its localisation and cell-cycle behaviour suggest that Ki-67 could be involved in the organisation of nucleolar chromatin during interphase in proliferating cells .",
"Recently , it was reported that Ki-67 also functions in mitosis in stabilisation and maintenance of the mitotic spindle by recruiting Hklp2 to mitotic chromosomes ( Vanneste et al . , 2009 ) .",
"In this study , we have identified Ki-67 as ancestral to the PP1 targeting subunit Repo-Man ( Trinkle-Mulcahy et al . , 2006 ) and have shown that Ki-67 is a PIP ( PP1 Interacting Protein ) that contributes to the phospho-regulation of nucleophosmin/B23 by CKII .",
"Ki-67 is also a major organiser of the perichromosomal layer , possibly acting as an interaction platform .",
"Remarkably , Ki-67 depletion prevents all nucleolar proteins tested from accumulating around the chromosomes in mitosis .",
"Thus , chromosomes in cells depleted of Ki-67 appear to lack most or all of their perichromosomal layer .",
"This enabled us to gain insights into the function of this enigmatic chromosomal compartment .",
"Interestingly , loss of the perichromosomal layer does not detectibly compromise the condensed morphology or intrinsic structure of mitotic chromosomes but does result in significant changes in nucleolar morphology and nuclear organization in the following interphase ."
],
[
"We used a phylogenetic approach to identify putative functional regions within the sequence of Repo-Man , a targeting protein that binds PP1 in a cell-cycle specific manner regulated by a phospho-switch ( Trinkle-Mulcahy et al . , 2006; Qian et al . , 2011; Vagnarelli et al . , 2011 ) .",
"A BLAST search revealed significant ( E = 5 × 10−4 ) similarity between a small region ( amino acids 388–420 ) of human Repo-Man and Ki-67 ( Figure 1A1 , 2 ) , a very large protein that exhibits strong links to cell proliferation ( Gerdes et al . , 1983 ) .",
"The region conserved between Repo-Man and Ki-67 contains the PP1 binding motif ( RVTF ) of Repo-Man , which is conserved as RVSF in human Ki-67 ( Figure 1C3 ) . 10 . 7554/eLife . 01641 . 003Figure 1 . Ki-67 is evolutionary related to Repo-Man but shows distinct behaviour during mitosis .",
"( A1 )",
"Schematic representations of evolutionarily conserved regions in human Repo-Man and Ki-67 proteins ( shown approximately to scale ) .",
"( A2 )",
"E-values corresponding to global profile-to-sequence ( HMMer3 ) comparisons between the PP1 binding conserved regions ( blue oval ) in Repo-Man and Ki-67 families .",
"Arrows indicate the profile search direction .",
"The Repo-Man profile identified Ki-67 proteins as homologous with a highly significant E-value of 4 . 9 × 10−11 .",
"Conversely , the profile of Ki-67 homologous sequences in animals identified Repo-Man proteins with a significant E-value of 9 . 9 × 10−13 .",
"( A3 )",
"Representative multiple sequence alignment of conserved regions from Repo-Man and Ki-67 families .",
"Important mitotic phospho-residues in Repo-Man ( T412 and T419 ) are indicated in blue .",
"The RVTF motif is indicated with an orange box .",
"The most parsimonious explanation of Repo-Man and Ki-67 evolution is shown to the left of the alignment .",
"Vertebrate branches are coloured in blue .",
"Sequences are named according to: Repo_Human , NCBI:NP_689775 , Homo sapiens; Repo_Frog , NCBI:ACR33033 , Xenopus laevis; Repo_Danre , UniProt:A2CEF0 , Danio rerio; Ki_Human , UniProt:P46013 , Homo sapiens; Ki_Frog , UniProt:Q0VA85 , Xenopus laevis; Ki_Fugu , UniProt:UPI00016EA029 , Takifugu rubripes; Ki_Cioin , UniProt:UPI000180CFDA , Ciona intestinalis; Ki_Sacko , Baylor College of Medicine genome and FGENESH+ , Saccoglossus kowalevskii; Ki_Hydra , UniProt:UPI0001926DD5 , Hydra magnipapillata; and , Ki_Triad , UniProt:B3SB24 , Trichoplax adhaerens .",
"The amino acid colouring scheme indicates average BLOSUM62 scores ( which are correlated with amino acid conservation ) for each alignment column: red ( greater than 3 . 5 ) , violet ( between 3 . 5 and 2 . 5 ) , and light yellow ( between 2 . 5 and 0 . 5 ) .",
"( B–C )",
"Ki-67 and PP1γ interact in vivo .",
"Ki-67301–700 fused to Lac repressor:GFP ( Lac repressor:Ki-67PP1BD_wt ) was transfected together with RFP:PP1γ into a DT40 cell line containing a LacO array integrated on a single chromosome .",
"Ki-67 was enriched at the LacO site ( panels 2 , 2′ ) where it recruited PP1γ ( 2 , 2″ ) .",
"However , neither Lac repressor:GFP ( panels 1–1″ ) or the Ki-67 PP1-non-binding mutant ( Lac repressor:Ki-67PP1BD_RASA ) ( panels 3–3″ ) caused PP1 accumulation at the LacO site .",
"( D ) Ki-67 recruits PP1γ more efficiently than PP1beta and more efficiently in interphase than in mitosis .",
"The experimental set up was as in ( B ) .",
"The enrichment of PP1 signal at the Lac repressor spot was compared to the background nuclear ( interphase ) or cytoplasmic ( mitosis ) signal within the same cell .",
"Scale bar 10 μm .",
"( E ) Direct and indirect interactors of Ki-67 .",
"( F ) The B23S125 phosphorylation level remains high in Ki-67 depleted mitotic cells .",
"Immunoblots of whole cell extracts of cycling ( interphase ) of Nocodazole-arrested ( mitotic ) HeLa cells transfected with Ki-67 RNAi oligo 5 or control oligos , were probed for Ki-67 , tubulin , B23T199ph , and B23S125ph .",
"Two exposures of the B23S125ph blot are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00310 . 7554/eLife . 01641 . 004Figure 1—figure supplement 1 . Characterisation of Ki-67 RNAi . Two siRNAs against Ki-67 deplete the protein efficiently in HeLa cells .",
"Top and middle panels show two different exposures of the same blot . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00410 . 7554/eLife . 01641 . 005Figure 1—figure supplement 2 . Distribution of PP1gamma in mitosis after the Ki-67 siRNA . In cells depleted of Ki-67 , PP1 is still recruited to the nucleolus in interphase ( panels 4–4′ ) and to kinetochores in metaphase ( panels 5–5′ ) but its levels are lower on anaphase chromosomes ( panels 6–6′ ) .",
"HeLa cells were transfected with RFP:PP1γ ( green ) and with oligo 5 ( panels 4–6 ) or control oligo ( panels 1–3 ) .",
"Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 005 The similarity between Repo-Man and Ki-67 within the PP1 binding domain suggested that Ki-67 could be a PP1-interacting protein ( PIP ) .",
"Indeed , a possible connection between Ki-67 and PP1 was previously identified in two large scale screens for PP1 interactors .",
"In one , in silico-screening based on a stringent definition of the RVxF motif ( the PP1 binding motif ) identified Ki-67 as putative interactor , however no in vivo interaction was demonstrated ( Hendrickx et al . , 2009 ) .",
"Another study identified Ki-67 in a displacement affinity chromatography analysis of PP1 nuclear interactors ( Moorhead et al . , 2008 ) .",
"To analyse if Ki-67 was a cell-cycle regulated PIP in vivo , we performed a tethering/recruitment experiment ( Vagnarelli et al . , 2011 ) .",
"Ki-67301–700 wild type ( wt ) and a PP1 non-binding RASA mutant were fused to GFP:Lac repressor ( Figure 1B ) and co-expressed with RFP:PP1γ or β in DT40 cells carrying a lacO array integrated on a single chromosome ( Vagnarelli et al . , 2011 ) .",
"GFP:Lac repressor on its own was used as a control in this experiment .",
"All GFP:Lac repressor fusion constructs accumulated at the LacO integration site ( Figure 1C1′–3′ ) .",
"Furthermore , the Ki-67 wt construct recruited PP1 to the same spot ( Figure 1C2″ ) .",
"Quantification of the PP1 signal intensity at the GFP:Lac repressor spot compared to the general nuclear distribution ( excluding nucleoli ) revealed that Ki-67 recruitment of PP1 is more efficient in interphase than in mitosis and that PP1γ is recruited more efficiently than PP1β during interphase ( Figure 1D ) .",
"These data suggest that Ki-67 is a PIP in vivo and the interaction with PP1γ is cell-cycle regulated .",
"In order to determine whether Ki-67 is required to target PP1γ to any of its known locations in vivo , we used RNAi to deplete Ki-67 in human cells and determined the effects that this had on the distribution of PP1γ .",
"A previous report described the successful depletion of Ki-67 ( Vanneste et al . , 2009 ) .",
"However , the target sequence recognised by the published siRNA lies within a repeated stretch and rescue experiments to validate the phenotype were not reported .",
"We therefore identified two new siRNAs that could deplete the protein as efficiently as the published siRNA ( Figure 1 , Figure 1—figure supplement 1 ) .",
"Both new siRNAs yielded comparable phenotypes and we used siRNA 5 ( Ki5 ) for the depletion experiments presented below .",
"This siRNA was validated in a rescue experiment that is discussed in a later section ( Figure 2B ) . 10 . 7554/eLife . 01641 . 006Figure 2 . Ki-67 is required for targeting of nucleolar proteins to the chromosome periphery in mitosis .",
"( A ) Localisation of endogenous nucleolin is aberrant in mitotic cells after Ki-67 depletion ( panels 2 , 3 , 5 ) .",
"HeLa cells were transfected with Ki-67 RNAi oligo 5 ( panels 2 , 3 ,",
"5 ) or control oligos ( panels 1 ,",
"4 ) and stained for nucleolin ( green ) .",
"Quantification of the phenotypes is indicated in the graph above the corresponding representative images .",
"Scale bar 5 μm .",
"( B ) RNAi rescue experiment .",
"HeLa cells depleted of Ki-67 were transfected with either mCherry:Ki-67wt , or mCherry:Ki-67RASA , together with Ki-67 RNAi oligo 5 or control oligo and stained for nucleolin .",
"The localisation of nucleolin in mitotic cells was quantified in the different experimental conditions .",
"See Figure 2—figure supplement 1 for representative images .",
"Scale bar 10 μm .",
"( C ) The mitotic chromosome peripheral localisation of NIFK is disrupted upon Ki-67 RNAi ( panels 5–6 ) .",
"HeLa cells were transfected with GFP:NIFK ( green ) and oligo 5 ( panels 5–8 ) or control oligo ( panels 1–4 ) .",
"Scale bar 10 μm .",
"( D ) All novel cPERPs tested failed to accumulate on the chromosome periphery in mitosis .",
"HeLa cells were co-transfected with GFP:cPERPs identified in an earlier study ( Ohta et al . , 2010 ) ( green ) and oligo 5 ( panels 3–4 , 7–8 , 11–12 , 15–16 ) or control oligo ( panels 1–2 , 5–6 , 9–10 , 13–14 ) : GFP:cPERP-B ( panels 1–4 ) , GFP:cPERP-C ( panels 5–8 ) , GFP:cPERP-D ( panels 9–12 ) , GFP:cPERP-F ( panels 13–16 ) .",
"Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00610 . 7554/eLife . 01641 . 007Figure 2—figure supplement 1 . Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides with and without rescue by Ki-67 cDNA . RNAi rescue experiment .",
"Representative microscopy images for quantifications shown in Figure 2B .",
"HeLa cells depleted of Ki-67 were transfected with either mCherry:Ki-67wt ( panels 2 ,",
"5 ) or mCherry:Ki-67RASA ( panels 3 ,",
"6 ) ( red ) together with Ki-67 RNAi oligo 5 ( panels 4 , 5 ,",
"6 ) or control oligo ( panels 1 , 2 ,",
"3 ) and stained for nucleolin ( green ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00710 . 7554/eLife . 01641 . 008Figure 2—figure supplement 2 . Distribution of nucleolin in mitosis following exposure of cells to different Ki-67 siRNA oligonucleotides . HeLa cells were transfected with Ki-67 RNAi oligo 1 , 2 or 5 or control oligos and stained for nucleolin .",
"Nucleolin localisation was classified as for Figure 2B ( diffuse , aberrant , and big foci ) and the graph represents the quantification of the phenotypes .",
"Scale bar 5 μm .",
"The three different oligos produce the same phenotype . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 00810 . 7554/eLife . 01641 . 009Figure 2—figure supplement 3 . Distribution of NIFK in mitosis following Ki-67 depletion . NIFK T234 phosphorylation is regulated normally in the presence and absence of Ki-67 .",
"Hela cells were transfected with Ki-67 RNAi oligo 5 ( panels 3 ,",
"4 ) or control oligos ( panels 1 ,",
"2 ) and stained with NIFK234ph antibody ( green ) .",
"Scale bar 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 009 Ki-67 depletion in a HeLa cell line has no effect on the accumulation of RFP:PP1γ in the nucleolus ( Figure 1 , Figure 1—figure supplement 2[1 , 4] ) .",
"Indeed , the targeting subunit for PP1 nucleolar localisation has been recently reported to be RRP1B ( Chamousset et al . , 2010 ) .",
"In early mitosis , PP1γ localised normally on the spindle and at kinetochores in both control and Ki-67 depleted cells ( Figure 1 , Figure 1—figure supplement 2[2 , 5] ) .",
"However , we observed a significant decrease in PP1 levels on anaphase chromatin in Ki-67 depleted cells ( Figure 1 , Figure 1—figure supplement 2[3′ , 6′] ) .",
"Previous reports identified Repo-Man and Sds22 as responsible for targeting PP1 to anaphase chromatin ( Trinkle-Mulcahy et al . , 2006; Wurzenberger et al . , 2013 ) .",
"Thus , Ki-67 is one of the several factors contributing to the accumulation of PP1γ on chromatin during mitotic exit .",
"Analysis of the phosphorylation status of several known direct and indirect Ki-67 interacting proteins ( Figure 1E ) in interphase and mitosis revealed that nucleophosmin/B23 phospho-regulation was dependent on Ki-67 .",
"B23 is phosphorylated both in interphase and in mitosis by several kinases ( Pfaff and Anderer , 1988; Jiang et al . , 2000; Louvet et al . , 2006; Krause and Hoffmann , 2010; Ramos-Echazabal et al . , 2012; Reboutier et al . , 2012 ) , including CyclinB/CDK1 at T199 ( Tokuyama et al . , 2001 ) in mitosis and by casein kinase II ( CKII ) on S125 during interphase ( Szebeni et al . , 2003 ) .",
"Use of phospho-specific antibodies revealed a reproducible difference in nucleophosmin/B23 phosphorylation on S125 in the presence and absence of Ki-67 exponential cultures and in prometaphase cells ( Figure 1F ) .",
"In both cases , the levels of S125ph were significantly increased following Ki-67 depletion .",
"This was particularly evident in prometaphase-arrested cells .",
"In contrast , we observed no significant difference in the phosphorylation status of B23 at T199 in the presence or absence of Ki-67 ( data not shown ) .",
"These experiments support the notion that Ki-67 is a functional PP1-targeting subunit in vivo .",
"Several aspects of mitotic chromosome structure remain relatively poorly understood , but amongst these , the perichromosomal compartment ( also known as the chromosome periphery ) stands out as a structure about which almost nothing is known .",
"This is remarkable , as an ever-increasing list of chromosome periphery proteins has been compiled over the years ( Chaly et al . , 1984; McKeon et al . , 1984; Gautier et al . , 1992b; Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005; Gassmann et al . , 2005; Ohta et al . , 2010 ) .",
"Some of these are among the most abundant proteins associated with mitotic chromosomes ( Ohta et al . , 2010 ) .",
"Despite their abundance , the mechanism of their localisation and the role that they play on the chromosomes , if any , is still not understood ( Hernandez-Verdun and Gautier , 1994; Van Hooser et al . , 2005 ) .",
"In a recent proteomic study , we identified a number of novel chromosome periphery proteins , which we termed cPERPs ( Ohta et al . , 2010 ) .",
"Ki-67 is one of many nucleolar proteins that are recruited to the chromosome periphery at the transition from prophase to prometaphase .",
"As Ki-67 is recruited to the perichromosomal compartment relatively early , we decided to examine whether its depletion affected the localisation of other known chromosome periphery proteins .",
"We first examined the localisation of the endogenous nucleolin using a specific antibody .",
"Indeed , nucleolin failed to accumulate at the chromosome periphery in the absence of Ki-67 ( Figure 2A ) .",
"The phenotype was observed using all of the siRNAs that we have tested and was rescued by an siRNA-resistant cDNA ( Figure 2B , Figure 2—figure supplements 1 , 2 ) .",
"To examine whether Ki-67 depletion affected the behaviour of other chromosome periphery proteins , we next examined the behaviour of NIFK , a known KI-67 interactor ( Takagi et al . , 2001 ) .",
"During normal mitotic exit , NIFK concentrates around the segregating chromosomes before associating with nucleolar-derived foci ( NDF–Dundr et al . , 1996; Figure 2C3 ) .",
"NDF formation varies between cell lines ( Dundr et al . , 2000 ) , but in the HeLa cells that we have used for RNAi studies , prominent NDFs are seldom observed during unperturbed mitosis ( Figure 2C1–4 ) .",
"To follow NIFK behaviour , we transiently expressed GFP-tagged hNIFK in HeLa cells after control or Ki-67 RNAi .",
"The nucleolar localisation of GFP-NIFK in interphase was unaltered after Ki-67 depletion ( Figure 2C8 ) , but in early mitosis the protein failed to properly accumulate around the mitotic chromosomes ( Figure 2C5–7 ) .",
"Instead , in metaphase cells , it accumulated in large cytoplasmic aggregates , one of which was frequently found to ‘cap’ one end of the cluster of chromosomes on the metaphase plate ( Figure 2C5 ) .",
"This extraordinary behaviour was also seen for endogenous NIFK phosphorylated on Thr234 using a phospho-specific antibody ( Figure 2—figure supplement 3[3′] ) .",
"During mitotic exit , in the absence of Ki-67 , GFP-NIFK accumulated in NDF-like cytoplasmic aggregates that persisted until the reformation of the nuclear membrane ( Figure 2C5–7 ) .",
"In view of the striking similarity of the behaviour of nucleolin and NIFK in the absence of Ki-67 , we tested the generality of this effect by localizing four novel cPERPs identified in our proteomics studies ( Ohta et al . , 2010 ) .",
"Remarkably , all were mislocalised in Ki-67-depleted cells , and all were frequently observed to ‘cap’ one end of the metaphase plate ( Figure 2D3 , 7 , 11 , 15 ) .",
"As in the case of nucleolin and NIFK , these aggregates dispersed into clusters of smaller cytoplasmic foci during mitotic exit ( Figure 2D4 , 8 , 12 , 16 ) .",
"We considered two hypotheses to explain the observed failure of nucleolar proteins to associate with the chromosome periphery in mitosis of Ki-67-depleted cells .",
"First , Ki-67 might be required for the complete disassembly of the nucleolus during mitotic entry .",
"In this case , the larger cytoplasmic aggregates observed during metaphase might be remnants of incompletely disassembled nucleoli .",
"Correlative light-microscopy/Electron microscopy ( CLEM ) analysis of cells transfected with both GFP-cPERP-C and the indicated siRNA oligos eliminated this hypothesis .",
"The GFP-containing aggregates observed in mitosis in Ki-67-depleted cells did not correspond to nucleolar remnants or any other recognisable electron-dense structures such as NDFs ( Dundr et al . , 2000; Figure 3A , Figure 3—figure supplement 1 ) . 10 . 7554/eLife . 01641 . 010Figure 3 . Ki-67 organizes the mitotic chromosome periphery .",
"( A ) CLEM of HeLa cells transfected with GFP-PerP-C and control oligos ( top panels ) or Ki-67 oligos ( bottom panels ) .",
"Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates ( arrow ) were identified and processed for CLEM using an adapted protocol ( Booth et al . , 2011 ) .",
"Appropriate light and electron micrographs , from the matching z position , were overlaid using Adobe Photoshop Elements .",
"Scale bar 5 μm .",
"( B ) EM of mitotic cells from Control ( top panels ) and Ki-67 RNAi ( bottom panels ) .",
"At higher magnification ( zoom",
"2 ) it is possible to note that the amorphous material surrounding the mitotic chromosomes in control cells ( arrow ) is substantially reduced around Ki-67 depleted chromosomes .",
"Scale bars ( left to right ) 4 , 2 , and 1 μm .",
"Far right panels show regions of interest for pixel density analysis ( Figure 3C ) .",
"( C ) Line scans of the peripheral regions of mitotic chromosomes in control ( blue line ) and Ki-67 depleted cells ( red line ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01010 . 7554/eLife . 01641 . 011Figure 3—figure supplement 1 . Depletion of Ki-67 does not leave electron-dense nucleolar remnants in the cytoplasm . CLEM of HeLa cells transfected with PERP-C and control oligos ( left panels ) or Ki-67 oligonucleotides ( right panels ) .",
"Mitotic cells from control or Ki-67 RNAi with visible GFP aggregates ( green arrow ) were identified and processed for CLEM using an adapted protocol ( Booth et al . , 2011 ) .",
"Panels from top to bottom show electron micrographs of consecutive serial sections , together with the appropriate fluorescence micrograph of the same z position .",
"No obvious electron-dense material can be seen colocalising with the fluorescent aggregates in the cytoplasm .",
"The white arrow shows regions of the metaphase plate where chromosomes appear abnormally closely clustered together after Ki-67 depletion .",
"Scale bar 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 011 A second hypothesis suggested that Ki-67 might function as a scaffolding platform for organising the perichromosomal compartment .",
"Indeed , all candidate proteins that we have tested to date have failed to localise to the chromosomal periphery in Ki-67-depleted cells .",
"Although analyses of individual proteins can provide part of the story , it is difficult or impossible to make conclusions about the compartment as a whole since the chromosome periphery has a complex and as-yet undefined protein and RNA composition .",
"Nonetheless , further analysis of our CLEM images strongly suggests that depletion of Ki-67 causes a loss of most or all of the perichromosomal compartment .",
"Examination of electron micrographs of control and Ki-67-depleted cells at higher magnification revealed a subtle difference in the appearance of the edge of the chromosomes .",
"In control cells there was a ‘fuzzy’ transition zone between the electron-dense chromatin and the cytoplasm ( Figure 3B ) .",
"This transition appeared to be more abrupt in Ki-67-depleted chromosomes .",
"This was confirmed by line scans across the chromosome-to-cytoplasm boundary , which showed that there is normally a gradual decrease in density at the edge of mitotic chromosomes .",
"This transition was significantly more abrupt in the absence of Ki-67 ( Figure 3C ) .",
"These experiments suggest that Ki-67 directs the binding of nucleolar components to the chromosome periphery , perhaps by acting as a scaffold .",
"Alternatively , the macromolecular network at the chromosomal periphery could be delicately balanced , such that removal of any single component causes the entire structure to fail .",
"However , depletion of cPERP-B , -D , or -E using RNAi ( Figure 4—figure supplement",
"1 ) failed to alter the striking perichromosomal localization of Ki-67 ( Figure 4 ) .",
"This is consistent with Ki-67 being upstream of the other components in the assembly of the perichromosomal compartment . 10 . 7554/eLife . 01641 . 012Figure 4 . Ki-67 localisation is not dependant on other c-PERPS tested .",
"( A ) Ki-67 localisation was analysed following the RNAi depletion of several novel c-PERPs ( PERP B , D , and E ) .",
"Following a 48 hr knock-down period , cells were fixed and labelled with anti-Ki67 ( green ) antibody and Dapi ( blue ) .",
"Examples shown include metaphase and anaphase cells .",
"Scale bar 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01210 . 7554/eLife . 01641 . 013Figure 4—figure supplement 1 . Depletion of cPERPs by RNAi . As no antibodies are available yet targeting these novel cPERPs , HeLa cells were transfected with GFP:PERP cDNA with or without the co-transfection of siRNA .",
"Cells were harvested after 48 hr and prepared for Western analysis .",
"Anti-GFP probing shows decreased levels of GFP-PERP expression in samples co-transfected with the appropriate siRNA . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 013 The observations presented above suggest two possible hypotheses for how Ki-67 could function in the assembly of the chromosome periphery .",
"First , the protein might comprise part of a PP1 holoenzyme whose removal of phosphates from chromosomal proteins , could be required for those proteins to assemble at the chromosome periphery .",
"Alternatively , Ki-67 , which is a very large protein with multiple repeated domains , might function as a scaffold linking together key components at the chromosome periphery .",
"To begin to distinguish between these two hypotheses , we generated constructs expressing mRFP coupled to either wild-type Ki-67 or a Ki-67 RASA mutant that is incapable of binding PP1 at the conserved site shared with Repo-Man ( Figure 1A3 ) .",
"Both constructs encoded mRNAs that were engineered to be resistant to siRNA-5 .",
"Both mRFP:Ki-67-wt and mRFP:Ki-67-RASA proteins rescued the localisation of endogenous nucleolin throughout mitosis in cells transfected with Ki-67 siRNAs .",
"In transfected cells , nucleolin was once again concentrated at the chromosome periphery from prometaphase through telophase ( Figure 2B , Figure 2—figure supplement 1 ) .",
"When combined with our previous observations these data support the hypothesis that Ki-67 acts as a scaffold for formation of the perichromosomal compartment .",
"Importantly , this rescue experiment also confirmed that the chromosome periphery phenotypes observed following Ki-67 depletion by siRNA-5 are not due to off-target effects .",
"Because depletion of Ki-67 appears to result in either a dramatic reduction , or even complete loss , of the perichromosomal compartment , these experiments offer a unique opportunity to investigate the function ( s ) of this mysterious structure .",
"If the perichromosomal compartment is a kind of pellicle or ‘skin’ on the surface of the chromosomes ( Schrader , 1944 ) , then two obvious potential functions come to mind .",
"First , the perichromosomal layer could protect the chromosomal DNA from damage once it is released into a potentially hostile cytoplasmic environment following nuclear envelope breakdown .",
"Immunostaining for 53BP1 , a protein that associates with DNA damage foci revealed that levels of intrinsic DNA damage are low in mitotic cells under our culture conditions ( Figure 5A , B ) .",
"These levels were not substantially increased in chromosomes lacking Ki-67 .",
"Thus , this hypothesis appears unlikely . 10 . 7554/eLife . 01641 . 014Figure 5 . Ki-67 is does not function to protect chromosomes from DNA damage or provide structural maintenance .",
"( A ) Representative overview images of HeLa cells transfected with control or Ki-67 specific siRNA oligos probed with anti-53bp antibodies to assess levels of DNA damage .",
"Scale bar 10 μm .",
"( B ) A bar graph showing quantification of the percentage of mitotic cells found with DNA damage , marked using an anti-53bp antibody .",
"( C ) Chromosome spreads from control RNAi ( panels 1–1″ ) and Ki-67 oligo 5 RNAi ( panels 2–2″ ) were stained for the chromosome scaffold protein KIF4A ( green ) and for the kinetochore with ACA ( red ) .",
"Ki-67 depleted chromosomes still maintain a proper localisation of the chromosome scaffold and kinetochore proteins .",
"Scale bar 2 μm .",
"( D ) IMS Assay ( Intrinsic Metaphase Structure Assay ) .",
"Chromosomes from HeLa cells after Ki-67 depletion ( panels 2 , 4 ,",
"6 ) and control depletion ( panels 1 , 3 ,",
"5 ) at the beginning of the assay ( panels 1 , 2 ) , after the first TEEN treatment ( panels 3 , 4 ) , and after the second RSB addition ( panels 5 , 6 ) .",
"Scale bar 10 μm .",
"( E ) Quantification of the experiment in ( D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 014 An alternative hypothesis is that by forming a layer coating each chromosome , the perichromosomal compartment could promote the formation of physically separated chromosomes that is characteristic of mitosis .",
"Our prior studies revealed that condensin , the chromokinesin Kif4 and DNA topoisomerase II all contribute to shaping mitotic chromosomes ( Hudson et al . , 2003; Green et al . , 2012; Samejima et al . , 2012 ) .",
"Furthermore , proteomic analysis revealed a substantial reduction in levels of Ki-67 associated with isolated mitotic chromosomes depleted of KIF4A ( Samejima et al . , 2012 ) .",
"The converse is not true , however , as neither the localisation nor abundance ( as determined by immunostaining ) of KIF4A was altered in the absence of Ki-67 ( Figure 5C ) .",
"Thus , Ki-67 depletion does not affect the known mitotic chromosome structural proteins .",
"Mitotic chromosomes have an intrinsic metaphase structure ( IMS ) that can be probed using a specialized assay , in which chromosomes are induced to unfold down to the level of 10 nm fibres by removal of divalent cations , and then induced to re-fold by re-addition of Mg2+ ( Earnshaw and Laemmli , 1983; Hudson et al . , 2003 ) .",
"Even though chromosomes lacking condensin or KIF4 appear morphologically normal when cells are processed under optimal conditions , those chromosomes are severely impaired in the IMS assay—failing to re-adopt a normal appearance after restoration of divalent cations ( Hudson et al . , 2003; Samejima et al . , 2012 ) .",
"Other abundant chromosomal proteins , including , cohesin and DNA topoisomerase II are not required for this aspect of mitotic chromosome structure ( Vagnarelli et al . , 2004; Johnson et al . , 2009 ) .",
"We transfected cells with either control or Ki-67 siRNAs and then performed the IMS assay .",
"This experiment showed clearly that chromosomes depleted of Ki-67 efficiently regain their normal morphology after two rounds of unfolding and refolding .",
"Thus , the perichromosomal layer is not required for maintenance of the intrinsic structure of mitotic chromosomes ( Figure 5D , E ) .",
"In summary , we could find no evidence for a role of Ki-67—and by extension much or all of the perichromosomal compartment—in mitotic chromosome structure or integrity under these conditions .",
"Given the abnormal localization of nucleolar components during mitosis , it is no surprise that the segregation of these components is perturbed in mitosis .",
"The detailed behaviour of the complex population of nucleolar components following abolishment of the perichromosomal compartment is a subject for a follow-up study , but here as an example , we have examined the segregation of the abundant component nucleophosmin/B23 .",
"We followed cell division in living HeLa cells expressing GFP-B23 after either control or Ki-67 siRNA .",
"In Ki-67-depleted cells , B23 was never observed to associate with the chromosomes during mitosis .",
"Instead , it started forming cytoplasmic aggregates just after anaphase onset ( Figure 6A , 45' ) .",
"We then analysed the distribution of endogenous nucleolin between daughter cells in cytokinesis .",
"While in control cells there was a relatively even distribution of chromatin-associated nucleolin between daughter cells , in Ki-67-depleted cells the nucleolin aggregates were often not chromatin bound and their distribution between daughter cells tended to be more asymmetric ( Figure 6B , C ) . 10 . 7554/eLife . 01641 . 015Figure 6 . Segregation of nucleolin .",
"( A ) In cells depleted of Ki-67 the nucleolus never disassembles completely during mitosis and B23 never accumulates around the mitotic chromosomes .",
"Time-lapse imaging of GFP:B23 in HeLa cells after control or Ki-67 RNAi .",
"Scale bar 10 μm .",
"( B ) Nucleolin localisation was analysed in cells in cytokinesis after control or Ki67 RNAi .",
"Nucleolin distribution between the two daughter cells is uneven following Ki-67 RNAi compared to the control and the protein is predominantly not associated with the chromatin .",
"( C ) Quantification of the experiment in ( B ) .",
"The graphs represent the ratio of the total nucleolin intensity between daughter cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01510 . 7554/eLife . 01641 . 016Figure 6—figure supplement 1 . Electron micrographs of interphase nuclei from Ki-67 RNAi cells . Three major components of the nucleolus are recognised: Fibrillar centres ( FC ) , dense fibrillar component ( DFC ) , and granular component ( GC ) .",
"Scale bar 2 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 016 Despite this perturbation of the behaviour of major nucleolar components during mitosis , nucleoli did indeed re-form during mitotic exit .",
"Furthermore , the ultrastructure of the reformed nucleoli appeared to be normal , with fibrillar centres , dense fibrillar component , and granular component all readily observed ( Figure 6—figure supplement 1 ) .",
"Thus , Ki-67 is apparently not required for the structural organisation within interphase nucleoli .",
"Human cells contain five chromosomes with ribosomal gene clusters that can form NORs .",
"Thus , human cells could in theory have 10 nucleoli .",
"This is almost never seen , presumably due to natural clustering of the NORs and the failure of all NORs to become activated .",
"In our line of HeLa cells , we observed 3 . 5 ± 0 . 3 nucleoli per nucleus ( Figure 7D , E ) .",
"This changed dramatically following Ki-67 depletion , when the number of nucleoli observed dropped to 1 . 6 ± 0 . 3 .",
"This strongly suggests that chromosomes lacking a perichromosomal layer might associate with one another more closely than normal , thus promoting NOR fusion during reactivation . 10 . 7554/eLife . 01641 . 017Figure 7 . The nucleoli of Ki-67 depleted cells are fewer , smaller , and functionally repressed . Normal cell , nucleus , and nucleolus size is compromised following depletion of Ki-67 .",
"( A ) Representative images showing a reduced cell size and nucleolar size phenotype in Ki-67 depleted cells , using Rhodamine Phalloidin and DAPI as markers .",
"Scale bar 5 μm .",
"( B and C )",
"Quantification of cell area and nuclear area in control ( white bar ) and Ki-67 depleted ( black bar ) cells .",
"Bars show mean ± SEM .",
"ncell = 100 .",
"( D ) Representative images showing the small nuclei phenotype , with single nucleoli following depletion of Ki-67 .",
"Scale bar 5 μm .",
"( E and F )",
"Quantification of nucleolar number and area in control ( white bar ) and Ki-67 depleted ( black bar ) cells .",
"Bars show mean ± SEM .",
"ncell = 50 .",
"( G ) A 2D scatter plot showing combined nucleolar area ( per cell ) on the Y axis , vs nuclear area on the X axis , for control ( green ) and Ki-67 depleted ( red ) cells .",
"Each individual translucent dot represents one cell .",
"Black squares represent the means .",
"( H ) Northern blot of RNA samples prepared from control ( C ) or Ki-67 depleted cells , for 24 , 48 , and 72 hr of Ki-67 knock-down .",
"Blot shows decreased signal for 47S bands and a time course dependent decrease for 26S signal ( red stars ) .",
"No clear change was seen with bands for 18SE ( black star ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01710 . 7554/eLife . 01641 . 018Figure 7—figure supplement 1 . Ki-67 depletion influences cell size , nuclei size , and nucleoli size . A 2D scatter plot showing combined nuclei area on the Y axis , vs cell area on the X axis , for control ( green ) and Ki-67 depleted ( red ) cells .",
"Each individual translucent dot represents one cell .",
"Black squares represent the means . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01810 . 7554/eLife . 01641 . 019Figure 7—figure supplement 2 . Ki-67 depletion has only minor effects on the cell cycle detected by FACS .",
"( top )",
"HeLa cells transfected with control or Ki-67 siRNA oligos were incubated for 48 , 72 , or 96 hr , before cell-cycle analysis using FACS .",
"Gated cells were manually categorised into cell-cycle stages using FACS histograms .",
"( bottom ) bar graph representation of the histogram data in the top panel . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 01910 . 7554/eLife . 01641 . 020Figure 7—figure supplement 3 . Ki-67 depletion influences nucleolar size . A bar graph showing the mean cross-sectional area measurements of nucleoli from control and Ki-67 depleted cells .",
"Nucleoli measurements are on a per cell basis and placed in ascending order , from the largest nucleolus , to the smallest . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 020 A second reason why Ki-67-depleted cells might have single large nucleoli could be due to a decreased efficiency of NOR reactivation during mitotic exit .",
"Ki-67-depleted cells tended to be smaller and to have smaller nuclei ( Figure 7A–C , Figure 7—figure supplement 1 ) .",
"This was not due to an accumulation of the cells in G1 , as shown by FACs analysis ( Figure 7—figure supplement 2 ) , but could potentially be due to ribosomal insufficiency .",
"We have made several observations that are consistent with this .",
"First , if we calculate the total nucleolar area in optical sections of Ki-67-depleted nuclei , we find that these nuclei tend to have a smaller aggregate nucleolar area ( Figure 7F , G , Figure 7—figure supplement 3 ) .",
"If we assume total nucleolar area to be a proxy for the number of genes involved in rDNA transcription , this suggests that reactivation of ribosomal clusters following mitotic exit may be less efficient in Ki-67-depleted cells .",
"This is consistent with the results of a Northern analysis of total rRNA in control and Ki-67-depleted cells , which revealed decreased levels of pre-rRNA species , consistent with lower levels of rRNA transcription in the depleted cells ( Figure 7H ) .",
"To further examine NOR reactivation following mitotic exit , we exploited the fact that chromosomes bearing re-activated NORs are associated with nucleoli .",
"We used a HT1080 cell line carrying a LacO array integrated on chromosome 13p next to the NOR and expressing GFP fused to Lac repressor ( Chubb et al . , 2002 ) .",
"In this cell line the 13p locus ( visualized as a GFP spot ) tends to localise in the nuclear interior ( Figure 8A1 ) where it is usually closely associated with a nucleolus ( Figure 8B1 ) .",
"A nuclear erosion script developed by Bickmore and Perry ( Croft et al . , 1999 ) was used to divide the nucleus into 5 concentric rings of equal area in order to score the position of the locus within the nucleus ( Figure 8A2 ) . 10 . 7554/eLife . 01641 . 021Figure 8 . Ki-67 depletion affects nuclear architecture .",
"( A ) The position of a chromosome 13p ( marked by a LacO array:LaciGFP ) in HT1080 cells ( 1 ) was assessed in control and Ki67 RNAi experiments .",
"150 nuclei from three independent experiments were imaged and analysed with an erosion script software to locate the position of the locus ( 2 ) .",
"The locus repositions from the interior toward the periphery after Ki-67 RNAi ( 3 ) .",
"( B ) The cells described in A were stained for nucleolin after control or Ki-67 RNAi .",
"The association with the nucleolus was recorded as proximal ( 1–1′ ) or distal ( 2–2′ ) .",
"( 3 ) Quantification of the analyses .",
"( C ) Model for Ki-67 function in mitosis; ( blue: chromatin; green perichromosomal proteins; grey: microtubules ) .",
"See text for details . DOI: http://dx . doi . org/10 . 7554/eLife . 01641 . 021 Depletion of Ki-67 had a strong effect on the nuclear localization of chromosome 13p .",
"In cells depleted of Ki-67 , the tagged 13p locus tended to move from the nuclear interior towards the nuclear periphery ( Figure 8A3 ) .",
"This reorganization of chromosome 13p was correlated with a disengagement from the nucleolus ( Figure 8B2 , 3 ) .",
"Both phenotypes are consistent with a decreased efficiency of reactivation of the 13p NOR .",
"Thus , although Ki-67 depletion and loss of much or all of the perichromosomal compartment has little obvious effect on chromosome structure or segregation in the first mitosis after depletion , after mitotic exit the nucleus undergoes fundamental changes consistent with decreased activity of the ribosomal gene clusters and with altered interactions between chromosomes .",
"More detailed examination of the link between Ki-67 , the perichromosomal compartment , and nucleolar organizer region ( NOR ) reactivation will be an important subject for future study ."
],
[
"Our studies have revealed for the first time that Ki-67 functions as a structural/scaffolding protein required for assembly of the perichromosomal compartment on condensed mitotic chromosomes .",
"Thus , Ki-67 has an important role in determining the behaviour of many nucleolar components during mitosis .",
"Interestingly , this method of segregating nucleolar components means that nucleolar reassembly can begin immediately after CDK activity declines and before the re-establishment of nuclear-cytoplasmic transport during mitotic exit .",
"Ki-67 is a PP1-interacting protein , however the biological role of this activity remains elusive and indeed the protein could execute this function during another phase of the cell cycle rather than mitosis .",
"How the various activities of Ki-67 combine to determine the nucleolar morphology in proliferating cells and whether the perichromosomal layer has a subtle role in chromosome dynamics during mitosis or nuclear reformation remain to be determined in future studies ."
],
[
"HeLa Kyoto and MRC5 cells were maintained in DMEM supplemented with 10% and 15% FBS respectively .",
"DT40 cells carrying a single integration of the LacO array ( Vagnarelli et al . , 2006 ) were cultured in RPMI1640 supplemented with 10% FBS and 1% chicken serum .",
"For RNAi treatments HeLa cells in exponential growth were seeded in six-well plates with or without polylysine-coated glass coverslips and grown overnight .",
"Transfections were performed using Polyplus jetPRIME ( PEQLAB , Southampton , UK ) with the indicated siRNA oligos and analysed 48 hr later as previously described ( Vagnarelli et al . , 2006 ) .",
"For the rescue experiments HeLa cells at 50% confluence were transfected with 400 ng of plasmid DNA and 50 nM of siRNA oligonucleotides and analysed 48 hr post-transfection .",
"The siRNA oligonucleotides against Ki67 are as follows: Ki-1 as published by Vanneste et al . ( 2009 ) ; Ki-2 :5′AAGCACCAGAGACCCUGUATT3′; Ki-5:5′GCAUUUAAGCAACCUGCAA3′; a 21-mer oligonucleotide ( CGUACGCGGAAUACUUCGAdTdT ) was used as a control ( Elbashir et al . , 2001 ) .",
"To test for successful depletion of cPerP proteins , HeLa cells were co-transfected with specific siRNA oligos together with the appropriate GFP-cPerP cDNA constructs .",
"Control samples were prepared in parallel via cells transfected with GFP-PerP cDNA only .",
"Following a 48 hr expression/knock-down period cells were harvested and processed routinely for Western analysis .",
"Knockdown was considered successful if Western analysis revealed decreased GFP expression levels in siRNA treated samples ( rabbit polyclonal anti-GFP , Invitrogen , Paisley , UK ) .",
"The primary antibodies were used as follows: Ki-67 ( mouse monoclonal BD Transduction laboratory , Oxford , UK ) 1:100; nucleolin ( rabbit polyclonal; Abcam ) 1:300; NIFK T234ph ( Rabbit polyclonal; Abcam , Cambridge , UK ) 1:100; Repo-Man ( Vagnarelli et al . , 2011 ) ; anti-alpha-tubulin antibody ( B512; SIGMA , Gillingham , UK ) , anti-B23 S125ph , ( Abcam ) ; anti-B23T199ph ( Abcam ) ; anti-nucleolin ( Abcam ) .",
"For immunofluorescence , cells were fixed in 4% PFA and processed as previously described ( Vagnarelli et al . , 2011 ) .",
"Fluorescence-labelled secondary antibodies were applied at 1:200 ( Jackson ImmunoResearch ) .",
"3D data sets were acquired using a cooled CCD camera ( CH350; Photometrics ) on a wide-field microscope ( DeltaVision Spectris; Applied Precision ) with a NA 1 . 4 Plan Apochromat lens .",
"The data sets were deconvolved with softWoRx ( Applied Precision ) .",
"3D data sets were converted to Quick Projections in softWoRx , exported as TIFF files , and imported into Adobe Photoshop for final presentation .",
"Live cell imaging was performed with a DeltaVision microscope as previously described ( Vagnarelli et al . , 2011 ) .",
"For quantification of PP1 binding in vivo , images of prometaphase and interphase transfected cells were acquired and the intensity of PP1 staining at the GFP spot was calculated relative to the average nuclear intensity .",
"The 3D data sets obtained at the same exposure were projected as mean intensities .",
"A 12 × 12 pixel area containing the GFP spot was used to measure the total intensity of the signal .",
"An area of the same size was used to identify the background signal in each cell , and this value was subtracted from the measurement of the nuclear and spot area .",
"The IMS assay was conducted as previously described ( Hudson et al . , 2003 ) .",
"For immunoblotting , whole cell lysates were loaded onto polyacrylamide gels .",
"SDS-PAGE and immunoblotting was performed following standard procedures .",
"Ki-67 ( aa 161–659 ) was obtained by RT-PCR from HeLa cDNA using the following primers: GGATCCGGCGCCACGTTTCCTCTC and CTCGAGTTTTACTACATCTGC and CLONED PGEX4T3 BamHi/XhoI .",
"The PP1-non binding mutant version was generated from this vector using a QuikChange Site-Directed Mutagenesis Kit ( Agilent Technologies , Edinburgh , UK ) .",
"The Lac repressor fusion constructs were obtained by cloning Ki-67 ( aa 161–659 ) into pEGFP:Lac repressor .",
"hPP1γ was cloned into PET28 EcoRI/HindIII .",
"hNIFK was cloned by RT PCR from HeLa cDNA using the primers CTCGAGGGATGGCGACTTTTTCTGGC and GAATTCTCACTGATTGCTGCTTCT and cloned into the XhoI/EcoR1 sites of pEGFPC1 .",
"The cPERPs were cloned into the gateway system by PCR as previously described ( Ohta et al . , 2010 ) .",
"Accession numbers for cPERPs are as follows: cPERP-A: ( C1orf131 ) –NM_152379 . 2 , cPERP-B: ( CCDC137 ) –NM_199287 . 2 , cPERP-C: ( KIAA0020 ) –NM_014878 . 4 , cPERP-D: ( DDX18 ) –NM_006773 . 3 , cPERP-E: ( CIRH1A ) –NM_032830 . 2 , cPERP-F: ( DDX27 ) –XM_006723815 . 1 .",
"The CLEM processing method was an adapted version of a previously established protocol ( Booth et al . , 2011 , Booth et al . , 2013 ) .",
"Cells were seeded onto glass-bottomed , gridded dishes ( MatTek Corporation , USA ) and co-transfected with GFP-cPerpC together with control or Ki-67 specific siRNA oligos .",
"Following a 48 hr expression period , cells of interest were identified using a wide-field epifluorescence microscope ( DeltaVision RT; Applied Precision ) .",
"GFP-expressing mitotic cells were located and their position mapped using transmitted light to visualise reference coordinates .",
"Cells were then fixed for 1 hr ( 3% glutaraldehyde , 0 . 5% paraformaldehyde in 0 . 2 M sodium cacodylate buffer containing 5 µg/ml Hoechst ) and washed in PBS ( 3 × 5 min ) .",
"Cells of interest were then re-imaged to acquire micrographs of Hoechst stained chromosomes .",
"Next , cells were osmicated ( 1% osmium tetroxide in PBS ) for 1 hr , washed with PBS ( 3 × 5 min ) , ddH2O ( 2 × 20 min ) , and then 30% ethanol ( 1 × 10 min ) before contrast staining with uranyl acetate ( 0 . 5% in 30% ethanol ) for 1 hr .",
"Cells were then dehydrated using a graded series of ethanol washes culminating in 2 × 10 min incubations with 100% ethanol , followed by infiltration with ethanol:resin mixtures ( at 2:1 and then 1:1 ) .",
"Finally , cells were embedded in 100% resin , with a gelatin capsule of resin covering the cells of interest , before curing at 60°C for 3 days .",
"Ultra-small resin blocks ( 50 µm2 ) were fine trimmed and serial sections ( 85 nm thickness ) taken at areas corresponding to previously chosen coordinate positions , before post-staining in Reynold's lead citrate and uranyl acetate ( 5% in 50% ethanol ) for 10 and 5 min , respectively .",
"Cells were visualised with a Phillips CM120 BioTwin transmission electron microscope ( FEI ) and micrographs acquired using a Gatan Orius CCD camera ( Gatan ) .",
"The appropriate Z position correlative light/EM images of a cell were concatenated using ImageJ and then analysed and overlaid using Photoshop Elements 6 ( Adobe ) .",
"Electron micrographs containing metaphase chromosomes from control or Ki-67 depleted cells were used for chromosome periphery analysis .",
"The pixel density of a 1 μm region of interest was measured using the raw data from the ‘plot profile’ function of imageJ .",
"For unbiased consistency , the 1 μm region of interest always started within the chromosome body and finished in the cytoplasm , with the halfway point lying at the expected origin of the chromosome periphery .",
"Approximately 200 individual pixel density measurements were taken within each 1 μm region .",
"The data were plotted as a line scan profile , with each data point representing the mean of 6 × 1 μm regions of interest per chromosome .",
"HeLa cells were seeded onto coverslips and transfected with control or Ki-67 specific siRNA oligonucleotides .",
"Following a 48 hr knock-down period , cells were fixed using 4% paraformaldehyde , blocked with 3% BSA , and either directly mounted onto slides using hard-setting Vectashield ( containing DAPI ) or first stained with Rhodamine Phalloidin ( Biotium , Inc . , Cambridge , UK ) , before mounting .",
"All measurements were scored using imageJ and area measurement tools .",
"Total cell cross-sectional area was measured using Phalloidin to reveal the cytoplasm and by extension a guide for cell periphery .",
"The area of individual cells or clustered groups of cells were traced and scored using freehand measurement tools .",
"Nuclei and nucleoli cross-sectional area was measured and nucleolar numbers scored using DAPI as a marker .",
"A contrast threshold was applied to micrographs allowing the semi-automated , unbiased measurement of nucleolar area and number using the ImageJ wand ( tracing ) tool .",
"Control or Ki-67 depleted HeLa cells were subjected to cell-cycle analysis by FACS .",
"Briefly , 1 × 106 cells , per condition , were fixed with cold ethanol ( 70% ) for 1 hr , centrifuged , and resuspended in PBS containing RNase A ( 0 . 2 mg/ml ) and Propidium Iodide ( 10 µg/ml ) .",
"Following a 20-min incubation , cells were analysed using FACS .",
"The channel FL2 was used to analyse 20 , 000 events per condition .",
"Gated cells were manually categorised into cell-cycle stages .",
"Cells were analysed following knock-down periods of 24 , 48 , 72 , and 96 hr .",
"HT1080 cell line carrying a LacO integration on chromosome 13p and expressing a Laci:GFP ( kindly provided by W Bickmore ) was used for depleting Ki-67 in an RNAi experiment as described for HeLa above .",
"At 48 hr the cells were fixed and processed for immunostaining with a nucleolin antibody as described before .",
"150 nuclei from three independent experiments were imaged and analysed for with the nuclear erosion scrip ( Croft et al . , 1999 ) to assign the position of the 13p locus .",
"Control or Ki-67 depleted HeLa cells were cultured in 10 cm dishes and processed for Northern analysis .",
"RNA was extracted using fresh TRIzol ( Ambion ) according to manufacturer’s guidelines .",
"3 µg of RNA per sample was separated using a standard 1 . 2% TBE agarose gel and transferred to Hybon N+ membrane overnight , by capillary action .",
"Membranes were hybridized in 6X SSPE , 5X Denhardts , 5X SDS at 37°C before washing with 2XSSPE .",
"Probes ( see below ) were 5′ labelled with y32P-ATP and T4PNK .",
"Probe_d_human AGACGAGAACGCCTGACACGCACGGCAC 5'ETS probe .",
"Probe_e_human CCTCGCCCTCCGGGCTCCGTTAATGATC 5′ end of ITS1 ( 21S and 18SE ) ."
]
] | [
"When the nucleolus disassembles during open mitosis , many nucleolar proteins and RNAs associate with chromosomes , establishing a perichromosomal compartment coating the chromosome periphery .",
"At present nothing is known about the function of this poorly characterised compartment .",
"In this study , we report that the nucleolar protein Ki-67 is required for the assembly of the perichromosomal compartment in human cells .",
"Ki-67 is a cell-cycle regulated protein phosphatase 1-binding protein that is involved in phospho-regulation of the nucleolar protein B23/nucleophosmin .",
"Following siRNA depletion of Ki-67 , NIFK , B23 , nucleolin , and four novel chromosome periphery proteins all fail to associate with the periphery of human chromosomes .",
"Correlative light and electron microscopy ( CLEM ) images suggest a near-complete loss of the entire perichromosomal compartment .",
"Mitotic chromosome condensation and intrinsic structure appear normal in the absence of the perichromosomal compartment but significant differences in nucleolar reassembly and nuclear organisation are observed in post-mitotic cells ."
] | [
"The genetic information of an organism is found in the nucleus of each cell in the form of DNA organised into chromosomes .",
"The exact structure of those chromosomes changes as the cell moves through the different stages of the cell division cycle .",
"During the stage called mitosis , where the DNA of a cell ( which has previously been duplicated ) is shared into two daughter cells , the chromosomes become tightly packed structures that can be readily moved through the cytoplasm .",
"Since the late nineteenth century , it has been known that a layer of proteins , called the perichromosomal layer , coats the condensed chromosomes .",
"However , virtually nothing was known about the role this layer performs .",
"One of the first proteins to join the perichromosomal layer after mitosis begins is called Ki-67 .",
"This is only found in the cell nucleus when a cell is actively growing and dividing , and so is widely used as a marker in experiments investigating these processes: for example , Ki-67 is used to detect growing tumour cells amongst the normal cells in tissues of the body , and to measure the effectiveness of drugs designed to stop the growth of tumours .",
"Again , however , little is known about what Ki-67 actually does .",
"Booth et al . now reveal that when Ki-67 is not present in a cell , chromosomes do not have a perichromosomal layer—or at best , have a small remnant of one .",
"This allowed Booth et al . to investigate the role of the perichromosomal layer as well .",
"When the chromosomes first go through mitosis without a perichromosomal layer , no changes to the shape or the behaviour of the chromosomes are seen .",
"However , the new nuclei are smaller than normal and their contents are arranged differently .",
"This causes problems with the ability of daughter cells to synthesise protein building blocks and leads to an increased rate of spontaneous cell death when daughter cells try to undergo the next mitosis .",
"Further research is needed to understand why this happens ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Contrasting roles for parvalbumin-expressing inhibitory neurons in two forms of adult visual cortical plasticity | elife-11450-v1 | [
[
"Understanding how brain synapses , cells , and circuits are persistently modified by experience to store information represents one of the great challenges in neuroscience .",
"Mouse visual cortex has proven to be an excellent model system in which to examine experience-dependent neural response modification .",
"One robust type of visual response plasticity is reliably elicited in adult ( > P60 ) mice by simply closing one eyelid .",
"Over the course of 5–7 days of monocular deprivation ( MD ) , the responses in visual cortex evoked by stimulation of the non-deprived eye progressively increase ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .",
"This deprivation-enabled response potentiation is driven by visual experience through the non-deprived eye , as only the responses through one eye are potentiated ( i . e . , it is input specific ) and it fails to occur if both eyelids are closed or if the animals are kept in a dark room ( Blais et al . , 2008 ) .",
"There is evidence that the response potentiation is mediated in part by “Hebbian” strengthening of excitatory synaptic transmission in visual cortex , as induction requires cortical NMDA receptor ( NMDAR ) activation ( Sawtell et al . , 2003 ) ( Sato and Stryker , 2008 ) and α-calcium/calmodulin-dependent protein kinase II ( αCAMKII ) expression in principal cells ( Ranson et al . , 2012 ) .",
"This form of ocular dominance ( OD ) plasticity is likely responsible for the increase in visual acuity that occurs through the non-deprived eye following adult monocular deprivation ( Iny et al . , 2006 ) , and is of particular interest in the context of recovery of brain function after deprivation , disease , or damage ( Cho and Bear , 2010 ) .",
"Another robust form of visual response plasticity is induced by exposure of awake mice to oriented visual grating stimuli .",
"Brief daily presentation of a phase-reversing grating of a single orientation causes a large and persistent increase in the peak cortical response to this orientation , as measured by visual evoked potentials ( VEPs ) or unit recordings ( Frenkel et al . , 2006; Cooke et al . , 2015 ) .",
"This phenomenon is termed stimulus-selective response potentiation ( SRP ) because only responses to the experienced orientation are increased .",
"Abundant evidence suggests that SRP is also mediated by “Hebbian” mechanisms , particularly those revealed by the study of long-term synaptic potentiation ( Cooke and Bear , 2010; 2014 ) .",
"The mechanisms utilized for SRP within V1 have also been shown to mediate a fundamental form of long-term visual recognition memory , manifested behaviorally as orientation-selective habituation ( OSH ) ( Cooke and Bear , 2015; Cooke et al . , 2015 ) .",
"Both monocular deprivation and selective visual experience trigger input-specific increases in the short latency VEP measured in layer 4 of mouse visual cortex and , as reviewed above , both OD plasticity and SRP share some molecular requirements ( e . g . , NMDAR activation ) .",
"Thus , it came as a surprise that response potentiation after monocular deprivation and SRP do not occlude one another ( Frenkel and Bear , 2004 ) , suggesting that they employ different mechanisms or are expressed by different synapses .",
"We became interested in the possibility of differential involvement of cortical inhibition mediated by the parvalbumin-expressing ( PV+ ) fast spiking neurons .",
"PV+ fast-spiking inhibitory neurons comprise the most numerous sub-class of GABAergic cortical neurons ( Xu et al . , 2010 ) and receive substantial feed-forward glutamatergic input from the thalamus ( Cruikshank et al . , 2007 ) .",
"These interneurons synapse preferentially on the somata and initial axon segments of principal cells , and therefore are in a position to strongly and precisely modulate even short latency visually evoked responses ( Cristo et al . , 2004; Markram et al . , 2004; Kepecs and Fishell , 2014 ) .",
"Moreover , previous studies have suggested that fast-spiking interneurons participate in the expression of cortical eye dominance and OD plasticity in juvenile mice ( Yazaki-Sugiyama et al . , 2009; Smith and Bear , 2010 ) .",
"The contribution of this class of neuron to cortical plasticity is also of particular interest given the evidence for cortical PV+ neuron dysfunction in various psychiatric disorders characterized by impaired cognitive function ( Gogolla et al . , 2009 ) .",
"In the current study we used a variety of approaches to understand the contribution of PV+ neurons to adult OD plasticity and SRP .",
"We find that neither the pharmacogenetic silencing of PV+ cells nor the specific deletion of NMDARs within them affect the relative eye dominance or the expression of deprivation-enabled potentiation of VEPs in adult mice .",
"In contrast , and quite unexpectedly , we discovered that the expression of SRP is dependent on PV+ cell activity , and that perturbations of PV+ neuron function , most notably cell-type specific ablation of NMDARs , disrupt the expression of both SRP and its behavioral correlate , familiarity recognition ."
],
[
"In order to understand the role of PV+ neurons in the expression of experience-dependent visual cortical plasticity , we selectively inactivated these cells using a Dreadd ( Designer Receptors Exclusively Activated by Designer Drugs ) pharmacogenetic system: Specifically , we expressed a re-engineered G-protein coupled receptor , hM4D ( Gi ) , which is activated exclusively by an otherwise inert small molecule , clozapine-N-oxide ( CNO ) ( Nichols and Roth , 2009 ) .",
"The binding of CNO to the hM4D ( Gi ) receptor activates intracellular Gi-mediated signaling and subsequent hyperpolarization of the cell in which the receptors are expressed .",
"We locally expressed hM4D ( Gi ) receptors in PV+ cells of binocular V1 using an adeno-associated viral vector containing a construct for Cre-selective expression ( AAV9-hSyn-DIO-HA-hM4D ( Gi ) -IRES-mCitrine ) in mice that express Cre recombinase only in PV+ cells ( B6;129P2-Pvalbtm1 ( cre ) Arbr/J , PV-Cre ) .",
"We confirmed the selective expression of hM4D ( Gi ) receptors in PV+ neurons in binocular V1 using immunohistochemistry ( Figure 1A–E ) .",
"In order to ensure that PV+ neurons could be inactivated by this method , we took slices of V1 for ex vivo intracellular recordings .",
"Bath application of CNO drastically inhibited current evoked action potential firing of PV+ neurons in Layer 4 ( 2-way repeated measures ANOVA , interaction of CNO x current injection , F ( 8 , 72 ) = 6 . 227 , P < 0 . 001 , n = 10 cells , significant at data points above 100pA: q ( 8 ) = 3 . 716 , P = 0 . 014 ) but had no effect on neighboring non-expressing cells ( Figure 1F–G ) .",
"Layer 4 recordings in vivo demonstrated that systemic administration of CNO resulted in the elevation of visually-evoked potential ( VEP ) magnitude , and an increase in the firing rate of excitatory single units ( Figure 1H–I ) , which are expected consequences of inactivating PV+ inhibitory neurons in binocular V1 . 10 . 7554/eLife . 11450 . 003Figure 1 . The hM4D ( Gi ) DREADD system locally inactivates parvalbumin+ neurons in binocular primary visual cortex ( V1 ) .",
"( A ) An example of V1 expression of hM4D ( Gi ) in a parvalbumin ( PV ) -Cre recombinase ( Cre ) mouse infected locally in binocular V1 with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine .",
"( B ) A DAPI stain for cell nuclei is shown in blue .",
"( C ) Infected cells expressing hM4D ( Gi ) are labeled in green .",
"( D ) Immuno-labeled PV+ cells are shown in red .",
"( E ) The merged image reveals that hM4D ( Gi ) -expressing cells are also PV+ .",
"( F ) Intracellular current clamp recordings of hM4D ( Gi ) -infected PV+ layer 4 neurons in ex vivo slices of V1 reveal that green-labeled infected cells exhibit a non-adapting fast-spiking phenotype typical of fast-spiking inhibitory neurons ( black ) .",
"These cells do not fire action potentials in the presence of CNO ( red ) , the exogenous ligand for hM4D ( Gi ) receptors , despite depolarizing current injection .",
"In contrast , neighboring cells that are not mCitrine+ show no impact of CNO application .",
"( G ) HM4D ( Gi ) -mediated inactivation of putative fast-spiking PV+ inhibitory neurons is here summarized as the number of action potentials resulting from a given current injection before ( black ) and after CNO application ( red ) .",
"( H ) The effects of hM4D ( Gi ) -mediated inactivation of putative PV+ fast-spiking inhibitory neurons in vivo are apparent from electrophysiological recordings from V1 of awake , head-fixed mice viewing phase-reversing sinusoidal grating stimuli .",
"Averaged Visually Evoked Potential ( VEP ) recordings recorded in layer 4 reveal increased VEP magnitude in the presence of CNO ( red ) , relative to pre-CNO ( baseline ) recordings ( black ) , indicative of reduced inhibition .",
"( I ) Phase reversal-evoked action potentials recorded from neurons in layer 4 also exhibit a similar effect with elevated firing rates in the presence of CNO ( red ) relative to the baseline recording ( black ) .",
"Labeled scale bars are presented throughout .",
"Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00310 . 7554/eLife . 11450 . 004Figure 1—source data 1 . Action potential number for current injection . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 004 Mouse binocular V1 is ~2–3 times more strongly activated by the contralateral ( contra ) eye than the ipsilateral ( ipsi ) eye , an inherent bias known as ocular dominance ( OD ) .",
"We probed the involvement of PV+ neuron activity in maintaining baseline OD .",
"PV-Cre mice were infected with AAV virus to deliver hM4D ( Gi ) Dreadd receptors to PV+ neurons in binocular V1 ( Figure 2A ) .",
"Electrodes were simultaneously implanted into layer 4 of V1 for VEP recordings .",
"VEPs elicited through just contralateral or ipsilateral eyes were acquired consecutively before and after CNO administration ( Figure 2A–B ) .",
"After CNO injection , VEPs driven through each eye increased significantly in magnitude ( 2-way repeated measures ANOVA , effect of CNO , F ( 7 ) = 75 . 986 , P < 0 . 001; contralateral eye: 222 . 18 ± 14 . 79 µV baseline vs . 679 . 81 ± 64 . 12 µV CNO , Student-Newman-Keuls ( SNK ) post hoc test , q ( 7 ) = 13 . 925 , n = 8 mice , P < 0 . 001; ipsilateral eye: 105 . 56 ± 6 . 78 µV baseline vs . 358 . 63 ± 35 . 79 µV CNO , SNK post hoc test , q ( 7 ) = 7 . 711 , n = 8 mice , P < 0 . 001 , ( Figure 2C ) , consistent with the occurrence of cortical disinhibition .",
"However , the OD of visual responses in V1 ( contra/ipsi ratio ) was not significantly altered by CNO injection ( 2 . 13 ± 0 . 12 baseline vs . 1 . 96 ± 0 . 18 CNO , student’s paired two-tailed t-test , t ( 7 ) = 0 . 859 , n = 8 mice , P = 0 . 42 , Figure 2D ) .",
"This result suggests that the inherent OD of binocular visual cortex is not dependent on PV+ neuron activity . 10 . 7554/eLife . 11450 . 005Figure 2 . Inactivation of parvalbumin+ neurons has no impact on expression of ocular dominance ( OD ) or the ocular dominance shift as a result of monocular deprivation ( MD ) in the adult mouse .",
"( A ) For experiments described in this figure mice were infected across all cortical depths bilaterally in binocular V1 ( green ) .",
"During the same surgical implantation procedure VEP recording electrodes were also positioned in layer 4 and chronically fixed .",
"( B ) Mice ( P45-60 ) were infected and implanted with electrodes and then left for 3 weeks for the AAV9 viral vector to reach maximal expression , after which they underwent habituation to head-fixation and a gray screen for two consecutive days .",
"Following this , on experimental day 0 , mice were presented with an Xo phase-reversing sinusoidal grating stimulus separately to the left and right eye .",
"VEPs were recorded from each hemisphere in order to determine ocular dominance in binocular V1 .",
"Mice were then removed from the recording apparatus and , CNO was delivered systemically half an hour prior to undertaking the same recording procedure , this time using an orthogonal X + 90° visual stimulus .",
"( C ) As is well documented , responses in V1 to stimuli viewed through the contralateral eye ( blue ) were greater in magnitude than those elicited through the ipsilateral eye ( yellow ) , as measured here by VEP magnitude .",
"After application of CNO ( red outlines ) , VEP magnitude dramatically increased .",
"( D ) This increase was scaled such that the ratio of Contralateral:Ipsilateral VEP magnitude was maintained before ( white ) and after CNO ( red ) .",
"( E ) In a second group of mice , a similar experimental protocol was observed prior to measuring ocular dominance by recording VEPs in each hemisphere elicited by an Xo stimulus through each eye .",
"One hemisphere was then selected and the contralateral eye was sutured closed .",
"After 7 days of monocular deprivation , the eye was opened and VEPs driven through either eye were again recorded , this time elicited by an X + 60° stimulus .",
"Mice were then systemically injected with CNO and , half an hour later , VEPs driven through either eye were recorded , this time elicited by an X - 60° stimulus .",
"( F ) After the adult mice underwent 7 days of MD , there was a significant potentiation of the V1 response to visual input through the ipsilateral eye ( yellow ) .",
"After application of CNO ( red outlines ) , VEPs driven through each eye were elevated in magnitude , but again the increase in VEP magnitude was scaled .",
"( G ) As a result of open eye potentiation after MD , the OD ratio shifted dramatically from the contralateral bias of pre MD ( white ) to an almost equal cortical response through contralateral and ipsilateral eyes ( black ) .",
"This shifted ratio was unaffected by hM4Di-mediated inactivation of putative fast-spiking inhibitory neurons during CNO application ( red ) , indicating that the expression of OD and its shift as a result of MD in adult mice do not require fast-spiking inhibition .",
"Significant comparisons are labeled with an asterisk and non-significant comparisons with n . s . throughout .",
"Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00510 . 7554/eLife . 11450 . 006Figure 2—source data 1 . Ocular dominance and deprivation effects are maintained after PV neuron inactivation . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 006 The normal OD ratio is altered in the adult mouse by MD of the contralateral eye over 7 days , resulting in an ocular dominance shift that features potentiation of response through the open ipsilateral eye ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .",
"In a new group of mice we assayed the effect of PV+ neuron inactivation on the expression of the shift in OD caused by MD .",
"We recorded VEPs elicited through each eye from PV-Cre mice expressing hM4D ( Gi ) receptors in binocular V1 ( Figure 2E ) .",
"After baseline recordings the mice underwent contralateral eyelid suture and 7 days of monocular deprivation .",
"Subsequently , the contralateral ( deprived ) eye was opened and VEPs were re-recorded in order to reveal the expression of an OD shift .",
"There was a significant potentiation of VEP magnitude driven through the ipsilateral eye following 7 days of MD ( 69 . 5 ± 5 . 81 µV pre MD vs . 139 . 5 ± 9 . 33 µV post MD , n = 10 mice , student’s paired one-tailed t-test , t ( 9 ) = -10 . 184 , P < 0 . 001 , Figure 2F ) .",
"This potentiation of response through the ipsilateral ( non-deprived ) eye resulted in a significant shift in the OD ratio ( contra/ipsi ratio , 3 . 08 ± 0 . 26 pre MD vs . 1 . 33 ± 0 . 10 post MD , 1-way repeated measures ANOVA , SNK post hoc test , q ( 2 ) = 11 . 77 , P < 0 . 001 , Figure 2G ) .",
"Mice were then injected with CNO and re-recorded to assess whether the OD shift would persist in the absence of PV+ neuron activity .",
"CNO injection resulted in an increase in VEP magnitudes ( Figure 2F ) , but importantly the shift in the contra/ipsi ratio was maintained ( 1 . 33 ± 0 . 10 post MD vs . 1 . 35 ± 0 . 18 post MD CNO , SNK post hoc test , q ( 2 ) = 0 . 127 , P = 0 . 93 , Figure 2G ) .",
"Therefore , the expression of the adult OD shift induced by 7 days of MD persists after a strong reduction in PV+ neuron inhibition .",
"We next assayed the role of PV+ neurons in the expression mechanism underlying stimulus-selective response potentiation ( SRP ) , a second form of experience-dependent visual cortical plasticity that also manifests as an increase in V1 responses ( Frenkel et al . , 2006; Cooke and Bear , 2010 ) .",
"Binocular VEPs were recorded from awake , head-fixed adult mice viewing phase-reversing gratings of a particular orientation ( Xo stimulus ) on each of 6 consecutive days ( Figure 3A and B ) .",
"On the 7th day mice viewed blocks of the now familiar visual stimulus interleaved with blocks of a novel oriented stimulus ( X +/- 60° ) .",
"To address whether PV+ neuron activity is required for the expression of SRP , familiar and novel oriented grating stimuli were presented before and after mice received CNO ( Figure 3A ) . 10 . 7554/eLife . 11450 . 007Figure 3 . The expression of Stimulus-selective Response Potentiation ( SRP ) requires activity in PV+ neurons in V1 . ( A ) Mice expressing hM4D ( Gi ) receptors in PV+ cells underwent a standard SRP induction protocol .",
"On day 7 , mice viewed a novel oriented stimulus in addition to the familiar stimulus; before and after CNO injection .",
"( B ) We acquired binocular VEPs from awake , head-fixed mice elicited by the same full-field oriented sinusoidal grating stimulus over several days .",
"( C ) As a result of multiple days of experience cortical response was dramatically potentiated such that the familiar stimulus evoked VEPs of significantly greater magnitude than the novel stimulus ( black bars ) .",
"( D ) After application of CNO , VEPs underwent a notable increase in magnitude as a result of disinhibition ( red bars ) .",
"Most notably , CNO rendered response to familiar and novel stimuli equivalent in magnitude .",
"( E ) This lost discrimination of familiar and novel stimuli is reflected as a drop in the ratio of response to familiar and novel stimuli from approximately 2:1 ( black ) to approximately 1:1 ( red ) .",
"Significant comparisons are labeled with an asterisk and non-significant comparisons with n . s . throughout .",
"Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00710 . 7554/eLife . 11450 . 008Figure 3—source data 1 . PV Dreadds SRP induction and expression . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00810 . 7554/eLife . 11450 . 009Figure 3—figure supplement 1 . CNO has no impact on SRP expression in WT mice .",
"( A ) C57BL/6 WT mice were bilaterally infected with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine in binocular V1 .",
"Because these mice did not express Cre recombinase there was no subsequent expression of hM4D ( Gi ) DREADDS receptors .",
"After 4 weeks , during which hM4D ( Gi ) was expressed at high levels in PV-Cre mice , WT mice were taken through a standard SRP protocol of electrode implantation , habituation and visual experience of a single oriented grating .",
"This progressed as usual , resulting in a significant increase in the magnitude of the VEP .",
"( B ) Expression of SRP was then tested on day 7 by interleaving presentations of familiar and novel orientations and VEPs elicited by the familiar orientation were of significantly greater magnitude than the novel .",
"After delivery of CNO , VEPs were again tested in response to the familiar orientation and a second novel orientation .",
"The drug had no impact and a similar degree of significant stimulus selectivity was present .",
"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 00910 . 7554/eLife . 11450 . 010Figure 3—figure supplement 1—source data 1 . CNO has no effect on SRP expression in WT mice . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 010 As is characteristic of SRP , there was a significant increase in the magnitude of the average VEP evoked by the familiar oriented grating stimulus over days ( Friedman 1-way repeated measures ANOVA on ranks , n = 10 mice , X2 ( 5 ) = 40 . 40 , P < 0 . 001 , Figure 3C ) .",
"This potentiation was evident by day 2 ( 263 . 3 ± 19 . 96 µV ) in comparison with day 1 ( 184 ± 17 . 13 µV , SNK post hoc test , q ( 9 ) = 4 . 472 , P < 0 . 05 ) .",
"The stimulus selectivity of VEP magnitude potentiation was apparent on day 7 and significantly affected by inactivating PV+ inhibitory neurons in V1 ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F = 78 . 927 ( 1 , 9 ) , P < 0 . 001 , Figure 3D ) : Prior to delivery of CNO the average VEP magnitude driven by the familiar stimulus ( 324 . 7 ± 22 . 18 µV ) was significantly greater than that driven by a novel oriented stimulus ( 162 . 9 ± 16 . 31 µV , SNK post hoc test , q ( 9 ) = 10 . 709 , P < 0 . 001 ) .",
"Following injection of CNO to inactivate hM4D ( Gi ) -infected neurons in V1 , there was no longer a significant difference in the magnitude of VEPs driven by the familiar stimulus ( 537 . 2 ± 66 . 69 µV ) compared with a novel stimulus ( 525 . 1 ± 68 . 30 µV , SNK post hoc test , q ( 9 ) = 0 . 804 , P = 0 . 58 ) .",
"The selectivity of the potentiation to a familiar stimulus can be summarized by plotting the ratio of VEP magnitudes driven by familiar and novel stimuli ( Figure 3E ) .",
"Mice expressed a significantly larger familiar/novel ratio before CNO injection ( 2 . 11 ± 0 . 17 ) , corresponding to a greater response to the familiar stimulus , compared to after CNO injection ( 1 . 03 ± 0 . 04 , Mann Whitney rank sum test , U = 0 . 00 , P < 0 . 001 ) .",
"CNO injection did not affect the stimulus selectivity of SRP in wild type animals infected with virus ( Figure 3—figure supplement 1A–B ) .",
"These animals underwent SRP ( Figure 3—figure supplement 1A ) and on test day showed significantly larger VEPs to the familiar stimulus , both prior to CNO injection ( familiar VEP: 361 . 06 ± 27 . 97 µV , novel VEP: 219 . 25 ± 17 . 9 µV , 2-way repeated measures ANOVA , n = 8 mice , SNK post hoc test , q ( 7 ) = 13 . 618 , P < 0 . 001 ) and following CNO injection ( familiar VEP: 369 . 00 ± 31 . 16 µV , novel VEP: 256 . 75 ± 19 . 59 µV , SNK post hoc test , q ( 7 ) = 10 . 779 , P < 0 . 001 , Figure 3—figure supplement 1B ) .",
"These results show that the inactivation of PV+ neurons in binocular V1 disrupts the expression of SRP .",
"Cortical neurons respond within a dynamic range , and it is possible that discrimination of familiar and novel stimuli would be lost as responses approach saturation .",
"Our observation that OD is maintained after PV+ neuron inactivation ( Figure 2 ) indicates that V1 can still respond selectively to a strong ( contralateral eye ) and weak ( ipsilateral eye ) input .",
"However , to address the possibility that a “ceiling effect” contributes to the disruption of SRP expression during PV+ cell inactivation , we conducted an additional experiment in which mice viewed sinusoidal grating stimuli across a range of contrast values ( 5 , 10 , 25 , 50 , 100% ) .",
"VEPs were progressively greater in magnitude the greater the contrast of the viewed stimulus ( 2-way repeated measures ANOVA , n = 4 mice , effect of contrast , F ( 4 , 12 ) = 25 . 908 , P < 0 . 001 ) both before and during PV+ neuronal activation using the hM4D ( Gi ) system ( SNK post hoc test , 5 vs . 100 percent contrast , baseline; q ( 4 ) = 5 . 178 , P = 0 . 013; CNO; q ( 4 ) = 17 . 595 , P < 0 . 001 , Figure 4A ) .",
"Preservation of the approximately linear relationship of contrast and response during PV+ neuron inactivation indicates that responses have not exceeded their dynamic range . 10 . 7554/eLife . 11450 . 011Figure 4 . Expression of SRP to two separate contrast values is blocked by hM4D ( Gi ) -mediated PV+ neuron inactivation , but differential response to contrast is maintained .",
"( A ) CNO delivery to PV-Cre mice that had been infected with AAV9-hSyn-DIO-hM4D ( Gi ) -mCitrine impacted VEP magnitude ( red ) significantly across a range of contrast compared to baseline ( black ) .",
"( B ) SRP was induced to two differently oriented stimuli , each at different contrast values: 50% ( gray ) and 100% ( black ) .",
"Modest but significant SRP was expressed at 50% contrast prior to CNO application .",
"After CNO application ( red outlines ) , VEPs increased significantly in magnitude but were no longer significantly different for familiar and a second novel orientation .",
"( C ) SRP was also expressed for a different orientation at 100% contrast and , again , VEP magnitude was increased and SRP blocked by delivery of CNO .",
"( D ) The blockade of SRP expression by CNO at 50% contrast was apparent in the reduction in the familiar/novel ratio for VEP magnitude .",
"( E ) The blockade of SRP expression at 100% contrast was also observed as a significant drop in familiar/novel ratio of VEP magnitude after CNO delivery .",
"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01110 . 7554/eLife . 11450 . 012Figure 4—source data 1 . PV-neuron inactivation does not produce ceiling effect . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 012 We then induced SRP to different orientations in a different set of mice , one stimulus at 50% contrast and the other stimulus at 100% contrast .",
"After 6 days of SRP at 50% , there was a modest but significant difference in VEP magnitude for familiar ( 188 . 81 ± 11 . 80 µV ) and novel orientations ( 145 . 06 ± 8 . 11 µV , 2-way repeated measures ANOVA , n = 8 mice , SNK post hoc test , q ( 7 ) = 3 . 608 , P = 0 . 023 , Figure 4B ) , just as there was for the familiar ( 259 . 81 ± 17 . 66 µV ) and novel orientations ( 192 . 81 ± 17 . 27 µV , SNK post hoc test , q ( 7 ) = 3 . 793 , P = 0 . 018 , Figure 4C ) at 100% contrast .",
"SRP expression was abolished by PV+ neuronal inactivation using the hM4D ( Gi ) system at 50% contrast as VEPs elicited by familiar ( 404 . 69 ± 59 . 17 µV ) and novel orientations ( 376 . 94 ± 56 . 79 µV ) were no longer significantly different ( SNK post hoc test , q ( 7 ) = 2 . 289 , P = 0 . 128 , Figure 4B ) .",
"The same was true at 100% contrast as VEPs evoked by the familiar ( 532 . 44 ± 63 . 82 µV ) and novel orientations ( 514 . 88 ± 40 . 38 µV ) were also no longer significantly different ( SNK post hoc test , q ( 7 ) = 0 . 994 , P = 0 . 494 , Figure 4C ) .",
"Again , the blockade of SRP expression was also clearly observed in the familiar/novel ratio , which dropped significantly from ( 1 . 31 ± 0 . 05 ) to ( 1 . 08 ± 0 . 08 ) with CNO application at 50% contrast ( student’s paired two-tailed t-test , t ( 7 ) = 2 . 983 , P = 0 . 02 , Figure 4D ) and from ( 1 . 40 ± 0 . 10 ) to ( 1 . 02 ± 0 . 05 ) with CNO application at 100% contrast ( student’s paired two-tailed t-test , t ( 7 ) = 2 . 955 , P = 0 . 021 , Figure 4E ) .",
"Thus , SRP could still be abolished through selective loss of PV+ neuron activity even at reduced contrasts eliciting submaximal responses .",
"The disruption of SRP expression by inactivation of PV+ neurons is not a trivial consequence of a “ceiling effect” .",
"PV+ neurons have been implicated in the sharpening of orientation selectivity in V1 ( Runyan et al . , 2010; Adesnik et al . , 2012; Atallah et al . , 2012; Lee et al . , 2012; Wilson et al . , 2012 ) .",
"If orientation selectivity were completely abolished by inactivation of PV+ neurons then the loss of SRP expression could be attributed to a failure of orientation discrimination rather than familiarity .",
"A previous experiment has indicated that activation of PV+ neurons in V1 actually mildly enhances orientation-selectivity and visual discrimination ( Lee et al . , 2012 ) .",
"Therefore , we used optogenetics to activate PV+ neurons while mice were presented with familiar and novel stimuli to test whether SRP expression would be enhanced , unaffected or disrupted .",
"PV+ neurons in binocular V1 of PV-Cre mice expressed Channel-rhodopsin 2 ( ChR2 ) as a result of infection with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP .",
"During the same surgery VEP recording electrodes were implanted and optic fibers chronically implanted to deliver light to the recording site ( Figure 5A ) .",
"After stable expression 4 weeks after infection , mice were habituated to head-fixation on each of 2 days before undergoing a standard SRP experiment over 6 days ( Figure 5B , C ) .",
"Mice showed a significant increase in the magnitude of the average VEP evoked by the familiar oriented grating stimulus over days ( Friedman repeated measures ANOVA on ranks , n = 11 mice , X2 ( 5 ) = 24 . 818 , P < 0 . 001 , Figure 5C ) .",
"This potentiation was evident by day 2 ( 237 . 55 ± 29 . 01 µV ) in comparison with day 1 ( 182 . 59 ± 21 . 97 µV , SNK post hoc test , q ( 10 ) = 7 . 675 , P < 0 . 05 ) .",
"On day 7 , mice were presented with interleaved blocks of familiar and novel stimuli .",
"Also interleaved were blocks of familiar and novel stimuli in which blue light ( 473 nm ) was continuously delivered via the optic fiber to the recording site in binocular V1 .",
"VEPs elicited by the novel X + 90° stimulus were significantly lower in magnitude ( 221 . 34 ± 33 . 55 µV ) than those elicited by the familiar stimulus prior to optogenetic activation of PV+ neurons ( 310 . 70 ± 36 . 70 µV , 2-way repeated measures ANOVA , n = 11 mice , SNK post hoc test , q ( 10 ) = 11 . 799 , P < 0 . 001 , Figure 5D ) .",
"Optogenetic activation of PV+ neurons significantly diminished the selectivity of SRP , as VEPs elicited by the familiar stimulus ( 181 . 23 ± 26 . 43 µV ) and novel stimulus ( 157 . 80 ± 21 . 05 µV ) were more similar in magnitude ( interaction of stimulus x laser , F ( 1 , 10 ) = 46 . 606 , P < 0 . 001; familiar vs . novel SNK post hoc test , q ( 10 ) = 3 . 094 , P = 0 . 045 , Figure 5D ) .",
"The reduction of SRP selectivity is most clearly observed in the significant difference in the familiar/novel ratio without ( 1 . 55 ± 0 . 14 ) and with optogenetic stimulation ( 1 . 16 ± 0 . 07 , student’s paired two-tailed t-test , t ( 10 ) = 4 . 423 , P = 0 . 001 , Figure 5E ) .",
"Laser stimulation did not affect the stimulus selectivity of SRP in wild type animals infected with virus ( Figure 5—figure supplement 1 ) , as there was no significant interaction between stimulus and laser ( 2-way repeated measures ANOVA , F ( 1 , 8 ) = 0 . 677 , P = 0 . 434 ) .",
"These mice underwent SRP ( Figure 5—figure supplement 1A ) and on test day showed significantly larger VEPs to the familiar stimulus , both with the laser off ( familiar VEP: 273 . 69 ± 28 . 25 µV , novel VEP: 200 . 19 ± 19 . 53 µV , 2-way repeated measures ANOVA , n = 9 mice , SNK post hoc test , q ( 8 ) = 5 . 234 , P = 0 . 005 ) and the laser on ( familiar VEP: 277 . 08 ± 29 . 80 µV , novel VEP: 194 ± 18 . 39 µV , q ( 8 ) = 5 . 916 , P = 0 . 002 , Figure 5—figure supplement 1B ) .",
"Thus , SRP expression was disrupted with activation of PV+ neurons , just as it was with inactivation ( Figure 3D–E ) .",
"This result implies that PV+ neurons play a specific role in SRP expression beyond any role in enhancing orientation tuning . 10 . 7554/eLife . 11450 . 013Figure 5 . Optogenetic stimulation of PV+ inhibitory neurons prevents SRP expression .",
"( A ) Blue light was delivered locally into V1 via optic fibers chronically implanted at a 45° angle to target the VEP recording site in layer 4 of binocular V1 of PV-Cre mice infected with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP .",
"( B ) Experimental timeline showing that after viral infection , electrode implantation , and ChR2 expression; mice were accustomed to head-fixation and gray screen viewing .",
"Subsequently , they underwent a standard SRP induction protocol over 6 days .",
"On day 7 , mice viewed a novel oriented stimulus in addition to the familiar stimulus and , on 50% of presentations of each stimulus , blue light ( 473 nm ) was delivered to cortex to optogenetically activate PV+ cells .",
"( C ) Significant SRP was induced over 6 days as VEPs underwent a typical potentiation .",
"( D ) On day 7 , SRP was expressed through significantly larger VEP magnitude in response to the familiar Xo orientation than a novel X + 90° stimulus when blue light was not delivered ( Black bars ) .",
"In the presence of blue light ( blue bars ) , VEPs were suppressed , and there was a significant reduction in the differential magnitude of VEPs driven by familiar and novel stimuli .",
"( E ) The ratio of VEP magnitude elicited by familiar/novel stimuli was significantly reduced by optogenetic activation of PV+ neurons , reflecting a decrement in SRP expression .",
"Significant comparisons are marked with an asterisk and post hoc test p values are reported in D to emphasize the impact of laser stimulation on SRP selectivity .",
"Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01310 . 7554/eLife . 11450 . 014Figure 5—source data 1 . PV-neuronal activation . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01410 . 7554/eLife . 11450 . 015Figure 5—figure supplement 1 . Blue light has no impact on SRP expression .",
"( A ) To check that light itself had no effect on cortical physiology in the optogenetic experiments , WT mice were infected bilaterally with AAV5-EF1α -DIO-hChR2 ( H134R ) -eYFP in binocular V1 .",
"After 4 weeks , during which ChR2 was expressed at high levels in PV-Cre mice , WT mice were taken through the standard SRP protocol described above .",
"Again , significant SRP occurred .",
"( B ) On test day , during interleaved presentations of a familiar and novel oriented stimulus together with either blue light ( 473 nm ) or no light stimulation delivery to V1 , VEPs were significantly greater in magnitude for the familiar than the novel stimulus , regardless of the presence of blue light .",
"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01510 . 7554/eLife . 11450 . 016Figure 5—figure supplement 1—source data 1 . Laser does not effect VEPs in WT animals . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 016 Multiple lines of evidence indicate that SRP is dependent upon the NMDA class of glutamate receptors ( Frenkel et al . , 2006; Cooke et al . , 2015 ) .",
"Given the additional clear requirement for normal PV+ inhibitory cell function in SRP ( Figure 3D , E ) we selectively ablated the NMDAR from just PV+ cells by crossing the PV-Cre line of mice with a line in which the mandatory GluN1 subunit of NMDAR is excised by Cre recombinase activity ( B6 . 129S4-Grin1tm2Stl/J , GluN1 fl/fl ) .",
"The progeny of this cross , in which both alleles of Grin1 ( the gene encoding GluN1 ) were floxed , are henceforth described as either PV-GluN1 KO or Wildtype ( WT ) -GluN1 fl/fl depending on whether , respectively , Cre was expressed or not .",
"We implanted PV-GluN1 KO ( n = 14 ) and littermate WT-GluN1 fl/fl mice ( n = 17 ) with VEP recording electrodes in layer 4 , binocular V1 .",
"After recovery and 2 daily sessions of habituation we recorded VEPs elicited by an Xo oriented grating stimulus .",
"Immediately apparent was the significantly greater basal magnitude of VEPs recorded in the PV-GluN1 mice ( 242 . 45 ± 20 . 20 µV ) relative to their littermate controls ( 130 . 88 ± 9 . 69 µV , student’s two-tailed t-test , t ( 29 ) = -5 . 269 , P < 0 . 001 ) , consistent with the occurrence of disinhibition as a result of reduced glutamatergic drive on cortical PV+ inhibitory cells ( Figure 6A ) .",
"We then presented the same stimulus to these mice over several consecutive days and observed a significant difference in SRP across genotypes ( 2-way repeated measures ANOVA , interaction of genotype x day , F ( 4 , 116 ) = 3 . 835 , P = 0 . 006 , Figure 6A ) .",
"Beyond day 3 , VEP magnitudes were no longer significantly different between the PV-GluN1 KO mice ( 289 . 15 ± 24 . 57 µV ) and WT-GluN1 fl/fl littermates ( 239 . 94 ± 20 . 72 µV , SNK post hoc test , q ( 29 ) = 2 . 242 , P = 0 . 119 ) , suggesting an occlusion of SRP by the already elevated basal VEP magnitude in the PV-GluN1 KO mice .",
"The significant deficit in SRP is clearly apparent when the data are normalized to day 1 values ( 2-way repeated measures ANOVA , interaction of genotype x day , F ( 4 , 116 ) = 12 . 326 , P < 0 . 001 , Figure 6B ) .",
"Again , a significant deficit in SRP emerged by day 3 in the PV-GluN1 KO mice ( 119 . 26 ± 10 . 13% day 1 ) compared with WT-GluN1 fl/fl littermates ( 183 . 33 ± 15 . 83% day 1 , SNK post hoc test , q ( 29 ) = 4 . 727 , P = 0 . 002 ) , demonstrating that SRP is compromised by a loss of NMDAR expressed in PV+ neurons . 10 . 7554/eLife . 11450 . 017Figure 6 . Loss of NMDA receptors selectively from parvalbumin+ cells impacts SRP but not adult OD plasticity .",
"( A ) VEPs recorded from mice in which the mandatory GluN1 subunit of the NMDA receptor was genetically ablated from PV+ cells using Cre recombinase technology ( PV GluN1 KO , gray ) were significantly greater in magnitude than those recorded from WT littermates ( black ) , suggesting disinhibition of the visual response .",
"In these same PV GluN1 KO mice , SRP was also significantly impacted as there is was significantly less gain in magnitude over days of repeated presentation of an X° stimulus than observed in WT littermates .",
"( B ) This significant reduction in the magnitude of SRP was most clearly observed if VEP magnitude was normalized to the magnitude on day 1 .",
"( C ) After SRP , both PV GluN1 KO mice and their WT littermates exhibited a significantly greater VEP magnitude elicited by the now familiar stimulus than interleaved presentations of a novel oriented stimulus .",
"However , consistent with the observed difference in magnitude on day 1 , VEPs elicited by a novel X + 90° stimulus in PV GluN1 KO mice were significantly greater in magnitude than those in WT littermate mice .",
"No significant difference was observed for VEPs elicited by the familiar stimulus .",
"( D ) A significant difference in the ratio of VEP magnitude elicited by the familiar and novel stimuli reveals a deficit in SRP expression in PV GluN1 KO mice .",
"( E ) In contrast , PV GluN1 KO mice exhibited a normal adult OD shift after 7 days of MD , resulting from open eye potentiation ( yellow outlines ) .",
"( F ) Wild-type ( WT ) littermates exhibited the same significant open eye potentiation ( yellow bars ) after 7 days of MD . ( G ) A comparison of the degree of OD shift as a result of 7 days of MD reveals a significant shift in OD ratio in both genotypes but no difference between genotypes , indicating that NMDA receptors in PV+ cells are not required for induction or expression of the OD shift .",
"( H ) To confirm genetic ablation of NMDARs selectively from parvalbumin ( PV+ ) neurons in the PV-GluN1 KO; mice were injected with an AAV5 vector to express GFP in a Cre-dependent fashion in PV+ cells only .",
"After 1 month , fluorescence-guided intracellular recordings were performed from ex vivo slices of visual cortex .",
"NMDAR-mediated synaptic transmission was normal in excitatory control cells but abolished in PV+ cells .",
"This is expressed here as the NMDAR EPSC/AMPAR EPSC ratio in excitatory cells ( black outline ) and PV+ cells ( green outline ) .",
"Sample EPSC traces mediated by the AMPAR ( downward ) and NMDAR ( upward ) are shown at the top of the panel .",
"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01710 . 7554/eLife . 11450 . 018Figure 6—source data 1 . PV-GluN1 KODOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 018 We also tested for the stimulus-selectivity of SRP expression by presenting both groups of animals with interleaved blocks of the familiar Xo stimulus and a novel X + 90° stimulus ( Figure 6C ) .",
"Significant stimulus selectivity was present in both genotypes ( 2-way repeated measures ANOVA , stimulus , F ( 1 ) = 67 . 397 , P < 0 . 001 , interaction of genotype x stimulus , F ( 1 , 29 ) = 2 . 359 , P = 0 . 135 , Figure 6C ) : Although PV-GluN1 KO mice showed significant differences in VEP magnitude for familiar ( 340 . 24 ± 27 . 96 µV ) and novel orientations ( 242 . 54 ± 24 . 40 µV , SNK post hoc test , q ( 13 ) = 6 . 372 , P < 0 . 001 ) the difference was more pronounced in the WT-GluN1 fl/fl mice ( SNK post hoc test , q ( 16 ) = 10 . 254 , P < 0 . 001 ) , in which the familiar stimulus elicited VEPs 318 . 74 ± 25 . 19 µV in magnitude and the novel stimulus elicited VEPs 176 . 06 ± 11 . 55 µV in magnitude .",
"The familiar stimulus evoked VEPs that were not significantly different in the WT-GluN1 fl/fl mice ( 318 . 74 ± 25 . 19 µV ) and the PV-GluN1 KO mice ( 340 . 24 ± 27 . 96 µV , SNK post hoc test , q ( 29 ) = 0 . 947 , P = 0 . 507 ) but those evoked in the WT-GluN1 fl/fl by the novel stimulus ( 176 . 06 ± 11 . 55 µV ) were significantly lower in magnitude than in the PV-GluN1 KO mice ( 242 . 54 ± 24 . 40 µV , SNK post hoc test , q ( 29 ) = 2 . 926 , P = 0 . 045 ) .",
"The significant deficit in SRP expression in the PV-GluN1 KO mice is most apparent when the familiar/novel ratio of VEP magnitude ( 1 . 48 ± 0 . 13 ) is compared with the WT-GluN1 fl/fl littermates ( 1 . 83 ± 0 . 13 , Mann Whitney rank sum test , U = 60 . 000 , P = 0 . 020 , Figure 6D ) .",
"Thus , loss of NMDAR function selectively within PV+ neurons impairs the full expression of SRP .",
"We also examined if loss of NMDAR from PV+ neurons has an effect on OD plasticity .",
"We implanted VEP recording electrodes in a second cohort of 11 PV–GluN1 KO and 7 WT-GluN1 fl/fl mice .",
"After recovery and habituation over 2 days , monocular VEPs were acquired through each eye .",
"After 7 days of MD , PV-GluN1 KO mice exhibited a significant potentiation of response through the open ipsilateral eye ( 187 . 91 ± 25 . 52 µV ) compared with day 0 ( 90 . 64 ± 10 . 69 µV , 2-way repeated measures ANOVA , SNK post hoc test , q ( 16 ) = 8 . 849 , P < 0 . 001 , Figure 6E ) .",
"Similarly , WT-GluN1 fl/fl mice also showed a significant potentiation of the open ipsilateral eye-response ( 154 . 57 ± 7 . 60 µV ) after 7 days of MD compared with responses measured on day 0 ( 97 . 57 ± 15 . 85 µV , SNK post hoc test , q ( 16 ) = 4 . 137 , P = 0 . 01 , Figure 6F ) .",
"Comparisons of OD ratios prior to MD in the adult PV-GluN1 KO ( 3 . 40 ± 0 . 29 ) and WT-GluN1 fl/fl mice ( 2 . 57 ± 0 . 33 ) did not reveal any significant difference ( 2-way repeated measures ANOVA , effect of genotype , F ( 1 ) = 3 . 424 , P = 0 . 083 ) .",
"Significant shifts in the OD ratio occurred as a result of 7 days of MD in the adult PV-GluN1 KO ( 1 . 28 ± 0 . 33 , SNK post hoc test , q ( 16 ) = 10 . 6 , P < 0 . 001 ) and WT-GluN1 fl/fl mice ( 1 . 19 ± 0 . 09 , SNK post hoc test , q ( 16 ) = 5 . 517 , P = 0 . 001 ) .",
"The shifted OD ratio also did not differ significantly across genotype ( 2-way repeated measures ANOVA , interaction of genotype and MD , F ( 1 , 16 ) = 2 . 638 , P = 0 . 124 , Figure 6G ) .",
"Thus , the loss of NMDAR function from PV+ neurons did not have a significant impact on either the induction or the expression of adult OD plasticity , in contrast to SRP .",
"In order to confirm the removal of NMDARs selectively from PV+ cells , the PV-GluN1 KO mice were injected with an AAV5 vector to express GFP in a Cre-dependent fashion in PV+ cells only .",
"After 1 month , fluorescence-guided intracellular recordings were performed from ex vivo slices of visual cortex .",
"NMDAR-mediated synaptic transmission was normal in excitatory control cells but abolished in PV+ neurons ( Figure 6H ) .",
"This is expressed as significantly reduced NMDAR EPSC/AMPAR EPSC ratio in PV+ cells ( 0 . 040 ± 0 . 011 , n = 8 cells from 5 mice ) compared to neighboring non-fluorescent excitatory control cells ( 0 . 345 ± 0 . 039 , n = 10 cells from 4 mice , student’s one-tailed t-test , t ( 16 ) = -6 . 819 , P < 0 . 001 ) .",
"A group of non-competitive , open-channel NMDAR blockers , including ketamine , PCP and MK801 are known to have the paradoxical impact of increasing net neuronal activity in the brain .",
"It is thought that this apparent disinhibition arises from the preferential impact of these molecules on fast-spiking neurons , due to the tonic activation and the increased open-time of NMDAR expressed within these cells ( Homayoun and Moghaddam , 2007; Seamans , 2008 ) .",
"Interestingly , these compounds are also psychotomimetic and can reproduce , at high sub-anesthetic doses , most of the symptoms of schizophrenia ( Krystal et al . , 1994 ) .",
"Here we tested the possibility that a single acute dose of one of these substances , ketamine , would have an impact on the expression of SRP due to its action on NMDAR expressed in PV+ fast spiking inhibitory neurons .",
"We implanted VEP recording electrodes in layer 4 of binocular V1 in a group of 10 C57BL/6 mice .",
"After recovery and a standard SRP protocol we recorded VEP magnitudes driven by familiar and novel stimuli before , during and 2 days after recovery from systemic injection ( i . p . ) of a high but sub-anesthetic dose of ketamine ( 50 mg/kg ) ( Figure 7A ) .",
"Ketamine had a significant effect on SRP expression ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F ( 2 , 18 ) = 29 . 479 , P < 0 . 001 , Figure 7B ) .",
"After SRP but prior to ketamine delivery the familiar Xo stimulus elicited VEPs of significantly greater magnitude ( 245 . 47 ± 21 . 00 µV ) than a novel X + 60° stimulus ( 138 . 86 ± 9 . 32 µV , SNK post hoc test , q ( 9 ) = 9 . 228 , P < 0 . 001 ) in these mice .",
"After an hour of respite from head-fixation , mice were injected with ketamine and , 15 min later , they were returned to head-fixation and VEP magnitudes were again recorded .",
"Under the influence of ketamine , the familiar Xo stimulus elicited VEPs of significantly increased magnitude ( 381 . 90 ± 37 . 25 µV ) relative to pre ketamine ( 245 . 47 ± 21 . 00 µV , SNK post hoc test , q ( 9 ) = 7 . 223 , P < 0 . 001 ) .",
"However , VEPs elicited by a second novel X - 60° stimulus were increased relative to pre-ketamine by an even greater extent ( 397 . 53 ± 37 . 62 µV ) relative to pre ketamine ( 138 . 86 ± 9 . 32 µV , SNK post hoc test , q ( 9 ) = 13 . 695 , P < 0 . 001 ) , such that VEPs were no longer significantly different in magnitude in response to familiar and novel stimuli ( SNK post hoc test , q ( 9 ) = 1 . 353 , P = 0 . 349 ) .",
"After 2 day’s rest , allowing for complete recovery from drug effects , the mice were returned to the head-fixation apparatus and exposed to the familiar Xo stimulus and a third novel stimulus , X + 90° .",
"Just as observed prior to the ketamine injection , the Xo stimulus evoked VEPs of significantly greater magnitude ( 267 . 46 ± 26 . 13 µV ) than the novel X + 90° stimulus ( 149 . 40 ± 9 . 85 µV , q ( 9 ) = 10 . 219 , P < 0 . 001 ) ( Figure 7A and B ) . 10 . 7554/eLife . 11450 . 019Figure 7 . Ketamine prevents expression of SRP through blockade of NMDA receptors expressed in parvalbumin+ neurons but does not impact expression of the adult OD shift .",
"( A ) Mice were bilaterally implanted with VEP recording electrodes in layer 4 of binocular V1 .",
"After habituation to head-fixation and a gray screen for 2 days , SRP was induced over 4 days by repeatedly presenting sessions of an X° stimulus .",
"On day 5 , SRP expression was tested by presenting interleaved blocks of the familiar X° stimulus and a novel X + 60° stimulus .",
"In order to test the acute impact of blocking NMDA receptors on SRP expression , 50 mg/kg of ketamine was then delivered systemically 15 min before re-acquiring VEPs elicited by the familiar X° stimulus and interleaved presentations of a second novel X - 60° stimulus .",
"Mice were then allowed 2 days recovery and a complete washout of ketamine before re-testing SRP expression by again testing VEP magnitude in response to the familiar X° stimulus and a third novel X + 90° stimulus on day 7 .",
"( B ) Significant SRP was expressed on experimental day 5 prior to ketamine delivery as the familiar X° stimulus elicited VEPs of greater magnitude than the novel stimulus .",
"Delivery of 50 mg/kg ketamine ( purple ) had two notable impacts on the VEP: First , the overall magnitude of the VEP increased .",
"Second , and most importantly , the significant difference in magnitude of VEPs elicited by familiar and novel stimuli was no longer present .",
"This effect was acute , as SRP expression was again significantly apparent 2 days later .",
"( C ) The ratio of VEP magnitude elicited by the familiar stimulus over the novel .",
"This ratio was close to 2 and not significantly different prior to or after recovery from ketamine administration but dropped significantly to approximately 1 during ketamine exposure .",
"( D ) We tested whether ketamine had a differential impact on VEP magnitude in PV GluN1 KO mice and WT littermate mice .",
"( E ) In the WT littermate mice ( white bars ) 50 mg/kg ketamine had a significant potentiating effect on VEP magnitude , consistent with our previous observation .",
"In contrast , ketamine had no significant impact on VEP magnitude in the PV GluN1 KO mice ( gray bars ) .",
"( F ) The selectivity of ketamine’s impact on the WT mice is observed by comparing the ratio of VEP magnitude during ketamine over baseline , which was significantly greater for WT mice than the PV GluN1 KO mice , in which the ratio was approximately 1 .",
"( G ) In a separate group of mice , a similar protocol was then used to determine whether the OD ratio is affected by ketamine .",
"( H ) Ketamine impacted both the VEPs driven through the contralateral eye ( blue ) and ipsilateral eye ( yellow ) equally .",
"( I ) This scaled effect is demonstrated by a lack of significant difference between OD ratios prior to ( white ) and during 50 mg/kg ketamine ( purple ) .",
"( J ) We next tested whether ketamine has any impact on the expression of adult OD plasticity by recording VEP magnitudes through either eye in a new group of adult mice before taking them through a standard 7 day MD protocol .",
"( K ) As anticipated , 7 days of contralateral eye MD induced a significant ipsilateral eye potentiation ( yellow ) and ketamine then further potentiated VEPs elicited through both contralateral ( blue ) and ipsilateral eyes .",
"( L ) The OD ratio shifts significantly from a ratio heavily biased towards the contralateral eye , to a less biased ratio .",
"Ketamine administration did not significantly affect the magnitude of the OD shift .",
"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 01910 . 7554/eLife . 11450 . 020Figure 7—source data 1 . Ketamine impact on cortical plasticity . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 020 This significant effect of ketamine on the discrimination of familiar and novel stimuli is summarized by the ratio of VEP magnitude driven by the familiar/novel stimulus ( 1-way repeated measures ANOVA , F ( 2 , 18 ) = 24 . 683 , P < 0 . 001 , Figure 7C ) .",
"This familiar/novel ratio dropped significantly from 1 . 76 ± 0 . 10 prior to ketamine to 0 . 96 ± 0 . 02 after ketamine ( SNK post hoc test , q ( 9 ) = 8 . 443 , P < 0 . 001 ) .",
"Upon recovery , the familiar/novel ratio significantly recovered ( 1 . 79 ± 0 . 16 , SNK post hoc test , q ( 9 ) = 8 . 759 , P < 0 . 001 ) and was not significantly different from the first test after SRP but prior to ketamine injection ( SNK post hoc test , q ( 9 ) = 0 . 316 , P = 0 . 826 ) .",
"Thus , the psychotomimetic non-competitive NMDAR antagonist ketamine disrupts SRP .",
"The fact that this is an acute effect on already established SRP that recovers after drug washout indicates that the role for NMDAR in PV+ fast-spiking GABAergic neurons may be in memory retrieval rather than learning .",
"Ketamine blocks NMDAR expressed in all cell types throughout the CNS and is also known to have targets other than the NMDAR ( Chen et al . , 2009 ) .",
"In order to determine if ketamine has its effect on V1 responses specifically through NMDAR expressed in PV+ cells , we tested if there was a differential effect of ketamine on VEP magnitude in PV-GluN1 KO mice and WT-GluN1 fl/fl mice .",
"After a typical implantation and habituation protocol ( Figure 7D ) we tested VEP magnitudes elicited by a novel oriented Xo stimulus in WT-GluN1 fl/fl mice ( 178 . 11 ± 38 . 41 µV , n = 8 ) and PV-GluN1 KO mice ( 260 . 79 ± 50 . 75 µV , n = 8 ) ( Figure 7E ) .",
"We then removed mice from head-fixation and allowed them to recover in their home-cage before delivering 50 mg/kg ketamine ( i . p . ) .",
"After 15 min , the mice were returned to head-fixation and we observed the impact of ketamine on VEPs elicited by a novel X + 90° oriented stimulus , which was significantly different in its effect on the two genotypes ( 2-way repeated measures ANOVA , interaction of genotype x treatment , F ( 1 , 14 ) = 18 . 454 , P < 0 . 001 ) .",
"In the control WT-GluN1 fl/fl mice there was a significant potentiation of VEP magnitude as a result of ketamine application ( 386 . 65 ± 70 . 75 µV ) in comparison to pre treatment ( 178 . 11 ± 38 . 41 µV , SNK post hoc test , q ( 7 ) = 8 . 505 , P < 0 . 001 , Figure 7E ) , replicating our previous finding ( Figure 7B ) .",
"However , in the PV-GluN1 KO mice , ketamine had no ostensible significant impact ( 258 . 67 ± 44 . 86 µV ) in comparison to pre treatment ( 260 . 79 ± 50 . 75 µV , SNK post hoc test , q ( 7 ) = 0 . 087 , P = 0 . 952 ) .",
"A ratio of VEP magnitudes pre and post ketamine treatment reveals the significant difference between ketamine’s action on WT-GluN1 fl/fl mice ( 2 . 40 ± 0 . 25 ) and PV-GluN1 KO mice ( 1 . 12 ± 0 . 13 , student’s two-tailed t-test , t ( 14 ) = 4 . 610 , P < 0 . 001 ) ( Figure 7F ) .",
"Thus , while ketamine has a wide range of effects in the CNS , it exerts itself on the response of V1 to visual input selectively through NMDAR expressed in PV+ cells .",
"We then tested whether or not ketamine would disrupt either the OD ratio or the shift induced by 7 days of MD in the adult mouse .",
"Again , C57BL/6 mice ( n = 8 ) were implanted with VEP electrodes and taken through a standard surgery recovery and habituation protocol before measuring the OD ratio prior to 50 mg/kg ketamine and 15 min after ketamine delivery ( Figure 7G and H ) .",
"The normal contra/ipsi OD ratio exhibiting contralateral eye dominance prior to ketamine ( 2 . 84 ± 0 . 18 ) was not significantly altered by ketamine ( 2 . 61 ± 0 . 19 , student’s paired two-tailed t-test , t ( 7 ) = 0 . 871 , P = 0 . 413 ) ( Figure 7I ) .",
"A separate group of mice ( n = 11 ) then underwent a standard 7 day MD protocol .",
"VEPs elicited by an Xo oriented stimulus were recorded at baseline , followed by the deprivation period .",
"After eye opening VEP magnitudes were re-tested with a novel X + 60°stimulus ( the standard protocol to avoid contamination of the OD shift by SRP [Frenkel and Bear , 2004] ) .",
"These mice were then tested again with a second novel X - 60°stimulus after 1 hr rest and an additional 15 min after systemic 50 mg/kg ketamine administration ( Figure 7J ) .",
"As expected , VEPs elicited through the open ipsilateral eye ( 93 . 73 ± 12 . 85 µV ) were significantly potentiated by 7 days of MD in the adult mouse ( 155 . 73 ± 15 . 86 µV , 2-way repeated measures ANOVA , SNK post hoc test , n = 11 mice , q ( 10 ) = 7 . 102 , P < 0 . 001 ) , reflecting the well-documented OD shift .",
"These same ipsilateral VEPs were then further potentiated by ketamine administration ( 232 . 03 ± 22 . 19 µV , SNK post hoc test , q ( 10 ) = 8 . 741 , P < 0 . 001 ) .",
"However , ketamine had a similar significant potentiating effect on the contralateral VEP ( 279 . 07 ± 14 . 53 µV ) , relative to pre-ketamine ( 180 . 14 ± 11 . 22 µV , SNK post hoc test , q ( 10 ) = 11 . 333 , P < 0 . 001 , Figure 7K ) , suggesting a uniform scaling of response through the two eyes .",
"This observation is confirmed by the fact that the OD ratio , significantly shifted from ( 2 . 84 ± 0 . 29 ) to ( 1 . 23 ± 0 . 11 , Friedman repeated measures ANOVA on ranks , n = 11 mice , X2 ( 2 ) = 16 . 545 , p<0 . 001 , SNK post hoc test , q ( 10 ) = 5 . 126 , P < 0 . 05 ) by 7 days of MD , was not further significantly affected by delivery of ketamine ( 1 . 28 ± 0 . 09 , SNK post hoc test , q ( 10 ) = 0 . 426 , P > 0 . 05 , Figure 7L ) .",
"Thus , while ketamine prevents SRP expression through action on NMDAR expressed in PV+ cells , it does not significantly affect the adult OD shift after 7 days of MD , consistent once again with PV+ cells contributing to the expression of SRP but not adult OD plasticity .",
"Given the clear involvement of PV+ neurons in the expression of SRP we wanted to determine if loss of PV+ neuronal function local to V1 would have any behavioral impact .",
"Head-restrained mice viewing a phase reversing visual grating stimulus are known to exhibit a stereotyped motor response called a visually-induced fidget , or vidget ( Cooke et al . , 2015 ) .",
"Vidgets can be measured via a piezoelectric sensor located beneath the forepaws of the mouse ( Figure 8A ) .",
"Importantly , it has been shown that the magnitude of the vidget response is inversely correlated to the familiarity of the visual stimulus .",
"That is , a visual stimulus that is very familiar to the animal will on average evoke a relatively weak vidget behavioral response .",
"In contrast , the presentation of a novel stimulus will on average evoke a vidget of significantly greater magnitude .",
"Therefore , this behavioral response reflects the ability of the animal to discriminate and respond to a novel visual stimulus in its environment .",
"Importantly , genetic and pharmacological manipulations local to V1 that inhibit SRP also disrupt the behavioral discrimination of familiar and novel stimuli ( Cooke et al . , 2015 ) , demonstrating that this differential vidget response to familiar and novel stimuli is dependent on the plasticity in visual cortex .",
"Since PV+ neuron inactivation disrupted the expression of SRP , we tested whether this manipulation would likewise disrupt visual novelty detection . 10 . 7554/eLife . 11450 . 021Figure 8 . Discrimination of familiar and novel oriented stimuli involves PV+ neurons in V1 and NMDAR expressed within PV+ neurons .",
"( A ) Using the same protocol as described in Figure 3B , mice were progressively familiarized with a specific oriented stimulus .",
"On the test day , as mice viewed familiar and novel stimuli , vidget behavioral responses were measured via a piezoelectric sensor located beneath the forepaws of the head-fixed mouse .",
"( B ) After a standard SRP protocol , mice expressing hM4D ( Gi ) receptors selectively within PV+ cells of binocular V1 were exposed to both familiar and novel stimuli .",
"Prior to application of CNO , mice exhibited significant behavioral evidence of discriminating this familiar orientation from interleaved presentations of a novel oriented stimulus ( black bars ) .",
"After application of CNO , there was no longer successful discrimination of familiar and novel stimuli ( red bars ) .",
"Averaged vidget responses are displayed at the top of this panel .",
"( C ) A significant difference was observed in the ratio of response to familiar and novel stimuli from pre CNO ( black ) to post CNO ( red ) .",
"( D ) A deficit in OSH was also apparent in PV GluN1 KO mice as vidget recordings demonstrated a failure to significantly discriminate familiar from novel orientations ( gray bars ) .",
"WT littermates exhibited significantly greater vidget magnitudes for novel than familiar stimuli , indicating unimpaired discrimination of familiarity from novelty ( white bars ) .",
"Averaged behavioral responses are displayed above with accompanying scale bars .",
"( E ) The significant deficit of PV GluN1 KO mice in discriminating familiar from novel stimuli is apparent in the ratio of behavior elicited by the familiar over the novel stimulus in comparison to WT littermates .",
"Significant comparisons are marked with an asterisk throughout while non-significant comparisons are marked with n . s . Error bars are standard error of the mean ( S . E . M . ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02110 . 7554/eLife . 11450 . 022Figure 8—source data 1 . Novelty detection after PV+ neuronal disruption . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02210 . 7554/eLife . 11450 . 023Figure 8—figure supplement 1 . Cumulative distributions of average vidget behavioral response to familiar and novel stimuli for each individual animal included in analysis presented in Figure 8D and Figure 8E .",
"( A ) Prior to CNO administration , average behavioral response to the familiar stimulus ( light gray ) and the novel stimulus ( black ) of each individual animal bilaterally expressing hM4D ( Gi ) in PV+ neurons of binocular V1 .",
"Dotted line represents behavior no greater than pre stimulus baseline .",
"( B ) Average behavioral response of the same animals to the familiar and a new novel stimulus during PV+ neuron inactivation via CNO delivery , revealing consequent deficit in discrimination of familiar and novel stimuli .",
"( C ) Cumulative distributions of average vidget behavioral response to the familiar stimulus ( light gray ) and the novel stimulus ( black ) of each individual WT GluN1 fl/fl mouse .",
"( D ) Average vidget behavioral response of each PV-GluN1 KO mouse to the familiar and a new novel stimulus , revealing deficit in discrimination of familiar and novel stimuli .",
"Dotted line represents behavior no greater than pre stimulus baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 02310 . 7554/eLife . 11450 . 024Figure 8—figure supplement 1—source data 1 . Per animal plots of Novelty detection after PV+ neuronal disruption . DOI: http://dx . doi . org/10 . 7554/eLife . 11450 . 024 A group of PV-Cre mice expressing hM4D ( Gi ) receptors in PV+ cells ( n = 19 ) , underwent a SRP protocol similar to the previous experiment ( Figure 3A ) in which mice viewed phase-reversing gratings of a particular orientation ( Xo stimulus ) each day for 6 days .",
"On the 7th day mice viewed blocks of the now familiar visual stimulus interleaved with blocks of a novel oriented stimulus ( X + 60° ) .",
"On day 7 , vidget behavioral responses were acquired via a piezoelectric device situated underneath the forepaws of the mice ( Figure 8A ) , in order to measure the animal’s discrimination of familiar and novel stimuli .",
"On day 8 , PV+ cells in V1 were then inactivated by systemic delivery of CNO ( i . p . ) and vidget responses were acquired to the familiar Xo stimulus and a second X - 60° novel stimulus .",
"Inactivation of PV+ neurons in V1 significantly affected stimulus discrimination ( 2-way repeated measures ANOVA , interaction of treatment x stimulus , F ( 1 , 18 ) = 13 . 644 , P = 0 . 002 , Figure 8B ) : Prior to CNO , mice exhibited significantly larger vidget responses to the novel visual stimulus ( 3 . 64 ± 0 . 32 a . u . ) than the familiar stimulus ( 1 . 95 ± 0 . 26 a . u . , SNK post-hoc test , q ( 18 ) = 9 . 237 , P < 0 . 001 ) .",
"During PV+ cell inactivation in V1 , behavioral responses to the familiar ( 2 . 01 ± 0 . 16 a . u . ) and novel visual stimuli ( 2 . 46 ± 0 . 22 a . u . ) were no longer significantly different ( SNK post-hoc test , q ( 18 ) = 2 . 503 , P = 0 . 086 ) .",
"The deleterious effect of PV+ cell inactivation in V1 on the animal’s ability to discriminate familiar and novel stimuli is most obvious when a ratio of response to familiar/novel visual stimuli is calculated .",
"Prior to CNO delivery this ratio was significantly lower ( 0 . 54 ± 0 . 04 ) than during CNO treatment ( 1 . 00 ± 0 . 15 , Wilcoxon signed rank test , W = 138 . 000 , Z = 2 . 777 , P = 0 . 004 , Figure 8C ) .",
"These findings indicate that PV+ neuron activity is required not only for the expression of SRP , but also for visual novelty detection .",
"Finally , given the observation that the discrimination of familiar and novel visual stimuli is diminished by inactivation of PV+ neurons in V1 , we tested whether a similar failure would occur in the PV-GluN1 KO mice ( n = 17 ) compared with WT-GluN1 fl/fl littermates ( n = 15 , 2-way repeated measures ANOVA , stimulus , F ( 1 ) = 14 . 632 , P < 0 . 001 , Figure 8D ) .",
"In the PV-GluN1 KO mice , the familiar stimulus produced less behavioral response ( 2 . 75 ± 0 . 42 a . u . ) than a novel stimulus ( 3 . 67 ± 0 . 79 a . u . ) .",
"However , the difference was not significant ( SNK post hoc test , q ( 16 ) = 2 . 570 , P = 0 . 079 ) .",
"In comparison , the concurrently tested WT-GluN1 fl/fl littermate mice showed a suppression of behavioral response to the familiar stimulus ( 1 . 76 ± 0 . 26 a . u . ) relative to the novel stimulus ( 4 . 08 ± 0 . 51 a . u . ) that was significant ( SNK post hoc test , q ( 14 ) = 5 . 008 , P = 0 . 001 ) .",
"This observation was reinforced by a comparison of the ratio of behavior produced by familiar and novel stimuli ( Figure 8E ) , in which there was a significant difference between the PV-GluN1 KO mice ( 0 . 59 ± 0 . 11 ) and their WT-GluN1 fl/fl littermates ( 0 . 94 ± 0 . 14 , student’s one-tailed t-test , t ( 30 ) = -1 . 992 , P = 0 . 028 ) , reflective of the deficit in discrimination of familiar and novel stimuli as a result of lost NMDAR function in PV+ cells .",
"Individual animals’ average vidget responses to familiar and novel stimuli ( Figure 8—figure supplement 1 ) reveal significantly decreased stimulus selectivity subsequent to CNO administration ( Figure 8—figure supplement 1A–B ) , and in the PV GluN1 KO mice as compared to WT-GluN1 fl/fl littermate controls ( Figure 8—figure supplement 1C–D ) .",
"Thus , not only do NMDAR in PV+ cells contribute to SRP expression but they are also involved in its behavioral correlate ."
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"There has been a long-standing interest in the possible roles of PV+ neurons in juvenile OD plasticity .",
"These include modulation of the sensitivity of visual cortex to the effects of MD ( Fagiolini et al . , 1994; Hanover et al . , 1999; Huang et al . , 1999; Chattopadhyaya et al . , 2004 ) and the functional expression of the OD shift ( Maffei et al . , 2006; Yazaki-Sugiyama et al . , 2009; Smith and Bear , 2010 ) .",
"Our findings indicate that expression of OD plasticity in adult mice does not require participation of the PV+ neurons .",
"In interpreting the current findings , it is important to recognize how juvenile and adult OD plasticity differ .",
"In juvenile mice ( < ~P35 ) , a rapid and reliable consequence of MD is depression in cortex of responses mediated by the deprived eye .",
"This is followed by a progressive compensatory increase in responses through the non-deprived eye ( Frenkel and Bear , 2004 ) .",
"In adult mice maintained under standard laboratory conditions , depression of deprived-eye responses can be a weak and variable consequence of MD , but potentiation of the non-deprived eye still occurs reliably ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) .",
"This deprivation-enabled response potentiation is not mediated by homeostatic synaptic scaling because it requires visual experience through the non-deprived eye ( Blais et al . , 2008 ) , activation of cortical NMDA receptors ( Sawtell et al . , 2003; Sato and Stryker , 2008 ) , and is unaffected ( in adults ) by genetic deletion of TNFα that abolishes scaling ( Ranson et al . , 2012 ) .",
"These features are consistent with an alternative hypothesis that the adult OD shift occurs because deprivation of the dominant contralateral eye causes a metaplastic shift in the LTP threshold , enabling visual experience through the weaker ipsilateral eye to drive “Hebbian” synaptic strengthening ( Cooper and Bear , 2012 ) .",
"This interpretation is supported by evidence that light deprivation promotes LTP in visual cortex ( Kirkwood et al . , 1996; Philpot et al . , 2001; 2003 ) and that αCaMKII mutants that lack LTP also lack adult OD plasticity ( Ranson et al . , 2012 ) , and it is compatible with our finding that expression of the adult OD shift is not dependent on the activity of PV+ inhibitory neurons .",
"Saiepour and colleagues also recently observed that ocular dominance index ( ODI ) values of V1 neurons are not altered by PV+ interneuron suppression in monocularly deprived adult mice ( Saiepour et al . , 2015 ) .",
"Using single unit recordings they first showed that monocular deprivation caused a shift in ODI values towards the non-deprived eye , as expected .",
"They then tested the dependence of this shift on PV+ cell activity by measuring ODI values of V1 neurons during optogenetic suppression of PV+ cells .",
"This optogenetic-suppression did not alter the deprivation-induced shift in ODI values .",
"Our results using VEP recordings and PV+ suppression via hM4Di Dreadd receptors are in agreement with these results from Saiepour and colleagues , demonstrating that suppression of PV+ cell activity after deprivation does not interfere with the expression of the adult OD shift .",
"Although the findings that PV+ neurons are not necessary for expression of OD plasticity do not rule out a modulatory role in the induction mechanisms , we note that the adult OD shift still occurred in mutant mice in which PV+ neurons lack NMDARs .",
"In contrast to the adult OD shift , we found that the expression of SRP relies on PV+ neuron circuitry .",
"This finding came as a surprise , as SRP reflects the potentiation of short-latency synaptic currents and increased spiking activity in layer 4 ( Cooke et al . , 2015 ) , and previous experiments had strongly indicated that SRP might be considered a naturally occurring form of LTP .",
"For example , induction of SRP is input-specific , dependent upon NMDA receptor activation and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ( AMPAR ) insertion in principal cells ( Frenkel et al . , 2006 ) , and reversed by infusion of the zeta-inhibitory peptide ( ZIP ) that also erases LTP ( Cooke and Bear , 2010 ) .",
"Furthermore , LTP elicited by theta-burst stimulation of the thalamus occludes and is occluded by SRP ( Cooke and Bear , 2010 ) .",
"Altogether , these findings suggest a modification of feed-forward excitatory synaptic transmission .",
"Before assigning a specific role to inhibition in the expression of SRP , two relatively trivial explanations must be addressed .",
"The first is that the loss of discrimination of familiar and novel orientations could be accounted for simply by a failure of orientation selectivity and not by a failure of memory .",
"Throughout the experiments presented here we have used orientations that are at least 60° and often 90° different , meaning that orientation discrimination should be as unchallenging as possible .",
"Given the modest impact on orientation tuning of suppressing PV+ interneuron activity in V1 ( Runyan et al . , 2010; Adesnik et al . , 2012; Wilson et al . , 2012 ) we believe that this explanation is improbable .",
"However , we took a direct approach to testing this possibility by stimulating activity in PV+ cells using channelrhodopsin-2 ( ChR2 ) while mice are viewing familiar and novel oriented stimuli .",
"Stimulation of PV+ inhibition has been shown to actually mildly enhance orientation-selectivity ( Lee et al . , 2012 ) and yet our experiments reveal that the difference between visual cortical response to familiar and novel orientations is significantly diminished by activation PV+ neurons .",
"This result casts PV+ neurons in a very specific role in the recognition of familiar stimuli in addition to modest participation in orientation-selectivity .",
"A second concern is that , after the suppression of PV+ inhibition , V1 may be operating outside the dynamic range where discrimination is possible .",
"However , we show that the cortex responds to input from the contralateral eye with a response of approximately double the magnitude of that elicited by input through the ipsilateral eye even after silencing the PV+ cells , suggesting that the response of the cortex is not saturated .",
"We also find that PV+ neuron silencing affects SRP even when it is induced using lower contrast stimuli that evoke submaximal responses .",
"Moreover , it is also worth noting that less specific pharmacological approaches to suppressing inhibition in V1 ( Khibnik et al . , 2010 ) result in much greater magnitude of response to visual input than we observe here for PV+ neuron inactivation , PV-GluN1 KO or ketamine application , indicating that our manipulations do not encroach on a physiological ceiling .",
"One relatively simple explanation for the current findings is that SRP results from lasting homosynaptic ( i . e . input-specific ) long-term depression ( LTD ) of glutamatergic input to PV+ inhibitory neurons .",
"Thereafter , presentation of a familiar stimulus evokes a greater response in V1 due to selective disinhibition of cortical circuitry .",
"This hypothesis is consistent with our observation that SRP is deficient if NMDARs are no longer expressed on PV+ cells .",
"However , because we used a non-reversible genetic modification in our initial PV-GluN1 KO experiment , we were initially unable to show if these receptors were important for the storage of information or the retrieval of information already stored .",
"Ketamine , which among other actions serves as a non-competitive NMDAR antagonist , has been shown to preferentially antagonize NMDARs on PV+ cells ( Homayoun and Moghaddam , 2007; Seamans , 2008 ) .",
"By using ketamine to induce a temporary blockade of these receptors , which we confirmed by showing that there was no impact of ketamine on V1 responses in the PV-GluN1 KO mice , we were able to demonstrate that interference with NMDARs on PV+ cells disrupts expression of SRP even after it has been induced and saturated .",
"Importantly , acute application of ketamine did not affect the maintenance or stability of stored information , because a strong bias for the familiar stimulus returned after ketamine washout .",
"If SRP is indeed accounted for by homosynaptic depression of excitatory synapses carrying information about the familiar stimulus orientation , this LTD would need to be expressed by a synapse-specific reduction in NMDAR-mediated responses in order to account for these findings .",
"However , this simple model is difficult to reconcile with the findings reviewed above that SRP depends on LTP-like mechanisms , e . g . , NMDAR-dependent AMPAR delivery in principal cells .",
"Considered together , the findings to date indicate that induction and maintenance of SRP occur by modification of excitatory synapses onto excitatory neurons , but that expression of SRP is mediated by stimulus-selective modulation of PV-cell activity in V1 .",
"Obviously a polysynaptic circuit is required to yield these properties , and dissecting this essential cortical circuitry will require much more work .",
"However , it is interesting to note a recent study by Makino and Komiyama who showed , in addition to a stimulus-selective reduction in PV+ neuron activity in V1 as mice were repeatedly exposed to a specific visual stimulus , an experience-dependent increase in the responsiveness of somatostatin-positive interneurons ( Makino and Komiyama , 2015 ) .",
"This result is particularly interesting as it is known that many interneuron types innervate one another and , therefore , that the activity of these cell types are interdependent .",
"It is not difficult to sketch circuits in which stimulus-selective , feed-forward polysynaptic inhibition of PV+ cells accounts for SRP expression .",
"Clearly , experiments aimed at teasing apart the involvement of other interneuron types in SRP should now be a high priority .",
"We note that other studies have suggested that PV+ neurons may be important for plasticity following repeated exposure to sensory stimuli .",
"For instance , Meyer and colleagues , studying infero-temporal cortex ( IT ) in monkeys , showed that familiarity resulted in a sharpening of cortical responses to dynamic visual stimuli , which was likely orchestrated by local fast-spiking putative PV+ cells ( Meyer et al . , 2014 ) .",
"Additionally , mice constitutively lacking neuronal activity-regulated pentraxin ( NARP ) , a synaptic protein specifically enriched at excitatory synapses on PV+ cells , displayed a reduction in excitatory synapses onto PV+ cells and impairment in SRP-like effects ( Chang et al . , 2010; Gu et al . , 2013 ) .",
"Intriguingly , however , the NARP KO mice also failed to exhibit both juvenile and adult OD plasticity .",
"In juvenile animals , it is well established that reducing inhibition can impair induction of OD plasticity ( Ramoa et al . , 1988 ) and delay the developmental decline in the mechanism of deprived-eye depression ( Hanover et al . , 1999; Huang et al . , 1999 ) .",
"We speculate that the loss of OD plasticity in the adult NARP KO may be related to disruption of the developmental switch from juvenile to adult-type OD plasticity ( Heimel et al . , 2011 ) .",
"Our findings indicate that although open-eye potentiation as a result of MD in the adult and SRP are superficially similar , the expression mechanisms of these two forms of plasticity are quite distinct .",
"Based on evidence provided here as well as by others ( Sawtell et al . , 2003; Ranson et al . , 2012; Saiepour et al . , 2015 ) , the deprivation enabled potentiation of the open eye is consistent with a potentiation of synapses between excitatory cells and is independent of PV+ cell inhibition .",
"However , further work will be necessary to confirm that open eye potentiation in the adult animal is expressed by synaptic alterations between excitatory cells , and to determine whether these processes are present at the level of thalamocortical synapses or intracortical synapses .",
"SRP correlates with OSH ( Cooke et al . , 2015 ) , a form of visual learning which is dependent upon NMDARs in V1 , and which enables the animal to make the critically important discrimination between familiarity and novelty .",
"We have shown here that modulation of PV+ neuron activity in V1 is required for this selective recognition of familiarity and consequent novelty detection .",
"Loss of NMDAR function in these same cells also impairs familiarity recognition .",
"The cognitive symptoms of schizophrenia , and other psychiatric disorders , are characterized by deficits in habituation and familiarity ( Braff et al . , 1995; Ramaswami , 2014 ) and individuals with schizophrenia display impairment in visual cortical plasticity ( Çavuş et al . , 2012 ) .",
"Dysfunction of PV+ inhibitory neurons and the NMDARs expressed on PV+ cells have also been implicated in these same psychiatric disorders through a range of approaches ( Lewis et al . , 2005; Gogolla et al . , 2009; Lewis et al . , 2011 ) .",
"This notably includes observations of the profound psychotomimetic effect in humans of non-competitive NMDAR antagonists such as ketamine ( Krystal et al . , 1994; Krystal , 2015 ) , a substance that , as we show here , prevents SRP expression .",
"We suggest that understanding the cortical physiology underlying the processes of familiarity recognition and novelty detection may yield great insight into dysfunction underlying some symptoms of these disorders and that , because these processes are as important to mice as they are to humans , they are likely to be experimentally tractable ."
],
[
"All procedures adhered to the guidelines of the National Institutes of Health and were approved by the Committee on Animal Care at MIT , Cambridge , MA , USA .",
"For all experiments mice were male , aged between P60-90 and on a C57BL/6 background ( Charles River laboratory international , Wilmington , MA ) .",
"They were housed in groups of 2–5 with food and water available ad libitum and maintained on a 12 hr light-dark cycle .",
"For hM4D ( Gi ) experiments , mice were Parvalbumin-Cre recombinase knock-in mice ( B6;129P2-Pvalbtm1 ( cre ) Arbr/J , PV-Cre ) on a C57BL/6 background .",
"For GluN1 knockdown experiments , these PV-Cre mice were bred with homozygous mice in which the Grin1 gene , which encodes the GluN1 sub-unit was flanked by LoxP sites ( B6 . 129S4-Grin1tm2Stl/J , GluN1 fl/fl ) , enabling Cre-dependent ablation of this mandatory subunit of the NMDA receptor .",
"Multiple generations were required to set up crosses yielding offspring that were homozygous GluN1 fl/fl and Cre-expressing .",
"Of these approximately 50% expressed Cre and 50% served as wild-type littermates on the GluN1 fl/fl background .",
"All experiments were conducted blind to genotype and treatment .",
"Mice were first injected with 0 . 1 mg/kg Buprenex sub-cutaneously ( s . c . ) to provide analgesia .",
"They were then anesthetized with an intraperitoneal ( i . p . ) injection of 50 mg/kg ketamine and 10 mg/kg xylazine .",
"Prior to surgical incision , 1% lidocaine hydrochloride anesthetic was injected under the mouse’s scalp .",
"The skull was then cleaned with iodine and 70% ethanol .",
"A steel head post was affixed to the skull anterior to bregma using cyanoacrylate glue .",
"Burr holes ( < 0 . 5 mm ) were then drilled in the skull over binocular V1 ( 3 . 1 mm lateral of lambda ) .",
"Tapered tungsten recording electrodes ( FHC , Bowdoinham , ME , US ) , 75 μm in diameter at their widest point , were implanted in each hemisphere , 450 μm below cortical surface .",
"Silver wire ( A-M systems , Sequim , WA , US ) reference electrodes were placed over prefrontal cortex .",
"Mice were allowed to recover for at least 24 hr prior to head-fixation .",
"For hM4D ( Gi ) experiments , we infected V1 of P30-60 PV-Cre mice or wild-type littermates with an AAV9-hSyn-DIO-HA-hM4D ( Gi ) -IRES-mCitrine virus ( UNC viral core – generated by Dr . Brian Roth’s laboratory ) and for optogenetic experiments we infected mice from the same line with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP ( UNC viral core – generated by Dr . Karl Deisseroth’s laboratory ) .",
"Using a glass pipette and nanoject system ( Drummond scientific , Broomall , PA , US ) , we delivered 81 nl of virus at each of 3 cortical depths: 600 , 450 , and 300 µm from the cortical surface , and allowed 5 min between re-positioning for depth .",
"Mice were allowed 3–4 weeks recovery for virus expression to peak before experiments were initiated .",
"Clozapine-N-oxide ( CNO , Enzo Life Sciences ) was diluted in saline and injected i . p . at a dosage of 5 mg/kg 30 min prior to stimulus delivery .",
"Ketamine hydrochloride ( Vedco ) was diluted in water and delivered i . p . at 50 mg/kg 15 min prior to stimulus delivery .",
"Visual stimuli consisted of full-field , 100% contrast , sinusoidal gratings that were presented on a computer monitor .",
"Visual stimuli were generated using custom software ( http://bearlab-s1 . mit . edu/supp6/PlxStimOne_Opto . zip ) written in either C++ for interaction with a VSG2/2 card ( Cambridge Research systems , Kent , U . K . ) or Matlab ( MathWorks , Natick , MA , U . S . ) using the PsychToolbox extension ( http://psychtoolbox . org ) to control stimulus drawing and timing .",
"The display was positioned 20 cm in front of the mouse and centered , thereby occupying 92° × 66° of the visual field .",
"Visual stimuli consisted of full-field sinusoidal grating stimuli phase reversing at a frequency of 2 Hz .",
"Mean stimulus luminance was 27 cd/m2 .",
"Grating stimuli spanned the full range of monitor display values between black and white , with gamma-correction to ensure constant total luminance in both gray-screen and patterned stimulus conditions .",
"For data described in Figures 1–3 and 6 , experiments were fully automated and each stimulus block consisted of 200 phase reversals with 30-s intervals between each stimulus presentation , during which the screen was gray but of equivalent luminance .",
"For experiments described in Figures 4 and 5 stimulus blocks were also 200 phase reversals in length but each was triggered directly by the experimenter , meaning that the interval was approximately 30-s .",
"Throughout , stimulus orientation varied such that a novel orientation was always a minimum of 25° different from any experienced previously by the individual mouse and was never within 20° of horizontal because these orientations are known to elicit VEPs of greater magnitude than vertical or oblique stimuli .",
"If more than 1 orientation was shown within a session , stimuli were pseudo-randomly interleaved such that 3 consecutive presentations of the same stimulus never occurred .",
"For monocular presentation an occluding paddle was positioned in front of one eye to limit stimulus presentation to the opposite eye .",
"VEP recordings were conducted in awake , head-restrained mice .",
"Prior to recording , mice were habituated to the restraint apparatus by sitting in situ in front of a gray screen for a 30-minute session on each of two consecutive days .",
"For experiments in which monocular VEPs were subsequently acquired , mice were also habituated to the occluding paddle positioned in front of each eye .",
"On stimulus presentation days , mice were presented with 5 blocks of 200 phase reversals of each oriented stimulus separated by gray screen presentation for ~30 s .",
"For monocular presentations , recordings were conducted in sequence for each eye .",
"VEP magnitude was then quantified by measuring trough-peak response magnitude averaged over all phase reversals .",
"Recordings presented in Figures 1–3 and 6 were amplified and digitized using the Recorder-64 system ( Plexon Inc . , Dallas , TX ) .",
"Two recording channels were dedicated to recording EEG/VEPs from V1 in each implanted hemisphere and a third recording channel was reserved for the Piezo-electrical input carrying the behavioral information .",
"Recordings presented in Figures 4 and 5 were amplified using DAM80 amplifiers ( World Precision Instruments , Florida , U . S . ) and digitized using a custom National Instruments system ( National Instruments , Texas , U . S . ) ) .",
"Local field potential was recorded from V1 with 1 kHz sampling and a 500 Hz low-pass filter .",
"Data was extracted from the binary storage files and analyzed using custom software ( http://bearlab-s1 . mit . edu/supp6/VEP_Analysis . zip ) written in C++ , Matlab and Labview .",
"VEPs were averaged across all phase reversals within a block and trough-peak difference measured during a 200-millisecond period from phase reversal .",
"For experiments concerning Dreadd receptor expression in PV+ cells presented in Figure 1: Three weeks after infection with AAV9-hSyn-DIO- HA-hM4D ( Gi ) -IRES-mCitrine virus , visual cortical slices were prepared from the injected mice as previously described ( Philpot et al . , 2001 ) .",
"After dissection 350-µm-thick coronal slices recovered for 30 min at 32°C and then for an additional 2 hr at room temperature , in a holding chamber filled with warmed artificial cerebrospinal fluid ( ACSF ) , which contained: 124 mM NaCl , 3 . 5 mM KCl , 1 . 25 mM Na2PO4 , 26 mM NaHCO3 , 1 . 2 mM MgCl2 , 2 mM CaCl2 , and 10 mM dextrose , saturated with 95% O2 , 5% CO2 .",
"Whole cell patch clamp recordings were performed at 30°C from the parvalbumin-positive interneurons in the layer 4 , identified by the ECFP fluorescence .",
"Pipette tip resistances were 3–5 MΩ .",
"Internal solution contained: 20 mM KCl , 100 mM Na-gluconate , 10 Hepes , 4 mM MgATP , 0 . 3 mM Na2GTP , 7 mM phosphocreatine-Tris , 0 . 2% biocytin with pH adjusted to 7 . 2 and osmolarity adjusted to 300 mOsm .",
"All recordings were made using Axopatch 200B ( Molecular Devices , Sunnyvale , CA ) at 10 kHz sampling rate .",
"Cells with the series resistance <30 MΩ were included for analysis .",
"Data analysis was done using pClamp ( Molecular Devices ) and custom-written python scripts .",
"For experiments concerning NMDAR EPSCs and AMPAR EPSCs in PV-GluN1 KO mice as presented in Figure 6H: 4 PV-GluN1 KO mice were infected with AAV5-EF1α-DIO-hChR2 ( H134R ) -eYFP , to allow for fluorescence-guided intracellular recordings of PV+ cells .",
"Four weeks after infection , visual cortical slices were prepared as describe above .",
"For fluorescently labeled PV+ cells and unlabeled neighboring excitatory cells: EPSCs were recorded at -70 mV and +40 mV with pipettes filled with balanced intracellular solutions ( in mM ) : 115 cesium methane-sulfonate ( CsMeSO3 ) , 2 . 8 NaCl , 0 . 4 EGTA , 4 ATP-Mg2+ , 10 Na+-phosphocreatine , 0 . 5 Na+-GTP , 5 TEA-Cl- , 5 QX-314 Br- buffered with 20 HEPES , pH 7 . 25 , osmolarity 290 mOsm .",
"Synaptic events were evoked by stimulation of the white mater ( 150 μs , 0 . 1 Hz , glass electrode , and stimulator A365 from WPI ) .",
"AMPA component was measured from EPSCs at -70 mV in the presence of PTX ( 100 μM ) and glycine ( 1 μM ) and NMDA component was measured from EPSCs at +40 mV in the presence of DNQX ( 20 μM ) .",
"All behavioral experiments were performed during the mouse subject’s light cycle .",
"A piezo-electrical recording device ( C . B . Gitty , Barrington , NH , USA ) was placed under the forepaws of head-restrained mice during all sessions .",
"Mice became accustomed to the apparatus by sitting in situ in front of a gray screen for a 30-min session on each of 2 days .",
"Before stimulus presentation on each day mice also underwent 5 min of gray screen presentation after the experimenter had left the room .",
"A continuous voltage signal was recorded from the piezo device for the entire session .",
"Movements were detected as a shift in the voltage signal .",
"The recording system was automated so that no one was ever present in the closed room for any of the recording period and white noise was played at 67 dB in order to mask outside noise .",
"For vidget scoring , the continuous voltage signal was down-sampled to 100 Hz .",
"The period of interest in the experiments described here lasted from 2 s prior to stimulus onset until 5 s after stimulus onset ( which was the first 10 phase reversals in a block ) .",
"Quantification of movement driven by the onset of the stimulus ( the vidget ) was calculated by taking the Root Mean Square ( SQRT ( X2 ) ) of the voltage signal .",
"Post-stimulus signal was then normalized to the average magnitude during the 2-s period prior to stimulus onset .",
"The average normalized magnitude across the 5-s period subsequent to stimulus presentation was then used to quantify the degree of stimulus-driven movement and this is described throughout in arbitrary units ( a . u . ) .",
"After viral infection mice were also bilaterally implanted with VEP recording electrodes positioned in layer 4 .",
"Ready-made optic fibres ( 200 µm girth ) mounted in stainless steel ferrules ( 1 . 25 mm diameter , 2 mm fibre projection , Thor labs , Newton , NJ , US ) were then implanted positioned lateral ( 3 . 5 mm lateral to lambda ) to the recording site and at a 45° angle to the recording electrode , 0 . 1 mm below surface in each hemisphere .",
"1 month later , after peak of viral expression , mice were habituated to the head-fixation apparatus over 2 days before conducting optogenetic experiments .",
"We delivered 31-s long continuous pulses of blue light ( 473 nm , 150 mW ) into V1 using a laser ( Optoengine LLC , Midvale , UT , US ) .",
"These light pulses were delivered simultaneous to 50% of the 30-s long visual stimulus presentations , commencing 30 ms prior to visual stimulus onset and ending 30 ms after offset .",
"Animals were sacrificed and perfused within a week after this experiment for histological analysis .",
"Mice were deeply anaesthetized with fatal plus ( pentobarbital ) and perfused with saline followed by 4% paraformaldehyde in 0 . 1 M phosphate buffer .",
"The brain was removed and post-fixed for 24 hr at room temperature .",
"After fixation , the brain was sectioned into 60 µm coronal slices using a vibratome .",
"Slices were incubated with blocking solution ( 10% fetal bovine serum in PBS with 0 . 2% Triton X-100 ) for 1 hr at room temperature and then with anti-Parvalbumin mouse primary antibody ( MAB1572 , Millipore , Billerica , MA; 1:1000 ) and anti-GFP antibody ( Ab290 , Abcam , Cambridge , United Kingdom; 1:7000 ) details diluted in blocking solution overnight at 4 degrees Celsius .",
"Slices were then washed three times with PBS and incubated with the secondary antibody for 1 hr at room temperature ( Alexa488-conjugated anti-rabbit IgG , Invitrogen , Carlsbad , CA; 1:500 , Alexa594-conjugated anti-mouse IgG , Invitrogen; 1:500 ) .",
"Slices were washed three times with PBS and mounted with 49 , 6-diamidino-2 phenylindole ( DAPI ) -containing Vectashield ( Vector Laboratories , Burlingame , CA ) .",
"Fluorescence images were taken with a confocal fluorescence microscope ( Olympus , Tokyo , Japan ) .",
"In the results section , all data is expressed as a mean ± standard error of the mean ( S . E . M ) .",
"Sigmaplot was used for statistical analysis .",
"Normality of distribution and homogeneity of variation was tested and parametric ANOVAs ( for multiple groups ) or t-tests ( for 2 groups ) were performed when data passed these tests .",
"Otherwise , non-parametric ANOVAs or t-tests on ranks were used .",
"If ANOVAs yielded significance , Student-Newman-Keuls post-hoc tests with adjustment for multiple comparisons were applied for individual comparisons .",
"Repeated measures ANOVAs or paired t-tests were applied for all within subject comparisons .",
"For other comparisons unpaired ANOVAs or t-tests were used .",
"Individual tests used are described in the results .",
"P < 0 . 05 is used as a threshold for significance throughout but exact P values are given for all comparisons for which the P value is above 0 . 001 .",
"Post-hoc power analyses were used to determine that comparisons reached a power of > 0 . 8 for an alpha value of 0 . 05 .",
"Technical replicates were only used as N for the ex vivo electrophysiological test of hM4D ( Gi ) efficacy , when 10 cells were recorded from 7 mice , and the test of NMDAR functional loss in PV-GluN1 KO mice , when 8 cells were recorded from 5 mice .",
"Throughout the remainder of the study we report the N is an individual animal ( biological replicate ) .",
"Technical replicates ( secondary samples within each animal ) were not undertaken for in vivo electrophysiology .",
"Although both hemispheres were implanted initially , a decision was made as to the best hemisphere , based on response magnitude , morphology and binocularity after the very first recording ( prior to any measure of plasticity ) .",
"Technical replicates were undertaken for the behavior , with 10 , 6 ( for PV-HM4D ( Gi ) experiments ) or 5 stimulus onsets ( for PV-GluN1 KO experiment ) being delivered per day , although we only report the daily average ( biological replicate ) .",
"There variations in protocol were due experiments being conducted by different experimenters at different times .",
"However , protocols were completely consistent across treatments/genotypes .",
"The individual technical replicates are available in the uploaded source data files ."
]
] | [
"The roles played by cortical inhibitory neurons in experience-dependent plasticity are not well understood .",
"Here we evaluate the participation of parvalbumin-expressing ( PV+ ) GABAergic neurons in two forms of experience-dependent modification of primary visual cortex ( V1 ) in adult mice: ocular dominance ( OD ) plasticity resulting from monocular deprivation and stimulus-selective response potentiation ( SRP ) resulting from enriched visual experience .",
"These two forms of plasticity are triggered by different events but lead to a similar increase in visual cortical response .",
"Both also require the NMDA class of glutamate receptor ( NMDAR ) .",
"However , we find that PV+ inhibitory neurons in V1 play a critical role in the expression of SRP and its behavioral correlate of familiarity recognition , but not in the expression of OD plasticity .",
"Furthermore , NMDARs expressed within PV+ cells , reversibly inhibited by the psychotomimetic drug ketamine , play a critical role in SRP , but not in the induction or expression of adult OD plasticity ."
] | [
"What we see or fail to see through our eyes leaves a lasting impression by changing the strength of connections between neurons in a part of the brain called the visual cortex .",
"These changes are referred to as synaptic plasticity .",
"One example of synaptic plasticity results in the visual cortex becoming more responsive to the stimulation of one eye when the other eye is patched for about a week .",
"This phenomenon is known as “ocular dominance plasticity” .",
"Another example is the increase in responsiveness that occurs in the visual cortex when animals repeatedly view stripes of a single orientation .",
"This phenomenon is known as “stimulus-selective response potentiation” .",
"Some previous studies had suggested that both kinds of plasticity might be induced in the same way .",
"However , both forms of plasticity can happen at the same time , suggesting that distinct mechanisms may be involved .",
"To tease out how these two kinds of plasticity work , Kaplan , Cooke et al . inactivated one particular type of neuron that is thought to be involved in triggering ocular dominance plasticity and is found in the visual cortex .",
"These inhibitory neurons produce a molecular marker called parvalbumin and are therefore referred to as “parvalbumin-expressing neurons” .",
"The experiments showed that ocular dominance plasticity could still be seen in adult mice when these parvalbumin-expressing neurons were inactivated .",
"However , when these same neurons were inactivated , the visual cortex no longer responded differently to lines with familiar or new orientations .",
"This was the case even when the mice had seen lines in a given orientation for long periods of time .",
"Similarly , observations of the behavior of the mice also showed that their ability to distinguish new from familiar stimuli was lost when the parvalbumin-expressing neurons were inactivated locally within part of the visual cortex .",
"The connections between neurons that bring information from the eyes to the visual cortex release a chemical neurotransmitter called glutamate .",
"One important protein that detects glutamate , called an NMDA receptor , is required for ocular dominance plasticity .",
"Further experiments showed that NMDA receptors within the parvalbumin-expressing neurons are needed for stimulus-selective response potentiation , but not for ocular dominance plasticity .",
"This indicates that although the two forms of plasticity may share molecular requirements , different connections and cells types are likely involved .",
"Changes in parvalbumin-expressing neurons and NMDA receptors have been implicated in disorders such as autism and schizophrenia .",
"To improve treatments for these disorders , it is crucially important to better understand the links between these neurons and the retrieval of memories ."
] | 2016 |
[
"Introduction",
"Materials and methods"
] | [
"short report",
"cell biology"
] | Degradation of Gadd45 mRNA by nonsense-mediated decay is essential for viability | elife-12876-v2 | [
[
"Maintaining proper gene expression is critical for normal development and physiology .",
"In addition to de novo transcription , mRNA stability substantially contributes to forming the landscape of expression in a cell .",
"The nonsense-mediated mRNA decay ( NMD ) pathway is a trans-acting mechanism that destabilizes mRNAs , and is best known for its well-described role as a quality control system , degrading abnormal mRNAs containing premature termination codons ( PTCs ) ( Celik et al . , 2015 ) .",
"NMD also degrades many wild-type endogenous mRNAs and thus is an important aspect of their post-transcriptional ( Peccarelli and Kebaara , 2014 ) .",
"Loss of either of the core NMD genes Upf1 ( Rent1 ) or Upf2 causes lethality in most eukaryotes ( Kerényi et al . , 2008; Medghalchi et al . , 2001; Metzstein and Krasnow , 2006; Weischenfeldt et al . , 2008; Wittkopp et al . , 2009 ) , indicating regulation of mRNA stability by NMD is critical for viability .",
"However , the relative contributions to lethality from ectopic stabilization of PTC-containing mRNAs or endogenous NMD targets in NMD mutants remains unclear ( Hwang and Maquat , 2011 ) .",
"To identify which ectopically stabilized mRNAs are responsible for inducing lethality in NMD mutants , we performed an unbiased genetic suppressor screen seeking to restore viability in a Drosophila NMD mutant .",
"To detect subtle increases in survival , we screened to suppress the lethality of animals mutant for the partially viable , hypomorphic Upf225G allele , of which 10% survive to adulthood ( Chapin et al . , 2014; Metzstein and Krasnow , 2006 ) .",
"We crossed this allele to heterozygous deficiencies to simultaneously reduce the mRNA abundance of several loci ( Figure 1A ) .",
"Of the 376 deficiencies tested , covering more than half the genome , ~10% suppressed NMD mutant lethality ( Figure 1B , Figure 1—figure supplement 1A ) .",
"The suppression effect could not be explained by a reduction in overall mRNA load , as there was only a weak correlation between the increase in mRNAs expressed from a genomic region upon loss of NMD function and the strength of suppression when that region was removed by a deficiency ( Figure 1—figure supplement 1B ) .",
"Rather , deficiencies that suppressed NMD-mutant lethality clustered in three genomic regions ( Figure 1—figure supplement 1A ) .",
"These findings suggest that NMD mutant lethality is not the result of a global excess of nonspecific mRNAs , but rather is mediated by specific genes residing within the few identified regions . 10 . 7554/eLife . 12876 . 003Figure 1 . Drosophila suppressor screen identifies the Gadd45 pathway as the inducer of NMD-mutant lethality .",
"( A ) Scheme to screen deficiencies for the suppression of Upf225G partial lethality .",
"The Deficiency Suppression Score ( DSS ) represents the relative difference in Upf225G viability when crossed to a heterozygous deficiency ( Df ) compared to when crossed to a balancer ( Bal ) ( See Methods ) .",
"( B ) DSS from 376 screened deficiencies ranked by score .",
"A DSS greater than 0 . 1 ( dotted line ) indicates that deficiency suppresses Upf225G lethality .",
"( C and E )",
"Candidate suppressing regions uncovering Gadd45 ( C ) and Mekk1 ( E ) .",
"DSSs are shown in parenthesis .",
"Dotted lines denote extent of regions deleted by suppressing deficiencies but not non-suppressing deficiencies .",
"Filled blocks on chromosomes indicate predicted gene spans , Gadd45 pathway genes are indicated in red; suppressing deficiencies indicated in green , sple-J1 has undefined breakpoints located within hashed regions .",
"( D and F )",
"NMD mutant adult viability in combination with Gadd45F17 ( D ) or Mekk1Ur36 ( F ) mutants .",
"Upf126A and Upf27-5A are null alleles ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .",
"p-value compared to controls determined by the test of equal or given proportions indicated .",
"Error bars represent 95% confidence interval of the binomial distribution .",
"n equals total number of animals scored in each cross . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00310 . 7554/eLife . 12876 . 004Figure 1—figure supplement 1 . Reduced expression of specific loci , not overall mRNA abundance , produces NMD mutant suppression by deficiencies .",
"( A ) Map of 376 autosomal DrosDel deficiencies with an isogenic background and molecularly defined breakpoints ( Ryder et al . , 2007 ) and eight other deficiencies used to further test candidate suppressing regions without overlapping DrosDel deficiencies .",
"39 total deficiencies suppress Upf225G lethality , shown in green .",
"Regions deleted by any non-suppressing deficiencies were eliminated as candidate suppressing regions , removing false positives and reducing the size of the candidate intervals .",
"The three candidate suppressing regions that are deleted only by suppressing deficiencies are indicated in black and labeled 1–3 .",
"( B ) Each deficiency’s Deficiency Suppression Score ( DSS ) compared to percent increase in RNA abundance in Upf225G compared to wild-type from loci removed by that deficiency according to RNA-seq from Chapin et al . ( 2014 ) .",
"Trend line in red; statistics calculated using Pearson correlation test . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00410 . 7554/eLife . 12876 . 005Figure 1—figure supplement 2 . Drosophila Gadd45 is an endogenous direct NMD target .",
"( A ) Gadd45 mRNA expression in adults of the given genotypes measured by qRT-PCR .",
"Gadd45 mRNA expression is increased 16 . 7-fold in Upf225G mutants , and is eliminated by Gadd45F17 mutants .",
"p-values display one-sided Student’s t-test of indicated condition compared to control .",
"Error bars represent 2 SEM .",
"( B ) Fluorescence of GFP transgenes with SV40 , Act5C , or Gadd45 3’ UTRs expressed by UAS driven by Actin:GAL4 in Upf2+ or Upf225G third instar larvae .",
"SV40 and Gadd45 3’ UTR constructs show significantly increased fluorescence in Upf225G animals compared to Upf2+ , indicating NMD-dependent post-transcriptional degradation of mRNAs containing these UTRs .",
"The Act5C 3’ UTR construct has similar fluorescence in both backgrounds , indicating NMD does not regulate the post-transcriptional stability of this UTR .",
"Micrographs show dorsal views with anterior at top .",
"( C ) MAL-A2 , traL , and Gadd45 5’ and 3’ fragment mRNA expression measured by qRT-PCR in pcm14 null mutants ( Waldron et al . , 2015 ) normalized to controls .",
"Transcript structures of a non-NMD-target , maltase A2 ( MAL-A2 ) ; a known NMD-targeted transcript , the non-sex specific isoform of transformer ( traL ) ( Rehwinkel , 2005; Metzstein and Krasnow , 2006 ) ; and the Gadd45 transcript ( note Gadd45 has no introns ) .",
"Open boxes indicate UTRs; grey boxes indicate coding regions .",
"NMD targeting initiates endonucleolytic cleavage near the stop codon ( Gatfield and Izaurralde , 2004 ) , producing 5’ and 3’ fragments with unprotected ends , which are then subjected to degradation by cytoplasmic 3’-to-5’ and 5’-to-3’ exonucleases , respectively .",
"qRT-PCR primer pairs 5’ ( red ) and 3’ ( blue ) to the cleavage site can be used to differentially measure the quantity of these fragments .",
"The Drosophila 5’-to-3’ exonuclease is encoded by the XRN1 homologue pacman ( pcm ) , and fragments 3’ to an endonucleolytic NMD cleavage accumulate in Drosophila cells with reduced XRN1 activity ( Gatfield and Izaurralde , 2004 ) .",
"The MAL-A2 3’ primers show no difference in relative expression in pcm14 mutants compared to the 5’ primers , while tra and Gadd45 have relatively increased levels of a 3’ fragment in pcm14 mutants , revealing endonucleolytic cleavage has occurred between the primer pairs , probably near the stop codon , indicative of NMD-initiated degradation .",
"p-value between indicated samples using a two-sided Student’s t-test are displayed .",
"ns indicates a p-value greater than 0 . 05 .",
"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00510 . 7554/eLife . 12876 . 006Figure 1—figure supplement 3 . F17 is a null allele of Gadd45 . ( A ) Gadd45F17 is an imprecise excision of the P-element P{EPg}HP20647 , deleting a 894 bp region that includes the entire Gadd45 coding region .",
"Gadd45E8 is a precise excision of the same P-element , leaving Gadd45 intact .",
"Coding region in grey; untranslated regions in white .",
"Arrowhead indicates direction of transcription .",
"( B ) Adult viability of control and Gadd45F17mutants .",
"p = 0 . 4463 between Gadd45F17 and controls , using the test of equal or given proportions .",
"Error bars represent 95% confidence interval of the binomial distribution .",
"n equals total number of animals scored in each cross . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00610 . 7554/eLife . 12876 . 007Figure 1—figure supplement 4 . Loss of Gadd45 does not restore NMD activity in NMD mutants .",
"( A ) Expression of the endogenous NMD target transcript traL ( Rehwinkel , 2005; Metzstein and Krasnow , 2006 ) in control male , Upf225G/Y , and Upf225G/Y; Gadd45F17animals , measured by qRT-PCR .",
"There is no significant difference in traL expression between Upf225G/Y and Upf225G/Y; Gadd45F17animals .",
"p-values determined by one-sided Student’s t-test between indicated conditions are displayed .",
"ns indicates a p-value greater than 0 . 05 .",
"( B ) Relative abundance of PTC-containing Adhn4 ( Chia et al . , 1987 ) and dHR783 ( Fisk and Thummel , 1998 ) allele mRNAs compared to wild-type allele mRNA abundance in animals heterozygous for Adhn4or dHR783in each indicated genotype ( stabilization of dHR783 was not determined in Upf225G/Y; Gadd45F17 animals ) .",
"Neither reduction nor elimination of Gadd45 restored destabilization of these alleles .",
"p-values determined by two-sided Student’s t-test between indicated conditions are displayed .",
"ns indicates a p-value greater than 0 . 05 .",
"Error represents 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 007 We expected that any specific genes mediating NMD-mutant lethality would have increased expression levels in an NMD mutant and be a direct NMD target .",
"The only gene located within the suppressing regions to fit these criteria is Gadd45 ( Figure 1C , Figure 1—figure supplement 2A–C ) ( Chapin et al . , 2014 ) .",
"To determine if NMD targeting of Gadd45 mRNA is critical for viability , we generated a Gadd45 null allele , F17 , which completely removes the Gadd45 coding region ( Figure 1—figure supplement 3A ) and eliminates Gadd45 mRNA expression ( Figure 1—figure supplement 2A ) .",
"As a heterozygote , Gadd45F17 suppressed Upf225Glethality as strongly as the corresponding deficiency identified by our screen ( Figure 1D ) .",
"We found that Gadd45F17 homozygous mutants are fully viable ( Figure 1—figure supplement 3B ) , allowing us to test complete loss of Gadd45 for the suppression of NMD-mutant lethality .",
"Homozygous Gadd45F17 restored full viability to Upf225Gmutants , and remarkably even partially suppressed the complete lethality observed in null Upf1 and Upf2 mutants ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) ( Figure 1D ) .",
"Importantly , neither reducing nor eliminating Gadd45 restored NMD function to Upf225G mutants , as measured by the expression of both an endogenous NMD target ( Figure 1—figure supplement 4A ) and PTC-containing mRNAs ( Figure 1—figure supplement 4B ) .",
"In mammals , GADD45 activates the MTK1/MEKK4 kinase in a well-defined stress response pathway ( Takekawa and Saito , 1998 ) .",
"Strikingly , the Drosophila MTK1 orthologue , Mekk1 , resides within another Upf225G suppressing region ( Figure 1E ) .",
"Similar to Gadd45 , we found that Mekk1 null mutants ( Inoue et al . , 2001 ) suppressed Upf1 and Upf2 mutant lethality ( Figure 1F ) .",
"This suppression was not as strong as that caused by a loss of Gadd45 , revealing that although MEKK1 mediates NMD mutant lethality , it is likely that GADD45 has additional downstream effectors that influence viability .",
"Overall , our findings reveal that increased Gadd45 mRNA stability is the major factor inducing NMD mutant lethality , primarily via increased MEKK1 activity .",
"Activation of MTK1 in mammals triggers a MAPK signaling cascade that promotes apoptosis ( Takekawa and Saito , 1998 ) .",
"Over-expression of Gadd45 in Drosophila also induces apoptosis ( Peretz et al . , 2007 ) .",
"Interestingly , Drosophila cells lacking NMD function show excess cell death in a variety of tissues ( Avery et al . , 2011; Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .",
"To test if increased Gadd45 contributes to this excess death , we used TUNEL staining to examine cell death in wing imaginal discs from Upf225G mutant third instar larvae .",
"This analysis revealed elevated levels of cell death compared to controls ( Figure 2A , B , E ) , and this defect was completely suppressed by Gadd45F17 ( Figure 2C–E ) .",
"To confirm that , this effect was not specific to the Upf2 gene or 25G allele , we examined the wing discs in mutants of another essential NMD gene , Smg5 .",
"We found that Smg5 discs also showed elevated TUNEL signal , which was eliminated by loss of Gadd45 ( Figure 2—figure supplement 1A–E ) .",
"These results demonstrate that excess Gadd45 accounts for ectopic cell death in NMD mutant tissues . 10 . 7554/eLife . 12876 . 008Figure 2 . Loss of Gadd45 suppresses NMD-mutant cell death .",
"( A to D )",
"DAPI ( blue ) and ( A’to D’ ) TUNEL ( red ) staining in late 3rd instar larval wing discs from control ( A ) ; Upf225G ( B ) ; Gadd45F17 ( C ) ; and Upf225G; Gadd45F17 ( D ) animals .",
"( A’’ to D’’ ) are 4x view of outlined section at the base of the blade of the wing disc from A’-D’ , respectively .",
"Scale bar represents 100 μm .",
"( E ) Relative TUNEL signal in control and mutant wing discs , normalized to control .",
"p-value between indicated samples using a two-sided Student’s t-test are displayed .",
"ns indicates a p-value greater than 0 . 05 .",
"Error bars represent 2 SEM .",
"n equals total number of discs scored .",
"( F to I ) w- eye clones in Gadd45+ and Gadd45F17 backgrounds .",
"Dashed lines indicate clone boundaries .",
"( J ) Quantification of the fraction of the eye composed of w- cells in control and mutant eyes .",
"p-values indicate differences between Gadd45 mutant and control in the same NMD background ( indicated by horizontal bars ) or NMD mutant and control in the same Gadd45 background ( indicated by value above each individual bar ) , using a two-sided Student’s t-test .",
"ns indicates a p-value greater than 0 . 05 .",
"Error bars represent 2 SEM .",
"n = 20 eyes for all conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 00810 . 7554/eLife . 12876 . 009Figure 2—figure supplement 1 . Loss of Gadd45 suppresses ectopic cell death in Smg5 mutant wing discs .",
"( A to D )",
"DAPI ( blue ) and ( A’ to D’ ) TUNEL ( red ) staining in late 3rd instar larval wing discs from Smg5G115/+ ( A ) ; Smg5G115/C391 ( B ) ; Smg5G115/+ Gadd45F17 ( C ) ; and Smg5G115/C391 Gadd45F17 ( D ) animals .",
"Smg5G115 and Smg5C391 are null Smg5 alleles ( J . O . N . and M . M . M . , unpublished ) .",
"Scale bar represents 100 μm .",
"( E ) Relative TUNEL signal in wing discs , normalized to Smg5G115/+ .",
"p-values determined by two-sided Student’s t-test between indicated conditions are displayed .",
"ns indicates a p-value greater than 0 . 05 .",
"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 009 To test if Gadd45-induced cell death is the only cellular defect in NMD mutants , we examined NMD function in the developing eye .",
"NMD is required for proper development of eye cells , as clonal patches of NMD mutant cells in eyes are reduced in size ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) .",
"We found that Gadd45 is partially responsible for this defect , as the size of eye-cell clones lacking NMD activity in a Gadd45F17 background was increased , although not fully restored ( Figure 2F–J ) .",
"These results indicate that some , but not all , defects associated with loss of NMD are dependent on Gadd45 .",
"Gadd45 is one of the few genes that is directly regulated by NMD in both flies and mammals ( Huang et al . , 2011; Tani et al . , 2012; Viegas et al . , 2007 ) , raising the possibility that excess Gadd45 abundance may also contribute to the NMD-mutant lethality observed in mammalian cells ( Azzalin and Lingner , 2006; Li et al . , 2015; Medghalchi et al . , 2001; Weischenfeldt et al . , 2008 ) .",
"To test this hypothesis , we analyzed the effects of Gadd45 and Upf1 depletion in mouse NIH-3T3 cells .",
"Gadd45b mRNA ( also known as MyD118 ) , which is expressed at least 10-fold higher than any other Gadd45 paralogue in these cells ( Yue et al . , 2014 ) , was degraded rapidly in a partially Upf1-dependent manner after transcription was blocked with actinomycin D ( Figure 3A ) , and had increased expression during Upf1 knockdown ( Figure 3D ) , confirming it is sensitive to NMD .",
"We found that transfection of 3T3 cells with siRNAs targeting Upf1 resulted in significant reduction in cell counts after 48 hr ( Figure 3B ) , but co-transfection with siRNAs targeting both Upf1 and Gadd45b largely reversed this effect ( Figure 3B ) .",
"The reduction in cell counts was primarily due to increased cell death , as we found that ~25% of cells transfected with Upf1 siRNA were undergoing apoptosis ( Figure 3C ) .",
"Co-transfection of siRNA targeting Gadd45b almost entirely eliminated this increase ( Figure 3C ) , indicating the excess apoptosis observed in Upf1-knockdown cells was mostly due to increased Gadd45 activity .",
"However , while Gadd45b knockdown very greatly suppresses this excess death , it does not as fully rescue cell numbers , suggesting loss of NMD may lead to both Gadd45b-dependent cell death as well as a Gadd45b-independent effect on proliferation .",
"This mirrors the conclusions we made about the partial suppression of cell number defects in the Drosophila eye .",
"Importantly , Upf1 mRNA expression was equivalently reduced and the expression of the mammalian endogenous NMD targets Rassf1 and CRCP ( Tani et al . , 2012 ) was equivalently increased in both the single and double knockdown experiments ( Figure 3D ) , indicating that the restoration of viability was not due to a recovery of NMD pathway activity . 10 . 7554/eLife . 12876 . 010Figure 3 . Gadd45b mediates cell lethality in Upf1 siRNA knockdown 3T3 mouse embryonic fibroblasts .",
"( A ) Relative Gadd45b mRNA expression measured by qRT-PCR in NIH-3T3 cells after 48 hr of control ( black ) or Upf1 ( red ) siRNA treatment and 0 to 2 hr of actinomycin D treatment , normalized to expression prior to actinomycin treatment .",
"The half-life calculated for each decay curve is indicated .",
"( B ) Relative viable cell count of Upf1 and Gadd45b single and double siRNA treatment normalized to control siRNA .",
"p-values display two-sided Student’s t-test between indicated conditions .",
"( C ) Quantification of apoptosis as measured by annexin V staining .",
"p-values display two-sided Student’s t-test between indicated conditions .",
"( D ) Relative mRNA expression of Upf1 , Gadd45b , and two mammalian endogenous NMD targets , Rassf1 and CRCP ( Tani et al . , 2012 ) measured by qRT-PCR in Gadd45b and Upf1 single and double siRNA knockdown cells , normalized to expression in the control siRNA condition .",
"p-values display one-sided Student’s t-test for each condition compared to control .",
"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 01010 . 7554/eLife . 12876 . 011Figure 3—figure supplement 1 . GADD45A mediates cell lethality in Upf1 knockdown HEK293 cells .",
"( A ) Relative GADD45A mRNA expression measured by qRT-PCR in UPF1 and GADD45A single and double siRNA knockdown 72 hr after siRNA transfection in HEK293 cells , normalized to expression in the control siRNA condition .",
"p-values display one-sided Student’s t-test for each condition compared to control .",
"( B ) Viable cell count of UPF1 and GADD45A single and double siRNA-treated cells normalized to control siRNA -treated cells .",
"p-values display two-sided Student’s t-test between indicated conditions .",
"( C ) Relative UPF1 mRNA expression measured by qRT-PCR in UPF1 and GADD45A single and double siRNA knockdown , normalized to expression in control siRNA condition .",
"p-values display one-sided Student’s t-test for each condition compared to control .",
"Error bars represent 2 SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 12876 . 011 To extend our analysis to other mammalian cells , we analyzed the role of Gadd45 mediating the effects of loss of NMD in HEK293 cells .",
"We found , similarly to 3T3 cells , that siRNA knockdown of UPF1 in HEK293 cells led to increased GADD45A expression and reduced cell numbers compared to control siRNA ( Figure 3—figure supplement 1A , B ) .",
"Although transfection of siRNA targeting GADD45A alone slightly reduced HEK293 cell numbers , co-transfection with UPF1 siRNA did not further reduce cell count ( Figure 3—figure supplement 1B ) , and UPF1 expression was equivalently reduced in the single and double knockdown conditions ( Figure 3—figure supplement 1C ) .",
"These results suggest that UPF1 knockdown is no longer detrimental to HEK293 cell viability in the absence of GADD45A expression .",
"We conclude that increased expression of mammalian Gadd45 genes contributes to lethality in NMD-deficient mouse and human cells , as Gadd45 does in Drosophila .",
"Deconvoluting the contributions to organismal viability of the PTC-surveillance versus gene-regulatory functions of NMD has been historically difficult ( Hwang and Maquat , 2011 ) .",
"Here , we show that viability can be restored to Drosophila lacking core NMD factors when a single endogenous NMD target , Gadd45 , is eliminated , and that the requirement for the regulation of Gadd45 by NMD is evolutionarily conserved from flies to mammals .",
"Although our data suggest that up-regulation of Gadd45 is a major factor contributing to lethality when NMD activity is lost , it is likely that other NMD targets also contribute to the observed lethality .",
"In particular , viability is not restored to 100% in null Upf1; or Upf2; Gadd45 double mutants .",
"In addition , loss of Gadd45 suppresses programmed cell death caused by defects in NMD , but not additional cell cycle defects , as implied by the incomplete suppression in the Drosophila eye and mammalian cell culture .",
"Such defects in the cell cycle may be particularly pronounced during the development of certain tissue , or specific developmental stages .",
"Indeed , NMD has been reported to have differing stage and tissue- specific activities ( Bao et al . , 2015; Bruno et al . , 2011; Colak et al . , 2013; Li et al . , 2015 ) .",
"Whether this is due to a role in surveillance or another specific target remains unclear , but examination of the effects of loss of NMD in Gadd45 mutants should allow exploration of these possibilities .",
"The benefit for such a mechanism regulating Gadd45 expression may lie in a function of NMD in restricting viral growth ( Balistreri et al . , 2014 ) .",
"Because viruses encode trans-acting factors to inhibit NMD ( Mocquet et al . , 2012 ) , the resulting accumulation of GADD45 in infected cells may act as a “molecular tripwire” that rapidly elicits a stress response and cell death .",
"This outcome suggests that regulating responses to infection may underlie a conserved essential function of NMD .",
"Intriguingly , restriction of pathogens via NMD extends to plants ( Garcia et al . , 2014 ) , where NMD mutant lethality in A . thaliana , which do not encode Gadd45 orthologues , may be caused by the overexpression of a subset of immune-related intracellular nucleotide-binding leucine-rich repeat receptors , some of which are endogenous NMD targets ( Gloggnitzer et al . , 2014 ) .",
"In contrast , eukaryotes that do not rely on the activation of programmed cell death to protect against viruses , such as S . cerevisiae , S . pombe , and C . elegans , do not require NMD for viability ( Hodgkin et al . , 1989; Leeds et al . , 1991; Mendell et al . , 2000 ) .",
"Together these observations suggest a potential novel role for NMD and Gadd45 in immune responses , triggering the death of infected cells during pathogenic challenges .",
"Restoring the expression of PTC-containing alleles via NMD inhibition has been proposed as a promising therapy for a wide range of recessive genetic diseases ( Keeling et al . , 2014 ) .",
"Translation of stable PTC-containing mRNAs would produce truncated proteins that may be partially functional and alleviate disease symptoms normally caused by complete loss of the protein .",
"However , the essential function for NMD in viability has raised the concern that these therapies may have prohibitive side effects .",
"Our findings reveal a molecular basis for dealing with this obstacle by suggesting that inhibiting both the NMD and Gadd45 pathways ( Tornatore et al . , 2014 ) in combination could provide an effective and safe treatment for patients with debilitating genetic disorders ."
],
[
"Drosophila melanogaster stocks were raised on standard cornmeal/dextrose food at 25° .",
"The NMD mutant alleles Upf225G , Upf27-5A , and Upf126A ( Frizzell et al . , 2012; Metzstein and Krasnow , 2006 ) are on y w FRT19A chromosomes .",
"These alleles were balanced over FM7i , P{ActGFP}JMR3 ( Reichhart and Ferrandon , 1998 ) .",
"Smg5G115 and Smg5C391 are null alleles of Smg5 ( J . O . N . , D . Förster , S . Luschnig , and M . M . M . , unpublished ) and will be described in detail later .",
"The Smg5 alleles are balanced over CyO , P{Dfd:eYFP w+} ( Le et al . , 2006 ) .",
"Other alleles used were P{w[+mC]=EPg}HP20647 ( Staudt et al . , 2005 ) , Mekk1Ur36 ( Inoue et al . , 2001 ) recombined on FRT82B by D . Ryoo , ey-FLP ( Newsome et al . , 2000 ) , pcm14 ( Waldron et al . , 2015 ) , Adhn4 ( Chia et al . , 1987 ) and DHR783 ( Fisk and Thummel , 1998 ) .",
"Control chromosomes were y w FRT19A ( for Upf1 and Upf2 ) and FRT82B ( for Mekk1 ) ( Xu and Rubin , 1993 ) .",
"For all experiments using Gadd45F17 we used the Gadd45E8 precise excision as a control .",
"For viability assays , we mated flies for 3 days and collected all progeny each day for 10 days , starting 10 days after the cross was initiated .",
"The total numbers of F1 mutant and balancer males were scored , and the ratio of mutant males to balancer males was used to determine mutant animal viability .",
"To control for balancer viability within each experiment , we normalized the ratio of mutant to balancer animals to a ratio of the appropriate control chromosome to balancer animals produced from a parallel cross .",
"We screened autosomal deficiencies from the DrosDel collection ( Ryder et al . , 2007 ) .",
"All deficiencies scored can be found in Supplementary file",
"1 . Deficiencies on chromosome 2 were balanced over CyO , and deficiencies on chromosome 3 were balanced over TM6C .",
"We mated males from each deficiency stock to y w Upf225G FRT19A/FM7i , P{ActGFP}JMR3 females and scored all F1 males for the presence or absence of each balancer .",
"For any given deficiency tested , the percentage of Deficiency / + males that are Upf225G mutants , less the percentage of Balancer / + males that are Upf225G mutants was calculated , producing a Deficiency Suppression Score ( DSS ) , which represents the effect of an individual deficiency on the increase or decrease in Upf225G viability , while controlling for each deficiency’s general influence on viability .",
"A DSS greater than 0 . 1 indicates suppression of lethality .",
"Supplemental deficiencies used were from the Exelixis collection ( Parks et al . , 2004 ) and Df ( 2R ) sple-J1 ( Heitzler et al . , 1993 ) .",
"Deficiency mapping to the Drosophila genome was performed using the 5 . 1 genome release .",
"RNA-seq data sets were acquired from Chapin et al . ( 2014 ) ( archives SRR896609 , SRR896616 , SRR503415 , and SRR503416 ) and aligned using Bowtie and TopHat alignment with standard remapping parameters to the 5 . 1 Drosophila genome release .",
"SAMtools accessory scripts were used to retrieve read counts for deficiency and control regions .",
"All read counts were normalized to reads per million within each data set .",
"Average normalized reads in Upf225G samples were normalized to the relative reads of 74 ribosomal proteins in Upf225G samples compared to control samples .",
"Total normalized reads within the regions removed by each deficiency were averaged between biological replicates , and the difference between the Upf225Gand control samples was divided by one million to determine percent increase in genomic load across each deficiency region .",
"We produced P-element excision lines from the P{w[+mC]=EPg}HP20647 P-element insertion line crossed to a Δ2–3 transposase stock .",
"We mated F1 males containing the P-element and transposase on a CyO balancer to w; Tft / CyO females .",
"Cy+ Tft white-eyed F2 males were then individually mated to w; Tft / CyO females .",
"We then collected Tft+ , Cy males and females to create an isogenic stock from each individually mated F2 male .",
"To identify precise excisions we used the primers Gadd45_F1 / Gadd45_R1 flanking the P-element insert site to amplify a region across the excised P-element .",
"Lines that failed to amplify with these primers were candidate imprecise excisions , which we then tested with Gadd45_F1 / Gadd45_R3 primers for deletions .",
"Any detected deletions were subsequently sequenced using these same primers .",
"Primer sequences are found in Supplementary file",
"2 . We generated eye clones with the FLP/FRT system using the ey-FLP driver ( Newsome et al . , 2000 ) to induce recombination .",
"We imaged eyes on a Leica MZ125 stereo microscope with a Retiga-2000R camera ( QImaging , Canada ) with QCapture 3 . 1 . 2 software ( QImaging ) .",
"We focused images using the ImageJ stack focuser plugin and quantified relative eye clone size using the ImageJ analyzer tools .",
"A total of 20 eyes from 20 individual animals were scored for each condition .",
"For TUNEL assays , third instar larval wing discs were dissected as described in Sullivan et al . ( Sullivan et al . , 2000 ) .",
"TUNEL staining was performed using the Apoptag Red in situ Apoptosis Detection Kit ( Chimicon International Inc . , Billerica , MA ) according to Chakraborty et al . ( Chakraborty et al . , 2015 ) .",
"We DAPI stained wing discs ( 1:5000 ) for 5 min prior to mounting .",
"Confocal images were acquired using a Zeiss LSM710 laser scanning confocal microscope ( Carl Zeiss AG , Germany ) .",
"3-dimensional datasets were acquired with a Plan-Apochromat 20X/0 . 8 lens , 1 . 34 μm z-step , using the Zeiss ZEN software .",
"To measure TUNEL signal intensity z-projections images were summed with ImageJ .",
"Background signal was removed by using the ImageJ MaxEntropy auto-threshold .",
"Relative total TUNEL signal intensity was calculated using the ImageJ analyzer tools to measure the total pixel intensity within the wing discs of TUNEL images and normalized to the average intensity in control conditions .",
"For annexin V staining , we collected media ( including floating cells ) from siRNA treated cells .",
"We spun down media at 950g for 4 min to pellet cells , and then aspirated remaining media .",
"Concurrently , we trypsinized siRNA-treated cells still on plates and added them to the same respective tube as previously spun-down media .",
"Following the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit ( Abcam , UK ) protocol , we stained for apoptotic cells .",
"We visualized cells on an Olympus IX51 microscope ( Olympus , Japan ) with 20X objective .",
"We collected bright field as well as fluorescent images using a FITC filter with a QImaging QICam Fast1394 camera and QCaptureP software ( QImaging ) .",
"We analyzed cells by counting all cells within a bright field image as well as the annexin V positive cells from the same image .",
"The number of annexin V positive cells was divided by total cell number to generate the fraction of apoptotic cells for each treatment .",
">3000 total cells were counted across three biological replicates for each treatment .",
"We cultured mouse NIH-3T3 cells ( ATCC ) or HEK293 cells ( ATCC ) in DMEM ( Thermo-Fisher , Waltham , MA ) supplemented with 10% fetal bovine serum and glutamine .",
"For siRNA experiments , we transfected cells using RNAiMax and 24 pmol of negative control siRNA ( Qiagen , Netherlands ) , Upf1 siRNA ( Qiagen ) , or Gadd45b siRNA ( Sigma-Aldrich ) for 3T3 cell experiments , or negative control siRNA ( Qiagen ) , UPF1 siRNA ( Qiagen ) , or GADD45A siRNA ( Sigma-Aldrich , St . Louis , MO ) for HEK293 experiments .",
"For double siRNA-treated cells , we used 24 pmol of each Upf1 and Gadd45b siRNA for 3T3 experiments or UPF1 and GADD45A siRNA for HEK293 experiments .",
"For actinomycin experiments , we incubated cells with siRNA for 48 hr before changing the media and then incubated with 2 μg/mL actinomycin ( Sigma-Aldrich ) for 1 or 2 hr . mRNA half-life was determined by fitting an exponential decay curve to the relative expression at each time point ( Tani et al . , 2012 ) .",
"t1/2 was calculated based on the average expression at each time point , and the mean t1/2 for each condition is represented .",
"For cell counting experiments , we trypsinized cells , incubated a small aliquot with Trypan Blue at a final concentration of 0 . 04% in complete media , and counted Trypan Blue negative cells .",
"RNA was collected from the remaining cells , and relative mRNA levels were measured as described below .",
"For Drosophila qRT-PCR analyses , we collected 5–10 adult animals frozen in liquid nitrogen .",
"We isolated total RNA using TRIzol reagent ( Invitrogen ) and phase-lock tubes ( 5-Prime ) , and the RNeasy mini kit ( Qiagen ) .",
"We used on-column RNase-free DNase treatment ( Qiagen ) to reduce genomic contamination .",
"We determined RNA concentration by spectrophotometer and normalized concentration for reverse transcription .",
"For reverse transcription , we used random decamers and MMLV8 reverse transcriptase ( Retroscript Kit , Thermo-FIsher ) .",
"We performed qRT-PCR analysis using the SYBR Green qPCR Supermix ( Bio-Rad , Hercules , CA ) and the Bio-Rad iCycler thermocycler .",
"All experimental reactions were performed using three technical replicates and a minimum of three biological replicates per condition , and the expression level of all experimental assays was normalized to RpL32 mRNA expression .",
"For cell culture qRT-PCR analyses , we collected RNA following the Zymo Research Quick RNA MiniPrep kits protocol , and synthesized cDNA using MMLV reverse transcriptase ( NEB , Ipswich , MA ) with a template of 1 µg of total RNA and priming with a T18 oligo .",
"We measured relative mRNA levels by qRT-PCR using the Masterplex ep realplex ( Eppendorf , Germany ) with SYBR green fluorescent dye .",
"Each sample was measured with technical triplicates and three biological replicates , and target mRNA levels were normalized to those of ribosomal protein 19 ( Rpl19 ) mRNA .",
"For all qRT-PCR analyses we also measured samples that had been made without reverse transcriptase to ensure that signal was not due to genomic DNA .",
"Primer sequences can be found in Supplementary file",
"2 . We cloned the UAS-GFP::Gadd45 3’ UTR and control UAS-GFP::Act5C 3’ UTR constructs using the primers G45_3U_X1_F / G45_3U_S1_R or Act5C_X1_F / Act5C_S1_R ( Supplementary file 2 ) to amplify the Gadd45 and Act5C 3’ UTRs , respectively , from genomic DNA .",
"PCR fragments were inserted into the Zero Blunt TOPO vector ( Thermo-Fisher ) , sequenced to assure fidelity , and digested and cloned into a pUAST-attB GFP vector using standard cloning procedures to replace the SV40 3’ UTR .",
"Plasmids were injected by BestGene ( Chino Hills , CA ) into a stock containing the VK00027 attP site ( Venken et al . , 2006 ) for phiC31 directed integration .",
"We used previously described UAS-GFP::SV40 3’ UTR animals ( Metzstein and Krasnow , 2006 ) .",
"For imaging , wandering late L3 larvae were collected and examined using a Leica MZ 16F microscope and the Leica DFC340 FX camera with the Leica Application Suite v3 . 3 . 0 software .",
"We collected adult F1 Upf2+; Gadd45E8/+ , Upf225G; Gadd45E8/+ , and Upf225G; Gadd45F17/+males that were also heterozygous for either the dHR783 or Adhn4 .",
"The Adhn4allele is a PTC-containing allele and has been demonstrated to be a direct NMD target based on cleavage by Smg6 ( Gatfield and Izaurralde , 2004 ) .",
"The dHR783 allele is also a PTC-containing allele and thus is presumably degraded by NMD ( Fisk and Thummel , 1998 ) .",
"At least three biological replicates were collected for each condition .",
"We isolated RNA and generated cDNA as described in methods above and used this cDNA as a template for PCR amplification of the dHR78 transcript with the DRH78_F3 / DHR78_R3 primers and the Adh transcript with the Adh_F and Adh_R primers ( Supplementary file 2 ) , which flank the nonsense mutation in the respective transcripts .",
"To compare the relative abundance of the dHR783 allele to the wild-type allele , PCR products were Sanger sequenced , and the relative peak intensity for a T ( dHR783 allele ) compared to a C ( wild-type allele ) at nucleotide 1063 was compared .",
"To compare the relative abundance of the Adhn4 allele to the wild-type allele , PCR products were digested with PvuII ( a site disrupted by the n4 mutation ) , separated on a 1% agarose gel and stained with ethidium bromide .",
"The relative intensity of the cut and uncut bands was determined using ImageJ and normalized for fragment length .",
"All samples were ran on the same gel and compared under identical conditions .",
"All ratios were normalized to the ratio in the Upf225G; Gadd45E8/+condition .",
"All figures displaying viability assays represent a proportion of animals of the indicated genotypes that survive to adulthood; error bars for these figures represent the 95% confidence interval of the binomial distribution , and the Test of qual or Given Proportions was used to determine significance difference in these proportions between genotypes .",
"All other figures represent the mean value of multiple replicates have error bars depicting ± 2 SEM , which is a close approximation of the 95% confidence interval ( Krzywinski and Altman , 2013 ) .",
"For tests between two variable measures , a two-sided paired Student’s t-test was used to determine significance difference between mean value data .",
"For most qPCR experiments , data was compared to a normalized control , set to a constant of 1 , so these tests were performed with a one-sided Student’s t-test ."
]
] | [
"The nonsense-mediated mRNA decay ( NMD ) pathway functions to degrade both abnormal and wild-type mRNAs .",
"NMD is essential for viability in most organisms , but the molecular basis for this requirement is unknown .",
"Here we show that a single , conserved NMD target , the mRNA coding for the stress response factor growth arrest and DNA-damage inducible 45 ( GADD45 ) can account for lethality in Drosophila lacking core NMD genes .",
"Moreover , depletion of Gadd45 in mammalian cells rescues the cell survival defects associated with NMD knockdown .",
"Our findings demonstrate that degradation of Gadd45 mRNA is the essential NMD function and , surprisingly , that the surveillance of abnormal mRNAs by this pathway is not necessarily required for viability ."
] | [
"Messenger RNA ( mRNA ) molecules act as the templates from which proteins are made , and so control the amount of protein in a cell .",
"Having too much of certain proteins can harm cells .",
"Additionally , some mRNAs contain errors , and so can create faulty proteins that may also harm the cell .",
"Cells have therefore developed ways to destroy excess or error-ridden mRNAs to avoid a deadly build up of proteins .",
"One such quality control mechanism is called nonsense-mediated decay ( NMD ) .",
"This mechanism is so important that cells that cannot perform nonsense-mediated decay die , although it is not clear exactly what kills the cells .",
"Now , Nelson et al . have found that fruit flies whose cells are unable to perform nonsense-mediated decay die because a harmful protein called Gadd45 builds up in the cells .",
"In normal cells , nonsense-mediated decay destroys the mRNA that relays the instructions for making Gadd45 , which keeps the amount of the Gadd45 protein in the cell low .",
"Further experiments show that removing Gadd45 from cells that lack nonsense-mediated decay saves the flies .",
"Removing Gadd45 from human and mouse cells that are unable to perform nonsense-mediated decay also allows these cells to survive .",
"These findings imply that the only nonsense-mediated decay function needed for cells to live is the destruction of Gadd45 mRNA .",
"This further implies that most faulty and normal mRNAs that are normally destroyed by nonsense-mediated decay do not cause the cells to die when nonsense-mediated decay is lost .",
"Learning that creating faulty proteins when nonsense-mediated decay is lost is not necessarily harmful to cells opens new possibilities to treating numerous genetic diseases .",
"In some diseases , cells can only produce faulty forms of a particular protein .",
"Nonsense-mediated decay normally destroys all of these mutant proteins , but it may sometimes be better to have faulty versions of a protein than to have none of it .",
"Safely getting rid of nonsense-mediated decay by also eliminating Gadd45 from cells may therefore be a treatment strategy worth exploring ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration | elife-16043-v2 | [
[
"FRDA , an inherited recessive ataxia , is caused by mutations in FXN ( Campuzano et al . , 1996 ) .",
"During childhood or early adulthood , FRDA patients show a progressive neurodegeneration of dorsal root ganglia , sensory peripheral nerves , corticospinal tracts , and dentate nuclei of the cerebellum ( Koeppen , 2011 ) .",
"FXN is evolutionarily conserved and the homologs have been identified in most phyla ( Bencze et al . , 2006; Campuzano et al . , 1996 ) .",
"It encodes a mitochondrial protein that is required for iron-sulfur cluster assembly ( Layer et al . , 2006; Lill , 2009; Muhlenhoff et al . , 2002; Rotig et al . , 1997; Yoon and Cowan , 2003 ) .",
"Once synthesized , iron-sulfur clusters are incorporated into a variety of metalloproteins , including proteins of the mitochondrial electron transport chain ( ETC ) complexes and aconitase , where they function as electron carriers , enzyme catalysts , or regulators of gene expression ( Lill , 2009 ) .",
"It has been proposed that loss of FXN leads to impaired ETC complex , which in turn triggers ROS production that directly contributes to cellular toxicity ( Al-Mahdawi et al . , 2006; Anderson et al . , 2008; Calabrese et al . , 2005; Schulz et al . , 2000 ) .",
"However , the ROS hypothesis has been questioned in several studies .",
"For example , loss of FXN only leads to a modest hypersensitivity to oxidative stress ( Macevilly and Muller , 1997; Seznec et al . , 2005; Shidara and Hollenbeck , 2010 ) .",
"In addition , several clinical trails based on antioxidant therapy in FRDA patients have shown no or limited benefits ( Lynch et al . , 2010; Parkinson et al . , 2013; Santhera Pharmaceuticals , 2010 ) .",
"Loss of yeast frataxin homolog results in iron accumulation ( Babcock et al . , 1997 ) , and this phenotype has also been reported in cardiac muscles of a Fxn deficiency mouse and FRDA patients ( Koeppen , 2011; Lamarche et al . , 1980; Michael et al . , 2006; Puccio et al . , 2001 ) .",
"However , whether iron accumulates in the nervous system upon loss of FXN remains controversial .",
"Furthermore , whether iron deposits contribute to the pathogenesis is not clear .",
"It has been reported that elevated iron levels were observed in the dentate nuclei or in glia cells of FRDA patients ( Boddaert et al . , 2007; Koeppen et al . , 2012 ) .",
"Contrary to these results , others suggested that there is no increase of iron in the nervous system of Fxn deficiency mice and FRDA patients ( Koeppen et al . , 2007; Puccio et al . , 2001; Solbach et al . , 2014 ) .",
"Taken together , current data provide insufficient evidence to establish that iron dysregulation contributes to neurodegeneration .",
"In addition , the mechanism underlying iron toxicity is still unclear .",
"In summary , the pathological interplay of mitochondrial dysfunction , ROS , and iron accumulation remains to be established .",
"We identified the first mutant allele of fh in an unbiased forward genetic screen aimed at isolating mutations that cause neurodegenerative phenotypes .",
"We show that loss of fh causes an age dependent neurodegeneration in photoreceptors and affects mitochondrial function .",
"Unlike other mitochondrial mutants with impaired ETC activity , we do not observe an increase in ROS .",
"However , loss of fh causes an iron accumulation in the nervous system , induces an up-regulation of sphingolipid synthesis , and activation of Pdk1 and Mef2 .",
"Reducing iron toxicity or inhibiting the sphingolipid/Pdk1/Mef2 pathway significantly suppresses neurodegeneration in fh mutants .",
"To our knowledge , this is the first evidence that sphingolipids , Pdk1 , and the transcription factor Mef2 are shown to play a primary role in FXN-induced neurodegeneration ."
],
[
"We identified the first EMS ( ethyl methanesulfonate ) induced missense mutation in fh , the Drosophila homolog of FXN , through a forward genetic screen on the X chromosome ( Figure 1—figure supplement 1A ) ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) .",
"The molecular lesion , S136R , is in a highly conserved region , which is required for the FXN binding to the iron-sulfur cluster assembly complex ( Tsai et al . , 2011 ) .",
"In addition , six point mutations adjacent to S136 ( S157 in human ) have been reported in FRDA patients ( Figure 1—figure supplement 1B ) ( Santos et al . , 2010 ) .",
"Hemizygous fh mutants are L3 to pupal lethal , and the lethality is not enhanced in transheterozygous mutants that carry a deficiency ( fh/Df ( 1 ) BSC537 ) , suggesting that S136R is a severe loss-of-function ( Figure 1—figure supplement 1C ) .",
"The lethality of fh can be rescued by a genomic fh construct ( gfh ) , or by ubiquitous expression ( da-GAL4 driver ) of fh cDNA using the UAS/GAL4 system ( Brand and Perrimon , 1993 ) ( Figure 1—figure supplement 1C ) .",
"The lethality cannot be rescued with tissue specific drivers , including neuronal ( nSyb-GAL4 ) , muscular ( Mef2-GAL4 ) , or glial ( repo-GAL4 ) drivers , suggesting a requirement for fh in multiple tissues ( Figure 1—figure supplement 1C ) .",
"In addition , fh mutants exhibit a smaller body size and prolonged larval stages ( 10 to 12 days instead of 5 days for wild type animals ) , similar to other mitochondrial mutants ( Sandoval et al . , 2014; Zhang et al . , 2013 ) .",
"Interestingly , removal of maternal wild type fh mRNA or protein in the egg exacerbates the lethal phase from pupal to embryonic lethality ( Figure 1—figure supplement 1C ) .",
"In essence , the residual maternally deposited wild type Fh not only extends the lifespan but also creates a partial loss of Fh condition in the fh mutants .",
"To bypass pupal lethality of fh mutants , mosaic mutant clones of adult photoreceptor neurons were generated with the eyeless-FLP/FRT system .",
"As a functional readout , we recorded and compared electroretinograms ( ERGs ) in young and aged flies .",
"In response to a light stimulus , the ERG amplitudes of newly eclosed fh mutants ( Day 1 ) are ~60% of control animals ( y , w , FRT19Aiso , the isogenized flies used in the screen ) ( Figure 1A ) .",
"The ERG amplitudes rapidly decline over a six day period ( Figure 1A ) .",
"Morphologically , the fh mutant clones contain a normal number of photoreceptors with a typical trapezoidal organization at Day 1 ( Figure 1B ) .",
"However , by using transmission electron microscopy ( TEM ) , we found that Day 1 fh mutant photoreceptors exhibit an expansion of the endoplasmic reticulum ( ER ) and a dramatic accumulation of lipid droplets in glial cells that are not observed in controls ( Figure 1—figure supplement 2A and B ) .",
"At Day 3 , photoreceptors in mutant clones exhibit aberrant elongated rhabdomeres ( Figure 1B ) .",
"By Day 6 , many of the photoreceptors are missing in the mutant clones , whereas control retinas exhibit intact morphology ( Figure 1B and Figure 1—figure supplement 2C ) .",
"These data show that the mutant photoreceptors undergo a rapid and severe functional as well as morphological degeneration . 10 . 7554/eLife . 16043 . 003Figure 1 . Loss of fh results in age dependent neurodegeneration in photoreceptors .",
"( A ) ERG of control ( y w FRT19A ) and fh ( y w fh FRT19A ) mosaic eyes .",
"The ERG amplitudes from Day 1 to Day 6 are indicated by red double arrows .",
"Quantification of the ERG amplitudes of control ( y w FRT19A ) and fh ( y w fh FRT19A ) is on the right .",
"( B ) Retina morphology of control ( y w FRT19A ) and fh mosaic eyes .",
"Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .",
"Neuronal nuclei are labeled by anti-Elav antibody ( blue ) .",
"Scale bar: 10 µm .",
"( C ) ERG of control ( y w FRT19A ) , fh mutants , fh mutants carrying a genomic fh construct ( fh; gfh ) , and fh mutants that express the fh cDNA ( fh; Rh1>fh ) or human FXN cDNA ( fh; Rh1>hFXN ) in photoreceptors using a Rh1-GAL4 driver .",
"The ERG amplitudes were recorded at Day 6 .",
"Data are presented as mean ± SEM .",
"**p<0 . 01 , ***p<0 . 001 , Student’s t-test .",
"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00310 . 7554/eLife . 16043 . 004Figure 1—figure supplement 1 . Mutation and lethal phase analysis of fh mutants .",
"( A ) Molecular lesion of fh mutants .",
"A 3 . 4 kb genomic rescue construct is shown below the gene .",
"( B ) FXN sequences alignment between fly , human , and mouse .",
"Mutation S136R identified in fh mutants is indicated by a box .",
"Black arrow heads indicate the point mutations identified in the FRDA patients .",
"( C ) Lethal phase analysis of fh mutants .",
"The lethality can be rescued by a genomic fh construct , or expressing fh cDNA by the ubiquitous driver daughterless-GAL4 .",
"Overexpression of fh cDNA in a tissue specific manner cannot rescue lethality .",
"Finally , removing maternal Fh proteins in the female germ line by using OvoD ( Perrimon and Gans , 1983 ) leads to embryonic to early L1 lethality . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00410 . 7554/eLife . 16043 . 005Figure 1—figure supplement 2 . Ultrastructure of adult eyes exhibits morphological defects in fh mutant clones .",
"( A ) Transmission electron microscopy ( TEM ) images of retina morphology in control ( y w FRT19A ) and fh mutant clones at Day 1 .",
"Lipid droplets are indicated by red arrow .",
"Scale bar: 2 µm .",
"( B ) Quantification of lipid droplet in Day 1 retina of control ( y w FRT19A ) and fh mutants .",
"n = 6 .",
"( C ) Retina morphology in control ( y w FRT19A ) and fh mutant clones at Day 6 .",
"Scale bar: 2 µm .",
"( D ) Retina morphology of fh mutants ( fh; Rh1-Gal4 ) , fh mutants carrying a genomic fh construct ( fh; gfh ) , and fh mutants that express the fh cDNA ( fh; Rh1>fh ) or human FXN cDNA ( fh; Rh1>hFXN ) .",
"Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .",
"The red eye marker ( w+ ) of Rh1-GAL4 creates autofluorescence background that makes imaging difficult .",
"Scale bar: 10 µm .",
"Data are presented as mean ± SEM .",
"***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 005 The neurodegeneration can be fully rescued by a genomic fh construct or by expressing fh cDNA in photoreceptors ( Figure 1C and Figure 1—figure supplement 2D ) , suggesting that photoreceptor degeneration is cell-autonomous .",
"Moreover , expression of the human FXN cDNA rescues neurodegeneration in fh mutants ( Figure 1C and Figure 1—figure supplement 2D ) , showing that fly and human FXN have conserved molecular function , and that our studies are relevant to the biological role of human FXN .",
"FXN has been reported to be localized to mitochondria ( Koutnikova et al . , 1997 ) .",
"Indeed , Fh colocalizes with mitoGFP in larval motor neuron , and removing the predicted mitochondrial targeting sequence ( MTS ) of fh cDNA ( Fh-∆MTS-V5 ) leads to a diffused signal in the cytosol ( Figure 2—figure supplement 1A ) .",
"Removal of the MTS leads to a dysfunctional protein as expression of Fh-∆MTS-V5 in the fh mutant clone is not able to rescue the ERG defect ( Figure 2—figure supplement 1B ) .",
"Loss of fh causes a highly aberrant mitochondrial morphology in one day old adult photoreceptors .",
"The mitochondria in fh mutants are larger and exhibit aberrant cristae and altered inner membrane morphology ( Figure 2A and Figure 2—figure supplement 1C ) .",
"Loss of FXN has been reported to cause various ETC and respiratory defects in different animal models , including yeast , fly , mouse , as well as FRDA patients ( Al-Mahdawi et al . , 2006; Anderson et al . , 2005; Carletti et al . , 2014; Koutnikova et al . , 1997; Puccio et al . , 2001; Rotig et al . , 1997; Wilson and Roof , 1997 ) .",
"We find that ETC Complex I ( CI ) activity is severely reduced in fh mutants when compared to control and genomic rescued animals ( Figure 2B ) .",
"Consistent with this data , the ADP/ATP ratio is increased in fh mutants ( Figure 2C ) , showing that energy production is impaired . 10 . 7554/eLife . 16043 . 006Figure 2 . Loss of fh causes impaired mitochondria , and reducing ROS levels fails to suppress degeneration in fh mutants .",
"( A ) TEM images of mitochondria in photoreceptors of control ( y w FRT19A ) and fh mutant clones .",
"The insets show a single mitochondrion ( red box ) .",
"Scale bar: 500 nm .",
"( B ) ETC complex activities in control ( y w FRT19A ) , fh mutant , and genomic rescued animal ( fh; gfh ) .",
"Complex activities are normalized to citrate synthase ( CS ) , which act as a control of mitochondrial mass . n = 3 . ( C ) ADP/ATP ratio in control ( y w FRT19A ) , fh mutant , and rescued animal ( fh; gfh ) .",
"n = 3 . ( D ) ROS levels are measured by DHE in the larval ventral nerve cord .",
"Neuronal down-regulation of ND42 ( nSyb>ND42 RNAi ) , a subunit of CI , acts as a positive control .",
"Quantification of DHE fluorescence is on the right .",
"n = 8 .",
"Scale bar: 50 µm .",
"( E ) Day 3 ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which ROS scavengers are overexpressed in photoreceptors .",
"Data are presented as mean ± SEM .",
"**p<0 . 01 , ***p<0 . 001 , Student’s t-test .",
"ND , no significant difference .",
"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00610 . 7554/eLife . 16043 . 007Figure 2—figure supplement 1 . Mitochondrial phenotype of fh mutants .",
"( A ) The immunostaining of larval motor neuron cell body with expression of fh or fh without the mitochondrial targeting sequence ( MTS ) ( Fh-∆MTS ) .",
"Fh and Fh-∆MTS are labeled by V5 ( red ) , and mitochondria are labeled by mitoGFP ( green ) .",
"The nuclei are marked by DAPI ( blue ) .",
"Scale bar: 5 µm .",
"( B ) Quantification of ERG amplitudes of fh mutants with different cDNA constructs at Day",
"3 . ( C ) Quantification of abnormal mitochondria in photoreceptor in TEM image .",
"n = 6 .",
"( D ) Day 6 ERG of fh mutants ( fh; Rh1-GAL4 ) in which yeast Ndi1p are overexpressed in photoreceptors .",
"( E ) Quantification of ERG amplitudes of fh mutants at Day",
"3 . Treating fh mutants with an antioxidant drug AD4 with 40 μg/ml concentration does not suppress degeneration .",
"Data are presented as mean ± SEM .",
"***p<0 . 001 , Student’s t-test .",
"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00710 . 7554/eLife . 16043 . 008Figure 2—figure supplement 2 . ROS/JNK/SREBP pathway does not contribute to degeneration in fh mutants .",
"( A ) Nile red staining of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants that overexpress lipases .",
"Quantification of ERG amplitudes at Day 3 is on the right .",
"Scale bar: 5 µm .",
"( B ) ROS/JNK/SREBP pathway causes accumulation of lipid droplets and degeneration in other mitochondrial mutants ( Liu et al . , 2015 ) .",
"( C ) The levels of puc-lacZ ( green ) in the larval ventral nerve cord of control ( y w FRT19A ) and fh mutants .",
"Neuronal membrane is marked by anti-DLG antibody ( blue ) .",
"Scale bar: 40 µm .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 008 It has been reported that expression of yeast Ndi1p , which is a single subunit of NADH dehydrogenase , can mitigate the deleterious effects of an ETC CI deficit in the fly ( Vilain et al . , 2012 ) .",
"However , overexpression of Ndi1p in fh mutant clones does not suppress neurodegeneration ( Figure 2—figure supplement 1D ) .",
"Impaired ETC CI activity has been linked to increased ROS production that in turn causes cellular stress or toxicity ( Sugioka et al . , 1988; Turrens and Boveris , 1980 ) .",
"We therefore evaluated ROS production in vivo with dihydroethidium ( DHE ) , a dye that emits fluorescence upon oxidation by ROS .",
"Neuronal down-regulation of ND42 ( nSyb>ND42 RNAi ) , a subunit of CI , was used as a positive control ( Owusu-Ansah et al . , 2008 ) .",
"As shown in Figure 2D , loss of ND42 exhibits strong DHE fluorescence in the larval ventral nerve cord , indicating elevated levels of ROS .",
"However , DHE levels are not obviously increased in fh mutants , suggesting no or a very mild increase in ROS ( Figure 2D ) .",
"Elevated ROS levels are proposed to be a major contributor to the pathogenesis in FRDA .",
"To further investigate the role of ROS in photoreceptor degeneration of fh mutants , we overexpressed several ROS scavengers , including Catalase , Super Oxide Dismutase 1 ( SOD1 ) , and SOD2 , which have all been shown to effectively reduce ROS in flies ( Orr and Sohal , 1992; Parkes et al . , 1998; Sun et al . , 2002 ) .",
"Consistent with the DHE data , overexpression of these ROS scavengers does not suppress neurodegeneration in fh mutants ( Figure 2E ) .",
"Furthermore , feeding fh mutants with AD4 , a potent antioxidant that dampens oxidative stress ( Amer et al . , 2008; Liu et al . , 2015 ) , does not suppress neurodegeneration ( Figure 2—figure supplement 1E ) .",
"Taken together , our findings argue that ROS does not contribute to the demise of neurons in fh mutants .",
"It has been proposed that lipid droplets play a role in the pathogenesis of FRDA as knockdown of fh in flies causes lipid droplet accumulation in glia ( Navarro et al . , 2010 ) .",
"From TEM and nile red staining , we observed a strong lipid droplet accumulation in glial cells of fh mutant clones ( Figure 1—figure supplement 2A and Figure 2—figure supplement 2A ) .",
"We recently reported that similar phenotype is observed in several mitochondrial mutants and promotes neurodegeneration via a ROS/JNK/SREBP pathway ( Liu et al . , 2015 ) ( Figure 2—figure supplement 2B ) .",
"However , we do not observe elevated ROS levels ( Figure 2D ) , and the level of a JNK pathway reporter , puc-lacZ , is not altered in fh mutants ( Figure 2—figure supplement 2C ) .",
"Finally , overexpression of brummer lipase ( Bmm ) or lipase 4 ( lip4 ) , homologs of human Adipose Triglyceride Lipase ( ATGL ) or acid lipase ( Gronke et al . , 2005 ) respectively , effectively reduces the number of lipid droplet but does not delay neurodegeneration of fh mutants ( Figure 2—figure supplement 2A ) , indicating that these lipid droplets do not contribute to neurodegeneration .",
"We recently discovered that some mutants that are required for mitochondrial function display an activity dependent degeneration of photoreceptors ( Jaiswal et al . , 2015 ) .",
"These mitochondrial mutants share several common phenotypes , including reduced ATP production , ERG rundown upon repetitive stimulation , and Rhodopsin1 ( Rh1 ) accumulation in the photoreceptor cytoplasm upon light exposure ( Jaiswal et al . , 2015 ) .",
"In these mutants , dysfunctional mitochondria cause reduced ATP production and decreased calcium influx , which not only leads to reduced photoreceptor depolarization , but also triggers an internalization of Rh1 leading to an overload of the endolysosomal system upon light stimulation ( Jaiswal et al . , 2015 ) .",
"The accumulation of Rh1 results in activity dependent degeneration , as the photoreceptors only degenerate when flies are raised in light ( light-induced neuronal activity ) but not in dark ( no neuronal activity ) ( Alloway et al . , 2000; Chinchore et al . , 2009; Jaiswal et al . , 2015 ) .",
"Since loss of fh leads to decreased CI activity and reduced energy production ( Figure 2B and C ) , we hypothesized that a similar activity dependent degeneration mechanism contributes to neurodegeneration in fh mutants .",
"To test this hypothesis , we first examined whether ERG traces rundown upon repetitive light stimulation in fh mutant photoreceptors .",
"As shown in Figure 3A , the ERG amplitudes of the first and last traces show a similar size in control animals .",
"In contrast , the ERG amplitude rapidly declines during repetitive light stimulation in fh mutants ( Figure 3A ) , similar to other mitochondrial mutants ( Jaiswal et al . , 2015 ) .",
"Therefore , we assessed whether Rh1 internalization is affected in fh mutants .",
"Wild type flies exposed to 12 hr of constant light typically exhibit low levels of Rh1 in photoreceptors ( Figure 3B ) .",
"However , in fh mutants , Rh1 accumulates in the cytoplasm of photoreceptors ( Figure 3B ) .",
"Since fh mutant photoreceptors share several features with other mitochondrial mutants , we hypothesized that reducing photoreceptor activity by raising fh mutants in the dark would suppress degeneration .",
"However , fh mutant flies that are raised in the dark still display severe photoreceptor degeneration ( Figure 3B ) .",
"Hence , we propose that in addition to the Rh1 accumulation , another pathogenic mechanism must contribute to neurodegeneration and mask the rescue effect when flies are raised in dark . 10 . 7554/eLife . 16043 . 009Figure 3 . Loss of fh causes Rh1 accumulation and iron deposit .",
"( A ) ERG traces upon repetitive light stimulation in both control ( y w FRT19A ) and fh mutants .",
"The double red arrows indicate ERG amplitudes of the first and last trace during the stimulation .",
"Quantification of the ratio of last/first ERG amplitude is on the right .",
"( B ) Rh1 distribution in control ( y w FRT19A ) and fh mutant clones after a 12 hr light exposure .",
"Rh1 is labeled in red , and rhabdomeres are labeled in green .",
"Scale bar: 5 µm .",
"Quantification of ERG amplitudes of control ( y w FRT19A ) and fh mutant clones in different light conditions is on the right .",
"L/D , flies are raised in 12 hr light and dark cycle .",
"D , flies are raised in dark .",
"( C ) Fe2+ levels are assessed by RPA in larval muscle and NMJ in fh mutants and rescued animals ( fh; gfh ) .",
"RPA fluorescence is represented as a heat map .",
"The NMJ boutons are indicated by white arrows .",
"Quantification of normalized RPA intensity is on the right .",
"n = 8 .",
"Scale bar: 20 µm .",
"( D ) Larval brain Perls’/DAB staining of control ( y w FRT19A ) , fh mutants , and rescued animals ( fh; gfh ) with regular food or low iron food conditions .",
"Scale bar: 100 µm .",
"( E ) Percent of animals that develop into pupae in control ( y w FRT19A ) and fh mutants when animals are raised by low iron food with different iron concentration .",
"n = 3 . Data are presented as mean ± SEM .",
"***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 00910 . 7554/eLife . 16043 . 010Figure 3—figure supplement 1 . Fe2+ staining control . RPAC intensity in larva muscle and NMJ of fh mutants and rescued animals ( fh; gfh ) .",
"Scale bar: 25 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 010 Whether iron accumulates in the nervous system and contributes to the pathogenesis in mice and FRDA patients has remained a topic of debate ( Boddaert et al . , 2007; Puccio et al . , 2001; Solbach et al . , 2014 ) .",
"We therefore investigated whether iron is involved in neurodegeneration of fh mutants .",
"To assess if iron homeostasis is affected in fh mutants , we first examined ferrous iron ( Fe2+ ) levels by Rhodamine B- ( ( 1 , 10-phenanthrolin-5-yl ) -aminocarbonyl ) benzyl ester ( RPA ) .",
"RPA is a cell-permeable fluorescent dye that selectively accumulates in mitochondria , and its fluorescence is stoichiometrically quenched by Fe2+ ( Petrat et al . , 2002 ) .",
"As RPA poorly crosses the blood brain barrier ( data not shown ) , we performed RPA staining of larval muscles and neuromuscular junctions ( NMJ ) .",
"In fh mutants rescued with the genomic fh , which serves as a negative control , the RPA signal exhibits strong fluorescence ( Figure 3C ) .",
"In contrast , the RPA intensity in fh mutants is dramatically reduced , in both muscle and NMJ boutons , suggesting a strong Fe2+ accumulation in mitochondria ( Figure 3C ) .",
"RPAC ( Rhodamine B- ( ( phenanthren-9-yl ) -aminocarbonyl ) benzyl ester ) , a dye that is structurally very similar to RPA , is also taken up by mitochondria but is insensitive to Fe2+ mediated fluorescence quenching .",
"Indeed , RPAC fluorescence is not quenched in rescued animals ( fh; gfh ) and fh mutants ( Figure 3—figure supplement 1 ) , suggesting that the decreased fluorescence of RPA in fh mutants is not due to impaired dye uptake .",
"To investigate whether ferric iron ( Fe3+ ) levels are increased in fh mutants , we performed Perls’ staining and used 3 , 3'-Diaminobenzidine ( DAB ) to enhance the signal ( Meguro et al . , 2007 ) .",
"In control and rescued animals , the DAB signal is most obvious in the neuropil of the ventral nerve cord ( Figure 3D ) , suggesting that Fe3+ is unevenly distributed in the nervous system .",
"In fh mutants , however , the DAB signal is dramatically increased ( Figure 3D ) , indicating that loss of fh leads to a severe accumulation of Fe3+ .",
"Increased Fe3+ levels are not only found in the larval brain , but can also be observed in fat body and gut ( data not shown ) .",
"Together , these results indicate that loss of fh leads to both Fe2+ and Fe3+ accumulation in multiple tissues , including the nervous system .",
"To determine if the iron accumulation contributes to neurodegeneration in fh mutant clones , we reduced iron levels in the fly food .",
"Our regular food contains iron-rich molasses .",
"To reduce iron levels , we replaced molasses with sucrose as the main carbohydrate source and refer to this food as 'low iron food' .",
"As shown in Figure 3D , feeding larvae with low iron food reduces iron levels in the brains of both wild type control and fh mutants .",
"In addition , the lethal phase of fh mutants is susceptible to iron levels in the food .",
"In low iron food , 80% of fh mutants develop into pupae , but addition of 10 mM Fe3+ to the low iron food reduces pupation to about 10% , whereas the pupation rate of controls is not affected ( Figure 3E ) .",
"These data indicate that fh mutants are highly sensitive to iron levels .",
"To determine if iron toxicity is underlying the observed neurodegeneration , we fed low iron food to fh mutants and examined photoreceptor degeneration .",
"Treating fh mutants with low iron food improves the ERG defects when compared to regular food condition ( Figure 4A ) , suggesting that iron mediated toxicity contributes to neurodegeneration in fh mutants . 10 . 7554/eLife . 16043 . 011Figure 4 . Neuronal activity and iron interact synergistically to contribute to photoreceptor degeneration in fh mutants .",
"( A ) Quantification of ERG amplitudes of control ( y w FRT19A ) and fh mutants under different light and iron conditions .",
"( B ) Retina morphology of control ( y w FRT19A ) and fh mutants under different food conditions at Day 6 .",
"All animals are raised in dark .",
"Regular , flies are raised in regular food .",
"Low iron , flies are raised in low iron food .",
"Scale bar: 5 µm .",
"( C ) Quantification of ERG amplitudes of fh mutants in low iron food with different iron concentrations .",
"( D ) ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which ferritins are overexpressed .",
"Fer1* , enzymatic dead form of Fer1HCH .",
"Data are presented as mean ± SEM .",
"***p<0 . 001 , Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01110 . 7554/eLife . 16043 . 012Figure 4—figure supplement 1 . Control of low iron food treatment . Quantification of ERG amplitudes of control animals ( y w FRT19A ) in different food and iron conditions .",
"Regular , flies are raised in regular food .",
"Low iron , flies are raised in low iron food .",
"Data are presented as mean ± SEM .",
"L/D , flies are raised in 12 hr light and dark cycle . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 012 We next assessed if excess neuronal activity and iron accumulations act synergistically to promote neurodegeneration in fh mutants .",
"Interestingly , reducing photoreceptor activity ( raising flies in dark ) and iron ( low iron food ) leads to a synergistic effect in restoring both photoreceptor function and morphology ( Figure 4A and B ) .",
"To exclude the possibility that changes in other compounds than iron in low iron food suppresses neurodegeneration in fh mutants , we reintroduced iron into the low iron food ( low iron/10 mM iron ) and found that this treatment again induces rapid degeneration in fh mutant clones even when animals are raised in dark ( Figure 4B and C ) .",
"The ERG amplitudes of the wild type controls are not affected by different iron concentrations in the food ( Figure 4—figure supplement 1 ) .",
"These data suggest that iron toxicity contributes to the activity independent degeneration , as photoreceptors degenerate in the absence of neuronal activity .",
"We also tested if feeding 100 mM iron to wild type controls is sufficient to trigger neurodegeneration .",
"This concentration of iron is toxic as most animals die as embryos or young larval instars , and the Fe3+ levels were not increased in the brains ( data not shown ) .",
"We next explored if genetic manipulations can suppress iron toxicity and neurodegeneration in fh mutants .",
"We expressed Ferritins , the iron storage proteins that chelate and sequester irons .",
"In Drosophila , Ferritin 1 heavy chain homologue ( Fer1HCH ) and Ferritin 2 light chain homologue ( Fer2LCH ) form a heteropolymeric complex , whereas Ferritin 3 heavy chain homologue ( Fer3HCH ) forms a homopolymeric complex to store irons ( Missirlis et al . , 2006 , 2007 ) .",
"In fh mutant clones , co-expression of Fer1HCH and Fer2LCH ( fh; Rh1>Fer1 , Fer2 ) or expression of Fer3HCH alone ( fh; Rh1>Fer3 ) significantly suppresses degeneration when flies are raised in dark ( Figure 4D ) .",
"Co-expression of a ferroxidase inactive form of Fer1HCH along with wild type Fer2LCH in fh mutants ( fh; Rh1>Fer1* , Fer2 ) did not rescue degeneration ( Figure 4D ) .",
"These results further confirm that iron toxicity contributes to neurodegeneration in fh mutant clones .",
"In summary , we conclude that excess neuronal activity ( activity dependent ) and accumulated iron ( activity independent ) synergize to promote photoreceptor degeneration in fh mutants .",
"Given that iron accumulates in fh mutants , we investigated the mechanism downstream of iron toxicity .",
"Previous studies proposed that highly active iron interacts with hydrogen peroxide to generate free radicals , the Fenton reaction .",
"However , we did not observe an increase in ROS ( Figure 2D ) , and expression of different ROS scavengers did not suppress degeneration in fh mutants ( Figure 2E ) .",
"It has been previously documented that wild type yeast grow in medium with high iron levels increases sphingolipid synthesis ( Lee et al . , 2012 ) .",
"However , this phenotype was not documented in the yeast frataxin homolog mutants or FXN mutants in other organisms .",
"As we observed a strong iron accumulation in fh mutants , we hypothesized that iron toxicity induces sphingolipid synthesis and causes neurodegeneration .",
"To test our hypothesis , we first assessed the de novo synthesis of sphingolipids by mass spectrometry .",
"Consistent to our hypothesis , several upstream metabolic intermediates of sphingolipids are increased in fh mutants ( Figure 5A ) .",
"Strikingly , dihydrosphingosine ( dhSph ) is increased more than 10 fold in fh mutants .",
"Other sphingolipids , such as dihydroceramide ( dhCer ) and sphingosine ( Sph ) , are also increased to different levels ( Figure 5A ) .",
"This data suggest that the de novo synthesis of sphingolipids is increased in fh mutants . 10 . 7554/eLife . 16043 . 013Figure 5 . Increased sphingolipids contribute to degeneration of fh mutants .",
"( A ) Mass spectrometry analysis of sphingolipids in control ( y w FRT19A ) , fh mutants , and rescued animals ( fh; gfh ) .",
"dhsph , dihydrosphingosine; dhCer , dihydroceramide; Cer , ceramide; Sph , sphingosine .",
"n = 2 . ( B ) ERG of control ( y w FRT19A; Rh1-GAL4 ) , and fh mutants in which an RNAi against lace is expressed .",
"( C ) ERG amplitude and retinal morphology of fh mutants treated with 100 μM myriocin .",
"Scale bar: 5 µm .",
"( D ) Quantification of Day 3 ERG amplitudes of fh mutants with low iron food and myriocin treatments .",
"Data are presented as mean ± SEM .",
"The error bars represent the range of data in ( A ) .",
"**p<0 . 01 , ***p<0 . 001 , Student’s t-test .",
"D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01310 . 7554/eLife . 16043 . 014Figure 5—figure supplement 1 . Control of myriocin treatment . Quantification of Day 6 ERG amplitudes of control animals ( y w FRT19A ) with myriocin treatment .",
"Data are presented as mean ± SEM .",
"D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 014 To determine if increased sphingolipid synthesis contributes to neurodegeneration in fh mutants , we performed RNAi experiments against the first and rate-limiting enzyme in the de novo synthesis pathway , lace , the fly homolog of Serine palmitoyltransferase .",
"This significantly improved the ERG defects of fh mutants ( Figure 5B ) .",
"In addition , feeding fh mutants Myriocin , a serine palmitoyltransferase inhibitor , suppressed both the functional and morphological photoreceptor defects of fh mutant clones ( Figure 5C ) .",
"Feeding myriocin to control animals does not adversely affect photoreceptor function ( Figure 5—figure supplement 1 ) .",
"To assess if iron accumulation and increased sphingolipid synthesis act in the same or a different pathway , we treated fh mutants with low iron food as well as myriocin .",
"As shown in Figure 5D , we did not observe an additive or synergistic effect .",
"These results suggest that iron toxicity induces sphingolipid synthesis which in turn causes neurodegeneration in fh mutants .",
"Increased sphingolipids in yeast have been shown to activate a kinase pathway ( Pkh1/Ypk1/Smp1 ) that converges on a transcription factor Smp1p ( Lee et al . , 2012 ) .",
"However , this pathway has not yet been studied in higher eukaryotic cells , or FXN mutant animal models .",
"The corresponding proteins in mammals are PDK1 , SGK ( serum- and glucocorticoid regulated kinase ) , and Mef2 transcription factor ( Casamayor et al . , 1999; Dodou and Treisman , 1997 ) .",
"The Drosophila homolog of SGK has not been identified , but both Pdk1 and Mef2 are present in the fly .",
"Mef2 is a well known transcription factor required for muscle differentiation , however , it is also expressed in certain neurons and photoreceptors ( Blanchard et al . , 2010 ) .",
"As we observed an increase in sphingolipids , we assessed if increased levels of sphingolipids activate the Pdk1/Mef2 pathway and underlie the neurodegenerative phenotype in fh mutants .",
"We first tested if Pdk1/Mef2 is activated in fh mutants .",
"As a proxy for Mef2 activation , we examined the mRNA levels of known downstream targets of Mef2 .",
"Seven Mef2 targets , which have been validated through in situ hybridization and qRT-PCR , are all ectopically expressed in response to ectopic expression of Mef2 in vivo ( Elgar et al . , 2008 ) .",
"We therefore examined these seven Mef2 targets in the nervous system of fh mutants .",
"As shown in Figure 6A , all of these Mef2 targets are up-regulated .",
"Several negative control genes , the downstream targets of other transcription factors , including Enhancer of split ( E ( Spl ) ) , senseless ( sens ) , and patched ( ptc ) , as well as several housekeeping genes that are not Mef2 targets ( Sandmann et al . , 2006 ) , are not changed in fh mutants ( Figure 6—figure supplement 1A ) .",
"These data indicate that Mef2 is abnomally activated in fh mutants . 10 . 7554/eLife . 16043 . 015Figure 6 . Activated Pdk1/Mef2 pathway causes degeneration in fh mutants .",
"( A ) mRNA levels of Mef2 downstream targets in fh mutants and rescued animals ( fh; gfh ) .",
"n = 3 . ( B ) Day 3 ERG of control ( y w FRT19A; Rh1-GAL4 ) and fh mutants in which an RNAi against Pdk1 or Mef2 is expressed .",
"( C ) ERG of flies with Mef2 overexpression in the eye .",
"( D ) Mef2 luciferase reporter assay in Neuro-2A cells with different concentration of dhSph treatment .",
"GSK 2334470 , a PDK1 inhibitor .",
"n = 4 . Data are presented as mean ± SEM .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 , Student’s t-test .",
"D , flies are raised in dark . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 01510 . 7554/eLife . 16043 . 016Figure 6—figure supplement 1 . Control of qRT-PCR .",
"( A ) mRNA levels of downstream targets of Notch , Wingless , and Hedgehog pathway as well as several ribosomal genes are not changed in fh mutants .",
"( B ) Retina morphology of fh mutants , and fh mutants that express Pdk1 or Mef2 RNAi .",
"Rhabdomeres are labeled by phalloidin ( green ) , whereas anti-Na/K ATPase antibody ( red ) marks the photoreceptor membranes .",
"Scale bar: 10 µm .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 16043 . 016 Next , we tested if the activated Pdk1/Mef2 pathway contributes to neurodegeneration of fh mutants .",
"We reasoned that if Pdk1 and Mef2 are activated and toxic , their down-regulation may suppress degeneration .",
"Consistent with our hypothesis , RNAi mediated knockdown of Pdk1 suppresses the ERG and morphological defects in fh mutants ( Figure 6B and Figure 6—figure supplement 1B ) .",
"Furthermore , knock down of Mef2 by two independent RNAi lines suppresses neurodegeneration of fh mutants ( Figure 6B and Figure 6—figure supplement 1B ) , suggesting that activated Pdk1 and Mef2 are toxic and contribute to the degeneration .",
"To determine if up-regulation of Mef2 in wild type photoreceptors is sufficient to induce neurodegeneration , we ectopically expressed Mef2 in the adult eye using Rh1-Gal4 .",
"Overexpression of Mef2 is sufficient to cause photoreceptor degeneration ( Figure 6C ) .",
"Furthermore , flies with neuronal expression of Mef2 ( nSyb>Mef2 ) die within a week , instead of 70–80 days of the wild type flies ( data not shown ) .",
"These data suggest that abnormal activation of Mef2 in neuron is sufficient to trigger its demise .",
"To assess if a similar pathway is affected in vertebrate cells , we tested if sphingolipids can induce Mef2 activation in Neuro-2A cells .",
"dhSph , the sphingolipid that is increased more than 10 fold in fly fh mutants ( Figure 5A ) , induces luciferase signals of a Mef2 reporter in a concentration dependent manner ( Figure 6D ) .",
"In addition , the increased luciferase signal is suppressed by adding the PDK1 inhibitor GSK2334470 ( Figure 6D ) .",
"This data indicate that dhsph activates Mef2 through PDK1 , consistent with what we observed in the fly .",
"Collectively , these data suggest that sphingolipids act as signaling molecules to activate the Pdk1/Mef2 pathway and that this pathway contributes to photoreceptor degeneration in fh mutants ."
],
[
"Here , we report the isolation of the first severe loss of function allele of fh in Drosophila .",
"This allowed us to identify a novel molecular pathway that contributes to neurodegeneration .",
"Loss of fh in Drosophila causes an age dependent degeneration of photoreceptors in mutant clones .",
"Mutant photoreceptors display abnormal mitochondrial cristae morphology , reduced ETC CI activity , and impaired ATP production .",
"However , we do not observe an increase in ROS .",
"Our data indicate that Rh1 trafficking defects caused by mitochondria dysfunction lead to an activity dependent degeneration in fh mutants .",
"However , the accumulation of Fe2+ and/or Fe3+ stimulates sphingolipid synthesis which in turn activates the Pdk1/Mef2 pathway to cause an activity independent degeneration .",
"Hence , mitochondrial dysfunction , and iron toxicity through activation of the sphingolipid/Pdk1/Mef2 pathway , synergistically contribute to the demise of neurons in fh mutants .",
"The data indicate that ectopic activation of the transcription factor Mef2 induced by loss of FXN may play a role in FRDA .",
"Elevated ROS levels , either through impaired ETC CI or CIII , or iron associated Fenton reaction , have been proposed to be the major driving force of disease pathogenesis in FRDA ( Bayot et al . , 2011; Santos et al . , 2010 ) .",
"However , we do not observe increased ROS levels in fh mutants ( Figure 2D ) .",
"In addition , overexpression of ROS scavengers or feeding antioxidant AD4 does not suppress degeneration ( Figure 2E and Figure 2—figure supplement 1E ) .",
"The lack of increased ROS is surprising as mitochondrial mutants in Drosophila that exhibit a severely reduced CI activity typically show elevated ROS levels ( Owusu-Ansah et al . , 2008; Zhang et al . , 2013 ) .",
"Numerous studies using different animal models or cell lines have established that a partial or complete loss of FXN leads to different degrees of defects in the ETC .",
"Importantly , several reports have also documented that loss of FXN leads to an hypersensitivity to ROS , rather than elevated ROS levels ( Babcock et al . , 1997; Llorens et al . , 2007; Macevilly and Muller , 1997; Seznec et al . , 2005; Shidara and Hollenbeck , 2010 ) .",
"Consistent with our data , overexpression of ROS scavengers in an RNAi mediated fh knock down did not improve viability or cardiac function , and no ROS could be detected ( Anderson et al . , 2005; Shidara and Hollenbeck , 2010; Tricoire et al . , 2014 ) .",
"Similarly , loss of Fxn in mice does not lead to an increase in ROS ( Puccio et al . , 2001; Seznec et al . , 2005 ) , although elevated ROS levels in another mouse model have been documented ( Al-Mahdawi et al . , 2006 ) .",
"Combined , these data suggest that ROS is not a prominent player in model organisms and a clinical trial designed to suppress ROS have not provided compelling data ( Santhera Pharmaceuticals , 2010 ) .",
"Iron accumulation in mitochondria was first described in yeast frataxin homolog mutants in yeast ( Babcock et al . , 1997 ) .",
"Although iron deposits have been reported in cardiac muscles of Fxn deficiency mice and FRDA patients , iron accumulation in the nervous system has been reported by some ( Boddaert et al . , 2007; Koeppen et al . , 2009 ) but not by others ( Puccio et al . , 2001; Simon et al . , 2004; Solbach et al . , 2014 ) .",
"More importantly , the role of iron in the pathophysiology of FRDA has not yet been determined , and mitochondrial defects are thought to presage iron accumulation in mammals ( Puccio et al . , 2001 ) .",
"It has been argued that Fe3+ accumulations are a non-reactive , intra-mitochondrial precipitates ( Seguin et al . , 2010; Whitnall et al . , 2012 ) .",
"Finally , others proposed that iron accumulation is toxic as it induces the Fenton’s reaction and promotes ROS production .",
"We find that in Drosophila fh mutants , both Fe2+ and Fe3+ accumulate in the nervous system ( Figure 3C and D ) .",
"As we observed no increased ROS , we argue that Fe2+ and Fe3+ accumulation is toxic because:",
"1 ) the lethality is enhanced when iron is increased;",
"2 ) reduction of iron in food suppresses the progression of photoreceptor degeneration; and",
"3 ) overexpression of Ferritin delays the demise of neurons .",
"Taken together , we argue that iron accumulation in neurons is toxic and plays a primary role in the pathogenesis in fh mutants .",
"Our data indicate that iron accumulation enhances the synthesis of sphingolipid , and that the excess amount of sphingolipids is toxic and promotes neurodegeneration in fh mutants .",
"How iron accumulation leads to an increase in sphingolipid synthesis is unknown .",
"Although we show that Fe2+ accumulates in mitochondria of the larval muscle and at the NMJ in fh mutants , it is still not clear whether iron accumulates in other cellular organelles .",
"The de novo synthesis of sphingolipids occurs in the ER , and mitochondria have close contacts with ER at the MAMs ( mitochondria-associated endoplasmic reticulum membrane ) .",
"Mitochondrial or ER localized iron may promote the enzymatic function of Lace or other proteins in the ER to enhance sphingolipid synthesis .",
"However , treatment with Myriocin which targets Lace to decrease sphingolipid synthesis or down-regulation of lace via RNAi mediated knockdown suppresses degeneration in fh mutants ( Figure 5B and C ) , suggesting that increased sphingolipid metabolites contribute to toxicity .",
"These data are also in agreement with the observation that overexpression of Sk2 ( sphingosine kinase",
"2 ) or feeding dhSph-1P to wild type flies promotes the degeneration of photoreceptors ( Yonamine et al . , 2011 ) .",
"An increase in sphingolipids may affect various processes , including membrane integrity , lipid metabolism , and signal transduction ( Brown and London , 2000; Hannun and Obeid , 2008; Worgall , 2008 ) .",
"We found that down-regulation of Pdk1 or Mef2 in mutant photoreceptor delays neurodegeneration in fh mutants , while overexpression of Mef2 in photoreceptor promotes the demise of neurons ( Figure 6B and C ) .",
"It has been reported that Sph or dhSph , two sphingolipids that are elevated in fh mutants , directly induce autophosphorylation of PDK1 or SGK in an in vitro kinase assay ( Caohuy et al . , 2014; King et al . , 2000 ) .",
"Although there is no evidence that PDK1 or SGK can directly activate Mef2 , our data clearly show that Mef2 activity is up-regulated by elevating dhSph in the medium of cultured Neuro-2A cells , and this increased activity is suppressed by a PDK1 inhibitor ( Figure 6D ) , indicating that Mef2 can be activated by PDK1 or a downstream substrate of PDK1 ( Mora et al . , 2004 ) .",
"The MEF2 family includes four vertebrate genes ( MEF2A–D ) that play a key role in muscle differentiation ( Molkentin et al . , 1995 ) .",
"However , the MEFs are also expressed in neurons where they are involved in neuronal development ( Akhtar et al . , 2012; Leifer et al . , 1994; Lyons et al . , 1995; Mao et al . , 1999 ) .",
"The MEFs are known to regulate the expression of numerous target genes , including genes required for muscle differentiation , immediate-early genes , neuronal-activity-regulated genes , as well as genes involved in energy storage and immune response ( Clark et al . , 2013; Dietrich , 2013 ) .",
"In flies , ectopic expression of Mef2 in ectoderm is sufficient to induce nearly half of its predicted downstream targets that are typically expressed in muscles ( Sandmann et al . , 2006 ) .",
"Hence , its unusual activity may trigger neurodegeneration via the activation of some of its downstream targets .",
"Importantly , it has been shown that the up-regulation of Mef2 is implicated in the cardiac hypertrophy and dilation ( Molkentin and Markham , 1993; Xu et al . , 2006 ) , which is the major cause of death in FRDA patients .",
"In sum , our data indicate that reducing the levels of sphingolipid synthesis with Myriocin , or antisense strategy against enzymes involved in sphingolipid synthesis , or reducing the levels of Pdk1or Mef2 , are options that should be explored in FRDA models ."
],
[
"Flies were obtained from the Bloomington Drosophila Stock Center at Indiana University ( BDSC ) unless otherwise noted .",
"The stocks were routinely maintained at room temperature .",
"For genetic interaction experiments , flies were raised at 28°C to enhance the GAL4 activity .",
"For all the larval experiments , flies were allowed to lay eggs for 48 hr on grape juice plates with yeast paste .",
"Hemizygous mutant larvae and wild type controls were isolated by GFP selection at the first instar phase and transferred to standard fly food for the duration of their development .",
"To create mosaic mutant clones in the adult eye , y w FRT19A and y w fh FRT19A/FM7c , Kr>GFP females were crossed with y w GMR-hid FRT19A; ey-GAL4 UAS-FLP or y w GMR-hid l ( 1 ) cl FRT19A/FM7c , Kr>GFP; eyFLP Rh1-GAL4/CyO flies .",
"The following strains were used to generate fly stocks in this study: y w FRT19A ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) y w fh1 FRT19A/FM7c , Kr>GFP ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) y w fh1 FRT19A/FM7c , Kr>GFP; genomic-fh ( Haelterman et al . , 2014; Yamamoto et al . , 2014 ) Df ( 1 ) BSC537 , w1118/FM7h/Dp ( 2;Y ) G , P{hs-hid}Y y w GMR-hid FRT19A; ey-GAL4 UAS-FLP y w GMR-hid l ( 1 ) cl FRT19A/FM7c , Kr>GFP; eyFLP Rh1-GAL4/CyO ( Jaiswal et al . , 2015 ) w1; P{UAS-fh . A}1 ( Anderson et al . , 2005 ) w1; P{UAS-Cat . A}2 ( Anderson et al . , 2005 ) w1; P{UAS-Sod . A}B37 ( Anderson et al . , 2005 ) w1; P{UAS-Sod2 . M}UM83 ( Anderson et al . , 2005 ) P{rh1-GAL4}3 , ry506 P{Gal4-da . G32} UH1 y1 w*; P{nSyb-GAL4 . S}3 w; repo-GAL4/TM3 , Sb w*; P{w+mC bmmScer\\UAS = UAS-bmm} ( Gronke et al . , 2005 ) ( gift from Ronald Kühnlein ) w*; UAS-Lip4/CyO ( Liu et al . , 2015 ) P{UAS-Mef2-HA} ( Sandmann et al . , 2006 ) ( gift from Eileen Furlong ) P{UAS-Fer1HCH} P{UAS-Fer2LCH} ( Missirlis et al . , 2006 , 2007 ) ( gift from Hermann Steller ) P{UAS-Fer1HCH . mut} P{UAS-Fer2LCH} ( Missirlis et al . , 2006 , 2007 ) ( gift from Hermann Steller ) P{UAS-Fer3HCH} ( Missirlis et al . , 2006 ) ( gift from Patrik Verstreken ) w1118; P{GawB}D42 , P{UAS-mito-HA-GFP . AP}3 e1/TM6B , Tb1 ( Horiuchi et al . , 2005 ) ( gift from Bill Saxton ) w*; cno3 P{A92}pucE69/TM6B , abdA-LacZ ( Ring and Martinez Arias , 1993 ) ( Drosophila Genetic Resource Center ) ND42 RNAi: y1 sc* v1; P{TRiP . HMS00798}attP2 ( Owusu-Ansah et al . , 2008 ) lace RNAi: P{KK102282}VIE-260B ( Ghosh et al . , 2013 ) ( VDRC ) Pdk1 RNAi: P{KK108363}VIE-260B ( Tokusumi et al . , 2012 ) ( VDRC ) Mef2 RNAi: w1118; P{GD5039}v15549/TM3 and w1118; P{GD5039}v15550 ( Clark et al . , 2013 ) ( VDRC ) ERG recordings were performed as described ( Verstreken et al . , 2003 ) .",
"Briefly , flies were glued on a glass slide .",
"A recording electrode filled with 3M NaCl was placed on the eye , and another reference electrode was inserted into the fly head .",
"Before the recording , photoreceptors were allowed to recover by keeping flies in the dark for 3 min .",
"During the recording , a 1 s pulse of light stimulation was given , and the ERG traces of five to ten flies were recorded and analyzed by AXON -pCLAMP 8 software .",
"To detect Fe2+ levels , L3 larvae were dissected in ice-cold 0 . 25 mM Ca2+ HL3 ( Stewart et al . , 1994 ) ( 70 mM NaCl , 5 mM KCl , 20 mM MgCl2 , 10 mM NaHCO3 , 5 mM trehalose , 5 mM HEPES , 115 mM sucrose , pH 7 . 2 ) and rinsed in HL3 with 1 mM Ca2+ .",
"The animals were then incubated with 10 µM RPA or RPAC ( Squarix Biotechnology , Germany ) in 1 mM Ca2+ HL3 for 20 min at room temperature in the dark .",
"The animals were washed subsequently three times with ice-cold 1 mM Ca2+ HL3 and then incubated in 1 mM Ca2+ HL3 with 7 mM L-glutamic acid for 15 min at room temperature in the dark .",
"The RPA or RPAC fluorescence was obtained with a Zeiss LSM 510 confocal microscope ( Carl Zeiss ) and quantified by Image J . To evaluate Fe3+ levels , L3 larval brains were dissected and fixed in 3 . 7% formaldehyde for 20 min .",
"Samples were quickly washed three times with 0 . 4% Triton-PBS and incubated with Perls solution ( 1% K4Fe ( CN ) 6 and 1% HCl in PBT ) for 5 min .",
"After 3 quick washes in PBT , samples were incubated with DAB solution ( 10 mg DAB with 0 . 07% H2O2 in 0 . 4% Triton-PBS ) to enhance the signal .",
"The brains were then quickly washed three times with 0 . 4% Triton-PBS and mounted .",
"The brain images were obtained with a Leica MZ16 stereomicroscope equipped with Optronics MicroFire Camera .",
"L3 larvae were dissected in ice-cold 0 . 25 mM Ca2+ HL3 and rinsed with 1 mM Ca2+ HL3 .",
"The animals were then incubated with 10 µM dihydroethidium ( DHE , Sigma-Aldrich ) in 1 mM Ca2+ HL3 for 20 min at room temperature in the dark .",
"The samples were quickly washed three times with 1 mM Ca2+ HL3 with 7 mM L-glutamic acid , and the DHE fluorescence in larval brains were live imaged by Zeiss 510 confocal microscope ( Carl Zeiss ) and quantified by Image J . Low iron food was made using 10% dry yeast , 10% sucrose , and 0 . 6% agar in w/v in water .",
"The solution was microwaved and dried at room temperature , divided into vials , and stored at 4°C for further use .",
"To increase iron levels in the food , ammonium iron ( Ш ) citrate ( Sigma-Aldrich ) was added to a final concentration of 1 mM or 10 mM in the low iron food .",
"To test if inhibition of sphingolipid synthesis suppresses neurodegeneration in fh mutants , myriocin from Mycelia sterilia ( Sigma-Aldrich ) was dissolved in regular fly food ( molasses based ) or low iron food , and vials were then stored immediately at −20°C for future use .",
"The flies were transfer to fresh food vials every two days .",
"For the fh genomic rescue construct , a DNA fragment containing Drosophila X: 9 , 147 , 070 . . 9 , 150 , 371 was retrieved from a 20 kb P[acman] construct that covers the entire fh genomic locus ( clone CH322-14E18 from BACPAC Resources Center ) ( Venken et al . , 2009 ) .",
"The fh genomic sequence was then subcloned into p{w+}attB using KpnI and NotI sites .",
"The construct was then injected into y w ΦC31;VK33 embryos , and transgenic flies were selected .",
"To generate pUAST-attB-UAS-hFXN construct , the primers 5’- TCCGAATTCGCCACCATGTGGACTCTCGGGCGCCGCGC-3’ and 5’-GCGGTACCTCAAGCATCTTTTCCGGAATAGGC-3’ were used to clone the full length FXN cDNA from pcDNA-hFXN-HA , a gift from Gino Cortopassi ( Shan et al . , 2007 ) .",
"The PCR product was then subcloned into the pUAST-attB vector .",
"The construct was then injected into y w ΦC31;VK33 or y w ΦC31;VK37 embryos , and the transgenic flies were selected .",
"For whole mount eye staining , we dissected fly heads in cold PBS and fixed with 4% paraformaldehyde at 4°C overnight .",
"The retinas were then dissected and fixed for an additional 30 min .",
"For larval ventral nerve cord staining , L3 instar larvae were dissected and fixed with 3 . 7% formaldehyde for 20 min .",
"The antibodies were used at the following concentrations: mouse anti-Na/K ATPase ( α5 , Developmental Studies Hybridoma Bank ( DSHB ) ) , 1:200; rat anti-Elav ( 7E8A10 , DSHB ) , 1:500; mouse anti-Rh1 ( 4C5 , DSHB ) , 1:100; mouse anti-V5 ( R96025 , Invitrogen ) , 1:1000; rabbit anti-HRP ( 323-005-021 , Jackson ImmunoResearch ) , 1:1000; chicken anti-GFP ( ab13970 , abcam ) , 1:1000; rabbit anti-β-Galactosidase ( Cappel ) , 1:1000; mouse anti-DLG ( 4F3 , DSHB ) , 1:200; Alexa 488-conjugated phalloidin ( Invitrogen ) , 1:100; Alexa 405- , Alexa 488- , Cy3- , or Cy5-conjugated secondary antibodies ( Jackson ImmunoResearch ) , 1:250 .",
"Samples were then mounted in Vectashield ( Vector Laboratories ) before being analyzed under a confocal microscope .",
"All the confocal scans were acquired with a confocal microscope ( model LSM 510 or LSM 710; Carl Zeiss ) and processed using LSM Image Browser ( Carl Zeiss ) and Photoshop ( Adobe ) .",
"The Nile red staining was performed as described ( Liu et al . , 2015 ) .",
"Briefly , the retina was dissected and fixed in 3 . 7%formaldehyde .",
"The samples were then incubated for 10 min at 1:1 , 000 dilution of PBS with 1 mg/ml Nile Red ( Sigma ) .",
"The retina was then washed with PBS and mounted in Vectashield ( Vector Laboratories ) .",
"Images were obtained with a Zeiss LSM 510 confocal microscope .",
"Mitochondria were extracted as previously described ( Zhang et al . , 2013 ) .",
"Mitochondrial ETC enzymatic activity assay and aconitase activity assay were performed as previously described ( Zhang et al . , 2013 ) .",
"To determine ADP/ATP ratio , L3 larvae were collected and the ratio was determined with an ADP/ATP Ratio Assay Kit ( ab65313 , abcam ) .",
"Briefly , 5–10 L3 instar larvae were collected and frozen with liquid nitrogen .",
"The samples were then homogenized and the ADP/ATP ratio was measured following manufacturer’s protocol .",
"The luminescence was measured by Synergy 2 Microplate Reader ( BioTek ) .",
"Drosophila retina ultrastructure was imaged following standard electron microscopy procedures using a Ted Pella Bio Wave processing microwave with vacuum attachments .",
"Briefly , fly heads were dissected and fixed at 4°C in fixative ( 2% paraformaldehyde , 2 . 5% glutaraldehyde , 0 . 1 M sodium cacodylate , and 0 . 005% CaCl2 , pH 7 . 2 ) overnight .",
"The samples were then postfixed in 1% OsO4 , dehydrated in ethanol and propylene oxide , and then embedded in Embed-812 resin ( Electron Microscopy Sciences ) under vacuum .",
"Photoreceptors were then sectioned and stained in 1% uranyl acetate and saturated lead nitrate .",
"TEM images of photoreceptor sections were taken using a JEOL JEM 1010 transmission electron microscope at 80 kV with an AMT XR-16 mid-mount 16 mega-pixel digital camera .",
"ESI/MS/MS analysis of endogenous sphingosine bases and ceramide species ( C14- and C16-Sphingoid Base ) were performed on a Thermo_Fisher TSQ Quantum triple quadrupole mass spectrometer , operating in a Multiple Reaction Monitoring ( MRM ) positive ionization mode , using modified version ( Bielawski et al . , 2009 ) .",
"Briefly , whole larvae were fortified with the internal standards ( ISs: C17 base D-erythro-sphingosine ( 17CSph ) , C17 sphingosine-1-phosphate ( 17CSph-1P ) , N-palmitoyl-D-erythro-C13 sphingosine ( 13C16-Cer ) and heptadecanoyl-D-erythro-sphingosine ( C17-Cer ) ) , and extracted with ethyl acetate/iso-propanol/water ( 60/30/10 v/v ) solvent system .",
"After evaporation and reconstitution in 100 μl of methanol samples were injected on the HP1100/TSQ Quantum LC/MS system and gradient eluted from the BDS Hypersil C8 , 150 × 3 . 2 mm , 3 μm particle size column , with 1 . 0 mM methanolic ammonium formate / 2 mM aqueous ammonium formate mobile phase system .",
"Peaks corresponding to the target analytes and internal standards were collected and processed using the Xcalibur software system .",
"Quantitative analysis was based on the calibration curves generated by spiking an artificial matrix with the known amounts of the target analyte synthetic standards and an equal amount of the internal standards ( ISs ) .",
"The target analyte/IS peak areas ratios were plotted against anlyte concentration .",
"The target analyte/IS peak area ratios from the samples were similarly normalized to their respective ISs and compared to the calibration curves , using a linear regression model .",
"Applying consistent mass spectral conditions of Collision Assistant Dessociation ( CAD ) ; 35 eV and Electron Spray Ionization ( ESI ) all sphingoid bases and related ceramides undergo uniform transition from initial molecular ion ( M+1 ) to the respective sphingoid backbone secondary ions .",
"Consequently , calibration curves , generated from authentic standards of the typical , 18C-sphingosine and ceramides , can be used for quantitation of other , e . g . 20C- counterparts .",
"The levels of sphingolipid species are normalized to the phosphate amount of the samples .",
"Although absolute concentrations determined for compounds without authentic standards ( 20C-LCB derivatives ) may not be precise , due to possible differences in instrument response for 18C- and 20C-LCB related compounds , for comparative study , where changes in sphingolipids level rather than absolute concentration , are most important this indirect methodology provide reliable results .",
"Total RNA from the fly larval brains were extracted by Trizol RNA Isolation Reagents ( Thermo Fisher Scientific ) follow the manufacturer’s instructions .",
"The cDNA was synthesized by High-Capacity cDNA Reverse Transcription Kit ( Applied Biosystems ) .",
"Real-time PCR was carried out using iQ SYBR Green Supermix ( Bio-Rad ) and performed in a thermal cycler ( iCycler; Bio-Rad Laboratories ) .",
"The data were collected and analyzed using the optical module ( iQ5; Bio-Rad Laboratories ) .",
"The following primer pairs are used ( 5’to 3’ ) : RP49 forward ( control primer ) , TCCTACCAGCTTCAAGATGAC; RP49 reverse ( control primer ) , CACGTTGTGCACCAGGAACT; Act57B forward , TTGAGACCTTCAACTCGCCC; Act58B reverse , CCATCACCGGAGTCCAGAAC; mib2 forward , CGCCAGAAAACACTGTCGTG; mib2 reverse , GACGAACTCCAACTTGAGCATTA; CG5080 forward , CGCCCTCTCCAATTAGTTCTCC; CG5080 reverse , CAGCGACTGGATAGTTCCGC; Mlc2 forward , TCGGGTCCGATCAACTTCAC; Mlc2 reverse , ATTTCGCGGAATTTGTCACCG; Mlc1 forward , CCGAGGATGATGAAGGATTT; Mlc1 reverse , CTGGTCTGTCACACATTCTGG; CG6972 forward , AGGGATCACACACTGATGAACT; CG6972 reverse , CAACAGCCATTCGGAGGGAC; Mhc forward , ATCAATCCTTACAAGCGTTACCC; Mhc reverse , CCGTCAGAGATGGCGAAAATATG; E ( Spl ) forward , ATGGAATACACCACCAAGACCC; E ( Spl ) reverse , GGCGACAAGTGTTTTCAGGTT; sens forward , TACTGTGGCCCCAATTTTTGT; sens reverse , AAGGCAAAGTCACGATCCCG; ptc forward , GGATCTTTACATACGCACCAGC; ptc reverse , GGACTGGAATACTGATCGCAG; Neuro-2A cells were seed on 12 well plates , and each well was transfected with 3XMef2-luc reporter ( Addgene ) and Renilla control pRL-TK vector ( gift from Huda Zoghbi ) .",
"After two days , DMSO , DL-Dihydrosphingosine ( Sigma ) , or GSK2334470 ( gift from Huda Zoghbi ) was added into the medium .",
"Cells were cultured one more day with drug treatment .",
"The luciferase assay was then performed by Dual-Luciferase Reporter Assay System ( Promega ) and followed manufacturer instruction ."
]
] | [
"Mutations in Frataxin ( FXN ) cause Friedreich’s ataxia ( FRDA ) , a recessive neurodegenerative disorder .",
"Previous studies have proposed that loss of FXN causes mitochondrial dysfunction , which triggers elevated reactive oxygen species ( ROS ) and leads to the demise of neurons .",
"Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants .",
"We show that loss of frataxin homolog ( fh ) in Drosophila leads to iron toxicity , which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 ( Pdk1 ) and myocyte enhancer factor-2 ( Mef2 ) .",
"Dampening iron toxicity , inhibiting sphingolipid synthesis by Myriocin , or reducing Pdk1 or Mef2 levels , all effectively suppress neurodegeneration in fh mutants .",
"Moreover , increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved .",
"Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA ."
] | [
"Friedreich’s ataxia is a disorder in which nerve cells in the spinal cord , cerebellum and dorsal root ganglia progressively die as a person ages .",
"People with this disorder often have difficulties with walking and can eventually develop other problems such as heart disease and diabetes .",
"Mutations in a gene called Frataxin , known as FXN for short , are the primary cause of the disorder .",
"The FXN gene encodes a protein normally found in mitochondria – the structures that are best known for providing energy inside cells .",
"Previous studies suggest that mutations in the FXN gene prevent mitochondria from working normally , which triggers the production of toxic chemicals called reactive oxygen species .",
"However , therapies based on antioxidants ( which combat reactive oxygen species ) only have limited benefits in patients with Friedreich’s ataxia; this suggests that other mechanisms contribute to the progression of the disease .",
"Mutations in the FXN gene also cause iron to accumulate inside cells , which can be toxic too .",
"However , it remains hotly debated whether or not iron toxicity contributes to Friedreich’s ataxia .",
"Chen et al . set out to identify other mechanisms that can explain the loss of nerve cells seen in Friedreich’s ataxia using fruit flies as an experimental system .",
"Flies without the equivalent of FXN gene accumulated iron in their nervous systems and other tissues , but did not produce more reactive oxygen species .",
"The experiments also revealed that this build-up of iron increased the production of fatty molecules ( called sphingolipids ) , which in turn triggered the activation of two proteins ( called Pdk1 and Mef2 ) .",
"Chen et al . then showed that blocking any of these effects could effectively delay the death of nerve cells in the mutant flies .",
"Further experiments showed that boosting the levels of the Mef2 protein in the nerve cells of otherwise normal flies was enough to cause these cells to die .",
"The next step is to see whether the pathway also operates in mice and humans .",
"Future studies could also see if dampening down this pathway could provide new treatments for Friedreich’s ataxia ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Material and methods"
] | [
"cell biology",
"neuroscience"
] | A novel synaptic plasticity rule explains homeostasis of neuromuscular transmission | elife-12190-v1 | [
[
"In the nervous system , presynaptic neurotransmitter release , postsynaptic receptors , and postsynaptic excitability can be modulated to bi-directionally and durably change synaptic efficacy .",
"There is a large diversity of plasticity processes , which together shape the neuronal network ( Changeux and Danchin , 1976; Goda and Davis , 2003; Munz et al . , 2014 ) and tune its properties ( Nelson and Turrigiano , 2008 ) .",
"In the brain , concomitantly to various forms of associative plasticity which sustain memory and learning , homeostatic plasticity processes are proposed to restrain the mean level of neuronal activity within a physiological regime , and to maintain the stability of recurrent network activity that can be challenged by associative plasticity ( Turrigiano and Nelson , 2004; Macleod and Zinsmaier , 2006; Marder and Goaillard , 2006; Turrigiano , 2007; Nelson and Turrigiano , 2008 ) .",
"Homeostatic plasticity has been extensively studied at the neuromuscular synapse , in particular at the glutamatergic neuromuscular junction ( NMJ ) in Drosophila ( Frank , 2014 ) , due to the robustness of the homeostatic control of synaptic transmission and the great accessibility of the experimental model to genetic manipulations .",
"In vertebrates , each skeletal muscle fiber is mono-innervated ( Sanes and Lichtman , 1999 ) , and each single presynaptic action potential ( AP ) induces one postsynaptic AP , corresponding to a unity synaptic gain ( ratio between the numbers of post- and presynaptic APs equal to 1 ) ( Wood and Slater , 2001 ) .",
"The stability of the gain implies that presynaptic neurotransmitter release and/or postsynaptic receptors adapt the effective synaptic strength to the excitability of the muscle fiber , which depends on the fiber characteristics , and presumably decreases with growth and exercise ( Turrigiano , 2007 ) .",
"In adult vertebrate skeletal muscles , cholinergic nicotinic receptors are clustered in the synaptic region .",
"Expression and location of nicotinic receptors have been shown to depend not only on agrin ( McMahan , 1990; Hall and Sanes , 1993; Gautam et al . , 1995; 1996; Sandrock et al . , 1997 ) but also on activity ( Lømo , 2003 ) , suggesting their possible role as adjustment variables in the control of synaptic strength .",
"In the recent years however , thorough studies of the glutamatergic NMJ in Drosophila revealed that presynaptic regulation of neurotransmitter release is certainly a major adjustment variable for synaptic homeostasis ( Davis and Müller , 2015 ) .",
"In such 'presynaptic homeostasis' , increase in neurotransmitter release counterbalances a genetically-induced decrease of the postsynaptic sensitivity to glutamate ( Petersen et al . , 1997; Davis et al . , 1998; DiAntonio et al . , 1999 ) , a genetically-induced increase of postsynaptic input conductance ( Paradis et al . , 2001 ) or a pharmacological blockade of postsynaptic receptors ( Frank et al . , 2006 ) , thus resulting in the strict maintenance of the level of evoked postsynaptic depolarization .",
"Presynaptic compensation of postsynaptic excitability changes implies the existence of a retrograde feedback process from the innervated muscle fiber to the motor neuron .",
"Particular attention was paid to the determination of the molecular targets of the homeostatic retrograde signaling and of the presynaptic cell signaling involved .",
"It appeared that both the readily releasable vesicle pool ( Müller et al . , 2012; 2015 ) and the presynaptic voltage-gated Ca2+ channels ( Müller and Davis , 2012 ) are targeted to promote glutamate release when postsynaptic excitability is reduced .",
"The abundance of voltage-gated Ca2+ channels can also be decreased in response to a vesicular content increase ( Gaviño et al . , 2015 ) , confirming the bi-directionality of the process .",
"In Drosophila , endostatin is a candidate trans-synaptic factor for the retrograde feedback targeting presynaptic neurotransmitter release ( Wang et al . , 2014 ) .",
"Upstream from the retrograde factors , the postsynaptic kinases , CAMKII ( Haghighi et al . , 2003 ) and TOR ( Penney et al . , 2012 ) , pathways are necessary for a functional homeostatic control at the Drosophila neuromuscular transmission .",
"An abundant literature is thus progressively assembling pieces of the signaling puzzle underlying the interactions between the motor neuron and the muscle cell for the control of synaptic strength .",
"However , besides the nature of the retrograde feedback and its presynaptic targets , a mechanism for the evaluation of the synaptic strength should be present at the postsynaptic level .",
"The nature and dynamics of these mechanisms , primarily sensing synaptic strength in the muscle cell and initiating retrograde feedback , are key issues of homeostatic plasticity that still remain enigmatic in both invertebrates and vertebrates .",
"Here we investigate this issue in vertebrate ( Xenopus and mouse ) cholinergic transmission .",
"Motor neurons and muscle cells of Xenopus laevis embryos establish functional neuromuscular synapses ( Tabti et al . , 1998 ) in primary co-culture .",
"Standard intracellular recording from muscle cells is possible in Xenopus culture owing to the small size of the cells ( Figure 1A , perforated patch-clamp ) , while it is not possible in muscle cells from adult mice , due to macroscopic movements during contraction .",
"To circumvent this issue and maintain intracellular recordings in moving tissue , we adapted a ‘floating electrode’ device from previous methods invented for muscles ( Woodbury and Brady , 1956 ) or neurons ( Kunze , 1998 ) .",
"We applied this method to mouse ex vivo nerve-skeletal muscle preparations ( Figure 1B , Methods , and Figure 1—figure supplement 1 ) .",
"This approach preserved the physiological conditions , necessary to reveal the control exercised by the muscle cell over the neuromuscular synaptic transmission , a role that might have been previously overlooked in vertebrates due to commonly used high concentrations of the nicotinic receptor antagonist curare to prevent contraction . 10 . 7554/eLife . 12190 . 003Figure 1 . Synaptic transmission is homeostatically regulated .",
"( A ) Perforated patch-clamp on Xenopus neuron ( N ) and muscle cell ( M ) in primary culture .",
"Presynaptic APs were triggered with current steps .",
"Postsynaptic APs were recorded under current-clamp and nicotinic synaptic currents under voltage-clamp ( -80 mV ) .",
"( B ) Intracellular recording in soleus muscle fibers from an adult mouse using a floating sharp electrode ( see Materials and Methods and Figure 1—figure supplement 1 ) .",
"( C ) Nicotinic conductance calculated from averaged ePSCs ( n = 30 ePSCs for each dot ) in different Xenopus muscle cells as a function of their input conductance .",
"The black line shows the linear regression .",
"( D ) In mice , membrane potential reached by the ePSP in individual FDB muscle fibers after treatment with µ-conotoxin GIIIB , in absence of burst stimulation of the nerve ( black dots , n= 88 fibers , 2 muscles , 2 mice ) , and in test preparations ( grey dots , n = 108 fibers , 2 muscles , 2 mice ) for which the nerve was burst stimulated prior to conotoxin treatment ( 15 bursts in 10 min , each of 120 events at 30 Hz ) .",
"( E ) Mean synaptic gain at Xenopus synapses ( dots , n = 5 synaptic connections ) and in mouse neuromuscular junctions ( squares , n = 4 muscle fibers from different mice ) during chronic bursts of presynaptic stimulation ( bursts of 80 to 120 pulses , 30 Hz for 30 min ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00310 . 7554/eLife . 12190 . 004Figure 1—figure supplement 1 . Floating electrode . In conventional electrophysiology , an intracellular electrode made from pulled glass is rigidly fixed to an amplifier headstage and/or to the micromanipulator by a holder preventing free movements .",
"To allow intracellular recordings in contracting mouse muscle , we cut off the tip of the pipette and used this as an electrode connected to the amplifier headstage by a loose , 5–10 cm length 50 µm diameter silver wire ( see Materials and methods ) .",
"The lower trace shows the force developed by the muscle during a train of nerve stimulations with a frequency close to the tetanus .",
"The contraction force was measured with a FT03 force transducer from Grass Technologies ( Astro-Med Inc . , West Warwick ) and expressed in Newton .",
"The middle trace shows a single muscle cell recording of the membrane potential with the floating electrode technique .",
"The upper trace shows a detail view of the ePSPs and action potentials . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00410 . 7554/eLife . 12190 . 005Figure 1—figure supplement 2 . Mouse muscles fibers have a wide range of input conductances . Input conductances ( G ) of muscle fibers were calculated from the depolarization induced by injection of a positive current and application of Ohm’s law .",
"The histogram represents the normalized count distribution of the input conductances for 33 fibers in an Extensor Digitorum Longus muscle , for 34 fibers in a Flexor Digitorum Brevis muscle , and for 50 fibers in a Soleus muscle .",
"Data were binned with a step increment of 0 . 2 µS . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00510 . 7554/eLife . 12190 . 006Figure 1—figure supplement 3 . Characterization of the K+ conductances that determine the Xenopus muscle cell input conductance .",
"( A ) A K+ inward rectifying ( Kir ) conductance dominates the input conductance of Xenopus muscle cells .",
"The upper panel shows membrane currents recorded under voltage-clamp during ramp potentials ( 125 mV/s ) , in control conditions ( grey trace ) and after addition of external Ba2+ 300 µM ( black trace ) .",
"The Kir component , sensitive to external Ba2+ , was obtained by the difference between the two traces .",
"The lower panel shows the isolated Kir conductance ( gray trace ) and the remaining conductances in presence of Ba2+ ( black trace ) , composed of a passive K+ leak conductance and of the voltage-activated K+ ( Kv ) conductances .",
"The Kir conductance dominates the input conductance around the resting potential , decreases with depolarization and becomes null at the excitability threshold .",
"( B ) A linear correlation between the Kir current and the membrane capacitance ( an estimation of the surface ) of the cells reveals the strong homogeneity of the membrane conductance density in the cells culture , and emphasizes the wide range of input conductances and excitabilities found in muscle cells ( i . e . an increase of surface with constant leak density implies an increase in the input conductance , and a decrease in excitability ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 006"
],
[
"Synaptic efficacy is linked to the ability of evoked postsynaptic potentials ( ePSP ) to reach firing threshold .",
"In vertebrate muscle cells , the amplitude of PSPs depends on the ratio between the nicotinic conductance activated by acetylcholine ( ACh ) and the muscle input conductance , which is largely due to resting K+ leak currents .",
"Fibers in flexor digitorum brevis ( FDB ) , extensor digitorum longus and soleus mouse muscles have a wide distribution of input conductances ( Figure 1—figure supplement 2 ) .",
"A similar distribution in Xenopus muscle cells ( Figure 1C , X axis ) is linked to the diversity of membrane surfaces with homogenous conductance density ( 196 ± 14 pS/pF , n=19 , Figure 1—figure supplement 3 ) .",
"Despite these differences in muscle cell excitability , a single presynaptic AP triggered a single postsynaptic AP , and a contraction , in all tested cells from Xenopus and mouse ( Figure 1 ) .",
"In Xenopus cultures , we found that the nicotinic conductance activated in muscle cells by spike-evoked ACh release varied linearly with the postsynaptic input conductances measured in a population of synaptic neuron/muscle cell pairs ( Figure 1C ) , and resulted in comparable ePSP ( evoked PSP ) voltage amplitudes .",
"Similarly , in mouse muscles , we assessed ePSP characteristics after exogenous application of µ-conotoxin GIIIB which selectively blocks Na+v1 . 4 channels involved in the muscle cell AP ( Cruz et al . , 1985; Li et al . , 2003 ) .",
"This manipulation does not affect nerve APs , since this Na+ channel subtype is not critical for spiking in motor neurons .",
"Single-spike-evoked PSPs exhibited a narrow range of voltage magnitudes ( Figure 1D , in FDB muscle ) .",
"The comparable amplitude of ePSPs , despite widely ranging resting conductances in the postsynaptic muscle cells , indicates that the neuromuscular synapse exhibits robust plasticity that regulates its functional strength within a narrow range . 10 . 7554/eLife . 12190 . 007Figure 2 . Calcium signaling in Xenopus muscle cell and synaptic homeostasis . The middle scheme depicts our model of homeostatic control of the synaptic strength .",
"The nicotinic Ca2+ influx elicits a positive retrograde feedback signal ( red arrow ) on the presynaptic neurotransmitter release .",
"The positive feedback is balanced by a negative retrograde feedback signal ( blue arrow ) triggered by the muscle AP-induced DICR .",
"( DICR = depolarization-induced calcium release , AP = action potential , RyR = ryanodine receptor , SR = sarcoplasmic reticulum , R nico = nicotinic receptors ) .",
"( A ) Independence of the DICR signal from external Ca2+ .",
"AP ( black trace , induced with a brief postsynaptic current step ) , Ca2+ dye ( Fluo4 ) relative fluorescence in standard external medium ( dark blue trace ) and in Ca2+ free medium ( light blue trace ) , and qualitative measure of the cell contraction ( green trace , see Materials and methods ) .",
"( B ) Voltage-dependence of the DICR signal .",
"The Fluo4 fluorescence was measured at the steady-state of the Ca2+ signal during a classical voltage-step protocol from a holding potential of -80 mV and averaged ( n = 3 cells ) .",
"( C ) Blockade of the DICR signal by loading the muscle cell with 100 µM ryanodine .",
"( D ) Representative example of the DICR signal ( blue trace ) during repetitive muscle APs ( black trace ) .",
"( E ) Ca2+ build-up upon nicotinic receptor activation .",
"Fluo4 signal ( upper traces ) in control and in Ca2+ free medium , during iontophoretic ACh applications ( 5 pulses ) under postsynaptic voltage-clamp ( -80 mV ) .",
"The lower trace shows the nicotinic currents .",
"( F ) Dependence of the nicotinic Ca2+ build-up on the nicotinic conductance with increasing iontophoretic ACh applications at a constant membrane potential ( holding potential –80 mV ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00710 . 7554/eLife . 12190 . 008Figure 2—figure supplement 1 . Ionic permeability of the Xenopus nicotinic receptor-channel . To determine the ionic selectivity of the Xenopus nicotinic receptor in cultured cells , the reversal potential of the nicotinic current induced by ionophoretic acetylcholine application was measured under voltage-clamp in solutions of various ionic compositions , with an intra-pipette medium of the following composition ( in mM ) : 110 KCl , 3 NaCl , 2 MgCl2 , 0 . 5 EGTA and 10 HEPES ( pH 7 . 2 ) .",
"( A ) Current-voltage relationship of the nicotinic current induced by ionophoretic application of acetylcholine , in a classical physiological medium .",
"Inset shows the current traces recorded at the different holding potentials .",
"( B ) Table summarizing the external media compositions and the corresponding reversal potentials ( Vr ) .",
"Concentrations are expressed in mM , and the mean ± SEM reversal potentials in mV .",
"Fitting the measured reversal potentials to the GHK voltage equation ( see Materials and methods ) gave: PK/PNa = 1 . 07 , PCa/PNa = 0 . 23 , and PMg/PNa = 0 . 31 , used to calculate the theoretical reversal potentials t-Vr . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 008 How does the neuromuscular synapse adjust its strength depending upon the excitability of the postsynaptic muscle fiber ?",
"Previous research reported that in Xenopus cell cultures , burst activation of presynaptic input results in marked enhancement of ACh release , in both voltage-clamp and current-clamp conditions ( Wan and Poo , 1999 ) .",
"We reproduced this result in the specific condition of post-synaptic voltage-clamp ( not shown ) but not in that of current-clamp where we found instead absence of potentiation .",
"To observe such absence in current-clamp , we believe that it is essential that all or most of the ePSPs evoked by the conditioning burst , reach threshold for spike initiation in the postsynaptic cell .",
"This was not the case in the only example of current-clamp presented by Wan and Poo 1999 ( see their Figure 1A ( i ) ) .",
"Homeostatic plasticity rules suggest in fact that this form of plasticity should not occur in the presence of synaptic transmission that is effective in generating a muscle AP .",
"To test this hypothesis , we initiated burst stimulation of motor neurons in both Xenopus and mice –during current-clamp recordings– in order to allow voltage responses and APs in the muscle cells .",
"In these physiological conditions and despite the chronic burst activity , no significant change in the synaptic gain was visible .",
"The probability for a synaptic event to induce a postsynaptic AP was close to unity and kept constant ( Figure 1E ) .",
"In Xenopus however , test evoked postsynaptic currents ( ePSCs ) were recorded under brief periods of postsynaptic voltage-clamp before and after the 30 min of chronic activity under current-clamp , and showed a slight relative decrease of 0 . 2 in the averaged ePSCs amplitude ( see first bar in Figure 3C ) .",
"In mice , the average ePSPs were not changed by high-frequency conditioning stimulations ( Figure 1D and 3F ) .",
"Altogether , these observations suggest that synapses able to trigger postsynaptic APs do not exhibit strong plasticity , and that the occurrence of muscle APs may be involved in the homeostatic mechanisms adjusting and maintaining the synaptic strength in concordance with the postsynaptic input conductance . 10 . 7554/eLife . 12190 . 009Figure 3 . Nicotinic receptor activity induces a positive feedback on ACh release .",
"( A ) In order to obtain the nicotinic calcium signal in absence of DICR in Xenopus , synaptic events were transiently kept subthreshold either with curare ( decreased nicotinic conductance , Gnico ) or by dynamic-clamp injection of gK+ leaks ( increased input conductance , Gin ) , or left suprathreshold while DICR was blocked by ryanodine .",
"( B ) Effect of presynaptic burst stimulation and curare on spontaneous ( upper trace , sPSC ) and evoked synaptic currents ( ePSC ) .",
"ePSCs recorded under voltage-clamp were evoked at low rate ( 0 . 03 Hz ) and averaged by 30–40 events ( lower traces ) .",
"During conditioning presynaptic stimulations ( 3 bursts of 5 events at 30 Hz ) , the postsynaptic potential was released from clamp and curare transiently applied ( middle trace ) .",
"Upper trace: for clarity , ePSCs were removed from the continuous trace in order to display the sPSCs only .",
"( C ) , In Xenopus , mean ePSC relative change 30 min after control chronic bursting activity ( 'chronic activity' , n = 5 , illustrated in Figure 1E ) , 45 min after subthreshold synaptic activity ( 'Curare' , n = 9 , illustrated in B; 'Dynamic-clamp' , n = 5 , illustrated in Figure 3—figure supplement 2A ) , transient curare application in absence of stimulation ( 'curare No Burst' , n = 3 ) , sub- ( 'curare-ryanodine' , n = 3 ) and suprathreshold synaptic activity ( 'ryanodine' , n = 6 , illustrated in Figure 3— figure supplement 3 ) in muscle cells preloaded with ryanodine .",
"( D ) , Relative change in amplitude and frequency of sPSCs after potentiation in Xenopus .",
"( E ) , In FDB mice muscles , voltage reached by ePSPs in ryanodine treated ( black dots , n = 60 fibers , 2 mice ) , and in ryanodine treated and burst stimulated preparations ( red dots , n = 70 fibers , 2 mice ) .",
"( F ) Mean ePSP amplitude in control ( 'no stim' ) and high frequency nerve stimulation ( 'Pre stim' ) shown in Figure 1D , in non-stimulated ( 'Ryanodine' ) and in high frequency stimulated ( 'Pre stim Rya' ) ryanodine treated preparations shown in E . * , p<0 . 05; ** , p<0 . 01; *** , p<0 . 001; t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 00910 . 7554/eLife . 12190 . 010Figure 3—figure supplement 1 . Decreasing muscle cell excitability by injection of artificial conductances .",
"( A ) We used the K+ currents data of Figure 1—figure supplement 3 to establish and inject models of leak conductances into the muscle cell with the dynamic-clamp technique ( see Materials and methods ) .",
"Trace is a negative of the current output of the Kir conductance model when a ramp potential ( 125 mV/s ) was used as input .",
"( B ) Time course of sPSPs in the presence of a simulated Kir conductance with dynamic-clamp ( Kir DC ) .",
"Artificial increase of the postsynaptic input conductance decreased the averaged sPSP amplitude . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01010 . 7554/eLife . 12190 . 011Figure 3—figure supplement 2 . LTP induction with dynamic-clamp or postsynaptic ryanodine .",
"( A ) Potentiating effect of maintaining synaptic efficacy subthreshold by dynamic-clamp injection of gK+ leak ( see Materials and methods ) .",
"Middle trace: subthreshold nicotinic ePSPs in presence of the dynamic-clamp current ( trace below ) .",
"( B ) .",
"Potentiating effect of intact synaptic activity generating muscle cell AP firing with DICR blocked ( 100 µM ryanodine ) .",
"The muscle cell was pre-loaded with ryanodine using the classical whole-cell configuration of the patch-clamp technique .",
"After removing the patch pipette , a perforated patch was performed for electrophysiological recordings . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 011 How might the strength of synaptic transmission depend upon the occurrence of an AP in the postsynaptic muscle cell ?",
"We reasoned that there may be two distinct postsynaptic Ca2+ signaling pathways involved in this homeostatic regulation: a postsynaptic reporter of the presynaptic activity that signals the occurrence of a synaptic event , and a postsynaptic reporter that signals the occurrence of a postsynaptic AP .",
"The postsynaptic Ca2+ build up ( Figure 2E–F ) due to a high Ca2+ permeability of the nicotinic receptor-channel ( Fucile et al . , 2006 ) ( PCa2+/PNa+=0 . 23 in Xenopus , Figure 2—figure supplement 1 ) is a good candidate for signaling the occurrence of synaptic events .",
"Furthermore , in vertebrates , the skeletal muscle cells exhibit a specific Ca2+ signal linked to the AP and involved in the excitation-contraction coupling , i . e . 'the depolarization-induced Ca2+ release' ( DICR ) .",
"The DICR signal ( Figure 2A–D ) is a Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors , triggered by plasma membrane depolarizations above –40 mV , and thus triggered by AP in physiological conditions .",
"Depolarization is detected by a voltage-sensor ( the dihydropyridine receptor ) located in the plasma membrane .",
"This voltage-sensor derives from an L-Type Ca2+ channel; it is voltage-dependent but not ion permeable ( Almers et al . , 1981; Melzer et al . , 1995 ) .",
"Functionally linked to the ryanodine receptor at the level of the T-tubule , its activation triggers the opening of the ryanodine receptor ( Rios and Brum , 1987; Catterall , 1991; Franzini-Armstrong and Protasi , 1997 ) .",
"The resulting Ca2+ release is independent of the extra-cellular Ca2+ ( Figure 2A ) , purely voltage-dependent , and its voltage-dependency follows the typical pattern of the activation curve of a 'High Voltage-Activated' Ca2+ channel ( Figure 2B ) .",
"Ryanodine fully blocks this Ca2+ signal ( Figure 2C ) .",
"We hypothesized that these two distinct Ca2+ signals , nicotinic Ca2+ and DICR , are used by the muscle cell to detect synaptic activity and its efficiency to elicit an AP , respectively , leading to retrograde control of the presynaptic ACh release ( Figure 2 scheme ) .",
"To validate this hypothesis , we examined each of these Ca2+ dependent mechanisms in isolation and their respective impact on synaptic efficacy .",
"We first examined the specific role of nicotinic Ca2+ influx in the regulation of synaptic transmission .",
"To trigger the nicotinic Ca2+ influx but not the DICR in Xenopus muscle cells , we transiently reduced synaptic strength below AP threshold during the conditioning presynaptic burst stimulations performed in postsynaptic current-clamp .",
"We monitored subsequent synaptic conductance changes under postsynaptic voltage-clamp during low-rate single-pulse intracellular stimulation of the neuron .",
"We reduced the synaptic efficacy either by decreasing the synaptic conductance with low-doses of the reversible nicotinic antagonist curare ( Figure 3A–C ) , or by artificially increasing the muscle cell input conductance using dynamic-clamp ( Sharp et al . , 1993; Robinson and Kawai , 1993; Prinz et al . , 2004 ) ( Figure 3A , C , and Figure 3—figures supplements 1 , 2A ) .",
"The lowered synaptic conductance mimicked the effects of low ACh release in developing synapses .",
"Simulation and dynamic-clamp injection of a combination of a linear and an inwardly rectifying K+ leak conductances ( Materials and methods and Figure 3—figure supplement 1 ) mimicked the increased endogenous input conductance associated with muscle cell growth .",
"In both cases , presynaptic burst-stimulation ( 3 bursts of 5 pulses at 30 Hz ) induced a strong , fast ( within minutes ) and long-term potentiation ( LTP ) of ePSCs ( Figure 3B , C , and Figure 3—figure supplement 2A ) , while the transient application of curare in absence of presynaptic stimulation did not induce potentiation ( Figure 3C , 'curare No Burst' bar ) .",
"This form of LTP induced by subthreshold synaptic events depends solely on nicotinic receptor activity and therefore was insensitive to DICR blockade obtained by pre-loading the muscle cell with ryanodine ( Figure 3C ) .",
"Furthermore , when ePSPs were left intact but in presence of postsynaptic ryanodine , presynaptic burst-stimulation produced postsynaptic APs and nonetheless a strong LTP of the ePSCs ( Figure 3C , and Figure 3—figure supplement 2B ) , while no potentiation occurred when DICR was functional ( Figure 1E and Figure 3C ) .",
"The LTP induced in our postsynaptic current-clamp protocols was accompanied with a strong increase of the frequency , but not amplitude , of spontaneous postsynaptic currents ( sPSCs ) ( Figure 3B , D ) .",
"As previously observed in voltage-clamp protocols ( Wan and Poo , 1999 ) , this indicates that LTP is due to an evoked ACh release increase and not to a postsynaptic receptor modulation .",
"We found similar results in ryanodine-treated mouse muscles ( Figure 3E , F ) : it is only when nicotinic channels were activated following conditioning bursts of nerve stimulations and ACh release —in the absence of DICR and contraction— that subsequent single test pulses revealed a strong LTP of the ePSPs .",
"In contrast , as shown above , when DICR was functional and the nerve was stimulated , nicotinic ePSPs did not change and remained identical to those in the absence of nerve stimulation ( Figure 1D and Figure 3F ) .",
"Therefore the simultaneous occurrence of postsynaptic nicotinic receptor activation and DICR is essential to the stability of functional synaptic gain .",
"Because the regulation targets ACh release , this implies the existence of retrograde feedback onto the presynaptic compartment .",
"In order to understand the specific role of DICR , we examined its effect on synaptic efficacy in isolation from the effect of nicotinic receptor activation .",
"Direct stimulation of the Xenopus muscle cell induces postsynaptic APs and DICR , with no ACh release and no involvement of nicotinic receptors ( Figure 4A ) .",
"Conditioning bursts of suprathreshold current steps injected in the muscle cell induced a strong , fast and long-term depression ( LTD ) of ePSCs ( Figure 4B , C ) .",
"The average amplitude of sPSCs did not change , confirming the presynaptic locus of gain control , for negative modulations of synaptic strength just like for the positive modulations described above ( Figure 4D ) .",
"LTD induced by repetitive postsynaptic depolarizations has been previously reported ( Lo et al . , 1994; Dan et al . , 1995 ) .",
"Here we show that this form of LTD relies strictly on DICR: it was insensitive to removal of external Ca2+ ( Figure 4C and Figure 4—figure supplement 1A ) , and blocked by postsynaptic pre-loading with ryanodine ( Figure 4C and Figure 4—figure supplement 1B ) .",
"In adult mouse nerve-muscle preparations , external electrical stimulation elicits an AP in both the nerve and the muscle fibers , a situation in which both DICR and nicotinic Ca2+ influx occur .",
"As expected , in the presence of external Ca2+ , the external burst stimulation did not change the average ePSP ( Figure 4E , F ) compared to nerve stimulation only ( Figure 1D and Figure 4F ) .",
"When the external Ca2+ was transiently removed , the external stimulation still elicited APs in both the nerve and the muscle , but neither the ACh release nor its associated postsynaptic nicotinic Ca2+ influx .",
"In these conditions , the external stimulation induced DICR alone and resulted in a strong LTD of the ePSPs ( Figure 4E , F ) .",
"This form of depression is reversible , since subsequent burst stimulation of the nerve induced a rapid potentiation of the ePSPs ( Figure 4—figure supplement 2 ) . 10 . 7554/eLife . 12190 . 012Figure 4 . Muscle DICR induces a negative feedback on ACh release .",
"( A ) In order to trigger the DICR signal in absence of nicotinic Ca2+ influx in Xenopus , postsynaptic APs were directly induced in the muscle cell with brief current steps , without presynaptic stimulation , in presence or absence of external Ca2+ ( upper right scheme ) and with ryanodine that blocks the DICR ( bottom right scheme ) .",
"( B ) Effect of selective postsynaptic firing on synaptic currents .",
"Same layout as in Figure 3B .",
"The middle trace shows the muscle APs firing in response to positive current steps injection .",
"( C ) In Xenopus , mean ePSC relative change 45 min after control chronic bursting synaptic activity ( 'chronic activity' , n = 5 , see 1E ) , direct triggering of the muscle APs shown in B ( 'Direct AP' , n = 5 ) , APs triggered in a Ca2+-free medium ( 'Direct AP Ca2+-free' , n = 6 , illustrated in Figure 4—figure supplement 1 ) , APs triggered in muscle cells loaded with ryanodine ( 'Direct AP Ryanodine' , n = 3 , illustrated in Figure 4—figure supplement 2 ) .",
"( D ) , Relative change in sPSCs amplitude and frequency in Xenopus after potentiation ( red ) or depression ( green ) .",
"( E ) , In FDB mouse muscles , voltage reached by ePSPs in externally stimulated preparations ( 15 bursts of 120 events at 30 Hz , 10 min ) in presence ( black dots , n = 75 fibers , 2 mice ) and in absence of external Ca2+ ( green dots , n = 120 fibers , 2 mice ) .",
"( F ) , Mean ePSP amplitude in control ( 'no stim' ) and high frequency nerve stimulation ( 'Pre stim' ) shown in Figure 1D , in externally stimulated preparations shown in E in presence ( 'External stim-Ca2+' ) and absence of external Ca2+ ( 'External stim-0Ca2+' ) .",
"*** , p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01210 . 7554/eLife . 12190 . 013Figure 4—figure supplement 1 . External calcium-independent LTD and blockade by postsynaptic ryanodine .",
"( A ) LTD induction in a Ca2+ free medium .",
"For the conditioning bursts , postsynaptic APs were triggered by postsynaptic injection of positive current steps , without presynaptic stimulation .",
"( B ) .",
"ePSC depression via muscle firing is prevented when DICR is blocked with 100 µM ryanodine pre-loaded into the muscle cell . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 01310 . 7554/eLife . 12190 . 014Figure 4—figure supplement 2 . Recovery from LTD . In adult mice preparations , external stimulation in a Ca2+ free medium is a situation comparable to direct stimulation of the Xenopus muscle cells , and induces a depression of the ePSPs .",
"Here , after a depression induced by 3 bursts of external stimulations in a Ca2+ free medium , subsequent bursts of nerve-stimulations ( arrow ) induced a partial recovery of the ePSPs amplitude .",
"We transiently increased further the external Ca2+ concentration to 8mM ( which increases the nicotinic Ca2+ influx ) , and this resulted in an increase of the potentiating effect of the bursting nerve stimulations ( arrow ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 014 In the previous experiments , we used the extreme cases of either fully subthreshold synaptic events or of postsynaptic APs in the absence of presynaptic neuron activity , to emphasize the roles of the nicotinic calcium and the DICR signals respectively .",
"However , during normal synaptic activity the two calcium signals are combined and a unique plasticity orientation rule , reflecting the interaction between the potentiating and depressing synaptic processes , should be possible to define in terms of synaptic efficacy .",
"Patch-clamp on Xenopus cells in culture allows the examining of changes in the synaptic strength in individual neuromuscular synapses , and the assessing of synaptic conductance .",
"In Figure 1E , we observed in non-treated Xenopus synapses that 30 min of chronic burst stimulation of the motor neuron did not drastically change the synaptic gain , but nevertheless induced a slight depression of the averaged ePSCs .",
"Next , we set to closely examine these small synaptic changes in individual synapses .",
"Given the variety of average synaptic conductance among neuromuscular synapses ( Figure 1C , Y axis ) , and its strong correlation with the muscle input conductance ( Figure 1C ) , the range of the ratio between the synaptic and the input conductances 'Gsyn/Gin' is much tighter than the synaptic conductance range .",
"We reasoned that because this ratio , and not the synaptic conductance alone , determines the synaptic efficacy ( ePSP amplitude ) , it should be the appropriate synaptic parameter targeted by the homeostatic process .",
"Therefore , we recorded the ePSCs under brief test periods of postsynaptic voltage-clamp , before and after 20–30 min of chronic burst stimulation of the motor-neuron under postsynaptic current-clamp , and normalized these averaged ePSCs by the input conductance of the muscle cell .",
"Figure 5A ( black dots ) shows that the slight depression was not homogenous among neuromuscular synapses .",
"The degree of depression seems to depend on the distance of the initial Gsyn/Gin ratio from the mean ratio obtained after chronic activity ( Figure 5A , dashed line ) .",
"Consequently , standard deviation from the mean is reduced after chronic activity ( Figure 5A , inset ) .",
"This synaptic depression behavior can be interpreted as the convergence of the Gsyn/Gin ratios towards the set point of the homeostasis . 10 . 7554/eLife . 12190 . 015Figure 5 . Homeostatic control of the synaptic efficacy .",
"( A ) In Xenopus , ratios between averaged synaptic conductance ( 'Gsyn' , calculated from 30–40 ePSCs ) and muscle cell input conductance ( 'Gin' ) before and after 20–30 min of chronic burst stimulation of the motor neuron ( burst of 20–60 events at a 20–30 Hz frequency , every 30–40 s ) under postsynaptic current-clamp in non-treated ( black dots ) and low curare-treated ( red dots ) synapses .",
"Green dots represent the Gsyn/Gin ratio before and after 1–3 bursts of 5 presynaptic stimulations at 30 Hz ( green dots ) in ryanodine loaded muscle cells ( same data than in Figure 3C , ryanodine bar ) .",
"Inset shows the mean ± standard deviation of the Gsyn/Gin ratios in the three conditions .",
"The dotted lines show the averaged Gsyn/Gin ratio after chronic activity in non-treated synapses .",
"( B ) Degree of plasticity ( relative change in Gsyn/Gin ratio ) shown in A expressed as a function of the difference between the initial individual Gsyn/Gin ratio and the averaged ratio after chronic burst activity ( 'Distance to the set point' ) , in non-treated ( black dots ) and curare-treated ( red dots ) synapses .",
"The solid line shows the theoretical homeostatic relationship between plasticity and the distance to a set point of 2 . 36 , calculated as the mean Gsyn/Gin ratio after chronic activity in non-treated synapses .",
"( C ) Apparent averaged Gsyn/Gin ratios calculated in mouse FDB muscles from the data of Figure 3E , in non-stimulated and non-treated synapses ( no burst , black dot ) , in burst-stimulated and non-treated synapses ( after chronic bursts , black dot ) , in non-stimulated and ryanodine-treated synapses ( no burst , green dot ) and in burst-stimulated and ryanodine-treated synapses ( after chronic bursts , green dot ) .",
"Dots represent the mean ± Standard Deviation .",
"( D ) Relative change of contraction force during 2s-30Hz bursts of nerve stimulations in mouse soleus muscles ( n=4 ) , before and during exposure to a low dose ( 0 . 1 µM ) of curare . DOI: http://dx . doi . org/10 . 7554/eLife . 12190 . 015 In order to test whether synapses also converge if the initial Gsyn/Gin ratio is below the set point , we applied the same chronic activity in the continuous presence of low doses of curare ( 0 . 1–2 µM ) .",
"At these concentrations , the bursts of evoked synaptic events are still composed of a majority of suprathreshold events , combining both potentiating and depressing processes .",
"Figure 5A ( red dots ) shows that , again , the degree of plasticity ( potentiation in this case ) depends on the distance of the initial Gsyn/Gin ratio to the set point .",
"The plasticity orientation rule may then be expressed in a way showing its homeostatic character .",
"We plotted the degrees of plasticity ( relative change in the Gsyn/Gin ratio ) in non-treated ( Figure 5B , black dots ) and curare treated ( Figure 5B , red dots ) synapses shown in Figure 5A , as a function of the difference between the initial Gsyn/Gin ratios and the mean Gsyn/Gin ratio after chronic activity ( distance to the set point ) .",
"In a perfect homeostatic process , with an attractor set point , the degree of plasticity can be expressed as a function of the distance to the set point: ( GsynGin ) after ( GsynGin ) before=1−11+set point ( GsynGin ) before−set point The solid line in Figure 5B shows this theoretical relationship with a set point of 2 . 36 , equal to the mean Gsyn/Gin ratio obtained after chronic activity .",
"The overlap between the data points and the theoretical relationship shows the homeostatic function of the neuromuscular plasticity .",
"In order to place the potentiation we obtained with ryanodine ( Figure 3C , 'ryanodine' bar ) in regard of this homeostatic rule , we normalized the averaged synaptic conductances by the input conductance and added the data to the Figure 5A ( green dots ) .",
"The ryanodine-treated synapses did not converge towards a set point .",
"The averaged Gsyn/Gin ratios after burst activity were above the set point , and the standard deviations from the mean were increased .",
"These data suggest that the ryanodine receptors-dependent calcium signal participates to the stabilization of the synaptic efficacy at the set point .",
"In adult mouse neuromuscular synapses , an apparent Gsyn/Gin ratio can be extrapolated from the ePSPs .",
"If we assume a linear passive leak , the Gsyn/Gin ratio can be expressed as: GsynGin=−80−VpVp with Vp the peak membrane potential reached by the ePSP and 0 and -80 mV the reversal potentials of the synaptic and the leak currents respectively .",
"These ratios are only apparent and underestimated given the effect of distance between the recording site and the neuromuscular junction .",
"Figure 5C shows the apparent Gsyn/Gin ratio in non-stimulated and in burst-stimulated preparations , both for non-treated ( black dots ) and ryanodine-treated ( green dots ) preparations ( same data as in Figure 1D and Figure 3D–E ) .",
"These data in mouse ( Figure 5C ) confirmed the results seen in Xenopus ( Figure 5A ) .",
"Finally , in mouse , we applied a continuous low dose of curare ( 0 . 1 µM ) on soleus muscles , together with chronic burst stimulation of the nerve , in order to show that the homeostatic processes are also able to compensate for a decrease in the postsynaptic sensitivity to the neurotransmitter .",
"At this curare concentration , the synaptic activity is still composed of a majority of suprathreshold events , combining both nicotinic and DICR calcium signals .",
"We took advantage of the fact that the muscle contraction force integrates the synaptic efficacy over all the fibers of the muscle and all the synaptic events of the bursts , and as such is an indicator of even small changes in the synaptic gain .",
"Figure 5D shows the 26% decrease in the contraction force due to low curare , followed by progressive recovery of the force back to a normal level in response to subsequent nerve burst stimulations .",
"We could thus directly observe the functional effect of the synaptic homeostasis mechanism we describe ."
],
[
"When selectively triggered , the nicotinic Ca2+ influx and the DICR signal induce a form of LTP and LTD respectively .",
"The causal links between the nicotinic Ca2+ influx and the synaptic events , and between the DICR signal and the muscle APs , allow the establishment of an orientation rule for neuromuscular synaptic plasticity: synaptic events-induced LTP versus muscle AP-induced LTD .",
"This orientation rule finds its equilibrium in the suprathreshold range of the synaptic strength and provides the specific 'homeostatic' function to this plasticity .",
"This neuromuscular plasticity differs from the other forms of long-term plasticity , and in particular should not be confounded with anti-Hebbian plasticity ( Kullmann and Lamsa , 2008; Roberts and Leen , 2010 ) .",
"Anti-Hebbian plasticity underlies synaptic strength modulations while the present homeostatic mechanism maintains the stability of synaptic efficacy .",
"Modulation of synaptic transmission has been extensively studied in vitro at the Xenopus neuromuscular synapse by Poo group ( Fu and Poo , 1991; Lo and Poo , 1991; 1994; Lohof et al . , 1993; Lo et al . , 1994; Dan et al . , 1995; Cash et al . , 1996; Wang and Poo , 1997; Wan and Poo , 1999 ) .",
"Our work , partly done on the same experimental model , suggests that a number of results obtained by Poo group might be interpreted in the more specific framework of homeostatic plasticity .",
"In vertebrates , neurotrophic factors such as brain-derived neurotrophic factor , neurotrophin 3 and neurotrophin 4 , are trans-synaptic factors considered candidates to mediate presynaptic release modulations in many forms of synaptic plasticity ( Schinder and Poo , 2000; Poo , 2001 ) .",
"Skeletal muscle synthesize and release neurotrophins in an activity-dependent manner ( Funakoshi et al . , 1995; Wang and Poo , 1997; Xie et al . , 1997 ) , and motor nerve terminals contain the receptors tyrosine kinase TrkB and C ( Henderson et al . , 1993; Koliatsos et al . , 1993; Wong et al . , 1993; Yan et al . , 1993 ) .",
"At the Xenopus neuromuscular synapse in vitro , Poo group has shown that the exogenous application ( Lohof et al . , 1993; Wang and Poo , 1997 ) of these factors reproduces the synaptic LTP induced in the present work with subthreshold synaptic events or postsynaptic ryanodine treatment .",
"Finally , upregulation of the ACh evoked release in α-bungarotoxin treated rat was markedly reduced by inhibition of the tyrosin kinase receptors of the neurotrophins ( Plomp and Molenaar , 1996 ) .",
"Therefore , neurotrophic factors should be considered for future investigations to determine whether they can be retrograde factors mediating homeostatic plasticity at the neuromuscular synapse .",
"The diversity of the effects of the calcium signaling comes from the variety of its temporal patterns , amplitude , and location .",
"The two antagonist calcium signals that we demonstrated here to be involved in the orientation of the neuromuscular plasticity exhibit different temporal patterns and amplitudes .",
"The AP-associated DICR signal exhibits fast transient large calcium concentration elevations for each AP .",
"The kinetics of the nicotinic calcium build-up is much slower and does not contain transients during repetitive nicotinic receptor stimulations .",
"Its amplitude under voltage-clamp is low compare to the DICR signal ( Figure 2F ) , and even lower under current-clamp given the reduced driving-force for the calcium ion due to the postsynaptic depolarization .",
"This dichotomy between fast large transient and slow low calcium build-up is known to trigger selectively , via calmodulin , the CAMKII kinase and the calcineurin phosphatase respectively .",
"This mechanism is considered in the central nervous system to implement Hebbian plasticity ( Malenka et al . , 1989; Malinow et al . , 1989; Mulkey et al . , 1993; 1994 ) by modulating the conductance and number of the postsynaptic receptors ( Barria et al . , 1997; Mammen et al . , 1997; Beattie et al . , 2000 ) .",
"The CAMKII signaling pathway was also shown to be required for normal 'presynaptic homeostasis' in Drosophila ( Haghighi et al . , 2003 ) .",
"Furthermore , Wan and Poo 1999 showed at the Xenopus synapse in vitro that induction of LTP and LTD can be blocked by pre-loading the muscle cell with peptide inhibitors of calcineurin and CAMKII respectively .",
"As noted by Wan and Poo 1999 , this situation is opposite to the central synapses , where calcineurin is associated to depression and CAMKII to potentiation .",
"Our results suggest that the low-calcium nicotinic signal and the calcineurin activation have a possible causal link with the detection of the synaptic events and the synaptic potentiation , while the high-calcium DICR signal and the CAMKII activation are associated with the detection of the postsynaptic APs and the synaptic depression .",
"The convergence of the synaptic efficacy towards a set point ( Figure 5 ) suggests that the potentiating and depressing synaptic processes balance each other in the suprathreshold range of the synaptic strength .",
"These observations strongly suggest that the set point of the homeostatic plasticity is sustained by a push-pull mechanism .",
"Our results , however , do not determine the stages where this push-pull mechanism could operate in the causal chain linking the evaluation of synaptic efficacy to the presynaptic modulation .",
"In particular , our findings do not necessarily imply that two independent antagonist trans-synaptic retrograde factors balance their effects at the presynaptic level .",
"The coexistence of potentiating and depressing trans-synaptic factors is a possibility among others .",
"The increased and decreased secretion of a single positive retrograde factor —like for example endostatin in Drosophila ( Wang et al . , 2014 ) and neurotrophins in vertebrates— could also bi-directionally regulate neurotransmitter release .",
"In this case , it could be hypothesized that a push-pull mechanism operates downstream of the calcium signaling , for example at the level of the calcineurin/CAMKII balance , ultimately determining the secretion level of a single factor .",
"Therefore , the use of blue and red arrows in Figure 2 is intended as a schematic representation of the retrograde mechanisms that can bi-directionally change neurotransmitter release , without presuming the existence of two distinct retrograde factors .",
"Most of what we know about synaptic homeostasis stems from experimental studies at the Drosophila larva neuromuscular junction .",
"Given the numerous differences between Drosophila and vertebrates the comparison between these systems is not straightforward .",
"Action potentials and the associated DICR signal in vertebrates being absent in Drosophila muscle cells ( Hong and Ganetzky , 1994 ) , the homeostatic mechanism we propose here cannot be directly transposed to Drosophila .",
"Since no orthologue of the tyrosine kinase receptors are found in Drosophila ( Frank , 2014 ) , the neurotrophin hypothesis in vertebrates cannot either be applied to Drosophila .",
"However , different actors could play similar roles in both systems .",
"The calcium permeability of glutamate receptors in Drosophila could have a similar role than the calcium permeability of nicotinic receptors in vertebrates .",
"The low-voltage activated calcium channels activated by the ePSPs and the 'calcium-induced calcium release' signal responsible for the excitation-contraction coupling in Drosophila ( Peron et al . , 2009 ) could be the orthologue of the DICR signal in vertebrates .",
"Finally , endostatin in Drosophila ( Wang et al . , 2014 ) may play the role of neurotrophins in vertebrates .",
"In Drosophila , Frank et al . ( 2006 ) found that spontaneous glutamate release was sufficient to induce a rapid potentiation in the absence of nerve activity , while we had to use a 30Hz motor command to induce rapid potentiation in vertebrates .",
"In order to establish the Figure 5A , cells were incubated in curare in absence of neurons spikes during 30–60 min before recording ( red dots ) .",
"Therefore , the spontaneous ACh release did not restore the normal Gsyn/Gin ratio ( found in non-treated synapses ) before chronic bursts were applied .",
"A possible explanation of this difference between the two systems is the high frequency of miniatures in Drosophila ( 10–20 Hz ) ( Frank et al . , 2006 ) , while the average frequency of spontaneous release in the Xenopus cells culture was 100 fold lower .",
"In vertebrates , evoked activity responsible for muscular tonus and voluntary motions represents most of the synaptic activity , and the 20–30 Hz frequencies we used in our experiments are in the normal range for a motor command ( Gorassini et al . , 2000 ) .",
"Therefore , the evoked activity in vertebrate is more likely able to rapidly mobilize the homeostatic machinery than the spontaneous miniatures .",
"In the central nervous system , homeostatic forms of synaptic plasticity have been proposed to restrain the mean level of neuronal activity within a physiological regime , and to maintain the stability of recurrent network activity challenged by associative plasticity .",
"Because of the robustness of its synaptic transmission , the neuromuscular junction is considered as a model of homeostasis .",
"However , the neuromuscular junction being the end effector of the motor network , its main function is the faithful translation of the motor command into muscle activity .",
"The mean level of muscle activity is not involved in any recurrent network that requires stability , and therefore does not need to be controlled .",
"While the synaptic strength is the adjustment variable for the control of the mean level of neuronal activity in the CNS , at the NMJ it is the efficacy of synaptic transmission itself that must be maintained constant for reliable relay of the motor command .",
"Therefore , homeostatic plasticity could be regarded as a functionally different phenomenon at the NMJ than in the CNS .",
"Nevertheless , the plasticity orientation rule described in the present work matches a synaptic 'relay' function .",
"Despite differences in the nature of the activity sensors , other 'relay' synapses in the CNS , such as those involved in sensory inputs to the thalamus ( Guido , 2008 ) , might share with the NMJ a comparable homeostatic control based on pre- and post- synaptic activity detection ."
],
[
"Myotomal and spinal tissues from 1-day-old Xenopus laevis embryos ( stages 23 to 25 ) were mechanically dissociated using a Ca2+- and Mg2+-free medium of the following composition ( in mM ) : 115 NaCl , 2 . 6 KCl , 0 . 4 EDTA , 10 HEPES ( pH = 7 . 6 ) .",
"Cells were directly plated in a plastic recording chamber , and grown at 19°C for 12 hr prior to the experiments .",
"The culture medium consisted of 50% Leibovitz L-15 medium ( Gibco , Invitrogen Corp . , Cergy-Pontoise , France ) , 1% fetal calf serum , 1% antibiotic mixture ( ibid . , final concentration: 100 units/mL penicillin G and 100 µg/mL streptomycin ) , and 48% physiological solution of the following composition ( in mM ) : 113 NaCl , 2 KCl , 0 . 7 CaCl2 , 5 HEPES ( pH=7 . 8 ) .",
"Perforated patch-clamp recordings , to preserve the cell integrity , were performed at room temperature ( 20–22°C ) .",
"Pipettes were made from borosilicate glass ( Clark Electromedical Instruments , Reading , England ) and pulled on a P-1000 puller ( Sutter Instrument Company , Novato , CA , U . S . A . ) .",
"Patch electrodes had a resistance of 2–3 MΩ when filled with internal physiological solution .",
"Membrane currents and potential were recorded using Axopatch200B patch-clamp amplifiers ( Axon Instruments , Union City , CA ) .",
"Access resistances were compensated at 80% .",
"Myocyte membrane currents were filtered with an integrated low-pass Bessel filter at 2 kHz .",
"The filtered signals were digitized by a 12 bit A/D converter ( Digidata 1200B , ibid . ) and stored using pCLAMP 8 software ( ibid . ) .",
"Recordings were analyzed using the Origin 7 software ( OriginLab Corp . , Northampton , MA ) .",
"Motor neurons were current-clamped through an amphotericin-perforated membrane patch ( 400 pA , 3 ms current step to induce the presynaptic AP ) ; the intra-pipette solution had the following composition ( in mM ) : 1 NaCl , 140 K-gluconate , 1 MgCl2 , 10 HEPES ( pH=7 . 2 ) , and amphotericin-B at 300 µg/ml .",
"Myocytes were either voltage- or current-clamped , through an amphotericin-perforated membrane patch , using the following intra-pipette composition ( in mM ) : 1 NaCl , 20 KCl , 125 K-gluconate , 1 MgCl2 , 10 HEPES ( pH=7 . 2 ) , and amphotericin-B at 300 µg/ml .",
"The external solution had the following composition ( in mM ) : 140 NaCl , 3 KCl , 1 MgCl2 , 2 CaCl2 , 10 HEPES ( pH=7 . 4 ) .",
"Pipettes made with borosilicate glass had a resistance of 80–120 MOhm when filled with 0 . 5 to 1 M ACh-Cl .",
"ACh+ efflux was induced by positive current steps , using a home-made constant current generator .",
"Xenopus muscle cells were loaded with the calcium indicator in whole-cell patch-clamp configuration for 10 min with an intra-pipette medium of the following composition ( in mM ) : 110 KCl , 1 NaCl , 2 Mg2+ , 10 HEPES ( pH 7 . 2 ) , and 0 . 2 Fluo4-pentapotassium salt .",
"The patch pipette was then removed and a perforated patch was performed as described above .",
"Fluorescence intensity was quantified with an Olympus photomultiplier ( forming part of the OSP system , Olympus , Japan ) , and the tension signal was digitized with the Digidata converter .",
"After background fluorescence subtraction , signals were normalized according to the baseline fluorescence .",
"The reversal potential of the nicotinic current induced by ionophoretic acetylcholine application was measured under voltage-clamp in solutions of various ionic compositions ( Figure 2—figure supplement 1 ) .",
"The following equation , derived from the Goldman-Hodgkin-Katz flux equation ( Goldman , 1943; Hodgkin and Katz , 1949 ) , was used to calculate the permeability ratios:Vrev=RTF[0 . 75×PK[K]o+0 . 75×PNa[Na]o+0 . 25×4PCa[Ca]o+0 . 25×4PMg[Mg]o0 . 75×PK[K]i+0 . 75×PNa×[Na]i+0 . 25×Mg[Mg]i] In this equation , the terms in square brackets are ion concentrations , PS is the permeability of the S ion species , T is the absolute temperature , R and F are the gas and Faraday constants , and i and o are the intra- and extracellular compartments , respectively .",
"Factors represent the ionic activity coefficients .",
"The dynamic-clamp technique ( Sharp et al . , 1993; Robinson and Kawai , 1993 ) was used to inject computer-generated conductances in Xenopus muscle cells .",
"Dynamic-clamp experiments were run using the hybrid RT-NEURON environment ( Le Franc et al . , 2001; Sadoc et al . , 2009 ) , a modified version of NEURON ( Hines and Carnevale , 1997 ) running under the Windows operating system ( Microsoft Corp . , Redmond , Washington ) , augmented with the capacity of simulating models in real time , synchronized with the intracellular recording .",
"A PCI DSP board with 16 bit A/D-D/A converters ( Innovative Integration , SimiValley ) was used to input the membrane potential into the equations of the model , and to output the current to be injected into the cell with a time resolution of 0 . 1 ms . Passive K+ leak and K+ inward rectifying ( Kir ) conductances were injected into the muscle cell to simulate an increase in the input conductance ( Figure 3—figure supplement 1 ) .",
"The passive K+ leak was expressed in the form: Ileak = gleak x ( V-Eleak ) .",
"Eleak was set to -80 mV , and gleak to 5–15 nS .",
"The Kir conductance was expressed in the form: IKir =gmax x m x ( V – EK ) where the maximal conductance gmax was set to 20–50 nS , and changes with time of the gating variable m were calculated by solving the differential equation:dmdt=m∞−mτmm ( t ) =m∞− ( m∞−m0 ) ×⟮−tτm⟯ where fitting voltage-clamp recordings of the real isolated Kir current of Xenopus muscle cells gave and m∞ ( v ) =11+exp ( −0 . 074× ( EK−V ) ) andτm=0 . 2ms The 'contraction' trace shown in Figure 2A represents a qualitative measurement of the contraction of a muscle cell in culture .",
"In addition to the membrane potential recording pipette , a second patch pipette vertically approached the membrane cell until a slight increase in the pipette electrical resistance became visible .",
"The pipette was then held in that position , and increase in the cell thickness accompanying contraction was monitored through further variations of the pipette resistance .",
"3- to 5-month-old Swiss mice were anesthetized with isoflurane , and cervical dislocated .",
"Dissections were performed within 15 min in an oxygenated Ringer solution of the following composition ( in mM ) : 145 NaCl , 3 KCl , 2 CaCl2 , 1 MgCl2 , 10 HEPES ( pH 7 . 4 ) and 11 glucose .",
"Intracellular recordings ( Figure 1—figure supplement 1 ) were performed at 34°C , in the oxygenated Ringer solution defined above .",
"Sharp pipettes were made from borosilicate glass ( Clark Electromedical Instruments ) , pulled on a P-1000 puller ( Sutter Instrument Company ) , and had a resistance of 40–60 MΩ when filled with a KCl 3 M solution .",
"The filled pipette tip was cut at the limit of the pulled zone , and used as electrode .",
"The chlorided end of a 10 cm long , 50 µm diameter , silver wire was introduced inside this electrode , and plugged by its opposite end to the headstage of an Axoclamp 2A amplifier ( Axon Instruments ) , where it hung loosely above the muscle .",
"A drop of mineral oil was added at the back of the intra-pipette medium to avoid evaporation .",
"The pipette pendulum was vertically manipulated to enter the muscle cells , and its flexibility allowed stable membrane potential recordings in contracting muscles .",
"Nerve APs were induced with 6 V , 30 µs voltage steps in a suction pipette .",
"2 µM of µ-conotoxin GIIIB ( 10 min ) was used to isolate the ePSPs .",
"Data acquisition and analysis were made as above .",
"Under µ-conotoxin the amplitudes of the recorded ePSPs depend on the distance between the recording site and the synaptic region .",
"With the floating electrode method , the placement of the electrode is not precisely controlled .",
"We limited the distance effect by using the FDB mouse muscle , where cells are shorter ( 300 µm ) than the muscle length ( 1 cm ) .",
"Therefore , blind placement of the electrode can be performed on this muscle , with a maximal 150 µm distance between the recording site and the end plate .",
"The specific cells size and organization in FDB limit the distance effect in our recordings , but this effect is presumably responsible for an underestimation of the ePSPs amplitudes and for most of the variability between the recorded cells .",
"Despite the underestimation of the ePSPs amplitudes , the recorded amplitudes were above the -63 mV AP threshold determined in muscle cells of rat fast and slow muscles ( Wood and Slater , 1995 ) .",
"The salts composing the media used for electrophysiological recordings , EGTA used to obtain Ca2+-free media , and Amphotericin B used for perforated patch-clamp were obtained from Sigma-Aldrich ( Sigma-Aldrich , Saint Quentin Fallavier , France ) .",
"Ryanodine used in some experiments was 'Ryanodine fractions' from Latoxan ( Latoxan , Valence , France ) .",
"The µ-conotoxin GIIIB used to block mouse muscle action-potentials was obtained from Alomone Labs ( Alomone Labs , Jerusalem , Israel ) .",
"The Ca2+ dye 'Fluo4-pentapotassium salt' was obtained from Molecular Probes ( Molecular Probes , Eugene , Oregon ) ."
]
] | [
"Excitability differs among muscle fibers and undergoes continuous changes during development and growth , yet the neuromuscular synapse maintains a remarkable fidelity of execution .",
"Here we show in two evolutionarily distant vertebrates ( Xenopus laevis cell culture and mouse nerve-muscle ex-vivo ) that the skeletal muscle cell constantly senses , through two identified calcium signals , synaptic events and their efficacy in eliciting spikes .",
"These sensors trigger retrograde signal ( s ) that control presynaptic neurotransmitter release , resulting in synaptic potentiation or depression .",
"In the absence of spikes , synaptic events trigger potentiation .",
"Once the synapse is sufficiently strong to initiate spiking , the occurrence of these spikes activates a negative retrograde feedback .",
"These opposing signals dynamically balance the synapse in order to continuously adjust neurotransmitter release to a level matching current muscle cell excitability ."
] | [
"Nerve cells communicate with each other , and with targets such as muscle cells , at junctions called synapses .",
"The nerve cell before the synapses releases a chemical called a neurotransmitter , which binds to receptors on the cell after the synapses .",
"However , the first cell cannot determine by itself whether it is releasing the correct amount of neurotransmitter to activate its partner .",
"For this , it requires feedback from the second cell .",
"This feedback is particularly important at synapses between nerve cells and muscle cells , which are known as neuromuscular junctions .",
"The likelihood that a given amount of transmitter will activate a muscle cell can vary with age and after exercise .",
"Muscle cells must therefore be able to instruct their nerve cell partners to increase or decrease neurotransmitter release to accommodate these changes .",
"Ouanounou et al . have now identified the mechanism by which muscle cells determine whether nerve cells are releasing an appropriate amount of neurotransmitter .",
"Experiments in two distantly related animals – mice and embryos from a frog called Xenopus – revealed that muscle cells use two calcium-based signals .",
"The first is the flow of calcium ions into the muscle cell in response to binding of neurotransmitter to receptors at the synapses: this tells the muscle cell how active the nerve cell is .",
"The second is the release of calcium ions from internal stores inside the muscle cell: this occurs whenever neurotransmitter release is sufficient to activate the muscle cell .",
"In response to the first calcium signal , the muscle cell sends positive feedback to the neuron , telling it to increase neurotransmitter release further .",
"In response to the second signal , the muscle cell sends negative feedback to reduce neurotransmitter release .",
"Thus , when neurotransmitter release is not enough to activate the muscle , positive feedback dominates and neurotransmitter release increases .",
"However , when the muscle is activated , the two types of feedback act in balance to maintain efficient communication across the synapse .",
"The next steps are to identify the cell signaling cascades that are mobilized by the two calcium signals , including the specific molecule ( or molecules ) that regulate neurotransmitter release ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"neuroscience"
] | Electrical synaptic transmission requires a postsynaptic scaffolding protein | elife-66898-v1 | [
[
"Synapses are specialized cellular adhesions between neurons that rapidly transfer information to facilitate neural network function .",
"There are two modalities of fast synaptic transmission , chemical and electrical , both found throughout animal nervous systems including in mammals ( Moroz and Kohn , 2016; Ryan and Grant , 2009 ) .",
"Chemical synapses are inherently asymmetric structures , derived from presynaptic specializations that regulate the release of neurotransmitters and postsynaptic specializations that contain neurotransmitter receptors and the machinery required to propagate signal transmission .",
"Both specializations require hundreds to thousands of proteins , which together tightly control the structure , function , and modulation of synaptic communication ( Ackermann et al . , 2015; Grant , 2019; Siddiqui and Craig , 2011 ) .",
"For example , intracellular scaffolding proteins of the Post Synaptic Density ( PSD ) at chemical synapses regulate the number and functional state of AMPA and NMDA receptors , which are ligand-gated ion channels , at glutamatergic synapses ( Zhu et al . , 2016 ) .",
"By contrast , electrical synapses are often perceived as simple aggregates of intercellular channels known as gap junctions ( GJs ) ( Goodenough and Paul , 2009 ) .",
"Intercellular GJ channels are formed by the docking of two hemichannels , composed of Connexin proteins in vertebrates and Innexins in invertebrates ( Bhattacharya et al . , 2019; Phelan , 2005; Shruti et al . , 2014; Söhl et al . , 2005 ) .",
"Each neuron contributes a hemichannel from each side of the synapse , which form a channel and support communication by allowing the spread of electrical currents and small metabolites between adjacent ‘coupled’ neurons .",
"While multiple Connexins and Innexins can contribute to individual electrical synapses ( Bhattacharya et al . , 2019; Miller et al . , 2017; Phelan et al . , 2008; Rash et al . , 2013 ) , the complexity of neuronal GJ cellular biology ( Lynn et al . , 2012; Martin et al . , 2020; Meyer et al . , 2014; Sigulinsky et al . , 2020 ) and the variety of mechanisms regulating their synaptic strength ( Arroyo et al . , 2016; Bloomfield and Völgyi , 2009; Marder , 1998; O'Brien and Bloomfield , 2018; Pereda , 2014 ) suggest they require complex multimolecular structures to support function .",
"Several Connexin-associated proteins have been identified ( Lynn et al . , 2012; Miller and Pereda , 2017 ) ; however , it remains undetermined whether such associated proteins are ancillary to the channels or requisite for electrical synapse function .",
"Perhaps the best characterized Connexin-associated protein is Zonula Occludens 1 ( ZO1 ) ( Bauer et al . , 2010; Willott et al . , 1993 ) , which is an intracellular scaffolding protein and a member of the membrane-associated guanylate kinase ( MAGUK ) family of proteins .",
"MAGUKs constitute a large family of multifunctional adaptor proteins that play key roles in scaffolding membrane channels and receptors to intracellular signaling complexes and the cytoskeleton ( González-Mariscal et al . , 2000 ) .",
"MAGUK proteins , including ZO1 , contain PSD95/Dlg/ZO1 ( PDZ ) protein-protein interaction domains , which bind to PDZ-binding motifs often located at the carboxy terminus of partner proteins , including Connexins ( Zhu et al . , 2016 ) .",
"The best studied ZO1/Connexin interaction is with Connexin 43 ( Cx43 ) , a widely expressed , non-neuronal , GJ-channel forming protein ( Giepmans and Moolenaar , 1998 ) .",
"The ZO1/Cx43 interaction is thought to play important functional roles in GJ regulation by facilitating the docking of newly inserted hemichannels , which promotes the formation of intercellular channels ( Hunter et al . , 2005 ) .",
"Moreover , the ZO1/Cx43 interaction is critical for channels to remain conductive prior to removal during channel turnover at epithelial GJs ( Hervé et al . , 2014; Thévenin et al . , 2017 ) .",
"While ZO1 is an important regulator of Cx43-contaning GJs , less is known about its role at neuronal GJs , which are primarily formed by the Cx36-family of proteins and mediate electrical synaptic transmission in vertebrate nervous systems ( Connors and Long , 2004; Miller et al . , 2017; Rash et al . , 2013; Söhl and Willecke , 2004 ) .",
"In neurons , ZO1 immunostaining correlates with synapses containing Cx36 and its fish homologs ( Flores et al . , 2008; Li et al . , 2004; Marsh et al . , 2017; Yao et al . , 2014 ) , and its presence at synapses may play regulatory roles ( Flores et al . , 2008 ) , the nature of its contributions to electrical transmission remains unknown .",
"Despite mounting evidence for the widespread dynamic functional contributions of electrical synapses to neural circuit function , the perception of the simplicity of their molecular organization remains .",
"We hypothesized that scaffolding molecules form part of a multimolecular structure that is required for channel function akin to that found at chemical synapses .",
"Here , we explore the functional role of ZO1 in zebrafish by examining identifiable synaptic contacts of the Mauthner cell ( Bartelmez , 1915; Bodian , 1937; Hildebrand et al . , 2017; Kimmel , 1982; Robertson et al . , 1963 ) , which forms stereotyped electrical synapses accessible to genetic , biochemical , cell biological , electrophysiological , and behavioral analyses .",
"We show that the presence ZO1 protein is critically required for the structure and function of the intercellular channels .",
"Moreover , we find that the localization of ZO1 is compartmentalized postsynaptically where it functions in the formation of neuronal GJs .",
"Our results stand in contrast with current views on electrical synapse organization centered solely on the proteins forming GJ channels .",
"Thus , our findings provide strong support to the notion that electrical synapses constitute complex and asymmetric synaptic structures at which intercellular channels are governed by multimolecular structures with features that parallel the molecular and functional organization of the PSD at chemical synapses ."
],
[
"We sought to examine the relationship between the intracellular scaffold ZO1 and neuronal Connexins ( Cxs ) by utilizing the stereotyped synapses of the zebrafish Mauthner cell .",
"This circuit drives a fast escape response to threatening stimuli using both electrical and chemical connections ( Eaton et al . , 1977; Jacoby and Kimmel , 1982; Liu and Fetcho , 1999; Wolman et al . , 2015 ) .",
"Each animal has two Mauthner cells that receive multimodal sensory input that relay information to the spinal cord to coordinate circuits to elicit fast turns .",
"We focus on two populations of stereotyped electrical synapses made by Mauthner cells: ( 1 ) ‘club ending’ ( CE ) synapses ( Bartelmez and Hoerr , 1933; Pereda et al . , 2004; Yao et al . , 2014 ) , which are mixed electrical/glutamatergic chemical synaptic contacts formed between auditory afferents of the eighth cranial nerve and the Mauthner cell's lateral dendrite ( Figure 1A , B ) and ( 2 ) en passant electrical synapses formed between the Mauthner axon and Commissural Local ( CoLo ) interneurons found in each spinal-cord segment ( Figure 1A , M/CoLo synapses ) ( Satou et al . , 2009 ) .",
"Neuronal GJs at both CEs and M/CoLo synapses are made of heterotypic channels formed by Cx35 . 5 , encoded by the gene gap junction delta 2a ( gjd2a ) , and Cx34 . 1 , encoded by gjd1a , both homologous to mammalian Cx36 ( gjd2 ) .",
"We previously found that Cx35 . 5 and Cx34 . 1 are localized asymmetrically to the pre- and postsynaptic sides at CE and M/CoLo synaptic contacts ( Figure 1B; Miller et al . , 2017 ) .",
"Throughout we use the terms pre- and postsynaptic to reference the neuronal , cell-biological compartment in which a Connexin is localized .",
"At CE synapses , the auditory afferent axons are presynaptic to the postsynaptic Mauthner lateral dendrite; while at M/CoLo synapses , the Mauthner axon is presynaptic to the postsynaptic CoLo .",
"We first determined the localization of ZO1 and the Connexin proteins at Mauthner electrical synapses using immunofluorescence and confocal imaging .",
"We stained 5 day post fertilization ( dpf ) larvae , a time at which the Mauthner circuit elicits a mature startle response , with antibodies against the human ZO1 protein and those that distinguish the zebrafish Cx35 . 5 and Cx34 . 1 ( Figure 1C–L; Miller et al . , 2017 ) .",
"We observed extensive colocalized signal for these three proteins at CE and M/Colo electrical synapses , with each protein apparent in the stereotyped shape and position of the neuronal gap junctions ( GJs ) at these contacts .",
"We identified CEs unambiguously as large ( 1 . 5–2 µm ) oval areas localized in the distal portion of the lateral dendrite of the Mauthner cell ( Figure 1C; Yao et al . , 2014 ) .",
"M/Colo synapses were identified by their regularly spaced sites of contact in the spinal cord ( Figure 1K ) .",
"Next , we examined the role of ZO1 at electrical synapses using CRISPR/Cas9-induced mutations to knock out gene function .",
"Mammalian ZO1 is encoded by the gene tight junction protein 1 ( tjp1 ) , while zebrafish have two homologous genes , tjp1a and tjp1b .",
"Using a CRISPR-based screen , we found that mutations in tjp1b/ZO1b , but not tjp1a/ZO1a , caused a failure of Connexin localization at M/CoLo synapses ( Figure 1; Figure 1—figure supplement 1; Marsh et al . , 2017; Shah et al . , 2015 ) .",
"We examined the effect of these mutations on CEs and found that tjp1b/ZO1b-/- mutants lack most of the detectable fluorescent staining for both Cx35 . 5 and Cx34 . 1 , as well as ZO1 , at the stereotyped synaptic contact sites ( Figure 1D , L ) .",
"Quantitation of Cx35 . 5 , Cx34 . 1 , and ZO1 fluorescence at CEs and M/CoLo contacts confirmed that staining for all three antibodies was greatly diminished in tjp1b/ZO1b-/- mutants ( Figure 1M , N ) .",
"By contrast , homozygous tjp1a/ZO1a -/- mutants had extensive Connexin and ZO1 staining at these contacts , while tjp1a-/-; tjp1b-/- double mutants were indistinguishable from tjp1b-/- single mutants ( Figure 1—figure supplement 1A–G ) .",
"We conclude that ZO1b protein , encoded by the tjp1b gene , is localized to electrical synapses and required for the robust localization of both Cx35 . 5 and Cx34 . 1 at synaptic contacts .",
"While tjp1b/ZO1b-/- mutants showed significantly diminished levels of ZO1 and Connexin staining at presumptive synaptic locations , we wondered whether neurons were still attempting to assemble GJs .",
"Indeed , we detected both Cx35 . 5 and Cx34 . 1 by western blot from brain homogenates of tjp1b/ZO1b-/- mutant animals ( Figure 1—figure supplement 1H ) .",
"We therefore examined CEs using higher contrast and magnification to assess GJ structure as detectable by immunolabeling and individually stained for ZO1 or Connexin to avoid confounding the image analysis due to bleed through of signal amongst stained proteins .",
"We found that in tjp1b/ZO1b-/- mutants , each of the three antibodies revealed structures located at the stereotyped position of CE contacts and had morphologies reminiscent of wild-type animals , albeit with much dimmer fluorescence intensity ( Figure 1E–J; note that image contrast was increased in mutants ( H-J ) ) .",
"While we observed the stereotypical oval-shaped CE structures in mutants , the staining for each protein was weak and irregular in its distribution , suggesting the residual staining in mutants might represent incomplete , abortive synaptic structures ( see electrophysiology below ) .",
"Consistent with a reduced presence of GJ proteins , we also observed a reduced number of CEs detected by immunolabeling in tjp1b/ZO1b-/- mutants ( Figure 1—figure supplement 1I ) .",
"We found similar results at M/CoLo synapses , although their smaller size precluded an analogous detailed analysis ( Figure 1K , L , N ) .",
"In contrast to tjp1b/ZO1b-/- , the staining of tjp1a/ZO1a-/- mutants was indistinguishable from wildtype ( Figure 1—figure supplement 1E–G , I ) .",
"These observations suggest that neurons of the Mauthner cell network in tjp1b/ZO1b-/- mutants persist in attempting to create electrical synapses , despite their inability to robustly localize neuronal Connexins at synaptic contacts .",
"We conclude that ZO1 is localized to electrical synapses where it plays a critical role in neuronal GJ formation .",
"Given that Connexin localization was dependent on ZO1b , we sought to determine if the converse was true – did ZO1 localization require Connexins ?",
"Using previously generated mutations in gjd2a/Cx35 . 5 and gjd1a/Cx34 . 1 ( Miller et al . , 2017 ) , we examined the localization of Connexin and ZO1 proteins in mutants by immunolabeling ( Figure 2A–L ) .",
"First , we found that gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant animals revealed a complete loss of detectable staining for the mutated protein .",
"In addition , there was a failure of the non-mutated Connexin protein to robustly localize to the electrical synapse although low levels of staining were present .",
"In line with these observations , by using brain homogenates and western blots , we found a complete loss of the Connexin affected by each mutation , but no effect on the non-mutated Connexin protein ( Figure 1—figure supplement 1H ) .",
"By contrast , ZO1 staining in the Connexin mutants was robust at the synaptic contact sites with the stereotyped appearance , distribution , and position clearly evident ( Figure 2A–L ) .",
"By comparing the relative ZO1 fluorescence between wild-type and Connexin mutant animals , we found that ZO1 was present at synaptic contacts at approximately half the normal level ( Figure 2M , N ) .",
"We examined gjd2a-/-; gjd1a-/- double mutants and found that ZO1 still robustly localized to CE and M/CoLo contact sites ( Figure 2M , N; Figure 2—figure supplement 1A–F ) .",
"These results reveal two critical organizational principles about electrical synapses in the Mauthner cell: ( 1 ) ZO1b localizes to putative electrical synaptic sites largely independent of Connexin proteins and ( 2 ) each Connexin requires the other for robust localization to the synapse .",
"Based on these data , we conclude that ZO1 can localize to neuronal GJs independent of the presence of channel-forming proteins , yet ZO1 is absolutely essential for proper Connexin localization .",
"To further examine the electrical synapse structure of Connexin mutants , we assessed CEs at higher contrast ( Figure 2D–I ) .",
"We found that: ( 1 ) immunofluorescence for the mutated Connexin is completely lost at the synapse , ( 2 ) the non-mutated Connexin is detectable but with weak and irregular labeling , suggestive of incomplete , abortive structures , and ( 3 ) ZO1 labeling resembles wildtype with a distribution and morphology that appears normal across the expanse of the putative synaptic contact .",
"Consistent with these findings , the number of CEs detected by ZO1 labeling in Connexin mutants was indistinguishable from that observed in wildtype , while those detected by antibodies for the mutated Connexin were significantly reduced ( Figure 1—figure supplement 1I ) .",
"In addition , the zebrafish genome contains two additional homologous Connexin genes , gjd2b/Cx35 . 1 and gjd1b/Cx34 . 7 .",
"We found that animals that were homozygous mutant for these two genes had normal Connexin and ZO1 labeling at CEs ( Figure 2—figure supplement 1G–L ) .",
"Similarly , we previously found there was no effect on M/CoLo synapses in the gjd2b/Cx35 . 1 and gjd1b/Cx34 . 7 mutants ( Miller et al . , 2017 ) .",
"Together , these results support a hierarchical relationship in the formation of neuronal GJs , in which ZO1 localizes to electrical synaptic contact sites where it is essential to robustly localize neuronal Connexins .",
"We next sought to examine the functional consequences of ZO1 and Connexin mutants , so we explored the properties of synaptic transmission at CEs during whole-cell recordings of the Mauthner cell .",
"In wildtype zebrafish , extracellular stimulation of CE afferents near the posterior macula where they contact hair cells ( Figure 3A ) evoked a mixed synaptic response in the Mauthner cell composed of an early and large GJ-mediated electrical component followed by a delayed and smaller glutamatergic chemical response ( Figure 3B; Yao et al . , 2014 ) .",
"We first aimed to establish the functional consequences of removing the Connexins on this mixed synaptic response .",
"Consistent with the presence of Cx35 . 5 and Cx34 . 1 at CEs , synaptic responses in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant zebrafish lacked a detectable electrical component , while exhibiting a response with the same delay as the chemical component of the mixed synaptic potential of wildtypes ( Figure 3C–E ) .",
"By contrast , electrical transmission was unaffected in gjd2b/Cx35 . 1-/- and gjd1b/Cx34 . 7-/- mutant zebrafish ( Figure 3E; Figure 3—figure supplement 1A-B ) , as expected given our immunolabeling results ( Figure 2—figure supplement 1G-L ) .",
"The apparent chemical response in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants was blocked by extracellular application of a combination of the AMPA and NMDA glutamate receptor ( GluR ) antagonists cyanquixaline ( CNQX ) and D-2-Amino-5-phosphonovaleric acid ( DAP5 ) ( gray traces in Figure 3C , D; Figure 3—figure supplement 1C ) .",
"No change in membrane potential was observed after application of the blockers .",
"To confirm that the chemical synaptic potential observed in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants arises from the stimulation of CEs lacking electrical transmission , we examined if blocking GJs with meclofenamic acid ( MA ) could reproduce the observed synaptic phenotype .",
"We found that application of MA to wildtype recapitulated the gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutant phenotype , as the characteristic mixed synaptic response was replaced by a larger chemical synaptic response that was sensitive to GluR antagonists ( Figure 3F; Figure 3—figure supplement 1C ) .",
"We also observed a small hyperpolarization of the Mauthner cell ( from −80 . 2 ± 0 . 7 mV in control to −84 ± 1 mV in MA; p=0 . 04 , n = 5 ) , likely resulting from the action of MA on other membrane channels .",
"We conclude that together Cx35 . 5 and Cx34 . 1 generate functional neuronal GJs at CEs .",
"We next examined the properties of synaptic transmission in ZO1 mutants .",
"Strikingly , and consistent with the requirement for Connexin localization at contact sites ( Figure 1 ) , tjp1b/ZO1b-/- mutants exhibited a failure in electrical transmission .",
"The synaptic phenotype was indistinguishable from gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants , and similarly consisted of a single , delayed response that was blocked by GluRs antagonists ( Figure 3G; Figure 3—figure supplement 1D ) .",
"This functional deficit was specific for ZO1b , as tjp1a/ZO1a-/- mutant fish exhibited normal mixed synaptic responses ( Figure 3H , I ) .",
"These findings indicate the specificity of the mutants to electrical synapses as glutamatergic transmission at CEs remained intact .",
"Accordingly , neurotransmitter release properties ( Figure 3—figure supplement 1E ) and the localization of GluR2/3 receptors at these terminals were not affected in mutant fish ( Figure 3—figure supplement 2 ) .",
"We conclude that the presence of the ZO1b scaffold protein is critical for electrical transmission at CEs , even though its absence does not prevent the formation of glutamatergic synapses coexisting within the same contact .",
"To investigate the extent of the deficit on electrical transmission within the brainstem network , we investigated whether Connexin and ZO1 mutations affected other synaptic contacts onto the Mauthner cell .",
"The Mauthner cell receives mixed synaptic inputs from a variety of descending and ascending sensory modalities , including visual information from the optic tectum and somatic information from the spinal cord ( Dunn et al . , 2016; Faber and Pereda , 2011; Kimmel et al . , 1981; Korn and Faber , 2005 ) .",
"For this purpose , we recorded spontaneous synaptic responses that represent the activity of most , if not all , synaptic inputs received by this cell .",
"Electrically and chemically mediated spontaneous responses are easily differentiated due to their dramatically different duration ( Figure 4A ) .",
"Thus , for automated detection purposes , we defined fast spontaneous events ( <1 . 1 ms ) , which represent the electrical coupling of presynaptic spikes , as electrical responses and slow spontaneous events ( >1 . 1 ms ) as chemical responses .",
"Fast events were nearly absent in tjp1b/ZO1b-/- , gjd2a/Cx35 . 5-/- , and gjd1a/Cx34 . 1-/- mutants ( Figure 4B , C ) , and were dramatically reduced in wild-type fish by application of MA ( Figure 4C ) , confirming that they represent electrical transmission .",
"Slow events that remained after losing the electrical component were dramatically reduced by application of GluRs antagonists ( Figure 4D ) , confirming that they represent chemical responses .",
"We conclude that the deficit in electrical transmission observed in mutant zebrafish is likely to be widespread within hindbrain and spinal cord circuits .",
"Given the primary role of the Mauthner cell in triggering escape responses , we also investigated possible changes in cellular excitability in mutant fish .",
"Interestingly , the frequency of slow , chemical responses was increased in mutants and MA-treated animals ( Figure 4D ) , even though we observed no changes on presynaptic neurotransmitter release properties ( Figure 3—figure supplement 1E ) .",
"This suggests that the lack of electrical transmission could have enhanced the detection of smaller amplitude chemical responses .",
"We posit this could arise from electrical coupling influencing neuronal excitability , as the loss of neuronal GJs in mutants would increase the input resistance of the Mauthner cell ( Alcamí and Pereda , 2019 ) .",
"Thus , during whole cell recordings , we determined the input resistance ( Rin ) , rheobase , resting potential ( Vrest ) , and firing threshold ( Vthreshold ) of the Mauthner cells from wildtype and mutant zebrafish ( Table 1 ) .",
"The input resistance , the main determinant of neuronal excitability , was increased in tjp1b/ZO1b-/- , gjd2a/Cx35 . 5-/- , and gjd1a/Cx34 . 1-/- mutants .",
"Accordingly , the rheobase , a parameter negatively correlated with neuronal excitability , was decreased in these mutant animals ( Table 1 ) .",
"We found that the change in Rin correlated with the lack of electrical transmission and was not observed in fish that retained electrical transmission ( Figure 4E , F; note however that Rin in tjp1a/ZO1a-/- was found to slightly increase ) .",
"Differences in magnitude of the effects observed on Rin could be due to distinct compensatory mechanisms in each case or to the mutations affecting channels contributing to leak conductance in the Mauthner cell .",
"Thus , the lack of electrical transmission in tjp1b/ZO1b-/- fish rendered synaptic transmission exclusively mediated by a relatively delayed glutamatergic response and more excitable Mauthner cells .",
"Both deficits likely influence the behavioral responses generated by the Mauthner cell and its associated network .",
"Since tjp1b/ZO1b-/- mutants had functional deficits in electrical transmission , but not chemical , we assessed the consequences on the behavioral output of the Mauthner cell network .",
"Animals were placed into individual chambers of a multi-well testing stage and presented with acoustic-vibrational stimuli to elicit Mauthner-dependent startle responses ( Wolman et al . , 2015 ) .",
"Movements were captured with a high-speed camera ( 1000 frames per second ) and analyzed with FLOTE software to automatically track and measure the kinematics ( body movements ) of responses ( Burgess and Granato , 2007 ) .",
"In this paradigm , wild-type fish exhibit two types of escape responses: ( 1 ) Mauthner-cell-dependent short-latency C-bends ( SLCs , hereafter referred to as ‘startles’ ) and ( 2 ) Mauthner-cell-independent long-latency C-bends ( LLCs ) .",
"These two behavioral responses were automatically distinguished using well-established kinematic parameters ( Burgess and Granato , 2007 ) .",
"Larvae generated from crossing tjp1b/ZO1b+/- heterozygous animals were tested and analyzed blind to genotype with subsequent post-hoc identification .",
"We found that tjp1b/ZO1b-/- mutants startled to strong acoustic stimuli ( 25 . 9 dB ) as frequently as their wildtype siblings ( Figure 5A ) .",
"While these turns were classified as startles , we found that the tjp1b/ZO1b-/- mutants initiated their responses ~ 2 ms slower than their wildtype siblings ( Figure 5B ) .",
"A delayed behavioral response in consistent with our electrophysiological findings indicating that the mutant escape network operates with longer synaptic delays due to the lack of electrical transmission ( Figure 3 ) .",
"In mutant animals , we found that the escapes were often performed with the normal startle kinematic parameters , particularly the maximum angle of the turn and the maximum angular velocity of the response , albeit occurring later than in wild-type siblings ( Figure 5C–H ) .",
"However , we found a subset of mutant responses ( ~15% ) that showed abnormally shallow and slow turns ( Figure 5C , D red arrows ) .",
"These abnormal responses suggested deficits in performing the stereotyped C-bend elicited by the Mauthner-cell network , and so we reanalyzed the video data from these responses .",
"In these startles , we observed that the mutants displayed abnormal postures where the body would bend slightly to one side , creating ‘kinked’ or ‘S-shaped’ postures ( Figure 5I–L ) .",
"We note that the phenotypes observed in the tjp1b/ZO1b-/- mutants are strikingly similar to those we previously observed in gjd2a/Cx35 . 5-/- and gjd1a/Cx34 . 1-/- mutants ( Miller et al . , 2017 ) .",
"Such kinked body shapes are reminiscent of startles following CoLo neuron ablation ( Satou et al . , 2009 ) .",
"Based on these data , we conclude that electrical synapses are essential for generating the speed and coordination of the Mauthner-induced startle response .",
"The electrophysiological analysis of tjp1b/ZO1b-/- mutants also revealed that Mauthner cells showed increased excitability ( Figure 4 ) , suggesting animals may be hypersensitive with a lower threshold of response for environmental stimuli .",
"To examine this possibility , we presented larvae with 60 pseudo-randomized stimuli , 10 at six different intensities with a 20 s inter-stimulus interval to eliminate habituation ( Wolman et al . , 2015 ) .",
"We then assessed the frequency with which animals responded to the stimuli with turns ( Figure 5M–O ) .",
"As stimulus intensity increased , both wildtype siblings and tjp1b/ZO1b-/- mutants increased the likelihood of performing a startle response .",
"However , across the mid-range of stimulus intensities , tjp1b/ZO1b-/- mutants were more likely to respond with a startle than their wild-type siblings ( Figure 5M , O ) .",
"The increased tendency of mutants to perform Mauthner-dependent startles came at the expense of Mauthner-independent LLC responses , which were nearly absent in mutants ( Figure 5N ) .",
"We conclude that electrical synapses alter the sensitivity of Mauthner cells to environmental stimuli and contribute to the innate startle threshold , which alters the probability of eliciting a startle or LLC response .",
"These results lend additional evidence for the critical role of ZO1b for creating functional electrical synapses , ultimately contributing to appropriately balanced neural network function and behavior .",
"Mutant zebrafish revealed a hierarchical relationship between ZO1b and neuronal Connexins ( Figures 1 and 2 ) , so we next investigated the mechanisms underlying this relationship .",
"ZO1 is a membrane-associated guanylate kinase ( MAGUK ) scaffold protein and contains PSD95/Dlg/ZO1 ( PDZ ) protein-protein interaction domains that bind to PDZ binding motifs ( PBMs ) ( Zhu et al . , 2016 ) .",
"Previous work demonstrated that the C-terminal four amino acids of mouse Cx36 and perch Cx35 compose PBMs that are essential for interacting with ZO1 ( Flores et al . , 2008; Li et al . , 2004 ) .",
"Given that the PBM sequence is conserved in zebrafish Cx35 . 5 and Cx34 . 1 proteins ( Figure 6A ) , we tested whether these Connexins could mediate binding to zebrafish ZO1b .",
"We cloned full-length sequences of tjp1b/ZO1b , gjd2a/Cx35 . 5 , and gjd1a/Cx34 . 1 and used heterologous expression to test for interactions between the scaffold and Connexins .",
"HEK293T cells were co-transfected with mVenus-ZO1b and full-length Cx35 . 5 or Cx34 . 1 .",
"Using western blot analysis with antibodies specific to each Connexin , we found that both Cx34 . 1 and Cx35 . 5 were individually detected in mVenus-ZO1b immune complexes ( Figure 6B , lanes 1 , 3 ) compared to control immunoprecipitates ( Figure 6—figure supplement 1A , lanes 1 , 2 , 5 , 6 ) .",
"We further found that removing the presumptive PBMs , by deleting the C-terminal four amino acids from both Connexins , resulted in a loss of co-purifying Connexins from mVenus-ZO1b immunoprecipitates ( Figure 6B , lanes 2 , 4; Figure 6—figure supplement 1A , lanes 4 , 8 ) .",
"Control blots demonstrated the ability of the Connexin antibodies to equally recognize both full-length and ∆PBM versions of the proteins ( Figure 6B; Figure 6—figure supplement 1A , bottom input panels ) .",
"We conclude that zebrafish Cx35 . 5 and Cx34 . 1 can interact with ZO1b in a PBM-dependent manner .",
"The ZO1b scaffold has three PDZ domains that could mediate the interaction with neuronal Connexins ( Figure 6A ) .",
"Previous studies testing the three mammalian ZO1 PDZ domains demonstrated that Cx35/36 PBMs exclusively bound to ZO1-PDZ1 , while PDZ2 and PDZ3 did not interact ( Flores et al . , 2008; Li et al . , 2004 ) .",
"Given the high degree of conservation of the zebrafish ZO1 PDZ1 domain , including the amino acids of the putative PBM binding pocket ( Figure 6A ) , we tested whether zebrafish ZO1b-PDZ1 could directly interact with zebrafish neuronal Connexins .",
"To examine this question , we isolated the minimal domains of each zebrafish protein , produced them in bacteria , and performed in vitro binding studies .",
"We found that purified ZO1b-PDZ1 could be pulled down with a GST-Cx34 . 1 or a GST-Cx35 . 5 C-terminal intracellular-tail ( Figure 6C , lanes 2 , 4 ) , but not with control GST protein ( Figure 6C , lane1 ) .",
"Further , this interaction was significantly decreased when the predicted PBM in the Connexin tails were removed ( Figure 6C , lanes 3 , 5 ) .",
"We next used an overlay assay to compare the ability of ZO1b-PDZ1 and ZO1b-PDZ2 to bind to immobilized GST-Connexin tails .",
"Similar to the binding assays , significant amounts of ZO1b-PDZ1 bound to GST-Cx34 . 1 and GST-Cx35 . 5 tails in a PBM-dependent manner ( Figure 6—figure supplement 1B , left panels ) .",
"By contrast , little ZO1b-PDZ2 bound to the GST-Cx34 . 1 or GST-Cx35 . 5 tails , particularly when compared to the non-neuronal Cx43 C-terminal tail ( Figure 6—figure supplement 1B , right panels ) , which is known to bind PDZ2 ( Flores et al . , 2008; Li et al . , 2004 ) .",
"We conclude that ZO1b utilizes the PDZ1 domain to directly interact with the neuronal Connexin PBMs .",
"Since ZO1b and the neuronal Connexins colocalized at electrical synapses ( Figure 1 ) , we next investigated whether these proteins interacted in vivo .",
"We utilized adult zebrafish brains that maintain widespread electrical synapses , including those in Mauthner cell ( Kimmel et al . , 1981 ) , and provide an abundant source to derive ZO1b immunoprecipitates .",
"Homogenates derived from wild-type fish brains were immunoprecipitated with anti-ZO1 and control antibodies ( mIgG ) .",
"Immunoprecipitates demonstrated that Cx34 . 1 copurified with ZO1 , whereas Cx35 . 5 did not copurify with the anti-ZO1 antibody ( Figure 6D , lanes 1 , 2 ) .",
"To confirm that copurification of Cx34 . 1 was dependent upon ZO1b , we replicated the experiment using homogenates from tjp1b/ZO1b-/- mutants and found that Cx34 . 1 was lost in these immmunocomplexes ( Figure 6D , lane4 ) .",
"We conclude that ZO1 preferentially interacts with Cx34 . 1 in vivo .",
"We observed multiple ZO1 bands upon Western analysis of the immunoprecipitates , several of which were lost in the tjp1b/ZO1b-/- mutants ( Figure 6D , lanes 2 , 4 ) .",
"Since the ZO1 antibody was made against human protein , we reasoned it might be detecting the ZO1b protein and the highly similar ZO1a protein , so we sought evidence to confirm that ZO1b was the primary scaffold for Cx34 . 1 in vivo .",
"Upon examining ZO1 immunocomplexes from wildtype , tjp1b/ZO1b-/- , and tjp1a/ZO1a-/- brains , we found that only ZO1b deficiency resulted in concomitant loss of Cx34 . 1 ( Figure 6—figure supplement 1C ) .",
"Taken together , we conclude that ZO1b preferentially interacts with Cx34 . 1 in vivo , despite the fact that the scaffold can interact with either neuronal Connexin .",
"Next , we determined the functional relevance for a preferential ZO1b/Cx34 . 1 interaction at zebrafish electrical synapses .",
"We previously observed that Cx35 . 5 localizes presynaptically in axons , while Cx34 . 1 localizes postsynaptically in dendrites ( Miller et al . , 2017 ) .",
"Since ZO1b preferentially interacts with Cx34 . 1 in vivo ( Figure 6 ) , we speculated that ZO1b might share a similar dendritically compartmentalized localization .",
"To directly examine this , we visualized the localization of ZO1b protein after inserting a V5 epitope at the N-terminus of the endogenous tjp1b locus ( Figure 7—figure supplement 1A ) .",
"We found that in transgenicV5-tjp1b larvae , V5 antibody staining colocalized with both Cx34 . 1 and Cx35 . 5 at CEs and M/CoLo synaptic contacts ( Figure 7—figure supplement 1B–E ) .",
"Moreover , we found no effect on the stereotyped patterns of Connexin staining at Mauthner electrical synaptic contact sites in homozygous V5-tjp1b/V5-tjp1b animals ( Figure 7—figure supplement 1D , E ) , suggesting that V5-ZO1b is functional .",
"While V5-tjp1b larvae permitted ZO1b visualization at electrical synapses , we could not discriminate whether it was localized asymmetrically at synaptic contacts due to the small size of these structures and the resolution limits of light microscopy .",
"To overcome this , we utilized an alternate approach to address ZO1b compartmentalization , exploiting the fact that the Mauthner cell is both the postsynaptic partner at CEs and the presynaptic partner at the M/CoLo synapses ( Figure 7A ) .",
"We generated chimeric embryos by blastula transplantation ( Kemp et al . , 2009 ) , extracted GFP and V5-ZO1b expressing transgenic cells from donor embryos , and transferred them to wild-type hosts ( Figure 7B ) , allowing us to address V5-ZO1b localization within the Mauthner cell ( Figure 7C–E ) .",
"In animals containing only a donor-derived , V5-ZO1b-expressing Mauthner cell , we found that V5 staining was present at the CEs with Connexin staining , demonstrating that ZO1b was within the dendrite of the Mauthner cell and localized postsynaptically at electrical synapses ( Figure 7D ) .",
"Conversely , when we examined the M/CoLo synapses of these same embryos , we found that V5 staining was not present at these synaptic contacts despite the Mauthner cell expressing V5-ZO1b protein ( Figure 7E ) .",
"We note that in the non-chimeric , V5-ZO1b transgenic animals , V5 staining was observed at the M/CoLo synapses ( Figure 7—figure supplement 1C , E ) , suggesting that ZO1b at these synapses derives from the postsynaptic CoLo .",
"Our transplant experiments produce chimeric larvae in which only Mauthner , only CoLo , or both cells are derived from the transgenic donor embryos ( Figure 7—figure supplement 1F–H ) .",
"In larvae in which only CoLo expresses V5-ZO1b , we found that V5 staining is present at M/CoLo synapses ( Figure 7—figure supplement 1G ) , confirming ZO1b’s postsynaptic localization .",
"We conclude that ZO1b is robustly compartmentalized within the somato-dendritic compartment of the neuron and asymmetrically localized on the postsynaptic side of the electrical synapse .",
"We then addressed whether ZO1b functions postsynaptically to facilitate Connexin localization at the electrical synapse .",
"We utilized chimeric animals and again took advantage of Mauthner cell morphology .",
"However , in these experiments , we transplanted GFP-expressing cells from tjp1b/ZO1b-/- mutant donors into wildtype hosts , producing animals in which ZO1b was specifically removed from the Mauthner cell ( Figure 7F–I ) .",
"At CEs , the removal of ZO1b exclusively from the Mauthner cell resulted in the loss of staining for ZO1 , Cx34 . 1 , and Cx35 . 5 ( Figure 7H; Figure 7—figure supplement 1I ) .",
"The data indicates that ZO1b is required postsynaptically for the cell autonomous localization of Cx34 . 1 and non-autonomously for presynaptic Cx35 . 5 localization .",
"By contrast , when we examined the same chimeric animals with ZO1b removed from the Mauthner cell but focused on the M/CoLo synapses , in which Mauthner is presynaptic , there was no effect on either Connexin or ZO1 staining ( Figure 7I; Figure 7—figure supplement 1J ) , indicating that ZO1b is dispensable presynaptically .",
"As further support for this compartmentalized scaffold function , we reasoned that resupplying ZO1b to the Mauthner cell in an otherwise tjp1b/ZO1b-/- mutant animal would be sufficient to rescue Connexin localization at CEs , but not at M/CoLo synapses .",
"To test this prediction , we transplanted GFP-expressing cells from wildtype donor embryos into tjp1b/ZO1b-/- mutant hosts and identified animals with donor-derived Mauthner cells .",
"In such chimeras , where the tjp1b/ZO1b gene is functional only in the Mauthner cell , CEs had normal staining for both postsynaptic ZO1 and Cx34 . 1 and also for presynaptic Cx35 . 5 .",
"By contrast , there was no rescue of staining for these proteins at M/CoLo synaptic contacts ( Figure 7J , K; Figure 7—figure supplement 1J ) .",
"We conclude that ZO1b is both necessary and sufficient postsynaptically for building the structure of the neuronal GJ channels .",
"Taken together , we find that ZO1b is exclusively localized to the postsynaptic compartment where it functions both cell-autonomously and non-autonomously to localize Connexins and build functional neuronal gap junctions ."
],
[
"Despite the continuous nature of electrical transmission , the thousands of channels that make up GJ plaques found at electrical synapses are maintained by the active turnover of Connexin proteins ( Flores et al . , 2012 ) , similar to neurotransmitter receptors at chemical synapses ( Carroll and Zukin , 2002; Chen et al . , 2000; Ehlers , 2000; Lüscher et al . , 1999 ) .",
"Our findings show that ZO1b is required for the robust localization of Connexins , suggesting it functions to stabilize Connexin hemichannels at the synaptic site .",
"Additionally , ZO1 can localize to electrical synapses in the absence of the Connexins , although its apparent concentration was diminished .",
"This suggests that building robust neuronal GJ structures involves a reciprocal interaction between ZO1 and the Connexins .",
"Strikingly , our results reveal that ZO1b is compartmentalized to the dendrite and functions asymmetrically at postsynaptic sites of the Mauthner cell circuit .",
"This localization is consistent with ZO1b’s preferential in vivo interaction with Cx34 . 1 , as this Connexin is also localized and required postsynaptically at Mauthner cell electrical synapses ( Miller et al . , 2017 ) .",
"Despite the apparent autonomous ZO1b/Cx34 . 1 postsynaptic interaction at the GJ hemiplaque , tjp1b/ZO1b-/- mutants revealed that presynaptic Cx35 . 5 localization is also affected non-autonomously in the neighboring cell .",
"This trans-synaptic interaction likely occurs via the Connexins themselves , as mutations to the postsynaptic Cx34 . 1 prevented the robust localization of the presynaptic Cx35 . 5 .",
"Our results are complementary to recent analysis of the mouse rod/cone network , where removing Cx36 from one neuron of a coupled pair results in the failure of Connexin localization in the adjacent neuron ( Jin et al . , 2020 ) .",
"Taken together , our results reveal that ZO1b acts as a postsynaptic molecular scaffold that localizes Cx34 . 1 to the GJ hemiplaque , which in turn ensures Cx35 . 5 stabilization at presynaptic hemiplaques ( Figure 7C ) .",
"Whether presynaptic Cx35 . 5 lacks an in vivo scaffold , or utilizes another unidentified presynaptic scaffolding protein , remains unresolved .",
"If the molecular function and organization of ZO1 revealed here applies to all electrical synapses , including those formed by homotypic channels between various homologous cellular processes ( dendro-dendritic , somato-somatic , or axo-axonic ) , remains to be determined in future studies .",
"Never-the-less , we posit that the molecular organization of the electrical synapse can be asymmetrically compartmentalized , thereby enabling preferential biochemical interactions at each side of the junction .",
"Our results support the prediction that ZO1 likely plays distinct functional roles at GJs in different tissue types .",
"ZO1 was first described at epithelial tight junctions and later shown to interact with various Connexins , notably with Cx43 , a widespread Connexin expressed in many non-neuronal cell types ( Giepmans , 2004 ) .",
"Evidence from cell expression systems suggested that ZO1 played critical functions on the periphery of Cx43-contaning GJ plaques forming part of the ‘perinexus’ to facilitate newly inserted hemichannels at each side of the junction ( Rhett and Gourdie , 2012 ) .",
"Further , the ZO1/Cx43 interaction was critical for GJ communication before the channel ‘ages’ and is subsequently removed during channel turnover , a process governed by Cx43 phosphorylation ( Laird , 1996; Laird , 2006; Márquez-Rosado et al . , 2012; Solan and Lampe , 2016; Thévenin et al . , 2017 ) .",
"However , preventing the ZO1/Cx43 interaction does not prevent GJ formation ( Hunter and Gourdie , 2008; Hunter et al . , 2003; Hunter et al . , 2005 ) .",
"By contrast , our results demonstrated that ZO1b’s presence was asymmetric and required for robust Connexin localization and synaptic function .",
"Beyond our observations here , ZO1 is likely to have homeostatic functions in the modulation of Connexin usage at electrical synapses .",
"For example , analysis of the interactions between ZO1 and Cx36 ( and its fish homologs ) revealed the interactions occurs via a different PDZ domain than the interaction with Cx43 and the interaction with Cx36 has lower affinity and faster kinetics than that of Cx43 ( Flores et al . , 2008; Li et al . , 2004 ) .",
"This suggests ZO1 has a more dynamic interaction with neuronal Connexins , which may serve the plastic , activity-dependent regulation of electrical transmission observed in fish and mammalian electrical synapses ( Haas et al . , 2011; Landisman and Connors , 2005; Mathy et al . , 2014; Pereda and Faber , 1996; Pereda et al . , 1998; Turecek et al . , 2014; Yang et al . , 1990 ) .",
"Thus , our findings suggest that ZO1’s function at electrical synapses differs from its role at Cx43-containing GJs , perhaps serving a specialized function in synaptic communication .",
"Our results highlight that neural circuit function requires functional electrical and chemical synapses to create an appropriate behavioral response .",
"Our data indicates that the lack of electrical transmission in ZO1b and Connexin mutants did not prevent the formation of co-existing glutamatergic synapses at CEs on the Mauthner cell .",
"The remaining chemical transmission supported a behavioral response organized by the Mauthner-cell network , although importantly , the response showed deficits in performance and altered sensitivity .",
"Given that the startle behavior mediates predator avoidance , these defects would likely be detrimental to survival ( Hecker et al . , 2020 ) .",
"The lack of effect on glutamatergic transmission contrasts a wealth of data supporting a strong , interdependent relationship between the formation of electrical and chemical synapses during development in both invertebrate and vertebrate nervous systems ( Jabeen and Thirumalai , 2018 ) .",
"For example , in the leech , knockdown of Innexins at a developmental stage where synaptic contacts are solely electrically coupled prevents the formation of later-forming chemical transmission ( Todd et al . , 2010 ) .",
"Similarly , in the developing mouse neocortex , dominant negative constructs of Cx26 prevent the initial electrical coupling and subsequent chemical synapse formation amongst sister excitatory neurons within ontogenetic columns ( Yu et al . , 2012 ) .",
"Our findings indicate that this deficit does not occur at zebrafish CEs when Cx35 . 5 , Cx34 . 1 , and ZO1b are removed .",
"Whether this is due to the differences in the GJ proteins used in the Mauthner cell or instead due to mechanisms that specify CE formation , remains unknown .",
"One intriguing possibility is that other proteins may act as a common synaptic control mechanism that is independent of GJ-forming proteins .",
"Indeed , mutations in the scaffold Neurobeachin caused parallel defects in the formation of both electrical and chemical synapses of the Mauthner circuit ( Miller et al . , 2015 ) , yet the mechanism by which this coordination occurs remains to be elucidated .",
"Alternatively , rather than functional ( conductive ) GJ channels , other structural components of the electrical synapse may be sufficient to trigger chemical synapse formation via protein-protein interactions .",
"We posit such interactions would apply to mammalian electrical synapses , which can co-exist with neighboring glutamatergic synapses at distances comparable to those found in fish mixed synapses ( Nagy et al . , 2018 ) and may mediate similar functional interactions .",
"Identifying the functional relevance of an intracellular scaffolding protein as a critical part of the electrical synapse draws parallels with our understanding of chemical transmission , where neurotransmitter receptors are clustered and modified by a rich network of postsynaptic proteins that dynamically shape synaptic structure and function .",
"This chemical synapse protein network is known as the ‘postsynaptic density’ ( PSD ) , a term resulting from its structural identification by EM ( Cohen , 2013; Palay , 1956 ) and is composed of hundreds of unique proteins ( Grant , 2019 ) .",
"Mirroring those findings , EM images of electrical synapses in mammals ( Llinas et al . , 1974 ) and fish ( Brightman and Reese , 1969 ) revealed the presence of clearly identifiable electrodense structures , first described as ‘semi-dense material’ by Sotelo and Korn , 1978 .",
"These electrodense structures are localized intracellularly and form an undercoating band at neuronal GJs .",
"The identified structures are presumably formed by a proteinaceous ‘organelle’ that resembles that found at PSDs ( Feng et al . , 2019 ) .",
"As evidence grows for an array of proteins localized to electrical synapses , we propose that ZO1 is member of such an organelle .",
"Our data provide enticing hints that the molecular framework of the electrical synapse extends beyond the ZO/Connexin interaction .",
"In particular , the fact that ZO1 can localize to sites of synaptic contact independent of the Connexins , and that there remains immunofluorescent staining at Mauthner cell synapses in tjp1b/ZO1b-/- mutants , both imply the existence of additional proteins that contribute to this synaptic structure .",
"We presume the remaining ZO1 staining in tjp1b/ZO1b-/- mutants comes from either the paralogous ZO1a protein ( tjp1a ) or from the related ZO2 ( tjp2a , tjp2b ) and ZO3 ( tjp3 ) proteins .",
"However , our previous analysis of ZO2/ZO3 mutants in zebrafish did not reveal overt defects in Connexin localization ( Marsh et al . , 2017 ) , yet both proteins are localized to mammalian electrical synapses ( Li et al . , 2009 ) .",
"Whether these related scaffolds have functional roles at electrical synapses that were undetected in our initial screen remains to be determined .",
"Beyond the ZO-family , other molecules can directly interact with Cx36 and/or localize at mammalian electrical synapses , such as cell adhesion molecules , cytoskeletal interacting proteins , and molecules that regulate Connexin post-translational modifications ( Lynn et al . , 2012; Martin et al . , 2020; O'Brien and Bloomfield , 2018 ) .",
"Yet , the molecular roles of these proteins at the electrical synapse remain poorly defined .",
"Therefore , as opposed to simple aggregates of intercellular channels , we propose that electrical synapses are complex synaptic structures at which communicating pre- and postsynaptic Connexin hemichannels are governed by a yet to be determined mechanism that builds an asymmetric molecular scaffold .",
"Based on its analogy to the known functions of glutamatergic PSDs , we propose to name this organizational organelle the ‘electrical synapse density’ , or ‘ESD’ ( Lynn et al . , 2012; Miller and Pereda , 2017; Figure 7C ) .",
"Chemical synapses are organized to match their specific functional requirements by combining presynaptic release properties with unique combinations of postsynaptic receptors .",
"Electrical synapses also provide a variety of specific synaptic functions , yet we know little about the source of their diversity .",
"Work in C . elegans exposed the large variety of Innexin expression in neurons contributing to neural circuits , with dozens of potentially unique cellular combinations that are altered during development and following environmental stress ( Bhattacharya et al . , 2019 ) .",
"These observations have greatly increased the appreciation of the incidence and potential functional diversity of electrical transmission in invertebrates .",
"Our results indicate that the complexity of electrical synaptic transmission must also include their molecular scaffolds .",
"Scaffold diversity may be more relevant for the function of vertebrate electrical synapses , which in contrast to C . elegans , are formed by a smaller number of GJ-forming proteins , with most electrical communication being reliant on Cx36-related proteins investigated here .",
"By promoting and regulating channel trafficking and regulatory molecules , ZO1 , and other scaffolding proteins , could support a variety of functions by governing channel function and the local regulatory environment .",
"Thus , future investigations will further expose the molecular composition and functional roles of ZO1 and the ESD .",
"Unraveling the functional complexity of the ESD will lead to a deeper understanding of the diversity of the molecular organization underlying electrical transmission and its contributions to brain function ."
],
[
"Fish were maintained in the University of Oregon’s and the Albert Einstein College of Medicine fish facilities with approval from Institutional Animal Care and Use Committees of each institution .",
"Zebrafish , Danio rerio , were bred and maintained at 28°C on a 14 hr on and 10 hr off light cycle .",
"Animals were housed in groups , generally of 25 animals per tank .",
"Development time points were assigned via standard developmental staging ( Kimmel et al . , 1995 ) .",
"All fish used for this project were maintained in the ABC background developed at the University of Oregon .",
"Most fish had the enhancer trap transgene zf206Et ( M/CoLo:GFP ) in the background ( Satou et al . , 2009 ) , unless otherwise noted .",
"Mutant lines were genotyped for all experiments .",
"All immunohistochemistry , electrophysiological , and behavioral experiments were performed at 5 dpf .",
"At this stage of development , zebrafish sex is not yet determined ( Wilson et al . , 2014 ) .",
"Protein extractions were performed from both male and female adult brains and combined .",
"HEK293T/17 verified cells were purchased from ATCC ( CRL-11268; STR profile , amelogenin: X ) .",
"Cells were expanded and maintained in Dulbecco's Modified Eagle's Medium ( DMEM , ATCC ) plus 10% fetal bovine serum ( FBS , Gibco ) at 37°C in a humidified incubator in the presence of 5% CO2 .",
"Low passage aliquots were cryopreserved and stored according to manufacturer’s instructions .",
"Cells from each thawed cryovial were monitored for mycoplasma contamination using the Universal Mycoplasma Detection Kit ( ATCC , 30–1012K ) .",
"Anesthetized , 5–6 days post fertilization ( dpf ) larvae were fixed for 3 hr in 2% trichloroacetic acid in PBS .",
"Fixed tissue was washed in PBS + 0 . 5% Triton X-100 , followed by standard blocking and antibody incubations .",
"Primary antibody mixes included combinations of the following: rabbit anti-Cx35 . 5 ( Fred Hutch Antibody Technology Facility , clone 12H5 , 1:800 ) , mouse IgG1 anti-Cx35 . 5 ( Fred Hutch Antibody Technology Facility , clone 4B12 , 1:250 ) , rabbit anti-Cx34 . 1 ( Fred Hutch Antibody Technology Facility , clone 3A4 , 1:250 ) , mouse IgG2A anti-Cx34 . 1 ( Fred Hutch Antibody Technology Facility , clone 5C10A , 1:350 ) , mouse IgG1 anti-ZO1 ( Invitrogen , 33–9100 , 1:350 ) , mouse IgG2a anti-V5 peptide ( Invitrogen , R960-25 , 1:50 ) , and chicken anti-GFP ( abcam , ab13970 , 1:350- 1:500 ) .",
"All secondary antibodies were raised in goat ( Invitrogen , conjugated with Alexa-405 , –488 , −555 , or −633 fluorophores , 1:500 ) .",
"Tissue was then cleared stepwise in a 25% , 50% , 75% glycerol series , dissected , and mounted in ProLong Gold antifade reagent ( ThermoFisher , P36930 ) .",
"Images were acquired on a Leica SP8 Confocal using a 405-diode laser and a white light laser set to 499 , 553/554/557 ( consistent within experiments ) , and 631 nm , depending on the fluorescent dye imaged .",
"Each laser line’s data was collected sequentially using custom detection filters based on the dye .",
"Quantitative images of the Club Endings ( CEs ) were collected using a 63x , 1 . 40 numerical aperture ( NA ) , oil immersion lens , and images of M/Colo synapses were collected using a 40x , 1 . 20 NA , water immersion lens .",
"For each set of images , the optimal optical section thickness was used as calculated by the Leica software based on the pinhole , emission wavelengths , and NA of the lens .",
"Within each experiment where fluorescence intensity was to be quantified , all animals ( including 3–5 wildtype controls ) were stained together with the same antibody mix , processed at the same time , and all confocal settings ( laser power , scan speed , gain , offset , objective , and zoom ) were identical .",
"Multiple animals per genotype were analyzed to account for biological variation .",
"To account for technical variation , fluorescence intensity values for each region of each animal were an average across multiple synapses .",
"For high-contrast imaging of the CEs , fixed samples were washed three times with PBS , incubated at 4°C overnight , and hindbrains were dissected out .",
"Dissected hindbrains were washed , blocked , and stained as above with the following primary antibodies: rabbit anti-Cx35 . 5 ( 12H5 , 1:500 ) , mouse anti-Cx35/36 ( EMD Millipore , MAB3045 , 1:250 ) , mouse IgG2A anti-Cx34 . 1 ( 5C10A , 1:200 ) , mouse IgG1 anti-ZO1 ( 33–9100 , 1:200 ) , rabbit anti-GFP ( Invitrogen , G10362 , 1:200 ) , and chicken anti-GFP ( Invitrogen , A10262 , 1:200 ) .",
"Secondary antibodies were raised in goat ( Invitrogen , conjugated with Alexa-405 , –488 , −546 , –555 , −633 , or −647 fluorophores , 1:200 ) .",
"Samples were then transferred onto a slide in the dark and mounted with Fluoromount-G ( Southern Biotech , 0100–01 ) , covered using the standard ‘bridge’ procedure , and sealed with nail polish .",
"Samples were imaged on LSM 710 and LSM 880 Zeiss microscopes using appropriate laser wavelengths and detection filters .",
"Image stacks of roughly 20 µm were collected using a 63x , 1 . 40 numerical aperture ( NA ) , oil immersion lens .",
"For each Mauthner , laser strengths and gains were adjusted to achieve maximum visualization of CE staining .",
"Electrophysiological responses were obtained during whole-cell recordings of Mauthner cells ( M-cells ) in wt , Cx and ZO-1 mutant zebrafish larvae ( 5–7 dpf ) .",
"For this purpose , fish were first anesthetized with a 0 . 03% solution of MS222 ( pH adjusted to 7 . 4 with NaHCO3 ) and later transferred to external solution containing d-tubocurarine ( 10 μM , Sigma ) .",
"The external solution ( in mM ) : 134 NaCl , 2 . 9 KCl , 2 . 1 CaCl2 , 1 . 2 MgCl2 , 10 HEPES , 10 Glucose , pH adjusted to 7 . 8 with NaOH ( Yao et al . , 2014 ) .",
"Zebrafish larvae were put on their backs onto a Sylgard-coated small culture dish ( FluoroDish , WPI ) and kept in place using fine tungsten pins .",
"The hindbrain was then exposed ventrally following the dissection approach previously described ( Koyama et al . , 2011 ) .",
"Following this procedure , the larvae were placed on an Axio Examiner upright microscope ( Carl Zeiss AG ) equipped with a recording set-up and superfused with external solution during the entire recording session .",
"The M-cells were identified by GFP expression and/or far-red DIC optics .",
"Patch pipettes ( 3–4 MΩ ) were filled with internal solution ( in mM ) : 105 K-Methanesulfonate , 10 HEPES , 10 EGTA , 2 MgCl2 , 2 CaCl2 , 4 Na2ATP , 0 . 4 Tris-GTP , 10 K2-Phosphocreatine , 25 mannitol , pH adjusted to 7 . 2 with KOH .",
"Whole-cell recordings under the current-clamp configuration were performed with a Multiclamp 700B amplifier and a Digidata 1440A ( Molecular Devices ) digitizer .",
"The liquid-liquid junction potential was estimated in −16 mV using Clampex 10 . 6 ( Molecular Devices ) and was subtracted from the measured values .",
"The electrode’s resistance was compensated using the bridge balance feature of the amplifier .",
"To activate the auditory afferents terminating as CEs on the M-cell , a septated ( theta ) glass pipette was filled with external solution and positioned near the posterior macula of the ear , where the dendritic processes of auditory afferents contact the hair cells ( Yao et al . , 2014 ) .",
"The maximal amplitude of the electrical and chemical components was estimated by applying shocks of increasing intensity until the amplitude of the electrical component did not further increase and before additional responses with longer latency were evoked .",
"To estimate the Paired-Pulse Ratio ( PPR ) of the chemical component one , a stimulating-pulse was applied to record 10–20 traces in basal conditions .",
"Then two-stimulating pulses ( 2 ms apart ) were applied to record ( 10–20 traces ) facilitation of the chemical component .",
"Traces that clearly showed a chemical component were averaged for basal and facilitated conditions .",
"The trace in basal conditions was subtracted from the facilitated trace .",
"The PPR was then calculated using the amplitude of the chemical component of the facilitated trace divided by the amplitude of the chemical component in basal conditions .",
"Spontaneous electrical and chemical synaptic responses were assessed during offline analysis of continuous ( 10 s long ) recordings using Clampfit ( Axon instruments ) to automatically identify events based on their duration ( <1 . 1 ms for electrical spontaneous events and >1 . 1 ms for chemical spontaneous events ) .",
"Potential erroneous identification of spontaneous events by the software was monitored manually by verifying the duration of the events .",
"The assignment of electrical vs . chemical nature of spontaneous synaptic events by their duration was confirmed pharmacologically .",
"For synaptic transmission blockade , CNQX was first dissolved in DMSO to have a stock solution of 10 mM .",
"The pharmacological agents used to block synaptic transmission were added to the external solution: Meclofenamic Acid ( 200 μM , Sigma ) , CNQX and DAP5 ( 20 μM , Tocris Biosciences ) .",
"The M-cell input resistance was estimated by applying a hyperpolarizing-current step of −1 nA and 20 ms in duration and measuring the voltage deflection caused , followed by derivation of resistance with Ohm’s law .",
"The rheobase , defined as the minimum depolarizing current of infinite duration necessary to evoke an action potential , was determined by delivering a 20 ms current pulse of increasing intensity .",
"Voltage threshold for action potential generation was determined by applying a depolarizing-current step .",
"Startle behavior of 5dpf larvae was analyzed as described previously ( Marsden et al . , 2018 ) .",
"Briefly , larvae were adapted to the testing temperature and lighting conditions for 30 min and then transferred to individual wells of a custom , laser-cut acrylic multi-well testing arena , illuminated from below with an infrared ( IR ) LED array and from above with a white light LED bulb to simulate daylight conditions .",
"A total of 60 acoustic stimuli , 10 at each of 6 intensities , were delivered pseudorandomly using an acoustic-vibrational shaker ( Bruel and Kjaer ) with an inter-stimulus interval of 20 s to eliminate habituation to repeated stimulation ( Wolman et al . , 2015 ) .",
"The intensity of each stimulus was calibrated using a PCB Piezotronics accelerometer ( model #355B04 ) and signal conditioner ( model #482A21 ) , and voltage outputs were converted to dB using the formula dB = 20 log ( V/0 . 775 ) .",
"Behavioral responses were captured at 1000 frames per second with an IR-sensitive Photron mini-UX50 high-speed camera .",
"After testing , larvae were fixed in methanol for subsequent genotyping , thus all testing and analysis was performed blind to genotype .",
"Behavioral responses were tracked and analyzed using FLOTE software ( Burgess and Granato , 2007 ) , with short and long latency C-bend responses ( SLCs and LLCs , respectively ) automatically defined based on the kinematic parameters of the response .",
"Startle sensitivity index was calculated by measuring the area under the curve of stimulus intensity versus SLC frequency for each larva .",
"Cell lines were obtained from ATCC , identity ensured by using exclusively low-passage cells , and were confirmed to be mycoplama free .",
"Full-length Cx34 . 1 and full-length Cx35 . 5 were cloned into the pCMV expression vector .",
"Full-length ZO1b was cloned into the pCMV expression vector with an NH2-terminal mVenus tag and a COOH-terminal 8xHIS tag .",
"Low passage HEK293T/17 cells were seeded 24 hr prior to transfection ( 1 × 106 cells/well of a six-well dish ) , and the indicated plasmids were co-transfected using Lipofectamine 3000 ( Invitrogen ) following the manufacturer’s instructions .",
"Cells were collected 36–48 hr post-transfection and lysed in 0 . 25 ml solubilization buffer ( 50 mM Tris [pH7 . 4] , 100 mM NaCl , 5 mM EDTA , 1 . 5 mM MgCl2 , 1 mM DTT and 1% Triton X-100 ) plus a protease inhibitor cocktail ( Pierce ) .",
"Lysates were centrifuged at 20 , 000 x g for 30 min at 4°C , and equal amounts of extract were immunoprecipitated with 0 . 5 ug rabbit anti-GFP ( Abcam , Ab290 ) overnight with rocking at 4°C .",
"Immunocomplexes were captured with 25 µl prewashed Protein A/G agarose beads for 1 hr with rocking at 4°C .",
"Beads were washed three times with lysis buffer , and bound proteins were boiled for 3 min in the presence of LDS-PAGE loading dye containing 200 mM DTT .",
"Samples were resolved by SDS-PAGE using a 4–15% gradient gel and analyzed by Western blot using the following primary antibodies: rabbit anti-GFP ( Abcam Ab290 ) , rabbit anti-Cx34 . 1 3A4-conjugated-680LT , and mouse anti-Cx35 . 5 4B12 .",
"Compatible near-infrared secondary antibodies were used for visualization with the Odyssey system ( LI-COR ) .",
"Brains from wildtype , tjp1a/ZO1a-/- , or tjp1b/ZO1b-/- euthanized adult fish ( 4–15 months old ) were removed , snap frozen in liquid nitrogen and stored at −80C until use .",
"Brains were homogenized in 1 ml of HSE buffer ( 20 mM Hepes [pH7 . 5] , 150 mM NaCl , 5 mM EDTA , 5 mM EGTA , and 1 mM DTT ) plus a protease inhibitor cocktail using a glass homogenizer .",
"Detergent was added to the homogenate ( final 2% octyl ß-D-glucopyranoside , Anatrace ) and solubilized overnight with rocking at 4°C .",
"Solubilized homogenate was cleared by centrifugation at 20 , 000 x g for 30 min at 4°C , then pre-cleared for 1 hr at 4°C with Protein A/G beads before immunoprecipitation .",
"The protein concentration for each homogenate was measured by Bradford assay .",
"Pre-cleared homogenates ( 2 mg/IP ) were immunoprecipitated with 0 . 5 µg mouse anti-ZO1 ( Life Technologies , 33–9100 ) , control mouse IgG ( Jackson ImmunoResearch ) , mouse anti-Cx34 . 1 5C10 , or mouse anti-Cx35 . 5 4B12 antibody overnight with rocking at 4°C .",
"Immunocomplexes were captured with 25 ul prewashed Protein A/G agarose beads for 1 hr with rocking at 4°C .",
"Beads were washed three times with HSE buffer , and bound proteins were boiled for 3 min in the presence of LDS-PAGE loading dye containing 200 mM DTT .",
"Immune complexes were examined by western analysis using the following primary antibodies: mouse anti-ZO1 , rabbit anti-Cx34 . 1 3A4-conjugated-680LT , rabbit anti-Cx35 . 5 12H5-conjugated-680LT , or mouse anti-Cx35 . 5 4B12 .",
"Compatible near-infrared secondary antibodies were used for visualization .",
"The Cx34 . 1-tail ( aa256-299 ) , Cx34 . 1-tail ∆PBM ( aa256-295 ) , Cx35 . 5-tail ( aa267-309 ) , and Cx35 . 5-tail ∆PBM ( aa267-305 ) were cloned into the pGEX expression vector allowing for an NH2-terminal GST tag .",
"ZO1b-PDZ1 ( aa105-207 ) and ZO1b-PDZ2 ( aa298-387 ) were cloned into a modified pET expression vector ( pBH ) to allow for an NH2-terminal 6xHis tag followed by a TEV cleavage site ( vectors kindly provided by Ken Prehoda ) .",
"Plasmids were transformed in E . coli BL21 ( DE3 ) cells and plated on selective LB plates .",
"Single colonies were picked to inoculate 2 ml starter cultures and grown overnight .",
"Overnight cultures were inoculated into 250 ml selective LB and grown for ~3 hr at 37°C with shaking until OD600 reached 0 . 8–1 followed by 4 hr induction with 0 . 4 mM IPTG .",
"Cell pellets were collected by centrifugation at 6000 RPM for 5 min at 4°C and frozen at −20°C until test samples confirmed expression .",
"Pellets were resuspended in sonication buffer ( 50 mM NaPO4 [pH7 . 4] , 300 mM NaCl , and 1 mM PMSF ) .",
"After adding a dash of lysozyme , the mixture was incubated on ice for 30 min .",
"Resuspended bacteria were sonicated on ice at 50% amplitude , 1 s/1 s pulse on/off , four times for 20 s .",
"Debris was cleared by centrifugation at 16 , 000 x g for 30 min at 4°C .",
"For GST fusions , supernatant was added to 200 ul pre-washed glutathione agarose resin and incubated overnight with rocking at 4°C .",
"Beads were washed three times with sonication buffer and stored at 4°C .",
"Purity and amount loaded onto resin was determined by SDS-PAGE followed by Coomassie stain .",
"For 6xHIS fusions , supernatant was brought to a final concentration of 20 mM imidazole and incubated with pre-washed His60 resin overnight with rocking at 4°C .",
"Resin was washed with sonication buffer containing 20 mM imidazole .",
"Protein was eluted from the resin with sonication buffer containing 250 mM imidazole .",
"The protein was concentrated and exchanged into imidazole-free buffer using an Amicon centrifugal filter unit ( 10K MWCO ) and stored at 4°C on ice .",
"Protein concentration was estimated by A205 ( https://spin . niddk . nih . gov/clore/ ) ( Anthis and Clore , 2013 ) , and purity was determined by SDS-PAGE followed by Coomassie stain .",
"Equal amounts of GST fusions ( 10 µl bed of resin ) were aliquoted and the storage buffer was removed .",
"To each sample 15 µl of 6xHIS-ZO1b-PDZ1 ( 7 mg/ml ) was added , gently mixed and incubated at room temperature for 15 min .",
"Resin was washed three times with cold wash buffer ( 50 mM NaPO4 [pH7 . 4] , 300 mM NaCl ) .",
"After the last wash , all buffer was removed and resin was resuspended in 10 µl LDS-PAGE dye with 200 mM DTT .",
"Samples were boiled for 3 min and resolved by SDS-PAGE using a 4–20% gradient gel .",
"Samples were analyzed by Western blot using rabbit anti-TEV cleavage site primary antibody ( ThermoFisher , PA1-119 ) and visualized with a compatible near-infrared secondary antibody .",
"A portion of the GST fusion resin was analyzed by Coomassie stain to demonstrate equal loading .",
"Equal amounts of GST fusion were resuspended in LDS-PAGE dye plus 200 mM DTT , boiled for 3 min , resolved by SDS-PAGE on a 4–15% gradient gel , transferred to nitrocellulose , and blocked with 5% milk in TBS ( 20 mM Tris [pH7 . 4] , 150 mM NaCl ) .",
"Blocked membranes were incubated with TBS-T buffer ( TBS + 0 . 1% Tween-20 ) alone ( mock overlay ) , or TBS-T containing 1 µM 6xHIS-ZO1b-PDZ1 or 1 uM 6xHIS-ZO1b-PDZ2 overnight with shaking at 4°C .",
"Membranes were processed for western analysis using rabbit anti-TEV cleavage site primary antibody and a compatible near-infrared secondary antibody to detect bound PDZ protein .",
"Equal loading of GST fusions was determined by Stain-Free imaging technology .",
"A single guide RNA ( sgRNA ) targeting the 5’ region of the endogenous tjp1b coding sequence ( sequence in Key Resources Table ) was designed using the CRISPRscan algorithm ( Moreno-Mateos et al . , 2015 ) and synthesized as previously described ( Shah et al . , 2015 ) .",
"The sgRNA was generated using the T7 megascript kit ( ThermoFisher , AMB13345 ) .",
"The V5-tjp1b single stranded donor oligo ( ssODN ) was designed to repair into the endogenous tjp1b locus and was synthesized complimentary to the coding strand .",
"The ssODN contained 40 bp homology arms which flanked an XbaI restriction site , V5 sequence , and a 5x glycine linker , respectively ( sequence in Key Resources Table ) .",
"Upon correct repair , the inserted sequence was designed to disrupt the endogenous sgRNA recognition site to prevent further double stranded breaks after repair .",
"Injection mixes were prepared in a pH 7 . 5 buffer solution of 300 mM KCl and 4 mM HEPES and contained a final concentration of 200 pg/nL ssODN , 200 pg/nL gRNA , and 1600 pg/nL Cas9 protein ( IDT , 1081058 ) .",
"Injection mixes were incubated at 37 °C for 5 min immediately prior to injection to promote formation of the Cas9 and sgRNA complex .",
"Finally , 1 nL of solution was injected into embryos at the one-cell stage .",
"Injected embryos were raised to adulthood and outcrossed to wild-type animals .",
"Animals carrying insertions were identified and verified using PCR and Sanger sequencing .",
"Cell transplantation was performed at the high stage approximately 3 . 3 hr into zebrafish development using standard techniques ( Kemp et al . , 2009 ) .",
"Embryos were chemically de-chorionated with protease ( Sigma Aldrich , 9036-06-0 ) prior to transplantation .",
"Cells were transplanted using a 50 mm wide glass capillary needle attached to an oil hydraulic .",
"For ‘V5-tjp1b+/- into wildtype’ transplants ( Figures 7C , D and Figure 7—figure supplement 1B-H ) cells from animals heterozygous for V5-tjp1b in the M/CoLo:GFP background were transplanted into non-transgenic wildtype hosts .",
"For ‘tjp1b-/- into wildtype’ transplants ( Figure 7G and H ) , genotyped animals homozygous for the tjp1bΔ16bp mutation in the M/CoLo:GFP background were crossed and progeny were transplanted into non-transgenic wildtype hosts .",
"For 'wildtype into tjp1b-/-' transplants ( Figure 7I and J ) , transgenic M/CoLo:GFP wildtype animals were crossed to use as donors , and non-transgenic , homozygous tjp1bΔ16bp animals were crossed to produce hosts .",
"Approximately 20 cells were deposited ∼10–15 cell diameters away from the margin , with a single donor embryo supplying cells to 3–5 hosts .",
"At 5 dpf , larvae were fixed in TCA and processed for immunohistochemistry .",
"For fluorescence intensity quantitation , confocal images were processed and analyzed ( in part or in full ) using FiJi ( Schindelin et al . , 2012 ) software .",
"To quantify staining at M/Colo synapses , a standard region of interest ( ROI ) surrounding each M/CoLo site of contact was drawn , and the mean fluorescence intensity was measured .",
"For the quantification of staining at the club endings , confocal z-stacks of the Mauthner soma and lateral dendrite were cropped to 36 . 08 µm x 36 . 08 µm centered around the lateral dendritic bifurcation .",
"Using the SciPy ( Virtanen et al . , 2020 ) and scikit-image ( van der Walt et al . , 2014 ) computing packages , the cropped stack was then cleared outside of the Mauthner cell , a 33 median filter was applied to reduce noise , and a standard threshold was set within each experiment to remove background staining .",
"The image was then transformed into a max intensity projection and the integrated density of each stain within the Mauthner cell was extracted .",
"Where counts were used to quantify antibody labeling of CEs in high-contrast images , CEs were identified as large fluorescently labeled oval areas of approximately 1 . 5–2 microns on the distal portion and fork of the Mauthner cell’s lateral dendrite , which were thoroughly examined with the confocal microscope .",
"For Figure 7—figure supplement 1I , J , the presence or absence of electrical synapses on Mauthner was quantified as counts of stereotyped electrical synapse structures dually labeled for Cx34 . 1 and Cx35 . 5 .",
"Standard deviations and errors were computed using Prism ( GraphPad ) or Excel ( Microsoft ) software .",
"Figure images were created using FiJi , Photoshop ( Adobe ) , and Illustrator ( Adobe ) .",
"Statistical analyses were performed using Prism ( GraphPad ) and either an unpaired t-test with Welch’s correction or a one-way analysis of variance with Bonferrroni’s multiple comparison test was performed .",
"For all experiments , values were normalized to wildtype control animals , and n represented the number of fish used .",
"Fish were excluded from analysis if Mauthner morphology/GFP staining was abnormal .",
"Electrophysiology traces of spontaneous synaptic events were analyzed using Clampfit 10 . 7 software .",
"Electrophysiology traces were transferred to Canvas for illustration and to OriginPro software for quantification and statistical analyses .",
"For the different parameters measured using electrophysiological recordings , means and standard error of the mean ( SEM ) were illustrated using Prism ( GraphPad ) and computed using OriginPro software .",
"For statistical analyses comparing electrophysiological recordings in wildtype and mutant zebrafish , Mann-Whitney and Kruskal-Wallis non-parametric tests were performed using OriginPro software ."
]
] | [
"Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins .",
"While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins , it is unknown if channels at electrical synapses require similar scaffold support .",
"Here , we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 ( ZO1 ) , an intracellular scaffolding protein localized to electrical synapses .",
"Using model electrical synapses in zebrafish Mauthner cells , we demonstrated that ZO1 is required for robust synaptic Connexin localization , but Connexins are dispensable for ZO1 localization .",
"Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses .",
"We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels .",
"Thus , forming functional neuronal gap junctions requires a postsynaptic scaffolding protein .",
"The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses ."
] | [
"Neurons ‘talk’ with each another at junctions called synapses , which can either be chemical or electrical .",
"Communication across a chemical synapse involves a ‘sending’ neuron releasing chemicals that diffuse between the cells and subsequently bind to specialized receptors on the receiving neuron .",
"These complex junctions involve a large number of well-studied molecular actors .",
"Electrical synapses , on the other hand , are believed to be simpler .",
"There , neurons are physically connected via channels formed of ‘connexin’ proteins , which allow electrically charged ions to flow between the cells .",
"However , it is likely that other proteins help to create these structures .",
"In particular , recent evidence shows that without a structurally supporting ‘scaffolding’ protein called ZO1 , electrical synapses cannot form in the brain of a tiny freshwater fish known as zebrafish .",
"As their name implies , scaffolding proteins help cells organize their internal structure , for example by anchoring other molecules to the cell membrane .",
"By studying electrical synapses in zebrafish , Lasseigne , Echeverry , Ijaz , Michel et al . now show that these structures are more complex than previously assumed .",
"In particular , the experiments reveal that ZO1 proteins are only present on one side of electrical synapses; despite their deceptively symmetrical anatomical organization , these junctions can be asymmetric , like their chemical cousins .",
"The results also show that ZO1 must be present for connexins to gather at electrical synapses , whereas the converse is not true .",
"This suggests that when a new electrical synapse forms , ZO1 moves into position first: it then recruits or stabilizes connexins to form the channels connecting the two cells .",
"In many animals with a spine , electrical synapses account for about 20% of all neural junctions .",
"Understanding how these structures form and work could help to find new treatments for disorders linked to impaired electrical synapses , such as epilepsy ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology",
"genetics and genomics"
] | Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2 | elife-72375-v2 | [
[
"N6-methyladenosine ( m6A ) is the most abundant modified nucleotide in eukaryotic mRNA bodies .",
"It is required for embryonic development and stem cell differentiation in several animals and plants ( Zhong et al . , 2008; Batista et al . , 2014; Ping et al . , 2014; Geula et al . , 2015; Zhang et al . , 2017 ) and for the control of the meiotic program in yeast ( Shah and Clancy , 1992; Clancy et al . , 2002; Agarwala et al . , 2012 ) .",
"Most N6-adenosine methylation of mRNA is catalyzed in the nucleus ( Salditt-Georgieff et al . , 1976; Ke et al . , 2017; Huang et al . , 2019 ) by a highly conserved , multimeric methylase ( the m6A ‘writer’; Balacco and Soller , 2019 ) whose catalytic core consists of the heterodimer METTL3/METTL14 ( MTA/MTB in plants; Bokar et al . , 1997; Zhong et al . , 2008; Liu et al . , 2014 ) .",
"In addition , a number of highly conserved proteins is required for N6-methylation in vivo ( Balacco and Soller , 2019 ) .",
"The strong conservation of these core factors suggests that the biochemical basis of N6-adenosine methylation is common in eukaryotes , and indeed , m6A occurs in the consensus site RR ( m6A ) CH ( R = G/A , H = A/C/U ) , primarily in 3′-UTRs in vertebrates , plants , and fungi that possess the canonical METTL3/METTL14 methyltransferase ( Bodi et al . , 2012; Dominissini et al . , 2012; Meyer et al . , 2012; Schwartz et al . , 2013; Luo et al . , 2014a; Zhao et al . , 2017; Miao et al . , 2020; Parker et al . , 2020 ) .",
"Conversely , the characteristic motif and gene body location is not detected in organisms that lack METTL3/METTL14 homologs , such as the nematode Caenorhabditis elegans ( Sendinc et al . , 2020 ) and bacteria ( Deng et al . , 2015 ) .",
"m6A may impact mRNA function by different mechanisms , including the creation of binding sites for reader proteins that specifically recognize m6A in mRNA ( Dominissini et al . , 2012; Fu et al . , 2014; Meyer and Jaffrey , 2014 ) .",
"The best understood class of readers contains a so-called YT521-B homology ( YTH ) domain ( Stoilov et al . , 2002 ) of which two phylogenetic groups , YTHDF and YTHDC , have been defined ( Patil et al . , 2018; Balacco and Soller , 2019 ) .",
"The YTH domain harbors a hydrophobic methyl-binding pocket that increases the affinity of m6A-containing RNA by more than 10-fold compared to unmethylated RNA ( Li et al . , 2014b; Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014; Zhu et al . , 2014 ) .",
"Apart from interactions with the methylated adenosine and the purine at the –1 position , YTH domain-RNA interactions mostly involve the sugar-phosphate backbone of the RNA ( Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014 ) .",
"That is consistent with only mild reductions in the binding affinity of the YTH domain of human YTHDC1 upon substitution of nucleotides −2 , +1 , and +3 that abrogate the canonical RR ( m6A ) CH motif ( Xu et al . , 2014 ) , and poor sequence specificity of RNA binding by isolated YTH domains of human YTHDF1 , YTHDF2 , and YTHDC1 ( Arguello et al . , 2019 ) .",
"Thus , the methyltransferase complex gives the sequence specificity , while YTH domain proteins may bind to m6A-containing RNA regardless of the identity of the immediately adjacent nucleotides .",
"YTHDF proteins are typically cytoplasmic and consist of a long N-terminal intrinsically disordered region ( IDR ) followed by the globular YTH domain ( Patil et al . , 2018 ) .",
"Because the affinity of isolated YTH domains for m6A-containing RNA is modest , typically with dissociation constants on the order of 0 . 1–1 μM ( Li et al . , 2014b; Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014; Zhu et al . , 2014 ) , it has been suggested that the IDR may participate in RNA binding ( Patil et al . , 2018 ) .",
"Nonetheless , the clearest evidence for functions of the IDRs in YTHDF proteins reported thus far includes direct interactions with effectors such as the CCR4-NOT complex in mammalian cells ( Du et al . , 2016 ) , and the ability to cause liquid-liquid phase transition when sufficiently high local concentrations are reached ( Arribas-Hernández et al . , 2018; Gao et al . , 2019; Ries et al . , 2019; Fu and Zhuang , 2020; Wang et al . , 2020 ) .",
"The YTHDF family comprises 11 proteins in Arabidopsis that are referred to as EVOLUTIONARILY CONSERVED C-TERMINAL REGION1-11 ( ECT1-11 ) ( Li et al . , 2014c; Scutenaire et al . , 2018 ) .",
"ECT2 , ECT3 , and ECT4 are expressed in rapidly dividing cells of root , leaf , and flower primordia , and genetic analyses have revealed their general importance in organogenesis ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .",
"Importantly , the biological functions of ECT2/ECT3/ECT4 described thus far are shared with those of m6A writer components and , where tested , have been shown to depend on intact m6A-binding pockets , strongly suggesting that the basis for the observed phenotypes in ect2/ect3/ect4 mutants is defective regulation of m6A-modified mRNA targets ( Bodi et al . , 2012; Shen et al . , 2016; Ružička et al . , 2017; Arribas-Hernández et al . , 2018; Scutenaire et al . , 2018; Wei et al . , 2018; Arribas-Hernández et al . , 2020 ) .",
"Despite the progress in identifying biological functions of plant m6A-YTHDF axes , a number of fundamental questions regarding their molecular basis remains unanswered .",
"For example , it is unclear whether sequence determinants in addition to m6A are important for mRNA target association of ECT proteins in vivo , the mRNA targets of ECT2/ECT3/ECT4 responsible for the developmental delay of ect2/ect3/ ( ect4 ) mutants have not been identified , and it is not clear what the effects of ECT2/ECT3/ECT4 binding to them may be ( Arribas-Hernández and Brodersen , 2020 ) .",
"Clearly , robust identification of the mRNA targets directly bound by ECT proteins is key to obtain satisfactory answers to all of these questions .",
"Towards that goal , formaldehyde crosslinking and immunoprecipitation ( FA-CLIP ) was used to identify mRNA targets of ECT2 ( Wei et al . , 2018 ) .",
"Nonetheless , because formaldehyde , in contrast to UV illumination , generates both protein-protein and protein-RNA crosslinks , it is not an ideal choice for identification of mRNAs bound directly by a protein of interest ( see Arribas-Hernández and Brodersen , 2020 for a discussion ) .",
"In particular , this problem concerns the unexpected conclusion that ECT2 binds to the ‘plant-specific consensus motif’ URU ( m6A ) Y ( Y = U/C ) , not RR ( m6A ) CH ( Wei et al . , 2018 ) .",
"Thus , the field of gene regulation via m6A-YTHDF modules in plants is in a state of confusion: on the one hand , m6A mapping ( Luo et al . , 2014a; Wan et al . , 2015; Shen et al . , 2016; Duan et al . , 2017; Anderson et al . , 2018; Miao et al . , 2020; Parker et al . , 2020 ) and phenotypes of mutants defective in m6A writing ( Bodi et al . , 2012; Shen et al . , 2016; Ružička et al . , 2017 ) or m6A binding of ECT2/ECT3/ECT4 ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) suggest that these YTHDF proteins should act via recognition of m6A in the RRACH context .",
"On the other hand , the only attempt at a mechanistic understanding of ECT2 function via mRNA target identification concluded that ECT2 binds to a sequence element different from RRACH ( Wei et al . , 2018 ) .",
"To complicate matters further , a number of motifs including not only URUAY , but also UGUAMM ( M = A/C ) , UGWAMH ( W = A/U ) , UGUAWA , and GGAU have been reported to be enriched around m6A sites in Arabidopsis and other plant species ( Li et al . , 2014a; Anderson et al . , 2018; Miao et al . , 2020; Zhang et al . , 2019; Zhou et al . , 2019 ) , but it remains unclear whether the adenosines in such motifs are methylated in vivo .",
"Alternatively , these sequence contexts may play a role in guiding m6A deposition or ECT recognition nearby , either directly by ECT interaction or indirectly via additional RNA-binding proteins assisting or competing with ECT binding .",
"To clarify principles underlying mRNA recognition by ECT2 , we undertook rigorous analysis of its mRNA-binding sites using two orthogonal methods , the proximity-labeling method HyperTRIBE ( targets of RNA-binding proteins identified by editing ) ( McMahon Aoife et al . , 2016; Xu et al . , 2018 ) and iCLIP ( individual nucleotide resolution crosslinking and immunoprecipitation ) ( König et al . , 2010 ) .",
"This resulted in identification of high-quality target sets as judged by mutual overlaps and by overlaps with previously reported m6A maps from plants at a similar developmental stage ( Shen et al . , 2016; Parker et al . , 2020 ) .",
"Relying on this high-quality target set , we used the position information inherent to iCLIP and a single-nucleotide resolution m6A dataset ( Parker et al . , 2020 ) to establish six properties of m6A-containing mRNA and mRNA targeting by ECT2 .",
"( 1 ) RRACH and its variant DRACH ( D = R/U ) are unequivocally the most highly enriched motifs at m6A sites in Arabidopsis .",
"( 2 ) ECT2 binds to m6A sites in the canonical RRACH context as ECT2 crosslinking sites are preferentially found immediately 5′ to m6A sites , and RRACH is enriched immediately 3′ to ECT2 crosslinking sites .",
"( 3 ) GGAU is a minor m6A consensus site in plants .",
"( 4 ) U- and U/C-rich motifs are enriched around , but not at , m6A sites , and , together with RRACH and GGAU , constitute core elements that distinguish m6A-containing 3′-UTRs from non-m6A-containing 3′-UTRs in plants .",
"( 5 ) The IDR of ECT2 participates in RNA binding as it crosslinks to target mRNAs at U-rich elements highly abundant upstream of m6A sites .",
"( 6 ) Although URUAY , URURU , and similar motifs may crosslink to ECT2 , their presence in m6A-containing mRNA disfavors ECT2 binding , consistent with those motifs acting predominantly as sites of interaction for RNA-binding proteins that may compete with ECT2 ."
],
[
"HyperTRIBE uses fusion of RNA-binding proteins to the hyperactive E488Q mutant of the catalytic domain of the Drosophila melanogaster adenosine deaminase acting on RNA ( DmADARE488Qcd ) ( Kuttan and Bass , 2012 ) to achieve proximity labeling in vivo ( McMahon Aoife et al . , 2016; Xu et al . , 2018 ) .",
"Targets are identified as those mRNAs that contain adenosine-inosine sites significantly more highly edited than background controls , measured as A-G changes upon reverse transcription and sequencing .",
"To develop material suitable for ECT2 HyperTRIBE , we expressed AtECT2pro:AtECT2-FLAG-DmADARE488Qcd-AtECT2ter ( henceforth ‘ECT2-FLAG-ADAR’ ) in the single ect2-1 and triple ect2-1/ect3-1/ect4-2 ( te234 ) knockout backgrounds ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .",
"We identified lines exhibiting nearly complete rescue of te234 mutant seedling phenotypes , indicating that the fusion protein was functional ( Figure 1A ) .",
"We then used the expression level in complementing lines as a criterion to select lines in the ect2-1 single mutant background , for which no easily scorable phenotype has been described ( Figure 1—figure supplement 1A ) .",
"Lines expressing free DmADARE488Qcd under the control of the endogenous ECT2 promoter ( AtECT2pro:FLAG-DmADARE488Qcd-AtECT2ter; henceforth FLAG-ADAR ) at levels similar to or higher than those of the fusion lines ( Figure 1—figure supplement 1A , B ) were used to control for background editing after verification that FLAG-ADAR expression did not result in phenotypic abnormalities in Col-0 WT plants ( Figure 1A ) .",
"To identify ECT2 HyperTRIBE targets ( HT-targets ) , we sequenced mRNA from dissected root tips and shoot apices of 10-day-old seedlings of ect2-1/ECT2-FLAG-ADAR and FLAG-ADAR transgenic lines using five independent lines of each type as biological replicates to prevent line-specific artifacts .",
"Next , we generated nucleotide base counts for all positions with at least one mismatch across the full set of samples of mapped reads ( Figure 1B ) , resulting in a raw list of potential editing positions .",
"This revealed that the amount of editing was clearly higher in the lines expressing the ECT2-FLAG-ADAR fusion protein than in the negative control lines ( Figure 1C , Figure 1—figure supplement 1C ) .",
"To identify positions with significantly higher editing rates in ECT2-FLAG-ADAR lines compared to controls , we developed a new approach to detect differential editing ( Figure 1B ) described in detail by Rennie et al . , 2021 .",
"Briefly , the hyperTRIBER method of detecting differential editing exploits the powerful statistical capabilities of a method originally designed to detect differential exon usage ( Anders et al . , 2012 ) .",
"It efficiently takes replicates and possible differences in expression into account , resulting in high power to detect sites despite the generally low editing proportions that we found in our data ( Figure 1D ) .",
"As expected , the tendency towards higher editing proportions in fusion lines compared to controls was even more pronounced after filtering nonsignificantly edited sites ( Figure 1C , Figure 1—figure supplement 1C ) .",
"Three additional properties of the resulting editing sites indicate that they are the result of ADARcd activity guided by its fusion to ECT2 .",
"First , the vast majority of significant hits corresponded to A-to-G transitions ( Figure 1—figure supplement 1D ) .",
"Second , the consensus motif at the edited sites matched the sequence preference of DmADARE488Qcd ( 5′ and 3′ nearest-neighbor preference of U>A>C>G and G>C>A~U , respectively [Xu et al . , 2018; Figure 1E , Figure 1—figure supplement 1E] ) , with highly edited sites more closely matching the optimal sequence context than lowly edited ones ( Figure 1—figure supplement 1F ) .",
"Third , principal component analysis of editing proportions at significant sites over the different lines clearly separated the ECT2-FLAG-ADAR fusion lines from the control lines ( Figure 1F , Figure 1—figure supplement 1G ) .",
"Application of subsequent filtering steps , including removal of non- ( A-to-G ) mismatches and of potential line-specific single-nucleotide variants ( see Materials and methods ) , resulted in a final list of 16 , 176 edited sites for aerial tissues and 19 , 242 for roots , corresponding to 4864 and 5052 genes ( ECT2 HT-targets ) , respectively ( Figure 1B , Supplementary file 1 ) .",
"In both cases , this represents 27% of all expressed genes .",
"We note that the editing proportions were generally low ( Figure 1D ) compared to previous work in Drosophila ( Xu et al . , 2018 ) , perhaps in part due to the limited number of cells that express ECT2 ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .",
"Indeed , the ADAR expression level ( TPMs ) correlated strongly with editing proportions among ECT2-FLAG-ADAR lines ( Figure 1G , Figure 1—figure supplement 1H ) , and editing proportions were higher for target mRNAs that are coexpressed with ECT2 in a large percentage of cells according to single-cell RNA-seq ( Denyer et al . , 2019; Figure 1H ) , lending further support to the conclusion that the observed editing is ADAR-specific and driven to target mRNAs by ECT2 .",
"Hence , HyperTRIBE can be used to identify targets of RNA-binding proteins in planta .",
"To evaluate the properties of ECT2 HT-targets , we first noted that most of them were common between root and aerial tissues ( Figure 2A ) , as expected given the recurrent function of ECT2 in stimulating cell division in all organ primordia ( Arribas-Hernández et al . , 2020 ) .",
"In agreement with this result , most of the targets specific to root or aerial tissues were simply preferentially expressed in either tissue ( Figure 2B ) .",
"Moreover , the significant editing sites in roots and aerial tissues had a considerable overlap ( Figure 2A ) , and their editing proportions were similar in the two tissues ( Figure 2C ) .",
"Of most importance , we observed a large overlap between the ECT2 HT-targets and m6A-containing transcripts mapped by different methods in seedlings ( Shen et al . , 2016; Parker et al . , 2020 ) as more than 76% of ECT2 HT-targets had m6A support by either study ( Figure 2D ) .",
"These results validate our HyperTRIBE experimental setup and data analysis , and confirm that ECT2 binds predominantly to m6A-containing transcripts in vivo .",
"We next moved on to independent target and binding site identification via iCLIP ( Figure 3A ) .",
"We used transgenic lines expressing functional ECT2-mCherry under the control of the endogenous ECT2 promoter in the ect2-1 knockout background ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) to co-purify mRNAs crosslinked to ECT2 for iCLIP .",
"Lines expressing the ECT2W464A-mCherry variant were used as negative controls because this Trp-to-Ala mutation in the hydrophobic methyl-binding pocket of the YTH domain abrogates the increased affinity for m6A-RNA ( Li et al . , 2014b; Xu et al . , 2014; Zhu et al . , 2014 ) .",
"Accordingly , the point mutant behaves like a null allele in plants despite its wild-type-like expression pattern and level ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .",
"To test the feasibility of iCLIP , we first assessed the specificity of RNA co-purified with ECT2-mCherry after UV illumination of whole seedlings by 5′-radiolabeling of the immunoprecipitated RNP complexes followed by SDS-PAGE .",
"These tests showed that substantially more RNA co-purifies with wild-type ECT2 than with ECT2W464A upon UV-crosslinking , and that no RNA is detected without UV irradiation or from irradiated plants of non-transgenic backgrounds ( Figure 3B , Figure 3—figure supplement 1A ) .",
"RNAse and DNAse treatments also established that the co-purified nucleic acid is RNA ( Figure 3—figure supplement 1B ) .",
"Thus , UV crosslinking of intact Arabidopsis seedlings followed by immunopurification successfully captures ECT2-RNA complexes that exist in vivo .",
"Curiously , although the pattern of ECT2-RNA complexes with bands migrating at ~110 and 55kDa is highly reproducible , it does not correspond to the majority of the purified ECT2-mCherry protein , which runs at ~125kDa in SDS-PAGE ( Figure 3B and C ) .",
"A variety of control experiments ( Figure 3—figure supplement 1C-E ) , most importantly the disappearance of additional bands with use of an N-terminal rather than a C-terminal tag ( Figure 3C and D ) , indicate that the band pattern arises as a consequence of proteolytic cleavage of the N-terminal IDR in the lysis buffer , such that fragments purified using the C-terminal mCherry tag include the YTH domain with portions of the IDR of variable lengths ( Figure 3—figure supplement 2 ) .",
"Comparative analysis of RNA in 55-kDa and 110–125-kDa complexes may , therefore , provide insight into the possible role of the N-terminal IDR of ECT2 in mRNA binding ( Figure 3E ) , an idea consistent with the comparatively low polynucleotide kinase labeling efficiency of full-length ECT2-mCherry-mRNA complexes ( ~125kDa ) ( Figure 3B , Figure 3—figure supplement 2 ) .",
"Thus , we prepared separate iCLIP libraries from RNA crosslinked to ECT2-mCherry/ECT2W464A-mCherry that migrates at ~110–280kDa ( ‘110-kDa band’ ) and at ~55–75kDa ( ’55-kDa band’ ) ( Figure 3—figure supplement",
"3 ) to investigate the possible existence of IDR-dependent crosslink sites , and thereby gain deeper insights into the mode of YTHDF binding to mRNA in vivo .",
"We identified a total of 15 , 960 iCLIP ‘peaks’ or crosslink sites ( i . e . , single-nucleotide positions called by PureCLIP from mapped iCLIP reads [Krakau et al . , 2017] ) in 2281 genes from the 110-kDa band of wild-type ECT2-mCherry ( henceforth referred to as ECT2 iCLIP peaks and targets , respectively ) .",
"In the corresponding 55-kDa band , 4549 crosslink sites in 1127 genes were called , 93% of them contained in the 110-kDa target set ( Figure 3F and G , Figure 3—figure supplement 4 , Supplementary file 2 ) .",
"We note that these numbers perfectly agree with the idea of the 55-kDa band containing only YTH domain crosslink sites , while the full length may also include IDR crosslink sites .",
"Importantly , for both libraries , the majority of crosslink sites mapped to the 3′-UTRs of mRNAs ( Figure 3H , see Figure 4A , and Figure 4—figure supplement 1 for more examples ) , coincident with the main location of m6A ( Figure 4B; Parker et al . , 2020 ) .",
"Accordingly , the 3′-UTR specificity was largely lost in RNA isolated from 55-kDa ECT2W464A ( Figure 3H ) , for which neither YTH domain nor IDR binding to RNA can be expected .",
"Finally , iCLIP targets in full-length ( 110-kDa band ) ECT2 WT and ECT2W464A overlapped only marginally ( Figure 3G ) , providing molecular proof of the dependence of m6A-binding activity for ECT2 function .",
"Nonetheless , the bias towards occurrence in the 3′-UTR was only reduced , not abolished , for crosslinks to the full-length ECT2W464A protein , providing another indication that the IDR itself is able to associate with RNA-elements in 3′-UTRs ( Figure 3H ) .",
"We elaborate further on this important point by analysis of IDR-specific crosslinks to wild-type ECT2 after in-depth validation of sets of ECT2 target mRNAs and determination of the sequence motifs enriched around m6A and ECT2 crosslink sites .",
"To evaluate the congruence of the results obtained by iCLIP and HyperTRIBE , we investigated the cumulative number of iCLIP sites as a function of distance to the nearest editing site determined by HyperTRIBE .",
"This analysis showed a clear tendency for iCLIP peaks called with ECT2WT-mCherry , but not for ECT2W464A-mCherry , to be in the vicinity of editing sites ( Figure 4C ) , indicating that the majority of called iCLIP peaks identify genuine ECT2-binding sites on mRNAs .",
"Similar tendencies of proximity between iCLIP peaks and HyperTRIBE editing sites were previously observed for a Drosophila hnRNP protein ( Xu et al . , 2018 ) .",
"Although manual inspection of individual target genes confirmed these tendencies , it also revealed that ADAR-edited sites are too dispersed around iCLIP peaks to give precise information on the actual ECT2-binding sites ( Figure 4A , Figure 4—figure supplement 1 ) .",
"Therefore , we used both HyperTRIBE and iCLIP for gene target identification , but relied on iCLIP peaks for motif analyses .",
"To examine the quality of our target identification in further detail , we analyzed the overlap between ECT2 targets identified by iCLIP and HyperTRIBE .",
"This analysis also included m6A mapping data obtained with either m6A-seq ( Shen et al . , 2016 ) or the single-nucleotide resolution methods miCLIP and Nanopore sequencing ( Parker et al . , 2020 ) as young seedlings were used in all cases .",
"ECT2 targets identified by iCLIP and HyperTRIBE showed clear overlaps , both with each other and with m6A-containing transcripts , further supporting the robustness of ECT2 target identification via combined iCLIP and HyperTRIBE approaches ( Figure 4D , upper panel , Figure 4—figure supplement 2 ) .",
"Importantly , although some m6A targets are expected not to be bound by ECT2 because of the presence of MTA in cells that do not express ECT2 ( Arribas-Hernández et al . , 2020 ) , only 18% of the high-confident set of m6A-containing genes ( with support from miCLIP and Nanopore ) did not overlap with either ECT2 iCLIP or HT target sets ( Figure 4—figure supplement 2 , arrow ) .",
"We also observed that HyperTRIBE identifies approximately three times more ECT2 targets than iCLIP , possibly because of the bias towards high abundance inherent to purification-based methods like iCLIP ( Wheeler et al . , 2018 ) .",
"To test this idea , we compared the distribution of target mRNAs identified by the different techniques across nine expression bins .",
"As expected , a bias towards highly abundant transcripts was evident for iCLIP-identified targets compared to HyperTRIBE ( Figure 4E ) .",
"We also observed a similar bias for m6A-containing transcripts detected by miCLIP , another purification-based method , and in the Nanopore dataset ( Figure 4E ) , probably explained by its relatively low sequencing depth ( Parker et al . , 2020 ) .",
"These observations also suggest that the higher sensitivity of HyperTRIBE ( analyzed in detail in Figure 4—figure supplement",
"3 ) explains the lack of m6A support ( by Nanopore or miCLIP ) for 28% of ECT2 HT-targets ( 1689 ) compared to only 4% ( 83 ) of ECT2 iCLIP targets ( Figure 4D , Figure 4—figure supplement 2 , upper row ) since HT-targets may simply include genes that escape detection by m6A mapping methods due to low expression .",
"Indeed , ECT2-HT targets without any m6A support were distributed in lower-expression bins compared to those with m6A support ( Figure 4F ) .",
"Intriguingly , ECT2 FA-CLIP targets ( Wei et al . , 2018 ) did not show a bias towards highly expressed genes as their distribution over expression bins largely reflected that of the total number of genes ( Figure 4E ) , and as many as 37% of FA-CLIP targets did not have m6A support ( Figure 4D , Figure 4—figure supplement 2 , upper row ) .",
"In summary , these analyses show that ECT2 iCLIP and HT target sets are in excellent agreement with each other and with independently generated m6A maps , and that HyperTRIBE identifies targets below the detection limit of other techniques .",
"To characterize the sequence composition and exact positions of ECT2-binding sites relative to m6A , we first used the high resolution of iCLIP data to examine the position of ECT2 crosslink sites relative to m6A sites , determined at single-nucleotide resolution ( Parker et al . , 2020 ) .",
"This analysis showed that ECT2 crosslinks in the immediate vicinity , but preferentially upstream ( ~11 nt ) of Nanopore-determined m6A sites , with a mild depletion at the exact m6A site ( Figure 5A , upper panel ) .",
"Furthermore , while m6A-miCLIP sites corresponded to m6A-Nanopore sites overall , a subset of m6A-miCLIP sites were located upstream of m6A-Nanopore sites and coincided well with ECT2-iCLIP peaks ( Figure 5A ) .",
"This pattern is probably explained by the fact that the UV illumination used in both iCLIP and miCLIP preferentially generates RNA-protein crosslinks involving uridine ( Hafner et al . , 2021 ) , also detectable in the datasets analyzed here ( Figure 5B and C ) .",
"Thus , the depletion of ECT2-iCLIP sites at Nanopore- , but enrichment at miCLIP-determined m6A sites ( Figure 5A ) , might be explained by the absence of uridine within the RRAC core of the m6A consensus motif , and perhaps also to some extent by reduced photoreactivity of the m6A base stacking with indole side chains of the YTH domain .",
"Furthermore , the fact that nucleotides at −2 , +1 , and +2 positions are only expected to contribute sugar-phosphate backbone interactions with the YTH domain ( Luo and Tong , 2014b; Theler et al . , 2014; Xu et al . , 2014 ) may also contribute to the absence of direct crosslinks at the m6A site relative to the adjacent bases .",
"The 5′ shift observed for iCLIP and miCLIP sites relative to Nanopore sites might be explained by a higher occurrence of uridines upstream of m6A sites , a particularly interesting possibility given the numerous reports of U-rich motifs enriched around m6A sites in plants ( Li et al . , 2014a; Anderson et al . , 2018; Miao et al . , 2020; Zhang et al . , 2019; Zhou et al . , 2019; Luo et al . , 2020 ) and animals ( Patil et al . , 2016 ) .",
"To investigate the sequence composition around m6A and ECT2 sites , we first performed exhaustive unbiased de novo motif searches using Homer ( Heinz et al . , 2010; Figure 5—figure supplement",
"1 ) and extracted all candidate motifs , including the m6A consensus motif RRACH , as well as GGAU ( Anderson et al . , 2018 ) , URUAY ( Wei et al . , 2018 ) , and several other U-rich sequences .",
"Combined with manually derived candidate motifs ( Figure 5—figure supplement 1B ) , we then calculated position weight matrices ( PWMs ) for a final set of 48 motifs and scanned for their occurrences genome-wide using FIMO ( Grant et al . , 2011; Figure 5—figure supplements 1 and 2 ) .",
"This allowed us to determine three key properties .",
"First , the global enrichment of the motifs at locations across the gene body .",
"Second , the total count of occurrences of each motif at m6A sites and ECT2-iCLIP crosslink sites compared to a set of sites in non-target mRNAs matching the location within gene bodies of m6A/ECT2-iCLIP sites ( expected background ) .",
"Third , the distribution of the motifs relative to m6A and ECT2 iCLIP sites .",
"The results of this systematic analysis ( Supplementary file",
"3 ) were used to select those motifs with a more prominent enrichment at or around m6A and ECT2 sites ( Figure 5D ) .",
"This approach defined two major categories of motifs of outstanding interest , RRACH-like and GGAU on the one side , and a variety of U/Y-rich motifs on the other .",
"Figure 5D shows a minimal selection of such motifs , while a more comprehensive compilation is displayed in Figure 5—figure supplements 3 and 4 .",
"Not surprisingly , RRACH-like motifs were the most highly enriched at m6A sites and showed a clear enrichment immediately downstream of ECT2 crosslink sites in our analyses , with the degenerate variant DRACH being the most frequently observed ( Figure 5D , Figure 5—figure supplement 3 ) .",
"Motifs containing GGAU behaved similarly to DRACH , with a sharp enrichment exactly at m6A sites and mild enrichment downstream of ECT2 peaks ( Figure 5D ) , supporting a previous suggestion of GGAU as an alternative methylation site ( Anderson et al . , 2018 ) .",
"The possible roles of the U/Y-rich motifs in m6A deposition and ECT2 binding are analyzed in the following sections .",
"We first noticed that several motifs retrieved around ECT2 crosslink sites by Homer constituted extended versions of DRACH/GGAU with Us upstream ( e . g . , UGAAC/UGGAU ) or remnants of DRACH with U/Cs ( Ys ) downstream ( e . g . , ACUCU ) .",
"To test whether these motifs are indeed located adjacent to m6A , we examined their distribution and enrichment around ECT2 and m6A sites .",
"The distributions showed a clear enrichment at m6A positions with a shift in the direction of the U/Y-extension ( see Figure 5D for ACUCU and Figure 5—figure supplement 4 for others ) .",
"An enrichment over location-matched background sites close to ECT2-iCLIP sites was also apparent ( see Figure 5D for ACUCU and Figure 5—figure supplement 4 for others ) , further supporting that ECT2 preferentially crosslinks to uridines located in the immediate vicinity of DRACH ( /GGAU ) .",
"Thus , several enriched motifs around ECT2 crosslink sites are DRACH/GGAU-derived , and their detection in unbiased searches simply reflects a tendency of methylated DRACH/GGAU sites to be flanked by U/Ys .",
"U/R-rich motifs without traces of adjacent DRACH ( e . g . , YUGUM , URUAY , URURU ) showed a characteristic enrichment around , but depletion at , m6A sites .",
"For some motifs , the enrichment was more pronounced 5′ than 3′ to m6A sites ( see Figure 5D for URUAY and Figure 5—figure supplement 4 for others ) .",
"The distance between the site of maximal motif occurrence and the m6A site roughly coincided with the shift observed in ECT2 crosslink sites relative to m6A ( Figure 5A , upper panel ) .",
"Accordingly , these motifs were enriched exactly at ECT2 crosslink sites ( see Figure 5D for URUAY and Figure 5—figure supplement 4 for others ) , suggesting that they may constitute additional m6A-independent sites of interaction with ECT2 .",
"We also observed that the 3′ enrichment of YYYYY was asymmetric and closer to m6A than that of UUUUU/URURU/URUAY ( Figure 5—figure supplement 4 , second row from the top ) , indicating a preference for hetero-oligopyrimidine tracts immediately downstream the m6A site , as suggested by the 3′-enrichment of DRACUCU-type motifs as described above .",
"Taken together , these results suggest that N6-adenosine methylation preferentially occurs in DRACH/GGAU sequences surrounded by stretches of pyrimidines , with a preference for YYYYY ( e . g . , CUCU ) immediately downstream , URURU ( including URUAY ) immediately upstream , and UUUUU/UNUNU slightly further away in both directions .",
"The enrichment of ECT2 crosslink sites at these motifs , and the fact that the m6A-binding-deficient mutant of ECT2 ( W464A ) crosslinks preferentially to 3′-UTRs through its N-terminal IDR , indicates IDR-mediated binding to U/R- and Y-rich motifs around m6A .",
"Since our analysis thus far uncovered several motifs of potential importance for m6A deposition and ECT2 binding , we employed machine learning to distinguish m6A and ECT2 iCLIP sites from random location-matched background sites using motif-based features .",
"Importantly , the underlying classification model includes all motif features within the same model , allowing an evaluation of the importance of the motifs relative to each other .",
"We used as features the number of matches to each of the 48 motifs ( Figure 5—figure supplement",
"2 ) in three distinct regions relative to the methylated site according to Nanopore sequencing ( Parker et al . , 2020 ) , defined as position 0: ‘at’ [–10 nt; +10 nt] , ‘down’ [–50 nt; –10 nt] , or ‘up’ [+ 10 nt; +50 nt] ( Figure 6A ) .",
"The model involving all motifs could successfully distinguish the methylated sites from the background as indicated by an area under the receiver operating characteristic ( ROC ) curve ( true positive rate versus false positive rate , area under the curve [AUC] ) of 0 . 93 , and even a reduced model incorporating only the top 10 features from the full model classified sites largely correctly ( AUC = 0 . 86; Figure 6—figure supplement 1 ) .",
"The top 16 features ordered by importance from the full model confirmed that RRAC/DRACH or GGAU at the site was indicative of the presence of m6A ( Figure 6B ) .",
"Interestingly , U/Y-rich sequences ( UNUNU and YYYYY in particular ) flanking the site were also strongly indicative ( Figure 6B ) .",
"Some motifs showed a skew in their feature importance score , with UNUNU and YUGUM showing a preference to be upstream , and YYYYY downstream ( Figure 6B ) , thus corroborating our previous observations ( Figure 6C ) .",
"We used a similar modeling approach to identify non-m6A determinants of ECT2 binding , in this case comparing m6A sites within 10 nt distance of ECT2-iCLIP sites to m6A sites without ECT2-iCLIP sites nearby ( AUC = 0 . 94 , and AUC = 0 . 84 using only the top 10 features , Figure 6—figure supplement 1 ) .",
"In agreement with previous observations , this model showed flanking U/Y-rich sequences as the main determinants for ECT2 crosslinking ( Figure 6D ) .",
"To investigate the idea of URURU-like motifs as additional sites of ECT2 binding upstream of the m6A-YTH interaction site , we split Nanopore-m6A sites according to two criteria: ( 1 ) whether they occur in ECT2-target transcripts ( both permissive and stringent sets analyzed separately ) , and ( 2 ) for ECT2 targets , whether there is an ECT2 crosslink site within 25 nt of the m6A site ( ‘near’ ) or not ( ‘far’ ) .",
"Although there was no obvious differences between these categories for most of the motifs ( Supplementary file 3 , page 2 ) , some U-rich sequences displayed distinctive features ( Figure 7A , Figure 7—figure supplement",
"1 ) that can be summarized as follows .",
"If a transcript has m6A and ECT2 sites in close proximity , it is ( 1 ) more likely to have UNUNU/UUUUU/YYYYY sequences upstream of the m6A site than targets with distantly located ECT2-binding sites or than non-ECT2 targets; ( 2 ) less likely to have UUUUU/URURU sequences downstream of the m6A site , possibly because ECT2 prefers CUCU-like sequences downstream; and ( 3 ) less likely to have URURU/URUAY-like motifs upstream of the m6A site .",
"The latter observation is striking because for the specific subset of ECT2-bound m6A sites with URURU/URUAY upstream of m6A , these sequences tend to crosslink to ECT2 , as seen by the enrichment spike at ECT2 crosslink sites ( Figure 5D , Figure 7—figure supplement 1 , bottom panels ) .",
"Although these two results seem contradictory at first glance , they may be reconciled by a model in which a URURU/URUAY-binding protein would compete with ECT2 for binding adjacent to m6A .",
"If that protein is absent , ECT2 may bind to the site , potentially via its IDR , to stabilize the low-affinity YTH-m6A interaction and crosslink efficiently due to the U-content .",
"Conversely , if occupied by the alternative interacting protein , the site might repel ECT2 ( see Discussion and Figure 7B ) .",
"We reasoned that insights into contacts between ECT2 and mRNA may be gained by analysis of the iCLIP libraries prepared with the ‘YTH-mCherry’ truncation devoid of the N-terminal IDR ( ‘55-kDa band’ ) compared to the full-length ECT2-mCherry ( ‘110-kDa band’ ) ( Figure 3 , Figure 3—figure supplements 2–4 ) .",
"Initial inspection of the distribution of ECT2 peaks relative to Nanopore-m6A sites showed that the 5′–3′ asymmetry observed with full-length ECT2 was largely reduced with the truncated protein ( Figure 7C ) , as was the bias towards uridines ( Figure 7—figure supplement 2A ) .",
"These observations suggest that the IDR indeed is implicated in binding to U-rich regions upstream of m6A .",
"We next split the full-length ECT2 iCLIP peaks according to whether they are present in libraries from both full-length and truncated forms ( ‘IDR-independent’ ) or exclusively in the full-length ( ‘IDR-dependent’ ) ( distance >10 nt ) and plotted the enrichment of the studied motifs relative to the crosslink site ( Figure 7D , Figure 7—figure supplement 2B; Supplementary file 3 , page 2 ) .",
"UUUUUU/UNUNU-like motifs were more enriched at and immediately upstream of IDR-dependent crosslink sites relative to the IDR-independent ones , supporting preferential crosslinking of the IDR to Us in this region .",
"Remarkably , the exact opposite was true for URURU/URUAY motifs that showed modest depletion 5′ to IDR-dependent crosslink sites relative to their IDR-independent counterparts ( Figure 7D ) .",
"These observations are consistent with a model of an RNA-binding protein competing with the ECT2 IDR for interaction with upstream URURU/URUAY motifs ( Figure 7B ) ."
],
[
"Our work establishes experimental and computational approaches to implement HyperTRIBE for unbiased and sensitive mapping of direct targets of RNA-binding proteins in plants .",
"Two points are particularly relevant in this regard .",
"First , the examples studied here show that stable transgenic expression of DmADARcd does not lead to detrimental phenotypes , perhaps because of the generally low editing proportions obtained in vivo .",
"Second , the rigorous statistical approach developed to call editing sites makes HyperTRIBE powerful , despite the low editing proportions observed .",
"We also note that ECT2 is well suited to verify that HyperTRIBE mostly recovers directly bound target RNAs because of the possibility to cross-reference the data with independently obtained m6A maps ( Parker et al . , 2020 ) .",
"The combination of iCLIP and HyperTRIBE for unbiased mapping of targets proved particularly attractive for at least two reasons .",
"First , the convergence on overlapping target sets by orthogonal methods strengthens the confidence that the identified targets are biologically meaningful .",
"Second , HyperTRIBE , especially with the novel computational approach for calling of editing sites ( Rennie et al . , 2021 ) , offers higher sensitivity than iCLIP , while iCLIP is unmatched in providing information on binding sites within target RNAs .",
"It is possible that better positional information on binding sites may be obtained from HyperTRIBE data using maximal editing proportions rather than statistical significance as the parameter to call editing sites .",
"Indeed , recent work on the use of HyperTRIBE to identify targets of the RNA-binding protein MUSASHI-2 ( MSI-2 ) in leukemic stem cells recovered the known MSI-2-binding site as enriched around editing sites in targets ( Nguyen et al . , 2020 ) .",
"Nonetheless , our data shows that highly edited sites match the ADAR substrate consensus site better than lowly edited sites , suggesting that site proximity to ADAR is not the only determinant of editing proportions .",
"Finally , our work also clearly indicates that FA-CLIP , now used in at least two studies involving YTH domain proteins ( Wei et al . , 2018; Song et al . , 2021 ) , is not a recommendable technique as it recovers many false positives and fails to include many genuine targets .",
"Thus , with the possible exception of cases in which evidence for indirect association is specifically in demand , such as the recent study in human cells of mixed tailing of viral RNA by the cellular terminal nucleotidyl transferase TENT4 ( Kim et al . , 2020 ) , FA-CLIP should not be used for identification of RNAs associating with a particular RNA-binding protein of interest .",
"Our analyses of motif enrichments around m6A and ECT2 crosslink sites clarify roles of previously reported motifs and uncover new motifs of importance in m6A writing and ECT2 binding .",
"Since m6A is a prerequisite for ECT2 binding , any analysis of determinants of ECT2 binding must consider determinants of N6-adenosine methylation separately .",
"Three conclusions stand out from our analysis in this regard .",
"First , the major N6-adenosine methylation site is DRACH , consistent with conclusions from multiple other studies .",
"Second , GGAU is a minor N6-adenosine methylation site , as seen by its enrichment directly at m6A sites .",
"Third , m6A occurs in DRACH/GGAU islands embedded in U-rich regions .",
"Such U-rich regions around m6A sites emerged from sorting of methylated from non-methylated transcripts by machine learning as being of similar importance for recognition of m6A-containing transcripts from sequence features as DRACH and GGAU at m6A sites , suggesting their implication in MTA/MTB-catalyzed adenosine methylation ( Figure 6C ) .",
"This , in turn , may also explain the pronounced 3′-UTR bias of m6A occurrence as extensive poly-U and poly-pyrimidine tracts are rare in coding regions ( Figure 5D , second row on the right-most column; Supplementary file 3 , page 1 ) .",
"As a special case in this context , our analyses suggest a simple explanation for the tendency of m6A to occur at stop codons .",
"UAA and UGA correspond to DRA , increasing the frequency of occurrence of DRACH directly at stop codons ( Figure 5D , second row on the left-most column ) , many of which have adjacent U-rich elements in the 3′-UTRs .",
"We note that the observed pattern is in agreement with a role of the poly ( U ) -interacting proteins RBM15A/B associated with the mammalian methyltransferase complex in guiding methylation ( Patil et al . , 2016 ) .",
"Whether a similar mechanism operates in plants , potentially via the distant RBM15A/B homologue FPA ( Arribas-Hernández and Brodersen , 2020 ) , remains to be investigated .",
"It is a major conclusion of the present work that ECT2 binds to m6A predominantly in the DR ( m6A ) CH sequence context in vivo , consistent with reading of m6A written by the conserved nuclear MTA/MTB methyltransferase .",
"This key conclusion refutes the claim by Wei et al . , 2018 that ECT2 binds to the supposedly plant-specific m6A-containing sequence motif URU ( m6A ) Y , and it thereby reconciles knowledge on m6A-YTHDF axes in plants specifically and in eukaryotes more broadly .",
"The phenotypic similarity of plants defective in MTA/MTB writer and ECT2/ECT3/ECT4 reader function is now coherent with the locations of MTA/MTB-written m6A and ECT2-binding sites transcriptome-wide , and it is now clear that plants do not constitute an exception to the general biochemical framework for eukaryotic m6A-YTHDF function in which YTHDF proteins read the m6A signal written by the MTA/MTB methyltransferase .",
"The pronounced protease sensitivity of IDRs , leading to limited proteolysis of ECT2 upon cell lysis after in vivo crosslinking , allowed us to extract information on the mode of ECT2-RNA binding from different observations , all converging on the conclusion that the IDR of ECT2 participates in RNA binding .",
"First , RNA complexes with YTH-mCherry were 5′-labeled by polynucleotide kinase much more efficiently than RNA complexes with full-length ECT2-mCherry , indicating that the IDR limits accessibility to the 5′ of bound mRNAs .",
"Second , in contrast to the m6A-binding-deficient YTHW464A-mCherry truncation , the full-length ECT2W464A-mCherry mutant retained an enrichment of crosslink sites in 3′-UTRs .",
"Third , crosslinks specific to the IDR ( i . e . , observed only with full-length ECT2-mCherry-RNA complexes , but not with YTH-Cherry-RNA complexes ) , could be assigned and have two notable properties .",
"They are mainly 5′ to m6A sites , and thereby cause a conspicuously asymmetric distribution of ECT2 crosslink sites around m6A sites , not seen with crosslinks to the YTH-mCherry fragment .",
"In addition , the IDR-specific crosslinks are specifically enriched in U-rich elements of the type UUUUUU and UNUNU immediately upstream .",
"Taken together , these observations suggest that the IDR of ECT2 participates in locating ECT2 to 3′-UTRs by association with U-rich elements .",
"Thus , ECT2 , and perhaps YTHDF proteins more generally given their highly similar YTH domains , appears to bind RNA through multivalent interactions among which the YTH domain is responsible for m6A binding , and the IDR is responsible for interaction with adjacent elements .",
"We note that the notion of RNA interaction by IDRs has precedent ( Corley et al . , 2020 ) , is consistent with the modest affinity of isolated YTHDF domains for m6A-containing oligonucleotides ( Patil et al . , 2018 ) , and is reminiscent of the recent demonstration that transcription factors use their globular DNA-binding domains to recognize core sequence elements of promoters , and their IDRs to provide additional DNA contacts , contributing to specificity ( Brodsky et al . , 2020 ) .",
"Similarly , it is possible that diverging IDRs among YTHDF paralogs could confer target specificity via binding to distinct motifs in the vicinity of m6A sites , such that specific YTHDF-target mRNA repertoires could exist even for YTHDF proteins coexpressed in the same cells .",
"Finally , we stress that although our data point to an important role of the IDR in RNA binding , it does not in any way suggest that this is the only function of the IDR , and protein-protein interactions involving the IDR are likely to be key to understanding YTHDF function molecularly .",
"Despite the conclusions that URUAY does not contain m6A in Arabidopsis , and that ECT2 binds to DR ( m6A ) CH , our detailed analysis of sequence motifs enriched around m6A and ECT2 iCLIP crosslink sites shows that additional motifs , including URUAY , are likely to be implicated in m6A reading by ECT2 , even if not directly .",
"In contrast to other m6A-proximal , pyrimidine-rich sequences ( e . g . , UNUNU , YYYYY ) that may be of importance for both m6A writing and ECT2 binding , URUAY appears to have ties more specifically to ECT2 binding thanks to three properties .",
"( 1 ) When present 5′ to m6A sites , it crosslinks to ECT2 , suggesting that some part of the protein can be in contact with URUAY .",
"( 2 ) URUAY is more enriched close to m6A sites for which there is no evidence of ECT2 binding , suggesting that it weakens ECT2 binding .",
"This latter point is also consistent with the distinction of ECT2-bound from non-ECT2-bound m6A sites by machine learning that did not find URUAY to be of importance for ECT2-bound sites .",
"( 3 ) The URUAY enrichment 5′ to ECT2 crosslink sites is observed only when crosslinks to both full-length protein and the YTH-mCherry fragment are considered ( IDR-independent ) , but disappears when crosslinks specific to the full-length protein ( IDR-dependent ) are analyzed .",
"Although these observations may be explained by multiple scenarios , we find a simple , yet at present speculative , model attractive: URUAY may be a site of competition between the IDR of ECT2 and another , as yet unknown , RNA-binding protein .",
"Such a competing factor could in theory be another YTHDF protein using higher-affinity IDR-URUAY contacts than ECT2 to achieve competitive binding .",
"Many other possibilities exist , however .",
"For example , it is intriguing that URUAY resembles part of a Pumilio-binding site ( Hafner et al . , 2010; Huh et al . , 2013 ) as it raises the tantalizing possibility of functional interaction between YTHDF and Pumilio proteins .",
"In any event , the functional dissection of the URUAY element in m6A reading now constitutes a subject of major importance , emphasized by the broad conservation of its enrichment around m6A sites across multiple plant species , including rice ( Li et al . , 2014a; Zhang et al . , 2019 ) , maize ( Luo et al . , 2020; Miao et al . , 2020 ) , tomato ( Zhou et al . , 2019 ) , and Arabidopsis ( Miao et al . , 2020 ) ."
],
[
"We use the term ‘biological replicate’ in the following way: plants were grown at the same time , under the same conditions , but in separate plates .",
"Each sample replicate contains pools of seedlings prepared in such a way that no two replicates contain seedlings grown on the same plates .",
"This sampling ensures that plate-to-plate variation in growth conditions , if any , will have an effect on measurements of gene expression within a single genotype , and hence minimize the risk that any differences due to such variation are called as significant in comparisons between genotypes .",
"‘Technical replicates’ are understood to be independently conducted measurements using the same technique on the same biological material ( e . g . , on one biological replicate as defined above ) .",
"Technical replicates were not carried out in this study , and the term ‘replicate’ refers to biological replicate as defined above .",
"In our definition , an ‘experiment’ results in generation and comparison of measurements arising from multiple biological replicates of different biological entities , in the present case often Arabidopsis seedlings differing in genotype with respect to the genes ECT2 , ECT3 , and ECT4 .",
"Thus , repetition of an experiment in our definition entails generation and analysis of the required biological replicates at different points in time .",
"All lines used in this study are in the Arabidopsis thaliana Col-0 ecotype .",
"The mutant alleles or their combinations – ect2-1 ( SALK_002225 ) ( Arribas-Hernández et al . , 2018; Scutenaire et al . , 2018; Wei et al . , 2018 ) , ect3-1 ( SALKseq_63401 ) , ect4-2 ( GK_241H02 ) , and ect2-1/ect3-1/ect4-2 ( te234 ) ( Arribas-Hernández et al . , 2018 ) – have been previously described .",
"The transgenic lines expressing ECT2pro:ECT2-mCherry-ECT2ter , ECT2pro:ECT2W464A-mCherry-ECT2ter , ECT2pro:3xHA-ECT2-ECT2ter , or ECT2pro:3xHA-ECT2W464A-ECT2ter in the ect2-1 background have also been described or generated by floral dip in additional mutant backgrounds using the same plasmids and methodology ( Arribas-Hernández et al . , 2018; Arribas-Hernández et al . , 2020 ) .",
"Seeds were surface-sterilized by 2 min incubation in 70% EtOH plus 10 min in sterilizing solution ( 1 . 5% NaOCl , 0 . 05% Tween-20 ) and 2 H2O washes .",
"After 2–5 days of stratification at 4°C in darkness , seeds were germinated and grown on plates containing Murashige and Skoog ( MS ) -agar medium ( 4 . 4 g/L MS , 10 g/L sucrose , 10 g/L agar ) pH 5 . 7 at 20°C , receiving ~70 μmol m–2 s–1 of light in a 16 hr light/8 hr dark cycle as default .",
"For HyperTRIBE and iCLIP experiments , the plates were placed vertically to facilitate root harvesting .",
"MS-agar media for HyperTRIBE T2 seedlings was supplemented with 7 . 5 mg/L of glufosinate ammonium ( Sigma ) to select plants expressing the ADAR-containing transgenes .",
"To assess phenotypes of adult plants , ~8-day-old seedlings were transferred from horizontal MS plates ( 4 . 4 g/L MS , 10 g/L sucrose , 8 g/L agar; pH 5 . 7 ) to soil and maintained in Percival incubators under 16 hr light/8 hr dark cycles , 21°C day/18°C night temperature , and ~100 μmol m–2 s–1 light intensity .",
"We used Philips fluorescent tubes TL-D 90 De Luxe 36 W as light source .",
"We employed USER cloning ( Bitinaite and Nichols , 2009 ) to generate ECT2pro:ECT2-FLAG- DmADARE488Qcd-ECT2ter and ECT2pro:FLAG-DmADARE488Qcd-ECT2ter constructs in pCAMBIA3300U ( pCAMBIA3300 with a double PacI USER cassette inserted between the PstI-XmaI sites at the multiple cloning site; Nour-Eldin et al . , 2006 ) .",
"Fragments containing ECT2 gDNA sequences were amplified by PCR ( KAPA HiFi Hotstart Uracil + ReadyMix , Roche ) from plasmids previously generated in our lab ( Arribas-Hernández et al . , 2018 ) .",
"The FLAG-DmADARE488Qcd fragment was produced in the same way using a pGEM-T Easy ( Promega ) plasmid containing FLAG-DmADARE488Qcd as template , previously subcloned to introduce the E488Q hyperactive mutation by site-directed mutagenesis ( QuickChange , Agilent Technologies ) with primers LA729-LA730 ( Phusion HF DNA Polymerase , NEB ) .",
"The E488Q mutation was detected by NlaIII ( ThermoFisher ) digestion of the PCR reaction ( DreamTaq , ThermoFisher ) obtained with primers LA660-LA735 .",
"Of note , the FLAG and DmADARcd sequences had been previously glued together by USER cloning to produce AGO1pro:FLAG-DmADARcd-AGO1ter in pCAMBIA3300U for unrelated purposes ( unpublished work ) , and subsequently amplified by PCR with primers LA696-615 for introduction into pGEM-T Easy .",
"To build AGO1pro:FLAG-DmADARcd-AGO1ter in the first place , the catalytic domain of the ADAR deaminase isoform N ( Y268-E669 ) was amplified from cDNA of D . melanogaster Canton-S wild-type flies and larvae with USER primers MVUSER12-22 .",
"The rest of the fragments were amplified from pCAMBIA3300U AGO1pro:FLAG-AGO1-AGO1ter ( Arribas-Hernández et al . , 2016 ) with primers MVUSER1-11 and MVUSER23-6 .",
"USER primers to amplify all fragments were designed to create overhangs compatible with either the PacI USER cassette present in the pCAMBIA3300U plasmid or the flanking sequences of the neighboring fragments .",
"All primer sequences , their combinations to produce PCR fragments , and the arrangement of the fragments for USER cloning can be found in Appendix 1 .",
"Kanamycin-resistant colonies of Escherichia coli DH5α ( NEB ) transformed with the constructs were analyzed by restriction digestion and sequencing prior introduction of the plasmids in Agrobacterium tumefaciens GV3101 ( Koncz and Schell , 1986 ) for plant transformation .",
"Arabidopsis stable transgenic lines were generated by floral dip transformation ( Clough and Bent , 1998 ) of Col-0 WT , ect2-1 , or te234 , and selection of primary transformants ( T1 ) was done on MS-agar plates supplemented with glufosinate-ammonium ( Sigma ) ( 10 mg/L ) .",
"We selected five independent lines of each type based on segregation studies ( to isolate single T-DNA insertions ) , phenotypic complementation ( in the te234 background ) , and transgene expression levels assessed by FLAG western blot .",
"Protein extraction from 10-day-old seedlings and western blotting with FLAG , HA , and mCherry antibodies were done as previously described ( Arribas-Hernández et al . , 2018 ) .",
"Loading was documented by amido black , Coomassie , or Ponceau staining of the total protein on the membrane .",
"We extracted total RNA from manually dissected root tips and apices ( removing cotyledons ) of five independent lines ( 10-day-old T2 seedlings ) of each of the lines used for ECT2-HT to use as biological replicates .",
"The tissue was flash-frozen in liquid nitrogen and ground into a fine powder using liquid nitrogen-cooled adaptors in a tissue homogenizer .",
"For RNA extraction , we added 1 mL of TRI Reagent ( Sigma ) to the frozen tissue ( <100 mg ) , mixed quickly by vortexing , added 0 . 2 mL of chloroform , and separated the two resulting phases by vigorous shaking and 10 min centrifugation at 4°C .",
"The RNA was then precipitated from the aqueous phase for 30 min at room temperature with 1 volume of isopropanol .",
"RNA pellets were solubilized in 300 μL of H2O to remove polysaccharides through a mild precipitation by addition of 1/10 vol .",
"99% EtOH and 1/30 vol .",
"of 3 M NaOAc ( pH 5 . 2 ) and incubation on ice for 30 min .",
"After 15 min of full-speed centrifugation at 4°C to pellet polysaccharides , we re-precipitated the RNA from the supernatant with 2 , 5× vol .",
"99% EtOH and 1/10 vol .",
"of 3 M NaOAc ( pH 5 . 2 ) , washed the pellet two times with 70% EtOH , and resuspended in 20–40 μL of H2O .",
"This highly pure total RNA was then used to produce mRNA libraries through enrichment of mRNA with oligo ( dT ) beads ( 18-mers ) , random fragmentation , cDNA synthesis with random hexamers , custom second-strand synthesis ( Illumina ) , terminal repair , A-ligation and sequencing adaptor ligation , size selection ( 250–300 bp insert ) , and PCR enrichment .",
"The libraries were prepared and sequenced ( Illumina PE150 , Q30 ≥ 80% ) as a service from Novogene .",
"The entire HyperTRIBE experiment was done once .",
"Significant differentially edited sites between ECT2-FLAG-ADAR ( fusion ) and FLAG-ADAR ( control ) samples for ECT2 HyperTRIBE ( ECT2-HT ) were called according to the hyperTRIBER pipeline ( Rennie et al . , 2021 ) .",
"First , reads were trimmed using trimmomatic ( Bolger et al . , 2014 ) and mapped to the Arabidopsis genome ( TAIR10 ) using STAR ( Dobin et al . , 2013 ) , according to parameters suggested in a previous HyperTRIBE analysis ( Xu et al . , 2018 ) .",
"All HyperTRIBE samples were also quantified using Salmon ( Patro et al . , 2017 ) , with appropriate settings for pair-end sequencing and non-stranded library setup and based on the transcriptome for Araport11 ( Cheng et al . , 2017 ) with manual addition of the FLAG-ADAR sequence .",
"A custom Perl script based on SAMtools mpileup ( Li et al . , 2009 ) returned base counts for all positions where there is a mismatch from the reference in at least one sample .",
"For running the hyperTRIBER analysis pipeline , we specified that any tested position must have a putative edit in at least four of the five replicates in the ECT2-FLAG-ADAR samples ( three of four in the case of roots since one of these samples , ‘L3 , ’ was deemed as low quality and subsequently removed from the significance calling pipeline ) .",
"Significant hits ( adjusted p-value<0 . 01 and log2FC > 1 ) were further filtered as follows: ( 1 ) hits that did not correspond to an A-to-G change ( or a T-to-C change for the negative strand ) , ( 2 ) hits that were likely SNPs arising specifically in either the ECT2-FLAG-ADAR or FLAG-ADAR line manifesting in an editing proportion at or close to 1 , and ( 3 ) hits where the coverage of tags at the edit base over the ECT2-FLAG-ADAR were fewer than 10 reads .",
"Specific scripts for the analysis of ECT2-HT data can be found at https://github . com/sarah-ku/targets_arabidopsis .",
"Editing proportions were calculated as G/ ( A + G ) ( alternatively C/ ( U + C ) for the negative strand ) for all significant sites , averaged over all samples , separately for the ECT2-FLAG-ADAR and FLAG-ADAR samples .",
"Significant sites were annotated to genes from Araport11 , prioritizing the gene with the highest expression ( annotated TPMs are based on Salmon quantifications of FLAG-ADAR control samples only ) in the given tissue in the case of multiple possibilities .",
"Possible transcripts were subsequently ordered by expression , along with gene body location along the transcript ( 5′-UTR , CDS , 3′-UTR ) .",
"Principal component analysis was carried out on the raw editing proportions per sample for all sites with significant evidence of editing .",
"For the comparison of sites between aerial tissues and roots , genes defined as commonly expressed in both types of tissues were considered in all gene-based comparisons .",
"For significant editing site-based comparison , we directly compared sites that were common and significant to both .",
"To calculate correlations between editing proportions and FLAG-ADAR expression levels among lines , transcripts per million ( TPM ) mapping to FLAG-ADAR were extracted from quantifications from Salmon ( Patro et al . , 2017 ) and correlated with the raw editing proportions per sample , separately for the fusion and control samples .",
"Background correlation estimates were calculated by first scrambling the order of the FLAG-ADAR TPM vector .",
"For motif identification at significant ECT2-HT sites , all sequences for bases at and -/+ 2 nt of the significant editing positions were derived from TAIR10 using the R package rtracklayer ( Lawrence et al . , 2009 ) in either aerial tissues or roots .",
"A matrix of nt frequencies ( A , C , G , or U ) was generated , and the R package ggseqlogo ( Wagih , 2017 ) was used to generate the final motif .",
"For the calculation of editing proportions as a function of the proportion of cells coexpressing ECT2 , we first downloaded the expression matrix based on a total of 4727 individual cells from scRNA-seq in roots from Denyer et al . , 2019 .",
"To estimate the relationship between coexpression of target genes with ECT2 and their average editing proportions , the expression matrix was used to calculate coexpression for each target gene as follows: ( # cells expressing ECT2 AND target gene ) / ( # cells expressing target gene ) .",
"These proportions were then split into groups and plotted against the maximum editing proportions from HyperTRIBE in the containing genes .",
"For expression binning , log2 ( TPM +1 ) values for all expressed genes in either aerial tissues , roots , or combined were split into nine bins of increasing expression , using the cut ( ) function from the R package Hmisc version 4 . 5-0 ( https://github . com/harrelfe/Hmisc/ ) .",
"For the proportion of target genes in every expression bin , we calculated the proportion of genes in each set ( ECT2 HT/iCLIP-targets or nontargets , ECT2 FA-CLIP; Wei et al . , 2018 ) or m6A sets ( Parker et al . , 2020 ) falling into each expression bin out of the total number of genes in that bin .",
"To demonstrate expression biases in unsupported ECT2-HT target genes , the genes were further split according to whether or not they had support from m6A ( Nanopore , miCLIP [Parker et al . , 2020] and m6A-seq [Shen et al . , 2016] ) .",
"In vivo UV crosslinking of 12-day-old seedlings and construction of iCLIP libraries were optimized for ECT2-mCherry from the method previously employed for Arabidopsis GRP7-GFP ( Meyer et al . , 2017; Köster and Staiger , 2020 ) as follows .",
"Crosslinked plant tissues ( see details below ) were finely ground in liquid nitrogen with mortar and pestle , homogenized in iCLIP buffer ( 50 mM Tris-HCl pH 7 . 5 , 150 mM NaCl , 4 mM MgCl2 , 5 mM DTT , 1% SDS , 0 . 25% sodium deoxycholate , 0 . 25% Igepal ) supplemented with protease inhibitors ( 4 mM PMSF , 1 tablet/10 mL of Complete Protease Inhibitor Cocktail [Roche] , and 1/30 vol . of Protease Inhibitor Optimized for Plant Extracts [Sigma P9599] ) , and cleared by centrifugation and filtration ( 0 . 45 μm pore ) of the supernatant .",
"RNP-complexes were then immunopurified with beads coupled to anti-RFP nanobodies ( ChromoTek RFP-Trap in our case ) for 1 hr at 4°C under constant rotation .",
"In particular , we used 20 μL of beads for 4 g of tissue in 6 mL of iCLIP buffer for every replicate .",
"After thorough washes with RIP-Wash Buffer ( 2 M urea , 50 mM Tris-HCl pH 7 . 5 , 500 mM NaCl , 4 mM MgCl2 , 2 mM DTT , 1% SDS , 0 . 5% sodium deoxycholate , 0 . 5% Igepal ) , RNP-complexes attached to the beads were subjected to treatment with DNase ( Turbo DNase [Ambion] , 4 U/100 μL ) and RNase I ( Ambion , 1 U/mL ) at 37°C for 10 min , dephosphorylation of RNA 3′ ends ( PNK [ThermoFisher] in pH 6 . 5 buffer ) , and 3′ RNA linker ligation ( L3-App linker [Huppertz et al . , 2014] and NEB HC RNA Ligase ) at 16°C overnight .",
"RNA was radioactively labeled at the 5′ end by PNK-mediated phosphorylation using γ-32P-ATP ( 20 min at 37°C ) .",
"The labeled RNP complexes were subjected to SDS-PAGE and blotting on a nitrocellulose membrane ( Protran BA-85 ) .",
"Pieces of membrane containing a size range of RNA species bound to the protein ( a smear above the expected molecular weight localized by autoradiography ) were excised and subjected to proteolysis ( 200 μg of Proteinase K [Roche] in 200 μL of PK buffer [100 mM Tris-HCl pH 7 . 4 , 50 mM NaCl , 10 mM EDTA] for 20 min at 37°C ) to release RNA bound to small peptides .",
"The RNA was then purified with TRI-Reagent ( Sigma ) and used to prepare sequencing libraries through the following steps: reverse transcription ( Superscript III , Invitrogen ) using a two-part cleavable DNA adapter complementary to the 3′ RNA linker as primer , gel purification and size selection of cDNA ( high , 120–200 nt; medium , 85–120 nt; low , 70–85 nt ) , circularization ( CircLigase II Epicentre ) , relinearization ( BamHI ) , and PCR amplification ( AccuPrime Supermix I , Invitrogen ) .",
"All steps were performed as described by Huppertz et al . , 2014 , and the amount of cycles in the final PCR was optimized to the amount of cDNA in each sample .",
"Notice that we introduced a few modifications in the original protocol ( Köster and Staiger , 2020 ) to account for ( 1 ) low abundance of ECT2 compared to AtGRP7 .",
"To obtain enough RNA , we increased the crosslinking energy and irradiated 12-day-old seedlings with 2000 mJ/cm2 of 254 nm UV light , harvesting roots and shoots ( 4 g of tissue per replicate ) to maximize the amount of purified ECT2-mCherry .",
"( 2 ) ECT2 sensitivity to proteolysis .",
"We did not pre-clear the lysates to reduce the incubation time , and we used high amounts of protease inhibitors during immunoprecipitation .",
"( 3 ) High molecular weight of ECT2-mCherry .",
"Due to the size of the protein , we required longer electrophoresis time and cooling ( 3 hr at 180 V with the tank on ice ) .",
"( 4 ) Different RNA-binding capacity of ECT2 .",
"Based on trials , we decided to adjust the RNase I treatment to 1 U/mL , incubating for 10 min at 37°C ( 5 μL of RNase I [Ambion , 100 U/μL pre-diluted 1:5000] in 100 μL ) .",
"Of note , the conditions indicated here were specifically used for library preparation .",
"Although we used the same conditions as default for CLIP experiments to assess ECT2 RNA-binding capacity , ECT2 sensitivity to proteolysis and ECT2-bound RNA sensitivity to RNase treatment , variations in buffer composition , incubation time , concentration of protease inhibitors , and/or RNase I are specified in the corresponding figure legends where necessary .",
"The entire iCLIP-seq experiment including three replicates of each group was done once .",
"Sequenced reads from all samples were investigated after each processing step with fastqc 0 . 11 . 5 ( https://www . bioinformatics . babraham . ac . uk/projects/fastqc/ ) .",
"Adapters at the 3′ end were trimmed using cutadapt version 1 . 16 ( Martin , 2011 ) .",
"The demultiplexing of the samples was performed using flexbar 3 . 4 . 0 with the -bk parameter to conserve the barcode information for further steps ( Roehr et al . , 2017 ) .",
"Reads with a length below 24 nucleotides were discarded .",
"Barcodes were trimmed and saved to the read_id field .",
"Processed reads were mapped to the TAIR10 genome with STAR version 2 . 6 . 0a allowing a maximum of two mismatches and soft clipping only at 3′ end ( Dobin et al . , 2013 ) .",
"PCR duplicates were removed by grouping the reads by their mapping start position .",
"Reads with the identical start position and random barcode were removed from the samples ( Python3 and pybedtools ) .",
"The peak calling of uniquely mapped reads was done using PureCLIP 1 . 0 . 4 , choosing the second peak-shape option to allow more broader peaks to be called ( Krakau et al . , 2017 ) .",
"For consistency with the ECT2-HT datasets , the ECT2-iCLIP datasets were annotated using the hyperTRIBER annotation ( Rennie et al . , 2021 ) , using quantifications based on the average of roots and aerial tissues from FLAG-ADAR samples in ECT2-HT ( to reflect that the ECT2-iCLIP data is based on whole seedlings ) .",
"To calculate the proportion of sites falling at each nucleotide , nucleotide sequences from the reference genome were first obtained from site coordinates for ECT2 iCLIP/m6A-Nanopore/ m6A-miCLIP using the R packages GenomicRanges ( Lawrence et al . , 2013 ) and rtracklayer ( Lawrence et al . , 2009 ) .",
"Nucleotide proportions were plotted using ggplot2 ( https://ggplot2 . tidyverse . org ) .",
"Single-cell expression data and marker genes associated with 15 clusters annotated to cell types in roots were downloaded from Denyer et al . , 2019 .",
"Single-nucleotide resolution locations of m6A sites ( defined according to Nanopore or miCLIP ) were downloaded from Parker et al . , 2020 .",
"Intervals defining m6A locations based on m6A-seq were downloaded from Shen et al . , 2016 , and intervals defining locations of ECT2-bound sites as determined by FA-CLIP were downloaded from Wei et al . , 2018 .",
"For consistency with HyperTRIBE and ECT2-iCLIP , all sets of m6A or ECT2-bound sites were gene annotated using the hyperTRIBER pipeline , based on genes and transcripts from Araport11 .",
"To remove redundancy after ECT2-iCLIP peak calling , directly adjacent peaks ( crosslink sites ) were grouped together and only the peak with the highest pureCLIP score ( dominant ) was kept .",
"The called peak position ( 1 nt resolution ) was extended by 4 nt up- and downstream to define a ‘collapsed crosslink site’ ( CSS ) with length 9 nt .",
"The center position marks the dominant called peak .",
"The extension of the peak positions was computed using bedtools version 2 . 27 . 1 ( Quinlan and Hall , 2010; Dale et al . , 2011 ) .",
"The collapsed ECT2-iCLIP crosslink sites and m6A-Nanopore sites ( Parker et al . , 2020 ) were used to find motifs significantly enriched by Homer ( Heinz et al . , 2010 ) using a variety of window sizes , settings , and backgrounds .",
"Motifs resulting from Homer searches were collated manually , and a range of variants of the consensus motif RRACH ( e . g . , RACH , DRAY , DRACH , URACH , DRACG ) were also added to the list , as well as various combinations of U-rich sequences ( e . g . , UUUUU , UNUNU , etc . ) , specific motifs found to be of interest in scientific literature ( e . g . , URUAY [Wei et al . , 2018] , GGAU [Anderson et al . , 2018] ) , and extra motifs that appeared of potential interest from manually browsing with IGV ( Robinson et al . , 2011 ) the sequence in the vicinity of iCLIP peaks ( e . g . , YYYYY , DRACUCU ) .",
"This resulted in a final list of 48 motifs for further analysis .",
"For each of the 48 motifs compiled from multiple sources , a custom PWM was generated based on local sequence frequencies around ECT2-iCLIP peaks and used as input to FIMO 5 . 1 . 1 ( Grant et al . , 2011 ) to detect genome-wide occurrences .",
"To generate PWMs , we used the formula PWMb , j = log2 [p ( b , j ) /p ( b ) ] , where p ( b ) is the background frequency of each nucleotide ( see further down ) , and p ( b , i ) is the frequency of the nucleotides in each position j .",
"We also included an extra small frequency count in the calculation to account for potential uncertainty in redundancies .",
"In order to account for location-specific sequence contexts ( typically 3′-UTR ) , each site from iCLIP or m6A ( Parker et al . , 2020 ) sets was assigned a random ‘matched background’ site , in a non-target gene , at the same relative location along the annotated genomic feature of the site ( 5′-UTR , CDS or 3′-UTR ) , according to a resolution of 10 bins per feature .",
"Logos for all motifs were generated using the R packages ggplot2 ( https://ggplot2 . tidyverse . org ) and ggseqlogo ( Wagih , 2017 ) .",
"To run the calculated PWMs through FIMO , we specified background letter frequencies ( A: 0 . 273 , C: 0 . 165 , G: 0 . 173 , U: 0 . 389 ) , a threshold of 0 . 05 , and scanning across the full TAIR10 Arabidopsis genome .",
"Sites were further filtered downstream to have a score of at least 4 – in the vast majority of cases corresponding to an exact match the ( short ) motif .",
"Distributions of motifs per 1000 sites over distance , centering on ECT2-iCLIP or m6A sites and the respective matched backgrounds , were generated using a custom R-script ( https://github . com/sarah-ku/targets_arabidopsis ) based on overlaps using GenomicRanges ( Lawrence et al . , 2013 ) .",
"At any given shift from the peak set , the raw number of overlaps of the motif ( at any point ) was calculated and normalized to give a motif count per 1000 peaks .",
"To adjust for the potential for downstream regions overshooting the end of the 3′-UTR , at each given distance only sites that continue to overlap an annotated gene ( Araport11 ) are counted .",
"For some analyses , peak sets were further split according to IDR-dependency or target status as indicated .",
"To calculate motif enrichment over the gene body , motifs were first annotated a value according to their relative position within the gene body regions: 5′-UTR , CDS or 3′-UTR .",
"In order to account for over-representation of counts within the CDS , due to greater sequence coverage within transcript annotations , a random background set of 10 million positions were generated from the transcript annotation file and annotated in the same way as the motif locations to obtain an expected distribution of all positions over the gene body regions .",
"This Expected distribution was used to normalize the Observed distribution of each motif , and O/E values were plotted as a metagene plot over the gene .",
"An enrichment of 1 suggests that the motif is neither over- or under-represented at that location .",
"Called positions from either Nanopore m6A data ( Parker et al . , 2020 ) or ECT2 iCLIP were first reduced to remove redundant regions of multiple peaks within the same window , then paired with matched background sets ( described above ) .",
"Windows representing ‘at’ ( ±10 nt ) the motif together with adjacent upstream ‘up’ and downstream ‘down’ windows of length 50 nt ( resulting in total window sizes of 120 nt ) around each position were annotated according to the number of each of the motifs overlapping ( truncated at 10 ) , and the final data set normalized .",
"To create a held-out set , 1/5th of the peaks were removed from the set , and the other 4/5th were used to build a random forest model using gradient boosting ( R package gbm version 2 . 1 . 8; https://github . com/gbm-developers/gbm ) , with settings specifying a shrinkage of 0 . 05 , an interaction . depth of 6 , cv . folds = 5 , and n . trees = 2000 .",
"For each model ( m6A Nanopore-based or ECT2 iCLIP-based ) , importance scores were extracted from the model and the top features were selected .",
"The held-out data was further used to estimate the predictive score of the model by calculating the AUC ( R package pROC; Robin et al . , 2011 ) .",
"Two further models were run – one involving the top 10 features from the full feature model , and ( only for the m6A-Nanopore set-up ) one involving features from only DRACH and UNUNU ( equating to six features in total ) , and AUC values were calculated and compared to that of the full feature model ."
]
] | [
"Specific recognition of N6-methyladenosine ( m6A ) in mRNA by RNA-binding proteins containing a YT521-B homology ( YTH ) domain is important in eukaryotic gene regulation .",
"The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU ( m6A ) Y sites , yet RR ( m6A ) CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity .",
"Here , we apply iCLIP ( individual nucleotide resolution crosslinking and immunoprecipitation ) and HyperTRIBE ( targets of RNA-binding proteins identified by editing ) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites .",
"Our analyses show that ECT2 does in fact bind to RR ( m6A ) CH .",
"Pyrimidine-rich motifs are enriched around , but not at m6A sites , reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions .",
"Such motifs , particularly oligo-U and UNUNU upstream of m6A sites , are also implicated in ECT2 binding via its intrinsically disordered region ( IDR ) .",
"Finally , URUAY-type motifs are enriched at ECT2 crosslink sites , but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins .",
"Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins ."
] | [
"Genes are strings of genetic code that contain instructions for producing a cell’s proteins .",
"Active genes are copied from DNA into molecules called mRNAs , and mRNA molecules are subsequently translated to create new proteins .",
"However , the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA .",
"Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production .",
"One of these methods involves adding a chemical tag , a methyl group , onto mRNA while it is being created .",
"These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation .",
"Most eukaryotic species – which include animals , plants and fungi – use the same system to add methyl tags to mRNA molecules .",
"One methyl tag in particular , known as m6A , is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants .",
"However , in the flowering plant Arabidopsis thaliana , ECT2 was thought to bind to an mRNA sequence different from the one normally carrying the chemical tag , creating obvious confusion about how the system works in plants .",
"Arribas-Hernández , Rennie et al . investigated this question using advanced large-scale biochemical techniques , and discovered that conventional m6A methyl tags are indeed used by ECT2 in Arabidopsis thaliana .",
"The confusion likely arose because the sequence ECT2 was thought bind is often located in close proximity to the m6A tags , possibly acting as docking stations for proteins that can influence the ability of ECT2 to bind mRNA .",
"Arribas-Hernández , Rennie et al . also uncovered additional mRNA sequences that directly interact with parts of ECT2 previously unknown to participate in mRNA binding .",
"These findings provide new insights into how chemical labels in mRNA control gene activity .",
"They have broad implications that extend beyond plants into other eukaryotic species , including humans .",
"Since this chemical labelling system has a major role in controlling plant growth , these findings could be leveraged in biotechnology applications to improve crop yields and enhance plant-based food production ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"cell biology"
] | A conserved function for pericentromeric satellite DNA | elife-34122-v2 | [
[
"Satellite DNA is AT-rich , non-coding , repetitive DNA that is abundant in centromeric and pericentromeric heterochromatin .",
"Unlike the satellite DNAs that comprise the vast majority of natural centromeres ( Willard , 1990; Sun et al . , 1997 , 2003 ) , the role of pericentromeric satellite DNA remains obscure: although function for a few satellite DNA repeats has been implied in certain cellular processes such as meiotic segregation of achiasmatic chromosomes , X chromosome dosage compensation and formation of lampbrush-like loops on the Y chromosome during male meiosis ( Yunis and Yasmineh , 1971; Bonaccorsi et al . , 1990; Dernburg et al . , 1996; Menon et al . , 2014 ) , a unifying theme for pericentromeric satellite DNA function remains elusive .",
"Moreover , highly divergent satellite DNA sequences even among closely related species has led to the idea that satellite DNA does not serve a conserved function and is mostly a selfish element or junk ( Doolittle and Sapienza , 1980; Walker , 1971 ) .",
"Pericentromeric satellite DNA repeats are proposed to be sources of genomic instability , as their misexpression is associated with the formation of genotoxic R-loops and DNA damage ( Zhu et al . , 2011; Zeller et al . , 2016; Zeller and Gasser , 2017 ) .",
"Most studies on pericentromeric heterochromatin have focused on the mechanisms to repress satellite DNA transcription , and accordingly , a clear rationale for the existence of most pericentromeric satellite DNA is still lacking .",
"Cytologically , it is well documented that pericentromeric satellite DNA from multiple chromosomes is clustered into chromocenters in interphase nuclei in diverse eukaryotes including Drosophila , mouse and plants ( Figure 1A ) ( Jones , 1970; Pardue and Gall , 1970; Gall et al . , 1971; Fransz et al . , 2002 ) .",
"While multiple factors such as epigenetic modifications and transcription of repetitive DNA from pericentromeric DNA sequences are known to be required for chromocenter formation ( Peters et al . , 2001; Probst et al . , 2010; Bulut-Karslioglu et al . , 2012; Pinheiro et al . , 2012; Hahn et al . , 2013 ) , the ultimate consequences of disrupted chromocenter formation has never been addressed , leaving the function of chromocenters unknown .",
"In this study , we explored the role of pericentromeric satellite DNA/chromocenters by studying multi-AT-hook proteins , D1 from Drosophila melanogaster and HMGA1 from mouse .",
"D1 and HMGA1 are known to bind specific pericentromeric satellite DNA , and we show that these proteins are required for chromocenter formation .",
"When chromocenters are disrupted in the absence of these proteins , cells exhibited a high frequency of micronuclei formation , leading to DNA breakage and cell death .",
"We show that micronuclei are formed during interphase by budding from the nucleus .",
"We further show that D1 binding to the target DNA sequence is sufficient to bring it to the chromocenter .",
"High-resolution imaging revealed chromatin threads positive for D1/HMGA proteins and satellite DNA that connect heterologous chromosomes .",
"Taken together , we propose that chromocenter formation via bundling of satellite DNA from heterologous chromosomes functions as a mechanism to encapsulate the full complement of the genome into a single nucleus .",
"We suggest that satellite DNA function as a critical constituent of chromosomes and may serve an evolutionarily conserved role across eukaryotic species ."
],
[
"D1 in Drosophila melanogaster and HMGA1 in mouse are multi-AT-hook proteins; D1 contains 10 AT-hooks , HMGA1 contains three AT-hooks and both proteins contain C-terminal acidic domains ( Aulner et al . , 2002 ) .",
"D1 and HMGA1 are known to bind the Drosophila {AATAT}n satellite DNA ( ~8% of the Drosophila male diploid genome ) and mouse major satellite DNA ( ~6% of the mouse genome ) , respectively ( Goodwin et al . , 1973; Rodriguez Alfageme et al . , 1980; Levinger and Varshavsky , 1982b; Levinger and Varshavsky , 1982a; Lund et al . , 1983 ) .",
"The {AATAT}n satellite is distributed across 11 loci on multiple chromosomes as visualized by DNA fluorescence in situ hybridization ( FISH ) on mitotic chromosome spreads ( Figure 1B ) ( Lohe et al . , 1993; Jagannathan et al . , 2017 ) .",
"However , it is typically clustered into a few foci in Drosophila interphase nuclei , colocalizing with the D1 protein ( Figure 1C ) .",
"The D1/{AATAT}n foci stained positively for H3K9me2 in interphase nuclei ( Figure 1C ) , a well-established characteristic of constitutive heterochromatin/chromocenters ( Guenatri et al . , 2004 ) .",
"Consistently , D1 localized near the centromere ( marked by Drosophila CENP-A , Cid ) on mitotic chromosome spreads ( marked by phospho-H3 S10 ) ( Figure 1D ) .",
"These results suggest that D1 is a chromocenter-localizing protein , via its binding to the {AATAT}n satellite DNA .",
"The mouse HMGA1 protein was originally identified as an abundant non-histone component of mammalian chromatin ( Goodwin et al . , 1973; Lund et al . , 1983 ) with subsequent studies demonstrating its binding to satellite DNA ( Strauss and Varshavsky , 1984; Radic et al . , 1992 ) .",
"Mouse major satellite , which is present in pericentromeric regions of all chromosomes ( Figure 1E ) ( Lyon and Searle , 1989 ) , clustered into DAPI-dense chromocenters positive for HMGA1 protein ( Figure 1F , Figure 1—figure supplement 1A , B ) , revealing an analogous relationship to D1/{AATAT}n satellite in Drosophila .",
"Interestingly , we found that Drosophila D1 protein localizes to major satellite/chromocenters when ectopically expressed in multiple mouse cell lines ( Figure 1G , Figure 1—figure supplement 1C , D ) , suggesting that D1 and HMGA1 may possess an orthologous and conserved function as satellite DNA/chromocenter-binding proteins .",
"We next examined the effects of D1 mutation and siRNA-mediated knockdown of HMGA1 on chromocenters .",
"We used two D1 alleles , D1LL03310 and D1EY05004 , which we show to be protein null alleles , evidenced by near-complete loss of anti-D1 antibody staining ( Figure 1—figure supplement 2A–C ) .",
"When these alleles were combined with the D1 deficiency allele , Df ( 3R ) BSC666 , it led to severe declustering of {AATAT}n satellite DNA ( Figure 1H–J , Figure 1—figure supplement 2D–E ) , suggesting that D1 is required for clustering of pericentromeric satellite DNA into chromocenters .",
"We observed D1’s requirement for chromocenter formation in multiple cell types ( Figure 1—figure supplement 2F–I ) , but we largely focused on spermatogonial cells , where the phenotypes ( such as cell death ) were most penetrant and severe .",
"We also examined the requirement for HMGA1 in mouse chromocenter formation .",
"Following siRNA-mediated knockdown of HMGA1 , which led to near complete loss of HMGA1 protein ( see Figure 2D , E and Figure 2—figure supplement 1A–B , D–E for efficiencies of HMGA1 knockdown ) , we observed chromocenter disruption in multiple mouse cell lines ( Figure 1K–M , Figure 1—figure supplement 2J–L ) .",
"These results suggest that D1 and HMGA1 have an orthologous function to organize pericentromeric satellite DNA into chromocenters .",
"To explore the function of chromocenters and satellite DNA , we examined the effects of D1 mutation/HMGA1 knockdown , which showed strikingly similar phenotypes .",
"We found that D1 mutation as well as siRNA-mediated HMGA1 knockdown in multiple mouse cell lines resulted in a significant increase in micronuclei formation ( Figure 2A–F , Figure 2—figure supplement 1A–F ) .",
"Micronuclei are known to have compromised nuclear envelope integrity , leading to DNA damage and catastrophic chromosomal rearrangement therein ( Crasta et al . , 2012; Hatch et al . , 2013 ) .",
"Therefore , we first examined a possible defect in nuclear envelope integrity in D1 mutant .",
"We found that loss of D1 led to breaching of the nuclear envelope both in major and micronuclei , visualized by the cytoplasmic leakage of nuclear GFP ( nlsGFP ) ( Figure 2G–I ) , suggesting that nuclear envelope integrity might be generally compromised .",
"Consistently , we observed mislocalization of nuclear envelope proteins in D1 mutant spermatogonia .",
"We frequently observed that lamin surrounded the nucleus incompletely in D1 mutant ( 1 . 9% in control ( n = 52 ) and 68 . 9% in D1 mutant ( n = 58 ) ) ( Figure 2J , K , arrows indicate lamin-negative regions on the nuclear membrane ) .",
"We also observed cytoplasmic ‘holes’ , which resemble the nucleus in that they exclude cytoplasmic proteins such as Vasa ( Figure 2K , arrowhead ) , but are devoid of nuclear lamin ( Figure 2K , arrowhead ) .",
"These ‘holes’ were often surrounded by an ER marker , which normally surrounds the nuclear envelope ( Figure 2J ) ( Dorn et al . , 2011 ) .",
"Similarly , Otefin , an inner nuclear membrane LEM-domain protein ( Barton et al . , 2014 ) , also showed perturbed localization ( 2 . 7% in control ( n = 109 ) and 24 . 5% in D1 mutant ( n = 106 ) ) ( Figure 2L , M , arrows indicate lamin/Otefin negative regions on the nuclear envelope while the arrowhead indicates Otefin-positive micronuclei ) .",
"Taken together , these results show that D1 mutant cells exhibit compromised nuclear envelope integrity , which is associated with micronuclei formation .",
"It has been shown that defects in nuclear envelope integrity can lead to extensive DNA damage in the major nucleus and micronuclei ( Crasta et al . , 2012; Hatch et al . , 2013; Zhang et al . , 2015; Denais et al . , 2016; Raab et al . , 2016 ) .",
"Nuclear envelope defects and extensive DNA damages therein lead to catastrophic chromosomal breaks/rearrangements termed chromothripsis ( Crasta et al . , 2012; Hatch et al . , 2013 ) .",
"Such catastrophic DNA breaks/rearrangements are speculated to lead to tumorigenesis ( Hatch and Hetzer , 2015 ) .",
"Consistent with defective nuclear envelope integrity , we observed extensive DNA damage ( revealed by γ-H2Av ) in both major and micronuclei ( Figure 3A–F , arrows point to damaged DNA in micronuclei in B and D ) .",
"Likely as a result of DNA damage and defective nuclear envelope integrity , D1 mutant testes rapidly degenerated ( Figure 3—figure supplement 1A , B ) .",
"When Omi , a gene required to promote germ cell death ( Yacobi-Sharon et al . , 2013 ) , was knocked down in D1 mutant testes , it restored the cellularity in D1 mutant testis ( Figure 3—figure supplement 1C–D ) , but the surviving cells showed a dramatic increase in DNA damage ( Figure 3—figure supplement 1E–F ) .",
"Under these conditions , we observed that surviving germ cells in D1 mutant testes showed a high frequency of chromosome breaks compared to control , revealed by FISH on metaphase chromosome spreads from spermatocytes ( 3 . 7% in control ( n = 27 ) vs . 15 . 8% in D1 mutant ( n = 57 ) ) ( Figure 3G , H , arrowheads indicate sites of chromosome breaks ) .",
"These results show that loss of D1/HMGA1 results in compromised nuclear envelope integrity , leading to extensive DNA damage and chromosomal breaks .",
"It has been shown that micronuclei form by lagging chromosomes ( Crasta et al . , 2012 ) .",
"Thus , we examined whether D1 mutation/HMGA1 knockdown resulted in mitotic chromosome segregation errors , causing micronuclei formation .",
"However , we did not observe an increase in lagging chromosomes in D1 mutant spermatogonia or HMGA1-depleted mouse cells ( Figure 4—figure supplement 1A–G ) , suggesting an alternative route for micronuclei formation .",
"Instead , time-lapse live observation showed that micronuclei formed by budding from the interphase nucleus both in Drosophila spermatogonia and mouse cells ( Figure 4A–D ) .",
"In Drosophila spematogonia , nuclear contents were visualized by a GFP-tagged nuclear protein , Df31 , and RFP-tagged histone H2Av .",
"Control cells stably maintained nuclear contents for a prolonged time period ( only 1 event of nuclear blebbing without concurrent micronuclei formation ( as detected by H2Av-RFP ) over 1552 min of live imaging ) ( Figure 4A ) .",
"In contrast , D1 mutant cells showed budding off of nuclear contents and micronuclei formation in interphase ( 15 nuclear breaches with eight micronuclei formed over 3427 min of live imaging with a total budding duration of 172 min ) ( Figure 4B ) .",
"Similarly , live imaging in mouse cells using the Hoechst DNA dye revealed that HMGA1 knockdown also resulted in micronuclei formation during interphase ( siControl – no micronuclei formation over 253 min of live observation , siHMGA1 – three micronuclei formed by budding over 5962 min of live imaging with a total budding duration of 310 min ) ( Figure 4C , D ) .",
"These results show that micronuclei in D1 mutant/HMGA1-knockdown cells are generated during interphase , via budding from the nucleus .",
"Based on the above results , we postulated that chromocenter formation , that is clustering of satellite DNA from multiple chromosomes , might be a mechanism to bundle heterologous chromosomes together to prevent individual chromosomes from floating out of the nucleus .",
"In this manner , the full set of chromosomes may be retained within a single nucleus .",
"In the absence of chromocenter formation , individual chromosomes may bud off the nucleus , leading to micronuclei formation .",
"Previous in vitro experiments indicated that HMGA1 is capable of crosslinking multiple DNA strands with individual AT-hooks binding AT-rich DNA strands ( Vogel et al . , 2011 ) .",
"Bundling of DNA in this manner by D1/HMGA1 could explain how pericentromeric satellite DNA from multiple chromosomes may be clustered to form chromocenters .",
"A few lines of evidence support this idea .",
"When Drosophila D1 was expressed in mouse cells , it localized to the chromocenter as described above ( Figure 1G ) , and its overexpression enhanced chromocenter formation in a dose-dependent manner ( Figure 5A–C ) : the higher the amount of D1 that was expressed in mouse cells , the fewer chromocenters per cell was observed ( i . e . more clustering ) .",
"These results suggest that D1 is sufficient to bundle its binding target , tethering it to chromocenter .",
"Consistent with this idea , we found that artificial tethering of D1 protein to euchromatic LacO repeat DNA sequences was sufficient to bring LacO repeats to the chromocenter .",
"D1 protein or D1-LacI fusion protein was expressed in a Drosophila strain in which LacO repeats are inserted in the distal regions of the 2nd chromosome ( Figure 5D , arrows ) .",
"In control spermatogonial cells expressing wild type D1 , LacO repeats were observed far away from the {AATAT}n satellite foci/chromocenters ( Figure 5E , G , arrow indicates site of LacO repeats in interphase nucleus ) .",
"However , in cells expressing the LacI-D1 chimeric protein , we observed recruitment of the LacO repeats close to {AATAT}n/chromocenters ( Figure 5F , G , arrow indicates site of LacO repeats recruited to the chromocenter ) , demonstrating that D1’s binding to a DNA sequence is sufficient to incorporate the target sequence into chromocenters .",
"Although it cannot be visualized how DNA strands from multiple chromosomes might be bundled in these interphase chromocenters , deconvolution microscopy of D1/HMGA1 proteins on early mitotic chromosomes revealed proteinaceous threads between chromatin in the process of condensation ( Figure 6A , B , arrows indicate D1/HMGA1 threads ) , which we speculate contributed to bundling of chromosomes in the previous interphase .",
"These threads were also detectable by DNA FISH against {AATAT}n and the mouse major satellite ( Figure 6C , D , dotted lines are alongside the satellite DNA threads ) , suggesting that satellite DNA bound by D1/HMGA1 can form threads .",
"These threads likely connect heterologous chromosomes , as we see threads between chromosomes that are clearly distinct in their morphology ( e . g . Figure 6C ) .",
"These D1/HMGA1 threads are reminiscent of ‘DNA fibers’ , which were observed among mitotic chromosomes , although their function has never been appreciated ( Takayama , 1975; Burdick , 1976; Kuznetsova et al . , 2007 ) .",
"Taken together , these results support a model , in which D1/HMGA1 bind their target sequences ( satellite DNA ) on multiple chromosomes and bundle them into chromocenters , likely via their multivalent DNA-binding domains ( multiple AT-hooks ) ( Figure 6E ) ."
],
[
"The function of chromocenters , as well as that of satellite DNA , has remained enigmatic , even though cytological association of pericentromeric satellite DNA into chromocenters was identified almost 50 years ago ( Jones , 1970; Pardue and Gall , 1970 ) .",
"Pericentromeric heterochromatin has most often been studied and discussed in the context of how to maintain its heterochromatic , repressed nature ( Nishibuchi and Déjardin , 2017 ) , based on the assumption that the underlying sequences are mostly selfish , which have negative phenotypic consequences when derepressed in cells ( Zeller and Gasser , 2017 ) .",
"Although satellite DNA’s function has been speculated and implicated in several examples ( Yunis and Yasmineh , 1971; Bonaccorsi et al . , 1990; Dernburg et al . , 1996; Menon et al . , 2014 ) , the non-coding nature and lack of conservation in repeat sequence among closely related species led to the idea that they are mostly junk DNA , serving no essential function ( Walker , 1971; Doolittle and Sapienza , 1980 ) .",
"Instead , we propose that satellite DNA is a critical constituent of eukaryotic chromosomes to ensure encapsulation of all chromosomes in interphase nucleus .",
"Our results may also explain why the sequences of pericentromeric satellite DNA are so divergent among closely related species , a contributing factor that led to their dismissal as junk .",
"Based on our model that pericentromeric satellite DNA serves as a platform for generating heterologous chromosome association to form chromocenters , the essential feature of satellite DNA is that they are bound by protein ( s ) capable of bundling multiple DNA strands .",
"If so , the underlying sequence does not have to be conserved .",
"Instead , the binding of satellite DNA by a chromocenter bundling protein may be a critical feature of pericentromeric satellite DNAs .",
"Based on this idea , chromocenter bundling proteins and pericentromeric satellite DNA may be co-evolving .",
"We observed perturbation of nuclear envelope integrity upon chromocenter disruption .",
"Understanding the mechanisms underlying perturbation of nuclear envelope integrity in D1 mutant awaits future investigation .",
"Previous studies have documented that cytoskeletal forces are transmitted to chromatin through nuclear envelope and external mechanical forces can cause temporary nuclear envelope breaches ( King et al . , 2008; Denais et al . , 2016; Hatch and Hetzer , 2016; Raab et al . , 2016 ) .",
"Therefore , we speculate that chromosome bundling in the form of chromocenter may help prevent cytoskeletal forces from shearing chromosomes and nuclear envelope: when chromosomes are not bundled , cytoskeletal forces may be transmitted to individual chromosomes and associated nuclear envelope , resulting in shearing of nuclear envelope , disrupting its integrity .",
"In summary , our study provides the first evidence for a conserved function of pericentromeric satellite DNA and chromocenters .",
"Our data suggest that the multi-AT-hook proteins , D1 and HMGA1 , play an evolutionarily conserved role in the formation of chromocenters , likely via their ability to bind and bundle satellite DNA from heterologous chromosomes .",
"Heterologous chromosome association , mediated by chromocenter-binding proteins , may represent a third mode of chromosomal ‘gluing’ after meiotic homologous pairing and sister chromatid cohesion .",
"Through heterologous association , the chromocenter plays a fundamental role in maintaining the full complement of the genome , which is divided into multiple chromosomes , into a single nucleus .",
"This function of the chromocenter may be conserved in eukaryotic species that contain pericentromeric satellite DNA , thereby bringing about a signature characteristic of eukaryotic cells ."
],
[
"All fly stocks were raised on standard Bloomington medium at 25°C .",
"The following fly stocks were used: D1EY05004 ( BDSC17340 ) , Df ( 3R ) BSC666 ( BDSC26518 ) , UAS-OmiRNAi ( BDSC55165 ) , UAS-GFP-nls ( BDSC4776 ) and UAS-GFP-ER-SR ( BDSC59042 ) were obtained from the Bloomington Drosophila stock center .",
"D1LL03310 ( DGRC140754 ) and Df31-GFP ( DGRC110806 ) were obtained from the Kyoto stock center .",
"nos-gal4 ( Van Doren et al . , 1998 ) , hs-flp;nos-FRT-stop-FRT-gal4 , UAS-GFP ( Salzmann et al . , 2013 ) , UAS-H2A-YFP ( Bellaïche et al . , 2001 ) and B1 LacO ( Vazquez et al . , 2002 ) have been previously described .",
"A stock containing B chromosomes , mtrm126 +B ( Bauerly et al . , 2014 ) , was a kind gift from Scott Hawley .",
"Chromocenter disruption was scored in Drosophila testes by assessing {AATAT}n morphology in GFP +cells that were generated as follows in control ( hs-flp; nos-FRT-stop-FRT-gal4 , UAS-GFP ) and D1 mutant ( hs-flp;nos-FRT-stop-FRT-gal4 , UAS-GFP;D1LL03310/Df ) flies .",
"Testes were dissected 24 hr following a 20 min heat shock at 37°C .",
"Chromocenters were considered disrupted in Drosophila and mouse when satellite DNA adopted thread-like morphology in interphase nuclei .",
"Micronuclei were scored in 0-3 d testes where early germ cell chromosomes were labeled with H2A-YFP .",
"The genotypes used were , control – nos > H2 A-YFP and D1 mutant – nos > H2 A-YFP; D1LL03310/Df .",
"For construction of UAS-GFP-D1 , the D1 ORF was PCR-amplified from cDNA using the following primer pair , 5’-GATCAGATCTATGGAGGAAGTTGCGGTAAAG-3’ and 5’-GATCCTCGAGTTAGGCAGCTACCGATTCGG-3’ .",
"The amplified fragment was subcloned into the BglII and XhoI sites of pUASt-EGFP-attB ( Salzmann et al . , 2013 ) resulting in UAS-GFP-D1 .",
"For UAS-GFP-LacI-D1 , the LacI ORF ( lacking 11 C-terminal residues ) ( Straight et al . , 1996 ) was synthesized using GeneArt ( Thermofisher ) and inserted into the BglII site of UAS-GFP-D1 resulting in UAS-GFP-LacI-D1 .",
"Transgenic flies were generated by PhiC31 integrase-mediated transgenesis into the attP40 site ( BestGene ) .",
"For expression of GFP and GFP-D1 in mouse cells , GFP and GFP-D1 was subcloned from pUASt-EGFP-attB into pCDNA3 ( gift from Cheng-Yu Lee ) using EcoRI and XhoI sites .",
"Mouse MOVAS cells were obtained from Daniel Eitzman .",
"Mouse C2C12 cells were obtained from David Bridges .",
"Mouse RAW264 . 7 cells were obtained from Dr . Harry Mobley .",
"Mouse C3H10T1/2 cells were obtained from Stephen Weiss .",
"MOVAS , C2C12 and RAW264 . 7 cells were maintained in Dulbecco's minimal essential medium ( DMEM ) ( Gibco ) supplemented with 10% fetal bovine serum ( FBS ) .",
"C3H10T1/2 cell line was maintained in alpha minimal essential media ( Gibco ) supplemented with 10% fetal bovine serum .",
"All cell lines used were authenticated as mouse cells by the presence of mouse-specific satellite DNA as is shown throughout the manuscript .",
"Two major cell lines used in this study , C2C12 and MOVAS cells , were treated with Plasmocin ( Invivogen ) prior to use as a precaution for mycoplasma infection .",
"RNA interference ( RNAi ) against HMGA1 was performed using ON-TARGET plus Mouse HMGA1 siRNA SMARTpool ( Dharmacon , L-049293–01 ) consisting of the following target sequences , CCAUUUAGCCGCAGCCCGA , AGGCAAACGGGCACCAACA , GGGCGCAGCAGACUGGUUA , GUUCAUUCUUAGAUACCCA .",
"ON-TARGET plus Non-targeting pool ( Dharmacon , D-001810–10 ) consisting of the following sequences , UGGUUUACAUGUCGACUAA , UGGUUUACAUGUUGUGUGA , UGGUUUACAUGUUUUCUGA , UGGUUUACAUGUUUUCCUA , was used as a negative control .",
"siRNA transfections were performed using DharmaFECT four reagent ( Dharmacon , Lafayette , CO ) according to the manufacturer's protocol .",
"25 nM of siControl and siHMGA1 were transfected using DharmaFECT 4 ( Dharmacon ) according to the manufacturer’s protocol .",
"Cells were fixed for immunostaining/in situ hybridization 6 days post-transfection .",
"Transient transfection of GFP and GFP-D1 was performed using Fugene HD ( Roche ) reagent according to the manufacturer’s protocol .",
"For Drosophila tissues , immunofluorescence staining was performed as described previously ( Cheng et al . , 2008 ) .",
"Briefly , tissues were dissected in PBS , transferred to 4% formaldehyde in PBS and fixed for 30 min .",
"Testes were then washed in PBS-T ( PBS containing 0 . 1% Triton-X ) for at least 60 min , followed by incubation with primary antibody in 3% bovine serum albumin ( BSA ) in PBS-T at 4°C overnight .",
"Samples were washed for 60 min ( three 20 min washes ) in PBS-T , incubated with secondary antibody in 3% BSA in PBS-T at 4°C overnight , washed as above , and mounted in VECTASHIELD with DAPI ( Vector Labs ) .",
"The following primary antibodies were used: rabbit anti-vasa ( 1:200; d-26; Santa Cruz Biotechnology ) , rabbit anti-H3K9 dimethyl ( 1:200; Abcam , ab32521 ) , guinea pig anti-Otefin ( gift from Georg Krohne , 1:400 ) , chicken anti-Cid ( 1:500 , generated using the synthetic peptide CDGENDANDGYVSDNYNDSESVAA ( Covance ) ) , mouse anti-LaminDm0 ( ADL84 . 12 , 1:200 , Developmental Studies Hybridoma Bank ) , mouse anti-γ−H2Av ( UNC93-5 . 2 . 1 , 1:400 , Developmental Studies Hybridoma Bank ) , Phalloidin-Alexa546 ( ThermoFisher , a22283 , 1:200 ) .",
"Adherent mouse cells were fixed in 4% formaldehyde in PBS for 20 min at room temperature on coverslips .",
"Cells were permeabilized in PBS-T for 5 min , rinsed three times with PBS , blocked using 3% BSA in PBS-T for 30 min at room temperature and incubated with primary antibody diluted in 3% BSA in PBS-T overnight at 4°C .",
"Cells were then washed for 30 min ( three 10 min washes ) , incubated with secondary antibody in 3% BSA in PBS-T for 2 hr at room temperature , washed as above and mounted in VECTASHIELD with DAPI ( Vector Labs ) .",
"For nucleoplasmic extraction , cells were incubated with CSK buffer ( 10 mM PIPES pH7 , 100 mM NaCl , 300 mM sucrose , 3 mM MgCl2 , 0 . 5% Triton X-100 , 1 mM PMSF ) for 10 min at room temperature .",
"After CSK extraction , cells were washed with PBS and fixed and immunostained as above .",
"The following antibodies were used: rabbit anti-HMGA1 ( 1:400 , Abcam , ab129153 ) , goat anti-LaminB ( C-20 ) ( 1:20 , Santa Cruz Biotechnology , sc-2616 ) , mouse anti-α-tubulin ( 4 . 3 , 1:100 , Developmental Studies Hybridoma Bank ) and γ−H2Ax S139 ( 2577 , 1:200 , Cell Signaling Technologies ) .",
"Images were taken using a Leica TCS SP8 confocal microscope with 63x oil-immersion objectives ( NA = 1 . 4 ) .",
"Deconvolution was performed when indicated using the Hyvolution package from Leica .",
"Images were processed using Adobe Photoshop software .",
"Testes from newly eclosed flies were dissected into Schneider’s Drosophila medium containing 10% fetal bovine serum .",
"The testis tips were placed inside a sterile glass-bottom chamber and were mounted on a three-axis computer-controlled piezoelectric stage .",
"An inverted Leica TCS SP8 confocal microscope with a 63 × oil immersion objective ( NA = 1 . 4 ) was used for imaging .",
"For mouse live cell imaging , transfected cells were seeded onto a sterile glass-bottom chamber coated with poly-lysine .",
"Cells were incubated with Hoechst 33342 for 10 min , rinsed with PBS and fresh medium was added to the chamber .",
"Cells were imaged using a stage-top Tokai-Hit incubator that was mounted on an inverted TCS SP5 confocal microscope with a 63x oil immersion objective ( NA = 1 . 4 ) .",
"All images were processed using Adobe Photoshop software .",
"Metrics used for quantification of live imaging were total imaging duration ( defined as number of cells x imaging duration ) , total budding duration ( defined as number of cells with micronuclei that formed by budding x time with budded micronuclei ) .",
"Whole mount Drosophila testes were prepared as described above , and optional immunofluorescence staining protocol was carried out first .",
"Subsequently , samples were post-fixed with 4% formaldehyde for 10 min and washed in PBS-T for 30 min .",
"Fixed samples were incubated with 2 mg/ml RNase A solution at 37°C for 10 min , then washed with PBS-T +1 mM EDTA .",
"Samples were washed in 2xSSC-T ( 2xSSC containing 0 . 1% Tween-20 ) with increasing formamide concentrations ( 20% , 40% and 50% ) for 15 min each followed by a final 30 min wash in 50% formamide .",
"Hybridization buffer ( 50% formamide , 10% dextran sulfate , 2x SSC , 1 mM EDTA , 1 μM probe ) was added to washed samples .",
"Samples were denatured at 91°C for 2 min , then incubated overnight at 37°C .",
"For mitotic chromosome spreads , testes and larval 3rd instar brains were squashed according to previously described methods ( Larracuente and Ferree , 2015 ) .",
"Briefly , tissue was dissected into 0 . 5% sodium citrate for 5–10 min and fixed in 45% acetic acid/2 . 2% formaldehyde for 4–5 min .",
"Fixed tissues were firmly squashed with a cover slip and slides were submerged in liquid nitrogen until bubbling ceased .",
"Coverslips were then removed with a razor blade and slides were dehydrated in 100% ethanol for at least 5 min .",
"After drying , hybridization mix ( 50% formamide , 2x SSC , 10% dextran sulfate , 100 ng of each probe ) was applied directly to the slide , samples were heat denatured at 95°C for 5 min and allowed to hybridize overnight at room temperature .",
"Following hybridization , slides were washed thrice for 15 min in 0 . 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .",
"For the in situ experiment described in Figure 4j–m , testes were dissected into PBS and fixed in 4% formaldehyde for 4 min .",
"Tips of fixed testes were firmly squashed with a cover slip and slides were submerged in liquid nitrogen until bubbling ceased .",
"Coverslips were removed with a razor blade and slides were subjected to 5 min washes in 2XSSC and 2XSSC with 0 . 1% Tween-20 .",
"The samples were denatured in freshly made 0 . 07N NaOH for 5 min , rinsed in 2X SSC .",
"Hybridization mix ( 50% formamide , 2x SSC , 10% dextran sulfate , 100 ng of each probe ) was added directly to the slide and allowed to hybridize overnight at room temperature .",
"Following hybridization , slides were washed three times for 15 min in 0 . 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .",
"The following probes were used for Drosophila in situ hybridization: {AATAT}6 , {AAGAG}6 , IGS and have been previously described ( Jagannathan et al . , 2017 ) .",
"LacO probe - 5’-Cy5-CCACAAATTGTTATCCGCTCACAATTCCAC-3’ .",
"For interphase mouse cells , optional immunostaining was carried out as above .",
"Subsequently , samples were post-fixed with 4% formaldehyde in PBS for 10 min and rinsed three times in PBS .",
"Post-fixed cells were incubated with 0 . 1 mg/ml RNase A solution at 37°C for 1 hr , rinsed three times in PBS and denatured in 1 . 9M HCl for 30 min .",
"After three rinses in ice-cold PBS , hybridization mix ( 2X SSC , 60% formamide , 5 µg/ml salmon sperm DNA and 500 nM probe ) was added to the samples and incubated overnight at room temperature .",
"Following hybridization , coverslips were washed three times for 15 min in 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .",
"For mouse mitotic cells , chromosomes were spread on slides as previously described .",
"Subsequently , chromosomes were denatured in 70% formamide in 2XSSC for 1 . 5 min at 70°C , dehydrated in 100% ethanol and hybridization mix ( 2X SSC , 60% formamide , 5 µg/ml salmon sperm DNA and 500 nM probe ) was added directly to the slide and incubated overnight at room temperature .",
"Following hybridization , slides were washed three times for 15 min in 2X SSC and mounted with VECTASHIELD with DAPI ( Vector Labs ) .",
"The following probe was used: Major satellite - 5’-Cy3-GGAAAATTTAGAAATGTCCACTG-3’ ."
]
] | [
"A universal and unquestioned characteristic of eukaryotic cells is that the genome is divided into multiple chromosomes and encapsulated in a single nucleus .",
"However , the underlying mechanism to ensure such a configuration is unknown .",
"Here , we provide evidence that pericentromeric satellite DNA , which is often regarded as junk , is a critical constituent of the chromosome , allowing the packaging of all chromosomes into a single nucleus .",
"We show that the multi-AT-hook satellite DNA-binding proteins , Drosophila melanogaster D1 and mouse HMGA1 , play an evolutionarily conserved role in bundling pericentromeric satellite DNA from heterologous chromosomes into ‘chromocenters’ , a cytological association of pericentromeric heterochromatin .",
"Defective chromocenter formation leads to micronuclei formation due to budding from the interphase nucleus , DNA damage and cell death .",
"We propose that chromocenter and satellite DNA serve a fundamental role in encapsulating the full complement of the genome within a single nucleus , the universal characteristic of eukaryotic cells ."
] | [
"On Earth , life is divided into three domains .",
"The smallest of these domains includes all the creatures , from sunflowers to yeasts to humans , that have the genetic information within their cells encased in a structure known as the nucleus .",
"The genomes of these organisms are formed of long pieces of DNA , called chromosomes , which are packaged tightly and then unpackaged every time the cell divides .",
"When a cell is not dividing , the chromosomes in the nucleus are loosely bundled up together .",
"It is well known that DNA is the blueprint for the building blocks of life , but actually most of the genetic information in a cell codes for nothing , and has unknown roles .",
"An example of such ‘junk DNA’ is pericentrometric satellite DNA , small repetitive sequences found on all chromosomes .",
"However , new experiments by Jagannathan et al . show that , in the nucleus of animal cells , certain DNA binding proteins make chromosomes huddle together by attaching to multiple pericentrometric satellite DNA sequences on different chromosomes .",
"In fact , if these proteins are removed from mice and fruit flies cells grown in the laboratory , the chromosomes cannot be clustered together .",
"Instead , they ‘float away’ , and the membranes of the nucleus get damaged , possibly buckling under the pressure of the unorganized DNA .",
"These events damage the genetic information , which can lead to the cell dying or forming tumors .",
"‘Junk DNA’ therefore appears to participate in fundamental cellular processes across species , a result that opens up several new lines of research ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | Epithelial magnesium transport by TRPM6 is essential for prenatal development and adult survival | elife-20914-v2 | [
[
"Mg2+ is the most abundant intracellular divalent cation and is essential for the regulation of a broad spectrum of metabolic and signalling pathways ( de Baaij et al . , 2015 ) .",
"In addition , direct association with Mg2+ fosters the structural integrity of key metabolites ( such as ATP ) , proteins , lipid membranes and nucleic acids ( de Baaij et al . , 2015 ) implying that organismal Mg2+ deficiency , a surprisingly common condition in humans ( King et al . , 2005; Rosanoff et al . , 2012 ) , may be especially harmful during prenatal development and early postnatal life , when the production of and the demand for Mg2+-bound metabolites is particularly high .",
"There is growing evidence to suggest that Mg2+ deprivation is accompanied by different types of metabolic , immune , cardiovascular and neurological disorders ( de Baaij et al . , 2015 ) .",
"However , mainly due to the lack of adequate mammalian genetic models , it still remains unclear whether an imbalance in Mg2+ metabolism is merely associated with or can directly trigger the latter pathophysiological processes .",
"Furthermore , it has recently been shown that cellular Mg2+ fluxes regulate the circadian rhythm and energy balance ( Feeney et al . , 2016 ) , CGRP-mediated osteogenic differentiation ( Zhang et al . , 2016 ) and synaptic plasticity ( Palacios-Prado et al . , 2014 ) , and that changes in the composition of brain interstitial Mg2+ concentrations participate in the control of the sleep-wake cycle ( Ding et al . , 2016 ) .",
"The remarkable recent progress in our understanding of the critical role of Mg2+ in health and disease contrasts with the dearth of knowledge about the mechanisms governing cellular and organismal Mg2+ balance .",
"Approximately 10 plasma membrane Mg2+ channels have been proposed ( Quamme , 2010 ) indicating a high degree of redundancy .",
"However , quite some controversy surrounds the biological role of many of these proteins , and the question whether there is a central gatekeeper responsible for organismal Mg2+ balance has not yet been answered .",
"The kinase-coupled ion channel TRPM7 has been proposed as a ubiquitous , indispensable cellular Mg2+ entry pathway ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010; Stritt et al . , 2016 ) .",
"However , studies with Trpm7 gene-deficient mice failed to confirm a corresponding in vivo role of Trpm7 .",
"Thus , constitutive inactivation of Trpm7 in mice entailed early embryonic lethality for as yet unknown reasons ( Jin et al . , 2008 ) .",
"Furthermore , tissue-specific deletions of Trpm7 in mouse embryos affected morphogenesis of internal organs apparently in a Mg2+-independent manner ( Jin et al . , 2008 , 2012; Sah et al . , 2013 ) .",
"More recently , it was suggested that the Mg2+ transporter MagT1 rather than TRPM7 might play a critical role for Mg2+ homeostasis in T lymphocytes ( Li et al . , 2011 ) and probably also in the whole embryo ( Zhou and Clapham , 2009 ) .",
"Hence , the biological role of TRPM7 requires further clarification .",
"In the present work , we focussed on the closest TRPM7 relative , TRPM6 , because loss-of-function mutations in TRPM6 cause hypomagnesemia ( low Mg2+ blood levels ) in human infants thought to mainly result from renal Mg2+ wasting ( Schlingmann et al . , 2002; Walder et al . , 2002; Voets et al . , 2004 ) .",
"However , deletion of Trpm6 in mice has resulted in neural tube closure defects and embryonic death ( Walder et al . , 2009 ) indicating a direct role of TRPM6 in developmental processes and calling into question the simplistic view on the human TRPM6 phenotype .",
"By integrating systematic phenotyping of Trpm6 gene-modified mice with biochemical analysis , gene expression , metabolomics , and cell biological approaches , we decipher the molecular and organismal roles of TRPM6 in prenatal development and postnatal survival ."
],
[
"To understand the role of Trpm6 in prenatal development , we determined the onset of embryonic lethality in Trpm6 null embryos and investigated the expression pattern of Trpm6 at this stage .",
"Using a mouse strain carrying a gene-trap mutation in Trpm6 ( Trpm6βgeo ) ( Table 1 ) , we found that Trpm6βgeo/βgeo embryos were present at embryonic days ( e ) 8 . 5–10 . 5 ( Figure 1A ) .",
"However , only a few mutants were found between e11 . 5–12 . 5 and no Trpm6βgeo/βgeo individuals were viable after e14 . 5 ( Figure 1A ) .",
"Compared to e9 . 5 C-shaped Trpm6+/+ individuals , all Trpm6βgeo/βgeo embryos isolated had not turned ( S-shaped ) and were smaller indicating a developmental retardation after e8 . 5 ( Figure 1B ) .",
"Consequently , we investigated the expression pattern of Trpm6 in e8 . 5 fetuses by in situ hybridization ( ISH ) and found that Trpm6 was specifically expressed in the visceral yolk sac endoderm and extraembryonic chorion ( Figure 1C ) and that Trpm6 was not detectable in the neural tube ( Figure 1—figure supplement 1 ) .",
"Within the placental labyrinth a network of maternal sinusoids are intertwined with fetal blood capillaries , separated by two layers of transporting trophoblast cells , syncytiotrophoblasts I ( SynT-I ) and II ( SynT-II ) ( Simmons and Cross , 2005; Simmons et al . , 2008 ) .",
"At e8 . 5 , morphogenesis of the labyrinth is in the initial stages and SynT-I/SynT-II cell layers are distinguishable ( Simmons and Cross , 2005; Simmons et al . , 2008 ) .",
"We observed that Trpm6 expression was restricted to SynT-I cells ( Figure 1D ) .",
"In the fully maturated labyrinth at e14 . 5 Trpm6 mRNA was detected in syncytiotrophoblasts as well ( Figure 1E ) . 10 . 7554/eLife . 20914 . 003Figure 1 . Assessment of Trpm6 function in extraembryonic tissues .",
"( A ) Survival of Trpm6βgeo/βgeo embryos obtained from Trpm6βgeo/+ intercrosses .",
"( B ) Representative images of e9 . 5 Trpm6+/+ ( +/+ , n = 13 ) and Trpm6βgeo/βgeo ( βgeo/βgeo , n = 5 ) embryos from dataset in ( A ) .",
"Dashed lines underline C-shaped versus S-shaped morphology of Trpm6+/+ and Trpm6βgeo/βgeo embryos , respectively .",
"( C ) ISH on serial paraffin sections obtained from wildtype n = 5 e8 . 5 fetus using antisense ( left ) and sense ( right ) probes for Trpm6 .",
"Boxes indicate the positions of the magnified images of the chorion ( ch ) and yolk sac ( yc ) .",
"Arrows indicate Trpm6-positive cells in the developing labyrinth ( chorion ) and the endoderm layer in the visceral yolk sac .",
"( D ) ISH on serial paraffin sections of wildtype e8 . 5 placenta using DIG-labelled probes for Trpm6 ( left ) , SynA ( middle ) and Gcm1 ( right ) , respectively .",
"Note: Trpm6 expression was restricted to cells positive for SynA , a marker of SynT-I , and absent in cells expressing Gcm1 , a marker of SynT-II .",
"Representative images of n = 2 independent tissues are shown .",
"( E ) ISH of WT e14 . 5 placenta with the antisense Trpm6 probe .",
"The box indicates the position of the magnified image .",
"The Trpm6 signal is restricted to the labyrinth ( lab ) and not detectable in the decidua ( dec ) and trophoblast giant cells ( GT ) .",
"Representative images of n = 8 independent placentas are shown .",
"( F ) Mg2+ levels in e9 . 5 Trpm6+/+ ( n = 4 ) and Trpm6βgeo/βgeo ( n = 3 ) embryos .",
"Distal segments of the embryos were used for genotyping , and the remaining parts were analysed by ICP-MS .",
"Elementary magnesium ( Mg ) contents were normalized to phosphorus ( P ) and sulfur ( S ) levels represented as mean±SEM .",
"*-p≤0 . 05 ( Student’s t-test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00310 . 7554/eLife . 20914 . 004Figure 1—figure supplement 1 . ISH on serial paraffin sections obtained from wildtype e8 . 5 fetus using antisense ( left ) and sense ( right ) probes for Trpm6 . Arrows indicate Trpm6-positive cells in the chorion ( blue arrows ) and the endoderm layer in the visceral yolk sac ( black arrows ) stained only by the antisense probe .",
"Note: Trpm6 was not detectable in embryonic tissues including the neural tube ( red arrows ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00410 . 7554/eLife . 20914 . 005Table 1 . Postnatal survival of the mice with global and tissue-restricted deletions of Trpm6 . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 005Targeted tissueBreeding strategyExpected F1 outcome* Survival of the mutantConstitutive mutagenesisWhole fetus♂Trpm6βgeo/+ x ♀Trpm6βgeo/+ 25%Trpm6βgeo/βgeo 50% Trpm6βgeo/+ 25% Trpm6+/+ noWhole fetus♂Trpm6Δ17/+ x ♀Trpm6 Δ17/+ 25% Trpm6 Δ17/Δ17 50% Trpm6 Δ17/+ 25% Trpm6+/+ noWhole fetus♂Trpm6Δ17/+ x ♀Trpm6βgeo/+ 25% Trpm6βgeo/Δ17 25% Trpm6βgeo/+ 25% Trpm6Δ17/+ 25% Trpm6+/+ noConditional mutagenesis using Cre/LoxP systemEpiblast♂Trpm6Δ17/+;Sox2-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/Δ17;Sox2-Cre 25% Trpm6Δ17/fl 25% Trpm6Δ17/+;Sox2-Cre 25% Trpm6fl/+ yesIntestine♂Trpm6Δ17/+;Villin1-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/fl;Villin1-Cre† 25% Trpm6Δ17/fl 25% Trpm6fl/+;Villin1-Cre 25% Trpm6fl/+ yesKidney♂Trpm6Δ17/+;Ksp-Cre x ♀Trpm6fl/fl 25% Trpm6Δ17/fl;Ksp-Cre† 25% Trpm6Δ17/fl 25% Trpm6fl/+;Ksp-Cre 25% Trpm6fl/+ yes*Genotypes were determined using genomic DNA extracted from tail fragments .",
"†Individuals were homozygous for Trpm6Δ17 allele in the targeted cells .",
"Syncytiotrophoblasts and endoderm cells of the yolk sac exchange metabolites between the maternal and fetal blood ( Simmons and Cross , 2005 ) .",
"To clarify whether TRPM6 is required for Mg2+ supply by extraembryonic tissues , we used inductively coupled plasma mass spectrometry ( ICP-MS ) and found that relative magnesium ( Mg2+ ) levels were reduced in the whole e9 . 5 Trpm6βgeo/βgeo embryos ( Figure 1F ) .",
"Thus , Trpm6 is specifically expressed in the placental labyrinth and the yolk sac at the stage when the Mg2+ deficiency and growth delay of Trpm6-deficient embryos become apparent .",
"To investigate whether TRPM6 activity in extraembryonic cells underlies the lethality of Trpm6 null embryos , we characterized a mouse strain with a ‘floxed’ ( Trpm6fl ) allele ( Table 1 ) .",
"Cre-mediated excision engendered viable mice heterozygous for the constitutive deletion mutation in Trpm6 ( Trpm6Δ17/+ ) .",
"However , we were unable to produce live Trpm6βgeo/Δ17 or Trpm6Δ17/Δ17 offspring , indicating that Trpm6∆17 is a true null mutation ( Table 1 ) .",
"The paternally inherited Sox2-Cre transgene drives recombination only in epiblast cells , but not in extraembryonic tissues ( Hayashi et al . , 2003 ) .",
"Notably , intercrosses of Trpm6Δ17/+;Sox2-Cre males and Trpm6fl/fl females resulted in viable Trpm6Δ17/Δ17 pups at the expected ratio ( Table 1 ) .",
"Therefore , the embryonic mortality of Trpm6-deficient mice appears to be caused by the loss of TRPM6 in extraembryonic tissues .",
"We next studied the impact of a global deletion of Trpm6 postnatally .",
"Examination of Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre ) mice at weaning did not reveal conspicuous abnormalities .",
"However , during the follow-up period , we observed the gradual development of pathologies .",
"Thus , Trpm6-deficient mice had a lifespan of no longer than 16 weeks ( Figure 2A ) .",
"Mutants were growth-delayed , and displayed a lighter fur colour and low night-time activity ( Figure 2B , C ) .",
"Weight gain and lean body mass of Trpm6-deficient mice were reduced ( Figure 2D , E ) , as was the muscle fibre area of the gastrocnemius muscle of 12–13 week-old Trpm6-deficient mice indicative of sarcopenia ( Figure 2F ) .",
"Mutant mice displayed kyphosis ( Figure 2G ) and completely lacked abdominal and subcutaneous fat depots ( Figure 2H , I ) indicative of catabolic metabolism .",
"However , the total amount of faeces ( Figure 2—figure supplement 1A ) and the calorimetrically determined faecal energy content , as a measure of energy excretion ( Figure 2—figure supplement 1B ) , were not altered in Trpm6 mutants , ruling out insufficient food intake .",
"Histological analysis of internal organs ( Figure",
"3 ) showed that Trpm6-deficient mice developed lung emphysema and degeneration of lymphoid organs .",
"Thus , the thymus of mutant mice was rudimentary and the cortex region was not distinguishable .",
"In the spleen of Trpm6-deficient mice , the red pulp was substantially reduced .",
"Hepatocytes of Trpm6-deficient mice were depleted of glycogen granules ( Figure 3 ) , corroborating catabolic metabolism .",
"It has been suggested that low serum Mg2+ and TRPM6 function are associated with atherosclerosis in humans ( Maier , 2012; Tin et al . , 2015 ) .",
"Therefore , we investigated whether such a phenotype would develop in our mouse model as well .",
"However , examination of thoracal aorta showed no signs of atherosclerosis development in mutant mice ( Figure 2—figure supplement 2 ) . 10 . 7554/eLife . 20914 . 006Figure 2 . Pathophysiological changes displayed by Trpm6-deficient adult mice . Unless stated otherwise , 10–12 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were studied .",
"( A–E )",
"Mice were examined for survival rate ( A ) , overall physical appearance ( B ) , day/night activity of 8 week-old individuals ( C ) , growth rate ( D ) and lean mass ( E ) .",
"( F ) Fibre size of the gastrocnemius muscle after hematoxylin-eosin staining .",
"( G ) X-ray images of mice .",
"The red arrow indicates the characteristic skeletal deformation ( kyphosis ) observed in Trpm6-deficient mice .",
"( H ) Assessment of abdominal fat .",
"Arrows indicate fat deposits observed only in control mice .",
"( I ) H and E staining of paraffin skin sections .",
"Arrows indicate a layer of fat cells present only in control mice .",
"Histological analysis was performed with three animals per group resulting in similar observations .",
"( J ) The levels of main elements in the serum of 8 week-old mice assessed by ICP-MS .",
"( K ) The survival rate of mice maintained on high Mg2+ ( 0 . 75% ) and regular ( 0 . 22% ) chows .",
"Data are represented as mean±SEM .",
"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00610 . 7554/eLife . 20914 . 007Figure 2—figure supplement 1 . Examination of energy balance in Trpm6-deficient mice . Eight week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were evaluated for ( A ) chow intake as assessed by feces production , ( B ) energy content of feces studied by bomb calorimetry and ( C ) serum levels of β-hydroxybutyrate .",
"Glucose ( D ) and insulin levels ( E ) in the serum of mice subjected to a glucose tolerance test .",
"Note: mutant mice had lower peripheral glucose concentrations than controls despite of a similar amount of insulin released , thus reflecting increased insulin sensitivity .",
"Data are represented as mean±SEM .",
"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00710 . 7554/eLife . 20914 . 008Figure 2—figure supplement 2 . Evaluation of atherosclerosis development in Trpm6-deficient mice . An assessment of 8 week-old control ( Control , n = 3 ) and Trpm6-deficient ( KO , n = 3 ) with ApoE-/- mice ( as a positive control , n = 1 ) using en-face thoracal aorta preparation and Oil-Red O staining ( A ) and hematoxylin-eosin staining ( B ) , Mac2-staining ( green ) ( C ) of aortic arches .",
"Representative images are shown .",
"Arrows indicate atherosclerosis lesions observed only in ApoE-/- mice .",
"( D ) Plasma cholesterol levels ( mean±SEM ) of control and Trpm6-deficient mice .",
"n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 00810 . 7554/eLife . 20914 . 009Figure 3 . Histology of internal organs of Trpm6-deficient mice . Hematoxylin-eosin staining of paraffin embedded tissue sections of 12–13 week-old control ( Control ) and Trpm6-deficient ( KO ) mice maintained either on regular ( 0 . 22% Mg2+ ) or Mg2+ supplemented ( 0 . 75% Mg2+ ) chows .",
"Trpm6-deficient mice maintained on the regular diet showed marked airspace enlargement ( indicated by stars ) mimicking lung emphysema , distortion of splenic red pulp ( rp ) / white pulp ( wp ) microarchitecture , thymic atrophy , and reduction of intracellular glycogen in hepatocytes ( indicated by arrows ) .",
"Histological analysis was performed with three animals per group resulting in similar observations . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 009 Next , we asked whether the phenotype of Trpm6-deficient mice is caused by Mg2+ deficiency .",
"We employed ICP-MS to compare the concentrations of main elements in serum from controls and Trpm6-deficient littermates .",
"We found that , similar to humans with mutations in the TRPM6 gene ( Schlingmann et al . , 2002; Walder et al . , 2002 ) , Trpm6-deficient mice developed hypomagnesemia ( Figure 2J ) .",
"Serum Mg2+ levels of mutant mice were only 0 . 58 mM ( 36% of control value , 1 . 58 mM ) , whereas concentrations of other elements were not changed ( Figure 2J ) .",
"A Mg2+-enriched diet is an efficient way to alleviate hypomagnesemia in humans lacking TRPM6 ( Schlingmann et al . , 2002; Walder et al . , 2002 ) .",
"Therefore , we asked whether the phenotypes of Trpm6-deficient mice were caused by Mg2+ deprivation and could be rescued by dietary supplementation .",
"To this end , we changed the regular chow ( 0 . 22% Mg2+ ) of 4 week-old mutant mice and control littermates for a Mg2+ enriched diet ( 0 . 75% Mg2+ ) .",
"Notably , none of the Trpm6-deficient mice died during the following 12 weeks of Mg2+ supplementation ( Figure 2K ) .",
"However , returning to regular chow resulted in 100% mortality of mutant mice within the following 13 weeks ( Figure 2K ) .",
"Mg2+ supplemented mutants neither exhibited kyphosis nor lipodystrophy ( data not shown ) .",
"Furthermore , the morphology of the lung , spleen , and thymus of Mg2+ supplemented mutants closely resembled that of control mice ( Figure 3 ) .",
"We asked whether dietary Mg2+ supplementation of Trpm6βgeo/+ parents would benefit the survival of Trpm6-deficient offspring .",
"However , similar to a previous study ( Walder et al . , 2009 ) , we found that this treatment was inefficient .",
"Trpm6-deficient adult mice phenocopy salient pathologies reported for a set of mouse strains advocated as genetic models of ‘accelerated’ or ‘premature’ aging ( Kuro-o et al . , 1997; Trifunovic et al . , 2004; Kujoth et al . , 2005; Varela et al . , 2005; Mostoslavsky et al . , 2006; Niedernhofer et al . , 2006; van der Pluijm et al . , 2007; López-Otín et al . , 2013 ) .",
"Similar to Trpm6-deficient mice , the latter mutants display short lifespan , growth failure , low physical activity , kyphosis , lung emphysema , sarcopenia , lipodystrophy and degeneration of lymphoid organs .",
"A characteristic feature of these mouse strains is suppression of the somatotropic axis accompanied by induction of xenobiotic detoxification gene networks in the liver ( Niedernhofer et al . , 2006; van de Ven et al . , 2006; van der Pluijm et al . , 2007; Schumacher et al . , 2008; Garinis et al . , 2009; Mariño et al . , 2010 ) , interpreted as a defensive organismal response , slowing down growth and metabolism in favor of somatic preservation ( López-Otín et al . , 2013 ) .",
"We asked whether Trpm6-deficient mice would also display such protective metabolic responses .",
"In fact , we found that serum IGF1 concentrations were reduced in Trpm6-deficient mice as well ( Figure 4A ) .",
"Mutant mice had a lower core body temperature ( Figure 4B ) and a profoundly reduced urinary content of major urinary proteins ( MUPs ) ( Figure 4C ) , two known features of supressed IGF1 signalling ( Mariño et al . , 2010; Bartke et al . , 2013 ) .",
"Even though mutant mice showed signs of overall energy shortage , circulating levels of ketone bodies ( ß-hydroxybutyrate ) were not elevated ( Figure 2—figure supplement 1C ) .",
"When subjected to an oral glucose tolerance test , mutant mice displayed lower peripheral glucose concentrations than controls despite of a similar amount of insulin released , thus reflecting increased insulin sensitivity ( Figure 2—figure supplement 1D , E ) , another hallmark of suppressed IGF1 signalling ( Bartke et al . , 2013 ) .",
"Notably , body weight and IGF1 serum levels were indistinguishable in Mg2+ supplemented mutant and control mice suggesting that the variations observed in mice maintained on a regular diet were induced by Mg2+ deficiency ( Figure 2—figure supplement 1F , G ) . 10 . 7554/eLife . 20914 . 010Figure 4 . Assessment of metabolic profiles of Trpm6-deficient mice .",
"( A–C ) 8 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermates were evaluated for ( A ) serum IGF1 , ( B ) body temperature , ( C ) urinary MUPs content in individual mice .",
"Data are represented as mean±SEM .",
"***-p≤0 . 001; *-p≤0 . 05 ( Student’s t-test ) ; n – number of mice examined .",
"( D ) IPA analysis of genome-wide hepatic transcriptome profiling of Trpm6-deficient ( n = 3 ) vs control ( n = 4 ) littermates .",
"The diagram shows the top 5 of IPA Canonical Pathways significantly changed in mutant mice ( Supplementary file 2 ) .",
"Numbers of the commonly changed transcripts are indicated close to the lines connecting the pathways .",
"( E ) Venn diagram for sets of metabolites significantly changed ( FDR p≤0 . 05 ) in serum , liver and gastrocnemius muscle Trpm6-deficient ( n = 6 ) vs control ( n = 8 ) littermates ( Supplementary file 3 ) .",
"Commonly changed metabolites are listed in different colours as outlined in the Venn diagram .",
"( F–J )",
"Levels of AC C18:1 ( F–H ) and AC C18 ( I–J ) examined in the serum ( F , I ) , liver ( G ) and gastrocnemius muscle ( H , J ) of Trpm6-deficient and control mice .",
"Data are represented as mean±SEM .",
"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different to control group maintained on a regular Mg2+ diet ( one-way ANOVA ) ; n – number of mice examined .",
"( K ) ATP production by mitochondria isolated from the liver of wildtype C57BL/6 mice with succinate , palmitoylcarnitine or octanoylcarnitine as energy sources .",
"ATP levels were determined after 30 min incubation of untreated ( - ) or treated ( + ) mitochondria by EDTA with or without Mg2+ .",
"Data are represented as mean±SEM of 4–5 independent isolations ( N ) .",
"##-p≤0 . 01; #-p≤0 . 05 significantly different to the control group; ***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05 significantly different to the EDTA treated group ( Student’s t-test ) .",
"n . s . – not significantly different . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01010 . 7554/eLife . 20914 . 011Figure 4—figure supplement 1 . Gene expression profiling of Trpm6-deficient and control mice . Heatmap diagram of differently expressed genes with FDR p≤0 . 1 in the liver of 12–13 week-old control ( Trpm6fl/+ , n = 4 ) vs Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 3 ) male littermates .",
"n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01110 . 7554/eLife . 20914 . 012Figure 4—figure supplement 2 . Metabolomic profiling of Trpm6-deficient and control mice . Profiling of metabolites in the serum , liver and gastrocnemius muscle samples from 8–10 week-old control ( Trpm6fl/+ , n = 8 ) and KO ( Trpm6Δ17/Δ17;Sox2-Cre , n = 6 ) male littermates were studied for a panel of 237 metabolites outlined in Supplementary file 3 .",
"The heatmap diagram shows concentration levels scaled to zero mean and unit standard deviation of significantly changed metabolites for control and KO genotypes in a colour-coded way .",
"n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01210 . 7554/eLife . 20914 . 013Figure 4—figure supplement 3 . Assessment of the membrane potential ( Δψm ) in isolated mitochondria .",
"( A ) Mitochondria isolated from the liver of wildtype C57BL/6 mice were incubated ( 30 min ) in the presence of succinate/rotenone , octanoylcarnitine/malate or palmitoylcarnitine/malate as energy source .",
"The left panel shows representative measurements of Δψm using Rh123 probe .",
"As a positive control , carbonyl cyanide 4- ( trifluoromethoxy ) phenylhydrazone ( FCCP ) was added at the end of recording to induce breakdown of Δψm .",
"The right panel shows the calculated start- and endpoints of Δψm elicited by the application of 3 mM EDTA in the absence or presence of 1–5 mM Mg2+ for traces shown in left panel .",
"Data are represented as mean±SD .",
"N – independent mitochondria isolations; n – independent measurements .",
"***-p≤0 . 001 , **-p≤0 . 01 , *-p≤0 . 05 significant to the untreated ( control ) group; ###-p≤0 . 001 , ##-p≤0 . 01 , #-p≤0 . 05 significant to 3 mM EDTA + 0 mM Mg2+ group; ‡‡‡-p≤0 . 001 , ‡‡-p≤0 . 01 , ‡-p≤0 . 05 significant to 3 mM EDTA + 1 mM Mg2+ group ( Student’s t-test ) .",
"( B ) Representative measurements of ψm in the presence of 3 mM EDTA with/without 3–5 mM Ca2+ or Zn2+ performed analogously to ( A ) .",
"Three independent experiments showed similar results . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 013 Finally , we performed whole-genome profiling of the liver transcriptome ( Supplementary file 1 ) .",
"Applying a cut-off value of p≤0 . 1 for the false discovery rate ( FDR ) , we identified 46 genes up- or down-regulated in the livers of Trpm6-deficient mice ( Figure 4—figure supplement 1 , Supplementary file 1 ) .",
"The majority of affected transcripts code for solute carrier transporters , cytochrome P450 metabolising enzymes , glutathione S-transferases and proteins metabolizing steroids .",
"As expected , Ingenuity Pathway Analysis ( IPA ) revealed that the inactivation of Trpm6 is associated with an induction of interconnected gene networks controlling toxicity responses and xenobiotic metabolism governed by nuclear receptors such as retinoid X receptors ( RXR ) , liver X receptor ( LXR ) and farnesoid X receptor ( FXR ) ( Figure 4D , Supplementary file 2 ) .",
"Hence , Trpm6-deficient mice were characterized by suppression of the somatotropic axis and induction of xenobiotic detoxification responses .",
"To gain mechanistic insight into the altered energy metabolism of mutant mice , we quantified serum , liver and skeletal muscle levels of 237 metabolites ( Supplementary file 3 , Figure 4—figure supplement 2 ) .",
"Unexpectedly , alterations in Trpm6-deficient mice ( FDR p≤0 . 1 ) were restricted to only several metabolites representing mainly long-chain acylcarnitines ( AC ) , phosphatidylcholines ( PC ) , and sphingomyelins ( SM ) ( Figure 4E ) .",
"These findings , as well as the results of gene array profiling ( Figure 4D ) suggest that sustained Mg2+ deficiency triggers a specific metabolic response rather than widespread unsystematic changes .",
"In line with this idea , we noted that AC C18:1 and the related AC C18 were consistently increased in tissues of Trpm6-deficient mice ( Figure 4F–J ) , whereas carnitine levels were not changed ( Supplementary file 3 ) .",
"In conjunction with lowered concentrations of glucose and unchanged ketogenesis ( Figure 2—figure supplement 1C ) , this constellation is a metabolic ‘signature’ of a frequent inherited human disorder characterized by inefficient β-oxidation of fatty acids due to mitochondrial carnitine palmitoyltransferase II deficiency ( Gempel et al . , 2002; Bonnefont et al . , 2004 ) .",
"AC C18:1 and C18 levels were normalized in serum and tissues obtained from Mg2+ supplemented mutants ( Figure 4F–J ) , implying that metabolic changes of AC were caused by Mg2+ deficiency .",
"Consequently , we asked whether Mg2+ would specifically affect mitochondrial ATP production ( Figure 4K ) and maintenance of the mitochondrial membrane potential ( MMP ) ( Figure 4—figure supplement 3A ) .",
"To address this question , wildtype liver mitochondria were incubated in a buffer containing 3 mM EDTA with or without 1–5 mM Mg2+ .",
"Such manipulations had no negative effect on mitochondrial respiration when succinate was offered as an energy source ( Figure 4K , Figure 4—figure supplement 3A ) .",
"In contrast , mitochondria failed to utilize octanoylcarnitine or palmitylcarnitine for ATP production ( Figure 4K ) and to maintain MMP ( Figure 4—figure supplement 3A ) in the presence of EDTA .",
"Importantly , ATP production and MMP could be fully rescued by the administration of 3–5 mM Mg2+ ( Figure 4K , Figure 4—figure supplement 3A ) , but not Zn2+ or Ca2+ ( Figure 4—figure supplement 3B ) .",
"Thus , sustained Mg2+ deprivation impairs energy homeostasis resulting in a catabolic metabolism in Trpm6-deficient mice , at least partially due to insufficient mitochondrial utilization of AC .",
"Next , we investigated the etiology of hypomagnesemia in Trpm6-deficient mice .",
"~50% of body Mg2+ content is stored in bones , ~30% in muscle tissues and only ~1% in the serum ( de Baaij et al . , 2015 ) .",
"Using ICP-MS we studied the Mg2+ content in bones ( right tibia ) and gastrocnemius muscle , and observed that Mg2+ levels in bones of Trpm6 null mice were only 24% of control values ( Figure 5A ) .",
"Furthermore , the Mg2+ content of muscle was also significantly reduced in Trpm6-deficient mice ( Figure 5B ) .",
"Hence , Trpm6-deficient mice develop a severe systemic Mg2+ deficit . 10 . 7554/eLife . 20914 . 014Figure 5 . Examining of Mg2+ balance in Trpm6-deficient adult mice .",
"( A–F )",
"Assessment of 8 week-old Trpm6fl/+ ( Control ) and Trpm6Δ17/Δ17;Sox2-Cre ( KO ) littermate males .",
"( A ) Mg2+ levels in bones and ( B ) gastrocnemius muscle assessed by ICP-MS .",
"( C ) Immunostaining of kidney cryosections using a TRPM6-specific antibody .",
"Representative images are shown ( n = 2 tissues per genotype ) .",
"The blue square indicates the position of the confocal and differential interference contrast magnified images acquired from control tissue .",
"Arrows indicate labelling of the apical surface of renal tubules .",
"( D ) 24 hr urinary and ( E ) fecal Mg2+ excretion rates .",
"( F ) ISH on paraffin sections obtained from the colon of control and Trpm6-deficient mice ( n = 2 tissues per genotype ) .",
"( G–J )",
"Examination of 6 month-old Trpm6fl/+ ( Control ) and Trpm6Δ17/fl;Ksp-Cre ( Kidney KO ) littermate males .",
"( G ) Immunostaining of TRPM6 in kidney cryosections .",
"Arrows indicate labelling of renal tubules .",
"( H–I )",
"Determination of Mg2+ in serum ( H ) and bones ( I ) .",
"( J ) 24 hr urinary Mg2+ excretion rate .",
"( K–N )",
"Assessment of 6 month-old Trpm6fl/+ ( Control ) and Trpm6Δ17/fl;Villin1-Cre ( Intestine KO ) littermate males .",
"( K ) ISH on paraffin sections of the colon using a Trpm6-specific probe ( n = 2 tissues per genotype ) .",
"( L , M )",
"Mg2+ levels in the serum ( L ) and bones ( M ) .",
"( N ) 24 hr urinary Mg2+ excretion rate .",
"Data are represented as mean±SEM .",
"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) ; n – number of mice examined .",
"Histological analysis in ( F ) and ( K ) was performed with n = 3 animals per group resulting in similar observations . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01410 . 7554/eLife . 20914 . 015Figure 5—figure supplement 1 . Expression pattern of Trpm6 in the intestine . ISH on serial paraffin sections obtained from wildtype intestine using sense ( left ) and antisense ( right ) DIG-labelled probes for Trpm6 .",
"Boxes indicate the positions of the magnified images of the proximal and distal colon .",
"Representative images of n = 3 tissues are shown .",
"Arrows indicate Trpm6-positive cells in the colon .",
"Note: Trpm6 transcripts were not detectable in the duodenum and ileum and specifically present in the absorptive epithelium cells of the proximal and distal colon . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 015 It is generally assumed that the distal convoluted tubule ( DCT ) of the kidney is critical for whole-body Mg2+ balance ( de Baaij et al . , 2015 ) .",
"Accordingly , immunostaining of control kidney cryosections with a TRPM6-specific antibody labelled nephron segments resembling DCT ( Figure 5C ) .",
"TRPM6 was not detectable in the kidneys of Trpm6-deficient mice .",
"Surprisingly , mutant mice exhibited substantially reduced urinary Mg2+ excretion ( only 14% of control values , Figure 5D ) , whereas fecal Mg2+ loss was significantly increased ( 166% , Figure 5E ) , indicating that mice lacking TRPM6 develop Mg2+ deficiency primarily due to impaired intestinal Mg2+ uptake .",
"Therefore , we studied the expression pattern of Trpm6 in the intestine .",
"Because the TRPM6-specific antibody did not efficiently and specifically detect TRPM6 protein in the intestine , we resorted to ISH ( Figure 5—figure supplement 1 ) .",
"Trpm6 transcripts were not detectable in the small intestine , but Trpm6-specific signal was observed in absorptive epithelial cells of the colon ( Figure 5—figure supplement 1 ) .",
"The colon of mutant mice was not stained by a Trpm6-specific ISH probe ( Figure 5F ) , consistent with the notion that systemic Mg2+ deficit in Trpm6-deficient mice was primarily caused by a defect of Mg2+ uptake in the colon .",
"To directly assess the contribution of the kidney versus intestine to the Trpm6 null phenotype , we employed Ksp-Cre ( Shao et al . , 2002 ) and Villin1-Cre ( Madison et al . , 2002 ) transgenic mice to specifically ablate floxed Trpm6 alleles in renal and intestinal epithelial cells , respectively ( Table 1 ) .",
"Surprisingly , conditional Trpm6 inactivation in the kidney neither impacted serum Mg2+ concentration nor urinary Mg2+ excretion , and bone Mg2+ content was only slightly reduced ( Figure 5G–J ) .",
"In contrast , disruption of Trpm6 in the intestine resulted in hypomagnesemia , reduced bone Mg2+ content and lowered urinary Mg2+ excretion ( Figure 5K–N ) , indicating that wildtype kidneys are not able to compensate for the ablation of intestinal TRPM6 .",
"Hence , in contrast to current thinking , our findings support the new concept that Trpm6-dependent Mg2+ uptake in the intestine plays an indispensable role for systemic Mg2+ balance .",
"TRPM6 is invariably co-expressed with the TRPM7 channel and the question as to why TRPM6 function is non-redundant remains central to a mechanistic understanding of the Trpm6 null phenotype .",
"Because Trpm6 expression levels in the epithelial cells of the colon ( Figure 5—figure supplement",
"1 ) and the kidney ( Figure 5C ) are highly heterogeneous , we searched for an alternative native cell model to dissect the functional interplay of TRPM6 and TRPM7 .",
"Trophoblast stem ( TS ) cells are widely used to study the transport function of placental trophoblasts ( Tanaka et al . , 1998; Simmons and Cross , 2005 ) and , as shown before ( Figure 1 ) , this cell type is crucial for the fetal Trpm6 phenotype .",
"Therefore , we derived Trpm6+/+ and Trpm6-deficient ( Trpm6βgeo/βgeo ) TS cells from e3 . 5 blastocysts isolated from Trpm6βgeo/+ parents ( Figure 6—figure supplement 1A ) .",
"We also produced Trpm7+/+ and Trpm7-deficient ( Trpm7Δ17/Δ17 ) TS cells ( Figure 6—figure supplement 2A ) using Trpm7Δ17/+ mice ( Jin et al . , 2008 ) .",
"As expected , RT-PCR analysis revealed that wildtype TS cells expressed TRPM6 and TRPM7 ( Figure 6—figure supplement 1B , Figure 6—figure supplement 2B ) .",
"Trpm6βgeo/βgeo TS cells were maintained in culture for >40 passages .",
"Furthermore , analysis of DNA content showed that the proportion of polyploidy was similar in Trpm6+/+ and Trpm6βgeo/βgeo TS cells ( Figure 6—figure supplement 1C , D ) , suggesting that inactivation of Trpm6 did not affect the self-renewal of Trpm6βgeo/βgeo stem cells .",
"In contrast , Trpm7Δ17/Δ17 TS cells did not proliferate , unless the cell culture medium was supplemented with additional Mg2+ ( Figure 6—figure supplement 2C ) , supporting the concept that TRPM7 plays a pivotal role in cellular Mg2+ uptake that cannot be maintained by TRPM6 alone ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010 ) .",
"TRPM6 and TRPM7 have been suggested as molecular correlates of MgATP- and Mg2+-regulated cation currents responsible for the cellular uptake of divalent cations including Mg2+ ( Aarts et al . , 2003; Nadler et al . , 2001; Schmitz et al . , 2003; Chubanov et al . , 2004; Voets et al . , 2004; Zhang et al . , 2014 ) .",
"Patch-clamp analysis showed that Trpm6βgeo/βgeo TS cells display substantially reduced TRPM6/M7-like outward currents at +80 mV ( Figure 6A ) .",
"Due to permeation block by extracellular divalent cations ( Nadler et al . , 2001; Fleig and Chubanov , 2014 ) , inward currents at physiological membrane potentials were very small ( Figure 6A; p≤0 . 001 , t-test ) .",
"However , exposure of TS cells to a divalent cation free ( DVF ) solution resulted in large monovalent cation currents ( Figure 6B ) .",
"Under these conditions , mutant TS cells exhibited a comparable reduction of inward ( p≤0 . 01 , t-test ) as well as outward ( p≤0 . 05 , t-test ) monovalent currents ( Figure 6B ) . 10 . 7554/eLife . 20914 . 016Figure 6 . Characterization of TRPM6/M7-like currents in Trpm6- and Trpm7-deficient TS cells .",
"( A ) Left panel: Whole-cell currents measured at −80 mV and +80 mV over time in Trpm6+/+ ( n = 22 ) and Trpm6βgeo/βgeo ( n = 22 ) TS cells .",
"Middle panel: Representative current-voltage relationships obtained at 12 s and 400 s .",
"Right panel: Bar graphs of current amplitudes at +80 mV ( 400 s ) .",
"( B ) Measurements were performed in control ( n = 16 ) and Trpm6-deficient ( n = 14 ) TS cells analogous to ( A ) except that the external saline ( containing 2 mM Mg2+ and 1 mM Ca2+ ) was exchanged with divalent-free ( DVF ) solution ( black bar ) .",
"Right panel shows currents measured before ( filled bars ) and after application of DVF solution ( open bars ) at 400 s and 600 s , respectively .",
"( C , D )",
"Dose-dependent inhibition of currents ( +80 mV , 400 s ) by [MgATP]i and [Mg2+]i , respectively ( n = 4–18 cells per concentration ) .",
"( E , F )",
"Whole-cell currents of Trpm7+/+ ( n = 15 ) and Trpm7Δ17/Δ17 ( n = 10 ) TS cells studied similar to ( A , B ) .",
"Data are represented as mean±SEM .",
"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( Student’s t-test ) .",
"n – number of cells examined .",
"( G ) A suggested model for the molecular role of TRPM6 in epithelial cells . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01610 . 7554/eLife . 20914 . 017Figure 6—figure supplement 1 . Characterization of Trpm6-deficient TS cells .",
"( A ) Phase-contrast images of Trpm6+/+ ( +/+ ) and Trpm6βgeo/βgeo ( βgeo/βgeo ) TS cells cultured in a regular cell culture medium .",
"( B ) Expression of Trpm6 and Trpm7 in TS cells assessed by RT-PCR .",
"( C , D )",
"Assessment of self-renewal of Trpm6+/+ and Trpm6βgeo/βgeo cells .",
"( C ) Diploid ( 2N ) , tetraploid ( 4N ) , and polyploid ( 8–16N ) DNA content was analysed by flow cytometry of Trpm6+/+ and Trpm6βgeo/βgeo TS cells stained with propidium iodide ( PI ) .",
"( D ) Bar graphs showing DNA contents ( mean+/-SEM ) calculated from three independent experiments outlined in ( A ) .",
"n . s . – not significantly different ( Student’s t-test ) .",
"Note: 2N DNA content ( diploid cells in G1 ) , 4N ( diploid cells in G2 or tetraploid cells in G1 ) and 8–16N ( spontaneously differentiated polyploid trophoblasts ) were not significantly altered in Trpm6-deficient TS cells indicating that self-renewal of Trpm6βgeo/βgeo cells was not affected . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01710 . 7554/eLife . 20914 . 018Figure 6—figure supplement 2 . Examination of TS cells deficient in Trpm7 . ( A ) Phase-contrast images of Trpm7+/+ ( +/+ ) and Trpm7Δ17/Δ17 ( Δ17/Δ17 ) TS cells cultured in a cell culture medium supplemented by 10 mM Mg2+ .",
"( B ) RT-PCR analysis of Trpm7 and Trpm6 in TS cells and mouse intestine ( positive control ) .",
"( C ) Proliferation rate of Trpm7+/+ ( +/+ ) and Trpm7Δ17/Δ17 ( Δ17/Δ17 ) TS cells in standard and Mg2+ ( 10 mM ) supplemented medium .",
"The experiment was repeated three times .",
"Student’s t-test was applied for comparison of Trpm7+/+versus Trpm7Δ17/Δ17 datasets . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 01810 . 7554/eLife . 20914 . 019Figure 6—figure supplement 3 . Evaluation of human haploid leukaemia ( HAP1 ) cells deficient in TRPM7 . ( A ) Phase-contrast images of parental ( WT ) and TRPM7-deficient ( KO ) HAP1 cells cultured in a cell culture medium supplemented with 10 mM Mg2+ .",
"( B ) Western-blot analysis of TRPM7 in WT and KO HAP1 cells .",
"( C ) Whole-cell currents in WT and KO HAP1 cells ( determined as described in Figure 6 ) .",
"Left panel: currents measured at −80 mV and +80 mV over time in WT ( n = 9 ) and KO ( n = 4 ) HAP1 cells .",
"Data are represented as mean±SEM .",
"Right panel: Representative current-voltage relationships obtained at 300 s .",
"( D ) Proliferation rate of WT and KO HAP1 cells either in standard or in Mg2+ ( 10 mM ) supplemented medium .",
"The experiment was repeated three times ( n = 3 ) .",
"Data are represented as mean±SEM .",
"Student’s t-test was applied for comparison of the growth rates of WT versus KO cells cultured in standard medium ( ***-p≤0 . 001 ) .",
"( E ) Determination of total Mg content in WT and KO HAP1 cells .",
"Dried cell pellets ( n = 4 for each genotype ) were obtained from WT and KO HAP1 cells cultured for 24 hr in standard medium and analysed by ICP-MS .",
"Elementary magnesium ( Mg ) content was normalized to sulfur ( S ) levels and represented as mean±SEM .",
"***-p≤0 . 001 ( Student’s t-test ) .",
"( F ) Assessment of total ATP levels in WT and KO HAP1 cells cultured for 24 hr in standard medium .",
"ATP-induced luminescent of luciferase ( CellTiter-Glo2 . 0 reagent ) was normalized to a number of viable cells ( Cell Counting Kit-8 ) .",
"The normalized luminescent signal in WT HAP1 cells was designated 100% .",
"The experiment was repeated six times ( n = 6 ) .",
"**-p≤0 . 01 ( Student’s t-test ) .",
"( G–H )",
"Routine respiration rate ( G ) and maximal respiration rate ( H ) analysed by Oxygraph-2k in WT and KO HAP1 cells cultured for 24 hr in standard cell culture medium ( n – number of independent experiments ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 019 Cytosolic levels of free Mg2+ ( [Mg2+]i ) and MgATP ( [MgATP]i ) have been suggested to exert a negative feedback mechanism on TRPM6/M7 channel activity ( Fleig and Chubanov , 2014 ) .",
"We observed ( Figure 6C ) that currents in Trpm6βgeo/βgeo TS cells were more susceptible to concentration-dependent inhibition by cytosolic MgATP ( p≤0 . 0001 , F-test ) .",
"The calculated IC50 value for Trpm6+/+ was 3 . 17 mM ( Hill ( h ) slope = −1 . 81 ) .",
"Currents in Trpm6βgeo/βgeo cells were inhibited by [MgATP]i with an IC50 value of 1 . 45 mM ( h = −1 . 95; p≤0 . 0015 , F-test ) .",
"These results suggest that physiological concentrations of [MgATP]i varying between 2–7 mM in mammalian cells ( Günther , 2006; Romani , 2011 ) will affect currents in Trpm6+/+ and Trpm6βgeo/βgeo cells differently .",
"In contrast , we observed no significant differences in [Mg2+]i concentration-response curves for Trpm6+/+ and Trpm6βgeo/βgeo currents ( p=0 . 62 , F-test ) ( Figure 6D ) .",
"The obtained IC50 value for Trpm6+/+ currents was 0 . 60 mM ( h = −1 . 14 ) and was not significantly altered in Trpm6βgeo/βgeo TS cells ( 0 . 72 mM , h = −1 . 18; p=0 . 22 , F-test ) , suggesting that physiological concentrations ( 0 . 3–1 mM , ( Günther , 2006; Romani , 2011 ) ) of [Mg2+]i will exert similar effects on ion currents in Trpm6+/+ and Trpm6βgeo/βgeo cells .",
"Next , we asked whether TRPM6 channel activity would be detectable in the absence of TRPM7 .",
"Remarkably , we observed that Trpm7Δ17/Δ17 TS cells completely lacked TRPM6/M7-like currents ( Figure 6E , F ) .",
"These findings cogently support our model ( Chubanov et al . , 2004 ) that native TRPM6 primarily functions in close cooperation with TRPM7 .",
"TRPM6 facilitates Mg2+ uptake by increasing the amplitude of TRPM7-like currents and relieving TRPM7 from the negative feedback by MgATP ( Figure 6G ) .",
"Finally , we studied whether Mg2+ deprivation may affect energy metabolism at the cellular level .",
"We chose the genetically tractable human haploid leukaemia cell line ( HAP1 cells ) ( Essletzbichler et al . , 2014; Blomen et al . , 2015; Wang et al . , 2015 ) as a new model system .",
"CRISPR/Cas9-mediated ablation of the TRPM7 protein in HAP1 cells ( Figure 6—figure supplement 3A , B ) completely abolished TRPM7-like currents ( Figure 6—figure supplement 3C ) .",
"When cultured in standard medium for 24 hr , TRPM7-deficient cells were characterized by a reduced total cellular Mg2+ content and a Mg2+-dependent proliferation defect ( Figure 6—figure supplement 3D , E ) .",
"In addition , TRPM7-deficient HAP1 cells had reduced intracellular ATP levels ( Figure 6—figure supplement 3F ) .",
"Finally , the respiration rate of TRPM7-deficient HAP1 cells was significantly lower when compared to control cells ( Figure 6—figure supplement 3G , H ) .",
"Our studies with Trpm6-deficient mice clearly demonstrated that a sustained disruption of Mg2+ homeostasis is detrimental for overall health and eventually reduces the lifespan of affected animals .",
"Conversely , we asked whether a Mg2+-enriched diet might exert a beneficial effect on the lifespan of wildtype mice .",
"Since there are no prior reports on lifespan extension of mice subjected to life-long dietary Mg2+ supplementation ( or any other mineral ) , we performed proof-of-principle experiments to investigate , if such an effect can be observed .",
"To this end , we used only the long-lived B6C3F1 hybrid mouse strain to avoid genotype-specific effects on disease susceptibility observed in longevity experiments with inbred strains ( Lipman et al . , 1999; Turturro et al . , 1999; Mitchell et al . , 2016 ) .",
"At this stage , we studied only females because of a larger number of early losses of males due to fighting ( Miller et al . , 2007 ) .",
"Finally , we studied animals only under pathogen-free conditions , since Mg2+ may elicit a protective effect via the immune system ( Brandao et al . , 2013; Chaigne-Delalande et al . , 2013 ) .",
"Consistent with published reports ( Turturro et al . , 1999 ) , the mean lifespan of B6C3F1 mice ( 886 days regarded as 100% ) was significantly extended ( 1100 days , 125% ) by dietary restriction ( DR ) ( Figure 7A , B , Table 2 ) .",
"Remarkably , supplementation with three Mg2+ salts ( Mg ( CH3COO ) 2 , Mg ( OH ) 2 and MgCl2 ) increased the mean lifespan of mice by approximately 10% ( 976 , 976 and 961 days , respectively ) .",
"The nutritional CaCl2 administration was without any significant effect ( 828 days ) .",
"In contrast to DR , animals supplemented with Mg2+ had a normal or even increased body weight ( Figure 7C ) , ruling out the possibility that high dietary Mg2+ affected the lifespan of mice due to reduced food intake .",
"Hence , opposite to Trpm6-dependent Mg2+ deprivation , dietary Mg2+ supplementation may have a beneficial effect on the lifespan of mice suggestive future large-scale longevity studies with varied conditions and species . 10 . 7554/eLife . 20914 . 020Figure 7 . Effects of whole-life Mg2+ dietary treatments on B6C3F1 mouse strain .",
"( A ) Mean survival ages of B6C3F1 mice maintained at a control diet ( Control , n = 335 ) , under dietary restriction ( DR , n = 60 ) , or supplemented by Mg ( CH3COO ) 2 ( MgAc , n = 15 ) , Mg ( OH ) 2 ( n = 15 ) , MgCl2 ( n = 15 ) and CaCl2 ( n = 15 ) in drinking water as outlined in Table 2 .",
"Pooled Mg shows results for all Mg2+ supplemented mice pooled within a common group ( n = 45 ) .",
"The obtained survival distributions were analysed by the MATLAB computing environment to calculate mean lifespans and corresponding P-values: ***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different .",
"Alternatively , survival data of control mice versus individually treated groups were assessed by log-rank test: ###-p≤0 . 001; ##-p≤0 . 01; #-p≤0 . 05; n . s . – not significantly different .",
"( B ) Kaplan-Meier survival distributions of B6C3F1 mice maintained on control diet ( Control ) , Mg2+ supplemented groups ( MgAc , MgCl2 , Mg ( OH ) 2 ) or mice under dietary restriction ( DR ) .",
"( C ) Body weights ( mean+/-SEM ) of control and nutritionally fortified mice studied in ( A ) .",
"***-p≤0 . 001; **-p≤0 . 01; *-p≤0 . 05; n . s . – not significantly different ( one-way ANOVA ) .",
"n – number of mice examined . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 02010 . 7554/eLife . 20914 . 021Table 2 . Dietary regimes used to maintain B6C3F1 mice . DOI: http://dx . doi . org/10 . 7554/eLife . 20914 . 021Experimental groupChow 5 K54 Drinking water ( ad libitum ) Number of miceControlad libitumRegular water* 335Dietary restriction60% of ad libitumRegular water60Magnesium acetate supplementedad libitum1 g/l Mg ( CH3COO ) 2 . ( H2O ) 4 in regular water15Magnesium chloride supplementedad libitum1 g/l MgCl2 in regular water15Magnesium hydroxide supplementedad libitum14 mg/l Mg ( OH ) 2 in regular water† 15Calcium chloride supplementedad libitum0 . 8 g/l CaCl2 in regular water15*Regular water contained 0 . 44 mg/l Mg2+ .",
"†Mg ( OH ) 2 concentration was lowered to prevent overall alkalization of the diet ."
],
[
"Here , we present a new mechanistic model of the regulation of Mg2+ homeostasis during development and postnatal life of mice .",
"Against current thinking we show in vivo that TRPM6 is not required for embryonic development per se , but primarily operates in placenta and intestine to regulate Mg2+ levels by transcellular transport , while TRPM6 function in the kidney – commonly thought to be essential – is expendable for organismal Mg2+ balance .",
"We demonstrate that ablation of TRPM6 in adult mice leads to reduced lifespan , growth defects and profoundly impaired health of mutant mice due to defective energy metabolism .",
"We also show that dietary Mg2+ supplementation is not only sufficient to prevent all Trpm6 null pathologies , but Mg2+ is the only mineral known so far able to extend the lifespan of wildtype mice .",
"It has been reported that constitutive Trpm6 inactivation leads to embryonic lethality , resulting in the generally accepted tenet that Trpm6 is required for the development of the embryo proper ( Walder et al . , 2009 ) .",
"On the contrary , we provide genetic evidence that the embryonic mortality of Trpm6-deficient mice is caused by the loss of TRPM6 activity in placental SynT-I and yolk sac endoderm cells , and that Trpm6-deficient embryos are depleted of Mg2+ .",
"Altogether , we postulate that Trpm6 controls the maternal Mg2+ supply to the fetus , and that growth failure and death are secondary phenotypes induced by Mg2+ deprivation .",
"Differences in the role of TRPM6 for embryonic survival of humans and mice may be attributable to different morphologies of the placental exchange interfaces , fetal growth rate , litter size and dietary preferences ( Simmons and Cross , 2005 ) .",
"However , at present we cannot exclude that loss-of-function mutations in TRPM6 might be associated with prenatal mortality in humans as well .",
"Pioneering positional cloning studies ( Schlingmann et al . , 2002; Walder et al . , 2002 ) and follow-up case reports ( Schlingmann et al . , 2005; Konrad and Schlingmann , 2014 ) focused on hospitalized human infants selected by the criterion of deleterious Mg2+ deficiency .",
"Hence , additional TRPM6 phenotypes , such as infertility or embryonic death , might have been overlooked .",
"Accordingly , it has recently been shown that single nucleotide polymorphisms in human TRPM6 are associated with a neural tube closure defect , i . e . meningomyelocele ( Saraç et al . , 2016 ) .",
"Of note , dietary Mg2+ supplementation is common practice of pregnant women ( Durlach et al . , 2004 ) .",
"Our work provides first mechanistic insight as to how this essential mineral is delivered to the fetus .",
"There is growing evidence to suggest that Mg2+ deprivation is involved in the development of metabolic , immune , cardiovascular and neurological disorders ( de Baaij et al . , 2015 ) .",
"However , due to the lack of adequate mammalian genetic models , it is still unclear whether impaired Mg2+ homeostasis can be regarded as the cause or the consequence of the latter pathophysiological processes .",
"Therefore , we studied the impact of TRPM6 deletion on postnatal mice .",
"Trpm6-deficient mice displayed shorter lifespans , failure to thrive , low physical activity , kyphosis , lung emphysema , sarcopenia and degeneration of lymphoid organs .",
"In addition , Trpm6-deficient animals developed signs of catabolic metabolism such as lipodystrophy , increased insulin sensitivity and hypothermia , and showed suppression of the somatotropic axis accompanied by induction of xenobiotic detoxification gene networks in the liver .",
"Altogether , we noted that the complex phenotype of Trpm6-deficient mice mirrors many phenotypic hallmarks of mutant mouse strains that are generally considered genetic models of ‘accelerated aging’ in the scientific literature ( López-Otín et al . , 2013 ) .",
"Furthermore , our unbiased screen for metabolic pathways dysregulated in Trpm6-deficient mice revealed an error in energy metabolism reminiscent of humans with mutations in the gene coding for mitochondrial carnitine palmitoyltransferase II , the most common inherited disorder of lipid metabolism in adult humans ( Bonnefont et al . , 2004; Gempel et al . , 2002 ) .",
"Interestingly , an early biochemical study revealed that Mg2+ and MgATP could regulate activity of mitochondrial carnitine acyltransferase activity ( Saggerson , 1982 ) .",
"Accordingly , in proof-of-concept experiments , we demonstrated that Mg2+ is specifically required for the utilization of acyl carnitines ( AC ) as an energy source in liver mitochondria .",
"Hence , these results suggest that insufficient mitochondrial utilization of AC represents a plausible mechanism contributing to the pathologies developed by Trpm6-deficient mice .",
"The exact role of Mg2+ in AC metabolism as well as the molecular pathophysiology of carnitine palmitoyltransferase II deficiency is still not completely understood and , being beyond of the scope of the current manuscript , has to be addressed in future studies .",
"Notably , if Trpm6-deficient mice were fed with a high Mg2+ diet , they were viable and displayed normal physical activity , morphology of internal organs and tissue levels of AC , indicating that the phenotypes in Trpm6-deficient mice were triggered by Mg2+ deficiency .",
"Therefore , we studied in more detail how mutant mice develop organismal Mg2+ deprivation .",
"According to current thinking , the active transport of Mg2+ in the kidney , in particular in DCT , determines the final urinary Mg2+ concentration and is central to whole-body Mg2+ balance ( de Baaij et al . , 2015 ) .",
"Contrary to this model , we demonstrate that global inactivation of Trpm6 leads to Mg2+ deficiency due to a defect in intestinal Mg2+ uptake .",
"To interrogate the renal role of TRPM6 , we conditionally inactivated Trpm6 in the kidney and , surprisingly , did not observe any changes in serum Mg2+ levels .",
"In contrast , intestine-specific disruption of Trpm6 resulted in hypomagnesemia indicating that wildtype kidneys were not able to counterbalance the ablation of TRPM6 in the intestine .",
"Taken together , our findings lend strong support to the new concept that Trpm6-dependent Mg2+ uptake by the colon plays an indispensable role for systemic Mg2+ balance in mice .",
"However , we assume that renal TRPM6 activity may have an important role under conditions of insufficient dietary Mg2+ intake , for instance , during prolonged fasting periods .",
"Currently there are two suggested mechanistic models of hypomagnesemia in humans carrying mutations in TRPM6 .",
"A pioneering study of patients with congenital hypomagnesemia anticipated that Mg2+ malabsorption in the intestine plays a key role in organismal Mg2+ deprivation ( Friedman et al . , 1967; Milla et al . , 1979 ) .",
"More recently , it was reported that in affected humans renal Mg2+ loss plays a key role ( Schlingmann et al . , 2002; Walder et al . , 2002 ) .",
"The results obtained with Trpm6-deficient mice are well compatible with the former model .",
"However , we cannot exclude physiological differences between humans and mice , an important issue to address in future studies .",
"In conclusion , our findings support the idea that TRPM6 is a central gatekeeper of organismal Mg2+ balance in mammals and that this role cannot be compensated for by any other Mg2+ channel and transporter .",
"What is the molecular mechanism underlying insufficient Mg2+ intake in Trpm6-deficient mice ?",
"TRPM6 and its close homolog TRPM7 have been invoked as molecular correlates of cation currents responsible for Mg2+ entry into cells .",
"In order to circumvent the limitations and pitfalls imposed by overexpression of recombinant proteins , we employed TS cells to compare the roles of native TRPM6 and TRPM7 .",
"We found that stem cells express both TRPM6 and TRPM7 , thus mimicking the in vivo situation .",
"Inactivation of Trpm6 did not affect the self-renewal of TS cells .",
"In contrast , Trpm7-deficient cells were not able to proliferate unless the cell culture medium was supplemented with additional Mg2+ , supporting the idea that TRPM7 plays a non-redundant role in cellular Mg2+ uptake ( Schmitz et al . , 2003; Chubanov et al . , 2004; Ryazanova et al . , 2010 ) .",
"We further showed that wildtype TS cells exhibited [Mg2+]i- and [MgATP]i-sensitive divalent cation currents , and that inactivation of Trpm6 reduced current amplitudes .",
"In contrast , deletion of TRPM7 caused complete ablation of these currents .",
"Remarkably , ion currents in Trpm6 deficient TS cells still expressing TRPM7 were substantially more sensitive to intracellular MgATP when compared to TRPM6/M7 co-expressing cells .",
"Thus , in a native environment the presence of TRPM6 reduces the sensitivity of TRPM6/M7-like currents to inhibition by intracellular MgATP .",
"It has recently been reported that the TRPM6/M7 heteromer is completely insensitive to MgATP after heterologous expression in HEK 293 cells ( Zhang et al . , 2014 ) .",
"Thus , native TRPM6/M7 currents do not fully recapitulate the latter findings obtained in a heterologous expression system and further studies are required to clarify these discrepancies .",
"Nevertheless , our results are concordant with our model ( Chubanov et al . , 2004 ) that native TRPM6 functions primarily as a subunit of heteromeric TRPM6/M7 complexes by increasing current amplitudes and relieving TRPM7 from inhibition by [MgATP]i ( Figure 6G ) .",
"Such facilitated Mg2+ entry is most probably not required for any cell autonomous function , but is necessary for epithelial Mg2+ transport and the maintenance of serum Mg2+ levels .",
"Inadequate nutritional Mg2+ intake is commonplace in Western societies ( in up to 68% of the US population [King et al . , 2005] ) .",
"In addition , a growing percentage of the population is exposed to drug-induced forms of hypomagnesemia ( de Baaij et al . , 2015 ) .",
"Consequently , we asked whether wildtype mice would benefit from extra Mg2+ supplementation .",
"Our proof-of-concept experiments suggest that life-long Mg2+ supplementation may extend the lifespan of mice .",
"This idea is concordant with the recent observation that Mg2+acting alone or in conjunction with dietary calorie restriction counteracts lifespan-shortening effects of RNA-DNA hybrid ( R loop ) accumulation in yeasts and human HeLa cells ( Abraham et al . , 2016 ) .",
"Hence , large-scale investigations of nutritional Mg2+ adjustments and their impact on health- and lifespan of different species should be enlightening in this regard ."
],
[
"Experiments involving animals were done in accordance with the EU Animal Welfare Act and were approved by the local councils on animal care ( permit No 55 . 2-1-54-2532-134-13 from the Government of Oberbayern , Germany , and permit No 2347-15-2014 from the State Ministry of Brandenburg , Germany ) .",
"Microarray data were deposited in NCBI Gene Expression Omnibus ( GEO ) ( GSE70457 ) .",
"Liver tissues were collected from 12–13 week-old Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 3 ) and control ( Trpm6+/fl , n = 4 ) male littermates , snap-frozen in liquid nitrogen and stored at −80°C .",
"Total RNA was extracted using the GenElute mammalian total RNA purification kit ( Sigma-Aldrich ) .",
"Whole genome profiling was performed using a GeneChip Mouse Gene 1 . 0 ST Array ( Affymetrix ) at Source Bioscience ( Berlin , Germany ) .",
"Biotinylated single-stranded DNA was prepared according to the standard Affymetrix protocol ( Whole Transcript Expression arrays ) from 100 ng total RNA using the WT terminal labelling kit .",
"2 . 5 µg of fragmented and labelled ssDNA were hybridized for 16–18 hr at 45°C .",
"GeneChips were washed and stained in an Affymetrix Fluidics Station 450 .",
"GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 .",
"Processing of the array data , including quality assessment , background correction , normalization and summarization was performed with the Affymetrix Expression Console ( version 1 . 4 . 0 ) .",
"All statistical analyses were carried out with the statistical computing environment R ( version 3 . 1 . 2 , www . R-project . org ) .",
"Differential expression analysis was performed with the R package limma ( version 3 . 22 . 4 ) ( Ritchie et al . , 2015 ) .",
"p-values were adjusted for multiple testing with the Benjamini-Hochberg method for controlling the false discovery rate ( FDR ) .",
"A heatmap was generated for a group of 46 transcripts differentially expressed at a level of FDR p≤0 . 1 .",
"Analysis of the affected pathways and causal transcriptional regulators was performed by Ingenuity pathway analysis ( IPA ) environment ( www . ingenuity . com , RRID:SCR_008653 ) using a set of 2443 transcripts changed at p≤0 . 05 confidence level ( t-test ) .",
"Serum , liver and gastrocnemius muscle samples were collected from 8–10 week-old control ( Trpm6+/fl , n = 8 ) and Trpm6-deficient ( Trpm6Δ17/Δ17;Sox2-Cre , n = 6 ) male littermates , flash frozen in liquid nitrogen and stored at −80°C .",
"Metabolomic analyses were performed at Biocrates Life Sciences AG ( Innsbruck , Austria ) .",
"Measurements comprised the quantification of 237 metabolites including 41 amino acids/biogenic amines , 40 acylcarnitines ( AC ) , 22 bile acids ( BA ) , 14 lysophosphatidylcholines ( LysPC ) , 77 phosphatidylcholines ( PC ) , 15 sphingomyelins ( SM ) , 17 eicosanoids/prostaglandins and 11 energy metabolism intermediates as outlined in Supplementary file 3 .",
"To extract metabolites , tissue samples were treated with corresponding extraction buffers and incubated in a chilled ultrasonic bath for 5 min .",
"Afterwards samples were centrifuged and the supernatant was used for analysis .",
"FIA- and LC-MS/MS measurement techniques were applied as described in detail previously ( Pena et al . , 2014 ) .",
"All statistical analyses have been applied by using the statistical computing environment R ( www . r-project . org ) .",
"Metabolite measurements containing more than 75% missing values or more than 75% of values below the limit of detection ( LOD ) across all samples per matrix were removed from analysis .",
"Measured concentrations of metabolites were log2-transformed for moderated statistical tests .",
"Measurements were scaled to µ = 0 mean and unit standard deviation for each biological matrix separately for heatmap visualization .",
"Changes in average metabolite levels between control and mutant individuals were tested using a linear model framework implemented in the R package Limma ( Smyth , 2004 ) .",
"Resulting P-values for moderated t-test were corrected for multiple testing by Benjamini and Hochberg approach .",
"A p-value threshold of 0 . 05 was considered as significant .",
"A Heatmap diagram for metabolites with significant changes was calculated with the R-package Heatmap using ward clustering and Euclidean distance measure and the R-package VennDiagram was applied to calculate a Venn diagram of significantly changed metabolites across sample matrices .",
"Mouse liver mitochondria were isolated by differential centrifugation from freshly prepared homogenates as previously described ( Springer et al . , 2002; Schulz et al . , 2015 ) .",
"Liver mitochondria were further purified by Percoll density gradient centrifugation ( Schulz et al . , 2013 ) .",
"Isolated mitochondria were subjected to quantification by the Bradford assay and kept on ice until use .",
"Assessment of the mitochondrial membrane potential Δψm ( MMP ) was followed by Rh123 fluorescence quenching ( Ex . 485 nm , Em . 528 nm ) in a 96-well plate reader ( BioTek ) and quantitatively evaluated by curve analysis as previously described ( Schulz et al . , 2013 ) ( set threshold slopes were ≥0 . 67 for start points and ≤0 . 67 for end points , respectively ) .",
"In order to exclude fluctuations at measurement start , slope calculations were started after 6 min measurement time with slope values ≤ 1 . 5 .",
"A kit-based assay ( ATP Bioluminescence Assay Kit , Roche ) was used to analyze the ATP content from cleared lysates after 30 min mitochondrial ATP synthesis at RT , initiated by the addition of 160 µM ADP and stopped at 95°C for 5 min .",
"For both analyses assay buffer composition was 0 . 2 M sucrose , 10 mM MOPS-Tris , 1 mM Pi and 10 µM EGTA .",
"Respiratory substrates were either succinate ( 25 mM ) /rotenone ( 2 µM ) , or DL-octanoylcarnitine ( 10 µM ) /malate ( 12 . 5 mM ) , or DL-palmitoylcarnitine ( 10 µM ) /malate ( 12 . 5 mM ) .",
"Buffers and solutions were essentially Mg²+-free , as determined by ICP-OES ( Ciros Vision , SPECTRO Analytical Instruments GmbH ) after wet ashing with 65% nitric acid ( Zischka et al . , 2011 ) .",
"EDTA , Mg2+ , Ca2+ , or Zn2+ was added at the concentrations indicated in the respective figures .",
"Mice were raised in a specific pathogen-free facility at Jackson Laboratory Sacramento ( Sacramento , CA , USA ) .",
"The long-lived B6C3F1 hybrid strain ( Lipman et al . , 1999; Turturro et al . , 1999 ) was used .",
"Specifically , F1 females were derived by crossing C57BL/6J females with C3H/HeJ males .",
"The produced B6C3F1 females were weaned at 3 weeks of age , and afterwards kept in individually ventilated polycarbonate cages ( Thoren Caging Systems ) on multigrain chow 5 K54 ( Purina ) containing 0 . 22% Mg2+ and drinking water containing 0 . 44 mg/l Mg2+ .",
"Five mice were housed per cage and cages were changed every two weeks .",
"The housing rooms were with artificial lighting and 12 hr light/dark cycle ( 6 am to 6 pm ) .",
"Temperature and relative humidity in animal rooms were 22 ± 4°C and 50 ± 15% , respectively .",
"At 5 months of age , mice were assigned to control , dietary restricted ( DR ) or supplemented cohorts as outlined in Table 2 .",
"For supplementation experiments , corresponding salts ( Sigma-Aldrich ) were diluted in drinking water that was administered ad libitum throughout the lifespan of all groups ( Table 2 ) .",
"To reduce bias , the supplemented and control groups were examined simultaneously and the study was performed as a blinded trial .",
"Mice were inspected daily .",
"Necropsies of randomly selected dead mice revealed that 58 of 88 control females ( 66% ) developed tumours .",
"8 of 11 females ( 73% ) from the three Mg2+ supplemented groups also had tumours suggesting that high dietary Mg2+ had no substantial effect on tumor rate at death .",
"Kaplan-Meier survival distributions were computed to illustrate survival times .",
"For statistical analysis , we used MATLAB computing environment and programming language ( MathWorks , www . de . mathworks . com ) , in particular its built-in fast convolution function that allows to find the distribution of the sum of independent random variables , given the distributions of individual variables .",
"To enable statistical comparisons between control and dosed groups , the distribution of the mean lifespan of 15 control mice was found as a convolution of the original distribution of lifespans of all control mice .",
"To calculate P-values of the mean lifespan from the dosed groups relative to the control group , we calculated the probability of the average of 15 control mice showing the same or more extreme ( away from control mean ) lifespan than the experimentally determined mean lifespan of the dosed mice .",
"A similar technique was used to find P-values for pooled Mg and dietary restriction groups relative to controls .",
"The MATLAB code is available from the Dryad Digital Repository ( Chubanov and Gudermann , 2016 ) .",
"In addition , the survival data of control mice versus individual treated groups were assessed by log-rank test using GraphPad Prism software .",
"P-values for both approaches are indicated in Figure 7A ."
]
] | [
"Mg2+ regulates many physiological processes and signalling pathways .",
"However , little is known about the mechanisms underlying the organismal balance of Mg2+ .",
"Capitalizing on a set of newly generated mouse models , we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6 .",
"We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development .",
"In adult mice , TRPM6 is required in the intestine to maintain organismal Mg2+ balance , but is dispensable in the kidney .",
"Trpm6 inactivation in adult mice leads to a shortened lifespan , growth deficit and metabolic alterations indicative of impaired energy balance .",
"Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice , but also may extend the lifespan of wildtype mice .",
"Hence , maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood ."
] | [
"A balanced diet contains a variety of minerals such as magnesium ions , which are required for many chemical reactions in our body .",
"A shortage of magnesium ions is linked to many diseases and is thought to be especially harmful to babies in the womb and shortly after birth .",
"Magnesium ion deficiency is widespread in human populations and in the US is thought to affect up to 68% of people .",
"Despite its prominent role in human health , our understanding of how the body maintains the right balance of magnesium ions remains extremely vague .",
"Magnesium ions can enter and leave a cell by passing through specific types of proteins that form channels in the membrane surrounding the cell .",
"There are thought to be around ten types of these magnesium ion channels in human cells , but we do not know what roles any of them perform in the body .",
"One such channel called TRPM6 may be particularly important because mutations in the gene that encodes this channel can cause magnesium ion deficiency in human infants .",
"However , the loss of TRPM6 in mice disrupts how mouse embryos develop , suggesting that our current view on the role that TRPM6 plays in regulating the magnesium ion balance in humans may be too simplistic .",
"To address this question , Chubanov et al . studied mice with mutations that disrupted the production of TRPM6 in specific tissues only .",
"The experiments show that TRPM6 primarily operates in the placenta and intestine to regulate the balance of magnesium ions in the body .",
"Further experiments show that the loss of TRPM6 in adult mice leads to reduced lifespan , growth defects and poor health by disrupting important biochemical reactions .",
"Supplying the mutant mice with magnesium ion supplements improved their health and could extend lifespans of normal animals .",
"The findings of Chubanov et al . demonstrate that TRPM6 plays a crucial role in regulating the levels of magnesium ions in mice before birth and into adulthood .",
"The next step is to carry out large-scale experiments to investigate the effects of altering the levels of magnesium ions in human diets ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Principles of operation of a cerebellar learning circuit | elife-55217-v2 | [
[
"A hallmark of brain function is the ability to learn and remember .",
"We can learn and successfully recall faces , events , language , concepts , places , facts , things that were frightening or rewarding , and the motor commands required to skillfully move our motor effectors .",
"The basic currency of learning and memory is ‘plasticity’ , changes in either the strength of synapses or the intrinsic excitability of a neuron’s membrane .",
"While decades of research have identified the basic rules that govern plasticity and , for some memory systems , have even defined the neural sites that undergo plasticity , behavioral learning is not solely a property of synapses , neurons , or individual brain sites .",
"Rather , it is the emergent property of a complete learning neural circuit in which the sites and plasticity mechanisms of learning are embedded .",
"Only when we incorporate our current knowledge about the specific sites of learning and the rules of plasticity with an understanding of circuit-level interactions can we truly understand learning and memory .",
"Here , our goal is to define the requisite set of circuit-level computational principles that operate during cerebellar-dependent motor learning .",
"Arguably , motor learning is the domain that affords the best chance of understanding the principles of learning and memory , due to the exquisite relationship between sensory stimuli and adaptive motor behavior , combined with the tight link from known neural circuits to the output motoneurons .",
"Across a wide range of movement modalities , motor learning depends crucially on the cerebellum ( Gilbert and Thach , 1977; Golla et al . , 2008; Herzfeld et al . , 2014a; Martin et al . , 1996; Medina and Lisberger , 2008; Smith and Shadmehr , 2005; Robinson , 1974 ) , providing a well-defined neural substrate to investigate how the fundamental principles of learning are implemented in a specific neural circuit .",
"In the motor learning paradigm we study , direction learning in smooth pursuit eye movements , neurophysiological data has demonstrated that short-term motor learning , on the order of a single learning trial , occurs at or upstream of Purkinje cells in the floccular complex of the cerebellum ( Medina and Lisberger , 2008; Yang and Lisberger , 2013; Yang and Lisberger , 2014 ) .",
"Single-trial learning is tightly linked to climbing fiber inputs , in agreement with the predictions of the classical theory of cerebellar learning ( Marr , 1969; Albus , 1971; Ito , 1984 ) .",
"Crucially , Purkinje cells are only two synapses away from the motoneurons that drive the adaptive motor response we measure , significantly constraining the circuit architecture that implements the transformation from sensory inputs to adapted behavior across short timescales .",
"However , recent behavioral results have suggested that longer term pursuit direction learning , on the order of hundreds to thousands of trials , may be mediated by multiple sites and/or learning mechanisms ( Hall et al . , 2018; Yang and Lisberger , 2010 ) .",
"The existence of multiple components in pursuit learning is consistent with behavioral and computational evidence from other cerebellar-dependent motor learning behaviors ( Boyden et al . , 2004; Ethier et al . , 2008; Kojima et al . , 2004; Kording et al . , 2007; Lee and Schweighofer , 2009; Medina et al . , 2000; Smith et al . , 2006; Kassardjian et al . , 2005; Shutoh et al . , 2006 ) .",
"While plasticity is possible across many synapses in the cerebellar circuit ( Carey , 2011; D'Angelo and De Zeeuw , 2009; Hansel et al . , 2001; McElvain et al . , 2010; Mittmann and Häusser , 2007 ) , a prime candidate for long-term learning is the deep cerebellar nucleus , one synapse downstream of the Purkinje cells .",
"Convergent neurophysiological and behavioral evidence across multiple learning paradigms lends credence to role of the deep cerebellar nucleus for long-term storage of cerebellar-dependent memories ( Raymond and Medina , 2018; Kassardjian et al . , 2005; Shutoh et al . , 2006; Lee et al . , 2015; Raymond and Lisberger , 1998; Popa et al . , 2016; Broussard and Kassardjian , 2004; Lisberger , 1994 ) .",
"Taken together , these results highlight the idea that multiple neurophysiological mechanisms within the cerebellar circuit may underlie behavioral motor learning across longer timescales .",
"Our goal in the present paper is to elucidate the operation of the full and well-characterized cerebellar learning circuit .",
"We identify the properties of the signals that guide acquisition and expression of motor learning across timescales through strategic manipulations of the experimental learning conditions coupled with quantitative measures of behavior .",
"Our results suggest four circuit-level principles that define pursuit direction learning .",
"First , rapid acquisition of learning occurs at the parallel fiber to Purkinje cell synapse , driven by climbing fiber responses , with limited retention .",
"Second , learned responses in Purkinje cells guide the slow acquisition of learning in the deep cerebellar nucleus , which learns slowly but has excellent retention .",
"Third , the functional properties of the inputs that are subject to learning are different in the cerebellar cortex and deep cerebellar nuclei .",
"Finally , the extent of learning is limited by negative feedback from the deep nucleus to the inferior olive , reducing the teaching signal that drives learning in the cerebellar cortex ."
],
[
"Given the substantial neurophysiological evidence suggesting the primary role of complex-spike-linked plasticity at the parallel fiber to Purkinje cell synapse for single-trial motor learning , our first objective was to fully characterize the properties underlying the acquisition of a motor memory following a single movement error .",
"To isolate the properties of short-term motor learning , we developed a ‘dual-trial’ experimental paradigm that is a small , but important , modification of our previous methods for studying single-trial learning ( see Materials and methods ) .",
"In each pair of trials , the first trial is defined as the ‘learning’ trial .",
"In the learning trial , the monkey begins to pursue a smoothly moving target in a randomly chosen ‘pursuit direction . ’ After 250 ms of target motion in the pursuit direction , the target abruptly changes direction due to the addition of a velocity component in an orthogonal ‘learning direction’ , either +90 or −90 degrees relative to the pursuit direction ( Figure 2A ) .",
"The discrepancy between animal’s smooth eye movement in the original pursuit direction and target’s net direction due to the addition of orthogonal target motion creates a movement error that serves as an instruction for motor learning ( Figure 2B ) .",
"We therefore refer to the addition of the target velocity component orthogonal to the original pursuit direction as the ‘instruction' . We measure motor learning in the subsequent ‘probe’ trial , where the target moves in the same pursuit direction as the learning trial but without any instruction .",
"In probe trials , the animal exhibits a ‘learned response’ due to the error induced by the instruction in the preceding learning trial .",
"The learned response ( Figure 2C , arrowhead ) starts about 200 ms after the onset of target motion in the pursuit direction , anticipating the change in target direction imposed in the preceding learning trial .",
"We measure learning in the 50 ms interval surrounding the time of the instruction , from 225 to 275 ms after the onset of pursuit target motion .",
"The measurement interval is chosen strategically to capture the anticipatory response of the pursuit system at the time of the instruction .",
"Later in the paper , it also allows us to measure the learned response even in learning trials , because the measurement interval precedes any visually driven eye movement resulting from retinal image motion caused by the instruction ( Hall et al . , 2018; Yang and Lisberger , 2017; Medina and Lisberger , 2008 ) .",
"We first characterized the dependence of single-trial learning on the magnitude of the error imposed in the learning trial .",
"To ensure that we were measuring only the learned response due to the occurrence of a single movement error , we prevented any long-term learning by choosing the pursuit direction for each learning-probe pair randomly from the cardinal axes ( see Materials and methods ) .",
"In each learning trial , we also chose the speed of the instruction randomly to be 0 , 5 , 10 , 15 , 20 , 25 , or 30 deg/s ( Figure 3A ) , and measured the effect of varying instruction speed on the learned response in the subsequent probe trial .",
"Single-trial learning using randomized pursuit directions creates a situation where the error magnitude experienced in the learning trial is identical to the imposed instruction magnitude , because the average eye speed in the learning direction during the learning trial is zero at the time of the instruction .",
"This allowed us to control the size of the error that drives learning and to measure directly the shape of the relationship between learning and error .",
"In both monkeys , learned responses in the probe trial increased as a function of the error magnitude experienced in the learning trial ( Figure 3B ) .",
"Quantitative analysis revealed that the learned response did not increase linearly as a function of error magnitude in the learning trial , but rather saturated as error increased ( Figure 3C ) .",
"We quantified the effect of error magnitude on single-trial learning as a sigmoid of the form: ( 1 ) Y2E1=a1+e-τE1-a2where E1 is the magnitude of the error imposed on the learning trial and Y2 is the measured learned response in the subsequent probe trial .",
"The parameters a and τ were obtained by fitting Equation 1 to the data for each monkey in Figure 3C ( dashed traces versus connected , filled , symbols ) .",
"Equation 1 fitted the data well for both monkeys ( Monkey RE: a = 1 . 35 , τ = 0 . 21 , R2 = 0 . 99; Monkey YO: a = 1 . 25 , τ = 0 . 118 , R2 = 0 . 98 ) .",
"The sigmoidal model of single-trial learning was superior to an alternative linear model ( Akaike’s information criterion ( AIC ) ( Akaike , 1974 ) , Monkey RE: -53 . 3 ( sigmoid ) versus -14 . 5 ( linear ) ; Monkey YO: -29 . 2 ( sigmoid ) versus -20 . 5 ( linear ) ) .",
"Our next step was the characterize the stability of short-term pursuit learning across trials that did not include a movement error .",
"To do so , we used a modified experimental design where the first 20 trials were standard learning trials with an instruction magnitude of 30 deg/s .",
"The subsequent 10 trials were ‘error-clamp’ trials ( Figure 3D ) that we used to estimate retention in the absence of movement error ( after Scheidt et al . , 2000 ) .",
"Error-clamp trials used the same pursuit target motion as in the instruction trials , but the motion of the target in the learning direction was yoked to the animal’s eye movement in that direction , to ensure that the animal experienced no visual error signals in the learning direction .",
"Thus , any trial-to-trial change in the animal’s learned response over the course of 10 error-clamp trials was due solely to forgetting of the previously acquired short-term motor memory .",
"We normalized the learned response in the 10 error-clamp trials by the magnitude of the animal’s learned response in the final learning trial of the 20-trial learning block .",
"Both monkeys demonstrated an exponential decay in the magnitude of the learned response across error-clamp trials ( Figure 3E ) .",
"Fitting an exponential model to each monkey’s retention data ( Figure 3E , black lines ) suggested a retention constant of approximately 0 . 85 ( 95% confidence intervals based on four experiments per monkey , Monkey RE: 0 . 80–0 . 86 , Monkey YO: 0 . 85–0 . 91 ) .",
"Therefore , in the absence of any error to drive learning , approximately 15% of the short-term learned response is forgotten on each trial .",
"We performed this experiment only for instruction speeds of 30 deg/s because the larger amplitude of learning provided more reliable estimates of retention .",
"We next sought to use generalization of learning to constrain the functional properties of the site of plasticity that causes single-trial motor learning .",
"Our strategy is based on the following line of reasoning .",
"Imagine , for example , that the input signals that are subject to single-trial learning are linearly related to eye speed in the pursuit direction ( ve ) and that plasticity operates as a scalar gain ( g ) .",
"Then , the learned change in firing of the relevant post-synaptic cells should be proportional to gve .",
"As a result , the learned eye speed should generalize linearly as a function of pursuit speed in the probe trial .",
"We can invert this logic to use the measured characteristics of generalization of learning to reveal the functional properties of signals that undergo plasticity .",
"Under the assumption that the site of plasticity for single-trial learning acts as a scalar gain , if learned eye speed generalizes linearly with pursuit eye speed in the probe trial then the population response of neurons upstream from the site of plasticity should be linearly related to eye speed in the pursuit direction .",
"We again employed our dual-trial paradigm with a randomized pursuit direction for each pair of learning and probe trials .",
"Here , the learning trials always had a pursuit speed of 20 deg/s and an orthogonal instruction speed of 30 deg/s .",
"The pursuit speed of the target in the probe trial was selected randomly from 5 , 10 , 15 , or 20 deg/s ( Figure 4A ) .",
"We found that average eye speed in the learning direction scaled with the speed of the probe target for both monkeys RE and YO , especially in the interval from 225 to 275 ms after the onset of target motion ( Figure 4B , shaded area ) .",
"We note that scaling is less clear earlier in the probe trial for Monkey YO , partly because the animal’s eye speed in the pursuit direction scales poorly with target speed during this earlier interval .",
"A RM-ANOVA showed a main effect of eye speed in the pursuit direction in the probe trial on the amount of measured behavioral learning for both monkeys ( Monkey RE: F ( 3 , 6 ) =24 . 3 , p<10−3 , Monkey YO: F ( 3 , 15 ) =13 . 7 , p<0 . 001 ) .",
"For both monkeys , the effect of eye speed in the pursuit direction on the learned response from 225 to 275 ms was highly linear ( Figure 4C , Monkey RE: R2 = 0 . 99 , Monkey YO: R2 = 0 . 97 ) .",
"These results suggest that signals related to the speed of eye motion in the pursuit direction may be part of the substrate that is subject to plasticity .",
"The linear generalization of single-trial learning with pursuit direction eye speed is consistent with prior observations that the eye speed at the time of the instruction linearly modulates the expression of behavioral learning ( Hall et al . , 2018 ) .",
"In contrast , target/eye speed in the pursuit direction during the learning trial did not affect the expression of single-trial learning on the subsequent probe trial .",
"Here , we again used the dual-trial paradigm with pursuit speed randomly chosen to be 5 , 10 , 15 , or 20 deg/s in the learning trials but always 20 deg/s in the probe trials .",
"The instruction occurred 250 ms after the onset of target motion and had a magnitude of 30 deg/s .",
"The direction of the instruction was chosen randomly to be either +90° or −90° relative to the pursuit direction .",
"Both Monkeys RE and YO ( Figure 4D ) showed no effect of pursuit speed in the learning trial on the magnitude of the learned response measured in the probe trials .",
"A RM-ANOVA failed to show a significant effect at 250 ms in the probe trial ( Figure 4E , Monkey RE: F ( 3 , 15 ) =0 . 56 , p=0 . 65 , Monkey YO: F ( 3 , 15 ) =0 . 39 , p=0 . 76 ) .",
"Thus , we can conclude that target/eye speed in the pursuit direction has distinct and very different effects on the acquisition of a motor memory during the learning trial versus on the expression of learning in the probe trial .",
"Previous neurophysiological results suggest that complex-spike-linked plasticity at the parallel fiber to Purkinje cell synapse in the cerebellar cortex is likely the primary driver of single-trial pursuit learning ( Medina and Lisberger , 2008; Yang and Lisberger , 2014 ) .",
"Combining these neurophysiological observations with our behavioral results thus far allows us to begin to constrain the parameters of a cerebellar model of motor learning .",
"A model of short-term pursuit learning appears in Figure 5A ( Source code 1 ) .",
"Here , we chose to model the activity of parallel fibers as linearly related to eye speed in the pursuit direction , to account for the generalization of single-trial learning ( Figure 4B–D ) .",
"We use only two parallel fibers with opposite preferred directions ( more parallel fibers are trivially possible but unnecessary ) with an arbitrary scaling parameter , r , that converts eye velocity to spikes/s: ( 2 ) PF1∝rE˙PF2∝−rE˙ In the absence of learning , the parallel fiber inputs cancel perfectly , ensuring that the firing of the post-synaptic Purkinje cell does not modulate in relation to eye movement in the pursuit direction , consistent with experimental data ( Medina and Lisberger , 2008 ) .",
"We can model Purkinje cell firing on the nth trial , PCn , as the weighted contributions of the pre-synaptic parallel fibers and a background response , PC0: ( 3 ) PCn=wn1PFn1+wn2PFn2+PC0 The synaptic weight for each parallel fiber , wi , is adaptable via the complex spikes caused by climbing fiber inputs .",
"The plasticity rule includes two terms: ( 1 ) a decay term , αPF , that allows the relaxation of any learned weight changes back to their baseline value in the absence of error and ( 2 ) an update term that depresses weights by a fractional amount ( β ) depending on the positive firing on the parallel fiber in question and the probability of a complex spike given the neural representation of the image motion from the instruction , PCSEn: ( 4 ) wn+1i={wni− ( 1−αPF ) ( wni−w0i ) −βP ( CS|En ) if PFi > 0wni− ( 1−αPF ) ( wni−w0i ) if PFi ≤ 0 When αPF=1 , the weights are maintained completely from one trial to the next and learning is allowed to perfectly accumulate; when αPF<1 , the weights decay back to their baseline levels , w0i , in the absence of any climbing fiber input .",
"We model only learning for instructions in the direction that causes complex spikes so that we do not need to explicitly simulate synaptic potentiation beyond the relaxation back toward the baseline weights provided by αPF .",
"Our results in Figures 3 and 4 suggest appropriate values for the unknown parameters of this simplified cerebellar model of learning .",
"First , we know that , in the absence of error , short-term motor learning decays across trials .",
"The time constant analysis in Figure 3E suggests that αPF≈0 . 85 for both monkeys .",
"In addition , Equation 1 describes how the parallel fiber to Purkinje cell weights are modified as a function of error during single-trial learning: Y2E1∝βPCSE1 .",
"Previous neurophysiological data has demonstrated that the probability of observing a complex spike following a 30 deg/s instruction is approximately 30% ( Yang and Lisberger , 2014; Yang and Lisberger , 2017 ) .",
"Given these observations , we can derive a rule linking the probability of observing a complex spike to a given error magnitude using a sigmoid function with the same form as Equation 1: ( 5 ) PCSEn=0 . 61+e-τEn-0 . 3 Here , the probability of observing a complex spike saturates at 30% for large error magnitudes .",
"The value of τ matches the values we obtained for each monkey in Equation 1 .",
"Purkinje cells inhibit the floccular target neurons ( FTNs ) in the vestibular nucleus which , in turn , modulate eye movements via their projections to motoneurons .",
"In the absence of other inputs to FTNs that are modulated by learning , the change in the firing of FTNs from their baseline activity is FTNn=PC0-PCn .",
"The resulting learned response , Yn , measured via generated eye movements is then Yn= cFTNn , where c is an arbitrary constant that scales FTN activity to deg/s .",
"Equations 2-5 describe a 'single-learning-process' model .",
"The constants , r , β , w0i and c are arbitrary and affect only the scaling of firing rates to eye movements .",
"The values we chose for these parameters are shown in Table 1 , but any number of parameter combinations would give qualitatively similar behavioral results .",
"The single-learning-process model appropriately estimated our behavioral observations for two different stimulus conditions , one that was more-or-less built into the model and one that matches our data as an emergent property of the model .",
"First , the trial-to-trial change in output following a range of imposed error magnitudes closely matched the learned behavior of both animals ( Figure 5B ) , because this relationship was built into the model based on the data in Figure 3C .",
"Second , the model reproduced the results of an experiment that was a superset of the conditions used in Figure 4A–C .",
"Here , we presented a series of dual-trial stimuli where the instruction trial used a randomly chosen pursuit speed of 0 , 10 , 15 , or 20 deg/s and delivered an instruction at 30 deg/s .",
"The probe trial also delivered a pursuit speed that was randomly chosen from 0 , 10 , 15 or 20 deg/s .",
"For each probe trial , we computed the ‘learning expression ratio’ as the magnitude of the learned response expressed in each probe trial normalized by the average learning expressed in probe trials with the same pursuit speed as the preceding learning trial .",
"When the pursuit speed in probe trial matched the pursuit speed in the learning trial , the learning expression ratio is ~1 . 0 .",
"We then plotted the learning expression ratio as a function of the ratio of the pursuit speeds of the probe trial and the preceding learning trial to assess the generalization function of learning across any combination of pursuit speeds in the learning and probe trials .",
"The model reproduces the results of this experiment on both monkeys ( Figure 5C ) , demonstrating strong linear generalization across probe pursuit speeds .",
"In summary , a cerebellar model with a single learning site where plasticity operates on signals linearly related to pursuit eye speed predicts all of our behavioral data for single-trial learning .",
"In the more natural situation where the same instruction for learning occurs on movement after movement and is corrected gradually , learning could be simply due to an accumulation of single-trial learning plus forgetting at the rate of 15% per trial .",
"At some point , the forgetting and learning on each trial would balance and the learning curve would reach an asymptote .",
"To provide a dataset to ask whether the ‘single-learning-process’ model of single-trial cerebellar learning with forgetting ( Equations 2-5 ) can explain the accumulation of behavioral learning across multiple learning trials , monkeys completed 100 consecutive learning trials .",
"Each learning trial used identical pursuit and learning directions and speeds .",
"Within each learning block of 100 trials , the instruction magnitude was either 0 , 5 , 10 , 15 , 20 , 25 , or 30 deg/s .",
"Note that during 100-trial learning blocks , we control the magnitude of the instruction , but the magnitude of the error on any given trial is the difference between the instruction and the monkey’s expression of behavioral learning on that trial .",
"When the same learning trial is repeated , acquisition of behavioral learning leads to a gradual reduction in the magnitude of the error for a given magnitude of instruction .",
"Therefore , we use the terms ‘instruction magnitude’ and ‘error magnitude’ carefully to convey this distinction .",
"For each instruction magnitude , the trial-course of learning revealed dual-exponential learning curves that largely saturated by the end of 100 learning trials ( Figure 6A ) .",
"Yet , even after 100 consecutive learning trials , the magnitude of the animal’s learned response was always much smaller than the speed of the imposed instruction: neither animal ever exhibited full compensation for the imposed instruction ( Figure 6B ) .",
"It is especially noteworthy that the system is able to achieve a learned response of 5 deg/s after 100 learning trials for an instruction at 30 deg/s , but not for an instruction at 5 deg/s .",
"The effect of instruction magnitude on the learned response was much more linear after 100 consecutive learning trials than after a single learning trial .",
"The learned eye speed in the last 25 trials of the learning epoch was linearly related to instruction magnitude for both monkeys ( Monkey RE: R2 = 0 . 99 , t-test on the slope of the regression fit: t ( 5 ) =21 . 9 , p<10−4; Monkey YO: R2 = 0 . 99 , t ( 5 ) =18 . 9 , p<10−4 ) .",
"After 100 learning trials , a linear model was preferred when compared against the best-fit saturating model from Equation 1 ( AIC , Monkey RE: 7 . 7 ( sigmoid ) versus 1 . 5 ( linear ) ; Monkey YO: 0 . 6 ( sigmoid ) versus 0 . 02 ( linear ) , Figure 6C ) .",
"The difference in the effect of instruction magnitude on learning after 1 versus 100 learning trials hinted that multiple plasticity mechanisms and/or sites within the cerebellar circuit could be driving learning across the different trial-courses .",
"Using the single-learning-process model , we simulated blocks of 100 consecutive learning trials .",
"In each trial , we drove learning with an error that was the difference between model output and In , the imposed instruction: En≡In-cFTN , simulating the gradual reduction in the error signal for a fixed instruction magnitude due to the acquisition of learning across trials .",
"The single-learning-process model failed to predict two crucial features of the true trial-course of learning .",
"First , it produced single exponential trial-courses that learned too quickly and reached asymptote within the first 20 learning trials ( Figure 7A ) .",
"Second , it systematically over-estimated the extent of asymptotic learning for instruction magnitudes between 5 and 20 deg/s ( Figure 7B , arrows ) and underestimated learning for instruction magnitudes of 30 deg/s .",
"Indeed , the model predicted a sigmoidal relationship between asymptotic learning and instruction magnitude ( Figure 7B ) , inherited from the relationship between single-trial learning and instruction magnitude , in contrast to the highly linear relationship in the data ( Figures 6C and 7B ) .",
"We note that we did not fit the model to the data .",
"Instead , we used the parameters derived from our behavioral experiments that allowed Equations 2-5 to predict the details of single-trial learning .",
"We therefore conclude that pursuit learning cannot be mediated solely by the accumulation of single-trial learning with forgetting ( Yang and Lisberger , 2010; Hall et al . , 2018 ) .",
"Our next step was to identify the minimal principles of a cerebellar circuit model that can explain behavioral learning across multiple timescales .",
"The features of behavioral learning in Figures 3 and 4 mandate several properties of a more complete cerebellar model: ( 1 ) acquisition of single-trial learning saturates as a function of the magnitude of the error according to a sigmoidal relationship dictated by the data in Figure 3C; ( 2 ) at least across the 100 learning trials we tested in Figure 6 but also after 2000 trials ( Hall et al . , 2018 ) , the asymptotic response for a consistent instruction magnitude falls short of completely correcting the movement error created by the imposed instruction; ( 3 ) the trial-course of learning across long bouts of consecutive learning trials ( Hall et al . , 2018 ) and the different relationship between learned response and error magnitude after 1 versus 100 trials suggest the presence of at least two learning processes ( Hall et al . , 2018; Kojima et al . , 2004; Lee and Schweighofer , 2009; Smith et al . , 2006 ) .",
"To test our conclusion about multiple learning processes contributing to cerebellar learning across longer learning sessions , we next asked whether the retention of the motor memory changed across long bouts of learning trials .",
"We used the same error-clamp strategy as before , but now to measure the retention constant after blocks of >1000 identical learning trials .",
"At the end of long blocks of learning trials , we alternated short blocks of 10 error-clamp trials with runs of 20 learning trials to ‘top up’ the asymptotic learning .",
"We found significantly better retention after 1000 trials than after 20 trials ( Figure 8A ) : the measured retention constant after 1000 trials was approximately 0 . 95 ( 95% confidence intervals , Monkey RE: 0 . 95–0 . 97 , Monkey YO: 0 . 94–0 . 96 ) ) .",
"These data suggested that the motor memory gradually transitions , at least conceptually , from a relatively labile , low-retention learning process early in learning to a second learning process with high retention ( Smith et al . , 2006; Herzfeld et al . , 2014a ) .",
"Armed with knowledge that learning appears to transition from a low-retention learning process to a higher retention process after extended learning , we expanded our circuit model of cerebellar learning to include a secondary site of plasticity ( Figure 8B , Source code 2 ) .",
"Given neurophysiological results from other cerebellar-dependent learning tasks ( Kassardjian et al . , 2005; Shutoh et al . , 2006; Lee et al . , 2015; Raymond and Lisberger , 1998; Lisberger , 1994 ) as well as the proximity of the cerebellar circuit to motoneurons , we view the floccular target neurons , or FTNs , in the vestibular nucleus as the most likely candidate for long-term storage of learning .",
"FTNs are immediately downstream of the Purkinje cells , the presumed site of rapid learning , and synapse onto output motoneurons .",
"We modeled the learned responses of FTNs on trial n as the sum of the firing of input axons , INni , weighted by the scalar synaptic weight , vni , minus the baseline-subtracted firing of the Purkinje cell: ( 6 ) FTNn=∑ivniINni- ( PCn-PC0 ) To account for a secondary site of plasticity within the learning circuit , we implemented Hebbian-style plasticity at the synapse from the eye movement inputs to FTNs .",
"Plasticity at the inputs to FTNs was driven by the learned responses of presynaptic Purkinje cells: ( 7 ) vn+1i=vni+η ( PC0-PCn ) INni Here , η determines the strength of the Purkinje cell’s teaching signal and the rate of plasticity in the inputs to FTNs .",
"The value of η was set to be small so that the FTNs represented the site of a slow-learning process , in contrast to the rapid learning at the parallel fiber to Purkinje cell synapse .",
"Based on our behavioral results in Figure 8A , plasticity in the slow-learning-process is retained almost perfectly , so that Equation 7 lacks the forgetting factor provided by αPF in Equation",
"4 . Together , Equations 6 and 7 assume that the learning in the Purkinje cell responses acts as a teacher for long-term plasticity at the inputs to the FTNs .",
"Simulation of Equations 2-7 for a variety of instruction magnitudes ( Figure 8C ) correctly demonstrates one crucial feature of our long-term learning data .",
"The simulated learning is dual-exponential , with initial fast acquisition and a second , slower , timescale of acquisition .",
"However , the simulations do not predict a crucial aspect of our behavioral data .",
"Due to the stability of the long-term memory at the FTN inputs , the asymptotic response of the model is much larger than is found in our data .",
"Also , Figure 8C shows that the slow-learning process produces a linear relationship between asymptotic learning and instruction magnitude , but only after 2000 trials and not after 100 trials ( vertical dashed line ) as seen in the data .",
"The excessive learning in the model of Figure 8B suggests the existence of inhibition within the learning circuit .",
"Multiple deficiencies of the model in Equations 2-7 are corrected when recurrent inhibition from the cerebellum to the inferior olive modulates the error signals used to drive learning at the parallel fiber to Purkinje cell synapse .",
"We modeled inhibition by reducing the magnitude of the error signal that drives climbing fiber responses in proportion to the size of the learned response: ( 8 ) En=In−cFTNn−γcFTNnE˙ Here , In is the speed of the instruction on the nth trial and cFTNn is the FTN firing converted to units of learned eye velocity .",
"The term ( In-cFTNn ) in Equation 8 represents the physical error that results from the difference between the instruction magnitude and the response of FTNs .",
"This formula makes the distinction between the physical error , ( In-cFTNn ) , and the learning system’s internal representation of this error used to drive learning in the cerebellar cortex ( En ) .",
"The last term in Equation 8 implements modulation of the internal representation of the physical image motion in proportion to γ and the learned response , cFTNn .",
"There is no recurrent control when the value of γ is zero .",
"The modulation is normalized by eye speed in the pursuit direction ( E˙ ) so that inhibition of the climbing fiber input occurs in proportion to the change in weight of the parallel fiber to Purkinje cell synapse .",
"The combination of the Purkinje cell modulation as a teacher ( Equation 7 ) as well as the presence of recurrent inhibition ( Equation 8 ) transfers the motor memory from the cerebellar cortex to the vestibular nucleus across long blocks of learning trials .",
"We note that the probability of complex spikes decreases considerably across 100 repetitions of the same learning trial ( Yang and Lisberger , 2017 ) , compatible with the mechanism implemented by Equation 8 .",
"We adjusted the strength of the teaching signal from the Purkinje cells to the FTN inputs ( η ) and the strength of recurrent inhibition of the instructive signal ( γ ) to allow the model in Figure 8B and Equations 2-8 to reproduce both the two-component trial-course of learning ( Figure 8D ) and the correct linear relationship between the asymptotic learning and instruction magnitude after 100 learning trials ( Figure 8E ) .",
"The best-fit model ( two unknowns , η and γ ) agrees well with the data for both monkeys ( Figure 8E , black curves , Monkey RE: R2 = 0 . 99; Monkey YO: R2 = 0 . 97 ) .",
"In addition , both monkeys had similar values for both estimated parameters ( 95% confidence intervals , Monkey RE: η = 5 . 1×10−4–9 . 2 × 10−4 , γ = 55 . 9–68 . 5 , Monkey YO: η = 4 . 5×10−4–9 . 4 × 10−4 , γ = 38 . 8–65 . 0 ) .",
"Linearity emerges after 100 trials because recurrent feedback switches the mechanism driving asymptotic behavior from a tradeoff between learning and forgetting to an emergent property of the operation of a dynamic feedback loop .",
"The successful model in Figure 8B has three crucial features: ( 1 ) a site of rapid learning with limited retention at the parallel fiber to Purkinje cell synapse , ( 2 ) gradual transfer of learning from the cerebellar cortex to a slower-learning process with excellent retention at the inputs to the FTNs , and ( 3 ) recurrent inhibition from FTNs to the inferior olive that limits learning and linearizes the stimulus-response function of learning by controlling the error signals available to drive learning at the parallel fiber to Purkinje cell synapse .",
"We next provide additional behavioral evidence for separate sites of learning within the cerebellar circuit .",
"By probing the expression of learning as a function of pursuit speed across the trial-course of learning , we show that the linear pattern of generalization to pursuit speed we observed following a single learning trial ( Figure 4 ) changes gradually but qualitatively across 1000 trials .",
"We suggest that the site and/or mechanism of learning changes across a long trial-course .",
"We again deployed the dual-trial paradigm , but with the modification that the instruction trial in each pair of trials repeated the same fixed parameters of pursuit ( 5 deg/s or 20 deg/s ) , instruction magnitude ( 30 deg/s ) , instruction direction , and pursuit direction for the entire experimental session .",
"We studied generalization across the gradual accumulation of learning by varying target speed in the pursuit direction randomly in the probe trial to be 5 , 10 , 15 or 20 deg/s , while keeping the pursuit direction the same as in the learning trials .",
"An example experiment demonstrates how generalization changed over a long trial-course when pursuit speed was 5 deg/s in the learning trials and was always equal or faster in the probe trials .",
"Averages of the trajectory of the learned eye velocity as a function of time during the first 250 trials confirmed that the learned response scaled with pursuit speed in the initial stages of learning ( Figure 9A , left ) , in agreement with the data for single-trial learning in Figure",
"4 . However , at the end of the same learning session , the expression of learning no longer scaled with pursuit speed in the probe trials ( Figure 9A , right ) .",
"The change in generalization across the session could not be explained by any systematic differences in the eye speed in the pursuit direction during the first versus last 250 trials ( Figure 9B , continuous versus dashed traces ) .",
"The change in the generalization of the expression of learning in the probe trials occurred gradually ( Figure 10A ) .",
"Across 2250 trials ( 1125 dual-trial stimuli ) in Monkey RE , the expression of learning declined for probes with pursuit speeds of 20 deg/s and remained constant for probes with pursuit speeds of 5 deg/s .",
"By 2250 trials , the learned response did not depend on the target speed in the probe , in agreement with the right graph of Figure 9A .",
"The observations were similar in Monkey YO , although he did not work for as many trials as Monkey RE ( note the shorter x-axis ) .",
"These results suggest that something about the inputs that are subject to plasticity or the mechanism of plasticity at the site of single-trial , short-term , learning may differ from those to the site of long-term pursuit learning .",
"We observed a qualitatively different pattern of generalization across learning blocks when pursuit speed was 20 deg/s in the learning trials and was always equal or slower in the probe trials .",
"Now , the expression of learning increased slightly for probe trials with a pursuit speed of 20 deg/s and decreased as a function of trial when the pursuit speed in the probe trial was 5 deg/s ( Figure 10B ) .",
"Together , the results of learning with pursuit target speeds in the learning trials of 5 and 20 deg/s show a general rule: learning generalizes approximately linearly for pursuit speed across all timescales of learning when the pursuit speed in the probe trial is slower than the learning trial; when pursuit speed in the probe trial is faster than that in the learning trial , linear generalization early in learning is replaced gradually with a learned response that appears largely independent of pursuit speed after long-term learning .",
"To verify the statistical veracity of the effects in Figure 10A and B , we performed a regression analysis on the trial-course of generalization of each experiment ( lines in Figure 10A and B ) .",
"For each experimental condition , we measured the rate of change of the learned response across trials ( Figure 10C ) .",
"Here , a negative slope indicated that the learned response for a particular probe pursuit speed decayed over the experiment ( e . g . 20 deg/s probe trials when the pursuit speed of the learning trial was 5 deg/s ) .",
"Positive slopes indicated that the learned response measured in the probe trials tended to increase across the experiment , as was the case when the pursuit speed in both the learning trial and the probe trial was 20 deg/s ( Figure 10B , red curves ) .",
"A two-way ANOVA confirmed the differential effect of pursuit speed in the learning trials on the generalization properties of the motor memory .",
"We tested for main effects of probe pursuit speed ( 5 , 10 , 15 or 20 deg/s ) , pursuit speed in the learning trials ( 5 deg/s or 20 deg/s ) , and their interaction .",
"For both monkeys , we observed a main effect of pursuit speed in the learning trials ( RE: F ( 1 , 32 ) =46 . 8 , p<10−7; YO: F ( 1 , 20 ) =31 . 6 , p<10−4 ) , no main effect of probe speed ( RE: F ( 3 , 32 ) =1 . 39 , p=0 . 26; YO: F ( 3 , 20 ) =0 . 33; p=0 . 81 ) , and crucially a significant learning pursuit speed by probe pursuit speed interaction ( RE: F ( 3 , 32 ) =14 . 2 , p<10−5 , YO: F ( 3 , 20 ) =9 . 0 , p<10−3 ) .",
"Thus , the relationship between pursuit speeds of the target in the learning and probe trials significantly affected the shape of generalization across trials .",
"To further validate the interaction between pursuit speed in the learning and probe trials , we conducted additional experiments in Monkey RE that tested other pursuit speeds in the learning trial ( not shown ) .",
"Here , different experiments used one of either 5 , 10 or 20 deg/s as the consistent pursuit speed in the learning trials and probed with pursuit speeds selected randomly from the same three speeds .",
"After 1000 learning trials , the expression of learning generalized approximately linearly as a function of slower pursuit speeds in the probe trial compared to the learning trial , but not for probe speeds faster than used in the learning trial , in agreement with Figure 10A and B . Thus , the rules for generalization depend not on whether the probe speed is the same or different from the pursuit speed in the instruction trials , but rather on whether it is faster or slower .",
"These preliminary experiments also served as a control to ensure that the change in generalization across many trials was not a result of the alternation of learning and probe trials , because probe trials with the other two pursuit speeds were interspersed randomly among the learning trials with a ratio of 10 learning trials to every probe .",
"In the previous section , we showed that learning generalizes differently in relation to pursuit target speed early versus late in a long learning block .",
"Next , we analyze our data in a way that quantifies the shape of the pursuit speed generalization function at different stages of learning .",
"We used the data from the long blocks of learning/probe pairs in Figure 10 , where the pursuit and instruction speed were fixed in the learning trial but pursuit speed in each interleaving probe trial was selected randomly among 5 , 10 , 15 , or 20 deg/s .",
"Here , we computed the ‘learning expression ratio’ for each pair of trials as the magnitude of the learned response in the probe trial divided by the learning expressed in the preceding learning trial .",
"The learning expression ratio is a single-trial metric of generalization , allowing us to probe the shape of generalization across long bouts of learning while controlling for the gradual accumulation of behavioral learning across the session .",
"We then binned the resulting learning expression ratio by the ratio between the target pursuit speed in each probe trial and learning trial .",
"When the probe trial and the preceding learning trial have the same pursuit speed ( probe speed to pursuit speed ratio of 1 . 0 ) , we would expect nearly complete generalization between the learning and probe trials ( a learning expression ratio ~1 . 0 ) .",
"We refer to the relationship between the learning expression ratio and the ratio of the probe and learning trial pursuit speeds as the ‘generalization function' .",
"In the first 250 trials of a long learning block ( blue curve , Figure 11A ) , both monkeys demonstrated an approximately linear generalization function , paralleling our finding of single-trial generalization in Figure",
"5 . Thus , early in learning , both monkeys expressed greater ( less ) learning in the probe trial than in the learning trial when pursuit speed in the probe trial was faster ( slower ) than in the preceding learning trial .",
"As learning progressed through a long block of trials ( Figure 11A , pink and purple traces ) , the shape of the generalization function changed gradually .",
"After 1000 trials , probe trials with pursuit target speed faster than in the learning trial ( probe speed to pursuit speed ratio >1 . 0 ) no longer showed enhancement of the expression of learning ( learning expression ratio ~1 . 0 ) .",
"The changing shape of the generalization function was further accentuated in Monkey RE after 2000 trials when probe trials expressed less behavioral learning ( learning expression ratio <1 . 0 ) if the pursuit speed was faster in the probe than in the learning trial .",
"That the generalization function changes across 2000 trials indicates either that the properties of a single site of learning change or that learning transfers between sites with different functional properties .",
"Cognizant that factors such as non-linear synapses and recurrent inhibition can control the properties of the signals at a site of learning , the simplest way to model generalization is in terms of the signals conveyed by the afferent fibers whose synapses are subject to plasticity .",
"Figure 11 suggests that we can model learning under the assumption that the motor memory gradually transitions from an early site of learning where the inputs are linearly related to pursuit speed to a secondary site/synapse where the inputs are unimodally tuned for pursuit speed .",
"We completed our model of cerebellar learning ( Figure 12A ) by defining the functional properties of non-Purkinje cell inputs to FTNs .",
"We simulated 50 inputs to the FTNs , each with a preferred direction either equal to or opposite the pursuit direction and a Gaussian tuned response for pursuit speed: ( 9 ) INni=cos ( θne−θi ) exp ( − ( E˙−si ) 22σ2 ) Here , for the ith input axon , θi is either 0 or 180 deg , si is the preferred eye speed ranging from 0 to 50 deg/s , and σ is the standard deviation of the Gaussian function .",
"The eye movement vector on the nth trial is defined as ( θne , E˙n ) .",
"Note that any unimodal function ( e . g . cosine ) would yield comparable results to the Gaussian function .",
"The circuit-level learning system defined by the schematic in Figure 12A and Equations 2-9 reproduces our data on both the trial-course of learning and the trial-course of the generalization of learning to pursuit speed .",
"The learned response , like the real data , shows a saturating relationship to instruction magnitude for single-trial learning ( Figure 12B ) and a linear relationship after 100 trials ( Figure 12C ) .",
"For learning with pursuit target motion at 5 deg/s , the expression of the learned response in the model generalizes differently early versus late in a 1000 trial learning block ( Figure 12D ) .",
"Early in the learning block , the learned response generalizes linearly to pursuit speeds regardless of the pursuit speed in the learning trials .",
"Late in the learning block , the learned response is largely identical across probe speeds .",
"For learning with pursuit target motion at 20 deg/s , the learned response generalizes linearly across probe speeds lower than 20 deg/s for the entire duration of the learning block ( Figure 12E ) .",
"The model also makes predictions .",
"It suggests that adaptive changes in the Purkinje cell responses should diminish over the course of thousands of trials ( Figure 12F , G ) as learning transfers to the FTN inputs ( Figure 12H , I ) .",
"We considered a number of other model forms besides our final cerebellar model ( Figure 12A ) .",
"The criteria for a plausible model were imposed by our data for single-trial learning ( Figures 3 and 4 ) and repeated presentation of the same learning trial ( Figure 6 ) .",
"Single-trial learning always showed a saturating relationship as a function of the error imposed by the instruction .",
"Therefore , all candidate models were constrained by the fits in Figure 3C , ensuring that the measured learned behavior on trial 2 as a function of the error magnitude experienced on the first trial , Y2e1 , was approximately equal to Equation 1 .",
"We then asked whether each candidate model could account for the nearly linear asymptotic learned response as a function of instruction magnitude after a block of 100 learning trials ( Figure 6C ) .",
"We began by assuming the simplest model capable of reaching an asymptotic learned response that was less than the imposed instruction .",
"This model featured a single learning process , x , with a retention parameter that allowed trial-to-trial forgetting: ( 10 ) xn+1=αxn+ΔYn ( en ) Yn=xn Here , α is the retention factor where α=1 indicates complete retention ( no forgetting ) and Yn is the measured behavioral response on trial n .",
"The change in the measured amount of learning on each trial , ∆Ynen , is given by extending Equation 1 from the case of single trial learning ( trials 1 and 2 ) to trial-over-trial learning at any stage ( trials n and n+1 ) .",
"The asymptotic response of this single learning process occurs when Yn+1≈Yn , which is given by: ( 11 ) Y∞=∆Y∞e∞1-α The form of the asymptotic response is a scaled version of the measured single-trial learning from Figure 3C and therefore cannot , by itself , reproduce the linear relationship between instruction magnitude and asymptotic learning that we measured after 100 consecutive learning trials .",
"Any single-learning-process model that ( 1 ) has a static relationship between learning and error and ( 2 ) predicts saturating single-trial learning in Figure 3 would fail to predict the linear asymptotic learning after 100 repeated learning trials in Figure 6 .",
"Pursuit learning might align with other models of motor learning that also have posited two ( Lee and Schweighofer , 2009; Smith et al . , 2006 ) or more ( Kording et al . , 2007 ) learning processes .",
"For our system , learning could be the net sum of two learning processes that run independently and in parallel: a fast-learning process , xf , with rapid learning from error but limited retention and a slow-learning process , xs , with strong retention: ( 12 ) xn+1f=αfxnf+ΔYn ( en ) xn+1s=αsxns+ηsenYn=xnf+ xns A central requirement is that the retention of the slower-learning process is greater than the retention of the faster process , namely αs≫αf , and the rate of learning is greater in the fast-learning process versus the slow-learning process , ∆Ynen≫ηsen .",
"As long as the fast-learning process shows a saturating relationship between error and single-trial learning , the model will reproduce the single-trial learning data in Figure 3C .",
"The two-independent process model in Equation 12 fails to predict a linear relationship between the asymptotic response after extended learning and instruction magnitude .",
"Given the retention values , we measured experimentally ( αf≈0 . 85 and αs>0 . 95 ) , the value of ηs must be very small to ensure that learning saturates at a level significantly below the imposed instruction after extended learning ( Figure 6A ) .",
"With this constraint ( ηs≈0 ) , we can derive the asymptotic response of the two-learning-process model from Equation 12: ( 13 ) Y∞=x∞f+x∞s=ΔYn ( en ) ( 1−αs ) ( 1−αf ) ( 1−αs+η ) + ηI ( 1−αf ) ( 1−αs+η ) ≈Δxf ( e∞ ) 1−αf Equation 13 represents a scaled version of the single-trial response shown in Figure 3 and is largely identical to the asymptotic response predicted by a single-learning-process in Equation 11 .",
"We expressly set out to model our data in a way that was driven by the known architecture of the cerebellar learning circuit .",
"Adherence to the known circuit and the ability to reproduce a wide set of behavioral data with relatively simple assumptions are virtues of the model in Figure 12A .",
"At the same time , we doubt that our successful model is unique and other strategies might ( or might not ) be able to reproduce our data as well .",
"We give two examples .",
"( 1 ) A single-site model might work if the rate of learning is not constant but rather changes from trial-to-trial .",
"It would be possible to account for some of our data in a single-state model where complex-spike-mediated depression of the parallel-fiber to Purkinje cell synapse intrinsically weakened or saturated across repeated identical learning trials .",
"( 2 ) Some models modulate learning based on Bayesian integration of noisy sensory feedback with internal estimates of the perturbation ( i . e . the Kalman filter ) , experienced errors ( Herzfeld et al . , 2014b ) , or environmental consistency ( Gonzalez Castro et al . , 2014 ) .",
"Each of these models predicts that learning rate should increase for repeated presentations of the same learning stimulus versus single-trial learning , whereas we observed that the learning rate decreases over trials .",
"However , one might contrive a model based on these principles where learning rate is lower when the stimulus regime is highly regular and consistent .",
"In addition to their other failings , none of these approaches have the advantage of working seamlessly within the constraint of the architecture of the cerebellar learning circuit ."
],
[
"Our data add to the evidence for a fast-learning process with poor retention based on climbing-fiber mediated plasticity at the parallel to Purkinje cell synapse in the cerebellar cortex ( Medina and Lisberger , 2008; Yang and Lisberger , 2014; Nguyen-Vu et al . , 2013; Kimpo et al . , 2014 ) .",
"Long-term depression at this particular synapse has been a staple of cerebellar learning for many years ( Ito , 2001; Marr , 1969; Albus , 1971 ) , and short-term forms of plasticity also occur at the same site ( Brenowitz and Regehr , 2005; Suvrathan et al . , 2016 ) .",
"Still , the tight link between the occurrence of a complex spike and neural learning in Purkinje cell simple-spike responses ( and behavior ) does not prove that the parallel fiber to Purkinje cell synapse is the mechanism of the fast-learning process and we need to remain open to the possibility that the actual site of plasticity may be upstream of the parallel fiber to Purkinje cell synapse .",
"We are able to measure the properties of the fast-learning process largely independent of other , slower , components of the recurrent learning circuit using the analysis of single-trial learning .",
"Thus , we effectively measured the ‘open-loop’ features of the fast-learning process and discovered that the fast-learning process features a non-linear , saturating relationship between the magnitude of learning and the size of the movement error .",
"We suspect that the non-linear relationship reflects the effects of the retinal slip on the probability and/or duration of the complex spikes that occur in Purkinje cells ( Mathy et al . , 2009; Najafi and Medina , 2013; Yang and Lisberger , 2014 ) .",
"However , we cannot exclude two alternatives .",
"The asymptote of single-trial learning for the highest instruction magnitude could reflect the influence of inhibitory neurons on the learning signal ( Rowan et al . , 2018 ) and/or a simple limit on the maximum amount of plasticity that can be induced by a single climbing fiber input to most Purkinje cells .",
"The saturating dependence of single-trial adaptation in our system on instruction magnitude or stimulus strength parallels findings of single-trial learning in other cerebellar-dependent adaptive behaviors ( Fine and Thoroughman , 2006; Herzfeld et al . , 2014b; Marko et al . , 2012; Hutter and Taylor , 2018 ) .",
"Our finding of qualitatively different generalization functions early versus late in learning provides experimental support for two distinct sites of learning , one that learns quickly and predominates early versus a second process that learns slowly and therefore predominates late in a 1000-trial learning block .",
"Evidence that learning transfers from the fast- to the slow-learning site , rather than proceeding independently in parallel , comes from our computational and theoretical analysis .",
"The cerebellar circuit model with recurrent inhibition of the error input to the cerebellar cortex can explain linearization of the relationship between the size of learning and instruction magnitude after 100 learning trials , whereas a standard model with independent fast and slow-learning processes would require considerable modification to do so ( see Equations 12 and 13 ) .",
"Thus , we think that the fast-learning process for pursuit learning occurs in the floccular complex of the cerebellum ( Medina and Lisberger , 2008 ) and that the floccular target neurons in the vestibular nucleus ( Lisberger et al . , 1994 ) are likely the site of the slow-learning process and long-term storage .",
"At this time , we cannot rule out the possible role of additional downstream areas .",
"There are multiple reasons to think that learning could transfer from the cerebellar cortex to the deep cerebellar nuclei , even though there is currently no direct neurophysiological evidence .",
"Mechanisms of plasticity are abundant in the deep cerebellar nuclei and optogenetic modulation of the activity of Purkinje cells can adapt the vestibulo-ocular reflex , presumably through Purkinje-cell induced plasticity in the deep cerebellar nuclei ( Jang et al . , 2020; Nguyen-Vu et al . , 2013 ) .",
"Recordings from Purkinje cells and deep cerebellar nucleus neurons following long-term vestibulo-ocular gain adaptation lead to the conclusion of neural learning in both sites ( Lisberger , 1994 ) and are entirely compatible with the suggestion that the learned responses in Purkinje cells instruct learning in the deep cerebellar nucleus ( Miles and Lisberger , 1981 ) .",
"For both eye movements ( Pastor , Pastor et al . , 1994 ) and classical conditioning of the eyelid response ( Garcia et al . , 1999 ) , inactivation or lesions of the cerebellar cortex prevents adaptation but does not fully abolish learned responses from previous adaptation ( McCormick and Thompson , 1984; Perrett et al . , 1993 ) , suggesting that the residual long-term learning resides outside of the cerebellar cortex .",
"Finally , for learning in the optokinetic response , lesions of the flocculus abolish the learned response completely after short-term learning but only partially after long-term learning ( Shutoh et al . , 2006 ) .",
"We think that the accessible neural substrate of pursuit learning affords the opportunity to study the neural mechanisms of memory transfer , a ubiquitous characteristic across memory systems .",
"For example , different sites of early acquisition versus long-term storage exist for fear memories ( Rogan et al . , 1997 ) , ( Do-Monte et al . , 2015 ) , and in bird song ( Bottjer et al . , 1984; Nordeen and Nordeen , 1993; Warren et al . , 2011 ) .",
"Indeed , the acquisition of a memory in one structure and the long-term storage of this memory in separate structures is clear from amnesic subjects , such as HM ( Zola-Morgan and Squire , 1990 ) , as well as from many more modern observations ( Tonegawa et al . , 2018 ) .",
"In our data , the early learned response generalizes linearly as a function of all pursuit speeds in the probe trials , even when the probe speed is faster than the pursuit speed in the learning trial .",
"After 1000 trials of learning , the learned response scales only for probe speeds slower than pursuit speed in the learning trial .",
"The literature contains other evidence of changes in the generalization of the expression of learning across timescales .",
"In birdsong , learned changes in the pitch of one syllable generalizes differently early versus late in learning ( Warren et al . , 2011 ) , suggesting that early learning in LMAN changes signals that are related to the overall song , while the late learning in the motor structure RA operates on signals related to the motor act of producing the syllable .",
"In the vestibulo-ocular reflex , Kimpo et al . , 2005 showed different generalization for stimulus frequency for gain-up versus gain-down learning , suggesting different sites of learning based on modification of different vestibular signals .",
"After Shadmehr , 2004 , we interpret the change in generalization of learning as possible evidence for a change in the properties of the neural signals that are subjected to learning .",
"Our circuit model accounts for the change in generalization by assuming that a different set of eye speed signals is learned at Purkinje cells versus FTNs .",
"The use in the model of different inputs to the sites of plasticity in the floccular complex and at the floccular target neurons suggests two features of the neural system that run counter to current doctrine .",
"First , the general rule is that Purkinje cells and their targets in the deep cerebellar nuclei receive identical inputs from mossy fibers .",
"In the floccular complex , however , this does not seem to be the case: brainstem areas that send afferent mossy fibers to the ventral paraflocculus region of the floccular complex send only sparse projections to the location of FTNs in the vestibular nucleus ( Osanai et al . , 1999; Nagao et al . , 1997 ) .",
"Second , the general rule in the oculomotor system is the firing rate is linearly related to eye position and velocity , and this is true for mossy fibers in the floccular complex ( Miles et al . , 1980; Lisberger and Fuchs , 1978 ) .",
"Thus , there is no obvious precedent for neural responses that are unimodally tuned for eye velocity .",
"Limited recordings from FTNs during pursuit learning Joshua et al . , 2013 demonstrate a diversity of FTN velocity-dependent responses to a 30 deg/s pursuit stimulus , perhaps consistent with a distribution of preferred speeds across the neural population .",
"We realize that the assumptions in the model may not reflect the actual implementation of the system in the brain , and that the differences in generalization at different sites could reflect properties of synapses or plasticity mechanisms rather than of the input signals that are subject to plasticity .",
"We have always been troubled by the fact that pursuit learning , like other forms of motor learning ( Krakauer et al . , 2000; Tseng et al . , 2007; Vaswani et al . , 2015 ) , never reaches an amplitude that completely eliminates the error created by the imposed instruction .",
"Even after more than 1000 learning trials , the adapted pursuit eye movements shows residual errors than are in excess of 50% of the imposed instruction ( Hall et al . , 2018 ) .",
"We suggest that motor learning never fully adjusts for the instruction because of active inhibition of the instruction for learning Kenyon et al . , 1998 ) .",
"Via the known double-inhibitory pathway from Purkinje cells to the inferior olive via the deep cerebellar nucleus ( Houck and Person , 2014; Najac and Raman , 2015; de Zeeuw et al . , 1988 ) , learned depression of Purkinje cell simple-spike firing could reduce the effectiveness of a given movement error by attenuating the probability and/or duration of climbing fiber inputs to the cerebellar cortex .",
"Previous neurophysiological observations over 100 trials of pursuit learning demonstrate a reduction in the probability of complex spikes , hinting at possible inhibition of the olive ( Medina and Lisberger , 2008; Yang and Lisberger , 2017 ) .",
"Our data and modeling render unlikely the alternate explanation that incomplete adaptation is the result of a competition between the learning signals and a restoring force due to forgetting ( Smith et al . , 2006; Albert et al . , 2019 ) .",
"During error-clamp trials , we measured the retention parameter after 1000 trials to be close to 1 . 0 , corresponding to almost complete retention .",
"We imagine that recurrent inhibition of the error inputs to the fast-learning process promotes slow , conservative learning that does not respond hastily to noisy inputs .",
"We do not understand fully why it is advantageous for the fast-learning process to remain inhibited after learning has been transferred from the cerebellar cortex to FTNs .",
"Perhaps the goal is to force other parts of the pursuit circuit to take over some of the learning under conditions where permanent changes seem desirable , by transferring some of the learning away even from the FTNs .",
"Alternatively , our somewhat rarified stimulus conditions may not be engaging the neural learning circuit fully so that we are seeing only a part of the full learning capability of the system .",
"The cerebellar learning circuit is stereotyped , well-known , accessible for analyses at the level of the activity of single neurons , and engaged in quantitatively-defined learning behavior .",
"We see it as an exemplar where we have the greatest potential to determine not just the sites and mechanisms of plasticity , but also how the essential neural circuit exploits the local mechanisms of plasticity and converts them into the global deliverable of an adaptive change in behavior .",
"Similar analysis and thinking are going to be essential in all other learning systems and neural circuits before we can say that we ‘understand’ any form of learning and memory .",
"The particular behavioral system we study affords the huge advantage that we can constrain models by both the architecture of the essential circuit and data on the operation of the system .",
"Our data and computational thinking assemble a statement of the principles of operation of this learning neural circuit .",
"Once additional behavioral and neurophysiological evidence further refine these principles of operation , we will have a strong statement of how one learning circuit works ."
],
[
"Monkeys were positioned with their heads fixed 30 cm in front of a CRT monitor ( resolution: 2304 × 1440 , framerate: 80 Hz ) .",
"The monitor subtended 58 × 46° of the monkey’s visual field .",
"All experiments took place while the monkey was in a dimly lit room .",
"The visual target for all experiments was a 0 . 5° diameter black dot presented on a light grey background ( approximate luminance 32 . 9 cd/m2 ) .",
"The motion of the target at each frame was controlled by our laboratory’s custom ‘Maestro’ software .",
"Separate voltage signals from the scleral coil system , corresponding to the monkey’s horizontal and vertical eye position , were digitized at 1 kHz .",
"We also digitized eye velocity signals obtained by passing the eye position voltages through an analog differentiator circuit with a low-pass filter that reduced the amplitude of signals above 25 Hz ( −20 dB/decade ) .",
"All signals were stored for later offline processing and analysis .",
"At the start of each trial , the monkey fixated within ±3° of a target at the center of the screen for a random interval , chosen from a uniform distribution of 400–800 ms . After the fixation period , the target instantaneously jumped eccentrically by 0 . 15 vt degrees , and then began moving at a constant speed , vt , in the opposite direction , termed the ‘pursuit direction’ ( step-ramp paradigm of Rashbass , 1961 ) .",
"The eccentric target jump minimizes the number of catch-up saccades during the initial tracking of the target .",
"During baseline , probe , and washout trials , the target proceeded at the constant ‘pursuit speed’ for 850 ms before stopping .",
"The monkey then fixated the target at its final position for an additional 200 ms . In exchange for appropriate tracking of the target as well as fixating the target within ±3° at the end of the trial , the monkey received a small fluid reward .",
"If the monkey broke fixation during the initial fixation period or failed to adequately track the moving target and fixate its final position , the trial was aborted immediately and the monkey received no reward .",
"Aborted trials were not included in the data analysis .",
"To induce cerebellar-dependent motor learning , we used a direction learning paradigm ( Medina et al . , 2005 ) .",
"In ‘learning trials’ ( Figure 2A , dotted line , Figure 2B , D ) , the target proceeded in the original pursuit direction for 250 ms , before the addition of an orthogonal velocity component , termed the ‘instruction . ’ We call the direction of the orthogonal instruction the ‘learning direction , ’ since the instruction induces visual motion that serves as an error and drives cerebellar-dependent motor learning .",
"During the instruction , we suspended the eye position requirements for liquid reward .",
"In some experiments , we varied the speed of the instruction .",
"In all experiments , the instruction had a duration of 400 ms , so that it was followed by 200 ms of target motion in the original pursuit direction and then 200 ms of fixation .",
"Reward was not contingent upon generation of a learned response .",
"Monkeys typically worked for 1500 to 3000 successful trials per experimental session .",
"To measure the amount of trial-to-trial forgetting in the absence of error , we used ‘error-clamp’ trials .",
"During error-clamp trials , the target proceeded in the original pursuit direction , as in a probe trial .",
"However , the location of the target in the direction orthogonal to the pursuit direction ( the learning direction ) was ‘clamped’ to the animal’s eye position , such that the position of the target on each of the monitor’s frames matched the animal’s current eye position in the learning direction ( Figure 3D ) .",
"Target stabilization in the learning direction removes any errors that could drive unlearning and allowed us to measure the intrinsic retention/decay of motor memories without any stimulus for learning .",
"To separately investigate the characteristics of the signals that drive learning of a motor memory from those that affect generalization on the timescale of a single-trial , we developed a ‘dual-trial’ experimental paradigm as a variant of our previously described paradigm for single-trial learning ( Yang and Lisberger , 2010 ) .",
"Our modified paradigm uses pairs of trials , where the first trial , n , is the learning trial that provides a direction-change instruction ( Figure 2A , dotted line , Figure 2B ) and the second trial , n+1 , is a probe trial without an instruction ( Figure 2A , solid line , Figure 2C ) .",
"Both the learning and probe trial in each dual-trial pair used the same pursuit direction , chosen randomly for each pair of trials from the cardinal axes ( 0° , 90° , 180° , or 270° ) .",
"For some experiments , the speed of the target in the pursuit axis ( i . e . , the pursuit speed ) was identical in both the learning and probe trials .",
"In other experiments , we strategically altered the pursuit speed in either the learning trial or the probe trial .",
"The direction of the instruction in the learning trial was chosen randomly to be either +90° or −90° relative to the pursuit direction .",
"Due to the random directions of the initial pursuit and the instruction within a daily experiment , learning did not accumulate across trials .",
"We explicitly prohibited the consecutive occurrence of pairs of trials with the same pursuit and instruction directions .",
"To quantify the behavioral learning expressed in the second trial of each pair , we measured the ‘learned response’ from the eye velocity in the direction of the instruction in the preceding learning trial ( i . e . , the learning direction ) .",
"Monkeys typically performed 750 to 1500 pairs of trials in a given experimental session .",
"We extended our dual-trial paradigm to investigate changes in the generalization of learning to pursuit speed across long blocks of trials .",
"Now , we allowed the learning to accumulate by using a consistent pursuit direction in both the learning and probe trials across the entire experimental session .",
"The pursuit direction for both the learning and probe trial was chosen randomly from the cardinal axes ( 0° , 90° , 180° , or 270° ) for each experimental session .",
"Within an experimental session , the pursuit speed during the learning trial was either 5°/s or 20°/s .",
"To measure the generalization of learning to pursuit speed , the speed of each probe trial was chosen randomly ( 5 , 10 , 15 , or 20 deg/s ) .",
"For all experiments that used the dual-trial paradigm , we report statistics for each monkey across days/experimental sessions .",
"To measure changes in the acquisition of a motor memory across timescales longer than a single-trial , we used blocks of trials with consistent instruction statistics .",
"For each ‘learning block , ’ we chose a single direction of pursuit for all trials from one of the cardinal axes .",
"Monkeys performed a series of baseline trials without an instruction , followed by a series of learning trials with an imposed instruction in a consistent learning direction that was orthogonal to the pursuit direction .",
"The behavioral learning was subsequently extinguished by presenting washout trials that did not feature an instruction .",
"The number of washout trials was always equal to or larger than the number of the preceding learning trials .",
"Because the direction of the instruction was consistent across the learning trials , we define the behavioral learning on each trial as the speed of the eye movement in the direction of the instruction experienced during the learning trials .",
"After a complete learning block , including washout , a new pursuit direction and instruction direction were chosen randomly for the subsequent block .",
"Monkeys typically performed multiple repetitions of repeated-instruction learning paradigms in a given experimental session .",
"No two consecutive learning blocks were allowed to have the same pursuit and instruction directions .",
"For all experiments in a repeated-instruction design , we report statistics for each monkey across repetitions of the learning paradigm .",
"To ensure that saccades did not contaminate our estimate of the learned pursuit response , we identified and removed saccades from eye velocity traces .",
"We identified saccades using a combined speed and acceleration threshold: any instance when the speed of the eye exceeded 20 deg/s and the eye acceleration exceeded 1250 deg/s/s was labeled as a saccade .",
"Time points were marked as a saccade from 10 ms before the first time point that exceeded joint velocity and acceleration thresholds to 10 ms after the final time point that exceeded both thresholds .",
"We eliminated these intervals from data analysis by treating them as missing data .",
"Because the target motions were designed to minimize their occurrence , saccades were typically infrequent in the analysis intervals and occurred at variable times .",
"To summarize the magnitude of behavioral learning on a single-trial , we averaged the speed of the eye in the learning direction from 25 ms before to 25 ms after the time of the instruction , 225–275 ms after the onset of pursuit-direction target motion ( grey shading in Figure 2B , C ) .",
"Even during trials that included an instruction ( e . g . during block-wise learning ) , this measurement provides an estimate of the learned response because the effects of visual feedback and online corrections in pursuit movements do not appear until at least 75 ms after the onset of the instruction .",
"This measurement of motor learning is consistent with previous pursuit direction learning experiments ( Hall et al . , 2018; Yang and Lisberger , 2010; Yang and Lisberger , 2017 ) .",
"We used two tailed t-tests to compare sample means for conditions that were not sampled within the same experimental session , with the significance level set at p=0 . 05 .",
"Remaining tests with conditions that were simultaneously sampled within an experimental session were performed using a repeated measures analysis of variance ( RM-ANOVA ) ."
]
] | [
"We provide behavioral evidence using monkey smooth pursuit eye movements for four principles of cerebellar learning .",
"Using a circuit-level model of the cerebellum , we link behavioral data to learning’s neural implementation .",
"The four principles are: ( 1 ) early , fast , acquisition driven by climbing fiber inputs to the cerebellar cortex , with poor retention; ( 2 ) learned responses of Purkinje cells guide transfer of learning from the cerebellar cortex to the deep cerebellar nucleus , with excellent retention; ( 3 ) functionally different neural signals are subject to learning in the cerebellar cortex versus the deep cerebellar nuclei; and ( 4 ) negative feedback from the cerebellum to the inferior olive reduces the magnitude of the teaching signal in climbing fibers and limits learning .",
"Our circuit-level model , based on these four principles , explains behavioral data obtained by strategically manipulating the signals responsible for acquisition and recall of direction learning in smooth pursuit eye movements across multiple timescales ."
] | [
"The human brain can do many things , from reading and remembering the words written on a page to adapting and improving movements .",
"When a movement misses its goal , the strength of the connections between cells in a part of the brain known as the cerebellum changes .",
"The cerebellum is important for coordinating movements , including eye movements .",
"When the connections between the cells in the cerebellum – known as neurons – strengthen or weaken , the cerebellum changes how it will respond in the future , leading to more accurate movements .",
"However , the speed of the changes in the connections and how the connections between different neurons evolve and coordinate were unknown .",
"Herzfeld et al . have now combined eye-tracking studies in monkeys with computer modeling based on what is known about the neural circuits in the cerebellum to learn more about the changes in these connections .",
"Monkeys watched a moving target that would abruptly change direction .",
"In the next movement , the eye-tracking equipment monitored how well the monkey’s eyes anticipated the unexpected change in the target’s direction – a form of motor learning .",
"Using the experimental data , Herzfeld et al . produced a model that outlines general principles of how the cerebellum might manage this process .",
"The model suggested that neurons in one region in the cerebellum , known as Purkinje cells , learn from mistakes quickly , but have poor long-term retention .",
"If the movement is repeated , Purkinje cells teach another area of the cerebellum , the cerebellar nucleus , which takes longer to learn but has much better retention .",
"Although these findings are based on a simple motor learning task , they are the first step to understanding how the brain forms memories and how we might learn more complex behaviors ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy | elife-38064-v2 | [
[
"Heritable hypertrophic cardiomyopathy ( HCM ) is a leading cause of death among young adults , particularly competitive athletes .",
"This heterogeneous and complex disease typically involves asymmetric growth of the heart , interventricular septum thickening , disorganized cellular architecture , diastolic dysfunction , arrhythmias , and an increased risk of sudden cardiac death ( Davis et al . , 2016; Maron , 2002; Maron and Maron , 2013; Masarone et al . , 2018 ) .",
"Diastolic dysfunction is characterized by impaired left ventricular relaxation , chamber stiffening , elevated filling pressures , and , consequently , reduced stroke volumes and cardiac output .",
"Enhanced cardiomyocyte contractile activity is a leading hypothesis for the underlying cause of HCM .",
"Increased Ca2+-sensitivity of the contractile apparatus as well as heightened function of the myosin molecular motor are potential molecular mechanisms .",
"The over-active contractile apparatus can prolong mechanical tension , which apparently initiates hypertrophy via activation of specific signaling cascades that regulate heart growth ( Davis et al . , 2016 ) .",
"Although therapies exist to treat problematic HCM abnormalities , there is no cure .",
"Thus , it is critical to better understand both the disease-causing mutations and the mechanism by which they produce HCM .",
"Over 1400 point mutations in sarcomeric proteins cause HCM ( Maron and Maron , 2013 ) , with the largest number ( >300 ) found in the β-cardiac myosin heavy chain .",
"This myosin II molecule forms sarcomere thick filaments and serves as a molecular motor to drive ATP-dependent movement along actin-containing thin filaments .",
"Mapping the locations of the mutations onto a three-dimensional structure of myosin reveals several hot spots .",
"These include the converter domain ( Colegrave and Peckham , 2014 ) and the recently described ‘myosin mesa’ ( Homburger et al . , 2016 ) .",
"The converter guides the myosin lever arm in its rotation during the power stroke that generates force and motion ( Mesentean et al . , 2007 ) .",
"The myosin mesa has been implicated in enabling formation of an interacting head motif ( Trivedi et al . , 2018 ) , wherein the heads of the dimeric molecule fold together to dampen motor function ( Woodhead et al . , 2005 ) .",
"Several molecular and physiological studies suggest that enhanced ATPase , in vitro actin sliding speed and muscle contractile force and/or velocity correlate with many , but not all , of the HCM-causing myosin mutations ( Adhikari et al . , 2016; Moore et al . , 2012; Sommese et al . , 2013; Spudich , 2014 ) .",
"For the domains cited above , this could occur via activation of the lever arm through enhanced converter function or stimulation of myosin cross-bridge initiation through inhibiting the formation of the interacting head motif .",
"The mechanism by which increased contractility results in development of HCM phenotypes remains an active area of investigation ( Davis et al . , 2016; Garfinkel et al . , 2018 ) .",
"In this communication , we model a human myosin HCM mutation in Drosophila melanogaster , which provides a highly integrative approach that yields unique insights into the disease .",
"The Drosophila heart , although a relatively simple tube-shaped structure , shares numerous gene expression patterns , as well as developmental and functional properties , with the human heart ( Bier and Bodmer , 2004 ) .",
"Further , the fly system allows evaluation of the effects of a mutation upon isolated myosin ( Swank et al . , 2000 ) , as well as on the myofibrillar structure and physiological function of both skeletal ( Maughan and Vigoreaux , 1999 ) and cardiac muscles ( Ocorr et al . , 2014 ) .",
"A major advantage of Drosophila is its simplified genetics , in that one Mhc gene gives rise to all myosin isoforms through alternative RNA splicing ( George et al . , 1989 ) .",
"Hence compensation by myosin multigene family members that can mask mutant protein effects does not occur .",
"Further , myosin expression levels and tissue-specificity can be controlled by combining Mhc transgenes with lines containing Mhc alleles that specifically knock out myosin heavy chain expression in the indirect flight muscles ( Collier et al . , 1990 ) or in all muscle types ( O'Donnell and Bernstein , 1988 ) .",
"Previously , using this approach , we showed that a mutant myosin with enhanced ATPase activity yielded disrupted skeletal muscle structure and function and restricted the Drosophila cardiac tube to reduce its output ( Cammarato et al . , 2008 ) .",
"This indicates that Drosophila models could yield insights into the basis of HCM through assessing human mutations that cause overactive myosin .",
"For the current study , we produced transgenic flies expressing the Drosophila equivalent of the K146N human HCM myosin mutation .",
"This mutation was identified in the family of an adult patient with increased left ventricular wall thickness in the absence of hypertension ( Ingles et al . , 2005 ) and as a spontaneous mutation in a child who displayed left ventricular hypertrophy ( Morita et al . , 2008 ) .",
"Residue 146 is particularly interesting in that it does not map to a well-studied HCM hot spot .",
"Instead , it maps near the N-terminus of the protein , which has few HCM-causing mutations ( Colegrave and Peckham , 2014 ) .",
"However , mutational analysis in Dictyostelium ( Ruppel et al . , 1994 ) and a domain swap study in Drosophila ( Swank et al . , 2003 ) suggest that effects on the mechanochemical cycle of myosin might be expected from alterations to the N-terminal region of myosin II molecules .",
"Our molecular modeling predicts that mutation of residue 146 of muscle myosin reduces its interaction with the myosin lever arm during the pre-power stroke state .",
"The myosin S1 head domain is in a cocked position during this state , so that it is prepared to drive the power stroke and muscle contraction upon actin binding during the mechanochemical cycle .",
"We found that the primary result of the 146N mutation is that myosin spends increased time in strongly bound states of the cross-bridge cycle .",
"This prolonged binding of myosin to actin increases force generation , but slows actin motility , decreases cyclical muscle power generation and reduces flight ability .",
"Higher force production and stiffness also cause progressive skeletal and cardiac muscle structural degeneration , and a restricted cardiac phenotype with diastolic dysfunction .",
"Some of our observations , such as the prolonged periods of systolic tension as well as increased ATPase rates , indicate this HCM mutation causes hyperdynamic contractile properties .",
"However , our use of the integrative approach in the Drosophila system shows that the mutant phenotype is complex , with reduced functions for a number of other parameters .",
"This emphasizes the need for a comprehensive analysis , from the molecular to organismal levels , which has allowed us to provide key insights into defining the molecular mechanism behind myosin-based HCM ."
],
[
"We determined the location and interactions for the amino acid residue corresponding to human K146 , which in its mutant form ( K146N ) causes HCM .",
"The Drosophila indirect flight muscle ( IFM ) myosin heavy chain sequence was modeled onto the scallop muscle myosin II crystal structure during the pre-power stroke state ( PDB 1QVI ) and the actin-detached post-power stroke state ( PDB 1KK8 ) ( Figure 1A and B ) .",
"Both scallop and Drosophila substitute an identically charged arginine residue for lysine at residue 146 , as do some human muscle myosins ( Rossi et al . , 2010 ) ( for clarity of comparison , the human β-cardiac myosin numbering system is used throughout ) .",
"The location of the modeled Drosophila residue , which is at the surface of the motor domain of the molecule , is shown in magenta .",
"In the pre-power stroke state , this residue is in close proximity to the lever arm ( Figure 1A ) , a domain that rotates to generate movement and force during muscle contraction .",
"As a result of configuration changes during the mechanochemical cycle , the N-terminus and R146 are distant from the lever arm in the post-power stroke state ( Figure 1B ) .",
"The Drosophila , scallop and human β-cardiac myosins show strong sequence similarity in the R146 region and also in the lever arm interacting region ( Figure 1B inset ) .",
"Analysis of the pre-power stroke model reveals specific charge interactions with residue 146 ( Figure 1C ) .",
"There is a 3 . 2 Å contact distance between positively charged R146 and negatively charged E774 , as well as a 3 . 0 Å contact distance between R146 and E775 .",
"These close contact distances permit the formation of salt bridges with E774 and E775 residues in the lever arm of the molecule .",
"In a model with HCM mutant residue R146N , the close contact and salt bridges with E774 and E775 are eliminated , with distances increased to 4 . 5 and 5 . 7 Å , respectively ( Figure 1D ) .",
"This could decrease the stability of the pre-power stroke state and alter rates of conformational changes required for the actin-myosin mechanochemical cycle to proceed normally .",
"A similar disruption may occur for the human β-cardiac mutant myosin as well ( Figure 1—figure supplement 1 ) .",
"To study the effects of the R146N mutation within myosin and Drosophila muscle , we constructed a mutant myosin transgene and obtained 29 transgenic lines by P element-mediated transformation .",
"Three independent transgenic inserts that mapped to the third chromosome ( PwMhcR146N-11 , PwMhcR146N-15 , PwMhcR146N-28; abbreviated hereafter with PwMhc deleted ) were crossed into the Mhc10 background , which is null for myosin heavy chain in IFM ( Collier et al . , 1990 ) .",
"RT-PCR of RNA isolated from dissected IFM verified that each transgenic line expresses only the mutant myosin in the IFM and that the normal alternative exon splicing pattern is not disrupted ( see Materials and methods ) .",
"SDS-PAGE analysis confirmed that upper thoraces of mutant 2-day-old adult female flies from lines 11 , 15 and 28 express normalized ratios of myosin to actin ( 1 . 06 ± 0 . 06 , 0 . 99 ± 0 . 02 , 0 . 98 ± 0 . 01 , respectively ) that are essentially identical to the wild-type control transgenic line PwMhc2 ( 1 . 00 ± 0 . 04 ) .",
"To explore the impact of the R146N mutation on the mechanochemical cycle of myosin , we assessed the steady-state ATPase parameters of myosin isolated from the IFM of R146N homozygotes ( Figure 2A–E ) .",
"Both Ca-ATPase ( 16 . 64 ± 1 . 87 vs . 9 . 51 ± 1 . 08 sec−1 ) and basal Mg-ATPase ( 0 . 62 ± 0 . 14 vs . 0 . 25 ± 0 . 04 sec−1 ) rates of R146N myosin increased significantly , about two-fold compared to wild-type myosin ( Figure 2A , B ) .",
"While Ca2+ typically yields higher in vitro ATPase activities , Mg2+ is the physiologically relevant cation that is employed for actin stimulation .",
"We found that actin-stimulated Mg-ATPase activity ( Vmax ) ( Figure 2C ) was not significantly different from the control ( 1 . 57 ± 0 . 27 vs . 1 . 62 ± 0 . 14 sec−1 ) .",
"The Km of actin concentration relative to ATPase activity was significantly increased , indicating that a 35% higher actin concentration is required to reach 50% Vmax ( 0 . 42 ± 0 . 06 vs . 0 . 31 ± 0 . 04 µM ) ( Figure 2D ) .",
"Further , the mutant’s catalytic efficiency ( defined as Vmax/Km ) was significantly lower than that of the control ( 3 . 80 ± 0 . 60 vs . 5 . 34 ± 1 . 07 sec−1/µM ) ( Figure 2E ) .",
"Analysis of in vitro actin-sliding velocity data showed that the mutant myosin possesses an ~two fold reduction in its ability to drive actin filament sliding compared to myosin isolated from control IFM ( 3 . 32 ± 0 . 21 vs . 6 . 71 ± 0 . 17 µm/s ) ( Figure 2F ) .",
"Thus the major effects of the R146N mutation on ATPase and in vitro motility are an enhancement of basal Mg- and Ca-ATPase rates and a decrease in actin filament sliding velocity .",
"To determine if the R146N mutation affects muscle structure and stability , we employed transmission electron microscopy to examine IFMs of homozygous female flies at late-pupal stage as well as fibers from 2-hr-old , 2-day-old and 1-week-old adults .",
"Transverse and longitudinal sections of R146N late-stage pupae and 2-hr-old adults ( Figure 3E , F ) showed normal sarcomeres containing double hexagonal arrays of thick and thin filaments , which are indistinguishable from those of the PwMhc2 homozygous control line at the same developmental stages ( Figure 3A , B ) .",
"Transverse and longitudinal sections of R146N 2-day-old adults showed minor disruptions of thick and thin filament packing ( Figure 3G ) compared to the control ( Figure 3C ) , with even more modest disruption seen in R146N/+ heterozygotes ( Figure 3—figure supplement 1 ) .",
"Transverse and longitudinal sections of R146N 7-day-old adults showed more disorder in myofibril morphology , with gaps in the hexagonal packing of thick and thin filaments and disruption in the Z- and M-lines ( Figure 3H ) compared to the control ( Figure 3D ) .",
"Hence R146N mutant myosin is capable of contributing to the formation of normal myofibrillar structures that begin to deteriorate at 2 days into adulthood , presumably as a result of mechanical stress during use .",
"We minimized any influence of structural changes in R146N adult IFMs on our muscle mechanical assays by employing fibers from 2-hr-old flies for these assays .",
"We also assessed heterozygous organisms ( R146N/+ ) to determine whether the dominant nature of the mutation in humans is mirrored in Drosophila .",
"We assessed the effects of R146N myosin on IFM function by testing flight ability .",
"Line 15 R146N homozygotes exhibited ~50% and ~70% decreases in flight index at 15°C ( temperature employed for flight muscle mechanics ) and 22°C ( ambient temperature ) , respectively , compared to the control line ( Table 4 ) .",
"The flight index of R146N/+ heterozygotes was significantly higher than that of homozygotes at both temperatures , with a statistically significant 15% reduction compared to the control line at 22°C and no significant difference with the control line at 15°C ( Table 4 ) .",
"The flight ability of the other two lines of homozygous R146N mutants also was severely diminished ( Supplementary file 2 ) .",
"Impairment for all lines increased with age in a statistically significant manner , as flight index at 7 days decreased to about half that at 2 days ( Supplementary file 2 ) .",
"In contrast , the flight index of control organisms showed a significant decrease of only 11% during the same timeframe ( Supplementary file 2 ) .",
"We also assessed wing beat frequency ( WBF ) at 2 days of age and found a significant reduction in the homozygous R146N mutants , with 10% ( 15°C ) and 7% ( 22°C ) decreases compared to controls ( Table 4 ) .",
"The R146N/+ heterozygous mutant showed no significant decrease in WBF at either temperature .",
"However , there was a significant difference between the WBFs of the heterozygous and homozygous mutants at both temperatures .",
"Thus , R146N myosin detrimentally affects WBF and flight ability .",
"Homozygotes display more severe phenotypes than heterozygotes .",
"We next assessed the structure of adult hearts of R146N/+ heterozygous flies by transmission electron microcopy to determine the effects of the dominant myosin mutation on cardiac ultrastructure .",
"Two distinct layers are observed in transverse sections taken between the second and third abdominal segments ( Figure 6 ) .",
"The lumen-facing layer is composed of contractile cardiomyocytes that contain a circumferential array of myofibrils oriented perpendicularly to the anterior-posterior axis of the heart .",
"A layer of structurally supportive ventral-longitudinal skeletal muscle is located ventral to the cardiomyocyte layer ( Figure 6 , VL ) .",
"In heterozygous controls ( PwMhc2/+ ) , both young 1-week-old and aged 3-week-old flies showed intact myofibrils with characteristic discontinuous Z-lines ( Achal et al . , 2016 ) ( Figure 6 left ) .",
"In contrast , R146N-15/+ heterozygotes displayed areas of myofibrillar discontinuity , where filamentous fields appear to have been pulled apart ( Figure 6 center , arrows ) and these defects worsened with aging .",
"Although R146N-28/+ hearts showed essentially normal myofibrillar integrity and organization in young flies , they displayed myofibrillar discontinuities in aged flies ( Figure 6 right , arrows ) .",
"This suggests that defects in older mutant flies are not due to gross assembly defects but occur during the aging process .",
"To assess whether concentric cardiac hypertrophy ( addition of myofibrils in parallel ) occurs in R146N/+ heterozygotes , we measured the average cardiomyocyte thickness in ventral and dorsal areas and compared values between young and aged flies for mutant lines and controls .",
"Cardiomyocyte thickness in both young and aged mutant heterozygotes showed no statistically significant differences compared to controls ( Supplementary file 3 ) .",
"Further , we observed no evidence of eccentric hypertrophy ( addition of sarcomeres in series ) , given that our cardiac physiological analysis showed no increases in chamber dimensions in young or old mutant hearts under basal conditions ( Figure 7A–C ) , and a dilated phenotype was not observed after complete relaxation with EGTA+ blebbistatin ( Figure 8A–C ) .",
"Hence the R146N myosin mutation caused progressive defects in myofibrillar continuity , but did not yield hypertrophy in the Drosophila heart .",
"To investigate the pathophysiological consequences of the dominant R146N HCM mutation on the adult Drosophila heart , we surgically exposed , imaged , and assessed wall movement in semi-intact R146N/+ heterozygous flies .",
"The effects of mutant myosin expression on cardiac dimensions and contractile performance , at 1- and 3-weeks of age , were quantified and compared to those resulting from wild-type transgenic myosin expression in age-matched PwMhc2/+ controls ( Figure 7A–C ) .",
"The diastolic and systolic diameters across the heart wall of 1-week-old control ( PwMhc2/+ ) Drosophila were 65 . 68 ± 0 . 73 and 39 . 60 ± 0 . 46 µm , respectively , and were 67 . 58 ± 0 . 80 and 43 . 49 ± 0 . 67 µm in 3-week-old animals ( Supplementary file 4 ) .",
"R146N myosin expression triggered a significant reduction in dimensions at both ages .",
"The diastolic and systolic diameters of R146N-15/+at 1 week were 52 . 13 ± 0 . 53 and 32 . 83 ± 0 . 45 µm , respectively , and were 48 . 62 ± 0 . 64 and 34 . 76 ± 0 . 53 µm at 3 weeks .",
"The greater mutational effect on diastolic vs . systolic diameter resulted in 7 . 5% and 22% decreases in fractional shortening at 1 and 3 weeks of age relative to controls ( Figure 7C ) .",
"Similar results were found for R146N-28/+ flies ( Supplementary file 4 ) .",
"The age-related decrease in fractional shortening suggests progressive remodeling in the mutant , which correlates with the decreasing myofibrillar continuity we observed .",
"The heart period , which is the time required for a complete cardiac cycle consisting of a diastolic and subsequent systolic phase , was not significantly different between mutant and control flies at either age tested ( Figure 7C ) .",
"The systolic interval , however , was 0 . 19 ± 0 . 01 and 0 . 21 ± 0 . 01 s for 1- and 3-week-old control flies and 0 . 21 ± 0 . 01 and 0 . 23 ± 0 . 01 s for R146N-15/+ heterozygotes .",
"Therefore , the proportion of time during the cardiac cycle that was spent generating tension , calculated by determining the ratio of systolic interval to heart period ( SI/HP ) , was significantly greater for the mutant heart tubes relative to controls at all ages studied ( Figure 7C ) .",
"Together , these findings illustrate that the R146N/+ hearts exhibit reduced cardiac diameters and potentially elevated tone during diastole , as well as prolonged periods of systolic tension generation , which are indicative of restrictive physiology , diastolic dysfunction , and impaired relaxation ( Cammarato et al . , 2008; Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .",
"To further investigate the molecular basis of diastolic restriction and potentially altered resting tone in the R146N/+ heterozygote hearts , we compared the responses of three-week-old control and mutant cardiac tubes to treatments with EGTA-AM , a cell permeable chelator of intracellular Ca2+ ( Johnson et al . , 1997 ) and blebbistatin , a small molecule myosin inhibitor ( Fedorov et al . , 2007; Limouze et al . , 2004 ) .",
"Upon incubation with artificial hemolymph supplemented with 10 mM EGTA and 100 µM EGTA-AM , which removes extra- and intracellular Ca2+ , the hearts ceased beating and ‘relaxed’ , as manifest by an increase in diameter across the wall of both control and R146N/+ hearts relative to diastole ( Figure 8A ) .",
"Interestingly , there was a significantly amplified relaxation response in R146N/+hearts ( Δ = 4 . 50 ± 0 . 13 µm ) relative to controls ( Δ = 3 . 77 ± 0 . 19 µm ) ( Figure 8B ) .",
"We previously demonstrated in vivo , that a small population of residual cross-bridges actively cycles and generates force , and helps establish basal mechanical tone in wild-type Drosophila myocardium during diastole ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .",
"In this regard , subsequent treatment of control and mutant hearts with artificial hemolymph containing 10 mM EGTA , 100 µM EGTA-AM , and 100 µM blebbistatin generated a second ‘relaxation’ response ( Figure 8A ) .",
"Comparing the cardiac diameters after extra- and intra-cellular Ca2+ chelation and following blebbistatin treatment revealed a significant increase of roughly 2 . 8% ( Δ = 2 . 34 ± 0 . 17 µm ) in control hearts ( Figure 8C ) .",
"Notably , there was a strikingly augmented response to blebbistatin treatment in the R146N/+ heterozygote hearts that led to a ~ 10% ( Δ = 6 . 14 ± 0 . 35 µm ) increase in heart tube diameter ( Figure 8C ) .",
"Comparing the change in diameters between the two genotypes revealed that the response to blebbistatin is significantly greater in the R146N/+ mutant hearts vs . that for controls ( Figure 8C ) .",
"Our observations suggest that , in addition to elevated diastolic Ca2+ and Ca2+-activated cross-bridge cycling , excessive Ca2+-independent cross-bridges contribute to diastolic dysfunction and inadequate relaxation in the R146N/+ mutant hearts .",
"Overall , we utilized an integrative approach in the Drosophila system to determine the effects of a myosin HCM mutation on myosin function , skeletal muscle structure , muscle mechanics and cardiac physiology .",
"We found that altering modeled interactions between the mutant R146N residue and the lever arm leads to increased basal ATPase activity and changes to myosin cross-bridge kinetics that disrupt actin motility , myofibril power output , myofibril stability , flight ability and cardiac structure and function .",
"Our analyses provide insight into how a myosin mutation translates into mutant phenotypes , thereby elucidating underlying mechanisms of HCM ."
],
[
"We leveraged the strengths of the Drosophila myosin system to determine new phenotypic and mechanistic insights into HCM , a common disease of young adults .",
"The Drosophila model is an outstanding tool for integrative analysis of contractile protein mutations and for dissecting the mechanistic basis of disease , allowing an understanding from the molecular through the whole organism level .",
"In our HCM model , the Drosophila heart tube shows a restricted phenotype , which , like abnormalities in the mutated IFM , develops from prolonged cross-bridge binding to actin .",
"The restricted cardiac tube and diastolic dysfunction in this model of human HCM is congruent with phenotypes observed when expressing myosin with enhanced function ( Cammarato et al . , 2008 ) or tropomyosin that is inefficient at blocking the myosin binding site on actin ( Viswanathan et al . , 2014 ) .",
"This suggests that Drosophila is an excellent analytical system for screening mutations to assess their potential for causing human HCM , which are typically expected to enhance contractility ( Adhikari et al . , 2016; Nag et al . , 2017; Spudich , 2014 ) .",
"Integrating the results from our ATPase , actin motility and muscle mechanical studies reveals that there are alterations to the myosin cross-bridge cycle caused by the R146N mutation .",
"Muscle mechanics measurements showed an increase in apparent rate constant 2πb , which , according to the modeling of Kawai and Brandt ( 1980 ) is influenced by rates associated with myosin attachment to actin , including binding rate , phosphate release and the power stroke ( Figure 5—figure supplement 1 ) .",
"The enhanced basal myosin ATPase rates suggest that phosphate release rate has been increased , since this parameter is rate limiting for basal ATPase rate .",
"This would yield an increased speed of weak to strong actin binding .",
"Our modeling of molecular interactions within the myosin molecule suggests a destabilization of the pre-power stroke state , which could account for the increased affinity of the R146N myosin ADP . Pi state for actin and faster phosphate release rate .",
"Drosophila myosin residue R146 normally has charge-based interactions with E774 and E775 of the myosin lever arm at the pre-power stroke state ( Figure 1C ) .",
"This interaction is abolished by the R146N mutation ( Figure 1D ) , which would reduce the stability of the pre-power stroke molecule .",
"This could drive the cycle forward by enhancing ATP hydrolysis and the production of cross-bridges , possibly contributing to prolonged actin binding .",
"A similar mechanism may occur for human β-cardiac myosin as well , since the analogous K146 residue is also predicted to interact with E775 in the pre-power stroke state ( Figure 1—figure supplement 1A ) and this contact is destroyed by the K146N mutation ( Figure 1—figure supplement 1B ) .",
"Interestingly , analysis of another HCM mutation suggests a similar mode of action as for R146N ( Guhathakurta et al . , 2017 ) .",
"Time-resolved fluorescence resonance energy transfer analysis of myosin containing the E56G mutation in the lever-arm-binding essential light chain showed a reduction in pre-power stroke state molecules and enhanced levels of those in the post-power stroke state in the presence of ATP .",
"As with R146N , this yielded greater actin attachment and an increased duty ratio ( Guhathakurta et al . , 2017 ) .",
"Recent studies of omecamtiv mecarbil , a compound that enhances cardiac myosin power output ( Liu et al . , 2015; Planelles-Herrero et al . , 2017; Shen et al . , 2010 ) , are also relevant to understanding the molecular basis of the R146N mutant phenotypes .",
"As shown in Figure 1—figure supplement 1C , the compound binds in a pocket at the nexus of the N-terminus ( including K146 ) , the relay helix and the converter domain/lever arm ( including residue E774 ) ( Planelles-Herrero et al . , 2017 ) .",
"Omecamtiv mecarbil enhances the actin attachment rate and the duty ratio and slows in vitro motility ( Liu et al . , 2015 ) , similar to the effects that we observed with R146N myosin .",
"In contrast to our findings , ATPase activity in the absence of actin is inhibited by the compound ( Liu et al . , 2015 ) .",
"However , the rate of phosphate release is increased upon actin binding .",
"While the R146N mutation enhances basal activity , which may arise from destabilization of the pre-power stroke state , omecamtiv mecarbil appears to stabilize this state prior to actin interaction , including facilitation of a weak charge-based interaction between K146 and E774 ( Figure 1—figure supplement 1C ) .",
"Further clinical evidence supports the importance of these interactions in relationship to HCM , in that mutation of E774 to V774 also yields HCM ( Moric et al . , 2003 ) .",
"More rapid cross-bridge formation due to the R146N or E774V mutations destabilizing the pre-power stroke state coupled with decreased detachment rates ( see below ) stabilize actin-myosin interaction , leading to enhanced force production and decreased actin filament in vitro motility .",
"A similar outcome could arise from omecamtiv mecarbil increasing the number of molecules in the pre-power stroke state , which would increase the number of attached cross-bridges as the myosin molecules move through the mechanochemical cycle .",
"Although myosin attachment ( Brizendine et al . , 2017 ) and detachment rates ( Harris and Warshaw , 1993 ) both influence the duty ratio ( the fraction of time myosin is strongly bound to actin during a mechanochemical cycle ) , the latter is thought to have the major influence on in vitro motility at high myosin concentrations .",
"Thus , the ~50% decrease in the rate of actin movement in vitro for R146N myosin ( Figure 2F ) is likely facilitated by the decreased actin detachment rate suggested by our fiber mechanics measurements .",
"We observed a decrease in apparent rate constant 2πc ( Table 3 ) , a measure of rates associated with myosin detachment from actin , which includes detachment induced by ADP release and ATP binding ( Kawai and Brandt , 1980 ) ( Figure 5—figure supplement 1 ) .",
"Further , our observed increase in ATP Km in the fiber studies ( Figure 5A ) suggests decreased ATP affinity , which would slow the ATP-induced detachment rate of myosin from actin .",
"This is supported by the trends showing decreases in fmax with increasing phosphate concentration for the mutant ( Figure 5B ) , which does not occur in control myosin .",
"fmax is most likely being slowed by phosphate competing with ATP for the rigor state ( Pate and Cooke , 1989 ) .",
"Normally , increasing phosphate either has no effect ( as seen in the control ) or causes fmax and 2πb to increase ( as seen in many slow vertebrate muscle types [Galler et al . , 2005; Kawai et al . , 1993; Siemankowski et al . , 1985] ) .",
"Overall , the increase in attachment and decrease in detachment rate , in concert with no change in actin-activated ATPase rate arising from the mutation , indicate an increase in duty ratio .",
"In addition to slowed in vitro motility , an increase in duty ratio would cause the increased fiber tension generation and stiffness that we observed from analysis of elastic modulus values ( Figure 5C , D ) .",
"The increased duty ratio is also expected to negatively influence mutant IFM power generation ( Figure 4A ) .",
"The major cause of power loss is likely decreased net work production , given that there was relatively little change in optimal frequency for muscle power generation ( fmax ) .",
"Lack of change in fmax may be due to increased actin attachment and phosphate release being balanced by reduced ATP-induced detachment rate .",
"Work production , however , was decreased proportionally to power for homozygous fibers examined via sinusoidal analysis , and for both homozygous and heterozygous fibers when measured using the work loop technique ( Figure 4B , Table 2 ) .",
"When amplitude and length change conditions were optimized for power generation , the ratio of work absorption to work generation was increased in the mutants compared to control fibers .",
"Tellingly , decreasing optimal muscle length change amplitude allowed the mutant fibers to generate more power ( compare optimal power work loops with work loops performed under the identical length change and frequency conditions ) .",
"This indicates that less muscle stretching was beneficial because it decreased work absorbed , resulting in more net work and hence more power .",
"It appears that increased time of cross-bridge attachment causes cross-bridge elements to act more like brakes or shock absorbers during cyclical power generation .",
"These abnormally high stresses on the myofilaments likely result in the disruption of sarcomere integrity that develops over time in the IFM expressing the mutant myosin ( Figure 3 ) , as well as the reduced flight ability and WBF ( Table 4 ) .",
"Our analysis of the heart tube in R146N/+ heterozygotes produced results that support the thesis that an increased myosin duty ratio yields significant mutant phenotypes .",
"While the hearts in the mutants did not show a change in heart period ( suggesting no overall change in kinetics , in agreement with no change for fmax in IFM and for actin-activated ATPase ) , the heart spent a greater portion of each heart period in systole compared to the control ( Figure 7 ) .",
"This is consistent with prolonged contractions arising from increased myosin attachment kinetics and a decreased detachment rate .",
"An enhanced attachment rate would also increase the rate of force generation , which may partially account for the restricted cardiac phenotype .",
"The reduced fractional shortening observed for the R146N/+ heart may correspond to its optimal pumping mode , mimicking the improved IFM fiber mechanical performance at shorter muscle length changes .",
"At the ultrastructural level , the observed myofibrillar discontinuity in cardiomyocytes ( Figure 6 ) could be caused by the same mechanism as IFM degeneration , that is , excessive tension generation disrupting the integrity of the sarcomeric ultrastructure .",
"We did not detect hypertrophy of the cardiomyocytes in R146N/+ heterozygotes , although it has been shown that hypertrophy can occur in the conical chamber of the Drosophila heart as a result of abnormal receptor tyrosine kinase pathway signaling ( Yu et al . , 2013 ) .",
"It is important to note , however , that there is significant variability in the presence and degree of hypertrophy detected in patients carrying the 146N mutation ( personal communication from Jodie Ingles , Sydney Medical School ) , suggesting that interacting genes or environment may play a role in hypertrophy .",
"Interestingly , our data suggest that cross-bridges in the Drosophila heart bind to thin filaments via Ca2+-dependent and Ca2+-independent mechanisms during diastole .",
"This contributes to impaired relaxation .",
"Our results imply that small amounts of diastolic Ca2+ promote contraction and yield slightly shortened cardiomyocytes at rest in both mutant and control lines .",
"However , based upon the greater diameter increase ( greater tension drop ) in R146N/+ hearts compared to control hearts upon extra- and intracellular Ca2+ chelation with EGTA/EGTA , AM ( Figure 8B ) , disruption of Ca2+-handling in the mutant appears likely , which may promote the restricted cardiac tube phenotype .",
"Therefore , it is possible that perturbation of Ca2+-handling in R146N/+ mutant fly hearts , as is frequently observed in human cardiomyopathies ( Kranias and Bers , 2007 ) , results in excessive diastolic Ca2+ levels , yielding enhancement of cell shortening and elevated basal tone .",
"Additionally , the higher duty ratio of R146N myosin could indirectly enhance the Ca2+ sensitivity of the cross-bridges through thin filament activation .",
"Full thin filament activation requires both Ca2+ binding to troponin and strong binding of cross-bridges .",
"Thus , a higher duty-ratio myosin will have a greater number of myosin molecules strongly bound at a lower Ca2+ concentration than a lower-duty-ratio myosin .",
"We experimentally demonstrated this when we expressed the high-duty-ratio EMB myosin in the jump muscle , which caused enhanced Ca2+ sensitivity for tension development compared to jump muscle myosin ( Eldred et al . , 2010 ) .",
"Thus , the same concentration of Ca2+ in the R146N/+ mutant heart would generate higher force than in the control , yielding decreased cardiac diameters .",
"Our analysis with blebbistatin , which impedes acto-myosin cycling ( Kovács et al . , 2004 ) , indicates that a portion of the cross-bridges responsible for diastolic tone is calcium-independent in both control and R146N/+ hearts .",
"Treatment with the drug ( Figure 8C ) shows that there is a greater diameter increase in R146N/+ hearts than in control hearts , suggesting an enhanced number of Ca2+-independent cross-bridges .",
"This enhancement likely arises from the higher on-rate ( actin affinity ) , which contributes to the restricted phenotype and impaired relaxation observed in R146N/+ mutant hearts .",
"Increased cross-bridge availability could also result from disruption of the super-relaxed state that has been documented in both skeletal and cardiac muscles ( Hooijman et al . , 2011; Stewart et al . , 2010 ) .",
"This state correlates with the presence of the interacting head motif , wherein the dimeric heads of myosin molecules interact to reduce their actin binding and ATPase activity ( Trivedi et al . , 2018; Woodhead et al . , 2005 ) .",
"Disruption of this motif by mutation is linked to myosin activation that correlates with HCM ( Alamo et al . , 2017b; Nag et al . , 2017 ) .",
"Interestingly , myosin is found in its pre-power stroke configuration in the interacting head motif ( Alamo et al . , 2017a; Alamo et al . , 2016 ) and disturbance of this state of filament packing by the R146N mutation could reduce the number of molecules in the super-relaxed state .",
"This would enhance myosin activity within the myofibril , leading to the phenotypes we observed .",
"Drosophila myosins are indeed capable of entering the interacting head motif ( Lee et al . , 2018 ) , but the presence of the super-relaxed state in thick filaments or muscle fibers is yet to be documented .",
"In summary , our integrative analysis of the Drosophila analogue of the K146N human HCM mutation revealed increases in several , but not all , contractile parameters , as posited for a general mechanism of action for HCM mutations in humans ( Spudich , 2014 ) .",
"Notably , enhancements in basal ATPase rate , length of systole and cross-bridge binding are concordant with these expectations .",
"Further , the restricted phenotype and reduced fractional shortening observed in the Drosophila heart mimic the diastolic dysfunction/impaired relaxation and reduced ejection fraction that can accompany HCM ( Davis et al . , 2016; Maron , 2002; Maron and Maron , 2013; Masarone et al . , 2018 ) .",
"The restricted phenotype observed in our model of an HCM mutant , as well as the restricted phenotypes arising from a hyperactive myosin ( Cammarato et al . , 2008 ) or mutant troponin T or actin that enhance myosin binding ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) , suggest that restriction is a common phenotype produced in the Drosophila cardiac tube due to over-active cross-bridges .",
"If all or most HCM mutations have increased force production as their basis , the restricted cardiac tube phenotype in Drosophila could serve as a useful screening tool for determining whether specific human mutations are causative of HCM .",
"The fly might also serve to illuminate conserved signaling pathways that lead from contractile protein mutations to HCM .",
"Further , the amelioration of the mutant phenotype through genetic or drug screens in Drosophila may yield insights into potential treatments for human HCM ."
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[
"Scallop muscle myosin II crystal structures in the pre-power stroke state ( PDB 1QVI ) ( Gourinath et al . , 2003 ) and the actin-detached post-power stroke state ( PDB 1KK8 ) were used as templates to predict Drosophila and human myosin structures ( Himmel et al . , 2002 ) .",
"Myosin S1 amino acid sequences were modeled using the SWISS-MODEL homology modeling server ( http://swissmodel . expasy . org/ ) ( Arnold et al . , 2006 ) .",
"Structures were viewed using PyMOL ( http://www . pymol . org; DeLano Scientific , Palo Alto , CA ) .",
"A P element-containing Mhc transgene with the R146N mutation was constructed using standard cloning techniques along with site directed mutagenesis .",
"The wild-type genomic construct PwMhc2 ( Swank et al . , 2000 ) was digested with Eag I to produced two subclones , PwMhc-5’ and pMhc3’ .",
"The PwMhc-5’ subclone contains an 11 . 3 kb Eag I Mhc fragment cloned into the pCaSpeR vector ( Thummel and Pirrotta , 1992 ) .",
"The PMhc-3’ subclone contains a 12 . 5 kb Eag I Mhc fragment cloned into pBluescriptKS Eag I site ( Stratagene , La Jolla , CA ) .",
"The PwMhc-5’ subclone was further digested with Xho I and Avr II .",
"A 6 . 8 kb Xho I-Avr II digested fragment from PwMhc-5’ was gel isolated and ligated into an Xho I-Avr II site in pLitmus 28I vector ( New England Biolabs , Ipswich , MA ) to produce pMhc-5’-XA .",
"The PwMhc-5’-XA subclone was further digested with Pst I and Avr II .",
"A 4 . 3 kb Pst I-Avr II digested fragment from PwMhc-5’-XA was gel isolated and ligated into an Pst I-Avr II site in pLitmus 28I vector , to produce pMhc-5’-PA .",
"The PwMhc-5’-PA subclone was further digested with Pst I and Age I .",
"A 1 . 6 kb Pst I-Age I digested fragment from PwMhc-5’-PA was gel isolated and ligated into an Pst I-Age I site in pLitmus 28I vector , to produce pMhc-5’-PA1 . 6 .",
"The pMhc-5’-PA1 . 6 subclone was subjected to site-directed mutagenesis using the QuickChange II kit ( Stratagene ) and exon specific-primer 5’-CCGTGGCAAGAACCGTAATGAGG-3’ containing the R146N nucleotide coding change ( bold ) to yield , pR146N-5’PA-1 . 6 .",
"Upon sequence confirmation of the R146N site-directed mutagenesis product , the pR146N-5’-PA-1 . 6 subclone was digested with Pst I and Age I .",
"The 1 . 6 kb R146N fragment from this digest was used to replace the wild-type Pst I-Age I fragment of pMhc-5’PA .",
"The resulting clone was digested with Pst I-Avr II and the Pst I-Avr II fragment was used to replace the wild-type Pst I-Avr II fragment of pMhc-5’-XA .",
"The resulting clone was digested with Xho I-Avr II and the isolated Xho I-Avr II fragment was used to replace the wild-type Xho I-Avr II fragment of PwMhc-5’ .",
"The resulting clone , PwMhcR146N-5’ , was digested with Eag I .",
"The wild-type 3’ end subclone pMhc-3’ also was digested with Eag I .",
"The 12 . 5 kb Eag I fragment was gel isolated and ligated into the Eag I of PwMhcR146N-5’ , to yield PwMhcR146N .",
"The entire coding region and all ligation sites of the final PwMhcR146N plasmid were confirmed by DNA sequencing ( Eton Bioscience , San Diego , CA ) prior to P element transformation .",
"Transgenic lines for PwMhcR146N were generated by P element-mediated germline transformation ( Rubin and Spradling , 1982 ) by BestGene , Inc . ( Chino Hills , CA ) .",
"BestGene injected 1200 embryos with the PwMhcR146N transgene , and 29 transgenic lines were obtained .",
"Each insert was mapped to its chromosomal location using balancer chromosomes and standard genetic crosses .",
"Ten inserts mapped to the second chromosome , 3 to the X chromosome and 16 to the third chromosome .",
"Three independent lines that mapped to the third chromosome were crossed into Mhc10 background , which is null for myosin heavy chain in IFM and TDT muscle due to mutation of the endogenous Mhc gene , which is located on the second chromosome ( Collier et al . , 1990 ) .",
"RT-PCR was employed to verify that Mhc transcripts from the PwMhcR146N transgenic lines contained the appropriate site-directed nucleotide changes in exon four and that flanking alternative exons 3 and 7 were spliced correctly .",
"RNA was prepared by LiCl2 extraction ( Becker et al . , 1992 ) from 2-day-old adult female transgenic flies .",
"cDNAs were generated for each line using the Protoscript cDNA synthesis kit ( New England Biolabs ) .",
"A reverse primer specific to exon 8 ( 5’-GTTCGTCACCCAGGGCCGTA-3’ ) and a forward primer specific to exon 2 ( 5’-TGGATCCCCGACGAGAAGGA-3’ ) were used to generate cDNA .",
"Briefly , 3 μmol of reverse specific primer were mixed with 0 . 5 μg of total RNA from each transgenic line .",
"PCR was performed using 1 μl of cDNA and 3 μmol of forward and reverse primers under the following conditions: 60 s at 94° C then 30 cycles of: 30 s at 94° C , 30 s at 55° C and 2 min at 68° C . RT-PCR products were sequenced by Eton Bioscience .",
"To determine myosin protein expression levels for each transgene in an Mhc10 background , SDS polyacrylamide gel electrophoresis was utilized as previously described ( O'Donnell et al . , 1989 ) .",
"Upper thoraces from six 2-day-old homozygous female flies were homogenized in 60 µl SDS gel buffer .",
"Six µl of sample were loaded on a 9% polyacrylamide gel .",
"This was repeated five different times each time using a freshly prepared sample .",
"Protein accumulation was determined using Coomassie blue stained gels that were digitally scanned and analyzed on NIH Image J software .",
"Values are expressed relative to the control transgene ±S . E . M . As previously reported , actin was prepared from chicken skeletal muscle after multiple steps of polymerization–depolymerization ( Kronert et al . , 2014; Kronert et al . , 2015 ) .",
"Myosin was isolated from dissected dorsolongitudinal IFMs of ~250 transgenic flies and its concentration was determined by spectrophotometry ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .",
"A final concentration of 2 . 0 µg/µl myosin was used for ATPase assays and 0 . 5 µg/µl was used for in vitro motility assays .",
"Steady-state calcium , magnesium and actin-stimulated magnesium ATPase activities of myosin were obtained using [γ-32P]-ATP as previously described ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .",
"Basal Mg-ATPase activities obtained in the absence of actin and actin-stimulated Mg-ATPase was determined using increasing concentrations of filamentous actin .",
"Basal Mg-ATPase values were subtracted from all data points , which were then fit with the Michaelis–Menten equation to determine actin-stimulated ATPase ( Vmax ) and actin affinity relative to ATPase ( Km ) .",
"Catalytic efficiency ( defined as Vmax/Km ) was determined as previously reported ( Kronert et al . , 2014; Kronert et al . , 2015 ) .",
"In vitro motility assays were carried out by adding ATP and filamentous actin labeled with fluorescent phalloidin to nitrocellulose treated cover slips coated with wild-type or mutant myosin ( Kronert et al . , 2014; Kronert et al . , 2015; Swank et al . , 2001 ) .",
"Video sequences captured under fluorescence optics were analyzed computationally to determine actin-sliding velocity .",
"Mean values from five independent experiments ( two assays for each ) for the mutant and wild-type ATPase or from three independent motility assays ( at least 30 filaments/assay ) were compared for statistically significant differences ( p<0 . 05 ) by Student’s t-tests .",
"To determine the effects of transgene expression on IFM structure in an Mhc10 null background , transmission electron microscopy was performed as previously described ( O'Donnell and Bernstein , 1988 ) .",
"We examined four different stages of development; late stage pupae , 2-hr-old adults , 2-day-old adults and 1-week-old adults .",
"Cross-sections and longitudinal sections were obtained from females for each homozygous transgenic line , with at least three different samples examined for each of the three transgenic lines .",
"One myofibril is shown in each panel and is representative of the entire population at the given stage of development .",
"Images were obtained using a FEI Tecnai 12 transmission electron microscope with a TVIPS 214 high-resolution camera .",
"For cardiac electron microscopy , heart samples at 1 or 3 weeks of age ( three per age per genotype ) were surgically exposed in an oxygenated artificial hemolymph solution and fixed using a modified procedure as described previously ( Achal et al . , 2016 ) .",
"Rhythmic beating was observed to confirm structural integrity of samples .",
"Hearts were relaxed in 10 mM EGTA , fixed in primary fixative ( 3% formaldehyde , 3% glutaraldehyde , 0 . 1 M cacodylate buffer pH 7 . 4 ) , washed 6x in 0 . 1 M cacodylate buffer pH 7 . 4 , then bathed in secondary fixative ( 1% OsO4 , 10 mM MgCl2 , 100 mM phosphate buffer pH 7 . 4 ) and washed 3x in 0 . 1 M cacodylate buffer pH 7 . 4 .",
"The samples were dehydrated in an acetone series , oriented and embedded in Epon-filled BEEM capsules , and polymerized at 60°C under vacuum .",
"Thin sections ( ≤50 nm ) were obtained using a Leica ultramicrotome , picked up on Formvar-coated grids , and stained with 4% uranyl acetate for 10 min .",
"Images were obtained at 120 kV on a FEI Tecnai 12 transmission electron microscope .",
"Transverse sections of the heart were utilized to determine average cardiac thickness between the second and third abdominal segments .",
"For each measurement , a roughly rectangular area of cardiomyocyte tissue ( defined as myofibrillar and mitochondrial material ) of 10 µm in length within the inner and outer edges of the cardiac tube was highlighted using Adobe Photoshop .",
"The image was imported into ImageJ to calculate the area of the highlighted region .",
"Dividing the resulting cardiomyocyte area by 10 µm yielded the average cardiac thickness for that highlighted section .",
"At least three images each for dorsal areas and for ventral areas per heart section were analyzed .",
"Three or more transverse sections along the anterior-poster axis at least 10 µm apart were assessed in the same way .",
"Cardiac thickness measurements from dorsal-side or ventral-side images were averaged per section and the data were then averaged for each biological replicate .",
"Data from at least three hearts were averaged per line to yield the final values , which are presented as mean ± SEM .",
"A one-way ANOVA with the Bonferroni correction determined statistical significance ( p<0 . 05 ) .",
"Newly eclosed female PwMhcR146N-15 homozygous or heterozygous flies were collected every half hour and allowed to mature for 2 hr .",
"Control flies were taken from the PwMhc2 stock .",
"Individual IFM fibers were isolated by dissection as previously described by Swank ( Swank , 2012 ) .",
"Briefly , the head , abdomen and wings were removed and the thorax was placed into dissection solution ( Swank , 2012 ) .",
"The thorax was split in half , and the IFM fiber bundle was removed .",
"The fibers were skinned in the dissection solution for 1 hr at 6°C before being transferred to storage solution ( same as the dissection solution but without Triton X-100 ) .",
"Individual fibers were then separated from the bundle and split in half with a long , thinly etched tungsten probe .",
"The split fiber was then fitted with a pair of aluminum foil t-clips .",
"The clipped fiber was mounted in relaxing solution ( pCa 8 . 0 , 12 mM MgATP , 30 mM creatine phosphate , 600 U/ml creatine phosphokinase , 1 mM free Mg2+ , 5 mM EGTA , 20 mM pH 7 . 0 BES , 200 mM ionic strength , adjusted with Na methane sulfonate , 1 mM DTT ) onto a mechanics apparatus by settling it onto two hooks attached to a motor arm and a force transducer .",
"The muscle was lengthened to be just taught , then stretched 5% beyond this length .",
"The resulting length , width and height were measured to obtain cross-sectional area .",
"Passive tension ( Po ) was recorded before adding activating solution ( the same as relaxing solution except that pCa was adjusted to 5 . 0 ) .",
"The maximum power of the muscle was determined by performing sinusoidal analysis ( see below ) , under low amplitude conditions ( 0 . 125% muscle length ) .",
"After every run , the muscle was stretched by 2% muscle length ( ML ) until the power did not increase by more than 3% .",
"The active tension ( Ao ) at the optimal muscle length was measured and the net tension ( Fo ) was calculated by Ao-Po .",
"Two- and 7-day-old homozygotes for each of the three transgenic lines and controls were tested for flight ability in an Mhc10 background at 22°C .",
"Flight was determined by the ability to fly up ( U ) , horizontal ( H ) , down ( D ) or not at all ( N ) ( Drummond et al . , 1991 ) .",
"As previously described ( Tohtong et al . , 1995 ) flight index is defined as: 6 U/T+ 4 H/T+ 2 D/T+ 0 N/T where T is the total number of flies tested .",
"Flight abilities and wing beat frequencies of two-day-old PwMhcR146N-15 homozygotes and heterozygotes were examined at 15°C and 22°C .",
"An optical tachometer was used to measure the wing beat frequency of tethered flies ( Tohtong et al . , 1995 ) .",
"All values represent mean ± SEM .",
"Significance was assessed by a Student’s t-test at p<0 . 05 .",
"Cardiac tubes of 1- and 3-week-old female adult flies were surgically exposed under oxygenated artificial hemolymph as described by Vogler and Ocorr ( 2009 ) .",
"High-speed videos of contracting hearts were taken at ~120 frames per second using a Hamamatsu Orca Flash 2 . 8 CMOS camera on a Leica DM5000B TL microscope with a 10x ( 0 . 30 NA ) immersion lens .",
"Physiological parameters were assessed from individual videos using the semiautomatic optical heartbeat analysis ( SOHA v . 3 ) program as previously outlined ( Cammarato et al . , 2015; Fink et al . , 2009; Viswanathan et al . , 2017 ) .",
"M-mode kymograms were generated to provide an edge trace documenting the movement of heart wall in the y-axis over time in the x-axis .",
"Values represent mean ± S . E . M . Two-way ANOVAs were employed to test if the effects of genotype and age ( or if an interaction exists ) were significant for each cardiac parameter investigated .",
"Significance was assessed at p<0 . 05 .",
"Beating hearts from 3-week-old heterozygous female PwMhc2/+ and PwMhcR146N-15/+ flies were imaged as described above using a 20x ( 0 . 50 NA ) immersion objective lens and analyzed as previously detailed ( Viswanathan et al . , 2014; Viswanathan et al . , 2017 ) .",
"The effects of EGTA/EGTA-AM and blebbistatin treatment on cardiac diameters were evaluated using paired t-tests of the matched groups .",
"Unpaired t-tests were used to distinguish significant differences in the cardiac responses to EGTA , EGTA . AM and blebbistatin between PwMhc2 and mutant MHC expressing hearts .",
"Significance was assessed at p<0 . 05 ."
]
] | [
"K146N is a dominant mutation in human β-cardiac myosin heavy chain , which causes hypertrophic cardiomyopathy .",
"We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical , physiological and mechanical foundations of the disease .",
"ATPase assays , actin motility , and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment , yielding prolonged binding .",
"This increases isometric force generation , but also resistive force and work absorption during cyclical contractions , resulting in decreased work , power output , flight ability and degeneration of flight muscle sarcomere morphology .",
"Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes , 146N/+ hearts are hypercontractile with increased tension generation periods , decreased diastolic/systolic diameters and myofibrillar disarray .",
"This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments ."
] | [
"Myosin is a motor protein that drives the contraction of muscles .",
"Filaments made from myosin molecules slide between filaments of another protein called actin , tugging the edges of the muscle cell inwards .",
"To achieve this , part of each motor protein – called the 'head' – grabs hold of actin and uses energy to pull on the filaments .",
"Small genetic mutations in the gene for myosin can change the shape of the protein .",
"This can change the way that it interacts with actin , altering the molecular machinery that makes muscles contract .",
"In some cases , gene errors can cause the heart muscle wall to thicken , a condition called hypertrophic cardiomyopathy .",
"Mapping the locations of known mutations revealed 'hot spots' on the myosin protein where these errors are likely to cause disease .",
"These include the part of the molecule that swings the myosin heads , and the heads themselves .",
"It only takes a change to a single letter in the DNA code to thicken the heart wall , but the impact of each possible change is not yet known .",
"Kronert et al . have now genetically modified fruit flies to give them one of the mutations that causes thickening of the heart wall in humans .",
"The mutation , known as K146N , does not appear in one of the well-known 'hot spots' .",
"The experiments revealed that the mutation causes myosin to remain attached to actin for longer than normal .",
"This increased the amount of force the myosin generated , but slowed down actin movement , causing muscle stiffness .",
"This resulted in less power for every cycle of muscle movement , and caused the muscles to degenerate over time .",
"As a result , the flies were less able to use their wings , and their hearts pumped less well .",
"Hypertrophic cardiomyopathy can cause death in young adults , particularly competitive athletes .",
"Yet studying the disease in humans is challenging .",
"Recreating myosin mutations in fruit flies provides a way to study hypertrophic cardiomyopathy in the laboratory .",
"In the future , extensions to this technique could allow researchers to examine the impact of other mutations .",
"Models like this one could also allow early testing of new drugs or genetic treatments to repair faulty myosin molecules ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"medicine",
"microbiology and infectious disease"
] | Mucosal effects of tenofovir 1% gel | elife-04525-v2 | [
[
"The HIV prevention field has invested considerable resources in testing the phosphonated nucleoside reverse transcriptase inhibitor ( NRTI ) tenofovir as a mucosally applied topical microbicide to prevent sexual HIV transmission .",
"In a phase 2B trial , CAPRISA 004 , pericoital tenofovir 1% gel was 39% efficacious in preventing vaginal HIV acquisition ( Abdool Karim et al . , 2010 ) .",
"However , in another phase 2B trial , the VOICE study ( MTN-003 ) , the daily vaginal tenofovir 1% gel arm was discontinued for futility ( Marrazzo et al . , 2015 ) .",
"Adherence to product use was low in VOICE , likely explaining the differences in findings between the two studies .",
"Currently , the CAPRISA 004 study is being repeated in a phase 3 trial ( FACTS 001 study ) .",
"A reduced glycerin formulation of the vaginal tenofovir 1% gel for use as a rectal microbicide appears safe when evaluated by epithelial sloughing , fecal calprotectin , inflammatory cytokine mRNA/protein levels , and cellular immune activation markers ( McGowan et al . , 2013 ) .",
"However , because topical application of an antiretroviral drug to the mucosa is a novel prevention strategy without clinical precedent , we conducted a comprehensive systems biology assessment of tenofovir gel's effects on the mucosa ."
],
[
"We measured mRNA expression changes across the complete human transcriptome by microarrays in rectal biopsies taken at 9 and 15 cm proximal to the anal margin .",
"Biopsies were obtained before treatment , after a single and after seven consecutive once-daily applications of reduced glycerin tenofovir 1% gel , nonoxynol-9 ( N-9 ) 2% gel , hydroxyethyl cellulose ( HEC ) placebo gel , or no treatment ( eight participants per arm were tested by microarrays ) .",
"The primary results of the clinical study , MTN-007 , a phase 1 , randomized , double-blind , placebo-controlled trial at three US sites were reported elsewhere ( McGowan et al . , 2013 ) .",
"Relative to enrollment biopsies , after 7 days of treatment , tenofovir 1% gel suppressed 505 genes and induced 137 genes in the 9 cm biopsies , whereas the detergent N-9 , a transient mucosal toxin , suppressed 56 genes and induced 60 genes ( log2 fold expression change ≥ 0 . 5 for induction or ≤ −0 . 5 for suppression , FDR ≤ 0 . 05 ) ( Figure 1A , B and Supplementary file 2 ) .",
"15 suppressed and 4 induced genes were common to tenofovir and N-9 ( Figure 1B ) .",
"In the HEC gel and no treatment arms , 16 and 23 genes changed ( Figure 1B ) .",
"Tenofovir 1% gel affected more genes after 7 days of treatment than after a single application and more genes in 9 cm than in 15 cm biopsies ( Figure 1B ) , with significant correlations between expression changes at 9 cm and 15 cm ( suppression: Spearman rho = 0 . 4775 [95% CI: 0 . 4051–0 . 5440]; p < 0 . 001 , Figure 1C; induction: Spearman rho = 0 . 427 [95% CI: 0 . 2840–0 . 5514]; p < 0 . 001 , Figure 1—figure supplement 1 ) .",
"By fold change of the individual genes , tenofovir suppressed genes more strongly than N-9 ( median 0 . 505 vs 0 . 627-fold; p < 0 . 001 ) , but induced genes less strongly ( 1 . 58 vs 1 . 69-fold; p < 0 . 001 ) ( Figure 1—figure supplement 2 ) . 10 . 7554/eLife . 04525 . 003Figure 1 . Tenofovir-induced gene expression changes in the human rectum .",
"( A ) Heat map of differentially expressed genes in eight participants after a single ( I ) and after seven consecutive once-daily ( VII ) rectal applications of tenofovir 1% gel compared to baseline in biopsies taken at 9 cm and 15 cm proximal to the anal margin .",
"Red and green bars signify strength of gene induction and suppression , respectively .",
"642 genes are shown , all of which exhibited an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≥ 0 . 5 ( induction ) or ≤ −0 . 5 ( suppression ) when evaluated jointly for all eight participants at time point VII in the 9 cm biopsies .",
"( B ) Numbers of significantly suppressed ( green borders and numbers ) and induced ( red borders and numbers ) genes after reduced glycerin tenofovir 1% gel ( TFV ) treatment and their overlap with nonoxynol-9 ( N-9 ) , hydroxyethyl cellulose ( HEC ) and no treatment ( No tx ) .",
"Circle area symbolizes the number of affected genes , overlap the number of genes independently affected by two or three conditions .",
"( C ) Correlation of log2 fold gene suppression from baseline to Day VII between 9 and 15 cm biopsies .",
"All 505 genes significantly suppressed at 9 cm are included .",
"Genes depicted as blue dots were significantly suppressed in both 9 and 15 cm biopsies ( 15 cm FDR < 0 . 05 ) , genes depicted as black dots were only significant in 9 cm biopsies ( 15 cm FDR ≥ 0 . 05 ) .",
"Spearman rho correlation between the 9 and 15 cm biopsies expression and the corresponding p-value of a Spearman rank correlation test are indicated on the plot .",
"Genes tested in Figure 2B by RT-ddPCR are specifically indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00310 . 7554/eLife . 04525 . 004Figure 1—figure supplement 1 . Correlation of log2 fold gene induction by tenofovir 1% gel from baseline to Day VII between 9 and 15 cm biopsies . All 137 genes at 9 cm significantly induced ( FDR < 0 . 05 ) and with log2 fold change > 0 . 5 are included .",
"Genes depicted as blue dots were significantly induced in both 9 and 15 cm biopsies ( 15 cm FDR < 0 . 05 ) , genes depicted as black dots were only significant in 9 cm biopsies ( 15 cm FDR ≥ 0 . 05 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00410 . 7554/eLife . 04525 . 005Figure 1—figure supplement 2 . Log2 fold gene suppression ( A ) and induction ( B ) from baseline to Day VII caused by N-9 and tenofovir in 9 cm biopsies .",
"( A ) All genes with an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≤ 0 . 5 are included ( N-9: 56 genes; tenofovir: 505 genes ) .",
"( B ) All genes with an estimated FDR ≤ 0 . 05 and a log2 fold expression change of ≥ 0 . 5 are included ( N-9: 60 genes; tenofovir: 137 genes ) .",
"Differences in magnitude of gene expression changes were statistically compared between N-9 and tenofovir by unpaired Mann–Whitney tests .",
"The box plots indicate medians and interquartile ranges and the whiskers indicate 10th–90th percentiles . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 005 To independently confirm the microarray results , we selected nine induced and six suppressed genes and performed reverse transcription digital droplet PCR ( RT-ddPCR ) assays with RNA from the 9 cm biopsies in the remaining seven individuals enrolled in the tenofovir 1% gel arm ( two men and five women ) , whose biopsies had not been analyzed by microarray ( Figure 2 ) .",
"The mRNA copy numbers of all 15 genes increased or decreased as predicted from the microarray data between baseline and time point VII ( Figure 2A , B ) .",
"Next , we combined the microarray and RT-ddPCR expression data , normalized fluorescence and copy number values over their respective baselines , and compared the fold change after 7 days of treatment with baseline ( Figure 2C ) .",
"Expression changes for all 15 genes assessed by microarray and RT-ddPCR were statistically significant ( p < 0 . 01 for all genes except TRIM5 [p = 0 . 02] ) . 10 . 7554/eLife . 04525 . 006Figure 2 . Confirmation of microarray data .",
"( A and B )",
"Quantification of mRNA copy numbers measured in 9 cm biopsies by reverse transcription ( RT ) droplet digital PCR ( ddPCR ) relative to the housekeeping gene hemoglobin beta ( HBB ) copy numbers in seven additional study participants .",
"( A ) Nine selected genes induced in the microarrays: CCL19 , CCL21 , CCL23 , CXCL9 , CCR7 , CD7 , CD19 , matrix metallopeptidase 12 ( MMP12 ) , and serine peptidase inhibitor of the Kazal type 4 ( SPINK4 ) .",
"Copy numbers at baseline ( 0 ) , after a single tenofovir gel application ( I ) and after seven consecutive once-daily applications ( VII ) are shown .",
"Line colors signify each of the seven study participants .",
"( B ) Six selected suppressed genes: p21-activated kinase ( PAK2 ) , nuclear factor of activated T cells 5 ( NFAT5 ) , desmoplakin ( DSP ) , TGF-β receptor associated protein ( TGFBRAP ) , interleukin 10 ( IL-10 ) , and tripartite motif-containing protein 5 ( TRIM5 ) .",
"( C ) Normalized fold changes of gene expression at Day VII over baseline in all 15 individuals treated with tenofovir 1% gel .",
"Red dots depict fold changes measured by microarray , blue dots depict fold changes measured by RT-ddPCR .",
"The boxes indicate median and 25th–75th percentiles and the whiskers indicate 10th–90th percentile .",
"Asterisks indicate statistical significance level relative to baseline ( one asterisk p < 0 . 05; two asterisks p < 0 . 01 , by one-sided Wilcoxon tests , adjusted for multiple testing ) .",
"( D ) Immunostaining of formalin-fixed 9 cm rectal biopsies from 10 participants for the proteins CD7 ( immunohistochemistry [IHC] ) , CD3 ( immunofluorescence ) and ubiquitin D ( UBD; IHC ) , predicted to be induced by the microarrays , and for IL-10 ( IHC ) , predicted to be suppressed .",
"For CD7 and CD3 , tissue sections were evaluated in their entirety and positive cells per mm2 are shown at baseline ( 0 ) and after seven consecutive once-daily applications ( VII ) .",
"Representative images are shown in Figure 2—figure supplement",
"1 . For UBD and IL-10 , only columnar epithelial cells were evaluated .",
"For UBD , the average mean staining intensities ( MSI ) per cell are shown .",
"Representative images are shown in Figure 2—figure supplement",
"2 . Colors signify each of the 10 study participants .",
"The boxes indicate median and 25th–75th percentiles and the whiskers indicate the range .",
"Paired Wilcoxon signed-rank test p values for differences between 0 and VII are listed . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00610 . 7554/eLife . 04525 . 007Figure 2—figure supplement 1 . Immunohistochemistry for CD7 , and immunofluorescence for CD3 , in rectal biopsies before ( 0 ) and after 7 days ( VII ) of daily tenofovir 1% gel use . Individual study participants are designated by letters .",
"Blue indicates nuclei , red indicates CD3 or CD7 . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00710 . 7554/eLife . 04525 . 008Figure 2—figure supplement 2 . Immunohistochemistry for IL-10 and ubiquitin D ( UBD ) in rectal biopsies before ( 0 ) and after 7 days ( VII ) of daily tenofovir 1% gel use . Individual study participants are designated by letters .",
"Blue indicates nuclei , brown indicates IL-10 or UBD . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 008 For additional confirmation , we selected three induced ( CD7 , CD3 and ubiquitin D ) and one suppressed gene ( IL-10 ) for immunohistochemical staining of the respective proteins in 9 cm rectal biopsies from 10 subjects ( Figure 2D and Figure 2—figure supplements 1 , 2 ) .",
"Consistent with the gene expression studies , infiltrating T lymphocytes increased twofold to fivefold in the mucosa ( mean fold change 5 . 0 , 95% CI 2 . 46–10 . 1 , p < 0 . 001 for CD7+; 2 . 44 , 1 . 17–5 . 11 , p = 0 . 023 for CD3+ ) , whereas IL-10+ columnar epithelial cells decreased by more than half ( 0 . 36 , 0 . 18–0 . 79 , p = 0 . 017 ) , between baseline and following 7 days of tenofovir 1% gel use .",
"Ubiquitin D was widely expressed in all biopsies , but tenofovir treatment increased the intensity of its expression ( p = 0 . 007 ) , as predicted by the microarrays .",
"Tenofovir 1% gel was more suppressive than stimulatory , with a ratio of induced to suppressed genes in the 9-cm rectal biopsies of 0 . 116 ( 17 genes up-regulated to 146 down ) for nuclear products .",
"However , genes encoding secreted proteins were more often induced than suppressed ( Figure 3A ) , with a ratio of 2 . 33 ( 35 genes up-regulated to 15 down; χ2 p < 0 . 001 ) .",
"Noteworthy among induced genes for secreted products were the chemokines CCL2 , CCL19 , CCL21 , CCL23 , CXCL9 , and CXCL13 ( Figure 3A ) .",
"Correspondingly , transcripts of a number of leukocyte-specific cell surface markers increased , specifically CD2 , CD3D , CD7 , CD8A , CD19 , CD52 , CD53 , CCR6 , and CCR7 .",
"The kinetics of gene induction is depicted in Figure 3—figure supplement 1 . 10 . 7554/eLife . 04525 . 009Figure 3 . Expression pattern and functional pathway analysis .",
"( A ) Ingenuity pathways analysis of tenofovir-induced effects in rectal biopsies , showing cellular localizations of and relationships between individual gene products .",
"Red symbols indicate induction and green symbols suppression at Day VII relative to baseline in 9 cm biopsies .",
"The diagram includes all significant genes identified as primarily located in the extracellular space and the cell nucleus .",
"A few selected significant genes with products localizing to the plasma membrane ( PM ) or cytoplasm ( CP ) are also shown based on their putative functional roles .",
"Direct ( solid lines ) and indirect ( dashed ) interactions between gene products are indicated .",
"Line color is arbitrary and meant to indicate relationships between groups of genes .",
"Yellow-shaded areas indicate zinc finger transcription factors .",
"( B ) Pathways analysis of tenofovir-induced effects in primary vaginal epithelial cells .",
"Only genes that were suppressed or induced by tenofovir both in 9 cm rectum in vivo and in vaginal epithelial cells in vitro are shown .",
"Primary vaginal epithelial cells derived from three healthy women were cultured with 50 or 500 μM tenofovir for 14 days .",
"Global gene expression microarrays at 4 , 7 , and 14 days of culture were evaluated in comparison to untreated epithelial cells .",
"Pre-processed microarray expression data were extremely consistent between the three vaginal cell cultures ( mean Pearson correlation coefficient 0 . 9912; Figure 3—source data 1 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 00910 . 7554/eLife . 04525 . 010Figure 3—source data 1 . Pearson correlation coefficients of pre-processed microarray probe expression values between the three primary vaginal cell cultures . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 01010 . 7554/eLife . 04525 . 011Figure 3—figure supplement 1 . Average strength of gene induction by tenofovir 1% gel across all 8 microarray study participants . Heat map colors depict fold-change at Day I and Day VII over baseline .",
"Baseline is depicted as the vertical bar labeled ‘0’ filled with the shade of blue corresponding to a fold-change of",
"1 . Genes included in the list exhibited ≥1 . 6-fold average induction on Day VII or were ≥1 . 1-fold induced in at least six of eight study participants , and some knowledge about the gene products exists in the literature .",
"The heat map divisions signify three different kinetic patterns of gene induction: flat at day I and then induced; induced at day I and then flat; or induced at day I and then more strongly induced at day VII . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 01110 . 7554/eLife . 04525 . 012Figure 3—figure supplement 2 . Selected biological processes defined in the InnateDB database with significant enrichment of genes suppressed or induced by tenofovir 1% gel at Day VII in 9 cm biopsies . Green bars depict the percentage of genes identified as suppressed in a particular process out of the total number of genes included by InnateDB in that process .",
"Red bars depict corresponding percentages for gene induction .",
"Numbers of suppressed and induced genes are indicated above the bars .",
"Gene enrichment in each biological process was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .",
"Not all processes with significant gene enrichment are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 012 Among suppressed genes with products localizing to the cell nucleus , we identified a large number of known or putative transcription factors and their co-factors , including CREB1 ( CAMP responsive element-binding protein 1 ) and CREBBP ( CREB-binding protein ) , both activators of IL-10 transcription ( Woodgett and Ohashi , 2005; Alvarez et al . , 2009 ) , NFAT5 ( nuclear factor of activated T cells 5 ) ( Neuhofer , 2010 ) , and many zinc finger proteins ( Figure 3A ) .",
"Tenofovir 1% gel also suppressed genes important for regulation of transcription and translation , as well as biological processes involving transforming growth factor beta ( TGF-β ) , epithelial structure organization , regulation of cell proliferation , and apoptosis ( Figure 3—figure supplement 2 ) .",
"Lastly , tenofovir 1% gel suppressed genes important for mitochondrial function , including PNPT1 ( polyribonucleotide nucleotidyltransferase 1 ) ( Wang et al . , 2010 ) and OPA3 ( optic atrophy 3 ) ( Misaka et al . , 2002 ) .",
"Among the few genes with nuclear products that were strongly induced , KIAA0101 and ubiquitin D were notable ( Yu et al . , 2001; Lee et al . , 2003; Hosokawa et al . , 2007 ) .",
"Tenofovir 1% gel is most advanced as a candidate product for vaginal HIV prevention .",
"We were therefore interested to extend our findings from the rectum to the vaginal mucosa .",
"We isolated epithelial cells from vaginal tissues donated by three healthy women and tested their response to 50 and 500 μM tenofovir in vitro for up to 14 days of culture using RNA expression analysis .",
"Preliminary tests showed that these dosages give roughly similar intracellular concentrations of the active drug ( tenofovir diphosphate ) as measured in the vagina after tenofovir 1% gel use ( not shown ) ( Hendrix et al . , 2013 ) .",
"Pre-processed microarray expression data were extremely consistent between the three vaginal cell cultures ( mean Pearson correlation coefficient 0 . 9912; Figure 3—source data 1 ) .",
"Tenofovir's effects on the purified vaginal epithelial cells ( Figure 3B ) were similar to its effects on the rectal mucosa ( Figure 3A ) , but , as expected , changes likely driven by leukocytes in the biopsies were not seen in the purified epithelial cells , or were differently regulated .",
"In the epithelial cells , tenofovir did not significantly change chemokine , chemokine receptor , and cluster of differentiation ( CD ) genes , and it suppressed ISG 15 ( interferon-stimulated gene 15 ) and MX1 ( myxovirus resistance 1 ) , which were induced in the biopsies .",
"The number of genes affected was initially higher with 500 μM than with 50 μM tenofovir , but equalized after 14 days of culture ( Figure 4A ) .",
"Next , we confirmed the expression changes of select genes by ddPCR with vaginal epithelial cells from four healthy women: mRNA copies of DSP ( desmoplakin ) and IL-10 significantly decreased , and KIAA0101 significantly increased during 7 days of tenofovir treatment , as seen in the microarray data ( Figure 4B ) .",
"In fact , IL-10 transcripts were virtually eliminated at 7 days ( p = 0 . 002 and p = 0 . 003 for 50 and 500 μM , respectively ) , and KIAA0101 increased more than 10-fold at 500 μM tenofovir ( p = 0 . 005 ) .",
"By ELISA of cell lysates , IL-10 protein also decreased significantly ( n = 4 cell lines; p = 0 . 007 and p < 0 . 001 for 50 and 500 μM , respectively ) ( Figure 4C ) . 10 . 7554/eLife . 04525 . 013Figure 4 . Effects of tenofovir on primary vaginal epithelial cells .",
"( A ) Number of suppressed ( green ) and induced ( red ) genes in response to treatment with 50 μM ( open circles ) or 500 μM ( filled circles ) tenofovir for 1 , 4 , 7 , or 14 days ( n = 3 cell lines from different women ) .",
"( B ) Quantification of mRNA copy numbers at days 1 and 7 of culture by RT-ddPCR assays for two selected genes identified as suppressed ( DSP and IL-10 ) and one induced ( KIAA0101 ) in the microarray data set ( n = 4 cell lines ) .",
"( C ) Quantification of IL-10 protein concentrations in vaginal epithelial cells at days 1 and 7 of culture by ELISA .",
"Mean ( ±standard deviation ) IL-10 concentrations in the untreated cultures were 5 . 65 pg/ml ( ±0 . 25 ) at day 1 and 5 . 86 pg/ml ( ±0 . 32 ) at day 7 of culture .",
"Boxes and error bars in ( B ) and ( C ) signify means and standard deviations with vaginal epithelial cell cultures derived from four healthy women .",
"Asterisks indicate statistical significance level relative to untreated ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001 ) .",
"( D ) Selected biological processes defined in the InnateDB and DAVID databases with significant enrichment of genes suppressed or induced by 50 μM tenofovir in vaginal epithelial cells after 7 days of culture .",
"Green bars depict the percentage of genes identified as suppressed in a particular process out of the total number of genes included in that process .",
"Red bars depict gene induction .",
"Numbers of suppressed and induced genes are indicated above the bars .",
"Gene enrichment in each biological process was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .",
"Not all processes with significant gene enrichment are shown .",
"( E ) Proliferation of vaginal epithelial cells without or with various concentrations of tenofovir ( n = 3 cell lines ) .",
"Boxes depict mean percent reduction of the alamarBlue reagent in comparison to the maximum reduction .",
"Error bars signify standard deviations . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 013 Tenofovir treatment of primary vaginal epithelial cells in vitro mostly impacted the same biological processes as it did in the rectum in vivo ( Figure 4D and Figure 3—figure supplement 2 ) .",
"Additionally , it suppressed genes important for keratinocyte differentiation and cellular innate immunity , and induced genes involved in DNA damage repair .",
"Furthermore , tenofovir enhanced vaginal epithelial cell proliferation and/or cell survival in vitro ( p = 0 . 02 ) ( Figure 4E ) .",
"Our microarray results indicated that tenofovir suppresses PNPT1 ( Figure 1C ) , which has been characterized as a master regulator of RNA import into mitochondria and whose deletion impairs mitochondrial function ( Wang et al . , 2010 ) .",
"To explore tenofovir's effects on mitochondria , we first confirmed its inhibition of PNPT1 in the 9 cm and 15 cm rectal biopsies of all 15 study participants by RT-ddPCR .",
"PNPT1 copy numbers decreased more than 10-fold at 9 cm ( mean fold change 0 . 09 , 95% CI 0 . 009–0 . 172 , p < 0 . 001 ) and by half at 15 cm ( 0 . 53 , 0 . 34–0 . 73 , p < 0 . 001 ) after 7 days of treatment ( Figure 5A ) .",
"To directly assess mitochondrial function , we picked one of the 13 genes encoded by mitochondrial DNA , ATP synthase F0 subunit 6 ( ATP6 ) , a key component of the proton channel ( Houstek et al . , 2006 ) , and measured its transcription by RT-ddPCR in the 9 cm biopsies of all 15 study participants in the tenofovir arm ( Figure 5B ) .",
"Because mitochondrial genes are not included on the microarray chips , we had no preexisting information on its expression .",
"ATP6 mRNA copy numbers decreased on average threefold ( p = 0 . 003 ) after a single application and sixfold after 7 days ( p < 0 . 001 ) .",
"In contrast , ATP6 copy numbers were stable in all 15 study participants treated with 2% N-9 gel ( p = 0 . 491 ) ( Figure 5B ) . 10 . 7554/eLife . 04525 . 014Figure 5 . Quantification of mitochondria-associated parameters .",
"( A ) PNPT1 mRNA copy numbers measured in 9 and 15 cm biopsies at baseline ( 0 ) , after a single tenofovir gel application ( I ) and after seven consecutive once-daily applications ( VII ) by RT-ddPCR assay .",
"( B ) Mitochondrial ATP6 mRNA copy numbers measured in 9 cm biopsies after tenofovir or N-9 treatment .",
"Line colors in ( A ) and ( B ) signify the 15 participants in the tenofovir arm .",
"Black lines signify the 15 participants in the N-9 arm .",
"Baseline values were compared between 9 and 15 cm biopsies by paired t-test and between tenofovir and N-9 by unpaired t-test .",
"Expression changes over time were tested for statistical significance by ANOVA with Bonferroni adjusted post-tests .",
"( C ) Assessment of mitochondrial density by electron microscopy of 9 cm biopsies in two study participants .",
"Each dot indicates the mean number of mitochondria per µm2 in a separate 2000× image .",
"( D ) Assessment of mitochondrial sizes by electron microscopy in the same biopsies .",
"Each dot depicts the size in µm2 of an individual mitochondrion measured at 5000× .",
"Dot colors in ( C ) and ( D ) correspond to the line colors of the same two study participants in ( A ) and ( B ) .",
"Density and size changes were tested for statistical significance by unpaired t-tests .",
"Horizontal lines and error bars depict means and standard deviations .",
"( E ) Representative electron microscopy images of normal mitochondria at baseline , and of enlarged and dysmorphic mitochondria at time point VII , in 9 cm biopsies of Subject Y . Fine structural detail is limited due to formalin fixation of biopsies .",
"( F ) PNPT1 and ATP6 gene expression in vaginal epithelial cell cultures in response to 1 and 7 days of 50 μM ( blue boxes ) or 500 μM ( green boxes ) tenofovir exposure in vitro .",
"Boxes and error bars signify means and standard deviations across four independent experiments with epithelial cell cultures derived from the vaginal mucosa of four healthy women .",
"Statistical significance levels in all figure panels are indicated by asterisks ( *p < 0 . 05; **p < 0 . 01; ***p < 0 . 001; ****p < 0 . 0001; ‘ns’ , not significant ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 014 Next , we evaluated changes in mitochondrial number and size between baseline and after 7 days of treatment in two study participants chosen for exhibiting pronounced PNPT1 suppression by tenofovir .",
"The number of mitochondria per µm2 decreased by more than half after 7 days of tenofovir treatment ( p < 0 . 001 for both subjects ) ( Figure 5C ) .",
"In the second participant , mitochondria also increased in size by 1 . 4-fold ( p < 0 . 001 ) ( Figure 5D ) and developed dysmorphic cristae during treatment ( Figure 5E ) .",
"In parallel , tenofovir also caused statistically significant inhibition of PNPT1 and ATP6 mRNA expression in vaginal epithelial cells ( Figure 5F ) .",
"Extensive protein studies were not an intended part of MTN-007 and samples were not preserved optimally for this purpose .",
"However , 483 proteins were detected consistently at baseline and time point VII by mass spectrometry .",
"382 proteins increased in the tenofovir arm vs 112 in the no treatment arm ( five participants/arm ) .",
"Among these , significant increases in individual protein expression were only seen in the tenofovir arm ( Figure 6 ) .",
"The top 100 proteins exhibited an average fold increase of 3 . 8 ( median 3 . 2 , range 1 . 6–128 . 7 ) in the tenofovir arm , listed by p value in Figure 6 .",
"Enrichment analysis primarily indicated enhancement of cell survival and induction of leukocyte migration by tenofovir ( Figure 6 ) , which is consistent with our other findings . 10 . 7554/eLife . 04525 . 015Figure 6 . Unbiased mass spectrometry proteomics in rectal secretions from five study participants each in the tenofovir and the no treatment arm .",
"( A ) 483 proteins were consistently detected in all 10 study participants .",
"Of these , 382 proteins in the tenofovir arm had log2 values > 0 for fold change between baseline and after seven daily gel applications , and 112 had log2 fold change values > 0 in the no treatment arm .",
"Log2 fold changes ( x axis ) and p values signifying the likelihood of change ( y axis ) for these proteins are shown .",
"Upper panel , tenofovir arm ( 382 proteins ) ; lower panel , no treatment arm ( 112 proteins ) .",
"Data for proteins with log2 fold values ≤ 0 were not interpretable and are not shown .",
"( B ) Log2 fold changes of the top 100 proteins upregulated between baseline and after 7 days of daily gel application in each of the five study participants in the tenofovir arm .",
"( C ) Selected biofunctional processes defined in the Ingenuity database with significant enrichment of proteins induced in rectal secretions by 7 days of daily tenofovir 1% gel use .",
"The red bars with arrow heads depict the z scores for these biofunctions , indicating the strength of the directionality of the effect .",
"Protein enrichment in each biofunction was tested for statistical significance as described in the ‘Materials and methods’ and the computed p values are depicted by the stars .",
"Numbers of induced proteins are indicated above/below the bars . DOI: http://dx . doi . org/10 . 7554/eLife . 04525 . 015"
],
[
"Our findings indicate that reduced glycerin rectal tenofovir 1% gel affects expression of a different and much broader range of genes than N-9 2% gel , potentially affecting mucosal immune homeostasis , mitochondrial function , and regulation of epithelial cell differentiation and survival .",
"These results make biological sense given that tenofovir is a DNA chain terminator , with possible off-target effects in human cells ( Lewis et al . , 2003 ) , and that topical application achieves at least 100-fold higher active drug concentrations in the mucosa than oral administration of 300 mg tenofovir disoproxil fumarate ( Anton et al . , 2012; Hendrix et al . , 2013 ) .",
"Moreover , tenofovir caused similar changes in primary vaginal epithelial cells cultured from several healthy women .",
"We did not find evidence that tenofovir directly causes inflammation , which is in keeping with our prior report that rectal tenofovir gel did not cause overt histological inflammation or increased mRNA/protein levels of a select panel of pro-inflammatory cytokines ( McGowan et al . , 2013 ) .",
"Rather , tenofovir dampened anti-inflammatory factors .",
"Most prominently , it strongly inhibited IL-10 gene and protein expression , likely via blocking of CREB1 and its coactivator CREBBP ( Martin et al . , 2005; Woodgett and Ohashi , 2005; Gee et al . , 2006; Alvarez et al . , 2009 ) .",
"In addition , it suppressed signaling pathways downstream of TGF-β , a central anti-inflammatory mediator in the gut ( Konkel and Chen , 2011; Surh and Sprent , 2012 ) .",
"Consequently , a number of chemokines were induced , such as the B lymphocyte chemoattractant CXCL13 ( Ansel et al . , 2002 ) , and CCL19 and CCL21 , both ligands of CCR7 on T lymphocytes and dendritic cells ( Forster et al . , 2008 ) .",
"Correspondingly , CCR7 , the B cell marker CD19 , and the T cell markers CD2 , CD3D and CD7 increased .",
"In keeping with this , we observed higher densities of CD3+ and CD7+ T lymphocytes in the rectal mucosa following 7 days of tenofovir 1% gel use .",
"In concert , these changes suggest that tenofovir creates a state of potential hyper-responsiveness to external inflammatory stimuli but does not itself cause inflammation .",
"In populations who , unlike our MTN-007 study cohort , have a high incidence of mucosal infections and associated immune activation , this could potentially diminish the anti-viral protective effect of topical tenofovir prophylaxis ( Naranbhai et al . , 2012 ) .",
"Mitochondrial toxicity of nucleotide/nucleoside reverse transcriptase inhibitors such as tenofovir is well described but the mechanism remains unclear ( Lewis et al . , 2003 ) .",
"We found that tenofovir consistently inhibited expression of PNPT1 , which encodes polynucleotide phosphorylase ( PNPASE ) .",
"PNPASE regulates nucleus-encoded RNA import into mitochondria ( Wang et al . , 2010 ) .",
"In PNPT1 knock-out mice , mitochondrial morphology and respiratory capacity are disrupted in a manner quite similar to the disruption in renal proximal tubular cells in patients with tenofovir-induced nephrotoxicity ( Perazella , 2010; Wang et al . , 2010 ) .",
"In our study , just 1 week of daily tenofovir 1% gel application lowered transcription of mitochondrial ATP6 by sixfold and caused visible ultrastructural mitochondrial changes .",
"These findings suggest that tenofovir's suppression of PNPT1 expression may underlie its reported , but heretofore unexplained , mitochondrial toxicity .",
"A number of changes in rectal biopsies and primary vaginal epithelial cells also suggested that tenofovir can cause increased epithelial proliferation .",
"Furthermore , tenofovir's negative effect on mitochondrial function could lead to impairment of tumor progenitor cell apoptosis ( Modica-Napolitano et al . , 2007; Ni Chonghaile et al . , 2011 ) , as has been specifically reported for loss-of-function mutations of mtATP6 , a mitochondrial gene strongly suppressed by tenofovir in our study ( Shidara et al . , 2005 ) .",
"Neoplastic pressure could also arise from the strong induction of KIAA0101 and UBD ( ubiquitin D ) .",
"KIAA0101 is important for regulation of DNA repair ( Simpson et al . , 2006 ) , is increased in tumor tissues ( Yu et al . , 2001 ) , and enhances cancer cell growth ( Jain et al . , 2011; Hosokawa et al . , 2007 ) .",
"UBD appears to increase mitotic non-disjunction and chromosome instability ( Ren et al . , 2006 , 2011 ) and is highly up-regulated in gastrointestinal cancers ( Lee et al . , 2003; Ren et al . , 2006 , 2011 ) .",
"Notably , though , these findings remain circumstantial , as there is no actual clinical evidence for carcinogenicity .",
"Nevertheless , they raise the question of whether the relatively high concentrations of tenofovir achieved in the mucosa during topical use could potentially lead to neoplastic lesions with continuous and long-term use .",
"According to Viread's Product Monograph , gastrointestinal tumorigenicity has been observed in mice after high oral dosing of tenofovir disoproxil fumarate .",
"Vaginal tumorigenicity has been documented for azido-thymidine , an NRTI and DNA chain terminator like tenofovir , which induced vaginal hyperplasia and carcinomas when delivered to mice intravaginally as a 2% solution ( ∼25% carcinoma rate ) ( Ayers et al . , 1996 ) .",
"This is the first time that a systems biology approach has been applied to a clinical trial of mucosal pre-exposure prophylaxis , and our study shows the value of using these technologies for comprehensive mucosal safety assessment .",
"Our findings raise concerns regarding the safety of topical tenofovir 1% gel in the rectum with long-term use .",
"Tenofovir's effects on vaginal epithelial cells suggest similar activities in the vagina , which we are currently verifying in MTN-014 , a phase I clinical trial comparing vaginal and rectal tenofovir 1% gel in a cross-over format .",
"Further studies are required to gauge whether tenofovir , which has become a valuable cornerstone drug in treating HIV infection , can also be safely and effectively used as a vaginal or rectal microbicide ."
],
[
"MTN-007 ( ClinicalTrials . gov registration NCT01232803 ) was a phase 1 , double blind , placebo-controlled trial in which participants were randomized to receive rectal reduced glycerin tenofovir 1% , nonoxynol-9 ( N-9 ) 2% or hydroxyethylcellulose ( HEC ) gels , or no-treatment ( 1:1:1:1 ) , at three clinical research sites ( Pittsburgh , PA; Birmingham , AL; Boston , MA ) .",
"The study protocol was approved by IRBs at all three sites .",
"All participants gave written informed consent .",
"The study details , and general safety and acceptability data , have been published elsewhere and showed that rectal tenofovir 1% gel was well tolerated and appeared safe by established safety parameters ( McGowan et al . , 2013 ) .",
"Each gel was administered as a single dose and then , after at least a 1-week recovery period , once daily for seven consecutive days .",
"The first dose of study product was self-administered under supervision by the clinic staff at the Treatment 1 Visit .",
"Subsequent administrations occurred at home , and study participants were instructed to insert one dose of gel into the rectum once daily throughout the 7-day period in the evening or before the longest period of rest .",
"A total of 65 study participants were enrolled and randomized in the study , 62 of whom completed it ( tenofovir , n = 15; N-9 , n = 16; HEC , n = 15; and no treatment , n = 16 ) .",
"43 ( 69% ) were male .",
"Microarray studies were performed on eight randomly selected male participants in each group , and confirmatory gene expression studies were done on the remaining participants .",
"The study population consisted of healthy , HIV-uninfected adults aged 18 or older who were required to abstain from receptive anal intercourse during the course of the clinical trial .",
"Female participants were required to use effective contraception .",
"Individuals with abnormalities of the colorectal mucosa , significant gastrointestinal symptoms ( such as a history of rectal bleeding or inflammatory bowel disease ) , evidence of anorectal Chlamydia trachomatis or Neisseria gonorrhea infection , hepatitis B infection , or who used anticoagulants were excluded from the study .",
"Reduced glycerin tenofovir 1% gel and HEC gel , known as the ‘Universal Placebo Gel’ ( Tien et al . , 2005 ) , were supplied by CONRAD ( Arlington , VA , USA ) .",
"2% N-9 gel was provided as Gynol II ( Johnson & Johnson ) .",
"All study products were provided in identical opaque HTI polypropylene pre-filled applicators ( HTI Plastics , Lincoln , NE ) containing 4 ml of study product .",
"Rectal biopsies for the microarray studies were obtained before treatment at enrollment ( time point ‘0’ ) , 30–60 min following application of the single gel dose ( time point ‘I’; to test acute single-dose effects ) , and again on the day following the last dose of the seven once-daily gel applications ( time point ‘VII’; to test multiple-dose effects ) .",
"Following an enema with Normosol-R pH 7 . 4 , a flexible sigmoidoscope was inserted into the rectum and biopsies were collected at 15 cm from the anal margin .",
"Following the sigmoidoscopy , a disposable anoscope was inserted into the anal canal for collection of rectal biopsies at 9 cm from the anal margin .",
"Immediately after harvest , biopsies were immersed in RNA later ( Qiagen , Germany ) , stored at 4°C overnight , and transferred to a −80°C freezer for long-term storage until shipping to Seattle and processing .",
"Tissues routinely discarded from vaginal repair surgeries were harvested from four otherwise healthy adult women , placed in ice-cooled calcium- and magnesium-free phosphate-buffered saline containing 100 U/ml penicillin , 100 μg/ml streptomycin , and 2 . 5 μg/ml Fungizone ( Thermo Fisher Scientific , Waltham , MA ) , and transported to the laboratory within 1 hr of removal from the donor .",
"Tissue harvesting and experimental procedures were approved by the Institutional Review Boards of the University of Washington and the Fred Hutchinson Cancer Research Center .",
"The deep submucosa was removed with surgical scissors and the remaining vaginal mucosa was cut into 5 × 5 mm pieces , which were incubated at 4°C for 18 hr in 5 ml of a 25 U/ml dispase solution ( 354235; BD Biosciences , Franklin Lakes , NJ ) .",
"The epithelial sheets were dissected off under a stereoscope and incubated for 10–12 min at 37°C in 2 ml 0 . 05% trypsin while gently shaking .",
"The dispersed cells were poured through a 100-μm cell strainer into a 50-ml tube , pelleted by centrifugation , and resuspended in F medium ( 3:1 [vol/vol] F12 [Ham]-DMEM [Thermo Fisher Scientific] , 5% fetal calf serum [Gemini Bio-Products , Calabasas , CA] , 0 . 4 μg/ml hydrocortisone [H-4001; Sigma-Aldrich , St . Louis , MO] , 5 μg/ml insulin [700-112P; Gemini Bio-Products] , 8 . 4 ng/ml cholera toxin [227036; EMD Millipore , Billerica , MA] , 10 ng/ml epidermal growth factor [PHG0311; Thermo Fisher Scientific] , 24 μg/ml adenine [A-2786; Sigma] , 100 U/ml penicillin , and 100 μg/ml streptomycin [Thermo Fisher Scientific] ) .",
"The keratinocytes were plated into culture flasks in the presence of ∼12 , 500/cm2 irradiated ( 6000 Rad ) 3T3-J2 feeder fibroblasts ( a kind gift by Cary A Moody ) and 10 μM of Rho kinase inhibitor Y27632 ( 1254; Enzo Life Sciences , Farmingdale , NY ) was added ( Rheinwald and Green , 1975; Chapman et al . , 2010; Liu et al . , 2012 ) .",
"Keratinocytes were fed every 2–3 days and passaged when around 80% confluent by 1 min treatment with 10 ml versene ( Thermo Fisher Scientific ) to remove the feeder cells , followed by 5 min treatment with trypsin/EDTA ( Thermo Fisher Scientific ) .",
"Dislodged keratinocytes were washed and re-plated at ∼2500 keratinocytes/cm2 with irradiated 3T3-J2 feeder fibroblasts .",
"Tenofovir ( CAS 147127-20-6; T018500 , Toronto Research Chemicals , Canada ) was dissolved in phosphate-buffered saline , 7% dimethyl sulfoxide , and 5% 5N sodium hydroxide to result in a 767 mM stock solution , and further diluted in culture media for addition to keratinocyte cultures in concentrations ranging from 0 . 05 to 32 mM .",
"Based on initial titration experiments in which we measured the intracellular concentration of tenofovir diphosphate , the active cellular metabolite of tenofovir , by liquid chromatography-tandem mass spectrometry ( performed in the laboratory of Dr Craig Hendrix , Johns Hopkins University ) , concentrations in the culture media at the lower end ( 0 . 05–0 . 5 mM range ) were estimated to be equivalent to the active concentrations likely achieved by topical tenofovir 1% gel ( 35 mM ) in mucosal epithelial cells in vivo ( Hendrix et al . , 2013; Louissaint et al . , 2013 ) .",
"In all experiments , control keratinocytes were cultured in parallel without tenofovir .",
"Keratinocytes were harvested at pre-determined time points , split into several aliquots , and pelleted .",
"Pellets were frozen and stored at −80°C either as dry pellets for protein assays or after suspension in RNAprotect Cell Reagent ( Qiagen ) for RNA assays .",
"Primary vaginal keratinocytes were cultured in six-well plates ( Corning ) with or without various concentrations of tenofovir for up to 14 days .",
"At day 1 , day 4 , day 7 , or day 14 , the alamarBlue reagent ( BUF012A; AbD Serotec ) was added .",
"After 5 hr , absorbance was measured at 570 and 600 nm on a Varioskan Flash Multimode reader ( Thermo Fisher Scientific ) .",
"Percentage reduction of alamarBlue , corresponding to the level of cell proliferation , was calculated as specified in the manufacturer's technical datasheet .",
"This was converted to percentage of maximal alamarBlue reduction by dividing each well by the well with the highest amount of alamarBlue reduction .",
"The IL-10 concentration in primary vaginal keratinocytes was measured after lysis of frozen cell pellets in Cell Lysis buffer ( R&D Systems , Minneapolis , MN ) using a commercial human IL-10 immunoassay ( Quantikine HS , HS100C; R&D Systems ) according to the manufacturer's specifications .",
"RNA was isolated from the biopsies using the RNeasy Fibrous Tissue Mini Kit ( Qiagen ) , and from the vaginal keratinocytes using the Direct-zol RNA MiniPrep kit ( Zymo Research , Irvine , CA ) , according to the manufacturer's instructions , treated with 27 Kunitz units of DNAse ( Qiagen ) to remove genomic DNA contamination , and evaluated for integrity using the Agilent RNA 6000 Nano Kit ( Agilent , Palo Alto , CA ) on an Agilent 2100 Bioanalyzer .",
"All samples had an RNA Integrity Number of 7 or greater .",
"500 ng of total RNA was amplified and labeled using the Illumina TotalPrep RNA Amplification kit ( Thermo Fisher Scientific ) .",
"cRNA from a total of 192 rectal biopsy samples ( eight men per study arm , four study arms , three time points , and two biopsy sites [9 cm and 15 cm] ) , and from a total of 36 primary vaginal keratinocyte cultures ( three tissue donors , three arms , and four time points ) , was hybridized to HumanHT12 v4 Expression BeadChips ( Illumina , San Diego , CA ) according to the manufacturer's protocols .",
"Each chip contains 47 , 323 probes , corresponding to 30 , 557 genes .",
"Sponges were obtained on the same visits as the biopsies , prior to the biopsy procedure , and processed as previously described ( Anton et al . , 2011 ) .",
"Protein concentration of sponge eluates was determined by BCA assay ( EMD Millipore ) .",
"Equal amounts of total protein from each sample ( 100 μg ) were then denatured in pH 8 . 0 urea exchange buffer ( 8 M Urea [GE HealthCare , United Kingdom] , 50 mM HEPES [Sigma-Aldrich] ) for 20 min at room temperature , and then placed into 10 kDa Nanosep filter cartridges .",
"After centrifugation , samples were treated with 25 mM dithiothreitol ( Sigma-Aldrich ) for 20 min , then 50 mM iodoacetamide ( Sigma-Aldrich ) for 20 min , followed by a wash with 50 mM HEPES buffer .",
"Trypsin ( Promega , Fitchburg , WI ) was added ( 2 μg/100 μg protein ) and samples were incubated at 37°C overnight in the cartridge .",
"Peptides were eluted off the filter with 50 mM HEPES , and the digestion was stopped with 1% formic acid .",
"Peptides were dried via vacuum centrifugation and cleaned of salts and detergents by reversed-phase liquid chromatography ( high pH RP , Agilent 1200 series micro-flow pump , Water XBridge column ) using a step-function gradient .",
"The fractions were then dried via vacuum centrifugation .",
"Equal amounts of peptides were re-suspended in 2% acetonitrile ( Thermo Fisher Scientific ) , 0 . 1% formic acid ( EMD , Canada ) and injected into a nano-flow LC system ( Easy nLC , Thermo Fisher Scientific ) connected in-line to a LTQ Orbitrap Velos mass spectrometer ( Thermo Fisher Scientific ) .",
"Mass spectrometry instrument settings were the same as described previously ( Burgener et al . , 2013 ) .",
"A two-step reverse transcription ( RT ) droplet digital PCR ( ddPCR ) was used to confirm microarray transcriptome data for selected genes of interest ( Hindson et al . , 2011; Pinheiro et al . , 2012 ) .",
"In a ddPCR assay , each sample is partitioned into ∼20 , 000 droplets representing as many individual PCR reactions .",
"The number of target DNA copies present per sample can be quantified based on Poisson distribution statistics , because each individual droplet is categorized as positive or negative for a given gene .",
"For rectal biopsies , cDNA was generated using the High Capacity cDNA Reverse Transcription Kit ( Thermo Fisher Scientific ) and the ddPCR was carried out using the 2× ddPCR Supermix for Probes ( BioRad , Hercules , CA ) .",
"For epithelial cell lines , these steps were combined using the 2× One-Step RT-ddPCR Kit for Probes ( BioRad ) .",
"ddPCR was performed on the QX100 droplet digital PCR system ( BioRad ) .",
"Reactions were set up with 20× 6-carboxyfluorescein ( FAM ) -labeled target gene-specific qPCR assay ( Integrated DNA Technologies , Coralville , IA; or Thermo Fisher Scientific ) and 20× VIC-labeled housekeeping hemoglobin B ( HBB ) gene-specific Taqman gene expression assay ( Thermo Fisher Scientific ) .",
"Each assembled ddPCR reaction mixture was loaded in duplicate into the sample wells of an eight-channel disposable droplet generator cartridge ( BioRad ) and droplet generation oil ( BioRad ) was added .",
"After droplet generation , the samples were amplified to the endpoint in 96-well PCR plates on a conventional thermal cycler using the following conditions: denaturation/enzyme activation for 10 min at 95°C , 40 cycles of 30 s denaturation at 94°C , and 60 s annealing/amplification at 60°C , followed by a final 10 min incubation step at 98°C .",
"After PCR , the droplets were read on the QX100 Droplet Reader ( BioRad ) .",
"Analysis of the ddPCR data was performed with QuantaSoft analysis software version 1 . 3 . 1 . 0 ( BioRad ) .",
"Four-micron sections were cut on a Leica RM2255 Automated Rotary Microtome ( Leica , Germany ) , mounted on positively charged EP-3000 slides ( Creative Waste Solutions , Tualatin , OR ) , dried for 1 hr at 60°C , and stored until staining at 4°C .",
"Slides were deparaffinized in xylene and rehydrated in graded dilutions of ethanol in water .",
"Antigen retrieval was performed by heating the slides in Trilogy Pretreatment Solution ( Sigma-Aldrich ) for 20 min at boiling temperature in a conventional steamer ( Black&Decker , Towson , MD ) .",
"Slides were then cooled for 20 min , rinsed three times in Tris-buffered 0 . 15 M NaCl solution containing 0 . 05% Tween 20 ( Wash Buffer; Dako , Santa Clara , CA ) , and stained at room temperature in an automated slide-processing system ( Dako Autostainer Plus ) .",
"Endogenous peroxidase activity was blocked using 3% H2O2 for 8 min , followed by 10 min in Serum-Free Protein Block ( Dako ) .",
"The slides were then stained for 1 hr with the primary antibodies .",
"Staining was performed with the following primary antibodies: anti-CD3 ( RM9107S , rabbit clone SP7; Thermo Fisher Scientific ) , anti-ubiquitin D ( NBP2-13498 , rabbit polyclonal anti-FAT10 antibody; Novus Biologicals , Littleton , CO ) , anti-CD7 ( M7255 , mouse clone CBC . 37; Dako ) , and anti-interleukin-10 ( sc-8438 , mouse clone E−10; Santa Cruz Biotechnology , Dallas , TX ) .",
"Negative control primary immunoglobulins were either whole rabbit IgG ( 011-000-003; Jackson ImmunoResearch Laboratories , West Grove , PA ) or mouse IgG ( I-2000; Vector Laboratories , Burlingame , CA ) .",
"For CD3 staining , the slides were incubated with anti-CD3 for 60 min at a dilution of 1:25 , washed in Wash Buffer , incubated for 30 min with sheep-anti-rabbit Dylight 649 ( 611-643-122; Rockland Immunochemicals , Limerick , PA ) , washed , and incubated for 30 min with donkey-anti-sheep Dylight 649 ( 613-743-168; Rockland ) .",
"Sections were counter-stained for 20 min with the nucleic acid-binding dye Sytox Orange ( S11368; Thermo Fisher Scientific ) at a dilution of 1:20 , 000 , and coverslipped in ProLong Gold antifade reagent ( P36930; Thermo Fisher Scientific ) .",
"For ubiquitin D ( UBD ) , CD7 and interleukin-10 ( IL-10 ) staining , the slides were incubated with 0 . 8 μg/ml ( UBD and CD7 ) or 4 μg/ml ( IL-10 ) of the primary antibody for 60 min .",
"After washing , the anti-UBD-stained slides were incubated for 30 min with Leica Power Vision HRP rabbit-specific antibody polymer ( PV6119; Leica ) ; and the anti-CD7 and anti-IL-10 stained slides were incubated for 30 min with LeicaPower Vision HRP mouse-specific antibody polymer ( PV6114; Leica ) .",
"After washing , staining was visualized with 3 , 3′-diaminobenzidine ( Liquid DAB+ Substrate Chromogen System; Dako ) for 7 min , and the sections were counter-stained for 2 min with hematoxylin ( Biocare , Concord , CA ) .",
"Controls for all antibodies were run with identical procedures but replacing the primary antibodies with either rabbit IgG or mouse IgG , as appropriate , at calculated matching concentrations .",
"Formalin-fixed paraffin-embedded rectal biopsies were de-paraffinized and fixed overnight in half-strength Karnovsky's fixative .",
"Staining , embedding , cutting , and viewing on a JEOL 1400 SX transmission electron microscope were performed as previously described ( Hladik et al . , 1999 , 2007 ) .",
"20 images per sample were acquired at 5 , 000× magnification .",
"Using ImageJ ( Collins , 2007 ) , the two-dimensional sizes in µm2 of all individual mitochondria with a circularity index of ≥0 . 9 were calculated ( for standardization purposes , only mitochondria cut near perfectly along their minor axis were evaluated ) .",
"10 images per sample were also acquired at 2 , 000× magnification , always including the epithelial cell brush border .",
"Using ImageJ , a grid of 1 . 32 µm2 squares of defined size was overlaid onto each image .",
"All mitochondria in the images were counted , except those in the first row of squares falling on the brush border and in squares along the image rims ( which only partially covered the tissue ) , and the mean numbers of mitochondria per µm2 were calculated for each of the acquired 2 , 000× images ( range of counted squares per image: 23–50 ) .",
"All p values reported were adjusted for multiple testing as appropriate , except for the exploratory protein mass spectrometry data .",
"Correlations of log2 fold gene expression changes between 9 cm and 15 cm biopsies in Figure 1C and Figure 1—figure supplement 1 were tested by Spearman's rank correlation coefficient .",
"Gene and protein expression changes over baseline were compared between study arms by two-tailed Mann–Whitney test ( Figure 1—figure supplement 2 and Figure 6 ) .",
"The combined expression data from the microarray and RT-ddPCR tests shown in Figure 2C were compared separately for log-fold induction or suppression over baseline using a one-tailed Wilcoxon signed-rank test with Bonferroni adjustment .",
"Immunohistology measurements in Figure 2D were compared between baseline and after 7 days of treatment by two-tailed paired t tests .",
"Due to high skewness of the CD7+ , CD3+ , and IL-10+ cell counts , these were tested after log10 transformation .",
"Ratios of induced to suppressed genes in Figure 3A were tested for a difference between cellular compartments using Chi-square statistics .",
"DSP , IL-10 , KIAA0101 , PNPT1 , and ATP6 copy numbers or protein concentrations in Figures 4B , C , 5A , B , F were tested for significant change across three time points by repeated measures ANOVA , and exact Sidak's ( Figures 4B , C , 5F ) or Tukey ( Figure 5A , B ) post-tests were run for comparisons between two time points .",
"Due to high skewness of the KIAA0101 copy numbers , these were tested after log10 transformation .",
"The effect of increasing tenofovir concentrations on the proliferation of primary vaginal epithelial cells in Figure 4E was assessed for significance by a linear model accounting for days and donors .",
"Copy numbers between 9 cm and 15 cm biopsies in Figure 5A were compared by two-tailed paired t test .",
"Copy numbers between baseline tenofovir and N-9 in Figure 5B were compared by a two-tailed unpaired t test .",
"Mitochondria counts and sizes in Figure 5C , D were compared between baseline and Day VII , separately for the two subjects evaluated by electron microscopy , by two-tailed unpaired t tests with Bonferroni adjustment .",
"Microarray chip probe expression values were tested for correlation between the three primary vaginal cell cultures by computing pairwise Pearson correlation coefficients ( Figure 3—source data 1 ) .",
"Statistics packages used were Prism 6 ( Graphpad Software , La Jolla , CA ) and Bioconductor/Lumi in R . Statistical results are summarized in Supplementary file 1 ."
]
] | [
"Tenofovir gel is being evaluated for vaginal and rectal pre-exposure prophylaxis against HIV transmission .",
"Because this is a new prevention strategy , we broadly assessed its effects on the mucosa .",
"In MTN-007 , a phase-1 , randomized , double-blinded rectal microbicide trial , we used systems genomics/proteomics to determine the effect of tenofovir 1% gel , nonoxynol-9 2% gel , placebo gel or no treatment on rectal biopsies ( 15 subjects/arm ) .",
"We also treated primary vaginal epithelial cells from four healthy women with tenofovir in vitro .",
"After seven days of administration , tenofovir 1% gel had broad-ranging effects on the rectal mucosa , which were more pronounced than , but different from , those of the detergent nonoxynol-9 .",
"Tenofovir suppressed anti-inflammatory mediators , increased T cell densities , caused mitochondrial dysfunction , altered regulatory pathways of cell differentiation and survival , and stimulated epithelial cell proliferation .",
"The breadth of mucosal changes induced by tenofovir indicates that its safety over longer-term topical use should be carefully monitored .",
"Clinical trial registration: NCT01232803 ."
] | [
"Tenofovir is a drug that can stop some viruses—including HIV—from multiplying .",
"It is commonly used in multidrug therapies to control HIV infection .",
"Clinical trials are underway to find out whether using the drug in the form of a gel applied to the vagina or rectum could be an effective way to prevent HIV transmission during sex .",
"Some of the clinical trials carried out so far have produced promising results .",
"However , since the use of gels containing anti-viral drugs is a new strategy for HIV prevention , there are limited data available about the safety of these products .",
"Previous studies have shown that the concentration of tenofovir in the vagina is much higher in individuals using the gel than in those taking the tablet form of the drug .",
"These high concentrations could lead to unexpected effects on the health of the cells exposed to the gel .",
"Here , Hladik , Burgener , Ballweber et al . used a systems biology approach to look at the broad effects of tenofovir gel on tissue from the rectum .",
"Tissue samples taken from the rectums of 15 patients who used tenofovir gel for seven days were compared with tissue samples taken from individuals who used a control gel that did not contain the drug or who did not use any gel .",
"Genes that regulate inflammation were suppressed in the rectal tissue from patients who used tenofovir , as were genes that help these tissues regenerate and produce energy .",
"The tissue from these patients also contained more immune cells , suggesting that their local immune systems were more active .",
"Additionally , Hladik , Burgener , Ballweber et al . observed changes that could potentially lead to the increased growth of cells .",
"Similar differences were also observed in vaginal cells that had been treated with tenofovir in the laboratory .",
"These findings suggest that tenofovir delivered directly to the vagina or rectum may have unintentional local side effects .",
"However , it is important to acknowledge that tenofovir gel has been evaluated in multiple studies that have not observed overt clinical adverse effects .",
"Therefore , the implication of these findings is currently unclear and warrants further study ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"microbiology and infectious disease"
] | Angiomotin functions in HIV-1 assembly and budding | elife-03778-v2 | [
[
"As obligate parasites , viruses must harness host cellular pathways to complete many different steps in viral replication .",
"For example , a large number of enveloped viruses , including HIV-1 , usurp the cellular ESCRT ( Endosomal Sorting Complexes Required for Transport ) pathway to bud from cells ( for recent reviews , see Meng and Lever , 2013; Votteler and Sundquist , 2013; Weissenhorn et al . , 2013 ) .",
"Viral structural proteins typically use one or more short peptide motifs , termed ‘late assembly domains’ , to bind and recruit early-acting ESCRT factors or ESCRT-associated E3 ubiquitin ligases to sites of viral assembly and budding .",
"The three best characterized viral late assembly domains are: ( 1 ) the ‘PTAP’ motif ( Pro-Thr/Ser-Ala-Pro ) , which binds the UEV domain of the TSG101 subunit of the ESCRT-I complex , ( 2 ) the ‘YPXnL’ motif ( Tyr-Pro-Xn-Leu , where X can vary in identity and sequence length ) , which binds the V domain of the ESCRT factor ALIX , and ( 3 ) the ‘PPXY’ motif ( Pro-Pro-X-Tyr ) , which binds the WW domains of NEDD4 family E3 ubiquitin ligases .",
"All three of these viral late assembly domains mimic cellular protein–protein interactions that facilitate ESCRT-dependent vesiculation processes such as multi-vesicular body ( MVB ) biogenesis and ectosome formation .",
"In addition to the three well-characterized late assembly domains described above , there are indications that enveloped viruses can also connect to the ESCRT pathway via alternative interactions that are not yet well-understood .",
"For example , efficient release of the model paramyxovirus , PIV5 , requires a non-canonical ‘FPIV’ late assembly domain motif , ubiquitylation of the matrix ( M ) protein , and host Angiomotin-like protein 1 ( AMOTL1 ) ( Schmitt et al . , 2005; Pei et al . , 2010; Harrison et al . , 2012 ) .",
"There is no evidence that AMOTL1 can bind the FPIV late assembly domain , however , and any relationships between ubiquitin , AMOTL1 and the FPIV late assembly domain activity are not yet understood ( Pei et al . , 2010 ) .",
"Owing to its medical importance , HIV-1 has become a leading model for understanding ESCRT pathway recruitment and viral budding .",
"The C-terminal p6 region of the viral Gag structural protein contains canonical PTAP and YPXnL motifs that serve as the major functional late assembly domains in most cell types .",
"Nevertheless , the release of ‘crippled’ HIV-1 Gag constructs that lack both of these late domains can still be stimulated by overexpression of the HECT E3 ubiquitin ligase , NEDD4L ( also known as NEDD4-2 ) ( Chung et al . , 2008; Usami et al . , 2008 ) .",
"Humans express nine different NEDD4 protein family members ( reviewed in Ingham et al . , 2004; Bernassola et al . , 2008 ) , but NEDD4L stimulates virus budding more potently than any other NEDD4 family member , implying that HIV-1 Gag preferentially engages this E3 ubiquitin ligase ( Chung et al . , 2008; Usami et al . , 2008 ) .",
"Previous studies have defined many of the requirements for NEDD4L-stimulated HIV-1 release .",
"NEDD4L has many different isoforms ( Chen et al . , 2001; Dunn et al . , 2002; Itani et al . , 2005 ) , but isoform 2 ( also called NEDD4-2s , and here termed NEDD4L ) potently stimulates the budding of crippled HIV-1 constructs , and uniquely rescues the Gag processing defects that accompany defective budding ( Chung et al . , 2008; Usami et al . , 2008 ) .",
"This NEDD4L isoform contains only the final 31 residues of the N-terminal C2 membrane binding domain , a central linker region that contains four WW domains , and a C-terminal HECT E3 ubiquitin ligase domain that forms Lys-63-linked poly-ubiquitin chains ( see Figure 1A ) ( Kim and Huibregtse , 2009; Weiss et al . , 2010 ) .",
"Stimulation of HIV-1 budding requires NEDD4L E3 ubiquitin ligase activity , indicating that Lys-63-linked poly-ubiquitin chains perform an essential function ( Chung et al . , 2008; Usami et al . , 2008 ) .",
"The functional target ( s ) of Lys-63 poly-ubiquitylation has not been established definitively , but HIV-1 Gag itself is the leading candidate because functional virus budding correlates with Lys-63-linked poly-ubiquitylation of Gag , and because NEDD4L truncation constructs that include the HECT domain can be functionally rescued by fusion to the HIV-1 Gag-targeting cyclophilin A domain ( Weiss et al . , 2010; Sette et al . , 2013 ) .",
"HIV-1 Gag lacks identifiable PPXY motifs , however , and there is no evidence that Gag can bind NEDD4L directly . 10 . 7554/eLife . 03778 . 003Figure 1 . AMOT p130 is a NEDD4L binding partner .",
"( A ) Schematic illustrations of the domain structures and motifs in NEDD4L , AMOT p130 and p80 , AMOTL1 , AMOTL2 and HIV-1 Gag .",
"Here and throughout , NEDD4L refers to a naturally occurring protein isoform ( isoform",
"2 ) that contains only the C-terminal 32 residues of the C2 domain ( Genebank accession number AAM46201 . 1 , denoted NEDD4Ls in other publications and NEDD4LΔC2 in our previous publication ( Chung et al . , 2008 ) ) .",
"To maintain consistency with that publication , the NEDD4L numbering scheme used here corresponds to NEDD4L isoform 1 , which is 121 residues longer at the N-terminus and contains the entire C2 domain ( denoted NEDD4LWT in reference Chung et al . , 2008 ) .",
"( B ) Affinity co-purification and identification of AMOT p130 as a binding partner of OSF ( One-STrEP-FLAG ) -tagged NEDD4L .",
"SDS-PAGE/Coomassie-stained gel showing STrEP-Tactin matrix affinity purified proteins from 293T cells transfected with an OSF-NEDD4L expression construct ( lane",
"2 ) or an empty vector control ( lane 1 ) .",
"Labels denote OSF-NEDD4L ( bait ) and AMOT p130 ( prey ) .",
"Trypsin-digested AMOT p130 peptides identified by mass spectrometric analyses are summarized in Figure 1—figure supplement 1 .",
"A representative image from three independent repetitions is shown .",
"( C ) NEDD4L WW domains are required for AMOT p130 binding .",
"Western blots showing co-immunoprecipitations of endogenous 293T cell AMOT proteins ( prey ) with OSF-NEDD4L ( bait ) .",
"Panel 1: endogenous AMOT proteins ( anti-AMOT blot ) co-immunoprecipitated with OSF-NEDD4L baits from cells that lacked exogenous OSF-NEDD4L ( lane 1 , control ) , expressed wild type OSF-NEDD4L ( lane 2 ) , expressed OSF-NEDD4LΔWW ( lane 3 , construct has inactivating point mutations in all four WW domains ) , expressed OSF-NEDD4LC942A ( lane 4 , construct has an inactivating point mutation in the HECT E3 domain ) , or expressed OSF-NEDD4LC942A/ΔWW ( lane 5 , construct has inactivating point mutations in the WW and HECT E3 domains ) .",
"Panel 2: same co-immunoprecipitation experiment blotted with anti-FLAG antibodies to detect OSF-NEDD4L proteins .",
"Panel 3: input levels of endogenous AMOT ( anti-AMOT blot , 10% of total ) .",
"Panel 4: input levels of exogenous OSF-NEDD4L ( anti-FLAG , 10% of total ) .",
"Note that endogenous AMOT p80 was present in the input lysate , but did not co-immunoprecipitate with OSF-NEDD4L .",
"Representative image from three independent repetitions . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00310 . 7554/eLife . 03778 . 004Figure 1—figure supplement 1 . Identification of AMOT p130 as a NEDD4L binding partner . The protein band denoted ‘AMOT p130’ in Figure 1B , lane 2 was excised , digested with trypsin and the eluted peptides were identified by mass spectrometry as described in ‘Materials and methods’ .",
"This analysis was performed three times and the identified peptides are mapped onto the AMOT p130 primary sequence , with peptides identified in experiments 1–3 color coded in black , blue and red , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00410 . 7554/eLife . 03778 . 005Figure 1—figure supplement 2 . The AMOT p130 PPXY motifs contribute to NEDD4L binding . Western blots show co-immunoprecipitations of OSF-NEDD4L ( bait , captured on STrEP-Tactin affinity matrices ) with endogenous 293T cells AMOT and exogenously expressed HA-AMOT p130 proteins ( prey ) .",
"Co-immunoprecipitations with OSF-NEDD4L were from cells that lacked exogenous AMOT p130 ( lane 1 ) , expressed wild type HA-AMOT p130 ( lane",
"2 ) or expressed a mutant HA-AMOT p130 protein carrying PP/AA mutations in the three N-terminal ‘PPXY’ motifs ( Wang et al . , 2012; Yi et al . , 2013 ) .",
"Panel 1: AMOT protein co-immunoprecipitations ( anti-AMOT blot ) with OSF-NEDD4L .",
"Note that OSF-NEDD4L co-immunoprecipitated low levels of endogenous AMOT p130 ( lanes 1 and 3 , denoted p130 ) but not endogenous AMOT p80 ( denoted p80 with a dashed line ) .",
"Panel 2: The same co-immunoprecipitation experiment blotted with anti-HA antibodies to detect exogenously expressed HA-AMOT p130 proteins .",
"Panel 3: The same co-immunoprecipitation experiment blotted with anti-FLAG antibodies to detect exogenously expressed OSF-NEDD4L .",
"Panels 4–6 show the input quantities ( 10% ) of endogenous AMOT and HA-AMOT p130 ( panel 4 , anti-AMOT ) , exogenous HA-AMOT p130 ( panel 5 , anti-HA ) , and exogenous OSF-NEDD4L ( panel 6 , anti-FLAG ) .",
"A representative image from three independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 005 These observations imply that NEDD4L may be recruited to sites of HIV-1 assembly through an alternative mechanism .",
"We undertook the present study with the goal of identifying co-factors that might help link NEDD4L to HIV-1 Gag and stimulate virus budding .",
"We identified Angiomotin ( AMOT ) as a protein that binds both NEDD4L and HIV-1 Gag , and functions in HIV-1 assembly and release .",
"AMOT is required to complete virion assembly and envelopment , and is therefore a new host factor that acts in a previously uncharacterized step of HIV-1 morphogenesis ."
],
[
"Affinity purification/mass spectrometry experiments were performed to identify candidate NEDD4L binding partners .",
"A number of cellular proteins co-purified with OSF ( One-STrEP-FLAG ) -tagged NEDD4L , as visualized by SDS-PAGE ( Figure 1B , compare lanes 1 and 2 ) .",
"Prominent co-purifying proteins were excised from the gel , digested with trypsin , and identified by mass spectrometry .",
"The p130 isoform of human Angiomotin was unambiguously identified as a protein that co-purified with OSF-NEDD4L in three independent repetitions of this experiment , ( lane 2 , labeled AMOT p130 , peptide coverage data are summarized in Figure 1—figure supplement 1 ) .",
"Smaller AMOT p130 fragments were also identified , as was the related AMOT family protein , Angiomotin-like protein 1 ( AMOTL1 , data not shown ) .",
"Cells express two AMOT isoforms , a longer p130 isoform , and a shorter C-terminal p80 isoform that is expressed from an alternatively spliced message ( Figure 1A ) ( Moreau et al . , 2005 ) .",
"AMOT p130 binds NEDD4 protein family members via interactions between the four central NEDD4 WW domains and three PPXY motifs located within the N-terminal extension that is unique to the AMOT p130 isoform ( Figure 1A ) ( Wang et al . , 2012 ) .",
"In good agreement with this report and with our affinity purification/mass spectrometry results , we found that AMOT p130 co-immunoprecipitated with exogenously expressed OSF-NEDD4L ( Figure 1C , panel 1 , lane 2 ) .",
"In contrast , AMOT p130 did not co-immunoprecipitate with a mutant OSF-NEDD4L protein that contained inactivating Trp to Ala substitutions in the key PPXY-binding ‘W39’ residues from each of the four WW domains ( denoted OSF-NEDD4LΔWW , panel 1 , compare lanes 2 and",
"3 ) ( Otte et al . , 2003 ) .",
"A previous study has demonstrated that NEDD4 proteins can ubiquitylate and reduce AMOT p130 levels ( Wang et al . , 2012 ) , and we also observed that OSF-NEDD4L overexpression modestly reduced AMOT p130 levels ( Figure 1C , panel 3 , compare lanes 1 and 2 ) .",
"We therefore tested whether AMOT p130 also co-precipitated with OSF-NEDD4L proteins that carried inactivating Cys942Ala point mutations in the active site cysteine of the HECT E3 ubiquitin ligase domain ( OSF-NEDD4LC942A ) .",
"This mutation increased OSF-NEDD4L levels in the lysate and immunoprecipitate ( panels 2 and 4 compare lanes 4 and 5 to lanes 2 and",
"3 ) and restored normal AMOT protein levels ( panel 3 , compare lanes 4 , 5 and 1 to lane 2 ) .",
"Once again , AMOT p130 ( and breakdown products ) co-immunoprecipitated with OSF-NEDD4LC942A ( panel 1 , lane 4 ) , but not with a protein that also contained inactivating WW domain mutations ( OSF-NEDD4LΔWW/C942A panel 1 , lane 5 ) .",
"AMOT p130 co-precipitation levels were higher with OSF-NEDD4LC942A than with wild type OSF-NEDD4L , presumably owing to the higher cellular levels of both OSF-NEDD4L and AMOT p130 ( compare lanes 2 and 4 ) .",
"Three additional observations confirmed that the NEDD4L WW domains and AMOT p130 PPXY motifs are critical for the NEDD4L-AMOT p130 interaction .",
"Firstly , mutating the three N-terminal AMOT p130 PPXY motifs strongly inhibited co-immunoprecipitation of HA-AMOT p130 with OSF-NEDD4L ( Figure 1—figure supplement 2 , panel 1 , compare lanes 2 and 3 ) .",
"Secondly , endogenous AMOT p80 , which lacks the N-terminal PPXY motifs , failed to co-immunoprecipitate with either OSF-NEDD4L or OSF-NEDD4LC942A ( Figure 1C , panel 1 , lanes 2 and 4 ) , even though AMOT p80 was present in the 293T cell extracts ( panel 3 , lanes 1–5 ) .",
"Thirdly , we observed that exogenously expressed , epitope-tagged NEDD4L and AMOT p130 co-localized well in HeLa-M cells , and this co-localization required both the AMOT PPXY and NEDD4L WW motifs ( data not shown ) , in good agreement with previous reports that motins co-localize with other NEDD4 family members ( Skouloudaki and Walz , 2012; Wang et al . , 2012 ) .",
"Taken together , these experiments demonstrate that NEDD4L interacts specifically with AMOT p130 in cells and that the interaction requires the WW domains of NEDD4L and the PPXY motifs of AMOT .",
"Pull-down experiments with purified recombinant proteins were performed to test whether AMOT p130 binds NEDD4L directly , and whether AMOT p130 can also bind HIV-1 Gag and thereby link NEDD4L to the viral Gag protein .",
"Wild type and mutant NEDD4L proteins and an HIV-1 Gag protein that lacked the flexible p6 domain ( GagΔp6 ) were expressed in Escherichia coli and purified to near homogeneity ( Figure 2A , lanes 1 and 2 , and Figure 2B , lane 1 , respectively ) .",
"OSF-AMOT p130 was overexpressed in SF-9 insect cells and substantially enriched by binding to STrEP-Tactin affinity resin ( Figure 2A , lane 6 and Figure 2B , lane 7 ) , although we were unable to purify the protein to homogeneity .",
"As shown in Figure 2A , NEDD4L did not bind an immobilized control OSF-GFP protein , but bound with at least 1:1 stoichiometry to immobilized OSF-AMOT p130 ( compare lanes 4 and 7 ) .",
"In contrast , binding of the NEDD4LΔWW mutant protein was nearly undetectable ( Figure 2A , compare lanes 7 and 8 , particularly in the western blot in panel 2 ) .",
"These experiments confirm that NEDD4L and AMOT p130 bind one another directly , with the WW domains of NEDD4L binding the PPXY motifs of AMOT p130 . 10 . 7554/eLife . 03778 . 006Figure 2 . AMOT p130 binds directly and specifically to NEDD4L and HIV-1 Gag∆p6 . ( A ) OSF-AMOT p130 binds NEDD4L .",
"Recombinant OSF-GFP ( specificity control , lanes 3–5 ) or OSF-AMOT p130 ( lanes 6–8 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 3 and",
"6 ) or buffers containing 0 . 5 µM wild type NEDD4L ( lanes 4 and",
"7 ) or NEDD4LΔWW ( lanes 5 and 8 , inactivating point mutations in all four WW domains ) .",
"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining ( panel",
"1 ) or by western blotting ( panel 2 , anti-NEDD4L ) to confirm the identities of the bound NEDD4L and help to distinguish background proteins from low-level binding in panel 1 .",
"Input levels of NEDD4L ( lane 1 , 1 . 5% of total ) and NEDD4LΔWW ( lane 2 , 1 . 5% of total ) are shown for reference .",
"A representative image from three independent repetitions is shown .",
"( B ) OSF-AMOT p130 binds HIV-1 Gag∆p6 .",
"Recombinant OSF-GFP ( control , lanes 3–6 ) or OSF-AMOT p130 ( lanes 7–10 ) were expressed , captured on STrEP-Tactin affinity matrices and incubated with a buffer control ( lanes 3 and",
"7 ) or with buffers containing 1 . 0 µM HIV-1 Gag∆p6 ( lanes 3 and 8 ) , 0 . 5 µM wild type NEDD4L ( lanes 5 and 9 ) or both proteins ( lanes 6 and 10 ) .",
"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining ( panel",
"1 ) or by western blotting to confirm the identities of the bound Gag ( panel 2 , anti-MA and anti-CA ) and NEDD4L ( panel 3 , anti-NEDD4L ) proteins and help to distinguish background proteins from low-level binding in panel 1 .",
"Input Gag∆p6 ( lane 1 , 2% of total ) and NEDD4L ( lane 2 , 1 . 5% of total ) are shown for reference .",
"A representative image from three independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00610 . 7554/eLife . 03778 . 007Figure 2—figure supplement 1 . AMOT p130 , AMOTL1 and AMOTL2 bind directly and specifically to NEDD4L and HIV-1 Gag∆p6 . ( A ) OSF-AMOT p130 , OSF-AMOTL1 , and OSF-AMOTL2 bind NEDD4L .",
"Recombinant OSF-GFP ( specificity control , lanes 2 and 6 ) , OSF-AMOT p130 ( lanes 3 and 7 ) , OSF-AMOTL1 ( lanes 4 and 8 ) , and OSF-AMOTL2 ( lanes 5 and 9 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 2–5 ) or with a buffer containing 0 . 5 µM wild type NEDD4L ( lanes 6–9 ) .",
"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining .",
"Input NEDD4L ( lane 1 , 1 . 5% of total ) is shown for reference .",
"Asterisk denotes an OSF-AMOTL2 breakdown product that co-migrates with NEDD4L .",
"A representative image from two independent repetitions is shown .",
"( B ) OSF-AMOT p130 , OSF-AMOTL1 , and OSF-AMOTL2 bind Gag∆p6 .",
"Recombinant OSF-GFP ( specificity control , lanes 2 and 6 ) , OSF-AMOT p130 ( lanes 3 and 7 ) , OSF-AMOTL1 ( lanes 4 and 8 ) , and OSF-AMOTL2 ( lanes 5 and 9 ) were expressed , captured on STrEP-Tactin affinity matrices , and incubated with either a buffer control ( lanes 2–5 ) or buffers containing 1 µM Gag∆p6 ( lanes 6–9 ) .",
"Matrix-bound proteins were released by boiling in denaturing buffer and detected by SDS-PAGE with Coomassie blue staining .",
"Input Gag∆p6 ( lane 1 , 2% of total ) is shown for reference .",
"A representative image from two independent repetitions is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 007 Analogous pull-down experiments were performed to test whether AMOT binds HIV-1 GagΔp6 .",
"Removing the p6 region facilitates Gag protein expression and purification , and is not expected to interfere with any biologically relevant interactions because NEDD4L potently stimulates HIV-1 GagΔp6 release from cells ( Chung et al . , 2008; Usami et al . , 2008 ) .",
"As shown in Figure 2B , HIV-1 GagΔp6 bound immobilized OSF-AMOT p130 , but did not bind an immobilized OSF-GFP control ( compare lanes 4 and 8 ) .",
"This interaction did not appear to be mediated by nucleic acids because the affinity-purified OSF-AMOT p130 protein was pre-washed with high salt solution to remove any bound nucleic acid ( see ‘Materials and methods’ ) , and because the interaction was maintained , albeit at slightly lower levels , even when immobilized OSF-AMOT p130 was incubated with high concentrations of RNase that were sufficient to eliminate other RNA-mediated Gag-protein interactions ( not shown ) .",
"GagΔp6 and NEDD4L appeared to bind OSF-AMOT p130 independently because their binding levels did not change significantly when all three proteins were incubated together ( compare lanes 8 and 9 to lane 10 ) .",
"AMOT is the founding member of the human motin family , which also includes Angiomotin-like protein 1 ( AMOTL1 ) and Angiomotin-like protein 2 ( AMOTL2 ) ( Moleirinho et al . , 2014 ) .",
"AMOTL1 and AMOTL2 share 52% and 45% pair-wise sequence identity with AMOT p130 , and both contain the N-terminal extension and PPXY motifs that are present in AMOT p130 but absent in AMOT p80 ( Figure 1A ) .",
"Pull-down experiments demonstrated that AMOTL1 and AMOTL2 also bind both NEDD4L and HIV-1 GagΔp6 ( Figure 2—figure supplement 1 ) .",
"Thus , AMOT p130 binds directly and specifically to both NEDD4L and HIV-1 Gag , and these binding activities are shared by the other two human motins .",
"Co-expression experiments were performed to test whether AMOT and NEDD4L can cooperate in stimulating HIV-1 release ( Figure 3 ) .",
"These experiments employed the crippled HIV-1ΔPTAP , ΔYP viral expression construct , which lacks functional PTAP and YPXnL late domains and is therefore particularly responsive to cellular NEDD4L activity ( Chung et al . , 2008; Usami et al . , 2008 ) .",
"As expected , HIV-1ΔPTAP , ΔYP was poorly released from control 293T cells , as measured by western blot that detected virion-associated CA and MA proteins ( Figure 3 , upper right panel , lane 1 ) , and low viral titers ( lower right panel , lane 1 ) .",
"Overexpression of HA-AMOT p130 increased the levels of the full-length protein ( and presumptive breakdown products were also evident , left panel 1 , compare lanes 1 and 2 ) .",
"AMOT p130 overexpression modestly increased HIV-1ΔPTAP .",
"ΔYP release and infectivity ( right panels compare lanes 1 and 2 , 1 . 7-fold increases in both release and infectivity ) , without significantly altering the intracellular levels of Gag ( left , panel 4 , compare lanes 1 and",
"2 ) or a control cellular protein ( GAPDH , left panel 3 , compare lanes 1 and 2 ) .",
"Thus , AMOT p130 overexpression stimulates HIV-1ΔPTAP , ΔYP release and infectivity , but the effect is modest . 10 . 7554/eLife . 03778 . 008Figure 3 . AMOT p130 stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , FLAG-NEDD4L proteins ( lanes 3–6 , with wild type ( WT ) FLAG-NEDD4L in lanes 3 and 4 and FLAG-NEDD4LΔWW , in lanes 5 and 6 ) , and wild type HA-AMOT p130 ( lanes 2 , 4 and",
"6 ) or an empty vector control ( lanes 1 , 3 , and 5 ) .",
"Right panels show corresponding levels of extracellular , virion-associated CAGag and MAGag proteins ( panel 1 , anti-MA and anti-CA ) and viral titers ( panel 2 , IU denotes ‘infectious units’ ) .",
"Here and in subsequent figures: ( 1 ) error bars denote standard deviations between independent repetitions of the experiment , n = 5 in this case , and ( 2 ) numbers within the blots show integrated intensities of the CA band intensities ( relative to the value in the control experiment , set to 1 . 0 ) .",
"Here and throughout significance is denoted by: NS , not significant ( p > 0 . 05 ) ; *0 . 05 > p > 0 . 01; **p < 0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00810 . 7554/eLife . 03778 . 009Figure 3—figure supplement 1 . Dose-dependent AMOT p130 stimulation of NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–8 ) , FLAG-NEDD4L ( lanes 2–8 ) , and increasing quantities of an AMOT p130 expression construct ( lanes 3–8 , 0 . 25–1 . 5 μg DNA ) .",
"Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 3 μg DNA total ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 00910 . 7554/eLife . 03778 . 010Figure 3—figure supplement 2 . The AMOT p130 isoform specifically stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT , exogenous p80 and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , AMOT p80 ( lanes 2 and 5 ) , AMOT p130 ( lanes 3 and",
"6 ) and FLAG-NEDD4L ( lanes 4–6 ) .",
"Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 2 . 5 μg DNA total ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 010 As seen previously , FLAG-NEDD4L overexpression also stimulated HIV-1ΔPTAP , ΔYP release ( Figure 3 , right panel 1 , compare lanes 1 and 3 , 3 . 2-fold stimulation ) and infectivity ( right panel 2 , compare lanes 1 and 3 , 5 . 3-fold stimulation ) , and also largely rescued the HIV-1ΔPTAP , ΔYP Gag processing defects ( left panel 4 , compare lane 3 to lanes 1 and 2 , and note the reduction in p41 and p25 Gag processing intermediates ) ( Chung et al . , 2008; Usami et al . , 2008 ) .",
"Co-overexpression of both HA-AMOT p130 and FLAG-NEDD4L further increased HIV-1ΔPTAP , ΔYP release ( right panel 1 , lane 4 , sixfold increase over basal levels ) and infectivity ( right panel 2 , lane 4 , 14-fold increase over basal levels ) .",
"As shown in Figure 3—figure supplement 1 , the degree of hyper-stimulation correlated positively with AMOT p130 expression levels , until an inhibitory level was reached .",
"In contrast to wild type NEDD4L , the NEDD4LΔWW mutant stimulated HIV-1ΔPTAP , ΔYP release and infectivity only very modestly ( Figure 3 , right panels , compare lanes 5 and",
"3 ) and did not synergize with AMOT p130 ( right panels , compare lanes 6 and 5 ) .",
"Similarly , the shorter AMOT p80 isoform , which lacks the PPXY motifs found in the N-terminal domain of AMOT p130 , failed to synergize with NEDD4L in stimulating virus release and infectivity , and instead modestly reduced NEDD4L stimulation , probably by dominantly inhibiting endogenous AMOT p130 activity ( Figure 3—figure supplement 2 , compare lanes 4–6 ) .",
"These data indicate that AMOT p130 and NEDD4L cooperate in stimulating HIV-1ΔPTAP , ΔYP release and infectivity .",
"siRNA depletion experiments were performed to determine whether AMOT was also necessary for NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release and infectivity .",
"We used an siRNA that targeted both AMOT isoforms and reduced cellular AMOT p130 and p80 levels by at least fivefold ( Figure 4 , left panel 1 , compare lanes 1 and 2 and lanes 3 and 4 ) .",
"AMOT depletion reduced the already low levels of virus release and infectivity even further , to near background levels ( Figure 4 , right panels , compare lanes 1 and 2 , 7 . 5-fold infectivity reduction ) .",
"Cellular levels of Gag and a GAPDH control were unaffected by this treatment ( left panels 3 and 4 , compare lanes 1 and 2 ) , although AMOT depletion reduced cellular protein levels slightly in some repetitions ( not shown ) . 10 . 7554/eLife . 03778 . 011Figure 4 . AMOT p130 is required for NEDD4L-stimulated release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were co-transfected with a control siRNA ( lanes 1 and",
"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–6 ) , with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–6 ) , and with FLAG-NEDD4L ( lanes 3–6 ) or an empty vector control ( lanes 1 and 2 ) , and with siRNA-resistant expression constructs for HA-AMOT p130 proteins ( with wild type HA-AMOT p130 in lane 5 and an HA-AMOT p130ΔPPXY protein carrying inactivating point mutations in the three PPXY motifs in lane 6 ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01110 . 7554/eLife . 03778 . 012Figure 4—figure supplement 1 . Dose-dependent AMOT p130 rescue of HIV-1 release from cells depleted of endogenous AMOT . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were co-transfected with a control siRNA ( lanes 1 and",
"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–10 ) , with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–10 ) , for FLAG-NEDD4L ( lanes 3–10 ) , and with increasing quantities of an siRNA-resistant AMOT p130 construct ( lanes 5–10 , 0 . 25–1 . 5 μg DNA ) .",
"Cells were also co-transfected with empty vectors as necessary to keep total DNA levels constant ( 3 μg DNA total ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01210 . 7554/eLife . 03778 . 013Figure 4—figure supplement 2 . The AMOT p130 isoform specifically stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 or AMOT p80 proteins ( panel 1 , anti-AMOT ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 GagΔPTAP , ΔYP proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were transfected with a control siRNA ( lanes 1 and",
"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–7 ) .",
"Cells were co-transfected with expression vectors for HIV-1ΔPTAP , ΔYP ( lanes 1–7 ) , FLAG-NEDD4L ( lanes 3–7 ) , and siRNA-resistant expression constructs for AMOT p80 ( lane 5 ) , HA-AMOT p130 ( lane",
"6 ) or both ( lane 7 ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 013 AMOT depletion blocked the ability of NEDD4L to stimulate HIV-1ΔPTAP , ΔYP release and infectivity ( Figure 4 and Figure 4—figure supplements 1 , 2 ) .",
"In the control case , NEDD4L overexpression stimulated HIV-1ΔPTAP , ΔYP release ( Figure 4 , right panels , compare lanes 1 and 3 , 4 . 1-fold infectivity increase ) .",
"However , virus release was reduced to the levels seen in untreated control cells when endogenous AMOT p80 and p130 proteins were simultaneously depleted ( compare lanes 2–4 ) .",
"Similar results were observed for a second siRNA that targeted both AMOT isoforms ( not shown ) .",
"Thus , AMOT is required for NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release .",
"Rescue experiments with exogenous siRNA-resistant AMOT expression constructs were performed to confirm the specificity of the AMOT depletion and to test the requirements for AMOT function .",
"Re-expression of AMOT p130 completely rescued the block to virus release and infectivity imposed by AMOT depletion ( Figure 4 , right panels , compare lanes 5 and 4 ) , restoring viral infectivity to a level that was actually slightly higher than the control ( compare lanes 3 and 5 ) .",
"The degree of rescue generally correlated positively with AMOT p130 re-expression levels , and peaked at levels that approximated those of the native endogenous protein ( Figure 4—figure supplement 1 , lane 8 ) .",
"In contrast , an AMOT p130 protein that lacked the three N-terminal PPXY motifs failed to rescue NEDD4L-dependent HIV-1ΔPTAP , ΔYP release ( Figure 4 , AMOT p130ΔPPXY , right panels , compare lanes 6 and 5 ) .",
"Similarly , re-expression of an siRNA-resistant AMOT p80 construct also failed to rescue virus release and infectivity ( Figure 4—figure supplement 2 , right panels , compare lanes 4 and 5 ) , unless exogenous AMOT p130 was present ( Figure 4—figure supplement 2 , right panels , lane 7 ) .",
"Hence , NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release and infectivity requires the presence of an AMOT p130 protein that can bind NEDD4L , but is not significantly affected by AMOT proteins that cannot bind NEDD4L , including AMOT p80 .",
"siRNA depletion and rescue experiments were also performed to test whether AMOT p130 is required for release and infectivity of wild type HIV-1 .",
"In control experiments , depletion of either of the well-characterized HIV-1 budding factors , ALIX and TSG101 , reduced virus release and infectivity , although the effects were much greater for TSG101 depletion , as reported previously ( Figure 5 , right panels , compare lanes 2 and 3 to lane",
"1 ) ( Fujii et al . , 2009 ) .",
"AMOT depletion also significantly reduced both HIV-1 release and infectivity ( Figure 5 , right panels , compare lane 4 to lane 1 , eightfold reduction in infectivity and 2 . 5-fold reduction in release ) .",
"The magnitudes of these effects were intermediate between those seen for depletion of TSG101 ( 20-fold reduction in infectivity and threefold reduction in virion release ) and ALIX ( 1 . 5-fold reduction in infectivity and insignificant reduction in virion release ) .",
"Importantly , the detrimental effects of AMOT p130 depletion were almost completely rescued by re-expression of wild type AMOT p130 ( compare lanes 1 , 4 and 5 ) but not by the mutant AMOT p130ΔPPXY protein ( lane 6 ) .",
"These experiments demonstrate that AMOT p130 is required for efficient release of wild type HIV-1 from 293T cells and mutations that abolish NEDD4L binding block the ability of AMOT p130 to function in virus release . 10 . 7554/eLife . 03778 . 014Figure 5 . AMOT p130 is required for efficient release of wild type HIV-1 . Left panels are western blots showing 293T cellular levels of endogenous ALIX ( panel 1 , anti-ALIX ) , TSG101 ( panel 2 , anti-TSG101 ) , AMOT ( panel 3 , anti-AMOT ) , GAPDH ( panel 4 , anti-GAPDH , loading control ) and levels of HIV-1 Gag proteins ( panel 5 , anti-MA and anti-CA ) .",
"Cells were co-transfected with a control siRNA ( lane",
"1 ) or with an siRNA that depleted ALIX ( lane 2 ) , TSG101 ( lane",
"3 ) or AMOT p130 and p80 ( lanes 4–6 ) , with expression vectors for wild type HIV-1 ( lanes 1–6 ) and with an siRNA-resistant expression constructs for HA-AMOT p130 proteins ( with wild type HA-AMOT p130 in lane 5 and an HA-AMOT p130ΔPPXY protein with inactivating point mutations in all three PPXY motifs in lane 6 ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 014 AMOT mRNA is expressed in 293T cells , as well as in white blood cells and lymphoid tissues that are the natural hosts for HIV-1 infection ( Moleirinho et al . , 2014 ) .",
"In contrast , HeLa cells reportedly express little or no AMOT mRNA , although they do express AMOTL2 mRNA ( Moleirinho et al . , 2014 ) .",
"We confirmed that endogenous AMOT protein levels are at least 10-fold lower in HeLa cells than in 293T cells ( Figure 6—figure supplement 1 ) .",
"This observation begs the question of how HIV-1 can be released from a cell type that lacks AMOT , and this issue is particularly relevant because HeLa cells are commonly used to study HIV-1 assembly .",
"We therefore tested the effects of AMOT p130 expression on HIV-1 release and infectivity in HeLa cells .",
"As shown in Figure 6A , AMOT p130 expression increased HIV-1 release from HeLa cells in a dose-dependent fashion , up to 15-fold at the highest levels of AMOT tested ( right panel 1 , compare lanes 1 and 5 ) .",
"Viral titers similarly increased with AMOT expression in a dose-dependent fashion , although the overall effects were less dramatic ( right panel 2 , compare lanes 1–5 , threefold infectivity increase ) .",
"Thus , HIV-1 release from wild type HeLa cells is suboptimal , and can be enhanced by expression of AMOT p130 . 10 . 7554/eLife . 03778 . 015Figure 6 . AMOT p130 stimulates release of HIV-1 from HeLa and Jurkat T cells .",
"( A ) Left panels are western blots showing HeLa cellular levels of endogenous AMOT p130 and exogenous HA-AMOT p130 ( panel 1 , anti-AMOT ) , endogenous GAPDH ( panel 2 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 3 , anti-MA and anti-CA ) .",
"HeLa cells were co-transfected with expression vectors for HIV-1 ( lanes 1–5 ) , and with increasing concentrations of an expression construct for HA-AMOT p130 ( 0–4 μg DNA in lanes 1–5 ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 .",
"( B ) Levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) .",
"Jurkat T cells were co-transfected with expression vectors for HIV-1 ( lanes 1–5 ) , together with a control vector ( lane",
"1 ) or expression vectors for wild type HA-AMOT p130 ( lanes 2 and 3 , 5 and 10 µg DNA respectively ) or an HA-AMOT p130ΔPPXY protein with inactivating point mutations in the three PPXY motifs ( lanes 4 and 5 , 5 and 10 µg DNA respectively ) , n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01510 . 7554/eLife . 03778 . 016Figure 6—figure supplement 1 . HeLa cells express little or no AMOT . Western blots showing endogenous AMOT p130 and p80 expression levels in 293T cells ( lanes 1–4 ) and in HeLa cells ( lanes 5–8 ) .",
"Increasing numbers of cell equivalents were added as indicated ( ranging from 1 . 5 × 105 cells in lanes 1 and 5 to 1 . 2 × 106 cells in lanes 4 and 8 ) .",
"Upper and lower panels show lighter and darker exposures of the same blot .",
"Image is representative of two independent repetitions of this experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 016 T cells are natural hosts for HIV-1 infection , and AMOT mRNA is present in these cells , including Jurkat T cells ( Yeung et al . , 2009; Sheynkman et al . , 2013 ) .",
"To determine whether AMOT p130 can function in virus release from Jurkat T cells , we tested whether expressing exogenous AMOT p130 expression stimulated HIV-1 release and infectivity .",
"As shown in Figure 6B , AMOT p130 expression increased HIV-1 release from Jurkat T cells in a dose-dependent fashion , with up to 16-fold stimulation observed at the highest levels of AMOT p130 tested ( panel 1 , compare lanes 1–3 ) .",
"Viral titers similarly increased in a dose-dependent fashion ( panel 2 , compare lanes 1–3 , sevenfold infectivity increase ) .",
"Western blots confirmed that virus expression levels were the same in all cases ( not shown ) , but AMOT p130 expression levels were below our detection limits in this cell type .",
"We therefore also tested the effects of overexpressing the mutant AMOT p130ΔPPXY protein .",
"Unlike wild type AMOT p130 , AMOT p130ΔPPXY failed to stimulate virus release , and actually reproducibly decreased viral titers as compared to the untreated control ( compare lanes 4 and 5 to lane 1 ) .",
"We speculate that this effect again reflects dominant inhibition of endogenous AMOT p130 .",
"Regardless , our data indicate that AMOT p130 also stimulates virus release and infectivity from T cells , which are natural targets of HIV-1 infection .",
"Consistent with this conclusion , a global shRNA screen for host cofactors revealed that Jurkat T cells transduced with an shRNA that targeted AMOT were protected against HIV cytotoxicity ( Yeung et al . , 2009 ) .",
"This observation implies that AMOT contributes to HIV-1 replication in cultured Jurkat T cells , although the role of AMOT in HIV-1 replication was not characterized further in that study .",
"( Yeung et al . , 2009 ) .",
"siRNA depletion/rescue experiments were performed to test whether other motin family members can substitute for AMOT p130 in supporting release of wild type HIV-1 ( Figure 7 ) .",
"In control experiments , HIV-1 release and infectivity were again decreased by depletion of endogenous AMOT from 293T cells ( right panels , compare lanes 1 and 2 , fivefold infectivity reduction ) .",
"As expected , these effects were largely rescued by expression of exogenous siRNA-resistant AMOT p130 ( right panels , compare lanes 2 and 3 ) .",
"Expression of either AMOTL1 or AMOTL2 also significantly rescued HIV-1 release and infectivity ( right panels , compare lanes 4 and 5 to lanes 2 and 3 ) .",
"Hence , both AMOT-like proteins can facilitate HIV-1 release in the absence of endogenous AMOT p130 .",
"Neither AMOTL1 nor AMOTL2 was quite as effective as AMOT p130 in rescuing HIV-1 infectivity in this assay , possibly because these proteins are intrinsically less active than AMOT p130 and/or because they were expressed at lower levels ( left panel 2 , compare lanes 3–5 ) . 10 . 7554/eLife . 03778 . 017Figure 7 . AMOTL1 and AMOTL2 can substitute for AMOT p130 in HIV-1 release . Left panels are western blots showing 293T cellular levels of endogenous AMOT and exogenous HA-AMOT p130 proteins ( panel 1 , anti-AMOT ) , exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 proteins ( panel 2 , anti-HA ) , endogenous GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were co-transfected with a control siRNA ( lane",
"1 ) or with an siRNA that depleted AMOT p130 and AMOT p80 ( lanes 2–5 ) , and with expression vectors for HIV-1 ( lanes 1–5 ) , with siRNA-resistant expression constructs for AMOT p130 ( lane 3 ) , AMOTL1 ( lane 4 ) or AMOTL2 ( lane 5 ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01710 . 7554/eLife . 03778 . 018Figure 7—figure supplement 1 . AMOTL2 can stimulate NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP and rescue HIV-1ΔPTAP , ΔYP release from cells depleted of endogenous AMOT .",
"( A ) AMOTL2 stimulates NEDD4L-dependent release of HIV-1ΔPTAP , ΔYP .",
"Left panels are western blots showing 293T cellular levels of exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 proteins ( panel 1 , anti-HA ) , exogenous FLAG-NEDD4L ( panel 2 , anti-FLAG ) , GAPDH ( panel 3 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 4 , anti-MA and anti-CA ) .",
"Cells were co-transfected with expression vectors for HIV-1ΔPTAP/ΔYP ( lanes 1–8 ) , FLAG-NEDD4L ( lanes 5–8 ) , and either HA-AMOT p130 ( lanes 2 and 6 ) , HA-AMOTL1 ( lanes 3 and",
"7 ) or HA-AMOTL2 ( lanes 4 and 8 ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel ) and viral titers ( lower panel ) , n = 3 .",
"( B ) AMOTL2 can rescue HIV-1ΔPTAP , ΔYP release from 293T cells depleted of endogenous AMOT .",
"Left panels are western blots showing cellular levels of endogenous AMOT and exogenous HA-AMOT proteins ( panel 1 , anti-AMOT ) , exogenous HA-AMOT p130 , HA-AMOTL1 or HA-AMOTL2 ( panel 2 , anti-HA ) , exogenous FLAG-NEDD4L ( panel 3 , anti-FLAG ) , GAPDH ( panel 4 , anti-GAPDH , loading control ) and HIV-1 Gag proteins ( panel 5 , anti-MA and anti-CA ) .",
"Cells were co-transfected with a control siRNA ( lanes 1 and",
"3 ) or with an siRNA that depleted both AMOT p130 and p80 ( lanes 2 and 4–7 ) , with expression vectors for HIV-1 ( lanes 1–7 ) , FLAG-NEDD4L ( lanes 3–7 ) , and either an siRNA-resistant HA-AMOT p130 expression construct ( lane 5 ) , or expression constructs for HA-AMOTL1 ( lane 6 ) , or HA-AMOTL2 ( lane 7 ) .",
"Right panels show corresponding levels of extracellular virion-associated Gag proteins ( upper panel , western blot , anti-MA and anti-CA ) and viral titers ( lower panel ) , n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 018 We also tested whether the other AMOT family members could cooperate with NEDD4L in stimulating release of the crippled HIV-1ΔPTAP , ΔYP virus ( Figure 7—figure supplement 1A ) and whether AMOTL1 and AMOTL2 could rescue the block to NEDD4L stimulation of HIV-1ΔPTAP , ΔYP release imposed by depletion of endogenous AMOT ( Figure 7—figure supplement 1B ) .",
"AMOTL2 strongly stimulated/rescued HIV-1ΔPTAP , ΔYP release in both of these assays , albeit slightly less efficiently than AMOT p130 ( Figure 7—figure supplement 1A , compare lane 8 to lanes 5 and 6 and Figure 7—figure supplement 1B , compare lane 7 to lanes 4 and 5 ) .",
"In contrast , AMOTL1 exhibited little or no activity in either assay ( Figure 7—figure supplement 1A , compare lane 7 to lanes 5 and 6 and Figure 7—figure supplement 1B , compare lane 6 to lanes 4 and 5 ) .",
"Thus , AMOTL2 could substitute for AMOT p130 in three different virus release assays , whereas AMOTL1 was slightly less active than AMOT in the release of wild type HIV-1 , and failed to stimulate release of the ‘crippled’ HIV-1ΔPTAP , ΔYP construct .",
"Scanning and transmission electron microscopy ( SEM and TEM , respectively ) were used to visualize the stage at which AMOT depletion inhibited HIV-1 assembly or release ( Figures 8 , 9 ) .",
"SEM imaging was initially performed because this method can survey the entire cell surface and thereby identify global changes in virion assembly and budding profiles .",
"SEM analyses were performed on 293T cells that expressed HIV-1 and were treated with either: ( 1 ) a control siRNA , ( 2 ) an siRNA that depleted TSG101 ( positive control ) , or ( 3 ) an siRNA that depleted AMOT .",
"As expected , viruses budding from control siRNA-treated cells were detectable but rare ( e . g . , Figure 8A–C ) .",
"In contrast , large numbers of assembling virions were visible on the surfaces of cells that lacked TSG101 ( Figure 8D–F ) or that lacked AMOT ( Figure 8G–I ) .",
"We also observed that the arrested virions tended to cluster in the center of cells lacking AMOT , whereas they were more evenly distributed across the surfaces of cells lacking TSG101 .",
"The dramatic increase in the numbers of arrested budding virions implies that AMOT , like TSG101 , must function following the initial stages of Gag assembly , but prior to viral membrane fission . 10 . 7554/eLife . 03778 . 019Figure 8 . AMOT p130 is required for HIV-1 assembly/budding . Scanning electron microscopic ( SEM ) images of HIV virions budding from 293T cells depleted with a control siRNA ( A–C ) , depleted of TSG101 ( D–F ) or depleted of AMOT ( G–I ) .",
"Successive images across each row show expansions of the adjacent boxed regions , and scale bars are 500 nm in all cases .",
"Arrows in panel ( C ) highlight budding wild type HIV-1 virions . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 01910 . 7554/eLife . 03778 . 020Figure 9 . AMOT p130 is required for an early stage of HIV-1 assembly/budding .",
"( A ) Transmission electron microscopic ( TEM ) images of HIV virions budding from 293T cells depleted of TSG101 .",
"Panel 1: wide view .",
"Panel 2: expanded view .",
"Scale bars here and in part B are 100 nm .",
"( B ) TEM images of HIV virions budding from 293T cells depleted of AMOT p130 and p80 .",
"Panel 1: wide view .",
"Panel 2: expanded view .",
"( C ) Quantification of the release and maturation status of HIV-1 virions associated with cells that were treated with a control siRNA ( black bars ) , depleted of TSG101 ( red ) , depleted of AMOT ( teal ) , or depleted of AMOT and re-expressing AMOT p130 from an siRNA-resistant construct ( light blue ) .",
"This experiment was repeated twice with similar results , and error bars were derived by quantifying three separate sets of >100 virions each from one of the experiments .",
"( D ) Quantification of the completeness of virions budding from cells treated with a control siRNA ( black triangles ) , depleted of TSG101 ( red squares ) , or depleted of AMOT ( teal diamonds ) .",
"The extent of the Gag shell arc ( in degrees ) was measured from TEM images of 500 budding virions of each type , the measurements were binned into the intervals shown below the x-axis , and the virion numbers in each bin are shown in the plot . DOI: http://dx . doi . org/10 . 7554/eLife . 03778 . 020 TEM experiments were performed to characterize how virus assembly/budding arrested in the absence of AMOT .",
"These experiments again visualized HIV-1 budding from 293T cells treated with a control siRNA , an siRNA that depleted TSG101 ( positive control ) , an siRNA that depleted AMOT , or an siRNA that depleted endogenous AMOT together with an siRNA-resistant construct that re-expressed wild type AMOT p130 ( rescue control ) .",
"Culture supernatants were removed from the virus producing cells and the adherent cells were fixed while still attached to the culture dish in order to maximize the numbers of cell-associated virions .",
"Fixed cells were harvested , embedded in resin , thin sectioned , stained , and visualized by TEM .",
"Cell-associated virions observed in these experiments were scored as being either: ( 1 ) mature ( discernable conical or condensed cores ) , ( 2 ) immature ( discernable immature Gag shells ) , or ( 3 ) budding ( contiguous cellular and viral membranes ) .",
"HIV-1 virions associated with wild type cells were usually mature ( 76 ± 5% , see Figure 9C ) , but immature and budding virions were also observed ( 9 ± 3% and 15 ± 5% , respectively ) .",
"As expected , TSG101 depletion induced a strong virus budding defect , with the majority of detected virions arrested at the budding stage ( 67 ± 7% , shown in Figure 9A and quantified in Figure 9C ) .",
"In addition , the ratio of mature ( 10 ± 6% ) to immature ( 23 ± 2% ) virions ( 1:2 . 3 ) was much lower than in the control case ( 1:0 . 1 ) , consistent with previous reports that TSG101 is also required for efficient Gag processing and maturation ( Göttlinger et al . , 1991; Garrus et al . , 2001; Martin-Serrano et al . , 2003 ) .",
"AMOT depletion also inhibited virus release , as reflected in a large increase in the percentage of virions detected at the budding stage ( 72 ± 6% , shown in Figure 9B and quantified in Figure 9C ) .",
"In this case , however , the virions that were released exhibited a normal ratio of mature:immature virions ( 25 ± 5% vs 3 ± 2%; 1:0 . 1 ) .",
"Importantly , the budding defect seen in cells lacking endogenous AMOT was almost completely rescued by re-expression of the siRNA-resistant AMOT p130 ( Figure 9C ) , so that the majority of virions were now mature ( 70 ± 5% vs 76 ± 5% in the control ) , and budding virion levels were nearly normal ( 26 ± 9% vs 15 ± 5% in the control ) .",
"Thus , AMOT depletion blocks virus release at the assembly/budding stage , but does not inhibit Gag processing or virion maturation .",
"It was also striking that AMOT depletion caused virion assembly/budding to arrest at an earlier stage than TSG101 depletion .",
"This difference can be seen in the EM images shown in Figure 9A , B , where most of the virions in TSG101-depleted cells exhibit the characteristic ‘lollipop’ morphology associated with a late assembly/membrane fission defect ( Figure 9A ) , whereas most of the virions budding from AMOT-depleted cells exhibit a ‘half-moon’ morphology in which the nascent viral membrane and Gag shell are incomplete ( Figure 9B ) .",
"This observation was quantified by imaging 500 budding HIV-1 virions from: ( 1 ) wild type cells ( control ) , ( 2 ) cells depleted of TSG101 , and ( 3 ) cells depleted of AMOT .",
"The arc formed by the Gag shell was measured manually for each budding virion , and the 500 measurements for each condition were then binned in 15° intervals and displayed graphically ( Figure 9D ) .",
"The Gag arc distribution of control virions budding from wild type cells exhibited two similarly sized peaks; one in which the Gag shells covered an arc of ∼155° , and another in which the Gag shells covered an arc of ∼320° .",
"These measurements indicate that virion assembly intermediates ( or possibly dead-end products ) appreciably accumulate at two different stages , suggesting that either stage can be rate limiting .",
"In cells lacking TSG101 , budding-arrested virions were much more prevalent and they typically arrested at very late stages of assembly , with the maximal population exhibiting a Gag shell arc of ∼320° .",
"In cells lacking AMOT , arrested virions were again prevalent but they arrested at a much earlier stage , with the maximal population exhibiting a Gag shell arc of ∼165° .",
"The dramatic increases in numbers of budding virions induced by depletion of TSG101 or AMOT can explain how the assembly phenotypes could distribute as single peaks in these cases , whereas virions budding from wild type cells ( which were much rarer ) distributed into two peaks .",
"Our experiments reveal that HIV budding arrests at an earlier stage in the absence of AMOT than in the absence of TSG101 , implying that AMOT acts at an earlier stage of virion morphogenesis ."
],
[
"We have identified Angiomotin ( AMOT ) as a host protein required for efficient HIV-1 release .",
"In the absence of AMOT , most virions arrested at a ‘half-moon’ stage in which a hemisphere of assembled Gag molecules distended the membrane , but did not form a spherical particle ( Figure 9 ) .",
"The AMOT depletion phenotype was distinct from the defect induced by the absence of ESCRT factors , where virus budding arrested at a ‘lollipop’ stage in which nearly complete immature virions remained tethered to the host cell via thin membrane stalks .",
"Thus , cellular factors facilitate ( at least ) two distinct steps in virion morphogenesis , with AMOT required to complete assembly/envelopment and the ESCRT machinery required for membrane fission .",
"HIV-1 virions budding from control cells accumulated at both stages , suggesting that both steps can be kinetic bottlenecks ( see Figure 9D and reference Ku et al . , 2013 ) .",
"The Gag lattice organizes the virion and undoubtedly helps to drive membrane envelopment because pure recombinant Gag proteins can form spherical particles in vitro ( Campbell et al . , 2001 ) .",
"In cells , however , Gag appears to require additional activities to bend the membrane , extrude the particle , and sever the bud neck .",
"The well-studied process of endocytic vesicle formation provides precedence for these additional requirements because clathrin alone can form spherical assemblies but nevertheless requires assistance from BAR domain-containing proteins to help bend membranes , and assistance from actin and dynamin to release the vesicle ( Weinberg and Drubin , 2012 ) .",
"It is intriguing that AMOT p130 also contains a candidate BAR-like domain ( Heller et al . , 2010 ) and can associate with F-actin ( Ernkvist et al . , 2006 , 2008; Chan et al . , 2013 ) .",
"The AMOT BAR-like domain can remodel membranes in vitro ( Heller et al . , 2010 ) , although it is not yet clear whether it functions as a classical or an inverse BAR domain .",
"This distinction is mechanistically significant because classical BAR domains stabilize positive membrane curvature whereas inverse BAR domains stabilize negative curvature ( Mim and Unger , 2012 ) , both of which are generated during enveloped virus assembly .",
"In principle , the BAR domain could mediate AMOT p130 recruitment at the half-moon stage , once the nascent viral membrane has reached the proper degree of curvature .",
"The BAR domain could also help drive the additional membrane curvature required to proceed to the lollipop stage .",
"Similarly , F-actin recruitment and polymerization could help ‘push’ the virion toward the lollipop stage , although several recent studies argue strongly against an essential role for F-actin in HIV-1 assembly ( Bharat et al . , 2014; Rahman et al . , 2014 ) .",
"Our experiments indicate that another important AMOT p130 function is to recruit NEDD4L to sites of virus budding .",
"In support of this idea , we have shown that: ( 1 ) the two proteins bind one another directly through interactions between the NEDD4L WW domains and PPXY elements present in the unique N-terminal region of the AMOT p130 isoform ( Figures 1 , 2 and references Wang et al . , 2012; Moleirinho et al . , 2014 ) , ( 2 ) AMOT p130 can recruit NEDD4L to cellular membranes , and this activity requires the NEDD4L WW-AMOT p130 PPXY interactions ( [Heller et al . , 2010] and our data not shown ) , ( 3 ) NEDD4L cannot stimulate release of ‘crippled’ HIV-1 constructs in the absence of AMOT ( Figure 4 ) , ( 4 ) NEDD4L mutants that cannot bind AMOT are also impaired in their ability to stimulate virus budding ( Figure 5 ) , ( 5 ) AMOT p80 and AMOT p130 PPXY mutants that cannot bind NEDD4L do not support virus budding ( Figures 3–6 ) and ( 6 ) recombinant AMOT p130 and HIV-1 Gag proteins can bind one another directly , albeit weakly in vitro ( Figure 2B ) , and could bind more robustly in cells owing to avidity and membrane topology effects .",
"Previous studies indicate that other retroviruses also employ distinct host factors to promote membrane envelopment and fission .",
"The clearest example is the D-type Betaretrovirus Mason-Pfizer Monkey Virus ( M-PMV ) , which forms spherical particles in the cytoplasm and then obtains its outer membrane and exits the cell by crossing the plasma membrane .",
"M-PMV Gag contains two distinct late assembly domains that function non-redundantly: a PPPY motif that binds NEDD4 ( and perhaps other NEDD4 family members ) , and a PSAP motif that binds TSG101/ESCRT-I ( Gottwein et al . , 2003 ) .",
"PPPY and PSAP mutations induce strikingly different phenotypes: mutation of the PPPY motif causes virions to stack up against the plasma membrane where they fail to initiate membrane envelopment whereas virions lacking the PSAP motif acquire an envelope , but accumulate at the lollipop stage or are released as chains of membrane-tethered particles .",
"These observations imply that Gag PPPY-NEDD4 complexes are required for early stages of membrane bending and envelopment , whereas Gag PSAP-ESCRT-I complexes recruit the ESCRT machinery to mediate membrane fission .",
"Like other family members , NEDD4 binds AMOT ( Moleirinho et al . , 2014 ) , and we therefore speculate that M-PMV may also recruit AMOT to facilitate membrane bending and particle envelopment .",
"This would imply that viruses can recruit NEDD4-motin complexes by binding either member of the complex .",
"The deltaretrovirus HTLV-I is another case where a PPXY motif appears to be required for an early step in particle envelopment and a PTAP motif may function at a later membrane fission step ( [Le Blanc et al . , 2002; Blot et al . , 2004; Heidecker et al . , 2004] , but also see reference [Wang et al . , 2004] ) .",
"Distinct roles for PPXY and PTAP motifs have not been delineated in other well-studied viruses that use both motifs , such as the gammaretrovirus Murine Leukemia Virus ( Yuan et al . , 2000 ) or the filovirus Ebola Virus ( Timmins et al . , 2003 ) .",
"Although many details of HIV-1 assembly and budding remain to be elucidated , our data are consistent with a stepwise pathway in which: ( 1 ) AMOT p130 is recruited at the half-moon stage of HIV-1 assembly , perhaps by binding cooperatively to Gag and to appropriately curved membranes , ( 2 ) AMOT p130 promotes further membrane curvature and recruits NEDD4L via PPXY-WW domain interactions , ( 3 ) NEDD4L builds Lys-63-linked ubiquitin chains on the viral Gag protein ( and/or other nearby substrates ) , ( 4 ) the juxtaposition of Gag late assembly domains and Lys-63-linked Ub chains creates high affinity binding sites for the early-acting ESCRT factors , TSG101 and ALIX , and ( 5 ) these factors then recruit the downstream ESCRT-III and VPS4 components required to effect membrane scission .",
"This model is attractive because: ( 1 ) our work shows that AMOT functions upstream of the ESCRT pathway , ( 2 ) AMOT appears to function as a NEDD4L adaptor ( see above ) , ( 3 ) ubiquitin transfer is required for efficient HIV-1 release ( Martin-Serrano , 2007 ) , ( 4 ) NEDD4L builds Lys-63-linked ubiquitin chains and can use HIV-1 Gag as a substrate ( Weiss et al . , 2010; Sette et al . , 2013 ) , and ( 5 ) both TSG101/ESCRT-I and ALIX have ubiquitin binding activities .",
"The ALIX activity is specific for Lys-63-linked ubiquitin chains ( Dowlatshahi et al . , 2012; Keren-Kaplan et al . , 2013; Pashkova et al . , 2013 ) , and ESCRT-I complexes also have multiple ubiquitin binding sites , although linkage specificity has not been observed ( Stefani et al . , 2011; Agromayor et al . , 2012; McCullough et al . , 2013 ) .",
"Cooperative binding to the late domains and ubiquitin could explain how ALIX and TSG101/ESCRT-I can be efficiently recruited , despite making intrinsically weak interactions with the p6Gag late assembly domains .",
"The proposed sequence of events could also provide a ‘timing’ mechanism for recruiting ( or activating ) these early-acting ESCRT proteins to function at a late stage of virion assembly , although the precise timing of early-acting ESCRT recruitment remains to be clarified because published reports differ on this issue ( Jouvenet et al . , 2011; Ku et al . , 2014 ) .",
"All enveloped viruses must bend and remodel membranes as they bud , and AMOT requirements may therefore be quite general .",
"Indeed , AMOTL1 has been shown to bind the M protein of PIV5 and to be required for the efficient , ubiquitin-dependent release of this virus ( Pei et al . , 2010 ) .",
"Motin family members therefore contribute to the assembly of at least two highly diverged enveloped viruses; the paramyxovirus PIV5 and the lentivirus HIV-1 .",
"This suggests that AMOT proteins , like the ESCRT machinery , may provide general activities required for the assembly and release of a variety of different enveloped viruses ."
],
[
"Expression constructs , siRNA sequences , and antibodies are described in detail in Supplementary file 1 .",
"Constructs for expressing wild type and mutant NEDD4L and AMOT proteins in E . coli , insect SF-9 cells , and human 293T cells were created by standard cloning and mutagenesis methods , with detailed methods available upon request .",
"All of the new expression constructs used in our studies have been deposited in the public DNASU plasmid repository ( http://dnasu . org/DNASU/Home . jsp ) .",
"For the experiment shown in Figure 1B , an empty vector control or a pCAG-OSF-NEDD4L expression vector ( Morita et al . , 2007 ) expressing NEDD4L with an N-terminal One-STrEP-FLAG ( OSF ) affinity tag was transfected into 293T cells ( calcium phosphate method , 2 × 10 cm plates each , 25 μg DNA/plate ) .",
"After 48 hr , cells from each sample were collected by scraping , pooled , washed three times with PBS ( 1 . 5 mM KH2PO4 , 155 mM NaCl , 2 . 7 mM Na2HPO4 , pH 7 . 4 ) , and lysed with 500 μl Triton lysis buffer ( 1% Triton X-100 , 50 mM Tris pH 8 . 0 , 150 mM NaCl , 150 μg/ml PMSF ) .",
"This , and all subsequent preparation steps , were performed at 4°C .",
"Soluble extracts were pre-incubated for 30 min with 5 μl Protein-A resin slurry ( Millipore , Temecula , CA ) to remove non-specific binding proteins .",
"Supernatants were collected and incubated for 2 hr with 10 μl STrEP-Tactin resin ( IBA-Lifesciences , Göttingen , Germany ) .",
"The resins were washed three times with 500 μl washing buffer ( 0 . 1% Triton-X100 , 50 mM Tris , 150 mM NaCl , pH 8 . 0 ) , resuspended in 35 μl SDS-PAGE loading buffer ( 2% SDS , 10% glycerol , 2% 2-mercaptoethanol ( BME ) , 0 . 002% bromophenol blue and 62 . 5 mM Tris , pH 6 . 8 ) , and boiled to release the bound proteins .",
"12 μl samples of each solution were electrophoresed ( 10% SDS-PAGE ) and proteins were visualized by staining with Coomassie brilliant blue .",
"This procedure was repeated in three independent experiments , and in each case the protein band corresponding to AMOT p130 was visible in the Coomassie-stained gel ( Figure 1B ) .",
"This band was excised ( and the equivalent region from the control lane was also excised in one of the repetitions ) , de-stained in 50% methanol with 50 mM ammonium bicarbonate ( 2 × 1 ml , 1 hr , 23°C , gentle vortexing ) , re-hydrated in 50 mM ammonium bicarbonate ( 1 ml , 30 min , 23°C ) , and cut into several pieces .",
"Each piece was dehydrated in acetonitrile ( 1 ml , 30 min , 23°C , gentle shaking ) , and the excess acetonitrile was removed .",
"Proteins were in-gel digested using 10–20 μl sequence-grade modified trypsin ( Promega , Madison , WI , 20 ng/μl ) in 50 mM ammonium bicarbonate ( 16 hr , 37°C ) , and the reaction was quenched by the addition of 20 μl 1% formic acid .",
"The solution was removed and the gel washed twice with 50% acetonitrile in 1% formic acid ( 20 μl , 20 min , 37°C , with sonication ) and once with 100% acetonitrile ( 20 min , 37°C , with sonication ) .",
"The wash solutions were combined , dried , and reconstituted in 100 μl 5% acetonitrile with 0 . 1% formic acid for LC/MS/MS analysis .",
"Peptides were analyzed using a nano-LC/MS/MS system comprising a nano-LC pump ( 2D-ultra , Eksigent ) and an LTQ-FT mass spectrometer ( ThermoElectron Corporation , San Jose , CA ) , equipped with a nanospray ion source ( ThermoElectron Corporation ) .",
"5–20 fmoles of each tryptic digest were injected onto a homemade C18 nanobore LC column ( C18 [Atlantis , Waters Corporation , Milford , MA] ) ; 3 μm particle; column ( 75 μm ID × 100 mm length ) , and eluted using a linear gradient of 5–70% solvent B in 78 min ( solvent B: 80% acetonitrile with 0 . 1% formic acid; solvent A: 5% acetonitrile with 0 . 1% formic acid , 350 nl/min ) .",
"The LTQ-FT mass spectrometer was operated in the data-dependent acquisition mode ( Xcalibur 1 . 4 software ) with the 10 most intense peaks in each FT primary scan determined on-the-fly and trapped for MS/MS analysis and CID peptide fragmentation/sequencing in the LTQ linear ion trap .",
"Spectra in the FT-ICR were acquired from m/z 350 to 1400 at 50 , 000 resolution ( ∼2 ppm mass accuracy ) with the following LTQ linear ion trap parameters: precursor activation time 30 ms and activation Q at 0 . 25; collision energy at 35%; dynamic exclusion width at 0 . 1 Da–2 . 1 Da , with one repeat count and 10 s duration .",
"Raw LTQ FT MS data files were converted to peak lists with BioworksBrowser 3 . 2 software ( ThermoElectron Corporation ) using the following processing parameters: precursor mass 351–5500 Da; grouping enabled , 5 intermediate MS/MS scans; precursor mass tolerance 5 ppm , minimum ion count in MS/MS = 15 , and minimum group count = 1 .",
"Resulting DTA files were merged , and searched to identify peptides/proteins against NCBInr using the MASCOT search engine ( Matrix Science Ltd . ; version 2 . 2 . 1 , Boston , MA ) .",
"Searches were done with tryptic specificity , allowing two missed cleavages , or ‘non-specific cleavage’ and a mass error tolerance of 5 ppm in MS spectra ( i . e . , FT-ICR data ) and 0 . 5 Da for MS/MS ions ( i . e . , LTQ linear ion trap ) .",
"Identified peptides were accepted when the MASCOT ion score value exceeded 20 .",
"In all three repetitions , at least two peptides corresponding to AMOT p130 were identified in the tryptic digest , and the maximal coverage was 33% ( see Figure 1—figure supplement 1 ) .",
"To assay OSF-NEDD4L protein binding to endogenous AMOT p130 ( Figure 1C ) , 293T cells ( 2 × 106 cells per 10 cm plate ) were transfected using Lipofectamine 2000 ( Invitrogen/Life Technologies , Carlsbad , CA ) with either 3 . 5 μg of an empty pCAG-OSF expression vector ( control ) or with pCAG-OSF vectors that expressed the designated OSF-NEDD4L proteins , using 3 . 5 μg of expression constructs for wild type NEDD4L or NEDD4LΔWW , or 2 μg each of expression constructs for NEDD4LC942A or NEDD4LΔWW/C942A .",
"In this case , and in all other experiments , empty vector was added to keep total DNA levels constant ( i . e . , 1 . 5 μg empty pCAG-OSF vector in the case of NEDD4LC942A or NEDD4LΔWW/C942A ) .",
"48 hr post transfection , cells were washed in PBS buffer and lysed ( 25 mM Tris , 150 mM NaCl , 1 mM EDTA , 0 . 5% CHAPS , pH 7 . 5 , 10 min , 4°C ) .",
"Soluble lysates were collected by centrifugation ( 5000×g , 5 min , 4°C ) and incubated with 50 μl STrEP-Tactin resin ( IBA-Lifesciences , Göttingen , Germany , 3 hr , 4°C ) equilibrated with lysis buffer .",
"The resin was washed ( 25 mM Tris-HCl , 150 mM NaCl , 1 mM EDTA , 0 . 1% CHAPS , pH 7 . 5 , 3 × 1 ml ) , resuspended in 50 μl 2× SDS-PAGE loading buffer , boiled , and the released proteins were analyzed by SDS-PAGE and western blotting .",
"Separated proteins were transferred to PVDF membranes , blocked with 5% non-fat dry milk for 45 min at RT , and incubated overnight at 4°C with primary antibodies ( see Supplementary file 1 ) .",
"Secondary antibodies were anti-rabbit IgG or anti-mouse IgG conjugated to IRdye800 or IRdye700 , respectively ( 1:10 , 000 and 1:20 , 000 respectively , Rockland Immunochemicals Inc . , Gilbertsville , PA ) .",
"All western blots were visualized using an Odyssey scanner ( Li-Cor Biosciences , Lincoln , NB ) .",
"To assay OSF-NEDD4L protein binding to exogenous HA-AMOT p130 proteins ( Figure 1—figure supplement 2 ) , 293T cells ( 2 × 106 cells per 10 cm plate ) were co-transfected using Lipofectamine 2000 ( Invitrogen/Life Technologies ) with 3 . 5 μg of a pCAG-OSF vector expressing wild type OSF-NEDD4L and 3 . 5 μg of an empty pCAG vector ( control ) , or pcDNA vectors expressing HA-AMOT p130 or HA-AMOT p130ΔPPXY .",
"Thereafter , the co-immunoprecipitation protocol was identical to the one described above .",
"Proteins were expressed in the Rosetta-pLysS bacterial strain grown in auto-induction media ZYP-5052 ( Studier , 2005 ) .",
"For the experiments described in Figure 2 and Figure 2—figure supplement 1 , SF9 cells ( 2 l ) were infected at an MOI of 5 with Bac-to-Bac baculoviruses expressing OSF-tagged GFP ( control ) , AMOT p130 , AMOTL1 or AMOTL2 .",
"Pellets from 200 ml of culture were lysed in 10 ml of lysis buffer ( 1% NP-40 , 150 mM NaCl , 20 mM Tris pH 8 . 0 , 0 . 5 mM EDTA supplemented with protease inhibitors ) .",
"All steps were performed at 4°C .",
"Supernatants were collected by centrifugation for 30 min at 13 , 000 RPM in a microcentrifuge and 0 . 5 ml of each lysate was incubated with 30 μl of a slurry of STrEP-Tactin resin ( 1 hr ) .",
"Resins were washed with high salt wash buffer ( 20 mM Tris , 1 M LiCl , 0 . 5 mM EDTA , 0 . 1% NP-40 , pH 8 . 0 ) , followed by low salt buffer ( 20 mM Tris , 150 mM NaCl , 0 . 5 mM EDTA , 0 . 1% NP-40 , pH 8 . 0 ) .",
"For NEDD4L binding experiments , the OSF-GFP or OSF-AMOT-bound resins were washed with binding buffer ( 150 mM NaCl , 50 mM Tris , 1 mM TCEP , 10 μM ZnCl2 , 0 . 1% NP-40 , pH 7 . 5 ) .",
"Pure recombinant NEDD4L or NEDD4LΔWW proteins ( 0 . 5 μM in 500 μl binding buffer ) were pre-incubated with washed resins for 1 hr , and then incubated for 1 hr with GFP- or AMOT-bound resins .",
"Resins were washed three times with 250 μl binding buffer , resuspended in 30 μl SDS-PAGE loading buffer , boiled , separated by SDS-PAGE , and visualized by Coomassie staining or by Western blotting ( with 1 μl samples used for Coomassie staining , and 1 μl samples of a 50-fold dilution used for Western blotting ) .",
"For GagΔp6 binding experiments , the OSF-GFP ( control ) - or OSF-AMOT-bound resins were washed with binding buffer .",
"Pure recombinant HIV-1 GagΔp6 ( 1 μM ) in 500 μl binding buffer was incubated with the washed resins for 1 hr .",
"Resins were washed with binding buffer , and processed as described above .",
"NEDD4L and AMOT protein family co-overexpression experiments shown in Figure 3 and Figure 3—figure supplement 1 , Figure 3—figure supplement 2 , and Figure 7—figure supplement 1A were performed following the protocol: 293T cells ( 4 × 105 cells/well in a 6-well plate ) were co-transfected ( Lipofectamine , 2000; Invitrogen/Life Technologies ) with 1 μg of an R9-based expression vector for the NL4-3 strain of HIV-1ΔPTAP , ΔYP ( Swingler et al . , 1997 ) , 0 . 5 μg of pCI-FLAG NEDD4L or NEDD4LΔWW ( Chung et al . , 2008 ) or pCI-FLAG empty vector in combination with either 0 . 5 μg of pCMV AMOT p80 ( Figure 3—figure supplement 2 ) or 1 μg pcDNA3 HA-AMOT p130 ( Figure 3 and Figure 3—figure supplement 2 ) or 0 , 0 . 25 , 0 . 5 , 0 . 75 , 1 , 1 . 25 or 1 . 5 μg pcDNA3 HA-AMOT p130 , ( Figure 3—figure supplement",
"1 ) or 1 μg pcDNA3 HA AMOTL1 or 1 μg pcDNA3 HA AMOTL2 ( Figure 7—figure supplement 1A ) .",
"48 hr post-transfection , media was harvested for titer measurements and virus purification , and cells were washed off the plate in PBS .",
"Viral titers were assayed in HeLa-TZM indicator cells using a β-galactosidase assay , and following the manufacturer's instructions ( Promega , Madison , WI ) .",
"Briefly , HeLa-TZM cells ( 5000 cells per well , 96-well plate ) were infected with four different dilutions of virus-containing culture media , harvested after 48 hr , washed once ( PBS , 4°C ) , and lysed in 25 mM Tris phosphate ( pH 7 . 8 ) , 2 mM DTT , 2 mM 1 , 2-diaminocyclohexane N , N , N′ , N′-tetraacetic acid ( DCTA ) , 10% glycerol and 1% Triton X-100 ( 10 min , 23°C ) .",
"Cell lysates were incubated in 200 mM sodium phosphate buffer ( pH 7 . 3 ) , 2 mM MgCl2 , 100 mM BME and 1 . 33 mg/ml ortho-Nitrophenyl-β-galactoside for 60 min at 37°C .",
"Reactions were terminated by addition of 200 μl 1 M Tris base and absorbance at 420 nm was read in a plate reader .",
"For western blot analyses , virions were pelleted by centrifugation through a 20% sucrose cushion ( 90 min , 15 , 000×g , 4°C ) and resuspended in SDS-PAGE loading buffer .",
"Cells were washed in PBS , lysed in RIPA buffer ( 10 mM Tris , 1 mM EDTA , 0 . 5 mM EGTA , 1% Triton X-100 , 0 . 1% sodium deoxycholate , 0 . 1% SDS , 140 mM NaCl , pH 8 . 0 ) supplemented with protease inhibitors ( cOmplete , Roche ) ( 10 min , 4°C ) , and the insoluble material was removed by centrifugation ( 10 min , 15 , 000×g , 4°C ) .",
"Primary antibodies and dilutions used for western blots are given in Supplementary file 1 .",
"Depletion/re-expression experiments ( Figure 4 , Figure 4—figure supplement 1 , Figure 4—figure supplement 2 , Figure 5 , Figure 7 , and Figure 7—figure supplement",
"1 ) were performed following the protocol: t = 0: 293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were transfected with 10 nM siRNA using 7 . 5 μl Lipofectamine RNAiMax ( Invitrogen/Life Technologies ) ; t = 24 hr: media changed and second siRNA co-transfection with 10 nM siRNA and 0 . 5 μg wild type HIV-1 R9 expression vector or 1 μg HIV-1ΔPTAP , ΔYPXL expression vector and 0 . 5 μg of pCI-FLAG NEDD4L ( 10 μl Lipofectamine , 2000; Invitrogen/Life Technologies ) ; t = 72 hr: media and cells harvested for titer measurements and western blot analyses as described above .",
"For the rescue experiments , siRNA-resistant expression constructs expressing AMOT p80 , AMOT p130 , AMOT p130ΔPPXY , AMOTL1 or AMOTL2 were co-transfected at t = 24 hr .",
"AMOT mRNA is expressed in 293T cells and in white blood cells and lymphoid tissues , but cannot be detected in HeLa cells ( Moleirinho et al . , 2014 ) .",
"Western blots were performed to compare AMOT protein expression levels in 293T vs HeLa cells ( Figure 6—figure supplement 1 ) .",
"293T ( 2 x 106 cells/10 cm plate ) and HeLa M ( 1 . 2 x 106 cells/10 cm plate ) cells were seeded , harvested 72 hr later by trypsin treatment , washed twice in PBS , counted , lysed in RIPA buffer ( 1 . 2 × 105 cells/μl ) for 15 min at 4°C , clarified by centrifugation ( 15 min , 15 , 000×g , 4°C ) and diluted 1:1 with 2× SDS loading buffer .",
"2 . 5 , 5 , 10 and 20 μl of each cell lysate mixture was loaded on a 4–15% gradient gel and AMOT proteins were detected by western blotting .",
"Experiments to test whether AMOT overexpression can stimulate HIV-1 release from HeLa cells ( Figure 6A ) were performed as follows: HeLa M cells ( 2 . 5 × 105 cells/well in a 6-well plate ) were co-transfected with 0 . 5 μg of wild-type R9 HIV-1 and 0 . 5 , 1 , 2 or 4 μg of pcDNA3-HA AMOT p130 expression vectors .",
"Cells and viruses were harvested 48 hr post-transfection .",
"To test whether AMOT could stimulate virus release from physiologically relevant T cells , AMOT was overexpressed in Jurkat T cells as follows: t = 0 , cells were harvested , washed in PBS and resuspended in Neon resuspension buffer R ( Invitrogen/Life Technologies ) .",
"5 × 106 cells were combined with 3 μg of the wild type R9 HIV-1 expression vector and 0 , 5 or 10 μg of pcDNA3-HA AMOT p130 or AMOT p130ΔPPXY expression vectors .",
"Cells were then electroporated in a 100 µl tip with the Neon Transfection System using three 10 ms pulses of 1325 V . Following electroporation , each sample was immediately transferred to a 10 cm plate containing 10 ml pre-warmed RPMI ( supplemented with 10% FBS and 2 mM glutamine ) .",
"t = 96 hr: media and cells were harvested .",
"Protocols for analyzing protein expression and viral titers were identical to those described for 293T cells experiments .",
"293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were seeded onto glass cover slips ( 24 hr prior transfection ) and co-transfected with the designated siRNAs and the R9 HIV-1 expression construct as described above .",
"48 hr post-transfection , the media was removed and cells were fixed in 1 ml fixation buffer ( 2 . 5% glutaraldehyde/1% paraformaldehyde in sodium cacodylate buffer; 50 mM sodium cacodylate , 18 mM sucrose , 2 mM CaCl2 , pH 7 . 4 , 10 min ) .",
"This and all subsequent steps were performed at 23°C .",
"Fixed cells were washed three times in sodium cacodylate buffer ( 5 min ) , and stained with 2% OsO4 for 1 hr .",
"Each sample was then dehydrated in a graded ethanol series , dried in HDMS ( hexamethyldisilazane ) , and sputter coated with platinum .",
"SEM images were collected on a FEI Quanta 600 FEG scanning electron microscope at a beam energy of 10 kV , a spot size of three , and magnifications of 10 , 000 , 30 , 000 or 65 , 000 X . 293T cells ( 2 . 5 × 105 cells/well , 6-well plate ) were co-transfected with the designated siRNAs and the R9 HIV-1 expression construct as described above .",
"48 hr post-transfection , the media was removed and cells were fixed in 1 ml fixation buffer ( 2 . 5% glutaraldehyde/1% paraformaldehyde in sodium cacodylate buffer [50 mM sodium cacodylate , 18 mM sucrose , 2 mM CaCl2 , pH 7 . 4 , 10 min] ) .",
"This and all subsequent steps were performed at 23°C .",
"Fixed cells were harvested , washed three times in sodium cacodylate buffer ( 5 min ) , and stained with 2% OsO4 for 1 hr .",
"The pellet was rinsed once in sodium cacodylate buffer and twice in water ( 5 min ) , followed by incubation in 100 μl of a 4% uranyl acetate solution ( 30 min ) .",
"Stained cells were dehydrated in a graded ethanol series followed by acetone , and embedded in epoxy resin EMBed-812 ( Electron Microscopy Sciences , Hatfield , PA ) .",
"Thin sections ( 80–100 nm ) were cut , post-stained with saturated uranyl acetate ( 20 min ) , rinsed with water , dried , stained with Reynolds' lead citrate ( 10 min ) and dried again .",
"TEM images were collected on a Hitachi H-7100 transmission electron microscope at an accelerating voltage of 75 kV , equipped with a Gatan Orius sc1000 camera .",
"Imaged virions were scored as ‘mature’ if they lacked an observable cellular tether and a condensed core was evident , as ‘immature’ if they were untethered but lacked a condensed core , and as ‘budding’ if a Gag layer was clearly evident and the nascent virion was assembling or was clearly tethered to the cell .",
"The degree of assembly was assessed by rendering a ( hypothetical ) spherical particle and measuring the arc angle of the nascent Gag layer .",
"To image 500 virions budding from wild type cells , it was necessary to screen about eight-times as many thin slices of wild type control cells ( ∼2500 cells ) vs TSG101- or AMOT-depleted cells ( 275 and 300 , respesctively ) ."
]
] | [
"Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 E3 ubiquitin ligase family to facilitate virus release .",
"Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif , suggesting that NEDD4L may function through an adaptor protein .",
"Here , we demonstrate that the cellular protein Angiomotin ( AMOT ) can bind both NEDD4L and HIV-1 Gag .",
"HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT depletion , whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release .",
"Electron microscopic analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle .",
"Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding ."
] | [
"To multiply and spread infections , viruses must enter and exit cells .",
"Once inside a cell , many viruses conscript the cell's machinery to produce new viral particles and release them into the surroundings .",
"Some viruses—like HIV-1—exit the cell in a way that leads to them being wrapped ( or ‘enveloped’ ) in membrane from the host cell .",
"A virus protein called Gag is required for the release of HIV-1 and other enveloped viruses .",
"In some cases , Gag proteins bind directly to members of the NEDD4 protein family to enable the viruses to be released .",
"However , the Gag protein from HIV-1 does not appear to be able to interact directly with NEDD4 proteins , so it was not clear how Gag works in this case .",
"Mercenne et al . studied how HIV-1 is released from human cells grown in the laboratory .",
"The experiments show that members of a human protein family called the Angiomotins bind to both Gag and NEDD4L ( a member of the NEDD4 family ) and are required for the efficient release of viruses .",
"Using a technique called electron microscopy , Mercenne et al . observed that when Angiomotins are present , Gag proteins assemble in spheres at the cell membrane and viruses are able to exit the cell .",
"However , when Angiomotins are depleted or absent , incomplete spheres of Gag proteins accumulate on the inner membrane surface and viruses are not released .",
"These findings show that Angiomotins act as a link between Gag and NEDD4L to promote the release of HIV-1 from human cells .",
"The next step will be to learn precisely how this works .",
"There are indications that the Angiomotins may also be involved in the release of other enveloped viruses , so the findings may be useful for the development of treatments for a variety of viral infections ."
] | 2015 |
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